Figure 4. Simultaneous analysis of ASR behavior and dorsal raphe nucleus (DRN) neuron calcium signals in 6 dpf zebrafish larvae.

(A) Image shows varicosities projecting from the DRN towards the Mauthner cell dendrites (maximum projection of a Z stack of confocal images, variegated individual shown for ease of demonstration). (B) Representative image of a confocal slice of DRN serotonergic neurons expressing GCaMP5. (C) Representative trace shows ROI normalized fluorescence in a single DRN neuron. (D) Probability of sound-evoked calcium responses for individual DRN neurons. Graph shows probability of sound-induced responses for individual cells during sound stimuli that did not induce escapes versus stimuli followed by escapes. (E) Acoustic startle responses of individual larvae ordered along y-axis by probability of escape over the stimulus series. Each dot represents a sound-induced escape. (F) Significant decrease in ASR probability (F-statistic vs constant model: 23, P < 10⁻⁵, adjusted R-squared = 0.06). (G) Fractional activation of DRN neurons in individual larvae ordered in the y-axis by probability of escape during stimulus series. The size of each dot is proportional to the number of cells activated by acoustic stimuli as a fraction of total number of analyzed individual neurons/fish. (H) Decrease in number of sound-activated DRN neurons as fraction of total number of analyzed individual neurons/fish during habituation (F-statistic vs constant model: 29.2, P < 10⁻⁶, adjusted R-squared = 0.073). Statistics: linear regression model analysis. 8–14 neurons/fish, n_{DRN neurons} = 244, n_larvae = 24. Error bars, s.e.m.. Scale bar 25 µm.