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Neuroscience 277 (2014) 506–521
GENETIC DELETION OF MT1/MT2 MELATONIN RECEPTORSENHANCES MURINE COGNITIVE AND MOTOR PERFORMANCE
G. O’NEAL-MOFFITT, * J. PILLI, S. S. KUMAR ANDJ. OLCESE
Florida State University Program in Neuroscience, College of
Medicine, Department of Biomedical Sciences, 1115 West
Call Street, Tallahassee, FL 32306, USA
Abstract—Melatonin, an indoleamine hormone secreted into
circulation at night primarily by the brain’s pineal gland, has
been shown to have a wide variety of actions on the devel-
opment and physiology of neurons in the CNS. Acting via
two G-protein-coupled membrane receptors (MT1 and MT2),
melatonin modulates neurogenesis, synaptic functions,
neuronal cytoskeleton and gene expression. In the present
studies, we sought to characterize the behavior and neuro-
nal biology of transgenic mice lacking both of these melato-
nin receptors as a way to understand the hormone’s
receptor versus non-receptor-mediated actions in CNS-
dependent activities, such as learning and memory, anxiety,
general motor performance and circadian rhythmicity.
Assessment of these behaviors was complemented by
molecular analyses of gene expression in the brain. Our
results demonstrate mild behavioral hyperactivity and a
lengthened circadian period of free-running motor activity
in melatonin receptor-deficient mice as compared to recep-
tor-intact control mice beginning at an early age. Significant
improvement in cognitive performance was found using the
Barnes Maze and the Y-Maze. No behavioral changes in
anxiety levels were found. Electrophysiological measures
in hippocampal slices revealed a clear enhancement of
http://dx.doi.org/10.1016/j.neuroscience.2014.07.0180306-4522/� 2014 Published by Elsevier Ltd. on behalf of IBRO.
*Corresponding author. Address: 1115 West Call Street, Florida StateUniversity, College of Medicine, Tallahassee, FL 32306, USA. Tel:+1-850-645-1479.
E-mail addresses: [email protected] (G. O’Neal-Moffitt), [email protected] (J. Pilli), [email protected] (S. S. Kumar),[email protected] (J. Olcese).Abbreviations: aCSF, artificial cerebrospinal fluid; ADHD, attentiondeficit/hyperactivity disorder; ANOVA, analysis of variance; C3B6, micewith a combination C3H/He and C57BL/6 background; cDNA,complementary deoxyribonucleic acid; CAT, catalase; CREB, cAMPresponse element-binding protein; DBL-KO, double-knockout; DD,dark–dark or constant dark; DNA, deoxyribonucleic acid; EDTA,ethylenediaminetetraacetic acid; EPM, elevated plus maze; EPSP,excitatory post-synaptic potential; ERK, extracellular signal-regulatedkinases; GAD1, glutamic acid decarboxylase 1; GluR1, glutamatereceptor subunit 1; GPx-1, glutathione peroxidase 1; HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HPC, hippocampus;LD, light–dark; LTP, long-term potentiation; MAPKs, mitogen-activatedprotein kinases; mRNA, messenger ribonucleic acid; MTNR, melatoninreceptor; MT1, melatonin receptor type 1; MT2, melatonin receptor type2; MTNR1a, gene encoding the MT1 receptor; MTNR1b, geneencoding the MT2 receptor; NIR, near-infrared; Nrf2, nuclear factorerythroid 2-related factor 2; NT, non-transgenic; PGK,phosphoglycerate kinase promoter; PCR, polymerase chain reaction;qPCR, quantitative polymerase chain reaction; SCN, suprachiasmaticnuclei; SOD1, superoxide dismutase 1; SDS–PAGE, sodium dodecylsulfate–polyacrylamide gel electrophoresis.
506
long-term potentiation in mice lacking melatonin receptors
with no significant differences in paired-pulse facilitation.
Quantitative analysis of brain protein expression levels of
phosphoCREB and phosphoERK1/2 and key markers of
synaptic activity (synapsin, glutamate receptor 1, spinophi-
lin, and glutamic acid decarboxylase 1) revealed significant
differences between the double-knockout and wild-type ani-
mals, consistent with the behavioral findings. Thus, genetic
deletion of melatonin receptors produces mice with
enhanced cognitive and motor performance, supporting
the view that these receptors play an important role in
neurobehavioral development. � 2014 Published by Elsevier
Ltd. on behalf of IBRO.
Key words: melatonin, learning, memory, activity, LTP, mice.
INTRODUCTION
Knockout mice lacking both the MT1 and the MT2
(encoded by the MTNR1a and MTNR1b genes
respectively) melatonin receptors have been used in a
variety of studies to examine the mechanisms of
melatonin action in specific disorders and conditions,
including focal cerebral ischemia (Kilic et al., 2012),
methamphetamine-induced locomotor sensitization
(Hutchinson et al., 2012), sleep–wake characterization
(Comai et al., 2013), nicotine sensitivity (Mexal et al.,
2012), and blood glucose regulation (Muhlbauer et al.,
2009); however, no comprehensive characterization of
baseline differences between these transgenic animals
and their wild-type counterparts has been published. Sim-
ilarly, aspects of neurodevelopment have not been
addressed before, despite the fact that these animals’
brains develop not only without the receptor-mediated
influence of melatonin, but they also lack the impact of
the normally constituently active MT1 receptor
(Dubocovich et al., 2010).
The genetic absence of specific hormone receptors
can result in a variety of neurobiological disorders, e.g.,
oxytocin receptors and social behavior (cf. Donaldson
and Young, 2008), vasoactive intestinal peptide receptors
and circadian behavior (Harmar et al., 2002), androgen
receptors and reproductive systems (Chang et al.,
2013). Functionally significant polymorphisms in the cod-
ing sequences of both the human MT1 and the human
MT2 melatonin receptors have been identified and found
to be associated with some diseases and disorders (Li
et al., 2013; Comai and Gobbi, 2014). A few of the
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 507
reported receptor polymorphism associated diseases
include, but are not limited to, breast cancer (MT1 and
MT2; Deming et al., 2012), type 2 diabetes mellitus
(MT2; Bonnefond et al., 2012) and multiple sclerosis
(MT2; Natarajan et al., 2012). As little is known about
the consequences of complete melatonin membrane
receptor deletion on brain function and behavior, our
primary objective was to identify any behavioral, neuro-
biological or molecular differences in melatonin receptor-
deficient mice as compared to mice expressing both
receptors. Our findings point to a significant role for mel-
atonin receptor signaling in brain development and in
the regulation of behavioral activity through actions on
major intracellular signal transduction pathways and on
both excitatory and inhibitory synaptic communication.
EXPERIMENTAL PROCEDURES
Ethics statement
All mice were housed and handled in accordance with
Federal animal welfare guidelines and in compliance
with the Public Health Service Policy on Humane Care
and Use of Laboratory Animals (2002) and the Guide for
the Care and Use of Laboratory Animals (8th Edition).
Experiments were reviewed and approved prior to being
carried out by the Institutional Animal Use and Care
Committee (IACUC) of the Florida State University
(Protocols #1016, 1135; Association for Assessment
and Accreditation of Laboratory Animal Care
International accreditation unit #001031; Office of
Laboratory Animal Welfare Assurance #A3854-01).
Animals
Mice in this study were progeny of a University of
Massachusetts Medical School mouse colony, where
the melatonin receptor knockout strains were generated
by a genetic mutation introduced by homologous
recombination (Liu et al., 1997; Jin et al., 2003). Sites
within exon 1 of both the melatonin type 1 receptor gene
(MT1) and the melatonin type 2 receptor gene (MT2) were
replaced with phosphoglycerate kinase promoter (PGK-
neomycin) cassettes. In each case, the clones carrying
the targeted allele were injected into C57BL/6 mouse
blastocytes. The resulting chimeric animals were bred to
C57BL/6 animals to obtain offspring that were heterozy-
gous for the knockout allele. These animals were then
backcrossed for 10 generations to C3H/He mice because
others have used the C3H strain extensively in examining
circadian behavioral responses to melatonin. The C3H
strain also has rhythmic melatonin production (Ebihara
et al., 1986) unlike most other inbred strains of mice,
including the C57BL/6 (Roseboom et al., 1998;
Kasahara et al., 2010). The resultant C3B6 (C3H/
He + C57BL/6) melatonin type 1 receptor-deficient
homozygous mice (MT1�/�) were crossed with C3B6
melatonin type 2 receptor-deficient mice (MT2�/�) to obtain
MT1�/�/MT2
�/� double-mutant mice, hereafter referred to
as double-knockout (DBL-KO) mice, and their non-trans-
genic (NT) wild-type counterparts, hereafter referred to
as NT mice.
