N-甲基-D-天冬氨酸受体拮抗剂类药物在脑缺血损伤中的应用进展

综述了近年来N-甲基-D-天冬氨酸受体拮抗剂治疗脑缺血的应用进展，并从不同作用位点分别对各类药物特点作了阐述。     

D-环丝氨酸促进大鼠条件性恐惧消退记忆的长期保持

目的研究D-环丝氨酸(D-serine,DCS)对条件性恐惧消退记忆的长时保持的影响。方法成年雄性SD大鼠40只,采用完全随机方法分为空白对照组、消退对照组、消退训练组、消退训练前DCS干预组、消退训练后DCS干预组,每组8只。在消退训练后3 d时进行僵立行为测试与旷场行为测试。结果①消退训练组的僵立时间百分比[(39.77±22.69)%]显著低于消退对照组[(61.46±11.10)%](P<0.05),但仍高于空白对照组[(2.82±1.27)%](P<0.01);旷场测试中消退训练组的中央路程百分比及直立次数[(2.82±2.89)%,(7±4)]显著高于消退对照组[(0.50±0.84)%,(3±4)],但低于空白对照组[(19.29±6.08)%,(20±6)](P<0.05);②消退训练后DCS干预组的僵立时间百分比与旷场行为测试成绩与其他组比较均有统计学差异(P<0.05)。结论消退训练后给予D-环丝氨酸能够促进条件化恐惧消退记忆的长时保持。     

Protective effect of gan mai da zao decoction in unpredictable chronic mild stress-induced behavioral and biochemical alterations

Aim: Growing evidence indicates that the glutamatergic system, especially the abnormalities of glutamate and N-methyl-D-aspartate (NMDA) receptors contribute to the pathophysiology and possibly the pathogenesis of major depressive disorders. This study is to evaluate the effect of gan mai da zao (GMDZ) decoction on glutamate and NMDA receptor in unpredictable chronic mild stress (UCMS) rats.

Materials and methods: Sucrose preference test and open field test were used to estimate the depressive-like behaviors of UCMS rats. Glutamate levels and NMDA receptor subunits (NR1, NR2A and NR2B) in the frontal cortex and hippocampus were determined by HPLC-FLD and by western-blot respectively.

Results: 32 days UCMS induced depressive-like behaviors, increased glutamate concentration and decreased NMDA receptor subunits NR2A and NR2B in the frontal cortex and hippocampus of rats. However, NR1 expression remained constant in stressed rats compared with normal. The GMDZ decoction alleviated the depressive-like behavior, decreased glutamate level, and increased expression of NMDA receptor subunit NR2A and NR2B in the frontal cortex and hippocampus of stressed rats.

Conclusions: These results suggest that GMDZ treatment reversed chronic unpredictable stress-induced depressive-like behaviors in UCMS rats, possibly via reducing glutamate levels and increasing the NMDA receptor subunits NR2A and NR2B in frontal cortex and hippocampus.     

N-甲基-D-天冬氨酸受体2B亚基拮抗剂对帕金森病运动并发症大鼠行为及相关信号蛋白的影响

目的 探讨N-甲基-D-天冬氨酸受体2B亚基(NR2B)拮抗剂CP-101.606对帕金森病运动并发症大鼠的行为效应及相关信号蛋白的影响.方法 将6-羟多巴胺立体定向注射于大鼠前脑内侧束建立帕金森病(PD)动物模型.对模型成功的PD大鼠接受每日2次左旋多巴(每公斤体质量50mg左旋多巴加每公斤体质量12.5mg苄丝肼)腹腔注射,持续22d.在第23天左旋多巴注射前,PD大鼠给予CP-101.606处理.评估旋转反应时间,采用蛋白印迹法检测纹状体区磷酸化NR2B、磷酸化钙调激酶Ⅱ( CaMKⅡ)及磷酸化谷氨酸受体1( GluR1 S831)表达情况.结果 NR2B拮抗剂CP-101.606逆转了左旋多巴所诱导的PD大鼠旋转时间的缩短;左旋多巴长期作用后引起磷酸化NR2B及下游相关信号蛋白磷酸化CaMKⅡ与磷酸化GluR1 S831升高至(145.3±6.5)％、(132.5±5.7)％及(105.6±6.3)％,而CP-101.606能下调三者磷酸化水平,其表达量分别为(102±4.9)％、(98.4±3.9)％与(49.5±4.2)％.结论 NR2B磷酸化可能通过介导N-甲基-D-天冬氨酸(NMDA)受体功能上调并激活其下游相关信号蛋白参与了帕金森病运动并发症的发生,抑制NR2B的药物可能是治疗PD运动并发症的一种新的治疗方式.     

