Differential expression of NMDA and AMPA receptor subunits in rat dorsal and ventral hippocampus

Several studies have demonstrated anatomical and functional segregation along the dorsoventral axis of the hippocampus. This study examined the possible differences in the AMPA and NMDA receptor subunit composition and receptor binding parameters between dorsal and ventral hippocampus, since several evidence suggest diversification of NMDA receptor-dependent processes between the two hippocampal poles. Three sets of rat dorsal and ventral hippocampus slices were prepared: 1) transverse slices for examining a) the expression of the AMPA (GluRA, GluRB, GluRC) and NMDA (NR1, NR2A, NR2B) subunits mRNA using in situ hybridization, b) the protein expression of NR2A and NR2B subunits using Western blotting, and c) by using quantitative autoradiography, c(1)) the specific binding of the AMPA receptor agonist [(3)H]AMPA and c(2)) the specific binding of the NMDA receptor antagonist [(3)H]MK-801, 2) longitudinal slices containing only the cornus ammonis 1 (CA1) region for performing [(3)H]MK-801 saturation experiments and 3) transverse slices for electrophysiological measures of NMDA receptor-mediated excitatory postsynaptic potentials. Ventral compared with dorsal hippocampus showed for NMDA receptors: 1) lower levels of mRNA and protein expression for NR2A and NR2B subunits in CA1 with the ratio of NR2A /NR2B differing between the two poles and 2) lower levels of [(3)H]MK-801 binding in the ventral hippocampus, with the lowest value observed in CA1, apparently resulting from a decreased receptor density since the B(max) value was lower in ventral hippocampus. For the AMPA receptors CA1 our results showed in ventral hippocampus compared with dorsal hippocampus: 1) lower levels of mRNA expression for GluRA, GluRB and GluRC subunits, which were more pronounced in CA1 and in dentate gyrus region and 2) lower levels of [(3)H]AMPA binding. Intracellular recordings obtained from pyramidal neurons in CA1 showed longer NMDA receptor-mediated excitatory postsynaptic potentials in ventral hippocampus compared with dorsal hippocampus. In conclusion, the differences in the subunit mRNA and protein expression of NMDA and AMPA receptors as well as the lower density of their binding sites observed in ventral hippocampus compared with dorsal hippocampus suggest that the glutamatergic function differs between the two hippocampal poles. Consistently, the lower value of the ratio NR2A/NR2B seen in the ventral part would imply that the ventral hippocampus NMDA receptor subtype is functionally different than the dorsal hippocampus subtype, as supported by our intracellular recordings. This could be related to the lower ability of ventral hippocampus for long-term synaptic plasticity and to the higher involvement of the NMDA receptors in the epileptiform discharges, observed in ventral hippocampus compared with dorsal hippocampus.     

In developing hippocampal neurons, NR2B-containing N-methyl-D-aspartate receptors (NMDARs) can mediate signaling to neuronal survival and synaptic potentiation, as well as neuronal death

It has been suggested that NR2B-containing N-methyl-d-aspartate (NMDA) receptors have a selective tendency to promote pro-death signaling and synaptic depression, compared with the survival promoting, synapse potentiating properties of NR2A-containing NMDA receptors. A preferential localization of NR2A-containing NMDA receptors at the synapse in maturing neurons could thus explain differences in synaptic vs. extrasynaptic NMDA receptor signaling. We have investigated whether NMDA receptors can mediate signaling to survival, death, and synaptic potentiation, in dissociated rat neuronal cultures at a developmental stage prior to significant NR2A expression and subunit-specific differences between synaptic and extrasynaptic NMDA receptors. We show that in developing hippocampal neurons, the progressive reduction in sensitivity of NMDA receptor currents to the NR2B antagonist ifenprodil applies to both synaptic and extrasynaptic locations. However, the reduction is less acute in extrasynaptic currents, indicating that NR2A does partition preferentially, but not exclusively, into synaptic locations at DIV>12. We then studied NMDA receptor signaling at DIV10, when both synaptic and extrasynaptic NMDA receptors are both overwhelmingly and equally NR2B-dominated. To analyze pro-survival signaling we studied the influence of synaptic NMDA receptor activity on staurosporine-induced apoptosis. Blockade of spontaneous NMDAR activity with MK-801, or ifenprodil exacerbated the apoptotic insult. Furthermore, MK-801 and ifenprodil both antagonized neuroprotection promoted by enhancing synaptic activity. Pro-death signaling induced by a toxic dose of NMDA is also blocked by NR2B-specific antagonists. Using a cell culture model of synaptic NMDA receptor-dependent synaptic potentiation, we find that this is mediated exclusively by NR2B-containing N-methyl-D-aspartate receptors, as implicated by NR2B-specific antagonists and the use of selective vs. non-selective doses of the NR2A-preferring antagonist NVP-AAM077. Therefore, within a single neuron, NR2B-NMDA receptors are able to mediate both survival and death signaling, as well as model of NMDA receptor-dependent synaptic potentiation. In this instance, subunit differences cannot account for the dichotomous nature of NMDA receptor signaling.     

