贝美格致痫大鼠皮层、海马γ—氨基丁酸和谷氨酸免疫反应细胞的改变

目的 γ－氨基丁酸（GABA）和谷氨酸（Glu）是脑内重要的抑制性和兴奋性神经递质，二者与癫痫发作密切相关，为了阐明癫痫发病的病理生理机制。方法 我们采用免疫细胞化学及PAP法，观察了贝美格（Bemegride,Be)腹腔致痫大鼠顶叶大脑皮层、海马CA1、CA3、齿状回Glu和GABA免疫反应细胞的改变。结果 图像分析结果显示：Be致痫组大脑皮层、海马Glu免疫反应平均阳性细胞数及光密度较正常对照组明显增加（P＜0．01）；GABA细胞数及光密度减少（P＜0．01）。结论 提示贝美格致痫作用与调节脑内GABA和Glu系统的兴奋性有关。     

中枢组胺对戊四氮点燃大鼠海马GABA能神经元的影响

目的:探讨中枢组胺抗癫痫的作用机制。方法:应用免疫组织化学方法研究中枢组胺对戊四氮(PTZ)致癫痫大鼠海马神经元GABA、GAD67表达的影响。结果:戊四氮致痫组大鼠海马神经元GABA、GAD67的表达量明显低于正常对照组,L-组胺酸干预组明显高于戊四氮致痫组,L-组胺酸干预组与正常对照组之间差异无显著性意义。结论:中枢组胺通过激活海马GABA能神经元来抑制癫痫的发生和发展。     

低剂量伽玛刀照射对致大鼠皮质及海马神经元c-fos和nNOS表达的影响

目的 通过观察低剂量伽玛刀照射对致痫大鼠大脑皮质及海马神经元c—fos和脑型一氧化氮合酶（nNOS）表达的影响，探讨伽玛刀治疗癫痫的作用机制。方法将44只青霉素致痫大鼠模型等分为实验组和实验对照组大鼠各22只，另取4只正常大鼠作为正常对照组。实验组行伽玛刀照射（周边剂量12Gy）后，应用免疫组化方法，观察大脑皮质及海马神经元c-fos和nNOS表达的变化。结果无论是皮质还是海马，c—fos和nNOS在实验组与实验对照组动物之间，表达均有明显的差别，实验组表达明显少于实验对照组，而后者呈现双高峰现象。结论c—fos和nNOS在伽玛刀治疗癫痫的机制中发挥了重要作用。     

癫痫状态大鼠海马HSP70mRNA及GAD67mRNA表达水平的研究

目的 研究癫痫演变过程中特定脑区选择性神经元损伤与修复机制。方法 采用分子杂交方法动态观察了马桑内酯（coriaria Lactone,CL)诱导的癫痫状态大鼠海马区早期神经元损伤标志物热休克蛋白（heat shock protein,HSP)70mRNA及谷氨酸脱羧酶（glutamin acid decarboxylase,GAD)67mRNA表达水平的变化,同时也观察了大鼠行为学及脑电图改变。结果 侧脑室注射CL后20分钟,大鼠出现连续发作的四肢抽搐伴有EEG持续性的棘慢波及尖波发放。注射CL后2小时组大鼠海马GAD67 mRNA表达水平有所降低,但HSP70mRNA变化不明显,注射CL后8小时组大鼠海马GAD67mRNA表达水平有所降低,但HSP70mRNA表达变化不明显,注射CL后8小时组大鼠海马GAD67mRNA表达水平明显低于对照组（P＜0．01）,HSP70mRNA表达水平较对照组明显增高（P＜0．01）。结论 癫痫状态期间大鼠海马区可能存在神经元损伤与修复过程,而且GABA能神经元受到不同程度的损伤。     

癫痫大鼠海马区白细胞介素1受体1免疫反应阳性细胞的观察

目的：探讨白细胞介素１受体１在海马区的表达与癫痫发病的关系。方法；用免疫细胞化学法，观察成年和美解眠致痫大鼠海马区ＩＬ－１Ｒ１免疫反应阳性细胞分布。结果：成年大鼠海马区存在ＩＬ－１Ｒ１－ＩＲ阳性细胞，美解眠致痫后，海马区该阳性细胞数显著增多；致痫前后给予尼莫地平，海马区该阳性细胞增较癫痫大鼠显著减少，与正常比较无显著差异。     

