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Funder: Howard Hughes Medical Institute; FundRef: http://dx.doi.org/10.13039/100000011, Neuroendocrine systems in animals maintain organismal homeostasis and regulate stress response. Although a great deal of work has been done on the neuropeptides and hormones that are released and act on target organs in the periphery, the synaptic inputs onto these neuroendocrine outputs in the brain are less well understood. Here, we use the transmission electron microscopy reconstruction of a whole central nervous system in the Drosophila larva to elucidate the sensory pathways and the interneurons that provide synaptic input to the neurosecretory cells projecting to the endocrine organs. Predicted by network modeling, we also identify a new carbon dioxide-responsive network that acts on a specific set of neurosecretory cells and that includes those expressing corazonin (Crz) and diuretic hormone 44 (Dh44) neuropeptides. Our analysis reveals a neuronal network architecture for combinatorial action based on sensory and interneuronal pathways that converge onto distinct combinations of neuroendocrine outputs.

Proprioception, the sense of self-movement and position, is mediated by mechanosensory neurons that detect diverse features of body kinematics. Although proprioceptive feedback is crucial for accurate motor control, little is known about how downstream circuits transform limb sensory information to guide motor output. Here we investigate neural circuits inDrosophilathat process proprioceptive information from the fly leg. We identify three cell types from distinct developmental lineages that are positioned to receive input from proprioceptor subtypes encoding tibia position, movement, and vibration. 13Bα neurons encode femur-tibia joint angle and mediate postural changes in tibia position. 9Aα neurons also drive changes in leg posture, but encode a combination of directional movement, high frequency vibration, and joint angle. Activating 10Bα neurons, which encode tibia vibration at specific joint angles, elicits pausing in walking flies. Altogether, our results reveal that central circuits integrate information across proprioceptor subtypes to construct complex sensorimotor representations that mediate diverse behaviors, including reflexive control of limb posture and detection of leg vibration.

Animal behavior is shaped both by evolution and by individual experience. In many species parallel brain pathways are thought to encode innate and learnt behavior drives and as a result may link the same sensory cue to different actions if innate and learnt drives are in opposition. How these opposing drives are integrated into a single coherent action is not well understood. In insects, the Mushroom Body Output Neurons (MBONs) and the Lateral Horn Neurons (LHNs) are thought to provide the learnt and innate drives, respectively. However their patterns of convergence and the mechanisms by which their outputs are used to select actions are not well understood. We used electron microscopy reconstruction to comprehensively map the downstream targets of all MBONs in Drosophila larva and characterise their patterns of convergence with LHNs. We discovered convergence neurons that receive direct input from MBONs and LHNs and compare opposite behaviour drives. Functional imaging and optogenetic manipulation suggest these convergence neurons compute the overall predicted value of approaching or avoiding an odor and mediate action selection. Our study describes the circuit mechanisms allowing integration of opposing drives from parallel olfactory pathways.

To integrate changing environmental cues with high spatial and temporal resolution is critical for animals to orient themselves. Drosophila larvae show an effective motor program to navigate away from light sources. How the larval visual circuit processes light stimuli to control navigational decision remains unknown. The larval visual system is composed of two sensory input channels, Rhodopsin5 (Rh5) and Rhodopsin6 (Rh6) expressing photoreceptors (PRs). We here characterize how spatial and temporal information are used to control navigation. Rh6-PRs are required to perceive temporal changes of light intensity during head casts, while Rh5-PRs are required to control behaviors that allow navigation in response to spatial cues. We characterize how distinct behaviors are modulated and identify parallel acting and converging features of the visual circuit. Functional features of the larval visual circuit highlight the principle of how early in a sensory circuit distinct behaviors may be computed by partly overlapping sensory pathways., The response of Drosophila larva to light depends on both spatial and temporal inputs. Here the authors show that Rhodopsin5 photoreceptors, but not Rhodopsin6 photoreceptors, are required for conveying spatial light cues.

