Download Deficiency of brain 5-HT synthesis but serotonergic neuron

J Neural Transm
DOI 10.1007/s00702-008-0096-6
BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - RAPID COMMUNICATION
Deficiency of brain 5-HT synthesis but serotonergic neuron
formation in Tph2 knockout mice
Lise Gutknecht Æ Jonas Waider Æ Stefanie Kraft Æ
Claudia Kriegebaum Æ Bettina Holtmann Æ
Andreas Reif Æ Angelika Schmitt Æ Klaus-Peter Lesch
Received: 30 June 2008 / Accepted: 6 July 2008
Ó Springer-Verlag 2008
Abstract The relative contribution of the two tryptophan
hydroxylase (TPH) isoforms, TPH1 and TPH2, to brain
serotonergic system function is controversial. To investigate the respective role of TPH2 in neuron serotonin
(5-HT) synthesis and the role of 5-HT in brain development, mice with a targeted disruption of Tph2 were
generated. The preliminary results indicate that in Tph2
knockout mice raphe neurons are completely devoid of
5-HT, whereas no obvious alteration in morphology and
fiber distribution are observed. The findings confirm the
exclusive specificity of Tph2 in brain 5-HT synthesis and
suggest that Tph2-synthesized 5-HT is not required for
serotonergic neuron formation.
Keywords Tryptophan hydroxylase 2 Tph2 Knockout Serotonin Development Depression Attention-deficit/hyperactivity disorder
L. Gutknecht (&) J. Waider S. Kraft C. Kriegebaum A. Reif A. Schmitt K.-P. Lesch (&)
Molecular and Clinical Psychobiology,
Department of Psychiatry, Psychosomatics,
and Psychotherapy, University of Wuerzburg,
Fuechsleinstrasse 15, 97080 Wuerzburg, Germany
e-mail: [email protected]
K.-P. Lesch
e-mail: [email protected]
B. Holtmann
Rudolf Virchow Center, DFG Research Center for Experimental
Biomedicine, University of Wuerzburg, Versbacherstrasse 9,
97078 Wuerzburg, Germany
Introduction
The two tryptophan hydroxylase (TPH) isoforms, TPH1
and TPH2, act as rate-limiting enzymes in serotonin
(5-hydroxytryptamine, 5-HT) synthesis (Walther and Bader
2003; McKinney et al. 2005). Based on evidence supporting exclusive brain specificity of TPH2, particularly
this isoform has been implicated in cognition and emotion
regulation in the general population (Gutknecht et al. 2007;
Canli et al. 2005; Herrmann et al. 2007) as well as in
the pathophysiology of syndromal dimensions of various
neuropsychiatric conditions, such as depression (Zhou et al.
2005), bipolar disorder (Harvey et al. 2004) and attentiondeficit/hyperactivity disorder (ADHD) (Walitza et al. 2005;
Baehne et al. 2008). However, there is persisting controversy regarding the respective roles of the two TPH
isoforms in brain 5-HT synthesis and the role of 5-HT in
neuron formation and brain development.
While the two initial studies revealing the existence of
TPH2 (Walther et al. 2003; Côté et al. 2003) concluded that
the expression of the two isoforms is mutually exclusive,
designating TPH1 as the peripheral form, extremely
abundant in the pineal gland and enterochromaffine cells,
and TPH2 representing the neuronal subtype, specifically
expressed in serotonergic neurons of the raphe nuclei,
several subsequent differential expression studies reported
detectable TPH1 expression in raphe neurons of the rat
(Patel et al. 2004; Malek et al. 2005), the mouse (Gundlah
et al. 2005) and humans (Zill et al. 2007). Nakamura et al.
