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Calcineurin, the predominant Ca2+/calmodulin-dependent serine/threonine protein phosphatase (also known as protein phosphatase 2B), is highly expressed in immune T cells and the nervous system, including the dorsal root ganglion and spinal cord. It controls synaptic transmission and plasticity by maintaining the appropriate phosphorylation status of many ion channels present at presynaptic and postsynaptic sites. As such, normal calcineurin activity in neurons and synapses is mainly involved in negative feedback regulation in response to increased neuronal activity and intracellular Ca2+ levels. Calcineurin inhibitors (e.g., cyclosporine and tacrolimus) are widely used as immunosuppressants in tissue and organ transplantation recipients and for treating autoimmune diseases but can cause severe pain in some patients. Furthermore, diminished calcineurin activity at the spinal cord level may play a major role in the transition from acute to chronic neuropathic pain after nerve injury. Restoring calcineurin activity at the spinal cord level produces long-lasting pain relief in animal models of neuropathic pain. In this article, we provide an overview of recent studies on the critical roles of calcineurin in regulating glutamate NMDA and AMPA receptors, voltage-gated Ca2+ channels, potassium channels, and transient receptor potential channels expressed in the spinal dorsal horn and primary sensory neurons. [ABSTRACT FROM AUTHOR]

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]

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]

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

Purpose The purpose of this study is to investigate the effect of the spacer length of zinc porphyrin-TiO2 hybrids by photodegradation of methyl orange (MO) in aqueous solution under visible light.Design/methodology/approach 5-Mono-[4-hydroxyphenyl]-10,15,20-triphenylporphyrin was synthesized using Alder method. A new series of porphyrins and their corresponding zinc complexes (ZnPp) were obtained from 5-mono-[4-hydroxyphenyl]-10,15,20-triphenylporphyrin via nucleophilic substitution reaction. The ZnPp-TiO2 photocatalysts were prepared by loading ZnPp onto TiO2 and characterized by scanning electron microscope, X-ray diffraction, UV-vis diffuse reflectance spectrum and X-ray photoelectron spectroscopy.Findings The results indicated that zinc porphyrins were successfully loaded on the surface of TiO2 microsphere, which is crucial to enhance the activity of the catalytic composite under visible light. All the novel photocatalysts showed much enhanced photoactivity than bare TiO2. Among all the prepared ZnPp-TiO2, 5,10,15-triphenyl-20-[4-(4-naphthoxy)-butoxy]phenyl zinc porphyrin-TiO2 (4b) showed the highest photocatalytic activity for the degradation of MO.Research limitations/implications Synthesis of these zinc porphyrins had never been reported previously.Originality/value Four novel zinc porphyrin-TiO2 photocatalysts which could response to visible light in degradation of MO were synthesized using Alder method. The results show that the photocatalytic activity of 5,10,15-triphenyl-20-[4-(4-naphthoxy)butoxy] phenyl zinc porphyrin- TiO2 is higher than others. [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]

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]

A new series of 4-azasteroidal-20-oxime derivatives have been synthesised from progesterone. All the new compounds have been characterised by analysis and spectroscopic data and subsequently evaluated for their anticancer activity in vitro against T24 cell. The studies show that the methyl and ethyl oxime-ethers exhibited higher activity than the aryl substituted oxime-esters. [ABSTRACT FROM AUTHOR]

Highlights: [•] Nitric oxide increases presynaptic glutamate release to baroreceptor NTS neurons. [•] Nitric oxide increases N-type Ca2+ currents in baroreceptor NTS neurons in nodose ganglion. [•] Cav2.2 subunit is present on the baroreceptor afferent nerve terminals. [ABSTRACT FROM AUTHOR]

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

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]

