Chemical Senses Advance Access originally published online on October 27, 2006
Chemical Senses 2007 32(1):65-89; doi:10.1093/chemse/bjl030
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The Drosophila Larva as a Model for Studying Chemosensation and Chemosensory Learning: A Review
1 Universität Würzburg, Biozentrum, Am Hubland, Lehrstuhl für Genetik und Neurobiologie, D-97074 Würzburg, Germany 2 University of Fribourg, Department of Biology, 10, chemin du Museé, CH-1700 Fribourg, Switzerland
Correspondence to be sent to either author: Bertram Gerber, e-mail: bertram.gerber{at}biozentrum.uni-wuerzburg.de; Reinhard F. Stocker, e-mail: reinhard.stocker{at}unifr.ch
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Abstract |
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 Top Abstract Smell and taste systemsLearningAcknowledgementsReferences |
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Understanding the relationship between brain and behavior is the fundamental challenge in neuroscience. We focus on chemosensation and chemosensory learning in larval Drosophila and review what is known about its molecular and cellular bases. Detailed analyses suggest that the larval olfactory system, albeit much reduced in cell number, shares the basic architecture, both in terms of receptor gene expression and neuronal circuitry, of its adult counterpart as well as of mammals. With respect to the gustatory system, less is known in particular with respect to processing of gustatory information in the central nervous system, leaving generalizations premature. On the behavioral level, a learning paradigm for the association of odors with food reinforcement has been introduced. Capitalizing on the knowledge of the chemosensory pathways, we review the first steps to reveal the genetic and cellular bases of olfactory learning in larval Drosophila. We argue that the simplicity of the larval chemosensory system, combined with the experimental accessibility of Drosophila on the genetic, electrophysiological, cellular, and behavioral level, makes this system suitable for an integrated understanding of chemosensation and chemosensory learning.
Key words: Drosophila, larva, learning, olfaction, taste
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Smell and taste systems |
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TopAbstractSmell and taste systemsLearningAcknowledgementsReferences |
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Chemosensory systems serve animals to cope with their chemical environment; this may sound trivial but has a rather different thrust than suggesting that chemosensory systems need to faithfully reflect the outside world. To be sure, neuronal activity along the chemosensory pathways must allow the animal to tell apart different chemicals—but only insofar as may be required to differentially behave toward them. Thus, depending on the behavioral demands, different inputs may or may not lead to different activity patterns in the chemosensory pathways; in turn, however, differences in activity pattern need to reflect differences in input. In other words, the discriminative demands stem from the potential behavioral matters of concern, not from the diversity of inputs: what is at issue is whether different chemicals should make a behavioral difference to the animal. Interestingly, sensory modalities, and in particular olfaction and taste, can differ tremendously in terms of this required discriminative ability.
This review focuses in its first part on chemosensory processing and asks how discriminative patterns of neuronal activity come about in chemosensory pathways. In the second part, it discusses whether and how odors can be recognized as the same as those odors that had been previously encountered. Given the diversity of chemicals involved, given the combinatorial possibilities with which these chemicals can occur in nature, and given the temporal variability of the chemical environment, understanding how these tasks can be managed is a real challenge. A breakthrough was achieved upon the identification of odorant receptor genes in rodents (Buck and Axel 1991
) and some years later in Caenorhabditis elegans (Sengupta et al. 1996) and Drosophila (Clyne et al. 1999; Vosshall et al. 1999). Their expression patterns in particular are useful for dissecting the circuits underlying olfaction (Ressler et al. 1994; Vassar et al. 1994; Gao et al. 2000; Vosshall et al. 2000). Remarkably, these studies confirm earlier convictions that the olfactory systems of mammals and insects are organized according to common principles (Hildebrand and Shepherd 1997; Strausfeld and Hildebrand 1999; Ache and Young 2005). Yet, insect chemosensory systems comprise only a fraction of the cell numbers involved in the vertebrate systems, providing an attractively simple option for investigating the chemical senses. What is more, in holometabolous insects, such as Drosophila, the larval chemosensory system offers an even simpler alternative. Apart from a study in larval Manduca sexta (Itagaki and Hildebrand 1990), little was known until recently about the organization of larval olfactory systems. Adults and larvae are anatomically and behaviorally much different, reflecting their different lifestyles. For example, adult Drosophila flies need to find food (as well as mates, egg-laying sites, etc.), which requires sophisticated odor-driven behavior. Fly larvae, in contrast, live on their food source and hence do not need long-range odor detection to find food. Although larvae respond to a variety of chemicals (Rodrigues 1980; Cobb 1999; Heimbeck et al. 1999; Cobb and Domain 2000), one may expect the chemosensory systems of both developmental stages to display significant differences in terms of cell number, organization, and behavioral function.
A number of recent papers promote the larval olfactory system of Drosophila as a "elementary" model system (Fishilevich et al. 2005; Kreher et al. 2005; Masuda-Nakagawa et al. 2005; Melcher and Pankratz 2005; Michels et al. 2005; Ramaekers et al. 2005) rendering a review on larval chemosensation timely.
Sensory level
Cephalic chemosensory organs
The cephalic chemosensory apparatus of the larva includes 3 external sense organs, dorsal organ (DO), terminal organ (TO), and ventral organ (VO), as well as 3 internal, pharyngeal organs (Figure 1C–H) (Singh RN and Singh K 1984; Singh 1997; Python and Stocker 2002a; Gendre et al. 2004). Each of them consists of several sensilla, a sensillum comprising one to several sensory neurons and 3 accessory cells, all housed below a common cuticular structure or terminal pore.
