The Interaction between Early Life Epilepsy and Autistic-
Like Behavioral Consequences: A Role for the
Mammalian Target of Rapamycin (mTOR) Pathway
Delia M. Talos1,2.¤, Hongyu Sun1,2., Xiangping Zhou1,2., Erin C. Fitzgerald1, Michele C. Jackson1,
Peter M. Klein1, Victor J. Lan1, Annelise Joseph1, Frances E. Jensen1,2,3*
1Department of Neurology, Children’s Hospital, Boston, Massachusetts, United States of America, 2Division of Neuroscience, Children’s Hospital, Boston, Massachusetts,
United States of America, 3Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, United States of America
Early life seizures can result in chronic epilepsy, cognitive deficits and behavioral changes such as autism, and conversely
epilepsy is common in autistic children. We hypothesized that during early brain development, seizures could alter
regulators of synaptic development and underlie the interaction between epilepsy and autism. The mammalian Target of
Rapamycin (mTOR) modulates protein translation and is dysregulated in Tuberous Sclerosis Complex, a disorder
characterized by epilepsy and autism. We used a rodent model of acute hypoxia-induced neonatal seizures that results in
long term increases in neuronal excitability, seizure susceptibility, and spontaneous seizures, to determine how seizures
alter mTOR Complex 1 (mTORC1) signaling. We hypothesized that seizures occurring at a developmental stage coinciding
with a critical period of synaptogenesis will activate mTORC1, contributing to epileptic networks and autistic-like behavior in
later life. Here we show that in the rat, baseline mTORC1 activation peaks during the first three postnatal weeks, and
induction of seizures at postnatal day 10 results in further transient activation of its downstream targets phospho-4E-BP1
(Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/236), as well as rapid induction of activity-dependent
upstream signaling molecules, including BDNF, phospho-Akt (Thr308) and phospho-ERK (Thr202/Tyr204). Furthermore,
treatment with the mTORC1 inhibitor rapamycin immediately before and after seizures reversed early increases in
glutamatergic neurotransmission and seizure susceptibility and attenuated later life epilepsy and autistic-like behavior.
Together, these findings suggest that in the developing brain the mTORC1 signaling pathway is involved in epileptogenesis
and altered social behavior, and that it may be a target for development of novel therapies that eliminate the progressive
effects of neonatal seizures.
Citation: Talos DM, Sun H, Zhou X, Fitzgerald EC, Jackson MC, et al. (2012) The Interaction between Early Life Epilepsy and Autistic-Like Behavioral Consequences:
A Role for the Mammalian Target of Rapamycin (mTOR) Pathway. PLoS ONE 7(5): e35885. doi:10.1371/journal.pone.0035885
Editor: Joshua L Bonkowsky, University of Utah, United States of America
Received January 17, 2012; Accepted March 23, 2012; Published May 2, 2012
Copyright: ? 2012 Talos et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by United States National Institutes of Health (NIH, www.nih.gov) Grants RO1 NS 31718 (to F.E.J.) and DP1 OD003347 (from
the Office of the Director) (to F.E.J.), Mental Retardation Developmental Disorders Research Center Grant P30 HD18655 (National Institute of Child Health and
Human Development, www.nichd.nih.gov) (to F.E.J.), Tuberous Sclerosis Alliance (www.tsalliance.org) (to D.M.T.), Ruth L. Kirschstein National Research Service
Award (NRSA, grants.nih.gov/training/nrsa.htm) T32HD007466 (to X.Z.), and Canadian Institutes of Health Research (www.cihr-irsc.gc.ca) MFE-115462 (to H.S.). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤ Current address: Department of Neurology, New York University Medical Center and New York School of Medicine, New York, New York, United States of
. These authors contributed equally to this work.
Epilepsy is the third most common major neurological disease
[1,2] and is increasingly recognized as a disease that reaches well
beyond seizures, with a high incidence of neuropsychiatric co-
morbidities not associated with abnormal electrographic activity.
Up to half of all epilepsy patients suffer cognitive and/or
neuropsychiatric disabilities [3,4]. Nowhere is this more prominent
than in early life, where the incidence of seizures is at one of
highest levels of the lifespan . In addition, early life epilepsy is
often accompanied by learning and neurocognitive disorders,
including autism [6,7]. In the case of autism, epilepsy is present in
up to 46% of patients, and correlates with lower IQ . Co-
occurrence of epilepsy and autism has been found in approxi-
mately 30% of children with either disorder [8,9,10] suggesting an
interaction between epilepsy and autism.
Rodent models of early life seizures also exhibit long term
consequences of epilepsy and altered synaptic plasticity .
Clinically, the most common cause of seizures in the neonatal
period is hypoxic/ischemic encephalopathy . The rat model of
neonatal hypoxia-induced seizures (HS) demonstrates features of
the human disease state, including post-seizure changes in
hippocampal and cortical excitability [11,13], increased later life
seizure susceptibility, cognitive deficits, mossy fiber sprouting, and
spontaneous seizures [14,15]. In addition, neonatal seizures cause
early post-translational modification and potentiation of the a-
Amino-3-hydroxy-5-Methyl-4-isoxazole-Propionic Acid (AMPA)
PLoS ONE | www.plosone.org1May 2012 | Volume 7 | Issue 5 | e35885
subtype of excitatory glutamate receptors similar to those observed
in physiologic synaptic plasticity [11,16].
We hypothesize that during a period of robust synaptogenesis,
seizures may cause age-specific alterations that result in both
epilepsy and neurobehavioral deficits. As synaptic plasticity models
demonstrate that long-lasting change requires new protein
synthesis , we investigated whether mTOR-dependent protein
translation was altered following neonatal seizures, thereby
contributing to epileptogenesis and behavioral deficits.
The mTOR Complex 1 (mTORC1) pathway regulates protein
translation via activation of the ribosomal S6 protein kinase
(p70S6K) and its downstream target ribosomal S6 protein, as well
as inactivation of the eukaryotic initiation factor 4E (eIF4E)-
binding protein 1 (4E-BP1). The mTORC1 is activated by many
factors, including ionotropic and metabotropic glutamate recep-
tors, trophic factors such as brain-derived neurotrophic factor
(BDNF), activity dependent extracellular signal-regulated kinase
(ERK) pathway, and phosphoinositide-39 kinase (PI3K)/Akt
pathway, and is constitutively suppressed by the TSC1 (hamartin)
and TSC2 (tuberin) complex [17,18]. Inactivating mutations in
either TSC1 or TSC2 result in Tuberous Sclerosis Complex (TSC),
characterized by abnormal cortical development and seizures .
Epilepsy occurs in up to 80% of TSC cases and intellectual
disability and/or autism is seen in up to 60% [7,20,21,22].
Mutations of Tsc1 or Tsc2 in the mouse are associated with
The mTORC1 pathway critically regulates neuronal growth,
synaptic plasticity and memory consolidation [28,29], and
inhibition of mTORC1 with rapamycin blocks long term synaptic
potentiation (LTP), a model of learning and memory . In adult
brain, recent studies have shown that the mTORC1 pathway can
be activated by seizures in the absence of a genetic mutation in
Despite the clinical link between epilepsy and neurobehavioral
deficits in TSC and the experimental evidence that mTORC1
plays a role in epileptogenesis in adulthood, little is known about
whether seizure-induced changes in mTORC1 signaling occur in
early life epilepsy, or whether these changes have behavioral and
epileptogenic consequences . To investigate the role of
mTORC1 in later epileptogenesis and behavioral deficits without
the confound of cell death seen in adult models, we used a model
of early life hypoxic seizures [15,36]. We first assessed baseline and
seizure-induced changes of critical downstream and upstream
components of mTORC1 signaling in the developing brain. Next,
we examined whether these changes were associated with altered
glutamate receptor function, later life epilepsy and social
behavioral deficits, given the high incidence of these abnormalities
in genetic diseases that upregulate mTORC1 pathway activity.
Finally, we determined whether early treatment with the
mTORC1 inhibitor rapamycin could attenuate subsequent
behavioral deficits and seizure-induced increases in neuronal
excitability in vivo and in vitro.
