The Interactive Fly
Zygotically transcribed genes
The central brain of Drosophila consists of the supraesophageal ganglion (SPG) and the subesophageal ganglion (SEG), both of which are generated by neural stem cell-like neuroblasts during embryonic and postembryonic development. Considerable information has been obtained on postembryonic development of the neuroblasts and their lineages in the SPG. In contrast, very little is known about neuroblasts, neural lineages, or any other aspect of the postembryonic development in the SEG. This study characterized the neuroanatomy of the larval SEG in terms of tracts, commissures, and other landmark features as compared to a thoracic ganglion. Then clonal MARCM labeling was used to identify all adult-specific neuroblast lineages in the late larval SEG, and a surprisingly small number of neuroblast lineages, 13 paired and one unpaired, were found. The Hox genes iDfd, Scr, and Antp are expressed in a lineage-specific manner in these lineages during postembryonic development. Hox gene loss-of-function causes lineage-specific defects in axonal targeting and reduction in neural cell numbers. Moreover, it results in the formation of novel ectopic neuroblast lineages. Apoptosis block also results in ectopic lineages suggesting that Hox genes are required for lineage-specific termination of proliferation through programmed cell death. Taken together, these findings show that postembryonic development in the SEG is mediated by a surprisingly small set of identified lineages and requires lineage-specific Hox gene action to ensure the correct formation of adult-specific neurons in the Drosophila brain (Kuert, 2014).
A total of 14 identified postembryonic neuroblast lineages generate the adult-specific secondary neurons in the larval SEG. This is a surprisingly small number compared with the approximately 80 neuroblast lineages in the embryonic SEG. Cell counts indicate that only about one fourth of these ~80 neuroblasts are reactivated postembryonically. This is markedly different in the supraesophageal ganglion (SPG), where about 85 of the 100 embryonically active neuroblasts are reactivated and proliferate in larval stages. The experiments indicate that the fate of half of the embryonic SEG neuroblasts that are not present postembryonically is programmed cell death. This situation is comparable to that of embryonic neuroblasts in the abdominal ganglia where the majority of neuroblasts undergo apoptosis at the end of embryogenesis. The molecular cues that trigger cell death in these embryonic neuroblasts have not been studied. The fate of the other half of the embryonic SEG neuroblasts is unknown. They may terminate proliferation through other reaper/hid/grim-independent cell death mechanisms or through cell cycle exit at the end of embryogenesis. Further experiments will be necessary to elucidate this (Kuert, 2014).
The low number of postembryonic SEG lineages has interesting consequences for the relationship between primary neurons and secondary neurons in the mature SEG. Most neuroblasts generate 10-20 neural cells embryonically and 100-150 neural cells postembryonically. Thus, the ~80 embryonic SEG neuroblasts should generate 800-1600 primary neural cells per hemiganglion while the 14 postembryonic neuroblasts generate approximately 900 secondary neural cells (as estimated by cell counts) per hemiganglion. Assuming that most of the primary neurons survive metamorphosis, this suggests that a substantial fraction of the neurons in the adult SEG could be primary neurons that comprise the functional larval SEG before their integration into the adult brain (Kuert, 2014).
Previous work has shown that 75 neuroblast lineages generate the secondary neurons of the three thoracic neuromeres. This is in striking contrast to the 14 neuroblast lineages that generate secondary neurons in the three SEG neuromeres. This reduction is most evident in the SA region, where only one commissure (ISA) is present which is also formed by only one lineage, SA3. The labial neuromere is also reduced but not as dramatically. Moreover, it retains the two commissures (aI, pI) which are also characteristic of the thoracic neuromeres. This relatively small number of postembryonic neuroblast lineages in the SEG neuromeres is likely to reflect the marked reduction and fusion of segmental appendages in the three gnathal segments that are innervated by the SEG. From an evolutionary perspective, a loss/reduction of gnathal appendages in insects such as flies would eliminate or reduce the need for corresponding neural control circuitry at least in the adult. Interestingly, and in contrast to the VNC, no evidence was found for the presence of postembryonically generated motoneurons in the SEG, indicating that all secondary neurons in the SEG are interneurons. This notion is supported by the fact that none of the 14 SEG neuroblast lineages join the labial or pharyngeal nerves (which contain the motor axons from the proboscis), but instead they project their secondary axon tracts (SATs) to areas within the CNS (Kuert, 2014).
During embryonic and postembryonic brain development, the Hox genes Dfd, Scr, and Antp are regionally expressed in discrete and largely non-overlapping domains in the neuromeres of the SEG. In both cases Dfd is expressed in an anterior domain, Scr is expressed in a posteriorly adjacent domain, and Antp expression begins in a small labial domain adjacent to the prothoracic neuromere. Moreover, while the total number of neuroblast lineages that express a given Hox gene may be different embryonically and postembryonically, most of the postembryonic neuroblast lineages do express one of these genes suggesting that Hox gene expression is a stable developmental feature of SEG lineages. Indeed, most if not all of the Hox genes that are expressed in the embryonic CNS, are re-expressed in the neuroblast lineages of the postembryonic CNS (Kuert, 2014).
Hox genes are known to be expressed during CNS development in a number of bilaterian animal groups, including vertebrates, hemichordates, insects, and annelids, and in all of these animal groups the order of Hox gene expression domains in the developing CNS appears to be conserved. For example, the order of expression of orthologous Hox genes in the developing CNS of Drosophila, mouse, and human is virtually identical. Taken together, these findings suggest that a conserved pattern of Hox gene expression domains may be a common feature in the developing CNS of all bilaterians (Kuert, 2014).
This study reveals two types of lineage-specific requirement for Hox genes during postembryonic SEG development. The first is a requirement of the Hox genes Dfd, Scr and Antp for correct postembryonic development of a subset of those lineages that are normally present in the wildtype SEG. Hox genes are required for correct SAT projections in the lineages SA1 (Dfd), SA5 (Scr) and LB3 (Antp). Interestingly, in all three cases the lineage-specific loss-of-function of these Hox genes results in specific, reproducible SAT misprojections and not in randomized axonal misprojections. While this could, in principle, be the result of a homeotic transformation phenotype, no evidence was found for such a transformation, since in terms of their projection patterns mutant SATs of these three lineages do not resemble any of the wildtype SATs present in the larval SEG (Kuert, 2014).
Hox genes are also required for correct cell number in the lineages LB5 (Scr) and LB3 (Antp). While these Hox mutant lineages lose about half of their cells, which would suggest the involvement cell death in a hemilineage-dependent manner, no evidence was found for hemilineage-specific Hox gene expression in these lineages. Thus, further studies of Hox gene action in the lineages LB3 and LB5 are necessary to dissect the functional requirement of Scr and Antp in lineage-specific cell survival (Kuert, 2014).
The second type of lineage-specific requirement for Hox genes during postembryonic SEG development is the prevention of ectopic lineage formation. Thus, in addition to their requirement for correct development of normal wildtype lineages, the genes Dfd and Scr are also required for suppressing the appearance of aberrant ectopic lineages that are not normally present in the wildtype SEG. When Dfd or Scr mutant neuroblast clones are induced at early larval stages and recovered at late larval stages, five distinct types of ectopic neuroblast clones are found. Each of these is identifiable based on reproducible neuroanatomical features such as position, secondary axon tract projection and cell number. These ectopic lineages do not represent homeotic transformations of other wildtype neuroblast lineages, since all other SEG neuroblast lineages are present. Whether these ectopic lineages become functionally integrated into the adult brain of Drosophila is currently unknown. Evidence for an integration of ectopic neuron groups into a mature brain comes from mammalian studies, which show that Hoxa1 mutant hindbrain progenitors can establish supernumerary ectopic neural cell groups that escape apoptosis and give rise to a functional circuit in the postnatal brain (Kuert, 2014).
The molecular regulators through which the Hox genes Dfd, Scr and Antp exert their diverse roles in lineage-specific SEG development are currently not known. In terms of the Hox gene requirement for correct development of wildtype lineages, only 4 of the 14 SEG lineages (11 of which express Hox genes) show misprojection or cell number mutant phenotypes. However, in these 4 lineages, the Hox gene mutant phenotypes are highly penetrant and reproducible. The lineage-restricted nature of these mutant phenotypes suggests that Hox genes interact with other lineally acting control elements to determine the developmental features of the affected lineages. While the ensemble of these control elements is currently unknown, there is increasing evidence for the importance of transcription factor codes in controlling the expression of axonal guidance molecules. In terms of the Hox gene requirement for preventing the formation of ectopic lineages, the data suggest that this involves lineage-specific programmed cell death of the corresponding postembryonic neuroblasts. Indeed, all Hox genes studied to date have been implicated in some aspect of programmed cell death in postembryonic neuroblasts. The lab gene is required for the termination of specific tritocerebral neuroblasts, Dfd and Scr are required for lineage-specific neuroblast termination in the SEG, Antp und Ubx can trigger neuroblast death if misexpressed in thoracic lineages, and abd-A induces programmed cell death in neuroblasts of the central abdomen. It is therefore concluded that a general function of Hox genes in postembryonic neural development is in the regionalized termination of progenitor proliferation as a key mechanism for neuromere-specific differentiation and specialization of the adult brain (Kuert, 2014).
Fruit flies taste compounds with gustatory neurons on many parts of the body, suggesting that a fly detects both the location and quality of a food source. For example, activation of taste neurons on the legs causes proboscis extension or retraction, whereas activation of proboscis taste neurons causes food ingestion or rejection. Whether the features of taste location and taste quality are mapped in the fly brain was studied using molecular, genetic, and behavioral approaches. Projections were found to be segregated by the category of tastes that they recognize: neurons that recognize sugars project to a region different from those recognizing noxious substances. Transgenic axon labeling experiments also demonstrate that gustatory projections are segregated based on their location in the periphery. These studies reveal the gustatory map in the first relay of the fly brain and demonstrate that taste quality and position are represented in anatomical projection patterns (Wang, 2004).
Sixty-eight gustatory receptor (GR) genes have been identified in the sequenced Drosophila genome. These receptors are likely to recognize subsets of taste cues and therefore serve as molecular markers to distinguish neurons recognizing different taste stimuli. To determine whether there is a map of taste quality in the fly brain, the distribution of GRs in sensory neurons was examined. The potential number of tastes that a neuron may recognize was investigated and then the projections of different taste neurons in the brain were examined (Wang, 2004).
One of the difficulties in determining receptor expression patterns in the Drosophila taste system is that GR genes are expressed at very low levels. Most GR genes are not detectable by in situ hybridization experiments, and it has been necessary to generate transgenic flies in which GR promoters drive expression of reporters using the Gal4/UAS system to determine receptor expression. Two transgenic reporter systems were used to simultaneously detect the expression of different receptors. The Gal4/UAS system was used to label one set of neurons, using Gr-Gal4 to drive expression of UAS-CD2. Nine different GR promoters were used that have been reported to drive robust reporter expression in subsets of taste neurons. To label the second set of GR-bearing neurons, transgenic flies were generated in which a GR promoter drives expression of multiple copies of GFP (e.g., Gr66a-GFP-IRES-GFP-IRES-GFP; for simplification, subsequently referred to as Gr-GFP). Although it is not known how much amplification multiple copies provide, this approach successfully allowed visualization of taste projections whereas direct promoter fusions to a single GFP did not. Transgenic flies for three different GR promoters (Gr32a, Gr47a, Gr66a) were generated and crossed to seven different Gr-Gal4, UAS-CD2 lines to generate a matrix of 21 double receptor combinations (Wang, 2004).
GRs are expressed in subsets of taste neurons, suggesting that one or a few receptors are expressed per cell. This hypothesis was tested by direct comparison of reporter expression for the matrix of three Gr-GFP by seven Gr-Gal4, UAS-CD2 receptor combinations. Focus was placed on the proboscis to compare reporter expression driven by different GR promoters. These studies revealed several surprising findings. (1) Many GR promoters drive reporter expression in partially overlapping cell populations. Gr66a-Gal4 drives expression in approximately 25 neurons per labial palp, in a single neuron in most or all sensilla. Of the six other GRs tested, five show expression in subsets of Gr66a-positive neurons. Of these five, four show largely overlapping expression with each other and one shows mostly non-overlapping expression. Therefore, Gr66a defines a population of gustatory neurons that express overlapping patterns of multiple receptors. (2) Some receptors are segregated into different cells. Gr5a-Gal4 drives reporter expression in approximately 30 neurons per labial palp, in one neuron in most sensilla. Gr5a is not expressed in Gr66a-positive cells. Thus, two non-overlapping neural populations can be identified by Gr66a and Gr5a. Together, these cells account for two of the four gustatory neurons in each taste sensillum (Wang, 2004).
