NMDA Receptor Subunit NR2B Research Articles

Acute administration of toluene affects memory retention in novel object recognition test and memory function‐related gene expression in mice

The present study was designed to investigate the acute effect of a single administration of toluene (300 mg kg(-1), i.p.) on memory retention in the hippocampus-dependent novel object recognition test and N-methyl-D-aspartate (NMDA) receptor subunit expression in the hippocampus of C3H/HeN female mice using real-time RT-PCR. We performed a novel object recognition test including a habituation phase, training phase and test phase in each mouse. Twenty-four hours after the training phase, to determine the effect of acute toluene administration on memory retention, half of the mice (n=10) were injected with toluene 60 min before the test phase. Toluene-injected mice did not prefer novel objects and showed poor discrimination between novel and familiar objects and decreased expression of NMDA receptor subunit NR2B mRNA in the hippocampus. This is the first study to show that acute toluene injection impairs hippocampus-dependent nonspatial memory retention accompanied by selective modulation of NMDA receptor subunit expression.

NR2B subunit of NMDA receptor at nucleus accumbens is involved in morphine rewarding effect by siRNA study

BackgroundChronic use of morphine causes rewarding effects and behavioral sensitization, which may lead to the development of craving for morphine. A number of studies indicate that the NMDA receptors may be involved in these effects, especially the NR2B-containing NMDA receptors. It is also well recognized that the nucleus accumbens (NAc) and the ventral tegmental area (VTA) are involved in drug addiction, including morphine addiction. AimsIn this study, we further investigate the role of the NR2B subunit of NMDA receptors at NAc or VTA in morphine rewarding effects and behavioral sensitization. Methods and ResultsThe siRNA against the NR2B subunit of NMDA receptors was locally injected to decrease the expression of NR2B at NAc or posterior VTA in male Sprague-Dawley (S.D.) rats in the present study. The rats were then treated with morphine chronically. A conditioned place preference (CPP) test was used to examine the rewarding effect, and locomotor activity was measured to determine the behavioral sensitization induced by chronic morphine treatment. Results showed that morphine-induced rewarding behavior but not behavioral sensitization was abolished when the NR2B subunit of NMDA receptors at the NAc were significantly decreased. The dopamine turnover rate was not altered by the decrease of NR2B subunit at NAc. ConclusionThese findings suggest that the NR2B subunit of NMDA receptors at the NAc is involved in morphine-induced rewarding effect and may not be through directly interacting with dopamine neurons.

The analgesic effects of intrathecal administration of Ro 25-6981 on incision pain in rats

Objective To investigate the analgesic effects of intrathecal administration of Ro 25-6981, a selective antagonist of the NR2B subunit of NMDA receptors (NR2B), in a rat model of incision pain. Methods Sixty SD rats were randomly divided into 4 groups: control group (C); incisional pain group (Ⅰ); intrathecal group of Ro 25-6981 at the dose of 50 μg/25 μl(R1);intrathecal group of Ro 25-6981 at the dose of 200 μg/25 μl(R2). Intrathecal injection of 5% DMSO solvent at the dose of 25 μl was administrated in group C and Ⅰ. Rat models of incisional pain on the right hind were prepared after intrathecal injection in group Ⅰ,R1 and R2. All rats were anesthetized with sevoflurane. The 50% paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were tested to evaluate the behavioral changes and measured at 24 h before incision and at 2,6,24 h after incision. And the locomotor functions were also examined at the same time points. Results Compared with group C and baseline,the decreases of 50% PWMT (7.0±0.9), (6.7±0.7), (6.4±1.2) g and PWTL (10.3±1.4), ( 10. 1±1.0), (10.0±1.0) s were observed at each time point after incision in group Ⅰ (P＜0.05); Compared with group Ⅰ, significant changes of 50% PWMT and PWTL were not observed at each time point in group R1 (P＞0.05); Compared with group Ⅰ, increases of both 50% PWMT (9.0±0.8) g and PWTL (13.7±1.0) s were observed at 2 h after incision in group R2 (P＜0.05), while no significant changes of 50% PWMT and PWTL were observed at 6 h and 24 h after incision in group R2. No significant changes in locomotor functions were observed before and after intrathecal administration in rats (P＞0.05). Conclusion Intrathecal injection of Ro 25-6981 at the dose of 200.0 μg, the selective antagonist of the NR2B, exerts significant analgesic effects on incision pain in rats without affecting of locomotor functions. Key words: Ro 25-6981; NMDA receptor subunit NR2B; Incision pain; Intrathecal injection

