Light Evokes Melanopsin-Dependent Vocalization and
Neural Activation Associated with Aversive Experience in
Anton Delwig1, Anne M. Logan2¤b, David R. Copenhagen1,3*¤a, Andrew H. Ahn2,4*¤a
1Department of Ophthalmology, University of California San Francisco, San Francisco, California, United States of America, 2Department of Anatomy, University of
California San Francisco, San Francisco, California, United States of America, 3Department of Physiology, University of California San Francisco, San Francisco, California,
United States of America, 4Department of Neurology, University of California San Francisco, San Francisco, California, United States of America
Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are the only functional photoreceptive
cells in the eye of newborn mice. Through postnatal day 9, in the absence of functional rods and cones, these ipRGCs
mediate a robust avoidance behavior to a light source, termed negative phototaxis. To determine whether this behavior is
associated with an aversive experience in neonatal mice, we characterized light-induced vocalizations and patterns of
neuronal activation in regions of the brain involved in the processing of aversive and painful stimuli. Light evoked distinct
melanopsin-dependent ultrasonic vocalizations identical to those emitted under stressful conditions, such as isolation from
the litter. In contrast, light did not evoke the broad-spectrum calls elicited by acute mechanical pain. Using markers of
neuronal activation, we found that light induced the immediate-early gene product Fos in the posterior thalamus, a brain
region associated with the enhancement of responses to mechanical stimulation of the dura by light, and thought to be the
basis for migrainous photophobia. Additionally, light induced the phosphorylation of extracellular-related kinase (pERK) in
neurons of the central amygdala, an intracellular signal associated with the processing of the aversive aspects of pain.
However, light did not activate Fos expression in the spinal trigeminal nucleus caudalis, the primary receptive field for
painful stimulation to the head. We conclude that these light-evoked vocalizations and the distinct pattern of brain
activation in neonatal mice are consistent with a melanopsin-dependent neural pathway involved in processing light as an
aversive but not acutely painful stimulus.
Citation: Delwig A, Logan AM, Copenhagen DR, Ahn AH (2012) Light Evokes Melanopsin-Dependent Vocalization and Neural Activation Associated with Aversive
Experience in Neonatal Mice. PLoS ONE 7(9): e43787. doi:10.1371/journal.pone.0043787
Editor: William Rowland Taylor, Oregon Health & Science University, United States of America
Received May 18, 2012; Accepted July 24, 2012; Published September 13, 2012
Copyright: ? 2012 Delwig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by funding from the National Institutes of Health NS066091 (AHA), EY01869 (DRC), March of Dimes (DRC) and Research to
Prevent Blindness (Department of Ophthalmology and DRC), Knights Templar Eye Foundation (AD), a supplement funded by the ARRA NS047113 (AHA) and
National Eye Institute (Core grant EY 002162). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (DRC); firstname.lastname@example.org (AHA)
¤a Current address: Department of Neurology, University of Florida College of Medicine, Gainesville, Florida, United States of America
¤b Current address: The Neuroscience Graduate Program, Oregon Health Sciences University, Portland, Oregon, United States of America
In neonatal rats and mice, light evokes negative phototaxis, a
stereotyped avoidance behavior, characterized by a vigorous
reorientation away from the light source [1,2]. In neonatal mice
between postnatal day 6 and 9 (P6 to P9), before the emergence of
rod and cone visual signaling, the melanopsin-expressing intrin-
sically photosensitive retinal ganglion cells (ipRGCs) mediate this
robust behavior . However, it is not known if light activation of
ipRGCs during negative phototaxis is associated with an aversive
In adults, even moderate levels of light can be aversive or
possibly even painful. In adult rats, bright light activates pain-
reactive neurons in the trigeminal nucleus caudalis (TNC) [4,5].
Also in adult rats, light activates dura-sensitive neurons in the
posterior thalamus (Po), where a convergent light-evoked signal
from ipRGCs has been implicated in a human clinical symptom
called photophobia, in which light exacerbates migraine headache
. Finally, adult mice genetically altered to have increased
sensitivity to calcitonin gene-related peptide (CGRP) show
increased avoidance of light [7,8]. The central role of CGRP in
pain processing , especially in the central nucleus of the
amygdala [10,11], suggested to us that cellular activation of this
area  could also reflect the aversiveness or negative salience of
bright light in these neonatal mice.
