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Associating stimuli with positive or negative reinforcement is essential for survival, but a complete wiring diagram of a higher-order circuit supporting associative memory has not been previously available. Here we reconstruct one such circuit at synaptic resolution, the Drosophila larval mushroom body. We find that most Kenyon cells integrate random combinations of inputs but that 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 found between output neurons could enhance the selection of learned behaviours. The complete circuit map of the mushroom body should guide future functional studies of this learning and memory centre.

Animal locomotion requires spatiotemporally coordinated contraction of muscles throughout the body. Here, we investigate how contractions of antagonistic groups of muscles are intersegmentally coordinated during bidirectional crawling of Drosophila larvae. We identify two pairs of higher-order premotor excitatory interneurons present in each abdominal neuromere that intersegmentally provide feedback to the adjacent neuromere during motor propagation. The two feedback neuron pairs are differentially active during either forward or backward locomotion but commonly target a group of premotor interneurons that together provide excitatory inputs to transverse muscles and inhibitory inputs to the antagonistic longitudinal muscles. Inhibition of either feedback neuron pair compromises contraction of transverse muscles in a direction-specific manner. Our results suggest that the intersegmental feedback neurons coordinate contraction of synergistic muscles by acting as delay circuits representing the phase lag between segments. The identified circuit architecture also shows how bidirectional motor networks could be economically embedded in the nervous system., Locomotion involves the coordinated contraction of antagonistic muscles. Here, the authors report that in Drosophila larvae a pair of higher-order feedback neurons temporally regulates the intersegmental coordination of contraction of synergistic muscles enabling bidirectional movement.

The insect juvenile hormone receptor is a basic helix-loop-helix (bHLH), Per-Arnt-Sim (PAS) domain protein, a novel type of hormone receptor. In higher flies like Drosophila, the ancestral receptor germ cell-expressed (gce) gene has duplicated to yield the paralog Methoprene-tolerant (Met). These paralogous receptors share redundant function during development but play unique roles in adults. Some aspects of JH function apparently require one receptor or the other. To provide a foundation for studying JH receptor function, we have recapitulated endogenous JH receptor expression with single cell resolution. Using Bacteria Artificial Chromosome (BAC) recombineering and a transgenic knock-in, we have generated a spatiotemporal expressional atlas of Met and gce throughout development. We demonstrate JH receptor expression in known JH target tissues, in which temporal expression corresponds with periods of hormone sensitivity. Larval expression largely supports the notion of functional redundancy. Furthermore, we provide the neuroanatomical distribution of JH receptors in both the larval and adult central nervous system, which will serve as a platform for future studies regarding JH action on insect behavior.

During metamorphosis of the tobacco hornworm Manduca sexta, the simple thoracic legs of the larva are remodeled into the more complex adult legs. Most of the adult leg epidermis derives from the adult primordia, small sets of epidermal cells located in specific regions of the larval leg, which proliferate rapidly in the final larval instar. In contrast, the contribution of the epidermal cells outside the primordia is unknown. In this study we have determined their contribution to the adult leg by labeling them with 5-bromodeoxyuridine (BUdR) and following their fate. Although the labeled cells diminished drastically in number, small groups of these cells persisted into the midpupal stage suggesting that they do contribute to the adult leg epidermis. We also found that during the wandering stage the adult primordia went through active proliferation and very little cell death, while the cells outside the primordia went through extensive cell death accounting for the decrease in their number. Our results indicate that two distinct cell populations exist outside the adult primordia. Most cells belong to the first population, which is larval-specific and disappears through apoptosis early in metamorphosis. The second population consists of polymorphic cells that contribute to the larval, pupal and adult leg epidermis.

Insect metamorphosis is a fascinating and highly successful biological adaptation, but there is much uncertainty as to how it evolved. Ancestral insect species did not undergo metamorphosis and there are still some existing species that lack metamorphosis or undergo only partial metamorphosis. Based on endocrine studies and morphological comparisons of the development of insect species with and without metamorphosis, a novel hypothesis for the evolution of metamorphosis is proposed. Changes in the endocrinology of development are central to this hypothesis. The three stages of the ancestral insect species-pronymph, nymph and adult-are proposed to be equivalent to the larva, pupa and adult stages of insects with complete metamorphosis. This proposal has general implications for insect developmental biology.

