周边γ-氨基丁酸通过GABA_B受体调控骶髓后联合核神经元谷氨酸能突触

目的研究突触周边γ-氨基丁酸(ambient GABA)通过GABAB受体调控骶髓后联合核(SDCN)神经元谷氨酸能突触的机制。方法在急性切取的骶段脊髓薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs),将GABAB受体用其特异性受体拮抗剂CGP52432阻断,观察谷氨酸突触终末上的GABAB受体被周边GABA作用的影响。结果在突触后GABAB受体被从胞内阻断的条件下,再灌流CGP52432阻断谷氨酸能突触前GABAB受体,可增加刺激引发的EPSCs(eEPSCs)幅度;改变配对刺激的两个EPSC比率(paired-pulse ratio,PPR),并激发沉默突触(silent synapse)。但CGP52432对微小兴奋性突触后电流(mEPSCs)无影响。结论位于SDCN神经元谷氨酸能突触前的GABAB受体受周边GABA调控。这种影响参与调节谷氨酸释放并可能参与痛觉信息在脊髓水平的传递。     

辣椒素受体参与骶髓后联合核神经元突触传递

目的研究辣椒素受体对大鼠骶髓后联合核(SDCN)神经元突触传递的影响。方法在脊髓骶段横切薄片上,利用全细胞膜片钳法记录骶髓后联合核神经元谷氨酸能兴奋性突触后电流(EPSCs)和γ-氨基丁酸(GABA)能抑制性突触后电流(IPSCs),比较激动辣椒素受体后上述突触电流的变化;观察激动辣椒素受体对SDCN神经元动作电位发放的影响。结果辣椒素受体被其特异性激动剂辣椒素(1μmol.L-1)激动后,自发EPSCs(sEPSCs)的频率和振幅均有明显增加(P<0.05,n=17)。在河豚毒素(0.5μmol.L-1)存在的条件下,辣椒素明显增加微小EPSCs(mEPSCs)的频率(P<0.01,n=13),但对mEPSCs的振幅无影响(P>0.05,n=13),提示辣椒素的作用在突触前。辣椒素也明显增加动作电位发放(P<0.05,n=19)。上述作用均可被辣椒素受体特异性拮抗剂capsazepine(10μmol.L-1)阻断。辣椒素也增加GABA能的自发IPSCs(sIPSCs)的频率(P<0.05,n=20),但对其不依赖动作电位的微小IPSCs(mIPSCs)的频率或振幅均无作用(P>0.05,n=9)。结论在SDCN,辣椒素受体主要表达于兴奋性突触终末;激动辣椒素受体影响兴奋性和抑制性突触活动,并可能参与痛觉信息在脊髓水平的传递和调制。     

氯苯氨丁酸抑制脊髓背角神经元谷氨酸量子释放的机制

目的　研究GABAB 受体特异性激动剂氯苯氨丁酸(baclofen)在脊髓背角神经元抑制谷氨酸量子释放的机制。方法　在脊髓薄片标本上 ,采用全细胞电压钳法记录脊髓背角神经元谷氨酸能的微兴奋性突触后电流 (miniatureexcita torypostsynapticcurrents;mEPSCs) ,通过分析这些电流的变化来研究baclofen影响谷氨酸量子释放的机制。结果　ba clofen抑制mEPSCs的发放频率 ,但对平均幅度无明显影响 ,表明baclofen抑制谷氨酸释放的作用部位在突触前。在无钙溶液或者K+ 通道阻滞剂 4 AP存在的条件下 ,baclofen对mEPSCs发放频率的抑制作用不受影响 ,但腺苷酸环化酶激动剂foskolin (可使cAMP保持在较高水平 )能降低其抑制作用。而蛋白激酶C (PKC)激动剂PDBu对baclofen的抑制作用无影响。用NEM破坏G蛋白 ,则可取消baclofen的抑制效果。结论　baclofen不是通过影响突触前Ca2 + 通道或K+通道 ,或PKC途径 ,而是通过作用于G蛋白和 (或 )cAMP途径抑制谷氨酸的释放 ;这种抑制作用可能参与baclofen在脊髓水平的镇痛     

