Abstract
Purpose :
In the retina, the slc32a1 gene or the vesicular inhibitory amino acid transporter (VIAAT) has been of great interest and is expressed at synaptic terminals of horizontal cells in the outer retina and amacrine cells in the inner retina; with GABA having a significant role in both feedback and feedforward signaling in the outer retina driven by horizontal cells, and GABAergic and glycinergic feedback signaling in the inner retina among amacrine cells. Zebrafish are a widely used model for retinal studies and have undergone a gene duplication event that has resulted in the duplication of ~ 50% of all genes. This duplication event has permitted the partitioning of gene function or expression patterns. The goal of this study is to explore the impact of this duplication event on the expression profile of VIAAT paralogues (slc32a1 and si:dkey-126h10.1) in the adult zebrafish retina.
Methods :
Adult zebrafish brains or retinas were used to extract RNA, and generate probes for in situ hybridization as described previously (Grillo et al., Purinergic Signal. 2019). Indirect immunofluorescence and confocal microscopy with antibodies targeted to VIAAT, GABA, glycine, and glutamic acid decarboxylase (GAD) were used to characterize the key components of the inhibitory signaling system in the inner and outer zebrafish retina. Ensembl and BLAST were used to identify slc32a1 members. All sequences were compiled and aligned using BLAST, and evolutionary analysis and the phylogenetic tree creation were completed using MEGA (ver. 11), with the human protein sequence serving as the reference in BLAST.
Results :
Unexpectedly, we found that the expression slc32a1, the 1st VIAAT homolog was expressed exclusively in the inner retina at amacrine cells, and si:dkey-126h10.1, the 2nd VIAAT homolog was restricted to horizontal cells in the outer retina. This novel observation may relate to the developmental changes that likely occurred during the gene duplication event in teleosts through the segregation of inner and outer retinal circuitry.
Conclusions :
This will also serve as an important tool for the development of gene targeted approaches involving VIAAT in the zebrafish retina. Our data suggest that these VIAAT expression patterns may provide the specificity necessary to dissect the cell type specific roles for VIAAT inhibitory neurotransmission in the outer and inner retina.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.