Crustacean Cardioactive Peptide Neurons Research Articles

Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion.

Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the interoceptive subesophageal zone neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. Here, we investigate the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, we find that the ISNs synapse with a novel cell-type bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPCs), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion.

Hunger- and thirst-sensing neurons modulate a neuroendocrine network to coordinate sugar and water ingestion

Consumption of food and water is tightly regulated by the nervous system to maintain internal nutrient homeostasis. Although generally considered independently, interactions between hunger and thirst drives are important to coordinate competing needs. In Drosophila, four neurons called the interoceptive subesophageal zone neurons (ISNs) respond to intrinsic hunger and thirst signals to oppositely regulate sucrose and water ingestion. Here, we investigate the neural circuit downstream of the ISNs to examine how ingestion is regulated based on internal needs. Utilizing the recently available fly brain connectome, we find that the ISNs synapse with a novel cell-type bilateral T-shaped neuron (BiT) that projects to neuroendocrine centers. In vivo neural manipulations revealed that BiT oppositely regulates sugar and water ingestion. Neuroendocrine cells downstream of ISNs include several peptide-releasing and peptide-sensing neurons, including insulin producing cells (IPCs), crustacean cardioactive peptide (CCAP) neurons, and CCHamide-2 receptor isoform RA (CCHa2R-RA) neurons. These neurons contribute differentially to ingestion of sugar and water, with IPCs and CCAP neurons oppositely regulating sugar and water ingestion, and CCHa2R-RA neurons modulating only water ingestion. Thus, the decision to consume sugar or water occurs via regulation of a broad peptidergic network that integrates internal signals of nutritional state to generate nutrient-specific ingestion.

Extracting temporal relationships between weakly coupled peptidergic and motoneuronal signaling: Application to Drosophila ecdysis behavior.

Neuromodulators, such as neuropeptides, can regulate and reconfigure neural circuits to alter their output, affecting in this way animal physiology and behavior. The interplay between the activity of neuronal circuits, their modulation by neuropeptides, and the resulting behavior, is still poorly understood. Here, we present a quantitative framework to study the relationships between the temporal pattern of activity of peptidergic neurons and of motoneurons during Drosophila ecdysis behavior, a highly stereotyped motor sequence that is critical for insect growth. We analyzed, in the time and frequency domains, simultaneous intracellular calcium recordings of peptidergic CCAP (crustacean cardioactive peptide) neurons and motoneurons obtained from isolated central nervous systems throughout fictive ecdysis behavior induced ex vivo by Ecdysis triggering hormone. We found that the activity of both neuronal populations is tightly coupled in a cross-frequency manner, suggesting that CCAP neurons modulate the frequency of motoneuron firing. To explore this idea further, we used a probabilistic logistic model to show that calcium dynamics in CCAP neurons can predict the oscillation of motoneurons, both in a simple model and in a conductance-based model capable of simulating many features of the observed neural dynamics. Finally, we developed an algorithm to quantify the motor behavior observed in videos of pupal ecdysis, and compared their features to the patterns of neuronal calcium activity recorded ex vivo. We found that the motor activity of the intact animal is more regular than the motoneuronal activity recorded from ex vivo preparations during fictive ecdysis behavior; the analysis of the patterns of movement also allowed us to identify a new post-ecdysis phase.

Drosophila Zic family member odd-paired is needed for adult post-ecdysis maturation.

Specific neuropeptides regulate in arthropods the shedding of the old cuticle (ecdysis) followed by maturation of the new cuticle. In Drosophila melanogaster, the last ecdysis occurs at eclosion from the pupal case, with a post-eclosion behavioural sequence that leads to wing extension, cuticle stretching and tanning. These events are highly stereotyped and are controlled by a subset of crustacean cardioactive peptide (CCAP) neurons through the expression of the neuropeptide Bursicon (Burs). We have studied the role of the transcription factor Odd-paired (Opa) during the post-eclosion period. We report that opa is expressed in the CCAP neurons of the central nervous system during various steps of the ecdysis process and in peripheral CCAP neurons innerving the larval muscles involved in adult ecdysis. We show that its downregulation alters Burs expression in the CCAP neurons. Ectopic expression of Opa, or the vertebrate homologue Zic2, in the CCAP neurons also affects Burs expression, indicating an evolutionary functional conservation. Finally, our results show that, independently of its role in Burs regulation, Opa prevents death of CCAP neurons during larval development.

