激动NMDA受体甘氨酸位点可改善皮质酮致海马长时程增强损伤

目的研究N-甲基-D-天冬氨酸(NMDA)受体甘氨酸位点与皮质酮(CORT)致长时程增强(LTP)损伤之间的关系。方法应用雄性BALB/c小鼠,采用皮下注射CORT50 mg·kg~(-1)造模,60 min后进行电生理实验。NMDA受体甘氨酸位点激动剂300 nmol/只D-丝氨酸,300 nmol/只L-丝氨酸,100 nmol/只甘氨酸(侧脑室注射)和100 mg·kg~(-1)D-氨基酸氧化酶抑制剂MPC(腹腔注射)在CORT前30 min注射,1 mg·kg~(-1)NMDA受体甘氨酸位点拮抗剂L-701,324在D-丝氨酸和MPC前30 min腹腔注射。结果与正常小鼠相比,CORT可显著损伤小鼠海马的LTP(P<0.01),D-丝氨酸、L-丝氨酸、甘氨酸和MPC均可显著改善CORT引起的海马LTP损伤(P<0.01),甘氨酸位点拮抗剂L-701,324可阻断D-丝氨酸和MPC对CORT致海马LTP损伤的改善作用(P<0.01)。结论激动NMDA受体甘氨酸位点可改善CORT致海马LTP损伤,提示CORT引起的LTP损伤可能与NMDA受体功能下降相关。     

异氟烷催眠、镇痛作用与NMDA受体甘氨酸位点的关系

目的分析异氟烷催眠、镇痛作用与NMDA受体甘氨酸位点的关系。方法建立小鼠异氟烷注射催眠、镇痛模型后,在小鼠催醒、甩尾、福尔马林实验中,观察侧脑室或鞘内注射NMDA受体甘氨酸位点的激动剂D-丝氨酸(D-Serine,D-Ser)后小鼠睡眠时间、甩尾潜伏期或累计舔足时间的变化;用免疫组化方法观察异氟烷及鞘内用药对福尔马林小鼠脊髓Fos蛋白表达的影响。结果侧脑室注射D-Ser对异氟烷的催眠时间无影响(P>0.05)。鞘内注射D-Ser(0.025、0.05、0.1ng)可拮抗甩尾实验、福尔马林实验Ⅰ相中异氟烷的镇痛作用(P<0.05,P<0.01),而对福尔马林实验Ⅱ相异氟烷的镇痛作用无影响(P>0.05)。鞘内注射D-Ser0.05ng可拮抗异氟烷对福尔马林小鼠脊髓Fos蛋白表达的抑制作用(P<0.01)。结论异氟烷催眠作用与脑内NMDA受体甘氨酸位点关系不大;脊髓NMDA受体甘氨酸位点介导异氟烷对热、化学刺激的镇痛作用,而与异氟烷对慢性炎性疼痛的镇痛作用无明显关系。     

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。     

D-丝氨酸在中枢神经系统的作用研究进展

长期以来,人们普遍认为D型氨基酸在高等生物中没有功能。但最近10年的研究表明,D丝氨酸不但能够被胶质细胞合成和分泌,而且能够作为共激活因子,作用于NMDA受体的“甘氨酸位点”,参与该受体的激活。该文对D丝氨酸在中枢神经系统的产生、代谢和作用进行了简要介绍。     

中央内侧丘脑核中 NMDA 受体参与丙泊酚致意识消失的作用

目的 探讨中央内侧丘脑核（CMT）中 N-甲基-D-天门冬氨酸（NMDA）受体在全身麻醉意识消失中的作用。方法 60 只大鼠微注射模型随机均分为 4 组： NMDA10 mmol/L 组、 NMDA20 mmol/L 组、 NMDA40 mmol/L 组及对照组 （C 组）, 丙泊酚麻醉后分别于前 3 组 CMT 内微量泵注 10、 20 和 40 mmol/L 的 NMDA 以及等体积生理盐水 （C组）。观察微注射后自主体动反应的发生率以及翻正反射恢复时间, 并通过组织学对微注射位点进行定位。结果 当注射位点在 CMT 中时, 与 C 组相比, 各浓度 NMDA 组大鼠麻醉后翻正反射恢复时间均明显缩短; 其中 NMDA20 mmol/L 与 40 mmol/L 组恢复时间短于 NMDA10 mmol/L 组, 且 NMDA20 mmol/L 与 40 mmol/L 组大鼠麻醉中自主体动的发生率显著高于 C 组 （均 P < 0.05）。当注射部位在 CMT 外时, 各浓度 NMDA 组翻正反射恢复时间均明显长于注射部位在 CMT 内者（P < 0.05）, 而各组间比较自主体动发生率与翻正反射恢复时间均无明显差异（P＞0.05）。结论 CMT 中的NMDA 受体参与了丙泊酚所导致的全身麻醉效应。     

