Search

Both inhibiting ascending nociceptive transmission and activating descending inhibition are involved in the opioid analgesic effect. The spinal dorsal horn is a critical site for modulating nociceptive transmission by descending pathways elicited by opioids in the brain. μ-Opioid receptors (MORs, encoded by Oprm1) are highly expressed in primary sensory neurons and their central terminals in the spinal cord. In the present study, we tested the hypothesis that MORs expressed in primary sensory neurons contribute to the descending inhibition and supraspinal analgesic effect induced by centrally administered opioids. We generated Oprm1 conditional knockout (Oprm1-cKO) mice by crossing Advillin Cre/+ mice with Oprm1 flox/flox mice. Immunocytochemical labeling in Oprm1-cKO mice showed that MORs are completely ablated from primary sensory neurons and are profoundly reduced in the superficial spinal dorsal horn. Intracerebroventricular injection of morphine or fentanyl produced a potent analgesic effect in wild-type mice, but such an effect was significantly attenuated in Oprm1-cKO mice. Furthermore, the analgesic effect produced by morphine or fentanyl microinjected into the periaqueductal gray was significantly greater in wild-type mice than in Oprm1-cKO mice. Blocking MORs at the spinal cord level diminished the analgesic effect of morphine and fentanyl microinjected into the periaqueductal gray in both groups of mice. Our findings indicate that MORs expressed at primary afferent terminals in the spinal cord contribute to the supraspinal opioid analgesic effect. These presynaptic MORs in the spinal cord may serve as an interface between ascending inhibition and descending modulation that are involved in opioid analgesia., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Neuronal hyperactivity in the spinal dorsal horn can amplify nociceptive input in diabetic neuropathic pain. The glutamate N-methyl-d-aspartate and α -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (NMDA receptors and AMPA receptors, respectively) are involved in spinal nociceptive transmission. It is unclear, however, whether painful diabetic neuropathy is associated with changes in the activity of synaptic NMDA receptors and AMPA receptors in spinal dorsal horn neurons. AMPA receptors lacking GluA2 are Ca 2+ -permeable (CP-AMPA receptors), and their currents display characteristic inward rectification. In this study, we showed that evoked excitatory postsynaptic currents (EPSCs), induced by streptozotocin, exhibited inward rectification in spinal dorsal neurons in diabetic rats. Presynaptic and postsynaptic NMDA receptor activity in the spinal dorsal horn was similar in diabetic and control rats. In the dorsal spinal cord, the membrane GluA2 protein level was significantly lower in diabetic than in control rats, whereas the cytosolic GluA2 level was greater in diabetic than in control rats. In contrast, the GluA1 subunit levels in the plasma membrane and cytosol did not differ between the two groups. Blocking CP-AMPA receptors significantly reduced the amplitude of EPSCs of dorsal horn neurons in diabetic but not in control rats. Furthermore, blocking spinal CP-AMPA receptors reduced pain hypersensitivity in diabetic rats but had no effect on nociception in control rats. Our study suggests that diabetic neuropathy augments CP-AMPA receptor activity in the spinal dorsal horn by causing intracellular retention of GluA2 and impairing GluA2 membrane trafficking. Increased prevalence of spinal CP-AMPA receptors sustains diabetic neuropathic pain. SIGNIFICANCE STATEMENT: This study demonstrates that the prevalence of synaptic calcium-permeable AMPA receptors is increased in the spinal dorsal horn, which mediates pain hypersensitivity in diabetic neuropathy. Thus, calcium-permeable AMPA receptors play an important role in glutamatergic synaptic plasticity in the spinal cord in painful diabetic neuropathy. This new knowledge improves our understanding of the mechanisms involved in central sensitization associated with diabetic neuropathic pain and suggests that calcium-permeable AMPA receptors are an alternative therapeutic target for treating this chronic pain condition., (Copyright © 2019 by The American Society for Pharmacology and Experimental Therapeutics.)

Key Points: The angiotensin AT1 receptor expression and protein kinase C (PKC)-mediated NMDA receptor phosphorylation levels in the hypothalamus are increased in a rat genetic model of hypertension. Blocking AT1 receptors or PKC activity normalizes the increased pre- and postsynaptic NMDA receptor activity of hypothalamic presympathetic neurons in hypertensive animals. Inhibition of AT1 receptor-PKC activity in the hypothalamus reduces arterial blood pressure and sympathetic nerve discharges in hypertensive animals. AT1 receptors in the hypothalamus are endogenously activated to sustain NMDA receptor hyperactivity and elevated sympathetic outflow via PKC in hypertension., Abstract: Increased synaptic N-methyl-d-aspartate receptor (NMDAR) activity in the hypothalamic paraventricular nucleus (PVN) plays a major role in elevated sympathetic output in hypertension. Although exogenous angiotensin II (AngII) can increase NMDAR activity in the PVN, whether endogenous AT1 receptor-protein kinase C (PKC) activity mediates the augmented NMDAR activity of PVN presympathetic neurons in hypertension is unclear. Here we show that blocking AT1 receptors with losartan or inhibiting PKC with chelerythrine significantly decreased the frequency of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of puff NMDA currents of retrogradely labelled spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Also, treatment with chelerythrine abrogated the potentiating effect of AngII on mEPSCs and puff NMDA currents of labelled PVN neurons in SHRs. In contrast, neither losartan nor chelerythrine had any effect on mEPSCs or puff NMDA currents in labelled PVN neurons in Wistar-Kyoto (WKY) rats. Furthermore, levels of AT1 receptor mRNA and PKC-mediated NMDAR phosphorylation in the PVN were significantly higher in SHRs than in WKY rats. In addition, microinjection of losartan or chelerythrine into the PVN substantially reduced blood pressure and renal sympathetic nerve discharges in SHRs but not in WKY rats. Chelerythrine blocked sympathoexcitatory responses to AngII microinjected into the PVN. Our findings suggest that endogenous AT1 receptor-PKC activity is essential for presynaptic and postsynaptic NMDAR hyperactivity of PVN presympathetic neurons and for the augmented sympathetic outflow in hypertension. This information advances our mechanistic understanding of the interplay between angiotensinergic and glutamatergic excitatory inputs in hypertension., (© 2019 The Authors. The Journal of Physiology © 2019 The Physiological Society.)

Chronic neuropathic pain is a debilitating condition that remains challenging to treat. Glutamate N-methyl-D-aspartate receptor (NMDAR) antagonists have been used to treat neuropathic pain, but the exact sites of their actions have been unclear until recently. Although conventionally postsynaptic, NMDARs are also expressed presynaptically, particularly at the central terminals of primary sensory neurons, in the spinal dorsal horn. However, presynaptic NMDARs in the spinal cord are normally quiescent and are not actively involved in physiological nociceptive transmission. In this review, we describe the emerging role of presynaptic NMDARs at the spinal cord level in chronic neuropathic pain and the implications of molecular mechanisms for more effective treatment. Recent studies indicate that presynaptic NMDAR activity at the spinal cord level is increased in several neuropathic pain conditions but not in chronic inflammatory pain. Increased presynaptic NMDAR activity can potentiate glutamate release from primary afferent terminals to spinal dorsal horn neurons, which is crucial for the synaptic plasticity associated with neuropathic pain caused by traumatic nerve injury and chemotherapy-induced peripheral neuropathy. Furthermore, α2δ-1, previously considered a calcium channel subunit, can directly interact with NMDARs through its C-terminus to increase presynaptic NMDAR activity by facilitating synaptic trafficking of α2δ-1-NMDAR complexes in neuropathic pain caused by chemotherapeutic agents and peripheral nerve injury. Targeting α2δ-1-bound NMDARs with gabapentinoids or α2δ-1 C-terminus peptides can attenuate nociceptive drive form primary sensory nerves to dorsal horn neurons in neuropathic pain.

Inflammatory pain is associated with peripheral and central sensitization, but the underlying synaptic plasticity at the spinal cord level is poorly understood. Transient receptor potential (TRP) channels expressed at peripheral nerve endings, including TRP subtypes ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), can detect nociceptive stimuli. In this study, we determined the contribution of presynaptic TRPA1 and TRPV1 at the spinal cord level to regulating nociceptive drive in chronic inflammatory pain induced by complete Freund's adjuvant (CFA) in rats. CFA treatment caused a large increase in the frequency of spontaneous excitatory postsynaptic currents (EPSCs) in lamina I, but not lamina II outer zone, dorsal horn neurons. However, blocking NMDA receptors had no effect on spontaneous EPSCs in lamina I neurons of CFA-treated rats. Application of a specific TRPA1 antagonist, AM-0902, or of a specific TRPV1 antagonist, 5'-iodoresiniferatoxin, significantly attenuated the elevated frequency of spontaneous EPSCs and miniature EPSCs, the amplitude of monosynaptic EPSCs evoked from the dorsal root in lamina I neurons of CFA-treated rats. AM-0902 and 5'-iodoresiniferatoxin had no effect on evoked or miniature EPSCs in lamina I neurons of vehicle-treated rats. In addition, intrathecal injection of AM-0902 or 5'-iodoresiniferatoxin significantly reduced pain hypersensitivity in CFA-treated rats but had no effect on acute nociception in vehicle-treated rats. Therefore, unlike neuropathic pain, chronic inflammatory pain is associated with NMDA receptor-independent potentiation in glutamatergic drive to spinal lamina I neurons. Endogenous presynaptic TRPA1 and TRPV1 activity at the spinal level contributes to increased nociceptive input from primary sensory nerves to dorsal horn neurons in inflammatory pain. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2019 International Society for Neurochemistry.)