孕激素对癫痫大鼠大脑皮层,海马FOS蛋白表达的影响

本实验采用流式细胞免疫荧光技术对马桑内酯（ＣＬ）致痫及给予孕激素后再致痫大鼠大脑皮层、海马细胞ＦＯＳ表达进行定量检测。结果显示：ＣＬ致痫组大鼠皮层、海马ＦＯＳ表达的荧光指数（ＦＩ）和阳性细胞数较正常对照组明显增高（Ｐ＜０．０１）；给予孕酮（Ｐ）后再致痫组皮层、海马ＦＯＳ表达较单纯ＣＬ致痫组减少（Ｐ＜０．０１，Ｐ＜０．０５）。     

低剂量伽玛刀对致痫大鼠皮层及海马神经元Fos表达的影响

目的通过观察低剂量伽玛刀照射对致痫大鼠大脑皮质及海马神经元cfos表达的影响,初步探讨伽玛刀治疗癫痫的作用机制,为临床治疗提供理论依据。方法以青霉素致痫模型为对象,应用免疫组化方法,检测低剂量伽玛刀照射(周边剂量12Gy)后,大脑皮质及海马神经元cfos表达的变化。实验组和对照组大鼠各22只,正常对照组大鼠4只。结果无论是皮层还是海马,伽玛刀照射后cfos在实验组动物和对照组动物中,表达均有明显的差别,并随时间呈现一定的规律性。结论cfos在伽玛刀治疗癫痫的机理中发挥重要作用。     

皮质醇对马桑内酯致痫大鼠大脑皮层、海马γ-氨基丁酸免疫组化分布的影响

为了解皮质醇对马桑内酯致痫大鼠大脑皮层、海马γ－氨基丁酸（ＧＡＢＡ）免疫组化分布的影响，应用免疫组化方法研究３组大鼠大脑皮层、海马γ－氨基丁酸免疫反应（ＧＡＢＡ－ＩＲ）阳性细胞的分布，以探讨马桑内酯致痫及人工合成皮质醇－地塞米松（Ｄｅｘ）的抗痫是否与ＧＡＢＡ系统相关。结果显示：马桑内酯致痫后大鼠大脑皮层、海马齿状回ＧＡＢＡ－ＩＲ阳性细胞较正常组明显减少，应用Ｄｅｘ后再致痫，大鼠大脑皮层、海马ＧＡＢＡ－ＩＲ细胞数居正常及致痫组之间，３组ＧＡＢＡ－ＩＲ细胞数经Ｆ检验有非常显著性差异（大脑皮层Ｆ值＝２０．９，Ｐ＜０．００１，海马Ｆ值＝４７．７，Ｐ＜０．０１），表明马桑内酯致痫可能有ＧＡＢＡ系统的参与，ＧＡＢＡ－ＩＲ阳性细胞的减少使大脑皮层及海马神经元脱抑制，临床表现抽搐。Ｄｅｘ可能通过抑制ＧＡＢＡ的再摄取、促使ＧＡＢＡ神经元蛋白合成，参与兴奋的抑制。     

青霉素致痫大鼠脑内HSP70的表达

目的 探讨应激蛋白HSP70在癫痫大鼠脑组织中的表迭及其意义。方法 采用腹腔注射青霉素法复制大鼠癫痫模型，免疫组织化学方法检测大脑皮层、海马组织中HSP70表迭的变化。结果 (1)实验组大鼠给药后均有程度不同的癫痫行为发生；(2)对照组大鼠脑内未见HSP70阳性细胞，青霉素致痈后大鼠大脑皮层、海马出现HSP70阳性细胞。在海马的CA1、CA3、CA4区和颞叶皮质HSP70阳性细胞较为密集。结论 青霉素致痫大鼠脑内可见HSP70的广泛表达，提示HSP70蛋白在癫痫发生过程中可能对神经元具有保护作用。     

