association of human hippocampal neurochemistry, serotonin transporter genetic variation, and...
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NeuroImage 26 (2005) 123–131
Association of human hippocampal neurochemistry, serotonin
transporter genetic variation, and anxiety
Jqrgen Gallinat,a,T Andreas Strfhle,a Undine E. Lang,b Malek Bajbouj,b Peter Kalus,a
Christiane Montag,c Frank Seifert,c Catrin Wernicke,b Hans Rommelspacher,b
Herbert Rinneberg,c and Florian Schubertc
aClinic for Psychiatry and Psychotherapy, Charite University Medicine, Campus Mitte, St. Hedwig Krankenhaus, Turmstrasse 21, 10559 Berlin, GermanybClinic for Psychiatry and Psychotherapy, Charite University Medicine, Campus Benjamin Franklin, GermanycLaboratory for Biomedical Optics and NMR-Measuring Techniques, Division of Medical Physics and Metrological Information Technology,
Physikalisch-Technische Bundesanstalt Berlin, Germany
Received 29 July 2004; revised 19 December 2004; accepted 8 January 2005
Available online 25 February 2005
The impact of the serotonin transporter (5-HTT) gene-linked
polymorphic region (5-HTTLPR) on anxiety-related behavior and
related cerebral activation has facilitated the understanding of
neurobiological mechanisms of anxiety. However, the influence of
the 5-HTTLPR genotype on hippocampal neuronal development and
neurochemistry, which is relevant to anxiety behavior, has not been
investigated. In 38 healthy subjects, absolute concentrations of N-
acetylaspartate (NAA) were measured as a main surrogate parameter
for hippocampal neurochemistry on a 3-T scanner. A significantly
lower hippocampal NAA concentration in s allele carriers was
observed as compared to l/l genotype. Other metabolites (choline,
creatine + phosphocreatine, glutamate) were unaffected by genotype.
The hippocampal NAA concentration was negatively correlated with
trait anxiety scores (STAI). Metabolites measured in the anterior
cingulate cortex (reference region) were not associated with genotype.
The results are in accordance with the recently reported relationship
between hippocampal neuronal development and anxiety behavior in
adult animals and show an association between human limbic
neurochemistry and genetically driven serotonergic neurotransmission
relevant to anxiety.
D 2005 Elsevier Inc. All rights reserved.
Keywords: Human hippocampal neurochemistry; Serotonin transporter
genetic variation; Anxiety; Magnetic resonance spectroscopy
Introduction
Several lines of evidence suggest that the central serotonergic (5-
HT) neurotransmission plays a crucial role in the modulation of
1053-8119/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.neuroimage.2005.01.001
T Corresponding author. Fax: +49 30 2311 2903.
E-mail address: [email protected] (J. Gallinat).
Available online on ScienceDirect (www.sciencedirect.com).
anxiety-related behavior (Charney and Deutch, 1996; Senkowski et
al., 2003). Moreover, there is a growing body of evidence that
serotonin plays a critical role in neuronal development including
cell proliferation, differentiation, and synaptogenesis (Magarinos et
al., 1999; McKittrick et al., 2000). Animal data show that
insufficient or excessive levels of serotonin during the postnatal
period lead to a long-lasting change in hippocampal plasticity,
including a decreased dendritic spine density (Haring and Yan,
1999; Yan et al., 1997). A recent study suggests that normal
anxiety-like behavior in the adult requires proper signaling by
serotonin via forebrain (hippocampus and cerebral cortex) 5-HT1a
receptors during the early postnatal period (Gross et al., 2002).
These findings indicate a relationship between hippocampal
development, serotonin, and anxiety-related behavior and are in
accordance with early neuroanatomical models of anxiety (Gray,
1988).