盐酸埃他卡林对大鼠海马神经元谷氨酸受体功能及突触活动的影响

目的 :研究盐酸埃他卡林 (Ipt)对脑神经元谷氨酸受体功能及突触活动的影响。方法 :采用原代培养的大鼠海马神经元 ,应用膜片钳全细胞记录技术 ,记录Ipt对培养的海马神经元谷氨酸或天冬氨酸(NMDA)诱发电流及神经元突触后电流的影响。结果 :Ipt(1～ 1 0 0 μmol·L- 1)可浓度依赖性地对抗培养的海马神经元谷氨酸或NMDA诱发电流 ,并为ATP敏感性钾通道拮抗剂格列本脲 30 μmol·L- 1所对抗。Ipt抑制培养的海马神经元之间突触联系形成的自发兴奋性突触后电流 ,降低其发放频率 ,抑制其电流幅度 ;但对微小兴奋性突触后电流无显著性影响。结论 :Ipt可阻断脑神经元谷氨酸受体功能 ,抑制脑神经元谷氨酸的兴奋性突触传递 ,其作用与ATP敏感性钾通道相关     

多巴胺激活Kv通道抑制胰岛素分泌的电生理机制

目的探讨多巴胺激活电压依赖性钾(Kv)通道抑制葡萄糖刺激胰岛素分泌的电生理机制。方法分离雄性SD大鼠胰岛和β细胞,根据实验使用多巴胺(DA)、D;样受体激动剂(SKF38393)和D;样受体激动剂(Quinpirole)及阻断剂(Eticlopride)进行干预。酶联放射免疫实验测定胰岛素含量;膜片钳电压钳记录β细胞膜上Kv通道电流的变化,电流钳记录动作电位时程的长短;DiBAC4(3)染色观察INS-1细胞膜电位的变化。结果 SKF38393对胰岛素分泌和Kv通道均没有明显影响;Quinpirole明显抑制胰岛素分泌,并且增加Kv通道电流;多巴胺明显抑制胰岛素分泌,增加Kv通道电流,缩短β细胞动作电位时程,且均可被Eticlopride逆转;此外,多巴胺降低INS-1细胞膜电位。结论多巴胺作用于D;样受体,活化Kv通道,缩短动作电位时程,降低β细胞膜电位,从而发挥抑制葡萄糖促进胰岛素分泌的作用。     

代谢型谷氨酸受体1缺失导致小鼠蛋白尿和肾小球损伤

谷氨酸是中枢神经系统中数量最多的神经递质。代谢型谷氨酸受体1(GRM1)表达于神经元突触前膜和突触后膜,并通过触发释放细胞内贮存的钙离子,经过一系列信号通路最终调节神经元兴奋性、突触可塑性以及参与神经递质释放     

几种静脉麻醉药对GABA能神经元的Phasic／Tonic抑制的影响

γ-氨基丁酸（gama-amino butyic acid ,GABA ）是广泛存在于中枢神经系统中一种重要的抑制性神经递质,参与调控睡眠和镇静等作用,主要通过作用于 GABAA 离子型亚基受体发挥生理效用。GABAA 受体属配体门控离子通道受体,通过增加神经细胞膜对氯离子和碳酸根离子的通透性,使细胞膜外高浓度的阴离子顺浓度梯度产生内向流动,从而引起突触后的超极化反应,导致神经元的兴奋性降低,这种由突触内的GABAA 型受体短暂的或“阶段性”的激活突触囊泡释放GABA ,从而产生的抑制,称为Phasic抑制,也是 GABAA 受体介导抑制性作用的经典途径。而近年来,研究者们提出了一种GABAA 受体介导的新型抑制作用即Tonic抑制［1］,它是由于轴突末梢保持一种持续少量的GA-BA释放,细胞外低浓度GABA 的环境能持续性的激活突触外的GABAA 受体引起的生理效应。本篇拟综述突触内和突触外GABAA 受体在控制神经元兴奋性的独特作用以及与全麻机制相关的最新研究进展。     

脊髓薄片制备方法的改进及其脊髓Ⅱ板层神经元突触后电流的记录

目的探讨记录脊髓Ⅱ板层神经元兴奋性和抑制性突触后电流的方法。方法SD雄性大鼠,160～180 g,2%氟烷和100%氧气吸入麻醉后快速取腰段脊髓,迅速放入冰冷充氧的人工脑脊液,将约1 cm长的脊髓粘立于振动切片机载物台上,同时覆以冰冷的人工脑脊液进行横切片,片厚350～450μm,Kreb’S液提前30 min用95%CO2和5%O2预充氧,将切好的脊髓薄片转移其中,34～35℃孵育至少1 h后,以备实验记录用。结果在倒置显微镜下可清楚分辨出脊髓Ⅱ板层神经元。采用全细胞电压钳记录技术,给予选择性受体阻断剂以分离不同的突触后受体电流。在钳制电压为-70 mV时,可以记录到谷氨酸能的兴奋性突触后电流（EPSCs）;而在钳制电压为0 mV时,可以记录到抑制性突触后电流（IPSCs）。结论该方法为研究脊髓Ⅱ板层神经元递质释放提供了有效的途径。     

