NR1 Protein Expression Research Articles

Distinct behavioral effects of short-term voluntary running in phosphatase and tensin homolog deleted on chromosome 10 neuronal haploinsufficient mice

Phosphatase and tensin homolog deleted on chromosome ten (PTEN) is a tumor suppressor with functions related to its phosphatase activity. PTEN plays various roles, such as cell proliferation, survival, and migration and is involved in neurogenesis and synaptic plasticity in the central nervous system. It has been reported that running could have protective effects against the aging process and neurodegenerative diseases. Therefore, we aimed to evaluate the effects of physical exercise on behavioral and biochemical aspects of PTEN-conditioned knockout female mice. We observed that 10 days of voluntary running positively affected fear memory but caused no changes in anxiety-like behavior. However, it was unable to counteract the social recognition memory deficit in PTEN neuronal haploinsufficient mice. In terms of biochemical aspects, we observed that short-term running reduced S6 phosphorylation in PTEN heterozygous mice and PTEN protein expression independent of the genotype. In addition, PTEN heterozygous mice presented reduced N-methyl-D-aspartate subunit NR1 protein expression. Our results regarding decreased S6 phosphorylation in HT mice suggest that short-term voluntary running could have induced a protective effect in reducing dysregulated cell growth, possibly related to the downregulation of tumor suppressor expression/activity such as PTEN. Moreover, running induced distinct behavioral effects in PTEN-conditioned knockout mice.

Developmental regulation and lateralization of N-methyl-d-aspartate (NMDA) receptors in the rat hippocampus

N-methyl-d-aspartate receptors (NMDARs) are expressed abundantly in the brain and play a crucial role in the regulation of central nervous system (CNS) development, learning, and memory. During early neuronal development, NMDARs modulate neurogenesis, neuronal differentiation and migration, and synaptogenesis. The present study aimed to examine the developmental expression of NMDARs subunits, NR1 and NR2B, in the developing hippocampus of neonatal rats during the first two postnatal weeks. Fifty-four male offspring were randomly divided into three age groups, postnatal days (P) 0, 7, and 14. Real-time-PCR, western blotting, and immunohistochemistry (IHC) analyses were employed to examine and compare the hippocampal expression of the NMDA receptor subunits. The highest mRNA expression of NR1 and NR2B subunits was observed at P7, regardless of its laterality. The mRNA expression of both subunits in the right hippocampus was significantly higher than that of the left one at P0 and P7. Similarly, the highest protein level expression of NR1 and NR2B subunits was also observed at P7 in both sides hippocampi. Although the protein expression of NR1 was significantly higher on the right side in all studied days, the NR2B was significantly higher in the right hippocampus only at P7. The analysis of optical density (OD) has shown a marked increase in the distribution pattern of the NR1 and NR2B subunits at P7 in all hippocampal subregions. In conclusion, there is a marked right-left asymmetry in the expression of NR1 and NR2B subunits in the developing rat hippocampus, which might be considered as a probable mechanism for the lateral differences in the structure and function of the hippocampus in rats.

An Effective Phytoconstituent Aconitine: A Realistic Approach for the Treatment of Trigeminal Neuralgia.

Trigeminal neuralgia pain remains a challenge to treat. Natural compounds may be promising options for relieving pain. This study was aimed at investigating the effects of aconitine in a rat model of trigeminal neuralgia pain. Infraorbital nerve chronic constriction injury was performed in adult Wistar Albino rats. After the neuropathic pain developed, the rats were assigned to one of the treatment groups: carbamazepine 40 or 80 mg/kg; aconitine 0.25, 0.50, or 0.75 mg/kg; or saline injection (control group). Behavioral testing with von Frey filaments and the rotarod test were carried out before the surgical procedure and on the 24th to 29th postoperative days. Following the completion of tests, ipsilateral and contralateral spinal cords were harvested for Western blot analyses to assess NR-1 protein expression. ANOVA followed by Mann-Whitney U test was performed for the statistical analyses. P values of <0.05 were considered significant. Aconitine significantly reduced mechanical sensitivity in a dose-dependent manner. A significant reduction in motor coordination was noted for the higher doses of aconitine which was similar with the 40 and 80 mg/kg doses of carbamazepine. NR-1 expression was reduced in the ipsilateral spinal cord, whereas no significant difference was noted between the groups in the expression of NR-1 in the contralateral spinal cord. Aconitine had a significant pain relieving effect, which was similar to carbamazepine, in a dose-dependent manner. Aconitine may be an alternative pharmacological agent for the control of trigeminal neuralgia pain.

