Guang Bai

Molecular basis of NMDA receptor-coupled ion channel modulation by S-nitrosylation.

Several ion channels are thought to be directly modulated by nitric oxide (NO), but the molecular basis of this regulation is unclear. Here we show that the NMDA receptor (NMDAR)-associated ion channel was modulated not only by exogenous NO but also by endogenous NO. Site-directed mutagenesis identified a critical cysteine residue (Cys 399) on the NR2A subunit whose S-nitrosylation (NO+ transfer) under physiological conditions underlies this modulation. In cell systems expressing NMDARs with mutant NR2A subunits in which this single cysteine was replaced by an alanine, the effect of endogenous NO was lost. Thus endogenous S-nitrosylation can regulate ion channel activity.

Acute Cannabinoids Impair Working Memory through Astroglial CB1 Receptor Modulation of Hippocampal LTD

Impairment of working memory is one of the most important deleterious effects of marijuana intoxication in humans, but its underlying mechanisms are presently unknown. Here, we demonstrate that the impairment of spatial working memory (SWM) and in vivo long-term depression (LTD) of synaptic strength at hippocampal CA3-CA1 synapses, induced by an acute exposure of exogenous cannabinoids, is fully abolished in conditional mutant mice lacking type-1 cannabinoid receptors (CB(1)R) in brain astroglial cells but is conserved in mice lacking CB(1)R in glutamatergic or GABAergic neurons. Blockade of neuronal glutamate N-methyl-D-aspartate receptors (NMDAR) and of synaptic trafficking of glutamate α-amino-3-hydroxy-5-methyl-isoxazole propionic acid receptors (AMPAR) also abolishes cannabinoid effects on SWM and LTD induction and expression. We conclude that the impairment of working memory by marijuana and cannabinoids is due to the activation of astroglial CB(1)R and is associated with astroglia-dependent hippocampal LTD in vivo.

Molecular Depletion of Descending Serotonin Unmasks Its Novel Facilitatory Role in the Development of Persistent Pain

Recent studies indicate that persistent pain after tissue or nerve injury is accompanied by an enhanced net descending facilitatory drive that contributes to an amplification and spread of pain. Although 5-HT-containing neurons in the rostral ventromedial medulla (RVM) provide the major descending serotonergic projection to the spinal cord, it is not clear whether the neurotransmitter 5-HT itself released from RVM-spinal neurons contributes to descending pain modulation. In the present study, we determined the role of the descending 5-HT in rat nocifensive behaviors after persistent pain by selectively depleting functional phenotypes of 5-HT in RVM neurons with regional shRNA interference (RNAi) of tryptophan hydroxylase-2 (Tph-2), the rate-limiting enzyme in the synthesis of neuronal 5-HT. Compared to negative control shRNA, Tph-2 shRNA induced significantly prolonged downregulation of Tph-2 in the RVM and 5-HT in spinal dorsal horn. The 5-HT-depleted rats showed normal pain sensitivity in responses to acute noxious stimulation. However, the same RNAi treatment attenuated formalin-induced spontaneous nocifensive responses and tissue or nerve injury-induced allodynia and hyperalgesia. Furthermore, in control shRNA-treated animals, intra-RVM microinjection of brain-derived neurotrophic factor produced a reversible hyperalgesia, which was completely prevented by Tph-2 RNAi pretreatment. Descending inhibition induced by intra-RVM electrical stimulation, but not microinjection of the μ- or κ-opioid receptor agonists in control shRNA-treated animals was eliminated in 5-HT-depleted rats. These results indicate that the descending 5-HT from the RVM is an important contributor to pain facilitation during the development of persistent pain, and may not mediate opioid-induced descending inhibition in acute pain.