The mTORC1 Pathway Activity is Upregulated in the
As the mTORC1 signaling is known to control neuronal
maturation and plasticity, we hypothesized that mTORC1 activity
is enhanced during early postnatal development. First, we
quantified the expression of total mTOR protein and found that
it gradually increased from postnatal day 3 (P3) to P10/11 in both
hippocampus and neocortex (p,0.05), before approaching almost
adult levels by P16–P21 (Figure 1A). Phospho-mTOR (Ser2448),
which can be associated with both mTORC1 and mTORC2 
(Figure 1B), demonstrated different trends in hippocampus and
neocortex: it progressively increased with age in the hippocampus
(p.0.05), while being transiently elevated between P3–P10/11
before dropping to adult levels in the neocortex (p,0.001).
Phospho-mTOR (Ser2448)/mTOR ratios demonstrated no mat-
urational changes in the hippocampus, but a significant upregula-
tion at P3–P10/11 in the neocortex (p,0.001), as compared to
adult standard. Due to the fact that phosphorylation of Ser2448
has been linked to both mTORC1 and mTORC2, in order to
assess the mTORC1 pathway specifically, we quantified the
phosphorylation status of two of its major downstream targets:
[17,38]. Unlike endogenous mTOR protein, total p70S6K and
S6 showed stable expression levels over time, however both were
highly phosphorylated at early ages (Figure 1C–D). Phospho-
p70S6K/p70S6K ratios showed a transient upregulation with
significantly increased levels at P6–P10/11 in hippocampus
(p,0.05), and at P3–P10/11 in neocortex (p,0.001) (Figure 1C).
Similarly, phospho-S6/S6 ratios in the hippocampus were
upregulated at P6 (p,0.001), P10/11 (p,0.001) and at P16
(p,0.01), and relatively delayed in neocortex, with highest levels
occurring at P10/11 (p,0.05), P16 (p,0.01) and P21 (p,0.001)
(Figure 1D). These results demonstrate that, although total mTOR
gradually increases with maturation, functional markers of
mTORC1 activity are transiently elevated during early brain
development coincident with the peak of synaptogenesis [16,39].
marker NeuN and phospho-S6 (Ser235/236) confirmed neuronal
co-localization, both in hippocampus (Figure 2A1–C1) and
neocortex (Figure 2A2–C2) (n=6). In contrast, there was no co-
localization of phospho-S6 with either the GABA-ergic neuronal
marker GAD-67 (Figure 2D1–F1, D2–F2), or the astrocytic
marker GFAP (Figure 2G1–I1, G2–I2). This demonstrates that the
developmental hyperactivation of mTORC1 pathway seen by
western blots predominates in excitatory principal neurons, with
little contribution of inhibitory neurons or astroglia.
mTORC1 Signaling Pathway is Further Transiently
Activated by Neonatal Seizures
Changes in the mTORC1 downstream targets phospho-4E-BP1
(Thr37/46), phospho-p70S6K (Thr389) and phospho-S6 (Ser235/
236) were quantified over the first 48 h after hypoxic seizures (HS)
induced at P10 (Figure 3). At 12 h post-HS, phospho-4E-BP1
increased in both hippocampus (p,0.001) and neocortex (p,0.05,
Figure 3A), with similar increases in phospho-p70S6K (p,0.05,
Figure 3B). Phospho-S6 upregulation followed later at 24 h after
HS in both hippocampus and neocortex (p,0.01, Figure 3C).
There were no significant changes in total levels of 4E-BP1,
p70S6K and S6 (p.0.05, Figure 3). These results demonstrate
that seizures in the developing brain cause acute increases in
downstream components of mTORC1 pathway.
Neonatal Seizures Induce Transient Upregulation of
BDNF Levels and Early Activation of PI3- and MAP- Kinase
Brain derived neurotrophic factor (BDNF), which can trigger
mTORC1-dependent protein translation in activity-dependent
neuronal plasticity [29,40], was transiently increased at 1 h
(p,0.05) and 12 h (p,0.01) following seizures in hippocampus,
and at 1 h in neocortex (p,0.005, Figure 4A). The kinases Akt
and ERK1/2, downstream from BDNF, and upstream from
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mTOR Role in Epilepsy and Associated Autism
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mTORC1  showed a rapid induction following neonatal
seizures. Phospho-Akt (Thr308) levels were transiently elevated at
1 h after HS in hippocampus (p,0.05) and 3 h in neocortex
(p,0.05), with later neocortical downregulation at 24 h post-HS
(p,0.01, Figure 4B). Phospho-ERK1/2 (Thr202/Tyr204) levels
were briefly increased at 3 h post-HS, more consistent in
hippocampus (p,0.05), compared to neocortex showing variable
responses (p=0.11) (Figure 4C). Importantly, total Akt and
ERK1/2 levels remained unaltered in hippocampus and in
neocortex compared to controls during
(p.0.05, Figure 4B-C). These data indicate that HS in the
immature brain induce a rapid and transient activation of BDNF,
followed by PI3K/Akt/mTORC1 and the ERK1/2 signaling
this time course
Cell-specific Upregulation of mTORC1 Signaling
Following Neonatal Seizures
We next examined the cellular localization of the mTORC1
pathway marker phospho-S6 at the 24 h peak of the post-seizure
activation (n=6/group). We observed an overall increase in
phospho-S6 expression in pyramidal neurons, most prominent in
the apical and basal dendrites (Figure 5). SMI 311 neurofilament
staining was unchanged post-HS, suggesting no changes in
dendrite morphology or integrity. In addition, no seizure-induced
increases in phospho-S6 were observed in either interneurons or
astrocytes (data not shown), suggesting that mTORC1 activation is
predominantly induced in the dendritic portion of glutamatergic
neurons, consistent with its known role in synaptic function .
Figure 1. Components of the mTORC1 pathway are developmentally regulated. A. Western blot quantification of total mTOR protein levels
in rat brain demonstrates a significant downregulation at P3–P11 in the hippocampus (32.066.1% of adult, n=5, p,0.01 at P3, 44.762.8 of adult,
n=4, p,0.05 at P6, 69.066.1% of adult, n=5, p,0.05 at P10/11) and P3–P16 in the neocortex (28.065.4% of adult, n=4, p,0.001 at P3, 36.462.1 of
adult, n=4, p,0.001, 54.065.2% of adult, n=5, p,0.001 at P10/11, 70.067.8% of adult, n=4, p,0.05 at P16), compared to adulthood (100%). B.
Phospho-mTOR (Ser2448)/mTOR ratios demonstrate a modest peak at P3 in the hippocampus (1.7660.14 fold, n=5, p.0.05), and a significant
upregulation at P3–P10/11 in the neocortex (28.864.5 fold increase, n=4, p,0.001 at P3, 21.062.5 fold increase, n=4, p,0.001 at P6, 17.761.7 fold
increase, n=5, p,0.001 at P10/11), as compared to adult standard (1). C. Phospho-p70S6K (Thr389)/p70S6K ratios demonstrate a significant
upregulation at P6–P11 in the hippocampus (54.2615.3 fold increase, n=4, p,0.05 at P6, 43.8612.3 fold increase, n=5, p,0.05 at P10/11) and at
P3–P11 in the neocortex (98.5612.6 fold, n=5, p,0.001 at P3, 130.3612.6 fold, n=4, p,0.001 at P6, 70.768.4 fold, n=7, p,0.001 at P10/11) as
compared to adult standard (1). D. Phospho-S6 (Ser235/236)/S6 ratios are significantly increased at P6–P16 in the hippocampus (2.960.2 fold
increase, n=5, p,0.001 at P6, 4.960.9 fold increase, n=5, p,0.001 at P10/11, 3.060.3 fold increase, n=4, p,0.01 at P16), and at P10–P21 in the
neocortex (3.160.4 fold, n=5, p,0.05 at P10/11, 8.561.7 fold, n=5, p,0.01 at P16, 4.960.3 fold, n=4, p,0.001 at P21), relative to adult standard (1).
Mean values for each age group are expressed relative to the mean adult levels, represented by the dotted line. *: p,0.05. Error bars indicate S.E.M.
Insets are the representative western blots for individual proteins.
Figure 2. Cell-specific expression of the phospho-S6 (Ser235/236) protein in the normal developing rat brain. Coronal sections double
labeled with NeuN (red) and phospho-S6 (Ser235/236) (green) demonstrate high phospho-S6 expression in hippocampal CA3 region (A1–C1) and
neocortical layer V (A2–C2) at P11. Double labeling with GAD-67 (red) in combination with phospho-S6 (green) demonstrates no co-expression in
hippocampal (D1–F1) or neocortical (D2–F2) interneurons. GFAP and phospho-S6 double labeling shows no phospho-S6 expression in the
hippocampal (G1–I1) and neocortical (G2–I2) astrocytes. Scale bars 50 mm.