Ultraspiracle-independent anti-apoptotic function of ecdysone receptors is required for the survival of larval peptidergic neurons via suppression of grim expression in Drosophila melanogaster.

In Drosophila melanogaster a significant number of heterogenous larval neurons in the central nervous system undergo metamorphosis-associated programmed cell death, termed metamorphoptosis. Interestingly distinct groups of doomed larval neurons are eliminated at different metamorphic phases. Although ecdysone hormonal signaling via nuclear ecdysone receptors (EcRs) is known to orchestrate the neuronal metamorphoptosis, little is known about how this signaling controls such diverse neuronal responses. Crustacean cardioactive peptide (CCAP)-producing neurons in the ventral nerve cord are developmentally programmed to die shortly after adult emergence. In this study, we show that disruption of endogenous EcR function by ectopic expression of dominant negative forms of EcRs (EcRDN) causes premature death of larval CCAP neurons in a caspase-dependent manner. This event is rescued by co-expression of individual EcR isoforms. Furthermore, larval CCAP neurons are largely normal in ecr mutants lacking either EcR-A or EcR-B isoforms, suggesting that EcR isoforms redundantly function to protect larval CCAP neurons. Of surprise, a role of Ultraspiracle (Usp), a canonical partner of EcR, is dispensable in the protection of CCAP neurons, whereas both EcR and Usp are required for inducing metamorphoptosis of vCrz neurons shortly after prepupal formation. As a downstream, grim is an essential cell death gene for the EcRDN-mediated CCAP neuronal death, while either hid or rpr function is dispensable. Together, our results suggest that Usp-independent EcR actions protect CCAP neurons from their premature death by repressing grim expression until their normally scheduled apoptosis at post-emergence. Our studies highlight two opposite roles played by EcR function for metamorphoptosis of two different peptidergic neuronal groups, proapoptotic (vCrz) versus antiapoptotic (CCAP), and propose that distinct death timings of doomed larval neurons are determined by differential signaling mechanisms involving EcR.

FUS-induced neurotoxicity in Drosophila is prevented by downregulating nucleocytoplasmic transport proteins.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases characterized by the progressive loss of specific groups of neurons. Due to clinical, genetic and pathological overlap, both diseases are considered as the extremes of one disease spectrum and in a number of ALS and FTD patients, fused in sarcoma (FUS) aggregates are present. Even in families with a monogenetic disease cause, a striking variability is observed in disease presentation. This suggests the presence of important modifying genes. The identification of disease-modifying genes will contribute to defining clear therapeutic targets and to understanding the pathways involved in motor neuron death. In this study, we established a novel in vivo screening platform in which new modifying genes of FUS toxicity can be identified. Expression of human FUS induced the selective apoptosis of crustacean cardioactive peptide (CCAP) neurons from the ventral nerve cord of fruit flies. No defects in the development of these neurons were observed nor were the regulatory CCAP neurons from the brain affected. We used the number of CCAP neurons from the ventral nerve cord as an in vivo read-out for FUS toxicity in neurons. Via a targeted screen, we discovered a potent modifying role of proteins involved in nucleocytoplasmic transport. Downregulation of Nucleoporin 154 and Exportin1 (XPO1) prevented FUS-induced neurotoxicity. Moreover, we show that XPO1 interacted with FUS. Silencing XPO1 significantly reduced the propensity of FUS to form inclusions upon stress. Taken together, our findings point to an important role of nucleocytoplasmic transport proteins in FUS-induced ALS/FTD.

Anatomical characterization of PDF-tri neurons and peptidergic neurons associated with eclosion behavior in Drosophila.

