An approach toward real-time imaging of protein phosphorylation in visual cortical neurons

视觉皮层神经元蛋白质磷酸化实时成像的方法

基本信息

批准号：
07558111
负责人：
TSUMOTO Tadaharu
金额：
$ 11.07万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1997
项目状态：
已结题

项目摘要

In hippocampus and developing visual cortex, phosphorylation of synapse-related proteins by Ca^<2+>/calmodu1in-dependent protein kinase II (CaMKII) or dephosphorylation by protein phosphatase IIb (calcineurin) have been proposed to play a role in long-term potentiation (LTP) or long-term depression (LTD) of synaptic transmission. However, there has been no information about where, when and how long such changes take place. The present study was designed to develop methods to obtain such information by real-time imaging of phosphorylation and dephosphorylation in cortical neurons.Thin slices of visual cortex were prepared from young rats. Under visual control, neurons in layer 111111 were loaded with a fluorescent indicator for CaMKII (LEAS2) or calcineurin (P-ARII) through micropipettes for patch-clamp recording. Changes in fluorescence intensity were recorded simultaneously with excitatory postsynaptic potentials (EPSPs) evoked by test stimulation of layer IV.Tetanic stimulation of the 0-burst type which is known to induce LTP led to a change in fluorescence intensity of LEAS2 in some neurons. In most neurons, however, the injection of LEAS2 led to depolarization of neurons so that stable measurements of EPSPs were not possible. In contrast, P-ARII did not induce such a depolarization in most neurons, and showed a change in its fluorescence intensity during the LTD-inducing type of layer IV stimulation (1 Hz for 15 mm). This change was blocked by FK506, an inhibitor selective for calcineurin, indicating that the fluorescence intensity reflects activity of calcineurin. In most cases, they change in fluorescence took place in proximal dendrites and somas of neurons with latency of several mm and lasted for 20-30 min. These results indicate that an activation of calcineurin in postsynaptic neurons is involved in LTD in visual cortex.

在海马和发展的视觉皮层中，已提出，已提出，已提出，已提出，已提出，已提出，已提出，通过蛋白质磷酸酶IIB（钙调蛋白）来对突触相关蛋白的磷酸化或cA^<2+>/calmodu1in依赖性蛋白激酶II（CAMKII）或去磷酸化的磷酸化，或者已提出在长期降低（LTP）中发挥作用。但是，没有关于此类变化发生时间，何时和多长时间的信息。本研究旨在开发通过在皮质神经元中的磷酸化和去磷酸化的实时成像来获取此类信息的方法。从幼鼠制备了视觉皮层的细切片。在视觉控制下，将111111层中的神经元加载到CAMKII（LEAS2）或钙调神经酶（P-ARII）的荧光指示器，通过微夹板进行贴片钳记录。通过测试刺激IV层IV刺激引起的荧光强度的变化同时记录了兴奋性的突触后电位（EPSP）。对0-肉体类型的二烷基刺激，已知LTP诱导LTP导致某些神经元的Leas2荧光强度的变化。然而，在大多数神经元中，LEAS2的注射导致神经元去极化，因此无法对EPSP进行稳定测量。相比之下，P-ARII在大多数神经元中没有诱导这种去极化，并且在LTD诱导类型的IV层刺激（1 Hz 1 HES 15 mm）期间显示出其荧光强度的变化。这种变化被FK506（一种抑制剂选择性的钙调神经酶选择性）阻止，表明荧光强度反映了钙调蛋白的活性。在大多数情况下，它们的荧光变化发生在近端树突和延迟的神经元中，持续了20-30分钟。这些结果表明，钙调神经素在突触后神经元中的激活参与了视觉皮层中的LTD。