Effect of acute exposure to hypobaric hypoxia on learning and memory in adult Sprague-Dawley rats

The present study aimed to observe the effects of acute exposure to hypobaric hypoxia on learning and memory in adult Sprague-Dawley (SD) rats as well as the changes in N-methyl-D-aspartate receptor (NMDAR) subunits. Learning and memory abilities were evaluated with Morris water maze following simulated hypoxia at 5000 m or 7000 m for 2, 4 or 6 days. The number of red blood cells, the level of hemoglobin, oxidative stress markers, the extent of neuronal damage in the hippocampal CA1, and the expression of NMDAR subunits were all measured. In place navigation test, the escape latency significantly increased only in the 5000 M 6Day (P < 0.05) group than the latency in the plain group on the fourth day. In the spatial probe test, the times of platform passing showed no significant difference between the hypoxia and plain groups. Polycythemia, an increased ratio of superoxide dismutase/malondialdehyde and degeneration of neurons appeared in an elevation-dependent and duration-dependent manner in those hypoxia groups. Furthermore, the protein expression of NR1 increased and the protein expression levels of NR2A, NR2B, and NR3A were all decreased in the 5000M 6Day and 7000M 6Day groups. In summary, the feeble effect of acute exposure to simulated hypobaric hypoxia on learning and memory in adult SD rats may be attributed to increased antioxidative capacities and decreased expression of NR3A. And moreover, differences in the expressions of NMDAR subunits were closely related to the altitude and duration.

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.

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.

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.

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.

A lasting effect of postnatal sevoflurane anesthesia on the composition of NMDA receptor subunits in rat prefrontal cortex

Sevoflurane is widely used in pediatric anesthesia and studies have shown that it is capable of inducing neurodegeneration and subsequent cognitive disorders in the developing brain. However, the evidence that anesthetics are toxic to the human brain is insufficient. N-Methyl-d-aspartate (NMDA) receptors, critical for learning and memory, display expression changes with age and can be modulated by inhalation anesthetics. Generally, NMDA receptor (NR) type 1 is expressed at birth, peaks around the third postnatal week, and then declines slightly to adult levels. NR2Bs slowly decrease and NR2As gradually increase during postnatal development. These developmental switches of NMDA receptor subunits composition mark the transition from immature to adult neural processing and allow for the final maturation of associative learning abilities. In this study, we aimed to evaluate the effect of repeated sevoflurane anesthesia on NMDA receptor subunits composition in the developing rat brain and related behavioral disorders. Six-day-old male Sprague Dawley rats were randomly allocated into either a control group (group con) or a sevoflurane group (group sevo). Group sevo inhaled 2.1% sevoflurane carried by 70% oxygen for 2h each day from postnatal day (PND) 6 to PND 8. The same procedure, without applying the sevoflurane, was executed in group con. The membrane protein expression of NR1, NR2A and NR2B in the prefrontal cortex (PFC) and hippocampus was assessed at the end of the three days of anesthesia and at PND 21. An open field test was carried out to assess spontaneous locomotion on PNDs 21, 28 and 35. Y maze performance was used to assess attention and working memory on PND 28. Sevoflurane induced upregulation of NR1 and NR2B in the PFC at the end of anesthesia. On PND 21, NR1 and NR2B receptors were significantly increased whereas NR2A receptors were significantly decreased in the PFC in group sevo. Sevoflurane-treated rats showed hyper-locomotion and impairment of working memory in the behavior tests. These results indicate that repeated sevoflurane anesthesia at early stage of life can induce a long lasting effect of interfering with NMDA receptor subunits composition in rat PFC. These changes may contribute to the effects of sevoflurane on neuronal development and subsequent neurobehavioral disorders.

Enhanced NMDA receptor-mediated modulation of excitatory neurotransmission in the dorsal vagal complex of streptozotocin-treated, chronically hyperglycemic mice.

