Autism gene variant causes hyperserotonemia,
serotonin receptor hypersensitivity, social impairment
and repetitive behavior
Jeremy Veenstra-VanderWeelea,b,c,d,e,1, Christopher L. Mullera, Hideki Iwamotoc, Jennifer E. Sauera,
W. Anthony Owensf, Charisma R. Shaha, Jordan Cohena, Padmanabhan Mannangattig, Tammy Jessenc,
Brent J. Thompsonh,i, Ran Yec, Travis M. Kerrc, Ana M. Carneiroc,d,e, Jacqueline N. Crawleyj,
Elaine Sanders-Busha,c,d,e,k, Douglas G. McMahonc,d,e,k,l, Sammanda Ramamoorthyg, Lynette C. Dawsf,i,
James S. Sutcliffea,d,e,m, and Randy D. Blakelya,c,d,e,k,1
Departments ofaPsychiatry,bPediatrics, andcPharmacology,dBrain Institute, andeKennedy Center for Research on Human Development, Vanderbilt
University, Nashville, TN 37232; Departments offPhysiology,gNeurosciences, andhCellular and Structural Biology, University of Texas Health Science Center,
San Antonio, TX 78229;iDepartment of Pharmacology, University of Texas Health Science Center, San Antonio, TX 78229;jLaboratory of Behavioral
Neuroscience, National Institute of Mental Health, Bethesda, MD 20892;kSilvio O. Conte Center for Neuroscience Research, Vanderbilt University, Nashville,
TN 37232;lDepartment of Biological Sciences, Vanderbilt University, Nashville, TN 37232; andmDepartment of Molecular Physiology and Biophysics,
Vanderbilt University, Nashville, TN 37232
Edited* by Susan G. Amara, University of Pittsburgh School of Medicine, Pittsburgh, PA, and approved February 14, 2012 (received for review July 28, 2011)
Fifty years ago, increased whole-blood serotonin levels, or hyper-
serotonemia, first linked disrupted 5-HT homeostasis to Autism
Spectrum Disorders (ASDs). The 5-HT transporter (SERT) gene
(SLC6A4) has been associated with whole blood 5-HT levels and
ASD susceptibility. Previously, we identified multiple gain-of-func-
tion SERT coding variants in children with ASD. Here we establish
that transgenic mice expressing the most common of these var-
iants, SERT Ala56, exhibit elevated, p38 MAPK-dependent trans-
porter phosphorylation, enhanced 5-HT clearance rates and
hyperserotonemia. These effects are accompanied by altered basal
firing of raphe 5-HT neurons, as well as 5HT1Aand 5HT2Areceptor
hypersensitivity. Strikingly, SERT Ala56 mice display alterations in
social function, communication, and repetitive behavior. Our ef-
forts provide strong support for the hypothesis that altered 5-HT
homeostasis can impact risk for ASD traits and provide a model
with construct and face validity that can support further analysis
of ASD mechanisms and potentially novel treatments.
and communication, as well as repetitive behavior (1). Hyperse-
rotonemia, or increased whole-blood serotonin [i.e., 5-hydroxy-
tryptamine (5-HT)], is a well replicated biomarker that is present
in approximately 30% of subjects with ASD (2, 3). Some data
suggest an association of hyperserotonemia with stereotyped or
self-injurious behavior, but results have been inconsistent (4, 5).
Despite the high heritability of whole-blood 5-HT levels (6), a
mechanistic connection between hyperserotonemia and specific
components of the pathophysiology of ASD remains enigmatic.
In blood, 5-HT is contained almost exclusively in platelets (7) that
lack 5-HT biosynthetic capacity but accumulate the monoamine
via the antidepressant-sensitive serotonin transporter (SERT;
5-HTT). A genome-wide study of whole-blood 5-HT as a quanti-
tative trait found association with the SERT-encoding gene
SLC6A4, as well as with ITGB3, which encodes the SERT-inter-
acting protein integrin β3. In both cases, the strongest evidence for
associationwasfoundinmales(8–10).Linkagestudies inASD also
implicate the 17q11.2 region containing SLC6A4, again with
stronger evidence in males (11, 12).
ASD(13), weandour colleagues previously screened SLC6A4 for
to 17q11.2. In this effort, we identified five rare SERT coding
variants, each of which confers increased 5-HT transport in
utism spectrum disorder (ASD) is a male-predominant dis-
order that is characterized by deficits in social interactions
transfected cells as well as in lymphoblasts derived from SERT
variant-expressing probands (11, 14, 15). We found the most
common of these variants, Ala56 (allele frequency in subjects of
European ancestry of 0.5–1%), to be overtransmitted to autism
probands, and to be associated with both rigid-compulsive be-
havior and sensory aversion (11, 16). No such trait association is
seen in families without linkage to this region (17). In transfected
cells, SERT Ala56 also demonstrates increased basal phosphor-
ylation and insensitivity to PKG- and p38 MAPK-linked signaling
that normally produce increased transporter trafficking and cat-
alytic activation, respectively (15). These findings suggest that
homeostatic control of 5-HT may be impaired in some children
with ASD. Importantly, model system studies indicate that 5-HT
and SERT are important determinants of normal brain devel-
opment and that early-life perturbations in 5-HT signaling can
have enduring effects on behavior (18–21).