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The DO is composed of the central "dome" (Figure 1E) and 6 peripheral sensilla. The dome, whose wall is perforated by thousands of pore tubules, is innervated by the profuse dendritic arbors of 21 olfactory receptor neurons (ORNs). An olfactory function of the dome is demonstrated by electrophysiological recordings (Oppliger et al. 2000
; Kreher et al. 2005) and by combined toxin expression and behavioral studies (Heimbeck et al. 1999; Larsson et al. 2004; Fishilevich et al. 2005). Indeed, by expressing diphtheria toxin or by genetically ablating the atypical odorant receptor OR83b that is required for normal ORN function in the 21 sensory neurons of the dome, both latter studies demonstrate that these neurons are the sole larval ORNs. In Musca, 5 of the 6 peripheral sensilla of the DO, most of the TO sensilla and 1 of the 4 VO sensilla are thought to be taste sensilla (Chu and Axtell 1971; Chu-Wang and Axtell 1972a, 1972b). TO sensilla belong to at least 6 different types, collected in a distal group of 11 sensilla and a dorsolateral group of 2 sensilla that are discernable from the outside (chevrons in Figure 1F), and, in analogy to the situation in Musca (Chu-Wang and Axtell 1972a), likely a third one that is not. Thus, the DO is a mixed smell and taste organ, whereas the TO, the VO, and the pharyngeal sensilla serve gustatory function. However, apart from chemosensory neurons, thermosensory (Liu, Yermolaieva, et al. 2003), mechanosensory, or hygrosensory neurons may be present.
The DO, TO, and VO all have their proper ganglion (Figure 1C,G). The ganglion of the DO contains 36–37 sensory neurons (Python and Stocker 2002a). The 21 ORNs among them extend their dendrites as 7 triplets into the dome. The dendrites of 3 other neurons project toward the dorsolateral sensilla of the TO (Kankel et al. 1980; Frederik and Denell 1982; Heimbeck et al. 1999; Python and Stocker 2002a), whereas the remaining cells innervate the 6 peripheral sensilla of the DO. The TO and VO ganglia include 32 and 7 sensory neurons, respectively (Python and Stocker 2002a).
The 3 pharyngeal sense organs consist of several sensilla each, comprising 1–9 sensory neurons (Singh RN and Singh K 1984; Python and Stocker 2002a; Gendre et al. 2004). Gustatory and mechanosensory function is suggested by the presence of pores or bristles, respectively. The dorsal and ventral pharyngeal sense organs, both of which are situated behind the mouth hooks, contain 17 and 16 neurons, respectively; the posterior pharyngeal sense organ consists of 2 sensilla with 3 neurons each (Figures 1C and 3).
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As in adult flies, central olfactory projections remain supraesophageal and are collected in the antennal lobe (AL), whereas taste information is sent to multiple target areas in the subesophageal ganglion (SOG [Figure 1C], which do not show any obvious glomerulus-like organization). Different from adults, however, all olfactory projections remain ipsilateral. Neurons from the DO ganglion, regardless of their olfactory or gustatory nature and regardless of whether their dendritic tips extend to the DO or TO, connect to the brain via the antennal nerve (Tissot et al. 1997
; Python and Stocker 2002a) (Figure 1C,I). The supraesophageal labral nerve carries the afferents from the dorsal pharyngeal organ and probably from the posterior pharyngeal organ, whereas the subesophageal maxillary and labial nerves comprise those from the TO and VO ganglia and from the ventral pharyngeal organ, respectively (cf., Schmidt-Ott et al. 1994; Campos-Ortega and Hartenstein 1997; Python and Stocker 2002a; Gendre et al. 2004) (Figure 1C).
Expression of olfactory receptors
Olfactory receptors (ORs) define the range of detectable odors. The OR expression pattern across the population of ORNs provides the basis for a combinatorial activation in their target areas, which allows the animal to discriminate a practically unlimited number of different odors (for mammals: Buck and Axel 1991; Ressler et al. 1994; Vassar et al. 1994; for fruitflies: Clyne et al. 1999; Vosshall et al. 1999, 2000; Gao et al. 2000).
In adult Drosophila, 2 subfamilies of 7-transmembrane chemosensory receptors are known, an OR family comprising 62 members (Clyne et al. 1999; Vosshall et al. 1999; Robertson et al. 2003; Hallem et al. 2006) and a family of gustatory receptors (GRs) with 60 members (Clyne et al. 2000; Dunipace et al. 2001; Scott et al. 2001; Robertson et al. 2003; Hallem et al. 2006). Similar to mammalian ORNs, ORNs of the adult fly typically express only a single "conventional" OR that is responsible for the ligand specificity of the ORN (Clyne et al. 1999; Vosshall et al. 1999; Dobritsa et al. 2003; Hallem et al. 2004; Goldman et al. 2005). For a substantial subset of these ORs, odorant response spectra as well as their expression in identified types of ORNs are reported (Dobritsa et al. 2003; Hallem et al. 2004). ORNs expressing a given OR converge onto 1 or 2 glomeruli in the AL (Gao et al. 2000; Vosshall et al. 2000; Bhalerao et al. 2003), a layout that is shared with the mammalian olfactory system. Thus, the chemical information conveyed by ORNs is translated into a pattern of glomerular activation (Fiala et al. 2002; Ng et al. 2002; Wang et al. 2003; Yu et al. 2004). Apart from the conventional ORs, one atypical OR, OR83b, is expressed in 70–80% of the antennal ORNs; it appears to be involved in proper localization and function of conventional ORs but does not seem to influence ligand specificity (Larsson et al. 2004; Neuhaus et al. 2004; Benton et al. 2005).
Three recent studies (Larsson et al. 2004; Fishilevich et al. 2005; Kreher et al. 2005) demonstrate that the logic of Or gene expression in the larval olfactory system, despite its simplicity, is surprisingly similar to the adult and mammalian design. For 25 Or genes, expression is shown in the DO both by RNA in situ hybridization and by Or-Gal4 transgene expression (Fishilevich et al. 2005). Evidence for a few additional larval Or gene candidates derives from reverse transcriptase–polymerase chain reaction amplification or from Or-Gal4 driver analysis (Couto et al. 2005; Fishilevich et al. 2005; Kreher et al. 2005). Each of the 21 larval ORNs expresses the atypical receptor gene Or83b (Larsson et al. 2004). The large majority of the neurons appear to express one conventional OR along with OR83b, whereas 2 ORNs were shown to express 2 conventional ORs together with OR83b (Fishilevich et al. 2005). Interestingly, of the 25 well characterized larval Or genes, 13 are larval specific (Or1a, Or22c, Or24a, Or30a, Or45a, Or45b, Or59a, Or63a, Or74a, Or83a, Or85c, Or94a, Or94b) (Fishilevich et al. 2005; Kreher et al. 2005), whereas the remaining 12 Or genes are expressed in adults as well (Or2a, Or7a, Or13a, Or33a, Or33b, Or35a, Or42a, Or42b, Or47a, Or67b, Or82a, Or83b) (Clyne et al. 1999; Vosshall et al. 1999, 2000; Robertson et al. 2003; Komiyama et al. 2004). Coexpression of 2 ORs in one ORN refers to the gene pairs Or33b/Or47a and Or94a/Or94b. Because the number of identified ORs exceeds the total number of ORNs, a few more cases of triple OR expression are to be expected; indeed, many combinations of different Or genes are not tested to date.