Inhibition of class II histone deacetylases in the spinal cord attenuates inflammatory hyperalgesia

BackgroundSeveral classes of histone deacetylases (HDACs) are expressed in the spinal cord that is a critical structure of the nociceptive pathway. HDAC-regulated histone acetylation is an important component of chromatin remodeling leading to epigenetic regulation of gene transcription. To understand the role of histone acetylation in epigenetic regulation of pathological pain, we have studied the impact of different classes of HDACs in the spinal cord on inflammatory hyperalgesia induced by complete Freunds adjuvant (CFA).ResultsWe intrathecally applied inhibitors specific to different classes of HDACs and evaluated their impact on inflammatory hyperalgesia. Pre-injected inhibitors targeting class I as well as II (SAHA, TSA, LAQ824) or IIa (VPA, 4-PB) HDACs significantly delayed the thermal hyperalgesia induced by unilateral CFA injection in the hindpaw. Existing hyperalgesia induced by CFA was also attenuated by the HDAC inhibitors (HDACIs). In contrast, these inhibitors did not interfere with the thermal response either in naïve animals, or on the contralateral side of inflamed animals. Interestingly, MS-275 that specifically inhibits class I HDACs failed to alter the hyperalgesia although it increased histone 3 acetylation in the spinal cord as SAHA did. Using immunoblot analysis, we further found that the levels of class IIa HDAC members (HDAC4, 5, 7, 9) in the spinal dorsal horn were upregulated following CFA injection while those of class I HDAC members (HDAC1, 2, 3) remained stable or were slightly reduced.ConclusionsOur data suggest that activity of class II HDACs in the spinal cord is critical to the induction and maintenance of inflammatory hyperalgesia induced by CFA, while activity of class I HDACs may be unnecessary. Comparison of the effects of HDACIs specific to class II and IIa as well as the expression pattern of different HDACs in the spinal cord in response to CFA suggests that the members of class IIa HDACs may be potential targets for attenuating persistent inflammatory pain.

SYNERGISTIC ACTIVATION OF THE N-METHYL-D-ASPARTATE RECEPTOR SUBUNIT 1 PROMOTER BY MYOCYTE ENHANCER FACTOR 2C AND SP1

The N-methyl-d-aspartate (NMDA) subtype of glutamate receptor plays important roles in neuronal development, plasticity, and cell death. NMDA receptor subunit 1 (NR1) is an essential subunit of the NMDA receptor and is developmentally expressed in postnatal neurons of the central nervous system. Here we identify on the NR1 promoter a binding site for myocyte enhancer factor 2C (MEF2C), a developmentally expressed neuron/muscle transcription factor found in cerebrocortical neurons, and study its regulation of the NR1 gene. Co-expression of MEF2C and Sp1 cDNAs in primary neurons or cell lines synergistically activates the NR1 promoter. Disruption of the MEF2 site or the MEF2C DNA binding domain moderately reduces this synergism. Mutation of the Sp1 sites or the activation domains of Sp1 protein strongly reduces the synergism. Results of yeast two-hybrid and co-immunoprecipitation experiments reveal a physical interaction between MEF2C and Sp1 proteins. The MEF2C DNA binding domain is sufficient for this interaction. Dominant-negative MEF2C interferes with expression of NR1 mRNA in neuronally differentiated P19 cells. Growth factors, including epidermal growth factor and basic fibroblast growth factor, can up-regulate NR1 promoter activity in stably transfected PC12 cells, even in the absence of the MEF2 site, but the Sp1 sites are necessary for this growth factor regulation, suggesting that Sp1 sites may mediate these effects.