Extracellular recordings of taste responses from proboscis chemosensory bristles have suggested that all taste sensilla are equivalent and that each of the four taste neurons within a sensillum recognizes a different taste modality, with one neuron responding to sugars, two to salts, and one to water. However, more recent experiments suggest a greater diversity of responsiveness. Because Gr5a and Gr66a are expressed in different cells in a sensillum, it was wondered whether they might mark neurons recognizing different classes of tastes. Interestingly, Drosophila defective in Gr5a show reduced responses to the sugar trehalose both in behavioral and electrophysiological studies, and heterologous expression of Gr5a in tissue culture cells confers trehalose responsiveness, strongly arguing that the ligand for Gr5a is trehalose. Given that Gr5a marks a cell that responds to a sugar, it was hypothesized that Gr66a might mark a cell responding to a different taste category. This type of segregation has been demonstrated in the mammalian taste system, where taste cells that respond to sweet are different from those responding to bitter or umami tastants. The taste ligands that Gr5a and Gr66a cells recognize were examined using genetic cell ablation and behavioral studies. It was discovered that Gr66a cells participate in the recognition of bitter compounds (Wang, 2004).
How is taste quality represented in the brain? Because Drosophila taste receptor neurons need not only recognize different tastes but most likely also the gustatory source (e.g., proboscis, internal mouthparts, legs, and wings), gustatory projections were examined to determine whether taste quality or location is represented in sensory projection patterns (Wang, 2004).
The adult Drosophila brain contains approximately 100,000 neurons, with cell bodies in an outer shell surrounding the dense fibrous core. The primary gustatory relay is the subesophageal ganglion/tritocerebrum (SOG) located in the ventral region of the fly brain. It receives input from three peripheral nerves. Neurons from the proboscis labellum project through the labial nerve; mouthpart neurons project through the pharyngeal/accessory pharyngeal nerve, and neurons from thoracic ganglia project via the cervical connective. Early studies employing cobalt backfills provided evidence that mouthpart neurons project more anteriorly in the SOG than proboscis neurons, suggesting that there might be a map of different taste organs in the fly brain (Wang, 2004).
To examine whether taste neurons in different locations project to different brain regions, GR promoters that drive reporter expression in different peripheral tissues were exploited to follow gustatory projections from the proboscis, mouthparts, or leg. Brains of Gr-Gal4, UAS-GFP were stained by anti-GFP immunohistochemistry, and a series of 1 μm optical sections through the SOG was collapsed to produce a two-dimensional representation of projections. These studies reveal differences in projections for neurons in different peripheral tissues. For instance, Gr2a is expressed only in the mouthparts and these neurons exit the pharyngeal nerve and arborize anteriorly. Gr59b, however, is expressed only in proboscis neurons that arborize in a ringed web. Notably, some receptors are expressed both in the proboscis and mouthparts. Interestingly, their neural projections seem to be the composite of Gr2a and Gr59b projections (Wang, 2004).
Two color labeling approaches were used to examine whether projections are segregated by peripheral tissue. For example, differential labeling of Gr2a neural projections, expressed in the mouthparts, and Gr66a neurons expressed in the proboscis, mouthparts, and legs illustrate overlap of the mouthpart projections but not of proboscis projections. Similarly, when the projections of Gr59b, expressed only in the proboscis, and Gr66a are differentially labeled, there is overlap of projections in the ventral proboscis region but not the dorsal mouthparts region (Wang, 2004).
The different axonal patterns from mouth, proboscis, and leg are also seen in different optical sections through the SOG of Gr32a-Gal4, UAS-GFP flies, arguing that projections are segregated by peripheral tissue even if they contain the same receptor. To better resolve the projections of individual neurons with the same receptor, taste neurons were labeled using a genetic mosaic strategy that relies on postmitotic recombination to induce expression of reporters in single cells. The Gr32a receptor is expressed in proboscis, mouthpart, and leg neurons. Single Gr32a-positive neurons from each tissue were labeled and their arborizations were examined in the SOG. A single mouthpart neuron sends an axon that arborizes in a discrete arbor in the most anterior aspect of the SOG. However, a proboscis neuron with the same receptor sends an axon that shows diffuse branching in the medial SOG, a region different from mouthpart projections. Gr32a-positive leg neurons project through the thoracic ganglia and directly terminate in the most posterior part of the SOG (Wang, 2004).
Overall, these studies demonstrate that taste neurons in different tissues project to different locations in the SOG, with mouthpart projections more anterior than proboscis projections, which are more anterior than leg projections. The demonstration that neurons that express the same receptor in different parts of the body project to distinct locations argues that they elicit different spatial patterns of brain activity and provide a means for encoding different behaviors in response to the same tastant (Wang, 2004).
It was next asked whether neurons from the same peripheral tissue that recognize different tastes project to the same or a different brain region to evaluate if taste quality is encoded in sensory projection patterns. Two-color labeling strategy was used to differentially label projections from Gr5a neurons that recognize sugars and Gr66a neurons that recognize bitter compounds. Remarkably, the projections of proboscis neurons with these receptors are clearly segregated in the SOG: Gr5a projections are more lateral and anterior to Gr66a projections. The Gr5a projections are ipsilateral and resemble two hands holding onto the medial, ringed web of Gr66a projections. Interestingly, leg taste projections for Gr5a and Gr66a neurons are segregated: Gr66a neurons project to the SOG whereas Gr5a neurons project to thoracic ganglia (Wang, 2004).
By contrast, when receptors are contained in partially overlapping populations, there is no obvious segregation of projections. For example, Gr32a is contained in a small fraction of Gr66a-positive cells in the proboscis, yet Gr32a-positive fibers colocalize with Gr66a-positive fibers in all optical sections. Moreover, Gr32a and Gr47a are expressed in mostly non-overlapping subsets of Gr66a-positive proboscis neurons, and their projections overlap, showing that smaller populations of Gr66a-positive cells are not spatially segregated. The lack of segregation suggests that these cell types are not functionally distinct (Wang, 2004).
These studies demonstrate that receptors that are expressed in subsets of cells that recognize bitter substances do not show segregated projections. However, different projection patterns are clearly discernible for proboscis neurons that recognize bitter compounds versus those that recognize sugars. The segregated projections from Gr5a and Gr66a cells reveal that there is a spatial map of taste quality in the brain (Wang, 2004).
The patterns of Drosophila taste receptor expression resemble those of the mammalian taste system and the C. elegans chemosensory system, where multiple receptors are also expressed per cell. In the mammalian taste system, multiple bitter receptors are coexpressed in one population of cells on the tongue whereas receptors for sugars are expressed in a different population of taste cells, arguing that different sensory cells recognize different taste modalities. Remarkably, the concept of distinct sweet and bitter cells also applies to the fly (Wang, 2004).
This study identified two populations of proboscis neurons that show spatially segregated projection patterns in the SOG. These different patterns correspond to different taste categories: neurons that recognize bitter substances are mapped differently in the fly brain from those that recognize sugars, suggesting that there is a map of taste modalities or behaviors in the fly brain. In addition, several subpopulations of Gr66a-positive cells show convergent projections. Two different models could account for this convergence. (1) In the simplest model, convergence could imply similar function. For example, all neurons mediating avoidance behaviors might project to the same region and synapse on a second order neuron that conveys avoidance. (2) The apparent convergence could still yield segregated gustatory information if there is a molecular identity code such that second order neurons synapse exclusively with gustatory neurons containing the same receptors. This second model is akin to what is seen in the mammalian pheromone system and sensory-motor connectivity in the spinal cord. Future experiments examining synaptic connectivity will be essential to determine how gustatory information is transmitted to higher brain centers. Nevertheless, the observation that there is spatial segregation of Gr5a sugar cells and Gr66a bitter cells, but not of smaller populations of Gr66a-positive cells, suggests that the diversity of recognition afforded by 68 or so receptor genes may be simplified into only a few different taste categories in the fly brain (Wang, 2004).
Gustatory projections are also segregated according to the peripheral position of the neuron. Early studies employing cobalt backfills argue that mouthpart neurons project more anteriorly in the SOG than proboscis neurons. The results are consistent with, and extend, these observations. Using genetic mosaic approaches, single taste neurons were labeled, and it was found that projections from different organs are segregated even from neurons containing the same receptor. These studies argue that the same taste stimulus will produce different patterns of brain activity depending on the stimulus' location in the periphery and may mediate different behaviors, consistent with the observation that sugar on the leg causes proboscis extension whereas sugar on the ovipositor causes egg laying. An organotopic map of gustatory projections may provide a means for the fly to distinguish different taste locations (Wang, 2004).
The reproductive and defensive behaviors that are initiated in response to specific sensory cues can provide insight into how choices are made between different social behaviors. This study manipulated both the activity and sex of a subset of neurons and found significant changes in male social behavior. Results from aggression assays indicate that the neuromodulator octopamine (OCT) is n for Drosophila males to coordinate sensory cue information presented by a second male and respond with the appropriate behavior: aggression rather than courtship. In competitive male courtship assays, males with no OCT or with low OCT levels do not adapt to changing sensory cues and court both males and females. A small subset of neurons was identified in the subesophageal ganglion region of the adult male brain that coexpress OCT and male forms of the neural sex determination factor, Fruitless (FruM). A single FruM-positive OCT neuron sends extensive bilateral arborizations to the subesophageal ganglion, the lateral accessory lobe, and possibly the posterior antennal lobe, suggesting a mechanism for integrating multiple sensory modalities. Furthermore, eliminating the expression of FruM by transformer expression in OCT/tyramine neurons changes the aggression versus courtship response behavior. These results provide insight into how complex social behaviors are coordinated in the nervous system and suggest a role for neuromodulators in the functioning of male-specific circuitry relating to behavioral choice (Certel, 2007).
To reduce or eliminate the function of OCT neurons, the Tyramine β-hydroxylase (Tβh) mutant lines were used. The Tβh gene encodes the enzyme necessary to convert TYR to OCT, and null mutants (TβhnM18) produce no detectable OCT, whereas the hypomorphic TβhMF372 strain generates low levels of OCT. The revertant TβhM6 allele was used as the control. The original alleles were generated by P-element manipulations on the same chromosome. Subsequent manipulations were performed to replace the w1118 allele and backcrossed to Canton-S (CS) to maintain comparable genetic backgrounds. OCT, dopamine, and serotonin levels were verified in each Tβh allele by using HPLC (Certel, 2007).
Modulation of classical neurotransmitter action on target neurons adds great flexibility to synaptic output between neurons and is suggested to be at the core of important behavioral processes like learning and memory. In vertebrates, amines like serotonin, dopamine, and norepinephrine; peptides like arginine vasopressin, and oxytocin; gonadal steroids; and various glucocorticoids serve as well known neuromodulatory substances. Through selective actions at individual synaptic sites, neuromodulators coordinate the output of neuronal ensembles to generate behavioral patterns of varying complexity (Certel, 2007).
An elegant example of coordinating network output comes from studies with the stomatogastric ganglion of crustaceans. In this small neuronal ensemble, neuromodulators function either singly or in various combinations at multiple sites in the ganglion to alter the patterned output of the ganglion and thereby the movement of food through the stomach. An example of changing network ensembles in vertebrates is seen in studies of vole social behavior. Here, the distribution of oxytocin, vasopressin, and dopamine receptors within different brain regions appears linked to the differences seen in social behavior between prairie voles and montane voles (Certel, 2007 and references therein).
