Locomotor behavior in TMZ-treated zebrafish larvae. (A) Distance traveled (mm) and (B) velocity (mm/s) of zebrafish larvae pre- and post-TMZ administration at increasing TMZ concentrations. (C) Representative movement track pre- and post-TMZ (200µM) administration. n range = 34–84 larvae/group; specifically indicated in each panel. Statistical analysis was performed using the paired Wilcoxon test- Asterisks denote significance; ****p ≤ 0.0001). Data are presented as Mean ± SEM.

Spinal cord Ca²⁺ imaging upon TMZ exposure. (A) Ca²⁺ imaging in spinal cord neurons of Tg(neurod1:GCaMP6f) larvae was performed 10 minutes (min) before 100 µM TMZ treatment for baseline recordings (PRE-TMZ). After TMZ administration and after a TMZ-diffusion time of 1 min, calcium imaging was resumed for other 10 min (POST-TMZ). (B) Representative z-projection images of the spinal cord Ca²⁺ imaging performed in the lateral caudal neurons of a single zebrafish larva before the treatment (PRE-TMZ) and after the treatment (POST-TMZ), the Region of Interest (ROI) is highlighted with the white dotted line). On the right side, the look up table color range of Ca²⁺ fluorescence is shown. (C) A representative graph from a single zebrafish larva shows the ∆F/F0 calcium fluorescent signal in spinal cord for each frame before and after TMZ treatment (D) The graph shows the full distribution of data with the median and quartiles of ∆F/F0 fluorescent signal in spinal neurons. (E) The graph shows full distribution of data, with median and quartiles of frequency fluorescent signal in spinal neurons n = 10 analyzed larvae. Statistical analysis was performed using the paired Wilcoxon test. Asterisks denote significance; *p ≤ 0.05, **p ≤ 0.01 (p of ∆F/F0 = 0.003; p of frequency = 0.01).

Whole brain Ca²⁺ imaging upon TMZ exposure. (A) Representative z-projection images of the whole brain Ca²⁺ imaging performed in the rostral-dorsally placed Tg(neurod1:GCaMP6f) larvae before the treatment (PRE-TMZ) and after the treatment (POST-TMZ). On the right side, the look up table color range of Ca²⁺ fluorescence is shown. Images were taken with 4X magnification objective. The dotted line indicates the analyzed ROI. (B) A representative graph from a single zebrafish larva shows the ∆F/F0 Ca²⁺ fluorescent signal in the whole brain for each frame before and after TMZ treatment (C) The graph shows full distribution of data, with median and quartiles of ∆F/F0 fluorescent signal in whole brain. (D) The graph shows full distribution of data, with median and quartiles of frequency fluorescent signal in whole brain. n = 10 analyzed larvae. Since the same individuals were analyzed before and after the treatment, statistical analysis was performed using the paired Wilcoxon test. ns: not significant, (p of ∆F/F0 = 0.19; p of frequency = 0.06;).

Hindbrain Ca²⁺ imaging upon TMZ exposure. (A) Representative z-projection images of the hindbrain calcium imaging performed in Tg(neurod1:GCaMP6f) larvae rostral-dorsally settled before the treatment (PRE-TMZ) and after the treatment (POST-TMZ). On the right side, the look up table color range of Ca²⁺ fluorescence is shown. The dotted line indicates the analyzed ROI, while the white arrows indicate the Mauthner cells localization. (B) A representative graph from a single zebrafish larva shows the ∆F/F0 Ca²⁺ fluorescent signal in hindbrain for each frame before and after TMZ treatment. (C) The graph shows full distribution of data, with median and quartiles of ∆F/F0 fluorescent signal in in the hindbrain. (D) The graph shows full distribution of data, with median and quartiles of frequency fluorescent signal in in the hindbrain n = 10 analyzed larvae. Statistical analysis was performed using the paired Wilcoxon test. *p ≤ 0.05, ns: not significant (p of ∆F/F0 = 0.13; p of frequency = 0.03).