Modulatory (e.g. dopaminergic) neurons provide “teaching signals” that drive associative learning across the animal kingdom, but the circuits that regulate their activity and compute teaching signals are still poorly understood. We provide the first synaptic-resolution connectome of the circuitry upstream of all modulatory neurons in a brain center for associative learning, the mushroom body (MB) of theDrosophilalarva. We discovered afferent pathways from sensory neurons, as well as an unexpected large population of 61 feedback neuron pairs that provide one- and two-step feedback from MB output neurons. The majority of these feedback pathways link distinct memory systems (e.g. aversive and appetitive). We functionally confirmed some of the structural pathways and found that some modulatory neurons compare inhibitory input from their own compartment and excitatory input from compartments of opposite valence, enabling them to compute integrated common-currency predicted values across aversive and appetitive memory systems. This architecture suggests that the MB functions as an interconnected ensemble during learning and that distinct types of previously formed memories can regulate future learning about a stimulus. We developed a model of the circuit constrained by the connectome and by the functional data which revealed that the newly discovered architectural motifs, namely the multilevel feedback architecture and the extensive cross-compartment connections, increase the computational performance and flexibility on learning tasks. Together our study provides the most detailed view to date of a recurrent brain circuit that computes teaching signals and provides insights into the architectural motifs that support reinforcement learning in a biological system.

The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. We generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. We did not find any cases of neurons using more than one neurotransmitter, but found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, our work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. Our detailed transmitter- usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior.

International audience; Animals use sensory information to move toward more favorable conditions. Drosophila larvae can move up or down gradients of odors (chemotax), light (phototax), and temperature (thermotax) by modulating the probability, direction, and size of turns based on sensory input. Whether larvae can anemotax in gradients of mechanosensory cues is unknown. Further, although many of the sensory neurons that mediate taxis have been described, the central circuits are not well understood. Here, we used high-throughput, quantitative behavioral assays to demonstrate Drosophila larvae anemotax in gradients of wind speeds and to characterize the behavioral strategies involved. We found that larvae modulate the probability, direction, and size of turns to move away from higher wind speeds. This suggests that similar central decision-making mechanisms underlie taxis in somatosensory and other sensory modalities. By silencing the activity of single or very few neuron types in a behavioral screen, we found two sensory (chordotonal and multidendritic class III) and six nerve cord neuron types involved in anemotaxis. We reconstructed the identified neurons in an electron microscopy volume that spans the entire larval nervous system and found they received direct input from the mechanosensory neurons or from each other. In this way, we identified local interneurons and first- and second-order subesophageal zone (SEZ) and brain projection neurons. Finally, silencing a dopaminergic brain neuron type impairs anemotaxis. These findings suggest that anemotaxis involves both nerve cord and brain circuits. The candidate neurons and circuitry identified in our study provide a basis for future detailed mechanistic understanding of the circuit principles of anemotaxis.

The development of the adult optic lobe (OL) of Drosophila melanogaster is directed by a wave of ingrowth of the photoreceptors over a 2-day period at the outset of metamorphosis, which is accompanied by the appearance of the pupal-specific transcription factor Broad-Z3 (Br-Z3) and expression of early drivers in OL neurons. During this time, there are pulses of ecdysteroids that time the metamorphic events. At the outset, the transient appearance of juvenile hormone (JH) prevents precocious development of the OL caused by the ecdysteroid peak that initiates pupariation, but the artificial maintenance of JH after this time misdirects subsequent development. Axon ingrowth, Br-Z3 appearance and the expression of early drivers were unaffected, but aspects of later development such as the dendritic expansion of the lamina monopolar neurons and the expression of late drivers were suppressed. This effect of the exogenous JH mimic (JHM) pyriproxifen is lost by 24 h after pupariation. Part of this effect of JHM is due to its suppression of the appearance of ecdysone receptor EcR-B1 that occurs after pupation and during early adult development., Summary: Developmental gradients and steroid surges interact during optic lobe development. Early, ectopic juvenile hormone treatment alters steroid receptor levels, suppresses late events but not early events linked to developmental gradients.

Neuroinformatics 16(1), 65-80 (2018). doi:10.1007/s12021-017-9349-6, Published by Springer, New York, NY

