Neuron 50, 377–388, May 4, 2006 ª2006 Elsevier Inc.DOI 10.1016/j.neuron.2006.03.023
Pten Regulates Neuronal Arborization
and Social Interaction in Mice
Chang-Hyuk Kwon,1,3,4Bryan W. Luikart,1,4
Craig M. Powell,2,4Jing Zhou,1Sharon A. Matheny,1
Wei Zhang,1Yanjiao Li,1Suzanne J. Baker,3
and Luis F. Parada1,*
1Center for Developmental Biology and
Kent Waldrep Foundation Center for Basic
Neuroscience Research on Nerve Growth and
2Departments of Neurology and Psychiatry
University of Texas Southwestern Medical Center
Dallas, Texas 75390
3Department of Developmental Neurobiology
St. Jude Children’s Research Hospital
Memphis, Tennessee 38105
CNS deletion of Pten in the mouse has revealed its
roles in controlling cell size and number, thus provid-
ing compelling etiology for macrocephaly and Lher-
mitte-Duclos disease. PTEN mutations in individuals
with autism spectrum disorders (ASD) have also been
reported, although a causal link between PTEN and
ASD remains unclear. In the present study, we deleted
Pten in limited differentiated neuronal populations in
the cerebral cortex and hippocampus of mice. Result-
ing mutant mice showed abnormal social interaction
and exaggerated responses to sensory stimuli. We ob-
served macrocephaly and neuronal hypertrophy, in-
tracts with increased synapses. This abnormal mor-
phology was associated with activation of the Akt/
mTor/S6k pathway and inactivation of Gsk3b. Thus,
our data suggest that abnormal activation of the
PI3K/AKT pathway in specific neuronal populations
can underlie macrocephaly and behavioral abnormali-
ties reminiscent of certain features of human ASD.
Phosphatase and tensin homolog on chromosome ten
(PTEN) is a tumor suppressor gene mutated in many hu-
man cancers (Ali et al., 1999). Individuals with germline
PTEN mutations are prone to tumors but also display
brain disorders, including macrocephaly, seizure, Lher-
mitte-Duclos disease, and mental retardation (Waite
and Eng, 2002). PTEN mutations also have been re-
ported in autistic individuals with macrocephaly (Butler
et al., 2005; Goffin et al., 2001; Zori et al., 1998).
PTEN has lipid phosphatase activity against the 30
phosphate of phosphatidylinositol 3,4,5 trisphosphate
(Maehama and Dixon, 1998). Phosphatidylinositol 3-ki-
nase (PI3K) catalyzes the reverse of this reaction, re-
sulting in AKT activation. Upon activation, AKT phos-
phorylates a diverse spectrum of substrates, including
tuberous sclerosis complex 2 (TSC2) gene product tu-
berin, glycogen synthase kinase 3b (GSK3b), and the
proapoptotic protein BAD (Luo et al., 2003).
Abnormalities in many components of the PI3K/AKT
pathway have been associated with diverse brain disor-
ders.For example, inactivating mutations in PTEN orac-
tivating mutations in PI3K are found in malignant brain
tumors (Ali et al., 1999; Broderick et al., 2004), low AKT
level is associated with schizophrenia (Emamian et al.,
orders, including autism (Wiznitzer, 2004). The PI3K/
plasticity processes in the brain. Activation of the path-
way was found in the amygdala in fear-conditioned rats
(Lin et al., 2001). Components of the mTOR/S6K path-
way, downstream of PI3K/Akt, are present in synapses
and mediate synaptic plasticity through local protein
synthesis (Tang et al., 2002).
Pten null mice die during embryogenesis, and hetero-
zygotes are prone to tumors in the prostate, endome-
trium, and lymphoid system (Stiles et al., 2004). Condi-
tional loss of Pten can have differing consequences
depending on the cell type or its state of differentiation.
Consistent with the frequent association of somatic mu-
tations with cancer, Pten deletion in dividing T-lympho-
cytes, mammary epithelial cells, neural stem cells, and
astrocytes induced hyperplasia (Fraser et al., 2004;
Groszer et al., 2001; Li et al., 2002; Suzuki et al., 2001).
Pten deletion in granule neurons, cardiomyocytes, and
et al., 2002; Kwon et al., 2001). In primary neuron cul-
tures, the PI3K pathway regulates cell survival, neurite
growth, and dendritic arborization (Crowder and Free-
man, 1998; Jaworski et al., 2005; Klesse and Parada,
1998; Markus et al., 2002). The above studies manipu-
lated either mitotic cells or immature neurons. The
in vivo role of the PI3K/PTEN/AKT pathway has been
poorly studied in mature neurons where specialized
properties including synapses and polarity are already
In the present study, we employed a Neuron-specific
enolase (Nse) promoter-driven cre transgenic mouse
line, in which cre activity is confined to discrete mature
neuronal populations in the cerebral cortex and hippo-
campus (Kwon et al., 2006). The resulting conditional
Pten mutant mice develop macrocephaly due to cre-
specific neuronal hypertrophy. We identified abnormal
dendritic and axonal growth and synapse number. The
mice exhibit altered social behavior and inappropriate
responses to sensory stimuli.
Pten Is Inactivated in Differentiated Neurons
Cre activity in the brain of Nse-cre mice was limited to
subsets of differentiated neurons, mostly in layers III to
V of the cerebral cortex and in the CA3, dentate gyrus
granular layer (GL), and polymorphic layer (PML) of the
hippocampal formation (Kwon et al., 2006). By 4 weeks
of age, roughly 30%–60% of the neurons in these
4These authors contributed equally to this work.
regions expressed functional b-galactosidase when
crossed with Rosa26-stop-lacZ cre reporter (Rosa26R
hereafter) (Soriano, 1999). A low level of sporadic cre
activity was observed in a few neurons in the olfactory
bulb, spinal cord, and cerebellar Purkinje cell layer. To
confirm cre activity in differentiated neurons, we stained
brain sections from 2-week-old mice for b-galactosi-
dase and either a dendritic marker, microtubule-associ-
atedprotein-2 (MAP2), oraneural stem cellmarker,nes-
tin. In the dentate gyrus at this age, active neurogenesis
is still occurring in the subgranular zone (SGZ) between
the GL and the PML (Shapiro and Ribak, 2005). Most
granule neurons in the dentate gyrus are born in the
SGZ and migrate and differentiate outward into the GL.
