This study is directed at understanding the morphological and physiological organization of the inner plexiform layer of the mammalian retina, and focuses on examining the synaptology related to physiologically identified ganglion cells in the rabbit retina. The present study intends to characterize the transmitter and receptors in the context of the microcircuits responsible for a specific physiological response. Direct information pertaining to the neurochemical characteristics of the synaptic profiles will be obtained by combining intracellularly HRP filled cell with electron microscopic post-embedding immunogold-cytochemistry. An analysis of transmitters and their receptor subtypes involved in microcircuits impinging on the direction selective (DS) and alpha ganglion cells will then be made. The DS and alpha ganglion cells will be injected with HRP, reacted to recover the stained cell, prepared for electron microscopy and embedded in plastic. The stained cell will be serially thin sectioned and two different antibodies will be used to label alternate sections from the continuous series. The GABA and ChAT antibodies will be used with the DS cell; GABA and glycine antibodies will be used on the alpha cell. The HRP stained dendrites with their associated synaptic profiles will be examined with electron microscopy, photographed, and printed to form a montage encompassing all the processes related to the HRP stained dendrite. The montage for each section containing its synaptic profiles will be traced into the computer, and the GABA, ChAT and glycine immunoreactive processes will be color coded. By 3-dimensionally reconstructing each synaptic profile, the serial sections containing immunoreactive positive processes will be superimposed, which allows the double labeled processes to be identified and demonstrate the synaptic interactions between differently labeled processes. In addition, the antibodies to the a- and b-subunits for the GABAA receptor and the a- and b-subunits for the acetylcholine receptor will be used to localize and determine the specific synaptology involved in the synaptic interactions between the cholinergic, starburst amacrine cell and the DS ganglion cell as well as between two cholinergic cells. Whole-cell patch-clamp recordings will be made from cone bipolar cells and alpha ganglion cells to correlate the immunocytochemical results to the presence or absence of GABA- and glycine-gated currents on these cells.
Showing the most recent 10 out of 27 publications
