Figure 2.

A Critical Period to Influence Seizure

(A and B) RTs measured from L3 that pan-neuronally expressed either ChR (λ470 nm, 100 ms/1 Hz) or eNpHR (λ565 nm, 600 ms/1 Hz) and exposed to light during 11–19 hr AEL of embryogenesis. In both manipulations, disturbing neural activity (+LED) is sufficient to increase the RT of L3 in response to electroshock. Controls were not exposed to light (−LED). The presence of AEDs, Phy (0.4 mg/ml) and Gbp (0.1 mg/ml), during embryogenesis prevents the induction of a seizure phenotype. In order to exclude an unspecific effect of the LED stimulation, embryos were optically manipulated in absence of all-trans-retinal (−R+LED, black dotted lines). The RT (mean ± SEM) for homozygous bss is shown for reference (red dotted lines).

(C) Seizure threshold is lower in L3 derived from manipulated embryos. Seizure response to varying voltages shows a lower threshold for bss [8, 19]. Similarly, larvae in which activity was manipulated during embryogenesis (+LED) require a lower voltage to exhibit a significant increase in RT compared to controls (−LED and WT, n = 20 in each group).

(D) The effect of ChR activation is independent of developmental time. To extend duration of larval stage, larvae were maintained at either 25°C (4 days) or 18°C (9 days) until L3. Two-way ANOVA shows a significant effect of LED treatment (F_(1,116) = 249.39, p < 0.001), but no effect for developmental time (F_(1,116) = 0.18, p = 0.67).

(E) Temporally controlled experiments indicate that manipulation of neuronal activity (ChR, λ470 nm, 100 ms/1 Hz) between 17 and 19 hr AEL is sufficient to induce maximal seizure duration at L3.

Data (A–E) are represented as mean ± SEM. ^∗p < 0.05 and ^∗∗∗p < 0.001, Bonferroni’s post hoc test. The number of tested larvae is shown above the x axis. Red and black dotted lines represent reference RTs (mean ± SEM) obtained from homozygous bss and WT, respectively.