Downregulation of selective microRNAs in trigeminal ganglion neurons following inflammatory muscle pain

Active regulation of gene expression in the nervous system plays an important role in the development and/or maintenance of inflammatory pain. MicroRNA (miRNA) negatively regulates gene expression via posttranscriptional or transcriptional inhibition of specific genes. To explore the possible involvement of miRNA in gene regulation during inflammatory pain, we injected complete Freunds adjuvant (CFA) unilaterally into the rat masseter muscle and quantified changes in neuron-specific mature miRNAs in the trigeminal ganglion (TG). Real-time reverse-transcription polymerase chain reaction revealed significant, but differential, downregulation of mature miR-10a, -29a, -98, -99a, -124a, -134, and -183 in the ipsilateral mandibular division (V3) of the TG within 4 hr after CFA. In contrast, levels of tested miRNAs did not change significantly in the contralateral V3 or the ipsilateral ophthalmic and maxillary divisions of the TG from inflamed rats, nor in the ipsilateral V3 of saline-injected animals. The downregulated miRNAs recovered differentially to a level equal to or higher than that in naive animals. Full recovery time varied with miRNA species but was at least 4 days. Expression and downregulation of some miRNAs were further confirmed by in situ hybridization of TG neurons that innervate the inflamed muscle. Although neurons of all sizes expressed these miRNAs, their signals varied between neurons. Our results indicate that miRNA species specific to neurons are quickly regulated following inflammatory muscle pain.

Effect of the ubiquitous transcription factors, SP1 and MAZ, on NMDA receptor subunit type 1 (NR1) expression during neuronal differentiation.

The silencer factor NRSF/REST has been reported to restrict expression to neurons of a variety of genes, including that encoding NMDA receptor subunit type 1 (NR1), by suppressing transcription in nonneuronal cells. However, we recently reported that in addition to the absence of NRSF/REST-binding activity, another neuron-specific mechanism is necessary for high level expression of the NR1 gene in neurons. In this study, we explored the mechanism of induction of NR1 promoter activity during neuronal differentiation of the P19 cell line. We identified a 27 base pair GC-rich region in the promoter as an important element responsible for induction of the NR1 gene after neuronal differentiation. We found that the ubiquitous transcription factors SP1 and MAZ bind to this GC-rich region. Surprisingly, the binding activities of SP1 and MAZ are not remarkably changed after neuronal differentiation. Mutations in the SP1 and MAZ sites impair binding of SP1 and MAZ proteins and also decrease NR1 promoter activity. These findings suggest that SP1 and MAZ mediate enhancement of NR1 promoter activity during neuronal differentiation despite the fact that their binding activity does not change.

MUSCLE INFLAMMATION INDUCES A RAPID INCREASE IN CALCITONIN GENE-RELATED PEPTIDE (CGRP) mRNA THAT TEMPORALLY RELATES TO CGRP IMMUNOREACTIVITY AND NOCICEPTIVE BEHAVIOR

Recent data support an important role for calcitonin gene-related peptide (CGRP) in deep tissue nociceptive processing. Using real-time reverse transcriptase polymerase chain reaction (RT-PCR), radioimmunoassay, immunohistochemistry and behavioral testing, we studied the early time course of CGRP mRNA and protein expression as well as nociceptive behavior following muscle inflammation. A rapid and significant increase in CGRP mRNA occurred in the mandibular division (V3) of the ipsilateral trigeminal ganglion at 30 minutes, 4 and 24 h after the injection of complete Freunds adjuvant as an inflammatory agent into rat masseter muscle. No change in mRNA occurred in the ipsilateral ophthalmic and maxillary divisions (V1/V2) or in the contralateral V3. The levels of immunoreactive calcitonin gene-related peptide (iCGRP) in the ipsilateral V3 significantly increased at 1, 4 and 24 h following muscle inflammation. In contrast, no change occurred in iCGRP levels in either the ipsilateral V1/V2 or contralateral V3. When saline was injected into the masseter muscle, the levels of mRNA or iCGRP did not change in the ipsilateral V3 suggesting that the biochemical changes are specific to CFA-induced muscle inflammation. The number of muscle afferent neurons immunoreactive for CGRP was significantly reduced compared with control at 1, 4 and 24 h in the ipsilateral but not in the contralateral trigeminal ganglion following inflammation. This decrease in the ipsilateral ganglion may indicate a loss of intrasomatic CGRP as a result of increased axonal transport away from the neuronal cell body and/or release of CGRP. Behavioral testing showed a reduction in head withdrawal thresholds bilaterally from 30 min through 24 h following muscle inflammation. Thus upregulation of CGRP mRNA and iCGRP levels are temporally related to the development of inflammation and lowered pain thresholds. The present data support the hypothesis that CGRP is upregulated during deep tissue inflammation and suggest that gene transcription is involved in this upregulation.

