Figure 2. Calcium imaging in paralyzed larvae confirms a larger recruitment of spinal motor neurons for fast escapes than for slow swimming.
(A) Calcium transients emitted from spinal motor neurons in Tg(mnx1:gal4;UAS:GCaMP6f,cryaa:mCherry) zebrafish larvae at 4 dpf were recorded simultaneously with ventral nerve root recordings (VNR) during fictive behaviors elicited by a water puff to the otic vesicle. (B) Expression pattern in a Tg(mnx1:gal4;UAS:GCaMP6f,cryaa:mCherry) larva at 4 dpf (R is rostral, V is ventral; scale bar is 50 µm). (C) VNR for each fictive behavior illustrates typical spontaneous fictive slow swims and induced fictive escapes: burst frequencies ranged between 20–30 Hz for slow swims (left panel) and 20–80 Hz for escapes (right panel). (D) GCaMP6f signals from individual motor neurons during spontaneous slow swimming (4 swims, left panel) and during an evoked escape response (right panel). For each recording, out-of-focus light was estimated within the spinal cord (black trace) and used as a criterion to determine the active vs inactive status of each cell. Pie charts represent the proportion of active cells in each behavior: 16/69 cells across 27 swims versus 61/69 cells across 12 escapes (n = 3 larvae). (E) Dorso-ventral (D–V) position of cells recruited during each maneuver shows dorsal motor neurons only active during escapes (the dorso-ventral axis within the spinal cord is normalized to 0 at the ventral limit and 1 the dorsal limit; mean D-V position for escapes = 0.41 + /- 0.02 versus 0.25 ± 0.01, p<0.001, n = 78 cells in n = 3 larvae). (F) Mean ΔF/F amplitude was higher during escapes compared to spontaneous swims across larvae (91.2 ± 4.7% versus 25.9 ± 3.8%, p<0.001, 12 escapes and 27 swims in n = 3 larvae).