The Interactive Fly

Zygotically transcribed genes

Olfaction in Drosophila

Olfactory receptors Olfactory neurons of the antenna and maxillary palp Olfactory glomeruli in the antennal lobe Mushroom body and olfactory learning

  • Genetic and functional subdivision of the Drosophila antennal lobe
  • The wiring diagram of a glomerular olfactory system
  • Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila
  • Activity-dependent plasticity in an olfactory circuit
  • Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly
  • Elucidating the neuronal architecture of olfactory glomeruli in the Drosophila antennal lobe
  • Synaptic and circuit mechanisms promoting broadband transmission of olfactory stimulus dynamics
  • Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe
  • Integration of chemosensory pathways in the Drosophila second-order olfactory centers
  • Serotonergic modulation differentially targets distinct network elements within the antennal lobe of Drosophila melanogaster
  • Identified serotonergic modulatory neurons have heterogeneous synaptic connectivity within the olfactory system of Drosophila
  • Decoding of context-dependent olfactory behavior in Drosophila
  • Embryonic origin of olfactory circuitry in Drosophila: contact and activity-mediated interactions pattern connectivity in the antennal lobe
  • Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits
  • Mechanisms underlying population response dynamics in inhibitory interneurons of the Drosophila antennal lobe
  • Circuit variability in the antennal lobe interacts with excitatory-inhibitory diversity of interneurons to regulate network encoding capacity
  • Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit
  • Identification and analysis of a glutamatergic local interneuron lineage in the adult Drosophila olfactory system
  • Early integration of temperature and humidity stimuli in the Drosophila brain
  • Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system
  • Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli
  • Synaptic spinules in the olfactory circuit of Drosophila melanogaster

    GABAergic neurons
  • Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe
  • A presynaptic gain control mechanism fine-tunes olfactory behavior
  • A single GABAergic neuron mediates feedback of odor-evoked signals in the mushroom body of larval Drosophila
  • Calcium imaging revealed no modulatory effect on odor-evoked responses of the Drosophila antennal lobe by two populations of inhibitory local interneurons

    Projection neurons
  • Target neuron prespecification in the olfactory map of Drosophila
  • Developmental origin of wiring specificity in the olfactory system of Drosophila
  • Integration of chemosensory pathways in the Drosophila second-order olfactory centers
  • A population of projection neurons that inhibits the lateral horn but excites the antennal lobe through chemical synapses in Drosophila
  • Developmentally programmed remodeling of the Drosophila olfactory circuit
  • Teneurins instruct synaptic partner matching in an olfactory map
  • Synaptic organization of the Drosophila antennal lobe and its regulation by the Teneurins
  • Dendritic Eph organizes dendrodendritic segregation in discrete olfactory map formation in Drosophila
  • Metamorphosis of an identified serotonergic neuron in the Drosophila olfactory system
  • Orthodenticle is required for the development of olfactory projection neurons and local interneurons in Drosophila
  • Convergence, divergence, and reconvergence in a feedforward network improves neural speed and accuracy
  • Calcium imaging revealed no modulatory effect on odor-evoked responses of the Drosophila antennal lobe by two populations of inhibitory local interneurons
  • Cav3-type α1T calcium channels mediate transient calcium currents that regulate repetitive firing in Drosophila antennal lobe PNs
  • Odor discrimination in Drosophila: from neural population codes to behavior
  • GABAergic projection neurons route selective olfactory inputs to specific higher-order neurons
  • Olfactory coding from the periphery to higher brain centers in the Drosophila brain
  • Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement
  • Wiring variations that enable and constrain neural computation in a sensory microcircuit
  • The organization of projections from olfactory glomeruli onto higher-order neurons

    Odor coding in the Drosophila maxillary palp

    Olfactory systems detect and differentiate among many kinds of odor stimuli. Olfactory receptor neurons encode qualitative, quantitative, temporal, and spatial information about odors. Elucidating the mechanisms by which olfactory information is encoded is an intriguing problem in contemporary neurobiology. A major difficulty has been the complexity of most olfactory systems. In the case of mammals, for example, the number of olfactory receptor neurons (ORNs) is very large, as is the number of distinct functional classes into which these neurons may fall. Moreover, in many systems it is difficult to measure systematically the physiological properties of individual ORNs in vivo. Insect ORNs are distributed in sensilla, usually in the form of sensory hairs that protrude from the cuticle, providing accessibility and ease of identification. Odorants pass through tiny pores in the walls of these sensilla and stimulate dendrites bathing in the lymph inside. Drosophila has a relatively simple olfactory system. Olfactory response can be measured in vivo, via either physiological or behavioral means, and a variety of genetic and molecular approaches are available to study its olfactory system (de Bruyne, 1999 and references).

    Each of the paired maxillary palps contains only 120 ORNs, housed in 60 sensilla of a single category, sensilla basiconica. The numerical simplicity of the maxillary palp makes it possible to perform an exhaustive study of its neuronal composition and to characterize in detail its functional organization. Extensive extracellular recordings from single sensilla reveal that the neurons fall into six functional classes. Each of the 60 sensilla houses two neurons (A and B), which observe a pairing rule: each sensillum combines neurons of two particular classes A or B in a fixed, not a random, configuration. Behaviorial studies reveal that there are three sensillum types (1, 2 or 3). The three sensillum types are intermingled on the surface of the palp, but their distribution is not random. Since there are two neurons in each sensilla, and three sensillum types, the cellular contents of each of the three sensilla are identified as follows: sensillum type 1 contains neurons 1A and 1B, sensillum type 2 contains neurons 2A and 2B and sensillum type 3 contains neurons 3A and 3B, thus accounting for the six functional classes). Identity of a sensillum is not strictly determined by its position. The neuron pairs in each sensillum exhibit different response characteristics, providing the basis for an olfactory code. A particular odor can excite one neuron and inhibit another; a particular neuron can be excited by one odor and inhibited by another. Some excitatory responses continue beyond the end of odor delivery, but responses to most odors terminate abruptly after the end of odor delivery, with some followed by a period of poststimulus quiescence. Adaptation and cross-adaptation have been documented, and cross-adaptation experiments demonstrate that the two neurons within one type of sensillum can function independently (de Bruyne, 1999).

    ORNs from single sensilla on the Drosophila maxillary palp exhibit spontaneous action potentials, clearly distinguishable from background noise. The spikes from an individual sensillum can be resolved into two distinct populations, based on their amplitudes (large and small). This bimodal distribution of spike amplitudes is interpreted as representing the activities of two distinct neurons, an interpretation that has been extensively supported in a wide variety of other insect sensory systems. The large and small spikes represent the output of the two neurons that make up each of the 60 sensilla of the maxillary palp. The two neurons in each sensilla account for the 120 ORNs in each maxillary palp. The two neurons of a sensillum are referred to as the A and B cells (de Bruyne, 1999).

    Diversity among the olfactory neurons of the maxillary palp can be observed by analysis of their spontaneous firing rates. The spontaneous action potential frequency of individual cells varies, with most neurons exhibiting frequencies between 3 and 13 spikes/sec. The spontaneous rate of one of these individual cells is relatively constant, however; it stays within a range of +/-3 spikes/sec over the course of recording periods lasting as long as 60 min. In addition to these neurons, some neurons have noticeably higher spontaneous rates of ~30 spikes/sec. Odor stimulation elicits a marked increase in firing frequency in many cases. In most cases one of the two neurons in a sensillum is clearly more excited than was the other by stimulation with a particular odor. The excited neuron can generally be identified by its spike height at the start of the response. In some cases the spike amplitude of the responding neuron gradually changes during the course of stimulation, a phenomenon widely observed in single-sensillum recordings from insects. In such cases, the identities of the neurons can often be confirmed if necessary by examination of the spike shapes, which differ between the two neurons. Excitatory responses start ~80 msec after odor is administered; calculations reveal that 50 msec is required for odor to reach the preparation, indicating a response latency of ~30 msec. The spike frequency rises to a maximum within 50-100 msec after response initiation, depending on the odor and dose, and then declines (de Bruyne, 1999).

    Excitatory responses terminate abruptly after the end of the odor stimulation period in most cases. This is consistent with the expected sharp decline in odor levels after the 500 msec delivery period. In these cases a brief period is often observed in which no action potentials are recorded from this neuron. The duration of this poststimulus quiescence appears to be dose-dependent, with higher doses producing longer periods of quiescence, although this relationship has not been examined quantitatively. Poststimulus quiescence is not, however, observed for all odors. In fact, in some cases excitation continues long past the end of odor stimulation. Thus the variations in response at the end of odor stimulation show that different odors may have different effects on the kinetics of excitation, thereby providing one putative mechanism for odor discrimination. Inhibitory responses were also observed in some sensilla. Interestingly, in these sensilla some odors inhibit one cell but excite the other; the cell with the large spikes is inhibited, and the cell with the small spikes is excited. Thus an individual odor can have opposite effects on the firing frequency of different neurons, and an individual neuron can respond oppositely (excitation vs. inhibition) to different odors (de Bruyne, 1999).

    To define the basic elements of the olfactory code, attempts were made to determine whether ORNs fall into discrete functional classes and, if so, to determine the number and odor specificity of such classes. This analysis was initiated with a chemically diverse group of 16 odorants selected on the basis of three criteria. Some odorants (ethyl acetate, 3-octanol, and benzaldehyde) were selected because they have been used extensively in previous research on Drosophila olfaction and are known to induce strong behavioral responses. Others were selected because they play important roles in the ecology of related dipteran insects. For example, 4-methylphenol is present in cattle urine and attracts tsetse flies to their hosts; E2-hexenal (leaf aldehyde) is a plant odor that attracts many insect species. Finally, odors were selected to represent certain chemical groups (e.g., ketones, aldehydes, alcohols, esters, and aromatics) (de Bruyne, 1999 and references).

    Initial recordings from individual sensilla clearly indicated that sensilla can be divided into three functional types, termed palpal basiconic 1 (pb1), pb2, and pb3. Although of the same morphological category, the three types of sensilla contain neurons with response spectra that are distinct from one another and from neurons of the other sensillum types. The two neurons of the pb1 sensillum are denote pb1A and pb1B. Of these two neurons, pb1B cell responds strongly to only one of the tested odorants, 4-methylphenol, to which it shows an increase in spike frequency of 178 +/- 47 spikes/sec (+/- SD; n = 13). The only other response from pb1B that is significantly different from that of the paraffin oil control is the response to 4-methylcyclohexanol, an odor molecule structurally similar to 4-methylphenol. The pb1A cell, by contrast, shows a broader range of responses to the tested stimuli: it responds most strongly to ethyl acetate, showing an increase of 138 +/- 32 spikes/sec (+/- SD; n = 13), but also responds to several other stimuli (de Bruyne, 1999).

    The different neurons of the maxillary palp could be distinguished using a diagnostic subset of 7 of the 16 odors. These 7 odors were therefore used to extend the analysis to a larger number of sensilla. The results confirm the presence of exactly three sensillum types, each containing two neurons, yielding a total of six types of neurons with distinguishable response properties: pb1A, pb1B, pb2A, pb2B, pb3A, and pb3B. The pb1 sensilla contain an A cell that responds strongly to ethyl acetate and a B cell that responds strongly to 4-methylphenol. In the pb2 sensilla, the A cell responds strongly to benzaldehyde. The other cell, pb2B, is excited by 4-methylphenol, although not as strongly as is pb1B. In addition, pb2B is strongly inhibited by 3-octanol and several other odors. Specifically, the spontaneous firing frequency of pb2B is 32 +/- 7 spikes/sec and is reduced 80-100% by 3-octanol. The pb3 sensillum contains two neurons that are both excited by 3-octanol and isoamyl acetate, but pb3B is more strongly stimulated by isoamyl acetate than is pb3A. In summary, this analysis reveals that the maxillary palp contains six distinguishable neuronal types. The responses of some neuronal types are overlapping (e.g., 4-methylphenol excites both pb1B and pb2B neurons), but comparisons of responses to multiple odors clearly shows the profiles to be distinct. In pb1 and pb3 sensilla, A neurons consistently have larger spike amplitudes than B neurons. pb2 sensilla differ from pb1 and pb3 sensilla, not only because the B neuron consistently has higher spontaneous activity than the other five neuronal types but also because pb2A has smaller spikes than pb2B in some sensilla. However, spikes of pb2A are consistently different in shape, having larger negative phases relative to the positive phase (de Bruyne, 1999).

    The two neurons in one sensillum observe a pairing rule; for example, the cell that is excited by benzaldehyde (pb2A) is always paired with a cell that is inhibited by 3-octanol (pb2B). Among 232 sensilla examined, the activity of two cells were recorded in 225 cases (in the 7 exceptional sensilla, one cell was absent or unresponsive, perhaps because of damage caused by the recording electrode in at least some cases); in 222 of the 225 cases, one of the three characteristic combinations of cells was found (de Bruyne, 1999).

    Although the existence of exactly six neuronal classes seemed clear from this physiological analysis, the classification scheme was tested more rigorously. A cluster analysis of the responses of 54 neurons in 27 sensilla to five of the odors was performed. This analysis yielded five clusters based on the response profiles. Cluster 1 groups cells with a strong response to ethyl acetate (corresponding to pb1A), and cluster 2 unites cells responding strongly to benzaldehyde (pb2A). Clusters 5 and 4 contain cells that respond to 4-methylphenol but that either are or are not inhibited by 3-octanol (pb2B and pb1B, respectively). Cluster 3 includes cells that are excited by both isoamyl acetate and 3-octanol but that show no strong response to the other odors; this cluster includes both pb3A and pb3B cells. This analysis confirms the initial observation that ORNs on the palp can reliably be assigned to cell classes that have clearly distinct response characteristics. This cluster analysis, however, did not resolve pb3A and pb3B. Therefore further evidence was sought that pb3A and pb3B are distinguishable by testing their responses to an additional set of odors. Significant responses from pb3 cells were only observed to 3 of the initial 16 odorants. A homologous series of esters was chosen because the previous analysis had revealed a difference in the responses of pb3A and pb3B to isoamyl acetate. Aliphatic esters with varying chain lengths of both the alcohol and acid moieties were tested. The responses of pb3A and pb3B are clearly distinguishable, in two respects. (1) For most of the esters there is a significant difference in the absolute responses of the two neurons; for example, for pentyl acetate, the response of pb3B is 99 +/- 12 spikes/sec, whereas the response of pb3A is only 9 +/- 2 spikes/sec. (2) The relative responses of the two neurons to different odors vary; for example, the pb3A neuron responds dramatically better to ethyl butyrate than to hexyl acetate, whereas for pb3B the converse is true. This analysis confirms that the response spectra of pb3A and pb3B are clearly different and that the pb3 sensillum, like pb1 and pb2, contains two distinguishable neurons. It is noted with interest that for pb3A, the length of the odorant molecule correlates with potency; odors that elicit the strongest mean responses are those with a total of 6 carbons, with the next most potent being those with 4, 5, or 7 carbons, followed by those with 8 or 10 carbons. However, none of the tested odors elicits more than ~30 spikes/sec from this neuron, leading to a suspicion that there are other molecules in odor space that are more effective stimuli for this neuron (de Bruyne, 1999).

    All three sensillum types are found on both male and female palps, and no evidence was found of sexual dimorphism of any kind. A quantitative analysis of spike frequency shows no differences between the sexes in the response of pb1 sensilla to any of seven odors tested (ethyl acetate, isoamyl acetate, 4-methylphenol, benzaldehyde, 3-octanol, E2-hexenal, and cyclohexanone); limited data reveal no differences between the sexes for pb2 or pb3 sensilla. These results are consistent with the lack of sexual dimorphism in the glomeruli of the antennal lobes and form a striking contrast with the dimorphism observed in the olfactory sensilla and glomeruli of moths (de Bruyne, 1999).

    The number of functional types of neurons on the maxillary palp (six) is of the same order as the estimated number of five glomeruli that receive afferent fibers from it. This approximate numerical equivalence, in which the number of neuronal classes is determined by direct physiological analysis, is consistent with the approximate equivalence of the number of glomeruli with the number of neuronal types in the mammalian main olfactory epithelium. In moths, there is also a well documented pattern of projection from particular functional classes of neurons, pheromone-sensitive neurons, to a cluster of specialized glomeruli, the macroglomerular complex. However, it remains to be seen whether the two neurons in each sensillum type project to one glomerulus, giving three palpal glomeruli, or to two glomeruli, which would give six palpal glomeruli. The total number of olfactory glomeruli reported for the antennal lobe of Drosophila is ~40 (Laissue, 1999). Hence the total peripheral input to the olfactory-processing centers in the CNS may consist of ~40 basic types of input elements (de Bruyne, 1999 and references).

    A major way in which the insect olfactory system differs from that of vertebrates is that the neurons of the sensory field are compartmentalized in sensilla. In this respect, the insect olfactory system can be considered (by analogy to the compound eye) as a 'compound nose'. Neurons of the palp are ordered with respect to this level of organization; the six types of neurons are distributed within sensilla in stereotyped pairs, with a neuron of one particular response spectrum cohabiting in a sensillum with a neuron of another particular response spectrum. Despite the intimate cohabitation of neurons within a sensillum, cross-adaptation experiments show that they are able to function primarily as independent units of perception. This independence and their distinct response spectra are observed despite the fact that both neurons in a sensillum share a common pool of binding proteins (Hekmat-Scafe, 1997; see Drosophila Lush for more information on odorant binding proteins or OBPs) and a common electrical circuit. In this sense, olfactory tissues in insects may be similar to those in mammals (de Bruyne, 1999 and references).

    The largely overlapping distribution of the three sensillum types across the sensory field shows that in the maxillary palp there is not an obvious odotopic layout at the primary neuron level. However, the presence of a pb2 'exclusion' zone provides some heterogeneity and likely points to zones that are developmentally distinct. This analysis of sensillum function and organization raises interesting questions about how such a sensory field develops. Olfactory sensilla develop from founder cells (Ray, 1995) that build different sensillum categories. The mixed distribution of functional types on the palp and the variability in the positions of individual sensilla suggest that sensilla are not committed to one of the three alternative fates strictly according to their position in the field. It is not known how the expression of genes encoding the choice between pb1, pb2, and pb3 is regulated. Moreover, further studies will be necessary to reveal the developmental logic by which the stereotyped pairing of neurons is produced. What is the mechanism that regulates expression of class-specific elements such as receptors and coordinates it between the two neurons of one sensillum? (de Bruyne, 1999).

    The olfactory system of adult Drosophila contains two organs, the antenna and the maxillary palp. Each antenna is covered with ~500 sensilla of three morphological types, whereas the maxillary palp is covered with 60 sensilla of a single morphological type. It is tempting to compare this arrangement with the presence of two olfactory organs in mammals, the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). Like the VNO, the maxillary palp has been associated with pheromone response and the modulation of sexual behavior. The VNO neurons project to an accessory olfactory bulb that does not overlap with the main olfactory bulb; likewise, projections from the palp have been reported to map to a subset of glomeruli distinct from those that receive input from the antenna. However, VNO neurons differ from MOE neurons in that they are microvillous rather than ciliate, whereas both palpal and antennal ORNs are ciliate. In addition, the VNO neurons probably perceive a different type of odorants (less volatile and larger molecules) from those perceived by the MOE, whereas the present study shows that the maxillary palp is sensitive to many small, volatile molecules that also stimulate the antenna. No evidence was found for a special role of the palp in pheromone perception. This is based on two findings: (1) cis-vaccenyl acetate, a compound reported previously as an inhibitory sex pheromone induces a response from pb1A. However, this neuron responds more strongly to many other odors, and cis-vaccenyl acetate has also been found to stimulate an antennal neuron. (2) No differences were found between male and female ORN populations, making it unlikely that sex-specific pheromonal receptor cells occur on the palps (de Bruyne, 1999 and references).

    Odor coding in the Drosophila antenna

    Odor coding in the Drosophila antenna is examined by a functional analysis of individual olfactory receptor neurons (ORNs) in vivo. Sixteen distinct classes of ORNs, each with a unique response spectrum to a panel of 47 diverse odors, were identified by extracellular recordings. ORNs exhibit multiple modes of response dynamics: an individual neuron can show either excitatory or inhibitory responses, and can exhibit different modes of termination kinetics, when stimulated with different odors. The 16 ORN classes are combined in stereotyped configurations within seven functional types of basiconic sensilla. One sensillum type contains four ORNs and the others contain two neurons, combined according to a strict pairing rule. A functional map of ORNs is provided, showing that each ORN class is restricted to a particular spatial domain on the antennal surface (de Bruyne, 2001).

    Electrophysiological recordings from basiconic sensilla on the antenna consistently show spontaneous action potentials. Spikes from most recordings could be resolved into two populations based on their amplitudes. The two populations of spikes in these sensilla indicate the presence of two neurons, as found in basiconic sensilla on the maxillary palp (de Bruyne, 1999). The neuron with the larger spike amplitude is referred to as the A neuron and the one with the smaller spikes as the B neuron (de Bruyne, 2001).

    Neurons show excitatory responses to odors, with different neurons excited by different odors. For instance, one A neuron increases its firing frequency in response to ethyl acetate, while the B neuron spike frequency is unaffected. By contrast, the B neuron in the same sensillum is excited by hexanol. In another sensillum, the A neuron is excited by 1-octen-3-ol, whereas the B neuron is unaffected. In this sensillum, the B neuron is excited by ethyl butyrate; the A neuron also shows a modest increase in firing frequency (de Bruyne, 2001).

    Recordings of spontaneous activity from some large basiconic sensilla reveal the presence of four neurons, whose spike amplitudes fell into four classes. These physiological results confirm ultrastructural observations that while most basiconic sensilla house two neurons, many large basiconic sensilla contain four neurons. Strikingly, one of these neurons, the C neuron, responds strongly to CO2 (de Bruyne, 2001).

    Analysis reveals that sixteen ORN classes are housed in stereotyped combinations in seven functional types of basiconic sensilla. A set of 47 odorants was chosen to characterize the responses of neurons in antennal basiconic sensilla. The odorants were chosen from a variety of chemical classes and include compounds that contain branched chains, double bonds, two functional groups, and other structural features. Most odorants were chosen because of evidence that they play a role in the chemical ecology of Drosophila or other flies. In addition to testing a large number of neurons with the entire set of 47 odorants, a subset of 12 diagnostic odorants was identified that is particularly useful in identifying and distinguishing among neuronal classes: these 12 odorants were used in most subsequent experiments (de Bruyne, 2001).

    Extensive recordings from large basiconic sensilla reveal that they fall into three distinct types based on the odor response spectra of their ORNs. These three types are referred to as antennal basiconic (ab) types ab1, ab2, and ab3, as distinct from pb sensilla (basiconic sensilla on the maxillary palp [de Bruyne, 1999]). Each ab type contains a stereotyped combination of neurons (de Bruyne, 2001).

    The ab1 sensillum is unique in that it houses four neurons. The A neuron within this sensillum type, referred to as ab1A, responds most strongly to ethyl acetate. This neuron also responds to several other odorants, notably ethyl butyrate, but it is unusual in responding strongly to the diluent control stimulus, paraffin oil. It is likely that for this particular neuron, much of the response observed to other odors represents a response to an element of the delivery system. The ab1B, C, and D neurons respond with a high degree of specificity to 2,3-butanedione, CO2, and methyl salicylate, respectively; they do not respond to the diluent control stimulus. The other two types of large basiconic sensilla, ab2 and ab3, contain only two neurons. ab2 sensilla contain an A neuron that responds strongly to ethyl acetate, with a somewhat weaker response to 2,3-butanedione, and a B neuron that responds moderately to ethyl butyrate and hexanol. ab3 contains an A neuron that is excited by ethyl butyrate and pentyl acetate and a B neuron that responds to heptanone and hexanol (de Bruyne, 2001).

    The functional classification of eight neuronal types in large basiconic sensilla was confirmed through a cluster analysis. The eight ORNs are distinct from the six of the maxillary palp. Furthermore, of the 145 large basiconic sensilla from which recordings were taken, all could be classified as either ab1, ab2, or ab3; no evidence was found for an additional type (de Bruyne, 2001).

    In addition to recording from large basiconic sensilla, recordings were taken from 128 small basiconic sensilla on the anterior and posterior surfaces of the third antennal segment. An additional four sensillum types, called ab4, ab5, ab6, and ab7, were identified based on odor response profiles. Each of the four sensillum types contains two neurons distinct from each other and from all other previously defined neuronal types, including those on the maxillary palp. Thus, an additional eight ORN classes housed within the small basiconic sensilla have been defined. Moreover, the neurons in the small basiconic sensilla, like those in the large basiconic sensilla, are housed in characteristic combinations; they observe a strict pairing rule (de Bruyne, 2001).

    The ab4 sensillum houses a neuron, ab4A, that responds strongly to E2-hexenal, a 'green leaf volatile'; other insects have ORNs that appear to be narrowly tuned to this molecule. Not only does this odor elicit a high frequency of firing from the ab4A neuron, but the neuron continues to fire above spontaneous levels for more than a minute following a 0.55 s pulse of E2-hexenal. ab5A responds strongly to geranyl acetate, a monoterpene ester, whereas ab5B responds strongly to pentyl acetate and heptanone. ab6A shows a strong response to a number of the selected subset of 12 odors, but 1-octen-3-ol is distinguished by evoking a long-lasting excitation such as that for ab4A. The ab7A neuron shows moderate responses to several odors. None of the 12 odors strongly excited ab4B, ab6B, or ab7B. However, ab6B is excited by 4-methylphenol and inhibited by ethyl acetate and pentyl acetate, and ab7B is mildly excited by ethyl butyrate (de Bruyne, 2001).

    Although all of the large basiconic sensilla from which recordings were taken could be clearly identified as ab1, 2, or 3, some of the small basiconic sensilla (23 of 128) did not fall into a well-defined category. The neurons in these sensilla exhibited spontaneous action potentials, and most yielded weak excitatory responses to at least some odors, but none responded strongly to any of the 47 odors in the full panel (de Bruyne, 2001).

    16 classes of ORNs have been found on the antenna. How are these classes distributed spatially? As useful coordinates in describing the distributions of these functional units, regions drawn on the basis of sensillar morphology are referred to. Specifically, the anterior and posterior surfaces of the third antennal segment are covered with olfactory sensilla, but the sensory field is heterogeneous in terms of sensillar morphology and can be divided accordingly into five regions. Regions I and II contain exclusively large basiconic sensilla. Region III is largely devoid of sensilla, except for a number of coeloconic sensilla. Region IV contains both small basiconic and coeloconic sensilla. Finally, region V contains a high density of trichoid sensilla, but also contains some small basiconic and coeloconic sensilla (de Bruyne, 2001).

    Each functional type of sensillum -- and consequently each functional class of neuron -- is restricted to a particular spatial domain of the antennal surface. Although certain types are intermingled, each is compartmentalized by particular spatial boundaries. Ab1, ab2, and ab3 are restricted to regions I and II, whereas the four remaining sensillar types are localized exclusively in regions IV and V (this segregation follows from the fact that ab1, ab2, and ab3 are the only large basiconic sensillar types, and from the definition of the regions). However, ab1, ab2, and ab3 do not show identical distributions. ab3 shows a more restricted distribution; it is limited to region I and, moreover, is restricted to the dorso-medial portion of region I. The distribution of ab1 overlaps closely with that of ab2. ab4 and ab6 are found in region IV but not region V, and their distributions appear similar. ab5 and ab7 are found in both regions IV and V. Thus, four functional domains can be distinguished: those for (1) ab1 and ab2; (2) ab3; (3) ab4 and ab6 and (4) ab5 and ab7. Within a functional domain the distributions of functional types may not be identical, but a more detailed analysis will be required to establish these distinctions conclusively (de Bruyne, 2001).

    How many neurons of each functional class are located on the antenna? Among the large basiconic sensilla, 50% were identified as ab1, with 30% as ab2 and 20% as ab3. The results suggest that there are on the order of 45 ab1A, B, C, and D neurons, 27 of the ab2 neurons, and 18 of the ab3 neurons. This estimate predicts that 50% of large basiconic sensilla would contain four neurons, which is in excellent agreement with the estimate of 52% from ultrastructural studies of males. Similar calculations suggest that there are 13-28 of the neuronal types in small basiconic sensilla, again assuming that the sampling bias is not large (de Bruyne, 2001).

    What relationship is there between the number of ORN classes and the number of glomeruli? The most informative data are available for the ORNs within large basiconic sensilla. Eight classes of these ORNs have been defined and no evidence has been found for any additional classes. Approximately seven glomeruli are indicated as receiving input from the region of the antenna containing large basiconic sensilla. While there are some uncertainties in comparing the two studies, it is nonetheless striking that the number of ORN classes is similar to the number of identified glomeruli. This numerical similarity is consistent with the finding that neurons expressing a particular odorant receptor project to one or two glomeruli, both in vertebrates and in Drosophila. Furthermore, the total number of ORN classes identified in this study, 16, when added to the 6 classes characterized in an extensive analysis of maxillary palp sensilla (de Bruyne, 1999) and the 7 classes for which evidence is found in a preliminary characterization of trichoid and coeloconic sensilla on the antenna, yields a total of 29, a number which approaches 41, the number of olfactory glomeruli that have been identified in Drosophila (de Bruyne, 2001).

    An approximate numerical correspondence is also observed between the number of neurons per ORN class and the number of ORNs expressing an individual odor receptor gene. Specifically, it is estimated that the number of neurons in a functional class ranges from 13 to 45. In situ hybridization experiments have shown that among four individual odor receptor genes tested quantitatively, each was expressed in 16 to 40 cells per antenna, a figure that is in reasonable agreement with data for a number of other receptor genes. This numerical correspondence is consistent with the concept that neurons of a particular functional class, i.e., neurons with a particular odor response spectrum, express the same odor receptor gene(s) (de Bruyne, 2001).

    Three variables describe an olfactory stimulus: odor identity (analogous to hue), odor concentration (intensity), and time. Each of these variables is represented in the action potential frequency of different receptor neurons in the Drosophila antenna. Different ORN classes show diverse odor response spectra, sensitivities, and dynamics. Correspondingly, a particular odor signal produces a different response among different ORN classes (de Bruyne, 2001).

    Although individual ORNs may have evolved to respond to particular ligands of ecological importance, 'cross-stimulation' of ORNs by other ligands is likely to be critical in the coding of odor identity. Of the 16 ORN classes defined in this study, 11 responded to more than one of the test stimuli. Conversely, most (73%) of the test stimuli that elicited a response from any ORN elicited a response from multiple ORNs, and 23% elicited a response from five or more ORN classes. The response of individual ORNs to multiple odors, and the response of multiple ORNs to individual odors, is consistent with a number of other studies of vertebrate ORNs and some other insect ORNs. An example of how odor identity could be encoded by integrating the responses of multiple ORNs is illustrated by the responses of ab1A and ab3A to ethyl acetate and ethyl propionate. The ORN that is most sensitive to ethyl acetate, ab1A, is also highly sensitive to ethyl propionate. How then can the fly discriminate between a high dose of ethyl acetate and ethyl propionate? One possible answer is by integrating the response of ab1A with that of ab3A, which responds to ethyl propionate but not ethyl acetate (de Bruyne, 2001).

    Is the structure of the code arbitrary? In principle, if each ORN class represented a binary coding unit, the 16 classes described here could encode 216, or some 65,000, different odors. However, in interpreting the response matrix, it is useful to consider that the ORN repertoire of Drosophila is not designed for maximal computational efficiency across the entirety of odor space, but rather has evolved to fit the organism's need for survival and reproduction. Esters and alcohols, which are commonly present in fermenting fruits, elicit responses from a relatively large number of neurons, which may thereby provide resolving power to aid in discrimination among these odors (de Bruyne, 2001).

    Coding of odor concentration is also likely to depend upon the multiplicity of responding ORNs. For example, ab1A is very sensitive to ethyl acetate, saturating at a 10-4 dilution. ab2A is less sensitive, showing a linear relationship that initiates at 10-4 and remains linear until 10-2; thus the presence of two ethyl acetate neurons of differing sensitivities effectively expands the dynamic range of the response. Low doses of ethyl acetate are likely to be encoded by ab1A; at higher doses, not only is a stronger signal sent by ab1A to a glomerulus in the antennal lobe, but an additional signal is transmitted by ab2A, presumably to an additional glomerulus. It has been noted that among the dose-response relationships characterized for ORNs of both the antenna and the maxillary palp (de Bruyne, 1999), the slopes are remarkably constant. Moreover, the linear phase of the curve in each case extends over at least a 100-fold range of odor dilutions (de Bruyne, 2001).

    Both excitation and inhibition of ORNs by odors have been documented. The ability of a neuron to exhibit two modes of response adds a degree of freedom that may be used in the coding of odor identity. Inhibition may provide a mechanism for enhancing signal recognition; for example, an odor may inhibit some ORNs while exciting others, which may enhance contrast in the glomeruli. In fact, linalool can inhibit firing of the ab2A neuron, but can excite the ab4A, ab6A, and ab7A neurons. Linalool is a terpene common among plants and excites ORNs from a variety of insect species; it has previously been shown to inhibit pheromone-sensitive ORNs in a moth (de Bruyne, 2001).

    In addition to odor quality and quantity, the temporal profile of an odor stimulus also must be encoded by spike trains. The temporal profile of an odor is critical to flying insects that seek odor sources. Such insects encounter intermittent 'odor pockets' while traversing an odor plume, and the frequency of such encounters is an essential parameter in navigation (de Bruyne, 2001).

    How is temporal information encoded? In addition to whether an individual ORN fires, and the frequency of firing, a third degree of freedom is the distribution of spikes in time. The spike frequency is not a simple reflection of the instantaneous odor concentration. For example, in some cases the spike train terminates abruptly after stimulus termination, and in other cases the spike train continues long after the odor stimulus has ceased; these radically different patterns can be observed for two neurons in the same sensillum, stimulated with the same odor. The underlying molecular basis of these kinetic differences is not known. One possibility is that they relate to differences in ligand-receptor binding affinities; another possibility is that they represent differences in the kinetics of receptor deactivation, with post-stimulus inhibition perhaps representing an extreme form of receptor deactivation. In any case, this third degree of freedom could provide a means of expanding the coding capacity of the system. Fast off-responses may convey information about rapid changes in odor concentration, such as those encountered by a flying insect. Slow off-responses may play another role in the fly's orientation behavior, perhaps by providing a 'memory' of a recently encountered odor stimulus. Thus, the two different response modes may simultaneously present a phasic and tonic representation of selected features of the odor environment (de Bruyne, 2001).

    The spatial organization of the antennal domains, as determined by physiological recording, are of interest in a developmental context. The boundaries of these domains are roughly perpendicular to the axis that extends from the dorso-medial portion of the antenna to the ventro-lateral portion. Thus, ab3 sensilla are the most dorso-medial, followed by ab1 and ab2, with ab4 and ab6 occupying an intermediate domain, and ab5 and ab7 located more ventro-laterally. This axis is parallel to the direction of many mitotic clones observed in developmental studies of the antenna and to the orientation of individual sensilla. It is perpendicular to the boundaries between a number of sensillar types as defined anatomically. Not only do large basiconic sensilla lie dorso-medial to the small basiconic sensilla, but at the dorso-medial extreme lies an ultrastructural subclass of large basiconic sensilla called LBI-1, which corresponds well in distribution and number to the ab3 sensilla. Another ultrastructural subclass of basiconic sensilla, TB-2, shows a distribution pattern similar to that of the ab4 and ab6 sensilla. It is noted also that the distribution of most of the sensilla that could not be classified is similar to that of 'sensilla intermedia,' a type of sensillum which, by electron microscopy, exhibits features intermediate between basiconic and trichoid sensilla. It is tempting to speculate that the different sensillar classes that have been defined functionally can also be distinguished anatomically, and that their distinct characteristics arise during development by differential expression of many genes, including receptor genes, along a morphogenetic axis (de Bruyne, 2001).

    Chemosensory coding by neurons in the coeloconic sensilla of the Drosophila antenna

    Odor coding is based on the diverse sensitivities and response properties of olfactory receptor neurons (ORNs). In the Drosophila antenna, ORNs are housed in three major morphological types of sensilla. Although investigation of the Drosophila olfactory system has been expanding rapidly, the ORNs in one of these types, the coeloconic sensilla, have been essentially unexplored. Four functional types of coeloconic sensilla were defined through extracellular physiological recordings. Each type contains at least two neurons, with a total of at least seven distinct ORN classes that vary remarkably in their breadth of tuning. Analysis of 315 odorant-ORN combinations reveals how these neurons sample odor space via both excitation and inhibition. A class of neurons was defined that is narrowly tuned to small amines, and humidity detectors were found that define a cellular basis for hygroreception in Drosophila. The temporal dynamics of responses vary widely, enhancing the potential for complexity in the odor code. Molecular and genetic analysis shows that a broadly tuned ORN, antennal coeloconic 3B (ac3B), requires the odor receptor gene Or35a for its response in vivo. The activity of ac3B is not required for the response of the other ORN within that sensillum, ac3A. The functional analysis presented here, revealing a combination of highly specialized neurons and a broadly tuned ORN, along with the ancient origin of coeloconic sensilla, suggests that the specificities of these ORNs may reflect basic needs of an ancestral insect (Yao, 2006; Full text of article).

