An olfactory sensory map in the fly brain

Although the size of the receptor gene family is similar in nematodes and mice, the logic of olfactory perception differs in the two organisms. The discrimination of olfactory information requires neural mechanisms capable of distinguishing which of the numerous receptors have been activated by a given odorant. In C. elegans, a family of 1000 receptor genes is expressed in only 16 pairs of sensory cells, and each neuron expresses multiple odorant receptors. Activation of any one of the multiple receptors expressed in one cell will lead to chemoattraction, whereas activation of a receptor in a different cell results in chemorepulsion. Thus, the behavioral response to a specific sensory input is a property of the neuron that is activated and not a function of the chemosensory receptor itself. This organization allows for the recognition of a diverse array of odorous ligands but diminishes the organism's discriminatory power (Vosshall, 2000 and references therein).

A different logic is employed to discriminate odors by the mammalian olfactory system. In mice, each of the two million olfactory receptor neurons expresses only one of a thousand odorant receptor genes. Neurons expressing a given receptor project axons with precision to two of 1800 discrete synaptic structures, the glomeruli, within the olfactory bulb. The pattern of projections is spatially invariant, providing a two-dimensional representation of receptor activation in the brain. The existence of a physical map that segregates the projections of neurons expressing a given receptor (and therefore responsive to given odors) is in accord with both electrophysiological and imaging studies that demonstrate that different odors elicit defined patterns of spatial activity within the olfactory bulb (Vosshall, 2000 and references therein).

How is olfactory information represented in the insect brain? Does the logic of olfactory discrimination in the fruit fly, Drosophila, more closely resemble that of the invertebrate C. elegans or the more complex olfactory system of vertebrates? The anatomy of the insect olfactory system is reminiscent of vertebrates. Olfactory recognition in Drosophila is accomplished by sensory hairs distributed over the surface of the third antennal segment and the maxillary palp. Olfactory neurons within sensory hairs send projections to one of 43 glomeruli within the antennal lobe of the brain. The glomeruli are innervated by dendrites of the projection neurons, the insect equivalent of the mitral cells in the vertebrate olfactory bulb. In turn, these antennal lobe neurons project to the mushroom body and lateral horn of the protocerebrum. 2-deoxyglucose mapping in the fruit fly demonstrates that different odorants elicit defined patterns of glomerular activity, suggesting that in insects, as in vertebrates, a topographic map of odor quality is represented in the antennal lobe. However, in the absence of the genes encoding the receptor molecules, it has not been possible to define a physical basis for this spatial map (Vosshall, 2000 and references therein).

The 'complete' family of Drosophila odorant receptors (DORs) has now been identified and these genes have been employed to visualize the projections of individual neurons to the fly brain. Fifty-seven DOR genes within the Drosophila genome have been described. Individual receptors are expressed in from 2 to 50 olfactory neurons in a conserved pattern that defines a topographic map on the surface of the antenna. Individual neurons are likely to express only one receptor gene. Neurons expressing a given receptor gene project with precision to spatially invariant glomeruli within the antennal lobe. In the fly, as in mammals, a topographic map of receptor activation in the peripheral sense organs is represented in the brain. These spatial patterns may then be decoded in higher sensory centers in the brain to translate stimulus features into meaningful neural information. These data suggest a logic of odor discrimination that has been maintained over the 500 million years of evolution separating insects from mammals, perhaps reflecting an efficient solution to the complex problem of olfactory sensory perception (Vosshall, 2000).

Analysis of the recently completed euchromatic genome sequence of Drosophila using BLAST searches with existing members of the DOR gene family indicates that a total of 57 DOR genes are present in the genome. This family of 57 genes is tentatively defined as the 'complete' complement of DOR genes, recognizing that 60 Mb of heterochromatic DNA remain to be analyzed. Each of the 57 genes encodes a putative seven transmembrane domain protein of ~380 amino acids. The family as a whole is extremely divergent and exhibits from 17% to 26% amino acid identity. However, each of the genes shares short common motifs in fixed positions that define these sequences as highly divergent members of a gene family. Analysis of the sequence of all 57 receptors reveals the existence of discrete subfamilies whose members exhibit significantly higher sequence identity, ranging from 40% to 60% (Vosshall, 2000).

The DOR genes are widely dispersed in the genome and most exist as single genes that distribute on each of the Drosophila chromosomes. There are two clusters of three-linked genes and eight examples of two-linked receptors. The approximate chromosomal position of each of the DOR genes was determined relative to sequence-tagged sites generated by the Berkeley Drosophila Genome Project. Thus, the 57 DOR genes identified within the euchromatic Drosophila genome may constitute the complete family of odorant receptor genes (Vosshall, 2000).

A profile of receptor expression has been obtained by performing in situ hybridization with digoxigenin-labeled RNA antisense probes to each of the 57 DOR genes. The expression of 32 of the DOR genes is restricted to the antenna; seven are expressed solely in the maxillary palp, and one is expressed in both olfactory organs. No expression of 17 DOR genes has been detected in embryonic, larval, or adult olfactory organs nor in other regions of the adult head. Each receptor is expressed in a spatially restricted subpopulation of neurons. The number of cells that express a given receptor gene, as well as the spatial pattern of expression, is conserved between individuals and is bilaterally symmetric in the two antennae. Or49b, for example, is expressed in two cells at the lateral edge of the antenna at the midpoint of the proximodistal axis. At the other extreme, Or47b is expressed in ~50 antennal neurons that reside in the lateral distal edge of the antenna. These patterns were conserved in over 30 individual flies examined for each gene (Vosshall, 2000).

In situ hybridization, coupled with immunocytochemistry with pan neuronal markers, demonstrates that this family of receptor genes is expressed in sensory neurons rather than support cells or glia within the antenna and the maxillary palp. Expression of this gene family is only observed in cells within the antenna and maxillary palp. No hybridization is observed in neurons of the brain, nor is hybridization observed elsewhere in the adult fly (including the taste cells of the proboscis) or in any tissue at any stage during embryonic or larval development. Thus, it seems likely that this receptor family is dedicated to the perception of volatile olfactory stimuli by adult sensory neurons in the antenna and maxillary palp (Vosshall, 2000).

The diversity of receptor expression in individual sensory neurons will have important implications for the logic of odor discrimination. In C. elegans, individual neurons may express up to 20 different receptor genes, whereas in mammals, olfactory sensory neurons transcribe only a single member of the gene family. Individual sensory neurons in Drosophila express different complements of receptors. At the extreme, these experiments are consistent with a model in which individual neurons express only a single receptor gene. The availability of the 'complete' repertoire of receptor genes has allowed this question of diversity of receptor expression to be examined in greater detail (Vosshall, 2000).

This problem is best addressed in the maxillary palp, which contains about 120 sensory neurons and expresses only seven members of the DOR gene family. Each of the seven DOR genes expressed in the palp identifies about 20 neurons, consistent with a model in which individual sensory neurons are functionally distinct. Two-color in situ hybridization experiments were performed in which the neurons expressing Or85e were identified with antisense RNA probes labeled with fluorescein. Neurons expressing Or46a in the maxillary palp were detected with RNA probes labeled with digoxigenin, and expression was visualized with fluorescent antibodies that distinguish the two probes. These two-color in situ hybridization experiments and additional experiments with mixed probes reveal that Or85e, Or46a, and Or59c are expressed in nonoverlapping subsets of cells (Vosshall, 2000).

Demonstrating the expression of a single receptor gene in individual sensory neurons within the antenna is more difficult since this olfactory organ expresses 32 members of the DOR gene family in ~1000 sensory cells. The lateral distal domain of the antenna contains about 300 neurons, 50 of which express Or47b. This same domain expresses 15 other receptors, including Or47a, Or23a, Or67a, Or43a, Or88a, Or49b, Or98a, and Or83c. In pairwise experiments, antennal sections were annealed with an Or47b antisense RNA probe and either individual or mixed probes for these eight additional receptors. RNA probes were labeled with either fluorescein or digoxigenin and visualized with antibodies that distinguish the two types of probes. Or47b is expressed in a subpopulation distinct from that expressing any of these eight receptors. A similar experiment was performed to examine DOR gene expression in the medial proximal region of the antenna. Or22a probes were labeled with fluorescein and a mix of probes complementary to five additional genes expressed in this domain (Or7a, Or56a, Or85b, Or42b, and Or59b) was labeled with digoxigenin. Despite the interspersion of Or22a cells with cells expressing other DOR genes in the domain, there is no overlap in the expression of Or22a with any of these five DOR genes. Taken together, these data provide strong support for a model reminiscent of the mammalian olfactory system, in which individual sensory neurons express only a single receptor. This conclusion must be tempered by the previous description of an odorant receptor gene, Or83b, that is expressed in all olfactory sensory neurons in both the antenna and maxillary palp. If Or83b does indeed recognize odorous ligands, then this data would indicate that all sensory neurons express two receptors rather than one: Or83b and one additional gene from the family of DOR genes (Vosshall, 2000).

These experiments suggest that there are 39 distinct neuronal cell types within the Drosophila olfactory organs. In the mammalian olfactory system, neurons expressing the same receptor project their axons to two spatially invariant glomeruli. It was therefore determined whether cells expressing a given receptor in Drosophila converge upon spatially defined loci in the antennal lobe, the first relay station for olfactory information in the fly brain. Axons from the antenna and maxillary palp have been found to synapse with the dendrites of projection neurons in the antennal lobe. There are 43 morphologically distinct synaptic structures or glomeruli in the antennal lobe that are invariant in position and size in individual flies. The number of antennal lobe glomeruli dedicated to olfactory input (41) approximates the number of different sensory neurons identified in the two olfactory organs (39), suggesting that olfactory neurons expressing a given receptor may converge on a single glomerulus (Vosshall, 2000).

Genetic experiments were performed that permit the visualization of the axonal projections from neurons expressing a given receptor. Transgenic flies were generated in which DOR gene promoters direct the expression of the yeast transcriptional activator, Gal4. These flies were then crossed with stocks bearing a transgene in which the Gal4-responsive promoter, UAS, drives the expression of either beta-galactosidase (LacZ) or a C-terminal fusion of green fluorescent protein (GFP) to neuronal synaptobrevin (nsyb-GFP). The expression of LacZ in specific subpopulations of sensory neurons allows the visualization of cell bodies, whereas the expression of nsyb-GFP, which selectively labels synaptic vesicles in nerve terminals, allows the visualization of terminal axonal projections (Vosshall, 2000).

Two to eight kilobases of DNA immediately upstream of the putative translation start from five DOR genes were fused to the coding sequence of Gal4. To demonstrate that these transgenes recapitulate the expression of the endogenous gene, the DOR-Gal4 strains were crossed with UAS-lacZ responders. The progeny of these crosses express LacZ in spatially defined subsets of cells in the antenna or maxillary palp that mirror the pattern of expression of the endogenous receptor. To confirm that these cells are sensory neurons, it was demonstrated that cells expressing LacZ are also labeled with an antibody that recognizes the neuron-specific RNA binding protein Elav. As a further test for the fidelity of expression of the promoter fusions, it was demonstrated that all cells expressing LacZ from the transgenes also express the corresponding endogenous odorant receptor RNA. In these experiments, RNA in situ hybridization was used to detect endogenous receptor RNA; immunofluorescence localized the expression of LacZ protein. Each of five DOR promoter fusions recapitulates the pattern of expression of the endogenous receptor gene (Vosshall, 2000).

The DOR-Gal4 transgenes were used to drive the expression of UAS-nsyb-GFP to visualize the projections of different populations of sensory neurons. Flies carrying five different DOR-Gal4 transgenes were crossed with animals bearing a UAS-nsyb-GFP transgene, and the brains of the adult progeny were then examined for localization of GFP. Immunofluorescence was performed on whole-mount brain preparations with antibody directed against GFP and with the monoclonal antibody, nc82, which labels neuropil in the fly brain and identifies the individual glomeruli in the antennal lobe. Four DOR-Gal4 promoter fusions (Or47a, Or47b, Or22a, and Or23a) are expressed in subpopulations of antennal neurons. When these flies are crossed with flies carrying the UAS-nsyb-GFP transgene, GFP labeling is seen in spatially invariant subsets of glomeruli within the antennal lobe. Or47a, for example, is expressed in 20 lateral distal neurons and their axonal projections converge on a single bilaterally symmetric glomerulus located at the dorsal medial limit of the antennal lobe. Or47b is expressed in 50 lateral distal neurons and these cells project axons that converge on a single large glomerulus that lies at the ventral lateral edge of the antennal lobe. Neurons expressing Or22a converge upon one dorsal glomerulus. The projections of one of the seven different subpopulations of sensory neurons from the maxillary palp have also been visualized. Sensory neurons expressing the palp receptor, Or46a, project to a single glomerulus that resides ventrally in the antennal lobe (Vosshall, 2000).

These four subpopulations of olfactory sensory neurons therefore project axons to single spatially invariant, bilaterally symmetric glomerulus within the antennal lobe. Cells expressing Or23a, however, send processes to two glomeruli. Or23a fibers enter the brain and initially converge upon a small dorsal glomerulus. Axons are seen extending more ventrally into the anterior of the antennal lobe where they converge upon a larger glomerulus. These two Or23a glomeruli are bilaterally symmetric and positionally invariant in multiple independent transgenic lines. It is not possible at present to determine whether individual fibers branch to form synapses within the two glomeruli nor if Or23a-expressing neurons sort such that individual subpopulations project to either one of the two glomeruli (Vosshall, 2000).

The topographic map of all five populations of olfactory sensory projections in the antennal lobe is unrelated to spatial domains of receptor expression in the antenna. For instance, while the 20 neurons that express Or22a in a proximal medial domain of the antenna converge upon a dorsal medial glomerulus, the Or47a glomerulus is situated directly adjacent to the Or22a glomerulus but receives input from cells in the lateral distal domain of the antenna. These precise patterns of glomerular convergence were observed in at least 20 individuals derived from multiple independent transgenic lines obtained for each of the four constructs. At a low frequency (1/100 flies), Or47a-expressing olfactory neurons project to two bilaterally symmetric medial glomeruli in addition to the OR47a glomerulus. The basis for this variation in axon targeting is unknown (Vosshall, 2000).

