NR1 Expression Research Articles

Alterations of NMDA and AMPA receptors and their signaling apparatus in the hippocampus of mouse offspring induced by developmental arsenite exposure.

Loss of cognitive function due to arsenic exposure is a serious health concern in many parts of the world, including China. The present study aims to determine the molecular mechanism of arsenic-induced neurotoxicity and its consequent effect on downstream signaling pathways of mouse N-methyl-D-aspartate receptors (NMDARs) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). Drinking water containing 0, 25, 50 or 100 mg/L arsenite was provided each day to mother mice throughout gestation period until postnatal day (PND) 35 to expose the newborn mice to arsenite during early developmental period. The effect of arsenite in the expressions of different components of NMDAR (NR1, NR2A, NR2B) and AMPAR (GluR1, GluR2, GluR3), including calcium/calmodulin-dependent protein kinase II (CaMKII) and phosphorylated-CaMKII (p-CaMKII), at PND 7, 14, 21 and 35 was estimated and analyzed from the hippocampus of mice. A significant inhibition in the protein and mRNA expressions of NR1, NR2A, NR2B and GluR1 was observed in mice exposed to 50 mg/L arsenite since PND 7. Down regulation of GluR2 and GluR3 both at mRNA and protein levels was observed in mice exposed to 50 mg/L arsenite till PND 14. Moreover, both CaMKII as well as p-CaMKII expressions were significantly limited since PND 7 in 50 mg/L arsenite exposed mice group. Findings form this study suggested that the previously reported impairment in learning and memorizing abilities in later stage due to early life arsenite exposure is associated with the alterations of NMDARs, AMPARs, CaMKII and p-CaMKII expressions.

Memantine ameliorates pulmonary inflammation in a mice model of COPD induced by cigarette smoke combined with LPS

An enhanced chronic inflammatory response in the airways has been regarded as a critical characteristic of chronic obstructive pulmonary disease (COPD). Memantine, an N-methyl-d-aspartate (NMDA) receptors antagonist, has been reported to alleviate lung inflammation. In this study, we investigated the effect and mechanism of memantine on the COPD model induced by cigarette smoke (CS) combined with LPS. Mice and RAW264.7 cells were treated with LPS in the presence or absence of CS. We performed H&E staining to analysis the lung histopathological characteristics. Cytokines (IL-6, TNF-α, and IFN-γ) levels in bronchoalveolar lavage fluid (BALF), lung tissue homogenates and RAW264.7 cell culture medium were determined. Glutamate levels in plasma and culture medium of RAW264.7 were determined. The intracellular Ca2+ flux in RAW264.7 cells was measured by fluo-3 AM staining. The protein levels of NR-1, xCT, ERK1/2, and AKT signaling in the lung tissue and cells were investigated. The result showed that CS and LPS stimulation caused inflammation response, a significant increase in the release of cytokines, including TNF-α, IL-6, and IFN-γ, the elevated release of glutamate and protein levels of NR-1 and xCT, increased Ca2+ influx, and the activation of the ERK1/2 pathway in vitro and in vivo. The above effects of CS and LPS stimulation could be significantly attenuated by memantine treatment. In conclusion, memantine can effectively ameliorate pulmonary inflammation in CS + LPS-induced COPD in mice via reducing NR-1 and xCT expression, glutamate release, Ca2+ influx, and the phosphorylation of Erk1/2. We provided a possible mechanism by which memantine ameliorates COPD in mice.

Prenatal propofol exposure downregulates NMDA receptor expression and causes cognitive and emotional disorders in rats

