Figure 1.
Gna11 and Gna14 are expressed in ipRGCs, often in combination.
A–E. Representative images of RT-PCR analysis of single ipRGCs for Opn4, Gnaq, Gna11, Gna14, and Gna15. All representative gels show RT-PCR analysis of single ipRGCs taken from P1 Opn4Cre/+; Z/EG mice. Each lane represents one cell, the positive control is whole retinal RNA, and the negative control is water. F–G Summary of expression of Gq/11 family members in the 16 ipRGCs obtained from P1 and P4 Opn4Cre/+; Z/EG mice. All cells expressed melanopsin. 15 cells expressed Gna11, 10 of which also expressed Gna14. H. Venn diagram showing the distribution of Gq/11 family member expression in all 32 ipRGCs sampled.
Figure 2.
Gq/11 mutant lines exhibit pupillary light reflex indistinguishable from WT.
A. Representative images of the pupil constriction in WT (16 animals), Opn4LacZ/LacZ (MKO, 7 animals), Gna11−/− (Gna11 KO, 4 animals), Gna14−/− (Gna14 KO, 5 animals), Gna15−/− (Gna15 KO, 7 animals), Gnaqflx/flx; Gna11−/−; Opn4Cre/+ (Gnaq; Gna11 DKO, 9 animals), and Gna11−/−; Gna14−/− (Gna11; Gna14 DKO, 7 animals) at both high (1.4×1016 photons/cm2/sec) and low (7.3×1013 photons/cm2/sec) light intensities. B–C. Quantification of the pupillary light reflex at low (7.3×1013 photons/cm2/sec) and high (1.4×1016 photons/cm2/sec) light intensities. All animals exhibited pupillary light reflex indistinguishable from WT. One-way ANOVA with Tukey post-hoc analysis. Error bars represent s.e.m.
Figure 3.
Gq/11 mutant lines exhibit circadian behaviors indistinguishable from WT.
A. Representative actograms of wheel running activity from WT (14 animals), MKO (9 animals), Gna15 KO (7 animals), Gnaq; Gna11 DKO (8 animals), and Gna11; Gna14 DKO (7 animals) mice under a 12∶12 LD cycle, constant darkness, and constant light. The white background indicates light, grey background indicates darkness, and the yellow asterisk indicates a 15-minute light pulse at circadian time (CT) 15. All mice photoentrained to the LD cycle. B. Quantification of free-running period under constant dark conditions. All animals exhibited circadian periods indistinguishable from WT. One-way ANOVA with Tukey post-hoc analysis. Error bars represent s.e.m. C. Quantification of phase shifting to a 15-minute light pulse given at CT 15. All animals exhibited phase shifting indistinguishable from WT. One-way ANOVA with Tukey post-hoc analysis. Error bars represent s.e.m. D. Quantification of free running period under constant light. As previously reported, MKO mice exhibited reduced lengthening of their circadian period under constant light conditions. Gnaq; Gna11 DKO exhibited a slight reduction in the lengthening of their circadian period in constant light, and their period length was significantly different from both WT and MKO. Gna15 KO and Gna11; Gna14 DKO exhibited lengthened periods that were indistinguishable from WT. One-way ANOVA with Tukey post-hoc analysis. Error bars represent s.e.m.
Figure 4.
ipRGC intrinsic phototransduction persists in Gna11; Gna14 DKO mice.
A. Representative voltage traces for ipRGC intrinsic light responses in WT and Gna11; Gna14 DKO retinas at two 480 nm light intensities (7×1012 and 7×1013 photons/cm2/sec). Horizontal bar represents light stimulation (60 sec). Vertical scale bar is 100 µV. B. Total number of spikes in ipRGCs light responses to two 480 nm light intensities (7×1012 and 7×1013 photons/cm2/sec) and white light (267 mW/cm2). ipRGC light responses in Gna11; Gna14 DKO were indistinguishable from WT. Student's t-test. Error bars represent s.e.m. C–D. Quantification of the number of spikes, in 1 second bins, during a 60 second pulse of either 7×1012 photons/cm2/sec or 7×1013 photons/cm2/sec 480 nm light. Photoresponses in Gna11; Gna14 DKO mice were indistinguishable from WT. Student's t-test. Error bars represent s.e.m.
Figure 5.
Gq/11 family members are upregulated in the retinas of some Gq/11 mutant lines.
A–D. Expression levels of Gnaq, Gna11, Gna14, and Gna15 in the retina relative to WT in Gna14 KO, Gna15 KO, Gnaq; Gna11 DKO, and Gna11; Gna14 DKO mice. Normalized to levels of 18S RNA. (N = 3 mice for each; 2 retinas per RNA sample). * indicates P<0.05 by one-way ANOVA with Tukey post-hoc analysis. Error bars represent s.e.m.