Receptors In Spinal Dorsal Horn Research Articles

AKAP150 and its Palmitoylation Contributed to Pain Hypersensitivity Via Facilitating Synaptic Incorporation of GluA1-Containing AMPA Receptor in Spinal Dorsal Horn.

The A-kinase anchoring protein 150 (AKAP150) organizes kinases and phosphatases to regulate AMPA receptors (AMPARs) that are pivotal for synaptic plasticity. AKAP150 itself undergoes S-palmitoylation. However, the roles of AKAP150 and its palmitoylation in spinal nociceptive processing remain unknown. In this study, we found that intraplantar injection of complete Freund's adjuvant (CFA) significantly increased the synaptic expression of AKAP150 and caused a reorganization of AKAP150 signaling complex in spinal dorsal horn. Knockdown of AKAP150 or interruption of its interactions with kinases effectively suppressed the CFA-induced synaptic expression of GluA1 subunit of AMPARs. Our data also showed that an upregulation of AKAP150 palmitoylation was involved in the synaptic redistribution of AKAP150. Inhibition of AKAP150 palmitoylation by expression of palmitoylation-defective mutant AKAP150 (C36, 123S) effectively repressed the CFA-induced phosphorylation and synaptic expression of GluA1 subunit, meanwhile, attenuated the development of mechanical allodynia and thermal hyperalgesia. Furthermore, we found that an increased expression of palmitoyl acyltransferase ZDHHC2 contributed to the upregulation of AKAP150 palmitoylation and GluA1 accumulation in inflamed mouse. These data indicated that AKAP150 and its palmitoylation were involved in AMPA receptor-dependent modification of nociceptive transmission, and the manipulations of AKAP150 signaling complex and palmitoylation might serve as potential therapeutic strategies for persistent pain after inflammation.

100 Hz Electroacupuncture Alleviated Chronic Itch and GRPR Expression Through Activation of Kappa Opioid Receptors in Spinal Dorsal Horn.

BackgroundClinical studies have shown that electroacupuncture (EA) alleviates chronic itch. Gastrin-releasing peptide receptor (GRPR) and dynorphin (DYN) in the spinal dorsal horn positively or negatively regulate itch, respectively. However, which frequency of EA is effective on relieving chronic itch and reducing the expression of GRPR, whether DYN-A in the spinal cord is involved in the underlying mechanism of the antipruritus effect of EA remains unknown.MethodsThe mixture of acetone and diethyl ether (1:1) [designated as AEW (acetone/diethyl ether and water) treatment] was used to induce the dry skin model of chronic itch. EA was applied to Quchi (LI11) and Hegu (LI4). Western blot was used to detect the expression of GRPR and DYN-A. Immunofluorescence was used to detect the expression of DYN-A.ResultsThe AEW administration induced remarkable spontaneous scratching, enhanced the expression of GRPR, and reduced the expression of DYN-A. Compared with the sham EA, 2 Hz EA, or 15 Hz EA group, 100 Hz EA was the most effective frequency for relieving chronic itch, reducing the expression of GRPR, and increasing the expression of DYN-A in the cervical dorsal horn. Furthermore, intraperitoneal injection of kappa opioid receptors (KORs) antagonist nor-Binaltorphimine dihydrochloride (nor-BNI) significantly reversed the effect of 100 Hz EA on the inhibition of both itching behavior and GRPR expression.ConclusionEA at 100 Hz is the most effective frequency that inhibits chronic itch and GRPR expression through activation of KORs in the spinal dorsal horn, which can effectively guide the clinical treatment and improve the antipruritic effect of acupuncture.

PD-1 Regulates GABAergic Neurotransmission and GABA-Mediated Analgesia and Anesthesia.

