Dysregulation of the Norepinephrine Transporter
Sustains Cortical Hypodopaminergia and Schizophrenia-
Like Behaviors in Neuronal Rictor Null Mice
Michael A. Siuta1., Sabrina D. Robertson1., Heidi Kocalis2, Christine Saunders3, Paul J. Gresch3, Vivek
Khatri4, Chiyo Shiota2,5, J. Philip Kennedy3, Craig W. Lindsley3, Lynette C. Daws6, Daniel B. Polley4,7,
Jeremy Veenstra-Vanderweele1,3,4,8, Gregg D. Stanwood3,4, Mark A. Magnuson2, Kevin D.
Niswender9,2,10*", Aurelio Galli1,2*"
1Center for Molecular Neuroscience, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 2Department of Molecular Physiology and
Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 3Department of Pharmacology, Vanderbilt University School of
Medicine, Nashville, Tennessee, United States of America, 4Vanderbilt Kennedy Center for Research on Human Development, Vanderbilt University School of Medicine,
Nashville, Tennessee, United States of America, 5Department of Surgery, Children’s Hospital of Pittsburgh of UPMC, and University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania, United States of America, 6Department of Pharmacology, University of Texas Health Science Center, San Antonio, Texas, United States of
America, 7Department of Hearing and Speech Sciences, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 8Department of
Psychiatry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America, 9Tennessee Valley Healthcare System, Nashville, Tennessee, United
States of America, 10Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America
The mammalian target of rapamycin (mTOR) complex 2 (mTORC2) is a multimeric signaling unit that phosphorylates protein
kinase B/Akt following hormonal and growth factor stimulation. Defective Akt phosphorylation at the mTORC2-catalyzed
Ser473 site has been linked to schizophrenia. While human imaging and animal studies implicate a fundamental role for Akt
signaling in prefrontal dopaminergic networks, the molecular mechanisms linking Akt phosphorylation to specific
schizophrenia-related neurotransmission abnormalities have not yet been described. Importantly, current understanding of
schizophrenia suggests that cortical decreases in DA neurotransmission and content, defined here as cortical
hypodopaminergia, contribute to both the cognitive deficits and the negative symptoms characteristic of this disorder.
We sought to identify a mechanism linking aberrant Akt signaling to these hallmarks of schizophrenia. We used conditional
gene targeting in mice to eliminate the mTORC2 regulatory protein rictor in neurons, leading to impairments in neuronal
Akt Ser473 phosphorylation. Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated
behavior. In addition, they show reduced prefrontal dopamine (DA) content, elevated cortical norepinephrine (NE),
unaltered cortical serotonin (5-HT), and enhanced expression of the NE transporter (NET). In the cortex, NET takes up both
extracellular NE and DA. Thus, we propose that amplified NET function in rictor KO mice enhances accumulation of both NE
and DA within the noradrenergic neuron. This phenomenon leads to conversion of DA to NE and ultimately supports both
increased NE tissue content as well as a decrease in DA. In support of this hypothesis, NET blockade in rictor KO mice
reversed cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in
NET expression, which we show is ultimately influenced by Akt phosphorylation status. These data illuminate a molecular
link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific
mechanism for cortical hypodopaminergia and hypofunction. Additionally, our findings identify Akt as a novel modulator of
monoamine homeostasis in the cortex.
Citation: Siuta MA, Robertson SD, Kocalis H, Saunders C, Gresch PJ, et al. (2010) Dysregulation of the Norepinephrine Transporter Sustains Cortical
Hypodopaminergia and Schizophrenia-Like Behaviors in Neuronal Rictor Null Mice. PLoS Biol 8(6): e1000393. doi:10.1371/journal.pbio.1000393
Academic Editor: Eric Nestler, Mount Sinai School of Medicine, United States of America
Received December 11, 2009; Accepted April 29, 2010; Published June 8, 2010
Copyright: ? 2010 Siuta 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 is supported by National Institutes of Health grants DA14684 (to AG and LCD), MH058921 (to AG), DK085712 (to KDN and AG), MH084755 (to
SDR), DK069927 (to KDN), and partially by DC009488 (to DBP), DK064857 (to KDN), resources of the Tennessee Valley Healthcare System, and by the Diabetes
Research and Training Center (DRTC; DK20593, to AG and KDN). This work is partially supported by resources of the Vanderbilt University Silvio O. Conte Center
for Neuroscience Research (MH078028, to PJG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: COMT, catechol-O-methyltransferase; DA, dopamine; KO, knockout; mTOR, mammalian target of rapamycin; mTORC2, mTOR complex 2;
mTORC1, mTOR complex 1; NE, norepinephrine; NET, norepinephrine transporter; PPI, prepulse inhibition; SN, substantia nigra; SPL, sound pressure level; TH,
tyrosine hydroxylase; VTA, ventral tegmental area.
* E-mail: firstname.lastname@example.org (AG); email@example.com (KDN)
. These authors contributed equally to this work.
" These authors also contributed equally to this work.
PLoS Biology | www.plosbiology.org1 June 2010 | Volume 8 | Issue 6 | e1000393
(mTORC2) is one of two highly conserved protein kinases that
are critical regulators of cell growth and metabolism. mTOR
complex 1 (mTORC1) and mTORC2 are functionally distinct
multiprotein complexes that are defined by their subunit
composition, rapamycin sensitivity, and substrate selectivity.
Raptor, mLST8, PRAS40, and mTOR comprise the rapamycin
sensitive mTORC1 while the rapamycin insensitive mTORC2
contains rictor, mSIN1, mLST8, and mTOR. Two key substrates
of mTORC1 are S6K and 4E-BP, which are important regulators
of translation, while protein kinase B, also known as Akt, is the
primary substrate of mTORC2 . Specifically, mTORC2 is the
kinase responsible for phosphorylation of Akt at serine residue 473,
one of two key phosphorylation sites . Akt is an extensively
studied kinase that has been implicated in numerous disorders
such as diabetes, obesity, cancer, and mental disorders such as
schizophrenia . Post-mortem, imaging, and genetic association
studies in humans [3,4,5] reveal that Akt deficiencies are
associated with schizophrenia. Genetic studies in rodents further
corroborate the relationship between dysregulation in Akt
signaling and disruptions in dopamine (DA)-associated behaviors
linked to schizophrenia [4,6].
Putative evidence for a role of defects in mTORC2 signaling in
mental illnesses preceded the discovery of the mTORC2 complex
itself. Indeed, lithium, used to treat bipolar disorder, stimulates
phosphorylation of Akt at Ser473, the mTORC2 phosphorylation
site . The link between mTORC2 signaling deficits and mental
illness has been strengthened by seminal work demonstrating that
certain antidepressants , along with both typical  and
atypical antipsychotics , increase Akt Ser473 phosphorylation.
