Characterization of the Gap Junction Gene Innexin in Lymnaea stagnalis

Connections between neurons called synapses are key components underlying the basic functioning of the nervous system. From simple reflexes to high cognitive functions, all nervous system operations rely on two main forms of synaptic communication to efficiently transmit signals: chemical (transmitter‐mediated) and electrical (gap junction protein‐mediated). Emerging research has proved the widespread expression of gap junction (GJ) channels in many species including humans and in a variety of brain regions, and recent studies have indicated that GJs play vital roles in the early development of brains; yet, little is known about the regulatory factors (both intrinsic and extrinsic) and mechanisms that control the development of electrical synapses. The current lack of fundamental knowledge in the area of electrical synaptogenesis and regulation is mainly attributed to the extreme complexity of the mammalian brain which does not permit long‐term, real‐time neuronal recordings throughout electrical synapse formation. Therefore, we opt to use an in vitro experimental system, the mollusc Lymnaea stagnalis , in which physically large, well‐defined neuronal networks and a published genome allow unique opportunities to study cell‐specific expression of genes throughout electrical synapse formation in the developing brain. The objective of this study was to characterize the expression, localization, and regulation of the GJ‐encoding gene innexin in L. stagnalis during development. To do this, the central nervous system (CNS) of L. stagnalis was dissected and underwent either subsequent RNA extraction (for cloning) or single‐cell extraction. Innexin was cloned utilizing degenerate primers and 3′ and 5′ rapid amplification of cDNA ends (RACE) systems, and, from the innexin sequence information, primers were designed for RT‐ and qPCR. The innexin cloning experiments revealed 10 isoforms of the gene in L. stagnalis , all with conserved transmembrane domain regions. To elucidate the tissue‐ and cell‐specific expression of innexin isoforms, RT‐PCR was performed on various L. stagnalis organs, and qPCR was performed on cells in the CNS known to form electrical synapses. Innexin isoforms are expressed ubiquitously throughout L. stagnalis but vary between individuals and across developmental time. In situ hybridization also demonstrated variable localization of one innexin isoform across the CNS, with only some electrical synapse‐forming cells localizing the isoform mRNA on the plasma membrane and others not expressing the mRNA at all. Extrinsic cellular molecules called trophic factors were found to regulate electrical synapse formation and innexin expression in vitro . Overall, the GJ gene innexin has variable expression and distribution throughout the life of L. stagnalis , indicating the critical role the gene plays in the proper development of invertebrates and, by conserved functional homology, vertebrates. Support or Funding Information This project was supported by the Saint Louis University Start‐up Fund, the President Research Fund, the Beaumont Faculty Development Fund, and Spark Microgrant awarded to Dr. Xu. This project was also supported by the Sigma Xi Grant‐In‐Aid of Research awarded to Ms. Brittany Mersman. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .