review paper

Article

Light Affects Mood and Learning through Distinct

Retina-Brain Pathways

Graphical Abstract

Highlights

d Distinct ipRGC subpopulations drive the effects of light on

learning and mood

d SCN-projecting ipRGCs affect learning without disrupting

the central pacemaker

d The ipRGC-PHb pathway drives the light-mediated mood

alterations

d Thalamic PHb is integrated in a distinctive circuitry with

mood-regulating centers

Fernandez et al., 2018, Cell 175, 71–84 September 20, 2018 Published by Elsevier Inc. https://doi.org/10.1016/j.cell.2018.08.004

Authors

Diego Carlos Fernandez,

P. Michelle Fogerson,

Lorenzo Lazzerini Ospri, ..., Haiqing Zhao,

David M. Berson, Samer Hattar

Correspondence [email protected] (D.C.F.), [email protected] (S.H.)

In Brief

The effects of light on learning and mood

via intrinsically photosensitive retinal

ganglion cells involve a pacemaker-

independent role for the suprachiasmatic

nucleus as well as distinct circuitry in a

region of the thalamus called the

perihabenular nucleus.

Article

Light Affects Mood and Learning through Distinct Retina-Brain Pathways Diego Carlos Fernandez,1,5,6,* P. Michelle Fogerson,2,6 Lorenzo Lazzerini Ospri,3,6 Michael B. Thomsen,1,5

Robert M. Layne,4 Daniel Severin,3 Jesse Zhan,1 Joshua H. Singer,4 Alfredo Kirkwood,3 Haiqing Zhao,1 David M. Berson,2

and Samer Hattar1,5,7,* 1Department of Biology, Johns Hopkins University, Baltimore, MD 21218, USA 2Department of Neuroscience, Brown University, Providence, RI 02912, USA 3Department of Neuroscience, Johns Hopkins University, Baltimore, MD 21205, USA 4Department of Biology, University of Maryland, College Park, MD 20742, USA 5Present address: Section on Light and Circadian Rhythms (SLCR), National Institute of Mental Health, NIH, Bethesda, MD 20892, USA 6These authors contributed equally 7Lead Contact

*Correspondence: [email protected] (D.C.F.), [email protected] (S.H.)

https://doi.org/10.1016/j.cell.2018.08.004

SUMMARY

Light exerts a range of powerful biological effects beyond image vision, including mood and learning regulation. While the source of photic information affecting mood and cognitive functions is well estab- lished, viz. intrinsically photosensitive retinal gan- glion cells (ipRGCs), the central mediators are unknown. Here, we reveal that the direct effects of light on learning and mood utilize distinct ipRGC output streams. ipRGCs that project to the suprachi- asmatic nucleus (SCN) mediate the effects of light on learning, independently of the SCN’s pacemaker function. Mood regulation by light, on the other hand, requires an SCN-independent pathway linking ipRGCs to a previously unrecognized thalamic region, termed perihabenular nucleus (PHb). The PHb is integrated in a distinctive circuitry with mood-regulating centers and is both necessary and sufficient for driving the effects of light on affective behavior. Together, these results provide new in- sights into the neural basis required for light to influ- ence mood and learning.

INTRODUCTION

The constancy of the solar cycle as a signal for the regulation of

mammalian behavior is manifested, among other things, in the

powerful effects that light exerts on mood and cognition. These

effects have been documented in laboratory animals (Bedrosian

et al., 2011; LeGates et al., 2012), as well as in humans (Vande-

walle et al., 2010). Depressive symptoms and cognitive dysfunc-

tion linked to light can be brought on by natural conditions or by

people’s own agency by traveling across time zones and expe-

riencing jet lag or in shift-work (Jaeger et al., 2006; Kurlansik

and Ibay, 2012; Roh et al., 2016; Scott et al., 1997). The magni-

tude of these mood effects is large as �20% of the general

population is affected (Melrose, 2015) with symptoms being

fairly common, including anhedonia and feelings of helplessness

(Russo and Nestler, 2013).

In mammals, light detection occurs exclusively in the retina

(Hattar et al., 2003; Wässle, 2004). In addition to classical rod

and cone photoreceptors, a subpopulation of retinal ganglion

cells (RGCs) that express the photopigment melanopsin

(Opn4), making them intrinsically photosensitive (ip)RGCs,

constitute the third type of photoreceptors (Berson et al.,

2002; Hattar et al., 2002; Provencio et al., 2000). At present,

5 subtypes (M1–M5) of ipRGCs have been described (Ecker

et al., 2010; Schmidt et al., 2011). Their central projections differ

amply, reflecting in this variety the multiform nature of the

non-image-forming visual functions they participate in such as

circadian photoentrainment and sleep regulation (Altimus

et al., 2008; Hattar et al., 2006). A major ipRGC target is the hy-

pothalamic suprachiasmatic nucleus (SCN), which houses a

central pacemaker that orchestrates peripheral clocks to drive

circadian rhythmic coherence (Morin, 2013). Genetic ablation

of ipRGCs completely abrogates light reception at the SCN

level, suggesting that ipRGCs constitute the only conduit for

light input to modulate circadian brain functions (Güler

et al., 2008).

Notably, the regulation of mood and learning by light is also

absent in mice where ipRGCs are ablated (LeGates et al.,

2012, 2014), indicating that ipRGCs are the main sensory chan-

nel driving these behavioral effects. The ipRGC central targets

mediating these effects, however, remain unknown. Here, we

show that a subset of ipRGCs, that are defined by the lack of

Brn3b expression and project to the SCN, are sufficient to drive

light-mediated cognitive deficits without disrupting the SCN

clockwork machinery. An SCN-independent pathway mediates

light-induced mood changes through ipRGC input to the periha-

benular nucleus (PHb) of the dorsal thalamus. PHb neurons proj-

ect to well-characterized mood-regulating centers. Furthermore,

the PHb is both necessary and sufficient for driving the effects of

light on affective behavior. These findings reveal two distinct

retinal-brain pathways that mediate the direct effects of light

on mood and cognition.

Cell 175, 71–84, September 20, 2018 Published by Elsevier Inc. 71

Figure 1. SCN-Projecting ipRGCs Drive Light-Induced Cognitive Deficits

(A) Schemes showing ipRGC projections in Opn4Cre/+ (control) and Opn4Cre/+;Brn3bzDTA mice.

(B and C) Retinal projections (Brn3b(�) ipRGCs) were retained in the SCN (B), but absent in most ipRGC targets (C) in Opn4Cre/+;Brn3bzDTA mice. (D and E) The pattern of locomotor activity (D) and circadian periods length (E) under the T7 cycle were similar between groups. Data are mean ± SEM, by

Student’s t test (n = 15).

(F) A significant reduction in the time exploring the novel object was observed in mice exposed to the T7 cycle versus T24-housed mice. The statistical analysis

compared to 50% (dotted lines) was Opn4Cre/+: T24 p < 0.01; T7 p = 0.716; Opn4Cre/+;Brn3bDTA: T24 p < 0.001; T7 p = 0.754; by one sample t test (n = 15–35).

(G and H) Using the MWM, we found significant deficits during learning (G) and test (H) trials in mice exposed to the T7 cycle versus T24-housed mice (n = 12–14).

(I) T7-housed Opn4Cre/+;Brn3bDTA mice showed significant LTP deficits versus T24-housed mice (n = 8–10 mice).

Data are mean ± SEM. *p<0.05, **p<0.01, by Tukey’s test. 3v, third ventricle; ox, optic chiasm; LGN, lateral geniculate nucleus; Hb, habenular complex; PT,

pretectum. Scale bars, 100 mm (B), 1 mm (C).

See also Figure S1.

RESULTS

The SCN-Projecting ipRGCs Drive the Effects of Light on Learning The use of an ultradian light cycle (T7 cycle; alternating 3.5-hr

periods of light and darkness) allowed us to determine the direct

effects of light on mood and learning (LeGates et al., 2012). To

investigate whether light input to the SCN is sufficient to drive

the behavioral alterations induced by the T7 cycle, we used a

previously described mouse line, Opn4Cre/+;Brn3bzDTA/+ (Chen

et al., 2011), in which Brn3b(�) ipRGCs that innervate the SCN survive, whereas Brn3b(+) ipRGCs projecting outside the

72 Cell 175, 71–84, September 20, 2018

SCN are substantially ablated (Figures 1A–1C and S1A–S1C).

Opn4Cre/+;Brn3bzDTA/+ mice photoentrained under a normal

light/dark cycle (T24 cycle), and their circadian periods were

lengthened when exposed to the T7 cycle, similar to Opn4Cre/+

(control) mice (Figures 1D and 1E). Thus, the general circadian

responses are intact in these animals.

Under the T24 cycle, Opn4Cre/+ and Opn4Cre/+;Brn3bzDTA/+

mice showed similar learning and cognitive functions (Figures

1F–1H; novel object recognition [NOR] test: p = 0.57; Morris wa-

ter maze [MWM], test trial: p = 0.5, by Tukey’s test), indicating

that the ablation of Brn3b(+) ipRGCs did not affect the cognitive

functions of these animals. When exposed to the T7 cycle for

2 weeks, Opn4Cre/+;Brn3bzDTA/+ animals displayed cognitive def-

icits, similar to those observed in T7 cycle-housed control

(Opn4Cre/+) mice (Figures 1F–1H). Specifically, T7 cycle-housed

mice spent significantly less time exploring the novel object than

mice housed under the T24 cycle (Figures 1F and S1D). In the

MWM, T7 cycle-housed mice exhibited significant deficits during

training, test, and reverse phases, relative to mice housed under

the T24 cycle (Figures 1G, 1H, S1E, and S1F). For both behav-

ioral tests, no significant differences were found in the results ob-

tained from Opn4Cre/+ and Opn4Cre/+;Brn3bzDTA/+ mice exposed

to the T7 cycle (Figures 1F–1H; NOR: p = 0.45; MWM, test trial:

p = 0.86; by Tukey’s test).

In line with the behavioral studies, hippocampal long-term

potentiation (LTP) was significantly attenuated in both Opn4Cre/+

(control) and Opn4Cre/+;Brn3bzDTA/+ mice when housed under

the T7 cycle, relative to T24-housed groups (Figures 1I and

S1G). The LTP alterations were specific to activity-dependent

potentiation by the theta-burst stimulus, because other aspects

of transmission were normal (Figures S1H and S1I). Together,

these results highlight the sufficiency of the Brn3b(�) ipRGCs in mediating light-induced LTP and cognitive deficits.

The T7 Cycle Alters the Photoresponsiveness of the SCN We previously found that the rhythmic expression of the clock

gene period2 (PER2) in the SCN was unperturbed by the T7 cycle

(LeGates et al., 2012). This raises a conundrum: if the SCN re-

mains responsive to light under the T7 cycle, why are PER2

levels unaffected under a recurring light activation? To address

this question, we first tested whether the photic responsiveness

of SCN neurons is affected under the T7 cycle, by evaluating the

phosphorylation of CREB (cAMP response element-binding pro-

tein), one of the first steps in gene induction in response to light

(Chawla, 2002). Light-induced pCREB levels were similar in the

SCN from animals maintained under T24 and T7 cycles (Figures

2A–2D), indicating that the SCN is still responsive to light in

T7 cycle-housed mice. Interestingly, it was recently shown that

the SCN can act not only as a circadian pacemaker, but also

as a relay for light input to peripheral oscillators (Izumo et al.,

2014). To explore the role of the SCN as a light relay versus a

circadian pacemaker, we analyzed the effects of T7 cycle on

Period1 (PER1), a clock gene that shows both a robust rhythmic

expression and a fast response to light (Travnickova-Bendova

et al., 2002). We found that under T7 cycle, the expression levels

of PER1 were rhythmic in the SCN (Figures 2E and 2F), whereas

the acute effects of light on PER1 induction were abrogated (Fig-

ures 2G and 2H). Combined, the effects of light on pCREB levels

and PER1 induction indicate that the photic responsiveness of

the SCN is distinctively altered by the T7 cycle.

To further investigate the overall changes in SCN photic

responsiveness, we performed RNA-sequencing (RNA-seq) on

isolated SCN samples (Figure 2A). We found that 29 genes

were differentially regulated between T24 and T7 cycle groups

(false discovery rate [FDR]-adjusted p < 0.01, Figure S2A)

including a striking depletion of classical immediate-early genes

in T7 cycle-housed animals (Figures 2I and 2J). Gene ontology

analysis of differentially regulated transcripts revealed a deple-

tion of genes involved in transcription as well as Ca2+ and

cAMP responses, further suggesting that the T7 cycle dramati-

cally alters the photic responsiveness of the SCN (Figure S2B).

In support of the RNA-seq data, we found a significant decrease

in light-mediated c-Fos induction in SCN neurons under the

T7 cycle, compared with T24-housed mice (Figures S3A and

S3B). c-Fos induction in the SCN requires the phosphorylation

of both CREB and ELK-1, leading to the recruitment of CREB-

binding protein, resulting in gene transcription through the acet-

ylation and phosphorylation of histones, including histone-3 (H3)

(Chawla, 2002; Kako and Ishida, 1998). In contrast to pCREB, the

light-induced pELK-1 and pH3 levels were significantly reduced

in SCN neurons from animals housed under the T7 cycle (Figures

S3A, S3C, and S3D). Combined, these data show that the SCN

becomes refractory to light-induced transcriptional changes

that would otherwise impinge on the clockwork machinery under

the T7 cycle.

The Effects of Light on Mood Are Independent of the SCN To investigate whether Brn3b(�) SCN-projecting ipRGCs also affect mood-related behaviors, mice were housed under the

T24 or T7 cycles, and the sucrose preference test (SPT),

tail suspension test (TST), and forced-swim test (FST) were

performed. Under normal light conditions, Opn4Cre/+ (control)

and Opn4Cre/+;Brn3bzDTA/+ mice showed no significant differ-

ences in their behavioral responses (Figure 3; SPT,

p = 0.600; TST, p = 0.921; FST, p = 0.859; by Tukey’s test).