Effects of NMDA receptor modulators on a blood–brain barrier in vitro model

Changes of the functionality of the blood–brain barrier (BBB) have been reported in the context of several brain related diseases such as multiple sclerosis, epilepsy, Alzheimer's disease and stroke. Several publications indicated the presence and functionality of the NMDA receptor (NMDAR) at the brain endothelium and a possible involvement of the NMDAR in the above-mentioned diseases. Recently, it was shown that the application of the NMDAR antagonist MK801 can block several adverse effects at the BBB in vitro, but also that MK801 can significantly change the proteome of brain endothelial cells without simultaneous stimulation of NMDAR by glutamate. Based on these reports we investigated if NMDAR antagonists MK801 and D-APV can affect the intracellular calcium level (Ca2+i) of an in vitro BBB model based on human cell line ECV304 on their own and compared these results to effects mediated by NMDAR agonists glutamate and NMDA. Treatment of ECV304 cells for 30min with glutamate resulted in no significant change of Ca2+i. On the contrary, application of NMDA and NMDAR antagonists D-APV and MK801 led to a significant and concentration dependent decrease of Ca2+i. Further studies revealed that glutamate was able to decrease the transendothelial electrical resistance (TEER) of the BBB in vitro model, whereas NMDA and D-APV were able to increase TEER. Analysis of the protein expression levels of tight junctional molecules ZO-1 and occludin showed a complex regulation after application of NMDAR modulators. In summary, it was shown that NMDAR antagonists can alter BBB key properties in vitro on their own. Moreover, although qPCR results confirmed the presence of NMDA receptor subunits NR1, NR2A, NR2B and NR2C, membrane binding studies failed to prove the typical plasma membrane localization and functionality in human BBB cell line ECV304.

Death-Associated Protein Kinase 1 Phosphorylates Pin1 and Inhibits Its Prolyl Isomerase Activity and Cellular Function

Pin1 is a phospho-specific prolyl isomerase that regulates numerous key signaling molecules and whose deregulation contributes to disease notably cancer. However, since prolyl isomerases are often believed to be constitutively active, little is known whether and how Pin1 catalytic activity is regulated. Here, we identify death-associated protein kinase 1 (DAPK1), a known tumor suppressor, as a kinase responsible for phosphorylation of Pin1 on Ser71 in the catalytic active site. Such phosphorylation fully inactivates Pin1 catalytic activity and inhibits its nuclear location. Moreover, DAPK1 inhibits the ability of Pin1 to induce centrosome amplification and cell transformation. Finally, Pin1 pSer71 levels are positively correlated with DAPK1 levels and negatively with centrosome amplification in human breast cancer. Thus, phosphorylation of Pin1 Ser71 by DAPK1 inhibits its catalytic activity and cellular function, providing strong evidence for an essential role of the Pin1 enzymatic activity for its cellular function.