Neonatal mice produce specific vocalizations in response to
distressful or painful stimuli, so we hypothesized that these
behaviors could be informative. Mouse pups emit ultrasonic
vocalizations in the 50–80 kHz range in response to a variety of
stressful events, including isolation from the home cage [13,14].
Additionally, neonatal mice respond to acutely painful stimuli,
such as tail pinch, with broadband vocalizations (squeals) heard
prominently in the 5-kHz range .
In this study we asked whether light activation of melanopsin-
expressing ipRGCs during negative phototaxis is associated with
aversive or even painful experience in neonatal mice. To answer
PLOS ONE | www.plosone.org1September 2012 | Volume 7 | Issue 9 | e43787
this question, we first tested whether pups vocalize in response to
light, and if so, whether these vocalizations are related to stress or
pain. Next, we characterized neural activation in three brain areas
involved in processing aversive and painful stimuli in adults:
posterior thalamus (Po), central amygdala (CeLC) and trigeminal
nucleus caudalis (TNC).
The results of our experiments could also inform the recent
debate as to whether lighting conditions affect outcomes in the
care of human preterm infants in neonatal intensive care units
[15,16]. In this brightly illuminated environment, preterm infants
can display what appear to be escape responses, including
squinting of the eyes, turning of the head away from light,
saluting, and finger splaying . Although there is general
agreement that pain and distress should be minimized in the care
of preterm infants , it is a challenge to determine in this clinical
setting whether these reactions to light are merely reflexive, akin to
pupillary responses to light, or whether bright light is truly aversive
or even painful to these infants.
Mice were housed in an AALAC-accredited pathogen-free
animal facility with ad libitum access to food and water, and with a
12-hour light-dark cycle with lights on at 7AM and off at 7PM.
Mice were used in accord with an experimental protocol that was
approved by an institutional review board (IACUC, UCSF), and
meets guidelines on the care and use of laboratory animals by the
U.S. Public Health Service.
The animals used in these experiments were C57/BL6J wild-
type (WT) mice obtained from a local vendor (Jackson Laboratory,
Sacramento CA). The mice lacking the melanopsin gene (Opn4)
are derived from the line described by Panda, et al , and were
kindly provided by Russ Van Gelder. This is also the mouse line
with which we previously described melanopsin-dependent pho-
totaxis . Melanopsin knockout (KO) pups used in the behavior
experiments were compared to both heterozygotes (Opn4+/2) as
well as the WT mice, the latter two groups showing no difference.
The KO mice used in the experiments of neuronal activation were
bred from crosses between KO heterozygotes and were compared
to heterozygote littermates and WT mice. Tail DNA for
genotyping was obtained on anesthetized pups immediately prior
to intracardiac perfusion. Histological analysis took place without
knowledge of the animals’ genotype.
All testing was performed within a 4-hour interval during the
subjective daytime. Possible changes in light ultrasonic vocaliza-
tions or neuronal activation during subjective night were not tested
here as our previous study of negative phototaxis revealed no
diurnal variations. . Animal behaviors were monitored with an
infrared camera and ultrasound detector as shown in Figure 1.
We monitored audible 5-kHz squeals and 62-kHz ultrasonic
vocalizations (USVs) from mouse pups using an ultrasound
detector (UltraSound Advice, UK; model: mini-3). This detector
has two channels: one was tuned to 62 kHz (detection range: 58 to
66 KHz) and the second monitored audible frequency range (20–
20,000 Hz). The audio output from the mini-3 detector was
recorded continuously onto a sound recorder (Sony PCM-M10).
Timing of USVs was detected by thresholding the root mean
square levels (5 msec bins; Matlab) as described previously with
minor modifications . The Matlab code used for analyses is
available from authors upon request. The experimenter quantified
5-kHz squeals manually.