A variety of metabolic disorders, including complications experienced by diabetic patients, have been linked to altered neural activity in the dorsal vagal complex. This study tested the hypothesis that augmentation of N-Methyl-D-Aspartate (NMDA) receptor-mediated responses in the vagal complex contributes to increased glutamate release in the dorsal motor nucleus of the vagus nerve (DMV) in mice with streptozotocin-induced chronic hyperglycemia (i.e., hyperglycemic mice), a model of type 1 diabetes. Antagonism of NMDA receptors with AP-5 (100 μM) suppressed sEPSC frequency in vagal motor neurons recorded in vitro, confirming that constitutively active NMDA receptors regulate glutamate release in the DMV. There was a greater relative effect of NMDA receptor antagonism in hyperglycemic mice, suggesting that augmented NMDA effects occur in neurons presynaptic to the DMV. Effects of NMDA receptor blockade on mEPSC frequency were equivalent in control and diabetic mice, suggesting that differential effects on glutamate release were due to altered NMDA function in the soma-dendritic membrane of intact afferent neurons. Application of NMDA (300 μM) resulted in greater inward current and current density in NTS neurons recorded from hyperglycemic than control mice, particularly in glutamatergic NTS neurons identified by single-cell RT-PCR for VGLUT2. Overall expression of NR1 protein and message in the dorsal vagal complex were not different between the two groups. Enhanced postsynaptic NMDA responsiveness of glutamatergic NTS neurons is consistent with tonically-increased glutamate release in the DMV in mice with chronic hyperglycemia. Functional augmentation of NMDA-mediated responses may serve as a physiological counter-regulatory mechanism to control pathological disturbances of homeostatic autonomic function in type 1 diabetes.

Suppression of neurite outgrowth of primary cultured hippocampal neurons is involved in impairment of glutamate metabolism and NMDA receptor function caused by nanoparticulate TiO2

Numerous studies have indicated that nano-titanium dioxide (TiO2) can induce neurotoxicity in vitro and in vivo, however, it is unclear whether nano-TiO2 affects neurite outgrowth of hippocampal neurons. In order to investigate the mechanism of neurotoxicity, rat primary cultured hippocampal neurons on the fourth day of culture were exposed to 5, 15, and 30 μg/mL nano-TiO2 for 24 h, and nano-TiO2 internalization, dendritic growth, glutamate metabolism, expression of N-methyl-d-aspartate (NMDA) receptor subunits (NR1, NR2A and NR2B), calcium homeostasis, sodium current (INa) and potassium current (IK) were examined. Our findings demonstrated that nano-TiO2 crossed the membrane into the cytoplasm or nucleus, and significantly suppressed dendritic growth of primary cultured hippocampal neurons in a concentration-dependent manner. Furthermore, nano-TiO2 induced a marked release of glutamate to the extracellular region, decreased glutamine synthetase activity and increased phosphate-activated glutaminase activity, elevated intracellular calcium ([Ca2+]i), down-regulated protein expression of NR1, NR2A and NR2B, and increased the amplitudes of the INa and IK. In addition, nano-TiO2 increased nitric oxide and nitrice synthase, attenuated the activities of Ca2+-ATPase and Na+/K+-ATPase, and increased the ADP/ATP ratio in the primary neurons. Taken together, these findings indicate that nano-TiO2 inhibits neurite outgrowth of hippocampal neurons by interfering with glutamate metabolism and impairing NMDA receptor function.

Rostrocaudal changes in the protein expression of NMDA receptor subunits within the rostral ventrolateral medulla of sedentary and active rats (875.12)

Recently we found enhanced sympathoexcitatory responses to glutamate in the RVLM following sedentary conditions. We have also reported increased dendritic branching in more rostral compared to caudal regions within the RVLM of sedentary rats. The mechanisms by which physical inactivity increases glutamatergic neurotransmission and produces structural changes in more rostral regions of the RVLM is unclear. The NMDA subtype of glutamate receptor has been associated with neuroplasticity in other brain regions of sedentary versus active rats. We hypothesized that sedentary conditions would alter protein expression of NMDA receptor subunits in a rostrocaudal manner within the RVLM. To test this hypothesis, male Sprague Dawley rats were allowed to remain sedentary (n=4) or exercise voluntarily via running wheels (n=4) for 14 weeks. Animals were then sacrificed and the RVLM cryosectioned (80 μm) and micropunched bilaterally. Sections were stained using cresyl violet to identify the rostrocaudal boundary of the RVLM. Based on their location relative to the caudal pole of the facial nucleus (FN), punches were pooled into four regions: caudal ‐480 µm (FN‐480), caudal ‐240 µm (FN‐240), rostral +240 µm (FN+240) and rostral +480 µm (FN+480). Preliminary western blot results showed that within the RVLM of sedentary rats, NR1 protein expression normalized to actin was significantly higher at FN‐480 (2.6 ± 0.2) compared to FN+240 (1.6 ± 0.2, p &lt; 0.005). Interestingly, within the caudal regions of the active rats, NR1 protein expression was significantly lower at FN‐480 (1.4 ± 0.2) compared to FN‐240 (2.3 ± 0.2, p &lt; 0.005). Changes were not observed for NR2A protein expression within both groups. Rostrocaudal differences observed in sedentary rats were not consistent with our structural findings. However, it is important to further investigate these novel findings, including examination of other receptor subunits which could relate to the structural neuroplasticity observed in sedentary rats.Grant Funding Source: R01HL096787