The impacts of prenatal propofol on cognition and emotion of offspring remain elusive. In the present study, pregnant rats in the second trimester were anesthetized with propofol. Neuronal apoptosis and proliferation was determined in fetuses and postnatal rats by detecting caspase-3 and BrdU expression. The offspring were subjected to several behavior tests. Then the pyramidal neurons in hippocampus and the expression of NR1, NR2A, and NR2B subunits of NMDA receptor and PSD-95 in frontal cortex were examined. Propofol exposure significantly increased the number of caspase-3+ cells in lateral ganglionic eminence and hippocampus compared with control group (P < 0.001). The number of BrdU+ cells in the subventricular zone (SVZ) and dentate gyrus (DG) was also reduced in propofol group (P < 0.001). In propofol group, the swimming distance and time in the quadrant were shorter,but longer out of the quadrant. The number of escapes was less than those of the control group. Prenatal propofol exposure also decreased the sucrose preference, reduced the numbers of grooming, crossing, and rearing of the pups, and increased the fecal particle numbers and immobility time (P < 0.05). There were fewer and shorter dendritic branches of pyramidal neurons in the CA1 and CA3 of offspring. The expression of NR1, NR2A, NR2B, and PSD-95 in the frontal cortex was downregulated by propofol exposure (P < 0.001). These data suggested that prenatal propofol exposure impairs neuronal development, which may be associated with depression- and anxiety-like behaviors and cognitive disorders in offspring.

Kaempferol Inhibits Extra-synaptic NMDAR-Mediated Downregulation of TRkβ in Rat Hippocampus During Hypoxia

Cognitive dysfunction on chronic exposure to hypobaric hypoxia has been attributed to a myriad of survival and degenerative factors. Downregulation of Trkβ and compromised survival signaling has been ascribed as a major contributing factor for hypoxic neurodegeneration. The mechanisms leading to downregulation of Trkβ in hypoxia, however, remain to be elucidated. The present study aimed at investigating the upstream signaling mechanisms leading to Trkβ downregulation in hypoxia and the potential of Kaempferol in ameliorating these changes. Our results showed a duration-dependent increase in hypoxic neurodegeneration as measured by Fluoro-Jade C staining of hippocampal CA3 neurons. Protein expression studies revealed strong correlation of Trkβ with NR1 and NR2b expression on exposure to hypoxic stress. Administration of Kaempferol during hypoxic stress revealed its neuroprotective effect and Morris Water Maze test also highlighted its efficacy in improving spatial learning and memory. Further elucidation of the signaling mechanisms using specific inhibitors and in vitro silencing experiments confirmed involvement of extra-synaptic N-methyl-d-aspartate receptor (NMDAR) i.e. NR2b receptor subunit in downregulation of Trkβ under hypoxic conditions. ChIP assay showed involvement of E47 transcription factor in NR2b mediated Trkβ downregulation. Selective inhibition of signaling intermediate MLK2 by CEP11004 and inhibition of extra-synaptic NMDAR during hypoxic stress prevented Trkβ downregulation in the hippocampus of hypoxic rats. Administration of Kaempferol also inhibited phosphorylation of E47 and hypoxia-induced downregulation of Trkβ. The present study establishes the role of extra-synaptic NMDAR in hypoxia-induced downregulation of Trkβ and the efficacy of Kaempferol in inhibiting extra-synaptic NMDAR-mediated signaling.

Role of Steroid Therapy after Ischemic Stroke by n-Methyl-d-Aspartate Receptor Gene Regulation

BackgroundStroke is the main cause of cerebrovascular disease mortality. Prolonged stimulation of n-methyl-d-aspartate (NMDA) receptor subtypes by the accumulation of glutamate neurotransmitter in the extracellular space after a stroke could activate cell death pathways. It is reported that progesterone provides different mechanisms of neuroprotection and could be considered as a candidate for stroke treatment. This study aimed to investigate progesterone impact on the expression of NMDA receptor subunits NR1, NR2 (A and B), NR3 (A and B) after an experimental model of ischemic stroke which is followed by an in silico analysis. MethodsProgesterone was introduced subcutaneously after transient middle cerebral artery occlusion in male rats. After a period of reperfusion, a set of behavioral tests was performed to evaluate the postischemic neurological deficits. The 2,3,5-triphenyltetrazolium chloride staining method was done for quantification of infarct volume and gene expression analysis was performed in the penumbra region using reverse transcription polymerase chain reaction for NMDA receptor subunits. An AutoDock tool was employed to perform molecular docking analyses for evaluation of progesterone interaction with NMDA receptor. ResultsCerebral ischemia caused a significant downregulation in NR1, NR2A, NR2B and a profound upregulation of NR3B in cortical penumbra region. Treatment with progesterone resulted in upregulation of NR1, NR2A, and NR3B which could explain a possible the neuroprotection of steroids via binding to NMDA glutamate receptor. In addition, in silico analysis revealed that progesterone could strongly interact with NR1/NR2B and NR2A. ConclusionThe findings elucidate a new aspect of the neuroprotective mechanism of progesterone via NMDA receptors gene regulation.