To explore the dependence of juvenile and adult behavior on
early-life 5-HT manipulation and further understand the impact
of the SERT Ala56 variant in vivo, we generated mice expressing
SERT Ala56 from the native mouse Slc6a4 locus (22). Although
SERT Ala56 mice exhibit normal growth and fertility (22), they
display significantly increased CNS 5-HT clearance, enhanced
5-HT receptor sensitivity, and hyperserotonemia. Even more strik-
ing, SERT Ala56 animals display alterations in a number of ASD-
SERT Function and Synaptic Homeostasis Is Altered in SERT Ala56
Mice. As predicted from studies of SERT Ala56 transfected cells
(11, 14, 15), midbrain SERT protein levels in Ala56 mice were
found to be identical to those of WT, Gly56 littermate controls
(Fig. S1A), results that are paralleled by the results of antagonist
Author contributions: J.V., H.I., A.M.C., J.N.C., E.S.-B., D.G.M., S.R., L.C.D., J.S.S., and R.D.B.
designed research; J.V., C.L.M., H.I., J.E.S., W.A.O., C.R.S., J.C., P.M., T.J., B.J.T., R.Y., T.M.K.,
and S.R. performed research; J.V., C.L.M., H.I., A.M.C., J.N.C., E.S.-B., D.G.M., S.R., L.C.D.,
J.S.S., and R.D.B. analyzed data; and J.V. and R.D.B. wrote the paper.
Conflict of interest statement: The authors have no direct competing financial interests.
J.V. receives research funding for nonoverlapping work from Seaside Therapeutics, Roche
Pharmaceuticals, and Novartis, and has served as a consultant to Novartis. R.D.B. receives
research funding for nonoverlapping work from Forest Pharmaceuticals and serves on the
Lundbeck Pharmaceuticals Advisory Board and as a consultant to JubilantInnovation.
*This Direct Submission article had a prearranged editor.
1To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or randy.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| April 3, 2012
| vol. 109
| no. 14
binding (Fig. S1B) and immunohistochemical studies (Fig. S2).
SERT proteins exhibit significant posttranslational regulation
(23), including Ser/Thr phosphorylation that involves PKG and
p38 MAPK-linked pathways (24, 25). Consistent with our find-
ings in transfected cells (14, 15), we found phosphorylation of
SERT Ala56 to be significantly elevated in midbrain synapto-
somes under basal conditions (P = 0.0002; Fig. 1A). Moreover,
we found that activation of PKG with 8-Bromo-cGMP (8-Br-
cGMP) fails to increase phosphorylation of SERT Ala56,
whereas a robust increase in phosphorylation is observed for WT
SERT (P = 0.0013; Fig. 1B). Basal SERT phosphorylation is
dependent on p38 MAPK activity (24), and PKG activation leads
to a p38 MAPK-dependent increase in SERT activity (26). The
gain of SERT activity following activation of p38 MAPK is
paralleled by an increased affinity for 5-HT that can support an
enhanced rate of transport at low 5-HT concentrations (14, 15).
When we incubated synaptosomes with the p38-MAPK inhibitor
PD169316, we found significant reductions in basal phosphory-
lation of SERT Gly56 mice (P = 0.018; Fig. 1C). Importantly,
the inhibitor also normalized the difference in phosphorylation
between the WT and mutant transporters. These findings suggest
that constitutive phosphorylation of SERT Ala56 precludes the
flexibility exhibited by WT SERT to move between low and high
activity states in parallel with changes in 5-HT release.
To complement our ex vivo phosphorylation studies and de-
termine whether SERT Ala56 mice exhibit constitutively en-
hanced SERT activity in vivo, we monitored hippocampal 5-HT
clearance by in vivo chronoamperometry (27). In these studies,
we observed a significant increase in the rate of 5-HT clearance
for Ala56 animals vs. WT littermates (P < 0.0001; Fig. 1D and
Fig. S3). Paralleling our findings in synaptosomes, we observed
a significant increase in 5-HT clearance following infusion of 8-
Br-cGMP in the WT animals but no significant response in
SERT Ala56 animals (P = 0.022 and P = 0.009 for time to clear
20% and 80% of maximum 5-HT signal, respectively; Fig. 1E).
Despite the significant increase in 5-HT clearance, no change
was observed in midbrain or forebrain tissue 5-HT levels (Fig.
S4 A and B). In contrast, in whole blood, in which 5-HT is se-
questered by platelets that lack the capability to offset 5-HT
accumulation with decreased 5-HT synthesis, SERT Ala56 mice
exhibited significantly increased 5-HT levels relative to WT lit-
termates (P = 0.02; Fig. 1F).
diminished sensitivity of multiple 5-HT receptors (28). Therefore,
we hypothesized that increased CNS 5-HT clearance could lead to
decreased synaptic 5-HT availability and a compensatory increase
in 5-HT receptor sensitivity. Consistent with this idea, enhanced 5-
HT receptor sensitivity occurs in mice overexpressing SERT (29).
