Exo- and Endocytosis at a Retinal Inhibitory Synapse during Crossover Inhibition

Abstract

The AII amacrine cell (AII) receives chemical and electrical synapses from rod bipolar cells (BC) and ON cone BCs. AIIs form glycinergic synapses at lobular varicosities near the soma, providing inhibitory output to the OFF pathway; this is called crossover inhibition. We performed whole-cell voltage clamp recordings and used the “sine + DC” method to make measurements of membrane capacitance (Cm) with high temporal resolution. With this approach we were able to investigate the cell membrane dynamics of this inhibitory synapse. AIIs were held at −80 mV and L-type Ca2+ currents were evoked with a 100 ms, 70 mV depolarizing step, triggering Ca2+ dependent vesicle fusion (exocytosis). Our observed Cm jumps ranged from 14.81-152.3 fF, corresponding to the fusion of 370-3800 vesicles (n=38). Presynaptic potentiation was also seen at this synapse after 4 minutes of treatment of 1 mM cAMP (n=22). Following a 50 ms depolarizing step, we saw a gradual decay in Cm, indicative of vesicular membrane retrieval (endocytosis). Tau ranged from 3.90 to 7.11 s (n=10), suggesting that AIIs are suited for fast membrane recovery. We propose that the variability in membrane dynamics of these interneurons is due to their diverse cell-to-cell lobular morphologies. In order to test this hypothesis, we performed Cm recordings in conjunction with wide-field optical sectioning of dye filled AIIs. We were able to compare Cm jumps to 3D reconstructions of the lobular varicosities from individual AIIs. Our results show that the magnitude of glycine release positively correlates to the number and overall size of these lobules (n=10). These results suggest that AIIs with larger lobules have more active zones and make more synaptic connections with OFF BCs and/or ganglion cells.