, 131ra51 (2012);
4 Sci Transl Med
et al.Jill L. Silverman
Repetitive Behaviors and Rescues Social Deficits in Mouse Models of
Negative Allosteric Modulation of the mGluR5 Receptor Reduces
point to develop a pharmacological therapy that alleviates many symptoms of autism spectrum disorders.
domains in two distinct mouse models is a promising beginning. This single biological target may offer a useful entry
discovery of therapeutic efficacy for an mGluR5 negative allosteric modulator in both the repetitive and the social
Although the path from target identification to effective human treatment is a long and winding road, the
replicated these beneficial actions of the mGluR5 compound in several separate groups of mice, in two laboratories.
one for social interactions between freely moving pairs of mice. A particular strength of this study is that the authors
GRN-529 also improved social behaviors in BTBR in two assays, one for social approach to an unfamiliar mouse and
GRN-529 reduced both the repetitive self-grooming in BTBR and the repetitive jumping in C58. Most intriguingly,
an mGluR5 compound might help autistic symptoms.
people with the fragile X mutation, who have both intellectual impairments and autism, so the authors reasoned that
the main excitatory neurotransmitter in the brain. Other mGluR antagonists are showing promise in clinical trials for
C58 repetitively jumps. They used GRN-529, a compound developed by Pfizer that reduces the actions of glutamate,
of autism. BTBR mice show deficits in many types of social interactions and high levels of repetitive self-grooming.
The authors used two inbred strains of mice that display robust behaviors relevant to the diagnostic symptoms
directed at a central glutamate receptor of the brain, mGluR5.
relevant to the diagnostic symptoms of autism and shown that some of these symptoms can be improved with a drug
Silverman and colleagues have used two inbred strains of mice that display well-replicated behavioral abnormalities
number of cases, and these genes point to connections between neurons as a vulnerable point in autism. Now,
anxiety, or intellectual impairment. A large number of genes can put people at risk for this disorder, each in a small
and body language. Most show repetitive motor behaviors and restricted interests and can have associated seizures,
communication. They may fail to develop relationships with their peers and be unable to interpret nuances of speech
When they are 2 to 5 years old, children with autism start to show unusual social interactions and impaired
Treatment of Autism Symptoms in Mice
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on April 26, 2012
Negative Allosteric Modulation of the mGluR5 Receptor
Reduces Repetitive Behaviors and Rescues Social
Deficits in Mouse Models of Autism
Jill L. Silverman,1Daniel G. Smith,2Stacey J. Sukoff Rizzo,2Michael N. Karras,1
Sarah M. Turner,1Seda S. Tolu,1Dianne K. Bryce,2Deborah L. Smith,2Kari Fonseca,2
Robert H. Ring,2* Jacqueline N. Crawley1†
Neurodevelopmental disorders such as autism and fragile X syndrome were long thought to be medically un-
treatable, on the assumption that brain dysfunctions were immutably hardwired before diagnosis. Recent revela-
tions that many cases of autism are caused by mutations in genes that control the ongoing formation and
maturation of synapses have challenged this dogma. Antagonists of metabotropic glutamate receptor subtype 5
(mGluR5), which modulate excitatory neurotransmission, are in clinical trials for fragile X syndrome, a major genetic
cause of intellectual disabilities. About 30% of patients with fragile X syndrome meet the diagnostic criteria for
autism. Reasoning by analogy, we considered the mGluR5 receptor as a potential target for intervention in autism.
We used BTBR T+tf/J (BTBR) mice, an established model with robust behavioral phenotypes relevant to the three
diagnostic behavioral symptoms of autism—unusual social interactions, impaired communication, and repetitive
behaviors—to probe the efficacy of a selective negative allosteric modulator of the mGluR5 receptor, GRN-529.
GRN-529 reduced repetitive behaviors in three cohorts of BTBR mice at doses that did not induce sedation in control
assays of open field locomotion. In addition, the same nonsedating doses reduced the spontaneous stereotyped
jumping that characterizes a second inbred strain of mice, C58/J. Further, GRN-529 partially reversed the striking
lack of sociability in BTBR mice on some parameters of social approach and reciprocal social interactions. These
findings raise the possibility that a single targeted pharmacological intervention may alleviate multiple diagnostic
behavioral symptoms of autism.
Autism spectrum disorders affect an estimated 1% of the population
(1–4). Intensive behavioral therapy is currently the only effective treat-
ment for the three diagnostic symptoms: qualitative impairment in
social interaction, deficits in communication, and stereotyped repeti-
tive behaviors with restricted interests (5–10). To date, only two drugs
have been approved by the U.S. Food and Drug Administration for
use in patients diagnosed with autism. These two agents, Risperdal
and Abilify (11), do not target the core symptoms, but rather treat
a cluster of associated symptoms referred to as irritability. Given the
high financial and emotional burden to the families and educational
and health care systems, affordable treatments for the core diagnostic
symptoms of autism represent a severe unmet medical need.
Mouse models of autism spectrum disorders can serve as tools to
evaluate the therapeutically relevant efficacy of experimental agents.
To incorporate construct validity for the various genetic mutations
identified in small numbers of people with autism (12–21), these mu-
tations have been generated in mice (22, 23). Behavioral phenotypes
with face validity for some of the diagnostic symptoms of autism have
been reported in some of these mutant mouse models (24–47). Exper-
imental interventions, working through diverse genetic and pharma-
cological mechanisms, rescue subsets of behavioral abnormalities in
these mouse models (31, 43, 48–52). Discovery of elevated metabotrop-
ic glutamate receptor subtype 5 (mGluR5)–mediated signaling and
protein synthesis in fragile X knockout mice (53–56) provided the ra-
tionale for testing mGluR5 antagonists in ongoing fragile X clinical
trials. A large subset of cases of fragile X meet the diagnostic criteria
for autism (57, 58). Because the primary symptoms of autism and
fragile X differ in qualitative features, and mouse models of autism
and fragile X display sharply divergent phenotypes, testing mGluR5
antagonists in specific assays of mouse behavioral phenotypes that op-
timize relevance to the diagnostic symptoms of autism would be most
informative for translational goals.
Several naturally occurring inbred strains of mice display behavioral
features that recapitulate diagnostic symptoms of neurodevelopmental
disorders (59–70). These inbred strains are genetically homogeneous
and commercially available, maximizing their feasibility as transla-
tional tools in medications development (71–73). Inbred strains with
robust behavioral phenotypes relevant to the defining symptoms of
autism, but without identified genetic mutations, are analogous to in-
dividuals with autism for whom the responsible genetic factor(s) re-
mains unknown; at present, this is more than 75% of the cases of
autism (21). In addition, inbred strains can incorporate multiple back-
ground genes that influence their behavioral deficits, allowing evaluation
of two-hit and multiple-hit hypotheses of autism spectrum disorders.
BTBR T+tf/J (BTBR) is a commercially available inbred strain of
mouse that displays behavioral phenotypes relevant to all three di-
agnostic symptoms of autism (60–62, 64, 66, 68–70, 72, 74–78).
BTBR engages in low levels of reciprocal social interactions as ju-
veniles and adults, minimal social approach by both males and
females, and low levels of ultrasonic vocalizations in response to social
1Laboratory of Behavioral Neuroscience, National Institute of Mental Health, Bethesda,
MD 20892–3730, USA.
*Present address: Autism Speaks, Princeton, NJ 08540, USA.
†To whom correspondence should be addressed. E-mail: firstname.lastname@example.org
2Pfizer Worldwide Research and Development, Groton, CT
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on April 26, 2012
olfactory cues and during reciprocal social interactions compared
to other standard inbred strains such as C57BL/6J (B6) and FVB
(60–62, 64, 66, 68–70, 74, 76–78). Social interaction and communica-
tion deficits in BTBR represent face validity to the first and second
diagnostic symptoms of autism, respectively. Long bouts of repetitive
self-grooming are the third major characteristic of BTBR, representing
face validity to the third diagnostic symptom of autism, spontaneous
stereotyped and repetitive patterns of behavior. Absence of the corpus
callosum connecting the right and left cortical hemispheres in BTBR
(79) is reminiscent of a small subset of individuals with autism who
are acallosal (80, 81) and may be a sign of more global abnormalities
in white matter connectivity in this mouse strain. Experimentally in-
duced postnatal lesions of the corpus callosum in B6 mice, however,
did not recapitulate the social deficits, or the repetitive self-grooming,
that characterize BTBR (82). C58/J (C58) is another commercially
available inbred strain of mouse that displays high levels of
stereotyped vertical jumping behaviors, relevant to the motor
stereotypies of the third diagnostic symptom category of
autism (65, 67). Unlike mouse models based on identified
genetic mutations, the background genes responsible for
autism-relevant behavioral traits in these inbred strains of
mice remain under investigation (64, 83–85).
