Increases in cyclic 3', 5'-guanosine monophosphate (cGMP) occur at ecdysis in an evolutionarily conserved crustacean cardioactive peptide-immunoreactive insect neuronal network.

At the end of each instar, insects shed their old cuticle by performing the stereotyped ecdysis behavior. In the month, Manduca sexta, larval ecdysis is accompanied by increases in intracellular cyclic 3', 5'-guanosine monophosphate (cGMP) in a small network of 50 peptidergic neurons within the ventral central nervous system (CNS). Studies on a variety of insects show that this cGMP response has been associated with ecdysis throughout most of insect evolution. In the mealbeetle (Tenebrio, Coleoptera) and the mosquito (Aedes, Diptera), all 50 neurons showed increases in cGMP immunoreactivity (-IR) at ecdysis, and all were immunopositive for crustacean cardioactive peptide (CCAP). Other insects varied with respect to their cGMP response at ecdysis and their CCAP-IR. In more primitive insects, such as the silverfish (Ctenolepisma, Zygentoma) and the grasshopper (Locusta, Orthoptera), an abdominal subset of these neurons did not show detectable cGMP-IR at ecdysis, although the neurons were CCAP-IR. Conversely, whereas CCAP-IR was severely reduced in the thoracic and subesophageal neurons of Lepidoptera larvae and may be absent in a subset of the corresponding abdominal neurons in crickets (Gryllus, Orthoptera), the ecdysial cGMP response occurred in all 50 neurons. The most extreme case was found in cyclorrhaphous flies, in which most of the 50 neurons were CCAP-IR, although none showed increases in cGMP at ecdysis. This situation in higher Diptera is discussed in terms of their highly modified ecdysis behaviors.

Dynamics of cyclic GMP levels in identified neurones during ecdysis behaviour in the locust Locusta migratoria

A grasshopper hatches from its egg, which is laid in soil, as a vermiform larva. This larva continues the stereotyped hatching behaviour as it digs through the egg pod, which provides a passageway to the soil surface. Once at the surface, shedding, or ecdysis, of the vermiform cuticle is initiated. When this process is complete, the first-instar cuticle is expanded to assume the form of the first-instar hopper. We have demonstrated, using immunocytochemical techniques, that these behaviour patterns are associated with dramatic increases in intracellular levels of cyclic GMP in sets of identified neurones in the ventral central nervous system. The most prominent cyclic-GMP-expressing cells are 34 neurones that appear to contain crustacean cardioactive peptide (CCAP). These CCAP cells show no detectable cyclic GMP at hatching or while the vermiform larva digs through the soil. Upon reaching the surface and freeing itself, the larva initiates ecdysis and associated air-swallowing and tracheal filling within about 1 min. These changes are immediately preceded by the appearance of cyclic GMP in the CCAP cells. Cyclic GMP levels in these neurones peak by 5 min and then decline back to basal levels by 20-30 min. Conditions that cause ecdysing animals to resume digging prolong the elevation of cyclic GMP levels. Once animals have assumed their 'hopper' form, however, external stimuli can no longer affect the time course of the cyclic GMP response. The neurones containing elevated cyclic GMP levels probably influence the air-swallowing, tracheal filling and circulatory changes that are associated with ecdysis behaviour. Pairs of descending midline neurones in abdominal segments 2-4 also become cyclic-GMP-immunoreactive, but they show peak expression after cyclic GMP levels in the CCAP cells have declined. Also, neurones in the caudolateral region of the abdominal ganglia often become cyclic-GMP-immunoreactive when ecdysing animals are forced to resume digging for an extended period.

Neuromodulation of the locust frontal ganglion during the moult: a novel role for insect ecdysis peptides.

In insects, continuous growth requires the periodic replacement of the exoskeleton during the moult. A moulting insect displays a stereotypical set of behaviours that culminate in the shedding of the old cuticle at ecdysis. Moulting is an intricate process requiring tightly regulated physiological changes and behaviours to allow integration of environmental cues and to ensure the proper timing and sequence of its components. This is under complex hormonal regulation, and is an important point of interaction between endocrine and neural control. Here, we focus on the locust frontal ganglion (FG), an important player in moulting behaviour, as a previously unexplored target for ecdysis peptides. We show that application of 10(-7) mol l(-1) ecdysis-triggering hormone (ETH) or 10(-7) mol l(-1) and 10(-6) mol l(-1) Pre-ecdysis-triggering hormone (PETH) to an isolated FG preparation caused an increase in bursting frequency in the FG, whereas application of 10(-6) mol l(-1) eclosion hormone (EH) caused an instantaneous, though temporary, total inhibition of all FG rhythmic activity. Crustacean cardioactive peptide (CCAP), an important peptide believed to turn on ecdysis behaviour, caused a dose-dependent increase of FG burst frequency. Our results imply a novel role for this peptide in generating air-swallowing behaviour during the early stages of ecdysis. Furthermore, we show that the modulatory effects of CCAP on the FG motor circuits are dependent on behavioural state and physiological context. Thus, we report that pre-treatment with ETH caused CCAP-induced effects similar to those induced by CCAP alone during pre-ecdysis. Thus, the action of CCAP seems to depend on pre-exposure to ETH, which is thought to be released before CCAP in vivo.