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

Abstract: Most δ-opioid receptors are located on the presynaptic terminals of primary afferent neurons in the spinal cord. However, their presence in different phenotypes of primary afferent neurons and their contribution to the analgesic effect of δ-opioid agonists are not fully known. Resiniferatoxin (RTX) is an ultra-potent transient receptor potential vanilloid type 1 channel (TRPV1) agonist and can selectively remove TRPV1-expressing primary afferent neurons. In this study, we determined the role of δ-opioid receptors expressed on TRPV1 sensory neurons in the antinociceptive effect of the δ-opioid receptor agonists [d-Pen2,d-Pen5]-enkephalin and [d-Ala2,Glu4]-deltorphin. Nociception was measured by testing the mechanical withdrawal threshold in the hindpaw of rats. Changes in the δ-opioid receptors were assessed using immunocytochemistry and the [3H]-naltrindole radioligand binding. In RTX-treated rats, the δ-opioid receptor on TRPV1-immunoreactive dorsal root ganglion neurons and afferent terminals in the spinal cord was diminished. RTX treatment also significantly reduced the maximal specific binding sites (31%) of the δ-opioid receptors in the dorsal spinal cord. Interestingly, intrathecal injection of [d-Pen2,d-Pen5]-enkephalin or [d-Ala2,Glu4]-deltorphin produced a large and prolonged increase in the nociceptive threshold in RTX-treated rats. These findings indicate that loss of TRPV1-expressing afferent neurons leads to a substantial reduction in presynaptic δ-opioid receptors in the spinal dorsal horn. However, the effect of δ-opioid agonists on mechano-nociception is paradoxically potentiated in the absence of TRPV1-expressing sensory neurons. This information is important to our understanding of the cellular sites and mechanisms underlying the spinal analgesic effect of δ-opioid agonists. [Copyright &y& Elsevier]

Abstract: The α2-adrenoceptors (α2-ARs) are located on primary afferent terminals and on neurons in the spinal cord dorsal horn. However, their relative contribution to the analgesic effect of the α2-AR agonists is not known. In this study, we determined the role of certain presynaptic α2-ARs in the antinociceptive effect produced by intrathecal administration of the α2-AR agonist clonidine. TRPV1-expressing sensory neurons were removed by resiniferatoxin (RTX). The effect of intrathecal injection of clonidine was measured by testing the paw withdrawal response to noxious mechanical or heat stimuli. In RTX-treated rats, the α2A-AR-immunoreactivity co-expressed with TRPV1-expressing terminals in the spinal cord was eliminated. However, the α2C-AR-immunoreactivity in the spinal cord was little changed. Surprisingly, intrathecal administration of clonidine produced a much greater increase in the mechanical withdrawal threshold in RTX- than in vehicle-treated rats. The duration of the clonidine effect was also significantly increased in RTX-treated rats. Furthermore, in the vehicle-treated group, although intrathecal injection of clonidine produced a large increase in the thermal withdrawal latency, it only had a small and short-lasting effect on the mechanical withdrawal threshold. This study provides new information that the antinociceptive effect of spinally administered α2-AR agonists is largely modality-specific. Loss of TRPV1-expressing sensory neurons leads to a reduction in presynaptic α2A-ARs but paradoxically potentiates the effect of clonidine on mechano-nociception. [Copyright &y& Elsevier]

Abstract: Systemically administered μ opioids may produce analgesia through inhibition of the ascending nociceptive transmission and activation of descending pathways. However, the relative importance of the spinal and supraspinal sites in the analgesic action of systemic opioids remains uncertain. It has been shown that systemic morphine can inhibit dorsal horn neurons independent of the descending system. In this study, we determined the extent to which spinal μ opioid receptors mediate the analgesic effect of systemic μ opioids. Rats were instrumented with an intrathecal catheter with the tip placed in the lumbar spinal cord. Nociception was measured by testing the paw withdrawal threshold in response to a noxious radiant heat or pressure stimulus. Surprisingly, intrathecal pretreatment with naloxone or H-D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP, a specific μ opioid receptor antagonist) completely blocked the inhibitory effect of intravenous morphine on mechanical nociception. Intrathecal naloxone or CTAP also abolished the effect of intravenous morphine on the withdrawal latency of the hindpaw, but not the forepaw, measured with a radiant heat stimulus. Furthermore, the inhibitory effect of subcutaneous fentanyl on mechanical nociception was eliminated by CTAP injected intrathecally. Intrathecal CTAP similarly abolished the effect of subcutaneous fentanyl on thermal nociception of the hindpaw but not the forepaw. Therefore, this study provides new information that when spinal μ opioid receptors are blocked, subsequent systemic administration of μ opioids fails to produce an analgesic effect. This finding highlights the important role of μ opioid receptors in the spinal cord in the antinociceptive action of opioids. [Copyright &y& Elsevier]

