LETTER

doi:10.1038/nature09972

A transient placental source of serotonin for the fetal
forebrain
Alexandre Bonnin1,2, Nick Goeden1, Kevin Chen3, Melissa L. Wilson4, Jennifer King4, Jean C. Shih3, Randy D. Blakely5,6,
Evan S. Deneris7 & Pat Levitt1,2

Serotonin (5-hydroxytryptamine or 5-HT) is thought to regulate
neurodevelopmental processes through maternal–fetal interactions that have long-term mental health implications. It is thoughtthat beyond fetal 5-HT neurons there are significant maternal
contributions to fetal 5-HT during pregnancy1,2 but this has notbeen tested empirically. To examine putative central and peripheral sources of embryonic brain 5-HT, we used Pet12/2 (alsocalled Fev) mice in which most dorsal raphe neurons lack 5-HT3.
We detected previously unknown differences in accumulation of
5-HT between the forebrain and hindbrain during early and late
fetal stages, through an exogenous source of 5-HT which is not ofmaternal origin. Using additional genetic strategies, a new technology for studying placental biology ex vivo and direct manipulationof placental neosynthesis, we investigated the nature of this exogenous source. We uncovered a placental 5-HT synthetic pathway
from a maternal tryptophan precursor in both mice and humans.
This study reveals a new, direct role for placental metabolic pathways in modulating fetal brain development and indicates thatmaternal–placental–fetal interactions could underlie the pronounced impact of 5-HT on long-lasting mental health outcomes.
Fetal 5-HT dysfunction is implicated in developmental programming by altering brain circuit formation4, which later translates into
abnormal adult behaviours5,6. In humans, risk alleles in genes involvedin 5-HT function combine with early adverse experiences during
development to affect adult-onset mental illnesses7. Furthermore,polymorphisms in the 5-HT transporter (SLC6A4, which encodes
SERT) and 5-HT receptors, which are expressed early in brain
development8, are associated with neurodevelopmental disorders suchas autism spectrum disorder and schizophrenia9,10. A puzzling issue
regarding the role of 5-HT in fetal brain development is that receptors,
transporters and degrading enzymes for 5-HT often appear before thedevelopment of 5-HT innervation11 itself, suggesting the existence ofan exogenous source of 5-HT at early stages of development. The most
biologically influential source of exogenous 5-HT is claimed to bematernal1,2 but there is a lack of experimental data to support such amechanism of developmental programming.
Analysis of fetal Pet12/2 mice provides an opportunity to assess
potential extra-embryonic sources of 5-HT, as only ,30% of dorsal
raphe 5-HT neurons can be detected in null mice, which also express
low levels of tryptophan hydrolase 2 (TPH2), aromatic l-amino aciddecarboxylase (AADC; also called dopa decarboxylase, Ddc), SERTand other markers of the serotonergic phenotype3. To assess whether
dorsal raphe neurons are also the sole source of brain 5-HT during
development, we compared the concentration of 5-HT in embryonicbrains collected from Pet12/2 and wild-type littermates from embryonic day (E) 10.5, the onset of 5-HT synthesis in dorsal raphe neurons,
to E17.5, when 5-HT axons are fully deployed throughout the forebrain. High-performance liquid chromatography (HPLC) was used tomeasure the concentration of 5-HT in the mid/hindbrain region(termed ‘hindbrain’), which contains 5-HT cell bodies and proximal
axons, and in the forebrain, which contains only distal 5-HT axons12,13.
Consistent with dorsal raphe neurons providing the main source of
5-HT in the hindbrain, the 5-HT concentration was lower in Pet12/2
hindbrains than in those of wild-type mice at every age tested (Fig. 1a).
Notably, in the Pet12/2 forebrain, 5-HT levels were statistically indistinguishable from wild type at E10.5 to E15.5; however, large differences emerged at E16.5 (Fig. 1b). This is consistent with dorsal rapheaxons being the major source of forebrain 5-HT at this and later ages
but not earlier (Supplementary Fig. 2 and ref. 3). Remarkably, even
before the arrival of 5-HT axons in the ventral forebrain (E10.5–
E12.5), low levels of 5-HT were detected (Fig. 1b). Normally, overthe next three embryonic days, progressively more 5-HT axons growinto the forebrain13 (Fig. 1c–e). In the Pet12/2 forebrain, however,there is a marked reduction in 5-HT axon density compared to wild
type (Fig. 1c–i), even though total tissue 5-HT concentrations arecomparable. The density and distribution of thalamocortical axons,
which also express SERT and can take up 5-HT14, are similar in
Pet12/2 and wild-type mice (Fig. 1c, f). These results reveal a complex
regulation of 5-HT in the fetal brain, with dorsal raphe serotonergic
neurons and axons representing the major source of 5-HT in thehindbrain and at later embryonic stages in the forebrain but not themain source of 5-HT in the early developing forebrain.
The greater decrease in total tissue 5-HT concentration in the hindbrain than in the forebrain in Pet12/2 mice suggests a differential contribution of non-dorsal-raphe sources in these regions. Alternatively,
because 5-HT degradation enzyme (monoamine oxydase A; MAO-A)
activity is higher in the hindbrain than in the forebrain at early stages ofdevelopment15, a differential degradation of 5-HT across the two brain
regions may account for the difference. Consistent with this latter
possibility, 5-hydroxyindoleacetic acid (5-HIAA) concentration in theE14.5 sMaoa2/2 mouse (which lacks MAOA enzymatic activity andcannot efficiently degrade 5-HT16) were decreased 3.4-fold in the forebrain, but 6.1-fold in the hindbrain compared to wild type littermates(Supplementary Fig. 3). In contrast, at E16.5, 5-HIAA concentrations
were decreased to a similar extent in the sMaoa2/2 forebrain and hindbrain (3.6- and 3.1-fold respectively). A downregulation of MAOA
activity in the Pet12/2 forebrain before E16.5 could explain the normal
concentrations of 5-HT measured in the region. To test this possibility,
we quantified MAOA activity in the forebrain at E14.5 and showed that
it was not different in Pet12/2, Pet11/2 and Pet11/1 forebrains (Supplementary Table 2). Furthermore, 5-HIAA concentrations were notdifferent in the Pet12/2 and Pet11/1 forebrains at E12.5 and E14.5(Supplementary Fig. 3c). The data demonstrate that MAO-A activity
is not downregulated in the Pet12/2 forebrain.
The non-dorsal-raphe origin of 5-HT in the early fetal forebrain could
arise from several sources. Because adult forebrain catecholaminergic

