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.
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.
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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.
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Glutamate NMDA receptors (NMDARs) in the nucleus accumbens (NAc) are critically involved in drug dependence and reward. α2δ‐1 is a newly discovered NMDAR‐interacting protein that promotes synaptic ...trafficking of NMDARs independently of its conventional role as a calcium channel subunit. However, it remains unclear how repeated opioid exposure affects synaptic NMDAR activity and α2δ‐1‐NMDAR interaction in the NAc. In this study, whole‐cell patch‐clamp recordings showed that repeated treatment with morphine in mice markedly increased the NMDAR‐mediated frequency of miniature excitatory postsynaptic currents (mEPSCs) and amplitude of puff NMDAR currents in medium spiny neurons in the NAc core region. Morphine treatment significantly increased the physical interaction of α2δ‐1 with GluN1 and their synaptic trafficking in the NAc. In Cacna2d1 knockout mice, repeated treatment with morphine failed to increase the frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Furthermore, inhibition of α2δ‐1 with gabapentin or disruption of the α2δ‐1‐NMDAR interaction with the α2δ‐1 C terminus–interfering peptide blocked the morphine‐elevated frequency of mEPSCs and amplitude of puff NMDAR currents in the NAc core. Correspondingly, systemically administered gabapentin, Cacna2d1 ablation, or microinjection of the α2δ‐1 C terminus–interfering peptide into the NAc core attenuated morphine‐induced conditioned place preference and locomotor sensitization. Our study reveals that repeated opioid exposure strengthens presynaptic and postsynaptic NMDAR activity in the NAc via α2δ‐1. The α2δ‐1‐bound NMDARs in the NAc have a key function in the rewarding effect of opioids and could be targeted for treating opioid use disorder and addiction.
Glutamate NMDA receptors (NMDARs) in the nucleus accumbens (NAc) are critically involved in opioid dependence and reward. Under normal conditions, NMDARs are not associated with the α2δ‐1 protein in the NAc. Repeated stimulation of the μ‐opioid receptor (MOR) increases the α2δ‐1–NMDAR interaction and their synaptic trafficking in the NAc, leading to increased presynaptic and postsynaptic NMDAR activity. As a result, glutamatergic synaptic transmission is potentiated in the NAc, which promotes the formation of opioid reward memory. Thus, the α2δ‐1–coupled NMDARs could be targeted for treating opioid use disorder and addiction.
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.
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Transient receptor potential subtypes ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) channels are expressed at the peripheral and central terminals of primary sensory neurons. However, their role in regulating pain transmission at the spinal cord level is unclear. We found that TRPA1 and TRPV1 at presynaptic terminals in the spinal dorsal horn are normally quiescent but become tonically activated during inflammatory pain. They are involved in potentiating excitatory glutamatergic input (via synaptic glutamate release and subsequent stimulation of AMPA and NMDA receptors) from primary sensory nerves to lamina I neurons to maintain central sensitization. Blocking TRPA1 or TRPV1 channels at the spinal cord level alleviates chronic inflammatory pain.
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The SARS-CoV-2-infected disease (COVID-19) outbreak is a major threat to human beings. Previous studies mainly focused on Wuhan and typical symptoms. We analysed 74 confirmed COVID-19 cases with GI ...symptoms in the Zhejiang province to determine epidemiological, clinical and virological characteristics.
COVID-19 hospital patients were admitted in the Zhejiang province from 17 January 2020 to 8 February 2020. Epidemiological, demographic, clinical, laboratory, management and outcome data of patients with GI symptoms were analysed using multivariate analysis for risk of severe/critical type. Bioinformatics were used to analyse features of SARS-CoV-2 from Zhejiang province.
Among enrolled 651 patients, 74 (11.4%) presented with at least one GI symptom (nausea, vomiting or diarrhoea), average age of 46.14 years, 4-day incubation period and 10.8% had pre-existing liver disease. Of patients with COVID-19 with GI symptoms, 17 (22.97%) and 23 (31.08%) had severe/critical types and family clustering, respectively, significantly higher than those without GI symptoms, 47 (8.14%) and 118 (20.45%). Of patients with COVID-19 with GI symptoms, 29 (39.19%), 23 (31.08%), 8 (10.81%) and 16 (21.62%) had significantly higher rates of fever >38.5°C, fatigue, shortness of breath and headache, respectively. Low-dose glucocorticoids and antibiotics were administered to 14.86% and 41.89% of patients, respectively. Sputum production and increased lactate dehydrogenase/glucose levels were risk factors for severe/critical type. Bioinformatics showed sequence mutation of SARS-CoV-2 with m
A methylation and changed binding capacity with ACE2.
