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Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin inhibitor‐induced hypertension (CIH) is linked to augmented sympathetic output from the hypothalamic paraventricular nucleus (PVN). GluA2‐lacking, Ca2+‐permeable AMPA receptors (CP‐AMPARs) are a key feature of glutamatergic synaptic plasticity, yet their role in CIH remains elusive. Here, we found that systemic administration of FK506 in rats significantly increased serine phosphorylation of GluA1 and GluA2 in PVN synaptosomes. Strikingly, FK506 treatment reduced GluA1/GluA2 heteromers in both synaptosomes and endoplasmic reticulum‐enriched fractions from the PVN. Blocking CP‐AMPARs with IEM‐1460 induced a larger reduction of AMPAR‐mediated excitatory postsynaptic current (AMPAR‐EPSC) amplitudes in retrogradely labelled, spinally projecting PVN neurons in FK506‐treated rats than in vehicle‐treated rats. Furthermore, FK506 treatment shifted the current–voltage relationship of AMPAR‐EPSCs from linear to inward rectification in labelled PVN neurons. FK506 treatment profoundly enhanced physical interactions of α2δ‐1 with GluA1 and GluA2 in the PVN. Inhibiting α2δ‐1 with gabapentin, α2δ‐1 genetic knockout, or disrupting α2δ‐1–AMPAR interactions with an α2δ‐1 C terminus peptide restored GluA1/GluA2 heteromers in the PVN and diminished inward rectification of AMPAR‐EPSCs in labelled PVN neurons induced by FK506 treatment. Additionally, microinjection of IEM‐1460 or α2δ‐1 C terminus peptide into the PVN reduced renal sympathetic nerve discharges and arterial blood pressure elevated in FK506‐treated rats but not in vehicle‐treated rats. Thus, calcineurin in the hypothalamus constitutively regulates AMPAR subunit composition and phenotypes by controlling GluA1/GluA2 interactions with α2δ‐1. Synaptic CP‐AMPARs in PVN presympathetic neurons contribute to augmented sympathetic outflow in CIH. Key points: Systemic treatment with the calcineurin inhibitor increases serine phosphorylation of synaptic GluA1 and GluA2 in the PVN.Calcineurin inhibition enhances the prevalence of postsynaptic Ca2+‐permeable AMPARs in PVN presympathetic neurons.Calcineurin inhibition potentiates α2δ‐1 interactions with GluA1 and GluA2, disrupting intracellular assembly of GluA1/GluA2 heterotetramers in the PVN.Blocking Ca2+‐permeable AMPARs or α2δ‐1–AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor. [ABSTRACT FROM AUTHOR]

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]

Glutamate AMPA receptors (AMPARs) lacking GluA2 subunit are calcium permeable (CP‐AMPARs), which are increased in the hypothalamic paraventricular nucleus (PVN) and maintain sympathetic outflow in hypertension. Here, we determined the role of α2δ‐1, an NMDA receptor–interacting protein, in regulating synaptic CP‐AMPARs in the hypothalamus in spontaneously hypertensive rats (SHR). Co‐immunoprecipitation showed that levels of GluA1/GluA2, but not GluA2/GluA3, protein complexes in hypothalamic synaptosomes were reduced in SHR compared with Wistar–Kyoto rats (WKY). The level of GluA1/GluA2 heteromers in endoplasmic reticulum‐enriched fractions of the hypothalamus was significantly lower in SHR than in WKY, which was restored by inhibiting α2δ‐1 with gabapentin. Gabapentin also switched AMPAR‐mediated excitatory postsynaptic currents (AMPAR‐EPSCs) from inward rectifying to linear and attenuated the inhibitory effect of IEM‐1460, a selective CP‐AMPAR blocker, on AMPAR‐EPSCs in spinally projecting PVN neurons in SHR. Furthermore, co‐immunoprecipitation revealed that α2δ‐1 directly interacted with GluA1 and GluA2 in the hypothalamus of rats and humans. Levels of α2δ‐1/GluA1 and α2δ‐1/GluA2 protein complexes in the hypothalamus were significantly greater in SHR than in WKY. Disrupting the α2δ‐1–AMPAR interaction with an α2δ‐1 C terminus peptide normalized GluA1/GluA2 heteromers in the endoplasmic reticulum of the hypothalamus diminished in SHR. In addition, α2δ‐1 C terminus peptide diminished inward rectification of AMPAR‐EPSCs and the inhibitory effect of IEM‐1460 on AMPAR‐EPSCs of PVN neurons in SHR. Thus, α2δ‐1 augments synaptic CP‐AMPARs by inhibiting GluA1/GluA2 heteromeric assembly in the hypothalamus in hypertension. These findings extend our understanding of the molecular basis of sustained sympathetic outflow in neurogenic hypertension. [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]

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]

