At the end of each molt, insects shed their old cuticle by performing the ecdysis sequence, an innate behavior consisting of three steps: pre-ecdysis, ecdysis, and postecdysis. Blood-borne ecdysis-triggering hormone (ETH) activates the behavioral sequence through direct actions on the central nervous system. To elucidate neural substrates underlying the ecdysis sequence, neurons expressing ETH receptors (ETHRs) have been identified in Drosophila. Distinct ensembles of ETHR neurons express numerous neuropeptides including kinin, FMRFamides, eclosion hormone (EH), crustacean cardioactive peptide (CCAP), myoinhibitory peptides (MIP), and bursicon. Real-time imaging of intracellular calcium dynamics revealed sequential activation of these ensembles after ETH action. Specifically, FMRFamide neurons are activated during pre-ecdysis; EH, CCAP, and CCAP/MIP neurons are active prior to and during ecdysis; and activity of CCAP/MIP/bursicon neurons coincides with postecdysis. Targeted ablation of specific ETHR ensembles produces behavioral deficits consistent with their proposed roles in the behavioral sequence. These findings offer novel insights into how a command chemical orchestrates an innate behavior by stepwise recruitment of central peptidergic ensembles (Kim, 2006).
Analysis of the pupal ecdysis behavioral sequence: In Drosophila, pupal ecdysis is preceded by pupariation, whereby the prepupa contracts its body into a barrel shape to form the puparium composed of the old larval cuticle. The underlying new pupal cuticle then separates from the puparium during pupal ecdysis ~12 hr later. The stereotypic nature of pupal ecdysis and reliable developmental markers make it a favorable model for the behavioral analysis and neural imaging (Kim, 2006).
Pupal ecdysis consists of three centrally patterned behavioral subunits performed sequentially: pre-ecdysis (~10 min), ecdysis (~5 min), and postecdysis (~60-70 min). The behavioral sequence was examined through the semitransparent puparium ('puparium-intact'), but it was found that the puparium obscures and places constraints on some movements. This made it particularly difficult to discriminate differences in abdominal swinging movements during ecdysis and postecdysis. Therefore, a 'puparium-free' preparation was used by surgically removing the puparium immediately after pre-ecdysis onset. The improved visibility and room for movement in this preparation allowed for a more complete analysis of natural and ETH-induced behavior. The following description of the natural pupal ecdysis sequence resulted from comparison of behaviors observed in both puparium-intact and puparium-free prepupae (Kim, 2006).
Pre-ecdysis: About 5 min after in vivo ETH release, preecdysis commences with the abrupt appearance of an air bubble at the posterior end of the prepupa (time zero). Pre-ecdysis involves anteriorly directed rolling contractions along the lateral edges of the abdomen, alternating on the left and right sides of the animal. These contractions move the air bubble anteriorly to separate pupal cuticle from the puparium. This behavior is completed within ~10 min and is followed by ecdysis behavior. Preecdysis behavior is the same in puparium-intact and puparium-free animals (Kim, 2006).
Ecdysis: In higher Diptera including Drosophila, the incipient adult head develops within the prepupal thorax. During pupal ecdysis, head eversion results from lateral swinging movements of the abdomen occurring along with anteriorly directed peristaltic contractions. In puparium-intact preparations, head eversion occurs ~1 min after the onset of ecdysis swinging and is completed within ~5s. After completion of head eversion, ecdysis contractions continue for ~15 min, facilitating expansion of wing pads and legs to their final size. The frequency of ecdysis swinging (~5 swings/min) decreases markedly after head eversion (~2 swings/min). In puparium-free animals, head eversion occurs sooner, and the duration of ecdysis behavior lasts only ~5 min. Later in ecdysis, anteriorly directed swinging contractions are often interrupted by posteriorly directed ones, indicating a transition to postecdysis (Kim, 2006).
