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

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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.