NR2A Expression Research Articles

Postsynaptic density-93 deficiency protects cultured cortical neurons from N-methyl- d-aspartate receptor-triggered neurotoxicity

It has been reported that N-methyl- d-aspartate receptor (NMDAR)-triggered neurotoxicity is related to excessive Ca 2+ loading and an increase in nitric oxide (NO) concentration. However, the molecular mechanisms that underlie these events are not completely understood. NMDARs and neuronal NO synthase each binds to the scaffolding protein postsynaptic density (PSD)-93 through its PDZ domains. In this study, we determined whether PSD-93 plays a critical role in NMDAR/Ca 2+/NO-mediated neurotoxicity. We found that the targeted disruption of the PSD-93 gene attenuated the neurotoxicity triggered by NMDAR activation, but not by non-NMDAR activation, in cultured mouse cortical neurons. PSD-93 deficiency reduced the amount of NMDAR subunits NR2A and NR2B in synaptosomal fractions from the cortical neurons and significantly prevented NMDA-stimulated increases in cyclic guanosine 3′,5′-monophosphate and Ca 2+ loading in the cortical neurons. These findings indicate that PSD-93 deficiency could block NMDAR-triggered neurotoxicity by disrupting the NMDAR-Ca 2+-NO signaling pathway and reducing expression of synaptic NR2A and NR2B. Since NMDARs, Ca 2+, and NO play a critical role during the development of brain trauma, seizures, and ischemia, the present work suggests that PSD-93 might contribute to molecular mechanisms of neuronal damage in these brain disorders.

Laser microdissection and pressure catapulting: Combining LMPC with IHC to investigate NMDA receptor subunit composition

Laser microdissection and pressure catapulting (LMPC) has allowed investigators the opportunity to look at enriched cell populations from heterogeneous tissues. Selection of cells is routinely based upon morphological or histological criteria. The inclusion of an immunohistochemistry step prior to LMPC potentially allows investigators to classify cell populations both morphologically and functionally. Practical limitations with regard to RNA integrity following IHC steps have meant that the value that this combination brings has been reduced. Here, through the use of RNA preserving buffers, we have been able to successfully label, dissect and extract RNA from NK1 expressing cells. RT-PCR was carried out to confirm the expression of NK1, NR2A and NR2B subunit expression. Our data confirmed the expression of NK1 in cells labelled with the antibody, and the absence of expression in cells absent of staining. As well as this, relative expression of both NR2A and NR2B was determined. Furthermore, the RNA was of high enough quality to allow these methods to be used for studies involving RNA amplification steps, such as microarray analysis.

Reduced expression of glutamate receptors and phosphorylation of CREB are responsible for in vivoΔ9‐THC exposure‐impaired hippocampal synaptic plasticity

Chronic use of marijuana impairs synaptic plasticity and cognitive function. However, the molecular mechanisms by which marijuana alters long-term synaptic plasticity are largely unknown. Here, we show that repeated in vivo exposures to Delta9-THC for 7 consecutive days significantly impaired hippocampal long-term potentiation (LTP) of excitatory glutamatergic synaptic transmission. The Delta9-THC exposure-induced decrease in LTP was prevented by pharmacological inhibition or deletion of the cannabinoid 1 receptor (CB1R). To determine the molecular mechanisms underlying Delta9-THC-altered LTP, we targeted expression and function of the glutamate receptors (GluR) and phosphorylation status of cAMP-response element-binding protein (CREB). Chronic in vivo exposure to Delta9-THC produced CB1R-dependent decreases in expression of hippocampal GluR1, NR2A, and NR2B, the ratio of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/NMDA receptor-gated currents, and phosphorylation of CREB. Our results suggest that reduced expression and function of the GluR subunits and phosphorylation of CREB may underlie the impaired long-term synaptic plasticity induced by repeated in vivo exposure to Delta9-THC.