海人酸致痫大鼠海马及颞叶皮质区域NMDA受体亚单位R1蛋白的表达

背景：研究表明天门冬氨酸受体亚单位R1与癫痫的发生有关,但其具体的表达情况与癫痫脑损伤的关系尚不明确。 目的：观察海人酸致痫大鼠海马及颞叶皮质区域天门冬氨酸受体亚单位1蛋白表达的变化。 设计、时间及地点：随机对照动物实验,于2002-03/2003-03在吉林大学基础医学院生理学和病理生物学教研室完成。 材料：兔抗天门冬氨酸受体受体亚单位1多克隆抗体购自武汉博士德生物科技公司。 方法：将80只雄性健康鼠龄22周的Wistar大鼠随机分为模型组70只和假手术组10只。模型组注射海人酸1 μL至右侧杏仁核,制备癫痫动物模型,分别于造模后2,6,24,72 h及7,15,30 d各取10只动物处死。假手术组在大鼠右侧杏仁核注入PBS 1 μL。 主要观察指标：致痫后2,6,24,72 h及7,15,30 d应用流式细胞仪技术及免疫组织化学的方法观察大鼠脑组织海马及颞叶皮质天门冬氨酸受体受体亚单位1蛋白表达的变化。 结果：假手术组海马各区及颞叶皮质有极少量的天门冬氨酸受体受体亚单位1蛋白阳性细胞分布,海人酸致痫后2 h大鼠海马各区及颞叶皮质天门冬氨酸受体受体亚单位1蛋白表达迅速增加,6 h明显升高,7 d略有下降（CA3区及颞叶皮质）但仍高于假手术组,并持续至30 d（P<0.01）。致痫后海马CA3区、齿状回与颞叶皮质相比,NMDAR1阳性细胞数增多 (P < 0.01)。 结论：海人酸致痫后大鼠海马及颞叶皮质天门冬氨酸受体受体亚单位1蛋白的表达水平均上调,在海马CA3及齿状回较为明显,并持续至致痫后的30 d。天门冬氨酸受体受体亚单位1可能参与了癫痫发生和癫痫脑的长时程的兴奋过程。     

GABAergic neurons in rat hippocampal culture

The experiments described here were designed to study biochemical and histological measures of gamma-aminobutyric acid (GABA) uptake and glutamic acid decarboxylase (GAD) in primary dissociated cell cultures prepared from 17-21-day fetal rat hippocampus. Preparations from all ages of animals, except 21-day fetuses, were enriched in GABAergic neurons, when compared to the adult hippocampus in situ. These cells comprise 30-50% of the large, phase-bright, process-bearing cells in hippocampal cultures as estimated by autoradiography of GABA uptake and GAD immunocytochemistry. Neurons concentrate GABA by a relatively slow but high-affinity process (Km = 2.6 microM) that has considerably higher maximum velocity than glial uptake (Vmax = 479 pmol/mg protein/min for neurons and 31 pmol/mg protein/min for glia). No low-affinity uptake process was noted in neurons or glia. GABA uptake into neurons was competitively inhibited by cis-4-OH-nipecotic acid (Ki = 39 +/- 11 microM). These cultures also possess considerable GAD activity, up to 6 nmol/mg protein/min in one-month-old cultures, which approximates that of the adult hippocampus. Both GABA uptake and GAD activity increased with time in culture. The enrichment of GABAergic markers indicates that this preparation may be useful for the detailed study of hippocampal GABAergic neurons.     

Insulin inhibits pyramidal neurons in hippocampal slices

The effect of bombesin, applied intraventricularly in the rat, was examined with regard to the central control of behavior and arousal. Infusion of 1.0 micrograms bombesin produced stereotypic grooming activity and completely eliminated observable sleep. EEG frequency analysis confirmed the absence of sleep and demonstrated normal waking patterns during behavioral stereotypy. These results support a possible physiologic function for bombesin-like peptides in grooming and the sleep-waking cycle.     

Isoflurane-induced neuronal apoptosis in developing hippocampal neurons

We hypothesized that the P2X7 receptor may be the target of isoflurane, so we investigated the roles of the P2X7 receptor and inositol triphosphate receptor in calcium overload and neuronal apoptosis induced by isoflurane in cultured embryonic rat hippocampal neurons. Results showed that isoflurane induced widespread neuronal apoptosis and significantly increased cytoplasmic Ca 2+ . Blockade of P2X7 receptors or removal of extracellular Ca 2+ combined with blockade of inositol triphosphate receptors completely inhibited apoptosis or increase in cytoplasmic Ca 2+ . Removal of extracellular Ca 2+ or blockade of inositol triphosphate receptor alone could partly inhibit these effects of isoflurane. Isoflurane could directly activate P2X7-gated channels and induce inward currents, but did not affect the expression of P2X7 receptor protein in neurons. These findings indicate that the mechanism by which isoflurane induced neuronal apoptosis in rat developing brain was mediated by intracellular calcium overload, which was caused by P2X7 receptor mediated calcium influx and inositol triphosphate receptor mediated calcium release.     

