The m5 muscarinic receptor: Labeling and localization studies

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)

First Committee Member

Donna D. Flynn - Committee Chair


The aims of this study were: (1) to develop binding conditions that labeled predominantly the m5 muscarinic receptor subtype; (2) to use m5-selective labeling conditions to study the distribution of the m5 receptor subtype in the rat brain and make regional comparisons to m1-m4 patterns and (3) to determine whether m5 receptors are expressed on nigrostriatal dopaminergic neurons and contribute to striatal m5 labeling.The m5 labeling conditions described here are the first method for the selective labeling and localization of the M5 (m5) muscarinic receptor subtype in the brain. Selective labeling of the m5 muscarinic receptor subtype with the non-selective muscarinic antagonist ($\sp3$H) NMS was based on the ability of toxins in green mamba venom to selectively occupy m1 and m4 receptor subtypes and on the selectivity profile of AQ-RA 741 for the m1-m5 receptor subtypes expressed in CHO-K1 cells. Green mamba venom (30 $\mu$g/ml) occluded essentially all m1 and 90% of m4 receptors, with little effect on ($\sp3$H) NMS binding to other subtypes. At 1 $\mu$M AQ-RA 741, all m2, $\geq$90% m1 and m4, $>$80% m3 and 40% m5 receptors were inhibited from binding ($\sp3$H) NMS. By combining the two strategies, 55% m5, 15% m3, and 0% m1, m2 or m4 receptors were labeled with ($\sp3$H) NMS.In vitro autoradiography, using these m5-selective labeling conditions, has provided the first map of the m5 receptor protein distribution in the brain. Distinct m5-labeling was found in the superficial layer of the cortex, discrete subfields of the hippocampus, striatum, specific subregions of the thalamus, and superior colliculus. Labeling was absent throughout most of the brainstem and cerebellum. This distribution was distinct from M1 (m1), M2 (m2), M3 (m3) and M4 (m4) localization.Animals received 6-OHDA lesions of the nigrostriatal dopaminergic neurons to permit determination of whether m5 receptors are present on dopaminergic neurons of the substantia nigra, pars compacta (SNc). In lesioned animals, a 70% and 34% decrease in m5 labeling of the SNc and striatum, respectively, was observed. These results suggested that m5 receptors were expressed both on dopaminergic cell bodies in the SNc and on dopaminergic terminals in the striatum.


Biology, Neuroscience; Health Sciences, Pharmacology

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