194 | Nature | Vol 581 | 14 May 2020
Article
Retinal innervation tunes circuits that drive
nonphotic entrainment to food
Diego Carlos Fernandez^1 ✉, Ruchi Komal^1 , Jennifer Langel^1 , Jun Ma^1 , Phan Q. Duy1,3,
Mario A. Penzo^1 , Haiqing Zhao^2 & Samer Hattar^1 ✉Daily changes in light and food availability are major time cues that influence
circadian timing^1. However, little is known about the circuits that integrate these time
cues to drive a coherent circadian output^1 –^3. Here we investigate whether retinal
inputs modulate entrainment to nonphotic cues such as time-restricted feeding.
Photic information is relayed to the suprachiasmatic nucleus (SCN)—the central
circadian pacemaker—and the intergeniculate leaflet (IGL) through intrinsically
photosensitive retinal ganglion cells (ipRGCs)^4. We show that adult mice that lack
ipRGCs from the early postnatal stages have impaired entrainment to time-restricted
feeding, whereas ablation of ipRGCs at later stages had no effect. Innervation of
ipRGCs at early postnatal stages influences IGL neurons that express neuropeptide Y
(NPY) (hereafter, IGLNPY neurons), guiding the assembly of a functional IGLNPY–SCN
circuit. Moreover, silencing IGLNPY neurons in adult mice mimicked the deficits that
were induced by ablation of ipRGCs in the early postnatal stages, and acute inhibition
of IGLNPY terminals in the SCN decreased food-anticipatory activity. Thus, innervation
of ipRGCs in the early postnatal period tunes the IGLNPY–SCN circuit to allow
entrainment to time-restricted feeding.The circadian system contains the SCN, the central circadian pace-
maker, that orchestrates rhythmic functions of peripheral clocks
located throughout the body^1. This system integrates multiple time
cues from sensory, as well as circadian and metabolic, systems to gen-
erate a coherent perception of the environment^2 ,^3. At present, little is
known about the brain circuits and mechanisms that integrate different
time cues to drive a coordinated circadian output.
In mammals, light is transmitted to circadian centres through a
subpopulation of retinal ganglion cells^4 that are intrinsically photo-
sensitive, owing to their expression of the photopigment melanopsin
(encoded by OPN4)^5 ,^6 to drive circadian photoentrainment. As a central
pacemaker, the SCN receives dense axonal projections from multiple
areas of the brain—particularly the thalamic IGL, which is thought to
be involved in the circadian entrainment to nonphotic cues (hereafter,
nonphotic entrainment)^7 –^10. Here we show that retinal input affects
circadian circuits that control nonphotic entrainment.
Ablation of ipRGCs attenuates timed feeding
We assessed the effect of ablating retinal input on the circuits that
control entrainment to nonphotic time cues. We used a mouse line
that removes ipRGCs during development up to early postnatal stages
through the expression of subunit A of diphtheria toxin (Opn4DTA mice)^11.
Nonphotic entrainment was evaluated by limiting the food access
to a 7-h period (Fig. 1a), in what is known as time-restricted feeding
(TRF)^12. We opted to keep mice under constant darkness; thus, the
time-restricted access to food constituted the only recurrent time cue
for the mice. Both female and male control (wild-type) and Opn4DTA mice
with ad libitum access to food (hereafter, free-running conditions)
showed robust rhythmic patterns of feeding that closely overlapped
with their patterns of locomotor activity (Extended Data Fig. 1a, b),
which confirms that early ablation of ipRGCs has no effect on locomo-
tor activity and rhythmic feeding pattern in adult mice.
Under TRF, control mice displayed a robust and sustained
food-anticipatory activity^13 ,^14 (Fig. 1b–d, Extended Data Fig. 1c).
Opn4DTA mice showed deficits in nonphotic entrainment to TRF
(Fig. 1b–d, Extended Data Fig. 1d), as reduced food-anticipatory activ-
ity was observed throughout the restriction paradigm (Fig. 1e, f). A
graded-score analysis system (Extended Data Fig. 1e; see Methods for a
full description of the system) similarly showed significant deficits in cir-
cadian anticipation to TRF in mice that lack ipRGC innervation (Fig. 1g).
We next evaluated the hormones involved in the control of feed-
ing. Levels of insulin, leptin and total ghrelin—as well as glucose—were
similar in control and Opn4DTA mice under free-running conditions
(Extended Data Table 1). Under TRF, control and Opn4DTA mice had
similar levels of glucose and anorexigenic hormones, leptin and insu-
lin (Fig. 1h, i, Extended Data Table 1). In addition, control and Opn4DTA
mice consumed similar amounts of food, and their feeding patterns,
body weight and body composition were indistinguishable (Extended
Data Fig. 1f–j). Together, these results indicate that the behavioural
alterations that we observed in mice that lack ipRGCs are not caused
by changes in food intake or caloric restriction.
The levels of total ghrelin—an orexigenic hormone known for its
stimulatory effects on food intake^15 ,^16 —were increased in anticipationhttps://doi.org/10.1038/s41586-020-2204-1
Received: 23 July 2019
Accepted: 21 February 2020
Published online: 22 April 2020
Check for updates(^1) National Institute of Mental Health (NIMH), National Institutes of Health (NIH), Bethesda, MD, USA. (^2) Department of Biology, Johns Hopkins University, Baltimore, MD, USA. (^3) Present address:
MSTP, Yale University, New Haven, CT, USA. ✉e-mail: [email protected]; [email protected]