Anatomical tracing experiments in Drosophila have defined two subtypes of olfactory neurons in the antenna: those that project solely to the ipsilateral antennal lobe and others that branch and project to bilaterally symmetric glomeruli. The axonal projections from neurons expressing a given receptor in a single antenna or maxillary palp were traced to both the left and right antennal lobe. Removal of both the antenna and maxillary palp from one side of the fly results in effective deafferentation (Vosshall, 2000).

When either the left or right sensory organs were removed singly, it was found that the projections from the remaining antenna or maxillary palp converge upon the correct glomerulus in both the left and right antennal lobes. The position and size of the glomerulus is unaffected by unilateral deafferentation, but the density of nsyb-GFP-labeled fibers is decreased by about half. These data indicate that each of the four distinct subsets of olfactory neurons extend axons that innervate both the ipsilateral and contralateral antennal lobes equally. It is not possible from these studies to discern if individual axons branch or if distinct populations of axons independently sort to the left and right antennal lobes. However, it is known that afferent axons split into ipsilateral and contralateral branches. Thus, neurons expressing a given receptor project axons both ipsi- and contra-laterally to one or two spatially invariant glomeruli, creating a topographic map of receptor activation in the antennal lobe (Vosshall, 2000).

The diversity of odors recognized by a species is likely to be a function of the number of different odorant receptors it expresses. Thus, the repertoire of odors detected by the fruit fly may be small and may reflect the diminished importance of olfaction in insect species with a highly developed visual system. Drosophila, with rotting fruit as its sole source of food, may have evolved a narrow DOR gene repertoire that adequately accommodates its ecological niche. However, the true range of odors detectable by fruit flies in their natural environment has not been examined, nor has there been an analysis of subtle aspects of odor discrimination. It should be noted that even a relatively small repertoire of receptors and an equivalent number of glomeruli could allow the discrimination of a vast array of odorants that numerically far exceed the size of the receptor repertoire. Imaging studies in both invertebrates and vertebrates reveal that a given odor, even if composed of a single molecular species, activates multiple glomeruli. This spatial pattern of glomerular activation may constitute a combinatorial code that would be read in higher sensory centers. If a given odor activates ten receptors and therefore ten glomeruli, then a repertoire of 40 receptors could generate over 10⁸ different spatial patterns. Moreover, the relatively small number of odorant receptor genes in Drosophila may not be representative of other insects. The honeybee, Apis mellifera, has about 160 glomeruli and by inference 160 different odorant receptors, suggesting that in this insect, olfaction may be a more central means of acquiring sensory information from the environment. Whatever the fruit fly's sensory capabilities, its olfactory sensory system is endowed with a relatively small number of receptor genes (Vosshall, 2000 and references therein).

Of the 57 DOR genes in the Drosophila genome, 39 are expressed in subpopulations of sensory neurons, and one gene (Or83b) is expressed in all olfactory sensory neurons. Expression of 17 genes was not detected in either embryonic, larval, or adult olfactory organs or in any other regions of the adult head. Despite the inability to detect expression of these 17 genes by in situ hybridization, analysis of their sequence does not reveal nonsense or frame shift mutations that characterize pseudogenes within families of odorant receptors in other species. If these sequences indeed represent pseudogenes, then the annotation generating an intact coding sequence may be incorrect. Alternatively, these 17 genes may be expressed either in olfactory organs or elsewhere at levels below that which can be detected by in situ hybridization techniques (5-10 mRNAs per cell). This level of expression is far below estimates of receptor mRNA content in olfactory neurons in any species examined. However, other studies have employed RT-PCR to demonstrate the expression within olfactory sensory organs of several of the 17 DOR genes is not detected by in situ hybridization. Experiments in which the putative promoter regions from these genes are used to drive the expression of Gal4 transgenes will provide more definitive information concerning these 17 genes (Vosshall, 2000 and references therein).

The diversity of receptor expression in individual sensory neurons has important implications for odor discrimination. In C. elegans it is estimated that chemosensory cells express up to 20 receptor genes, whereas in mammals neurons express only a single receptor. Discrimination requires that the brain determine which receptors have been activated by an odorant. If a sensory neuron expresses only one receptor, then this problem reduces to the simpler requirement that the brain discern which neurons have been activated by a given odorant. In the fly, individual neurons express the Or83b along with only one of the remaining DOR genes. This is perhaps easiest to demonstrate in the maxillary palp, which expresses seven DOR genes and Or83b. The seven DOR genes expressed in the maxillary palp together hybridize with about 120 cells. This value is close to the total number of neurons in the palp, suggesting that every neuron in the palp can be identified with seven genes from the DOR gene family. Moreover, two-color in situ hybridization with either pairwise combinations or mixes of these genes as probes consistently reveals that a sensory neuron expresses a single receptor gene. The data suggesting that neurons express only a single receptor are difficult to reconcile with electrophysiological recordings that have been interpreted to indicate that individual palp neurons express multiple receptors (Vosshall, 2000 and references therein).

What is the role of Or83b, the only DOR gene family member expressed in all olfactory sensory neurons? Or83b might encode a receptor capable of binding odorous ligands, thus affording each cell a common and a unique specificity. One model would argues that Or83b does not serve as an odorant receptor but performs a function in sensory neurons independent of ligand binding. For example, recent studies reveal that the metabotropic GABA_B receptor is composed of a heterodimeric pair of seven transmembrane domain proteins. Surface expression and functional ligand binding of the GABA_BR1 subunit is obtained only upon coexpression of a second GABA_B receptor, GABA_BR2. Thus, Or83b might perform a function essential for chemosensory signaling, independent of odor binding, thereby retaining the one neuron-one receptor rule (Vosshall, 2000 and references therein).

In situ hybridization with 39 of the 57 DOR genes reveals that each receptor is expressed in a spatially restricted subpopulation of cells in the antenna and palp. Both the pattern of receptor expression and the number of neurons expressing a given receptor are conserved in different individuals. Some genes are expressed in as few as two spatially defined cells in the antenna, whereas other genes are expressed in as many as 50 neurons. These experiments with the 'complete' set of receptor genes define a neural epithelium in which each of 1000 cells in the antenna is marked by the expression of one of 32 unique genes. It is presumed that this spatial patterning is a consequence of axes of positional information that ultimately dictate the expression of specific genes. At present, it is not possible to determine the precision of the pattern of specific receptor expression. Nonetheless, the fine control of neural identity reflected by receptor expression is likely to be distinct from the more coarse patterning that generates the three morphologically distinct sensilla (trichoid, basiconic, and coeloconic) that populate the olfactory sensory organs (Vosshall, 2000 and references therein)

If graded positional information governs a spatial map of receptor expression in the periphery, why is there interdigitating pattern in which two cells expressing a given receptor are interspersed by a cell expressing a different receptor? It is possible that precise patterning to the level of individual cells indeed occurs early in development and neural migration results in local disruptions in the peripheral map. Alternatively, positional information may dictate a set of defined spatial domains within which the expression of not one but a subset of receptors is permitted. This latter suggestion is reminiscent of a model of receptor expression in mammals in which a given receptor gene is expressed within one of four broad but circumscribed zones within the olfactory epithelium of the nose. Within a zone, however, neurons expressing a given receptor appear randomly dispersed. Such a mechanism would also be consistent with the patterns of expression of receptor genes in the fly antenna. More precise reconstructions of the spatial patterns of expression of multiple different receptors will be required to distinguish between these alternatives (Vosshall, 2000 and references therein).

One distinction between receptor expression in mice and flies is that in mice neurons express a single receptor gene from either the maternal or paternal allele, but never from both alleles (monoallelic expression). Moreover, in mice, neurons that express an odorant receptor transgene do not express the same gene from its endogenous locus. In contrast, in Drosophila, sensory neurons that express a transgene driven by a DOR promoter always express the appropriate endogenous DOR gene. In the fly, therefore, two DOR promoters can be activated in the same cell, arguing that monoallelic expression is not occurring in Drosophila olfactory neurons (Vosshall, 2000).

The organization of the peripheral olfactory sensory system in the fruit fly is remarkably similar to that of vertebrates. In mammals, olfactory neurons express only one of a thousand odorant receptor genes, the organization and functional logic of the complex process of olfactory sensory perception has been maintained over five hundred million years of evolution despite dramatic differences in other aspects of neural function. It is suggested that this evolutionary conservation reflects the maintenance of an efficient solution to the complex problem of recognition and discrimination of a vast repertoire of odors in the environment (Vosshall, 2000).

Genetic and functional subdivision of the Drosophila antennal lobe

Olfactory systems confer the recognition and discrimination of a large number of structurally distinct odor molecules. Recent molecular analysis of odorant receptor (OR) genes and circuits has led to a model of odor coding in which a population of olfactory sensory neurons (OSNs) expressing a single OR converges upon a unique olfactory glomerulus. Activation of the OR can thus be read out by the activation of its cognate glomerulus. Drosophila is a powerful system in which to test this model because the entire repertoire of 62 ORs can be manipulated genetically. However, a complete understanding of how fly olfactory circuits are organized is lacking. A nearly complete map is presented of OR projections from OSNs to the antennal lobe (AL) in the fly brain. Four populations of OSNs coexpress two ORs along with Or83b, and a fifth expresses one OR and one gustatory receptor (GR) along with Or83b. One glomerulus receives coconvergent input from two separate populations of OSNs. Three ORs label sexually dimorphic glomeruli implicated in sexual courtship and are thus candidate Drosophila pheromone receptors. This olfactory sensory map provides an experimental framework for relating ORs to glomeruli and ultimately behavior (Fishilevich, 2005).

This study presents the results of a large-scale genetic effort to label OSNs expressing each of the 62 known OR genes and map their projections to approximately 50 morphologically defined glomeruli in the adult antennal lobe. Putative regulatory regions upstream of 49 ORs were cloned in front of the Gal4 transcription factor, and transgenic flies carrying these OR-Gal4 transgenes were crossed to cytoplasmic (UAS-lacZ) and synaptic (UAS-nsyb-GFP) reporters. Of these 49 OR-Gal4 transgenes, 30 (comprising 25 antennal and five maxillary palp ORs) produce appropriate gene expression in subpopulations of adult OSNs and are presented in this study. Of the remaining 19 OR-Gal4 constructs, 11 are selectively expressed in the larval olfactory system, one (Or83b) is broadly expressed in most OSNs, where it plays an essential role in olfaction, and seven either show no expression or are ectopically expressed. The remaining 13 OR-Gal4 lines were not generated as a result of design constraints imposed by the tight linkage of these ORs to unrelated genes or technical difficulties. Patterns of OR-Gal4:UAS-lacZ gene expression of 30 transgenes in the antenna and maxillary palp are similar to those obtained by an in situ hybridization screen and follow the same strict segregation of ORs expressed in the antenna and maxillary palp, with no OR-Gal4 lines expressed in both organs. These same lines were later used to determine the map of connectivity to the antennal lobe. Double-RNA in situ hybridization was performed to verify that OR-Gal4 lines reflect the expression of the endogenous OR mRNA. Although levels of staining varied, all cells appeared to have both endogenous OR and Gal4 transcripts. At least two OR-Gal4 lines were examined for each OR (Fishilevich, 2005).

To determine how OSNs expressing different ORs connect to the brain, genetically labeled axons were traced to their termini in the adult-fly antennal lobe. Analysis of glomerular projections of the 25 antennal OR-Gal4:UAS-nsyb-GFP strains reveals that 23 populations of OSNs expressing different ORs target a single glomerulus, whereas two (Or33b and Or67d) project to two glomeruli. Both Or33b-Gal4 and Or67d-Gal4 are coexpressed with their respective endogenous ORs, suggesting that these OSN populations indeed innervate two glomeruli. There is some apparent redundancy in the map, which was investigated further: six glomeruli are independently labeled by two different OR-Gal4 lines (Fishilevich, 2005).

In some cases, weak and variable labeling was observed, in secondary glomeruli, that reflects either variability in the expression levels of ORs in different subpopulations of neurons or transgene variability. Distinguishing between these possibilities is constrained by detection thresholds of in situ hybridization, which may not detect OR transcripts in cells weakly positive for the OR-Gal4 transgene. Some Or23a-Gal4 lines mark a second, ventrally located glomerulus (possibly DP1m). Other cases of weak and variable secondary innervation include the following: Or65a-Gal4, in the vicinity of D; Or85a-Gal4, in the vicinity of DM3; Or56a-Gal4, in the vicinity of DL4; Or10a-Gal4, in the vicinity of VA7m, and Or33b-Gal4, variable ectopic expression in multiple glomeruli. The general conclusions below are based on those glomeruli that show strong and reproducible labeling, although it is recognized that the weakly labeled glomeruli may also contribute to the odor code (Fishilevich, 2005).

Axonal projections of the five maxillary palp OR-Gal4 lines were examined in brain whole mounts. All palp neurons target the same ventral-medial region in the antennal lobe that does not receive projections from antennal OSNs. Or33c and Or85e are coexpressed, and these OSNs target the VC1 glomerulus. Or46a-expressing OSNs target an AL region with diffuse glomerular boundaries, and thus no glomerular identity could be assigned. To clarify the position of Or46a relative to the assigned palp glomeruli, the projections of Or46a-expressing neurons compared to other palp OSNs were examined simultaneously. The Or71a glomerulus is located in the same anterior-posterior plane but medial to Or46a, whereas the Or33c/Or85e glomerulus is located posterior and slightly medial to Or46a. The segregation of antennal and maxillary palp projections in Drosophila has been seen in anatomical tracing studies that preceded the advent of OR markers. The functional significance of antennal and maxillary palp segregation remains obscure because no exclusive function has been ascribed to either olfactory organ (Fishilevich, 2005).