大鼠鞘内注射AP5的抗内脏伤害作用

目的观察鞘内注射竞争性N-甲基-D-天冬氨酸（NMDA）受体拮抗剂2-氨基-5-磷酰基戊酸酯（AP5）对大鼠结直肠扩张（CRD）诱发的内脏伤害性刺激的影响及剂量关系。方法利用大鼠CRD模型诱导的内脏伤害性刺激,经鞘内引入药物,观察对腹壁撤回反射（AWR）达到3级时痛阈的影响。结果AP5剂量依赖地抑制CRD诱导的内脏伤害性刺激,并有时间作用特点。结论竞争性NMDA受体拮抗剂AP5可以抑制CRD诱导的内脏伤害性刺激,NMDA受体在内脏伤害性刺激脊髓水平的传递中起重要作用。     

吗啡依赖大鼠脊髓和脑干μ受体和κ受体mRNA表达的研究

目的 观察吗啡依赖戒断时大鼠脊髓和脑干μ受体和Κ受体mRNA表达以及毒蕈碱受体拮抗剂、NMDA受体拮抗剂和NOS抑制剂对些基因表达的影响。方法 用逆转录聚合酶链反应（RT－PCR）,以β-actin mRNA为内标检测μ受体和Κ受体mRNA的表达水平。结果 吗啡依赖大鼠脊髓和脑干μ受体mRNA表达明显升高,纳洛酮激发大鼠戒断反应1h后μ受体基因表达降低,4h后接近正常组,而脊髓和脑干Κ受体基因的变化与μ受体基因表达趋势相反。鞘内注射PKA抑制剂Rp-cAMPs和蛋白磷酸酶抑制剂calyculinA可以明显减少脊髓和脑干中μ受体和Κ受体基因表达,而PKA激活剂Sp-cAMPs则无明显影响;经NOS抑制剂l-N-硝基精氨酸甲酯（l-NAME)处理后,脊髓μ受体和Κ受体基因表达明显减少,经毒蕈碱（M）受体拮抗剂甲基东莨菪碱处理后,脊髓μ受体和脑干Κ受体基因表达也明显减少,而脊髓Κ受体和脑干μ受体基因表达变化不明显;经NMDA拮抗剂MK801和M1受体拮抗剂哌拉唑嗪处理后,脊髓和脑干μ受体和Κ受体基因表达较戒断1h组无明显差异。脊髓和脑干中β-actin基因表达各处理组之间没有差别。结论 吗啡依赖和戒断动物脊髓和脑干中μ受体和Κ受体基因表达发生改变,M受体拮抗剂和NOS抑制剂在吗啡戒断反应时减少μ受体和Κ受体基因表达可能是它们有效控制吗啡戒断症状的机制之一。     

强啡肽A（1－17）对大鼠脊髓组织钙调蛋白含量和磷酸二酯酶活性的影响

观察强啡肽Ａ（１－１７）经大鼠蛛网膜下腔注射后对胞内Ｃａ２＋受体钙调蛋白（ＣａＭ）含量及其依赖于Ｃａ２＋／ＣａＭ的磷酸二酯酶（ＰＤＥ）活性的影响。结果表明：强啡肽Ａ（１－１７）１０、２０ｎｍｏｌ给药１０ｍｉｎ可使脊髓组织ＣａＭ含量和ＰＤＥ活性明显下降，呈量效依赖关系，２ｈ后均有不同程度的恢复。选择性ｋ型阿片受体拮抗剂ｎｏｒ－ＢＮＩ３０ｎｍｏｌ、兴奋性氨基酸ＮＭＤＡ受体特异性拮抗剂ＡＰＶ１０ｎｍｏｌ可显著对抗强啡肽Ａ（１－１７）２０ｎｍｏｌ降低脊髓组织ＣａＭ含量的作用并完全阻断强啡肽Ａ（１－１７）对ＰＤＥ活性的抑制；Ｌ型Ｃａ２＋通道阻断剂异搏定１００ｎｍｏｌ亦可部分阻断强啡肽Ａ（１－１７）２０ｎｍｏｌ对脊髓组织ＣａＭ含量和ＰＤＥ活性的影响。     