To explore this hypothesis in our mice, we first examined the
cumulative graph of basal phosphorylation of SERT [Bartlett test statistic, 26.49, showing unequal variances (P < 0.0001); therefore, nonparametric Mann–
Whitney test was used, U = 6.00, P = 0.0002, n = 12 per genotype]. (B) Representative gel and cumulative graph of 8-Br-cGMP–induced phosphorylation (two-
way repeated-measures ANOVA interaction of cGMP treatment by genotype, F = 17.51, P = 0.0013; Bonferroni post-test in WT for cGMP treatment, t = 3.66,
P < 0.01; Bonferroni post-test in Ala56 for cGMP, t = 2.26, P > 0.05; WT, n = 7; Ala56, n = 7). (C) Representative gel and cumulative graph of PD169316
inhibition of phosphorylation (two-way repeated-measures ANOVA interaction of PD169316 treatment by genotype, F = 8.815, P = 0.018; Bonferroni post-test
of genotype difference in basal condition, t = 4.83, P < 0.01; Bonferroni post-test of genotype difference after PD169316 treatment, t = 0.63, P > 0.05; WT, n =
5; Ala56, n = 5). (D) 5-HT clearance rates in the CA3 region of the hippocampus as a function of increasing extracellular 5-HT concentrations. Mean clearance
values from multiple 5-HT pulses ± SEM with three to six mice per point. For purposes of clarity, the SEMs for signal amplitudes are not shown, but they were
always within 10% of the mean. Clearance of 5-HT was significantly faster in Ala56 mice than that in WT controls (main effect genotype, F1,73= 64.69, P <
0.0001; main effect 5-HT concentration, F7,73= 15.86, P < 0.0001, two-way ANOVA with Bonferroni post hoc comparisons). Kinetic analysis reveals an ap-
proximate twofold increase in the apparent Vmaxfor 5-HT clearance (t10= 7.248, P < 0.0001; Ala56, 82 ± 20 nM/s vs. Gly56, 41 ± 15 nM/s) with no change in
apparent transporter affinity (KT) (corrected for volume fraction, α = 0.02; Ala56, 64 ± 42 nM vs. Gly56, 64 ± 28 nM). (E) Time to clear 20% (T20) and 80% (T80)
of the peak 5-HT signal amplitude 10 min after application of 0.5 pmol of 8-Br-cGMP, normalized to baseline 5-HT clearance. T20reflects 5-HT at a concen-
tration at which SERT-mediated 5-HT clearance is near Vmax, whereas T80provides an index of 5-HT clearance at a concentration approximating the Kmfor
SERT-mediated 5-HT uptake. 8-Br-cGMP significantly shortened both T20and T80for 5-HT clearance in WT mice but was without effect in Ala56 mice (T20, t =
2.65, P = 0.022; T80: t = 3.195, P = 0.009; WT, n = 7; Ala56 n = 6). (F) HPLC measurement of 5-HT in whole blood. Unpaired t test revealed a significant increase in
whole-blood 5-HT in the Ala56 animals compared with WT controls (t = 2.55, P = 0.02; WT, n = 11; Ala56, n = 9).
Dysregulated SERT phosphorylation, increased 5-HT uptake, and hyperserotonemia in the SERT Ala56 knock-in mice. (A) Representative gel and
| www.pnas.org/cgi/doi/10.1073/pnas.1112345109Veenstra-VanderWeele et al.
sensitivity of animals to the 5-HT2A/2Creceptor agonist 1-(2,5-
dimethoxy-4-iodophenyl)-2-aminopropane (DOI), which produces
a stereotyped head twitch response mediated by postsynaptic,
cortical 5-HT2Areceptors (30). We observed a significantly ele-
vated head twitch response in SERT Ala56 mice compared with
WT littermate controls (P < 0.01; Fig. 2A). Next, we treated ani-
mals with the 5-HT1A/7agonist 8-hydroxy-2-(di-n-propylamino)-
tetraline (8-OH-DPAT), which leads to hypothermia in mice, me-
diated by 5-HT1Aautoreceptors located on raphe 5-HT neurons
(31). As with DOI studies, SERT Ala56 mice displayed a signifi-
cantly increased sensitivity to 8-OH-DPAT–induced hypother-
mia compared with WT controls (P < 0.0001; Fig. 2B; baseline
temperature shown in Fig. S5A).
To establish a physiological consequence of altered 5-HT
receptor signaling in SERT Ala56 mice, we used loose-patch
recordings of dorsal raphe 5-HT neurons in midbrain slices to
examine basal firing rates and 5HT1A-mediated suppression
of raphe neuron excitability. Location of recordings was first
two observers blind to genotype over 15 min following injection of saline solution, the 5-HT2agonist DOI, or the specific 5-HT2Aantagonist M-100907 fol-
lowed by DOI. Two-way repeated-measures ANOVA revealed a significant genotype–drug interaction (F = 6.88, P = 0.0029, n = 10 per genotype), with
a significant Bonferroni posttest result only for the difference between WT and SERT Ala56 animals in the DOI condition (P < 0.01). (B) Change in rectal
temperature from baseline after administration of the 5-HT1A/5-HT7receptor agonist 8-OH-DPAT. Piecewise mixed linear model analysis revealed a significant
genotype–drug–time interaction over the 30 min from baseline to maximal hypothermia response (F1,177, P < 0.0001, n = 12 per genotype), reflecting
a steeper slope in the SERT Ala56 animals compared with the WT controls. (C) Example traces with basal firing rates are shown for cell-attached extracellular
recordings of dorsal raphe neurons in midbrain slices (n = 16 per genotype). Unpaired t test with Welch correction for unequal variances (F test to compare
variances, F15,15= 4.345, P = 0.0036) revealed a significant decrease in firing rate in the Ala56 animals compared with the WT controls (t = 2.92, P = 0.032). (D)
Percent inhibition of firing of dorsal raphe neurons as a function of varying, bath-applied 5-HT concentration. Curve fit analysis against log(5-HT concen-
tration) with variable slope reveals a significant increase in sensitivity to inhibition of firing by 5-HT in the Ala56 animals compared with the WT controls (F2,6=
292.3, P < 0.0001). (E) Pup vocalizations upon separation from the dam for 5 min at postnatal day 7. Mann–Whitney test revealed a significant decrease in
ultrasonic vocalizations in the SERT Ala56 animals in contrast with WT littermate controls (U = 85.5, P = 0.015; WT, n = 15; Ala56, n = 22). (F) Time in each
chamber of the three-chamber Crawley sociability test is shown. Animals with four or fewer total entries were excluded from the analysis as a result of
inactivity (Fig. S5C). Two-way repeated measures ANOVA revealed a main effect for chamber (F = 23.25, P = 0.0006) and a trend for an interaction between
genotype and stimulus (F = 3.92, P = 0.058; WT, n = 11; Ala56, n = 17). Bonferroni posttest revealed a significant preference for the social chamber in the WT
(P < 0.01) but not the SERT Ala56 animals (P > 0.05). (G) Wins (frontward exit) for male animals on the tube test. McNemar exact test revealed a significant
decrease in wins in the SERT Ala56 animals in contrast with WT littermate controls (P < 0.0001, n = 140 pairings). (H) Time spent performing individual
behaviors over 24 h in the home cage. To allow better visualization, time spent sleeping is not shown, but did not differ by genotype. Two-way repeated
measures ANOVA of log10(time) revealed a significant genotype effect (F = 5.84, P = 0.027, n = 10 per genotype), with Bonferroni posttest showing a sig-
nificant genotype difference only for time spent hanging (P < 0.05). (I) Number of bouts of hanging behavior in 24 h in the home cage. t test of log10(bouts)
revealed a significant increase in bouts of hanging in Ala56 SERT animals in comparison with WT littermate controls (t = 2.567, P = 0.019), with a significant
correlation between log10(time) and log10(bouts) (Pearson R = 0.749, P < 0.001).