SummaryThe immune checkpoint inhibitor programmed cell death protein 1 (PD-1) plays a critical role in immune regulation. Recent studies have demonstrated functional PD-1 expression in peripheral sensory neurons, which contributes to neuronal excitability, pain, and opioid analgesia. Here we report neuronal expression and function of PD-1 in the central nervous system (CNS), including the spinal cord, thalamus, and cerebral cortex. Notably, GABA-induced currents in spinal dorsal horn neurons, thalamic neurons, and cortical neurons are suppressed by the PD-1-neutralizing immunotherapeutic Nivolumab in spinal cord slices, brain slices, and dissociated cortical neurons. Reductions in GABA-mediated currents in CNS neurons were also observed in Pd1−/− mice without changes in GABA receptor expression. Mechanistically, Nivolumab binds spinal cord neurons and elicits ERK phosphorylation to suppress GABA currents. Finally, both GABA-mediated analgesia and anesthesia are impaired by Pd1 deficiency. Our findings reveal PD-1 as a CNS-neuronal inhibitor that regulates GABAergic signaling and GABA-mediated behaviors.

The role of P2Y6 receptors in the maintenance of neuropathic pain and its improvement of oxidative stress in rats.

To explore the role of P2Y6 receptors in the maintenance of neuropathic pain and progression of oxidative stress, we investigated the efficacy of the selective P2Y6 receptors antagonist MRS2578 on the antiallodynic effects and improvement of pathological neuropathic pain-induced oxidative stress, thereby finding a potential therapeutic target in neurological disease. The mechanical allodynia in the ipsilateral spinal dorsal horn (SDH) of rats was observed in rats after chronic constriction injury (CCI). Meanwhile, the messenger RNA (mRNA) levels of biological parameters, including superoxide dismutase (SOD), glutathione (GSH), and heme oxygenase-1 (HO-1) in the SDH of rats were measured by real-time polymerase chain reaction (RT-PCR). In addition, the mRNA expression and protein levels of P2Y6 were measured by RT-PCR and Western blot assay, respectively. Next, the rats subjected to CCI were intrathecally infused with MRS2578 to block the expression of P2Y6 receptors. The positive expression of P2Y6 receptors was examined by immunohistochemistry. In the present study, the results revealed that the P2Y6 expression in the ipsilateral SDH of CCI rats was significantly upregulated. In addition, inhibition of the P2Y6 receptor in SDH increased CCI-induced tactile allodynia. Furthermore, the levels of SOD, GSH, and HO-1 which were correlated with oxidative stress produced by CCI were also decreased. The results demonstrated that inhibition of the P2Y6 receptor can generate antiallodynic effects and improved the pathological neuropathic pain-induced oxidative stress. Thus, this study provides a potential approach for the therapy of neurological disease.

The effect of exercise frequency on neuropathic pain and pain-related cellular reactions in the spinal cord and midbrain in a rat sciatic nerve injury model

BackgroundExercise regimens are established methods that can relieve neuropathic pain. However, the relationship between frequency and intensity of exercise and multiple cellular responses of exercise-induced alleviation of neuropathic pain is still unclear. We examined the influence of exercise frequency on neuropathic pain and the intracellular responses in a sciatic nerve chronic constriction injury (CCI) model.Materials and methodsRats were assigned to four groups as follows: CCI and high-frequency exercise (HFE group), CCI and low-frequency exercise (LFE group), CCI and no exercise (No-Ex group), and naive animals (control group). Rats ran on a treadmill, at a speed of 20 m/min, for 30 min, for 5 (HFE) or 3 (LFE) days a week, for a total of 5 weeks. The 50% withdrawal threshold was evaluated for mechanical sensitivity. The activation of glial cells (microglia and astrocytes), expression of brain-derived neurotrophic factor (BDNF) and μ-opioid receptor in the spinal dorsal horn and endogenous opioid in the midbrain were examined using immunohistochemistry. Opioid receptor antagonists (naloxone) were administered using intraperitoneal injection.ResultsThe development of neuropathic pain was related to the activation of glial cells, increased BDNF expression, and downregulation of the μ-opioid receptor in the ipsilateral spinal dorsal horn. In the No-Ex group, neuropathic pain showed the highest level of mechanical hypersensitivity at 2 weeks, which improved slightly until 5 weeks after CCI. In both exercise groups, the alleviation of neuropathic pain was accelerated through the regulation of glial activation, BDNF expression, and the endogenous opioid system. The expression of BDNF and endogenous opioid in relation to exercise-induced alleviation of neuropathic pain differed in the HFE and LFE groups. The effects of exercise-induced alleviation of mechanical hypersensitivity were reversed by the administration of naloxone.ConclusionThe LFE and HFE program reduced neuropathic pain. Our findings indicated that aerobic exercise-induced alleviated neuropathic pain through the regulation of glial cell activation, expression of BDNF in the ipsilateral spinal dorsal horn, and the endogenous opioid system.