The robustness and reproducibility of autism-relevant
phenotypes in BTBR and C58 provide an attractive transla-
tional platform for the preclinical evaluation of intervention
therapies (72, 73). A treatment that attenuates different forms
of repetitive behaviors in two different inbred strains is likely
to generalize across a range of repetitive behaviors and po-
tentially to generalize across species. Here, we test the hy-
pothesis that a selective negative allosteric modulator of the
mGluR5 receptor, GRN-529, will ameliorate autism-relevant
behavioral abnormalities in mouse models of autism.
GRN-529 brain penetration and mGluR5
We measured the plasma and brain exposure levels of GRN-
529 (Fig. 1, B to D) and the relationship between unbound
brain levels of GRN-529 and mGluR5 occupancy (Fig. 1F)
after systemic administration. The relationship between
GRN-529 brain exposure and mGluR5 occupancy was sim-
ilar in B6, BTBR, and C58 mouse strains (Fig. 1F). A 30- to
60-min timeframe was chosen for occupancy and behavior-
al experiments because a 2-hour time course of plasma and
brain exposure after GRN-529 administration revealed that
peak concentrations occurred between 0 and 60 min in B6
mice (Fig. 1E).
Amelioration of repetitive and stereotyped behaviors
GRN-529 and the prototypic mGluR5 antagonist 2-methyl-
6-(phenylethynyl)pyridine (MPEP) reduced the high levels of
repetitive self-grooming that characterizes the BTBR strain.
treated with vehicle engaged in much longer bouts of self-
grooming than did B6 (Fig. 2 and fig. S1). In three replications
by two laboratories, acute administration of GRN-529 signifi-
cantly reduced repetitive self-grooming scores in BTBR (Fig. 2, B and D,
3-((2-Methyl-4-thiazolyl)ethynyl)pyridine (MTEP), another standard
mGluR5 antagonist, similarly reduced repetitive self-grooming in BTBR
at some doses (fig. S1D) and similarly had no effect in B6 mice (fig.
tive mGluR5 antagonist MPEP (fig. S1, A and B) (72).
C58 mice displayed high levels of stereotyped vertical jumping (Fig.
2E), consistent with previous findings (67). GRN-529 dose-dependently
reduced jumping in C58, almost completely abolishing this repetitive
behavior at 3.0 mg/kg, a dose that achieved ~90% occupancy (Fig. 1F).
The effects in C58 were not attributable to reduced locomotion or seda-
tion (Fig. 2F and fig. S6).
Complete statistical analyses of all behavioral experiments appear
in the Supplementary Materials.
(mg/kg, 60 min, s.c.)
(mg/kg, 30 min, i.p.)
(mg/kg, 30 min, i.p.)
1 mg/kg (plasma)
10 mg/kg (brain)
1 mg/kg (brain)
10 mg/kg (plasma)
Cb,u [GRN-529] (nM)
mGluR5 occupancy (%)
mGluR5 Ki: 5.4 nM
mGluR5 IC50: 3.1 nM
Fig. 1. GRN-529 chemical structure, plasma and brain concentrations, and receptor
occupancy in B6, BTBR, and C58 mice. (A) Chemical structure of GRN-529 and binding
properties [Ki= 5.4 nM and median inhibitory concentration (IC50) = 3.1 nM] at rat
mGluR5. (B to D) Unbound plasma and brain concentrations of GRN-529 30 or
60 min after systemic administration in B6, BTBR, and C58 mice. s.c., subcutaneously;
i.p., intraperitoneally. (E) Time course of unbound plasma and brain concentrations
(nM) of GRN-529 for 2 hours after systemic intraperitoneal administration in B6 mice.
(F) Relationship of the concentration of unbound (Cb,u) GRN-529 concentrations (nM)
and mGluR5 occupancy in brains from BTBR, B6, and C58 mice. n = 3 to 5 per dose
and strain. Data are expressed as the mean for each group.
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on April 26, 2012
Improvement in social behaviors
Two parameters of social behavior were
scored in our automated three-chambered
social approach task (86) as described
(72, 87). B6 control mice spent more time
in the side chamber containing a novel
mouse than in the side chamber contain-
ing a novel object, meeting the definition
of normal sociability in this task, as exten-
sively reported (22, 62, 64, 72–74, 87–89).
GRN-529 did not affect the sociability in
B6 controls at any dose (Fig. 3, A and C).
BTBR mice displayed lack of sociability,
defined as not spending more time in the
side chamber with the novel mouse than in
the side chamber with the novel object, as
reported (22, 62, 64, 72–74, 87). GRN-529
reversed sociability deficits in BTBR at a
dose of 3.0 mg/kg, as measured by time
in the chamber (Fig. 3D). For time spent
sniffing the novel mouse versus the novel
object, which is a more precise and sen-
sitive measure of true social interaction
(87, 90), GRN-529 reversed the deficit in
BTBR, again with no detrimental effect
on B6 sociability (Fig. 3, A and C). BTBR
mice spent significantly more time sniffing
the novel mouse than the novel object after
doses of 0.3, 1.0, and 3.0 mg/kg (Fig. 3B).
The number of entries between chambers,
an internal control for general exploration,
was not significantly affected by the lower
doses of GRN-529 in either strain, but ele-
vated numbers of entries in both strains
appeared at a dose of 3.0 mg/kg (Fig. 3,
Eand F).A second cohortdisplayed a sim-
ilar pattern of responses (fig. S2).
Multiple parameters of social behav-
iors were scored during a freely moving,
dyadic reciprocal social interaction test
in adult B6 and BTBR mice treated with
vehicle or the most effective dose of GRN-
529 in the social approach test, 3.0 mg/kg.
These detailed interactive social param-
eters were collected with 129/SvImJ mice
as stimulus partners, chosen for their in-
herently low spontaneous locomotion and
aggression, as reported (60, 61, 91). During
the reciprocal interaction test, B6 mice con-
sistently exhibited high levels of nose-to-
nose sniffing, front approach, and total time
spent in social contact (Fig. 4, A and C, and
fig. S3A), whereas BTBR mice displayed
lack ofsociabilityonthesestandard param-
eters (Fig. 4, B and D, and fig. S3B), con-
sistent with previous reports (60, 78, 91).
GRN-529 increased sociability in BTBR
on some of these parameters, particularly
Fig. 2. Effect of GRN-529 on repetitive self-grooming in BTBR and stereotyped jumping in C58 mice.
Cumulative time spent self-grooming by BTBR and B6 mice was scored over a 10-min session in a clean,
closed, empty cage after a 10-min acclimation period. Observations of stereotyped jumping behavior in
C58 mice were quantified for a period of 10 min. GRN-529 was tested in two independent laboratory
environments across three cohorts. (A) B6 mice did not display any significant differences in the amount
of time spent self-grooming after treatment with vehicle (10% Tween 80/saline) or GRN-529 at doses of
0.3, 1.0, or 3.0 mg/kg intraperitoneally (n = 8 to 10 per dose, cohort 1, tested at NIMH, *P < 0.05 versus
vehicle). (B) BTBR displayed significant reductions in their innately high levels of repetitive self-grooming
after treatment with GRN-529 at doses of 1.0 and 3.0 mg/kg (n = 11 to 14 per dose, cohort 1, tested at
NIMH, *P < 0.05 versus vehicle). (C) B6 mice displayed significant reductions in the amount of time spent
self-grooming after treatment with GRN-529 at doses of 1.0 and 3.0 mg/kg compared to vehicle (cohort 2,
tested at Pfizer). (D) BTBR again displayed significant reductions in high levels of repetitive self-grooming
after treatment with GRN-529 at doses of 1.0 and 3.0 mg/kg intraperitoneally (n = 17 to 25 per dose for
each strain, cohort 2, tested at Pfizer, *P < 0.05 versus vehicle). (E) Stereotyped vertical jumping in C58 mice
was significantly reduced after GRN-529 administration at doses of 0.3, 1.0, and 3.0 mg/kg intraperitoneally
versus vehicle (*P < 0.05, tested at Pfizer). (F) No adverse or sedating effects on the general activity of C58
mice were observed during open field locomotion (P > 0.05, tested at Pfizer).