Remarkably, ablating ipRGC projections outside the SCN

abolished mood alterations induced by the T7 cycle. Specif-

ically, Opn4Cre/+;Brn3bzDTA/+ mice exposed to the T7 cycle

showed no significant differences in the sucrose preference

index (Figure 3A), immobility time during the TST (Figure 3B)

and FST (Figure 3C), compared to mice housed under the

T24 cycle. In addition, under the T7 cycle, results obtained

from Opn4Cre/+;Brn3bzDTA/+ mice were significantly different

compared with Opn4Cre/+ mice (Figure 3; SPT, p < 0.001;

TST, p < 0.05; FST, p < 0.001; by Tukey’s test). Thus, ipRGC

pathways, distinct from the SCN, must be responsible for

driving light regulation of mood.

PHb Neurons Receive Direct Input from ipRGCs Among the ipRGC targets that might mediate the perturbation of

affective behavior by unnatural light cycles, a region of the dorsal

thalamus, adjacent to the epithalamic lateral habenula (LHb), is

of particular interest. This region, which was previously termed

perihabenular region (PHb), has been shown to receive retinal

innervation (Hattar et al., 2006; Morin and Studholme, 2014).

We first evaluated the light-responsiveness of the PHb. Wild-

type (WT) mice showed a robust light-mediated c-Fos induction

throughout the PHb (Figures 4A and 4C), whereas mice lacking

ipRGC innervation to the PHb (Opn4Cre/+;Brn3bzDTA/+) showed

no induction of c-Fos (Figure S3E). We found that T7-housed

WT mice showed a normal c-Fos induction in the PHb after a light

pulse exposure (Figures 4B and 4C), suggesting that, contrary to

the SCN, the PHb photoresponsiveness is unaltered under

T7 cycle.

PHb neurons show a robust rhythmic expression of the clock

gene PER2 under normal light conditions (Figure 4D). The c-Fos

induction observed in PHb neurons raises the possibility that

Cell 175, 71–84, September 20, 2018 73

Figure 2. Photic Responsiveness and Gene Expression in the SCN

(A) T24- or T7-housed mice were kept in darkness for 1 day, exposed to a light pulse, and immediately perfused for assess pCREB levels (green star), perfused

after 90 min for PER1 levels (red star), or after 60 min for SCN dissections (blue star).

(B) Scheme showing a coronal brain section at SCN level.

(C and D) A significant increase in pCREB levels were observed in both light pulse (LP)-treated groups, versus no light pulse (noLP) controls. Data are mean ±

SEM. **p<0.01; by Tukey’s test (n = 5–6 mice).

(E and F) No significant differences in the rhythmicity of PER1 levels were observed between groups (F). Data are mean ± SEM; by Student’s t test (n = 4–5 mice).

(GandH)TheexposureofmicetotheT7cycleaffectedthelight-mediatedinductionofPER1intheSCN.Dataaremean± SEM.**p<0.01;byTukey’stest(n=5–6mice).

(I) Heatmap of relative gene expression levels for selected immediate-early genes.

(J) Fold change in expression level of immediate-early genes in T7 versus T24 samples (T24: n = 3, T7: n = 4 replicates; SCN tissue was pooled from 3 mice per

replicate, FDR-adjusted p < 0.01 for all samples).

3v, third ventricle; ox, optic chiasm; DD, constant darkness; ZT, zeitgeber time. Scale bars, 100 mm.

See also Figures S2 and S3.

74 Cell 175, 71–84, September 20, 2018

Figure 3. ipRGC-SCN Pathway Is Not Involved In Light-Mediated Mood Alterations

(A–C) Opn4Cre/+ mice housed under the T7 cycle showed mood alterations, as shown in the SPT (A), TST (B), and FST (C). These mood-related deficits induced by

the T7 cycle were abolished in Opn4Cre/+;Brn3bzDTA mice. For the SPT, the total volume consumed (in mL) was Opn4Cre/+, T24 = 5.3 ± 1.2 and T7 = 5.1 ± 0.8 and

Opn4Cre/+;Brn3bDTA, T24 = 5.1 ± 1.1 and T7 = 5.8 ± 0.8 by Tukey’s test. In addition, the statistical analysis versus 50% (dotted lines, A) was Opn4Cre/+,

T24 p < 0.01 and T7 p = 0.30 and Opn4Cre/+;Brn3bDTA, T24 p < 0.01 and T7 p < 0.01 by one sample t test.

Data are mean ± SEM. ***p<0.001, **p<0.01, by Tukey’s test. Opn4Cre/+ mice, n = 15–16; Opn4Cre/+;Brn3bDTA mice, n = 29–42.

under the T7 cycle, neurons may be abnormally (and chronically)

activated by the appearance of light every 3.5 hr, which would

lead to alterations in PER2 rhythmic expression. Indeed, when

WT mice were housed under the T7 cycle, PER2 rhythmicity

was abolished in the PHb, maintaining high PER2 levels

throughout (Figure 4D). These results suggest that light input

has a direct impact over clock genes expression, as revealed

by alterations in PER2, known to show a slow response to light

(Travnickova-Bendova et al., 2002; Wilsbacher et al., 2002). To

further evaluate whether ipRGCs are involved in this process,

we tested mice in which ipRGCs are ablated by the expression

of the attenuated diphtheria toxin (Opn4aDTA/aDTA). We previously

found a drastic reduction of ipRGC innervation in these animals

at 6 months of age (Ecker et al., 2010; Güler et al., 2008; LeGates

et al., 2012). Specifically, only sparse retinal fibers were

observed in the most caudal PHb region in Opn4aDTA/aDTA mice

(Figures S4A and S4B). Importantly, in the absence of the ipRGC

projections, PER2 rhythmicity in the PHb was maintained in

Opn4aDTA/aDTA mice housed under the T7 cycle (Figures 4E,

S4C, and S4D). These findings reveal that irregular light/dark cy-

cles perturb the circadian clock machinery selectively in the PHb

through an ipRGC-dependent circuit.

Retinal innervation to the PHb was precisely mapped using

intravitreal injections of the tracer cholera toxin b-subunit

(CTb). Retinal axons labeled a wedge-shaped zone near the

boundary between the dorsal thalamus and the LHb, especially

caudally, in regions typically included in the central lateral (CL)

or the lateral posterior (LP) thalamic nuclei (Figures 4F and

S4A). A few retinal fibers penetrate into the most lateral edge

of the LHb (Figure S4A). The retina-PHb circuit was further char-

acterized by mapping the presynaptic terminals of ipRGC axons

by using Opn4CreERT/+;ROSASynaptophysin-tdTomato mice (Fig-

ure S4E). Labeled puncta, marking ipRGC output synapses,

were densely distributed throughout the PHb (Figures 4G

and S4F). To determine the identity of the RGCs that project to

this area, we injected the tracer CTb into the PHb in WT mice

(Figure 4H). In the contralateral retina, all retrolabeled RGCs

(70.6 ± 5.2 cells/retina, n = 4 mice) were melanopsin immunopos-

itive (Figures 4I and 4J). Thus, the PHb receives its retinal input

exclusively from ipRGCs.

Next, we sought functional evidence for retinal influence on

PHb neurons. For that, we expressed channelrhodopsin-2

(ChR2) in retinal axons by intravitreal injections of an AAV2/

ChR2-EYFP (Figures 4K, S4G, and S4H). PHb cells recorded in

current clamp exhibited bursting responses when relatively

hyperpolarized (�70 mV), whereas a tonic mode of firing was observed when cells were relatively depolarized (�60 mV) (Fig- ures S4I and S4J), similar to previous descriptions of thalamic

neurons (Kim et al., 2001; Sherman, 2001), and a subpopulation

of LHb neurons (Wagner et al., 2017). Activation of local ChR2-

positive retinal axons by flashes of blue light (2 ms) evoked

cationic excitatory postsynaptic currents (EPSCs) in a subset

(9.5%, n = 63 cells) of patch-recorded PHb neurons (Figures

4L, 4M, and S4K–S4M). Together, these data indicate that

ipRGCs provide excitatory synaptic input to PHb neurons.

PHb Is a Distinct Thalamic Region that Projects to Mood- Regulating Centers The location of the PHb was delineated using markers of gene

expression exclusively associated with thalamic or epithalamic

areas. For that, the PHb area was outlined based on the light-

mediated c-Fos induction. We found that most c-Fos(+) cells

were co-labeled with GRID2IP (Figure 5A), a marker for dorsal

thalamic nuclei (Nagalski et al., 2016). Only a minor fraction of to-

tal PHb c-Fos(+) cells co-localized with Brn3a (also known as

POU4F1, Figure 5B), a marker for the epithalamic LHb (Quina

et al., 2015). Additionally, we observed that most ipRGC termi-

nals were in close apposition with thalamic neurons immunopos-

itive for GRID2IP or PKCd (Figures S5A and S5B), another

marker used for delineating the dorsal thalamus (Nagalski

et al., 2016). In sum, these results assign the PHb as part of

the dorsal thalamus.

We then investigated the PHb downstream circuitry using CTb

injections to uncover targets potentially implicated in mood

regulation. We found that the most prominently labeled region

was the ventral medial prefrontal cortex (vmPFC) (Figure S5C).

Cell 175, 71–84, September 20, 2018 75

Figure 4. Characterization of Retinal Input to the PHb

(A–C) A significant increase in the number of light-induced c-Fos(+) cells in PHb were observed in both groups, versus no light pulse controls. Data are mean ±

SEM. **p<0.01; by Tukey’s test (n = 5 mice).

(D and E) Immunohistochemical evidence for the rhythmic PER2 expression in the PHb (white arrows) in mice housed under the T24 or T7 cycles. In WT mice,

PER2 levels exhibited circadian rhythmicity in the PHb under the T24 cycle, but the T7 cycle exposure significantly raised PER2 expression levels (D). In mice

lacking ipRGC projections (Opn4aDTA/aDTA), PER2 rhythms in the PHb (E) became impervious to the effects of the T7 cycle. Data are mean ± SEM. *p < 0.05,

**p < 0.01, by Student’s t test (n = 4–5 mice).

(F) Retinal projections to the PHb traced by CTb.

(G) ipRGC afferents to PHb contain a synaptophysin fusion protein (Opn4CreERT;ROSASyn-tdTomato).

(H–J) CTb was stereotaxically injected in the PHb (H), and all retrolabeled RGCs (I) were melanopsin(+) (J). From a total of 49 ipRGCs analyzed, 46 cells were

identified as M1 and 3 were M3 (n = 8 mice).

(legend continued on next page)

76 Cell 175, 71–84, September 20, 2018

Figure 5. Thalamic PHb Projects to Mood-Regulating Centers

(A and B) Light induced cFos(+) cells in the PHb were colocalized with GRID2 (A) or Brn3a (B) markers. Data are mean ± SEM (n = 3 mice).

(C–F) HSV/cre was injected into the vmPFC, while AAV/DIO-synaptophysin-tdTomato was injected into the PHb. Labeled somas were exclusively found in the

PHb (C). PHb neurons have three targets: the vmPFC, including the IL (D), the dorsomedial striatum (E), and the NAc (F) (n = 12 mice).

(G) These experiments revealed a thalamocortical loop, represented here diagrammatically.

cc, corpus callosum; M2, secondary motor cortex; AC, anterior cingulate cortex; Str, striatum; aco, anterior commissure; Sep, septal nuclei; Orb, orbitofrontal

cortex, Hip, hippocampus. Scale bars, 50 mm (D, inset); 100 mm (A–C); 200 mm (D–F).

See also Figure S5.

In particular, there were retrogradely labeled somas in layers V

and VI of the infralimbic (IL) and prelimbic (PL) cortices bilaterally

with respect to injection site and anterograde-labeled fibers in

(K–M) Optogenetic evidence for functional connections between ipRGCs and PH

A subpopulation of PHb neurons showed a ChR2-induced response to light puls

3v, third ventricle; ox, optic chiasm; no LP, no light pulse; LP, light pulse. Scale

See also Figures S3 and S4.

layers I and III-IV of the IL ipsilaterally. This result pointed to

the existence of a thalamocortical loop between the PHb and

vmPFC that is distinct from the CL or the LP nuclei, which are

b neurons (K). Current response in a representative PHb neuron is shown (L).

es (M) (n = 63 cells).

bars, 100 mm (J); 200 mm (B and D–H).

Cell 175, 71–84, September 20, 2018 77

Figure 6. Disynaptic Circuits Connect ipRGCs to Mood-Regulating Centers

(A–D) A three-virus retrograde transynaptic tracing system was used to identify the retino-PHb-vmPFC pathway (A). Injection sites were confirmed in the vmPFC

(B) and PHb (C). EnvA-GDRabies-mCherry expresses mCherry in PHb neurons expressing both Cre-EYFP and the Cre-dependent helper AAV (starter neurons)

and presynaptic inputs to these neurons. A retrolabeled RGC expressing mCherry counterstained for melanopsin is shown (D).

(E) The PHb was transfected with a fluorescent Ca2+ sensor by injecting a cre-dependent GCaMP6m vector in the PHb region and a retrogradely transported cre

virus in the vmPFC.

(F) Average response to a 2-s light pulse delivered at zeitgeber time (ZT) 18 to a mouse kept under the T24 cycle. The Ca2+ transient comprises a fast-rising

component plateauing for 1.1 s before a second phase supervenes to generate a higher peak 3 s after light presentation. A biphasic response could bespeak

either the transfection in our experimental design of two distinct (sub)populations that respond to light with different time courses or a functional property of a

single neuronal population.

(G) A map of the 14 individual trials averaged in (F) is shown.

(H) PHb activity following regularly scheduled lights-on event in T24 cycle.

(legend continued on next page)

78 Cell 175, 71–84, September 20, 2018

not known to project to the vmPFC (Noseda et al., 2010; Voorn

et al., 2004). To increase the precision of our projection tracing,

we used a dual injection strategy with a retrogradely transported

Cre-carrying vector injected in the IL and a Cre-dependent vec-

tor carrying synaptophysin-tdTomato injected in the PHb (Fig-

ures 5C and 5D). We found that one of the PHb targets is indeed

the vmPFC (Figure 5D). To determine which vmPFC subdivisions

are innervated, we used vGluT2 staining to reveal the ventral

boundary of the cortex (Figure S5D). PHb terminals extended

ventrally within 200 mm of the ventral cortical boundary (Fig-

ure 5D), which is assigned IL territory. Furthermore, PHb termi-

nals revealed a compression of cortical layers characteristic of

the cytoarchitecture of IL. Based on the same factors, the

most dorsal extent of the PHb’s cortical innervation field may

fall within the ventral PL. The second and third targets of PHb

neurons are the dorsomedial striatum (Figure 5E) and the nu-

cleus accumbens (NAc) (Figure 5F). It should be noted that the

dorsomedial striatum also receives input from the same cortical

areas targeted by the PHb, indicating that PHb is part of a tha-

lamo-frontocortico-striatal loop (Figures 5G, S5E, and S5F).