Glutamate receptor composition of the post-synaptic density is altered in genetic mouse models of NMDA receptor hypo- and hyperfunction

The N-methyl- d-aspartate receptor (NMDAR) and α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) are ionotropic glutamate receptors responsible for excitatory neurotransmission in the brain. These excitatory synapses are found on dendritic spines, with the abundance of receptors concentrated at the postsynaptic density (PSD). We utilized two genetic mouse models, the serine racemase knockout (SR−/−) and the glycine transporter subtype 1 heterozygote mutant (GlyT1+/−), to determine how constitutive NMDAR hypo- and hyperfunction, respectively, affect the glutamate receptor composition of the PSD in the hippocampus and prefrontal cortex (PFC). Using cellular fractionation, we found that SR−/− mice had elevated protein levels of NR1 and NR2A NMDAR subunits specifically in the PSD-enriched fraction from the hippocampus, but not from the PFC. There were no changes in the amounts of AMPAR subunits (GluR1, GluR2), or PSD protein of 95 kDa (PSD95) in either brain region. GlyT1+/− mice also had elevated protein expression of NR1 and NR2A subunits in the PSD, as well as an increase in total protein. Moreover, GlyT1+/− mice had elevated amounts of GluR1 and GluR2 in the PSD, and higher total amounts of GluR1. Similar to SR−/− mice, there were no protein changes observed in the PFC. These findings illustrate the complexity of synaptic adaptation to altered NMDAR function.

Prenatal stress in rat causes long-term spatial memory deficit and hippocampus MRI abnormality: Differential effects of postweaning enriched environment

Prenatal stress (PS) can cause long-term hippocampus alternations in structure and plasticity in adult offspring. Enriched environment (EE) has an effect in rescuing a variety of neurological disorders. Pregnant dams were left undisturbed (prenatal control, PC) or restrained 6h per day from days 14 to 21 (prenatal stress, PS). Control and prenatal stressed offspring rats were subjected to a standard rearing environment (SE) or an EE on postnatal days 22–120 (PC/SE PC/EE, PS/SE, and PS/EE; n=5, each group). At ∼4 months of age, all rats underwent Morris water maze test and brain MRI examination. Hippocampi were then dissected for biochemical analyses, including, Western blot for NMDA receptor (NR) subunits and synaptophysin and RT-PCR forβ1 integrin and tissue-plasminogen activator (t-PA). MRI showed all 5 rats in the PS/SE group and 5 in the PS/EE group exhibited increased signals in bilateral hippocampus and increased T2 time in the PS/SE group. Exposure to EE treatment on postnatal days 22–120 counteracted the deficit in spatial memory and increased NR1 protein expression, but it did not affect the rate of high signals and increased T2 time, decreased NR2, synaptophysin, β1 integrin and t-PA mRNA expressions in PS adult offspring. The results of this study indicate PS in rats causes long-term spatial memory deficits and gross hippocampus pathology. Postnatal EE treatment has differential benefits in terms of spatial learning, signaling molecules, and gross hippocampus pathology.

