Structure and development of the subesophageal zone of the Drosophila brain. I. Segmental architecture,compartmentalization, and lineage anatomy

The subesophageal zone (SEZ) of the Drosophila brain houses the circuitry underlying feeding behavior and is involved in many other aspects of sensory processing and locomotor control. Formed by the merging of four neuromeres, the internal architecture of the SEZ can be best understood by identifying segmentally reiterated landmarks emerging in the embryo and larva, and following the gradual changes by which these landmarks become integrated into the mature SEZ during metamorphosis. In previous works, the system of longitudinal fibers (connectives) and transverse axons (commissures) has been used as a scaffold that provides internal landmarks for the neuromeres of the larval ventral nerve cord. We have extended the analysis of this scaffold to the SEZ and, in addition, reconstructed the tracts formed by lineages and nerves in relationship to the connectives and commissures. As a result, we establish reliable criteria that define boundaries between the four neuromeres (tritocerebrum, mandibular neuromere, maxillary neuromere, labial neuromere) of the SEZ at all stages of development. Fascicles and lineage tracts also demarcate seven columnar neuropil domains (ventromedial, ventro‐lateral, centromedial, central, centrolateral, dorsomedial, dorsolateral) identifiable throughout development. These anatomical subdivisions, presented in the form of an atlas including confocal sections and 3D digital models for the larval, pupal and adult stage, allowed us to describe the morphogenetic changes shaping the adult SEZ. Finally, we mapped MARCM‐labeled clones of all secondary lineages of the SEZ to the newly established neuropil subdivisions. Our work will facilitate future studies of function and comparative anatomy of the SEZ.     

The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva

Understanding neuronal function at the local and circuit level requires understanding astrocyte function. We have provided a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, our data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. Our data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits—the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron–glia interactions in this neuropil.     

Characterization of Drosophila octopamine receptor neuronal expression using MiMIC-converted Gal4 lines

Octopamine, the invertebrate analog of norepinephrine, is known to modulate a large variety of behaviors in Drosophila including feeding initiation, locomotion, aggression, and courtship, among many others. Significantly less is known about the identity of the neurons that receive octopamine input and how they mediate octopamine-regulated behaviors. Here, we characterize adult neuronal expression of MiMIC-converted Trojan-Gal4 lines for each of the five Drosophila octopamine receptors. Broad neuronal expression was observed for all five octopamine receptors, yet distinct differences among them were also apparent. Use of immunostaining for the octopamine neurotransmitter synthesis enzyme Tdc2, along with a novel genome-edited conditional Tdc2-LexA driver, revealed all five octopamine receptors express in Tdc2/octopamine neurons to varying degrees. This suggests autoreception may be an important circuit mechanism by which octopamine modulates behavior.     

Developmental analysis of the dopamine‐containing neurons of the Drosophila brain

The Drosophila dopaminergic (DAergic) system consists of a relatively small number of neurons clustered throughout the brain and ventral nerve cord. Previous work shows that clusters of DA neurons innervate different brain compartments, which in part accounts for functional diversity of the DA system. We analyzed the association between DA neuron clusters and specific brain lineages, developmental and structural units of the Drosophila brain that provide a framework of connections that can be followed throughout development. The hatching larval brain contains six groups of primary DA neurons (born in the embryo), which we assign to six distinct lineages. We can show that all larval DA clusters persist into the adult brain. Some clusters increase in cell number during late larval stages, whereas others do not become DA positive until early pupa. Ablating neuroblasts with hydroxyurea (HU) prior to onset of larval proliferation (generates secondary neurons) confirms that these added DA clusters are primary neurons born in the embryo, rather than secondary neurons. A single cluster that becomes DA positive in the late pupa, PAM1/lineage DALcm1/2, forms part of a secondary lineage that can be ablated by larval HU application. By supplying lineage information for each DA cluster, our analysis promotes further developmental and functional analyses of this important system of neurons. J. Comp. Neurol. 525:363–379, 2017. © 2016 Wiley Periodicals, Inc.     