Light exposure chamber
We used a monitoring chamber (106364 cm; L6W6H) made
of clear acrylic warmed by a heating pad to 35uC. Using the same
chamber, we previously showed that a single light source evoked
negative phototaxis . In the present experiment, we fixed two
LED light sources (Philips Lumileds Lighting Company; model:
Luxeon III star, LXHL-LB3C, wavelength=470 nm) at 5 cm
from the ends of the testing chamber as shown in Figure 1. The
measured power flux at each end of the chamber was 40 mW/cm2
(UDT Instruments, San Diego, CA; model S471). The photon flux
at the 470 nm wavelength was 961016photons/sec/cm2, which is
roughly equivalent to the amount of blue light in the direct
sunlight at midday. Taking into account that eyelids are closed at
this age (about 100 fold attenuation of light ) and that pups are
free to move inside the testing chamber (4 fold difference in light
intensity depending on the location inside the chamber), we
estimate that the amount of light that reached their corneas ranged
from 100 to 400 mW/cm2(2.2 to 961014photons/sec/cm2).
Mice were kept in darkness for at least one hour before the
experiment. Neonatal pups at ages P7 to P9 were tested
individually, and transferred to the testing chamber under dim
red light. Pups were allowed to acclimate to the chamber until the
isolation-induced 62-kHz USVs calls  ceased (10–15 minutes).
A recording trial began with a 60 second baseline in the dark, a
60 second exposure to light, followed by an additional 60 seconds
of recording in the dark. We quantified the percentage of pups that
showed locomotor and vocal responses to light as evidenced by
turning away from light and increase in the rate of 62-kHz USVs.
Vocal responses were further quantified as the number of 62-kHz
vocalizations during each 1-minute interval, and are presented as a
mean value 6 SEM. The scorer was blinded to the genotype of
pups when quantifying vocalizations from mixed genotype litters
(Opn4+/2, Opn4+/+, and Opn42/2) obtained from the mating of
Opn4+/2parents. Tests of statistical significance were determined
Figure 1. Schematic diagram of testing chamber. Mouse pups
(P7–P9) were placed in a clear plastic enclosure with a blue LED
positioned 5 cm from each end of the chamber. The pup’s behavior was
monitored using an infrared camera to record motor responses and a
broadband microphone to detect 62-kHz ultrasonic vocalizations (USVs)
and 5-kHz squeals.
Light-Evoked Vocalizations and Neural Activation
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by pair wise comparisons between WT and KO mice, using
Student’s t-test, with the criteria of significance set at p,0.05.
Controls of specificity
Several control experiments excluded the possibility that sound,
smell, vibration or heat, related to the light source was the relevant
stimulus for USVs. Simply blocking the light paths between the
LEDs and the pups, or turning the illuminated LEDs away from
the pups eliminated the USVs when the light was on. All olfactory
and auditory cues were unchanged under these experimental
conditions. Additionally, light delivered via fiber optic cable, which
does not carry smell, sound or vibrations, from the light source
positioned 10 m away elicited the same locomotor responses and
USVs. Blocking the light paths with infrared blocking filters had
no discernible effect on the light-evoked USVs or negative
To determine vocal responses to acute pain, the pup was
allowed to become quiet after the end of light pulse (typically about
5 minutes) after which time its tail was lightly pinched for
5 seconds. This pinching produced not only 62-kHz USVs but
also audible 5-KHz squeals and a robust escape motor response.
The injection of dilute formalin was a positive control stimulus
for nociceptive stimulation. We diluted formalin (37% formalde-
hyde, Fisher) in isotonic saline and injected 10 ml of 2% formalin
or control saline into the supraorbital skin using a 30-gauge needle
mounted on a 50-ml glass Hamilton syringe. All injections were
performed under dim red-light conditions to avoid extraneous
Light exposure and tissue preparation for
On the morning of the experiment, the home cage was
maintained in the dark for 1 to 3 hours. To minimize spurious
exposure to ambient light, animals were transferred to the light
chamber under dim red light conditions. P8 pups were transferred
to the warmed light chamber and stimulated for 30 minutes with
15-second pulses of blue light followed by 15 seconds of darkness.
Following 90 min recovery in the darkened home cage, animals
were euthanized and perfused intracardially for tissue sections.
