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Aberrant activation of presynaptic NMDARs in the spinal dorsal horn is integral to opioid-induced hyperalgesia and analgesic tolerance. However, the signaling mechanisms responsible for opioid-induced NMDAR hyperactivity remain poorly identified. Here, we show that repeated treatment with morphine or fentanyl reduced monomeric mGluR5 protein levels in the dorsal root ganglion (DRG) but increased levels of mGluR5 monomers and homodimers in the spinal cord in mice and rats of both sexes. Coimmunoprecipitation analysis revealed that monomeric and dimeric mGluR5 in the spinal cord, but not monomeric mGluR5 in the DRG, directly interacted with GluN1. By contrast, mGluR5 did not interact with μ-opioid receptors in the DRG or spinal cord. Repeated morphine treatment markedly increased the mGluR5-GluN1 interaction and protein levels of mGluR5 and GluN1 in spinal synaptosomes. The mGluR5 antagonist MPEP reversed morphine treatment-augmented mGluR5-GluN1 interactions, GluN1 synaptic expression, and dorsal root-evoked monosynaptic EPSCs of dorsal horn neurons. Furthermore, CRISPR-Cas9-induced conditional mGluR5 knockdown in DRG neurons normalized mGluR5 levels in spinal synaptosomes and NMDAR-mediated EPSCs of dorsal horn neurons increased by morphine treatment. Correspondingly, intrathecal injection of MPEP or conditional mGluR5 knockdown in DRG neurons not only potentiated the acute analgesic effect of morphine but also attenuated morphine treatment-induced hyperalgesia and tolerance. Together, our findings suggest that opioid treatment promotes mGluR5 trafficking from primary sensory neurons to the spinal dorsal horn. Through dimerization and direct interaction with NMDARs, presynaptic mGluR5 potentiates and/or stabilizes NMDAR synaptic expression and activity at primary afferent central terminals, thereby maintaining opioid-induced hyperalgesia and tolerance. SIGNIFICANCE STATEMENT Opioids are essential analgesics for managing severe pain caused by cancer, surgery, and tissue injury. However, these drugs paradoxically induce pain hypersensitivity and tolerance, which can cause rapid dose escalation and even overdose mortality. This study demonstrates, for the first time, that opioids promote trafficking of mGluR5, a G protein-coupled glutamate receptor, from peripheral sensory neurons to the spinal cord; there, mGluR5 proteins dimerize and physically interact with NMDARs to augment their synaptic expression and activity. Through dynamic interactions, the two distinct glutamate receptors mutually amplify and sustain nociceptive input from peripheral sensory neurons to the spinal cord. Thus, inhibiting mGluR5 activity or disrupting mGluR5-NMDAR interactions could reduce opioid-induced hyperalgesia and tolerance and potentiate opioid analgesic efficacy., (Copyright © 2023 the authors.)

Glutamate NMDA receptors (NMDARs) in the nucleus accumbens (NAc) are critically involved in drug dependence and reward. α2δ-1 is a newly discovered NMDAR-interacting protein that promotes synaptic trafficking of NMDARs independently of its conventional role as a calcium channel subunit. However, it remains unclear how repeated opioid exposure affects synaptic NMDAR activity and α2δ-1-NMDAR interaction in the NAc. In this study, whole-cell patch-clamp recordings showed that repeated treatment with morphine in mice markedly increased the NMDAR-mediated frequency of miniature excitatory postsynaptic currents (mEPSCs) and amplitude of puff NMDAR currents in medium spiny neurons in the NAc core region. Morphine treatment significantly increased the physical interaction of α2δ-1 with GluN1 and their synaptic trafficking in the NAc. In Cacna2d1 knockout mice, repeated treatment with morphine failed to increase the frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Furthermore, inhibition of α2δ-1 with gabapentin or disruption of the α2δ-1-NMDAR interaction with the α2δ-1 C terminus-interfering peptide blocked the morphine-elevated frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Correspondingly, systemically administered gabapentin, Cacna2d1 ablation, or microinjection of the α2δ-1 C terminus-interfering peptide into the NAc core attenuated morphine-induced conditioned place preference and locomotor sensitization. Our study reveals that repeated opioid exposure strengthens presynaptic and postsynaptic NMDAR activity in the NAc via α2δ-1. The α2δ-1-bound NMDARs in the NAc have a key function in the rewarding effect of opioids and could be targeted for treating opioid use disorder and addiction., (© 2022 International Society for Neurochemistry.)

