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Fig. 2 Figure 2. th2+ Neuron Activity Is Highly Correlated with Spontaneous Behavior (A) Experimental setup. Behavior recording was accomplished using a ring of oblique 830-nm LEDs mounted to the fluorescence objective. Fish behavior was recorded with a high-speed camera below the stage at 500 Hz. (B) Examples of forward swim, turn, and struggle regressors (orange, blue, and brown) and significantly correlated calcium traces (green). (C) Calcium traces aligned to spontaneous behavior onset, separated by anatomical nucleus and behavior type. Gray traces show the behavior-triggered average for individual units (mean dF/F). Green traces are mean ± SEM across animals (n = 20 fish). Activity in Hc, Hi, and PT neurons was recorded using Tg(th2:gcamp5); activity in PON was recorded using transient expressors of th2:jgcamp7s. Example recording is shown in Video S2. (D) Percentage of units in each nucleus significantly correlated with swim (top), turn (middle), and struggle (bottom) regressors. Red bar indicates the mean; gray boxes, 95% CI; gray lines, SD; ∗p < 0.05, Bonferroni-corrected ANOVA (n = 20 fish). (E) Scatterplot of the maximum tail bend angle of individual bouts versus the peak calcium signal of th2+ neurons in each nucleus within 1 s of the behavior. Red line, fitted regression. From left to right: n = 3,833, 3,883, 3,152, and 1,797 behaviors; Pearson’s R = 0.38, 0.35, 0.12, and 0.05. (F) Average length of significant calcium transients from cells in each nucleus of Tg(th2:gcamp5) animals. Bonferroni-corrected ANOVA; n = 1,154 cells; p < 0.001. (G) Pearson’s R values between units within individual nuclei. Dots represent single neuron pairs. n = 200, 28, and 414 pairs. (H) Pearson’s R values between units in different nuclei. Dots represent single neuron pairs. n = 31, 263, and 89 pairs.