In Nse-cre; Rosa26R brains, b-galactosidase colocal-
ized with MAP2-expressing cells in the outer GL and
the PML, but not in the inner GL or the SGZ. No b-galac-
tosidase was detected in nestin-positive cells (Fig-
ure 1A). Consistent with this, postnatal day 15 (P15)
Nse-cre; Rosa26R brains pulsed with bromodeoxyuri-
dine (BrdU) at P14 did not show colocalization of BrdU
and b-galactosidase (Figure 1B), indicating the absence
of cre activity in dividing neuronal precursors. Chasing
BrdU signal 4 weeks after the pulse revealed partial
colocalization of BrdU and b-galactosidase in dentate
granule neurons. Thus, cre activity is confined to sub-
sets of differentiated neurons in Nse-cre mice.
Nse-cre; PtenloxP/loxPmice (mutant mice hereafter)
were viable at birth and appeared normal until 4–5
weeks of age. Consistent with a previous report (Peran-
dones et al., 2004), control mice showed Pten signal in
most differentiated neurons, including those in the GL
and PML of the dentate gyrus and cerebral cortex (Fig-
ures 1C and 1D, respectively). At 2 weeks of age, mutant
dentate gyrus exhibited Pten-negative cells at the PML
and the outer GL, similar to the cre activity of Nse-cre;
Rosa26R brains. The same regions also showed in-
creased phospho-Ser473-Akt (P-Akt), a marker for Akt
activation. At 4 weeks of age, Pten-negative and P-
Akt-positive cells increased in mutant dentate GL, indi-
cating that Pten deletion accompanies differentiation
of dentate granule neurons. Pten deletion and increased
P-Akt signal were found in all sites of Nse-cre activity
(Kwon et al., 2006), and the ratios of Pten deletion
(55.89% 6 6.62% in dentate granule neurons; 44.88 6
1.62% in sensory cortical layers III to V) coincided with
the ratios predicted by the Rosa26R assays, indicating
similar recombination efficiency for the loxP-Pten and
Behavioral Abnormalities in Social Interaction
and Social Learning
individuals with germline PTEN mutations (Butler et al.,
2005; Goffin etal., 2001; Zori etal., 1998). Since the adult
Pten mutant mice tended to be isolated from their litter-
mates within the cage, we examined the colony using
a series of established behavioral paradigms. We found
that mutant mice exhibit a distinct pattern of behavioral
abnormalities reminiscent of ASD.
Mice are a social species and display behavioral so-
cial interaction (Murcia et al., 2005). Thus, social interac-
been used to measure autism-like behaviors in other
mutant mouse models (Lijam et al., 1997; Moretti et al.,
2005). As anticipated, control mice exposed to a novel
conspecific juvenile exhibited typical behavior of ap-
proaching and sniffing, but such initial social interaction
was profoundly decreased in mutant mice (Figure 2A).
When re-exposed to the same juvenile after 3 days, con-
trol mice exhibited a typical decrease in social inter-
action compared to the initial interaction, indicating
Figure 1. Pten Deletion Occurred in Differentiated Neurons
(A) In 2-week-old Nse-cre; Rosa26R mice, b-galactosidase signal
was detected in MAP2-expressing neurons in the polymorphic layer
(PML) and the outer granular layer (GL), close to the molecular layer
(ML), in the dentate gyrus, but not in the inner GL, subgranular zone
(SGZ), or in cells expressing nestin. Although a similar level and pat-
tern of MAP2 or nestin immunoreactivity appeared in control (either
without cre or Rosa26R), b-galactosidase was not detected (data
not shown). Scale bar, 200 mm.
(B) P15 Nse-cre; Rosa26R mice injected with BrdU at P14 retained
the BrdU signal mainly in the SGZ, which was absent in b-galactosi-
dase-expressing cells in the outer GL and PML. Four weeks after the
BrdU injection, BrdU signal was detected in the GL, and some of
them colocalized with b-galactosidase-expressing granule neurons
(arrow, for example). Scale bar, 100 mm.
(C) In mutant tissue at 2 weeks of age, similar to b-galactosidase ac-
tivity in Nse-cre; Rosa26R brain, Pten-negative (blue), and P-Akt-
positive signals (brown) were detected in the PML and the outer
GL. At 4 weeks of age, the number of Pten-negative, P-Akt-positive
cells increased in mutant dentate gyrus. Scale bar, 200 mm.
(D) Inthe sensory cortex layersIII to VI,Pten (brown) was detected in
most neurons in control. In mutant cortex, Pten-negative cells were
mainly detected at layers III to V. Scale bar, 100 mm.
recognition of the familiar juvenile and normal social
learning. The mutant mice, however, did not decrease
their interaction, indicating impaired social learning or
inability to identify the juvenile due to the low level of ini-
tial interaction. We next examined nest formation, a test
for home cage behavior (Lijam et al., 1997; Moretti et al.,
2005). In contrast to the immediate activity of nest for-
mation in control mice, mutant mice showed little nest-
forming activity (Figure 2B). We do not attribute the ab-
normalities in social interaction and nest formation to
deficiencies in general interest in novelty or olfactory
sensation, since we did not detect significant difference
between groupsintestsfor novel inanimate objectinter-
action (Figure 2C) or olfaction (Figure 2D). Each of the
above tests was fully replicated with equivalent signifi-
cant results using a separate cohort of mice and per-
formed by a different investigator, demonstrating the re-
producibility of the results (data not shown).
