Light enhances learned fear

Author(s): Daniel M. Warthen, Brian J. Wiltgen and Ignacio Provencio

Source: Proceedings of the National Academy of Sciences of the United States of America,
Vol. 108, No. 33 (August 16, 2011), pp. 13788-13793
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/27979276 .
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learned fear

Light enhances

Brian J.Wiltgenb'\

Daniel M. Warthen3,

University of Virginia, Charlottesville, VA 22903

of aBiology and Psychology,

Departments

and Ignacio Provencio3,1

Edited by Joseph S. Takahashi, Howard Hughes Medical

(received for review February 25, 2011)

Institute, University of Texas Southwestern Medical

events
is
and respond to emotional
behavioral
However,
strategy.
dysregulated
are maladaptive.
to these memories
To
responses

to learn, remember,

The ability
a powerful

survival

and physiological
fully understand

learned

fear and

the pathologies
that arise during
the environmental
variables

we must reveal
response malfunction
that influence learned fear responses.

Light, a ubiquitous

environ

to

cue

conditioned

significant freezing during

intertriai intervals indicated

mice,
a 2-wk

The

habituation

absence
and

period
light specifically modulates
cue rather than the context

that

ing to the learned acoustic

chamber.
Repeating
experimental
mutant models,
Pde6brd1/rd1 and
enhancement
dependent
the rods and/or cones.

in darkness.

than

of

during
freez
of

the

our assay
in two photoreceptor
~
4~
that light
mice, revealed

of conditioned
By repeating

fear

is driven

our protocol

primarily by
with an altered

lightingregimen,we found that lightingconditionsacutelymodu

late responses
ismanifested

when

altered

either

as an enhancement

between

and testing. This

conditioning
of freezing when
light is

of freezing when
light is
during testing or as a depression
but not depression,
removed during testing. Acute enhancement,
and melanopsin-dependent
photorecep
requires both rod/cone-

added

results

tion. Our

demonstrate

to learned

responses

a modulation

by

light of behavioral

fear.

learning | Pavlovian | posttraumatic

retinal ganglion cell

stress disorder

| retina |

qualities:

of objects
their color,

to physical
according
and motion.
The
parallel

in the environment

form,

texture,

non-image forming (NIF) pathway enables light to exert nu

merous effects on physiology and behavior independently of
image formation,such as pupil constriction,modulation of heart
rate,

the

and

synchronization

of circadian

rhythms

and

sleep

wake cycles to the daily light-dark cycle (2). In addition to the

effectsof lighton basic physiological functions, lightalso mod
ulates higher-order cognitiveprocesses, includinganxiety,mood,
and alertness/awakeness (3, 4). The retina, the sole photosensory
organ inmammals, projects directly to brain regions involved in
emotional responses. Among these are the amygdala, the bed
nucleus of the stria termin?lis,and the periaqueductal gray (5).
Activity in some of these regions is known to be acutelymodu
lated by light in a wavelength-dependent manner (3, 6), whereas
the link between photoreception and function in other retino
recipient emotional processing areas remains to be elucidated.
Brain sites involved in emotional processing participate in the
critical function of learning and remembering emotionally
arousing

events.

This

function

enables

an

organism

to deal

ef

with similar situationswhen they arise

fectivelyand efficiently
again. Although fear of learned stimuli can be advantageous,
disproportionate fear can lead to pathological states in humans.
In fact, an estimated 40 million Americans over the age of 18 y
sufferfrom an anxiety disorder, a hallmark of which is dysre
gulation of fear (7). A complete understanding of fear and fear
13788-13793

| PNAS

| August 16,2011

| vol.108

| no. 33

that modulate

fear responses.

On thebasis of theknown role of lightinmodulating cognitive

functionand the suggestiveanatomical evidence,we hypothesized
that lightmay influence learning,memory, and the expression of
determine

whether

an

light has

on

effect

learning,

re

membering, and responding to learned emotionally arousing

we

events,

used

a well-established

for the assessment

assay

of

learningandmemory, tone-cued fear conditioning (Fig. 1). Tone

cued fearconditioningconsistsof repeated presentations of a tone
(the conditioned stimulus), paired with a mild shock (the un
conditioned

After

stimulus).

several

of the tone

presentations

shock pair, the subject learns to associate the shockwith the tone
andwill subsequentlyrespond topresentations of the tone alone as
though the shockwere imminent (8). In rodents, the response is
a

cessation

complete

of locomotor

termed

activity,

"freezing"

(9).