Early effects of 16O radiation on neuronal morphology and cognition in a murine model

Astronauts exposed to high linear energy transfer radiation may experience cognitive injury. The pathogenesis of this injury is unknown but may involve glutamate receptors or modifications to dendritic structure and/or dendritic spine density and morphology. Glutamate is the major excitatory neurotransmitter in the central nervous system, where it acts on ionotropic and metabotropic glutamate receptors located at the presynaptic terminal and in the postsynaptic membrane at synapses in the hippocampus. Dendritic spines are sites of excitatory synaptic transmission, and changes in spine structure and dendrite morphology are thought to be morphological correlates of altered brain function associated with hippocampal-dependent learning and memory. The aim of the current study is to assess whether behavior, glutamate receptor gene expression, and dendritic structure in the hippocampus are altered in mice after early exposure to 16O radiation in mice. Two weeks post-irradiation, animals were tested for hippocampus-dependent cognitive performance in the Y-maze. During Y-maze testing, mice exposed to 0.1 Gy and 0.25 Gy radiation failed to distinguish the novel arm, spending approximately the same amount of time in all 3 arms during the retention trial. Exposure to 16O significantly reduced the expression of Nr1 and GluR1 in the hippocampus and modulated spine morphology in the dentate gyrus and cornu Ammon 1 within the hippocampus. The present data provide evidence that 16O radiation has early deleterious effects on mature neurons that are associated with hippocampal learning and memory.

The effects of electroacupuncture at shu and mu points of stomach on gastric motility, the NMDA of vagus nerve dorsal nucleus and serum NO expression in functional dyspepsia rats

To research the central molecular mechanism of gastric motility in functional dyspepsia (FD) rats treated with electroacupuncture (EA) at shu and mu points of stomach. A total of 30 SD rats were randomized into a blank group, a model group, a Zhongwan+Weishu group, a Weishu group and a Zhongwan group, 6 rats in each group. FD rats were established by moderate clipping tail infuriation and irregular feeding except in the blank group. EA was used at "Zhongwan"(CV 12),"Weishu"(BL 21), and"Zhongwan"(CV 12) +"Weishu"(BL 21) in the corresponding groups for 7 days, once a day, and 20 min a time. No intervention was used in the blank and model groups. Grabbing and fixation were applied in the model group. Gastric antrum motion range and frequency were recorded by gastrointestinal pressure transducer. The expression of subunit NR1 of N-methyl-D-aspartate recepter (NMDAR) in dorsal motor nucleus of the vagus (DMV) was determined by Western blotting. The content of serum nitric oxide (NO) was measured by ELISA. Compared with the blank group, the gastric antrum motion range and NR1 in the DMV decreased and the serum NO content increased in the model group (all P<0.05). Compared with the model group, the gastric antrum motion range and NR1 in the DMV increased and the serum NO content decreased in the three EA groups (all P<0.05). Compared with the Zhongwan and Weishu groups, the gastric antrum motion range and NR1 in the DMV increased in the Zhongwan + Weishu group (all P<0.05). Compared with Zhongwan + Weishu and Zhongwan groups, the expression of NO in the Weishu group decreased (both P<0.05). The gastric antrum motion frequency among the 5 groups had no statistical significance (all P>0.05). EA at the shu and mu points can regulate the gastric motility in FD rats which may be by modulating the activity of NMDAR in the central DMV region, thus regulating the serum NO content.

Chronic ethanol exposure induces SK-N-SH cell apoptosis by increasing N-methyl-D-aspartic acid receptor expression and intracellular calcium.

