Figure 1.

Nerve-evoked Ca²⁺ entry during a single action potential at the frog neuromuscular junction. A, Gray-scale (top) and pseudocolor representation (bottom) of a difference image generated by subtraction of the mean resting fluorescence from the fluorescence observed in a single trial in the absence of nerve stimulation. B, Gray-scale (top) and pseudocolor representation (bottom) of a difference image generated by subtraction of the mean resting fluorescence from the fluorescence observed immediately after nerve stimulation in a single trial. Images in A and B were collected using 1 msec illumination (in B, this began 1.5 msec after nerve stimulation). The white boxes in the bottom panels of B and C indicate the area of this terminal that is shown in D and used to generate the histogram shown in E. C, The top panel shows a sample single resting fluorescence image of the nerve terminal (same illumination conditions as in A; gray-scale bar in photoelectron counts). The bottom panel shows an image mask (red) indicating pixels in the resting fluorescence image that have intensities between 50 and 100% of maximum. This mask identifies those pixels that sample light from the nerve terminal portion of the image. D, Enlarged region of the stimulus-evoked difference image (from the white box in the bottom panel of B) representing the spatial distribution of Ca²⁺ entry (left). The right panel shows the same region of the neuromuscular junction labeled with rhodamine-α-BTX to show the location of postsynaptic acetylcholine receptors. E, Histogram showing the distribution of pixel intensities taken from those pixels in the masked nerve terminal portion (red pixels in the bottom panel of C) in the presence and absence of nerve stimulation. A pseudocolor difference image from this same region is shown in D, but the histogram does not include data from pixels outside the nerve terminal (masked region), because these off-nerve data would dominate the distribution. The distribution shown in black represents pixel intensities measured in 10 images recorded in the absence of nerve stimulation (defined as “resting fluctuations”), the distribution shown in red represents pixel intensities measured in 10 images recorded after nerve stimulation, and the dotted blue line represents the limit of two SDs above the resting fluctuation distribution after fit to a single Gaussian. The pseudocolor scale bar is the same for the bottom panels in A and B and the left panel in D, and is expressed as ΔF/F (%). Scale bars, 2 μm.