Background: Chronic use of μ-opioid receptor agonists paradoxically causes both hyperalgesia and the loss of analgesic efficacy. Opioid treatment increases presynaptic N-methyl-D-aspartate receptor activity to potentiate nociceptive input to spinal dorsal horn neurons. However, the mechanism responsible for this opioid-induced activation of presynaptic N-methyl-D-aspartate receptors remains unclear. α2δ-1, formerly known as a calcium channel subunit, interacts with N-methyl-D-aspartate receptors and is primarily expressed at presynaptic terminals. This study tested the hypothesis that α2δ-1-bound N-methyl-D-aspartate receptors contribute to presynaptic N-methyl-D-aspartate receptor hyperactivity associated with opioid-induced hyperalgesia and analgesic tolerance., Methods: Rats (5 mg/kg) and wild-type and α2δ-1-knockout mice (10 mg/kg) were treated intraperitoneally with morphine twice/day for 8 consecutive days, and nociceptive thresholds were examined. Presynaptic N-methyl-D-aspartate receptor activity was recorded in spinal cord slices. Coimmunoprecipitation was performed to examine protein-protein interactions., Results: Chronic morphine treatment in rats increased α2δ-1 protein amounts in the dorsal root ganglion and spinal cord. Chronic morphine exposure also increased the physical interaction between α2δ-1 and N-methyl-D-aspartate receptors by 1.5 ± 0.3 fold (means ± SD, P = 0.009, n = 6) and the prevalence of α2δ-1-bound N-methyl-D-aspartate receptors at spinal cord synapses. Inhibiting α2δ-1 with gabapentin or genetic knockout of α2δ-1 abolished the increase in presynaptic N-methyl-D-aspartate receptor activity in the spinal dorsal horn induced by morphine treatment. Furthermore, uncoupling the α2δ-1-N-methyl-D-aspartate receptor interaction with an α2δ-1 C terminus-interfering peptide fully reversed morphine-induced tonic activation of N-methyl-D-aspartate receptors at the central terminal of primary afferents. Finally, intraperitoneal injection of gabapentin or intrathecal injection of an α2δ-1 C terminus-interfering peptide or α2δ-1 genetic knockout abolished the mechanical and thermal hyperalgesia induced by chronic morphine exposure and largely preserved morphine's analgesic effect during 8 days of morphine treatment., Conclusions: α2δ-1-Bound N-methyl-D-aspartate receptors contribute to opioid-induced hyperalgesia and tolerance by augmenting presynaptic N-methyl-D-aspartate receptor expression and activity at the spinal cord level.

Key Points: μ-Opioid receptors (MORs) are expressed peripherally and centrally, but the loci of MORs responsible for clinically relevant opioid analgesia are uncertain. Crossing Oprm1 flox/flox and Advillin Cre/+ mice completely ablates MORs in dorsal root ganglion neurons and reduces the MOR expression level in the spinal cord. Presynaptic MORs expressed at primary afferent central terminals are essential for synaptic inhibition and potentiation of sensory input by opioids. MOR ablation in primary sensory neurons diminishes analgesic effects produced by systemic and intrathecal opioid agonists and abolishes chronic opioid treatment-induced hyperalgesia. These findings demonstrate a critical role of MORs expressed in primary sensory neurons in opioid analgesia and suggest new strategies to increase the efficacy and reduce adverse effects of opioids., Abstract: The pain and analgesic systems are complex, and the actions of systemically administered opioids may be mediated by simultaneous activation of μ-opioid receptors (MORs, encoded by the Oprm1 gene) at multiple, interacting sites. The loci of MORs and circuits responsible for systemic opioid-induced analgesia and hyperalgesia remain unclear. Previous studies using mice in which MORs are removed from Nav1.8- or TRPV1-expressing neurons provided only an incomplete and erroneous view about the role of peripheral MORs in opioid actions in vivo. In the present study, we determined the specific role of MORs expressed in primary sensory neurons in the analgesic and hyperalgesic effects produced by systemic opioid administration. We generated Oprm1 conditional knockout (Oprm1-cKO) mice in which MOR expression is completely deleted from dorsal root ganglion neurons and substantially reduced in the spinal cord, which was confirmed by immunoblotting and immunocytochemical labelling. Both opioid-induced inhibition and potentiation of primary sensory input were abrogated in Oprm1-cKO mice. Remarkably, systemically administered morphine potently inhibited acute thermal and mechanical nociception and persistent inflammatory pain in control mice but had little effect in Oprm1-cKO mice. The analgesic effect of intrathecally administered morphine was also profoundly reduced in Oprm1-cKO mice. Additionally, chronic morphine treatment-induced hyperalgesia was absent in Oprm1-cKO mice. Our findings directly challenge the notion that clinically relevant opioid analgesia is mediated mostly by centrally expressed MORs. MORs in primary sensory neurons, particularly those expressed presynaptically at the first sensory synapse in the spinal cord, are crucial for both opioid analgesia and opioid-induced hyperalgesia., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Opioid-induced hyperalgesia and analgesic tolerance can lead to dose escalation and inadequate pain treatment with μ-opioid receptor agonists. Opioids cause tonic activation of glutamate NMDA receptors (NMDARs) at primary afferent terminals, increasing nociceptive input. However, the signaling mechanisms responsible for opioid-induced activation of pre-synaptic NMDARs in the spinal dorsal horn remain unclear. In this study, we determined the role of MAPK signaling in opioid-induced pre-synaptic NMDAR activation caused by chronic morphine administration. Whole-cell recordings of excitatory post-synaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine administration markedly increased the frequency of miniature EPSCs, increased the amplitude of monosynaptic EPSCs evoked from the dorsal root, and reduced the paired-pulse ratio of evoked EPSCs. These changes were fully reversed by an NMDAR antagonist and normalized by inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2), p38, or c-Jun N-terminal kinase (JNK). Furthermore, intrathecal injection of a selective ERK1/2, p38, or JNK inhibitor blocked pain hypersensitivity induced by chronic morphine treatment. These inhibitors also similarly attenuated a reduction in morphine's analgesic effect in rats. In addition, co-immunoprecipitation assays revealed that NMDARs formed a protein complex with ERK1/2, p38, and JNK in the spinal cord and that chronic morphine treatment increased physical interactions of NMDARs with these three MAPKs. Our findings suggest that opioid-induced hyperalgesia and analgesic tolerance are mediated by tonic activation of pre-synaptic NMDARs via three functionally interrelated MAPKs at the spinal cord level. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)

Painful peripheral neuropathy is a severe and difficult-to-treat neurological complication associated with cancer chemotherapy. Although chemotherapeutic drugs such as paclitaxel are known to cause tonic activation of presynaptic NMDA receptors (NMDARs) to potentiate nociceptive input, the molecular mechanism involved in this effect is unclear. α2δ-1, commonly known as a voltage-activated calcium channel subunit, is a newly discovered NMDAR-interacting protein and plays a critical role in NMDAR-mediated synaptic plasticity. Here we show that paclitaxel treatment in rats increases the α2δ-1 expression level in the dorsal root ganglion and spinal cord and the mRNA levels of GluN1, GluN2A, and GluN2B in the spinal cord. Paclitaxel treatment also potentiates the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. Strikingly, inhibiting α2δ-1 trafficking with pregabalin, disrupting the α2δ-1-NMDAR interaction with an α2δ-1 C-terminus-interfering peptide, or α2δ-1 genetic ablation fully reverses paclitaxel treatment-induced presynaptic NMDAR-mediated glutamate release from primary afferent terminals to spinal dorsal horn neurons. In addition, intrathecal injection of pregabalin or α2δ-1 C-terminus-interfering peptide and α2δ-1 knockout in mice markedly attenuate paclitaxel-induced pain hypersensitivity. Our findings indicate that α2δ-1 is required for paclitaxel-induced tonic activation of presynaptic NMDARs at the spinal cord level. Targeting α2δ-1-bound NMDARs, not the physiological α2δ-1-free NMDARs, may be a new strategy for treating chemotherapy-induced neuropathic pain. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/., (© 2018 International Society for Neurochemistry.)

The striatum receives extensive cortical input and plays a prominent role in motor learning and habit formation. Glutamate N -methyl-d-aspartate (NMDA) receptor (NMDAR)-mediated long-term potentiation (LTP) is a major synaptic plasticity involved in learning and memory. However, the molecular mechanism underlying NMDAR plasticity in corticostriatal LTP is unclear. Here, we show that theta-burst stimulation (TBS) consistently induced corticostriatal LTP and increased the coincident presynaptic and postsynaptic NMDAR activity of medium spiny neurons. We also found that α2δ-1 (previously known as a subunit of voltage-gated calcium channels; encoded by the Cacna2d1 gene) physically interacted with NMDARs in the striatum of mice and humans, indicating that this cross-talk is conserved across species. Strikingly, inhibiting α2δ-1 trafficking with gabapentin or disrupting the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide abolished TBS-induced LTP. In Cacna2d1 -knockout mice, TBS failed to induce corticostriatal LTP and the associated increases in presynaptic and postsynaptic NMDAR activities. Moreover, systemic gabapentin treatment, microinjection of α2δ-1 C terminus-interfering peptide into the dorsomedial striatum, or Cacna2d1 ablation impaired the alternation T-maze task and rotarod performance in mice. Our findings indicate that the interaction between α2δ-1 and NMDARs is of high physiological relevance and that a TBS-induced switch from α2δ-1-free to α2δ-1-bound NMDARs is critically involved in corticostriatal LTP and LTP-associated learning and memory. Gabapentinoids at high doses may adversely affect cognitive function by targeting α2δ-1-NMDAR complexes., (© 2018 Zhou et al.)