A map of sensilla and neurons in the taste system of drosophila larvae

In Drosophila melanogaster larvae, the prime site of external taste reception is the terminal organ (TO). Though investigation on the TO's implications in taste perception has been expanding rapidly, the sensilla of the TO have been essentially unexplored. In this study, we performed a systematic anatomical and molecular analysis of the TO. We precisely define morphological types of TO sensilla taking advantage of volume electron microscopy and 3D image analysis. We corroborate the presence of five external types of sensilla: papilla, pit, spot, knob, and modified papilla. Detailed 3D analysis of their structural organization allowed a finer discrimination into subtypes. We classify three subtypes of papilla and pit sensilla, respectively, and two subtypes of knob sensilla. Further, we determine the repertoire of receptor genes for each sensillum by analyzing GAL4 driver lines of Ir, Gr, Ppk, and Trp receptor genes. We construct a map of the TO, in which the receptor genes are mapped to neurons of individual sensilla. While modified papillum and spot sensilla are not labeled by any GAL4 driver, neurons of the pit, papilla, and knob type are labeled by partially overlapping but different subsets of GAL4 driver lines of the Ir, Gr, and Ppk gene family. The results suggest that pit, papilla and knob sensilla act in contact chemosensation. However, they likely do these employing different stimulus transduction mechanisms to sense the diverse chemicals of their environment.     

Neuropeptides in the desert ant Cataglyphis fortis: Mass spectrometric analysis,localization, and age‐related changes

Cataglyphis desert ants exhibit an age‐related polyethism, with ants performing tasks in the dark nest for the first ～4 weeks of their adult life before they switch to visually based long‐distance navigation to forage. Although behavioral and sensory aspects of this transition have been studied, the internal factors triggering the behavioral changes are largely unknown. We suggest the neuropeptide families allatostatin A (AstA), allatotropin (AT), short neuropeptide F (sNPF), and tachykinin (TK) as potential candidates. Based on a neuropeptidomic analysis in Camponotus floridanus, nano‐LC‐ESI MS/MS was used to identify these neuropeptides biochemically in Cataglyphis fortis. Furthermore, we show that all identified peptide families are present in the central brain and ventral ganglia of C. fortis whereas in the retrocerebral complex only sNPF could be detected. Immunofluorescence staining against AstA, AT, and TK in the brain revealed arborizations of AstA‐ and TK‐positive neurons in primary sensory processing centers and higher order integration centers, whereas AT immunoreactivity was restricted to the central complex, the antennal mechanosensory and motor center, and the protocerebrum. For artificially dark‐kept ants, we found that TK distribution changed markedly in the central complex from days 1 and 7 to day 14 after eclosion. Based on functional studies in Drosophila, this age‐related variation of TK is suggestive of a modulatory role in locomotion behavior in C. fortis. We conclude that the general distribution and age‐related changes in neuropeptides indicate a modulatory role in sensory input regions and higher order processing centers in the desert ant brain. J. Comp. Neurol. 525:901–918, 2017. © 2016 Wiley Periodicals, Inc.     

The distribution of mechanosensory hair afferents within the locust central nervous system

The sensory projections from mechanosensory hairs on different body segments of the locust were filled with cobalt and their distribution within the central nervous system was described. A common feature of all projections was that in all ganglia axons were observed only in the median ventral tract and in the neuropil area known as the ventral association center. Within these restricted regions, axons from different segments or from different locations within a segment showed specific differences in the extent of their projection.     

Drosophila ammonium transporter Rh50 is required for integrity of larval muscles and neuromuscular system

Rhesus glycoproteins (Rh50) have been shown to be ammonia transporters in many species from bacteria to human. They are involved in various physiological processes including acid excretion and pH regulation. Rh50 proteins can also provide a structural link between the cytoskeleton and the plasma membranes that maintain cellular integrity. Although ammonia plays essential roles in the nervous system, in particular at glutamatergic synapses, a potential role for Rh50 proteins at synapses has not yet been investigated. To better understand the function of these proteins in vivo, we studied the unique Rh50 gene of Drosophila melanogaster, which encodes two isoforms, Rh50A and Rh50BC. We found that Drosophila Rh50A is expressed in larval muscles and enriched in the postsynaptic regions of the glutamatergic neuromuscular junctions. Rh50 inactivation by RNA interference selectively in muscle cells caused muscular atrophy in larval stages and pupal lethality. Interestingly, Rh50-deficiency in muscles specifically increased glutamate receptor subunit IIA (GluRIIA) level and the frequency of spontaneous excitatory postsynaptic potentials. Our work therefore highlights a new role for Rh50 proteins in the maintenance of Drosophila muscle architecture and synaptic physiology, which could be conserved in other species.     

