Figure 3
Nitric oxide mediates activity perturbation during the critical period. (A) Schematic representation of the experimental procedure. Early exposure to NOS drugs were achieved by feeding gravid females. We manipulated neuronal activity in embryos pan-neuronally expressing ChR by exposure to light between 17 and 19 h AEL (blue bar) which spans the identified critical period5. The resulting L3 were tested by either electrophysiological recording from aCC motoneurons or by electroshock. (B) Voltage-clamp recordings from L3 aCC show an increase in SRC duration following pan-neuronal activation of ChR during the critical period (elavC115 > ChR: + LEDλ470nm 17–19 h AEL, blue trace, vs. -LED, black trace). Inhibiting NOS (0.1 M L-NAME, see Figure S2A), prior to optogenetic manipulation, blocks this effect, while exposure to the NO donor (1.5 mM SNP, see Figure S2B) potentiates. (C) Quantitative analysis of SRC duration, n = 10 in each group. A two-way ANOVA shows significance for the + LED treatment (F(1, 54) = 52.63, p < 0.001), NOS manipulation (F(2, 54) = 13.16, p < 0.001), and interaction (F(2, 54) = 16.4, p < 0.001). (D) RT to electroshock measured at L3 following the same stimulation protocol. Exposure to L-NAME blocked the ChR-induced increase in RT, while the NO donor SNP potentiated this effect, n = 30 in each group. A two-way ANOVA shows significance for the + LED treatment (F(1, 174) = 86.43, p < 0.001), NOS manipulation (F(2, 174) = 23.54, p < 0.001), and interaction (F(2, 174) = 15.1, p < 0.001). **p < 0.01 and ***p < 0.001 are significant to + LED vs. -LED within each group. +p < 0.05, ++p < 0.01 and +++p < 0.001 show significance between NOS drugs and CTRL (+ LED groups vs. CTRL + LED group), Bonferroni’s post-hoc test. (E) RT from electroshock of L3 pan-neuronally co-expressing various transgenes together with ChR. Embryos were exposed to light during the critical period (+ LED group: λ470 nm, 100 ms, 17–19 h AEL). NOS inhibition (NOSRNAi) reduced the expected ChR-induced increase in RT. By contrast, up-regulation of NOS-signalling (NOS and macNOS) potentiated the effect of ChR activation compared to control (elavC155 > ChR). Co-expression of GFPRNAi, an additional control, showed no effect. One-way ANOVA (F(4, 145) = 25.38, p < 0.001) followed by Bonferroni’s post-hoc test, n = 30 in each group, *p = 0.0312 and 0.0324, respectively, ***p < 0.001.
