We are using 3D electron microscopic techniques to examine membrane fine structure in the vicinity of nicotinic receptor clusters previously described as being """"""""perisynaptic"""""""" on chick ciliary ganglion neurons. The receptors are composed of alpha 7 subunits and bind alpha-bungarotoxin with high affinity. The hypothesis being tested is whether the receptor clusters may in large part represent receptors distributed over pseudodendrites compressed against the cell surface in patches. Neurons both freshly dissociated and in whole ganglia will be labeled with either HRP conjugated or biotinylated alpha-bungarotoxin and either examined straightaway with high voltage EM or will be examined first by confocal fluorescence microscopy to locate receptor clusters and them examined by EM to identify related fine structure. The biotinylated bungarotoxin will be localized by 10 nm colloidal gold. Work on this project is underway. Several thick sections of HRP-labeled ciliary ganglion cells have been examined which indicated that these neurons possess somatic spines that are heavily labeled with bungarotoxin. The pattern of bungarotoxin binding sites is being examined further using electron tomography and colloidal gold labeled specimens. In the tomographic reconstructions, we can easily resolve the complex tangle of somatic spines as well as quantify the labeling over spiny and non-spiny regions of membrane.
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