Expression of galanin and nitric oxide synthase in subpopulations of serotonin neurons of the rat dorsal raphe nucleus

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Abstract

The dorsal raphe nucleus (DR) of the rat was studied with triple labeling immunofluorescence histochemistry to evaluate the quantitative relationships between neurons expressing 5-hydroxytryptamine (5-HT), the neuropeptide galanin (GAL) and the nitric oxide (NO) synthesizing enzyme NO synthase (NOS). In addition retrograde tracing studies were performed. It could be established that a high percentage (between 40 and 60%) of the 5-HT neuron profiles in the ventromedial and dorsomedial subgroups of the DR contained both GAL and NOS after colchicine treatment. This triple coexistence was lower in the dorso-lateral subgroup and much lower in the lateral subgroup (down to 5%). All GAL neuron profiles contained 5-HT, and they constituted up to 80% of all labeled profiles in the rostral ventromedial and caudal dorsomedial subgroups, with the lowest percentage in the lateral subgroup (45%). The percentage of 5-HT-alone neuron profiles in these four subgroups varied between 15–40%. The proportion of 5-HT/NOS neurons was low (a few percent) at all levels, and this was the case also for NOS-alone neuron profiles except in the lateral subgroup, where 10–20% were of this type. It could be established that some 5-HT/GAL/NOS neurons project to the striatum. These retrogradely labeled cells were mainly found in the mid-line subgroups. In the striatum a moderately dense 5-HT fiber network and numerous NOS-positive cell bodies and fibers could be observed. However, only a few, weakly fluorescent GAL fibers were found and in a small number of cases it could be shown or was likely that 5-HT and GAL coexisted. No evidence for coexistence between 5-HT and NOS was obtained. The present findings strongly suggest that a large proportion of the DR 5-HT neurons can synthesize and release two additional messenger molecules, GAL and NO. Furthermore, even if the 5-HT/GAL/NOS neurons project to the striatum, the amounts of GAL and NOS transported to the terminal ramifications in this area are very low. This is in agreement with a very low GAL synthesis in the DR under normal circumstances, which is also indicated by the fact that colchicine treatment is needed to visualize GAL-like immunoreactivity in DR cell bodies. It is possible that NO, and perhaps GAL, in 5-HT neurons exert their main actions at the somatic and dendritic level in the DR.

Introduction

The raphe regions of the brain stem harbour the major serotonin 5-hydroxytryptamine (5-HT) containing cell groups in the rat brain. Thus, these neurons and their location and distribution have been extensively described using several histochemical methods, first by Dahlström and Fuxe (1964) using the Falck-Hillarp formaldehyde-induced fluorescence method and subsequently with autoradiography (Chan-Palay, 1977) and immunohistochemistry (Steinbusch, 1981;Takeuchi et al., 1982;Jacobs et al., 1984;Clements et al., 1985). The dorsal raphe nuclei (DR) in the periaqueductal central grey harbour the highest density of 5-HT-containing neurons, and they send ascending projections to the di- and telencephalon in the rat brain (Fuxe, 1965;Andén et al., 1966;Steinbusch, 1981; see Törk, 1988). The serotoninergic systems arising from the raphe nuclei are implicated in a wide range of functions (for review, see Whitaker-Azmitia and Peroutka, 1990).

Galanin (GAL) is a 29 amino acid peptide first isolated from the porcine gut (Tatemoto et al., 1983). Based on immunocytochemical studies, GAL has been shown to occur in many areas of the central nervous system (CNS) (Rökaeus et al., 1984;Skofitsch and Jacobowitz, 1985;Melander et al., 1986a) and may represent a putative peptide messenger molecule (for review, see Skofitsch and Jacobowitz, 1990;Hökfelt et al., 1991;Bartfai et al., 1993;Merchenthaler et al., 1993). GAL is co-localized with a variety of classical transmitters and other peptides (Melander et al., 1986b) and modulates 5-HT neurotransmission in the CNS (Fuxe et al., 1988;Sundström and Melander, 1988;Ögren and Fuxe, 1989;Fuxe et al., 1990;Hedlund and Fuxe, 1991;Hedlund et al., 1991a, Hedlund et al., 1991b). Thus, GAL may have a role in regulating the activity of DR neurons, a view supported by electrophysiological results showing that GAL hyperpolarizes 5-HT-containing neurons in DR slices (Xu et al., 1994a).