Multiple cohorts of 6–10 male mice were randomly
selected from breeders of the DBL-KO and NT lines for
multiple aspects of this study: circadian, behavioral, a
melatonin challenge, or serum collection. Some animals
were sacrificed at specific ages for tissue analysis (e.g.,
long-term potentiation (LTP) at 6 months of age), while
other cohorts were maintained for behavioral testing at
3, 6, 12, and 15 months of age. Prior to experimentation
at each age, animal weights in grams were 24.2 ± 2.4,
29.8 ± 3.8, 33.6 ± 4.9, 32.9 ± 3.5 respectively, and
there were no significant differences between genotype
groups at any age. The animals were housed
individually in a polycarbonate cage (Ancare, Bellmore,
NY; 19 cm � 29 cm � 13 cm) with hardwood laboratory
bedding chips (Nepco Beta Chip�, Warrensburg, NY),
nesting material (Ancare Nestlet), and a polycarbonate
mouse igloo (Bio-Serv, Flemington, NJ) for enrichment.
They were maintained under a 12-h light–dark cycle
(7 am to 7 pm) at 21.1 �C and given ad libitum access to
LabDiet� 5001 Rodent Chow and water. Daily water
intake was averaged from bottle weight measurements
every 5–7 days for 4 weeks.
Genotyping by polymerase chain reaction (PCR)
At approximately 21 days of age, an IACUC and
veterinarian recommended inhalation anesthetic,
isoflurane (Butler Schein, Dublin, OH; 029405) was used
to sedate the animal. Once anesthetized, 2-mm terminal
segment of tail was removed with a sterile scissors.
Hemostasis was achieved using a silver nitrate applicator
stick (Butler Schein; 005383) and potential pain and
discomfort were alleviated by applying bupivacaine
hydrochloride in sterile isotonic solution (Sigma, St.
Louis, MO; B5274; 2.5 lg/mL) locally to the excision site
for long-acting pain management. Post-surgical excision
site monitoring occurred for 10 days. The tail biopsy was
placed in 250 lL of DirectPCR Tail lysis buffer (Viagen
101-T) with 10 lL Proteinase K solution (Viagen 501-K)
and lysed for 6 h at 55 �C. The sample was then
incubated at 85 �C for 45 min and briefly centrifuged. The
supernatant containing the genetic material was
collected and stored at �20 �C for subsequent PCR
analysis. Primers used to amplify the MT1 receptors
included mMT1R-FW-WT (50-GAAGTTTTCTCAGTGTC
CCGCAATGG-30), mMT1R-REV-WT (50-GAGTCCAAGT
TGCTGGGCAGTGGA-30), and mMT1R-NEO-FW-KO
(50-CCAGCTCATTCCTCCACTCATGATCTA-30). The
genomic DNA (deoxyribonucleic acid) was subjected to a
3-min hot start at 94 �C and then 35 cycles of 45 s at
94 �C, 45 s at 60 �C, and then 3 min at 72 �C, with a final
stage of 7 min at 72 �C. Primers used to detect the MT2
receptor alleles included a common forward primer
(mMT2R-FW-WT 50-CCAGGCCCCCTGTGACTGCCCG
GG-30), a gene-specific reverse primer from intron 1
(mMT2R-REV-WT 50-CCTGCCACTGAGGACAGAACAG
GG-30), and a reverse primer based on the PGK-neo
cassette (mMT2R-NEO-REV-KO 50-TGCCCCAAAGGC
CTACCCGCTTCC-30). These genomic DNA samples
were subjected to a 3-min hot start at 94 �C, 35 cycles of
30 s at 94 �C, 30 s at 68 �C, and 1 min at 72 �C, with a
final 7-min stage at 72 �C. The resulting samples were
508 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
loaded on to a 1.5% agarose gel (EMDMillipore OmniPur�
2120-OP) for 25 min of electrophoresis at 110 V and
subsequently imaged on a Bio-Rad Gel Doc� XR (Fig. 1).
Melatonin determination
To validate that both the DBL-KO and NT mice used in
this study were endogenously synthesizing melatonin in
a rhythmic fashion, blood serum was collected for
analysis by radioimmunoassay. Collection occurred
during the night under red light conditions at 7 pm,
10 pm, 1 am, and 4 am and during the day in white light
at 7 am and 1 pm. Three littermates of each genotype
were assigned to a single time point. Mice were
anesthetized with isoflurane and blood was collected via
a puncture to the vascular bed at the back of the jaw
where the orbital veins, the submandibular veins, and
the other veins draining the facial area coalesce to form
the jugular vein. The exuding droplets of blood (�0.3–0.4 mL) were collected into an EDTA tube, after which,
a sterile gauze compress was applied to the puncture
site for 20 s. This rapid blood collection method is more
humane than retro-orbital or cardiac puncture, leaving
the animals unaffected with no signs of distress (Golde
et al. 2005). Whole blood was centrifuged at 2000g for
3 min to separate the serum, which was sent frozen in
light-protective vials to a commercial diagnostics com-
pany (Pharmasan, Inc. Osceola, WI, USA) for melatonin
radioimmunoassay.
Behavioral testing
DBL-KO and NT mice were tested in either six-week
circadian running wheel study or in a behavioral battery,
which included Open Field activity, elevated plus maze
(EPM), Rota-rod, Y-Maze, Platform Recognition, and
Barnes Maze. For all behavioral testing, testers were
blinded to the animal identification number and their
respective genotypes and were only allowed to know
the artificial 5-digit alphanumeric scheme created to
identify the animals. Behavioral tests were performed
either during the 12-h light phase in white light or during
the 12-h dark phase, corresponding to the active phase
for mice, under red light or white light conditions.
Fig. 1. PCR analysis of mouse genomic DNA from wild-type, heterozygous,
PCR using a cocktail of three primers: mMT1R-REV-WT (primer A), mMT1R
distinct bands representing the wild-type and disrupted alleles. The wild-type
allele (primers A–C) produces a 240-bp product. (MT2) Genomic DNA PCR
(primer B), and mMT2R-NEO-REV-KO (primer C) to produce distinct bands
(primers A–B) produces a 272-bp product, while the disrupted allele (primer
Light-phase testing occurred during the hours of 8 am to
noon. Dark-phase testing occurred during the hours of
7 pm to midnight, and the rodents were shielded from all
non-red light sources during transport to and from the
testing chamber during the dark phase. For any
behavioral tests conducted both during the active and
inactive phases at a single age, the same mice were
used for testing in both the phases. Video recording
and/or computer monitoring was utilized to provide
accurate analysis of results at a later date. Testing was
done in male mice at multiple ages in independent
cohorts (3mo n= 3–5; 6mo n= 5–6; 12mo n= 10–11;
15mo n= 4–6).
Circadian behavior. For assessment of circadianrhythmicity, 6-month-old mice of both genotypes were
singly housed in cages equipped with running wheels.
All mice were entrained in a cycle of 12-h light and 12-h
dark (LD) for at least 1 week before they were released
into constant darkness (DD). After 2 weeks in DD mice
were returned to LD for 1 week before another exposure
to DD. Circadian period duration, or tau, was
determined by using a best-fit regression line followed
by a chi-squared (v2) periodogram analysis function in
Clocklab (Actimetrics, Evanston, IL, USA). Tau was
calculated from activity onset during the final DD
episode. Time to re-entrain was determined by number
of days required to obtain a 24-h rhythm in LD
conditions after the first DD exposure.
Anxiety behavior. To assess anxiety, animals were
evaluated using a near-infrared (NIR) backlit Elevatedplus maze (EPM; Med Associates, Inc., St. Albans, VT,
USA) consisting of an elevated plus-shaped maze with
two opposite open arms (50 cm � 10 cm) and two
opposite closed arms (50 cm � 10 cm with 40-cm walls).
The tests were conducted at night from 7:30 pm to
10 pm under red lighting, with NIR backlight for animal
tracking. The task was initiated by placing the test
subject into the center of the maze facing an open arm.
Activity was monitored via a mounted overhead camera
and video tracking system for a 5-min period. Entries
and homozygous mutant mice. (MT1) Genomic DNA was subjected to
-FW-WT (primer B), and mMT1R-NEO-FW-KO (primer C) to produce
allele (primers A–B) produces a 480-bp product, while the disrupted
with three primers: mMT2R-FW-WT (primer A), mMT2R-REV-WT
representing the wild-type and disrupted alleles. The wild-type allele
s A–C) produces a 550-bp product.
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 509
into each arm, and the time spent in open arms, closed
arms, and center of maze were evaluated.
Sensorimotor/locomotor behavior. An accelerating
Rota-rod treadmill (Med Associates, Inc., St. Albans,
VT, USA) was used to assess motor and coordination
deficits. In this test, the mouse was placed on a rotating
horizontal rod, which was 3.2 cm in diameter. The test
was conducted at night from 8 pm to 10:30 pm under
red light conditions. In each trial, the animal was placed
on the rod and allowed to acclimate while the rod
rotated at a constant 4 rotations per minute (rpm).
Subsequently, the speed was accelerated from 4 to
40 rpm. Latency in seconds was recorded when either
the mouse dropped to the platform (16.5 cm) or until
5 min elapsed.