Finally, we noticed that a subset of labeled PHb neurons appear

to send collaterals into the lateral part of the LHb (Figure S5G).

Control injections of HSV-cre in the dorsomedial prefrontal cor-

tex did not result in any labeled cells in the PHb proper (Fig-

ure S5H). Together, these results demonstrate that the PHb is

incorporated into a loop reminiscent of limbic thalamic nuclei

(Vertes et al., 2015). As a cohesive neuronal cluster with distinc-

tive connectivity, genetic, and functional properties (including

being a circadian oscillator), the PHb must be properly reckoned

as an independent, previously unrecognized thalamic nucleus.

Disynaptic Circuits Connect ipRGCs to Specific Mood- Regulating Regions To determine whether the PHb links ipRGCs to vmPFC through a

disynaptic circuit, we used a triple-virus trans-synaptic retro-

grade tracing strategy (Schwarz et al., 2015): a retrogradely

transported AAV/Cre-YFP was injected into the vmPFC (Figures

6A and 6B). Then, presynaptic partners of these thalamocortical

relay neurons were labeled transynaptically by injecting a Cre-

dependent helper virus followed by EnvA-pseudotyped

gDRabies-mCherry in the PHb (Figure 6C). Finally, labeled

PHb-projecting RGCs were found, and all of them were mela-

nopsin(+) (Figure 6D). Using this strategy, we labeled from one

to seven ipRGCs per animal; the low number of labeled ipRGCs

could be due to the low probability requirement for the three

viruses to infect the same cells in the PHb. Therefore, we also

deployed a dual-virus strategy: a retrograde helper virus was

injected into the vmPFC, followed by EnvA-pseudotyped

G-deleted rabies injected into the PHb (Figures S6A–S6D). Using

this strategy, 25 ± 7.1 labeled ipRGCs were found per retina. In

(I) In T7 cycle, the PHb responds to dark-to-light transitions with a biphasic Ca2+

(J) Comparison of peak fluorescence in T24- versus T7-housed mice recorded o

Student’s t test).

(K and L) Time course of peak fluorescence (calculated as the difference between

in T24 (K) or T7 (L) cycles. Dashed orange lines indicate light transitions.

Data are mean ± SEM (n = 3 mice). Scale bars, 100 mm (D); 200 mm (C); 500 mm

See also Figure S6.

sum, these results confirm the existence of a disynaptic pathway

linking ipRGCs through PHb to vmPFC.

Next, we sought to characterize the physiology of the PHb

in vivo. The target cell population was transfected with a fluores-

cent Ca2+ indicator, GCaMP6m, by injections of a cre-depen-

dent vector in the PHb and a retrogradely transported

cre-carrying vector in the vmPFC; an optical fiber was implanted

above the PHb to deliver and collect light locally in awake, free-

moving mice (Figure 6E). We first confirmed that vmPFC-projec-

ting PHb neurons responded to brief light pulses delivered in the

active phase of mice (Figure 6F) with a biphasic Ca2+ transient

characterized by a fast component rising 90 ± 3 ms after external

light presentation and by a subsequent slower further rise peak-

ing 3 s thereafter. Return to the pre-stimulus baseline occurs with

high temporal variability and irrespective of continued environ-

mental illumination (Figure 6G). To analyze the PHb physiology

over longer time-spans, we exposed injected mice to the T24

or T7 cycles, and the PHb activity was recorded. Under both

lighting conditions, we observed a fast response of PHb neurons

to each dark-light transition (Figures 6H and 6I). A comparison of

the magnitude of the peak fluorescence in the PHb in the course

of exposure to the two light cycles revealed a significant increase

of such activity under the T7 cycle (Figures 6J–6L). In sum, these

results indicate that light input modulates the Ca2+ dynamics of

vmPFC-projecting PHb neurons in vivo, with the T7 cycle specif-

ically shown to increase activity.

Chronic Activation of PHb Neurons Is Sufficient to Regulate Mood The PHb is well positioned to link environmental lighting to affec-

tive behavior. If the PHb is required for the effects of light on

mood, then chronic activation of the PHb should induce mood

disorders in animals housed in a normal light/dark cycle,

whereas silencing PHb neurons should block the dysphoric

effects induced by the unnatural light cycle. To achieve long-

lasting activation of light-responsive PHb neurons, we used a

chemogenetic strategy based on designer receptors exclusively

activated by designer drugs (DREADDs) (Roth, 2016) combined

with c-FosCreERT2 mice. A Cre-dependent AAV encoding an

excitatory (Gq) was bilaterally injected in the PHb (Figure 7A).

One week later, mice received a light pulse (circadian time

[CT] 14), leading to the expression of the tamoxifen (Tam)-sensi-

tive Cre recombinase only in light-responsive neurons. At the

end of the light pulse, mice were injected with 4-OH-Tam. Injec-

tion sites were invariably restricted to PHb and immediately adja-

cent thalamic nuclei (Figures 7A and S6E). In AAV-injected

c-FosCreERT2 mice that received 4-OH-Tam, but without light

pulse exposure, only sparse mCherry(+) cells or no detectable

mCherry expression was observed in the PHb (Figure S6F).

As a control group, littermate Cre-negative mice received the

transient analogous to the one observed in T24 cycle.

ver 72 hr. Peak fluorescence in T7 cycle is significantly higher (**p < 0.001; by

maximum and median for each sample recording) recorded over multiple days

(B).

Cell 175, 71–84, September 20, 2018 79

Figure 7. Manipulation of PHb Neuronal Function Alters Affective Behavior

(A) c-FosCreERT/+ mice injected with an AAV5/DIO-hM3D-mCherry in the PHb received a light pulse and 4-OH-Tam. Injection sites were evaluated for mCherry,

and c-Fos staining was used to confirm CNO-neuronal activation.

(B and C) Chronic activation of light-responsive PHb neurons leads to behavioral alterations, as shown in the TST (B) and FST (C) (n = 10–16).

(D) The SPT was evaluated in c-FosCreERT/+ and control-injected mice that received CNO twice a day (see STAR Methods). c-FosCreERT/+-injected mice displayed

a significant reduced preference for sucrose versus control group (n = 5–7).

(E) vmPFC-projecting PHb cells were chronically activated in mice that were bilaterally injected in the vmPFC with a retrogradely transported AAV/Cre (J and K)

and an AAV5/DIO-hM3D-mCherry in PHb.

(F and G) CNO treatment in DREADD-injected mice caused a significant increased in the immobility time in the TST (F) and FST (G) versus control mice (n = 12–15).

(H and I) CNO treatment in DREADD-injected mice had no effect on cognitive functions. For the NOR test (H), the statistical analysis versus 50% (dotted lines) was

Control, p < 0.05 and DREADD, p < 0.05 by one sample t test. Using the MWM test, we found no significant deficits during the test (I) trial (n = 12–14).

(J) AAV5/Cre-GFP was bilaterally injected into the PHb of mice that express tetanus toxin (tetX) in a Cre-dependent manner. GFP expression confirms injection

site that included the PHb and was largely restricted to immediately adjoining thalamic nuclei.

(K–M) Suppressing PHb synaptic output eliminated any statistical difference in the TST (K), FST (L), and SPT (M) between mice housed under the T24 or T7 cycles.

For the SPT, the statistical analysis compared to 50% (dotted lines, M) was: T24 p < 0.001 and T7 p < 0.001 by one sample t test (n = 14–15).

Data are mean ± SEM. *p< 0.05; ***p<0.001 by Student’s t test. Hb, habenular complex. Scale bars, 200 mm.

See also Figures S6 and S7.

same treatment and viral injections. Beginning 4 weeks post-

Tam injections, mice continuously received the designer ligand

clozapine-N-oxide (CNO) in their drinking water to selectively

and tonically activate neurons expressing the designer receptor

(hM3D) (Figures S6G–S6I). After 2 weeks of chronic CNO treat-

ment, virally injected c-FosCreERT mice displayed mood alter-

ations, spending significantly more time immobile in both the

TST and FST, compared with control mice (Figures 7B and

7C). The SPT was not evaluated in this case out of concern

that CNO treatment could affect water palatability. To avoid

issues with palatability, in a different set of experiments, AAV-

injected c-FosCreERT and Cre-negative mice received two

(intraperitoneal [i.p.]) injections of CNO (1 mg/kg) per day,

during 2 weeks. We found that this CNO treatment induced a sig-

nificant reduction in the sucrose preference index in c-FosCreERT-

injected mice, compared to the control group (Figures 7D

and S6J).

80 Cell 175, 71–84, September 20, 2018

To determine whether the PHb-vmPFC circuit is sufficient for

driving mood alterations, we performed a long-lasting activation

of vmPFC-projecting PHb neurons. WT mice were injected with a

retrograde transported AAV/Cre into the vmPFC, and a Cre-

dependent AAV encoding excitatory(Gq)-mCherry was injected

in the PHb (Figure 7E). As a result, mCherry(+) neurons were

restricted exclusively to the PHb (Figures 7E and S7A). Control

mice received the same treatment, except that an AAV1/GFP

was injected in the vmPFC. Four weeks after injections, mice

kept in T24 cycle (Figure S7B) received CNO in their drinking

water for 2 weeks, as described above. Chronic activation of

vmPFC-projecting PHb neurons induced significant alterations

in mood, as evaluated using the TST and the FST (Figures 7F

and 7G).

We then investigated whether the chronic activation of

DREADD-controlled PHb neurons could also cause cognitive

deficits. Using the same protocol of chronic activation of

mPFC-projecting PHb neurons, we found that the CNO treat-

ment has no significant effect on learning and cognitive functions

(Figures 7H and 7I). In the NOR test, both control and DREADD-

controlled mice spent significantly more time exploring the novel

object (Figures 7H and S7C). During the MWM, both group of

mice displayed similar responses during the training and test

phases (Figures 7I and S7D). In sum, these results suggest that

the PHb is specific for driving the effects of light on mood.

PHb Neurons Are Necessary for the Effects of Light on Mood We next evaluated whether the PHb is necessary for the mood

alterations induced by the unnatural light cycle by inhibiting syn-

aptic release specifically from PHb neurons. Tetanus toxin light

chain subunit (tetX) was expressed in PHb neurons by injecting

an AAV/Cre-GFP in mice that express tetX in a Cre-dependent

manner (R26tetX-Z/EG) (Zhang et al., 2008) (Figure 7J). Injection

sites were confirmed post hoc by assessing GFP expression

(Figures 7J and S7E). Four weeks after AAV injections, mice

were housed in either T24 or T7 cycles for 2 weeks (Figure S7F).

Remarkably, expressing tetX in PHb neurons prevented the T7

cycle-induced mood alterations as tetX-AAV-injected mice

housed under the T7 cycle were statistically indistinguishable

from those housed under the T24 cycle on the TST, FST, and

SPT (Figures 7K–7M and S7G). These results demonstrate that

inhibiting PHb neurons blocks the mood alterations triggered

by abnormal light cycles.

Next, we explore whether the PHb is specific in mediating

the effects of light on mood or whether it is also activated by

non-light-dependent treatments that induce mood changes.

For that, WT mice were exposed to three different paradigms

known to cause mood changes in rodents (see STAR Methods),

and the induction of c-Fos was evaluated in PHb neurons, as well

as in other brain regions known to be involved in mood control

(Choi et al., 2013; Huang et al., 2004; Matsuda et al., 1996).

For all the paradigms used, a significant increase in the number

of c-Fos(+) cells were found in the LHb and prefrontal cortex,

compared with control mice (Figures S7H and S7I), confirming

the effectiveness of the paradigms used. However, these same

stimuli did not cause a significant induction of c-Fos in PHb neu-

rons. This is in distinct contrast to the robust c-Fos induction

mediated by light (Figure S7J). Therefore, the PHb nucleus is

part of a circuitry that specifically routes photic stimuli to influ-

ence mood.

DISCUSSION

We have previously uncovered that ipRGCs are the sole retinal

conduit responsible for signaling light information to modulate

mood and cognitive functions (LeGates et al., 2012). Here,

we reveal that (1) distinct ipRGC subpopulations drive the

effects of light on learning and mood, (2) Brn3b(�) SCN-projec- ting ipRGCs relay light information to influence cognitive func-

tions without disrupting the central pacemaker, and (3) the

thalamic PHb mediates the ipRGC-driven effects of light on

mood. Together, these results delineate two distinct retina-

brain pathways that mediate the effects of light on learning

and mood.

Light Routed through SCN-Projecting ipRGCs Affects Cognitive Functions The use of the Opn4Cre/+;Brn3bzDTA mouse line allowed us to

implicate the ipRGC-SCN pathway in driving the effects of light

on learning and memory, which is consistent with previous

reports showing that a functional SCN is required for hippocam-

pal learning (Fernandez et al., 2014; Ruby et al., 2008). This ani-

mal model, however, still retains minor ipRGC innervation to

other brain regions, including the intergeniculate leaflet (IGL).

This minor innervation is most likely arising from collaterals of

the �200 SCN-projecting ipRGCs (Fernandez et al., 2016) that remain in the Opn4Cre/+;Brn3bzDTA mice. It is possible that these

minor innervations to the non-SCN targets could somehow

contribute to the learning deficits observed in mice housed under

the T7 cycle. However, based on the previous literature (Fernan-

dez et al., 2014; Ruby et al., 2008) and the substantial innervation

to the SCN, the most parsimonious explanation is that the SCN is

the main driver of the effects of light on learning and memory.

Recent evidence showed that the SCN can act as a relay for

light input to peripheral oscillators, even in the absence of a func-

tional molecular clockwork machinery (Izumo et al., 2014). We

found that the SCN remains responsive to light under the T7

cycle, as observed with the robust phosphorylation of CREB,

solidifying the role of the SCN as a relay for light input. Addition-

ally, we observed that SCN neurons become refractory to light-

induced transcriptional changes, as suggested by the reduced

levels of the immediate-early genes, including c-Fos and the

light-responsive clock gene, PER1. Light-induction of c-Fos

and PER1 depends on two regulatory regions: CRE and SRE,

through pCREB and pELK-1 binding, respectively (Chawla,

2002; Coogan and Piggins, 2003). Our results show that under

the T7 cycle, the transcription factor ELK-1 is not phosphory-

lated in response to light, whereas the phosphorylation of

CREB was unaffected. This result suggests that CREB and

ELK-1 activation pathways are differentially affected by the

T7 cycle, consistent with published reports indicating that

different intracellular cascades cause phosphorylation of these

activators (Coogan and Piggins, 2003; Xia et al., 1996).

