Fig. 4

Augmentation of descending excitatory glutamatergic synaptic inputs to primary motoneurons expressing mutTARDBP. (A) Example bright field of image of a 56 hpf transgenic Chx10:ChRWF-EGFP zebrafish larva (left, scale bar represents 1 mm). Bright field image showing a section of the zebrafish trunk showing 6 somites (middle, scale bar represents 50 μm) with corresponding EGFP fluorescence image depicting spinal V2a interneurons expressing Chx10:ChRWF-EGFP (right, scale bar represents 50 μm and in inset 25 μm). (B) Example still frames from high speed video recordings of optogenetic stimulation of locomotor tail-beat patterns in zebrafish larvae partially embedded in agarose. (C) Proportion of larvae exhibiting different patterns of optogenetic-evoked activity, with the majority of larvae expressing mutTARDBP displaying tail thrashing. Sample sizes for optogenetically-evoked patterns of activity for wild type, wtTARDBP and mutTARDBP larval groups were 17, 21, and 22 respectively. (D) Example whole-cell voltage clamp recordings of optogenetically-evoked synaptic currents in CaP motoneurons (above) and EPSC raster plots from individual larvae (below). Recorded EPSC were binned into three groups, representing: 5 seconds before optogenetic stimulation, 5 seconds during light exposure, and the following 5 seconds after stimulation. Both the mean (E) and maximum amplitudes (F) of EPSCs recorded in CaP motoneurons in larvae expressing mutTARDBP were found to be higher than EPSCs recorded in wild type CaP motoneurons. Furthermore, the mean frequency of optogenetically driven EPSCs was found to be higher in larvae expressing mutTARDBP when compared to wild type EPSCs in CaP motoneurons (G). When the total number of evoked EPSCs was examined over the 5 second stimulus, larvae expressing mutTARDBP were found to generate more EPSCs than both wild type and wtTARDBP expressing larvae (H). Asterisks and daggers represent statistical differences from wild type (p < 0.05) and wtTARDBP (p < 0.05) larvae respectively.