In the past few years, the genetic background of serotonergic
neurotransmission has gained interest with respect to anxiety-
related traits (Lesch et al., 1996) and activation of brain circuits
relevant to anxiety behavior (Gallinat et al., 2003; Hariri et al.,
2002). In humans, a 5-HT transporter (5-HTT) gene-linked
polymorphic region (5-HTTLPR) resulting in allelic variation of
5-HTT expression has been investigated extensively. Among the
relatively large trials, 5 studies did find evidence for a weak
association between the 5-HTTLPR and anxiety-related traits
(Greenberg et al., 2000; Lesch et al., 1996; Mazzanti et al., 1998;
Melke et al., 2001; Murakami et al., 1999), and three did not
(Flory et al., 1999; Jorm et al., 1998; Lang et al., 2004), and
several smaller studies have also been published with conflicting
results.
Using transfected cells (Lesch et al., 1996) and human platelets
(Greenberg et al., 1999), it was shown that the short (s) allele of
this polymorphism is linked to a lower transcriptional efficiency
of the promoter which may be associated with a higher availability
J. Gallinat et al. / NeuroImage 26 (2005) 123–131124
of 5-HT in the synaptic cleft than the long (l) allele. Using in vivo
single photon emission computed tomography (SPECT) imaging
in healthy subjects, serotonin transporter availability was reported
to be higher for l/l individuals compared to s carriers (Heinz et al.,
2000), which was not replicated in another SPECT investigation
with a larger sample size (Jacobsen et al., 2000). A more complex
relationship was found in a sample of 96 healthy subjects with a
greater transporter availability in l/l (trend) and s/s subjects (sig-
nificance) compared to s/l individuals (van Dyck et al., 2004). A
post-mortem ligand binding studies in human brain showed
increased 5-HTT binding levels and serotonin transporter mRNA
density in individuals with the l/l versus l/s or s/s genotypes (Little
et al., 1998). Experiments in 5-HTT knock out mice, which show
enhanced synaptic 5-HT concentrations, provided evidence for
abnormalities in hippocampal neurochemistry (Fabre et al., 2000;
Mossner et al., 2002). We therefore hypothesized that the 5-
HTTLPR genotype plays a role in the integrity of the human
hippocampus.
In the present study, the hippocampus was investigated in vivo
with single voxel proton magnetic resonance spectroscopy (1H-
MRS). Due to the important role of the hippocampus in emotional
processing including anxiety (Fish et al., 1993; Kim and
Fanselow, 1992; Seidenbecher et al., 2003) together with its
larger volume (compared to the amygdala), which provides a
better signal-to-noise ratio in voxel based MRS, it defines this
structure as an interesting target region for MRS. Absolute
concentrations of N-acetylaspartate (NAA) were determined in 38
healthy volunteers. NAA is considered to be a neuronal as well as
axonal marker (Ross and Bluml, 2001), and some authors have
speculated that NAA may also reflect a decreased synaptic
density (Li et al., 1999). As a reference region, the anterior
cingulate cortex (ACC) was investigated. In order to assess
anxiety behavior, the self-rating Spielberger State Trait Anxiety
Inventory (STAI) (Laux et al., 1981) was applied and genotyping
of the 5-HTTLPR was performed.
Table 1
Clinical data for subjects separated by genotype
Total
N 38
Age F SD (years) 35.9 (9.9)
Gender (m/f) 19/19
Handedness (r/l) 33/5
Education years 13.6 (2.5)
Smokers 14
STAI state score 35.1 (8.7)
STAI trait score 33.9 (7.5)
NAA concentration ACC (mmol/l) 13.6 (1.2)
NAA concentration Hip (mmol/l) 11.6 (1.0)
Percentage of gray matter volume Hip 64.5 (10.3)
Percentage of white matter volume Hip 32.5 (10.4)
Percentage of CSF volume Hip 3.0 (3.3)
Percentage of gray matter volume ACC 60.0 (9.4)
Percentage of white matter volume ACC 28.1 (6.0)
Percentage of CSF volume ACC 12.0 (8.2)
SD in parentheses.a Independent t test.b Chi-square test.