A complete list of ORs and the glomeruli they target is presented in this study. Five cases were found in which two OR-Gal4 lines label the same glomerulus and one case in which a single glomerulus is marked by an OR-Gal4 and a GR-Gal4 line. Two-color RNA in situ hybridization was carried out to distinguish between two possibilities that would lead to two different OR-Gal4 lines apparently labeling the same glomerulus -- either the same population of OSNs is labeled by different OR-Gal4 lines, or two separate OSN populations marked by two different OR-Gal4 lines coconverge to the same glomerulus. Endogenous mRNAs for Or33a/Or56a, Or10a/Gr10a, Or33b/Or47a, Or33b/Or85a, and Or33c/Or85e are coexpressed. Whereas Or33b is coexpressed with both Or47a and Or85a, Or47a and Or85a are expressed in different OSNs. Or67d and Or82a are not coexpressed. Therefore, glomerulus VA6 receives input from two separate populations of OSNs expressing different ORs (Fishilevich, 2005).

The availability of a more complete map of AL projections allowed an examination of the organizational logic of this first olfactory synapse. There is a general trend for OSNs located in lateral/distal positions in the antenna to project to lateral AL glomeruli and for medial/proximal OSNs to target medial AL glomeruli. This segregation is most likely related to the topographic segregation of trichoid and basiconic classes of sensilla on the surface of the antenna. Or47b, Or88a, and Or67d are examples of the former, and Or42b, Or33b, and Or22a are examples of the latter. There are exceptions, notably Or19a, which targets a dorsal/medial glomerulus, although Or19a OSNs are located in the lateral/distal domain in the antenna. Of the 47 distinct glomerular compartments described, this study assigns a genetic OR identity to 26. No name could be assigned to Or46a, which may be a previously unnamed glomerulus. Other studies mapped Gr21a OSNs to V and Or59c OSNs to 1, bringing the total known number of OR assignments to AL glomeruli in the present literature to 29. Eighteen glomeruli remain to be associated with chemosensory receptors, and 20 ORs were not included in the mapping in this study. Thus, it is likely that projections of distinct OSNs can account for the remaining uncharacterized glomeruli (Fishilevich, 2005).

Available knowledge of OSN odor-response profiles were synthesized with their glomerular identity to determine whether there is any obvious chemotopic organization in the fly AL. This analysis was constrained by the limited and nonoverlapping collections of odorants used by different groups as well as differences in experimental techniques that make it difficult to compare across studies. Each OR/glomerulus was screened with a small subset of the 76 odors used across these six studies, and thus no comprehensive survey of the ligand specificity of a given OR/glomerulus exists. Nevertheless, a greater tendency was found for OSNs expressing broadly responsive ORs to project to dorsal/medial glomeruli, whereas the more selective glomeruli are located at ventral/lateral positions. However, there are many exceptions to this rule, and the ordered chemotopy described in the mouse olfactory bulb (Fishilevich, 2005).

Five subpopulations of OSNs are described that express multiple receptors along with the universal coreceptor Or83b. What might be the function of such OR coexpression? On the basis of previously published odor-response profiles for Or33b, Or47a, and Or85a, it is suggested that OR coexpression could modulate ligand-response profiles. Both Or47a and Or85a respond to more odors when ectopically expressed in the ab3A 'empty' neuron than the native neurons, which coexpress Or33b/Or47a or Or33b/Or85a. Or33b expressed alone in the 'empty' neuron responds weakly and with inhibition to most odors, giving a weak excitatory response only to ethyl propionate. Thus, Or33b coexpression in the native OSN could function to temper the relatively broad tuning of Or47a and Or85a. Confirmation of such a role awaits further genetic analysis of these ORs in vivo (Fishilevich, 2005).

An intriguing OSN population was identified that coexpresses members of the OR and GR families, along with Or83b. The role of GRs in the antenna is poorly understood, although Gr21a is expressed in neurons that respond to carbon dioxide. It will be interesting to determine whether Gr10a contributes to the detection of odors along with Or10a and subserves an olfactory instead of gustatory function (Fishilevich, 2005).

Recent work examining the expression of the male-specific isoform of the fruitless (fru) transcription factor implicates two large, sexually dimorphic glomeruli (VA1lm and DA1) in male courtship behavior. This study has revealed the molecular identity of the OSNs projecting to these glomeruli as Or47b and Or67d, respectively. Other glomeruli that receive input from fru-expressing OSNs are VL2a and, occasionally, VA6, which is identified in this study as receiving coconvergent input from Or82a- and Or67d-expressing OSNs. The identity of the VL2a-projecting OSNs remains obscure. Interestingly, these OSNs and the glomeruli to which they project show little or no activation in response to general odors. The exception is Or82a, which responds very selectively to geranyl acetate, a green-leaf volatile that is also a major component of medfly male sex pheromone. On the basis of anatomical tracing studies, it is suggested that Or82a- and Or67d-expressing OSNs target the same glomerulus. Whether they synapse uniformly upon the same population of postsynaptic projection neurons or whether the glomerulus has functional subcompartments is not known. In either scenario, the glomerulus might act as a coincidence detector that would require both the Or82a ligand, geranyl acetate, and the unknown Or67d ligand for activation (Fishilevich, 2005).

Courtship behavior in Drosophila involves multimodal input from visual, gustatory, auditory, and olfactory cues. The involvement of volatile pheromones in Drosophila sexual behavior has long been inferred, but neither the putative pheromones nor the receptors that detect them are known. It is suggested that these ORs are pheromone receptors that respond to volatile pheromones. In support of this, silencing or reprogramming these OSNs leads to selective disruption in male sexual behavior (Fishilevich, 2005)

This paper presents a nearly complete map of olfactory projections to the fly AL. From this map, five populations of OSNs were identified that express multiple receptors and two populations of OSNs expressing different ORs that coconverge upon a common glomerulus. An analysis of published odor-response profiles for these ORs and their glomeruli suggests that more broadly tuned neurons map to the dorsal/medial domain, whereas more restricted OSNs map to ventral/lateral glomeruli. Candidate Drosophila pheromone receptors were identified by virtue of their innervation of sexually dimorphic fru-positive glomeruli. A number of intriguing questions follow from this study. (1) What is the genetic identity of the projections that target the posterior face of the antennal lobe? These glomeruli may receive input from OSNs expressing OR or GR genes not examine in this study. (2) It will be of interest to understand in greater detail what effects receptor coexpression and OSN coconvergence have on the capacity of the fly to detect and discriminate odors. (3) The availability of candidate pheromone receptors in Drosophila will make it possible to study sex pheromones in a genetically tractable organism from the circuits they activate to the stereotyped behaviors they elicit (Fishilevich, 2005).

Target neuron prespecification in the olfactory map of Drosophila

In Drosophila and mice, olfactory receptor neurons (ORNs) expressing the same receptors have convergent axonal projections to specific glomerular targets in the antennal lobe/olfactory bulb, creating an odor map in this first olfactory structure of the central nervous system. Projection neurons of the Drosophila antennal lobe send dendrites into glomeruli and axons to higher brain centers, thereby transferring this odor map further into the brain. The MARCM method has been used to perform a systematic clonal analysis of projection neurons, allowing the correlation of lineage and birth time of projection neurons with their glomerular choice. Projection neurons are prespecified by lineage and birth order to form synapses with specific incoming ORN axons, and therefore to carry specific olfactory information. This prespecification could be used to hardwire the fly's olfactory system, enabling stereotyped behavioral responses to odorants. Developmental studies have led to the hypothesis that recognition molecules ensure reciprocally specific connections of ORNs and projection neurons. These studies also imply a previously unanticipated role for precise dendritic targeting by postsynaptic neurons in determining connection specificity (Jefferis, 2001).

A common process in neural network formation is the establishment of one-to-one corresponding connections between two groups of neurons in two different locations, thereby generating a neural map. Three basic mechanisms for the formation of such neural maps can be proposed. In the first two mechanisms, either input or target neurons are genetically prespecified, whereas neurons of the remaining field are naive until specified by the identity of their partners during the connection process. In the third mechanism, input and target neurons are independently specified. This problem has been explored in the wiring of the Drosophila olfactory system. The organization of the Drosophila peripheral olfactory pathway is very similar to that of mammals. About 1,300 ORNs expressing 40-60 different receptors project their axons to 40-50 individually identifiable glomeruli of the antennal lobe (equivalent to the mammalian olfactory bulb). Information leaves the antennal lobe through an estimated 150 projection neurons (equivalent to mammalian mitral/tufted cells), whose cell bodies are located at the periphery of the antennal lobe. Projection neurons project their dendrites to glomeruli and their axons to higher brain centers, including the mushroom bodies and the lateral horn. As in mice, each ORN probably expresses one specific receptor, and the axons of ORNs expressing the same receptors converge at the same morphologically and spatially distinct glomeruli. In mice, ORNs seem to be genetically programmed to project to specific glomeruli, instructed by the receptors that they express; indeed, this convergence seems to be independent of the presence of the target neurons of the olfactory bulb. Although analogous experiments have not been reported in flies, ORNs expressing a particular receptor reside in stereotypic and discrete zones of the antennae and maxillary palps -- the fly's olfactory appendages. Assuming that ORN cell bodies do not relocate after their axons reach the antennal lobe, it seems probable that their glomerular targets are prespecified. Thus, of the three models for formation of the neural map, the second model seems unlikely in both mice and Drosophila. It was of interest to distinguish whether Drosophila projection neurons are specified by virtue of their connection with ORNs or are independently specified (Jefferis, 2001).

The MARCM (mosaic analysis with a repressible cell marker) system can be used to determine neuronal lineage and the projection patterns of individual neurons. A typical neuroblast in the Drosophila brain undergoes asymmetric division to regenerate a new neuroblast and a ganglion mother cell, which divides once more to generate two postmitotic neurons. Using MARCM, one can generate positively-labelled single-cell clones as well as neuroblast clones. By controlling the timing of mitotic recombination using heat-shock-induced FLP recombinase, one can produce labelled clones of cells born at different developmental times. To study projection neurons of the antennal lobe, use was made of a GAL4 line, GAL4-GH146, which drives marker expression in a large subset of projection neurons (approximately 90). By crossing GH146 with a membrane marker, UAS-mouse CD8-green fluorescent protein (GFP), the cell body and dendrites of projection neurons in the antennal lobe can be visualized in the anterior part of the brain, as well as their axonal projections in the posterior part of the brain. When the MARCM technique is applied using GAL4-GH146, subsets of these projection neurons can be selectively visualized as either neuroblast clones or single-cell clones (Jefferis, 2001).

Systematic clonal analysis revealed that GH146-positive projection neurons (referred to as projection neurons) are derived from three neuroblasts: an anterodorsal, a lateral and a ventral neuroblast. When neuroblast clones are induced in the early embryo and then examined in adults, the anterodorsal, lateral and ventral neuroblasts give rise to approximately 50, 35 and 6 projection neurons, respectively. These three numbers correspond well to the number of projection neurons present in the three GH146- positive cell groups. Since no clones can be induced by applying heat shock after puparium formation (APF), and neuroblast clones generated in late larvae contain 3-5 cells, it is inferred that all projection neurons are born well before the arrival of pioneering adult ORN axons in the antennal lobe around 20-24 h APF. Subsequent analyses focused on anterodorsal projection neurons and lateral projection neurons since most of these neurons have uniglomerular dendritic projections, whereas some ventral projection neurons have diffuse dendritic arborizations, and all project by means of a different path to the higher brain centers, bypassing the mushroom bodies (Jefferis, 2001).

When glomerular projections of neuroblast clones generated in early larvae were examined, it was found that anterodorsal and lateral neuroblast clones appear to innervate stereotypical, intercalated but non-overlapping glomeruli. By contrast, an anterodorsal neuroblast clone and a lateral neuroblast clone examined at the same depth, projected to complementary glomeruli. In the 54 anterodorsal neuroblast and 25 lateral neuroblast clones examined, no exception to this rule was found, despite the fact that there is no obvious relationship between the positions of projection neuron cell bodies and their glomeruli. These observations indicate that the neuroblast from which a projection neuron is derived restricts its glomerular choice and consequently the subset of olfactory information that it can carry further into the brain. It was next asked whether projection neurons are further specified within a neuroblast lineage. Because labelled, single-cell MARCM clones are born shortly after heat-shock induction of mitotic recombination, it was possible to test whether projection neurons born during specific developmental periods would project to specific glomeruli by using the time of heat shock as a variable. It was found that anterodorsal single-cell projection neuron clones with particular glomerular projections were generated within restricted and characteristic developmental windows. Notably, single-cell clones induced by early larval heat shock (0-36 h) exclusively produced projection neurons projecting to glomerulus DL1 (Jefferis, 2001).

Because individual larvae develop at different rates, and FLP recombinase can persist for some time after heat-shock induction, single-cell clone analysis cannot distinguish unequivocally the birth order of projection neuons that are born immediately after each other. Therefore a complementary approach was taken in which multicellular neuroblast clones generated at different developmental periods were examined, and it was determined whether they included certain landmark glomeruli. If projection neurons innervating different glomeruli are produced in a defined sequence, then multicellular neuroblast clones induced at progressively later times during development should innervate a subset of the glomeruli in neuroblast clones generated at earlier times. Eventually the last-born, smallest clones should contain projection neurons innervating only one glomerulus. Such a nested set is exactly what was observed when anterodorsal neuroblast clones were scored for the presence or absence of projection neurons innervating ten landmark glomeruli. An ordered birth sequence of projection neurons (VA2, DL1, DC2, D, VA3, VA1d, VM7, VM2, DM6, VA1lm) could be inferred from the 54 clones analysed. No neuroblast clone was found in which projection neurons projecting to a particular glomerulus were altered from this order; for example, neuroblast clones containing projection neurons projecting to VM7 always additionally contained all three of the later-born types of projection neuron, VM2, DM6 and VA1lm. Since, on average, about three projection neurons innervate each glomerulus, the fact that an order can be inferred implies that projection neurons innervating a common glomerulus are likely to be born at a similar time. Together with the single-cell clone analysis, it can be concluded that, at least for these ten glomeruli, there is a strict order of generation of projection neurons that can predict future glomerular targets (Jefferis, 2001).