    The first step in the coding of an olfactory stimulus is the activation of ORNs in olfactory organs. This study examined in detail the coding of olfactory information by ORNs in a major class of olfactory sensilla, the coeloconic sensilla, whose function in Drosophila has until now been essentially unexplored (Yao, 2006).

    Physiological recordings identified four distinct functional types of coeloconic sensilla on the antennal surface. Each type contains at least two ORNs, and the data show that there are at least seven distinguishable classes of ORNs; it was not possible to distinguish with confidence the activities of the different ORNs in ac4 sensilla, but it is clear from the response to phenylacetaldehyde, for example, that at least one of the ORNs in ac4 is distinct from those in ac1, ac2, and ac3. It is noted that an anatomical study of coeloconic sensilla revealed two subtypes: one containing two neurons and the other containing three neurons. It thus seems likely that at least one functional type, such as ac4, contains three ORNs (Yao, 2006).

    Neurons were identified in the Drosophila antenna that respond strongly to humidity. Hygroreception is critical in the insect world. Many insects, such as mosquitoes, lay eggs in water, and dry environments can lead to desiccation and death. Drosophila exhibits preferences when given a choice between environments of different humidities; however, the molecular and genetic basis of hygroreception is not understood, and even its cellular basis has until now been unknown in Drosophila (Yao, 2006).

    In defining a cellular basis of hygroreception in Drosophila, a focus is provided for a molecular and genetic analysis of the mechanism of transduction. It is noted that ablation experiments had provided evidence in a previous study that hygroreception localizes to the arista, a feathery projection extending from the third antennal segment. However, the current study also found that spinelessaristapedia, a homeotic mutant in which aristae are transformed into leg-like structures, had normal hygroreception. These studies do not exclude the possibility of hygroreceptive cells associated with the arista or elsewhere; however, identification of hygroreceptors in coeloconic sensilla is consistent with studies of other insects in which hygroreceptive cells were identified in morphologically similar sensilla (Yao, 2006).

    The temporal dynamics of humidity responses appear largely tonic. The responses to odors vary in their dynamics. The diverse temporal dynamics shown by coeloconic ORNs may reflect another degree of freedom used by the antenna in odor coding: the temporal structure of olfactory information has been shown to be critical in odor coding in a variety of systems (Yao, 2006).

    It is noted with interest that, although the transduction mechanisms underlying hygroreception and olfaction may differ, they are likely to be housed in a common cell (i.e., in a multimodal neuron: isoamylamine inhibits all neuronal activity in ac2 sensilla), including that of humidity-sensitive cells, suggesting that hygroreceptive cells contain a receptor for isoamylamine (Yao, 2006).

    Amine detectors were also detected. One class of ORN, ac1A, is highly sensitive to ammonia, and another, ac2A, responds strongly to 1,4-diaminobutane. A particularly important example of amine detection in insects is the attraction of mosquitoes to ammonia, a component of human sweat. Ammonia-sensitive cells have been detected in double-walled, grooved-peg sensilla on the antenna of Anopheles mosquitoes and Triatoma infestans, a vector of trypanosomiasis, or Chagas disease. Identification of these cells in Drosophila lays a foundation for examining the molecular genetics of amine detection, which, in turn, could be useful in designing new means of pest control (Yao, 2006).

    The response spectra of the coeloconic ORNs vary remarkably in their apparent breadth of tuning. At one extreme is ac3B, which reveals exceptionally broad tuning. ac3B is excited by 36 of 45 odors at the test concentrations; there is evidence that some coeloconic sensilla in the moth Bombyx mori yield responses to a variety of acids and alcohols. ac1B, in contrast, is excited by none of these 45 odors; it may be the cell in ac1 that responds to humidity. ac1A responds strongly to ammonia and weakly to two other amines, but shows no responses to the other 42 tested odors. The profile of ac2A suggests that it may have evolved to signal the presence of 1,4-diaminobutane. No strong excitatory responses were detected among the neurons of ac4, other than a modest response to phenylacetaldehyde, and it seems likely that they detect the presence of biologically significant stimuli that are not included in the odor set. Conclusions about the breadth of ORN tuning are limited by the number of odors tested. Odor space is vast and discontinuous, and the sampling of odors in this study is necessarily limited. Nonetheless, the pattern that emerges from this analysis is one of a single broadly tuned ORN, ac3B, and others that are excited by only one or a small number of stimuli (Yao, 2006).

    In light of the widely varying tuning breadths of these ORNs, it seems plausible that the coeloconic sensilla have evolved in large part to signal the presence of a small number of specific chemosensory stimuli, such as water vapor, ammonia, and 1,4-diaminobutane, also known as putrescine. The exceptionally broad spectrum of ac3B could have evolved as a more general sensor to signal the simultaneous presence of food sources. The coeloconic sensilla have an ancient origin, and perhaps their specificities reflect the most basic needs of an ancestral insect (Yao, 2006).

    The specificities of these neurons are also of interest in light of the recent construction of a receptor-to-neuron map for the Drosophila antenna. Or genes were individually expressed in a mutant in which a particular ORN, ab3A, loses its odorant response because of a deletion that removes two odorant receptor genes, Or22a and Or22b. When particular Or genes were expressed in this mutant ORN, they conferred in many cases an odor response spectrum that matched that of a defined ORN. In this way, individual Or genes could be assigned to particular ORNs. Testing of virtually the entire repertoire of antennal Or genes mapped many receptor genes to basiconic ORNs, but only one, Or35a, to a coeloconic ORN. Because a number of receptors in that study conferred response profiles that did not match those of any defined ORNs, these unmapped receptors were further tested, using a panel of odorants from that the present study has identified as diagnostic for particular coeloconic ORN classes. However, it was not possible to map any of these receptors to coeloconic ORNs (Yao, 2006).

    Why has only a single class of coeloconic ORN been found to derive its odor response profile from an Or gene? One possible interpretation is that some coeloconic ORNs, for example those sensitive to humidity or ammonia, do not rely on receptors of the Or family to detect the molecules that activate them. It is noted that no Or gene was mapped to ab1C, a basiconic ORN that is a sensor of CO2. A Gr (Gustatory receptor) gene has been mapped to this neuron, although at present there is no evidence that it is a CO2 receptor. In the present study, it was shown that Or35a is necessary for the response of ac3B. An important goal now is to determine which receptors are necessary for the responses of the other coeloconic neurons. The analysis of coeloconic ORNs presented in this study provides a foundation for investigating the molecular and genetic basis of the mechanisms by which coeloconic ORNs transduce chemosensory signals. It also provides a basis for investigating the roles of these neurons in olfactory-driven behaviors (Yao, 2006).

    Measuring activity in olfactory receptor neurons in Drosophila: Focus on spike amplitude

    Olfactory responses at the receptor level have been thoroughly described in Drosophila melanogaster by electrophysiological methods. Single sensilla recordings (SSRs) measure neuronal activity in intact individuals in response to odors. For sensilla that contain more than one olfactory receptor neuron (ORN), their different spontaneous spike amplitudes can distinguish each signal under resting conditions. However, activity is mainly described by spike frequency. Some reports on ORN response dynamics studied two components in the olfactory responses of ORNs: a fast component that is reflected by the spike frequency and a slow component that is observed in the LFP (local field potential, the single sensillum counterpart of the electroantennogram, EAG). However, no apparent correlation was found between the two elements. This report shows that odorant stimulation produces two different effects in the fast component, affecting spike frequency and spike amplitude. Spike amplitude clearly diminishes at the beginning of a response, but it recovers more slowly than spike frequency after stimulus cessation, suggesting that ORNs return to resting conditions long after they recover a normal spontaneous spike frequency. Moreover, spike amplitude recovery follows the same kinetics as the slow voltage component measured by the LFP, suggesting that both measures are connected. These results were obtained in ab2 and ab3 sensilla in response to two odors at different concentrations. Both spike amplitude and LFP kinetics depend on odorant, concentration and neuron, suggesting that like the EAG they may reflect olfactory information (Martin, 2016).

    Hedgehog signaling regulates the ciliary transport of odorant receptors in Drosophila

    Hedgehog (Hh) signaling is a key regulatory pathway during development and also has a functional role in mature neurons. This study shows that Hh signaling regulates the odor response in adult Drosophila olfactory sensory neurons (OSNs). This is achieved by regulating odorant receptor (OR) transport to and within the primary cilium in OSN neurons. Regulation relies on ciliary localization of the Hh signal transducer Smoothened (Smo). This study further demonstrates that the Hh- and Smo-dependent regulation of the kinesin-like protein Cos2 acts in parallel to the intraflagellar transport system (IFT) to localize ORs within the cilium compartment. These findings expand knowledge of Hh signaling to encompass chemosensory modulation and receptor trafficking (Sanchez, 2016).

    This study demonstrates that the Hh pathway modulates the magnitude of the odorant response in adult Drosophila. The results show that the Hh pathway determines the level of the odorant response because it regulates the response in both the positive and negative directions. Loss of Ptc function increases the odorant response and the risk for long sustained responses, which shows that the Hh pathway limits the response potential of the OSNs and is crucial for maintaining the response at a physiological level. In addition, it was shown that the OSNs produce Hh protein, which regulates OR localization, which is interesting because autoregulation is one of the prerequisites for an adaptive mechanism. It was further shown that Hh signaling regulates the responses of OSNs that express different ORs, which demonstrates that the regulation is independent of OSN class and suggests that Hh signaling is a general regulator of the odorant response. It has been shown previously that Hh tunes nociceptive responses in both vertebrates and Drosophila (Babcock, 2011). It is not yet understood how Hh regulates the level of nociception. However, the regulation is upstream of the nociceptive receptors, which indicates that the Hh pathway is a general regulator of receptor transport and the level of sensory signaling (Sanchez, 2016).

    The results show that OSN cilia have two separate OR transport systems, the Hh-regulated Cos2 and the intraflagellar transport complex B (IFT-B) together with the kinesin II system. The results show that Cos2 is required for OR transport to or within the distal cilium domain and suggest that the IFT system regulates the inflow to the cilium compartment. The two transport systems also are required for Smo cilium localization (Kuzhandaivel, 2014). This spatially divided transport of one cargo is similar to the manner in which Kif3a and Kif17 regulate distal and proximal transport in primary cilia in vertebrates. However, Cos2 is not required for the distal location of Orco or tubulin (Kuzhandaivel, 2014), indicating that, for some cargos, the IFT system functions in parallel to Cos2 (Sanchez, 2016).

    Interestingly, the vertebrate Cos2 homolog Kif7 organizes the distal compartment of vertebrate primary cilia (He, 2014). Similar to the current results, Kif7 does so without affecting the IFT system, and its localization to the cilia is dependent on Hh signaling. However, the Kif7 kinesin motor function has been questioned (He, 2014). Therefore, it will be interesting to analyze whether Kif7-mediated transport of ORs and other transmembrane proteins occurs within the primary cilium compartment and whether the ciliary transport of ORs is also regulated by Hh and Smo signaling in vertebrates. To conclude, these results place the already well-studied Hh signaling pathway in the post-developmental adult nervous system and also provide an exciting putative role for Hh as a general regulator of receptor transport to and within cilia (Sanchez, 2016).

    Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search

    Internal physiological states influence behavioral decisions. This study investigated the underlying cellular and molecular mechanisms at the first olfactory synapse for starvation modulation of food search behavior in Drosophila. It was found that a local signal by short neuropeptide F (sNPF) and a global metabolic cue by insulin are integrated at specific odorant receptor neurons (ORNs) to modulate olfactory sensitivity. Results from two-photon calcium imaging show that starvation increases presynaptic activity via intraglomerular sNPF signaling. Expression of sNPF and its receptor (sNPFR1) in Or42b neurons is necessary for starvation-induced food search behavior. Presynaptic facilitation in Or42b neurons is sufficient to mimic starvation-like behavior in fed flies. Furthermore, starvation elevates the transcription level of sNPFR1 but not that of sNPF, and insulin signaling suppresses sNPFR1 expression. Thus, starvation increases expression of sNPFR1 to change the odor map, resulting in more robust food search behavior (Root, 2011).

    This study reports that a state of starvation modulates olfactory sensitivity at the first synapse in a form of presynaptic facilitation. Starvation increases sNPFR1 transcription in ORNs, which is both necessary and sufficient for presynaptic facilitation. It has been well established that fluctuation of insulin is a key metabolic cue to maintain energy homeostasis. This study implicates that a low insulin signal via the PI3K pathway increases sNPFR1 expression. Interestingly, a subset of glomeruli exhibit starvation-dependent presynaptic facilitation that depends on intraglomerular sNPF signaling, while selective knockdown of sNPF or sNPFR1 in only the DM1 glomerulus affects food search behavior. This finding corroborates previous work revealing that the DM1 glomerulus is hardwired for innate odor attraction (Semmelhack, 2009). Thus, an internal state of starvation, with insulin as a global satiety signal acting on sensory neurons through a local sNPF signal, shifts the odor map. Starvation modulation of the odor map increases the saliency of glomerular activity to match the changing physiological needs of an organism (Root, 2011).

    The Or42b sensory neurons may be considered as a neural substrate for appetitive choices because they integrate internal and external cues to influence an important innate behavior. In this integration a highly conserved neuropeptide plays an important role in the peripheral olfactory system. A similar presynaptic facilitation mechanism may exist in vertebrates as well. In an aquatic salamander, NPY has been shown to enhance electrical responses of cells in the olfactory epithelium to a food related odorant in starved animals. In addition, NPY immunoreactivity has been observed in the olfactory epithelium of mouse and zebrafish. In the nematode C. elegans, elevated activity levels of an NPY-like receptor cause a change in foraging pattern (Macosko, 2009). This study demonstrates that a fluctuating metabolic cue controls sNPFR1 levels in Or42b neurons, which in turn modulates appetitive behavior. However, it remains to be determined whether other ORNs mediate attraction behavior and whether they are subject to sNPF mediated modulation. Given the ubiquitous use of insulin as a metabolic cue, modulation by NPY/sNPF receptors in the early olfactory system could be a conserved mechanism between different animal species (Root, 2011).

    The internal state of an organism influences its behavior. There is abundant evidence indicating that the global metabolic cue, insulin, works together with local neuropeptides in specific neural circuits to generate state-dependent behavioral responses. In Drosophila, the tolerance of a noxious food source is suppressed by insulin signaling and enhanced by NPF signaling such that these two peptides exert their opposing effects on the same neurons that mediate the behavior. In the mammalian hypothalamus, expression of the orexigenic NPY is suppressed in the satiety state via insulin signaling. Results from this study indicate that olfactory response in the periphery is reduced in the satiety state, in which insulin suppresses NPFR1 expression to alter neuronal excitability. Insulin's upstream control over sNPFR1 expression, however, appears to be specific to select neuronal types. Previous work in Drosophila has shown that sNPFR1 signaling exerts upstream control of insulin production in the Dilp2 neuroendocrine cells (Lee, 2008). In C. elegans, the release of an insulin-like peptide in an interneuron is downstream of a neuropeptide involved in promoting behavioral adaptation to food odors (Chalasani, 2010). Thus, different neuronal subtypes may adopt the same neuropeptides for unique and divergent molecular responses. Peptidergic modulation provides a rich repertoire of functional states for the same neural circuit to meet the demand of different internal states (Root, 2011).

    Central mechanisms to control appetitive behavior, similar to the well-documented modulation of the hypothalamus by NPY, also appear to be important in Drosophila. A recent study demonstrates that appetitive memory requires the NPF receptor in the dopaminergic neurons that innervate specific mushroom body lobes (Krashes, 2009). This poses the question: what functions are subserved by starvation modulation of multiple neural substrates? It is interesting to note that sensitization of Or42b ORNs is sufficient to enhance food search behavior in fed flies. Perhaps central modulation by starvation is not necessary for food search behavior. Modulation in the periphery may serve to gate an animals’ sensitivity to specific food odorants, while central modulation may serve to enhance an animal’s ability to remember the relevant cues in finding a particular food source (Root, 2011).

    Multiple sites of adaptation lead to contrast encoding in the Drosophila olfactory system

    Animals often encounter large increases in odor intensity that can persist for many seconds. These increases in the background odor are often accompanied by increases in the variance of the odor stimulus. Previous studies have shown that a persistent odor stimulus (odor background) results in a decrease in the response to brief odor pulses in the olfactory receptor neurons (ORNs). However, the contribution of adapting mechanisms beyond the ORNs is not clear. Thus, it is unclear how adaptive mechanisms are distributed within the olfactory circuit and what impact downstream adaptation may have on the encoding of odor stimuli. In this study, adaptation to the same odor stimulus is examined at multiple levels in the well studied and accessible Drosophila olfactory system. The responses of the ORNs are compared to the responses of the second order, projection neurons (PNs), directly connected to them. Adaptation in PN spike rate was found to be much greater than adaptation in the ORN spike rate. This greater adaptation allows PNs to encode odor contrast (ratio of pulse intensity to background intensity) with little ambiguity. Moreover, distinct neural mechanisms contribute to different aspects of adaptation; adaptation to the background odor is dominated by adaptation in spike generation in both ORNs and PNs, while adaptation to the odor pulse is dominated by changes within olfactory transduction and the glomerulus. These observations suggest that the olfactory system adapts at multiple sites to better match its response gain to stimulus statistics (Cafaro, 2016).

    Embryonic origin of olfactory circuitry in Drosophila: contact and activity-mediated interactions pattern connectivity in the antennal lobe

    Olfactory neuropiles across different phyla organize into glomerular structures where afferents from a single olfactory receptor class synapse with uniglomerular projecting interneurons. In adult Drosophila, olfactory projection interneurons, partially instructed by the larval olfactory system laid down during embryogenesis, pattern the developing antennal lobe prior to the ingrowth of afferents. In vertebrates it is the afferents that initiate and regulate the development of the first olfactory neuropile. This study investigated the embryonic assembly of the Drosophila olfactory network. Dye injection and genetic labelling was used to show that during embryogenesis, afferent ingrowth pioneers the development of the olfactory lobe. With a combination of laser ablation experiments and electrophysiological recording from living embryos, it was shown that olfactory lobe development depends sequentially on contact-mediated and activity-dependent interactions, and an unpredicted degree of similarity was revealed between the olfactory system development of vertebrates and that of the Drosophila embryo. Electrophysiological investigation is also the first systematic study of the onset and developmental maturation of normal patterns of spontaneous activity in olfactory sensory neurons, and some of the mechanisms regulating its dynamics were uncovered. It was found that as development proceeds, activity patterns change, in a way that favours information transfer, and that this change is in part driven by the expression of olfactory receptors. These findings show an unexpected similarity between the early development of olfactory networks in Drosophila and vertebrates and demonstrate developmental mechanisms that can lead to an improved coding capacity in olfactory neurons (Prieto-Godino, 2012).

    A striking feature of olfactory system organization is the conserved arrangement of olfactory sensory neuron (OSN) terminals and uniglomerular projections neurons (PNs) into an odotopic glomerular map. Previous studies lead to the conclusion that the sequence of events and developmental mechanisms patterning connectivity among OSNs and PNs in vertebrates and in insects are radically different. However, most studies of the development of the olfactory network in insects have focused on adult development. This study uncovered developmental events and mechanisms leading to the embryonic assembly of the Drosophila olfactory network from the beginning, before contacts are made, until functional maturity at hatching. It was found that afferent ingrowth pioneers AL development and that contact and activity-dependent interactions among the components of the circuit are essential for appropriate patterning of connectivity in the larval AL. This study provides insights into axon-to-dendrite and axon-to-axon interactions in neural circuit assembly and reveals an unexpected degree of similarity with other embryonically developing vertebrate olfactory systems. Furthermore, this paper provides systematic study of the onset and developmental maturation of normal patterns of spontaneous activity in OSNs. The implications of these findings is discussed in the context of general principles of neural network development and more specifically with a focus on the development of connectivity in olfactory circuits (Prieto-Godino, 2012).

    A key finding in this study is the interdependence of OSNs and PNs for the proper development of the larval antennal lobe (AL). Although at early stages of embryogenesis OSN and PN axons approach the site of the future AL independently of each other, once PN dendrites penetrate the emerging AL, interactions with OSN regulate the patterning of connectivity (Prieto-Godino, 2012).

    Embryonic development of the Drosophila AL begins with OSN terminals targeting distinct territories that probably represent the origins of AL glomeruli. At this stage PN axons turn away from this site and continue growing towards higher brain centres. By the time growth cones of OSN axons contact the proximal region of PNs axons, the PNs have not yet extended any dendrites. Hours later, PN extend dendrites directed towards particular territories within the emerging AL, possibly guided by the same cues that direct OSN terminal targeting. The early arrival of OSNs in the future region of the AL before PN dendrite extension suggested a possible role for OSNs in the development of the AL. Indeed, this study found that PNs require presynaptic innervation for their survival, although innervation does not necessarily have to come from OSNs. Additionally, there is no specific requirement for OSN terminals in promoting sprouting of PN dendrites since in the absence of OSNs, surviving PNs have dendrites. These dendrites are normally longer than controls, suggesting they elongate until they find presynaptic partners, with the implication that OSNs normally give PN dendrites a stop growth signal. This effect is both contact and activity dependent, because PNs in animals where all OSNs had been silenced have overgrown dendrites that do not extend beyond the AL. A similar effect has been found in the dendrites of motorneurons in Drosophila embryos, where the removal of presynaptic terminals induces an overgrowth of postsynaptic motorneuron dendrites that anticipates the dendritic overgrowth induced by the lack of pre-synaptic activity at later developmental stages (Prieto-Godino, 2012).

    Independently of whether PNs survive or not, in all cases the AL is lost when OSNs are ablated. Loss of the AL has also occurred on an evolutionary scale in terrestrial isopods, which in the process of colonising the land have secondarily lost their olfactory sensilla in the main olfactory appendage, together with the corresponding olfactory deutocerebral structures (second neuromere of the supraesophageal ganglion where the olfactory lobe is located). Furthermore, in some species the tritocerebrum (posteriorly adjacent neuromere to the deutocerebrum) seems to have acquired additional neuropile structures. The findings show that there is an interdependence in the development of the Drosophila embryonic olfactory system that results in the loss of deutocerebral olfactory structures (the AL) in response to the ablation of OSNs. At the same time the finding of occasional ectopic tritocerebral and subesophageal innervation of PNs indicates a possible developmental route for the evolutionary acquisition of additional tritocerebral structures (Prieto-Godino, 2012).

    These results contrast with previous studies in adult Drosophila, which show that PNs pioneer development of the adult AL independently from adult OSN development. Why is development of the olfactory system in Drosophila different during embryogenesis and metamorphosis? Interestingly, experiments in other embryonically developing olfactory systems, in both vertebrates and invertebrates, also demonstrate an essential role for OSN ingrowth in the development of their first olfactory centres. Experiments in Xenopus where OSNs were removed unilaterally at early embryonic stages showed that an olfactory bulb fails to develop on the ablated side, but is present on the control side. Similarly, an experiment in cockroaches where most, but not all, OSNs were unilaterally removed during embryogenesis before they innervate the AL showed that the deafferented lobe was severely disrupted, its characteristic glomeruli were missing, and it was markedly reduced in volume. Furthermore, as with the current findings, PNs in these partially deafferented lobes were sparsely branched and had elongated dendrites instead of their characteristic uniglomerular tufts. In contrast, when OSNs were ablated early in adult development in insects (Manduca and Drosophila adult) an AL still formed, and PN dendrites arborized in their glomerular territories. It is concluded that the differences found in the development of the Drosophila larval and adult olfactory systems probably arise from fundamental differences between embryonic development and metamorphosis. In embryos (vertebrate or Drosophila) there is no preexisting network to guide development, whereas during metamorphosis the adult olfactory system makes use of cues derived from the larval olfactory system. Thus its wiring seems to rely more on external cues and less on interactions among its network components than the wiring of the larval network (Prieto-Godino, 2012).

    The method allows spontaneous activity to be recorded from OSNs developing in vivo in the Drosophila embryo. Although it has been assumed that OSNs in mice and insects may be active during development , and there is a previous report of activity recorded from the antennal nerve of Manduca during adult development, this is the first systematic description of the onset and developmental maturation of normal patterns of spontaneous activity in OSNs (Prieto-Godino, 2012).

    The results reveal three important features about the development of activity patterns in OSNs:

  • As in other developing systems, the earliest action potentials generated by OSNs are different from mature ones, with smaller amplitude and a longer duration. Such changes in spike shape seem to be a general feature of emerging activity as ionic conductances are acquired and mature (Prieto-Godino, 2012).
  • At early stages, intermittent bursts of activity are recorded in the OSNs. Activity patterns that consist of spontaneous bursts are common to many developing neural networks, including the auditory, visual, motor, and olfactory systems, and their time course is remarkably similar across different neural systems, with inter-burst intervals varying between 0.5 and 2 mi. Such activity may be an inevitable consequence of cells acquiring mature excitable properties, but it is also possible that the generality of these activity patterns, and the diversity of mechanisms by which they are generated and terminated, is an indication of an essential and significant role in the development of neural networks (Prieto-Godino, 2012).
  • As development proceeds, variability of the spike train diminishes, which is predicted according to information theory to increase signal (odour) detectability.
  • A previous in vitro study of locust frontal ganglion neurons showed that there is a transient period during the wiring process when activity is irregular, but as the network matures, regularity increases. This is the first direct statistical analysis of the transition from immature to mature spike-trains in vivo and allows leads to the suggestion that the coding capabilities of the network improve as it develops. It seems likely that a change towards patterns that would be expected to increase signal detectability, and thus network functionality, would be a general feature in neural networks as they mature (Prieto-Godino, 2012).

    The mechanisms by which this immature activity is generated, shaped, and terminated vary from system to system. In the embryonic OSNs, the transition from irregular spike-trains to continuous discharge may require the expression of olfactory receptors (OR), because in larvae mutant for the co-receptor Orco Or83b, necessary for OR function, this transition does not occur normally. Since Orco is expressed before the onset of spontaneous activity, it is suggested that the change in the pattern of OSN spontaneous activity is likely to be driven, at least in part, by the onset and level of expression of specific ORs. However, this might not be the only factor shaping spontaneous activity patterns over development, and other factors such as expression of other ion channels may also play a role. This might explain why 16 h AEL Orco mutants have indistinguishable levels of activity when compared with controls, yet the variability in their spike train is significantly increased (Prieto-Godino, 2012).

    Previous studies have suggested that spontaneous activity is essential for the normal development of vertebrate OSNs, but that there is no such requirement in insects. However, this study found that there is a role for OSN activity in the development of the larval olfactory network. OSN activity regulates the morphology of OSN terminals independently of activity in neighbouring axons, and without activity terminals appear immature and occupy larger territories. This is similar to what has been described in zebrafish and mouse OSN terminals devoid of activity. There is also a report of a similar phenotype found in the AL of third instar Drosophila larvae after synaptic release was blocked in a large subset of OSNs. The results show that while immature terminal morphology is a cell autonomous phenotype that is independent of activity levels in neighbouring OSN axons, the expansion of OSN terminals is limited by interactions among the OSN terminals. Interestingly a similar process has been found to regulate the morphology and terminal expansion of retinotectal axons. Thus the control of axonal terminal extension via activity-dependent interactions may be a general process in the wiring of nervous systems. The nature of inter-axonal interactions that limit terminal growth remains unknown and is one example of how future work using amenable experimental systems such as the one provided by the larval olfactory network in Drosophila larvae may reveal general mechanisms operating during the assembly of neural circuitry (Prieto-Godino, 2012).

    Mechanisms underlying population response dynamics in inhibitory interneurons of the Drosophila antennal lobe

    Local inhibitory neurons control the timing of neural activity in many circuits. To understand how inhibition controls timing, it is important to understand the dynamics of activity in populations of local inhibitory interneurons, as well as the mechanisms that underlie these dynamics. This study describes the in vivo response dynamics of a large population of inhibitory local neurons (LNs) in the Drosophila melanogaster antennal lobe, the analog of the vertebrate olfactory bulb, and dissects the network and intrinsic mechanisms that give rise to these dynamics. Some LNs respond to odor onsets ("ON" cells) and others to offsets ("OFF" cells), whereas still others respond at both times. Moreover, different LNs signal odor concentration fluctuations on different timescales. Some respond rapidly, and can track rapid concentration fluctuations. Others respond slowly, and are best at tracking slow fluctuations. A continuous spectrum of preferred stimulation timescales was found among LNs, as well as a continuum of ON-OFF behavior. Using in vivo whole-cell recordings, it was shown that the timing of an LN's response (ON vs OFF) can be predicted from the interplay of excitatory and inhibitory synaptic currents that it receives. Meanwhile, the preferred timescale of an LN is related to its intrinsic properties. These results illustrate how a population of inhibitory interneurons can collectively encode bidirectional changes in stimulus intensity on multiple timescales, and how this can arise via an interaction between synaptic and intrinsic mechanisms (Nagel, 2016).

    Circuit variability in the antennal lobe interacts with excitatory-inhibitory diversity of interneurons to regulate network encoding capacity

    Local interneurons (LNs) in the Drosophila olfactory system exhibit neuronal diversity and variability, yet it is still unknown how these features impact information encoding capacity and reliability in a complex LN network. This study used two strategies to construct a diverse excitatory-inhibitory neural network beginning with a ring network structure and then introduced distinct types of inhibitory interneurons and circuit variability to the simulated network. The continuity of activity within the node ensemble (oscillation pattern) was used as a readout to describe the temporal dynamics of network activity. Inhibitory interneurons were found to enhance the encoding capacity by protecting the network from extremely short activation periods when the network wiring complexity is very high. In addition, distinct types of interneurons have differential effects on encoding capacity and reliability. Circuit variability may enhance the encoding reliability, with or without compromising encoding capacity. Therefore, this study has described how circuit variability of interneurons may interact with excitatory-inhibitory diversity to enhance the encoding capacity and distinguishability of neural networks. This work has evaluate the effects of different types and degrees of connection diversity on a ring model, which may simulate interneuron networks in the Drosophila olfactory system or other biological systems (Tsai, 2018).

    Diverse populations of local interneurons integrate into the Drosophila adult olfactory circuit

    Drosophila olfactory local interneurons (LNs) in the antennal lobe are highly diverse and variable. How and when distinct types of LNs emerge, differentiate, and integrate into the olfactory circuit is unknown. Through systematic developmental analyses, this study found that LNs are recruited to the adult olfactory circuit in three groups. Group 1 LNs are residual larval LNs. Group 2 are adult-specific LNs that emerge before cognate sensory and projection neurons establish synaptic specificity, and Group 3 LNs emerge after synaptic specificity is established. Group 1 larval LNs are selectively reintegrated into the adult circuit through pruning and re-extension of processes to distinct regions of the antennal lobe, while others die during metamorphosis. Precise temporal control of this pruning and cell death shapes the global organization of the adult antennal lobe. These findings provide a road map to understand how LNs develop and contribute to constructing the olfactory circuit (Liou, 2018.

    Early integration of temperature and humidity stimuli in the Drosophila brain

    The Drosophila antenna contains receptor neurons for mechanical, olfactory, thermal, and humidity stimuli. Neurons expressing the ionotropic receptor IR40a have been implicated in the selection of an appropriate humidity range, but although previous work indicates that insect hygroreceptors may be made up by a 'triad' of neurons (with a dry-, a cold-, and a humid-air-responding cell), IR40a expression included only cold- and dry-air cells. This study reports the identification of the humid-responding neuron that completes the hygrosensory triad in the Drosophila antenna. This cell type expresses the Ir68a gene, and Ir68a mutation perturbs humidity preference. Next, the projections of Ir68a neurons were followed to the brain, and they were shown to form form a distinct glomerulus in the posterior antennal lobe (PAL). In the PAL, a simple sensory map represents related features of the external environment with adjacent 'hot,' 'cold,' 'dry,' and 'humid' glomeruli - an organization that allows for both unique and combinatorial sampling by central relay neurons. Indeed, flies avoided dry heat more robustly than humid heat, and this modulation was abolished by silencing of dry-air receptors. Consistently, at least one projection neuron type received direct synaptic input from both temperature and dry-air glomeruli. These results further understanding of humidity sensing in the Drosophila antenna, uncover a neuronal substrate for early sensory integration of temperature and humidity in the brain, and illustrate the logic of how ethologically relevant combinations of sensory cues can be processed together to produce adaptive behavioral responses (Frank, 2017).

    Glutamate is an inhibitory neurotransmitter in the Drosophila olfactory system

    Glutamatergic neurons are abundant in the Drosophila central nervous system, but their physiological effects are largely unknown. This study investigated the effects of glutamate in the Drosophila antennal lobe, the first relay in the olfactory system and a model circuit for understanding olfactory processing. In the antennal lobe, one-third of local neurons are glutamatergic. Using in vivo whole-cell patch clamp recordings, this study found that many glutamatergic local neurons are broadly tuned to odors. Iontophoresed glutamate hyperpolarizes all major cell types in the antennal lobe, and this effect is blocked by picrotoxin or by transgenic RNAi-mediated knockdown of the GluClα gene, which encodes a glutamate-gated chloride channel. Moreover, antennal lobe neurons are inhibited by selective activation of glutamatergic local neurons using a nonnative genetically encoded cation channel. Finally, transgenic knockdown of GluClα in principal neurons disinhibits the odor responses of these neurons. Thus, glutamate acts as an inhibitory neurotransmitter in the antennal lobe, broadly similar to the role of GABA in this circuit. However, because glutamate release is concentrated between glomeruli, whereas GABA release is concentrated within glomeruli, these neurotransmitters may act on different spatial and temporal scales. Thus, the existence of two parallel inhibitory transmitter systems may increase the range and flexibility of synaptic inhibition (Liu, 2013).

    Although glutamatergic neurons are abundant in the Drosophila brain, the role of glutamate as a neurotransmitter in the Drosophila CNS has received little study. In the antennal lobe, where approximately one-third of LNs are glutamatergic, the physiological effects of glutamate have never been characterized. This study shows that glutamate is an inhibitory transmitter that shapes the responses of PNs to olfactory stimuli (Liu, 2013).

    In the past, glutamate has been proposed to mediate lateral excitation between olfactory glomeruli. The results of this study demonstrate that the main effect of glutamate is inhibition, not excitation. The possibility cannot be ruled out that glutamate has small excitatory effects, but no evidence was found of excitation even when GluClα was knocked down genetically or inhibited pharmacologically. It is noted that there is in fact lateral excitation in the antennal lobe, which exists in parallel with lateral inhibition. However, lateral excitation is mediated not by glutamate, but by electrical coupling between LNs and PNs (Liu, 2013).

    All of the effects of glutamate on PNs were eliminated by knocking down GluClα. The dominant role for GluClα is notable, given how many other glutamate receptors are in the genome. The results are particularly surprising in light of two recent studies that have reported behavioral effects of knocking down an NMDA receptor subunit (NR1) in PNs. Further experiments will be needed to clarify the role of NR1 (Liu, 2013).

    There is a precedent for the idea that glutamate can be an inhibitory neurotransmitter in the Drosophila brain. Specifically, several studies have reported that bath-applied glutamate inhibits the large ventrolateral neurons of the Drosophila circadian clock circuit. Collectively, these studies suggest roles for both ionotropic and metabotropic glutamate receptors in glutamatergic inhibition. Regardless of which glutamate receptors are involved, these studies are consistent with the conclusion that glutamate is an important mediator of synaptic inhibition (Liu, 2013).

    The idea that glutamate can be inhibitory has important implications for neural coding. One particularly interesting case is the motion vision circuit of the Drosophila optic lobe. Two neuron types, L1 and L2, both receive strong synaptic inputs from photoreceptors, and they respond equally to contrast increments (“on”) and decrements (“off”). However, based on conditional silencing experiments, L1 is thought to provide input to an on pathway, and L2 to an off pathway. Therefore, opponency must arise downstream from L1 and L2. According to recent evidence, L1 is glutamatergic, whereas L2 is cholinergic. In light of the current data, that result suggests that L1 may actually be inhibitory, which would be sufficient to create opponency in the on and off pathways (Liu, 2013).

    Glutamate can act as an inhibitory neurotransmitter in the Caenorhabditis elegans olfactory circuit, and this fact too has implications for neural coding of odors in this organism. In the worm, a specific type of glutamatergic olfactory neuron inhibits one postsynaptic neuron via GluCl, while also exciting another postsynaptic neuron via an AMPA-like receptor. This arrangement creates a pair of opponent neural channels that respond in an anticorrelated fashion to odor presentation or odor removal, analogous to opponent channels in the visual system (Liu, 2013).

