~ Pergamon 0306-4522(94)00451-X Neuroscience Vol. 64, No. 4, pp. 875 891, 1995 Elsevier Science Ltd Copyright © 1995 IBRO Printed in Great Britain. All rights reserved 0306-4522/95 $9.50 + 0.00 ULTRASTRUCTURAL STUDIES ON PEPTIDES IN THE DORSAL HORN OF THE RAT SPINAL CORD--II. CO-EXISTENCE OF GALANIN WITH OTHER PEPTIDES IN LOCAL NEURONS X. ZHANG, A. P. NICHOLAS and T. HOKFELT* Department of Neuroscience, Karolinska Institute, S-171 77, Stockholm, Sweden Abstract--Using light microscopic immunoperoxidase and immunofluorescence histochemistry, double- staining methodology, and electron microscopic pre-embedding and post-embedding immunocytochem- istry, 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 ~ terminals were most common; they contained densely packed synaptic vesicles and many large dense-core vesicles, were strongly immunos- tained and most frequently made synaptic contact with galanin-negative dendrites. Type fl terminals contained loosely packed synaptic vesicles and a few large dense-core vesicles, and were weakly immunostained. Axosomatic synaptic contact were sometimes found betwen type fl 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. Peptide-containing neuronal cell bodies and nerve endings are present in the superficial layers of the dorsal horn of the spinal c o r d . 32'40'79'99'100'109 In addition to the terminals of primary afferents (for references, see Ref. 116), many small local neurons and their axonal processes in the superficial dorsal horn of the spinal cord of rat and other species contain substance P (SP), somatostatin, neurotensin, enkephalin (ENK), cholecystokinin (CCK) and/or neuropeptide Y (NPY) and their mRNAs ( S P ; 15'17'22'35'37'54'64'85'86'96'97 s o m a t o s t a t i n ;2,19,33,61,64, 72,75.82 CCK ;12,20,22,39,83,103 *To whom correspondence should be addressed. Abbreviations: BSA, bovine serum albumin; CCK, chole- cystokinin; ENK, enkephalin; FITC, fluorescein isothiocyanate; GAD, glutamate decarboxylase; IR, immunoreactive; LDCV, large dense-cone vesicle; LI, like immunoreactivity; LRSC, lissamine rhodamine; NPY, neuropeptide Y; PB, phosphate buffer; PBS, phosphate-buffered saline; SP, substance P. NSC64/4~B n e u r o t e n s i n ; l 8,22,35,60,66.87 ENK;3,7A 7,23,3%35.37.45.60,63,80.91 NpyI'I6'21'33'35'38'55'56'69). It has been established that some of thes e peptides co-exist in local neurons which often also contain the principal transmitter GABA and its synthesizing enzyme glutamate decarboxylase (GAD) (see Ref. 99 and references below). On morphological grounds, several types of local neuron have been described in substantia gelatinosa, including islet cells, stalked cells, spiny cells and arboreal cells 2526,29(see Refs 8, 27, 109). Islet cells and stalked cells appear to be the most common local neurons. The islet cell somata have rostrocaudally enlongated dendritic arbors, which are largely confined to either lamina II0 or IIi, and their axons ramify within the region of the dendritic tree. The stalked cells have their cell bodies at the outer edge of lamina II near the border to lamina I. Their dendritic trees are directed mainly ventrally and to the medial and lateral sides. In the cat stalked cells and those islet cells with their dendritic arbors largely 875