The peptidergic Pigment-dispersing factor (PDF)-Tri neurons are a group of non-clock neurons that appear transiently around the time of adult ecdysis (=eclosion) in the fruit fly Drosophila melanogaster. This specific developmental pattern points to a function of these neurons in eclosion or other processes that are active around pupal-adult transition. As a first step to understand the role of these neurons, we here characterize the anatomy of the PDF-Tri neurons. In addition, we describe a further set of peptidergic neurons that have been associated with eclosion behavior, eclosion hormone (EH), and crustacean cardioactive peptide (CCAP) neurons, to single cell level in the pharate adult brain. PDF-Tri neurons as well as CCAP neurons co-express a classical transmitter indicated by the occurrence of small clear vesicles in addition to dense-core vesicles containing the peptides. In the tritocerebrum, gnathal ganglion and the superior protocerebrum PDF-Tri neurites contain peptidergic varicosities and both pre- and postsynaptic sites, suggesting that the PDF-Tri neurons represent modulatory rather than pure interneurons that connect the subesophageal zone with the superior protocerebrum. The extensive overlap of PDF-Tri arborizations with neurites of CCAP- and EH-expressing neurons in distinct brain regions provides anatomical evidence for a possible function of the PDF-Tri neurons in eclosion behavior.

Stereotyped responses of Drosophila peptidergic neuronal ensemble depend on downstream neuromodulators.

Neuropeptides play a key role in the regulation of behaviors and physiological responses including alertness, social recognition, and hunger, yet, their mechanism of action is poorly understood. Here, we focus on the endocrine control ecdysis behavior, which is used by arthropods to shed their cuticle at the end of every molt. Ecdysis is triggered by ETH (Ecdysis triggering hormone), and we show that the response of peptidergic neurons that produce CCAP (crustacean cardioactive peptide), which are key targets of ETH and control the onset of ecdysis behavior, depends fundamentally on the actions of neuropeptides produced by other direct targets of ETH and released in a broad paracrine manner within the CNS; by autocrine influences from the CCAP neurons themselves; and by inhibitory actions mediated by GABA. Our findings provide insights into how this critical insect behavior is controlled and general principles for understanding how neuropeptides organize neuronal activity and behaviors.

The study of the Bithorax-complex genes in patterning CCAP neurons reveals a temporal control of neuronal differentiation by Abd-B.

ABSTRACTDuring development, HOX genes play critical roles in the establishment of segmental differences. In the Drosophila central nervous system, these differences are manifested in the number and type of neurons generated by each neuroblast in each segment. HOX genes can act either in neuroblasts or in postmitotic cells, and either early or late in a lineage. Additionally, they can be continuously required during development or just at a specific stage. Moreover, these features are generally segment-specific. Lately, it has been shown that contrary to what happens in other tissues, where HOX genes define domains of expression, these genes are expressed in individual cells as part of the combinatorial codes involved in cell type specification. In this report we analyse the role of the Bithorax-complex genes – Ultrabithorax, abdominal-A and Abdominal-B – in sculpting the pattern of crustacean cardioactive peptide (CCAP)-expressing neurons. These neurons are widespread in invertebrates, express CCAP, Bursicon and MIP neuropeptides and play major roles in controlling ecdysis. There are two types of CCAP neuron: interneurons and efferent neurons. Our results indicate that Ultrabithorax and Abdominal-A are not necessary for specification of the CCAP-interneurons, but are absolutely required to prevent the death by apoptosis of the CCAP-efferent neurons. Furthermore, Abdominal-B controls by repression the temporal onset of neuropeptide expression in a subset of CCAP-efferent neurons, and a peak of ecdysone hormone at the end of larval life counteracts this repression. Thus, Bithorax complex genes control the developmental appearance of these neuropeptides both temporally and spatially.

Specification of neuronal subtypes by different levels of Hunchback.

During the development of the central nervous system, neural progenitors generate an enormous number of distinct types of neuron and glial cells by asymmetric division. Intrinsic genetic programs define the combinations of transcription factors that determine the fate of each cell, but the precise mechanisms by which all these factors are integrated at the level of individual cells are poorly understood. Here, we analyzed the specification of the neurons in the ventral nerve cord of Drosophila that express Crustacean cardioactive peptide (CCAP). There are two types of CCAP neurons: interneurons and efferent neurons. We found that both are specified during the Hunchback temporal window of neuroblast 3-5, but are not sibling cells. Further, this temporal window generates two ganglion mother cells that give rise to four neurons, which can be identified by the expression of empty spiracles. We show that the expression of Hunchback in the neuroblast increases over time and provide evidence that the absolute levels of Hunchback expression specify the two different CCAP neuronal fates.