单次可卡因注射对VTA区DA神经元兴奋性突触传递和内在兴奋性的影响

目的:研究单次可卡因注射24 h后VTA区多巴胺能(DA)神经元兴奋性突触传递强度和内在兴奋性的变化。方法:采用离体膜片钳技术,检测单次可卡因注射24 h后VTA区DA神经元自发性慢性内向流(slow inwardcurrents,SICs)、内在兴奋性以及兴奋性突触后电流(EPSCs)的变化。结果:DA神经元的SICs、内在兴奋性和EPSCs均有显著增强。结论:单次可卡因注射后VTA区DA神经元兴奋性突触传递强度和内在兴奋性呈现协同增强效应。     

GABA_B受体对牛磺酸调节突触体氨基酸释放的影响

目的　牛磺酸 (Tau)调节大鼠大脑皮层突触体Asp、Glu、GABA的释放的机制尚不清楚 ,本研究从GABA受体的角度进行探讨。方法　将 phaclofen、biculline、baclofen加入悬浮有突触体的KRB液中 ,氨基酸测定采用Dans Cl柱前衍生 ,高效液相测定。结果　Tau对GABA释放的抑制作用能有效被Phaclofen所拮抗 ,而它们对GluAsp的释放均无影响。结论　Tau抑制去极化引发GABA的释放是通过激发突触体前膜GABAB 受体而起作用的 ,同时还作用于Asp、Glu神经末梢的突触体前膜 ,从而抑制去极化引发的Asp、Glu的释放。     

Cannabidiol-caused depression of spinal motoneuron responses in cats

Intracellular recording techniques were used on spinal motoneurons in the cat in order to define the synaptic pharmacology of cannabidiol (CBD). The cannabinoid produces only depression of electrophysiological responses of the motoneurons: For instance, the drug decreases the amplitude of excitatory postsynaptic potentials (EPSPs); this reduction does not appear to be the result of a change in the afferent input. In addition, CBD raises the firing threshold and decreases the amplitude of motoneuron action potentials; the effects on action potentials are related to changes in postsynaptic membrane conductances, probably involving at least sodium conductance. The spinal motoneuron effects provide potential electrophysiological mechanisms for CBD's central depressant actions.     

Electrophysiological actions of GABAB agonists and antagonists in rat dorso-lateral septal neurones in vitro.

1. The actions of GABAB-receptor agonists and antagonists on rat dorso-lateral septal neurones in vitro were recorded with intracellular microelectrodes. 2. In the presence of 1 microM tetrodotoxin to prevent indirect neuronal effects caused by action potential-dependent neurotransmitter release, bath application of baclofen (0.1-30 microM) or SK&F 97541 (0.01-3 microM) evoked concentration-dependent hyperpolarizations which reversed close to the potassium equilibrium potential; the EC50S were 0.55 and 0.05 microM, respectively. No significant desensitization was observed during prolonged agonist exposure (< or = 10 min). 3. Hyperpolarizations induced by baclofen were antagonized in a competitive manner by the following GABAB-receptors antagonists (calculated pA2 values in parentheses): CGP 36742 (4.0), 2-OH saclofen (4.2), CGP 35348 (4.5), CGP 52432 (6.7) and CGP 55845A (8.3). Responses to SK&F 97541 were also antagonized by CGP 55845A (pA2 = 8.4). 4. The amplitude of the late, GABAB receptor-mediated inhibitory postsynaptic potential (i.p.s.p.) was reduced by the GABAB antagonists as follows (means +/- s.e.mean): CGP 55845A (1 microM) 91 +/- 5%, CGP 52432 (1 microM) 64 +/- 5%, CGP 35348 (100 microM) 82 +/- 5%, CGP 36742 (100 microM) 76 +/- 8%, and 2-OH saclofen (100 microM) 68 +/- 3%. 5. It is concluded that neurones in the rat dorso-lateral septal nucleus express conventional GABAB receptors, which are involved in the generation of slow inhibitory postsynaptic potentials. CGP 55845A is the most potent GABAB receptor antagonist described in this brain area.     

The actions of 3-aminopropanephosphinic acid at GABAB receptors in rat hippocampus.