Abstract: Patients with postherpetic neuralgia often have an increased sensitivity to a tactile stimulus but impaired thermal sensitivity in the same affected dermatomes. We recently found that depletion of capsaicin-sensitive afferents by systemic treatment with a potent TRPV1 agonist, resiniferotoxin, in adult rats produces long-lasting paradoxical changes in mechanical and thermal sensitivities, which resemble the unique clinical features of postherpetic neuralgia. The anticonvulsant gabapentin is effective in reducing the subjective pain score in patients with postherpetic neuralgia. In this study, we quantified the potential effect of systemic and intrathecal gabapentin on tactile allodynia induced by resiniferotoxin in rats. Intraperitoneal injection of 200 μg/kg resiniferotoxin produced a rapid and sustained increase in the paw withdrawal latency to a radiant heat stimulus. Profound tactile allodynia developed in all the resiniferotoxin-treated rats within 3 weeks. Intraperitoneal injection of 30–60 mg/kg of gabapentin in resiniferotoxin-treated rats significantly increased the withdrawal threshold in response to von Frey filaments. Furthermore, intrathecal administration of 10–30 μg of gabapentin also produced a significant effect on the mechanical withdrawal threshold in all resiniferotoxin-treated rats. These data provide complementary new information that gabapentin administered systemically and spinally can effectively relieve tactile allodynia in this animal model of postherpetic neuralgia. [Copyright &y& Elsevier]

Neurons in the paraventricular nucleus (PVN) are important in regulating autonomic function through projections to the brain stem and spinal cord. Although the vanilloid receptors (VR(1)) are present in the PVN, their physiological function is scarcely known. In this study, we determined the role of VR(1) receptors in the regulation of synaptic inputs and the excitability of spinally projecting PVN neurons. Whole cell patch-clamp recordings were performed on the PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Capsaicin significantly increased the frequency of glutamatergic miniature excitatory postsynaptic currents (mEPSCs) without changing the amplitude and decay time constant of mEPSCs. On the other hand, capsaicin had no effect on GABAergic miniature inhibitory postsynaptic currents (mIPSCs). The effect of capsaicin on mEPSCs was abolished by a specific VR(1) antagonist, iodo-resiniferatoxin (iodo-RTX), or ruthenium red. Importantly, iodo-RTX per se significantly reduced the amplitude of evoked EPSCs and the frequency of mEPSCs. Removal of extracellular Ca(2+), but not Cd(2+) treatment, also eliminated the effect of capsaicin on mEPSCs. Furthermore, capsaicin caused a large increase in the firing rate of PVN neurons, and such an effect was abolished in the presence of ionotropic glutamate receptor antagonists. Additionally, the double-immunofluorescence labeling revealed that all of the VR(1) immunoreactivity was colocalized with a presynaptic marker, synaptophysin, in the PVN. Thus this study provides the first evidence that activation of VR(1) receptors excites preautonomic PVN neurons through selective potentiation of glutamatergic synaptic inputs. Presynaptic VR(1) receptors and endogenous capsaicin-like substances in the PVN may represent a previously unidentified mechanism in hypothalamic regulation of the autonomic nervous system. [ABSTRACT FROM AUTHOR]

Spinally administered muscarinic receptor agonists or acetylcholinesterase inhibitors can produce antinociception. However, the mechanisms of the action of cholinergic agents in the spinal cord are not fully understood. Activation of spinal muscarinic receptors evokes γ-aminobutyric acid (GABA) release, which reduces the glutamatergic synaptic input to dorsal horn neurons through GABAB receptors. In this study, we determined the functional role of spinal GABAB receptors in the antinociceptive action of intrathecal cholinergic agents in normal rats and in a rat model of diabetic neuropathic pain. Diabetes was induced by intraperitoneal streptozotocin in rats. The intrathecal catheter was inserted with its tip positioned at the lumbar spinal level. Nociceptive threshold was measured by the paw withdrawal latency in response to a radiant heat stimulus in normal rats. Mechanical allodynia in diabetic rats was determined by von Frey filaments applied to the hindpaw. The effect of intrathecal muscarine or neostigmine was examined through pretreatment with the specific GABAB receptor antagonist, CGP55845, or its vehicle. Intrathecal injection of muscarine or neostigmine significantly increased the withdrawal latency in response to a heat stimulus in normal rats and the withdrawal threshold in response to application of von Frey filaments in diabetic rats. Intrathecal pretreatment with CGP55845 significantly attenuated the effect of both muscarine or neostigmine in normal rats. Furthermore, the antiallodynic effect of intrathecal neostigmine and muscarine was largely eliminated by CGP55845 in diabetic rats. These data suggest that the GABAB receptors in the spinal cord mediate both the antinociceptive and antiallodynic actions of intrathecal muscarine or neostigmine in normal rats and in a rat model of diabetic neuropathic pain. This study provides new functional evidence that activation of spinal GABAB receptors is one of the important mechanisms underlying the antinociceptive action of intrathecal cholinergic agents. [Copyright &y& Elsevier]