Platform Recognition is a swimming-based
sensorimotor task often used to measure the animal’s
ability to visually identify/recognize a variably placed
elevated platform, and its ability to approach and
ascend on to the surface of the platform. Specifically,
this test was utilized as a means of determining if any
animal had visual deficits that could impair their ability to
perform in later cognitive tests, as mice on a combined
C3H background can have retinal degeneration. The
test was conducted at night from 8 pm to 11 pm under
red lighting in a pool that measured 88 cm in diameter
and 21 cm deep and was separated into four equal
quadrants by lines suspended above the pool. The
temperature of the water was held at 26 �C. A 9 cm
circular platform with a prominent black ensign attached
was raised 0.8 cm above the surface of the water. The
platform had a textured surface to aid the mouse in
climbing onto the surface and to encourage the mouse
to stay on the platform. A 61-cm high circular barrier
was placed around the pool to lessen the escape of the
mice. The mice were given four, 60-s maximum trials
per day for 4 days. On each day, the first two trials were
consecutive and were separated by a 30-min gap before
the next two consecutive trials were performed. The
mice began each trial facing the wall of the pool in
the same quadrant for each trial for that particular day.
The platform was placed in a different quadrant at the
start of each day and the mice were started from a
different quadrant than the previous day. For each trial,
the escape latency was recorded in seconds, concluding
when the mouse obtained the platform. A 30-s stay on
the platform between consecutive trials was
encouraged. A full 60 s was recorded for any animal
that did not obtain the platform and the mouse was
gently guided toward the platform and encouraged to
remain on the platform for 30 s. During the 30-min gap
between consecutive trials and after daily testing, the
mice were washed and placed under warming lamps to
dry. For statistical analysis, escape latencies were
averaged for the first two trials and the second two trials
per day.
An Open Field activity test was used to measure
exploratory behavior and general activity. The mice
were individually placed into an open field box (45 cm
long � 43.9 cm wide � 30 cm high). The area was
divided into a 4 � 4 grid (16 squares) with each square
measuring 11.8 cm � 11.2 cm. Testing was conducted
during both the active dark phase (7 pm–10 pm) and the
inactive light phase (10 am–12 pm). Mice were given
one 4-min trial in which they were free to move around
the box. Activity was scored as the number of line
crossings by the mouse during the trial. A line crossing
was defined as all four limbs entering into a new square.
The Y-Maze was used also to measure general
activity. The maze consisted of three equal arms (47 cm
long � 16 cm wide � 30.1 cm high) with an equilateral
19-cm triangular center compartment. The center could
be blocked off from all three arms by the insertion of
sliding panels. Visual cues were placed around the
room as well as on the maze arms. Each mouse was
given one minute in the blocked-off center compartment
to acclimate to the maze. At the end of one minute, the
panels blocking entry into the three arms were removed
to allow the mice full access to the maze for 8 min. The
number of entries into each arm and the sequence of
entries were recorded. An entry was defined as all four
limbs entering an arm. Testing was conducted during
both the active dark phase (7:30 pm–11:30 pm) and the
inactive light phase (8 am–11 am). Total number of
entries was used to determine activity.
Cognitive behavior. The Barnes Maze (Med
Associates, Inc., St. Albans, VT, USA) is a type of
delayed match-to-place experiment used to assess
spatial/reference learning and memory. Visual objects
arranged around the maze served as spatial cues and
testing occurred from 7:30 pm to 10 pm. The circular
platform was 121.8 cm in diameter and 140.2 cm high
with 40 equally spaced holes on the periphery of the
platform. A dark box filled with bedding was positioned
under one of the holes on the platform to allow the
mouse to escape from aversive stimuli. Each mouse
was assigned a specific unique escape hole for the
duration of the experiment. For each trial, the mouse
was placed in the center of the platform facing away
from its target escape hole. The aversive stimuli
included one high-intensity fan blowing at the level of
the platform and two 120-watt flood lamps hung from
the ceiling near to and aimed at the platform. Each
mouse was given one 600-s trial per day for 4 days and
the escape latency in seconds was recorded for each
trial. A full 600 s was recorded for any animal that did
not find its target escape hole during a trial.
The number of alternating entries in the Y-Maze was
also used to assess basic memory function (refer to
protocol above). For this, the number of entries that
were different from the two preceding entries was
divided by the total number of entries. This normalizes
the results to account for differences in speed through
the maze during the 8-min trials.
LTP
Hippocampal slices (350-lm thick) were cut from brains
of C3B6 mice at 6–7 months of age in a chilled (4 �C)low-Ca2+, low-Na+ ‘‘cutting solution’’ containing the
following (in mM): 230 sucrose, 10 D-glucose, 26
510 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, and 0.5
CaCl2 (equilibrated with 95% O2/5% CO2). Slices were
allowed to equilibrate in oxygenated artificial
cerebrospinal fluid (aCSF; in mM: 126 NaCl, 26
NaHCO3, 3 KCl, 1.25 NaH2PO4, 2 MgSO4, 2 CaCl2, and
10 D-glucose, pH 7.4), first at 32 �C for 1 h and
subsequently at room temperature before being
transferred to the recording chamber. Recordings were
obtained at 32 ± 1 �C from neurons of the CA1 region,
visualized through a 63� objective under Infra-red
Differential Interference Contrast (IR-DIC) optics.
Recording electrodes (1.2–2-lm-tip diameters; 3–6 MX)
contained the following (in mM): for current-clamp
experiments, 105 potassium gluconate, 30 KCl, 10
HEPES, 10 phosphocreatine, 4 MgATP, 0.3 GTP, and
20 biocytin (adjusted to pH 7.3 with KOH). Slices were
maintained in oxygenated (95% O2/5% CO2) aCSF.
Stimulating electrodes placed on the Schaffer collaterals
(S: bipolar CE-2C75, FHC) delivered constant current
pulses 50 ls in duration and 100–500 lA in amplitude
at low frequencies (0.1–0.3 Hz) to activate the Schaffer
collaterals. Tetanus was applied in bursts consisting of
5–8 stimuli at 100 Hz, burst interval ranged from 250 ms
to 5 s. Excitatory postsynaptic responses (EPSPs) were
recorded with an Axon� MultiClamp� 700B amplifier and
pClamp� software (Molecular Devices), filtered at
1–2 kHz (10 kHz for current-clamp), digitized at
10–20 kHz, and stored digitally.
Tissue collection for protein and transcript analysis
All mice were euthanized between the hours of 8am and
11am by sterile intraperitoneal overdose injection of
ketamine (Butler Schein; 100 mg/kg) and xylazine
(Vedco, St. Joseph, MO; 10 mg/kg), followed by
transcardial 0.9% sterile saline perfusion. Brains were
quickly removed and placed on an ice-cold plate for
dissection. The brain was initially bisected along the
sagittal plane and the left hemisphere was dissected for
the hippocampus (HPC) and frontal cortex. The
dissected samples were then flash frozen for
subsequent protein analysis. The right hemisphere was
similarly dissected and preserved in QIAGEN RNAlater�
RNA stabilization reagent for later mRNA (messenger
ribonucleic acid) analysis by quantitative polymerase
chain reaction (qPCR).
Isolation of protein from brain tissue andimmunoblotting
Hippocampi or frontal cortices (�10 mg per sample) were
disrupted and homogenized in 300 lL standard HEPES
protein extraction buffer containing Thermo Scientific
Halt� phosphatase inhibitor cocktail and Roche
Complete protease inhibitor cocktail. Samples were
centrifuged at 10,000g for 10 min at 4 �C. The
supernatant containing the solubilized proteins was
transferred to a new tube for a second spin. The final
supernatant was analyzed on a Thermo Scientific
Nanodrop photometer to determine protein
concentration. 2� SDS (sodium dodecyl sulfate) dye
was added in an equal volume and samples were
stored at �80 �C for later immunoblotting. SDS–PAGE
(sodium dodecyl sulfate–polyacrylamide gel
electrophoresis) immunoblotting was performed and
proteins were detected via the LI-COR Odyssey�
infrared imaging system with secondary antibodies
sensitive to 685- and 785-nm excitation wavelengths. All
primary antibodies were rabbit polyclonals from Cell
Signaling Technologies, with the exception of goat anti-
b-actin (Abcam, Cambridge, MA), rabbit anti-GluR1
(Abcam) and rabbit anti-spinophilin, courtesy of
Dr. Charles Ouimet (Allen et al., 1997). Levels of b-actinremained consistent for both DBL-KO and NT samples.
Isolation of mouse brain mRNA and real-time qPCR
Total cellular RNA was extracted from homogenized
frontal cortices with QIAShredder� columns in
QIAGEN’s buffer RLT and b-mercaptoethanol in a 100:1
ratio followed by processing with the QIAGEN� RNeasy
Kit� and DNase Set treatment kits according to the
manufacturer’s protocols. The RNA concentration was
measured with a Thermo Scientific Nanodrop
photometer. For analysis of transcript levels, one lg of
total RNA was reverse-transcribed to cDNA
(complementary deoxyribonucleic acid) by means of the
Bio-rad iScript� reverse-transcription system.