In the moth, Manduca sexta, the neuropeptide, eclosion hormone, triggers a dramatic rise in the levels of intracellular cGMP within a group of 50 neurons. The cells within this group include the segmentally repeated neurosecretory cell, Cell 27. In this study the effect of cGMP on the excitability of Cell 27 was investigated using intracellular recordings. Prior to its normal elevation in cGMP, Cell 27 exhibited a high spike threshold, but this was lowered dramatically when intracellular cGMP levels increased. The latter was also associated with spontaneous action potentials. This change in excitability did not correspond with changes in either resting potential, input resistance, or action potential amplitude. A similar lowering of threshold was induced by perfusion of 8-bromo-cGMP, whereas 8-bromo-cAMP caused the threshold to increase. Intracellular recordings using various ion substitution paradigms and channel blockers provided evidence which suggests indirectly that Ca2+ is mostly responsible for the depolarizing phase of the action potential while a Ca(2+)-activated K+ current contributes to the hyperpolarization. The results of these manipulations are consistent with the hypothesis that cGMP may partially increase excitability in Cell 27 by enhancing an inward Ca2+ current.

The hawkmoth, Manduca sexta, undergoes periodic molts during its growth and metamorphosis. At the end of each molt, the old cuticle is shed by means of a hormonally-activated ecdysis behavior. The pharate adult, however, must not only shed its old cuticle but also dig itself out from its underground pupation chamber. To accomplish this, the adult performs a series of abdominal retractions and extensions; the extensions are coupled with movements of the wing bases. This ecdysis motor pattern is distinct from the slowly progressing, anteriorly-directed, abdominal peristalses expressed by ecdysing larvae and pupae. We have found that the ability to produce the larval-like ecdysis pattern is retained in the adult. Although this behavior is not normally expressed by the adult, larval-like ecdysis could be unmasked when descending neuronal inputs, originating in the pterothoracic ganglion, were removed from the unfused abdominal ganglia. Transformation of the adult-specific ecdysis pattern to the larval-like pattern was accomplished by transecting the connectives between the pterothorax and the abdomen, or by reversibly blocking neuronal activity with a cold-block. A comparative analysis of the ecdysis motor patterns expressed by larvae and by isolated adult abdomens indicates that the two motor patterns are indistinguishable, suggesting that the larval ecdysis motor pattern is retained through metamorphosis. We speculate that its underlying neural circuitry is conserved through development and later modulated to produce the novel ecdysis pattern expressed in the adult stage.

The final step in the moulting of all insects is ecdysis, the shedding of the cuticle of the previous instar, which is triggered in Lepidop-tera by the neurosecretory peptide eclosion hormone1. This hormone acts directly on the nervous system to release the appropriate motor patterns for larval, pupal and adult ecdyses2–5, but there are only brief periods near the end of each moult when the nervous system is competent to respond to the hormone. Previous experiments have shown that the action of eclosion hormone on the nervous system at pupal ecdysis in the tobacco hornworm, Manduca sexta, is mediated by the second messenger cyclic GMP1. Here we report that the hormone-stimulated increase in cGMP results in the phosphorylation of two proteins, each with an apparent relative molecular mass (Mr) of 54,000. Moreover, the brief periods during which the central nervous system (CNS) is responsive to eclosion hormone seem to result from the transient presence of these substrate proteins within the nervous system. This provides a novel mechanism by which hormonal responsiveness can be regulated.