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Rapamycin Treatment Attenuates Seizure-induced
Increases in 4E-BP1, p70S6K and S6 Phosphorylation
Rapamycin (3 mg/kg i.p.) was administered 24 h before and 1h
post-HS in order to achieve trough levels of phospho-S6 at the
time of exposure to hypoxia at P10 (see Methods). This treatment
markedly suppressed the post-HS increases in phospho-4E-BP1,
phospho-p70S6K and phospho-S6 in both hippocampus and
neocortex (Figure 6A). Specifically, rapamycin-treated HS rats
demonstrated a significant reduction of phospho-4EBP1/4EBP1
(p,0.05 in hippocampus, and p,0.01 in neocortex), phospho-
p70S6K/p70S6K (p,0.05 in hippocampus, and p,0.001 in
neocortex), and phospho-S6/S6 ratios (p,0.001 in hippocampus,
and p,0.001 in neocortex), relative to vehicle-treated controls. In
addition, consistent with prior reports in normal brain [42,43], this
dose also suppressed baseline activity of mTORC1 downstream
markers in naı ¨ve control rat pups (Figure 6A), confirming
pharmacologic efficacy and brain penetration of this agent.
Rapamycin-treated control rats demonstrated a significant de-
crease in phospho-4EBP1/4EBP1 (p,0.05 in hippocampus, and
p,0.01 in neocortex), phospho-p70S6K/p70S6K (p,0.05 in
hippocampus, and p,0.001 in neocortex), and phospho-S6/S6
ratios (p,0.001 in hippocampus and neocortex), compared to
vehicle- treated sham controls.
Rapamycin Treatment Protects against Subacute
Increases in Seizure Susceptibility and Enhanced AMPA
Neonatal seizures are characterized by subacute increases in
susceptibility to kainic acid (KA) seizures [11,36], and by
subsequent development of chronic epilepsy . As there is no
treatment to date to prevent the long term epileptogenesis after
early life seizures [10,44], we examined whether mTORC1
inhibition may have a protective effect. Control and HS rats were
treated with rapamycin or vehicle as previously described, and
then seizures were induced by systemic KA injection at P13 (72 h
post-HS, 2 mg/kg, i.p.). Consistent with previous reports [32,33],
rapamycin had no acute effects on the initial HS at P10, as it did
not alter either the number of generalized tonic-clonic seizures per
rat pup (vehicle: 9.8461.07, n=13; rapamycin: 8.1361.16,
n=15; p=0.29), or the average duration of these seizures (vehicle:
17.0663.59 sec, n=12; rapamycin: 15.3762.37 sec, n=12;
p=0.70). However, rapamycin treatment reversed the enhanced
susceptibility to KA seen in HS rats at P13 (Figure 6B). As we have
previously shown [11,36], KA-induced seizure severity (maximal
seizure stage reached) at P13 was increased in vehicle-treated HS
rats relative to vehicle-treated littermate controls (p,0.05)
(Figure 6B). In contrast, rapamycin-treated HS animals exhibited
a significantly lower seizure severity score, compared to the
vehicle-treated HS rats (p,0.05), and this was similar to the
vehicle-treated naı ¨ve control group (Figure 6B). The KA seizure
severity was unchanged in the rapamycin-treated control group
(p.0.05). Additionally, we found no relationship between the
number of HS and the severity of KA-induced seizures in either
the vehicle- treated or rapamycin-treated HS rats (p=0.72; two-
Concurrent with increased seizure susceptibility at P13 in vivo,
we have also reported a subacute increase in AMPA receptor
function in hippocampal CA1 neurons in ex vivo slices removed at
48–72 h post-HS . As mTORC1 activation upregulates
Figure 3. Neonatal seizures transiently activate the mTORC1 downstream targets phospho-4E-BP1 (Thr37/46), phospho-p70S6K
(Thr389), and phospho-S6 (Ser235/236). A. Phospho-4E-BP1 levels significantly increase in both hippocampus (1.4760.13 fold, n=12, p,0.001)
and neocortex (1.5560.19 fold, n=11, p,0.05) at 12 h after HS induction in P10 rat pups. B. Phospho-p70S6K is simultaneously significantly
upregulated in both hippocampus (1.3460.14 fold, n=7, p,0.05) and neocortex (1.8160.34 fold, n=7, p,0.05) 12 h following HS onset. C.
Subsequent increases in phospho-S6 are observed in both hippocampus (1.8860.31 fold, n=12, p,0.01) and neocortex (1.3560.08 fold increase,
n=9, p,0.01) at 24 h post-HS. Histograms represent averaged optical density normalized to actin, and expressed relative to the mean control values
(represented by the dotted line). No significant difference is observed in the total levels of any of these proteins (p.0.05). Insets are the
representative western blots for individual proteins. C: control; HS: hypoxic seizures. *: p,0.05, **: p,0.01. Error bars indicate S.E.M.
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AMPA receptor function in vitro , we tested whether HS-
induced enhancement of receptor function could be prevented by
in vivo rapamycin treatment. Spontaneous (s) AMPA receptor-
mediated EPSCs (pharmacologically isolated by blocking GABA
and NMDA receptors with 60 mM picrotoxin and100 mM DL-AP-
5, respectively) were recorded in hippocampal CA1 pyramidal
neurons in slices removed 48 h post-HS. Neurons from vehicle-
treated HS group showed significantly larger sEPSCs amplitude
compared to vehicle-treated littermate controls (p,0.001), as well
as a trend toward higher frequencies (p=0.16) (Figure 6C–F),
consistent with our prior results . In vivo treatment with
rapamycin reversed seizure-induced enhancement of AMPA
receptor-mediated sEPSC amplitude (p,0.01) in slices removed
from HS rats, but had no significant effect in the naı ¨ve littermate
controls (p.0.05, Figure 6C–F). AMPA receptor-mediated
miniature (m) EPSCs, isolated by adding TTX (1 mM) to the
bath solution, demonstrated a similar change at 48 h post-HS.
The amplitude of AMPA mEPSCs was significantly increased in
vehicle-treated HS animals compared to the vehicle-treated naı ¨ve
controls (139.968.1% of vehicle-treated controls, p,0.01, n=5).
Rapamycin treatment significantly attenuated the increased
mEPSC amplitude in HS rats (103.565.4% of vehicle-treated
controls, p,0.01, n=5), but showed no effects in the naı ¨ve
controls (101.264.1% of vehicle-treated controls, p.0.05, n=5).
These results suggest a potential role of mTORC1 activation in
the increased seizure susceptibility and enhancement of AMPA
receptor function observed following early life seizures.
mTORC1 Inhibition Prevents the Development of Chronic
Epilepsy Following Neonatal Seizures
Given these subacute protective effects of rapamycin, we further
evaluated possible protective effects of mTORC1 inhibition on
later life spontaneous seizures development. HS seizures were
induced at P10, and rapamycin was administered as previously
described. Video-EEG recordings were performed in young adult
rats at P36–38. The fairly brief electrographic seizures (all groups:
6.460.71 sec; vehicle-treated HS group: 7.661.37 sec; Figure 7A)
were accompanied by sudden behavioral arrest, staring episodes,
head jerking, and facial automatisms. Consistent with our previous
reports , we found that both the number of spontaneous
seizure per hour and the cumulative time seizing per hour were
significantly increased in vehicle-treated HS rats, relative to
normoxic vehicle-treated littermates (p,0.01) (Figure 7B,C). Rats
that had been previously treated with rapamycin around HS
demonstrated significantly decreased later life spontaneous seizure
frequency and reduced cumulative seizure time (p,0.01), relative
to vehicle-treated HS rats. Importantly, treatment with rapamycin
at P10 in normoxic rats did not cause any later life EEG changes
compared to vehicle-treated controls, which at baseline have very
low rates of EEG abnormalities (0.1260.07 seizures/h, p.0.05)
(Figure 7B,C), consistent with prior reports in normal Long-Evans
rats [15,45]. These data indicate that seizure-induced mTORC1
activation plays a critical role in causing later epileptogenesis after
early life seizures, and that rapamycin treatment, associated with
post-seizure suppression of mTORC1 activation, not only results
Figure 4. Rapid seizure-induced alterations in mTORC1 upstream regulators BDNF, phospho-Akt (Thr308) and phospho-ERK1/2
(Thr202/Tyr204). A. Quantification of BDNF protein levels at 1, 3, 12, 24, 48 h post-HS shows a bi-phasic upregulation in the hippocampus at 1 h
(1.3160.12 fold, n=7, p,0.05) and 12 h (1.2560.08 fold, n=7, p,0.01), and a single increase at 1 h (1.2760.06 fold, n=10, p,0.01) in the neocortex.