Altered effects of ethanol in NR2A(DeltaC/DeltaC) mice expressing C-terminally truncated NR2A subunit of NMDA receptor

Phosphorylation of C-termini of receptor subunits is thought to play a significant role in modulation of N-methyl-D-aspartic acid (NMDA) receptor function. To investigate whether the C-terminus of the NR2A subunit is involved in determining the sensitivity of NMDA receptors to ethanol we compared the effects of ethanol in vitro on NMDA-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 and dentate gyrus (DG) of adult male NR2A(DeltaC/DeltaC) mice lacking the C-terminus of NR2A subunit and in their parental strain C57Bl/6. We also tested the in vivo effects of a hypnotic dose of ethanol in C57Bl/6 and NR2A(DeltaC/DeltaC) mice and their F2 offspring. Ifenprodil (10 microM) was used to distinguish between the NR2A and NR2B components of NMDA fEPSPs. Ethanol (100 mM) in the presence of ifenprodil inhibited the CA1 NR2A-mediated component of NMDA fEPSPs two times more in NR2A(DeltaC/DeltaC) than in C57Bl/6. Ethanol inhibition of the CA1 NR2B-mediated component was five to seven times lower in NR2A(DeltaC/DeltaC) than in C57Bl/6. In the DG ethanol had similar effects in the two strains. In vivo administration of ethanol (4 g/kg) induced sedation of similar duration in both strains of mice. A second administration of ethanol 7 days after the initial injection revealed an increased ethanol sensitivity of NR2A(DeltaC/DeltaC) and F2(DeltaC/DeltaC) mice including a shortened time to loss of righting reflex and an increased sleep time. The sensitization of NR2A(DeltaC/DeltaC) mice to alcohol was not accompanied by an altered ethanol sensitivity of NMDA fEPSPs recorded in vitro.Our data are consistent with the inhibitory action of ethanol on NMDA receptors being mediated by a site other than the intracellular C-terminus of the NR2A subunit. The altered sensitivities to ethanol of both NR2A- and NR2B-mediated responses in the CA1 of NR2A(DeltaC/DeltaC) imply that NR2A- and NR2B subunit-containing NMDA receptors may be linked by a common target of ethanol.     

Differential NR2B subunit expression at dorsal root and ventrolateral funiculus synapses on lumbar motoneurons of neonatal rat

Synapse specific differences in NR2 subunit expression exist in several systems within the mammalian CNS. Here we have studied such differences on motoneurons in the neonatal rat cord using ifenprodil known to inhibit voltage-, use- and glycine-independent responses mediated by NR2B-containing N-methyl-d-aspartate receptors (NMDARs) with high specificity. In neonatal rats (P1–P9), the synapses made by the dorsal root (DR) fibres were more sensitive to ifenprodil than ventrolateral funiculus (VLF) connections on the same motoneuron. DR connections exhibited very little additional blockade to bath-applied MK-801 whereas VLF connections displayed a further decrease in amplitude. This suggests that at this immediate postnatal age, DR synapses on motoneurons contain a higher proportion of ifenprodil-sensitive diheteromeric NR1/NR2B receptors than VLF synapses. Since DR synapses have been shown in other studies to be less mature than VLF synapses on the same motoneuron at this developmental stage, these data are interpreted as indicating that less mature NMDA receptors feature a higher proportion of NR2B subunits which declines as the synapse matures. This novel finding of staggered development of NMDA receptors from different synaptic inputs on the same motoneuron is discussed in the context of its developmental and functional implications.     