Combined actions of Na+/K+-ATPase, NCX1 and glutamate dependent NMDA receptors in ischemic rat brain penumbra

Instrumental role of Na+ and Ca2+ influx via Na+/K+ adenosine triphosphatase (Na+/K+-ATPase) and Na+/Ca2+ exchanger 1 (NCX1) is examined in the N-Methyl-D-aspartate (NMDA) receptor-mediated pathogenesis of penumbra after focal cerebral ischemia. An experimental model of 3, 6, and 24 h focal cerebral ischemia by permanent occlusion of middle cerebral artery was developed in rats. The changes in protein expression of Na+/K+-ATPase and NCX1 as well as functional subunits of NMDA receptor 2A and 2B (NR2A and NR2B) in the penumbra were assessed using by quantitative immunoblottings. The most prominent changes of Na+/K+-ATPase (78±6%, n=4, *P<0.05) and NCX1 (144±2%, n=4, *P<0.05) in the penumbra were developed 24 h after focal cerebral ischemia. The expression of NR2A in the penumbra was significantly increased (153±9%, n=4, *P<0.05) whereas the expression of NR2B was significantly decreased (37±2%, n=4, *P<0.05) as compared with sham-operated controls 3 h after focal cerebral ischemia. However, the expression of NR2A and NR2B in the penumbra was reversed 24 h after focal cerebral ischemia (NR2A: 40±7%; NR2B: 120±16%, n=4, *P<0.05). Moreover, the decreased expression of neuronal nuclei (NeuN) in the penumbra was most prominent than that of glial fibrillary acidic protein (GFAP) 24 h after focal cerebral ischemia. These findings imply that intracellular Na+ accumulation via decreased Na+/K+-ATPase exacerbate the Ca2+ overload cooperated by the increased NCX1 and NR2B-containing NMDA receptor which may play an important role in the pathogenesis of the penumbra.

Dysbindin regulates hippocampal LTP by controlling NMDA receptor surface expression

Abnormalities in NMDA receptor (NMDAR) function have been implicated in schizophrenia. Here, we show that dysbindin, a schizophrenia-susceptibility gene widely expressed in the forebrain, controls the surface expression of NMDARs in a subunit-specific manner. Imaging analyses revealed a marked increase in surface NR2A, but not NR2B, in hippocampal neurons derived from dysbindin-null mutant mice (Dys-/-). Exogenous expression of dysbindin reduced NR2A surface expression in both wild-type and Dys-/- neurons. Biotinylation experiments also revealed an increase in surface expression of endogenous NR2A in Dys-/- neurons. Disruption of the dysbindin gene dramatically increased NR2A-mediated synaptic currents, without affecting AMPA receptor currents, in hippocampal CA1 neurons. The Dys-/- hippocampal slices exhibited an enhanced LTP, whereas basal synaptic transmission, presynaptic properties, and LTD were normal. Thus, dysbindin controls hippocampal LTP by selective regulation of the surface expression of NR2A. These results reveal subunit-specific regulation of NMDARs by dysbindin, providing an unexpected link between these two proteins implicated in schizophrenia.

Differential Effects of Chronic Ethanol Consumption and Withdrawal on Homer/Glutamate Receptor Expression in Subregions of the Accumbens and Amygdala of P Rats

Homer proteins are constituents of scaffolding complexes that regulate the trafficking and function of central Group1 metabotropic glutamate receptors (mGluRs) and N-methyl-d-aspartate (NMDA) receptors. Research supports the involvement of these proteins in ethanol-induced neuroplasticity in mouse. In this study, we examined the effects of short versus long-term withdrawal from chronic ethanol consumption on Homer and glutamate receptor protein expression within striatal and amygdala subregions of selectively bred, alcohol-preferring P rats. For 6 months, male P rats had concurrent access to 15% and 30% ethanol solutions under intermittent (IA: 4 d/wk) or continuous (CA: 7 d/wk) access conditions in their home cage. Rats were killed 24 hours (short withdrawal: SW) or 4 weeks (long withdrawal: LW) after termination of ethanol access, subregions of interest were micropunched and tissue processed for detection of Group1 mGluRs, NR2 subunits of the NMDA receptor and Homer protein expression. Within the nucleus accumbens (NAC), limited changes in NR2a and NR2b expression were detected in the shell (NACsh), whereas substantial changes were observed for Homer2a/b, mGluRs as well as NR2a and NR2b subunits in the core (NACc). Within the amygdala, no changes were detected in the basolateral subregion, whereas substantial changes, many paralleling those observed in the NACc, were detected in the central nucleus (CeA) subregion. In addition, most of the changes observed in the CeA, but not NACc, were present in both SW and LW rats. Overall, these subregion specific, ethanol-induced increases in mGluR/Homer2/NR2 expression within the NAC and amygdala suggest changes in glutamatergic plasticity had taken place. This may be a result of learning and subsequent memory formation of ethanol's rewarding effects in these brain structures, which may, in part, mediate the chronic relapsing nature of alcohol abuse.