Wiring patterns from auditory sensory neurons to the escape and song-relay pathways in fruit flies

Many animals rely on acoustic cues to decide what action to take next. Unraveling the wiring patterns of the auditory neural pathways is prerequisite for understanding such information processing. Here, we reconstructed the first step of the auditory neural pathway in the fruit fly brain, from primary to secondary auditory neurons, at the resolution of transmission electron microscopy. By tracing axons of two major subgroups of auditory sensory neurons in fruit flies, low-frequency tuned Johnston's organ (JO)-B neurons and high-frequency tuned JO-A neurons, we observed extensive connections from JO-B neurons to the main second-order neurons in both the song-relay and escape pathways. In contrast, JO-A neurons connected strongly to a neuron in the escape pathway. Our findings suggest that heterogeneous JO neuronal populations could be recruited to modify escape behavior whereas only specific JO neurons contribute to courtship behavior. We also found that all JO neurons have postsynaptic sites at their axons. Presynaptic modulation at the output sites of JO neurons could affect information processing of the auditory neural pathway in flies.     

Morphology and physiology of abdominal projection interneurones in the locust with mechanosensory inputs from ovipositor hair receptors

The anatomy and physiological properties of eight non-giant projection interneurones which originate from the locust terminal abdominal ganglion and receive wind and tactile inputs from ovipositor hair receptors are described. Their cell bodies (diameter 25–40 μm) are clustered in the anterolateral region of the eighth abdominal neuromere, and their axons ascend through either the contralateral or the ipsilateral connective to more anterior abdominal ganglia. In contrast to the giant interneurones, they have small-diameter axons and are not sensitive to cercal hair wind inputs. According to their arborisation pattern within the terminal abdominal ganglion, the non-giant projection interneurones can be divided into those with main central arborisations in the ventral neuropil (anterolateral interneurones 1–6, ALIN1–ALIN6) and those with arborisations in the dorsal neuropil (ALIN7 and ALIN8). Interneurones of the first type possess four to six secondary neurites, which form a dense dendritic field in the ventral neuropil, either contralaterally or ipsilaterally to their soma. Two interneurones have contralaterally ascending axons and main dendritic fields contralateral to their soma. Two interneurones have contralaterally ascending axons and ipsilateral main dendritic fields. One interneurone has an ipsilaterally ascending axon and an ipsilateral main dendritic field. The primary neurites of interneurones with contralateral axons transverse the ganglion through dorsal commissure 1. Five interneurones have unilateral ventral dendritic fields. One interneurone posseses bilateral ventral branches. Some interneurones project only in the eighth abdominal neuromere, whereas others send branches posteriorly into the neuropil of the ninth abdominal neuromere. Interneurones of the second type send three to four secondary neurites to the dorsal neuropil of the eighth and ninth abdominal neuromeres. One interneurone has an ascending axon in the ipsilateral connective and the other in the contralateral connective. The axons of the projection interneurones pass through a lateral or dorsal tract to the seventh abdominal ganglion. Their axonal projections are sparse, remain ipsilateral to the axons, and are confined to the dorsomedial neuropil. ALIN1-ALIN7 are depolarised and spike in response to wind and direct mechanical deflection of trichoid sensilla on both left and right ovipositor valves. They respond with more spikes to stimulation of hairs on the ventral valve ipsilateral to their main dendritic field. ALIN8, in contrast, shows a delayed inhibitory/excitatory response. © 1996 Wiley-Liss, Inc.     

