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Changes in ambient temperature affect all biological processes. However, these effects are process specific and often vary non-linearly. It is thus a non-trivial problem for neuronal circuits to maintain coordinated, functional output across a range of temperatures. The cardiac nervous systems in two species of decapod crustaceans, Homarus americanus and Cancer borealis , can maintain function across a wide but physiologically relevant temperature range. However, the processes that underlie temperature resilience in neuronal circuits and muscle systems are not fully understood. Here, we demonstrate that the non-isolated cardiac nervous system (i.e., the whole heart: neurons, effector organs, intrinsic feedback systems) in the American lobster, H. americanus , is more sensitive to warm temperatures than the isolated cardiac ganglion (CG) that controls the heartbeat. This was surprising as modulatory processes known to stabilize the output from the CG are absent when the ganglion is isolated. One source of inhibitory feedback in the intact cardiac neuromuscular system is nitric oxide (NO), which is released in response to heart contractions. We hypothesized that the greater temperature tolerance observed in the isolated CG is due to the absence of NO feedback. Here, we demonstrate that applying an NO donor to the isolated CG reduces its temperature tolerance. Similarly, we show that the NO synthase inhibitor L-nitroarginine (LNA) increases the temperature tolerance of the non-isolated nervous system. This is sufficient to explain differences in temperature tolerance between the isolated CG and the whole heart. However, in an intact lobster, the heart and CG are modulated by an array of endogenous peptides and hormones, many of which are positive regulators of the heartbeat. Many studies have demonstrated that excitatory modulators increase temperature resilience. However, this neuromuscular system is regulated by both excitatory and inhibitory peptide modulators. Perfusing SGRNFLRFamide, a FLRFamide-like peptide, through the heart increases the non-isolated nervous system’s tolerance to high temperatures. In contrast, perfusing myosuppressin, a peptide that negatively regulates the heartbeat frequency, decreases the temperature tolerance. Our data suggest that, in this nervous system, positive regulators of neural output increase temperature tolerance of the neuromuscular system, while modulators that decrease neural output decrease temperature tolerance. 

Post-translational modifications (PTMs) diversify peptide structure and allow for greater flexibility within signaling networks. The cardiac neuromuscular system of the American lobster, Homarus americanus, is made up of a central pattern generator, the cardiac ganglion (CG), and peripheral cardiac muscle. Together, these components produce flexible output in response to peptidergic modulation. Here, we examined the role of PTMs in determining the effects of a cardioactive neuropeptide, myosuppressin (pQDLDHVFLRFamide), on the whole heart, the neuromuscular junction/muscle, the isolated CG, and the neurons of the CG. Mature myosuppressin and noncyclized myosuppressin (QDLDHVFLRFamide) elicited similar and significant changes in whole heart contraction amplitude and frequency, stimulated muscle contraction amplitude and the bursting pattern of the intact and ligatured neurons of the ganglion. In the whole heart, nonamidated myosuppressin (pQDLDHVFLRFG) elicited only a small decrease in frequency and amplitude. In the absence of motor neuron input, nonamidated myosuppressin did not cause any significant changes in the amplitude of stimulated contractions. In the intact CG, nonamidated myosuppressin elicited a small but significant decrease in burst duration. Further analysis revealed a correlation between the extent of modulation elicited by nonamidated myosuppressin in the whole heart and the isolated, intact CG. When the neurons of the CG were physically decoupled, nonamidated myosuppressin elicited highly variable responses. Taken together, these data suggest that amidation, but not cyclization, is critical in enabling this peptide to exert its effects on the cardiac neuromuscular system. NEW & NOTEWORTHY Myosuppressin (pQDLDHVFLRFamide), a well-characterized crustacean neuropeptide, and its noncyclized (QDLDHVFLRFamide) and nonamidated (pQDLDHVFLRFG) isoforms alter the output of the cardiac neuromuscular system of the American lobster, Homarus americanus. Mature myosuppressin and noncyclized myosuppressin elicited similar and significant changes across all levels of the isolated system, whereas responses to nonamidated myosuppressin were significantly different from other isoforms and were highly variable. These data support the diversity of peptide action as a function of peptide structure. 

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.