NEURONAL NICOTINIC ACETYLCHOLINE RECEPTORS

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The long-range objective of this research is to understand how innervation regulates the number and distribution of nicotinic acetylcholine receptors (AChRs) on neurons. Our approach has been to use a simple and tractable system, the frog cardiac ganglion, where AChRs on parasympathetic neurons can be analyzed by immunocytochemistry, autoradiography, and biochemistry, and where the influence of innervation upon AChRs can be monitored during denervation and reinnervation in adult frogs (Rana) as well during initial innervation in embryos (Xenopus). In the past award period we found that denervation does not increase the number of AChRs on the surface of cardiac ganglion cells, measured by the binding of 125I-neuronal bungarotoxin, a snake toxin that blocks AChR function. Denervation does increase the sensitivity of cardiac ganglion cells to acetylcholine (ACh) applied via a micropipet; this effect appears to be due to a reduction in the effectiveness of the hydrolytic enzyme acetylcholinesterase (AChE) and not to a change in the number of functional AChRs on the cell surface. We have three specific aims. In the first, we will examine the consequences of denervation and reinnervation upon the distribution of AChRs and AChR clusters in adult Rana pipiens. In the second aim, we will determine whether denervation supersensitivity can be explained by a reduction in the number of AChE molecules in the extracellular space surrounding ganglion cells and/or a change in their kinetic properties. We will also examine whether the reduction in ACh sensitivity that occurs during the first stages of reinnervation is due to a change in AChE, in AChRs, or in both. In the third aim we will examine whether AChRs are expressed on developing cardiac ganglion cells in Xenopus laevis before, during, or after cells are contacted by preganglionic axons. We will determine whether AChRs appear on neurons that have never been innervated by preganglionic axons, and, if so, whether innervation enhances AChR expression. These studies should increase our understanding of how neuronal nicotinic AChRs and ACh sensitivity are regulated by innervation.