谷氨酸单钠透过盘屏障对仔鼠特写脑区的结构和功能的影响

＾３Ｈ－Ｇｌｕ示踪实验表明,谷氨酸可以透过胎屏障进入胎鼠体内,较均一地分布于中枢神经系统及内脏各器官。经母鼠隔日注射高,低剂量的谷氨酸单钠（ＭＳＧ,２．５ｍｇ／ｇ,１．０ｍｇ／ｇ）至分娩,对成年后的仔鼠进行检测发现,高剂量组仔鼠记忆能力及Ｙ－迷宫空间分辨学习能力均严重受损,下丘脑弓状核及腹内侧核神经元被明显破坏,出现胞质肿胀、核固缩、神经元数目减少。孕期ＭＳＧ处理还可以使仔鼠正丘脑和海马＾３Ｈ－Ｇ     

外源性神经节苷脂对人－肝癌培养细胞内钙的作用及其方式

本工作采用荧光探针Ｆｕｒａ－２ＡＭ观察了外源性神经节苷脂ＧＭ３和ＧＤ３对ＳＭＭＣ－７７２１人肝癌培养细胞钙的影响,证明ＧＭ３和ＧＤ３均能升高细胞内钙浓度（［Ｃａ２＋］ｉ）,但程度上有极大差异。１０ｎｍｏｌ／ｍＬＧＭ３或１．０ｎｍｏｌ／ｍＬＧＤ３可使［Ｃａ２＋］ｉ上升高是明显,与对照相比［Ｃａ２＋］ｉ分别增加２１５～２５０％和４２％。进一步用Ｖｅｒａｐａｍｉｌ阻断钙通道和内质网钙释放、去除细胞外Ｎａ＋以抑制Ｎａ＋－Ｃａ２＋交换以及去除细胞外Ｃａ２＋在无外钙内流等系统观察了ＧＭ３和ＧＤ３的作用方式,结果提示ＧＭ３升高［Ｃａ２＋］ｉ的机制是一个同时增加内质网钙释放、激活钙通道并伴有质膜Ｃａ２＋－ＡＴＰ酶激活的综合结果;而ＧＤ３则主要抑制Ｎａ＋－Ｃａ２＋交换系统。     

SNP抑制5-HT诱导的胞内游离钙浓度升高和内钙释放

用Ｆｕｒａ － ２／ＡＭ 荧光测量技术研究了５ － 羟色胺（５－ ＨＴ） 诱导的大鼠尾动脉平滑肌细胞胞内钙升高和一氧化氮（ＮＯ） 的抑制效应。实验表明, 胞外０ｍ ｍｏｌ／ Ｌ Ｃａ２ ＋ 时胞内静息［Ｃａ２ ＋ ］ ｉ 为２０ ．２±８ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ８） 。１０μｍｏｌ／Ｌ ５－ ＨＴ 可诱导出胞内钙库释放引起的瞬态［Ｃａ２ ＋］ｉ 升高,其峰值达２４５ ．７ ±７１．６ｎｍｏｌ／ Ｌ（ｎ ＝ ６） 。１０ － ７ ｍｏｌ／Ｌ 硝普钠（ＳＮＰ） 可抑制５－ ＨＴ 诱导的［Ｃａ２ ＋］ｉ 升高,其峰值浓度降为７５．１±３５ ．９ｎｍｏｌ／Ｌ（ｎ ＝ ５） 。当细胞浴液含２．５ｍ ｍｏｌ／Ｌ Ｃａ２ ＋ 时,静息［Ｃａ２ ＋］ｉ为１１２ ．８ ±１０ ．３ｎｍｏｌ／ Ｌ（ｎ ＝ ５） , 这时１０μｍｏｌ／ Ｌ ５ － ＨＴ 可诱导［Ｃａ２ ＋ ］ ｉ 的峰值为２５２ ．３ ±８０ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,以及其后平台浓度为１４３ ．０ ±３７ ．６ｎｍｏｌ／Ｌ（ｎ ＝ ４） ,略大于［Ｃａ２ ＋］ｉ 为１１２．８ ±１０ ．３ｎｍｏｌ／Ｌ 的静息浓度,为外钙内流引起。１０ － ７ ｍｏｌ／Ｌ ＳＮＰ 也可抑制５－ ＨＴ 诱导［Ｃａ２ ＋ ］ｉ 平台相浓度。平台浓度由１４３ ±４７     