Amplification of the cDNA was performed on a Bio-Rad
CFX96 Touch� Real-Time PCR Detection System using
the iQ SYBR� Green Supermix protocol according to
the manufacturer’s specifications. The following thermal
cycling parameters were used: initial heat activation of
the DNA-polymerase was performed at 95 �C for 5 min.
Thereafter, 40 cycles of 95 �C (10 s), 57–60.5 �C (10 s)
and 72 �C (30 s) were run. The primer sequences and
annealing temperatures for the gene transcripts that
were analyzed are listed in Table 1.
Statistics
All calculations, comparisons and statistical analysis were
performed using GraphPad Prism version 6.0a software
for Mac OS X (GraphPad Software, La Jolla, California,
USA, www.graphpad.com). The values shown on the
graphs represent the means ± S.E.M. from independent
experiments. Molecular or behavioral differences in
genotype for a single test conducted only at night or
only in the day for a specific age were computed with
an independent (between subjects) Student’s t-test.Behavioral tests comparing differences at night versus
differences during the day were conducted with the
same animals and computed with a 2-way analysis of
variance (ANOVA) followed by Bonferroni’s multiple
comparison of means. Tests conducted with multiple
trials or time points were analyzed with a 2-way ANOVA
for repeated measures (mixed-design) and followed by
the Bonferroni test for post hoc comparisons. For
behavioral tests conducted longitudinally with different
cohorts (3mo, 6mo, 12mo, 15mo), Student’s t-test wasused to evaluate each age independently. Statistical
differences for the LTP data were measured with the
t-test (paired where indicated). Statistical values
reaching p 6 0.05 were considered significant.
Table 1. Gene-specific primers used for qPCR experiments
Gene Gene-specific primers 50–30 GenBank# Amplicon size Annealing
SOD1 S: ATG GCG ATG AAA GCG GTG TG NM_011434 460 59 �CAS: GCG CAA TCC CAA TCA CTC CA
GPx1 S: ATG TGT GCT GCT CGG CTC TC NM_008160 590 60 �CAS: TGC TGG GAC AGC AGG GTT TC
CAT S: AGG TTT GGC CTC ACA AGG AC NM_009804 239 58 �CAS: GCG GTA GGG ACA GTT CAC AG
Nrf2 S: AAC GAC AGA AAC CTC CAT CTA C NM_010902 94 57 �CAS: AGT AAG GCT TTC CAT CCT CAT C
Rp27 S: CCA GGA TAA GGA AGG AAT TCC TCC TG NM_024277 297 59 �CAS: CCA GCA CCA CAT TCA TCA GAA GG
MT1R S: GGA TAT GGG TCC TGG TCC TT NM_008639.2 215 58.5 �CAS: ACT AGC CAC GAA CAG CCA CT
MT2R S: ACA TCA CAG CCA TTG CCA TCA ACC NM_145712.2 186 60.5 �CAS: AGG TGC AGG AAT AGA TGC GTG GAT
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 511
RESULTS
Genotyping
Confirmation of genotypes for the MT1�/�/MT2
�/� DBL-KO
mice and their NT wild-type counterparts was performed
on tail snips of each mouse at approximately 21 days of
age (Fig. 1). Subsequently, RNA-Sequencing was
performed on the mouse frontal cortex and hippocampal
samples per previously published protocols (Mortazavi
et al., 2008). Sequencing was performed on an Illumina
HiSeq 2500 and confirmed that no melatonin receptor
expression was present in the DBL-KO mice, while both
MT1 and MT2 expression were clearly evident in NT sam-
ples. The RNA-Sequencing data were confirmed by
qPCR for both MT1 and MT2.
To further confirm a phenotype difference between the
two groups, we utilized a melatonin or vehicle challenge.
As part of a parallel study, we administered either
melatonin (100 lg/mL; Sigma M5250) or vehicle (0.1%
ethyl alcohol; Electron Microscopy Sciences 15058) for
10 months in the drinking water of both NT and DBL-KO
mice (n= 4–6). No differences were seen in health,
weight, or appearance post-treatment. For tissues
collected at 12 months, expression levels of superoxide
dismutase 1 (SOD1), glutathione peroxidase 1 (GPx-1),
and catalase (CAT) were determined in the frontal cortex
by quantitative PCR. Additionally, we measured the
transcript levels of nuclear factor erythroid 2-related
factor 2 (Nrf2), a transcription activator that binds to
antioxidant response elements in many genes involved
in cellular responses to oxidative stress. Fig. 2 shows
that, in all cases, the strong melatonin-receptor mediated
effects of melatonin treatment versus vehicle (0.1%
ethanol) on antioxidant signaling which are present in
wild-type mice are absent in melatonin receptor-deficient
mice. All evaluations: a 2-way ANOVA using between-
group experimental design; genotype � treatment;
n= 4–6 for all groups. Fig. 2A SOD1: genotype:
F(1,15) = 16.07, p= 0.0011; treatment: F(1,15) = 8.91,
p= 0.0093; interaction: F(1,15) = 19.50, p= 0.0005;
Bonferroni post hoc analysis revealed a difference
between Mel and EtOH treatment only in animals with
their receptors (p= 0.0001). Fig. 2B GPx-1: genotype:
F(1,17) = 1.644, p= 0.2170; treatment: F(1,17) = 7.507,
p= 0.0140; interaction: F(1,17) = 10.77, p= 0.0044;
Bonferroni post hoc analysis revealed a difference
between Mel and EtOH treatment only in animals with
their receptors (p= 0.0013). Fig. 2C CAT: genotype:
F(1,16) = 0.08874, p= 0.7696; treatment: F(1,16) =
5.412, p= 0.0335; interaction: F(1,16) = 3.190, p=
0.0931; Bonferroni post hoc analysis revealed a
difference between Mel and EtOH treatment only in
animals with their receptors (p= 0.0273). Fig. 2D Nrf2:
genotype: F(1,18) = 18.75, p= 0.0004; treatment:
F(1,18) = 14.00, p= 0.0015; interaction: F(1,18) = 3.948,
p= 0.0624; Bonferroni post hoc analysis revealed a
difference between Mel and EtOH treatment only in
animals with their receptors (p= 0.0022).
Serum melatonin determination by commercial RIA
Commercial radioimmunoassay of blood serum from both
DBL-KO and NT mice (n= 3 per genotype per time point)
confirmed that endogenous melatonin was rhythmic,
ranging from 10 pg/mL in the light to 100 pg/mL in the
dark (a 2-way mixed-design ANOVA for repeated
measures; time: F(5,20) = 4.940, p= 0.0042) and
present in similar levels in both genotypes at each time
point (genotype F(1,4) = 0.00046, p= 0.9839; Fig. 3).
The peak value for melatonin was at Zeitgeber time 15,
corresponding to 3 h after lights off. Lowest melatonin
levels were recorded between Zeitgeber time 6 and 12,
or 6–12 h after lights came on as reported by others
(Vivien-Roels et al. 1998; Kennaway et al. 2002).
Behavior
Circadian behavior. Six-month male NT and DBL-KO
mice were assessed for free-running periods under
constant dark conditions (DD) and for their ability to
entrain to LD cycles (Fig. 4). The free-running period
(tau) for NT mice was determined to be 23.25 h as
compared to 23.73 h for the DBL-KO mice (NT n= 4
and DBL-KO n= 5; Student’s t-test; p= 0.028).
Re-entrainment to LD for DBL-KO mice was
Fig. 2. qPCR analysis after melatonin or vehicle challenge in the frontal cortex of 12-month NT and DBL-KO mice. Four key elements of the
antioxidant pathway respond to melatonin administration (MEL; shaded) vs. vehicle (EtOH; unshaded) in mice expressing the melatonin receptors
(NT; blue) but not in mice lacking the receptors (DBL-KO; red). The results show RP27 normalized mRNA expression levels of (A) superoxide
dismutase (SOD1), (B) glutathione peroxidase (GPx-1), (C) catalase and (D) nuclear factor erythroid 2-related factor 2 (Nrf2) in NT vs DBL-KO mice
(a 2-way ANOVA, p 6 0.05 for all comparisons; error bars represent SEM; n= 4–6 for all groups). (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
512 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
1.80 ± 0.84 days compared to 3.75 ± 2.22 days for NT
mice (Student’s t-test; p= 0.109). Light onset for re-
entrainment was initiated during the subjective day for
both the DBL-KO mice and NT mice to compensate for
differences in phase after 2 weeks in DD.
