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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. [ABSTRACT FROM AUTHOR]

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. 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. 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 FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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/. [ABSTRACT FROM AUTHOR]

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/. Under the normal condition, NMDA receptors at the pre‐synaptic terminals in the spinal cord are not phosphorylated or functionally active. However, pre‐synaptic NMDA receptors in the spinal dorsal horn become tonically activated during chronic morphine treatment. Here, we found that chronic treatment with morphine increases the association of NMDA receptors with three MAPKs (ERK1/2, p38, and JNK) in the spinal cord. This increased interaction leads to NMDA receptor phosphorylation at pre‐synaptic terminals. As a result, pre‐synaptic NMDARs are endogenously activated to augment synaptic glutamate release to post‐synaptic neurons in the spinal dorsal horn to cause opioid‐induced hyperalgesia and analgesic tolerance. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

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/. Although NMDA receptors are expressed presynaptically in the spinal dorsal horn, they are not bound to α2δ‐1 proteins and are in a quiescent state under the normal condition. Treatment with paclitaxel increases the expression of α2δ‐1 in primary sensory neurons and the interaction between α2δ‐1 and NMDA receptors, which promote the synaptic trafficking of α2δ‐1–bound NMDA receptor complexes and lead to presynaptic NMDA receptor hyperactivity in the spinal dorsal horn. The increased presynaptic NMDA receptor activity results in sustained potentiation in glutamate release to spinal dorsal horn neurons, which constitutes a critical mechanism of chemotherapy‐induced chronic pain. Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Volume-regulated anion channels (VRACs) are critically involved in regulating cell volume, and leucine-rich repeat-containing protein 8A (LRRC8A, SWELL1) is an obligatory subunit of VRACs. Cell swelling occurs early after brain ischemia, but it is unclear whether neuronal LRRC8a contributes to ischemia-induced glutamate release and brain injury. We found that Lrrc8a conditional knockout (Lrrc8a -cKO) mice produced by crossing Nestin Cre+/− with Lrrc8a flox+/+ mice died 7–8 weeks of age, indicating an essential role of neuronal LRRC8A for survival. Middle cerebral artery occlusion (MCAO) caused an early increase in LRRC8A protein levels in the hippocampus in wild-type (WT) mice. Whole-cell patch-clamp recording in brain slices revealed that oxygen-glucose deprivation significantly increased the amplitude of VRAC currents in hippocampal CA1 neurons in WT but not in Lrrc8a -cKO mice. Hypotonicity increased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) in hippocampal CA1 neurons in WT mice, and this was abolished by DCPIB, a VRAC blocker. But in Lrrc8a -cKO mice, hypotonic solution had no effect on the frequency of sEPSCs in these neurons. Furthermore, the brain infarct volume and neurological severity score induced by MCAO were significantly lower in Lrrc8a -cKO mice than in WT mice. In addition, MCAO-induced increases in cleaved caspase-3 and calpain activity, two biochemical markers of neuronal apoptosis and death, in brain tissues were significantly attenuated in Lrrc8a -cKO mice compared with WT mice. These new findings indicate that cerebral ischemia increases neuronal LRRC8A-dependent VRAC activity and that VRACs contribute to increased glutamatergic input to hippocampal neurons and brain injury caused by ischemic stroke. Schematic drawing outlines the potential role of neuronal LRRC8A-containing volume-regulated anion channels (VRACs) in ischemic brain damage. Neuronal VRACs are not activated or involved in glutamate release in the normal brain. Cerebral ischemia causes rapid cell swelling and neuronal depolarization to increase the activity of VRACs expressed in presynaptic and postsynaptic neurons. Augmented VRAC activity in ischemic neurons leads to chloride influx and glutamate release and subsequent activation of glutamate NMDA receptors, which can induce excitotoxicity and neuronal death through calplain/caspase-mediated signaling pathways. Unlabelled Image • Cerebral ischemia transiently increases LRRC8A protein levels in the hippocampus. • LRRC8A is essential for hippocampal neuronal VRAC activity increased by ischemia. • LRRC8A is required for hypotonicity-potentiated glutamatergic input to neurons. • LRRC8A-dependent VRACs contribute to brain injury caused by cerebral ischemia. [ABSTRACT FROM AUTHOR]