Sensory navigation results from coordinated transitions between distinct behavioral programs. During chemotaxis in the Drosophila melanogaster larva, the detection of positive odor gradients extends runs while negative gradients promote stops and turns. This algorithm represents a foundation for the control of sensory navigation across phyla. In the present work, we identified an olfactory descending neuron, PDM-DN, which plays a pivotal role in the organization of stops and turns in response to the detection of graded changes in odor concentrations. Artificial activation of this descending neuron induces deterministic stops followed by the initiation of turning maneuvers through head casts. Using electron microscopy, we reconstructed the main pathway that connects the PDM-DN neuron to the peripheral olfactory system and to the pre-motor circuit responsible for the actuation of forward peristalsis. Our results set the stage for a detailed mechanistic analysis of the sensorimotor conversion of graded olfactory inputs into action selection to perform goal-oriented navigation. This work was initiated as part of the multi-lab Larval Olympiad conducted at the Janelia Research Campus. We are in debt to the work of S Reid (Louis lab) during the initial phase of the screen. We thank I Andrade, A Fushiki, J Jonaitis, I Larderet, P Schegel, C Schneider-Mizell and M Zwart for contributing to the EM reconstruction. We thank H Aberle for glutamate antibodies, as well as V Jayaraman, A Nern, S Pulver, G Rubin and J. Simpson for sharing fly lines. We thank V Jayaraman and R Francoville for training and access to the functional imaging setup. ML and DT acknowledges support of the Spanish Ministry of Economy and Competitiveness (MICINN and BFU2011-26208), ‘Centro de Excelencia Severo Ochoa 2013–2017’, the CERCA Programme/Generalitat de Catalunya, the EMBL/CRG Systems Biology Program and the University of California, Santa Barbara. IT was supported by the Marie Curie FP7 Programme through FLiACT (ITN). AK was supported by the ‘La Caixa’ International PhD Programme. JT, MZ and AC acknowledge funding from the Howard Hughes Medical Institute.

Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higherorder circuit supporting associative memory has not been previously available. We reconstructed one such circuit at synaptic resolution, theDrosophilalarval mushroom body, and found that most Kenyon cells integrate random combinations of inputs but a subset receives stereotyped inputs from single projection neurons. This organization maximizes performance of a model output neuron on a stimulus discrimination task. We also report a novel canonical circuit in each mushroom body compartment with previously unidentified connections: reciprocal Kenyon cell to modulatory neuron connections, modulatory neuron to output neuron connections, and a surprisingly high number of recurrent connections between Kenyon cells. Stereotyped connections between output neurons could enhance the selection of learned responses. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory center.

Neuromedin U (NMU) is a potent regulator of food intake and activity in mammals. While some downstream targets of NMU have been localized, connectivity of the neural circuits employing this neuropeptide is largely unknown. In Drosophila, neurons producing the homologous neuropeptide hugin regulate feeding and locomotion in a similar manner and project to structures of the central nervous system analogous to those in which NMU is found. Here, we use EM reconstruction and receptor expression analysis to map the connectome of hugin-producing neurons in the Drosophila larval central nervous system. We show that hugin-producing neurons establish distinct units that are reciprocally connected and share connectivity motifs. One of these units simultaneously employs synaptic as well as peptide-receptor connections to target neuroendocrine cells (NSCs) of the pars intercerebralis, the Drosophila analog of the hypothalamus. These NSCs produce CRH- and insulin-like peptides which are homologs of downstream targets of NMU. Furthermore, most of the hugin-producing neurons, including those that target the NSCs, receive inputs from chemosensory neurons in the subesophageal zone, the brain stem analog in Drosophila. Our data positions hugin neurons as part of a novel sensory-to-endocrine network that may reflect the way NMU operates in mammals. We propose that the hugin neurons interconnecting chemosensory and neuroendocrine organs are part of a physiological control system that has been conserved not only at functional and molecular levels, but at the network architecture level as well.

During larval life most of the thoracic neuroblasts (NBs) in Drosophila undergo a second phase of neurogenesis to generate adult-specific neurons that remain in an immature, developmentally stalled state until pupation. Using a combination of MARCM and immunostaining with a neurotactin antibody, Truman et al. (2004; Development 131:5167-5184) identified 24 adult-specific NB lineages within each thoracic hemineuromere of the larval ventral nervous system (VNS), but because of the neurotactin labeling of lineage tracts disappearing early in metamorphosis, they were unable extend the identification of these lineages into the adult. Here we show that immunostaining with an antibody against the cell adhesion molecule neuroglian reveals the same larval secondary lineage projections through metamorphosis and bfy identifying each neuroglian-positive tract at selected stages we have traced the larval hemilineage tracts for all three thoracic neuromeres through metamorphosis into the adult. To validate tract identifications we used the genetic toolkit developed by Harris et al. (2015; Elife 4) to preserve hemilineage-specific GAL4 expression patterns from larval into the adult stage. The immortalized expression proved a powerful confirmation of the analysis of the neuroglian scaffold. This work has enabled us to directly link the secondary, larval NB lineages to their adult counterparts. The data provide an anatomical framework that 1) makes it possible to assign most neurons to their parent lineage and 2) allows more precise definitions of the neuronal organization of the adult VNS based in developmental units/rules. J. Comp. Neurol. 524:2677-2695, 2016. © 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

