NR2B mRNA Expression Research Articles

Carveol mitigates the development of the morphine anti-nociceptive tolerance, physical dependence, and conditioned place preference in mice

Emergence of analgesic tolerance and dependence to morphine is frequently the limiting factor in the use of this agent in the management of pain. Hence, this study aimed to investigate the beneficial effects of the natural compound carveol (CV) against morphine antinociceptive tolerance, dependence and conditioned place preference (CPP) in mice. Behavioural paradigms included hot plate and tail-flick (for tolerance), observation of withdrawal signs (for dependence) while biochemical tests involved the assays for mRNA expression, nitrite levels, antioxidants, and immunohistochemistry studies. Behavioural tests indicated that treatment with CV significantly attenuated the morphine analgesic tolerance, physical dependence and CPP in mice. It was observed during biochemical analysis that CV-treated animals exhibited reduced mRNA expression of inducible nitric oxide synthase (iNOS) and NR2B (an NMDA subtype). In addition, decreased levels of nitrite were observed in mouse hippocampus following CV treatment than morphine administration only. Further, CV enhanced the neuronal innate antioxidants including Glutathione-S-Transferase (GST), glutathione (GSH) and catalase (CAT), while curtailed lipid peroxidase (LPO) levels in mice brain tissues. Moreover, CV exerted significant anti-inflammatory effects as evidenced by reduced expression of TNF-α and p–NF–κB in these animals than with morphine treatment only. Together, anti-inflammatory and antioxidant effects might confer needed neuro-protection following morphine administration. These observations warrant further investigations of the beneficial role of CV as a novel agent in overcoming the development of tolerance and physical dependence following morphine use.

Doxorubicin impairs cognitive function by upregulating AMPAR and NMDAR subunit expression and increasing neuroinflammation, oxidative stress, and apoptosis in the brain

Introduction: The anticancer drug doxorubicin (DOX) is used for various malignancies. However, it also causes cognitive impairment in cancer survivors. In order to determine the mechanisms underlying the acute effects of DOX, we assessed the mRNA and protein expression of glutamate receptors and proteins involved in cognitive function and apoptosis.Methods: Fear-conditioning memory tests were performed in rats after a single intraperitoneal injection of DOX (25 mg/kg) to evaluate short-term memory function. Rat brain samples were collected, and GluA1 mRNA and protein expression; NR2A and NR2B mRNA expression; and COX-2, NF-kB, TNF-α, and MDA, Bax, and caspase-3 levels were assessed via reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assays.Results: We observed a decreased number of entries in Y-maze, decreased exploration time to the novel object in the novel object recognition (NOR), and decreased freezing time in the fear-conditioning memory tests in DOX-treated rats relative to those in control rats, demonstrating cognitive impairment. GluA1, NR2B, and NR2A expression and MDA, NF-κB, Bax, COX-2, TNF-α, and caspase-3 levels in the brain were significantly elevated in DOX-treated rats.Conclusion: DOX induced cognitive impairment in the rats via neuronal toxicity by upregulating AMPAR and NMDAR expression and increasing neuroinflammation, oxidative stress, and apoptosis in the brain.

Combined use of hair follicle stem cells and CEPO (carbamylated erythropoietin)-Fc in a rat model of chronic cerebral hypoperfusion: A behavioral, electrophysiological, and molecular study

BackgroundIn dementia, synaptic dysfunction appears before neuronal loss. Stem cell therapy could potentially provide a promising strategy for the treatment of dementia models. The carbamylated erythropoietin fusion protein (CEPO-Fc) has shown synaptotrophic effects. This study aimed to determine the efficiency of the combined use of hair follicle stem cells (HFSC) and CEPO-Fc in the basal synaptic transmission (BST) and long-term plasticity (LTP) of chronic cerebral hypoperfusion (CCH) rats. MethodsWe divided 64 adult rats into control, sham, CCH+vehicle, CCH+CEPO, CCH+HFSC, and CCH+HFSC+CEPO groups. The CEPO-Fc was injected three times/week for 30 days. HFSC transplantation was done on days 4, 14, and 21 after surgery. The Morris water maze test and passive avoidance were used to assess memory. BST and LTP were assessed by a field-potential recording of the CA1 region. The hippocampal mRNA expression of IGF-1, TGF-β1, β1-Catenine, NR2B, PSD-95, and GSk-3β was evaluated by quantitative RT-PCR. ResultsFollowing combination therapy, spatial memory retention, and BST showed significant improvement relative to HFSC and CEPO-Fc groups. These effects were also confirmed by recovered mRNA expression of β1-catenin, TGF-β1, and NR2B. GSK-3β expression was downregulated in all treatment groups. The upregulated PSD-95 was identified in HFSC and combination groups compared to the vehicle group. ConclusionsThese findings indicate that the combined use of HFSC and CEPO-Fc may be more advantageous for treating memory disruption in the CCH model than CEPO-Fc or HFSC alone. This type of combination therapy may hopefully lead to a new approach to treatment for dementia.