Using a strategy based on the expression of single Or genes in adult dysfunctional mutant ORNs, that is, the "empty neuron approach" (Dobritsa et al. 2003; Hallem et al. 2004; Goldman et al. 2005), the odor response spectra of 11 larval ORs were studied by testing electrophysiological responses to a panel of 29 odorants (Kreher et al. 2005). These odors, that include different chemical classes, are known as adult or larval stimulants (Monte et al. 1989; Cobb 1999; Heimbeck et al. 1999; Hallem et al. 2004; Goldman et al. 2005). The response spectra of these ORs are very diverse, ranging from OR94b that responds to a single tested odorant, 4-methylphenol, to OR42a and OR85c each responding to 9 odorants (Kreher et al. 2005). Odorants that elicit strong responses usually do so in multiple receptors. Some receptors respond most strongly to aliphatic compounds (OR42a, OR74a, OR85c), whereas others seem to be tuned to aromatic compounds (OR30a, OR45b, OR59a, OR94b). Most of the recorded responses are excitatory, but some receptors are strongly inhibited by one compound and excited by another. Finally, response dynamics and odor sensitivities vary largely among different receptors. Yet, it should again be noted that these data are obtained by recording from "empty" adult antennal ORNs in which the larval Or genes were ectopically expressed.
Expression of gustatory receptors
The gustatory system appears to have a lower dimensionality than olfaction. In the mouse, for example, receptor cells expressing specific heterodimer combinations of the taste receptor family T1R are tuned to sugars or amino acids (Montmayeur and Matsunami 2002), but the diversity of these compounds compared with the spectrum of odorants is probably much smaller. The much larger T2R family, responsible for detecting bitter compounds, is expressed in other cells. Each of them expresses multiple T2Rs, suggesting that its capacity to distinguish between different bitter substances is limited (Montmayeur and Matsunami 2002). Thus, in comparison with the discrimination-optimized olfactory system, the taste system seems to be designed to classify the substances involved into a handful of behavioral matters of concern, for example, "nonedible" versus "edible"; this is in accordance with the much closer association of gustatory sensory neurons with motor centers. This association with motor centers may correspondingly explain the apparent lack of a specific, unified first-order gustatory neuropil that could integrate all gustatory input; this, again, is striking when compared with the role of the AL in olfaction. In short, smell may be for discrimination—to potentially be linked to many kinds of behavior—whereas taste may be for classification that already is hooked up to rather specific behaviors.
In adult Drosophila, the available evidence suggests that the GR family mediates both sweet and bitter responses. The expression pattern of the members of this family in the adult was studied exclusively by Gr gene promoter-Gal4 analysis (Dunipace et al. 2001; Scott et al. 2001) due to low levels of Gr gene expression. Whether the observed reporter patterns faithfully reflect Gr expression remains to be shown. Similar to mammals, neurons responding to sugars (the best studied example being Gr5a, expressed in cells that respond to trehalose [Chyb et al. 2003]) and neurons responding to "bitter" substances express different sets of Gr genes (Thorne et al. 2004; Wang, Singhvi, et al. 2004). This design allows eliciting attractive or aversive behaviors, respectively. Surprisingly, 3 GRs are expressed in neurons of the antenna, suggesting that smell and taste functions may not strictly be associated with OR/GR family membership, respectively (Scott et al. 2001). One among these Gr genes, Gr21a, is expressed in CO2-sensitive cells of the antenna that are necessary for behavioral responses of the flies to CO2 (Suh et al. 2004).
Gal4 expression driven by Gr gene promoters is also used in the larva (Scott et al. 2001; Fishilevich et al. 2005; Colomb et al., in preparation), but so far data are relatively scarce. Gr2a, Gr21a, Gr22e, Gr28be, Gr32a, and Gr66a, all of which drive expression also in the adult, are expressed in neurons of the TO. Gr2a labels in addition 2 neurons in the DO that are nonolfactory. GR22e, GR28be, GR32a, and GR66a are suspected to represent "bitter" receptors in the adult, as they are coexpressed in many neurons (Thorne et al. 2004; Wang, Singhvi, et al. 2004). However, when studying Gal4 driver lines, no coexpression was observed in the larva between Gr66a on the one hand and Gr2a, Gr21a, Gr32a, and Gr59f on the other (Scott et al. 2001, Colomb J, Grillenzoni N, Ramaekers A, Stocker RF, in preparation). Yet, drawing general conclusions about the numbers of GRs expressed by individual neurons seems premature. Finally, it is worth mentioning that Gr21a, which in adults is expressed in CO2-responsive cells, is expressed in the TO in cells necessary for the behavioral response of the larvae to CO2 (Faucher et al. 2006) and that several members of the Or gene family (Or30a, Or42a, Or49a, Or63a) seem to be expressed in both DO and TO (Scott et al. 2001; Fishilevich et al. 2005; Kreher et al. 2005). Thus, as in adults, gene family membership, involvement in processing of airborne versus nonairborne chemicals and site of expression are not strictly linked, reflecting the notion that Gr and Or genes belong to a single large superfamily (Scott et al. 2001; Robertson et al. 2003).
Salt detection is not mediated by GRs but by degenerin/epithelial Na+ channels, which are expressed in the TO as well as in adult taste bristles (Liu, Leonard, et al. 2003, Colomb et al., in preparation).