Invariant association of ecdysis with increases in cyclic 3',5'-guanosine monophosphate immunoreactivity in a small network of peptidergic neurons in the hornworm, Manduca sexta.

At the end of each molt insects shed their old cuticle by performing the stereotyped behavior of ecdysis. In the moth, Manduca sexta, this behavior is triggered by the neuropeptide eclosion hormone (EH). Insights into the mechanism of action of EH have come from the identification of a small network of peptidergic neurons that shows increased cyclic 3',5'-guanosine monophosphate (cGMP) immunoreactivity at ecdysis in insects from many different orders. Here we present further evidence that strengthens the association between ecdysis and the occurrence of this cGMP response in Manduca. We found that the cGMP increases occurred at every ecdysis, although some of the neurons that showed a response at larval ecdysis did not participate at pupal and adult ecdysis. Both ecdysis and the cGMP increases only required an intact connection with the brain for the first 30 min after EH injection. Interestingly, ecdysis in debrained animals only occurred if the cGMP response had been initiated, suggesting that the onset of this response marks the time at which the central nervous system is first able to drive ecdysis. Finally, we found that the appearance of sensitivity to EH for triggering the cGMP response coincided with the time at which EH first triggers ecdysis.

Neuropeptide induction of cyclic GMP increases in the insect CNS: resolution at the level of single identifiable neurons.

In insects, the neuropeptide eclosion hormone (EH) acts on the CNS to evoke the stereotyped behaviors that cause ecdysis, the shedding of the cuticle at the end of each molt. Concomitantly, EH induces an increase in cyclic GMP (cGMP). Using antibodies against this second messenger, we show that this increase is confined to a network of 50 peptidergic neurons distributed throughout the CNS. Increases appeared 30 min after EH treatment, spread rapidly throughout these neurons, and were extremely long lived. We show that this response is synaptically driven, and does not involve the soluble, nitric oxide (NO)-activated, guanylate cyclase. Stereotyped variations in the duration of the cGMP response among neurons suggest a role in coordinating responses having different latencies and durations.

An inducible promoter fused to the period gene in Drosophila conditionally rescues adult per-mutant arrhythmicity.

The period (per) gene of Drosophila melanogaster is involved in the expression of circadian rhythms of locomotor activity in adult flies. Molecular studies of per (reviewed in ref. 2) have shown that the transcribed and translated products of this gene are present primarily at the embryonic, pupal and adult stages. Here we describe experiments with arrhythmic per mutants bearing an inducible form of this gene which indicate that strongly rhythmic adult behaviour can be obtained only if per expression is induced in the adult, independent of its history of expression earlier in development. Thus per-mutant locomotor-activity phenotypes seem not to result from abnormalities in the development of neural structures or in physiological processes that may be required at pre-adult stages for the expression of this circadian rhythm. Moreover, the action of per after light:dark cycle entrainment seems to be sufficient for activity rhythms to be exhibited in constant darkness; this suggests further that the per product is required only during the time that the rhythmic behaviour is being manifested. Our strategy used a heat-shock gene promotor fused to per coding sequences to obtain conditional gene expression. Heat-shock promoter-driven genes have previously been used to study the mode of action and tissue specificity of a variety of Drosophila genes; our experiments on circadian rhythms demonstrate the use of such gene constructions for the temporal manipulation of genes whose phenotypes, behavioural and otherwise, affect whole organisms.

Control of insect ecdysis by a positive-feedback endocrine system: roles of eclosion hormone and ecdysis triggering hormone.