Effect of NMDA receptor antagonist on proliferation of neurospheres from embryonic brain

The N-methyl-D-aspartate (NMDA) receptor, a subtype of ionotropic glutamate receptors, plays an important role in the regulation of neuronal development, learning and memory, and neurodegenerative diseases. NMDA receptor blockade enhances neurogenesis in the hippocampal dentate gyrus in vivo. The effect of NMDA receptor antagonist on proliferation of neural progenitor cells, however, remains to be determined. We investigated changes in the diameter and number of neurospheres derived from the embryonic rat brain after NMDA receptor blockade. Cortical progenitor cells were isolated from gestational day 18 fetal rats according to the Percoll density gradient method. Cultured spheres expressed neural progenitor markers, musashi-1 and nestin. Immunohistochemical analysis demonstrated that cells in Dulbecco's modified Eagle medium/F12 containing 1% fetal bovine serum on day 8 differentiated to MAP-2-positive neurons and GFAP-positive astrocytes. The expression of NR1 and NR2B subunits of the NMDA receptor in neurospheres was detected. Neither brief nor sustained exposure to NMDA altered the diameter and number of neurospheres. Brief exposure to 30 μM MK-801, an NMDA receptor antagonist, decreased the diameter of neurospheres. Sustained exposure to 30 μM MK-801 decreased the diameter and number of neurospheres. Our results provide evidence that MK-801 directly decreased proliferation of neural progenitor cells.     

Functional NR2B- and NR2D-containing NMDA receptor channels in rat substantia nigra dopaminergic neurones

NMDA receptors regulate burst firing of dopaminergic neurones in the substantia nigra pars compacta (SNc) and may contribute to excitotoxic cell death in Parkinson's disease (PD). In order to investigate the subunit composition of functional NMDA receptors in identified rat SNc dopaminergic neurones, we have analysed the properties of individual NMDA receptor channels in outside-out patches. NMDA (100 n m ) activated channels corresponding to four chord conductances of 18, 30, 41 and 54 pS. Direct transitions were observed between all conductance levels. Between 18 pS and 41 pS conductance levels, direct transitions were asymmetric, consistent with the presence of NR2D-containing NMDA receptors. Channel activity in response to 100 n m or 200 μ m NMDA was not affected by zinc or TPEN ( N,N,N',N '-tetrakis-[2-pyridylmethyl]-ethylenediamine), indicating that SNc dopaminergic neurones do not contain functional NR2A subunits. The effect of the NR2B antagonist ifenprodil was complex: 1 μ m ifenprodil reduced open probability, while 10 μ m reduced channel open time but had no effect on open probability of channels activated by 100 n m NMDA. When the concentration of NMDA was increased to 200 μ m , ifenprodil (10 μ m ) produced the expected reduction in open probability. These results indicate that NR2B subunits are present in SNc dopaminergic neurones. Taken together, these findings indicate that NR2D and NR2B subunits form functional NMDA receptor channels in SNc dopaminergic neurones, and suggest that they may form a triheteromeric NMDA receptor composed of NR1/NR2B/NR2D subunits.     

Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block

We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC₅₀ values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo . Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of p K _a values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (−)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.     

Synaptic and extrasynaptic NMDA receptor NR2 subunits in cultured hippocampal neurons