Dissecting the Contribution of Individual Receptor Subunits to the Enhancement of N-methyl-d-Aspartate Currents by Dopamine D1 Receptor Activation in Striatum

Neurons in the primary visual cortex receive subliminal information originating from the periphery of their receptive fields (RF) through a variety of cortical connections. In the cat primary visual cortex, long-range horizontal axons have been reported to preferentially bind to distant columns of similar orientation preferences, whereas feedback connections from higher visual areas provide a more diverse functional input. To understand the role of these lateral interactions, it is crucial to characterize their effective functional connectivity and tuning properties. However, the overall functional impact of cortical lateral connections, whatever their anatomical origin, is unknown since it has never been directly characterized. Using direct measurements of postsynaptic integration in cat areas 17 and 18, we performed multi-scale assessments of the functional impact of visually driven lateral networks. Voltage-sensitive dye imaging showed that local oriented stimuli evoke an orientation-selective activity that remains confined to the cortical feedforward imprint of the stimulus. Beyond a distance of one hypercolumn, the lateral spread of cortical activity gradually lost its orientation preference approximated as an exponential with a space constant of about 1 mm. Intracellular recordings showed that this loss of orientation selectivity arises from the diversity of converging synaptic input patterns originating from outside the classical RF. In contrast, when the stimulus size was increased, we observed orientation-selective spread of activation beyond the feedforward imprint. We conclude that stimulus-induced cooperativity enhances the long-range orientation-selective spread.

Prenatal PCBs disrupt early neuroendocrine development of the rat hypothalamus

Neonatal exposure to endocrine disrupting chemicals (EDCs) such as polychlorinated biphenyls (PCBs) can interfere with hormone-sensitive developmental processes, including brain sexual differentiation. We hypothesized that disruption of these processes by gestational PCB exposure would be detectable as early as the day after birth (postnatal day (P) 1) through alterations in hypothalamic gene and protein expression. Pregnant Sprague–Dawley rats were injected twice, once each on gestational days 16 and 18, with one of the following: DMSO vehicle; the industrial PCB mixture Aroclor 1221 (A1221); a reconstituted mixture of the three most prevalent congeners found in humans, PCB138, PCB153, and PCB180; or estradiol benzoate (EB). On P1, litter composition, anogenital distance (AGD), and body weight were assessed. Pups were euthanized for immunohistochemistry of estrogen receptor α (ERα) or TUNEL labeling of apoptotic cells or quantitative PCR of 48 selected genes in the preoptic area (POA). We found that treatment with EB or A1221 had a sex-specific effect on developmental apoptosis in the neonatal anteroventral periventricular nucleus (AVPV), a sexually dimorphic hypothalamic region involved in the regulation of reproductive neuroendocrine function. In this region, exposed females had increased numbers of apoptotic nuclei, whereas there was no effect of treatment in males. For ERα, EB treatment increased immunoreactive cell numbers and density in the medial preoptic nucleus (MPN) of both males and females, while A1221 and the PCB mixture had no effect. PCR analysis of gene expression in the POA identified nine genes that were significantly altered by prenatal EDC exposure, in a manner that varied by sex and treatment. These genes included brain-derived neurotrophic factor, GABAB receptors-1 and -2, IGF-1, kisspeptin receptor, NMDA receptor subunits NR2b and NR2c, prodynorphin, and TGFα. Collectively, these results suggest that the disrupted sexual differentiation of the POA by prenatal EDC exposures is already evident as early as the day after birth, effects that may change the trajectory of postnatal development and compromise adult reproductive function.

Cognitive dysfunction and prefrontal synaptic abnormalities in a mouse model of fragile X syndrome

Among the hallmark phenotypes reported in individuals with fragile X syndrome (FXS) are deficits in attentional function, inhibitory control, and cognitive flexibility, a set of cognitive skills thought to be associated with the prefrontal cortex (PFC). However, despite substantial clinical research into these core deficits, the PFC has received surprisingly little attention in preclinical research, particularly in animal models of FXS. In this study, we sought to investigate the molecular, cellular, and behavioral consequences of the loss of the fragile X mental retardation protein in the PFC of Fmr1 KO mice, a mouse model of FXS. We identify a robust cognitive impairment in these mice that may be related to the deficits in cognitive flexibility observed in individuals with FXS. In addition, we report that levels of proteins involved in synaptic function, including the NMDA receptor subunits NR1, NR2A, and NR2B; the scaffolding proteins PSD-95 and SAPAP3; and the plasticity-related gene Arc, are decreased in the prefrontal cortex of Fmr1 KO mice and are partly correlated with behavioral performance. Finally, we report that expression of c-Fos, a marker of neuronal activity, is decreased in the PFC of Fmr1 KO mice. Together, these data suggest that Fmr1 KO mice may represent a valuable animal model for the PFC-associated molecular, cellular, and behavioral abnormalities in FXS and that this model may be useful for testing the efficacy of therapeutic strategies aimed at treating the cognitive impairments in FXS.