    This study has shown that the cellular actions of Glu-LNs are broadly similar to the actions of GABA-LNs. Specifically, both types of LNs inhibit PNs and other LNs. In addition, both GABA and glutamate inhibit neurotransmitter release from ORNs. Thus, both neurotransmitters inhibit all of the major cell types in the antennal lobe circuit. However, Glu-LNs and GABA-LNs are not functionally identical. In particular, it was found that the vesicular glutamate transporter is mainly confined to the spaces between glomeruli, whereas the vesicular GABA transporter is abundant within glomeruli. This finding implies that glutamate and GABA are released in largely distinct spatial locations. Consistent with this implication, no individual synaptic connections from Glu-LNs onto PNs were found, whereas a substantial rate of connections was found from GABA-LNs onto PNs. Nevertheless, PNs were found to be hyperpolarized by coactivation of multiple Glu-LNs, and PNs are disinhibited by knockdown of GluCl specifically in PNs (Liu, 2013).

    These results can be reconciled by a model where the sites of glutamate release are distant from PN glutamate receptors. As a result, glutamate would need to diffuse some distance to inhibit PNs. Coactivation of multiple Glu-LNs would increase extracellular glutamate concentrations, overwhelming uptake mechanisms and allowing glutamate to diffuse further. In this scenario, glutamatergic inhibition should be most important when LN activity is intense and synchronous. By comparison, GABAergic inhibition of PNs does not require LN coactivation, implying a comparatively short distance between presynaptic and postsynaptic sites. There is a precedent in the literature for the idea that different forms of inhibition can be differentially sensitive to LN coactivation, due to the spatial relationship between presynaptic and postsynaptic sites. In the hippocampus, GABAA receptors are closer than GABAB receptors to sites of GABA release, and so activation of individual interneurons produces GABAA but not GABAB currents, whereas coactivation of many interneurons produces both GABAA and GABAB currents (Liu, 2013).

    The pharmacology of glutamate-gated conductances in antennal lobe neurons is similar to the pharmacology of GABAA conductances in these neurons. This result should prompt a reevaluation of studies that used picrotoxin to block inhibition in the antennal lobe. Given the current results, it seems likely that these studies were reducing both glutamatergic and GABAergic inhibition (Liu, 2013).

    It is perhaps surprising that knocking down GluClα in PNs had such a substantial effect on PN odor responses, given that picrotoxin alone has comparatively modest effects. The solution to this puzzle may lie in the finding that glutamate regulates not only PNs but also GABA-LNs. Importantly, GABA-LNs are spontaneously active and provide tonic inhibition to PNs. Hence, in the intact circuit, glutamatergic inhibition of GABA-LNs should tend to disinhibit PNs. Picrotoxin prevents Glu-LNs from inhibiting GABA-LNs and should tend to potentiate GABAergic inhibition. The effects of GABA are mediated in part by GABAB receptors, which are not sensitive to picrotoxin. Thus, picrotoxin likely has bidirectional effects on the total level of inhibition in the circuit. By contrast, knockdown of GluClα specifically in PNs should not directly affect GABA-LNs and should not produce these complex effects. These results illustrate how a cell-specific genetic blockade of a neurotransmitter system can have more dramatic effects than a global pharmacological blockade of the same system (Liu, 2013).

    This study reveals that an LN can have push-pull effects on a single population of target cells. For example, Glu-LNs directly inhibit PNs, but they should also disinhibit PNs, via the inhibition of GABA-LNs. This architecture may allow for more robust gain control and rapid transitions between network states and is similar to the wiring of many cortical circuits, where corecruitment of excitation and inhibition is a common motif (Liu, 2013).

    Why might the existence of two parallel inhibitory transmitters be useful? The data argue that GABA and glutamate may act on different spatial and temporal scales. Because these two inhibitory systems comprise different cells, receptors, and transporters, they can be modulated independently. Because their properties are encoded by different genes, they can also evolve independently. This organization should confer increased flexibility in adapting synaptic inhibition to a changing environment (Liu, 2013).

    Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli

    The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. This study shows that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation (Wu, 2017)

    The use of discrete neuropil compartments for organizing and signaling information is widespread in invertebrate and vertebrate nervous systems. In both the fly antennal lobe and vertebrate olfactory bulb, axons from different ORN classes are segregated into distinct glomeruli. The rodent barrel cortex also uses discrete compartments, the barrels, to represent individual whiskers. This study shows that FGF signaling between neurons and glia mediates neural compartment formation in the Drosophila antennal lobe (Wu, 2017)

    Members of the FGF family have diverse functions in a variety of tissues in both vertebrates and invertebrates. Vertebrate FGFs regulate not only neural proliferation, differentiation, axon guidance, and synaptogenesis but also gliogenesis, glial migration, and morphogenesis. Many of these roles are conserved in invertebrates. For example, Ths and Pyr induce glial wrapping of axonal tracts, much like the role other FGF members play in regulating myelin sheaths in mammals. Ths and Pyr also control Drosophila astrocyte migration and morphogenesis; likewise, FGF signaling promotes the morphogenesis of mammalian astrocytes. Therefore, studying the signaling pathways in Drosophila will extend understanding of the principles of neural development (Wu, 2017)

    In ensheathing glia, whose developmental time course and mechanisms have not been well documented before this study, a glial response was observed to FGF signaling reminiscent of the paradigm shown previously; however, the exquisite compartmental structure of the Drosophila antennal lobe and genetic access allowed this study to scrutinize further the changes of neuropil structure and projection patterns that occurred alongside morphological phenotypes in ensheathing glia. The requirement for Ths in LNs was demonstrated, although it is possible that ORNs and PNs also contribute. The function was tested of the other ligand, Pyr, in antennal lobe development. No change was detected in ensheathing glia morphology with pyr RNAi, and double RNAi against ths and pyr did not enhance the phenotype compared with ths knockdown alone (Wu, 2017)

    FGF signaling in glomerular wrapping appears to be highly local. In overexpression experiments, the hyperwrapping effect was restricted to the glomerulus where the ligand is excessively produced and did not spread to nearby nonadjacent glomeruli. These experiments suggest that Ths communicates locally to instruct glial ensheathment of the glomeruli rather than diffusing across several microns to affect nearby glomeruli. Because heparan sulfate proteoglycans are known to act as FGF coreceptors by modulating the activity and spatial distribution of the ligands, it is speculated that Ths in the antennal lobe may be subject to such regulation to limit its diffusion and long-range effect (Wu, 2017)

    The data showed that deficient ensheathment of antennal lobe glomeruli is accompanied by imprecise ORN axon targeting. However, it was not possible to determine whether these targeting defects reflect initial axon-targeting errors or a failure to stabilize or maintain the discrete targeting pattern. Previous models for the establishment of antennal lobe wiring specificity suggested that the glomerular map is discernible by the time glia processes start to infiltrate the antennal lobe. Because of a lack of class-specific ORN markers for early developmental stages, the relative timing between when neighboring ORN classes refine their axonal targeting to discrete compartments and when ensheathing glia barriers are set up still remains unclear. Nevertheless, this discovery that FGF signaling functions in the formation of discrete neuronal compartments in the antennal lobe highlights an essential role for glia in the precise assembly of neural circuits (Wu, 2017)

    Synaptic spinules in the olfactory circuit of Drosophila melanogaster

    This study reports on ultrastructural features of brain synapses in the fly Drosophila melanogaster and outline a perspective for the study of their functional significance. Images taken with the aid of focused ion beam-scanning electron microscopy (EM) at 20 nm intervals across olfactory glomerulus DA2 revealed that some synaptic boutons are penetrated by protrusions emanating from other neurons. Similar structures in the brain of mammals are known as synaptic spinules. A survey with transmission EM (TEM) disclosed that these structures are frequent throughout the antennal lobe. Detailed neuronal tracings revealed that spinules are formed by all three major types of neurons innervating glomerulus DA2 but the olfactory sensory neurons (OSNs) receive significantly more spinules than other olfactory neurons. Double-membrane vesicles (DMVs) that appear to represent material that has pinched-off from spinules are also most abundant in presynaptic boutons of OSNs. Inside the host neuron, a close association was observed between spinules, the endoplasmic reticulum (ER) and mitochondria. It is proposed that by releasing material into the host neuron, through a process triggered by synaptic activity and analogous to axonal pruning, synaptic spinules could function as a mechanism for synapse tagging, synaptic remodeling and neural plasticity (Gruber, 2018).

    Role of GABAergic inhibition in shaping odor-evoked spatiotemporal patterns in the Drosophila antennal lobe

    Drosophila olfactory receptor neurons project to the antennal lobe, the insect analog of the mammalian olfactory bulb. GABAergic synaptic inhibition is thought to play a critical role in olfactory processing in the antennal lobe and olfactory bulb. However, the properties of GABAergic neurons and the cellular effects of GABA have not been described in Drosophila, an important model organism for olfaction research. Whole-cell patch-clamp recording, pharmacology, immunohistochemistry, and genetic markers have been used to investigate how GABAergic inhibition affects olfactory processing in the Drosophila antennal lobe. This study shows that many axonless local neurons (LNs) in the adult antennal lobe are GABAergic. GABA hyperpolarizes antennal lobe projection neurons (PNs) via two distinct conductances, blocked by a GABAA- and a GABAB-type antagonist, respectively. Whereas GABAA receptors shape PN odor responses during the early phase of odor responses, GABAB receptors mediate odor-evoked inhibition on longer time scales. The patterns of odor-evoked GABAB-mediated inhibition differ across glomeruli and across odors. LNs display broad but diverse morphologies and odor preferences, suggesting a cellular basis for odor- and glomerulus-dependent patterns of inhibition. Together, these results are consistent with a model in which odors elicit stimulus-specific spatial patterns of GABA release, and as a result, GABAergic inhibition increases the degree of difference between the neural representations of different odors (Wilson, 2005).

    Smell begins when odor molecules interact with olfactory receptor neurons (ORNs). ORNs then project to the brain following anatomical rules common to species as evolutionarily distant as flies and rodents. Briefly, the odor sensitivity of a particular ORN is specified by the expression of a single olfactory receptor gene. All the ORNs that express a particular receptor send their axons to the same glomeruli in the brain. There, ORNs make synapses with second-order neurons [mitral cells (in vertebrates) or projection neurons (in insects)] (Wilson, 2005).

    What happens when signals reach these second-order olfactory neurons is determined by complex local circuitry. One obstacle to understanding this circuitry is the sheer number of input channels in the mammalian olfactory system. The rat olfactory bulb contains ~1000 glomeruli; in contrast, the Drosophila antennal lobe contains just ~40 glomeruli. This, along with the genetic advantages of Drosophila, makes the fruit fly a useful model for investigating olfactory processing (Wilson, 2005).

    A given odor excites many Drosophila antennal lobe projection neurons (PNs) but inhibits others. These odor-evoked inhibitory epochs can last from ~100 ms to several seconds. Similar odor-evoked inhibition has also been observed in other insects and in olfactory bulb mitral cells. Some odor responses of mitral cells and PNs are purely inhibitory. Other responses are multiphasic, in which an inhibitory epoch follows or precedes an excitatory epoch. These temporal patterns are cell and odor dependent and have been proposed to encode information about the stimulus. However, the mechanism of these 'slow' patterns is not fully understood (Wilson, 2005).

    One possibility is that inhibitory epochs represent periods when principal neurons are synaptically inhibited by GABAergic local neurons (LNs). GABA-immunoreactive LNs are present in the adult antennal lobe of several species and in the larval Drosophila antennal lobe (Python, 2002). Antennal lobe LNs can synaptically inhibit PNs, and the antennal lobe is strongly immunoreactive for GABAA receptors (Harrison, 1996). However, GABAA antagonists do not block odor-evoked slow inhibition or slow temporal patterns in PNs. Therefore, these inhibitory epochs have been hypothesized to reflect a metabotropic conductance or the action of a different inhibitory neurotransmitter. Alternatively, inhibition of PNs could be caused by inhibition of ORNs (Wilson, 2005).

    This study investigated the mechanisms of odor-evoked inhibition in PNs. It was confirmed that many Drosophila antennal lobe LNs are GABAergic. GABA receptors contribute to odor-evoked inhibition of PNs on both fast and slow time scales, and GABA-mediated slow inhibition increases the diversity of odor-evoked responses among PNs. This is consistent with models that invoke GABAergic inhibition to increase the discriminability of olfactory representations (Wilson, 2005).

    As in the olfactory bulb, each glomerulus in the Drosophila antennal lobe contains four main classes of neurons: (1) the axon terminals of ORNs, (2) the dendrites of PNs that convey information from ORNs to higher brain centers, (3) neurites from LNs that interconnect glomeruli, and (4) the centrifugal axonal projections of neurons that relay information to the antennal lobe from higher brain centers. Recent studies have illuminated the development, morphology, and physiology of Drosophila ORNs and PNs. Drosophila LNs, in contrast, have not received much attention. LNs have been noted in Golgi-impregnated antennal lobes, but remarkably little is known about the number, morphology, and connectivity of these cells or about their impact on other antennal lobe neurons. If adult LNs are also GABAergic, and if GABA is inhibitory (as it is in other insects), then LNs could participate in sculpting the inhibitory epochs prominent in many PN odor responses. In the larval Drosophila antennal lobe, many LNs are immunopositive for GABA. In the adult, it has been shown that many somata around the antennal lobes express the GABA biosynthetic enzyme glutamic acid decarboxylase (Wilson, 2005).

    This study confirms that many adult Drosophila antennal LNs are GABAergic. Using confocal immunofluorescence microscopy with an anti-GABA antibody, many GABA-positive somata were observed in the vicinity of the antennal lobe neuropil. To identify LNs, flies were used in which a large subpopulation of these cells were genetically labeled. In these flies (GAL4 enhancer trap line GH298), reporter gene activity labels a cluster of somata lateral to the antennal lobe neuropils. The neurites of these neurons collectively fill the antennal lobes, reminiscent of the morphology of LNs identified in Golgi impregnations. When whole-cell patch-clamp recordings were made from the somata of GFP-positive cells in GH298-GAL4, UAS-CD8GFP flies, intrinsic properties characteristic of LNs were observed, namely high input resistances and action potentials with amplitude >40 mV. It was also confirmed with single-cell biocytin fills that these GFP-positive neurons were indeed LNs. When GH298-GAL4,UAS-CD8GFP brains stained for GABA were visualized using dual-channel confocal microscopy, it was found that most GFP-positive somata were also GABA positive. About one-fifth of the GFP-positive somata did not stain for GABA. These neurons may contain a different neurotransmitter, or the staining may not have been sensitive enough to detect low levels of GABA. The possibility cannot be excluded that these GABA-negative neurons are not LNs (Wilson, 2005).

    It was then confirmed that GABA hyperpolarizes antennal lobe neurons. In LNs, these results imply that inhibition is mediated entirely by GABAA receptors. In contrast, GABAergic inhibition of PNs is mediated by both GABAA and GABAB receptors. Thus, synaptic inhibition onto PNs and LNs is functionally specialized (Wilson, 2005).

    How might GABAergic inhibition contribute to olfactory processing in the Drosophila antennal lobe? Recent studies using optical measurements of neural activity have concluded that ORN and PN odor responses are very similar and that the antennal lobe is merely a relay station that faithfully transmits ORN signals to PNs without alteration. These conclusions imply that synaptic inhibition in the antennal lobe may exist merely to control global excitability and may not play an important role in representing information about the stimulus. However, the optical reporters used in these studies lack temporal resolution, have limited dynamic range, and may not be sensitive to inhibitory events. Whole-cell patch-clamp recordings from Drosophila PNs show prominent inhibitory epochs in many odor responses, generating odor-dependent spatiotemporal response patterns. Such complex temporal patterns are not present in the responses of ORNs, implying that they arise in the antennal lobe and thus represent a transformation of the olfactory code between the first and second layers of olfactory processing. These temporal patterns are reminiscent of those seen in olfactory bulb mitral cells and in other insects (Wilson, 2005).

    A common notion in olfaction is that such spatiotemporal patterns represent lateral interactions, the net effect of which is to amplify contrast. This idea has taken two main forms. The first proposes a contrast-enhancement mechanism akin to that seen in the retina. According to this model, specific mutual inhibitory interactions exist between principal neurons in nearby glomeruli with similarly tuned ORN inputs. When a principal neuron is activated strongly by an odor, it will trigger lateral inhibition of its neighbors to suppress weak responses to that odor, sharpening the difference between their tuning curves. A different hypothesis is that lateral interactions exist in a more distributed manner. Odors are represented as stimulus-specific sequences of neuronal ensembles. The stimulus is represented both by the identity of the active neurons and the time when they are active. According to this model, the net effect of interglomerular interactions is not to prune away weak responses. Rather, inhibitory interactions may coexist with excitatory interactions (or relief-of-inhibition mechanisms), such that new principal neuron responses appear as others disappear. Because each stimulus is represented by an evolving neural ensemble, the available coding space is expanded. Again, the outcome of this process is thought to be a progressive decorrelation, such that overlap is reduced between stimulus representations (Wilson, 2005).

    Both these models predict that eliminating odor-evoked inhibitory epochs in second-order olfactory neurons will increase the similarity between the spatiotemporal activity patterns produced in these neurons by different odors. This study reports that odor-evoked inhibitory epochs in Drosophila PNs are mostly suppressed by a GABAB receptor antagonist and that blocking GABAB receptors decreases the coefficient of variation among PN peristimulus-time histograms. These results are consistent with models in which lateral interactions between principal and local neurons increase the degree of difference between the neural representations of different odors (Wilson, 2005).

    It is important to point out that the effect of the GABAB antagonist on PN odor responses may be mediated partly by presynaptic effects on ORN axon terminals or by indirect effects via other excitatory inputs to PNs. Determining the locus of this effect will require additional experiments using cell type-specific genetic manipulations. However, because GABAB receptors mediate much of the direct effect of GABA on PNs, it seems likely that the effect of CGP54626, a compound that blocks the late inhibitory epoch in a PN odor response, on odor-evoked PN activity is attributable at least in part to postsynaptic GABAB receptors (Wilson, 2005).

    Finally, it should be noteed that two conceptually distinct kinds of temporal patterns can in principle coexist among second-order olfactory neurons. Slow temporal patterns are punctuated by inhibitory epochs on the timescale of tens to thousands of milliseconds. In this study, it was shown that these slow patterns in the Drosophila antennal lobe are sensitive to a GABAB antagonist. Distinct from this is fast inhibition, which synchronizes the firing of principal neurons on time scales of several milliseconds and is sensitive to picrotoxin. Fast, odor-evoked synchronous oscillations occur in the olfactory systems of many organisms and are required for fine olfactory discrimination in the honeybee. There is little evidence for such oscillatory synchronization among Drosophila PNs. These observations deserve additional investigation but suggest that different organisms may emphasize different strategies for olfactory processing (Wilson, 2005).

    Theoretical models of olfactory processing that invoke synaptic inhibition to increase the contrast between different stimulus representations presume nonuniform connectivity between inhibitory and principal neurons. In insects, a GABAergic LN can arborize across the entire antennal lobe, and so it is not obvious that single LNs will make connections preferentially with particular glomeruli. In this study, it was found that the neurites of single LNs form spatially heterogeneous patterns in the antennal lobe. This finding alone does not prove that individual LNs make connections preferentially in the glomeruli in which their dendrites are most dense; for example, average synaptic strength could be higher in glomeruli with fewer neurites. However, individual LNs also displayed specific odor preferences. This supports the idea that the odor tuning of individual LNs might be correlated with which glomeruli were preferentially innervated by that LN. According to this model, LN odor tuning would be biased toward the tuning of the excitatory neurons innervating those glomeruli. Drosophila LNs receive excitatory input from PNs. In other insect species, LNs are also known to receive direct input from ORNs (Wilson, 2005).

    Consistent with these conclusions, a functional imaging study of the Drosophila antennal lobe has found that each odor stimulus evokes GABA release in some glomeruli more than others. Furthermore, these spatial patterns of GABA release are odor dependent (Ng, 2002). That study measured synaptic release from all GABAergic neurons simultaneously. This investigation has now been extended to single LNs, the morphological and functional diversity of which suggests a cellular mechanism for how the pattern of GABA release can be nonuniform and odor dependent. Ultimately, a test of this idea should come from correlating the morphology of single LNs with their odor preferences. Recent studies have reported the odor tuning of a large subset of Drosophila olfactory receptors and the mapping of each receptor to a specific ORN type. Once it is know which ORN type corresponds to each glomerulus, it should be possible to design experiments of this type more systematically (Wilson, 2005).

    A presynaptic gain control mechanism fine-tunes olfactory behavior

    Early sensory processing can play a critical role in sensing environmental cues. This study investigated the physiological and behavioral function of gain control at the first synapse of olfactory processing in Drosophila. Olfactory receptor neurons (ORNs) express the GABAB receptor (GABABR) and its expression expands the dynamic range of ORN synaptic transmission that is preserved in projection neuron responses. Strikingly, it was found that different ORN channels have unique baseline levels of GABABR expression. ORNs that sense the aversive odorant CO2 do not express GABABRs nor exhibit any presynaptic inhibition. In contrast, pheromone-sensing ORNs express a high level of GABABRs and exhibit strong presynaptic inhibition. Furthermore, a behavioral significance of presynaptic inhibition was revealed by a courtship behavior in which pheromone-dependent mate localization is impaired in flies that lack GABABRs in specific ORNs. Together, these findings indicate that different olfactory receptor channels may employ heterogeneous presynaptic gain control as a mechanism to allow an animal's innate behavioral responses to match its ecological needs (Root, 2008).

    The stereotypic organization of the Drosophila olfactory system and the identification of the family of odorant receptor genes make the fly an attractive system to study olfactory mechanisms. An adult fly expresses about 50 odorant receptor genes and each ORN typically expresses just one or a few receptor genes. ORNs detect odors in the periphery and send axons to glomeruli in the antennal lobe. Each glomerulus receives axons from about 20 ORNs expressing the same receptor genes and dendrites of a few uniglomerular projection neurons (PNs), which propagate olfactory information to higher brain centers. This numerically simple olfactory system coupled with genetic markers to label most of the input channels provides an opportunity to dissect synaptic function and information processing (Root, 2008).

    The Drosophila antennal lobe is populated with GABAergic local interneurons (LNs) that release GABA in many if not all glomeruli. GABA exerts its modulatory role via two distinct receptor systems, the fast ionotropic GABAA receptor, which is sensitive to the antagonist picrotoxin, and the slow metabotropic GABAB receptor, which is sensitive to the antagonist CGP54626. Pharmacological blockade of the GABA receptors demonstrate that GABA-mediated hyperpolarization suppresses PN response to odor stimulation in a non-uniform fashion. Electron microscopy studies of the insect antennal lobe show that GABAergic LNs synapse with PNs, which support the well established olfactory mechanism of lateral inhibition. GABAergic LNs also synapse onto ORNs and imaging studies in mouse suggest that activation of GABABRs in ORN terminals suppress neurotransmitter release in ORNs (Root, 2008).

    It was hypothesized that setting the appropriate olfactory gain for environmental cues is important for adjusting an organism's sensitivity to its environment. A recent study shows that GABABR mediated presynaptic inhibition provides a mechanism to modulate olfactory gain. Electrical recordings show that interglomerular presynaptic inhibition suppresses the olfactory gain of PNs to potentially increase the dynamic range of the olfactory response. Likewise, gain modulation may not be uniform among different glomeruli, which could reflect a tradeoff between sensitivity and dynamic range in different olfactory channels. For example, high sensitivity may be crucial for some environmental cues, such as those that require an immediate behavioral response, whereas a larger dynamic range may be more advantageous for other odors where precise spatial and temporal information may be critical for optimal performance (Root, 2008).

    This study investigated the physiological and behavioral function of gain control in early olfactory processing. Interneuron-derived GABA was shown to activate GABABRs on ORN terminals, reducing the gain of ORN-to-PN synaptic transmission. Different types of ORNs exhibit different levels of presynaptic inhibition and this heterogeneity in presynaptic inhibition is preserved in antennal lobe output projection neurons. Interestingly, pheromone-sensing ORNs exhibit high levels of GABABR expression and behavioral experiments indicate that GABABR expression in a population of pheromone ORNs is important for mate localization, suggesting that presynaptic gain control is important for the olfactory channel-specific fine-tuning of behavior (Root, 2008).

    Two-photon imaging was used to monitor activity in selective neural populations in the antennal lobe. Specific blockade of GABABRs reveals a scalable presynaptic inhibition to suppress olfactory response at high odor concentrations. Pharmacological and molecular experiments suggest that GABABRs are expressed in primary olfactory receptor neurons. Furthermore, the level of presynaptic inhibition is different in individual glomerular modules, which is tightly linked to the level of GABABR expression. The importance of presynaptic GABABRs in olfactory localization was investigated, and it was found that reduction of GABABR expression in the presynaptic terminal of ORNs impairs the ability of male flies in locating potential mates (Root, 2008).

    Heterogeneity was found in the levels of presynaptic inhibition among different glomeruli. Varying GABABR2 expression level in ORNs with molecular manipulations is sufficient to produce predictable alterations in presynaptic inhibition in specific glomeruli. Together these experiments argue that presynaptic GABABR expression level is a determinant of glomerulus-specific olfactory gains in the antennal lobe. A recent report revealed that there is a non-linear transformation between ORNs and PNs that is heterogeneous between glomeruli. In other words, PNs innervating a given glomerulus have a unique response range for its ORN input. Given that ORNs are the main drivers of PN response, it is plausible that the heterogeneity in presynaptic inhibition contributes to the heterogeneity in ORN to PN transformations observed by Bhandawat and colleagues. Additionally, heterogeneity in GABA release by LNs could also contribute to heterogeneity in presynaptic inhibition. It is interesting to note that when presynaptic inhibition is abolished, heterogeneity remains in the input-output curves of PN response to the four different odors in these experiments, suggesting that other mechanisms such as probability of vesicle release contribute to the heterogeneity as well (Root, 2008).

    Theoretical analysis of antennal lobe coding has recently suggested that the non-linear synaptic amplification in PNs provides an efficient coding mechanism for the olfactory system. According to this model, the optimal distribution of firing rates across a range of odorants should be flat without clusters. Firing rates of a given ORN responses cluster in an uneven distribution. Conversely, PNs exhibit a more equalized firing rate distribution than ORNs. According to the optimum coding theory, the high amplification of ORN to PN transformation generates a more even distribution of PN firing rates that should facilitate odor discrimination. However, this model of olfactory coding poses a potential problem. The high gain in this synaptic amplification reduces the dynamic range of PNs, causing a loss of information about concentration variation that could be important for an animal to localize odor objects. Presynaptic inhibition may provide a mechanism to expand the dynamic range of the olfactory system. For some glomerular modules that mediate innate behaviors such as avoidance of the stress odorant CO2, there is a potential trade off for odor sensitivity and dynamic range. The lack of GABABR in the CO2 sensing ORNs could be important to maintain high sensitivity (Root, 2008).

    Pheromones play an important role in Drosophila mating behaviors and the current results indicate that pheromone sensing ORNs have high levels of GABABR, which is correlated with a high level of presynaptic inhibition in these ORNs. Mate localization is impaired in the absence of presynaptic inhibition in one pheromone sensing glomerulus. It is interesting to note that in addition to the pheromone sensing ORNs, the palpal ORNs also exhibit high GABABR expression level. Although the behavioral role of the palpal ORNs has not been determined, it is possible that they are also important for odor object localization. There are two potential mechanisms for the role of GABABR in olfactory localization. GABABR-mediated activity-dependent suppression of presynaptic transmission on a short time scale provides a mechanism for dynamic range expansion. On a longer time scale, activity-dependent suppression provides a mechanism for adaptation, hence a high pass filter to allow the detection of phasic information. Further experiments will be necessary to determine which property is important for olfactory localization (Root, 2008).

    Intraglomerular and interglomerular presynaptic inhibition mediated by GABABRs have been described in the mammalian olfactory system. Intraglomerular presynaptic inhibition was suggested as a mechanism to control input sensitivity while maintaining the spatial maps of glomerular activity. Interglomerular presynaptic inhibition was proposed as a mechanism to increase the contrast of sensory input. A recent report revealed a similar gain control mechanism by interglomerular presynaptic inhibition in the Drosophila olfactory system where GABABR expression in ORNs was shown to scale the gain of PN responses. Interestingly, most if not all of the presynaptic inhibition was suggested to be lateral. In contrast, this study study does not seek to distinguish between intra- and interglomerular presynaptic inhibition, however evidence was found that the VA1lm glomerulus receives significant intraglomerular presynaptic inhibition. Thus, despite significant differences between the insect and mammalian olfactory systems, the inhibitory circuit in the first olfactory processing center appears remarkably similar (Root, 2008).

    Based on whole cell recordings of PNs in response to ORN stimulation, Olsen (2008) suggests that both GABAAR and GABABR are expressed in ORNs to mediate presynaptic inhibition and that GABAAR signaling is a large component of lateral presynaptic inhibition. In contrast, this study, which employed direct optical measurements of presynaptic calcium and synaptic vesicle release, suggests that GABABRs but not GABAARs are involved in presynaptic inhibition. To resolve these discrepancies further molecular experiments will be important to determine conclusively whether ORNs express GABAAR and whether the receptor contributes to gain control. Furthermore, the antennal lobe is a heterosynaptic system comprised of at least three populations of neurons that include ORNs, LNs and PNs. Therefore, how these different populations of neurons respond to GABA signaling and what contribution they make to olfactory processing in the antennal lobe is a critical question for future investigation (Root, 2008).

    This study has demonstrated differential presynaptic gain control in individual olfactory input channels and its contribution to the fine-tuning of physiological and behavioral responses. Synaptic modulation by the intensity of receptor signaling is reminiscent of the mammalian nervous system where expression levels of AMPA glutamate receptors play an important role in regulating synaptic efficacy. Furthermore, presynaptic regulation of GABABR signaling provides a mechanism to modulate the neural activity of individual input channels without much interference with overall detection sensitivity because this mechanism of presynaptic inhibition would only alter responses to high intensity stimuli. In parallel, it is tempting to speculate that global modulation of interneuron excitability should alter the amount of GABA release across channels, thus providing a multi-channel dial of olfactory gain control that may reflect the internal state of the animal (Root, 2008).

    A single GABAergic neuron mediates feedback of odor-evoked signals in the mushroom body of larval Drosophila

    Inhibition has a central role in defining the selectivity of the responses of higher order neurons to sensory stimuli. However, the circuit mechanisms of regulation of these responses by inhibitory neurons are still unclear. In Drosophila, the mushroom bodies (MBs) are necessary for olfactory memory, and by implication for the selectivity of learned responses to specific odors. To understand the circuitry of inhibition in the calyx (the input dendritic region) of the MBs, and its relationship with MB excitatory activity, the simple anatomy of the Drosophila larval olfactory system was used to identify any inhibitory inputs that could contribute to the selectivity of MB odor responses. A single neuron was found to account for all detectable GABA innervation in the calyx of the MBs, and this neuron has pre-synaptic terminals in the calyx and post-synaptic branches in the MB lobes (output axonal area). This neuron was called the larval anterior paired lateral (APL) neuron, because of its similarity to the previously described adult APL neuron. GFP reconstitution across synaptic partners (GRASP) suggests that the larval APL makes extensive contacts with the MB intrinsic neurons, Kenyon Cells (KCs), but few contacts with incoming projection neurons (PNs). Using calcium imaging of neuronal activity in live larvae, this study shows that the larval APL responds to odors, in a manner that requires output from KCs. These data suggest that the larval APL is the sole GABAergic neuron that innervates the MB input region and carries inhibitory feedback from the MB output region, consistent with a role in modulating the olfactory selectivity of MB neurons (Masuda-Nakagawa, 2014).

    A single GABAergic neuron, the larval APL neuron, accounts for all the GABAergic innervation that were detected in the larval MB calyx. This neuron is highly polarized, with overwhelmingly dendritic processes in the MB lobes and pre-synaptic processes in the calyx. It makes extensive contacts with KCs in both the calyx and lobes. Its polarity suggests that it receives input from KCs in the MB lobes and releases GABA onto KC dendrites in the calyx. These features strongly support a role for it as a feedback neuron, mediating inhibition of KC depolarization across the whole calyx, in response to KC outputs (Masuda-Nakagawa, 2014).

    Imaging of odor-evoked activity in the larval APL further supports its role as a feedback neuron. It responds to at least two different odors, ethyl acetate and pentyl acetate, in both cases with activity throughout the calyx. The odor responses of the larval APL appear to be evoked by KC output. Larval APL activity is inhibited by blocking KC output, and both the polarity of the larval APL, and its extensive contacts with KCs in the MB lobes, suggest direct synaptic transmission from KCs to larval APL dendrites in the lobes. Therefore, the larval APL, by releasing GABA in response to odor-evoked activity in KCs, would mediate negative feedback from KC output to the calyx, in a manner that is neither odor-selective nor KC subset-selective (Masuda-Nakagawa, 2014).

    While KCs appear to be the main target of the larval APL in the calyx, there are occasional sites of contact of larval APL terminals with PNs. Therefore some pre-synaptic inhibition of PN activity could also contribute to a general inhibition of neuronal activity in the calyx. Other synaptic partners of the larval APL in the calyx might include as yet non-characterized non-PN and non-KC extrinsic neurons (Masuda-Nakagawa, 2014).

    Some larval APL boutons in the calyx are relatively large (4-5?m diameter) and lie between glomeruli. The larval APL, like the adult APL and locust GGN, is presumably non-spiking, and so large GABA stores may be required for continual GABA release, as well as to provide additional GABA on olfactory stimulation. The extraglomerular sites of these boutons hint that GABA might diffuse within the calyx and create a general inhibitory environment; the calyx is surrounded by glia but has no obvious internal glial barriers to neurotransmitter diffusion. However, the large boutons are surrounded by KCs, despite their extraglomerular locations. The main barrier to diffusion of GABA in the calyx must therefore be the localization and activity of plasma membrane GABA transporters within it, about which nothing is currently known (Masuda-Nakagawa, 2014).

    The larval APL might potentially be activated in the calyx by PNs, or from pre-synaptic specializations on KC dendrites. However, no evidence has been seen for this; PN activity and KC excitatory post-synaptic potentials are expected to show odor-specific localization in the calyx and this study observeded only broadly uniform responses of the larval APL terminals across the entire calyx (Masuda-Nakagawa, 2014).

    Despite the obvious similarities, there are some major differences between the larval and adult APL neurons. Whereas the larval APL innervates the entire calyx but only certain regions of the lobes, the adult APL innervates the whole of the calyx, lobes and pedunculus. Furthermore, the larval APL is strongly polarized, appearing overwhelmingly dendritic in the MB lobes and pre-synaptic in the calyx, albeit with some dendritic label in the calyx and a small amount of pre-synaptic marker in the lobes. By contrast, the adult APL appears completely non-polarized, strongly expressing both pre-synaptic Syt::HA and dendritic DenMark in both the calyx and the lobes; therefore, in addition to being a feedback neuron, it might be able to mediate local inhibitory circuits within both the calyx and lobes. Other fly neurons that anatomically appear highly polarized can also have mixed axodendritic projections; these include some classes of adult KCs that have pre-synaptic as well as post-synaptic specializations in the calyx, although the post-synaptic targets of these pre-synaptic specializations, and whether the calycal microcircuits that they could mediate include the APL, are unknown (Masuda-Nakagawa, 2014).

    Octopamine has recently been reported in the adult APL, together with evidence that knocking down its synthesis can impair anesthesia-resistant memory. However, no specific octopamine immunoreactivity was detected in the larval APL, even when image intensity is saturating in most other octopaminergic termini in the calyx; this makes it unlikely that weak immunoreactivity similar to that reported in the adult APL was missed. Whether this reflects an absence of anesthesia-resistant memory in larvae is unknown (Masuda-Nakagawa, 2014).

    The larval APL appears broadly similar to GABAergic MB neurons in a number of other insects. The locust GGN has projections in the MB lobes and calyx that resemble that of the Drosophila larval APL, and consistent with the current findings, it receives monosynaptic inputs from KCs. Like the Drosophila larval APL and unlike the adult APL, it shows extensive arborization in restricted areas of the KC lobes and unlike either stage of the Drosophila APL, the locust GGN also innervates the lateral horn. There is not yet any molecular evidence concerning GGN axo-dendritic polarity. GABAergic “feedback neurons” that connect subregions of the MB lobes and calyces, and whose processes in the lobes appear to be post-synaptic, are also been seen in other insects including honeybee or the moth Manduca, where there are about 50 or 150, respectively, rather than just one as in Drosophila. The honeybee feedback neurons respond to various sensory stimuli via input from the MB lobes, and their activity can be influenced by learning (Masuda-Nakagawa, 2014).