The Thalamic PHb Is a Relay for Light-Mediated Mood Control ipRGCs show widespread projections throughout the brain,

including areas controlling sleep and general activity, as well

as limbic regions (Hattar et al., 2006). A principal target of

ipRGCs revealed in this study is an area of the dorsal thalamus,

the PHb, which expresses a circadian clock and shows distinc-

tive connectivity. Our results indicate that the PHb constitutes a

distinct thalamic region, with a different pattern of projections

compared with the thalamic nuclei into which it has heretofore

been subsumed. Specifically, the CL is connected to premotor

frontocortical regions and to the dorsolateral striatum, (Voorn

et al., 2004), and the thalamic LP nucleus projects to a diverse

array of sensory and motor cortices (Noseda et al., 2010). We

revealed that PHb neurons send collateral projections to vmPFC,

dorsal, and ventral striatum. Prefrontal cortex is fundamental to

mood regulation and has been consistently implicated in major

depressive disorders (MDD) by imaging studies of patients (Frodl

et al., 2009; Meng et al., 2014; Shen et al., 2017) and using animal

Cell 175, 71–84, September 20, 2018 81

models of mood disorders (Shrestha et al., 2015). Another target

of PHb, the dorsomedial striatum, is integrated in a thalamo-

frontocortical loop, which may be involved in affective-emotional

processing. Reduced caudate and putamen volumes were

described in MDD patients (Kerestes et al., 2014). The third

target of PHb neurons is the ventral striatum, particularly the

NAc, which has been extensively implicated in mood control

and depression (Francis and Lobo, 2017). In sum, our results

provide evidence for a previously unrecognized thalamic

nucleus, the PHb, which is both necessary and sufficient for

relaying the effects of light on mood.

Perspective The retino-PHb pathway presented here can satisfactorily

account for mood changes wrought by light and represents,

therefore, a new general mechanism for mood regulation

(Lazzerini Ospri et al., 2017; LeGates et al., 2014). Irregular

light stimulation leads to mood alterations associated with

changes in the thalamic PHb, including increased activity of

PHb neurons, sustained light-mediated induction of the imme-

diate-early gene c-Fos, and a breakdown of clock gene rhyth-

micity. We speculate that in humans, chronic exposure to light

at night could cause similar neuronal changes leading to mood

deficits, highlighting the negative impact of irregular light

stimulation during the normal day/night cycle. This occurs

in a context of lamentable stagnation in the development

of effective treatments for mood disorders. While promising

drug candidates currently undergoing trials exist, the

sad truth is that no radically new class of antidepressants

has reached the clinic since the introduction of selective sero-

tonin reuptake inhibitors (SSRIs) in the 1980s (Hillhouse and

Porter, 2015).

Reduced cognitive functions have been extensively reported

in patients suffering from MDD (Hammar and Ardal, 2009;

Jaeger et al., 2006; Roh et al., 2016). Although a direct causal

link is missing, current theories suggest that neuroanatomical

changes in MDD patients may be the cause of cognitive deficits

(Davidson et al., 2002). Our results demonstrate that anatomi-

cally distinct neuronal circuits are involved in different light-

mediated behavioral deficits and suggest that learning and

mood deficits are affected independently. Understanding the

neuronal basis for mood and learning regulation by light consti-

tutes a promising step toward new treatments for neuropsychi-

atric disorders.

STAR+METHODS

Detailed methods are provided in the online version of this paper

and include the following:

d KEY RESOURCES TABLE

d CONTACT FOR REAGENT AND RESOURCE SHARING

d EXPERIMENTAL DESIGN AND SUBJECT DETAILS

82 C

B Animals

B Rhythmic activity measurement

B Chronic control of PHb function

B Behavioral test

B Light-independent paradigms of mood alterations

ell 175, 71–84, September 20, 2018

d METHOD DETAILS

B Light-mediated gene induction/ protein

phosphorylation

B PER1/2 rhythmic expression

B Immunofluorescence

B SCN dissection and RNA-sequencing

B Retinal injections

B Stereotaxic injections

B Electrophysiology

B Hippocampal recordings

B Fiber Photometry

d QUANTIFICATION AND STATISTICAL ANALYSIS

B Morphometric analysis

B Statistical Analysis

d DATA AND SOFTWARE AVAILABILITY

SUPPLEMENTAL INFORMATION

Supplemental Information includes seven figures and can be found with this

article online at https://doi.org/10.1016/j.cell.2018.08.004.

ACKNOWLEDGMENTS

We would like to thank the Johns Hopkins Biology Mouse Tri-Lab—especially

Dr. Rejji Kuruvilla—for support and discussion; the Department of Biology at

the University of Maryland and Dianne and Kimberly Boghossian for assis-

tance maintaining and genotyping mice; Dr. Antonello Bonci, Dr. Yeka Aponte,

and their lab members for help addressing reviewers’ concerns; and Elaine

Nguyen for help with tracer injections. We would like to thank Mio Akasako

for her key assistance with the retrolabeling experiments. This work was sup-

ported by the NIH (GM076430, EY027202, EY012793, and EY017137), the

generous contributions of the Alcon Research Institute (to D.M.B.), PEW Char-

itable Trusts (to D.C.F.), the NSF (I2011104359 to P.M.F.), and the intramural

research at the National Institute of Mental Health.

AUTHOR CONTRIBUTIONS

D.C.F. performed behavioral tests, circadian analysis, and immunohistochem-

ical analysis of gene expression. P.M.F. and L.L.O. carried out tracing exper-

iments. D.C.F. and M.B.T. did RNA-seq analysis. L.L.O. and J.Z. performed

fiber photometry recording. D.C.F., R.M.L., D.S., J.H.S., and A.K. performed

electrophysiological experiments. D.C.F., P.M.F., L.L.O., D.M.B., H.Z., and

S.H. wrote the paper.

DECLARATION OF INTERESTS

The authors declare no competing interests.

Received: December 12, 2017

Revised: May 3, 2018

Accepted: August 2, 2018

Published: August 30, 2018

REFERENCES

Altimus, C.M., Güler, A.D., Villa, K.L., McNeill, D.S., Legates, T.A., and Hattar,

S. (2008). Rods-cones and melanopsin detect light and dark to modulate sleep

independent of image formation. Proc. Natl. Acad. Sci. USA 105, 19998–

20003.

Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq-A Python framework to

work with high-throughput sequencing data. Bioinformatics 31, 166–169.

Bedrosian, T.A., Fonken, L.K., Walton, J.C., Haim, A., and Nelson, R.J. (2011).

Dim light at night provokes depression-like behaviors and reduces CA1

dendritic spine density in female hamsters. Psychoneuroendocrinology 36,

1062–1069.

Berson, D.M., Dunn, F.A., and Takao, M. (2002). Phototransduction by retinal

ganglion cells that set the circadian clock. Science 295, 1070–1073.

Can, A., Dao, D.T., Terrillion, C.E., Piantadosi, S.C., Bhat, S., and Gould, T.D.

(2012). The tail suspension test. J. Vis. Exp. (59), 3769.

Charan, J., and Kantharia, N.D. (2013). How to calculate sample size in animal

studies? J. Pharmacol. Pharmacother. 4, 303–306.

Chawla, S. (2002). Regulation of gene expression by Ca2+ signals in neuronal

cells. Eur. J. Pharmacol. 447, 131–140.

Chen, S.-K., Badea, T.C., and Hattar, S. (2011). Photoentrainment and pupil-

lary light reflex are mediated by distinct populations of ipRGCs. Nature

476, 92–95.

Choi, S.H., Chung, S., Cho, J.H., Cho, Y.H., Kim, J.W., Kim, J.M., Kim, H.J.,

Kim, H.J., and Shin, K.H. (2013). Changes in c-Fos expression in the forced

swimming test: common and distinct modulation in rat brain by desipramine

and citalopram. Korean J. Physiol. Pharmacol. 17, 321–329.

Coogan, A.N., and Piggins, H.D. (2003). Circadian and photic regulation of

phosphorylation of ERK1/2 and Elk-1 in the suprachiasmatic nuclei of the Syr-

ian hamster. J. Neurosci. 23, 3085–3093.

Davidson, R.J., Pizzagalli, D., Nitschke, J.B., and Putnam, K. (2002). Depres-

sion: perspectives from affective neuroscience. Annu. Rev. Psychol. 53,

545–574.

Dobin, A., Davis, C.A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut,

P., Chaisson, M., and Gingeras, T.R. (2013). STAR: Ultrafast universal RNA-

seq aligner. Bioinformatics 29, 15–21.

Ecker, J.L., Dumitrescu, O.N., Wong, K.Y., Alam, N.M., Chen, S.-K., LeGates,

T., Renna, J.M., Prusky, G.T., Berson, D.M., and Hattar, S. (2010). Melanopsin-

expressing retinal ganglion-cell photoreceptors: cellular diversity and role in

pattern vision. Neuron 67, 49–60.

Faul, F., Erdfelder, E., Buchner, A., and Lang, A.-G. (2009). Statistical power

analyses using G*Power 3.1: tests for correlation and regression analyses.

Behav. Res. Methods 41, 1149–1160.

Fernandez, F., Lu, D., Ha, P., Costacurta, P., Chavez, R., Heller, H.C., and

Ruby, N.F. (2014). Dysrhythmia in the suprachiasmatic nucleus inhibits mem-

ory processing. Science 346, 854–857.

Fernandez, D.C., Chang, Y.-T., Hattar, S., and Chen, S.-K. (2016). Architecture

of retinal projections to the central circadian pacemaker. Proc. Natl. Acad. Sci.

USA 113, 6047–6052.

Francis, T.C., and Lobo, M.K. (2017). Emerging role for nucleus accumbens

medium spiny neuron subtypes in depression. Biol. Psychiatry 81, 645–653.

Franklin, K., and Paxinos, G. (2012). Paxinos and Franklin’s the Mouse Brain in

Stereotaxic Coordinates, Fourth Edition (Academic Press).

Frodl, T., Scheuerecker, J., Albrecht, J., Kleemann, A.M., Müller-Schunk, S.,

Koutsouleris, N., Möller, H.-J., Brückmann, H., Wiesmann, M., and Meisen-

zahl, E. (2009). Neuronal correlates of emotional processing in patients with

major depression. World J. Biol. Psychiatry 10, 202–208.

Güler, A.D., Ecker, J.L., Lall, G.S., Haq, S., Altimus, C.M., Liao, H.-W., Barnard,

A.R., Cahill, H., Badea, T.C., Zhao, H., et al. (2008). Melanopsin cells are the

principal conduits for rod-cone input to non-image-forming vision. Nature

453, 102–105.

Hammar, A., and Ardal, G. (2009). Cognitive functioning in major depression–a

summary. Front. Hum. Neurosci. 3, 26.

Hattar, S., Liao, H.W., Takao, M., Berson, D.M., and Yau, K.W. (2002). Mela-

nopsin-containing retinal ganglion cells: architecture, projections, and intrinsic

photosensitivity. Science 295, 1065–1070.

Hattar, S., Lucas, R.J., Mrosovsky, N., Thompson, S., Douglas, R.H., Hankins,

M.W., Lem, J., Biel, M., Hofmann, F., Foster, R.G., and Yau, K.W. (2003). Mel-

anopsin and rod-cone photoreceptive systems account for all major acces-

sory visual functions in mice. Nature 424, 76–81.

Hattar, S., Kumar, M., Park, A., Tong, P., Tung, J., Yau, K.-W., and Berson,

D.M. (2006). Central projections of melanopsin-expressing retinal ganglion

cells in the mouse. J. Comp. Neurol. 497, 326–349.

Hillhouse, T.M., and Porter, J.H. (2015). A brief history of the development of

antidepressant drugs: from monoamines to glutamate. Exp. Clin. Psychophar-

macol. 23, 1–21.

Huang, Y.-H., Cheng, C.-Y., Hong, C.-J., and Tsai, S.-J. (2004). Expression of

c-Fos-like immunoreactivity in the brain of mice with learned helplessness.

Neurosci. Lett. 363, 280–283.

Huang, W., Sherman, B.T., and Lempicki, R.A. (2009). Systematic and integra-

tive analysis of large gene lists using DAVID bioinformatics resources. Nat.

Protoc. 4, 44–57.

Izumo, M., Pejchal, M., Schook, A.C., Lange, R.P., Walisser, J.A., Sato, T.R.,

Wang, X., Bradfield, C.A., and Takahashi, J.S. (2014). Differential effects of

light and feeding on circadian organization of peripheral clocks in a forebrain

Bmal1 mutant. eLife 3. https://doi.org/10.7554/eLife.04617.

Jaeger, J., Berns, S., Uzelac, S., and Davis-Conway, S. (2006). Neurocognitive

deficits and disability in major depressive disorder. Psychiatry Res. 145,

39–48.

Kako, K., and Ishida, N. (1998). The role of transcription factors in circadian

gene expression. Neurosci. Res. 31, 257–264.

Kerestes, R., Harrison, B.J., Dandash, O., Stephanou, K., Whittle, S., Pujol, J.,

and Davey, C.G. (2014). Specific functional connectivity alterations of the dor-

sal striatum in young people with depression. Neuroimage Clin. 7, 266–272.

Kim, D., Song, I., Keum, S., Lee, T., Jeong, M.J., Kim, S.S., McEnery, M.W.,

and Shin, H.S. (2001). Lack of the burst firing of thalamocortical relay neurons

and resistance to absence seizures in mice lacking alpha(1G) T-type Ca(2+)

channels. Neuron 31, 35–45.

Kim, Y., Perova, Z., Mirrione, M.M., Pradhan, K., Henn, F.A., Shea, S., Osten,

P., and Li, B. (2016). Whole-brain mapping of neuronal activity in the learned

helplessness model of depression. Front. Neural Circuits 10, 3.

