A requirement for mushroom body signaling during olfactory memory retrieval

The mushroom bodies of the Drosophila brain are important for olfactory learning and memory. To investigate the requirement for mushroom body signaling during the different phases of memory processing, neurotransmission was transiently inactivated through this region of the brain by expressing a temperature-sensitive allele of the shibire dynamin guanosine triphosphatase, which is required for synaptic transmission. Inactivation of mushroom body signaling through alpha/beta neurons during different phases of memory processing reveal a requirement for mushroom body signaling during memory retrieval, but not during acquisition or consolidation (McGuire, 2001).

Genetic and chemical disruption of the MBs produces flies that are normal for general behaviors but are defective in olfactory learning. Many genes involved in olfactory learning and memory show enriched expression in the MBs, particularly those encoding components of the cyclic adenosine monophosphate signaling pathway. Targeting of a constitutively active G-protein alpha subunit to the MBs disrupts olfactory learning, and restoring the rutabaga-encoded adenylyl cyclase specifically to the MBs of rutabaga mutants is sufficient to restore short-term memory in these flies. The model that has emerged from these experiments posits the MBs as important centers in olfactory associative learning and the likely site of convergence of the conditional (CS) and unconditioned (US) stimuli in classical conditioning (McGuire, 2001 and references therein).

A limitation of the previous experiments is that they all involve permanent alterations to the fly's brain throughout development, leading to the possibility that some of the effects on learning might reflect developmental perturbations rather than modifications of the physiology of these neurons that subserve learning and memory processes. Additionally, the irreversible nature of these interventions has made it impossible to dissect the roles of the MBs at the different stages of memory acquisition, consolidation, and retrieval (McGuire, 2001).

To explore the roles of the MBs in the different phases of memory processing, an approach was used that allows transient inactivativation of synaptic transmission from the MBs by targeting expression of a temperature-sensitive shibire^ts1 transgene to the MBs by the GAL4/UAS system. The shibire gene encodes a dynamin guanosine triphosphatase (GTPase) that is essential for synaptic vesicle recycling and maintenance of the readily releasable pool of synaptic vesicles. The temperature-sensitive allele shibire^ts1 bears a mutation in the GTPase domain, which renders the protein inactive at restrictive temperatures (>29°C) and causes a rapid inactivation of synaptic transmission and subsequent paralysis. Restricted expression of the shibire^ts1 transgene in specific cells produces blindness and paralysis at restrictive temperatures. Recently, the transgene was used to demonstrate the role of the dorsal paired medial neurons in memory formation (McGuire, 2001).

A number of GAL4 lines that exhibited enriched MB expression patterns were screened for 3-min memory performance when driving the UAS-shi^ts1 transgene at both permissive (25°C) and restrictive (32°C) temperatures in an olfactory classical conditioning paradigm. In this assay, flies are conditioned by exposure to one odor paired with electric shock (CS+) and subsequent exposure to a second odor in the absence of electric shock (CS-). Memory is then assayed at predetermined time points after training by forcing the flies to choose between the CS+ and CS-. Several MB GAL4 lines demonstrate significant memory impairment at 3 min when tested at the restrictive temperature. These lines were next analyzed for sensorimotor functions required for the conditioning assay, including locomotion, odor avoidance, and electric shock avoidance, at both the permissive and restrictive temperatures. Subsequently, focus was placed on the GAL4 lines, c739 and 247, which demonstrate intact sensorimotor functions when driving the UAS-shi^ts1 at both permissive and restrictive temperatures. For these MB GAL4 lines, memory at 3 min at the permissive temperature is indistinguishable among flies bearing both the GAL4 element and the UAS-shi^ts1 transgene in combination and control flies bearing the GAL4 element or the UAS-shi^ts1 element alone. At the restrictive temperature, however, the combination of c739 or 247 with the UAS-shi^ts1 transgene results in a significant impairment of performance. The line 201Y; UAS-shi^ts1 shows a slight but nonsignificant decrease in memory performance under these conditions. These data indicate that the inactivation of MB neurotransmission disrupts the processes underlying the encoding, storage, or retrieval of memory tested 3 min after training (McGuire, 2001).

These data were analyzed relative to the expression patterns of the three GAL4 lines to gain insights into possible functional subdivisions of the MBs. The GAL4 line 247, in which GAL4 is under the control of a 247-base pair (bp) enhancer fragment isolated from the D-mef2 gene, drives reporter gene expression in all lobes of the MB. In the line 201Y, the gamma lobe is preferentially marked, along with a small subset of the alpha/beta neurons. In contrast, the GAL4 c739 element drives reporter gene expression preferentially in the alpha/beta lobes. The expression overlap between the two GAL4 lines that disrupt 3-min memory when combined with UAS-shi^ts1 at the restrictive conditions is within the alpha/beta lobes, suggesting the importance of this subset of MB neurons for the expression of memory. At the restrictive temperature, the UAS-shi^ts1 in combination with 201Y, which preferentially drives reporter gene expression principally in the gamma lobes, does not significantly impair 3-min memory. The mild memory impairment in this line could be due to insufficient levels of expression of the UAS-shi^ts1 transgene or rather it could reflect the possibility that the neurons in which this line drives the UAS-shi^ts1 are not necessary for memory expression at this time point (McGuire, 2001).

To determine whether the deficient performance of these flies arises from a defect in memory acquisition, consolidation, or retrieval, memory was examined at a later time point (3 hours). Prior research has shown that most of the memory measured at this time point has been consolidated into an anesthesia-resistant form. The separation of training and testing also allows MB signaling to be reversibly inactivated separately during each phase and then it can be asked whether memory performance is affected. Three-hour memory was examined at the permissive temperature throughout the experiment. Under these conditions, the performance of the c739;UAS-shi^ts1 flies is indistinguishable from flies bearing either the c739 element or the UAS-shi^ts1 element. The lines 247 and 201Y in combination with UAS-shi^ts1 disrupted 3-hour memory at the permissive temperature and were not analyzed further (McGuire, 2001).

To examine the requirement for signaling through the MBs during the retrieval of olfactory memory, training was performed under permissive conditions and the flies were maintained under these conditions until just before testing, at which point they were shifted to the restrictive temperature. When the performance of these flies was examined at 3 hours under these conditions, memory was abolished in the c739;UAS-shi^ts1 flies, whereas the memory of the control groups was intact. Whether the acquisition of olfactory memory shares a similar requirement for MB signaling was examined. Training was performed under the restrictive conditions and immediately the flies were cooled to the permissive temperature. When the performance of these flies was examined at 3 hours under these conditions, a difference was observed between the c739;UAS-shi^ts1 flies and the control line c739 but no difference between c739;UAS-shi^ts1 and the UAS-shi^ts1 control, indicating a general effect of heat on lines carrying the UAS-shi^ts1 element, but no specific disruption of memory when UAS-shi^ts1 is combined with c739. Subsequently it was investigated whether the interval between training and testing, during which memories are consolidated and stored, would require signaling through the MBs to observe normal memory performance at 3 hours. Flies were trained and tested under permissive conditions and given a temperature shift to restrictive conditions during the interval between these events. Under these conditions, a general effect of heat on the performance of all of the lines was observed, but no significant difference between any of the groups was observed (McGuire, 2001).

By transiently blocking synaptic transmission from the MBs during memory formation, consolidation, and retrieval, the temporal requirements of MB signaling during the different phases of memory processing could be examined. The results suggest quite unexpectedly that signaling through the MB alpha/beta neurons is required during olfactory memory retrieval, but not during memory acquisition or storage. It is proposed that, in Drosophila, olfactory memory retrieval requires signaling through the alpha/beta lobes to downstream neurons for expression. This does not preclude, however, a role for other MB lobes in memory formation, consolidation, or retrieval. A recent study demonstrating the sufficiency of rutabaga expression in the MBs for rescue of the short-term memory defect in rutabaga mutants has suggested that the gamma lobes might be of particular importance in the formation of short-term memories. Recent studies have also demonstrated that fasciclinII mutants are defective in memory acquisition and this protein is predominantly expressed in the alpha/beta neurons, although it is expressed at lower levels in the gamma lobe. One hypothesis to explain the combined observations is that memory formation occurs in the gamma neurons, or in both gamma and alpha/beta neurons simultaneously, but that memory retrieval occurs principally through the output of the alpha/beta neurons. Indeed, such a scenario involving a partial redundancy of function can explain why a subset of neurons might be sufficient, but not necessary, for memory expression. However, the observation that rutabaga and fasciclinII flies are only partially impaired in short-term memory indicates the likelihood that other mechanisms and perhaps locations of signal convergence, such as the antennal lobe or the lateral protocerebrum, may additionally mediate memory acquisition or storage. Taken together, these data suggest that acquisition and consolidation occur upstream of the MB synapse upon follower neurons, either in the MB neurons themselves or in upstream circuits. Retrieval of these memories within 3 hours would then engage signaling through a subset of the MB neurons, involving the alpha/beta lobes. It remains to be determined whether long-term memories (>24 hours) are dependent on the MBs (McGuire, 2001).

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. 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).

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).

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 GABA_A-like and GABA_B-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 GABA_A nor GABA_B receptors can mediate the lateral depolarization observed. Both GABA_A and GABA_B conductances are hyperpolarizing in PNs. And although GABA_A and GABA_B 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).

Altered representation of the spatial code for odors after olfactory classical conditioning. Memory trace formation by synaptic recruitment

In the olfactory bulb of vertebrates or the homologous antennal lobe of insects, odor quality is represented by stereotyped patterns of neuronal activity that are reproducible within and between individuals. Using optical imaging to monitor synaptic activity in the Drosophila antennal lobe, classical conditioning is shown to rapidly alter the neural code representing the learned odor by recruiting new synapses into that code. Pairing of an odor-conditioned stimulus with an electric shock-unconditioned stimulus causes new projection neuron synapses to respond to the odor along with those normally activated prior to conditioning. Different odors recruit different groups of projection neurons into the spatial code. The change in odor representation after conditioning appears to be intrinsic to projection neurons. The rapid recruitment by conditioning of new synapses into the representation of sensory information may be a general mechanism underlying many forms of short-term memory (Yu, 2004).

Drosophila can develop a robust association between an odor, the conditioned stimulus (CS), and electric shock, the unconditioned stimulus (US), if the CS and the US are paired. Flies display their memory of this association by avoiding the odor CS during a test, after previously experiencing the pairing of the CS and the US. The number, nature, and the locations of the cellular memory traces that guide this acquired avoidance behavior are unknown, but significant evidence suggests that some cellular memory traces are formed in mushroom body neurons, higher-order neurons that form part of the olfactory nervous system. Furthermore, the evidence indicates that the memory traces are formed in part by the activation of the cyclic AMP signaling system. However, the memory traces that underlie insect odor memory are probably formed in many different areas of the olfactory nervous system and in other areas of the brain as well (Yu, 2004).

Optical imaging of synaptic activity in Drosophila brains coupled with behavioral conditioning has been used to visualize and study a cellular memory trace. This trace is established as new synaptic activity after conditioning in the antennal lobe projection neurons of the olfactory system. A concept established from these results, that may generalize to other forms of memory, is that memories form by the rapid recruitment of relatively inactive synapses into the representation of the sensory information that is learned. In other words, the synaptic representation of the odor CS is changed by learning, with new synaptic activity added to the representation after learning (Yu, 2004).

