Function of voltage-dependent Ca^<2+> channels in central nervous system neurons.

本课题的目的是利用Ca^<2+>成像技术，研究中枢神经系统神经元树突中功能电压门控Ca^<2+>通道的空间分布，以了解Ca^<2+>通道的作用。几种不同类型的电压门控Ca^<2+>已被证明分布在中枢神经系统神经元中，并根据其药理和动力学特性进行了分类。这些通道包括T、N、L和p型Ca^<2+>通道。利用离体或培养神经元的研究报道，小脑浦肯野神经元富含p型Ca^<2+>通道，低阈值Ca^<2+>通道也分布于浦肯野神经元中。我们制作了大鼠的小脑切片，并使用全细胞吸液器用荧光Ca^<2+>指示染料对单个浦肯野神经元进行染色。在TTX、Na^+通道b…More locker存在的情况下，通过胞体去极化刺激浦肯野神经元树突，并用冷却CCD相机测量胞内Ca^<2+>水平的变化。添加p型通道阻滞剂消除了Ca^<2+>依赖的作用峰和伴随的瞬态Ca^<2+>增加，使树突中的Ca^<2+>显著上升。Ca^<2+>成像研究表明，Ca^<2+>升高是由于低阈值Ca^<2+>通道激活所致，在电压门控K^+通道被药理学阻断的情况下，Ca^<2+>升高被低阈值Ca^<2+>通道阻滞剂Ni^<2+>抑制。基于这些发现，我们认为低阈值Ca^<2+>通道和低阈值K^+通道沿小脑浦肯野神经元树突分布。一项利用室室模型计算重建浦肯野神经元的模拟研究表明，低阈值Ca^<2+>和K^+通道是Ca^<2+>依赖平台电位的原因。需要进一步的研究来了解树突中的这些低阈值离子通道如何促进中枢神经系统神经元突触输入的整合机制。少

The objective of this project was to study spatial distribution of functional voltage-gated Ca^<2+> channels in the dendrites of central nervous system neurons in slice preparations using a Ca^<2+> imaging technique in order to understand the roles of Ca^<2+> channels. Several different types of voltage-gated Ca^<2+> has been shown to be distributed in central nervous system neurons and classified according to their pharmacological and kinetic properties. Those include T, N, L, and P-type of Ca^<2+> channels.It has been reported from the studies using isolated or cultured neurons that cerebellar Purkinje neurons are rich in P-type Ca^<2+> channels, and that low-threshold Ca^<2+> channels are also distributed in Purkinje neurons. We made cerebellar slices from rats and stained individual Purkinje neurons with fluorescent Ca^<2+> indicator dye using whole-cell pipetts. The dendrites of Purkinje neurons were stimulated by depolarizing the cell body in the presence of TTX, a Na^+ channel b … More locker, and the change in intracellular Ca^<2+> level was measured with a cooled CCD camera. Addition of a blocker for P-type channel abolished Ca^<2+> dependent action spikes and accompanying transient Ca^<2+> increase leaving a significant Ca^<2+> rise in the dendrites. Ca^<2+> imaging study showed that this Ca^<2+> rise was due to activation of low-threshold Ca^<2+> channels and that under a condition where voltage-gated K^+ channels were pharmacologically blocked, this Ca^<2+> rise was suppressed by Ni^<2+> , a blocker for low-threshold Ca^<2+> channels. Based on these findings we concluded that low-threshold Ca^<2+> channels and low-threshold K^+ channels are distributed along the dendrites of cerebellar Purkinje neurons. A simulation study to computationally reconstruct Purkinje neurons using a compartmental model suggested that those low-threshold Ca^<2+> and K^+ channels are responsible for the Ca^<2+>-dependent plateau potential.Further studies are needed to understand how these low-threshold ion channels in the dendrites contribute the mechanisms for integrating synaptic inputs in central nervous system neurons. Less