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on April 26, 2012
on the most sensitive measures, nose-to-
nose sniffing (Fig. 4B) and total time spent
in social contact (Fig. 4D), while having no
effect in B6 controls (Fig. 4, A and C, and
fig. S3, A, C, and E). GRN-529 did not af-
fect general exploratory locomotion in B6
and BTBR mice during the reciprocal in-
teraction session (fig. S3, G to J). Further,
interaction task, BTBR mice treated with
vehicle engaged in much longer bouts of
self-grooming and repetitive digging than
did B6 mice (Fig. 4, E to H). Acute admin-
istration of GRN-529 significantly reduced
these spontaneous repetitive behaviors with-
in a social context (Fig. 4, F and H).
Absence of sedation
stereotyped jumping such as thosethatwe
saw could be caused by sedative actions of
a pharmacological agent. Conversely, in-
creased numbers of entries in the social
approach apparatus could be caused by
stimulant actions of a pharmacological
of GRN-529 on general activity, we tested
the same doses at the 30-min time point
in B6, BTBR, and C58 mice on open field
locomotion. No evidence of sedation was
detected in any strain at any dose during
a 30-min test session (Fig. 5 and figs. S4
to S6). Higher total distances traveled were
seen in both strains at a dose of 3.0 mg/kg,
and higher vertical and center time scores
were seen in BTBR at the higher doses in
one cohort tested at Pfizer, indicating a
moderate increase in general exploratory
locomotion. No increases in total distance
traveled were observed in C58 mice at any
itatively unusual behaviors were observed
after GRN-529 treatments during any of
the grooming, social, or open field test
Autism is a behaviorally diagnosed, life-
time neurodevelopmental disorder. Bio-
logical abnormalities have been reported
in eye tracking, neuroanatomical path-
way connectivity, brain regional volumes,
cortical activation during social and com-
munication tasks as measured with func-
tional magnetic resonance imaging and
magnetoencephalography, serotonin lev-
els, and other biological assays, but not
Fig. 3. Effect of GRN-529 on social approach in adult BTBR mice. Social approach was assessed in an
automated photocell-equipped three-chambered arena with observer scoring of direct sniffing inter-
actions from videotapes of the social approach. (A) B6 mice displayed sociability on the more sensi-
tive parameter, time spent sniffing the novel mouse compared to time spent sniffing the novel object,
at each dose of GRN-529 and vehicle. (B) BTBR exhibited its characteristic lack of sociability on the
sniff parameter after vehicle administration. BTBR treated with a single acute dose of GRN-529, 0.3,
1.0, or 3.0 mg/kg intraperitoneally, exhibited significant sociability on the sniff time parameter. (C) The
B6 control strain displayed normal sociability, defined as spending more time in the chamber with the
novel mouse than in the chamber with the novel object, after a single intraperitoneal dose of vehicle
(10% Tween 80/saline) or GRN-529 at doses of 0.3, 1.0, and 3 mg/kg. (D) BTBR exhibited its char-
acteristic lack of sociability, that is, did not spend more time in the novel mouse chamber than in
the novel object chamber, after treatment with vehicle or the two lower doses of GRN-529. At the
highest dose, 3.0 mg/kg, BTBR displayed significant sociability. (E and F) B6 (E) and BTBR (F) displayed
a greater number of entries into the side chambers after treatment with GRN-529 at the highest dose,
3.0 mg/kg intraperitoneally, indicating a general increase of exploratory activity during the social ap-
proach task at that dose. *P < 0.05, novel mouse versus novel object in (A) to (D); *P < 0.05 versus
vehicle in (E) and (F). n = 10 per dose for each strain, cohort 1, assayed at NIMH. See fig. S2 for rep-
licated findings in cohort 2.
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on April 26, 2012
with sufficient consistency for these to constitute uniform diagnostic
biomarkers (92–94). Therefore, therapeutic efficacy is currently evaluated
by improvement in the diagnostic behavioral symptoms (11, 50, 95).
Compelling neuropharmacological targets for autism remain to
be identified. Target strategies draw from therapeutics under inves-
tigation for other neurodevelopmental disorders and hypotheses
emerging from mutations in synaptic genes identified in small num-
bersofindividualswithautismspectrumdisorders(43, 49, 52). One such
strategy is modulation of glutamatergic neurotransmission through
the mGluR5 receptor (96), which is under investigation for the treat-
ment of fragile X (55).
Using the BTBR mouse model, which recapitulates endophenotypic
analogies to the diagnostic social deficits, impaired communication, and
repetitive behavioral symptoms of autism, we evaluated GRN-529, a
compound with high specificity for the mGluR5 receptor. As demon-
strated by Hughes and colleagues (97), in competition binding experi-
ments, GRN-529 competes for [3H]MPEP binding at mGluR5 with high
affinity [inhibitionconstant (Ki) = 5.4 ±0.43nM],antagonizesglutamate-
induced increases in calcium signaling, but does not directly bind to the
orthosteric binding site or affect the affinity of glutamate for this site,
consistent with negative allosteric modulation (97). Using pharmaco-
kinetic and ex vivo receptor occupancy studies, we demonstrated brain
exposure and target engagement for GRN-529 across the efficacious
dose range (0.3 to 3.0 mg/kg) in B6, BTBR, and C58 mice. Treatment
with GRN-529 at these doses reversed the social approach deficits in
BTBR on two standardized mouse assays for sociability. In particular,
the more ethologically meaningful parameters of time engaged in in-
teractive sniffing of a novel mouse versus time spent sniffing a novel
object during social approach, bouts of nose-to-nose sniffing and time
in social contact during reciprocal interactions, were restored in BTBR
by the acute pharmacological intervention, which had no effect in the
normal control B6 strain.
We discovered strong reductions in the repetitive self-grooming
phenotype in the BTBR mouse model of autism after GRN-529 treat-
ment, replicated in three cohorts of mice at two research sites. Mag-
nitudes of reduction were consistent with a previous report with the
prototypic mGluR5 antagonist MPEP, which required higher doses and
is known to also act at N-methyl-D-aspartate (NMDA) receptors (72).
Parallel attenuation of self-grooming in BTBR was confirmed at the
high dose of MPEP, and for the more brain penetrant and selective
mGluR5 analog MTEP, although its dose-response curve was nonlinear.
Further, GRN-529 markedly reduced a stereotyped behavior in another
inbred strain, vertical jumping in C58.
Lower self-grooming and jumping scores in the treated mice were
administration. Small increases in open field scores, and on number of
chamber entries in the three-chambered apparatus, were detected in B6
ry locomotion was not significantly affected by GRN-529 in B6 and
Fig. 4. Effect of GRN-529 on dyadic reciprocal social interactions in BTBR
mice. Social interactions were digitally recorded in dyads of mice in the
Noldus PhenoTyper 3000 arena. Coded videos were subsequently scored
by an observer uninformed of the treatment condition using Noldus Ob-
server 8.0XT software. (A) The B6 control strain displayed normal sociabil-
ity, as illustrated by high levels of nose-to-nose sniffing with the 129/SvImJ
partner stimulus mouse, after a single intraperitoneal dose of vehicle (10%
Tween 80/saline) or GRN-529 (3.0 mg/kg). (B) BTBR treated with vehicle
exhibited its characteristic low sociability, displaying fewer bouts of
nose-to-nose sniffing with the 129/SvImJ partner stimulus mouse. GRN-
529 (3.0 mg/kg) increased nose-to-nose sniffing bouts in the BTBR. (C)
B6 displayed high sociability on the parameter, time spent in social contact
after GRN-529 or vehicle. (D) BTBR exhibited its characteristic low sociabil-
ity on time spent in social contact after vehicle administration. BTBR treated
with a single acute dose of GRN-529 (3.0 mg/kg) exhibited increased time
in social contact. (E) Cumulative time spent self-grooming was calculated
during the 10-min reciprocal social interaction test session. B6 mice treated
with either vehicle or GRN-529 (3.0 mg/kg) did not display any significant
differences in the amount of time spent self-grooming during the session.