Smell and taste centers
A glomerular map in the AL
The architecture of the larval olfactory pathway is surprisingly similar to its adult counterpart and to the situation in mammals, but much simpler. Olfactory afferents terminate in the larval antennal lobe (LAL) (Figure 1I,J,M) targeting 2 types of interneurons: local interneurons, which establish lateral connections in the LAL, and projection neurons (PNs) that connect the LAL with higher order olfactory centers, the mushroom body (MB) calyx and the lateral horn (Figures 1C,K,L,N and 2) (Python and Stocker 2002a; Marin et al. 2005). As in the adult fly, immunoreactivity against choline acetyl transferase (ChAT) suggests that ORNs and PNs are cholinergic. In contrast, most or even all local interneurons express 
-aminobutyric acid (GABA) (Python and Stocker 2002b), indicating that, as in adults, they probably have inhibitory effects.
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The expression patterns of ORN-specific and PN-specific Gal4 driver lines reveal the presence of glomerulus-like subregions in the LAL (Python and Stocker 2002a
). To study whether the terminals of individual ORNs target a single glomerulus or extend throughout the entire LAL, the flipase-out technique (FLP-out: Wong et al. 2002) was applied in the ORN-specific Or83b-Gal4 line (Ng et al. 2002; Larsson et al. 2004). This allows visualizing individual ORNs in the background of the remaining, differently labeled ORNs (Ramaekers et al. 2005). Clearly, each ORN projects to a single LAL glomerulus (Figure 2). Because in all the 84 studied cases, FLP-out and background labels were mutually exclusive, any given glomerulus must be the target of only a single ORN. Hence, each of the 21 ORNs is unique in projecting to its one and only glomerulus; thus, the "odor space" of the larva has 21 dimensions, as defined by the number of functional types of ORNs and LAL glomeruli. As in the adult AL (Laissue et al. 1999), the relative size, shape, and position of individual glomeruli are quite conserved, allowing the establishment of a glomerular terminology in the LAL (Ramaekers et al. 2005) (Figure 1M).
In parallel studies, Fishilevich et al. (2005) and Kreher et al. (2005) report the central projections of ORNs expressing a given "conventional" OR, using Gal4 transgene expression under the control of the Or gene promoters. For each of the 22 ORs studied, the corresponding axon terminals target a different glomerulus, except of course for OR33b and OR47a, that are coexpressed in a common ORN and which thus target a common glomerulus (the ORN coexpressing the Or94a/Or94b combination of genes was not studied with respect to its axon terminals). Moreover, in 42 cases using 2 Or-Gal4 driver constructs in the same animal, 2 labeled ORNs were found, each projecting to its genuine glomerulus. This confirms that indeed each of the ORNs has its one and only proper glomerulus. Having identified ligands for some of the ORs, a preliminary spatial map of odor representation in the LAL could be established (Kreher et al. 2005). Accordingly, target glomeruli of receptors tuned to aliphatic compounds and target glomeruli of receptors tuned to aromatic compounds appear to cluster at distinct sites of the LAL.
Are these glomeruli recognized also by the dendritic arbors of PNs? PNs in third instar larvae belong to 2 classes, that is, immature, adult-specific PNs with incomplete dendrites and axons (Jefferis et al. 2004) and mature larval PNs (Marin et al. 2005). As in adults, the latter establish dendritic arbors in the LAL and send their axons through the inner antennocerebral tract into the MB calyx and the lateral horn (Figures 1C,K,L and 2) (Python and Stocker 2002a). Using the same 2-label strategy as explained above for the ORNs, but in the PN-specific GH146-Gal4 driver (Stocker et al. 1997), the dendrites of these mature PNs were found to be restricted to single LAL glomeruli (Ramaekers et al. 2005). Using the technique of mosaic analysis with a repressible cell marker (MARCM) (Lee and Luo 1999), a minority of PNs were found to be biglomerular (Marin et al. 2005). Because in each of the 100 analyzed PN FLP-out cases, the FLP-out and background labels were mutually exclusive, each glomerulus seems to be innervated by a single GH146-Gal4–positive PN only (Ramaekers et al. 2005). The glomeruli recognized by PNs correspond to the ones identified on the basis of the ORN terminals, indicating that the glomeruli of the LAL meet the wiring criteria of typical insect glomeruli, that is, an overlap of ORN terminals and dendritic PN arbors (Figures 1M and 2). Even though the 16–18 larval PNs labeled by GH146 may not comprise all PNs, the total PN number may not be much higher than the number of LAL glomeruli.
Finally, shown by single-cell FLP-out labelings with the c739-Gal4 driver, the arborizations of at least one type of local interneurons cover the entire LAL, similar to the common type of adult local interneurons (Ramaekers et al. 2005).
A glomerular map also in the MB calyx
How do the axons of PNs convey the activation pattern of the LAL glomeruli to higher brain centers, such as the MB calyx? The adult calyx is composed of hundreds of glomeruli (Yasuyama et al. 2002). Adult PNs establish 1–11 terminal boutons in variable calyx regions (Wong et al. 2002), each bouton probably corresponding to a single glomerulus (Yasuyama et al. 2002). In contrast, the larval MB calyx consists of a small number of well-defined, relatively large glomeruli (Figure 1N) (Marin et al. 2005). Two parallel approaches provide glomerular maps of the calyx. By expressing the reporter green fluorescent protein under the control of the MB-specific line OK107, Masuda-Nakagawa et al. (2005) identify 34 stereotypic calyx glomeruli. Based on strong ChAT immunoreactivity—which is very likely located in the terminals of PNs—Ramaekers et al. (2005) identify 28 calyx glomeruli from a somewhat larger total. Eighteen to 23 glomeruli are found to be targets of GH146-positive PNs (Masuda-Nakagawa et al. 2005; Ramaekers et al. 2005), and 23 glomeruli are found to be targets of another PN-specific driver line, NP225 (Masuda-Nakagawa et al. 2005). Fine structural data suggest that each calyx glomerulus is occupied by a swollen, bouton-like terminal of a single PN (Marin et al. 2005). Most of the PNs choose single calyx glomeruli as targets, but in a minority of cases, PNs establish terminals in 2 calyx glomeruli (Marin et al. 2005; Masuda-Nakagawa et al. 2005; Ramaekers et al. 2005). Again, calyx glomeruli seem to be innervated by single GH146-positive PNs (Ramaekers et al. 2005).
