Loss of MeCP2 in aminergic neurons causes
cell-autonomous defects in neurotransmitter
synthesis and specific behavioral abnormalities
Rodney C. Samacoa, Caleigh Mandel-Brehma, Hsiao-Tuan Chaob, Christopher S. Warda, Sharyl L. Fyffe-Maricicha,1,
Jun Renc, Keith Hylandd,2, Christina Thallere, Stephen M. Maricichf,1, Peter Humphreysg, John J. Greerc, Alan Percyh,
Daniel G. Glazef, Huda Y. Zoghbia,b,f,i,3, and Jeffrey L. Neula,f,3
Departments ofaMolecular and Human Genetics,bNeuroscience,eBiochemistry,fPediatrics, Section of Neurology,iHoward Hughes Medical Institute, Baylor
College of Medicine, Houston, TX 77030;cDepartment of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7;dInstitute of Metabolic
Disease, Baylor University Medical Center, Dallas, TX 75226;gChildren’s Hospital of Eastern Ontario, Ottawa, Ontario, Canada K1H 8L1; andhDepartments
of Pediatrics, Neurology, Neurobiology and Genetics, University of Alabama, Birmingham, AL 35284
Contributed by Huda Y. Zoghbi, October 26, 2009 (sent for review August 24, 2009)
behavioral deficits. Because several of these abnormalities occur in
other disease states associated with alterations in aminergic neuro-
transmitters, we investigated the contribution of such alterations to
RTT pathogenesis. We found that both individuals with RTT and
Mecp2-null mice have lower-than-normal levels of aminergic metab-
olites and content. Deleting Mecp2 from either TH-positive dopami-
nergic and noradrenergic neurons or PET1-positive serotonergic
neurons in mice decreased corresponding neurotransmitter concen-
tration and specific phenotypes, likely through MeCP2 regulation of
rate-limiting enzymes involved in aminergic neurotransmitter pro-
duction. These data support a cell-autonomous, MeCP2-dependent
mechanism for the regulation of aminergic neurotransmitter synthe-
sis contributing to unique behavioral phenotypes.
dopamine ? norepinephrine ? Rett syndrome ? serotonin
mutations in Methyl-CpG Binding Protein 2 (MECP2) (1). RTT
primarily affects females, with a milder clinical phenotype corre-
lating with late truncating and some missense mutations (2, 3). It
has been suspected that aminergic neurons malfunction in RTT,
because the heightened anxiety, aggression, and mood alterations
seen in RTT and MECP2 disorders are associated with abnormal-
ities of the serotonergic system in other human disorders (4–6).
Rigidity and movement abnormalities are associated with dysfunc-
tion of dopaminergic system (7–9). Autonomic dysfunction under-
lying breathing irregularities could be attributable to decreased
norepinephrine (7, 10). Although clinical studies have explored the
hypothesis that the aminergic neurotransmitter systems might be
suggested decreased aminergic metabolite levels in the spinal fluid
of individuals with RTT (11, 12), but subsequent reports failed to
identify such changes (13, 14). More recently, one study reported
instances of both decreased and increased aminergic metabolite
levels in a few individuals with RTT (15). The interpretation of
these clinical data are hindered by clinical heterogeneity, small
sample sizes, and lack of a sufficient number of controls. To
circumvent these problems, we explored the role of the aminergic
neurotransmitter system by performing both clinical and animal
studies. To determine whether the aminergic neurotransmitter
system is altered in RTT, we analyzed the levels of the dopamine
metabolite homovanillic acid (HVA) and the serotonin metabolite
5-hydroxyindoleacetic acid (5-HIAA) in spinal fluid from women
who both met the clinical criteria for RTT and had a disease-
causing MECP2 mutation. We also evaluated neurotransmitter
changes in a RTT murine model and studied the neurochemical,
molecular, and behavioral consequences of deleting Mecp2 from
ett syndrome (RTT, MIM 312750) is an X-linked neurodevel-
opmental disorder caused, in the vast majority of cases, by
either TH-positive dopaminergic and noradrenergic neurons or
PET1-positive serotonergic neurons in the mouse.