降钙素基因相关肽对缺氧时海马细胞内游离Ca^2＋的影响

本实验用Ｆｕｒａ－２荧光测定技术直接监测了缺氧时大鼠海马细胞内游离Ｃａ２＋浓度（［Ｃａ２＋］ｉ）的变化,并观察了降钙素基因相关肽（ＣＧＲＰ）对这种变化的影响。结果发现,缺氧可使海马细胞［Ｃａ２＋］ｉ显著增高;４或８ｎｍｏｌ／ＬＣＧＲＰ能明显地降低缺氧引起的［Ｃａ２＋］ｉ增高,但在无胞外Ｃａ２＋的情况下,ＣＧＲＰ的作用消失。结果表明,ＣＧＲＰ的降钙作用是通过抑制缺氧时胞外Ｃａ２＋的内流来实现的。     

糖皮质激素对肝细胞内游离钙的作用

应用钙离子荧光指示剂ｆｕｒａ－２,对糖皮质激素（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ,ＧＣ）是否影响肝细胞内游离钙（Ｉｎｔｒａｃｅｌｌｕｌａｒ　ｆｒｅｅ　ｃａｌｃｉｕｍ,［Ｃａ２＋］ｉ作了初步探讨。结果发现,ＧＣ在短期内能升高肝细胞［Ｃａ２＋］ｉ,水平,并具有明显的量效关系。以１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏｌ和１０．０μｍｏｌ／Ｌ的Ｄｅｘａｍｅｔｈａｓｏｎｅ效果最好。加入１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏｌ０．２５ｍｉｎ即可引起肝细胞［Ｃａ２＋］ｉ的明显升高,到１０分钟时效应达高峰。此时与静息状态的肝细胞［Ｃａ２＋］ｉ水平相比,胞浆内游离钙升高了近３倍;与相应对照组比较,胞浆内游离钙升高具有明显的统计学意义,Ｐ＜０．０１。ＲＵ４８６为一种人工合成的糖皮质激素受体（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ　ｒｅｃｅｐｔｏｒ,ＧＲ）的拮抗剂,它可以取消ＧＣ升高肝细胞［Ｃａ２＋］ｉ的效应,提示ＧＣ升高肝细胞内［Ｃａ２＋］ｉ可能与ＧＲ介导有一定关系。鉴于ＧＣ升高［Ｃａ２＋］ｉ时间较短,推测与肝细胞膜ＧＲ的非基因快速调节作用影响钙离子通道有关。     

γ—氨基丁酸抑制缺氧所致神经元钙超载

本文以体外分散培养的新生大鼠海马ＣＡ１区神经细胞为标本,分别采用激光扫描共聚集显微镜动态监测单个细胞［Ｃａ＾２＋］ｉ和膜片箝全细胞记录的电生理技术检测细胞的ＮＭＤＡ电流和电压依赖性Ｃａ＾２＋电流等方法,较为深入地研究了抑制性神经递质γ－氨基丁酸（ＧＡＢＡ）及ＧＡＢＡ－Ａ受体激动剂蝇蕈醇对急性缺氧时海马ＣＡ１神经元［Ｃａ＾２＋］ｉ升高过程的影响方式及其作用机制。结果表明：对照组细胞缺氧后比缺氧前［Ｃ     

兴奋性氨基酸加强高原低氧大鼠下丘脑前生长抑素原mRNA的表达

目的和方法：应用大鼠高原低氧模型及原位杂交技术和氨基酸测定法,研究下丘脑前生长抑素原（ＰＰＳ）ｍＲＮＡ表达和谷氨酸（Ｇｌｕ）、天门冬氨酸（Ａｓｐ）含量的变化。结果：高原低氧组大鼠下丘脑Ｇｌｕ和Ａｓｐ的含量明显增多,室周核、室旁核、弓状核ＰＰＳ－ｍＲＮＡ阳性神经元数目显著增加;而ＮＭＤＡ受体拮抗抗剂氯铵酮,虽然对Ｇｌｕ和Ａｓｐ含量无明显影响,但可使高原低氧大鼠下丘脑ＰＰＳ－ｍＲＮＡ阳性神经元数目减少     