(F) BTBR treated with GRN-529 (3.0 mg/kg) displayed significant reductions
in their high levels of repetitive self-grooming versus BTBR treated with
vehicle. (G) Cumulative time spent digging in the arena floor bedding dur-
ing the social task was calculated during the 10-min test session. B6 mice
treated with either vehicle or GRN-529 (3.0 mg/kg) displayed similar time
spent digging during the session. (H) BTBR treated with GRN-529 (3.0 mg/kg)
displayed significant reductions in their high levels of repetitive digging
behavior versus BTBR treated with vehicle. n = 9 to 11 per treatment
group, GRN-529 (3.0 mg/kg) and vehicle, for each strain, *P < 0.05 ver-
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on April 26, 2012
BTBR mice engaged in social interaction in the PhenoTyper arena.
Total distance traversed in the open field was not affected by GRN-
529 in C58, did not reach the range considered hyperactive for B6,
and did not mimic the qualitative type of fast, unstructured activity
that characterizes rodent responses to psychostimulants. Nevertheless,
it remains conceptually possible that increased general exploration
ther, these results may suggest that mGluR5
treatment could prove helpful for the subset
of individuals with autism and attention deficit
Our results show that negative allosteric
modulation of the mGluR5 receptor improves
social interactions, reduces high levels of repet-
itive behaviors in BTBR, and reduces stereo-
typed behaviors in C58, relevant to the first and
third diagnostic symptoms of autism. Early clin-
ical indications of beneficial actions of mGluR5
antagonists in fragile X syndrome make this
class of therapeutic targets of particular interest
(55). The present preclinical findings on rever-
sal of features relevant to autism in two mouse
models convey promise for the mGluR5 strat-
egy as a therapeutic intervention for two core
diagnostic symptoms of autism.
MATERIALS AND METHODS
Adult male and female C57BL/6J (B6) and
BTBR T+tf/J (BTBR) mice tested at the Na-
tional Institute of Mental Health (NIMH) in
Bethesda,Maryland, were bred from adult pairs
originally purchased from The Jackson Labora-
tory (JAX). B6, BTBR, and C58/J (C58) tested
at Pfizer in Groton, Connecticut, were pur-
chased as adults from JAX. Behavioral param-
eters were scored with automated equipment
or from digital videotapes by investigators un-
informed of treatment. To further ensure ab-
sence of unconscious bias by the raters, we
recorded identities of subject mice from paw
tattoos only after the behavioral test session
ended. All procedures were approved by the
NIMH and the Pfizer Inc. Animal Care and
Use Committees. Complete behavioral and
biochemical methods appear in the Supple-
Mice were injected with GRN-529 and
killed at the time points indicated for pharma-
cokinetic and ex vivo receptor occupancy assays
(Fig. 1, B to F). Plasma and forebrain sam-
ples were collected for analysis of drug con-
centrations by liquid chromatography–mass
spectrometry, and of receptor occupancy by
binding of 1 nM [3H]MPEPy, similar to the
procedures used by Hughes et al. (97). Results
from the pharmacokinetic, ex vivo receptor
occupancy and/or previous behavioral assays in B6, BTBR, and C58
mice were used to select the doses and posttreatment interval for the
present behavioral studies. Complete methods and results appear in
the Supplementary Materials.
Repetitive self-grooming was scored from digital videotapes with
methods previously published (22, 62, 64, 72, 74, 89) and described
Fig. 5. Effect of GRN-529 on open field locomotion at doses that reversed repetitive and so-
cial deficits. Exploratory locomotion was assayed in a standard automated open field arena,
in 5-min time bins across a 30-min session after the identical GRN-529 treatments. (A) B6
displayed a significant increase in total distance traversed after GRN-529 at the highest dose,
3.0 mg/kg, intraperitoneally, compared to vehicle. (B and C) GRN-529 administration had no
significant effect on (B) time spent in the center of the arena or (C) vertical activity in B6
tested at NIMH. n = 9 to 10 per dose. (D) BTBR displayed significant increases in total distance
traversed after GRN-529 at doses of 1.0 and 3.0 mg/kg intraperitoneally compared to vehicle,
indicating increased exploratory activity. (E and F) GRN-529 administration had no significant
effect on (E) time spent in the center of the arena or (F) vertical activity in BTBR. *P < 0.05, n =
10 per dose, cohort 1 tested at NIMH. See fig. S4 for open field results replicated at NIMH and
fig. S5 for open field results replicated at Pfizer. See fig. S6 for additional open field param-
eters in C58 mice.
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on April 26, 2012
in the Supplementary Materials. Briefly, each subject mouse was
placed in a bare, empty cage for a 10-min habituation session and then
video was recorded for a 10-min test session. Cumulative time spent
self-grooming was scored from the videos using a high-accuracy Trace-
able stopwatch (Thomas Scientific) with the auditory component
Sociability in the automated three-chambered apparatus developed by
our group (86) was conducted as published (22, 32, 62, 64, 72, 74, 87, 89)
and described in the Supplementary Materials. Briefly, each subject
mouse was placed in the bare, empty three-chambered apparatus for
a 10-min habituation session. A novel object, an inverted wire cup,
was then placed in one side chamber, and a novel mouse was placed
inside a second inverted wire cup in the other side chamber. The sub-
ject mouse was given a 10-min test session, offering the choice of
spending time in the vicinity of a novel social partner or a novel in-
animate object. Time in each chamber, representing proximity to a
social partner, was scored by the automated software. Time spent in
directly sniffing the novel mouse, representing actual reciprocal social
interactions, was subsequently scored from digital videos of the ses-
sions by an observer with a stopwatch.
Reciprocal social interaction was tested in adult B6 and BTBR sub-
ject mice during a 10-min session in a Noldus PhenoTyper 3000 arena,
as described (91). 129/SvImJ mice were used as interaction stimulus
partners to evaluate social behavior in response to social cues from a
uniform stimulus mouse during a 10-min session between freely mov-
ing dyads (60, 64, 91). Standard interaction parameters, time spent in
repetitive behaviors, and arena exploration were simultaneously scored
as published (91) and described in the Supplementary Materials.
Open field locomotor activity was evaluated in a standard AccuScan
photocell-equipped open field over a 30-min test session, using methods
previously published (32, 72, 89, 98) and described in the Supplemen-
tary Materials. Automated parameters including total distance, vertical
activity, and center time were generated by the VersaMax software.
Material and Methods
Fig. S1. mGluR5 antagonists reduced repetitive self-grooming in B6 and BTBR.
Fig. S2. GRN-529 partially ameliorated social approach deficits in BTBR.
Fig. S3. GRN-529 partially ameliorated reciprocal interaction social deficits in BTBR without
sedation or hyperactivation in BTBR.
Fig. S4. GRN-529 did not induce sedation at doses that reversed repetitive and social deficits in
cohort 2 B6 and BTBR mice at NIMH.
Fig. S5. GRN-529 did not induce sedation in cohort 3 B6 and BTBR mice at doses that reversed
repetitive behaviors in cohort 3 at Pfizer.
Fig. S6. GRN-529 did not induce sedation in C58 mice at Pfizer.
REFERENCES AND NOTES
1. M. D. Kogan, S. J. Blumberg, L. A. Schieve, C. A. Boyle, J. M. Perrin, R. M. Ghandour, G. K. Singh,
B. B. Strickland, E. Trevathan, P. C. van Dyck, Prevalence of parent-reported diagnosis of
autism spectrum disorder among children in the US, 2007. Pediatrics 124, 1395–1403
2. B. J. Kim, W. S. Cheon, H. C. Oh, J. W. Kim, J. D. Park, J. G. Kim, Prevalence and risk factor of
erosive esophagitis observed in Korean National Cancer Screening Program. J. Korean
Med. Sci. 26, 642–646 (2011).