The actions of 3-aminopropanephosphinic acid (APPA) were examined using whole-cell patch-clamp recording in rat hippocampal slice. In recordings from neurons in subfield CA1 of slices from young (2-4 weeks) and adult (greater than 2 month) rats, APPA (0.5-50 microM) produced membrane hyperpolarization and outward current under voltage-clamp. APPA also inhibited excitatory postsynaptic potentials with an IC50 of 2.3 microM, and reduced inhibitory postsynaptic potentials at concentrations from 0.1 to 1 microM. The hyperpolarizing and synaptic depressant effects of APPA were reduced by 2-OH-saclofen an antagonist at the B-type receptor for the neurotransmitter gamma-aminobutyric acid (GABA). In this preparation APPA exhibited potencies similar to those previously reported for the GABAB receptor agonist baclofen. APPA was much less effective in inhibiting synaptic transmission measured using field potential recordings. The observations made with whole-cell patch-clamp recording indicate that in hippocampus APPA acts as a potent agonist at presynaptic GABAB receptors associated with both excitatory and inhibitory synapses, and also activates postsynaptic GABAB receptors.     

Actions of the GABAB agonist, (-)-baclofen, on neurones in deep dorsal horn of the rat spinal cord in vitro.

1. The electrophysiological actions of the GABAB agonist, (-)-baclofen, on deep dorsal horn neurones were studied using an in vitro preparation of the spinal cord of 9-16 day old rat. 2. On all neurones tested, (-)-baclofen (100 nM-30 microM) had a hyperpolarizing action which was associated with a reduction in apparent membrane input resistance. The increase in membrane conductance was dose-dependent and had a Hill coefficient of 1.0. 3. The (-)-baclofen-activated hyperpolarization persisted in the presence of bicuculline (50 microM) and Mg2+ (20 mM). 4. The reversal potential of the hyperpolarizing event was estimated at 102 mV and was made less negative by increasing the external concentration of potassium ions. 5. Over the same concentration range, (-)-baclofen also depressed the polysynaptic composite excitatory postsynaptic potentials (e.p.s.ps) evoked in these neurones by electrical stimulation of the dorsal root entry zone. 6. The potassium channel blockers caesium, applied intracellularly, and barium, applied extracellularly, depressed the postsynaptic response to baclofen but not its effect on e.p.s.ps. 7. We propose that (-)-baclofen has more than one mechanism of action in spinal dorsal horn: a postsynaptic action mediated via an increase in potassium conductance and a presynaptic action that is not associated with potassium channels and may be mediated via calcium channels. Since previous studies have demonstrated little effect of (-)-baclofen on transmitter release in spinal cord, it is possible that the postsynaptic hyperpolarizing action of (-)-baclofen may account for its clinical potency as an anti-spastic agent.     

Effects of dithiothreitol, 5,5''-dithiobis(2-nitrobenzoic acid) and n-ethylmaleimide on synaptic transmission at sympathetic ganglion cells of frog

The effects of disulfide bond and sulfhydryl group reagents on synaptic transmission were studied in the bullfrog sympathetic ganglion. Ten millimoles of dithiothreitol (DTT). a disulfide bond reducing agent, decreased the amplitude of the transmitted postganglionic compound action potential (CAP) to 41% of control. More than one hour wash-out of DTT with normal Ringer's solution did not reverse the block, but 1 mM 5.5'-dithiobis(2-nitrobenzoic acid) (DTNB), an oxidizing agent, rapidly restored the compound action potential amplitude. N-Ethylmalemide (NEM), an alkylating agent, added before DTNB, prevented the restoration of transmission by DTNB. Block of transmission induced by dithiothreitol was also reversed rapidly by 50 μm 3,4-diaminopyridine (3,4-DAP), to 87% of control compound action potential amplitude, and subsequent addition of DTNB restored transmission completely and this was accompanied by the stimulus-bound repetitive firing (SBR) which diaminopyridine produced in otherwise untreated ganglia.

One to 3 mM dithiothreitol decreased the amplitude of excitatory post-synaptic potentials (EPSP), and greater concentrations slightly depolarized the cell membrane and decreased membrane resistance. Washout with Ringer's solution reversed the depolarization, but not the block of excitatory postsynaptic potentials. The amplitude of excitatory post-synaptic potentials was restored to threshold by 1 mM DTNB, which also slightly increased membrane resistance and the resting potential.

It is concluded that the actions of dithiothreitol and DTNB in the bullfrog sympathetic ganglion are generally similar to those previously observed in other junctional tissues. Interactions between these drugs and 3,4-diaminopyridine in the present study also raise the question whether dithiothreitol and DTNB have any presynaptic effects.     

