氯硝安定治疗TD

对TD仍没有有效的药物治疗方法,虽然DA阻滞剂对抑制运动障碍症状有效,但可能会加重症状或妨碍TD的自然缓解。近年来对TD的治疗集中注意于γ-氨酪酸(GABA)的激动类药物。据动物实验研究认为,GABA能神经传出障碍可产生多动性不自主运动,黑质GABA减少对TD的病理生理起着重要作用。现已知GABA_B受体与抗运动障碍作用无关。有人首次(1979)对7例TD病人使用GABA_A激动剂蝇蕈醇(muscimol)后,运动障碍的症状平均减轻了45%。随后,使用其他直接或间接的GABA激动剂药物均获     

大鼠生后MGBv神经元主动膜特性的变化及GABA受体激动剂的影响

目的观察大鼠生后不同发育阶段内侧膝状体腹侧部(ventral of medial geniculate body，MGBv)神经元的主动膜特性的变化及GABA受体激动剂的影响。方法依大鼠MGB的发育特点将实验大鼠按出生后日龄分为出生后(postnatal day，P)3-7，8-11，12—20，21-30，四组，采用脑片膜片钳全细胞记录技术获得MGBv神经元的全细胞记录，电流钳下给予去极化电流刺激，记录神经元动作电位(action potential，AP)发放的变化及GABA受体激动剂的影响。结果出生后不同日龄大鼠MGBv神经元AP的发放模式不同，在出生后不同日龄大鼠MGBv神经元静息电位基础上注入去极化电流脉冲，P3的MGBv神经元只记录到低阈值Ca^2 峰(low threshold Ca^2 spikes，LTS)，而P6-30的MGBv神经元可在LTS的基础上记录到AP的发放；P8的MGBv神经元的AP在脉冲起始处出现，其神经元AP的峰后超极化电位(after hyperpolarization potential，AHP)的持续时间较P20神经元的长，而P20神经元经过明显的延搁后，在去极化斜坡上引起了AP发放；GABA受体激动剂，氯苯氨丁酸((2-ABA。受体激动剂)和毒蕈醇(GABAA受体激动剂)均引起P6的MGBv神经元膜的去极化和AP数目的增加，而氯苯氨丁酸对P15的MGBv神经元AP幅度和时程无影响，毒蕈醇则完全抑制了P15的MGBv神经元AP的发放。结论大鼠MGBv神经元的AP波形成熟较快，可在中枢神经系统的神经元环路中产生协调性的活动；在MGBv神经元发育早期，GABA主要作为兴奋性神经递质而发挥作用，它可使P6的MGBv神经元产生去极化，AP发放频率显著增加，随着出生后日龄的增加，GABA受体的抑制作用才逐渐发育成熟。     

介导NA抑制螺旋神经元GABA电流作用受体的研究

目的探讨何种肾上腺素（NA）受体亚型介导NA抑制耳蜗螺旋神经元γ-氨基丁酸（GABA）诱发电流的作用。方法离体培养耳蜗螺旋神经元，利用穿孔膜片钳技术，记录不同肾上腺素受体激动剂及拮抗剂对NA诱发耳蜗螺旋神经元GABA电流的影响。结果α1肾上腺素受体激动剂苯肾上腺素（Phe）和β肾上腺素受体激动剂异丙肾上腺素（Iso），均不能模拟出NA的作用。而α2肾上腺素受体激动剂（Clo）能模拟出NA的作用。α1肾上腺素受体拮抗剂哌唑嗪（Pra）和β肾上腺素受体拮抗剂心得安（Pro），对NA的抑制效应没有明显影响。而α2肾上腺素受体拮抗剂育亨宾（Yoh），能完全阻断NA对，ICABA的抑制作用。结论这些结果表明以肾上腺素受体的激活介导NA对ICABA的抑制效应。     