Lack of Beneficial Effect for Preemptive Analgesia in Postoperative Pain Control

In previous animal studies, preemptive treatments with N-methyl-d-aspartate (NMDA) antagonists were ineffective at preventing incision-induced allodynia. It is very likely that the model was not clinically relevant for testing treatment effects on postoperative pain. The beneficial effects of preemptive treatment can be verified only by treatments with a pharmacologically proven effect in a specific pain type or animal model. We previously showed that NMDA receptor antagonists effectively alleviate enhanced mechanical hyperalgesia after plantar incision in adult rats that had been given an intraplantar injection of carrageenan as neonates. Here, using this modified model, we tested the efficacy of preemptive treatment with the NMDA antagonist MK-801. We injected rat pups subcutaneously with 0.25% carrageenan or saline in the plantar surface of one hindpaw on postnatal day 1. On postnatal day 50, rats were killed and the ipsilateral side of the lumbar spinal cords were harvested for biochemical analysis of the expression of NR2A and NR2B at baseline, 2 hours, 4 hours, 8 hours, and 24 hours after plantar incision (n = 5 per group for each time point). For pharmacological study, rats were allocated into one of the following groups: 1 intrathecal injection of 40 nmol MK-801 15 minutes before plantar incision, 1 intrathecal injection 30 minutes after plantar incision, or 2 injections of 20 nmol or 40 nmol given at 15 minutes and 60 minutes after plantar incision (n = 10 per group for neonatally saline-treated and 12 for carrageenan-treated rats). Paw withdrawal thresholds were measured with von Frey filaments, and weight-bearing percentages were measured hourly after plantar incision. Expressions of NMDA receptor subunits NR2A and NR2B were increased maximally 4 hours postoperatively and were significantly greater in carrageenan-treated rats than in saline-treated rats. Tests of pain sensitivity showed that MK-801 significantly alleviated the incision-induced mechanical hyperalgesia and increased weight-bearing percentage on the injured paw in carrageenan-treated rats. However, preincisional treatment was not superior to postincisional treatment as assessed during the 6-hour postoperative observation period. Groups with 2 successive postoperative injections exhibited prolonged analgesic effects. Only the group that received 2 postoperative injections and increased total dosage had improved analgesic indices. Under conditions of proven analgesic action of an NMDA antagonist, we demonstrated that preincisional treatment is not more beneficial than postincisional treatment for postoperative pain relief in the modified animal model. Increasing the duration of administration and/or total dosage had an incremental analgesic effect in comparison with a single injection.

Dissecting the contribution of individual receptor subunits to the enhancement of N-methyl-D-aspartate currents by dopamine D1 receptor activation in striatum

Dopamine, via activation of D1 receptors, enhances N-methyl-d-aspartate (NMDA) receptor-mediated responses in striatal medium-sized spiny neurons. However, the role of specific NMDA receptor subunits in this enhancement remains unknown. Here we used genetic and pharmacological tools to dissect the contribution of NR1 and NR2A/B subunits to NMDA responses and their modulation by dopamine receptors. We demonstrate that D1 enhancement of NMDA responses does not occur or is significantly reduced in mice with genetic knock-down of NR1 subunits, indicating a critical role of these subunits. Interestingly, spontaneous and evoked α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptor-mediated responses were significantly enhanced in NR1 knock-down animals, probably as a compensatory mechanism for the marked reduction in NMDA receptor function. The NMDA receptor subunits NR2A and NR2B played differential roles in D1 modulation. Whereas genetic deletion or pharmacological blockade of NR2A subunits enhanced D1 potentiation of NMDA responses, blockade of NR2B subunits reduced this potentiation, suggesting that these regulatory subunits of the NMDA receptor counterbalance their respective functions. In addition, using D1 and D2 receptor EGFP-expressing mice, we demonstrate that NR2A subunits contribute more to NMDA responses in D1-MSSNs, whereas NR2B subunits contribute more to NMDA responses in D2 cells. The differential contribution of discrete receptor subunits to NMDA responses and dopamine modulation in the striatum has important implications for synaptic plasticity and selective neuronal vulnerability in disease states.