Freezing is robustand readilyquantifiable.The percentage of time

spent freezingduring the tone presentation is a reliable indicator
of the strengthof the association that the subject has formedbe
the tone

tween

ject's

learning

Responses
photoreceptor

and shock,
and memory.

in more

or,

terms, of the sub

general

to light inmammals are mediated by the three

classes

of the retina:

the rods,

the cones,

and

the

intrinsicallyphotosensitive retinal ganglion cells (ipRGCs) (1).

The rods and cones are the primary photoreceptors for image
formation,whereas the ipRGCs are implicated primarily inNIF
responses. To identifythe retinal photoreceptors mediating ob
served

is a pervasive feature of the environment and exerts

broad effects on behavior and physiology via two parallel
Light
pathways (1). The familiar image-formingvisual pathway allows
discernment

related pathologies requires an accounting of environmental

factors

fear. To

cognition and anxiety. We hypothesized

to learned fear. Using tone-cued
fear
that lightmodulates
responses
to
behavioral
responses
conditioning, we found that light enhances
in light freeze more
in response
learned fear in C57BL/6J mice. Mice
feature, modulates

mental

July8, 2011

Center, Dallas, TX, and approved

of

light modulation

fear

responses,

we

our

performed

assay in three lines ofmice all on theC57BL/6J strain:wild-type

(WT) mice, which have the full complement of photoreceptors;
Pde6brdllrdlmice, which lack functional rods and cones (10) but
retain intrinsicipRGC photoresponses; and Opn4~f~ mice, which
have

rods

and

cones

but

lack

intrinsic

ipRGC

photoresponses

owing to loss of the photopigmentmelanopsin (11).

Here,

using

a tone-cued

fear conditioning

assay, we

show

that

lightdoes indeedmodulate behavioral responses to conditioned

an increase
In mice
in the percentage
of
light causes
time spent freezing to a conditioned
fear stimulus. This enhance
ment
to the learned stimulus. Furthermore,
is specific to responses
fear stimuli.

using several retinal mutant

are
the rods and/or cones

mouse

models

the dominant

we

demonstrate

photoreceptors

that

driving

lightenhancement of conditioned fear.Finally, by repeating our

fear conditioningassay with an altered lightingregimenwe show
that light or darkness

can

acutely modulate

responses

to a condi

tioned fear stimuluspreviouslyacquired under a differentlighting

condition.

This

work

has

far-reaching

implications

for the treat

ment of fearand anxietydisorders and forpractical applications for

themodulation of learningand memory.
Author contributions:D.M.W., B.J.W.,and I.P.designed research;D.M.W. performed re
search; B.J.W.contributednew reagents/analytic
tools; D.M.W., B.J.W.,and I.P.analyzed
data; and D.M.W., B.J.W.,and I.P.wrote the paper.
The authors declare no conflictof interest.
This article isa PNAS Direct Submission.
1Towhom correspondence may be addressed. E-mail: ?p7m@virginia.eduor bw4fh@
virginia.edu.
10.
This articlecontains supporting information
online atwww.pnas.org/lookup/suppl/doi:
1073/pnas.1103214108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1103214108

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BL

ITM

ITI2

ITI4

ITI3

ITI5

?-"

Train
Time (min) *?

11

15

= Tone

23

19

24/48 hrs

Test

=Shock

Time (min)1

11

23

19

15

30 min per day for 14 d before conditioning. On the

Fig. 1. Tone-cued fear conditioning protocol. Mice were preexposed to the conditioning chamber for
tone-shock pairs was presented. The tone-shock
day of conditioning (Train) activitywas recorded for a 3-min baseline period (BL). At 3 min, the firstof five
was separated by a 3-min intertriai interval (ITI). A fifth ITI
a
s
were
Each
tone-shock
a
with
shock.
2
of
60-s
which
the
final
of
consisted
tone,
pair
paired
pair
followed the final tone-shock pair. Twenty-four hours and 48 h after testing mice were returned to the conditioning chamber and monitored during a tone
no shock was paired with the tone.
only test (Test). The tone-only test was identical to the tone-shock conditioning, except that

Results
Light Enhances

Behavioral

to Conditioned

Responses

Fear. We

ini

tiallyperformed our experiments in both lightand darkness in

WT C57BL/6J mice, which have normal retinas.Before condi
tioning,mice were preexposed to the fear conditioning chamber
for 30 min each day for2 wk (Fig. 1). This 2-wk latent inhibition
period

was

sufficient

to minimize

fear associations,

contextual

as

discussed below. During conditioning (Training) therewas an

increase in freezinginWT mice inboth lightand darkness across
the session, revealed by a main effect of trial, indicating suc
cessful conditioning to the tone (Fig. 24) [F(4,128) = 83.74, F <
0.0001]. Freezing during conditioningwas enhanced inWT mice

Testing Day 1

Training
19 3 ?WT:Light
-*-WT:Dark

100
90?
80
j?70
? 60
% 50
?L 40
5* 30
20

\?