Anatomy of the lobula complex in the brain of the praying mantis compared to the lobula complexes of the locust and cockroach

The praying mantis is an insect which relies on vision for capturing prey, avoiding being eaten and for spatial orientation. It is well known for its ability to use stereopsis for estimating the distance of objects. The neuronal substrate mediating visually driven behaviors, however, is not very well investigated. To provide a basis for future functional studies, we analyzed the anatomical organization of visual neuropils in the brain of the praying mantis Hierodula membranacea and provide supporting evidence from a second species, Rhombodera basalis, with particular focus on the lobula complex (LOX). Neuropils were three‐dimensionally reconstructed from synapsin‐immunostained whole mount brains. The neuropil organization and the pattern of γ‐aminobutyric acid immunostaining of the medulla and LOX were compared between the praying mantis and two related polyneopteran species, the Madeira cockroach and the desert locust. The investigated visual neuropils of the praying mantis are highly structured. Unlike in most insects the LOX of the praying mantis consists of five nested neuropils with at least one neuropil not present in the cockroach or locust. Overall, the mantis LOX is more similar to the LOX of the locust than the more closely related cockroach suggesting that the sensory ecology plays a stronger role than the phylogenetic distance of the three species in structuring this center of visual information processing.     

Development of the anterior visual input pathway to the Drosophila central complex

The anterior visual pathway (AVP) conducts visual information from the medulla of the optic lobe via the anterior optic tubercle (AOTU) and bulb (BU) to the ellipsoid body (EB) of the central complex. The anatomically defined neuron classes connecting the AOTU, BU, and EB represent discrete lineages, genetically and developmentally specified sets of cells derived from common progenitors (Omoto et al., Current Biology, 27, 1098–1110, 2017). In this article, we have analyzed the formation of the AVP from early larval to adult stages. The immature fiber tracts of the AVP, formed by secondary neurons of lineages DALcl1/2 and DALv2, assemble into structurally distinct primordia of the AOTU, BU, and EB within the late larval brain. During the early pupal period (P6–P48) these primordia grow in size and differentiate into the definitive subcompartments of the AOTU, BU, and EB. The primordium of the EB has a complex composition. DALv2 neurons form the anterior EB primordium, which starts out as a bilateral structure, then crosses the midline between P6 and P12, and subsequently bends to adopt the ring shape of the mature EB. Columnar neurons of the central complex, generated by the type II lineages DM1‐4, form the posterior EB primordium. Starting out as an integral part of the fan‐shaped body primordium, the posterior EB primordium moves forward and merges with the anterior EB primordium. We document the extension of neuropil glia around the nascent EB and BU, and analyze the relationship of primary and secondary neurons of the AVP lineages.     

Visual pathways in the brain of the jumping spider Marpissa muscosa

Some animals have evolved task differentiation among their eyes. A particular example is spiders, where most species have eight eyes, of which two (the principal eyes) are used for object discrimination, whereas the other three pairs (secondary eyes) detect movement. In the ctenid spider Cupiennius salei, these two eye types correspond to two visual pathways in the brain. Each eye is associated with its own first- and second-order visual neuropil. The second-order neuropils of the principal eyes are connected to the arcuate body, whereas the second-order neuropils of the secondary eyes are linked to the mushroom body. We explored the principal- and secondary eye visual pathways of the jumping spider Marpissa muscosa, in which size and visual fields of the two eye types are considerably different. We found that the connectivity of the principal eye pathway is the same as in C. salei, while there are differences in the secondary eye pathways. In M. muscosa, all secondary eyes are connected to their own first-order visual neuropils. The first-order visual neuropils of the anterior lateral and posterior lateral eyes are connected with a second-order visual neuropil each and an additional shared one (L2). In the posterior median eyes, the axons of their first-order visual neuropils project directly to the arcuate body, suggesting that the posterior median eyes do not detect movement. The L2 might function as an upstream integration center enabling faster movement decisions.     

A population of descending tyraminergic/octopaminergic projection neurons of the insect deutocerebrum

In this study, we describe a cluster of tyraminergic/octopaminergic neurons in the lateral dorsal deutocerebrum of desert locusts (Schistocerca gregaria) with descending axons to the abdominal ganglia. In the locust, these neurons synthesize octopamine from tyramine stress-dependently. Electrophysiological recordings in locusts reveal that they respond to mechanosensory touch stimuli delivered to various parts of the body including the antennae. A similar cluster of tyraminergic/octopaminergic neurons was also identified in the American cockroach (Periplaneta americana) and the pink winged stick insect (Sipyloidea sipylus). It is suggested that these neurons release octopamine in the ventral nerve cord ganglia and, most likely, convey information on arousal and/or stressful stimuli to neuronal circuits thus contributing to the many actions of octopamine in the central nervous system.     