It has been identified that chronic ethanol exposure damages the nervous system, particularly neurons. There is scientific evidence suggesting that neuronal loss caused by chronic ethanol exposure has an association with neuron apoptosis and intracellular calcium oscillation is one of the primary inducers of apoptosis. Therefore, the present study aimed to investigate the inductive effects of intracellular calcium oscillation on apoptosis in SK-N-SH human neuroblastoma cells and the protective effects of the N-methyl-D-aspartic acid receptor (NMDAR) antagonist, memantine, on SK-N-SH cell apoptosis caused by chronic ethanol exposure. SK-N-SH cells were treated with 100 mM ethanol and memantine (4 µM) for 2 days. Protein expression of NR1 was downregulated by RNA interference (RNAi). Apoptosis was detected by Annexin V/propidium iodide (PI) double-staining and flow cytometry and cell viability was detected using an MTS kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration and the levels of NR1 and caspase-3 were detected using western blotting. NR1 mRNA levels were also detected using qPCR. It was found that chronic ethanol exposure reduced neuronal cell viability and caused apoptosis of SK-N-SH cells, and the extent of damage in SK-N-SH cells was associated with ethanol exposure concentration and time. In addition, chronic ethanol exposure increased the concentration of intracellular calcium in SK-N-SH cells by inducing the expression of NMDAR, resulting in apoptosis, and memantine treatment reduced ethanol-induced cell apoptosis. The results of the present study indicate that the application of memantine may provide a novel strategy for the treatment of alcoholic dementia.

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.

Effect of decabrominated diphenyl ether exposure on spatial learning and memory, the expression and phosphorylation of hippocampal glutamate receptor subunits in adult Sprague-Dawley rats.

Previous studies have reported the potential developmental neurobehavioral effects of decabrominated diphenyl ethers (BDE 209) on developing animals, but the effects on adult animals are rare or controversial and the mechanism is not fully understood. In the present study, male adult Sprague-Dawley rats performed poor spatial learning and memory in Morris water maze after exposure to BDE 209 by gavage for 30 days. The expression of hippocampal glutamate receptor subunits NR1, NR2B and GluR1, the phosphorylation of NR2B subunit at Ser1301 (p-NR2B Ser1303) and GluR1 subunit at Ser831 (p-GluR1 Ser831) were all decreased, and the phosphorylation ratio of NR2B revealed an increasing trend after BDE 209 exposure. The present study provided evidence that BDE 209 could induce spatial learning and memory deficits in adult rats, and further explored the potential mechanism.

The protective effect of the activation of NMDAR1/ERK1/2 signal pathway induced by levodopa on visual cortical neurons in monocular deprivation rats