    Inhibitory feedback neurons therefore appear to be an ancient component of MBs, dating back at least some 300 M years to the divergence of Diptera and Orthoptera. However, the detailed specifications of these neurons differ in properties such as the numbers of neurons, in the regions of the lobes and calyx, and hence in the individual KCs innervated, and in the potential for local inhibitory circuits within the lobes or calyx (Masuda-Nakagawa, 2014).

    KC responses to odors are both sparse, with a high input threshold for firing, and transient, consisting of only around 1-10 spikes. This is achieved by the organization of the calyx: KCs possess a dendritic organization that makes them combinatorial integrators of olfactory inputs, and concomitant activation of multiple PN inputs is required to make KCs fire; KCs are also the major target of the larval APL inhibitory feedback neuron, which might contribute to the high threshold of firing of KCs. In the adult APL, blocking RDL (GABAA) receptor expression in KCs, or reducing GABA synthesis in the APL using GH146-GAL4, increased the numbers of KCs firing in response to odors. The inhibitory feedback provided by the larval APL neuron from KC pre-synapses to KC dendrites is also likely to make KC responses transient (Masuda-Nakagawa, 2014).

    In adult Drosophila, knockdown of GABA synthesis in the adult APL neuron increases associative olfactory learning, implying that adult APL activity inhibits learning. However in reversal learning using a similar olfactory choice paradigm, but with a second round of training in which the conditioned stimulus is swapped, knockdown of GABA synthesis in the adult APL impairs learning. How could these observations be explained and reconciled, in light of the inhibitory feedback circuit just discussed? When APL inhibitory activity in the calyx is impaired, KCs would have lower firing thresholds and hence increased sensitivity to conditioned stimuli; associative learning could thus in some circumstances be enhanced . However, higher sensitivity will lower discrimination, since there will be greater overlap in the populations of KCs responding to different odors. Therefore in other circumstances, impaired ability to discriminate odors could lead to lower learning scores (Masuda-Nakagawa, 2014).

    The larval calyx is a microcircuit for odor discrimination, receiving olfactory input from PNs to form neural representations in the KCs. This study has characterized a key component of the circuit, the larval APL neuron, a feedback inhibitory neuron that innervates the MB lobes post-synaptically and the calyx pre-synaptically. The larval APL neuron has a clear polarity, with dendrites in the MB lobes that can be activated by KC output, and with GABA-containing terminals in the calyx, where KC dendrites are its main post-synaptic targets. It is the only GABAergic neuron that arborizes throughout the calyx, and therefore is the only inhibitory component that can regulate the sparseness of KC olfactory responses, and their transientness, both essential for odor discrimination. Identification of the larval APL as the sole channel for inhibitory feedback in the larval MB advances system-level understanding of how specific sensory qualities can be selectively encoded in a memory center in the higher brain (Masuda-Nakagawa, 2014).

    Calcium imaging revealed no modulatory effect on odor-evoked responses of the Drosophila antennal lobe by two populations of inhibitory local interneurons

    Although considerable knowledge is available about how odors are represented in the antennal lobe (AL), the insects' analogue to the olfactory bulb, how the different neurons in the AL network contribute to the olfactory code is not fully understood. In Drosophila it is possible to selectively manipulate specific neuronal populations to elucidate their function in odor processing. This study silenced the synaptic transmission of two distinct subpopulations of multiglomerular GABAergic local interneurons (LN1 and LN2) using shibire (shits) and analyzed their impact on odor-induced glomerular activity at the AL input and output level. Notably, selective silencing of both LN populations did not significantly affect the odor-evoked activity patterns in the AL. It is therefore concluded that these LN subpopulations, which cover one third of the total LN number, are not predominantly involved in odor identity coding per se. As suggested by their broad innervation patterns and contribution to long-term adaptation, they might contribute to AL-computation on a global and longer time scale (Strube-Bloss, 2017).

    Convergence, divergence, and reconvergence in a feedforward network improves neural speed and accuracy

    One of the proposed canonical circuit motifs employed by the brain is a feedforward network where parallel signals converge, diverge, and reconverge. This study investigated a network with this architecture in the Drosophila olfactory system. Focus was placed on a glomerulus whose receptor neurons converge in an all-to-all manner onto six projection neurons that then reconverge onto higher-order neurons. Both convergence and reconvergence were found to improve the ability of a decoder to detect a stimulus based on a single neuron's spike train. The first transformation implements averaging, and it improves peak detection accuracy but not speed; the second transformation implements coincidence detection, and it improves speed but not peak accuracy. In each case, the integration time and threshold of the postsynaptic cell are matched to the statistics of convergent spike trains (Jeanne, 2015).

    Orthodenticle is required for the development of olfactory projection neurons and local interneurons in Drosophila

    The accurate wiring of nervous systems involves precise control over cellular processes like cell division, cell fate specification, and targeting of neurons. The nervous system of Drosophila melanogaster is an excellent model to understand these processes. Drosophila neurons are generated by stem cell like precursors called neuroblasts that are formed and specified in a highly stereotypical manner along the neuroectoderm. This stereotypy has been attributed, in part, to the expression and function of transcription factors that act as intrinsic cell fate determinants in the neuroblasts and their progeny during embryogenesis. This study focuses on the lateral neuroblast lineage, ALl1, of the antennal lobe and shows that the transcription factor-encoding cephalic gap gene orthodenticle is required in this lineage during postembryonic brain development. Immunolabelling was used to demonstrate that Otd is expressed in the neuroblast of this lineage during postembryonic larval stages. Subsequently, MARCM clonal mutational methods were used to show that the majority of the postembryonic neuronal progeny in the ALl1 lineage undergoes apoptosis in the absence of orthodenticle. Moreover, it was demonstrated that the neurons that survive in the orthodenticle loss-of-function condition display severe targeting defects in both the proximal (dendritic) and distal (axonal) neurites. These findings indicate that the cephalic gap gene orthodenticle acts as an important intrinsic determinant in the ALl1 neuroblast lineage and, hence, could be a member of a putative combinatorial code involved in specifying the fate and identity of cells in this lineage (Sen, 2014).

    During early embryogenesis, the cephalic gap gene otd is expressed in a broad stripe in the anterior most domain of the cephalic region of the embryo where it is known to specify the entire segment, including the anterior brain that derives from this segment. Studies that have analysed the expression of otd in the later stages of embryonic brain development have shown that otd continues to be expressed in specific neuroblasts. For example, in the protocerebral part of the embryonic brain, otd is expressed in about 70% of the neuroblasts. Interestingly, 15% of the embryonic neuroblasts that express otd co-express the cephalic gap gene ems (Sen, 2014).

    This study reports that otd is also co-expressed with ems in a neuroblast lineage during postembryonic brain development. This study focused on the ALl1 neuroblast, which has been shown to express ems during larval development. While these findings indicate that the expression of otd is relatively low compared to the level of ems expression in the ALl1 neuroblast, mutant analysis indicates that otd is essential for the development of the neurons in this lineage. It will be interesting to see if otd might be similarly involved in the development of the other neuroblast lineages in the brain (Sen, 2014).

    Mutant analysis of the function of otd in the ALl1 lineage revealed several distinct requirements for this gene. The first, most evident defect observed in clonal loss-of-function experiments was the reduction in cell number of the ALl1 lineage; only 20% of the cells present in the wild-type adult brain were seen in the mutant condition. This phenotype is reminiscent of, but not exactly like, the phenotypes observed in this lineage due to the loss of function of three other genes, empty spiracles (ems), homothorax (hth) and extradenticle (exd). Upon the loss of function of any of these genes, the entire lineage is eliminated. In contrast, upon the loss of function of otd, 20% of the neural cells (~40 cells) survive and are present in the adult brain. This suggests that the mechanism of action of these genes might be different. In this respect, it is interesting to note that accompanied with the loss of function of ems, hth or exd a severe reduction in the size of the antennal lobe results, whereas following otd loss of function, the lobe size and its general glomerular organization remains largely unaffected (Sen, 2014).

    A different requirement for otd in the ALl1 lineage, determined by mutational analysis, was in the targeting of the dendrites and the arborization of the axons of the 20% of the cells that do survive to adulthood. Upon the loss of function of otd, ALl1 PNs displayed a variety of targeting defects including diffuse and disorganised dendritic arbours, innervations in non-antennal neuropiles, as well as extensive, premature defasciculation and misprojections of the axonal terminals. This suggests that patterning of the PNs at both the proximal and the distal terminals might be coupled. Such coupling of PN pattering has been uncovered for other genes as well, including other transcription factors like acj6, drifter, hth, exd and lola (Sen, 2014).

    It has been postulated that the identity of a NB and its lineage depends upon a certain constellation of transcription factors that acts as a code of identity. Expression analysis of NBs in the embryo has revealed that there do exist unique combinations of transcription factors in specific NBs. Moreover, recent studies, which are largely limited to a few well-described lineages in the brain, are beginning to identify the elements of putative 'combinatorial codes' of NB specification. Results from this study imply that the two cephalic gap genes otd and ems are included among the set of intrinsic cell fate determinants for the ALl1 lineage. As most postembryonic lineages have now been identified in both the larval and adult brains, such molecular genetic analyses can now be extended to other brain lineages. It is noteworthy that although analyses such as these have uncovered genes that are required in NB lineages for their survival or local targeting, none, so far, have identified genes that can actually switch the identity of one NB lineage into that of other. It will be interesting to see if future studies uncover such important factors that determine the identities of lineages (Sen, 2014).

    Molecular, anatomical, and functional organization of the Drosophila olfactory system

    Olfactory receptor neurons (ORNs) convey chemical information into the brain, producing internal representations of odors detected in peripheral receptors. A comprehensive understanding of the molecular and neural mechanisms of odor detection and processing requires complete maps of odorant receptor (Or) expression and ORN connectivity, preferably at single-cell resolution. Near-complete maps have been constructed of Or expression and ORN targeting in the Drosophila olfactory system. These maps confirm the general validity of the 'one neuron-one receptor' and 'one glomerulus-one receptor' principles and reveal several additional features of olfactory organization. ORNs in distinct sensilla types project to distinct regions of the antennal lobe, but neighbor relations are not preserved. ORNs grouped in the same sensilla do not express similar receptors, but similar receptors tend to map to closely appositioned glomeruli in the antennal lobe. This organization may serve to ensure that odor representations are dispersed in the periphery but clustered centrally. Integrated with electrophysiological data, these maps also predict glomerular representations of specific odorants. Representations of aliphatic and aromatic compounds are spatially segregated, with those of aliphatic compounds arranged topographically according to carbon chain length. These Or expression and ORN connectivity maps provide further insight into the molecular, anatomical, and functional organization of the Drosophila olfactory system. These maps also provide an essential resource for investigating how internal odor representations are generated and how they are further processed and transmitted to higher brain centers (Couto, 2005).

    The 60 Or genes of Drosophila are predicted to encode a total of 62 odorant receptors, from transcripts originating from 62 distinct Or promoters. A set of mCD8-GFP reporter lines was constructed for all 62 promoters, as well as 59 GAL4 reporter lines. In parallel, a set of in situ hybridization experiments was performed to detect mRNA for 54 distinct Or genes in sections of the adult olfactory organs, the antennae and maxillary palps. For all Or genes for which expression was detected by both of these methods, sections were also probed simultaneously for the Or mRNA and the corresponding Or transgenic reporter. In all, 42 Or genes were detected for which the reporter line and in situ hybridization labeled identical subsets of ORNs. None of these Or genes is sex specific. In addition, the Or83b reporter is broadly expressed in ORNs, as is Or83b itself. No validated reporter was detected for five of the Or genes known to be expressed in the antenna or maxillary palp (Or1a, Or33a, Or71a, Or85b, and Or98b); a validated reporter for Or71a has already been obtained in another study. Thus, the expression of 44 Or genes can be mapped in the antenna and maxillary palp (Couto, 2005).

    A related family of 60 Gr genes encodes 68 predicted gustatory receptors, some of which may actually function as odorant receptors. A systematic survey of Gr expression in the olfactory system was performed, preparing mCD8-GFP reporter lines for 67 of 68 Gr promoters. Only Gr21a could be mapped to specific ORNs by both reporter expression and in situ hybridization. Several other Gr reporters are also expressed in the antenna, but their expression is generally either weak or broad or could not be confirmed by in situ hybridization. It is therefore concluded that few if any of the other Gr genes are likely to encode odorant receptors (Couto, 2005).

    The larval olfactory system comprises just 21 ORNs, which also expresses members of the Or gene family. Since 14 of the Or reporters could not be detected in the adult, it is suspected that these Or genes may in fact encode larval odorant receptors. Indeed, 11 of these reporters are expressed specifically in the larval olfactory system. This search was expanded to cover all Or reporters and another seven Or genes were identified that are expressed in the larval as well as the adult olfactory system. This analysis confirmed 8 of the 10 larval Or genes previously identified by both RT-PCR and reporter experiments, but only 3 of the additional 13 receptors with RT-PCR evidence alone. Thus, a total of 20 larval Or genes have been identified (in addition to Or83b). This is a close match to the 21 ORNs, consistent with the notion that, in the larva, each ORN expresses a single and distinct odorant receptor. This analysis also confirms that the larval and adult odorant receptors are encoded by phylogenetically dispersed members of the same Or family, some of which are stage specific whereas others are common to both stages (Couto, 2005).

    In the adult, ORNs are housed in sensory sensilla of four distinct morphological types: basiconic, trichoid, coeloconic, and intermediate sensilla (in order of decreasing abundance). All four sensilla types are found on the antenna. The maxillary palp contains only basiconic sensilla. Each adult Or gene was mapped to a specific sensillum type simply by noting the morphology of the sensilla innervated by GFP-positive dendrites in Or-mCD8-GFP or Or-GAL4, UAS-mCD8-GFP flies (Couto, 2005).

    Most sensilla contain 2-4 ORNs, so attempts were made to identify pairs of Or genes expressed in distinct ORNs of the same sensillum. For this, double labelings were performed for most possible pairings of Or genes within both the basiconic and trichoid classes. This generally involved immunofluorescence detection of one Or transgenic reporter and in situ hybridization to detect the second Or, although in some cases either double in situ hybridization or double immunofluoresence were performed (using the mCD8-GFP reporter for one Or and the GAL4 reporter and UAS-τlacZ for the other). These methods allowed the mapping of 44 Or genes and Gr21a to 38 ORN classes that innervate 19 distinct and highly stereotyped sensilla types (Couto, 2005).

    The basiconic sensilla of the antenna comprise three large (ab1-3), three thin (ab4-6), and four small sensilla (ab7-10), following the nomenclature used for their morphological and physiological classification . The sensilla classes define as ab9 and ab10 were not identified in the physiological studies, possibly because they are located more laterally on the antenna. The expression was confirmed of Or genes in 11 antennal basiconic ORN classes, most of which had previously only been inferred from functional data, and Or genes were identified for another 11 ORN classes. The map of the maxillary palp comprises three thin basiconic sensilla (pb1–3), with Or genes assigned to all six ORN classes. In summary, 31 receptor genes expressed in the basiconic ORNs were identified, defining a total of 28 distinct ORN classes and 13 sensilla classes. With the exception of one neuron in the ab6 sensillum, the receptor-to-neuron map of the basiconic sensilla of the antenna and palp is now most likely complete (Couto, 2005).

    The trichoid sensilla are innervated by one, two, or three neurons (T1, T2, or T3 sensilla, respectively). There are only very limited physiological data available for the trichoid sensilla, and no receptor-to-neuron assignments have previously been made. Twelve Or genes expressed in 9 ORN classes in 4 distinct classes of trichoid sensilla were identified: a single T1 sensillum (which is referred to as at1), a single T2 sensillum (at2), and two distinct classes of T3 sensillum (at3 and at4). The compositions of at2 and at3 seem to be strictly stereotyped, but at4 may be slightly variable. The total numbers of trichoid sensilla and ORNs that were identified closely match the numbers reported in the morphological survey, and since at least one Or was assigned to each ORN class, it is anticipated that this receptor-to-neuron map of the trichoid sensilla is also likely to be complete (Couto, 2005).

    Coeloconic sensilla house either two or three ORNs, and based on the number of glomeruli innervated by coeloconic ORNs, it is anticipated that there are eight distinct classes of coeloconic ORNs. However, only a single Or gene, Or35a, could be assigned to the coeloconic sensilla. Or or Gr genes might be expressed only at very low levels in the coeloconic ORNs, making them difficult to detect by these methods. Alternatively, these neurons may express some other type of chemoreceptor (Couto, 2005).

    The intermediate sensilla number only about 20-30, and also contain either 2 or 3 ORNs. Or13a was tentatively assigned to the intermediate sensilla, although it is also possible that one or another of the sensilla classes identified as basiconic or trichoid by light microscopy may in fact correspond to sensilla described as intermediate by electron microscopy (Couto, 2005).

    What logic, if any, guides the selection and pairing of Or genes in individual sensilla? With regard to sensillum type, one might predict that the different sensilla types would express different subfamilies of Or genes, as defined either by the sequences of the receptors they encode or their chromosomal locations. However, no obvious pattern emerged when either a phylogenetic tree or a genomic map of Or genes was annotated with the corresponding sensillum type. With regard to the specific combinations of receptors expressed in ORNs of the same sensilla, two extreme possibilities are that paired receptors might be closely related (because the odorants they detect must pass through a shared extracellular enviroment, and so may be chemically related) or maximally divergent (in order to minimize passive interference between ORNs. To test these hypotheses, the sequence distance was determined between two receptors for each of the 990 possible pairs of the 45 odorant receptors on the map, and each pair was binned into one of four categories: pairs expressed in the same ORN class (e.g., both in ab3A), in distinct ORNs of the same sensillum (e.g., ab3A and ab3B), in different sensilla classes of the same type (e.g., ab3A and ab4A), or in different sensilla types (e.g., ab3A and at1A). It was found that those receptors expressed in different neurons of the same sensillum are, on average, no more and no less closely related to each other than those expressed in different sensilla, different types of sensilla, or indeed any receptor pair chosen at random. Thus, although highly stereotyped, the selection and pairing of Or genes into distinct sensilla types and classes does not seem to follow any particular logic with regard to either the sequence of the receptor or the location of its gene (Couto, 2005).

    Anatomical studies using general synaptic markers have defined some 40-50 glomeruli in the Drosophila antennal lobe, with some minor discrepancies between different studies. The Or transgenic reporter lines provided a set of molecular markers for individual glomeruli; these reporters can be used individually or in combination to refine existing maps and establish an atlas of 49 glomeruli. To assign receptors to individual glomeruli, ORN projections were examined for each of the promoter-mCD8-GFP fusions. Previously, this approach had been used to map receptors to 13 glomeruli. The set of 44 verified adult Or reporters facilitated correction of 4 of these earlier assignments and to extend the coverage to a total of 37 glomerul. For each Or, the projections were identical in each animal examined, with no differences between the sexes. Each Or reporter also labels just a single glomerulus (although a few reporters are weakly or ectopically expressed in additional ORN classes that target other glomeruli). Through a series of unilateral deafferentation experiments, it was determined that only the V glomerulus is innervated unilaterally; all other glomeruli on the map receive bilateral innervation (Couto, 2005).

    It was anticipated that many of the glomeruli that remained unassigned were likely to be innervated by coeloconic ORNs. To identify these glomeruli, an ato-GAL4 reporter was used: this reporter is expressed in all coeloconic ORNs of the antenna and the basiconic ORNs of the maxillary palp. A total of 14 glomeruli are labeled in ato-GAL4, UAS-mCD8-GFP flies: the six glomeruli already assigned to the six ORN classes of the maxillary palp, the one glomerulus assigned to an antennal coeloconic ORN (VC3), and seven still unassigned glomeruli (DC4, DL2, VL1, VL2a, VM1, VM4, and VM6). It is therefore inferred that VC3 and these seven additional glomeruli are the targets of the antennal coeloconic ORNs (Couto, 2005).

    The ORN connectivity map reveals a spatial organization in the antennal lobe that was not apparent from the few ORN classes previously examined. Specifically, afferents from ORNs in distinct sensilla types project to distinct regions of the antennal lobe: ORNs in antennal trichoid sensilla project to the lateral anterior region, antennal basiconic sensilla to the medial region, palp basiconic sensilla to the central-medial region, and antennal coeloconic sensilla to the posterior (Couto, 2005).

    This segregation of sensory input according to sensillum type does not extend down to the level of individual sensilla classes. Specifically, ORNs that are neighbors in the same sensillum do not always project to neighboring glomeruli. Nevertheless, ORNs in the same sensilla class might still innervate glomeruli that are generally close to each other within the antennal lobe. To test this possibility, the distances between all 666 pairs of the 37 assigned glomeruli was determined and then it was asked whether glomeruli innervated by ORNs in the same sensilla are generally closer to each other than those innervated by ORNs in different sensilla. Distances were calculated in two different ways for each pair. An average physical distance between the geometric centers of the two glomeruli was determined from 3D reconstructions of four male antennal lobes. In addition, the 'degrees of separation' between each pair was determined: 1 for neighboring glomeruli, 2 for glomeruli that are not themselves neighbors but have a common neighbor, and so on. It was found that, by either distance measure, pairs of glomeruli innervated by ORNs in the same sensilla are, on average, no closer together or further apart than those innervated by ORNs from different sensilla of the same type. Thus, sensory inputs from the different sensilla types are segregated into distinct regions of the antennal lobe, but within each of these regions the arrangement of glomeruli bears no obvious relationship to the location or pairing of ORNs in the periphery (Couto, 2005).

    It was next asked whether receptors that are more closely related by sequence tend to map to glomeruli that are physically closer within the antennal lobe. To test this, all 938 possible pairs of the 44 odorant receptors on the map were examined (excluding the eight pairs that are coexpressed in the same neuron). For each pair, the sequence divergence of the two receptors and the separation of the corresponding glomeruli in the antennal lobe were determined. There is a strong positive correlation between the two, using either the actual distances between pairs of glomeruli or their degrees of separation. This correlation can be attributed entirely to the antennal basiconic sensilla, as it is not observed at all for the other two sensilla types . Thus, among the antennal basiconic sensilla, pairs of ORNs that express more closely related receptors tend to map to more closely positioned glomeruli (Couto, 2005).

    By integrating the molecular and anatomical maps with existing electrophysiological data, odor-evoked activity patterns could be predicted for a total of 29 glomeruli. Each of these 29 glomeruli were classified according to whether the test odorants that elicited a strong response (above a threshold of 50 spikes/second) were linear aliphatic compounds or aromatic compounds containing a benzene ring. This analysis suggested a spatial separation of aliphatic and aromatic odor representations in the antennal lobe. Glomeruli that respond primarily to aromatic odorants are clustered in a ventral-central region of the antennal lobe, whereas those that respond preferentially to aliphatic odorants are clustered in the medial region. This clustering does not bear any relationship to the clustering of inputs from the different sensilla types (Couto, 2005).

    The test odorants used in these physiological studies had been selected primarily in order to maximize their chemical diversity, rather than to systematically sample 'odor space'. Esters are, however, particularly well represented in these data sets and range in size from 4 to 12 carbons. Collectively, the odorant receptors or ORNs that are activated above a threshold of 50 spikes/second by these esters map to 16 of the 20 'aliphatic' glomeruli on the map. For each of these 16 glomeruli, the carbon number of the ester that gave the maximum response was noted. This revealed a broad ordering of glomeruli along the posterior to anterior axis, with more anterior glomeruli generally preferring larger esters. A similar trend was also observed for alcohols and ketones, but for these compounds the data are too sparse to draw any strong conclusions (Couto, 2005).

    In both mammals and insects, individual ORNs are thought to express only a single functional odorant receptor, although exceptions have been documented in both rats and Drosophila. The critical test of this hypothesis is to map, at single-cell resolution, the expression of the entire family of odorant receptor genes. This task has now been almost completed for the adult olfactory system of Drosophila melanogaster; this study mapped 45 odorant receptors to 38 distinct ORN classes (Couto, 2005).

    Only six ORN classes express more than one receptor (excluding the widely expressed Or83b, which heterodimerizes with other odorant receptors but is not functional by itself; as well as the low levels of some additional Or or Gr genes in some neurons). In four of these six cases, the coexpressed Or genes are closely linked and highly conserved, suggesting that they arose through a relatively recent gene duplication. These pairs of coexpressed receptors are likely to detect the same odorants, and so do not represent a meaningful exception to the one neuron-one receptor principle. The two cases of coexpressed but unrelated and unlinked Or genes are Or33c and Or85e in pb2A and Or49a and Or85f in an ab10 neuron. For the Or33c/Or85e pair, both receptors are functional when ectopically expressed, but the response profile of the pb2A neuron in which they are endogenously coexpressed can be attributed to Or85e alone. Similar comparisons are not yet possible for the Or49a/Or85f pair, since there are no electrophysiological data available for Or49a or the ab10 sensillum (Couto, 2005).

    In mammals, a negative feedback mechanism ensures that only one functional receptor is expressed. The choice of a specific receptor is largely stochastic, although each ORN is somehow restricted to selecting from a large subset of 'available' receptor genes according to its position in the olfactory epithelium. In contrast, receptor choice in Drosophila appears to be entirely deterministic, as indicated by the highly stereotyped patterns of Or expression in olfactory sensilla. There is also no evidence for any negative-feedback mechanism in Drosophila; the loss of an endogenous receptor does not lead to the expression of an alternative receptor, nor does ectopic expression of a second receptor block the expression of an endogenous receptor (Couto, 2005).

    The 45 receptors that were mapped can be paired in nearly 1000 different ways, yet less than 20 distinct combinations are actually deployed in olfactory sensilla. Why have these specific combinations been selected? One possibility is that ORNs compartmentalized into the same sensillum might express closely related receptors, as the odorants they detect are transported and processed by the same set of molecules in their common sensillar lymph, and so may be chemically related. However, this study found that pairs of Or genes expressed in the same sensillum are no more closely related to each other than any randomly selected pair. Similarly, electrophysiological surveys of a more limited set of basiconic sensilla have shown that ORNs housed in the same sensilla tend to have distinct rather than similar response spectra. These observations are more readily explained by a model in which ORNs housed in the same sensilla instead express divergent receptors, so as to minimize their functional overlap and afford each ORN a greater dynamic range (Couto, 2005).

    Nevertheless, there are still many different ways in which pairs of divergent odorant receptors could be combined, so this consideration alone cannot completely explain the specific combinations deployed. An additional factor may be that two odorants could be discriminated with a higher spatial and temporal resolution if the ORNs that detect them are placed in the same rather than distinct sensilla, possibly even allowing the insect to discern whether two odorants are present in the same or different filaments of an odor plume. This might be particularly relevant for odorants such as pheromones, for which it may be critically important to distinguish whether the individual components of a blend originate from a single source (a potential mate) or from two closely spaced sources (Couto, 2005).

    Whatever the logic behind these pairings, it will be of great interest to determine how they are programmed developmentally. At present, little is known of these mechanisms. ORNs in the same sensillum are likely to be related by lineage, and so selection of a specific Or gene might be part of the instrinsic mechanisms that generate diverse cell fates within each lineage. Alternatively, by analogy to the signaling mechanisms that coordinate rhodopsin gene selection between R7 and R8 cells in the same ommatidium in the eye, one ORN in each sensillum might choose its Or first and then instruct the Or choice of its neighbor(s). The promoter regions that were defined will be a valuable guide in computational and experimental approaches aimed at defining the cis-acting determinants of Or choice, while the transgenic reporters should facilitate genetic screens to identify the trans-acting factors (Couto, 2005).

    In Drosophila, axons of ORNs that express the same odorant receptor are thought to converge upon a single glomerulus, with each glomerulus receiving input from just a single class of ORN. The Or axonal reporters generated prior to this study all label a single glomerulus per antennal lobe, as does each of the 45 verified reporters in this study. The few cases in which Or reporters have been observed targeting multiple glomeruli can most likely be explained by low levels of 'ectopic' Or expression or by reporters that do not faithfully mimic the endogenous Or expression. Innervation of a single glomerulus thus appears to be a strict rule (Couto, 2005).

    More difficult to verify, in any species, has been the postulate that each glomerulus receives input only from a single class of ORNs (the one glomerulus-one receptor hypothesis). No exceptions have yet been reported in the main olfactory systems of mice or Drosophila. However, with only a small fraction of odorant receptors examined in each case, the chances of detecting a glomerulus with multiple inputs had, until now, been vanishingly small. With the map of ORN connectivity now almost complete for Drosophila, it can now be confirmed that most, and probably all, glomeruli do indeed receive input from just a single class of ORN. Specifically, each of 38 glomeruli could be assigned to a single and distinct ORN class and most of the remaining glomeruli to a nonoverlapping set of unidentified coeloconic ORNs (Couto, 2005).

    The connectivity map also reveals an unanticipated topographic organization of the antennal lobe, with ORNs in distinct sensilla types projecting into distinct regions of the antennal lobe. A similar topographic organization may apply in the vertebrate main olfactory system. Within these regions, however, neighbor relationships are not preserved -- ORNs that are neighbors in the same sensillum do not target neighboring glomeruli, or indeed even closely positioned glomeruli (Couto, 2005).

    For the antennal basiconic sensilla, the distance between glomeruli does, however, correlate with the sequence distance between the corresponding odorant receptors. Thus, whereas ORNs are grouped into sensilla in ways that appear to favor combinations of divergent receptors, their target glomeruli may be arranged in part in ways that tend to juxtapose ORNs that express similar receptors. This redistribution of ORNs between the periphery and the antennal lobe may contribute to the formation of chemotopic maps in the brain (Couto, 2005).

    The peripheral and central mechanisms of Drosophila olfaction have been well described. What has been missing until now is the causal link between the two. The expression and connectivity maps of this study provide this link. The internal representations of specific odorants can now be explained and predicted from the knowledge of the receptors they activate, the ORNs that express these receptors, and the glomeruli that these ORNs target (Couto, 2005).

    This study has predicted odor respresentations covering 29 glomeruli for the diverse set of odorants used in the physiological studies. These odor maps reveal a functional organization of the antennal lobe that is not apparent from imaging studies with Drosophila but is consistent with imaging and electrophysiological data from other insects. Specifically, aromatic and aliphatic compounds are predicted to activate spatially distinct regions of the antennal lobe. A similar segregation of aromatic and aliphatic representations has also been suggested for the larval olfactory system. It was also found that, within the 'aliphatic cluster' of the adult antennal lobe, compounds of increasing carbon chain length are predicted to successively shift the activity pattern in an anterior direction (Couto, 2005).

    These features are also not unique to insect olfactory systems. Accumulating evidence points to a similar functional organization of the mammalian olfactory bulb, with distinct chemical classes activating distinct glomerular clusters and carbon chain length represented topographically within each cluster. Thus, the chemotopic organization of the antennal lobe that emerges from the map of the Drosophila olfactory system appears to be a common feature of both insect and mammalian olfactory systems (Couto, 2005).

    In the antennal lobe, ORN axons synapse with second order projection neurons (PNs), which extend axons to the protocerebrum. Since high-resolution anatomical maps are beginning to emerge for PN axons, it may soon be possible to predict odor representations at higher brain levels as well. It will be fascinating to learn to what extent this chemotopic map is retained or transformed at higher levels. Before doing so, however, it will be essential to understand the transformations that take place within the antennal lobe itself. Imaging studies have indicated a high degree of correlation between the ORN and PN responses for individual glomeruli, suggesting that the antennal lobe is primarily a relay station with little transformation of olfactory information. In contrast, electrophysiological data suggest that PNs are more broadly tuned and dynamic in their responses than the corresponding ORNs. A caveat to this result, however, was that ORN and PN responses could be compared only for a single glomerulus, DM2 (Couto, 2005).

    The connectivity map will now allow more systematic comparisons of ORN input and PN output in the antennal lobe. The electrophysiological data for PNs are still too limited to significantly extend this analysis. Nevertheless, ORN versus PN comparisons can be made for two additional glomeruli: VA7l and DM1. The VA7l PNs appear to be more broadly tuned than their presynaptic pb2B ORNs, whereas the DM1 PNs seem to be a much closer match to the corresponding ORNs, most likely of the ab1B class. Thus, some odors may undergo complex transformations in the antennal lobe, whereas others may be transmitted to higher brain centers with little further processing (Couto, 2005).

    In conclusion, these detailed maps of Or expression and ORN connectivity have not only confirmed and extended understanding of the basic molecular and anatomical principles of the olfactory system, they also provide a framework for understanding its functional organization. With these maps, it is now possible to explain and predict how the peripheral activation of odorant receptors produces a chemotopic represention in the antennal lobe. In future, these maps can also be used to determine precisely how olfactory information is further processed in the antennal lobe and transmitted to higher brain centers (Couto, 2005).

    Precise and fuzzy coding by olfactory sensory neurons

    The exact nature of the olfactory signals that arrive in the brain from the periphery, and their reproducibility, remain essentially unknown. In most organisms, the sheer number of olfactory sensory neurons (OSNs) makes it impossible to measure the individual responses of the entire population. The individual in situ electrophysiological activity of OSNs in Drosophila larvae were measured in response to stimulation with 10 aliphatic odors (alcohols and esters). Control larvae (a total of 296 OSNs) and larvae with a single functional OSN were studied, created using the Gal4-upstream activator sequence system. Most OSNs showed consistent, precise responses (either excitation or inhibition) in response to a given odor. Some OSNs also showed qualitatively variable responses ('fuzzy coding'). This robust variability was an intrinsic property of these neurons: it was not attributable to odor type, concentration, stimulus duration, genotype, or interindividual differences, and was seen in control larvae and in larvae with one and two functional OSNs. It is concluded that in Drosophila larvae the peripheral code combines precise coding with fuzzy, stochastic responses in which neurons show qualitative variability in their responses to a given odor. It is hypothesized that fuzzy coding occurs in other organisms, is translated into differing degrees of activation of the glomeruli, and forms a key component of response variability in the first stages of olfactory processing (Hoare, 2008).

    Against expectations, it was found that in whole Drosophila larvae, responses to a narrow range of ecologically significant odors involved a mixture of (1) precise, reproducible neuronal coding in which specific neurons were consistently excited, inhibited, or never responded, and (2) variable, fuzzy coding, in which some neurons responded inconsistently to certain odors. The result is a noisy peripheral signal, in which patterns of OSN activity are rarely repeated, even when an identical stimulus is presented. The first assumption was that these responses must reflect some kind of artifact. However, control experiments showed that these phenomena were not a function of odor, of concentration, of stimulus duration, or of stimulus delivery, nor were they related to differences in responsiveness shown by OSNs or by individual larvae. Strong evidence for the existence of fuzzy coding is provided by the existence of such profiles in [OrX-Gal4/UAS-Or83b; Or83b-/-] larvae in which only one OSN is functional and in which the activity of the given OSN is readily identifiable (this also shows that the procedure for detecting the activity of single OSNs in a multiunit recording was not the cause of the observation of qualitative response variability). Following the prediction, the typical response profiles seen in single-functional OSN larvae of two different OSN classes were also found in larvae in which these two OSNs were expressed together. Gal4 lines can vary in penetrance across a population or between lines, thus the existence of fuzzy responses could be produced by low or absent OR83b levels in particular individuals or lines. It found that given single-functional OSN lines consistently showed both precise and fuzzy responses and that all individuals of that genotype showed both types of response, strongly suggesting that the results are not caused by differential penetrance of Or83b across or within lines. Finally, it should be recalled that stochastic activity in response to certain odors was also observed in the full complement of larval OSNs in two unmanipulated control strains: this was not a phenomenon that was limited to genetically manipulated strains (Hoare, 2008).

    Faced with this accumulation of evidence it was conclude that, as expected, OSNs can show precise, consistent responses to odors, but also that they can show stochastic, fuzzy responses to other odors. Responses of mouse OSNs expressing MOR71 shows consistent calcium increases when stimulated with acetophenone, but only 33% showed such a response to benzaldehyde. Another study found that response thresholds to lyral in MOR23 mouse OSNs varied over three log units of odor concentration. A third study found an intraclass correlation of only 0.65 in the responses of adult Drosophila OSN types and also described heterogeneity in the response profile of OSNs projecting to the same glomerulus. Finally, in a clear parallel to the current findings, it has been reported that Anopheles gambiae TE1A OSNs responded >80% of the time to 4-ethylphenol but <20% of the time to pentatonic acid. Overall, these studies support the current findings and are given coherence by the interpretation that some OSNs, when presented with some odors, can show stochastic or fuzzy responses (Hoare, 2008).