Increased receptor sensitivity and altered social, communication, and repetitive behavior in SERT Ala56 knock-in mice. (A) Head twitches recorded by
Veenstra-VanderWeele et al. PNAS
| April 3, 2012
| vol. 109
| no. 14
established by using ePET-1:EYFP transgenic mice (32), cen-
tering on neurons of the medial division of the dorsal raphe.
Under basal conditions, we observed a decrease in the firing rate
of these neurons in SERT Ala56 brain slices (P = 0.0036; Fig.
2C), potentially arising from increased firing suppression by in-
hibitory 5-HT1Aautoreceptors (33). Consistent with this hypoth-
esis, dose–response studies of raphe neuron inhibition produced
by bath-applied 5-HT revealed an enhanced inhibitory potency of
bath-applied 5-HT (P < 0.0001; Fig. 2D).
SERT Ala56 Mice Show Abnormal Social, Communication, and
Repetitive Behavior. Impaired social communication is often the
first sign of ASD (34). To obtain a measure of early social
communication, we measured ultrasonic vocalizations in pups
that were separated from their dam at postnatal day 7. We ob-
served a twofold decrease in vocalizations in SERT Ala56 pups
in contrast to Gly56 littermate controls (P = 0.015; Fig. 2G). As
body temperature could affect vocalization, we measured body
temperatures in 7-day-old pups and found no differences be-
tween SERT Ala56 pups and Gly56 littermate controls (Fig.
S5B). As adults, SERT Ala56 and WT littermates exhibit a low
baseline level of ambulatory activity in novel environments (Fig.
S6 A–C), typical of 129S substrains (35, 36). Thus, in analyses of
adult animals that are dependent upon exploratory behavior, we
included only data from mice with sufficient activity levels to
allow a valid comparison between time spent in different arms or
chambers (Fig. S6 B and C). In these studies, we observed no
differences in anxiety-like behavior on the elevated plus-maze
among mice with more than four arm entries (Fig. S6 D–F; pooled
results from active and inactive animals are shown in Fig. S6G). In
cognitive or behavioral assays dependent on forced movement,
including the Morris water maze test of spatial learning (Fig. S7
A–C), the rotarod test (Fig. S7D), and the forced swim test (Fig.
S7E), no significant differences were observed. However, when we
tested SERT Ala56 mice for potential social interaction deficits in
the three-chamber test of sociability (36, 37), these animals, un-
like their WT littermates, failed to demonstrate preference for
another mouse vs. an inanimate object (Fig. 2E; pooled results
from active and inactive animals are shown in Fig. S6H).
To evaluate adult social interaction in a task that does not re-
quire high levels of ambulatory activity, we implemented the tube
test of social dominance (38). Mouse models of other disorders
that display ASD traits, including Rett and Fragile X syndromes
(38, 39), show altered behavior on this task. After being trained to
progress forward through an empty tube to be returned to their
home cage, mice encounter an unfamiliar mouse that has entered
from the opposite endof the tube. In theseexperiments, we found
that SERT Ala56 animals more often withdrew from the tube
upon encountering an age- and sex-matched WT littermate
control (P < 0.0001; Fig. 2F).
In our studies that identified multiple, gain-of-function SERT
variants in ASD subjects, we found the SERT Ala56 variant to be
associated with rigid-compulsive behavior and sensory aversion in
ASD (11). Several tests of sensorimotor function display deficits
in subjects with ASD, including prepulse inhibition (40), a sen-
sorimotor gating test that can be applied in mice. In a compari-
son with WT littermate controls, SERT Ala56 mice displayed
a genotype by prepulse amplitude interaction effect on acoustic
startle amplitude and prepulse inhibition of startle, reflecting
an increased startle response at baseline that attenuated with
increasing prepulse amplitudes (Fig. S8 A and B). To assess
spontaneous repetitive behavior, we performed noninvasive, au-
tomated monitoring of animals in the home cage. Although many
behaviors were found to be normal in these studies, we observed
that SERT Ala56 mice spent a significantly greater time hanging
from the wire cage lid relative to WT littermates (P < 0.05; Fig.
2H). Time hanging was significantly correlated with the number
of bouts of hanging (Pearson R = 0.749, P < 0.001). Indeed,
examination of recordings revealed that SERT Ala56 animals
climbed up to hang briefly on the wire lid and then returned back
to the floor of the cage, repeating this behavior an average of
approximately 1,000 times over a 24-h recording period (P =
0.019; Fig. 2I). Other potential repetitive behaviors, including
grooming, were not found in the home cage (Fig. 2H). We also
did not see abnormalities in marble burying (Fig. S9), a test of
repetitive behavior proposed to be relevant to ASD (41).
