Calcitonin gene-related peptide and its receptor plays important role in nociceptive regulation in the arcuate nucleus of hypothalamus of rats with inflammatory pain.

The present study has explored the role of calcitonin gene-related peptide (CGRP) and its receptor in inflammatory pain modulation in arcuate nucleus of hypothalamus (ARC). Our study demonstrated that intra-ARC injection of CGRP induced antinociceptive effects to naïve rats and rats with inflammatory pain, the effect could be inhibited by the selective CGRP receptor antagonist CGRP8-37. Interestingly, the CGRP-induced antinociception effect was decreased in rats with inflammatory pain compared to naïve rats. Similarly, we found that calcitonin receptor like receptor (CLR), a main component of CGRP receptor, had a low decreased expression levels in the ARC regions of rats with inflammatory pain. The CGRP-induced antinociceptive effect was significantly impaired after reducing CLR expression by intra-ARC administration of CLR targeted siRNA. These findings demonstrated that CGRP might play a crucial role in nociceptive modulation in the ARC during inflammatory pain, which was mediated by CGRP receptor in the ARC. This study shed light upon CGRP and its receptor interaction might be valuable strategies for the alleviation of inflammatory pain.

Role of calcitonin gene-related peptide in pain regulation in the parabrachial nucleus of naive rats and rats with neuropathic pain.

Researches have shown that calcitonin gene-related peptide (CGRP) plays a pivotal role in pain modulation. Nociceptive information from the periphery is relayed from parabrachial nucleus (PBN) to brain regions implicated involved in pain. This study investigated the effects and mechanisms of CGRP and CGRP receptors in pain regulation in the PBN of naive and neuropathic pain rats. Chronic sciatic nerve ligation was used to model neuropathic pain, CGRP and CGRP 8-37 were injected into the PBN of the rats, and calcitonin receptor-like receptor (CLR), a main structure of CGRP receptor, was knocked down by lentivirus-coated CLR siRNA. The hot plate test (HPT) and the Randall Selitto Test (RST) was used to determine the latency of the rat hindpaw response. The expression of CLR was detected with RT-PCR and western blotting. We found that intra-PBN injecting of CGRP induced an obvious anti-nociceptive effect in naive and neuropathic pain rats in a dose-dependent manner, the CGRP-induced antinociception was significantly reduced after injection of CGRP 8-37, Moreover, the mRNA and protein levels of CLR, in PBN decreased significantly and the antinociception CGRP-induced was also significantly lower in neuropathic pain rats than that in naive rats. Knockdown CLR in PBN decreased the expression of CLR and the antinociception induced by CGRP was observably decreased. Our results demonstrate that CGRP induced antinociception in PBN of naive or neuropathic pain rats, CGRP receptor mediates this effect. Neuropathic pain induced decreases in the expression of CGRP receptor, as well as in CGRP-induced antinociception in PBN.

Role of Calcitonin Gene-Related Peptide in Nociceptive Modulationin Anterior Cingulate Cortex of Naïve Rats and Rats With Inflammatory Pain.

It is known that calcitonin gene-related peptide (CGRP) plays a key role in pain modulation in the brain. There are high expressions of CGRP and CGRP receptor in anterior cingulate cortex (ACC), an important brain structure in pain modulation. The present study explored the role and mechanisms of CGRP and CGRP receptor in nociceptive modulation in ACC in naïve rats and inflammatory rats. Administration of different does of CGRP in ACC induced significant antinociception in a dose-dependent manner in both naïve rats and rats with inflammatory pain. The CGRP-induced antinociception was attenuated by injection of the CGRP receptor antagonist CGRP8-37 in ACC. Interestingly, both CGRP-induced antinociception and CGRP receptor expression decreased in ACC in rats with inflammatory pain compared with naïve rats. Knockdown of CGRP receptor in ACC by siRNA targeting to CGRP receptor attenuated both the CGRP receptor expression and the CGRP-induced antinociception significantly in rats. These findings demonstrate that CGRP and CGRP receptor participate in nociceptive modulation in ACC in rats, inhibiting CGRP receptor expression induces decrease in CGRP-induced antinociception in ACC.