    The mechanism underlying fuzzy coding is unknown. It is speculated that fuzzy conding may reflect less effective receptor-ligand binding at the 'edge' of the molecular receptive range of a receptor, or that lateral peripheral interactions between OSNs may shape the response. No evidence was found for such peripheral interactions in [Or42a-Gal4/UAS-Or83b; Or83b-/-] larvae (stimulation with an odor led to no significant change in the activity of the nonresponsive Or83b-/- OSNs), but this may merely indicate that such peripheral interactions require the presence of a functioning receptor in the OSN membrane. In this case, it is possible that fuzzy coding in wild-type 21-OSN larvae reflects the existence of lateral interactions between OSNs, whereas fuzzy coding in single OSN larvae would be attributable to the lack of such interactions because the nonfunctional OSNs present a constant signal. Further electrophysiological, genetic, and pharmacological investigations will be required to test this possibility (Hoare, 2008).

    The function of fuzzy coding is also unclear, although behavioral data suggest it may form part of the peripheral code in this organism. Recent studies of central processing have suggested that the introduction of noise via lateral inhibition and excitation may be involved in gain control of faint signals. The existence of noise in a subset of OSNs that respond to a particular odor may increase this effect. In organisms in which there is more than one OSN for each Or (that is, for the vast majority of organisms but not the Drosophila larva), the consequence of fuzzy coding would presumably be an intermediate level of excitation in the appropriate glomerulus, since only a proportion of the OSNs would respond to a given presentation of an odor. In this way, fuzzy coding may represent a means by which greater signal variability is introduced into central processing by peripheral events. This is reinforced by the growing appreciation that variability (noise) may be an essential component of neuronal function, in particular for synchronizing the activity of groups of neurons and by data from models of the activity of sensory neuronal networks, which suggest that fuzzy coding may be involved in processing incomplete or variable data (Hoare, 2008).

    In Drosophila larvae the combinatorial code involves not only lock and key-style precise coding but also patterns of stochastic, fuzzy activity. Previous data showing heterogeneous responses in mouse OSNs, in Drosophila and in Anopheles gambiae mosquitoes, can be explained by the current findings, suggesting that this may be a general feature of the peripheral olfactory code. In organisms in which a particular OSN class consists of more than one cell, it is speculated that fuzzy coding would lead to only a subset of that class responding to a given odor; as a result the glomerulus to which they projected might show lower levels of excitation than in response to precise coding. In Drosophila adults, the antennal lobe carries out nonlinear transformation of OSN inputs, amplifying weak but not strong signals, introducing noise by excitatory lateral neurons, carrying out gain control by lateral presynaptic inhibition, and detecting synchrony in patterns of projection neuron output. These processes may be necessary at least partly because there is variability in the responses of classes of OSN; in the larva, in which the olfactory system is reduced to a single pair of each type of OSN, it is more difficult to see how animal copes with the unreliability of aspects of the peripheral signal. More extensive research on a wider range of organisms will be necessary to subject this radical hypothesis to rigorous testing (Hoare, 2008).

    Central peptidergic modulation of peripheral olfactory responses

    Drosophila neuropeptide F (NPF) modulates the responses of a specific population of antennal olfactory sensory neurons (OSNs) to food-derived odors. Knock-down of NPF in NPF neurons specifically reduces the responses of the ab3A neurons to ethyl butyrate, a volatile ester found in apples and other fruits. Knock-down of the NPF receptor (NPFR) in the ab3A neuron reduces their responses and disrupts the ability of the flies to locate food. A sexual dimorphism was identified in ab3A responsiveness: ab3A neurons in females immediately post-eclosion are less responsive to ethyl butyrate than those of both age-matched males and older females. Not only does this change correlate with brain NPF levels, but also NPFR mutants show no such sexual dimorphism. Finally, by way of mechanism, mutation of NPFR seems to cause intracellular clustering of OR22a, the odorant receptor expressed in the ab3A neurons. This modulation of the peripheral odorant responsiveness of the ab3A neurons by NPF is distinct from the modulation of presynaptic gain in the ab3A neurons previously observed with the similarly named but distinct neuropeptide sNPF. Rather than affecting the strength of the output at the level of the first synapse in the antennal lobe, NPF-NPFR signaling may affect the process of odorant detection itself by causing intracellular OR clustering (Lee, 2017).

    This study has identified a role in Drosophila for neuropeptide F (NPF) and its receptor NPFR in modulating the peripheral responses of the ab3A class of olfactory sensory neurons (OSNs). These neurons detect a range of fruity-smelling esters associated with the fruits that provide Drosophila food and a place to lay their eggs. Loss of NPF in NPF neurons reduces odor-evoked spiking of ab3A neurons in response to apple odors (e.g., ethyl butyrate and methyl butyrate) without affecting their spontaneous activity. It does not, however, affect the responses of ab1A/B or ab2A neurons to their preferred ligands or several other neuron classes to ethyl butyrate (i.e., ab2B, ab8A/B, and pb1A). The study has shown that ab3A neurons must express NPFR themselves to exhibit this increase in olfactory responsiveness; ab3A-specific expression of NPFR rescues the reduced ab3A responses of NPFR c01896 mutant flies. This modulation of olfactory responses by NPF and NPFR also affects olfactory-guided behaviors, as ab3A neuron-specific knock-down of NPFR reduces attraction of flies to apple juice baits (Lee, 2017).

    Root (2011) found that hunger enhances the responses of DM1, DM2, and DM4-the antennal lobe glomeruli that receive input from ab1A, ab3A, and ab2A neurons, respectively. They found that these neurons produce sNPF, which when released, alters the calcium responses of their own presynaptic terminals. This acts as a sort of gain control, enhancing the activation of the corresponding second-order olfactory projection neurons. In the ab1A neurons, Root unambiguously attributed this presynaptic gain control to the sNPF receptor sNPFR1 and showed that insulin signaling and starvation both alter sNPFR1 expression. This means that the ab1A neurons, which respond to food-related esters like the ab3A neurons, induce larger responses in their associated projection neurons in times of starvation, enhancing foraging behavior. The NPF-mediated modulation of ab3A neurons discovered in this study seems to differ from this sNPF-mediated gain control. Rather than magnifying presynaptic calcium responses as sNPFR activation seems to do, NPFR activation increases the number of odor-evoked action potentials in the ab3A neurons. It is unclear how these two pathways relate and what other implications these distinctions may have, but they suggest that the modulation of ab3A neurons by NPF and sNPF act through different molecular mechanisms. It is distinct from NPF's modulation of sugar taste detection, which was discovered by Inagaki (2014). That study found that hunger and NPF increase sugar responses indirectly by modulating the influence of upstream dopaminergic neurons on the GR5a-positive GRNs in the labellum (Lee, 2017).

    Lin (2016) recently reported evidence that juvenile hormone (JH), which is secreted into the circulating hemolymph by the corpora allata, acts on its receptor Methoprene-tolerant (Met) in the pheromone-sensitive antennal trichoid at4 neurons to sensitize them. While hormones are secreted into the circulation to act on distant target tissues, neuropeptides are typically secreted from peptide-producing neurons onto neighboring cells. No one had previously reported NPFergic innervation of the antennae or antennal lobes, it was initially suspected that NPF may be similarly secreted into the circulation. But by combining two copies of NPF-GAL4, it was possible to visualize NPF-GAL4-positive innervation of the antennal lobes. Since this study also found that knock-down of NPF in the ab3A neurons themselves reduces their responses to EB, NPF seems to be acting locally (Lee, 2017).

    To address the mechanism by which NPF-NPFR signaling modulates ab3A neurons, NPFR mutant antennae were stained with an antiserum that recognizes OR22a, the odorant receptor expressed by the ab3A neurons. No difference was detected in OR22a staining in the outer ab3A dendrites where odorant binding takes place. Still, compared to control antennae, NPFR mutant antennae show dramatically more OR22a-positive puncta near the ciliary dilations that separate the inner and outer ab3A dendritic segments. It is unclear what these OR22a-positive puncta are, but it is expected that they represent either OR22a molecules whose trafficking to the ciliated outer dendrites is being blocked or those whose internalization from the periphery is being enhanced. These puncta strongly resemble the OR22a-positive puncta that appear in the absence of the olfactory co-receptor Orco required for OR trafficking to the ciliated outer dendrites and co-localize with markers of the endoplasmic reticulum. This could support the former hypothesis-a specific reduction in dendritic OR22a trafficking in the absence of NPFR-but the OR22a staining in the outer dendrites of NPFR c01896 antennae seems to be unaffected (Lee, 2017).

    Although the molecular dynamics of OR dendritic surface localization, odorant binding, internalization, deactivation, and recycling remain somewhat unclear, it is speculated that NPFR may modulate OR recycling. If NPFR acts to stabilize active odorant/OR complexes at the cell surface, effectively delaying receptor inactivation, each odorant stimulus would elicit more action potentials. Reduced signaling through NPFR could accelerate the internalization of the active odorant/OR complexes, effectively reducing the length of time they spend on the outer dendritic membrane. If this speculation proves true, the wild-type levels of OR22a in the NPFR c01896 mutant antennae suggest that there may be a compensatory increase in the trafficking of new OR complexes to the outer segment. In other words, if this speculation is true, there should be a tight coupling between OR externalization and internalization. Since NPFR typically inhibits adenylyl cyclase and reduces neuronal activity, it is unclear how such a modulation of OR recycling would occur. Future studies should address the precise mechanisms that guide the movement of ORs in and out of the outer dendrites and how the various peptidergic signaling pathways may modulate those movements (Lee, 2017).

    This study also found that female flies show lower ab3A responses immediately post-eclosion than male flies, but this difference disappears as the flies age. A clear correlation was found between ab3A responses and NPF staining in young female versus male brains, and this sexual dimorphism was shown to be absent in NPFR c01896 mutants. Unfortunately, it was not possible to directly compare males and females in the trap assay, especially when they were young. Young males and females have dramatically different levels of body fat and appetites. Because of this, females require much longer periods of starvation to motivate them to move through the trap than males. This is why only males were used for this behavioral assay and why it was only possible to focus on the role of NPFR in the OSNs rather than on sex-specific differences in behavior. The function of this sexual dimorphism is unclear, but it may enhance dispersal of young females to new and more palatable food sources. Once Drosophila larvae reach their final larval instar, they stop foraging and move out of their food to find a dry location to pupate. A piece of fruit suitable for laying eggs before a single round of the 10- to 14-day Drosophila life cycle may be less suitable for a second. Thus, the reduction in olfactory responses to fruit odors observe in young female flies may help encourage them to find new food sources for egg laying. It will be interesting to test this hypothesis in future studies (Lee, 2017).

    The majority of the impact insects have on human society stems from their feeding behaviors (e.g., destroying crops or transmitting disease through infectious bites). Since insect feeding behaviors are guided by olfaction, this study focused in how insect olfactory systems change in response to internal and external cues. This showed that in genetic model insect Drosophila melanogaster NPF acts on its receptor NPFR to sensitize a specific population of antennal olfactory neurons that detect an important food-related odorant. This peripheral olfactory modulation by NPF and NPFR is sexually dimorphic in young adult flies and it affects olfactory-guided attraction to food odors. Since homologues of NPF and NPFR exist across insect species, it will be interesting to see whether these homologues also modulate olfactory food detection in these species. If so, this modulation may represent another potential target for future pest control strategies (Lee, 2017).

    Excitatory interactions between olfactory processing channels in the Drosophila antennal lobe

    Each odorant receptor gene defines a unique type of olfactory receptor neuron (ORN) and a corresponding type of second-order neuron. Because each odor can activate multiple ORN types, information must ultimately be integrated across these processing channels to form a unified percept. This study shows that, in Drosophila, integration begins at the level of second-order projection neurons (PNs). All the ORNs that normally express a particular odorant receptor were genetically silenced and it was found that PNs postsynaptic to the silent glomerulus receive substantial lateral excitatory input from other glomeruli. Genetically confining odor-evoked ORN input to just one glomerulus reveals that most PNs postsynaptic to other glomeruli receive indirect excitatory input from the single ORN type that is active. Lateral connections between identified glomeruli vary in strength, and this pattern of connections is stereotyped across flies. Thus, a dense network of lateral connections distributes odor-evoked excitation between channels in the first brain region of the olfactory processing stream (Olsen, 2007).

    The goal of this study was to observe the synaptic inputs to PNs arising from local antennal lobe circuits. A variety of complementary strategies were used to remove direct ORN input to the PN that were recording from, meanwhile leaving other ORNs intact. These manipulations allowed direct observation of lateral excitatory input to a PN originating from other glomeruli (Olsen, 2007).

    It is important to emphasize that this lateral excitation cannot be ascribed purely to compensatory rearrangement of the antennal lobe circuitry. This point is most forcefully demonstrated by experiments in which most or all ORNs are normal and active until the antennal nerves were severed immediately before recording. In these experiments recordings were performed from PNs 10-20 min after removing the antennae and odor-evoked lateral depolarizations were always observed. Hence, the circuitry mediating these responses must exist in normal flies prior to removing antennal input (Olsen, 2007).

    Excitatory connections between glomeruli appear to be very dense, perhaps all-to-all. This conclusion is supported by four pieces of evidence. (1) The magnitude of the depolarization observed when almost all ORNs are intact is larger than that observed when only the maxillary palp ORNs are intact, which in turn is larger than that observed when only a single ORN type is intact. This argues that most PNs receive indirect input from many ORN types. (2) When ORN input was restricted to a single glomerulus, every PN recorded from (87 of 87 cells) received at least weak lateral input from that glomerulus. This implies that each ORN type broadcasts indirect input to most or all glomeruli. (3) The odor tuning of the total lateral input to a glomerulus is much broader than the odor tuning of a typical ORN. (4) The lateral input to VM2 PNs and DL1 PNs has a relatively similar (though not identical) odor-tuning profile. This suggests that large and overlapping populations of ORNs provide indirect input to these two types of PNs. All-to-all connectivity is a parsimonious explanation for all these observations (Olsen, 2007).

    It should be noted that although lateral excitatory connectivity is dense and perhaps all-to-all, it is nevertheless selective. When a single ORN type was stimulated and recordings were sequentially made from PNs in different glomeruli, it was found that each PN type receives a characteristically strong or weak lateral input from that ORN type. Furthermore, these characteristic connection strengths are relatively stereotyped across flies. This suggests that the synaptic connectivity of local interneurons in the antennal lobe may be genetically hardwired (Olsen, 2007).

    Notably, the strength of these lateral excitatory connections is not correlated with the distance between the target glomerulus and the location of the ORN inputs. This means that the spatial relationship between glomeruli does not limit the strength of their lateral interactions. This finding also argues that lateral excitation does not reflect spillover of excitatory neurotransmitter from the glomerulus receiving active ORN input, since in this case PNs closer to the active glomerulus would be expected to see a larger depolarization (Olsen, 2007).

    There is some tension between the idea that excitatory connection strengths between glomeruli are varied and the finding that VM2 and DL1 PNs see similarly tuned total lateral excitatory input. One possibility is that the lateral inputs to VM2 and DL1 PNs just happen to be unusually well correlated. Another possibility is that a given target glomerulus receives characteristically strong (or characteristically weak) indirect inputs from all ORN types. In this latter scenario, the strength of the lateral depolarization would vary across glomeruli, but its odor tuning would not (Olsen, 2007).

    The lateral excitatory circuits of the antennal lobe are remarkably sensitive to small levels of afferent input. Activating ORNs presynaptic to a single glomerulus produces a substantial lateral depolarization in many or all PNs. Moreover, the magnitude of the lateral depolarization arising from a single ORN type is extremely sensitive to small increases in ORN firing rate. Even an odor that evokes a very weak response in these ORNs (e.g., 1-butanol or geranyl acetate) still evokes substantial lateral excitation (Olsen, 2007).

    Another striking feature of lateral excitatory circuits is their saturability. In experiments where only one ORN type was stimulated, increasing the rate of incoming ORN spikes from 50 to 150 spikes/second had little effect on the amount of lateral excitatory input that was broadcast to other glomeruli. Furthermore, in experiments where two ORN types were stimulated, the combined effect of these two input channels was substantially less than the sum of each channel when stimulated individually. This type of saturation should tend to limit the magnitude of lateral excitatory synaptic input to a PN (Olsen, 2007).

    Together, these results suggest that the impact of lateral excitatory connections might be strongly dependent on odor concentration. Testing this hypothesis will require comparing the sensitivity of direct and lateral inputs to a range of concentrations and understanding how these inputs are integrated by PNs (Olsen, 2007).

    While this manuscript was under review, a report appeared that identified a novel population of cholinergic local neurons in the Drosophila antennal lobe (Shang, 2007). There is no direct evidence that these local neurons mediate the local excitatory connections observed, but this hypothesis seems plausible. Each cholinergic local neuron reportedly innervates most glomeruli, and this morphology could easily explain the observation that a single ORN type broadcasts excitatory input to most or all PNs. Interestingly, excitatory (glutamatergic) local neurons were also recently identified in the mammalian olfactory bulb (Aungst, 2003), although it is not known whether these cells make synapses onto mitral cells, the analog of antennal lobe PNs (Olsen, 2007).

    Shang (2007) also independently provided evidence that PNs receive lateral excitatory input. As in this study, Shang measured activity in PNs whose presynaptic ORNs have been silenced by an odorant receptor gene mutation. Complementary to the electrophysiological approach, Shang used a genetically-encoded ecliptic pHluorin to monitor the balance of synaptic vesicle exocytosis and endocytosis at presynaptic sites in PN dendrites. That study found that PNs whose presynaptic ORNs were silent still showed odor-evoked dendritic synaptopHluorin signals, implying that these PNs receive indirect excitatory input from other ORNs (Olsen, 2007).

    Models of olfactory processing in the insect antennal lobe and the vertebrate olfactory bulb stress the importance of inhibitory connections between glomeruli. What about lateral inhibition in the Drosophila antennal lobe? It is known that GABAergic interneurons ramify throughout the Drosophila antennal lobe and release GABA in response to odor stimulation. Drosophila antennal lobe PNs have GABAA-like and GABAB-like receptors, and antagonists of these receptors disinhibit PN odor responses. Given this, it is perhaps surprising that lateral synaptic inhibition was not observed in PNs (Olsen, 2007).

    Two considerations put this finding in perspective. (1) Although the lateral inputs observed are dominated by excitation, it is possible that these responses reflect the integration of both excitatory and inhibitory inputs. As a result, inhibition could be masked by a larger postsynaptic excitation. (2) Although the results are inconsistent with a dominant role for interglomerular postsynaptic inhibition of PNs, the findings do not preclude a role for interglomerular presynaptic inhibition of ORN axon terminals. Presynaptic inhibition of neurotransmitter release from ORN axons is a well-known phenomenon in the mammalian olfactory bulb and in the crustacean olfactory lobe. In this study, direct ORN input to the PNs that were recorded from were abolished or severely reduced; this necessarily prevents observation of any substantial presynaptic inhibition (Olsen, 2007).

    It is worth noting that neither GABAA nor GABAB receptors can mediate the lateral depolarization observed. Both GABAA and GABAB conductances are hyperpolarizing in PNs. And although GABAA and GABAB receptor antagonists together completely block GABA-evoked hyperpolarizations in PNs, they do not diminish the lateral depolarization described in this study. This result also demonstrates that the lateral depolarization does not represent disinhibition (inhibition of inhibitory input to PNs) (Olsen, 2007).

    A significant transformation in odor responses occurs between the ORN and PN layer in the Drosophila olfactory system. First, the odor tuning of PNs can be broader than the odor tuning of their presynaptic ORNs. This may reflect, in part, the effects of the lateral excitatory connections described in this study. Because it was observed that the odor tuning of lateral input to a PN is different from the odor tuning of its direct ORN input, it seems likely that these lateral inputs promote excitatory responses to odors that would not have otherwise excited that PN. A second feature of the ORN-to-PN transformation is that the rank order of PN odor preferences can differ from the odor preferences of their presynaptic ORNs. Again, because the odor tuning of lateral input to a PN is different from the odor tuning of its direct ORN input, it seems likely that lateral excitatory connections between glomeruli contribute to this phenomenon (Olsen, 2007).

    However, it would be misleading to neatly assign different components of a PN's odor response to direct versus lateral excitatory inputs. Direct and lateral excitation may coexist with pre- and postsynaptic inhibition, and all these inputs are likely to be integrated by PNs in a nonlinear fashion. Broad tuning in PNs could also reflect some nonlinearity in ORN-to-PN connections (Olsen, 2007).

    In general, bridging the gap between cellular and systems neuroscience will require a deeper understanding of how neurons integrate complex synaptic inputs in vivo. Using a combination of genetic techniques and in vivo electrophysiology, this study has begun to dissect the various synaptic interactions involved in odor processing in the Drosophila antennal lobe. The strategy has been to eliminate one input to an identified neuron in order to unmask other relevant interactions. Here, this approach has revealed broadly distributed but specific excitatory connections between glomeruli. Although the behavior of a neural circuit is ultimately a complex product of its components, some insight can nevertheless be gained by manipulating one element at a time, provided that appropriate genetic tools are available. In this respect, the Drosophila olfactory circuit represents a powerful system for understanding the synaptic and cellular computations performed on sensory stimuli that ultimately produce perception and behavior (Olsen, 2007).

    Non-synaptic inhibition between grouped neurons in an olfactory circuit

    Diverse sensory organs, including mammalian taste buds and insect chemosensory sensilla, show a marked compartmentalization of receptor cells; however, the functional impact of this organization remains unclear. This study shows that compartmentalized Drosophila olfactory receptor neurons (ORNs) communicate with each other directly. The sustained response of one ORN is inhibited by the transient activation of a neighbouring ORN. Mechanistically, such lateral inhibition does not depend on synapses and is probably mediated by ephaptic coupling. Moreover, lateral inhibition in the periphery can modulate olfactory behaviour. Together, the results show that integration of olfactory information can occur via lateral interactions between ORNs. Inhibition of a sustained response by a transient response may provide a means of encoding salience. Finally, a CO2-sensitive ORN in the malaria mosquito Anopheles can also be inhibited by excitation of an adjacent ORN, suggesting a broad occurrence of lateral inhibition in insects and possible applications in insect control (Su, 2012).

    Integration of olfactory information has long been known to occur in the CNS, and has more recently been shown to occur in individual ORN. This study has demonstrated that integration also occurs at a third level, the sensillum, via lateral inhibition between ORNs responding to different components of a mixture. The sensillum thus acts as a processing unit in olfactory computation (Su, 2012).

    Lateral inhibition of a prolonged signal by a transient signal may provide a neural representation of the salience of an odour that has recently reached the fly. Sustained responses were inhibited more strongly by stronger transient pulses. This graded pattern of lateral inhibition may give rise to a potent form of contrast enhancement in which the output of a sensillum is dominated by a pulse of a strong odour. Graded lateral inhibition may provide a peripheral mechanism for evaluating countervailing signals and allowing one to prevail. It is noted that in Drosophila, an ORN that responds to a pheromone is the only ORN that does not have a neighbour, as if to ensure that its sustained response is not inhibited by a pulse of any other odorant (Su, 2012).

    The finding that lateral inhibition does not require synapses is consistent with anatomical data. Electron microscopy in Drosophila has not revealed synaptic structures or gap junctions between ORNs housed in the same sensillum. Rather, as detailed here, the physiological features of olfactory sensilla suggest another mechanism of lateral information flow: ephaptic transmission, which refers to non-synaptic communication between adjacent neurons through an extracellular electrical field. The ability of either neuron in a two-neuron sensillum to inhibit the other, as well as the grossly similar temporal dynamics of activation and lateral inhibition, are consistent with ephaptic transmission (Su, 2012).

    In insect olfactory sensilla, a substantial electrical potential exists between two isolated compartments: the sensillum lymph, which bathes the dendrites, and the haemolymph, which surrounds the somata. This 'transepithelial' potential serves as the primary driving force for odorant-induced transduction currents of the ORNs. Elaboration of an established electrical circuit model based on these physiological features predicts that strong activation of one ORN will hyperpolarize the soma of a co-compartmentalized ORN, resulting in a reduced firing rate. This prediction is consistent with the results of molecular genetic analysis and with the interpretation that lateral inhibition is due to ephaptic interactions (Su, 2012).

    The model further predicts that the magnitude of the hyperpolarization of the neighbouring neuron, and hence its reduction in firing rate, is reflected by the change in the transepithelial potential (VA), measured experimentally as a local field potential (LFP). Although strong activation of an ORN can influence the LFP in a neighbouring sensillum, this study found that the magnitude of the LFP change in nearby unstimulated sensilla is small. Consistent with this observation, lateral inhibition does not spread among homotypic sensilla that are in close proximity to one another. These results further support the conclusion that the lateral inhibition is due to local electrical interactions between neighbouring ORNs within a sensillum (Su, 2012).

    The two-odour paradigm used in this analysis, in which a transient odour is superimposed upon a sustained odour, differs from the classic one-odour paradigm in which a transient pulse of a single odour is delivered. A priori one might expect to observe ephaptic effects in the one-odour paradigm if one ORN were excited sufficiently strongly, but the effects may be expected to be less pronounced than in the two-odour paradigm. ORN spike frequency is determined not only by the somatic transmembrane potential Vm, but also by its rate of change, dVm/dt. According to the model, transient activation of ORN2 reduces the depolarizing current of ORN1. In the two-odour paradigm, activation of ORN2 has a marked effect on the value of dVm1/dt, which changes from 0 to a negative value. By contrast, in the one-odour paradigm, the activation of ORN2 has a more subtle effect on dVm1/dt when the sensillum is stimulated with an odour that activates both neurons: dVm1/dt is positive either in the presence or absence of ORN2 activation, only somewhat less positive when ORN2 is activated. The more subtle influence of ORN2 activation on dVm1/dt in the one-odour paradigm may explain why in the one-odour paradigm, the excitatory responses of an ORN containing an ectopically expressed receptor were markedly similar to those of the ORN that endogenously expresses the same receptor, despite major differences in the response profiles of their neighbours (Su, 2012).

    It is noted that the results indicate the possibility of a new approach to insect control: the inhibition of key insect ORNs by activation of their neighbours with odorant (Su, 2012).

    Synaptic and circuit mechanisms promoting broadband transmission of olfactory stimulus dynamics

    Sensory stimuli fluctuate on many timescales. However, short-term plasticity causes synapses to act as temporal filters, limiting the range of frequencies that they can transmit. How synapses in vivo might transmit a range of frequencies in spite of short-term plasticity is poorly understood. The first synapse in the Drosophila olfactory system exhibits short-term depression, but can transmit broadband signals. This study describes two mechanisms that broaden the frequency characteristics of this synapse. First, two distinct excitatory postsynaptic currents transmit signals on different timescales. Second, presynaptic inhibition dynamically updates synaptic properties to promote accurate transmission of signals across a wide range of frequencies. Inhibition is transient, but grows slowly, and simulations reveal that these two features of inhibition promote broadband synaptic transmission. Dynamic inhibition is often thought to restrict the temporal patterns that a neuron responds to, but these results illustrate a different idea: inhibition can expand the bandwidth of neural coding (Nagel, 2014).

    Synergism and combinatorial coding for binary odor mixture perception in Drosophila

    Most odors in the natural environment are mixtures of several compounds. Olfactory receptors housed in the olfactory sensory neurons detect these odors and transmit the information to the brain, leading to decision-making. But whether the olfactory system detects the ingredients of a mixture separately or treats mixtures as different entities is not well understood. Using Drosophila melanogaster as a model system, this study has demonstrated that fruit flies perceive binary odor mixtures in a manner that is heavily dependent on both the proportion and the degree of dilution of the components, suggesting a combinatorial coding at the peripheral level. This coding strategy appears to be receptor specific and is independent of interneuronal interactions (Kundu, 2016).

    Functional analysis of a higher olfactory center, the lateral horn

    The lateral horn (LH; see Anatomical organization of the olfactory nervous system in Drosophila) of the insect brain is thought to play several important roles in olfaction, including maintaining the sparseness of responses to odors by means of feedforward inhibition, and encoding preferences for innately meaningful odors. Yet relatively little is known of the structure and function of LH neurons (LHNs), making it difficult to evaluate these ideas. This study surveyed >250 LHNs in locusts using intracellular recordings to characterize their responses to sensory stimuli, dye-fills to characterize their morphologies, and immunostaining to characterize their neurotransmitters. A great diversity of LHNs was found, suggesting this area may play multiple roles. Yet, surprisingly, no evidence was found to support a role for these neurons in the feedforward inhibition proposed to mediate olfactory response sparsening; instead, it appears that another mechanism, feedback inhibition from the giant GABAergic neuron, serves this function. Further, all LHNs observed responded to all odors tested, making it unlikely these LHNs serve as labeled lines mediating specific behavioral responses to specific odors. The results rather point to three other possible roles of LHNs: extracting general stimulus features such as odor intensity; mediating bilateral integration of sensory information; and integrating multimodal sensory stimuli (Gupta, 2012).

    Compared to the well-studied roles of the mushroom bodies (MB) in olfactory coding and learning, the role of the LH in general olfaction remains unclear. Dye-fills of individual LHNs in the locust revealed a surprising diversity of neurons, comprising at least 10 distinct morphological classes. A class of LHNs, the LHIs, was previously proposed to provide feed-forward inhibition to regulate the firing of KCs based on two sets of observations: intracellular dye-fills showing these LHIs send processes to the MB calyx, and GABA immunostains showing a cluster of ~60 GABAergic neurons in the area of somata of LHIs and a GABAergic fiber tract between the LH and the MB calyx. Whether the dye-filled LHIs labeled positively for GABA was not tested (Gupta, 2012).

    With dye-fill experiments this study identified LHNs matching morphological and physiological descriptions of the LHIs (C1). However, following the same immunostaining technique as the earlier authors, these dye-filled neurons were found not to be GABAergic (although strongly-stained GABAergic neurons were observed adjacent to themB). Using this techniqud the cluster of GABAergic neurons reported earlier was also observed. However, intracellular fills showed this cluster contained GABA-positive C8 neurons that do not project to the calyx. Recent work shows β-LNs with somata near those of the LHIs are inhibitory (Cassenaert, 2012) suggesting the GABAergic cluster may also contain these β-LNs. The current study also found that the GABAergic tract between the LH and the MB, thought by the earlier authors to carry feed-forward inhibition from LHIs, is actually a branch of giant GABAergic neuron (GGN). Moreover, most and perhaps all of the GABAergic fibers in the calyx appear to be branches of GGN. It remains unknown what functions are served by the LH-branch of GGN. Although a blind-stick sampling procedure cannot definitively rule out the existence of any given neuron, and unknown neurons or neurotransmitters may provide additional inhibitory pathways, neither published observations nor the new results provide evidence that GABAergic LHIs exist, or that any LHNs provide feed-forward inhibition to the KCs (Gupta, 2012).

    The current results are consistent with anatomical findings from other insect species. In Drosophila, the anterior paired lateral (APL) neuron, which may contribute to olfactory memory, is the only known GABAergic neuron projecting to the calyx; it is morphologically and physiologically similar to the locust GGN, although its influence on KCs remains to be tested. In cockroach and honeybee only large GABAergic neurons, with GGN-like connectivity, appear to provide substantial inhibitory input to the calyx. The moth Manduca sexta has a single cluster of GABAergic cells that arborizes in the calyx and the lobe areas of the MB (Gupta, 2012).

    Periodic phase-locked inhibition from LHIs was proposed to help the KCs function as coincidence-detectors. This idea has also influenced thinking about the role of inhibition in the mammalian cortex. This study found that the graded output of GGN is tightly phase-locked to the olfactory system's oscillatory cycle with a phase-lag suitable for defining an input integration window in KCs within each cycle. Thus, although GGN differs from the presumed LHIs in its population size (1 versus 60), output properties (graded versus spiking) and the mechanism of inhibition (feed-back versus feed-forward), it may also promote coincidence-detection in the KCs by imposing brief, stable integration windows in each oscillatory cycle. The results are consistent with earlier finds that infusing a GABA receptor-blocker to the MB reduces the specificity of olfactory responses in the KCs; the current results suggest the source of GABA is GGN rather than the LHIs. Other factors are also thought to contribute to the sparseness of KCs, including their high firing thresholds and specialized nonlinear membrane conductances. A theoretical model suggested that adaptive regulation of the strength of the PN-KC synapse could also help maintain this sparseness (Gupta, 2012).

    Increasing concentrations of odors elicit increasingly coincident spiking across PNs, yet the odor-elicited responses of KCs remain sparse across a broad range of odor concentrations. A computational model suggested GABAergic LHIs could help maintain this sparseness by advancing the spiking phase of feed-forward inhibition they provide to KCs as the odor concentration increases. Could GGN instead play this role? This study found the output phase of GGN remains invariant with concentration, ruling out this mechanism. However, GGN appears able to offset increasingly coincident input from PNs in a different way: it counters increasing firing in KCs by feeding back to them stronger inhibition. Because strong IPSPs rise faster than weak ones, rhythmic inhibition from GGN could regulate the duration of integration windows (Gupta, 2012).

    Neurons of the LH have been proposed to mediate specific responses to odorants that are innately meaningful to the animal. Some LHNs in Drosophila express sex-specific transcripts of the fruitless gene implicated in courtship behaviors (Yu, 2010). Further, recent physiological work identified a cluster of neurons in the LH that responded only to the pheromone cVA. These results suggest pheromones, and possibly other odors involved in sexual behaviors, may be processed by specifically-tuned subpopulations of LHNs (Jefferis, 2007; Touhara, 2009; Yamagata, 2010). Whether the LH also provides specialized pathways for specific odors processed by the general (non-pheromonal) olfactory system has been unclear. In Drosophila, some anatomical evidence suggested stereotypy and clustering in the projections of genetically labeled PNs in the LH, but whether dendrites of LHNs are restricted to these clusters remains controversial. Nevertheless, the LH, in general, is thought to mediate very specific responses to odors. Surprisingly, this study found in locust that all LHNs tested (classes C1-C10) responded to all odors. Anatomical and electrophysiological results show dense convergence of PNs converge onto LHNs, providing no support for the idea that LHNs, in general, contribute to a labeled-line-like coding of innate preferences for specific odors in the general olfactory system. It remains possible the LH could mediate innate behaviors through specifically-tuned neurons that were not observed, or by responding preferentially to categories of odors that were not test, or through another mechanism, such as the combinatorial coding scheme used by PNs (Gupta, 2012).

    The great diversity of LHNs observed suggests the LH may perform other sensory functions, though. These observations lead to a speculation about three possible roles: representing general odor properties such as intensity; beginning bilateral olfactory integration; and participating in multimodal integration. These possible functions could all contribute to unlearned olfactory tasks such as odor-tracking. A previous study has proposed that phase-coding may allow extremely fast decoding of stimulus intensity by downstream cells. Class C3 neurons, which contain information about the odor concentration in their spike-phases and show bilateral projections, could assist in bilateral integration in the locust by allowing rapid comparison of odor intensity across the midline, a feature potentially important for odor-tracking in some animals (Gupta, 2012).

    Previous work in Drosophila identified neurons connecting the LH with different parts of the brain, including those serving other senses (Tanaka, 2004; Tanaka, 2008; Jefferis, 2007; Ruta, 2010). It was found that several neurons responded to both visual and olfactory stimuli, providing direct evidence for multimodal responses in the LH. Behavioral studies have shown that visual input contributes to odor-tracking (Frye, 2003). Thus, in addition to possibly integrating input from the two antennae, neurons of the LH may mediate integration of olfactory and visual cues for odor-tracking behaviors in insects (Duistermars, 2010). The current results provide specific neuronal targets, from the variety of LHN classes, for further investigation into mechanisms underlying odor-tracking. If the term 'innate behaviors' can be used broadly to encompass all memory-independent behaviors such as odor-tracking regardless of odor identity, these results support the role of LH in these behaviors (Gupta, 2012).

    Olfactory coding in the honeybee lateral horn

    Olfactory systems dynamically encode odor information in the nervous system. Insects constitute a well-established model for the study of the neural processes underlying olfactory perception. In insects, odors are detected by sensory neurons located in the antennae, whose axons project to a primary processing center, the antennal lobe. There, the olfactory message is reshaped and further conveyed to higher-order centers, the mushroom bodies and the lateral horn. Previous work has intensively analyzed the principles of olfactory processing in the antennal lobe and in the mushroom bodies. However, how the lateral horn participates in olfactory coding remains comparatively more enigmatic. This work studied odor representation at the input to the lateral horn of the honeybee, a social insect that relies on both floral odors for foraging and pheromones for social communication. Using in vivo calcium imaging, consistent neural activity was shown in the honeybee lateral horn upon stimulation with both floral volatiles and social pheromones. Recordings reveal odor-specific maps in this brain region as stimulations with the same odorant elicit more similar spatial activity patterns than stimulations with different odorants. Odor-similarity relationships are mostly conserved between antennal lobe and lateral horn, so that odor maps recorded in the lateral horn allow predicting bees' behavioral responses to floral odorants. In addition, a clear segregation of odorants based on pheromone type is found in both structures. The lateral horn thus contains an odor-specific map with distinct representations for the different bee pheromones, a prerequisite for eliciting specific behaviors (Roussel, 2014).