关于NMDA受体亚型介导的兴奋性毒性与神经保护剂的研究

兴奋性毒性涉及多种神经病理过程，理解这一涉及细胞死亡的途径对于许多疾病的临床治疗具有关键性作用。早期研究显示拮抗N－甲基－D－天冬氨酸（NMDA）受体具有神经保护作用，使得该受体已成为兴奋性毒性研究的重点。运用分子生物学方法发现NMDA受体是一个由多种不同亚基组成的杂合体，多种新的潜在治疗靶点已被揭示，并且发展了几个模型用于研究NMDA受体介导的细胞死亡。本文讨论了这些模型以及NMDA受体亚型与兴奋性毒性之间关系的新认识。     

鞘内注射NMDA拮抗吸入麻醉药的镇痛作用

目的探讨脊髓NMDA受体与吸入麻醉药安氟醚、异氟醚、七氟醚镇痛作用的关系。方法建立小鼠注射吸入麻醉药镇痛模型,用热板法和扭体法实验分别观察鞘内注射(it)不同剂量的NMDA对其痛阈的影响。结果NMDA2.5、5、10 ng it对清醒小鼠热板法痛阈(Pain threshold in hot-p late test,HPPT)和扭体次数无明显影响(P>0.05);NMDA2.5、5、10 ng it可剂量依赖性地减少安氟醚、异氟醚、七氟醚镇痛小鼠的HPPT(P<0.05)和增加扭体反应的次数(P<0.05)。结论脊髓的NMDA受体是吸入麻醉药安氟醚、异氟醚、七氟醚镇痛作用的重要靶位。     

Activation of supraspinal NMDA receptors by both D-serine alone or in combination with morphine leads to the potentiation of antinociception in tail-flick test of rats

Although there is a variety of information concerning the effects of the N-methyl-D-aspartate (NMDA) receptor on opioid-induced antinociception at the spinal level, little is known about the effects at the supraspinal level. To clarify the role of the NMDA receptor on the morphine-induced antinociception at the supraspinal level, we investigated the effects of the intracerebroventricular (i.c.v.) administration of D-serine, a selective agonist for the glycine site of the NMDA receptors, alone or in combination with morphine using the tail-flick test. The i.c.v. administration of D-serine, but not L-serine, alone produced a dose-dependent antinociception in the tail-flick response. D-Serine also dose-dependently potentiated the antinociceptive effect induced by the i.c.v. administration of morphine and the simultaneous administration produced an additive effect. The potentiation of the antinociception produced by both D-serine alone or in combination with morphine was dose-dependently attenuated by the i.c.v. administration of L-701,324, a selective antagonist for the glycine site of the NMDA receptors. In addition, the potentiation of the D-serine-induced antinociception was antagonized by the i.c.v. administration of naloxone, a nonselective opioid receptor antagonist. These observations, together with the fact that D-serine is an endogenous and selective co-agonist for the glycine site of the NMDA receptors, strongly suggested that the activation of the supraspinal NMDA receptors by D-serine leads to the potentiation of the antinociception in the tail-flick test and that endogenous D-serine could modulate the mu-opioid receptor mediated antinociception via the glycine site of the NMDA receptors at the supraspinal level.     

Modulation of the NMDA receptor by D-serine in the cortex and the spinal cord, in vitro

We present a comparative study of the modulation of the N-methyl-D-aspartate (NMDA) receptor at the strychnine-insensitive glycine site in the spinal cord and in the cortex. The excitatory effect of NMDA was potentiated by D-serine (a glycine mimetic) in the hemisected rat spinal cord. The non-competitive NMDA antagonists 7-chlorokynurenic acid (7-Cl KYNA; 10 microM) and 3-amino-1-hydroxypyrrolid-2-one (HA-966; 100 or 200 microM) antagonized the effect of NMDA in the spinal cord and cortical wedge preparation. The antagonism was reversed by the addition of D-serine. This effect was strychnine-insensitive and hence not related to the inhibitory glycine receptor known to be present in the spinal cord. Our results suggest strongly that glycine positively modulates the NMDA system not only at a supraspinal level but also at the spinal level. As the positive modulation of NMDA responses by D-serine was also seen in the presence of tetrodotoxin, we conclude that the NMDA/glycine complex is (also) located on motoneurones in addition to the known glycine-mediated inhibitory system.     