1

Zilkha Neurogenetic Institute, Keck School of Medicine of USC, Los Angeles, California 90089, USA. 2Department of Cell & Neurobiology, Keck School of Medicine of USC, Los Angeles, California 90089,
USA. 3Department of Pharmacology & Pharmaceutical Sciences, USC School of Pharmacy, Los Angeles, California 90089, USA. 4Departments of OB/GYN & Preventive Medicine, Keck School of Medicine of
USC, Los Angeles, California 90089, USA. 5Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37221, USA. 6Center for Molecular Neuroscience, Vanderbilt University
Medical Center, Nashville, Tennessee 37221, USA. 7Department of Neuroscience, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
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RESEARCH LETTER

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Figure 1 | Comparison of fetal 5-HT concentrations in the hindbrain andforebrain of Pet12/2 and wild type embryos from E10.5 to E17.5. a, 5-HT
concentration in the Pet12/2 (KO) hindbrain is significantly lower than in wildtype (WT) littermates at every age tested. b, In contrast, 5-HT concentrations in the
Pet12/2 forebrain are not significantly different from wild-type littermates fromE10.5 to E15.5 but become significantly lower at E16.5 and E17.5 (n $ 6 embryos
per genotype per age; *, P , 0.005; one-way analysis of variance (ANOVA); data
are presented as means 6 s.e.m.). c–h, Serotonergic axons (SERT1) and dorsal
thalamic (DT) axons (NetG1a1) immunostained on sagittal sections at E14.5 inwild-type (c) and Pet12/2 (f) embryos (regions shown correspond to the red box in
the drawings). In wild-type E14.5 embryos (d) SERT1 axons grow ventrally into
the forebrain through the medial forebrain bundle (mfb, white arrowheads); SERT
also labels dorsal thalamic axons at this age (open arrowhead). In comparison, very
few SERT1 axons remain in the Pet12/2 (g). The pattern and density of SERT1
DT axons appear to be unaffected in the Pet12/2 forebrain. Scale bars: 50 mm. The
rostral-most extent of ingrowing serotonergic axons immunolabelled with 5-HTin the wild type (e) shows numerous 5-HT1 axons, some of which diverge towards
the hypothalamus (Hyp). In contrast, only few 5-HT1 axons remain in the
Pet12/2 forebrain (h). Scale bars: 20 mm. i, Densitometric analysis of 5-HT1 axonsin the most rostral part of the medial forebrain bundle (region indicated in the
diagram) at E14.5 confirms fewer axons in the Pet12/2. AU, arbitrary units.
j, AADC staining identifies dopaminergic neurons in the substantia nigra pars
compacta (SNC), along with dopaminergic and serotonergic axons coursing
through the ventral forebrain at E14.5, and also AADC1 catecholaminergic
neurons present in the hypothalamus (Hyp, black box). Scale bar: 100 mm.
k, AADC1 neurons in the hypothalamic region (red box in bottom right drawing;
middle panel, white arrowheads) are 5-HTP-negative. Open arrowheads indicate
fluorescence from blood vessels. Scale bar: 25 mm.

