Figure 4.

(A) Schematic of TBI using CaMPARI (Calcium Modulated Photoactivatable Ratiometric Integrator) to optogenetically quantify neuronal excitability. Three dpf CaMPARI larvae were freely swimming while subjected to TBI, coincident with exposure to 405 nm photoconversion light. CaMPARI fluorescence permanently photoconverts from green to red emission only if the photoconversion light is applied while neurons are active (high intracellular [Ca²⁺]). The ratio of red:green emission is stable such that it is quantifiable via subsequent microscopy. (B) Increased neural activity during TBI is represented by increased red:green emission (red pseudocolored to magenta) in the hindbrain of larvae (B”), compared to larvae not receiving TBI (B’) or fish not exposed to photoconverting light (‘no PC’ in panel B). These representative maximum intensity projection images show dorsal view of zebrafish brain (anterior at top), including merged, or red or green channels alone. (C) Heatmaps encode the CaMPARI signal (higher neural activity = higher red:green = hotter colors), highlighting location of increased neural activity during TBI relative to control larvae not receiving TBI. (D) Quantification of CaMPARI output in the hindbrain area reveals a significant increase in the neuronal excitability during TBI compared to control group not receiving TBI (**p=0.0087, Mann-Whitney test. Each data point is an individual larva). Scale bars = 100 μm.