(2006), who described preferential expression of Tph1
mRNA at postnatal day 21 (P21) in the murine raphe,
assumed that Tph1 plays a critical role during late developmental stages of the brain. Recently, we have conducted
a systematic analysis of Tph1 and Tph2 expression in adult
mouse and human brain, as well as during pre- and
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L. Gutknecht et al.
postnatal development of the murine brain (L. Gutknecht
et al., submitted). Four complementary approaches (qRTPCR, in situ hybridization, immunohistochemistry, and
Western blot) did not reveal significant Tph1 expression in
the raphe of both species. These results indicate exclusive
Tph2 expression in raphe neurons in adulthood as well as
during development.
To further confirm the role of TPH2 in raphe neuron
5-HT synthesis and eventually the role of 5-HT in brain
development and function, we inactivated Tph2 in mice by
homologous recombination and Cre-assisted deletion
resulting in constitutive Tph2 null mutants with a complete
loss of 5-HT immunodetection in raphe serotonergic
neurons.
Methods
Generation of Tph2 knockout mice
Genomic contigs of Tph2 encompassing exon 5 and
flanking sequence were obtained by screening of a 129/Ola
mouse Cosmid library (library number 121, RZPD, Berlin,
Germany). Positive clones were further characterized by
restriction mapping and Southern blot analysis. For the
gene targeting construct, a *4.4 kb XhoI-XhoI fragment
located upstream of exon 5 was selected as the 50 flank,
while a *3.4 kb XhoI-XhoI fragment containing exon 5
was projected to induce the targeted deletion (Fig. 1a). The
elimination of Tph2 exon 5, which codes for an amino acid
sequence at the start of the catalytic domain and ends with
a partial codon, was predicted to create a shift in the
reading frame resulting in a truncated non-functional Tph2
protein. A LoxP site was inserted between these two
fragments by cloning into pKSLox (modified pKS bluescript including one LoxP site). A *2.0 kb XhoI-XmnI
fragment, serving as 30 flank, was cloned into pKSLNL
Fig. 1 Generation and genotyping of Tph2 null mutants. a Targeting
strategy. Top: targeting vector designed to remove exon 5 and
flanking sequences (XhoI-XhoI deletion, pink) by homologous
recombination in wildtype (WT) genomic DNA (middle). Exons 2–
6 are depicted by vertical black boxes. XhoI-XhoI 50 and XhoI-XmnI 30
flanks are indicated in green and blue, respectively. Bottom: predicted
structure of the Tph2 null allele (KO) after excision by Nestin-Cre
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(modified pKS bluescript including a neomycine-resistance
(NEO) cassette flanked by two LoxP sites) downstream of
the NEO cassette. Then, the NotI-ApaI fragment comprising 50 flank-LoxP-deletion was excised from the pKSLox
vector and inserted 50 of the NEO cassette of 30 flankcontaining pKSLNL. After verification by restriction
analysis and sequencing, the targeting construct was linearized with NotI and electroporated into 129 R1 embryonic
stem (ES) cells which were subjected to G418 selection.
Targeted homologous recombination was confirmed by
PCR and Southern blot analysis. The NEO cassette was
removed by transient expression of Cre recombinase in the
recombinant ES cells resulting in a Tph2 allele with exon 5
flanked by LoxP sites. After verification of NEO cassette
excision by PCR, Southern blot and sequencing, an ES
clone was injected into C57BL/6 blastocysts and implanted
into pseudopregnant mice. A chimeric male displaying
germ-line transmission was then used to propagate the
floxed Tph2 allele on a C57BL/6 background for several
generations. Tph2+/- were obtained by breeding Tph2+/flox
mice with Nestin-Cre transgenics (Tronche et al. 1999)
maintained on a C57BL/6 background. Several intercrossing of Tph2+/flox mice with Tph2+/flox/cre eventually
resulted in Tph2 inactivation in the germline with a transmittable Tph2 null allele in Cre transgene-devoid Tph2-/mice, equivalent to constitutive knockout.