Mechanisms of Postecdysis‐Associated Programmed Cell Death of Peptidergic Neurons in Drosophila melanogaster

Crustacean cardioactive peptide (CCAP)-expressing neurons undergo programmed cell death (PCD) within 24 hours after adult eclosion. A subset of the doomed CCAP neurons in the ventral nerve cord also expressed the neuropeptide bursicon and thus are referred to as bursCCAP neurons. In this study, we undertook comprehensive genetic and transgenic analyses to dissect the PCD mechanisms of bursCCAP neurons. Expression of a versatile caspase inhibitor, p35, blocked PCD of bursCCAP neurons, suggesting caspase-dependent apoptosis. Further genetic analyses showed that Dronc/Dark and Drice are key caspases, but they are not sufficient to carry out the PCD fully. We did not find a role for other known caspases, Strica, Dredd, Damm, or Decay. Of interest, Dcp-1 is required not for the death of bursCCAP neurons per se but for the removal of neural projections. DIAP1 is an important survival factor that inhibits premature death of bursCCAP neurons. We found that grim functions as a principal death inducer, whereas other death genes, hid, reaper, and sickle, show no endogenous function. Taken together with other studies, our work supports the role of grim as a major death inducer particularly for the removal of obsolete larval neurons during CNS metamorphosis. Results from the ectopic expression of the mutant grim lacking either N-terminal IBM or internal GH3 domain indicated that both domains are necessary to induce CCAP cell death.

Temporally tuned neuronal differentiation supports the functional remodeling of a neuronal network in Drosophila

During insect metamorphosis, neuronal networks undergo extensive remodeling by restructuring their connectivity and recruiting newborn neurons from postembryonic lineages. The neuronal network that directs the essential behavior, ecdysis, generates a distinct behavioral sequence at each developmental transition. Larval ecdysis replaces the cuticle between larval stages, and pupal ecdysis externalizes and expands the head and appendages to their adult position. However, the network changes that support these differences are unknown. Crustacean cardioactive peptide (CCAP) neurons and the peptide hormones they secrete are critical for ecdysis; their targeted ablation alters larval ecdysis progression and results in a failure of pupal ecdysis. In this study, we demonstrate that the CCAP neuron network is remodeled immediately before pupal ecdysis by the emergence of 12 late CCAP neurons. All 12 are CCAP efferents that exit the central nervous system. Importantly, these late CCAP neurons were found to be entirely sufficient for wild-type pupal ecdysis, even after targeted ablation of all other 42 CCAP neurons. Our evidence indicates that late CCAP neurons are derived from early, likely embryonic, lineages. However, they do not differentiate to express their peptide hormone battery, nor do they project an axon via lateral nerve trunks until pupariation, both of which are believed to be critical for the function of CCAP efferent neurons in ecdysis. Further analysis implicated ecdysone signaling via ecdysone receptors A/B1 and the nuclear receptor ftz-f1 as the differentiation trigger. These results demonstrate the utility of temporally tuned neuronal differentiation as a hard-wired developmental mechanism to remodel a neuronal network to generate a scheduled change in behavior.

The control of anterior foregut motility during a larval molt of the moth Manduca sexta involves the modulation of presynaptic activity

In the moth, Manduca sexta, anterior foregut motility is modulated during the larval-larval molts in order to control the timing of molting fluid (MF) ingestion. MF is the enzymatic mixture that destroys the outer cuticle so that it can be shed at the end of the molt. The onset of the larval-larval molt is characterized by a dramatic decline in the amplitude of the anterior foregut contractions so that MF is not prematurely ingested. As the end of the molt approaches, the robust contractions of the anterior foregut return and the MF is ingested, enabling the larva to free itself from its old cuticle. In the present study we examine possible mechanisms involved in modulating anterior foregut motility during a larval-larval molt. Our results reveal that the release of a blood-borne factor plays a role in the decline in anterior foregut peristaltic activity during the molt. This blood-borne factor reduces the efficacy of the presynaptic endings of the motorneurons, resulting in a reduction in the amplitude of the excitatory junctional potential (EJP) recorded from the anterior foregut musculature. We also present evidence that crustacean cardioactive peptide (CCAP) targets the motorneuron terminals and its actions are sufficient to trigger the dramatic increase in EJP amplitude and anterior foregut contractions. Finally, the surgical ablation of the subesophageal ganglion, which has been previously described to be a source of CCAP neurons and the CCAP projections to the anterior foregut region, blocks both the increase in anterior foregut motility and the ingestion of MF that normally occur at the end of a larval-larval molt.

