Abstract
Purpose :
AII amacrine cells (AIIs) receive excitation at their distal arboreal dendrites from rod bipolar cell glutamatergic terminals during night and from electrical synapses with ON cone bipolar cells during daytime. This excitation is translated by AIIs into graded synaptic release of inhibitory glycine from their proximal dendritic lobules onto OFF cone bipolar cell (OFF-BC) terminals, termed crossover inhibition. AIIs operate across a wide range of light intensities from night to day. This is partially attributed to the functional modulation of homologous AII-AII gap junction coupling— dependent on the second messenger cAMP. Since synaptic potentiation is one of the many cellular processes that are mediated by cAMP, we examined the role of cAMP at the AII to OFF-BC inhibitory synapse.
Methods :
Presynaptic capacitance (Cm) recordings using the “sine +DC” method in whole-cell voltage clamp were performed to measure net vesicle fusion (exocytosis) in AIIs. Exocytosis was evoked with a 70 mV, 100 ms depolarizing pulse (Vhold = -80 mV). In between the step depolarization two voltage-clamped sine waves (2kHz; 30mV peak to peak) were applied to calculate time-resolved Cm. Whole-cell inhibitory postsynaptic currents were recorded in OFF-BCs (Vhold= -70mV). Electrophysiological recordings were performed at 33°C using Cs-based internal solutions. Results displayed as mean ± SEM.
Results :
Cm jumps were recorded with 1 mM cAMP in the patch pipette solution. Unlike our controls, Cm jumps increased on average after the dialysis of cAMP for 4 min (1 min: 47.3 ± 4.9 fF and at 4 min: 73.9 ± 7.53 fF; n=20). Potentiation was blocked using 1µM HJC 0350, a selective EPAC antagonist. Cm jumps were enhanced by increasing intracellular cAMP with 25 µM forskolin. Forskolin also increased the frequency of glycinergic sIPSCs in OFF-BCs.
Conclusions :
Our findings suggest that cAMP performs dual roles in the AII through different molecular pathways. Previous reports show that a cAMP/PKA pathway regulates coupling in the distal dendritic compartment; however, our study indicates that cAMP also interacts with EPAC in the proximal dendritic compartment (the lobular appendages). This molecular mechanism may potentiate glycine release from AIIs by modulating proteins involved with vesicle fusion (e.g. SNARE, RIMs, or MUNC13), and may provide a way for this synapse to enhance the tonic inhibition of OFF-BCs across a broader range of light intensities.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.