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

在海马区和发育中的视皮层，钙/钙调蛋白依赖性蛋白激酶II(CaMKII)或蛋白磷酸酶IIb(Calcineurin)去磷酸化突触相关蛋白在突触传递的长时程增强(LTP)或长时程抑制(LTD)中起作用。然而，目前还没有关于这种变化发生的地点、时间和时间的信息。本研究旨在开发通过对皮层神经元的磷酸化和去磷酸化进行实时成像来获取此类信息的方法。在视觉控制下，111111层神经元通过微管加载CaMKII(Leas2)或CaCineurin(P-ARII)荧光指示剂，用于膜片钳记录。同时记录刺激IV层诱发的兴奋性突触后电位(EPSP)的荧光强度变化。强直刺激0-爆发型LTP可引起部分神经元LEAS2的荧光强度变化。然而，在大多数神经元中，注射LEAS2导致神经元去极化，因此不可能稳定地测量EPSPS。相反，P-ARII不能在大多数神经元上引起这种去极化，并且在LTD诱导的IV层刺激(1 Hz，15 mm)时，其荧光强度发生变化。这种变化可被钙调神经磷酸酶选择性抑制剂FK506阻断，表明荧光强度反映了钙调神经磷酸酶的活性。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胞体，潜伏期为几毫米，持续20-30分钟。这些结果表明，LTD与视皮层突触后神经元钙调神经磷酸酶的激活有关。

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.