Furthermore, findings of diminished Ser473 phosphorylation and/
or activity in post-mortem brains of patients with schizophrenia
 and depression  potentially fortify the association between
dysregulation of mTORC2-Akt signaling and development of
psychiatric illnesses, although these findings may be confounded
by perimortem artifacts . Recent observations that blunted
Ser473 phosphorylation occurs in lymphocytes derived from
patients with schizophrenia and psychosis-prone normal individ-
uals  also support the plausibility that mTORC2-Akt deficits
are involved in schizophrenia.
While human imaging and animal studies implicate a
fundamental role for Akt signaling in prefrontal DA networks,
the molecular mechanisms linking mTORC2/Akt to schizophre-
nia-related neurotransmission abnormalities have been elusive
[3,6]. Importantly, models of schizophrenia suggest that cortical
deficits in DA neurotransmission and content, defined here as
cortical hypodopaminergia, contribute to both the cognitive
deficits and the negative symptoms characteristic of this disorder
. Consistent with this hypothesis, imaging studies reveal that
genetic variation associated with low activity Akt alleles interact
epistatically with catechol-O-methyltransferase (COMT), a gene
responsible for degradation of prefrontal synaptic DA. Together,
these interactions ultimately affect the fidelity of prefrontal
networks in humans  by decreasing DA availability at prefrontal
synapses . Thus, a compelling hypothesis in schizophrenia is
that impaired mTORC2/Akt signaling triggers aberrant regula-
tion of DA homeostasis.
Termination of DA signaling at prefrontal synapses involves two
mechanisms: degradation via enzymes including COMT, and
clearance via the norepinephrine (NE) transporter (NET)
[16,17,18], which takes up both major brain catecholamines,
DA and NE [17,19]. Interestingly, insulin administration, which
stimulates mTORC2/Akt signaling, decreases NET transcription
in brain, while hypoinsulinemia and decreased mTORC2/Akt
signaling increases NET transcription . Therefore, we
hypothesized that dysregulation of mTORC2/Akt signaling may
provide a mechanistic link to cortical hypodopaminergia.
Specifically, we propose that reduced Akt activity mediates
increased NET expression and increased DA clearance by
noradrenergic neurons in cortex, a novel molecular mechanism
that explains how Akt dysfunction contributes to a reduction in
To test this hypothesis, we have generated an animal model in
which mTORC2/Akt signaling down-regulation is achieved by
neuronal deletion of a key mTORC2 regulatory subunit, rictor.
We used a Cre-lox strategy to restrict the genetic deletion to
neurons and bypass embryonic lethality associated with whole
body deletion . The goal of the present study is to test how
alteration in Akt signaling affects DA homeostasis in the prefrontal
Rictor Deletion Attenuates Akt Ser473 Phosphorylation
Akt deficiency is mechanistically linked to prefrontal cortex
abnormalities and schizophrenia-linked phenotypes in several
mouse models , although a clear molecular mechanism for how
Akt regulates cortical function remains elusive. Here, we
investigate the dopaminergic consequences of abolishing Akt
phosphorylation at Ser473 in neurons by utilizing the Cre/LoxP
system to delete rictor specifically in neurons. Mice were
engineered with a floxed rictor allele, as previously described
, and crossed with neuron-specific nestin gene (NES mice) Cre
driver line. Validating our approach, rictor knockout (KO) mice
lack rictor mRNA expression and rictor protein expression in a
gene-dosage dependent manner within the brain and cortex
(Figure S1; p,0.01 and p,0.05, respectively, by one-way
Schizophrenia is a disorder caused by multiple genetic and
environmental variables. Despite the disease’s heteroge-
neous causes, current hypotheses suggest that dysfunc-
tion of dopamine signaling in the brain is one of the final
common pathways involved. One gene that may be
involved encodes the protein kinase Akt, which is
regulated by hormones, growth factors, and neurotrans-
mitter receptors. In this study, we examined the potential
molecular mechanisms linking Akt dysregulation to cortical
hypodopaminergia, and ultimately to the pathology of
schizophrenia. Using transgenic technology, we generated
a mouse model with defective neuronal Akt signaling.
Neurochemical and behavioral phenotypes associated
with schizophrenia include decreases in prefrontal dopa-
mine signaling and deficits in sensorimotor gating, two
phenotypes we observed in our transgenic animals.
Further, we observed that impaired cortical Akt activity
significantly enhanced norepinephrine transporter func-
tion. Interestingly, we found that by blocking this
transporter, we could reverse the cortical hypodopami-
nergia and behavioral deficits seen in our transgenic mice.
The norepinephrine transporter is a presynaptic mem-
brane protein that is critical for maintaining both
norepinephrine and cortical dopamine homeostasis. Taken
together, this work supports the potential for targeting
both Akt and the norepinephrine transporter for treating
dopamine-related mood disorders.
Akt Deficits Sustain Cortical Hypodopaminergia
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ANOVA followed by Dunnett’s test). Importantly for the current
hypothesis, neuronal rictor deletion abolishes Akt phosphorylation
at Ser473 within the cortex of rictor KO mice (Figure 1A;
***p,0.001 by one-way ANOVA Dunnett’s test) compared to
FLOX (floxed allele(s) in the absence of Cre), NES (Cre allele in
the absence of a FLOX allele), and heterozygous rictor neuronal
KO mice (HET). Phosphorylation of Akt at Thr308 (Figure 1B)
and total levels of Akt (Figure 1C) within the cortex are not
different among the genotypes, allowing a direct evaluation of the
effects of Ser473 phosphorylation. Similarly, rictor deletion also
abolishes Akt phosphorylation at Ser473 in other brain regions,
such as the substantia nigra (SN)/ventral tegmental area (VTA) of
rictor KO mice, while Thr308 phosphorylation and total levels
of Akt are unaltered (data were normalized to control (FLOX)
and reported as mean6s.e.m., p values by Student’s t test; pAkt
Ser473 FLOX=100613%, KO=661%, p,0.001; pAkt Thr308
FLOX=100613%, KO=101610%, p=0.96). Furthermore,
total protein levels of mTOR in the cortex are not altered by
neuron-specific rictor KO (data were normalized to control
(FLOX) and reported as mean6s.e.m.; FLOX=10069%,
NES=130612%, HET=118612%, KO=116612%; p=0.32
by one-way ANOVA followed by Dunnett’s test).
Neuronal Rictor KO Mice Display Sensorimotor Gating
Prepulse inhibition (PPI) behavior has long been identified as a
the direct parallels in expression between rodent and human subjects.
