Extended Data Fig. 2: Early ablation of ipRGCs causes alterations in the IGL^NPY–SCN circuit.

a–c, FOS induction in the arcuate nucleus mediated by the expected food access. Mice were exposed to TRF and perfused on the 21st day at the expected food time (immediately before food delivery). As controls, mice with ab libitum access to food were perfused at circadian time 12. All mice were housed under constant darkness. The area analysed is shown in a diagram of a representative coronal brain section (a). Representative images of control and Opn4^DTA mice exposed to TRF are shown (b); the number of FOS⁺ cells in the arcuate nucleus was quantified (c). Data are mean ± s.e.m. (n = 4 mice for each genotype), two-tailed Tukey’s tes,. d–h, Projection pattern of IGL^NPY cells. Npy^cre/+ mice were unilaterally injected in the IGL using a Cre-dependent AAV encoding tdTomato (AAV2/9-phSyn1(S)-Flex-tdTomato-T2A-SynEGFP-WPRE). IGL^NPY neurons send dense and bilateral projections to the SCN (d) and, to a least extent, unilateral projections to other brain targets, including the dorsal geniculate and dorsal thalamus (e and f, respectively), and the superior colliculus (g). The complete pattern of IGL^NPY projections is shown in a diagram of representative coronal brain sections (h). Three independent experiments were performed with similar results. i, Representative SCN sections obtained from control and Opn4^DTA mice are shown. Marked alterations in the pattern of NPY staining in the SCN were observed in Opn4^DTA mice, compared to control mice. j, Correlation between NPY level in somas and axonal terminals, measured in the IGL and SCN, respectively. Results obtained from control and Opn4^DTA mice are shown. Pearson r values were measured for both groups (control = 0.728; Opn4^DTA = 0.389). A linear regression was applied, and the comparison of slope fits was not significantly different (slope ± s.e., control = 0.136 ± 0.052; Opn4^DTA = 0.100 ± 0.096). The asymptotic normal 95% confidence interval is shown for both groups. (n =7 control mice, 8 Opn4^DTA mice). k, Brain targets that are not innervated by ipRGCs and that express NPY were studied in control and Opn4^DTA mice. No obvious changes in NPY expression levels were observed in the paraventricular hypothalamic nucleus, arcuate nucleus, paraventricular nucleus of the thalamus or hypothalamic dorsomedial nucleus. Three independent experiments were performed with similar results. CL, centrolateral nucleus of the thalamus; LA, lateroanterior hypothalamic nucleus; LHb, lateral habenula LP, lateral posterior thalamic nucleus; SO, supraoptic nucleus. Scale bar, 100 μm (b, i, k), 200 μm (d, f), 400 μm (e, g).