These studies describe biochemical, physiological and behavioral
traits thatderivefromtheconversionofa single amino acid, Gly56,
in SERT. Although conversion of Gly to Ala is a relatively minor
protein associations that may be impacted (23, 42–44). In this re-
gard, SERT associates with proteins that influence the trans-
Ser/Thr phosphatase PP2A (45) and PKG1 (46); although sites
supporting these associations have not been defined. As the Ala56
suspect that the alteration modifies the secondary structure of the
N terminus to permit enhanced access of one or more kinases (or
restrictedaccessofa proteinphosphatase)toeitherthe Nterminus
itself or a nearby cytoplasmic phosphorylation site. The SERT N
terminus is directly connected to transmembrane domain 1, a do-
main that participates in 5-HT binding during the translocation
process (47, 48). We hypothesize therefore that SERT Ala56-in-
duced changes in N-terminal structure, protein associations, or
phosphorylation, lock the transporter in a high-affinity conforma-
tion for 5-HT. Such an effect could lead to diminished availability
for SERT activity to track changes in 5-HT release. As proper
control of 5-HT availability is vital to normal brain development
(18–21), constitutively diminished 5-HT availability could lead to
alterations in brain wiring and enduring changes in behavior.
Thepatternofalterations in wholeblood5-HTlevels,midbrain
5-HT neuron firing, and receptor sensitivities in the SERT Ala56
mouse reflects homeostatic changes in response to the primary
change in SERT. Hyperserotonemia in the Ala56 mouse is con-
sistent with the role of SERT in platelet 5-HT uptake and with
prior studies showing that mice lacking SERT show essentially no
whole-blood 5-HT (9, 49). Although no changes were found in
brain tissue 5-HT levels in the SERT Ala56 mice, we suspect that
tryptophan hydroxylase activity can be readily modified to reduce
5-HT biosynthetic capacity. Platelets lack this mechanism of ho-
meostatic control, as they do not synthesize 5-HT but rather ac-
cumulate 5-HT released by duodenal enterochromaffin cells as
they circulate through the gut (49). Moreover, tissue levels are
a poor correlate of the synaptic availability of 5-HT, which likely
depends more on smaller, readily releasable pools of neuro-
transmitter and the inherent excitability of 5-HT neurons. Our
findings of altered basal firing of raphe neurons in vitro and in-
creased sensitivity of SERT Ala56 animals to challenge with
5HT1Aand 5HT2Areceptor agonists provides critical evidence
that the changes seen in 5-HT clearance translate into behav-
iorally relevant changes in 5-HT signaling.
Substantial ethological differences exist between mice and
humans, and scientists have, to date, generated only a few mouse
models derived from gene variants identified in ASD (50–54). It is
thus not possible to assert that a particular set of behavioral ab-
normalities directly models ASD in a mouse. We believe that, at
this time, it is more reasonable to identify how genetic variation
impacts mouse behavior, with the goal of identifying underlying
changes in brain function that may be conserved in man and which
that SERT Ala56 represents a susceptibility variant, rather than
a highly penetrant, monogenic cause of ASD (11, 17), we do not
expect animals expressing the variant to model all aspects of ASD.
| www.pnas.org/cgi/doi/10.1073/pnas.1112345109Veenstra-VanderWeele et al.
on the presence of other genetic or environmental factors. Thus,
the biochemical, physiological, and behavioral changes seen in an
animal model of a susceptibility variant could offer many, or few,
parallels to the human disorder. Further, individuals with ASD
show considerable heterogeneity in clinical symptoms and genetic
susceptibility, and an animal model of a susceptibility variant
could therefore show some features that arise in only a subset of
individuals (55). Further research is needed to understand how
other genetic or environmental factors modulate the phenotypes
that we observed in the SERT Ala56 mice, as well as the in-
teraction between this variant and other risk factors in individuals
with ASD (11).
We find potential parallels of ASD-associated deficits in the
SERT Ala56 mice. ASD is a disorder of pediatric onset. The
decrease in ultrasonic vocalizations we observe in SERT Ala56
pups suggests an early emergence of the impact of 5-HT on the
capacity or drive for communication. Social interaction deficits in
ASD persist into adulthood. The tube test represents an etho-
logically valid mouse social interaction with a binary outcome that
may be particularly sensitive to changes in social proficiency (39).
Interestingly, Duvall and colleagues (56) identified a male-pre-
dominant, quantitative trait locus for social responsiveness
in multiplex ASD families on chromosome 17q, including the
SLC6A4 gene region. Consistent with this, we observed a lack of
preference by adult SERT Ala56 mice for a social stimulus in
the three-chamber test. Finally, SERT Ala56 has been associ-
ated with sensory aversion in ASD subjects (11). Although we
observed only a modest enhancement in the startle response
during prepulse inhibition tests, more sensitive studies are
needed that examine the physiological properties of the sensory
fields of mice and the ability of animals to integrate sensory
information as these functions are known to be under the in-
fluence of 5-HT during development and in the adult (19–21).
Rigid-compulsive behavior (57) is significantly elevated in
SERT Ala56 carriers and in a combined group comprising SERT
Ala56 carriers and other carriers of rare, hyperfunctional SERT
coding variants (11). It is difficult to predict how a genetic de-
terminant of rigid-compulsive behavior in humans will manifest
in an animal model. The repetitive bouts of hanging from the
wire cage lid we observe in these mice may represent a novel
parallel of the repetitive, nonfunctional routines that are com-
mon in ASD (1), although other interpretations are possible.