-s0

0> \?

Total ITIFreezing

cP cP *e
\? \?

of a significant

by the absence

modulate

behavioral

responses

to learned

fear

inWT

mice.

To support the conclusion that light is the exclusive driver of

modulation

of freezing, we

our

repeated

experiment

with a less intense lightsource (Dim Light;Mater?als andMeth

ods). Dim lightwas not sufficientto enhance freezing levels rel
ative to levelsobserved indarkness on any day of theprotocol, as
shown by an absence of a main effectof lighton the day of con
=
= 1.95, =
0.1741], testingday 1 [F(l,104)
ditioning [F(l,104)
- 0.15, =
=
2
and
0.6983]
0.22,
testingday [F( 1,104)
0.6396],
was sufficientto enhance
(Fig. SI ). Conversely, our standard light
freezingrelative to dim lighton all days of theprotocol, as shown
=
=
by amain effectof lightduringconditioning [F( 1,104) 8.15,F
=
and
F
1
5.41,
testing
0.0281],
0.0084], testingday [F( 1,104)
- 4.42, F =
0.0453] (Fig. SI). These data suggest
day 2 [F(l,104)
that there isa threshold intensityfor the enhancing effectof light
the intensities

tested

here.

Rods and/orConesAre theDominantPhotoreceptorsforDrivingLight

Enhancement
Training Test Day 1 Test Day 2

< 0.05 considered

measures ANOVA, and inD evaluations are t tests, with
as average percentage
significant. *P < 0.05, **P < 0.01. Data are presented
freezing ? SEM.

of Conditioned

Fear. To

determine

the contributions

of the rods, cones, and ipRGCs to the observed light-dependent

enhancement

WT mice. [A
Fig. 2. Light enhances conditioned fear responses inC57BL/6J
C) InWT mice, light significantly enhances freezing to a conditioned cue
=
during both conditioning (A) and 2 subsequent days of testing (? and C) (n
17 in light, = 17 in dark). (D) Freezing during ITIswas not significantly
different between
light and dark groups on any day, indicating that con
textual fear associations were minimized. InA-C evaluations are repeated

Warthen et al.

as shown

trial number,

=
=
1.88,
0.1173]. The en
light trial interaction [F(4,128)
over
2
of
effect
subsequent days of test
lightpersisted
hancing
ing,as revealed by a main effectof lighton testingday 1 (Fig. 2B)
=
= 11.55,
0.0018] and testingday 2 (Fig. 2C)
[F(l,128)
=
=
8.09,
0.0077]. Extinction was minimal in both
[F(l,128)
groups on testingday 1, as shownby the absence of a main effect
of trialon freezing [F(4,128) = 1.36, = 0.2519]. Extinctionwas
observed on testingday 2, however, revealed by a main effectof
trial [F(4,128) = 15.99, < 0.0001]. Interestingly,the rate of
extinctionon testingday 2 was influencedby lightingconditions,
=
revealed by a light trial interaction [F(4,128) = 2.90,
can
indeed
that
demonstrate
results
therefore
light
0.0245]. Our

that lies between

a
\?

vary across

the observed

<P
^
<P ^0 \?

in light,as revealed by a main effectof light [F( 1,128) = 4.16,

?
0.0498]. This light-dependent increase in freezingdid not

tioning assay

of

learned

in two mutant

our fear
fear, we performed
mouse
lines: Pde6brdllrdl mice,

condi

which

lack functional rods and cones beyond 4 wk of age (rodless

coneless) (10); and Opn4~f~ mice, which have functional rods
and cones but lackmelanopsin and thereforelack intrinsicipRGC

photoresponses (melanopsin-knockout) (11). During conditioning,

mice in both light (n = 14) and darkness (n = 10)
Pde6brditrdl
successfullyacquired a fearfulassociationwith the tone, indicated
=
86.69,
by a main effectof trialon freezing (Fig. 3/4) [F(4, 88)
PNAS

j August 16, 2011

| vol. 108

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All use subject to JSTOR Terms and Conditions

| no. 33

| 13789

< 0.0001]. However, no main effectof lighton freezingwas ob

served during conditioning [F(l,88) = 0.87, = 0.3610], nor was
a main effectof lighton freezingobserved during testingday 1
=
2.11,
(Fig. 3B) [F(l,88)
0.1605] or testingday 2 (Fig. 3C)
=
=
1.76,
[F(l,88)
0.1984]. Although rodless-conelessmice in
light tended to freezemore than theircounterparts in darkness
duringboth conditioningand testing,the effectwas not statistically
significantduringanyportion of theprotocol. The lackof an effect
mice indicates that the rods and/
of lighton freezing inPde6brdllrdl
or cones play a criticalrole in thenormal enhancing effectof light
on learned fear. It should be noted that the lack of an observed
difference