View-responsive neurons in the primate hippocampal complex

Recordings were made from single neurons in the hippocampus and parahippocampal gyrus while macaques were moved on a platform mounted on a free-moving robot or on wheels in a cue-controlled 2 m × 2 m × 2 m environment, in order to investigate the representation of space and of spatial memory in the primate hippocampus. The test conditions allowed factors that might account for spatial firing of the cells, including the spatial location where the monkey looked, the place were the monkey was, and the head direction of the monkey, to be identified. The responses of some (“view”) neurons depended on where the monkey was looking in the enviornment, but not on the place of the monkey in the environment. The responses of one other neuron depended on a combination of where the monkey was facing and his place in the test chamber. The response of view-dependent neurons was affected by occlusion of the visual field. It was possible to show for one neuron that its “view” response rotated with rotation of the test chamber. Some neurons responded to a combination of whole-body motion and view or place, and one neuron responded in relation to whole-body movement to a particular place. One neuron responded depending on the place where the monkey was in the environment and relatively independently of view. The representations of space provided by hippocampal view-responsive neurons may be useful in forming memories of spatial environments (for example, of where an object has been seen and of where the monkey is as defined by seen views) and, together with whole-body motion cells, in remembering trajectories through environments, which is of use, for example, in short range spatial navigation. © 1995 Wiley-Liss, Inc.     

Serotonin stimulates mitochondrial transport in hippocampal neurons

Axonal transport of mitochondria is critical for proper neuronal function. However, little is known about the extracellular signals that regulate this process. In the present study, we show that the neuromodulator serotonin (5-HT) greatly enhances mitochondrial movement in the axons of rat hippocampal neurons in vitro. Administration of a 5-HT1A receptor antagonist inhibited mitochondrial movement, whereas addition of fluoxetine, a selective serotonin reuptake inhibitor, promoted mitochondrial movement. 5-HT receptors are known to activate the Akt/Protein kinase B pathway. Consistent with this, directional mitochondrial movement was almost completely blocked by a specific Akt inhibitor. Moreover, an inhibitor of glycogen synthase kinase-3beta (GSK3beta), a kinase whose activity is blocked by Akt-mediated phosphorylation, promoted mitochondrial movement. These findings show that 5-HT1A receptor activation stimulates mitochondrial movement in hippocampal neurons by inhibiting GSK3beta activity via Akt. Our findings suggest that 5-HT may mediate the redistribution of energy sources within responsive neurons, a possibility that has significant implications for understanding the global biological effects of this important neuromodulator.     

Vasopressin-induced calcium signaling in cultured hippocampal neurons

We recently demonstrated that the neural peptide vasopressin (AVP) can act as a neurotrophic factor for hippocampal nerve cells in culture. Because the neurotrophic effect of vasopressin is mediated by the V₁ receptor [11], we investigated AVP activation of calcium signaling pathways in cultured hippocampal neurons. Results of this investigation demonstrate that exposure of cultured hippocampal neurons prelabeled with [³H]myo-inositol to vasopressin induced a significant accumulation of [³H]inositol-1-phosphate ([³H]IP₁). The selective V₁ vasopressin receptor agonist, [Phe², Orn²]vasotocin, induced a s significant accumulation of [³H]IP₁ whereas a selective V₂ vasopressin receptor agonist, [deamino¹, d-Arg⁸]-vasopressin, did not. Moreover, V₁ agonist-induced accumulation of [³H]IP₁ was blocked by the selective V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ agonist-induced accumulation of [³H]IP₁ was concentration dependent and exhibited a steep inverted U-shaped curve that included both stimulation and inhibition of [³H]IP₁ accumulation. Time course analysis of V₁ agonist-induced accumulation of [³H]IP₁ revealed significant increase by 20 min which continued to be significantly elevated for 60 min. Investigation of the effect of closely related peptides on [³H]IP₁ accumulation indicated that the vasopressin metabolite peptide AVP_4–9 and oxytocin significantly increased [³H]IP₁ accumulation whereas the vasopressin metabolite peptide AVP_4–8 did not. AVP_4–9 and oxytocin induced [³H]IP₁ accumulation were blocked by the V₁ vasopressin receptor antagonist d(CH₂)₅[Tyr(Me)²]-vasopressin. V₁ receptor activation was associated with a pronounced rise in intracellular calcium. Results of calcium fluorometry studies indicated that V₁ agonist exposure induced a marked and sustained rise in intracellular calcium that exhibited oscillations. Interestingly, absence of calcium in the extracellular medium abolished both the rise in intracellular calcium and the appearance of oscillations. The loss of the intracellular calcium signal is not due to a failure to induce PIP₂ hydrolysis since activation of the phosphatidylinositol pathway occurred in the absence of extracellular calcium. V₁ agonist (250 nM) induced a highly significant increase in⁴⁵Ca²⁺ uptake from the extracellular medium within 5 sec of exposure.⁴⁵Ca²⁺ uptake remained significantly greater than basal for 300 sec. The increase in⁴⁵Ca²⁺ uptake was followed by a significant inhibition of uptake by 20 min of exposure. These results indicate that in cultured hippocampal neurons, V₁ vasopressin receptor activation leads to activation of the phosphatidylinositol signaling pathway, uptake of calcium from the extracellular medium and induction of complex intracellular calcium signals. These data provide the first step in delineating the biochemical mechanism that underlies vasopressin-induced neutrophism.     