Docosahexaenoic acid promotes hippocampal neuronal development and synaptic function

Docosahexaenoic acid (DHA, 22:6n-3), the major polyunsaturated fatty acid accumulated in the brain during development, has been implicated in learning and memory, but underlying cellular mechanisms are not clearly understood. Here, we demonstrate that DHA significantly affects hippocampal neuronal development and synaptic function in developing hippocampi. In embryonic neuronal cultures, DHA supplementation uniquely promoted neurite growth, synapsin puncta formation and synaptic protein expression, particularly synapsins and glutamate receptors. In DHA-supplemented neurons, spontaneous synaptic activity was significantly increased, mostly because of enhanced glutamatergic synaptic activity. Conversely, hippocampal neurons from DHA-depleted fetuses showed inhibited neurite growth and synaptogenesis. Furthermore, n-3 fatty acid deprivation during development resulted in marked decreases of synapsins and glutamate receptor subunits in the hippocampi of 18-day-old pups with concomitant impairment of long-term potentiation, a cellular mechanism underlying learning and memory. While levels of synapsins and NMDA receptor subunit NR2A were decreased in most hippocampal regions, NR2A expression was particularly reduced in CA3, suggesting possible role of DHA in CA3-NMDA receptor-dependent learning and memory processes. The DHA-induced neurite growth, synaptogenesis, synapsin, and glutamate receptor expression, and glutamatergic synaptic function may represent important cellular aspects supporting the hippocampus-related cognitive function improved by DHA.

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain-derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson's disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole-cell patch-clamp recordings, l-glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration-response plots of differentiated NPCs yielded an EC(50) of 2.2 microM for glutamate and an EC(50) of 36 microM for NMDA. Glutamate-induced currents were markedly inhibited by memantine in contrast to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggesting a higher density of functional NMDA than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. NMDA-evoked currents and calcium signals were blocked by the NR2B-subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage-gated calcium channels by verapamil abolished AMPA-induced calcium responses but only partially reduced NMDA-evoked transients suggesting the expression of calcium-impermeable, GluR2-containing AMPA receptors. Quantitative real-time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 microM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase-immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.

NMDA NR2A and NR2B receptors in the rostral anterior cingulate cortex contribute to pain-related aversion in male rats

NMDA receptors, which are implicated in pain processing, are highly expressed in forebrain areas including the anterior cingulate cortex (ACC). The ACC has been implicated in the affective response to noxious stimuli. Using a combination of immunohistochemical staining, Western blot, electrophysiological recording and formalin-induced conditioned place avoidance (F-CPA) rat behavioral model that directly reflects the affective component of pain, the present study examined formalin nociceptive conditioning-induced changes in the expressions of NMDA receptor subunits NR1, NR2A, and NR2B in the rostral ACC (rACC) and its possible functional significance. We found that unilateral intraplantar (i.pl.) injection of dilute formalin with or without contextual conditioning exposure markedly increased the expressions of NMDA receptor subunits NR2A and NR2B but not of NR1 in the bilateral rACC. NMDA-evoked currents in rACC neurons were significantly greater in formalin-injected rats than in naïve or normal saline-injected rats. Selectively blocking either NR2A or NR2B subunit in the rACC abolished the acquisition of F-CPA and formalin nociceptive conditioning-induced Fos expression, but it did not affect formalin acute nociceptive behaviors and non-nociceptive fear stimulus-induced CPA. These results suggest that both NMDA receptor subunits NR2A and NR2B in the rACC are critically involved in pain-related aversion. Thus, a new strategy targeted at NMDA NR2A or NR2B subunit might be raised for the prevention of pain-related emotional disturbance.

Hemispheric lateralization of the corticostriatal glutamatergic system in the rat

Little is known about hemispheric lateralization of subcortical structures. Here, we show a higher expression of the subunit NR2A of the NMDA receptor mRNA in the striatum and of vGluT1 mRNA in the cingulate cortex, in the left hemisphere compared to the right one. This suggests a lateralization of the glutamatergic cortico-subcortical system, at the level of postsynaptic receptors as well as at the level of corticostriatal projections. Such lateralization could play a role in asymmetric diseases like Parkinson's disease.