and dopaminergic neurons express the AADC enzyme, these cells canectopically synthesize 5-HT, albeit after administration of large dosesof the precursor 5-HTP17. We tested the possibility that embryonic

AADC-expressing neurons could ectopically produce 5-HT in theearly Pet12/2 forebrain. Consistent with measures of unaltered dopamine concentration (Supplementary Fig. 1), AADC immunostaining
revealed normal catecholaminergic and dopaminergic neuron andaxon density in the Pet12/2 forebrain (Fig. 1j). Furthermore, catecholaminergic neurons present in the Pet12/2 hypothalamus do not exhibit
ectopic 5-HTP or 5-HT immunoreactivity (Fig. 1k), consistent withthere being no local cellular source of 5-HT in the Pet12/2 forebrain.
In the developing forebrain, E10.5–E15.5 is the period of pronounced neurogenesis and axon growth. As 5-HT modulates bothprocesses4,10, it is essential that its availability be regulated during thistime. It is remarkable, therefore, that over this time period, even in theabsence of 5-HT axons, the concentration of 5-HT in the Pet12/2
forebrain is normal. Possible exogenous sources include the embryonicgut, the maternal blood through the placenta, or the placenta itself. We
ruled out the embryonic gut as a source because expression of the 5-HT
biosynthetic enzyme tryptophan hydroxylase (TPH1), which provides
blood 5-HT, begins late (E15.5) in fetal enterochromaffin cells1,18. To
test the possibility that maternal 5-HT is transferred to the fetal brain,
we examined brains from fetuses of Sert knockout (Sert2/2) dams;
total blood and platelets in these dams contain virtually no 5-HT19.
This absence of blood 5-HT is attributed to a failure of uptake byplatelets, to rapid degradation of the remaining free plasma 5-HT in
the liver and to compensatory uptake by other transporters in the gut19.
Despite this, the concentration of 5-HT is not different in the forebrainof Sert1/2 E12.5 embryos from Sert2/2 or wild-type dams (Supplementary Table 1), indicating that maternal blood 5-HT is not themain source of fetal blood and forebrain 5-HT at early stages ofdevelopment.
We considered the alternative hypothesis that the essential aminoacid tryptophan, originating from the pregnant dam, would be converted to 5-HT in the placenta and delivered to the fetal circulation.
Injection of tryptophan in pregnant dams increases 5-HT concentration in the fetal brain20 but the precise location of the synthetic conversion has never been identified. Quantitative RT-PCR of placental tissue
detected transcripts of Tph1 and Aadc, but not Pet1 (Supplementary
Fig. 4a). Immunocytochemistry confirmed that TPH1 and AADC
proteins are expressed in the syncytiotrophoblastic cell layer of theplacenta at E10.5–E14.5 (Fig. 2c–h and Supplementary Fig. 4e–j).
The placenta thus has the necessary machinery to synthesize 5-HT.
We tested for placental 5-HT neosynthesis and showed that both
5-HTP and 5-HT neosyntheses occur in placenta and fetal hindbrain
extracts incubated with tryptophan (Fig. 2a) as early as E10.5 (Supplementary Fig. 4b). Placental 5-HT synthesis capacity was greater at
E14.5 than at E18.5, whereas the converse was true in the hindbrain,consistent with changes in Tph1 transcript expression (data notshown). The capacity for placental 5-HT synthesis at E14.5 was notaffected by the absence of embryonic Pet1 gene expression (Supplementary Fig. 4c). This synthetic capacity is not unique to mice, as
human placental fetal villi at 11 weeks of gestation showed robust 5-HT
neo-synthesis (Fig. 2b), indicating that a placental source of 5-HT isimportant for human fetal development.
Although not providing conclusive proof, these data are consistent
with the possibility that neosynthesis and transport of 5-HT from amaternal tryptophan precursor occurs in an intact placenta. We
addressed this with two strategies. First, we developed a novel ex vivo
technology for regulating placental organ perfusion, thus allowing thepresentation of maternal precursor and the collection of fetal perfusate
in intact, live murine placentas (Fig. 2i). Within 15 min of tryptophan
injection through the maternal uterine artery, there was a large accumulation of newly synthesized 5-HT that passes through the fetal
placental circulation (Fig. 2j), demonstrating that the live placenta isable to convert tryptophan to 5-HT and release the neurotransmitter
into the fetal circulation. In contrast, when 5-HT (1.5 nM) was injected
into the uterine artery, only 0.32 6 0.16% of the maternal free 5-HT
was transferred to the fetal umbilical vein during a 30 min perfusion

