Figure 3: Proximal thinning defines dendrites with higher excitability.

(a) A schematic of the photostimulation experiment. In C4da neurons expressing channelrhodopsin-2 fused with GFP (ChR2-EGFP), blue light pulses drive concomitant VGCCs-mediated Ca²⁺ responses in dendrites, which are detected by the Ca²⁺ indicator RGECO1, depending on the stimulation light intensity. (b) Live confocal images of an identical neuron at the white pupal stage (left, WP) and 3.5 h a.p.f. (right) labelled with channelrhodopsin-2 fused with GFP (ChR2-EGFP). Magenta arrowheads mark the proximal dendrites with thinning. Orange and grey squares correspond to the regions from which fluorescent traces of RGECO1 in c were obtained. Scale bar, 100 μm. (c) RGECO1 fluorescence changes in response to various intensities of blue light. Time traces obtained from the orange and grey square regions in b at 3.5 h a.p.f. are shown on the left and right, respectively. Traces at the threshold intensities of blue light for inducing dendritic Ca²⁺ responses are indicated in red. The threshold intensity was lower in the orange region (0.73 mW mm⁻²) compared with that in the grey region (1.2 mW mm⁻²), indicating that intrinsic excitability is elevated by a factor of 2 in the orange region (that is, dendrites distal from the thinning region). Note that no ‘spontaneous’ Ca²⁺ transients were generated during the photostimulation experiments, indicating that compartmentalized Ca²⁺ transients had not started yet. (d) Quantification of dendritic branch width at the proximal edge of dendrites with elevated excitability. Top, a live confocal image corresponding to a dashed rectangle in b. The proximal edge of dendrites with elevated excitability is shown with a green line. Quantification was done along dashed arrows. Values are relative to an unthinned dendritic region in the same neuron. Representative result from five experiments is shown in a–d. (e) Quantification of branch width at the proximal regions of dendrites with (orange) and without (grey) increased excitability. Ten single dendrites were analysed. *P<0.01; t-test. Error bars, s.d.