Fig. 4.

Strong activation of synaptic NMDARs induced hippocalcin-YFP translocation to dendritic spines. (A) An overlay of morphological (white) and hippocalcin-YFP translocation (red) images of neuron during a spontaneous burst of synaptic NMDAR-dependent currents at the time indicated as d in Ba. All synapses that were active during the burst appear in red. Panels b–e demonstrate overlays of morphological (white) and translocation images taken at the times indicated by respective letters in italic in Ba. Colour arrows indicate spines for which time courses of hippocalcin-YFP translocation are demonstrated in Ba. NMDAR-dependent currents were simultaneously recorded in whole-cell voltage clamp mode (holding potential −70 mV) and shown in Ba (black trace). (Bb) Values of hippocalcin-YFP translocation to spines compared with those in a dendritic tree at 1 μm from the respective spines. (C) Hippocalcin-YFP translocation to spines in response to membrane depolarization and to NMDAR-dependent synaptic current. An example of hippocalcin-YFP fluorescence changes and simultaneously recorded transmembrane current is shown in Ca. In this example, a spontaneous NMDAR-dependent postsynaptic current developed due to a network burst immediately after the train of depolarizing pulses (seven pulses from −70 to 0 mV; 50 ms at 7 Hz). It is clear that the current via synaptic NMDARs rather than the train induced hippocalcin-YFP translocation. In all neurons tested with this particular protocol no translocation was observed as a result of the trains. (Cb) Pooled results showing that a vigorous bpAP train (100 APs at 20 Hz) did not lead to hippocalcin-YFP translocation to sites where bursting-induced translocation was observed. At the same time, the trains did induce hippocalcin-YFP translocation to neighbouring sites in the dendritic shaft. ROIs were only placed over sites where bursting-induced hippocalcin-YFP translocation was observed. Experiments were conducted with CNQX, gabazine and glycine and without Mg²⁺.