3. C. Lord, Epidemiology: How common is autism? Nature 474, 166–168 (2011).
4. J. A. Pinto-Martin, S. E. Levy, J. F. Feldman, J. M. Lorenz, N. Paneth, A. H. Whitaker, Prev-
alence of autism spectrum disorder in adolescents born weighing <2000 grams. Pediatrics
128, 883–891 (2011).
5. American Psychiatric Association, Diagnostic Criteria from DSM-IV (American Psychiatric
Association, Washington, DC, 1994).
6. C. Lord, S. Risi, L. Lambrecht, E. H. Cook Jr., B. L. Leventhal, P. C. DiLavore, A. Pickles, M. Rutter,
The autism diagnostic observation schedule—Generic: A standard measure of social and
communication deficits associated with the spectrum of autism. J. Autism Dev. Disord. 30,
7. L. Krasny, B. J. Williams, S. Provencal, S. Ozonoff, Social skills interventions for the autism
spectrum: Essential ingredients and a model curriculum. Child Adolesc. Psychiatr. Clin. N. Am.
12, 107–122 (2003).
8. R. J. Landa, Diagnosis of autism spectrum disorders in the first 3 years of life. Nat. Clin.
Pract. Neurol. 4, 138–147 (2008).
9. L. Zwaigenbaum, S. Bryson, C. Lord, S. Rogers, A. Carter, L. Carver, K. Chawarska, J. Constantino,
G. Dawson, K. Dobkins, D. Fein, J. Iverson, A. Klin, R. Landa, D. Messinger, S. Ozonoff, M. Sigman,
W. Stone, H. Tager-Flusberg, N. Yirmiya, Clinical assessment and management of toddlers
with suspected autism spectrum disorder: Insights from studies of high-risk infants. Pediatrics
123, 1383–1391 (2009).
10. G. Dawson, S. Rogers, J. Munson, M. Smith, J. Winter, J. Greenson, A. Donaldson, J. Varley,
Randomized, controlled trial of an intervention for toddlers with autism: The Early Start
Denver Model. Pediatrics 125, e17–e23 (2010).
11. M. L. McPheeters, Z. Warren, N. Sathe, J. L. Bruzek, S. Krishnaswami, R. N. Jerome,
J. Veenstra-Vanderweele, A systematic review of medical treatments for children with autism
spectrum disorders. Pediatrics 127, e1312–e1321 (2011).
12. S. Jamain, H. Quach, C. Betancur, M. Råstam, C. Colineaux, I. C. Gillberg, H. Soderstrom,
B. Giros, M. Leboyer, C. Gillberg, T. Bourgeron; Paris Autism Research International Sibpair
Study, Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are asso-
ciated with autism. Nat. Genet. 34, 27–29 (2003).
13. B. S. Abrahams, D. H. Geschwind, Advances in autism genetics: On the threshold of a new
neurobiology. Nat. Rev. Genet. 9, 341–355 (2008).
14. M. Alarcón, B. S. Abrahams, J. L. Stone, J. A. Duvall, J. V. Perederiy, J. M. Bomar, J. Sebat,
M. Wigler, C. L. Martin, D. H. Ledbetter, S. F. Nelson, R. M. Cantor, D. H. Geschwind, Linkage,
association, and gene-expression analyses identify CNTNAP2 as anautism-susceptibility gene.
Am. J. Hum. Genet. 82, 150–159 (2008).
15. C. W.Brune, E.Korvatska, K. Allen-Brady, E. H.Cook Jr., G.Dawson, B.Devlin, A.Estes,M.Hennelly,
S. L. Hyman, W. M. McMahon, J. Munson, P. M. Rodier, G. D. Schellenberg, C. J. Stodgell, H. Coon,
Heterogeneous association between engrailed-2 and autism in the CPEA network. Am. J.
Med. Genet. B Neuropsychiatr. Genet. 147B, 187–193 (2008).
16. J. D. Buxbaum, Multiple rare variants in the etiology of autism spectrum disorders. Dialogues
Clin. Neurosci. 11, 35–43 (2009).
17. J. Gauthier, D. Spiegelman, A. Piton, R. G. Lafrenière, S. Laurent, J. St-Onge, L. Lapointe,
F. F. Hamdan, P. Cossette, L. Mottron, E. Fombonne, R. Joober, C. Marineau, P. Drapeau,
G. A. Rouleau, Novel de novo SHANK3 mutation in autistic patients. Am. J. Med. Genet. B
Neuropsychiatr. Genet. 150B, 421–424 (2009).
18. J. T. Glessner, K. Wang, G. Cai, O. Korvatska, C. E. Kim, S. Wood, H. Zhang, A. Estes, C. W. Brune,
J. P. Bradfield, M. Imielinski, E. C. Frackelton, J. Reichert, E. L. Crawford, J. Munson, P. M. Sleiman,
R. Chiavacci, K. Annaiah, K. Thomas, C. Hou, W. Glaberson, J. Flory, F. Otieno, M. Garris, L. Soorya,
L. Klei, J. Piven, K. J. Meyer, E. Anagnostou, T. Sakurai, R. M. Game, D. S. Rudd, D. Zurawiecki,
C.J.McDougle,L.K.Davis,J.Miller,D.J.Posey,S.Michaels,A.Kolevzon,J. M. Silverman, R. Bernier,
S. E. Levy, R. T. Schultz, G. Dawson, T. Owley, W. M. McMahon, T. H. Wassink, J. A. Sweeney,
J. I. Nurnberger, H. Coon, J. S. Sutcliffe, N. J. Minshew, S. F. Grant, M. Bucan, E. H. Cook,
J. D. Buxbaum, B. Devlin, G. D. Schellenberg, H. Hakonarson, Autism genome-wide copy
number variation reveals ubiquitin and neuronal genes. Nature 459, 569–573 (2009).
19. R. A. Kumar, C. R. Marshall, J. A. Badner, T. D. Babatz, Z. Mukamel, K. A. Aldinger, J. Sudi,
C. W. Brune, G. Goh, S. Karamohamed, J. S. Sutcliffe, E. H. Cook, D. H. Geschwind, W. B. Dobyns,
S. W. Scherer, S. L. Christian, Association and mutation analyses of 16p11.2 autism candidate
genes. PLoS One 4, e4582 (2009).
20. S. Berkel, C. R. Marshall, B. Weiss, J.Howe, R. Roeth, U. Moog, V. Endris, W. Roberts, P. Szatmari,
D. Pinto, M. Bonin, A. Riess, H. Engels, R. Sprengel, S. W. Scherer, G. A. Rappold, Mutations in
the SHANK2 synaptic scaffolding gene in autism spectrum disorder and mental retardation.
Nat. Genet. 42, 489–491 (2010).
21. J. H. Miles, Autism spectrum disorders—A genetics review. Genet. Med. 13, 278–294 (2011).
22. J. L. Silverman, M. Yang, C. Lord, J. N. Crawley, Behavioural phenotyping assays for mouse
models of autism. Nat. Rev. Neurosci. 11, 490–502 (2010).
23. E. Ey, C. S. Leblond, T. Bourgeron, Behavioral profiles of mouse models for autism
spectrum disorders. Autism Res. 4, 5–16 (2011).
24. J. Molina, P. Carmona-Mora, J. Chrast, P. M. Krall, C. P. Canales, J. R. Lupski, A. Reymond,
K. Walz, Abnormal social behaviors and altered gene expression rates in a mouse model
for Potocki-Lupski syndrome. Hum. Mol. Genet. 17, 2486–2495 (2008).
25. P. Moretti, H. Y. Zoghbi, MeCP2 dysfunction in Rett syndrome and related disorders.
Curr. Opin. Genet. Dev. 16, 276–281 (2006).
