Ethanol-related behaviors in mice lacking the NMDA receptor NR2A subunit

Rationale The ionotropic NMDA glutamate receptor is composed of NR1 and NR2 (NR2A-D) subunits. While there is compelling evidence that NMDA receptors modulate behavioral effects of ethanol, there is little understanding of how the subunit composition of the NMDA receptor mediates these effects.Objectives In the current study, we assessed the relative roles of NMDA subunits via phenotypic assessment of ethanol-related behaviors in NR2A knockout (KO) mice.Results Results demonstrated that NR2A KO and heterozygous mice failed to show evidence of ethanol-induced conditioned place preference. As compared to wild-type (WT) controls, KO mice showed impaired motor coordination at baseline and, in some instances, following ethanol treatment on the accelerating rotarod, balance beam, and wire-hang tests. By contrast, open field locomotor-stimulant, sedative/hypnotic, and hypothermic responses to ethanol were not different between genotypes, nor was voluntary ethanol consumption and preference in a two-bottle choice paradigm. Blood ethanol concentrations were lower in KO than WT mice following intraperitoneal ethanol injection.Conclusions Results suggest that the loss of NR2A subunit-containing NMDA receptors impairs the ability to form or express learned reward-related responses to ethanol and causes deficits in motor coordination. However, the loss of NR2A does not alter other measures of acute ethanol intoxication or ethanol consumption, possibly implicating other NMDA subunits in these effects. These data provide novel insight into the role of NMDA receptors in modulating the behavioral effects of ethanol.Research supported by the National Institute on Alcohol Abuse and Alcoholism Intramural Research Program.     

Role of the NMDA receptor NR2B subunit in the discriminative stimulus effects of ketamine

The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, ketamine, is a dissociative anesthetic with antihyperalgesic properties. However, its clinical use is compromised by psychotomimetic side-effects. As ketamine and other noncompetitive NMDA antagonists, such as phencyclidine and dizocilpine, are not selective for the NR2A-2D subunits of the NMDA receptor, it is unclear which of these subunits is responsible for the psychotomimetic side-effects. This study investigated the role of the NR2B subunit in the ketamine drug discrimination model, a possible correlate for such side-effects. In a first experiment aimed at assessing general potency and time dependency, ketamine, dizocilpine, phencyclidine and the NR2B-selective antagonists ifenprodil and Ro 25-6981, dose-dependently suppressed fixed ratio 10 food-reinforced responding in rats, with peak efficacy obtained around 15-40 min. In rats trained to discriminate ketamine from vehicle in a two-lever fixed ratio 10 food-reinforced procedure, ketamine, dizocilpine, phencyclidine and Ro 25-6981 induced complete generalization (>80%); whereas ifenprodil induced partial generalization (33%). These findings suggest that the NR2B subunit is involved in the discriminative stimulus effects of noncompetitive NMDA antagonists, and that selective NR2B antagonists may also induce psychotomimetic side-effects.     

Genetic inactivation of the NMDA receptor NR2A subunit has anxiolytic- and antidepressant-like effects in mice.

There is growing evidence implicating the glutamate system in the pathophysiology and treatment of mood and anxiety disorders. Glutamatergic neurotransmission is mediated by several receptor subfamilies including multiple NMDA receptor subunits (NR2A-D). However, little is currently understood about the specific roles of NMDA subunits in the mediation of emotional behavior due to a lack of subunit-specific ligands. In the present study, we employed a mouse gene-targeting approach to examine the role of the NR2A subunit in the mediation of anxiety- and depressive-related behaviors. Results showed that NR2A knockout (KO) mice exhibit decreased anxiety-like behavior relative to wild-type littermates (WT) across multiple tests (elevated plus maze, light-dark exploration test, novel open field). NR2A KO mice showed antidepressant-like profiles in the forced swim test and tail suspension test, as compared to WT controls. Locomotor activity in the nonaversive environments of the home cage or a familiar open field were normal in the NR2A KO mice, as were gross neurological and sensory functions, including prepulse inhibition of startle. Taken together, these data demonstrate a selective and robust reduction in anxiety- and depression-related behavior in NMDA receptor NR2A subunit KO mice. Present results support a role for the NR2A subunit in the modulation of emotional behaviors in rodents and provide insight into the role of glutamate in the pathophysiology and treatment of mood and anxiety disorders.     