Impacts of anti-nerve growth factor antibody on pain-related behaviors and expressions of opioid receptor in spinal dorsal horn and dorsal root ganglia of rats with cancer-induced bone pain.

ObjectiveTo investigate the impacts of anti-nerve growth factor antibody on pain-related behaviors and expressions of μ-opioid receptor in spinal dorsal horn and dorsal root ganglia of rats with cancer-induced bone pain.MethodsThe rats were randomly grouped and then injected with 10 μl of phosphate buffer saline or Walker256 tumor cells into the upper segment of left tibia. Thirteen days after the injection, the intrathecal catheterization was performed, followed by the injection of saline, anti-nerve growth factor, nerve growth factor, and naloxone twice a day. The pain ethological changes were measured at the set time points; the expression changes of μ-opioid receptor protein and mRNA in spinal dorsal horn and dorsal root ganglia were detected on the 18th day.ResultsAfter the tumor cells were injected into the tibia, hyperalgesia appeared and the expression of μ-opioid receptor protein and mRNA in spinal dorsal horn and dorsal root ganglia was increased, compared with the sham group; after intrathecally injected anti-nerve growth factor, the significant antinociceptive effects appeared, and the μ-opioid receptor expression was increased, compared with the cancer pain group; the μ-opioid receptor expressions in the other groups showed no statistical significance. The naloxone pretreatment could mostly inverse the antinociception effects of anti-nerve growth factor.ConclusionsAnti-nerve growth factor could reduce hyperalgesia in the cancer-induced bone pain rats, and the antinociceptive effects were related with the upregulation of μ-opioid receptor.

Noradrenergic α2 receptor attenuated inflammatory pain through STEP61/ERK signalling.

Activation of noradrenergic α2 receptor in spinal dorsal horn effectively alleviates the pathological pain. However, the precise mechanisms underlying noradrenergic pain suppression are not fully understood. Convincing evidence has indicated that extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a key role in spinal sensitization. The present study investigated the potential influence of noradrenergic α2 receptor agonist clonidine on ERK1/2 activity. Clonidine was intrathecally given after intraplantar injection of complete Freund's adjuvant (CFA) in mice. The possible changes ofERK1/2 signalling were detected by Western blot, immunohistochemistry, co-immunoprecipitation and behavioural tests. CFA significantly enhanced ERK1/2 activity in spinal dorsal horn, which was, however, greatly attenuated by clonidine application. Pretreatment with pertussis toxin abolished the inhibitory effect of clonidine on ERK1/2, suggesting the involvement of Giα subunit (Gi protein). Noradrenergic α2 receptor/Gi protein might repress ERK1/2 through cAMP-dependent protein kinase (PKA) pathway, because direct ERK1/2 activation by PKA agonist forskolin was also suppressed by clonidine. We found that 61kD isoform of striatal-enriched protein phosphatase (STEP61) was a key intermediary for α2 receptor/Gi protein/PKA signalling to manipulate ERK1/2 activity. By reducing PKA-mediated phosphorylation of STEP61 at Ser221, clonidine significantly resumed the inhibition conferred by STEP61 on ERK1/2. Direct expression of STEP61 mutant devoid of Ser221 phosphorylation mimicked clonidine by inhibiting ERK1/2 and pain sensitization in CFA-injected mice. The analgesic action produced by noradrenergic α2 receptor agonist clonidine involved the reversal of ERK1/2 hyperactivity in spinal dorsal horn of inflamed mice.