Early in development, neurons only express NR1/NR2B-containing N-methyl-d-aspartate (NMDA) receptors. Later, NR2A subunits are upregulated during a period of rapid synapse formation. This pattern is often interpreted to indicate that NR2A-containing receptors are synaptic and that NR2B-containing receptors are extrasynaptic. We re-examined this issue using whole cell recordings in cultured hippocampal neurons. As expected, the inhibition of whole cell currents by the NR2B-specific antagonist, ifenprodil, progressively decreased from 69.5 +/- 2.4% [6 days in vitro (DIV)] to 54.9 +/- 2.6% (8 DIV), before reaching a plateau in the second week (42.5 +/- 2%, 12-19 DIV). In NR2A-/- neurons, which express only NR1/NR2B-containing NMDA receptors, autaptic excitatory postsynaptic currents (EPSCs; > or =12 DIV) were more sensitive to ifenprodil and decayed more slowly than EPSCs in wild-type neurons. Thus NR2B-containing receptors were not excluded from synapses. We blocked synaptic NMDA receptors with MK-801 during evoked transmitter release, thus allowing us to isolate extrasynaptic receptors. Ifenprodil inhibition of this extrasynaptic population was highly variable in different neurons. Furthermore, extrasynaptic receptors in autaptic cultures were only partially blocked by ifenprodil, indicating that NR2A-containing receptors are not exclusively confined to the synapse. Extrasynaptic NR2A-containing receptors were also detected in NR2A(-/-) neurons transfected with full-length NR2A. Truncation of the NR2A C terminus did not eliminate synaptic expression of NR2A-containing receptors. Our results indicate that NR2A- and NR2B-containing receptors can be located in either synaptic or extrasynaptic compartments.     

Alterations in NMDA receptor subunit densities and ligand binding to glycine recognition sites are associated with chronic anxiety in Alzheimer's disease

Glutamatergic deficits are established neuropathological features of Alzheimer's disease (AD) and are known to correlate with cognitive impairments. In contrast, the role of glutamatergic alterations in behavioral and psychological symptoms of dementia (BPSD) is unclear. There is considerable preclinical evidence for the importance of glycine recognition sites (GlyRS) of N-methyl-d-aspartate (NMDA) receptors in the regulation of anxiety behaviors. This study aimed to correlate several glutamatergic measures with chronic anxiety in AD. Twenty-one AD patients assessed by the Neuropsychiatric Inventory (NPI) were divided into low anxiety (LA) and high anxiety (HA) subgroups. GlyRS and NMDA channel were measured by brain homogenate binding with [³H]MDL105,519 and [³H]MK-801, respectively. Densities of NMDA receptor NR2A, NR2B and alternate spliced NR1 subunits were quantified by immunoblotting. We found that the binding affinity to GlyRS was significantly higher in HA compared to LA, and this higher GlyRS affinity correlated with selective reduction of NR2A density as well as with elevated anxiety scores. Our observations suggest a novel mechanism whereby subunit specific changes in the NMDA receptor complex may be linked to chronic anxiety in AD via effects on GlyRS function. We propose that NR2A and GlyRS should be further assessed as novel targets of behavioral pharmacotherapy in AD.     

The role of NMDA receptor binding sites in ethanol place conditioning

Little is known about the specific role of glutamate, in particular its actions at N-methyl-D-aspartate (NMDA) receptors, in ethanol reward. Pretreatment with channel blockers MK-801 and ketamine, NMDA NR2B receptor subunit antagonists ifenprodil and CP-101,606, and the glycine(B) partial agonist (+)-HA-966 did not alter acquisition of ethanol-induced conditioned place preference (CPP) in mice. However, pretreatment with the competitive antagonist CGP-37849 attenuated acquisition of ethanol-induced CPP. Follow-up experiments indicated that CGP-37849 also blocked acquisition of ethanol-induced and lithium chloride-induced conditioned place aversion but did not produce rewarding or aversive effects on its own. These results suggest that the NMDA receptor glutamate binding site is important for ethanol place conditioning. Moreover, these results suggest CGP-37849 modulates ethanol place conditioning by impairing the ability to learn these tasks.     

