The GLP-1 Receptor Agonist Exendin-4 and Diazepam Differentially Regulate GABAA Receptor-Mediated Tonic Currents in Rat Hippocampal CA3 Pyramidal Neurons

Glucagon-like peptide-1 (GLP-1) is a metabolic hormone that is secreted in a glucose-dependent manner and enhances insulin secretion. GLP-1 receptors are also found in the brain where their signalling affects neuronal activity. We have previously shown that the GLP-1 receptor agonists, GLP-1 and exendin-4 enhanced GABA-activated synaptic and tonic currents in rat hippocampal CA3 pyramidal neurons. The hippocampus is the centre for memory and learning and is important for cognition. Here we examined if exendin-4 similarly enhanced the GABA-activated currents in the presence of the benzodiazepine diazepam. In whole-cell recordings in rat brain slices, diazepam (1 μM), an allosteric positive modulator of GABA_A receptors, alone enhanced the spontaneous inhibitory postsynaptic current (sIPSC) amplitude and frequency by a factor of 1.3 and 1.6, respectively, and doubled the tonic GABA_A current normally recorded in the CA3 pyramidal cells. Importantly, in the presence of exendin-4 (10 nM) plus diazepam (1 μM), only the tonic but not the sIPSC currents transiently increased as compared to currents recorded in the presence of diazepam alone. The results suggest that exendin-4 potentiates a subpopulation of extrasynaptic GABA_A receptors in the CA3 pyramidal neurons.     

Medial and Lateral Entorhinal Cortex Differentially Excite Deep versus Superficial CA1 Pyramidal Neurons

1. Download high-res image (151KB)
2. Download full-size image

     

Glycine Induces Bidirectional Modifications in N-Methyl-d-aspartate Receptor-mediated Synaptic Responses in Hippocampal CA1 Neurons

Glycine can persistently potentiate or depress AMPA responses through differential actions on two binding sites: NMDA and glycine receptors. Whether glycine can induce long-lasting modifications in NMDA responses, however, remains unknown. Here, we report that glycine induces long-term potentiation (LTP) or long-term depression (LTD) of NMDA responses (Gly-LTP_NMDA or Gly-LTD_NMDA) in a dose-dependent manner in hippocampal CA1 neurons. These modifications of NMDA responses depend on NMDAR activation. In addition, the induction of Gly-LTP_NMDA requires binding of glycine with NMDARs, whereas Gly-LTD_NMDA requires that glycine bind with both sites on NMDARs and GlyRs. Moreover, activity-dependent exocytosis and endocytosis of postsynaptic NMDARs underlie glycine-induced bidirectional modification of NMDA excitatory postsynaptic currents. Thus, we conclude that glycine at different levels induces bidirectional plasticity of NMDA responses through differentially regulating NMDA receptor trafficking. Our present findings reveal important functions of the two glycine binding sites in gating the direction of synaptic plasticity in NMDA responses.     

Bradykinin Postconditioning Protects Pyramidal CA1 Neurons Against Delayed Neuronal Death in Rat Hippocampus

Aims The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. Methods Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 μg/kg) applied 48 h after ischemia. Results We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. Conclusions Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.     

乌拉坦对大鼠海马CA1区锥体神经元自发放电的抑制作用

为了深入研究麻醉药乌拉坦对大鼠海马CA1锥体神经元自发放电的作用及其机制,分析了10 mmol/L乌拉坦对自发放电、电压门控钠通道、电压门控钾通道的作用．从自发放电信号中计算了放电频率、提取了峰峰间隔序列(ISI)并利用样品熵和去趋势波动法对ISI进行了非线性分析．结果表明,乌拉坦不仅抑制了自发放电的频率,而且降低了自发放电ISI序列的复杂度并弱化了其长时程相关性．离子通道研究结果表明,乌拉坦显著地抑制了钠通道电流(I_Na),对延迟整流钾通道电流(I_K)和瞬时外向钾通道电流(I_A)虽然也有抑制作用但无统计学意义．由于乌拉坦不影响突触传递,因此它可能通过抑制I_Na使自发放电的阈值升高而降低放电频率,同时,由于参与的通道数量或活性降低而使得ISI的复杂度下降,长时程相关性弱化．     