急性缺氧对大鼠海马神经元钙激活钾通道的作用

目的:本实验采用膜片钳单通道技术研究缺氧条件下对培养新生大鼠海马神经元钙激活钾通道的作用;方法:取1 ～3天的SD大鼠海马神经元组织,制成细胞悬液,加入含80 %DMEM(dulbecco' s modified eagle' s medium)、20 %小牛血清培养基进行培养,将培养3 ～5天的形态典型的神经元置于对称性高钾溶液中,用膜钳技术记录单通道电流,其通道电流通过膜片钳放大器(CEZ -2200)放大,滤波(3KHz)后,经A/D、D/A转换器输入计算机,采用pClamp6 .0 .6软件采样分析;结果:在细胞贴附式下,应用氰化钠(20μmol/L)造成细胞急性缺氧,在缺氧早期(5 ～20 min)通道开放概率增加(P<0 .01或P<0 .05 ,n=5) ,而缺氧后期(20 ～30 min) ,通道开放概率明显降低,表明缺氧时间对通道的开放概率有明显影响。结论:缺氧早期神经元钙激活钾通道有自身激活作用,当钙激活钾通道激活时,钾离子外流增加,产生膜的复极化,使去极化不易产生,从而稳定细胞膜及降低细胞的兴奋性,这可能是缺氧早期神经元自身的一种代偿机制。     

快速老化小鼠海马神经元电压门控离子通道特点

目的:观察快速老化小鼠(Senescence-accelerated mouse,SAM)海马神经元的基本离子通道特点,并对抗快速老化亚系(SAM-resistance/1,SAMR1)与快速老化亚系(SAM-prone/8,SAMP8)的基本离子通道特点进行了比较,探讨离子通道变化在衰老中的可能角色.方法:应用全细胞记录方式,观察并比较原代培养SAMR1和SAMP8海马神经元的电压门控离子通道及膜参数.结果:原代培养SAMR1和SAMP8海马神经元电压门控Na+通道电流(INa)和电压门控延迟整流K+通道电流(Ik)的电学特点和幅度基本一致.SAMP8的电压门控Ca2+通道电流(ICa)和瞬时外向K+通道电流(IA)的幅值则大于相同培养天数的SAMR1.经膜电容校正所得的ICa电流密度也表现出增大的变化规律.结论:SAMP8与SAMR1神经元间IA和ICa的差异可能与其神经系统变异而产生的学习记忆功能下降有关.     

快速老化小鼠海马神经元电压门控离子通道特点

目的：观察快速老化小鼠（Senescence-accelerated mouse,SAM)海马神经元的基本离子通道特点，并对抗快速老化亚系（SAM－resistance/1,SAMR1)与快速老化亚系（SAM－prone/8,SMAP8)的基本离子通道特点进行了比较，探讨了离子通道变化在衰老中的可能角度，方法：应用全细胞记录方式，观察并比较原代培养SAMR1和SAMP8海马神经元的电压门控离子通道及膜参数。结果：原代培养SAMR1和SAMP8海马神经元电压门控Na^2 通道电流（INa)和电压门控延迟整流K^ 通道电流（IK）的电学特点和幅度基本一致。SAMP8的电压门控Ca^2 通道电流（ICa)和瞬时外向K^ 通道电流（IA）的幅值则大于相同培养天数的SAMR1。经膜电容校正所得的ICa电流密度也表现出增大的变化规律。结论：SAMP8与SAMR1神经元间IA和ICa的差异可能与其神经系统变异而产生的学习记忆功能下降有关。     

幼年非洲爪蟾视顶盖区突触前、后膜上受体间的相互作用

目的　 记录幼年非洲爪蟾视顶盖区第六层神经元的自发性微突触后电流 (mPSCs)。 方法 　应用盲法电压膜片钳全细胞记录技术。 结果　 观察到用谷氨酸受体激动剂NMDA灌流脑片后先引起mIPSCs的频率明显增加并出现内向膜电流及高频的mEPSCs,经一段时间洗脱后mIPSCs和mEPSCs又均完全消失 ,而膜电流恢复到原来未加NMDA前的水平。GABA受体的激动剂GABA可诱发明显的外向膜电流。GABAa受体拮抗剂荷包牡丹碱 (bicucullin ,BM)不仅能将mIPSCs全部抑制掉 ,并且还可以诱发mEPSCs。谷氨酸受体的拮抗剂APV对mPSCs亦有类似的作用 ,不仅可以抑制顶盖神经元的mEPSCs,而且可以使原有的mIPSCs的频率和振幅均增加。 结论　幼年期的突触前、后膜上既有兴奋性谷氨酸能受体也有抑制性γ 氨基丁酸能受体 ,而且在突触前膜上受体可以调制突触末梢神经递质的释放。因此突触前、后膜上的受体间存在相互作用 ,以确保突触前后活动和功能上的稳定 ,从而达到神经网络的平衡。     