Deals with a study which examined the transmural release of bradykinin during myocardial ischemia and the influence of ischemic preconditioning on bradykinin release during myocardial ischemia. Hemodynamic data profile; Transmural myocardial bradykinin release during ischemia-reperfusion; Effect of ischemic preconditioning on myocardial interstitial bradykinin levels during ischemia.

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]

3,3,17,17-Diethylenedioxyandrost-5-ene was obtained by ketalisation of androstenedione, which was oxidised with PDC and t-BuOOH to form 3,3,17,17-diethylene-dioxyandrost-5-ene-7-one. Stereoselective reduction of 3,3,17,17-diethylene-dioxyandrost-5-ene-7-one by NaBH4 in the presence of CeCl3 .6H2 O gave 3,3,17,17-diethylenedioxy 7β-hydroxy-androst-5-ene, which was deprotected with p-toluenesulfonic acid to gave 3,3-ethylenedioxyandrost-5-ene-7β-hydroxy and androst-4,6-dien-3,17-dione. A series of androstenedione derivatives were obtained from 3,3-ethylenedioxyandrost-5-ene-7β-hydroxy by the esterification reaction. Their structures were confirmed by MS,1 H NMR, 13C NMR and HRMS. [ABSTRACT FROM AUTHOR]

A simple and efficient method has been developed for stereoselective one-pot synthesis of (E)-3-(4-arylpiperazin-1-yl)-1-arylprop-2-en-1-ones by condensation of aryl ketones with arylpiperazines and N, N-dimethylformamide dimethyl acetal in acetic acid with 54-69% yields. Compared with reported methods, this one-step procedure appears to be a useful approach to prepare (E)-β-enaminoketones with good stereoselectivity, simple operation and moderate yields. [ABSTRACT FROM AUTHOR]

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

R601 --> 715.1 --> Calcineurin is a calcium/calmodulin‐dependent serine/threonine protein phosphatase, and hypertension is a major side effect of clinically used calcineurin inhibitors. Although increased sympathetic nerve discharges are associated with calcineurin inhibitor‐induced hypertension, the sources of sympathetic outflow are unknown. In this study, we determined the role of the paraventricular nuclear (PVN) of the hypothalamus in calcineurin inhibitor‐induced hypertension. Immunocytochemistry labeling showed that calcineurin was expressed on spinally projecting PVN neurons. Radiotelemetry recordings showed that systemic administration of tacrolimus (FK506), a specific calcineurin inhibitor, for 14 days caused a gradual and significant increase in arterial blood pressure in rats, which lasted at least another 7 days after discontinuing FK506 treatment. FK506 significantly decreased calcineurin activity in the PVN and increased the protein level of GluN1, the NMDA receptor obligatory subunit, and serine/threonine phosphorylation levels of GluN1 in the PVN synaptosomes. Electrophysiological recordings revealed that the spontaneous firing rate, frequency of mEPSCs, and amplitude of puff NMDA‐elicited currents in spinally projecting PVN neurons were significant greater in FK506‐treated rats than in vehicle‐treated rats. Remarkably, treatment with the NMDA receptor antagonist AP5 normalized the stimulatory effects of FK506 on PVN neurons of FK506‐treated rats. Furthermore, microinjection of AP5 into the PVN decreased renal sympathetic nerve discharges and blood pressure increased in FK506‐treated rats. Systemic administration of memantine, a clinically used NMDA receptor antagonist, also significantly lowered the high blood pressure in FK506‐treated rats. Collectively, our findings indicate that calcineurin inhibitor‐induced persistent hypertension results from augmented sympathetic output through increased NMDA receptor phosphorylation and activity in the PVN. [ABSTRACT FROM AUTHOR]

