大脑皮层神经元NMDA受体的单通道特性

本文用膜片箝技术对机械分离培养的大鼠大脑皮层神经元胞体上的ＮＭＤＡ受体的单通道特性进行了研究,实验用细胞贴附和内面向外两种形式记录单离子通道的活动。电极液内含有ＮＭＤＡ或Ｌ－门冬氨酸时,在皮层神经元上常见电导为３５ｐＳ的离子通道。通道对Ｎａ＋,Ｋ＋非选择性通透,对Ｃｌ－不通透,其平均开放时间和开放概率随超极化程度增大而降低。开放、关闭时间及ｂｕｒｓｔ时程的分布直方图均需双指数拟合。Ｍｇ２＋以电压和浓度依赖性的方式减小通道开放时间,ＡＰＶ能阻断通道活动,温度降低使通道开放时间延长及电流幅度减小。本文结果表明大脑皮层神经元上ＮＭＤＡ受体通道活动自身具有电压依赖性,因此提示ＮＭＤＡ受体通道的正常功能活动可能依赖于某些细胞内调控过程的存在。     

低压低氧对胎鼠海马神经元NMDA受体影响的实验研究

目的：观察低压低氧环境对胎鼠海马神经元NMDA受体数目和通道特性的影响。方法：采用原位杂交和膜片钳观察NMDA受体的数量和功能。结果：胎鼠低压低氧后,NMDA受体数量和通道开放机率减少,通道开放时间常数减少,通道关闭时间常数增加。结论：低压低氧影响到胎鼠NMDA受体的发育,提示低压低氧环境下大鼠的学习记忆可能受到影响。     

大脑皮层神经元NMDA受全的单通道特性

本文和膜片箝技术对机械分离培养的大鼠大脑皮层神经元胞ＮＭＤＡ受体的单通道特性进行了研究,实验用细胞贴附和内面向外两种形式记录单离子通道的活动。电极液内含有ＮＭＤＡ或Ｌ－门冬氨酸时,在皮层神经元上常见电导为３５ｐＳ的离子通道。通道对Ｎａ＾＋,Ｋ＾＋非选择必通透,对Ｃｌ＾－不通透,其平均开放时间和开放概率随超极化程度增大而降低。开放、关闭时间及ｂｕｒｓｔ时程的分布直方图均需双指数拟合。Ｍｇ＾２＋以电压     

烟碱和槟榔碱对离体大鼠海马脑片CA1锥体细胞外诱发电位…

在离全Ｗｉｓｔａｒ大鼠海马脑片上,用微电极记录刺激顶树突层Ｓｃｈａｆｆｅｒ侧支引起的ＣＡ１区锥体细胞外群峰电位（ＰＳ）。观察用半浸式浴槽或多管微电极气压微量注射给予Ｎ受体激动剂烟碱（Ｎｉｃ）和Ｍ受体激动剂槟碱（Ａｒｅ）对ＰＳ的影响,探讨Ｍ和Ｎ受体功能。结果表明：Ｎｉｃ对ＰＳ幅度无明显影响,浓度过．０ｍｍｏｌ／Ｌ时约１／４脑片诱发出第二个群峰电位（ＰＳ２）,浓度增加,ＰＳ２发生率增加,ＰＳ２幅度较Ｐ     

棉铃虫核型多角体病毒p35基因的PCR扩增和克隆及其在原核细胞中的表达

用苜蓿银纹夜蛾核型多角体病毒（ Ａｃ Ｎ Ｐ Ｖ） 凋亡抑制基因ｐ３５ 的合成引物,从棉铃虫核型多角体病毒（ Ｈａ Ｎ Ｐ Ｖ） 基因组中用 Ｐ Ｃ Ｒ 扩增到了１ｋｂ 片段。采用 Ｐ Ｃ Ｒ－ 双脱氧核苷酸链终止银染法,测定了所扩增到的１ ０１０ｂｐ 全序列,发现其开放阅读框完整,长８９７ｂｐ ,编码２９９ 个氨基酸。用 Ｄ Ｎ Ａ Ｓ Ｉ Ｓ 和 Ｐ Ｒ Ｏ Ｓ Ｉ Ｓ 软件分析发现,此片段与 Ａｃ Ｍ Ｎ Ｐ Ｖ ｐ３５ 基因同源区核苷酸同源性为９１ ％ ,氨基酸同源性也达８１ ％ ,证明了所扩增的片段是 Ｈａ Ｎ Ｐ Ｖ 的ｐ３５ 基因。为了研究此ｐ３５ 基因的功能及其效应机制,克隆了这一基因并在大肠杆菌细胞中实现了表达。     

