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

Abstract

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.

Acetylcholine efflux from retrosplenial areas and hippocampal sectors during maze exploration

“…Indeed, the PPT and LDT mainly project to the posterior subdivision of the posterior anteroventral thalamic nucleus (AVp) and less so to the medial anteroventral thalamic nucleus (AVm). Axons projecting from the AV terminate in layer Ia and IV of area 29 and provide an important source of excitation that is driven by muscarinic receptor activation [75, 77, 78]. This organization provides activation of area 29 neurons through presynaptic regulation of M2 receptors located on glutamatergic axon terminals that project to layers Ia and IV.…”

“…Neuropharmacological assays of both nicotinic and muscarinic receptor subtypes place a significant density within the granular layers of the retrosplenial cortex [59, 62]. Furthermore, various studies have placed a high concentration of muscarinic receptors in area 29, suggesting a substantial degree of cholinergic activity in this region [75, 77, 78]. Both areas 29a/b and 30 display similar baseline levels of ACh, as observed by fentomole measurements, but only area 29a/b underwent a significant behaviorally evoked rise in ACh efflux during maze traversal.…”

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). As shown in Fig.…”

“…The ml, m3, and m4 receptors are expressed by cortical neurons, binding of the antagonist pirenzepine does not distinguish among them (Brann et ai., 1987;Buckley et ai., 1988), and these sites are together referred to as the pharmacological M} subtype (Birdsall and Hulme, 1983). The m2 receptor has a low affinity for pirenzepine, is optimally labeled in rat brain with 0.1 nM [3H]oxotremorine-M in the presence of 50 nM unlabeled pirenzepine (Vogt and Burns, 1988) and has been termed the pharmacological M2 subtype. The laminar distribution of pH]pirenzepine and [3H]oxotremorine-M binding in area 29c is quite different as can be seen in Fig.…”

“…The proportion of specific binding of [3HJpirenzepine (PZ) and [3HJoxotremorine-M in the presence of 50 nM unlabeled PZ (OXO/PZ) was calculated for each layer of rat area 29c. Experimental studies of the binding of these ligands have shown that the peaks in OXO/PZ binding in layers Ia and IV are associated mainly with anterior thalamic axons and to a lesser extent the apical and basal dendrites of pyramidal neurons in layers II -IV, while PZ binding is to all dendrites of pyramidal neurons (Vogt and Burns, 1988). slight elevation in layer Ic, while that of oxotremorine-M is heterogeneous with pronounced peaks in layers la and IV.…”