RAGE signaling pathway is involved in CUS-induced depression-like behaviors by regulating the expression of NR2A and NR2B in rat hippocampus DG.

NMDA receptors play pivotal roles in the neurobiology of chronic stress-induced mood disorders. But the mechanism for chronic stress to disturb the expression of NMDA receptor subunits is still unclear. Recent researches indicated the involvement RAGE signaling pathway in regulation of glutamate system functions. In this study, we hypothesized RAGE signaling pathway mediated chronic stress-induced alteration in the expression of NMDA receptor subunits, leading to depressive-like behaviors. CUS decreased the expression of RAGE, NR2A, and NR2B, inhibited the phosphorylation of transcript factor ERK and CREB in rat hippocampus DG. RAGE knockdown in hippocampus DG by RAGE shRNA lentiviral particles induced depressive-like behaviors, reduced the mRNA and protein expression of NR2A and NR2B, and inhibited the phosphorylation of ERK and CREB. RAGE over-expression in hippocampus DG by RAGE adenovirus particles reversed the effects of CUS on depressive-like behaviors, ERK and CREB phosphorylation, and NR2A and NR2B expression. Our findings suggests that RAGE signaling pathway at least partially participates in the regulation of NR2A and NR2B expression, which mediates the effects of chronic stress on the depressive-like behaviors. These data provide evidence for RAGE signaling as a possible new pathway through which chronic stress results in the maladaptation of NMDA receptors.

The effects of sodium benzoate exposure on learning and neurobehavior during the prepubertal period in rats

Abstract Objectives Exposure to food additives, especially during the critical development period, has been associated with some behavioral disorders and learning difficulties in childhood. In this study, we aimed to evaluate the impact of sodium benzoate (NaB) on learning and neurobehavior in prepubertal rats. Methods A total of male rats (4-week-old) were divided into three groups as the control (CG), experiment 1 (NaB-ADI (acceptable daily intake) dose, Exp1G), and experiment 2 (NaB-NOAEL (no observed adverse effect level) dose, Exp2G) groups and NaB was administered by oral gavage for six weeks. Changes in learning, memory, motor activity and neurobehavior were evaluated by Morris water maze and open field tests. At the end of the experiment, rats’ hippocampi were extracted; NR1, NR2A, NR2B, and nAChRα7 receptor concentrations and mRNA expressions were determined. Results Neurobehavioral tests supported the possibility of increased anxiety and decreased motivation in the Exp1G. Also, molecular tests showed that NR2B receptor protein and mRNA expression levels significantly increased in Exp2G compared to CG. Conclusions Our study showed that NaB administration in the prepubertal period may cause increased anxiety, decreased motivation, and attention with ADI dose. Furthermore, NOAEL dose administration may lead to an increase in NR2B receptor levels. In light of these results, NaB consumption in childhood may affect hippocampal functions related to learning and neurobehavior.

Tooth Loss Suppresses Hippocampal Neurogenesis and Leads to Cognitive Dysfunction in Juvenile Sprague-Dawley Rats.

BackgroundBoth animal studies and prospective observational studies on patients with neurodegenerative disease have reported a positive link between oral diseases and cognitive function. However, the effect of early tooth loss on hippocampal morphology remains unknown.MethodsIn this study, 6-week-old, male, juvenile Sprague–Dawley (SD) rats were randomized into the control (C) and tooth loss (TL) groups. In the TL group, all right maxillary molars of SD rats were extracted, while in the C group, no teeth were extracted. After 3 months, the learning and memory behavior were examined by Morris Water Maze (MWM), and the protein expression and mechanic signaling pathways were analyzed by real-time polymerase chain reaction, and cresyl violet staining.ResultsTwo days after the operation, the body weight of both groups recovered and gradually returned to the level before operation. Three months after tooth extraction, the completion time of the C group in the MWM was significantly shorter than the TL group. The mRNA expression of BDNF, TrkB, AKT1, and NR2B in the C group were significantly higher than in the TL group. The pyramidal neurons in the TL group was fewer than in the C group.ConclusionTooth loss in the juvenile SD rats will reduce the number of pyramidal neurons in the hippocampus, inhibit the expression of BDNF, TrkB, AKT1, and NR2B, and eventually lead to cognitive dysfunction.

