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A biologically active lipid, sphingosine-1-phosphate (S1P) is highly abundant in blood, and plays an important role in regulating the growth, survival, and migration of many cells. Binding of the endogenous ligand S1P results in activation of various signaling pathways via G protein-coupled receptors, some of which generates Ca2+ mobilization. In astrocytes, S1P is reported to evoke Ca2+ signaling, proliferation, and migration; however, the precise mechanisms underlying such responses in astrocytes remain to be elucidated. Transient receptor potential canonical (TRPC) channels are Ca2+ -permeable cation channels expressed in astrocytes and involved in Ca2+ influx after receptor stimulation. In this study, we investigated the involvement of TRPC channels in S1P-induced cellular responses. In Ca2+ imaging experiments, S1P at 1 μM elicited a transient increase in intracellular Ca2+ in astrocytes, followed by sustained elevation. The sustained Ca2+ response was markedly suppressed by S1P2 receptor antagonist JTE013, S1P3 receptor antagonist CAY10444, or non-selective TRPC channel inhibitor Pyr2. Additionally, S1P increased chemokine CXCL1 mRNA expression and release, which were suppressed by TRPC inhibitor, inhibition of Ca2+ mobilization, MAPK pathway inhibitors, or knockdown of the TRPC channel isoform TRPC6. Taken together, these results demonstrate that S1P induces Ca2+ signaling in astrocytes via Gq -coupled receptors S1P2 and S1P3 , followed by Ca2+ influx through TRPC6 that could activate MAPK signaling, which leads to increased secretion of the proinflammatory or neuroprotective chemokine CXCL1.

Valuable synthetic routes to the Lycopodium alkaloid lycodine (1) and its unsymmetric dimers, complanadines A (4) and B (5), have been developed. Regioselective construction of the bicyclo[3.3.1]nonane core structure of lycodine was achieved by a remote functionality-controlled Diels-Alder reaction and subsequent intramolecular Mizoroki-Heck reaction. A key coupling reaction of the lycodine units, pyridine N-oxide (66) and aryl bromide (65), through C-H arylation at the C1 position of 66 provided the unsymmetric dimer structure at a late stage of the synthesis. This strategy greatly simplified the construction of the dimeric architecture and functionalization. Complanadines A (4) and B (5) were synthesized by adjusting the oxidation level of the bipyridine mono-N-oxide (67). The diverse utility of this common intermediate (67) suggests a possible biosynthetic pathway of complanadines in Nature. Both enantiomers of lycodine (1) and complanadines A (4) and B (5) were prepared in sufficient quantities for biological evaluation. The effect on neuron differentiation of PC-12 cells upon treatment with culture medium, in which human astrocytoma cells had been cultured in the presence of 1, 4, or 5 was evaluated.

Microglia, the resident immune cells in the brain, survey the environment of the healthy brain. Microglial migration is essential for many physiological and pathophysiological processes. Although microglia express some members of the transient receptor potential (TRP) channel family, there is little knowledge regarding the physiological roles of TRP channels in microglia. Here, we explored the role of TRP vanilloid 1 (TRPV1), a channel opened by capsaicin, heat, protons, and endovanilloids, in microglia. We found that application of capsaicin induced concentration-dependent migration in microglia derived from wild-type mice but not in those derived from TRPV1 knockout (TRPV1-KO) mice. Capsaicin-induced microglial migration was significantly inhibited by co-application of the TRPV1 blocker SB366791 and the Ca(2+) chelator BAPTA-AM. Using RT-PCR and immunocytochemistry, we validated that TRPV1 was expressed in microglia. Electrophysiological recording, intracellular Ca(2+) imaging, and immunocytochemistry indicated that TRPV1 was localized primarily in intracellular organelles. Treatment with capsaicin induced an increase in intramitochondrial Ca(2+) concentrations and mitochondrial depolarization. Furthermore, microglia derived from TRPV1-KO mice showed delayed Ca(2+) efflux compared with microglia derived from wild-type mice. Capsaicin-induced microglial migration was inhibited by membrane-permeable antioxidants and MAPK inhibitors, suggesting that mitochondrial TRPV1 activation induced Ca(2+) -dependent production of ROS followed by MAPK activation, which correlated with an augmented migration of microglia. Moreover, a mixture of three endovanilloids augmented microglial migration via TRPV1 activation. Together, these results indicate that mitochondrial TRPV1 plays an important role in inducing microglial migration. Activation of TRPV1 triggers an increase in intramitochondrial Ca(2+) concentration and following depolarization of mitochondria, which results in mtROS production, MAPK activation, and enhancement of chemotactic activity in microglia.

Microglia are intrinsic immune cells in the brain. In response to neurodegenerative events, excessively activated microglia change their shapes and release various cytokines leading to the pathogenesis of central nervous system (CNS) disease. Because the intracellular mechanisms of this process are still unclear, we have evaluated the functional roles of transient receptor potential vanilloid 4 (TRPV4) channel expressed in the microglia. Robust microglial activation after an injection of lipopolysaccharide (LPS) into the mouse cerebral ventricle was suppressed by concurrent administration of a selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD). When the mechanism was further investigated using cultured rat microglia intrinsically expressing functional TRPV4, release of tumor necrosis factor-α (TNF-α) and expression of galectin-3 were both increased by LPS. These increases were significantly suppressed by cotreatment with 4α-PDD, and the inhibitory effects of 4α-PDD were abolished by knockdown of TRPV4 or TRPV4 antagonists. The amplitude of voltage-dependent K+ current, which is augmented during microglial activation, was also suppressed by 4α-PDD treatment. Opening of TRPV4 channels with 4α-PDD induced membrane depolarization mainly by increasing Na+ influx. In addition, mimicking depolarization with a high-K+ solution suppressed LPS-induced TNF-α release and galectin-3 upregulation. Both depolarizing treatments with 4α-PDD and high-K+ solution decreased store-operated Ca2+ influx caused by thapsigargin. These results suggest that depolarization in response to opening of the TRPV4 channel attenuates the driving force for extracellular Ca2+ and suppresses microglial activation. © 2012 Wiley Periodicals, Inc.