How can the birth time of a projection neuron predict which glomerulus it will eventually innervate? One possibility is that the ordered generation of projection neurons could result in the ordered differentiation of their dendrites, and that temporally ordered availability of proto-glomeruli for innervation restricts projection neurons born at a certain time to a particular glomerulus. However, it was found that at around 22 h APF, when pioneering ORN axons just start to invade the antennal lobe, projection neurons born at different times had similar dendritic differentiation statuses, having already initiated their dendritic branches in the vicinity of the antennal lobe region. The axons of the projection neurons had already reached the mushroom body and the lateral horn. These observations argue against the hypothesis of differentiation timing. Instead the hypothesis is favored that individual projection neurons and ORNs are independently specified to carry molecular signals that allow them to recognize either each other or a common set of cues located at the developing antennal lobe. Although it is assumed that stereotyped ORN axon projections depend on a guidance map in the developing antennal lobe, these results imply that such cues may also be used for precise dendritic targeting of projection neurons, or that projection neuron dendrites actively participate in creating the guidance map (Jefferis, 2001).

Independent pre-patterning of input and target fields has been demonstrated in the formation of vertebrate retinotectal projections along the anterior-posterior axis, and even implicated in the development of ocular dominance columns. In both systems, activity-dependent processes have important roles in refining the coarse map at the level of the single cell. This study shows the independent specification of projection neurons at the single-cell level, matching the precision of the ORN identities. These experiments support the importance of independent specification in formation of the neural map. Moreover, since no obvious logic correlates cell body position of the projection neuron, birth time and the location of the glomerular projection, simple molecular gradient/counter-gradient models as used for axon guidance in the retinotectal system are unlikely to suffice. Instead, it is proposed that dendrites of projection neurons use a combination of recognition molecules specific to each eventual glomerulus; mechanisms that generate a complex repertoire of cell-surface molecules have recently been described. Furthermore, this study uncovers an elegant mechanism for specifying different projection neurons: the precisely ordered generation by a single neuroblast of a large number of distinct neurons. Currently it is being determined whether timer mechanism intrinsic to the neuroblast, cues from neighboring cells at the time of birth, or a combination of such mechanisms are used, perhaps to specify the expression of recognition molecules (Jefferis, 2001).

Given the similarities in organization of the Drosophila and mammalian peripheral olfactory systems, it will be of great interest to test whether and to what extent the mitral/tufted cells in the mammalian olfactory system are independently specified. It is conceivable that the information used to pattern the olfactory bulb for ORN axon targeting could be used to prespecify mitral/tufted cells, thereby coordinating their dendritic targets in the olfactory bulb and axonal projections in higher brain centers. Such prespecification mechanisms may also be used in neural map formation in other parts of the developing brain (Jefferis, 2001).

Developmental origin of wiring specificity in the olfactory system of Drosophila

In both insects and mammals, olfactory receptor neurons (ORNs) expressing specific olfactory receptors converge their axons onto specific glomeruli, creating a spatial map in the brain. Second order projection neurons (PNs) in Drosophila are prespecified by lineage and birth order to send their dendrites to one of ~50 glomeruli in the antennal lobe. How can a given class of ORN axons match up with a given class of PN dendrites? The cellular and developmental events that lead to this wiring specificity have been examined. Before ORN axon arrival, PN dendrites have already created a prototypic map that resembles the adult glomerular map, by virtue of their selective dendritic localization. Positional cues that create this prototypic dendritic map do not appear to be either from the residual larval olfactory system or from glial processes within the antennal lobe. It is proposed instead that this prototypic map might originate from both patterning information external to the developing antennal lobe and interactions among PN dendrites (Jefferis, 2004).

A key finding in this study is that PN dendritic development is surprisingly independent of presynaptic ORN axons. It is generally thought that dendritic growth and maturation are coupled with presynaptic axon invasion and synapse formation, which might be significantly shaped by electrical activity. Before ORN axons reach the developing AL, PN dendrites, however, have undergone significant growth and branching. More strikingly, dendrites of different PN classes have created a pattern in the AL at 18 hours APF that resembles the adult glomerular map, in the complete absence of their presynaptic partners. This observation is in contrast to the prevailing view of olfactory development and underlines a very significant dendritic contribution to the origins of wiring specificity (Jefferis, 2004).

It is important to realize that this finding is not a natural prediction of previous findings that PNs are prespecified to synapse with a specific class of ORNs by lineage and birth order. The current study clearly favours the idea that PN dendrites are patterned first. It is probable that gradual invasion of ORN axons from 24 hours APF refines and consolidates this prototypic dendritic map, allowing rapid development of the AL to essentially adult form by 50 hours APF (Jefferis, 2004).

Site selection of one synaptic partner before the other has also been described recently in Caenorhabditis elegans. Interestingly, in that case, the presynaptic partner selects the synaptic site in the absence of its postsynaptic partner by interacting with a third party guidepost cell (Jefferis, 2004 and references therein).

What positional cues allow dendritic patterning before ORN axon arrival? The first leading candidate to provide positional cues is the larval lobe because it is presumably patterned for larval olfaction, albeit in a form simpler than that of the adult AL. However, at early pupal stages, the developing adult AL is clearly distinct from the larval lobe and is minimally invaded by remnants of the degenerating larval lobe. Glia are a second candidate and, indeed, glia have been suggested to play important roles in sorting ORN axons into individual classes in the moth and are the leading candidates in the mouse olfactory bulb to provide positional cues for ORN axon targeting. However, no significant glial processes are found within the developing AL between 0 hours and 18 hours APF, the critical period for PN dendritic patterning. Although it is likely that glial cells and processes surrounding the AL contribute to PN dendritic patterning, it is difficult to imagine that the surrounding tissues could contribute all the positional cues that allow dendrites of a specific class to occupy specific regions of the AL, which is a three-dimensional sphere (Jefferis, 2004).

These analyses have led to the speculation that PN dendrite-dendrite interaction might contribute significantly to the eventual patterning of PN dendrites for the following reasons. (1) PN dendrites appear to be a major constituent of the developing adult-specific AL, which is composed predominantly, if not exclusively, of neuronal processes. Indeed, preliminary electron microscopic analysis using genetically encoded electron microscopy markers suggests that the developing AL is composed entirely of neuronal profiles and that GH146-positive PN dendritic profiles (enhancer trap GAL4-GH146 labels ~90 of the estimated 150 PN) are clustered together without intermingling with other neuronal profiles. (2) Different classes of PNs are probably endowed with molecular differences because of their lineage and birth order -- these differences could be used to create heterogeneities in the developing lobe. LN processes do remain a possible source of information for which neither positive evidence could be found nor could it be ruled out. However, it is more difficult to imagine how heterogeneity is created by LNs, because processes from each LN occupy a large proportion of the AL, rather than individual glomeruli (Jefferis, 2004).

Based on these considerations, how is it envisaged that PN patterning could occur in early pupa before ORN arrival? A hierarchy of positional cues is proposed. Global cues, which could be diffusible or contact-mediated guidance molecules from outside the lobe, could allow dendrites to target to an approximate region of the developing lobe with respect to the body axes, causing, for example, glomerulus V to form ventrally and glomerulus D to form dorsally. Local dendrite-dendrite interactions would then allow PN dendrites of the same class to adhere tightly; dendrites destined to occupy neighboring glomeruli would associate more weakly and/or be repelled. This hierarchy of cues seems to be essential for generating a structure that has global order and highly reproducible local spatial relationships among neighboring PN classes. The observations at 18 hours APF suggest that although the global targeting of dendrites to specific regions of the AL is largely complete, the sorting process is not, because dendrites from neighboring classes can still exhibit significant overlap. Continuing dendrite-dendrite interaction after 18 hours APF and interaction with ORN axons would further refine this prototypic dendritic map (Jefferis, 2004).

The observation that in Drosophila PN patterning precedes that of ORNs seems at odds with existing observations in insects and vertebrates, emphasizing the primary role of ORN axons in organizing glomerular development. In all these studies, glomerular formation was first evident in the accumulation of ORN axons in protoglomeruli. This organizational function of ORNs is further supported by several perturbation experiments. For instance, in the moth Manduca sexta, developmental deantennation prevents normal glomerular formation, whereas surgical removal of a subset of PNs still permits ORNs to form relatively normal glomerular terminations outlined by glial cells. Similarly, genetic ablation of most mitral or granule cells in mutant mice still permits axonal convergence of specific ORN classes, although the structure of the olfactory bulb is disorganized. In Drosophila, it was reported that in atonal mutants, formation of a proposed pioneer class of ORNs is disrupted, axonal targeting of other ORNs is delayed and glial processes are disturbed. It was further suggested that PN development must depend on ORNs because, in atonal mutants, PN patterning [as visualized by GH146 staining (labelling ~90 PNs)] is disrupted (Jefferis, 2004 and references therein).

Many of these apparent contradictions can be resolved by treating spatial patterning and glomerular formation as two distinct processes. An important technical improvement of this study is that it was possible to visualize the dendritic fields of identifiable PNs down to the single cell level with MARCM or small groups of cells with GAL4-Mz19. It was therefore possible to describe much earlier developmental events with higher anatomical resolution, demonstrating the existence of spatial patterning well before glomeruli are morphologically distinct and, indeed, before one major component of the glomeruli -- the ORN axons -- is even present. For example, dendrites of DL1 PNs occupy a discrete and specific location in the developing lobe. However, because their dendrites still overlap with other PN classes, this patterning is not that obvious when looking at larger numbers of neurons and is scarcely apparent at all when more cells are visualized. Indeed, some existing single cell labelling studies have shown that PNs in Manduca have restricted dendritic arborizations before any glomerular patterning is evident. However, without being able to label specific classes of PNs, such studies cannot determine whether PN dendrites are in a spatially appropriate location (Jefferis, 2004).

How do ORN axons come into register with the prototypic PN map when they reach the lobe? At one extreme, ORN patterning might be completely dependent on PN patterning. ORN axons could simply recognize specific classes of pre-patterned PN dendrites through receptor-ligand or homophilic interactions. Alternatively, PN patterning could generate a third-party map, which is recognized by ORNs. Although certainly consistent with the developmental studies described here, these models are not supported by experiments in other organisms. In addition, transplantation experiments in moth or the formation of a novel glomerulus upon expression of a rat olfactory receptor in mice, indicate that ORNs might have substantial autonomous patterning ability. At the other extreme, patterning of ORN axons could be completely independent of the prototypic map created by PN dendrites. In theory, ORN axons could recognize third-party cues previously used to pattern PN dendrites. However, such third-party cues could not be found within the developing AL. Of course, ORNs could still be patterned by PN independent cues external to the lobe and axon-axon interactions, in a manner directly analogous to the hypothesis for PN patterning. In the case of such strict independence, ORNs expressing a particular receptor would form specific connections with partner PNs, rather than inappropriate adjacent PNs, only because both sets of neurons target with great precision to exactly the same spatial location. This degree of independence seems both implausible and inefficient (Jefferis, 2004).

Instead, it is proposed that both ORN axons and PN dendrites have substantial autonomous patterning ability -- for instance through PN-PN or ORN-ORN mutual interactions and interactions with cellular cues surrounding the developing AL -- but that the two resultant proto-maps interact during development to generate the final mature glomerular organization. This proposal is the most parsimonious explanation for the existing data supporting the organizational functions of ORNs. This model also has the advantage of robustness -- each map could reinforce and refine the other, resulting in a precise match between pre- and postsynaptic partners without the necessity for extreme targeting precision. Furthermore, although two maps might seem to be more complicated than one, they need not be molecularly more complex. Many of the molecules used for PN patterning could also be used for ORN patterning or for interactions between ORNs and PNs. Having described with great precision the cellular and developmental events that led to the patterning of the AL, the molecular basis of wiring specificity in the Drosophila olfactory system can now be attacked (Jefferis, 2004).

Developmentally programmed remodeling of the Drosophila olfactory circuit

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input; their dendrites synapse with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to receive input from a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons (Marin, 2005).

One of the best-studied examples of neuronal reorganization in an insect brain is the gamma neuron of Drosophila mushroom bodies (MBs). MB gamma neurons are born during embryonic and early larval stages. They send dendrites into the MB calyx and axons into larval medial and dorsal MB axon lobes. During early metamorphosis, gamma neurons prune their larva-specific dendrites and axon branches before re-extending adult-specific processes. What happens to their synaptic partners while MB gamma neurons reorganize their dendrites and axons? In this study, it was shown that a subset of olfactory projection neurons -- the major presynaptic partners of MB gamma neurons -- are also morphologically differentiated to function in both larva and adult. The reorganization of these neurons during metamorphosis is independently controlled by some of the same molecular mechanisms as that of the MB gamma neurons (Marin, 2005).

In the adult fly, odors are detected by olfactory receptors (ORs) on the dendrites of about 1300 olfactory receptor neurons (ORNs) in the antennae and maxillary palps. In general, each ORN appears to express one of ~45 possible OR types, and the axons of all ORNs expressing a given OR converge to one of ~45 stereotypical glomeruli in the antennal lobe (AL), the equivalent of the mammalian olfactory bulb. From there, 150-200 projection neurons (PNs) relay olfactory activity to higher brain centers, the MB calyx and the lateral horn (LH) of the protocerebrum. Systematic clonal analysis using the MARCM method to label single and clonally related clusters of PNs that express the GAL4 driver GH146 revealed that these PNs are prespecified by lineage and birth order to receive input via their dentrites from particular glomeruli in the adult AL. Moreover, each glomerular class of PNs exhibits a characteristic axon branching pattern in the LH, suggesting stereotyped targets in at least one higher olfactory center (Marin, 2005).