    A population of projection neurons that inhibits the lateral horn but excites the antennal lobe through chemical synapses in Drosophila

    In the insect olfactory system, odor information is transferred from the antennal lobe (AL) to higher brain areas by projection neurons (PNs) in multiple AL tracts (ALTs). In several species, one of the ALTs, the mediolateral ALT (mlALT), contains some GABAergic PNs; in the Drosophila brain, the great majority of ventral PNs (vPNs) are GABAergic and project through this tract to the lateral horn (LH). Most excitatory PNs (ePNs), project through the medial ALT (mALT) to the mushroom body (MB) and the LH. Recent studies have shown that GABAergic vPNs play inhibitory roles at their axon terminals in the LH. However, little is known about the properties and functions of vPNs at their dendritic branches in the AL. This study used optogenetic and patch clamp techniques to investigate the functional roles of vPNs in the AL. Surprisingly, the results show that specific activation of vPNs reliably elicits strong excitatory postsynaptic potentials (EPSPs) in ePNs. Moreover, the connections between vPNs and ePNs are mediated by direct chemical synapses. Neither pulses of GABA, nor pharmagological, or genetic blockade of GABAergic transmission gave results consistent with the involvement of GABA in vPN-ePN excitatory transmission. These unexpected results suggest new roles for the vPN population in olfactory information processing (Shimizu, 2017).

    Integration of chemosensory pathways in the Drosophila second-order olfactory centers

    Behavioral responses to odorants require neurons of the higher olfactory centers to integrate signals detected by different chemosensory neurons. Recent studies revealed stereotypic arborizations of second-order olfactory neurons from the primary olfactory center to the secondary centers, but how third-order neurons read this odor map remained unknown. Using the Drosophila brain as a model system, the connectivity patterns between second-order and third-order olfactory neurons was analyzed. Three common projection zones were isolated in the two secondary centers, the mushroom body (MB) and the lateral horn (LH). Each zone receives converged information via second-order neurons from particular subgroups of antennal-lobe glomeruli. In the MB, third-order neurons extend their dendrites across various combinations of these zones, and axons of this heterogeneous population of neurons converge in the output region of the MB. In contrast, arborizations of the third-order neurons in the LH are constrained within a zone. Moreover, different zones of the LH are linked with different brain areas and form preferential associations between distinct subsets of antennal-lobe glomeruli and higher brain regions. MB is known to be an indispensable site for olfactory learning and memory, whereas LH function is reported to be sufficient for mediating direct nonassociative responses to odors. The structural organization of second-order and third-order neurons suggests that MB is capable of integrating a wide range of odorant information across glomeruli, whereas relatively little integration between different subsets of the olfactory signal repertoire is likely to occur in the LH (Tanaka, 2004).

    A smell usually comprises a mixture of odorants, which are initially detected by the array of olfactory receptor neurons (ORNs, also termed first-order olfactory neurons). For the perception of a particular smell, information carried by each type of ORN must be integrated and then further categorized within the brain. ORNs expressing the same olfactory receptor send their axons to topographically fixed glomeruli in the primary olfactory center of the brain (olfactory bulb in mammals, antennal lobe [AL] in insects). Representation of odor at this level is thus a dynamic combination of active glomeruli (Tanaka, 2004 and references therein).

    Projection neurons (PNs, also termed second-order olfactory neurons, mitral/tufted cells in mammals, and projection neurons in insects) convey this information from AL glomeruli to secondary olfactory centers (e.g., piriform cortex, olfactory tubercle, and entorhinal cortex in mammals; mushroom body [MB] and lateral horn [LH] in insects. Because most PNs are uniglomerular and receive signals from a single type of ORNs, information detected by different ORN channels is not likely to be fully integrated at the level of the PNs. Supporting this contention, PN activities visualized by functional imaging and with the recording of characteristic synchronized oscillatory spikes show a clear correlation between the ensemble of activated PNs and the types of odor applied (Tanaka, 2004 and references therein).

    Integration among different ORN channels must therefore occur in secondary or even higher-order olfactory centers. If this process is to be understood, important insight must be gained from the connectivity patterns, namely those between PNs and third-order neurons, in the next synaptic level of the olfactory pathway. The projection pattern of PNs has recently been reported both in mammals and insects. In Drosophila melanogaster, for example, PNs from each glomerulus of the AL terminate in a stereotypic manner at the LH -- one of the two target neuropils of the PNs. The distribution of terminals in the other target (the calyx region of MB) remains unclear. The relationships between these PN terminals and the dendritic arborizations of third-order neurons of both LH and MB remain essentially unknown (Tanaka, 2004 and references therein).

    This study is the first systematic comparison of arborization patterns between PNs and third-order neurons. In the MB, they are organized such that the MB's output region (called lobes) can read olfactory information conveyed via all types of PNs. In the LH, however, third-order neurons link segregated subgroups of PNs exclusively with specific brain areas. This latter result is unexpected because it suggests the existence of parallel but separated channels between distinct subsets of olfactory sensory neurons and higher brain regions (Tanaka, 2004).

    PNs have stereotypic arborizations in the LH. Comparing the localization of PN terminals in the cross-section, there are at least three zones in the LH and each of these receives different sets of olfactory input via PNs. Previous studies, which classified PNs according to their branching patterns, did not identify zonations in the MB. The branching patterns of PNs are much more variable in the MB than in the LH. The area of their arborizations, however, is strikingly consistent. Mapping these areas, it was possible to identify clear concentric zones in the MB (Tanaka, 2004).

    Because these zones receive information from different sets of AL glomeruli, a particular odor would evoke different activity between them. Indeed, optical imaging with a calcium-sensitive fluorescence reporter, cameleon, revealed different activity patterns between the center and periphery of the calyx (Tanaka, 2004).

    The zonal projections identified in the LH and MB are highly correlated. This suggests that glomeruli in the AL can be categorized into discrete functional groups not only according to (1) the identity of the ORNs they receive but also (2) whether their PNs converge to the same or different zones of secondary olfactory centers (Tanaka, 2004).

    Attempts were made to reveal the connectivity pattern between second-order olfactory neurons (PNs) and third-order olfactory neurons by comparing their areas of arborization. Combinations of PNs and LHNs that share the same arborization field in the LH were detected. Simultaneous visualization showed that, at least in the cases tested, the arborizations of these neurons contact each other. This would strongly suggest that there are synaptic connections between them. Even in the case when they actually had intersected without making synapses, interaction between these intertwined arborizations was much more intense than between the neurons whose arborizations were completely segregated (Tanaka, 2004).

    Precise synaptic connection between PNs and third-order neurons could, in principle, be analyzed more directly via targeted expression of a trans-synaptic marker such as wheat germ agglutinin (WGA). The system, however, does not work reliably in most neurons of the Drosophila central brain, where WGA spreads into adjacent neurons nonspecifically. Unless a more specific technique is developed, the approach taken in this study would be the best alternative (Tanaka, 2004).

    The distributions of PN terminals are essentially similar between the two secondary olfactory centers. Thus, the functional differences between these centers are likely to be reflected by the differences in how third-order neurons are associated with the zonal arborizations of PNs (Tanaka, 2004).

    Behavioral and molecular analyses suggest that the information pathway involving the MB is crucial for the associative processing of olfactory signals. MBNs have been suggested to function as coincidence detectors. Although PNs convey activity information from a specific group of AL glomeruli to a specific zone of the MB calyx, dendrites of MBNs that contribute to each lobe collectively cover these zones. At the single-cell level, an MBN extends its dendrites either within a single zone or in two or three zones, suggesting that different MBNs contact PNs from diverse combinations of glomeruli. Axons of this heterogeneous population of MBNs all converge at the lobe region. Thus, each lobe could, in principle, read information sent from the entire AL. Such convergence might be important for the associative function of this olfactory center (Tanaka, 2004).

    In the LH of the Drosophila brain, the arborizations of the third-order neurons were identified for the first time. Their arborizations are constrained within zones that are defined by PN terminals. Thus, each group of LHNs has access to only a limited repertoire of olfactory information. Furthermore, LHNs originating from different zones of the LH innervate different areas of the brain. One consequence of such connectivity pattern is the hitherto unexpected existence of separated parallel channels between olfactory sensory neurons and higher processing sites. These channels are made before eclosion and maintained without olfactory input. This might suggest that keeping such neural circuits would be important when insects mediate olfactory responses to odors they have never experienced (Tanaka, 2004).

    The different zones of the LH are also associated with the sensory pathways of other modalities in a different way. The ventral region of LH is linked with the vlpr and ammc, which are, respectively, the major target of visual neurons from the optic lobe and the sole target of the mechanosensory antennal neurons, including the auditory sensory organ. The brain regions connected with the dorsal LH, in contrast, lack major input from the visual and mechanosensory pathways. There is thus a significant difference in the degree of sensory convergence between odorant information associated with the ventral and dorsal halves of the LH (Tanaka, 2004).

    Information pathways via the LH must be sufficient for nonassociative odor-related behavior because the ablation of the MB causes no effect on these functions. Structural organization of PNs and LHNs suggests that the LHNs and presumably higher centers in the brain linked with these segregated LH zones read only a subset of olfactory glomeruli. Such a limited level of integration seems sufficient for mediating animals' direct behavioral responses to odors (Tanaka, 2004).

    From the sensory organs to the primary and secondary centers, the structure and topology of olfactory neural networks are strikingly similar between insects and mammals. Like information from the LH and MB of insects, information from a single type of mammalian ORN is conveyed to only a small part of each secondary center, such as the piriform cortex and olfactory tubercle. If the similarity between insect and mammalian olfactory systems can further be extrapolated, similarly separated channels from the ORN to higher cortical areas might play important roles in mediating the direct olfactory response of mammals (Tanaka, 2004).

    The present analysis has provided an important perspective about the structural relationships between second-order and third-order olfactory neurons of Drosophila. Arborizations of second-order neurons from distinct subgroups of AL glomeruli form essentially similar zonations in the two secondary olfactory centers, the LH and MB. In the MB, which is important for olfactory learning and memory, dendrites of third-order neurons show diverse distributions across zones. Axons of these heterogeneous neurons converge at each MB lobe, suggesting that extensive integration across a wide range of olfactory signals would occur. In the LH, which is important for immediate responses to odors, arborization of each type of third-order neurons is limited within one of these zones, suggesting limited integration among small subsets of odorant repertoire. Further physiological analyses of the uniquely identified second- and third-order neurons will provide vital information for understanding how olfactory information is received and integrated in the two secondary olfactory centers (Tanaka, 2004).

    Stereotyped connectivity and computations in higher-order olfactory neurons.

    In the first brain relay of the olfactory system, odors are encoded by combinations of glomeruli, but it is not known how glomerular signals are ultimately integrated. In Drosophila melanogaster, the majority of glomerular projections target the lateral horn. Lateral horn neurons (LHNs) receive input from sparse and stereotyped combinations of glomeruli that are coactivated by odors, and certain combinations of glomeruli are over-represented. One morphological LHN type is broadly tuned and sums input from multiple glomeruli. These neurons have a broader dynamic range than their individual glomerular inputs do. By contrast, a second morphological type is narrowly tuned and receives prominent odor-selective inhibition through both direct and indirect pathways. This wiring scheme confers increased selectivity. The biased stereotyped connectivity of the lateral horn contrasts with the probabilistic wiring of the mushroom body, reflecting the distinct roles of these regions in innate as compared to learned behaviors (Fisek, 2013).

    To understand higher olfactory processing, it is fundamentally important to know how many glomeruli provide input to a typical higher-order neuron. It is also important to know whether these connections are stereotyped, whether different glomerular inputs are associated with different synaptic weights and whether some combinations of glomeruli occur preferentially. In this study, paired recordings were used to map the connectivity of representative LHN projection neurons and to address these questions. Because connectivity was found to be stereotyped, large numbers of paired recordings could be used to build a cumulative picture of the connectivity of these neurons (Fisek, 2013).

    Random samples of PN-LHN pairs allowed estimation the number of input glomeruli for each LHN type. For representative type I neurons (Mz671 neurons), 120 paired recordings with random PNs were performed, and five connections were found. Given 49 glomeruli, binomial statistics would indicate with ~95% confidence that there are at most four connected glomeruli. Indeed, four inputs for these neurons (DM1, DM2, DM4 and VA7l) were found. This calculation assumes that there are equal numbers of PNs in all glomeruli. In total, there are ~150 PNs, which would predict three PNs per glomerulus, but it is known that some glomeruli contain more than three PNs (for example, glomerulus DA1 and some contain only one PN. If a glomerulus contained only one PN, then it would be likely to be missed, and indeed, glomerulus DM1 was a near miss: it contains one PN<, and it did not turn up in the random screen. DM1 was identified only as a result of targeted paired recordings. Thus, four glomeruli might be an underestimate. Nonetheless, it seems likely that each type I neuron receives input from fewer than ten glomeruli (Fisek, 2013).

    For representative type II neurons (NP6099 neurons), 82 paired recordings were performed with random PNs, and no connections were found. Binomial statistics would indicate that there are at most two connected glomeruli, with the same caveats as those listed above. However, for these neurons, there is independent evidence arguing that DP1m is the only excitatory input. Specifically, all the odors that activate these neurons also activate DP1m PNs, and the firing rates of DP1m PNs are sufficient to predict the strongest odor responses in these neurons. It will be interesting to learn whether all type II neurons receive excitatory input from a single glomerulus. It is notable that VA2 PNs do not connect to these neurons despite substantial axon-dendrite overlap. These results raise the question of how an LHN reliably forms a connection with one axon but avoids forming a connection with another axon in a case where the two axons are overlapping (Fisek, 2013).

    A notable conclusion of this study is that some glomerular combinations are substantially over-represented in the lateral horn. Consider the fact that there are three Mz671 neurons per lateral horn, but there are only several hundred LHNs in total (on the basis of cell counts in experiments in which PA-GFP was expressed pan-neuronally, and a large volume of the lateral horn was photoactivated. Four glomeruli connected to Mz671 neurons were identified. Given that there are 49 glomeruli in total, there are >200,000 possible combinations of four glomeruli. This is far larger than the total number of neurons in the lateral horn. Moreover, the particular glomerular combination sampled by the Mz671 neurons occurs not once but at least three times in every lateral horn. Therefore, the space of possible glomerular combinations is sampled nonrandomly (Fisek, 2013).

    Paired recordings also showed that different glomerular inputs to an LHN can be associated with nonuniform and stereotyped synaptic weights. This idea has been proposed previously as a way to render LHNs more selective for a particular olfactory feature. This result also indicates a high level of precision in the development of this circuit (Fisek, 2013).

    In many of these respects, the results show that the lateral horn differs radically from the mushroom body, which is the other third-order olfactory region in insects. In the mushroom body, the pattern of glomerular inputs appears to be different in different individuals. And although there are regional biases in connections from glomeruli to the mushroom body, and glomeruli with similar tuning tend to wire together, connectivity in the mushroom body nonetheless seems to be probabilistic rather than deterministic. This contrasts with the highly stereotyped wiring that this study found in the lateral horn. Disrupting the mushroom body impairs learned but not unlearned olfactory discriminations, implying that the lateral horn is sufficient for innate olfactory behavior. Thus, the mushroom body and lateral horn serve different behavioral functions, and the results demonstrate that they also sample differently from olfactory glomeruli (Fisek, 2013).

    It should be noted that the stereotypy observed may be specified entirely by the genetic inheritance of these organisms, but this is not necessarily the case. All the flies were raised in these experiments in a similar environment. Future studies will be needed to determine whether there is any experience-dependent element in these connections or their weights (Fisek, 2013).

    The results demonstrate that different types of LHNs carry out distinct computations on the information they receive from olfactory glomeruli. Type I neurons are broadly tuned to odors, and Mz671 neurons are typical of type I neurons in this respect. Consonant with this, it was found that Mz671 neurons pool excitation from a handful of coactivated glomeruli, and input from even a single glomerulus can be sufficient to drive postsynaptic spiking. For this reason, it might be expected that these LHNs are more broadly tuned to odors than are PNs. Broad odor tuning to a group of related chemicals might be a useful way to link a large region of chemical space (for example, odors associated with fruit) with an innate behavioral program (for example, feeding) (Fisek, 2013).

    In addition, this study observed that the Mz671 neurons have a broader dynamic range for concentration encoding as compared to their presynaptic PNs. Drosophila can generalize across different concentrations of the same odor, and this behavioral performance requires integrating activity across multiple glomeruli that are coactivated by some odors but with different sensitivities to those odors. Whereas each individual glomerulus can only encode concentration over roughly two orders of magnitude, summing several glomeruli that have different dynamic ranges can yield a broader range of sensitivity. This is precisely what the Mz671 neurons do. Thus, type I LHNs might have a role in concentration generalization (Fisek, 2013).

    In contrast to type I neurons, type II neurons are narrowly tuned, and NP6099 neurons are typical of type II neurons in this respect. Again, consonant with their narrow tuning, this study showed that NP6099 neurons combine excitation from one (or a few) glomeruli with tuned inhibition from coactivated glomeruli, yielding greater selectivity. This computation is distinct from that performed by the Mz671 neurons. On theoretical grounds, combining excitation and inhibition from coactivated glomeruli has been proposed as a way to generate selectivity. Behavioral data show that Drosophila can perform fine discriminations among odor stimuli with different chemical compositions. Neurons with high selectivity might be a useful way to link specific odor stimuli with behavioral programs (Fisek, 2013).

    In other sensory systems, the receptive field of a neuron can be described as a set of positive and negative weights over stimulus space. This study shows that this framework can be extended to higher-order olfactory receptive fields, which are essentially a set of positive and negative weights over olfactory glomeruli. Each glomerulus corresponds to an odorant receptor, and each receptor is selective for a molecular feature. Thus, higher-order olfactory receptive fields represent weighted sums of molecular features. In other sensory systems, receptive field structures are nonrandom insofar as they have a strong tendency to sample from overlapping regions of stimulus space, reflecting the statistical regularities of the environment. Analogous to this, neurons were described that sample from glomeruli with overlapping chemical tuning, and it was shown that the sampling is highly nonrandom. It will be interesting to investigate how the computations that occur in the lateral horn might relate to the statistical distribution of odors in the environment, as well as their ecological relevance to the organism (Fisek, 2013).

    GABAergic projection neurons route selective olfactory inputs to specific higher-order neurons

    This study characterizes an inhibitory circuit motif in the Drosophila olfactory system, parallel inhibition, which differs from feedforward or feedback inhibition. Excitatory and GABAergic inhibitory projection neurons (ePNs and iPNs) each receive input from antennal lobe glomeruli and send parallel output to the lateral horn, a higher center implicated in regulating innate olfactory behavior (see Anatomical organization of the olfactory nervous system in Drosophila). Ca2+ imaging of specific lateral horn neurons as an olfactory readout revealed that iPNs selectively suppress food-related odor responses, but spare signal transmission from pheromone channels. Coapplying food odorant did not affect pheromone signal transmission, suggesting that the differential effects likely result from connection specificity of iPNs, rather than a generalized inhibitory tone. Ca2+ responses in the ePN axon terminals show no detectable suppression by iPNs, arguing against presynaptic inhibition as a primary mechanism. The parallel inhibition motif may provide specificity in inhibition to funnel specific olfactory information, such as food and pheromone, into distinct downstream circuits (Liang, 2013).

    Two general circuit motifs involving inhibitory neurons are widely used in vertebrate and invertebrate nervous systems. In feedback inhibition, inhibitory neurons are locally activated by excitatory neurons. In turn, they inhibit a broad array of excitatory neurons, including those that excite them. In feedforward inhibition, excitatory input activates both excitatory and inhibitory target neurons, and the activated inhibitory target neurons further inhibit the excitatory target neurons. The mammalian olfactory bulb, for instance, provides examples of both motifs. As an example of feedback inhibition, granule cells are activated by mitral cells in response to odor stimuli. In turn, they inhibit the same and neighboring mitral cells. As an example of feedforward inhibition, ORN axons excite periglomerular cells and mitral cells in parallel; some periglomerular cells inhibit mitral cells in the same and adjacent glomeruli. Both granule cells and periglomerular cells contribute to the lateral inhibition and sharpening of the olfactory signals that mitral cells deliver to the olfactory cortex. Similarly, the fly antennal lobe, the equivalent of the mammalian olfactory bulb, has a diversity of GABAergic local interneurons (LNs). Some LNs are excited by ORNs and subsequently provide feedback inhibition onto ORN axon terminals for gain control. Other LNs may act on PN dendrites for feedforward inhibition. This study describes an inhibitory circuit motif that differs from classic feedforward and feedback inhibition, which is termed parallel inhibition, wherein excitatory and inhibitory projection neurons receive parallel input and send parallel output to a common target region (the lateral horn) (Liang, 2013).

    What are the possible roles of iPNs, and what advantages might the parallel inhibition motif confer? By monitoring olfactory responses of a subset of putative third-order lateral horn neurons (the vlpr neurons) and by laser transecting the ascending mACT input from iPNs while sparing ePNs, this study has shown that iPNs selectively route olfactory input to vlpr neurons. Specifically, the vlpr responses to the food odors are inhibited by the iPNs, but the response to the cVA pheromone-processing channel is not subjected to this inhibition. Previous anatomical studies revealed highly stereotyped branching and terminal arborization patterns for uniglomerular ePNs and iPNs (Jefferis, 2007: Lai, 2008). Results in this study provide functional demonstration that GABAergic iPNs regulate olfactory inputs to the lateral horn neurons. Indeed, the fact that removing iPN inhibition allows isoamyl acetate and vinegar signals to activate vlpr neurons suggests that anatomical segregation of PN axon terminals representing food and pheromone (Jefferis, 2007) alone is not sufficient to prevent food odors to activate vlpr neurons, at least some of which are normally activated by pheromones. iPN inhibition provides another level of specificity of the higher-order neuronal responses to olfactory input (Liang, 2013).

    This specificity of inhibition provides a special feature of parallel inhibition in comparison with feedforward and feedback inhibition. Feedforward and feedback inhibition tend to be nonspecific with respect to their target population within the same neuronal type, which is optimal for certain functions these motifs serve, such as lateral inhibition and gain control. In the Drosophila antennal lobe, for example, while exhibiting a large variety of arborization patterns, most local interneurons (LNs) innervate many to all glomeruli, where they both receive input and send output. By contrast, the specific dendritic glomerular innervation of individual iPNs in the antennal lobe, as well as their stereotyped axonal arborization patterns in the lateral horn, enable iPNs to selectively inhibit some olfactory-processing channels, but not others. It is speculated that food odors should activate other lateral horn higher-order neurons relevant to foraging and that such activation is not strongly inhibited by iPNs, perhaps also due to inhibition specificity (Liang, 2013).

    Another interesting feature of parallel inhibition is the timing of inhibition. Inhibition from feedforward and certainly feedback motifs arrive later than excitation due to transmission through an extra synapse, which is used to confine the magnitude and/or duration of excitation. The parallel inhibition motif in principle allows for simultaneous arrival of excitation and inhibition at the postsynaptic neurons, potentially enabling inhibition to completely suppress excitation, and is ideally suited for information gating. This study provides evidence that the primary action of iPNs is unlikely through presynaptic inhibition of ePNs, as ePN presynaptic Ca2+ signals in response to olfactory stimuli were not elevated by middle antennocerebral tract (mACT) transection. A caveat of this interpretation is that some forms of presynaptic inhibition can bypass Ca2+ entry, for instance through Gβγ action on the release machinery; however, GABAergic inhibition that acts in this manner has not currently been identified. Thus, the idea is favored that iPNs act directly on postsynaptic third-order neurons under the experimental conditions. Due to the limited temporal resolution of Ca2+ imaging, the temporal property of parallel inhibition has not been explored in this study. It will be interesting for future research to measure the arrival time of both excitatory and inhibitory input directly with more sensitive and temporally precise electrophysiological methods (Liang, 2013).

    This study describes the use of the parallel inhibition motif in sensory systems. Long-distance GABAergic projections are prevalent in the mammalian brain. Specifically, some GABAergic neurons in the hippocampus and cortex have recently been identified that send long-distance projections, sometimes to the same area as the glutamatergic projection neurons. Thus, parallel inhibition can potentially be a widely used mechanism in the nervous system (Liang, 2013).

    This study has identified a unique class of higher-order neurons that respond to Or67d [and presumably the pheromone 11-cis-vaccenyl acetate (cVA)] activation. Or67d ORNs and their postsynaptic partner DA1 excitatory PNs express FruM, a male-specific transcription factor that is a key regulator of sexual behavior. A previous study identified a number of Fru+ higher-order cVA-responsive neurons whose cell bodies reside dorsal and lateral to the lateral horn (Ruta, 2010). Indeed, the analyses of Fru+ neurons have so far provided many examples where Fru+ neurons are connected with each other to regulate different aspects of sexual behavior. However, lateral horn-projecting Mz699+ vlpr neurons do not appear to express FruM, despite their robust activation by Fru+ Or67d ORNs. This may reflect a broad function of cVA as a pheromone that regulates not only mating but also aggression and social aggregation (Liang, 2013).

    This study revealed a difference between food- and pheromone-processing channels in their susceptibility to inhibition by iPNs and suggests that pheromone channels may be insulated from general inhibition by iPNs. It is almost certain that iPNs play additional functions than reported in this study, as iPN function was studied only from the perspective of their effect on the olfactory response of a specific subset of higher-order neurons. Indeed, in a companion manuscript, Parnas (2013) showed that iPNs play an instrumental role in facilitating the discrimination of mostly food odors, as assayed by quantitative behavioral experiments. Taken together, these studies uncovered two distinct aspects of iPN function: increased discrimination of diverse food odors and information gating between qualitatively different olfactory stimuli (Liang, 2013).

    Finally, it is notable that of the two major ePN targets, iPN axons only project to the lateral horn but spare the mushroom body. The mushroom body is a well-documented center for olfactory learning and memory, whereas PN projections to the lateral horn are implicated in regulating innate olfactory behavior (see Parnas, 2013). ePN axons exhibit striking stereotypy in their terminal arborization patterns in the lateral horn, but not in the mushroom body. Recent anatomical tracing in mice also revealed differential input organization in distinct olfactory cortical areas, suggesting a common principle in olfactory systems of insects and mammals. The selective innervation by iPNs of targeting neurons in the lateral horn suggests that regulation of innate olfactory behavior engages an additional level of specific inhibition to ensure that olfactory information carrying different biological values, such as food and pheromone, is funneled into distinct downstream circuits, resulting in the activation of distinct behavioral outputs (Liang, 2013).

    Cav3-type α1T calcium channels mediate transient calcium currents that regulate repetitive firing in Drosophila antennal lobe PNs

    Projection neurons (PNs), located in the antennal lobe region of the insect brain, play a key role in processing olfactory information. To explore how activity is regulated at the level of single PNs within this central circuit recordings were made from these neurons in adult Drosophila melanogaster brains. A previous study demonstrated that PNs express voltage-gated calcium currents with a transient and sustained component. This study found that the sustained component is mediated by cac gene-encoded Cav2-type channels involved in regulating action potential-independent release of neurotransmitter at excitatory cholinergic synapses. The function of the transient calcium current and the gene encoding the underlying channels, however, were unknown. This study reports that the transient current blocked by prepulse inactivation is sensitive to amiloride, a vertebrate Cav3-type channel blocker. In addition PN-specific RNAi knockdown of Ca2+-channel protein α1 subunit T (Ca-alpha1T), the Drosophila Cav3-type gene, caused a dramatic reduction in the transient current without altering the sustained component. These data demonstrate that the α1T gene encodes voltage-gated calcium channels underlying the amiloride-sensitive transient current. Alterations in evoked firing and spontaneous burst firing in the α1T knockdowns demonstrate that the Cav3-type calcium channels are important in regulating excitability in adult PNs (Iniguez, 2013).

    A wide variety of insect behaviors are driven or modulated by olfactory input and the ensemble of neurons involved in processing olfactory information is well defined. Olfactory perception begins when odorant molecules bind to receptors in olfactory receptor neurons (ORNs) located in the antennae and the maxillary palps. ORNs project to the antennal lobes, the insect equivalent of the vertebrate olfactory bulb, where they synapse onto the dendrites of projection neurons (PNs), the principal output cells that extend axons to higher order processing centers in the mushroom bodies and lateral horn. In Drosophila melanogaster, where genetic manipulations and behavioral assessment are routine, it is now feasible to record from identified neurons within this circuit in the adult brain. This has made it possible to explore the mechanisms that regulate activity in a circuit important in generating specific components of an adult behavior at the single cell level. Whole cell recordings from single PNs have demonstrated that olfactory processing begins in the antennal lobe where both intra- and interglomerular interactions influence activity of these cells (Iniguez, 2013).

    To understand the molecular mechanisms underlying regulation of neuronal activity in individual PNs in the olfactory circuit requires identification of the ion channel subtypes that govern excitability and synaptic transmission in these cells. One important class of channels present in all neurons are voltage-gated calcium channels that mediate depolarization-induced calcium influx that influences a number of cellular processes including excitability and release of neurotransmitters at chemical synapses. The α1-subunit of these multimeric proteins forms the ion-conducting pore that defines many of the functional properties characteristic of the distinct calcium channel subtypes. There are three families of genes encoding α1 subunits and in the Drosophila genome there is one α1 subunit gene in each family: α1D (Cav1), cac (Cav2), and α1T (Cav3). A recent study found that voltage-gated calcium currents recorded from the cell bodies of PNs in the adult brain could be separated into two kinetically distinct components: a rapidly decaying transient current and a slowly decaying sustained current. Using a combination of pharmacological and molecular genetic strategies, this study has demonstrated that the Cav2-type cac gene encodes calcium channels that mediate PLTXII-sensitive sustained calcium currents. These studies show that the CAC channels regulate action potential-independent release of neurotransmitter at excitatory cholinergic synapses in the adult brain, a novel role not predicted from previous studies at peripheral synapses (Iniguez, 2013 and references therein).

    While a recent study suggests that Cav2-type CAC channels (aka Dmca1A) also contribute to the transient calcium currents in adult motor neurons, the current results indicated that neither PLTXII nor mutations in the cac gene reduced the transient currents in PNs. This suggests that a distinct calcium channel subtype gives rise to the transient current in adult PNs. In vertebrates, previous studies have demonstrated that Cav3 genes encode channels underlying transient calcium currents (Iniguez, 2013).

    This study reports that amiloride, a vertebrate Cav3-type channel blocker, reduces the transient calcium current without significantly altering the sustained Cav2-type CAC channel-mediated current in adult PNs. In addition, RNAi mediated knockdown of the α1T gene, the Drosophila Cav3-type homolog, in PNs reduced the transient component significantly but the sustained component was not affected. Alterations in evoked and spontaneous firing were observed in the α1T knockdowns. These data demonstrate that α1T-encoded Cav3-type channels mediate transient calcium currents that are important in shaping the PN firing properties and, therefore, play an important role in regulating olfactory signal processing (Iniguez, 2013).

    Projection neurons in the adult Drosophila antennal lobe process olfactory input from olfactory receptor neurons and relay this to neurons in the mushroom body and lateral horn. This well-defined neural pathway and access to these neurons in the whole brain preparation provide an excellent model system to explore how voltage-gated calcium channels regulate signaling in an identified neuronal subtype within the adult olfactory circuit. The results of this study demonstrate that the transient calcium current in the adult Drosophila antennal lobe PNs is mediated by amiloride-sensitive α1T-encoded channels. Furthermore, the α1T channels were found to modulate cell excitability, a novel role in the Drosophila central nervous system (Iniguez, 2013).

    A recent analysis of adult Drosophila motor neurons reported three distinct calcium current phenotypes in an α1T null mutant: complete loss of all calcium currents (56%), reduction of both sustained and transient currents (28%), and no effect on calcium currents (16%). In contrast, whena PN-specific Gal4 driver was used in conjunction with two independently generated UAS-RNAi lines targeted to the α1T gene, this resulted in specific reductions in the transient calcium current. No significant change in the sustained calcium current was observed. Since the two RNAi lines were targeted to distinct regions of the α1T gene, this is compelling evidence that the α1T gene encodes channels underlying the transient current in PNs. The varied effects of an α1T null mutant of calcium currents previously reported in motor neurons may be associated with activation of homeostatic regulatory mechanisms caused by elimination of the channel in all cell types (Iniguez, 2013).

    Similar to currents mediated by T-type channels in vertebrates, the transient calcium currents in adult PNs are inhibited by amiloride, a vertebrate Cav3 T-type calcium channel blocker. Amiloride has also been reported to block a portion of the calcium current that could be inactivated by depolarization in Drosophila larva body wall fibers and embryo motor neurons. While the underlying gene encoding the current in muscle and motor neurons was not identified, the current data suggest the α1T gene may also encode the channels in these cells. In a recent study it appears that α1T channels also underlie an amiloride-sensitive transient current that represents a relatively small component of the total calcium current in adult motor neurons. The majority of transient current in the motor neurons, however, appears to be mediated by Cav2-type CAC channels. This suggests that, similar to finding in the mammalian central nervous system, stage and/or cell-specific splicing events give rise to calcium channels with distinct functional properties (Iniguez, 2013 and references therein).

    In contrast to T-type currents in vertebrates that typically activate at low voltages, the PN transient calcium currents are first activated at a membrane potential of between -50 and -40 mV. This activation profile is similar to that reported for transient calcium currents in neurons cultured from brains of late stage wild-type pupae, Drosophila embryonic motor neurons, Drosophila muscle fibers, and Drosophila larval motor neurons. In contrast, studies in cytokinesis-arrested neuroblasts, embryonic motor neurons, and adult motor neurons reported both low- and high-voltage-activated calcium currents. The discrepancies in the studies could arise from the differences in cell types and developmental stages. It is also possible that contributing to the differences noted is the use of barium vs. calcium as the charge carrier for studying calcium influx. Replacing calcium with barium substantially increases the current amplitude . Barium is also known to suppress Ca2+-dependent inactivation of calcium channels. In larval motor neurons when barium was used to replace calcium as the charge carrier, this caused a substantial increase in current amplitude and altered the kinetics of calcium current decay. All of the recordings in PNs in the present study were conducted in physiological concentrations of Ca2+, and therefore, calcium-dependent inactivation of the current could contribute to the transient nature of the current (Iniguez, 2013).

    The increase in evoked firing frequency in PNs reported in the RNAi-1T knockdowns demonstrates that the α1T channels are important in regulating excitability in adult Drosophila neurons. The increased excitability caused by reducing expression of Cav3-type channels in the adult PNs is similar to the upregulation of firing frequency documented in larval motor neurons following genetic reduction of Cav1 or Cav2 type calcium channels. In the wild-type motor neurons, the increase in firing frequency was mimicked by acute removal of calcium from the recording solution suggesting the change in excitability reflects reduced activation of Ca2+-activated K+ channels. However, the possibility has not been ruled out that there was compensatory downregulation of other K+ channel genes in the Cav1 and Cav2 mutants that could have the same effect of increasing motor neuron-evoked firing frequency (Iniguez, 2013).

    To avoid the possible contribution of developmental regulation of other channel types, it would be helpful to explore the role of α1T channels in regulating excitability by acutely blocking these channels in wild-type PNs. Unfortunately Drosophila express nonvoltage-gated sodium channels that are also sensitive to amiloride, and preliminary studies indicate these can affect membrane potential depolarizations directly. Therefore, additional experiments with double mutant combinations will be necessary to determine if reduced activation of Ca-activated K channels and/or reduced expression of other K channels contribute to the increase in firing frequency seen in adult PNs in α1T knockdowns (Iniguez, 2013).

    Low-voltage-activated T-type Ca2+ channels in mammals have been shown to be important in pacemaking activity in sino-artrial node of the heart, and they are crucial for generation of rhythmic bursts of action potentials in thalamic relay neurons of the thalamus. In PNs there was no significant change in the burst firing frequency, but the burst duration in α1T knockdowns was significantly increased. This suggests that while T-type calcium channels do have a role in regulating spontaneous burst firing in Drosophila PNs, their relatively high activation voltage may limit their contribution to pacemaking type activities (Iniguez, 2013).