Control wildtype (WT) and null alleles were detected by
duplex PCR using the oligonucleotide 50 - tggggcctgcc
gatagtaacac as reverse primer located in the 50 side of the
30 flank (position of the primers are indicated in Fig. 1a).
Paired to this reverse primer, the forward primer
50 -tggggcatctcaggacgtagtagt, located in the 30 end of the
targeted deletion led to the amplification of a 437 bp
product in the presence of the WT allele, while the forward
primer 50 -caccccaccttgcagaaatgttta, located in the 30 side of
the 50 flank permits amplification of a 387 bp amplicon
indicative of the Tph2 null allele (Fig. 1b). The reliability
recombinase of the sequence flanked by LoxP sites (red triangles).
Horizontal lines illustrate the position of primers used for genotyping.
b Duplex PCR genotyping using 2 forward and 1 reverse primers
indicated in a. Size of the amplicons from homozygous KO (387 bp,
Tph2-/-) and WT (437 bp, Tph2+/+) mice are shown. SM DNA size
marker
Tph2 knockout mouse model
of the PCR-based genotyping procedure was verified by
Southern blot analysis using standard procedures with
genomic DNA derived from ES cells or tail cuts using
cDNA probes (data not shown).
Generation of Tph2-specific antibodies
Tph2-specific antibodies were generated as described in
detail (L. Gutknecht et al., submitted). Briefly, for antiTph2 specific antibodies generation, two peptides were
selected within the murine Tph2 protein based on their
internal characteristics and specificity for this isoform. The
peptides comprising the murine amino acids 458–472
(TRSIENVVQDLRSDL) and 476–488 (CDALNKMNQYLGI) corresponding to the C-terminal portion were
chosen as immunogens. Peptide synthesis, coupling to
keyhole limpet haemocyanin (KLH) and immunization of
rabbits were carried out at Eurogentec (Seraing, Belgium)
following their AS-DOUB-LX protocol. Hyperimmune
serum samples were tested for reactivity and specificity
using Western blot analysis and immunohistochemistry.
Immunohistochemistry for Tph2, 5-HT and 5-HT
transporter
Dissected brains with attached pineal gland from Tph2-/mice and WT littermates (3.5 months of age, 6th generation,
with an estimated *94% C57BL/6 background) were fixed
by immersion in 4% PFA in PBS, cryoprotected in 20%
sucrose, frozen in dry ice-cooled 2-methylbutane and stored
at -80°C until 12 lm cryostat sections were prepared and
mounted on glass slides. Immunohistochemistry of Tph2
was performed using the avidin-biotin method whereas a
polymer-based detection system (EnVision + SystemHRP, DAKO, Glostrup, Denmark) was used for 5-HT and
5-HT transporter (Sert), with diaminobenzidine (DAB) as
chromogen. Frozen sections were allowed to dry at room
temperature (RT) and heat-induced unmasking of epitope
were performed for each antibody at 96°C for 10 min in
10 mM citrate buffer (pH 6.0) containing 0.05% Tween.
Washings were performed between each step 3 9 5 min in
Tris-buffered saline (TBS, pH 7.5). After 40 min of subsequent cooling at RT, and blocking of endogenous
peroxidase activity for 30 min with 0.6% H2O2 in TBS in
case of Tph2, and 10 min using peroxidase blocking solution (DAKO) for 5-HT and Sert, sections were preincubated
for 1 h at RT in blocking solution (BL) containing 5%
normal goat serum (NGS), 2% BSA and 0.25% Triton
X-100 diluted in TBS to prevent non-specific binding.
