Immunohistochemical localization of serotonin transporter in normal and colchicine treated rat brain
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.
Section snippets
Animals
Adult female Japanese white rabbits weighing 2.5–3.5 kg and adult male Wistar rats weighing 250–350 g (Clea, Osaka, Japan) were used in this study. They were fed and housed under regular conditions. Care of animals was conducted in accordance with the Guide to the Care and Use of Experimental Animals (Shiga University of Medical Science).
Antiserum production
The carboxyl terminal region (residues 617-630; CTPTEIPCGDIRMNAV) and amino terminal region (residues 1-14; CMETTPLNSQKVLSE) were selected as specific
Immunoblotting
Normal rabbit serum failed to detect any bands in the frontal cortex (Fig. 1, lane 1). SET C antibody stained the dense 56 kDa and faint 66 kDa bands in the frontal cortex with PNGase treatment (Fig. 1, lane 3), but only 66 kDa band was detected by the same antibody in the frontal cortex without PNGase treatment (Fig. 1, lane 2). The 56 kDa band was also detected in the PNGase-treated samples from COS-7 cells transfected with SET cDNA (Fig. 1, lane 5), while 85 kDa band was detected in the same
Specificity of serotonin transporter antibody
In the immunoblot analysis, the antibodies against C terminal region of SET detected two different bands of 66 and 85 kDa in the rat frontal cortex and COS-7 cells expressing recombinant SET, respectively. However, the molecular size of the both immunoreactive bands was reduced to 56 kDa by PNGase treatment, suggesting that the difference of the apparent molecular size of SET was due to the different glycosylation in the rat brain and COS-7 cells. Qian et al. (1995) also reported that 56 kDa
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