缺氧星形胶质细胞产生的脑源性神经营养因子对海马正常神经元γ-氨基丁酸A受体功能的影响

缺氧星形胶质细胞释放的脑源性神经营养因子是否可对正常海马神经元γ-氨基丁酸A受体功能产生影响？实验结果发现，100μmol/L的γ-氨基丁酸可引起经缺氧星形胶质细胞培养液孵育的神经元钙离子浓度增高，此种作用可被γ-氨基丁酸A受体拮抗剂bicuculline或脑源性神经营养因子受体拮抗剂k252a完全抑制。在缺氧星形胶质细胞培养液的影响下，100μmol/L的γ-氨基丁酸诱发的神经元γ-氨基丁酸A受体门控电流由生理条件下的内向电流转为外向电流，可被k252a所抑制。其反转电位左移至-93 mV，并可被k252a和Na-K-Cl转运蛋白抑制剂布美他尼可完全抑制。提示缺氧星形胶质细胞释放的脑源性神经营养因子可能通过提高Na+-K+-Cl-转运蛋白功能，使得γ-氨基丁酸A受体门控电流反转电位左移，并导致γ-氨基丁酸激活γ-氨基丁酸A受体后引起细胞内钙超载而发挥兴奋作用，从而导致神经元损伤。     

Baclofen对大鼠脊髓薄片胶状质神经元的抑制作用

用脊髓薄片全细胞电压钳法观察激活γ－氨基丁酸Ｂ亚型受体（ＧＡＢＡＢR)对大鼠脊髓背角胶状质（ＳＧ）神经元活动的影响,选择性ＧＡＢＡＢＲ激动剂baclofen减少所有被记录ＳＧ神经元（n＝１５）的微小兴奋性突触后电流（mEPSGs）的发放频率。同时引起一缓慢的抑制性（外向）膜电流并伴有膜电导增加,细胞内电泳Ｇ蛋白偶联受体抑制剂（ＧＤＰ－β－Ｓ）抑制被钳制神经元的Ｇ蛋白偶联受体后,baclofen引起的缓慢外向电流被抑制,但对mEPSC频率的减低作用依然存在。结果提示脊髓内baclofen敏感的ＧＡＢＡＢ受体被激活后直接引起ＳＧ神经元超极化并能减少突触前递质的释放。     

依托咪酯激活大鼠骶髓后连合核神经元GABAA门控氯通道电流

目的　 研究依托咪酯 (etomidate ,ET)在急性分离的大鼠骶髓后连合核 (sacraldorsalcommissuralnucleus,SDCN)神经元的药理学特性。方法　采用制霉菌素穿孔膜片钳全细胞记录。结果　在大鼠SDCN神经元 ,ET(10～ 30 0 0 μmol/L)在钳制电位为 - 40mV时 ,可引起内向电流 (IET) ,EC50 为 (33.35± 3.0 7) μmol/L ,该电流可被GABAA受体拮抗剂荷包牡丹碱 (bicuculline)及氯通道阻滞剂印防己毒素 (picrotoxin)以浓度依赖方式所阻断。结论　ET在大鼠SDCN神经元可通过作用于GABAA受体而诱发氯通道电流 ,此作用可能和全麻状态下脊髓水平的麻醉效应有关     