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LETTER RESEARCH

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Figure 2 | Placental synthesis of 5-HT in vitro and ex vivo. a, Placental andhindbrain extracts were incubated with the cofactor tetrahydrobiopterin (BH4;
control) or BH4 and tryptophan (1Trp1BH4) and 5-HT neosynthesis wasmeasured after 30 min. b, Similar experiments conducted using human placenta
tissue collected at 11 weeks of gestation (11 GW) show that human fetal villi
synthesize 5-HTP and 5-HT (statistical significance versus control analysed byStudent’s t-test; *, P , 0.005; n 5 3; data are presented as means 6 s.e.m.).
c, Immunostaining for the monocarboxylate transporter MCT1, a marker ofsyncytiotrophoblastic cells (sc) in the labyrinth (la) region on the fetal side (fet)of an E14.5 mouse placenta. On the maternal side (mat) the decidua (dc) is
devoid of staining. d–h, Higher magnifications of the region boxed in c; MCT1 isexpressed on the apical side (arrows) of syncytiotrophoblasts facing the maternal
blood space (mbs) (d, e). The 5-HT synthetic enzymes TPH1 (f, g) and AADC(h) are expressed in overlapping patterns in the cytoplasm ofsyncytiotrophoblastic cells (arrows). Asterisks (e, g, h) indicate red blood cells ofmaternal origin. Scale bars: c, 20 mm; d, f: 500 mm; e, g, h: 150 mm. i, Schematicsof the ex vivo dual perfusion system for the mouse placenta. UA, umbilical artery;
UtA, uterine artery; UV, umbilical vein. j, 5-HT neosynthesis in ex vivo dually
perfused mouse placentas at E17.5. L-tryptophan (100 mM) was injected throughthe uterine artery. 5-HT, neo-synthesized from maternal tryptophan andreleased into the umbilical vein, is evident within 15 min of precursor injection(statistical significance of 5-HT levels variation across time was analysed by oneway ANOVA; *, P , 0.05; **, P , 0.01; data from three independent
experiments are presented as means 6 s.e.m.).

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Percentage of control (×100)areduced 5-HT levels in the placenta but did not reduce 5-HT in the
fetal hindbrain, indicating that exposure to PCPA was too short toinhibit TPH2 activity in dorsal raphe serotonergic neurons (Fig. 3b).
Nevertheless, the brief exposure to PCPA resulted in a significant
decrease in fetal forebrain 5-HT levels (Fig. 3b). The data demonstratethat an exogenous source of 5-HT produced in the placenta is required
to maintain normal levels of forebrain 5-HT during early stages offorebrain development (Supplementary Fig. 5).
The concept that 5-HT from maternal blood could be transferred to
the fetal circulation after crossing the placenta is widely accepted1,2 but
direct or indirect transfer of the molecule has never been demonstrated. Although uptake of exogenous 5-HT by syncytiotrophoblasts
in the human and mouse placenta was demonstrated in vitro2,21, theconcomitant high level of Maoa expression suggests that the placenta
would prevent the vasoconstrictive effect of any free maternal blood
5-HT (a small fraction of total blood 5-HT19) rather than transfer it to
the fetus21. This potentially lethal bioactivity and the quasi-absence ofmaternal blood 5-HT transfer to the fetus has been demonstrated
previously20,22 and also in this study. A recent study concluded thatmaternal 5-HT is crucial for early fetal development (before E111)
based on abnormal phenotypes that emerge in embryos of Tph12/2
pregnant dams. Indirect effects of the Tph12/2 maternal mutation,
however, cannot be excluded as Tph12/2 mice are diabetic23, a pathological condition that affects fetal development independently of adirect maternal 5-HT effect24. Although the importance of a maternalsource of 5-HT before and during placentation remains an open question, our results provide the first direct evidence for maternal influenceson fetal brain development through a precursor that is metabolized
directly by the placenta. Given our demonstration of synthetic capability
5-HT (ng mg–1 protein)period. In a second strategy, we specifically blocked placental TPH1
enzyme activity by microinjecting small volumes of the TPH inhibitor
p-chlorophenyalanine (PCPA) directly into the labyrinth zone of
E14.5 placentas in utero (Fig. 3a). To minimize nonspecific effects
due to diffusion of the drug into the maternal and fetal blood compartments, placental and fetal brain tissues were collected after a short
30-min period of drug exposure. This pharmacological manipulation