Of note, only the mature 14 kD BDNF band is quantified because of the relevance of this isoform for synaptic plasticity and epileptogenesis. B.
Phospho-Akt (Thr308) is significantly increased at 1 h post-HS in the hippocampus (1.2460.09 fold, n=8, p,0.05), while in the cortex levels are
upregulated at 3 h (1.360.11 fold, n=7, p,0.05) and downregulated at 24 h after HS (0.760.04 fold, n=7, p,0.01). C. Phospho-ERK1/2 expression is
significantly higher at 3 h post-HS in the hippocampus (1.4660.18 fold, n=7), but not in the neocortex (1.3760.22 fold, n=11, p=0.11). Averaged
optical density normalized to actin are expressed relative to the mean control values (dotted line). No significant difference is observed in the total
levels of phospho-Akt (Thr308) or phospho-ERK1/2 (Thr202/Tyr204) (p.0.05). Insets are the representative western blots for individual proteins. C:
control; HS: hypoxic seizures. *: p,0.05, **: p,0.01. Error bars indicate S.E.M.
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in a decrease in subacute network hyperexcitability, but also can
attenuate long term risk of epilepsy.
Autistic-like Social Deficits Induced by Neonatal Hypoxic
Seizures are Reversed by mTORC1 Inhibition
Given the link between epilepsy and autism, we aimed to
identify whether altered social behavior could be detected
following early life seizures, and secondly whether this could be
modified by rapamycin treatment. Standardized behavioral assays
were used to measure social behavior (Figure 8A) [46,47,48,49].
First, open field and olfactory habituation/dishabituation tests
were performed and confirmed no baseline differences between
groups, ruling out any primary differences or alterations in motor
and sensory function in the treated HS and control rats
(Figure 8B,C). Next, social behavior assessed using a three-
chamber social choice test showed a significant preference for
sociability in all four groups, indicated by greater time spent with a
social stimulus than a non-social object (p,0.0001, Figure 8D). In
contrast, the preference for interacting with a novel rat over a
familiar rat was significantly reduced in vehicle-treated HS
animals (p=0.14) compared with the significant preference for
the novel rat exhibited by naı ¨ve controls (p,0.002, Figure 8E),
indicating that early life seizures can impair later social behavior to
an extent similar to other models of autism [47,50]. Furthermore,
rats that had been treated with rapamycin were protected against
exhibiting later deficits, and showed significant levels of preference
for social novelty (p,0.002), suggesting an important role of
mTORC1 pathway activation in inducing altered social behavior
following early life HS. Interestingly, the brief treatment of sham
control rats at P10–11 with the same doses of rapamycin appeared
to cause a loss of preference for the social novelty (p=0.49),
suggesting that even a 24 h disruption of mTORC1 activity during
the critical period is important for normal social development.
Early life epilepsy is associated with increased risk of intellectual
disability and autism  and epilepsy occurs at a significantly
higher rate in autistic patients relative to the general population
[7,8]. The interaction between epilepsy and autism is likely to be
multifactorial, but the present study supports a significant role for
perturbations in mTORC1 signaling pathway in linking the
pathogenesis of these two disorders, both of which have been
attributed at least in part to synaptic dysfunction. Early life is
characterized by enhanced synaptogenesis and synaptic plasticity,
and here we show that there is a commensurate increase in
activation of mTORC1-dependent downstream signaling in the
immature brain. While mTORC1 has been shown to regulate
synaptic function and plasticity in vitro [51,52], we now demon-
strate that seizures sequentially activate up- and downstream
components of plasticity related mTORC1 signaling, including
upstream activators PI3K/Akt, MAPK/ERK and BDNF and
downstream effectors 4E-BP1, p70S6K and ribosomal protein S6.
Figure 5. Phospho-S6 (Ser235/236) expression is highly upregulated in the cell bodies and dendrites of hippocampal and
neocortical pyramidal neurons at 24 h following neonatal seizures. A-B. Confocal images of CA1 hippocampal region double labeled with
NeuN (red) and phospho-S6 (green) show a moderate increase in phospho-S6 expression in pyramidal neuron cell bodies and a marked phospho-S6
upregulation in the dendrites (arrows) in the post-HS group (B, B1), relative to controls (A, A1). C-D. Confocal microscopy of neocortical layer V
demonstrates a similar predominant increase in phospho-S6 (green) expression in the apical (arrows) and basal (arrowheads) dendrites of NeuN (red)
positive neurons in the HS group (D, D1), relative to controls (C, C1). Confocal images represent z-stacks composed of multiple plane images collected
at 1 mm intervals. Scale bars are 100 mm for A-D. Insets (A1–D1) show corresponding higher magnification of individual cells.
mTOR Role in Epilepsy and Associated Autism
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The mTORC1 pathway is therapeutically targetable, and a major
finding of this study is that pharmacologic suppression of
mTORC1 activity with acute rapamycin treatment disrupts
subsequent development of epilepsy as well as autistic-like
behavior, suggesting that the mTORC1 pathway may be a
common, and reversible, mediator for the interaction between
early life epilepsy and autism.
Figure 6. Rapamycin attenuates seizure-induced mTORC1 pathway activation, subsequent subacute increases in seizure
susceptibility, and enhanced AMPA receptor function. A. Representative western blots of hippocampal and neocortical tissue, collected at
12–24 h post-HS, show that rapamycin treatment almost completely blocks phospho-4EBP1, phospho-p70S6K and phospho-S6 in HS and normoxic
control animals, with no effect on total protein levels. The rapamycin-treated HS rats (HS+R) demonstrate a significant reduction of phospho-4EBP1/
4EBP1 (0.4860.07, n=8, p,0.05 in hippocampus, and 0.5260.04, n=8, p,0.01 in neocortex), phospho-p70S6K/p70S6K (0.4360.04, n=4, p,0.05 in
hippocampus, and 0.1960.06, n=8, p,0.001 in neocortex), and phospho-S6/S6 (0.0360.02, n=4, p,0.001 in hippocampus, and 0.0860.02, n=4,
p,0.001 in neocortex), relative to vehicle-treated controls (C+V) (1). Sham control rats treated with rapamycin (C+R) also show a significant decrease
in phospho-4EBP1/4EBP1 (0.5660.06, n=8, p,0.05 in hippocampus, and 0.5360.04, n=8, p,0.01 in neocortex), phospho-p70S6K/p70S6K
(0.4560.05, n=4, p,0.05 in hippocampus, and 0.1660.01, n=8, p,0.001 in neocortex), and phospho-S6/S6 (0.160.02, n=4, p,0.001 in
hippocampus, and 0.0860.03, n=4, p,0.001 in neocortex), compared to vehicle-treated sham controls (C+V). B. Rapamycin treatment reduces
subacute increases in seizure susceptibility induced by kainic acid (KA) administration. When exposed to KA at P13, vehicle-treated HS rats (HS+V)
demonstrate a significant increase in the average seizure severity (2.860.12, n=13; modified Racine scale, see methods), compared to vehicle-treated
normoxic control littermates (C+V), (2.260.11, n=27, p,0.05). Rapamycin-treated HS rats (HS+R) show a significant decrease in KA seizure severity,
relative to the HS+V group (2.1360.09, n=15; p,0.01). C. Representative traces of AMPA receptor-mediated spontaneous excitatory post-synaptic
currents (sEPSCs), pharmacologically isolated by blocking NMDA and GABAAreceptors, in hippocampal ex vivo slices from control and HS rats treated
in vivo with either vehicle (C+V, HS+V) or rapamycin (C+R, HS+R) and removed at 48 h post-HS (P12). D. Normalized cumulative distribution of AMPA
receptor sEPSCs, recorded at 48 h post-HS demonstrates a significant increase in amplitude in the vehicle-treated HS (HS+V) animals compared to
vehicle (C+V) or rapamycin-treated controls (C+R), and this is significantly attenuated by in vivo rapamycin treatment (HS+R), (p,0.001, Kolmogorov–
Smirnov test). E. AMPA receptor sEPSCs amplitude is significantly higher in the vehicle-treated HS group (HS+V), relative to vehicle-treated controls
(C+V), (137.6263.39% of vehicle-treated controls, n=6; p,0.001), and reduced significantly in the rapamycin-treated HS rat pups (HS+R),
(107.9364.64% of vehicle-treated controls, n=6; p,0.01). In vivo rapamycin treatment has no effect on AMPA receptor sEPSCs amplitude in the
normoxic control rats (C+R), (102.2865.4% of vehicle-treated controls; n=6, p.0.05). F. AMPA receptor sEPSCs show a trend of increased frequency
in the HS+V group, compared to C+V and C+R groups (164.98637.98% of vehicle-treated controls, n=6, p=0.16), which is attenuated by in vivo
rapamycin treatment (122.35626.5% of vehicle-treated controls, n=6). In vivo rapamycin treatment has no effect on AMPA receptor sEPSCs
frequency in the normoxic control rats (C+R) (108.39618.6% of vehicle-treated controls, n=6, p.0.05). *: p,0.05; **: p,0.01. Error bars indicate
mTOR Role in Epilepsy and Associated Autism
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This is the first report to show that markers of mTORC1
activity are transiently increased at baseline during the first three
postnatal weeks in rodents, consistent with heightened synaptic
plasticity. The expression of phosphorylated p70S6K (Thr389),
one of the best-characterized downstream targets of mTORC1
, was highest during the first postnatal weeks in both
hippocampus and neocortex. The increased mTORC1 signaling
appears to be occurring coincident with the developmental
upregulation of known upstream activators of this pathway,
including glutamate receptors, BDNF and Rheb [39,53,54,55],
which supports its critical involvement in synaptic and network
development . Interestingly, we found a developmental time
lag between the increase in phospho-p70S6K and phospho-S6. To
date, the timing of activation of these pathways has only been
studied in cell cultures or acute slices, and our in vivo results are
especially intriguing given the reported rapid kinetics of these
phosphorylation events in vitro [41,57]. The activity of mTORC1 is
highly regulated and balanced: for example, activation of p70S6K
blocks further activation of this pathway by inhibiting PI3K/Akt
signaling . During development, this negative feedback loop
might be initially stronger then the ability of p70S6K to activate
S6, at least to a level detectable by western blot in brain tissue.