Frozen brain and spinal cord sections were cut in the coronal
plane on a sliding microtome at 40 mm and collected in 0.1 M
phosphate buffer (pH 7.4). We examined every other section
within the region from the C2 spinal cord to the trigeminal nucleus
caudalis at the level of the area postrema and decussation of the
Free-floating sections were pre-incubated for one hour at room
temperature (RT) in phosphate-buffered saline with 0.3% Triton
X-100 (PBST) and 10% normal goat serum (10% NGST). Within
the trigeminal nucleus caudalis and the posterior thalamus, the
expression of the immediate-early gene Fos was used as a reporter
of neuronal activation, using a rabbit anti-Fos antiserum
(Oncogene, San Diego, CA 1:30,000). The phosphorylation of
the extracellular related kinase (pERK) in the lateral and capsular
portions of the central amygdala, the so-called nociceptive
amygdala, is associated with the negative and aversive aspects of
painful stimulation . To detect pERK we used the anti-
Phospho-p44/42 kinase rabbit mAB (1:1000 Cell Signaling
Technology, catalog # 4376, clone 20G11, Danvers, MA.) at
room temperature (RT) overnight. Primary and secondary
antisera were diluted in PBST with 2% NGS (2% NGST).
Sections were then washed three times in 2% NGST for ten
minutes each and incubated for one hour at RT with biotinylated
goat anti-rabbit antibody (Vector Labs, Burlingame, CA) in 2%
NGST, and washed three times in PBST for ten min each at RT.
To localize the secondary antibody we used an avidin-biotin HRP
complex (ExtrAvidin Peroxidase, Sigma, St. Louis, MO), with
glucose oxidase (Sigma, St. Louis, MO) as the substrate and nickel-
enhanced 3, -39diaminobenzidine (DAB, Sigma, St. Louis, MO) as
the chromogen. Sections were then mounted on gelatin-coated
glass slides and cover slipped under DPX mounting media (EM
Sciences, Fort Washington, PA).
We stained and counted alternate sections from the TNC at the
level of the decussation of the pyramids to the C2 cervical spinal
cord for Fos-immunoreactive nuclei. After staining and mounting
the sections, we binned the sections into four regions, the rostral
portion at the level of the decussation of the pyramids (PYX), the
caudal portion of TNC adjacent to C1 (TNC) and the two upper
cervical levels C1 and C2. This arbitrary division yielded
approximately 5 sections in each of the four bins. We counted
the Fos-positive neurons in the first 3–4 fully intact sections from
each bin. In addition, we determined the location of the Fos-
positive neurons within superficial (laminae I–II) or the deeper
layers (laminae III–V), using the dark field image of the tissue as
the anatomical landmark. We counted Fos-positive nuclei within
the posterior thalamic group in a similar manner, using the
hippocampus and the lateral ventricle as the anatomical landmark.
The individual thalamic nuclei are indistinct at this stage, but the
formalin- and light-induced Fos-stained neurons appeared in a
consistent posterior thalamic region that includes the lateral
posterior (LP), central lateral (CL) and posterior (Po) thalamic
nuclei, to which we refer as Po or the posterior thalamic group. To
count pERK-positive cells within the central amygdala (CeA), we
stained alternate (coronal) sections through this region and
identified three sections from within a 240-mm rostrocaudal span
within the middle of the CeA, where central lateral (CeL), central
central (CeC), and central medial (CeM) parts of CeA are all
clearly present, and counted all pERK positive cells in the CeA
versus basolateral amygdala. The scorer manually counted all
sections in all experiments while blinded to the experimental group
and genotype of the animal. The data are presented as the mean of
the average cell counts per section from each animal 6 SEM. We
made pair wise comparisons between dark and light conditions,
using Student’s t-test, with the criteria of significance set at
Light evokes aversive but not acute pain-related
vocalizations in neonatal mice
We recorded vocalizations from neonatal mouse pups, placed
individually into a testing chamber (Figure 1). A majority of pups
emitted ultrasonic vocalizations at 62-kHz initially, upon separa-
tion from their littermates (USVs; Videos S1 and S2) . A
period of 10–15 minutes in the dark was sufficient for pups to
acclimate to the testing chamber and cease making isolation-
induced 62-kHz USVs. A recording trial then consisted of a
60 second baseline in the dark, a 60 second exposure to light,
followed by an additional 60 seconds of recording in the dark
(Videos S3 and S4). We recorded both 62-kHz USVs distress calls,
Light-Evoked Vocalizations and Neural Activation
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and audible squeals at 5-kHz . We also monitored locomotor
activity (head pivoting and complete turnarounds).