Can we increase the speed and efficacy of antidepressant treatments? Part II. Glutamatergic and RNA interference strategies

In the second part we focus on two treatment strategies that may overcome the main limitations of current antidepressant drugs. First, we review the experimental and clinical evidence supporting the use of glutamatergic drugs as fast-acting antidepressants. Secondly, we review the involvement of microRNAs (miRNAs) in the pathophysiology of major depressive disorder (MDD) and the use of small RNAs (e.g.., small interfering RNAs or siRNAs) to knockdown genes in monoaminergic and non-monoaminergic neurons and induce antidepressant-like responses in experimental animals.The development of glutamatergic agents is a promising venue for antidepressant drug development, given the antidepressant properties of the non-competitive NMDA receptor antagonist ketamine. Its unique properties appear to result from the activation of AMPA receptors by a metabolite [(2 S,6 S;2 R,6 R)-hydroxynorketamine (HNK)] and mTOR signaling. These effects increase synaptogenesis in prefrontal cortical pyramidal neurons and enhance serotonergic neurotransmission via descending inputs to the raphe nuclei. This view is supported by the cancellation of ketamine's antidepressant-like effects by inhibition of serotonin synthesis.We also review existing evidence supporting the involvement of miRNAs in MDD and the preclinical use of RNA interference (RNAi) strategies to target genes involved in antidepressant response. Many miRNAs have been associated to MDD, some of which e.g., miR-135 targets genes involved in antidepressant actions. Likewise, SSRI-conjugated siRNA evokes faster and/or more effective antidepressant-like responses. Intranasal application of sertraline-conjugated siRNAs directed to 5-HT_1A receptors and SERT evoked much faster changes of pre- and postsynaptic antidepressant markers than those produced by fluoxetine.     

Involvement of adenosine and glutamate receptors in the induction of c-fos in the striatum by haloperidol

The psychostimulant drugs amphetamine and cocaine induce the expression of immediate early genes, such as c-fos, in the striatum via D₁ dopamine receptor activation. This occurs primarily in the striato-nigral neurons. Conversely, neuroleptic drugs, such as haloperidol, which block D₂-type dopamine receptors, induce c-fos expression in striatal neurons projecting to the globus pallidus. In order to gain insight into the neurochemical substrates of neuroleptic-induced c-fos expression, we examined the effects of adenosine A₂ and N-methyl-D-aspartate (NMDA) receptor antagonists as well as inhibition of nitric oxide synthase, on haloperidol-induced Fos immunoreactivity in the striatum. While blockade of D₁ receptors had no effect on haloperidol-induced Fos expression, adenosine A₂ receptor antagonists decreased the number of neurons in the striatum expressing haloperidol-induced Fos by half. NMDA receptor antagonists also potently blocked the induction of Fos immunoreactivity by haloperidol, while inhibition of nitric oxide synthase activity had no effect. These results indicate that in the presence of a dopamine D₂ antagonist, Fos expression in striato-pallidal neurons is mediated in part through activation of A₂ receptors by adenosine, and via NMDA receptor activation by glutamate. © 1996 Wiley-Liss, Inc.     