Antagonist properties of eliprodil and other NMDA receptor antagonists at rat NR1A/NR2A and NR1A/NR2B receptors expressed in Xenopus oocytes

We have studied the effects of a variety of N-methyl-

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-aspartate (NMDA) antagonists acting at different sites of the NMDA receptor complex on NMDA-induced currents in Xenopus oocytes expressing heteromeric NR1A/NR2A and NR1A/NR2B receptors. The polyamine site antagonists eliprodil (IC₅₀=3.0 μM) and ifenprodil (IC₅₀=0.27 μM) antagonized NMDA responses at NR1A/NR2B receptors but not at NR1A/NR2A receptors (IC₅₀>100 μM). The channel blockers dizocilpine, memantine and phencyclidine (PCP) were equally potent antagonists at both receptor subtypes whereas dextromethorphan was four times more potent at NR1A/NR2A receptors. The glycine site antagonists

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-689,560 and 7-Cl-kynurenate were 10 times more potent at NR1A/NR2A than at NR1A/NR2B receptor subtypes. The selectivity of eliprodil and ifenprodil for the NR1A/NR2B receptor subtype may, at least partially, explain their favorable side effect profile.     

The NR2B subunit in NMDA receptors is functionally important during cerebellar granule cell migration

Migration of cerebellar granule cells (CGCs) from the external germinal cell layer (EGL) to the internal granule cell layer (IGL) within the cerebellar cortex is a crucial developmental process. Antagonists to NMDA receptors impair CGC migration significantly, but studies to determine which subunit subtypes control or affect migration have been controversial. Migrating CGCs transiently express NMDA receptor subunit subtypes NR1a plus NR2B. Grafted NR1-/- subunit knockout cells continue to migrate, indicating that the NR1 subunit is not necessary for migration. In the present study, the functional importance of the NR2B subtype in developing cerebellum was investigated using organotypic slice cultures prepared from postnatal day 8 (P8) rats. Slice cultures were labeled with bromodeoxyuridine (BrdU) during the first 20h and then continuously treated with the NR2B-subtype-specific NMDA antagonist, ifenprodil, or the non-specific NMDA antagonist, APV, for 7 days. Cultures were incubated with fluorescently tagged anti-BrdU IgG and the percent of BrdU-labeled CGCs that migrated from the EGL to the IGL during treatment was analyzed using laser confocal microscopy. Migration into the IGL was significantly impaired by treatment with 0.5 and 1.0 microM ifenprodil. Fewer cells had migrated to the IGL in 1.0 microM ifenprodil than in 0.5 microM ifenprodil; there was no significant difference between the percent impairment caused by 1.0 microM ifenprodil and 50 microM APV. Untreated controls had few, if any, CGCs in the EGL at DIV 8. The percent of CGCs remaining in the EGL following treatment with antagonists significantly increased, indicating impairment of migration. In conclusion, the NR2B subunit appears to be necessary for CGC migration.     

NMDA receptors are involved in upstream of the spinal JNK activation in morphine antinociceptive tolerance

N-methyl-d-aspartate (NMDA) receptors and c-Jun N-terminal kinase (JNK) have been shown to be involved in morphine antinociceptive tolerance. However, whether chronic morphine-induced activation of the spinal JNK is NMDA receptor-dependent is unknown. The present study investigated the link between the spinal NMDA receptor NR2B subunit and the JNK activation during morphine antinociceptive tolerance in rats. Our results showed that chronic morphine treatment induced upregulation of the NR2B expression and activation of JNK in the spinal cord. Moreover, the increased NR2B-immunoreactivity (IR) and phosphorylated JNK-IR were observed mainly at the superficial dorsal horn laminae of the spinal cord; the spinal p-JNK was mainly expressed in astrocytes and NR2B in neurons. SP600125, a selective inhibitor of JNK, significantly attenuated morphine tolerance. MK-801, a noncompetitive NMDA receptor antagonist, not only suppressed morphine antinociceptive tolerance and the increase in NR2B, but also reduced the spinal JNK activation induced by chronic morphine treatment. These findings demonstrated for the first time that NMDA receptor-dependent activation of the spinal JNK contributes to morphine antinociceptive tolerance and that MK-801 attenuates morphine tolerance partly due to its inhibition on the spinal JNK activation.     

