Lanzenberger et al. 2011, Supplementary Information
SUPPLEMENTARY INFORMATION
Progesterone level predicts serotonin-1A
receptor binding in the male human brain
Rupert Lanzenberger1, Markus Mitterhauser2, Georg S. Kranz1,
Christoph Spindelegger1, Wolfgang Wadsak2, Patrycja Stein1,
Ulrike Moser1, Markus Savli1, Kurt Kletter2, Siegfried Kasper1
1 Department
of Psychiatry and Psychotherapy
2 Department
of Nuclear Medicine
Medical University of Vienna, Austria
Submission to:
Neuroendocrinology
March, 2011
1/13
Lanzenberger et al. 2011, Supplementary Information
Supplementary figure (see legend at page 3/14)
2/13
Lanzenberger et al. 2011, Supplementary Information
3/13
Supplementary Figure Legend: Schematic of the 5-HT1A receptor transrepression
mechanism. Progesterone increases the mineralocorticoid receptor expression via
the progesterone receptor (PR). Cortisol-activated transcription factors – the
mineralocorticoid (MR) and glucocorticoid (GR) receptors – bind to the promoter
region of the 5-HT1A receptor gene and suppress tonically the transcription of the 5HT1A receptor protein.
In detail: 1, Progesterone and cortisol diffuse freely from the intercellular space into
the cytoplasm. In the cytoplasm, the hormones bind to their respective receptors and
activate them. 2, Activated progesterone receptors translocate into the nucleus and
bind to hormone response elements(1). 3, The binding of activated PR at the
promotor region of the MR gene induces the MR promoter activity and thereby
increases the MR transcription(2). 4, The mineralocorticoid receptor mRNA
translocates to the ribosomes, where it is translated into MR proteins. The formed
MRs remain in the cytoplasm as inactive cytosolic complexes. 5, In the presence of
cortisol, the MRs are freed from their complexes and can translocate into the
nucleus(3-5). Before they interact with DNA they form homodimers (MR-MR) or
heterodimers (MR-GR) with activated glucocorticoid receptors(4, 6). 6, Within the
nucleus, the MR-GR heterodimers bind to the 5-HT1A promotor region thereby
transrepressing the 5-HT1A transcription. 7, As the transcription of the 5-HT1A
receptor decreases, the 5-HT1A mRNA translation also diminishes. 8, This results in
an overall reduction of 5-HT1A receptor expression at the neuronal membrane.
Lanzenberger et al. 2011, Supplementary Information
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Supplementary tables
Demographic characteristics
Mean
±
SD
Age (years)
27.0
±
5.8
Body weight (kg)
75.6
±
10.9
Body mass index (BMI)
23.4
±
2.5
5.7
±
0.8
Radiochemical purity (%)
97.8
±
1.4
Specific activity (GBq/μmol)
29.7
±
21.7
Injected volume (mL/kg)
0.07
±
0.03
Weight of precursor WAY100634 (μg/kg)
0.10
±
0.05
Weight of unlabeled WAY100635 (µg/kg)
0.15
±
0.15
Injected dose (MBq/kg body weight)
Supplementary Table 1: Demographic characteristics and radiochemical variables
of the study sample. Values are given as mean value ± standard deviation (SD) at
the application date.
Hormone
Cortisol
Progesterone
Limit of sensitivity
Interassay CV
0.4
ng/mL
6
%
0.0002
ng/mL
4-7
%
Supplementary Table 2: Lower limit of sensitivity (LOS), Interassay coefficient of
variation (CV). To facilitate the comparability to table 1 in the main paper, the limits of
sensitivity are given in ng/mL.
Lanzenberger et al. 2011, Supplementary Information
Region
Partial R2 for
5/13
Uncorrected p
Corrected p*
p value of global model
Progesterone
Amygdala
0.46
0.0008
0.0069
0.001
Retrosplenial cortex
0.65
0.0009
0.0063
0.013
Orbitofrontal cortex
0.41
0.0024
0.0147
0.016
Anterior cingulate cortex
0.32
0.0111
0.0555
0.025
Hippocampus
0.30
0.0131
0.0525
0.022
Insula
0.35
0.0152
0.0455
0.078
Raphe nuclei
0.28
0.0315
0.0629
>0.1
Posterior cingulate cortex
0.20
0.0412
0.0412
0.055
Supplementary Table 3: Results of the multiple regression analyses using region
specific 5-HT1A BP as dependent variable and mean progesterone plasma level, age,
specific activity and injected dose as independent variables. Adjusted R squared in
the second column represents the explained variance of 5-HT1A BP which can be
attributed to progesterone (calculated as the difference of adjusted R2 between the
model including progesterone and the model excluding progesterone). *indicates P
values after correction for multiple testing using Bonferroni-Holm (8 regions of
interest).
