HIUROSCIUIC[ IEIT[IIS ELSEVIER Neuroscience Letters 187 (1995) 119-122 Galanin is contained in GABAergic neurons in the rat spinal dorsal horn D.R. Simmons, R.C. Spike, A.J. Todd* Laboratory of Human Anatomy, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK Received 11 January 1995; revisedversionreceived25 January 1995; accepted27 January 1995 Abstract In order to determine which types of neuron in laminae I-III of the rat spinal dorsal horn contain the peptide galanin, pre-embedding immunocytochemistry with antiserum to galanin was combined with post-embedding detection of GABA- and glycine-like immunoreactivities. Sixty-eight galanin immunoreactive neurons in laminae I-III selected from four rats were examined, and in each case semi-thin sections through the cell body were tested with a monoclonal antibody to GABA and an antiserum to glycine. All of the 68 galanin-immunoreactive neurons tested were GABA-immunoreactive, while only one of them (in lamina III) was glycine- immunoreactive. This suggests that galanin is contained in inhibitory intemeurons, and that (like enkephalin, neuropeptide Y and thyrotropin-releasing hormone) it is mainly restricted to GABAergic neurons which do not use glycine as a co-transmitter. Keywords: Immunocytochemistry; Spinal cord; Glycine; Peptides; Transmitter co-localisation Galanin is a peptide containing 29 amino acids, which was originally isolated from pig intestine [14], and has since been found in various parts of the central nervous system [1,7,8,10]. Many galanin-immunoreactive axons are present in laminae I and II of the rat spinal dorsal horn [1,7,8,10,21,23,24], and these are thought to be derived from both dorsal root ganglion cells and local interneu- rons [1,5,11,17,21,23,24]. Galanin-immunoreactive neu- rons in the superficial laminae of the dorsal horn were initially observed after intrathecal administration of col- chicine [1,7,8], however with more sensitive immunocy- tochemical techniques they can be detected without appli- cation of colchicine [23,24]. At present, relatively little is known about the types of dorsal horn neuron that contain galanin. Attempts have been made to classify neurons in the superficial dorsal horn on both morphological and neurochemical grounds (for review, see Ref. [17]). Within lamina II, two main types of neuron have been identified: stalked and islet cells [4]. Stalked cells have their cell bodies in the dorsal part of the lamina and dendrites that pass ventrally, while islet cells have long, rostrocaudally orientated dendrites. There is a relationship between mor- phology and transmitter content, since large islet cells in lamina II are GABA-immunoreactive, whereas stalked * Corresponding author. Tel.: +44 41 3398855; Fax: +44 41 3304299. cells and a population of small islet cells are not [15,17]. Glycine-like immunoreactivity (-LI) is also observed in cell bodies in laminae I-III, but is virtually restricted to cells that are GABA-immunoreactive, and it has been suggested that these neurons release both inhibitory transmitters [20]. Zhang et al. [24] have recently reported that galanin-immunoreactive neurons in lamina II of rat dorsal horn resemble islet cells, and since many of these are thought to be inhibitory interneurons which release GABA (with or without glycine), the present study was carried out to determine whether galanin co-exists with GABA or glycine in cells of the superficial dorsal horn. Pre-embedding immunocytochemistry to detect gala- nin in cell bodies was combined with post-embedding detection of GABA- and glycine-LI as described previ- ously [3,9]. Four adult Albino-Swiss rats (either sex; 220- 280 g) were deeply anaesthetised and perfused with a fixative containing 1% glutaraldehyde/l% formaldehyde in 0.1 M phosphate buffer (PB). Segments of mid-lumbar spinal cord were removed, stored in the same fixative overnight and cut into 70-/tm transverse sections with a Vibratome. The sections were treated in 1% sodium bo- rohydride for 30 min, rinsed extensively in phosphate buffered saline (PBS), incubated for 1 h in 10% goat se- rum in PBS and then overnight in rabbit antiserum to rat galanin (Peninsula) diluted 1:10000, and processed by 0304-3940/95/$09.50 © 1995 ElsevierScience IrelandLtd. All rights reserved SSDI 0304-3940(95)11358-Z