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. 2017 Nov 22;6:e29797. doi: 10.7554/eLife.29797

Figure 5. Rk-expressing neurons act in central pattern generation.

(A–C) Analysis of ETH1-induced Ca++ activity in VNC-Rk neurons. (A) Images from two complete cycles of alternating Ca++ signal in VNC-Rk neurons during Phase 2. Images correspond to the indicated times of peak signal (brackets) on each side of the midline. Scale bar: 100 μm. (B) Phase 2 Ca++ signals from the preparation shown in (A) measured on the right (magenta) and left (green) sides of the midline (as shown in Figure 3B) oscillate in anti-phase. (C) Ca++ signals corresponding to Phase 1 (top) measured on either side of the midline are poorly correlated, whereas those corresponding to Phase 3 (bottom) are in phase. (For more detailed analysis of Ca++ signals from selected small ROIs within the VNC-Rk neurons see Figure 5—figure supplement 1.). (D–F) Analysis of Ca++ activity in abdominal muscles during the pupal ecdysis sequence. (D) Images from two complete cycles of alternating Ca++ signal in the abdominal musculature during Phase 2. Times and brackets indicate peak signal on each side of the midline, as in (B). Scale bar: 500 μm. (E) Phase 2 Ca++ signals measured on the right (magenta) and left (green) sides of the midline. (F) Ca++ signals corresponding to Phase 1 (top) measured on either side of the midline are poorly correlated, whereas those corresponding to Phase 3 (bottom) are in phase. (G) Timecourse of Ca++ activity in VGlut-expressing (motor) neurons of the abdominal ganglia in preparations in which the Rk-expressing neurons were activated (black trace) by ATP (arrow), or not activated because the P2X2 channel was not expressed (gray trace). Traces shown are representative of n = 6 experimental and n = 6 control preparations.

Figure 5.

Figure 5—figure supplement 1. VNC-Rk responses to ETH1 differ.

Figure 5—figure supplement 1.

(A) A Snapshot of ETH1-induced GCaMP6s activity (green) in VNC-Rk neurons. Circles indicate the 95 regions of interest (ROIs) selected for analysis of their response to ETH1. These regions were selected because their signals were spatially separated in the X-Y plane during the timecourse and conformed in size to individual cell bodies. Labeled ROIs in magenta are those whose Ca++ traces are shown in B. (B) Timecourse of ETH1-induced Ca++ activity of ROIs indicated in A. The timecourse of the global Ca++ signal (calculated for the entire set of VNC-Rk neurons, as shown in Figure 4A) is shown at the bottom. Dotted red lines indicate the time periods representing the three phases of Ca++ activity derived from the global signal. (C) Heatmaps showing the average oscillation frequencies for each phase for each of the 95 ROIs in A. Average frequencies were calculated for each trace using PhaseFinder to define peaks and phase durations determined from the global VNC-Rk signal (with Phase 3 subdivided into the Transition Phase and a ‘late’ Phase 3, as described in Figure 4C). The average Phase frequencies for the global VNC-Rk signal is shown at the top for reference and arrows indicate ROIs that showed a distribution of Phase frequencies that approximated that of the global signal, including ROI #74 from B.