Fyn kinase‐regulated NMDA receptor‐ and AMPA receptor‐dependent pain sensitization in spinal dorsal horn of mice

Inhibition of Src-family protein tyrosine kinases (SFKs) in spinal dorsal horn has been established as an effective strategy for the alleviation of chronic pathological pain. As one of the important SFKs members, Fyn kinase is critical for synaptic plasticity and many pathophysiological processes. However, whether Fyn is involved in spinal sensitization is far from being elucidated. We manipulated Fyn activity by expressing a constitutively active Fyn mutant [Fyn(Y528F) ] or a catalytically null mutant [Fyn(K296M) ] in the spinal dorsal horn of mice, and performed behavioural and biochemical experiments to investigate the role of Fyn in regulating the nociceptive responses and the synaptic expression of ionotropic glutamate receptors. Spinal expression of Fyn(Y528F) alone in intact mice was sufficient to elicit persistent mechanical allodynia and thermal hyperalgesia, which lasted for at least 12 days. Fyn(Y528F) simultaneously enhanced the concentrations of N-methyl-D-aspartic acid (NMDA)-subtype and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype glutamate receptors at synaptosomal membrane fraction. Pharmacological inhibition of NMDA receptors or AMPA receptors greatly alleviated Fyn(Y528F)-induced pain hypersensitivity. To evaluate the contribution of Fyn to inflammatory pain, we expressed Fyn(K296M) before intradermal injection of complete Freund's adjuvant (CFA), finding that Fyn(K296M) had no effect on the induction of inflammatory pain within 3 h post-CFA injection, which, however, repressed the synaptic accumulation of NMDA receptors and AMPA receptors to attenuate the maintenance of chronic pain states. Fyn played a key role in the sustained sensitization of nociceptive behaviours by up-regulating the functions of ionotropic glutamate receptors in spinal dorsal horn.

α2 noradrenergic receptor suppressed CaMKII signaling in spinal dorsal horn of mice with inflammatory pain

Intrathecal application of α2 noradrenergic receptor agonists effectively alleviates the pathological pain induced by peripheral tissue injury. However, the spinal antinociceptive mechanisms of α2 noradrenergic receptors remain to be characterized. The present study performed immunohistochemistry and western blot to elucidate the signaling pathway initiated by α2 noradrenergic receptors in spinal dorsal horn of mice, and identified calcium/calmodulin-dependent protein kinase II (CaMKII) as an important target for noradrenergic suppression of inflammatory pain. Our data showed that intraplantar injection of Complete Freund's Adjuvant (CFA) substantially enhanced CaMKII autophosphorylation at Threonine 286, which could be abolished by intrathecal administration of α2 noradrenergic receptor agonist clonidine. Gi protein-coupled α2 noradrenergic receptor might inhibit cAMP-dependent protein kinase (PKA) to disturb CaMKII signaling. We found that pharmacological activation of PKA in intact mice also enhanced spinal CaMKII autophosphorylation level, which was completely antagonized by clonidine. Moreover, direct PKA inhibition in CFA-injected mice mimicked the suppressive effect of α2 noradrenergic receptors on CaMKII. PKA inhibition has been shown to downregulate CaMKII by enhancing protein phosphatase activity. Consistent with this notion, spinal treatment with protein phosphatase inhibitor okadaic acid ruled out clonidine-mediated CaMKII dephosphorylation in CFA-injected mice. Through PKA/protein phosphatase/CaMKII pathway, clonidine noticeably decreased CFA-evoked phosphorylation of N-methyl-d-aspartate subtype glutamate receptor GluN1 and GluN2B subunit as well as α-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid subtype glutamate receptor GluA1 subunit. These data suggested that interference with CaMKII signaling might represent an important mechanism underlying noradrenergic suppression of inflammatory pain.

Pregabalin Alters Nociceptive Behavior and Expression Level of P2X3 Receptor in the Spinal Dorsal Horn in a Rat Model Induced by Chronic Compression of the Dorsal Root Ganglion