A comparison of the input and output sites of PNs so far reveals 7 types of PNs, which stereotypically connect a specific LAL glomerulus with a specific calyx glomerulus (Ramaekers et al. 2005). Thus, the combinatorial activity pattern set up in LAL glomeruli, which is a result of ORN input and modulation by local interneurons, seems to be rather faithfully conveyed to the calyx. Whether such strict input–output correlations apply to all PNs remains to be shown. In adults, the terminals of PNs establish stereotypical patterns in the lateral horn (Marin et al. 2002; Wong et al. 2002; Tanaka et al. 2004), whereas in the MB calyx, only concentric target zones can be defined for PNs deriving from specific AL glomeruli (Tanaka et al. 2004). Clearly, the straightforward connectivity of larval PNs seems well suited for analyzing calyx function.
Single-cell clones produced in MB-expressing Gal4 lines by FLP-out and MARCM methods (Lee et al. 1999) (for a discussion of these methods, see supplement of Ramaekers et al. 2005), allow classifying MB neurons according to their dendritic connectivity in the calyx. MB neurons differ with respect to the number of calyx glomeruli they innervate: embryonic-born MB neurons typically establish dendritic projections in 1 or 2–3 calyx glomeruli (Ramaekers et al. 2005); larval-born MB neurons, however, may have either one (Ramaekers et al. 2005) or multiple dendritic termini in usually 6 glomeruli (Masuda-Nakagawa et al. 2005). Depending on larval stage, it may therefore be that both types of neurons contribute to MB function. In any event, the populations of single-cell clones generated from Gal4 lines labeling subsets of MB neurons do not reveal any preferential innervation of particular glomeruli by these subsets of MB neurons (Masuda-Nakagawa et al. 2005). Thus, in principle, the MB neurons marked in a given Gal4 line receive the complete PN-mediated olfactory representation. Interestingly, MB neurons from different MB-expressing Gal4 lines seem to supply different regions of the peduncle and lobes (Kurusu et al. 2002), suggesting that genetically defined subsets of MB neurons, although drawing upon the same calycal input, are multiplexed in terms of their output. From a developmental perspective, however, the progeny deriving from each of the 4 MB neuroblasts appears to have a loosely defined preference to target-specific subsets of calyx glomeruli (Masuda-Nakagawa et al. 2005) and would thus each receive slightly different facets of the PN-mediated olfactory information. In any event, the hardly more than 21 larval PNs are confronted with an estimated 600 functional MB neurons, functionality being assumed by the presence of dendritic arborizations (Lee et al. 1999; L Luo, personal communication; for an estimation of a total of 1800 MB neurons in the late third larval instar, based on fiber counts in electron micrograph sections, see Technau and Heisenberg 1982). Hence, the larval calyx, like its adult homologue, is a site of divergence: One can estimate that each PN connects to 30–180 MB neurons. That is, if all MB neurons were uniglomerular, the chance for any of the 600 MB neurons to connect to any of the approximately 20 PNs is 1/20 x 1/600. As any given PN has 600 such "chances" because there are 600 MB neurons, it should connect to 600 x 1/20 x 1/600 = 1/20 of all MB neurons, that is, 30 neurons. If all MB neurons were multiglomerular and connecting to 6 glomeruli, the chance for any of the 600 MB neurons to connect to any of the approximately 20 PNs is 1/20 x 1/600 x 6. As any given PN has 600 such "chances," it should connect to 600 x 1/20 x 1/600 x 6 = 3/10 of all MB neurons, that is, 180 neurons. By the same token, considering all MB neurons (1800: Technau and Heisenberg 1982), divergence from PNs to MB neurons would be ranging between 1:90 and 1:420. Obviously, these estimates of divergence differ by an order of magnitude (minimum 1:30, maximum 1:420), suggesting that more quantitative studies on the development, number, functionality, and dendritic connectivity of larval MB neurons are needed. Finally, one must not forget that to the extent that MB neurons receive input from more than one PN, these MB neurons are a point of convergence (Perez-Orive et al. 2002).
The larval olfactory circuit: functional implications
As discussed above, larval ORNs express only 1 or 2 conventional Or genes along with the atypical Or83b (Larsson et al. 2004; Fishilevich et al. 2005; Kreher et al. 2005). This is similar to adult flies and to mammals but differs from C. elegans, in which ORNs express multiple ORs (Troemel et al. 1995). By using "subtractive" and "additive" ORN strategies, possible rules of olfactory coding were investigated in chemotaxis assays (Fishilevich et al. 2005). In the subtractive strategy, selected ORNs are genetically ablated via toxin expression, whereas in the additive strategy, animals are created with only 1 or 2 pairs of functional ORNs.
In the subtractive approach, distinct types of results were obtained. Animals in which the OR1a-expressing neuron or the OR49a-expressing neuron is ablated show reduced chemotaxis to only 1 of the 20 odors tested. This mild effect is consistent with the broad and overlapping ligand tuning of many ORNs in adults (Hallem et al. 2004) and larvae (Kreher et al. 2005). In contrast, loss of the neuron expressing OR42a results in a lack of behavioral responses to 4 of the 20 odors. In the additive approach, larvae with 1 or 2 functional ORNs are generated using Or1a, Or42a, or Or49a driver lines (Fishilevich et al. 2005). Consistent with the stronger OR42a-ablated phenotype, OR42a-functional larvae behaviorally respond to 22 of the 53 odors tested (compared with 36 odors in the wild type), including 3 of the 4 odors to which OR42a-ablated animals are unresponsive. The broad behavioral response profile for OR42a-functional larvae is in agreement with the broad ligand tuning of this receptor (Goldman et al. 2005; Kreher et al. 2005). In contrast, OR1a- and OR49a-functional larvae do not exhibit significant chemotaxis to any of the 53 odors, consistent with the weak phenotype of the corresponding ablated larvae and with electrophysiological responses (Kreher et al. 2005). Animals with 2 pairs of functional ORNs respond to a somewhat different subset of odors, or with enhanced chemotaxis, than larvae having a single functional pair of neurons alone, suggesting cooperativity (Fishilevich et al. 2005).