Anxiety behavior. Assessment of anxiety/emotionality
in rodents is commonly made using the EPM, which
incorporates both open and closed arms, the mice
generally preferring the closed arms. Thus, the greater
the time spent in the open arms is considered a
measure of decreased anxiety. As seen in Fig. 5, mice
tested at 6, 12 and 15 months of age showed no
significant differences (p> 0.05), despite a clear trend
to less anxiety (more time spent in the open arms) in
the DBL-KO mice at 6 months of age. Striped bars
indicate nighttime testing. (Student’s t-test; 6mo: NT
n= 6 and DBL-KO n= 5, p= 0.1818; 12mo
NT n= 11 and DBL-KO n= 10, p= 0.7311; 15mo NT
n= 6 and DBL-KO n= 4, p= 0.1725.) There were
also no significant group differences seen in the ratio of
open arm entries to closed arm entries.
Sensorimotor behavior. No evidence of sensorimotor
deficits was seen on the Platform Recognition or Rota-
rod tests at any of the ages tested (Fig. 6). There were no
statistical differences between groups, trials or the
interaction in the Platform Recognition test and no
significant differences (p> 0.05) on Rota-rod
performance between the two genotypes at any age. It is
worth noting that these mice clearly had no visual deficits,
despite their C3H background, most likely because they
were out crossed for multiple generations to the C57BL/6
line. A single mouse with acute ophthalmological
problems was excluded from final analysis. Fig. 6A
Platform Recognition: a 2-way mixed-design ANOVA for
repeated measures; genotype: F(1,8) = 1.393,
p= 0.2718; trial: F(3,24) = 1.822, p= 0.1701;
interaction: F(3,24) = 0.7244, p= 0.5473; 6mo NT n= 6
and DBL-KO n= 5. Fig. 6B Rota-rod: Student’s t-test;6mo: NT n= 6 and DBL-KO n= 5, p= 0.6356; 12mo
NT n= 10 and DBL-KO n= 10, p= 0.2396; 15mo NT
n= 6 and DBL-KO n= 4, p= 0.1506.)
Increased locomotor activity in DBL-KO mice. Assess-
ment of locomotor activity beginning at 3 months of age
Serum Melatonin (RIA)
0 6 12 15 18 210
20
40
60
80
100
120
ZT (Hours)
Mel
aton
in (p
g/m
L)
NTDBL-KO
Fig. 3. Radioimmunoassay (RIA) for serum melatonin in 6-month NT
and DBL-KO mice. Commercial radioimmunoassay of blood serum
from both DBL-KO (red) and NT (blue) mice confirmed that endo-
genous melatonin was rhythmic and present in similar levels (n= 3
mice per genotype per time point). Zeitgeber time 0 corresponds to
light onset (a 2-way RM ANOVA, p > 0.05). (For interpretation of the
references to color in this figure legend, the reader is referred to the
web version of this article.)
Elevated Plus Maze
6 mo 12 mo 15 mo0
50
100
150
Tim
e(s
)
DBL-KONT
Fig. 5. Analysis of anxiety in NT and DBL-KO mice at multiple ages.
The elevated plus maze (EPM) revealed no significant difference
between the NT (blue) and DBL-KO (red) mice in the time spent in
open arms at any age (Student t-test, p 6 0.05; error bars represent
SEM). Striped bars indicate nighttime testing. 6mo n= 6 (NT) and 5
(DBL-KO); 12mo n= 11 (NT) and 10 (DBLO); 15mo n= 6 (NT) and
4 (DBL-KO). (For interpretation of the references to color in this figure
legend, the reader is referred to the web version of this article.)
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 513
was performed using both the Y-Maze and the Open Field
tests. Initially, at 3 months of age, a trend toward a lower
total number of entries was seen in the Y-Maze by the
DBL-KO mice as compared to control NT mice,
irrespective of time of day (NT n= 3 and DBL-KO
n= 5; Fig. 7A – 3mo: a 2-way ANOVA using between-
group experimental design; genotype � time; genotype:
F(1,12) = 7.292, p= 0.0193; time: F(1,12) = 0.0582,
p= 0.8135; interaction: F(1,12) = 0.7892, p= 0.3918;
Fig. 7B – 3mo: a 2-way ANOVA using between-group
experimental design; genotype � time; genotype:
F(1,12) = 1.335, p= 0.2705; time: F(1,12) = 0.3202,
p= 0.5819; interaction: F(1,12) = 0.5782, p= 0.4617;).
By 6 months of age, the control mice were making
substantially fewer entries in the Y-Maze than they were
Fig. 4. Circadian behavior in 6-month NT and DBL-KO mice. Representative
mice maintained under 12-h light:12-h dark (LD), then constant darkness (DD
running period in constant darkness was 23.25 h for the NT mice, and 23.73
indicate wheel running and recordings from consecutive days are stacked in
second day of each horizontal bar were re-plotted as those for the first day
when younger such that their performance was then
significantly below that of the DBL-KO mice, also
irrespective of time of day. NT n= 6 and DBL-KO
n= 4; a 2-way ANOVA using between-group
experimental design; genotype � time; genotype:
F(1,13) = 14.55, p= 0.0021; time: F(1,13) = 1.113,
p= 0.3106; interaction: F(1,13) = 0.1394, p= 0.7149;
Bonferroni post hoc analysis revealed a significant
difference between genotypes during the day
(p= 0.0388) and at night (p= 0.0336). The activity
level of juvenile male mice is known to be higher than
that of adult male mice (Weinert and Waterhouse,
1999). At 12-months of age, the DBL-KO mice continued
to make significantly more entries in the Y-Maze than the
controls (NT n= 11 and DBL-KO n= 10; Student’s
t-test, p= 0.0460). Similar to the Y-Maze, a significantly
higher level of activity was exhibited by the DBL-KO
in the Open Field test (Fig. 7B) at 6 months of age
double-plotted wheel-running actograms for NT (A) and DBL-KO (B)
), followed by a repeat of both photoperiods to calculate tau. The free-
h for the DBL-KO mice (Student t-test, p= 0.028). Black markings
rows. Each horizontal bar represents 2 days (48 h). The data for the
of the next horizontal bar. n= 4 (NT) and 5 (DBL-KO).
Platform Recognition
1 2 3 40
10
20
30
40
Trial
Late
ncy
to P
latfo
rm (s
)
NTDBL-KO
Rota-rod Balance
6 mo 12 mo 15 mo0
100
200
300
Bal
ance
Mai
ntai
ned
(s)
DBL-KONT
A
B
Fig. 6. Analysis of sensorimotor behavior in 6-, 12-, or 15-month NT
and DBL-KO mice. (A) A test of visual and motor skills in a water
maze Platform Recognition test revealed no significant differences
between the NT (blue) and DBL-KO (red) mice at 6 months of age.
Animals in both groups were able to see, swim to, and climb onto the
platform and trended toward improvement between the first and last
trials. (B) The Rota-rod revealed no significant difference at any age
between the NT (blue) and DBL-KO (red) mice in the length of time
balance was maintained on a rotating horizontal rod that accelerated
from 4 to 40 rpm (a 2-way RM ANOVA for A and Student t-test for B;p 6 0.05; error bars represent SEM). Striped bars indicate nighttime
testing. Platform Recognition and Rota-rod 6mo n= 6 (NT) and 5
(DBL-KO); Rota-rod 12mo n= 10 (NT) and 10 (DBL-KO); Rota-rod
15mo n= 6 (NT) and 4 (DBL-KO). (For interpretation of the
references to color in this figure legend, the reader is referred to
the web version of this article.)
514 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
(NT n = 6 and DBL-KO n = 4; Student’s t-test, p =
0.0284) and 12 months of age (NT n= 11 and DBL-KO
n= 10; Student’s t-test, p= 0.0238). By 15-months of
age these differences are attenuated in both testing mea-
sures (15mo NT n= 6 and DBL-KO n= 4; Student’s
t-test, p= 0.3112 for Y-Maze and p= 0.2444 for Open
Field).
Increased water consumption in DBL-KO mice. In
accordance with the increased free-running period
(Fig. 4) and increased locomotor activity (Fig. 7) noted
in the DBL-KO mice, daily water consumption was also
found to be significantly higher in the DBL-KO mice as
compared to control NT which consumed close to the
daily averages reported for mice (5.8 ± 0.2 mL/day)
(Bachmanov et al., 2002). Six-month old NT mice con-
sumed 5.77 ± 0.10, mL/day (n= 10), while the DBL-
KO mice consumed 7.29 ± 0.15 mL/day (n= 10;
Student’s t-test, p< 0.0001).