While PPI deficits are present in psychiatric disorders other than
schizophrenia, they have a clear heritable component in schizophrenic
families and these deficits can be attenuated by antipsychotic drug
administration. Furthermore, PPI deficits are also linked to the
hypofunction of corticostriatal forebrain circuits that is characteristic of
schizophrenia . The PPI behavioral assay measures the degree to
when immediately preceded by a non-startling ‘‘prepulse’’ sound. As
such, it assays the degree to which a brief sensory trace can rapidly
modify a subsequent motoric response, thereby representing a
straightforward approach towards quantifying sensorimotor dysregu-
lation, which is generally regarded as an endophenotype of
schizophrenia. Since evidence suggests a role for Akt in schizophrenia,
and rictor KO mice demonstrate profound deficits in Akt phosphor-
ylation, we tested whether rictor KO mice display impaired PPI
relative to FLOX control mice. No differences in startle responses
elicited by a 94 dB sound pressure level (SPL) noise burst were
observed, suggesting that hearing and gross motor function were
similar between groups (Figure 2A; Student’s t test; p=0.44). By
contrast, analysis of PPI behavior revealed clear differences between
genotypes; startle reflex amplitude was inhibited at all prepulse
intensities in FLOX control mice, with the amount of PPI increasing
monotonically from 40 to 75 dB SPL (Figure 2B). In rictor KO mice,
prepulse sound levels between 40 and 55 dB did not appreciably
attenuate the startle reflex. PPI was not observed in rictor KO mice
until prepulse levels .55 dB, albeit at a weaker level in comparison to
FLOX mice. These differences gave rise to a significant reduction of
PPI across prepulse sound levels (Figure 2B; *p,0.05 by ANOVA).
This PPI deficit can also be expressed as a significant decrease in
average PPI across all sound levels in rictor KO mice (Figure 2C;
Student’s t test, *p,0.05). Thus neuronal rictor deletion, with loss of
Akt Ser473 phosphorylation, impairs the forebrain circuits critically
involved with sensorimotor integration. Given the strong evidence for
PPI deficits in schizophrenic patients, we hypothesize that this mouse
model has the potential to lend novel insight into the molecular
mechanisms by which Akt deficits contribute to the schizophrenic
Rictor KO Mice Display Hypodopaminergia in Rostral
For almost 50 years, schizophrenia research has centered on
dopaminergic signaling as a crucial component of the etiology of
the disease [23,24,25]. In particular, the original ‘‘DA hypothesis’’
heavily supported the notion of excessive DA neurotransmission
and DA content, defined here as hyperdopaminergia, within the
brain [26,27]. Subsequent revision of the hypothesis, however, has
Figure 1. Neuronal rictor deletion specifically abolishes Akt
phosphorylation at Ser473 in the cortex. (A) Phosphorylation of
Akt on residue Ser473 in the cortex. (B) Phosphorylation of Akt at
Thr308 and (C) total Akt in the cortex are similar for all genotypes.
Shown are mean6s.e.m of optical densities as a percentage of FLOX
control mice. Genotypes shown include animals expressing only nestin-
CRE (NES), mice expressing two copies of the ‘‘floxed’’ rictor allele
(FLOX) only, heterozygous mice which express nestin-CRE and a single
copy of the ‘‘floxed’’ rictor allele (HET), and knockout mice expressing
both nestin-CRE and two copies of the ‘‘floxed’’ rictor allele (KO). Total
cortical protein extract was loaded in each lane. Representative
immunoblots are shown, as probed with antibodies to phosphorylated
Akt at Ser473 (A) Thr308 (B), total Akt (C), and actin to serve as a loading
control. Samples n=9–16. ***p,0.001 one-way ANOVA.
Akt Deficits Sustain Cortical Hypodopaminergia
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transformed thinking from a global hyperdopaminergia to a
regional hyperdopaminergia within the striatum and dopaminer-
gic hypofunction within the cortex [14,28,29]. While this
conceptualization is an oversimplification of a highly complex
disorder, we utilized this hypothesis to hone in on molecular
mechanisms that contribute to cortical hypodopaminergia.
Furthermore, previous studies have linked pre-frontal DA deficits
with PPI deficits in animal models, and perhaps the PPI deficits
observed in the rictor KO mice could be partially explained by
alterations in cortical DA content [30,31]. Thus, we investigated
steady state levels of DA, serotonin (5-HT), and NE in a region of
mouse brain that is roughly analogous to the human prefrontal
cortex and contains areas such as the intralimbic, prelimbic, and
anterior cingulate cortex. Interestingly, HPLC with electrochem-
ical detection of DA, NE, and 5-HT in the PFC of rictor KO mice
revealed striking alterations in the DA and NE tissue content of
these animals, while 5-HT levels remained unchanged (Figure 3).
While both DA and NE levels are significantly different in rictor
KO mice, they change in opposite directions; NE tissue content is
significantly increased (Figure 3A; **p,0.01 by one-way ANOVA
followed by Dunnett’s test) while DA levels are significantly
decreased (Figure 3B; Student’s t test, *p,0.05). In addition, NES
mice show similar PFC DA content levels (6.460.2 ng/mg
protein) as FLOX mice. Thus, rictor KO mice display a key
feature of the ‘‘dopamine hypothesis’’ of schizophrenia, namely
hypodopaminergia in the rostral cortex, which may explain the
sensorimotor gating deficits described earlier. Importantly, 5-HT
levels are unaltered in the cortex (Figure 3C; Student’s t test,
p=0.26) indicating that rictor deletion does not simply result in
global monoaminergic alterations but rather specific changes in
the dopaminergic and noradrenergic systems. In addition,
extracellular levels of DA were determined in the PFC of rictor
KO mice by microdialysis. Under basal conditions, extracellular
DA is not significantly different in rictor KO mice compared to
FLOX controls (data are reported as pg of DA/mL, mean6s.e.m.;
FLOX=0.5460.15, KO=0.7660.18, n=4–5; p=0.35 by Stu-
dent’s t test). While basal extracellular levels of DA are unaltered in
rictor KO mice, these animals do display significant deficits in DA
tissue content, suggesting that maintenance of DA homeostasis is
While prefrontal hypodopaminergia has been linked to PPI
deficits, other studies clearly demonstrate a link between striatal
hyperdopaminergia and PPI deficits [32,33]. Thus, we sought to
determine if rictor deletion increases tissue levels of DA in the
Figure 2. Neuronal rictor deletion results in sensorimotor gating deficits as assayed by PPI. (A) No difference in startle reflex elicited by a
94 decibel (dB) sound pressure level (SPL) noise was observed between rictor KO and littermate FLOX control mice. (B) The percentage of PPI was
reduced significantly across the entire dB range in rictor KO mice. (C) The average PPI across the entire dB range reveals a significant deficit in PPI in
rictor KO mice; n=6–7 animals. *p,0.05 Student’s t test.
