Our work focuses on specialized synapses in the inner retina. We have expanded our study of inhibitory synaptic connections made by amacrine cells within the inner retina, to understand how feedforward and feedback inhibition contributes to signal processing in this network. We previously discovered that A17 amacrine cells provide rapid GABAergic feedback to rod bipolar cell ribbon synapses via a release process that is independent of membrane depolarization or voltage-gated calcium channels (Chavez, et al., 2006). This rapid feedback may be essential to prevent the rapid depletion of readily-releasable vesicles from the rod bipolar cell synaptic terminal (Singer and Diamond, 2006). Our most recent work indicates that reciprocal feedback from A17s extends the range over which these synapses encode luminance and compute contrast (manuscript nearing submission). Feedback from other amacrine cells weakly modulates the synaptic gain but does not change the operating range. We also find that A17-mediated feedback inhibition enhances the gain of synaptic responses in the rod pathway to the absorption of single photons (manuscript nearing submision). Publication of this work has been delayed by the difficulty of confirming the subsynaptic localization of different GABA receptors (on the presynaptic rod bipolar cell terminal) and AMPA receptors and BK channels on the A17 synaptic varicosity. Those data have been collected and are being incorporated into a greatly improved manuscript. Our understanding of ribbon synaptic physiology in bipolar cells is limited to rod bipolar cells. We don't know as much about synaptic transmission from cone bipolar cells, because it is very difficult to obtain synaptically coupled cone bipolar - ganglion cell pairs. We have crossed mouse lines with genetically encoded markers identifying specific types of bipolar and ganglion cells that are very likely to be connected. Preliminary experiments suggest that this approach will enable us to study synaptic release consistently from single cone bipolar types, an essential feature to avoid likely variations between cell types. We also plan to compare ON and OFF bipolar cell inputs to the same ganglion cell type to compare the dynamics of transmission in these parallel channels. We are also working to understand how NMDA receptors contribute to the developmental refinement of ganglion cell dendrites, with are precisely stratified within the inner plexiform layer, the synaptic neuropil of the inner retina. We have successfully combined multiple genetic tools to acquire a mouse in which the NMDA receptors can be knocked out in a single type of ganglion cell at any time during development. This project has taken a long time to prepare, but experiments now are providing exciting new insights in the role of NMDA receptors in retinal development. We find that ganglion cells do not require NMDA receptors to find their way to the proper region of the inner plexiform layer, but finer stratification, essential for precise connectivity, is compromised. Finally, we are extending our electron microscopy studies, in collaboration with Tom Reese and Richard Leapman, to explore the detailed ultrastructure of synaptic ribbons in photoreceptors and rod bipolar cells. So far, EM tomography enables us to detect protein filaments that tether synaptic vesicles to the ribbon and the presynaptic membrane. This approach may enable us to discern morphologically, for the first time, docked and primed synaptic vesicles. We have obtained detailed reconstructions of entire, intact ribbons (Graydon, et al., 2014) and are now working to obtain higher resolution images to examine quantitatively the tethers that connect synaptic vesicles to the ribbon and to the presynaptic membrane. In addition, we are continuing our efforts to obtain high-resolution images of ribbon synapses using high-pressure freezing EM techniques. This approach has been used to great effect in cultured mammalian neurons, but it has proved quite difficult in intact tissue. A great deal of effort has finally enabled us to obtain high-quality freezing the in the inner plexiform layer of intact retina, which will enable us to address numerous questions of synaptic function.
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