烟碱和槟榔碱对离体大鼠海马脑片CA_1锥体细胞外诱发电位的影响

在离体Ｗｉｓｔａｒ大鼠海马脑片上,用微电极记录刺激顶树突层Ｓｃｈａｆｆｅｒ侧支引起的ＣＡ＿１区锥体细胞外群峰电位（ＰＳ）。观察用半浸式浴槽或多管微电极气压微量注射给予Ｎ受体激动剂烟碱（Ｎｉｃ）和Ｍ受体激动剂槟榔碱（Ａｒｅ）对ＰＳ的影响,探讨Ｍ和Ｎ受体功能。结果表明：Ｎｉｃ对ＰＳ幅度无明显影响,浓度１．０ｍｍｏｌ／Ｌ时约１／４脑片诱发出第二个群峰电位（ＰＳ＿２）,浓度增加,ＰＳ＿２发生率增加,ＰＳ＿２幅度较ＰＳ低。美加明能部分对抗烟碱诱发的ＰＳ＿２,表明ＰＳ＿２出现与Ｎ受休关系密切。Ａｒｅ降低ＰＳ幅度,当浓度达到０．００１ｍｍｏｌ／Ｌ即能使ＰＳ幅度降低为正常的７４％,ＰＳ幅度降低存在有效量关系,低浓度阿托品能选择性地对抗之,表明Ａｒｅ所致ＰＳ幅度下降是通过兴奋Ｍ受体的结果。     

NO对大鼠睡眠-觉醒的调节

目的和方法：通过对大鼠侧脑室微量注射ＮＯＳ抑制剂Ｌ－ＮＡＭＥ及ＮＯ的前体Ｌ－精氨酸（Ｌ－Ａｒｇ）观察两种物质对大鼠睡眠－觉醒的影响。结果：注射１ｍｇ Ｌ－ＮＡＭＥ（５μＬ）后４ｈ觉醒（Ｗ）明显增加,尤以注射后第１￣２ｈ显著;４ｈ慢波睡眠（ＳＷＳ）明显减少,该效应同样以注射后第１￣２ｈ显著;异相睡眠（ＰＳ）无明显变化。小剂量Ｌ－ＮＡＭＥ（０．２ｍｇ,５μｌ）对大鼠的Ｗ、ＳＷＳ、ＰＳ无明显影响;同样方     

单耳气传导阻滞对下丘脑NMDA受体各亚基mRNA基因表达的影响

Ｎ 甲基 Ｄ 门冬氨酸 (Ｎ ｍｅｔｈｙｌ Ｄ ａｓｐａｒｔａｔｅ,ＮＭＤＡ)受体是中枢兴奋性氨基酸中兴奋性极强的一种。它与神经系统的一些最重要功能如突触可塑性、学习记忆、再生修复等有密切关系。本研究采用研究者本人设计的大鼠单侧气传导持续阻滞模型 (单侧外耳道皮下缝合术 ) ,应用ＲＴ ＰＣＲ技术 ,研究环境变化对生后发育过程中听觉中枢神经系统下丘ＮＭＤＡ受体ＮＲ1、ＮＲ2Ａ、ＮＲ2Ｂ和ＮＲ2ＣｍＲＮＡ基因表达的影响 ,以期为在分子水平上揭示神经系统功能发育可塑性的本质提供依据。1　方法(1)样本制备　怀孕ＳＤ大鼠取自上海…     

海马神经元乙酰胆碱激活通道在不同培养期的功能特性

用膜片箝技术对不同培养期的新生大鼠海马神经元上乙酰胆碱受体单通道特性进行了研究,结果表明不同培养期ＡＣｈ激活通道的电学特性不同。培养早期（１－２ｄ）,２０ｐｓ通道占优势,开放以单个短开放事件为主,平均开放时间小于２ｍｓ．培养后期（１８－２１ｄ）３１,ｐＳ通道为主,开放随膜片的不同可分成两类,即单个短开放（时间常数为０．３５ｍｓ和１．２９ｍｓ）和簇状开放（时间常数为１．１５ｍｓ和９．６ｍｓ）,同时也     