This paper focuses on the roles of octopamine, a phenolamine structurally related to the catecholamine norepinephrine, in modulating the choice between courtship and aggression in male flies. Norepinephrine has been shown to be important in many aspects of vertebrate behavior, including arousal, anxiety, learning and memory, opiate reward, and aggression. Among invertebrates, OCT influences foraging behavior in honey bees; resets aggressive motivation in crickets; and functions in appetitive associative learning, ethanol tolerance development, and possibly aggression levels in Drosophila. Like their vertebrate amine neuron counterparts, OCT neurons in Drosophila (1) are few in number but have enormous fields of innervation covering essentially all neuropil areas in the fly brain and (2) function by activating multiple G protein-coupled receptors (Certel, 2007).
Aggression and courtship usually are mutually exclusive behaviors. By examining the choices made between these behaviors by male flies, a powerful approach is offered with which to study the genetic and neural basis of complex behaviors. Multiple decision-making actions are required for each of these behaviors, including the processing of chemosensory and visual information and deciding whether another fly is a potential opponent or a potential mate. Using aggression and competitive courtship assays, OCT was found to be necessary for pairs of Drosophila males to respond to the sensory cues presented and to coordinate expression of the appropriate response: aggression. Feminizing OCT/tyramine (TYR) neurons in males also changes the aggression vs. courtship response behavior. Because the gene fruitless directs both courtship and aggression in flies, the expression patterns of OCT and the male forms of Fruitless (FruM) was analyzed and the were found to be coexpressed in distinct subesophageal ganglion (SOG) neurons in the male brain. This region receives the contact gustatory pheromone information thought to facilitate sex and species discrimination. The arborizations of one of the FruM-octopaminergic neurons were found to project bilaterally and appear to ramify in the posterior antennal lobe, multiple SOG layers, as well as the lateral accessory lobe (ventral body). These results offer insight into how sensory cues are integrated and modulated in the nervous system to direct sex-specific complex behaviors and indicate a role for the neuromodulator OCT in the functioning of the male-specific circuitry relating to behavioral choice (Certel, 2007).
Males and females react to environmental cues with distinct sex-specific innate behaviors particularly in the areas of courtship/reproduction and aggression/defense. Results from a number of studies have demonstrated that functional and structural sex differences in the brain can influence and direct these behaviors, but how sensory cues contribute to the appropriate response of one of these two mutually exclusive behaviors remains unclear. This study presents evidence that the neuromodulator OCT functions within a defined circuit to provide at least one means of regulating the choice between courtship and aggression. The results of these aggression studies indicate that male flies require OCT to respond with an appropriate aggressive response to another male. The results of the male-female courtship assays suggest that normal OCT function provides increased behavioral response confidence about the sensory cues being presented (Certel, 2007).
Identifying a potential mate or opponent relies on discriminating specific stimuli from background and then integrating this information with other sensory modalities. Anatomically, the extensive arrays of OCT-immunoreactive processes that are found throughout the Drosophila brain offer one such overlying integration network that may fine-tune sensory input and activate sex-specific behavioral subcircuits. In Drosophila, male-specific behavioral circuits are specified by the male-specific products of the fruitless gene. In this study, it was demonstrated that three VUM neurons in the male SOG coexpress FruM and OCT. The SOG is the primary taste-processing center in the fly. The sensory information sent to this neuropil includes the female pheromone recognition cues necessary for male courtship behavior. Therefore, an intriguing possibility is that OCT is necessary in the subset of FruM-positive SOG neurons to accurately relay contact gustatory pheromone information (Certel, 2007).
Morphological results suggest that a single neuron can provide a simple integration network of multisensory cues. The arborizations of one of the VUM 1 FruM-positive OCT neurons extensively ramify throughout multiple neuropil regions, including the SOG, posterior antennal lobe, and the lateral accessory lobe (ventral body), suggesting a link between various sensory modalities. Gustatory information from OCT/FruM SOG neurons could also be linked to higher-order processing centers through synaptic contacts with the male-specific SOG projections of FruM-expressing mAL neurons identified. The superior lateral protocerebrum has been proposed to be the output site of these interneurons. Linkages of this type may be of particular significance because FruM-expressing neurons play critical roles in two sex-specific social behaviors: aggression and courtship. Thus, the same circuits may need to integrate the context-specific sensory information necessary to direct the output of appropriate behavioral patterns (Certel, 2007).
How might OCT modify distinct SOG neurons to regulate behavioral choice by males? In the spider, OCT increases the overall sensitivity of mechanosensory neurons by local release from efferent endings. This local release suggests that sensory input from specific sensilla relative to others can be emphasized depending on behavioral circumstances. In the silkworm moth, OCT specifically increases the sensitivity of male pheromone-sensitive receptor neurons but not general odorant-sensitive responses. Recent modeling studies in vertebrates suggest that neuromodulators can play a key role at specific times in decision-making tasks by regulating competition between populations of neurons that represent choices. This regulation may allow an organism to integrate noisy sensory information and past experience to make optimal decisions (Certel, 2007).
Although the mouse neural pathways that mediate the output of two sex-specific behaviors, reproduction and defense, are anatomically segregated, a recent study identified a hypothalamic point of convergence that may function as a choice selection mechanism for sensory activation of defensive responses over reproduction. The results suggest that whether an individual male mouse responds with the appropriate behavior depends on the coordinated activation of the appropriate subcircuits by amygdalo-hypothalamic projections. Likewise the different behavioral outputs of Drosophila males and females could be generated through the activation of sex-specific segregated neural ensembles. However, behavioral differences also could emerge through sex-specific modulation of circuits that are common to both sexes. In males, FruM proteins are expressed in small groups of neurons throughout the CNS, and eliminating FruM expression in a neuronal subset has profound effects on the progression of male courtship behaviors. At the gross level almost all of the FruM-producing neurons have counterparts in the female and in terms of function, a recent report indicates that the sex-specific reproductive behaviors of females and males involve shared neural circuits. The splicing of fruM-specific transcripts have been proposed to modify neurons common in both sexes for male-specific functions through differences in neuron morphology and/or physiology (Certel, 2007).
In addition to changing the activity of OCT neurons, OCT/TYR neurons were feminized in an otherwise masculine brain and altered male behavioral choice was demonstrated. The results from OCT immunostaining do not indicate any sex-dependent changes in SOG neuron number. The identification of a sex-independent marker for the FruM-positive OCT neurons should allow determination of whether feminizing these neurons changes either their branching patterns, their synaptic connections, or their OCT-related biochemical properties. Further examination of these OCT/FruM SOG neurons should offer a behaviorally relevant ensemble with which to address questions of sex-specific morphology and function-related physiology (Certel, 2007).
This study examined the molecular and cellular basis of taste perception in the Drosophila larva through a comprehensive analysis of the expression patterns of all 68 Gustatory receptors (Grs). Gr-GAL4 lines representing each Gr are examined, and 39 show expression in taste organs of the larval head, including the terminal organ (TO), the dorsal organ (DO), and the pharyngeal organs. A receptor-to-neuron map is constructed. The map defines 10 neurons of the TO and DO, and it identifies 28 receptors that map to them. Each of these neurons expresses a unique subset of Gr-GAL4 drivers, except for two neurons that express the same complement. All of these neurons express at least two drivers, and one neuron expresses 17. Many of the receptors map to only one of these cells, but some map to as many as six. Conspicuously absent from the roster of Gr-GAL4 drivers expressed in larvae are those of the sugar receptor subfamily. Coexpression analysis suggests that most larval Grs act in bitter response and that there are distinct bitter-sensing neurons. A comprehensive analysis of central projections confirms that sensory information collected from different regions (e.g., the tip of the head vs the pharynx) is processed in different regions of the subesophageal ganglion, the primary taste center of the CNS. Together, the results provide an extensive view of the molecular and cellular organization of the larval taste system (Kwon, 2011).
Of the 67 Gr-GAL4 transgenes, 43 showed expression in the larva, of which 39 were expressed in the major taste organs of the head. The 39 Gr-GAL4 drivers are expressed in combinatorial fashion. Individual Gr-GAL4 drivers are expressed in up to 12 cells, in the case of Gr33a- and Gr66a-GAL4; approximately one-half, however, are expressed in only one cell (Kwon, 2011).
For some Gr-GAL4 drivers the observed pattern of expression may not be identical with that of the endogenous Gr gene. It was precisely with this concern in mind that a mean of 7.6 independent lines were analyzed for each of the 67 Gr drivers, and a rigorous, quantitative protocol was establised for identifying a representative line for each gene. In the absence of an effective in situ hybridization protocol, the approach used here seemed likely to be the most informative in providing a comprehensive systems-level view of larval taste reception (Kwon, 2011).
The Gr receptor-to-neuron map of the dorsal and terminal organs identified 10 neurons. Two neurons have cell bodies in the DOG and innervate the DO, two have cell bodies in the DOG and innervate the TO, and six have cell bodies in the TOG and innervate the TO (Kwon, 2011).
28 receptors were mapped to these 10 neurons. All of these neurons express at least two Gr-GAL4 drivers. Two receptors, Gr21a and Gr63a, are coreceptors for CO2; neither is sufficient to confer chemosensory function alone. It is conceivable that many other Grs may also require a coreceptor, which may explain the lack of neurons expressing a single Gr-GAL4. The number of receptors per neuron ranges up to 17, in the case of C1. This number is comparable with the maximum number of Gr-GAL4s observed in a labellar neuron (29), and much greater than the number of Ors observed in individual neurons of either the larval or adult olfactory system (Kwon, 2011).
Among the 10 identified cells, individual Gr-GAL4 drivers are expressed in as few as one cell and as many as six cells. Most of the drivers are expressed in only one of these 10 cells. The drivers expressed in six cells, Gr33a-GAL4 and Gr66a-GAL4, are expressed in all bitter neurons of the adult labellum. It is noted that Gr33a-GAL4 and Gr66a-GAL4 are the only drivers expressed in B1, arguing against the possibility that both of these receptors function exclusively as chaperones or as coreceptors that require another Gr for ligand specificity (Kwon, 2011).
There is little cellular redundancy. Only two neurons, A1 and A2, express the same complement of receptors. All other neurons contain a unique subset of the Gr repertoire. In this respect, the larval taste system differs from the adult taste system but is similar to the larval olfactory system, which also contains little if any cellular redundancy (Kwon, 2011).
Analysis of the central projections of all 39 Gr-GAL4 drivers provided evidence for a systematic difference among projection patterns between TO/DO neurons and pharyngeal neurons. These results support the conclusion that sensory information collected from the tip of the head is processed in different regions of the SOG than information collected in the pharynx, i.e., that evaluation of a potential food source before ingestion and the testing of food quality during ingestion are functionally partitioned in the brain. Similar inferences were drawn in an elegant study of a limited number of Gr-GAL4 transgenes (Kwon, 2011 and references therein).
Conspicuously absent from the list of Gr-GAL4 drivers expressed in the larval taste system are those representing the eight members of the sugar receptor subfamily (Gr5a, Gr61a, Gr64a-f). The founding member of this family, Gr5a, mediates response to the sugar trehalose, and two other members of the subfamily have been shown to encode sugar receptors as well. No GFP expression for these genes was observed in cells of the taste organs or in neural fibers in the brain or ventral ganglion. Most of these Gr-GAL4 transgenes drive expression in the adult, but it is acknowledged that these transgenes may not faithfully reflect expression in the larva (Kwon, 2011).
Given that Drosophila larvae respond to sugars, as do larvae of other insect species, how do they detect them without members of the sugar receptor subfamily? Other Grs, including the recently identified fructose receptor Gr43a, may underlie sugar detection in the larva. It is noted that Gr59e-GAL4 and Gr59f-GAL4 are coexpressed in a cell that does not express the bitter cell markers Gr33a-GAL4 or Gr66a-GAL4. Sugar reception may also be mediated by other kinds of receptors, such as those of the TRPA family (Kwon, 2011).
In adult Drosophila, Gr33a-GAL4 and Gr66a-GAL4 are coexpressed with other Gr-GAL4s in bitter neurons; the simplest interpretation of expression and functional analysis is that multiple bitter receptors are coexpressed (Kwon, 2011).
In the larva, it ws found that most larval Gr-GAL4s are coexpressed with Gr33a- and Gr66a-GAL4, suggesting the possibility that most larval Grs act in bitter response. It is noted that, of the 17 Gr-GAL4s coexpressed in the C1 neuron, 15 are coexpressed in a bitter neuron of the labellum. It was also establish that there are distinct molecular classes of Gr33a-GAL4, Gr66a-GAL4-expressing neurons. The simplest interpretation of these results is that there are distinct bitter-sensing neurons in larvae (Kwon, 2011).