1. Metamorphosis in the mothManduca sexta culminates with the secretion of the peptide eclosion hormone (EH), which triggers the stereotyped behavior of adult emergence (eclosion) from the pupal cuticle. In restrained but spontaneously behaving animals, the release of EH occurred shortly before the onset of subjective night (Fig. 3) and coincided with a depletion of EH from the neurohemal organs of the brain, the corpora cardiaca-corpora allata complex (CC-CA; Fig. 4). 2. EH is produced by neurons within a bilaterally paired group of brain neurosecretory cells (Group Ia) which project to the CC-CA via the nervi corporis cardiaci-1+2 (NCC-1+2; Fig. 1). Electrical stimulation of the NCC-1+2 caused a marked increase in the levels of EH secreted from isolated CC-CA (Fig. 2), while stimulation of the other nerves innervating the neurohemal organs did not. 3. Electrical activity in the NCC-1+2 paralleled that of the cerebral neurosecretory cells (Fig. 1). Chronic extracellular recordings revealed a sudden increase in the tonic firing of several units within this nerve approximately 2 to 3 h before normal eclosion (Fig. 5), coincident with the release of EH bioactivity from the CC-CA (Fig. 6). 4. The Group Ia neurons were electrically inactive on the day before eclosion (Day −1), but on the day of eclosion (Day 0) a subgroup of these cells exhibited both enhanced synaptic input and elevated rates of tonic firing during the normal time of EH release (Fig. 7). 5. No significant differences in resting membrane potential or spike waveform characteristics were detected among the various subsets of Group Ia cells on either Day −1 or Day 0, while a significant increase in the resting input resistance was seen in the active subgroup on Day 0 (Fig. 8). This increase may be due to the regulatory effects of the steroid 20-hydroxyecdysone, which inhibits the release of EH and may act by preventing the synaptic activation of the EH neurons until the final day of adult development.

1. When the pupal cuticle was peeled from aroundAntheraea pernyi andHyalophora cecropia moths on the last day of adult development, the animals showed reduced levels of adult behavior. Clawing and extension movements of the legs as well as wing flapping were seen, but complex behaviors such as the righting response and walking were absent (A. pernyi) or only occasionally observed (H. cecropia). 2. Injection of the eclosion hormone into “peeled” moths was followed 2 to 3 hours later by the onset of the eclosion movements. Walking behavior and the righting response appeared abruptly at the time of eclosion. 3. The development of certain adult behaviors was examined by peelingA. pernyi moths that were in the 14th through 19th (last) day of adult development. Animals peeled on days 16 or 17 showed distinct eclosion movements which started within a few minutes after peeling. But on day 18, this behavior became suppressed and could no longer be elicited by peeling. 4. Sensitivity to the eclosion hormone did not appear until day 18 and became fully developed on day 19. 5. Injection of picrotoxin into peeled day 19A. pernyi resulted in the abrupt onset of walking behavior. 6. It was concluded that certain adult behaviors such as eclosion and walking become complete and functional during adult development. But as development progresses, these then become repressed—presumably through neural inhibition. The eclosion hormone apparently acts by turning off this inhibition.

1. At the culmination of each molt, insects shed their old cuticle by a behavior called ecdysis, which generally involves rhythmic peristaltic body movements. We have compared the abdominal peristalsis behaviors produced by the tobacco hornworm,Manduca sexta, during larval-larval molts (‘larval ecdysis’) and at the larval-pupal molt (‘pupal ecdysis’), to see whether the morphological changes during metamorphosis were accompanied by alterations in the motor patterns. 2. At larval ecdysis, caterpillars shed their old cuticle by means of rhythmic peristaltic contractions which run anteriorly along the body, accompanied by coordinated retractions and extensions of the abdominal prolegs (Fig. 2). During the larval-pupal transformation the prolegs are lost, so that at pupal ecdysis the cuticle is shed by peristalsis alone (Fig. 1). 3. Cinematographic analysis of ecdysing larvae and pupae revealed that the peristaltic movements resulted from each abdominal segment being sequentially pulled forward and partially telescoped into the next anterior segment, due to the contraction of intersegmental muscles (ISMs) in the more anterior segment. Despite the marked changes in body morphology which occurred during the larval-pupal transformation, the pattern of the peristaltic contractions produced at larval and pupal ecdysis was the same (Table 1). 4. The neural basis of these behaviors was investigated in semi-intact, deafferented preparations which produced ecdysis motor patterns in response to a peptide hormone, eclosion hormone (EH), which is the normal blood-borne trigger for ecdysis (Fig. 3). The motoneurons which generated the peristaltic motor patterns in larvae and pupae were identified by recording extracellularly from various nerve branches (Figs. 4, 5, 6, 8) and intracellularly from neuronal somata (Figs. 7, 9). 5. The pattern of the motoneuron bursts was the same during both larval and pupal ecdysis (Table 2). The only apparent difference at the two stages was that pupae exhibited a lower level of tonic background motor activity than did larvae (e.g., Figs. 4, 8). 6. Thus, both behavioral and electrophysiological data indicated that the pattern-generating circuitry underlying abdominal peristalsis remained stable during the larval-pupal transformation. In contrast, the loss of the prolegs at pupation and their consequent absence from pupal ecdysis behavior did suggest a possible stage-specific change in the neural circuitry; this possibility is investigated in an accompanying paper (Weeks and Truman 1984).