中缝大核在刺激视上核镇痛中的作用

运用核团灌流液的放免测定和高压液相色谱法以及核团内注射拮抗剂,观察了化学刺激下丘脑视上核（ＳＯＮ）对中缝大核（ＮＲＭ）灌流液内催产素（ＯＴ）、精氨酸加压素（ＡＶＰ）和５－羟色胺（５－ＨＴ）含量的影响以及ＮＲＭ内注射ＡＶＰ、５－ＨＴ或ＯＴ受体拮抗剂对痛阈（ＰＴ）的影响。结果表明：ＳＯＮ内注射Ｌ－谷氨酸（Ｌ－Ｇｌｕ）１０μｇ后动物痛阈明显升高,ＮＲＭ灌流液中ＯＴ和５－ＨＴ的含量明显高于对照组水平,ＡＶＰ的含量仅有一过性增加。ＮＲＭ内注射ｏＴ或５－ＨＴ拮抗剂可逆转化学刺激ＳＯＮ引起的镇痛作用;而ＡＶＰ的Ｖ＿(１／２)受体拮抗剂也轻度抑制这种镇痛作用,但Ｖ＿１拮抗剂对此作用无影响。以上结果提示：在刺激ＳＯＮ镇痛中,ＯＴ起着重要作用,Ｌ－Ｇｌｕ刺激ＳＯＮ的ＯＴ细胞释放ＯＴ,作用于ＮＲＭ细胞的ＯＴ受体和Ｖ＿２受体而产生镇痛作用,５－ＨＴ在此过程中也发挥重要作用。     

北京中医药大学基础医学院

目的:通过对更年期抑郁症模型大鼠行为学以及学习记忆的观察,探讨中药复方更逍遥冲剂的作用机制 方法:SD雌性大鼠40只,随机分为假手术组、更年期抑郁症模型组、中药中剂量组、中药大剂量组和西药对照组,观察开野实验中大鼠活动度、强迫游泳中大鼠的不动时间、以及通道式水迷宫检测大鼠学习记忆能力.结果:模型大鼠经过21d治疗后,开野实验水平积分、垂直积分明显增加,强迫游泳不动时间缩短,水迷宫实验游出时间缩短,错误次数减少.结论:更逍遥冲剂能够明显的改善更年期抑郁症大鼠的行为学评分,并具有改善其学习记忆能力的作用.     

Developmental regulation of NMDA receptor 2B subunit mRNA and ifenprodil binding in the zebra finch anterior forebrain

In passerine songbirds, song learning often is restricted to an early sensitive period and requires the participation of several discrete regions within the anterior forebrain. Activation of N-methyl-D-aspartate (NMDA) receptors is implicated in song learning and in one forebrain song region, the lateral magnocellular nucleus of the anterior neostriatum (IMAN), NMDA receptors decrease in density, their affinity for the antagonist MK-801 increases, and their currents decay more quickly as young male zebra finches lose the ability to imitate new song elements. These developmental changes in NMDA receptor pharmacology and physiology suggest that the subunit composition of NMDA receptors changes developmentally. Here, we have used in situ hybridization and [3H]ifenprodil receptor autoradiography to study the developmental regulation of the NMDA receptor 2B subunit (NR2B) within the anterior forebrain of male zebra finches. NR2B mRNA expression within the IMAN was twice as great in 30-day-old males (early in the sensitive period for song learning) as in adult males, and this developmental decrease in NR2B mRNA expression was mirrored by a decrease in high-affinity (NR2B-associated) [3H]ifenprodil binding within this song region. In another anterior forebrain song region, Area X, NR2B mRNA also declined significantly after 30 days posthatch, but this decline was not accompanied by a significant decrease in [3H]ifenprodil binding. The results are consistent with the hypothesis that developmental changes in NMDA receptor function mediated by regulation of subunit composition contribute to the sensitive period for vocal learning in birds.     

Ethanol potentiates and blocks NMDA-activated single-channel currents in rat hippocampal pyramidal cells

Single-channel currents activated by N-methyl-D-aspartate (NMDA) were characterized using the outside-out patch clamp technique in cultured hippocampal cells from the rat. Several conductance states were observed, and the main one of 47 pS was further analyzed for channel lifetime and frequency. Open times decreased with hyperpolarization of the membrane. In view of recent evidence linking NMDA receptors to central nervous system processes such as learning and memory and ethanol (EtOH) tolerance, the effects of EtOH (0.01-1%, v/v, or congruent to 1.74-174 mM) were studied in this preparation. Two effects of EtOH could be discerned: (i) at low concentrations (1.74-8.65 mM) an increase in the probability of opening (p open) of the NMDA-activated channel currents, without change in the mean channel open time, and (ii) at higher concentrations (86.5-174 mM) a decrease in p open with a concomitant decrease in the mean open time. It is suggested that EtOH, even at rather low concentrations, may affect important brain functions.     