Role of the neuropeptide CCAP inDrosophila cardiac function

The heartbeat of adult Drosophila melanogaster displays two cardiac phases, the anterograde and retrograde beat, which occur in cyclic alternation. Previous work demonstrated that the abdominal heart becomes segmentally innervated during metamorphosis by peripheral neurons that express crustacean cardioactive peptide (CCAP). CCAP has a cardioacceleratory effect when it is applied in vitro. The role of CCAP in adult cardiac function was studied in intact adult flies using targeted cell ablation and RNA interference (RNAi). Optical detection of heart activity showed that targeted ablation of CCAP neurons selectively altered the anterograde beat, without apparently altering the cyclic cardiac reversal. Normal development of the abdominal heart and of the remainder of cardiac innervation in flies lacking CCAP neurons was confirmed by immunocytochemistry. Thus, in addition to its important role in ecdysis behavior (the behavior used by insects to shed the remains of the old cuticle at the end of the molt), CCAP may control the level of activity of the anterograde cardiac pacemaker in the adult fly. Expression of double stranded CCAP RNA in the CCAP neurons (targeted CCAP RNAi) caused a significant reduction in CCAP expression. However, this reduction was not sufficient to compromise CCAP's function in ecdysis behavior and heartbeat regulation.

Identification of the Gene Encoding Bursicon, an Insect Neuropeptide Responsible for Cuticle Sclerotization and Wing Spreading

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon [1, 2], but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon [3], we identified the Drosophila melanogaster gene CG13419 as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-β (TGF-β) and glycoprotein hormones [4]. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased bursicon bioactivity. In situ hybridization and immunocytochemistry revealed that bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons [5] also lacked bursicon bioactivity. Our results indicate that CG13419 encodes bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to have a defined function in invertebrates. Mutants show that the spectrum of bursicon actions is broader than formerly demonstrated.

Neuroendocrine control of larval ecdysis behavior in Drosophila: complex regulation by partially redundant neuropeptides.

To complete each molting cycle, insects display a stereotyped sequence of behaviors to shed the remains of the old cuticle. These behavioral routines, as well as other related physiological events, are critical for proper development and are under the control of several neuropeptides. Their correct deployment and concatenation depends on the complex actions and interactions among several peptide hormones: ecdysis triggering hormone (ETH), eclosion hormone (EH), and crustacean cardioactive peptide (CCAP). Numerous theories, some in conflict, have been proposed to define the functional hierarchies by which these regulatory factors operate. Here we use wild-type Drosophila and transgenic flies bearing targeted ablations of either EH or CCAP neurons, or ablations of both together, to reevaluate their roles. Consistent with findings in moths, our results suggest that EH and ETH affect the release of each other via a positive feedback, although ETH can also be released in the absence of EH. We show that EH and ETH both contribute to the air filling of the air ducts (trachea) of the next stage but that EH may play a primary role in this process. We present evidence that EH, whose actions have always been placed upstream of CCAP, may also regulate ecdysis independently of CCAP. Finally, we confirm that flies lacking EH neurons do not ecdyse prematurely when injected with ETH peptides. These findings are surprising and not easily explained by currently available hypotheses. We propose that important additional neuropeptides, and additional interactions between known regulators, contribute to the mechanisms underlying insect ecdysis behaviors.

Targeted ablation of CCAP neuropeptide-containing neurons of Drosophila causes specific defects in execution and circadian timing of ecdysis behavior.