Interestingly, the delay in S6 activation was more pronounced in
the neocortex, where phospho-p70S6K levels were highest.
Another interesting finding was the apparent disconnect between
the steady increase in total mTOR protein and the downregula-
tion of mTORC1 signaling with increasing age, which would
suggest that in the adult brain, the activity of mTORC2
predominates over mTORC1. This hypothesis is supported by
the developmental regulation of phospho-mTOR (Ser2448)
showing a progressive increase with maturation, at least in the
hippocampus. Although the functional relevance of this phos-
phorylation site in neurons remains to be determined, it has been
previously reported that phospho-mTOR (Ser2448) can be
associated with both mTORC1 and mTORC2 complexes .
Future analyses, beyond the scope of this study, will need to
examine the developmental regulation of specific mTORC2
targets, including phospho-Akt (Ser 473) .
This study also reveals upregulation of multiple mTORC1
pathway components following neonatal seizure induction in both
the hippocampus and neocortex. Specifically, we found that
mTORC1 downstream signaling is transiently activated, likely
through induction of the putative upstream activators PI3K/Akt,
MAPK/ERK and BDNF pathways (Figure 9). This is consistent
with synaptic plasticity models [40,41], and status epilepticus in
adult rats [34,60]. We show a sequential transient increase in
BDNF, phospho-Akt and phospho-ERK between 1 and 3 h after
seizure induction, followed by upregulation of the mTORC1
downstream effectors phospho- 4E-BP1, phospho-p70S6K and
phospho-S6 at 12–24 h post-seizure (Figure 9).
Figure 7. Rapamycin treatment at HS attenuates later epilepsy. A. Representative EEG traces from HS+R and HS+V rats, with HS+V showing
an example of an observed seizure. Scale bar =1 sec, 50 mV for both traces. Inset shows an expanded view of the EEG trace in the HS+V group. B.
Subdermal wire electrode EEG recordings show that rats that had received vehicle with HS at P10 (HS+V) have significantly more spontaneous
seizures at P36–38 (0.8960.25 seizures/h, n=15) than control normoxic litter mates (C+V), (0.1260.07 seizures/h, n=16, p,0.01). Rapamycin
treatment at HS (HS+R) attenuates the HS induced later life epilepsy (0.2560.10 seizures/h, n=15, p,0.05). Rapamycin treatment of sham P10–11
littermates (C+R) has no significant effect on the EEG pattern (0.3060.08 seizures/h, n=28, p=0.70), as compared to the C+V. C. Rapamycin also
reverses the effects of HS on cumulative ictal EEG activity/h. Rats with prior HS treated with vehicle (HS+V) exhibit significantly greater ictal activity
per hour (6.3063.31sec/h, n=16 rats) compared to naı ¨ve control rats treated with either vehicle (C+V), (0.6060.37 sec/h, n=16), or rapamycin (C+R),
(1.4560.58 sec/hr, n=15). In contrast, rapamycin treatment in HS rats (HS+R) shows significant protection (1.4860.42sec/h, n=28, p,0.01).
*: p,0.05, **: p,0.01, ***: p,0.001. Error bars indicate S.E.M.
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Seizures can rapidly elevate both glutamate and BDNF levels in
adult and immature animal models of epilepsy [61,62,63], which is
in agreement with our findings. Interestingly, in addition to the
early increases of BDNF in hippocampus and neocortex, we found
a later upregulation at 12 h in hippocampus, which is consistent
with the fact that multiple waves of activity-induced BDNF-
Figure 8. Rapamycin attenuates long term behavioral deficits after neonatal seizures. A. Schematic of behavioral testing protocol B.
Distance traveled and time spent exploring the center zone during a 30-minute open field test does not vary significantly between the treatment
groups (n=16–29, p.0.05) (C+V: vehicle-treated sham control litter mates; C+R: rapamycin-treated sham control litter mates; HS+V: vehicle-treated
HS rats; HS+R: rapamycin-treated HS rats). C. The olfactory habituation/dishabituation assay confirms no significant differences in the time spent
sniffing non-social odors or social odors, with similar habituation and dishabituation, as the time spent sniffing the first swab of each new odor
increases (two-way ANOVA for treatment [F(3,420)=1.18, p=0.32], odor [F(14,420)=22.59, p,0.0001] and interaction [F(42,420)=0.33, p=1.00]). D.
All groups spent significantly more time interacting with the novel rat than with a novel object in the three-chamber sociability test (two-way ANOVA
for treatment [F(3,146)=0.58, p=0.63], stimulus [F(1,146)=255.9, p,0.0001] and interaction [F(3,146)=1.32, p=0.27]). E. In contrast, there were
significant differences in the preference for social novelty among treatment groups (Bonferroni post-tests). C+V rats had a significant preference for
social novelty, spending greater time interacting with the novel rat (90.88615.93 vs. 32.1366.09 sec, n=16, p=0.0013), while the HS+V rats lacked a
preference for social novelty (76.97614.11 vs. 50.7369.76 sec, n=16, p=0.14). Importantly, rapamycin treatment reversed the deficits in social
novelty in HS rats (HS+R), (113.24610.67 vs. 70.7468.41 sec, n=29, p=0.0017). Rapamycin-treated control rats (C+R) did not significantly shift their
preference for the social novelty (82.46612.99 vs. 69.96613.52 sec, n=16, p=0.49) (two-way ANOVA for treatment [F(3,146)=3.41, p=0.02],
stimulus [F(1,146)=17.21, p,0.0001] and interaction [F(3,146)=1.29, p=0.28]).
mTOR Role in Epilepsy and Associated Autism
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Figure 9. Potential mTORC1-dependent mechanisms involved in epileptogenesis and altered social behavior following neonatal
seizures. Neonatal seizures induce intense synaptic activity followed by rapid increases in extracellular glutamate and BDNF levels (within 1 h post-
HS; Figure 4A). Activation of glutamate receptors, including NMDA receptors (NMDARs) and Ca2+-permeable AMPA receptors (CP-AMPARs) ,
followed by opening of L-type voltage-gated Ca2+channels (VGCCs), results in increased Ca2+influx. Enhanced expression and function of BDNF
activates the membrane-bound tyrosine kinase B receptors (TRKBRs) and, together with Ca2+influx, leads to the recruitment of the PI3K/PDK1/Akt
and the Ras/MEK/ERK signaling pathways (within 1–3 h post-HS; Figure 4B–C). Akt- and ERK-dependent inactivation of Tsc1/Tsc2 complex results in
activation of Rheb GTP-ase, and subsequent induction of mTORC1 activity (within 12–24 h post-HS; Figure 3). In turn, the mTORC1 may inhibit 4EBP1
and enhance the activity of p70S6K at 12 h post-HS, leading to enhanced eIF4E and S6 signaling, and increased protein translation at 24 h post-HS.