Postecdysis: Postecdysis behavior consists of two behavioral subroutines: postecdysis swinging and stretch-compression movements of the abdomen. Postecdysis swinging occurs along with posteriorly directed peristaltic contractions and alternates with longitudinal movements of the abdomen, referred to as 'stretch-compression.' The frequency and intensity of postecdysis contractions wane gradually until they are detected mainly in the anterior part of the abdomen; they cease w100 min after pre-ecdysis onset. Postecdysis behavior concludes with compression of the pupa at the posterior end of the puparium (Kim, 2006).
ETH release coincides with initiation of the ecdysis sequence: To confirm the role of ETH in initiation of the pupal ecdysis sequence, its release from endocrine Inka cells was monitored in vivo by using time-lapse EGFP fluorescence imaging in pharate pupae (prepupae) carrying the chimeric transgene 2eth3-egfp. In this transgenic fly, EGFP is expressed as part of a fusion protein with the ETH propeptide precursor, and loss of EGFP fluorescence indicates ETH release. Because pharate pupae generally are immobile and Inka cells are located immediately below the semitransparent puparium, in situ imaging of Inka cell in intact pharate pupae is feasible. Two to three Inka cells were monitored simultaneously in each experiment (Kim, 2006).
Depletion of ETH-EGFP occurs in about 50% of monitored Inka cells shortly before pre-ecdysis onset (time zero). The time course of secretory activity for each Inka cell was variable. The mean value for the timing of ETH release was 4.5 min prior to pre-ecdysis onset, and the duration of ETH secretion was 4.4. In contrast, 40% of Inka cells showed no sign of secretory activity by pre-ecdysis onset. After initiation of pre-ecdysis contractions, it was usually impossible to continue monitoring loss of EGFP fluorescence as a result of movement artifacts. All Inka cells are depleted of ETH-EGFP by the end of ecdysis sequence (Kim, 2006).
Injection of ETH induces the ecdysis sequence: Because ETH release coincides with onset of pupal preecdysis, it was of interest to determine whether ETH injection would trigger the pupal ecdysis sequence. The Drosophila gene eth encodes a precursor producing one copy each of two peptides, ETH1 and ETH2, which share similar structure and biological activity. In vivo experiments were carried out primarily in puparium-free preparations (Kim, 2006).
Injection of ETH1 alone into pharate pupae (~1-2 hr prior to natural ecdysis) induced within 1-3 min strong pre-ecdysis contractions followed by ecdysis and postecdysis contractions sequentially. ETH-induced pre-ecdysis showed a strong dose dependence, with higher doses inducing shorter pre-ecdysis duration and higher frequency of contractions. Similar but somewhat less pronounced dose-dependent effects were observed during ecdysis behavior, whereas the frequency of postecdysis contractions showed little or no dose dependence during the first 10 min of behavior (Kim, 2006).
Injection of ETH2 was less efficacious for induction of the behavioral sequence compared to ETH1. ETH2 generated prolonged pre-ecdysis behavior lasting to 50 min or more, but no ecdysis behavior. In contrast, injection of the same dose of ETH1 (0.4 pmol) produced the complete behavioral sequence consisting of pre-ecdysis, ecdysis, and postecdysis. Higher doses of ETH2 (20 pmol) generated a behavioral sequence comparable to that induced by 4 pmol ETH1 in terms of pre-ecdysis duration and frequency of contractions. Behaviors after injecting a cocktail of ETH1 and ETH2 (0.4 pmol of each peptide) were also examined. Because the two peptides are processed from the same precursor, it is likely that these peptides are coreleased under natural conditions. The duration of the behavioral sequence induced by injection of the cocktail was similar to the naturally occurring sequence or one induced by 0.4 pmol ETH1 alone. It is estimated that a 0.4 pmol ETH injection into a prepupa w10 hr after puparium formation results in a concentration of ~300 nM in vivo (Kim, 2006).