HVA and 5-HIAA Are Decreased in RTT Individuals and Mecp2null/y
Mouse Brain. Weanalyzedthemetabolitesofaminergicneurotrans-
samples used to define the reference ranges using HPLC (16). A
significant fraction of RTT individuals had HVA levels below the
reference range (12/64, ?19%, Fig. 1A). Similarly, a significant
fraction of individuals with RTT had 5-HIAA levels below the
reference range (15/64, ?23%, Fig. 1B). In contrast, only ?1% of
control individuals (3/209) had low HVA levels, and ?1% of
control individuals (3/258) had 5-HIAA levels that were slightly
below the threshold level. These numbers were significantly differ-
HVA and 5-HIAA demonstrated a significant decrease in individ-
uals with RTT compared with controls (Fig. 1C). A comparison of
the metabolite levels in RTT individuals carrying two specific
mutations that confer either severe (p.Arg168X, n ? 12/64 RTT
individuals) or mild (p.Arg133Cys, n ? 7/64 RTT individuals)
phenotypes (2) revealed that 33% (n ? 4/12) and 50% (n ? 6/12)
of the individuals with the p.Arg168X mutation had HVA and
5-HIAA levels below the reference range, respectively, whereas
only one person with the p.Arg133Cys mutation had HVA and
5-HIAA levels below the reference range. Interestingly, further
comparison of the age-adjusted mean metabolite levels in individ-
p.Arg168X mutation had a significantly greater reduction in HVA
normative control samples. In fact, the age-adjusted mean values
for the metabolite levels of individuals with the p.Arg133Cys
To understand the mechanism underlying these neurotransmit-
ter abnormalities, we analyzed aminergic content in brains of male
Author contributions: R.C.S., H.Y.Z., and J.L.N. designed research; R.C.S., C.M.-B., H.-T.C.,
C.S.W., S.L.F.-M., J.R., K.H., C.T., S.M.M., P.H., J.J.G., A.P., D.G.G., and J.L.N. performed
research; R.C.S. and J.L.N. analyzed data; and R.C.S., H.Y.Z., and J.L.N. wrote the paper.
Conflict of interest statement: Keith Hyland is co-owner of Medical Neurogenetics, L.L.C.
The authors do not advocate or recommend ongoing cerebrospinal fluid (CSF) analysis in
girls with Rett syndrome, as it does not contribute to the treatment or diagnosis of the
Medicine, Cleveland, OH 44106.
2Present address: Medical Neurogenetics, Atlanta, GA 30338.
3To whom correspondence may be addressed. E-mail: email@example.com or firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/cgi/content/full/
December 22, 2009 ?
vol. 106 ?
mice that completely lack MeCP2 function (Mecp2null/y). As previ-
ously observed (17), their concentrations of dopamine (DA),
norepinephrine (NE), and serotonin (5HT) were lower than those
of littermate control animals (Fig. 1E). This confirms that the
alterations identified in individuals with RTT result from MeCP2
dysfunction and that the reduced levels seen in humans may be
caused by decreased biogenic amine production rather than alter-
ations in the degradative pathways for these amines.
Loss of MeCP2 Causes a Decrease in Expression of Biosynthetic
Enzymes Th and Tph2. We reasoned that one potential cause of the
neurochemical changes that we observed in RTT spinal fluid and
Mecp2null/ybrain could be defects in the pathways responsible for
neurotransmitter synthesis. Tyrosine hydroxylase (Th) and Trypto-
phan hydroxylase 2 (Tph2) are the key rate-limiting enzymes in the
catalysis of tyrosine and tryptophan to produce DA and NE, and
5HT, respectively (16). Defects in the level of these enzymes could
cause a concomitant decrease in neurotransmitters; therefore, we
assessed the RNA levels of Th and Tph2 by quantitative real-time
corresponding protein levels by Western analysis in Mecp2-null
mice. qPCR analysis showed that the levels of Th and Tph2 were
reduced in Mecp2null/yanimals compared with control littermates
(Fig. 2A). Moreover, quantitative ISH analysis (18) of the signal
intensity showed a reduction in the total signal intensity of Th and
Tph2 in Mecp2null/ywhole brain compared with control littermate
brain (Fig. 2B). Examination of signal intensities in different brain
regions showed that the Th signal intensities in the locus ceruleus
and the midbrain region containing the ventral tegmental area and
substantia nigra were reduced compared with control littermate
Th signal intensity in the medulla was decreased but failed to reach
statistical significance (Fig. 2C). Examination of the Tph2 signal
intensities in the hindbrain region containing raphe nuclei B1
MeCP2 function. (A) Levels of the dopamine and norepinephrine metabolite,
normative reference ranges are indicated by the solid lines. (C) Age-adjusted
reduced in the spinal fluid of girls with RTT. Values were normalized to control
samples. (D) Age-adjusted mean values for HVA (mean age ? 7.16) and 5-HIAA
(mean age ? 7.66) in individuals with the p.Arg168X mutation compared to the
values are provided in Table S1.