一种稳定阿尔茨海默病细胞模型的建立方法

目的旨在应用不同浓度Aβ_(1-42)诱导新生SD大鼠海马神经元,建立稳定阿尔茨海默病细胞模型。方法分离并培养新生24 h内SD大鼠原代海马神经细胞;分别加入不同浓度Aβ_(1-42)诱导,诱导后采用MTT法观察细胞活力,选取理想Aβ_(1-42)诱导的海马神经细胞作为阿尔茨海默病细胞模型;通过免疫荧光技术(IF)鉴定神经元特异性烯醇化酶(NSE)在海马神经元细胞的表达。结果 Aβ_(1-42)诱导后的海马神经细胞存活率下降,可导致海马神经元细胞胞体变形、收缩和细胞膜完整性破坏,胞间网络结构消失或断裂,细胞外基质乱不清,神经元大量凋亡;Aβ_(1-42)诱导后的海马神经细胞存活率下降与Aβ_(1-42)浓度成线性关系;加入不同浓度Aβ_(1-42)(0μmol/L、10μmol/L、20μmol/L、30μmol/L、40μmol/L和50μmol/L)后实验各组细胞存活率分别为(100.00±0.00)%、(98.55±1.42)%、(90.37±3.25)%,(66.34±2.76)%、(53.23±3.41)%和(41.55±2.37)%,其中20μmol/L组与0μmol/L组相比P<0.05,30μmol/L、40μmol/L和50μmol/L组与对照组相比P<0.01。结论 Aβ_(1-42)可导致海马神经元大量凋亡;Aβ_(1-42)浓度为20μmol/L诱导的SD大鼠海马神经元可作为理想的阿尔茨海默病细胞模型。     

Complex responses activated by ibotenate in postnatal rat hippocampal neurons

The actions of the excitatory amino acid ibotenate were investigated in postnatal rat hippocampal neurons. In neurons voltage clamped at negative membrane potentials using low-chloride internal solutions, ibotenate responses consist of an inward cationic current and two outward currents. The inward current is inhibited by 2-amino-5-phosphonovalerate (APV), a selectiveN-methyl-d-aspartate (NMDA) antagonist. The two outward currents consist of a picrotoxin and bicuculline-sensitive chloride current and a slowly developing calcium activated potassium current. The bicuculline sensitive current appears to be the product of contamination of ibotenate samples with the γ-aminobutyric acid (GABA) agonist muscimol and not the result of a direct action of ibotenate.     

Mechanism of modulation of GABA-activated current by internal calcium in rat central neurons

GABA-activated currents in Purkinje cells isolated from rat cerebellum were investigated. Increase of intracellular Ca2+ in the physiological range of concentrations caused a decrease in GABA-activated chloride currents. This effect resulted from a decrease of both the maximal values of GABA-activated currents and possibly from the affinity of GABAA receptors. Therefore, the mechanism of Ca2+ effect on GABAA receptors in central rat neurons differs from that in bullfrog sensory neurons.     

Androgens predispose males to GABAA-mediated excitotoxicity in the developing hippocampus

Clinical evidence and animal models indicate greater brain damage in newborn males following injury. In adults, glutamate is the primary source of excitotoxic cell death and the steroid, estradiol, is neuroprotective. In neonatal brain, membrane depolarization following activation of GABAA receptors is the major source of excitation. Consequent influx of calcium via L-type channels is normally trophic, but becomes excitotoxic during periods of excessive activation of GABAA receptors, such as hypoxia-ischemia, alcohol exposure and seizures. The use of sex-specific hippocampal cultures revealed greater cell death induced by the GABAA agonist, muscimol, in male- versus female-derived cultures. Pretreatment with the androgen, dihydrotestosterone (DHT) increased muscimol-induced death in both sexes. Exploration of calcium dynamics indicated that, counter to expectation, female neurons achieved higher [Ca2+]i than male, but the calcium transient duration was shorter due to faster rise and decay. However, a second exposure to muscimol within minutes of the first, caused significant attenuation of [Ca2+]i in female neurons. In contrast, while male neurons exposed to muscimol for the first time exhibited lower maximal [Ca2+]i, when exposed to muscimol again there was no attenuation in [Ca2+]i. The latter effect was induced in females by DHT, and inversely correlated with the amount of gamma2 subunit of the GABAA receptor. This novel effect of androgen on GABA-mediated excitotoxicty suggests a unique opportunity for a sex-specific therapeutic approach involving antagonism of the androgen receptor in neonatal males at risk for brain injury.     