Dopaminergic neurons serve multiple functions, including reinforcement processing during associative learning [1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12]. It is thus warranted to understand which dopaminergic neurons mediate which function. We study larval Drosophila, in which only approximately 120 of a total of 10,000 neurons are dopaminergic, as judged by the expression of tyrosine hydroxylase (TH), the rate- limiting enzyme of dopamine biosynthesis [ 5 and 13]. Dopaminergic neurons mediating reinforcement in insect olfactory learning target the mushroom bodies, a higher-order “cortical” brain region [ 1, 2, 3, 4, 5, 11, 12, 14 and 15]. We discover four previously undescribed paired neurons, the primary protocerebral anterior medial (pPAM) neurons. These neurons are TH positive and subdivide the medial lobe of the mushroom body into four distinct subunits. These pPAM neurons are acutely necessary for odor-sugar reward learning and require intact TH function in this process. However, they are dispensable for aversive learning and innate behavior toward the odors and sugars employed. Optogenetical activation of pPAM neurons is sufficient as a reward. Thus, the pPAM neurons convey a likely dopaminergic reward signal. In contrast, DL1 cluster neurons convey a corresponding punishment signal [5], suggesting a cellular division of labor to convey dopaminergic reward and punishment signals. On the level of individually identified neurons, this uncovers an organizational principle shared with adult Drosophila and mammals [ 1, 2, 3, 4, 7, 9 and 10] (but see [6]). The numerical simplicity and connectomic tractability of the larval nervous system [ 16, 17, 18 and 19] now offers a prospect for studying circuit principles of dopamine function at unprecedented resolution.

The sense of smell enables animals to react to long-distance cues according to learned and innate valences. Here, we have mapped with electron microscopy the complete wiring diagram of the Drosophila larval antennal lobe, an olfactory neuropil similar to the vertebrate olfactory bulb. We found a canonical circuit with uniglomerular projection neurons (uPNs) relaying gain-controlled ORN activity to the mushroom body and the lateral horn. A second, parallel circuit with multiglomerular projection neurons (mPNs) and hierarchically connected local neurons (LNs) selectively integrates multiple ORN signals already at the first synapse. LN-LN synaptic connections putatively implement a bistable gain control mechanism that either computes odor saliency through panglomerular inhibition, or allows some glomeruli to respond to faint aversive odors in the presence of strong appetitive odors. This complete wiring diagram will support experimental and theoretical studies towards bridging the gap between circuits and behavior. DOI: http://dx.doi.org/10.7554/eLife.14859.001, eLife digest Our sense of smell can tell us about bread being baked faraway in the kitchen, or whether a leftover piece finally went bad. Similarly to the eyes, the nose enables us to make up a mental image of what lies at a distance. In mammals, the surface of the nose hosts a huge number of olfactory sensory cells, each of which is tuned to respond to a small set of scent molecules. The olfactory sensory cells communicate with a region of the brain called the olfactory bulb. Olfactory sensory cells of the same type converge onto the same small pocket of the olfactory bulb, forming a structure called a glomerulus. Similarly to how the retina generates an image, the combined activity of multiple glomeruli defines an odor. A particular smell is the combination of many volatile compounds, the odorants. Therefore the interactions between different olfactory glomeruli are important for defining the nature of the perceived odor. Although the types of neurons involved in these interactions were known in insects, fish and mice, a precise wiring diagram of a complete set of glomeruli had not been described. In particular, the points of contact through which neurons communicate with each other – known as synapses – among all the neurons participating in an olfactory system were not known. Berck, Khandelwal et al. have now taken advantage of the small size of the olfactory system of the larvae of Drosophila fruit flies to fully describe, using high-resolution imaging, all its neurons and their synapses. The results define the complete wiring diagram of the neural circuit that processes the signals sent by olfactory sensory neurons in the larva’s olfactory circuits. In addition to the neurons that read out the activity of a single glomerulus and send it to higher areas of the brain for further processing, there are also numerous neurons that read out activity from multiple glomeruli. These neurons represent a system, encoded in the genome, for quickly extracting valuable olfactory information and then relaying it to other areas of the brain. An essential aspect of sensation is the ability to stop noticing a stimulus if it doesn't change. This allows an animal to, for example, find food by moving in a direction that increases the intensity of an odor. Inhibition mediates some aspects of this capability. The discovery of structure in the inhibitory connections among glomeruli, together with prior findings on the inner workings of the olfactory system, enabled Berck, Khandelwal et al. to hypothesize how the olfactory circuits enable odor gradients to be navigated. Further investigation revealed more about how the circuits could detect slight changes in odor concentration regardless of whether the overall odor intensity is strong or faint. And, crucially, it revealed how the worst odors – which can signal danger – can still be perceived in the presence of very strong pleasant odors. With the wiring diagram, theories about the sense of smell can now be tested using the genetic tools available for Drosophila, leading to an understanding of how neural circuits work. DOI: http://dx.doi.org/10.7554/eLife.14859.002