Total 5-HT
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Figure 3 | HPLC measures of 5-HT concentrations in E14.5 hindbrain,
forebrain and placenta of in utero PCPA-injected mice. a, Illustration of thein vivo injection procedure: PCPA or vehicle solution was injected into thelabyrinth zone of the placenta. After 30 min, dams were killed and tissue
collected and processed for HPLC. Uc, umbilical cord; Ut, uterus; UtA, uterine
artery. b, Compared to vehicle injection (untreated, n 5 6), placental PCPA
injection (1PCPA, n 5 4) has no significant (NS) effect on 5-HT concentrationin the hindbrain, whereas it significantly lowers 5-HT concentration in theforebrain and the placenta (statistical significance versus 5-HT levels in
untreated tissue was analysed by Student’s t-test; *, P , 0.05; data are presented
as means 6 s.e.m.). c, Model of the progressive switch of the source of 5-HT in
the fetal forebrain, from an early exogenous (placental, blue line) to a later
endogenous (5-HT axons, red line) source. The green boxes represent theamount of 5-HT measured in the Pet12/2 mice, which lack most of theendogenous neuronal source. ctx, cortex; DR, dorsal raphe; DT, dorsal thalamus.
2 1 A P R I L 2 0 1 1 | VO L 4 7 2 | N AT U R E | 3 4 9©2011 Macmillan Publishers Limited. All rights reserved

RESEARCH LETTER
in the early human placenta, it will be clinically important to define thespecific time period during human pregnancy for this placental influence on brain development. The present results also emphasize the needto examine fetal and placental tryptophan availability. Mutations intryptophan 2,3-dioxygenase degrading enzymes (TDO1 and TDO2,
which are expressed in the placenta25) affect neurogenesis, produce
anxiety-related behaviour in mice26 and are associated with increased
risks of schizophrenia, bipolar disorder and autism27,28. Our results provide a mechanism through which alterations of tryptophan metabolic
pathways in the placenta would affect placental 5-HT synthesis and fetal
forebrain development.
Our study provides a new framework for understanding the complexity of maternal-fetal relationships that can influence brain structureand function. We focused on the 5-HT system and showed that there isa progressive switch from an early dependence on an exogenous(placental) source of 5-HT to a later endogenous brain source(Fig. 3c). The exogenous source of 5-HT is provided to the forebrain
during developmental epochs that include cortical neurogenesis, migration and initial axon targeting10. These events can be negatively affectedby disrupting 5-HT signalling: we demonstrated that a forebrainspecific disruption of 5-HT signalling in vivo affects axon guidance,
leading to abnormal thalamocortical axon trajectories4. This phase in
the mouse corresponds to the first and early second trimesters in thehuman: prenatal periods that are associated with greater risk for mental
illnesses due to maternal perturbations29,30. Thus, translation of ourfindings to those corresponding periods in the human will be of significant clinical relevance.

METHODS SUMMARY
Animals and reagents. Timed-pregnant CD-1 mice were purchased from theCharles River Laboratory. Plug date was considered E0.5 and the age of individual
embryos was confirmed by measuring the crown–rump length and checking fordevelopmental landmarks such as digits and eye formation. Pet1 (ref. 3) knockout(2/2), heterozygotes (1/2) and wild-type (1/1) littermate embryos were generated
by crossing Pet11/2 males and females. Sert1/2 embryos were obtained by crossing
Sert2/2 females19 with wild-type C57BL/6 males. The Pet1 and Sert knockout lines
have been backcrossed on the C57Bl/6J background for .10 generations. The Maoa
spontaneous knockout mouse (sMaoa KO) was described earlier16. All procedures
using mice were approved by the Institutional Animal Care and Use Committee atUniversity of Southern California and conformed to NIH guidelines. Unless otherwise
noted, all reagents and antibodies were purchased from Sigma. Protocols for HPLC,
tissue staining, in vitro 5-HT synthesis assays, MAOA enzymatic activity assays, invivo synthesis inhibition assays and ex vivo placental perfusions are described in
Methods.
Human placenta samples. Normal human placental villi samples were obtained
from women undergoing elective pregnancy terminations at the Reproductive
Options Clinic at the Los Angeles County (LAC) and University of Southern
California (USC) Medical Center. For the purposes of this study, the tissue wascarefully dissected by the pathologist and immediately flash-frozen in liquid nitrogen. No patient characteristics other than gestational age were recorded. The
University of Southern California Health Science Campus Institutional Review
Board approved this study.
Full Methods and any associated references are available in the online version of
the paper at www.nature.com/nature.
Received 24 August 2010; accepted 24 February 2011.
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19. Chen, J. J. et al. Maintenance of serotonin in the intestinal mucosa and ganglia ofmice that lack the high-affinity serotonin transporter: Abnormal intestinal motility
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20. Howd, R. A., Nelson, M. F. & Lytle, L. D. L-tryptophan and rat fetal brain serotonin.
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21. Balkovetz, D. F., Tiruppathi, C., Leibach, F. H., Mahesh, V. B. & Ganapathy, V.
Evidence for an imipramine-sensitive serotonin transporter in human placental
brush-border membranes. J. Biol. Chem. 264, 2195–2198 (1989).
22. Robson, J. M. & Senior, J. B. The 5-hydroxytryptamine content of the placenta andfoetus during pregnancy in mice. Br. J. Pharmacol. Chemother. 22, 380–391 (1964).
23. Kim, H. et al. Serotonin regulates pancreatic beta cell mass during pregnancy.
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25. Suzuki, S. et al. Expression of indoleamine 2,3-dioxygenase and tryptophan 2,3dioxygenase in early concepti. Biochem. J. 355, 425–429 (2001).
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1756-6606-2-8 (2009).
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank H.H. Wu and K. Eagleson for discussions and commentson the manuscript. We thank R. Johnson (Vanderbilt HPLC core facility), L. Zhang fortechnical help and E. Meng for discussions. This work was supported by the NICHD(grant 5R21HD065287 to A.B.), NARSAD (A.B.) and the NIMH (grant R01MH39085 to
J.C.S. and 1P50MH078280A1 to R.D.B. and P.L.).
Author Contributions A.B. conducted the experiments with assistance from N.G. inplacenta studies, K.C. and J.C.S. in providing mutant mouse strains and in MAOA
enzymatic assays. R.D.B. and E.S.D. provided mutant mouse strains and M.L.W. and J.K.
provided human tissue. A.B. and P.L. conceived this study, interpreted the data andwrote the manuscript. All authors commented on the paper.
Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.
Readers are welcome to comment on the online version of this article atwww.nature.com/nature. Correspondence and requests for materials should be
addressed to A.B. (bonnin@usc.edu) or P.L. (plevitt@usc.edu).

