A phosphomimetic Cx36 mutant mouse displays excess neuronal coupling and photopic visual deficits

Abstract

Purpose : Electrical synapses made of Connexin 36 (Cx36) display striking functional plasticity in retinal neurons during light adaptation to optimize neural network function. Coupling is dramatically reduced in photoreceptor and AII amacrine cell networks in daytime, light-adapted conditions, and changes in other cell types as well. It is not possible to evaluate the necessity for such plasticity with a Cx36 knockout model, so we developed a mutant mouse in which Cx36 is locked in an open state.

Methods : Functional regulation of Cx36 mutants was assessed by tracer coupling in HeLa cells with activation or inhibition of PKA signaling. A conditional knock-in phosphomimetic mutant Cx36 mouse in C57Bl6 background was developed through Cyagen. This mouse, termed Cx36-DEDD, was crossed with Six3-Cre transgenic mice. Retinal morphology and Cx36 expression were assessed by immunofluorescence. Coupling of AII amacrine cells was assessed by Neurobiotin microinjection. Contrast sensitivity and visual acuity were assessed through optokinetic response.

Results : We systematically replaced each phosphorylatable serine or threonine residue in mouse Cx36 that is known to regulate coupling with either aspartate (D) or glutamate (E). In general, aspartate residues performed better than glutamate to support functional coupling in HeLa cells, and it was necessary to replace all four regulatory residues to obtain a mutant that displayed enhanced coupling in control conditions and did not uncouple with PKA activation. The saturated mutant, Cx36-DEDD, formed gap junctions with a normal distribution in knock-in mouse retina after Six3-Cre-mediated recombination, even in the homozygous condition. Cells expressing the mutant were marked by expression of tdTomato via an IRES sequence following the mutant exon. AII amacrine cells in the homozygous mutant, but not in the heterozygous mutant, were significantly more coupled than wild-type. Homozygous mutant animals displayed reduced photopic visual acuity compared to wild-type.

Conclusions : We have developed a conditional knock-in phosphomimetic Cx36 mouse that permits study of the role played by electrical synapse plasticity in neural functions. Expression of Cx36-DEDD widely in the retina led to excessive coupling in the daytime, as predicted, and compromised photopic visual acuity.

This is a 2020 ARVO Annual Meeting abstract.

 

AII amacrine cell tracer coupling in Cx36-DEDD heterozygote (A) and homozygote (B).