Treatment with opioids not only inhibits nociceptive transmission but also elicits a rebound and persistent increase in primary afferent input to the spinal cord. Opioid-elicited long-term potentiation (LTP) from TRPV1-expressing primary afferents plays a major role in opioid-induced hyperalgesia and analgesic tolerance. Here, we determined whether opioid-elicited LTP involves vesicular glutamate transporter-2 (VGluT2) or vesicular GABA transporter (VGAT) neurons in the spinal dorsal horn of male and female mice and identified underlying signaling mechanisms. Spinal cord slice recordings revealed that µ-opioid receptor (MOR) stimulation with DAMGO initially inhibited dorsal root-evoked EPSCs in 87% VGluT2 neurons and subsequently induced LTP in 49% of these neurons. Repeated morphine treatment increased the prevalence of VGluT2 neurons displaying LTP with a short onset latency. In contrast, DAMGO inhibited EPSCs in 46% VGAT neurons but did not elicit LTP in any VGAT neurons even in morphine-treated mice. Spinal superficial laminae were densely innervated by MOR-containing nerve terminals and were occupied by mostly VGluT2 neurons and few VGAT neurons. Furthermore, conditional Grin1 knockout in dorsal root ganglion neurons diminished DAMGO-elicited LTP in lamina II neurons and attenuated hyperalgesia and analgesic tolerance induced by repeated treatment with morphine. In addition, DAMGO-elicited LTP in VGluT2 neurons was abolished by protein kinase C inhibition, gabapentin, Cacna2d1 knockout, or disrupting the α2δ-1-NMDA receptor interaction with an α2δ-1 C terminus peptide. Thus, brief MOR stimulation distinctively potentiates nociceptive primary afferent input to excitatory dorsal horn neurons via α2δ-1-coupled presynaptic NMDA receptors, thereby causing hyperalgesia and reducing analgesic actions of opioids. SIGNIFICANCE STATEMENT Opioid drugs are potent analgesics for treating severe pain and are commonly used during general anesthesia. However, opioid use often induces pain hypersensitivity, rapid loss of analgesic efficacy, and dose escalation, which can cause dependence, addiction, and even overdose fatality. This study demonstrates for the first time that brief opioid exposure preferentially augments primary sensory input to genetically identified glutamatergic excitatory, but not GABAergic/glycinergic inhibitory, neurons in nociceptive dorsal horn circuits. This opioid-elicited synaptic plasticity is cell type specific and mediated by protein kinase C-dependent and α2δ-1-dependent activation of NMDA receptors at primary sensory nerve terminals. These findings elucidate how intraoperative use of opioids for preemptive analgesia paradoxically aggravates postoperative pain and increases opioid consumption and suggest new strategies to improve opioid analgesic efficacy., (Copyright © 2022 the authors.)

Rationale: Hypertension is a common and serious adverse effect of calcineurin inhibitors, including cyclosporine and tacrolimus (FK506). Although increased sympathetic nerve discharges are associated with calcineurin inhibitor-induced hypertension, the sources of excess sympathetic outflow and underlying mechanisms remain elusive. Calcineurin (protein phosphatase-2B) is broadly expressed in the brain, including the paraventricular nuclear (PVN) of the hypothalamus, which is critically involved in regulating sympathetic vasomotor tone., Objective: We determined whether prolonged treatment with the calcineurin inhibitor causes elevated sympathetic output and persistent hypertension by potentiating synaptic N-methyl-D-aspartate (NMDA) receptor activity in the PVN., Methods and Results: Telemetry recordings showed that systemic administration of FK506 (3 mg/kg per day) for 14 days caused a gradual and profound increase in arterial blood pressure in rats, which lasted at least 7 days after discontinuing FK506 treatment. Correspondingly, systemic treatment with FK506 markedly reduced calcineurin activity in the PVN and circumventricular organs, but not rostral ventrolateral medulla, and increased the phosphorylation level and synaptic trafficking of NMDA receptors in the PVN. Immunocytochemistry labeling showed that calcineurin was expressed in presympathetic neurons in the PVN. Whole-cell patch-clamp recordings in brain slices revealed that treatment with FK506 increased baseline firing activity of PVN presympathetic neurons; this increase was blocked by the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist. Also, treatment with FK506 markedly increased presynaptic and postsynaptic NMDA receptor activity of PVN presympathetic neurons. Furthermore, microinjection of the NMDA or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist into the PVN of anesthetized rats preferentially attenuated renal sympathetic nerve discharges and blood pressure elevated by FK506 treatment. In addition, systemic administration of memantine, a clinically used NMDA receptor antagonist, effectively attenuated FK506 treatment-induced hypertension in conscious rats., Conclusions: Our findings reveal that normal calcineurin activity in the PVN constitutively restricts sympathetic vasomotor tone via suppressing NMDA receptor activity, which may be targeted for treating calcineurin inhibitor-induced hypertension.