Aminergic metabolite levels and content are reduced by the loss of
whole Mecp2null/ybrain shows a reduction in both Th and Tph2 expression. (C)
signal intensity is reduced in the locus ceruleus (LC) and midbrain (MB) but not
of the hindbrain. Total Tph2 signal intensity is reduced in the hindbrain region
intensity is observed in the hindbrain region that encompasses B4 through B9
raphe nuclei (HB B4–9). (E) Representative pseudocolored images are shown for
ored images are shown for Tph2 expression in the inferior olivary complex (IO),
raphe nucleus pallidus (RPA), and raphe nucleus obscurus (RO). (G) The protein
and TPH2 are reduced in Mecp2null/yanimals.*, P ? 0.05;**, P ? 0.001. ns, Not
mean ? SEM.
Expression of Th and Tph2 is reduced in Mecp2null/yanimals. (A) qPCR
Samaco et al. PNAS ?
December 22, 2009 ?
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through B3 (HB B1–3) revealed a significant reduction in signal
intensity (Fig. 2D, representative image shown in Fig. 2F). For
regions containing the raphe nuclei B4 through B9 (HB B4–9), we
detected a trend toward decreased signal intensity of Tph2 (P ?
0.06) (Fig. 2D). Similar reductions in Th and Tph2 expression levels
total ISH signal intensity into strong, medium, and weak signal
intensities (18) [Supporting Information (SI) Text, Fig. S1 and Fig.
S2). Furthermore, analysis of TH and TPH2 protein levels by
Western blot analysis showed reduced expression of both synthetic
enzymes in Mecp2null/ybrain compared with those of wild-type
controls (Fig. 2 G and H).
Deletion of Mecp2 from Either TH-Positive or PET1-Positive Neurons
Results in Specific Phenotypes.Animportantquestioniswhetherthe
amines or whether it is a non-autonomous effect of disrupting
MeCP2 function within other cell populations. Another question is
whether the neurochemical changes, possibly caused by abnormal
cell-autonomous neurotransmitter synthesis, contribute to specific
clinical features of RTT. To address these questions, we removed
Mecp2 specifically from either TH-expressing dopaminergic and
noradrenergic neurons or PC12 ets factor 1 (PET1)-expressing
serotonergic neurons using Cre-lox technology to generate condi-
tional knock-out (CKO) animals. The resulting TH-CKO and
PET1-CKO mice allowed characterization of the molecular and
phenotypic consequences of deleting Mecp2 in these particular
We bred male transgenic mice that express Cre recombinase
from either the TH or PET1 regulatory regions with female mice
that were wild-type, TH- or PET1-Cre, Mecp2flox/y(Flox), or
TH-Cre; Mecp2flox/y(TH-CKO) or PET1-Cre; Mecp2flox/y(PET1-
CKO). The TH-Cre transgenic mouse line expresses Cre in all TH
expressing regions of the brain, including the substantia nigra/
ventral tegmental area, locus ceruleus, and medullary norepineph-
rine neurons, as well as in the peripheral nervous system and the
adrenal medulla (19). In contrast, the PET1-Cre transgenic mouse
line expresses Cre driven by the PET1 enhancer element in the
dorsal and medial raphe nuclei (20). The recombination efficiency
of each Cre line was tested by immunofluorescence, showing that
Mecp2 was effectively deleted from the specified brain regions (SI
Text and Fig. S3).
Whole-brain HPLC analysis of neurotransmitter levels in each
CKO line showed neurochemical changes influenced by the pres-
ence of the Mecp2floxallele. This was expected, given the recent
report that the Mecp2floxallele is a hypomorphic allele (21).