Neuropathic pain is associated with persistent changes in gene expression in primary sensory neurons, but the underlying epigenetic mechanisms that cause these changes remain unclear. The muscarinic cholinergic receptors (mAChRs), particularly the M2 subtype (encoded by the cholinergic receptor muscarinic 2 ( Chrm2 ) gene), are critically involved in the regulation of spinal nociceptive transmission. However, little is known about how Chrm2 expression is transcriptionally regulated. Here we show that nerve injury persistently increased the expression of RE1-silencing transcription factor (REST, also known as neuron-restrictive silencing factor [NRSF]), a gene-silencing transcription factor, in the dorsal root ganglion (DRG). Remarkably, nerve injury-induced chronic but not acute pain hypersensitivity was attenuated in mice with Rest knockout in DRG neurons. Also, siRNA-mediated Rest knockdown reversed nerve injury-induced chronic pain hypersensitivity in rats. Nerve injury persistently reduced Chrm2 expression in the DRG and diminished the analgesic effect of muscarine. The RE1 binding site on the Chrm2 promoter is required for REST-mediated Chrm2 repression, and nerve injury increased the enrichment of REST in the Chrm2 promoter in the DRG. Furthermore, Rest knockdown or genetic ablation in DRG neurons normalized Chrm2 expression and augmented muscarine's analgesic effect on neuropathic pain and fully reversed the nerve injury-induced reduction in the inhibitory effect of muscarine on glutamatergic input to spinal dorsal horn neurons. Our findings indicate that nerve injury-induced REST up-regulation in DRG neurons plays an important role in the acute-to-chronic pain transition and is essential for the transcriptional repression of Chrm2 in neuropathic pain., (© 2018 Zhang et al.)

Vesicular glutamate transporter-2 (VGluT2) mediates the uptake of glutamate into synaptic vesicles in neurons. Spinal cord dorsal horn interneurons are highly heterogeneous and molecularly diverse. The functional significance of VGluT2-expressing dorsal horn neurons in physiological and pathological pain conditions has not been explicitly demonstrated. Designer receptors exclusively activated by designer drugs (DREADDs) are a powerful chemogenetic tool to reversibly control neuronal excitability and behavior. Here, we used transgenic mice with Cre recombinase expression driven by the VGluT2 promoter, combined with the chemogenetic approach, to determine the contribution of VGluT2-expressing dorsal horn neurons to nociceptive regulation. Adeno-associated viral vectors expressing double-floxed Cre-dependent Gαq-coupled human M3 muscarinic receptor DREADD (hM3D)-mCherry or Gαi-coupled κ-opioid receptor DREADD (KORD)-IRES-mCitrine were microinjected into the superficial spinal dorsal horn of VGluT2-Cre mice. Immunofluorescence labeling showed that VGluT2 was predominantly expressed in lamina II excitatory interneurons. Activation of excitatory hM3D in VGluT2-expressing neurons with clozapine N-oxide caused a profound increase in neuronal firing and synaptic glutamate release. Conversely, activation of inhibitory KORD in VGluT2-expressing neurons with salvinorin B markedly inhibited neuronal activity and synaptic glutamate release. In addition, chemogenetic stimulation of VGluT2-expressing neurons increased mechanical and thermal sensitivities in naive mice, whereas chemogenetic silencing of VGluT2-expressing neurons reversed pain hypersensitivity induced by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2-expressing excitatory neurons play a crucial role in mediating nociceptive transmission in the spinal dorsal horn. Targeting glutamatergic dorsal horn neurons with inhibitory DREADDs may be a new strategy for treating inflammatory pain and neuropathic pain., (© 2018 International Society for Neurochemistry.)

Background and Purpose- Glutamate NMDARs (N-methyl-D-aspartate receptors) play a major role in the initiation of ischemic brain damage. However, NMDAR antagonists have no protective effects in stroke patients, possibly because they impair physiological functions of NMDARs. α2δ-1 (encoded by Cacna2d1) is strongly expressed in many brain regions. We determined the contribution of α2δ-1 to NMDAR hyperactivity and brain injury induced by ischemia and reperfusion. Methods- Mice were subjected to 90 minutes of middle cerebral artery occlusion followed by 24 hours of reperfusion. Neurological deficits, brain infarct volumes, and calpain/caspase-3 activity in brain tissues were measured. NMDAR activity of hippocampal CA1 neurons was measured in an in vitro ischemic model. Results- Middle cerebral artery occlusion increased α2δ-1 protein glycosylation in the cerebral cortex, hippocampus, and striatum. Coimmunoprecipitation showed that ischemia rapidly enhanced the α2δ-1-NMDAR physical interaction in the mouse brain tissue. Inhibiting α2δ-1 with gabapentin, uncoupling the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide, or genetically ablating Cacna2d1 had no effect on basal NMDAR currents but strikingly abolished oxygen-glucose deprivation-induced NMDAR hyperactivity in hippocampal CA1 neurons. Systemic treatment with gabapentin or α2δ-1 C-terminus-interfering peptide or Cacna2d1 genetic knock-out reduced middle cerebral artery occlusion-induced infarct volumes, neurological deficit scores, and calpain/caspase-3 activation in brain tissues. Conclusions- α2δ-1 is essential for brain ischemia-induced neuronal NMDAR hyperactivity, and α2δ-1-bound NMDARs mediate brain damage caused by cerebral ischemia. Targeting α2δ-1-bound NMDARs, without impairing physiological α2δ-1-free NMDARs, may be a promising strategy for treating ischemic stroke.

Key Points: α2δ-1 is upregulated, promoting the interaction with NMDA receptors (NMDARs), in the hypothalamus in a rat model of hypertension. The prevalence of α2δ-1-bound NMDARs at synaptic sites in the hypothalamus is increased in hypertensive animals. α2δ-1 is essential for the increased presynaptic and postsynaptic NMDAR activity of hypothalamic neurons in hypertension. α2δ-1-bound NMDARs in the hypothalamus are critically involved in augmented sympathetic outflow in hypertensive animals., Abstract: Increased glutamate NMDA receptor (NMDAR) activity in the paraventricular nucleus (PVN) of the hypothalamus leads to augmented sympathetic outflow in hypertension. However, the molecular mechanisms underlying this effect remain unclear. α2δ-1, previously considered to be a voltage-activated calcium channel subunit, is a newly discovered powerful regulator of NMDARs. In the present study, we determined the role of α2δ-1 in regulating synaptic NMDAR activity of rostral ventrolateral medulla (RVLM)-projecting PVN neurons in spontaneously hypertensive rats (SHRs). We show that the protein levels of α2δ-1 and NMDARs in synaptosomes and the α2δ-1-NMDAR complexes in the hypothalamus were substantially higher in SHRs than in normotensive control rats. The basal amplitude of evoked NMDAR currents and NMDAR-mediated synaptic glutamate release in RVLM-projecting PVN neurons were significantly increased in SHRs. Strikingly, inhibiting α2δ-1 activity with gabapentin or disrupting the α2δ-1-NMDAR association with an α2δ-1 C-terminus peptide completely normalized the amplitude of evoked NMDAR currents and NMDAR-mediated synaptic glutamate release in RVLM-projecting PVN neurons in SHRs. In addition, microinjection of the α2δ-1 C-terminus peptide into the PVN substantially reduced arterial blood pressure and renal sympathetic nerve discharges in SHRs. Our findings indicate that α2δ-1-bound NMDARs in the PVN are required for the potentiated presynaptic and postsynaptic NMDAR activity of PVN presympathetic neurons and for the elevated sympathetic outflow in hypertension. α2δ-1-bound NMDARs may be an opportune target for treating neurogenic hypertension., (© 2018 The Authors. The Journal of Physiology © 2018 The Physiological Society.)

Both the sympathetic nervous system and the renin-angiotensin system are critically involved in hypertension development. Although angiotensin II (Ang II) stimulates hypothalamic paraventricular nucleus (PVN) neurons to increase sympathetic vasomotor tone, the molecular mechanism mediating this action remains unclear. The glutamate NMDAR in the PVN controls sympathetic outflow in hypertension. In this study, we determined the interaction between α2δ-1 (encoded by Cacna2d1 ), commonly known as a Ca 2+ channel subunit, and NMDARs in the hypothalamus and its role in Ang II-induced synaptic NMDAR activity in PVN presympathetic neurons. Coimmunoprecipitation assays showed that α2δ-1 interacted with the NMDAR in the hypothalamus of male rats and humans (both sexes). Ang II increased the prevalence of synaptic α2δ-1-NMDAR complexes in the hypothalamus. Also, Ang II increased presynaptic and postsynaptic NMDAR activity via AT1 receptors, and such effects were abolished either by treatment with pregabalin, an inhibitory α2δ-1 ligand, or by interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide. In Cacna2d1 knock-out mice (both sexes), Ang II failed to affect the presynaptic and postsynaptic NMDAR activity of PVN neurons. In addition, the α2δ-1 C terminus-interfering peptide blocked the sympathoexcitatory response to microinjection of Ang II into the PVN. Our findings indicate that Ang II augments sympathetic vasomotor tone and excitatory glutamatergic input to PVN presympathetic neurons by stimulating α2δ-1-bound NMDARs at synapses. This information extends our understanding of the molecular basis for the interaction between the sympathetic nervous and renin-angiotensin systems and suggests new strategies for treating neurogenic hypertension. SIGNIFICANCE STATEMENT Although both the sympathetic nervous system and renin-angiotensin system are closely involved in hypertension development, the molecular mechanisms mediating this involvement remain unclear. We showed that α2δ-1, previously known as a calcium channel subunit, interacts with NMDARs in the hypothalamus of rodents and humans. Angiotensin II (Ang II) increases the synaptic expression level of α2δ-1-NMDAR complexes. Furthermore, inhibiting α2δ-1, interrupting the α2δ-1-NMDAR interaction, or deleting α2δ-1 abolishes the potentiating effects of Ang II on presynaptic and postsynaptic NMDAR activity in the hypothalamus. In addition, the sympathoexcitatory response to Ang II depends on α2δ-1-bound NMDARs. Thus, α2δ-1-NMDAR complexes in the hypothalamus serve as an important molecular substrate for the interaction between the sympathetic nervous system and the renin-angiotensin system. This evidence suggests that α2δ-1 may be a useful target for the treatment neurogenic hypertension., (Copyright © 2018 the authors 0270-6474/18/386388-11$15.00/0.)