Neuropharmacological characterization of 1-aminocyclopropane-1-carboxylate and 1-aminocyclobutane-1-carboxylate, ligands of the N-methyl-D-aspartate-associated glycine receptor

Following intravenous administration, 1-aminocyclobutane-1-carboxylate (ACBC, 100 mg/kg), a N-methyl-D-aspartate (NMDA)-associated glycine receptor antagonist, was eliminated with a T1/2 of 5 min in mouse brain and 4 min in rat cerebrospinal fluid (CSF). 1-Aminocyclopropane-1-carboxylate (ACC), a NMDA-associated glycine receptor agonist, was found to have a T1/2 of less than 5 min in mouse brain. ACC and ACBC did not alter basal cerebellar cGMP. Glycine and D-serine increased cGMP, and 1-hydroxy-3-aminopyrrolidone-2 (HA-966), a glycine antagonist, reversed the D-serine-induced increases in cGMP. In contrast, ACBC did not reverse the D-serine-induced increases in cGMP. These data suggest that despite their brain bioavailability and marked potency at the glycine receptor in vitro, ACC and ACBC are rapidly inactivated and thus have limited in vivo utility.     

D-丝氨酸对N-甲基-D-天冬氨酸诱导大鼠海马cGMP释放作用的影响

The microdialysis technique was utilized to study the effects of exogenous D-serine on the cGMP level in the perfusate of rat hippocampus. Addition of N-methyl-D-aspartate (NMDA) 250 μmol·L^-1 to the perfusate resulted in a striking increase of cGMP in perfusate. The cGMP response elicited by NMDA was prevented by 7-chlorokynurenic acid (7-CKA), a selective antagonist at the glycine site of the NMDA receptor. D-serine completely reversed the antagonism by 7-CKA of the NMDA-evoked cGMP response. The NMDA-evoked cGMP response was enhanced by D-serine. D-serine alone was inactive. The data show that D-serine, administered to freely-moving rats, behaves as a selective agonist at the glycine site of hippocampus NMDA receptor. Furthermore, these results suggest that the glycine modulatory site may not be fully saturated in vivo, and thus can serve to regulate NMDA receptor function.     

Both agonists and antagonists of the strychnine-insensitive glycine site of N-methyl-D-aspartate receptors modulate polysynaptic excitations in slices of mouse olfactory cortex

Summary In this study, it is reported that bath application of D-serine and, to a lesser extent glycine, potentiated polysynaptic but not monosynaptic excitations evoked in slices of mouse olfactory cortex perfused with solution containing Mg²⁺ (1 mmol/l), picrotoxin and strychnine (both 25 μmol/l). Effects were largely confined to the longer latency components of the field potentials and occurred at amino acid concentrations of between 0.01 and 1 mmol/l. The effects of D-serine and glycine were antagonized by 7-chlorokynurenate and indole-2-carboxylate, antagonists of the glycine regulatory site of the N-methyl-D-aspartate (NMDA) receptor complex. D-Serine (glycine not tested) also potentiated, and 7-chlorokynurenate partially inhibited the longer latency components of the polysynaptic field potentials evoked in slices perfused in the absence of picrotoxin and strychnine. However, neither D-serine nor glycine potentiated responses evoked by the bath application of NMDA. It is concluded that under the present experimental conditions, the glycine regulatory sites of those NMDA receptor involved in the mediation of polysynaptic excitations in the mouse olfactory cortex are not saturated with endogenous glycine.     

D-serine modulates the NMDA receptor/nitric oxide/cGMP pathway in the rat cerebellum during in vivo microdialysis

Several lines of investigation indicate that D-serine may be an endogenous ligand for the glycine site of N-methyl-D-aspartate (NMDA) receptors in some CNS regions. We here studied the in vivo effects of D-serine on the NMDA receptor/nitric oxide/cGMP pathway by monitoring extracellular cGMP in the cerebellum of freely-moving rats subjected to transcerebral microdialysis. Local application of NMDA (200, 500 μM) through the dialysis probe for 20 min evoked transient, concentration-dependent cGMP responses which peaked in the fraction of drug administration, the nucleotide levels returning to basal values after 40 min. The NMDA-induced elevation of the extracellular nucleotide was completely inhibited by the selective receptor channel blocker dizocilpine (MK-801) locally co-perfused at the concentration of 10 μM. The non-competitive antagonist had no effect on its own suggesting that endogenous glutamic acid does not tonically activate NMDA receptors. The effect of 200 μM NMDA was largely attenuated by 30 μM 7-chloro-kynurenic acid and completely abrogated when the concentration of the strychnine-insensitive glycine receptor antagonist was raised to 100 μM. D-serine (300 μM), perfused in the presence of 7-chloro-kynurenate (30 μM), was able to fully restore the NMDA (200 μM)-induced increase of cGMP extracellular levels. On the other hand, the D-amino acid directly potentiated in a concentration-dependent manner (0.3, 1 and 10 mM) the NMDA (200 μM)-evoked cGMP production whereas it was inactive on its own. These data show that in vivo the activation of the strychnine-insensitive glycine site is essential for the functioning of the NMDA receptor complex and can be activated by the selective agonist D-serine. They also confirm that cerebellar NMDA receptors do not have their glycine sites saturated. Received: 25 June 1996 / Accepted: 23 September 1996     