NR1、NR2A与PSD-95在生后大鼠海马发育中的表达

目的:探讨N-甲基-D-天冬氨酸受体亚单位1(N-methy1-D-aspartate receptor subunit1,NR1)、N-甲基-D-天冬氨酸受体亚单位2A(N-methy1-D-aspartate receptor subunit2A,NR2A)与突触后密度蛋白-95(Postsynapticdensity protein 95,PSD-95)在Wistar大鼠海马生后发育过程中的表达。方法:应用免疫荧光染色方法检测NR1、NR2A与PSD-95在生后不同时期大鼠海马CA1、CA3区和齿状回(DG)中的表达情况。结果:NR1于生后各期海马CA1、CA3区和DG的表达均增强,P14~P21达高峰期后减弱。NR2A于生后在海马CA1和CA3区的表达略有减弱,P4后增强至高峰期P14,然后轻微减弱,在DG中NR2A的表达生后略有减弱,P4后在增强的趋势中于P7、P14和P28后均有不同程度的减弱,高峰期在P28。PSD-95于生后各期海马CA1、CA3区和DG的表达均增强,P21~P28达高峰期后轻微减弱。结论:NR1、NR2A和PSD-95在CA1、CA3区和DG中的表达具有特异的时空分布模式,此模式可能与其在生后发育中发挥的不同生理功能相关。     

Ionotropic and metabotropic glutamate receptor mediation of glucocorticoid-induced apoptosis in hippocampal cells and the neuroprotective role of synaptic N-methyl-D-aspartate receptors

Glutamate receptors have been proposed to mediate the apoptotic actions of glucocorticoids in hippocampal cells. To further analyze the role of glutamate receptors in this process, we pretreated primary hippocampal cells from neonatal (postnatal day 4) rats with antagonists of ionotropic glutamate receptor (iGluR) and metabotropic glutamate receptor (mGluR) antagonists before exposure to the specific glucocorticoid receptor agonist dexamethasone (DEX) at a dose of 1 microM. Dizocilpine (MK801; a general N-methyl-D-aspartic acid [NMDA] receptor antagonist, NMDAR antagonist) and ifenprodil (a specific ligand of the NMDAR 2B subunit, NR2B), were used to block iGluR; (RS)-alpha-ethyl-4-carboxyphenylglycine (E4CPG) and (RS)-alpha-cyclopropyl-4-phosphonophenyl-glycine (CPPG) were employed as I/II (E4CPG) and II/III (CPPG) mGluR antagonists. Blockade of iGluR resulted in a significant attenuation of DEX-induced cell death; the finding that ifenprodil exerted a similar potency to MK801 demonstrates the involvement of NR2B receptors in glucocorticoid-induced cell death. Apoptosis accounted for a significant amount of the cell loss observed, as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling histochemistry for the in situ labeling of DNA breaks; apoptotic cells were distinguished from necrosis on the basis of morphological criteria, including chromatin condensation, membrane blebbing and presence of apoptotic bodies. Treatment with E4CPG and CPPG completely abolished the apoptotic response to DEX, thus showing the additional contribution of mGluR to the phenomenon. Further, dose-response studies with NMDA revealed that whereas high (10 microM) doses of NMDA themselves elicit cytotoxic responses, low (1-5 microM) concentrations of NMDA can effectively oppose DEX-induced cell death. Interestingly, the neuroprotective actions of low dose NMDA stimulation were abolished when either synaptic or extrasynaptic NMDA receptors were blocked with MK801 in combination with the GABA receptor antagonist bicuculline (synaptic) or ifenprodil (extrasynaptic). In summary, the present data show that both iGluR and mGluR mediate the neurotoxic effects of glucocorticoids on hippocampal cells and that pre-treatment with low doses of NMDA, by acting on synaptic and extrasynaptic receptors, render hippocampal cells less vulnerable to glucocorticoid insults.     

Zinc,magnesium and NMDA receptor alterations in the hippocampus of suicide victims

Background

There is evidence for an association between suicidal behavior and depression. Accumulating data suggests that depression is related to a dysfunction of the brain's glutamatergic system, and that the N-methyl-d-aspartate (NMDA) receptor plays an important role in antidepressant activity. Zinc and magnesium, the potent antagonists of the NMDA receptor complex, are involved in the pathophysiology of depression and exhibit antidepressant activity.