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. Many excitatory interneurons in the spinal cord dorsal horn express vesicular glutamate transporter‐2 (VGluT2), but their functional significance in physiological and pathological pain has not been shown specifically. Using chemogenetic approaches, we showed that stimulating VGluT2‐expressing dorsal horn neurons with hM3Dq‐CNO elicits pain hypersensitivity. Conversely, inhibiting VGluT2‐expressing dorsal horn neurons with KORD‐SALB attenuates persistent pain hypersensitivity caused by tissue inflammation and peripheral nerve injury. These findings indicate that VGluT2‐expressing neurons in the spinal dorsal horn play a pivotal role in transmitting painful information. [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]

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. [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]

Summary 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. [ABSTRACT FROM AUTHOR]

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. [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]

δ-Opioid receptors (DORs, encoded by the Oprd1 gene) are expressed throughout the peripheral and central nervous system, and DOR stimulation reduces nociception. Previous studies suggest that DORs promote the development of analgesic tolerance of μ-opioid receptor (MOR) agonists. It is uncertain whether DORs expressed in primary sensory neurons are involved in regulating chronic pain and MOR agonist-induced tolerance. In this study, we generated Oprd1 conditional knockout (Oprd1 -cKO) mice by crossing Advillin-Cre mice with Oprd1 -floxed mice. DOR expression in the dorsal root ganglion was diminished in Oprd1 -cKO mice. Systemic or intrathecal injection of the DOR agonist SNC-80 produced analgesia in wild-type (WT), but not Oprd1 -cKO, mice. In contrast, intracerebroventricular injection of SNC-80 produced a similar analgesic effect in WT and Oprd1 -cKO mice. However, morphine-induced analgesia, hyperalgesia, or analgesic tolerance did not differ between WT and Oprd1 -cKO mice. Compared with WT mice, Oprd1 -cKO mice showed increased mechanical and heat hypersensitivity after nerve injury or tissue inflammation. Furthermore, blocking DORs with naltrindole increased nociceptive sensitivity induced by nerve injury or tissue inflammation in WT, but not Oprd1 -cKO, mice. In addition, naltrindole potentiated glutamatergic input from primary afferents to spinal dorsal horn neurons increased by nerve injury or CFA in WT mice; this effect was absent in Oprd1 -cKO mice. Our findings indicate that DORs in primary sensory neurons are critically involved in the analgesic effect of DOR agonists but not morphine-induced analgesic tolerance. Presynaptic DORs at primary afferent central terminals constitutively inhibit inflammatory and neuropathic pain by restraining glutamatergic input to spinal dorsal horn neurons. • δ-opioid receptors (DORs) in primary sensory neurons restrain pain hypersensitivity caused by inflammation or nerve injury. • Presynaptic DORs tonically inhibit glutamatergic input to spinal dorsal horn neurons induced by inflammation or nerve injury. • The analgesic effect produced by systemic or intrathecal DOR agonists critically depends on DORs in primary sensory neurons. • Morphine produces analgesia, hyperalgesia, and tolerance independent of DORs expressed in primary sensory neurons. [ABSTRACT FROM AUTHOR]

The hypothalamic paraventricular nucleus (PVN) plays a major role in generating increased sympathetic output in hypertension. Although group III metabotropic glutamate receptors (mGluRs) are expressed in the hypothalamus, little is known about their contribution to regulating PVN presympathetic neurons in hypertension. Here we show that activating group III mGluRs with L-2-amino-4-phosphonobutyric acid (L-AP4) consistently inhibited the firing activity of spinally projecting PVN neurons in normotensive rats. However, in spontaneously hypertensive rats (SHRs), L-AP4 inhibited 45% of PVN neurons but excited 37%. L-AP4 significantly reduced glutamatergic and GABAergic input to PVN neurons in both groups. Blocking postsynaptic G protein signaling eliminated the excitatory but not the inhibitory effect of L-AP4 on PVN neurons in SHRs. Remarkably, prior activation of group I mGluRs converted the L-AP4 effect from inhibitory to excitatory in PVN neurons, and L-AP4 consistently inhibited PVN neurons when mGluR5 was blocked in SHRs. Furthermore, the expression level of mGluR4 and mGluR6 in the PVN was significantly higher in SHRs than in normotensive rats. Microinjection of L-AP4 into the PVN decreased blood pressure and lumbar sympathetic nerve discharges in normotensive rats and SHRs. Additionally, blocking group I mGluRs in the PVN potentiated L-AP4's sympathoinhibitory effect in SHRs. Therefore, activation of presynaptic group III mGluRs inhibits the excitability of PVN presympathetic neurons to attenuate sympathetic vasomotor activity. Through crosstalk with mGluR5, postsynaptic group III mGluR stimulation paradoxically excites PVN presympathetic neurons in SHRs. Concurrently blocking mGluR5 and activating group III mGluRs in the PVN can effectively reduce sympathetic outflow in hypertension. • Group III mGluR stimulation inhibits hypothalamic neurons in normotension but excite some of these neurons in hypertension. • Group III mGluR activation reduces excitatory and inhibitory synaptic inputs to hypothalamic neurons in both conditions. • Group III mGluR stimulation paradoxically excites some hypothalamic neurons in hypertension through postsynaptic mGluR5. • Hypothalamic group III mGluR activation reduces sympathetic output, which is enhanced by blocking mGluR5 in hypertension. [ABSTRACT FROM AUTHOR]