Larvae must determine whether to accept or reject a food source, and in principle this determination could be made by a simple binary decision-making circuit. However, the existence of six Gr33a-GAL4, Gr66a-GAL4-expressing neurons expressing distinct subsets of Gr-GAL4s suggests a greater level of complexity in the processing of gustatory information. One possibility is that C1, which expresses the largest subset of drivers among the TO/DO neurons, may activate an aversive behavior in response to many of the bitter compounds that the larva encounters, while C2, C3, C4, or B2 either potentiates the response or activates a different motor program in response to chemical cues of particular biological significance or exceptional toxicity. The existence of heterogenous bitter-sensing cells, some more specialized than others, is a common theme in insect larvae. In particular, many species contain a taste cell that responds physiologically to many aversive compounds and whose activity deters feeding. C1 could be such a cell, and its coexpression of many receptors may provide the molecular basis of a broad response spectrum (Kwon, 2011).
It is striking that the number of TO/DO neurons that express Gr-GAL4s is small compared with the total number of TO/DO neurons. Gr-GAL4 expression was mapped to only 10 cells in the TO/DO (although Gr2a-GAL4 and Gr28a-GAL4 were each expressed in two TO neurons that were not mapped). The DOG and TOG contain 36-37 and 32 sensory neurons, respectively, among which 21 in the DOG are olfactory. Thus, of the nonolfactory cells, on the order of 20%-30% express Gr-GAL4 drivers. It will be interesting to determine how many of the other DOG/TOG cells express other chemoreceptor genes, such as Ppk, Trp, or IR genes, and how many of the other neurons have mechanosensory, thermoreceptive, hygrosensory, or other sensory functions (Kwon, 2011).
The role of Gr genes in the larval pharyngeal organs is unknown. In adult pharyngeal sensilla, the TRPA1 channel, which detects irritating compounds, regulates proboscis extension. It is possible that Grs expressed in larval pharyngeal organs may also play a role in modulating feeding behavior. Of the 24 Gr-GAL4 drivers expressed in the larval pharyngeal organs, 9 are coexpressed with Gr33a-GAL4 and Gr66a-GAL4 in the TO/DO; it seems plausible that they may monitor ingested food for the presence of aversive compounds (Kwon, 2011).
In summary, this study has analyzed essential features of the molecular and cellular organization of a numerically simple taste system in a genetic model organism. Ten gustatory receptor neurons were described and evidence was provided that they express Grs in combinatorial fashion, with most of these neurons and receptors acting in the perception of bitter compounds. The results lay a foundation for a molecular and genetic analysis of how these receptors and neurons, and the downstream circuitry, underlie a critical decision: whether to accept or reject a food source (Kwon, 2011).
The decision to engage in one behavior often precludes the selection of others, suggesting cross-inhibition between incompatible behaviors. For example, the likelihood to initiate feeding might be influenced by an animal's commitment to other behaviors. This study examined the modulation of feeding behavior in the fruit fly, Drosophila melanogaster, and identified a pair of interneurons in the ventral nerve cord that is activated by stimulation of mechanosensory neurons and inhibits feeding initiation, suggesting that these neurons suppress feeding while the fly is walking. Conversely, inhibiting activity in these neurons promotes feeding initiation and inhibits locomotion. These studies demonstrate the mutual exclusivity between locomotion and feeding initiation in the fly, isolate interneurons that influence this behavioral choice, and provide a framework for studying the neural basis for behavioral exclusivity in Drosophila (Mann, 2013).
The neurons that inhibit proboscis extension (which are named
PERin) have cell bodies and processes in the first leg neuromeres
of the VNC and projections to the SOG, the brain region that contains
gustatory sensory axons and proboscis motor neuron
dendrites. Labeling with the presynaptic synaptotagmin-
GFP marker and the postsynaptic
DenMark marker indicated that the dendrites
of PERin neurons are restricted to the first leg neuromeres,
whereas axons are found in both the SOG and the first leg neuromeres. The anatomy of these neurons suggests
that they convey information from the leg neuromeres to
a region of the fly brain involved in gustatory processing and proboscis
extension. Anatomical studies examining the proximity of
PERin fibers to gustatory sensory dendrites or proboscis motor
axons revealed that PERin neurons do not come into close
contact with known neurons that regulate proboscis extension (Mann, 2013).
Many behaviors are mutually exclusive, with the decision to
commit to one behavior excluding the selection of others.
This study shows that feeding initiation and locomotion are mutually
exclusive behaviors and that activity in a single pair of interneurons
influences this behavioral choice. PERin neurons are
activated by stimulation of mechanosensory neurons and activation
of PERin inhibits proboscis extension, suggesting that they
inhibit feeding while the animal is walking. Consistent with this,
leg removal or immobilization enhances proboscis extension
probability and this is inhibited by increased PERin activity. The
opposite behavior is elicited upon inhibiting activity in PERin neurons:
animals show constitutive proboscis extension at the
expense of locomotion. This work shows that activity in a single
pair of interneurons dramatically influences the choice between
feeding initiation and movement (Mann, 2013).
The precise mechanism of activation of PERin neurons
remains to be determined. PERin dendrites reside in the first
leg neuromere, suggesting that they process information from
the legs. Stimulation of leg chemosensory bristles with sucrose
or quinine or activation of sugar, bitter, or water neurons using
optogenetic approaches did not activate PERin neurons, nor
did satiety state change tonic activity. Stimulation of sensory
nerves into the ventral nerve cord and stimulation of mechanosensory
neurons, using a nompC driver, activated PERin. In addition,
by monitoring activity of PERin while flies moved their legs,
it was demonstrated that activity was coincident with movement (Mann, 2013).
These studies argue that PERin is activated by nongustatory
cues in response to movement, likely upon detection of mechanosensory
cues. Additional cues may also activate PERin.
Studies of behavioral exclusivity in other invertebrate species
suggest two mechanisms by which one behavior suppresses
others. One strategy is by competition
between command neurons that activate dedicated circuits
for different behaviors. More common is a strategy in which decision-
making occurs by distributed activity changes across
neural populations. Although this studies are a starting point to
begin to examine these models in Drosophila, the circuits for proboscis
extension and locomotion drive different motor neurons,
muscles, and behaviors, suggesting that they may be connected
by a few links rather than largely overlapping circuitry. PERin is
likely to inhibit feeding initiation while the animal is moving and
is one critical link. The observation that simply gluing the proboscis
in an extended state, but not in a retracted state, inhibits
locomotion suggests that motor activity or proprioceptive feedback
from the proboscis acts as a reciprocal link to locomotor circuits (Mann, 2013).
Neurons act over different timescales and in response to
different sensory cues to influence behavior. The powerful molecular
genetic approaches available in Drosophila enable the
precise manipulation of individual neurons and allow for the examination
of their function in awake, behaving animals. Modulatory
neurons such as PERin are difficult to identify by calcium
imaging or electrophysiological approaches because they influence
gustatory-driven behavior but are not activated by gustatory
stimulation. The ability to probe the function of neurons in
unbiased behavioral screens facilitates the identification of neurons
that act as critical nodes to influence behavior. The identification
and characterization of PERin as a significant modulator of
feeding initiation provides a foundation for future studies determining
how PERin influences proboscis extension circuits to alter
behavioral probability and how mechanosensory inputs activate
PERin. In addition, examining how proboscis extension suppresses
locomotion will provide important insight into the links
between different behaviors (Mann, 2013).
Neural circuits for a given behavior do not work in isolation.
Information from multiple sensory cues, physiological state,
and experience must be integrated to guide behavioral decisions.
This work uncovers a pair of interneurons that influences
the choice between feeding initiation and locomotion. The
discovery of the PERin neurons will aid in examining the neural
basis of innate behaviors and the decision-making processes
that produce them (Mann, 2013).
A small subset of two to six subesophageal neurons, expressing the male products of the male courtship master regulator gene products fruitlessMale (fruM), are required in the early stages of the Drosophila melanogaster male courtship behavioral program. Loss of fruM expression or inhibition of synaptic transmission in these fruM(+) neurons results in delayed courtship initiation and a failure to progress to copulation primarily under visually-deficient conditions. A fruM-dependent sexually dimorphic arborization was identified in the tritocerebrum made by two of these neurons. Furthermore, these SOG neurons extend descending projections to the thorax and abdominal ganglia. These anatomical and functional characteristics place these neurons in the position to integrate gustatory and higher-order signals in order to properly initiate and progress through early courtship (Tran, 2014).
Initiation of unilateral wing extension is heavily dependent on visual, olfactory, and gustatory cues. By forcing males to depend on non-visual pathways for courtship and co-expressing tissue-specific fruM RNAi, this study screened for fruM(+) neurons that likely regulate chemosensory-dependent processes in courtship, which manifested as infrared-specific courtship latency defects. The P[GawB]4-57 line driving UAS-fruMIR possessed normal courtship latency in ambient light and significant infrared-specific delays. Notably fruM overlap was strongest in the SOG, while lacking any detectable peripheral expression. Behavioral and anatomical studies using Cha-Gal80, to subdivide the P[GawB]4-57 expression pattern, highlighted a small subpopulation of fruM(+) neurons in the SOG, two-four anterior SG x 4-57 neurons (marked by the intersection of two drivers) and two medial SG x 4-57 neurons as responsible for the courtship defects (Tran, 2014).
Several lines of evidence suggest a direct role for the mSG x 4-57 neurons in regulating the initiation of wing extension and copulatory behaviors. First, expression of fluorescent markers was detected in the mSG x 4-57 neurons driven by P[GawB]4-57 in all brains, whereas fluorescence was only detected in a subset of animals for the other fruM x 4-57 subpopulations. The mSG x 4-57 neurons made sexually dimorphic arbors in the tritocerebrum, where male arbors were significantly larger than in wild type female and fru mutant male brains. The mSG x 4-57 neuronal tracts extended into the VNC where presynaptic innervation of the mesothoracic triangle was seen. The mesothoracic triangle is a target of descending command neurons that control wing song. Faint projections were detected in the posterior metathoracic/anterior abdominal ganglia, which suggest possible regulation of motor circuitry needed for abdominal curling during copulatory behaviors (Tran, 2014).
The sexually dimorphic projections of the mSG x 4-57 suggest sex-specific roles in receiving tritocerebral signals in males. In males, fruM knockdown and silencing of fruFLP x 4-57 neurons resulted in a failure to progress to copulation, a behavior that follows proboscis contact with a female ('licking'). The internal mouthparts house gustatory sensilla that likely detect contact female pheromones accessed via licking behavior (Tran, 2014).
Functions for the non-mSG x 4-57 neurons cannot be ruled out, particularly the DT6 x 4-57 (aSG) neurons in regulating courtship initiation, however. The current approach infers, but does not conclusively demonstrate that the mSG x 4-57 neurons are responsible for the courtship initiation and copulation defects. Further studies are required to conclusively identify the neurons responsible for each behavioral phenotype and their exact roles (Tran, 2014).
Several studies have examined the projections of fruM(+) neurons in the SOG. Antibody staining using anti-FruM identified 12±2 total FruM(+) nuclei in the SOG in the 2-day pupal brain. An intersectional study, using 131 Gal4 lines with sparse overlap with fruFLP, identified 8 fruFLP(+) SOG neuronal classes divided into six anterior, aSG1-6, and two posterior neuronal types, pSG1-2. At least one aSG x 4-57 neuron’s projection pattern, identified in this study, is consistent with the aSG5 class identified in that larger-scale study. Cachero (2010) used mosaic analyses of fruGal4 to identify larval neuroblast clonal populations of fruGal4 (+) neurons. Cachero identified six clones in SOG, however, none appear to correspond to neurons identified in this study. It appears that these broad mapping studies, while extensive, have not exhaustively identified fru-expressing neurons in the SOG (Tran, 2014).
