The Journal of neuroscience : the official journal of the Society for Neuroscience
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In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. ⋯ In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI.
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We investigated the mechanisms by which activation of group I metabotropic glutamate receptors (mGluRs) and CB1 cannabinoid receptors (CB1Rs) leads to inhibition of synaptic currents at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of the rat auditory brainstem. In approximately 50% of the MNTB neurons tested, activation of group I mGluRs by the specific agonist (s)-3,5-dihydroxyphenylglycine (DHPG) reversibly inhibited AMPA receptor- and NMDA receptor-mediated EPSCs to a similar extent and reduced paired-pulse depression, suggestive of an inhibition of glutamate release. Presynaptic voltage-clamp experiments revealed a reversible reduction of Ca2+ currents by DHPG, with no significant modification of the presynaptic action potential waveform. ⋯ The inhibition of EPSCs by DHPG, but not by WIN, was abolished after dialyzing 40 mm BAPTA into the postsynaptic cell, suggesting that DHPG activated postsynaptic mGluRs. Light and electron microscopy immunolabeling indicated a presynaptic expression of CB1Rs and postsynaptic localization of mGluR1a. Our data suggest that activation of postsynaptic mGluRs triggers the Ca2+-dependent release of endocannabinoids that activate CB1 receptors on the calyx terminal, which leads to a reduction of presynaptic Ca2+ current and glutamate release.
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We used a model system in which dopaminergic (DA) neurons from embryonic rat mesencephalon undergo spontaneous and selective degeneration as they develop in culture. Here, we show that DA cell loss can be prevented efficiently by low concentrations of the Na+ channel agonist veratridine. The survival promoting effect of veratridine was reproduced by, but independent of, glial cell line-derived neurotrophic factor. ⋯ Calcium measurements showed that veratridine-induced Na+ influx was necessary to maintain intracellular Ca2+ within a neuroprotective range through the stimulation of low-voltage activated T-type calcium channels, a mechanism that was distinct from that elicited by high K+-evoked depolarization. Interestingly, increasing neuronal excitability by treatment with apamin, an inhibitor of Ca2+-activated K+ channels, or with ouabain, a blocker of the Na+/K+-ATPase pump, was also neuroprotective by a mechanism involving T-type calcium channel activation. These results support the idea that mesencephalic DA neurons depend primarily on excitatory input for their survival during development.
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An in vivo fluorescent deltorphin (Fluo-DLT) internalization assay was used to assess the distribution and regulation of pharmacologically available delta opioid receptors (deltaORs) in the rat lumbar (L4-5) spinal cord. Under basal conditions, intrathecal injection of Fluo-DLT resulted in the labeling of numerous deltaOR-internalizing neurons throughout dorsal and ventral horns. The distribution and number of Fluo-DLT-labeled perikaryal profiles were consistent with that of deltaOR-expressing neurons, as revealed by in situ hybridization and immunohistochemistry, suggesting that a large proportion of these cells was responsive to intrathecally administered deltaOR agonists. ⋯ Unilateral dorsal rhizotomy also resulted in increased Fluo-DLT internalization in the ipsilateral dorsal horn when compared with the side contralateral to the deafferentation or to non-deafferented controls, suggesting that deltaOR trafficking in dorsal horn neurons may be regulated by afferent inputs. Furthermore, morphine treatment no longer increased Fluo-DLT internalization on either side of the spinal cord after unilateral dorsal rhizotomy, indicating that microOR-induced changes in the cell surface availability of deltaOR depend on the integrity of primary afferent inputs. Together, these results suggest that regulation of deltaOR responsiveness through microOR activation in this region is linked to somatosensory information processing.
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TRPM8 is a nonselective cation channel activated by cold and the cooling compounds menthol and icilin (Peier et al., 2002). Here, we have used electrophysiology and the calcium-sensitive dye Fura-2 to study the effect of pH and interactions between temperature, pH, and the two chemical agonists menthol and icilin on TRPM8 expressed in Chinese hamster ovary cells. Menthol, icilin, and cold all evoked stimulus-dependent [Ca2+]i responses in standard physiological solutions of pH 7.3. ⋯ In voltage-clamp experiments in which the intracellular pH was buffered to different levels, acidification reduced the current amplitude of icilin responses and shifted the threshold for cold activation to lower values with half-maximal inhibition at pH 7.2 and pH 7.6. The results demonstrate that the activation of TRPM8 by icilin and cold, but not menthol, is modulated by intracellular pH in the physiological range. Furthermore, our data suggest that activation by icilin and cold involve a different mechanism to activation by menthol.