Cognitive enhancement in male DBL-KO mice. Mice
were tested for cognitive performance with a standard
behavioral test for spatial/reference learning (Barnes
Maze) and basic working memory (Y-Maze). While both
groups of mice showed learning in the Barnes Maze (day
4 was significantly different than day 1), the DBL-KO
mice had a significantly lower latency to find the target
escape hole on days two and three compared to their NT
counterparts (Fig. 8A Barnes Maze: a 2-way mixed-
design ANOVA for repeated measures; NT n= 6 and
DBL-KO n= 4; genotype: F(1,8) = 0.6253, p= 0.4519;
days: F(3,24) = 22.20, p< 0.0001; interaction: F(3,24) =
4.781, p= 0.0095). For the DBL-KO, Bonferroni post hoc
analysis revealed significant differences between day 1
vs. 2 (p< 0.0001), day 1 vs. 3 (p< 0.0001), and day 1
vs. 4 (p< 0.0001). For the NT, Bonferroni post hoc
analysis revealed no significant differences between day
1 vs. 2 (p= 0.1663) or between day 1 vs. 3
(p= 0.0595), but a significant difference between day 1
vs. 4 (p= 0.0008). DBL-KO mice also showed a
statistically significant elevation in the number of arm
alternations in the Y-Maze when assessed during the day
and a similar trend when assessed at night (Fig. 8B
Y-Maze: a 2-way ANOVA using between-group
experimental design; genotype � percent alternation; NT
n= 7 and DBL-KO n= 9; genotype: F(1,24) = 12.81,
p= 0.0015; percent alternation: F(1,24) = 1.868,
p= 0.1844; interaction: F(1,24) = 0.1719, p= 0.6821;
Bonferroni post hoc analysis between genotypes during
the day (p= 0.0143) and at night (p= 0.0829).
LTP heightened in male DBL-KO mice
Behavioral data from both the Barnes Maze and the
Y-Maze alternation task revealed enhanced cognition in
the DBL-KO mice compared to the NT mice. Hence, in
order to determine whether a neurobiological correlate
might exist, we assayed for alterations in synaptic
efficacy following tetanization. Tetani were applied to
the Schaffer collateral pathway in the HPC while
recording from pyramidal neurons in the CA1 (Fig. 9A).
Changes in amplitude of the minimally evoked EPSPs
were monitored before and after tetanization. We found
EPSPs in both groups to be significantly potentiated, but
potentiation in DBL-KO mice was greater compared to
the NT mice (p 6 0.001, t-test) (Fig. 9B). At �5 min
post-tetanization NT and DBL-KO EPSP amplitudes
averaged 195 ± 29% and 287 ± 25% of their pre-
tetanus baseline values respectively (100%, n= 9, 8
cells; p 6 0.05, paired t-test) (Fig. 9C). Potentiation was
observed in �90% of all trials. The potentiation in both
groups was long lasting (�45 min) and series
resistance, which was monitored periodically, remained
unchanged throughout the course of the experiments.
The averaged paired-pulse ratio (PPR), defined as the
Y-Maze Day v Night - 3mo
NT Day
NT Night
DBL-KO D
ay
DBL-KO Night0
20
40
60
Tota
l Ent
ries ns
Y-Maze Day v Night - 6mo
NT Day
NT Night
DBL-KO D
ay
DBL-KO Night0
20
40
60
Y-Maze Day - 12mo
NT
DBL-KO
0
20
40
60 p=.0460
Y-Maze Day - 15mo
NT
DBL-KO
0
20
40
60 ns
ns
Open Field Day v Night - 3mo
NT Day
NT Night
DBL-KO D
ay
DBL-KO Night0
50
100
150
200
Line
s C
ross
ed
ns
Open Field Night - 6mo
NT
DBL-KO
0
50
100
150
200 p=.0284
Open Field Day - 12mo
NT
DBL-KO
0
50
100
150
200 p=.0238
Open Field Day - 15mo
NT
DBL-KO
0
50
100
150
200
A
B
* *
Fig. 7. Analysis of locomotor activity in NT and DBL-KO mice at multiple ages. Locomotor activity was assessed by Y-Maze (A) and Open Field (B)
tests at four different ages. Juvenile NT mice (3mo) showed a trend toward higher activity levels as compared to DBL-KO mice in both tests. From
6mo onward, the DBL-KO (red) were significantly more active than the NT (blue) in both tests (a 2-way ANOVA or Student t-test, p 6 0.05; error
bars represent SEM). Where evaluated, striped bars indicate nighttime testing. Y-Maze and Open Field: 3mo = 3 (NT) and 5 (DBL-KO); 6mo n= 6
(NT) and 4 (DBL-KO); 12mo n= 11 (NT) and 10 (DBL-KO); 15mo n= 6 (NT) and 4 (DBL-KO). (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 515
ratio of the second to the first response, (50-ms interval)
remained invariant following tetanus in DBL-KO mice
(1.7 ± 0.1 vs. 1.4 ± 0.1, n= 6; p= 0.1, paired t-test)and in NT mice (1.4 ± 0.2 vs. 1.2 ± 0.1, n= 10;
p= 0.3, paired t-test), suggests that the potentiation in
both groups is mediated post-synaptically (Fig. 9D).
Protein analysis by immunoblotting
In view of our behavioral and electrophysiological data
pointing to neurobiological differences between control
and melatonin receptor-deficient mice (see above), we
decided to perform quantitative infrared fluorescence
immunoblotting for several key signaling and synaptic
markers in the brain. Levels of phosphoCREB
(normalized to constitutive CREB (cAMP response
element-binding protein) levels) were significantly
elevated in the frontal cortices of DBL-KO mice with a
similar non-significant trend in the HPC (n= 4–6 for all
analyses; Fig. 10A: t-test, p= 0.0056; Fig. 10G: t-test,p= 0.4325). Conversely, expression of phosphoERK1/2
(normalized to ERK1/2) was significantly lower in the
DBL-KO brain frontal cortex with a similar trend in the
HPC (Fig. 10B: t-test, p= 0.0404; Fig. 10H: t-test,p= 0.2574). PhosphoSynapsin levels (normalized to
synapsin) were also significantly lower in the DBL-KO
frontal cortex but not in the HPC (Fig. 10C: t-test,p= 0.0226; Fig. 10I: t-test, p= 0.4834). Protein
expression levels for the dendritic spine marker,
spinophilin was statistically less in both the frontal cortex
and HPC of DBL-KO mice (Figs. 10D: t-test, p= 0.0403
and 10J: t-test, p= 0.0007), as was the key enzyme of
GABA synthesis, glutamic acid decarboxylase 1 (GAD1;
Figs. 10E: t-test, p= 0.0464 and 10K: t-test,p= 0.0494). A significant elevation of the ionotropic
glutamate AMPA receptor subunit 1 (GluR1) was also
noted in the frontal cortex (Fig. 10F: t-test, p= 0.0417)
with a similar trend in the HPC (Fig. 10L: p= 0.4204).
DISCUSSION
The results of the present investigations demonstrate
molecular, electrophysiological, and behavioral
alterations in mice genetically lacking all known
G-protein-coupled membrane melatonin receptors. The
behavioral phenotype includes mild hyperactivity in DBL-
KO mice, a longer free-running circadian clock, and
enhanced cognition. No evidence of altered anxiety or
changes in sensorimotor performance was apparent.
Hippocampal function at the level of LTP was found to
be significantly enhanced in the DBL-KO animals.
Alterations in several key signaling and synaptic
markers also confirm the reorganizational outcome in
the brain of mice lacking melatonin receptors.
The two known mammalian melatonin receptors
(MTNRs) were cloned in the mid-1990s (Reppert et al.,
1994, 1995, 1996) and subsequently shown to be unique
G-protein-coupled membrane receptors based on the
molecular structure and chromosomal localization (cf.
Dubocovich et al., 2010). Both receptors signal via multi-
ple intracellular pathways, including inhibition of cyclic
AMP and cyclic GMP, activation of protein kinase C and
Barnes Maze
1 2 3 40
200
400
600
Days
Late
ncy
to E
scap
e H
ole
(s)
NTDBL-KO
Y-Maze Alternations Day v Night
NT Day
NT Night
DBL-KO D
ay
DBL-KO Night
0
20
40
60
80
Alte
rnat
ion
%
A
B
*
* ***
Fig. 8. Cognitive performance of 6-month NT and DBL-KO mice.
Barnes Maze results (A) revealed that the DBL-KO (red) mice learned
the testing paradigm by day two as compared to NT (blue) which
required until day four to demonstrate significant learning. Results
from the Y-Maze (B) revealed a greater percent alternation in the
DBL-KO mice as compared to NT in the day (p= 0.024) but not at
night. A 2-way RM ANOVA for A and a 2-way ANOVA for B; error
bars represent SEM; Barnes n= 6 (NT) and 4 (DBL-KO); Y-Maze
n= 7 (NT) and 9 (DBL-KO). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of
this article.)
516 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
extracellular signal-regulated protein kinase (ERK1/2),
and many other pathways (cf. Dubocovich et al., 2010).
Among the numerous downstream targets, we demon-
strate here (Fig. 2) that components of the neuronal anti-
oxidant response, i.e. Nrf2, SOD1, GPx-1 and CAT,
respond to melatonin treatment in an MTNR-dependent
manner.