ETH receptors are expressed in diverse ensembles of peptidergic neurons: ETH acts directly on the CNS to initiate the ecdysis behavioral sequence in moths and flies via unknown signaling pathways within the CNS. A starting point for elucidation of these downstream signaling pathways is identification of primary neuronal targets of ETH. The ETH receptor gene (CG5911), first identified in Drosophila, encodes two G protein-coupled receptors, ETHR-A and ETHR-B, via alternative splicing. In situ hybridization was used for identification of central neurons expressing ETHR-A and ETHR-B by using DNA probes specific for each receptor subtype. ETHR-A and ETHR-B transcripts were located in mutually exclusive populations of neurons distributed throughout the CNS, suggesting that two subtypes of ETH receptors likely mediate different functions. Further analysis revealed that most ETHR-A neurons are peptidergic. Neurons expressing ETHR-B have not been identified thus far (Kim, 2006).
Multiple ensembles of ETHR-A neurons are classified according to specific neuropeptides they express. Peptides expressed in these ensembles were identified by using GAL4 transgenes under control of neuropeptide promoters to drive UAS-GFP or UAS-GCaMP expression (GAL4::GFP or GAL4::GCaMP). Expression of neuropeptides in these cells was confirmed by combining immunohistochemical staining and in situ hybridization. The first ETHR-A ensemble comprises six pairs of lateral abdominal neurons producing kinin, also known as drosokinin. These cells project axons posteriorly along the lateral edge of the neuropile and then turn anteriorly along the midline of ventral nerve cord, where they arborize and form possible central release sites. Axons of these cells also exit the CNS through nerve roots, suggesting peripheral kinin release. The second ETHR-A ensemble contains three pairs of ventrolateral FMRFamide neurosecretory cells (Tv1-3 or T1-3) in the thoracic neuromeres TN1-3. These cells project axons into the dorsomedial neurohemal organs (NHOs) specialized for peptide release into the hemolymph (Kim, 2006).
The third class of ETHR-A neurons comprises the eclosion hormone (EH)-producing VM neurons in the brain, which project one axonal branch anteriorly into the neurohemal ring gland and a second posteriorly along the dorsal midline of the entire ventral nerve cord. The fourth ETHR-A ensemble is composed of paired dorsolateral neurons producing CCAP, MIPs, and bursicon in subesophageal, thoracic, and abdominal neuromeres (SN1-3, TN1-3, AN1-7, respectively). These cells are likely homologs of moth neurons 27/704, on the basis of their anatomy, peptide coexpression profile, and functional roles during pupal ecdysis. These neurons are referred to as Drosophila neurons 27/704 and them were subdivided on the basis of peptide coexpression. In AN1-4, CCAP is colocalized with MIPs and the heterodimeric peptide hormone bursicon (composed of burs and pburs subunits). In TN2-3 and AN5- 9, CCAP is colocalized with burs, but pburs is not expressed in these neurons. Finally, CCAP is colocalized with MIPs in large paired neurons of AN8,9, but ETHR-A expression has not been confirmed in these cells. The presence of MIP mRNA in abdominal neurons 27/704 was further confirmed by in situ hybridization (Kim, 2006).
Ca2+ imaging of primary ETH targets in transgenic flies: Having shown that ETH receptors occur in diverse ensembles of peptidergic neurons, it was asked whether these cells are activated by ETH and whether this activity coincides with specific behavioral steps of the ecdysis sequence. Calcium dynamics in each group of ETHR-A neurons was monitored by driving expression of the GFP-based Ca2+ sensor, GCaMP [23, 24], in genetically defined sets of neurons with the binary GAL4/UAS system. Ca2+ elevation induces a conformational change of GCaMP, increasing its GFP fluorescence. Using optical imaging of GFP fluorescence, [Ca2+]i dynamics of ETHR neurons were monitored, and these events were associated with each behavioral phase induced by ETH (Kim, 2006).
An abundance of evidence indicates that the ecdysis behavioral sequence in insects is centrally patterned. In particular, the onset and duration of each behavior in the sequence (pre-ecdysis I, pre-ecdysis II, ecdysis) is the same whether observed in vivo or as fictive behavior recorded from the isolated CNS in vitro. On the basis of this evidence, [Ca2+]i dynamics of ETHR-A neuron ensembles of the isolated CNS were associated with behaviors observed in puparium-free preparations (Kim, 2006).