26. T. M. DeLorey, GABRB3 gene deficient mice: A potential model of autism spectrum disorder.
Int. Rev. Neurobiol. 71, 359–382 (2005).
www.ScienceTranslationalMedicine.org25 April 2012Vol 4 Issue 131 131ra51
on April 26, 2012
27. W. Shu, J. Y. Cho, Y. Jiang, M. Zhang, D. Weisz, G. A. Elder, J. Schmeidler, R. De Gasperi,
M. A. Sosa, D. Rabidou, A. C. Santucci, D. Perl, E. Morrisey, J. D. Buxbaum, Altered ultra-
sonic vocalization in mice with a disruption in the Foxp2 gene. Proc. Natl. Acad. Sci. U.S.A. 102,
28. M. A. Cheh, J. H. Millonig, L. M. Roselli, X. Ming, E. Jacobsen, S. Kamdar, G. C. Wagner, En2
knockout mice display neurobehavioral and neurochemical alterations relevant to autism
spectrum disorder. Brain Res. 1116, 166–176 (2006).
29. C. H. Kwon, B. W. Luikart, C. M. Powell, J. Zhou, S. A. Matheny, W. Zhang, Y. Li, S. J. Baker,
L. F. Parada, Pten regulates neuronal arborization and social interaction in mice. Neuron
50, 377–388 (2006).
30. Y. S. Mineur, L. X. Huynh, W. E. Crusio, Social behavior deficits in the Fmr1 mutant mouse.
Behav. Brain Res. 168, 172–175 (2006).
31. J. Guy, J. Gan, J. Selfridge, S. Cobb, A. Bird, Reversal of neurological defects in a mouse
model of Rett syndrome. Science 315, 1143–1147 (2007).
32. K. K. Chadman, S. Gong, M. L. Scattoni, S. E. Boltuck, S. U. Gandhy, N. Heintz, J. N. Crawley,
Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice. Autism Res.
1, 147–158 (2008).
33. S. Jamain, K. Radyushkin, K. Hammerschmidt, S. Granon, S. Boretius, F. Varoqueaux,
N. Ramanantsoa, J. Gallego, A. Ronnenberg, D. Winter, J. Frahm, J. Fischer, T. Bourgeron,
H. Ehrenreich, N. Brose, Reduced social interaction and ultrasonic communication in a
mouse model of monogenic heritable autism. Proc. Natl. Acad. Sci. U.S.A. 105, 1710–1715 (2008).
34. M. R. Etherton, C. A. Blaiss, C. M. Powell, T. C. Südhof, Mouse neurexin-1a deletion causes
correlated electrophysiological and behavioral changes consistent with cognitive impair-
ments. Proc. Natl. Acad. Sci. U.S.A. 106, 17998–18003 (2009).
35. J. Nakatani, K. Tamada, F. Hatanaka, S. Ise, H. Ohta, K. Inoue, S. Tomonaga, Y. Watanabe,
Y. J. Chung, R. Banerjee, K. Iwamoto, T. Kato, M. Okazawa, K. Yamauchi, K. Tanda, K. Takao,
T. Miyakawa, A. Bradley, T. Takumi, Abnormal behavior in a chromosome-engineered
mouse model for human 15q11-13 duplication seen in autism. Cell 137, 1235–1246 (2009).
36. D. T. Page, O. J. Kuti, C. Prestia, M. Sur, Haploinsufficiency for Pten and Serotonin trans-
porter cooperatively influences brain size and social behavior. Proc. Natl. Acad. Sci. U.S.A.
106, 1989–1994 (2009).
37. K. Radyushkin, K. Hammerschmidt, S. Boretius, F. Varoqueaux, A. El-Kordi, A. Ronnenberg,
D. Winter, J. Frahm, J. Fischer, N. Brose, H. Ehrenreich, Neuroligin-3-deficient mice: Model
of a monogenic heritable form of autism with an olfactory deficit. Genes Brain Behav. 8,
38. O. Bozdagi, T. Sakurai, D. Papapetrou, X. Wang, D. L. Dickstein, N. Takahashi, Y. Kajiwara,
M. Yang, A. M. Katz, M. L. Scattoni, M. J. Harris, R. Saxena, J. L. Silverman, J. N. Crawley,
Q. Zhou, P. R. Hof, J. D. Buxbaum, Haploinsufficiency of the autism-associated Shank3
gene leads to deficits in synaptic function, social interaction, and social communication.
Mol. Autism 1, 15 (2010).
39. D. M. Young, A. K. Schenk, S. B. Yang, Y. N. Jan, L. Y. Jan, Altered ultrasonic vocalizations in
a tuberous sclerosis mouse model of autism. Proc. Natl. Acad. Sci. U.S.A. 107, 11074–11079
40. M. D. Carter, C. R. Shah, C. L. Muller, J. N. Crawley, A. M. Carneiro, J. Veenstra-VanderWeele,
Absence of preference for social novelty and increased grooming in integrin b3 knockout
mice: Initial studies and future directions. Autism Res. 4, 57–67 (2011).
41. D. Ehninger, A. J. Silva, Increased levels of anxiety-related behaviors in a Tsc2 dominant
negative transgenic mouse model of tuberous sclerosis. Behav. Genet. 41, 357–363 (2011).
42. J. Peça, C. Feliciano, J. T. Ting, W. Wang, M. F. Wells, T. N. Venkatraman, C. D. Lascola, Z. Fu,
G. Feng, Shank3 mutant mice display autistic-like behaviours and striatal dysfunction.
Nature 472, 437–442 (2011).
43. O. Peñagarikano, B. S. Abrahams, E. I. Herman, K. D. Winden, A. Gdalyahu, H. Dong,
L. I. Sonnenblick, R. Gruver, J. Almajano, A. Bragin, P. Golshani, J. T. Trachtenberg, E. Peles,
D. H. Geschwind, Absence of CNTNAP2 leads to epilepsy, neuronal migration abnormalities,
and core autism-related deficits. Cell 147, 235–246 (2011).
44. S. E. Smith, Y. D. Zhou, G. Zhang, Z. Jin, D. C. Stoppel, M. P. Anderson, Increased gene
dosage of Ube3a results in autism traits and decreased glutamate synaptic transmission
in mice. Sci. Transl. Med. 3, 103ra97 (2011).
45. X. Wang, P. A. McCoy, R. M. Rodriguiz, Y. Pan, H. S. Je, A. C. Roberts, C. J. Kim, J. Berrios,
J. S. Colvin, D. Bousquet-Moore, I. Lorenzo, G. Wu, R. J. Weinberg, M. D. Ehlers, B. D. Philpot,
A. L. Beaudet, W. C. Wetsel, Y. H. Jiang, Synaptic dysfunction and abnormal behaviors in
mice lacking major isoforms of Shank3. Hum. Mol. Genet. 20, 3093–3108 (2011).
46. M. Wöhr, F. I. Roullet, A. Y. Hung, M. Sheng, J. N. Crawley, Communication impairments in
mice lacking Shank1: Reduced levels of ultrasonic vocalizations and scent marking be-
havior. PLoS One 6, e20631 (2011).
47. M. Yang, M. L. Scattoni, K. K. Chadman, J. M. Silverman, J. N. Crawley, in Behavioral Evaluation
of Genetic Mouse Models of Autism, D. G. Amaral, G. Dawson, D. H. Geschwind, Eds. (Oxford
Univ. Press, Oxford, UK, 2010).
48. M. L. Hayashi, B. S. Rao, J. S. Seo, H. S. Choi, B. M. Dolan, S. Y. Choi, S. Chattarji, S. Tonegawa,
Inhibition of p21-activated kinase rescues symptoms of fragile X syndrome in mice.
Proc. Natl. Acad. Sci. U.S.A. 104, 11489–11494 (2007).
49. D. Ehninger, S. Han, C. Shilyansky, Y. Zhou, W. Li, D. J. Kwiatkowski, V. Ramesh, A. J. Silva,
Reversal of learning deficits in a Tsc2+/−mouse model of tuberous sclerosis. Nat. Med. 14,
50. D. Ehninger, W. Li, K. Fox, M. P. Stryker, A. J. Silva, Reversing neurodevelopmental disorders in
adults. Neuron 60, 950–960 (2008).
51. S. Cobb, J. Guy, A. Bird, Reversibility of functional deficits in experimental models of Rett
syndrome. Biochem. Soc. Trans. 38, 498–506 (2010).
52. J. Zhou, J. Blundell, S. Ogawa, C. H. Kwon, W. Zhang, C. Sinton, C. M. Powell, L. F. Parada,
Pharmacological inhibition of mTORC1 suppresses anatomical, cellular, and behavioral ab-
normalities in neural-specific Pten knock-out mice. J. Neurosci. 29, 1773–1783 (2009).