The Drosophila larval olfactory system is much smaller and simpler by comparison, shown to consist of only 21 ORNs in the dorsal organ and believed to include ~50 PNs relaying information to the larval MB and LH. Developmental analysis has shown that the PNs born during larval stages exhibit only a single unbranched process from the cell body to the MB calyx until early metamorphosis, when dendrites and axon terminal branches start to elaborate. Thus, larval-born PNs do not participate in the larval olfactory circuit (Marin, 2005).

What, then, is the origin of the relay interneurons that connect the larval AL to higher olfactory centers? Do they contribute to the adult olfactory system as well? This study shows that, in contrast to the larval-born PNs, PNs generated during embryogenesis exhibit morphologically differentiated dendrites and axons in both larva and adult. These neurons prune their processes locally during the first few hours of metamorphosis and later re-extend them to innervate developing adult structures. This pruning process is regulated by ecdysone and TGFß signaling, as has been demonstrated previously for MB gamma neurons. Thus, developmentally programmed remodeling allows these embryonic-born PNs to participate in two distinct olfactory circuits at two different stages in the Drosophila life cycle (Marin, 2005).

The MARCM method allows the labeling of a single neuron, or all neurons born from the same neuroblast, that express a particular GAL4 driver. These studies focus on the ~90 (out of an estimated total 150-200) PNs that express GAL4-GH146. A heatshock-promoter-driven FLP recombinase was used to control the timing of the mitotic recombination that results in labeled MARCM clones. In a previous study (Jefferis, 2004), at least one GAL4-GH146-positive PN was identified that could only be labeled by heatshock-induced mitotic recombination during embryogenesis. This embryonic-born PN specifically targeted its dendrites to glomerulus VA2, one of many glomeruli innervated when labeling the entire population of GH146 PNs, yet never by PN single-cell or neuroblast clones labeled by heatshock during larval stages. This discrepancy in the number of innervated glomeruli suggested that a fraction of adult AL glomeruli were being targeted by a subset of PNs born during embryogenesis (Marin, 2005).

To study the embryonic-born neurons labeled by the GH146 driver, MARCM clones were systematically generated by heatshock induction at embryonic stages. Large anterodorsal neuroblast clones labeled by heatshock early in embryogenesis innervated at least 15 glomeruli not targeted by either the anterodorsal or lateral neuroblast clones labeled by heatshocking newly hatched larvae. MARCM single-cell clones were used to characterize embryonic-born PNs that innervate eight different landmark glomeruli in the adult AL. The gross morphology of these PNs in the adult brain is quite similar to that of the larval-born PNs previously described: each PN generally innervates a single glomerulus in the antennal lobe (distinct from those innervated by larval-born PNs), then sends its axon via the inner antennocerebral tract to display a characteristic terminal branching pattern in the LH according to its glomerular class, along with a number of collaterals in the MB calyx that end in prominent boutons (Marin, 2005).

By comparing the specific glomeruli innervated in each partial anterodorsal neuroblast clone generated by heatshock at different times during embryogenesis, it was ascertained that: (1) these embryonic-born PNs were generated in the order DP1m, VL2p, VA6, VA2, DL5, DM3, VM3 and finally DL6, and (2) every clone labeled by embryonic heatshock included all of the larval-born anterodorsal PNs analyzed in the previous study, indicating that both PN subsets originate from the same neuroblast. Upon generation of the DL6 PN(s), the anterodorsal neuroblast apparently arrests, producing additional projection neurons later only in larval life (as indicated by heatshock-induced labeling of just a single anterodorsal glomerular class, DL1, until about 36 hours after larval hatching) (Marin, 2005).

In summary, embryonic-born PNs look just like larval-born PNs with regard to both their dendritic and axonal projections in the adult brain. Moreover, since their dendrites target a distinct subset of AL glomeruli and their axons exhibit characteristic terminal branching patterns in the LH according to their glomerular classes, these embryonic-born PNs serve to expand the repertoire of odor representation in adults beyond the larval-born PNs previously characterized (Marin, 2005).

Given their early origin, these GH146-positive embryonic-born PNs may participate in the larval olfactory circuit as well. Indeed, examining third instar larval brains reveals that GH146 is strongly expressed in presumptive projection neurons that appear to innervate the larval AL and to send axons up to the MB calyx and larval equivalent of the adult LH. These projections appear to be contributed by about 16 to 18 clustered neurons that are presumably derived from the anterodorsal neuroblast (Marin, 2005).

To examine the morphology and connectivity of these PNs in the larval olfactory system with greater resolution, the MARCM method was used to specifically label PNs generated prior to larval hatching and brains were dissected from wandering third instar larvae. In contrast to the larval-born PNs analyzed in earlier studies (Jefferis, 2004), all anterodorsal embryonic-born PNs exhibited densely branched dendrites in the larval AL and axons with large synaptic structures targeting glomerulus-like subregions in the MB calyx as well as branches in the presumptive LH. The large majority of the anterodorsal embryonic-born PNs each targeted a single glomerulus in the LAL and/or in the MB. In some cases, individual PNs targeted two glomeruli in one or both structures (Marin, 2005).

Several lines of evidence suggest that the embryonic-born PNs observed in the larval olfactory system are the same cells as the PNs that contribute to the much larger and more complex adult circuit. (1) The frequencies of labeled single-cell clones are comparable between the two stages, arguing against the possibilities that embryonic-born PNs are either dying off during metamorphosis or remaining quiescent and undetected through larval life. (2) The numbers of GH146-positive PNs observed at the time of puparium formation and in the adult are similar. (3) Most importantly, each embryonic-born PN undergoes characteristic morphological changes during metamorphosis. Therefore, the PNs labeled by embryonic heatshock are referred to as persistent projection neurons (PPNs). However, at this point, the methods do not allow correlation of specific glomerular classes in larva with those observed later in adulthood (Marin, 2005).

Prior studies have used MB gamma neurons as a model system to study the molecular mechanisms of axon pruning. γ neuron pruning depends on cell-autonomous reception of the steroid hormone ecdysone; single neurons that are homozygous mutant for the ecdysone co-receptor ultraspiracle (usp) in an otherwise heterozygous brain fail to reorganize their processes and retain both dorsal and medial axon lobes in the adult brain. In addition, gamma neurons must upregulate the expression of ecdysone receptor isoform B1 (EcRB1) prior to axon pruning. This upregulation requires TGFß signaling; MB gamma neurons that are mutant for the TGFß/Activin Type I receptor baboon (babo) or its downstream effector mad do not upregulate EcRB1 expression and consequently fail to prune (Marin, 2005 and references therein).

It was asked whether a similar molecular pathway is utilized during PPN reorganization. To ascertain whether the pruning of PPNs is also regulated by ecdysone, EcRB1 expression patterns were analzyed. At puparium formation, only 20 of the ~90 GH146+ projection neurons present were strongly positive for EcRB1. These strongly stained PNs, ~18 of which belonged to the anterodorsal cluster, also had noticeably larger and brighter cell bodies than surrounding PNs, which were probably immature larval-born PNs. Single-cell MARCM clones generated by embryonic heatshock were also strongly positive for EcRB1 at puparium formation. Thus, it is concluded that EcRB1 expression is highly expressed in PPNs at the onset of metamorphosis (Marin, 2005).

Is TGFß signaling generally required to regulate expression of EcRB1 for neuronal pruning during metamorphosis? MARCM was used to label cells that were homozygous for the strongest baboon allele, babo^Fd4, in a heterozygous background to test whether PPNs also require TGFß reception for normal pruning. At the wandering third instar stage, PPNs homozygous for babo^Fd4 appeared to have normal dendritic and axonal projections. However, babo^Fd4 PPNs failed to show high-level expression of EcRB1 by the onset of puparium formation. This result indicates that, as for the MB gamma neurons, high-level expression of EcRB1 in remodeling PPNs depends on TGFß signaling (Marin, 2005).

Consistent with the loss of EcRB1 expression, babo^Fd4 PPNs fail to reorganize their processes normally during the first few hours of metamorphosis. For wild-type PPNs at 8 hours APF, approximately 95% of dendrites, 80% of MB calyx processes, and 85% of LH processes are in the final two stages of pruning. However, most of the embryonic-born babo^Fd4 PPNs still retain dendrites and axons with larval morphology at this time. Dense dendritic processes were visible in the larval AL for 100% of PN clones examined, and only 14% of axon branches in the LH appeared to resemble the final two stages of pruning. The degree of pruning in the calyx was more difficult to estimate, due to the concurrent degeneration of gamma MB neuron dendrites and loss of glomerular organization, but disappearance of synaptic boutons still seemed inhibited (Marin, 2005).

To confirm that this failure to prune resulted from loss of ecdysone signal reception, MARCM was used to label PPNs that were homozygous for a well-characterized mutant allele of the ecdysone co-receptor, usp³. At the wandering third instar stage, usp³ PPNs exhibit normal morphology, and, as expected, EcRB1 was expressed at wild-type levels at the time of puparium formation (Marin, 2005).

However, when these brains were examined at 8 hours APF, a significant defect in dendrite and axon pruning was observed. In the majority of cases, both dendritic densities in the location of the larval antennal lobe and axon branches in the MB calyx and LH had been retained. Taken together, these mosaic experiments suggest that PPN dendritic and axonal pruning require cell-autonomous function of EcRB1/USP, as has been shown previously for MB gamma neurons (Marin, 2005).

What are the consequences for the adult olfactory circuit when larval circuits fail to prune? PPNs homozygous for usp³ or babo^Fd4 that failed to prune their dendrites and axons during metamorphosis allowed investigation of this question. When examined in adults, wild-type PPN dendrites were confined to a single glomerulus in the adult AL with the exception of the VL2p+ class. Dendrites of single-cell PPN clones homozygous for usp³ generally appeared to target glomeruli in the adult AL appropriate for PPNs; however, ectopic processes in additional areas of the AL, which could be interpreted as persisting larval dendrites, were often present. In a few cases, usp³ PPN dendrites were sparser and less specifically targeted to particular glomeruli, but still remained somewhat confined to certain regions of the AL. Likewise, whereas wild-type PPNs always exhibit terminal swellings on short side branches, about 40% of usp³ PPNs retained larval-like boutons directly on their main trunks in the MB calyx; however, they always had side branches with terminal swellings as well, implying that re-extension and adult-specific outgrowth were not completely impaired. In addition, the main axon trunk often diverted conspicuously from the inner antennocerebral tract in the MB calyx, presumably to maintain contact with the larval boutons. Nearly all usp³ PPN axons exhibited grossly wild-type morphologies in adult LH; only one usp³ PPN axon in the sample failed to enter the LH. In summary, usp³ PPNs display ectopic processes in AL and MB that appear to be due to defects in pruning during early metamorphosis; these pruning defects do not seem to interfere with the growth or even targeting (in the case of AL) of adult-specific processes (Marin, 2005).

In comparison, babo^Fd4 PPNs exhibited more severe dendritic and axonal phenotypes in the adult brain. In a few cases, these PPNs had targeted an appropriate glomerulus but also featured ectopic processes. More commonly, sparse diffuse processes were observed in the AL that were somewhat localized but did not appear to target any specific glomerulus. Processes also occasionally strayed to arborize outside the ventral AL. In the most severe cases, sparse dendrites were distributed broadly throughout the AL. In the MB calyx, all babo^Fd4 PPN axons appeared to have retained large larval-like boutons directly on their main trunks, rather than exclusively terminal swellings on short side branches as in wild type; in 64% of cases, there were no MB collaterals with wild-type adult appearance at all. The main axon trunk often diverged dramatically from the inner antennocerebral tract in the MB calyx. Finally, in the LH, the majority of babo^Fd4 PPNs featured significant aberrations including unusually profuse swellings along the branches, failure to enter the LH and/or failure to elaborate higher order branches in the LH. These phenotypes imply an axon re-extension, pathfinding and/or targeting defect in addition to the impaired pruning observed at 8 hours APF (Marin, 2005).

In summary, both usp³ and babo^Fd4 PPNs exhibit phenotypes in the adult brain consistent with blockage of pruning during early metamorphosis, including extraglomerular processes in the AL as well as large larval-like boutons on the main trunk and diversion from the inner antennocerebral tract as the axon passes through the MB calyx. However, babo^Fd4 PPNs also feature more severe phenotypes, particularly a complete lack of glomerular innervation and of adult-like axon collaterals with terminal swellings in the MB calyx, as well as failure to enter the LH and/or to elaborate higher order terminal branches. These latter phenotypes appear to be qualitatively different from those attributable to a simple loss of pruning, suggesting that TGFß signaling via baboon may have an additional role in re-extension and/or adult-specific targeting during metamorphosis (Marin, 2005).

In this study, the PPNs of the Drosophila olfactory system have been shown to play analogous functions in two neural circuits at different life stages. They do so by developmentally programmed disassembly and reassembly of synaptic connections during metamorphosis. The implications of these findings for the larval and adult olfactory systems and to neural circuit reorganization are discussed below (Marin, 2005).

Therefore, PPNs serve as relay interneurons connecting the antennal lobe to the MB calyx and the presumptive LH in larvae, just as previously characterized larval-born projection neurons do in adults. Each PPN generally targets its dendrites to one glomerular substructure in the larval AL, probably receiving input from one of the 21 olfactory receptor neurons of the dorsal organ. From there, the PPN's axon extends to higher brain centers, forming one or two large synaptic structures en passant on its way through the MB calyx to the LH. Electron microscopy studies with genetically encoded markers expressed separately in PPNs or in MB gamma neurons established that PPNs form functional synapses in the larval circuit and that MB gamma neurons are among their postsynaptic partners (Marin, 2005).

This analysis of these PPNs in the adult olfactory circuit confirmed and extended the developmental and wiring logic derived from previous analysis of larval-born PNs. Just like larval-born PNs, embryonic-born PPNs are prespecified to target their dendrites to particular glomeruli according to their birth order. Specifically, most PPNs are derived from the same anterodorsal neuroblast that later gives rise to about half the GH146-positive PNs. Like the larval-born PNs, PPNs exhibit stereotyped terminal arborization patterns in the LH. Interestingly, in the adult AL, PPNs innervate a distinct subset of glomeruli from either their larval-born anterodorsal cousins or the projection neurons generated by the lateral neuroblast. This indicates that, in addition to relaying activity from larva-specific olfactory receptor neurons earlier in development, PPNs expand the olfactory repertoire of the adult circuit (Marin, 2005).