However, the respective biogenic amines in CKO animals were
markedly reduced in comparison to Flox littermate animals. TH-
CKO animals displayed reduced DA and NE levels but 5HT levels
that were similar to those of Flox animals (Fig. 3A), whereas
PET1-CKO animals displayed reduced 5HT levels but DA and NE
levels that were similar to those of Flox animals (Fig. 3B). To probe
the mechanism underlying these neurochemical changes, we as-
sessed the expression levels of Th and Tph2. Th was reduced in the
TH-CKO animals, whereas Tph2 was reduced in the PET1-CKO
animals (Fig. 3C and D). Thus, the loss of MeCP2 causes a
cell-autonomous reduction of biogenic amines comparable to the
decreased neurotransmitter content observed in Mecp2null/yani-
mals, and this reduction is associated with decreased levels of
rate-limiting enzymes responsible for the production of those
amines. We also found, by chromatin immunoprecipitation (ChIP)
of these genes, suggesting that MeCP2 may directly regulate the
expression of these genes (Fig. 3 E and F).
To determine the behavioral consequences of removing MeCP2
from these specific neuronal populations, we performed a detailed
behavioral analysis of both the TH-CKO and the PET1-CKO
animals, selecting behavioral tests relevant to phenotypes seen in
RTT and MeCP2 mouse models (see SI Text and Table S2 for the
list of the specific behavioral assays, the age of the animals at the
time of each test, and a complete summary of statistical analyses).
TH-CKO animals showed a specific alteration in locomotor activ-
in the open field and poor performance on the dowel walking task
(Fig. 4 A–C). We investigated the breathing phenotypes of TH-
CKO animals because previous work proposed that the breathing
in the noradrenergic system (22). The hypomorphic Flox animals
had an increased number of apnea episodes (Fig. 4D), possibly
because of the decrease in biogenic amine levels (Fig. 3A), but we
did not detect a more severe breathing phenotype in the TH-CKO
animals beyond those observed in the hypomorphic Flox animals,
despite further reduction of NE levels in the TH-CKO animals and
S3). We also did not detect abnormalities in motor learning,
anxiety, social interaction or learning and memory that were
specific to the loss of Mecp2 in TH-positive neurons (Fig. 4 E–H).
In contrast to TH-CKO animals, PET1-CKO animals did not
effects on biogenic amines and expression of synthetic enzymes. (A) Selective
removal of MeCP2 from TH neurons (TH-CKO) caused a reduction in DA and NE
levels. 5HT levels were unchanged compared with Flox animals. (B) Selective
removal of MeCP2 from serotonergic neurons (PET1-CKO) caused a reduction in
5HT levels. DA and NE levels were unchanged compared with the Flox animals.
Values were normalized to wild-type samples. (C and D) The expression levels of
5HT were reduced in a cell-autonomous fashion. Th levels were decreased in
were normalized to wild-type samples. (E) ChIP-PCR showed that MeCP2 was
bound to the Th and Tph2 promoters. Representative gel image is shown. (F)
ChIP-qPCR showed that MeCP2 is significantly enriched within 1kb of the pro-
moter regions of Th and Tph2. The fold enrichment of chromatin fragments
immunoprecipitated with anti-MeCP2 antibody compared with a control anti-
body (normal rabbit IgG), relative to input samples is shown (ddCT method).*, P ?
Removing MeCP2 from TH or PET1-neurons causes cell-autonomous
www.pnas.org?cgi?doi?10.1073?pnas.0912257106Samaco et al.
show decreased motor function. Similar to TH-CKO animals,
PET1-CKO animals showed an altered pattern of social interest
influenced by the Flox allele (Fig. 5A). Defects in 5HT levels have
been linked to aggression in other murine models (4, 6). To test for
the presence of increased aggressive behavior, we performed the
resident intruder test. PET1-CKO animals were more aggressive
when exposed to conspecific partner mice (Fig. 5B). The seroto-
nergic system has also been implicated in anxiety, repetitive be-
havior, and hyperactivity; therefore, we also tested PET1-CKO
animals using assays that would address these behavioral domains
(23–25). We found no evidence of altered anxiety in the light–dark
box exploration task, altered self-grooming behavior in the splash
test, or abnormal repetitive behavior in the marble-burying task
that was specific to the deletion of Mecp2 in PET1-positive sero-
tonergic neurons (Fig. 5 C–E). Although the selective deletion of
MeCP2 in PET1-positive neurons appeared to increase activity
based on total distance traveled, we believe that this resulted from
increased baseline activity in mice carrying both the Flox allele and
the PET1-Cre transgene. Consistent with this interpretation, a
one-way ANOVA among the four genotypes showed a statistically
significant increase in distance traveled in PET1-CKO animals
compared with littermate control animals, but a two-way ANOVA
(MeCP2-Flox allele X PET1-Cre allele) of these data does not
support genetic interaction between the two alleles (Fig. 5F, Table
S2). Given that the loss of serotonergic neurons causes increased
and their wild-type littermates. We found a significant effect of the
Flox allele on the distribution of respiratory frequency with Flox
animals showing a greater percentage of time breathing faster than
control animals (Fig. S4). However, a further specific reduction of
5HT in the PET1-CKO animals (Fig. 3B) did not worsen the
breathing phenotype. PET1-CKO animals showed no specific ab-
normalities in motor coordination, motor learning or learning and
memory (Fig. 5 G–I). Both CKO animals lived until at least 18
months of age; in comparison, the constitutive Mecp2null/ymice on
an F1 hybrid background live until 69 days of age (median age, n ?
