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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]

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]

Key points: μ‐Opioid receptors (MORs) are expressed peripherally and centrally, but the loci of MORs responsible for clinically relevant opioid analgesia are uncertain.Crossing Oprm1flox/flox and AdvillinCre/+ mice completely ablates MORs in dorsal root ganglion neurons and reduces the MOR expression level in the spinal cord.Presynaptic MORs expressed at primary afferent central terminals are essential for synaptic inhibition and potentiation of sensory input by opioids.MOR ablation in primary sensory neurons diminishes analgesic effects produced by systemic and intrathecal opioid agonists and abolishes chronic opioid treatment‐induced hyperalgesia.These findings demonstrate a critical role of MORs expressed in primary sensory neurons in opioid analgesia and suggest new strategies to increase the efficacy and reduce adverse effects of opioids. The pain and analgesic systems are complex, and the actions of systemically administered opioids may be mediated by simultaneous activation of μ‐opioid receptors (MORs, encoded by the Oprm1 gene) at multiple, interacting sites. The loci of MORs and circuits responsible for systemic opioid‐induced analgesia and hyperalgesia remain unclear. Previous studies using mice in which MORs are removed from Nav1.8‐ or TRPV1‐expressing neurons provided only an incomplete and erroneous view about the role of peripheral MORs in opioid actions in vivo. In the present study, we determined the specific role of MORs expressed in primary sensory neurons in the analgesic and hyperalgesic effects produced by systemic opioid administration. We generated Oprm1 conditional knockout (Oprm1‐cKO) mice in which MOR expression is completely deleted from dorsal root ganglion neurons and substantially reduced in the spinal cord, which was confirmed by immunoblotting and immunocytochemical labelling. Both opioid‐induced inhibition and potentiation of primary sensory input were abrogated in Oprm1‐cKO mice. Remarkably, systemically administered morphine potently inhibited acute thermal and mechanical nociception and persistent inflammatory pain in control mice but had little effect in Oprm1‐cKO mice. The analgesic effect of intrathecally administered morphine was also profoundly reduced in Oprm1‐cKO mice. Additionally, chronic morphine treatment‐induced hyperalgesia was absent in Oprm1‐cKO mice. Our findings directly challenge the notion that clinically relevant opioid analgesia is mediated mostly by centrally expressed MORs. MORs in primary sensory neurons, particularly those expressed presynaptically at the first sensory synapse in the spinal cord, are crucial for both opioid analgesia and opioid‐induced hyperalgesia. Key points: μ‐Opioid receptors (MORs) are expressed peripherally and centrally, but the loci of MORs responsible for clinically relevant opioid analgesia are uncertain.Crossing Oprm1flox/flox and AdvillinCre/+ mice completely ablates MORs in dorsal root ganglion neurons and reduces the MOR expression level in the spinal cord.Presynaptic MORs expressed at primary afferent central terminals are essential for synaptic inhibition and potentiation of sensory input by opioids.MOR ablation in primary sensory neurons diminishes analgesic effects produced by systemic and intrathecal opioid agonists and abolishes chronic opioid treatment‐induced hyperalgesia.These findings demonstrate a critical role of MORs expressed in primary sensory neurons in opioid analgesia and suggest new strategies to increase the efficacy and reduce adverse effects of opioids. [ABSTRACT 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]

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

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

Key points We developed an animal model to study the mechanisms underlying the pain syndrome commonly seen in organ transplant patients receiving calcineurin inhibitors., Systemic treatment with the calcineurin inhibitor induces long-lasting pain hypersensitivity and increases glutamate receptor activity in the spinal cord., Blocking glutamate receptor activity at the spinal cord level effectively reduces pain hypersensitivity induced by the calcineurin inhibitor., This information advances our understanding of the molecular basis of pain caused by calcineurin inhibitors and identifies new strategies for treating such pain syndrome in transplant patients., Abstract 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. [ABSTRACT FROM AUTHOR]

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

Background and Purpose: Sirtuin 6 (SIRT6) is involved in regulation of glucose and fat metabolism. However, its possible contribution to cardiac dysfunction remains to be determined. In the present study, the effect of SIRT6 on cardiac hypertrophy induced by angiotensin II (AngII) and the underlying molecular mechanisms were investigated.Experimental Approach: The expression and deacetylase activity of SIRT6 were measured in hypertrophic cardiomyocytes induced by AngII. After SIRT6 overexpression by transfection, or depletion by RNA interference in neonatal rat cardiomyocytes, cellular hypertrophy was monitored by measuring cell surface area and the mRNA levels of hypertrophic biomarkers. Further, the interaction between SIRT6 and the transcription factor NF-κB was investigated by co-immunoprecipitation, confocal immunofluorescence microscopy and luciferase reporter gene assay. The expression and deacetylase activity of SIRT6 were measured in vivo, using the abdominal aortic constriction (AAC) model of cardiac hypertrophy in rats.Key Results: In AngII-induced hypertrophic cardiomyocytes and also in AAC-induced hypertrophic hearts, the expression of SIRT6 protein was upregulated, while its deacetylase activity was decreased. Overexpression of wild-type SIRT6 but not its catalytically inactive mutant, attenuated AngII-induced cardiomyocyte hypertrophy. We further demonstrated a physical interaction between SIRT6 and NF-κB catalytic subunit p65, whose transcriptional activity could be repressed by SIRT6 overexpression.Conclusions and Implications: Our findings suggest that SIRT6 suppressed cardiomyocyte hypertrophy in vitro via inhibition of NF-κB-dependent transcriptional activity and that this effect was dependent on its deacetylase activity. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 944-953. Abstract Abnormal hyperexcitability of primary sensory neurons contributes to neuropathic pain development after nerve injury. Nerve injury profoundly reduces the expression of big conductance Ca2+ -activated K+ (BK) channels in the dorsal root ganglion (DRG). However, little is known about how nerve injury affects BK channel activity in DRG neurons. In this study, we determined the changes in BK channel activity in DRG neurons in a rat model of neuropathic pain and the contribution of brain-derived neurotrophic factor (BDNF) to reduced BK channel activity. The BK channel activity was present predominantly in small and medium DRG neurons, and ligation of L5 and L6 spinal nerves profoundly decreased the BK current density in these neurons. Blocking BK channels significantly increased neuronal excitability in sham control, but not in nerve-injured, rats. The BDNF concentration in the DRG was significantly greater in nerve-injured rats than in control rats. BDNF treatment largely reduced BK currents in DRG neurons in control rats, which was blocked by either anti-BDNF antibody or K252a, a Trk receptor inhibitor. Furthermore, either anti-BDNF antibody or K252a reversed reduction in BK currents in injured DRG neurons. BDNF treatment reduced the mRNA levels of BKα1 subunit in DRG neurons, and anti-BDNF antibody attenuated the reduction in the BKα1 mRNA level in injured DRG neurons. These findings suggest that nerve injury primarily diminishes the BK channel activity in small and medium DRG neurons. Increased BDNF levels contribute to reduced BK channel activity in DRG neurons through epigenetic and transcriptional mechanisms in neuropathic pain. [ABSTRACT FROM AUTHOR]

