Abstract
Purpose :
Neural networks electrically coupled via connexin 36 (Cx36) gap junctions have been found to be modifiable by the activity of Ca2+/calmodulin-dependent kinase II. Plasticity has been previously shown to depend on activity of NMDA receptors in AII amacrine cells, but it is unclear whether NMDA receptors are the sole source for calcium that may drive plasticity and it is unknown whether calcium-dependent plasticity may be widespread.
Methods :
Retina slice preparations were made from transgenic mice expressing Cx36 fused to GCaMP3 driven by the Cx36 promoter (Cx36-GCaMP). Slices were perfused with oxygenated Ames media and imaged with an EMCCD camera using Superresolution Radial Fluctuations (SRRF) employing 30 frames per SRRF image with an effective time resolution of 1.03 sec/image. Slices were transiently puffed with glutamate + glycine, AMPA, or NMDA + glycine in the presence or absence of CPP, CdCl2, or nifedipine. GCaMP fluorescence baselines were fit with an exponential decay model for each individual gap junction. Responses were considered positive by having at least two points of DF measurements in the responding time window greater than two SD of the baseline measurements. Response amplitude was assessed by area under the curve (AUC) within the responding window.
Results :
Cx36-GCaMP gap junctions responded to puffs of glutamate + glycine with robust, transient increases in fluorescence; approximately 80% of gap junctions displayed a positive response. Mean response amplitude was significantly reduced 85% by NMDA receptor antagonist CPP. Response amplitude was decreased to a smaller extent by CdCl2, a generalized inhibitor of calcium channels. NMDA + glycine induced very small responses, which nonetheless were partially reduced by CdCl2. AMPA puffs also produced robust responses which were partially reduced by L-type calcium channel blocker nifedipine.
Conclusions :
Glutamate drives transient Ca2+ increases in the vicinity of Cx36 gap junctions, in keeping with activity-dependent plasticity mechanisms. Both NMDA and AMPA receptors contribute to local Ca2+ dynamics, and voltage-dependent calcium channels are an important source of the local Ca2+.
This is a 2020 ARVO Annual Meeting abstract.