Figure 2A shows examples of 62-kHz USVs from 6 different
wild type (WT) and 6 different melanopsin knockout (KO)
animals. Figures 2B and 2C show the group data. During the
60-second exposure to light, 100% of WT pups (n=22) exhibited
increased locomotor activity and 91% exhibited increased number
of 62-kHz USVs. These light-induced 62-kHz USVs were
indistinguishable from isolation-induced USV calls. In contrast,
only 8% of KO pups (n=13) exhibited increased locomotor
activity or 62-kHz USVs during light stimulation (Figure 2B,C).
The absence of light-induced locomotor responses and USVs in
KO pups is consistent with the melanopsin-expressing intrinsically
photosensitive retinal ganglion cells (ipRGCs) being the photo
sensors for these light induced behaviors .
We also determined whether light evokes audible 5-kHz squeals.
We confirmed that tail pinching evoked 5-kHz squeals in both WT
and KO pups (Figure 2D). However, neither WT nor KO pups
produced 5-kHz squeals before, during or after the light
stimulation. Altogether, these results show that in neonatal mice,
bright light evokes melanopsin-dependent 62-kHz distress calls but
does not evoke 5-kHz pain-related audible squeals.
Light does not activate neurons in spinal trigeminal
In adult rats, bright light increases the number of Fos
immunoreactive neurons and the electrophysiological activity of
defined neurons in superficial laminae of the trigeminal nucleus
caudalis (TNC) [4,5]. We tested whether light elicits activity in the
pain-related areas of the TNC in neonatal mice, by trying to detect
of Fos expression in the upper cervical spinal cord covering C2
and C1, and rostrally through the trigeminal nucleus caudalis to
the level of the area postrema and the decussation of the pyramids.
To establish whether TNC could be activated in neonatal mice,
we injected formalin, a well-recognized nociceptive stimulus, into
the supraorbital skin. We saw a robust increase of Fos positive
neurons in the TNC (2465 cells per section) from a baseline of
361 cells per section, confirming the existence of Fos-reactive pain
pathways in these neonatal mice. Following a light stimulation
procedure akin to that by Okamoto and colleagues , we
stimulated awake, unanesthetized pups for 30 minutes, at a light
intensity at least 1 log unit above the threshold required to elicit
negative phototaxis and USVs. Light-induced Fos immunoreactive
cells were not detectable in the TNC nuclei above the
unstimulated baseline of 361 cells per section. Thus conditions
of bright illumination sufficient to produce negative phototaxis
behaviors were not sufficient to produce evidence for the
activation of pain-reactive circuits in the trigeminal nucleus
caudalis by Fos immunohistochemistry.
Figure 2. Light evokes aversive but not pain-associated vocalizations in neonatal mice. (A) Raster plots of the 62-kHz USVs from six wild
type (WT) and six melanopsin-knockout (KO) pups during one-minute periods before, during and after light exposure. Each tick represents a 62-kHz
USV of an individual pup. (B) Percent responders to one-minute light were qualitatively assessed during the experiments. Locomotor response was
considered positive if a pup turned around or produced vigorous head pivoting in response to light. No or very little movement was present in either
WT or KO pups during one minute prior to light onset. Vocalization response was positive if a pup noticeably increased the number of 62-kHz USVs
during exposure to light as compared to baseline. (C) Average number of 62-kHz USVs in the minute prior to turning light on (Dark) and during
1 minute of light (Light) in WT (n=22) and KO (n=13,) pups. The asterisk (*) indicates p,1025. (D) Average number of 5-kHz squeals in the minute
before and 1 minute after the onset of light or tail pinch in WT (n=8) and KO (n=10) pups. No 5-kHz squeals were present at any time during
isolation, acclimation and light stimulation. Data are presented as mean values 6 SEM.
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Light induces pERK expression in central amygdala
The lateral and capsular portion of the central amygdala
(CeLC) is critical for the affective processing of aversive and
painful stimuli . In fact, Carrasquillo and Gereau  showed
in adult rats that the phosphorylation of ERK (pERK) in the
CeLC is necessary and sufficient for the expression of pain-related
behaviors after the injection of formalin into the hind paw.
Therefore, we chose to use pERK instead of Fos as the marker of
neural activation in this region of amygdala. Our control injections
of formalin into supraorbital skin of neonatal mice resulted in a 2-
fold increase in the number of pERK-positive neurons in the
CeLC (Figure 3; (WT Dark pups n=8, WT Light pups n=9).).