Additional social tests gave similar results. For exam-
ple, another social interaction test presents a test
mouse with a caged adult mouse (social target) and an
empty cage (inanimate target) in an open field. Control
mice spent significantly more time interacting with the
social target than with the inanimate target (Figure 2E).
Incontrast, Ptenmutants showeddecreasedinteraction
with the social target compared to controls and spent
a similar amount of time interacting with both targets.
Another test for social novelty uses a three-roomed
chamber. Initial interaction with the empty cage was
similar in both groups, while interaction with the social
target was significantly decreasedin mutantscompared
to controls (Figure 2F). Subsequently, when mice were
exposed to the familiar mouse versus a novel mouse,
control mice showed a clear preference for the novel
mouse over the familiar mouse as expected, while mu-
tant mice did not show a preference for social novelty
(Figure 2G). Additionally, mutants showed significantly
less interaction with the novel target mouse compared
to controls. In summary, the mutant mice exhibited de-
creased social interaction without change in novel ob-
ject exploration or a preference for social novelty.
Thus, the Pten-deficient mice display deficiencies in
classic social interaction paradigms.
We also measured sexual and maternal behavior.
While all naive control males made female mice preg-
nant, none of naive mutant males did. Although we do
not exclude potential reproductive defects, we did not
observe any active sexual behavior, such as mounting,
from the mutant males. While mutant females could be
fertilized by normal males, mortality of pups by P5 was
higher than control group (Figure 2H), indicating defects
in maternal care. Taken together, the Pten mutant mice
exhibited abnormalities in social interaction, memory
and preference, sexual behaviors, and maternal care in
several different social paradigms.
Behavioral Abnormalities in Response to Sensory
Stimuli, Anxiety, and Learning
By 6 weeks of age, mutant mice were also distinguish-
stimulation. When investigators handled mutant mice,
they were unusually resistant to handling (n = 52). Con-
sistent with this subjective observation, mutant mice
exhibited normal locomotor activity in less stressful
Figure 2. The Pten Mutant Mice Were Abnormal in Behavioral Tests
for Social Interaction and Social Learning
(A) At day 1, mutants spent significantly less time interacting with
a conspecific juvenile compared to controls (n = 12). Control mice
spent significantly less time interacting with the same juvenile 3
days hence (p < 0.05), yet mutants did not decrease their interaction
time (p = 0.8). Legend in this panel applies to all bar graphs.
ANOVA revealed a significant effect of genotype (F1,22= 7.97, p =
0.01), time (F3,66= 11.37, p < 0.00001), and an interaction between
genotype and time (F3,66= 6.26, p < 0.001).
(C) Time spent interacting with a novel inanimate object under the
same conditions as in (A) was not significantly affected by genotype
(n = 12).
(D) Mutant mice did not show significant difference from control in
latency to find a buried treat following overnight food deprivation
(n = 12).
(E) When exposed to caged social and inanimate targets in an open
field, controls showed normal preference for a social target over an
inanimate target, while mutants spent similar time interacting with
both targets (n = 12). Furthermore, mutant mice spent significantly
less time interacting with a social target compared to controls (p <
0.00001). In this task, there was also a significant decrease in in-
animate object interaction time between genotypes (p < 0.01),unlike
(F) In a social preference task, mutants spent less time with a social
target compared to controls (n = 12). Time spent with an inanimate
object was not significantly different in both groups.
(G) In a preference for social novelty task, controls showed a prefer-
ence for social novelty, while mutants showed no preference be-
tween the social targets (n = 12). Mutants spent significantly less
time interacting with a novel social target compared to controls.
(H) Mutant female mice delivered normal-sized pups. Mortality of
pups between P0 and P5 was significantly higher in mutants (n = 6)
compared to controls (n = 8). *p < 0.05 to controls and < 0.005 to P0.
Pten Regulates Social Interaction in Mice
environments but hyperactivity under more stressful
conditions. In the bright environment of the open field,
mutants were hyperactive, traveling further (Figure 3A)
at an increased average speed. However, in the dark/
light boxes and in the enclosed, darker environments
of the locomotor apparatus, locomotor activity was nor-
mal (see Figures S1A and S1B in the Supplemental Data
available online). Additionally, mutants exhibited in-
creased initial startle responses to a 120 dB white noise
stimulus (Figure 3B). Upon repeated startle stimulation,
mutants showed similar startle responses to controls,
indicating normal habituation (Figure S1C). Sensorimo-
tor gating, as measured with a prepulse inhibition para-
digm, was also significantly impaired in mutant mice
(Figure 3C). Thus, the mutants showed quantifiably in-
creased activity in response to sensory stimuli.
Consistent with exaggerated response to stressful
sensory stimuli, mutant mice also showed increased
anxiety-like behavior in the open field test where they
spent significantly less time in the center zone (Fig-
time (data not shown, p = 0.02). Similarly, in the dark/
light apparatus, mutants showed longer latencies to
enter the light side, spending the majority of their time
on the dark side (Figure 3E). In a third anxiety-related
test, the elevated plus maze, mutants did not show the
increased anxiety-like behavior (Figure 3F). Indeed,
there was an opposite effect in both the time spent in
the open arms and the ratio of open arm entries to total
arm entries (data not shown).
Because of the profound abnormalities in the dentate
gyrus (described below), we tested the Pten mutant
mice in the Morris water maze. Because of the abnormal
anxiety, we habituated the mice to swimming in the
maze for 4 days using a visible platform task. Mutant
mice learned the visible platform task as well as con-
trols, though there was a trend toward slower acquisi-
tion (Figure S1D). In the submerged platform version,
measuring both latency to reach the platform and dis-
tance traveled to reach the platform, mutant mice did
trial, control mice spent significantly more time in the
target quadrant than the opposite quadrant, while
mutants showed no significant preference (Figure 3H).
