Axotomy causes loss of muscarinic receptors and loss of synaptic contacts in the hypoglossal nucleus

Abstract

MUSCARINIC receptors in the brain and ileum can be studied by means of the binding of radiolabelled cholinergic antagonists^1,2. The basic criteria for receptor-specific labelling are that there should be a saturable component of binding and that the binding of the radiolabelled ligand should be inhibited by pharmacologically effective concentrations of drugs which are known to act at muscarinic receptors and not by drugs that act at different receptors. In addition, the saturable binding component should be restricted to tissues known from pharmacological experiments to contain muscarinic receptors. These criteria are satisfied by tritiated propylbenzilylcholine mustard (³H-PrBCM)³, a potent irreversible muscarinic antagonist the binding of which to rat brain sections can be demonstrated autoradiographically. The evidence which suggests that the bulk of tissue radioactivity is associated with muscarinic receptors is that in autoradiographs the regional variation of autoradiographic grain density parallels the intensity of specific ³H-PrBCM binding observed by in vitro assay on brain homogenates from selected regions, and that in the homogenates 65–85% of the bound radioactivity is blocked by pretreatment with the classical muscarinic antagonist, atropine (10⁻⁶M). In the forebrain the general features of the distribution of ³H-PrBCM binding to thin sections correspond closely with the findings of Kuhar and Yamamura^4,5 who used the non-covalently bound antagonist ³H-3-quinuclidinylbenzilate. We report here, however, that in the brain stem the hypoglossal nuclei are heavily labelled with ³H-PrBCM. In light microscopic autoradiographs (Fig. 1a, left side) the grains are distributed throughout the neuropil of the hypoglossal nucleus. The cytoplasm and nuclei of the large hypoglossal neuronal somata (Fig. 1b) are unlabelled. The density of the silver grains falls abruptly at the borders of the hypoglossal neuropil. This clear demarcation of the labelled hypoglossal neuropil is abolished by atropine pretreatment (Fig. 1c).