The anatomical organization of the Drosophila olfactory nervous system shares many fundamental similarities to that of vertebrates, suggesting that the mechanisms for odor perception, discrimination, and learning are shared. Olfactory receptor neurons (ORNs), distributed near the surface of the antenna and maxillary palp on each side of the head, project axons to the antennal lobe, where they terminate in morphologically discrete and synapse-dense areas known as glomeruli. The projection patterns of the ORNs are stereotyped between animals; ORNs that express the same olfactory receptor gene, although distributed across the surface of the antenna and maxillary palps, project their axons to the same glomerular target in the antennal lobe. There they are thought to form excitatory synapses with at least two classes of neurons: the local interneurons (LNs), a large fraction of which are GABAergic inhibitory neurons, and the projection neurons (PNs). A unique feature of the circuitry within the insect antennal lobe is the apparent existence of reciprocal dendrodendritic connections between the PNs and the LNs. The presence of these unique junctions with both transmissive and receptive specializations indicates that each glomerulus processes and makes computations that may underlie odor perception, discrimination, and learning, rather than being a simple transit station for the throughput of olfactory information. Individual PNs generally extend dendrites into a single antennal lobe glomerulus and then convey the processed olfactory information to two higher brain centers: the mushroom bodies and the lateral protocerebrum (Yu, 2004).

The neuroanatomy thus suggests that distinct odors are represented (1) by the stimulation of distinct sets of ORNs; (2) by spatial patterns of glomerulus activation within the antennal lobe, and (3) by a distinct set of synaptic fields activated in the mushroom bodies and the lateral protocerebrum. Functional imaging experiments have suggested the existence of a spatial code for odors within the antennal lobe of insects and the olfactory bulb of vertebrates. Calcium dyes, voltage-sensitive dyes, transgenically supplied fluorescent proteins, and intrinsic optical signals have been used to visualize odor-specific patterns of glomerulus activation in Drosophila, honeybee, zebrafish, salamander, and rat (Yu, 2004 and references therein).

The search for cellular memory traces began by first asking whether synaptic transmission could be detected in antennal lobe glomeruli of intact but immobilized adult flies after stimulation with pure odors that are frequently used as conditioned stimuli in behavioral learning experiments. It is possible to detect olfactory responses with optical reporters in the antennal lobes using reduced preparations of either isolated adult heads or dissected adult brains. Intact Drosophila adults are used, immobilized in a pipette tip, with their heads and antennae exposed. A small square of cuticle is removed from the dorsal head of each animal. Flies are mounted under a laser-scanning confocal microscope to detect basal fluorescence and the change in fluorescence induced with the application of odor. Initially flies were used carrying the GH146-GAL4 transgene to drive expression of a reporter of synaptic transmission; UAS-synapto-pHluorin (UAS-spH) was used to reveal PN presynaptic specializations within antennal lobe glomeruli (Yu, 2004).

A brief application of odor through a glass micropipette directed at the antennae produced a rapid, quantifiable, and stereotypic response in glomeruli between animals. For instance, the odor 3-octanol (OCT) produced a rapid burst of fluorescence in several glomeruli that occurred with the presentation of odor. Responses were quantified as the average percent change in the intensity of the pixels that represent each glomerulus during stimulation. A spatial response observed to OCT was observed as a pseudocolor image over eight glomeruli that were unambigously identified and that formed the focus of this study. Four of the eight glomeruli were activated reproducibly by OCT, whereas four others remained unchanged. These responses were quantitatively similar at two different odor concentrations. The increased responses of the four glomeruli at the higher odor concentration indicated that responses at lower odor concentrations fell well below the dynamic response ceiling for spH. The remarkably small standard errors that were obtained for glomerular responses between flies indicate that the procedures and standards that were employed were highly consistent and accurate. The many variables that could influence reproducibility include fly dissection, fly mounting, odor application, confocal scanning, glomerulus identification, and glomerulus circumscription during data analysis (Yu, 2004).

A stimulus that is used frequently as the unconditioned stimulus for olfactory classical conditioning is mild electric shock. This shock is normally delivered to flies as they stand on an electrified grid while also being in the presence of an odor. This US is effective at conditioning flies when presented along with an odor, although the identity of the neurons within the olfactory pathway that are stimulated by both odor and shock is unknown. Neurons that can function as cellular coincidence detectors must be activated either directly or indirectly by both stimuli (Yu, 2004).

It was therefore asked whether the PNs that responded to the odor CS could also respond to the US of electric shock. Pulses of electric shock were applied (at an intensity and frequency used to behaviorally condition adult Drosophila) to the abdomens of flies that were immobilized under the microscope. Synaptic transmission was activated in all glomeruli that expressed UAS-spH. The synaptic transmission events occurred with a periodicity that matched the 5 s interstimulus interval of the electric shock. When these time-based signals were converted to the frequency domain by Fourier transformation, a major component with a frequency matching the frequency of shock delivery (0.2 Hz) was extracted. These data indicate, therefore, that PNs are activated by electric shock stimuli applied to the abdomen. The neural pathways that carry the electric shock stimulus from the abdomen to the brain and the identity of the neurons immediately presynaptic to the PNs in this pathway have not yet been identified (Yu, 2004).

Since some PNs responded to both OCT and the US of electric shock when presented separately, it was of interest to ask whether these neurons could be conditioned by simultaneously presenting both odor and shock (forward conditioning). To test this, individual flies were conditioned either with OCT paired with electric shock or with one of a series of control protocols, including the odor only, shock only, and odor with shock but separated by 30 s to 2 min (trace conditioning). The optical response of the PNs to an odor test stimulus was then monitored 3 min after these treatments. The delay of 3 min was chosen, since for normal behavioral conditioning experiments it takes 3 min after training flies to test their choice behavior in a T-maze. Focus was placed on the effect of forward conditioning compared to other conditioning protocols, since the protocols of CS only, US only, and trace conditioning failed to produce behavioral conditioning (Yu, 2004).

The responses of most PNs to the test odor of OCT after the various conditioning protocols were similar or identical to the naive response. For instance, PNs innervating glomerulus DM2 responded to OCT with a 6% increase. Conditioning with the CS only, US alone, CS and US paired, or CS + US trace did not significantly alter this response. Similarly, most PNs that failed to respond to the odor CS by itself failed to exhibit any change in response after the conditioning protocols. Surprisingly, however, there was one notable exception. PNs innervating glomerulus D responded after forward conditioning to OCT with a %deltaF/F (fluorescence within each glomerulus relative to the basal fluorescence measured prior to the test with OCT) of 7%, while the responses of these PNs after US only, CS only, or trace conditioning protocols were similar to the naive response, which was not significantly different from zero. These data indicate, therefore, that forward pairing of the odor CS and the shock US rapidly awakens the PN synapses in the D glomerulus within 3 min after conditioning. The failure to observe a conditioning effect on the OCT-responsive glomeruli -- DM6, DM2, DM3, and DL3 -- cannot be due to a ceiling effect, since the odor concentration used for conditioning was well below the ceiling of spH's dynamic range. Thus, additional PN synapses in the antennal lobe are recruited rapidly to represent the odor CS after forward conditioning (Yu, 2004). These conclusions were reproduced and extended with a second type of experimental design. Since the response of the D PNs during the CS test was not affected by prior exposure of the CS when compared between flies, a 'within-animal' design was used for the next set of experiments. Each fly was presented the odor CS for 3 s, during which PN responses were monitored. After a rest of 5 min, the fly was then conditioned, and 3 min after conditioning, the response to a 3 s odor test was again monitored. The response before conditioning was compared with the response after conditioning. As before, the response of the D PNs to OCT alone was undetectable. However, the response after forward conditioning with OCT reached a %deltaF/F of 6% when measured 3 min later (Yu, 2004).

Is the memory trace in the D PNs specific for OCT as a CS, with other odors recruiting other sets of neurons and synapses, or is the change in D PNs a general property of learning something about any odor? To address this issue, within-animal conditioning experiments were carried out using methylcyclohexanol (MCH) as the CS, a second odor that is used frequently for odor learning in Drosophila (Yu, 2004).

The responses of some PNs to MCH before any conditioning were more variable between flies than for OCT. However, PNs innervating the three glomeruli DM6, DM2, and DM3 exhibited significant responses to MCH alone applied before conditioning. Forward conditioning, however, recruited the activity of glomerulus VA1 (both VA1l and VA1m) into the representation of MCH. Like the D glomerulus for OCT responses, VA1 was insensitive to MCH prior to conditioning. Therefore, different odors recruit normally insensitive PNs into their spatial representation after conditioning (Yu, 2004).

Behavioral memories can be very short or quite enduring, depending on the nature of the task learned, the strength of the training, the saliency of the cues, and undoubtedly the nature and number of the cellular memory traces that underlie the behavioral memory trace. The stability of the cellular memory trace that was established by forward pairing with OCT and shock in the D glomerulus PNs was probed by testing at different times after conditioning. This conditioned response waned rapidly. When tested at 5 min after conditioning, the increased response at 3 min had decayed to 5%, and by 7 min after conditioning the cellular memory trace was not significantly different from zero. Attempts to extend the duration of this memory trace with multiple and spaced conditioning trials have not been successful (Yu, 2004).

The recruitment of PN synapses of the D glomerulus into the representation of OCT and those of the VA1 glomerulus into the representation of MCH after conditioning suggests that synaptic recruitment was odorant specific. Nevertheless, the conditioned animals were conditioned and challenged with only one of the two odorants. To further explore the specificity of synaptic recruitment, a discriminative, within-animal experimental design was employed in which each animal was challenged with both odors prior to and after conditioning with either OCT or MCH (Yu, 2004).

PN synapses innervating glomeruli DM6, DM2, and DM3 showed significant responses to MCH before conditioning, while the remaining glomeruli failed to show significant responses. Most importantly, there were no significant differences in the responses to MCH after conditioning compared to those before conditioning. However, the conditioning recruited the PN synapses of the D glomerulus into the naive representation of OCT, which consisted of significant responses from glomeruli DM6, DM2, DM3, and DL3. In the reciprocal experiment, conditioning with MCH did not alter the representation of OCT by glomeruli DM6, DM2, DM3, and DL3 but selectively recruited PN synapses of VA1 into the MCH representation. These results, obtained with animals that were presented with two different odors in a discriminative protocol, strongly support the contention that the recruitment is odor specific (Yu, 2004).

Since the D PNs receive synaptic inputs from ORNs and LNs, it was of interest to ask whether the memory trace induced by OCT conditioning in D PN synapses was intrinsic to these neurons or whether the trace was established in one of the presynaptic partners so that the increase in D PN synaptic activity was only a reflection of an upstream memory trace. To test whether a synaptic memory trace was established in ORNs, UAS-spH was expressed using the ORN driver OR83b-GAL4. Using imaging conditions that were designed to identify glomerulus D and other glomeruli visible with GH146-GAL4, six glomeruli, along with D, were reproducibly discernable using this driver. Stimulation of flies with OCT produced a synaptic response in three of the six identified glomeruli, but these did not include D. Thus, PNs that innervate D do not receive excitatory input from the OR83b-expressing ORNs that form synapses in D (Yu, 2004).

