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-beta-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.

The ultrapotent transient receptor potential vanilloid 1 (TRPV1) agonist resiniferatoxin (RTX) induces small-fiber sensory neuropathy, which has been widely used model of postherpetic neuralgia to study mechanisms of neuropathic pain and new analgesics. The long non-coding RNA (lncRNA) and mRNA expression profiles in spinal dorsal horn tissues of rats six weeks after RTX injection to identify new RNAs related to neuropathic pain.

Chronic itch severely reduces the quality of life of patients. Electroacupuncture (EA) is widely used to treat chronic itch. However, the underlying mechanism of this therapeutic action of EA is largely unknown. Cannabinoid CB1 receptors in the ventrolateral periaqueductal gray (vlPAG) mediate the analgesic effect of EA. Using a dry skin-induced itch model in mice, we determined whether EA treatment reduces chronic itch via CB1 receptors in the vlPAG. We showed that the optimal inhibitory effect of EA on chronic itch was achieved at the high frequency and high intensity (100 Hz and 3 mA) at "Quchi" (LI11) and "Hegu" (LI14) acupoints, which are located in the same spinal dermatome as the cervical skin lesions. EA reversed the increased expression of CB1 receptors in the vlPAG and decreased the concentration of 5-hydroxytryptamine (5-HT) in the medulla oblongata and the expression of gastrin-releasing peptide receptors (GRPR) in the cervical spinal cord. Furthermore, knockout of CB1 receptors on GABAergic neurons in the vlPAG attenuated scratching behavior and the 5-HT concentration in the medulla oblongata. In contrast, knockout of CB1 receptors on glutamatergic neurons in the vlPAG blocked the antipruritic effects of EA and the inhibitory effect of EA on the 5-HT concentration in the medulla oblongata. Our findings suggest that EA treatment reduces chronic itch by activation of CB1 receptors on glutamatergic neurons and inhibition of CB1 receptors on GABAergic neurons in the vlPAG, thereby inhibiting the 5-HT release from the medulla oblongata to GRPR-expressing neurons in the spinal cord. Our findings suggest that EA attenuates chronic itch via activating CB1 receptors expressed on glutamatergic neurons and downregulating CB1 receptors on GABAergic neurons in the vlPAG, leading to the reduction in 5-HT release in the rostroventral medulla and GRPR signaling in the spinal cord. Our study not only advances our understanding of the mechanisms of the therapeutic effect of EA on chronic itch but also guides the selection of optimal parameters and acupoints of EA for treating chronic itch.

Inflammatory bowel disease (IBD) results in chronic abdominal pain in patients due to the presence of inflammatory responses in the colon. Electroacupuncture (EA) is effective in alleviating visceral pain and colonic inflammation associated with IBD. Cannabinoid CB2 receptor agonists also reduce colonic inflammation in a mouse model of IBD. However, whether EA reduces visceral pain and colonic inflammation via the CB2 receptor remains unknown. Here, we determined the mechanism of the antinociceptive effect of EA in a mouse model of IBD induced by rectal perfusion of 2,4,6-trinitrobenzenesulfonic acid solution (TNBS). EA or sham EA was performed at the bilateral Dachangshu (BL25) point for seven consecutive days. The von Frey and colorectal distension tests were performed to measure mechanical referred pain and visceral pain. Western blotting and immunohistochemistry assays were carried out to determine the expression of IL-1β and iNOS and activation of macrophages in the colon tissues. We found that EA, but not sham EA, attenuated visceral hypersensitivity and promoted activation of CB2 receptors, which in turn inhibited macrophage activation and the expression of IL-1β and iNOS. The effects of EA were blocked by AM630, a specific CB2 receptor antagonist, and by CB2 receptor knockout. Our findings suggest that EA attenuates mechanical allodynia and visceral hypersensitivity associated with IBD by activating CB2 receptors and subsequent inhibition of macrophage activation and expression of IL-1β and iNOS.

Both the sympathetic nervous system and the renin-angiotensin system are critically involved in hypertension development. Although angiotensin II (Ang II) stimulates hypothalamic paraventricular nucleus (PVN) neurons to increase sympathetic vasomotor tone, the molecular mechanism mediating this action remains unclear. The glutamate NMDAR in the PVN controls sympathetic outflow in hypertension. In this study, we determined the interaction between α2δ-1 (encoded by Cacna2d1), commonly known as a Ca2+ channel subunit, and NMDARs in the hypothalamus and its role in Ang II-induced synaptic NMDAR activity in PVN presympathetic neurons. Coimmunoprecipitation assays showed that α2δ-1 interacted with the NMDAR in the hypothalamus of male rats and humans (both sexes). Ang II increased the prevalence of synaptic α2δ-1-NMDAR complexes in the hypothalamus. Also, Ang II increased presynaptic and postsynaptic NMDAR activity via AT1 receptors, and such effects were abolished either by treatment with pregabalin, an inhibitory α2δ-1 ligand, or by interrupting the α2δ-1-NMDAR interaction with an α2δ-1 C terminus-interfering peptide. In Cacna2d1 knock-out mice (both sexes), Ang II failed to affect the presynaptic and postsynaptic NMDAR activity of PVN neurons. In addition, the α2δ-1 C terminus-interfering peptide blocked the sympathoexcitatory response to microinjection of Ang II into the PVN. Our findings indicate that Ang II augments sympathetic vasomotor tone and excitatory glutamatergic input to PVN presympathetic neurons by stimulating α2δ-1-bound NMDARs at synapses. This information extends our understanding of the molecular basis for the interaction between the sympathetic nervous and renin-angiotensin systems and suggests new strategies for treating neurogenic hypertension.SIGNIFICANCE STATEMENT Although both the sympathetic nervous system and renin-angiotensin system are closely involved in hypertension development, the molecular mechanisms mediating this involvement remain unclear. We showed that α2δ-1, previously known as a calcium channel subunit, interacts with NMDARs in the hypothalamus of rodents and humans. Angiotensin II (Ang II) increases the synaptic expression level of α2δ-1-NMDAR complexes. Furthermore, inhibiting α2δ-1, interrupting the α2δ-1-NMDAR interaction, or deleting α2δ-1 abolishes the potentiating effects of Ang II on presynaptic and postsynaptic NMDAR activity in the hypothalamus. In addition, the sympathoexcitatory response to Ang II depends on α2δ-1-bound NMDARs. Thus, α2δ-1-NMDAR complexes in the hypothalamus serve as an important molecular substrate for the interaction between the sympathetic nervous system and the renin-angiotensin system. This evidence suggests that α2δ-1 may be a useful target for the treatment neurogenic hypertension.