Involvement of galanin and galanin receptor 1 in nociceptive modulation in the central nucleus of amygdala in normal and neuropathic rats.

The present study was performed to explore the role of galanin and galanin receptor 1 (GalR 1) in nociceptive modulation in the central nucleus of amygdala (CeA) in normal rats and rats with neuropathy, and the involvement of GalR 1 and PKC was also investigated. The hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations were increased in a dose-dependent manner after intra-CeA injection of galanin in both normal rats and rats with neuropathy. The increased HWLs were significantly attenuated by intra-CeA injection of galanin receptor antagonist M40, indicating an involvement of galanin receptor in nociceptive modulation in CeA. Furthermore, intra-CeA administration of the GalR 1 agonist M 617 induced increases in HWLs in normal rats, suggesting that GalR 1 may be involved in galanin-induce antinociception in CeA. Additionally, intra-CeA injection of the PKC inhibitor inhibited galanin-induced antinociception, showing an involvement of PKC in galanin-induced antinociception in CeA of normal rats. Moreover, there was a significant increase in GalR1 content in CeA in rats with neuropathy than that in normal rats. These results illustrated that galanin induced antinociception in CeA in normal rats and rats with neuropathy, and there is an up-regulation of GalR1 expression in rats with neuropathy.

Involvement of galanin and galanin receptor 2 in nociceptive modulation in anterior cingulate cortex of normal rats and rats with mononeuropathy.

The present study was performed to explore the role of galanin and galanin receptor 2 in nociceptive modulation in anterior cingulate cortex (ACC) of normal rats and rats with mononeuropathy. Intra-ACC injection of galanin induced significant increases in hindpaw withdrawal latencies (HWLs) to thermal and mechanical stimulations in both normal rats and rats with mononeuropathy, the increased HWLs were attenuated significantly by intra-ACC injection of galanin receptor 2 antagonist M871, indicating an involvement of galanin receptor 2 in nociceptive modulation in ACC. Interestingly, the galanin-induced HWL was significant higher in rats with mononeuropathy than that in normal rats tested by Randall Selitto test. Furthermore, both the galanin mRNA expression and galanin content increased significantly in ACC in rats with mononeuropathy than that in normal rats. Moreover, both the mRNA levels of galanin receptor 2 and the content of galanin receptor 2 in ACC increased significantly in rats with mononeuropathy than that in normal rats. These results found that galanin induced antinociception in ACC in both normal rats and rats with mononeuropathy. And there may be plastic changes in the expression of galanin and galanin receptor 2 in rats with mononeuropathy, as well as in the galanin-induced antinociception.

Antinociception induced by galanin in anterior cingulate cortex in rats with acute inflammation.

The present study was performed to explore the role of galanin in nociceptive modulation in anterior cingulate cortex (ACC) of rats with acute inflammation, and the changes in galanin and galanin receptor 2 (Gal R2) expressions in rats with acute inflammation. Intra-ACC injection of galanin induced antinociception in rats with acute inflammation, the antinociceptive effects induced by galanin were attenuated significantly by intra-ACC injection of the Gal R2 antagonist M871, indicating an involvement of Gal R2 in nociceptive modulation in ACC in rats with acute inflammation. Furthermore, we found that both the galanin mRNA expression and galanin content increased significantly in ACC in rats with acute inflammation than that in normal rats. Moreover, both the mRNA levels of Gal R2 and the content of Gal R2 in ACC increased significantly in rats with acute inflammation than that in normal rats. These results demonstrated that galanin induced antinociception in ACC in rats with acute inflammation. And there were changes in the expression of galanin and Gal R2 in rats with acute inflammation.

GluR2-3Y Inhibits the Acquisition and Reinstatement of Morphine-Induced Conditioned Place Preference in Rats.