Developmental organization of central neurons in the adult Drosophila ventral nervous system

We have used MARCM to reveal the adult morphology of the post embryonically produced neurons in the thoracic neuromeres of the Drosophila VNS. The work builds on previous studies of the origins of the adult VNS neurons to describe the clonal organization of the adult VNS. We present data for 58 of 66 postembryonic thoracic lineages, excluding the motor neuron producing lineages (15 and 24) which have been described elsewhere. MARCM labels entire lineages but where both A and B hemilineages survive (e.g., lineages 19, 12, 13, 6, 1, 3, 8, and 11), the two hemilineages can be discriminated and we have described each hemilineage separately. Hemilineage morphology is described in relation to the known functional domains of the VNS neuropil and based on the anatomy we are able to assign broad functional roles for each hemilineage. The data show that in a thoracic hemineuromere, 16 hemilineages are primarily involved in controlling leg movements and walking, 9 are involved in the control of wing movements, and 10 interface between both leg and wing control. The data provide a baseline of understanding of the functional organization of the adult Drosophila VNS. By understanding the morphological organization of these neurons, we can begin to define and test the rules by which neuronal circuits are assembled during development and understand the functional logic and evolution of neuronal networks.     

Organization of the antennal lobes in the praying mantis (Tenodera aridifolia)

Olfaction in insects plays pivotal roles in searching for food and/or for sexual partners. Although many studies have focused on the olfactory processes of nonpredatory insect species, little is known about those in predatory insects. Here, we investigated the anatomical features of the primary olfactory center (antennal lobes) in an insect predator whose visual system is well developed, the praying mantis Tenodera aridifolia. Both sexes of T. aridifolia were found to possess 54 glomeruli, and each glomerulus was identified based on its location and size. Moreover, we found a sexual dimorphism in three glomeruli (macroglomeruli) located at the entrance of the antennal nerves, which are 15 times bigger in males than their homologs in females. We additionally deduced the target glomeruli of olfactory sensory neurons housed in cognate types of sensilla by degenerating the sensory afferents. The macroglomeruli received sensory inputs from grooved peg sensilla, which are present in a large number at the proximal part of the males' antennae. Furthermore, our findings suggest that glomeruli at the posteriodorsal part of the antennal lobes receive sensory information from putative hygro‐ and thermosensitive sensilla. The origins of projections connected to the protocerebrum are also discussed. J. Comp. Neurol. 525:1685–1706, 2017. © 2016 Wiley Periodicals, Inc.     

Neuroarchitecture of the tritocerebrum of Drosophila melanogaster

The organisation of the tritocerebrum of Drosophila melanogaster was studied by Bodian-Protargol reduced silver staining, Golgi-silver impregnation, horseradish peroxidase (HRP), and cobalt-chloride labelling of neurones and transmission electron microscopy. Nerve fibres of six categories were found to project to the tritocerebrum. (1 and 2) The sensory fibres from the internal mouthpart sensilla known to course along pharyngeal and accessory pharyngeal nerves were found to project in mainly two tiers, in the tritocerebrum. (3) Stomodaeal nerve fibres also project along the pharyngeal nerve, to the tritocerebrum. (4) Cells of the pars intercerebralis (PI) project along the median bundle and arborise in the tritocerebrum. HRP labelling and subsequent examination by transmission electron microscopy indicated their neurosecretory nature. (5 and 6) Two tracts of ascending fibres, designated as dorsal and ventral ascending tracts, were found to project to the tritocerebrum. Some of the sensory fibres from the labial nerve extend close to the sensory projections of the tritocerebrum, suggesting a possible convergence of the two sensory inputs. In the tritocerebrum, the sensory input, the stomodaeal input, the neurosecretory fibres of PI, and the ascending fibres were found to have overlapping fields, suggesting mutual interaction. The medial subesophageal ganglion and the tritocerebrum may interact through the ventral ascending tract. © 1994 Wiley-Liss, Inc.     