P2X3 receptors are present in the spinal dorsal horn (SDH) and play an essential role in the regulation of nociception and pain. Pregabalin (PGB) has been used as a new antiepileptic drug in the treatment of neuropathic pain. However, it is unclear whether PGB-induced analgesia was associated with the P2X3 receptor in SDH. Here, rats were randomly divided into four groups (n = 12 per group), including 2 sham operation groups, which was treated by normal saline (Sham + NS group) or PGB (Sham + PGB group), other 2 groups with chronic compression of the dorsal root ganglion, a normal saline-treated CCD group (CCD+NS group), and a PGB-treated CCD group (CCD + PGB group). A rat model of neuropathic pain was used by compressing the right L4 and L5 dorsal root ganglia. Each group was evaluated using the mechanical withdrawal threshold (MWT). The mRNA and protein levels of the P2X3 receptor in the ipsilateral SDH were measured by RT-PCR, western blot, and immunofluorescence on 14 day after CCD operation. CCD rats showed the highest mechanical hyperalgesia and the lowest pain threshold in the four groups. Simultaneously, CCD rats showed higher P2X3 mRNA and protein expression in ipsilateral side of the SDH than the sham operation rats. However, the MWT was increased and expression of P2X3 mRNA and protein in the ipsilateral SDH in CCD rats was decreased 3 days after PGB treatment. Thus, PGB may partially reverse mechanical hyperalgesia in CCD rats by inhibiting P2X3 receptor expression in the ipsilateral SDH.

Inhibition of protein tyrosine phosphatases in spinal dorsal horn attenuated inflammatory pain by repressing Src signaling

Tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) subtype glutamate receptors by Src-family protein tyrosine kinases (SFKs) plays a critical role in spinal sensitization. Besides SFKs, the tyrosine phosphorylation levels of proteins are also determined by protein tyrosine phosphatases (PTPs). However, whether PTPs are involved in spinal nociceptive processing is largely unknown. The present study found that intrathecal application of broad-spectrum PTPs inhibitors orthovanadate or Bpv (phen) generated little effects on the paw withdrawal thresholds of intact rats to Von Frey filament stimuli. Although the basal nociceptive responses didn't require the involvement of PTPs, the mechanical allodynia evoked by intrathecal injection of NMDA was greatly attenuated by orthovanadate and Bpv (phen), suggesting that PTPs activity, once stimulated by NMDA receptors, became essential for spinal sensitization. Biochemical analysis demonstrated that PTPs functioned to activate SFKs member Src and promote Src interaction with NR2B subunit-containing NMDA receptors (NR2B receptors). As a result, PTPs inhibition largely suppressed Src-mediated NR2B phosphorylation at Tyr1472 and reduced the synaptic concentration of NR2B receptors in spinal dorsal horn of NMDA-treated rats. Importantly, intraplantar injection of Complete Freund's Adjuvant (CFA) naturally activated spinal PTPs to initiate Src signaling, because PTPs inhibition significantly repressed Src activity, reduced Src phosphorylation of NR2B, decreased NR2B synaptic accumulation and eventually ameliorated inflammatory pain. These data indicated an important role played by spinal PTPs in inducing Src-dependent NR2B receptor hyperfunction and suggested that PTPs inhibition might represent an effective strategy for the treatment of inflammatory pain.

Peripheral inflammation increased the synaptic expression of NMDA receptors in spinal dorsal horn

Considerable evidence has indicated that the aberrant, sustained enhancement of spinal NMDA receptors (NMDARs) function is closely associated with behavioral sensitization during inflammatory pain. However, the molecular mechanisms underlying inflammation-induced NMDARs hyperfunction remain poorly understood. The present study performed immunoblotting analysis to evaluate the possible changes in the protein expression of spinal NMDARs after injection of complete Freund’s adjuvant (CFA) in mice. We found that CFA did not affect the total protein level of NMDARs subunit NR1 in spinal dorsal horn. However, NR1 immunoreactivity at synapses significantly increased after CFA injection, which was correlated in the time course with the development of mechanical allodynia. Inhibition of spinal NMDARs with D-APV completely eliminated the CFA-induced increase in NR1 immunoreactive density at synapses, and direct application of NMDA onto the spinal cord of naïve mice mimicked the effects of CFA, suggesting the importance of NMDARs activity in regulating the synaptic content of NR1 during inflammatory pain. Moreover, cAMP-dependent protein kinase (PKA) downstream to NMDARs was also required for NR1 synaptic expression because inhibition of PKA activity abolished the enhancement of synaptic NR1 immunoreactivity evoked by either CFA or NMDA. Thus, our data suggested that NMDARs- and PKA-dependent increase in NR1 synaptic expression represented an important mechanism for the hyperfunction of spinal NMDARs following peripheral inflammation.