Using tdc2-Gal4, three studies characterized three octopaminergic FruM(+) neurons in the SOG: designated VPM1 and VPM2 (ventral paired median) and one VUM1 (ventral unpaired median) neuron. Expression of tdc2-Gal4-driven UAS-fruMIR leads to courtship latency delays but no copulation defect. The VUM1 neuron tritocerebral projections appear similar to the mSG x 4-57 projections, however, no descending tracts to the VNC were reported. The VPM1 and VPM2 appear to correspond to the DT8 neurons Repression of fruM using tdc2-Gal4 appeared to primarily disrupt male-female discrimination, resulting in significant male-male courtship, whereas no significant male-male courtship was detected using P[GawB]4-57 (Tran, 2014).
Given the extensive projections of fruM(+) innervations, the tritocerebrum appears to be a site of gustatory integration with higher-order information in male courtship. The extensive, sexually dimorphic arbors from the mSG x 4-57 receive signals in the tritocerebrum that serve to regulate the progression to copulation in males and the performance of courtship. The tritocerebrum is targeted directly by gustatory afferents from the mouthparts via the pharyngeal nerves, indirectly via the SOG interneurons, which could relay signals from proboscis gustatory afferents entering via the labial nerve, and by descending tracts from the par interecerebralis of the superior medial protocerebrum (SMPR), which contains many neurosecretory cells. These mSG x 4-57 cells could then relay signals to circuitry controlling wing extension/song in the metathoracic triangle and copulation/abdominal curling in the anterior abdominal ganglia (Tran, 2014).
The decision to perform courtship by males likely weighs the receptivity of the female versus the cost of female rejection via escape, with greater costs associated with later steps in the ritual, i.e. copulation. In open environs, escape behaviors exhibited by rejecting females likely results in the cessation of the courtship unless the male correctly gauges receptivity. It is proposed that the fruM(+) SOG neurons identified in this study play a vital link between detection of female receptivity cues and integration of higher-order signals in order to appropriate initiate wing extension and copulatory behaviors (Tran, 2014).
Bitter is a taste modality associated with toxic substances evoking
aversive behaviour in most animals, and the valence of different taste
modalities is conserved between mammals and Drosophila. Despite
knowledge gathered in the past on the peripheral perception of taste,
little is known about the identity of taste interneurons in the brain.
This study shows that hugin
neuropeptide-containing neurons in the Drosophila larval subesophageal zone are necessary for avoidance behaviour to caffeine, and when activated,
result in cessation of feeding and mediates a bitter taste signal within
the brain. Hugin neuropeptide-containing neurons project to the
neurosecretory region of the protocerebrum and functional imaging
demonstrates that these neurons are activated by bitter stimuli and by
activation of bitter sensory receptor neurons. The study proposes that
hugin neurons projecting to the protocerebrum act as gustatory
interneurons relaying bitter taste information to higher brain centres in
Drosophila larvae (Hückesfeld, 2016). The bitter taste rejection response is important for all animals that encounter toxic or harmful food in their environment. This study showed that the hugin neurons in the Drosophila larval brain function as a relay between bitter sensory neurons and higher brain centres. Strikingly, activation of the hugin neurons, located in the subesophageal zone, made the animals significantly more insensitive to substrates with negative valence like bitter (caffeine) and salt (high NaCl), as well as positive valence like sweet (fructose). In other words, when the hugin neurons are active these animals 'think' they are tasting bitter and therefore become insensitive to other gustatory cues. This is in line with observations made in mice, in which optogenetically activating bitter cortex neurons caused animals to avoid an empty chamber illuminated with blue light. In this situation, although mice do not actually taste something bitter, they avoid the empty chamber since the bitter perception has been optogenetically induced in the central nervous system (CNS) and the mice 'think' they are tasting a bitter substance (Hückesfeld, 2016). In previous work, activation of all hugin neurons led to behavioural and physiological phenotypes such as decreased feeding, decrease in neural activity of the antennal nerve (AN), and induction of a wandering-like behaviour (Schoofs, 2014). The neurons responsible specifically to those that project have now been pinpointed to the protocerebrum. These neurons not only respond to bitter stimuli, but also show a concentration dependent increase in calcium activity in response to caffeine. Dose dependent coding of bitter taste stimuli was previously shown to occur in peripheral bitter sensory neurons, where bitter sensilla exhibit dose dependent responses to various bitter compounds. Larvae in which the huginPC neurons have been ablated still showed some avoidance to caffeine. Whether this implies the existence of other interneurons being involved in caffeine taste processing remains to be determined. Interestingly, the huginPC neurons are inhibited when larvae taste other modalities like salt (NaCl), sugar (fructose) or protein (yeast). This may indicate that taste pathways in the brain are segregated, but influence each other, as previously suggested (Hückesfeld, 2016). Bitter compounds may be able to inhibit the sweet-sensing response to ensure that bitter taste cannot be masked by sweet tasting food. This provides an efficient strategy for the detection of potentially harmful or toxic substances in food. For appetitive tastes like fructose and yeast, bitter interneurons neurons like the huginPC neurons in the CNS may become inhibited to ensure appropriate behaviour to pleasant food. Salt is a bivalent taste modality, that is, low doses of salt drive appetitive behaviour, whereas high doses of salt are aversive to larval and adult Drosophila. Inhibition of huginPC neurons when larvae are tasting salt might be due to a different processing circuit for different concentrations of salt and the decision to either take up low doses or reject high doses (Hückesfeld, 2016). Taken together, it is proposed that hugin neuropeptide neurons projecting to the protocerebrum represent a hub between bitter gustatory receptor neurons and higher brain centres that integrate bitter sensory information in the brain, and through its activity, influences the decision of the animal to avoid a bitter food source. The identification of second order gustatory neurons for bitter taste will not only provide valuable insights into bitter taste pathways in Drosophila, but may also help in assigning a potentially novel role of its mammalian homologue, Neuromedin U, in taste processing (Hückesfeld, 2016). Drosophila has become an excellent model system for investigating the organization and function of the gustatory system due to the relatively simple neuroanatomical organization of its brain and the availability of powerful genetic and transgenic technology. Thus, at the molecular and cellular levels, a great deal of insight into the peripheral detection and coding of gustatory information has already been attained. In contrast, much less is known about the central neural circuits that process this information and induce behaviorally appropriate motor output. This study combined functional behavioral tests with targeted transgene expression through specific driver lines to identify a single bilaterally homologous pair of bitter-sensitive interneurons that are located in the subesophageal zone of the brain. Anatomical and functional data indicate that these interneurons receive specific synaptic input from bitter-sensitive gustatory receptor neurons. Targeted transgenic activation and inactivation experiments show that these bitter-sensitive interneurons can largely suppress the proboscis extension reflex to appetitive stimuli, such as sugar and water. These functional experiments, together with calcium-imaging studies and calcium-modulated photoactivatable ratiometric integrator (CaMPARI) labeling, indicate that these first-order local interneurons play an important role in the inhibition of the proboscis extension reflex that occurs in response to bitter tastants. Taken together, these studies present a cellular identification and functional characterization of a key gustatory interneuron in the bitter-sensitive gustatory circuitry of the adult fly (Bohra, 2018).
The gustatory and olfactory systems of Drosophila represent powerful models for analyzing the neuronal organization of chemosensory systems. In the adult olfactory system, a great deal is now known about the structure and function of the circuitry that detects and processes olfactory information. There are approximately 50 different classes of olfactory sensory neurons, each with a specific olfactory receptor type. Each olfactory sensory neuron type projects its axon to a single glomerulus in the antennal lobe of the brain, where synaptic interactions with local interneurons and projection neurons take place. Projection neurons convey processed sensory information from the glomeruli to higher order brain centers in the mushroom bodies and lateral horn, which process olfactory information further for behavioral functions, such as learning and memory or appetitive and aversive response control.
In the adult gustatory system, considerable insight into the molecular and cellular mechanisms of taste perception has also been attained. Functionally distinct classes of gustatory receptor neurons (GRNs) have been identified, including GRNs for bitter tastants such as those labeled by the Gr66a receptor and GRNs for sweet tastants such as those labeled by the Gr5a and Gr64f receptors; GRNs for salt, water, and pheromone detection have also been identified. Moreover, the gustatory receptor molecules expressed in these different GRN classes have also been characterized. Tastant-driven GRN activation results in modality-specific behavioral responses. Thus, activation of sweet GRNs stimulates feeding behavior such as the proboscis extension reflex (PER) and ingestion, while activation of bitter GRNs promotes aversive behavior such as PER inhibition and avoidance of noxious compound mixtures (Bohra, 2018).
The axons of the GRNs in legs and wing margins project to the thoracic ganglion, with the exception of Gr43a-expressing neurons in legs. By contrast, axons of the GRNs, which are located on the proboscis, project into discrete regions of the subesophageal zone (SEZ) in the central brain, which is the initial processing center for gustatory information. The SEZ also comprises the motor neurons, which control the PER that occurs in response to appetitive gustatory sensory input, as well as motor neurons that control ingestion. Moreover, the SEZ also contains a pair of local interneurons that have command function in the feeding motor program . Thus, local circuits might exist within the SEZ that mediate the transformation of the appropriate sensory input from GRNs into the proboscis motor neuron output required for feeding. In support of this notion, recent large-scale calcium imaging studies indicate that approximately 70 neurons located in the SEZ respond to either sweet or bitter gustatory input and that the majority of these cells are not motor neurons but rather modality-specific interneurons. Interestingly, these studies also suggest that the gustatory input to motor neurons is similarly modality specific (i.e., sweet and bitter tastants activate different motor neurons), implying that neural circuitry for sweet and bitter gustatory information processing in the SEZ is largely segregated from sensory input to motor output (Bohra, 2018).
Given the large number of interneurons in the SEZ that appear to respond to gustatory input, it is remarkable that only very few of these have been identified at the cellular level. Several gustatory interneuron types that respond to sweet taste input have been identified in the adult SEZ. All are thought to receive direct synaptic input from sweet-sensitive GRNs, implying that they are first-order gustatory interneurons. In contrast, with the exception of two identified bitter-sensitive projection interneuron types, information about first-order interneurons that receive and process gustatory information about other tastants categories such as bitter, salt, and water is largely lacking. This paucity of information has been a major stumbling block for unraveling gustatory circuits in the SEZ and understanding the interneuronal processing of different taste modalities in the gustatory system of the adult fly (Bohra, 2018).
This study used functional behavioral screening with transgenic driver lines to identify and characterize bitter-sensitive gustatory interneurons in the adult SEZ. By screening Gal4 lines for their ability to inhibit sugar-induced appetitive PER by targeted transgenic activation via TrpA1, this study identified the VGN 6341 line as a marker for interneurons implicated in aversive gustatory responses. Further experiments that limit the targeted transgenic activation to specific CNS regions show that the corresponding VGN 6341 interneurons are located in the SEZ. GRASP (GFP reconstitution across synaptic partners) experiments indicate that these interneurons receive direct synaptic input from Gr66a-labeled bitter-sensitive GRNs. The combination of VGN 6341 cis-flipout with a functional behavior assay identifies a single bilaterally symmetric pair of SEZ interneurons as the first-order gustatory interneurons responsible for the inhibition of the appetitive PER. Further activity assays using calcium imaging provide functional confirmation for the existence of the VGN 6341 interneuron pair in the SEZ that is activated by natural or transgenic stimulation of bitter GRNs (Bohra, 2018).
The experiments reported in this study identify a single pair of bilaterally homologous local interneurons in the SEZ of the adult fly. Anatomical, behavioral, and functional data indicate that these gustatory local interneurons (bGLN) cells receive specific synaptic input from bitter-sensitive GRNs. Moreover these data show that activation of these interneurons has a marked inhibitory effect on appetitive PER. From this, it is concluded that the bGLN cells are first-order gustatory interneurons that have an important role in the aversive bitter-sensitive gustatory circuitry of the adult fly (Bohra, 2018).
While it is certain that there are other candidate bitter-sensitive interneurons in the SEZ, this study focused only on the interneurons that are labeled by VGN 6341 Gal4. Among these, it was found that a single interneuron among the 4 medial cluster VGN 6341 interneurons can mediate the inhibitory effects on appetitive PER. Further, it was found that a single interneuron among the 4 medial cluster VGN 6341 interneurons receives functional input from bitter-sensitive GRNs. While it is, in principle, conceivable that there are two different medial cluster VGN 6341 interneurons implicated-one that receives bitter-sensitive input but does not mediate inhibitory effects on appetitive PER and another that mediates inhibitory effects on appetitive PER but does not receive bitter-sensitive input- this is considered to be highly unlikely. Thus, the most reasonable explanation for these findings is that one and only one of the four VGN 6341 interneurons in the medial cluster (namely, the cell with the features of the anatomically characterized bGLN) receives bitter-sensitive input and mediates PER inhibition (Bohra, 2018).