Nerve injury-induced hyperactivity of primary sensory neurons in the dorsal root ganglion (DRG) contributes to chronic pain development, but the underlying epigenetic mechanisms remain poorly understood. Here we determined genome-wide changes in DNA methylation in the nervous system in neuropathic pain. Spinal nerve ligation (SNL), but not paclitaxel treatment, in male Sprague Dawley rats induced a consistent low-level hypomethylation in the CpG sites in the DRG during the acute and chronic phases of neuropathic pain. DNA methylation remodeling in the DRG occurred early after SNL and persisted for at least 3 weeks. SNL caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands, in introns, intergenic regions, and repetitive sequences. In contrast, SNL caused more gains of methylation in the spinal cord and prefrontal cortex. The DNA methylation changes in the injured DRGs recapitulated developmental reprogramming at the neonatal stage. Methylation reprogramming was correlated with increased gene expression variability. A diet deficient in methyl donors induced hypomethylation and pain hypersensitivity. Intrathecal administration of the DNA methyltransferase inhibitor RG108 caused long-lasting pain hypersensitivity. DNA methylation reprogramming in the DRG thus contributes to nerve injury-induced chronic pain. Restoring DNA methylation may represent a new therapeutic approach to treat neuropathic pain. SIGNIFICANCE STATEMENT Epigenetic mechanisms are critically involved in the transition from acute to chronic pain after nerve injury. However, genome-wide changes in DNA methylation in the nervous system and their roles in neuropathic pain development remain unclear. Here we used digital restriction enzyme analysis of methylation to quantitatively determine genome-wide DNA methylation changes caused by nerve injury. We showed that nerve injury caused DNA methylation changes at 8% of CpG sites with prevailing hypomethylation outside of CpG islands in the dorsal root ganglion. Reducing DNA methylation induced pain hypersensitivity, whereas increasing DNA methylation attenuated neuropathic pain. These findings extend our understanding of the epigenetic mechanism of chronic neuropathic pain and suggest new strategies to treat nerve injury-induced chronic pain., (Copyright © 2018 the authors 0270-6474/18/386090-12$15.00/0.)

α2δ-1, commonly known as a voltage-activated Ca 2+ channel subunit, is a binding site of gabapentinoids used to treat neuropathic pain and epilepsy. However, it is unclear how α2δ-1 contributes to neuropathic pain and gabapentinoid actions. Here, we show that Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. Conversely, Cacna2d1 knockdown or ablation normalizes synaptic NMDAR activity increased by nerve injury. α2δ-1 forms a heteromeric complex with NMDARs in rodent and human spinal cords. The α2δ-1-NMDAR interaction predominantly occurs through the C terminus of α2δ-1 and promotes surface trafficking and synaptic targeting of NMDARs. Gabapentin or an α2δ-1 C terminus-interfering peptide normalizes NMDAR synaptic targeting and activity increased by nerve injury. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery in neuropathic pain. Gabapentinoids reduce neuropathic pain by inhibiting forward trafficking of α2δ-1-NMDAR complexes., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Chemotherapeutic drugs such as paclitaxel cause painful peripheral neuropathy in many cancer patients and survivors. Although NMDA receptors (NMDARs) at primary afferent terminals are known to be critically involved in chemotherapy-induced chronic pain, the upstream signaling mechanism that leads to presynaptic NMDAR activation is unclear. Group I metabotropic glutamate receptors (mGluRs) play a role in synaptic plasticity and NMDAR regulation. Here we report that the Group I mGluR agonist ( S )-3,5-dihydroxyphenylglycine (DHPG) significantly increased the frequency of miniature excitatory postsynaptic currents (EPSCs) and the amplitude of monosynaptic EPSCs evoked from the dorsal root. DHPG also reduced the paired-pulse ratio of evoked EPSCs in spinal dorsal horn neurons. These effects were blocked by the selective mGluR5 antagonist 2-methyl-6-(phenylethynyl)-pyridine (MPEP), but not by an mGluR1 antagonist. MPEP normalized the frequency of miniature EPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats but had no effect in vehicle-treated rats. Furthermore, mGluR5 protein levels in the dorsal root ganglion and spinal cord synaptosomes were significantly higher in paclitaxel- than in vehicle-treated rats. Inhibiting protein kinase C (PKC) or blocking NMDARs abolished DHPG-induced increases in the miniature EPSC frequency of spinal dorsal horn neurons in vehicle- and paclitaxel-treated rats. Moreover, intrathecal administration of MPEP reversed pain hypersensitivity caused by paclitaxel treatment. Our findings suggest that paclitaxel-induced painful neuropathy is associated with increased presynaptic mGluR5 activity at the spinal cord level, which serves as upstream signaling for PKC-mediated tonic activation of NMDARs. mGluR5 is therefore a promising target for reducing chemotherapy-induced neuropathic pain., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

The role of nitric oxide (NO) in nociceptive transmission at the spinal cord level remains uncertain. Increased activity of spinal N-methyl-d-aspartate (NMDA) receptors contributes to development of chronic pain induced by peripheral nerve injury. In this study, we determined how endogenous NO affects NMDA receptor activity of spinal cord dorsal horn neurons in control and spinal nerve-ligated rats. Bath application of the NO precursor l-arginine or the NO donor S-nitroso-N-acetylpenicillamine (SNAP) significantly inhibited NMDA receptor currents of spinal dorsal horn neurons in both sham control and nerve-injured rats. Inhibition of neuronal nitric oxide synthase (nNOS) or blocking the S-nitrosylation reaction with N-ethylmaleimide abolished the inhibitory effects of l-arginine on NMDA receptor currents recorded from spinal dorsal horn neurons in sham control and nerve-injured rats. However, bath application of the cGMP analog 8-bromo-cGMP had no significant effects on spinal NMDA receptor currents. Inhibition of soluble guanylyl cyclase also did not alter the inhibitory effect of l-arginine on spinal NMDA receptor activity. Furthermore, knockdown of nNOS with siRNA abolished the inhibitory effects of l-arginine, but not SNAP, on spinal NMDA receptor activity in both groups of rats. Additionally, intrathecal injection of l-arginine significantly attenuated mechanical or thermal hyperalgesia induced by nerve injury, and the l-arginine effect was diminished in rats treated with a nNOS inhibitor or nNOS-specific siRNA. These findings suggest that endogenous NO inhibits spinal NMDA receptor activity through S-nitrosylation. NO derived from nNOS attenuates spinal nociceptive transmission and neuropathic pain induced by nerve injury., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Phosphodiesterase-9A (PDE9A) expression is upregulated during cardiac hypertrophy and heart failure. Accumulating evidence suggests that PDE9A might be a promising therapeutic target for heart diseases. The present study sought to investigate the effects and underlying mechanisms of C33(S), a novel selective PDE9A inhibitor, on cardiac hypertrophy in vitro and in vivo. Treatment of neonatal rat cardiomyocytes (NRCMs) with PE (100 μmol/L) or ISO (1 μmol/L) induced cardiac hypertrophy characterized by significantly increased cell surface areas and increased expression of fetal genes (ANF and BNP). Furthermore, PE or ISO significantly increased the expression of PDE9A in the cells; whereas knockdown of PDE9A significantly alleviated PE-induced hypertrophic responses. Moreover, pretreatment with PDE9A inhibitor C33(S) (50 and 500 nmol/L) or PF-7943 (2 μmol/L) also alleviated the cardiac hypertrophic responses in PE-treated NRCMs. Abdominal aortic constriction (AAC)-induced cardiac hypertrophy and ISO-induced heart failure were established in SD rats. In ISO-treated rats, oral administration of C33(S) (9, 3, and 1 mg·kg -1 ·d -1 , for 3 consecutive weeks) significantly increased fractional shortening (43.55%±3.98%, 54.79%±1.95%, 43.98%±7.96% vs 32.18%±6.28%), ejection fraction (72.97%±4.64%, 84.29%±1.56%, 73.41%±9.37% vs 49.17%±4.20%) and cardiac output (60.01±9.11, 69.40±11.63, 58.08±8.47 mL/min vs 48.97±2.11 mL/min) but decreased the left ventricular internal diameter, suggesting that the transition to heart failure was postponed by C33(S). We further revealed that C33(S) significantly elevated intracellular cGMP levels, phosphorylation of phospholamban (PLB) and expression of SERCA2a in PE-treated NRCMs in vitro and in ISO-induced heart failure model in vivo. Our results demonstrate that C33(S) effectively protects against cardiac hypertrophy and postpones the transition to heart failure, suggesting that it is a promising agent in the treatment of cardiac diseases.