Kurlansik, S.L., and Ibay, A.D. (2012). Seasonal affective disorder. Am. Fam.

Physician 86, 1037–1041.

Lazzerini Ospri, L., Prusky, G., and Hattar, S. (2017). Mood, the circadian sys-

tem, and melanopsin retinal ganglion cells. Annu. Rev. Neurosci. 40, 539–556.

LeGates, T.A., Altimus, C.M., Wang, H., Lee, H.-K., Yang, S., Zhao, H., Kirk-

wood, A., Weber, E.T., and Hattar, S. (2012). Aberrant light directly impairs

mood and learning through melanopsin-expressing neurons. Nature 491,

594–598.

LeGates, T.A., Fernandez, D.C., and Hattar, S. (2014). Light as a central modu-

lator of circadian rhythms, sleep and affect. Nat. Rev. Neurosci. 15, 443–454.

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold

change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.

Matsuda, S., Peng, H., Yoshimura, H., Wen, T.C., Fukuda, T., and Sakanaka,

M. (1996). Persistent c-fos expression in the brains of mice with chronic social

stress. Neurosci. Res. 26, 157–170.

Melrose, S. (2015). Seasonal affective disorder: an overview of assessment

and treatment approaches. Depress. Res. Treat. 2015, 178564.

Meng, C., Brandl, F., Tahmasian, M., Shao, J., Manoliu, A., Scherr, M.,

Schwerthöffer, D., Bäuml, J., Förstl, H., Zimmer, C., et al. (2014). Aberrant

topology of striatum’s connectivity is associated with the number of episodes

in depression. Brain 137, 598–609.

Morin, L.P. (2013). Neuroanatomy of the extended circadian rhythm system.

Exp. Neurol. 243, 4–20.

Morin, L.P., and Studholme, K.M. (2014). Retinofugal projections in the mouse.

J. Comp. Neurol. 522, 3733–3753.

Nagalski, A., Puelles, L., Dabrowski, M., Wegierski, T., Kuznicki, J., and Wis-

niewska, M.B. (2016). Molecular anatomy of the thalamic complex and the un-

derlying transcription factors. Brain Struct. Funct. 221, 2493–2510.

Noseda, R., Kainz, V., Jakubowski, M., Gooley, J.J., Saper, C.B., Digre, K., and

Burstein, R. (2010). A neural mechanism for exacerbation of headache by light.

Nat. Neurosci. 13, 239–245.

Cell 175, 71–84, September 20, 2018 83

Provencio, I., Rodriguez, I.R., Jiang, G., Hayes, W.P., Moreira, E.F., and Rollag,

M.D. (2000). A novel human opsin in the inner retina. J. Neurosci. 20, 600–605.

Quina, L.A., Tempest, L., Ng, L., Harris, J.A., Ferguson, S., Jhou, T.C., and

Turner, E.E. (2015). Efferent pathways of the mouse lateral habenula.

J. Comp. Neurol. 523, 32–60.

Roh, S.-C., Park, E.-J., Shim, M., and Lee, S.-H. (2016). EEG beta and low

gamma power correlates with inattention in patients with major depressive dis-

order. J. Affect. Disord. 204, 124–130.

Roth, B.L. (2016). DREADDs for neuroscientists. Neuron 89, 683–694.

Ruby, N.F., Hwang, C.E., Wessells, C., Fernandez, F., Zhang, P., Sapolsky, R.,

and Heller, H.C. (2008). Hippocampal-dependent learning requires a functional

circadian system. Proc. Natl. Acad. Sci. USA 105, 15593–15598.

Russo, S.J., and Nestler, E.J. (2013). The brain reward circuitry in mood disor-

ders. Nat. Rev. Neurosci. 14, 609–625.

Schmidt, T.M., Chen, S.-K., and Hattar, S. (2011). Intrinsically photosensitive

retinal ganglion cells: many subtypes, diverse functions. Trends Neurosci.

34, 572–580.

Schwarz, L.A., Miyamichi, K., Gao, X.J., Beier, K.T., Weissbourd, B., DeLoach,

K.E., Ren, J., Ibanes, S., Malenka, R.C., Kremer, E.J., and Luo, L. (2015). Viral-

genetic tracing of the input-output organization of a central noradrenaline

circuit. Nature 524, 88–92.

Scott, A.J., Monk, T.H., and Brink, L.L. (1997). Shiftwork as a risk factor for

depression: a pilot study. Int. J. Occup. Environ. Health 3 (Suppl 2 ), S2–S9.

Shen, X., Reus, L.M., Cox, S.R., Adams, M.J., Liewald, D.C., Bastin, M.E.,

Smith, D.J., Deary, I.J., Whalley, H.C., and McIntosh, A.M. (2017). Subcortical

volume and white matter integrity abnormalities in major depressive disorder:

findings from UK Biobank imaging data. Sci. Rep. 7, 5547.

Sherman, S.M. (2001). Tonic and burst firing: dual modes of thalamocortical

relay. Trends Neurosci. 24, 122–126.

Shrestha, P., Mousa, A., and Heintz, N. (2015). Layer 2/3 pyramidal cells in the

medial prefrontal cortex moderate stress induced depressive behaviors. eLife

4. https://doi.org/10.7554/eLife.08752.

84 Cell 175, 71–84, September 20, 2018

Travnickova-Bendova, Z., Cermakian, N., Reppert, S.M., and Sassone-Corsi,

P. (2002). Bimodal regulation of mPeriod promoters by CREB-dependent

signaling and CLOCK/BMAL1 activity. Proc. Natl. Acad. Sci. USA 99, 7728–

7733.

Vandewalle, G., Schwartz, S., Grandjean, D., Wuillaume, C., Balteau, E.,

Degueldre, C., Schabus, M., Phillips, C., Luxen, A., Dijk, D.J., and Maquet,

P. (2010). Spectral quality of light modulates emotional brain responses in

humans. Proc. Natl. Acad. Sci. USA 107, 19549–19554.

Venzala, E., Garcı́a-Garcı́a, A.L., Elizalde, N., Delagrange, P., and Tordera,

R.M. (2012). Chronic social defeat stress model: behavioral features, antide-

pressant action, and interaction with biological risk factors. Psychopharmacol-

ogy (Berl.) 224, 313–325.

Vertes, R.P., Linley, S.B., and Hoover, W.B. (2015). Limbic circuitry of the

midline thalamus. Neurosci. Biobehav. Rev. 54, 89–107.

Voorn, P., Vanderschuren, L.J.M.J., Groenewegen, H.J., Robbins, T.W., and

Pennartz, C.M.A. (2004). Putting a spin on the dorsal-ventral divide of the stria-

tum. Trends Neurosci. 27, 468–474.

Wagner, F., Weiss, T., and Veh, R.W. (2017). Electrophysiological properties of

neurons and synapses in the lateral habenular complex (LHb). Pharmacol. Bio-

chem. Behav. 162, 38–45.

Wässle, H. (2004). Parallel processing in the mammalian retina. Nat. Rev. Neu-

rosci. 5, 747–757.

Wilsbacher, L.D., Yamazaki, S., Herzog, E.D., Song, E.-J., Radcliffe, L.A., Abe,

M., Block, G., Spitznagel, E., Menaker, M., and Takahashi, J.S. (2002). Photic

and circadian expression of luciferase in mPeriod1-luc transgenic mice invivo.

Proc. Natl. Acad. Sci. USA 99, 489–494.

Xia, Z., Dudek, H., Miranti, C.K., and Greenberg, M.E. (1996). Calcium influx via

the NMDA receptor induces immediate early gene transcription by a MAP

kinase/ERK-dependent mechanism. J. Neurosci. 16, 5425–5436.

Zhang, Y., Narayan, S., Geiman, E., Lanuza, G.M., Velasquez, T., Shanks, B.,

Akay, T., Dyck, J., Pearson, K., Gosgnach, S., et al. (2008). V3 spinal neurons

establish a robust and balanced locomotor rhythm during walking. Neuron

60, 84–96.

STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies

anti-Brn3a Millipore Cat #MAB1585; RRID: AB_94166

anti-cFos Calbiochem Cat #Ab-5; RRID: AB_2106755

anti-cFos EnCor Cat #MCA-2H2; RRID: AB_2571561

anti-GFP Abcam Cat #Ab13970; RRID: AB_300798

anti-GRID2IP Bioss Cat #bs-11347R

anti-Melanopsin Advanced Targeting Cat #AB-N38; RRID: AB_1608077

Anti-NPY Peninsula Lab Cat #T-4070; RRID: AB_518504

anti-pCREB-ser133 Cell signaling Cat #9198; RRID: AB_2561044

anti-pELK-1-Ser383 Cell Signaling Cat #9181; RRID: AB_2099016

anti-PER1 A generous gift from Dr. David Weaver N/A

anti-PER2 Alpha Diagnostic Cat #PER21-A; RRID: AB_1875482

anti-pH3-Ser10 Cell signaling Cat #D2C8; RRID: AB_10694226

anti-PKC delta Abcam Cat #ab182126

anti-RFP MBL Cat #PM005; RRID: AB_591279

anti-Vglut2 Millipore Cat #AB2251; RRID: AB_2665454

Bacterial and Virus Strains

AAV1/hSyn-HI-eGFP-Cre-WPRE.SV40 Penn Vector Core Cat #AV-1-PV1848

AAV1/CAG-Flex-GCaMP6f-WPRE.SV40 University of Pennsylvania viral core Cat #AV-1-PV2816

AAV1/hSyn-HI-eGFP-Cre-WPRE.SV40 Penn Vector Core N/A

AAV1/Syn-GCaMP6m-WPRE.SV40 University of Pennsylvania viral core Cat #AV-1-PV2823

AAV2/synP-DIO-sTpEpB.WPRE.bGH Penn Vector Core N/A

AAV5/CAG-Flex-GCaMP6f.WPRE.SV40 University of Pennsylvania viral core Cat #AV-5-PV2816

AAV8.2/EF1a-DIO-synaptophysin-tdTomato McGovern Institute Viral core Cat #AAV1

AAVDJ/EF1a-DIO-GCaMP6f Stanford University viral core Cat #GVVC-AAV-093

CAV2/cre Institut de Génétique Moléculaire

de Montpellier CNRS UMR

N/A

EnvA G-deleted Rabies-mCherry Salk Vector Core N/A

HSV/cre McGovern Institute Viral core Cat #RN425

rAAV2/hSyn-hChR2(H134R)-YFP-WPREpA UNC Vector Core N/A

rAAV5/hSyn-DIO-hm3D-mCherry UNC Vector Core N/A

rAAV5/Syn-Cre-GFP UNC Vector Core N/A

Chemicals, Peptides, and Recombinant Proteins

3,30-diaminobenzidine Sigma Cat #D8001

4OH-tamoxifen Sigma Cat #H7904

AntiFade medium Molecular Probes Cat #P36980

Avertin (2, 2, 2-Tribromoethanol Sigma Cat #T48402

Biocytin Sigma Cat #B4261

Cholera toxin b-subunit (CTb) fluorescently conjugated ThermoFisher Cat #C22842; C22841

Clozapine-N-oxide Sigma Cat #C0832

Normal Goat Serum Vector Labs Cat #S-1000

(Continued on next page)

Cell 175, 71–84.e1–e7, September 20, 2018 e1

Continued

REAGENT or RESOURCE SOURCE IDENTIFIER

Paraformaldehyde Electron Microscopy Sciences N/A

Tamoxifen Sigma Cat #T5648

Critical Commercial Assays

Ribo-Zero Gold rRNA Removal Kit Illumina Cat #MRZG126

RNeasy Lipid Tissue Mini Kit QIAGEN Cat #74804

TruSeq RNA Library Prep Kit v2 Illumina Cat #RS-122-2001

Vectastain HRP kit Vector Labs Cat #PK-6101

Deposited Data

Raw and analyzed (SCN) RNA-seq data This paper GSE113875 N/A

Mus musculus reference genome (mm10) UCSC Genome Browser

https://genome.ucsc.edu/cgi-bin/

hgGateway?db=mm10

N/A

Experimental Models: Organisms/Strains

Mouse: C57BL/6J The Jackson laboratory Cat #000664

Mouse: CD-1 Charles River labs Cat #022

Mouse: cFosCreERT2 The Jackson laboratory Cat #021882

Mouse: Opn4aDTA/aDTA Generated in the laboratory of Dr. Hattar N/A

Mouse: Opn4Cre/+;Brn3bzDTA/+ Generated in the laboratory of Dr. Hattar N/A

Mouse: Opn4CreERT2 Generated in the laboratory of Dr. Hattar N/A

Mouse: ROSASynaptophysin-tdTomato The Jackson laboratory Cat #012570

Software and Algorithms

Adobe Photoshop Adobe Systems N/A

ANY-maze Behavioral tracking software Stoelting Co. N/A

ClockLab Actimetrics, IL N/A

Database for Annotation, Visualization and Integrated

Discovery (DAVID)

Huang et al., 2009 https://david.ncifcrf.gov N/A

DESeq2 Love et al., 2014 https://bioconductor.org/

packages/release/bioc/html/DESeq2.html

N/A

G*Power Faul et al., 2009 Version 3.1 N/A

GRAPHIC STATE Software for Behavioral Research Coulbourn Version 2.101 N/A

GraphPad Prism 7.04 GraphPad software N/A

HTSeq-count Anders et al., 2015 https://github.com/

simon-anders/htseq

N/A

IgorPro Wavemetrics N/A

ImageJ software NIH, USA N/A

MAtlab Mathworks N/A

Origin OriginLab N/A

STAR v2.4 Dobin et al., 2013 https://github.com/

alexdobin/STAR

N/A

VitalView software Mini Mitter, OR N/A

Zeiss Zen software Zeiss N/A

Other

23-Watt compact fluorescent light bulbs GE Daylight Cat #FLE10HT3/2/D

Coronal slice brain matrix Kent Scientific Cat #RBMA-200C

Coulbourn precision animal shocker Coulbourn Cat #H13-17A

Hamilton syringe Hamilton Cat #701 LT SYR

Light meter EXTECH Cat #401025

Microcapillary tube Sigma Cat #P0674

e2 Cell 175, 71–84.e1–e7, September 20, 2018

CONTACT FOR REAGENT AND RESOURCE SHARING

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Samer

Hattar ([email protected]).