53. M. F. Bear, K. M. Huber, S. T. Warren, The mGluR theory of fragile X mental retardation.
Trends Neurosci. 27, 370–377 (2004).
54. G. Dölen, E. Osterweil, B. S. Rao, G. B. Smith, B. D. Auerbach, S. Chattarji, M. F. Bear, Correction
of fragile X syndrome in mice. Neuron 56, 955–962 (2007).
55. D. D. Krueger, M. F. Bear, Toward fulfilling the promise of molecular medicine in fragile X
syndrome. Annu. Rev. Med. 62, 411–429 (2011).
56. S. Jacquemont, A. Curie, V. des Portes, M. G. Torrioli, E. Berry-Kravis, R. J. Hagerman,
F. J. Ramos, K. Cornish, Y. He, C. Paulding, G. Neri, F. Chen, N. Hadjikhani, D. Martinet, J. Meyer,
J. S. Beckmann, K. Delange, A. Brun, G. Bussy, F. Gasparini, T. Hilse, A. Floesser, J. Branson,
G. Bilbe, D. Johns, B. Gomez-Mancilla, Epigenetic modification of the FMR1 gene in fragile
X syndrome is associated with differential response to the mGluR5 antagonist AFQ056.
Sci. Transl. Med. 3, 64ra1 (2011).
57. D. D. Hatton, J. Sideris, M. Skinner, J. Mankowski, D. B. Bailey Jr., J. Roberts, P. Mirrett,
Autistic behavior in children with fragile X syndrome: Prevalence, stability, and the
impact of FMRP. Am. J. Med. Genet. A 140A, 1804–1813 (2006).
58. S. W. Harris, D. Hessl, B. Goodlin-Jones, J. Ferranti, S. Bacalman, I. Barbato, F. Tassone,
P. J. Hagerman, H. Herman, R. J. Hagerman, Autism profiles of males with fragile X syndrome.
Am. J. Ment. Retard. 113, 427–438 (2008).
59. G. M. Sankoorikal, K. A. Kaercher, C. J. Boon, J. K. Lee, E. S. Brodkin, A mouse model system
for genetic analysis of sociability: C57BL/6J versus BALB/cJ inbred mouse strains.
Biol. Psychiatry 59, 415–423 (2006).
60. V. J. Bolivar, S. R. Walters, J. L. Phoenix, Assessing autism-like behavior in mice: Variations
in social interactions among inbred strains. Behav. Brain Res. 176, 21–26 (2007).
61. S. S. Moy, J. J. Nadler, N. B. Young, A. Perez, L. P. Holloway, R. P. Barbaro, J. R. Barbaro,
L. M. Wilson, D. W. Threadgill, J. M. Lauder, T. R. Magnuson, J. N. Crawley, Mouse behav-
ioral tasks relevant to autism: Phenotypes of 10 inbred strains. Behav. Brain Res. 176, 4–20
62. M. Yang, V. Zhodzishsky, J. N. Crawley, Social deficits in BTBR T + tf/J mice are un-
changed by cross-fostering with C57BL/6J mothers. Int. J. Dev. Neurosci. 25, 515–521
63. A. H. Fairless, H. C. Dow, M. M. Toledo, K. A. Malkus, M. Edelmann, H. Li, K. Talbot, S. E. Arnold,
T. Abel, E. S. Brodkin, Low sociability is associated with reduced sizeof the corpus callosum in
the BALB/cJ inbred mouse strain. Brain Res. 1230, 211–217 (2008).
64. H. G. McFarlane, G. K. Kusek, M. Yang, J. L. Phoenix, V. J. Bolivar, J. N. Crawley, Autism-like
behavioral phenotypes in BTBR T+tf/J mice. Genes Brain Behav. 7, 152–163 (2008).
65. S. S. Moy, J. J. Nadler, M. D. Poe, R. J. Nonneman, N. B. Young, B. H. Koller, J. N. Crawley,
G. E. Duncan, J. W. Bodfish, Development of a mouse test for repetitive, restricted behaviors:
Relevance to autism. Behav. Brain Res. 188, 178–194 (2008).
66. M. L. Scattoni, S. U. Gandhy, L. Ricceri, J. N. Crawley, Unusual repertoire of vocalizations in
the BTBR T+tf/J mouse model of autism. PLoS One 3, e3067 (2008).
67. B. C. Ryan, N. B. Young, J. N. Crawley, J. W. Bodfish, S. S. Moy, Social deficits, stereotypy
and early emergence of repetitive behavior in the C58/J inbred mouse strain. Behav.
Brain Res. 208, 178–188 (2010).
68. E. B. Defensor, B. L. Pearson, R. L. Pobbe, V. J. Bolivar, D. C. Blanchard, R. J. Blanchard, A
novel social proximity test suggests patterns of social avoidance and gaze aversion-like
behavior in BTBR T+ tf/J mice. Behav. Brain Res. 217, 302–308 (2011).
69. B. L. Pearson, R. L. Pobbe, E. B. Defensor, L. Oasay, V. J. Bolivar, D. C. Blanchard, R. J. Blanchard,
Motor and cognitive stereotypies in the BTBR T+tf/J mouse model of autism. Genes Brain
Behav. 10, 228–235 (2011).
70. M. Wöhr, F. I. Roullet, J. N. Crawley, Reduced scent marking and ultrasonic vocalizations in
the BTBR T+tf/J mouse model of autism. Genes Brain Behav. 10, 35–43 (2011).
71. G. C. Wagner, N. Avena, T. Kita, T. Nakashima, H. Fisher, A. K. Halladay, Risperidone re-
duction of amphetamine-induced self-injurious behavior in mice. Neuropharmacology 46,
72. J. L. Silverman, S. S. Tolu, C. L. Barkan, J. N. Crawley, Repetitive self-grooming behavior in
the BTBR mouse model of autism is blocked by the mGluR5 antagonist MPEP. Neuropsycho-
pharmacology 35, 976–989 (2010).
73. M. Yang, K. Perry, M. D. Weber, A. M. Katz, J. N. Crawley, Social peers rescue autism-relevant
sociability deficits in adolescent mice. Autism Res. 4, 17–27 (2011).
74. M. Yang, M. L. Scattoni, V. Zhodzishsky, T. Chen, H. Caldwell, W. S. Young, H. G. McFarlane,
J. N. Crawley, Social approach behaviors are similar on conventional versus reverse
www.ScienceTranslationalMedicine.org25 April 2012 Vol 4 Issue 131 131ra51
on April 26, 2012
lighting cycles, and in replications across cohorts, in BTBR T+ tf/J, C57BL/6J, and vaso-
pressin receptor 1B mutant mice. Front. Behav. Neurosci. 1, 1 (2007).
75. J. L. Silverman, M. Yang, S. M. Turner, A. M. Katz, D. B. Bell, J. I. Koenig, J. N. Crawley, Low
stress reactivity and neuroendocrine factors in the BTBR T+tf/J mouse model of autism.
Neuroscience 171, 1197–1208 (2010).
76. K. K. Chadman, Fluoxetine but not risperidone increases sociability in the BTBR mouse
model of autism. Pharmacol. Biochem. Behav. 97, 586–594 (2011).
77. G. G. Gould, J. G. Hensler, T. F. Burke, R. H. Benno, E. S. Onaivi, L. C. Daws, Density and function
of central serotonin (5-HT) transporters, 5-HT1Aand 5-HT2Areceptors, and effects of their
targeting on BTBR T+tf/J mouse social behavior. J. Neurochem. 116, 291–303 (2011).
78. R. L. Pobbe, B. L. Pearson, E. B. Defensor, V. J. Bolivar, D. C. Blanchard, R. J. Blanchard,
Expression of social behaviors of C57BL/6J versus BTBR inbred mouse strains in the visible
burrow system. Behav. Brain Res. 214, 443–449 (2010).
79. D. Wahlsten, P. Metten, J. C. Crabbe, Survey of 21 inbred mouse strains in two laboratories
reveals that BTBR T/+ tf/tf has severely reduced hippocampal commissure and absent corpus
callosum. Brain Res. 971, 47–54 (2003).