We report COVID-19 cases with GI symptoms with novel features outside Wuhan. Attention to patients with COVID-19 with non-classic symptoms should increase to protect health providers.
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.
α2δ-1, commonly known as a voltage-activated Ca2+ channel subunit, is a binding site of gabapentinoids used to treat neuropathic pain and epilepsy. However, it is unclear how α2δ-1 contributes to ...neuropathic pain and gabapentinoid actions. Here, we show that Cacna2d1 overexpression potentiates presynaptic and postsynaptic NMDAR activity of spinal dorsal horn neurons to cause pain hypersensitivity. Conversely, Cacna2d1 knockdown or ablation normalizes synaptic NMDAR activity increased by nerve injury. α2δ-1 forms a heteromeric complex with NMDARs in rodent and human spinal cords. The α2δ-1-NMDAR interaction predominantly occurs through the C terminus of α2δ-1 and promotes surface trafficking and synaptic targeting of NMDARs. Gabapentin or an α2δ-1 C terminus-interfering peptide normalizes NMDAR synaptic targeting and activity increased by nerve injury. Thus, α2δ-1 is an NMDAR-interacting protein that increases NMDAR synaptic delivery in neuropathic pain. Gabapentinoids reduce neuropathic pain by inhibiting forward trafficking of α2δ-1-NMDAR complexes.
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•α2δ-1 forms a heteromeric complex with NMDARs, mainly through its C terminus domain•α2δ-1 is essential for nerve injury-induced pre- and postsynaptic NMDAR hyperactivity•α2δ-1 promotes synaptic and surface expression of α2δ-1-NMDAR complexes•α2δ-1-bound NMDARs are critical for neuropathic pain development and gabapentin actions
Chen et al. show that α2δ-1, through its C terminus, physically interacts with NMDA receptors and promotes synaptic expression of α2δ-1-NMDA receptor complexes in neuropathic pain. Gabapentin reduces neuropathic pain primarily by targeting α2δ-1-bound NMDA receptors.
K
-Cl
cotransporter-2 (KCC2) critically controls neuronal chloride homeostasis and maintains normal synaptic inhibition by GABA and glycine. Nerve injury diminishes synaptic inhibition in the spinal ...cord via KCC2 impairment. However, how KCC2 regulates nociceptive input to spinal excitatory and inhibitory neurons remains elusive. Here, we show that basal GABA reversal potentials were significantly more depolarized in vesicular GABA transporter (VGAT)-expressing inhibitory neurons than those in vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in spinal cords of male and female mice. Strikingly, inhibiting KCC2 with VU0463271 increased currents elicited by puff NMDA and the NMDAR-mediated frequency of mEPSCs in VGluT2, but not in VGAT, dorsal horn neurons. Notably, VU0463271 had no effect on EPSCs monosynaptically evoked from the dorsal root in VGluT2 neurons. Furthermore, VU0463271 augmented α2δ-1-NMDAR interactions and their protein levels in spinal cord synaptosomes. In
KO mice, VU0463271 had no effect on puff NMDA currents or the mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with α2δ-1 C-terminus mimicking peptide diminished VU0463271-induced potentiation in the mEPSC frequency and puff NMDA currents in VGluT2 neurons. Additionally, intrathecal injection of VU0463271 reduced mechanical and thermal thresholds in wild-type mice, but not in
KO mice. VU0463271-induced pain hypersensitivity in mice was abrogated by co-treatment with the NMDAR antagonist, pregabalin (an α2δ-1 inhibitory ligand), or α2δ-1 C-terminus mimicking peptide. Our findings suggest that KCC2 controls presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons. KCC2 impairment preferentially potentiates nociceptive transmission between spinal excitatory interneurons via α2δ-1-bound NMDARs.
Impaired function of potassium-chloride cotransporter-2 (KCC2), a key regulator of neuronal inhibition, in the spinal cord plays a major role in neuropathic pain. This study unveils that KCC2 controls spinal nociceptive synaptic strength via NMDA receptors in a cell type- and synapse type-specific manner. KCC2 inhibition preferentially augments presynaptic and postsynaptic NMDA receptor activity in spinal excitatory interneurons via α2δ-1 (previously known as a calcium channel subunit). Importantly, spinal KCC2 impairment triggers pain hypersensitivity through α2δ-1-coupled NMDA receptors. These findings pinpoint the cell and molecular substrates for the reciprocal relationship between spinal synaptic inhibition and excitation in chronic neuropathic pain. Targeting both KCC2 and α2δ-1–NMDA receptor complexes could be an effective strategy in managing neuropathic pain conditions.
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.
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.
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