is not

in these mice

to saturated

due

responses

(i.e.,

a "ceiling effect").When subjected to a more intense training

foot shock under "bright light" conditions),
protocol (0.75-mA
1
enhanced freezingduring the
Pde6brdllr mice exhibitsignificantly
testingphase, relative toPde6brd1 mice conditionedwith a 0.40
mA foot shock indarkness [F(l,48) = 6.48, = 0.0257] (Fig. S2).
The observation thatfreezingcan be driven significantly
higher in
these mice

mental

that

indicates

are not

paradigm

to our

the responses
saturated.

standard

during

the 2

subsequent

days

of

tone-only

testing,

Opn4~!~ mice in lightdid freezemore in response to the tone

thanmice in darkness, as indicated by a main effectof lighton
=
=
9.45,
freezing during testingday 1 (Fig. AB) [F(l,72)
=
and
2
7.87,
testingday (Fig. AC) [F(l,72)
0.0065]
0.0117].
This

indicates

that

the rods

and/or

cones

are

sufficient

to drive

this response during the recall testingphase. Furthermore, the

rate of extinctionwas influencedby lightingconditions on testing
day 1, as indicated by a significantlight trial interaction [F
=
=
3.46,
(4,72)
0.0122]. This effectwas not observed on

Training

f
Testing Day 2

'

100
90
80-I
?70
? 60?
S 50
? 40
S? 30
20
10
o

100
90-I
80?
=?70
? 60
S 50
? 40
5? 30
20
10
0

A?

centage

13790

< 0.05

considered
freezing ? SEM.

significant Data

Testing Day 2

'

100
90
80'
?70
? 60
% 50?
? 40?
55 30?
20
10-1
0?

100
90
80
?70
? 60
% SO
LL40
30
20
10
0

<S>

Total ITIFreezing

mm

mm

Light Light
Light
?EfflJ
I?flfl ||

Training Test Day 1 Test Day 2

Fig. 4. Light enhances conditioned fear responses inOpn4~'~ mice. (A-C) in

Opn4~'~ mice light does not significantly enhance freezing to a conditioned
cue during conditioning (A) but does enhance freezing during 2 subsequent
~ 14 in
= 6 indark). (D)
days of testing (? and C) (
light,
Freezing during ITIs
was not significantly different between
light and dark groups during con
ditioning and testing day 2, but a slight elevation was observed during
testing day 1. InA-C evaluations are repeated-measures ANOVA, and inD
< 0.05 considered significant. *P < 0.05, **P <
evaluations are t tests, with
0.01. Data are presented as average

testing day 2 [Light

contrast

to WT

mice,

percentage

Trial, F(4,72)

extinction

was

freezing ? SEM.

1.17,

apparent

=
on

0.3326]. In

both

days

of

testing,as indicated by a main effectof trial on testing day 1

=
=
=
2.72,
9.54,
[F(4,72)
0.0362] and testingday 2 [F(4,72)
< 0.0001]. Taken together, these data indicate that the rods
and/or cones are the dominant photoreceptor class(es) driving
light enhancement

of learned

Enhancement

fear.

of Fear

Is Specific

to the Learned

Cue.

0.3733).

Furthermore,

on

the day of conditioning

and

both

days of testing,freezingduring the 3 min before the first tone

presentation (Baseline, Fig. 5 B-D) and during the intertriai
intervals (ITIs) (Fig. 2D) was negligible, indicating that contex

<e <P
^?

<

Importantly,freezingduring a 2-wk preexposure period (Mate

dais andMethods; Fig. 1) was found to be similar between WT
mice in light (n ? 17) and WT mice in darkness (n = 17), in
dicating that lightalone does not cause freezing (Fig. 5/1) (t test,

tual

\?

fear

associations

with

the fear conditioning

chamber

were

successfullyminimized by the preexposure period and that fear

associations

Total m Freezing

were

predominantly

made

with

the

tone.

Fear

associations with the conditioning apparatus itself could rea

sonably have been expected to differbetween the groups, owing
to the differentiallightconditions, but the data indicate that this

was

not

the case.