大鼠海马CA1区锥体细胞上一种 Ca2+依赖性KATP通道

KATP通道在细胞的新陈代谢与膜兴奋性的耦联中起重要作用.采用膜片钳的内面向外式记录方法,在成年大鼠海马CA1区锥体细胞上记录到一种被胞浆侧ATP和甲糖宁(tolbutamide,一种KATP通道阻断剂)抑制的Ca2+依赖性钾离子通道.在细胞膜内外的K+浓度均为140 mmol/L时,通道的电导为(204±21) pS,翻转电位为(3.57±1.13) mV,通道无整流性.通道开放概率及ATP对通道的抑制作用均呈现电压依赖性.该KATP通道与以往报道的"经典"KATP通道有显著不同,其活动受膜电位、胞内Ca2+和ATP三重调节,表明这是一种新型的KATP通道.上述结果表明在海马神经元上至少有两种性质不同的KATP通道,提示神经元可能通过不同性质的KATP通道感受细胞内的代谢状态,进而调节细胞膜的兴奋性.     

Regulation of GABA Equilibrium Potential by mGluRs in Rat Hippocampal CA1 Neurons

The equilibrium potential for GABA-A receptor mediated currents (E_GABA) in neonatal central neurons is set at a relatively depolarized level, which is suggested to be caused by a low expression of K⁺/Cl^- co-transporter (KCC2) but a relatively high expression of Na⁺-K⁺-Cl^- cotransporter (NKCC1). Theta-burst stimulation (TBS) in stratum radiatum induces a negative shift in E_GABA in juvenile hippocampal CA1 pyramidal neurons. In the current study, the effects of TBS on E_GABA in neonatal and juvenile hippocampal CA1 neurons and the underlying mechanisms were examined. Metabotropic glutamate receptors (mGluRs) are suggested to modulate KCC2 and NKCC1 levels in cortical neurons. Therefore, the involvement of mGluRs in the regulation of KCC2 or NKCC1 activity, and thus E_GABA, following TBS was also investigated. Whole-cell patch recordings were made from Wistar rat hippocampal CA1 pyramidal neurons, in a slice preparation. In neonates, TBS induces a positive shift in E_GABA, which was prevented by NKCC1 antisense but not NKCC1 sense mRNA. (RS)-a-Methyl-4-carboxyphenylglycine (MCPG), a group I and II mGluR antagonist, blocked TBS-induced shifts in both juvenile and neonatal hippocampal neurons. While blockade of mGluR1 or mGluR5 alone could interfere with TBS-induced shifts in E_GABA in neonates, only a combined blockade could do the same in juveniles. These results indicate that TBS induces a negative shift in E_GABA in juvenile hippocampal neurons but a positive shift in neonatal hippocampal neurons via corresponding changes in KCC2 and NKCC1 expressions, respectively. mGluR activation seems to be necessary for both shifts to occur while the specific receptor subtype involved seems to vary.     

氧化还原对成年大鼠海马CA1区锥体神经元外向整流氯通道的调控作用

采用膜片钳内面向外式记录技术,研究急性分离成年大鼠海马CAl区锥体神经元外向整流氯离子通道的氧化还原调控。发现细胞内侧给予氧化剂DTNB(5,5'-dithiobis-2-nitrobenzoic acid),可显著减弱氯通道的活动,IC50值为(28.05±2.42)μmol/L;还原剂DTT(dithiothreitol)对氯通道没有明显影响,但可逆转DTNB引起的抑制效应。说明DTNB不改变通道电导,其引起的通道活动减弱是由氯通道关闭时间延长而开放时间缩短所致。研究还发现,另一对氧化型和还原型谷胱甘肽具有与DTNB和DTT同样的效应。本研究结果显示,成年大鼠海马CA1区锥体神经元外向整流氯通道可以被细胞内氧化还原剂所调控。     

Elevation of Basal Protein Kinase C Activity Increases Ethanol Sensitivity of GABAA Receptors in Rat Hippocampal CA1 Pyramidal Neurons

Abstract: The ability of ethanol to enhance GABA_A receptor function remains controversial; conflicting observations have been made even in the same brain region, and when using apparently similar methodologies. In this study we characterized a single protocol variable, the initial incubation temperature of brain slices, that had dramatic effects on the ethanol sensitivity of GABA_A inhibitory postsynaptic currents (IPSCs) recorded from rat hippocampal CA1 pyramidal neurons. Incubation of hippocampal slices at relatively low temperatures (11–15°C) immediately after slice preparation significantly affected a number of physiological and biochemical parameters. Such slices showed a decrease in extracellular inhibitory postsynaptic potential amplitude, a significant increase in the ethanol sensitivity of GABA_A IPSCs in CA1 pyramidal neurons, no change in pentobarbital or flunitrazepam potentiation of IPSCs, and an increase in basal protein kinase C (PKC) activity relative to slices incubated at 31–33°C. In addition, the increase in ethanol sensitivity of GABA_A IPSCs was blocked by chelerythrine, a selective inhibitor of PKC. These results suggest that differences in hippocampal slice incubation protocols may have contributed to the disparate results of previous investigations of ethanol modulation of GABA_A receptor-mediated synaptic transmission in the rat hippocampus. In addition, these findings provide further evidence that PKC activity positively modulates the interaction between ethanol and GABA_A receptors in the mammalian brain.     