1. A preliminary analysis of the silkmoth eclosion clock demonstrated that the endogenous, free-running cycle of the clock could be divided into two parts: an initial synchronization period and a dark decay period. 2. A 1/2 hour light pulse applied during the synchronization period served to restart the free-running cycle. By proper timing of light pulses, a given cycle of the eclosion clock could be restarted ad infinitum. 3. When the length of the interrupting pulse was progressively increased up to 24 hours, the subsequent eclosion rhythms showed a constant phase relationship to the end of the interruption, irrespective of its duration. This phase relationship was the same as observed when the eclosion clock was started by transfer of moths from continuous light to continuous darkness. 4. It is concluded that the synchronization period involves a photoreversible process. During this period light rapidly stops the clock and the cycle can begin only with the onset of darkness.

1. Motor neurons in the fourth abdominal ganglion were identified by diffusing cobalt into the ganglion through the cut ends of the peripheral nerves. 2. The ganglion from the last instar larva had a total of 74 motor neurons. These were divided into 10 groups which were characterized by the position of the cell body in the ganglion and the root through which the axon travelled to the periphery. 3. In the transition to the pupal stage, 12 larval neurons were lost and 8 new motor neurons appeared. 4. There was no change in the number of motor neurons during adult development. Most of the larval neurons persist through metamorphosis and are used by the adult at emergence. At this stage there are 70 motor neurons. 5. After adult eclosion there was a loss of 40 motor neurons. Only “larval” neurons degenerated. Those which remained included both those which were larval in origin and those which appeared in the pupa. After emergence the interneuron number dropped from approximately 380 to 200. 6. At the time of emergence the eclosion hormone “turns-off” pupal behavior and “turns-on” adult behavior. It also triggers the breakdown of various muscle groups in the abdomen. It is concluded that the neural death was the end result of this aspect of eclosion hormone action.

1. In silkmoths the gating of adult eclosion by light-dark cycles persists after extirpation of the compound eyes, frontal ganglion, subesophageal ganglion, or corpora allata-corpora cardiaca complex. Removal of the brain abolishes gating and such moths emerge irrespective of time of day or night. 2. Implantation of a brain into the abdomen of a debrained animal restores the ability both to entrain to a light-dark cycle and to free-run under conditions of continuous darkness. 3. Implantation of pieces of brain which contain the cerebral lobes likewise restored to debrained moths the ability to entrain and to free-run. But the synchrony was not as sharp as that observed in moths which had received implants of the entire brain. 4. Transection of the brain lateral to each median neurosecretory cell cluster yields pieces which are apparently incapable of gating the eclosion of debrained moths. Both moths receiving the median piece and those receiving the lateral pieces showed a randomized distribution of eclosion. 5. Experiments involving brain transplantation and selective illumination of parts of the body showed that the brain directly receives the light information which is necessary for the entrainment of the clock. 6. In theCecropia moth, the lights-on signal serves to stimulate eclosion and thus partially masks the output of the clock. This effect is mediated solely by light which is perceived by the compound eyes.

Ultrastructural aspects of the natural degeneration of a group of six motor neurons in the fourth abdominal ganglion of Manduca sexta are described. These motor neurons innervate intersegmental muscles that degenerate and disappear immediately after adult eclosion. The first detectable changes in the cell bodies appear 12 h after eclosion and include disruption of the endoplasmic reticulum and an increase in the size and number of lamellar bodies. At 32 h the nuclear membranes rupture, and the membranous and granular cytoorganelles segregate in different parts of the cell. At that stage the surrounding glial cells participate in the digestion of material from the degenerating neurons. From 72 h onward the remaining neuronal structures become disrupted, and are finally transformed into a single, large lamellar body (residual body) within the glial profile. The degeneration pattern differs significantly from that of embryonic vertebrate neurons.

Invertebrate Endocrinology and Hormonal Heterophylly. Edited by Walter J. Burdette. Pp. xviii + 438. (Springer: Berlin and New York, 1974.) $21.50.