Genetic Inactivation of the NMDA Receptor NR2A Subunit
has Anxiolytic- and Antidepressant-Like Effects in Mice
Janel M Boyce-Rustay*,1and Andrew Holmes1
1Section on Behavioral Science and Genetics, Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism,
National Institutes of Health, Rockville, MD, USA
There is growing evidence implicating the glutamate system in the pathophysiology and treatment of mood and anxiety disorders.
Glutamatergic neurotransmission is mediated by several receptor subfamilies including multiple NMDA receptor subunits (NR2A-D).
However, little is currently understood about the specific roles of NMDA subunits in the mediation of emotional behavior due to a lack
of subunit-specific ligands. In the present study, we employed a mouse gene-targeting approach to examine the role of the NR2A subunit
in the mediation of anxiety- and depressive-related behaviors. Results showed that NR2A knockout (KO) mice exhibit decreased
anxiety-like behavior relative to wild-type littermates (WT) across multiple tests (elevated plus maze, light–dark exploration test, novel
open field). NR2A KO mice showed antidepressant-like profiles in the forced swim test and tail suspension test, as compared to WT
controls. Locomotor activity in the nonaversive environments of the home cage or a familiar open field were normal in the NR2A KO
mice, as were gross neurological and sensory functions, including prepulse inhibition of startle. Taken together, these data demonstrate a
selective and robust reduction in anxiety- and depression-related behavior in NMDA receptor NR2A subunit KO mice. Present results
support a role for the NR2A subunit in the modulation of emotional behaviors in rodents and provide insight into the role of glutamate in
the pathophysiology and treatment of mood and anxiety disorders.
Neuropsychopharmacology (2006) 31, 2405–2414. doi:10.1038/sj.npp.1301039; published online 8 February 2006
Keywords: NMDA; NR2A; glutamate; anxiety; depression; knockout
There is growing evidence in support of an important role
for the glutamate system in the mediation of emotion, and
in the pathophysiology of mood and anxiety disorders.
Some but not all studies have found altered levels of
glutamate and glutamate receptor binding in the brains of
depressed patients and suicide victims (Auer et al, 2000;
Holemans et al, 1993; Law and Deakin, 2001; Meador-
Woodruff et al, 2001; Nowak et al, 1995; Palmer et al, 1994;
Pfleiderer et al, 2003). In rodents, exposure to stress
produces a robust increase in glutamate release in regions
mediating emotional behavior, such as the locus coeruleus,
hippocampus and prefrontal cortex (Moghaddam, 1993;
Moghaddam et al, 1994; Singewald et al, 1995, 1996).
Additionally, exposure to stress increases NMDA receptor
expression and mRNA levels in the rat ventral tegmental
area and hippocampus (Bartanusz et al, 1995; Fitzgerald
et al, 1996).
While these data point to glutamatergic involvement in
emotional regulation, defining this role is difficult because
glutamate exerts its effects on neural function in a highly
mediated via ionotropic (N-methyl-D-aspartate (NMDA),
(AMPA), kainate) and metabotropic receptors. However,
possible differential functional roles of these receptor
subfamilies in the mediation of behavioral processes such
as emotion remains incompletely understood. Recent
studies in rodents have demonstrated that blockade of
glutamate’s actions at metabotropic and AMPA receptors
exerts antidepressant-like and anxiolytic-like effects in
various tasks, leading to the clinical development of
metabotropic glutamate receptors for emotional disorders
(for reviews, see (Javitt, 2004; Spooren and Gasparini, 2004;
Swanson et al, 2005)). Manipulation of NMDA receptor
function has also been found to affect emotion-related
behaviors in rodent models. In rats, treatment with
competitive NMDA receptor antagonists produces anxioly-
tic- and antidepressant-like effects in various tasks, such as
the elevated plus-maze and forced swim test, at doses that
Online publication: 4 January 2006 at http://www.acnp.org/citations/
Received 16 September 2005; revised 11 December 2005; accepted
20 December 2005
*Correspondence: Dr JM Boyce-Rustay, Section on Behavioral Science
and Genetics, Laboratory for Integrative Neuroscience, National
Institute on Alcohol Abuse and Alcoholism, 5625 Fishers Lane Rm
2N-09, Rockville, MD 20892, USA, Tel: +1 301 443 4052, Fax:
+1 301 480 1952, E-mail: email@example.com
Neuropsychopharmacology (2006) 31, 2405–2414
& 2006 Nature Publishing GroupAll rights reserved 0893-133X/06 $30.00
do not produce nonspecific changes in locomotor activity
(Martinez et al, 2002; Molchanov and Guimaraes, 2002;
Przegalinski et al, 2000; Trullas and Skolnick, 1990; Wiley
et al, 1995). Moreover, some but not all, noncompetitive
NMDA receptor channel blockers exert anxiolytic- and
antidepressant-like effects in these models (Silvestre et al,
1997; Wiley et al, 1995).
