Figure 5. Electrical stimulation of the nMLF elicits swim-like behavior and neural responses modulated by the stimulation intensity.

(A) Behavioral responses during midbrain electrical stimulation. (A, left) Image of a larva responding to midbrain electrical stimulation. Electrode position can be seen as well as border of agarose removed to free the tail. (A, right) Recorded locomotor responses elicited by nMLF stimulation. (B) Calcium activity in the nMLF during electrical stimulation. (B, left). Reference image of nMLF neurons, labeled with Texas Red Dextran, viewed with both bright-field and epifluorescence illumination. Stimulation pipette is outlined and the left MeLc neuron (calcium response shown in panel E) circled. (B, right) Raw calcium trace from nMLF left MeLc cell during midbrain electrical stimulation and accompanying stimulus of five trains. (C) Average bout durations from swim-like behavioral responses to midbrain electrical stimulation (0.45 μA) over five different stimulation frequencies (2, 4, 6, 8, 10 pulses per second). n= 13 larvae and 4,373 bouts. Error bars indicate SEM. A three-way ANOVA was used to determine statistical significance: frequency of stimulation was significant (p<0.0001). Across larvae, behavior was always significantly different from each other (p<0.0001). (D) The average maximum TBFs calculated per bout and averaged across larvae during bouts elicited by electrical stimulation (n= 13 larvae and 4,373 bouts). Frequency of stimulation significantly modulated maximum TBF (p<0.0003). Stimulation parameters and statistical methods identical to those in C. (E) Calcium responses to midbrain electrical stimulation from identifiable nMLF cells for 3 different currents over 5 different stimulation frequencies. Maximum Δf/f response for the average of all cells across all larvae (n=16 larvae, 101 cells). Error bars indicate SEM.