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Tobacco consumption is the leading cause of premature deaths in the United States. Nicotine has been identified as the principal addictive component in tobacco smoke causing this pathological behavior. In all known cases nicotine exerts its actions in the body by acting on a family of specific nicotinic receptors. These receptors are proteins on the surface of neuronal cells that bind nicotine and cause an electrical event to occur inside. These electrical events represent one of the basic signaling mechanisms used for information processing in the nervous system. One of the most abundant nicotinic receptors in our nervous system is the one containing the a7-receptor, which has the unusual feature of allowing large amounts of calcium to enter cells. Calcium is a critically important element that regulates fundamental processes in the brain. Recent evidence from a number of sources indicate that a7-receptors are capable of many functions in the brain as a result, and that, when inappropriately activated or regulated, may contribute to diverse pathologies including schizophrenia, lung cancer, and degeneration of cells in the brain. How the a7-receptors achieve their effects, what their physiological significance is normally for brain function, and how they are influenced by prolonged exposure to nicotine are questions that remain largely unanswered.
The fact that a7-receptors can display multiple functions depending on their location on the cell surface, directs attention to the mechanisms responsible for the delivery of receptors to specific sites. To address this issue, the experiments proposed here will test the effect that intercellular interactions have on the distribution and function of nicotinic receptors expressed on the cell surface.
Because a7-receptors – possibly one of the most important nicotinic acetylcholine receptors in the nervous system – can quickly elevate intracellular calcium levels and because these receptors appear early and in a variety of places, they are positioned to influence many events during development. The expected results arising from this project will provide new information about the signaling mechanisms of a7-receptors and may suggest development events at risk by prolonged exposure to nicotine, e.g. during pregnancy. The outcome will be a better understanding of the consequences of smoking, new insights into mechanisms that may contribute to the nicotinic response, and may suggest intervention strategies of biomedical relevance. |
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PROPOSED RESEARCH PLAN: OVERVIEW
Presynaptic a7-nicotinic acetylcholine receptors ((x7-nAChRs) can influence transmitter release and modulate synaptic transmission (e.g. Gray et al., 1996). Postsynaptically they generate substantial synaptic currents (e.g. Frazier et al., 1998). Because a7-nAChRs can quickly elevate intracellular Ca 2+ levels and because the receptors appear early and in a variety of places, they are suitable candidates to influence many events during development. Therefore, identifying the mechanisms responsible for targeting a7-nAChRs to specific locations on the cell surface constitutes a major challenge.
Ciliary neurons concentrate large numbers of a7-nAChRs on clusters of somatic spines (Shoop et al., 1999). Though, these somatic receptors are excluded from postsynaptic densities, they contribute importantly to synaptic transmission (e.g. Chang and Berg, 1999). The experiments proposed in this project, took advantage of the embryonic chick ciliary ganglion (CG) as a model for examining how nAChRs become concentrated and maintained at specific neuronal domains in order to exert their function.
Specific aims:
I. Identify cell-cell interactions in vivo that induce or stabilize somatic spines with a7-nAChRs on ciliary ganglion neurons.
II. Test candidates in cell culture for regulatory roles in spine formation and receptor accumulation.
III. Determine whether ciliary ganglion a7-nAChRs also get exported to presynaptic sites in target muscle and are subject to the same regulatory controls as spine receptors.
RESULTS: SUMMARY
In order to manipulate the cellular interactions that induce or stabilize a7-nAChRs clusters on CG neurons in the developing embryo, we used surgical interventions (removal of either the CG source of innervation or the CG target) and chronically treated the embryos with cholinergic drugs (d-tubocurarine, (x-bungarotoxin, (-)-nicotine, atropine). To evaluate the consequences on cytoskeleton organization, somatic spine formation and a7-nAChRs clustering on neurons in situ, we used fluorescence imaging as well as electron microscopy.
From these studies we report:
• chick CG neurons cluster a7-nAChRs and these clusters colocalize with cytoskeletal elements such as F-actin and drebrin (marker for somatic spines)
• the formation of a7-nAChRs clusters on CG neurons and their colocalization with cytoskeletal components (i.e. F-actin and drebrin) is not a phenomenon driven by cholinergic activity.
target innervation does not induce the formation of a7-nAChRs clusters on CG neurons.
• target innervation deprivation neither alters the distribution of cytoskeletal components (i.e. F-actin and drebrin) on CG neurons nor their spatial association with a7-nAChRs clusters.
• CG presynaptic input deprivation causes major cytoskeletal reorganization without significantly altering the formation of a7-nAChRs clusters on CG neurons.
FINAL REMARKS
Aim I was largely completed during the first 10 months of the project. The results and the conclusions derived from this study will be included in a manuscript that we will submit for publication before the end of year 2001. The results obtained in this project provide new information about the mechanisms involved in a7-nAChRs subcellular targeting and may suggest development events at risk e.g. by prolonged exposure to nicotine during pregnancy. The outcome contributes to a better understanding of the consequences of smoking, new insights into mechanisms that may contribute to the nicotinic response, and may suggest intervention strategies of biomedical relevance.
REFERENCES
Chang K and Berg DK (1999). Nicotinic acetylcholine receptors containing 0 subunits are required for reliable synaptic transmission in situ. Journal of Neuroscience 19: 3701-3710.
Gray R, Rajan AS, Radcliffe KA, Yakehiro M and Dani JA (1996). Hippocampal synaptic transmission enhanced by low concentrations of nicotine. Nature 383: 713-716.
Frazier CJ, Rollins YD, Breese CR, Leonard S, Freedman R and Dunwiddie TV (1998). Acetylcholine activates an a-bungarotoxin-sensitive nicotinic current in rat hippocampal interneurons, but not pyramidal neurons. Journal of Neuroscience 18: 1187-1195.
Shoop RD, Martone ME, Yamada N, Ellisman MH and Berg DK (1999). Neuronal acetylcholine receptors with 0 subunits are concentrated on somatic spines for synaptic signaling in embryonic chick ciliary ganglia. Journal of Neuroscience 19: 692-704. |
