Title

Identification and localization of muscarinic receptors in the dorsal horn of the human and rat spinal cord

Date of Award

1998

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Molecular and Cellular Pharmacology

First Committee Member

Lincoln T. Potter, Committee Chair

Abstract

The research in this dissertation followed evidence that muscarinic neurotransmission can inhibit nociception. Specifically: (a) the intrathecal administration of muscarinic agonists and acetylcholinesterase inhibitors in rodents and humans was found to reduce nociception, (b) nerve terminals containing cholineacetyltransferase in the dorsal horn of the rat spinal cord were found to be presynaptic to primary afferent nerve terminals, (c) 3H-pirenzepine (which binds with highest affinity to the m1 and m4 muscarinic receptor subtypes) was found to be sharply localized in the superficial dorsal horn of the human and rat spinal cord, and (d) Eli Lilly & Co developed a systemic drug, which may be an m4 agonist, that was analgesic in rats and mice. On the basis of these observations, I formulated the following question: Which subtypes of muscarinic receptors are localized in the dorsal horn of the human and rat spinal cord at sites where they may be involved in the modulation of nociception? This question was investigated using radioligand binding assays, light microscopic autoradiography, light microscopic immunocytochemistry and electron microscopic immunocytochemistry.1. Muscarinic receptors were labeled with one nM 3H-N-methylscopolamine (3H-NMS). Membranes from whole rat spinal cord contained 6.9 fmol of receptors per mg; of tissue, while human spinal cord contained 1.47 fmol/mg.2. Right dorsal rhizotoiny in rats reduced the binding of 3H-NMS by 10% at one day, with no effect after 7 days. It was concluded that most muscarinic receptors are not on primary afferent nerve terminals.3. m1-Toxin was used as a completely selective ligand for in m1 receptors. It reduced the binding of 3H-NMS by 6% in the dorsal horn of the human spinal cord and 10% in the ventral horn of the rat spinal cord. It was concluded that m1 receptors were a small proportion of the muscarinic receptors of the dorsal horn of the human spinal cord, while they were not found in the dorsal horn of the rat spinal cord.4. m4-Toxin was used as a completely selective ligand for m4 receptors (after the block of m1 receptors). It reduced the binding of 3H-NMS by 30% in the dorsal horn of the human spinal cord and 15% in the dorsal horn of the rat spinal cord. It was concluded that m4 receptors composed a significant proportion of the muscarinic receptors of the dorsal horn of the human and rat spinal cord.5. Gallamine was used as a highly selective ligand for m2 receptors. At concentrations that blocked m2 receptors selectively, gallamine reduced the binding of 3H-NMS by 89% in the rat spinal cord and 67% in the human spinal cord. It was concluded that m2 receptors were the predominant subtype of muscarinic receptor in the rat and human spinal cord. m2 Receptors were localized most densely to the superficial dorsal horn of the rat and lamina II of the dorsal horn of the human spinal cord with anti-m2 antibodies. The ultrastructural location of m2 receptors was established by electron microscopy with anti-m2 antibodies. Immunoreactivity was localized in synapses on dendrites, with little or no reactivity in synapses on nerve terminals. It was concluded that the activation of m2 receptors probably modulates the reception of sensory neurotransmitters.

Keywords

Biology, Molecular; Biology, Neuroscience

Link to Full Text

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