Tissue sections were then exposed to primary antibodies
diluted 1:900 for Tph2 in TBST for 2 h at RT and overnight
at 4°C, 1:10,000 for 5-HT (rabbit anti-5-HT, Incstar, Stillwater, USA) in BL for 90 min at RT and 1:750 for Sert
(rabbit anti-Sert, Calbiochem, Darmstadt, Germany) overnight at 4°C. For negative control of Tph2 immunostaining,
primary antibodies were omitted from the incubation and
1:900 dilution of the rabbit preimmune serum was used. The
detection of bound anti-Tph2 antibodies was carried out
with biotinylated secondary antibodies (goat anti-rabbit
IgG, 1:900; Vector Laboratories, Wiesbaden, Germany)
diluted in 2% NGS, 2% BSA and 0.25% Triton-X100 in
TBS for 90 min, followed by incubation in streptavidinbiotin-peroxidase complex (Vector Laboratories, Wiesbaden, Germany) for 1 h at RT. Anti-5-HT and anti-Sert
detection was achieved with secondary anti-rabbit antibody
coupled to horse radish peroxidase (DAKO) for 40 min at
RT. Peroxidase activity was visualized using DAB substrate
(metal enhanced; Roche, Penzberg, Germany) or DAB
substrate (DAKO) for 4 min.
Results
Tph2 null mutant mice (Tph2-/-) are viable and do not
show apparent increase in premature lethality during
development or adulthood. Despite a tendency toward a
lower body weight compared to their WT littermates,
Tph2-/- mice do not display obvious developmental
defects including overall brain structure. While Tph2-specific antibodies detect the expected expression pattern of
Tph2 in the soma and fibers of raphe serotonergic neurons
of WT control mice, complete absence of Tph2 immunostaining was observed in Tph2-/- mice (Fig. 2a, b).
Immunohistochemistry of 5-HT in raphe serotonergic
neurons revealed that constitutive gene inactivation is
associated with a loss of Tph2 enzymatic activity. Typical
distribution of 5-HT positive neurons was observed in the
WT control brain, whereas no trace of 5-HT immunoreactivity was detected in the raphe nuclei of Tph2-/- mice
(Fig. 2c, d). In contrast, a comparably strong 5-HT-specific
signal was observed in the pineal gland of both WT and
null mutant mice (Fig. 2, inserts in c and d). Immunodetection of the Sert, located on the soma, dendrites and
terminals of serotonergic neurons, failed to reveal obvious
differences in serotonergic neuron morphology including
fiber distribution in Tph2-/- mice compared to WT controls. A similar pattern of innervation by Sert-positive
soma, dendrites and terminal was observed in the raphe
nuclei as well as across several target regions of the brain,
including the hippocampus. (Fig. 2, e and f, inserts).
Discussion
Tph2 was inactivated in mice using the Cre/LoxP strategy
resulting in viable Tph2 null mutants. The preliminary
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L. Gutknecht et al.
Fig. 2 Immunohistochemical
analysis of homozygous WT
(top panels) and KO (bottom
panels) mice. Representative
pictures from raphe nuclei (a–d)
and hippocampus (e, f) coronal
sections are displayed. a, b
Detection of Tph2 with isoformspecific Tph2 antibody at the
level of dorsal (DR) and median
(MnR) raphe. c, d Detection of
5-HT at the level of DR, MnR
and pineal gland (PG, inserts).
e, f Detection of Sert in different
layers of the hippocampus (H
hilus; GL granule cell layer; ML
molecular cell layer; LM
lacunosum-molecular layer; RL
radiatum layer) and DR
(inserts). All scales bars
represent 200 lm
results indicate that raphe serotonergic neurons in mice
with a targeted disruption of Tph2 are completely devoid of
5-HT immunoreactivity, whereas no alterations in morphology and fiber distribution appear to occur. Our findings
therefore confirm the exclusiveness of Tph2 in raphe
neuron 5-HT synthesis in mice and provide preliminary
evidence that Tph2-synthesized 5-HT may not be required
for raphe serotonergic neuron development, maturation,
maintenance, and survival.
