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

Abstract

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.

Localization of acetylcholine receptors on isolated CNS neurons: cellular and subcellular differentiation

“…Previous studies have shown that neuroreceptor function, as well as various other aspects of cellular physiology, are well preserved (Sarthy, 1986). Ultrastructural studies have shown that papain-dissociated neurons have no presynaptic terminals adhering to them (Vogt et al, 1987;Lankford and Klein, unpublished data).…”

The Role of Layer I in Cortical Function

“…First, dissociated pyramidal neurons have specific binding of the nonselective antagonist propylbenzilylcholine mustard to their somata and 1°, 2°, and 3° basal and apical dendrites, i.e., the proximal integrative region including all proximal dendrites (Vogt et at., 1987). Second, ibotenic acid lesions of layers II -IV, which are composed primarily of small and fusiform pyramidal neurons , greatly reduce specific binding of pirenzepine in layers II-IV (i.e., the proximal compartment) as well as in all divisions of layer I (i.e., the distal dendritic compartment; Vogt and Burns, 1988).…”

Experimental localization of muscarinic receptor subtypes to cingulate cortical afferents and neurons

“…Second, ACh-evoked, slow depolarization in pyramidal neurons is PZ sensitive Prince, 1985, 1986). Third, high-resolution autoradiographic localization of PZ-sensitive sites to dendrites and somata has been made with dissociated cortical neurons (Vogt et al, 1987).…”

“…Although cells can be dissociated and specific binding determined autoradiographically for identified types of adult neurons (Vogt et al, 1987) there are limitations to this approach. First, the only ligands appropriate for wet emulsion autoradiography are those that are irreversible, but propylbenzilylcholine mustard (PrBCM), which is irreversible, is not selective for a receptor subtype.…”