Neuroprotective effect of sumatriptan in pentylenetetrazole-induced seizure is mediated through N-methyl-D-aspartate/nitric oxide and cAMP response element-binding protein signaling pathway.

Seizure occurs as a result of uncontrolled electrical disturbances within the brain. Various biomolecules such as N-methyl-D-aspartate (NMDA), nitric oxide (NO), and cAMP response element-binding protein (CREB) have been implicated in the pathophysiology of seizure. Sumatriptan is a specific 5-Hydroxytryptamine 1B/1D receptor agonist and has neuroprotective effects in various neuropsychiatric disorders. In the current study, we tried to investigate the possible interaction of sumatriptan with NMDA/NO and CREB signaling pathway in PTZ induced seizure. For this purpose, various agonist and antagonist of NMDA such as MK-801 and Ketamine, NO precursor L-ARG, and NOS inhibitors L-NAME and 7-NI were co-administered with sumatriptan in PTZ induced seizure model. The level of nitrite in mice hippocampus was determined by Griess reaction. The gene expression of NR1, NR2A, NR2B, and CREB were quantified by quantitative real time-polymerase chain reaction (qRT-PCR). Furthermore, the involved neuronal nitric oxide synthase (nNOS) protein expression was examined via western blot analysis. Effective dose of sumatriptan (1.2 mg/kg) alone and subeffective dose of sumatriptan (0.3 mg/kg) in combination with NMDA and/or NO antagonist showed significant (P < 0.001) anticonvulsant activity in mice. Furthermore, sumatriptan significantly inhibited the PTZ-induced mRNA expression of NR2A (P < 0.0001), NR2B (P < 0.05), and CREB (P < 0.01). Also, the expression of nNOS protein in PTZ treated group was reversed by sumatriptan (P < 0.01). Hence, current findings suggest that the anticonvulsant effect of sumatriptan was due to down regulation of NMDA/NO and CREB signaling pathway.

Effects of ketogenic diet on cognitive function in pentylenetetrazol-kindled rats

Although the ketogenic diet (KD) is known to control seizures and improve cognition function in patients with drug-refractory epilepsy, the underlying mechanism remains unknown. In the present study, using pentylenetetrazol (PTZ)-induced and kindled rats, we found that KD significantly improved the impaired spatial reference memory of PTZ-kindled rats in the Morris water maze. To explore the mechanism underlying the action of KD in PTZ-kindled rats, quantitative real-time PCR (qRT-PCR) and immunohistochemical analysis were used to detect the expression of GluR1 and NR2B. The results showed that both the mRNA and protein expression of GluR1 and NR2B were significantly downregulated in the hippocampus of PTZ-kindled rats, while KD could observably improve both the mRNA and protein expression of GluR1 and NR2B in the hippocampus of PTZ-kindled rats. Additionally, KD improved the over-activated MAPK in PTZ-kindled rats, but not CAMKII, as detected by enzyme-linked immuno sorbent assay (ELISA), suggesting that the MAPK signaling pathway might be involved in the memory improvement of KD in PTZ-kindled rats. In conclusion, these results demonstrate that KD can indeed improve impaired spatial reference memory in PTZ-kindled rats, and KD can improve the expression of NR2B and GluR1.

Sesame cake hydrolysates improved spatial learning and memory of mice

Age-related decrease in cognition function was shown to be related to oxidative stress, and the study investigated the effect of sesame cake hydrolysates (SCH) on the improvement of learning and memory in mice using the Morris water maze (MWM) test. The mice administered a medium dose (20 mg/kg/d) of SCH did better in learning and memory function compared with the low-dose (10 mg/kg/d), and high-dose (30 mg/kg/d) treated animals with no significant differences in body weight. Biochemical analysis (SOD, GSH-Px, MDA, NO, and AChE) showed SCH improved the antioxidant and AChE activity and reduced the oxidative stress status, although no obvious differences in NO were observed between the control and test groups. qRT-PCR results showed that mRNA expression of CREB, NR2A, and NR2B improved in the test groups, which was consistent with the change of antioxidant capacity. All these results indicated a potential mRNA mechanism for SCH to improve learning and memory ability that was associated with the changes of antioxidant capacity, oxidative stress, and the cholinergic system.