In addition to serving larval-specific functions, one proposed function for larval circuits is to provide a foundation upon which adult circuits can be built. In the case of the olfactory circuit, however, previous analysis indicates that the adult-specific antennal lobes form adjacent to, but spatially distinct from, the larval antennal lobe (Jefferis, 2004). Analysis of PPN remodeling supports the notion that the adult circuit is constructed de novo rather than upon the larval circuit. A developmental timecourse analysis revealed that PPNs prune their dendrites and axon branches during early metamorphosis, so that only the main unbranched process from the cell body to the distal edge of the calyx remains by 12 hours APF. By contrast, the larval-born PNs begin to elaborate dendrites at the onset of puparium formation and restrict their processes to specific regions of the developing AL between 6 and 12 hours APF (Jefferis, 2004). Persistent projection neurons start exhibiting this type of localized dendritic outgrowth in the adjacent but distinct adult AL site only at 18 hours APF, around the time that adult-specific ORN axons arrive but prior to their invasion of the AL. This strongly implies that, far from providing contact-mediated cues for differentiating larval-born PNs, PPNs target glomeruli in the developing AL only after the larval-born PNs have established their dendritic target domains. The finding that PPN-specific glomeruli are intercalated with those targeted by dendrites of larval-born PNs, rather than occupying a spatially segregated domain in the adult antennal lobe, implies complex targeting rules in the establishment of wiring specificity of the adult circuit (Marin, 2005).

The fact that PPNs have clearly identifiable addresses for their dendritic targeting in the adult circuit suggested an interesting question: does assembly of the adult circuit depend on the disassembly of the larval circuit? The data suggest that neuronal reorganization appears to be separable into two at least partially independent events, pruning and re-extension. Even usp³ PPNs whose larva-specific dendrites and axons appear unpruned still exhibit the random fine filopodial extensions characteristic of wild-type neurons at 8-12 hours APF, and moreover target their new dendrites to appropriate adult antennal lobe glomeruli, as well as exhibiting adult-specific axon collaterals in the MB calyx and grossly wild-type terminal branches in the LH (Marin, 2005).

The fact that most usp³ persistent PNs still innervate appropriate glomeruli in the adult antennal lobe and have axons with adult characteristics would suggest that ultraspiracle-mediated execution of ecdysone signaling is required for pruning but not for responding to re-extension and/or targeting cues in the developing brain. However, most babo^Fd4 PPNs failed to target appropriately in the adult olfactory system. This difference in phenotypes may be due to differential perdurance of wild-type Usp versus Babo protein in single-cell MARCM clones and/or to differences in the severity of the alleles examined, consistent with the observation that babo^Fd4 PPNs show slightly more homogeneous pruning phenotypes at 8 hours APF. However, usp³ carries a missense mutation that alters an invariant arginine in the DNA-binding domain and blocks MB gamma neuron pruning completely. Thus, the possibility is favored that baboon is required for additional ultraspiracle-independent functions during metamorphosis, in the initiation of pruning, re-extension and/or targeting of adult olfactory structures (Marin, 2005).

Presynapses in Kenyon cell dendrites in the mushroom body calyx of Drosophila

Plastic changes at the presynaptic sites of the mushroom body (MB) principal neurons called Kenyon cells (KCs) are considered to represent a neuronal substrate underlying olfactory learning and memory. It is generally believed that presynaptic and postsynaptic sites of KCs are spatially segregated. In the MB calyx, KCs receive olfactory input from projection neurons (PNs) on their dendrites. Their presynaptic sites, however, are thought to be restricted to the axonal projections within the MB lobes. This study shows that KCs also form presynapses along their calycal dendrites, by using novel transgenic tools for visualizing presynaptic active zones and postsynaptic densities. At these presynapses, vesicle release following stimulation could be observed. They reside at a distance from the PN input into the KC dendrites, suggesting that regions of presynaptic and postsynaptic differentiation are segregated along individual KC dendrites. KC presynapses are present in γ-type KCs that support short- and long-term memory in adult flies and larvae. They can also be observed in α/β-type KCs, which are involved in memory retrieval, but not in α'/β'-type KCs, which are implicated in memory acquisition and consolidation. It is hypothesized that, as in mammals, recurrent activity loops might operate for memory retrieval in the fly olfactory system. The newly identified KC-derived presynapses in the calyx are, inter alia, candidate sites for the formation of memory traces during olfactory learning (Christiansen, 2011).

This study used several approaches to provide evidence that the KC dendrites within the calyx of larval and adult Drosophila are not exclusively postsynaptic. They also form presynaptic active zones (AZs), which was named KCACs. These findings are supported by data from two previous studies (Rolls, 2007; Pauls, 2010), which reported the presence of a presynaptic vesicle protein, Synaptobrevin, in KCs within the calyx. This study used a functional imaging approach to show that KCACs are able to release SVs. Furthermore, which different KC subtypes form KCACs was examined and a detailed description of the KCAC location within the calyx is provided. The presence of these previously undescribed KC-intrinsic presynaptic elements adds a new layer of complexity to the MB microcircuitry (Christiansen, 2011).

Within KC dendrites, AZs and PSDs are clearly organized into discrete subdomains. Here, the question emerges whether a given KC dendrite is either exclusively presynaptic or postsynaptic, or whether both presynaptic and postsynaptic domains can be present within the same KC dendrite in a consecutive fashion. MARCM identified single KCs, which showed Bruchpilot (BRP) puncta spatially segregated from claw-like regions that are thought to harbor the postsynaptic specializations of cholinergic PN::KC synapses. In parallel experiments, these claws were shown clustered the acetylcholine receptor Dα7, as was expected from previous work (Kremer, 2010). Thus, it appears likely that presynaptic and postsynaptic domains can be present within the same KC dendrite (Christiansen, 2011).

Based on BRP-RNAi analysis, it is estimated that ~20%-30% of all presynapses in the calyx are KCACs, in both adults and larvae. These synapses are apparently part of the general calyx microcircuitry. They might synapse onto PNs, KCs themselves, the anterior paired lateral (APL) neuron, modulatory neurons, or so-far-undescribed cells. From this analysis, it appears unlikely that PN boutons are direct postsynaptic partners of the KCACs, as the KCACs appear to be clearly physically segregated from the PN boutons. KCACs might, however, project onto PN axons (Christiansen, 2011).

It appears well possible that KCACs project onto the GABAergic APL neuron, which arborizes in the whole calyx. Within the insect antennal lobe, reciprocal dendrodendritic connections between the PNs and the partially GABAergic local interneurons (LNs) have been described. The PN neurites and the LNs are both transmissive and receptive in the antennal lobe, suggesting a computation between them. KCACs might be involved into similar computations in the calyx. This would be in accordance with EM studies in crickets that suggest presynapses in KCs that connect to GABAergic fibers in the MB calyx (Christiansen, 2011).

KCACs might also mediate KC::KC communication. In fact, dendritic segments of KCs that harbor presynapses appear to run in a parallel fashion. This arrangement could promote the communication between dendritic segments of KCs via dendrodendritic synapses. Such KC::KC synapses could therefore modulate signals originating from the distal segments of the arborizations, which carry odor-evoked signals. By these means, an effective computation between KCs could be accomplished before they transmit their input signals downstream (Christiansen, 2011).

Unfortunately, at the moment no general PSD markers are available in Drosophila. Moreover, the neurotransmitter used by KCs remains unknown. With a general postsynaptic marker or knowledge about the KC transmitter, it would have been possible to generated tools to identify the postsynaptic partners of KCACs. Yet currently, despite efforts, it is only possible to speculate about the postsynaptic partners of KCs in the calyx (Christiansen, 2011).

Memory traces are typically thought to be manifested as plastic changes in neuronal anatomy and physiology that occur in specific brain regions. Several lines of evidence indicate that MBs are causally involved in associative learning of olfactory stimuli. Flies with chemically ablated KCs or mutants lacking the MBs are deficient in olfactory learning. Learning was investigated in flies mutant for the adenylyl cyclase rutabaga (rut), which is suggested to act as a coincidence detector between conditioned stimulus (odor) and unconditioned stimulus (e.g., electric shock). Reexpression of a rut cDNA in a rut^- background within a subpopulation of KCs sufficed to restore odor learning. For appetitive learning, reexpression of rut in either PNs or KCs is sufficient to restore the mutant defect, whereas aversive learning is rescued only by rut reexpression in KCs. Reversible disruption of transmitter release in Drosophila KCs, using a temperature-sensitive dynamin transgene, UAS-shibire^ts1, was shown to block memory retrieval in α/β neurons and acquisition and stabilization of memory in α'/β' neurons. Together, these data imply that MBs play a major role in learning and memory. To form, stabilize, and retrieve memory, KCs use their presynapses. The KC presynapses are so far believed to reside in the lobes (Christiansen, 2011 and references therein).

The biogenic amines octopamine and dopamine are thought to mediate the unconditioned stimulus signal for learning olfactory associations, with octopamine representing appetitive stimuli and dopamine representing aversive stimuli. It has been shown that, in honeybees, sugar can be replaced by octopamine application to the calyx to trigger the conditioned proboscis-extension reflex. In the fruit fly, the amines octopamine and dopamine are released onto MB lobes and calyx. This holds also true for the larva. Therefore, the KCACs might be involved in appetitive learning as well as in aversive learning in fly and larva (Christiansen, 2011).

Notably, this study found that the KC subpopulations α and α/β, but not α'/β', form KCACs. This dichotomy correlates with functional differences in learning and memory that have been assigned to these KC classes in previous studies. For example, α'/β' KCs were reported to be required during and after training to acquire and stabilize olfactory memory, whereas output from α/β neurons was postulated to be required to retrieve memory. It has been proposed that, during acquisition, olfactory information received from PNs is first processed in parallel by the α/β and α'/β' KCs. Notably, activity in α'/β' KCs (which do not form KCACs) is supposed to trigger a recurrent loop between α'/β' KCs and dorsal paired medial neurons, which project to the MB lobes. This loop, in turn, might be necessary for memory consolidation in α/β neurons. Subsequently, memories could be stored in α/β neurons, whose activity is required during recall. As α'/β' neurons are devoid of KCACs, KCACs cannot be involved in the circuit described above. Instead, it is likely that additional, similar recurrent loops exist, which are mediated via KCACs. However, it remains unresolved how exactly KC::KC communication is organized anatomically and functionally. This study now proposes a newly discovered synapse population as candidate sites for KC::KC communication (Christiansen, 2011).

In the mammalian olfactory system, major feedback pathways exist, which project onto neurons one level lower in hierarchy. It has been shown that likewise in Drosophila activation of KCs induced a depolarization in cell bodies of PNs and LNs within the antennal lobes. It was thus suggested that MB lobes provide feedback to the ALs. Moreover, an additional memory trace appears to exist in the antennal lobe, in the PNs. It may therefore well be the case that KCs project onto PNs or onto feedback neurons via their KCACs (Christiansen, 2011).

An urgent question of the field concerns the identification of the postsynaptic partner cells of KC presynapses, which harbor memory traces during olfactory conditioning. It is generally assumed that MB-extrinsic downstream neurons involved in behavioral execution of learned behavior serve as postsynaptic partners here. The current findings raise the possibility that microcircuits inside the MB could be places for further modulation and computation of olfactory processing and/or memory formation and modulation. As a consequence, not only the communication to downstream neurons but also the representation of sensory information within the MB circuitry might be changed by experience. Future analysis using optophysiological tools at the KCACs, together with further anatomical work, should provide answers to these questions (Christiansen, 2011).

Metamorphosis of an identified serotonergic neuron in the Drosophila olfactory system

Odors are detected by sensory neurons that carry information to the olfactory lobe where they connect to projection neurons and local interneurons in glomeruli: anatomically well-characterized structures that collect, integrate and relay information to higher centers. Recent studies have revealed that the sensitivity of such networks can be modulated by wide-field feedback neurons. The connectivity and function of such feedback neurons are themselves subject to alteration by external cues, such as hormones, stress, or experience. Very little is known about how this class of central neurons changes its anatomical properties to perform functions in altered developmental contexts. A mechanistic understanding of how central neurons change their anatomy to meet new functional requirements will benefit greatly from the establishment of a model preparation where cellular and molecular changes can be examined in an identified central neuron. This study examined a wide-field serotonergic neuron in the Drosophila olfactory pathway and mapped the dramatic changes that it undergoes from larva to adult. Expression of a dominant-negative form of the ecdysterone receptor prevents remodeling. Different transgenic constructs were used to silence neuronal activity, and defects are reported in the morphology of the adult-specific dendritic trees. The branching of the presynaptic axonal arbors is regulated by mechanisms that affect axon growth and retrograde transport. The neuron develops its normal morphology in the absence of sensory input to the antennal lobe, or of the mushroom bodies. However, ablation of its presumptive postsynaptic partners, the projection neurons and/or local interneurons, affects the growth and branching of terminal arbors. These studies establish a cellular system for studying remodeling of a central neuromodulatory feedback neuron and also identify key elements in this process. Understanding the morphogenesis of such neurons, which have been shown in other systems to modulate the sensitivity and directionality of response to odors, links anatomy to the development of olfactory behavior (Singh, 2007).

Changes in the pattern of arborization of a mature neuron can come about as a consequence of removal of its afferent inputs or targets, chronic stress or other environmental inputs, such as delivered during learning or exercise. Many of these changes are effected through the action of growth factors and developmental signals acting in concert with steroid hormones and neuronal activity to modify the cytoskeleton or synaptic properties relevant to an altered functional setting. Metamorphosis in Drosophila (a period during which mature larval neurons are often altered to take on new adult functions) provides a context where the mechanistic underpinnings of such neuronal change can be genetically dissected (Singh, 2007).