A successful ecdysis in insects requires the precise coordination of behaviour with the developmental changes that occur late in a moult. This coordination involves two sets of endocrine cells: the peripherally located Inka cells, which release ecdysis triggering hormone (ETH), and the centrally located neurosecretory neurones, the VM neurones, which release eclosion hormone (EH). These two sets of endocrine cells mutually excite one another: EH acts on the Inka cells to cause the release of ETH. ETH, in turn, acts on the VM neurones to cause the release of EH. This positive-feedback relationship allows the Inka cells and the VM neurones to be the peripheral and central halves, respectively, of a decision-making circuit. Once conditions for both halves have been satisfied, their reciprocal excitation results in a massive EH/ETH surge in the blood as well as a release of EH within the central nervous system. This phasic signal then causes the tonic activation of a distributed network of peptidergic neurones that contain crustacean cardioactive peptide. The relationship of the latter cells to the subsequent maintenance of the ecdysis motor programme is discussed.

Genetic and hormonal regulation of the death of peptidergic neurons in the Drosophila central nervous system.

To understand the role apoptosis plays in nervous system development and to gain insight into the mechanisms by which steroid hormones regulate neuronal apoptosis, we investigated the death of a set of peptidergic neurons in the CNS of the fruitfly Drosophila melanogaster. Typically, apoptosis in Drosophila is induced by the expression of the genes reaper, grim, or head involution defective (hid). We provide genetic evidence that the death of these neurons requires reaper and grim gene function. Consistent with this genetic analysis, we demonstrate that these doomed neurons accumulate reaper and grim transcripts prior to the onset of apoptosis. These neurons also accumulate low levels of hid, although the genetic analysis suggests that hid may not play a major role in the induction of apoptosis in these neurons. We show that the death of these neurons is dependent upon the fall in the titer of the steroid hormone 20-hydroxyecdysone that occurs at the end of metamorphosis, and demonstrate that the accumulation of both reaper and grim transcripts is inhibited by this steroid hormone. These observations support the notion that 20E controls apoptosis by regulating the expression of genes that induce apoptosis.

Embryonic expression of the period clock gene in the central nervous system of Drosophila melanogaster.

We have examined the temporal and spatial expression of the 4.5-kb mRNA that is transcribed from the period locus of Drosophila melanogaster and is the best candidate for the per gene product. Both Northern blot analyses and hybridizations in situ to tissue sections reveal significant expression of the 4.5-kb mRNA in embryos. This expression is limited to the central nervous system of the developing embryo and is localized within the brain and ventral ganglia. The 4.5-kb mRNA is enriched in adult heads (by Northern blotting) although we were not able to detect specific localization (in situ). In addition to the physiological role the 4.5-kb mRNA might have in maintaining biological rhythms, we now suggest that it has a developmental role for establishing mechanisms that are necessary for eventual expression of clock functions.

Requirement for period gene expression in the adult and not during development for locomotor activity rhythms of imaginal Drosophila melanogaster.

Mutations at the period (per) locus of Drosophila melanogaster disrupt the circadian rhythm of adult locomotor activity. Molecular studies have shown that this gene is expressed primarily at the embryonic, pupal and adult stages. We have used conditional per mutants to infer the stages of development during which per expression is required for adult rhythmicity. In experiments carried out with germline transformants in which the arrhythmic per01 allele has been transformed with a heat-shock protein 70 promoter-driven per gene (hsp-per transformants) we find that per expression in the adult is both necessary and sufficient for imaginal rhythms. Results obtained with existing per alleles and other per transformant strains that behave as conditional per mutants are consistent with those obtained with these molecularly engineered conditional mutants. Using hsp-per transformants we have found that the per gene product is apparently required only at the time of manifestation of rhythmicity, and can rescue the host's arrhythmic phenotype even when supplied many days after transfer to constant darkness. We present evidence suggesting that it is necessary for pacemaker function itself, rather than being involved in a process that couples the activity of the pacemaker to the output pathway. The levels of per transcript and the abundance and tissue distribution of its protein product observed in hsp-per transformants exposed to different temperature regimes are described. An initial report of some of these results has been published previously (Ewer et al., 1988).

Isolation, characterization and expression of the eclosion hormone gene of Drosophila melanogaster.

Eclosion hormone (EH) is a neuropeptide that triggers the performance of ecdysis behaviors at the end of a molt. We have isolated the EH gene from Drosophila melanogaster, and localized the gene to the right arm of chromosome 3 at band position 90B1-2. The 97-amino-acid translation product contains a signal peptide followed by a 73-amino-acid prohormone. The N-terminus of the prohormone has diverged from lepidopteran EH both in its length and amino acid composition, and contains a potential endoproteolytic cleavage site. The deduced sequence of Drosophila EH is 58% identical (36 of 62 amino acids) to that of Manduca EH. The EH gene is expressed as a 0.8-kb transcript in a single pair of brain neurons which extend their processes the entire length of the central nervous system and also to the corpora cardiaca portion of the ring gland. These cells show massive depletion of immunoreactive EH at ecdysis.