Our recent systematic analysis of Tph1 and Tph2
expression in adult mouse and human brain, as well as
during pre- and postnatal development of the murine brain,
did not reveal significant Tph1 expression in the raphe of
both species, whereas the pineal gland displayed high
concentrations of this isoform. Based on these findings, we
concluded that Tph1 is unlikely to contribute to brain 5-HT
synthesis neither in adulthood nor during development
(L. Gutknecht et al., submitted). This view of exclusive
Tph2 specificity in brain 5-HT synthesis is strongly supported by the complete absence of 5-HT immunostaining
in serotonergic neurons of the raphe nuclei of Tph2-/mice as compared to WT controls and the unchanged
strong Tph1-dependent 5-HT immunoreactivity in the
pineal gland of both the Tph2-/- and WT control mice.
Moreover, the lack of Tph2-dependent 5-HT synthesis
during early brain development does not seem to reverse
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repression of Tph1 transcription as an adaptive mechanism
to initiate and maintain physiological 5-HT system function. The apparent failure of Tph1 to compensate for Tph2related 5-HT deficiency in brain also underscores the relevance of allelic variation in TPH2 function resulting in
alterations of brain 5-HT system function associated with
cognitive and emotional processes as well as the pathophysiology of affective disorders and ADHD.
Although Tph2 inactivation leads to stalled 5-HT synthesis in the brain, the virtually unaltered immunostaining
of the serotonergic cell-specific marker Sert on the soma,
dendrites, and fibers of raphe neural cells suggests the
machinery of specification and other differentiation process,
including neurite outgrowth towards projection areas of
serotonergic neurons is not dependent on the synthesis or
release of 5-HT. Extensive evidence supports a morphogenic effect of 5-HT on proliferation, differentiation,
migration, and survival of neural cells (Di Pino et al. 2004;
Gaspar et al. 2003). During ontogeny, 5-HT appears long
before maturation of raphe serotonergic neurons suggesting
a fundamental role in embryonic brain development as well
as on the development and maturation of the serotonergic
system itself. Although in vivo studies generally underscore
this notion (Gaspar et al. 2003; Vitalis et al. 2007), several
recently generated mouse models for 5-HT deficiency lead
to mice with no apparent developmental abnormalities.
Tph2 knockout mouse model
Among these models are Nkx2.2 knockout mice in
which 5-HT neurons are preserved only in the dorsal raphe
(Briscoe et al. 1999), conditional Lmx1b knockout mice
lacking most 5-HT neuron in adult brain (Ding et al. 2003;
Zhao et al. 2006), Pet1-deficient mice displaying a 70%
loss of differentiated raphe serotonergic neurons (Hendricks et al. 2003), BALB/cJ mouse strain which present a
*40% decreased 5-HT concentration, compared to 129X1/
SvJ strain, in frontal cortex and striatum linked to proline
substitution at amino acid position 447 of the Tph2 protein
(Zhang et al. 2004), and mutant Tph2 mice, carrying the
rare human variant R441H, which show a *80% reduced
brain 5-HT (Beaulieu et al. 2008). In contrast to Tph2-/mice however, none of these mouse models permit investigation of the consequences of a complete loss of brain
5-HT synthesis, either during development or across the
life span.
In Lmx1b and Pet1 knockout mice 5-HT neurons seem
to be generated but fail to further differentiate. Remaining
5-HT-positive neurons in Pet1-deficient mice display
reduced expression of Tph2 and Sert, whereas in conditional Lmx1b knockouts, early transient raphe neuron 5-HT
synthesis up to embryonic day E14.5 is not maintained in
later developmental stages. Taken together, these and our
findings suggest that differentiation and maintenance of a
serotonergic phenotype is independent on the production of
serotonin by the maturating neurons. Nevertheless, further
detailed studies are required to evaluate to which degree
the serotonergic phenotype of these neurons is complete,
specifically how the expression pattern and function of
markers, such as 5-HT1A and 5-HT1B autoreceptors as
well as the function of these neurons and their electrophysiological properties such as firing rate, among
numerous other parameters that reflect 5-HT system function, are altered by a deficiency of 5-HT synthesis.