Methods

The present study investigated the potency of Zn²⁺ and Mg²⁺ to [³H] MK-801, which binds to the NMDA receptor channel in the hippocampus of suicide victims (n=17) and sudden death controls (n=6). Moreover, the concentrations of zinc and magnesium (by flame atomic absorption spectrometry) and levels of NMDA subunits (NR2A and NR2B) and PSD-95 protein (by Western blotting) were determined.

Results

Our results revealed that there was a statistically significant decrease (by 29% and 40%) in the potency of zinc and magnesium (respectively) to inhibit [³H] MK-801 binding to NMDA receptors in the hippocampus in suicide tissue relative to the controls. These alterations were associated with increased NR2A (+68%) and decreases in both the NR2B (−46%) and PSD-95 (−35%) levels. Furthermore, lower concentrations (−9%) of magnesium (although not of zinc) were demonstrated in suicide tissue.

Conclusions

Our findings indicate that alterations in the zinc, magnesium and NMDA receptor complex in the hippocampus are potentially involved in the pathophysiology of suicide-related disorders (depression), which may lead to functional NMDA receptor hyperactivity.     

髓鞘形成相关NMDA-R亚基在精神分裂症小鼠模型中的变化

目的:探讨髓鞘形成相关N-甲基-D-天冬氨酸受体(NMDA-R)亚基在MK-801诱导的精神分裂症小鼠模型大脑中的变化。方法:35只雄性C57BL/6J小鼠,随机分为:对照组(control)和地卓西平马来酸盐处理组(MK-801),利用旷场实验、探孔实验及高架十字迷宫检测小鼠行为学改变,免疫荧光染色观察小鼠大脑NMDAR亚基NR1的表达,real time RT-PCR检测髓鞘形成相关NMDA-R亚基NR1、NR2C和NR3A mRNA的表达。结果:与对照组小鼠相比,MK-801处理组在旷场内10 min总运动路程较对照组显著增加;3 min及5 min内探孔次数较对照组显著降低;模型组小鼠在开臂内停留总时间较对照组显著降低。NR1在小鼠神经细胞胞膜、轴突周围和髓鞘部位均有表达。大脑皮层、海马内的NMDA-R亚基NR1,NR2C及NR3A mRNA的表达在两组间均无显著差异;胼胝体区域NR1和NR2C mRNA表达在两组间同样无显著差异;但相较于对照组,MK-801慢性给药组小鼠胼胝体区域NMDA-R亚基NR3A mRNA表达显著上调。结论:NMDA-R在少突胶质细胞、髓鞘等部位确有表达;胼胝体部位NR3A亚基上调可能在MK-801诱导的精神分裂症中发挥作用。     

Selective effects of neonatal handling on rat brain N-methyl-d-aspartate receptors

Neonatal handling, an experimental model of early life experiences, is known to affect the hypothalamic–pituitary–adrenal axis function thus increasing adaptability, coping with stress, cognitive abilities and in general brain plasticity-related processes. A molecule that plays a most critical role in such processes is the N-methyl-d-aspartate (NMDA) receptor, a tetramer consisting of two obligatory, channel forming NR1 subunits and two regulatory subunits, usually a combination of NR2A and NR2B. Since the subunit composition of the NMDA receptor affects brain plasticity, in the present study we investigated the effect of neonatal handling on NR1, NR2A and NR2B mRNA levels using in situ hybridization, and on NR2B binding sites, using autoradiography of in vitro binding of [³H]-ifenprodil, in adult rat limbic brain areas. We found that neonatal handling specifically increased NR2B mRNA and binding sites, while it had no effect on the NR1 and NR2A subunits. More specifically, neonatally handled animals, both males and females, had higher NR2B mRNA and binding sites in the dorsal CA1 hippocampal area, as well as the prelimbic, the anterior cingulate and the somatosensory cortex, compared to the non-handled. Moreover NR2B binding sites were increased in the dorsal CA3 area of handled animals of both sexes. Furthermore, neonatal handling had a sexually dimorphic effect, increasing NR2B mRNA and binding sites in the central and medial amygdaloid nuclei only of the females. The neonatal handling-induced increase in the NR2B subunit of the NMDA receptor could underlie the higher brain plasticity, which neonatally handled animals exhibit.