Expression of the period clock gene within different cell types in the brain of Drosophila adults and mosaic analysis of these cells' influence on circadian behavioral rhythms.

The product of the period (per) gene of Drosophila melanogaster is continuously required for the functioning of the circadian pacemaker of locomotor activity. We have used internally marked mosaics to determine the anatomical locations at which per expression is required for adult rhythmicity, and thus where the fly's circadian pacemaker is likely located in this holometabolous insect. We first provide a detailed description of the distribution and nature of per-expressing cells in the fly's CNS. Using an antibody to the per gene product, or to that of a reporter of per expression, in conjunction with an antibody to the embryonic lethal-abnormal visual system (elav) gene product--which is used as a marker of neuronal identity--we have experimentally confirmed previously proposed assignments of per-expressing cells to the neuronal and glial classes. Thus, we found that per expression and elav immunoreactivity colocalized in large cells located in the lateral cortex of the central brain, as well as in more dorsally located cells in the posterior central brain. In contrast, we found that cells located at the margins of the cortex and the neuropil, and within the neuropil, as well as smaller cortical cells found throughout the brain's cortex, were elav negative, supporting the notion that they are glial in nature. Using internally marked mosaics, we find that the pacemaker is located in brain but is not exclusive to the eyes, the ocelli, or the optic lobes, which is consistent with previous reports obtained in this and other insects of this class. Although the pacemaker may be a paired structure, we show that the functioning of one of them is sufficient for rhythmicity. Finally, we report that glial expression is sufficient for some behavioral rhythmicity to be manifest. However, the rhythmicities of animals for which per expression was confined to glia were weak, suggesting that neuronal per expression as well may be required for normal pacemaker function.

The innervation of the pyloric region of the crab, Cancer borealis: homologous muscles in decapod species are differently innervated.

The muscles of the pyloric region of the stomach of the crab, Cancer borealis, are innervated by motorneurons found in the stomatogastric ganglion (STG). Electrophysiological recording and stimulating techniques were used to study the detailed pattern of innervation of the pyloric region muscles. Although there are two Pyloric Dilator (PD) motorneurons in lobsters, previous work reported four PD motorneurons in the crab STG (Dando et al. 1974; Hermann 1979a, b). We now find that only two of the crab PD neurons innervate muscles homologous to those innervated by the PD neurons in the lobster, Panulirus interruptus. The remaining two PD neurons innervate muscles that are innervated by pyloric (PY) neurons in P. interruptus. The innervation patterns of the Lateral Pyloric (LP), Ventricular Dilator (VD), Inferior Cardiac (IC), and PY neurons were also determined and compared with those previously reported in lobsters. Responses of the muscles of the pyloric region to the neurotransmitters, acetylcholine (ACh) and glutamate, were determined by application of exogenous cholinergic agonists and glutamate. The effect of the cholinergic antagonist, curare, on the amplitude of the excitatory junctional potentials (EJPs) evoked by stimulation of the pyloric motor nerves was measured. These experiments suggest that the differences in innervation pattern of the pyloric muscles seen in crab and lobsters are also associated with a change in the neurotransmitter active on these muscles. Possible implications of these findings for phylogenetic relations of decapod crustaceans and for the evolution of neural circuits are discussed.

Programmed cell death of identified peptidergic neurons involved in ecdysis behavior in the Moth, Manduca sexta.

The eclosion of the adult Manduca sexta moth is followed by a wave of cell death that eliminates up to 50% of the neurons of the central nervous system within the first few days of imaginal life. While the identity of some of the dying motoneurons has been established, that of most doomed neurons is unknown. Here, we show that the dying cells include peptidergic neurons involved in the control of ecdysis behavior. These cells belong to a small population of 50 neurons that express crustacean cardioactive peptide (CCAP), a potent regulator of the ecdysis motor program, and show increases in cyclic 3',5'-guanosine monophosphate at each ecdysis. First, we describe new markers for these neurons and show that they are expressed in these CCAP-immunoreactive neurons in a complex temporal pattern during development. We then show that these neurons die within 36 h after adult eclosion, the last performance of ecdysis behavior in the life of the animal, via the active, genetically determined process of programmed cell death. The death of these neurons supports the hypothesis that outmoded or unused neurons are actively eliminated.