The synthesis of novel trispiropyrrolidine derivatives has been achieved by a one-pot, three-component 1,3-dipolar cycloaddition reaction of 3-aryl-5-arylmethylenespiro[indole-3′,2-[1,3]thiazolane]-2′(1H),4-dione, isatin and sarcosine in refluxing toluene which produced the corresponding cycloadducts in good yields. The structures were determined by 1 H NMR, IR and elementary analysis. [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]

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

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]

Abstract: Flavonoids are a class of compounds that exist in nature with the structure of 2-phenyl-chromone. In Chinese traditional medicine, herbal drugs containing flavonoids are widely used for the treatment of inflammation, cardiovascular disease, tumor and so on. In this study, we investigated the anti-inflammatory effect and related mechanisms of a novel synthetic flavonoid, (E)-1-(4-ethoxyphenyl)-3-(4-nitrophenyl)-prop-2-en-1-one (ETH) in lipopolysaccharide (LPS) stimulated macrophages. Our results showed that ETH inhibited LPS-induced TNF-α and IL-6 release in a dose-dependent manner, and decreased TNF-α, IL-1β, IL-6 and iNOS mRNA production. LPS-induced expression of cyclooxygenase-2 (COX-2) was also significantly attenuated by ETH. Pretreatment with ETH reduced the I-κBα phosphorylation, p65 nuclear translocation as well as NF-κB-dependent transcriptional activity. In addition, ETH exhibited a significant protection against LPS-induced inflammatory mortality in mice. Taken together, these findings suggest that ETH can inhibit LPS-induced inflammation via suppressing NF-κB signaling pathway, indicating that ETH may be a potential anti-inflammatory agent. [Copyright &y& Elsevier]

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]

Background: The efflux of cellular cholesterol mediated by apolipoprotein (apo)A-I and ATP-binding cassette transporter A1 (ABCA1) is a major pathway of reverse cholesterol transport. We investigated the effect of aspirin on this process. Methods: The expression levels of ABCA1 in RAW264.7 cells were determined using reverse transcription polymerase chain reaction and Western blot analysis. 3H-cholesterol efflux was measured by scintillation counting. Results: 0.5 mmol/l aspirin increased apoA-I-mediated cholesterol efflux and increased the expression of ABCA1. By increasing the dose of aspirin higher than 0.5 mmol/l, ABCA1 expression and function were significantly decreased. In cells transfected with a specific peroxisome proliferator-activated receptor (PPAR)-α small interfering RNA, the induction of ABCA1 expression and apoA-I-mediated 3H-cholesterol efflux by aspirin were substantially suppressed. Conclusions: The data demonstrate that low-dose aspirin increases ABCA1 expression via a PPAR-α-dependent mechanism and increases apoA-I-mediated cholesterol efflux. Copyright © 2010 S. Karger AG, Basel [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]

Neurons in the paraventricular nucleus (PVN) that project to the brain stem and spinal cord are important for autonomic regulation. The excitability of preautonomic PVN neurons is controlled by the noradrenergic input from the brain stem. In this study, we determined the role of alpha(2) adrenergic receptors in the regulation of excitatory and inhibitory synaptic inputs to spinally projecting PVN neurons. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were recorded using whole cell voltage-clamp techniques on PVN neurons labeled by a retrograde fluorescence tracer injected into the thoracic spinal cord of rats. Bath application of 5-20 muM clonidine, an alpha(2) receptor agonist, significantly reduced the amplitude of evoked GABAergic IPSCs in a dose-dependent manner. Also, 10 microM clonidine significantly decreased the frequency (from 2.68 +/- 0.41 to 1.22 +/- 0.40 Hz) but not the amplitude of miniature IPSCs (mIPSCs), and this effect was blocked by the alpha(2) receptor antagonist yohimbine. Furthermore, clonidine increased the paired-pulse ratio of evoked IPSCs from 1.25 +/- 0.05 to 1.61 +/- 0.08 (P < 0.05). On the other hand, clonidine had little effect on evoked glutamatergic EPSCs, mEPSCs, and the paired-pulse ratio of evoked EPSCs in most labeled cells examined. Additionally, immunofluorescence labeling revealed that the alpha(2A) receptor and GABA immunoreactivities were co-localized in close apposition to labeled PVN neurons. Collectively, these data suggest that stimulation of alpha(2) adrenergic receptors primarily attenuates GABAergic inputs to PVN output neurons to the spinal cord. The presynaptic alpha(2) receptors function as heteroreceptors to modulate synaptic GABA release and contribute to the hypothalamic regulation of sympathetic outflow. [ABSTRACT FROM AUTHOR]