缺氧时大鼠红细胞变形性损伤的机制研究

本实验通过测定平原和模拟高原减压缺氧３０天大鼠红细胞滤过指数（ＩＦ）、红细胞内［ｐＨ］ｉ、［Ｋ＋］ｉ／［Ｎａ＋］ｉ比值、［Ｃａ２＋］ｉ、［Ｍｇ２＋］ｉ、平均红细胞体积（ＭＣＶ）及平均红细胞血红蛋白浓度（ＭＣＨＣ）,从而探讨缺氧条件下大鼠红细胞变形性损害的机制。结果发现：１．缺氧组大鼠红细胞［Ｃａ２＋］ｉ明显升高,且与ＩＦ呈显著正相关,但［Ｍｇ２＋］ｉ无明显差异;２．缺氧组［Ｋ＋］ｉ／［Ｎａ＋］ｉ值较平原组明显降低,且与ＩＦ呈显著负相关;３．缺氧组ＭＣＨＣ与平原组无明显差异,但ＭＣＶ显著升高;４．缺氧组红细胞内［ｐＨ］ｉ较平原组明显升高。提示：缺氧时红细胞［Ｃａ２＋］ｉ升高,［Ｋ＋］ｉ／［Ｎａ＋］ｉ值降低,ＭＣＶ增大以及红细胞［ｐＨ］ｉ值的改变在其变形性损伤中起重要作用。     

培养乳鼠心肌细胞自发性〔Ca~（2＋）〕_i瞬间变化的实验模型

使用Ｃａ￣（２＋）敏感的荧光指示剂Ｆｕｒａ－２／ＡＭ,对培养的心肌细胞测定细胞内Ｃａ￣（２＋）的瞬间变化。结果为,在一个心搏周期中,经过大约１８０ｍｓ的时间,［Ｃａ￣（２＋）］＿ｉ瞬间变化达到最大值,然后恢复到基线水平（最小值）,经历了２６０ｍｓ。测得平均［Ｃａ￣（２＋）］＿ｉ瞬间变化的最大值及最小值分别为３４６±３８ｎｍｏｌ／Ｌ和１０２±１８ｎｍｏｌ／Ｌ。血管紧张素Ⅱ１００ｎｍｏｌ／Ｌ引起［Ｃａ￣（２＋）］＿ｉ瞬间变化最大值明显增加,但对［Ｃａ￣（２＋）］＿ｉ瞬间变化的基线水平没有明显的影响。ｒｙａｎｏｄｉｎｅ３μｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化的幅度明显降低。ＫＣｌ５０ｍｍｏｌ／Ｌ使［Ｃａ￣（２＋）］＿ｉ瞬间变化完全停止,并明显增加［ｃａ￣（２＋）］＿ｉ的基线水平。结果提示,本实验模型可用来观察［Ｃａ￣（２＋）］＿ｉ的瞬间变化。     

糖皮质激素对肝细胞内游离钙的作用

应用钙离子荧光指示剂ｆｕｒａ－２,对糖皮质激素（Ｇｌｕｃｏｃｏｒｔｉｃｏｉｄ,ＧＣ）是否影响肝细胞内游离钙（Ｉｎｔｒａｃｅｌｌｕａｌａｒ ｆｒｅｅ ｃａｌｃｉｕｍ,［Ｃａ＾２＋］ｉ作了初步探讨,结果发现,ＧＣ在短期内能升高肝细胞［Ｃａ＾２＋］水入１．０μｍｏｌ／Ｌ的Ｃｏｒｔｉｓｏ０．２５ｍｉｎ即可引起肝细胞［Ｃａ＾２＋］的明显升高,到１０分钟时效应达高峰。此时与静息状态的肝细胞水平相比胞浆内游离钙     

Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in motor learning and memory

Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.     