Denervation induced abnormal phosphorylation in hippocampal neurons

This study demonstrates that combined dopaminergic and cholinergic denervation of the hippocampus results in the appearance of morphologically altered, Tau reactive, apical dendrites of granule cells in the rat dentate gyrus. The denervated granule cells and their apical dendrites also display immunoreactivity to a mitogen-activated protein kinase, ERK-1, and also evidence of abnormal phosphorylation of these dendrites as revealed by SMI-31 immunoreactivity. Dopaminergic denervation alone also causes mitogen activated protein kinase reactivity without the Tau-reactive apical dendrites. These results suggest an analogy to synaptophysin loss and the appearance of dendritic threads described in Alzheimer's disease (AD), as an early stage in the formation of neurofibrillary tangles (NFT). This is the first animal model in which abnormal phosphorylation of Tau has been shown to be produced experimentally in vivo.     

Inhibition in synchronously firing human hippocampal neurons

In order to study the extent of inhibition in human epileptic hippocampus, we recorded extracellular unit activities of human hippocampal neurons and their responses to single pulse stimulation in temporal lobe epilepsy patients during interictal periods. The criteria for diagnosing the hippocampus as epileptic were: (1) all seizures originated in that one hippocampus, (2) surgical removal of that hippocampus resulted in seizure relief, and (3) the surgically excised hippocampus was sclerotic. Analysis of firing pattern by cross-correlation showed that synchronized firing between neurons occurred only in the epileptic hippocampus. However, synchronized firing was not limited to only bursting neurons, as previously reported in some animal models of epilepsy, but was also observed among non-bursting neurons in the epileptic hippocampus. Furthermore, no significant difference in distribution of burst-discharge neurons was found between epileptic and non-epileptic hippocampi. In response to single pulse stimulation, neurons in both 'normal' (contralateral hippocampus) and epileptic hippocampus showed a rapid increase of firing (excitation), cessation of firing (inhibition), or a sequence of both (initial excitation followed by inhibition). However, a significant difference was found in the duration of the inhibition between synchronously firing neurons and non-synchronously firing neurons: the inhibition evoked by a single stimulation in synchronously firing epileptic neurons was significantly longer (373.8 msec +/- 35.9 S.E.M., P less than 0.005) than that of non-synchronously firing neurons (83.9 msec +/- 8.9 S.E.M.). Moreover, prolonged inhibition in synchronously firing epileptic neurons could occur with little or no prior excitation, suggesting that this inhibition does not necessarily depend on an intrinsic Ca2+-dependent K+-mediated after-burst hyperpolarization but is rather likely to be synaptic. As this inhibition was longer when epileptic neurons fired in synchrony, it could be interpreted that principal neurons recruited more recurrent inhibitory circuits by firing synchronously. By taking into account the previously reported neurophysiological evidence in human in vitro epileptic tissue showing GABA-mediated inhibition and the neuroanatomical evidence in excised human epileptic hippocampus showing GAD-positive neurons and synapses, our data suggest that, in human chronic epileptic hippocampus, recurrent inhibition remains functional, and alterations in GABA-mediated inhibition may not represent the critical change responsible for seizure generation.     

Rat hippocampal neurons in dispersed cell culture

An in vitro system has been developed for the study of isolated hippocampal neurons from 18- or 19-day rat featuses. Following trypsinization the cells are plated out at low density on polylysine-treated coverslips in an enriched medium. The isolated neurons rapidly attach to the substrate and initiate process extension. Little reaggregation occurs and the number of non-neuronal cells present is minimal. Unless co-cultured with tissue explants the neurons survive for only a few days; in the presence of hippocampal explants the initial growth of the isolated cells is improved and their survival in culture is extended to about two weeks. Some of the cells in such cultures develop a characteristic branching pattern closely resembling that of maturing hippocampal pyramidal cells in vivo. There is a clear relationship between the stage of the cells' development and their growth in culture. Cells which had completed DNA synthesis about 48 h before dissociation, and which were in the process of migration to the cortical plate, survived best in our cultures. Early post-mitotic cells which were still within the ventricular zone and cells which had already reached the cortical plate grew poorly. This system should permit the study not only of process formation by these cells, but also of their capacity to form specific synapses in vitro and of the biochemical constituents of their surfaces.