It was asked, nevertheless, whether the ORNs that project to D responded to the US of electric shock and whether forward conditioning could recruit the OCT-blind D ORNs into being OCT sensitive. Electrical stimulation of flies carrying both OR83b-GAL4 and UAS-spH produced no increase in fluorescence of D or other glomeruli in response to shock pulses. Furthermore, forward pairing failed to produce any detectable change in synaptic activity within the identified glomeruli (Yu, 2004).

A GAD-GAL4 driver was used to direct expression of UAS-spH in LNs to address the same issues for these neurons. LNs that innervate glomeruli DM6, DM2, DM3, and DL3 all responded to the odor CS, whereas those innervating D, DL2, DA1, and VA1 failed to respond. The sets of responding and nonresponding glomeruli matched exactly those observed using the PN GAL4 driver. However, electric shock pulses to the body failed to stimulate synaptic responses in the LNs innervating D, and the synaptic responses of these neurons also could not be conditioned. The failure of the D PN synaptic trace to be transmitted to the LNs, which may be both presynaptic and postsynaptic to PNs, may indicate that the recruited D PNs may synapse on other PNs or interneurons rather than on the GAD-expressing LN or that the threshold for LN activation is simply too high for the memory trace to be transferred from the D glomerulus PNs (Yu, 2004).

Therefore, forward conditioning directly recruits D PNs into the representation of the CS of OCT. This recruitment is not the manifestation of a conditioned memory trace in the presynaptic ORNs or the LNs, since neither the ORNs nor the LNs that are presynaptic to the D PNs responded to the shock US, and neither neuron type exhibited a conditioned response (Yu, 2004).

The forward conditioning protocol used for most of the imaging experiments employed a single odor as the CS, paired with the US of electric shock pulses. Behavioral conditioning experiments, however, have often employed discriminative conditioning protocols with two different odors. The two-odor, discriminative, behavioral conditioning paradigm was modified into a single-odor classical conditioning paradigm to test the behavioral effects of the various conditioning protocols used for imaging. Flies were presented with CS only, US only, CS + US paired, or CS + US with a trace interval of 30 s, 1 min, or 2 min. They were then tested for their avoidance of the odor CS in a T-maze against a second odor to which they were naive and under conditions in which animals naive to any conditioning protocol distribute equally between the two odors (Yu, 2004).

The GH146-GAL4/UAS-spH flies were behaviorally conditioned using the new single-odor conditioning protocol and the effects of this behavioral conditioning was compared at 3 min posttraining to the conditioned synaptic responses of D PNs. The CS only, US only, or trace conditioning protocols produced small or no behavioral changes, similar to the lack of effect at the synaptic level. In contrast, forward conditioning produced a high behavioral performance score, similar to the robust synaptic change observed in D PNs. Therefore, the synaptic changes that were observed in D PNs produced by the conditioning protocols correlate well with the behavioral changes produced by the same protocols at 3 min after conditioning. Although the relative effectiveness of the various conditioning protocols correlated well between the imaged memory trace and behavioral performance, the duration of the behavioral memory after single-odor CS/US coincidence was much more enduring (>2 hr) than the enhanced synaptic activity of the D glomerulus PNs. Therefore, the D glomerulus memory trace would be capable of driving behavior for only the first few minutes after conditioning. Other memory traces of longer duration must be formed for more enduring behavioral performance (Yu, 2004).

The results offer two main conceptual advances. First, it is shown that forward conditioning of living Drosophila alters the representation of the odor CS in the PN synapses in the antennal lobe. Prior studies with the honeybee have suggested that memory traces are laid down in the antennal lobes, but these studies have employed pharmacological manipulations, calcium imaging, or physical insults to the entire antennal lobe without discriminating the roles of individual glomeruli, specific neuron types, or their synapses. In this study the GAL4 system of Drosophila to drive reporter expression in subsets of neurons, which provided resolution between types of neurons, and the reporter synapto-pHluorin, which provided a specific readout of synaptic activity in response to odorants. This approach was extended by imaging living flies before and after conditioning. This extension led to the specific finding that a short-lived cellular memory trace forms in Drosophila PNs after conditioning (Yu, 2004).

The existence of the short-term cellular memory trace in PNs and the correlated behavioral responses lends strong support to the idea that transient olfactory memories are formed in the insect antennal lobe. Much evidence has now accumulated to support the hypothesis that mushroom body neurons are centrally involved in odor learning, using the cAMP signaling cascade, in part, for the integration of sensory information. However, memories are distributed, and neurons other than mushroom body neurons are clearly involved in olfactory learning. The data provide evidence that the distributed memory system in Drosophila includes the antennal lobes. An attractive hypothesis is that the antennal lobes and the mushroom bodies are both sites for memory formation but that the earliest memories are formed in the antennal lobes by altering the representation of the sensory stimulus and that this altered representation is then transferred to and perhaps strengthened by the mushroom bodies (Yu, 2004).

The evidence offers the surprising conclusion that the PNs likely function as integrators of the CS and US. The ORNs, LNs, and PNs that innervate glomeruli recruited by conditioning did not respond to the odor CS. Of the three, only the PNs responded to the US of electrical shock. Thus, the available evidence suggests that PNs are the first point in the CS pathway that intersects functionally with the US pathway, although the possibility cannot be eliminated that ORNs and LNs receive US information via neuromodulatory rather than excitatory inputs, nor can the possibility be eliminated that some unknown neuron presynaptic to the recruited PNs integrates the CS and US. There is no neuroanatomical information about the US pathway from peripheral receptors or the identity of the presynaptic neurons providing US input to the PNs. However, it seems likely that the stimulus of electric shock must itself be processed by higher-order neurons in order to acquire its negative value attribute, which can then be stamped onto the PNs as associated with the CS. The CS pathway to the recruited PNs also remains unknown, since the odor CS (OCT or MCH) does not appear to be conveyed to glomerulus D or VA1 via the OR83b-expressing ORNs. It is possible that some ORNs that fail to express OR83b may project to these glomeruli and convey the CS stimulus. An alternative and more attractive possibility is that some local interneurons may convey the CS information from other glomeruli by synapsing on PNs innervating the recruited glomeruli. Such excitatory, interglomerular local interneurons have been discovered in the vertebrate olfactory bulb (Yu, 2004).

The second major conceptual advance is that the evidence suggests that memory traces are formed by the recruitment of synapses that are relatively silent to the odor CS, within the sensitivity of optical imaging, into the ensemble of synapses whose activity represents the odor CS in naive animals and that the selection of recruited synapses is odor specific. The possibility cannot be excluded, however, that some synaptic activity exists within the recruited PNs that is below the sensitivity of that detectable by optical imaging. Nevertheless, the results and the emerging evidence that cellular synaptic plasticity may occur from the activation of normally silent synapses suggest that some forms of behavioral memory may occur through a large synaptic gain mechanism, perhaps approaching an 'off-on' switch mechanism, rather than through smaller graded changes in synapses that represent the stimuli in naive animals. Thus, memory formation involves the recruitment of synapses to represent the sensory cues that are learned (Yu, 2004).

In addition to these advances, these findings also pose new and intriguing puzzles. Is the short-term memory trace established in the PNs independent of other memory traces, so as to directly guide behavior for a short period after learning, or is it transferred to the mushroom bodies or the lateral protocerebrum, perhaps to be consolidated there into a more enduring trace, with behavior being guided from these higher-ordered brain centers? A related question is whether the PN synaptic memory trace is specific to the connections made in the antennal lobe or whether this occurs on a cell-wide basis, with conditioning also stamping its effects on PN synapses made in the mushroom bodies and the lateral protocerebrum. Do any of the known memory mutants disrupt the formation or stability of the PN memory trace? How is it that the recruitment of new synapses in the antennal lobe produces a new representation of the learned odor? Is it just simply that more activated synapses represent the learned odor, or does the synaptic activation of PNs alter the coding of the odor CS, perhaps by influencing the coherency or timing of PN and LN oscillations that may contribute to odor encoding (Yu, 2004)?

Drosophila DPM neurons form a delayed and branch-specific memory trace after olfactory classical conditioning

Formation of normal olfactory memory requires the expression of the wild-type amnesiac gene in the dorsal paired medial (DPM) neurons. Imaging the activity in the processes of DPM neurons revealed that the neurons respond when the fly is stimulated with electric shock or with any odor that was tested. Pairing odor and electric-shock stimulation increases odor-evoked calcium signals and synaptic release from DPM neurons. These memory traces form in only one of the two branches of the DPM neuron process. Moreover, trace formation requires the expression of the wild-type amnesiac gene in the DPM neurons. The cellular memory traces first appear at 30 min after conditioning and persist for at least 1 hr, a time window during which DPM neuron synaptic transmission is required for normal memory. DPM neurons are therefore 'odor generalists' and form a delayed, branch-specific, and amnesiac-dependent memory trace that may guide behavior after acquisition (Yu, 2005).

Memory traces together represent the memory engram that directs behavior of the organism after learning or conditioning events. Classical conditioning is one form of learning whereby a conditioned stimulus (CS) becomes predictive of an unconditioned stimulus (US) when the two stimuli are paired in an appropriate way. The prototypic example of classical conditioning stems from studies on dogs conducted by Ivan Pavlov in which tone cues (CS) paired with a food reward (US) became predictive of the food reward, shown by the dog's salivation upon hearing the tone cue after conditioning. In Drosophila, olfactory classical conditioning is a robust and well-studied type of learning in which olfactory cues (CS) are usually paired with electric shock (US), such that conditioning leads to learned avoidance behavior of the CS. Learning to associate two forms of sensory information likely involves specific neurons that respond to both sensory cues and can integrate the information to produce learning. Thus, memory traces for olfactory classical conditioning in Drosophila are expected to form in neurons positioned at the intersections of the olfactory nervous system, the pathway that conveys and processes the CS (CS pathway), and the pathways that convey and process the US (US pathway) (Yu, 2005 and references therein).

The insect olfactory nervous system begins with olfactory receptor neurons (ORNs) distributed between the antennae and maxillary palps. The ORNs project axons to the antennal lobe, where they terminate in morphologically discrete and synapse-dense areas known as glomeruli. There, the ORNs are thought to form excitatory synapses with at least two classes of neurons, one of these being the projection neurons. The projection neurons then convey the olfactory information along their axons in the antennal cerebral tract to at least two higher brain centers, the mushroom bodies and the lateral horn. A large body of evidence has accumulated indicating the importance of the mushroom bodies for olfactory learning. Thus, odors are represented first in the olfactory nervous system by the activation of overlapping sets of ORNs; second by the activation of overlapping sets of projection neurons, and third by the activation of mushroom body and lateral horn neurons (Yu, 2005 and references therein).

An olfactory memory trace forms in the projection neurons after olfactory classical conditioning. Synaptic release of neurotransmitter from presynaptic specializations of projection neurons in the antennal lobe was monitored optically using the transgenically supplied indicator of synaptic transmission, synapto-pHluorin (spH). The memory trace was detected in this case by a rapid but short-lived recruitment of new synaptic activity into the representation of the learned odor. More specifically, distinct odors stimulate distinct sets of projection neurons in naive animals. Within 3 min after conditioning, additional sets of projection neurons become activated by the learned odor. This recruitment is odor specific; different odors recruit different sets of projection neurons into the representation of the learned odor. The recruitment of new sets of projection neuron synapses into the representation of the learned odor, however, is short lived, lasting only 5 min before the synaptic release from the recruited sets of projection neurons decays to the undetectable levels observed prior to conditioning. The short-lived memory trace of projection neurons, although potentially important for guiding behavior for a few minutes after conditioning, cannot account for the time course for behavioral memory, which can last for days. Thus, memory traces in other areas of the nervous system must provide for the persistence of behavioral memory (Yu, 2005).

