Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Preferential synaptic relationships between substance P-immunoreactive boutons and neurokinin 1 receptor sites in the rat spinal cord

Departments of ^*Pharmacology and Therapeutics and ^‡Anatomy and Cell Biology, McGill University, Montreal, Quebec H3G 1Y6, Canada; and ^†Neurogen Corporation, Branford, CT 06405.

Edited by Tomas Hökfelt, Karolinska Institutet, Stockholm, Sweden, and approved October 26, 1998 (received for review July 22, 1998)

Abstract

Substance P plays an important role in the transmission of pain-related information in the dorsal horn of the spinal cord. Recent immunocytochemical studies have shown a mismatch between the distribution of substance P and its receptor in the superficial laminae of the dorsal horn. Because such a mismatch was not observed by using classical radioligand binding studies, we decided to investigate further the issue of the relationship between substance P and its receptor by using an antibody raised against a portion of the carboxyl terminal of the neurokinin 1 receptor and a bispecific monoclonal antibodies against substance P and horseradish peroxidase. Light microscopy revealed a good correlation between the distributions of substance P and the neurokinin 1 receptor, both being localized with highest densities in lamina I and outer lamina II of the spinal dorsal horn. An ultrastructural double-labeling study, combining preembedding immunogold with enzyme-based immunocytochemistry, showed that most neurokinin 1 receptor immunoreactive dendrites were apposed by substance P containing boutons. A detailed quantitative analysis revealed that neurokinin 1 receptor immunoreactive dendrites received more appositions and synapses from substance P immunoreactive terminals than those not expressing the neurokinin 1 receptor. Such preferential innervation by substance P occurred in all superficial dorsal horn laminae even though neurokinin 1 receptor immunoreactive dendrites were a minority of the total number of dendritic profiles in the above laminae. These results suggest that, contrary to the belief that neuropeptides act in a diffuse manner at a considerable distance from their sites of release, substance P should act on profiles expressing the neurokinin 1 receptor at a short distance from its site of release.

Footnotes

↵ § To whom reprint requests should be addressed at: Department of Pharmacology and Therapeutics, McGill University, 3655 Drummond Street, Room 1325, Montreal, Quebec H3G 1Y6, Canada. e-mail: aribeiro{at}pharma.mcgill.ca.
This paper was submitted directly (Track II) to the Proceedings Office.
ABBREVIATIONS:

IR,

immunoreactive;

NK-1r,

neurokinin-1 receptor;

PB,

phosphate buffer;

SP,

substance P;

PBS+T,

PBS with 0.2% Triton X-100