Figure 3. Monoamine content in the rostral cortex is significantly altered in rictor KO mice. Tissue content of (A) NE, (B) DA, and (C)
serotonin (5-HT) in rostral cortical homogenates. Results are presented as mean6s.e.m ng/mg of protein, n=4–10. *p,0.05; **p,0.01 Student’s t
Akt Deficits Sustain Cortical Hypodopaminergia
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striatum or in projecting DA neurons from the SN and VTA. DA
levels in the SN/VTA are not altered in rictor KO mice (data are
reported as ng of DA/mg protein, mean6s.e.m.; FLOX=4.460.5,
KO=4.860.4, p=0.60 by Student’s t test). Importantly, similar to
the cortex, DA tissue content in the striatum of rictor KO mice is
significantly decreased (data are reported as ng of DA/mg protein,
mean6s.e.m.; FLOX=101.267.7, KO=80.163.4, *p,0.05 by
Student’s t test). Thus, our data indicate that the PPI deficits
observed in rictor KO mice are likely to arise from impairments in
Rictor Deletion Increases NET Expression and Function
It is intriguing that DA content is decreased while NE content is
signaling induced by hypoinsulinemia have been shown to increase
NET transcription . Moreover, early studies and unpublished data
from our laboratory implicate deficits in Akt signaling with not only
increases in NET transcription but also acute increases in NET cell
surface expression (i.e. intact Akt signaling decreases NET availability
at the plasma membrane) [20,34,35,36]. Thus, we predict that altered
DA homeostasis in rictor KO mice is due to changes in NET cell
surface expression mediated by impaired Akt phosphorylation.
Importantly, unlike other brain regions where DAT is the primary
mechanism for removing DA from the synapse, in cortex DAT
contributes relatively little and NET performs the majority of DA
clearance. Indeed NET has a higher affinity for DA than NE itself, but
DAcan alsobedegraded in the synapse byCOMT [16,17]. Giventhe
pivotal role of rictor in Akt regulation and the role of Akt signaling in
determining NET availability, we hypothesize thatrictorKOmice will
DA levels seen in the rostral cortex.
As hypothesized, total cortical NET protein is increased
approximately 2-fold in rictor KO mice compared to all other
genotypes (Figure 4A; ***p,0.0001 by one-way ANOVA
followed by Dunnett’s test). Furthermore, biotinylation assays
reveal that cell surface levels of NET are also significantly
increased (Figure 4B; Student’s t test, ***p,0.0001). Tyrosine
hydroxylase (TH), a cytosolic protein, was detected exclusively in
the total protein fraction but not in the surface fraction,
indicating that the biotinylated fraction represents exclusively
cell surface proteins. Finally, the striking enhancement in surface
NET detected in rictor KO mice results in a significant increase
in NET function as assayed by cortical synaptosomal NE uptake
(Figure 4C; Student’s t test, *p,0.05). The nearly 2-fold increase
in cortical synaptosomal NE uptake was also observed for DA
(Figure 4C; Student’s t test, *p,0.05) indicating that rictor
deletion increases DA clearance by NET in noradrenergic
neurons and as a consequence reduces cortical DA content.
Furthermore, DA content is not decreased due to increased
degradation since COMT levels were not different in cortex
compared to FLOX control mice (data were normalized to
FLOX=10068%, NES=78611%, HET=130624%, KO=
80611%; p=0.10 by one-way ANOVA followed by Dunnett’s
test). Thus, the increase in NET expression and function within
the cortex of rictor KO mice has the potential to mechanistically
explain both the increased NE tissue content and decreased
cortical DA tissue content described earlier (Figure 3). Interest-
ingly, we did not find a significant difference in serotonin
transporter expression within the PFC of the rictor KO mice, as
measured by citalopram binding (unpublished data).
While our data indicate that global neuronal mTORC2
dysfunction enhances NET function and induces cortical
hypodopaminergia, we sought to demonstrate more specifically
that these alterations could arise from downregulation of cortical
Akt activity. Thus, we utilized the isoform specific Akt1 inhibitor
[37,38,39] in cortical slices to show that Akt inhibition is capable
of directly determining NET surface availability (Figure S2).
Surface levels of NET are significantly enhanced in biotinylated
cortical slices treated with the Akt1 inhibitor (Figure S2A;
Figure 4. Neuronal rictor deletion results in increased NET
expression and function. (A) NET protein levels in the cortex.
Mean6s.e.m optical densities are shown as a percentage of FLOX
control mice. Representative immunoblots are shown, as probed with
antibodies to NET, and actin (loading control); n=10. (B) Levels of
surface NET as measured from the biotinylated fraction of cortical slices.
Mean6s.e.m optical density is shown as a percentage of FLOX control
mice. Representative immunoblots are shown, as probed with
antibodies to NET, Na+/K+ATPase to serve as plasma membrane/
loading control (n=3–5), and TH which is absent in the biotinylated
fractions since it is a cytosolic protein. (C) [3H]NE and [3H]DA uptake into
cortical synaptosomes of FLOX and KO mice. Mean6s.e.m uptake is
shown as a percentage of uptake in FLOX control mice; n=12–18.
*p,0.05; ***p,0.001 Student’s t test.
Akt Deficits Sustain Cortical Hypodopaminergia
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*p,0.05 by Student’s t test). Importantly, the levels of Akt Ser473
phosphorylation are substantially diminished in samples of these
inhibitor treated slices (Figure S2B; ***p,0.0001). Together,
these data support the notion that Akt stimulated regulation of
the transporter occurs not only at the level of transcription, as is
seen in rictor KO mice, but also at the level of transporter
trafficking. Furthermore, the ability of Akt inhibition to enhance
NET surface expression in cortical slices indicates that all the
molecular machinery necessary for this rictor/Akt regulation of
NET is intact within the PFC and thus is consistent with our
hypothesis that altered cortical monoamine homeostasis via
aberrant NET regulation underlies PPI deficits in rictor KO
We hypothesize that amplified NET function in rictor KO mice
enhances the accumulation of both NE and DA within the
noradrenergic neuron leading to conversion of DA to NE and
ultimately supporting both increased NE tissue content and a state
of hypodopaminergia. Such a mechanism within the prefrontal
cortex provides an elegant molecular mechanism linking Akt
hypophosphorylation to both cortical hypodopaminergia and PPI
deficits, two key hallmarks of schizophrenia.
Midbrain Dopaminergic Neurons and Cortical
Monoaminergic Projections Are Unaltered in Rictor KO
Considering the widespread function of Akt and its role in cell
growth and proliferation, we next sought to demonstrate that the
changes in DA and NE levels within the cortex were specifically due
to increased NET expression rather than global changes in the
number or projections of dopaminergic and noradrenergic neurons.
While rictor KO mice do display a gross reduction in brain size,
similar to what is seen for Akt3 deficient mice (brain weight
normalized to body weight; FLOX 2.4960.18% compared to KO
1.6360.10%;p,0.0005 byone-wayANOVA followed by Dunnett’s
test), coronal brain sections stained for TH revealed no significant
alterations in dopaminergic cell number within the VTA or SN
(Figure 5A and 5C–5D; VTA p=0.82 by one-way ANOVA, SN
p=0.53 by one-way ANOVA). Furthermore, TH staining of
dopaminergic and noradrenergic projections within the cortex do
not reveal any gross alterations among the groups (Figure 5B). The
immunostaining was confirmed with Western blot analysis of total
corticalTHproteinlevels(Figure5E;p=0.20 by one-way ANOVA).