Sudden unexplained death in epilepsy (SUDEP) is the most common cause of premature mortality in epilepsy and was linked to mutations in ion channels; however, genes within the channel protein interactome might also represent pathogenic candidates. Here we show that mice with partial deficiency of Sentrin/SUMO-specific protease 2 (SENP2) develop spontaneous seizures and sudden death. SENP2 is highly enriched in the hippocampus, often the focus of epileptic seizures. SENP2 deficiency results in hyper-SUMOylation of multiple potassium channels known to regulate neuronal excitability. We demonstrate that the depolarizing M-current conducted by Kv7 channel is significantly diminished in SENP2-deficient hippocampal CA3 neurons, primarily responsible for neuronal hyperexcitability. Following seizures, SENP2-deficient mice develop atrioventricular conduction blocks and cardiac asystole. Both seizures and cardiac conduction blocks can be prevented by retigabine, a Kv7 channel opener. Thus, we uncover a disease-causing role for hyper-SUMOylation in the nervous system and establish an animal model for SUDEP.

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.

Knee osteoarthritis (KOA) is a highly prevalent, chronic joint disorder, which can lead to chronic pain. Although electroacupuncture (EA) is effective in relieving chronic pain in the clinic, the involved mechanisms remain unclear. Reduced diffuse noxius inhibitory controls (DNIC) function is associated with chronic pain and may be related to the action of endocannabinoids. In the present study, we determined whether EA may potentiate cannabinoid receptor-mediated descending inhibitory control and inhibit chronic pain in a mouse model of KOA. We found that the optimized parameters of EA inhibiting chronic pain were the low frequency and high intensity (2 Hz + 1 mA). EA reversed the reduced expression of CB1 receptors and the 2-arachidonoylglycerol (2-AG) level in the midbrain in chronic pain. Microinjection of the CB1 receptor antagonist AM251 into the ventrolateral periaqueductal gray (vlPAG) can reversed the EA effect on pain hypersensitivity and DNIC function. In addition, CB1 receptors on GABAergic but not glutamatergic neurons are involved in the EA effect on DNIC function and descending inhibitory control of 5-HT in the medulla, thus inhibiting chronic pain. Our data suggest that endocannabinoid (2-AG)-CB1R-GABA-5-HT may be a novel signaling pathway involved in the effect of EA improving DNIC function and inhibiting chronic pain.

Activation of spinal muscarinic acetylcholine receptors (mAChRs) inhibits nociception. However, the cellular mechanisms of this action are not fully known. In this study, we determined the role of mAChR subtypes in regulation of synaptic glycine release in the spinal cord. Whole-cell voltage-clamp recordings were performed on lamina II neurones in the rat spinal cord slices. The mAChR agonist oxotremorine-M significantly increased the frequency of glycinergic sIPSCs but not mIPSCs. Surprisingly, the effect of oxotremorine-M on sIPSCs was largely attenuated at a higher concentration. On the other hand, 1-10 microm oxotremorine-M dose-dependently increased the frequency of sIPSCs in rats pretreated with intrathecal pertussis toxin. Furthermore, oxotremorine-M also dose-dependently increased the frequency of sIPSCs in the presence of himbacine (an M2/M4 mAChR antagonist) or AF-DX116 (an M2 mAChR antagonist). The M3 mAChR antagonist 4-DAMP abolished the stimulatory effect of oxotremorine-M on sIPSCs. Interestingly, the GABA(B) receptor antagonist CGP55845 potentiated the stimulatory effect of oxotremorine-M on sIPSCs. In the presence of CGP55845, both himbacine and AF-DX116 similarly reduced the potentiating effect of oxotremorine-M on sIPSCs. Collectively, these data suggest that the M3 subtype is present on the somatodendritic site of glycinergic neurones and is mainly responsible for muscarinic potentiation of glycinergic input to spinal dorsal horn neurones. Concurrent stimulation of mAChRs on adjacent GABAergic interneurones attenuates synaptic glycine release through presynaptic GABA(B) receptors on glycinergic interneurones. This study illustrates a complex dynamic interaction between GABAergic and glycinergic synapses in the spinal cord dorsal horn.

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/.

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/.

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

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.