Long-term exposure to endogenous levels of tributyltin decreases GluR2 expression and increases neuronal vulnerability to glutamate

Tributyltin (TBT), an endocrine-disrupting chemical, has been used commercially as a heat stabilizer, agricultural pesticide and component of antifouling paints. In this study, we investigated the effect of long-term exposure to endogenous levels of TBT on neuronal glutamate receptors. Cultured rat cortical neurons were exposed to 1–50 nM TBT for 9 days (from day 2 to day 10 in vitro). The number of neurons was reduced by long-term exposure to 50 nM TBT, but not to 1–20 nM TBT. Long-term exposure to 20 nM TBT decreased the mRNA expression of glutamate receptors NR1, NR2A, GluR1 and GluR2, and increased that of NR2B, GluR3 and GluR4. GluR2 protein was also reduced by long-term exposure to TBT. Because AMPA receptor lacking GluR2 exhibits Ca 2+ permeability, we investigated whether Ca 2+ influx or glutamate toxicity was affected. Indeed, glutamate-induced Ca 2+ influx was increased in TBT-treated neurons. Consistent with this, neurons became more susceptible to glutamate toxicity as a result of long-term exposure to TBT and this susceptibility was abolished by an antagonist of GluR2-lacking AMPA receptor. Thus, it is suggested that long-term exposure to endogenous levels of TBT induces a decrease of GluR2 protein, causing neurons become more susceptible to glutamate toxicity.

Isolation rearing induces social and emotional function abnormalities and alters glutamate and neurodevelopment-related gene expression in rats

Isolation rearing induces various cognitive abnormalities such as reversal learning deficits and reduced prepulse inhibition in rats. However, there are few reports in the literature on its effects on social and emotional functions. In the current study we aimed to address these issues and demonstrated that isolation rearing induced aggression and impaired social recognition, produced moderate anxiogenic effects in the elevated-plus maze, and resulted in hyperactivity in a novel open field. We also found NR2A, NR2B, PSD-95 and SAP-102 mRNA expression were significantly up-regulated in the hippocampus while NR2B was down-regulated in prefrontal cortex in response to isolation rearing. This study advances the use of social isolation as an animal model for studying etiological mechanisms of various neuropsychiatric disorders.

Dizocilpine (MK-801) induces distinct changes of N-methyl-d-aspartic acid receptor subunits in parvalbumin-containing interneurons in young adult rat prefrontal cortex

N-methyl-D-aspartic acid receptor (NMDAR) hypofunction has long been implicated in schizophrenia and NMDARs on gamma-aminobutyric acid (GABA)ergic interneurons are proposed to play an essential role in the pathogenesis. However, controversial results have been reported regarding the regulation of NMDAR expression, and direct evidence of how NMDAR antagonists act on specific subpopulations of prefrontal interneurons is missing. We investigated the effects of the NMDAR antagonist dizocilpine (MK-801) on the expression of NMDAR subtypes in the identified interneurons in young adult rat prefrontal cortex (PFC) by using laser microdissection and real-time polymerase chain reaction, combined with Western blotting and immunofluorescent staining. We found that MK-801 induced distinct changes of NMDAR subunits in the parvalbumin-immunoreactive (PV-ir) interneurons vs. pyramidal neurons in the PFC circuitry. The messenger RNA (mRNA) expression of all NMDAR subtypes, including NR1 and NR2A to 2D, exhibited inverted-U dose-dependent changes in response to MK-801 treatment in the PFC. In contrast, subunit mRNAs of NMDARs in PV-ir interneurons were significantly down-regulated at low doses, unaltered at medium doses, and significantly decreased again at high doses, suggesting a biphasic dose response to MK-801. The differential effects of MK-801 in mRNA expression of NMDAR subunits were consistent with the protein expression of NR2A and NR2B subunits revealed with Western blotting and double immunofluorescent staining. These results suggest that PV-containing interneurons in the PFC exhibit a distinct responsiveness to NMDAR antagonism and that NMDA antagonist can differentially and dose-dependently regulate the functions of pyramidal neurons and GABAergic interneurons in the prefrontal cortical circuitry.

Effects of postnatal exposure to methylmercury on spatial learning and memory and brain NMDA receptor mRNA expression in rats

