BackgroundThe glial glutamate transporter GLT-1 is abundantly expressed in astrocytes and is crucial for glutamate removal from the synaptic cleft. Decreases in glutamate uptake activity and expression of spinal glutamate transporters are reported in animal models of pathological pain. However, the lack of available specific inhibitors and/or activators for GLT-1 makes it difficult to determine the roles of spinal GLT-1 in inflammatory and neuropathic pain. In this study, we examined the effect of gene transfer of GLT-1 into the spinal cord with recombinant adenoviruses on the inflammatory and neuropathic pain in rats.ResultsIntraspinal infusion of adenoviral vectors expressing the GLT-1 gene increased GLT-1 expression in the spinal cord 2–21 days after the infusion. Transgene expression was primarily localized to astrocytes. The spinal GLT-1 gene transfer had no effect on acute mechanical and thermal nociceptive responses in naive rats, whereas it significantly reduced the inflammatory mechanical hyperalgesia induced by hindlimb intraplantar injection of carrageenan/kaolin. Spinal GLT-1 gene transfer 7 days before partial sciatic nerve ligation recovered the extent of the spinal GLT-1 expression in the membrane fraction that was decreased following the nerve ligation, and prevented the induction of tactile allodynia. However, the partial sciatic nerve ligation-induced allodynia was not reversed when the adenoviruses were infused 7 or 14 days after the nerve ligation.ConclusionThese results suggest that overexpression of GLT-1 on astrocytes in the spinal cord by recombinant adenoviruses attenuates the induction, but not maintenance, of inflammatory and neuropathic pain, probably by preventing the induction of central sensitization, without affecting acute pain sensation. Upregulation or functional enhancement of spinal GLT-1 could be a novel strategy for the prevention of pathological pain.

Glutamate uptake by the Na(+)-dependent glutamate transporter GLT-1, which is predominantly expressed in astrocytes, is crucial for regulating glutamate concentration at the synaptic cleft and achieving proper excitatory neurotransmission. A body of evidence suggests that GLT-1 constitutively traffics between the plasma membrane and endosomes via an endocytosis/recycling pathway, and forms a cluster. Here, we report substrate transport via GLT-1-induced formation of GLT-1 cluster accompanied by intracellular trafficking in rat astroglial-neuronal cultures. We constructed a recombinant adenovirus expressing enhanced green fluorescence protein (EGFP)-tagged GLT-1. Adenoviral infection resulted in the expression of functional GLT-1-EGFP preferentially in astrocytes, partly as clusters. Treatment with glutamate, but not N-methyl-D-aspartate, dramatically increased the number of GLT-1 clusters within 1 h. The estimated EC(50) value of glutamate was 240 microm. In addition, glutamate decreased the cell surface expression and increased the intracellular expression of GLT-1. The GLT-1 clusters were found in early and recycling endosomes and partly in lysosomes, and were inhibited by blockade of endocytotic pathways. Ionotropic and metabotropic glutamate receptor antagonists had no effect on glutamate-induced GLT-1 clustering. The non-transportable glutamate uptake inhibitors (2S,3S)-3-[3-[4-(trifluoromethyl)benzoylamino]benzyloxy]aspartate and dihydrokainate, as well as Na(+)-free conditions, prevented the glutamate-induced GLT-1 clustering, whereas the competitive substrates, aspartate and L-trans-pyrrolidine-2,4-dicarboxylate, induced GLT-1 clustering. Furthermore, the Na(+)/K(+)-ATPase inhibitor, ouabain, and the Na(+) ionophores, gramicidin and monensin, produced GLT-1 clustering. Modulators of intracellular Ca(2+)signaling or membrane depolarization had no effect on GLT-1 clustering. Taken together, these results suggest that Na(+) influx associated with GLT-1 substrate transport triggers the formation of GLT-1 clusters accompanied by intracellular trafficking via endocytotic pathways in astrocytes.