Interindividual differences in neuronal wiring may contribute to behavioral individuality and affect susceptibility to neurological disorders. To investigate the causes and potential consequences of wiring variation in Drosophila melanogaster, we focused on a hemilineage of ventral nerve cord interneurons that exhibits morphological variability. We find that late-born subclasses of the 12A hemilineage are highly sensitive to genetic and environmental variation. Neurons in the second thoracic segment are particularly variable with regard to two developmental decisions, whereas its segmental homologs are more robust. This variability “hotspot” depends on Ultrabithorax expression in the 12A neurons, indicating variability is cell-intrinsic and under genetic control. 12A development is more variable and sensitive to temperature in long-established laboratory strains than in strains recently derived from the wild. Strains with a high frequency of one of the 12A variants also showed a high frequency of animals with delayed spontaneous flight initiation, whereas other wing-related behaviors did not show such a correlation and were thus not overtly affected by 12A variation. These results show that neurodevelopmental robustness is variable and under genetic control in Drosophila and suggest that the fly may serve as a model for identifying conserved gene pathways that stabilize wiring in stressful developmental environments. Moreover, some neuronal lineages are variation hotspots and thus may be more amenable to evolutionary change.

Natural events present multiple types of sensory cues, each detected by a specialized sensory modality. Combining information from several modalities is essential for the selection of appropriate actions. Key to understanding multimodal computations is determining the structural patterns of multimodal convergence and how these patterns contribute to behaviour. Modalities could converge early, late or at multiple levels in the sensory processing hierarchy. Here we show that combining mechanosensory and nociceptive cues synergistically enhances the selection of the fastest mode of escape locomotion in Drosophila larvae. In an electron microscopy volume that spans the entire insect nervous system, we reconstructed the multisensory circuit supporting the synergy, spanning multiple levels of the sensory processing hierarchy. The wiring diagram revealed a complex multilevel multimodal convergence architecture. Using behavioural and physiological studies, we identified functionally connected circuit nodes that trigger the fastest locomotor mode, and others that facilitate it, and we provide evidence that multiple levels of multimodal integration contribute to escape mode selection. We propose that the multilevel multimodal convergence architecture may be a general feature of multisensory circuits enabling complex input-output functions and selective tuning to ecologically relevant combinations of cues.

SummaryAn often-overlooked aspect of neural plasticity is the plasticity of neuronal composition, in which the numbers of neurons of particular classes are altered in response to environment and experience. The Drosophila brain features several well-characterized lineages in which a single neuroblast gives rise to multiple neuronal classes in a stereotyped sequence during development [1]. We find that in the intrinsic mushroom body neuron lineage, the numbers for each class are highly plastic, depending on the timing of temporal fate transitions and the rate of neuroblast proliferation. For example, mushroom body neuroblast cycling can continue under starvation conditions, uncoupled from temporal fate transitions that depend on extrinsic cues reflecting organismal growth and development. In contrast, the proliferation rates of antennal lobe lineages are closely associated with organismal development, and their temporal fate changes appear to be cell cycle-dependent, such that the same numbers and types of uniglomerular projection neurons innervate the antennal lobe following various perturbations. We propose that this surprising difference in plasticity for these brain lineages is adaptive, given their respective roles as parallel processors versus discrete carriers of olfactory information.

The ability to specify the expression levels of exogenous genes inserted in the genomes of transgenic animals is critical for the success of a wide variety of experimental manipulations. Protein production can be regulated at the level of transcription, mRNA transport, mRNA half-life, or translation efficiency. In this report, we show that several well-characterized sequence elements derived from plant and insect viruses are able to function in Drosophila to increase the apparent translational efficiency of mRNAs by as much as 20-fold. These increases render expression levels sufficient for genetic constructs previously requiring multiple copies to be effective in single copy, including constructs expressing the temperature-sensitive inactivator of neuronal function Shibire ts1 , and for the use of cytoplasmic GFP to image the fine processes of neurons.