Importantly, the repetitive hanging phenotype was identified in
the context of many normal behaviors and in the animals’ home
cage, suggesting that we detected a spontaneous, rather than
experimentally induced, repetitive behavior.
The biochemical, physiological, and behavioral results in the
SERT Ala56 animals also have some important limitations. First,
it is not clear how the specific change from Gly56 to Ala56 leads
to increased p38-MAPK–sensitive basal phosphorylation, whether
by way of altered SERT tertiary structure or disrupted protein–
protein interactions in the N-terminal domain where Gly56 is
expressed. Further work is needed to understand these mecha-
nisms, including identifying the residues at which phosphorylation
occurs and the kinases or phosphatases that act at these residues.
Second, we studied only homozygous animals to maximize our
ability to detect phenotypes. Future studies will be needed to
understand whether similar biochemical, physiological, and be-
SERT Ala56 variant. Third, the low activity seen in some animals
in the elevated plus-maze and three-chamber sociability test limit
the interpretation of these data. This low activity level appears to
be a result of the inbred strain background (35, 36) and does not
differ by genotype. When coupled with the ultrasonic vocalization
and tube test results, however, the overall data are consistent with
a change in social function. Further experiments conducted on
other inbred strain backgrounds may clarify the lack of preference
for social stimuli in the SERT Ala56 mice. Finally, in contrast
with increased grooming behavior observed in some other mouse
models of ASD-associated genetic variation (52, 54, 58), the in-
creased climbing/hanging behavior we observed in the SERT
Ala56 mice is a repetitive behavior that has not previously been
described to our knowledge. The difference in the pattern of ob-
served repetitive behavior in the SERT Ala56 mice, in contrast
to other models of ASD susceptibility, could reflect the fact that
expression of this susceptibility variant is limited to a single, neu-
rochemically defined pathway. Further experiments will be nec-
essary to connect this behavior to changes in underlying circuits.
As autism is a neurodevelopmental disorder, it will be impor-
tant to now investigate the temporal profile and developmental
requirements for constitutively elevated 5-HT transport in the
changes we observe in the SERT Ala56 mice. Excess 5-HT clear-
ance during early stages of development could influence neuronal
migration, axonal projections, and synapse development in these
mice, as indicated by other developmental manipulations that
target 5-HT signaling (19, 20, 59). Our constitutively expressed
variant also does not speak to the important sites of expression of
SERT Ala56 in dictating phenotypes. SERT is not only expressed
gland, immune cells, pancreas, and lung. Moreover, Bonnin and
coworkers have shown that the placenta, which expresses high
levels of SERT (60, 61), is a source of forebrain 5-HT during
gestation and is important for normal axonal trajectories (62).
Modulating 5-HT levels or transporter function to assess the re-
versibility of these phenotypes could yield insight into the de-
velopmental impact ofincreased and dysregulated SERT function.
Ultimately, studies that allow for temporal and spatial control of
the SERT Ala56 variant are needed to answer these questions.
Finally, ASD is a disorder with few therapies and none that con-
sistently reverse major deficits. We believe that the SERT Ala56
model offers an opportunity to pursue genetic and pharmacolog-
ical studies that can both probe ASD mechanisms and possibly
identify novel therapeutic targets.
Materials and Methods
All animal procedures were in accordance with the National Institutes of
Vanderbilt University, Medical University of South Carolina, or University of
Ala56 knock-in mice were constructed as previously described (22). Details of
methods related to synaptosome preparation, Western blotting, citalopram
binding, SERT phosphorylation, HPLC, immunohistochemistry, in vivo elec-
trochemical recordings, slice preparation, electrophysiological recordings, and
behavioral experiments are provided in SI Materials and Methods.
ACKNOWLEDGMENTS. Michelle Carter, Lauren Huntress, and Clinton Canal
provided technical assistance; Mu Yang and Jill Silverman provided advice on
behavioral data analysis; and Warren Lambert provided statistical assistance.
The authors thank the families who participated in the original genetic study.
This work was supported by National Institutes of Health (NIH) Grants
MH081066 (to J.V.), MH094604 (to J.V.), DA07390 (to R.D.B.), MH078098 (to
R.D.B.), HD065278 (to R.D.B.), and MH62612 (to S.R.); an Autism Speaks Pilot
Award (to J.V.), an American Academy of Child and Adolescent Psychiatry
Pilot Research Award (to J.V.), and NIH Grants HD15052 (to the Vanderbilt
Kennedy Center) and RR024975 (to the Vanderbilt Institute for Clinical and
1. American Psychiatric Association (2000) Diagnostic and Statistical Manual of Mental
Disorders-Text Revision (DSM-IV-TR) (American Psychiatric Association Press, Wash-
ington, DC), 4th Ed.
2. Schain RJ, Freedman DX (1961) Studies on 5-hydroxyindole metabolism in autistic and
other mentally retarded children. J Pediatr 58:315–320.
3. Mulder EJ, et al. (2004) Platelet serotonin levels in pervasive developmental disorders
and mental retardation: Diagnostic group differences, within-group distribution, and
behavioral correlates. J Am Acad Child Adolesc Psychiatry 43:491–499.
4. Kolevzon A, et al. (2010) Relationship between whole blood serotonin and repetitive
behaviors in autism. Psychiatry Res 175:274–276.
Veenstra-VanderWeele et al. PNAS
| April 3, 2012
| vol. 109
| no. 14
5. Sacco R, et al. (2010) Principal pathogenetic components and biological endophe- Download full-text
notypes in autism spectrum disorders. Autism Res 3:237–252.
6. Abney M, McPeek MS, Ober C (2001) Broad and narrow heritabilities of quantitative
traits in a founder population. Am J Hum Genet 68:1302–1307.