Likewise, freezing during the preexposure period in both

melanopsin-knockout
Light
Light
Light
l^^l
|^^|
Training Test Day 1 Test Day 2

Fig. 3. Light does not significantly enhance fear responses in Pde6brdl/rd1

mice. (A-C) InPde6brdl/rd1mice, light does not significantly enhance freez
ing to a conditioned cue during conditioning (A) or 2 subsequent days of
~ 14 in
= 10 indark). (D)
testing (? and C) {
light,
Freezing during ITIswas
not significantly different between lightand dark groups on any day. InA-C
evaluations are repeated-measures ANOVA, and inD evaluations are t tests,

with

<

<?

Testing Day 1

100
90
80
?70
? 60
8 50
? 40
S? 30
20
10
O1

f 4? 4? 4?

Light-Dependent

Testing Day 1

100
90
80
70
60
50
40
30
20
10

Training
Opn4"A:Light

experi

During conditioning,melanopsin-knockout mice (Opn4~l~) in

?
=
light (n
14) and darkness (n
6) also successfullyacquired
a fearfulassociation with the tone, as indicated by a significant
< 0.0001].
main effect of trial on freezing [F(4,72) = 32.34,
modulate freezing inOpn4~!~ mice
Light did not significantly
during conditioning,as indicatedby the absence of a main effect
=
of lighton freezing (Fig. 44) [F(l,72) - 0.90,
0.3553].
However,

-?-

are presented

as average

per

and

rodless-coneless

mice

was

near

zero,

and freezing levels did not differsignificantlybetween lightand

dark groups, demonstrating that light alone does not induce
in either of our mutant genotypes (Fig. 5A) (t test,
freezing
- =
=
40.5675; Pde6brd?lrdl
0.1155). As withWT mice,
the
3-min
baseline
freezingduring
period was negligible (Fig. 5
B-D), and freezingduring the ITIs was low relative to tone-cued
freezing in both lines during conditioning and both days of
testing(Figs. 3D and AD). Freezing was similarbetween lightand
dark groups in both genotypes during all baseline and ITI peri
ods (t test, > 0.05) with the exception ofmodest elevations in
Opn4~!~ mice in lightduring the baseline on the day of condi

I www.pnas.org/cgi/doi/10.1073/pnas.1103214108

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All use subject to JSTOR Terms and Conditions

Warthen et al.

Latent

Training: Baseline

Inhibition (First 3 Min Each Day)

100?

100-1

Wildtype

Opnf''

PdeBb"111

Wildtype

Pde6b'

Opnt1'

Fig. 5. Light alone does not cause elevated freezing. (A) Freezing during the preexposure period was near zero for all genotypes, indicating that light alone
does not induce freezing. (B) Freezing during the first3 min {before tone presentation) on the day of conditioning was near zero for all genotypes. (C and D)
Freezing during the first3 min on both days of testing was negligible relative to tone-cued freezing, indicating that fear associations were made primarily
< 0.05 considered significant. *P < 0.05, **P < 0.01. Data are presented as average percentage freezing ? SEM.
with the tone. All evaluations are ttests, with

=
tioning (/ test,
0.0309) and during the ITIs during testingday
1 0 test, = 0.0264).
to Fear
Behavioral
Responses
Lighting Conditions Acutely Modulate
that light enhances
Cues. Our
learned
fear
results demonstrate
en
feature of the conditioning
when
it is a constant
responses

vironment.We
behavioral
whether

then asked whether lightcould acutely enhance

to fear cues
in darkness
learned
responses
to conditioned
could
darkness
suppress
responses

and

in darkness,
of fear cues learned
expression
fear conditioning
with one important
protocol
mice
in darkness,
and conditioned
then,
preexposed

the behavioral

we

our

repeated
change: we

during the testingperiod 24 h later,we exposed themice to light

(Fig. 6/4).As shown inFig. 6B, when WT mice were tested in
=
lightafter conditioning in darkness (n
10) their freezingwas
to
elevated
relative
freezing levels during testing
significantly

when

mice

were

preexposed,

and

conditioned,

tested

in darkness

=
=
only (n
17) (t test,
0.0198). This demonstrates that light
does not have to be present during the acquisition of a fear as
sociation

to have

an

effect

enhancing

on

the response

during

subsequent testinginWT mice. In otherwords, lightcan acutely

enhance

freezing

responses

to cues

learned

in darkness.

Freezing

inWT mice tested in lightwas similar whether conditioning

occurred in light (n = 17) or in darkness (n - 16) (Fig. 6B)
=
0.6197). This result shows that a maximal effectof
(t test,
on
light freezingbehavior can be attained regardlessof whether
lightwas present during conditioning and also reveals that light
must be present during the testingphase tomaintain high levels
of freezing induced by light during the acquisition phase.
Moreover,

this result underscores

that contextual

conditioning

is

not behind the lightenhancement of conditioned fear responses.