The dorsal paired medial (DPM) neurons are large neurons that express neuropeptides encoded by the amnesiac (amn) gene and are critical for normal memory. The encoded neuropeptides are related to pituitary adenylyl cyclase-activating peptide (PACAP). The DPM neurons have been widely hypothesized to be part of the US pathway through the release of the expressed modulatory neuropeptides, in part because their processes invade the mushroom body neuropil and are thought to intersect the olfactory nervous system. There is also evidence that these neurons release acetylcholine as a co-neurotransmitter along with neuropeptides (Yu, 2005).

The hypothesis that DPM neurons are solely part of a US pathway predicts that the US but not the CS should activate them and that their response properties should not change after olfactory classical conditioning. A change in their response pattern after conditioning would indicate the presence of a memory trace. A surprising observation is reported that not only do DPM neurons respond to the US of electric shock -- predicted by the hypothesis that they are part of the US pathway -- but they are odor generalists, responding to all odors that were tested. Moreover, they form odor-specific memory traces as registered by increased odor-evoked calcium influx and synaptic transmission. In contrast to the memory trace that forms immediately after conditioning in projection neurons, the memory trace that forms in the DPM neurons is delayed, appearing at 30 min after olfactory classical conditioning. Temporally distinct memory traces that form within projection neurons and DPM neurons after classical conditioning may be partly responsible for guiding behavior during different time windows after learning (Yu, 2005).

There are two DPM neurons, each with a large cell body residing in the dorsal aspect of each brain hemisphere. They have no obvious dendritic field and extend a single neurite in an anterior direction toward the neuropil regions (lobes) that contain the axons of the mushroom body neurons. The neurite from each DPM neuron splits, and one branch broadly innervates the vertical mushroom body lobes while the other innervates the horizontal mushroom body lobes. Careful examination of 12 different confocal stacks highlighting the DPM neurons with the DPM neuron driver, c316-GAL4, revealed that all of the fluorescence in the vertical and horizontal lobes of the mushroom bodies in c316-GAL4/UAS-mCD8GFP flies can be traced to the DPM neuron cell bodies rather than other c316-GAL4-expressing neurons in the brain (Yu, 2005).

It was first asked whether DPM neurons respond to electric-shock pulses delivered to the abdomen of living flies. The processes of DPM neurons in the mushroom body lobes of flies carrying c316-GAL4 and the synaptic transmission reporter UAS-synapto-pHluorin (UAS-spH) or the calcium reporter UAS-G-CaMP were visualized before and during the application of electric-shock pulses delivered to the abdomen. Electric-shock pulses were used of the same intensity, duration, and frequency as those used for behavioral conditioning. The calcium influx in the DPM neuron processes innervating the vertical mushroom body lobes that occurs with electric shock was examined. There was a dramatic response in the processes at the distal tip of the vertical lobes as well as at the vertical-lobe stalk. The change in fluorescence (ΔF/F_o) was examined that occurs with 12 shock pulses delivered at a rate of 1 shock pulse every 5 s. There was an increase in ΔF/F_o coincident with each shock pulse in the vertical lobes and the horizontal lobes with both G-CaMP and spH. These data indicate therefore that electric-shock pulses to the abdomen produce both calcium influx into the DPM neuron processes and synaptic release from their terminals, observations consistent with the possibility that DPM neurons provide US input to the mushroom body neurons (Yu, 2005).

The hypothesis that DPM neurons provide US input into the mushroom body neurons for olfactory memory formation predicts that these neurons should not be activated by the CS of an olfactory stimulus. To test this prediction, DPM neuron calcium influx and synaptic transmission was examined in flies presented with odor stimuli to their antennae and maxillary palps. Stimulation with pure odors like 3-octanol (OCT), 4-methylcyclohexanol (MCH), and benzaldehyde (BEN) elicited robust calcium influx into the DPM neuron processes innervating the vertical mushroom body lobes. The magnitude of the response was dependent on the odor concentration; no responses were observed using air blown over mineral oil, which was used as an odorant diluent. Moreover, these pure odors elicited synaptic activity of the DPM neuron as well as increased calcium influx. Odor responses were also observed in the DPM neuron processes innervating the horizontal mushroom body lobes. Finally, the generality of the DPM odor-evoked response was tested using a battery of 17 different odorants, ranging from pure odors to complex odors such as apple, banana, and grape. In all cases, the DPM neurons responded with increased calcium influx into their processes. Therefore, the DPM neurons are odor generalists in the sense that they apparently respond to all odors administered to the fly. However, the circuitry that provides odorant information to the DPM neurons is unknown (Yu, 2005).

Since the DPM neurons responded to both an odor conditioned stimulus and an electric-shock unconditioned stimulus, the possibility was considered that the neurons might form a memory trace and exhibit a changed response to the CS after olfactory classical conditioning. Either calcium influx into the DPM neuron processes or synaptic release after olfactory classical conditioning were measured. For all experiments, each animal was used for only one measurement in order to avoid potential complications produced by odor habituation, adaptation, or generalization that could occur with multiple exposures (Yu, 2005).

A within-animal experimental design was employed in which the response of the DPM neuron processes within each animal was first evaluated with a single, 3 s presentation of odor. This was followed by forward conditioning, in which a 60 s odor stimulus was presented simultaneously with 12 electric-shock pulses or by backward conditioning in which the 60 s odor stimulus was presented after the onset of the electric-shock stimuli. Forward conditioning leads to robust behavioral conditioning, whereas backward conditioning does not. The response of the DPM neuron processes was then tested at various times after conditioning, again within each animal, and the postconditioning response was compared to the preconditioning response (Yu, 2005).

The postconditioning responses of the DPM neurons to any of three conditioned odors, OCT, MCH, or BEN, did not differ from the preconditioning responses when assayed at 3 min after forward conditioning. This is in marked contrast to the odor-specific memory trace responses that occur in the projection neurons of the antennal lobe within 3 min after conditioning. However, amn mutant flies are only slightly impaired in 3 min memory and have a more pronounced impairment at later times beginning 10-60 min after training. Furthermore, synaptic transmission from DPM neurons is not required for 3 min memory and is dispensable during acquisition and retrieval for robust 3 hr behavioral memory. DPM synaptic transmission is instead required during the interval between training and testing. These observations led to a consideration of the possibility that the DPM neurons might form a memory trace with a delayed onset (Yu, 2005).

The postconditioning responses at 30 min after forward conditioning to three different odors compared to the preconditioning responses revealed that a delayed memory trace registered by increased calcium influx was detectable at this time. The increase in calcium influx was not observed at 30 min after backward conditioning, indicating that the memory trace is dependent on the order in which the CS and US are presented, like behavioral conditioning. Furthermore, the increased calcium influx after forward conditioning was also detectable at 60 min after conditioning but not at 15 or 120 min after forward conditioning. However, the variability of the postconditioning responses at 120 min was larger than at other time points, probably because the physiological state of the flies becomes compromised and more variable from the prolonged immobilization. Thus, the DPM neuron memory trace, detectable first at 30 min postconditioning, extends to at least 1 hr and perhaps 2 hr after conditioning. Moreover, the delayed memory trace registered by increased calcium influx into the DPM neuron processes at 30 min after conditioning was also registered as increased synaptic transmission using spH as a reporter. Therefore, odor evokes both increased calcium influx and increased synaptic transmission from DPM neurons 30 min after forward conditioning (Yu, 2005).

Two general models were considered for the role of the amn gene and the DPM neurons in the process of olfactory memory formation in Drosophila. The first model, the possibility that the amn gene and the DPM neurons provide solely US information for the process of acquisition, is unlikely for several different reasons. (1) amn mutants have normal levels of memory acquisition, shown by memory growth curves with multiple training trials relative to control flies. Impairment in the processing of the US information would likely cause the mutant flies to exhibit performance scores that reach asymptote at levels lower than controls. (2) The parallel nature of the memory growth curves also suggests that the processing of CS information is unimpaired since CS impairment should slow the memory growth rate relative to control flies. (3) These data suggest that the association process itself, or acquisition, is unimpaired since a defect in the association of the CS with the US would also alter the memory growth curve. (4) The discovery of a delayed olfactory memory trace within the DPM neurons themselves, unless fortuitous, is inconsistent with a role specific to US processing. Rather, the data are strongly consistent with a second model alternative envisioning amn and DPM neuron involvement in the formation of intermediate-term memory. The amn mutants exhibit no obvious deficit in acquisition but are impaired in memory. Synaptic transmission is required from the DPM neurons during the interval between training and testing but not at the time of training or testing. The latter observation indicates either that DPM neurons are chronically active or that acquisition itself leads to sustained DPM neuron activity since blocking synaptic activity after acquisition produces a memory impairment at 3 hr. The delayed memory trace formed in DPM neurons that is coincident in time with their requirement for normal memory formation argues for their involvement in an intermediate stage of memory (Yu, 2005).

The delayed olfactory memory trace that forms in the DPM neurons is different from previously observed projection neuron memory trace in several interesting ways. (1) The memory trace formed by antennal-lobe projection neurons occurs by the recruitment of new synaptic activity into the representation of the learned odor. In other words, there is a qualitative change in the brain's representation of the learned odor as represented by projection neuron activity. The memory trace formed by DPM neurons, in contrast, is a quantitative one, being manifest as an increase in calcium influx and synaptic release with CS stimulation after acquisition. Despite this, the trace formed in the DPM neurons is odor specific. (2) The memory trace formed by projection neurons is detectable very early (as little as 3 min) after training, whereas the memory trace formed by DPM neurons is delayed, forming between 15 and 30 min after training. (3) The memory trace formed by projection neurons is very short lived, existing for about 5 min after training. The memory trace established in DPM neurons persists for at least 2 hr after training. The existence of multiple memory traces in distinct areas of the olfactory nervous system with different times of formation and duration leads to the interesting hypothesis that memory of a singular event over time is due to multiple and distinct memory traces that guide behavior during different windows of time after learning, a conclusion also reached from studies with the honeybee (Menzel, 2001; Yu, 2005).

These observations show that the delayed olfactory memory trace is established in the DPM neuron branch that innervates the vertical mushroom body lobes and not in the branch that innervates the horizontal mushroom body lobes. Thus, there exists an intriguing branch specificity to the formation of the delayed olfactory memory trace. The significance of this observation is not yet clear. However, other studies have pointed to the possibility that mushroom body neurons have branch-specific information processing. Some flies mutant for the α lobes absent (ala) gene lack the vertical branch or the horizontal branch of the mushroom body neurons. Intriguingly, mutant animals missing only the vertical branch of the mushroom body neurons have been reported to exhibit normal short-term memory but no long-term memory. Thus, long-term memory may form only in the vertical branch of the mushroom body neurons or be retrieved specifically from this branch. The formation of a delayed olfactory memory trace in the DPM neuron branch that innervates the vertical mushroom body lobes is consistent with the possibility that branch-specific long-term memory processes occurring in the vertical branch of the mushroom body lobes are dependent on the delayed memory trace that forms in this DPM neuron branch (Yu, 2005).

