Nature | Vol 581 | 14 May 2020 | 197at the SCN level or a combination of both. To test these possibilities, we
used a mouse line (Opn4cre/+ Brn3bDTA/+) (Brn3b is also known as Pou4f2)
in which Brn3b− ipRGCs that innervate the SCN survive (Extended Data
Fig. 4a), whereas Brn3b+ ipRGCs that mostly project to non-SCN regions
(including the IGL) are ablated during early postnatal stages^20 ,^21. In
Opn4cre/+ Brn3bDTA /+ mice, NPY immunostaining was unaffected in both
the IGL and SCN (Extended Data Fig. 4a–c), and these mice showed
sustained food-anticipatory activity to TRF (Extended Data Fig. 4d–h).
Thus, innervation by ipRGCs to the SCN is sufficient for tuning the
functional assembly of the IGLNPY–SCN circuit.
ipRGCs establish NPY levels in IGL
The reduced NPY levels in fibres that innervate the SCN suggest that
there is either a marked depletion of the neuropeptide or a lack of
axonal innervation from the IGL. To test this, we enucleated Npycre/+
mice at P0, and three months later mice were injected in the IGL with a
Cre-dependent AAV to trace NPY+ projections and their synaptic termi-
nals (Extended Data Fig. 5a, b). We found that the innervation pattern
and the density of synaptic terminals at the SCN level were not affected
in adult Npycre/+ mice that were enucleated at P0 (Extended Data Fig. 5c,
d), demonstrating that early ablation of ipRGCs affects the level of NPY,
but not IGLNPY axonal projections.
IGLNPY neurons affect entrainment to TRF
Adult mice that lack NPY (NPY-knockout mice) showed reduced entrain-
ment to TRF (Extended Data Fig. 6a–d). In addition, NPY-knockout mice
showed a reduction in the amount of food they consumed during the
TRF paradigm (Extended Data Fig. 6e), reflecting the critical role that
NPY signalling has in overall feeding behaviour^22.
To silence the synaptic release of IGLNPY neurons, a Cre-dependent
AAV encoding the light chain subunit of tetanus toxin (TenT) fused to
green fluorescent protein (GFP) was injected into adult Npycre/+ mice
(Fig. 4a). Npycre/+ mice injected with an AAV encoding DIO–GFP were
used as a control group. Injection sites were confirmed post hoc by
assessing GFP expression (Fig. 4a). Four weeks after the AAV injections,
mice were exposed to the TRF paradigm. The inhibition of synaptic
release specifically in IGLNPY neurons abolished food-anticipatory
activity (Fig. 4b–f, Extended Data Fig. 6f, g). It is important to note
that, similar to NPY-knockout mice, the amount of food consumed by
Npycre/+ mice was reduced when housed with ad libitum access to food
(Extended Data Fig. 6e). However, this reduction was exacerbated dur-
ing the time-restricted access to food in Npycre/+ mice injected with the
AAV encoding TenT (Extended Data Fig. 6e).
To specifically manipulate the IGLNPY–SCN circuit, we used a virally
delivered optogenetic strategy to transiently silence IGLNPY projections
that innervate the SCN. A Cre-dependent AAV encoding archaerhodop-
sin TP009 fused with tdTomato (DIO–ArchT–tdTomato) was bilaterally
injected in adult Npycre/+ mice (hereafter, Npycre/+ ArchT mice), and opti-
cal fibres were implanted just above the SCN (Fig. 4g). As control group,
Npycre/+ mice were injected with an AAV encoding DIO–tdTomato (here-
after, Npycre/+ sham mice) (Extended Data Fig. 7a). Mice were allowed to
recover for two weeks, and then placed under TRF. Both Npycre/+ sham
mice and Npycre/+ ArchT mice showed similar food-anticipatory activity
and food consumption (Extended Data Fig. 7b–d). Next, neural silenc-
ing was optogenetically induced in the IGLNPY–SCN circuit starting
2 h before food delivery by applying 3 pulses of 20 min of light, with
20-min intervals (Fig. 4h). Locomotor activity was measured starting
3 h before food delivery, and the total activity was recorded before and
after neuronal silencing, and compared in both groups of mice. Control
Npycre/+ sham mice displayed no significant changes in the locomotor
activity (Fig. 4i, Extended Data Fig. 7e, g, h). By contrast, Npycre/+ ArchT
mice displayed reduced exploratory activity during the optical silenc-
ing of NPYArchT-positive fibres (Fig. 4j, Extended Data Fig. 7f–h), without
affecting subsequent feeding behaviour (Extended Data Fig. 7c, d).Control TenTControl TenT**Percentage of activity 3 hbefore food access3020100 5 4 3 2 1 0***SCNIGLAAV-DIO-TenT-GFPNpycre/+mouseNpycre/+mouse0 8 16 0 8 16 0 h 0 8 16 0 8 16 0 hdLGNGFPIGLvLGNDays DaysDaysScore (AU)Control-injected Npycre/+
Te nT-injected Npycre/+***(P = 0.925)(P = 0.0418)Hours before food accessRelative locomotor activitySCNIGLLight stimulationFAA FAA + light Food access350
300
250
200
150
100
50
0350
300
250
200
150
100
50
0NS*FAA FAA + light FAA FAA + lightFAA (counts per h) FAA (counts per h)3.5
3.0
2.5
2.0
1.5
1.0
0.5
0–3 –2 –1 0 hDays0 8 16 0 8 16 0 hab cd ef gh i j–9 –8 –7 –6 –5 –4 –3 –2 –1 0AAV/DIO-ArchT-tdTomatoFig. 4 | NPY signalling in the IGL–SCN circuit regulates entrainment to TRF.
a, A Cre-dependent A AV encoding TenT–GFP was bilaterally injected into the
IGL of three-month-old Npycre/+ mice. Top, schematic. Bottom, injection site.
b, c, Representative actograms obtained from Npycre/+ mice exposed to TRF
that were injected with a control A AV (b) and A AV encoding TenT (c).
d–f, Locomotor activity before food access (d) and food-anticipatory activity
(e) were measured for control and Npycre/+ mice. Data are mean ± s.e.m.
(n = 5 mice for each genotype), *P < 0.001, *P = 0.0029, two-tailed Student’s
t-test. A score analysis was performed for all actograms obtained (f); data are
mean ± s.e.m. (n = 5 mice for each genotype); P = 0.0079, Student’s
t non-parametric (Mann–Whitney) test, two-tailed. g–h, Optogenetic silencing
of the IGLNPY–SCN circuit during food anticipatory activity. Schematic showing
the AAV injections and cannula implantation (g). Schematic showing the
optical stimulation protocol (h). i, j, Locomotor activity was measured for 3 h
(in 5-min bins) before food access in Npycre/+-sham (i) and Npycre/+ A rc hT (j) mice
exposed to TRF and optogenetic stimulation. Data are expressed as activity
(total beam break counts per hour) measured during food anticipatory activity,
with or without optical stimulation. Data are mean ± s.e.m. (n = 5 mice for each
condition), *P = 0.0418, two-tailed paired Student’s t-test. Scale bar, 200 μm (a).