6,7-Dinitroquinoxaline-2,3-dione and 6-nitro,7-cyanoquinoxaline-2,3-dione antagonize responses mediated by N-methyl-D-aspartate and NMDA-associated glycine recognition sites in vivo: measurements of cerebellar cyclic-GMP

Direct intracerebellar administration of quisqualate resulted in marked increases in levels of cGMP in the cerebellum of the mouse, with a Hill number of 2.0. Quinoxalinediones, DNQX (6,7-dinitroquinoxaline-2,3-dione) and CNQX (6-nitro,7-cyanoquinoxaline-2,3-dione) attenuated the quisqualate-induced response. 6,7-Dinitroquinoxaline-2,3-dione also attenuated the D-serine-induced increases in levels of cGMP in a competitive manner. Intracerebellar injection of DNQX also antagonized the response to parenterally-administered harmaline. Similar results were also obtained with CNQX. These results indicate that these quinoxalinediones can attenuate the responses, mediated through the NMDA-associated glycine recognition sites, as well as the NMDA receptor complex. However, the glycine antagonist HA-966 (3-amino-1-hydroxypyrrolidone-2), at doses which completely reversed the increases induced by D-serine, failed to alter the response to quisqualate, indicating a lack of effect of glycine antagonists on quisqualate-mediated synaptic events. These results further support the interaction of the quinoxalinediones, DNQX and CNQX, with the NMDA receptor complex as established in receptor binding and electrophysiological studies.     

Effect of 7-chloro kynurenic acid on glycine modulation of the N-methyl-D-aspartate response in guinea-pig myenteric plexus

The glycine modulation of the N-methyl-D-aspartate (NMDA) response in guinea-pig myenteric plexus was investigated by using D-serine and 7-chloro kynurenic acid as a glycine agonist and antagonist, respectively. D-serine caused a concentration-dependent enhancement of the NMDA response, an effect which was competitively inhibited by 7-chloro kynurenic acid (pA2 = 6.0). In addition, 7-chloro kynurenic acid induced a concentration-dependent, non-competitive inhibition of the NMDA response per se, even in the absence of added D-serine. This inhibition was fully reversed by exogenous D-serine, suggesting that this effect was also due to the occupancy of the glycine site. These results emphasize the usefulness of the guinea-pig myenteric plexus for studying the function of the NMDA receptor complex.     

Intrathecally administered D-cycloserine produces nociceptive behavior through the activation of N-methyl-D-aspartate receptor ion-channel complex acting on the glycine recognition site

Intrathecal (i.t.) administration of D-cycloserine (100 and 300 fmol), a partial agonist of the glycine recognition site on the N-methyl-D-aspartate (NMDA) receptor ion-channel complex, produced a behavioral response mainly consisting of biting and/or licking of the hindpaw and the tail along with slight hindlimb scratching directed toward the flank in mice, which peaked at 5 - 10 min and almost disappeared at 15 min after the injection. The behavior induced by D-cycloserine (300 fmol) was dose-dependently inhibited by an intraperitoneal injection of morphine (0.5-2 mg/kg), suggesting that the behavioral response is related to nociception. The nociceptive behavior was also dose-dependently inhibited by i.t. co-administration of 7-chlorokynurenic acid (0.25-4 nmol), a competitive antagonist of the glycine recognition site on the NMDA receptor ion-channel complex; D-(-)-2-amino-5-phosphonovaleric acid (62.5-500 pmol), a competitive NMDA receptor antagonist; MK-801 (62.5-500 pmol), an NMDA ion-channel blocker; ifenprodil (0.5-8 nmol); arcaine (31-125 pmol); and agmatine (0.1-10 pmol), all being antagonists of the polyamine recognition site on the NMDA receptor ion-channel complex. However, [D-Phe7,D-His9]-substance P(6-11), a specific antagonist for substance P (NK1) receptors, and MEN-10,376, a tachykinin NK2-receptor antagonist, had no effect on D-cycloserine-induced nociceptive behavior. These results in the mouse spinal cord suggest that D-cycloserine-induced nociceptive behavior is mediated through the activation of the NMDA receptor ion-channel complex by acting on the glycine recognition site and that it does not involve the tachykinin receptor mechanism.