Chemotherapeutic drugs, including bortezomib, often cause painful peripheral neuropathy, which is a severe dose-limiting adverse effect experienced by many cancer patients. The glutamate N-methyl-d-aspartate receptors (NMDARs) at the spinal cord level are critically involved in the synaptic plasticity associated with neuropathic pain. In this study, we determined whether treatment with bortezomib, a proteasome inhibitor, affects the NMDAR activity of spinal dorsal horn neurons. Systemic treatment with bortezomib in rats did not significantly affect postsynaptic NMDAR currents elicited by puff application of NMDA directly to dorsal horn neurons. Bortezomib treatment markedly increased the baseline frequency of miniature excitatory postsynaptic currents (EPSCs), which was completely normalized by the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5). AP5 also reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation in bortezomib-treated, but not vehicle-treated, rats. Furthermore, inhibition of protein kinase C (PKC) with chelerythrine fully reversed the increased frequency of miniature EPSCs and the amplitude of evoked EPSCs in bortezomib-treated rats. Intrathecal injection of AP5 and chelerythrine both profoundly attenuated mechanical allodynia and hyperalgesia induced by systemic treatment with bortezomib. In addition, treatment with bortezomib induced striking membrane translocation of PKC-βII, PKC-δ, and PKC-ε in the dorsal root ganglion. Our findings indicate that bortezomib treatment potentiates nociceptive input from primary afferent nerves via PKC-mediated tonic activation of presynaptic NMDARs. Targeting presynaptic NMDARs and PKC at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Ghrelin increases food intake and body weight by stimulating orexigenic agouti-related protein (AgRP)/neuropeptide Y (NPY) neurons and inhibiting anorexic pro-opiomelanocortin (POMC) neurons in the hypothalamus. Growth hormone secretagogue receptor (Ghsr) mediates the effect of ghrelin on feeding behavior and energy homeostasis. However, the role of Ghsr in the ghrelin effect on these two populations of neurons is unclear. We hypothesized that Ghsr mediates the effect of ghrelin on AgRP and POMC neurons. In this study, we determined whether Ghsr similarly mediates the effects of ghrelin on AgRP/NPY and POMC neurons using cell type-specific Ghsr-knockout mice. Perforated whole-cell recordings were performed on green fluorescent protein-tagged AgRP/NPY and POMC neurons in the arcuate nucleus in hypothalamic slices. In Ghsr +/+ mice, ghrelin (100 nM) significantly increased the firing activity of AgRP/NPY neurons but inhibited the firing activity of POMC neurons. In Ghsr -/- mice, the excitatory effect of ghrelin on AgRP/NPY neurons was abolished. Ablation of Ghsr also eliminated ghrelin-induced increases in the frequency of GABAergic inhibitory postsynaptic currents of POMC neurons. Strikingly, ablation of Ghsr converted the ghrelin effect on POMC neurons from inhibition to excitation. Des-acylated ghrelin had no such effect on POMC neurons in Ghsr -/- mice. In both Ghsr +/+ and Ghsr -/- mice, blocking GABA A receptors with gabazine increased the basal firing activity of POMC neurons, and ghrelin further increased the firing activity of POMC neurons in the presence of gabazine. Our findings provide unequivocal evidence that Ghsr is essential for ghrelin-induced excitation of AgRP/NPY neurons. However, ghrelin excites POMC neurons through an unidentified mechanism that is distinct from conventional Ghsr., (© 2017 International Society for Neurochemistry.)

We previously identified AG-690/11026014 (6014) as a novel poly(ADP-ribose) polymerase-1 (PARP-1) inhibitor that effectively prevented angiotensin II (Ang II)-induced cardiomyocyte hypertrophy. In the present study, we reported a new synthesis route for 6014, and investigated its protective effects on Ang II-induced cardiac remodeling and cardiac dysfunction and the underlying mechanisms in mice. We designed a new synthesis route to obtain a sufficient quantity of 6014 for this in vivo study. C57BL/6J mice were infused with Ang II and treated with 6014 (10, 30, 90 mg·kg -1 ·d -1 , ig) for 4 weeks. Then two-dimensional echocardiography was performed to assess the cardiac function and structure. Histological changes of the hearts were examined with HE staining and Masson's trichrome staining. The protein expression was evaluated by Western blot, immunohistochemistry and immunofluorescence assays. The activities of sirtuin-1 (SIRT-1) and the content of NAD+ were detected with the corresponding test kits. Treatment with 6014 dose-dependently improved cardiac function, including LVEF, CO and SV and reversed the changes of cardiac structure in Ang II-infused mice: it significantly ameliorated Ang II-induced cardiac hypertrophy evidenced by attenuating the enlargement of cardiomyocytes, decreased HW/BW and LVW/BW, and decreased expression of hypertrophic markers ANF, BNP and β-MHC; it also prevented Ang II-induced cardiac fibrosis, as implied by the decrease in excess accumulation of extracellular matrix (ECM) components collagen I, collagen III and FN. Further studies revealed that treatment with 6014 did not affect the expression levels of PARP-1, but dose-dependently inhibited the activity of PARP-1 and subsequently restored the activity of SIRT-1 in heart tissues due to the decreased consumption of NAD+ and attenuated Poly-ADP-ribosylation (PARylation) of SIRT-1. In conclusion, the novel PARP-1 inhibitor 6014 effectively protects mice against AngII-induced cardiac remodeling and improves cardiac function. Thus, 6014 might be a potential therapeutic agent for heart diseases..

Aristoyunnolin H is a novel aristophyllene sesquiterpenoid isolated from the traditional Chinese medicine Aristolochia yunnanensis Franch. The present research was designed to explore the anti-fibrotic effects of aristoyunnolin H in adult rat cardiac fibroblasts (CFs) stimulated with angiotensin II (Ang II). Western blot analysis data showed that aristoyunnolin H reduced the upregulation of fibronectin (FN), connective tissue growth factor and collagen I(Col I) production induced by Ang II in CFs. By studying the dynamic intracellular changes of Ca 2+ , we further found that while aristoyunnolin H relieved the calcium influx, it has no effect on intracellular calcium store release. Meanwhile, aristoyunnolin H also inhibited the Ang II-stimulated phosphorylation of Ca 2+ /calmodulin-dependent protein kinase II. In conclusion, aristoyunnolin H may attenuate extracellular matrix secretion in vitro by inhibiting Ang II-induced calcium signaling.

Painful peripheral neuropathy is a severe adverse effect of chemotherapeutic drugs such as paclitaxel (Taxol). The glutamate N-methyl-d-aspartate receptors (NMDARs) are critically involved in the synaptic plasticity associated with neuropathic pain. However, paclitaxel treatment does not alter the postsynaptic NMDAR activity of spinal dorsal horn neurons. In this study, we determined whether paclitaxel affects presynaptic NMDAR activity by recording excitatory postsynaptic currents (EPSCs) of dorsal horn neurons in spinal cord slices. In paclitaxel-treated rats, the baseline frequency of miniature EPSCs (mEPSCs) was significantly increased; the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) completely normalized this frequency. Also, AP5 significantly reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation and reversed the reduction in the paired-pulse ratio of evoked EPSCs in paclitaxel-treated rats. Blocking GluN2A-containing, but not GluN2B-containing, NMDARs largely decreased the frequency of mEPSCs and the amplitude of evoked EPSCs of dorsal horn neurons in paclitaxel-treated rats. Furthermore, inhibition of protein kinase C fully reversed the increased frequency of mEPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats. Paclitaxel treatment significantly increased the protein level of GluN2A and phosphorylated GluN1 in the dorsal root ganglion. In addition, intrathecal injection of AP5 or systemic administration of memantine profoundly attenuated pain hypersensitivity induced by paclitaxel. Our findings indicate that paclitaxel treatment induces tonic activation of presynaptic NMDARs in the spinal cord through protein kinase C to potentiate nociceptive input from primary afferent nerves. Targeting presynaptic NMDARs at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Chronic neuropathic pain is a debilitating condition that remains difficult to treat. Diminished synaptic inhibition by GABA and glycine and increased NMDA receptor (NMDAR) activity in the spinal dorsal horn are key mechanisms underlying neuropathic pain. However, the reciprocal relationship between synaptic inhibition and excitation in neuropathic pain is unclear. Here, we show that intrathecal delivery of K(+)-Cl(-) cotransporter-2 (KCC2) using lentiviral vectors produces a complete and long-lasting reversal of pain hypersensitivity induced by nerve injury. KCC2 gene transfer restores Cl(-) homeostasis disrupted by nerve injury in both spinal dorsal horn and primary sensory neurons. Remarkably, restoring Cl(-) homeostasis normalizes both presynaptic and postsynaptic NMDAR activity increased by nerve injury in the spinal dorsal horn. Our findings indicate that nerve injury recruits NMDAR-mediated signaling pathways through the disruption of Cl(-) homeostasis in spinal dorsal horn and primary sensory neurons. Lentiviral vector-mediated KCC2 expression is a promising gene therapy for the treatment of neuropathic pain., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

The μ-opioid receptor (MOR, encoded by Oprm1) agonists are the mainstay analgesics for treating moderate to severe pain. Nerve injury causes down-regulation of MORs in the dorsal root ganglion (DRG) and diminishes the opioid effect on neuropathic pain. However, the epigenetic mechanisms underlying the diminished MOR expression caused by nerve injury are not clear. G9a (encoded by Ehmt2), a histone 3 at lysine 9 methyltransferase, is a key chromatin regulator responsible for gene silencing. In this study, we determined the role of G9a in diminished MOR expression and opioid analgesic effects in animal models of neuropathic pain. We found that nerve injury in rats induced a long-lasting reduction in the expression level of MORs in the DRG but not in the spinal cord. Nerve injury consistently increased the enrichment of the G9a product histone 3 at lysine 9 dimethylation in the promoter of Oprm1 in the DRG. G9a inhibition or siRNA knockdown fully reversed MOR expression in the injured DRG and potentiated the morphine effect on pain hypersensitivity induced by nerve injury. In mice lacking Ehmt2 in DRG neurons, nerve injury failed to reduce the expression level of MORs and the morphine effect. In addition, G9a inhibition or Ehmt2 knockout in DRG neurons normalized nerve injury-induced reduction in the inhibitory effect of the opioid on synaptic glutamate release from primary afferent nerves. Our findings indicate that G9a contributes critically to transcriptional repression of MORs in primary sensory neurons in neuropathic pain. G9a inhibitors may be used to enhance the opioid analgesic effect in the treatment of chronic neuropathic pain., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We found that activation of pre-synaptic M2 muscarinic receptors inhibit glutamatergic input from vestibular primary afferents, whereas stimulation of post-synaptic M3 muscarinic receptors increases the firing activity of cerebellum-projecting MVN neurons. This new information advances our understanding of the cholinergic mechanism regulating the vestibular system., (© 2016 International Society for Neurochemistry.)