Metamorphosis is one of the most common, yet dramatic of life history strategies. In insects, complete metamorphosis with morphologically distinct larval stages arose from hemimetabolous ancestors that were more direct developing. Over the past century, several ideas have emerged that suggest the holometabolous pupa is developmentally homologous to the embryonic stages of the hemimetabolous ancestor. Other theories consider the pupal stage to be a modification of a hemimetabolous nymph. To address this question, we have isolated an ortholog of the pupal determinant, broad (br), from the hemimetabolous milkweed bug and examined its role during embryonic development. We show that Oncopeltus fasciatus br (Of’br) is expressed in two phases. The first occurs during germ band invagination and segmentation when Of’br is expressed ubiquitously in the embryonic tissues. The second phase of Of’br expression appears during the pronymphal phase of embryogenesis and persists through nymphal differentiation to decline just before hatching. Knock-down of Of’br transcripts results in defects that range from posterior truncations in the least-affected phenotypes to completely fragmented embryonic tissues in the most severe cases. Analysis of the patterning genes engrailed and hunchback reveal loss of segments and a failure in neural differentiation after Of’br depletion. Finally, we show that br is constitutively expressed during embyrogenesis of the ametabolous firebrat, Thermobia domestica. This suggests that br expression is prominent during embryonic development of ametabolous and hemimetabolous insects but was lost with the emergence of the completely metamorphosing insects.

SUMMARYLight signals can entrain circadian clocks, but they can also mask aspects of the circadian output. We have analyzed the masking effects of a lights-on(LOn) signal on Drosophila eclosion. The LOn response results in 12–21% of the flies that emerge on a given day eclosing within 10 min of the LOn signal. Flies that lack the neuropeptide eclosion hormone (EH), or in which its release is inhibited by the tetanus toxin light chain, lack the response. Optic photoreceptors in both the ocelli and the compound eyes appear to be required for the response. The LOn signal has two effects: (1) it drastically reduces the interval between EH release and eclosion, presumably by suppressing a transient descending inhibition that immediately follows EH release, and (2) it stimulates premature EH release. The LOn signal does not influence the latency of wing spreading, an EH-regulated post-ecdysis behavior.

The control of molting and metamorphosis in insects first captured the attention of physiologists in the 1920s, when the Polish physiologist Stephan Kopec showed that the brain stimulated molting in larvae of the gypsy moth (1). However, 70 years elapsed before the hormone from the brain, prothoracicotropic hormone (PTTH), was finally sequenced by groups led by H. Ishizaki and A. Suzuki (2). During the interim, it was shown that the brain’s role in causing molting was not direct but was mediated through its action on paired endocrine glands in the thorax, the prothoracic glands (PGs), that then secreted a steroid molting hormone, ecdysone. Circulating PTTH acts on these glands to stimulate steroidogenesis via calcium entry and cAMP generation (3) in a manner similar to that seen in some vertebrate steroidogenic systems. Although the control of ecdysone secretion by a circulating tropic hormone has been a central concept in comparative endocrinology for >40 years, a series of recent articles have revealed that negative factors are also at play (4–6). The article by Yamanaka et al . (7) in a recent issue of PNAS described some novel peptides that exert inhibitory control on the PGs and showed that the pathway by which they exert their effects is through the direct innervation of the glands. The simple picture of PG control in insects arose from the simplicity of the system first used to elucidate the brain–PG axis. Pioneering work by Carroll Williams (8) exploited diapausing pupae of the giant silkmoth, Hyalophora cecropia , whose development shuts down early in metamorphosis to deal with the rigors of winter. To then activate adult differentiation in the spring, the dormant PGs required PTTH released from the brain, and diapausing pupae whose brains were surgically removed never resumed development, although they survived for months (8 … *E-mail: jwt{at}u.washington.edu

A requirement for nitric oxide (NO) in visual system development has been demonstrated in many model systems, but the role of potential downstream effector molecules has not been established. Developing Drosophila photoreceptors express an NO-sensitive soluble guanylate cyclase (sGC), whereas the optic lobe targets express NO synthase. Both of these molecules are expressed after photoreceptor outgrowth to the optic lobe, when retinal growth cones are actively selecting their postsynaptic partners. We have previously shown that inhibition of the NO-cGMP pathway in vitro leads to overgrowth of retinal axons. Here we examined flies mutant for the alpha subunit gene of the Drosophila sGC (Gcalpha1). This mutation severely reduced but did not abolish GCalpha1 protein levels and NO-stimulated sGC activity in the developing photoreceptors. Although few mutant individuals possessed a disorganized retinal projection pattern, pharmacological NOS inhibition during metamorphosis increased this disorganization in mutants to a greater degree than in the wild type. Adult mutants lacked phototactic behavior, and the off-transient component of electroretinograms was frequently absent or greatly reduced in amplitude. Normal phototaxis and off-transient amplitude were restored by heat shock-mediated Gcalpha1 expression applied during metamorphosis but not in the adult. We propose that diminished sGC activity in the visual system during development causes inappropriate or inadequate formation of first-order retinal synapses, leading to defects in visual system function and visually mediated behavior.

