Abstract
Purpose :
Intrinsic membrane property (IMP) has been successfully used to categorize the Tbr2-expressing retinal neurons such as intrinsically photosensitive retinal ganglion cells. However, the expression of voltage gated cation channels (VGCC) in the retina has not been systematically examined. To explore its potential in classifying additional retinal cells, we characterized the retinal expression patterns of all VGCC genes in adult mouse retinas.
Methods :
NCBI database search identified candidate VGCC genes in the mouse (Mus musculus) genome. Retinas of adult C57BL/6J mice of both genders were isolated and total RNA extracted by using the Trizol reagent. First strand cDNA were synthesized by using the Applied Biosystems HC RT kit. Two sets of gene-specific primers were designed to assess which set would better minimize primer dimer formation. One primer set was designed by hand based on several considerations including primer length, GC content, and similarity to sequence of related genes in the same family. The second primer set was generated by using the NCBI Primer-BLAST tool. Both sets of primers were synthesized and tested against primer dimer formation using qPCR, and fourteen primer sets were subsequently redesigned. Conventional and quantitative RT-PCR was used to test expression levels of each VGCC gene with reference to the housekeeping GAPDH gene.
Results :
A total of 143 candidate VGCC genes were screened. Currently, the expression of 44 VGCC genes in the adult mouse retina has been confirmed.
Conclusions :
This study shows that not all, but rather a select set of VGCC genes are expressed in adult mouse retinas. This suggests that VGCC expression patterns may be utilized as markers for classifying inner retinal neurons. Additionally, the primer sequences used in this survey can be further developed to help distinguish randomly sampled retinal neurons based on unique resting membrane potential profiles, analyze variability of VGCC expression in cells of the same neuronal type, and ultimately allow for further exploration of unique cell functions.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.