Phenylbutyrate rescues dendritic spine loss associated with memory deficits in a mouse model of Alzheimer disease

Alzheimer's disease (AD) and ageing are associated with impaired learning and memory, and recent findings point toward modulating chromatin remodeling through histone acetylation as a promising therapeutic strategy. Here we report that systemic administration of the HDAC inhibitor 4-phenylbutyrate (PBA) reinstated fear learning in the Tg2576 mouse model of AD. Tg2576 mice develop age-dependent amyloid pathology and cognitive decline that closely mimics disease progression in humans. Memory reinstatement by PBA was observed independently of the disease stage: both in 6-month-old Tg2576 mice, at the onset of the first symptoms, but also in aged, 12- to 16-month-old mice, when amyloid plaque deposition and major synaptic loss has occurred. Reversal of learning deficits was associated to a PBA-induced clearance of intraneuronal Aβ accumulation, which was accompanied by mitigation of endoplasmic reticulum (ER) stress, and to restoration of dendritic spine densities of hippocampal CA1 pyramidal neurons to control levels. Furthermore, the expression of plasticity-related proteins such as the NMDA receptor subunit NR2B and the synaptic scaffold SAP102 was significantly increased by PBA. Our data suggest that the beneficial effects of PBA in memory are mediated both via its chemical chaperone-like activity and via the transcriptional activation of a cluster of proteins required for the induction of synaptic plasticity and structural remodeling.

Cyclic Adenosine Monophosphate–Independent Tyrosine Phosphorylation of NR2B Mediates Cocaine-Induced Extracellular Signal-Regulated Kinase Activation

Activation of the extracellular signal-regulated kinase (ERK) in the striatum is crucial for long-term behavioral alterations induced by drugs of abuse. In response to cocaine, ERK phosphorylation (i.e., activation) is restricted to medium-sized spiny neurons expressing dopamine D1 receptor (D1R) and depends on a concomitant stimulation of D1R and glutamate N-methyl-D-aspartate receptor (NMDAR). However, the mechanisms responsible for this activation, especially the respective contribution of D1R and NMDAR, remain unknown. We studied striatal neurons in culture stimulated with D1R agonist and/or glutamate and wild-type or genetically modified mice treated with cocaine. Biochemical, immunohistochemical, and imaging studies were performed. Mice were also subjected to behavioral experiments. Stimulation of D1R cannot activate ERK by itself but potentiates glutamate-mediated calcium influx through NMDAR that is responsible for ERK activation. Potentiation of NMDAR by D1R depends on a cyclic adenosine monophosphate-independent signaling pathway, which involves tyrosine phosphorylation of the NR2B subunit of NMDAR by Src family kinases. We also demonstrate that the D1R/Src family kinases/NR2B pathway is responsible for ERK activation by cocaine in vivo. Inhibition of this pathway abrogates cocaine-induced locomotor sensitization and conditioned place preference. Our results show that potentiation of NR2B-containing NMDAR by D1R is necessary and sufficient to trigger cocaine-induced ERK activation. They highlight a new cyclic adenosine monophosphate-independent pathway responsible for the integration of dopamine and glutamate signals by the ERK cascade in the striatum and for long-term behavioral alterations induced by cocaine.