Previous studies have provided evidence for the notion that bitter and sweet gustatory stimuli are processed by different sensory interneuron populations in the SEZ of the adult fly, implying a segregation of the circuitry for aversive and appetitive taste modalities. Several SEZ sensory interneurons involved in the circuitry for appetitive sweet taste processing have been characterized; these comprise both local and projection interneurons. In contrast, prior to this report, only two types of bitter taste projection interneuron have been identified, and in terms of sensory local interneurons in the SEZ circuitry for bitter taste processing, nothing was known. Thus, the identification of the first bitter-sensitive gustatory local interneuron in the SEZ of the adult fly represents a significant step toward understanding how bitter taste modalities are processed by the gustatory circuitry in the SEZ of the brain (Bohra, 2018).
Functional experiments involving targeted activation of the identified bitter-sensitive gustatory interneurons indicate that the bGLN cells are important elements of the circuitry for aversive gustatory stimulus processing. Indeed, the strong PER response elicited by tarsal stimulation with high sucrose concentrations is all but eliminated by concomitant activation of these local interneurons. In wild-type animals, this type of powerful inhibition of PER responses is generally only seen if highly aversive and potentially toxic substances, such as quinine or strychnine, are mixed with the appetitive sugar stimulus. In this respect, the important and, possibly, vital role of the identified SEZ interneurons in bitter avoidance is underscored by experiments in which they are functionally inhibited. Remarkably, in this case, the PER response to an otherwise aversive mixture of sucrose and quinine is significantly increased from the very low response level that occurs if the interneurons are functional to a level that could result in the ingestion of toxic substance mixes in the wild. Indeed, without the concomitant existence of the mechanism for peripheral modulation of taste input by which the sweet taste neurons are directly inhibited by bitter tastants via odorant-binding proteins in accessory cells, positive PER responses to such harmful mixes would be even larger. From this point of view, the dual mechanisms for suppressing the response to mixtures of sweet and bitter compounds via central bitter-sensitive interneuronal circuitry and via peripheral modulation provide a vital safeguard against intoxication (Bohra, 2018).
VGN 6341-based genetic access to the identified bGLN cells sets the stage for future investigations focused on identifying their postsynaptic targets in the bitter gustatory circuitry of the SEZ. Several neuron types with neural processes in the SEZ that are known to be involved in gustatory/feeding circuitry might represent putative target neurons for these identified bGLNs. (Given that the first-order local interneurons have all of their neural arbors located within the SEZ, their target neurons must also arborize, at least in part, in the SEZ). These include local interneurons with command function in the feeding program, projection interneurons that modulate feeding in the adult and that are thought to combine feeding modulation with bitter taste detection in the larva, and motor neurons that control proboscis extension as well as food ingestion. Moreover, the bGLNs could conceivably be involved in the postulated circuitry for presynaptic inhibition of sweet GRNs, although in this case, the circuitry would have involved at least one further intercalated interneuron type, given the lack of a GRASP signal between the bGLNs and the sweet GRNs. Whether these or other as-yet-unidentified SEZ neurons with roles in gustation or feeding are, indeed, postsynaptic targets of the first-order bitter-sensitive interneurons and whether they receive excitatory or inhibitory input from these cells must await further investigation (Bohra, 2018).
It is remarkable that the bitter taste modality is conserved in insects and mammals and that bitter gustatory information plays a key role in evoking aversive behavior in these and many other animals. From this point of view, future studies of the structure, function, and behavioral role of the bitter-sensitive gustatory circuitry in Drosophila are likely to be of general significance for understanding gustation in all animals, including humans (Bohra, 2018).
Internal sensory neurons innervate body organs and provide information about internal state to the CNS to maintain physiological homeostasis. Despite their conservation across species, the anatomy, circuitry, and development of internal sensory systems are still relatively poorly understood. A largely unstudied population of larval Drosophila sensory neurons, termed tracheal dendrite (td) neurons, innervate internal respiratory organs and may serve as a model for understanding the sensing of internal states. This study characterized the peripheral anatomy, central axon projection, and diversity of td sensory neurons. Evidence for prominent expression of specific gustatory receptor genes in distinct populations of td neurons, suggesting novel chemosensory functions. This study identified two anatomically distinct classes of td neurons. The axons of one class project to the subesophageal zone (SEZ) in the brain, whereas the other terminates in the ventral nerve cord (VNC). This study identified expression and a developmental role of the POU-homeodomain transcription factor Pdm3 in regulating the axon extension and terminal targeting of SEZ-projecting td neurons. Remarkably, ectopic Pdm3 expression is alone sufficient to switch VNC-targeting axons to SEZ targets, and to induce the formation of putative synapses in these ectopic target zones. These data thus define distinct classes of td neurons, and identify a molecular factor that contributes to diversification of axon targeting. These results introduce a tractable model to elucidate molecular and circuit mechanisms underlying sensory processing of internal body status and physiological homeostasis (Qian, 2018).
High-resolution studies of sensory axon morphology in embryos identified unusual axon projections of td neurons beyond their segment of origin to a common target in thoracic neuromeres. Whether this neuromere represented an intermediate or terminal axon target was unknown because mature td axon projections in the third instar larva were not described. This study shows that all td neurons make long-range projections but have dichotomous terminal zones anteriorly in the SEZ and in the VNC. The SEZ receives chemosensory inputs and contains numerous peptidergic fibers. Based on their location along trachea, td neurons were proposed to function as proprioceptors or gas sensors, although the function of td neurons is as yet unknown. Anatomical data from this study are more consistent with roles for td neurons as internal chemosensors. It is noted that axons that project to the SEZ form en passant synapses throughout the VNC, suggesting distributed input to central circuits. SEZ- and VNC-targeting axons could conceivably share postsynaptic partners in the VNC, with SEZ-targeting axons connecting with an additional population of targets in the SEZ, although precise connectivity remains to be determined. A recent electron microscopic study of the SEZ identified ascending sensory projections that form synapses with a subset of peptidergic Hugin neurons (Schlegel, 2016). These sensory projections likely correspond to a subset of td neurons. Functional interrogation of this Hugin circuit and reconstruction of additional downstream targets of SEZ- and VNC-projecting td neurons will provide insights into possible roles for the td system in behavior and physiology (Qian, 2018).
This study identified expression of multiple gustatory and ionotropic receptor (GR and IR) reporters in td neurons. These findings, together with anatomical data, suggests that td neurons may function to sense internal chemical stimuli. In Drosophila, the combinatorial coexpression of specific GRs determines the tuning of gustatory neurons to specific ligands. The patterns of coexpressed GRs that were observed in td neurons have not been observed in other gustatory neurons, suggesting possible tuning to novel ligands. Two GRs that appear to be expressed in td neurons, Gr33a and Gr89a, are expressed in all adult bitter neurons, and Gr33a is broadly required for responses to aversive cues in the context of feeding. These GRs have been proposed to function as 'core bitter coreceptors'. It is possible that at least a subset of td neurons may detect aversive chemical stimuli. Given that td dendrites appear to be bathed in hemolymph and associated with the trachea, td neurons may detect both dissolved circulating stimuli (e.g., ingested toxins, metabolites, electrolytes) and gaseous stimuli (e.g., CO2, O2). The expression of a reporter for Ir76b, a detector of low salt, and oxygen-sensitive guanylyl cyclase in different subsets of td neurons is consistent with this idea. It is speculated that td neurons may detect chemical imbalances and relay signals to the SEZ and VNC to elicit behavioral or physiological responses to restore homeostasis. Neurons in the SEZ could regulate feeding, and neurons in the VNC could regulate locomotion or fluid balance. In mammals, lung-innervating sensory neurons comprise molecularly distinct subtypes with different anatomical projections and functions. This study shows that larval Drosophila trachea-innervating sensory neurons similarly comprise molecularly distinct subtypes with distinct axon projections. Future studies to image and manipulate td activity, and disrupt chemosensory receptor gene function, should clarify the sensory functions of td neurons and the underlying molecular mechanisms (Qian, 2018).
This study uncovered multiple levels of specificity of td neuron dendrite-substrate relationships, including strict association with a tracheal substrate, arborization across specific tracheal branches, and dendritic specializations at tracheal fusion cells. The factors that specify sensory dendrite organization of td neurons are unknown and do not appear to include Pdm3. Whether dendrite specializations are important for detection of chemicals in the tracheal lumen or whether trachea merely serve as an attachment site to allow sensing of abdominal hemolymph status is not clear. The positioning of td dendrites may place them out of direct contact with the tracheal interior; however, association across tracheal cells could still permit sensing of tracheal physiology. Future studies to monitor tracheal system and td dendrite development will help to sort out mechanisms of dendrite-substrate interactions and the importance of this association for td neuron function (Qian, 2018).
Many of the guidance decisions made by sensory axons involve decisions to terminate at specific mediolateral and dorsoventral positions or in specific neuropil layers. For td axons, the guidance decisions are complex. Single td axons switch between medial and lateral positions, and dorsal and ventral positions and do so at specific locations along their length. Moreover, the terminal position of td axons varies according to cell identity and segment of origin. It is predicted that studies of td neurons may be especially useful for understanding sequential and regionally restricted guidance switches in axons, a model more akin to long-range projections, such as vertebrate corticospinal tract axons that navigate multiple choice points, than other locally projecting Drosophila sensory axons (Qian, 2018).
This study provides initial insight into one major choice of td axons: the choice to project, or not, to far anterior regions of the CNS (SEZ). The Pdm3 transcription factor is expressed in most, but not all, td neurons that project to the SEZ and is expressed in none of the td neurons that terminate in the VNC. Ectopic Pdm3 expression promoted anterior axon growth along the canonical td axon path, indicating that Pdm3 expression is sufficient for SEZ projections. This effect depends on sensory context because misexpression of Pdm3 in cIV dendritic arborization neurons did not convert axons to a td-like projection, but rather led to axon defasciculation, overgrowth, and axon straying, occasionally into the SEZ. Loss of Pdm3 led to modest disruptions of terminal targeting in SEZ-projecting tds, suggesting sufficiency, but redundancy with other factors, in SEZ targeting. This study noted specific patterns of axon-axon segregation among axons that project to the SEZ and those that project to the VNC. Thus, in addition to the possibility that Pdm3 functions as a growth-promoting factor, other explanations could account for Pdm3 misexpression phenotypes, such as promoting specific patterns of axon-axon interactions that underlie pathfinding to anterior CNS (Qian, 2018).
These results extend the roles for Pdm3 in axon targeting and chemosensory receptor expression. Prior studies identified roles for Pdm3 in targeting of olfactory sensory neurons, in olfactory receptor expression and in ellipsoid ring (R) neuron axon targeting (Chen, 2012). In R neurons, Pdm3 controls axon terminal targeting, without impacting dendritic arborization, cell fate determination, or initial axon outgrowth. The results for td neurons support a role in axon terminal growth and targeting, or maintenance, and in regulation of GR expression. Thus, this study demonstrates that Pdm3 regulates multiple aspects of td cellular identity, consistent with prior findings in the olfactory system. With respect to fine terminal targeting, one potential role for Pdm3 may be to inhibit midline contact of sensory axon terminals, which could account for the Pdm3 loss-of-function phenotype in td neurons and part of the Pdm3 misexpression phenotype in cIV neurons. The normal functions of Pdm3 in different cell types suggest context-dependent roles to promote terminal targeting. Identifying whether conserved transcriptional targets are shared between these different systems will be an important future step. Studies of Pdm3 might reveal how axon initial growth, pathfinding, terminal targeting, and maintenance are regulated in a modular fashion across different neurons, which could be important not only for axon wiring during development but also for regeneration (Qian, 2018).
