Interaction between cholinergic neurons and substance P or calcitonin gene-related peptide terminals of the rat sacral intermediolateral nucleus: Double immunostaining at the light and electron microscopic levels

Abstract

The relationships both between cholinergic neurons and substance P (SP) and between cholinergic neurons and calcitonin gene‐related peptide (CGRP) terminals were examined in the rat sacral intermediolateral nucleus at the light and electron microscopic levels by means of double‐immunostaining methods. Cholinergic neurons were labeled by a monoclonal antibody to choline acetyltransferase (CAT) with the avidin‐biotin technique and stained bluish‐green by indolyl‐β‐galactoside reaction products with β‐galactosidase as a marker. On the same sections, SP or CGRP fibers were labeled by polyclonal antisera to SP or CGRP after application of the peroxidase‐antiperoxidase (PAP) method and stained brown by the p‐dimethylaminoazobenzene (DAB) reaction. After embedding in Epon, light and electron microscopic sections were examined. At the light microscopic level, CGRP‐like immunoreactive (CGRP‐I) fibers and SP‐like immunoreactive (SP‐I) fibers were found to pass through the lateral edge of the dorsal horn and then into the dorsal region of the sacral intermediolateral nucleus. In addition, SP‐I fibers also extend from the dorsolateral funiculus into the entire sacral intermediolateral region. At the electron microscopic level, many axosomatic and axodendritic synapses were found between CAT‐I structures and SP‐I terminals in the intermediolateral nucleus, whereas most of the CGRP‐I terminals in this area made axodendritic synapses with CAT‐I dendrites. These results indicate that cholinergic neurons in the sacral intermediolateral nucleus receive direct synaptic input from SP‐I and CGRP‐I terminals. Therefore, it is likely that both SP‐I and CGRP‐I fibers from dorsal root ganglia and intrinsic SP‐I fibers make direct synapses with sacral parasympathetic preganglionic neurons and thus have great importance in the control of bowel and bladder reflexes.