Changes in expression of nociceptin/orphanin FQ and its receptor in spinal dorsal horn during electroacupuncture treatment for peripheral inflammatory pain in rats

The neuropeptide nociceptin/orphanin FQ (N/OFQ), the endogenous agonist of the N/OFQ peptide receptor (NOP receptor), has been demonstrated to be involved in many physiological and pathological functions including pain modulation. It was reported that electroacupuncture (EA) had a potent analgesic effect on inflammatory pain by activating various endogenous transmitters such as the opioid peptides. In the present study, we investigated the effect of EA on peripheral inflammatory pain and the expression of N/OFQ and the NOP receptor in the spinal dorsal horn of rats, using a behavioral test, RT-PCR, immunohistochemistry and Western blot analysis techniques. The results showed: (1) EA had an accumulative analgesic effect on chronic inflammatory pain; (2) in the superficial layers of the spinal dorsal horn, the level of mRNA of the precursor protein for N/OFQ (preproN/OFQ, ppN/OFQ) was increased and the N/OFQ immunoreactivity was decreased after peripheral inflammation, and could be significantly increased by EA treatment; (3) both mRNA and protein levels of the NOP receptor in the spinal dorsal horn were significantly increased after chronic inflammatory pain and could be further enhanced by EA treatment. The present data demonstrated that EA could activate the endogenous N/OFQ–NOP receptor system, and this might underlie the effectiveness of EA in the treatment of inflammatory pain.

Baclofen inhibition of dorsal root-evoked inhibitory postsynaptic currents in substantia gelatinosa neurons of rat spinal cord slice

We investigated the action of baclofen, an agonist for GABA B receptor, on the dorsal root-evoked inhibitory postsynaptic currents (IPSCs) in substantia gelatinosa (SG) neurons in the adult rat spinal cord slice. Most of the dorsal root-evoked IPSCs in SG were mediated by Aδ fibers. Baclofen (10 μM) reduced the amplitude of Aδ fiber-evoked glycine receptor-mediated IPSCs to 20.9±3.0% ( n=13), and GABA A receptor-mediated IPSCs to 18.6±3.4% ( n=12) of the controls, respectively. The results further suggest modulatory role of GABA B receptor in spinal dorsal horn.

Neurotropin induces antinociceptive effect by enhancing descending pain inhibitory systems involving 5-HT 3 and noradrenergic α2 receptors in spinal dorsal horn

Neurotropin, a non-protein extract from the inflamed skin of rabbits inoculated with vaccinia virus, has been clinically used as an analgesic drug in Japan. Its analgesic effect has been demonstrated by reduced mechano-nociception in hyperalgesic rats exposed to SART-stress (a repeated cold stress) for 5 days. In order to clarify the mechanism of the analgesic effect of neurotropin at the spinal cord level, we examined the effects of several neurotransmitter receptor antagonists given by intrathecal (i.t.) injection on the antinociceptive effect of intraperitoneally (i.p.) injected neurotropin [100 and 200 Neurotropin Unit (NU)/kg]. The analgesic effect of neurotropin was significantly inhibited not only by methysergide (100 nmol Rat , i.t.), a non-selective antagonist against serotonin (5-HT), but also MDL 72222 (30 nmol Rat , i.t.), a selective 5-HT 3 antagonist, but not influenced by ketanserin (100 nmol Rat , i.t.), a 5-HT 2A antagonist. The antinociceptive effect of neurotropin (200 NU Kg , i.p.) was significantly inhibited also by yohimbine (30 nmol Rat , i.t.), a noradrenergic α 2 antagonist. However, the analgesic effect of neurotropin (100 and 200 NU Kg , i.p.) was not influenced by naloxone (30 nmol Rat , i.t.), an opioid antagonist. These results suggest that the mechanism of the antinociceptive effect of neurotropin is via enhancement of endogenous descending pain inhibitory pathways of the serotonergic and noradrenergic systems, especially involving 5-HT 3 and noradrenergic α 2 receptors in spinal dorsal horn in which these neurons terminate. No influence of opioid receptors at the spinal cord level is indicated.