Materials and methods
Subjects
The study was approved by the ethics committee of the
University Hospital Benjamin Franklin, Free University Berlin
(Germany). The subjects were healthy individuals of German
descent who were recruited through newspaper advertisements. All
subjects gave written informed consent. Somatic as well as
psychiatric health status was evaluated by a structured telephone
questionnaire specifically designed for this purpose. In a second
step just before the MRS, a structured interview (Mini-Interna-
tional Neuropsychiatric Interview, MINI) (Sheehan et al., 1998)
was performed by a psychiatrist. Subjects were excluded when
fulfilling the criteria for an axis I disorder or being likely to have an
axis II disorder (Cluster A, B, or C) according to DSM-IV criteria.
Family history (first degree) of Axis I disorder was also an
exclusion criteria. Further reasons for exclusion were neurological
and general medical disorders or clinically relevant abnormalities.
For further methodological details, see Gallinat et al. (2002) and
Lang et al. (in press). Anxiety related behavior was measured using
the widely accepted STAI (Laux et al., 1981). Out of 40 subjects, 2
were excluded because of motion artifacts during the spectroscopic
measurement. Demographic and clinical data of the participants are
given in Table 1.
Magnetic resonance spectroscopy
Magnetic resonance measurements were carried out on a 3-T
scanner (MEDSPEC 30/100, Bruker Biospin, Ettlingen, Germany)
using a circularly polarized head coil. After automated global shim
of the linear, xz, z2, and x2�y2 field components using a procedure
developed inhouse, T1-weighted images (MDEFT, TE = 5.5 ms,
TR = 23.4 ms, 64 contiguous slices of 2 mm thickness, 1 mm
inplane (x�y) resolution) were acquired. After localized shim-
l/l s/l and s/s Test result
17 21 (16/5)
34.2 (10.0) 37.4 (9.8) n.s.a
7/10 12/9 n.s.b
14/3 19/2 n.s.b
14.2 (2.1) 13.0 (2.6) n.s.a
5 9 n.s.b
33.8 (6.6) 36.1 (10.1) n.s.a
32.8 (5.6) 34.8 (8.7) n.s.a
13.6 (1.3) 13.5 (1.2) n.s.a
12.0 (0.9) 11.3 (0.9) t = �2.601,
df = 36,
P = 0.013
62.6 (11.0) 66.1 (9.8) n.s.a
33.8 (10.9) 31.6 (10.1) n.s.a
3.7 (4.4) 2.4 (1.9) n.s.a
60.0 (10.8) 60.0 (8.4) n.s.a
28.0 (7.7) 28.1 (4.5) n.s.a
12.0 (8.2) 11.9 (8.3) n.s.a
Fig. 2. MR spectra (thin line: original) in the frequency range of interest
including fit (. . .), fitted baseline (——), and residual (thick line) as they are
returned by the TDFD method. Left: hippocampus voxel; right: anterior
cingulate voxel. Phantom spectra for NAA, glutamate, and glutamine were
included in the analysis. Note that no zero filling and line broadening of raw
data were performed.