Regional distribution and kinetics of three sites on the GABAA receptor: lack of effect of portacaval shunting.

The regional distribution of binding sites on the GABAA receptor and their kinetic parameters were measured by quantitative autoradiography in brains from normal rats and rats with a portacaval shunt, a model of portal systemic encephalopathy in which GABA neurotransmission may be altered. The ligands used were [3H]flunitrazepam (a benzodiazepine-site agonist), [3H]-Ro15-1788 (a benzodiazepine-site antagonist), [3H]muscimol (a GABA-site agonist), and [35S]t-butylbicyclophosphorothionate (35S-TBPS, a convulsant that binds to a site near the chloride channel). Some brains were analyzed by computerized image analysis and three-dimensional reconstruction. The regional distribution of binding of the benzodiazepines was very similar, but the patterns obtained with [3H]muscimol and [35S]TBPS were different in many areas, suggesting a heterogeneous distribution of several subtypes of the GABAA receptor. The kinetic parameters were determined in brain regions for [3H]flunitrazepam, [3H]Ro15-1788, and [3H]muscimol. For each ligand, the Kd showed a significant heterogeneity among brain regions (at least threefold), contrary to conclusions drawn from earlier studies. In portacaval shunted rats, binding of all four ligands was essentially unchanged from that in control rats, indicating that, if there was an abnormality in GABA neurotransmission during portal systemic shunting, it was not reflected by altered binding to the main sites on the GABAA receptor.     

Disinhibitory actions of the GABAA agonist muscimol in immature hippocampus

Bath application of muscimol (10-20 microM) to hippocampal slices obtained from rats on postnatal days 10-15 produced epileptiform activity in the form of multiple population spikes in 20% of slices tested, concurrent with marked disinhibition. The disinhibition occurred in nearly 100% of cases tested at muscimol concentrations that produced epileptiform activity. Paired pulse analysis of GABAergic recurrent inhibition revealed a muscimol-induced disinhibitory effect involving a decrease in maximum possible inhibition. Spontaneous and antidromically elicited inhibitory postsynaptic potentials (IPSPs) recorded intracellularly were suppressed by muscimol. Current-voltage analysis of the recurrent IPSPs suggests that muscimol acted at a number of sites to produce disinhibition. The input conductance of the postsynaptic pyramidal cell increased due to muscimol, creating a current shunt which likely decreased the efficacy of synaptic currents. Muscimol also caused a decrease in the conductance due to the IPSP as well as a shift in the depolarizing direction of the equilibrium potential of the IPSP. The data indicate that muscimol, a GABAA agonist, can produce disinhibition resulting from the multiple consequences of its action. We conclude that the physiologic consequences of GABAA agonist treatment are complex. On the other hand, neurons are likely to be inhibited by a tonic increase in membrane conductance. However, since recurrent inhibition is simultaneously compromised, excitatory vollies of sufficient intensity may overcome the tonic inhibition and produce a hyperexcitable state. In some cases, this disinhibition may induce epileptiform activity. These observations are relevant in light of the proposed use of GABA agonists clinically to control seizures.     

GABAA receptor agonists modulate K+ currents in adult hippocampal glial cells in situ.

Glial cells are known for their role in development and expression of GABA receptors. However, there seems to be a lack of in situ studies characterizing GABA receptor expression and function in glial cells from early development to adulthood. Consequently, we examined GABA receptor expression on rat hippocampal glial cells in both neonatal and adult slices using the whole-cell patch-clamp technique. Glial cells in adult and neonatal slices exhibit responses to muscimol (1 mM; GABAA), but not baclofen (1 mM; GABAB), demonstrating that receptor electrophysiology remains qualitatively similar in glial cells throughout development. Adult muscimol current densities however, do show a decrease in size to approximately 36% of the neonatal response. Muscimol responses were found to be sensitive to bicuculline, suggesting that they are mediated by GABAA receptors. In addition to receptor currents, muscimol causes a concomitant long-term blockade of outward K+ currents in glial cells of both neonatal and adult slices. Comparisons of percentage peak blockade in adult and neonatal glial cells show no significant difference. However, when comparing average absolute conductance blockade, we see that adult glial cells display a significantly smaller response than neonatal and cultured astrocytes. Therefore, although the percentage blockade of outward currents remains consistent throughout development, neonatal glial cells display a larger physiological effect. Thus, it can be concluded that, although the complex GABA response in glial cells is affected by development, the receptor current and secondary blockade are a basic mechanism for neuronal-glial interaction throughout life.     