The delayed memory trace that forms in the DPM neurons is dependent on the normal function of the amn gene product since the trace fails to form in amn mutants but can be rescued by expression of the wild-type amn gene in the DPM neurons. This observation raises at least three possibilities for the role of the amn-encoded neuropeptides in the formation of the delayed olfactory memory trace. (1) It is possible that the released neuropeptides exert their effects in an autocrine fashion, interacting with neuropeptide receptors on the DPM neurons themselves in order to initiate the formation of the memory trace. (2) It is also possible that the released neuropeptides interact with receptors on postsynaptic neurons, such as mushroom body neurons, and that this stimulates a retrograde signal that leads to the formation of the DPM neuron memory trace. (3) It is possible that the amn-encoded neuropeptides are not employed for physiological changes in the adult brain but are required in a developmental capacity for DPM neurons to be competent to form the memory trace (Yu, 2005).

There exist at least two broad explanations for the role of the DPM neurons and the amn-encoded neuropeptides in olfactory learning. One possibility is that the DPM neurons integrate CS and US information independently of integration events that occur elsewhere in the nervous system. In this scenario, the CS information may be transmitted to the DPM neurons via unknown interneurons from the antennal lobe or lateral horn, or, alternatively, the DPM neurons might receive CS information from the mushroom body axons. In other words, DPM neurons may be postsynaptic to the mushroom body neurons. This could explain why the DPM neurons are odor generalists since their broad innervation of the mushroom body lobes would allow them to sample the odorant-stimulated activity of many or all mushroom body neurons. This possibility predicts that the DPM neurons should exhibit postsynaptic specializations on some of their processes -- perhaps those that innervate the horizontal lobes, as one possibility. The strengthening of specific mushroom body-DPM neuron synapses after olfactory learning could explain how the DPM neurons form odor-specific memory traces despite being odor generalists. Other DPM neuron processes may be presynaptic to the mushroom bodies such that the CS/US integration events that occur within the DPM neurons might be passed on to the mushroom bodies to reinforce their output. The presynaptic interactions may be through synapses onto the mushroom body fibers in the vertical lobes, reinforcing mushroom body output over the intermediate term and perhaps establishing the permissive signaling events for long-term memories to form in the vertical lobes. The DPM neurons may also receive US information indirectly from the mushroom body neurons or from other neurons. The contributions of the two putative DPM neuron neurotransmitters -- acetylcholine and neuropeptides -- to these processes remain to be clarified. Both acetylcholine and amn neuropeptides are required for behavioral memory (from experiments with Shibire and amn mutants, respectively). The amn neuropeptides are also required autonomously for the formation of the DPM neuron memory trace (Yu, 2005).

The second broad explanation envisions the DPM neurons as maintaining already integrated information through a networked association with the mushroom bodies. The complete integration of CS and US information may occur in the projection neurons and mushroom body neurons. DPM neurons, in a postsynaptic role to the mushroom bodies, would receive integrated information leading to increased excitability. The transfer of the CS/US-integrated information from the mushroom bodies to the DPM neurons may occur immediately after learning, initiating a process intrinsic to the DPM neurons that produces a delayed increase in odor-evoked transmission 30 min later, or the transfer of the integrated information itself from the mushroom bodies to the DPM neurons may occur through a delayed process after learning. In either case, the increased excitability of the DPM neurons would feed back onto and strengthen the output of the mushroom body neurons, leading to robust intermediate-term memory (Yu, 2005).

Sequential use of mushroom body neuron subsets during Drosophila odor memory processing

Drosophila mushroom bodies (MB) are bilaterally symmetric multilobed brain structures required for olfactory memory. Previous studies suggested that neurotransmission from MB neurons is only required for memory retrieval. An unexpected observation that Dorsal Paired Medial (DPM) neurons, which project only to MB neurons, are required during memory storage but not during acquisition or retrieval, led to a revisiting of the role of MB neurons in memory processing. Neurotransmission from the α′β′ subset of MB neurons is required to acquire and stabilize aversive and appetitive odor memory, but is dispensable during memory retrieval. In contrast, neurotransmission from MB αβ neurons is only required for memory retrieval. These data suggest a dynamic requirement for the different subsets of MB neurons in memory and are consistent with the notion that recurrent activity in an MB α′β′ neuron-DPM neuron loop is required to stabilize memories formed in the MB αβ neurons (Krashes, 2007).

It is often said that form follows function. According to this postulate, the striking multilobed arrangement of the insect MBs would imply functional differences between the different types of MB neurons: αβ, α′β′, and γ, but very limited data describing the individual function of these anatomical subdivisions exists. Although several complex behaviors in insects appear to require the MBs and a differential role for distinct MB neuron groups has been suggested, most conceptual models of memory treat the MBs as a single unit (Krashes, 2007).

One of the most detailed examinations of MB function has been in the context of Drosophila aversive olfactory memory, where flies are trained to associate specific odors with the negative reinforcement of electric shock. Genetic studies over the last thirty years have suggested that the MBs play an essential role in fly olfactory memory, but the role of the MBs in memory acquisition, storage, and retrieval has only been examined recently. Taking advantage of a dominant, temperature-sensitive dynamin transgene, uas-shi^ts1, a number of laboratories concluded that MB output was required only for recall, but not for acquisition or storage. These and other findings have led to a simple model wherein Drosophila olfactory memory is formed and “stored” at MB output synapses (Krashes, 2007).

Functional studies of DPM neurons, MB extrinsic neurons that ramify throughout the MB lobes, demonstrated they were specifically required during consolidation, but not acquisition or storage. Furthermore, genetically modified DPM neurons that primarily innervate the MB α′β′ lobes retain function, implying that MB α′β′ neurons might also have a similar function in memory consolidation (Krashes, 2007).

Examination of the GAL4 enhancer-trap lines used to express the uas-shi^ts1 transgene in the earlier MB studies revealed that c309, c747, and MB247 only express in a few MB α′β′ neurons compared to αβ and γ neurons, while c739 expresses exclusively in αβ neurons. Thus, it seems likely that prior studies utilizing these drivers did not observe requirements for MB activity during either olfactory memory acquisition or storage because of insufficient expression in α′β′ neurons (Krashes, 2007).

Subsequently two GAL4 enhancer-traps that strongly express in MB α′β′ neurons were identified to test this hypothesis. The expression of c305a appears to be entirely restricted to α′β′ neurons within the MBs whereas c320 expresses in α′β′, αβ, and a few γ neurons. Both of these lines also express in additional non-MB neurons, so MB{GAL80} tool was employed to more rigorously test the requirement for MB activity in these {GAL4} lines. With these reagents the role of MB α′β′ neurons in memory was investigated and it was found that MB α′β′ neuron output during and after training is critical for the formation and consolidation of both appetitive and aversive odor memory from a labile to a more stable state. For comparison the requirements were also examined for MB αβ neurons using c739, confirming previous results. Thus, output from the MB α′β′ neuron subset is required for memory acquisition and stabilization, whereas output from αβ neurons is apparently dispensable during training and consolidation but is required for memory retrieval (Krashes, 2007).

Based on c305a and c739 data, it is recognized that c320 flies, which express in both α′β′ and αβ neurons, might be expected to exhibit memory loss if MB neuron output was blocked during both the consolidation and recall time windows. However, it is possible that a retrieval effect was not observed with c320 because it expresses GAL4 in fewer αβ neurons, or is in a different subset of αβ neurons relative to the c739 driver (Krashes, 2007).

Despite this caveat, these data suggest that different lobes of the MB have different roles in memory and provide a significant shift in the current understanding of the role of the MB in memory. Older models implied that MB αβ, α′β′, and γ neurons were largely interchangeable, and that each of the MB neurons that responded to a particular odor received coincident CS and US input and modified their presynaptic terminals to encode the memory. The data presented in this study suggest that MB αβ and α′β′ neurons are functionally distinct (Krashes, 2007).

In this study, the role of the unbranched γ lobe neurons was not investigated. Previous work with c309, c747, and MB247 suggests that neurotransmission from γ neurons is likely dispensable for acquisition and consolidation. In addition, a prior study indicated that γ neurons are minimally involved in middle-term memory (MTM) and anesthesia-resistant memory (ARM). However, it is possible that experiments to date have not employed odors that require γ neuron activation. The response of γ neurons may be tailored to ethologically relevant odors such as pheromones. It is notable that fruitless, a transcription factor required for male courtship behavior, is expressed in MB γ neurons, and blocking expression of the male-specific fru^M transcript in the MB γ neurons impairs courtship conditioning. If the relevant odors can be identified, it will be interesting to determine if MB α′β′ neurons and Dorsal Paired Medial (DPM) neurons are required to stabilize these odor memories in the γ neurons. Recent work is supportive of the idea that odor identity may be a factor in determining the requirement for the subsets of MB neurons in olfactory learning (Krashes, 2007).

Stable aversive and appetitive odor memory requires prolonged DPM neuron output during the first hour after training, and DPM neuron output is dispensable during training and retrieval. DPM neurons ramify throughout the MB lobes, but DPM neurons that have been engineered to project mostly to the MB α′β′ lobes retain wild-type capacity to consolidate both aversive and appetitive odor memory. This study has demonstrated that, similar to wild-type DPM neurons, blocking output from these modified DPM neurons for 1 hr after training abolishes memory. Thus, finding a specific role for both DPM neuron output to MB α′β′ lobes and MB α′β′ neuron output during the first hour after training is consistent with the notion that a direct DPM-MB α′β′ neuron synaptic connection is important for memory stability. It should be reiterated that the focus of this paper has been on protein synthesis-independent memory, and whether or not a similar processing circuit is utilized for protein synthesis-dependent LTM remains an open question (Krashes, 2007).

Beyond simply attributing an additional function to the MBs, when taken in conjunction with work on the role of DPM neurons in memory, the data presented here suggest a new model for how olfactory memories are processed within the MBs. It is proposed that olfactory information received from the second-order projection neurons (PNs) is first processed in parallel by the MB αβ and α′β′ neurons during acquisition. Activity in α′β′ neurons establishes a recurrent α′β′ neuron-DPM neuron loop that is necessary for consolidation of memory in αβ neurons, and subsequently, memories are stored in αβ neurons, whose activity is required during recall. It is plausible that MB α′β′ neurons are directly connected to MB αβ neurons and/or that DPM neurons provide the conduit between MB neurons. However, the finding that DPM neurons that project primarily to MB α′β′ neurons are functional implies that only a few connections from DPM neurons to MB αβ neurons are necessary (Krashes, 2007).

The requirement for α′β′ neuron output during training also potentially provides a source for the activity that drives DPM neurons. DPM neuron activity is not required during training, and the current data are consistent with the idea that olfactory conditioning triggers activity in MB α′β′ neurons, which in turn elicits DPM neuron-dependent activity. It is proposed that after training, recurrent MB α′β′ neuron-DPM neuron activity is self-sustaining for 60-90 min. This recurrent network mechanism is similar to models for working memory in mammals. It is also conceivable that MB α′β′ neurons receive prolonged input after training from the antenna lobes via the PNs. Olfactory conditioning has been reported to alter the odor response of Drosophila PNs in the AL, but the observed effects were short-lived. Nevertheless, AL plasticity for a few minutes after training could contribute to the required MB α′β′ neuron activity. If continued activity from the AL is required for consolidation, blocking PN transmission with shi^ts1 for 1 hr after training should abolish memory. The bee AL and MB are clearly involved in olfactory memory and may function somewhat independently in learning and memory consolidation, respectively. However, biochemical manipulation of the bee AL can also induce LTM, and therefore it is possible that either plasticity in the AL alone can support LTM, or that the AL and MB interact during acquisition and consolidation. A differential role for the AL and MBs has also been suggested from neuronal ablation studies of courtship conditioning in Drosophila. Short-term courtship memory can be supported by the AL, but memory lasting longer than 30 min requires the MBs (Krashes, 2007).