Other markers of dopaminergic neurons in the cortex were not
significantly altered in the rictor KO mice such as total levels of D2
DAreceptors (data werenormalizedto control(FLOX)and reported
as mean6s.e.m.; FLOX=10065%, NES=95613%, HET=
9669%, KO=108618%; p=0.85 by one-way ANOVA followed
by Dunnett’s test). These data demonstrate that the DA and NE
systemsinthe rictor KO mice arenot globally altered. Therefore, we
primarily accounted for by the specific enhancement of NET
expression in noradrenergic neurons.
NET Blockade Reverses the Sensorimotor Gating Deficits
and Hypodopaminergia in Rictor KO Mice
We hypothesize that aberrant Akt phosphorylation in rictor
KO mice results in enhancement of NET function within the
cortex, resulting in changes in both DA and NE homeostasis. If
this model is valid, then NET inhibition should reverse both PPI
behavioral deficits and cortical deficits in DA tissue content. In
order to test this hypothesis, we treated rictor KO mice with
nisoxetine (NET specific blocker) or saline. Prior to treatment,
both groups of rictor KO mice demonstrated comparable startle
reflex amplitude similar to Figure 2B (unpublished data). The
average PPI across all sound levels also were not different
between the two rictor KO groups prior to treatment (Figure 6A;
Student’s t test, p=0.95). Following the initial PPI trial, the rictor
KO mice received i.p. injections of either saline or nisoxetine
(30 mg/kg) and 30 min later began a second PPI trial. Nisoxetine
reversed rictor KO PPI deficits compared to saline treated
animals with a ,5-fold increase of average PPI (Figure 6B
Student’s t test, ***p,0.0001). Similar to FLOX control mice
(Figure 2B), nisoxetine treated rictor KO mice display startle
reflex amplitudes that are inhibited by all prepulse intensities,
with the amount of PPI increasing monotonically from 40–75 dB
SPL (Figure 6C). Importantly, saline treated animals exhibited
PPI at prepulse levels.55 dB, albeit at a weaker level as
comparedto nisoxetine treated
Figure 2B. Thus, nisoxetine treatment significantly reverses the
PPI deficits observed in rictor KO mice (Figure 6C; p,0.0001 by
two-way ANOVA) providing support for our model of mecha-
nistic linkage between Akt, NET, and cortical DA and NE
In addition to nisoxetine treatment, we sought to determine if
traditional antipsychotics were capable of reversing the PPI deficits
displayed in rictor KO mice. Unlike nisoxetine, acute clozapine
treatment (i.p. 3 mg/kg for 30 min prior to PPI) did not rescue
PPI deficits in rictor KO mice (data are reported as average
percent PPI mean6s.e.m. as in Figure 2C; saline-KO 24.368.6%,
clozapine-KO 20.566.4%; Student’s t test, p=0.73). Interestingly,
previous studies have shown that antipsychotic treatment enhances
activity and phosphorylation of Akt at Ser473, and this increase is
hypothesized to be important for the efficacy of such drugs
[4,40,41]. Consistently, FLOX mice subjected to the same
clozapine treatment as described above show significantly
enhanced cortical phosphorylation of Akt at Ser473 (data are
normalized to total levels of Akt and expressed as percent of
control mean6s.e.m.; saline-FLOX 100620%, clozapine-FLOX
215650%; *p,0.05 by one-way ANOVA). Importantly, the same
treatment does not alter Ser473 phosphorylation of Akt in rictor
KO mice (data are normalized to total levels of Akt and expressed
as percent of control (saline treated FLOX) mean6s.e.m.; saline-
KO 561%, clozapine-KO 562%; p.0.05 by one-way ANOVA).
Thus, the inability of clozapine to rescue PPI deficits in rictor KO
mice may be partially due to the genetic neuronal deletion of
rictor, which abolishes the ability of clozapine to enhance Akt
Ser473 phosphorylation in these animals. These data are
consistent with the hypothesis that Akt deficits play an important
role in the etiology of schizophrenia and that perhaps some degree
of antipsychotic efficacy is due to their ability to enhance Akt
The success of nisoxetine treatment in rescuing the PPI deficits
in rictor KO mice was consistent with our model and led us next
to determine if NET inhibition could normalize prefrontal cortex
DA tissue content. In this experiment, rictor KO and FLOX
control mice received either nisoxetine (30 mg/kg) or saline
injections i.p. daily for 8 consecutive days and were euthanized
30 min following their last i.p. injection. DA levels were
measured in the rostral cortex by electrochemical detection.
Similar to the results seen in Figure 3, saline treated rictor KO
mice had reduced DA tissue content levels in comparison to
saline treated controls (Figure 6D; Student’s t test, *p,0.04) and
8 d of nisoxetine treatment significantly enhanced DA content
(Figure 6D; Student’s t test, *p,0.03) while the same treatment in
FLOX control mice did not have a significant impact on DA
levels (Figure 6D; Student’s t test, p=0.21). Moreover, chronic
nisoxetine treatment does not significantly alter cortical NE tissue
animals, consistent with
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content in either FLOX control or rictor KO mice (data are
reported as ng of NE/mg protein, mean6s.e.m.; FLOX-
saline=5.760.4, FLOX-nisoxetine=6.560.5, Student’s t test,
p=0.29; KO-saline=8.360.7, KO-nisoxetine=8.660.4, Stu-
dent’s t test, p=0.74). These data further support the model that
enhanced NET expression alters DA content in the rictor KO
mice and indicate that hypodopaminergia (and PPI deficits) can
be rectified with NET specific inhibition via nisoxetine. The
observations that chronic nisoxetine administration rescues DA
content and acute treatment reverses impaired PPI are consistent
with a model whereby impairment of PPI is due to decreased
intra-synaptic DA in rictor KO mice. Our data suggest that
reduced intra-synaptic DA content could arise from enhanced
clearance of DA via NET.
NET specific inhibition with nisoxetine has been utilized to
rectify PPI deficits in some animal models of schizophrenia .
Only a small number of studies, however, have investigated the
utility of NET specific inhibition in schizophrenic patients with a
particular focus on symptoms related to cortical hypofunction,
and the efficacy of such treatment is still controversial .
Figure 5. TH staining and expression in the midbrain and cortex is similar in NES, FLOX, and KO mice. TH immunoreactivity in the (A)
midbrain substantia nigra (SN) and ventral tegmental area (VTA) and the (B) cortex. Scale bars=50 mm. Coronal brain sections were stained with TH
antibody and cell counts of TH+cells were taken. Cell counts are similar in NES, FLOX, and KO matched mice in both the (C) VTA and the (D) SN.
Mean6s.e.m TH+cells/mm2are shown; n=6. (E) TH protein levels in the cortex. Mean6s.e.m optical densities are shown as a percentage of FLOX
control mice; n=19–22. One-way ANOVA analysis reveals no significant difference between genotypes.