Objective: To investigate the protection effects of the activation of NMDAR1(NMDA receptor 1)/ERK1/2 signal pathway on visual cortex nerve cells induced by levodopa in amblyopia rats. Methods: SD rats of SPF grade, were randomly divided into for groups of 9, group A. Control, B. MD group, C. L-levodopa(20 mg·kg(-1))+MD group, D. H-levodopa (80 mg·kg(-1))+MD group, E. H-levodopa+MD+MK801 group, F. H-levodopa+MD+PD98059 group, G. MD+MK801 group, H. MD+PD98059 group, I. MD+DMSO group. Amblyopia rats were made by suture of the right eye. Levodopa was used to treatment amblyopia by gavage, and intervened by intracerebroventricular injection of MK801 and PD98059 respectively. The expression of NR1, p-ERK1/2, ERK1/2, NGF and c-FOS were detected by Western blotting. Nissl staining was used to detect morphological changes of neurons. Neuronal apoptosis was detected by TUNEL method, and detected the expression of Caspase-3, NGF and c-FOS by immunohistochemical staining. One/Two way Chi-square analysis was used for data analysis. LSD-t test was used as comparison between every two groups. Results: The morphology of Nissl bodies in neurons was complete and clear in A group, and the size of Nissl bodies got smaller, and caused karyopyknosis and loss of neurons in visual cortex of B group. Compared with A group, the apoptosis of visual cortical neurons(23.09±2.00 vs. 2.20±0.35, t=12.120, P=0.000) and the number of Caspase-3 postive cells (22.70±1.50 vs. 3.30±0.54, t=12.120, P=0.000)were significantly increased, the expression of NGF(0.31±0.04 vs. 0.74±0.09, t=7.674, P=0.000) and c-FOS(0.25±0.03 vs. 0.57±0.07, t=5.919, P=0.000) and the rats of NGF(8.30±0.82 vs. 35.18±2.01, t=12.37, P=0.0000) and c-FOS (10.84±1.02 vs. 35.68±2.55, t=9.056, P=0.0001) postive cell were decreased significantly in B group. After treatment with levodopa, the morphology of neurons recovered, the apoptosis of visual cortical neurons relieved, the expression of NR1(0.75±0.09 vs. 0.40±0.05, t=8.528, P=0.001) and p-ERK1/2(2.13±0.26 vs. 0.68±0.17, t=3.488, P=0.008) were increased significantly, and the rats of NGF (18.07±0.87 vs. 8.30±0.82, t=8.18, P=0.0000) and c-FOS (19.78±0.91 vs. 10.84±1.02, t=6.543, P=0.0001) postive cells were significantly increased. MK-801 or PD98059 intervention could effectively attenuate the effect of levodopa. It could effectively down-regulated the expression of NR1 (0.53±0.06 vs. 0.95±0.12, t=5.647, P=0.005) and p-ERK1/2(1.52±0.18 vs. 2.58±0.30, t=3.091, P=0.013) interference with MK801 or PD98059 in MD rats. MK-801 or PD98059 intervention further promote the Nissl body volume reduced, neurons karyopyknosis, the apoptosis of visual cortical neurons and Caspase-3 expression, and restrain the expression of NGF and c-FOS. Conclusion: Levodopa played a protective role in visual cortex nerve cells of amblyopia rats at least partially through activation of NMDA-ERK1/2 signal pathway. (Chin J Ophthalmol, 2017, 53: 931-940).

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.

CART neurons in the lateral hypothalamus communicate with the nucleus accumbens shell via glutamatergic neurons in paraventricular thalamic nucleus to modulate reward behavior.

Paraventricular thalamic nucleus (PVT) serves as a transit node processing food and drug-associated reward information, but its afferents and efferents have not been fully defined. We test the hypothesis that the CART neurons in the lateral hypothalamus (LH) project to the PVT neurons, which in turn communicate via the glutamatergic fibers with the nucleus accumbens shell (AcbSh), the canonical site for reward. Rats conditioned to self-stimulate via an electrode in the right LH-medial forebrain bundle were used. Intra-PVT administration of CART (55-102) dose-dependently (10-50ng/rat) lowered intracranial self-stimulation (ICSS) threshold and increased lever press activity, suggesting reward-promoting action of the peptide. However, treatment with CART antibody (intra-PVT) or MK-801 (NMDA antagonist, intra-AcbSh) produced opposite effects. A combination of sub-effective dose of MK-801 (0.01µg/rat, intra-AcbSh) and effective dose of CART (25ng/rat, intra-PVT) attenuated CART's rewarding action. Further, we screened the LH-PVT-AcbSh circuit for neuroadaptive changes induced by conditioning experience. A more than twofold increase was noticed in the CART mRNA expression in the LH on the side ipsilateral to the implanted electrode for ICSS. In addition, the PVT of conditioned rats showed a distinct increase in the (a) c-Fos expressing cells and CART fiber terminals, and (b) CART and vesicular glutamate transporter 2 immunostained elements. Concomitantly, the AcbSh showed a striking increase in expression of NMDA receptor subunit NR1. We suggest that CART in LH-PVT and glutamate in PVT-AcbSh circuit might support food-seeking behavior under natural conditions and also store reward memory.