7. Anderson GM, Feibel FC, Cohen DJ (1987) Determination of serotonin in whole blood,
platelet-rich plasma, platelet-poor plasma and plasma ultrafiltrate. Life Sci 40:
8. Weiss LA, Abney M, Cook EH, Jr., Ober C (2005) Sex-specific genetic architecture of
whole blood serotonin levels. Am J Hum Genet 76:33–41.
9. Carneiro AM, Cook EH, Murphy DL, Blakely RD (2008) Interactions between integrin
alphaIIbbeta3 and the serotonin transporter regulate serotonin transport and
platelet aggregation in mice and humans. J Clin Invest 118:1544–1552.
10. Weiss LA, et al. (2004) Genome-wide association study identifies ITGB3 as a QTL for
whole blood serotonin. Eur J Hum Genet 12:949–954.
11. Sutcliffe JS, et al. (2005) Allelic heterogeneity at the serotonin transporter locus
(SLC6A4) confers susceptibility to autism and rigid-compulsive behaviors. Am J Hum
12. International Molecular Genetic Study of Autism Consortium (IMGSAC) (2001) A ge-
nomewide screen for autism: Strong evidence for linkage to chromosomes 2q, 7q, and
16p. Am J Hum Genet 69:570–581.
13. Devlin B, et al.; CPEA Genetics Network (2005) Autism and the serotonin transporter:
The long and short of it. Mol Psychiatry 10:1110–1116.
14. Prasad HC, Steiner JA, Sutcliffe JS, Blakely RD (2009) Enhanced activity of human
serotonin transporter variants associated with autism. Philos Trans R Soc Lond B Biol
15. Prasad HC, et al. (2005) Human serotonin transporter variants display altered sensi-
tivity to protein kinase G and p38 mitogen-activated protein kinase. Proc Natl Acad
Sci USA 102:11545–11550.
16. Glatt CE, et al. (2001) Screening a large reference sample to identify very low fre-
quency sequence variants: Comparisons between two genes. Nat Genet 27:435–438.
17. Sakurai T, et al. (2008) A large-scale screen for coding variants in SERT/SLC6A4 in
autism spectrum disorders. Autism Res 1:251–257.
18. Ansorge MS, Zhou M, Lira A, Hen R, Gingrich JA (2004) Early-life blockade of the 5-HT
transporter alters emotional behavior in adult mice. Science 306:879–881.
19. Salichon N, et al. (2001) Excessive activation of serotonin (5-HT) 1B receptors disrupts
the formation of sensory maps in monoamine oxidase a and 5-ht transporter knock-
out mice. J Neurosci 21:884–896.
20. Bonnin A, Torii M, Wang L, Rakic P, Levitt P (2007) Serotonin modulates the response
of embryonic thalamocortical axons to netrin-1. Nat Neurosci 10:588–597.
21. Jitsuki S, et al. (2011) Serotonin mediates cross-modal reorganization of cortical cir-
cuits. Neuron 69:780–792.
22. Veenstra-Vanderweele J, et al. (2009) Modeling rare gene variation to gain insight
into the oldest biomarker in autism: construction of the serotonin transporter
Gly56Ala knock-in mouse. J Neurodev Disord 1:158–171.
23. Steiner JA, Carneiro AM, Blakely RD (2008) Going with the flow: Trafficking-de-
pendent and -independent regulation of serotonin transport. Traffic 9:1393–1402.
24. Samuvel DJ, Jayanthi LD, Bhat NR, Ramamoorthy S (2005) A role for p38 mitogen-
activated protein kinase in the regulation of the serotonin transporter: Evidence for
distinct cellular mechanisms involved in transporter surface expression. J Neurosci 25:
25. Ramamoorthy S, Samuvel DJ, Buck ER, Rudnick G, Jayanthi LD (2007) Phosphorylation
of threonine residue 276 is required for acute regulation of serotonin transporter by
cyclic GMP. J Biol Chem 282:11639–11647.
26. Zhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD (2004) Adenosine receptor,
protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation
of serotonin transporters involves both transporter trafficking and activation. Mol
27. Daws LC, Toney GM, Davis DJ, Gerhardt GA, Frazer A (1997) In vivo chronoampero-
metric measurements of the clearance of exogenously applied serotonin in the rat
dentate gyrus. J Neurosci Methods 78:139–150.
28. Fox MA, et al. (2007) A pharmacological analysis of mice with a targeted disruption of
the serotonin transporter. Psychopharmacology (Berl) 195:147–166.
29. Jennings KA, Sheward WJ, Harmar AJ, Sharp T (2008) Evidence that genetic variation
in 5-HT transporter expression is linked to changes in 5-HT2A receptor function.
30. Willins DL, Meltzer HY (1997) Direct injection of 5-HT2A receptor agonists into the
medial prefrontal cortex produces a head-twitch response in rats. J Pharmacol Exp
31. Rusyniak DE, Zaretskaia MV, Zaretsky DV, DiMicco JA (2007) 3,4-Methylenediox-
ymethamphetamine- and 8-hydroxy-2-di-n-propylamino-tetralin-induced hypother-
mia: Role and location of 5-hydroxytryptamine 1A receptors. J Pharmacol Exp Ther
32. Scott MM, et al. (2005) A genetic approach to access serotonin neurons for in vivo and
in vitro studies. Proc Natl Acad Sci USA 102:16472–16477.
33. Wischmeyer E, Karschin A (1996) Receptor stimulation causes slow inhibition of IRK1
inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.
Proc Natl Acad Sci USA 93:5819–5823.