3 5 0 | N AT U R E | VO L 4 7 2 | 2 1 A P R I L 2 0 1 1©2011 Macmillan Publishers Limited. All rights reserved

LETTER RESEARCHMETHODS
Animals and reagents. Timed-pregnant CD-1 mice were purchased from theCharles River Laboratory. Plug date was considered E0.5 and the age of individual
embryos was confirmed by measuring the crown–rump length and checking fordevelopmental landmarks such as digits and eye formation. Pet1 (ref. 3) knockout(2/2), heterozygotes (1/2) and wild-type (1/1) littermate embryos were generated by crossing Pet11/2 males and females. Sert1/2 embryos were obtained bycrossing Sert2/2 females19 with wild-type C57BL/6 males. The Pet1 and Sert
knockout lines have been backcrossed on the C57Bl/6J background for .10 generations. The Maoa spontaneous knockout mouse (sMaoa KO) was describedearlier16. All procedures using mice were approved by the Institutional Animal
Care and Use Committee at University of Southern California and conformed to
NIH guidelines. Unless otherwise noted, all reagents and antibodies were purchased from Sigma.
Human placenta samples. Normal human placental villi samples were obtained
from women undergoing elective pregnancy terminations at the Reproductive
Options Clinic at the Los Angeles County (LAC) and University of Southern
California (USC) Medical Center. For the purposes of this study, the tissue wascarefully dissected by the pathologist and immediately flash-frozen in liquid nitrogen. No patient characteristics other than gestational age were recorded. The
University of Southern California Health Science Campus Institutional Review
Board approved this study.
HPLC measures. Hindbrains and forebrains were homogenized, using a tissue
dismembrator, in 100–750 ml of 0.1 M trichloroacetic acid TCA, which contains
1022 M sodium acetate, 1024 M EDTA, 5 ngml21 isoproterenol (as internal standard)and 10.5% methanol (pH 3.8). Samples were spun at 10,000g for 20 min. The
supernatant was removed and stored at 280 uC. The pellet was used for protein
analysis; total protein concentrations were determined using the BCA Protein
Assay Kit (Pierce Chemical Company). The supernatant was then thawed andspun for 20 min. The concentration of biogenic amines in each sample was determined by a specific HPLC assay using an Antec Decade II (oxidation, 0.5 V)
electrochemical detector operated at 33 uC. 20 ml samples of supernatant were
injected using a Water 7171 autosampler onto a PhenomenexNucleosil (5 u,
100 A) C18 HPLC column (150 3 4.60 mm). Biogenic amines were eluted with amobile phase consisting of 89.5% 100 mM TCA, 10 mM sodium acetate, 0.1 mM
EDTA and 10.5% methanol (pH 3.8). Solvent was delivered at 0.6 mlmin21 using a
Waters 515 HPLC pump. Using this HPLC solvent, the biogenic amines eluted in
the following order: noradrenaline, adrenaline, DOPAC, dopamine, 5-HIAA,
HVA, 5-HT and 3-MT. HPLC control and data acquisition were managed byMillennium 32 software.
Statistical analysis. Measures in Pet12/2 and wild-type littermate embryos(n $ 6 per genotype) are presented as mean 6 s.e.m. Statistical comparison of
measures between genotypes across development was performed using a oneway ANOVA (Fig. 1). Differences were considered statistically significant at
P , 0.05. Single statistical comparison between two groups was performed using
a two-tailed Student’s t-test (for example, comparison of measures in Pet12/2
and wild-type littermates or sMaoa2/2 and wild-type littermates at a single age
or in utero PCPA- versus vehicle-injected mice; see Fig. 3 and Supplementary
Tables 1 and 2).
In vitro 5-HT synthesis assays. Pregnant CD-1 mice were anaesthetized by an
intraperitoneal injection of 0.2 ml pentobarbital and perfused through the left
ventricle with 100 ml of phosphate buffered saline (PBS). The uterus was immediately dissected and the resulting embryos were placed on ice in PBS. The placenta, forebrain and hindbrain were removed and kept on ice in 1.5 ml eppendorf
tubes. The collected tissue was homogenized in 3 volumes of 0.05 M Tris buffer,
pH 7.5, containing 1 mM dithiothreitol and 1 mM EGTA. Homogenates were
centrifuged at 29,000g for 15 min at 4 uC and the supernatants were placed onice; tryptophan hydroxylase assays were performed as previously described31.
Briefly, 70 mg of protein from the supernatant was added to tubes containing
100 ml of: 0.05 M Tris buffer, pH 7.5, 1 mM EGTA, 15 mg catalase, 250 mM
L-tryptophan, and 50 mM tetrahydrobiopterin (BH4; a cofactor required for
TPH1 and TPH2 activity32). Reactions were performed for 10 and 30 min at
37 uC and stopped by flash freezing in liquid nitrogen. L-tryptophan metabolism
was determined by measuring 5-hydroxytryptophan (5-HTP), 5-hydroxytyptamine(5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) using HPLC with electrochemical
detection (see above). Protein concentrations were determined using a DC protein
assay (Bio-Rad).
In vitro MAO-A activity measures. MAO-A activity was determined by incubating forebrain and hindbrain homogenates with [14C]-5-HT (1 mM) at 37 uC for
20 min. At the end of the incubation, the products were extracted and the radioactivity determined as described previously33.
Immunohistochemistry. Brain sections were processed for immunohistochemistry using the following primary antibodies: anti-5-HT (rabbit, 1:1,000; goat,