Melatonin MT1 and MT2 receptors are expressed at
high levels in the embryonic brain (Rivkees and
Reppert, 1991; Seron-Ferre et al., 2007) and are presum-
ably activated by circulating maternal melatonin, which
crosses the placenta to affect the developing fetus
(Klein, 1972). For example, administration of melatonin
to pregnant, suprachiasmatic nuclei (SCN)-lesioned
female Syrian hamsters entrains circadian activity
rhythms in fetal and neonatal pups (Davis and Mannion,
1988; Grosse et al., 1996). However, only a very few
investigations have explored the role for MTNRs in neuro-
development. Melatonin treatment of mammalian neural
stem cells promotes their differentiation (Moriya et al.,
2007; Kong et al., 2008), while treatment of zebrafish
embryos with melatonin or MTNR antagonists modulates
the timing of neuronal differentiation (Danilova et al.,
2004; de Borsetti et al., 2011).
Although MTNRs are expressed in many areas of the
adult brain, most attention over the past years has been
aimed at MTNR expression by the cells of the
hypothalamic SCN, which serve to mediate the
feedback effects of circulating melatonin, whose
secretory rhythm is generated by the circadian activity
of the central clock in the SCN (cf. Dubocovich et al.,
2010). Melatonin acts on SCN neurons via MT1 receptors
to acutely hyperpolarize them (Liu et al., 1997) and via
MT2 (and possibly MT1) receptors to induce phase shifts
in both molecular and electrophysiological rhythms
(Sugden, 1983; Dubocovich et al., 2010). By inhibiting
SCN neurons, and probably other target neurons else-
where in the brain, melatonin is thought to modulate the
sensitivity of these cells to other inputs, such as activity-
dependent neurotransmitter secretion.
In the present study, we determined that animals
lacking MTNRs throughout development and into
adulthood do not show locomotor arrhythmia, i.e. the
basic mechanisms of circadian oscillation in the SCN
remains intact. This is also evident at the level of
melatonin secretory rhythms, which are similar in both
NT and DBL-KO mice (Fig. 3). However, the
endogenous free-running circadian period of the DBL-
KO mice is significantly lengthened as compared to NT
mice expressing both MTNRs (Fig. 4). We interpret
these results to indicate that circadian melatonin signals
from the maternal system, either during embryonic/fetal
neurodevelopment and/or during early postnatal life
(e.g. maternal milk) may play an important role in
shaping the molecular and cellular maturation of the
SCN clockwork and its output. Numerous mechanisms
could be envisioned, such as altered gene expression in
developing SCN neurons, or modulation of innervation
patterns; however, this remains to be explored more
fully. A limitation of the current investigation is the global
and irreversible nature of the melatonin receptor
deletion. In this regard, future studies with conditional
and tissue-targeted melatonin receptor silencing would
overcome this hurdle.
Melatonin, when administered acutely, has been
demonstrated to have sedative/hypnotic properties in
rodents (Sugden, 1983, 1995) and humans (Lieberman
et al., 1984; Dollins et al., 1993; Tzischinsky and Lavie,
1994). Acute administration to mice also decreases spon-
taneous motor activity and at very high doses produces
motor incoordination (Sugden, 1995). Our present find-
ings of significantly enhanced locomotor activity in both
the Y-Maze and Open Field tests (Fig. 7) is consistent
with the view that melatonin, even though it is released at
night during the mouse’s active phase, contributes net
Fig. 9. Long-term potentiation in hippocampi of 6-month NT and DBL-KO mice. (A) Hippocampal slice preparation showing placement of
stimulating electrode (S) on the Schaffer collaterals and recording electrode (R) in the CA1 region. (B) Plot of the ensemble data showing time
course of changes in EPSP amplitude (percentage of baseline, 100%= average of all control values) following tetanus (indicated by an arrow) of
DBL-KO (red) and NT (blue) mice. DBL-KO mice had significantly elevated EPSP compared to NT. For both, each point (closed circles) representsthe ensemble average across the indicated number of experiments of 5–7 consecutive measurements of EPSP amplitude. Error bars represent
SEM, where this is greater than the size of the symbols. Traces (B, inset) are averaged consecutive records (P15) of minimally evoked EPSPs
taken from 20 min post-tetanus region of the time course. Time course of changes in Rs (± SEM) (closed diamonds), a measure of series
resistance (% baseline) is also shown. (C) Bar plots representing the mean (±SEM) percentage potentiation of NT and DBL-KO EPSPs following
tetanus. (D) Traces are averaged consecutive records of EPSPs evoked during paired stimulation of NT and DBL-KO mice before and after tetanus.
Bar plots showing no significant changes in the mean (± SEM) paired-pulse ratio following tetanus for both NT (n= 10) and DBL-KO (n= 6) mice
measured across experiments. Statistically significant change (⁄, p< 0.05; ⁄⁄⁄, p< 0.001) with the t-test when comparing between NT and
DBL-KO mice or with the paired t-test when comparing before and after effects within groups; ns (no significant difference) p> 0.05. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 517
inhibitory signals to the brain’s motor system via binding to
the MTNRs. When these receptors are absent, such as in
the current studies, the animals show meaningfully higher
levels of locomotor activity. Consistent with our findings,
DBL-KO mice were reported to spend more of the 24-h
cycle awake as compared to mice with both MT1 and
MT2 receptors (Comai et al., 2013), which the authors pro-
pose is due to a reduction of REM sleep that accompanies
deletion of the MT1 receptor in combination with reduction
of non-REM sleep arising from loss of the MT2 receptor.
Recently, in a mutation screening of humans with attention
deficit/hyperactivity disorder (ADHD) a loss-of-function
mutation of MT1 was reported (Chaste et al., 2011). Addi-
tionally, damped rhythms of serum melatonin have also
been associated repeatedly with both pediatric and adult
ADHD (Van der Heijden et al., 2005; Van Veen et al.,
2010; Baird et al., 2012), and the clinical data thus far –
while still limited (cf. Bendz and Scates, 2010) – suggests
that melatonin administration may be effective in the treat-
ment of ADHD-associated insomnia, consistent with the
hypothesis that melatonin generally serves to inhibit motor
activity. Based on our results presented here, the DBL-KO
mouse may provide a useful model for understanding how
melatonin regulates locomotor activity.
Acute melatonin administration has been reported to
have anxiolytic effects in both animal models (Papp
et al., 2006; Crupi et al., 2010) and in human trials
(Srinivasan et al., 2006; Caumo et al., 2009). Recently,
Ochoa-Sanchez and colleagues (2012) reported that the
novel MT2-selective agonist, UCM765, showed anxiolytic
properties in the Open Field test and EPM. However, in
contrast to the prediction that DBL-KO mice might be
subject to a general anxiety disorder, our results find no
significant change – either during the day time (data not
shown) or night time – in the time spent in open arms of
the EPM or in the center portion of an Open Field test
(data not shown) when compared to the control NT mice.
These results largely conform to those recently published
by Comai and Gobbi (2014) using the MT2 knockout
mouse model, but they would appear to contrast with
the results of Larson et al. (2006) who reported signifi-
cantly greater time spent in the center of the Open Field
test in mice lacking only the MT2 receptor. Possibly the
complete lack of MT receptors in our DBL-KO mice
eliminated this MT2-specific effect. Alternatively, the time
spent in the center of the Open Field test arena may sim-
ply reflect altered decision-making functions rather than
anxiety per se (Rodgers and Johnson, 1995). Though
p-CREB / CREB
0.0
0.2
0.4
0.6
% R
elat
ive
IR F
luor
esce
nce p=.0056
p-ERK / ERK
0.00
0.05
0.10
0.15
% R
elat
ive
I R F
luo r
esce
n ce p=.0404
p-Synapsin / Synapsin
0.00
0.02
0.04
0.06
0.08
0.10
% R
elat
ive
IR F
luor
esce
nce p=.0226
Spinophilin / Actin
0.00
0.02
0.04
0.06
0.08
% R
elat
ive
IR F
luor
esce
nce p=.0403
GAD1 / Actin
% R
ela t
ive
IR F
luor
e sce
nce
0.00
0.02
0.04
0.06
0.08
0.10 p=.0464
GluR1/Actin
% R
ela t
i ve
IR F
luo r
esce
nce
0.0
0.2
0.4
0.6
0.8 p=.0417
Frontal Cortex Hippocampus
p-CREB / CREB
% R
elat
ive
IR F
luor
e sce
nce
0.0
0.5
1.0
1.5
2.0
p-ERK / ERK
0.0
0.2
0.4
0.6
0.8
% R
elat
ive
IR F
luor
esce
nce
p-Synapsin / Synapsin
0.000
0.002
0.004
0.006
0.008
0.010
% R
elat
ive
IR F
luor
esce
nce
Spinophilin / Actin
0.0
0.1
0.2
0.3
0.4
0.5
% R
elat
ive
IR F
luor
e sce
nce p=.0007
GAD1 / Actin
0.00
0.05
0.10
0.15
0.20
% R
elat
ive
IR F
luor
esce
nce p=.0494
GluR1 / Actin
0
1
2
3
4
% R
elat
ive
IR F
luo r
esce
nce
A B G H
I J
K L
C D
E F*
Representative dual-labeled fluorescent Western
(Green GluR1/Red Actin)
* NT DBL-KO100 kDa
42 kDa
DBL-KONT
Fig. 10. Immunoblotting results from frontal cortex and hippocampus of 6-month NT and DBL-KO mice using quantitative infrared
immunofluorescence. (A, G) Ratio of phosphoCREB to CREB signal. (B, H) Ratio of phosphoERK1/2 to ERK1/2 signal. (C, I) Ratio of
phosphoSynapsin to Synapsin signal. (D, J) GAD1 expression normalized to b-actin. (E, K) Spinophilin expression normalized to b-actin. (F⁄, L)GluR1 expression normalized to b-actin. ⁄Representative dual-labeled LI-COR fluorescent Western blot for GluR1/b-actin in frontal cortex. Student
t-test, p 6 0.05 for all comparisons; error bars represent SEM; n= 4–6 for all analyses.
