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.

在海马和发育中的视觉皮层中，Ca^2+/钙调蛋白依赖性蛋白激酶 II (CaMKII) 对突触相关蛋白的磷酸化或蛋白磷酸酶 IIb（钙调神经磷酸酶）的去磷酸化已被认为在长期发挥作用。突触传递的增强（LTP）或长期抑制（LTD）。然而，目前还没有关于此类变化发生的地点、时间和持续时间的信息。本研究旨在开发通过皮层神经元磷酸化和去磷酸化实时成像来获取此类信息的方法。从年轻大鼠身上制备视觉皮层薄片。在视觉控制下，通过微量移液管将 CaMKII (LEAS2) 或钙调神经磷酸酶 (P-ARII) 的荧光指示剂加载到 111111 层的神经元上，以进行膜片钳记录。荧光强度的变化与第四层测试刺激引起的兴奋性突触后电位（EPSP）同时记录。已知可诱导LTP的0-突发型强直刺激导致一些神经元中LEAS2荧光强度的变化。然而，在大多数神经元中，注射 LEAS2 会导致神经元去极化，从而无法稳定测量 EPSP。相比之下，P-ARII 不会在大多数神经元中诱导这种去极化，并且在 LTD 诱导类型的 IV 层刺激（1 Hz，15 mm）期间显示出其荧光强度的变化。这种变化被 FK506（一种钙调磷酸酶选择性抑制剂）阻断，表明荧光强度反映了钙调磷酸酶的活性。在大多数情况下，它们的荧光变化发生在神经元的近端树突和体细胞中，潜伏期为数毫米，持续20-30分钟。这些结果表明突触后神经元中钙调神经磷酸酶的激活与视觉皮层的LTD有关。