Activation of Class II or III Metabotropic Glutamate Receptors Protects Cultured Cortical Neurons Against Excitotoxic Degeneration

Trans-1-aminocyclopentane-1,3-dicarboxylic acid, a mixed agonist of all metabotropic glutamate receptor (mGluR) subtypes, is known to produce either neurotoxic or neuroprotective effects. We have therefore hypothesized that individual mGluR subtypes differentially affect neurodegenerative processes. Selective agonists of subtypes which belong to mGluR class II or III, such as (2_s, 1′_R,2′_R,3′_R)-2-(2,3-dicarboxycyclopropyl)-glycine (DCG-IV) (specific for subtypes mGluR2 or 3) or L-2-amino-4-phosphonobutanoate and L-serine-O-phosphate (specific for subtypes mGluR4, 6 or 7), were highly potent and efficacious in protecting cultured cortical neurons against toxicity induced by either a transient exposure to N-methyl-D-aspartate (NMDA) or a prolonged exposure to kainate. In contrast, agonists that preferentially activate class I mGluR subtypes (mGluR1 or 5), such as quisqualate or trans-azetidine-2,3-dicarboxylic acid, were inactive. DCG-IV was still neuroprotective when applied to cultures after the toxic pulse with NMDA. This delayed rescue effect was associated with a reduction in the release of endogenous glutamate, a process that contributes to the maturation of neuronal damage. We conclude that agonists of class II or III mGluRs are of potential interest in the experimental therapy of acute or chronic neurodegenerative disorders.     

Autoradiographic localization of dopamine uptake sites in the rat brain with3H-GBR 12935

Summary The firing rate and terminal excitability of identified nigrostriatal dopamine (DA) neurons was determined before, and over a 10–15 min period following, direct intrastriatal administration of the glutamate (GLU) agonist NMDA, or saline. NMDA (0.025 and 0.075 mol) produced a short latency increase in DA cell firing rate. In 7/8 cases, this increase in firing rate was accompanied by a profound reduction in terminal excitability. The decrease in excitability usually outlasted the increase in firing rate (sometimes by more than 8 min), and was superseded at a later stage by a marked increase in excitability. None of these effects were seen with saline (n=5), and they could all be blocked by preadministration of the competitive NMDA antagonist AP-7 (0.025 mol; n=6). The sequence of events leading to the observed results is argued to be as follows; NMDA initially excites striatal efferents to the DA cell, which through disinhibition and direct stimulation increase DA cell firing rate. Increased firing rate leads to enhanced striatal DA release. Dopamine's inhibitory influence pre-empts any effect NMDA itself may have on the terminals of nigrostriatal neurons, and counteracts NMDA's stimulatory effect on striatal output cells. Furthermore, the marked reduction in terminal excitability suggests that DA becomes the dominant influence in the striatum for a time. Hence, the net outcome of the injection is augmented striatal DA tone. Later, the effect of residual NMDA becomes predominant once more.     

The Role of N-Methyl-D-Aspartate-Type Glutamatergic Neurotransmission in the Photic Induction of Immediate-Early Gene Expression in the Suprachiasmatic Nuclei of the Syrian Hamster