FMRFamide neurons and their neurohemal endings become active early in pre-ecdysis: [Ca2+]i levels were monitored in ETHR-A/FMRFamide Tv neurons by preparing transgenic flies doubly homozygous for FMRFa-GAL4 and UAS-GCaMP. Prior to ETH1 exposure (4-6 hr prior to ecdysis), Tv cell bodies and neurohemal endings in the dorsomedial NHO exhibit low levels of basal GCaMP fluorescence (Kim, 2006).
Exposure of the CNS to ETH1 (600 nM) elicits robust increases in calcium-associated fluorescence in cell bodies and axon terminals of all Tv neurons. At this concentration of ETH1, calcium dynamics typically are characterized by transient, spike-shaped fluctuations superimposed upon a slow upward shift of the baseline, beginning ~8 min after exposure to the peptide. This response lasts ~10-15 min, after which weaker spike-like fluctuations continue without baseline changes until the end of recordings (~40 min). It is estimated that a concentration of 600 nM ETH1 results from a dose of w0.4 pmol of the peptide in vivo. Thus the major calcium response of Tv neurons coincides with the early phase of pre-ecdysis, and weaker activity persists through ecdysis and postecdysis. In contrast, ETH2 alone (600 nM) generates Ca2+ responses after a longer delay comparable to one following exposure to 60 nM ETH1. The longer delay of Ca2+ responses after ETH2 fits with the observations of in vivo behavior, where ETH2 is a less potent agonist than ETH1. The cocktail of ETH1 and ETH2 (600 nM each) evokes Ca2+ dynamics after a delay similar to that induced by ETH1 alone. Overall, [Ca2+]i dynamics observed in Tv neurons are synchronized. In many preparations, Tv neurons from the same neuromere appear to be strongly coupled, given that they produce precisely synchronized Ca2+ dynamics. Transient Ca2+ signals are obvious in the terminal processes of Tv neurons in NHO, the release sites of FMRFamides (Kim, 2006).
Lower concentrations of ETH1 elicit calcium dynamics after a somewhat longer delay. Interestingly, calcium dynamics are obvious first in neurohemal endings of the NHO, followed by fluctuations in cell bodies. This was particularly evident at 60 nM ETH1, where a robust calcium response in the NHO was accompanied by only a weak response in the Tv2 cell body. No calcium responses are observed in Tvs exposed to 6 nM ETH1 (Kim, 2006).
EH neurons reach peak activity at ecdysis: VM neurons producing eclosion hormone (EH) have been implicated as primary ETH targets during fly and moth ecdysis. Expression of ETHR-A was demonstrated in VM neurons, confirming that they are primary ETH targets. To determine whether ETH elicits activity in EH neurons, transgenic flies were prepared doubly homozygous for EHup-GAL4 and UASGCaMP, that show GCaMP fluorescence only in these cells (Kim, 2006).
EH neurons are highly sensitive to ETH1, exhibiting robust [Ca2+]i dynamics upon exposure to concentrations as low as 6 nM. No detectable fluorescence responses are observed after exposure to 0.6 nM ETH1 over a period of 50-60 min. The latency to Ca2+ responses is inversely proportional to the concentration of ETH1; higher ETH1 concentrations evoke faster responses. The cocktail of ETH1 and ETH2 (600 nM each) elicited Ca2+ responses after a ~10-15 min delay (Kim, 2006).
Close examination of these ETH-evoked fluorescence responses reveals two components distinguished by slow and fast kinetics. The slow component is characterized by a gradual increase in baseline levels of Ca2+ followed by a decrease over 20-30 min, whereas the fast component is composed of transient, spike-like activity. Fast components have durations ranging from 5-20 s. Peak DF/F responses are quite variable, even among a group of neurons exposed to the same ETH1 concentration. No significant concentration dependence could be detected in peak response (Kim, 2006).