Accumulating evidence indicates that α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) are involved in the relapse to abused drugs. However, the role of AMPARs containing the GluR2 subunit in opiate addiction is still unclear. GluR2-3Y, an interfering peptide, prevents the endocytosis of AMPARs containing the GluR2 subunit. In this study, we explored the effect of intravenous injection of GluR2-3Y on the acquisition, expression, and reinstatement of morphine-induced conditioned place preference (mCPP) in rats. We found that infusion of GluR2-3Y (1.5 nmol/g) one hour before morphine during the conditioning phase inhibited the acquisition of mCPP, while an identical injection one hour before the post-conditioning test had no influence on the expression of mCPP. Injection of GluR2-3Y (1.5 nmol/g) after mCPP extinction blocked the morphine-induced reinstatement of mCPP. Our results strongly support the hypothesis that inhibition of AMPAR endocytosis provides a new target for the treatment of opiate addiction.

Pharmacologically inhibiting GluR2 internalization alleviates neuropathic pain.

Neuropathic pain is of serious clinical concern and only about half of patients achieve partial relief with currently-available treatments, so it is critical to find new drugs for this condition. Recently, the cellsurface trafficking of pain-related receptors has been suggested as an important mechanism underlying persistent neuropathic pain. Here, we used the short peptide GluA2-3y, which specifically inhibits the GluA2-dependent endocytosis of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and tested its anti-nociceptive effect in the periaqueductal grey (PAG) of intact rats and rats with neuropathic pain. Intra-PAG injection of 0.15, 1.5, 7.5, and 15 pmol of GluA2-3y induced dose-dependent increases in hindpaw withdrawal latencies to noxious thermal and mechanical stimuli in intact rats, suggesting that GluA2 cell-surface trafficking in the PAG is involved in pain modulation. Furthermore, GluA2-3y had much stronger anti-nociceptive effects in rats with neuropathic pain induced by sciatic nerve ligation. Interestingly, the intra-PAG injection of 15 pmol GluA2-3y had an analgesic effect similar to 10 µg (35 nmol) morphine in rats with neuropathic pain. Taken together, our results suggested that GluA2 trafficking in the PAG plays a critical role in pain modulation, and inhibiting GluA2 endocytosis with GluA2-3y has potent analgesic effects in rats with neuropathic pain. These findings strongly support the recent hypothesis that targeting receptor trafficking could be a new strategy for the treatment of neuropathic pain.

Involvement of galanin receptor 2 and CaMKII in galanin-induced antinociception in periaqueductal grey of rats.

The present study was performed to explore the effect of the galanin receptor 2 (GalR2) antagonist M871 on the galanin-induced antinociception in periaqueductal grey (PAG), and an involvement of Ca(2+)/calmodulin-dependent kinase II (CaMKII) in the galanin-induced antinociception. Intra-PAG injection of galanin induced marked increases in HWLs to noxious thermal and mechanical stimulation. The increased HWLs to thermal and mechanical stimulation decreased significantly after intra-PAG administration of the GalR2 antagonist M871, indicating an involvement of GalR2 in the galanin-induced antinociception in PAG of rats. Furthermore, rats received intra-PAG injection of galanin, followed 5min later by intra-PAG administration of the CaMKII inhibitor MAP. The galanin-induced increases in HWLs to thermal and mechanical stimulation decreased significantly after intra-PAG administration of MAP, indicating that there is an involvement of CaMKII in the galanin-induced antinociception in PAG, blockade the activity of CaMKII by MAP inhibits the galanin-induced antinociception in PAG of rats. Our results strongly indicate that the galanin-induced antinociception is mediated by GalR2 in the PAG, and CaMKII may be involved in the galanin-induced antinociception in PAG of rats.