GABA- and glutamate-activated currents in glial cells of the mouse corpus callosum slice.

Whole-cell transmitter-activated currents were recorded with the patch-clamp technique from glial cells in thin frontal brain slices of the corpus callosum. In slices from 6- to 8-day-old mice, glioblasts were predominantly found, while oligodendrocytes were predominant in slices from 10- to 13-day-old mice. These developmental stages could be readily distinguished by their K+ channel pattern and their morphology and ultrastructural features. Both cell types expressed GABA and glutamate receptors in this in situ preparation. GABA responses showed similarities to those described for GABAA receptors, i.e., they were mimicked by muscimol, blocked by bicuculline, and enhanced by pentobarbital. Glutamate responses showed similarities to those of the kainate/quisqualate receptor subtype. The amplitude of GABA-activated currents recorded in oligodendrocytes was significantly smaller than that from glioblasts, while glutamate responses did not show marked differences in either cell type.     

Muscimol-induced reduction of GABAA receptor alpha 1-subunit mRNA in primary cultured cerebral cortical neurons.

The expression of mRNA for GABAA receptor alpha 1-subunit in mouse cerebral cortical neurons in primary culture was examined using RNA blot analysis and ribonuclease protection assay following the treatment of neurons with muscimol, a selective agonist of GABAA receptor. The level of mRNA for GABAA receptor alpha 1-subunit showed a decrease in comparison with that in non-treated cells, whereas no changes in the level of beta-actin mRNA were noted under the same experimental conditions. This muscimol-induced reduction in GABAA receptor alpha 1-subunit mRNA was counteracted by the simultaneous exposure of neurons to both bicuculline, an antagonist of GABAA receptor, and muscimol. The expression of mRNA for GABAA receptor alpha 1-subunit also showed a decline by the treatment of cells with flunitrazepam alone, an agonist of benzodiazepine receptor, and this change was also abolished by the simultaneous exposure of cells to flunitrazepam and Ro15-1788, an antagonist for central benzodiazepine receptor. These results suggest that the continuous stimulation of cerebral GABAA receptor complex may induce the reduced expression of mRNA for the receptor complex.     

Partial hippocampal kindling increases GABAB receptor-mediated postsynaptic currents in CA1 pyramidal cells

In previous studies, we showed that partial hippocampal kindling decreased the efficacy of the presynaptic GABAB receptors on both GABAergic and glutamatergic terminals of CA1 neurons in hippocampal slices in vitro. In this study, GABAB receptor-mediated inhibitory postsynaptic currents (GABAB-IPSCs) were assessed by whole-cell recordings in CA1 pyramidal neurons in hippocampal slices of male Long-Evans rats. The peak GABAB-IPSC evoked by a brief train of supramaximal stratum radiatum stimuli (20 pulses of 300 Hz) in the presence of picrotoxin (0.1 mM) and kynurenic acid (1 mM) was larger in neurons of kindled (65.9 +/- 5.2 pA, N=42 cells) than control (45.8 +/- 4.8 pA, N=32 cells) rats (P<0.01). Adding GABA uptake blocker nipecotic acid (1 mM) or GABAB receptor agonist baclofen (0.01 mM) in the perfusate induced outward currents that were blocked by GABAB receptor antagonist CGP 55845A (1 microM). The peak outward current induced by nipecotic acid was larger in neurons of the kindled (55.4 +/- 5.7 pA, N=30) than the control group (39.8 +/- 4.5 pA, N=28) (P<0.05). However, the magnitude of the baclofen-induced current was not different between kindled (90.8 +/- 6.9 pA, N=29) and control (87.2 +/- 5.9 pA, N=21) groups (P>0.05). We concluded that partial hippocampal kindling increased GABAB-IPSCs in hippocampal CA1 pyramidal cells via multiple presynaptic mechanisms.