Figure 3. The Pten Mutant Mice Showed Abnormalities in Responses to Sensory Stimuli, Anxiety, and Learning
(A) Mutants exhibited increased locomotor activity in an open field test (n = 16 mutants, 17 controls). Average speed was 11.30 6 0.84 cm/s for
mutants and 8.51 6 0.47 cm/s for controls (p = 0.006). Legend in this panel applies to all bar graphs.
(B) Initial startle response was significantly increased in mutants (n = 11 mutants, 14 controls). Data represent the average startle response to the
first six presentations of a 40 ms, 120 dB white noise stimulus.
(C) In a prepulse inhibition test, mutants showed significantly impaired sensorimotor gating (n = 11 mutants, 14 controls).
(D) Mutants exhibited anxiety-like behavior as they spent significantly less time in the center zone of the open field apparatus (n = 16 mutants,
(E) The latency to enter the light side of the dark/light boxes was significantly elevated in mutants (n = 16 mutants, 17 controls).
(F) In elevated plus maze test, mutants exhibited significantly increased duration in open arm (n = 16 mutants, 17 controls).
(G) Mutants exhibited a significantly decreased learning curve in the submerged platform version of the water maze when latency to reach the
platform was measured (n = 9 mutants, 12 controls). ANOVA revealed a main effect of genotype (F1,17= 11.17, p < 0.01) and day number (F10,170=
2.21, p< 0.05). Similar effect was seen indistance traveled toreach the platform(data not shown). Threemutant mice died during the watermaze
task due to seizure activity during training.
(H) Controls showed clear preference for target quadrant versus opposite whereas mutants showed no preference (n = 9 mutants, 12 controls).
(I) Mutant mice spent significantly increased time along the edge of the water maze (thigmotaxis, n = 9 mutants, 12 controls). ANOVA revealed
a main effect of genotype (F1,17= 39.76, p < 0.00001).
In addition, mutant mice exhibited a significantly greater
tendency to swim along the edge of the maze (thigmo-
taxis) (Figure 3I), which is similar to their behavior in
the open field test.
In addition to the behavioral abnormalities described,
6 of 52 mutant mice studied, including 3 of 12 during the
water maze test, showed sporadic seizures (subjective
observation). Further analysis using electroencephalo-
gram/electromyogram (EEG/EMG) recording revealed
that all mutant mice analyzed (n = 3) developed sponta-
neous seizures during the light phase, but no seizures
were observed in any of control mice (Table S1). Repet-
itive spike-wave patterns were noted, sometimes
accompanied by rhythmic slow activity. Continuous
spike-wave bursting could also be seen. The incidence
of seizures was 0.67 per mouse per day, and the mean
duration was 10 min 50 s. Sound and tactile stimuli did
not induce seizures in any mouse (data not shown).
The relatively low incidence and short duration of sei-
zure recorded in the subset of mutant mice is consistent
with the low frequency by subjective observation. Given
the relatively low incidence and short duration of sei-
zures, it is unlikely that seizures bear on the robust be-
havioral abnormalities seen in all 52 mice. Consistent
with this view, as described below, mutant mice did
not show any deficit in tests for locomotor activity and
strength. In the accelerating rotarod test, mutant mice
exhibited normal coordination during the initial trials
(Figure S1E). Interestingly, while both groups showed
significant motor learning, during subsequent rotarod
trials, mutant mice actually performed better on this
repetitive test of motor coordination compared to
controls. In vertical pole and dowel tests to measure
strength and endurance, mutant mice performed as
well as controls (Figures S1F and S1G). In addition,
mutant mice did not show deficits in context- and cue-
dependent fear conditioning (Figure S1H).
Progressive Macrocephaly and Soma Hypertrophy
in Mutant Brain
occasionally with reduced body weight (Kwon et al.,
2001, and unpublished observations). In the present
study, the mutant mice appear to have a normal life
span, presumably due to more restricted Pten deletion
in subsets of postmitotic neurons. They also showed
progressive macrocephaly, but without significant
rocephaly was confined to the forebrain (cortex and hip-
pocampus; Figures 4B and 4C), where most cre-medi-
ated Pten deletion occurs (Kwon et al., 2006). Analysis
of aging mice indicated eventual foliation of the DG and
compression of the CA1 region (Figure 4C; lower right
panel). Most dentate gyrus granule cells from wild-type
mice expressed Pten and sustain relatively even soma
diameter (Figure S2A). In contrast, Pten-negative neu-
rons were larger than Pten-positive neurons at 4 weeks
of age. The progressive increase in soma diameter and
disorganized GL continued in mutant aging mice (Fig-
ures S2A and S2B). Such soma hypertrophy was also
observed in the cortex and CA3 (Figure S2C) where cre
activity is also present.
Regulation of Axonal Growth In Vivo
The Pten pathway is known to regulate neurite out-
growth in cell culture (Markus et al., 2002). Since Nse-
cre drives Pten ablation only in differentiated neurons
with established polarity, we examined the effects of
Pten loss on existing neuronal processes. In the dentate
Figure 4. Progressive Macrocephaly and Re-
gional Hypertrophy in the Pten Mutant Mice
(A) Relative sizes of mutant brain to control at
different ages indicate progressive macroce-
phaly in mutant mice (n = 4 or more per
group). *p < 0.005 versus control.
(B) A representative mutant brain at 10
months of age (right) is bigger than that from
littermate control. Scale bar, 4 mm.