Three major conclusions can be drawn from these data. First, the minimal effects on chemotaxis after ablating the OR1a or OR49a neuron may suggest that either these neurons do not participate in processing the tested panel of odors or there is functional redundancy with respect to these odors. Second, the OR42a neuron plays a particularly important role in odor detection: It is sufficient to initiate chemotaxis to a high fraction of odors, and its loss leads to severe behavioral defects. Why there is functional heterogeneity between the OR42a neuron and the OR1a or OR49a neuron remains unclear. Finally, cooperative action is suggested by the overadditive chemotactic responses of OR1a/OR42a-functional animals compared with the single functional animals. Olfactory coding thus does not simply rely on additive activation of 21 parallel pathways but involves horizontal interactions as well. Such cross talk may occur at many levels of the circuit, from the sensory neurons themselves to olfactory target neurons in the brain. The primary candidates are local interneurons in the LAL that provide lateral connections among glomeruli (Ramaekers et al. 2005). Significant transformation of olfactory signals between sensory neurons and PNs is indeed known from the AL of both adult (Sachse and Galizia 2002; Lei et al. 2004; Wilson et al. 2004; Wilson and Laurent 2005) and larval insects (Itagaki and Hildebrand 1990). Such a transformation of olfactory signals may relate to quantitative and qualitative parameters, such as detection threshold and odor discrimination ability, respectively, and indeed integrative processes may be particularly crucial if very few channels have to cope with many odors.
Further processing of olfactory signals occurs in higher brain centers, such as the MBs. The different classes of larval MB neurons, innervating various numbers of calyx glomeruli, obviously allow different modes of signal transfer. Uniglomerular MB neurons receiving input from a single PN may be involved in elementary, labeled-line coding of odor features. In contrast, multiglomerular MB neurons receive input from, on average, 6 PNs, and if activation of 4–6 PNs were required for driving them, these MB neurons may act as coincidence detectors for interpreting combined activity as an odor (Perez-Orive et al. 2002; Heisenberg 2003; Wang, Guo, et al. 2004; Masuda-Nakagawa et al. 2005; Ramaekers et al. 2005). Thus, although both LAL and larval calyx are glomerular, the logic of connectivity is different: LAL glomeruli have stereotypic connectivity between defined ORNs and PNs, whereas calyx glomeruli exhibit stereotypic PN input but, with respect to the multiglomerular MB neurons, provide mostly nonstereotypic, highly combinatorial output (Masuda-Nakagawa et al. 2005) (Figure 2).
Larval versus adult olfactory circuits
The recent reports overall show that the design of the larval olfactory pathway is very similar to the one of adults. However, every ORN and most (if not all) PNs appear to be unique, leading to an almost complete lack of cellular redundancy at the first 2 relay stations in the olfactory pathway (Figure 2). Consequently, any loss of these cells should affect olfactory function more severely than in the adult system. Moreover, the presence of only 21 ORNs and 21 LAL glomeruli suggests that the number of primary olfactory dimensions is reduced in the larva compared with adults with about 50 glomeruli (Laissue et al. 1999). Also, given the uniglomerular patterns of ORNs and PNs and the almost equal number of ORs, ORNs, LAL glomeruli, PNs, and calyx glomeruli, the early levels of the larval olfactory pathway lack convergent and divergent connectivity and are organized in a 1:1:1:1:1 manner (Ramaekers et al. 2005). This contrasts with the adult olfactory pathway, where 1300 ORNs express at least 43 ORs and converge onto about 50 glomeruli, which diverge again to approximately 150 PNs and hundreds of calyx glomeruli (Stocker 1994, 2001) (Figure 2). Finally, distinct from the adult situation, ORN projections remain ipsilateral in the larva; whether and which kind of functional consequences this entails is unclear.
Convergence and cellular redundancy in sensory systems increase the signal-to-noise ratio, whereas divergent connections can expand the dimensionality of the signals to make them more discriminable (Nowotny et al. 2005). In the larva, the lack of cellular redundancy, the low number of input channels, and the absence of a convergence/divergence architecture in the LAL are likely to reduce the signal-to-noise ratio, discriminative power, and the sensitivity of their olfactory system. However, its performance seems still adequate for an animal that lives on its food supply and obviously is good enough to solve simple discrimination learning tasks (see Learning).
Primary gustatory centers
In the adult, gustatory afferents from the pharynx, labellum, and legs travel through different nerves and terminate in distinct regions of the SOG (Stocker and Schorderet 1981; Thorne et al. 2004; Wang, Singhvi, et al. 2004). Interestingly, neurons that are located on different positions and project to different regions may express the same receptor, suggesting that the same tastants may trigger different behaviors, depending on the stimulation site. Moreover, labellar neurons expressing putative "bitter" receptors such as GR66a project bilaterally along the midline of the SOG, whereas terminals of labellar neurons expressing the trehalose receptor gene Gr5a extend to additional, lateral regions and do not cross the midline (Thorne et al. 2004; Wang, Singhvi, et al. 2004). Hence, the 2 types of neurons that mediate aversive and attractive responses, respectively (Marella et al. 2006), establish distinct but partially overlapping projections.
A number of recent data allow to draw some conclusions also about the organization of larval taste centers (Scott et al. 2001; Colomb et al., in preparation). Using Gal4 driver lines in conjunction with FLP-outs, 4 major target subregions were identified in the larval SOG (Colomb et al., in preparation) that seem to be correlated primarily with the nerve through which the afferents travel and less with the Gr gene expressed. As in the adult, neurons in different sense organs but expressing the same gene, such as Gr2a, may have different central targets (Scott et al. 2001; Colomb et al., in preparation). Circumstantial evidence suggests that afferents involved in attractive responses (Heimbeck et al. 1999) may project to a region slightly different from the 4 subregions mentioned (Colomb et al., in preparation). Finally, it should be noted that Gal4 lines driven by Or30a, Or42a, and Or49a promoters that show additional expression in the TO also label sensory terminals in the SOG (Kreher et al. 2005).
