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Antisera against the circadian clock proteins Period (PER) and Timeless (TIM) were used to construct a detailed time course of PER and TIM expression and subcellular localization in a subset of the ventrolateral neurons (vLNs) in theDrosophilaaccessory medulla (AMe). These neurons, which express pigment-dispersing factor, play a central role in the control of behavioral rhythms. The data revealed several unexpected features of the circadian clock inDrosophila. First, TIM but not PER was restricted to the cytoplasm of vLNs throughout most of the early night. Second, the timing of TIM and PER nuclear accumulation was substantially different. Third, the two subsets of vLNs, the large and small vLNs, had a similar timing of PER nuclear accumulation but differed by 3–4 hr in the phase of TIM nuclear accumulation. These aspects of PER and TIM expression were not predicted by the current mechanistic model of the circadian clock inDrosophilaand are inconsistent with the hypothesis that PER and TIM function as obligate heterodimers. The differing profiles of TIM and PER nuclear accumulation suggest that PER and TIM have distinct functions in the nuclei of vLNs.

In insects, the shedding of the old cuticle at the end of a molt involves a stereotyped sequence of distinct behaviors. Our studies on the isolated nervous system ofManduca sextashow that the peptides ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP) elicit the first two motor behaviors, the pre-ecdysis and ecdysis behaviors, respectively. Exposing isolated abdominal ganglia to ETH resulted in the generation of sustained pre-ecdysis bursts. By contrast, exposing the entire isolated CNS to ETH resulted in the sequential appearance of pre-ecdysis and ecdysis motor outputs. Previous research has shown that ETH activates neurons within the brain that then release eclosion hormone within the CNS. The latter elevates cGMP levels within and increases the excitability of a group of neurons containing CCAP. In our experiments, the ETH-induced onset of ecdysis bursts was always associated with a rise in intracellular cGMP within these CCAP neurons. We also found that CCAP immunoreactivity decreases centrally during normal ecdysis. Isolated, desheathed abdominal ganglia responded to CCAP by generating rhythmical ecdysis bursts. These ecdysis motor bursts persisted as long as CCAP was present and could be reinduced by successive application of the peptide. CCAP exposure also actively terminated pre-ecdysis bursts from the abdominal CNS, even in the continued presence of ETH. Thus, the sequential performance of the two behaviors arises from one modulator activating the first behavior and also initiating the release of the second modulator. The second modulator then turns off the first behavior while activating the second.

Programmed cell death occurs in the nervous and muscular system of newly emerged adult Drosophila melanogaster. Many of the abdominal muscles that were used for eclosion and wing-spreading behavior degenerate by 12 hr after eclosion. Related neurons in the ventral ganglion also die within the first 24 hr. Ligation experiments showed that the muscle breakdown is triggered by a signal from the anterior region, presumably the head, that occurs about 1 hr before adult emergence. The timing of this signal suggests that eclosion hormone may be involved. Although muscle death is triggered prior to ecdysis, it can be delayed, at least temporarily, by forcing the emerging flies to show a prolonged ecdysis behavior. In contrast to the muscles, the death of the neurons is triggered after emergence. The signal for neuronal degeneration is closely correlated with the initiation of wing inflation behavior. Ligation and digging experiments and behavioral manipulations that either blocked or delayed wing expansion behavior had a parallel effect in suppressing or delaying neuronal death.

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.

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 emergence of the adult Manduca sexta moth is followed by the programmed degeneration of about 50% of the insect's abdominal interneurons and motoneurons. Neurons in implanted ganglia undergo degeneration in concert with neurons of the host, thereby indicating that a hormonal cue is important. The neuronal death follows the normal decline in the levels of the steroid hormones, ecdysteroids, that occurs at the end of metamorphosis. Manipulations that cause a precocious ecdysteroid decline result in an early death of the neurons. Also, prolongation of the ecdysteroid titer extend the life of the neurons. By properly timed steroid application it was possible to dissociate the neuronal degeneration from the behavioral events and muscle degeneration that also occur at emergence. Also the stereotyped sequence of death among identified motoneurons could be stopped at various points in the sequence by the carefully timed application of steroid. It was concluded that the signal for neuronal death was the withdrawal of ecdysteroids at the end of metamorphosis and that the death was probably a direct response of the individual neurons to the hormone withdrawal.

The present study describes 2 phosphoproteins, both with an apparent molecular weight of 54 kDa, in the CNS of the tobacco hornworm, Manduca sexta. Their phosphorylation is regulated by a neuropeptide, eclosion hormone (EH), and the second messenger cGMP, which thus have been named the EGPs (eclosion hormone- and cGMP-regulated phosphoproteins). Although cAMP was more effective than cGMP at stimulating the phosphorylation of the EGPs in CNS homogenates, in the intact CNS cGMP was more effective. Since cGMP mediates the action of EH, this strongly suggests that cGMP is the second messenger that stimulates the phosphorylation of the EGPs in vivo. The EGPs can only be phosphorylated in vitro during discrete time periods during the life of Manduca. During the larval and pupal molts, the EGPs can first be phosphorylated just prior to ecdysis. Their ability to be phosphorylated is correlated with the time when the insect is sensitive to EH. This close temporal correlation suggests that the ability to phosphorylate the EGPs determines when the insect can first respond to EH. During adult development, the EGPs first appeared on fluorograms 6 d before sensitivity to EH, suggesting that at this stage other factors may also be involved in the regulation of sensitivity. For the ecdyses of all 3 stages, EH appeared to stimulate the phosphorylation of the EGPs at ecdysis. The EGPs were found in all regions of the prepupal nervous system that were investigated, but only in the abdominal and pterothoracic ganglia of the developing adult. Fractionation of nervous system homogenates by ultracentrifugation revealed that one of the EGPs was present only in the pellet fraction, whereas the other was approximately equally distributed between pellet and supernatant. Furthermore, the EGPs in the pellet fraction could be partially solubilized with detergents and high salt concentrations.

The nervous system of an adult moth is comprised of retained larval neurons that are remodeled during metamorphosis and a set of new adult specific neurons. The new neurons arise from a stereotyped array of stem cells (neuroblasts) that divide during larval life to generate nests of up to 100 arrested postmitotic immature neurons, the imaginal nest (IN) cells. At the onset of metamorphosis, some of the IN cells die while the remainder differentiate into mature functional neurons. Metamorphosis in insects is regulated by 2 classes of hormones, the ecdysteroids and the juvenile hormones. The transition from larva to pupa requires the disappearance of juvenile hormones followed by 2 releases of ecdysteroids: a small "commitment peak" and a larger "prepupal peak." Through a series of endocrine manipulations, we demonstrate that the death and differentiation observed among the abdominal IN cells at metamorphosis are both influenced by these hormonal cues. If the abdomen was isolated from the hormonal sources in the anterior half of the larva before the onset of metamorphosis, death and differentiation of the IN cells were prevented. Infusion of ecdysteroids into such abdomens, to mimic the prepupal peak, resulted in the IN cells showing the same fate as seen in control animals during the early phases of metamorphosis. The response of the IN cells to the small commitment peak of ecdysteroids was heterogeneous. Exposure to this small peak of steroids caused some cells to become committed to resume their development, making them resistant to juvenile hormone application.(ABSTRACT TRUNCATED AT 250 WORDS)

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.