The role and mechanism of glutamic NMDA receptor in the mechanical hyperalgesia in diabetic rats

Objectives: Some studies have shown that painful neuropathy is a common and costly complication of both type 1 and type 2 diabetes mellitus, and glutamate is involved in the process although the mechanisms are not clear. The purpose of the present study was to investigate the effect of N-methyl-D-aspartate (NMDA) receptor on mechanical hyperalgesia in diabetic rats and the possible mechanism. Methods: Diabetic rat model was established by intraperitoneal injection of streptozotocin (STZ, 1%, 70 mg/kg) once, and evaluated by the change in the fasting blood glucose. The mechanical hyperalgesia was estimated by mechanical withdrawal threshold (MWT) using a set of calibrated Von Frey’s filaments. In addition, the expressions of phosphorylated NMDA NR1 and phosphorylated cAMP response element binding protein (pCREB) in L4/L5 dorsal horns of spinal cord were observed. Results: Behavioral results showed that MK-801, an antagonist of NMDA receptor, could reduce the proportion of mechanical hyperalgesia in diabetic rats from 76.67 to 20.00%. Meanwhile, the mean MWTs in STZ group or saline-treated STZ group decreased significantly at 3–8 week, while, the MWTs in MK-801 treated STZ group were significant higher than those in STZ or saline-treated STZ group. In addition, the expressions of NMDA NR1 and pCREB in L4/5 dorsal horns of spinal cord were significant higher in diabetic rats, and MK-801 down-regulated their expressions partly. Conclusion: All these results suggested that NMDA receptor and pCREB in the spinal cord were involved in the regulation of mechanical hyperalgesia in diabetic rats.

Memantine Can Reduce Ethanol-Induced Caspase-3 Activity and Apoptosis in H4 Cells by Decreasing Intracellular Calcium.

Caspase-3 activation and apoptosis are associated with various neurodegenerative disorders. Calcium activation is an important factor in promoting apoptosis. We, therefore, assessed the role of intracellular calcium in ethanol-induced activation of caspase-3 in H4 human neuroglioma cells and the protective effect of the NMDA receptor antagonist, memantine, on ethanol-induced apoptosis in H4 cells. H4 cells were treated with 100mM EtOH (in culture medium) for 2days. For interaction studies, cells were treated with memantine (4μM), EDTA (1mM), or BAPTA-AM (10μM) before treatment with EtOH. Knockdown of the gene encoding the NR1 subunit of the NMDA receptor was performed using RNAi. Apoptosis was detected by Annexin V-FITC/PI staining and flow cytometry. Cell viability was detected using an MTS cell proliferation kit. Fluorescence dual wavelength spectrophotometry was used to determine the intracellular calcium concentration. The levels of NR1, caspase-3, IP3R1, and SERCA1 proteins were detected by western blotting. NR1, IP3R1, and SERCA1 mRNA levels were detected by qPCR. We observed increased expression of NR1, IP3R1, SERCA1, and increased intracellular levels of calcium ions in H4 cells exposed to ethanol. In addition, the calcium chelators, EDTA and BAPTA, and RNAi disruption of the NMDA receptor reduced ethanol-induced caspase-3 activation in H4 cells. Memantine treatment reduced the ethanol-induced increase of intracellular calcium, caspase-3 activation, apoptosis, and the ethanol-induced decrease in cell viability. Our results indicate that ethanol-induced caspase-3 activation and apoptosis are likely to be dependent on cytosolic calcium levels and that they can be reduced by memantine treatment.

The change in retinoic acid receptor signaling induced by prenatal marginal vitamin A deficiency and its effects on learning and memory

This study investigated the prenatal marginal vitamin A deficiency (mVAD)-related impairment in learning and memory and the interactions between RARα, Src and NR1. Learning and memory were assessed in adult rats that were exposed to prenatal mVAD with Morris water maze. The average escape time was longer in mVAD rats, and they passed the hidden platform fewer times during the memory retention test than normal vitamin A intake (VAN) rats. The mRNA and protein levels of RARα, Src and NR1 in mVAD rats were significantly lower than those in VAN rats. RARα and Src, but not NR1, were in the same protein complex. RA treatment-induced increase in RARα, Src and NR1 expressions in mVAD neurons was much lower than that in VAN neurons. Overexpression of RARα gene in VAN neurons induced an increase in RARα, Src and NR1 expressions, while silencing of RARα gene induced a decrease in expressions of RARα and Src, but not that of of NR1. In mVAD neurons, however, overexpression of RARα did not induce an increase in NR1 expression, while silencing of RARα gene had no effect on Src and NR1 expressions. Furthermore, inhibition of Src was associated with a decrease in NR1 expression but not that of RARα. Prenatal mVAD was associated with impaired learning and memory in adult rats. It is possible that mVAD-related decrease in RARα led to a decrease in Src expression, which in turn down-regulated NR1 expression and Ca2+ influx and eventually caused learning and memory deficits.