1:1,000, Sigma), anti-NetrinG1a (goat, 1:250, RnD), anti-SERT (rabbit, 1:200,
Sigma), anti-5-HTP (rabbit, 1:1,500; ImmunoStar) and anti-AADC (rabbit,
1:500, Abcam). The secondary antibodies (Jackson ImmunoResearch) were:Cy2- or Cy3-conjugated donkey anti-rabbit IgG (1:800), rhodamin-X conjugateddonkey anti-goat (1:200) and biotin-conjugated anti-rabbit (1:1,000). 20 mm brain
sections were permeabilized, and pre-incubated with blocking solution (0.1%
Triton X-100, 2% fetal bovine serum) in PBS for 2 h and then incubated overnightwith a primary antibody diluted in the same blocking solution at 4 uC. Sections
were washed with PBS 1 0.1% Triton X-100 and incubated with appropriate
secondary antibodies diluted in same blocking solutions for a minimum of 2 h
at room temperature (22-25 uC). Sections were then washed with PBS 1 0.1%
Triton X-100 and mounted using a fluorescence anti-fade medium (Prolong
Gold, Vector Laboratories). For non-fluorescent immunohistochemistry (forexample, AADC staining), similar procedures were followed, except that thesections were incubated in methanol with 3% H2O2 for 15 min to quench endogenous peroxidase activity. The reaction product was visualized using a Vector
ABC Elite Kit (Vector Laboratories). Images were acquired on a Zeiss AxoplanII
microscope coupled to an AxioCamHRc camera using Axiovision 4.1 software(Zeiss). For placental immunochemistry, 5 mm frozen sections were thawed for
15 min at room temperature then washed in a mixture of 13 PBS containing 0.1%
Triton X-100 (PBS-T) for 15 min at room temperature to allow for membrane
permeabilization. Endogenous HRP was quenched by incubating the slides in asolution of methanol containing 3% hydrogen peroxide for 15 min at room temperature. The slides were then incubated for 2 h in a solution of PBS-T containing
2% Fetal Bovine Serum (Blocking Solution). The slides were then incubatedovernight with anti-MCT1 (MCT1 is a marker of syncytiotrophoblasts34; chicken;
0.5 mgml21) or anti-AADC or anti-TPH1 (rabbit; 1:500) overnight at room temperature. Slides were then washed 3 3 10 min in PBS-T and incubated withappropriate secondary antibodies (biotin-conjugated anti-chicken; 1:1,000 or biotin-conjugated anti-rabbit; 1:1,000; Jackson ImmunoResearch) for 2 h at room
temperature. After 6 3 5 min washes, slides were processed for HRP visualization
as described above.
In utero inhibition of placental TPH1. Pregnant CD-1 mice were anaesthetizedby an intraperitoneal injection of 0.5–0.7 ml of 10% Nembutal and uterine horns
exposed as previously described4. Glass micropipettes were filled with a solutionof 10 mM PCPA (4-Chloro-DL-phenylalanine; an inhibitor of TPH1 enzymatic
activity35) in PBS and 0.1% fast green dye (FCF; Sigma). FCF was used to monitor
visually the diffusion of solution into the fetal villi of the placenta. 2–5 ml were
injected through the uterine wall into the labyrinth region in half of the placentas(n 5 4–6) for each pregnant mouse (n 5 3); remaining placentas were injectedwith vehicle only. After 30 min, the dam was killed and placentas and corresponding forebrains and hindbrains collected and quickly frozen in liquid N2. Samples
were then processed for HPLC analysis as described above.
Ex vivo dual perfusion of the mouse placenta. A single placenta with uterine andumbilical arteries and veins attached was harvested from a pregnant CD-1 mouseat E17.5 in warm PBS. The placenta was rapidly transferred to a thermostatedtissue culture chamber (60 mm diameter, Warner Instruments) to which warm
(37 uC) and oxygenated (95% O2, 5% CO2) PBS solution was constantly provided.
Using a dissection microscope, micro-catheters were carefully attached to theuterine artery on the maternal side and umbilical artery and vein on the fetal sideof the placenta. The uterine artery at E17.5 (internal diameter is approximately
150 mm) was cannulated using a 150 mm outside diameter polyethylene microcatheter. Micro-catheters were kept solidly attached to the arteries or veins using
micro-clamps (F.S.T.) and suture. The other end of the uterine artery microcatheter was connected to an 8-channel micro-manifold (Automate Scientific);each channel’s polyethylene tubing was then connected to an individual fastswitching valve (1.5 ms response time; Automate Scientific); the artificial maternal
solution (warm oxygenated PBS) was delivered to each individual channel througha pressurized cylinder connected to each valve. An electronic valve controller
enabled timed sequential switching of maternal solutions. The input pressure
was calibrated to allow a maximal flow rate of 10–20 mlmin21 through the uterine
artery micro-catheter. On the fetal side, the umbilical artery and vein were similarly cannulated; for E17.5 placentas polyethylene tubing with 100 mm internal
diameter was used. The other end of the umbilical artery micro-catheter was
directly connected to a 5 ml syringe containing the artificial fetal solution (PBS
with 0.01% fast green dye FCF, Sigma) and mounted on a precision, computercontrolled syringe pump (Braintree Scientific); the infusion flow rate was set at
6 mlmin21. For fetal eluate collection, the umbilical vein was cannulated to 100 mm
internal diameter polyethylene tubing; this tubing was then connected to a speciallymodified SmartSquirt system (Automate Scientific) which consisted of a micromanifold connected to eight independent 3-ml reservoirs in which a controlled
negative pressure (215 PSI) was individually applied. The integrated fast-switchingvalves system allowed the application of negative pressure to each reservoir©2011 Macmillan Publishers Limited. All rights reserved