The extreme vulnerability of developing nervous system to methylmercury (MeHg) is well documented. Still unclear is the consequence of different postnatal period exposure to MeHg. We investigated the critical postnatal phase when MeHg induced neurotoxicity in rats and the underlying mechanism. Rats were given 5mg/(kg day) methylmercury chloride (MMC) orally on postnatal day (PND) 7, PND14, PND28, and PND60 for consecutive 7 days. A control group was treated with 0.9% sodium chloride solution 5 ml/(kg day) instead. On PND69, spatial learning and memory was evaluated by Morris water maze test. Behavior deficits were found in MMC-treated rats of PND7 and PND14 groups (p<0.01). N-methyl-D-aspartate (NMDA) receptor 2 subunits mRNA expressions were evaluated 3 days after the last administration. In hippocampus, the mRNA expression of NR2A and NR2B decreased, but the NR2C expression increased in PND14 group following MMC-treatment (p<0.01). In cerebral cortex, mRNA expression of NR2A decreased, with NR2C expression elevating in PND14 group following MMC-treatment (p<0.05). These observations suggest that the postnatal exposure to MeHg during PND7-20 could cause neurobehavioral deficits which extend to adulthood. Furthermore, the abnormal expression of NMDAR 2 subunits might associate with the impairment.

The Serine Protease Plasmin Cleaves the Amino-terminal Domain of the NR2A Subunit to Relieve Zinc Inhibition of the N-Methyl-d-aspartate Receptors

Zinc is hypothesized to be co-released with glutamate at synapses of the central nervous system. Zinc binds to NR1/NR2A N-methyl-d-aspartate (NMDA) receptors with high affinity and inhibits NMDAR function in a voltage-independent manner. The serine protease plasmin can cleave a number of substrates, including protease-activated receptors, and may play an important role in several disorders of the central nervous system, including ischemia and spinal cord injury. Here, we demonstrate that plasmin can cleave the native NR2A amino-terminal domain (NR2A(ATD)), removing the functional high affinity Zn(2+) binding site. Plasmin also cleaves recombinant NR2A(ATD) at lysine 317 (Lys(317)), thereby producing a approximately 40-kDa fragment, consistent with plasmin-induced NR2A cleavage fragments observed in rat brain membrane preparations. A homology model of the NR2A(ATD) predicts that Lys(317) is near the surface of the protein and is accessible to plasmin. Recombinant expression of NR2A with an amino-terminal deletion at Lys(317) is functional and Zn(2+) insensitive. Whole cell voltage-clamp recordings show that Zn(2+) inhibition of agonist-evoked NMDA receptor currents of NR1/NR2A-transfected HEK 293 cells and cultured cortical neurons is significantly reduced by plasmin treatment. Mutating the plasmin cleavage site Lys(317) on NR2A to alanine blocks the effect of plasmin on Zn(2+) inhibition. The relief of Zn(2+) inhibition by plasmin occurs in PAR1(-/-) cortical neurons and thus is independent of interaction with protease-activated receptors. These results suggest that plasmin can directly interact with NMDA receptors, and plasmin may increase NMDA receptor responses through disruption or removal of the amino-terminal domain and relief of Zn(2+) inhibition.

Prenatal NAP+SAL prevents developmental delay in a mouse model of Down syndrome through effects on N-methyl-D-aspartic acid and γ-aminobutyric acid receptors

Down syndrome (DS) affects 1/800 infants. Prenatal NAPVSIPQ (NAP) and SALLRSIPA (SAL) (NAP+SAL) prevent developmental delay in Ts65Dn mice, a mouse model of DS. We investigated whether this finding involves N-methyl-D-aspartic acid and gamma-aminobutyric acid (GABA) receptor subunits. Pregnant Ts65Dn mice were treated with placebo or NAP+SAL on gestational days 8-12. After developmental delay prevention was shown, 4 trisomic (Ts), 4 control, and 3 Ts+NAP+SAL adult offspring brains (from 3 litters) were collected. Calibrator-normalized real-time polymerase chain reaction was performed using primers for N-methyl-D-aspartic acid subunits NR2A and NR2B, and for GABA subunits GABA(A)alpha5 and GABA(A)beta3 with glyceraldehyde-3-phosphate dehydrogenase standardization. Statistics included analysis of variance and Fisher PLSD with P < .05 as significant. NR2A, NR2B, and GABA(A)beta3 levels were decreased in Ts vs control (all P < .05). Prenatal NAP+SAL increased NR2A, NR2B, and GABA(A)beta3 to levels similar to control (all P < .05). A significant difference in GABA(A)alpha5 levels was not found. Prenatal NAP+SAL increases NR2A, NR2B, and GABA(A)beta3 expression in adult DS mice to levels similar to controls. This may explain how NAP+SAL improve developmental milestone achievement.

