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Seasonal changes in brain serotonin transporter binding in short 5-

HTTLPR-allele carriers but not in long-allele homozygotes

Jan Kalbitzer

1,2*

, David Erritzoe

1,2

, Klaus K. Holst

2,3

, Finn Å. Nielsen

2,4

, Lisbeth

Marner

1,2

, Szabolcs Lehel

2,5

, Tine Arentzen

1,2

, Terry L. Jernigan

2,6

, and Gitte M.

Knudsen

1,2

Neurobiology Research Unit, Copenhagen University Hospital Rigshospitalet,

Copenhagen, Denmark

Center for Integrated Molecular Brain Imaging, Copenhagen, Denmark

Department of Biostatistics, University of Copenhagen, Denmark

Department of Informatics and Mathematical Modelling, Technical University of

Denmark, Lyngby, Denmark

PET and Cyclotron Unit, Copenhagen University Hospital Rigshospitalet, Copenhagen,

Denmark

Danish Research Center for Magnetic Resonance, Copenhagen University Hospital

Hvidovre Hospital, Copenhagen, Denmark

Light deprivation is a known environmental stressor in Scandinavia and other regions of

high latitude; in a large fraction of the population, behavioral changes such as reduced

activity, higher food-intake, and increased urge to sleep take place during months with

few or even no daylight hours

. In some people, this environmental stress can provoke a

particular form of mood disorder, termed seasonal affective disorder (SAD), which is

characterized by the occurrence of depressive symptoms in winter. This observation led

to the successful development of the rational and efficient treatment strategy of bright

light therapy

Several findings suggest that SAD is mediated through the serotonin (5-HT) system. In

post mortem human brain samples, 5-HT concentrations are lowest in people dying in the

winter

. Also, the concentration of the serotonin metabolite 5-HIAA is lower in jugular

blood samples collected in winter

. These biomarkers may be associated with seasonal

changes in the activity of plasma-membrane serotonin transporter (5-HTT) that serves a

central role in neural serotonin transmission by regulating synaptic interstitial 5-HT

levels

5, 6

. However, previously there has been no clear evidence for seasonal variation in

the 5-HTT binding in brain in vivo. Two contradictory brain molecular imaging studies,

one reporting lower binding

and one higher

binding in winter compared to summer,

suffered from small sample sizes, questionable criteria for the cut-offs between summer

and winter, and radioligands less specific than those available today.

However, the link between 5-HTT and SAD is also pointed out by the observation that

carriers of a 44-base pair deletion in the 5-HTT-linked polymorphic region (short 5-

HTTLPR allele = S-allele) generally are more vulnerable to the disorder than carriers of

the insertion (long 5-HTTLPR allele = L-allele)

. This polymorphism influences 5-HTT

expression, with lower expression in carriers of the S-allele

. In comparison to L-allele

homozygotes, S-allele carriers have increased propensity to develop mood disorders in

response to stressful environmental cues

10, 11

, in particular in response to seasonal

changes

9, 12

. Further, in functional magnetic resonance (fMRI) studies exposure to

stressful and fearful environmental cues evoke greater limbic activation in S-allele

carriers

. However, there was no difference in cerebral 5-HTT binding between carriers

of the S-allele and L-allele homozygotes in a two large samples of 96 healthy

volunteers

measured in vivo with single photon emission computed tomography

(SPECT) and of 42 healthy volunteers

measured with positron emission tomography

(PET). Only when subjects were stratified according to an additional single nucleotide

polymorphism (SNP) on the L-allele, which has not been taken into account by most

activation studies, were differences in in vivo 5-HTT binding identified

16, 17

. Furthermore,

phasic 5-HTT changes, which would be the molecular equivalent to the endophenotype

identified by activation studies, in terms of a different molecular response to

environmental cues in S-allele carriers compared to L-allele homozygotes, have to our

knowledge - not yet been studied with PET.

We sought evidence for seasonal variation in the 5-HTT binding data from a sample of

54 healthy subjects studied with the highly specific radioligand [

C]DASB

. To avoid

arbitrary classification, we predicted [

C]DASB binding with the number of daylight

minutes

the

latitude

Copenhagen

(http://aa.usno.navy.mil/data/docs/Dur_OneYear.php/), and incorporated adjustment for

age and gender in a general linear model. We found that length of daylight time in

minutes correlates negatively with BP

for [

C]DASB in the putamen and the caudate

(putamen: -0.0438 BP

/(100 minutes) [-0.0689; -0.0186], p=0.001, caudate: -0.0363 [-

0.0702; -0.0023] BP

/(100 minutes), p = .037), with a similar tendency in the thalamus

(-0.0299 BP

/(100 minutes) [-0.0656; 0.00581, p=0.099), but found no such association

in the midbrain (0.0192 BP

/(100 minutes) [-0.0858; 0.1242], p=0.715). The findings

were also reproduced when data were modeled to a harmonic function that allows for a

time delay in the seasonal effect. Correction for age and gender revealed a marked gender

effect (higher binding in men) in the caudate (p = 0.0002), and decreasing [

C]DASB

with age in the putamen (p = 0.034) and the thalamus (p = 0.043). It could be

argued that seasonal changes in availability of food selection and seasonal changes in

food preferences might affect neural serotonin levels through variations in tryptophan-

intake. However, in our sample there was no correlation between [

C]DASB BP

and

plasma tryptophan, neither in terms of absolute concentration nor relative to large neutral

amino acids with which tryptophan is competing at the blood-brain barrier

. Also, there

was no seasonal variation of absolute or relative tryptophan concentrations in our sample.

