Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain

Abstract

Distribution of serotonin transporter (SET) was examined immunohistochemically in the rat brain using two specific polyclonal antibodies raised against oligopeptides corresponding with 15 amino acids of carboxyl terminus and 14 amino acids of amino terminus of rat SET. The distribution and density of SET immunoreactive varicose fibers were quite similar to those of serotonin immunoreactive fibers, however no neuronal cell bodies in the brainstem raphe nuclei was stained in normal rat brain. Electron microscopic study showed that SET immunoreactivity was predominantly localized in the presynaptic terminals. After intraventricular infusion of colchicine, neuronal perikarya of dorsal, median, and pontine raphe nuclei became visible. These results suggest that SET is likely present at the synaptic terminals of serotonergic neurons and such localization may be in good agreement with its pharmacological action which includes reuptake of serotonin at presynaptic nerve terminals.

Introduction

Serotonin transporter (SET) has been considered to exist in the presynaptic membrane of the serotonin (5HT) neurons and play an important role in terminating the 5HT transmission (Fuller and Wong, 1990, Amara and Kuhar, 1993, Rudnick and Clark, 1993, Schloss et al., 1994, Worrall and Williams, 1994). Previously the presence of SET in the brain has been shown indirectly by the uptake of radiolabeled 5HT into the brain slices (Blackburn et al., 1967, Bjökland et al., 1973, Azmitia and Marovitz, 1980) or isolated neurons (Bruns et al., 1993) in addition to the autoradiographic or binding studies of radiolabeled SET antagonists (Sette et al., 1981, Hrdina et al., 1990, Barone et al., 1994). Recently, cDNAs clone for SET have been isolated from the rat brain (Blakely et al., 1991) as well as from the rat basophilic leukemia cells (Hoffman et al., 1991). In situ hybridization histochemistry revealed that SET mRNA is located in neuronal cell bodies in the raphe nuclei (Fujita et al., 1993) and more sensitive reverse transcription/polymerase chain reaction have shown the wide distribution of SET mRNA in the rat brain including frontal cortex, hippocampus and neostriatum (Lesch et al., 1993). Direct evidence for the localization of SET in the rat brain has been shown by using the specific antibody against SET. In previous immunohistochemical studies, SET immunoreactivity was found in nerve fibers widely distributed in the brain as well as in neuronal cell bodies in the raphe nuclei and its distribution was in good agreement with the serotonergic innervation (Qian et al., 1995, Sur et al., 1996, Zhou et al., 1996). However positive staining of SET in neuronal cell bodies is not consistent with the role of this transporter including uptake of 5HT from the synaptic cleft at nerve terminals. In fact, other sodium and chloride ion-dependent transporters, GABA (GAT1) or glycine transporter (GLYT2) was shown to locate in nerve terminals but not in neuronal cell bodies (Ikegaki et al., 1994, Minelli et al., 1995, Zafra et al., 1995, Itouji et al., 1996, Ribak et al., 1996). In the present study, we raised two specific antibodies against SET and immnohistochemical localization of SET was thoroughly examined, with special reference to the staining pattern between normal and colchicine treated animals.