Conclusion
The Tph2 knockout mouse represents a novel rodent model
which will be useful for investigations of the developmental, adaptive, and functional consequences of altered
brain-specific 5-HT synthesis and their role in cognition,
emotion regulation, and neuropsychiatric disease risk.
Acknowledgments The authors are grateful to S. Wiese for providing cloning vectors and construct advise as well as to M. Sendtner,
C. Gross and P. Gaspar for helpful discussions and continuing support. The authors also thank N. Steigerwald for the genotyping of
mice and H. Brunner for expertise in animal husbandry. This research
is supported by the Deutsche Forschungsgemeinschaft (SFB 581;
KFO 125; GRK 1253 fellowship to CK., GSLS fellowship to J.W.),
European Commission (NEWMOOD LSHMCT-2003-503474),
and the Bundesministerium für Bildung und Forschung (IZKF 01
KS 9603).
References
Baehne CG, Ehlis AC, Plichta MM, Conzelmann A, Pauli P, Jacob C,
Gutknecht L, Lesch KP, Fallgatter AJ (2008) Tph2 gene variants
modulate response control processes in adult ADHD patients and
healthy individuals. Mol Psychiatry (Online 22 April 2008)
Beaulieu JM, Zhang X, Rodriguiz RM, Sotnikova TD, Cools MJ,
Wetsel WC, Gainetdinov RR, Caron MG (2008) Role of GSK3
beta in behavioral abnormalities induced by serotonin deficiency.
Proc Natl Acad Sci USA 105:1333–1338
Briscoe J, Sussel L, Serup P, Hartigan-O’Connor D, Jessell TM,
Rubenstein JL, Ericson J (1999) Homeobox gene Nkx2.2 and
specification of neuronal identity by graded Sonic hedgehog
signalling. Nature 398:622–627
Canli T, Congdon E, Gutknecht L, Constable RT, Lesch KP (2005)
Amygdala responsiveness is modulated by tryptophan hydroxylase-2 gene variation. J Neural Transm 112:1479–1485
Côté F, Thevenot E, Fligny C, Fromes Y, Darmon M, Ripoche MA,
Bayard E, Hanoun N, Saurini F, Lechat P, Dandolo L, Hamon M,
Mallet J, Vodjdani G (2003) Disruption of the nonneuronal tph1
gene demonstrates the importance of peripheral serotonin in
cardiac function. Proc Natl Acad Sci USA 100:13525–13530
Di Pino G, Moessner R, Lesch P, Lauder J, Persico A (2004) Roles for
serotonin in neurodevelopment: more than just neural transmission. Curr Neuropharmacol 2:403–418
Ding YQ, Marklund U, Yuan W, Yin J, Wegman L, Ericson J,
Deneris E, Johnson RL, Chen ZF (2003) Lmx1b is essential for
the development of serotonergic neurons. Nat Neurosci 6:933–
938
Gaspar P, Cases O, Maroteaux L (2003) The developmental role of
serotonin: news from mouse molecular genetics. Nat Rev
Neurosci 4:1002–1012
Gundlah C, Alves SE, Clark JA, Pai LY, Schaeffer JM, Rohrer SP
(2005) Estrogen receptor-beta regulates tryptophan hydroxylase–
1 expression in the murine midbrain raphe. Biol Psychiatry
57:938–942
Gutknecht L, Jacob C, Strobel A, Kriegebaum C, Muller J, Zeng Y,
Markert C, Escher A, Wendland J, Reif A, Mossner R, Gross C,
Brocke B, Lesch KP (2007) Tryptophan hydroxylase–2 gene
variation influences personality traits and disorders related to
emotional dysregulation. Int J Neuropsychopharmacol 10:309–
320
Harvey M, Shink E, Tremblay M, Gagne B, Raymond C, Labbe M,
Walther DJ, Bader M, Barden N (2004) Support for the