(C) H/E staining on coronal sections shows
that the thickness of the cerebral cortex (ar-
rows), the length between the pial surface
and the corpus callosum (CC), increased in
adult mutant brain (upper panels). In the hip-
pocampus, progressively enlarged dentate
gyri (DG) and compressed or absence of
CA1 were seen in mutant brains (lower
panels). Scale bars, 200 mm.
Pten Regulates Social Interaction in Mice
gyrus, granule neuron axons form the mossy fiber tract
that projects from the GL through the PML to synapse
with CA3 dendrites (Amaral, 1978). The extent of the
mossy fiber tract was visualized by immunohistochem-
istry (IHC) using antibodies to synapsin I, a presynaptic
marker, and to calbindin, which is expressed in the
soma and processes of dentate granule neurons (Fig-
ure S3A). In adult mutants, the dentate gyrus showed
a marked enlargement of the mossy fiber tract that
progressed over time (Figures S3A and S3B). Confocal
microscopy revealed that mutant axonal processes
were more abundant and projected to a broader area
We also observed changes in the synapses of the mu-
tant dentate gyrus. Synapsin I staining was increased in
Figure 5A). Normally, most synapses in this region are
derived from mossy cells of the PML and express calre-
tinin (Blasco-Ibanez and Freund, 1997). The increased
synapsin I staining of the inner ML did not completely
overlap with calretinin staining (Figure S3C). Instead,
the abnormally localized axonal projections appear to
push mossy cell axons out of the inner ML. In fact, the
abnormal axonal projections were positive for Timm’s
staining (Figure 5B), which is specific for the mossy
fiber tract of the dentate gyrus (Danscher et al., 2004),
demonstrating ectopic positioning of granule axons in
the mutant mice.
Ultrastructural examination of the inner ML of mutant
animals indicated a dramatic increase in presynaptic
In support of the Timm’s staining data, the large vesicle
pools were apposed to multiple postsynaptic densities,
thus exhibiting the morphological appearance of gran-
ule neuron mossy fiber synapses in the CA3 region with
characteristic enlarged presynaptic terminals. Taken to-
gether, these results suggest that Pten inactivation in
differentiated neurons causes increased axonal growth,
ectopic axonal projections, and abnormal synapses.
Regulation of Dendrite Growth and
Spine Density In Vivo
To more closely examine the morphology of dendrites in
mutant brains, we used Golgi staining. In the cerebral
cortex from 3-month-old mutant mice, we observed
thickened or elongated processes (Figure 6A). There
was obvious dendritic hypertrophy in adult mutant den-
tate gyrus (Figure 6B). Estimation of the thickness of
MAP2-positive dentate ML revealed a significant in-
crease in mutants that progressed with age (Figure 6C).
We also observed a 24.9% increase in dendritic spine
density within the ML of mutant versus control mice
Ectopic neuronal processes extending from the cell
bodies into the PML were observed in mutant dentate
gyri at 3 months of age (data not shown) when the be-
havioral phenotypes were fully developed. The ectopic
neuronal processes were thin and spiny but could not
be unambiguously distinguished as dendrites or axons
cessesin Ptennullneurons extendingintothePMLwere
longer and thicker with obvious spines (arrows in Fig-
ures 6B and 7A). IHC for MAP2 revealed that the ectopic
processes were molecularly discernable as dendrites at
10 months of age (Figure 7B). Double labeling for MAP2
and P-Akt showed a layer of ectopic dendrites between
the GL and the PML with increased P-Akt in mutant den-
tate gyrus at 10 months of age. Furthermore, confocal
images showed that most granule neurons displaying
dendritic ectopia had increased P-Akt, while neurons
lacking P-Akt signal did not display dendritic ectopia
(Figure 7C), indicating that the ectopia was due to cell-
autonomous Pten deletion.
Figure 5. Hypertrophic and Ectopic Axonal Tract with Increased
Synapses in Pten-Deleted Dentate Gyrus
(A) Horizontal floating sections from 10-month-old brains were
stained for synapsin I (red) and calbindin (green). Confocal images
showed that elongated and dispersed mossy fiber tract from the
granular layer (GL) of mutant dentate gyrus and an ectopic layer of
axonal signals (arrows) in the molecular layer (ML), compared to
those of control (upper panels). Scale bar, 500 mm. High-magnifica-
area in mutant versus control animals (lower panels; from the boxes
in upper panels).
(B) The inner ML of 7-month-old mutant dentate gyrus was positive
for Timm staining (arrows), while such signals were absent in control
ML. Scale bar, 100 mm.
(C) Electron microscopic analysis of the inner ML (IML) of dentate
gyri revealed that the increased axonal staining in mutant was due
to enlarged presynaptic varicosities (red highlight). The varicosities
ofmutantcontained alargenumber ofdenselypackedsynaptic ves-
icles. Scale bar, 0.5 mm.
Molecular Correlates of Neuronal Hypertrophy
and Abnormal Polarity
We next examined the status of downstream signaling
ported that earlier Pten deletion in neurons increased
P-Akt and phospho-Ser235/236-S6 (P-S6), markers for
activation of the Akt and mTor/S6k pathways, respec-
tively (Kwon et al., 2003). Here we show similar results
when Pten is inactivated in differentiated neurons. We
observed increased P-Akt and P-S6 in Pten-deleted,
hypertrophic neurons, including the granule cells in the
rect target for Akt phosphorylation at Ser9, leading to
functional inactivation (Cross et al., 1995). Both IHC
and Western blot analysis demonstrate increased phos-
pho-Ser9-Gsk3b (P-Gsk3b) in mutant tissues (Figure 8).
Tuberin, the Tsc2 product, is another direct target for
Akt phosphorylation at Ser939 and an upstream regula-
tor of the mTor/S6k pathway (Manning and Cantley,
2003). We detected increased phospho-Tuberin Ser939
(P-Tuberin) in the same tissues (Figure 8B). Thus, all
tested downstream targets of Akt displayed increased
phosphorylation in the Pten-deficient tissues.