When we performed our altered lightingprotocol with WT
mice that had already been through the protocol once in all
Warthen et al.

and

conditioning,

(preexposure,

testing,

16),

we

found that the responses of thesemice during the testingportion

of the altered lightingprotocol were similar to responses of na?ve
mice (n = 10) during the same period (t test, = 0.2872). Be
cause

no

difference

was

in these

observed

mice,

subsequent

experimentsusing the altered lightingregimenwere performed

with mice already exposed to the original protocol.
To

fear

cues learned in light.To assess whether lightcould acutely en

hance

darkness

the precise

determine

roles of rods, cones,

and melanopsin

inmediating the acute effectsof lighton fear conditioning,we

repeated our altered lightingregimen experiment in rodless

coneless

mice.

and melanopsin-knockout

As

mentioned

above,

themice used in these experimentshad previouslybeen through

theprotocol once in theoriginal lightingconditions.When either
strainofmouse was conditioned in lightthen tested indarkness
(Opn4~?~

14, Pde6brdllrd?

14),

levels

freezing

during

testingdeclined radically relative to freezing levelsduring testing

=
of mice conditioned and tested in light (Opn4~f~
14,
4~'- < 0.0001;
Pde6brdllrdI = 14) (Fig. 6 C and D) (t test,
Pde6brdllrdl

0.0003),

However,

when

mice

were

conditioned

af
in darkness and tested in light,freezingwas not significantly
fected in either genotype relative to conditioning and testing in
darkness (Opn4-j- = 6,Pde6brdIlrd} = 3) (Fig. 6 C andD) (t
=
=
test,Opn4~f0.7570; Pde6brd?lrdl
0.9623). Taken to
indicate
that
these
data
photoreception by either the rods
gether,
and/orcones or bymelanopsin is sufficientto enhance behavioral
responses to learned fear,but onlywhen light is present during
both the acquisition and recall phase. For light to acutely en
hance

both

a cue learned
in darkness,
fear responses
during testing of
the rod-cone
and melanopsin
systems must be intact, sug

gesting a synergisticaction by these two pathways.

Discussion
In these experimentswe have shown that lightalters behavioral
responses to conditioned fear. This demonstration of light
PNAS

I August 16, 2011

| vol.108

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[ no. 33

| 13791

box, rodents are allowed to explore a divided chamber

light/dark
inwhich one half is illuminated and the other half is kept in

-WT: Light-Dark
-WT: Dark-Light

darkness.

the acoustic

of anxiety

an

(4),

and

in the frequency

arm

of open

duration

explora

tion (20) (but see ref.21 for a contradictoryview).

The reduction of exploratorybehavior in lighthas been sug

SO
|S 40'
it
SS 30

gested

a mechanism

to be

from

avoidance

for pr?dation

the

rodents'highlyvisual predators (18). By avoiding brightlylitareas,

detection

prey avoid

conditioned fear responses

Lighting conditions acutely modulate
in light. Dark
during testing. (B-D) Light-Dark indicates mice conditioned
Light indicates mice conditioned in dark. Test 1 indicates that conditioning
and testing occurred inthe same lighting condition. Test 2 indicates that the
conditioning and testing. (A)
lighting condition was switched between
in light and
Schematic of altered lighting protocol. Mice were conditioned
tested in dark, or conditioned in dark and tested in light, (?) InWT mice,
removing the lightduring testing results in a significant decline in freezing,
whereas
light during testing results in a significant increase in
adding
Fig. 6.

freezing. InOpn4~'~ mice (C) and inPde6brd1/rd1mice (D), removing the light
during testing results in a significant decline in freezing, but adding light
during testing does not increase freezing in either genotype. Data are pre
sented as average percentage freezing + SEM.

dependent

modulation

of responses

to learned

fear cues

repre

sents an unappreciated effect of light on behavior. Light

enhances freezing inWT mice in response to a conditioned fear
cue during both acquisition and recall (Fig. 2). Furthermore,our
experiments have shown that at the light intensitiesused here,
themodulation of conditioned fearby lightisdrivenprimarilyby
the rods and/or cones (Figs. 3 and 4). A primacy of rod-cone
input during conditioned responses is supported by previous
work showing that rodless-coneless mice show deficits [rd/rdcl
strain (12)] or
as a conditioned
The

elevated

are unresponsive
[Pde6brdl/rdlstrain
in a learned avoidance
stimulus
freezing

seen

levels

in our

(13)]
task.