Elevated levels of NR2A and PSD-95 in the lateral amygdala in depression

Compelling evidence suggests that major depression is associated with dysfunction of the brain glutamatergic transmission, and that the glutamatergic N-methyl-d-aspartate (NMDA) receptor plays a role in antidepressant activity. Recent post-mortem studies demonstrate that depression is associated with altered concentrations of proteins associated with NMDA receptor signalling in the brain. The present study investigated glutamate signalling proteins in the amygdala from depressed subjects, given strong evidence for amygdala pathology in depression. Lateral amygdala samples were obtained from 13-14 pairs of age- sex-, and post-mortem-interval-matched depressed and psychiatrically healthy control subjects. Concentrations of NR1 and NR2A subunits of the NMDA receptor, as well as NMDA receptor-associated proteins such as post-synaptic density protein-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) were measured by Western immunoblotting. Additionally, levels of enzymes involved in glutamate metabolism, including glutamine synthetase and glutamic acid decarboxylase (GAD-67), were measured in the same amygdala samples. NR2A protein levels were markedly and significantly elevated (+115%, p=0.03) in depressed subjects compared to controls. Interestingly, PSD-95 levels were also highly elevated (+128%, p=0.01) in the same depressed subjects relative to controls. Amounts of NR1, nNOS, glutamine synthetase, and GAD-67 were unchanged. Increased levels of NR2A and PSD-95 suggest that glutamate signalling at the NMDA receptor in the amygdala is disrupted in depression.     

Antinociceptive activity of CP-101,606, an NMDA receptor NR2B subunit antagonist

1. The analgesic activity of CP-101,606, an NR2B subunit-selective N-methyl-D-aspartate (NMDA) receptor antagonist, was examined in carrageenan-induced hyperalgesia, capsaicin- and 4β-phorbol-12-myristate-13-acetate (PMA)-induced nociceptive tests in the rat.
2. CP-101,606 30 mg kg⁻¹, s.c., at 0.5 and 2.5 h after carrageenan challenge suppressed mechanical hyperalgesia without any apparant alternations in motor coordination or behaviour in the rat.
3. CP-101,606 also inhibited capsaicin- and PMA-induced nociceptive responses (licking behaviour) with ED₅₀ values of 7.5 and 5.7 mg kg⁻¹, s.c., respectively.
4. These results suggest that inhibition of the NR2B subunit of the NMDA receptor is effective in vivo at modulating nociception and hyperalgesia responses without causing the behavioural side effects often observed with currently available NMDA receptor antagonists.

     

NR2B selective NMDA receptor antagonists

NR2B antagonists have received considerable attention in recent years. In this class of excitatory amino acid receptor antagonists NR2B antagonists have shown efficacy in neuroprotection, anti-hyperalgesic and anti-Parkinson animal models. Several groups are involved in developing these compounds as therapeutic agents and evaluating newer therapeutic targets for these agents. Until recently benzylpiperidine and phenylpiperidine templates, which were based on the structures of Ifenprodil and Eliprodil, formed the basis of most SAR in this area. A few chemical leads in this class such as CP-101,606, Ro25,6981 and PD0196860 have been identified as possible development leads which have generated significant interest in this area. In addition to the efforts of Pfizer (Parke-Davis), Roche and E.Merck, several other industrial and academic research groups have continued to work in the NR2B area and recently Merck and Roche have reported new chemical leads as NR2B antagonists with significantly different biaryl templates. These new advances have raised hope, for potential success of the NR2B antagonists as new therapeutic agents, for the treatment of several pathophysiological indications.     