灵芝对小鼠空间分辨学习与记忆的影响

本文用Y-型迷宫法测试小鼠空间分辨行为。实验结果表明,每日ig灵芝2.58/kg共7d,有明显促进学习的作用。每日ig灵芝2.5g/kg共7d或ig灵芝5g/kg共7d都能显著地拮坑东莨菪碱所致学习障碍的作用。此外,学习训练后立即ig灵芝2.5g/kg或ig灵芝5g/kg也有明显地改善东莨菪碱损害记忆巩固的作用。     

Facilitation of NMDAR-independent LTP and spatial learning in mutant mice lacking ryanodine receptor type 3

To evaluate the role in synaptic plasticity of ryanodine receptor type 3 (RyR3), which is normally enriched in hippocampal area CA1, we generated RyR3-deficient mice. Mutant mice exhibited facilitated CA1 long-term potentiation (LTP) induced by short tetanus (100 Hz, 100 ms) stimulation. Unlike LTP in wild-type mice, this LTP was not blocked bythe NMDA receptor antagonist D-AP5 but was partially dependent on L-type voltage-dependent Ca2+ channels (VDCCs) and metabotropic glutamate receptors (mGluRs). Long-term depression (LTD) was not induced in RyR3-deficient mice. RyR3-deficient mice also exhibited improved spatial learning on a Morris water maze task. These results suggest that in wild-type mice, in contrast to the excitatory role of Ca2+ influx, RyR3-mediated intracellular Ca2+ ([Ca2+]i) release from endoplasmic reticulum (ER) may inhibit hippocampal LTP and spatial learning.     

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A–E), receptor types (VEGFR1–3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.     

Effects of Scopolamine and Melatonin Cotreatment on Cognition,Neuronal Damage,and Neurogenesis in the Mouse Dentate Gyrus

It has been demonstrated that melatonin plays important roles in memory improvement and promotes neurogenesis in experimental animals. We examined effects of melatonin on cognitive deficits, neuronal damage, cell proliferation, neuroblast differentiation and neuronal maturation in the mouse dentate gyrus after cotreatment of scopolamine (anticholinergic agent) and melatonin. Scopolamine (1 mg/kg) and melatonin (10 mg/kg) were intraperitoneally injected for 2 and/or 4 weeks to 8-week-old mice. Scopolamine treatment induced significant cognitive deficits 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly improved spatial learning and short-term memory impairments. Two and 4 weeks after scopolamine treatment, neurons were not damaged/dead in the dentate gyrus, in addition, no neuronal damage/death was shown after cotreatment of scopolamine and melatonin. Ki67 (a marker for cell proliferation)- and doublecortin (a marker for neuroblast differentiation)-positive cells were significantly decreased in the dentate gyrus 2 and 4 weeks after scopolamine treatment, however, cotreatment of scopolamine and melatonin significantly increased Ki67- and doublecortin-positive cells compared with scopolamine-treated group. However, double immunofluorescence for NeuN/BrdU, which indicates newly-generated mature neurons, did not show double-labeled cells (adult neurogenesis) in the dentate gyrus 2 and 4 weeks after cotreatment of scopolamine and melatonin. Our results suggest that melatonin treatment recovers scopolamine-induced spatial learning and short-term memory impairments and restores or increases scopolamine-induced decrease of cell proliferation and neuroblast differentiation, but does not lead to adult neurogenesis (maturation of neurons) in the mouse dentate gyrus following scopolamine treatment.     

Steroid-monoamine feedback interactions in discrete brain regions using as a model the monosodium glutamate (MSG)-lesioned rat

The present studies examine the effects of neonatal treatment with monosodium glutamate (MSG) on dopamine (DA), 5-hydroxytryptamine (5-HT) and norepinephrine (NE) metabolism in discrete brain regions and correlate them with steroid receptor kinetics in the anterior pituitary (PIT), preoptic hypothalamus (POA) and caudal hypothalamus (HYP), and with steroid negative and positive feedback effects on luteinizing hormone (LH) secretion. Substantial decreases in the neuronal activity of all three amines in the arcuate nucleus, decreased DA and 5-HT metabolism in the suprachiasmatic nucleus and, surprisingly, increased metabolism of 5-HT and NE in the median eminence was observed in adult ovariectomized (OVX), MSG-treated versus OVX, vehicle-treated litter mate controls. Measurement of estradiol receptors in the nuclear and cytosolic fractions of the POA, HYP and PIT from MSG- and vehicle-treated rats killed during diestrus or 2 weeks after OVX revealed no differences. Similarly, no differences in cytosolic progestin receptors between control and MSG unprimed or estradiol-primed, OVX rats or on progestin receptor translocation induced by progesterone in Eb-primed rats were observed. Negative and positive feedback effects of estradiol or the positive feedback of progesterone on LH secretion were not significantly impaired in MSG rats, and indeed, MSG animals actually were hyper-responsive to the administration of the steroids or of luteinizing hormone-releasing hormone. These results indicate that the MSG-induced damage to DA, 5-HT and NE elements observed within several preoptic and hypothalamic nuclei does not impair estrogen and progestin receptor kinetics, nor does it prevent adequate negative or positive steroid feedback responses, if appropriate steroid regimens are employed, and that the impaired gonadal function reported in these animals does not result primarily from inadequate steroid feedback mechanisms.     

Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells

Recently, candidates for umami receptors have been identified in taste cells, but the precise transduction mechanisms of the downstream receptor remain unknown. To investigate how intracellular Ca(2+) increases in the umami transduction pathway, we measured changes in intracellular Ca(2+) levels in response to umami stimuli monosodium glutamate (MSG), IMP, and MSG + IMP in mouse taste receptor cells (TRCs) by Ca(2+) imaging. Even when extracellular Ca(2+) was absent, 1/3 of umami-responsive TRCs exhibited increased intracellular Ca(2+) levels. When intracellular Ca(2+) was depleted, half of the TRCs retained their response to umami. These results suggest that umami-responsive TRCs increase their intracellular Ca(2+) levels through two pathways: by releasing Ca(2+) from intracellular stores and by an influx of Ca(2+) from extracellular sources. We conclude that the Ca(2+) influx from extracellular source might play an important role in the synergistic effect between MSG and IMP.     

Neuronal calcium stores

Neuronal calcium stores associated with specialized intracellular organelles, such as endoplasmic reticulum and mitochondria, dynamically participate in generation of cytoplasmic calcium signals which accompany neuronal activity. They fulfil a dual role in neuronal Ca2+ homeostasis being involved in both buffering the excess of Ca2+ entering the cytoplasm through plasmalemmal channels and providing an intracellular source for Ca2+. Increase of Ca2+ content within the stores regulates the availability and magnitude of intracellular calcium release, thereby providing a mechanism which couples the neuronal activity with functional state of intracellular Ca2+ stores. Apart of 'classical' calcium stores (endoplasmic reticulum and mitochondria) other organelles (e.g. nuclear envelope and neurotransmitter vesicles) may potentially act as a functional Ca2+ storage compartments. Calcium ions released from internal stores participate in many neuronal functions, and might be primarily involved in regulation of various aspects of neuronal plasticity.     

Characterization of N-methyl-D-aspartate receptor subunits involved in acute ammonia toxicity

Rapid administration of large doses of ammonia leads to death of animals, which is largely prevented by pretreatment with N-methyl-D-aspartate (NMDA) receptor antagonists. The present study focuses on a subunit(s) of NMDA receptor involved in ammonia-induced death by use of NMDA receptor GluRepsilon subunit-deficient (GluRepsilon(-/-)) mice and the selective GluRepsilon2 antagonist CP-101,606. Acute ammonia intoxication was induced in mice (eight per group) by a single intraperitoneal (i.p.) injection of ammonium chloride. Appearance of neurological deteriorations depended on the doses of ammonium chloride injected. While wild-type, GluRepsilon1(-/-), GluRepsilon4(-/-), and GluRepsilon1(-/-)/epsilon4(-/-) mice all died by ammonium chloride at 12 mmol/kg during the first tonic convulsions, two of eight GluRepsilon3(-/-) mice survived. Pretreatment of wild-type mice with CP-101,606 prevented two mice from ammonia-induced death. Pretreatment of GluRepsilon3(-/-) mice with CP-101,606 prevented the death of three mice and prolonged the time of death of non-survivors. Similarly, the neuronal form of nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI) as well as the nonselective NOS inhibitor L-NMMA, but not the inducible NOS inhibitor 1400W, partially prevented the death of mice and prolonged the period of death. Furthermore, ammonium chloride prolonged the increase in intracellular free Ca2+ concentration ([Ca2+]i) and subsequent NO production induced by NMDA in the cerebellum. These results suggest that activation of NMDA receptor containing GluRepsilon2 and GluRepsilon3 subunits and following activation of neuronal NOS are involved in acute ammonia intoxication which leads to death of animals.