A technique was developed to evaluate the potency of ligand binding at M2 ACh receptors and to experimentally localize the M1 and M2 subtypes to specific neuronal processes. Normal and experimental material was prepared with tritiated ligand binding to cryostat-sectioned area 29c of posterior cingulate cortex in rat, coverslip autoradiography, and single-grain-counting techniques. Three fundamental issues were addressed. 1. A morphological criterion termed an index of heterogeneity was developed by which the specificity of M2 binding by different ligands could be assessed. The index was calculated by first determining the laminar distribution of pirenzepine (PZ) binding sites and then summing absolute laminar variations from this distribution for each ligand. According to this measure the most efficient protocol for assaying M2 sites was tritiated oxotremorine-M (OXO) coincubated in unlabeled PZ (5 x 10(-8) M). The classical muscarinic antagonist propylbenzilylcholine mustard (Pr-BCM), however, when coincubated in PZ, was almost as efficient as PZ-blocked OXO binding. 2. Terminal axons of neurons in the anterior thalamic nuclei (ATN) have M2 receptors based on the following observations. First, specific binding of the M1 ligand PZ was unaffected by ATN lesions. Second, tritiated OXO and PrBCM binding blocked with unlabeled PZ, conditions favoring M2 receptor binding, showed significant reductions in binding in layers la, lb, and IV following ATN ablations. Third, IC50 values as determined by competition of PZ for PrBCM binding sites were shifted to lower concentrations in superficial layers by ATN lesions but not in deep layers where the thalamus does not terminate. Finally, in contrast to PZ-blocked OXO and PrBCM binding, binding of PZ-blocked 3H- quinuclidinyl benzilate (QNB) was reduced to homogeneity following ATN lesions. 3. Cortical pyramidal neurons have dendritic receptors that are primarily of the M1 subtype but may also include M2 sites. Thus, full depth ibotenic acid lesions reduced PZ binding by almost 70%. Neurotoxin lesions of neurons in layers II-IV or Vb-VI were followed by degeneration of the apical dendrites of pyramids in layer I and 78 and 15% reductions, respectively, in PZ binding. Also, full-depth neurotoxin lesions combined with ATN ablations completely abolished heterogeneities in PrBCM and PZ-blocked OXO binding. These data demonstrate that experimental techniques can be used in conjunction with normal material to make morphological assessments of the efficiency of binding of putative M2 ligands.

Identifiable cortical neurons were obtained from area 29c of rat cingulate cortex using enzymatic and mechanical dissociation techniques. Dissociated neurons were either analyzed morphologically with the electron microscope or processed autoradiographically to evaluate the distribution of specific 3H-propylbenzilylcholine mustard (PrBCM) binding. Ultrastructurally, neurons appeared healthy and contained a full complement of cytoplasmic organelles. Membranes were intact and no presynaptic endings adhered to cell bodies or dendrites. Dendritic spines were not observed in these dissociations and serial sections of identified neurons indicated that all dendritic processes were smooth. Receptor binding studies were conducted on small and medium-to-large pyramidal neurons and multipolar cells. Specific binding of PrBCM was determined by calculating the mean number of grains/10 micron somal perimeter or dendritic length and subtracting mean values from a matched series of neurons that were coincubated in atropine. Specific binding was to somata and dendrites of all neurons. Nonspecific binding was an average of 33% of total binding. A 2 X 2 factorial analysis of variance comparing total and nonspecific binding for pairs of processes indicated that there were no regional differences in dendritic binding, either by cell type or by order of dendritic branching. Both somatic and dendritic PrBCM binding was antagonized by pirenzepine (PZ); however, PZ appeared to be more effective at secondary dendritic, rather than at somatic and primary apical dendritic sites. Thus, the IC50 values for somata and primary apical dendrites of small pyramids were 6 X 10(-7) and 9 X 10(-7) M PZ, respectively, while that for secondary basal dendrites of the same neurons was 5.8 X 10(-8) M. Morphological and pharmacological results together suggest that (1) muscarinic receptors are present on the smooth surfaces of all pyramidal and multipolar neurons; (2) many of the binding sites are high affinity, PZ-sensitive, M1 receptors; and (3) this binding is associated with the postsynaptic specialization of symmetric, cholinergic synapses.