We herein report a water-stable three-dimensional Cu-based metal-organic framework (MOF) 1 supported by a tritopic quaternized carboxylate and 4,4'-dipyridyl sulfide as an ancillary ligand. This MOF exhibits unique pore shapes with aromatic rings, positively charged pyridinium and unsaturated Cu(II) cation centers, free carboxylates, tessellating H2O, and coordinating SO4(2-) on the pore surface. Compound 1 can interact with two carboxyfluorescein (FAM)-labeled single-stranded DNA sequences (probe ss-DNA, delineated as P-DNA) through electrostatic, π-stacking, and/or hydrogen-bonding interactions to form two P-DNA@1 systems, and thus quench the fluorescence of FAM via a photoinduced electron-transfer process. These P-DNA@1 systems can be used as effective fluorescent sensors for human immunodeficiency virus 1 double-stranded DNA and Sudan virus RNA sequences, respectively, with detection limits of 196 and 73 pM, respectively.

Neuropathic pain is a debilitating clinical problem and difficult to treat. Nerve injury causes a long-lasting reduction in K(+) channel expression in the dorsal root ganglion (DRG), but little is known about the epigenetic mechanisms involved. We found that nerve injury increased dimethylation of Lys9 on histone H3 (H3K9me2) at Kcna4, Kcnd2, Kcnq2 and Kcnma1 promoters but did not affect levels of DNA methylation on these genes in DRGs. Nerve injury increased activity of euchromatic histone-lysine N-methyltransferase-2 (G9a), histone deacetylases and enhancer of zeste homolog-2 (EZH2), but only G9a inhibition consistently restored K(+) channel expression. Selective knockout of the gene encoding G9a in DRG neurons completely blocked K(+) channel silencing and chronic pain development after nerve injury. Remarkably, RNA sequencing analysis revealed that G9a inhibition not only reactivated 40 of 42 silenced genes associated with K(+) channels but also normalized 638 genes down- or upregulated by nerve injury. Thus G9a has a dominant function in transcriptional repression of K(+) channels and in acute-to-chronic pain transition after nerve injury.

Pannexin-1 (Panx1) is a large-pore membrane channel involved in the release of ATP and other signaling mediators. Little is known about the expression and functional role of Panx1 in the dorsal root ganglion (DRG) in the development of chronic neuropathic pain. In this study, we determined the epigenetic mechanism involved in increased Panx1 expression in the DRG after nerve injury. Spinal nerve ligation in rats significantly increased the mRNA and protein levels of Panx1 in the DRG but not in the spinal cord. Immunocytochemical labeling showed that Panx1 was primarily expressed in a subset of medium and large DRG neurons in control rats and that nerve injury markedly increased the number of Panx1-immunoreactive DRG neurons. Nerve injury significantly increased the enrichment of two activating histone marks (H3K4me2 and H3K9ac) and decreased the occupancy of two repressive histone marks (H3K9me2 and H3K27me3) around the promoter region of Panx1 in the DRG. However, nerve injury had no effect on the DNA methylation level around the Panx1 promoter in the DRG. Furthermore, intrathecal injection of the Panx1 blockers or Panx1-specific siRNA significantly reduced pain hypersensitivity induced by nerve injury. In addition, siRNA knockdown of Panx1 expression in a DRG cell line significantly reduced caspase-1 release induced by neuronal depolarization. Our findings suggest that nerve injury increases Panx1 expression levels in the DRG through altered histone modifications. Panx1 up-regulation contributes to the development of neuropathic pain and stimulation of inflammasome signaling., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: α-Enolase is a glycolytic enzyme with "second jobs" beyond its catalytic activity. However, its possible contribution to cardiac dysfunction remains to be determined. The present study aimed to investigate the role of α-enolase in doxorubicin (Dox)-induced cardiomyopathy as well as the underlying mechanisms., Experimental Approaches: The expression of α-enolase was detected in rat hearts and primary cultured rat cardiomyocytes with or without Dox administration. An adenovirus carrying short-hairpin interfering RNA targeting α-enolase was constructed and transduced specifically into the heart by intramyocardial injection. Heart function, cell apoptosis and mitochondrial function were measured following Dox administration. In addition, by using gain- and loss-of-function approaches to regulate α-enolase expression in primary cultured rat cardiomyocytes, we investigated the role of endogenous, wide type and catalytically inactive mutant α-enolase in cardiomyocyte apoptosis and ATP generation. Furthermore, the involvement of α-enolase in AMPK phosphorylation was also studied., Key Results: The mRNA and protein expression of cardiac α-enolase was significantly upregulated by Dox. Genetic silencing of α-enolase in rat hearts and cultured cardiomyocytes attenuated Dox-induced apoptosis and mitochondrial dysfunction. In contrast, overexpression of wide-type or catalytically inactive α-enolase in cardiomyocytes mimicked the detrimental role of Dox in inducing apoptosis and ATP reduction. AMPK dephosphorylation was further demonstrated to be involved in the proapoptotic and ATP-depriving effects of α-enolase., Conclusion: Our findings provided the evidence that α-enolase has a catalytically independent role in inducing cardiomyocyte apoptosis and mitochondrial dysfunction, which could be at least partially contributed to the inhibition of AMPK phosphorylation., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Adipose triglyceride lipase (ATGL), the rate-limiting enzyme of triglyceride (TG) hydrolysis, plays an important role in TG metabolism. ATGL knockout mice suffer from TG accumulation and die from heart failure. However, the mechanisms underlying cardiac hypertrophy caused by ATGL dysfunction remain unknown. In this study, we found that ATGL expression declined in pressure overload-induced cardiac hypertrophy in vivo and phenylephrine (PE)-induced cardiomyocyte hypertrophy in vitro. ATGL knockdown led to cardiomyocyte hypertrophy, while ATGL overexpression prevented PE-induced hypertrophy. In addition, ATGL downregulation increased but ATGL overexpression reduced the contents of ceramide, which has been proved to be closely associated with cardiac hypertrophy. Moreover, the accumulation of ceramide was due to elevation of free fatty acids in ATGL-knockdown cardiomyocytes, which could be explained by the reduced activity of peroxisome proliferator-activated receptor (PPAR) α leading to imbalance of fatty acid uptake and oxidation. These observations suggest that downregulation of ATGL causes the decreased PPARα activity which results in the imbalance of FA uptake and oxidation, elevating intracellular FFA contents to promote the accumulation of ceramides, and finally inducing cardiac hypertrophy. Upregulation of ATGL could be a strategy for ameliorating lipotoxic damage in cardiac hypertrophy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

