Figure 5. DN₁s release glutamate to inhibit light avoidance.

For all RNAi experiments UAS-dcr-2 was co-expressed to improve efficacy. The Gal4 control lines shown also express UAS-dcr-2. Statistical comparisons are as stated below. *p<0.05

(A–C) Larval light avoidance was measured as in Fig 2.

(A) Expression of a VGlut-RNAi transgene (GD2574) in all clock neurons (tim > VGlut^RNAi increased light avoidance at 150lux compared to control larvae. These data are significantly different (ANOVA p<0.005). Tukey's post-hoc comparison gives a significant difference only between tim > VGlut^RNAi and UAS-VGlut^RNAi / +. However, light avoidance in tim > VGlut^RNAi is higher than tim > + by t-test (p<0.05) and tim > VGlut^RNAi larvae also lose circadian rhythms in light avoidance (Fig S4A).

(B) Expression of Glutamate decarboxylase (UAS-Gad1) in DN₁s (DN₁ > Gad1) significantly increased light avoidance at 150lux compared to UAS-Gad1 / + control larvae (Gad1 / +, t-test p<0.05). See also Fig S4B.

(C) A GluCl-RNAi transgene expressed in LN_vs (Pdf > GluCl^RNAi) significantly increased light avoidance at 150lux compared to control larvae (ANOVA p<0.05). An mGluRA-RNAi transgene expressed in LN_vs (Pdf > mGluRA^RNAi) had no effect on light avoidance compared to controls (ANOVA). See also Fig S4C.

(D) Light avoidance was assayed in DD at 150lux as in Fig 4. Light avoidance is higher at CT24 than CT12 in control larvae (UAS-GluCl^RNAi / +, which also contain a UAS-dcr-2 transgene, t-test p<0.05). No rhythms in light avoidance were detectable when GluCl-RNAi was expressed in LN_vs (Pdf > GluCl^RNAi, ANOVA). Rhythmic light avoidance was still detectable in larvae expressing mGluRA-RNAi in LN_vs (Pdf > mGluRA^RNAi, ANOVA p<0.05). By 2 Way ANOVA comparison of CT12 and 24 time points, Pdf > GluCl^RNAi is different to control (F_1,22=9.17, p<0.01) whilst Pdf > mGluRA^RNAi is not (F_1,24=0.00, p=0.9547)

(E) Glutamate-mediated inhibition of ACh-stimulated Ca²⁺ transients in dissociated larval LN_vs. Representative relative fluorescence (F/Fo) recordings are shown from dissociated larval LN_vs expressing UAS-GCaMP1.6. Solution changes, including neurotransmitter applications, are indicated by black bars. Lowering extracellular Cl⁻ to 13.6mM completely relieved glutamate-dependent inhibition. Glutamate completely blocked ACh-stimulated transients when physiological Cl⁻ was restored.

(F) A 2min incubation of a larval LN_v with 500nM ivermectin irreversibly blocks subsequent ACh-induced Ca²⁺ transients.