115). This indicates that MeCP2 function is not required in either
TH- or PET1-positive neurons for normal lifespan.
Before the discovery of MECP2 mutations, one of the early
hypotheses regarding RTT pathogenesis ascribed a role for defects
in aminergic neurotransmitter systems. This was a reasonable
and 5HT (4–9, 27). Also, the first study that examined neurotrans-
activity in an open field. (C) TH-CKO animals had fewer side touches on a dowel
walking task, a measure of motor coordination. (D) Population data of plethys-
mographic recordings showed a significant increase in the frequency of apneas
was observed on days 3 and 4 of testing. (F) No significant differences were
observed in the amount of time mice explored the lit side of the light–dark box.
showed a significant increase in the time spent at the partition during a novel
encounter and during a second encounter with a familiar partner. (H) TH-CKO
represent mean ? SEM.
Removing MeCP2 from TH-neurons causes motor abnormalities. (A)
partition test for social interaction, PET1-CKO animals, like Flox animals, spent
(B) PET1-CKO animals were more aggressive in the resident intruder task as
with Cre animals. (E) PET1-CKO and Flox animals buried fewer marbles in a task
for repetitive behavior compared with littermate controls (F) PET1-CKO animals
traveled more in the open field compared to littermate animals because of an
additive effect of both the PET1-Cre and Flox alleles. (G) No difference was
observed between PET1-CKO and littermate animals in the number of side
touches in the dowel walking task. (H) No difference was observed between
PET1-CKO and littermate animals in motor learning; Flox animals show a signif-
to the loss of MeCP2 in PET1-positive neurons was observed in fear conditioning
Removing MeCP2 from PET1-neurons causes aggression. (A) In the
Samaco et al.PNAS ?
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mitter abnormalities in RTT supported such a hypothesis (11).
Subsequent studies ranged from inconclusive to contradictory
(13–15). Murine models of MECP2 disorders recapitulated behav-
ioral features that could be linked to neurotransmitter abnormal-
question in a controlled fashion. In this study, we investigated the
role of the aminergic neurotransmitter systems in RTT by first
settling the issue on aminergic metabolite levels in human samples
and then exploring the biological relevance of these aminergic
deficiencies in a RTT murine model. We also investigated the role
of MeCP2 in different types of aminergic systems and found that
MeCP2 regulates the functionality of aminergic neuronal subtypes
in a cell-autonomous manner.
Starting with clinical studies, we show that the loss of MeCP2
function causes a decrease in aminergic metabolites in humans.
Because of the large sample size and rigorous methodology used in
in RTT is strong. Furthermore, we found that individuals carrying
the p.Arg168X mutation, who have a more severe RTT phenotype
(2), had even more remarkable reductions in HVA and 5-HIAA,
whereas individuals with the milder p.Arg133Cys mutation had
metabolite levels similar to those of controls. The clinical and
aminergic metabolite data support the notion that missense muta-
tions such as the p.Arg133Cys mutation retain some MeCP2
function. Nevertheless, by comparing the age-adjusted mean levels
of these metabolites in RTT with reference ranges for the norma-
tive samples, we identified an overall significant decrease in the
levels regardless of the type of MECP2 mutation. To determine
whether this reduction was caused by a primary defect in the
production of amines rather than a secondary defect in aminergic
metabolite degradation, we analyzed aminergic neurotransmitter
content in Mecp2null/ymouse brain and showed that DA, NE and
in the Mecp2-null mice are concordant with observations made in
a previous study (17). Together, the results from both human and
murine biological specimens clearly implicate MeCP2 in the regu-
lation of aminergic neurotransmitters.
