Figure 6.
The DiO/DPA FRET pair reports simple and complex cerebellar Purkinje neuron activity in brain slices. A, B, DIC (A) and confocal fluorescence (B) images of a Purkinje neuron located in a brain slice preincubated and superfused with 1 μm DPA and 1 min after cell-attached labeling with DiO-C16. C, Single sweep AP-associated DiO/DPA responses (left) and their average (n = 19; bottom right) obtained from the Purkinje neuron displayed in A and B. Single APs were elicited by whole-cell current injection (2500 pA, 2 ms). Red traces are the current-clamp voltage recordings. Confocal image shows decreased membrane staining 24 min after DiO labeling, and yellow cross indicates the location of optical recordings. Right middle, Two plots of the amplitudes and widths, as a function of time, of the individual electrical (gray) and optical (black) AP recordings (scale of bottom plot is in milliseconds). D, DiO/DPA optical responses to single APs elicited by whole-cell current injection in the presence of 2 μm DPA. Bottom right, An average of 19 responses superimposed on the average electrical recording (red). E, Single and averaged (n = 10) optical responses to complex spike-like bursts elicited by larger current injections (3500 pA, 10 ms) from a different cell. All optical traces shown were filtered offline at 3.5 kHz.