(A) Left: Scheme showing the strategy for selecting frames for fast time-lapse acquisition of calcium indicator dye-filled NG2 cells stimulated in current clamp mode (inset on top shows waveform of injected current, as used for Figure 3). Frame-scans include very proximal and terminal dendrites of NG2 cells. Right: Sequence of image acquisition (100 @ 30 Hz). Curly brackets denote frames being averaged for the analysis shown in panels (B–F). (B) Average frame-scan of Fluo-4 fluorescence before (left) and after (middle) mock PSPs. Note the increase in brightness along the entire dendrite. This is better seen in the right image displaying the normalized difference in fluorescence (∆F/F). (C) Corresponding frame-scans of the tracer tetramethylrhodamine-biocytin (TMR). ROIs used for analysis are drawn consecutively with 5 µm increments along the dendrite. ROIs (numbered sequentially) with similar distances to the soma edge are binned for quantification in (E). (D) Calcium signals rise rapidly and with undiminished amplitude at proximal and distal locations. Traces show the time course of the mean fluorescence of ROIs at three different distances expressed as a volume-normalized signal (Fluo-4/TMR) (n=13, 34 and 5 ROIs, respectively). Asterisks indicate time of current injection. Scaling: 0.02 Fluo-4/TMR, 500 ms. (E) Average Ca2+ transient amplitudes slightly increase with the distance from the soma across the dendritic tree (linear correlation test, n=141 ROIs from 12 time-lapse series of seven NG2 cells, R2=0.08; dashed grey line). Peak amplitude values of ∆Fluo-4/TMR were binned according to distance from soma (n=13, 22, 34, 28, 25, 13 and 5 ROIs, respectively). (F) Minimal delay of calcium signals even distally from the soma. Onset of Ca2+ signals was determined for each individual ROI using a template-fitting algorithm (see Materials and methods). If the calcium signal were to propagate by pure diffusion from the somatic site of current injection, substantial delays for remote positions would be expected as denoted by the grey line (see Materials and methods). In order to obtain a precise estimate of the onset of the signal, a detection threshold was chosen (3.0, see Materials and methods) to select signals very clearly standing out of the recording noise (44 ROIs from nine time-lapse series of seven cells). (G) Primary dendrite of a CA1 pyramidal neuron used for a comparative analysis of the dendritic occurrence of Ca2+ signals. ROIs were defined as in (C). Scaling: 5 µm. (H) Ca2+ signals generated in dendrites of CA1 pyramidal neurons, which are known to express dendritic VGCCs, display fast rise times and minimal delays similar to those observed for NG2 cells. Note, however, that neuronal Ca2+ signals decay more rapidly. Neurons were bathed in TTX to prevent action potentials and to make the experiment more comparable to NG2 cells (showing no action potentials). As for NG2 cells, neurons were stimulated with 1 10 pulse 100 Hz train of mock PSPs (n=3). Asterisks indicate time of current injection. Scaling: 1.0 ∆(Fluo-4/TMR)/(Fluo-4/TMR), 500 ms.