EXPERIMENTAL DESIGN AND SUBJECT DETAILS

Animals Mice were housed under a 12 h light:12 h dark (T24) cycle at a temperature of 22�C with food and water ad libitum. Wild-type mice of a mixed background (B6/129 F1 hybrid; Jackson Laboratory) were used in this study. Opn4Cre/+;Brn3bzDTA/+ mouse line was previously

described (Chen et al., 2011). Briefly, we have shown that �200 M1 ipRGCs, which are the only ipRGCs that do not express the transcription factor Brn3b (POU4F2) (Schmidt et al., 2011), exclusively innervate the SCN (Chen et al., 2011). Mice that express

the Cre-dependent diphtheria toxin (DTA) under the control of the Brn3b promoter (Brn3bzDTA/+), were mated with Opn4Cre/Cre

mice to obtain Opn4Cre/+;Brn3bzDTA/+ mice, where Brn3b-negative cells are the only ipRGCs to survive. Opn4aDTA/aDTA mouse line

was previously described (Güler et al., 2008). ROSASynaptophysin-tdTomato mice (Jackson laboratory, Stock No: 012570) were mated

with Opn4CreERT2 mice (Ecker et al., 2010) to generate a mouse line (Opn4CreERT2/+;ROSASynaptophysin-tdTomato) in which most ipRGCs

expresses the fused protein (induced by high doses of Tam) in axonal terminals. Mice that express tetX in a Cre-dependent manner

(R26tetX) were previously described (Zhang et al., 2008). All animals were handled in accordance with guidelines of the Animal

Care and Use Committees of Johns Hopkins University, University of Maryland, Brown University, and the National Institute of

Mental Health (NIMH). All efforts were made to minimize the pain and the number of animals used. Calculation of sample size per

experiment was determined, or confirm by post hoc analyses, using a G*Power 3 software (Charan and Kantharia, 2013; Faul

et al., 2009).

Rhythmic activity measurement Single-housed mice were exposed to the T24 and/or T7 cycles, and light intensity was kept �500 lux (provided by Philips Daylight deluxe fluorescent lamps), measured using a light meter (EXTECH Foot Candle/Lux Light Meter, 401025). The general activity of mice

was monitored using infrared motion detectors from Mini Mitter (Respironics) mounted to the top of the cages. Data was collected

in 10-min bins using VitalView software (Mini Mitter, OR). The total activity and period lengths were calculated using ClockLab

(Actimetrics, IL).

Chronic control of PHb function Silencing of PHb neurons

Mice that express tetX/GFP in a Cre-dependent manner, and littermate controls, were stereotaxically injected with an AAV5/Cre in

the PHb region (bilateral injections). Four weeks post injection, mice were exposed to T24 or the T7 cycles.

Chronic DREADD strategy

c-FosCreERT2 and littermate Cre-negative (Control) mice were stereotaxically injected with a Cre-dependent AAV5/DIO-hM3D-

mCherry in the PHb region (bilateral injections). A week later, mice were subjected to a light pulse (15-min, 1000 lux, CT14), and imme-

diately injected with 4OH-tamoxifen (50 mg/kg i.p., H7904 Sigma) to induce Cre-recombination. After the light pulse, mice were in

darkness for 24 hours, and then kept under T24 cycle for 4 weeks, before starting with the experiments. c-FosCreERT2 mice injected

with the AAV that received 4OH-Tam, but without the light pulse exposure, were used as control of non-light-mediated c-Fos

induction.

CNO in drinking water

The effectiveness of clozapine-N-oxide (CNO, C0832 Sigma) dissolved in drinking water (5 mg/ml) was compared to CNO i.p. injec-

tions. For that, c-Fos induction (in AAV-infected neurons) was immunohistochemically evaluated in mice that were perfused either

12 hours after been exposed (and having access) to CNO dissolved in drinking water, or 2 hours after a single i.p. injection of

CNO (1mg/kg); in both cases, mice were kept in constant darkness during the experiment. To achieve chronic DREADD control,

water + CNO was given to mice during 14 days, and fresh solution was daily replaced (10 mL per day). The consumption of water +

CNO was daily measured.

CNO i.p. injections

In a different set of experiments, 4 weeks after light pulse + 4OH-Tam administration, CNO was intraperitoneally (i.p.) injected (1mg/kg)

twice a day (CT 13, and CT 18), during 14 days. To minimize the stress effect, tuberculin syringes were used.

Behavioral test Single-housed mice were kept under the T24 or T7 cycles for 2 weeks before testing, as previously described (LeGates et al., 2012).

The general activity of mice was monitored using infrared sensors and behavioral tests were performed between CT 12 - 14 (during

active phase). In all cases, room was illuminated using dim red lamps.

Cell 175, 71–84.e1–e7, September 20, 2018 e3

Mood-related behavior

Opn4Cre/+ and Opn4Cre/+;Brn3bzDTA/+ mice were tested for either the sucrose preference, TST or the FST. TetX mice (and respective

controls) were first evaluated for sucrose preference followed by either TST or FST. Mice with chronic activation of PHb neurons (AAV

DREADD and their respective controls) that received CNO in drinking water were exposed to either the TST or FST.

Sucrose Preference

Mice were acclimated to the 2-bottle water delivery system for 3 days. Sucrose preference was then assessed over two consecutive

days. Each day, one bottle containing 1% sucrose and one bottle containing water was introduced at the beginning of the active

phase of mice. Sucrose preference was calculated by dividing the amount of sucrose consumed by the total volume consumed

(water + sucrose).

Tail suspension

Mice were suspended by the tail using tape. White chambers prevented interaction between them. A maximum of six animals were

tested simultaneously. Mouse activity was recorded during 6 min. At the end of the session, mice were placed back in their home

cages. Immobility time was analyzed manually following the criteria described in Can et al. (2012).

Forced swim test

Mice were placed in an inescapable container of water (25�C) for 6 min. Behavioral response was monitored using a video camera positioned in front of the apparatus and scored using a computerized video tracking system (ANYmaze). Time spent immobile for the

last 4 min of the test was calculated.

Cognitive function tests

mice were tested for both the NOR test and the MWM. All behavioral tests were performed in a blind manner by a single observer.

Novel Object recognition test

On day 1, mice were placed in an open arena (40 cm x 30 cm) and were allowed to explore during 10 minutes before being returned to

their home cage. On day 2, mice were placed in the same open arena with two identical objects. After 10 minutes, mice were returned

to their home cage. After an hour, mice were placed back in the arena for 5 minutes with the familiar object and a novel object. Mice

were monitored and tracked for object exploration and the time was calculated for the percentage of time spent with the novel versus

the familiar object (ANYmaze software). At the end of the experiment, mice were placed back in their home cages and the arena and

objects were sanitized with 70% v/v ethanol. The objects used in this test had been previously validated using an independent cohort

of wild-type mice, which did not display significant preference for any of them.

Morris Water Maze

Mice were placed in a circular pool (150 cm diameter) to swim to a hidden platform (submerged in the pool such that 1cm of water

covered the platform hiding it from sight). Four different (in shape and color) cues were attached to the side of the pool equidistant

from one another, and the pool was surrounded by a plain curtain to block any other visual cues. Water was mixed with non-toxic

white paint to render it opaque. Water temperature was maintained between 20-25�C during experiments by water heaters designed for fish aquariums. On day 0, mice were trained to escape the pool using a visual cue located on top of the platform to familiarize them

with the test. Once the mouse either finds the island or swims for 90 s without finding the island, it was removed from the pool and

placed back in its cage. This was performed four times with an inter-trial interval of 30 min, and the platform was moved between each

trial. During the acquisition phase, mice were trained (one trial per day for 12 days) to find the hidden platform using the four visual

distal cues surrounding the pool. The mouse was randomly placed in a different area of the pool at the start of each trial with the

platform maintained in the same quadrant (target quadrant). Mice were allowed to swim for a maximum of 90 s and supervised

for the full time in the pool. The platform was removed on day 13 in the probe trial. The swimming in each quadrant (T: target, A1:

adjacent 1, A2: adjacent 2, O: opposite, quadrants) and specifically the preference for the target quadrant was measured to evaluate

spatial memory using a computerized video tracking system (ANYmaze). Reversal training began on day 14 when the platform was

moved to the quadrant opposite the original target quadrant. Mice were trained as described for the acquisition phase. During

acquisition phase and reversal training, the latency to find the platform was measured; probe trial was analyzed by calculating the

percentage time spent in the target quadrant.

Light-independent paradigms of mood alterations WT mice (3 months old) individually housed in T24 light cycle were exposed to a learned helplessness, forced-swim, or social defeat

paradigms at CT 14 (two hours after the beginning of active phase). During the sessions mice were kept in darkness to avoid any light-

mediated effect on c-Fos induction. Control mice were perfused at similar CTs.

Learned helplessness paradigm

Mice were exposed to sessions of inescapable foot shocks on two consecutive days, as previously described (Huang et al., 2004;

Kim et al., 2016), with minor modifications. Each session consisted of a total of 300 randomized electric foot shocks (300 mA intensity,

1-3 s duration, 5-15 s inter-shock intervals; Coulbourn precision animal shocker) applied during a period of �60 min. After the second session, mice were kept in darkness for 30 min, and perfused for histochemical analysis.

Forced-swim paradigm

Mice were exposed to two sessions of inescapable swimming on two consecutive days, following Choi et al. (2013), with minor

modifications. On day 1, mice were individually placed in a container of water (25�C) for 15 min; after that, mice were returned to their home cages. On day 2, mice were placed in the same pool for 5 min, then kept in darkness for 80 min, and finally perfused.

e4 Cell 175, 71–84.e1–e7, September 20, 2018

Social defeat paradigm

Cages with a plastic mesh dividing the arena into two similar areas were used. The protocol used was following Matsuda et al. (1996)

and Venzala et al. (2012), with minor modifications. On day 1, an intruder C57B6/J mouse was placed into the compartment housing a

resident CD1 mouse, allowing physical interaction for 5 minutes. After that, intruder mouse was placed on the next compartment of

the same cage, allowing sensory contact for 24 hours. This procedure was repeated during 4 consecutive days. On day 4, intruder

mice were exposed to physical interaction, returned to their home cages and perfused 80 min later.

METHOD DETAILS

Light-mediated gene induction/ protein phosphorylation T24 and T7 cycles-housed mice (for 2 weeks) were kept in darkness for 1 day to avoid any prior light exposure effect. Two hours after

the onset of activity (CT 14), mice were subjected to a 15-min light pulse (1000 lux), using 23-Watt compact fluorescent light bulbs (GE

Daylight FLE10HT3/2/D) with a color temperature of 6500 K to simulate natural sunlight. For CREB, ELK-1 and H3 phosphorylation

analysis, mice were intracardially perfused with 4% paraformaldehyde immediately after the end of the light pulse, while for PER1 and

c-Fos induction, mice were kept in darkness for 90 min after light pulse and then perfused. Coronal brain sections were blocked for

2 hours in 1x PBS containing 3% heat-inactivated goat serum and 0.3% Triton X-100, and then incubated using the following primary

antibodies (for 1 day at 4�C): rabbit antibody a-cFos (Calbiochem Ab-5; 1:20000); rabbit antibody a-PER1 (a generous gift from Dr. David Weaver); rabbit a-pCREB-Ser133 (Cell signaling 9198, 1:500); rabbit a-pELK-1-Ser383 (Cell Signaling 9181; 1:1000); rabbit

a-pH3-Ser10 (Cell signaling D2C8; 1:1000). The immunohistochemical detection was performed using a Vectastain HRP kit (Vector

Labs) based on biotin-streptavidin-peroxidase, and visualized using 3,30-diaminobenzidine (DAB) as chromogen. Finally, sections were mounted on microscope slides, dehydrated, and coverslipped. For characterization of PHb identity (thalamus versus epitha-

lamus), immunofluorescence analyses were performed as described below. The quantification analyses were performed as

described in sections Optical density analysis and c-Fos induction.

PER1/2 rhythmic expression After T24 or T7 cycles exposure, mice were kept in constant darkness for at least 24 hours to avoid any light mediated gene-induction

effects. The general activity of mice was monitored, and animals were perfused at CT 1, 7, 13, or 19. Coronal brain sections were used

to assess the levels of clock gene expression in the SCN and PHb. A rabbit antibody a-PER1 or a rabbit a-PER2 (Alpha Diagnostic

PER21-A, 1:4000) antibody were used, and the immunohistochemical detection was performed as described above. The analysis

was performed as described in the section Optical density analysis.

Immunofluorescence Brain sections and retinas were incubated in 0.1M PBS with 3% Goat serum (Vector Labs) and 0.3% Triton X-100 (Sigma-Aldrich) for

2 hours, and then incubated using the following antibodies (1-2 days, at 4�C): rabbit a-Melanopsin (Advanced Targeting AB-N38, 1:500); rabbit a-RFP (MBL PM005, 1:1000); chicken a-GFP (AbCam Ab13970, 1:2000); guinea pig IgG a-VGlut2 (Millipore

AB2251, 1:1000); mouse IgG1 a-c-Fos (EnCor MCA-2H2, 1:1000); rabbit a-c-Fos (Calbiochem AB-5, 1:1000); mouse IgG1

a-Brn3a (brain-specific homeobox/POU domain protein 3A, Millipore MAB1585, 1:250); rabbit a-GRID2IP (glutamate receptor ion-

otropic delta 2-interacting protein 1, Bioss bs-11347R, 1:1000); rabbit a-PKCd (AbCam, 1:2000). GRID2IP immunostaining required

an antigen retrieval step (citrate buffer, at 85�C for 30 min). After several washing steps, Alexa-conjugated secondary antibodies were used (Molecular Probes, 1:500, for 2 hours at room temperature). Finally, slides were mounted using AntiFade medium (Molecular

Probes). Images were acquired using an LSM-510 confocal microscope (Zeiss).

Melanopsin immunohistochemistry and dendritic reconstruction

Retrolabeled RGCs were imaged on a Zeiss LSM 510 confocal microscope. Images were taken at 40x magnification across the depth

of the inner plexiform layer (IPL) for each cell (Z stack) to capture dendritic stratification revealed by melanopsin expression. Stacks

were imported into Photoshop (Adobe) and reconstructed by tracing immunolabeled dendrites from retrolabeled somas through

adjacent planes of the Z stack. Cells with dendrites that branched in only the inner ON sublamina of the IPL were classified as

M1 ipRGCs; those with dendrites in both the ON and OFF IPL sublaminae were classified as M3 ipRGCs.