CP-101,606, an NR2B subunit selective NMDA receptor antagonist, inhibits NMDA and injury induced c-fos expression and cortical spreading depression in rodents

(1S, 2S)-1-(4-hydroxyphenyl)-2-(4-hydroxy-4-phenylpiperidino)-1-propanol (CP-101,606) is a noncompetitive antagonist of N-methyl-D-aspartate (NMDA) receptors containing the NR2B subunit. This compound was used to investigate the role of NR2B containing receptors in three responses to NMDA receptor activation in vivo. In mouse, CP-101,606 completely inhibited increases in fos-like immunoreactivity in dentate gyrus caused by a subconvulsant intraperitoneal dose of NMDA. In rat, the compound completely blocked cortical c-fos mRNA induction following focal injury in parietal cortex and the initiation and propagation of electrically induced cortical spreading depression. Inhibition of these responses by CP-101,606 indicates that c-fos induction and cortical spreading depression are dependent on activation of NMDA receptors containing the NR2B subunit. Since NMDA receptor dependent c-fos induction and cortical spreading depression may contribute to neuron loss after focal CNS injury, inhibition of these responses by CP-101,606 may contribute to the neuroprotective efficacy of the compound.     

Characterisation of the human NMDA receptor subunit NR3A glycine binding site

In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277-793nM)). Eight amino acids, which correspond to amino acids that are critical for ligand binding to other NMDA receptor subunits, situated within the S1S2 predicted ligand binding domain of hNR3A were mutated, which resulted in complete or near complete loss of [(3)H]-glycine binding to hNR3A. The NMDA NR1 glycine site agonist d-serine and partial agonist HA-966 (3-amino-1-hydroxypyrrolid-2-one), similarly to glycine displaced [(3)H]-glycine monophasically, suggesting a single common binding site. However, neither the partial agonist d-cycloserine nor the antagonist 7-chlorokynurenic acid displaced [(3)H]-glycine. Using homology modelling, a model of the NR3A binding pocket was generated which we suggest can be used to identify candidate agonists and antagonists. Our data show that glycine is a ligand, and most probably the endogenous ligand, for native NR3A at a binding site with unique pharmacological characteristics.     

Elevated concentrations of CRF in the locus coeruleus of depressed subjects.

Research evidence that corticotropin-releasing factor (CRF) plays a role in the pathophysiology of major depressive disorder (MDD) has accumulated over the past 20 years. The elevation of lumbar cerebrospinal fluid (CSF) concentrations of CRF decreased responsiveness of pituitary CRF receptors to challenge with synthetic CRF, and increased levels of serum cortisol in MDD subjects support the hypothesis that CRF is chronically hypersecreted in at least the endocrine circuits of the hypothalamic-pituitary-adrenal (HPA) axis and may also involve other CRF brain circuits mediating emotional responses and/or arousal. One such circuit includes the excitatory CRF input to the locus coeruleus (LC), the major source of norepinephrine in the brain. Furthermore, there are now reports of decreased levels of CRF in lumbar CSF from MDD patients after symptom relief from chronic treatment with antidepressant drugs or electroconvulsive therapy. Whether this normalization reflects therapeutic effects on both endocrine- and limbic-associated CRF circuits has not yet been effectively addressed. In this brief report, we describe increased concentrations of CRF-like immunoreactivity in micropunches of post-mortem LC from subjects with MDD symptoms as established by retrospective psychiatric diagnosis compared to nondepressed subjects matched for age and sex.     

Heterozygous deletion of NR1 subunit of the NMDA receptor alters ethanol-related behaviors and regional expression of NR2 subunits in the brain

NMDA receptors have been hypothesized to play a role in various aspects of ethanol-related phenotypes, notably in ethanol withdrawal. However, the role of each of the specific subunits remains unclear. To address this issue, mice that are heterozygous for the NR1 deletion, and thus have a reduction in functional NMDA receptors, were examined for ethanol consumption and acute ethanol withdrawal. Additionally, mice were examined for the level of vocalization following footshock, and behavior in an elevated plus maze, to determine their responses to stress. In these behavioral tests, NR1 heterozygous mice were shown to consume significantly higher levels of ethanol in the two bottle-choice test showing a possible role for this receptor in ethanol consumption. Analysis of acute withdrawal found that the heterozygous mice exhibit lower levels of handling-induced convulsions consistent with a role in ethanol sensitivity or withdrawal. In contrast, no effects on stress-related phenotypes were detected. Levels of NR2A-NR2D subunits of the NMDA receptor in specific brain regions were compared between NR1+/- mice and wild-type controls to assess whether the behavioral responses were specific to the diminution in NR1 expression or whether these changes could be due to secondary changes in expression of other NMDA subunits. Real-time quantitative PCR, Western blot and immunohistochemistry were used to examine expression levels in the hippocampus, neocortex, striatum and cerebellum. For the majority of the subunits, no differences were found between the wild-type and heterozygous mice in any of the brain regions. However, the NR2B subunit exhibited differences in expression of RNA in the hippocampus and protein levels in multiple brain regions, between wild-type and NR1+/- mice. These results show that NR1 plays a role, through mechanisms as yet unknown, in the expression of NR2 subunits in a region and subtype specific manner. This provides evidence of the effects of altered levels of NR1 expression on ethanol withdrawal and consumption, and suggests that concomitant changes in the levels of NR2B may contribute to that effect.     