Animals typically avoid unwanted situations with stereotyped escape behavior. For instance, Drosophila larvae often escape from aversive stimuli to the head, such as mechanical stimuli and blue light irradiation, by backward locomotion. Responses to these aversive stimuli are mediated by a variety of sensory neurons including mechanosensory class III da (C3da) sensory neurons and blue-light responsive class IV da (C4da) sensory neurons and Bolwig's organ (BO). How these distinct sensory pathways evoke backward locomotion at the circuit level is still incompletely understood. This study shows that a pair of cholinergic neurons in the subesophageal zone, designated AMBs, evoke robust backward locomotion upon optogenetic activation. Anatomical and functional analysis shows that AMBs act upstream of MDNs, the command-like neurons for backward locomotion. Further functional analysis indicates that AMBs preferentially convey aversive blue light information from C4da neurons to MDNs to elicit backward locomotion, whereas aversive information from BO converges on MDNs through AMB-independent pathways. This study also found that, unlike in adult flies, MDNs are dispensable for the dead end-evoked backward locomotion in larvae. These findings thus reveal the neural circuits by which two distinct blue light-sensing pathways converge on the command-like neurons to evoke robust backward locomotion, and suggest that distinct but partially redundant neural circuits including the command-like neurons might be utilized to drive backward locomotion in response to different sensory stimuli as well as in adults and larvae (Omamiuda-Ishikawa, 2020).
Discrimination of sensory signals is essential for an organism to form and retrieve memories of relevance in a given behavioral context. Sensory representations are modified dynamically by changes in behavioral state, facilitating context-dependent selection of behavior, through signals carried by noradrenergic input in mammals, or octopamine (OA) in insects. To understand the circuit mechanisms of this signaling, this study characterized the function of two OA neurons, sVUM1 neurons, that originate in the subesophageal zone (SEZ) and target the input region of the memory center, the mushroom body (MB) calyx, in larval Drosophila. sVUM1 neurons were found to target multiple neurons, including olfactory projection neurons (PNs), the inhibitory neuron APL, and a pair of extrinsic output neurons, but relatively few mushroom body intrinsic neurons, Kenyon cells. PN terminals carried the OA receptor Oamb, a Drosophila α1-adrenergic receptor ortholog. Using an odor discrimination learning paradigm, this study showed that optogenetic activation of OA neurons compromised discrimination of similar odors but not learning ability. These results suggest that sVUM1 neurons modify odor representations via multiple extrinsic inputs at the sensory input area to the MB olfactory learning circuit (Wong, 2021).
Neural circuits carry out complex computations that allow animals to
evaluate food, select mates, move toward attractive stimuli, and move away from threats. In insects, the subesophageal zone (SEZ)
is a brain region that receives gustatory, pheromonal, and
mechanosensory inputs and contributes to the control of diverse
behaviors, including feeding, grooming, and locomotion. Despite its
importance in sensorimotor transformations, the study of SEZ circuits
has been hindered by limited knowledge of the underlying diversity of
SEZ neurons. This study generate a collection of split-GAL4 lines that
provides precise genetic targeting of 138 different SEZ cell types in
adult D. melanogaster, comprising approximately one third of all SEZ
neurons. The single cell anatomy of these neurons was characterized, and
they were found to cluster by morphology into six supergroups that
organize the SEZ into discrete anatomical domains. The majority of local
SEZ interneurons are not classically polarized, suggesting rich local
processing, whereas SEZ projection neurons tend to be classically
polarized, conveying information to a limited number of higher brain
regions. This study provides insight into the anatomical organization of
the SEZ and generates resources that will facilitate further study of
SEZ neurons and their contributions to sensory processing and behavior (Sterne, 2021).
This study describes the SEZ Split-GAL4 Collection, a library of 277 split-GAL4 lines covering 138 SEZ cell types, which affords unprecedented genetic access to SEZ neurons for behavioral and functional study. These studies provide insight into the diversity of SEZ cell types and their organization into discrete anatomical domains. The SEZ Split-GAL4 Collection will enable further investigation of how local SEZ circuitry and ascending SEZ paths process sensory inputs and control specific behaviors (Sterne, 2021).
Most of the SEZ Split-GAL4 lines are specific, with 149/277 lines classified as ideal or excellent. These lines will be useful to manipulate individual SEZ cell types for behavioral, functional, and imaging experiments. The remaining, less specific, lines (those belonging to the good or poor categories) will still be useful for imaging and as starting points for creating more specific reagents. Good and poor lines may be used to generate CDM masks to search for new hemidrivers to make further split-GAL4 lines. Alternatively, their expression patterns may be refined using Killer Zipper or three-way intersections with LexA or QF lines. All lines in the SEZ Split-GAL4 Collection may be used to generate further tools including complementary split-LexA and split-QF reagents. Split-LexA and split-QF lines may be used in concert with the split-GAL4 lines reported here to simultaneously manipulate two independent neuronal populations for advanced intersectional experiments, including behavioral epistasis (Sterne, 2021).
By combining insights from a single-cell transcriptome atlas with direct cell counts of SEZ neuromeres, it is estimated that the SEZ Split-GAL4 Collection labels 30% of the ~1700 neurons in the SEZ. Because of the lack of stereotyped neuronal cell body positions in D. melanogaster, it is not possible to assign cell bodies to defined neuropil regions without a genetic marker. The advantage of this method of estimating SEZ neuron number is that it is based on analysis of the four genetically defined SEZ neuromeres, the tritocerebral, the mandibular, the maxillary, and the labial neuromeres. However, previous reports demonstrate that some deutocerebral commissures cross below the esophageal foramen, and therefore an unknown number of deutocerebral cell bodies may be part of the SEZ. The limitations of this estimate of SEZ neuron number therefore include the inability to directly count cells derived from the tritocerebral neuromere, the inability to directly count neurons rather than glia, and the inability to assess deutocerebral contributions. Thus, the estimate of SEZ cell number is likely an underestimate. Once all SEZ neurons are densely reconstructed in an EM volume, direct counts of SEZ neuronal cell bodies obtained by EM will provide a more accurate assessment of SEZ neuron number. Regardless, the SEZ Split-GAL4 Collection targets 510 neuronal cell bodies, which represents a substantial improvement in the ability to precisely target SEZ cell types for functional and morphological analysis. This study did not ascertain the neuromere or neuroblast of origin of the SEZ cell types in the SEZ Split-GAL4 Collection. However, recent work has established reliable anatomical criteria that define the boundaries between the four SEZ neuromeres and has mapped all secondary lineages of the SEZ (Hartenstein, 2018). Future efforts should focus on bridging previously identified fascicle, neuropil, and sensory domains into a common template or coordinate space to determine the neuromere and neuroblast origin of SEZ cell types (Sterne, 2021).
Discovering and genetically targeting SEZ cell types required the use of registered light-level imagery and computer-assisted searching. Four distinct strategies were used to identify 129 novel and 9 previously reported SEZ cell types in registered light-level imagery. Critically, each of these strategies allowed use of CDM mask searching to identify additional hemidrivers with which to target each cell type of interest. CDM mask searching enabled combing of large datasets and greatly increased the ease and speed of split-GAL4 generation over previous methods. The same strategies can be leveraged to gain genetic access to yet-undiscovered SEZ cell types. The recent electron microscopy (EM) volumes of the D. melanogaster brain provide an avenue for identifying SEZ cell types that are not covered by the SEZ Split-GAL4 Collection. Notably, this approach awaits comprehensive reconstruction of the SEZ, a region that is not included in the recently published dense reconstruction of the 'hemibrain' volume. Another EM volume, 'FAFB,' provides imagery of an entire adult female fly brain at synaptic resolution and includes the SEZ. Improvements in automated reconstruction of EM volumes coupled with large-scale human annotation should soon provide exhaustive reconstruction of the SEZ from which to identify additional SEZ cell types. Furthermore, available bridging registrations between EM volumes and light-level imagery should facilitate the identification of hemidrivers to target SEZ cell types discovered from EM reconstructions. Even without identifying additional SEZ cell types, the split-GAL4 reagents described will allow behavioral and functional evaluation of circuit hypotheses derived from EM imagery (Sterne, 2021).
These analyses of the SEZ Split-GAL4 Collection provide insight into the cellular architecture of the SEZ. To computationally probe the organization of the SEZ, 121 SEZ cell types were morphologically clustered using NBLAST. This approach reveals six cellular domains in the SEZ that are organized in a largely layered fashion from anterior to posterior. This layered structure is also hinted at by the recent description of SEZ neuropil domains throughout development from the larva to the adult (Kendroud, 2018). Based on anatomical position and the known function of a few SEZ neurons, it is tempting to speculate that different morphological clusters may participate in different behavioral functions. Group 1 contains projection neurons that innervate the region of the SMP surrounding the pars intercerebralis (PI), suggesting that group 1 neurons may impinge on neurosecretory neurons or function in energy and fluid homeostasis circuits. The proximity of group 1 interneurons to previously described interoceptive SEZ neurons (ISNs) and ingestion neurons (IN1) supports this hypothesis. Group 2 contains Fdg, a feeding-related neuron, as well as cell types (indigo, tinctoria) that are located near pumping motor neurons, suggesting that group 2 neurons have roles in feeding sequence generation. Group 3 contains G2N-1, a candidate second-order gustatory neuron, and projection neurons that innervate recently described taste-responsive SLP regions, suggesting that group 3 may, in part, be composed of taste-responsive neurons. Many interneurons in group 4 are located near proboscis motor neurons that control rostrum protraction, haustellum extension, and labellar spreading, indicating that group 4 members function in proboscis motor control. The proximity of neurons in group 5 to previously described stopping neuron MAN, and the inclusion of an antennal grooming neuron, suggests that group 5 neurons may participate in circuits that control grooming and stopping behaviors. Group 6 is located in the posterior SEZ and posterior slope, regions implicated in flight behaviors, including wing and neck control. While potential behavioral functions are hypothesized for each supergroup, it is readily acknowledged that the roles of the neurons described in this study are likely more diverse (Sterne, 2021).
These studies also shed light on information flow both within the SEZ and out of the SEZ to the higher brain. 91 local interneurons, 30 projection neurons, 16 descending neurons, and 1 sensory neuron were identified. Polarity analysis of 121/138 of the SEZ cell types covered by the SEZ Split-GAL4 Collection revealed that SEZ interneurons tend to have mixed or biased polarity while SEZ projection neurons tend to be classically polarized. Polarity analyses of the lateral horn, mushroom body, descending neurons, and protocerebral bridge identified few neurons with completely mixed polarity. Unlike these brain regions, the SEZ contains a large number of local interneurons. The mixed polarity of the SEZ interneurons argues for local and reciprocal connectivity between neurons, with information flowing in networks rather than unidirectional streams. Projection neurons, in contrast, may serve chiefly to pass information from highly interconnected SEZ circuits to other brain regions in a unidirectional manner. Notably, many SEZ projection neurons were identified that innervate the SMP-a region known to contain neurosecretory cell types. This may betray a role for acute taste detection or feeding circuit activation in the regulation of hormone secretion. In addition, the frequent innervation of the superior lateral protocerebrum and lateral horn by SEZ projection neurons may hint at the site of olfactory-gustatory synthesis. In contrast, this study did not identify projection neurons that link the SEZ directly to the central complex or mushroom body. If dense reconstruction of EM volumes corroborates the lack of direct connectivity between the SEZ and these regions, information must be conveyed through indirect pathways. As an example, taste information influences local search behaviors during foraging, a task that is expected to involve the central complex. Indirect relay of taste information to the central complex to inform foraging behavior would be consistent with previous anatomical studies suggesting that the central complex receives diverse indirect sensory inputs. Furthermore, the mushroom body is known to respond to taste, raising the possibility that taste information from gustatory sensory neuron axons in the SEZ must be relayed through yet another brain region before reaching mushroom body cell types. Thus, this analysis of SEZ neuron polarity indicates local SEZ processing and demonstrates direct pathways to a subset of higher brain regions (Sterne, 2021).
Overall, the SEZ Split-GAL4 Collection represents a valuable resource that will facilitate the study of the SEZ. This analysis of the collection reveals the cellular anatomy and polarity of individual SEZ neurons and their organization into six discrete domains in the SEZ. Coupled with emerging insights from reconstruction of EM volumes, the SEZ Split-GAL4 Collection will allow the use of genetic dissection to test circuit-level hypotheses about sensory processing and motor control in the SEZ (Sterne, 2021).