The nucleus locus coeruleus (LC) plays an important role in analgesia produced by opioids and by modulation of the descending noradrenergic pathway. The functional role of μ-opioid receptors (μOR) in regulation of the excitability of spinally projecting LC neurons has not been investigated. In the present study, we tested the hypothesis that activation of presynaptic μ-opioid receptors excites a population of spinally projecting LC neurons through attenuation of γ-aminobutyric acid (GABA)-ergic synaptic inputs. Spinally projecting LC neurons were retrogradely labeled by a fluorescent dye injected into the spinal dorsal horn of rats. Whole-cell current- and voltage-clamp recordings were performed on labeled LC neurons in brain slices. All labeled LC noradrenergic neurons were demonstrated by dopamine-β-hydroxylase (DβH) immunofluorescence. In 37 labeled LC neurons, (d-Ala2,N-Me-Phe4,Gly-ol5)-enkephalin (DAMGO) significantly increased the discharge activity of 17 (45.9%) neurons, but significantly inhibited the firing activity of another 15 (40.5%) cells. The excitatory effect of DAMGO on seven labeled LC neurons was diminished in the presence of bicuculline. DAMGO significantly decreased the frequency of GABA-mediated miniature inhibitory postsynaptic currents (mIPSCs) in all nine labeled LC neurons. However, DAMGO had no effect on glutamate-mediated miniature excitatory postsynaptic currents (mEPSCs) in 12 of 15 neurons. Furthermore, DAMGO significantly inhibited the peak amplitude of evoked inhibitory postsynaptic currents (eIPSCs) in all 11 labeled neurons, but had no significant effect on the evoked excitatory postsynaptic currents (eEPSCs) in 10 of these 11 neurons. Thus, data from this study suggest that activation of μ-opioid receptors excites a population of spinally projecting LC neurons by preferential inhibition of GABAergic synaptic inputs. These findings provide important new information about the descending noradrenergic modulation and analgesic mechanisms of opioids. [Copyright &y& Elsevier]

Although uterine distension in rats results in an escape reflex, there exists no model of uterine cervical distension (UCD), the pain stimulus during the first stage of labor. The aims of this study were to develop such a model in virgin rats and to test whether peripherally restricted kappa opioid receptor (KOR) agonists (ADL 10-0101, ADL 10-0102, ADL 10-0116) inhibit responses to UCD. Under intravenous (i.v.) pentobarbital and alpha-chloralose anesthesia, fine metal rods were inserted in both uterine cervical osses through a small midline laparotomy. UCD was performed by manual separation of the rods (25–100 g). Single-unit afferent responses in hypogastric nerve or reflex rectus abdominis electromyographic (EMG) activity were determined before and after i.v. KOR agonists. UCD resulted in a stimulus-dependent increase in single-unit afferent activity. Units could be characterized as low threshold (mean threshold 6.6±2.7 g), or high threshold (mean threshold 55±8.8 g); all were C fibers, all responded to topical bradykinin. ADL 10-0116 (10 mg/kg) reduced the afferent response to UCD. Reflex EMG response occurred over a distension force range of 25–100 g, unaffected by i.v. saline. All three KOR agonists produced a dose-dependent, naloxone-reversible inhibition of the EMG response with a potency relationship of ADL 10-0102 (ED50 0.04 mg/kg)>ADL 10-0101 (ED50 0.65 mg/kg)=ADL 10-0116 (ED50 0.60 mg/kg). These data support the use of acute UCD as a noxious stimulus, inducing afferent and reflex activity. Like other visceral stimuli, UCD is sensitive to inhibition by KOR agonists. [Copyright &y& Elsevier]

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]