Antagonists selective for NMDA receptors containing the NR2B subunit

In the late 1980s, a new class of N-methyl-D-aspartate (NMDA) receptor antagonists, exemplified by the phenylethanolamine ifenprodil (1), was identified. Initially, the mechanism of action of ifenprodil was a mystery as it was not a competitive antagonist at the glutamate or glycine (co-agonist) binding sites, nor was it a blocker of the calcium ion channel associated with the NMDA receptor. Early studies with a novel polyamine binding site associated with the NMDA receptor and functional studies in various brain regions suggested a unique and selective activity profile for 1. However, it was not until the NMDA receptor subunits were identified and expressed that ifenprodil was shown to be a selective antagonist for a subset of NMDA receptors containing the NR2B subunit. The wide range of potential therapeutic targets for NMDA antagonists coupled with the hope that NR2B selective agents might possess an improved clinical safety profile compared to non-selective compounds has supported an aggressive effort to develop the structure-activity relationships (SAR) of NR2B selective antagonists. This SAR and the basic physiology of the NMDA receptor form the basis of this review.     

The NMDA receptor NR2B subunit: a valid therapeutic target for multiple CNS pathologies

The NMDAR2B subunit is the focus of increasing interest as a therapeutic target in a wide range of CNS pathologies, including acute and chronic pain, stroke and head trauma, drug-induced dyskinesias, and dementias. Due to significant pharmaceutical endeavor, an impressive collection of chemical leads has been developed which target the NR2B subunit, some of which appear to discriminate between closely related subtypes. We now have the benefit of a structural template for the ifenprodil binding site which should further improve future structure activity relationships. A growing appreciation of the likely extrasynaptic localisation of the NR2B receptor subtype and importance of NR2B protein modification, notably tyrosine phosphorylation, may explain its therapeutic importance. The apparent superior preclinical and clinical data for the second and third generation NR2B compounds is likely to reflect subtype selectivity, a unique mode of action and cellular location of the NR2B receptors in the CNS.     

脊髓c-Jun在NMDA受体NR2B亚基介导的大鼠吗啡镇痛耐受中的作用

目的探讨脊髓c-Jun在N-甲基-D-天冬氨酸(N-meth-yl-D-aspartate,NMDA)受体NR2B亚基介导的吗啡镇痛耐受中的作用。方法取Sprague-Dawley(SD)成年大鼠连续7 d鞘内注射吗啡10μl(1.5 g.L-1)建立慢性吗啡镇痛耐受模型。应用热水甩尾法测定甩尾潜伏期(疼痛指标)以观察痛反应变化。应用免疫组织化学染色法检测磷酸化c-Jun(p-c-Jun)和总c-Jun(t-c-Jun)的表达。结果鞘内注射吗啡7 d,可激活大鼠脊髓的c-Jun,表现为神经元内p-c-Jun表达升高;鞘内注射NR2B选择性拮抗剂10μl Ro256981(2 g.L-1)可以抑制慢性吗啡镇痛耐受时脊髓神经元c-Jun的激活,并明显拮抗吗啡镇痛耐受的形成。结论脊髓神经元c-Jun的磷酸化参与NMDA受体NR2B亚基介导的吗啡镇痛耐受。     