Prenatal exposure to lambda-cyhalothrin impairs memory in developing rats: Role of NMDA receptor induced post-synaptic signalling in hippocampus

Effect of prenatal exposure to lambda-cyhalothrin (LCT) has been assessed on the integrity of NMDA receptors and associated post-synaptic signalling in hippocampus of developing rats. Decrease in the binding of [3H]-MK 801, known to label NMDA receptors was observed in hippocampus of rats prenatally exposed to LCT (1 and 3mg/kg body weight) on PD22, compared to controls. Consistent with this, decrease in the mRNA and protein expression of NR1 and NR2B subunits of NMDA receptors was evident in rats prenatally exposed to LCT (1 and 3mg/kg body weight) on PD22. There was no change in mRNA and protein expression of NR2A subunit of NMDA receptors. Prenatal exposure to LCT (1 and 3mg/kg body weight) decreased the expression of positive regulators (PSD95, pERK1/2, CaMKIIα & pCREB) and increased the expression of negative regulators (Cdk5 & SynGAP) associated with NMDA receptor dependent synaptic plasticity in hippocampus and impaired learning and memory of rats on PD22. The neurobehavioral changes continued to persist in rats exposed to LCT at high dose (3mg/kg body weight) while exhibited trend of recovery in those exposed at moderate dose (1mg/kg body weight) on PD45, compared to controls. No change in any of the neurobehavioral endpoint was observed in developing rats prenatally exposed to LCT at low dose (0.5mg/kg body weight) on PD22 and PD45. The results exhibit that alterations in NMDA receptors on prenatal exposure to LCT may affect postsynaptic signalling associated with impaired learning and memory in developing rats.

The regulatory effect of electro-acupuncture on the expression of NMDA receptors in a SCI rat model

BackgroundIn early spinal cord injury (SCI), glutamate receptors, including N-methyl-d-aspartate (NMDA) receptors (NMDARs), are over-stimulated by excessively released glutamate. The enhanced activity of NMDARs may cause cell death by overloading calcium (Ca2+) into cells based on their high permeability to Ca2+. Studies in SCI animals have shown that treatment with electro-acupuncture (EA) is able to reduce cell death and to improve functional recovery. One possible mechanism of this neuroprotective effect is that EA has regulatory effect on NMDARs. AimsTo test whether EA could protect the spinal cord after SCI by decreasing the expression levels of NR1 and NR2A. Main methodsWe conducted EA treatment on a rat SCI model produced with a New York University (NYU) Impactor and measured hindlimb locomotor function by Basso, Beattie and Bresnahan Locomotor Rating Scale (BBB Scale). The expression of NR1 and NR2, the subunits of NMDARs, in the injured spinal cord was measured by Immunofluorescence stainings, western blot and real-time quantitative PCR (RT-qPCR). Key findingOur results showed that two days after the SCI the expression of NR1 and NR2 were dramatically enhanced at both protein and mNRA levels, which were significantly reduced by EA treatment at two specific acupoints, Dazhui (DU14) and Mingmen (DU4). SignificanceEA is a potential therapeutic method for treating early SCI in human.