Prophylactic Repetitive Treatment with the Herbal Medicine Kei-kyoh-zoh-soh-oh-shin-bu-toh Attenuates Oxaliplatin-Induced Mechanical Allodynia by Decreasing Spinal Astrocytes.

Chemotherapeutic drugs typically induce peripheral neuropathy, which is a major dose-limiting side effect of these drugs and is difficult to manage. In this study, we examined whether the traditional herbal formulation Kei-kyoh-zoh-soh-oh-shin-bu-toh (KSOT) could relieve the mechanical allodynia induced by chemotherapeutic drugs (oxaliplatin, paclitaxel, vincristine, and bortezomib) in mice. A single intraperitoneal injection of oxaliplatin, paclitaxel, vincristine, and bortezomib was used to induce mechanical allodynia, which peaked on days 10, 14, 14, and 12 after the injection, respectively. A single oral administration of KSOT did not inhibit mechanical allodynia after any of the treatments. However, prophylactic repetitive oral administrations of KSOT inhibited the exacerbation of mechanical allodynia induced by oxaliplatin but were not effective for allodynia induced by the other drugs. A single intraperitoneal injection of oxaliplatin did not alter the mRNA expression of the NMDA receptor NR2B in the spinal cord and that of neuregulin-1 in the sciatic nerve. In addition, the number of microglia in spinal dorsal horn did not increase in oxaliplatin-treated mice. However, the number of reactivated astrocytes in the spinal dorsal horn increased, which could be inhibited by repetitive administration of KSOT. These results suggest that prophylactic repetitive treatment of KSOT attenuates oxaliplatin-induced mechanical allodynia by decreasing the number of spinal astrocytes.

Influence of electroacupuncture of "Dazhui" (EX-B2) and "Mingmen" (GV4) on NR2B expression in anterior horns of spinal cord in rats with acute spinal cord injury

To observe the influence of eletroacupuncture (EA) at "Dazhui" (EX-B2) and "Mingmen" (GV4) on expression of NR2B subunit of N-methyl-D-aspartate receptor (NMDA) in the injured anterior horn (AH) area of rats with acute spinal cord injury (SCI), so as to explore its mechanisms underlying improvement of neural repair. A total of 96 male Sprague-Dawley (SD) rats were randomly and equally divided into four groups: sham operation (sham), model, medication (Methyl-prednisone, MP) and EA (n＝24 in each group). The acute SCI model was established by using a MASCIS spinal cord impactor. EA (2 Hz, 0.5 mA) was applied to EX-B2 and GV4 for 30 min, once at 0.5 h, 12 and 24 h after SCI. Rats of the medication group were treated by tail intravenous injection of MP 30 mg/kg within 15 min (impact therapy) and 5.4 mg•kg－1•h－1 (maintaining treatment) 45 min thereafter for 23 h. The Basso, Beattie and Bresnahan (BBB) rating scale (0 to 21 points) was used to assess changes of locomotor function 6, 24 and 48 h after SCI. Histopathological changes of the injured spinal cord AH region were observed after sectioning and hematoxylin-eosin (H.E.) staining, and the expression levels of NR2B mRNA and protein of AH were measured by quantitative real-time PCR, Western blot and immunofluorescence, respectively. After SCI, the BBB scores at 6, 24 and 48 h were significantly decreased in the model group compared with those of the sham group (P<0.001), but had no remarkable changes in both medication and EA groups after the intervention in comparison with those of the model group (P>0.05). After modeling, the histopathological changes (blurred border of the grey-white matter, cellular karyopyknosis, deepening of the cytoplasmic red stain, and rupture, dissolution and disordered arrangement of myelinated nerve fibers) in the injury area of the spinal cord in the model group were apparent, the number of NR2B positive neurons and the relative expression levels of NR2B mRNA and protein were significantly increased in the model group relevant to the sham group (P<0.05). In contrast to the model group, the injured severity of the spinal cord AH region was relatively milder, and the expression levels of NR2B mRNA and protein were considerably down-regulated in both EA and MP groups (P<0.05). However, there were no significant differences between the EA and MP groups in the expression levels of NR2B mRNA and protein (P>0.05). EA at EX-B2 and GV4 may inhibit the expression of NR2B mRNA and protein in acute SCI rats, which may contribute to its action in promoting nerve repair of the injured ventricolumna area of the thoracic spinal cord.