Distinct subsets of neurons 27/704 are active during different phases of the ecdysis sequence: ETH-evoked Ca2+ signals of neurons 27/704 were examined in transgenic flies carrying CCAP-GAL4 and UAS-GCaMP. Use of the CCAP promoter to drive GCaMP expression resulted in a reporter pattern identical to that described previously. Upon exposure to 600 nM ETH1, distinct subsets of neurons 27/704 exhibited reproducible, stereotypic Ca2+ responses in terms of peak intensity, latency, and termination of Ca2+ dynamics. According to the magnitude of peak fluorescence intensity (peak DF/F), neurons 27/704 fall into three major groups: strong responders, weak responders, and nonresponders. The strong-responder group includes neurons 27/704 in AN1-4 (CCAP/MIPs/bursicon), AN8,9 (CCAP/ MIPs), and TN3 (CCAP). Weak responders are neurons 27/704 in SN2-3, TN1-2, and AN7 producing CCAP only. Neurons in the brain, SN1, and AN5,6 showed no reproducible Ca2+ dynamics in response to 600 nM ETH1 (Kim, 2006).
In response to ETH1, neurons 27/704 in TN3 and AN8,9 become active within 10-15 min, whereas neurons 27/704 in AN1-4 are activated after a 15-25 min delay. Neurons in TN3 and AN8,9 are therefore activated just prior to ecdysis onset, indicating their possible roles in initiation and maintenance of ecdysis behavior. In addition, Ca2+ dynamics observed in AN8,9 neurons terminated early in postecdysis, supporting this interpretation. In contrast, Ca2+ dynamics of neurons in AN1-4 begin during ecdysis and increase in intensity during the entire postecdysis period, suggesting their roles in these events. The cocktail of ETH1 and ETH2 (600 nM each) evoked Ca2+ dynamics similar to those induced by ETH1 alone. Two groups of neurons 27/704 in abdominal neuromeres (AN1-4 versus AN8,9) exhibit differences in sensitivity to ETH and in their patterns of [Ca2+]i dynamics. It was found that 6-60 nM ETH1 activates neurons in AN1-4 (n = 4), whereas higher concentrations of ETH1 (R600 nM) are required to activate neurons in AN8,9. In addition, neurons in AN8,9 generate transient (1-2 min) Ca2+ spikes over a 15-20 min period after ETH1 activation, whereas neurons in AN1-4 generally produce slower, more persistent Ca2+ dynamics. These differences among subgroups of neurons 27/704 suggest their different functional roles during the ecdysis sequence (Kim, 2006).
Targeted ablations of apecific ETHR neurons have behavioral consequences: To evaluate behavioral roles of specific ETHR neurons, phenotypes of the pupal ecdysis sequence were investigated in transgenic flies bearing targeted ablations of ETHR neurons, including Tv FMRFamide neurons, EH neurons, and CCAP neurons (27/704 homologs). In control flies carrying UAS-reaper and UAS-GCaMP, but lacking the GAL4 driver, pupal ecdysis was executed as in wild-type flies: pre-ecdysis (0-10 min), ecdysis (10-23 min), and postecdysis (23-100 min) (Figure 7). Given that puparium-intact animals were used, the duration of ecdysis behavior may have been overestimated. Transgenic flies bearing targeted ablations of Tv FMRFamide neurons (FMRF-KO) were generated by crossing females doubly homozygous for FMRFa-GAL4, UAS-GCaMP with homozygous UAS-reaper males. Pupal ecdysis of FMRFa-KO flies is very similar to that of control flies. Because FMRFa-GAL4 drives expression of GAL4 only in three pairs of thoracic Tv neurons and one pair of unidentified neurons in SN, FMRFa-KO flies lost only Tv neurons and not other FMRFamide neurons in the CNS. FMRFa-KO flies complete pupal ecdysis without any detectable abnormality, except that pre-ecdysis contractions appear weaker than in control flies. Pupal ecdysis of VM neuron knockout flies (EH-KO) was then examined. Behavioral analysis showed that, although they complete pupal ecdysis without any severe defects or lethality, ecdysis onset is delayed w4 min. As a result of this delay, EH-KO flies show longer pre-ecdysis than control flies. Additional parameters governing pre-ecdysis, ecdysis, and postecdysis are indistinguishable between EH-KO and control flies (Kim, 2006).
Finally, CCAP-KO flies were generated in order to examine the functional roles of neurons 27/704 (CCAP neurons) in pupal ecdysis. As expected, CCAP-KO flies failed to initiate ecdysis contractions and could not complete head eversion. Instead, they show prolonged pre-ecdysis contractions for ~25 min, followed by weak random contractions of the abdomen (different from ecdysis and postecdysis contractions of control flies) for the next 50 min (Kim, 2006).
Conclusions: This study has described orchestration of an innate behavior, the Drosophila pupal ecdysis sequence, by the endocrine peptide ETH. ETH release coincides with onset of behavior, and injection of ETH triggers the complete behavioral sequence, consistent with its role in ecdysis activation previously established in the moths Manduca sexta and Bombyx mori and in Drosophila larvae and adults. Absence of ETH causes lethal ecdysis deficiency, a phenotype that is rescued by ETH injection. ETH therefore functions as a 'command chemical' to orchestrate an innate behavior. Primary CNS targets of ETH was identified by using ETHR-specific in situ hybridization. ETHR-A occurs in multiple classes of peptidergic neurons producing EH, CCAP/MIPs/bursicon, FMRFamides, or kinin (Kim, 2006).
Expression of ETHR-A was shown in VM neurons, which release EH. In response to ETH, VM neurons become active prior to ecdysis behavior and reach peak levels of activity during ecdysis. These results provide further support for a previously described positive-feedback signaling pathway between VM neurons and Inka cells. This feedback is thought to ensure depletion of ETH from Inka cells. These findings are striking because independent evidence indicates that homologous VM neurons in the moth Manduca are direct targets of ETH and that their secretory products regulate ecdysis behaviors downstream of ETH. For example, isolated EH neurons of Manduca respond to direct action of ETH with increased excitability and spike broadening. In response to ETH action, these neurons release EH, causing cGMP elevation and increased excitability in CCAP-containing neurons 27/704 of the thoracic and abdominal ganglia. CCAP and MIPs, cotransmitters produced by neurons 704, are implicated in eliciting ecdysis behavior (Kim, 2006).
A homologous role for EH in activation of Drosophila 27/704 neurons has not been clearly demonstrated. For example, no cGMP elevation is observed in these neurons during the natural ecdysis sequence. This lack of cGMP elevation suggests that CCAP neurons are not directly targeted by EH in Drosophila. Nevertheless, EH-knockout flies exhibit a delay in ecdysis initiation, suggesting that EH may modulate excitability in 27/704 cells indirectly through release of additional factors within the CNS. It is therefore proposed that activation of EH neurons by ETH serves two purposes: (1) release of EH into the hemocoel functions as part of a positive-feedback pathway to ensure ETH depletion from Inka cells; (2) release of EH within the CNS synergizes direct ETH actions on different subsets of neurons 27/704 producing CCAP, MIPs, and bursicon, perhaps indirectly through release of downstream signals within the CNS (Kim, 2006).
Neurons 27/704 expressing ETHR-A respond to ETH with unique patterns of Ca2+ dynamics. These neurons are subdivided by pattern of transmitter expression: CCAP/MIPs/bursicon in AN1-4; CCAP/MIPs in AN8,9; and CCAP in SN1-3, TN1-3, and AN5-7. Temporal patterns of Ca2+ dynamics were determined in each neuronal subset relevant to the behaviors observed. On the basis of these temporal patterns, it is proposed that direct action of ETH on neurons 27/704 in TN3 and in AN8,9 induces initiation and execution of ecdysis contractions and head eversion. In support of this, it is shown that ablation of CCAP neurons abolishes ecdysis contractions and head eversion. Parallel study in Manduca showed that neurons 704 expressing ETHR-A and their peptide cotransmitters, CCAP and MIPs, are implicated in control of the ecdysis motor pattern, supporting the homologous function of 27/704 neurons in Drosophila. Neurons 27/704 in AN1-4 produce CCAP, MIPs, and bursicon, and therefore a cocktail of these peptides is likely released within the CNS and into the hemolymph during postecdysis. It is suggested that centrally released peptides control postecdysis movements, whereas blood-borne CCAP/MIPs regulate heart beat and blood pressure for cuticle expansion and bursicon controls sclerotization of expanded new cuticle. Bursicon was recently identified as a heterodimeric peptide hormone regulating cuticle plasticization, sclerotization, and melanization (Kim, 2006).
The Drosophila FMRFamide gene (FMRFa) encodes multiple FMRFamide-related neuropeptides, which are expressed in many different cell types, including neuroendocrine cells, interneurons, and perhaps motoneurons. Among these diverse FMRFamide-producing neurons, ETHR-A expression is confined to three pairs of thoracic neurosecretory neurons, Tv1-3. Results of the present study show that the Tv neurons are activated early in pre-ecdysis and that they remain active during ecdysis and postecdysis. However, FMRFa-KO flies show no differences in timing of the ecdysis behavioral sequence. Because FMRFamides enhance twitch tension of larval body-wall muscles through synaptic modulation at the neuromuscular junction, blood-borne FMRFamides released from Tv neurons likely facilitates pre-ecdysis, ecdysis, and postecdysis contractions. Thus the role of Tv neurons as primary ETH targets may be enhancement of muscle contraction during the behaviors. Further work to substantiate this is in progress (Kim, 2006).
Expression of ETHR-A occurs in in kinin neurons of abdominal neuromeres of Drosophila. Drosophila kinin is known to be involved in water balance, but its central functions have not been described or considered. Expression of ETHR-A in kinin neurons appears to be a conserved mechanism in fly and moth; the Manduca ETHR-A is expressed in abdominal neurosecretory cells (L3,4), which produce kinins and diuretic hormones (DHs). It was further found that the isolated Manduca CNS generates the fictive pre-ecdysis motor pattern upon exposure to a cocktail of kinin and DHs. These findings suggest that ETH activates L3,4 neurons in Manduca to release kinins and DHs centrally, which initiate and execute pre-ecdysis. On the basis of the conservation between Drosophila and Manduca in spatial expression pattern of ETHR, it is proposed that ETH initiates pre-ecdysis behavior indirectly via central release of kinin in Drosophila (Kim, 2006).
In Drosophila, pupal ecdysis is accomplished by sequential recruitment of three major behavioral units: pre-ecdysis (0-10 min), ecdysis (10-15 min), and postecdysis (15-100 min). Each behavioral unit is programmed in the CNS and sequentially activated by direct actions of ETH, which is synthesized and released from peripheral endocrine Inka cells. Around 4-5 min before pre-ecdysis onset, a sizeable portion (~50%) of Inka cells initiates secretion of ETH into the hemolymph, whereas the remaining portion completes secretion after onset of pre-ecdysis. Appearance of ETH in the hemolymph activates ETHR-A in neurons expressing neuropeptides including kinin, FMRFamides (Tv1-3), EH, or CCAP, MIPs, and bursicon, but they are not released until descending inhibition is removed at key times during the ecdysis sequence. Upon activation of ETHR, the central release of kinin initiates pre-ecdysis contractions, whereas Tv neurons secrete FMRFamides to enhance neuromuscular transmission. ETH activates neurons producing EH, CCAP, CCAP/MIPs, and CCAP/MIPs/bursicon at different times. EH cells in the brain and neurons producing CCAP in TN3 and CCAP/MIPs in AN8,9 become active ~10-13 min after pre-ecdysis initiation. EH participates in timing the activation of ecdysis neurons, whereas CCAP and MIPs from TN3 and AN8,9 control initiation and execution of the ecdysis motor program. At the end of ecdysis (25 min after pre-ecdysis onset), neurons in AN1-4 secrete a cocktail of CCAP, MIPs, and bursicon, which likely regulate postecdysis contractions and processes associated with cuticle expansion, hardening, and tanning (Kim, 2006).