J. Gallinat et al. / NeuroImage 26 (2005) 123–131 125
ming, MR spectra were acquired from 2 � 3 � 2 cm3 voxels
including the left hippocampus, and from 2.5 � 4 � 2 cm3 voxels
including the anterior cingulate of the volunteer brains (Fig. 1). No
other cerebral regions were investigated. For metabolite quantifi-
cation (vi), spectra were acquired from equal voxels in the center
of metabolite phantoms (0.1 M metabolite, pH 7.2, 378C). Aftermanual shimming to water line widths (FWHM) of 7–9 Hz and 6–
7 Hz for the two voxels, and determination of the radio frequency
power needed for a 908 excitation pulse, calibration of water
suppression (3 Gauss CHESS pulses of 25.6 ms duration) was
carried out, followed by acquisition of spectra with the PRESS
(point resolved spectroscopy) sequence using Shinnar–LeRoux-
optimized 908 and Mao refocusing pulses applied at the water
resonance frequency. To obtain one MR spectrum, 8 subspectra of
16 phase cycled scans each were recorded with TR = 3 s and TE =
80 ms, giving 128 averages. Subsequently, the water suppression
pulses were switched off to acquire a water-unsuppressed spectrum
(n = 8). Before further processing, the 8 individual metabolite
subspectra were corrected for eddy currents using the water-
unsuppressed spectrum and automatically frequency aligned
(Schubert et al., 2000) to correct for frequency shifts during the
scan time (of more than 6 min) caused by unvoluntary subject
motion and system instabilities. The resulting spectrum was
quantified using a program package that relies on a time
domain–frequency domain (TDFD) fitting procedure and involves
inclusion of phantom basis spectra and prior knowledge into the
fit, and background estimation by regularization (Elster et al.,
2000; Schubert et al., 2000, 2004). Extensive tests yielded mean
uncertainties (corresponding to Cramer–Rao lower bounds with
added uncertainties from background modeling) for the fitting of
NAA of 2.4–2.7% (Schubert et al., 2004). In the present spectra,
total choline (tCho), total creatine (tCr), NAA, glutamate, and
glutamine resonances were fitted by inclusion of phantom spectra
for the latter three and imposing the following prior knowledge:
constant frequency differences for glutamate, glutamine, and
NAA, equal line widths, adjustment of signal line shape to purely
Lorentzian. Typical spectra from hippocampus as well as cingulate
voxels including the fit result, background, and residual are shown
in Fig. 2. Metabolite amplitudes returned by the fitting procedure
were corrected for different coil loading by the phantoms and the
individual subject’s head (principle of reciprocity, (Danielsen and
Henriksen, 1994)) and for relaxation effects, assuming no differ-
Fig. 1. Voxel positions shown on typical brain MDEFT image
ences in relaxation behavior between the subjects. The transverse
relaxation time of NAA was determined using echo times of 50,
80, 135, 250, and 330 ms from 3 healthy volunteers to be 267 F15 ms for the hippocampus and 278 F 31 ms for the anterior
cingulate voxel. Longitudinal relaxation effects for NAA were
neglected because its T1 was estimated to be similar in the aqueous
phantom and in brain tissue. To correct the in vivo concentrations
for the content of cerebrospinal fluid of the voxels studied,
segmentation of the T1-weighted images was performed using
spm99 (Ashburner and Friston, 1997). Providing the highest retest
reliability (Schubert et al., 2004), a pixel was classified dependent
on which spm99 tissue classification had the greatest probability.
The csf fractions determined in the cingulate and hippocampus
voxels are presented in Table 1. As shown before (Schubert et al.,
2004), the segmentation performance did not suffer from the
incomplete brain coverage in the images used. Note that the
possible error introduced by erroneous estimation of csf fraction is
small given the small absolute fractions. Therefore, errors in csf
estimation caused by the small chemical shift displacement of
s; hippocampus (left), anterior cingulate cortex (right).
J. Gallinat et al. / NeuroImage 26 (2005) 123–131126
NAA and the deviation of the radio frequency pulses from perfect
rectangularity were regarded negligible.
Genetic analysis
Genomic DNAwas extracted from anticoagulated venous blood
samples using a salting out method (Miller et al., 1988). The
VNTR polymorphism in the promoter region of the serotonin
transporter gene was assessed in all participants of this study. The
PCRs were performed in a total volume of 20 Al containing 100 ng
DNA, primers each 10 pmol (Sander et al., 1997), 0.4 mM dNTPs,
0.5 U Taq polymerase (Invitek, Berlin, Germany) in 1 � NH4
buffer (Invitek, Berlin, Germany), and 1.5 mM MgCl2. PCRs were
done in a Perkin-Elmer system PE 9700 (Perkin-Elmer, Ueberlin-
gen, Germany). After denaturation at 988C for 3 min, the cycling
was performed 35 times at 948C for 30 s, at 638C for 60 s, and at
728C for 60 s, followed by a final elongation step at 728C for 10
min. The PCR products were separated in a 2.5% agarose gel
(Serva, Heidelberg, Germany) in 1 � Tris–Borate–EDTA buffer.
