Association of m1 and m2 muscarinic receptor proteins with asymmetric synapses in the primate cerebral cortex: morphological evidence for cholinergic modulation of excitatory neurotransmission.

Abstract

Muscarinic ml receptors traditionally are considered to be postsynaptic to cholinergic fibers, while m2 receptors are largely presynaptic receptors associated with axons. We have examined the distribution of these receptor proteins in the monkey cerebral cortex and obtained results that are at odds with this expectation. Using immunohistochemistry with specific antibodies to recombinant ml and m2 muscarinic receptor proteins, we have demonstrated that both ml and m2 receptors are prominently associated with noncholinergic asymmetric synapses as well as with the symmetric synapses that characterize the cholinergic pathways in the neocortex. At asymmetric synapses, both ml and m2 receptor immunoreactivity is observed postsynaptically within spines and dendrites; the m2 receptor is also found in presynaptic axon terminals which, in the visual cortex, resemble the parvicellular geniculocortical pathway. In addition, m2 labeling was also found in a subset of nonpyramidal neurons. These rmdings establish that the m2 receptor is located postsynapticafly as well as presynapticafly. The association of ml and m2 receptors with asymmetric synapses in central pathways, which use excitatory amino acids as neurotransmitters, provides a morphological basis for cholinergic modulation of excitatory neurotransmission.Muscarinic receptors in the central nervous system have been implicated in normal learning and memory (1-3), arousal (4-6), and motor and sensory modulation (4, 5, 7) as well as in conditions such as normal aging, Alzheimer and Parkinson diseases, schizophrenia, and depression (8)(9)(10)(11). Recently, the cloning of five different genes encoding structurally similar muscarinic receptor proteins (ml-m5) (12, 13) has led to production of subtype-specific antibodies against recombinant muscarinic receptor proteins (14, 15). These antibodies have enabled us to identify the neuronal elements containing particular receptors in the cerebral cortex at a level of resolution and specificity not possible in previous autoradiographic studies of these receptors (16, 17) or in previous immunohistochemical analyses using nonselective muscarinic antibodies (18). Our findings have revealed an unexpected synaptic association ofml and m2 receptors with fiber systems utilizing excitatory transmitters in the primate cerebral cortex. MATERIALS AND METHODSTissue and Fixation. The results of this study are based on light and electron microscopic analysis of frontal and occipital lobe tissue from three adult macaque monkeys (Macaca mulatta). The monkeys were anesthetized with an intraveThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. nous overdose of sodium pentobarbital (Nembutal) (100 mg/kg) and after initial saline perfusion, one monkey was perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4) and two monkeys were perfused with 4% paraformaldehyde/0.08...

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