This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Nicotinic acetylcholine receptors (nAChRs) are pentameric membrane proteins that function as cation selective, ligand-gated ion channels and are widely distributed throughout the vertebrate nervous system. One of the most abundant nAChRs is a species that contains the a7 gene product (a7-AChRs), binds the snake venom a-bungarotoxin, and rapidly desensitizes. These receptors have been shown to function at presynaptic sites to modulate transmitter release1, as well as on postsynaptic cells where they mediate transmission. Interestingly, these receptors have an exceptionally high relative permeability to calcium. In the chick ciliary ganglion, the a7-AChRs play a prominent role, by generating large synaptic currents, but the receptors appear to be excluded from postsynaptic densities on the cell. Immunohistochemical studies have shown that the receptors form large clusters on the surface of the ciliary ganglion neurons. We have recently shown that the a7-containing receptors are concentrated on mats of somatic spines in close proximity to putative sites of presynaptic transmitter release. Intermediate voltage electron microscopy (IVEM) together with tomographic reconstruction has permitted a three-dimensional analysis of the finger-like projections emanating from the cell bodies and the relationship of AchRs to the spines and pre-synaptic calyx. These projections were identified as somatic spines based on their morphology, cytoskeletal content and proximity to presynaptic elements. Both in situ and after ganglionic dissociation the spines were grouped on the cell surface and tightly folded into mats. Quantitative estimations suggested that these mats of spines effectively doubled the surface area of the neuron. Immunogold labeling of receptors containing a7 subunits showed them to be preferentially concentrated on the somatic spines. When combined with electron tomography, we were able to visualize the 3D distribution of gold particles over the surface of the neuron. Serial sections through spine mats in vivo revealed that less than half of the somatic spines have postsynaptic densities, however densities were often present on the soma near spines. High resolution tomographic reconstructions of the somatic spines in intact ganglia showed that the synaptic vesicle-filled projections of the presynaptic membrane were interdigitated among the spines. Moreover, the synaptic vesicles often abutted the membrane as though docked for release even when no obvious postsynaptic densities were juxtaposed on the spine. This work was recently published in the Journal of Neuroscience (Shoop et al., J. Neurosci., 19: 692-704, 1999; other refs here). Work is continuing to determine mechanisms of spine growth and retraction and to determine calcium dynamics in the spine mats using the high-speed multiphoton microscope recently developed at NCMIR.
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