脊髓切片中运动神经元的膜片箝技术研究:一种分析药物作用的高效工具(英文)

AIM: To develop a tool for detailed analysis of spirally acting anesthetic and analgesic agents. METHODS: Studies were done on visually identified motor neurons in 400 uuuuuuuum thick spinal cord slices from 14-23 d old rats using patch clamp techniques. Ethanol was used as a prototype general anesthetic agent. RESULTS: Cell bodies in the ventrolateral horn identified as motor neurons by retrograde fluorescent labeling had a mean dimension of 32 ± 5 /Mm (x ± s, n = 25). Mean resting potential was - 62. 8 ± 2. 4 mV; input resistance was 44±24 MΩ (n = 19). Threshold was -44± 7 mV, and action potential amplitude 101 ± 9 mV from baseline. Ethanol concentrations at and below 50-200 mmol/L decreased motor neuron excitability to the injected current; there was no effect on resting potential, but a variable reversible increase in input resistance. Ethanol re-versibly depressed the excitatory postsynaptic potential, with a dose-response relationship similar to that previously observed for the population e     

Electrophysiological characterization of potent agonists and antagonists at pre- and postsynaptic GABAB receptors on neurones in rat brain slices.

1. Intracellular recordings were made from neurons in striatum (caudate-putamen) and substantia nigra pars compacta in rat brain slices. Three GABAB agonists, baclofen, 3-aminopropylphosphinic acid (3-APPA) and 3-aminopropyl(methyl)phosphinic acid (SK&F 97541), depressed excitatory postsynaptic potentials (e.p.s.ps) mediated by glutamate in the striatum, and hyperpolarized neurones in the substantia nigra. The ability of 3-aminopropyl(diethyoxymethyl)phosphinic acid (CGP 35348), 3-aminopropyl (hexyl)phosphinic acid (3-APHPA) and phaclofen to antagonize these responses was assessed. 2. Striatal e.p.s.ps, studied in the presence of bicuculline (30 microns), were reduced in amplitude by 92% with 6,7-dinitroquinoxaline-2,3-dione (DNQX; 30 microns). These e.p.s.ps were depressed by up to 95% by SK&F 97541 and baclofen with EC50s of 0.092 microns and 1.25 microns respectively. The maximal effect of 3-APPA was 67% with an EC50 of 0.83 microns. Agonist concentration-effect data fitted a single-site logistic model. GABAB agonists were without effect on striatal neurone membrane potential, input resistance or depolarizations induced by applied glutamate. 3. The depression of striatal e.p.s.ps by SK&F 97541 was reversibly antagonized by CGP 35348, 3-APHPA and phaclofen with estimated equilibrium dissociation constants (KB) of 11.2 +/- 1.7 microns (n = 4), 13.3 +/- 0.4 microM (n = 3) and 405 +/- 43 microM (n = 3) respectively. CGP 35348 and 3-APHPA appeared to act competitively (Schild plot slopes of 0.99 and 1.01 respectively). 4. Nigral neurones were hyperpolarized by up to 25 mV by SK&F 97541 and baclofen with EC50s of 0.15 microns and 3.6 microns respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

An in vitro electrophysiological study on the effects of phenytoin, lamotrigine and gabapentin on striatal neurons

We performed intracellular recordings from a rat corticostriatal slice preparation in order to compare the electrophysiological effects of the classical antiepileptic drug (AED) phenytoin (PHT) and the new AEDs lamotrigine (LTG) and gabapentin (GBP) on striatal neurons. PHT, LTG and GBP affected neither the resting membrane potential nor the input resistance/membrane conductance of the recorded cells. In contrast, these agents depressed in a dose-dependent and reversible manner the current-evoked repetitive firing discharge. These AEDs also reduced the amplitude of glutamatergic excitatory postsynaptic potentials (EPSPs) evoked by cortical stimulation. However, substantial pharmacological differences between these drugs were found. PHT was the most effective and potent agent in reducing sustained repetitive firing of action potentials, whereas LTG and GBP preferentially inhibited corticostriatal excitatory transmission. Concentrations of LTG and GBP effective in reducing EPSPs, in fact, produced only a slight inhibition of the firing activity of these cells. LTG, but not PHT and GBP, depressed cortically-evoked EPSPs increasing paired-pulse facilitation (PPF) of synaptic transmission, suggesting that a presynaptic site of action was implicated in the effect of this drug. Accordingly, PHT and GBP, but not LTG reduced the membrane depolarizations induced by exogenously-applied glutamate, suggesting that these drugs preferentially reduce postsynaptic sensitivity to glutamate released from corticostriatal terminals. These data indicate that in the striatum PHT, LTG and GBP decrease neuronal excitability by modulating multiple sites of action. The preferential modulation of excitatory synaptic transmission may represent the cellular substrate for the therapeutic effects of new AEDs whose use may be potentially extended to the therapy of neurodegenerative diseases involving the basal ganglia.