Akt Deficits Sustain Cortical Hypodopaminergia
PLoS Biology | www.plosbiology.org7 June 2010 | Volume 8 | Issue 6 | e1000393
Currently, a number of additional clinical trials with substantial
sample sizes are ongoing and promise to reveal if NET specific
cognitive symptoms such as poor concentration and memory
The ‘‘DA hypothesis’’ for schizophrenia has enjoyed a
resurgence of interest, and increasing evidence links Akt to DA
related behaviors, yet key molecular mechanisms linking Akt to
DA homeostasis have been elusive. Given evidence for a role of
Akt in the regulation of NET expression and function in cortex
and evidence that NET transports DA in this brain area, it has
been our priority to develop a compelling experimental model to
uncover a molecular link between Akt and cortical DA
homeostasis. Here, we conducted the first studies, to our
knowledge, in an animal model where a genetic deletion that
disrupts Akt phosphorylation enhances expression of NET and
leads to a cortical hypodopaminergic phenotype with schizophre-
nia-linked behavioral consequences.
Our data are consistent with pathophysiological models of
schizophrenia that emphasize ‘‘hypofrontality’’ of DA systems.
Given the role of NET in DA clearance in prefrontal synapses
[16,17,44], Akt-linked changes in NET expression may thereby
translate to cognitive deficits and negative symptoms . These
data, as well as our proof-of-principle results in mice, lead to the
compelling hypothesis that NET inhibition would have therapeu-
tic potential to selectively enhance DA tone in the prefrontal
cortex and perhaps alleviate negative symptoms. Consistent with
this reasoning, several clinical trials are currently investigating
NET blockers for cognitive deficits in patients with schizophrenia.
Our data demonstrate that neuronal mTORC2 dysfunction is
sufficient to generate cortical hypodopaminergia and schizophre-
nia-linked behaviors. In particular, we show that genetic
mTORC2 disruption impairs PPI, a schizophrenia-linked pheno-
type, which has been validated in genetic mouse models of the
disorder . An emerging body of evidence associates Akt
phosphorylation deficits with mental illness in humans, giving our
findings of impaired Akt phosphorylation in mice more transla-
tional viability [4,13]. For example, studies show diminished Akt1
protein content in lymphocytes of patients with schizophrenia
[4,13]. In concert with our findings, candidate gene approaches
aimed at identifying genetic variation in proteins associated with
mTORC2/Akt signaling pathways, including rictor and other
mTORC2 subunit proteins like mSin1 , may yield new
insights into the genetic basis of mental illness.
While DA is classically implicated in the pathogenesis of
schizophrenia, concepts of neurotransmitter dysfunction in this
disease process are constantly evolving. This is reflected by
popular glutamatergic and recently proposed revisions to dopa-
minergic hypotheses of schizophrenia . However, the role of
elevated cortical NE, while not typically emphasized, should not
be overlooked. Indeed, findings of elevated NE in CSF of patients
with schizophrenia, which led to noradrenergic hypotheses of
schizophrenia in the early 1980s , support our model by which
increased cortical NET expression leads to increased NE content
but decreased DA content. While the sensitivity of these
measurements to acute stressors made the reproducibility and
reliability of these methods in the aforementioned studies
questionable, a role for elevated NE in the development of
schizophrenia-like phenotypes in humans cannot be completely
ruled out. Indeed, our current findings, as well as others,
intimately and mechanistically link DA alterations together with
NE changes . As a recent revision to the DA hypothesis of
schizophrenia suggests, schizophrenia could be conceptualized as a
disease where DA dysfunction is a ‘‘final common pathway’’ that
can be elicited by a number of more proximal causes, including
both genetic and epigenetic factors and disruption in other
neurotransmitter systems , and including, as we propose here,
increased NET function.
Schizophrenia is thought to arise from rather complex gene-
environment interactions, and therefore, acquired (rather than
monogenetic) dysfunction in mTORC2/Akt signaling is a partic-
ularly intriguing mechanism. For example, acquired Akt defects are
associated with impaired regulation of blood glucose and diabetes,
which is overrepresented in first episode, medication-naive patients
with schizophrenia . mTORC2/Akt signaling also provides a
promisingportal into‘‘multiple hit’’ models of schizophrenia, as Akt
is positioned to interact with other candidate genes such as
neuregulin-1 and COMT [3,49] as well as environmental risk
life stressors [8,50]. The effects of neuronal mTORC2 dysfunction
on NET expression observed in our study ultimately illustrates a
potential molecular mechanism to link disparate genetic and
environmental factors (i.e. obesity/diabetes/insulin resistance) to
dysfunction in a putative ‘‘final common pathway’’ of schizophre-
nia, namely alterations in DA signaling . Indeed, while more
remainsto belearnedabout mTORC2,deficienciesinAktsignaling
even in ‘‘healthy’’ individuals are associated with impaired
prefrontal cortex activation on working memory tasks  and
proneness to psychosis, suggesting a subtle influence on brain
function and behavior that may require other genetic and
environmental hits to result in clinical disease. Thus, our data
Figure 6. Nisoxetine restores PPI deficits and DA levels in the
rostral cortex of rictor KO mice. (A) No differences are observed in
the average %PPI across the experimental range of dB in KO mice prior
to treatment with saline or nisoxetine. (B) Average %PPI 30 min after i.p.
injection of either saline or nisoxetine (30 mg/kg) in KO mice reveals a
significant increase in %PPI in nisoxetine treated KO mice. (C) The mean
PPI across all sound levels in KO mice treated with either saline or
nisoxetine for 30 min; n=5–6, ***p,0.001 Student’s t test. (D) 8 d of
nisoxetine treatment restores DA levels in the rostral cortex of rictor KO
mice; n=4–6. *p,0.05 Student’s t test.
Akt Deficits Sustain Cortical Hypodopaminergia
PLoS Biology | www.plosbiology.org8 June 2010 | Volume 8 | Issue 6 | e1000393
provide one molecular mechanism, NET regulation, towards a
frameworklinking environmental stressorsand/orlifestylefactorsto
mTORC2/Akt signaling and ultimately to DA-dependent behav-
iors. Our studies provide a potential molecular mechanism linking
Akt dysfunction to a schizophrenia-like phenotype and suggest the
viability of targeting both Akt phosphorylation and NET as
pharmacotherapies for schizophrenia.
All procedures were performed according to Vanderbilt
University Institutional Animal Care and Use Committee
Generation of Mice
All mice were fully backcrossed to C57Bl6 background. Mice
homozygous for an allele containing LoxP sites flanking exon 3 of
the rictor gene (rictor f/f Nes2/2; FLOX) were crossed with
neuron specific Nestin cre transgenic mice (rictorw/w Nes+/+;
NES) obtained from Jackson Laboratories to create double
heterozygous (rictorf/w NesCre+/2; HET) offspring (w is the
wildtype allele). HET mice were then crossed to produce neuron
specific rictor KO mice (rictor f/f Nes+/+ or +/2; KO) and HET
KO mice. Control animals were of the following genotypes
(rictorf/f Nes2/2 FLOX, rictorf/w Nes2/2 and rictorw/w
Nes+/+, Nes+/2 or Nes2/2). Subsequent crossings between
FLOX and HET mice were used to generate additional study
animals. Genotyping was performed by PCR using DNA obtained
from tail clippings with primers for the floxed, nestin, and
recombined alleles as described .