Fragments showed a length of 484 bp for the short allele (14
repeats) and of 528 bp for the long allele (16 repeats). GenBank
accession number: X76753.
Apart from the 5-HTTLPR genotype, the G1947A COMT
(Val108/158Met) gene polymorphism has been tested with respect
to an association with NAA (manuscript in preparation). In the
future, it is planned to test further genetic polymorphisms in a
larger sample of healthy subjects including the present sample.
Statistical analyses
Statistical comparisons between the two genotypes (s carriers
versus non-s carriers) were performed by analysis of variance for
metric demographic data or v2 test for dichotomous variables. The
effect of 5-HTTLPR on NAA concentration was assessed by
ANOVA using 5-HTTLPR genotype as factor and age as covariate.
The association between NAA concentration and STAI trait
anxiety scores was investigated with the Pearson correlation
analysis. All tests were performed at a level of significance equal
to 0.05.
1 ANOVA with the 5-HTTLPR as three-step factor (s/s, s/l, l/l) revealed
also a significant result: F = 3.296, df = 2.35, P = 0.048.
Results
The allele frequency was 0.66 (l) and 0.34 (s) and the genotype
frequency was 44.7% (l/l), 42.1% (s/l), and 13.2% (s/s). There was
no significant deviation according to the Hardy–Weinberg equili-
brium (v2 = 0.159, P b 0.690). Subjects were divided into two
equal groups on the basis of their 5-HTTLPR genotype according
to a previous report (Lesch et al., 1996) showing that lymphoblasts
with one or two s alleles (group 1) possess similar 5HT-uptake,
which is lower than that of cells with the l/l genotype (group 2).
However, conflicting results on this topic have been published
(Willeit et al., 2001).
Imprecision of NAA determination including individual CSF
correction was 1.23 and 0.57 mM for the two voxels. Kolmo-
gorov–Smirnov test showed that the NAA concentrations in the
hippocampus (D = 0.094, df = 38, P = 0.200) and in the ACC (D =
0.124, df = 38, P = 0.266) were normally distributed. The between-
genotype comparison (l/l genotype versus s containing genotype)
revealed no significant differences for age, gender, handedness,
education years, or smoking status (Table 1). Higher STAI trait
scores were observed in subjects carrying the short allele as
compared to the subjects homozygous for the l allele but this
difference was not statistically significant. The average gray matter,
white matter, and CSF fractions for the hippocampus voxel and for
the ACC voxel were not different for the two genotype groups
(Table 1).
ANOVA showed a significant effect of the factor 5-HTTLPR
genotype on the hippocampal NAA concentration (dependent
variable; F = 6.765, df = 1.36, P = 0.013)1. The inclusion of age
and STAI trait score as covariates into the ANOVA showed no
age effect (F = 1.598, df = 1.34, P = 0.215) but a significant
effect of the anxiety score on the hippocampal NAA concentration
(F = 6.084, df = 1.34, P = 0.019) while the genotype effect
remained significant (F = 5.086, df = 1.34, P = 0.031). The
analysis of other hippocampal metabolites like tCho (F = 0.161,
df = 1.36, P = 0.690), tCr (F = 1.731, df = 1.36, P = 0.197), and
glutamate (F = 1.720, df = 1.35, P = 0.198) did not show a
significant association with the factor 5-HTTLPR. Fig. 3 shows
that subjects carrying the s allele have lower concentrations of
NAA in the hippocampus than subjects homozygous for the l
allele (t = 2.601, df = 36, P = 0.013, unpaired t test). An
additional ANOVA with the dependent variable NAA concen-
tration in the ACC did show neither a statistically significant
genotype effect (F = 0.001, df = 1.34, P = 0.971) nor an effect of
the covariates age (F = 0.390, df = 1.34, P = 0.536) or STAI trait
score (F = 0.321, df = 1.34, P = 0.575). No significant
association between the factor 5-HTTLPR and tCho (F =
2.916, df = 1.36, P = 0.096), tCr (F = 1.602, df = 1.36, P =
0.214), or glutamate (F = 0.133, df = 1.36, P = 0.717) in the
ACC was observed.