Figure 3 also shows that light increased the number of pERK
positive cells in CeLC. By contrast, light did not significantly
change pERK expression in CeLC of the KO mice (KO Dark
n=5, KO Light n=9). Light therefore produced a melanopsin-
dependent activation of a cellular signal in the central amygdala
similar to that of formalin, a known nociceptive stimulus.
Light induces Fos expression in the posterior thalamic
group of neonatal mice
To further investigate how light could be involved in the
processing of aversive or painful stimuli, we looked at the pattern
of neuronal activation in the posterior thalamic nuclear group (Po),
as summarized in Figure 4. In adult rats, light-responsive neurons
in the posterior thalamic group (Figure 4A) are activated by
ipRGCs . Animals maintained in the dark show low baseline
numbers of Fos-reactive nuclei (Figure 4B). Exposure of normal
wild-type pups to light produced an almost 3-fold increase in the
number of neurons with Fos positive nuclei (Figure 4C; WT Dark
n=8, Light n=9).). Control injection of formalin into the
supraorbital skin of neonatal mice resulted in a 2-fold increase of
Fos positive cells in Po (Figure 4D; n=4 both saline and formalin
groups), consistent with this region being responsive to nociceptive
To consider the melanopsin-dependence of this circuit, we
conducted parallel experiments in melanopsin knock out mice
(KO Dark n=5, KO Light n=9), and found that light failed to
induce the high numbers of Fos-reactive nuclei in the posterior
thalamic group (Figure 4D). Overall, the number of Fos positive
cells in light-stimulated KO pups was comparable to WT pups in
darkness. However, it was unexpected that the baseline number of
Fos positive neurons in unstimulated animals was much lower in
KO than in WT pups. Also unexpected, light did increase the
overall number of Fos positive neurons in the melanopsin KO
animals. These findings demonstrate that light does activate
thalamic neurons in the posterior thalamic group, but that they
Figure 3. Light activates neurons in central amygdala. (A) Line drawing indicating the region of interest within the basolateral (BLA), the
medial (CeM) and lateral and capsular portions of the central (CeLC) amygdala. The boxed outline indicates the region of the amygdala represented in
the micrographs to the right. (B) Example of pERK staining in a sham-treated P8 mouse pup that remained in the dark (WT Dark). (C) Example of pERK
staining in P8 mouse pup exposed to light for 30 min (WT Light). (D) Quantification of the number of pERK-expressing neurons in CeLC and BLA
areas. There were many fewer pERK-labeled cells within the CeM and BLA, and the BLA was counted as an out of region control. In WT pups, light
produced a 1.6-fold increase in the number of pERK-expressing neurons (WT Dark pups n=8 and WT Light pups n=9). Supraorbital formalin
injections produced a 2-fold increase in Fos staining compared to saline injections. (n=4, both saline and formalin groups). Light did not induce pERK
cells in KO mice (KO Dark n=5, KO Light n=9). Data are presented as means of the average number of pERK staining neurons in each animal 6 SEM.
Asterisks (*) indicate p,0.01.
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may engage both melanopsin-dependent and melanopsin-inde-
pendent signaling pathways (see Discussion).
Previous studies revealed that light evokes an avoidance
response during which rodent pups turn away from light .
Melanopsin-expressing intrinsically photosensitive retinal ganglion
cells (ipRGCs) mediate this negative phototaxis between postnatal
days 6 and 9 (P6–P9) . Here we report that in neonatal mice of
this age group, light also evokes melanopsin-dependent distress-
like ultrasonic vocalizations (USVs). Light-induced USVs and
negative phototaxis are accompanied by neural activation of the
central amygdala (CeLC) and the posterior thalamic group (Po),
brain regions involved in pain processing. In contrast, light does
not evoke audible squeals, a behavioral marker of acute
mechanical pain, and does not activate Fos expression in the
trigeminal nucleus caudalis (TNC). We conclude that light evokes
melanopsin-dependent vocalizations and patterns of neural
activation associated with aversive but not acutely painful
experience in neonatal mice.