In the neural circuits that comprise the crustacean stomatogastric nervous system (STNS), synaptically delivered neurotransmitters and circulating neurohormones elicit a wide range of rhythmic motor outputs. However, functional roles for second messengers in this system are poorly understood. Here we demonstrate two different signaling pathways that control the synthesis of 3′,5′-cGMP in the crab STNS. One pathway is activated by nitric oxide (NO) and is mediated by a cytoplasmic guanylate cyclase. A second pathway is stimulated by peptide-containing extracts from a crab neurohemal organ that activate a membrane-associated guanylate cyclase. Using whole-mount immunocytochemistry to localize individual cGMP-containing cells, we find that NO elevates intracellular cGMP in a small subset of STNS neurons. Immunopositive cells are found predominantly in the stomatogastric ganglion, with a few additional cells located in the oesophageal and commissural ganglia. Crab tissues differ in their sensitivities to NO and to the peptide-containing extract. The NO- mediated pathway is apparently restricted to the nervous system, whereas the peptidemediated pathway is present in every tissue tested. The results of these experiments demonstrate that multiple signaling pathways involving cGMP are present in the STNS and suggest that this second messenger may help control the metabolic and physiological status of these motor circuits.

The motor system of the moth Manduca sexta is completely remodeled during the pupal-adult transformation (PAT). It is stable until the formation of the pupal stage (0% PAT), but larval motor end plates become disrupted by 5% PAT and are lost by 10% PAT, at the time that the muscle has begun to degenerate. Most of the axonal arbor is retracted by 15% with the first signs of adult sprouts appearing by 20% PAT, coinciding with proliferative activity in the remains of the larval muscle. Extensive growth of the axonal arbor begins after 30% PAT, with an initial phase of rapid longitudinal growth (35–50% PAT) and then the production of short transverse branches that then form sprays of end plates (50–70% PAT). Growth and maturation of the end plates occupies the remainder of metamorphosis. Neuromuscular metamorphosis was interfered with by systemic or local treatment with a mimic of the insect juvenile hormone. The results of these treatments suggest that some aspects of the removal of larval axonal branches requires cues from the target. For the sprouting response, the rapid longitudinal growth over the muscle appears to be due to ecdysteroids acting directly on the cell body of the motoneuron. By contrast, the subsequent production and maintenance of transverse sprouts and the corresponding end plates may be an indirect response to stimulation of muscle growth and differentiation by ecdysteroids.

n the moth Manduca sexta, the declining ecdysteroid titer on the final day of the molt from the fourth to the fifth larval instar acts on the ventromedial neurosecretory cells (VM cells) to stimulate the release of eclosion hormone (EH). EH then triggers the motor programs involved in ecdysis behavior. Intracellular recordings that were made from the VM cells throughout the intermolt and molting stages showed no spontaneous action potentials until 0.9 hr before ecdysis (during the expected time of EH release), when 50% of the VM cells fired tonically at rest. This change was associated with a marked reduction in VM cell threshold without alteration of input resistance, resting potential, or synaptic drive. The increase in VM cell excitability was dependent on the declining ecdysteroid titer, because the injection of 20- hydroxyecdysone (20-HE) 11.5 hr before ecdysis significantly delayed the expected decrease in VM cell threshold. Since the same steroid treatment given 4.6 hr before ecydysis did not delay the subsequent increase in VM cell excitability, the inhibitory actions of 20-HE appear not to be mediated by a rapid membrane mechanism. The possible involvement of genomic events in the steroid-dependent increase in VM cell excitability was examined using the RNA synthesis inhibitor actinomycin D (AcD). When injected 6.3 hr before ecdysis, AcD blocked EH release without altering the response of the nervous system to exogenous peptide. Such AcD treatments also prevented the increase in VM cell excitability. These results suggest that the declining ecdysteroid titer increases the excitability of the VM cells via a transcription-dependent process.