Through the use of cre-mediated recombination, we
have deleted Pten and thus deregulated the PI3K path-
way in subsets of differentiated neurons in the cortex
Deregulation of Postmitotic Growth and Polarity
of Neuronal Processes
Atthe timeofPten deletion, thedentate granule neurons
already have established dendrites, extending over the
full length of the ML, and axons projecting into the
CA3 region. Furthermore, these neurons have already
established synaptic connectivity. Our current findings
indicate that Pten inactivation results in continued axo-
nal and dendritic growth, with ectopic positioning of
dentate axons to the ML and dendrites to the PML.
Molecular markers for activation of the PI3K pathway
werepredictably detected. These dataadd topreceding
reports of soma hypertrophy in Pten-deleted neurons
(Backman et al., 2001; Kwon et al., 2001) and demon-
strate that growth regulation by Pten extends to axons
Gsk3b was recently reported to be pivotal in control-
neurons (Jiang et al., 2005; Yoshimura et al., 2005). In
those studies, neurites destined to axons had more P-
Gsk3b (inactive), and exogenous inactivation of Gsk3b
resulted in formation of multiple axons. Conversely, ex-
pression of mutant Gsk3b insensitive to inactivation by
Akt inhibited axon formation. Similar to the abnormal
polarity in immature neurons, we observed hypertrophic
axonal tracts and evidence of altered polarity of neuro-
nal processes associated with inactivated Gsk3b in
Pten-deleted, differentiated granule neurons. These
results indicate that the PI3K/AKT/GSK3b pathway is
critical not only for the establishment of polarity in
Figure 6. Golgi Stain Revealed Dendritic Hy-
pertrophy, Ectopy, and Increased Spine Den-
sity in Pten-Deleted Brain
(A) Thickened or elongate neuronal pro-
cesses (arrow and arrow heads, respectively)
were present in mutant cerebral cortex com-
pare to control at 3 months of age. Scale bar,
dendritic arbors in the molecular layer (ML)
and ectopic neuronal processes (arrow) in
the polymorphic layer (PML) were observed
in mutant compare to control (upper panels).
Reconstructions of single neurons made
from image stacks emphasize the dendritic
hypertrophy of the mutant neurons compare
to control (lower panels). An axon can be
seen emanating from both mutant and con-
trol neurons (arrowheads). Scale bar, 100 mm.
(C) Mutant ML was significantly thicker than
that of control at all adult ages tested (p <
(D) Higher-magnification images of dendrites
in the ML revealed increased thickness and
spine density in mutant (1.434 6 0.064
spines/mm) versus control (1.077 6 0.033
spines/mm) brains (p < 0.000005, n = 23 and
26 dendritic branches from three brains, re-
spectively). Scale bar, 10 mm.
Pten Regulates Social Interaction in Mice
undifferentiated neurons in culture but also for mainte-
nance in differentiated neurons in vivo.
Behavioral Abnormalities in Social Interaction
In our mouse model, cre-mediated recombination af-
fects subsets of differentiated neurons in the hippocam-
pus and cerebral cortex. This process is reproducible,
and the anatomical and behavioral phenotypes are ro-
bust and fully penetrant. The Pten mutant mice appear
to have normal life span, which allowed us to test para-
digms proposed for autistic behaviors in mice (Crawley,
2004). Indeed, the mutant mice exhibited deficits in all
social paradigms tested and also showed exaggerated
reaction to sensory stimuli, anxiety-like behaviors, sei-
zures, and decreased learning, which are features asso-
ciated with ASD (American Psychiatric Association,
2000). The elevated plus maze gave different results
compared to open field and dark/light boxes. This may
reflect different aspects of anxiety-like behaviors and
their controlling neural networks (Dawson and Trickle-
bank, 1995). Nonetheless, two of three tests clearly
showed anxiety-like behavior in the mutant mice. Nor-
mal behaviors in many paradigms, including locomotor,
motor coordination, and fear conditioning, indicate that
the mutant mice were not globally impaired. It is worth
noting that the behavioral abnormalities of the mutant
mice appeared at a time when morphological abnormal-
ities were subtle (6 weeks of age).
A Potential Link with Autism
ASD is a neuropsychiatric disorder characterized pri-
marily by deficits in social interaction and repetitive
netic factors have been implicated in ASD, including
15q11-q13 duplication and mutations responsible for
fragile X mental retardation syndrome (FMR1) and Rett
syndrome (Andres, 2002; Muhle et al., 2004). Intrigu-
ingly, a few neurological disorders related to one an-
other by harboring mutations in the PI3K pathway are
also atypically associated with ASD. Individuals with tu-
berous sclerosis have mutations in the TSC1/2 complex
Figure 7. Ectopic Dendrites in Pten-Deleted Granule Neurons
(A) High-magnification images on Golgi-stained granule neurons in
8-month-old dentate gyri showed the presence of ectopic, spiny
neuronal processes (arrows) in mutant, but not in control. Scale
bar, 10 mm.
(B) Coronal floating sections were stained for MAP2 (green) and
P-Akt (red). Increased P-Akt was apparent in the granular (GL) and
molecular layers (ML) of mutant dentate gyri at all ages, but not in
controls. Mutant dentate gyrus at 10 months of age had an ectopic
layer of dendrites (arrow heads) between the GL and polymorphic
layer (PML), which was absent at 4 weeks of age. Scale bar, 200 mm.
(C) Higher-magnification confocal images revealed that the ectopic
dendrites were from P-Akt-positive granule neurons (light blue ar-
rows, for example), but not from P-Akt-negative neurons (white
arrows, for example). Scale bar, 50 mm.