518 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
previous reports show melatonin administration to have
anxiolytic effects, it should be remembered that compar-
ing acute hormonal effects in an intact animal is not inver-
sely equivalent with the consequences of genetic deletion
of that hormone’s receptor. The latter is most likely asso-
ciated with significant alterations in neurodevelopment
(see Discussion above).
Numerous previous studies have documented
melatonin effects on hippocampal functions (Collins and
Davies, 1997; Hogan et al., 2001; El-Sherif et al., 2002,
2003) including a concentration-dependent inhibition of
LTP, which could be blocked by selective melatonin
receptor antagonists (Wang et al., 2005) supporting
involvement of melatonin receptor signaling in the HPC.
Larson and colleagues (Larson et al., 2006) subsequently
reported that mice deficient in only the MT2 melatonin
receptor have significantly attenuated LTP responses as
compared to control animals. Additionally, this group
found that whereas control mice quickly developed a pref-
erence for the closed arms of an EPM, the MT2-deficient
G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521 519
mice showed significantly less preference for the closed
arms. Our results with the EPM (Fig. 5) are in fact consis-
tent with the findings of Larson et al. [ibid] although we
interpret the data as a trend toward less anxiety in the
DBL-KO mice. On most measures of cognitive perfor-
mance in the present investigation (Fig. 8) mice lacking
all melatonin receptors performed significantly better than
the intact NT mice. The Barnes Maze data are consistent
with the strong differences in hippocampal LTP (Fig. 9)
such that the DBL-KO mice showed enhanced LTP. Indi-
rect support of our data may be found in the results of
O’Leary et al. (2011) who examined learning performance
on the Barnes Maze in 13 mouse strains and reported that
among the highest performing strains was the C57BL/6 J
mouse, which is genetically compromised in its ability to
synthesize melatonin (Roseboom et al., 1998; Kasahara
et al., 2010), i.e. they develop in uterowithout the maternal
melatonin signal. Overall, our data support the hypothesis
that neurodevelopment is significantly influenced by
chronic input signaling of the melatonin receptors. This
likely involves site-specific alterations in gene expression,
intracellular signaling, and synaptic function consistent
with the recent work of Vilches and colleagues (2014),
who suppressed maternal melatonin in pregnant rats and
demonstrated impaired hippocampal gene expression
and spatial memory in their offspring, which could be res-
cued by melatonin supplementation of the pregnant dams.
To more fully characterize the molecular
consequences of MNTR deficient mice in light of the
behavioral results, we examined protein expression in
the frontal cortex and HPC of NT and DBL-KO mice
(Fig. 10). Signaling through cyclic AMP/protein kinase A
and cyclic GMP/protein kinase G activates constitutive
CREB via phosphorylation and has been implicated in
both locomotor activity and cognition (Bliss and
Collingridge, 1993; Lu et al., 1999). CREB activates
numerous target genes through cyclic AMP response ele-
ments, thus mediating signals from numerous physiologi-
cal stimuli, resulting in regulation of a broad array of
cellular responses. It plays a large regulatory role in the
nervous system and is believed to promote neuronal sur-
vival, precursor proliferation, neurite outgrowth, and neu-
ronal differentiation in certain neuronal populations.
Additionally, CREB signaling is involved in learning and
memory in several organisms. Melatonin’s classic signal-
ing signature – via both MT1 and MT2 – is to inhibit cAMP
generation and thereby protein kinase activation in target
tissues (Reppert et al., 1996; cf. Dubocovich et al., 2010).
Our data indicate significantly increased levels of cortical
phosphoCREB expression relative to CREB levels in the
DBL-KO (Fig. 10A) implying enhanced signaling through
this pathway. Similar results were found in hippocampal
tissue (although the latter did not reach statistical signifi-
cance), consistent with enhanced LTP in the DBL-KO
brain (Fig. 9).
Mitogen-activated protein kinases (MAPKs) are a
widely conserved family of serine/threonine protein
kinases involved in many cellular programs, such as cell
proliferation, differentiation, motility, and death. The p44/
p42 MAPK (ERK1/2) signaling pathway can be activated
in response to a diverse range of extracellular stimuli
including mitogens, growth factors, and cytokines.
Melatonin has generally been reported to activate
ERK1/2 signaling mechanisms (cf. Dubocovich et al.,
2010). The decreased levels of pERK1/2 expression rela-
tive to ERK1/2 levels in the frontal cortex (and a similar
trend in the HPC) of DBL-KO mice (Fig. 10B) are consis-
tent with this and may account for the presumptive differ-
ences in the neurodevelopmental program in the brains of
these mice as compared to NT control mice that devel-
oped in the presence of functional MTNRs.
Synapsin is a synaptic vesicle-associated protein,
participating in synapse formation, regulation of the
synthesis of other synaptic vesicle proteins, and
promotion of neurotransmitter release. Phosphorylation
of the synapsin by protein kinase A (PKA) or calcium
calmodulin-dependent protein kinases (CaMK) inhibits
its binding to phospholipids and dissociates synapsin
protein from synaptic vesicles, ultimately leading to
more efficient neurotransmitter release at the
presynaptic terminal (Bonanomi et al., 2005). Expression
levels of phosphoSynapsin were reduced in the DBL-KO
mouse as compared to control animals (Fig. 10C).
Although this suggests reduced synaptic activity (at least
for the frontal cortex) it remains unclear whether the effect
is a global one, i.e. affecting excitatory and inhibitory neu-
rotransmission equally. Consistent with the synapsin
data, spinophilin expression data reveal significantly
lower levels in the DBL-KO mice (Fig. 10D). Spinophilin
is an actin-associated scaffolding protein mainly localized
to the heads of dendritic spines and these spines are
known to receive excitatory input (Harnett et al., 2012).
To better understand the increased activity seen in the
DBL-KO mice, we also examined the expression levels of
the key GABA-synthesizing enzyme, GAD1. As shown in
Fig. 10E, GAD1 protein levels were significantly reduced
in the DBL-KO frontal cortex, consistent with the overall
trend toward hyperactivity in the DBL-KO mice (Fig. 7).
Conversely, expression of the post-synaptic
glutamate AMPA receptor subunit 1 (GluR1) was
significantly elevated in the frontal cortex of DBL-KO
mice as compared to NT mice (Fig. 10F) with a similar
trend in the HPC (Fig. 10L). This is consistent with
higher levels of LTP in the DBL-KO mice.
CONCLUSIONS
The finding that genetic deletion of melatonin receptors
produces mice with enhanced cognitive and motor
performance supports the emerging view that these
receptors play an important role in neurobehavioral
development. Future investigations on innate melatonin
receptor dysfunctions in the context of human cognitive
and motor development will likely provide new horizons
for treating a diverse variety of behavioral disorders.
AUTHOR CONTRIBUTIONS
G. O’Neal-Moffitt, J. Olcese, J. Pilli, and S.S. Kumar
designed the experiments; G. O’Neal-Moffitt and J. Pilli
performed the experiments, analyzed the data
and performed statistical analysis; G. O’Neal-Moffitt,
520 G. O’Neal-Moffitt et al. / Neuroscience 277 (2014) 506–521
J. Olcese, J. Pilli, and S.S. Kumar wrote and approved the
final manuscript.
Acknowledgments—The authors thank the following for their
respective contributions: M. D’Alessandro and Dr. S. Beesley
for circadian protocols and data analysis; Dr. D. Weaver for the
mice; Drs. Y. Zhou and C. Ouimet for antibodies; Drs. Y. Zhou
and J. Rodefer for use of behavioral equipment and constructive
advice on testing; J. Wolfson and M. de Jesus for assistance with
behavioral testing; C. Badland for assistance in generating fig-
ures. This work was supported by grants to J. Olcese from the
Florida State University Council on Faculty Research & Creativ-
ity, and the FSU College of Medicine (Division of Research).
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(Accepted 12 July 2014)(Available online 18 July 2014)