Abstract: The discovery of sirtuins (SIRT), a family of nicotinamide adenine dinucleotide (NAD)-dependent deacetylases, has indicated that intracellular NAD level is crucial for the hypertrophic response of cardiomyocytes. Nicotinamide mononucleotide adenylyltransferase (Nmnat) is a central enzyme in NAD biosynthesis. Here we revealed that Nmnat2 protein expression and enzyme activity were down-regulated during cardiac hypertrophy. In neonatal rat cardiomyocytes, overexpression of Nmnat2 but not its catalytically inactive mutant blocked angiotensin II (Ang II)-induced cardiac hypertrophy, which was dependent on activation of SIRT6 through maintaining the intracellular NAD level. Our results suggested that modulation of Nmnat2 activity may be beneficial in cardiac hypertrophy. [Copyright &y& Elsevier]

J. Neurochem. (2011) 119, 594-603. Abstract Painful neuropathy is one of the most serious complications of diabetes and remains difficult to treat. The muscarinic acetylcholine receptor (mAChR) agonists have a profound analgesic effect on painful diabetic neuropathy. Here we determined changes in T-type and high voltage-activated Ca2+ channels (HVACCs) and their regulation by mAChRs in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathy. The HVACC currents in large neurons, T-type currents in medium and large neurons, the percentage of small DRG neurons with T-type currents, and the Cav3.2 mRNA level were significantly increased in diabetic rats compared with those in control rats. The mAChR agonist oxotremorine-M significantly inhibited HVACCs in a greater proportion of DRG neurons with and without T-type currents in diabetic than in control rats. In contrast, oxotremorine-M had no effect on HVACCs in small and large neurons with T-type currents and in most medium neurons with T-type currents from control rats. The M2 and M4 antagonist himbacine abolished the effect of oxotremorine-M on HVACCs in both groups. The selective M4 antagonist muscarinic toxin-3 caused a greater attenuation of the effect of oxotremorine-M on HVACCs in small and medium DRG neurons in diabetic than in control rats. Additionally, the mRNA and protein levels of M4, but not M2, in the DRG were significantly greater in diabetic than in control rats. Our findings suggest that diabetic neuropathy potentiates the activity of T-type and HVACCs in primary sensory neurons. M4 mAChRs are up-regulated in DRG neurons and probably account for increased muscarinic analgesic effects in diabetic neuropathic pain. [ABSTRACT FROM AUTHOR]

Hyperactivity of spinal dorsal horn neurons plays an important role in the development of diabetic neuropathic pain. However, little is known as to whether synaptic input to spinal dorsal horn neurons is altered in diabetic neuropathy. Also, the function of GABAB receptors in the control of synaptic input to dorsal horn neurons in diabetes remains poorly understood. To determine the changes in synaptic input to dorsal horn neurons and the GABAB receptor function in streptozotocin-induced diabetes, we performed whole-cell recording (GDP-β-S included in the internal solution) on lamina II neurons in rat spinal cord slices. The frequency of glutamatergic mEPSCs and the amplitude of monosynaptic EPSCs evoked from the dorsal root were significantly higher in diabetic than in control rats. On the other hand, the basal frequency and amplitude of GABAergic spontaneous IPSCs and mIPSCs and those of glycinergic spontaneous IPSCs and mIPSCs did not differ significantly between control and diabetic rats. The GABAB agonist baclofen produced a significantly greater reduction in dorsal root-evoked EPSCs and the frequency of mEPSCs in control than in diabetic rats. However, the inhibitory effect of baclofen on GABAergic and glycinergic spontaneous IPSCs and mIPSCs was not significantly different in the two groups. These findings suggest that increased glutamatergic input from primary afferents to dorsal horn neurons may contribute to synaptic plasticity and central sensitization in diabetic neuropathic pain. Furthermore, the function of presynaptic GABAB receptors at primary afferent terminals, but not that on GABAergic and glycinergic interneurons, in the spinal cord is reduced in diabetic neuropathy. [ABSTRACT FROM AUTHOR]