Immunolocalization suggests a role of the histamine-gated chloride channel PxHCLB in spectral opponent processing in butterfly photoreceptors

Spectrally opponent responses, that is, wavelength-dependent inversions of response polarity, have been observed at the level of photoreceptors in butterflies. As inter-photoreceptor connections have been found in the butterfly Papilio xuthus, and histamine is the only neurotransmitter so far identified in insect photoreceptors, we hypothesize that histaminergic sign-inverting synapses exist in the lamina between different spectral receptors as a mechanism for spectral opponency as in the medulla of Drosophila. Here, we localized two histamine-gated chloride channels, PxHCLA (Drosophila Ort homolog) and PxHCLB (Drosophila HisCl1 homolog), in the visual system of Papilio xuthus by using specific antisera. The antiPxHCLA labeling was associated with the membrane of nonphotoreceptor cells that are postsynaptic to photoreceptors, while the antiPxHCLB labeling overlapped with photoreceptor axons, indicating that PxHCLB is expressed at inter-photoreceptor synapses: PxHCLB is likely involved in producing spectral opponency at the first visual synapses. Color processing in Papilio may appear earlier than previously hypothesized in insect visual systems.     

Olfactory ensheathing cells from the nasal mucosa and olfactory bulb have distinct membrane properties

Transplantation of olfactory ensheathing cells (OECs) is a potential therapy for the regeneration of damaged neurons. While they maintain tissue homeostasis in the olfactory mucosa (OM) and olfactory bulb (OB), their regenerative properties also support the normal sense of smell by enabling continual turnover and axonal regrowth of olfactory sensory neurons (OSNs). However, the molecular physiology of OECs is not fully understood, especially that of OECs from the mucosa. Here, we carried out whole-cell patch-clamp recordings from individual OECs cultured from the OM and OB of the adult rat, and from the human OM. A subset of OECs from the rat OM cultured 1–3 days in vitro had large weakly rectifying K⁺ currents, which were sensitive to Ba²⁺ and desipramine, blockers of Kir4-family channels. Kir4.1 immunofluorescence was detectable in cultured OM cells colabeled for the OEC marker S100, and in S100-labeled cells found adjacent to OSN axons in mucosal sections. OECs cultured from rat OB had distinct properties though, displaying strongly rectifying inward currents at hyperpolarized membrane potentials and strongly rectifying outward currents at depolarized potentials. Kir4.1 immunofluorescence was not evident in OECs adjacent to axons of OSNs in the OB. A subset of human OECs cultured from the OM of adults had membrane properties comparable to those of the rat OM that is dominated by Ba²⁺-sensitive weak inwardly rectifying currents. The membrane properties of peripheral OECs are different to those of central OECs, suggesting they may play distinct roles during olfaction.     

Central projections of Drosophila sensory neurons in the transition from embryo to larva

Sensory axons of different sensory modalities project into typical domains within insect ganglia. Tactile and gustatory axons project into a ventral layer of neuropil and proprioceptive afferents, including chordotonal axons, into an intermediate or dorsal layer. Here, we describe the central projections of sensory neurons in the first instar Drosophila larva, relating them to the projection of the same sensory afferents in the embryo and to sensory afferents of similar type in other insects. Several neurons show marked morphologic changes in their axon terminals in the transition between the embryo and larva. During a short morphogenetic period late in embryogenesis, the axon terminals of the dorsal bipolar dendrite stretch receptor change their shape and their distribution within the neuromere. In the larva, external sense organ neurons (es) project their axons into a ventral layer of neuropil. Chordotonal sensory neurons (ch) project into a slightly more dorsal region that is comparable to their projection in adults. The multiple dendrite (md) neurons show two distinctive classes of projection. One group of md neurons projects into the ventral-most neuropil region, the same region into which es neurons project. Members of this group are related by lineage to es neurons or share a requirement for expression of the same proneural gene during development. Other md neurons project into a more dorsal region. Sensory receptors projecting into dorsal neuropil possibly provide proprioceptive feedback from the periphery to central motorneurons and are candidates for future genetic and cellular analysis of simple neural circuitry.