A correlation was calculated for the concentration of NAAversus
the STAI trait scores. A significantly negative relationship was ob-
served for the two parameters in the hippocampus of subjects (r =
�0.447, P = 0.005) indicating low NAA concentrations in subjects
with high anxiety scores (Fig. 4). A similar result was found when
the 5-HTTLPR genotype was partialled out in the correlation
analysis (r = �0.431, P = 0.008), indicating that the significant
correlation is not a genotype group effect. No statistically significant
correlation between the NAA concentration in the ACC and the
STAI trait score was observed (r = �0.129, P = 0.442, and r =
�0.125, P = 0.462 when 5-HTTLPR genotype was partialled out).
Discussion
The present study indicates an association between the hippo-
campal NAA concentration and the genotype of the 5-HTTLPR,
with significantly lower NAA concentrations in healthy subjects
carrying the s allele as compared to subjects with the l/l genotype.
None of the other metabolites determined was associated with the
5-HTTLPR genotype.
Serotonin is an evolutionary ancient signaling molecule that
plays several roles in various species and tissues (Turlejski, 1996;
Whitaker-Azmitia, 1991), including the induction of mitosis in the
central nervous system (Malberg et al., 2000). Serotonergic
innervation of the hippocampus is dense and highly organized
(Moore and Halaris, 1975; Tork, 1985) with very dense plexus of
Fig. 3. Subjects carrying the s allele (s/l and s/s) showed significant lower
hippocampal NAA concentrations as compared to subjects homozygous for
the l allele (TP = 0.013).
J. Gallinat et al. / NeuroImage 26 (2005) 123–131 127
serotonergic fibers in the dentate gyrus (Halasy and Somogyi,
1993; Tork, 1985), an area in which neurogenesis was observed in
the adult brain (Jacobs et al., 2000). Glucocorticoids (Cameron et
al., 1993) and glutamate (Cameron et al., 1995) are known to
influence neurogenesis, but an important factor regulating pro-
liferation in the dentate gyrus is serotonin (Brezun and Daszuta,
1999, 2000). A number of investigations demonstrated the role of
5-HT synaptic availability for dendritic atrophy, reduced dendritic
branch points, and neurogenesis in the hippocampus (Czeh et al.,
2001; Magarinos et al., 1999; McEwen et al., 1997; McKittrick et
al., 2000). The 5-HT1a receptor is most likely involved in the
regulation of hippocampal neurogenesis since different antagonists
Fig. 4. Scatter plot of hippocampal NAA conce
of the 5-HT1a postsynaptic receptors reduced the number of newly
generated dentate gyrus cells in the adult rat (Radley and Jacobs,
2002) while agonists had the opposite effect (Santarelli et al.,
2003). However, the dual function of the 5-HT1a as both a
presynaptic autoreceptor, negatively regulating serotonin activity,
and a postsynaptic heteroreceptor, inhibiting the activity of
nonserotonergic neurons, has complicated the interpretation.
Moreover, activation of the 5-HT2a and 5-HT2c receptors has also
been shown to affect hippocampal neurogenesis (Banasr et al.,
2004).