Role of peripheral μ-opioid receptors in inflammatory orofacial muscle pain

The aims of this project were to investigate whether inflammation in the orofacial muscle alters mu opioid receptor (MOR) mRNA and protein expressions in trigeminal ganglia (TG), and to assess the contribution of peripheral MORs under acute and inflammatory muscle pain conditions. mRNA and protein levels for MOR were quantified by reverse-transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively, from the TG of naïve rats, and compared with those from the rats treated with complete Freunds adjuvant (CFA) in the masseter. TG was found to express mRNA and protein for MOR, and CFA significantly up-regulated both MOR mRNA and protein by 3 days following the inflammation. The MOR protein up-regulation persisted to day 7 and returned to the baseline level by day 14. We then investigated whether peripheral application of a MOR agonist, D-Ala2, N-Me-Phe4, Gly5-ol-enkephalin acetate salt (DAMGO), attenuates masseter nociception induced by masseteric infusion of hypertonic saline (HS) in lightly anesthetized rats. DAMGO (1, 5, 10 microg) or vehicle was administered directly into the masseter 5-10 min prior to the HS infusion. The DAMGO effects were assessed on mean peak counts (MPC) and overall magnitude as calculated by the area under the curve (AUC) of the HS-evoked behavioral responses. Under this condition, only the highest dose of DAMGO (10 microg) significantly reduced MPC, which was prevented when H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP), a selective MOR antagonist, was co-administered. DAMGO pre-treatment in the contralateral masseter did not attenuate MPC. The same doses of DAMGO administered into CFA-inflamed rats, however, produced a greater attenuation of both MPC and AUC of HS-evoked nocifensive responses. These results demonstrated that activation of peripheral MOR provides greater anti-nociception in inflamed muscle, and that the enhanced MOR effect can be partly explained by significant up-regulation of MOR expression in TG.

The role of the RE1 element in activation of the NR1 promoter during neuronal differentiation.

To understand the genetic mechanism controlling the expression of the NMDA subtype of glutamate receptors during neuronal differentiation, we studied activation of the N‐methyl‐D‐aspartate receptor subunit 1 (NR1) gene and the role of the repressor element‐1 (RE1) element in NR1 promoter activation. Following neuronal differentiation of P19 embryonic carcinoma cells, the NR1 transcription rate and mRNA level were significantly increased, while the nuclear level of the repressor RE1 silencing transcription factor (REST)/neuron‐restriction silencer factor (NRSF) was reduced. Nuclear REST/NRSF from undifferentiated cells formed a large complex with the NR1 RE1 element. While this complex was significantly reduced after the differentiation, REST/NRSF from differentiated cells formed a new, faster migrating complex. In transient transfections, deletion of the RE1 element increased activity of the 5.4‐kb NR1 promoter sixfold in undifferentiated cells, but only induced approximately 1.4‐fold increase in differentiated cells. Forced expression of REST/NRSF in differentiated cells suppressed the promoter, while forced expression of a dominant‐negative REST/NRSF induced promoter activity as well as the mRNA of the NR1 gene in undifferentiated cells. In stable transfectants, the wild‐type promoter showed a robust increase in activity following differentiation in a pattern similar to the NR1 mRNA increase. Conversely, the promoter lacking the RE1 element showed only a moderate increase. Our data suggest that the NR1 gene up‐regulation during neuronal differentiation is controlled by its promoter activation, which is largely determined by the interaction between the RE1 element and the repressor REST/NRSF.