Intravenous Injection of GluR2-3Y Inhibits Repeated Morphine-Primed Reinstatement of Drug Seeking in Rats.

Studies have demonstrated that the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor is essential to drug addiction. In this study, we explored the influence of GluR2-3Y, an interfering peptide to prevent the endocytosis of AMPA receptors containing the GluR2 subunit, on morphine-seeking behavior in the rat self-administration model. After self-administration was established, the rats received intravenous injections of GluR2-3Y during the extinction sessions. There were no significant differences in both active and inactive pokes compared to the control group of rats that received GluR2-3S, indicating that GluR2-3Y has no significant influences on the extinction of morphine self-administration. The other two groups of rats were trained, extinguished, and reinstated by repeated morphine priming (respectively, called Prime 1, Prime 2, and Prime 3). Only one intravenous injection of GluR2-3Y was performed before Prime 1. Compared to the control group, GluR2-3Y did not affect Prime 1, but significantly attenuated the morphine-seeking behavior during repeated morphine-primed reinstatement, indicating an inhibitory after effect of GluR2-3Y on morphine-seeking behavior in rats. The long-term depression (LTD) in the nucleus accumbens (NAc) shell was also assessed. Pretreatment with GluR2-3Y altered the ability of LTD induction to the level of that in the naive group, while pretreatment with GluR2-3S had no effects on LTD. Our results demonstrated that the intravenous injection of GluR2-3Y, to block the endocytosis of AMPA receptors, inhibited the reinstatement of morphine-seeking behavior, which may be induced by modulating the neuronal plasticity in the NAc shell of rats.

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.

Time Is a Critical Factor When Evaluating Oligonucleotide Therapeutics in hERG Assays.

In accord with International Conference on Harmonization S7B guidelines, an in vitro human ether-a-go-go-related gene (hERG) assay is one component of an integrated risk assessment for delayed ventricular repolarization. Function of hERG could be affected by direct (acute) mechanisms, or by indirect (chronic) mechanisms. Some approved oligonucleotide therapeutics had submitted hERG data to regulatory agents, which were all collected with the same protocol used for small-molecule testing (incubation time <20 min; acute), however, oligonucleotides have unique mechanisms and time courses of action (indirect). To reframe the hERG testing strategy for silencing RNA (siRNA), an investigation was performed to assess the time course for siRNA-mediated inhibition of hERG function and gene expression. Commercially available siRNAs of hERG were evaluated in a stable hERG-expressed cell line by whole-cell voltage clamp using automated electrophysiology and polymerase chain reaction. In the acute hERG study, no effects were observed after treatment with 100 nM siRNA for 20 min. The chronic effects of 100 nM siRNAs on hERG function were evaluated and recorded over 8-48 h following transfection. At 8 h there was no significant effect, whereas 77% reduction was observed at 48 h. Measurement of hERG mRNA levels demonstrated a 79% and 93% decrease of hERG mRNA at 8 and 48 h, respectively, consistent with inhibition of hERG transcription. The results indicate that an anti-hERG siRNA requires a long exposure time (48 h) in the hERG assay to produce a maximal reduction in hERG current; short exposures (20 min-8 h) had no effect. These findings imply that off-target profiling of novel oligonucleotides could benefit from using hERG protocol with long incubation times to de-risk potential off-target (indirect) effects on the hERG channel. This hERG assay modification may be important to consider if the findings are used to support an integrated nonclinical-clinical risk assessment for QTc (the duration of the QT interval adjusted for heart rate) prolongation.

A Novel CaMKII Inhibitory Peptide Blocks Relapse to Morphine Seeking by Influencing Synaptic Plasticity in the Nucleus Accumbens Shell.

Drugs of abuse cause enduring functional disorders in the brain reward circuits, leading to cravings and compulsive behavior. Although people may rehabilitate by detoxification, there is a high risk of relapse. Therefore, it is crucial to illuminate the mechanisms of relapse and explore the therapeutic strategies for prevention. In this research, by using an animal model of morphine self-administration in rats and a whole-cell patch-clamp in brain slices, we found changes in synaptic plasticity in the nucleus accumbens (NAc) shell were involved in the relapse to morphine-seeking behavior. Compared to the controls, the amplitude of long-term depression (LTD) induced in the medium spiny neurons increased after morphine self-administration was established, recovered after the behavior was extinguished, and increased again during the relapse induced by morphine priming. Intravenous injection of MA, a new peptide obtained by modifying Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor "myr-AIP", decreased CaMKII activity in the NAc shell and blocked the reinstatement of morphine-seeking behavior without influence on the locomotor activity. Moreover, LTD was absent in the NAc shell of the MA-pretreated rats, whereas it was robust in the saline controls in which morphine-seeking behavior was reinstated. These results indicate that CaMKII regulates morphine-seeking behavior through its involvement in the change of synaptic plasticity in the NAc shell during the relapse, and MA may be of great value in the clinical treatment of relapse to opioid seeking.

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 mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats.