    Is the third calcium channel subtype encoded by the DMCA1D gene also expressed in adult PNs? Preliminary studies revealed that calcium currents examined in PNs from hypomorphic α1D mutant AR66 and α1D-knockdowns (VDRC no.51491) had both transient and sustained currents that were not significantly different than wild-type. However, alteration in the firing properties of DMCA1D knockdown and mutant indicates that these channels are expressed in PNs. This suggests that the currents mediated by these channels are located in the axonal compartment, electrically distant from the soma. If calcium influx is initiated at some distance from the electrode, this current change will be substantially attenuated when it reaches the soma. Unfortunately, the soma of PNs is the only location in these cells that is accessible to the patch-clamp electrode (Iniguez, 2013).

    In conclusion, these results demonstrate for the first time that α1T-encoded voltage-gated calcium channels are expressed in adult PNs where they are important in regulating excitability. Further studies on how these channels regulate olfactory related behavior will be an important contribution to understanding of how activity within specific neurons in a well-defined circuit guides behavior (Iniguez, 2013).

    Activity-dependent plasticity in an olfactory circuit

    Olfactory sensory neurons (OSNs) form synapses with local interneurons and second-order projection neurons to form stereotyped olfactory glomeruli. This primary olfactory circuit is hard-wired through the action of genetic cues. It was asked whether individual glomeruli have the capacity for stimulus-evoked plasticity by focusing on the carbon dioxide (CO2) circuit in Drosophila. Specialized OSNs detect this gas and relay the information to a dedicated circuit in the brain. Prolonged exposure to CO2 induced a reversible volume increase in the CO2-specific glomerulus. OSNs showed neither altered morphology nor function after chronic exposure, but one class of inhibitory local interneurons showed significantly increased responses to CO2. Two-photon imaging of the axon terminals of a single PN innervating the CO2 glomerulus showed significantly decreased functional output following CO2 exposure. Behavioral responses to CO2 were also reduced after such exposure. It is suggested that activity-dependent functional plasticity may be a general feature of the Drosophila olfactory system (Sachse, 2007).

    Neuroanatomical, functional, and behavioral analysis suggests that the Drosophila olfactory system has the capacity for reversible activity-dependent plasticity. Evidence of this plasticity is readily seen by measuring the volume of the V glomerulus. Because the volume increase can be induced by odor activation of ORs ectopically expressed in the CO2-activated OSNs, it is concluded that persistent stimulus-evoked activity in these neurons underlies these anatomical changes. It has been shown that stimulus-evoked plasticity is a general feature of the Drosophila olfactory system and not a peculiarity of the CO2 circuit. For instance, the volume of DM2 is increased by chronic exposure to ethyl butyrate, a ligand for the Or22a-expressing neurons that target DM2 (Sachse, 2007).

    Drosophila, CO2 is detected by a population of approximately 25-30 OSNs in the antenna that express the chemosensory receptor Gr21a, which along with Gr63a comprises the Drosophila CO2 receptor. These OSNs project axons that terminate in the V glomerulus in the ventral antennal lobe. The Drosophila CO2 circuit is ideal for studying odor-evoked plasticity because Gr21a-expressing OSNs are the only neurons in the fly that respond to CO2, and they do not respond to any other stimuli. In this work, stimulus-evoked changes in the anatomy and function were examined of the Drosophila CO2 circuit. The results provide functional evidence that a primary olfactory center is capable of activity-dependent plasticity (Sachse, 2007).

    The data are consistent with a model in which one class of inhibitory LNs and the output of the V glomerulus are the major targets of plasticity induced by sensory exposure. Under conditions of ambient CO2, the Gr21a circuit forms normally and small amounts of CO2 produce robust behavioral responses. When flies are exposed to elevated CO2 early in life, it is postulated that chronic activation of Gr21a neurons promotes functional changes in the LN2 subtype of inhibitory local interneurons without affecting either the functional properties of the OSNs or the CO2-evoked response of the LN1 neurons. It is suggested that the volume increases seen with CO2 exposure may result from neuroanatomical changes in the LNs, although their extensive glomerular arborization made this hypothesis difficult to test experimentally. Since a majority of the LN2 population in Drosophila has been shown to be GAD1 positive and thus to release GABA, as known for antennal lobe LNs in other insects, greater CO2-evoked activity of LN2s may lead to an increased inhibition of the PN postsynaptic to Gr21a OSNs. The finding of reduced activity in the output region of the PN innervating the V glomerulus supports this hypothesis. Thus, CO2-evoked activity would be attenuated in the antennal lobe circuit in these animals, producing a corresponding decrease in the intensity of the behavioral response (Sachse, 2007).

    It has recently been shown that LNs are not only inhibitory, as has been assumed so far. A newly described population of excitatory cholinergic LNs forms a dense network of lateral excitatory connections between different glomeruli that may boost antennal lobe output (Olsen, 2007; Shang, 2007). Future studies are necessary to investigate if excitatory LNs are also subject to activity-dependent plasticity (Sachse, 2007).

    Stimulus-dependent plasticity can be induced and reversed in a critical period early in the life of a fly. Similar critical periods have been documented in selective deafferentation periods in mammalian somatosensory and visual cortex. In all these model systems, the critical period likely allows the animal to compare the genetically determined network template with external conditions and make activity-dependent adjustments that reflect the external environment. For instance, visual cortex 'expects' binocular input when it is wired in utero. If monocular input is experimentally imposed, the system is rewired to reflect this. The same rewiring occurs in the barrel cortex, in which the receptive fields of missing whiskers are invaded by neighboring whiskers, allowing the animal to maintain a continuous representation of external somatosensory space. Drosophila pupae have no sensory input during development and develop an olfactory system that relies neither on evoked activity nor the expression of ORs. The time following adult eclosion may represent a period in which the functional set point of the Drosophila olfactory system is evaluated and adapted to the local environment (Sachse, 2007).

    What elements of the antennal lobe circuit are responsible for the stimulus-dependent volume increases seen here? No evidence was found that OSNs modulate their number, morphology, branching pattern, or functional properties in response to CO2 exposure. The same neuroanatomical properties of single LNs or PNs could not be assayed due to the dense processes of these neurons in a given glomerulus. Since the observed net increase in volume cannot be ascribed to anatomical changes in OSNs, morphological plasticity is most likely occurring either at the level of LN or PN. A model is favored in which changes in the LNs underlie the observed volume increases because clear functional differences were found in LN2 responsivity in CO2-exposed animals and because PN dendrites and axons have been shown to be extremely stable in size and morphology when deprived of OSN input. Similar stability in mitral/tufted cells has been shown in rodent olfactory bulb. The possibility that other cells, such as glia, contribute to these activity-dependent volume changes cannot be excluded (Sachse, 2007).

    This work suggests that antennal lobe LNs marked with two different Gal4 enhancer traps, Gal4-LN1 and Gal4-LN2, are functionally distinct. The arborization of LN1 and LN2 processes in the V glomerulus suggests that they interact differentially with the antennal lobe circuitry. LN1 processes appear to innervate the core of a given glomerulus, while LN2 processes innervate the glomerulus more uniformly. Both LN1 and LN2 neurons show weakly concentration-dependent tuning to odor stimuli. Thus, compared to the OSNs or PNs, which transmit a precise spike-timing code that reflects absolute CO2 concentration, these LNs appear to respond in a binary fashion, showing similar levels of activity regardless of stimulus concentration (Sachse, 2007).

    There is a clear difference in how the responses of these two LN populations are modulated by CO2 exposure. While the activity of LN1 neurons was not significantly affected by CO2 exposure, LN2 neurons exhibited robust and significant increases in CO2-evoked activity after CO2 exposure. It will be of interest to examine the functional properties of these neurons in greater detail using electrophysiological approaches. It is plausible that circuit plasticity as evidenced in the LN2 neurons can be detected with electrophysiology at even lower CO2 concentrations for shorter exposure periods (Sachse, 2007).

    How might chronic activation of CO2-sensitive OSNs specifically affect the physiology of LN2 neurons? It is speculated that due to the broader innervation of LN2 processes, these neurons would receive greater presynaptic innervation from Gr21a-expressing OSNs. Thus, with chronic CO2 exposure, the LN2 neurons would be chronically activated. This might cause long-term plasticity leading to greater GABA release from LN2 neurons. In cerebellar stellate cells, such an increase in inhibitory transmitter release has been documented and coined 'inhibitory-long term potentiation' (I-LTP). I-LTP is induced in stellate cells by glutamate released from parallel fibers acting on presynaptic NMDA receptors in these inhibitory interneurons and producing a long-lasting increase in the release of GABA from these cells. Like stellate neurons, at least one population of Drosophila LNs is pharmacologically GABAergic (Sachse, 2007).

    How might alterations in LN2 pharmacology affect downstream circuit elements and ultimately CO2-evoked behavior? Drawing on the same cerebellar analogy discussed above, it is plausible that PNs exhibit a type of 'rebound potentiation' that has been observed in Purkinje cells responding to inhibitory input. GABA released from LNs would regulate the excitability of PNs, such that greater GABA release from LN2 would tend to decrease the excitability of CO2-specific PNs. The finding that the output from the V glomerulus to the lateral horn is reduced following CO2 exposure supports the idea that downstream activity in higher processing centers is modulated by the antennal lobe network. However, it still needs to be shown that LN2 neurons form direct inhibitory synapses onto PNs in the V glomerulus. Reduced PN activity in the lateral horn in turn may produce a reduced behavioral sensitivity to this stimulus. Future experiments that examine this stimulus-dependent plasticity at the cellular level using pharmacology and electrophysiology will be necessary to test this model (Sachse, 2007).

    The wiring diagram of a glomerular olfactory system

    The sense of smell enables animals to react to long-distance cues according to learned and innate valences. Using electron microscopy, this study mapped the complete wiring diagram of the Drosophila larval antennal lobe, an olfactory neuropil similar to the vertebrate olfactory bulb. A canonical circuit with uniglomerular projection neurons (uPNs) relaying gain-controlled ORN activity to the mushroom body and the lateral horn was found. A second, parallel circuit with multiglomerular projection neurons (mPNs) and hierarchically connected local neurons (LNs) selectively integrates multiple ORN signals already at the first synapse. LN-LN synaptic connections putatively implement a bistable gain control mechanism that either computes odor saliency through panglomerular inhibition, or allows some glomeruli to respond to faint aversive odors in the presence of strong appetitive odors. This complete wiring diagram will support experimental and theoretical studies towards bridging the gap between circuits and behavior (Berck, 2016).

    The wiring diagram of a glomerular olfactory system

    The sense of smell enables animals to react to long-distance cues according to learned and innate valences. Using electron microscopy, this study mapped the complete wiring diagram of the Drosophila larval antennal lobe, an olfactory neuropil similar to the vertebrate olfactory bulb. A canonical circuit with uniglomerular projection neurons (uPNs) relaying gain-controlled ORN activity to the mushroom body and the lateral horn was found. A second, parallel circuit with multiglomerular projection neurons (mPNs) and hierarchically connected local neurons (LNs) selectively integrates multiple ORN signals already at the first synapse. LN-LN synaptic connections putatively implement a bistable gain control mechanism that either computes odor saliency through panglomerular inhibition, or allows some glomeruli to respond to faint aversive odors in the presence of strong appetitive odors. This complete wiring diagram will support experimental and theoretical studies towards bridging the gap between circuits and behavior (Berck, 2016).

    This study mapped the wiring diagram of the first olfactory neuropil of the larva by reconstructing the left and right ORNs and all their synaptic partners. A complete volume was used of the central nervous system (CNS) of a first instar larva, imaged with serial section electron microscopy. 160 neuronal arbors were reconstructed using the software CATMAID. All together, the 160 neurons add up to a total of 38,684 postsynaptic sites and 55 millimeters of cable, requiring about 600,000 mouse clicks over 736 hr of reconstruction and 431 hr of proofreading. Only 136 of 14,346 (0.9%) postsynaptic sites of ORNs remained as small arbor fragments (comprising a total of 0.25 millimeters of cable, or 0.5% of the total reconstructed) that could not be assigned to any neuron (Berck, 2016).

    The 160 reconstructed neurons were sorted into 78 pairs of bilaterally homologous neurons and 4 ventral unpaired medial (VUM) neurons (2 are mPNs and 2 are octopaminergic 'tdc' neurons. These 78 pairs were further subdivided into 21 pairs of ORNs (see the complete wiring diagram of the Drosophila larval antennal lobe), 21 pairs of uPNs, 13 pairs of mPNs (plus 2 additional VUM mPNs), 14 pairs of LNs, 6 pairs of neurons projecting to the SEZ ('SEZ neurons'), 1 pair of descending neurons from the brain, 1 pair of serotonergic neurons, and 1 pair of octopaminergic non-VUM neurons (Berck, 2016).

    The 14 pairs of LNs originate in 5 different lineages. The same name was assigned to neurons of the same lineage, and numbered each when there is more than one per lineage. LNs connect to other neuron classes stereotypically in the two antennal lobes (see complete wiring diagram of the Drosophila larval antennal lobe). Names were selected that were reminiscent of either their circuit role or anatomical feature, including 'Broad' to refer to panglomerular arbors; 'Picky' and 'Choosy' for LNs of two different lineages (and different neurotransmitter; see below) with arbors innervating select subsets of glomeruli; 'Keystone' for a single pair that mediate interactions between LNs of different circuits; and 'Ventral LN' for a single pair of LNs with ventral cell bodies. The neurotransmitters of LNs that were previously unknown were determined. The properties of each LN type are described along with the olfactory circuits that they participate in (Berck, 2016).

    Parallel to the uniglomerular readout by the 21 uPNs, 14 multiglomerular PNs were found (mPNs; see The multiglomerular circuit consists of 14 mPNs that project to the brain and 5 Picky LNs, each an identified neuron). Each mPN receives unique and stereotyped inputs from multiple ORNs or at least from one ORN and multiple unidentified non-ORN sensory neurons in the SEZ . The mPNs originate in multiple neuronal lineages and project to multiple brain regions; most commonly the lateral horn (LH) but also regions surrounding the MB calyx. Of the 14 mPNs, three project to the calyx itself (mPNs) and another (mPN cobra) to the MB vertical lobe. In addition to the 14 mPNs that project to the brain, an extra 6 oligoglomerular neurons were detected that project to the SEZ. A class of mPNs has been described in the adult fly but their projection pattern does not match any of the larval mPNs. In strong contrast to uPNs, mPNs are very diverse in their lineage of origin, their pattern of inputs, and the brain areas they target (Berck, 2016).

    The multiglomerular circuit consists of 14 mPNs that project to the brain and 5 Picky LNs, each an identified neuron. In addition to inputs from Broad LNs, mPNs also receive up to 26% of inputs from 5 stereotypically connected, oligoglomerular LNs that were called Picky LNs. While both Choosy LNs and Picky LNs are oligoglomerular and present distinct axons, the Choosy LNs are GABAergic whereas at least 4 of the 5 Picky LNs are instead glutamatergic. The difference in neurotransmitter is consistent with Picky LNs deriving from a different lineage than Choosy LNs. In addition, the two Choosy LNs present indistinguishable connectivity, whereas each Picky LN has its own preferred synaptic partners. Additionally, unlike the Choosy LNs, Picky LNs rarely target uPNs. Glutamate has been shown to act as a postsynaptic inhibitory neurotransmitter in the adult fly antennal lobe for both PNs and LNs (Liu, 2013), and therefore in larva, Picky LNs may provide inhibition onto both mPNs and other LNs. Unlike the Broad LNs, which are panglomerular and axonless, the Picky LNs present separated dendrites and axons. Collectively, Picky LN dendrites roughly tile the antennal lobe. While some Picky LN axons target select uPNs, about 40% of Picky LN outputs are dedicated to mPNs or each other. Similarly to the mPNs, Picky LNs 2, 3, and 4 receive inputs from unidentified non-ORN sensory neurons in the SEZ (Berck, 2016).

    The glomerular olfactory system of the larva develops in a similar fashion to the vertebrate olfactory bulb where the afferents (i.e. ORNs) organize the central neurons, unlike in the adult fly. In zebrafish, GABAergic LNs provide depolarizing currents to PNs (mitral cells) via gap junctions at low stimulus intensities, enhancing low signals, and inhibit the same PNs at high stimulus intensity via GABA release, implementing a form of gain control. This role is played by a class of panglomerular excitatory LNs in the adult fly that make gap junctions onto PNs and excite inhibitory LNs (Yaksi, 2010). In the larva, all panglomerular neurons are GABAergic; if any were to present gap junctions with uPNs, a cell type for gain control in larva would be equivalent to the one in zebrafish. Particularly good candidates are the Broad LN Duet, which provide the bulk of feedforward inhibitory synapses onto uPNs in larva. Interestingly, postsynaptic inhibition might not be mediated by GABA in the adult fly, rendering olfactory circuits in larva more similar to vertebrates. Presynaptic inhibition exists both in the adult fly and, as suggested by the present work, in larva, and is mediated by the same kind of panglomerular GABAergic neurons (the Broad Trio LNs in larva; and see (Berck, 2016).

    The uniglomerular circuit is the most studied in all species both anatomically and physiologically. This study found that each uPN receives an unusually large number of inputs from an individual ORN compared to other sensory systems in the larva (Ohyama, 2015). This large number of morphological synapses could be interpreted as a strong connection, which would support faster or more reliable signal transmission. In the adult fly, the convergence of multiple ORNs onto an individual PN enables both a fast and reliable PN response to odors. The temporal dynamics of crawling are far slower than that of flying, and therefore it is speculated that the integration over time of the output of a single ORN might suffice for reliability, demanding only numerous synapses to avoid saturation (Berck, 2016).

    Positive, appetitive chemotaxis involves odor gradient navigation, leading to a goal area where food is abundant which may overwhelm olfaction. It is postulated that navigation and feeding correspond to the homogeneous and heterogeneous states of presynaptic inhibition that are described in this study. During navigation, homogeneous presynaptic inhibition (via Broad LN Trio) could best enhance salient stimuli and therefore chemotaxis, enabling the olfactory system to operate over a wide range of odorant intensities. During feeding, strongly stimulated ORNs could scale down the inputs provided by other, less stimulated, ORNs. In other words, if homogeneous presynaptic inhibition persisted during feeding, the larvae would lose the ability to detect important odorants that are likely to be faint, for example the scent of a predator such as a parasitic wasp via 49a. The larva can selectively release presynaptic inhibition via Keystone, which provides presynaptic inhibition to appetitive glomeruli while also inhibiting the Broad LN Trio-the major providers of panglomerular presynaptic inhibition. So the larva could feed and remain vigilant to evolutionarily important cues at the same time. Not surprisingly, the switch might be triggered by neuromodulatory neurons and non-ORN sensory neurons, potentially gustatory, that synapse onto Keystone (Berck, 2016).

    In addition to the uniglomerular system that is present across multiple vertebrate and invertebrate species, this study found, in the Drosophila larva, a multiglomerular system that presumably performs diverse processing tasks already at the first synapse. One such task could be the detection of concentration gradients for some odorant mixtures, suggesting an explanation for the observation that some ORNs can only drive chemotaxis when co-activated with other ORNs. Similar glomerular-mixing circuits have been described in higher brain areas (lateral horn) of the fly and of mammals. It is hypothesized that in the larva, the morphological adaptations to a life of burrowing might have led to specific adaptations, relevant to an animal that eats with its head, and therefore the dorsal organ housing the ORNs, immersed in food. It is perhaps not surprising that this study found multisensory integration across ORNs and non-ORNs (likely gustatory) already at the first synapse. And it is hypothesized that the pooling of chemosensors (ORNs and non-ORNs) onto mPNs and Picky LNs may be related to the reduction in the number of ORNs relative to insects with airborne antennae (Berck, 2016).

    With the complete wiring diagram of this tractable, transparent model system and genetic tools for manipulating and monitoring the activity of single identified neurons, the opportunity is now available to bridge the gap between neural circuits and behavior (Berck, 2016).

    Transmission of olfactory information between three populations of neurons in the antennal lobe of the fly

    Three classes of neurons form synapses in the antennal lobe of Drosophila brain, the insect counterpart of the vertebrate olfactory bulb: olfactory receptor neurons, projection neurons, and inhibitory local interneurons (see Anatomical organization of the olfactory nervous system in Drosophila). A genetically encoded optical reporter of synaptic transmission has been targeted to each of these classes of neurons and population responses to natural odors has been visualized. The activation of an odor-specific ensemble of olfactory receptor neurons leads to the activation of a symmetric ensemble of projection neurons across the glomerular synaptic relay. Virtually all excited glomeruli receive inhibitory input from local interneurons. The extent, odor specificity, and partly interglomerular origin of this input suggest that inhibitory circuits assemble combinatorially during odor presentations. These circuits may serve as dynamic templates that extract higher order features from afferent activity patterns (Ng, 2002).

    Composed of processes extended by ~1,500 neurons, the antennal lobe of the fly duplicates in miniature most characteristics of the vertebrate olfactory bulb. Individual olfactory receptor neurons (ORNs, ~1,200 on each side) expressing a limited repertoire of olfactory receptors, which probably includes a single cognate specificity, project their axons to stereotyped groups of target neurons in the antennal lobe. The neurites of these target neurons, and all synaptic connections they receive, are condensed in sharply demarcated, morphologically identifiable regions of neuropil, termed glomeruli. ORN targets within the antennal lobe consist of two types of neurons: inhibitory, GABAergic local interneurons (LNs, ~100 per lobe) and excitatory projection neurons (PNs, ~160 per lobe). The vertebrate homologs of these two classes of neurons are axonless granule cells and mitral and tufted cells, respectively. Localized interneurons form widespread inhibitory connections among many or all glomeruli; PNs serve as the relay neurons between the antennal lobes and higher brain centers. Most PNs innervate single, stereotypically located glomeruli in the antennal lobes and project to stereotyped target fields in the mushroom bodies and lateral protocerebra (Ng, 2002 and references therein).

    Genetic control over the expression of synapto-pHluorin permitted the activities of the three principal classes of neurons [ORNs, PNs, and localized interneurons] to be distinguished, interleaved in the glomerular neuropil. Flies carrying a Gal4-responsive UAS-synapto-pHluorin (UAS-spH) transgene were generated and crossed with driver lines that provided the transcription factor Gal4 in spatially restricted patterns that marked ORNs, PNs, and localized interneurons as selectively and comprehensively as could be achieved. Where labeling was partial (i.e., in the cases of ORNs and PNs), the possibility must be borne in mind that the genetically marked populations might represent functional subsets (Ng, 2002).

    Gal4 expression in ORNs was controlled by a promoter/enhancer sequence obtained from OR83b, an olfactory receptor gene that is expressed in a large (~70%) upopulation of ORNs but in no other structure in the brain. Expression in PNs relied on unknown regulatory sequences that drive Gal4 expression in the well-characterized enhancer trap line GH146-GAL4. This enhancer element is active in ~60% of PNs. Expression in all GABAergic localized interneurons was directed by the promoter/enhancer element of GAD1, the gene encoding the key enzyme in GABA biosynthesis, glutamic acid decarboxylase. Gad1-positive cell bodies occupy several circumscribed regions in the central nervous system, including the cortices of the antennal lobes, where localized interneuron and PN somata reside, but spared antennae and maxillary palps. Despite the anatomical proximity of their cell bodies, PNs and localized interneurons were genetically distinct (Ng, 2002).

    When synapto-pHluorin was expressed under the control of OR83b-GAL4, GH146-GAL4, and GAD1-GAL4, the protein appeared in the known synaptic target fields of ORNs, PNs, and localized interneurons, respectively. Or83b-expressing ORNs projected axons to 29 of the 43 glomeruli in the antennal lobe, where their synaptic terminals clustered in thick shells surrounding the glomerular cores. PNs formed extensive three-dimensional lattices of synaptic contacts in the mushroom body and the lateral protocerebrum. In addition, ~30 glomeruli in the antennal lobe were brightly fluorescent, suggesting the existence of local or recurrent PN synapses. GABAergic synapses originating from neurons expressing Gad1 were detected in many regions of the brain, including the antennal lobes, where they innervated all glomeruli (Ng, 2002).

    Of the three classes of neurons, two (ORNs and PNs) are synaptically coupled in precise anatomical register. To a first approximation, the symmetry of coupling between these two classes of neurons is reflected in symmetrical odor representations. Each representation consists of a combination of active and inactive ORNs and PNs, grouped by glomerular projection and origin, respectively. Odor representations are sparse at the lowest concentrations of odorant, with only a few glomeruli responding to each test fragrance, but become highly combinatorial as the concentration of odorous ligand increases. At the highest concentrations used in these experiments, the probability of any given odor to elicit an ORN or PN response in any given glomerulus approximates 0.7; the average response probability, determined over a concentration range spanning 6 orders of magnitude, is 0.38 for ORNs and 0.37 for PNs (Ng, 2002).

    Information is transmitted not only vertically across the glomerular relay between ORNs and PNs, but also horizontally through inhibitory localized interneuron connections that are activated in odor-specific patterns. These localized interneuron connections arguably constitute the computational core of the antennal lobe: while ORNs and PNs are constrained by synaptic connectivity that segregates information from different olfactory receptors into separate channels. Localized interneurons possess the anatomical freedom to bridge multiple glomeruli. They can, therefore, implement neural operations that require access to more than one channel (Ng, 2002).

    The functional outlines of the localized interneuron network are visible in recordings of odor-evoked activity across and within glomeruli. The majority of glomeruli transmitting information between ORNs and PNs receive coincident localized interneuron input; usually, the active PN ensembles originate in their entirety from glomeruli supplied simultaneously by active localized interneuron synapses. The simplest circuit model to account for the high degree of overlap between the excitatory and inhibitory odor maps is one in which ORN afferents, PN recurrences, or both directly excite localized interneurons forming synapses within the same target glomerulus. Recurrent coupling through excitatory and inhibitory synapses between PNs (or mitral and tufted cells) and localized interneurons (or granule cells) within a glomerulus can synchronize the action potentials of these neurons and may enhance the impact of PN discharges on detectors attuned to temporal coincidences of their synaptic inputs (Ng, 2002).

    Similar mechanisms are likely to operate between glomeruli, but with the important difference that localized interneuron-mediated synchrony (or other spike timing relations among PNs originating from two or more different glomeruli) could now be used not only to raise the detectability of individual glomerular signals, but also to encode second and higher order features of olfactory stimuli. The occurrence of specific constellations of co-active ORNs, for instance, could be detected by interglomerular localized interneuron circuits that enable synchronized PN ensemble responses to matching ORN inputs. Of varying complexity and stringency with respect to what they accept as matching input structures, the topologies of these circuits may range from reciprocal couplings that mutually enhance PN synchrony in two glomeruli, to inhibitory networks that link many glomeruli, to directed multiglomerular cycles whose PNs fire in phase only if all participating glomeruli receive simultaneous ORN input (Ng, 2002).

    The existence of specific inhibitory connections that link different glomeruli is consistent with this idea. Odor-specific localized interneuron activity patterns always extend over a significantly larger number of glomeruli than their ORN and PN counterparts, suggesting a divergence of localized interneuron connections from source to target glomeruli. Some glomeruli are found during each odor presentation that lack direct ORN input but nevertheless show inhibitory localized interneuron activity. These terminal 'leaves' on the graph of active inhibitory connections provide the most direct functional examples of interglomerular localized interneuron interactions. Numerous additional interglomerular links are likely to exist, but these may not be easily recognizable as such in standard recordings if they connect glomerular endpoints that are both supplied by active ORN afferents. Indeed, at higher resolution, and from the vantage point of individual target glomeruli, inhibitory input appears to converge from multiple sources (Ng, 2002).

    The matrix of inhibitory couplings among glomeruli could add a second combinatorial layer to the representation of olfactory information. Depending on which ORN afferents are stimulated and which interglomerular localized interneuron connections become active as a result, different topologies of inhibitory circuits are expected to assemble. Two combinatorial encoders would then operate in tandem: a primary encoder, consisting of the olfactory receptor repertoire expressed by the ORN ensemble, that transduces receptor occupancy patterns into glomerular 'odor images', and a secondary encoder, consisting of the matrix of localized interneuron couplings among glomeruli, that extracts higher order features from these odor images and represents them as timing relationships across the active PN ensemble. Because these feature-extracting localized interneuron circuits are expected to affect the function of the ORN–PN relay only subtly, by resetting PN spike times without altering firing frequencies, they could be flexibly tuned to specific stimulus features without compromising the primary representational capabilities of the system. Intriguing evidence indeed exists for experience-dependent plasticity at the level of the antennal lobe, but the neuronal substrate for change has remained elusive. A testable prediction of this notion is that the localized interneuron network constitutes this site of experience-dependent change (Ng, 2002).

    Odor discrimination in Drosophila: from neural population codes to behavior

    Taking advantage of the well-characterized olfactory system of Drosophila, a simple quantitative relationship was derived between patterns of odorant receptor activation, the resulting internal representations of odors, and odor discrimination. Second-order excitatory and inhibitory projection neurons (ePNs and iPNs) convey olfactory information to the lateral horn, a brain region implicated in innate odor-driven behaviors. The distance between ePN activity patterns is the main determinant of a fly's spontaneous discrimination behavior. Manipulations that silence subsets of ePNs have graded behavioral consequences, and effect sizes are predicted by changes in ePN distances. ePN distances predict only innate, not learned, behavior because the latter engages the mushroom body, which enables differentiated responses to even very similar odors. Inhibition from iPNs, which scales with olfactory stimulus strength, enhances innate discrimination of closely related odors, by imposing a high-pass filter on transmitter release from ePN terminals that increases the distance between odor representations (Parnas, 2013).

    The experiments reported in this study form the basis of a distance-discrimination model of innate olfactory behavior. The central tenet of this model is that the magnitude of spontaneous responses to odors, mediated by the LH, is bounded by a logistic function of distance between the corresponding patterns of odor-evoked activity across the ePN population. The larger this difference in ePN activity is, and, therefore, the more dissimilar the neuronal signals representing the two alternatives in the choice task, the more pronounced is the behavioral bias elicited by these alternatives. The distance-discrimination function is logistic, similar to many other examples in the statistical analysis of binary choices where the logistic function serves as the link between a continuous predictor variable, such as the spike rate of a neuron, and a categorical outcome, such as a decision between two alternatives (Parnas, 2013).

    From the viewpoint of a fly, the odor-evoked activity of its PNs provides noisy evidence from which the identity of the odors in the left and right arms of the chamber must be judged. To decide whether these odors are different or the same, the fly uses the distance between odor representations as its decision variable (Figure 2D). A decision variable quantifies the weight of evidence supporting a hypothesis (here, that the odors in the two halves of the chamber are different) over its negation (here, that the odors are the same); mathematically, the decision variable gives the log odds that the hypothesis is true. The logistic dependence of performance on the distance between ePN activity vectors indicates that the fly decides on the weight of the sensory evidence. If evidence that two odors are different is lacking (that is, if the ePN distance is small), then the fly displays indiscrimination; if the evidence is ambiguous, then the best attainable odds of correct choices are given by the distance-discrimination function; if the evidence is compelling, then performance plateaus (Parnas, 2013).

    The distance-discrimination model gives equal weight to signals carried by all types of ePNs and only takes average firing rates into account; there is no need to consider information encoded in timing relationships among spikes or invoke privileged receptor channels propagating signals with special behavioral significance. Although dedicated channels undoubtedly exist for mediating stereotyped responses to mating pheromones, the stress odorant CO2, or the microbial odorant geosmin, it remains unresolved whether innate odor responses in general reflect the activation of labeled lines that trigger hardwired behaviors. In this study, experimental manipulations that silence subsets of ePNs have graded, context-specific behavioral consequences; the same manipulation affects responses to different odor pairs differently, and effect sizes depend not only on the overall change but also on the initial distance between the respective ePN activity vectors. This finding suggests that innate responses to odors draw on many glomerular channels and not just a select few. If attraction and aversion to the test stimuli were driven by signals in single dedicated channels, as has been suggested for some generalist odors, then the consequences of manipulating ePN output should be all or nothing: eliminating transmission in an essential channel should abolish all behavioral bias, whereas interference with a nonessential channel should have no effect. The data presented in this study are difficult to reconcile with such a scenario (Parnas, 2013).

    The two brain regions targeted by ePNs employ distinct mechanisms for improving the contrast of the activity patterns projected onto them: expansion recoding in the MB and input gain control in the LH (Parnas, 2013).

    Olfactory signals from ~150 ePNs are projected onto ~2,500 KCs and an unknown, though, in all likelihood, significantly smaller, number of intrinsic LH neurons. Thus, the MB recodes compact, dense ePN activity patterns into a much larger ensemble of KCs. Consistent with the idea that expansion recoding facilitates stimulus separation, the significant performance benefit of training can be attributed entirely to the MBs, given that interrupting transmission through the MB loop occludes the effects of learning. The finding that spontaneous behavioral bias is identical regardless of whether MB output is blocked or intact indicates that untrained flies do not access discrimination information that is presumably always available in the MB (Parnas, 2013).

    In the LH, a group of ~40 GABAergic iPNs provide presynaptic inhibition to ePN terminals. iPN output improves innate performance when the distance between two odor representations is small, but it has no effect in the plateau regions of the distance-discrimination function. Consistent with previous results, this study found that input gain control, which selectively attenuates low-frequency ePN signals but transmits high-frequency signals in full, can amplify large differences in firing rate and thereby increase the separation between two sensory images. Because the high-pass filter must operate on the individual components of the ePN activity vector in order to achieve the desired effect, the likely target of inhibition in the LH is the presynaptic terminals of ePNs, which each represent a single activity vector component rather than the postsynaptic dendrites of intrinsic LH neurons, which may combine several activity vector components after synaptic integration. The experimental evidence supports all aspects of this mechanism. It was found that GABA modulates synaptic vesicle exocytosis at ePN terminals in the LH; GABAergic modulation was shown to convert these terminals to high-pass filters, and iPN projections were identified as the source of modulatory GABA (Parnas, 2013).

    The arrangement of parallel ePN and iPN projections to the LH appears to result in a tunable filter whose transmission characteristics adjust to the level of activity in the olfactory system. What might be the reason for scaling the strength of iPN inhibition with the overall level of ORN input? One possible advantage is to balance competing demands of sensitivity and contrast. At low levels of ORN input, ePN activity would be weak; therefore, in order to detect odors with maximal sensitivity, iPN activity would be curbed to allow the unimpeded transmission of low-frequency spike trains by ePN terminals. Only at higher levels of ORN input, where sensitivity to ePN spikes is a less pressing need, would the iPN high-pass filter be engaged in order to block the transmission of low-frequency spike trains and thereby enhance discrimination (Parnas, 2013).

    What the fly's nose tells the fly's brain

    The fly olfactory system has a three-layer architecture: The fly's olfactory receptor neurons send odor information to the first layer (the encoder) where this information is formatted as combinatorial odor code, one which is maximally informative, with the most informative neurons firing fastest. This first layer then sends the encoded odor information to the second layer (decoder), which consists of about 2,000 neurons that receive the odor information and "break" the code. For each odor, the amplitude of the synaptic odor input to the 2,000 second-layer neurons is approximately normally distributed across the population, which means that only a very small fraction of neurons receive a large input. Each odor, however, activates its own population of large-input neurons and so a small subset of the 2,000 neurons serves as a unique tag for the odor. Strong inhibition prevents most of the second-stage neurons from firing spikes, and therefore spikes from only the small population of large-input neurons is relayed to the third stage. This selected population provides the third stage (the user) with an odor label that can be used to direct behavior based on what odor is present (Stevens, 2015)

    Compound valence is conserved in binary odor mixtures in Drosophila melanogaster

    Most naturally occurring olfactory signals do not consist of monomolecular odorants but, rather, are mixtures whose composition and concentration ratios vary. While there is ample evidence for the relevance of complex odor blends in ecological interactions and for interactions of chemicals in both peripheral and central neuronal processing, a fine-scale analysis of rules governing the innate behavioral responses of Drosophila melanogaster towards odor mixtures is lacking. This study examined whether the innate valence of odors is conserved in binary odor mixtures. Binary mixtures of attractants are more attractive than individual mixture constituents. In contrast, mixing attractants with repellents elicits responses which are lower than the responses towards the corresponding attractants. This decrease in attraction is repellent-specific, independent of the identity of the attractant and more stereotyped across individuals than responses towards the repellent alone. Mixtures of repellents are either less attractive than the individual mixture constituents or these mixtures represent an intermediate. Within the limits of the data set, most mixture responses are quantitatively predictable on the basis of constituent responses. In summary, the valence of binary odor mixtures is predictable on the basis of valences of mixture constituents. These findings will further understanding of innate behavior towards ecologically relevant odor blends and will serve as a powerful tool for deciphering the olfactory valence code (Thoma, 2014).