We then asked if the seasonal effect was more pronounced in S-alleles carriers, given the

evidence from activation and population studies. We stratified our sample according to 5-

HTTLPR-allelic status, and searched for a gene-daylight interaction effect; 19 subjects

were homozygote L-allele carriers and 35 subjects were S-allele carriers. Again in

putamen, we found a significant gene*daylight effect (p, corrected for age and gender =

0.0448) with a negative correlation between the [

C]DASB BP

and daylight time in

carriers of the S-allele, but not in carriers of the L-allele (Figures 1 and 2).

Consistent with prior studies in which SAD patients had a higher likelihood of being S-

allele carriers

9, 12

, and having higher 5-HTT binding during depressive episodes

, we

found that the 5-HTT binding in carriers of the S-allele is affected by seasonal changes,

but not in carriers of the L-allele. We found the strongest seasonal effect in the putamen,

an anatomical region with a dense serotonin innervation

, that is implicated in motor

functions, but also in processing of aversive stimuli

21, 22

. We did not find any seasonal

variation in midbrain, where the serotonergic cell bodies are located

. Possibly, [

C]-

DASB is merely reflecting the number of neurons than 5-HTT tonus in this region and

thus not sensitive to seasonal effects.

By examining the effect of daylight hours on the serotonergic system, we identified a

distinct neurobiological endophenotype for the short 5-HTTLPR-allele with dynamic

seasonal changes in 5-HTT binding. The neurobiological endophenotype identified here

directly links activation studies, showing responses on the neural circuit level, with

dynamic changes in transporter expression measured in vivo.

Acknowledgements

We thank CL Licht for continuous inspiration and critical reviewing of the manuscript.

Funding

The study was funded by the Lundbeck Foundation and the Capital Region Hospital

Corporation. The John and Birthe Meyer Foundation donated the Cyclotron and the PET-

scanner.

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Figure 1. Left figure illustrates the seasonal effect on [

C]DASB BP

(putamen) with

pointwise confidence limits, modeled as a harmonic function with period 1 year

(estimated peak in the middle of December, SE = 21 days, in good agreement with the

model using daylight minutes as a predictor) adjusting for age and gender. The plotted

points are the partial residuals (male of mean age). The functional form was validated by

including additional frequency components and by comparison with estimates from an

additive model. The right figure displays the interaction between number of daylight

minutes and HTTLPR-allelic status adjusting for age and gender. For comparison with

the left figure the estimated linear response as a function of daylight minutes was

transformed to a function of calendar time.

Supplemental information

Subjects

Fifty-four healthy volunteers were recruited by advertisement for a research protocol

approved by the Ethics Committee of Copenhagen and Frederiksberg, DK [(KF) 01-

156/04, (KF) 01-124/04, and (KF) 11-283038]. After complete description of the study to

the subjects, written informed consent was obtained from 37 males with a mean age of 34

yrs (SD 18 yrs), and from 17 females with a mean age of 34 yrs (SD 20 yrs). Exclusion

criteria for all test persons included medical history, drug abuse or psychiatric disorders.

All subjects had a normal neurological examination and a magnetic resonance (MR)

image of the brain without pathological findings.

PET scans

PET scans were performed with an 18-ring GE-Advance scanner (General Electric,

Milwaukee, WI, USA), operating in 3D acquisition mode, and

producing 35 image slices

with an interslice distance of 4.25 mm. Following a 10 min transmission scan, a dynamic

90 minute long emission recording was initiated upon intravenous injection during 12 sec

of mean 487 (SD 90) MBq (range: 246 - 601) [

C]DASB with mean specific activity of

32 (SD 16) GBq/µmol (range: 9 - 82). The emission

recording consisted of 36 frames,

increasing progressively in duration from 10 sec to 10 min. The attenuation and decay

corrected recordings were reconstructed by filtered back projection using a 6 mm Hann

filter.