Intra-nucleus accumbens administration of the calcium/calmodulin-dependent protein kinase II inhibitor AIP induced antinociception in rats with mononeuropathy.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine- dependent protein kinase, which has been implicated in pain modulation at different levels of the central nervous system. The present study was performed in rats with mononeuropathy induced by left common sciatic nerve ligation. Unilateral sciatic nerve loose ligation produced decreases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation. Intra-nucleus accumbens (NAc) injection of 3 µg, 6 µg and 12 µg of myristoylated autocamtide-2-inhibitory peptide (AIP), the CaMKII inhibitor, dose-dependently increased the HWL to noxious thermal and mechanical stimulation in rats with mononeuropathy. Furthermore, intra-NAc administration of morphine, the HWL to noxious thermal and mechanical stimulation increased markedly, and there were no significant differences between morphine group and AIP group. Taken together, the results showed that intra-NAc injection of AIP induced significant antinociceptive effects in rats with mononeuropathy, indicating that CaMKII may play an important role in the transmission and/or modulation of nociceptive information in the NAc in rats with mononeuropathy.

Galanin up-regulates the expression of M1 muscarinic acetylcholine receptor via the ERK signaling pathway in primary cultured prefrontal cortical neurons.

The expression of galanin and galanin receptors are up-regulated in the brains from patients with Alzheimer's disease (AD). However, the role of galanin in the progress of AD is still controversial. Here we demonstrated that galanin increased the protein expression of M1 muscarinic acetylcholine receptor (M1) in the primary cultured prefrontal cortical neurons by ELISA and Western Blot. Moreover, we showed that the mRNA expression of M1 was also up-regulated by galanin treatment. We further explored the mechanism of the galanin induced up-regulation of M1. We found that galanin activated the ERK signaling pathway in the primary cultured prefrontal cortical neurons. In addition, our results showed that the up-regulation of M1 mRNA was blocked by an ERK inhibitor, U0126. Taken together, our results demonstrated that the ERK signaling pathway mediated the galanin induced up-regulation of M1 in the primary cultured prefrontal cortical neurons, supporting the hypothesis that galanin plays a beneficial role in the development of AD.

Intra-nucleus accumbens administration of the calcium/calmodulin-dependent protein kinase II inhibitor KN93 induced antinociception in rats with mononeuropathy.

The present study was conducted on rats with mononeuropathy induced by left common sciatic nerve ligation. Unilateral sciatic nerve loose ligation produced decreases of the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation. Intra-nucleus accumbens (NAc) injection of 1µg, 3µg and 6µg of KN93, the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor, dose-dependently increased the HWL in mononeuropathic rats. Furthermore, intra-NAc administration of morphine, the HWL to noxious thermal and mechanical stimulation increased markedly, and there were no significant differences between morphine group and KN93 group. The results demonstrated that intra-NAc injection of KN93 induced significant antinociceptive effects in rats with mononeuropathy, indicating CaMKII may play important roles in transmission of nociceptive information in the NAc of mononeuropathic rats.

Potential protection of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside against staurosporine-induced toxicity on cultured rat hippocampus neurons.

The present study explored the effect of 2,3,5,4'-tetrahydroxystilbene-2-O-ß-d-glucoside (THSG) on the staurosporine (STS)-induced toxicity in cultured rat hippocampal neurons. The results showed that administration of 200µM of THSG significantly protected against 0.3µM of STS-induced apoptosis in cultured rat hippocampal neurons tested by methyl thiazolyl tetrazolium (MTT) and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays. Furthermore, when the Akt signaling pathway was blocked by LY294002, an inhibitor of Phosphatidyl Inositol 3-kinase (PI3K), the protective effects of THSG against STS-induced neurotoxicity were abrogated. We further examined the involvement of PI3K/Akt signaling pathway in THSG protection against STS-induced cytotoxicity on cultured neurons and found that administration of THSG significantly inhibited the STS-induced decreases in the content of phosphorylated AKt (p-Akt). Moreover, we found that THSG rescued the down-regulation of B cell lymphoma/lewkmia-2 (Bcl2) and pro-caspase-3 (pro-Csp3) caused by STS in the neurons. These results indicate that THSG protect the cultured rat hippocampal neurons against STS-induced cytotoxicity and the PI3K/Akt signaling and mitochondrial apoptotic pathways are involved in the THSG-induced protective effects.