In Drosophila melanogaster, gustatory receptor neurons (GRNs) for sugar taste coexpress various combinations of gustatory receptor (Gr) genes and are found in multiple sites in the body. To determine whether diverse sugar GRNs expressing different combinations of Grs have distinct behavioral roles, this study examined the effects on feeding behavior of genetic manipulations which promote or suppress functions of GRNs that express either or both of the sugar receptor genes Gr5a (Gr5a+ GRNs) and Gr61a (Gr61a+ GRNs). Cell-population-specific overexpression of the wild-type form of Gr5a (Gr5a(+)) in the Gr5a mutant background revealed that Gr61a+ GRNs localized on the legs and internal mouthpart critically contribute to food choice but not to meal size decisions, while Gr5a+ GRNs, which are broadly expressed in many sugar-responsive cells across the body with an enrichment in the labella, are involved in both food choice and meal size decisions. The legs harbor two classes of Gr61a expressing GRNs, one with Gr5a expression (Gr5a+/Gr61a+ GRNs) and the other without Gr5a expression (Gr5a-/Gr61a+ GRNs). Blocking the Gr5a+ class in the entire body reduced the preference for trehalose and blocking the Gr5a- class reduced the preference for fructose. These two subsets of GRNs are also different in their central projections: axons of tarsal Gr5a+/Gr61a+ GRNs terminate exclusively in the ventral nerve cord, while some axons of tarsal Gr5a-/Gr61a+ GRNs ascend through the cervical connectives to terminate in the subesophageal ganglion. It is proposed that tarsal Gr5a+/Gr61a+ GRNs and Gr5a-/Gr61a+ GRNs represent functionally distinct sensory pathways that function differently in food preference and meal-size decisions (Kohatsu, 2022).
Swallowing is an essential step of eating and drinking. However, how the quality of a food bolus is sensed by pharyngeal neurons is largely unknown. This study finds that mechanical receptors along the Drosophila pharynx are required for control of meal size, especially for food of high viscosity. The mechanical force exerted by the bolus passing across the pharynx is detected by neurons expressing the mechanotransduction channel NOMPC (no mechanoreceptor potential C) and is relayed, together with gustatory information, to IN1 neurons in the subesophageal zone (SEZ) of the brain. IN1 (ingestion neurons) neurons act directly upstream of a group of peptidergic neurons that encode satiety. Prolonged activation of IN1 neurons suppresses feeding. IN1 neurons receive inhibition from DSOG1 (descending subesophageal neurons) neurons, a group of GABAergic neurons that non-selectively suppress feeding. These results reveal the function of pharyngeal mechanoreceptors and their downstream neural circuits in the control of food ingestion (Yang, 2021).
Overconsumption is harmful for animals. Although the drive to ingest can be overwhelming for a hungry animal in the initial stage of a meal, inhibition becomes more dominant with the processes of food intake. This study found that food flowing across the pharynx accumulates the satiety state in the brain, demonstrating that multiple strategies are used by the nervous systems to avoid overeating. These pharyngeal sensory neurons are sensitive to sugar and mechanical force, serving as a flow meter that monitors food quality and amount so that the brain knows how much food is ingested even before the food reaches the intestine. This circuit may coordinate with other satiety signals, such as those conveyed by mechanical feedback from the intestine, to control feeding (Yang, 2021).
Gustatory and mechanosensory neurons are well separated on the fly labellum before their axons reach the SEZ, where they interact with each other to regulate the perception of food quality. In contrast, the sensory neurons in the pharynx seem to adapt a different coding mechanism. Some of the pharyngeal neurons are polymodal because they respond to chemical and mechanical stimuli, with PM neurons being an example. A 'generalist' versus 'specialist strategy has been found in other sensory organs too. Being able to evaluate multiple properties of a bolus in the pharynx allow the animals to effectively control the feeding amount. There are sensory neurons in the pharynx that may be tuned to gustatory or mechanosensory cues. For example, the R41E11-GAL4 and nompC-QF labeled approximately 10 pairs of neurons in LSOs along the pharynx, similar to the number observed for mechanosensory neurons. Most of those neurons are likely 'generalist' and are tuned to mechanical stimuli only. It would be valuable to determine the full repertoire of these sensory neurons to understand how the swallowing maneuver is initiated, sustained, and terminated (Yang, 2021).
It has been proposed that IN1 neurons may function as a key node of the feeding control circuits to govern rapid feeding decisions. Previous studies have revealed that IN1 neurons are directly downstream of pharyngeal GRNs and that activation of IN1 neurons to sugar stimulation is correlated with a fly's motivation to feed. Because activation of IN1 neurons triggers proboscis extension to food, they are likely upstream of the motor circuit that controls feeding. IN1 neurons thus appear to function as a hub that integrates sensory information to initiate food ingestion. This study found that IN1 neurons' activity is under control of the fly's feeding states. IN1 neurons are directly downstream of DSOG1 neurons that non-selectively suppress ingestion. In fed flies, DSOG1 neurons impart inhibition on IN1 neurons, resulting in a transient and moderate response to a sugar sip that triggers a robust and sustained calcium response in fasted flies (Yang, 2021).
It has been proposed that DSOG1 neurons impart constant inhibition on the neuronal circuits that initiate food ingestion. However, the upstream circuit of DSOG1 neurons has not been identified. A cohort of neuropeptide receptor genes has been screened, but none of them seemed to function on DSOG1 neurons in feeding control. This study found that interrupting signaling of the neuropeptide MIP phenocopied overfeeding in flies with silenced DSOG1 neurons. It is tantalizing to hypothesize that MIP neurons are upstream of the DSOG1 circuit, either directly or indirectly. Because the receptors of MIP have not yet been identified, further experiments are need to differentiate between the two possibilities (Yang, 2021).
Besides PM neurons, there are many NOMPC-expressing mechanosensory neurons along the fly pharynx. Because of the lack of specific driver lines and the technique to record a single neuron's activity during feeding, their roles in feeding regulation are interesting open questions and await in-depth investigation. Moreover, the receptors of MIP peptide have not been identified, especially the ones involved in feeding regulation, making it difficult to establish a connection between MIP neurons and DSOG1 neurons (Yang, 2021).
Bohra, A. A., Kallman, B. R., Reichert, H. and VijayRaghavan, K. (2018). Identification of a single pair of interneurons for bitter taste processing in the Drosophila brain. Curr Biol 28(6): 847-858.e843. PubMed ID: 29502953
Cachero, S., Ostrovsky, A.D., Yu, J.Y., Dickson, B.J., Jefferis, GSXE (2010). Sexual Dimorphism in the Fly Brain, Curr. Biol. 20: 1589-1601. PubMed ID: 20832311
Certel, S. J., Savella, M. G., Schlegel, D. C. and Kravitz, E. A. (2007). Modulation of Drosophila male behavioral choice. Proc. Natl. Acad. Sci. 104: 4706-4711. PubMed ID: 17360588
Chen, C. K., Chen, W. Y. and Chien, C. T. (2012). The POU-domain protein Pdm3 regulates axonal targeting of R neurons in the Drosophila ellipsoid body. Dev Neurobiol 72(11): 1422-1432. PubMed ID: 22190420
Hartenstein, V., Omoto, J. J., Ngo, K. T., Wong, D., Kuert, P. A., Reichert, H., Lovick, J. K. and Younossi-Hartenstein, A. (2018). Structure and development of the subesophageal zone of the Drosophila brain. I. Segmental architecture, compartmentalization, and lineage anatomy. J Comp Neurol 526(1): 6-32. PubMed ID: 28730682
Huckesfeld, S., Schoofs, A., Schlegel, P., Miroschnikow, A. and Pankratz, M. J. (2015). Localization of motor neurons and central pattern generators for motor patterns underlying feeding behavior in Drosophila larvae. PLoS One 10: e0135011. PubMed ID: 26252658
Hückesfeld, S., Peters, M. and Pankratz, M.J. (2016). Central relay of bitter taste to the protocerebrum by peptidergic interneurons in the Drosophila brain. Nat Commun 7: 12796. PubMed ID: 27619503
Kendroud, S., Bohra, A. A., Kuert, P. A., Nguyen, B., Guillermin, O., Sprecher, S. G., Reichert, H., VijayRaghavan, K. and Hartenstein, V. (2018). Structure and development of the subesophageal zone of the Drosophila brain. II. Sensory compartments. J Comp Neurol 526(1): 33-58. PubMed ID: 28875566
Kohatsu, S., Tanabe, N., Yamamoto, D. and Isono, K. (2022). Which Sugar to Take and How Much to Take? Two Distinct Decisions Mediated by Separate Sensory Channels. Front Mol Neurosci 15: 895395. PubMed ID: 35726300
Kuert, P. A., Hartenstein, V., Bello, B. C., Lovick, J. K. and Reichert, H. (2014). Neuroblast lineage identification and lineage-specific Hox gene action during postembryonic development of the subesophageal ganglion in the Drosophila central brain. Dev Biol 390: 102-115. PubMed ID: 24713419
Kwon, J. Y., Dahanukar, A., Weiss, L. A. and Carlson, J. R. (2007). The molecular basis of CO2 reception in Drosophila. Proc. Natl. Acad. Sci. 104(9): 3574-8. Medline abstract: 17360684
Kwon, J. Y., Dahanukar, A., Weiss, L. A. and Carlson, J. R. (2011). Molecular and cellular organization of the taste system in the Drosophila larva. J. Neurosci. 31(43): 15300-9. PubMed Citation: 22031876
Mann, K., Gordon, M. D. and Scott, K. (2013). A pair of interneurons influences the choice between feeding and locomotion in Drosophila. Neuron 79: 754-765. PubMed ID: 23972600
Omamiuda-Ishikawa, N., Sakai, M. and Emoto, K. (2020). A pair of ascending neurons in the subesophageal zone mediates aversive sensory inputs-evoked backward locomotion in Drosophila larvae. PLoS Genet 16(11): e1009120. PubMed ID: 33137117
Qian, C. S., Kaplow, M., Lee, J. K. and Grueber, W. B. (2018). Diversity of internal sensory neuron axon projection patterns is controlled by the POU-domain protein Pdm3 in Drosophila larvae. J Neurosci 38(8): 2081-2093. PubMed ID: 29367405
Schlegel, P., Texada, M. J., Miroschnikow, A., Schoofs, A., Huckesfeld, S., Peters, M., Schneider-Mizell, C. M., Lacin, H., Li, F., Fetter, R. D., Truman, J. W., Cardona, A. and Pankratz, M. J. (2016). Synaptic transmission parallels neuromodulation in a central food-intake circuit. Elife 5. PubMed ID: 27845623
Schoofs, A., Hückesfeld, S., Schlegel, P., Miroschnikow, A., Peters, M., Zeymer, M., Spiess, R., Chiang, A. S. and Pankratz, M. J. (2014). Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain. PLoS Biol 12: e1001893. PubMed ID: 24960360
Sterne, G. R., Otsuna, H., Dickson, B. J. and Scott, K. (2021). Classification and genetic targeting of cell types in the primary taste and premotor center of the adult Drosophila brain. Elife 10. PubMed ID: 34473057
Tran, D. H., Meissner, G. W., French, R. L. and Baker, B. S. (2014). A small subset of fruitless subesophageal neurons modulate early courtship in Drosophila. PLoS One 9: e95472. PubMed ID: 24740138
Wang, Z., Singhvi, A., Kong, P. and Scott, K. (2004). Taste representations in the Drosophila brain. Cell 117(7): 981-91. 15210117
Wong, J. Y. H., Wan, B. A., Bland, T., Montagnese, M., McLachlan, A. D., O'Kane, C. J., Zhang, S. W. and Masuda-Nakagawa, L. M. (2021). Octopaminergic neurons have multiple targets in Drosophila larval mushroom body calyx and can modulate behavioral odor discrimination. Learn Mem 28(2): 53-71. PubMed ID: 33452115
Yang, T., Yuan, Z., Liu, C., Liu, T. and Zhang, W. (2021). A neural circuit integrates pharyngeal sensation to control feeding. Cell Rep 37(6): 109983. PubMed ID: 34758309
date revised: 22 February 2022
Genes involved in organ development
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