Abstract
Escape responses are highly stereotyped behaviors that enable organisms to avoid threats in their environment. To ensure the rapid and robust execution of these behaviors, they are often mediated by dedicated neuronal circuits with fast feed-forward signal propagation. Rectifying electrical synapses, which allow electrical current to preferentially flow only in one direction, are a hallmark of such circuits, and facilitate rapid and stereotyped neuronal signaling for fast, reflexive behaviors. In vitro studies have suggested that it is the heterotypic distribution of the gap junction proteins (called innexins in invertebrates), i.e., possessing different innexins in pre- and postsynaptic neurons, that enables the rectification of the electrical synapse. However, the presence of distinct pre- and postsynaptic gap junction proteins and the functional roles of these proteins have not been established in escape circuits. I am using the tail-flip escape behavior of crayfish, a classical behavioral model for understanding escape responses, to study gap junction proteins. The neuronal circuitry of the crayfish tail-flip behavior has been largely worked out, with specialized giant neurons identified for the two major types of escape modes in the animal – the lateral giant (LG) and medial giant (MG) tail-flip. In both MG and LG escape circuits, rectifying electrical synapses facilitate rapid signal transmission from primary afferents to the
motor neurons. However, the innexin proteins expressed in the crayfish nervous system and contributing to these rectifying synapses are unknown. To address this gap in knowledge, I use the marbled crayfish (Procambarus virginalis), the only crayfish species with identified genome and transcriptome. Employing bioinformatics, I identified five putative innexin genes (named Inx1 - Inx5), four of which were expressed in the nervous system and likely contribute to tail flip escape responses. Four of the five putative innexins (Inx2 – 5) were expressed in the ventral nerve cord and three of them (Inx2, 3 and 5) were also expressed in the brain. To test the contribution of these innexins to the escape behavior, I used RNA interference to reduce innexin expression. This was followed by behavioral assays to test whether MG and LG tail flips were altered by the RNAi treatment. My results indicate that reduction in expression of two of the five identified innexins, i.e., Inx2 and Inx3, using RNAi resulted in a significant delay in the onset of the LG tail-flip. This suggests that these two innexin proteins contribute to the formation of gap junction channels in the LG tail-flip circuit. In contrast, no significant effect was found for the MG tail-flip following the same RNAi approach. From these results, I conclude that there are four innexin proteins that are expressed in marbled crayfish nervous system and are homologousto other invertebrate innexins. Moreover, marbled crayfish innexin 2 and 3 constitute the gap junction channels that form electrical synapses in the LG tail-flip circuit and are important for robust signal transmission.
Keywords: electrical synapses • giant neurons • gap junctions • innexins • escape response •
tail-flip • RNA interference
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