Several lines of evidence suggest the involvement of the raphe‐serotonergic neurons in addiction to psychostimulants and some recreational drugs. In this study, we established rat organotypic mesencephalic slice cultures containing the raphe nuclei and examined the effects of sustained exposure to 3,4‐methylenedioxymethamphetamine (MDMA) and methamphetamine (METH). Immunostaining for tryptophan hydroxylase (TPH) studies revealed that serotonergic neurons were abundant in the slice cultures. Sustained exposure to MDMA and METH (1–1000 μM) for 4 days had little effect on the serotonin tissue content, [3H]citalopram binding, or expression/phosphorylation of TPH. Treatment with MDMA or METH for 30 min increased serotonin release in a concentration‐dependent manner. Slice cultures were exposed to MDMA for 4 days following a 1‐day withdrawal period and then challenged with MDMA (10 μM). Sustained MDMA exposure augmented MDMA‐induced serotonin release in a concentration‐dependent manner, indicating serotonergic sensitization. Similar serotonergic sensitization was observed for METH. The development of MDMA‐induced serotonergic sensitization was attenuated by the NMDA receptor antagonist, MK‐801 (10 μM). These results suggest that in mesencephalic slice cultures sustained MDMA or METH exposure induces serotonergic sensitization through activation of NMDA receptors without serotonergic neurotoxicity. The in vitro model system could help to elucidate the mechanisms underlying drug addiction.

Selective 5-hydroxytryptamine (5-HT, serotonin) reuptake inhibitors (SSRIs) are widely used antidepressants and their therapeutic effect requires several weeks of drug administration. The delayed onset of SSRI efficacy is due to the slow neuroadaptive changes of the 5-hydroxytryptaminergic (5-HTergic) system. In this study, we examined the acute and chronic effects of SSRIs on the 5-HTergic system using rat raphe slice cultures. EXPERIMENTAL APPROACHFor organotypic raphe slice cultures, mesencephalic coronal sections containing dorsal and median raphe nuclei were prepared from neonatal Wistar rats and cultured for 14-16 days. KEY RESULTSAcute treatment with citalopram, paroxetine or fluoxetine (0.1-10 mM) in the slice cultures slightly increased extracellular 5-HT levels, while sustained exposure for 4 days augmented the elevation of 5-HT level in a time-dependent manner. Sustained exposure to citalopram had no effect on tissue contents of 5-HT and its metabolite, expression of tryptophan hydroxylase or the membrane expression of 5-HT transporters. The augmented 5-HT release was attenuated by Ca 2+ -free incubation medium or treatment with tetrodotoxin. Experiments with 5-HT1A/B receptor agonists and antagonists revealed that desensitization of 5-HT1 autoreceptors was not involved in the augmentation of 5-HT release. Finally, co-treatment with an a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, but not an N-methyl-D-aspartate, receptor antagonist, suppressed this augmentation. CONCLUSION AND IMPLICATIONSThese results suggest that sustained exposure to SSRIs induces the augmentation of exocytotic 5-HT release, which is caused, at least in part, by the activation of AMPA/kainate receptors in the raphe slice cultures.Abbreviations 5-HIAA, 5-hydroxyindolacetic acid; 8-OH-DPAT, 8-hydroxy-2-(di-n-propylamino)tetralin; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; DMSO, dimethyl sulfoxide; GTPgS, guanosine 5′- [g-thio]triphosphate; KRH, Krebs-Ringer-Henseleit; MDMA, 3,4-methylenedioxymethamphetamine; NMDA, N-methyl-D-aspartate; PBS, phosphate-buffered saline; SERT, 5-HT (serotonin) transporter; SSRI, selective 5-HT (serotonin) reuptake inhibitor; TPH, tryptophan hydroxylase; TTX, tetrodotoxin BJP British Journal of Pharmacology DOI:10.1111DOI:10. /j.1476DOI:10. -5381.2010 British Journal of Pharmacology (2010) IntroductionDepression is a major health problem around the world, and the lifetime prevalence of major depressive disorder is 10 to 20% (Kessler et al., 2003). Because the sites of action of early types of antidepressant drugs, such as the tricyclic antidepressants and monoamine oxidase inhibitors, lie mostly in the monoaminergic system, major depressive disorder has been associated with hypofunction of the central monoaminergic system, in particular the 5-hydroxytryptaminergic (5-HTergic) system (Owens and Nemeroff, 1994;Belmaker and Agam, 2008). Selective 5-hydroxytryptamine (5-HT, serotonin) reuptake inhibitors (SSRIs) are now one ...