Prior to CNO injection, both a- and b-wave amplitudes showed typical irradiance response functions (
Fig. 2A–C), which were statistically similar in hM3Dq
Opn4Cre/+ and control
Opn4Cre/+ animals (a-wave: 2-way ANOVA with post hoc Bonferroni correction;
P > 0.99;
F test:
P = 0.96; b-wave: 2-way ANOVA with post hoc Bonferroni correction;
P > 0.99;
F test:
P = 0.67). Next, while maintaining dark-adapted conditions, we injected mice with CNO (5 mg/kg) to excite ipRGCs. Following a period of 30 minutes for stabilization/further dark adaptation, we repeated the above protocol (
Fig. 2A). Following CNO injection, we found a marked difference between the ERGs recorded in hM3Dq
Opn4Cre/+ and control
Opn4Cre/+animals. Statistical analyses revealed a significant decrease in a-wave amplitudes in hM3Dq
Opn4Cre/+ mice compared to that in controls injected with CNO (
Fig 2D) (2-way ANOVA finds significant effect of irradiance (
P < 0.0001), treatment (
P < 0.0001), and interaction (
P < 0.05);
F test comparing sigmoidal dose response curves finds data are better fit with two separate functions;
P < 0.0001). These effects were found to be significantly divergent at irradiances >10
13 photons/cm
2/s (post hoc Bonferroni correction,
P < 0.01). Similarly, there was a significant reduction in b-wave amplitudes in hM3Dq
Opn4Cre/+ mice compared to controls following CNO injection (
Fig. 2E) (2-way ANOVA finds significant effect of irradiance (
P < 0.0001), treatment (
P < 0.0001), and interaction (
P < 0.05);
F test, comparing sigmoidal dose response curves:
P < 0.0001). Bonferroni post hoc analyses revealed these effects were significantly different at irradiances >10
10 photons/cm
2/s.