Synaptic activity-dependent developmental regulation of NMDA receptor subunit expression in cultured neocortical neurons

The biophysical properties of NMDA receptors are thought to be critical determinants involved in the regulation of long-term synaptic plasticity during neocortical development. NMDA receptor channel properties are strongly dependent on the subunit composition of heteromeric NMDA receptors. During neocortical development in vivo, the expression of the NMDA receptor 2A (NR2A) subunit is up-regulated at the mRNA and protein level correlating with changes in the kinetic and pharmacological properties of functional NMDA receptors. To investigate the developmental regulation of NMDA receptor subunit expression, we studied NR2 mRNA expression in cultured neocortical neurons. With increasing time in culture, they showed a similar up-regulation of NR2A mRNA expression as described in vivo. As demonstrated by chronic blockade of postsynaptic glutamate receptors in vitro, the regulation of NR2A mRNA was strongly dependent on synaptic activity. In contrast, NR2B mRNA expression was not influenced by activity blockade. Moreover, as shown pharmacologically, the regulation of NR2A mRNA expression was mediated by postsynaptic Ca(2+) influx through both NMDA receptors and L-type Ca(2+) channels. It is interesting that even relatively weak expression of NR2A mRNA was correlated with clearly reduced sensitivity of NMDA receptor-mediated whole-cell currents against the NR2B subunit-specific antagonist ifenprodil. Developmental changes in the expression of NR1 mRNA splice variants were also strongly dependent on synaptic activity and thus might, in addition to regulation of NR2 subunit expression, contribute to developmental changes in the properties of functional NMDA receptors. In summary, our results demonstrate that synaptic activity is a key factor in the regulation of NMDA receptor subunit expression during neocortical development.     

海马内生长抑素与γ－氨基丁酸在大鼠穿梭箱主动回避反应中的作用

本实验以大鼠穿梭箱主动回避反应（ＡＡＲ）的习得和消退为学习记忆的指标,研究了海马内生长抑素（ＳＳ）和γ－氨基丁酸（ＧＡＢＡ）在学习记忆中的作用。结果如下：（１）经训练而建立了ＡＡＲ的大鼠,其海马内ＳＳ较对照组显著增高,而海马内ＧＡＢＡ含量却明显降低;（２）海马内注入ＳＳ的耗竭剂半胱胺（Ｃｙｓ,２０ｇ／Ｌ）使大鼠ＡＡＲ的习得受到明显损害,ＡＡＲ的消退显著加速,海马内ＳＳ明显降低,而ＧＡＢＡ含量却显著升高;（３）海马内注入ＧＡＢＡ（２００ｇ／Ｌ）使大鼠ＡＡＲ的消退显著加速的同时,其海马内ＳＳ含量亦显著降低。由此表明,海马内ＳＳ可能有促进学习记忆的作用,而海马内ＧＡＢＡ升高则有相反的效应;二者在海马调控学习记忆过程中具有重要作用。     

Effects of the neurogenesis stimulator Ro 25-6981 upon formation of spatial skill in adult rats depend on the term of its administration and the animals' ability to learn

Effect of administration of selective N-methyl-D-aspartate (NMDA) receptor antagonist Ro 25-6981 on learning and memory in a dose which is known to stimulate neoneurogenesis was assessed in adult rats with different abilities to formation of spatial skills in different time periods after the antagonist injection. Wistar male rats were trained to find hidden platform in the Morris water maze for 5 consecutive days. Rats' learning ability for spatial skill formation was evaluated depending on platform speed achievements. In re-training sessions (cues and platform location changed), it was found that all rats received Ro 25-6981 13 days before the re-training demonstrated impaired spatial memory. At the same time the inhibitor injected 29 days before re-training selectively facilitated the formation of spatial skill in animals with initially low learning abilities.     

适用于膜片钳研究的成年大鼠脑神经元急性分离法

研究一种结合酶消化和机械分离的方法,用于快速、可靠地急性分离成年大鼠海马神经元。此法可将实验大鼠的年龄提高到５００－６００ｄ,不损伤神经元的突触结构和细胞膜的电学特性。形态上有差异的细胞易于分辨。用膜片钳技术证实,链霉蛋白酶未破坏神经元上的Ｎ－甲基－Ｄ－门冬氨酸和乙酰胆碱受体通道,约有９５％的神经元可观测到内向电流活动,单通道电流的电导值分别为２０ｐＳ和３０ｐＳ。