Regarding second-order gustatory neurons, the discovery of a genetically defined set of approximately 20 neurons in the larval SOG was an important step; these neurons seem to receive input from GR neurons and provide output to the ring gland, the protocerebrum near yet outside the MB calyces, pharyngeal muscles, and ventral nerve cord (Melcher and Pankratz 2005). They express the hugin gene, which generates 2 neuropeptides; these are upregulated in the absence of the feeding-regulatory transcription factor klumpfuss (P[9036]) and downregulated by amino acid–deficient conditions. On the cellular level, blocking synaptic output from hugin-expressing neurons increases feeding (in the adult fly). These data, together with their connectivity, may suggest that these first-order gustatory interneurons integrate taste processing, the endocrine system, higher order brain centers, and motor output. Given that a subset of hugin-expressing neurons is likely also dopaminergic (Melcher and Pankratz 2005), it will be interesting to extend these studies to see how feeding behavior in the Drosophila larva, an indeed notorious feeder, is neuronally orchestrated by biogenic amines and how gustatory input drives associative reinforcement signals as carried by these amines (Schroll et al. 2006).
Larval contributions to the adult chemosensory system
Olfactory system
In holometabolous insects, larval sensilla are embryonic born and are typically lost during metamorphosis; they become replaced by postembryonic born, adult-specific sensilla that derive from imaginal discs (reviews: Levine et al. 1995; Truman 1996; Tissot and Stocker 2000). However, the recent demonstration of a persisting subset of larval visual sensory neurons and their integration into adult visual pathways (Helfrich-Förster et al. 2002; Malpel et al. 2002) already prepared for surprises. Metamorphosis of central circuits, as shown by a recent larval brain atlas (Pereanu and Hartenstein 2006), involves essentially the integration of a set of secondary neuronal lineages into a preexisting, embryonic-born tract system.
Concerning chemosensation, the metamorphic fate of DO and TO is not the same. Whereas the TO undergoes early apoptosis, the ganglion of the DO moves progressively backward from its peripheral site (N Gendre, personal communication). The larval ORNs become intimately associated with the antennal imaginal disc, that is, the origin of adult ORNs. Adult ORN afferents join and extend through the larval antennal nerve (Tissot et al. 1997) and reach the brain by 16–20 h after puparium formation (Jhaveri et al. 2000). A number of studies have focused on the ingrowth of adult olfactory afferents and their role in adult glomeruli formation (Jhaveri et al. 2000, 2004; Jhaveri and Rodrigues 2002; Hummel et al. 2003; Hummel and Zipursky 2004; Komiyama et al. 2004; Sen et al. 2005).
The adult AL derives from a brain region distinct from the LAL (Jefferis et al. 2004). In the LAL, the larval ORN terminals become gradually pruned (Jefferis et al. 2004). Many, perhaps all, larval olfactory interneurons become integrated in the adult system. Early reports referred to a persisting serotonergic neuron in Manduca with arborizations in the AL and protocerebrum (Kent et al. 1987; Oland et al. 1995). Local GABAergic interneurons occur both in the LAL and adult AL of several species, but it is not certain whether they are identical at both stages (Homberg and Hildebrand 1994; Stocker et al. 1997; Python and Stocker 2002b). Larval PNs in Drosophila obviously survive through metamorphosis (Stocker et al. 1997; Jefferis et al. 2001; Marin et al. 2005). In the adult AL, at least 15 glomeruli are innervated by embryonic-born PNs. These glomeruli are distinct from those innervated by larval-born PNs. The embryonic-born PNs in the adult circuit are very likely the same cells as the mature larval PNs (Marin et al. 2005). Until 12 h after puparium formation, their dendritic processes in the LAL are gradually pruned and ultimately disappear, together with LAL neuropil as a whole. At the same time, new dendritic arbors grow from the main PN process at a novel site, dorsal and posterior to the LAL. This secondary area then develops into the adult AL (Jefferis et al. 2004; Marin et al. 2005). The distinct site of the adult AL neuropil from the LAL and the minimal invasion of the former by larval ORN terminals or the "original" larval PN dendrites suggests that larval elements do not supply crucial patterning information for the adult AL (Jefferis et al. 2004; Marin et al. 2005).
The postembryonically born, adult-specific PNs extend their axons during the third larval instar, and only by the wandering stage, they have reached the MB calyx and lateral horn (Jefferis et al. 2004). Dendritic arborizations do not appear before puparium formation, demonstrating that these PNs are not functional in the larval system. Interestingly, the dendrites of the adult-specific PNs invade the prospective adult AL a few hours before those of the persisting larval PNs (Jefferis et al. 2004), further demonstrating that larval elements are not crucially involved in patterning the adult lobe.
Hence, the persisting, embryonic-born PNs are functionally integrated in both larval and adult olfactory pathways. It will be interesting to compare whether these PNs serve similar function at both stages, in particular, with respect to response spectra and the kinds of behavioral responses supported.
Concerning larval MB intrinsic neurons, their persistence through metamorphosis is well documented (Technau and Heisenberg 1982; Armstrong et al. 1998). A set of 4 MB neuroblasts divides continuously from embryonic to late pupal stages, giving rise first to the larval-type MB neurons and then successively to the adult-specific 
'/ß' and /ß neurons (Lee et al. 1999; Jefferis et al. 2002). Similar to the situation in PNs, the embryonic-born MB neurons prune their larval dendrites and axons to some extent before reextending them in modified form.
Gustatory system
A few hours after puparium formation, the cells of the external taste sensilla, that is, of the TO and very likely also of the VO, lose coherence, accumulate caspases, and disintegrate, suggesting that they undergo apoptosis (N Gendre, unpublished data). They are replaced by an entire set of adult-specific labellar taste bristles and taste pegs, which derive from the labial imaginal disc (Ray et al. 1993; Ray and Rodrigues 1994).
In contrast, lineage tracing with horseradish peroxidase (Technau 1986) and FLP-out induction (Wong et al. 2002) at embryonic stages in the neuron-specific driver line MJ94 demonstrates that most pharyngeal sensilla of the larva survive (Figure 3) (Gendre et al. 2004). This is surprising because the pharynx itself undergoes extensive reorganization (Gehring and Seippel 1967; Struhl 1981). An exception is the ventral pharyngeal sense organ, which disintegrates and likely undergoes apoptosis. The posterior pharyngeal organ on the other hand, including its central projections, survives through metamorphosis and becomes an anatomically almost identical adult organ, the dorsal cibarial sense organ (Gendre et al. 2004). A yet different developmental route is taken by the dorsal pharyngeal organ, which undergoes a complex transformation and splits into 2 parts, becoming the ventral cibarial sense organ and the labral sense organ in the adult. The latter organ is further characterized by the addition of new sensilla during pupal life, as shown by the application of the mitotic marker bromo-deoxyuridine. Thus, from the 32 sensory neurons of the 3 adult pharyngeal organs, 23 are likely persisting larval neurons, whereas the remaining ones arise during metamorphosis (Figure 3). In conclusion, metamorphosis of the pharyngeal sensory system is an intricate process involving neuronal death, generation of new neurons, and, surprisingly, persistence of many embryonic-born neurons.