Long-term potentiation (LTP) and long-term depression (LTD) in the spinal dorsal horn reflect activity-dependent synaptic plasticity and central sensitization in chronic pain. Tetanic high-frequency stimulation is commonly used to induce LTP in the spinal cord. However, primary afferent nerves often display low-frequency, rhythmic bursting discharges in painful conditions. Here, we determined how theta-burst stimulation (TBS) of primary afferents impacts spinal cord synaptic plasticity and nociception in male and female mice. We found that TBS induced more LTP, whereas tetanic stimulation induced more LTD, in mouse spinal lamina II neurons. TBS triggered LTP, but not LTD, in 50% of excitatory neurons expressing vesicular glutamate transporter-2 (VGluT2). By contrast, TBS induced LTD and LTP in 12-16% of vesicular GABA transporter (VGAT)-expressing inhibitory neurons. Nerve injury significantly increased the prevalence of TBS-induced LTP in VGluT2-expressing, but not VGAT-expressing, lamina II neurons. Blocking NMDARs, inhibiting α2δ-1 with gabapentin, or α2δ-1 knockout abolished TBS-induced LTP in lamina II neurons. Also, disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide prevented TBS-induced LTP in VGluT2-expressing neurons. Furthermore, TBS of the sciatic nerve induced long-lasting allodynia and hyperalgesia in wild-type, but not α2δ-1 knockout, mice. TBS significantly increased the α2δ-1-NMDAR interaction and synaptic trafficking in the spinal cord. In addition, treatment with NMDAR antagonists, gabapentin, or α2δ-1Tat peptide reversed TBS-induced pain hypersensitivity. Therefore, TBS-induced primary afferent input causes a neuropathic pain-like phenotype and LTP predominantly in excitatory dorsal horn neurons via α2δ-1-dependent NMDAR activation. α2δ-1-bound NMDARs may be targeted for reducing chronic pain development at the onset of tissue/nerve injury. SIGNIFICANCE STATEMENT Spinal dorsal horn synaptic plasticity is a hallmark of chronic pain. Although sensory nerves display rhythmic bursting discharges at theta frequencies during painful conditions, the significance of this naturally occurring firing activity in the induction of spinal synaptic plasticity is largely unknown. In this study, we found that theta-burst stimulation (TBS) of sensory nerves induced LTP mainly in excitatory dorsal horn neurons and that the prevalence of TBS-induced LTP was potentiated by nerve injury. This TBS-driven synaptic plasticity required α2δ-1 and its interaction with NMDARs. Furthermore, TBS of sensory nerves induced persistent pain, which was maintained by α2δ-1-bound NMDARs. Thus, TBS-induced LTP at primary afferent-dorsal horn neuron synapses is an appropriate cellular model for studying mechanisms of chronic pain., (Copyright © 2022 the authors.)

The interplay between genetic and environmental factors is critically involved in hypertension development. The paraventricular nucleus (PVN) of the hypothalamus regulates sympathetic output during stress responses and chronic hypertension. In this study, we determined mechanisms of synaptic plasticity in the PVN in chronic stress-induced persistent hypertension in male borderline hypertensive rats (BHR), the first offspring of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats. In Wistar-Kyoto rats, chronic unpredictable mild stress (CUMS) increased arterial blood pressure (ABP) and heart rate, which quickly returned to baseline after CUMS ended. In contrast, in BHR, CUMS caused persistent elevation in ABP, which lasted at least 2 weeks after CUMS ended. CUMS also increased the mRNA level of α2δ-1 and synaptic protein levels of GluN1, α2δ-1, and α2δ-1-GluN1 complexes in the PVN in BHR. Furthermore, CUMS significantly increased the frequency of miniature EPSCs and the amplitude of NMDAR currents in spinally projecting PVN neurons in BHR; these increases were normalized by blocking NMDARs with AP5, inhibiting α2δ-1 with gabapentin, or disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide. Microinjection of AP5 or α2δ-1Tat peptide into the PVN normalized elevated ABP and renal sympathetic nerve activity in stressed BHR. In addition, systemically administered gabapentin or memantine attenuated higher ABP induced by CUMS in BHR. Our findings indicate that chronic stress-induced persistent hypertension is mediated by augmented sympathetic outflow via α2δ-1-bound NMDARs in the PVN. This new information provides a cellular and molecular basis for how the genetic-environment interactions cause persistent hypertension. SIGNIFICANCE STATEMENT Chronic stress is a major risk factor for hypertension development, especially for individuals with a genetic predisposition to hypertension. Using a rat model of borderline hypertension, we showed that chronic stress induced long-lasting hypertension and sympathetic nerve hyperactivity, which were maintained by NMDAR activation in the hypothalamus. Chronic stress also increased the expression of α2δ-1, previously regarded as a Ca 2+ channel subunit, promoting physical interaction with and synaptic trafficking of NMDARs in the hypothalamus. Inhibiting α2δ-1, blocking NMDARs, or disrupting α2δ-1-bound NMDARs reversed chronic stress-induced sympathetic outflow and persistent hypertension. Thus, α2δ-1-dependent NMDAR activity in the hypothalamus is an effector of genetic-environment interactions and may be targeted for treating stress-induced neurogenic hypertension., (Copyright © 2021 the authors.)

N -methyl-d-aspartate receptors (NMDARs) are important for synaptic plasticity associated with many physiological functions and neurologic disorders. Protein kinase C (PKC) activation increases the phosphorylation and activity of NMDARs, and α2δ-1 is a critical NMDAR-interacting protein and controls synaptic trafficking of NMDARs. In this study, we determined the relative roles of PKC and α2δ-1 in the control of NMDAR activity. We found that α2δ-1 coexpression significantly increased NMDAR activity in HEK293 cells transfected with GluN1/GluN2A or GluN1/GluN2B. PKC activation with phorbol 12-myristate 13-acetate (PMA) increased receptor activity only in cells coexpressing GluN1/GluN2A and α2δ-1. Remarkably, PKC inhibition with Gӧ6983 abolished α2δ-1-coexpression-induced potentiation of NMDAR activity in cells transfected with GluN1/GluN2A or GluN1/GluN2B. Treatment with PMA increased the α2δ-1-GluN1 interaction and promoted α2δ-1 and GluN1 cell surface trafficking. PMA also significantly increased NMDAR activity of spinal dorsal horn neurons and the amount of α2δ-1-bound GluN1 protein complexes in spinal cord synaptosomes in wild-type mice, but not in α2δ-1 knockout mice. Furthermore, inhibiting α2δ-1 with pregabalin or disrupting the α2δ-1-NMDAR interaction with the α2δ-1 C-terminus peptide abolished the potentiating effect of PMA on NMDAR activity. Additionally, using quantitative phosphoproteomics and mutagenesis analyses, we identified S929 on GluN2A and S1413 (S1415 in humans) on GluN2B as the phosphorylation sites responsible for NMDAR potentiation by PKC and α2δ-1. Together, our findings demonstrate the interdependence of α2δ-1 and PKC phosphorylation in regulating NMDAR trafficking and activity. The phosphorylation-dependent, dynamic α2δ-1-NMDAR interaction constitutes an important molecular mechanism of synaptic plasticity. SIGNIFICANCE STATEMENT A major challenge in studies of protein phosphorylation is to define the functional significance of each phosphorylation event and determine how various signaling pathways are coordinated in response to neuronal activity to shape synaptic plasticity. PKC phosphorylates transporters, ion channels, and G-protein-coupled receptors in signal transduction. In this study, we showed that α2δ-1 is indispensable for PKC-activation-induced surface and synaptic trafficking of NMDARs, whereas the α2δ-1-NMDAR interaction is controlled by PKC-induced phosphorylation. Our findings reveal that α2δ-1 mainly functions as a phospho-binding protein in the control of NMDAR trafficking and activity. This information provides new mechanistic insight into the reciprocal roles of PKC-mediated phosphorylation and α2δ-1 in regulating NMDARs and in the therapeutic actions of gabapentinoids., (Copyright © 2021 the authors.)

Calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, are widely used as standard immunosuppressants in organ transplantation recipients. However, these drugs can cause severe pain in patients, commonly referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin inhibition increases NMDAR activity in the spinal cord, the underlying mechanism remains enigmatic. Using an animal model of CIPS, we found that systemic administration of FK506 in male and female mice significantly increased the amount of α2δ-1-GluN1 complexes in the spinal cord and the level of α2δ-1-bound GluN1 proteins in spinal synaptosomes. Treatment with FK506 significantly increased the frequency of mEPSCs and the amplitudes of monosynaptic EPSCs evoked from the dorsal root and puff NMDAR currents in spinal dorsal horn neurons. Inhibiting α2δ-1 with gabapentin or disrupting the α2δ-1-NMDAR interaction with α2δ-1Tat peptide completely reversed the effects of FK506. In α2δ-1 gene KO mice, treatment with FK506 failed to increase the frequency of NMDAR-mediated mEPSCs and the amplitudes of evoked EPSCs and puff NMDAR currents in spinal dorsal horn neurons. Furthermore, systemic administration of gabapentin or intrathecal injection of α2δ-1Tat peptide reversed thermal and mechanical hypersensitivity in FK506-treated mice. In addition, genetically deleting GluN1 in dorsal root ganglion neurons or α2δ-1 genetic KO similarly attenuated FK506-induced thermal and mechanical hypersensitivity. Together, our findings indicate that α2δ-1-bound NMDARs mediate calcineurin inhibitor-induced tonic activation of presynaptic and postsynaptic NMDARs at the spinal cord level and that presynaptic NMDARs play a prominent role in the development of CIPS. SIGNIFICANCE STATEMENT Calcineurin inhibitors are immunosuppressants used to prevent rejection of transplanted organs and tissues. However, these drugs can cause severe, unexplained pain. We showed that calcineurin inhibition enhances physical interaction between α2δ-1 and NMDARs and their synaptic trafficking in the spinal cord. α2δ-1 is essential for calcineurin inhibitor-induced aberrant activation of presynaptic and postsynaptic NMDARs in the spinal cord. Furthermore, inhibiting α2δ-1 or disrupting α2δ-1-NMDAR interaction reduces calcineurin inhibitor-induced pain hypersensitivity. Eliminating NMDARs in primary sensory neurons or α2δ-1 KO also attenuates calcineurin inhibitor-induced pain hypersensitivity. This new information extends our mechanistic understanding of the role of endogenous calcineurin in regulating synaptic plasticity and nociceptive transmission and suggests new strategies for treating this painful condition., (Copyright © 2020 the authors.)

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.

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.

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.)

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.)

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.)

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.)

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.)

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.)

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.

Loss of synaptic inhibition by γ-aminobutyric acid and glycine due to potassium chloride cotransporter-2 (KCC2) down-regulation in the spinal cord is a critical mechanism of synaptic plasticity in neuropathic pain. Here we present novel evidence that peripheral nerve injury diminishes glycine-mediated inhibition and induces a depolarizing shift in the reversal potential of glycine-mediated currents (E(glycine)) in spinal dorsal horn neurons. Blocking glutamate N-methyl-D-aspartate (NMDA) receptors normalizes synaptic inhibition, E(glycine), and KCC2 by nerve injury. Strikingly, nerve injury increases calcium-dependent calpain activity in the spinal cord that in turn causes KCC2 cleavage at the C terminus. Inhibiting calpain blocks KCC2 cleavage induced by nerve injury and NMDA, thereby normalizing E(glycine). Furthermore, calpain inhibition or silencing of μ-calpain at the spinal level reduces neuropathic pain. Thus, nerve injury promotes proteolytic cleavage of KCC2 through NMDA receptor-calpain activation, resulting in disruption of chloride homeostasis and diminished synaptic inhibition in the spinal cord. Targeting calpain may represent a new strategy for restoring KCC2 levels and tonic synaptic inhibition and for treating chronic neuropathic pain.