NMDA受体NR2B亚单位在慢性内脏痛觉敏化中的作用

目的探讨外周与脊髓NMDA受体NR2B亚单位在慢性内脏痛觉敏化中的作用。方法模型组大鼠出生后d8～15,每天接受一次伤害性结直肠扩张刺激,8wk龄后用腹壁撤退反射(AWR)评估大鼠肠道敏感性。对腰骶背根神经节及胸腰与腰骶脊髓背角神经元进行免疫组织化学染色,比较对照与模型大鼠NR2B的表达。并比较对照与模型两组大鼠腹腔注射AP-7(NMDA受体拮抗剂)前后原发传入神经对结直肠扩张刺激的反应。结果①模型组大鼠AWR评分显著增高。②模型大鼠脊髓背根神经节NR2B亚单位表达增强。③模型大鼠脊髓内脏相关神经元NR2B亚单位表达增强。④AP-7显著抑制模型大鼠腰骶传入神经纤维对结直肠刺激的反应。结论NMDA受体NR2B亚单位可能参与慢性内脏痛外周与脊髓痛觉敏化的过程。     

NMDA receptor 2C subunit is selectively decreased by MK-801 in the entorhinal cortex

Administration of the non-competitive NMDA receptor antagonist MK-801 (5.methyl-10,11-dihydro-5H-dibenzo[1, d]cyclohepten-5-10-imine) produces paradoxicl neurotoxicity in limbic cortical regions which incluide the entorhinal cortex. The expression of NMDAR-2C but not -2A, -2B, or -2D subunits was significantly decreased in rat entorhinal cortex layer III following MK-801 administration. These results suggest an important role for the NMDAR-2C subunit in the response to MK-801-induced neurotoxicity in brain regions highly vulnerable to injury.     

Activation of NR2B-containing NMDA receptors is not required for NMDA receptor-dependent long-term depression

The triggering of both NMDA receptor-dependent long-term potentiation (LTP) and long-term depression (LTD) in the CA1 region of the hippocampus requires a rise in postsynaptic calcium. A prominent hypothesis has been that the detailed properties of this postsynaptic calcium signal dictate whether LTP or LTD is generated by a given pattern of synaptic activity. Recently, however, evidence has been presented that the subunit composition of the NMDA receptor (NMDAR) determines whether a synapse undergoes LTP or LTD with NR2A-containing NMDARs triggering LTP and NR2B-containing NMDARs triggering LTD. In the present study, the role of NR2B-containing synaptic NMDARs in the induction of LTD in CA1 pyramidal cells has been studied using the selective NR2B antagonists, ifenprodil and Ro25-6981. While both antagonists reduced NMDAR-mediated synaptic currents, neither prevented induction of LTD. These results demonstrate that activation of NR2B-containing NMDARs is not an absolute requirement for the induction of LTD in the hippocampus.     

Recent advances in the development of NR2B subtype-selective NMDA receptor antagonists

Over activation of the NMDA receptor complex has been implicated in a number of neurological conditions. The use of NMDA antagonists as therapeutic agents has been limited by serious cognitive and motor side effects. Significant efforts have been reported in the development of NR2B subtype-selective antagonists, which have shown efficacy without the side effects observed with nonspecific NMDA antagonists. Classical ifenprodil-like molecules containing benzyl- and phenylpiperidines attached to a phenol or an appropriate isostere by a linker have provided valuable chemical leads as potential therapeutic agents. In this review, recent efforts in the discovery and development of structurally unique NR2B subtype-selective NMDA antagonists that do not fit the classical "ifenprodil-like" pharmacophore will be discussed.     

选择性N-甲基-D-天门冬氨酸受体NR2B亚型阻滞剂的研究进展

选择性N-甲基-D-天门冬氨酸受体NR2B亚型阻滞剂近年来受到了越来越多的关注。目前，已报道的NR2B亚型阻滞剂的结构类型绝大多数仍为艾芬地尔发展而来的哌啶衍生物，另外还有酰胺、脒、氨基喹啉等结构类型；NR2B亚型阻滞剂在神经元保护、镇痛、抗药物依赖、抗帕金森病等方面表现了潜力。现根据不同结构类型综述NR2B亚型阻滞剂的结构及药理活性，并探讨其毒副作用较低的可能原因和临床应用潜力。