    Chemotaxis behavior mediated by single larval olfactory neurons in Drosophila

    Odorant receptors (ORs) are thought to act in a combinatorial fashion, in which odor identity is encoded by the activation of a subset of ORs and the olfactory sensory neurons (OSNs) that express them. The extent to which a single OR contributes to chemotaxis behavior is not known. This question was investigated in Drosophila larvae, which represent a powerful genetic system to analyze the contribution of individual OSNs to odor coding. Twenty-five larval OR genes expressed in 21 OSNs were identified and genetic tools were generate that allow engineering of larvae missing a single OSN or having only a single or a pair of functional OSNs. Ablation of single OSNs disrupts chemotaxis behavior to a small subset of the odors tested. Larvae with only a single functional OSN are able to chemotax robustly, demonstrating that chemotaxis is possible in the absence of the remaining elements of the combinatorial code. Behavioral evidence is provided that an OSN not sufficient to support chemotaxis behavior alone can act in a combinatorial fashion to enhance chemotaxis along with a second OSN. It is concluded that there is extensive functional redundancy in the olfactory system, such that a given OSN is necessary and sufficient for the perception of only a subset of odors. This study is the first behavioral demonstration that formation of olfactory percepts involves the combinatorial integration of information transmitted by multiple ORs (Fishilevich, 2005).

    The 'nose' of the Drosophila larva resides in a pair of dorsal organs at the anterior tip of the animal, each containing 21 OSNs. Previous studies showed that up to 23 of the 61 Drosophila ORs are expressed in larvae by PCR and transgenic analysis. RNA in situ hybridization was performed to provide direct evidence that OR genes are expressed in larval OSNs. Or83b, which is necessary for the proper localization and function of conventional ORs, is broadly expressed throughout the dorsal-organ ganglion. Twenty-four of the 30 ORs tested in this study are expressed in a single larval neuron in the dorsal organ. The expression of Or10a, Or43b, or Or49a mRNA or OR43b protein was not detected, although RT-PCR analysis detects these transcripts in larvae. Or92a and Or98b are also not detected by RNA in situ hybridization. Most larval OSNs express a single OR along with Or83b, but two OSNs coexpress a pair of ORs along with Or83b: Or33b/Or47a and Or94a/Or94b. Such OR coexpression has also been documented in the adult olfactory system (Fishilevich, 2005).

    In parallel with the RNA in situ hybridization analysis, a collection of 42 different OR-Gal4 transgenes were examined that drive the expression of Gal4 under the control of OR promoter elements. To visualize gene expression in the dorsal organ, individual OR-Gal4 lines were crossed to UAS-GFP, encoding cytoplasmic green fluorescent protein (GFP) and the olfactory-neuron marker Or83b-Myc. Or83b-Gal4 labels all 21 larval OSNs. Per dorsal organ, 19 of the remaining 41 OR-Gal4 transgenes label a single larval OSN that is also positive for Or83b-Myc. Although Or49a mRNA was not detected in larvae, Or49a-Gal4 labels one dorsal-organ OSN along with a single terminal-organ gustatory neuron. Gustatory receptor (GR) genes are expressed in both olfactory and gustatory organs of the adult fly. GR-Gal4 transgenes are expressed only in the gustatory terminal organ or in nonolfactory dorsal-organ neurons that do not express Or83b-Myc. A total of 25 Drosophila ORs expressed in the larval dorsal organ were identified and direct evidence is provided that 24 of these OR mRNAs are expressed in situ. Of these, 14 are expressed only at the larval stage, whereas 11 are utilized by both larval and adult olfactory systems (Fishilevich, 2005).

    Larval OSNs project axons to the larval antennal lobe of the brain. Patterns of axonal projections to the larval antennal lobe were examined in larvae carrying each of 20 larval OR-Gal4 transgenes along with UAS-GFP or UAS-CD8-GFP. Each OR-Gal4 line reveals a single labeled axonal arbor that terminates in an antennal-lobe glomerulus whose position is conserved between animals (Fishilevich, 2005).

    The availability of genetic tools that uniquely label 19 of the 21 larval OSNs allows manipulation of the odor code by deconstructing the peripheral olfactory input and examining effects on behavioral output. Toward this end, a chemotaxis assay was establised of sufficient sensitivity to quantify differences in odor-evoked behavior. Chemotaxis of wild-type larvae was measured in response to 53 synthetic monomolecular odorants and three natural Drosophila attractants. The assay involves single-animal analysis in which the position of individual chemotaxing larvae is tracked over the course of a 5 min experiment (Fishilevich, 2005).

    This assay was used to screen larval chemotaxis to a panel of 53 synthetic odors and quantified the median distance to odor for Or83b−/− and Or83b+/+ larvae. Forty of the 53 odors are naturally present in fruit, and of these 40, 13 are known to elicit behavioral and electrophysiological responses in Drosophila. Anosmic Or83b−/− larvae do not respond to any odors, but wild-type (yw) larvae respond to many odors with strong chemotaxis (Fishilevich, 2005).

    It was next asked how sensitive larvae are to odors by performing chemotaxis experiments at various odor concentrations. The responses to 1-hexanol are weak and not statistically different from anosmic controls for low dilutions, whereas responses increase steeply between 0.02 μl and 0.2 μl doses and appear to reach a plateau for higher concentrations. No evidence was found that higher concentrations elicit repulsion. Response thresholds to heptanal and isoamyl acetate are one and two log orders, respectively, below that of 1-hexanol (Fishilevich, 2005).

    To test whether the weak responses observed for some odors at 2 μl could be explained by high detection thresholds, seven of these odors were further tested with 20 μl. Under these conditions, 1-butanol and 2,3-butanediol elicit chemotaxis, whereas the remaining five odors do not. Thus the 2 μl stimulus dose elicits robust chemotaxis across a large group of different odors, in accord with previous behavioral studies (Fishilevich, 2005).

    Upon loading of an odorant stimulus in the closed-dish assay, the spatial distribution and average airborne concentration of this odor in the dish will be partly determined by the odor's vapor pressure. Vapor pressure is thus likely to affect the behavioral response observed for a particular odorant stimulus. In addition to this factor, it is anticipated that the olfactory system of the larva may be differentially tuned to different stimuli. In the initial phases of this study, no clear correlation was found between the vapor pressure of a given odor and its corresponding behavioral efficacy. It was therefore decided to avoid any normalization of stimulus concentration and used the same quantity of odor (2 μl) for all 53 stimuli tested (Fishilevich, 2005).

    Whether chemotaxis elicited by single odors is comparable to that obtained with natural stimuli was examined. Chemotaxis was measured in the same assay to banana mush, balsamic vinegar, and yeast paste at different concentrations. It was found that attraction elicited by single synthetic odors is qualitatively similar to that obtained with natural odor blends and that the same steep threshold and stable plateau properties are seen for both stimulus types (Fishilevich, 2005).

    The relative contribution of any given OSN to the formation of an odor percept was examined. Diphtheria toxin (DTI), an attenuated version of the cell-autonomous protein-translation inhibitor diphtheria toxin, was used to ablate identified OSNs selectively. Most but not all larval OSNs are ablated by the expression of DTI along with GFP under control of the Or83b-Gal4 driver in all 21 larval OSNs. In Or83b-ablated animals, GFP expression is not detected, and sensory dendrites are severely atrophied but not completely absent. In Or49a-ablated animals, the Or49a-GFP marker is not visible, and expression of other ORs is not perturbed (Fishilevich, 2005).

    Chemotaxis of animals with single neurons ablated (Or1a, Or42a, or Or49a) was measured with a panel of 20 odors and compared to results obtained with the Or83b-ablation. Or83b-ablated larvae fail to respond to 17/20 odors. If a single false discovery (FD) is allowed for, Or83b-ablated animals fail to respond to 19/20 odors. Or1a-ablated and Or49a-ablated animals each show reduced chemotaxis to a single different odor, (E)-2-hexenal and 1-hexanol, respectively, but show normal chemotaxis to the other 19 odors. In contrast, ablation of the Or42a OSN causes decreases in chemotaxis to four of 20 odors. If FD = 1 is allowed, Or1a-ablated animals are impaired in responses to three of 20 and Or42-ablated animals to five of 20 odors (Fishilevich, 2005).

    It was next asked which OSNs are sufficient to produce chemotaxis to a given odor by constructing animals with only one or combinatorials of two functional OSNs. This was achieved by exploiting the Or83b mutation, which prevents OR trafficking to the sensory dendrite. Or83b function was restored in individual OSNs by crossing animals with specific OrX-Gal4 drivers to UAS-Or83b animals, allowing assessment of the contribution of single neurons to odor-evoked behavior in the OrX-functional progeny (Fishilevich, 2005).

    Only a single OR83b-expressing neuron is seen in Or42a-functional, Or49a-functional, and Or1a-functional animals, whereas two OR83b-positive neurons are visible in Or1a-/Or42a-functional and Or1a-/Or49a-functional animals. The remaining OSNs are present but unlabeled in these animals because the Or83b mutation eliminates OR83b protein expression. No evidence was found that the glomerular map is distorted by the activation of a single OSN in a background of nonfunctional neurons as evidenced by the normal position and volume of the Or1a glomerulus in Or1a-functional and Or83b mutant larvae (Fishilevich, 2005).

    These animals along with genetically matched control animals were screened for chemotaxis to 53 odors by using the same behavioral assay and nonparametric statistical analysis employed for the ablation experiments. Consistent with the strong Or42a-ablated phenotypes, Or42a-functional animals respond to 22 odors compared to 36 odors in Or83b+/+ controls possessing 21 functional OSNs. Or42a-functional animals respond to three of four odors to which Or42a-ablated animals are anosmic. The broad behavioral response profile observed for Or42a-functional larvae is in agreement with the broad ligand specificity of this OR as defined by electrophysiological experiments (Fishilevich, 2005).

    In contrast to the broad odor response profile of Or42a-functional larvae, Or1a- and Or49a-functional animals do not show significant chemotaxis to any of the 53 odors tested, consistent with the weak phenotype of ablating either the Or49a-expressing or Or1a-expressing neuron. These behavioral results are in accord with the ligand profiling of Or49a, which does not show strong electrophysiological responses to any of 27 odors tested (Fishilevich, 2005).

    Although Or1a- and Or49-functional larvae do not chemotax to any odors tested, it was asked whether these neurons contribute to chemotaxis in concert with the Or42a neuron. Chemotaxis performance of larvae with two functional neurons was compared to data from animals with only a single functional neuron. Larvae with two functional neurons respond to a somewhat different subset of odors than animals having either single functional neuron alone (Fishilevich, 2005).

    To examine the existence of interactions between these neurons and identify cases of combinatorial enhancement, a linear regression model was developed to compare chemotaxis data across genetically matched controls for larvae with one or two functional OSNs. The model was designed to identify potential cases where single-neuron chemotaxis behavior differs from two-neuron behavior. The linear model suggests six cases of potential positive cooperativity between Or1a and Or42a chemotaxis that merited further experimental investigation. Additional chemotaxis experiments were carried out with four odors (1-pentanol, 2-pentanol, 2-hexanol, and 3-octanone) at three concentrations. 1-pentanol shows significantly stronger chemotaxis in Or1a/Or42a-functional animals than Or42a-functional or Or1a-functional animals at all three concentrations. A qualitative view of this behavioral enhancement is seen in the sector-plot distributions comparing the anosmia of Or83b mutants to the progressive increase in chemotaxis to 1-pentanol of Or1a-functional or Or42a-functional compared to Or1a/Or42-functional. The Or1a/Or42-functional animals spend comparatively more time in the sector containing the odor than animals having either single functional neuron alone. For the other three odors, this cooperative effect is significant at a single odor concentration (Fishilevich, 2005).

    This study has used behavioral analysis to measure the contribution of individual neurons to the odor code and provide a missing link between the understanding of the molecular biology of ORs, the neurophysiological properties of the olfactory network, and complex odor-evoked behaviors. The goal was to approach the question of how the combinatorial activation of ORs encodes odor stimuli and elicits olfactory behavior. The results suggest that there is a high level of redundancy in the larval olfactory system, such that ablating a single neuron has minimal effects on odor detection. Among these olfactory inputs, the Or42a neuron plays a more important role in odor detection than the Or1a or Or49a neuron. Animals engineered to have the Or42a neuron functional are able to chemotax to multiple odors. The addition of a second OSN to such animals results in enhanced chemotaxis for several odors. Whereas Or1a-functional animals show no significant responses to any odor tested, it was observed that responses of Or1a/Or42a-functional animals to four odors are enhanced relative to Or42a-functional animals. This suggests that although olfactory input contributed by the Or1a-expressing OSN is not sufficient alone to elicit robust chemotaxis, it enhances the perception of odors in conjunction with the information transmitted by the Or42a-expressing OSN. (Fishilevich, 2005).

    Behavior is the ultimate output of a sensory system that integrates all aspects of external-information processing. These experiments demonstrate the feasibility and value of integrating behavioral analysis into the study of odor coding. It is proposed that the simple olfactory system of Drosophila larvae will be an invaluable model in any attempt to correlate the cellular basis of the odor code with its behaviorally relevant output (Fishilevich, 2005).

    Drosophila is a holometabolous insect that undergoes dramatic changes in lifestyle from the larval to adult stage. In a sense, these animals can be considered to occupy completely separate ecological niches. Larvae maintain constant contact with food until pupation, whereas adults are flying insects that use their sense of smell to identify suitable food sources and appropriate sites for egg-laying. In essence, larvae are specialized for feeding and growth, whereas adults are devoted to breeding and dispersal. To what extent have these two life stages of the same species evolved a different chemosensory system? This study shows 14 of 25 larval OR genes are stage specific and not used again by the adult animal. All larval OSNs are histolyzed in metamorphosis and replaced in the adult by newly differentiated antennal and maxillary palp OSNs. Perhaps this developmental changeover has led to largely separate OR genes with transcriptional regulatory regions specific for either larval or adult olfactory organs. Alternatively, the segregation of larval- and adult-expressed ORs could be functional and relate to the different ecological niches that these life stages occupy: larvae may cope with much higher odor concentrations because of their direct contact with food (Fishilevich, 2005).

    Odor processing occurs at various levels in the nervous system, from peripheral sensory neurons to primary processing centers, such as the olfactory bulb in vertebrates and the antennal lobe in insects, and further to higher brain centers of the olfactory cortex in vertebrates and mushroom body and lateral horn in insects. How the combinatorial code established by the ORs at the periphery is transmitted through this olfactory circuitry to produce the perception of an odor in any species is unknown. The data support the notion that peripheral sensory neurons constitute information channels that are not independent but subject to interactions in the olfactory circuit. Otherwise, one would expect that the behavioral response profile observed for the Or1a/Or42a-functional genotype be given by the union of the best performances of the single Or1a- and Or42a-functional genotypes. Where and how the information is processed remains unclear, but part of this transformation may occur in the antennal lobe (Fishilevich, 2005).

    A number of conclusions about odor coding in the Drosophila larva can be drawn from this work. There appears to be no clear structural relationship between the odors that elicit chemotaxis mediated by a given OSN, as has been previously shown in an analysis of the ligand response properties of ORs in the adult fly. The Or42a-expressing neuron differs from other neurons studied here in the large number of odors that attract animals having only this neuron active. Interestingly, the behavioral response profile of the Or42a-functional genotype indicates that an OR may not need to be strongly activated by a given odor to allow for chemotaxis toward the odor source. This point is best illustrated by 3-octanol and anisole, which both elicit strong chemotaxis in Or42a-functional animals whereas they seem to induce relatively weak electrophysiological activity (Fishilevich, 2005).

    Finally, the behavioral receptive field of animals having combinatorials of functional neurons cannot be predicted from a simple model where the responses of animals having either single OSN functional are added. The chemotaxis results reported in this study highlight the existence of strong nonlinearities in the processing of olfactory information in such a way that in the arithmetic of sensory coding, the whole is greater than what the parts can produce independently. Such a scheme would be consistent with the extraordinary needs of the olfactory system to detect numbers of odors that greatly exceed the number of OR genes in any given animal. The functional redundancy observed here could buffer the olfactory system against mutations and allow animals to adapt to changing or new odor environments (Fishilevich, 2005).

    The genetic tools presented in this study should permit a systematic analysis of the peripheral and central components that generate an odor response in the Drosophila larva. A number of key unanswered questions remain for future studies. Electrophysiological or optical imaging tools must be used to analyze the neuronal correlates of the observed behavior. Greater understanding of the second- and third-order neurons that communicate information from the antennal lobe to eventual motor output is needed. This study has been restricted to simple chemotaxis assays, and no attempt has been made to query larvae for their powers of odor discrimination. Animals missing a single OSN may chemotax normally but experience olfactory-perception not uncovered in these chemotaxis assays. By coupling associative learning of odors in intact animals followed by generalization tests in the same animals that conditionally lack a single OSN, it should be possible to determine whether odor salience is altered in larvae missing a single OSN. Finally, it will be important to determine whether the phenomena reported in this study can be considered general olfactory-coding principles that also apply to more complex animals (Fishilevich, 2005).

    Olfactory channels associated with the maxillary palp mediate short- and long-range attraction
    The vinegar fly Drosophila melanogaster is equipped with two peripheral olfactory organs, antenna and maxillary palp. The antenna is involved in finding food, oviposition sites and mates. However, the functional significance of the maxillary palp remains unknown. This study screened the olfactory sensory neurons of the maxillary palp (MP-OSNs) using a large number of natural odor extracts to identify novel ligands for each MP-OSN type. Each type was found to be the sole or the primary detector for a specific compound, and detects these compounds with high sensitivity. Next the contribution of MP-OSNs to behaviors evoked by their key ligands was dissected and MP-OSNs were found to mediate short- and long-range attraction. Furthermore, the organization, detection and olfactory receptor (Or) genes of MP-OSNs are conserved in the agricultural pest D. suzukii. The novel short and long-range attractants could potentially be used in integrated pest management (IPM) programs of this pest species (Dweck, 2016).

    Odor-evoked inhibition of olfactory sensory neurons drives olfactory perception in Drosophila

    Inhibitory response occurs throughout the nervous system, including the peripheral olfactory system. While odor-evoked excitation in peripheral olfactory cells is known to encode odor information, the molecular mechanism and functional roles of odor-evoked inhibition remain largely unknown. This study examined Drosophila olfactory sensory neurons and found that inhibitory odors triggered outward receptor currents by reducing the constitutive activities of odorant receptors, inhibiting the basal spike firing in olfactory sensory neurons. Remarkably, this odor-evoked inhibition of olfactory sensory neurons elicited by itself a full range of olfactory behaviors from attraction to avoidance, as did odor-evoked olfactory sensory neuron excitation. These results indicated that peripheral inhibition is comparable to excitation in encoding sensory signals rather than merely regulating excitation. Furthermore, it was demonstrated that a bidirectional code with both odor-evoked inhibition and excitation in single olfactory sensory neurons increases the odor-coding capacity, providing a means of efficient sensory encoding (Cao, 2017).

    Artificial selection for odor-guided behavior in Drosophila reveals changes in food consumption

    Appropriate behavioral responses to the chemical cues of predators are important for organismal survival and can influence traits such as organismal life span and food consumption. However, understanding the genetic mechanisms underlying odor-guided behavior, correlated responses in other traits, and how these constrain or promote their evolution, remain an important challenge. This study performed artificial selection for attractive and aversive behavioral responses to four chemical compounds, two aromatics (4-ethylguaiacol and 4-methylphenol) and two esters (methyl hexanoate and ethyl acetate), for thirty generations. Artificial selection for odor-guided behavior revealed symmetrical responses to selection for each of the four chemical compounds. Next, whether selection for odor-guided behavior resulted in correlated responses in life history traits and/or food consumption. Changes were found in food consumption upon selection for behavioral responses to aromatics was tested. In many cases, lines selected for increased attraction to aromatics showed an increase in food consumption. RNA sequencing of lines selected for responses to 4-ethylguaiacol was performed to identify candidate genes associated with odor-guided behavior and its impact on food consumption. The study detected 91 genes that were differentially expressed among lines, many of which were associated with metabolic processes. RNAi-mediated knockdown of select candidate genes further supports their role in odor-guided behavior and/or food consumption. This study identifies novel genes underlying variation in odor-guided behavior and further elucidates the genetic mechanisms underlying the interrelationship between olfaction and feeding (Brown, 2017).

    Olfactory coding from the periphery to higher brain centers in the Drosophila brain

    Odor information is processed through multiple receptor-glomerular channels in the first order olfactory center, the antennal lobe (AL), then reformatted into higher brain centers and eventually perceived by the fly. To reveal the logic of olfaction, it is fundamental to map odor representations from the glomerular channels into higher brain centers. This study characterized odor response profiles of AL projection neurons (PNs) originating from 31 glomeruli using whole cell patch-clamp recordings in Drosophila melanogaster. Odor representation from olfactory sensory neurons to PNs is generally conserved, while transformation of odor tuning curves is glomerulus-dependent. Reconstructions of PNs reveal that attractive and aversive odors are represented in different clusters of glomeruli in the AL. These separate representations are preserved into higher brain centers, where attractive and aversive odors are segregated into two regions in the lateral horn and partly separated in the mushroom body calyx. This study reveals spatial representation of odor valence coding from the AL to higher brain centers. These results provide a global picture of the olfactory circuit design underlying innate odor-guided behavior (Seki, 2017).

    Fragile X mental retardation protein requirements in activity-dependent critical period neural circuit refinement

    Activity-dependent synaptic remodeling occurs during early-use critical periods, when naive juveniles experience sensory input. Fragile X mental retardation protein (FMRP) sculpts synaptic refinement in an activity sensor mechanism based on sensory cues, with FMRP loss causing the most common heritable autism spectrum disorder (ASD), fragile X syndrome (FXS). In the well-mapped Drosophila olfactory circuitry, projection neurons (PNs) relay peripheral sensory information to the central brain mushroom body (MB) learning/memory center. FMRP-null PNs reduce synaptic branching and enlarge boutons, with ultrastructural and synaptic reconstitution MB connectivity defects. Critical period activity modulation via odorant stimuli, optogenetics, and transgenic tetanus toxin neurotransmission block show that elevated PN activity phenocopies FMRP-null defects, whereas PN silencing causes opposing changes. FMRP-null PNs lose activity-dependent synaptic modulation, with impairments restricted to the critical period. It is concluded that FMRP is absolutely required for experience-dependent changes in synaptic connectivity during the developmental critical period of neural circuit optimization for sensory input (Doll, 2017).

    Wiring variations that enable and constrain neural computation in a sensory microcircuit

    Neural network function can be shaped by varying the strength of synaptic connections. One way to achieve this is to vary connection structure. To investigate how structural variation among synaptic connections might affect neural computation, primary afferent connections were examined in the Drosophila olfactory system. Large-scale serial section electron microscopy was used to reconstruct all the olfactory receptor neuron (ORN) axons that target a left-right pair of glomeruli, as well as all the projection neurons (PNs) postsynaptic to these ORNs. Three variations were found in ORN --> PN connectivity. First, a systematic co-variation was found in synapse number and PN dendrite size, suggesting total synaptic conductance is tuned to postsynaptic excitability. Second, PNs were found to receive more synapses from ipsilateral than contralateral ORNs, providing a structural basis for odor lateralization behavior. Finally, evidence was found of imprecision in ORN --> PN connections that can diminish network performance (Tobin, 2017).

    An olfactory glomerulus represents a discrete neural network. The small spatial scale of this network allowed comprehensive reconstruction of the connections between every excitatory principal cell (i.e., every ORN and PN). This study also benefitted from existing in vivo electrophysiological measurements of these neurons. In particular, because the specific resistance and capacitance of the PN membrane is known, as well as the amplitudes of uEPSPs and mEPSPs, it was possible to construct highly-constrained compartmental models based on the EM reconstructions. These models served as analytical tools to examine the functional impact of structural variations among synaptic connections. Conclusions arising from these models were robust to measured variations in the model parameters derived from electrophysiological experiments (Tobin, 2017).

    The models considered in this study have several limitations. One limitation is that the models include only ORN->PN synapses. ORNs contribute almost 75% of the synaptic input to PNs, with the remaining 25% arising mainly from local neurons. Local neuron synapses could change the integrative properties of PN dendrites, but this would be difficult to capture in a model at present, even if all the local neurons in the circuit had been reconstructed, because local neurons have diverse and complicated spiking, and it seems likely that only a subset of local neurons provide most of the input to PN dendrites (Berck, 2016; Tobin, 2017 and references therein).

    A second limitation that was faced was the lack of knowledge about the active properties of PN dendrites, if any. In principle, voltage-gated conductances in PN dendrites might alter the integrative properties of PNs. However, active properties are unlikely to play an especially large role in PN synaptic integration, because the current-voltage relationships in PNs are fairly linear, whereas in other Drosophila neurons these relationships can be strongly nonlinear. Thus, passive models are good first approximations in this case (Tobin, 2017).

    Yet another limitation is the inability to directly measure the conductance at individual synapses. The models assume that conductance is identical at every ORN->PN synapse. This is a reasonable assumption, given that candidate anatomical proxies for synaptic conductance (T-bar volume and postsynaptic contact area) turned out to be relatively uniform across connections (i.e., the CV of these measurements across connections was relatively low). In the future, it will be interesting to investigate the anatomical correlates of synaptic conductance in more detail (Tobin, 2017).

    A final limitation is that PN dendrites may be distorted by sample preparation. EM fixation and preparation tends to reduce neuropil volume, but this effect is likely isotropic throughout the sample and mainly diminishes extracellular space. It is therefore likely that the estimated dendritic diameters are good first approximations (Tobin, 2017).

    Connection strength can be thought of as being determined by three major factors. The first is the conductance at each synapse within the connection. The second is the total number of synapses in the connection. The third is the filtering of those conductances by the postsynaptic dendrite. The conductance at each synapse cannot be directly measured, but anatomical measurements suggest that synapse-averaged conductance is relatively uniform across connections. The reconstruction provides direct information about the other two factors (synapse number and dendritic filtering), and the results imply that the first of these is particularly important (Tobin, 2017).

    Notably, it was found that the number of synapses per connection was strongly correlated with the strength of ORN->PN connections. The comparison between ipsi- and contralateral synapses represents the clearest example of this correlation, because it is known from prior electrophysiology experiments that ipsilateral connections are 30%-40% stronger. This study discovered that ipsilateral connections contain 35% more synapses per connection. This result argues that the difference in the number of synapses per connection is the main difference between ipsi and contra connections. By extension, it can be inferred that there is not a sizeable ipsi-contra difference in synaptic conductance. No ipsi-contra difference was found in the way that synapses are filtered by the PN dendritic tree, as evidenced by the fact that there is no ipsi-contra difference in simulated mEPSP amplitudes. In short, the number of synapses per connection is the dominant mechanism underlying the systematic functional difference between ipsi- and contralateral connections (Tobin, 2017).

    Interestingly, from the perspective of an individual PN, there was little variation across ORN connections in the average strength of the synapses that comprised each connection. The efficacy of mEPSP summation at the level of uEPSPs was also notably consistent across these connections. Thus, insofar as the models accurately represent the structure of each PN dendrite, it predicts that dendritic filtering has an essentially uniform effect on all ORN->PN connections. This uniformity arises because each connection is composed of many synapses, and synapses made by a given ORN axon tend to be placed onto the dendrite in a relatively unbiased fashion. In essence, each connection is composed of many quasi-random 'samples' of dendritic filtering properties, and so the average effect of dendritic filtering is similar across connections (Tobin, 2017).

    Although Drosophila neural networks are sometimes regarded as highly stereotyped, the number of neurons in such a network is actually variable. In the optic lobe, a recent EM study analyzed 7 repetitions of a modular neural network that normally contains 23 uniquely identifiable cells. In 3 of the 7 networks, one cell that ought to be present was in fact missing. In the case of one missing cell, a homologous cell in a neighboring column sent an extra branch into the vacated space, where it received synapses from the normal presynaptic partners of the missing cell. Thus, when a cell is missing, there can be compensatory changes in wiring (Tobin, 2017).

    Variations in antennal lobe PN numbers have been inferred previously based on Gal4 expression patterns. Indeed, based on Gal4 expression, it was found that there can be two, three, or four PNs in glomerulus DM6, with the 'typical' situation (three PNs) occurring only 67% of the time. The brain selected for large-scale serial section EM turned out to contain three PNs on the left side and two PNs on the right side. In the glomerulus with fewer PNs, it was found that PN dendrites were larger, perhaps because they had more space to fill. Moreover, there was also a compensatory increase in synapse numbers per PN, so that the total number of synapses per glomerulus was similar on the left and right (Tobin, 2017).

    The compartmental models allowed inferring the functional consequences of these concerted changes in PN dendrite morphology and ORN->PN synapse numbers. Remarkably, in the glomerulus with only two PNs, the up-regulation in synapse number was neatly balanced by the increased size of PN dendrites. As illustrated by classic work at the neuromuscular junction, increasing the size of a postsynaptic compartment produces a lower input resistance, and so each quantum of neurotransmitter produces a smaller depolarization. In each of the larger PNs, a presynaptic ORN spike should release more quanta than normal, but the postsynaptic voltage response to each quantum will be smaller. As a result, each PN has the same average response to an ORN spike (Tobin, 2017).

    Counterbalanced effects like this can result from homeostatic mechanisms. For example, in the larval ventral nerve cord, a decrease in presynaptic neurotransmitter release can elicit compensatory growth in postsynaptic dendrites. Of course, it cannot be certain that this is a case of homeostasis; perhaps there is just a fixed allocation of ORN synapses per glomerulus, and this is why the number of these synapses is equal on the left and the right. However, there is direct evidence that the electrical properties of the dendrites of antennal lobe PNs can instruct changes in ORN->PN connections. A previous study used cell-specific K+ channel overexpression to decrease a PN's input resistance, and found a compensatory increase in unitary excitatory synaptic currents at ORN connections onto that PN. That result demonstrated that PN dendrites can up-regulate synaptic currents to compensate for reduced dendritic excitability (Tobin, 2017).

    Together, these findings suggest the following scenario. When one PN failed to develop, the remaining PNs grew larger dendrites, and then synapse number increased to compensate for increased dendrite size. This scenario is consistent with the well-described instructive role of PN dendrites in ORN axon development: PN dendrites form a glomerular map prior to the arrival of migrating ORN axon terminals (Tobin, 2017).

    This scenario is reminiscent of the 'size matching' principle that governs the development of vertebrate neuromuscular junctions, where the size of a muscle is matched to the size of the axon's terminal arborization, thereby ensuring that large muscles (with low input resistance) receive a larger quantal content per presynaptic spike. At the developing vertebrate neuromuscular junction, the expansion of the postsynaptic cell seems to be primary, with the elaboration of the presynaptic arbor occurring in response (Tobin, 2017).

    This study found that the number of synapses per ORN->PN connection was quite variable. As a result, ORN->PN connections gave rise to simulated uEPSP amplitudes ranging from 1.6 to 10 mV. If all ORNs were functionally identical, this sort of variation would be non-optimal, because each PN's response would be dominated by only a fraction of its presynaptic ORN axons. Indeed, simulations showed that this sort of variation can substantially impair a PN's ability to accurately transmit information about total ORN spike counts, as well as right-left differences in ORN spiking. The simulations suggest that connection noise may be a factor limiting perceptual acuity (Tobin, 2017).

    The discovery of variability per se is not surprising: it was already clear that connections in the Drosophila brain can be variable. In the medulla of the optic lobe, the CV of synapse number per connection ranges from 0.08 to 0.87 (computed across all connections of a given type). In principle, variation across connections of a given type may be taken as evidence of developmental noise (imprecision), or else evidence of adaptive plasticity (precision). For example, systematic variations in upstream input (inherited from earlier layers of visual processing) might drive adaptive activity-dependent changes in the number of synapses per connection in the optic lobe. This study focused on primary afferent synapses rather than synapses deep in the circuit, so any systematic upstream variations are limited to variations in ORN spike trains. Moreover, it is knowm that all DM6 PNs witness identical ORN spike trains, because ORN spikes travel faithfully across the midline to invade both ipsi- and contralateral glomeruli. This fact allowed the hypothesis of adaptive plasticity to be tested by reconstructing all the synapses that each ORN axon made onto all PNs. It was found that the variation in synapse numbers was not faithfully correlated across all PNs, and so some of this variation is likely random - that is, unrelated to ORN activity, and caused by a fundamental imprecision in the processes that specify the number of synapses per connection (Tobin, 2017).

    Intriguingly, it was found that synapse number variations were correlated across sister PNs on the same side of the brain, even though they were uncorrelated on the opposite sides of the brain. In principle, this might be evidence of incomplete adaptive plasticity -- plasticity that works at ipsilateral connections but somehow fails at contralateral connections. More likely is the scenario of correlated developmental noise -- e.g., some ORN axons may simply arrive sooner at the ipsilateral glomerulus, and so may form more physical contact with ipsilateral PNs, and thus more synapses. This sort of correlated developmental noise may be one reason why sister PNs on the same side of the brain display such high levels of correlated electrical noise. As is shown in this study, sister PNs on the same side of the brain are dominated by the same pool of ORNs. These ipsilateral sister PNs converge onto higher-order neurons, which are especially sensitive to correlations in sister PN spike times. As a result, sister PN spike timing correlations represent a functionally-relevant constraint on circuit function which can affect both the speed and accuracy of odor stimulus responses (Jeanne and Wilson, 2015) (Tobin, 2017).

    Previous studies have highlighted other examples of seemingly non-optimal neural wiring patterns. These have generally been examples of individual neurons following highly tortuous paths. Tortuosity is energetically costly, and it is difficult to see any functional benefit to tortuosity in these cases, suggesting that it may simply be the result of an imprecise developmental process. This study extends this idea by providing evidence of imprecision at the level of synaptic connectivity, not just imprecision in the path that a neuron takes to find a synaptic target (Tobin, 2017).

    In the larval antennal lobe, there are 21 glomeruli, as compared to ~50 in the adult. Moreover, each glomerulus in the larva is relatively simple: it contains just one ORN axon and one uniglomerular PN dendrite. The larval antennal lobe connectome has just been reconstructed (Berck, 2016), and it is instructive to note the differences between the adult and the larval versions of the same circuit (Tobin, 2017).

    One difference concerns the structure of individual synaptic connections. In the larva, the average ORN->PN connection contains ~70 synapses. whereas in the adult, it contains 23 synapses. Thus, the increased number of ORNs in the adult is partly compensated by a decrease in the number of synapses per connection (Tobin, 2017).

    Another difference is in the control of ORN output. In the larva, almost all synapses onto ORNs arise from multiglomerular neurons. In the adult, most synapses onto ORNs arise from multiglomerular neurons, but a substantial minority arise from PNs and ORNs. This suggests that the adult network may exert more complicated control of ORN neurotransmitter release (Tobin, 2017).

    The brain's computational power would be substantially reduced if all synaptic connections were identical. From this perspective, systematic variations in synaptic connections are evidence of the brain's functional capacity -- the capacity to match a connection's strength to its required function. In this study, there was an unusual opportunity to discover systematic variations in a particular connection type, because there are many ORN->PN connections per brain (260 connections in the glomeruli that were reconstructed). By studying many instances of the same connection type, it was possible to discover several systematic variations. The results indicate that systematic variations in connection strength arise largely as a result of differences in the number of synapses per connection. This study shows how systematic ipsi-contra differences can enable odor lateralization, while systematic correlations between synapse number and dendrite size can equalize the response of different PNs to an average ORN spike (Tobin, 2017).

    On the other hand, unsystematic wiring variations ('connection noise') must limit the capacity of every neural system. Some of this imprecision can be balanced by homeostatic changes to other parameters, including synaptic parameters. The findings provide insight into the mechanisms underlying such compensatory changes, but the results also argue for the existence of residual non-optimal wiring variations that can impair neural computations (Tobin, 2017).

    Large-scale EM offers an unprecedented opportunity to study all these variations - and co-variations - in neural network wiring. Drosophila melanogaster is likely to be the next organism whose brain is fully mapped at the connectomic level. As such, it provides an opportunity to gain insight into the causes and consequences of systematic and noisy variations in network architecture (Tobin, 2017).

    The organization of projections from olfactory glomeruli onto higher-order neurons

    Each odorant receptor corresponds to a unique glomerulus in the brain. Projections from different glomeruli then converge in higher brain regions, but the logic governing which glomeruli converge and which do not is not understood. This study used two-photon optogenetics to map glomerular connections onto neurons in the lateral horn, the region of the Drosophila brain that receives the majority of olfactory projections. This study has identified 39 morphological types of lateral horn neurons (LHNs) and shows that different types receive input from different combinations of glomeruli. Different LHN types do not have independent inputs; rather, certain combinations of glomeruli converge onto many of the same LHNs and so are over-represented. Notably, many over-represented combinations are composed of glomeruli that prefer chemically dissimilar ligands whose co-occurrence indicates a behaviorally relevant "odor scene." The pattern of glomerulus-LHN connections thus represents a prediction of what ligand combinations will be most salient (Jeanne, 2018).


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    date revised: 20 June 2016

    Zygotically transcribed genes

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