196 | Nature | Vol 581 | 14 May 2020
Article
NPY staining (AU) NPY+ cells per sectionFOS+ cells per IGL sectionFOSFOSSFood ab libitum TRF CTB (retina) NPY MergeCTB (retina) NPY MergedLGNvLGNIGLOpn4DTA^ControlOpn4DTA^ControlOpn4DTAControlControl Opn4DTA Control Opn4DTAControl Opn4DTA0102030405012
10
8
6
4
2
0**^20
15
105
0NS
(P = 0.611)DAPI+ cells per section7,000
6,000
5,000
4,000
3,000
2,000
1,000(^0) Control Opn4DTA
(P = 0.769)NS^50
40
30
20
10
0
NPY staining (% of total SCN ar
ea)
ox
3v
(P < 0.0001)
(P= 0.988)NS
IGL
SCN
Control Opn4DTA
Ad libitum TRF Ad libitum TRF
dLGN
vLGN
IGL
ab cd(P < 0.0001)
efghij
Fig. 2 | Early ablation of ipRGCs alters connectivity between IGLNPY and
SCN. a, Schematic brain section, highlighting the location of the IGL.
b, c, Representative images showing FOS induction in IGL neurons in response
to ad libitum food or TRF in three-month-old control and Opn4DTA mice (b),
quantified in c. Data are mean ± s.e.m. (n = 4 mice for each genotype),
*P < 0.001, two-tailed Tukey’s test. d–g, Morphological characterization of
retinal innervation (CTB, red) and NPY staining (green) in the IGL in
three-month-old control and Opn4DTA mice. Representative coronal sections
are shown (d). The levels of NPY staining (e), number of NPY+ somas (f) and
DAPI+ nuclei (g) were quantified. Data are mean ± s.e.m. (n = 5 control mice,
8 Opn4DTA mice), *P = 0.0022, two-tailed Student’s t-test. h, Schematic brain
section, highlighting the location of the SCN. i, j, Retinal input to the SCN (CTB,
red) and NPY from IGL (NPY, green). Representative images are shown (i). NPY
staining in the SCN was quantified (j). Data are mean ± s.e.m. (n = 8 mice for
each genotype), P < 0.001, two-tailed Student’s t-test. dLGN, dorsal lateral
geniculate; vLGN, ventral lateral geniculate; 3v, third ventricle; ox, optic
chiasm. Scale bars, 100 μm (i), 200 μm (b, d).
50
40
30
20
10
0 Opn4
attnDT
A
Opn4
attnDT
A^
Contr
ol
NPY staining (% of total SCN arControl Opn4attnDTA
ea)
0 8 16 0 8 16 0 h
Contr
ol
Days
pp
NS
(P = 0.194)
NS
(P = 0.568)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
En P0
En P40
En P90
Days
0 8 16 0 8 16 0 h
0 8 16 0 8 16 0 000000 8 16 0 8 16 0 hh 0 8 16 0 8 16 0 h 0 8 16 0 8 16 0 h
WT enucleated P0 WT enucleated P40 WT enucleated P90
50
40
30
20
10
0
NS
**
(P = 0.0001)
(P = 0.995)
(P = 0.002)
(P = 0.165)
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0
NS
Per
centage of activity 3 hbefore
food access
Relative locomotor activity
Hours before food access Control En P0 En P40 En P90
Control En P0 En P40 En P90
Relative locomotor activity
Hours before food access
Control
Opn4attnDTA^
WT En P90 WT En P40 WT En P0 Contr
ol
Days Days Days
(P = 0.017)**
NS
(P = 0.613) 40
30
20
10
0
(P = 0.298)
40
30
20
10
0
3v CTB NPY
3v
ox
ox
Control Opn4attnDTA
Per
centage of activity 3 hbefor
e food access
NS
NS
NPY staining (% of total SCN ar
ea)
(P = 0.999)
abcd e
fghij
kl
–9 –8 –7 –6 –5 –4 –3 –2 –1 0
DAPI NPY
–9 –8 –7 –6 –5 –4 –3 –2 –1 0
Fig. 3 | Critical time window for assembly of the IGLNPY–SCN circuit.
a, b, Retinal (CTB, red) and IGL (NPY, green) innervation to SCN in
nine-month-old control and Opn4a t t n DTA mice. Representative SCN sections are
shown (a). NPY staining was analysed (b). Data are mean ± s.e.m. (n = 5 mice for
each genotype), two-tailed Student’s t-test. c–e, Locomotor activity was
measured in control and Opn4a t t n DTA mice exposed to TRF. Representative
actograms are shown (c). The locomotor activity before food access (d) and the
food-anticipatory activity (e) were measured. Data are mean ± s.e.m.
((n = 5 control mice, 8 Opn4a t t n DTA mice), two-tailed Student’s t-test. f, g, IGL
(NPY, green) innervation to SCN in 3–5-month-old in wild-type (WT) control
and enucleated (En) mice. Representative sections are shown (f). NPY staining
in the SCN was analysed (g). Data are mean ± s.e.m. (n = 5 control, 7 En P0,
4 En P40 and 5 EnP90 mice); ***P < 0.001, two-tailed Tukey’s test. h–l, Wild-type
mice, enucleated at different stages, were exposed to TRF. Representative
actograms are shown (h–j). The locomotor activity before food access (k) and
the food-anticipatory activity (l) were measured in intact and enucleated mice.
Data are mean ± s.e.m. (n = 11 control, 7 En P0, 12 En P40 and 11 En P90 mice),
two-tailed Tukey’s test. Scale bars, 100 μm (a, f).