Related to Fig. 3. a-c. SCN-projecting ipRGCs are not sufficient to induce daytime phase delays. a. Schematic representations of ipRGC projections in Gq-Opn4Cre (top panel) and Gq-Opn4Cre;Brn3bDTA (bottom panel) mice. In Gq-Opn4Cre;Brn3bDTA mice, where the extra-SCN-projecting Brn3b-positive ipRGCs are ablated, the surviving M1Brn3b− ipRGCs predominantly project to the SCN with minor projections to several other brain regions, including the intergeniculate leaflet (IGL), ventral lateral geniculate nucleus (vLGN), and the olivary pretectal nucleus (OPN). b. Immunohistochemistry staining shows that chemogenetic activation of the surviving M1Brn3b− ipRGCs in Gq-Opn4Cre;Brn3bDTA mice (bottom panels) does not induce H3 phosphorylation (green) in the SCN at CT6. The SCN (dashed outline) was visualized with DAPI (blue). ipRGC projections are visible with mCherry (magenta) within the SCN. Scale bars 100 µm. c. Quantification of H3 phosphorylation cell density in the SCN shown in b. H3 phosphorylation cell density was significantly smaller in Gq-Opn4Cre;Brn3bDTA mice (n=4) compared to Gq-Opn4Cre mice (n=5) (p < 0.0001, unpaired two-tailed t-test). Plotted data shows mice as individual points with mean ± SEM. Gq-Opn4Cre data (b and c) are re-shown from Fig. 1 for comparison. d. In Gq-Opn4Cre mice (upper panel) and Gq-Opn4Cre;Brn3bDTA mice (lower panel) ipRGC projections are visible with mCherry (magenta) within the suprachiasmatic nucleus (SCN), the olivary pretectal nucleus (OPN), the lateral geniculate nucleus (LGN), and the superior colliculus (SC). In Gq-Opn4Cre;Brn3bDTA mice ipRGC projections are visible with mCherry within the SCN. Minimal projections are visible in the OPN, LGN, and SC. The remaining projections to the LGN are restricted to the intergeniculate leaflet and the ventral lateral geniculate nucleus. All panels are from one animal. n = 4 mice. Scale bars 100 µm. e-f. Actograms for Gq-Opn4Cre;Brn3bDTA mice injected with CNO during the subjective day and subjective night. e. The double-plotted actograms of all Gq-Opn4Cre;Brn3bDTA mice (n = 9) during daytime activation of ipRGCs. On day 8 the mice received a CNO injection at CT4 and a 15-min LP at CT6. f. The double-plotted actograms of all Gq-Opn4Cre;Brn3bDTA mice (n = 9) during nighttime activation of ipRGCs. On day 8 the mice received a CNO injection at CT12 and a 15-min LP at CT14. Quantifications of observed phase shifts in e-f are shown in Fig. 3. g-m. Silencing of LGN neuron g. Schematic representations of ipRGC projections in Gq-Opn4Cre (top panel) and the LGN-silenced Gq-Opn4Cre mice (bottom panel). h. Immunohistochemistry staining shows the cFos expression (green) in the LGN following chemogenetic activation of ipRGCs in Gq-Opn4Cre (top panel) and the LGN-silenced Gq-Opn4Cre mice (bottom panel). ipRGC projections are visible with mCherry (magenta) within the LGN. Scale bars 100 µm. i. Magnified view of marked LGN in h, cFos expression is visible in green and ipRGC projections are visible with mCherry (magenta) in Gq-Opn4Cre (top panel) and the LGN-silenced Gq-Opn4Cre mice (bottom panel). Scale bars 100 µm. j. Quantification of cFos expression cell density in the LGN shown in i. cFos expression cell density was significantly reduced in the LGN-silenced Gq-Opn4Cre mice (n=3) compared to Gq-Opn4Cre mice (n=3) after a CNO. Plotted data shows mice as individual points with mean ± SEM. p = 0.0020, unpaired two-tailed t-test. k. Schematic representations of silencing of LGN in Opn4Cre mice. l. Immunohistochemistry staining shows the inhibitory DREADD expression that are visible with mCherry (magenta) within the LGN in LGN-silenced Opn4Cre mice. LGN was visualized with DAPI (blue). Scale bars 1000 µm. Magnified view is shown in right. n = 4 mice. Scale bars 100 µm. m. Whole mount retina (Scale bars 1000 µm) immunohistochemistry staining of LGN-silenced Opn4Cre mice. Melanopsin (Opn4) are visible in green. There was no mCherry (magenta) expression was observed in retina. n = 3 mice. Magnified view, Scale bars 100 µm. n-o. Actograms for LGN-silenced Gq-Opn4Cre mice injected with CNO during the subjective day and subjective night. n. The double-plotted actograms of all LGN-silenced Gq-Opn4Cre mice (n = 11) during daytime activation of ipRGCs. On day 8 the mice received a CNO injection at CT4 and a 15-min LP at CT6. o. The double-plotted actograms of all LGN-silenced Gq-Opn4Cre mice (n = 11) during nighttime activation of ipRGCs. On day 8 the mice received a CNO injection at CT12 and a 15-min LP at CT14. Quantifications of observed phase shifts in n-o are shown in Fig. 3. p-s. Chemogenetic activation of only Brn3b-positive ipRGCs. p. The double-plotted actograms of all Gq-Brn3b Cre;Opn4flpo mice (n = 4) during daytime activation of Brn3b-positive ipRGCs. On day 8 the mice received a CNO injection at CT4 and a 15-min LP at CT6. q. Quantifications of observed phase shifts in p and comparison of daytime phase shift by chemogenetic activation of all ipRGCs (Gq-Opn4Cre mice, n = 13) vs only brn3b-positive ipRGCs (Gq-Brn3b Cre;Opn4flpo mice n = 4). Plotted data shows mice as individual points with mean ± SEM. p < 0.0001, unpaired two-tailed t-test. r. Pupillary response after saline and CNO injection in Gq-Brn3b Cre;Opn4flpo mice (n = 3). s. Quantifications of observed PLR in r (n = 3 per condition). Plotted data shows mice as individual points with mean ± SEM. p = 0.0303, paired two-tailed t-test.