This work also has significant implications for the organization of aversive and appetitive odor memories in the fly brain. Stability of both appetitive and aversive memory is dependent on DPM neurons and MB α′β′ neurons. It therefore appears that processing of aversive and appetitive odor memories may bottleneck in the MBs. It has been demonstrated that aversive memory formation requires dopaminergic neurons whereas appetitive memory relies on octopamine to provide a possible mechanism to distinguish valence (see Tyramine β hydroxylase). However, it was also found that MB output is required to retrieve aversive and appetitive odor memory, suggesting that both forms of memory involve MB neurons and that both US pathways may converge on MB neurons. It will be important to understand how the common circuitry is organized to independently process the different types of memory and to establish if, and how, such memories coexist (Krashes, 2007).

These data imply that stable memory may reside in MB αβ neurons because blocking output from MB αβ neurons impairs retrieval of MTM and ARM (both components of 3 hr memory). It has been proposed that AMN peptide(s) released from DPM neurons cause prolonged cAMP synthesis in MB neurons that is required to stabilize memory. The finding that genetically engineered DPM neurons mostly projecting to the MB α′β′ lobes are functional, taken with the idea that stable memory resides in MB αβ neurons, is somewhat inconsistent with the notion that crucial AMN-dependent memory processes occur in MB αβ neurons. However, it is plausible that AMN, or another DPM product that is released in a shibire-dependent manner, could diffuse locally from the aberrant DPM neurons to MB αβ neurons (Krashes, 2007).

This work demonstrates that MB αβ neurons and α′β′ neurons have different roles in memory. Beyond gross structural and gene expression differences, it will be essential to establish the precise connectivity, relative excitability, and odor responses of the different MB neurons. Future study may also reveal further functional subdivision within the MB lobes, and it should be possible to refine the current MB α′β′ neuron GAL4 lines with appropriate GAL80 transgenes and FLP-out technology (Krashes, 2007).

In the mammalian brain memories that initially depend on the function of the hippocampus lose this dependence when they are consolidated. This transient involvement of the hippocampus has led to the idea that consolidation of memory results in the transfer of memory from the hippocampal circuits to the cortex. An alternate view is that aspects of the memory are always in the cortex but are dependent on the hippocampus because recurrent activity from cortex to hippocampus to cortex is required for consolidation. Hence, disrupting hippocampal activity during consolidation leads to memory loss (Krashes, 2007).

The current data suggest the simpler fruit fly brain similarly employs parallel and sequential use of different regions to process memory. MB α′β′ neuron activity is required to form memory, MB α′β′ neurons and DPM neurons are transiently required to consolidate memory, and output from αβ neurons is exclusively required to retrieve memory. It is therefore propose that aversive and appetitive odor memories are formed in MB αβ neurons and are stabilized there by recurrent activity involving MB α′β′, DPM neurons, and the MB αβ neurons themselves (Krashes, 2007).

It is becoming increasingly apparent that neural circuit analysis will play an important role in understanding how the brain encodes memory. The ease and sophistication with which one can manipulate circuit function in Drosophila, combined with the relative simplicity of insect brain anatomy, should ensure that the fruit fly will make significant contributions to this emerging discipline (Krashes, 2007).

Mapping olfactory representation in the Drosophila mushroom body

The first olfactory relay in the brain contains a spatial map. Olfactory receptor neurons (ORNs) expressing a specific odorant receptor (and therefore having precisely defined olfactory tuning properties) send axon projections to discrete and reproducibly positioned glomeruli in the vertebrate olfactory bulb or insect antennal lobe. In Drosophila, most ORN classes express one specific odorant receptor and send axons to one of ~50 glomerular targets (Jefferis, 2007 and references therein).

Persistent spatial organization deep within the brain is a motif in many sensory systems. For example, adjacent regions of the somatosensory cortex respond to stimuli from neighboring body parts. Does the spatial organization evident in the first olfactory relay also persist at deeper levels? In flies, the branching patterns have been described of the axons of second order projection neurons (PNs, equivalent to vertebrate mitral cells) in higher olfactory centers: the mushroom body (MB) and lateral horn (LH) of the protocerebrum. In the LH axon branching patterns of PNs of the same glomerular class were highly stereotyped across animals, while such stereotypy was less evident in the MB. Several putative output neurons of the LH have been described. Understanding how these neurons integrate olfactory information is a key problem in the neural basis of olfactory perception. In mice, the existence of some spatial organization in higher olfactory centers has been reported by following the targets of 2 of the 1000 ORN classes to the olfactory cortex. The integrative properties of olfactory cortical neurons have also been studied. However, the anatomical basis of this integration remains challenging because of the numerical complexity of the rodent olfactory system (Jefferis, 2007 and references therein).

Neuroanatomy is the foundation of both developmental and functional studies of the brain. In order to understand the development of neuronal wiring, it is necessary to describe the degree of wiring precision across individuals. Similarly, high-resolution neuroanatomy makes predictions about information transfer and transformation, constraining models of neural processing. Two anatomical approaches have been particularly influential in constructing wiring diagrams. The first is exemplified by the classic work of Cajal using the Golgi method. A small fraction of the neurons within a piece of tissue are stained to reveal their dendritic and axonal projection patterns; the information from many specimens is compared and integrated to give a global picture of the circuit. While this approach was enormously successful in defining the basic logic of connectivity, it lacks comprehensiveness and precision: comprehensiveness because only a small fraction of the neuronal elements are used to construct the global picture; precision because integrating information across sample brains has allowed only qualitative comparisons. The second method is a complete reconstruction of all the connections in a small number of specimens through serial electron microscopy. While new EM technologies are under development, traditional serial section transmission EM approaches are so labor intensive that this has only been achieved once - the reconstruction of the nervous system of C. elegans hermaphrodites (Jefferis, 2007 and references therein).

This study describes an approach that has merits of both methods. By combining genetic single-cell labeling with state-of-the-art image registration techniques, comprehensive maps have been produced of the LH and MB, the two higher olfactory centers of Drosophila. Projections of individual neuronal classes with their neighbors can be visualized and directly compared. These three dimensional maps directly demonstrate the spatial stereotypy of input to the LH and MB. Probabilistic synaptic density maps have been devised and used to identify and quantify the organizational principles of these two centers. It was found, for example, that fruit odors and pheromones are represented in distinct compartments of the LH. Finally, postsynaptic neurons of the LH have been characterized at the single-cell level and the density maps have been used to predict connectivity with input PNs. All the raw and derived data and the necessary software tools are available on the project website, providing a resource that will be integrated with future anatomical, physiological and behavioral data to understand the neural basis of olfactory perception in Drosophila (Jefferis, 2007).

Previous studies have revealed aspects of the spatial organization of higher olfactory centers - the MB calyx and the LH. Of particular relevance to the principles of olfactory information processing, single PNs of different classes have highly stereotyped LH projections. Using five Gal4 enhancer trap lines each labeling 1-3 PN classes, it has been found that PNs from 9 glomeruli project to 3 corresponding zones in the MB calyx and LH; MB output neurons integrate information from each of these zones whereas 6 groups of putative LH output neurons maintain the segregation of these 3 zones (Jefferis, 2007).

This study contains several advances over previous approaches: (1) the projection patterns of 11 new PN classes are described at single-cell resolution, qualitatively extending previous results; (2) all single neuron tracings were digitized, and transformed onto a common reference brain; (3) at the single neuron level, the distribution was determined of PN presynaptic terminals in the MB and LH. Fourth and most importantly, combining the above information allowed generation of quantitative synaptic density maps for 35 PN classes, representing 32 of ~50 unique olfactory channels defined by the projection of ORN classes to antennal lobe glomeruli. This allowed decomposition of MB and LH input into individual channels and then the reassemblage for most of the olfactory system, providing a global view of these higher order centers. Lastly, projection patterns are described of three groups of LHNs at single-cell resolution, and predictions are made about their physiological properties based on their potential connectivity with specific PN classes (Jefferis, 2007).

The concentric zonal organization of PN input into the MB calyx was quantitatively confirmed. However, LH organization is more complex and cannot simply be described as zonal, with the exception of the segregation of pheromone projections from the rest of the channels. This is evident from the single neuron projections of many classes that send stereotyped and divergent branches to multiple areas of the LH, as well as the synaptic density maps. Together with the extensive branching of individual LHNs, characterizing the LH as providing relatively little integration across glomeruli is now considered inaccurate (Jefferis, 2007).

Comparing PN branching patterns in the LH and MB suggests that the LH is likely to support more stereotyped integration. This proposal is consistent with the view that the LH mediates innate olfactory behaviors while the MB participates in odor-mediated learning. However, a clear stereotypy of PN terminals in the MB calyx has now been demonstrated. This is likely to explain observations that certain odors can evoke spatially stereotyped activity in MB neurons. Thus the MB calyx and LH receive different levels of stereotyped input that can be integrated by third order coincidence detectors that combine information from different input channels (Jefferis, 2007).

The most striking biological insight obtained from this study is the segregation in the LH between putative pheromone representing PNs and almost all other PNs in the apparently homogeneous LH neuropil. Interestingly, the highest degree of LH volumetric sexual dimorphism that was quantified coincides with the presynaptic terminals of the GABAergic vVA1lm and vDA1 PNs. It is important to note that in addition to the PNs that express the GAL4 driver GH146 that were characterized in this study, there may be other PNs that relay pheromone information from VA1lm and DA1 glomeruli to higher brain centers and contribute to the sexual dimorphism that was found in the LH (Jefferis, 2007).

The convergence of excitatory and inhibitory projections from these putative pheromone representing glomeruli at overlapping or adjacent locations may allow postsynaptic neurons to respond to the presence of a signal that activates these two glomeruli in a particular ratio or to allow signals from these two glomeruli to have opposing effects on LH neurons that initiate particular behaviors. Behaviorally, male flies appear to integrate information both from attractive and inhibitory pheromones produced by other males. Furthermore, new data show that Fru+ Or67d ORNs innervating the DA1 glomerulus detect a male sex pheromone that has a negative effect on other males and a positive effect on females. It is speculated that balanced excitation and inhibition in these pathways may regulate LHNs that contribute to the appropriate behavioral alternative. Sex-specific integration in the lateral horn may underlie sex-specific behaviors (Jefferis, 2007).

The spatial segregation of pheromone representation contrasts with the representation of glomeruli that receive input from ORNs of the basiconic sensilla, which are generally activated by fruit odorants. Many of these PN classes have extensive overlap in their LH synaptic density maps. This property, coupled with the fact that many fruit odorants activate multiple classes of basiconic ORNs, makes the representations of different fruit odorants and natural fruit odors quite overlapping. These data thus support the following principles: olfactory information concerning food has extensive structural intermixing at the LH compared to the glomerular organization of the antennal lobe, but rather discrete channels are retained for pheromones all the way from the sensory periphery to the LH. It is proposed that the LH is globally organized according to biological values rather than chemical nature of the odorant information (Jefferis, 2007).