The negative feedback inhibition between p70S6K and PI3K may account for the time lag between p70S6K and S6 phosphorylation changes. Altered
TORC1-dependent translation of synaptic plasticity proteins (such as PSD-95, CamKII, Arc, GluR1) [40,91], among with other important cellular
processes regulated by the mTORC1 kinase (autophagy, ribosomal biogenesis and cellular metabolism) , may lead to both short-term and long-
term neuronal hyperexcitability (Figure 6B), enhancement of synaptic function (Figure 6C-F), epilepsy (Figure 7), and autism-like behaviors (Figure 8).
Treatment with the mTORC1 inhibitor rapamycin immediately before and after neonatal seizures reverses both early and late neurological and
behavioral consequences (Figures 6–8).
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dependent protein synthesis are required for hippocampal
memory consolidation, beyond the first few hours post-training
[29,64]. BDNF, in addition to its role in neuronal proliferation,
differentiation and survival, represents a crucial regulator of
synaptic function and plasticity, in particular, it can activate the
PI3K/Akt and MAPK/ERK pathways promoting dendritic
growth, dendritic branching, and spine formation [65,66].
Phospho-Akt and phospho-ERK may be important in promot-
ing epileptogenesis, beyond their function of activating mTORC1.
Akt has an important role in cell proliferation and cell survival 
and may have a neuroprotective role in this model. ERK signaling
can enhance protein synthesis by directly phosphorylating S6 at
Ser235/236  as well as p70S6K at multiple sites . In
addition, ERK can modulate the transcription of multiple
plasticity genes by activating the transcriptional regulator cAMP
response element-binding CREB . Interestingly, we found that
phospho-ERK responses were more robust in the hippocampus,
consistent with its critical role in hippocampal synaptic plasticity,
learning and memory [70,71,72].
Notably, the activation of downstream mTORC1 signaling in
our mild neonatal seizures model occurs somewhat later than the
reported phospho-S6 upregulation following induction of status
epilepticus in adult rats (1–6 h post) [32,34]. In addition to more
severe seizures, the adult models also cause neuronal death,
whereas our neonatal model exhibits no cell death and shows
ongoing subclinical excitability over the first 24 to 48 h after
seizure onset [15,16]. Furthermore, hypoxia was used to induce
neonatal seizures in this study, and it is known that hypoxia itself
can suppress mTORC1 signaling [73,74,75]. Although the
duration of hypoxia used for seizure induction was only 15 min,
which is much less then needed to induce a marked inhibition of
phospho-p70S6K , we cannot exclude that this may have
prolonged the time lag between the seizure-induced activation of
mTORC1 upstream and downstream components. Nevertheless,
the lag in activation may present a window of opportunity for
Enhancing the significance of seizure-induced activation of
mTORC1 in neonatal seizures, we found that in vivo rapamycin
treatment attenuated a number of epileptogenic consequences
previously reported in this model [11,15,36]. Effective inhibition of
mTORC1 signaling at P10 during HS protected against enhanced
susceptibility to KA-induced seizures at P13, but also reversed the
long term effects of HS in inducing later life spontaneous seizures.
Importantly, these effects were not due to suppression of acute HS,
as their severity and duration were unaffected by rapamycin
treatment. This is consistent with previous studies in adult rats that
show little or no immediate effects of rapamycin on acute status
epilepticus in vivo [32,33], and in vitro studies showing no direct
effect of rapamycin on excitatory synaptic transmission or
neuronal excitability [76,77].
Rapamycin treatment not only blocked the increases in early
mTORC1 activity markers and later seizure susceptibility, but also
prevented seizure-induced changes in synaptic function. We have
previously shown that epileptogenesis in this model is selectively
mediated by enhanced activation and modification of AMPA
receptor subtype of ionotropic glutamate receptors, as evidenced
by HS-induced increases in AMPA receptor mEPSCs and sEPSCs
[11,16]. Importantly, treatment with AMPA receptor antagonists
in the subacute period following HS prevents long term increased
network excitabilityand impaired
[16,36,78]. Similarly, we found that rapamycin significantly
attenuated the increased AMPA receptor mEPSCs and sEPSCs
amplitude in HS rats, without affecting the basal synaptic
transmission. Notably, components of the mTORC1 pathway
co-localize with post-synaptic scaffolding protein post-synaptic
density 95 (PSD95), facilitating local long-lasting changes in
synaptic function . mTORC1 regulates translation of imme-
diate early genes, and of genes encoding ion channels, protein
kinases or cytoskeletal proteins critical to spine and synapse
function [29,40,79,80]. As the mRNAs encoding glutamate
receptor subunits are localized in dendrites and can undergo local
translation in an activity-dependent manner , activation of
mTORC1 could serve to increase local translation of AMPA
receptor subunits themselves, or of related proteins involved in
their trafficking and insertion . This is consistent with our data
showing the co-occurrence of increased phospho-S6 in dendrites of
pyramidal neurons at 24 h post-HS and subacute increases in
AMPA receptor-mediated sEPSC and mEPSCs amplitude.
This study provides the first evidence for development of
autistic-like social behavior deficits following neonatal seizures in
wild type animals, manifested as a lack of preference for social
novelty. Similar social novelty deficit have been shown in several
autism mouse models [50,82,83,84], including genetic models that
involve dysregulation of mTORC1 pathway components [25,47].
Although currently there is no known casual relationship between
epilepsy and autism , the high association of these two disorders
suggests that they may share some anatomical and molecular
mechanisms [7,9,44,85]. The same in vivo rapamycin treatment
that prevented subacute changes in seizure susceptibility and long
term epilepsy also ameliorated these autistic-like behavioral
deficits. Thus the present study implicates the mTORC1 pathway
not only in epileptogenesis, but also in the behavioral consequenc-
es of early life seizures. These results raise the possibility that
modifying epileptogenesis in the immature brain may also prevent
other manifestations of network dysfunction involved in later life
neurobehavioral co-morbidities. While these results suggest a
disease modifying effect of rapamycin for both epilepsy and
secondary autism, caution must be placed on potential clinical
translation. While rapamycin showed a beneficial effect in post-
seizure animals, there was a mild effect on later social preferences
in control rats that had been treated at P10. Hence the protection
here is predicated on pathological increases in mTORC1 activity
by early life seizures, and in the absence of this pathological
upregulation, suppression of the mTORC1 pathway may have
actually caused subtle developmental abnormalities in the naı ¨ve
controls . Safety studies of mTORC1 inhibition must address
In conclusion, these results show a time-dependent activation of
up and downstream components of mTORC1 pathway, begin-
ning within an hour after seizure onset and lasting up to 24 h in
the immature rat brain (Figure 9). Activation of the mTORC1
pathway, and subsequent increased AMPA receptor function, may
have a critical role in epilepsy as well as autistic-like behavior as a
consequence of early life seizures and may be one of probably
multiple convergence points underlying an interaction between
autism and neonatal seizures (Figure 9). These results suggest that
this interaction is not simply limited to TSC patient population,
and that mTORC1 inhibition may have more widespread
application as a preventative, disease modifying therapeutic
strategy following refractory early life seizures with impact on
subsequent brain function.
Materials and Methods
Male Long-Evans rat pups (Charles River Laboratories,
Wilmington, MA) were maintained in a 12 h light/dark cycle
facility. All experiments were approved by the Animal Care and
mTOR Role in Epilepsy and Associated Autism
PLoS ONE | www.plosone.org12 May 2012 | Volume 7 | Issue 5 | e35885
Use Committee at Children’s Hospital (Boston, MA), and were in
accordance with the National Institutes of Health guidelines.