Interestingly, serotonergic neurotransmission in the context of
psychosocial stress has been linked to reduced hippocampal NAA
concentrations and volume reduction in animal experiments (Czeh
et al., 2001). While psychosocial or restraint stress was shown to
enhance the extracellular level of 5-HT in the hippocampus of rats
(Thorre et al., 1997), the reduction of the synaptic availability of 5-
HTwas found to prevent stress-induced dendritic atrophy (Luine et
al., 1994; Magarinos et al., 1999; McEwen et al., 1997). Moreover,
the reduction of the NAA concentration, which was found in
animals exposed to stress, was restored to normal values when
synaptic 5-HT was reduced (Czeh et al., 2001). Assuming
enhanced extracellular levels of 5-HT in s carriers as compared
to the l/l genotype, our 1H-MRS results are compatible with the
role of 5-HT in hippocampal neurogenesis.
On the other hand, recent preclinical evidence has shown that
selective serotonin reuptake inhibitors (SSRI) promote neuro-
genesis (Malberg et al., 2000) and reverse the effects of stress on
hippocampal atrophy in patients with PTSD (Vermetten et al.,
2003). This effect seems to be contradictory to our results since
both SSRI treatment and the presence of the s allele (compared to
the l allele) have been associated with a higher availability of 5-HT
in the synaptic cleft. However, the presence of the s allele is not
comparable to the pharmacological effect of SSRI: clinical
evidence indicates that s allele carriers or individuals with the s/s
genotype compared to l/l genotype seem to have in fact more
depressive symptoms in relation to stressful life events (Caspi et
al., 2003), an increased risk for suicide attempts (Caspi et al., 2003;
Gorwood et al., 2000), and a poorer response to SSRI treatment in
ntrations and trait anxiety scores (STAI).
J. Gallinat et al. / NeuroImage 26 (2005) 123–131128
major depression (Arias et al., 2003). This indicates that 5-HTT
genotype and SSRI effects are not simply resulting in higher
synaptic 5-HT availability but represent complex conditions.
Moreover, the time point when 5-HT is acting on the brain seems
to be important. Remarkably, a recent report indicates that
serotonergic neurotransmission in the forebrain during the early
postnatal period is essential for anxiety-like behavior throughout
life: rats lacking the 5-HT1a receptor show increased anxiety-like
behavior (Gross et al., 2000). This behavioral phenotype of the
adult animal occurs only in the absence of the receptor in the
forebrain during the early postnatal period whereas the shut off of
the receptor in the adult animal did not affect behavior (Gross et al.,
2002). Other authors demonstrated that a postnatal treatment with
5-HT1a receptor antagonists leads to a permanently decreased
dendritic spine density in the dentate gyrus (Yan et al., 1997),
indicating a relationship between serotonergic neurotransmission,
hippocampal neurogenesis, and anxiety behavior in later life.
Interestingly, a recent animal investigation supports the notion that
also the genetic polymorphism of the 5-HTT may exert its effects
during early development of the CNS by altering maturation of
circuits that modulate emotional responses to novelty and stress
(Ansorge et al., 2004). Transient inhibition of the 5-HTT during
early development with fluoxetine produced abnormal emotional
behaviors related to anxiety in adult mice. This effect mimicked the
behavioral phenotype of mice genetically deficient in 5-HTT
expression. The result provides a potential explanation for the
increased susceptibility of individuals carrying one or two s alleles
to depression in the context of multiple life stressors (Caspi et al.,
2003), and the paradox that SSRI treatment, also producing a
reduction of 5-HTT function, ameliorates anxiety- and depression-
related symptoms in the mature organism.
Assuming that NAA reflects aspects of altered hippocampal
neurogenesis (Czeh et al., 2001), the observed negative correlation
between anxiety scores and hippocampal NAA concentration is in
accordance with the role of hippocampal development for anxiety
in the adult. However, correlation may also result from other
factors which are not related to the investigated parameters.
Therefore, this association should be viewed with caution and as
a preliminary result.