Total brain RNA was extracted from Trizol reagent (Invitro-
gen). cDNA was synthesized with a High Capacity cDNA reverse
transcription kit (Applied Biosystems). rictor mRNA was quanti-
fied with real time RT PCR on a Bio-Rad iCycler using iQ SYBR
green Supermix reagent (Bio-Rad) and primer pairs as reported in
. Expression was normalized to levels of the housekeeping
Mice were anesthetized with volatile isoflurane after which
rapid decapitation allowed brain removal. Brains were chilled on
ice and dissected using a scope for either total cortex, rostral
cortex, or caudal cortex. Tissue was homogenized and lysed in a
Triton based buffer that contained a cocktail of protease inhibitors
plus NaF (2 mM) and sodium orthovanadate (2 mM). After
homogenization samples were centrifuged at 17,0006 g for
30 min, and the supernatant was collected and processed for
protein concentration determination. For Western blotting,
<30 mg of protein per sample was run on a 10%–12% acrylamide
gel, transferred to a PVDF membrane blocked with 5% milk, and
incubated with primary antibodies to a variety of proteins. Akt,
rictor, and mTOR antibodies were obtained from Cell Signaling.
We also used actin (Sigma), Na+/K+ ATPase (DBH), NET (Mab
Technologies Inc. NET05-2), and TH (Chemicon/Millipore)
antibodies. Secondary antibodies were obtained from Santa Cruz
Biotechnology. After chemiluminescent visualization on Hyper-
film ECL film, protein band densities were quantified and
analyzed (Scion Image; http://www.scioncorp.com).
Startle responses were elicited with a 94 dB SPL broadband
noise burst (2–75 kHz, 50 ms duration, 0 ms rise/fall time) and
measured through a floor plate mounted on piezo force
transducers (PCB Piezotronics). In two-thirds of the trials, the
noise burst (pulse) was preceded by a prepulse (2.4–78.2 kHz
frequency-modulated sweep, 40 octaves/s, 0.14 ms stimulus onset
asynchrony) ranging from 40–75 dB SPL. Startle responses were
measured across 297 trials (2066.4 s intertrial interval, initial 27
trials discarded to minimize within-session habituation), as max–
min of the force signal within 400 ms following onset of the noise
burst. Presence of a normal startle response (significant difference
between force plate amplitude 2600 to 2200 ms (baseline) versus
0–400 ms (startle) from noise burst onset) was a prerequisite for
subsequent analysis to minimize possible contributions of gross
sensorimotor deficits in either genotype. This criterion excluded
2/8 WT and 0/7 rictor KO mice from further analysis. Inhibition
of the startle reflex was quantified for each prepulse intensity as
Tissue Extraction for Neurochemistry
Brain sections were homogenized in 100–750 ul of 0.1 M TCA,
which contains 10-2 M sodium acetate, 10-4 M EDTA, 5 ng/ml
isoproterenol (as internal standard), and 10.5% methanol (pH 3.8).
Samples were spun in a microcentrifuge at 10,000 g for 20 min, the
supernatant removedandstored at 280uC. Thepellet was saved for
protein analysis. Supernatant was then thawed and spun for 20 min
and then analyzed for biogenic monoamines and/or amino acids by
a specific HPLC assay (Vanderbilt Neurochemistry Core). Biogenic
amines were eluted in the following order: NE, MHPG,
epinephrine, DOPAC, DA, 5-HIAA, HVA, 5-HT, and 3-MT (2).
In Vivo Microdialysis
Mice were anesthetized with isoflurane and placed ina stereotaxic
frame using mouse-specific ear bars (Kopf Instruments, Tujunga,
CA). A guide cannula (CMA7 microdialysis, USA) was placed above
the medial prefrontal cortex (+2.0 AP, 60.7 ML from Bregma and
21.0 DV from skull for FLOX or NES mice and +1.9 AP,
60.6 ML from Bregma and 21.0 DV from skull for rictor KO
mice) and secured to the skull with epoxy adhesive (Plastics one).
Animals were allowed to recover from the surgery (1–3 d). The day
before the experiment, animals were placed in individual dialysis
chambers and the microdialysis probe (CMA7 microdialysis, USA)
with the active length of 2 mm was inserted into the guide cannula.
One end of a tether (Plastics One) is attached to a harness and the
other end attached to a swivel (Instech) that is mounted on a
counterbalanced arm above the dialysis chamber. The probe was
perfused overnight at a flow rate of 0.5 mL/min with artificial
cerebral spinal fluid containing 149 mM NaCl, 2.8 mM KCl,
1.2 mM CaCl2, 1.2 mM MgCl2, 5.4 mM d-glucose, pH 7.2. On
the day of the experiment the flow rate was changed to 1.0 mL/min
and after equilibration dialysis fractions (20 min each) were collected
to establish baseline concentrations of neurotransmitter efflux.
Dialysate samples were stored at 280uC and analyzed by HPLC-
EC for DA levels. Probe placement was verified after collection of
slices by Nissl staining of coronal slices.
Brain Slice Preparation and Biotinylation
Brain slices were prepared from 6- to 10-wk-old mice that were
anesthetized with isoflurane and rapidly decapitated. Following
brain removal, the brain was chilled in oxygenated <4uC sucrose
solution (sucrose 210 mM; NaCl 20 mM; KCl 2.5 mM; MgCl2
1 mM; NaH2PO4NH2O 1.2 mM), and then while in sucrose
solution 300 mm coronal slices were made using a vibratome. Slices
were then collected in oxygenated artificial cerebral spinal fluid
(ACSF) (NaCl 125 mM, KCl 2.5 mM, NaH2PO4NH2O 1.2 mM,
MgCl2 1 mM, CaCl2N2H2O 2 mM). For in vitro drug treatments
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PLoS Biology | www.plosbiology.org9June 2010 | Volume 8 | Issue 6 | e1000393
slices were then allowed to recover for 1 h at 37uC in oxygenated
ACSF (NaCl 125 mM, KCl 2.5 mM, 1 nM insulin, NaH2-
PO4NH2O 1.2 mM, MgCl2 1 mM, CaCl2N2H2O 2 mM) with
either vehicle or Akt1 inhibitor and following recovery slices were
then biotinylated. For non-treated slices, slices were immediately
washed twice with oxygenated 4uC ACSF following collection, and
then incubated with 4uC ACSF solution containing 1 mg/mL of
EZ-Link Sulfo-NHS-SS-Biotin (Pierce/ThermoScientific; Rock-
ford, IL) for 45 min. Slices were then washed twice with oxygenated
4uC ACSF, and then incubated with 4uC ACSF solution containing
1 mg/mL of EZ-Link Sulfo-NHS-SS-Biotin (Pierce Chemical;
Rockford, IL) for 45 min. After biotin incubation, the slices were
rinsed twice quickly and for two 10 min washes in oxygenated 4uC
ACSF. The reaction was quenched by washing twice for 20 min
each with oxygenated 4uC ACSF containing glycine. Following
quenching, slices were frozen on dry ice and the cortex was cut out
and frozen at 280uC until used. Slices were lysed in 1% Triton
buffer (25 mM Hepes, 150 mM NaCl, 2 mM sodium orthovana-
date, 2 mM NaF, plus a cocktail of protease inhibitors) and
centrifuged at 17,000 g for 30 min at 4uC. After isolation of
supernatant 0.1% Triton pulldown buffer (25 mM HEPES,
150 mM NaCl, 2 mM sodium orthovanadate, 2 mM NaF, plus a
cocktail of protease inhibitors) was added. Protein concentration
was determined using Bio-Rad’s protein concentration kit.
