The overall objective of this research is to understand the neuronal circuitry that processes information through the inner plexiform layer (IPL) of the mammalian retina. Because our knowledge of cone bipolar cell connectivity is insufficient, as is the knowledge of rabbit ganglion cell synaptology, the experimental design is focused on specific cone bipolar cell circuits, the excitatory input they receive from cone pedicles and the excitatory input they exert onto ganglion cell dendrites Therefore, our network analysis involves exploiting the cytoarchitectural organization of the retina, which is the keystone to understanding retinal function by examining the connectivity of identified cone bipolar cell axon terminals that excite ON-OFF directional selective (DS) and OFF-alpha ganglion cells. We have described a lateral feed-forward inhibitory microcircuit involving two different bipolar cells; the dyad of one bipolar cell directly excites the DS cell, while the dyad of the other excites a starburst amacrine cell which feeds forward to synapse with the DS cell. lmmunolabeling and intracellular staining, in combination with confocal microscopy, are used to identify bipolar cells synapsing with the DS cell in order to focus the electron microscopy (EM) analysis. After the bipolar cells are identified, the axon terminals of these intracellular!y stained cells are serially reconstructed with EM to examine their synaptology; two constrasting electron dense markers, pro embedding gold-substituted silver intensified peroxidase and DAB/peroxidase, are used to establish pro- and postsynaptic interactions between two cells. Combined microwave and chemical fixation and/or rapid-freezing are used with various pro- and postembedding EM immunolabeling techniques involving DAB/peroxidase and immunogold to identify the GABA and cholinergic synapses and determine how these receptors are used by the starburst amacrine cell to communicate with the DS cell and each other. Whole cell recordings are used to characterize the ionotropic glutamate receptors on the dendrites of OFF-bipolar cell(s) synapsing with OFF-alpha ganglion cells and to determine whether the bipolar cell(s) primarily uses the same glutamate receptor to synapse with the OFF-ganglion cell. Once the functional glutamate receptors are characterized, double immunolabeling is used to confirm their presence on the dendrites of the ganglion cell where it receives excitation from dyad synapses.
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