4-Aminopyridine (4-AP, fampridine) is used clinically to improve neuromuscular function in patients with multiple sclerosis, spinal cord injury, and myasthenia gravis. 4-AP can increase neuromuscular and synaptic transmission by directly stimulating high voltage-activated (HVA) Ca(2+) channels independent of its blocking effect on voltage-activated K(+) channels. Here we provide new evidence that the potentiating effect of 4-AP on HVA Ca(2+) channels depends on the specific combination of voltage-activated calcium channel α1 (Cavα1) and voltage-activated calcium channel β (Cavβ) subunits. Among the four Cavβ subunits examined, Cavβ3 was the most significant subunit involved in the 4-AP-induced potentiation of both L-type and N-type currents. Of particular note, 4-AP at micromolar concentrations selectively potentiated L-type currents reconstituted with Cav1.2, α2δ1, and Cavβ3. In contrast, 4-AP potentiated N-type currents only at much higher concentrations and had little effect on P/Q-type currents. In a phrenic nerve-diaphragm preparation, blocking L-type Ca(2+) channels eliminated the potentiating effect of low concentrations of 4-AP on end-plate potentials. Furthermore, 4-AP enhanced the physical interaction of Cav1.2 and Cav2.2 subunits to Cavβ3 and also increased their trafficking to the plasma membrane. Site-directed mutagenesis identified specific regions in the guanylate kinase, HOOK, and C-terminus domains of the Cavβ3 subunit crucial to the ability of 4-AP to potentiate L-type and N-type currents. Our findings indicate that 4-AP potentiates HVA Ca(2+) channels by enhancing reciprocal Cav1.2-Cavβ3 and Cav2.2-Cavβ3 interactions. The therapeutic effect of 4-AP on neuromuscular function is probably mediated by its actions on Cavβ3-containing L-type Ca(2+) channels., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) and K(+)-Cl(-) cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Sudden unexplained death in epilepsy (SUDEP) is the most common cause of premature mortality in epilepsy and was linked to mutations in ion channels; however, genes within the channel protein interactome might also represent pathogenic candidates. Here we show that mice with partial deficiency of Sentrin/SUMO-specific protease 2 (SENP2) develop spontaneous seizures and sudden death. SENP2 is highly enriched in the hippocampus, often the focus of epileptic seizures. SENP2 deficiency results in hyper-SUMOylation of multiple potassium channels known to regulate neuronal excitability. We demonstrate that the depolarizing M-current conducted by Kv7 channel is significantly diminished in SENP2-deficient hippocampal CA3 neurons, primarily responsible for neuronal hyperexcitability. Following seizures, SENP2-deficient mice develop atrioventricular conduction blocks and cardiac asystole. Both seizures and cardiac conduction blocks can be prevented by retigabine, a Kv7 channel opener. Thus, we uncover a disease-causing role for hyper-SUMOylation in the nervous system and establish an animal model for SUDEP., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Small conductance calcium-activated K(+) (SK) channels regulate neuronal excitability. However, little is known about changes in SK channel activity of pre-sympathetic neurons in the hypothalamic paraventricular nucleus (PVN) in essential hypertension. SK channels, calmodulin, and casein kinase II (CK2) form a molecular complex. Because CK2 is up-regulated in the PVN in spontaneously hypertensive rats (SHRs), we hypothesized that CK2 increases calmodulin phosphorylation and contributes to diminished SK channel activity in PVN pre-sympathetic neurons in SHRs. Perforated whole-cell recordings were performed on retrogradely labeled spinally projecting PVN neurons in Wistar-Kyoto (WKY) rats and SHRs. Blocking SK channels with apamin significantly increased the firing rate of PVN neurons in WKY rats but not in SHRs. CK2 inhibition restored the stimulatory effect of apamin on the firing activity of PVN neurons in SHRs. Furthermore, apamin-sensitive SK currents and depolarization-induced medium after-hyperpolarization potentials of PVN neurons were significantly larger in WKY rats than in SHRs. CK2 inhibition significantly increased the SK channel current and medium after-depolarization potential of PVN neurons in SHRs. In addition, CK2-mediated calmodulin phosphorylation level in the PVN was significantly higher in SHRs than in WKY rats. Although SK3 was detected in the PVN, its expression level did not differ significantly between SHRs and WKY rats. Our findings suggest that CK2-mediated calmodulin phosphorylation is increased and contributes to diminished SK channel function of PVN pre-sympathetic neurons in SHRs. This information advances our understanding of the mechanisms underlying hyperactivity of PVN pre-sympathetic neurons and increased sympathetic vasomotor tone in hypertension. Small conductance calcium-activated K(+) (SK) channels, calmodulin, and protein kinase CK2 form a molecular complex and regulate neuronal excitability. Our study suggests that augmented CK2 activity in hypertension can increase calmodulin (CaM) phosphorylation, which leads to diminished SK channel function in pre-sympathetic neurons. Diminished SK channel activity plays a role in hyperactivity of pre-sympathetic neurons in the hypothalamus in hypertension., (© 2014 International Society for Neurochemistry.)

Increased N-methyl-d-aspartate receptor (NMDAR) activity and phosphorylation in the spinal cord are critically involved in the synaptic plasticity and central sensitization associated with neuropathic pain. However, the mechanisms underlying increased NMDAR activity in neuropathic pain conditions remain poorly understood. Here we show that peripheral nerve injury induces a large GluN2A-mediated increase in NMDAR activity in spinal lamina II, but not lamina I, neurons. However, NMDAR currents in spinal dorsal horn neurons are not significantly altered in rat models of diabetic neuropathic pain and resiniferatoxin-induced painful neuropathy (postherpedic neuralgia). Inhibition of protein tyrosine kinases or protein kinase C has little effect on NMDAR currents potentiated by nerve injury. Strikingly, casein kinase II (CK2) inhibitors normalize increased NMDAR currents of dorsal horn neurons in nerve-injured rats. In addition, inhibition of calcineurin, but not protein phosphatase 1/2A, augments NMDAR currents only in control rats. CK2 inhibition blocks the increase in spinal NMDAR activity by the calcineurin inhibitor in control rats. Furthermore, nerve injury significantly increases CK2α and CK2β protein levels in the spinal cord. In addition, inhibition of CK2 or CK2β knockdown at the spinal level substantially reverses pain hypersensitivity induced by nerve injury. Our study indicates that neuropathic pain conditions with different etiologies do not share the same mechanisms, and increased spinal NMDAR activity is distinctly associated with traumatic nerve injury. CK2 plays a prominent role in the potentiation of NMDAR activity in the spinal dorsal horn and may represent a new target for treatments of chronic pain caused by nerve injury., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Stimulation of muscarinic acetylcholine receptors (mAChRs) inhibits nociceptive transmission at the spinal level. However, it is unclear how each mAChR subtype regulates excitatory synaptic input from primary afferents. Here we examined excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation in spinal cord slices from wild-type and mAChR subtype knock-out (KO) mice. In wild-type mice, mAChR activation with oxotremorine-M decreased the amplitude of monosynaptic EPSCs in ∼67% of neurons but increased it in ∼10% of neurons. The inhibitory effect of oxotremorine-M was attenuated by the M2/M4 antagonist himbacine in the majority of neurons, and the remaining inhibition was abolished by group II/III metabotropic glutamate receptor (mGluR) antagonists in wild-type mice. In M2/M4 double-KO mice, oxotremorine-M inhibited monosynaptic EPSCs in significantly fewer neurons (∼26%) and increased EPSCs in significantly more neurons (33%) compared with wild-type mice. Blocking group II/III mGluRs eliminated the inhibitory effect of oxotremorine-M in M2/M4 double-KO mice. In M2 single-KO and M4 single-KO mice, himbacine still significantly reduced the inhibitory effect of oxotremorine-M. However, the inhibitory and potentiating effects of oxotremorine-M on EPSCs in M3 single-KO and M1/M3 double-KO mice were similar to those in wild-type mice. In M5 single-KO mice, oxotremorine-M failed to potentiate evoked EPSCs, and its inhibitory effect was abolished by himbacine. These findings indicate that activation of presynaptic M2 and M4 subtypes reduces glutamate release from primary afferents. Activation of the M5 subtype either directly increases primary afferent input or inhibits it through indirectly stimulating group II/III mGluRs., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Nitric oxide (NO) increases glutamate release to the second-order neurons in the nucleus tractus solitarius (NTS). N-type Ca(2+) channel is essential for triggering glutamate release at synaptic terminals. In this study, we determined the role of Cav2.2 subunit in NO-induced increase in glutamate synaptic inputs to NTS neurons. The second-order NTS neurons and nodose ganglionic (NG) neurons were identified by applying DiA, a fluorescent lipophilic tracer, on aortic depressor nerve in rats. NO donor DEA/NO significantly increased tractus solitarius (TS)-evoked excitatory postsynaptic currents (EPSCs) in second-order NTS neurons, an effect was abolished by pretreatment of slice with ODQ, an inhibitor for soluble isoform of guanylyl cyclase. DEA/NO decreased the paired-pulse ratio of TS-evoked EPSCs, while increased the frequency, but not the amplitude, of miniature EPSCs in second-order NTS neurons. Furthermore, DEA/NO significantly increased Ba(2+) currents in identified baroreceptor NG neurons. However, DEA/NO had little effect on the Ba(2+) currents in the presence of specific N-type Ca(2+) blocker ω-conotoxin GVIA. In addition, immunocytochemistry staining revealed that Cav2.2 subunit immunoreactivates were colocalized with DiA-labeled baroreceptor nerve terminals in the NTS. Collectively, these findings suggest that NO stimulates glutamatergic synaptic inputs to second-order NTS neurons through augmentation of Cav2.2-mediated N-type Ca(2+) currents., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Clinically used calcineurin inhibitors, including tacrolimus (FK506) and cyclosporine A, can induce calcineurin inhibitor-induced pain syndrome (CIPS), which is characterized as severe pain and pain hypersensitivity. Increased synaptic N-methyl-D-aspartate receptor (NMDAR) activity in the spinal dorsal horn plays a critical role in the development of CIPS. Casein kinase II (CK2), a serine/threonine protein kinase, can regulate synaptic NMDAR activity in the brain. In this study, we determined whether spinal CK2 is involved in increased NMDAR activity and pain hypersensitivity caused by systemic administration of FK506 in rats. FK506 treatment caused a large increase in the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) evoked by primary afferent stimulation and in the frequency of miniature EPSCs of spinal dorsal horn neurons. CK2 inhibition with either 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or 4,5,6,7-tetrabromobenzotriazole (TBB) completely normalized the amplitude of evoked NMDAR-EPSCs of dorsal horn neurons in FK506-treated rats. In addition, DRB or TBB significantly attenuated the amplitude of NMDAR currents elicited by puff application of N-methyl-D-aspartate to dorsal horn neurons in FK506-treated rats. Furthermore, treatment with DRB or TBB significantly reduced the frequency of miniature EPSCs of spinal dorsal horn neurons increased by FK506 treatment. In addition, intrathecal injection of DRB or TBB dose-dependently reversed tactile allodynia and mechanical hyperalgesia in FK506-treated rats. Collectively, our findings indicate that CK2 inhibition abrogates pain hypersensitivity and increased pre- and postsynaptic NMDAR activity in the spinal cord caused by calcineurin inhibitors. CK2 inhibitors may represent a new therapeutic option for the treatment of CIPS.

Although postsynaptic glycine receptors (GlyRs) as αβ heteromers attract considerable research attention, little is known about the role of presynaptic GlyRs, likely α homomers, in diseases. Here, we demonstrate that dehydroxylcannabidiol (DH-CBD), a nonpsychoactive cannabinoid, can rescue GlyR functional deficiency and exaggerated acoustic and tactile startle responses in mice bearing point mutations in α1 GlyRs that are responsible for a hereditary startle-hyperekplexia disease. The GlyRs expressed as α1 homomers either in HEK-293 cells or at presynaptic terminals of the calyceal synapses in the auditory brainstem are more vulnerable than heteromers to hyperekplexia mutation-induced impairment. Homomeric mutants are more sensitive to DH-CBD than are heteromers, suggesting presynaptic GlyRs as a primary target. Consistent with this idea, DH-CBD selectively rescues impaired presynaptic GlyR activity and diminished glycine release in the brainstem and spinal cord of hyperekplexic mutant mice. Thus, presynaptic α1 GlyRs emerge as a potential therapeutic target for dominant hyperekplexia disease and other diseases with GlyR deficiency.