MR scans

Structural

brain scans were acquired on a Siemens Magnetom Trio 3T MR scanner with

an eight-channel head coil (In vivo, FL, USA). Thirty-five subjects underwent a high-

resolution 3D T1-weighted, sagittal, magnetization prepared rapid gradient echo

(MPRAGE) scan of the head (MPRAGE1: echo time (TE)/repetition time (TR)/inversion

time(TI)=3.93/1540/800 ms; slice resolution=75%; Bandwidth=130 Hz/Px; Echo

spacing=9.8 ms) and fifteen subjects underwent a 3D T1-weighted, sagittal, MPRAGE

(MPRAGE2: TE/TR/TI=3.04/1550/800 ms; slice resolution=100%; Bandwidth=170

Hz/Px; Echo spacing=7.7ms). Common to both MPRAGE's was a flip angle of 9°, a

FOV of 256 mm, a matrix of 256x256, 1x1x1mm voxels and 192 slices.

MR/PET co-registration

All time-frames of the attenuation-corrected emission recording were automatically

aligned to frame 26 using the AIR algorithm

. In a next step we used a mean PET image,

averaging time-frames 10-36 for co-registration to the individual MR image using the

AIR algorithm

; the quality of each co-registration was controlled visually.

VOI analysis

The VOI were delineated automatically as described in Svarer et al

in order to identify

the volumes in a user-independent fashion. For each of the 10-template VOI sets, a 12-

parameter affine transformation and a warping field was calculated

between the template

MR image and the individual MR image for a subject. Having obtained the MR/PET co-

registration for the same individual as described above, the template VOI sets are then

transferred to the dynamic PET image space for each subject, using the identified

transformation parameters. From the VOI sets, a probability map was created for each

subject, and a common VOI set was threshold-generated. These VOI sets were then used

for automatic extraction of time activity curves (TAC) for midbrain and volume-weighted

(left-right) averages for bilateral thalamus, caudate, putamen and cerebellum for all

subjects. The

midbrain, the caudate, the putamen and the thalamus were selected as

representative brain regions of homogenous, high 5-HTT binding

since we expected that

a genetic predisposition would lead to a global scaling effect. The TAC extracted for the

cerebellum was used as the reference tissue input for kinetic modeling.

Quantification of non-displaceable tracer uptake

The outcome parameter of the [

C]DASB binding within a brain region is the non-

displaceable binding potential, designated BP

. The BP

was calculated for the four

VOI using the cerebellum input as reference region with non-specific binding. We used a

modified reference tissue model designed specifically for quantification of [

C]DASB

(MRTM/MRTM2) as described and evaluated by Ichise et al.

using the PKIN tool of the

software PMOD version 2.9 : A fixed washout constant, designated k2', was calculated

for each individual as an average of k2 in caudate, putamen and thalamus relative to

cerebellum using MRTM. Subsequently, k2' was used as a constrained input parameter

for the calculation of BP

in the four VOIs relative to cerebellum.

Parametric images

For the voxel-based analysis, parametric images representing BP

for each voxel were

calculated for all subjects from the dynamic PET images. Using the PXMOD tool of the

PMOD software, we imported the VOI, generated as described above, and re-calculated

k2' relative to cerebellum using MRTM. As described by Ichise et al.

, we applied an R1

(tracer delivery to the VOI relative to cerebellum, to some extent reflecting blood flow)

threshold of 0.3 to exclude noisy voxels, particularly seen in regions with low tracer

clearance. Additionally, we restricted the BP

outcome to range between 0 and 10. The

resulting parametric images were filtered with a 3 mm median filter in their native space.

SPM5 (http://www.fil.ion.ucl.ac.uk/spm/software/spm5/) was used to normalize the

parametric images to Montreal Neurological Institute (MNI) space using a warping field

estimated by normalization of each subject's co-registered MR image. Subsequently, a 12

mm Gaussian filter was applied to all normalized images.

Genotyping

Blood samples for DNA analysis were taken during the scanning and immediately frozen

and stored at -20°C until further analysis. DNA was extracted from the blood using a

Qiagen Mini kit using the guidelines included in the kit (Qiagen, Valencia, CA, USA) 5-

HTTLPR (SLC6A4; 17q11.1-q12) genotyping was performed using a TaqMan 5'-

exonuclease allelic discrimination assay according to the instructions provided by the

manufacturer (Applied Biosystems, Foster City, California, Assay-on-Demand). The ABI

7500 multiplex PCR machine (Applied Biosystems, Foster City, California, USA) was

used for this analysis.

Effect of plasma tryptophan

Plama amino acids Blood samples were collected in heparinized vials and put on ice until

centrifuged. Plasma was stored at -80 degrees until analyzed. Immediately after the

samples were thawn sulphosalicylic acid was added to precipitate protein and norleucine

was added as internal standard. The samples were then measured with HPLC using a

Lithium Cation Exchange Column (Pickering,

www.pickeringlabs.com

). Tryptophan

competes in particular with leucine, isoleucine, valine, tyrosine, and phenylalanine at the

blood-brain barrier. We therefore measured concentrations of all these LNAAs and

calculated both absolute plasma tryptophan measures as well as tryptophan load relative

to the other LNAAs and searched for correlations of boths with daylight hours and with

[

C]DASB BP

. We found no significant correlations (p-value > 0.1).

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