SGK1 Phosphorylation of IκB Kinase α and p300 Up-regulates NF-κB Activity and Increases N-Methyl-D-aspartate Receptor NR2A and NR2B Expression

Serum- and glucocorticoid-inducible kinase 1 (SGK1) is a downstream target of phosphatidylinositol 3-kinase signaling, and it regulates various cellular and physiological functions, but the SGK1 substrate proteins and genes regulated by SGK1 are less known. Here we have identified IkappaB kinase alpha (IKKalpha) as a novel substrate of SGK1 by using biochemical and bioinformatic approaches. SGK1 directly phosphorylates IKKalpha at Thr-23 and indirectly activates IKKalpha at Ser-180. Furthermore, SGK1 enhanced nuclear factor kappaB (NF-kappaB) activity and up-regulated N-methyl-d-aspartate receptor NR2A and NR2B expression through activation of IKKalpha at Thr-23 and Ser-180, and these two residues play an equally important role in mediating these effects of SGK1. Although SGK1 does not phosphorylate IKKbeta, IKKbeta activity is still required for IKK complex activation and for SGK1 phosphorylation and activation of NF-kappaB. In addition, SGK1 increased the acetylation of NF-kappaB through phosphorylation of p300 at Ser-1834, and this also leads to NF-kappaB activation and NR2A and NR2B expression. Moreover, an endogenous stimulus of SGK1, insulin, increased IKKalpha and NF-kappaB phosphorylation as well as NF-kappaB acetylation and NF-kappaB activity, but SGK1 small interfering RNA transfection blocked these effects of insulin. In examination of the functional significance of the SGK1-IKKalpha-NF-kappaB signaling pathway, we found that transfection of the IKKalpha double mutant (IKKalphaT23A/S180A) to rat hippocampus antagonized SGK-1-mediated spatial memory facilitation. Our results together demonstrated novel substrate proteins of SGK1 and novel SGK1 signaling pathways. Activation of these signaling pathways enhances NR2A and NR2B expression that is implicated in neuronal plasticity.

Interaction of dopamine D1 with NMDA NR1 receptors in rat prefrontal cortex

Despite the tremendous importance of D1 and NMDA receptors to cognition (working memory, executive functions) and synaptic plasticity in the prefrontal cortex (PFC), little is known about the molecular mechanisms underlying D1–NMDA receptors interactions in this brain area. Here, we show that D1 receptors and the NMDA receptor co-localize in single pyramidal neurons and interneurons in adult rat PFC. NR1 and NR2A expression are found in different neuronal types. Conversely, D1 receptors are predominantly localized in pyramidal-like cells and parvalbumin positive cells. NR1 co-immunoprecipitates with D1 receptor in adult medial PFC. In prefrontal primary cultures, NMDA does not affect the D1 receptor dependent-cAMP production. In contrast, activation of D1 receptor potentiates the NMDA mediated increase in cytosolic Ca 2+, an effect that was blocked by a PKA inhibitor. We conclude that D1 receptor potentiates the NMDA-Ca 2+ signal by a PKA-dependent mechanism.

Expression of NMDA receptor subunit 2A in the spinal cord of morphine tolerant rats

ObjectiveTo study the distribution and expression of NMDA receptor subunit 2A in the spinal cord of morphine tolerant rats. MethodsTwelve Sprague-Dawley rats were randomly divided into two groups with 6 rats each: control group (C) were intrathecally administrated 0.9% NaCl 10μl and morphine group(M) received 10μg morphine (i.t.). Drugs were administrated twice daily for 7 consecutive days. Tail flick latency (TFL) in the hot water immersion test was used to evaluate changes of thermal hyperalgesia latency of each group before and 30min after administration every morning. Rats were killed the day after last administration and L4～5 segment of the spinal cord was removed for determination the expression of NR2A by immunofluorescence method. ResultsTFL of group M was decreased gradually after chronic administration of morphine intrathecally. There was no significant difference between group M[(3.25±0.93)s] and group C[(2.66±0.27)s] on the 7th day (P＞0.05). A morphine tolerant model was established successfully. NR2A was distributed throughout the rat spinal cord. The expression of NR2A in group M(OD:9617±1233) was increased compared with group C(OD:2.66±0.93) (t=3.133,P＜0.05).ConclusionThe expression of NR2A was upregulated after repeated administration of morphine intrathecally in the superficial dorsal horn of spinal cord of morphine tolerant rats,which may be part of the mechanisms of morphine tolerance. Key words: Morphine tolerance; NR2A subunit; Spinal cord

Src‐protein tyrosine kinases are required for cocaine‐induced increase in the expression and function of the NMDA receptor in the ventral tegmental area

Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization.