C-type allatostatins (AST-Cs) are pleiotropic neuropeptides that are broadly conserved within arthropods; the presence of three AST-C isoforms, encoded by paralog genes, is common. However, these peptides are hypothesized to act through a single receptor, thereby exerting similar bioactivities within each species. We investigated this hypothesis in the American lobster, Homarus americanus, mapping the distributions of AST-C isoforms within relevant regions of the nervous system and digestive tract, and comparing their modulatory influences on the cardiac neuromuscular system. Immunohistochemistry showed that in the pericardial organ, a neuroendocrine release site, AST-C I and/or III and AST-C II are contained within distinct populations of release terminals. Moreover, AST-C I/III-like immunoreactivity was seen in midgut epithelial endocrine cells and the cardiac ganglion (CG), whereas AST-C II-like immunoreactivity was not seen in these tissues. These data suggest that AST-C I and/or III can modulate the CG both locally and hormonally; AST-C II likely acts on the CG solely as a hormonal modulator. Physiological studies demonstrated that all three AST-C isoforms can exert differential effects, including both increases and decreases, on contraction amplitude and frequency when perfused through the heart. However, in contrast to many state-dependent modulatory changes, the changes in contraction amplitude and frequency elicited by the AST-Cs were not functions of the baseline parameters. The responses to AST-C I and III, neither of which is COOH-terminally amidated, are more similar to one another than they are to the responses elicited by AST-C II, which is COOH-terminally amidated. These results suggest that the three AST-C isoforms are differentially distributed in the lobster nervous system/midgut and can elicit distinct behaviors from the cardiac neuromuscular system, with particular structural features, e.g., COOH-terminal amidation, likely important in determining the effects of the peptides.NEW & NOTEWORTHY Multiple isoforms of many peptides exert similar effects on neural circuits. In this study we show that each of the three isoforms of C-type allatostatin (AST-C) can exert differential effects, including both increases and decreases in contraction amplitude and frequency, on the lobster cardiac neuromuscular system. The distribution of effects elicited by the nonamidated isoforms AST-C I and III are more similar to one another than to the effects of the amidated AST-C II.

Central pattern generators produce rhythmic behaviors independently of sensory input; however, their outputs can be modulated by neuropeptides, thereby allowing for functional flexibility. We investigated the effects of C-type allatostatins (AST-C) on the cardiac ganglion (CG), which is the central pattern generator that controls the heart of the American lobster, Homarus americanus, to identify the biological mechanism underlying the significant variability in individual responses to AST-C. We proposed that the presence of multiple receptors, and thus differential receptor distribution, was at least partly responsible for this observed variability. Using transcriptome mining and PCR-based cloning, we identified four AST-C receptors (ASTCRs) in the CG; we then characterized their cellular localization, binding potential, and functional activation. Only two of the four receptors, ASTCR1 and ASTCR2, were fully functional GPCRs that targeted to the cell surface and were activated by AST-C peptides in our insect cell expression system. All four, however, were amplified from CG cDNAs. Following the confirmation of ASTCR expression, we used physiological and bioinformatic techniques to correlate receptor expression with cardiac responses to AST-C across individuals. Expression of ASTCR1 in the CG showed a negative correlation with increasing contraction amplitude in response to AST-C perfusion through the lobster heart, suggesting that the differential expression of ASTCRs within the CG is partly responsible for the specific physiological response to AST-C exhibited by a given individual lobster.

Central pattern generators (CPGs) are neural networks that produce consistent, rhythmic patterned outputs to reliably activate specific muscles, resulting in repeated and rhythmic movements. The cardiac ganglion (CG) is a CPG in the American lobster, and is responsible for producing a consistent rhythmic heartbeat. Flexibility of rhythmic motor outputs arises from modulation of CPGs, largely by neuropeptides. The lobster heart is modulated by C‐type allatostatins (AST‐C), two of which [pQIRYHQCYFNPISCF, now AST‐C I, and SYWKQCAFNAVSCFamide, now AST‐C II] were previously known; both have been shown to modulate the lobster heart. Using transcriptomes generated from nervous system tissues, we identified a third isoform of AST‐C, GNGDGRLYWRCYFNAVSCF, or AST‐C III, in the lobster. All three AST‐C isoforms consistently lead to decreases in contraction frequency when perfused through an isolated lobster heart; in contrast, all can elicit variable responses in contraction amplitude, with amplitude increasing in some lobsters and decreasing in others. The responses to AST‐C I and AST‐C III are more similar to one another than to AST‐C II in any given lobster.We are currently testing the hypothesis that differences in AST‐C receptor expression among individuals underlie these varying responses. Mining of a Homarus americanus transcriptome has led to the identification of three full‐length and one partial putative AST‐C receptors. We are currently conducting experiments to determine whether and to what extent the four putative AST‐C receptors bind the three AST‐C isoforms. Understanding these receptor‐peptide relationships will allow us to elucidate the role the receptors play in the observed differential response to AST‐C.Support or Funding InformationNSF (IOS‐1353023, IOS‐1354567), NIH (8P20GM103423‐12), Cades Foundation, Doherty Foundation gift to Bowdoin College