The major goal of this proposal is to determine the neuroanatomical relationships between muscarinic receptor proteins m1-m5,their respective mRNAs and cholinergic innervation in brain. Cholinergic neurons project widely in the brain and, via stimulation of muscarinic receptors, participate in important neural mechanisms such as learning, memory, arousal, and movement. A family of five muscarinic receptor genes (m 1 -m5) has recently been cloned and sequenced, and mRNAs of four of these receptor subtypes are expressed in brain. Their varied distributions suggest that they are involved in different neural functions. Presently, pharmacological agents are not able to discriminate subtypes of muscarinic receptors clearly. Knowledge of detailed localization of muscarinic receptor proteins m1-m5 is fundamental to understanding cholinergic function in the brain; however, currently there are no methods available for this purpose. This proposal describes the production and characterization of antisera reactive specifically with synthetic oligopeptide sequences and their use in localization of muscarinic receptor proteins m 1 -m5 in rat hippocampus. Oligopeptides corresponding to three predicted unique regions of each receptor subtype will be synthesized and used to immunize rabbits. Antisera will be characterized for ability to bind to receptor subtypes by use of a sensitive coprecipitation assay, immunoblotting, radioligand binding, and immunohistochemistry. These studies will employ homogeneous sources of each receptor subtype, available from cloned cell lines transfected with muscarinic cDNAs ml-m5, in order to define fully the specificities of antisera for each subtype. The ml -m5 receptor proteins will be localized in hippocampus by using monospecific antisera and immunohistochemistical techniques. The distribution of receptor proteins will be compared to that of in situ hybridization of respective mRNAs, as well as the cholinergic nerve terminals by means of immunohistochemistical localization of choline acetyltransferase. Preliminary studies confirm the feasibility of proposed studies by demonstrating specific reactivity of and-peptide antisera against m3 and m4 receptor subtypes in a coprecipitation assay. In situ hybridization studies localizing mRNAs ml, m3, and m4, and immunohistochemical studies of choline acetyltransferase immunoreactivity in hippocampus have also been demonstrated. The anti-muscarinic ml-m5 antisera should be valuable for localizing receptor subtypes in brain, and determining their relationships to central cholinergic systems. This information will provide a rational basis for pharmacological intervention of Alzheimer's Disease, Parkinson's Disease, and other movement disorders as selective muscarinic agents become available.
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