SCN dissection and RNA-sequencing Mice housed under T24 or T7 cycles were kept in darkness for 1 day, and a light pulse was applied (15 min) at CT14. An hour later,

brains were isolated under red dim light and placed ventral side up on an ice-cold coronal slice brain matrix (Kent Scientific). A 1 mm

coronal slice of the brain was made with ice-cold razor blades positioned surrounding the optic chiasm. The slice was laid flat on one

of the blades and the SCN was collected using a 1mm diameter sample corer (FST) and immediately placed in Qiazol chilled on ice

and stored at �80C. SCN tissue samples from 3 mice were pooled for each biological replicate in all conditions. Following dissection, RNA was extracted using a QIAGEN RNeasy lipid tissue mini kit and processed with an Illumina RiboZero Gold kit. Sequencing

libraries were prepared with the Illumina TrueSeq RNA-v2 kit. Libraries were multiplexed and sequenced on a Hiseq4000 using

75bp paired-end reads to a depth of at least 130 million reads per sample. Reads were mapped to the mouse reference genome

(mm10) with STAR (v2.4.2a) and read counts were generated with htseq-count from the HTSeq python package (v0.9.1). Differential

Cell 175, 71–84.e1–e7, September 20, 2018 e5

expression analysis was performed with DESeq2. Gene ontology (GO) analysis was performed using the Database for Annotation,

Visualization and Integrated Discovery (DAVID) (Huang et al., 2009).

Retinal injections Retinal projections were visualized using intravitreal injections (1 ml) of the tracer cholera toxin b-subunit (CTb) fluorescently conju-

gated (Alexa Fluor 488 or 594, Thermofisher). AAVs (1 ml) were also injected in order to infect RGCs. Mice were anesthetized by i.p.

injections of avertin (2, 2, 2-Tribromoethanol) and placed under a stereo microscope. A glass needle (pulled 10 mL microcapillary tube,

Sigma P0674) and a 10 mL Hamilton syringe were used to drive the solution into the vitreous chamber of the eye to ensure delivery

specifically to the retina. Mice recovered from injections on a heating pad until they woke from anesthesia. After injections,

animals were given 3 to 4 days recovery period. Mice were deeply anesthetized by intraperitoneal (i.p.) injection of avertin (2, 2, 2-Tri-

bromoethanol), and perfused intracardially with 4% paraformaldehyde. Brains were post-fixed overnight in the same fixative, and

brain sections were obtained using a cryostat. In some cases, eyes were also removed and retinas were dissected in oxygenated

Ames medium (Sigma-Aldrich). Relieving cuts were made to mount retinas flat on a piece of lens paper. Retinas were then incubated

in 4% paraformaldehyde (Electron Microscopy Sciences) overnight at 4�C.

Stereotaxic injections Mice were deeply anesthetized, and CTb, AAVs or gDRabies were stereotaxically delivered. All coordinates used follows the Paxinos

and Franklin mouse atlas (Franklin and Paxinos, 2012). A 10 mL microcapillary pipette was pulled and loaded with the solution;

Injections were performed using a microinjector (Nanojector II, Drummond Scientific Company). A heating pad was used to maintain

the body temperature. Before and after surgery, systemic analgesics (buprenorphine, 0.1 mg/kg) were administrated. For anatomical

analysis mice were perfused at different times post injection (3-4 days after CTb injection, 4 weeks after AAVs injection, or 5 days

after gDRabies injection) and the brains were subsequently sectioned on a cryostat. In specific experiments, retinas were also

dissected out after perfusion and post-fixed for 1 hour.

Electrophysiology Mice were intravitreally injected with an AAV2/hSyn-hChR2(H134R)-EYFP (University of North Carolina Vector Core). Three to four

weeks post injection, mice were anesthetized and decapitated. Brains were removed into an iced cutting solution, oxigenated

with 95% O2/5% CO2 containing: (in mM): 234 sucrose, 26 NaHCO3, 11 dextrose, 10 MgCl2, 2.5 KCl, 1.25 NaH2PO4, and

0.5 CaCl2. Coronal slices (300 mm) were prepared using a VT1200S vibratome (Leica Microsystems Inc., Buffalo Grove, IL); slices

were then incubated for 20 minutes at 34�C immersed in oxygenated artificial cerebrospinal fluid (ACSF) solution containing (in mM): 126 NaCl, 26 NaHCO3, 10 dextrose, 2.5 KCl, 1.5 MgCl2, 1.25 NaH2PO4, and 1.15 CaCl2. Slices were then held at room tem-

perature in the same ASCF, and recordings commenced at least thirty minutes after high-temperature incubation. For recordings,

slices were perfused with ACSF at �34◦C. Labeled retinal fibers were visualized using oblique deep-red illumination under a 60X water-immersion lens (Olympus) mounted on an Axioskop 2 FS plus upright microscope (Carl Zeiss Microscopy, LLC,

Thornwood, NY) equipped with Plan N 4x and LUMFLN 60XW objectives (Olympus, Tokyo, Japan), part 91015 filter cube with set

number 49002 EGFP bandpass filter set (Chroma Technology Corporation, Bellows Falls, VT), M470L3 mounted LED controlled

by a LEDD1B driver (Thorlabs, Inc., Newton, NJ), and Ximea MQ013MG-E2 digital camera (XIMEA, Münster, Germany). Recording

pipettes contained (in mM): 95 CsCH3SO3 (voltage-clamp recordings) or 95 K D-gluconate (current-clamp recordings), 20 TEA-Cl or

20 mM NaCl (voltage-or current-clamp recordings, respectively), 10 HEPES, 8 PO4-creatine, 4 Mg-ATP, 1 4-AP, 1 BAPTA, and

0.4 GTP Tris, pH 7.2; 2 QX314 was sometimes added to block voltage-gated Na+ channels. Neurons were filled with biocytin

(0.1%) during the recording session for post hoc morphological reconstruction. Membrane potentials and currents were recorded

using a MultiClamp 700B amplifier (Molecular Devices, Sunnyvale, CA). Amplifier output was filtered at 4 kHz and digitized at

20 kHz using an ITC-18 data acquisition interface controlled by custom-written software (Igor Pro; Wavemetrics). Offline analysis

was performed using Igor Pro. Holding potentials were corrected for a �10 mV junction potential. Access resistances were typically �20 MU, and were compensated by �15%–30%. For ChR2 stimulation, the M470L3 mounted LED and LEDD1B driver were triggered using a TTL wave consisting of 2 ms pulses at varying frequencies generated in IGOR Pro. PHb neurons considered to

receive retinal input were defined according to their location in an area innervated by retinal fibers (YFP expression), and due to

the time-locked nature of the evoked EPSC relative to laser onset (averaged of several trials).

Hippocampal recordings Opn4Cre/+ and Opn4Cre/+;Brn3bzDTA/+ mice were exposed to the T24 or T7 cycles for 2 weeks. Mice activity was monitored and sam-

ples were obtained at the beginning of the active phase of mice (CT 12-14). Coronal hippocampal slices (0.4 mm) were used to eval-

uate hippocampal LTP, as previously described (LeGates et al., 2012). Slices were obtained and collected in ice-cold dissection

buffer (2.6mM KCl, 1.23mM NaH2PO4, 26mM NaHCO3, 212.7mM sucrose, 10mM dextrose, 3mM MgCl2 and 1mM CaCl2, bubbled

with 5% CO2, 95% O2). For recording, the same buffer was used, except that sucrose was replaced by NaCl, and the temperature

raised to 30�C. LTP protocol was delivered after 20 min of stable baseline transmission. Schaffer collaterals were stimulated to ob- tained synaptic responses in CA1 stratum radiatum (0.33 Hz stimulation, 0.2-ms pulses (concentric bipolar electrodes, FHC)). One or

four theta epochs (each epoch consisted of 10 trains of four pulses (at 100 Hz) delivered at 5 Hz) were used to obtain LTP responses.

e6 Cell 175, 71–84.e1–e7, September 20, 2018

Fiber Photometry The fiber photometry apparatus consists of two LEDs (Doric Lenses) emitting 405 and 465 nm light respectively delivering light

through a rotary joint to a 200 um optical fiber (Doric Lenses or ThorLabs) implanted in the region of interest in the brain of a freely

moving animal; light collected by the fiber passes through a filter/beam splitter (Doric Lenses) capable of separating light in the

420-450 nm range from light in the 500-550 nm range, and is detected by two H10722 photomultiplier tubes (Hamamatsu), one

for each frequency range. The signal is digitized and acquired by a computer. Data analysis was carried out with MATLAB

(Mathworks) and Origin (OriginLab). Briefly, both the GCaMP6 emission signal and the auto-fluorescence from the tissue were re-

corded at the same time at a sampling rate of 0.3 kHz. Both were processed with a FFT filter to reduce noise. Auto-fluorescence

intensity was transformed by constants determined for each experiment by linear regression of the GCaMP6 signal; it was then

subtracted from the GCaMP6 signal in order to remove artifacts. For all the experiments, the same mice were tested for both the

T24 and T7 cycles. This avoids any potential variations due to differential transfection and expression levels. In all cases the excitation

light was the same between T24 and T7 cycles, and different animal signals were normalized to baseline for averaging.

Optic fiber implantation

After isoflurane anesthesia, the mouse’s head was fixed to a stereotaxic apparatus; the skull was exposed after application of

povidone/iodine and 70% ethanol; one anchor screw was driven into the skull; a hole was drilled over the region of interest

(with coordinates calculated from bregma), and after thorough removal of bone debris, the optical fiber (Doric Lenses, inner

diameter = 200 mm, NA = 0.37; or Optogenix, inner diameter = 200 mm, NA = 0.66) was lowered into the brain at a speed of

�200 mm/min. On reaching the appropriate dorsoventral coordinate, the fiber was secured in place by application of dental cement around its exposed portion, encased in a metal ferrule.

QUANTIFICATION AND STATISTICAL ANALYSIS

Morphometric analysis For all morphometric image processing, digitalized captured TIF-images were assembled and processed with Zeiss Zen software

(Zeiss) and/or Adobe Photoshop (Adobe Systems), and transferred to ImageJ software (NIH, USA). Sample analysis was performed

with the experimenter blind to condition. All the nomenclature used in the manuscript follows that of Franklin and Paxinos (2012).

Optical density analysis

Digital images were obtained using an inverted microscope (Zeiss Axioimager.M1), converted to 8-bits gray scale and the optic

density (relative to total area) was measured. For both the SCN and PHb, total area of analysis was manually outlined in coronal brain

sections. In all cases, 3 independent sections per animal were quantified and averaged.

Light-mediated c-Fos induction

For both the SCN and PHb, total area of analysis was manually outlined in coronal brain sections. For the SCN, bilateral nuclei were

evaluated; and for the PHb only one side of the coronal section was evaluated. Results obtained from 3 separate sections/nucleus

were averaged per animal.

Characterization of light-induced c-Fos expression in PHb neurons

Total area of analysis was manually outlined in coronal brain sections. Double positive PHb cells (c-Fos(+), Brn3a(+) or c-Fos(+),

GRID2(+)) were quantified relative to total number of c-Fos(+) cells. Results obtained from 5 separate sections/animal were averaged.

c-Fos induction in light-independent models of depression

The number of c-Fos(+) cells were evaluated in the PHb, LHb, and vmPFC. As described above, three different protocols known

to induce mood alterations were used. For the PHb, total area of analysis was manually outlined; the total LHb area was used for

quantification; whereas an area of 1.21 3 0.64 mm was used for the vmPFC. In all cases, the number of c-Fos(+) cells was obtained

from only one side of these bilateral structures. Results obtained from 5-6 separate sections/nucleus were averaged per animal.

Statistical Analysis Statistical analysis of results was made by using unpaired Student’s t test, analysis of variance (ANOVA), followed by Tukey’s or

Dunnett’s tests, as stated. For the Sucrose preference and NOR tests, a One sample t test compared to a ‘test value’ = 50 was

also performed. All the analyses were done using GraphPad Prism, version 7

RNA-seq differential expression testing was performed in DESeq2 with a Wald test followed by p value adjustment for false

discovery rate using the Benjamini-Hochberg procedure, as described (Love et al., 2014).

DATA AND SOFTWARE AVAILABILITY

The accession number for the raw and processed gene expression data from the RNA-sequencing experiments reported in this

paper is GEO: GSE113875. Data from this study are available from the corresponding author upon request.

Cell 175, 71–84.e1–e7, September 20, 2018 e7

Supplemental Figures

Figure S1. T7 Cycle-Housed Opn4Cre;Brn3bzDTA Mice Showed Hippocampal Learning and Functional Deficits, Related to Figure 1

(A-C) CTb was intravitreally injected in Opn4Cre/+ and Opn4Cre/+;Brn3bzDTA/+ mice in order to trace retinal fibers. A complete serial sectioning of the SCN (A) and

PHb region (B), and a reconstruction of retinal innervation (B, green), are shown. A dense retinal innervation was observed throughout the SCN in both groups of

mice (A), whereas a drastic reduction in retinal innervation in the PHb was observed in Opn4Cre/+;Brn3bzDTA/+ mice. C: Neuropeptide Y (NPY) staining was used for

delineating the intergeniculate leaflet (IGL). As shown in the representative images, a substantial reduction in the retinal innervation to the IGL, compared with the

dorsal and ventral LGN, was found in Opn4Cre/+;Brn3bzDTA/+ mice. Four adult male mice per group were used to characterize the pattern of retinal projections.

(D) For the NOR, the total distance traveled during the first session (familiarization session, mice have 10 min to freely explore two similar objects) and the test

session (5 min exploratory time, where one of the objects was replaced by a novel object) were similar for both groups of mice exposed to T24 or T7 cycles. Data

(legend continued on next page)

are mean ± SEM, by Tukey’s test. Representative traces of the exploratory activity of mice during the test trial for all groups are shown in the lower panel. Blue line

represents the area of the familiar (stable) object, whereas the green line indicates the area with the novel object.

(E) For the MWM, representative traces obtained during the test trial are shown. Target area is represented in yellow.

