Expression of GABA_A receptor subunit mRNAs in horizontal cells of mouse retina

Abstract

Purpose : Horizontal cells are involved in feedback inhibition to photoreceptor terminals in mammalian retina, in which TPMPA-sensitive, rho-subunit-containing GABA_A receptors (GABA_AR) play a critical role. Horizontal cells also exhibit non-TPMPA-sensitive GABA evoked currents. The purpose was to identify the GABA_A receptor (GABAR) subunits, typically pentameric ion channels, expressed in mouse horizontal cells to ascertain whether and which other GABARs may be present.

Methods : The highly sensitive HiPlex RNAscope (ACD Bio) fluorescent in situ hybridization (FISH) assay (ACD Bio) was used to visualize the expression of GABA_AR message and horizontal cell markers in fixed cryostat sections of mouse retina. In some RNAscope experiments, the retina was also immunostained for calbindin. The images were collected on a Zeiss LSM 880 confocal microscope with Airyscan using a C-Apochromat 40x objective.

Results : Expression of calbindin (Calb1), synaptotagmin-2 (syt2) and Slc32a1 (VGAT), 3 markers of horizontal cells, identified large somata in the outer retina, with calbindin mRNA being the most abundant, delineating horizontal cell somata. Using the Calb1 probe, the GABA_AR subunit-specific probes were localized to cell bodies of horizontal cells. We observed fluorescent puncta corresponding to GABAR alpha subunits (α1 and α6), beta subunits (β1 and β3), delta (δ), and gamma2 (γ2) subunits, in addition to rho subunits (ρ1, ρ2 and ρ3), co-localized with the Calb1-identified horizontal cells. The α6 and rho subunits (ρ1-3) localizations were confirmed with calbindin immunolabeling. In addition, fine, less intense puncta for most of the subunits (α1, α6, β1, β3, δ, ρ1, ρ2, ρ3) were observed in the outer plexiform layer (OPL).

Conclusions : The expression of the three rho subunits likely comprise the GABAR rho subunit-containing receptors localized at horizontal cell endings. The presence of α6 and δ, components of extrasynaptic GABARs in other systems, suggest that other tonic GABARs may be used by these cells. The localization of many GABAR subunit mRNAs to the OPL suggests local mRNA translation occurs in dendrites and axons of outer retinal neurons. The subcellular localization and subunit composition of the GABAR subtypes remain for future research.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.