Region-specific alterations in glutamate receptor 1 phosphorylation during context-induced drug seeking after withdrawal from morphine self-administration.

Re-exposure to morphine-associated environments elicits morphine-seeking behavior after a long period of withdrawal in rats with a history of morphine dependence. Adaptations in glutamate receptor 1 (GluR1) phosphorylation in limbic brain regions have been shown to occur during withdrawal from addictive drugs, such as cocaine, methamphetamine, and heroin. However, whether similar adaptations exist after spontaneous withdrawal from repeated morphine intake has not been studied. In our present study, alterations in GluR1 expression and GluR1 phosphorylation at serine 845 (Ser845) and serine 831 (Ser831) in multiple limbic brain regions of rats were measured following context-induced drug craving after 1 or 10 days of withdrawal from intravenous morphine self-administration. Phosphorylation of GluR1 at Ser845, but not Ser831, increases in the nucleus accumbens and central amygdala from 1 to 10 days of withdrawal, and there were no changes in GluR1 phosphorylation at Ser845 or Ser831 in the hippocampal CA1 subregion from 1 to 10 days of withdrawal. Significant positive correlations between numbers of drug-seeking responses and GluR1 phosphorylation at Ser845 in the nucleus accumbens were found in individual animals. These results suggest that time-dependent and region-specific changes in phosphorylation of GluR1 at Ser845, but not Ser831, are involved in the drug-seeking behavior elicited by re-exposure to the morphine-associated context.

Antinociceptive effects induced by intra-periaqueductal grey injection of the galanin receptor 1 agonist M617 in rats with morphine tolerance.

The present study was performed to investigate the antinociceptive effects of M617, a selective galanin receptor 1 agonist, and M1145, a selective galanin receptor 2 agonist, in the periaqueductal grey (PAG) in rats with morphine tolerance. Intra-PAG injection of 0.1 nmol, 0.5 nmol and 1 nmol of M617 induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulations in rats with morphine tolerance. Nevertheless, intra-PAG injection of 5 nmol of the selective galanin receptor 2 agonist M1145 showed no significant influences on HWLs to noxious thermal and mechanical stimulations in rats with morphine tolerance. The results demonstrated that it is the selective galanin receptor 1 agonist M617, not the selective galanin receptor 2 agonist M1145, induced significant antinociceptive effects in morphine-tolerant rats, indicating that galanin receptor 1 is involved in nociceptive modulation in the PAG of morphine-tolerant rats.

Expression of calcitonin gene-related peptide receptor subunits in cultured neurons following morphine treatment.

Calcitonin gene-related peptide (CGRP) has been implicated in pain transmission and morphine tolerance. Although the release of CGRP is well observed in pain-related regions after chronic morphine treatment, a lack of evidence is obvious for the expression of CGRP-receptor subunits, calcitonin receptor-like receptor (CRLR) and receptor activity-modifying protein 1 (RAMP1) in the brain. In this study, we investigated in vitro the time-course of the expression of CRLR and RAMP1 following morphine administration, and compared their changes among striatal, cerebellar, cortical and the spinal cord neurons. The levels of CRLR showed no significant changes in striatal cultures during morphine treatment, while the levels of RAMP1 time-dependently increased. Decrease in the content of RAMP1 was detected in cerebellar cultures. Apart from these facts, no significant changes of CRLR or RAMP1 were detected in any other cultures. The changes in the level of CRLR on the cell membrane were examined. Significant increases of surface CRLR were detected in both striatal and cerebellar neurons after morphine treatment, while cortical neurons not. It is concluded that the expression of gross and surface CRLR and RAMP1 were differentially regulated in primary neuronal cultures following morphine treatments, indicating the involvement of CGRP receptor in morphine associated functions.

Influences of calcitonin gene-related peptide on mu opioid receptors in nucleus accumbens neurons of rats.

