Fig. 5.

Activation of synaptic and total pools of NMDARs resulted in differential hippocalcin-YFP signalling in spines. Spatial patterns of hippocalcin-YFP translocation induced by activation of synaptic and total (synaptic and extrasynaptic) pools of NMDARs. The synaptic pool was activated during spontaneous network bursts whereas the total pool was stimulated by iontophoretic glutamate application to a neuronal dendritic branch. (A) A fluorescent image (a) was taken using the YFP filter set. The position of the iontophoretic pipette is indicated by dashed lines. (Ab) A higher magnification image of a dendritic branch shown in the boxed area in Aa. Ac and Ad demonstrate translocation images taken at the times indicated by respective letters in italic in B. These differential pseudocolour images were taken after an onset of network burst (c) and of iontophoretic glutamate application (0.5 s, 100 nA) (d). An outline of the dendritic tree is shown in each pseudocolour image for better visualization of translocation sites. A green colour represents a decrease and red represents an increase in hippocalcin-YFP fluorescence. Colour arrows in Ab indicate sites where ROIs were placed. Time courses of fluorescence changes in these ROIs are shown in B. Colours of traces match arrow colours in Ab. An onset of iontophoretic glutamate application is shown by a black arrow in B. The postsynaptic current (black trace) was recorded in voltage clamp mode at −70 mV to abolish Ca²⁺influx via VOCC and leave NMDARs as the only source of Ca²⁺influx. (C) Pooled results demonstrating that hippocalcin-YFP translocates to dendritic spines during synaptic rather than both synaptic and extrasynaptic NMDAR activation. Experiments were conducted with CNQX, gabazine and glycine and without Mg²⁺in an extracellular solution in order to isolate NMDAR-dependent currents and to relieve them from Mg²⁺block.