Lots of studies have demonstrated that anterior cingulate cortex plays important roles in the pain perception and pain modulation. The present study explored the role of mu-opioid receptor in nociceptive modulation in anterior cingulate cortex of rats with neuropathic pain. Neuropathic pain model was set up by chronic constriction injury of the left sciatic nerve of rats. The hindpaw withdrawal latency to thermal and mechanical stimulation, by hot plate and Randall Selitto Test respectively, was used to evaluate the rat's responses to noxious stimulation. Results showed that intra-anterior cingulate cortex injection of morphine could induce the antinociception dose-dependently. By intra-anterior cingulate cortex injection of opioid receptor antagonist, the morphine-induced antinociception could be attenuated by naloxone, as well as much significantly by the selective mu-opioid receptor antagonist ß-funaltrexamine, indicating that mu-opioid receptor is involved in the morphine-induced antinociception in anterior cingulate cortex of rats with neuropathic pain. The morphine-induced antinociception was much more decreased in rats with neuropathic pain than that in normal rats, and there was a significant decrease in mu-opioid receptor messenger RNA levels in anterior cingulate cortex of rats with neuropathic pain, indicating that there may be a down-regulation in mu-opioid receptor expression in anterior cingulate cortex of rats with neuropathic pain. To further confirm the role of mu-opioid receptor in morphine-induced antinociception in anterior cingulate cortex, normal rats were received intra-anterior cingulate cortex administration of small interfering RNA targeting mu-opioid receptor and it was found that there was a down-regulation in mu-opioid receptor messenger RNA levels, as well as a down-regulation in mu-opioid receptor expression in anterior cingulate cortex tested by real-time polymerase chain reaction and western blotting. Furthermore, the morphine-induced antinociceptive effect decreased significantly in rats with small interfering RNA targeting mu-opioid receptor, which indicated that knockdown mu-opioid receptor in anterior cingulate cortex could also attenuate morphine-induced antinociceptive effect. These results strongly suggest that mu-opioid receptor plays a significant role in nociceptive modulation in anterior cingulate cortex of rats.

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.

Etelcalcetide, A Novel Calcimimetic, Prevents Vascular Calcification in A Rat Model of Renal Insufficiency with Secondary Hyperparathyroidism.

Etelcalcetide, a novel peptide agonist of the calcium-sensing receptor, prevents vascular calcification in a rat model of renal insufficiency with secondary hyperparathyroidism. Vascular calcification occurs frequently in patients with chronic kidney disease (CKD) and is a consequence of impaired mineral homeostasis and secondary hyperparathyroidism (SHPT). Etelcalcetide substantially lowers parathyroid hormone (PTH) and fibroblast growth factor-23 (FGF23) levels in SHPT patients on hemodialysis. This study compared the effects of etelcalcetide and paricalcitol on vascular calcification in rats with adenine-induced CKD and SHPT. Uremia and SHPT were induced in male Wistar rats fed a diet supplemented with 0.75% adenine for 4 weeks. Rats were injected with vehicle, etelcalcetide, or paricalcitol for 4 weeks from the beginning of adenine diet. Rats fed an adenine-free diet were included as nonuremic controls. Similar reductions in plasma PTH and parathyroid chief cell proliferation were observed in both etelcalcetide- and paricalcitol-treated rats. Serum calcium and phosphorus were significantly lower in etelcalcetide-treated uremic rats and was unchanged in paricalcitol-treated rats. Both serum FGF23 and aortic calcium content were significantly lower in etelcalcetide-treated uremic rats compared with either vehicle- or paricalcitol-treated uremic rats. The degree of aortic calcium content for etelcalcetide-treated rats was similar to that in nonuremic controls and corroborated findings of lack of histologic aortic mineralization in those groups. In conclusion, etelcalcetide and paricalcitol similarly attenuated progression of SHPT in an adenine rat model of CKD. However, etelcalcetide differentially prevented vascular calcification, at least in part, due to reductions in serum FGF23, calcium, and phosphorus levels.

Etelcalcetide (AMG 416), a peptide agonist of the calcium-sensing receptor, preserved cortical bone structure and bone strength in subtotal nephrectomized rats with established secondary hyperparathyroidism.

Sustained elevation of parathyroid hormone (PTH) is catabolic to cortical bone, as evidenced by deterioration in bone structure (cortical porosity), and is a major factor for increased fracture risk in chronic kidney disease (CKD). Etelcalcetide (AMG 416), a novel peptide agonist of the calcium-sensing receptor, reduces PTH levels in subtotal nephrectomized (Nx) rats and in hemodialysis patients with secondary hyperparathyroidism (SHPT) in clinical studies; however, effects of etelcalcetide on bone have not been determined. In a rat model of established SHPT with renal osteodystrophy, etelcalcetide or vehicle was administered by subcutaneous (s.c.) injection to subtotal Nx rats with elevated PTH (>750pg/mL) once per day for 6weeks. Sham-operated rats receiving vehicle (s.c.) served as non-SHPT controls. Prior to treatment, significant increases in serum creatinine (2-fold), blood urea nitrogen (BUN, 3-fold), PTH (5-fold), fibroblast growth factor-23 (FGF23; 13-fold) and osteocalcin (12-fold) were observed in SHPT rats compared to non-SHPT controls. Elevations in serum creatinine and BUN were unaffected by treatment with vehicle or etelcalcetide. In contrast, etelcalcetide significantly decreased PTH, FGF23 and osteocalcin, whereas vehicle treatment did not. Cortical bone porosity increased and bone strength decreased in vehicle-treated SHPT rats compared to non-SHPT controls. Cortical bone structure improved and energy to failure was significantly greater in SHPT rats treated with etelcalcetide compared to vehicle. Mineralization lag time and marrow fibrosis were significantly reduced by etelcalcetide. In conclusion, etelcalcetide reduced bone turnover, attenuated mineralization defect and marrow fibrosis, and preserved cortical bone structure and bone strength by lowering PTH in subtotal Nx rats with established SHPT.

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.