involvement of TPH2 gene in affective disorders. Mol Psychiatry 9:980–981
Hendricks TJ, Fyodorov DV, Wegman LJ, Lelutiu NB, Pehek EA,
Yamamoto B, Silver J, Weeber EJ, Sweatt JD, Deneris ES
(2003) Pet-1 ETS gene plays a critical role in 5-HT neuron
development and is required for normal anxiety-like and
aggressive behavior. Neuron 37:233–247
Herrmann MJ, Huter T, Muller F, Muhlberger A, Pauli P, Reif A,
Renner T, Canli T, Fallgatter AJ, Lesch KP (2007) Additive
effects of serotonin transporter and tryptophan hydroxylase-2
gene variation on emotional processing. Cereb Cortex 17:1160–
1163
Malek ZS, Dardente H, Pevet P, Raison S (2005) Tissue-specific
expression of tryptophan hydroxylase mRNAs in the rat
midbrain: anatomical evidence and daily profiles. Eur J Neurosci
22:895–901
McKinney J, Knappskog PM, Haavik J (2005) Different properties of
the central and peripheral forms of human tryptophan hydroxylase. J Neurochem 92:311–320
Nakamura K, Sugawara Y, Sawabe K, Ohashi A, Tsurui H, Xiu Y,
Ohtsuji M, Lin QS, Nishimura H, Hasegawa H, Hirose S (2006)
123
L. Gutknecht et al.
Late developmental stage-specific role of tryptophan hydroxylase 1 in brain serotonin levels. J Neurosci 26:530–534
Patel PD, Pontrello C, Burke S (2004) Robust and tissue-specific
expression of TPH2 versus TPH1 in rat raphe and pineal gland.
Biol Psychiatry 55:428–433
Tronche F, Kellendonk C, Kretz O, Gass P, Anlag K, Orban PC, Bock
R, Klein R, Schutz G (1999) Disruption of the glucocorticoid
receptor gene in the nervous system results in reduced anxiety.
Nat Genet 23:99–103
Vitalis T, Cases O, Passemard S, Callebert J, Parnavelas JG (2007)
Embryonic depletion of serotonin affects cortical development.
Eur J Neurosci 26:331–344
Walitza S, Renner TJ, Dempfle A, Konrad K, Wewetzer C, Halbach
A, Herpertz-Dahlmann B, Remschmidt H, Smidt J, Linder M,
Flierl L, Knolker U, Friedel S, Schafer H, Gross C, Hebebrand J,
Warnke A, Lesch KP (2005) Transmission disequilibrium of
polymorphic variants in the tryptophan hydroxylase-2 gene in
attention-deficit/hyperactivity disorder. Mol Psychiatry 10:1126–
1132
Walther DJ, Bader M (2003) A unique central tryptophan hydroxylase
isoform. Biochem Pharmacol 66:1673–1680
123
Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H,
Bader M (2003) Synthesis of serotonin by a second tryptophan
hydroxylase isoform. Science 299:76
Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG
(2004) Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217
Zhao ZQ, Scott M, Chiechio S, Wang JS, Renner KJ, Gereau RWT,
Johnson RL, Deneris ES, Chen ZF (2006) Lmx1b is required for
maintenance of central serotonergic neurons and mice lacking
central serotonergic system exhibit normal locomotor activity. J
Neurosci 26:12781–12788
Zhou Z, Roy A, Lipsky R, Kuchipudi K, Zhu G, Taubman J, Enoch
MA, Virkkunen M, Goldman D (2005) Haplotype-based linkage
of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4
populations. Arch Gen Psychiatry 62:1109–1118
Zill P, Buttner A, Eisenmenger W, Moller HJ, Ackenheil M, Bondy B
(2007) Analysis of tryptophan hydroxylase I and II mRNA
expression in the human brain: a post-mortem study. J Psychiatr
Res 41:168–173