Opioids are the most effective analgesics for the treatment of moderate to severe pain. However, chronic opioid treatment can cause both hyperalgesia and analgesic tolerance, which limit their clinical efficacy. In this study, we determined the role of pre- and postsynaptic NMDA receptors (NMDARs) in controlling increased glutamatergic input in the spinal cord induced by chronic systemic morphine administration. Whole-cell voltage clamp recordings of excitatory postsynaptic currents (EPSCs) were performed on dorsal horn neurons in rat spinal cord slices. Chronic morphine significantly increased the amplitude of monosynaptic EPSCs evoked from the dorsal root and the frequency of spontaneous EPSCs, and these changes were largely attenuated by blocking NMDARs and by inhibiting PKC, but not PKA. Also, blocking NR2A- or NR2B-containing NMDARs significantly reduced the frequency of spontaneous EPSCs and the amplitude of evoked EPSCs in morphine-treated rats. Strikingly, morphine treatment largely decreased the amplitude of evoked NMDAR-EPSCs and NMDAR currents of dorsal horn neurons elicited by puff NMDA application. The reduction in postsynaptic NMDAR currents caused by morphine was prevented by resiniferatoxin pretreatment to ablate TRPV1-expressing primary afferents. Furthermore, intrathecal injection of the NMDAR antagonist significantly attenuated the development of analgesic tolerance and the reduction in nociceptive thresholds induced by chronic morphine. Collectively, our findings indicate that chronic opioid treatment potentiates presynaptic, but impairs postsynaptic, NMDAR activity in the spinal cord. PKC-mediated increases in NMDAR activity at nociceptive primary afferent terminals in the spinal cord contribute critically to the development of opioid hyperalgesia and analgesic tolerance.

Neuropathic pain remains a major clinical problem and a therapeutic challenge because existing analgesics are often ineffective and can cause serious side effects. Increased N-methyl-d-aspartate receptor (NMDAR) activity contributes to central sensitization in certain types of neuropathic pain. NMDAR antagonists can reduce hyperalgesia and allodynia in animal models of neuropathic pain induced by nerve injury and diabetic neuropathy. Clinically used NMDAR antagonists, such as ketamine and dextromethorphan, are generally effective in patients with neuropathic pain, such as complex regional pain syndrome and painful diabetic neuropathy. However, patients with postherpetic neuralgia respond poorly to NMDAR antagonists. Recent studies on identifying NMDAR-interacting proteins and molecular mechanisms of increased NMDAR activity in neuropathic pain could facilitate the development of new drugs to attenuate abnormal NMDAR activity with minimal impairment of the physiological function of NMDARs. Combining NMDAR antagonists with other analgesics could also lead to better management of neuropathic pain without causing serious side effects.

Diabetic neuropathic pain remains an unmet clinical problem and is poorly relieved by conventional analgesics. N-methyl-D-aspartate (NMDA) receptors play an important role in central sensitization in neuropathic pain. Although NMDA antagonists are highly effective in reducing neuropathic pain, these agents cause severe side effects at therapeutic doses, which limit their clinical uses. Neramexane and memantine are uncompetitive NMDA antagonists with minimal side effects at therapeutic doses. Here we determined the antinociceptive effect of chronic administration of neramexane and compared its effect with that of memantine and gabapentin in a rat model of diabetic neuropathic pain. Mechanical hyperalgesia was measured with a noxious pressure stimulus, and tactile allodynia was assessed with von Frey filaments in diabetic rats induced by streptozotocin. Compared with vehicle-treated rats, treatment with neramexane (12.3, 24.6, and 49.2 mg/kg/day) for 2 weeks via an osmotic minipump produced dose-dependent and sustained effects on mechanical hyperalgesia and allodynia. Administration of memantine (20 mg/kg/day) or gabapentin (50 mg/kg/day) for 2 weeks also produced significant and persistent antinociceptive effects on mechanical hyperalgesia and allodynia. The magnitude of the antinociceptive effect produced by the intermediate and high doses of neramexane was comparable to that of gabapentin and memantine. The plasma level achieved by neramexane at 12.3, 24.6, and 49.2 mg/kg/day was 0.26 +/- 0.04, 0.50 +/- 0.05, and 1.21 +/- 0.16 microM, respectively. These data suggest that neramexane at therapeutically relevant doses attenuates diabetic neuropathic pain. Our study provides valuable information about the therapeutic potential of chronic administration of neramexane and memantine for painful diabetic neuropathy.