Insect growth and metamorphosis is punctuated by molts, during which a new cuticle is produced. Every molt culminates in ecdysis, the shedding of the remains of the old cuticle. Both the timing of ecdysis relative to the molt and the actual execution of this vital insect behavior are under peptidergic neuronal control. Based on studies in the moth, Manduca sexta, it has been postulated that the neuropeptide Crustacean cardioactive peptide (CCAP) plays a key role in the initiation of the ecdysis motor program. We have used Drosophila bearing targeted ablations of CCAP neurons (CCAP KO animals) to investigate the role of CCAP in the execution and circadian regulation of ecdysis. CCAP KO animals showed specific defects at ecdysis, yet the severity and nature of the defects varied at different developmental stages. The majority of CCAP KO animals died at the pupal stage from the failure of pupal ecdysis, whereas larval ecdysis and adult eclosion behaviors showed only subtle defects. Interestingly, the most severe failure seen at eclosion appeared to be in a function required for abdominal inflation, which could be cardioactive in nature. Although CCAP KO populations exhibited circadian eclosion rhythms, the daily distribution of eclosion events (i.e., gating) was abnormal. Effects on the execution of ecdysis and its circadian regulation indicate that CCAP is a key regulator of the behavior. Nevertheless, an unexpected finding of this work is that the primary functions of CCAP as well as its importance in the control of ecdysis behaviors may change during the postembryonic development of Drosophila.

Circadian regulation of the lark RNA-binding protein within identifiable neurosecretory cells.

Molecular genetic analysis indicates that rhythmic changes in the abundance of the Drosophila lark RNA-binding protein are important for circadian regulation of adult eclosion (the emergence or ecdysis of the adult from the pupal case). To define the tissues and cell types that might be important for lark function, we have characterized the spatial and developmental patterns of lark protein expression. Using immunocytochemical or protein blotting methods, lark can be detected in late embryos and throughout postembryonic development, from the third instar larval stage to adulthood. At the late pupal (pharate adult) stage, lark protein has a broad pattern of tissue expression, which includes two groups of crustacean cardioactive peptide (CCAP)-containing neurons within the ventral nervous system. In other insects, the homologous neurons have been implicated in the physiological regulation of ecdysis. Whereas lark has a nuclear distribution in most cell types, it is present in the cytoplasm of the CCAP neurons and certain other cells, which suggests that the protein might execute two different RNA-binding functions. Lark protein exhibits significant circadian changes in abundance in at least one group of CCAP neurons, with abundance being lowest during the night, several hours prior to the time of adult ecdysis. Such a temporal profile is consistent with genetic evidence indicating that the protein serves a repressor function in mediating the clock regulation of adult ecdysis. In contrast, we did not observe circadian changes in CCAP neuropeptide abundance in late pupae, although CCAP amounts were decreased in newly-emerged adults, presumably because the peptide is released at the time of ecdysis. Given the cytoplasmic localization of the lark RNA-binding protein within CCAP neurons, and the known role of CCAP in the control of ecdysis, we suggest that changes in lark abundance may regulate the translation of a factor important for CCAP release or CCAP cell excitability.

Eclosion hormone provides a link between ecdysis-triggering hormone and crustacean cardioactive peptide in the neuroendocrine cascade that controls ecdysis behavior.

Three insect peptide hormones, eclosion hormone (EH), ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP), have been implicated in controlling ecdysis behavior in insects. This study examines the interactions between these three peptides in the regulation of the ecdysis sequence. Using intracellular recordings, we found that ETH is a potent activator of the EH neurons, causing spontaneous action potential firing, broadening of the action potential and an increase in spike peak amplitude. In turn, electrical stimulation of the EH neurons or bath application of EH to desheathed ganglia resulted in the elevation of cyclic GMP (cGMP) levels within the Cell 27/704 group (which contain CCAP). This cGMP production increases the excitability of these neurons, thereby facilitating CCAP release and the generation of the ecdysis motor program. Extracellular recordings from isolated nervous systems show that EH has no effect on nervous systems with an intact sheath. In desheathed preparations, in contrast, EH causes only the ecdysis motor output. The latency from EH application to ecdysis was longer than that after CCAP application, but shorter than that when ETH is applied to a whole central nervous system. These data, along with previously published results, support a model in which ETH causes pre-ecdysis behavior and at higher concentrations stimulates the EH neurones. EH release then facilitates the onset of ecdysis by enhancing the excitability of the CCAP neurons.

Neuropeptide Hierarchies and the Activation of Sequential Motor Behaviors in the Hawkmoth,Manduca sexta

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 of Manduca sexta show 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.