Dissociated cingulate cortical neurons: morphology and muscarinic acetylcholine receptor binding properties

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.