While these findings suggest NMDA receptor modulation
of emotional behaviors, it remains unclear whether these
effects result from antagonism of specific NMDA receptor
subunits. NMDA receptors are tetramers composed of two
NR1 and two NR2 (NR2A-NR2D) subunits (Laube et al,
1998; Rosenmund et al, 1998; Schorge and Colquhoun,
2003). The NR2 subunit present in a functional NMDA
receptor determines the physiological characteristics of the
NMDA receptor; for example, NMDA receptors containing
NR2A mediate faster neurotransmission than NR2B-con-
taining NMDA receptors (Cull-Candy et al, 2001). In
addition, NR2A and NR2B subunits are differentially
expressed over the course of development, with NR2B
predominating in the mouse brain until the end of the
second postnatal week (Liu et al, 2004b). In the adult mouse
brain, NR2A- and NR2B-containing NMDA receptors are
both highly expressed in brain regions implicated in
emotion (Law et al, 2003; Magnusson et al, 2002; Sah and
Lopez De Armentia, 2003). Determining the differential
roles for specific subunit-containing receptors in behavior
is hampered by the lack of NR2A subunit selective
pharmacological ligands. In this context, gene-targeting
in mice provides a valuable approach to studying the
molecular basis of emotional behavior (Holmes, 2001;
Holmes et al, 2003a,b,c), and the behavioral functions of
NMDA subunits (Boyce-Rustay and Holmes, 2005; Tang
et al, 1999; Tsien et al, 1996).
In the current study, we used a mouse model in which the
gene encoding the NR2A subunit (Grin2a) was functionally
inactivated to investigate the role of NR2A in regulating
emotional behavior. Homozygous NR2A null mutant
(‘knockout,’ KO) and heterozygous mutant (HET) mice
were compared to wild-type (WT) littermate controls on a
battery of tests for anxiety-related behaviors (elevated plus
maze, light/dark exploration test, novel open field) and
depression-related behaviors (forced swim test, tail suspen-
sion test). To control for nonspecific phenotypic alterations
that could confound interpretation of anxiety- and depres-
sion-related behaviors in NR2A KO mice, such as motoric
abnormalities (Holmes and Cryan, 2005), NR2A KO mice
were assessed for locomotor activity in nonaversive
environments (home cage, familiar open field) and exam-
ined on a screen for gross behavior, neurological and
sensory functions (Crawley and Paylor, 1997; Holmes et al,
MATERIALS AND METHODS
NR2A KO mice were generated as previously described
(Sakimura et al, 1995; Watanabe et al, 1993). Briefly, the
targeting vector (pTVGRe1) was a 1.3kb blunted EcoRI-
BamHI fragment containing the neomycin phosphotrans-
ferase gene under the control of the phosphoglycerate
kinase promoter. This was then inserted into the Xcml site
of the 10.0kb GluRe1 DNA from a C57BL/6 genomic library
and a synthetic adaptor carrying Sa/I, HindIII and SmaI
sites replaced NotI-Xhol segment (16.5kb) located between
the ClaI and SacI sites. DNA was digested with NotI. TT2.
C57BL/6?CBA F1 hybrid ES cells were cultured, selected,
and the target clones were identified by G418 selection, PCR
and South blot hybridization. Embryonic stem cell clones
were microinjected into ICR blastocysts to obtain chimeric
progeny. Chimeric males were mated to C57BL/6 female
mice. Offspring were genotyped by Southern blot analysis of
tail biopsies to confirm germline transmission.
For the current study the NR2A null mutation was
backcrossed into the C57BL/6J strain for 410 generations
to produce a congenic C57BL/6J genetic background.
Analysis of 150 SNP markers at B15–20 megabase intervals
across all autosomal chromosomes confirmed 499%
C57BL/6J congenicty in the mutant line (JRS Allele
Typing Services, The Jackson Laboratory, Bar Harbor, ME,
USA). To avoid potential phenotypic abnormalities result-
ing from genotypic differences in maternal behavior and
early life environment, NR2A KO, NR2A HET and WT mice
were all generated from HET?HET matings. Mice were
bred and maintained at The Jackson Laboratory (Bar
Harbor, ME, USA) and shipped to the NIH at 7–9 weeks
of age and testing began at a minimum of 10 weeks of age.
On arrival at NIH, mice were housed in groups of 2–4 with
same-sex littermates in a temperature and humidity
controlled vivarium under a 12h light/dark cycle (lights
Testing was conducted using five separate cohorts of mice.
One cohort (11 male WT, three female WT, 12 male HET,
six female HET, seven male KO, seven female KO) was
evaluated for physical heath, sensory and neurological
functions, and then tested on the novel open field, elevated
plus maze, light–dark exploration, and tail suspension test,
in that order, with at least 1 week between tests. A second
cohort (second male WT, four female WT, two male HET,
four female HET, two male KO, one female KO) was tested
on the forced swim test. A third cohort (six male WT, four
female WT, six male HET, six female HET, six male KO, six
female KO) was tested on the forced swim test and home
cage activity with 410 weeks between experiments. A
fourth cohort (six male WT, six female WT, seven male
HET, eight female HET, 11 male KO, four female KO) was
tested for acoustic startle and prepulse inhibition of the
startle response. A fifth cohort (four male WT, four female
WT, six male HET, five female HET, six male KO, three
female KO) was tested on the repeated open field test. With
the exception of the home activity, all testing was conducted
during the light phase of the light/dark cycle. Where
appropriate, apparatuses were cleaned between subjects
with a 70% ethanol (v/v) solution. The experimenter
remained blind to genotype during testing; animals were
identified by subcutaneously implanted microchips. All
experimental procedures were approved by the National
Institute on Alcohol Abuse and Alcoholism Animal Care
and Use Committee and strictly followed the NIH guidelines
‘Using Animals in Intramural Research’.
NR2A KO emotionality
JM Boyce-Rustay and A Holmes
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