Our results provide evidence that in neonatal mice responses to
bright light are not processed neurally as mere reflexes, akin to
pupillary constriction to light. First, light activates both the
amygdala and Po, areas associated with the processing of the
aversive and emotionally negative aspects of pain [21–23]. Second,
light evokes avoidance locomotor responses and distress vocaliza-
tions, also associated with a range of aversive experiences.
Therefore, responses to bright light are accompanied by aversive
experience that is likely to include awareness and emotion.
We speculate that in neonatal mice, light-induced USVs and
negative phototaxis are adaptive behaviors. The survival value of
negative phototaxis, which would direct a pup that wandered into
a lighted environment back to its nest, presumably located in a
darkened area, would minimize exposure to predation. In
addition, light-induced USVs appear to be indistinguishable from
the isolation-induced USVs, which are known to elicit search and
retrieval behaviors in both parents .
A novel finding of our study is that aversive behavioral
responses to light in neonatal mice are accompanied by increased
number of pERK positive neurons in CeLC. It would be of further
interest to determine whether the light-aversive circuit involving
CeLC is also retained in the adult rodents. Several lines of
evidence in adult rodents support a strong connection between
light and pain processing in the amygdala, and possibly through
the pERK signaling pathway that we examined in the present
experiments. First, the enhancement of CGRP signaling in
transgenic mice overexpressing the human CGRP receptor
component increases light avoidance [7,8] and pain processing
 in adult mice. Second, the central amygdala is a major target
of CGRP signaling in the brain [10,11], where it is an important
modulator of behavioral responses to aversive and painful stimuli
through its actions in the central amygdala . Finally, activation
of pERK in the central amygdala appears to be a critical signal in
Figure 4. Light activates neurons in the posterior thalamic group. (A) Line drawing indicating the region of interest within the adult
thalamus, which encompasses a group of nuclei that we refer to here as the posterior thalamic group (Po – see Methods). The boxed outline indicates
the region of the thalamus shown in the micrographs to the right. (B) An example of Fos expression in a sham-treated P8 mouse pup that remained
in the dark (WT Dark). (C) An example of Fos expression in a P8 mouse pup exposed to light for 30 min (WT Light). (D) Quantification of the number of
Fos expressing neurons in Po. In WT pups, light increased the density of Fos cells 3 fold (Dark n=8 and Light n=9). Formalin injection increased the
density of Fos stained cells by 1.9-fold compared to saline injections (n=4 both saline and formalin groups). In KO pups, light increased Fos staining
(KO Dark n=5, KO Light n=9). Data are presented as means of the average number of Fos staining neurons per section in each animal 6 SEM.
Asterisks indicate (*) p,0.05 and (**) p,0.001, respectively.
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processing behavioral responses to painful stimuli . To the
extent that our findings in mice are transferrable to humans, we
might expect amygdala to be also activated in both adult and
neonatal human patients during episodes of photophobia.
The absence of a light-induced activation of the TNC in
neonatal mice diverges from findings in adult rats. Okamoto et al.
 reported that bright light stimulates Fos expression in pain-
receptive areas of the TNC and proposed that light-induced
dilation of the eye vasculature could be responsible for the
activation of pain-responsive neurons within the TNC via afferents
within the trigeminal nerve. The lack of a corresponding activation
of the TNC in neonatal mice could be due to differences in species
(mice vs. rats) or age (neonates vs. adults). At the age that we tested
(P7–P9), trigeminal afferents or eye vasculature might be still
immature. We ruled out the possible immaturity of pain-related
trigeminal afferents by showing that supraorbital injection of
formalin did stimulate the induction of Fos in the TNC of P8
neonatal mice, though this study did not formally exclude the
electrophysiological activation of pain-receptive neurons in the
TNC. On the other hand, the vasculature within the eye is still
developing as late as P16 , which may account for our inability
to observe light-induced activation of the TNC of neonatal mice.
Our experiments also revealed two seemingly perplexing results.
First, the baseline of neural activity in Po is significantly lower in
KO than in WT pups. The reason for this difference is currently
unknown. The retinal ipRGCs could provide tonic excitation to
Po or ipRGCs could modulate activity of other brain areas that
relay their signals via Po. Further experiments are needed to
determine whether melanopsin has a role in setting the basal level
of neural activity in Po.
