REGULATION OF THE INTRACELLULAR FREE CALCIUM-CONCENTRATION IN ACUTELY DISSOCIATED NEURONS FROM RAT NUCLEUS BASALIS
REGULATION OF THE INTRACELLULAR FREE CALCIUM-CONCENTRATION IN ACUTELY DISSOCIATED NEURONS FROM RAT NUCLEUS BASALIS
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DOI：
10.1113/jphysiol.1993.sp019628
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发表时间：
1993-05-01
影响因子：
5.5
通讯作者：
KATAYAMA, Y
中科院分区：
文献类型：
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作者：
TATSUMI, H;KATAYAMA, Y
1.从大鼠基底核急性分离神经元。采用全细胞膜片钳记录钙电流（I（Ca））和Fura-2微荧光法记录细胞内游离Ca ~（2+）浓度（[Ca ~（2+）]i）.从-60至0 mV去极化引起I（Ca）和[Ca ~（2+）]i逐渐增加。复极化后，I（Ca）在0.7ms内终止，[Ca ~（2+）]i指数恢复至对照水平（1- 5s）.在无Ca ~（2+）的细胞外液和含Cd ~（2+）的细胞外液中，阶跃去极化引起的I（Ca）和瞬时[Ca ~（2+）]i增加均被消除.从-90 mV到小于-40 mV的膜电位去极化诱导I（Ca）和[Ca ~（2+）]i增加。去极化至0 mV引起最大I（Ca），并产生最大的[Ca 2 +]i增加。[Ca 2 +]i的增加与I（Ca）的大小呈平行关系。[Ca 2 +]i增加与总Ca 2+内流的增加时，阶跃去极化的持续时间是不同的。当总Ca 2+内流超过200 pC时，[Ca 2 +]i瞬时峰值与总Ca 2+内流的关系逐渐接近。当超过30个动作电位用于增加[Ca 2 +] i时，也有类似的发现。[Ca 2 +]i恢复的过程减慢通过降低温度，通过细胞内透析与钒酸盐，通过细胞外应用线粒体抑制剂，羰基氰化物间氯苯腙（CCCP），和通过Na+-免费的外部解决方案。它不受膜电位（-50至-130 mV）的影响。当移液器溶液含有高浓度的fura-2（200 μ m）时，每1 pC的Ca 2+内流的[Ca 2 +]i增加减少，并且[Ca 2 +]i恢复缓慢。结果表明，I（Ca）通过电压依赖性Ca ~（2+）通道升高[Ca ~（2+）]i。神经元对Ca ~（2+）具有很大的缓冲能力，[Ca ~（2+）]i的恢复需要Ca ~（2+）泵和膜Na ~+-Ca ~（2+）交换。Meynert基底核在过去10年中一直是备受关注的焦点，因为它被发现是大脑皮层胆碱能传入的主要来源（约翰斯顿，McKinney & Coyle，1979; Lamour，Dutar，Rascol & Jobert，1986）。已经提出皮质血流由该核调节（Hallstrom，Sato，Sato & Ungerstedt，1990）。Nakajima，Nakajima，Obata，Carlson & Yamaguchi（1985）对前脑细胞核培养的胆碱能神经元进行了细胞内记录，并在电流钳条件下检查了这些细胞的基本电生理特性。这些细胞的变性被认为在与记忆障碍相关的疾病中是重要的（Oyanagi，Takahashi，Wakabayashi & Ikuta，1989）。在某些神经元中，变性可能与过量的钙内流有关（韦斯，Hartley，Koh & Choi，1990; Kudo，Takeda & Yamazaki，1990）。本实验采用全细胞膜片钳法和fura-2荧光法测量从大鼠基底核急性分离的神经元的Ca 2+电流和[Ca 2 +]i的相关变化。
1. Neurones were acutely dissociated from the rat nucleus basalis. Whole-cell patch clamp recordings of calcium currents (I(Ca)) and fura-2 microfluorimetric recordings of intracellular free Ca2+ concentration ([Ca2+]i) were made simultaneously.2. Depolarization from - 60 to 0 mV elicited I(Ca) and a gradual increase in [Ca2+]i. After repolarization, I(Ca) terminated in 0.7 ms, and [Ca2+]i recovered to control exponentially (1-5 s).3. Both I(Ca) and the transient [Ca2+]i increase in response to step depolarizations, were abolished in Ca2+ free extracellular solution and in Cd2+-containing solution.4. Depolarizations from - 90 mV to membrane potentials less negative than - 40 mV induced I(Ca) and an increase in [Ca2+]i. Depolarization to 0 mV elicited the maximum I(Ca), and produced the largest increase in [Ca2+]i. There was a parallel relationship between the [Ca2+]i increase and the magnitude of the I(Ca).5. The [Ca2+]i increase was associated with an increase in total Ca2+ influx when the duration of the step depolarization was varied. The relationship between the total Ca2+ influx and the peak of [Ca2+]i transient reached an asymptote as total Ca2+ influx exceeded 200 pC. A similar finding was made when more than thirty action potentials were used in increasing [Ca2+]i.6. The process of the [Ca2+]i recovery was slowed down by lowering the temperature, by an intracellular dialysis with vanadate, by extracellular application of a mitochondrial inhibitor, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), and by Na+-free external solution. It was unaffected by membrane potential (- 50 to - 130 mV).7. When pipette solution contained a high concentration of fura-2 (200 mum), the [Ca2+]i increase per 1 pC of Ca2+ influx decreased, and the [Ca2+]i recovery was slowed.8. The results indicate that the I(Ca) through voltage-dependent Ca2+ channels elevates [Ca2+]i. The neurones possess a large capacity for Ca2+ buffering, and the recovery of [Ca2+]i requires both the Ca2+ pump and membrane Na+-Ca2+ exchange. The nucleus basalis of Meynert has been the focus of much attention over the last 10 years, since it was found to be the major source of cholinergic afferents to the cerebral cortex (Johnston, McKinney & Coyle, 1979; Lamour, Dutar, Rascol & Jobert, 1986). It has been suggested that cortical blood flow is regulated by this nucleus (Hallstrom, Sato, Sato & Ungerstedt, 1990). Nakajima, Nakajima, Obata, Carlson & Yamaguchi (1985) have made intracellular recordings from cholinergic neurones in culture from forebrain nuclei and examined the basic electrophysiological properties of these cells in the current-clamp condition. Degeneration of these cells is thought to be important in diseases associated with memory impairment (Oyanagi, Takahashi, Wakabayashi & Ikuta, 1989). In some neurones, degeneration may be related to excessive calcium entry (Weiss, Hartley, Koh & Choi, 1990; Kudo, Takeda & Yamazaki, 1990).Present experiments were undertaken to measure Ca2+ currents and associated change in the [Ca2+]i of neurones acutely isolated from the rat nucleus basalis using the whole-cell patch clamp method and the fura-2 fluorescence method.