Ultrastructural studies on peptides in the dorsal horn of the rat spinal cord—II. Co-existence of galanin with other peptides in local neurons

Abstract

Using light microscopic immunoperoxidase and immunofluorescence histochemistry, double-staining methodology, and electron microscopic pre-embedding and post-embedding immunocytochemistry, we studied galanin-immunoreactive neurons in the superficial dorsal horn of the rat spinal cord. Co-existence of galanin with other neuropeptides was also analysed. The lumbar 4 and 5 segments of normal rats and after rhizotomy or spinal cord transection were studied. Galanin-positive local neurons in lamina II were often islet cells and could be classified as type A, which had abundant electron-dense cytoplasm containing many large dense-core vesicles, and type B, which had electron-lucent cytoplasm with only a few large dense-core vesicles. Galanin-positive and -negative peripheral afferent terminals made synaptic contact mostly with galanin-negative dendrites and cell bodies, but also with type B galanin cell bodies and with galanin-positive dendrites of unidentified type. Galanin-immunoreactive terminals from local neurons could also be classified into two types. Type alpha terminals were most common; they contained densely packed synaptic vesicles and many large dense-core vesicles, were strongly immunostained and most frequently made synaptic contact with galanin-negative dendrites. Type beta terminals contained loosely packed synaptic vesicles and a few large dense-core vesicles, and were weakly immunostained. Axosomatic synaptic contact were sometimes found between type beta terminals and type B galanin-positive cell bodies, but were most often associated with galanin-negative dendrites. Double immunostaining showed that galanin-like immunoreactivity co-localized mainly with enkephalin-like, but sometimes also with neuropeptide Y-like immunoreactivity in some local neurons in lamina II. Galanin-like and substance P-like immunoreactivities were identified in the same neurons in deeper layers of the dorsal horn. Coexistence of these neuropeptides and neurotensin with galanin was demonstrated not only in terminals in lamina II but also in large dense-core vesicles, as revealed by post-embedding immunocytochemistry. These results show that galanin-immunoreactive neurons in lamina II receive inputs directly from primary afferents and frequently make synaptic contacts with other intrinsic neurons. Galanin in the superficial dorsal horn may be released both from primary afferents and local neurons to modulate sensory processing in many different ways, including interacting with enkephalin, neuropeptide Y, neurotensin and substance P released from the same and/or other local neurons.