experiments

to light

are

not

due to a general inductionof freezingby light,as evidenced by

the similar freezing levels observed in mice in light and in
darkness during a 2-wk preexposure period (Fig. 5A). Freezing
was also similar between lightand dark groups during the ITIs
in all genotypes on both days of testing,with the exception of
a moderate elevation in
4~!~ mice in lighton the firstday of
and
2D,
3D,
AD). Furthermore, freezingduring the
testing(Figs.
3-min baseline period was negligible relative to tone-cued
freezing on both days of testing (Fig. 5 C and D). These data
support thehypothesis that lightspecificallyenhances freezing to
a conditioned cue. Finally, lightenhanced freezing inWT mice
during a recall test24 h after tone-cued conditioning indarkness
(Fig. 6/1), demonstrating that lightcan acutely enhance behav
responses

to conditioned

fear

stimuli

learned

in darkness.

together, these data strongly argue against the in

terpretation that contextual conditioning is responsible for the
increase in freezing seen inmice in light.
Light has long been recognized as an anxiogenic/aversive

stimulus

for nocturnal

rodents..

The

simplest

example

is the

suppression of locomotor activity exhibited by nocturnal mice

during a nighttime lightpulse (although under some circum
stances lightduring the nightmay increase locomotor activity)
(see ref. 14 for review). In a slightlymore complex assay, the
13792

for the dark

open field (19). Finally, darkness increases entry into the open
arms of an elevated plus maze (19), whereas bright lightcauses

:Light-Dark
:Dark-Light

Opnf'': Light-Dark
Dark-Light
Opn4'/m:

u. 40
55 30?
20

ioral

as a measure

reflex, used

startle

a decrease

Taken

a preference

show

a
to increased
also attributed
anxiety. A comple
phenomenon
to sudden
after exposure
effect is observed
darkness,
mentary
in the
a sudden
in measures
of anxiety
decrease
which
induces

D
-

rats

and

effect that the authors attribute to an anxiogenic effectof the

illumination.Exploratory behavior in both rats and mice is de
creased under high illumination in the open field test (16-18),

?> LIGHT

DARK

mice

Both

portion, avoiding the illuminatedside (15). Light also potentiates

fitness. The

increase

therefore

and

freezing

responsemeasured inour study isone of a suite of behaviors and

an im
physiological responses initiatedwhen an animal detects
minent threat(22). In fact,a recent studyshowed that thisbehavior
can be recapitulated in ratswhen facedwith a predator-like robot
in a seminaturalisticenvironment (23). The freezing response in
serves

particular

to make

the

animal

less

detectable,

thereby

avoiding pr?dation from the threat. It stands to reason that an

be
similar to decreased
exploratory
freezing response,
are
in light, when
be advantageous
havior, would
prey animals
on and extend prior
results therefore expand
to spot. Our
easier

enhanced

reports of the anxiogenic/aversiveeffects of lightby providing

a definitivedemonstrationof light-dependentincrease invigilance
and defensive behavior in response to a learned threat (a fear
conditioned tone), a modulation of a basic survival strategythat
has been

across

conserved

In humans,
anxiety. This

animal

(22).
species
an increase
than light, causes
in the laboratory as an enhancement

in

rather

darkness,
ismanifested

of

the acoustic startle reflex in darkness (24). This effectwas en

hanced furtherinpatientswith posttraumatic stressdisorder (25).
Furthermore, patients sufferingfrompanic disorder exhibit am
plification of freezing-likebehaviors under stressfulconditions
(26). Although lightingconditionswere not explored in thisstudy,
itstands to reason thatdarkness-induced anxietywould constitute
a

stressful

that lighting
from our results
It follows
to stressful cir
responses
likely alter behavioral
to
related
and others with disorders
in these patients

condition.

will

conditions
cumstances

fear. Light

learned

has

already

clinical

demonstrated

benefits:

bright lighttherapyis an accepted and widely used treatmentfor

seasonal affectivedisorder (27). In combinationwith the known
effectsof light(or darkness) on affectand anxiety,our resultsraise
the possibility that light therapy could be part of an effective
treatment

for other

regimen

affective

disorders

involving

dysre

gulated fear,broadly classified as anxietydisorders.

Anxiety and fear share overlapping but distinctpathways (28).
Anxiety is hypothesized to derive from a state of heightened
vigilance to a generalized, unperceived threat,whereas fear is
a rapid-onset and -offsetresponse to a specific threat (29). The
phenomenon we report here, lightmodulation of conditioned
fear,builds a bridge between the twowell-studied, distinctfields.
Similar to the light-modulation of anxiety-related behaviors
mentioned

above,

we

report

an

enhancement

of

fear-related

behaviors in the presence of light.Our data cannot be explained

simply

by

an

increase

terpretation would

exposure,

baseline,

in anxiety,

because

however,

such

an

in

imply increases in freezing during pre

and

ITI

periods.