Cathepsin C promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca2+-dependent PKC/p38MAPK/NF-\u03baB pathway

BackgroundMicroglia-derived lysosomal cathepsins are important inflammatory mediators to trigger signaling pathways in inflammation-related cascades. Our previous study showed that the expression of cathepsin C (CatC) in the brain is induced predominantly in activated microglia in neuroinflammation. Moreover, CatC can induce chemokine production in brain inflammatory processes. In vitro studies further confirmed that CatC is secreted extracellularly from LPS-treated microglia. However, the mechanisms of CatC affecting neuroinflammatory responses are not known yet.MethodsCatC over-expression (CatCOE) and knock-down (CatCKD) mice were treated with intraperitoneal and intracerebroventricular LPS injection. Morris water maze (MWM) test was used to assess the ability of learning and memory. Cytokine expression in vivo was detected by in situ hybridization, quantitative PCR, and ELISA. In vitro, microglia M1 polarization was determined by quantitative PCR. Intracellular Ca2+ concentration was determined by flow cytometry, and the expression of NR2B, PKC, p38, IkBα, and p65 was determined by western blotting.ResultsThe LPS-treated CatCOE mice exhibited significantly increased escape latency compared with similarly treated wild-type or CatCKD mice. The highest levels of TNF-α, IL-1β, and other M1 markers (IL-6, CD86, CD16, and CD32) were found in the brain or serum of LPS-treated CatCOE mice, and the lowest levels were detected in CatCKD mice. Similar results were found in LPS-treated microglia derived from CatC differentially expressing mice or in CatC-treated microglia from wild-type mice. Furthermore, the expression of NR2B mRNA, phosphorylation of NR2B, Ca2+ concentration, phosphorylation of PKC, p38, IκBα, and p65 were all increased in CatC-treated microglia, while addition of E-64 and MK-801 reversed the phosphorylation of above molecules.ConclusionThe data suggest that CatC promotes microglia M1 polarization and aggravates neuroinflammation via activation of Ca2+-dependent PKC/p38MAPK/NF-κB pathway. CatC may be one of key molecular targets for alleviating and controlling neuroinflammation in neurological diseases.

Role of NR2B in rostal anterior cingulate cortex in development of pain-related aversion in a rat model of bone cancer pain: siRNA technique

Objective To evaluate the role of 2B subunit-containing NMDA receptors(NR2B) in the rostal anterior cingulate cortex (rACC) in development of pain-related aversion in a rat model of bone cancer pain using siRNA technique. Methods Forty-five healthy male Wistar rats, weighing 220-250 g, were divided into 3 groups (n=15 each) using a random number table method: normal saline blank control group (group NS), NR2B-siRNA lentivirus group (group LV-NR2B) and pGC-FU-siRNA lentivirus group (group LV-NC). A total volume of MADB-106 cells 3 μl(4.8×109 cells/ml) was inoculated into the bone marrow cavity of the right tibia of rats.At day 2 after inoculation, NR2B/siRNA recombinant lentivirus 0.2 μl was injected into rACC in group LV-NR2B, and pGC-FU-siRNA negative recombinant lentivirus 0.2 μl was injected into rACC in group LV-NC.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before inoculation and 3, 7, 14 and 21 days after inoculation.Conditioned place avoidance test was performed at 1 day before inoculation and 14 days after inoculation, and the percentage of residence time in room A was calculated.The rats were sacrificed at 21 days after inoculation and the rACC was removed for detecting NR2B protein and mRNA expression by Western blot or real-time polymerase chain reaction. Results Compared with the baseline at 1 day before inoculation, the MWT was significantly decreased at 7, 14 and 21 days after inoculation, and the percentage of residence time in room A was decreased at 14 days after inoculation in NS and LV-NC groups (P 0.05). Compared with group NS, the MWT was significantly increased at 14 and 21 days after inoculation, the percentage of residence time in room A was increased at 14 days after inoculation, and the expression of NR2B protein and mRNA was down-regulated in group LV-NR2B (P 0.05). Conclusion Up-regulated expression of NR2B in rACC is involved in development of pain-related aversion in a rat model of bone cancer pain. Key words: Receptors, N-methyl-D-aspartate; Bone neoplasms; Pain; Emotions

Neuroprotection of Cytisine Against Cerebral Ischemia-Reperfusion Injury in Mice by Regulating NR2B-ERK/CREB Signal Pathway.

