Roles of syntaxin3 in the inner retina

Abstract

Purpose : Syntaxin3B (STX3) is a Ca²⁺-dependent phospho-protein that has been ascribed multiple roles in the mammalian retina. To elucidate the roles of STX3 in the inner retina, we monitored neurotransmitter release at rod bipolar cell (RBC) synapses in STX3-deficient RBCs and under conditions that perturb STX3 phosphorylation.

Methods : A RBC-specific knock-out mouse (rbSTX3KO) was created by crossing our Cre-dependent STX3 knockout mouse (Sanchez et al 2018) with a Pcp2-Cre mouse (Zhang et al 2005). Spatial acuity was measured using the optokinetic reflex. Histological approaches were used to assess retinal integrity. Mini-like EPSCs and light-evoked EPSCs were recorded from AII amacrine cells (AIIs) in retinal slices using electrophysiological approaches. To study exocytosis from a single RBC, stimulus-evoked changes in membrane capacitance were monitored. Results from adult rbSTX3KO mice were compared to age-matched controls.

Results : The retinae of rbSTX3KO mice were grossly normal except for the lack of STX3-immunoreactivity in RBC synaptic terminals. The spatial acuity of rbSTX3KO mice was normal under photopic conditions but significantly impaired under scotopic conditions (control: 0.31±0.02 c/d; rbSTX3KO: 0.12±0.07 c/d; n=4,6; p=0.0138), consistent with a selective defect in rod pathway vision. Light-evoked EPSCs in AIIs were absent in rbSTX3KO slices, but some mini-like EPSCs of normal amplitude were observed. In controls, the amplitude distribution of the inter-event interval (IEI) of mini-like events showed discrete peaks at ≈25ms and ≈80ms with virtually no values >200ms (n=17,476events). In rbSTX3KO AIIs, the first peak was shifted to ≈90ms and numerous IEIs were observed in the 200-500ms range (n=6,888events). Whether these minis arise from a few RBCs that still express STX3 or from an alternate fusion pathway is not known. At the single cell level, total exocytosis evoked by a stimulus that triggered multiple bouts of release was reduced 10-fold when STX3 phosphorylation was inhibited (control: 310±65fF; with inhibitor: 27±13fF; n=11,6; p=0.0013). There was no effect on I_Ca.

Conclusions : Our results demonstrate that STX3 is required for RBC to AII synaptic transmission and rod pathway vision. Inhibition of STX3 phosphorylation blocked the recruitment and/or fusion of newcomer vesicles, but not the fusion of those vesicles already in the releasable pool. Thus, a regulatory role for STX3 upstream of the final fusion step is also proposed.

This is a 2020 ARVO Annual Meeting abstract.