Delayed dye diffusion identifies MSN axons. A. A single MSN was loaded with OG1 and Alexa594 through the patch pipette and imaged by 2PLSM at 5 and 30 min after entering whole cell mode. Fluorescence intensity is shown in pseudocolor (ImageJ). Projection images (A1) were created from a z series of confocal Alexa594 images. At 5 min, the dendrites and proximal axon were filled. At 30 min (A2), dendritic labeling showed no significant change, while the axon was now clearly labeled. Dendritic processes were studded with spines, in contrast to the single axonal process, which was free of spines. B. The recorded MSN is shown in a projection image (B1) made from the full confocal z series of Alexa594 images (50 sections at 1 μm intervals). The area outlined in panel B1 (white rectangle) is shown enlarged in the inset (B2). This stretch of axon contained one hot spot, indicated by the arrowhead (B2). C. Presynaptic Ca2+ responses were evoked by trains of 5, 10 or 20 AP's. The raw Ca2+ responses showed the typical waveform of AP-induced Ca2+ responses, with a fast rising and slow decay phase. The decay followed a single exponential curve; the decay time constant τ (in msec) is indicated on each panel. Raw traces of evoked AP's are shown in the bottom panel (red trace). Note that the 20 depolarizing pulses evoked 20 AP's without failures. The last AP is shown magnified in the inset (right bottom), with the depolarizing pulse below; the end of the stimulus artifact (S) is seen as an inflection preceding the unclamped axon spike (AP).