Figure 3. The light-induced decrease in the frequency of the rhythm and the phase changes are not due to changes in pH.

(A) Locomotor-like activity recorded from the right and left L2 and the left L5 ventral roots (black traces) in a P1 ChAT-eNpHR animal. The superimposed green traces are the slow potentials obtained by low pass filtering the raw signals. The activity was evoked by applying 6 μM NMDA and 8 μM 5-HT and the green bar indicates the duration of the light (60 s). (B) Bar plot showing the average locomotor-like frequency in wild type (n = 28, black circles), ChAT-Arch (n = 25, light green circles) and ChAT- eNpHR (n = 12, dark green circles) cords before the light (ANOVA, p=0.0533, two-stage linear step-up procedure, *: p=0.0141). (C–D) Time series of the change in absolute phase (C) and frequency (D) averaged for all experiments for the bilateral flexor (C1–D1) and ipsilateral flexor-extensor roots (C2–D2). (E) Averaged integrated ventral root discharge (Change in MN firing) for the ipsilateral flexor (E1) and extensor (E2) ventral roots. The statistics are obtained using a bootstrap t-test between ChAT- eNpHR (n = 12) and wild type cords (n = 28). The statistics are color-coded as indicated in the box below the records. The green rectangles indicate the duration of the light (60 s). (F–G–H) Bar plots showing the average change in the absolute phase (F), frequency of the bilateral flexors (G) for the 10 s just before and just after the light is turned on (Start Light, circles) and the 10 s just before and just after the light is turned off (After Light, squares) for wild type (black), ChAT-Arch (light green) and ChAT- eNpHR (dark green) animals. (H) Bar plots showing the average change of the ipsilateral flexor root for the 10 s just before and just after the light is turned on (Start Light, circles). Using a two-way ANOVA, we calculated the statistical differences between the three groups of animals (genetic Identity, shown above the bars) and the differences between light on and light off (light status; shown below the bars). The results of the ANOVA for the frequency changes were (Light status: F(3, 248) p<0.0001, Genetic identity F(2,248) p=0.0276, Interaction F(6,248): p<0.0001), for the absolute phase changes were (Light status: F(3, 248) p<0.0001, Genetic identity F(2,248) p<0.0001, Interaction F(6,248): p=0.8341), and for the changes in motoneuron firing were (Light status: F(3, 248) p<0.0001, Genetic identity F(2,248) p<0.0001, Interaction F(6,248): p<0.0001*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

DOI: http://dx.doi.org/10.7554/eLife.26622.006

Figure 3—figure supplement 1. Effect of dopamine of the light-induced effects during drug-induced locomotor-like activity in ChAT-Arch animals.

(A) Ventral root recordings (Right L2 and Left L1 and L5) showing the effect of hyperpolarizing ChAT-positive neurons during the locomotor-like rhythm evoked by 5 μM NMDA, 10 μM 5-HT and 50 μM DA. The superimposed green traces are the slow potentials obtained by low pass filtering the raw signal. The green bar indicates the timing of the light. Before the light, the left L5 slow potentials are alternating with those in the left L1 ventral root. Once the light is turned on, they are reduced in amplitude but are now in phase (*) with the left L1 ventral root. (B–C) Time series of the changes in phase (Abs φ) and frequency for the bilateral flexors (B1–C1) and the ipsilateral flexor/extensors (B2–B3). The color-coded regions on the traces (key beneath the records) indicate the statistically different portions of the record when compared to wild-type animals using a bootstrap t-test (ChAT-Arch: n = 14, WT: n = 8). Note that, except for the phase changes in the ipsilateral flexor/extensor roots, most of the changes are not significant. Note also that the frequency increase during the light contrasts with the light-induced decrease in the frequency of the locomotor-like rhythm when dopamine is omitted from the drug cocktail, consistent with the earlier finding that dopamine blocks ventral root activation of the locomotor rhythm (Humphreys and Whelan (2012). (D1-D2) Changes in the integrated neurogram (Change in MN Firing %) during the light. (E) Ventral root recordings (Left L1 and Left L5) together with an intracellular recording from an extensor motoneuron (V_m = −42 mV) firing in phase with the left L5 ventral root during the locomotor-like rhythm evoked by 5 μM NMDA, 10 μM 5-HT and 50 μM DA. The superimposed green traces show the integrated neurogram. The green bar indicates the timing of the light. In both the L5 ventral root and the intracellular recording, the integrated neurogram and the locomotor drive potentials - which are alternating with the Left L1 ventral root activity - change their phasing and become synchronous with the L1 ventral root activity during the light. However, when the intracellular membrane potential is restored to the pre-light level by current injection (300 pA) as indicated by the current trace beneath the green bar, the intracellularly recorded drive potentials return to their pre-light out of phase activity whereas the Left L5 root recording does not. The grey boxes highlight the phasing between the roots and the intracellular recording. This suggests that during the light the membrane potential falls below the chloride equilibrium potential thereby rendering the inhibitory locomotor drive potentials depolarizing.

DOI: http://dx.doi.org/10.7554/eLife.26622.011