Chronic AT1receptor blockade normalizes NMDA-mediated changes in renal sympathetic nerve activity and NR1expression within the PVN in rats with heart failure

Exercise training normalizes enhanced glutamatergic mechanisms within the paraventricular nucleus (PVN) concomitant with the normalization of increased plasma ANG II levels in rats with heart failure (HF). We tested whether ANG II type 1 (AT(1)) receptors are involved in the normalization of PVN glutamatergic mechanisms using chronic AT(1) receptor blockade with losartan (Los; 50 mg.kg(-1).day(-1) in drinking water for 3 wk). Left ventricular end-diastolic pressure was increased in both HF + vehicle (Veh) and HF + Los groups compared with sham-operated animals (Sham group), although it was significantly attenuated in the HF + Los group compared with the HF + Veh group. The effect of Los on cardiac function was similar to exercise training. At the highest dose of N-methyl-d-aspartate (NMDA; 200 pmol) injected into the PVN, the increase in renal sympathetic nerve activity was 93 +/- 13% in the HF + Veh group, which was significantly higher (P < 0.05) than the increase in the Sham + Veh (45 +/- 2%) and HF + Los (47 +/- 2%) groups. Relative NMDA receptor subunit NR(1) mRNA expression within the PVN was increased 120% in the HF + Veh group compared with the Sham + Veh group (P < 0.05) but was significantly attenuated in the HF + Los group compared with the HF + Veh group (P < 0.05). NR(1) protein expression increased 87% in the HF + Veh group compared with the Sham + Veh group but was significantly attenuated in the HF + Los group compared with the HF + Veh group (P < 0.05). Furthermore, in in vitro experiments using neuronal NG-108 cells, we found that ANG II treatment stimulated NR(1) protein expression and that Los significantly ameliorated the NR(1) expression induced by ANG II. These data are consistent with our hypothesis that chronic AT(1) receptor blockade normalizes glutamatergic mechanisms within the PVN in rats with HF.

The N-methyl- d-aspartate receptor in heart development: A gene knockdown model using siRNA

Antagonists of the N-methyl- d-aspartate receptor (NMDAR) may disrupt the development of the cardiac neural crest (CNC) and contribute to conotruncal heart defects. To test this interaction, a loss-of-function model was generated using small interfering RNAs (siRNA) directed against the critical NR1-subunit of this receptor in avian embryos. The coding sequence of the chicken NR1 gene and predicted protein sequences were characterized and found to be homologous with other vertebrate species. Analysis of its spatiotemporal expression demonstrated its expression within the neural tube at pre-migratory CNC sites. siRNA targeted to the NR1-mRNA in pre-migratory CNC lead to a significant decrease in NR1 protein expression. However, embryo survival and heart development were not adversely affected. These results indicate that the CNC may function normally in the absence of functional NMDAR, and that NMDAR antagonists may have a complex impact upon the CNC that transcends impairment of a single receptor type.

Oxidative stress upregulates the NMDA receptor on cerebrovascular endothelium

N-methyl- d-aspartate receptor (NMDA-R)-mediated oxidative stress has been implicated in blood–brain barrier (BBB) disruption in a variety of neuropathological diseases. Although some interactions between both phenomena have been elucidated, possible influences of reactive oxygen species (ROS) on the NMDA-R itself have so far been neglected. The objective of this study was to examine how the cerebroendothelial NMDA-R is affected by exposure to oxidative stress and to assess possible influences on BBB integrity. RT-PCR confirmed several NMDA-R subunits (NR1, NR2B–D) expressed in the bEnd3 cell line (murine cerebrovascular endothelial cells). NR1 protein expression after exposure to ROS was observed via in-cell Western. The functionality of the expressed NMDA-R was determined by measuring DiBAC fluorescence in ROS-preexposed cells upon stimulation with the specific agonist NMDA. Finally, the effects on barrier integrity were evaluated using the ECIS system to detect changes in monolayer impedance upon NMDA-R stimulation after exposure to ROS. The expression of NR1 significantly ( p < 0.001) increased 72 h after 30 min exposure to superoxide (+ 33.8 ± 7.5%), peroxynitrite (+ 84.9 ± 10.7%), or hydrogen peroxide (+ 92.8 ± 7.6%), resulting in increased cellular response to NMDA-R stimulation and diminished monolayer impedance. We conclude that oxidative stress upregulates NMDA-R on cerebrovascular endothelium and thus heightens susceptibility to glutamate-induced BBB disruption.