Figure 8. MolecularSignalingDownstreamofPten-DeletedNeurons
(A) At 2 months of age, mutant dentate gyri exhibited increased sig-
nal (brown) for P-Akt, P-S6 and P-Gsk3b, compared to those in con-
trol. The increased staining in mutants was apparent in the granular
layer (GL)andinnermolecular layer(ML)for P-S6andinthe GLforP-
Gsk3b, whereas both the GL and ML displayed increased P-Akt. All
sections were counterstained with hematoxylin, except for P-Gsk3b
panels that were stained with methyl green. Scale bar, 200 mm.
(B) Western blot analysis showed decreased Pten and increased
P-Akt, P-S6, P-Gsk3b, and P-Tuberin in mutant versus control at
all ages tested. p < 0.005 for Pten at 2 months and P-Akt; p < 0.1
for P-S6, P-Gsk3b and P-Tuberin.
and have a high incidence of ASD (25%–50%) (Asano
et al., 2001; de Vries et al., 2005; Wiznitzer, 2004). In ad-
dition, the incidence of ASD patients who are subse-
quently diagnosed with neurofibromatosis type 1 (NF1)
greatly exceeds epidemiological prediction (Marui
et al., 2004; Mbarek et al., 1999). Finally, the recent pre-
liminary association of PTEN mutations in ASD with
macrocephaly (Butler et al., 2005) further points to ab-
normal activation of the PI3K pathway as one possible
etiology. NF1, PTEN, and the TSC complex are negative
regulators of the PI3K pathway, and inactivation of any
of the proteins results in a hyperactive signal trans-
duction in many circumstances (Hay, 2005; Klesse and
Parada, 1998; Manning and Cantley, 2003).
Diverse anatomical and cellular abnormalities have
been reported in brains from ASD individuals. For exam-
ple, both macrocephaly and microcephaly have been
described in autistic individuals, with a ratio of 15%
enlarged amygdala in children and enlarged hippocam-
pus in children and adolescents have been associated
with ASD (Schumann et al., 2004). The sum of recent
findings seems to approach a consensus that increased
brain volume is a relatively common feature of autism
(Cody et al., 2002). There have also been reports in au-
tism cases of enlarged or abnormally oriented neurons,
densely packed neuronal regions and isolated regions
of atrophic neurons with reduced dendritic arborization
(Bauman and Kemper, 2005; Murcia et al., 2005). Thus,
diverse genetic and cellular changes are present in
autistic brains. It is important to emphasize that causal
versus ancillary abnormalities remain to be rigorously
determined (Baron-Cohen and Belmonte, 2005; Cody
et al., 2002; Dakin and Frith, 2005).
Similar to our mouse model, mice null for FMR1 dis-
played deficits in social and anxiety behaviors and in-
creased spine density, which is also reported in fragile
X syndrome (Bagni and Greenough, 2005). Both FMR1
and the mTOR/S6K pathway regulate protein translation
and synaptic plasticity (Tang et al., 2002; Zalfa et al.,
2003). Given the known association of fragile X syn-
drome in ASD (Andres, 2002), aberrant regulation of
synaptic protein translation and abnormal synaptic con-
nectivity may be a common theme affecting social inter-
action and anxiety in the mouse models and potentially
in subsets of ASD as well. Conversely, in Rett Syndrome
and some reports of autism, neuronal atrophy and re-
duced dendritic spines have been reported (Zoghbi,
2003). It may bepossible that synaptic imbalance result-
ing from excess or reduced connectivity could result in
the similar abnormalities found in ASD.
Mice and Histology
of Toronto), and Rosa26R mice were from Jackson Lab (Bar Harbor,
maintained Nse-cre; Rosa26R or PtenloxPmice in C57/BL6 inbred
background for at least three generations. For BrdU chasing, we in-
jected subsets of 2-week-old cre; Rosa26R mice with BrdU as de-
between PtenloxP/loxPmouse and cre; PtenloxP/+mouse or between
cre; PtenloxP/+mice. Littermate controls used for this study were
with a genotype of cre; Pten+/+or PtenloxPmice without cre. For par-
affin sectioning, we dissected out, processed, and sectioned brains
asdescribed (Fraseretal.,2004).Forvibratome sectioning, weintra-
cardially perfused the mice with ice-cold PBS followed by 4% (w/v)
paraformaldehyde (PFA) in PBS. We dissected out the brain, post-
fixed it in 4% PFA for overnight, and embedded it into 3% agarose.
tome.Allmouse protocols wereapproved bythe InstitutionalAnimal
Care and Research Advisory Committee at University of Texas
Southwestern Medical Center.
We performed all IHC on triplicate sections per group. Based on
anatomy, we chose matched sections from control and mutant. An-
tibodies used for IHC were against b-galactosidase (ICN, Aurora,
OH), MAP2 (Sternberger Monoclonals, Lutherville, MA), nestin (BD
Bioscience, San Jose, CA), BrdU (Dako, Carpinteria, CA), Pten (Neo-
Markers, Winchester, MA), P-Akt, P-S6 (Cell Signaling, Beverly, MA),
calbindin (Swant, Bellinzona, Switzerland), synapsin I, calretinin
(Chemicon, Temecula, CA) or P-Gsk3b (Biosource, Camarillo, CA).
For paraffin sections, we used microwave antigen retrieval for all
antibodies, except that against P-Gsk3b. We visualized the primary
antibodies by treating the sections with biotinylated secondary an-
tibody and followed by amplification with peroxidase-conjugated
avidin and DAB substrate. DAB-stained sections were counter-
stained with hematoxylin or methyl green (Vector Labs, Burlingame,
CA). Alternatively, we detected the primary antibodies by secondary
antibodies conjugated with Cy3 (Jackson Immunoresearch, West
Grove, PA) or Alexa Fluor 488 (Molecular Probe, Eugene, OR) fol-
lowed by counterstaining with DAPI (Vector Labs).