This study has mapped central ETH receptor neurons, and discovered that they comprise multiple peptidergic ensembles, which are recruited sequentially to generate each phase of the ecdysis sequence. Ensemble-specific knockout analysis supports this interpretation. Each step of the ecdysis sequence (pre-ecdysis, ecdysis, postecdysis) is driven by a central pattern generator (CPG) within the CNS in the absence of sensory input. It is known that amines and peptides can modulate and reconfigure neuronal circuits comprising CPGs so as to elicit a variety of motor patterns. It seems likely that the multiple peptidergic ensembles described in this study as targets for ETH may be involved in configuring and activating CPGs underlying each step of the ecdysis sequence (Kim, 2006).
Processes in the brain that govern behaviors over longer time frames such as sleep, mood, sexual activities, and even learning and memory could be associated with coordinated release of neuromodulators such as peptides. Further work on activation of central peptidergic ensembles in the CNS may shed light on mechanisms underlying release of a variety of behaviors (Kim, 2006).
Drosophila EH mRNA is confined to one pair of ventromedial cells in the brains of wandering stage larvae. These same two cells are also found in brains of pharate adults, at pupariation and at various times during pupal and adult development. The cellular localization is similar to that found in Manduca and Bombyx except that the moths show two pairs of cells rather than one (Horodyski, 1993).
These cells are immunopositive to an antiserum raised against Manduca EH. In the larval stage these cells possess a bifurcating axon, one branch of which extends peripherally to the corpora cardiaca portion of the ring gland. The other branch projects through a medial tract along the length of the ventral CNS, typically terminating in the terminal abdominal neuromere. In occasional individuals a branch is seen which extends out of the CNS and into the terminal nerve. Such a branch is frequently observed in pharate adults. At ecdysis in Manduca, the arbors of the EH neurons show a massive reduction in EH immunoreactivity, coincident with the release of EH into the circulation. Likewise, the ventromedial cells in Drosophila show maximal levels of EH immunoreactivity in the terminal processes just prior to ecdysis but then show an almost total loss of staining from the processes at the time of ecdysis (Horodyski, 1993).
In insects, ecdysis is thought to be controlled by the interaction between peptide hormones; in particular between ecdysis-triggering hormone (ETH) from the periphery and eclosion hormone (EH) and crustacean cardioactive peptide (see Cardioacceleratory peptide) from the central nervous system. The behavioral and physiological functions of the first two of these peptides was studied in Drosophila melanogaster using wild-type flies and knockout flies that lacked EH neurons. ETH from Manduca sexta (MasETH) was used to induce premature ecdysis and the responses of the two types of flies were compared. The final release of EH normally occurs approximately 40 min before ecdysis. It is correlated with cyclic guanosine monophosphate (cGMP) production in selected neurons and tracheae, by an elevation in the heart rate and by the filling of the new tracheae with air. Injection of developing flies with MasETH causes all these events to occur prematurely. In EH cell knockouts, none of these changes occurs in response to MasETH, and these flies show a permanent failure in tracheal filling. This failure can be overcome in the knockouts by injecting them with membrane-permeant analogs of cGMP, the second messenger for EH. The basis for the 40 min delay between EH release and the onset of ecdysis was examined by decapitating flies at various times relative to EH release. In flies that had already released EH, decapitation is always followed within 1 min by the start of ecdysis. Immediate ecdysis is never observed when the EH cell knockout flies were decapitated. It is proposed that EH activates both ventral central nervous system elements necessary for ecdysis (possibly the CCAP cells) and descending inhibitory neurons from the head. This descending inhibition establishes a delay in the onset of ecdysis that allows the completion of EH-activated physiological processes such as tracheal filling. A waning in the inhibition eventually allows ecdysis to begin 30-40 min later (Baker, 1999).
Programmed cell death occurs in the nervous and muscular system of newly emerged adult Drosophila. Many of the abdominal muscles that are 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 show 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 block or delay wing expansion behavior have a parallel effect in suppressing or delaying neuronal death (Kimura, 1990).
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date revised: 10 April 2010
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