(F) Results obtained during the reversal phase of the MWM. A comparison between learning (day 12) and reversal (day 14) results was also performed for all

groups (Opn4Cre/+: T24 p < 0.01, T7 p = 0.12; Opn4Cre/+;Brn3bzDTA/+: T24 p < 0.001, T7: p = 0.81). Data are mean ± SEM. *p< 0.05, by Tukey’s test.

(G) LTP responses in the hippocampus obtained from Opn4Cre/+ mice housed under the T24 or T7 cycles (n = 8-12 mice). Data are mean ± SEM. **p< 0.01, by

Tukey’s test.

(H-I) Opn4Cre/+;Brn3bzDTA/+ mice housed under the T24 or T7 cycles showed no significant differences in the field potential slope, fiber volley amplitude, input–

output function (field potential slope normalized to fiber volley (FV) amplitude) (H), or pair pulse facilitation (PPF) responses (I). ISI, inter-stimulus intervals. Data are

mean ± SEM. *p<0.05, **p<0.01, by Student’s t test.

Scale bars: (A) 100 mm; (B, C) 200 mm.

Figure S2. Gene Expression Profile of the SCN, Related to Figure 2

(A) Volcano plot illustrating gene expression differences between SCN samples obtained from T24 versus T7 cycles. All genes with FDR-adjusted p < 0.01 are

colored in red (9 upregulated genes, 20 downregulated genes). 7 classical immediate early genes are highlighted.

(B) Gene ontology (GO) analysis of differentially expressed genes (80 genes, FDR-adjusted p < 0.1) in T7 cycle-housed SCN samples compared to T24-housed

samples. Genes included in the top category for each GO domain are shown in boxes. No GO categories were significantly enriched in the set of upregu-

lated genes.

T24 cycle: n = 3, T7 cycle: n = 4 replicates; SCN tissue was pooled from 3 mice per replicate.

Figure S3. Light Responsiveness in the SCN and PHb, Related to Figures 2 and 4

(A) T24 and T7 cycle-housed WT mice were kept in darkness for 1 day, and received a light pulse (15 min) at CT14. For cFos analysis, after the light pulse mice

were kept in darkness for 90 min, and perfused. For measuring phosphorylation of ELK-1 and H3, mice were perfused immediately after the light pulse.

(B-D) A significant reduction in cFos induction (B), and in the phosphorylation levels of both ELK-1 (C) and H3 (D) were observed in T7 cycle-housed mice,

compared to T24-housed mice. n = 5-6 mice.

(E) Light-mediated c-Fos induction in the PHb. Opn4Cre/+;Brn3bDTA mice showed no significant induction of c-Fos in PHb neurons, compared to control (Opn4Cre/+)

mice. n = 3-4 mice.

Data are mean ± SEM. **p<0.01, by Tukey’s test.

DD: constant darkness; ZT: Zeitgeber time; no LP: no light pulse; LP: light pulse. 3v, third ventricle; ox, optic chiasm.

Scale bars: (B, C, D) 100 mm, (E) 200 mm.

(legend on next page)

Figure S4. Retinal Input to PHb, Related to Figure 4

(A-B) CTb was intravitreally injected in WT (A) and Opn4aDTA/aDTA (B) mice in order to trace retinal fibers. A complete serial sectioning of the PHb region and a

reconstruction of retinal innervation (green) is also shown. In WT mice, a dense retinal innervation was observed throughout the PHb region of the dorsal thalamus.

Mice lacking ipRGC innervation showed a drastic reduction in retinal innervation in the PHb. Four adult male mice were used to characterize the pattern of retinal

projections.

(C-D) The rhythmic expression of the clock gene PER2 in SCN neurons was unaffected after the T7 cycle exposure in both control (C) and Opn4aDTA/aDTA (D) mice.

Data are mean ± SEM, by Student’s t test.

(E-F) A schematic representation of the mouse genetic lines used is shown (E). Opn4CreERT mice were crossed with ROSASynaptophysin-tdTomato mice. Finally,

Opn4CreERT;ROSASynaptophysin-tdTomato mice were used to study the pattern of ipRGC presynaptic terminals. tdTomato puncta expression was observed

throughout the PHb region (F). A reconstruction of ipRGC presynaptic terminals (F, red) is also shown. Four adult mice were used to characterize the pattern of

retinal projections and synaptic connectivity.

(G-H) An AAV2/ChR2-YFP was intravitrally injected (G), and retinal fibers GFP(+) were observed in the PHb (H).

(I) Electrophysiological characterization of PHb neurons. Cells were recorded in whole-cell current-clamp mode at two holding potentials. Left panel: When

relatively hyperpolarized (�70mV), cells showed a burst mode of firing responses to depolarizing rectangular current pulses (black trace). Red trace represents the same cell after the application of TTX; note that the action potentials disappeared, but a depolarizing waveform persisted, known as the low threshold spike

(LTS). This inward Ca2+ current, known as IT, involves voltage- (and time-) dependent T-type Ca 2+ channels and usually reaches the threshold for activating

Na+/K+ action potentials, producing a brief burst of firing. Right panel: cells were then relatively depolarized (�60mV), IT is inactivated, and a tonic mode of firing was observed in response to depolarizing steps (black trace). A hyperpolarizing current injection de-inactivates IT and evoked a rebound burst response (blue

trace). (n = 45 cells).

(J) Representative image showing two filled cells in the PHb region.

(K) A map showing the location of PHb neurons that displayed a response to ChR2 stimulation is shown.

(L-M) Adeno-asociated viruses have been shown to be extremely efficient for inducing stable and prolonged expression of genes of interest in the retina. Among

the broad variety of AAVs described to induce gene expression in many retinal cells after sub-retinal or intravitreal injections, AAV2 constitutes the most reliable

option for infecting retinal ganglion cells (RGCs). When AAV2/ChR2-EYFP was intravitreally injected in WT mice, a strong gene induction (YFP) was observed in

most RGCs. However, AAV2/ChR2-EYFP only infected a faction of ipRGCs, as reflected in most of their brain targets, including the SCN (L). Note the dense retinal

innervation to LGN in (M). In AAV2/ChR2-EYFP injected mice that were also injected with the tracer CTb, a quantification analysis revealed that only a fraction of

axons colocalized for both markers (SCN: 23.5 ± 8.7%, PHb: 16.3 ± 9.8%, n = 3 mice). In sum, these results indicate that ipRGCs are particularly resistant to AAV

infections, suggesting that the number of responder cells found in the PHb region could be underestimated due to the reduced infectivity of ipRGCs.

Hb: habenula; R: rostral; C: caudal; ox: optic chiasm; 3v: third ventricle,.

Scale bars: (C, D, G, L) 100 mm; (A, B, F, H, J, M) 200 mm.

Figure S5. Thalamic PHb Projections to Brain Targets, Related to Figure 5

(A-B) Retinal fibers (CTb) were observed in close apposition with thalamic neurons that express GRID2 (A) or PKCd (B).

(C) CTb was injected into the PHb, and retrogradely labeled somas were clustered in layers V and VI of the IL and PL, bilaterally with respect to the injection site in

the PHb; anterogradely labeled axons were evident in the IL ipsilaterally.

(D) vGluT2 staining was used to reveal the ventral boundary (dashed line) of the cortex. The ventral boundary of the cortex is pinpointed on the basis of striped

vGluT2 expression pattern (arrows).

(legend continued on next page)

(E-F) CTb was injected into the dorsomedial striatum (dmSrt) to determine, by retrograde tracing, the afferents of this PHb target (E). Labeled somas are also

present in layers III-IV of the IL and PL, with sparse cells in layer V as well (F).

(G) Using the origin-controlled conditional tracing of the PHb downstream circuitry described, we found that a subset of labeled PHb neurons have collaterals

penetrating into the lateral habenula (arrows).

(H) The PHb does not project to the dmPFC, unlike surrounding thalamic nuclei. HSV-cre was injected into the dorsal mPFC (specifically, in the anterior cingulate),

while AAV-DIO-synaptophysin-tdTomato was injected into the PHb region (n = 3 mice). Sparse tdTomato+ somas were present mostly in the CL, with scattered

neurons in the MD and rostromedial LP nuclei.

cc: corpus callosum; CL: centrolateral nucleus; MD: mediodorsal nucleus; PVT: paraventricular nucleus of the thalamus; Hb: habenula; dmPFC: dorsomedial

prefrontal cortex.

Scale bar: (G) 25 mm; (A, B, C, E, F, H) 100 mm; (D) 200 mm.

Figure S6. ipRGCs-PHb-vmPFC Pathway Drives Mood Alterations, Related to Figure 6

(A-D) A two-virus retrograde transsynaptic tracing system was used to identify RGCs feeding the retino-PHb-mPFC pathway. A retrograde helper vector (HSV-

TVA-rabiesG-mCherry) was injected into the vmPFC, while EnvA-GDRabies-EGFP was injected into the PHb (A). Only neurons transfected with the helper virus,

and their immediate presynaptic neurons, can be infected by the rabies virus. The number of GDRabies-EGFP+ starter neurons and ipRGCs found is shown (B,

n = 3 mice). C-D: Retrolabeled RGCs (green) in the retina are predominantly melanopsin-immunoreactive (red).

(E) cFosCreERT2 mice were bilaterally injected in the PHb region with an AAV5/DIO-hM3D-mCherry. At the end of the behavioral tests, injection sites were

evaluated. Schemes of coronal brain sections for three representative injected-mice are shown. Dark red areas represent regions of dense number of mCherry(+)

cells; whereas light red regions represent areas with lower number of stained cells.

(F) cFosCreERT2 injected-mice exposed to the same protocol, but without light pulse exposure, showed only sparse (arrow, upper panel) or non mCherry(+) cells

(lower panel) in the PHb region (n = 3 mice).

(G) The effects of clozapine-N-oxide (CNO) added to the drinking water were first compared with intraperitoneal (i.p.) CNO injections in WT mice stereotaxically

injected with a Cre-dependent AAV encoding an excitatory(Gq). c-Fos induction was observed in most infected neurons in mice that received CNO in the drinking

water (5 mg/ml), similar to results obtained from mice that received i.p. CNO injections (1mg/kg). Number of c-Fos(+) cells/ PHb section: Control: 0.4 ± 0.2; CNO

(i.p.) injection: 56.3 ± 13.9; CNO in drinking water: 48.8 ± 15.3; data are mean ± SEM n = 3 mice.

(H) The daily consumption of water+CNO was evaluated in cFosCreERT mice, and no differences were observed throughout the experiment, and compared with

control mice. n = 8-12 mice.

(I) The locomotor activity was monitored using infrared sensors in mice housed under the T24 cycle. A representative actogram is shown; red arrow indicates the

first day of CNO treatment. No significant alterations in the photoentrainment to a 12:12 hr light/dark cycle were observed (% activity during dark (active) phase:

cFosCreERT-injected mice = 97.3 ± 2.1; Control-injected mice = 96.5 ± 3.6). The total locomotor activity of mice was not affected by the CNO treatment, as

observed in cFosCreERT-injected (red) and control-injected (blue) mice.

(J) During the SPT, the total amounts consumed (water + sucrose) were comparable between groups.

Data are mean ± SEM, by Student’s t test.

Scale bar: (C) 25 mm; (G) 50 mm; (D) 100 mm; (F) 200 mm.

Figure S7. PHb Neuronal Function Has a Direct Impact on Light-Induced Mood Deficits, Related to Figure 7

(A) vmPFC-projecting PHb neurons were chronically activated using DREADD control. WT mice were bilaterally injected in the vmPFC with a retrogradely

transported AAV1/cre and in the PHb region with an AAV5/DIO-hM3D-mCherry. At the end of the behavioral tests, injection sites were evaluated. Schemes of

coronal brain sections for three representative injected-mice are shown. Dark green and red areas represent regions of dense number of GFP(+) or mCherry(+)

cells, respectively; whereas light green and red regions represent areas with lower number of stained cells.

(B) The locomotor activity was monitored in T24 cycle-housed mice. A representative actogram is shown; red arrow indicates the first day of CNO treatment. No

significant alterations in the photoentrainment to a 12:12 hr light/dark cycle were observed. The total locomotor activity of mice was not affected by the CNO

treatment (% activity during dark (active) phase: AAV-injected mice = 92.6 ± 4.5; control-injected mice = 90.3 ± 4.2). Data are mean ± SEM, by Student’s t test.

(C) Total distance traveled during the NOR test (First trial: familiarization session, mice have 10 min to freely explore two similar objects; test: 5 min exploratory

time, where one of the objects was replaced by a novel object. n = 12-14 mice. Data are mean ± SEM, by Student’s t test.

(legend continued on next page)

(D) Results obtained during the learning phase (12 days) of the MWM. Data are mean ± SEM n = 12-14 mice.

(E) Mice expressing tetanus toxin (tetX) in a Cre-dependent manner were bilaterally injected in the PHb region with an AAV5/Cre-GFP. At the end of the behavioral

tests, injection sites were evaluated. Schemes of coronal brain sections for three representative injected-mice are shown. Dark green areas represent regions of

dense number of GFP(+) cells, whereas light green regions represent areas with lower number of stained cells.

(F) tetX-injected mice were exposed to the T7 cycle, and the daily locomotor activity was monitored using infrared sensors. Under the T7 cycle, tetX-injected mice

showed period lengthening, similar to T7 cycle-housed control (sham) mice. Data are mean ± SEM, by Student’s t test. n = 14-15 mice.

(G) During the SPT, the total amounts consumed (water+sucrose) by tetX-injected mice housed under the T24 or T7 cycles were comparable. Data are mean ±

SEM, by Student’s t test.

(H-J) WT mice were exposed to a repeated forced-swim (FST), learned helplessness (LH), or chronic social defeat (SD) paradigms, and the induction of c-Fos was

evaluated in the lateral habenula, vmPFC, and PHb. In all cases, stimuli were applied during the active phase of mice (�CT 14), and during the sessions mice were kept in constant darkness to avoid any light-mediated effect on c-Fos induction. FST, LH and SD paradigms induced a significant increase in the number

of c-Fos(+) cells in the lateral habenula (H) and vmPFC (I), whereas no significant induction of c-Fos was observed in PHb neurons (J), compared with control

mice. Finally, we also found a significant difference in the effect of light pulse activation on PHb neurons, compared with all the paradigms used (J). Data are

mean ± SEM, *p<0.05, **p<0.01, ***p<0.001, by Dunnett test. n = 4 mice.