To gain insight into how MeCP2 dysfunction leads to alterations
the RNA and protein levels of the rate-limiting synthetic enzymes
levels of both synthetic enzymes are indeed reduced in Mecp2null/y
An important question is whether MeCP2 regulates the levels of
Th and Tph2 in a cell-autonomous manner. Previous studies have
reported both cell-autonomous and non-autonomous effects of
MeCP2 in murine models (34–37). To address the role of MeCP2
in aminergic neurons, we selectively deleted MeCP2 in either
TH-positive or PET1-positive neurons. Although we cannot fully
exclude our results as a consequence of both cell-autonomous and
non-autonomous effects, and we cannot exclude the effect of
deleting MeCP2 in select neuronal populations in the background
of the hypomorphic Flox allele, our data provide strong evidence
that the functional consequences of deleting Mecp2 in biogenic
amine neurons are specific. A significant finding from our work is
that the reduction in the expression levels of Th and Tph2 and their
corresponding neurotransmitter levels in the respective aminergic
MeCP2-CKO animals are comparable to those observed in
Mecp2null/yanimals, fully recapitulating the neurochemical and
molecular defects. In addition, the unique behavioral phenotypes
that we observed in the TH- and PET1-CKO mice are due to the
absence of MeCP2 function in either dopaminergic and noradren-
ergic or serotonergic neurons, respectively, and these phenotypes
have been linked to defects in the respective neurotransmitter
systems in both humans and mice (4–9, 27). To explore the
mechanism underlying the decrease in expression of the synthetic
enzymes, we tested whether MeCP2 occupied the Th and Tph2
promoter regions and found that the promoter regions of both
neurotransmitter synthesis genes are occupied by MeCP2. These
data, combined with our expression data, point to a potential role
with recent reports (38, 39, 40). Importantly, these data demon-
strate that MeCP2 is critical for the in vivo regulation of neuro-
transmitter synthesis genes.
A notable observation in the TH-CKO and PET1-CKO animals
is the absence of a specific breathing problem. In the hypomorphic
Flox animals, decreased NE and 5HT levels may contribute to the
breathing phenotype observed in these animals. However, the
breathing abnormalities are not worsened in either TH-CKO and
PET1-CKO animals that have a further specific reduction of NE or
5HT, respectively. One interpretation is that NE and 5HT levels
On the other hand, the effect caused by abnormal aminergic
neurotransmitter levels is due to the slight reduction in amine
concentration seen in the hypomorphic Flox animals but is not
worsened by a further reduction. Because breathing defects are
prominent in RTT, it will be interesting to determine whether
from a combination of various neurons, such as both TH- and
PET1-positive neurons, can recapitulate the more robust and
complex breathing defects in Mecp2null/yanimals. Furthermore,
neither the TH-CKO, PET1-CKO nor the Flox animals show the
dramatically shortened lifespan seen in the Mecp2-null animals,
despite having breathing abnormalities qualitatively similar to the
Mecp2null/yanimals. We conclude that the reduction in NE and
5HT, and the breathing abnormalities potentially caused by alter-
ations in MeCP2 function is not sufficient to cause death. It is
with the breathing abnormality are needed to cause death, or that
the breathing abnormality is not the underlying cause of death.