The Influences of I h on Temporal Summation in Hippocampal CA1 Pyramidal Neurons: A Modeling Study

Recent experimental and theoretical studies have found that active dendritic ionic currents can compensate for the effects of electrotonic attenuation. In particular, temporal summation, the percentage increase in peak somatic voltage responses invoked by a synaptic input train, is independent of location of the synaptic input in hippocampal CA1 pyramidal neurons under normal conditions. This independence, known as normalization of temporal summation, is destroyed when the hyperpolarization-activated current, I _h, is blocked [Magee JC (1999a), Nature Neurosci. 2: 508–514]. Using a compartmental model derived from morphological recordings of hippocampal CA1 pyramidal neurons, we examined the hypothesis that I _h was primarily responsible for normalization of temporal summation. We concluded that this hypothesis was incomplete. With a model that included I _h, the persistent Na⁺ current (I _NaP), and the transient A-type K⁺ current (I _A), however, we observed normalization of temporal summation across a wide range of synaptic input frequencies, in keeping with experimental observations.     

Hippocampal CA1 Pyramidal Cell Size is Reduced in Bipolar Disorder

1. Schizophrenia and bipolar disorder are neurodevelopmental disorders with significant genetic vulnerabilities. Several trophic genes and/or proteins have been implicated in the causation for both disorders. 2. We hypothesized that these genes and/or proteins may impact neuronal growth in both disorders. 3. Hippocampal tissue sections from CA1 area of schizophrenic, bipolar, depressed, and controls subjects, matched for age, sex, PMI, drug exposure, and brain pH were prepared for cell size determination using the Stanley Medical Research Foundation postmortem brain collection. 4. Quantification of hippocampal CA1 pyramidal neuron size showed a significant 12% reduction in cell size (p < 0.05) in bipolar subjects vs. controls. There were nonsignificant trends for reduction in cell size in both schizophrenic and depressed subjects vs. controls. 5. These results indicate for the first time that pyramidal cell atrophy is present in hippocampus of subjects with bipolar disorder.     

成年大鼠海马CA1区锥体神经元外向整流氯离子单通道特性

采用膜片钳内面向外式技术,在急性分离成年大鼠海马CAl区锥体细胞上记录到了外向整流氯离子通道（outwardly rectifying chloride channel,ORCC）.长时间去极化（≥60 mV）刺激后,在30%的游离膜片上记录到有外向整流特性的单通道氯电流,膜电位在－60 mV到0 mV之间的单通道电导为(16.58±1.54) pS（n=10）,而在0 mV到+60 mV之间电导为（40.92±3.17） pS.通道开放概率有明显的电压依赖性(膜电位－60 mV时,P_o=0.44±0.12;膜电位为+60 mV时,P_o=0.86±0.06, n=10).在对称Cl^－浓度（150 mmol/L）时,通道翻转电位为(－4.17±1.84) mV.当溶液中部分NaCl被葡萄糖酸钠替代后,翻转电位为：(－34.23±4.86) mV (［Cl^－］_i/［Cl^－］_o=(30 mmol/L)/(150 mmol/L)),接近氯离子通道的理论值,这表明通道具有氯离子选择性.浴槽液中分别加入氯通道阻断剂DIDS和SITS可以使+40 mV的通道开放概率从(0.83±0.06)和(0.86±0.06)分别降低到(0.12±0.05)和(0.13±0.04)（n=5）,冲洗后可使开放概率基本恢复.上述研究结果显示,在成年大鼠海马CA1神经元上存在外向整流氯离子通道.     

