Nature | Vol 581 | 14 May 2020 | 195of food in control, but not Opn4DTA, mice (Fig. 1j, Extended Data Table 1).
The differences in total ghrelin levels between groups were observed
as early as day 7 after TRF and persisted throughout the experiments
(Extended Data Fig. 1k). These results indicate that the lack of antici-
patory ghrelin responses in Opn4DTA mice correlate with the impaired
anticipatory activity to timed feeding.
ipRGCs influence the IGL–SCN circuit
Several brain and peripheral areas exhibit changes in activity in response
to TRF^17. The reduced nonphotic entrainment displayed by Opn4DTA mice
suggests the involvement of a target of ipRGCs. The IGL receives dense
innervation from ipRGCs and is implicated in driving photic and non-
photic signals to modulate circadian processes^7 ,^18. We found that mice
exposed to TRF showed a substantial induction of the immediate-early
gene Fos (also known as c-Fos) in neurons of the IGL (Fig. 2a–c). However,
Opn4DTA mice exposed to the same paradigm showed a reduced FOS
induction in the IGL (Fig. 2b, c). Importantly, in control and Opn4DTA mice,
FOS induction was not observed in the hypothalamic arcuate nucleus—
an area known to be involved in the homeostatic control of hunger and
food intake^19 (Extended Data Fig. 2a–c). These results implicate the IGL
as a brain region involved in circadian entrainment to TRF.
The IGL contains NPY-expressing neurons that project to the SCN^7 –^9. To
better characterize the innervation pattern of IGLNPY neurons, we injected
a Cre-dependent adeno-associated virus (AAV), AAV-DIO-tdTomato,
into the IGL of Npycre/+ mice. We found that IGLNPY neurons innervate
both the ipsi- and contralateral SCN and, to a lesser extent, send unilat-
eral projections to other regions of the brain (Extended data Fig. 2d–h).
Early ablation of ipRGCs causes a significant reduction in the NPY immu-
noreactivity in IGL neurons (Fig. 2d, e), whereas the number of NPY+
somas and DAPI+ nuclei were unaffected (Fig. 2f, g). Consistent with the
reduction in NPY levels in the IGL, we also found a significant reduction
in NPY+ reactivity in the SCN of Opn4DTA mice (Fig. 2h–j, Extended data
Fig. 2i, j, Supplementary Videos 1, 2). However, when we correlated the
NPY levels from the SCN and the IGL, we found that the NPY staining in
the IGL covers a larger percentage of the leaflet compared to the SCN
nuclei in Opn4DTA mice (Extended data Fig. 2j), suggesting that NPY axonal
transport could also be affected. Structures that are not innervated by
ipRGCs and that express high levels of NPY showed normal patterns of
NPY immunostaining (Extended data Fig. 2k).Time window for IGL–SCN circuit assembly
We next evaluated whether ablation of ipRGCs at adult stages causes
similar alterations to responses to TRF. We used a mouse line that
expresses an attenuated form of the diphtheria toxin (attnDTA, also
known as aDTA) controlled by the melanopsin promoter (hereafter,
Opn4attnDTA mice), inducing substantial ablation of ipRGCs that pro-
ject to the SCN and IGL by six months of age^4. Eliminating the innerva-
tion by ipRGCs in adult mice had no significant effect on NPY levels in
fibres innervating the SCN (Fig. 3a, b). In addition, adult Opn4attnDTA
mice exposed to the TRF protocol showed robust food-anticipatory
activity, comparable to age-matched controls (Fig. 3c–e, Extended
Data Fig. 3a, b). These results indicate that innervation by ipRGCs has
an important role in circuits that control entrainment to TRF, specifi-
cally during early postnatal stages.
To determine the critical period for the influence of innervation
by ipRGCs on the assembly of the IGLNPY–SCN circuit and nonphotic
entrainment, we enucleated wild-type mice at different postnatal
stages. We found that the IGLNPY–SCN circuit was disrupted in adult mice
that were enucleated at postnatal day (P)0 to up to P40, an early adult-
hood stage. However, enucleation of mice at P90, a mature adulthood
stage had no significant effect (Fig. 3f, g, Extended Data Fig. 3c, Supple-
mentary Videos 3–6). Concordantly, wild-type mice enucleated at P0
and P40 showed significant deficits in the entrainment to TRF, whereas
mice enucleated at P90 displayed robust food-anticipatory responses
(Fig. 3h–l, Extended Data Fig. 3d–g). These results indicate that there
is a critical time window for the innervation by ipRGCs to influence
the assembly of the IGLNPY–SCN circuit and nonphotic entrainment.ipRGC–SCN axons regulate the IGL–SCN circuit
SCN and IGL receive dense input from ipRGCs^8 ,^9 , which suggest that
ipRGCs could directly affect IGLNPY neurons, their axonal projections***081600816 hConstant darknessTRFAd libitumfoodFood restriction
(7-h food access)Food access
No food access
Expected FAA
(no food access)Percentage of activity
3 h before food accessOpn4DTAControlFeeding activity Locomotor activity CombinedDays
Control Opn4DTAControl Opn4DTA Control Opn4DTAControl Opn4DTA403020
1005 4 3 2 1 0Score (AU)***1.51.00.50.0503 0.2787 0NS (P = 0.674)NS
(P = 0.523)Fold changeFold change<0.0001 0.0002
Control Opn4DTA**Foldchange0.0023 0.04225 4 3 2 1 0
5 4 3 2 1 0acb de fghij3.5
3.0
2.5
2.0
1.5
1.0
0.5
0**Relative locomotor activity
–9 –8 –7 –6–5 –4 –3–2 –1 0
Hours before food accessControl
Opn4DTAFig. 1 | Early ablation of ipRGCs affects circadian entrainment to TRF.
a, Schematic illustrating the TRF paradigm. FA A, food-anticipatory activity.
b–d, Representative actograms for feeding (b), locomotor activity (c) and
combined actograms (d), measured in three-month-old control and Opn4DTA
mice. e, f, The locomotor activity before food access (e), and relative
food-anticipatory activity (f) were measured as described in Methods. Data are
mean ± s.e.m. (n = 12 control mice, 13 Opn4DTA mice), *P = 0.0001, P = 0.0042,
two-tailed Student’s t-test. g, A score analysis was performed for all actograms
obtained from control and Opn4DTA mice as described in Methods. Data are
mean ± s.e.m. (n = 12 control mice, 13 Opn4DTA mice), ***P = 0.003, Student’s
t non-parametric (Mann–Whitney) test, two-tailed. AU, arbitrary units.
h–j, Hormonal signals were measured in control and Opn4DTA mice. The levels of
leptin (h), insulin (i), and total ghrelin (j) were measured. Data are expressed as
the level of the hormone during restricted access to food relative to
free-running conditions. Data are mean ± s.e.m. (n = 10 control mice, 13 Opn4DTA
mice), **P = 0.0038, two-tailed Student’s t-test. NS, not significant. In addition,
the statistical analysis versus a hypothetical value = 1 (dotted lines) was
performed, and is shown per column; by one-sample t-test.