21 August 2020

Benson 2020

Illinois State UNiversity seal
Benson AM. 2020. Identification of innexins contributing to giant-fiber escape responses in marbled crayfish. Master's thesis, School of Biological Sciences, Illinois State University. Stein W, Vidal-Gadea A, advisors. http://doi.org/10.30707/ETD2020.Benson.A

Abstract

Gap junctions form intercellular pores that coordinate the flow of electrical signals between adjacent cells in the nervous system. While the physiology of electrical synapses has been investigated in sophisticated detail, the molecular underpinnings of electrical signal spread between neurons are not well understood. Even in the crayfish tail flip escape circuit, where electrical synapses have been studied for more than six decades, the gap junction proteins underlying electrical synaptic transmission are unknown. Invertebrate gap junctions are assembled from a diverse family of proteins called innexins (inx), and previous studies have suggested that in each species multiple innexins can contribute to electrical signal spread between cells. In this study, I used the genome and transcriptome assembly of the marbled crayfish, Procambarus virginalis, to identify which innexins are present and expressed in crayfish, and which contribute to the giant fiber tail flip escape response.

My bioinformatics analyses identified 8 putative innexin genes (termed inx1- inx8), only five of which were present in the transcriptome, suggesting that inx6-8 are not necessary for the tail flip. A conserved domain search of inx1 - 5 revealed that only inx1 - 3 contained the sequence signature common to innexins, indicating that inx4 and 5 may not contribute to the functioning of electrical synapses. RNA isolation from the ventral nerve cord (VNC) and brain, which contain distinct giant neurons that mediate the two major crayfish tail-flip responses, further suggested that inx1 - 3 could contribute to the tail flip: the brain expressed two innexins (inx2- 3), whereas the ventral nerve cord expressed three innexins (inx1-3). Basic Local Alignment Search Tool (BLAST) comparisons additionally revealed that inx1 and 2 were homologous to two innexins previously identified to contribute to giant fiber escape responses in insects. To test whether inx1 or 2 contribute to giant fiber tail flips in crayfish, I reduced inx1 and 2 gene expression through RNA interference (RNAi) and measured the behavioral consequences of this diminishment on the tail flip escape response. To elicit RNAi, I created innexin-specific double-stranded RNA (dsRNA) and verified the presence of intact innexin-½ dsRNA at the expected product size of 547 bp.

Animals receiving treatment were injected with 3 µg dsRNA/ g of body weight. A comparison of innexin expression levels between untreated (n = 2), control dsRNA (n = 1), and RNAi (n = 10) treatment groups using quantitative real-time PCR (qPCR) revealed that innexin expression levels diminished two days post-treatment. Behavioral measurements showed that the response latency onset of the tail flip response correlated with innexin expression levels (n = 6). A linear regression revealed a significant correlation between innexin expression and differences in response latency onset for abdominal flexion (p < 0.05, R2 = 0.77) and subsequent extension (p < 0.05, R2 = 0.82). In contrast, the tail flip strength was unaffected by the RNAi treatment (Fig. 20; p > 0.05, R2 = 0.27). Thus, at least one of the two innexins contributes to electrical synaptic transmission in the crayfish tail flip circuit.

Keywords: None provided.

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