Arrested Glutamatergic Synapse Development in Human Partial Epilepsy

While studying the brain function of the human partial epilepsy gene, leucine-rich glioma-inactivated 1 (LGI1), a new mechanism of human epileptogenesis was revealed-persistent immaturity of glutamatergic circuitries. LGI1, a novel secreted protein, was found to be increased during the postnatal period; when glutamatergic synapses both downregulate their presynaptic vesicular release probability and reduce their postsynaptic NMDA-receptor subunit NR2B. During this same period, the dendritic arbor and spines are pruned and remodeled. Using bacterial artificial chromosome transgenic mouse techniques, excess wild-type LGI1 was shown to magnify these critical brain developmental events in the hippocampal dentate gyrus; while an epilepsy-associated, truncated, dominant-negative form of LGI1 blocked them. By contrast, the hippocampal dentate granule neuron GABAergic synapses and intrinsic excitability were unaltered. A role for LGI1 in downregulating glutamate synapse function was confirmed by germline gene deletion; this intervention also revealed a selective increase of glutamatergic synaptic transmission with unaltered GABAergic synapses and intrinsic excitability of hippocampal CA1 pyramidal neurons. Interestingly, the role of LGI1 in neurological disease was further expanded when a subset of patients with limbic encephalitis (an autoimmune disorder with memory loss in 100% and seizures in 80% of individuals) were discovered to carry autoantibodies to LGI1.

Mutations in GRIN2A and GRIN2B encoding regulatory subunits of NMDA receptors cause variable neurodevelopmental phenotypes

N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly Ca²(+)-permeable cation channels which are blocked by extracellular Mg²(+) in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2A(N615K) (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg²(+) block and a decrease in Ca²(+) permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.

Allosteric modulation of the NMDA receptor by neurosteroids in rat brain and the impact of long term morphine administration

This study examined the allosteric modulation of the NMDA receptor by nanomolar concentrations of neurosteroids in rats treated long term with morphine. The neurosteroids dehydroepiandrosterone sulfate (DHEAS), pregnenolone sulfate (PS) and pregnanolone sulfate (3α5βS) are important mediators in the central nervous system. They induce rapid responses by non-classical steroidal mechanisms, e.g. via interaction with the N-methyl- d-aspartate (NMDA) receptor, and are known to modify the binding of ifenprodil to the NMDA receptor subunit NR2B. The NMDA receptor is involved in several processes, including memory, learning, synaptic plasticity and neuronal development. Morphine, a μ-opioid receptor agonist, has an important role in the clinical treatment of pain. The main drawback of morphine treatment is the associated development of dependence and tolerance. The mechanisms behind these phenomena are still to be elucidated, but several reports suggest the involvement of the NMDA receptor. The results of the present study indicate that the allosteric modulation induced by the neurosteroids DHEAS, PS and 3α5βS was similar in all tested brain regions. This suggests that the NR2B receptor subunit behaves independently of its site of expression. Moreover, the NR2B subunit was up-regulated in the frontal cortex but not in the hippocampus or hypothalamus. It is concluded that morphine does not affect the neurosteroid modulatory effect on ifenprodil binding in the rat hippocampus or hypothalamus but does significantly affect both the expression of the NR2B subunit and the 3α5βS modulatory effect on ifenprodil binding in the frontal cortex. It is suggested that the observed effect of long term morphine on the properties of NR2B in the frontal cortex may be associated with the mechanism underlying the development of opiate dependence.

The basolateral amygdala is critical for the acquisition and extinction of associations between a neutral stimulus and a learned danger signal but not between two neutral stimuli.