Crack cocaine inhalation induces schizophrenia-like symptoms and molecular alterations in mice prefrontal cortex

Crack cocaine (crack) addiction represents a major social and health burden, especially seeing as users are more prone to engage in criminal and violent acts. Crack users show a higher prevalence of psychiatric comorbidities – particularly antisocial personality disorders – when compared to powder cocaine users. They also develop cognitive deficits related mainly to executive functions, including working memory. It is noteworthy that stimulant drugs can induce psychotic states, which appear to mimic some symptoms of schizophrenia among users. Social withdraw and executive function deficits are, respectively, negative and cognitive symptoms of schizophrenia mediated by reduced dopamine (DA) tone in the prefrontal cortex (PFC) of patients. That could be explained by an increased expression of D2R short isoform (D2S) in the PFC of such patients and/or by hypofunctioning NMDA receptors in this region. Reduced DA tone has already been described in the PFC of mice exposed to crack smoke. Therefore, it is possible that behavioral alterations presented by crack users result from molecular and biochemical neuronal alterations akin to schizophrenia. Accordingly, we found that upon crack inhalation mice have shown decreased social interaction and working memory deficits analogous to schizophrenia's symptoms, along with increased D2S/D2L expression ratio and decreased expression of NR1, NR2A and NR2B NMDA receptor subunits in the PFC. Herein we propose two possible mechanisms to explain the reduced DA tone in the PFC elicited by crack consumption in mice, bringing also the first direct evidence that crack use may result in schizophrenia-like neurochemical, molecular and behavioral alterations.

Influence of occupational aluminum exposure on cognitive function and glutamate receptor protein expression in workers

Objective: To investigate the influence of occupational aluminum exposure on cognitive function and glutamate receptor protein expression in peripheral blood lymphocytes in workers and the possibility of glutamate receptor being used as a biomarker for cognitive impairment in aluminum workers. Methods: From October to December, 2014, cluster sampling was performed to select 121 workers in aluminum electrolysis workshop as exposure group and 231 workers in thermoelectric workshop and logistics department as control group. Mini-Mental State Examination, clock drawing test, digit span test (DST) , verbal fluency test (VFT) , and Fuld Object-Memory (FOM) Evaluation were used to analyze cognitive function. Graphite furnace atomic absorption spectrophotometry was used to measure plasma aluminum level as an exposure indicator. Enzyme-linked immunosorbent assay was used to measure the content of glutamate receptor proteins in peripheral blood lymphocytes, including the subunits of N-methyl-D-aspartate receptor NR1, NR2A, and NR2B and metabotropic glutamate receptor 1 (mGluR1) . The correlation between cognitive function indices and the content of glutamate receptor proteins was analyzed. Results: There was no significant difference in plasma aluminum level between the control group and the exposure group (132.52±80.40 μg/L vs 182.88±72.32 μg/L, P>0.05) . According to the plasma aluminum level, the study subjects were divided into control group and low-, medium-, and high-level plasma aluminum groups, and there were significant differences in plasma aluminum level between these groups (all P<0.01) . The high-level plasma aluminum group had a significantly lower memory ability score than the control group and the low- and medium-level plasma aluminum groups (all P<0.05) . The high-level plasma aluminum group had lower DST and digital span forward (DSF) scores than the control group and the low-and medium-level plasma aluminum groups. The low-, medium-, and high-level plasma aluminum groups had lower digital span backward (DSB) scores than the control group. The medium-and high-level plasma aluminum groups had lower VFT scores than the control group and the low-level plasma aluminum group. The high-level plasma aluminum group had significantly lower expression of NR1 and NR2A proteins than the control group and the low-and medium-level plasma aluminum groups, and the medium- and high-level plasma aluminum groups had significantly higher expression of mGluR1 protein than the control group and the low-level plasma aluminum group (all P<0.05) . The expression of NR1 and NR2A proteins was negatively correlated with plasma aluminum level (r=-0.475 and -0.692, both P<0.05) , andthe expression of mGluR1 protein was positively correlated with plasma aluminum level (r=0.756, P<0.05) . The expression of NR1 protein was positively correlated with DSF, DSB, DST, and VFT scores (r(s)=0.213, 0.249, 0.271, and 0.228, all P<0.05) , and the expression of NR2A protein was positively correlated with VFT score (r(s)=0.206, P<0.05) . Conclusion: Occupational aluminum exposure may affect workers' memory function, and the expression of NR1 and NR2A in peripheral blood lymphocytes is correlated with cognitive function indices and can be used as biomarkers for cognitive impairment in aluminum workers.