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Date: 2020-01-01

Creator: Audrey J. Muscato

Access: Open access

Central pattern generators (CPGs) produce patterned outputs independent of sensory input. The cardiac neuromuscular system of the American lobster (Homarus americanus) is driven by a CPG called the cardiac ganglion (CG), which is composed of nine neurons, making it a model system of study. Modulation of CPGs allows for functional flexibility. One neuropeptide family that modulates the CG is C-type allatostatin (AST-C I-III). Previous research has shown variation in the responses of the CG across the three isoforms and among individuals. First, we investigated why AST-C I and III elicit responses that are more similar to each other than they are to the responses elicited by AST-C II. We hypothesized that an amino acid difference in the conserved sequence was responsible for the observed variation in responses. We synthesized isoforms of AST-C that replaced the endogenous amino acid and recorded responses to these isoforms. The identity of one particular amino acid in the conserved sequence seems to be responsible for variations in responses in frequency. Next, we focused on variation among individuals in their responses to AST-C I and III. We hypothesized that the mechanism behind this individual variation is differential expression of AST-C receptors and/or their downstream targets. We recorded physiological responses of the cardiac system to AST-C and then sequenced CG RNA from the same lobsters. Differential expression of one of the AST-C receptors and a number of downstream factors is correlated with physiological response. These findings inspire further experimentation investigating molt cycle as the underlying cause.

Date: 2021-08-02

Creator: Audrey J. Muscato, Patrick Walsh, Sovannarath Pong, Alixander Pupo, Roni J., Gross, Andrew E. Christie, J. Joe Hull, Patsy S. Dickinson

Access: Open access

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.