To

the contrary,

we

have

observed significant increases in freezing specifically during

presentation

of a

learned

fear-inducing

cue.

Our results have far-reaching implications.Although further

research isneeded to determine the full extent of the effectsof

I www.pnas.org/cgi/doi/10.1073/pnas.1103214108

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Warthen et al.

our results show that

can in
light on learning and memory,
light
deed modulate
to learned stimuli. It is likely
behavioral
responses
re
that light can modulate
and behavioral
learning, memory,

sponses to learned cues in other paradigms. Although there is

discrepancy in thefield,several independent researchgroups have
that a switch from regular fluorescent
lighting, which emits
to full-spectrum
light at only a few wavelengths,
lighting, which
emits light across the visual spectrum, can result in improved mood

posited

and performance in theworkplace and classroom (30). It iswithin

results, that such a switch could have
given our present
effects on learning and memory
performance.
our results also draw attention
to the influence
of
Finally,

reason,
similar

lightingconditions inbehavioral assays. Care should be taken in

to assess
assays
of environmental
light
founding effects.

all behavioral
sition

the intensity and

sources
to avoid

compo
spectral
con
unrealized

Prior evidence has hinted that lightmay influence learning,

memory, and fear, including themodulatory effect of lighton
cognition, the role of light in anxiety, the role of light in circu
lating hormones

that feed

into memory

systems,

and

the central

projections of ipRGCs. Our results show definitivelythata per

vasive

environmental

variable,

fear responses.

light,

can modulate

conditioned

Materials and Methods

Animals. Male mice on a C57BL/6J background were used in this study.WT
mice were purchased from Jackson Labs. Pde6brd1/rd1mice were purchased
from Jackson Labs or bred at the University of Virginia. Opn4~!~ mice were
bred at the University of Virginia. Animals were housed in individual cages

and kept under a 12-h light/darkcyclewith lightson at 0500 hours (ZTO). All
of the experimental procedures were carried out in accordance with Asso
ciation for Assessment of Laboratory Animal Care policies and approved by
the University of Virginia Animal Care and Use Committee.

Tone-Cued Fear Conditioning. The fear conditioning and monitoring system

used in these studies has been described indetail previously (31). Particulars
of our study follow and are represented in Fig. 1. Before the first day of
conditioning, mice were

allowed

to explore the conditioning

apparatus

for

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30 min each day for at least 12 d. Preexposure days were not always con
secutive. On the day of conditioning, mice were placed in the conditioning
apparatus, and baseline activitywas recorded for 3 min. At 3 min, the firstof
five tone presentations began (2,800-kHZ pure tone, 85 dB). The tone per
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lowed by the second tone-shock. This pattern persisted through five tone
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testing), and freezing was scored using the Video Freeze system (Med
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modulation on fear conditioning (32, 33), preexposure,

testing were all performed between ZT7 and ZT12.

conditioning, and

Lighting. For mice in "light," blue light-emitting diodes (LEDs) with a peak
emission wavelength
of ?470 nm (Super Bright LEDs, catalog #E27-x8-G)
were placed adjacent to the fear-conditioning chambers. A neutral density

filterwas used to achieve a light intensity inside the chamber of 0.7 pW/cm2
at the point nearest to the light and 0.4
/cm2 at the point farthest from
the light, corresponding to a photon flux of ?9.5
1011 to 1.6 x 1012 pho
tons/s per cm2. For mice in "dark" the light fixtureswere removed. The light
intensity in the chambers in "dark" was <0.0001 pW/cm2. For mice in "dim
light," additional neutral density filters were used to achieve a light in

/cm2 at the center of the chamber, corresponding to a

tensity of ?0.01
1010 photons/s per cm2. For mice in "bright light" a blue
photon flux of ?3
LED (Super Bright LEDs, catalog #PAR20-B36) was used without neutral
/cm2at the center of the
density filters to achieve a light intensityof 165
chamber, corresponding

to a photon flux of ?3.9

1014 photons/s per cm2.

Statistical Analyses. Except where noted, all experiments were analyzed by

repeated-measures ANOVA. Significance was defined as a value <0.05.
ACKNOWLEDGMENTS. We thank Kaycie Tayler for technical assistance. This
work was supported by National Institutes of Health Grant NS052112 (to f.P.)
at the
and by the Biology (I.P.) and Psychology (B.J.W.) Departments
University of Virginia.
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Warthenet al.

PNAS

| August 16, 2011

| vol.108

This content downloaded from 152.118.24.10 on Thu, 27 Nov 2014 09:55:44 AM

All use subject to JSTOR Terms and Conditions

| no. 33

| 13793