The aim of the study was to elucidate the therapeutic effects of Cytisine (CYT) on cerebral ischemia-reperfusion injury in mice. Male ICR mice were pretreated with reagents (drug), and then subjected to 2h focal cerebral ischemia and 24h reperfusion. Morphologically, the histopathological impairment were estimated by the TTC, HE and TUNEL staining. The expression of GluN2B-containing NMDA receptor, phosphorylation of extracellular regulated protein kinases, total ERK, phosphorylation of cAMP-response element binding protein and total CREB were determined by immunofluorescence and Western blot assay, respectively. The mRNA expression of NR2B, ERK and CREB were quantified by the real-time RT-PCR. CYT significantly diminished the infarct size and neuronal apoptosis. Additionally, it ameliorated histopathological lesion dramatically. CYT promoted the phosphorylation of ERK, CREB and their mRNA expression. In contrast, the expression of NR2B was suppressed in concomitant with the down-regulation of genes. The overall results thus far suggest that CYT confers the neuroprotection against cerebral I/R injury by regulating the NR2B-ERK/CREB signal pathway.

High and low nightly running behavior associates with nucleus accumbens N-Methyl-d-aspartate receptor (NMDAR) NR1 subunit expression and NMDAR functional differences

The extent to which N-Methyl-d-aspartate (NMDA) receptors facilitate the motivation to voluntarily wheel-run in rodents has yet to be determined. In so, we utilized female Wistar rats selectively bred to voluntarily run high (HVR) and low (LVR) nightly distances in order to examine if endogenous differences in nucleus accumbens (NAc) NMDA receptor expression and function underlies the propensity for high or low motivation to voluntarily wheel-run. 12–14 week old HVR and LVR females were used to examine: 1.) NAc mRNA and protein expression of NMDA subunits NR1, NR2A and NR2B; 2.) NMDA current responses in isolated medium spiny neurons (MSNs) and 3.) NMDA-evoked dopamine release in an ex vivo preparation of NAc punches. Expectedly, there was a large divergence in nightly running distance and time between HVR and LVR rats. We saw a significantly higher mRNA and protein expression of NR1 in HVR compared to LVR rats, while seeing no difference in the expression of NR2A or NR2B. There was a greater current response to a 500 ms application of 300 μM of NMDA in medium-spiny neurons isolated from the NAc HVR compared to LVR animals. On average, NMDA-evoked punches (50 μM of NMDA for 10 min) taken from HVR rats retained ∼54% of the dopamine content compared to their bilateral non-evoked sides, while evoked punches from LVR animals showed no statistical decrease in dopamine content compared to their non-evoked sides. Collectively, these data suggest a potential link between NAc NR1 subunit expression as well as NMDA function and the predisposition for nightly voluntary running behavior in rats. In light of the epidemic rise in physical inactivity, these findings have the potential to explain a neuro-molecular mechanism that regulates the motivation to be physically active.

Expression of high-mobility group box-Ⅰ and N-methyl-D-aspartate receptor in status epilepticus