The neuropeptide eclosion hormone (EH) is a key regulator of insect ecdysis. We tested the role of the two EH-producing neurons in Drosophila by using an EH cell-specific enhancer to activate cell death genes reaper and head involution defective to ablate the EH cells. In the EH cell knockout flies, larval and adult ecdyses were disrupted, yet a third of the knockouts emerged as adults, demonstrating that EH has a significant but nonessential role in ecdysis. The EH cell knockouts had discrete behavioral deficits, including slow, uncoordinated eclosion and an insensitivity to ecdysis-triggering hormone. The knockouts lacked the lights-on eclosion response despite having a normal circadian eclosion rhythm. This study represents a novel approach to the dissection of neuropeptide regulation of a complex behavioral program.

With the exception of the wing imaginal discs, the imaginal discs of Manduca sexta are not formed until early in the final larval instar. An early step in the development of these late-forming imaginal discs from the imaginal primordia appears to be an irreversible commitment to form pupal cuticle at the next molt. Similar to pupal commitment in other tissues at later stages, activation of broad expression is correlated with pupal commitment in the adult eye primordia. Feeding is required during the final larval instar for activation of broad expression in the eye primordia, and dietary sugar is the specific nutritional cue required. Dietary protein is also necessary during this time to initiate the proliferative program and growth of the eye imaginal disc. Although the hemolymph titer of juvenile hormone normally decreases to low levels early in the final larval instar, eye disc development begins even if the juvenile hormone titer is artificially maintained at high levels. Instead, creation of the late-forming imaginal discs in Manduca appears to be controlled by unidentified endocrine factors whose activation is regulated by the nutritional state of the animal.

SummaryWe report the larval CNS expression patterns for 6,650 GAL4 lines based on cis-regulatory regions (CRMs) from the Drosophila genome. Adult CNS expression patterns were previously reported for this collection, thereby providing a unique resource for determining the origins of adult cells. An illustrative example reveals the origin of the astrocyte-like glia of the ventral CNS. Besides larval neurons and glia, the larval CNS contains scattered lineages of immature, adult-specific neurons. Comparison of lineage expression within this large collection of CRMs provides insight into the codes used for designating neuronal types. The CRMs encode both dense and sparse patterns of lineage expression. There is little correlation between brain and thoracic lineages in patterns of sparse expression, but expression in the two regions is highly correlated in the dense mode. The optic lobes, by comparison, appear to use a different set of genetic instructions in their development.

Two proteins (the EGPs) in the CNS of the tobacco hornworm Manduca sexta are phosphorylated by the action of the neuropeptide eclosion hormone (EH), which triggers ecdysis behavior (Morton and Truman, 1986, 1988). The onset of sensitivity to EH requires prior exposure to the steroid hormone 20-hydroxyecdysone (20-HE). A series of steroid removal and replacement experiments indicates that the EGPs are also regulated by 20-HE with a time course that is similar to that seen for the 20-HE regulation of behavioral sensitivity to EH. This suggests that the steroid regulation of EH sensitivity is due to the regulation of the EGPs. The appearance of the EGPs requires not only the presence of 20- HE, but also its subsequent removal. The appearance of the EGPs can be blocked in vivo and in vitro by maintaining artificially elevated levels of 20-HE, but only up to a particular time in development. The ending of this steroid-sensitive period occurs 4 hr before the normal appearance of the EGPs, consistent with the hypothesis that the EGPs are synthesized de novo in response to the removal of 20-HE.

Background In holometabolous insects such as Drosophila melanogaster, neuroblasts produce an initial population of diverse neurons during embryogenesis and a much larger set of adult-specific neurons during larval life. In the ventral CNS, many of these secondary neuronal lineages differ significantly from one body segment to another, suggesting a role for anteroposterior patterning genes. Results Here we systematically characterize the expression pattern and function of the Hox gene Ultrabithorax (Ubx) in all 25 postembryonic lineages. We find that Ubx is expressed in a segment-, lineage-, and hemilineage-specific manner in the thoracic and anterior abdominal segments. When Ubx is removed from neuroblasts via mitotic recombination, neurons in these segments exhibit the morphologies and survival patterns of their anterior thoracic counterparts. Conversely, when Ubx is ectopically expressed in anterior thoracic segments, neurons exhibit complementary posterior transformation phenotypes. Conclusion Our findings demonstrate that Ubx plays a critical role in conferring segment-appropriate morphology and survival on individual neurons in the adult-specific ventral CNS. Moreover, while always conferring spatial identity in some sense, Ubx has been co-opted during evolution for distinct and even opposite functions in different neuronal hemilineages.