We studied the neural substrates of higher-order conditioned fear in rats. We first studied acquisition and extinction of second-order fear. One stimulus (S1) was paired with a footshock unconditioned stimulus (US), a second stimulus (S2) was paired with S1, and, finally, fear of S2 was extinguished by S2 alone exposures. Reversible inactivation of the basolateral amygdala (BLA) via muscimol, systemic injection of the NMDA receptor (NMDAr) antagonist MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate], or intra-amygdala infusion of the NMDAr NR2B subunit selective antagonist ifenprodil impaired both acquisition and extinction of fear to S2. We then studied acquisition and extinction of sensory preconditioned fear. S2 was first paired with S1, S1 was then paired with the US, and, finally, fear of S2 was extinguished by S2 alone exposures. Extinction of sensory preconditioned fear was impaired by a BLA infusion of muscimol or ifenprodil and by MK-801. Acquisition of the S2-S1 association and extinction of this association by S2 alone exposures before conditioning of S1 was impaired by MK-801 but not by a BLA infusion of muscimol. These results show that NMDAr activation in the BLA is critical for acquisition of second-order conditioned fear and for the extinction of both second-order and sensory preconditioned fear just as it is for acquisition and extinction of first-order conditioned fear. Acquisition and extinction of an association between two neutral stimuli also require NMDAr activation. However, the present results show that the acquisition and extinction of these associations do not require the BLA.

Bisphenol-A rapidly promotes dynamic changes in hippocampal dendritic morphology through estrogen receptor-mediated pathway by concomitant phosphorylation of NMDA receptor subunit NR2B

Bisphenol-A (BPA) is known to be a potent endocrine disrupter. Evidence is emerging that estrogen exerts a rapid influence on hippocampal synaptic plasticity and the dendritic spine density, which requires activation of NMDA receptors. In the present study, we investigated the effects of BPA (ranging from 1 to 1000 nM), focusing on the rapid dynamic changes in dendritic filopodia and the expressions of estrogen receptor (ER) β and NMDA receptor, as well as the phosphorylation of NMDA receptor subunit NR2B in the cultured hippocampal neurons. A specific ER antagonist ICI 182,780 was used to examine the potential involvement of ERs. The results demonstrated that exposure to BPA (ranging from 10 to 1000 nM) for 30 min rapidly enhanced the motility and the density of dendritic filopodia in the cultured hippocampal neurons, as well as the phosphorylation of NR2B (pNR2B), though the expressions of NMDA receptor subunits NR1, NR2B, and ERβ were not changed. The antagonist of ERs completely inhibited the BPA-induced increases in the filopodial motility and the number of filopodia extending from dendrites. The increased pNR2B induced by BPA (100 nM) was also completely eliminated. Furthermore, BPA attenuated the effects of 17β-estradiol (17β-E 2) on the dendritic filopodia outgrowth and the expression of pNR2B when BPA was co-treated with 17β-E 2. The present results suggest that BPA, like 17β-E 2, rapidly results in the enhanced motility and density of dendritic filopodia in the cultured hippocampal neurons with the concomitant activation of NMDA receptor subunit NR2B via an ER-mediated signaling pathway. Meanwhile, BPA suppressed the enhancement effects of 17β-E 2 when it coexists with 17β-E 2. These results provided important evidence suggesting the neurotoxicity of the low levels of BPA during the early postnatal development of the brain.

Alterations of NMDA receptor subunits NR1, NR2A and NR2B mRNA expression and their relationship to apoptosis following transient forebrain ischemia

Glutamate excitotoxicity mediated by NMDA receptor activation plays a key role in many aspects of ischemic brain injury, but the expression of NMDA receptor subunits NR1, NR2A and NR2B mRNA and their relationship to apoptosis is still unclear. In this study, we applied in situ hybridization and TUNEL staining to investigate the expression of NMDA receptor subunit mRNA and apoptosis in hippocampus of rats after transient forebrain ischemia. The results showed that in the CA1 region, NR1 mRNA expression was significantly increased following ischemia–reperfusion (IR), reaching peak levels at IR 24 h, and then gradually decreasing until IR 7 days. NR2A and NR2B mRNA expression dropped to lowest levels at IR 6 h and IR 12 h, respectively, and then started to recover. The mRNA expression of both NR2A and NR2B then increased to peak levels at IR 48 h, followed by a sustained decline until IR 7 days. In the CA3 region and dentate gyrus the range of variation in mRNA expression was significantly reduced gradually. At IR 24 h, apoptosis-positive cells were observed mainly in the CA1 region. The number of apoptosis-positive cells continuously grew and showed a dramatic increase at IR 48 h and peaked at IR 72 h. Then, the number of apoptosis-positive cells started to decrease, but at IR 7 days the apoptosis-positive cells still remained. These results indicate that the alterations of NMDA receptor subunit mRNA expression may contribute to the ischemic apoptosis of hippocampus after transient forebrain ischemia.