Hypoxic seizures (HS) were induced in Long Evens rats at
postnatal day (P) 10. Briefly, pups were exposed to graded global
hypoxia for 15 min (7% O2for 8 minutes, 5% O2for 6 minutes,
4% O2for 1 minute), as described previously . Under hypoxic
conditions, over 95% pups experience tonic-clonic seizures,
automatisms followed by head and limb movements, and
myoclonic jerks, while only a very small number of animals
(,5%) that are exposed to hypoxia do not respond with seizures.
Seizures develop gradually during the 15 min hypoxia, however
rat pups continue to present short spontaneous seizures over 48 h
following the initial hypoxic insult . All experimental groups
have experienced the same level of hypoxia for the same period of
time. Littermate controls went through the same procedure, but
exposed to normal air.
Administration of Rapamycin
Rapamycin (LC Laboratories, Woburn, MA) was dissolved in
100% ethanol and diluted in 5% Tween 80, 5% polyethylene
glycol 400 (Sigma) and 4% ethanol before use. To time rapamycin
administration, we first established its pharmacokinetics in P9 rats,
based on the effect of a single dose (3 mg/kg) to suppress the
mTORC1 pathway marker phospho-S6 (Ser235/236). Western
blots performed on cortical tissue to assess levels of phospho-S6 at
1 h, 6 h, 12 h, 24 h, and 48 h following a single injection of
rapamycin revealed that maximal phospho-S6 suppression was
obtained at 24 h following injection (1.760.4%, p,0.05), with
partial reduction as early as 1 h post-injection (19.764.8%), and
almost restored levels at 48 h. These data correlate with
pharmacokinetic studies in solid tumors after systemic rapamycin
administration, where trough levels of phospho-S6 were found at
24–48 h [87,88]. These data suggest that pre-treatment is
necessary in order to achieve optimal mTORC1 pathway
inhibition at the time of insult and/or over the first 24 h of a
seizure induction. This is also suggested by prior publications
demonstrating the increased efficacy of rapamycin in a pre-
treatment paradigm, as opposed to post-seizure treatment, for
suppression of long term epileptogenesis [43,89]. Therefore,
control and HS rats were treated with rapamycin (3 mg/kg i.p.)
or vehicle 24 h before and 1 h after exposure to hypoxia at P10.
Kainic Acid (KA)-induced Seizures
At P13 (72 h post-HS), control and HS rat pups previously
treated with either vehicle or rapamycin were administered KA
i.p. (2 mg/kg, Cayman Chemical, Ann Arbor, MI). Behavioral
seizures were videotaped for a 3 h period. Seizure severity
(maximal seizure stage reached graded from 0 to 5) was evaluated
blinded by two independent investigators, as described previously
[11,36]. Differences in seizure severity among groups were
analyzed by one-way ANOVA followed post hoc Bonferroni
multiple comparison procedures. Statistical significance was
defined as p,0.05.
Western Blot Analysis
For time course studies, male Long-Evans rats were euthanized
at P3, P6, P10, P11, P16, P21 and adulthood (P50 and older,
average weight 300–400 g) (n=3–7/age group). HS and litter-
mate control pups were euthanized at 1 h, 3 h, 12 h, 24 h, and
48 h after seizures (n=4–21/group). Western blots were per-
formed as previously described . Briefly, hippocampal and
cortical tissue was dissected, rapidly frozen in ethanol and stored at
–80uC until used for whole-cell protein preparations. Tissue was
homogenized in lysis buffer containing a Complete Mini Protease
Inhibitor Cocktail Tablet (Roche, Germany) and the phosphatase
inhibitors phenylmethanesulfonyl fluoride (1 mM), sodium-ortho-
vanadate (1 mM) and okadaic acid (0.1 mM) to block proteases
and phosphatases. Total protein concentrations were measured
using a Bradford protein assay (Bio-Rad, Hercules, CA), and
samples were diluted for equal amounts of protein in each lane.
Whole-cell proteins were electrophoresed onto 4–20% Tris-HCl
gels, 12% Bis-Tris gels or 3–8% Tris-Acetate gels and were
subsequently transferred to polyvinylidene difluoride membranes
(Bio-Rad). Immunoblots were incubated with primary antibodies
at 4uC overnight. Except for BDNF (1:100, Santa Cruz
Biotechnology, Santa Cruz, CA), all antibodies were purchased
from Cell Signaling (Beverly, MA): phospho-Akt (Thr308) (1:500),
Akt (1:500), phospho-Erk1/2 (Thr202/Tyr204) (1:500; 1:1000),
Erk1/2 (1:500; 1:1000), phospho-4E-BP1 (Thr37/46) (1:100;
(1:500; 1:1000), S6 (1:500; 1:1000) and mTOR (1:500). Mem-
branes were then incubated with horseradish peroxidase -
conjugated anti-rabbit IgG secondary antibodies (1:5000, Pierce,
Rockford, IL). Protein bands were visualized with enhanced
chemiluminescence (Pierce) and measured with the Image Reader
LAS-3000 system and Image Gauge v3.0 software (Fujifilm). To
control for differences in protein loading, raw values were
normalized to corresponding b-actin (Sigma-Aldrich Corp., St.
Louis, MO) within an individual age group or time point.
Normalized values were expressed as a percent or ratio of the
mean expression level of controls (either adult or normoxic control
tissue run on the same blot). Phospho-protein/total protein ratios
were calculated in a similar fashion. One-way ANOVA followed
by post hoc Bonferroni procedures were used for multiple
comparisons across development. For the post-seizure time-course
experiments two tailed t-tests were used, as each time point was
statistically its own experiment. Statistical significance was defined
Control and HS pups were sacrificed at 24 h post-seizures.
Brains were collected and processed following standard protocols
[24,39]. Briefly, 4% paraformaldehyde-fixed brains were sectioned
at 50 mm on a Microm freezing microtome and stained with
neuronal markers NeuN (1:100, Millipore, Billerica, MA) and non-
phosphorylated neurofilament (SMI 311, 1:1000, Covance,
Princeton, NJ), GABA-ergic marker GAD67 (1:1000, Millipore),
astrocytic marker GFAP (SMI 22, 1:1000, Covance) and phospho-
S6 (Ser235/236) (1:500, Cell Signaling) antibodies. Fluorescent
conjugated secondary antibodies (Alexa Fluor 568 goat anti-mouse
IgG and Oregon Green 488 goat anti-rabbit IgG, Molecular
Probes, Eugene, OR) were used at concentrations of 1:1000.
Additional sections were incubated with omission of one or both
primary antibodies to exclude false-positive labeling. Slides were
viewed with an epifluorescence microscope (Nikon Eclipse 80i).
Images were captured with a Q Imaging digital camera and the
NIS-Elements BR software 3.0 (Micro Video Instruments).
Confocal multiple plane images, collected at 1 mm intervals, were
captured using a Zeiss LSM 510 scanning laser microscope
(Germany) to acquire z-stacks.
Hippocampal Slice Preparation
Control and HS male Long-Evans rat pups previously treated
with either vehicle or rapamycin were decapitated at 48 h post-
mTOR Role in Epilepsy and Associated Autism
PLoS ONE | www.plosone.org13 May 2012 | Volume 7 | Issue 5 | e35885
seizure. Hippocampal slices were prepared and maintained as
previously described . The brains were rapidly removed, glued
to the stage of a vibrating blade vibratome (LEICA VT1000S,
Leica Microsystem Inc., Bannockburn, USA), and submerged in
ice-cooled (0–4uC), oxygenated (95% O2and 5% CO2) cutting
solution containing (mM) 210 sucrose, 2.5 KCl, 1.02 NaH2PO4,
0.5 CaCl2, 10 MgSO4, 26.19 NaHCO3, and 10 D-glucose,
pH 7.4. Coronal slices (300 mm thickness) of the middle third of
the hippocampus were sectioned in cutting solution. Slices were
then incubated in oxygenated artificial cerebrospinal fluid (ACSF)
at 35uC for 30 min and subsequently maintained at room
temperature for at least 1 h before electrophysiological recordings.
The ACSF contained (in mM): NaCl, 124; KCl, 5; NaH2PO4,
1.25; MgSO4, 1.2; NaHCO3, 26; CaCl2, 2; and glucose, 10,
pH 7.4. For recordings, the slices were transferred to a 2.5 ml
recording chamber placed in an upright Nikon Eclipse E600FN
microscope equipped with infrared and differential interference
contrast imaging devices and with a 40x water immersion
objective, and superfused (1.0–1.5 ml/min) with gassed ACSF at
room temperature (22–24uC).