Accumulating evidence suggests that nitric oxide (NO), originally discovered as an endothelial derived relaxing factor by Furchgott and Zawadzki (1980), acts as a short-lived intercellular messenger that can strongly influence cellular functions in the nervous system (Dawson et al., 1994;Vincent, 1994;Garthwaite and Boulton, 1995). Nitric oxide synthase (NOS) is the key enzyme for NO biosynthesis and forms NO together with citrulline from L-arginine (Palmer et al., 1987, Palmer et al., 1988;Moncada, 1992). A constitutive, calcium/calmodulin-dependent NOS present in neurons (nNOS) has been purified (Bredt and Snyder, 1990) and cloned (Bredt et al., 1991b), and a wide spread distribution of NOS-expressing neurons has been demonstrated in the brain using both immnohistochemistry and in situ hybridization (Bredt et al., 1990;Bredt et al., 1991a). It has been recognized that the nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemical method, first described by Thomas and Pearse (1964), can be used to demonstrate NOS containing neurons (Dawson et al., 1991;Hope et al., 1991). The results with this method (Vincent and Kimura, 1992) agree well with the above mentioned in situ and immunohistochemical analyses, including the demonstration of NOS in the DR region.

The occurrence of GAL, NOS and 5-HT in many neurons in the DR nuclei suggests a possible colocalization of these messenger molecules. In fact, GAL has been shown to be present in many 5-HT neurons in the DR (Melander et al., 1986b;Fuxe et al., 1990;Priestley et al., 1993), and more recently NOS has also been shown to coexist with 5-HT in DR neurons using the NADPH-d method (Johnson and Ma, 1993;Wotherspoon et al., 1994) and NOS immunohistochemistry (Wang et al., 1995). However, so far there are no studies on the colocalization of all three of these messengers in the rat DR. In this study we used a triple-labeling technique to study quantitatively and in detail the distribution of GAL and NOS and their colocalization with 5-HT in the DR. NADPH-d histochemistry was also combined with immunofluorescence histochemistry for GAL, NOS and 5-HT. In most instances an antiserum raised against galanin message-associated peptide (GMAP) was used (Hökfelt et al., 1992). GMAP is a 60 amino acid peptide expressed in a 1:1 ratio on the GAL precursor (Rökaeus and Brownstein, 1986), and the GMAP antiserum has been shown to label all GAL neurons in the rat nervous system (Hökfelt et al., 1992;Xu et al., 1995). Finally, the projection of the 5-HT, GAL and NOS neurons to the striatum was analysed using Fluoro-Gold (FG) as retrograde tracer. These studies form a basis for a neurophysiological analysis of the effects of GAL and NO on 5-HT neurons in the DR (Xu et al., 1994a).

Section snippets

Animals

Male Sprague-Dawley rats (bwt. 150–300 g; B&K, Stockholm, Sweden) were used in the present study. All animals were treated according to guidelines approved by a local ethical committee (Stockholms Norra Djuförsöksetiska Nämnd).

Tracer injection

Five rats were deeply anesthetized by intraperitoneal injection of sodium pentobarbital (65 mg/kg) and placed in a stereotaxic frame. The stereotaxic coordinates used for retrograde tracing from the striatum were determined from the atlas of Paxinos and Watson (1986). The

Results

The results described below were, unless otherwise stated, collected from the eight colchicine treated rats. In all these animals the patterns of distribution of, respectively, 5-HT-, NOS-, GMAP/GAL-, NADPH-d-positive neurons and fibers in the DR was similar. Colchicine induced an increase in the levels of 5-HT-LI but had no distinct effect on NOS-LI or NADPHd staining, which thus were similar to those seen in untreated rats. GMAP/GAL-LI could only be observed in cell bodies of colchicine

Discussion

Early pharmacological evidence indicated that serotonin has a transmitter role in the brain (Pletscher et al., 1955; see Carlsson, 1966). This was supported by the first histochemical description of descrete groups of 5-HT neurons in various brain stem raphe regions, including the DR, as well as widespread distribution patterns of nerve terminals (Dahlström and Fuxe, 1964;Dahlström and Fuxe, 1965;Fuxe, 1965;Steinbusch, 1981; see Törk, 1988). Since then the ascending 5-HT systems originating in

Acknowledgements

This study was supported by the Swedish MRC (04X-2887), Marianne and Marcus Wallenbergs Stiftelse, Konung Gustav V:e och Drottning Victorias Stiftelse, Astra Arcus AB and the National Institute of Aging (AG-10491). We thank Dr K. Bedecs and Dr T. Bartfai, Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden (GMAP), Dr P. Emson, Department of Neurobiology, The Babraham Institute, Babraham, Cambridge, UK (NOS), Dr E. Theodorsson, Department of Clinical

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