In the current literature, the association between amygdala and
anxiety processing is a major topic (e.g., Hariri et al., 2002).
However, there are several lines of evidence indicating the
important role of the hippocampal formation in anxiety processing.
For instance, hippocampus and parahippocampus have been
associated with the experience of fear and anxiety during the
electrical stimulation of these structures in the context of the focus
localization in patients with intractable epilepsy (Fish et al., 1993).
Imaging studies of patients with anxiety disorders reported
structural (Fontaine et al., 1990; Ontiveros et al., 1989), functional
(Bisaga et al., 1998; Reiman et al., 1986), and neurochemical
(Massana et al., 2002) abnormalities in the medial temporal lobe.
Brain imaging studies in healthy controls indicate an important role
of the hippocampus in the regulation of unpleasant emotions (Lane
et al., 1997). Moreover, a close functional connection between
hippocampus and amygdala has been stressed especially in the
trace variant of fear conditioning (Buchel et al., 1999), probably
via descending hippocampal projections to the amygdala (Maren
and Fanselow, 1995). In a broader view, hippocampal inputs (from
the amygdala, hypothalamus, anterior/middle thalamic nuclei) and
the hippocampal output (from the subiculum to the cingulate
cortex) form a strong basis for participation of the hippocampal
formation in the contextual associations of strong emotions,
especially in fear conditioning (Buchel et al., 1999; Kim and
Fanselow, 1992; Phillips and LeDoux, 1992; Seidenbecher et al.,
2003). Recent work indicates that the so-called bflashbulbmemoriesQ of events associated with strong emotions, including
both fear and positive emotions, involve the amygdala as well as
the hippocampus (de Quervain et al., 1998; McGaugh et al., 1996).
This role of the hippocampus in emotional processing together
with its larger volume (compared to the amygdala), which provides
a better signal-to-noise ratio in voxel based MRS, defines this
structure as an interesting target region for MRS.
No genotype effect on metabolites and no correlation with
anxiety scores were found in regard to the NAA concentration in
the ACC, although 5-HT effects on cortical morphology have been
described in animal experiments (Goldman-Rakic and Brown,
1982; Killackey et al., 1995; Rhoades et al., 1994). For instance,
the reduction of the level of serotonin in the rat fetus resulted in
microencephaly in the newborn pups and the thickness of their
cerebral cortex was significantly reduced, while postnatal treatment
of these pups with the 5-HT1a agonist ipsapirone increased the
thickness of their cortex (Varrault et al., 1992). Moreover, the
behavioral phenotype of animals lacking the 5-HT1a receptor
mentioned above (Gross et al., 2000) may depend not only on the
receptors located in the hippocampus but also in the frontal cortex
since the investigated forebrain of rats includes both structures.
However, in the present study only a small part of the frontal lobe
(the anterior cingulate cortex) was investigated and it cannot be
ruled out that the other frontal lobe structures are affected by the 5-
HTTLPR genotype. Furthermore, the sample size may be too small
to detect small effects and further investigations have to be carried
out to clarify this issue.
We also observed a higher score on STAI trait in subjects with
the short allele of the 5-HTTLPR as compared to those with the
long allele. This result, although not statistically significant in this
and another sample from our laboratory (Lang et al., 2004), is in
agreement with several investigations of anxiety-related traits in
relatively large trials (Lesch et al., 1996; Mazzanti et al., 1998;
Melke et al., 2001; Murakami et al., 1999). However, neuro-
chemical parameters such as NAA with a relationship to anxiety-
related behavior (Tupler et al., 1997) may represent more proximal
effects of relevant gene variants than complex behavioral
categories (Tsuang and Faraone, 2000). As 1H-MRS is a powerful
tool to quantify such neurochemical parameters, this technique
may be employed to advance studies of the genetic basis of
psychiatric disorders related to serotonergic dysfunctions.
Acknowledgment
The authors thank the BMBF for support within the framework
of the Berlin Neuroimaging Center.
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