RESEARCH LETTER
independently and sequentially, thus allowing individual fraction collection overtime. The addition of fast green dye to the fetal perfusate allowed monitoring for any
leak through and around the placenta during the perfusion.
31. Johansen, P. A., Jennings, I., Cotton, R. G. & Kuhn, D. M. Tryptophan hydroxylase isphosphorylated by protein kinase A. J. Neurochem. 65, 882–888 (1995).
32. Kuhn, D. M., Ruskin, B. & Lovenberg, W. Tryptophan hydroxylase. The role ofoxygen, iron, and sulfhydryl groups as determinants of stability and catalytic
activity. J. Biol. Chem. 255, 4137–4143 (1980).

33. Chen, K. et al. Forebrain-specific expression of monoamine oxidase A reduces
neurotransmitter levels, restores the brain structure, and rescues aggressive
behavior in monoamine oxidase A-deficient mice. J. Biol. Chem. 282, 115–123(2007).
34. Nagai, A., Takebe, K., Nio-Kobayashi, J., Takahashi-Iwanaga, H. & Iwanaga, T.
Cellular expression of the monocarboxylate transporter (MCT) family in theplacenta of mice. Placenta 31, 126–133 (2010).
35. Koe, B. K. & Weissman, A. p-Chlorophenylalanine: a specific depletor of brain
serotonin. J. Pharmacol. Exp. Ther. 154, 499–516 (1966).

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