Relationship between NR1 subunit of N-methyl-D-aspartate receptor and visceral hypersensitivity caused by acute restraint stress: experiment with rats

To investigate the response of NR1 subunit of N-methyl-D-aspartate (NMDA) receptor to restraint stress in intestinal tract. Thirty Sprague-Dawley rats were randomly divided into 3 equal groups: acute stress group undergoing intraperitoneal injection of normal saline (NS), acute stress + MK-801 group, undergoing intraperitoneal injection of MK-801, a NMDA receptor inhibitor, and then acute restraint for 1 h, 30 min after the injection. Control group underwent intraperitoneal injection of NS without acute restraint to be used as. Then all rats underwent colorectal distension (CRD) at the pressure levels of 20, 40, 60, and 80 mm Hg respectively for 10 s 3 times with an interval of 40 s so as to establish visceral hypersensitivity models. Electromyography was performed at the external oblique muscle of abdomen to record the frequency of discharge to evaluate the visceral sensitivity. Then the rats were killed and specimens of ileocecum and the proximal and distal ends of colon were obtained. RT-PCR and Western blotting were used to detect the mRNA and protein expression of NMDA receptor NR1 subunit in intestinal tract. (1) The frequencies of discharge of external oblique muscle of abdomen responding to CRD of the acute stress group at the pressure levels of 40, 60, and 80 mm Hg were 925 +/- 217, 1480 +/- 347, and 1732 +/- 344 respectively, all significantly higher than those of the control group (188 +/- 31, 510 +/- 68, and 765 +/- 103, all P < 0.01) and the acute stress + MK-801 group (210 +/- 47, 525 +/- 97, 841 +/- 156, all P < 0.05). But no significantly different from the acute stress + MK-801 group and the control group (all P > 0.05). (2) The A values of the mRNA expression of NR1 in the ileocecal junction, proximal colon end, and distal colon end of the acute stress group were 1.57 +/- 0.20, 2.00 +/- 0.20, and 1.36 +/- 0.17 respectively, all significantly higher than those of the control group (0.68 +/- 0.10, 0.87 +/- 0.19, and 0.74 +/- 0.15, all P < 0.01) and the acute stress + MK-801 group (0.84 +/- 0.13, 0.91 +/- 0.16, 0.79 +/- 0.13, all P < 0.05). But no significantly different from the acute stress + MK-801 group and the control group (all P > 0.05). (3) The A values of the protein expression of NR1 in the ileocecal junction, proximal colon end, and distal colon end of the acute stress group were 1.69 +/- 0.20, 1.41 +/- 0.12, and 1.63 +/- 0.15 respectively, all significantly higher than those of the control group (0.54 +/- 0.11, 0.71 +/- 0.06, 0.71 +/- 0.07, all P = 0.000) and the acute + MK-801 group (0.75 +/- 0.09, 0.70 +/- 0.11, 0.63 +/- 0.11, all P = 0.000). But no significantly different from the acute stress + MK-801 group and the control group (all P > 0.05). NMDA receptor plays an important role in the visceral sensitivity induced by acute restraint stress.

Estrogen alters spinal NMDA receptor activity via a PKA signaling pathway in a visceral pain model in the rat

Pain symptoms in several chronic pain disorders in women, including irritable bowel syndrome, fluctuate with the menstrual cycle suggesting a gonadal hormone component. In female rats, estrogens modulate visceral sensitivity although the underlying mechanism(s) are unknown. In the present study the effects of 17-β estradiol on N-methyl- d-aspartate (NMDA) receptor signaling of colorectal nociceptive processing in the spinal cord were examined. Estrogen receptor alpha and the NR1 subunit of the NMDA receptor are co-expressed in dorsal horn neurons, supporting a direct action of estradiol on NMDA receptors. Intrathecal administration of the NMDA receptor antagonist d(−)-2-amino-5-phosphonopentanoic acid (APV) dose-dependently attenuated the visceromotor response with greater potency in ovariectomized (OVx) rats compared to OVx with estradiol replacement (E2) rats. Estradiol significantly increased protein expression of NR1 in the lumbosacral spinal cord compared to OVx rats. Colorectal distention significantly increased phosphorylation of NR1ser-897, a PKA phosphorylation site on the NR1 subunit in E2, but not OVx rats. Intrathecal administration of a PKA inhibitor significantly attenuated the visceromotor response, decreased NR1 phosphorylation and increased the potency of APV to attenuate the visceromotor response compared to vehicle-treated E2 rats. These data suggest that estradiol increases spinal processing of visceral nociception by increasing NMDA receptor NR1 subunit expression and increasing site-specific receptor phosphorylation on the NR1 subunit contributing to an increase in NMDA receptor activity.