槲皮素对谷氨酸诱导的PC12细胞损伤的保护作用及对海马CA1锥体神经元钾通道的影响(英文)

槲皮素广泛存在于许多药用植物中,属黄酮类化合物,在临床上常用于心血管疾病的治疗。采用四甲基偶氮唑盐比色法(MTT法)及DAPI染色,研究槲皮素(0.5、1、5、10μmol/L)对谷氨酸(10mmol/L)诱导的PC12细胞损伤作用的影响;并进一步研究槲皮素(0.3、3、30μmol/L)对急性分离的海马CA1锥体神经元离子通道的作用。MTT实验结果显示,槲皮素可提高谷氨酸处理组PC12细胞的存活率,并呈现为浓度和时间依赖性(P0.05);而槲皮素(5μmol/L)与谷氨酸(10mmol/L)共孵育PC12细胞后,DAPI染色结果表明槲皮素可减弱谷氨酸对PC12细胞的损伤。对电生理结果显示,槲皮素对瞬时外向钾电流(IA)和延迟整流钾电流(IK)有显著的抑制作用(P0.05),表现为浓度依赖性。以上结果提示,槲皮素可能通过抑制海马锥体神经元的外向钾电流进而对谷氨酸诱导的神经损伤起保护作用,这也说明了槲皮素对缺血样损伤的神经具有保护作用。     

Electrophysiological Effects of SKF83959 on Hippocampal CA1 Pyramidal Neurons: Potential Mechanisms for the Drug's Neuroprotective Effects

Although the potent anti-parkinsonian action of the atypical D₁-like receptor agonist {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 has been attributed to the selective activation of phosphoinositol(PI)-linked D₁ receptor, whereas the mechanism underlying its potent neuroprotective effect is not fully understood. In the present study, the actions of {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 on neuronal membrane potential and neuronal excitability were investigated in CA1 pyramidal neurons of rat hippocampal slices. {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent depolarization, associated with a reduction of input resistance in CA1 pyramidal neurons. The depolarization was blocked neither by antagonists for D₁, D₂, 5-HT_2A/2C receptors and α₁-adrenoceptor, nor by intracellular dialysis of GDP-β-S. However, the specific HCN channel blocker ZD7288 (10 µM) antagonized both the depolarization and reduction of input resistance caused by {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959. In voltage-clamp experiments, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (10–100 µM) caused a concentration-dependent increase of Ih current in CA1 pyramidal neurons, which was independent of D₁ receptor activation. Moreover, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 (50 µM) caused a 6 mV positive shift in the activation curve of Ih and significantly accelerated the activation of Ih current. In addition, {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 also reduced the neuronal excitability of CA1 pyramidal neurons, which was manifested by the decrease in the number and amplitude of action potentials evoked by depolarizing currents, and by the increase of firing threshold and rhoebase current. The above results suggest that {"type":"entrez-protein","attrs":{"text":"SKF83959","term_id":"1155968032","term_text":"SKF83959"}}SKF83959 increased Ih current through a D₁ receptor-independent mechanism, which led to the depolarization of hippocampal CA1 pyramidal neurons. These findings provide a novel mechanism for the drug''s neuroprotective effects, which may contributes to its therapeutic benefits in Parkinson''s disease.     

The Nootropic Drug Vinpocetine Inhibits Veratridine-Induced [Ca2+]i Increase in Rat Hippocampal CA1 Pyramidal Cells

The alkaloid derivative vinpocetine (14-ethoxycarbonyl-(3,16-ethyl)-14,15-eburnamine; Cavinton) has a well known beneficial effect on brain function in hypoxic and ischemic conditions. While it increases CNS blood flow and improves cellular metabolism, relatively little is known about vinpocetine's underlying molecular mechanisms on the single cell level. Since apoptotic and necrotic cell damage is always preceded by an increase in [Ca²⁺]_i, this study investigated the effect of vinpocetine on [Ca²⁺]_i increases in acute brain slices. Sodium influx is an early event in the biochemical cascade that takes place during ischemia. The alkaloid veratridine can activate this Na⁺ influx, causing depolarization and increasing [Ca²⁺]_i in the cells. Therefore, it can be used to simulate an ischemic attack in brain cells. Using a cooled CCD camera-based ratio imaging system and cell loading with fura 2/AM, the effect of vinpocetine on [Ca²⁺]_i changes in single pyramidal neurons in the vulnerable CA1 region of rat hippocampal slices was investigated. Preperfusion and continuous administration of vinpocetine (10 M) significantly inhibited the elevation in [Ca²⁺]_i induced by veratridine (10 M). When the drug was administered after veratridine, it could accelerate the recovery of cellular calcium levels. Piracetam, another nootropic used in clinical practice, could attenuate the elevation of [Ca²⁺]_i only at a high, 1 mM, concentration. We have concluded that vinpocetine, at a pharmacologically relevant concentration, can decrease pathologically high [Ca²⁺]_i levels in individual rat hippocampal CA1 pyramidal neurons; this effect might contribute to the neuroprotective property of the drug.