The persistence of sensory neurons and their central projections through metamorphosis suggests some continuity of gustatory target regions. This may aid navigating imaginal afferents toward and inside the central nervous system (Usui-Ishihara et al. 2000; Williams and Shepherd 2002) or allow the persistence of specific feeding-associated gustatory tasks through metamorphosis. Alternatively, "recycling" of neurons might simply be due to reasons of economy (Tissot and Stocker 2000). In any event, the survival of GR neurons suggests similarity of pharyngeal taste function between larval and adult stages.
Smell and taste systems: outlook
The usefulness of adult Drosophila as a model system in olfactory research is evident, given the genetic and molecular tools available in this species, the simplicity of its olfactory system in terms of cell number, and—last but not least—the striking similarities with the mammalian olfactory system with respect to OR expression patterns, glomerular OR convergence, and coding principles in the primary odor center. Surprisingly, the olfactory system of the larva also shows the same basic design as the mammalian system, but almost in the simplest conceivable form. Excitingly, larvae with a single pair of functional ORNs can be generated. In such animals, OR expression, electrophysiological, and behavioral responses can be directly correlated, allowing the analysis of olfactory behavior at the level of single, identified receptor cells. Thus, the larva may turn into an attractive model for olfactory studies.
Concerning gustation, the model character of Drosophila as compared with mammals is less obvious, both with respect to adults and larvae. Anatomically, the taste systems of mammals and insects are different. Nevertheless, there are a number of interesting parallels. In both insects and mammals, taste receptor cells seem to be tuned to classify inputs as either attractive or aversive. Moreover, many more of the taste receptor proteins seem dedicated to repulsive ligands than to attractive ones. Strikingly also, in both phyla cells responding to bitter substances express multiple receptors.
The parallels in the chemosensory systems of mammals and insects are not necessarily an argument in favor of their common ancestry. This is reflected, for example, by the nonhomology of the receptor gene families in both phyla (Benton et al. 2005). Rather, the similarities may reflect common functional constraints, both for smell and to a lesser extent also for taste. Understanding these constraints may aid our understanding of chemosensory function. In this sense, the simplicity of the olfactory and gustatory systems of Drosophila and the wealth of available molecular tools may contribute to our comprehension of smell and taste in general.
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Learning |
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TopAbstractSmell and taste systemsLearningAcknowledgementsReferences |
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Unlike a computer hard disk, the function of biological memory is not to faithfully document the past; rather, associative memory uses past experience to predict the future—be it the consequences of an animal's own actions or upcoming external events. These predictions then can contribute to the selection of behavior. Given the larva's cellular simplicity and experimental accessibility, a multilevel understanding of such learning should be comparably easy.
Two larval learning paradigms will be presented here, one for associating olfactory stimuli and the other for associating visual stimuli with gustatory reinforcement. We provide some detail about the used methods and then review recent findings, mainly concerning the olfactory learning paradigm. As a first step, however, we consider the responses of experimentally naive larvae to odors, as well as the consequences of odor exposure on subsequent odor responses; this seems important to appreciate the kinds of behavioral control procedures one needs to use when investigating associative learning.
Odor responses and behavioral consequences of odor exposure
The best starting point to understand how the olfactory system works is to watch it at work, that is, to observe the animal's responses to odors. The typical setup for larval Drosophila is to place them in the middle of an agarose-filled petri dish, which provides a solid, smooth, and slightly lubricated surface on which the larvae readily move around. On one side of the petri dish, an odor source is placed. After some minutes, the number of animals located on the odor side minus those on the odorless side is determined, and this difference is divided by the total number of animals. This provides a normalized odor preference score ranging from full attraction (+1) to full repulsion (–1). The salient feature of such experiments is that larvae are attracted by odors; however, closer inspection (Boyle and Cobb 2005) reveals that, similar to adult flies (Rodrigues 1980), this is a concentration-dependent effect. At very low concentrations, larvae behave indifferently, at low-to-medium concentrations they are attracted, but as concentration further increases, they eventually are repelled by the odor. In cases where only attraction or repulsion is observed, this may be due to testing a too restricted range of concentrations; this is understandable as generating very high or very low odor concentration is technically challenging.
Obviously, if larvae move toward or away from an odor, they can detect that odor. This, however, does not address the question whether they can distinguish it from another one. Similarly, if larvae show a relative preference for one odor over another, discrimination cannot be inferred. This is because both odors may drive the same set of ORNs, but to a different extent (because they differ in e.g., vapor pressure or in affinity to the ORs); consequently, one odor may be quantitatively a stronger attractant without being perceived as qualitatively different. Are cross-adaptation experiments suitable to tackle this issue? Such experiments exploit the observation that, after exposure to a stimulus A, behavioral responses to this stimulus decrease and may eventually cease; if this effect can be shown to be due to an effective shutdown of the respective sensory input lines, one speaks of sensory adaptation. Provided that such adaptation does occur and provided that the peripheral nature of this effect can be shown, the experiment can be modified to first expose to stimulus A and then to test for the behavioral response to stimulus B (and in independent, complementary experiments to expose to B first to then test A). If this cross-exposure remains without effect in both cases (symmetrical lack of cross-adaptation), one can safely conclude that there must be at least 2 functionally independent input lines in the sensory system. If in both cases there is a full abolishment of the responses to the nonexposed odor (symmetrical cross-adaptation), it is parsimonious to argue that only one input line exists, which can be driven by either stimulus (the most frequent cases of partial or of asymmetrical cross-adaptation are difficult to interpret). This approach can thus be used to determine the minimal number of independent input lines that a given sensory system must possess and thus the potential (