Fig. 3.
In vivo imaging of spontaneous premotor interneuron activity state transitions. Three-dimensional volumetric time-lapse images of the head region were acquired in the transgenic C. elegans strain QW1574 during early adulthood using dual inverted selective plane illumination microscopy. Displayed in (A) are maximum intensity projections28 of volumetric data sets at three consecutive time points, showcasing GCaMP6s expression in seven pairs of premotor interneurons that are centrally involved in locomotive and reversal behavior (AVA, AVB, AVD, AVE, AIB, RIM, and RIV).17,27 The neurons were identified and tracked via nuclear red fluorescent protein NLSwCherry expression, and their GCaMP signals were subsequently extracted (see Light-sheet Imaging). The anatomical right AVA is circled in the second and third panels of (A), with its GCaMP fluorescence intensity over a 10-min period displayed in (B). Using custom MATLAB scripts, which identified substantial shifts in fluorescence intensity (red and black vertical lines), individual calcium transient onsets and offsets were automatically extracted from these 14 interneurons and fitted to hyperbolic tangent functions (C; see Statistical Methods). For each tangent function, the scripts then calculated the rise or fall time. These average values are plotted in (D) for control animals and those that had been exposed to isoflurane during the first larval stage (for on transitions: isoflurane-exposed and control early adulthood n = 1,049 and 1,032 traces from 40 animals each, respectively; for off transitions: isoflurane-exposed and control early adulthood n = 441 and 471 traces from 40 animals each, respectively). Solid error bars denote 95% CI, and dashed error bars show the SD. *P < 0.05 unpaired two-tailed t test.