Objective: To research pharmacodynamic effect of Yangganjiejiu prescription on alcoholic fatty liver and its mechanism., Methods: The alcoholic fatty liver models of rats was set up by feeding high fat diet and alcohol. Sixty male SD rats were randomly divided into 6 groups: normal group, model group, low-dose Yangganjiejiu prescription group (0.7 g/kg), medial-dose group (1.4 g/kg), high-dose group (2.8 g/kg) and positive control drug Dongbao Gantai group, 10 rats were in each group., Results: Yangganjiejiu prescription (1.4 and 2.8 g/kg) could significantly reduce alcoholic fatty liver rats' serum and liver tissues of total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol( LDL-C) levels (compared with the model group, P < 0.05) and lower serum free fatty acids (FFA) content (compared with the model group, P < 0.05), and to some extent increase the serum and liver tissues of high-density lipoprotein (HDL-C) levels (but compared with the model group, there was no statistical significance, P > 0.05); It could improve liver function, lower serum AST, ALP and GGT content (compared with the model group, P < 0.05 or P < 0.01), significantly decrease serum and liver tissue MDA (P < 0.01), increase SOD in serum and liver tissue (P < 0.01), alleviate alcoholic fatty on the liver secondary liver pathology, and reduce fatty liver., Conclusion: Yangganjiejiu prescription has preventive and therapeutic effect on alcoholic fatty liver. Its mechanism is closely related to resistance to oxidative damage, reducing blood lipid, protecting liver and lowering transaminase.

α-Enolase is a metabolic enzyme in the catabolic glycolytic pathway. In eukaryotic cells, the subcellular compartmentalization of α-enolase as well as its multifaceted functions has been identified. Here, we report that α-enolase is a regulator of cardiac mitochondria; it partially located in the mitochondria of rat cardiomyocytes. Doxorubicin treatment displaced α-enolase from mitochondria, accompanied by activation of mitochondrial cell death pathway. Furthermore, in isolated mitochondria, recombinant α-enolase significantly alleviated Ca(2+)-induced loss of membrane potential, swelling of matrix and permeabilization of membrane. In contrast, mitochondria from α-enolase knockdown H9c2 myoblasts underwent more severe membrane depolarization and swelling after Ca(2+) stimulation. In addition, α-enolase was further identified to interact with voltage dependent anion channel 1 in the outer membrane of mitochondria, which was weakened by doxorubicin. Collectively, the present study indicates that mitochondria-located α-enolase has a beneficial role in stabilizing mitochondrial membrane. In cardiomyocytes, the displacement of α-enolase from mitochondria by doxorubicin may involve in activation of the intrinsic cell death pathway., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Transient receptor potential melastatin 7 (TRPM7), a Ca(2+)-nonselective cation channel, plays a key role in the pathophysiological response of multiple cell types. However, the role of TRPM7 channels in hydrogen peroxide (H2O2)-induced cardiac fibrosis remains unclear. This study aimed to explore whether TRPM7 channels are involved in H2O2-induced cardiac fibrosis and the underlying mechanisms. Our results showed that 2-aminoethoxydiphenylborate (2-APB), which is commonly used to block TRPM7 channels, inhibited H2O2-induced cardiac fibrosis via attenuating the overexpression of important fibrogenic biomarkers and growth factors in cardiac fibroblasts, including collagen type I (Col I), fibronectin (FN), smooth muscle α-actin (α-SMA), connective tissue growth factor (CTGF), and transforming growth factor-β1 (TGF-β1). In addition, 2-APB also decreased H2O2-mediated elevation of the concentration of intracellular Ca(2+) ([Ca(2+)]i). Meanwhile, silencing TRPM7 channels by shRNA interference also impaired the increased [Ca(2+)]i and upregulation of Col I, FN, α-SMA, CTGF, and TGF-β1 induced by H2O2. Furthermore, we found that H2O2-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2) decreased in TRPM7-shRNA cells and Ca(2+)-free culture media. These results demonstrated that TRPM7 channels contributed to H2O2-induced cardiac fibrosis and suggested that this contribution may be through mediating Ca(2+) influx and phosphorylation of ERK1/2.

Calcineurin inhibitors, such as cyclosporin A and tacrolimus (FK506), have played a pivotal role in the preservation of allograft function. However, these drugs can cause unexplained severe pain in patients, often referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin can regulate NMDA receptor (NMDAR) activity, the causal relationship between spinal synaptic plasticity and CIPS remains unknown. In this study, we showed that systemic administration of FK506 (1.5 mg kg(-1) day(-1)) for 7 days in rats led to long-lasting nociceptive and mechanical hypersensitivity. Whole-cell patch-clamp recordings in spinal cord slices revealed that FK506 treatment caused a large increase in the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation. The amplitude of NMDAR currents elicited by puff NMDA application to dorsal horn neurons was also significantly greater in FK506-treated than in vehicle-treated rats. The frequency of spontaneous and miniature EPSCs in most dorsal horn neurons was profoundly increased in FK506-treated rats and was reduced by blocking NMDARs. Furthermore, blocking GluN2A or GluN2B subunits similarly reduced the amplitude of evoked EPSCs and the frequency of miniature EPSCs in dorsal horn neurons of FK506-treated rats. In addition, intrathecal injection of an NMDAR antagonist or systemic administration of memantine effectively reversed nociceptive and mechanical hypersensitivity in FK506-treated rats. Our findings indicate that calcineurin inhibition increases glutamate-mediated nociceptive input by potentiating presynaptic and postsynaptic NMDAR activity in spinal cords. NMDAR antagonists may represent a new therapeutic option for the treatment of CIPS.

The potential involvement of nitric oxide (NO), a diffusible gaseous signaling messenger, in nociceptive transduction and transmission has been extensively investigated. However, there is no consistent and convincing evidence supporting the pronociceptive action of NO at the physiological concentration, and the discrepancies are possibly due to the nonspecificity of nitric oxide synthase inhibitors and different concentrations of NO donors used in various studies. At the spinal cord level, NO predominantly reduces synaptic transmission by inhibiting the activity of NMDA receptors and glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated calcium channels. NO also promotes synaptic glycine release from inhibitory interneurons through the cyclic guanosine monophosphate/protein kinase G signaling pathway. Thus, NO probably functions as a negative feedback regulator to reduce nociceptive transmission in the spinal dorsal horn during painful conditions., (© 2014 Elsevier Inc. All rights reserved.)

There is growing interest in using cannabinoid receptor 2 (CB2) agonists for the treatment of neuropathic pain and other indications. In continuation of our ongoing program aiming for the development of new small molecule cannabinoid ligands, we have synthesized a novel series of carbazole and γ-carboline derivatives. The affinities of the newly synthesized compounds were determined by a competitive radioligand displacement assay for human CB2 cannabinoid receptor and rat CB1 cannabinoid receptor. Functional activity and selectivity at human CB1 and CB2 receptors were characterized using receptor internalization and [(35)S]GTP-γ-S assays. The structure-activity relationship and optimization studies of the carbazole series have led to the discovery of a non-selective CB1 and CB2 agonist, compound 4. Our subsequent research efforts to increase CB2 selectivity of this lead compound have led to the discovery of CB2 selective compound 64, which robustly internalized CB2 receptors. Compound 64 had potent inhibitory effects on pain hypersensitivity in a rat model of neuropathic pain. Other potent and CB2 receptor-selective compounds, including compounds 63 and 68, and a selective CB1 agonist, compound 74 were also discovered. In addition, we identified the CB2 ligand 35 which failed to promote CB2 receptor internalization and inhibited compound CP55,940-induced CB2 internalization despite a high CB2 receptor affinity. The present study provides novel tricyclic series as a starting point for further investigations of CB2 pharmacology and pain treatment., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Barrington's nucleus (BN), commonly known as the pontine micturition center, controls micturition and other visceral functions through projections to the spinal cord. In this study, we developed a rat brain slice preparation to determine the intrinsic and synaptic mechanisms regulating pre-sympathetic output (PSO) and pre-parasympathetic output (PPO) neurons in the BN using patch-clamp recordings. The PSO and PPO neurons were retrogradely labeled by injecting fluorescent tracers into the intermediolateral region of the spinal cord at T13-L1 and S1-S2 levels, respectively. There were significantly more PPO than PSO neurons within the BN. The basal activity and membrane potential were significantly lower in PPO than in PSO neurons, and A-type K(+) currents were significantly larger in PPO than in PSO neurons. Blocking A-type K(+) channels increased the excitability more in PPO than in PSO neurons. Stimulting μ-opioid receptors inhibited firing in both PPO and PSO neurons. The glutamatergic EPSC frequency was much lower, whereas the glycinergic IPSC frequency was much higher, in PPO than in PSO neurons. Although blocking GABAA receptors increased the excitability of both PSO and PPO neurons, blocking glycine receptors increased the firing activity of PPO neurons only. Furthermore, blocking ionotropic glutamate receptors decreased the excitability of PSO neurons but paradoxically increased the firing activity of PPO neurons by reducing glycinergic input. Our findings indicate that the membrane and synaptic properties of PSO and PPO neurons in the BN are distinctly different. This information improves our understanding of the neural circuitry and central mechanisms regulating the bladder and other visceral organs., (© 2013 International Society for Neurochemistry.)