This study used a genetic method to mark the serotonin-immunoreactive deutocerebral interneurons (CSDn), recently identified on the basis of serotonin immunoreactivity. While this preparation identifies a central neuron, it also has an important feature that allows the analysis of mechanisms underlying the changes it undergoes during remodeling. This system, because of the random nature of the RN2-FLP action, results in bilateral, unilateral or no excision of the FRT element in the Tub-FRT-CD2-FRT-Gal4 construct in the CSDn. Thus, it was possible to choose and analyze preparations where the CSDn from only one hemisphere was labeled: this facility is vital as it allows the analysis of contralateral and ipsilateral projections of the CSDn, without this being obscured by projections of the neuron from the other hemisphere to the same target sites. The GFP reporter in the RN2-Flp, Tub-FRT-CD2-FRT-Gal4, UAS mCD8-GFP strain is first detected very late in embryogenesis (stage 20), after the neuron has acquired its mature larval pattern. These features thus provide a preparation where an identified central neuron, whose function is known, can be followed and genetically manipulated as it changes its form in response to external and internal cues during metamorphosis (Singh, 2007).

The neuron, present during the larval stages, undergoes well-defined changes during pupation to give rise to a more complex adult architecture. What are the factors that regulate the stereotyped pruning and re-growth of arbors in the CSDn during metamorphosis? The results suggest that the interaction of external factors and autonomous properties (some of which could be identified) establish the homeostasis required during branching and establishment of the adult form (Singh, 2007).

Arbors from the larval neuron are removed by pruning over the first 20 hours of pupation before the adult pattern is elaborated. The EcR-B1 isoform, whose expression is typically seen in neurons that alter their larval form and contribute to the circuitry in the adult, is detected in CSDn. Down-regulating EcR in the CSDn during metamorphosis results in a failure of remodeling and the 'adult' neuron retains a larval morphology. The detailed mechanisms by which EcR signaling acts to bring about sculpting of cell shape are not totally understood and reports on Manduca sexta indicate that steroid-induced modifications in dendritic shape can be regulated by activity-dependent mechanisms (Singh, 2007).

Studies on the cellular and molecular mechanisms of pruning events during metamorphosis could provide valuable insights into understanding of degeneration in higher systems. These events require ubiquitin-mediated proteolysis, and it is known that local activity of caspases is involved in dendritic pruning in an identified sensory neuron. Degeneration of specific branches is followed by migration of glial cells into the site of activity. The role of these glia in bringing about pruning and in clearing debris from the vicinity requires further study (Singh, 2007).

The assembly of complex circuits is dependent on a carefully orchestrated interplay of intrinsic and extrinsic cues. Does activity play a role in determining neuronal shape? Spontaneous and evoked activity in the CSDn were silenced using different methods and changes were observed in the dendritic arbors as well as in presynaptic terminals. The effects on the terminals and dendrites are possibly due to distinct mechanisms and will be discussed separately (Singh, 2007).

The strongest effects on presynaptic terminal branching were produced by expression of TeTxLC, which blocks synaptic release, and a dominant-negative Shi protein, which affects receptor-mediated endocytosis. Apart from blocking neuronal activity by abrogating synaptic vesicle release, both treatments could potentially affect axon growth. Consistent with this is the observation that TeTxLC expression affects re-growth of CSDn terminals during metamorphosis, while pruning occurred normally. Weak anatomical defects have also been described in other, non-modulatory neurons, some of which could be explained by a role in the regulation of levels of cell adhesion molecules (Singh, 2007).

Increases in size and branching pattern of the dendritic trees is a robust effect occurring notably when neuronal activity was silenced by Kir_2.1expression. In the third instar larva, expression of TNT-G leads to an increase in dendritic arbors with no significant effect on the presynaptic terminals. Expression using the RN2-Flp, Tub-FRT-CD2-FRT-Gal4, stock initiates in the fully developed larval neuron; hence, the changes in dendritic branches are likely to be a consequence of lack of neuronal activity, rather than a developmental effect. What are the mechanisms by which neuronal activity can alter morphologies of neurons? It has been demonstrated that tetanus toxin expression in motorneurons not only affects its presynaptic release because of cleavage of synaptobrevin, but also alters synaptic input by an as yet unknown mechanism. The finding of altered dendritic morphology supports the possibility that homeostatic alterations occur to compensate for a lack of activity (Singh, 2007).

A large body of data provides evidence for retrograde signaling in the development and consolidation of synapses. The observation of expanded dendritic trees upon expression of a dominant negative form of Glued, while intriguing, is difficult to explain in this light. The changes that were seen are in the dendritic (post-synaptic) field when retrograde transport is blocked cell-autonomously. While this needs further investigation, a possible explanation is that these effects are an indirect consequence of physiological alterations at the presynaptic terminals. Local morphological changes in neurons can be effected by sequestration of proteosomes and other molecules at different regions of the cell in response to activity, which could result in sculpting of cellular architecture due to altered protein composition at different cellular regions (Singh, 2007).

Defects in branching observed by abrogation of vesicle release at the synapse in a serotonergic neuron could implicate this modulator in paracrine or autocrine signaling in regulation of neuronal outgrowth, target selection and synapse formation. Such effects have been demonstrated in the gastropod Helisoma , as well as in Drosophila, where serotonin levels regulate neuronal branching and modulate the development of neuronal varicosities in the central nervous system. In these experiments, no significant changes were detected in the branching pattern of CSDn upon strong reduction of serotonin (and dopamine) using a temperature sensitive allele of dopa decarboxylase. Furthermore, unlike in M. sexta, where afferents are necessary for the formation of glomerular tufts of the serotonergic neuron within the antennal lobe, development of the CSDn occurs normally in the absence of sensory input from the antenna (Singh, 2007).

The olfactory pathway consists of afferent sensory neurons, local integrating neurons and projection neurons. Circuitry for an additional level of integration exists in the atypical projection neurons (aPNs), the antennal posterior superior protocerebral neuron (APSP), the giant symmetric relay interneurons (GSI) and the bilateral ACT relay interneurons (bACT). The architecture as well as the serotonergic nature of the CSDn closely resembles the S1 neuron in M. sexta, which receives input from bilateral projections in the protocerebrum and terminates in the lobe contralateral to the soma to modulate the activity of interneurons. It is proposed that the ipsilateral dendrites receive input from as-yet unidentified neural elements in the antennal lobe, while some axonal arbors are postsynaptic to interneurons in the calyx of the mushroom bodies and the lateral horn. It is speculated that the targets of the terminal arbors are either the PNs or the LNs since their ablation results in a reduction in branching. This architecture, which needs to be confirmed by electron microscopic analysis, provides circuitry for 'top-down' regulation of the primary olfactory center. It seems very likely that the CSDn, like its counterpart in the moth, responds to mechanosensory stimulation, providing an important role in responses to odor stimulation coupled with airflow, as would be expected in insects during flight. The modulatory effects of this large field neuron on its partners in the antennal lobe needs to be investigated by high-resolution functional imaging (Singh, 2007).

This study describes a serotonergic neuron whose anatomy suggests feedback integration within the antennal lobe of insects. The neuron undergoes remodeling during pupal life from a simple larval to a more complex adult pattern. These studies suggest that the morphology of the dendritic arbors that terminate in the lobe ipsilateral to the soma is regulated by neuronal activity. The arborization of terminal arbors depends on vesicle recycling, endocytosis and Dynein-dependant retrograde transport. These findings demonstrate a useful identified-neuronal preparation where developmental mechanisms and remodeling can be studied in the context of olfactory behavior (Singh, 2007).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Central synaptic mechanisms underlie short-term olfactory habituation in Drosophila larvae

Naive Drosophila larvae show vigorous chemotaxis toward many odorants including ethyl acetate (EA). Chemotaxis toward EA is substantially reduced after a 5-min pre-exposure to the odorant and recovers with a half-time of ~20 min. An analogous behavioral decrement can be induced without odorant-receptor activation through channelrhodopsin-based, direct photoexcitation of odorant sensory neurons (OSNs). The neural mechanism of short-term habituation (STH) requires the (1) Rutabaga adenylate cyclase; (2) transmitter release from predominantly GABAergic local interneurons (LNs); (3) GABA-A receptor function in projection neurons (PNs) that receive excitatory inputs from OSNs; and (4) NMDA-receptor function in PNs. These features of STH cannot be explained by simple sensory adaptation and, instead, point to plasticity of olfactory synapses in the antennal lobe as the underlying mechanism. These observations suggest a model in which NMDAR-dependent depression of the OSN-PN synapse and/or NMDAR-dependent facilitation of inhibitory transmission from LNs to PNs contributes substantially to short-term habituation (Larkin, 2010).

Experience-induced plasticity of synapses is believed to be a fundamental mechanism of learning and memory. However, central synaptic changes that underlie memory have not been clearly defined, even for relatively simple nonassociative learning processes such as habituation (Larkin, 2010).

During habituation, unreinforced exposure to a repeated or prolonged stimulus results in a reversible decrease in response to that stimulus. Habituation probably serves as an important building block for more complex cognitive function. By allowing unchanging or irrelevant stimuli to be ignored, it allows cognitive resources to be focused on more salient stimuli (Larkin, 2010 and references therein).

The neural basis of short-term habituation (STH) is best studied in the marine snail, Aplysia californica. Here STH (lasting ~30 min) of the defensive gill-withdrawal reflex in response to tactile stimulation of the siphon is thought to arise from presynaptic depression of transmitter release at sensorimotor synapses. However, even here, presynaptic plasticity may not be cell-autonomous, potentially requiring, for instance, activity of yet-to-be-identified interneurons (Larkin, 2010).

Several forems of habituation have been described in Drosophila and are often shown to require the function of genes that regulate cAMP-dependent forms of associative memory. For instance, habituation of proboscis extension reflex as well as odor-evoked startle reflex in adult Drosophila requires rutabaga (rut)-encoded Ca2+/calmodulin-sensitive adenylyl cyclase. In addition, habituation of the ethanol-induced startle response requires the shaggy/GSK-3 signaling pathway. Despite such pioneering observations, the mechanisms of these various forms of habituation, even whether the primary neuronal changes are purely sensory or involve plasticity of central synapses (involving centrally located interneurons that may integrate various different kinds of modulatory, inhibitory, and excitatory inputs), remain poorly understood (Larkin, 2010).

Recent advances in understanding the circuitry that underlies Drosophila olfactory behavior, as well as the development of new tools to perturb identified neurons in vivo, has opened the opportunity for understanding mechanisms of olfactory habituation at the level of the underlying neural circuitry (Larkin, 2010).

In the larval olfactory system, 21 olfactory sensory neurons (OSNs), each expressing a single odorant receptor (together with the broadly expressed Or83b co-receptor), synapse, respectively, onto 21 cognate projection neurons (PNs) within 21 glomeruli in the larval antennal lobe (AL). Local, predominantly GABAergic interneurons (LNs) synapse widely within the antennal lobe, interlinking different glomeruli. Various neuromodulatory synapses also form on the larval antennal lobe and mushroom body. Thus, odorant-stimulated signals in sensory neurons are processed in the antennal lobe, modulated by motivational or emotional states, and relayed through projection neurons to higher brain centers (Larkin, 2010).

Previous work has shown that in Drosophila larvae, olfactory chemotaxis decreases after odorant pre-exposure. This study shows that this behavioral habituation, alternatively referred to as 'adaptation' by some previous investigators, arises from mechanisms of synaptic plasticity. This study demonstrates that odorant receptor activation is not necessary for olfactory habituation; however, local interneuron activity and projection neuron signaling is necessary. These observations suggest a model in which habituation occurs by a pathway in which NMDA receptors in projection neurons signal depression of OSN-PN synapses and/or facilitation of LN-PN synapses (Larkin, 2010).

Previous studies have not clearly discriminated between peripheral and central mechanisms. Indeed, the term 'adaptation,' better applied to sensory neuron changes such as receptor desensitization, has often been used interchangeably with the term 'habituation', which is usually restricted to behavioral changes arising from central synaptic mechanisms (Larkin, 2010). .

The form of larval olfactory STH characterized in this study displays at least some of the defining behavioral characteristics of habituation. First, there is a behavioral decrement in response to repeated or sustained application of a particular stimulus. Second, STH shows spontaneous recovery with time in the absence of the habituating stimulus. And third, STH is susceptible to dishabituation when habituated larvae are presented with of a strong or noxious stimulus. The property of dishabituation is particularly significant, as an important way of distinguishing between habituation and either fatigue or sensory adaptation. Dishabituation shows that the habituated animal retains the capability to respond and suggests that the attenuated behavioral response arises from some form of active suppression. Thus, the behavioral data suggest (1) that the term 'habituation' may be better used in place of 'adaptation,' while referring to the behavioral phenomenon that was studied; and (2) that STH probably arises from central synaptic mechanisms, rather than sensory neuron adaptation (Larkin, 2010).

Three main lines of data support the conclusion that STH arises from a central synaptic mechanism that resides in the antennal lobe, rather than from adaptation of olfactory receptor signaling in the OSN. First, behavioral decrements similar to STH can be induced by direct depolarization of OSNs, indicating that STH may potentially be induced by processes stimulated by activation action-potential firing in OSNs, independently of olfactory receptor activation. Second, and more striking, STH requires synaptic-vesicle exocytosis from local interneurons during the process of odorant exposure, when STH is being established. This requirement is incompatible with an exclusively sensory mechanism. Third, STH requires the function of NMDA receptors on postsynaptic projection neurons. This last observation also provides a particularly strong argument for a synaptic mechanism, indicating a need for plasticity of OSN and/or LN synapses made onto dendrites of projection neurons in the antennal lobe. Given that OSNs are excitatory and LNs are primarily inhibitory, it appears most likely that NMDAR functions in PNs to depress excitatory OSN-PN synapses and/or to potentiate inhibition by strengthening the LN-PN synapse. It is suggestd that the LN-PN mechanism may be involved because (1) LN transmission seems necessary for both induction and expression of habituation; and (2) the process of dishabituation could be attractively explained as arising from the inhibition of local inhibitory synapses through descending neuromodulation. A requirement for facilitation of the LN-PN synapse would be consistent with previous studies (Sachse, 2007) showing that adult-long-term olfactory habituation is associated with an increase in odor-evoked calcium fluxes in GABAergic processes within the Drosophila antennal lobe (Larkin, 2010).

