ABSTRACT
Muscarinic receptors play a role in normal physiologic processes such as learning, memory, and arousal. This chapter introduces the molecular and pharmacological classification of muscarinic receptor subtypes. It presents a detailed protocol useful for identifying and quantifying the muscarinic receptor subtypes present in central nervous system (CNS) tissue homogenates. Muscarinic receptors are also classified into subtypes based on their interactions with antagonists, as studied using classical radioligand binding techniques. The chapter presents a detailed protocol for competition of [3H]-NMS binding in tissue homogenates using pirenzepine, AF-DX 116, methoctramine, and methoctramine plus atropine as unlabeled competitors. This protocol is based upon the original studies of Waelbroeck and colleagues. This protocol assumes that 500 mg wet weight of CNS tissue is available so that all four assays can be run concurrently. The protocol can easily be modified to run one assay at a time. Polyethyleneimidine minimizes nonspecific binding of the radioligand to the harvester filters.