This study investigated the role of N-methyl-D-aspartate (NMDA)-type glutamatergic neurotransmission in mediating the photic induction of immediate-early gene expression in the Suprachiasmatic nucleus (SCN) of the Syrian hamster. Activation of c-fos, c-jun and egr-1 was assessed by immunocytochemical detection of their protein products. To characterize the circadian basis to the inductive effects of light, hamsters were allowed to free-run in constant dim red light and received a 1 h light pulse at different circadian phases relative to activity onset (defined as CT 12). In control animals which did not receive light pulses, c-fos and egr-1 expression was absent or restricted to a small area of the dorsolateral region of the SCN, and expression of c-jun could not be detected in the SCN. In hamsters killed after presentation of a light pulse at either CT 14 or CT 20, there was a marked increase in c-fos and egr-1 immunoreactivities throughout the ventrolateral division of the SCN. In contrast, light pulses given at CT4 or CT 8 failed to activate immediate-early gene expression. Light pulses did not induce c-jun immunoreactivity at any circadian phase tested. Staining for c-fos was maximal 1 h after the start of the light pulse and had started to decline by 2 h. At this later time, c-jun expression was still undetectable. To compare the distribution of retinal afferents with that of c-fos induction, hamsters held on a light schedule of 16 h light: 8 h dark received an intraocular injection of cholera toxin-horseradish peroxidase conjugate 3 days before exposure to a 1 h light pulse given 2 h after lights off. Comparison of adjacent sections processed for c-fos immunoreactivity or for cholera toxin-horseradish peroxidase revealed that light-induced c-fos expression was precisely restricted to retinal terminal fields in the SCN. Light pulses also induced c-fos expression in the retinoreceptive ventral lateral geniculate nucleus and intergeniculate leaflet but not in the retinal fields of the dorsal lateral geniculate nucleus, indicating that the expression of cfos in response to light is spatially specific. The aim of the subsequent experiments was to investigate the role of NMDA-type glutamatergic neurotransmission in mediating the effects of light on c-fos expression in the SCN. To determine whether NMDA had the potential to activate c-fos expression in the SCN, hamsters were infused with 2.5 nmol NMDA or vehicle via an intracerebroventricular (icv) cannula positioned adjacent to the nuclei. In contrast to the effects of light, icv NMDA activated c-fos expression at both CT8 and CT 14. The distribution of immunoreactivity was more widespread than that observed after light, extending throughout the SCN and adjacent hypothalamus. To test whether NMDA receptors had a physiological role in the photic response, hamsters were treated systemically with the non-competitive NMDA antagonist MK801 (dose range 0.6 to 6.0 mg/kg body wt, ip) or vehicle prior to exposure to a 1 h light pulse given at CT 14 or CT 20. Expression of c-fos was still detectable in the dorsolateral SCN but MK801 blocked expression in the ventral portion of the retinoreceptive zone of the SCN. MK801 (10 or 100 nmol) delivered centrally (icv) also prevented light-induced c-fos expression in the ventral region of the SCN bordering the optic chiasm, though staining again persisted in the dorsolateral region. The induction of c-fos by icv NMDA, and the partial blockade of light-induced c-fos expression by the antagonist MK801, are consistent with the hypothesis that glutamate mediates the effects of light on SCN activity. However, the persistent photic induction of c-fos expression in a subfield of retinal afferents following treatment with MK801 suggests that other, non-NMDA-type mechanisms may contribute to photic entrainment.     

Interleukin-1beta exacerbates and interleukin-1 receptor antagonist attenuates neuronal injury and microglial activation after excitotoxic damage in organotypic hippocampal slice cultures

The effects of interleukin (IL)-1beta and IL-1 receptor antagonist (IL-1ra) on neurons and microglial cells were investigated in organotypic hippocampal slice cultures (OHSCs). OHSCs obtained from rats were excitotoxically lesioned after 6 days in vitro by application of N-methyl-D-aspartate (NMDA) and treated with IL-1beta (6 ng/mL) or IL-1ra (40, 100 or 500 ng/mL) for up to 10 days. OHSCs were then analysed by bright field microscopy after hematoxylin staining and confocal laser scanning microscopy after labeling of damaged neurons with propidium iodide (PI) and fluorescent staining of microglial cells. The specificity of PI labeling of damaged neurons was validated by triple staining with neuronal and glial markers and it was observed that PI accumulated in damaged neurons only but not in microglial cells or astrocytes. Treatment of unlesioned OHSCs with IL-1beta did not induce neuronal damage but caused an increase in the number of microglial cells. NMDA lesioning alone resulted in a massive increase in the number of microglial cells and degenerating neurons. Treatment of NMDA-lesioned OHSCs with IL-1beta exacerbated neuronal cell death and further enhanced microglial cell numbers. Treatment of NMDA-lesioned cultures with IL-1ra significantly attenuated NMDA-induced neuronal damage and reduced the number of microglial cells, whereas application of IL-1ra in unlesioned OHSCs did not induce significant changes in either cell population. Our findings indicate that: (i) IL-1beta directly affects the central nervous system and acts independently of infiltrating hematogenous cells; (ii) IL-1beta induces microglial activation but is not neurotoxic per se; (iii) IL-1beta enhances excitotoxic neuronal damage and microglial activation and (iv) IL-1ra, even when applied for only 4 h, reduces neuronal cell death and the number of microglial cells after excitotoxic damage.