Cell Counting, Size Measurement, Golgi Staining,
and Electron Microscopy
To measure the ratio of Pten deletion, we counted Pten-positive
or-negative neurons ina 73 5 mm2area inthe sensory cortex layers
III to V and dentate GL by using MetaMorph software (Universal Im-
aging Corporation, West Chester, PA). We measured soma diameter
of dentate granule neurons as described (Kwon et al., 2003), except
for using the MetaMorph software. To estimate the length of the
mossy fiber tract, we measured the center path of double-labeled
signals for synapsin I and calbindin from the CA3 to the point at
which two blades of dentate granular layer meet. Similarly, we esti-
mated the thickest region of MAP2-positive dentate ML of either
blade. We performed Golgi staining, image analysis, quantification
of spine density, and electron microscopy as described (Luikart
et al., 2005). Data were analyzed by Student’s t test, except where
noted, and displayed as mean 6 SEM.
Mutant mice were studied along with littermate controls in four co-
horts of mice. Order of tests and cohorts are displayed in Table S2.
olfaction, strength tests by using vertical pole and dowel (Moretti
et al., 2005), and nest formation (Lijam et al., 1997) were measured
as described. Exceptions were performing social learning 3 days af-
Student’s t test in strength tests. Caged social interaction for social
versus inanimate target (Moy et al., 2004) was performed in a 48 3
48 cm2white plastic arena using two 6.0 3 9.5 cm rectangular cages
imal tactile interaction. Social preference for novelty was performed
as described (Moy et al., 2004), except room and door dimensions
were different (15 3 90 3 18.5 cm divided into three rooms of 15 3
29 cm separated by dividers with a central 3.8 3 3.8 cm door), and
video tracking software from Noldus (Ethovision 2.3.19) replaced
lowed to explore the room for 10 min. Then mice were allowed to in-
miliar caged target mouse versus a novel caged target mouse. The
open field test was performed for 10 min in a brightly lit (w800 lux),
48 3 48 cm2white plastic arena using video tracking software with
a center zone defined as a 15 3 15 cm2square. Elevated plus
maze, dark/light behavior, locomotor activity, accelerating rotarod,
Pten Regulates Social Interaction in Mice
and Morris water maze were measured as described (Powell et al.,
2004). Exceptions were scoring parameters by video tracking in ele-
vated plus maze and allowing each mouse three sets of three trials
per day with w10 min between trials and 3 hr between sets in accel-
erating rotarod test. A variation on the startle reflex and prepulse in-
jected to five pseudorandomly presented trial types in a 22 min
session with an average of 15 s (7–23 s) between trials: Pulse alone
(40 ms,120dB, whitenoisepulse), threedifferent Prepulse/Pulsetri-
als (20 ms prepulse of 4, 8, or 16 dB above background noise level of
70 dB precedes the 120 dB pulse by 100 ms, onset to onset), and no
stimulus. Context and cue-dependent fear conditioning was per-
formed as described (Powell et al., 2004), except a different plexi-
glass box with clear walls (MedAssociates) was used and two pair-
ings of a 2 s, 0.8 mA footshock and tone were delivered with 60 s
between pairings. Freezing behavior was monitored at 10 s intervals
by an observer blind to the genotype. To measure sexual behavior,
naive male mice (n = 4) were caged with two sexually experienced
female mice up to 30 days. We observed their sexual behavior about
1 hr at the first day and 10 min at next 4 days. To measure maternal
behavior, we mated one or two naive female mice with a sexually ex-
perienced male mouse up to 30 days. We counted pups at P0, P5,
P10, and P15.
Pten mutants (n = 3) and controls (n = 4) at 8–9 months of age were
anesthetized and surgically implanted for long-term EEG/EMGmon-
otherwise disturbed. They were habituated to the recording condi-
tions for 2 weeks before EEG/EMG signals were recorded over a pe-
riod of 3 days, beginning at lights-off (CT 12:00). During the light pe-
riod on the third day, sound and tactile stimuli were used to examine
companied by atonic periods or sustained rhythmic contractions on
the EMG. Each seizure lasting for 2 s or more was noted.
For Timm’s staining, we intracardially perfused the mice with ice-
cold 0.37% (w/v) sodium sulfide followed by 4% PFA. We dissected
out the brain, postfixed it in 4% PFA for overnight, and cryopro-
tected in 30% (w/v) sucrose in PBS for 2–3 days. We made 16 mm
thick coronal sections by using a cryostat. We performed a modified
Timm staining as described (Danscher et al., 2004).
We performed Western blotting as described (Kwon et al., 2003), ex-
Western blotting were against Pten (NeoMarkers, Winchester, MA),
P-Akt, P-S6, P-Gsk3b, P-Tuberin, Tuberin (Cell Signaling), or b-actin
(Sigma, St. Louis, MO). We statistically analyzed chemilumines-
cence signals by using Kodak Image Station 2000R (Rochester, NY).
The Supplemental Data for this article can be found online at http://
The authors thank Christopher Sinton and Shiori Ogawa for EEG/
EMG analysis; Tak Mak for PtenloxPmice; Junyuan Zhang, Xiaoyan
Zhu, Steve McKinnon, Arash Khatami, Phillip Williams, David Theo-
bald, Yajuan Liu, Ki-Woo Kim, and Jian Chen for technical assis-
tance; and Gorm Danscher, Meredin Stoltenberg, Mark Lush,
Douglas Benson, and Gary Westbrook for suggestions and helpful
discussions. This work was supported in part by the American and
Lebanese Associated Charities, NIH grant NS44172 (to S.J.B), and
MH06597503 (to C.M.P.) NIH grant R37NS33199 and the American
Cancer Society (to L.F.P.).
Received: August 12, 2005
Revised: February 15, 2006
Accepted: March 16, 2006
Published: May 3, 2006
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