This finding is reminiscent of the male silkworm moth, Bombyx mori, where PNs from the macroglomeruli representing sex pheromones send axon projections to a discrete area in the lateral protocerebrum defined by a high level of anti-cGMP staining. Spatial segregation of the pheromone representation in higher olfactory centers may therefore be a conserved feature in insects. This segregation is exaggerated into two entirely separate pathways in mammals, where the nasal epithelium and main olfactory bulb process general odorants and some pheromones, while the vomeronasal organ and accessory olfactory bulb are more specific to pheromone sensation. Furthermore, mitral cells originating from the main and accessory olfactory bulbs project to distinct areas of the cortex (Jefferis, 2007).

Having generated a comprehensive and quantitative map of PN input to the LH, a future challenge is to identify and characterize third order LHNs: where are their dendritic fields in the LH, with which PNs do they form synapses, where do they send their axonal outputs, and what are their physiological properties and functions in olfactory behavior? This effort has been started by identification of Gal4 lines labeling neurons with projections in the vicinity of the LH. Three groups of LHN were characterized at single-cell resolution and their potential connectivity with different PN classes was predicted. However this is clearly only a beginning. The widespread distribution of LHN cell bodies and their potential output to different parts of the brain along with the difficulty of identifying large groups of LHNs labeled by new Gal4 enhancer traps suggest that LHNs are heterogeneous genetically, anatomically and, in all likelihood, functionally. One tractable avenue will be to find LHNs that send dendrites to DA1/VA1lm PN target areas and may therefore respond to pheromones and instruct mating behavior. Two LHN groups that were characterized project to this LH region, and single-cell and potential synapse analyses indicate that some of these LHNs may form strong connections with pheromone responsive PN channels. Further characterization of these and other LHNs will bring an understanding the neural circuit basis of olfactory perception and behavior (Jefferis, 2007).

Neural coding for olfactory sensory stimuli has been mapped near completion in the Drosophila first-order center, but little is known in the higher brain centers. This study reports that the antenna lobe (AL) spatial map is transformed further in the calyx of the mushroom body (MB), an essential olfactory associated learning center, by stereotypic connections with projection neurons (PNs). Kenyon cell (KC) dendrites are segregated into 17 complementary domains according to their neuroblast clonal origins and birth orders. Aligning the PN axonal map with the KC dendritic map and ultrastructural observation suggest a positional ordering such that inputs from the different AL glomeruli have distinct representations in the MB calyx, and these representations might synapse on functionally distinct KCs. These data suggest that olfactory coding at the AL is decoded in the MB and then transferred via distinct lobes to separate higher brain centers (Lin, 2007).

The greatest challenge facing the field of sensory biology at present is to address how sensory coding is represented from the first-order center to the higher brain centers, where neuronal activity must be computed to elicit appropriate behavior responses. This study reports a spatial map of olfactory representations in the MBs of adult Drosophila brains. It was shown that KC dendrites are segregated into 17 complementary domains defined by both clonal origins and birth orders. When viewed from the posterior, PN axonal termini of DL3/D/DA1/VA1d, of DM2, and of DM1/VA4 form three concentric zones corresponding to KC dendrites from early α/β, late α/β, and α'/β' neurons in the posterior calyx, respectively. The spatial organization of PN-to-KC connectivity suggests that olfactory coding in the AL is maintained in the MB calyx where signal processing is more versatile (Lin, 2007).

One question that now can be addressed is, are odorants carrying similar biological information processed by the same class of KCs? A chemotopic map of OR responses to 110 odorants indicates that excitatory and inhibitory responses are chemical-class dependent. By integrating the PN-to-KC map with existing electrophysiological data, it was observe that KC responses to chemicals are likely also class specific. For example, many aromatic odorants are excitatory to Or10a and many terpenes are inhibitory to Or49b, which connects via DL1 and VA5, respectively, to γ neurons. Since MBs are essential for odor discrimination, it is predicted that a fly will find it more difficult to discriminate two odorants that are relayed by the PNs to the same class(es) of KCs. It has been observed that odors of a particular chemical class are often clustered, as shown by the 'odor space' constructed from the response of 24 ORs to 110 odorants. It was shown that three odorants in different chemical classes mapped to three distinct points in this space: pentyl acetate (an ester) and 2-hepatanone (a ketone) elicited similar patterns of activation maps together, distant and different from that elicited by methyl salicylate (an aromatic compound). Consistently in the anatomical study, it was found that pentyl acetate- and 2-hepatanone-induced signals are likely processed by the same class of KCs (i.e., late α/β neurons), while those induced by methyl salicylate reach other classes of KCs, excluding late α/β neurons. It is noted that this is a working model of odor discrimination, since it does not yet include all PN-to-KC connections in the adult olfactory system and only six ORs have yet been linked with the PN-to-KC map. Additional functional subsets of KCs are expected in each of the five classes. While a more complete PN-to-KC map is needed, the model of odor space versus KC class provides an important advance in the understanding of neural computation underlying behavioral responses to odors (Lin, 2007).

The findings are in congruence with functional imaging studies, which indicate that odor-evoked activity occurs in specific regions in the calyx. As odor concentration increases, more glomeruli are activated in the AL and more KCs are activated in the calyx. The anatomical data suggest that perception of odor identity may require integration among five classes of KCs, while the number of responsive KCs may reflect the perception of odor intensity (Lin, 2007).

Confocal imaging of specific GAL4-driven reporter expression patterns reveals axonal segregation for each of the five KC classes. This topology implies that stereotyped olfactory representation in the AL glomeruli received from OSNs (first order) are further relayed by PNs (second order) to a fixed combination of five KC classes (third order) in the calyx; such an implication is supported by findings on connectivity. It is surmised that information processing is further achieved by segregating axon bundles to different MB lobes, where output neurons (fourth order) diverge the processed information to separate higher brain centers (fifth order). Incomplete as this model may be, the possibility is acknowledged of (1) crosstalk among KCs and (2) modulatory innervation of MB calyx and lobes. Because this study was focused only on a subset of PNs, the possibility cannot be ruled out that further functionally important differences in PNs may yet be discerned. Nonetheless, this anatomical model serves to advance the notion that an olfactory map in the MBs helps to guide olfactory-driven behaviors (Lin, 2007).

All animals are born with a set of innate behavioral responses, 'hardwired' in the nervous system. In Drosophila, innate behaviors such as sleep and courtship require proper functioning of the MBs. It is notable that Fruitless, a transcription factor required for male courtship behavior, is expressed in OSNs and PNs innervating the same set of AL glomeruli (VL2a, DA1, and VA1), suggesting interconnections between these two sets of olfactory neurons. Intriguingly, fruitless expresses also in the MBs of the ? and α/β lobes, and courtship conditioning is impaired when expression of the male-specific fru transcript is disrupted in MB γ neurons. In likelihood connectivity assignment, VL2a- and DA1-PNs connect with γ and early α/β lobes. It is possible that the fru-expressing PNs and KCs also are interconnected in the MB calyx, as are the fru-expressing OSNs and PNs in the AL. These data suggest that stereotyped connectivity in the PN-to-KC map is likely involved in fru-expressing circuits, which are essential for proper behavioral responses to volatile sex pheromones (Lin, 2007).

A central question in olfaction is how the brain discriminates different odors to elicit an appropriate behavioral response. Stereotypic connectivity maps of odorant-to-OR, OSN-to-PN, and PN-to-KC at three consecutive levels allow further construction of a neural computation of odor discrimination in the adult Drosophila brain. Stereotypic PN-to-KC connectivity and functional imaging suggest differential representation of the odors in the AL is maintained in the MB calyx and possibly further processed in the different MB neurons/lobes. If so, how does the same class of KCs discriminate odorants carrying different biological information, such as a sex pheromone and an aggregation pheromone? A single class of KCs might be sufficient to discriminate two different odors in some cases, since Drosophila larvae can discriminate different odors with only γ neurons. Thus, additional spatial and/or temporal complexity for neural computation must exist among KCs of the same birth-order class. Consistent with this notion, the data show that PNs connecting with the same class of KCs may have different projecting patterns among K1-K5 dendritic divisions, suggesting differential functions for each of the four KC clones. Even with the same developmental history of clonal origin and birth order, KCs are likely divided into different identities further based on differential gene expression. For example, Gal4 line G0050 labels the entire α'/β' lobe but c305a labels only the frontal-half α'/β' lobe. Even with such developmental specification, odor discrimination also may require additional integration among different classes of KCs (Lin, 2007).

Although stereotypic connectivity maps from ORNs to PNs to KCs give the impression of a straight and simple path, olfactory coding clearly will be modulated by both stimulatory and inhibitory signals as it makes its way through the brain. A single ORN can exhibit both excitatory and inhibitory responses to different odorants. In the ALs, odor responses of the PNs are reshaped by inhibition from local neurons. In the MBs, KCs may receive both stimulatory and inhibitory stimuli from PNs, since most of them are cholinergic but some of them are GABAergic. Immunohistochemical labeling and GFP expression patterns in Cha-GAL4 and GAD-GAL4 lines indicate that KCs are also composed of both cholinergic and GABAergic neurons. The distribution of odor responses across different classes of KCs and the imposition of odor-sensitive excitatory and inhibitory responses both appear to enhance distinct neural representations of different odors. Such complexity of odor representations greatly reduces the possibility of overlap between spatiotemporal patterns elicited by two different odorants, making them easier to discriminate or to memorize and recall (Lin, 2007).

In conclusion, the data offer specific and testable hypotheses that olfactory coding at the ALs is likely further represented and decoded in the MBs and then transferred via distinct lobes to separate higher brain centers. It would be important now to complete the PN-to-KC map, to identify further subclasses within each of the five KC classes, and to answer how different classes of KCs communicate with each other during olfactory neural computation (Lin, 2007).

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).

Hebbian STDP in mushroom bodies facilitates the synchronous flow of olfactory information in locusts

Odour representations in insects undergo progressive transformations and decorrelation from the receptor array to the presumed site of odour learning, the mushroom body. There, odours are represented by sparse assemblies of Kenyon cells in a large population. Using intracellular recordings in vivo, this study examined transmission and plasticity at the synapse made by Kenyon cells onto downstream targets in locusts. It was found that these individual synapses are excitatory and undergo hebbian spike-timing dependent plasticity (STDP) on a +-25 ms timescale. STDP is a phenomenon in which the gain of a connection can be changed according to the temporal relationship between pre- and post-synaptic spikes. When placed in the context of odour-evoked Kenyon cell activity (a 20-Hz oscillatory population discharge), this form of STDP enhances the synchronization of the Kenyon cells' targets and thus helps preserve the propagation of the odour-specific codes through the olfactory system (Cassenaer, 2007).