Whole-cell Patch-clamp Recordings
Whole-cell voltage clamp recordings were obtained from CA1
pyramidal neurons in hippocampal brain slices by using an
Axopatch 200A amplifier (Molecular Devices, Silicon Valley, CA)
and were performed at room temperature (22–24uC). Patch
electrodes were prepared from borosilicate glass capillaries with a
Flamming/Brown micropipette puller (Model P-87, Sutter Instru-
ments Co., Novato, CA, USA). The patch-pipettes had a
resistance of 3–6 MV when filled with the internal solution that
contained (in mM): 110 Cs-methanesulfonate, 10 TEA-Cl, 4
NaCl, 2 MgCl2, 10 EGTA, 10 HEPES, 4 ATP-Mg, and 0.3 GTP,
with 5 QX-314 (N-(2,6-dimethylphenylcarbamoylmethyl)-triethy-
lammonium chloride) and creatine phosphokinase (17 unit/ml).
The pH of the pipette solution was adjusted to 7.2–7.3 with CsOH
and the osmolarity was 280–290 mOsm/kg. AMPA-receptor-
mediated spontaneous EPSCs (sEPSCs) were pharmacologically
isolated by adding picrotoxin (60 mM) and DL-AP-5 (100 mM) to
perfusion solution to block GABA and NMDA receptors,
respectively. To record AMPA receptor-mediated miniature
EPSCs (mEPSCs), TTX (1 mM) was added to the ACSF. Series
resistance was compensated 70–80% and monitored online. Only
recordings in which the series resistance was ,25 MV and less
than 15% change during the whole recording period were
included in the data analysis. Signals were filtered at 2 KHz,
digitized at 20 kHz by a Digidata 1320A interface, acquired by the
pClamp 9.2 software, and analyzed with the Clampfit 9.2 program
(Molecular Devices). sEPSC and mEPSC events at the holding
potential of -60 mV were detected automatically with a threshold
of 5–6 pA, depending on the noise level. All events were
confirmed visually on the basis of the rise and decay times. The
cumulative distributions of the sEPSCs were constructed from at
least 10 min of recording from each cell, using a bin width of 1 pA
for amplitude. Statistical significant differences were established at
p,0.05 using the Kolmogorov-Smirnov test, or ANOVA followed
by post hoc Bonferroni multiple comparison procedures.
Video-EEG Recordings with Implanted Subcutaneous
Video-EEG recordings 3–4 h in duration were acquired from
P36–38 rats previously assayed in the behavioral testing. A total of
over 280 rat-hours of video-EEG data were analyzed, which
provided a good aggregate profile of spontaneous seizure activity,
despite the short recording time in each individual animal (vehicle-
treated control: 52 h, n=16; rapamycin-treated control: 60 h,
n=15; vehicle-treated HS: 57 h, n=16; rapamycin-treated HS:
115 h, n=28). Teflon-coated silver/silver chloride subdermal wire
electrodes were implanted subcutaneously in rats lightly anesthe-
tized with 2–4% isoflurane. The rats were connected to a low
torque commutator (Dragonfly Inc, Ridgeley, VA, USA) and
connector assembly (John Ives, Manitock, ON, Canada) and were
able to move freely around the enclosure . Seizures were
defined by the appearance of sustained polyspike activity,
significantly different than background rhythm, longer than
3 sec, and associated with a behavioral correlate on video (sudden
behavioral arrest, staring episodes, head-jerking, and facial
automatisms). Average seizure frequency was calculated per hour
of video-EEG recording, and seizure duration was measured as the
time from first spike to last spike. Statistical significant differences
were established at p,0.05 using non-parametric Kruskal-Wallis
and Dunn’s multiple comparison tests.
Control and HS rat pups treated with rapamycin or vehicle
were tested as adults in the following three assays: 1) open field
locomotion, 2) three-chamber social choice test and 3) olfactory
habituation/dishabituation. Behavioral experiments were con-
ducted in a standard behavioral testing room during the light
phase (0700–1900 h) of a 12 h light-dark cycle . All
investigators were blind to subject treatment groups. Statistically
significant differences were established at p,0.05 using ANOVA
followed by post hoc Bonferroni tests or non-parametric Kruskal-
Open field locomotion: The day before testing a P29–30 rat was
habituated for three hours in a 100x100x35 cm opaque Plexiglas
chamber (Stoelting, Co., Wood Dale, IL, USA). The following
day, each P30–31 rat was placed in the center of the open field,
illuminated at 120–129 lux, for 30 minutes and was tracked using
ANY-maze Video Tracking System (Stoelting, Co.). Data on
distance traveled and time spent in the center 80x80 cm area was
automatically calculated by the software. The chamber was
cleaned with ClidoxH-S, (PRL Pharmacal Research Laboratories,
Inc., Naugatuck, CT, 1:18:1) and 70% ethanol and was allowed to
dry completely between test sessions .
Three-chamber social choice test: On the day of testing, an
experimental P33–34 rat was individually housed in the behavior
room 30 minutes prior to testing. The rat was placed in the center
chamber of a 100x100x35 cm clear Plexiglas three-chamber maze
(Stoelting, Co.), illuminated at 35–55 lux, and was allowed to
habituate for ten minutes. The doors to both side chambers were
then opened for the rat to explore all three chambers for ten
minutes. The sociability test commenced with a stranger rat social
stimulus (novel rat) confined within a wire cage in one side
chamber and a nonsocial stimulus of an identical empty wire cage
in the opposite side chamber. The experimental rat was then
allowed to explore all three chambers for ten minutes. The social
novelty test began immediately afterwards and the experimental
rat was allowed to approach and sniff the previously investigated
social stimulus (familiar rat) and a novel social stimulus of a second
stranger rat (novel rat) in the side chambers for ten minutes.
Transitions between chambers and time spent in each chamber
were automatically recorded using the ANY-maze System. Time
spent sniffing the empty cage, novel rat, and familiar rat were each
recorded by an investigator in ANY-maze based on the duration of
a keypress on a computer keyboard. The stranger rats were age-
and gender-matched to the experimental rats, and were habitu-
ated to the wire cages for at least an hour on the day prior to
testing [46,90]. The three-chamber maze and wire cages were
mTOR Role in Epilepsy and Associated Autism
PLoS ONE | www.plosone.org 14May 2012 | Volume 7 | Issue 5 | e35885
cleaned with ClidoxH-S (1:18:1) and 70% ethanol and were
allowed to dry completely between test subjects.
Olfactory habituation/dishabituation: P36–38 rats were acclimated to
testing conditions in a new, empty cage (45x24x20 cm) with clean
bedding. A sterile cotton swab (15 cm, Puritan Medical Products
Company LLC, Guilford, MN, USA) was dipped in water,
inserted though the lid of the cage and stabilized with a binder clip
so that the moistened cotton tip was positioned 9 cm above the
floor of the cage. The rat was observed for two minutes and the
amount of time spent sniffing the cotton swab was quantified with
a stopwatch. Sniffing was defined as the rat positioning its nose
within 1 cm of the cotton tip. This was repeated for two further
two-minute trials with fresh water dipped swabs. Consistent with
this method, each rat was exposed in triplicate to two additional
nonsocial and two social odors on cotton swabs in the following
order: 1) water, 2) almond extract, 3) imitation banana extract, 4)
social odor #1 (soiled rat cage that contained unfamiliar male
rats), 5) social odor #2 (soiled rat cage that contained different
unfamiliar male rats). The almond and banana extracts (McCor-
mick & Co., Inc. Hunt Valley, MD, USA; 1:100 in tap water) were
diluted freshly the morning of testing. The social odors were
prepared by wiping the cotton swab along the perimeter and down
the center of a rat cage that contained soiled bedding and
unfamiliar male rats . Video of each experiment was recorded
and reviewed by two independent observers.
The authors would like to thank Ms. Kathia Cordero for
technical assistance. We would like to thank Dr. Jacquelyn
Crawley for her advice regarding socialization testing in rats.
Conceived and designed the experiments: DT HS XZ FJ. Performed the
experiments: DT HS XZ EF MJ PK VL AJ. Analyzed the data: DT HS
XZ EF MJ PK VL AJ. Contributed reagents/materials/analysis tools: DT
HS XZ FJ. Wrote the paper: DT HS XZ MJ PK FJ.
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