CHOLINERGIC MECHANISMS IN AGING AND AD

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This laboratory is using m1-toxin and m4-toxin as highly specific ligands to identify clinically relevant steps in muscarinic neurotransmission that can be controlled by specific agonists or antagonists for m1 or m4 receptors. Aim 1 concerns direct studies of these receptors. (1a) Human AD-m1 receptors will be isolated using biotinylated m1-toxin and a monoavidin affinity resin. Biochemical studies of the receptor protein should explain its defective coupling to G protein and, incomplete immunoprecipitation with anti-m1 antibodies, and may show ways to improve the effectiveness of esterase inhibitors in AD. (1b) 125I-m1- Toxin and 125I-m4-toxin will be used to establish the distribution of m1+m4 receptors in the rat, to focus attention on sites where new drugs can act, either beneficially or to produce side effects. (1c) Autoradiography will be used to localize 125I-m4-toxin in the dorsal horns of the human and rat spinal cord, and dorsal root rhizotomy in the rat will indicate whether m4 receptors are on afferent nerve terminals, like opiate receptors. The results will show whether m4 agonists are likely to prove useful for pain. (1d) 125I-Toxins will be used to study toxin-receptor complexes, and the kinetics of binding of m4-toxin at 4 degrees and 37 degrees C. (1e) Changes of m1+m4 receptors in hemi- Parkinson (hemi-PD) rat brains will be studied by autoradiography with 125I-toxins. Aim 2 concerns the effects of unilateral striatal m4-blockade on movement in rats. Optimum conditions for achieving specific m4- blockade will be assessed with 125I-m4-toxin by autoradiography 0.2-2 hours after right intrastriatal infusion of m4-toxin. Then rats +/- hemi-PD will be studied for altered spontaneous forearm use. Specific right m4 antagonism should increase left movement and correct the defective left movement of rats with right hemi-PD. Aim 3 concerns the effects pf established unilateral striatal m1-blockade ("ml knockdown") on movement in rats. Right intrastriatal m1-toxin is not expected to affect spontaneous left forearm use by itself or in right hemi-PD, but is expected to alter responses to apomorphine (dopamine agonist), pilocarpine and xanomeline (muscarinic agonists). At the least, the results will show whether a specific m1 antagonist can be used to control seizures. Aim 4 concerns mutant m1-toxins. The unique amino acids of the m1-isotoxins will be changed by site-directed mutagenesis to residues characteristic of toxins that bind to m2-m5 receptors, to produce mutant toxins with new selectivity profiles, to determine which residues confer the remarkable affinity and selectivity of m1-toxins, and to begin studies of the molecular fit between mutant m1-toxins and mutant m1 receptors.