Olfactory processing in insects begins in an array of receptor neurons that express collectively many tens of olfactory receptor genes (~60 in Drosophila; ~150 in honeybees). The representations of general odours are then decorrelated by local circuits of projection neurons and local neurons in the antennal lobe. In locusts and other insects, the antennal lobe output is distributed in space and time and can be described as stimulus-specific time-series of projection-neuron activity vectors, updated at each cycle of a 20-Hz collective oscillation. Distributed projection-neuron activity is then projected to Kenyon cells, the intrinsic neurons of the mushroom body. In contrast to projection neurons, Kenyon cells respond very specifically and fire extremely rarely. The mechanisms underlying this sparsening are starting to be understood. Such sparse representations are advantageous for memory and recall, consistent with established roles of the mushroom bodies in learning. In Drosophila, experiments combining molecular inactivation with behaviour indicate that synaptic output from Kenyon cells in the lobes is required for memory retrieval. Little is known, however, about the electrophysiological properties of these synapses (Cassenaer, 2007).

The connections made by Kenyon cells onto a small population of extrinsic neurons have been studied in the β-lobe of the locust mushroom body, using an intact, in vivo preparation. β-lobe neurons (β-LNs) respond to odours; their responses are odour-specific and their tuning is sensitive to input synchrony. This study recorded intracellularly from pairs of Kenyon cells and β-LNs: randomly selected Kenyon cells were impaled in their soma; β-LNs were impaled in a dendrite in the β-lobe. Focused was placed on one β-LN anatomical subtype, which comprises many individual neurons. Neurons of this subtype, called β-LNs here, could be recognized also by their physiological characteristics. Each β-LN has extensive dendrites that intersect many of 50,000 Kenyon cell axons. Monosynaptic connections were found in ~2% of tested Kenyon cell (KC)-β-LN pairs. All were excitatory. The delay between Kenyon cell spike and β-LN-excitatory post-synaptic potential (EPSP) onset was 6.5 +- 0.70 ms, including 5.4 +- 0.25 ms for spike propagation from Kenyon cell soma to the β-lobe. The remaining (synaptic) delay (~1 ms) is similar to that at another chemical synapse in the locust brain. Unitary EPSPs were large (1.58 mV +- 1.11), in contrast to those generated in Kenyon cells by individual projection neurons (86 microV +- 44). The fact that Kenyon cell outputs are powerful is consistent with Kenyon cell spikes being rare and therefore highly informative. EPSP amplitude varied greatly across connected pairs (0.55-4 mV). This could reflect a distribution of electrotonic distances between synapses and recording sites. Simultaneous impalements of different dendrites in the same β-LN, however, show that the amplitudes of most events were the same across recording sites. Consistent with this, unitary EPSP kinetics (10-90% rise time, 8.3 ms +- 2.3; time to 1-(1/e) of peak, 13.2 ms +- 4.4) were independent of the β-LN recorded and, thus, of the impalement site. Simultaneous dendritic recordings of different β-LNs, however, revealed that their synaptic backgrounds overlapped only partly. Common EPSPs rarely had the same amplitude. Hence, β-LNs may each receive inputs from hundreds to thousands (~2% of 50,000 Kenyon cells) of Kenyon cells, in overlapping subsets; KC-β-LN connections are strong on average, with target-specific strength (Cassenaer, 2007).

Odour-evoked activity in projection neurons and Kenyon cells consists principally of sequential volleys of synchronized spikes-generally, one spike per responding neuron per oscillation cycle. β-LN responses to odours also consisted typically of sequences of single phase-locked spikes, timed around the trough of several local field potential (LFP) oscillation cycles. The cycles when a spike was produced (usually with probability <1) depended on β-LN and stimulus identity. It is concluded that, to each oscillation cycle corresponds a particular activity vector in the projection neuron, Kenyon cell and β-LN populations. By recording from pairs of β-LNs simultaneously during odour trials, it was also observed that, when the two β-LNs fired one action potential during the same oscillation cycle, those action potentials were tightly synchronized (+-2 ms) (Cassenaer, 2007).

A fortuitous observation provided hints of plasticity at the KC-β-LN synapse. At trial 4 of a Kenyon cell stimulus sequence intended to explore β-LN integration, the β-LN fired a spontaneous action potential roughly at the time of the first (of 2) Kenyon-cell-evoked EPSP. At trial 5, 10 seconds after this single fortuitous pairing, the first EPSP of the pair was greatly enhanced. This suggested the possibility of spike-timing-dependent plasticity (STDP), a phenomenon thus far unknown in invertebrates but well characterized in vertebrates, in which the gain of a connection can be changed according to the temporal relationship between pre- and post-synaptic spikes. The consequence of pre-post temporal relationships was explored on the KC-β-LN synapse. A β-LN was impaled and stimulated alternately by two independent Kenyon cell pathways-one for pairing, one for unpaired control. Each stimulus was repeated every 10 s, with a 5-s delay between pairing and control stimuli. Pairing consisted of a single Kenyon cell (pre) stimulus and a 5-ms supra-threshold β-LN (post) current pulse, timed such that the delay (dt = t_post-t_pre) between pre- and post-synaptic spikes varied between -60 and +50 ms. Test trials, used to measure connection strength before and after pairing, were identical to the pairing trials in all respects except in the temporal relationship between pre- and post-synaptic spike times (2.5 s apart). Two examples (for dt = 10 ms and -4 ms, 25 pairings each) are given. For dt = 10 ms, the paired input underwent potentiation; for dt = -4 ms, it underwent depression. For both conditions, the control pathway (same β-LN, different Kenyon cell input) remained unchanged. The changes were thus input-specific; they were often detectable after a single pairing, and could be maintained for up to 25 min. 26 values of dt between -60 and +50 ms were tested. The resulting changes define a classical hebbian profile: the synapse is potentiated when pre- precedes post-, and depressed when post- precedes pre-, with symmetrical profiles. The changes could be fitted well with two exponential decays flanking a narrow linear range around t = +4 ms. Several connections were tested successively with two (or more) values of dt (some positive, others negative): the same connections could undergo both depression and potentiation, depending on the value of dt. The STDP profile thus seems to be a property of each connection and not only a collective one (Cassenaer, 2007).

It was observed that the values of dt over which synaptic weights change correspond to the period of single odour-evoked oscillation cycles; hence, only within-cycle 'coincidences' may modify the connections between a Kenyon cell and its targets. The features of the STDP curve, when considered together with the timing of Kenyon cells and β-LNs during odour-evoked activity, have interesting consequences. Consider the phases of Kenyon cell and β-LN spikes. Owing to propagation delays, Kenyon cell spikes reach their targets just before the trough of the LFP, a little before β-LN firing. Consider a cycle in which a β-LN spikes early: some KC-β-LN connections will undergo depression; at the next trial, β-LN spike time at this cycle should be delayed. If, in contrast, a β-LN spikes late, STDP should potentiate Kenyon cell drive to it, and thus advance spike time for that cycle. In short, the cycle-by-cycle action of STDP suggests adaptive control of β-LN spike phase. The need for such regulation is not unique to this system: models of cortical networks indicate that, as activity propagates through successive 'layers', accumulating noise can rapidly smear the temporal structure that may exist. Modelling studies predict that STDP, given appropriate parameters, could preserve the temporal discretization of activity through such layers (Cassenaer, 2007).

A reduced model was generated of the KC-β-LN circuit, and the STDP rule derived from these experiments was introduced. To control the relative phases of Kenyon cells and β-LNs, Kenyon cell spike phases were drawn from experiments and input weights from uniform distributions with different means: with low weights, β-LN spikes tended to occur late (dt > 0); with larger weights, they occurred early (dt < 0). After several trials (each with a random draw of inputs from the same distribution), STDP was allowed to modify synaptic weights for the following trials: when β-LN spikes occurred late (dt > 0), Kenyon cell outputs became potentiated and β-LN spikes were advanced; for dt < 0, time shifts were inverted. The histograms shown represent spike-time distributions for 1,000 trials before and after STDP, for each of three conditions. These simulations were repeated 200 times (50 trials each), with 11 different Kenyon cell input distributions. Once STDP was turned on (trial 1), the evolution was systematic and rapid, leading to the adaptive up- or downregulation of input weights, firing phase and response intensity. Given that the model is entirely constrained by experiments, it is noteworthy that the mean phase of the first β-LN spike at steady state, matches precisely that measured experimentally (Cassenaer, 2007).

To test directly the effect of STDP on β-LN output, β-LN spike timing was manipulated during responses to odours in vivo: if the model is correct, such manipulations should change the output of the odour-activated Kenyon cells onto that β-LN and, thus, generate predictable shifts in its spike phase. During odour stimuli, short current pulses locked to selected cycles of the LFP were injected in a β-LN: a negative pulse was injected during the cycles and phase when the β-LN would naturally fire (to prevent stimulus-evoked spikes), and a positive pulse was injected at a desired phase, for those same cycles (that is, at an abnormal time relative to the Kenyon cell inputs that would normally drive the recorded β-LN). An example is shown for four consecutive cycles. After several such pairing trials, current injection was terminated and β-LN-firing phase over the next trials was compared to that before pairing. The effects of one such manipulation (dt > 0) were plotted: as predicted, an artificial phase-delay caused a corrective phase-advance. Twenty distinct experiments were carried out in six β-LNs; the expected phase shifts were observed in 16 of those 20. This is consistent with an adaptive role for STDP in the fine-tuning of β-LN spike-phase, and may explain the tight synchronization of β-LNs. Hence, STDP helps preserve the discrete and periodic structure of olfactory representations as they flow through the mushroom bodies (Cassenaer, 2007).

This study showed that the connections made by Kenyon cells to β-LNs are excitatory, strong on average, variable across pairs, and plastic. Plasticity follows time-sensitive hebbian associativity rules and is constrained to within-cycle interactions between pre-and post-synaptic neurons. STDP is therefore not specific to vertebrates or cortical architectures. The molecular underpinnings of STDP in this system, or whether STDP might confer the associative features usually ascribed to mushroom bodies, are not known. The fly and honeybee genomes both reveal coding sequences for N-methyl-D-aspartate (NMDA) receptor subunits and some Drosophila behavioural results are compatible with STDP learning rules. One hypothesis, readily testable, is that STDP provides associativity by tagging transiently the subset of synapses activated simultaneously by the odour, before the conditional arrival of a slower, non-specific reward signal (Cassenaer, 2007).

The results reinforce the proposed importance of spike timing for this, and possibly other, olfactory system(s): Kenyon cells act as coincidence detectors for synchronized projection neuron input, β-LNs act as coincidence detectors for Kenyon cell input; because STDP helps enhance β-LN synchronization, it is inferred that spike timing must be relevant also for the processing of β-LN output. These results indicate that the oscillation cycle-a temporal unit of processing first defined by negative feedback in the antennal lobe-is actively preserved in at least three successive layers of processing (projection neurons, Kenyon cells and β-LNs). It will be interesting to assess whether all Kenyon cell outputs obey the same STDP rules, and if these rules are themselves subject to learning-related modifications. Indeed, Kenyon cells seem to communicate with one another through axo-axonal chemical synapses. Given the dynamics of projection neuron/Kenyon cell activity vectors in response to odours, the possibility that Kenyon cell-Kenyon cell synapses also undergo STDP suggests a mechanism for sequence learning, similar to principles proposed for spatial map formation in rodents; in this study, however, the learned sequences have no relation to movement in physical space. The existence of such similarities (synaptic learning rules, and synchronized and sequential neural activity patterns) may bring us closer to understanding the relationships between circuit dynamics, architecture and learning in the brain (Cassenaer, 2007).

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date revised: 25 August 2009

Zygotically transcribed genes

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