34. Landa RJ, Holman KC, Garrett-Mayer E (2007) Social and communication de-
velopment in toddlers with early and later diagnosis of autism spectrum disorders.
Arch Gen Psychiatry 64:853–864.
35. Moy SS, et al. (2009) Social approach in genetically engineered mouse lines relevant
to autism. Genes Brain Behav 8:129–142.
36. Moy SS, et al. (2007) Mouse behavioral tasks relevant to autism: Phenotypes of 10
inbred strains. Behav Brain Res 176:4–20.
37. Yang M, Clarke AM, Crawley JN (2009) Postnatal lesion evidence against a primary
role for the corpus callosum in mouse sociability. Eur J Neurosci 29:1663–1677.
38. Spencer CM, Alekseyenko O, Serysheva E, Yuva-Paylor LA, Paylor R (2005) Altered
anxiety-related and social behaviors in the Fmr1 knockout mouse model of fragile X
syndrome. Genes Brain Behav 4:420–430.
39. Moretti P, Bouwknecht JA, Teague R, Paylor R, Zoghbi HY (2005) Abnormalities of
social interactions and home-cage behavior in a mouse model of Rett syndrome. Hum
Mol Genet 14:205–220.
40. Perry W, Minassian A, Lopez B, Maron L, Lincoln A (2007) Sensorimotor gating deficits
in adults with autism. Biol Psychiatry 61:482–486.
41. Thomas A, et al. (2009) Marble burying reflects a repetitive and perseverative be-
havior more than novelty-induced anxiety. Psychopharmacology (Berl) 204:361–373.
42. Müller HK, Wiborg O, Haase J (2006) Subcellular redistribution of the serotonin
transporter by secretory carrier membrane protein 2. J Biol Chem 281:28901–28909.
43. Binda F, Lute BJ, Dipace C, Blakely RD, Galli A (2006) The N-terminus of the norepi-
nephrine transporter regulates the magnitude and selectivity of the transporter-as-
sociated leak current. Neuropharmacology 50:354–361.
44. Ciccone MA, Timmons M, Phillips A, Quick MW (2008) Calcium/calmodulin-dependent
kinase II regulates the interaction between the serotonin transporter and syntaxin
1A. Neuropharmacology 55:763–770.
45. Bauman AL, et al. (2000) Cocaine and antidepressant-sensitive biogenic amine
transporters exist in regulated complexes with protein phosphatase 2A. J Neurosci 20:
46. Steiner JA, et al. (2009) cGMP-dependent protein kinase Ialpha associates with the
antidepressant-sensitive serotonin transporter and dictates rapid modulation of se-
rotonin uptake. Mol Brain 2:26.
47. Yamashita A, Singh SK, Kawate T, Jin Y, Gouaux E (2005) Crystal structure of a bac-
terial homologue of Na+/Cl—dependent neurotransmitter transporters. Nature 437:
48. Adkins EM, Barker EL, Blakely RD (2001) Interactions of tryptamine derivatives with
serotonin transporter species variants implicate transmembrane domain I in substrate
recognition. Mol Pharmacol 59:514–523.
49. Chen JJ, et al. (2001) Maintenance of serotonin in the intestinal mucosa and ganglia
of mice that lack the high-affinity serotonin transporter: Abnormal intestinal motility
and the expression of cation transporters. J Neurosci 21:6348–6361.
50. Tabuchi K, et al. (2007) A neuroligin-3 mutation implicated in autism increases in-
hibitory synaptic transmission in mice. Science 318:71–76.
51. Jamain S, et al. (2008) Reduced social interaction and ultrasonic communication in
a mouse model of monogenic heritable autism. Proc Natl Acad Sci USA 105:
52. Etherton MR, Blaiss CA, Powell CM, Südhof TC (2009) Mouse neurexin-1alpha deletion
causes correlated electrophysiological and behavioral changes consistent with cog-
nitive impairments. Proc Natl Acad Sci USA 106:17998–18003.
53. Kwon CH, et al. (2006) Pten regulates neuronal arborization and social interaction in
mice. Neuron 50:377–388.
54. Peca J, et al. (2011) Shank3 mutant mice display autistic-like behaviours and striatal
dysfunction. Nature 472:437–442.
55. Silverman JL, Yang M, Lord C, Crawley JN (2010) Behavioural phenotyping assays for
mouse models of autism. Nat Rev Neurosci 11:490–502.
56. Duvall JA, et al. (2007) A quantitative trait locus analysis of social responsiveness in
multiplex autism families. Am J Psychiatry 164:656–662.
57. Tadevosyan-Leyfer O, et al. (2003) A principal components analysis of the Autism
Diagnostic Interview-Revised. J Am Acad Child Adolesc Psychiatry 42:864–872.
58. Peñagarikano O, et al. (2011) Absence of CNTNAP2 leads to epilepsy, neuronal mi-
gration abnormalities, and core autism-related deficits. Cell 147:235–246.
59. Riccio O, et al. (2009) Excess of serotonin affects embryonic interneuron migration
through activation of the serotonin receptor 6. Mol Psychiatry 14:280–290.
60. Ramamoorthy S, et al. (1993) Antidepressant- and cocaine-sensitive human serotonin
transporter: molecular cloning, expression, and chromosomal localization. Proc Natl
Acad Sci USA 90:2542–2546.
61. Prasad PD, et al. (1996) Functional expression of the plasma membrane serotonin
transporter but not the vesicular monoamine transporter in human placental
trophoblasts and choriocarcinoma cells. Placenta 17:201–207.
62. Bonnin A, et al. (2011) A transient placental source of serotonin for the fetal
forebrain. Nature 472:347–350.
| www.pnas.org/cgi/doi/10.1073/pnas.1112345109Veenstra-VanderWeele et al.