Biotinylated proteins were then isolated using ImmunoPure
immobilized streptavidin beads (Pierce) overnight at 4uC with
agitation. Beads were washed three times with 0.1% Triton
pulldown buffer and biotinylated proteins were then eluted in
50 mL of 26SDS-PAGE sample loading buffer at 95uC and then
cooled to room temperature. Total slice lysates and the biotinylated
(slice surface) fraction underwent immunodetection for NET
(MabTechnoligies Inc., Stone Mountain, GA) as described previously.
Mice were sacrificed by rapid decapitation and cortex dissected
using an ice cold metal block and then homogenized at 400 rpm in
at least 10 volumes (w/v) of ice-cold 0.32 M glucose with a Teflon-
glass homogenizer (Wheaton Science Products, Millville, NJ). After
centrifugation of the homogenate at 800 g for 10 min at 4uC, the
supernatant was again centrifuged at 10,000 g for 15 min. The
final pellet was gently resuspended in Krebs-Ringer HEPES
(KRH) medium containing 118 mM NaCl, 4.8 mM KCl, 25 mM
NaHCO3, 1 mM NaH2PO4, 1.3 mM CaCl2, 1.4 mM MgSO4,
10 mM glucose, 1 mM tropolone, 0.1 mM pargyline, and
0.1 mM ascorbic acid, pH 7.4, and protein concentration assayed.
Aliquots (0.2 ml) of synaptosomal preparations (50 ng of protein)
were prepared on ice. DA and NE transport assays (5 min at 37uC)
were initiated by the addition of [3H]DA or [3H]NE (,100 Ci/
mmol, Amersham) and were terminated by immediate filtration
over 0.3% polyethylenimine-coated glass fiber filters using a cell
harvester (Brandel Inc., Gaithersburg, MD). The filters were
washed three times with 1.5 ml of ice-cold phosphate-buffered
saline (PBS) and incubated overnight in Ecoscint H (National
Diagnostics, Atlanta, GA). Radioactivity bound to filters was
counted using a Beckman LS 6,000 liquid scintillation counter.
Nonspecific uptake, defined as the accumulation of [3H]DA or
[3H]NE in the presence of 1 mM desipramine, was subtracted
from total uptake values to obtain specific uptake values.
Mice (n=6/genotype) were anesthetized and transcardially
perfused with 4% paraformaldehyde. Brains were removed and
cryopreserved, and coronal sections were cut on a microtome at
40 mm and stained using minor modifications of published
protocols. Sections were blocked in 4% Blotto (Nestle ´ Carnation
dried milk), 0.2% Triton-X 100 in PBS, and incubated at 4uC for
3 d with a monoclonal anti-TH antibody (Sigma, 1:4,000).
Sections were thoroughly washed and then incubated in
biotinylated anti-mouse IgG (Jackson Immunoresearch, 1:1,000)
for 60 min. Avidin-biotin amplification (Vectastain ABC Stan-
dard, Vector Labs) and 3-39-diaminobenzidine reactions were
used to visualize labeled proteins. Sections from all genotypes were
processed in parallel to minimize variability between groups.
Negative controls in which primary antibody was omitted revealed
no specific labeling. Slides were coded so that the investigator was
blinded to the genotype and sections were imaged using a Zeiss
Axioskop microscope and Axiocam HR. TH-immunoreactive (IR)
cells were counted in images obtained with a 406objective in two
fields per hemisphere derived from two to three sections per
animal. Values were corrected for cell diameter, but no differences
in cell diameter were found across genotypes.
All data are expressed as the mean6s.e.m. Statistical signifi-
cance between groups was determined using t tests or one- and
two-way ANOVAs followed by post hoc tests when the main effect
or interaction was significant at p,0.05. Statistical analyses were
conducted using software from Graph-Pad Prism. The number of
animals and specific statistical analyses used in each experiment
are indicated in the results and figure legend sections.
the brain are reduced in a gene-dosage dependent
manner. (A) qRT-PCR confirms down-regulation of the rictor
gene in HET and KO mice. Mean6s.e.m relative expression
shown as a percentage of FLOX control mice; n=4–5 animals. (B)
Rictor protein levels in the cerebral cortex. Mean6s.e.m optical
densities are shown as a percentage of FLOX control mice; n=5,
*p,0.05; **p,0.01 one-way ANOVA.
Found at: doi:10.1371/journal.pbio.1000393.s001 (0.40 MB TIF)
Rictor mRNA levels and protein expression in
ability in cortical slices. (A) Levels of NET as measured from
the different fractions (surface, total) of cortical slices from NES
control mice. Slices were treated with either vehicle-DMSO (CTR)
or 12 mM of the Akt1 inhibitor. Mean6s.e.m optical density were
normalized to total NET and are shown as a percentage of CTR.
Representative immunoblots are shown, as probed with antibodies
to NET, Na+/K+ATPase to serve as plasma membrane/loading
control; n=4, *p,0.05 Student’s t test. (B) Phosphorylation of Akt
on residue Ser473 measured from the same samples as (A).
Mean6s.e.m of optical densities normalized to total Akt and shown
as a percentage of CTR; n=4, ***p,0.001 Student’s t test.
Found at: doi:10.1371/journal.pbio.1000393.s002 (0.67 MB TIF)
Akt1 inhibition enhances NET surface avail-
We would like to acknowledge Daniel R. Weinberger for helpful discussion
and insights during the preparation of these studies.
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: MAS SDR CS
VK DBP JVV GDS KDN AG. Performed the experiments: MAS SDR
HK CS PJG VK LCD JVV GDS. Analyzed the data: MAS SDR HK CS
PJG VK DBP JVV GDS. Contributed reagents/materials/analysis tools:
HK CS JPK CWL MAM. Wrote the paper: MAS SDR KDN AG.
Akt Deficits Sustain Cortical Hypodopaminergia
PLoS Biology | www.plosbiology.org10June 2010 | Volume 8 | Issue 6 | e1000393
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Akt Deficits Sustain Cortical Hypodopaminergia
PLoS Biology | www.plosbiology.org11 June 2010 | Volume 8 | Issue 6 | e1000393