80. L. K. Paul, W. S. Brown, R. Adolphs, J. M. Tyszka, L. J. Richards, P. Mukherjee, E. H. Sherr,
Agenesis of the corpus callosum: Genetic, developmental and functional aspects of
connectivity. Nat. Rev. Neurosci. 8, 287–299 (2007).
81. R. Booth, G. L. Wallace, F. Happé, Connectivity and the corpus callosum in autism
spectrum conditions: Insights from comparison of autism and callosal agenesis. Prog.
Brain Res. 189, 303–317 (2011).
82. M. Yang, A. M. Clarke, J. N. Crawley, Postnatal lesion evidence against a primary role for
the corpus callosum in mouse sociability. Eur. J. Neurosci. 29, 1663–1677 (2009).
83. V. Bolivar, M. Solanki, W. Du, S. Day, K. Manley, Autism-Like Behaviors in Mice Are Modified
by Genetic Background, Sex and Testing Protocol (Society for Neuroscience, Washington,
84. G. Bothe, M. Solanki, W. Du, G. Kusek, R. Auerbach, K. Manley, V. Bolivar, Genetic Investigations
of Corpus Callosum Abnormalities in BTBR T+ tf/J Mice (Society for Neuroscience, Washington,
85. D. Jones-Davis, M. Yang, E. Rider, S. Sen, J. Crawley, E. Sherr, Identification of Loci Associated
with Autism-Relevant Behavioral Traits in the BTBR Strain of Mouse (Society for Neuroscience,
Washington, DC, 2011).
86. J. J. Nadler, S. S. Moy, G. Dold, D. Trang, N. Simmons, A. Perez, N. B. Young, R. P. Barbaro,
J. Piven, T. R. Magnuson, J. N. Crawley, Automated apparatus for quantitation of social ap-
proach behaviors in mice. Genes Brain Behav. 3, 303–314 (2004).
87. M. Yang, J. L. Silverman, J. N. Crawley, Automated three-chambered social approach task
for mice. Curr. Protoc. Neurosci. Chapter 8, Unit 8.26 (2011).
88. E. S. Brodkin, BALB/c mice: Low sociability and other phenotypes that may be relevant to
autism. Behav. Brain Res. 176, 53–65 (2007).
89. J. L. Silverman, S. M. Turner, C. L. Barkan, S. S. Tolu, R. Saxena, A. Y. Hung, M. Sheng, J. N. Crawley,
Sociability and motor functions in Shank1 mutant mice. Brain Res. 1380, 120–137 (2011).
90. A. H. Fairless, R. Y. Shah, A. J. Guthrie, H. Li, E. S. Brodkin, Deconstructing sociability, an
autism-relevant phenotype, in mouse models. Anat. Rec. 294, 1713–1725 (2011).
91. M. Yang, D. N. Abrams, J. Y. Zhang, M. D. Weber, A. M. Katz, A. M. Clarke, J. L. Silverman,
J. N. Crawley, Low sociability in BTBR T+tf/J mice is independent of partner strain.
Physiol. Behav. 10.1016/j.physbeh.2011.12.025 (2012).
92. S. E. Levy, D. S. Mandell, R. T. Schultz, Autism. Lancet 374, 1627–1638 (2009).
93. S. Bent, R. L. Hendren, Improving the prediction of response to therapy in autism. Neurother-
apeutics 7, 232–240 (2010).
94. J. Veenstra-Vanderweele, R. D. Blakely, Networking in autism: Leveraging genetic, biomarker
and model system findings in the search for new treatments. Neuropsychopharmacology 37,
95. G. Dawson, Early behavioral intervention, brain plasticity, and the prevention of autism
spectrum disorder. Dev. Psychopathol. 20, 775–803 (2008).
96. F. Gasparini, G. Bilbe, B. Gomez-Mancilla, W. Spooren, mGluR5 antagonists: Discovery,
characterization and drug development. Curr. Opin. Drug Discov. Devel. 11, 655–665 (2008).
97. Z. Hughes, S. J. Neal, D. L. Smith, S. J. Sukoff Rizzo, C. M. Pulicicchio, S. Lotarski, S. Lu,
J. M. Dwyer, J. Brennan, M. Olsen, C. N. Bender, E. Kouranova, T. H. Andree, J. E. Harrison,
G. T. Whiteside, D. Springer, S. V. O’Neil, S. K. Leonard, L. E. Schechter, J. Dunlop,
S. Rosenzweig-Lipson, R. H. Ring, Negative allosteric modulation of metabotropic gluta-
mate receptor 5 results in broad spectrum activity relevant to treatment resistant depres-
sion. Neuropharmacology 10.1016/j.neuropharm.2012.04.007 (2012).
98. K. R. Bailey, M. N. Pavlova, A. D. Rohde, J. G. Hohmann, J. N. Crawley, Galanin receptor
subtype 2 (GalR2) null mutant mice display an anxiogenic-like phenotype specific to the
elevated plus-maze. Pharmacol. Biochem. Behav. 86, 8–20 (2007).
99. J. J. Anderson, M. J. Bradbury, D. R. Giracello, D. F. Chapman, G. Holtz, J. Roppe, C. King,
N. D. Cosford, M. A. Varney, In vivo receptor occupancy of mGlu5 receptor antagonists
using the novel radioligand [3H]3-methoxy-5-(pyridin-2-ylethynyl)pyridine). Eur. J. Pharmacol.
473, 35–40 (2003).
100. M. L. Terranova, G. Laviola, Scoring of social interactions and play in mice during adoles-
cence. Curr. Protocols Toxicol. 26, 13.10.1–13.10.11 (2005).
101. M. L. Terranova, G. Laviola, Scoring of Social Interactions and Play in Mice During Adoles-
cence (John Wiley & Sons Inc., Hoboken, NJ, 2005).
102. S. M. Hamilton, C. M. Spencer, W. R. Harrison, L. A. Yuva-Paylor, D. F. Graham, R. A. Daza,
R. F. Hevner, P. A. Overbeek, R. Paylor, Multiple autism-like behaviors in a novel transgenic
mouse model. Behav. Brain Res. 218, 29–41 (2011).
103. M. A. Bangash, J. M. Park, T. Melnikova, D. Wang, S. K. Jeon, D. Lee, S. Syeda, J. Kim, M. Kouser,
J. Schwartz, Y. Cui, X. Zhao, H. E. Speed, S. E. Kee, J. C. Tu, J. H. Hu, R. S. Petralia, D. J. Linden,
C. M. Powell, A. Savonenko, B. Xiao, P. F. Worley, Enhanced polyubiquitination of Shank3
and NMDA receptor in a mouse model of autism. Cell 145, 758–772 (2011).
Funding: This work was supported by the NIMH Intramural Research Program (J.L.S., M.N.K.,
S.M.T., S.S.T., and J.N.C.) and Pfizer Global Research (D.G.S., S.J.S.R., D.K.B., D.L.S., K.F., and R.H.R.). Au-
thor contributions: J.N.C., J.L.S., R.H.R., D.G.S., and S.J.S.R. designed the study; J.N.C., J.L.S., D.G.S., and
S.J.S.R. wrote the paper; J.L.S., M.N.K., S.M.T., and S.S.T. conducted the behavioral experiments and
statistical analyses at NIMH; S.J.S.R. and D.K.B. conducted the behavioral experiments and statistical
analyses at Pfizer; D.L.S. and K.F. conducted the pharmacokinetics and receptor occupancy at Pfizer.
Competing interests: The authors declare that they have no competing interests. GRN-529 (PF-
05212391) is published as international patent application publication number WO2010/124047.
Submitted 22 November 2011
Accepted 6 March 2012
Published 25 April 2012
Citation: J. L. Silverman, D. G. Smith, S. J. S. Rizzo, M. N. Karras, S. M. Turner, S. S. Tolu,
D. K. Bryce, D. L. Smith, K. Fonseca, R. H. Ring, J. N. Crawley, Negative allosteric modulation
of the mGluR5 receptor reduces repetitive behaviors and rescues social deficits in mouse
models of autism. Sci. Transl. Med. 4, 131ra51 (2012).
www.ScienceTranslationalMedicine.org25 April 2012 Vol 4 Issue 131 131ra51
on April 26, 2012