Objective: To investigate the expression changes of high mobility group box-1 (HMGB1) and the 2B receptor of N- methyl -D- aspartate receptor (NR2B) in status epilepticus (SE). Methods: (1) Primary hippocampal neurons from SD rats with 16 to 18 days of fetal age were cultured in vitro for 7 days, and exposed to Mg(2+) free media for 3 hours. Those cultured neurons were randomly divided into control group and intermittent hypoxia group. (2) SD rats with similar weight were selected and randomly divided into control group and SE model group. The rat model with SE was established by an intraperitoneal injection of lithium chloride-piloearpine (LI-PILO). Real-time PCR technique was used to detect the expression of HMGB1 and NR2B mRNA. Results: In Sombati's cell model cultured in normal concentration of oxygen, the HMGB1 mRNA expression levels were 0.005 01±0.000 54, 0.026 76±0.003 75, 0.003 52±0.000 33, and the NR2B mRNA expression levels were 0.008 84±0.000 69, 0.012 23±0.000 90, 0.029 11±0.000 71, respectively, at 2, 4 and 6 h; compared with the expressions of HMGB1 and NR2B mRNA at the same time points of Sombatis cell model groups, the differences were also significant (all P<0.05). After the successful establishment of epilepsy model, the HMGB1 mRNA expression levels were 0.000 11±0.000 09, 0.000 18±0.000 01, 0.000 11±0.000 01, and the NR2B mRNA expression levels were 0.196 12±0.009 41, 0.232 11±0.006 27, 0.272 48±0.005 84, respectively, at 6, 8 and 10 h; compared with the expressions of HMGB1 and NR2B mRNA at the same time points of control groups, the differences were all significant (all P<0.05). Conclusion: HMGB1 mRNA expression levels increase at 2, 4 h, decrease at 6 h in the Sombati's cell model in normal oxygen culture, while increase at 6, 8 h, and decrease at 10 h in LI-PILO induced rat model with SE; the NR2B mRNA relative expression increases with time in both the Sombati's cell model in normal oxygen culture and rat model of SE.

The effect of postnatal manganese exposure on the NMDA receptor signaling pathway in rat hippocampus.

Overexposure to manganese (Mn) is associated with neurological disorders in children. Evidence indicated that N-methyl-d-aspartate (NMDA) receptor signaling pathway was critical for neurobehavioral function. However, whether NMDA receptor signaling pathway contributes to Mn-induced neurotoxicity remains unknown. In this study, newborn Sprague-Dawley rats were randomly assigned to four groups exposed to 0, 10, 20, and 30mg/kg of Mn2+ by intraperitoneal injection (n=10/group: five males and five females). After 3weeks of Mn exposure, messenger RNA (mRNA) and protein expression of NMDA receptor subunits (NR1, NR2A, and NR2B), cAMP-response element binding protein (CREB), and brain-derived neurotrophic factor (BDNF) in hippocampus were measured by real-time quantitative RT-PCR and Western blot. In Mn-exposed rats, decreased mRNA and protein expression of NR1, NR2A, and NR2B, CREB, and BDNF was observed. The results imply that downregulated NMDA receptor signaling pathway may be of vital importance in the neuropathological process of Mn-induced neurotoxicity.

The effects of sodium butyrate on ethanol-seeking behavior and H3K9 acetylation in NMDA receptor 2B subunit promotor region in the hippocampus of rats

Objective To investigate the effects of sodium butyrate on ethanol-seeking behavior and H3K9 acetylation levels in NMDA receptor 2B subunit(NR2B)promoter region in the hippocampus of Wistar rats. To explore the epigenetic mechanism underlying ethanol-seeking behavior. Methods According to random number table, 48 male Wistar rats were divided into saline group, sodium butyrate group, ethanol group and sodium butyrate + ethanol group, with 12 rats in each group and administered by intraperitioneal injection respectively. Conditioned place preference(CPP)was used to evaluate the ethanol-seeking behavior. Using Western-blot, real-time PCR and chromatin immunoprecipitation assays, the expression of NR2B protein, NR2BmRNA and the relative level acetylated H3K9 in NR2B promoter region in the hippocampus were determined respectively. Results The CPP test and the CPP score in each group were different(P 0.05). Compared with the ethanol group, CPP test of sodium butyrate + ethanol group was significantly higher(P 0.05). Compared with the ethanol group, the expression of NR2B protein, NR2BmRNA and acetylated level H3K9 in NR2B promoter region in the hippocampus of ethanol group, these of sodium butyrate + ethanol group were significantly higher(P<0.05). The CPP score were positively correlated with the expression of NR2B protein(r=0.474, P<0.05). The expression of NR2B protein were positively correlated with the expression of NR2BmRNA(r=0.468, P<0.05). The expression of NR2BmRNA were positively correlated with the expression of H3K9 acetylation in NR2B promoter region(r=0.596, P<0.05), and the CPP score were positively correlated with the expression of H3K9 acetylation in NR2B promoter region (r=0.542, P<0.05). Conclusion The increasing acetylation level of H3K9 in NR2B promoter region in the hippocampus may be one of the epigenetic mechanisms of promoting ethanol-seeking behavior, and H3K9 deacetylation in NR2B promoter region in the hippocampus is likely to be a new target for controlling ethanol dependence. Key words: Alcohol; Sodium butyrate; Histone acetylation; NMDA receptor 2B subunit