BACKGROUND AND PURPOSE 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 APPROACH For 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 RESULTS Acute treatment with citalopram, paroxetine or fluoxetine (0.1–10 µM) 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 Ca2+-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 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate, but not an N-methyl-d-aspartate, receptor antagonist, suppressed this augmentation. CONCLUSION AND IMPLICATIONS These 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.

Activated astrocytes show various patterns of Ca(2+) mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca(2+) dynamics through investigation of thrombin-induced unique Ca(2+) entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03-3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca(2+) concentration ([Ca(2+)](i)) showed that thrombin induced heterogeneous Ca(2+) responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca(2+) release from intracellular stores, followed by refilling of Ca(2+) from the extracellular region without a direct [Ca(2+)](i) increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca(2+) mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca(2+) entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca(2+) responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca(2+) influx. Moreover, TRPC3-mediated Ca(2+) dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca(2+) dynamics, including Ca(2+) entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca(2+) channel and might modulate astrocytic activation in response to brain hemorrhaging.

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 microM) for 4 days had little effect on the serotonin tissue content, [(3)H]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 microM). 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 microM). 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.

Aminoglutethimide is a clinically available drug that suppresses steroid biosynthesis by inhibiting enzymes such as cytochrome P450scc and aromatase. Because several members of neurosteroids regulate glutamate receptors, we investigated the effect of aminoglutethimide on cell death induced by overactivation of glutamate receptors in CNS neurons. Long-term pretreatment of organotypic cerebrocortical slice cultures with aminoglutethimide (100–1000 μM) for 6 days or over resulted in concentration-dependent suppression of neuronal cell death induced by NMDA. Aminoglutethimide (1000 μM) also inhibited neurotoxicity of AMPA and kainate, but not of ionomycin or staurosporine. The protective effect of aminoglutethimide against NMDA cytotoxicity was not mimicked by other steroid synthesis inhibitors including trilostane and exemestane, and was not reversed by concurrent application of steroids such as pregnenolone, estrone, 17β-estradiol and estriol. In dissociated rat cerebrocortical cell cultures, long-term treatment with aminoglutethimide (10–1000 μM) attenuated NMDA receptor-mediated glutamate cytotoxicity but produced no significant effect on glutamate-induced increases in intracellular Ca2+. Brief as well as long-term pretreatment with aminoglutethimide (30–1000 μM) prevented NMDA receptor-dependent ischemic neuronal injury in organotypic cerebrocortical slice cultures, which was associated with suppression of glutamate release during the ischemic insult. These results indicate that aminoglutethimide, irrelevant to its actions on neurosteroid synthesis, protects CNS neurons from excitotoxic and ischemic injuries. Development of aminoglutethimide analogs possessing neuroprotective properties may be of therapeutic value. British Journal of Pharmacology (2006) 147, 729–736. doi:10.1038/sj.bjp.0706636

Neuroactive steroids can modulate brain excitability by interaction with several neurotransmitter receptor-associated channels. These compounds may thus exert profound influences on excitotoxic injury, i.e. neuronal cell death triggered by over-activation of glutamate receptors. It has been reported that pregnenolone sulphate (PS) and pregnenolone hemisuccinate (PHS) augment N-methyl-D-aspartate (NMDA) neurotoxicity in rat cultured neurons. Here we show that the effects of neuroactive steroids on AMPA cytotoxicity display features distinct from those on NMDA cytotoxicity. Concomitant application of PS (30-300 microm) attenuated, rather than augmented, AMPA neurotoxicity in cortical slice cultures in a concentration-dependent manner, whereas various other steroids including pregnenolone and PHS had no effect. Inhibition of steroid sulphatase by estrone-3-O-sulphamate led to a shift of the minimum effective concentration of PS against AMPA cytotoxicity from 30 to 10 microm. The protective action of PS was not affected by inhibition of protein synthesis or by blockade of glucocorticoid receptors, GABAA receptors or sigma-receptors. In dissociated cortical neurons, PS attenuated AMPA-induced inward currents whereas pregnenolone and PHS exhibited no significant effect. Thus, with strict structural specificity, PS but not pregnenolone or PHS attenuates AMPA cytotoxicity, probably by inhibiting activities of AMPA receptor-associated channels.

We have previously demonstrated that dopaminergic neurons in midbrain-striatum slice co-cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain-striatum co-cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) and 3-morpholinosydnonimine (SIN-1). The cytotoxicity of NMDA (30 microM) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (100 microM), whereas the toxicity in co-cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co-cultures than those in single cultures. Single cultures and co-cultures did not show appreciable differences in the number or distribution of NOS-containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn-SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co-cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.