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Fig. 3
CSF-cNs respond to active muscle contraction as well as to passive mechanical bending of the spinal cord.
(a) Schematic describing 2-photon imaging experiments used to record simultaneously from CSF-cNs expressing tagRFP (magenta) and GCaMP5 (green) in head-embedded Tg(pkd2l1:GCaMP5, pkd2l1:tagRFP) larvae. Infrared illumination combined with high-speed video recording shows unidirectional tail deflections induced by a water jet to the otic vesicle. Sample traces for ΔF/F of tagRFP and GCaMP shown with tail deflection during escape (note: vertical scale is 10 times larger for GCaMP compared to tagRFP signals). Subtracting the tagRFP signal from the GCaMP signal removed motion artifacts; breaks in the trace arise from frames when cells escaped from the focal plane. Scale bar: 10 µm. (b) Quantification of calcium transient amplitude in response to muscle contraction (n=11 larvae) in dorsal CSF-cNs either ipsilateral (red, 31 cells) or contralateral (yellow, 19 cells) or ventral (purple, 44 cells). Only dorsal ipsilateral cells exhibited responses greater than baseline (P=9.51 × 10-4) and all other cell types responded significantly less than dorsal ipsilateral cells (dorsal contralateral: P=2.43 × 10-3, ventral: P=4.85 × 10-5). (c) Passive mechanical stimulation of CSF-cNs in paralyzed larvae (n=5) was implemented with mechanical pressure exerted by pushing a glass probe laterally against the fish tail. Scale bar: 50 µm. (d) Response of proximal (<100 µm) and distal (>100 µm) CSF-cNs. Inset: average calcium response of proximal (red) versus distal (blue) CSF-cNs. (e) Response of dorsal ipsilateral (red) CSF-cNs as function of distance from the probe. (f) Response of dorsal ispsilateral (red, 28 cells), dorsal contralateral (yellow, 16 cells) and ventral (purple, 36 cells) CSF-cNs relative to the location of mechanical stimulation. Inset: Average calcium response of dorsal ispsilateral versus dorsal contralateral and ventral CSF-cNs. All cell types show a response different from 0 (dorsal ipsilateral: P<1.0 × 10-8, dorsal contralateral: P=5.95 × 10-5, ventral: P<1.0 × 10-8) and all other cell types responded significantly less than dorsal ipsilateral cells (dorsal contralateral: P=1.06 × 10-3, ventral: P=7.22 × 10-3).