Amphetamine regulates NR2B expression in Go2α knockout mice and thereby sustains behavioral sensitization

The α-subunit of Go2 is a regulator of dopamine (DA) homeostasis. Deletion of the protein results in an imbalance of the direct and indirect DA pathway by reducing D1 and increasing D2 receptors. As a result, cocaine-induced behavioral sensitization is abolished. Here we show that repeated amphetamine injections in Go2α-/- mice induced a similar D1/D2 receptor ratio shift as cocaine but surprisingly the knockouts developed normal behavioral sensitization. DA receptor signaling following either cocaine or amphetamine treatment was also similar in Go2α-/- mice suggesting another mechanism involved in the differential behavioral response. Evidence is increasing that DA-glutamate interactions in the striatum determine psychostimulant action. In this line, repeated amphetamine injections led to a twofold increase in the amount of the NMDA receptor subunit NR2B in Go2α-/- mice resulting in an enhanced inhibition of the indirect DA pathway. This effect is not seen after cocaine treatment. Furthermore, amphetamine but not cocaine treatment maintained the ratio between the glutamate receptor mGluR1/5 interacting proteins Homer and Homer1a in the knockouts thereby sustaining the direct pathway. We conclude that amphetamine provokes behavioral sensitization in Go2α-/- mice by an enhanced inhibition of the indirect pathway without disturbing the direct pathway thereby overcoming the imbalance in the DArgic system.

Exercise increases insulin signaling in the hippocampus: Physiological effects and pharmacological impact of intracerebroventricular insulin administration in mice

Increasing evidence indicates that physical exercise induces adaptations at the cellular, molecular, and systemic levels that positively affect the brain. Insulin plays important functional roles within the brain that are mediated by insulin-receptor (IR) signaling. In the hippocampus, insulin improves synaptic plasticity, memory formation, and learning via direct modulation of GABAergic and glutamatergic receptors. Separately, physical exercise and central insulin administration exert relevant roles in cognitive function. We here use CF1 mice to investigate (i) the effects of voluntary exercise on hippocampal insulin signaling and memory performance and (ii) whether central insulin administration alters the effects of exercise on hippocampal insulin signaling and memory performance. Adult mice performed 30 days of voluntary exercise on running wheel and afterward both, sedentary and exercised groups, received intracerebroventricular (icv) injection of saline or insulin (0.5-5 mU). Memory performance was assessed using the inhibitory avoidance and water maze tasks. Hippocampal tissue was measured for [U-(14)C] glucose oxidation and the immunocontent of insulin receptor/signaling (IR, pTyr, pAktser473). Additionally, the phosphorylation of the glutamate NMDA receptor NR2B subunit and the capacity of glutamate uptake were measured, and immunohistochemistry was used to determine glial reactivity. Exercise significantly increased insulin peripheral sensitivity, spatial learning, and hippocampal IR/pTyrIR/pAktser473 immunocontent. Glucose oxidation, glutamate uptake, and astrocyte number also increased relative to the sedentary group. In both memory tasks, 5 mU icv insulin produced amnesia but only in exercised animals. This amnesia was associated a rapid (15 min) and persistent (24 h) increase in hippocampal pNR2B immunocontent that paralleled the increase in glial reactivity. In conclusion, physical exercise thus increased hippocampal insulin signaling and improved water maze performance. Overstimulation of insulin signaling in exercised animals, however, via icv administration impaired behavioral performance. This effect was likely the result of aberrant phosphorylation of the NR2B subunit.