Effect of GLYX-13 on cognitive function after long-time isoflurane anesthesia in mice

Objective To evaluate the effects of GLYX-13 on cognitive function after long-time isoflurane anesthesia in mice. Methods A total of 192 healthy male C57/B6J mice, aged 8 weeks, weighing 22-25 g, were divided into 4 groups (n=48 each) using a random number table: control group (group C), isoflurane anesthesia group (group I), GLYX-13 group (group G), and isoflurane anesthesia plus GLYX-13 group (group IG). The animals were exposed to 1.5% isoflurane for 6 h in I and IG groups.GLYX-13 1 mg/kg was injected via the caudal vein at 2 h before anesthesia in G and IG groups.Novel object recognition test and contextual fear conditioning test were performed on 1st, 3rd and 7th days after anesthesia.The expression of 2B subunits-containing NMDA receptor (NR2B) and cyclic adenosine monophosphate response element-binding protein (CREB) mRNA in the hippocampus was detected by quantitative real-time polymerase chain reaction after the end of behavioral tests on 1st, 3rd and 7th days after anesthesia. Results Compared with group C, the percentage of time spent in exploring a novel object, discrimination index and percentage of freezing time were significantly decreased, and the expression of NR2B and CREB mRNA in the hippocampus was down-regulated in group I (P <0.05). Compared with group I, the percentage of time spent in exploring a novel object, discrimination index and percentage of freezing time were significantly increased, and the expression of NR2B and CREB mRNA in the hippocampus was up-regulated in group IG (P <0.05). Conclusion GLYX-13 can significantly improve the cognitive function after long-time isoflurane anesthesia in mice. Key words: Excitatory amino acid agonists; Isoflurane; Cognition disorders

NADPH Oxidase Isoform 2 (NOX2) Is Involved in Drug Addiction Vulnerability in Progeny Developmentally Exposed to Ethanol.

Ethanol exposure increases oxidative stress in developing organs, including the brain. Antioxidant treatment during maternal ethanol ingestion improves behavioral deficits in rodent models of fetal alcohol spectrum disorder (FASD). However, the impact of general antioxidant treatment in their adult offspring and the Specific Reactive Species (ROS)-dependent mechanism, are not fully understood. We hypothesized that pre and early postnatal ethanol exposure (PEE) modifies redox homeostasis, in particular NOX2 function during reward signaling in the mesocorticolimbic pathway, which reinforces the effects of alcohol. We developed a FASD rat model which was evaluated during adolescence (P21) and adulthood (P70). We first studied whether redox homeostasis is affected in PEE animals, by analyzing mRNA expression of SOD1, CAT, and Gpx1. We found that PEE reduced the mRNA levels of these three anti-oxidant enzymes in PFC and HIPP at P21 and in the VTA at P70. We also analyzed basal mRNA and protein expression of NOX2 subunits such as gp91phox, p22 phox, and p47 phox, in mesocorticolimbic brain areas of PEE rat brains. At P21, gp91 phox, and p47 phox levels in the VTA were decreased. At P70, gp91 phox mRNA levels was decreased in HIPP and both mRNA and protein levels were decreased in PFC. Since NOX2 is regulated by the N-methyl-D-aspartate Receptor (NMDAR), we analyzed NMDAR mRNA expression and found differential expression of NMDAR subunits (NR1 and NR2B) in the PFC that was age dependent, with levels decreased at P21 and increased at P70. The analysis also revealed decreased NR2B mRNA expression in HIPP and VTA at P70. Offspring from maternal ethanol users consumed 25% more ethanol in a free choice alcohol consumption test than control rats, and showed place preference for an alcohol-paired compartment. In vivo inhibition of NOX2 using apocynin in drinking water, or infusion of blocked peptide gp91 phox ds in the VTA normalized alcohol place preference, suggesting that NOX2 plays an important role in addictive like behavior. Taken together, PEE significantly affects the expression of antioxidant enzymes, NOX2, NMDAR in an age, and brain region dependent manner. Moreover, we demonstrate that NOX2 regulates alcohol seeking behavior.