The Mu opioid receptor (MOR) has been shown to participate in the analgesic effect of the calcitonin gene-related peptide (CGRP) in the nucleus accumbens (NAc) of adult rats. However, it is not clear whether and how CGRP regulates the MOR at the molecular levels. In the present study, it is found that the level of MORs on the cell membrane of NAc neurons was increased twice more than the control level following CGRP treatment (1µM, 30min), which is a phenomenon that was blocked by the peptidergic antagonist CGRP8-37. No direct physical interaction was observed between MORs and CGRP receptors, and neither brefeldin A nor dynosore preincubation affected such effects of CGRP. However, addition of 20µM monensin 1h before CGRP treatment significantly blocked the action of CGRP on surface MORs. In living animals, microinjection of CGRP (1nmol in 1µl) into the NAc partially restored morphine antinociception in morphine-tolerant rats, and the effect of CGRP on surface MORs extended beyond normal NAc neurons to chronic morphine-treated NAc neurons. To conclude, these results demonstrate that CGRP can act on MORs and increase the number of surface MORs in NAc neurons, partially explaining the involvement of opioid receptors in CGRP-induced antinociception in the rat NAc.

Increases in αCaMKII phosphorylated on Thr286 in the nucleus accumbens shell but not the core during priming-induced reinstatement of morphine-seeking in rats.

Addiction is a pathological usurpation of the neural processes that normally serve reward-related learning and memory. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an important molecule involved in the mechanisms of learning and memory, suggesting its roles in drug addiction. In this study, we detected the changes of CaMKII protein levels in the nucleus accumbens (NAc), a key nucleus involved in drug-reward, during the reinstatement of morphine-seeking behavior with animal model of morphine self-administration in rats. Moreover, considering that the NAc is also involved in the natural reward-related learning and memory, we detected the changes of CaMKII protein levels in the NAc during the reinstatement of natural reward-seeking with animal model of saccharin self-administration as a control. We found that the level of αCaMKII phosphorylated on Thr286 increased in the NAc shell subregion but not the NAc core during the reinstatement of morphine-seeking, compared with that after extinction. However, during the reinstatement of saccharin-seeking, the protein level of αCaMKII phosphorylated on Thr286 did not change in the NAc shell. Surprisingly, both αCaMKII phosphorylated on Thr286 and ßCaMKII phosphorylated on Thr287 decreased in the NAc core during the reinstatement of saccharin-seeking. These results suggest that increased phosphorylation of CaMKII (Thr286) in the NAc shell is involved in the relapse to opioids-seeking and the mechanisms underlying the reinstatement of morphine-seeking are different from those involved in the reinstatement of natural reward-seeking.

Antinociceptive effects induced by injection of the galanin receptor 1 agonist M617 into central nucleus of amygdala in rats.

The present study was performed to explore the antinociceptive effects of M617, a selective galanin receptor 1 agonist, in the central nucleus of amygdala (CeA) of rats. Intra-CeA injection of 0.1 nmol, 0.5 nmol and 1 nmol of M617 induced dose-dependent increases in hindpaw withdrawal latencies (HWLs) to noxious thermal and mechanical stimulations in rats. Furthermore, rats received intra-CeA administration of M617 and galanin. The HWL to noxious thermal and mechanical stimulations increased markedly, and there were no significant differences in HWLs of rats received intra-CeA administration of M617 and galanin. The results demonstrated that intra-CeA injection of M617 induced significant antinociceptive effects in CeA of rats, indicating that galanin receptor 1 may be involved in M617-induced antinociception in the CeA of rats.

Antinociceptive effects of galanin in the nucleus accumbens of rats.

It has been demonstrated that galanin plays important roles in the modulation of nociceptive information in rats. The present study is performed to investigate the regulating role of galanin in nociception in the nucleus accumbens (NAc) of rats. Intra-NAc administration of galanin induces dose-dependent increases in the hindpaw withdrawal latency (HWL) to noxious thermal and mechanical stimulation in rats. Furthermore, the galanin-induced antinociceptive effects are blocked by following intra-NAc injection of the galanin receptor antagonist galantide. The results demonstrate that galanin induces antinociceptive effects in the NAc of rats, and galanin receptors are involved in the galanin-induced antinociception effects.