CKO mice suggest etiologies for specific symptoms of the human
disorder. For example, it is well established that decreased dopa-
mine levels are linked to motor abnormalities in humans and in
mice (9, 41). Motor deficits are also prominent in girls and women
with RTT (8, 42). Our TH-CKO animals displayed motor defects
(41). Hypoactivity in the open field and abnormal motor coordi-
nation on the dowel walking task in TH-CKO animals were
independent of innate anxiety-like behavior in a novel environ-
ment, as exploratory behavior in the light–dark box exploration
task was normal. Our PET1-CKO animals also provide insight into
clinical features of RTT. Heightened anxiety and repetitive stereo-
typed behavior are commonly observed in RTT (43), and a large
body of evidence supports the notion that 5HT modulates both of
these behaviors (4, 5). Although the levels of 5HT are specifically
decreased in PET1-CKO animals, our data suggest that innate
altered in comparison with littermate controls. Moreover, in the
analysis of hyperactive behavior, a feature also attributed to defects
in 5HT levels (41), the increase in distance traveled was significant
only in a one-way ANOVA, as the hyperactivity that we observed
was an additive effect of both the PET1-Cre and Flox alleles in the
PET1-CKO animals. This is a significant finding given that TH-
CKO animals display hypoactivity. The presence of opposing
phenotypes in the two different aminergic-CKO animals (hypoac-
tivity in TH-CKO animals and hyperactivity in PET1-CKO ani-
mals) provides further evidence of a cell-autonomous role for
MeCP2 in these particular neurons. The most striking phenotype
unfamiliar conspecific animals. Although aggression is not a well-
documented symptom of RTT, there are some typically higher-
functioning RTT individuals that do display aggressive behavior
(44). Our group previously reported aggression in mice that lack
www.pnas.org?cgi?doi?10.1073?pnas.0912257106Samaco et al.
MeCP2inSim1-positivehypothalamicneurons(45).Thisstudynow Download full-text
reveals another anatomical origin of aggression in RTT, the dys-
function of MeCP2 in serotonergic neurons. It is interesting that
5HT reuptake inhibitor treatment in an individual with RTT
decreased self-abusive behaviors (15). Formal studies that investi-
gate aggressive behavior in RTT and potential interventions using
5HT reuptake inhibitors might prove clinically useful.
CKO animals support a critical role for MeCP2 in the aminergic
to these neurons. We propose that the overall neurochemical and
likely the cumulative result of MeCP2 dysfunction within a variety
of cells. The work reported here underscores the value of the
conditional knockout approach in dissecting the role of MeCP2 in
normal brain function and indicates that MeCP2 regulates genes
and/or molecules in specific neuronal populations to subsequently
function in additional neuronal subtypes will likely yield additional
deficits, autonomic dysfunction, and impaired learning. Impor-
tantly, the discovery that specific behavioral phenotypes in the
mouse may be a direct consequence of abnormal MeCP2-
that targeting therapies to modulate these specific systems may
mitigate specific deficits in individuals with RTT.
Materials and Methods
amine metabolites in cerebrospinal fluid were measured after isocratic HPLC
separation by electrochemical detection as previously described (16). For
qPCR, total RNA was isolated from brain (three to four animals per genotype)
(Superarray Biosciences). qPCR was performed as previously described (21)
using commercially available primers (Superarray Biosciences). Nonradioac-
tive ISH and quantification of ISH signal intensity were performed as previ-
ously described (18) on 25-?M, serially sectioned brain samples (three to four
animals per genotype). Western blot analyses of Mecp2null/yand wild-type
brain protein extracts (three animals per genotype) were performed as pre-
viously described (21) using 50 ?g of protein per sample and anti-TH (1:500,
Abcam), anti-TPH (1:500, Sigma), and anti-VCL (1:2000, Sigma) antibodies.
Quantification of signal intensity was performed using ImageJ (rsbweb.nih.
gov/ij/). For ChIP-PCR and ChIP-qPCR, chromatin was cross-linked and immu-
noprecipitated from brain tissue samples from wild-type and Mecp2null/y
tion of spinal fluid collection, probe sequences for ISH, primer sequences,
conditions for ChIP-PCR/qPCR amplification, and behavioral tests is provided
in Materials and Methods in the SI Text Data from statistical analyses for
behavioral tests are provided in Table S2.
ACKNOWLEDGMENTS. We thank the following individuals for their contribu-
tions to this work: the members of the Zoghbi laboratory and Vicky Brandt for
for the PET1-Cre mouse line; Corinne Spencer and Richard Paylor for advice on
neurobehavioral tasks; Agnes Liang, Ying Liu, and Ron Kao for technical assis-
tance with ISH experiments; Chris McGraw for assistance with protein analysis;
Melissa Arvide for tissue sectioning; and the Baylor College of Medicine Intellec-
NS052240 [to J.N.], and MH078678 [to H.T.C.]), the International Rett Syndrome
Foundation (to J.N.), and the Simons Foundation (to H.Z.). Huda Zoghbi is a
Howard Hughes Medical Institute investigator.
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Samaco et al.PNAS ?
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vol. 106 ?
no. 51 ?