Second, there is a significant light-induced activation of Po in
KO pups. This finding has not been reported previously. Since
neonatal melanopsin knockout pups (,P10) have no rod or cone-
based visual signaling in the eye and no intrinsic photoresponses
from the melanopsin-expressing retinal ganglion cells or photo-
sensitive cells in the iris, our finding that light exposure increases
neuronal activation in Po implies the possibility of alternative
photosensitive cells in the eye or brain. Expression of alternative
opsin genes in the eye and brain has been reported (OPN3
encoding encephalopsin  and OPN5 encoding neuropsin
). We can’t presently rule out the idea that one or both of these
uncharacterized opsins are capable of signaling to Po at early
Even though our study did not attempt to identify the circuit
linking ipRGCs to Po and CeLC, previous studies revealed the
possibility of both direct and indirect pathways from ipRGCs in
the retina to pain-responsive areas of the amygdala and thalamus.
By selectively labeling axons of ipRGCs, Hattar et al.  showed
that ipRGCs send direct axonal projections to the amygdala. In
addition, Noseda et al.  provided anatomical and electrophys-
iological evidence that ipRGCs make direct connections with
neurons in the posterior thalamic nuclei. Light signals from
ipRGCs can also reach CeLC and Po indirectly via other relay
nuclei such as lateral geniculate nucleus [29,30]. Thus, further
work is warranted to more precisely identify the neural pathways
linking ipRGCs to CeLC and Po.
It remains to be tested whether light sensitizes responses to other
sensory stimuli in neonates. In adult human migraineurs with
photophobia, light can exacerbate cutaneous pain [31,32]. In
adult rats, light potentiates air puff-induced blink reflex .
However, light can also desensitize other responses. For example,
in healthy human adults, light distracts from cutaneous pain in the
Further studies are needed to determine the role of melanopsin
photopigment in neonatal mice older than P9, which have
developed functional rod and cone visual signaling. It is possible
that once rods and cones start signaling, they can relay signal for
aversive responses to light via either ipRGCs or other retinal
ganglion cells. However, it is also possible that melanopsin
photopigment itself is important for initiating the aversive
responses. For example, melanopsin photopigment is required
for light-induced induction of sleep in mice at night .
We speculate that by analogy to developing mice, developing
human fetuses and preterm infants could also exhibit melanopsin-
dependent aversive responses to light. Melanopsin is expressed as
early as embryonic day 11 (E11) in mice  and gestational week
9 (GW9) in humans , well before the emergence of visual
signaling from cones and rods, which is P10 in mice [3,37] and
GW30 in humans . Since neonatal mice rely on melanopsin
photopigment to initiate aversive responses to light, it is reasonable
to suggest that even preterm human infants can also have
melanopsin-dependent aversion to bright light. Therefore, modest
accommodations to reduce overall distress in preterm infants being
cared for in neonatal intensive care units would include limiting
lighting conditions with significant power in the blue end of the
visible spectrum (natural light and fluorescent lights ), which
preferentially activates the melanopsin photopigment in the eye
(maximum spectral sensitivity of human melanopsin is at the
wavelength of 480 nm ).
kHz USVs in a P7 WT mouse pup. Recording duration is 5 sec.
Ultrasonic calls are detected at 62-kHz and shifted to lower
frequency range by heterodyne circuitry. Video recordings were
done with an infrared camera.
Video and audio recording of isolation-induced 62-
kHz USVs in a P7 KO mouse pup. Recording duration is 4 sec.
Video and auditory recording of isolation-induced 62-
from a P7 WT mouse pup. First minute of recording shows the
pup in darkness. Second minute is during light stimulation. Third
segment shows next minute in darkness. Total duration is
Video and audio recording of locomotor and USVs
from a P7 KO mouse pup. Dark, stimulation and subsequent dark
periods began at 0:00, 1:17 and 2:17 min, respectively. Total
duration is 3:15 min.
Video and audio recording of locomotor and USVs
The authors thank Patrick McQuillen and Jan Rodrigo Hoffmann for
helpful comments on the manuscript.
Conceived and designed the experiments: AD AML DRC AHA.
Performed the experiments: AD AML DRC AHA. Analyzed the data:
AD AML DRC AHA. Contributed reagents/materials/analysis tools:
DRC AHA. Wrote the paper: AD DRC AHA. Designed software used in
analysis: AD. Obtained mouse line: DRC. Fabricated stimulator: DRC
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