Based both on experimental and theoretical arguments, a simple model is suggested for short-term olfactory habituation. Since this is a model, no claim is being made to to having ruled out additional major contributing mechanisms, It is suggested that during initial odorant pre-exposure, dendritic NMDA receptors on projection neurons detect and respond to membrane depolarization occurs coincident with transmitter release from LNs. Calcium entry through dendritic NMDA receptors may trigger a local retrograde signal required for facilitation of transmitter release from the LNs. Although existing data do not rule out functions for rutabaga in higher larval brain centers, it is suggested that either the generation of a retrograde signal in PN dendrites or the presynaptic response of LNs to this signal could be dependent on the rut adenylate cyclase. In habituated animals, facilitation of GABA release would reduce odor-evoked projection neuron outputs to higher brain centers, thereby reducing olfactory behavior. As NMDAR signaling would only occur at active glomeruli, this mechanism can account not only for the observed odor selectivity of habituation, but also the instances of cross-habituation (Larkin, 2010).

Such a model also naturally suggests a hypothesis for the mechanism of dishabituation: namely, that dishabituating stimuli cause release of neuromodulators that act to reduce GABA release from local inhibitory synapses (Larkin, 2010).

Given the remarkable similarities in the anatomical organization of insect and mammalian olfactory systems, a significant conservation of olfactory mechanisms would be expected. In rodents, at least two forms of habituation have been described, lasting 2-3 and 30-60 min, respectively: the latter equivalent in timescale to larval STH described in this study. Consistent with a similar underlying mechanism, the more persistent form of olfactory habituation can be blocked by an N-methyl-D-aspartate (NMDA) receptor antagonist in the olfactory bulb, a structure homologous to the insect antennal lobe. Thus, larval STH described in this study has some similarities to a previously characterized form of mammalian olfactory habituation. Analysis of the underlying mechanisms is therefore likely to provide directly transferable insights in mammalian olfaction. The data make the prediction that the activity of mammalian olfactory interneurons, either periglomerular or granule cells, is critical for the establishment and display of at least one timescale of olfactory habituation (Larkin, 2010).

In addition to providing some insight into mechanisms of olfactory habituation in mammals, it possible that circuit mechanisms of larval olfactory habituation are relevant to other forms of behavioral habituation. In at least three previous instances, increased inhibition has been associated with attenuated behavior. For example, habituation of an escape reflex mediated by the lateral giant fibers in the crayfish has been associated with enhanced GABAergic transmission onto giant fibers. Similarly, LTP of inhibitory synapses controlling excitability of the Mauthner cell has been associated with reduced escape behavior in goldfish. Furthermore, ethanol, a potentiator of GABA synapses, has been shown to enhance habituation of a motor pathway in the frog spinal cord. Could these different instances of habituation all involve circuit mechanisms similar to those used in Drosophila larval olfactory behavior (Larkin, 2010)?

In all brain regions, principal/projection neurons are subject to inhibitory feedback modulation and a pathway that has been appreciated as potentially essential for neuronal homeostasis. Potentiation of inhibitory feedback triggered by the pattern of principle cell activation would be predicted to preferentially dampen this particular output pattern. Thus, the circuit mechanism suggest in this study is theoretically generalizable to other and more complex forms of habituation. Further experiments will be required to determine the validity of this very testable hypothesis (Larkin, 2010).

The importance of habituation has been underlined by the fact that deficits in sensory gating and pre-pulse inhibition (PPI), processes with similarities to habituation, have been linked with various neurological problems, including autism and schizophrenia. Indeed, a circuit model for understanding schizophrenia has specifically proposed that altered negative feedback in the hippocampus may underlie both positive and negative symptoms of schizophrenia (Larkin, 2010).

In addition, defects in habituation or habituation-like processes have been described in Fragile X syndrome and migraines. It has also been shown to have important effects relating to learning disabilities, age-related changes in learning, and substance abuse. If mechanisms of olfactory habituation prove to be general, then studies of olfactory plasticity may prove relevant for other forms of cognition as well as for human neurological disease (Larkin, 2010).

Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila

The Drosophila antennal lobe is organized into glomerular compartments, where olfactory receptor neurons synapse onto projection neurons. Projection neuron dendrites also receive input from local neurons, which interconnect glomeruli. This study investigated how activity in this circuit changes over time when sensory afferents are chronically removed in vivo. In the normal circuit, excitatory connections between glomeruli are weak. However, after receptor neuron axons projecting to a subset of glomeruli were chronically severed by removal of antennae, it was found that odor-evoked lateral excitatory input to deafferented projection neurons was potentiated severalfold. This was caused, at least in part, by strengthened electrical coupling from excitatory local neurons onto projection neurons, as well as increased activity in excitatory local neurons. Merely silencing receptor neurons was not sufficient to elicit these changes, implying that severing receptor neuron axons is the relevant signal. When the neuroprotective gene Wallerian degeneration slow (Wld^S; Hoopfer, 2006) was expressed in receptor neurons before severing their axons, this blocked the induction of plasticity. Because expressing WldS prevents severed axons from recruiting glia, this result suggests a role for glia. Consistent with this, it was found that blocking endocytosis in ensheathing glia blocked the induction of plasticity. In sum, these results reveal a novel injury response whereby severed sensory axons recruit glia, which in turn signal to central neurons to upregulate their activity. By strengthening excitatory interactions between neurons in a deafferented brain region, this mechanism might help boost activity to compensate for lost sensory input (Kazama, 2011).

The results demonstrate that when all the ORN afferents to a subset of glomeruli are removed, excitatory interactions between glomeruli become stronger. As a result, deafferented PNs acquire robust responses to odors. Whereas normal PNs respond selectively to different odor stimuli, deafferented PNs respond nonselectively. This presumably reflects the fact that each excitatory LN (eLN; excitatory input to projection neurons) arborizes in most or all glomeruli. Thus, these PNs likely pool indirect excitatory input from all surviving ORNs (Kazama, 2011).

The key finding of this study is that that removing ORN input causes an upregulation of excitatory connections between glomeruli. Previously, it was shown that overstimulating one ORN type causes an upregulation of inhibitory input to a glomerulus (Sachse, 2007). Both of these phenomena may be seen as forms of compensatory plasticity. Compensatory plasticity also occurs in the mammalian olfactory bulb at several synaptic sites (Kazama, 2011).

Silencing electrical activity in ORNs was not sufficient to induce the same functional changes produced by severing ORN axons. This implies that the trigger is not the loss of electrical activity, but rather a molecular signal that is produced by severed axons. Mis-expressing WldS in ORNs blocks induction, and this implies that WldS suppresses the signal that severed axons produce. Suppressing endocytosis in ensheathing glia also blocks induction. This suggests that the signal produced by severed axons acts on glial receptors that require endocytosis for signal transduction. It is interesting that blocking endocytosis in astrocytes had no effect, because astrocytes interact with neurons in other systems. It is possible that astrocytes are involved in this process, but astrocytic endocytosis is not required (Kazama, 2011).

It is notable that both the manipulations that blocked the induction of plasticity (mis-expressing WldS in ORNs, or blocking endocytosis in ensheathing glia) also block the recruitment of ensheathing glia into deafferented glomeruli after ORNs are removed. This would appear to suggest that the same signal triggers both neural plasticity and morphological changes in glia. However, these signaling cascades clearly diverge: the recruitment of glial membranes to degenerating neurons is blocked by mutating the glial transmembrane receptor draper, whereas draper is not required for the plasticity described in this study. Interestingly, removing only one antenna was not sufficient to induce plasticity in glomerulus VM2 PNs. This manipulation kills half the ORNs that target these PNs. It should be noted that removing both palps kills fourfold fewer ORNs than removing one antenna, and this manipulation also affects fewer glomeruli, yet this was sufficient to induce plasticity in palp PNs. Removing both palps is also sufficient for glial mobilization and phagocytosis in the palp glomeruli (Doherty, 2009). The current results argue that the relevant factor is not the total number of afferents that are killed, but the proportion of live and dead axons in a given glomerulus. However, it also seems that killing all the ORNs that target a single glomerulus is not sufficient. This conclusion arises from the finding that removing the ipsilateral antenna did not produce potentiation in glomerulus V PNs, which receive strictly ipsilateral antennal input. This result implies that some minimum number of glomeruli must be completely deafferented to trigger the described phenomenon (Kazama, 2011).

The results indicate that after some ORNs are chronically removed, several changes occur in the antennal lobe circuit over time. First, depolarization propagates more effectively from eLNs to PNs. This could reflect increased gap junctional conductance from eLNs onto PNs. However, the possibility cannot be excluded that it is the result of a change in the intrinsic properties of eLNs that produces better propagation of voltages from the eLN soma to the site of the eLN-PN gap junctions. In this latter scenario, there would not necessarily be a change in gap junction conductance. Because good voltage clamp in eLNs cannot be achieved, these alternatives could not be evaluated directly, but two pieces of evidence argue for a change in the gap junction itself. First, the gap junction subunit composition of these electrical connections is evidently changed, because it was observed that electrical coupling from eLNs onto PNs is no longer completely dependent on the ShakB.neural subunit. Whereas in normal flies odor-evoked lateral excitation is abolished by the shakB2 mutation, which eliminates ShakB.neural, odor-evoked lateral excitation is not abolished in mutant antennal PNs after chronic antennal removal. Second, no significant change was found in any intrinsic properties of eLNs, including input resistance, resting potential, or excitability (Kazama, 2011).

A second change that occurs in chronically deafferented PNs is that spontaneous membrane potential fluctuations are larger in these PNs compared with acutely deafferented PNs. This may result from the increased input from eLNs onto PNs (Kazama, 2011).

A third change is that odors elicit stronger depolarization in eLNs. The intrinsic excitability of eLNs does not significantly increase, and therefore this change is likely caused by increased synaptic drive to eLNs. This potentiated synaptic drive may originate from PNs: because odor responses in deafferented PNs become larger after the induction of plasticity, and because PNs make chemical as well as electrical synapses onto eLNs, a net increase in the synaptic drive that PNs provide onto eLNs would be expected. In addition, it is possible that ORN-to-eLN synapses are potentiated (Kazama, 2011).

In sum, the net effect of these changes is to produce more robust activity in chronically deafferented PNs, compared with acutely deafferented PNs. These findings also help explain why plasticity is expressed globally rather than locally: if eLNs are responding more robustly to odors, and each eLN innervates all glomeruli, then this increased excitation should propagate across the antennal lobe (Kazama, 2011).

Whereas normal PNs are selective for odor stimuli, the potentiated odor responses of deafferented PNs are comparatively nonspecific. This presumably reflects the fact that each eLN arborizes in most or all glomeruli and so likely pools input from all surviving ORN types. Nevertheless, the odor responses of deafferented PNs may still be useful from the perspective of higher olfactory brain regions. Because acutely deafferented PNs regain normal levels of activity over time, this type of plasticity should tend to restore normal levels of activity in higher olfactory regions. This might help maintain the sensitivity of these regions to sensory signals, or maintain tropic support to these regions (Kazama, 2011).

More broadly, it is speculated that the phenomenon describe in thes study might reflect a general injury response in the Drosophila nervous system, and perhaps also a phenomenon that occurs during normal nervous system development. By triggering the upregulation of specific interactions between surviving neurons following the death of other neurons, this mechanism might help increase the number of neurons that are driven by active afferents. This could be a generally useful adaptation to neuronal death because it should tend to maintain total neural activity within a normal dynamic range (Kazama, 2011).

The reorganization of central sensory representations following changes in sensory input is generally thought to reflect changes in the strength of chemical synapses. The results suggest that central electrical synapses can also be persistently altered following sensory deafferentation. It is well known that neuromodulators can produce short-term changes in the strength of electrical synapses, as illustrated by studies in the vertebrate retina and crustacean stomatogastric ganglion. There are fewer examples of long-term changes in electrical synapse strength, but a growing literature suggests that this may be a fundamental mechanism of neural plasticity (Kazama, 2011).

The reorganization of central sensory representations following sensory deafferentation is sometimes assumed to be triggered by reduced electrical activity, not cell death. However, there is growing evidence that changes in electrical activity may produce synaptic plasticity via signaling pathways that are also linked to injury and inflammation. Thus, changes in electrical activity can produce synaptic plasticity by 'co-opting' signaling systems that are involved in injury responses. The results show that, in the Drosophila antennal lobe, some functional rearrangements following deafferentation can be specific responses to cell death signals, and are not necessarily induced by electrical silencing. In this study, reduced electrical activity was disambiguated from cell death because genetic tools were used to create 'undead' severed axons. The results are reminiscent of studies in vertebrates showing that sensory afferent death can produce changes in target brain regions that are not mimicked by electrical silencing using pharmacological manipulations (Kazama, 2011).

Finally, these findings provide a new window on neural-glial interactions. In mammals, there is good evidence that glia can modulate synaptic transmission and neural excitability. In both mammals and in Drosophila, glia also play important roles following injury. In particular, there are many instances of sensory afferent injury causing morphological changes in glia and glial proliferation in target brain regions . However, it is not entirely clear how such glial responses might affect neuronal physiology and sensory codes in these brain regions. The results illustrate specific cellular and synaptic changes in a sensory circuit that result from glial responses to sensory afferent injury. More broadly, the results illustrate the power of Drosophila as a genetically tractable model for studying neural-glial interactions in vivo (Kazama, 2011).

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date revised: 5 August 2011

Zygotically transcribed genes

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