Lanzenberger et al. 2011, Supplementary Information
Region
Partial R2 for
6/13
Uncorrected p
Corrected p
p value of global model
Progesterone
Amygdala
0.49
0.0014
0.0113
0.005
Retrosplenial cortex
0.68
0.0015
0.0105
0.030
Orbitofrontal cortex
0.54
0.0036
0.0219
0.038
Anterior cingulate cortex
0.32
0.0175
0.0876
0.054
Hippocampus
0.29
0.0196
0.0784
0.049
Insula
0.29
0.0306
0.0919
>0.1
Raphe nuclei
0.31
0.0328
0.0657
>0.1
Posterior cingulate cortex
0.08
>0.1
>0.1
>0.1
Supplementary Table 4: Results of the multiple regression analyses using region
specific 5-HT1A BP as dependent variable and mean progesterone plasma level, age,
specific activity, injected dose and ROI volume as independent variables (see legend
to Supplementary Table 1 for further explanation).
Lanzenberger et al. 2011, Supplementary Information
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Partial R2 for
Percent
Uncorrected
Corrected
p value of global
Progesterone
reduction
p
p
model
Amygdala
0.29
40
0.011
0.088
0.011
Retrosplenial cortex
0.42
38
0.010
0.070
0.057
Orbitofrontal cortex
0.39
28
0.015
0.090
0.081
Anterior cingulate cortex
0.11
66
>0.1
>0.1
0.064
Hippocampus
0.19
35
0.061
>0.1
0.099
Insula
0.05
83
>0.1
>0.1
>0.1
Raphe Nuclei
0.20
34
0.082
>0.1
>0.1
Posterior cingulate cortex
0.05
37
>0.1
>0.1
>0.1
Region
Supplementary Table 5: Results of the multiple regression analyses using region
specific 5-HT1A BP as dependent variable and mean progesterone plasma level, age,
specific activity, injected dose, ROI volume and plasma cortisol level as independent
variables. The third column indicates the reduction of the explained variance of
progesterone after including cortisol into the model (see legend to Supplementary
Table 1 for further explanation).
Lanzenberger et al. 2011, Supplementary Information
ROI volumes (cm3)
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Mean
±
SD
Min.
-
Max.
Hippocampus
3.16
±
0.61
2.10
-
4.30
Insula
5.90
±
1.18
3.81
-
8.49
Anterior cingulate cortex
2.18
±
0.54
1.33
-
3.16
Amygdala
1.61
±
0.22
1.29
-
2.04
Orbitofrontal cortex
9.06
±
2.55
5.77
-
14.53
Retrosplenial cortex
1.28
±
0.27
0.91
-
1.92
Posterior cingulate cortex
1.43
±
0.07
1.20
-
1.50
Raphe nuclei
0.75
±
0.00
0.75
-
0.75
Supplementary Table 6: The volumes of eight regions of interest (ROI) in cm3 of the
study sample. Values are given as mean value ± standard deviation (SD) and range
(minimum, maximum).
Lanzenberger et al. 2011, Supplementary Information
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Supplementary methods
Subjects:
Eighteen male (25.6 ± 5.8 years) healthy subjects participated in the study.
Characteristics of the study sample including demographic data are given in the
supplementary table 1. The absence of medical history including drug abuse was
assessed by psychiatric interview, history, physical examination, routine blood tests,
electrocardiogram, and further psychological tests including Spielberger State-Trait
Anxiety Inventory (STAI) and the Mini International Neuropsychiatric Interview
(M.I.N.I.) as described recently (7). Exclusion criteria were the use of psychotropic or
hormonal drugs during the past 6 months including anabolic steroids. The study was
approved by the Ethics Committee of the Medical University of Vienna, and all
voluntary participants provided written informed consent after receiving written and
verbal explanation of the study. The subjects were compensated for their
participation. Recruiting was done by advertisement at the Medical University
building.
Positron emission tomography (PET):
[Carbonyl-11C] WAY-100635 was prepared using a
11C
methylation synthesizer from
Nuclear Interface (GE Medical Systems) as we have reported in detail (8). PET
measurements were done using a GE Advance Scanner. To minimize motion, we
used a head fixation device with foam pads and restraining straps. Procedures were
done as published elsewhere (7). To shortly summarize, dynamic scans (threedimensional mode) started simultaneously with bolus injection of [carbonyl-11C] WAY100635 in the right antecubital vein. A series of 30 time frames (15x1min, 15x5min)
was collected resulting in a total acquisition time of 90 minutes. The spatial resolution
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of the final attenuation corrected and reconstructed image (filtered back-projection)
was FWHM = 4.36mm at the center of the FOV (matrix 128x128, 35 slices).
Radiochemical variables are given in the table 1. No realignment for head movement
or partial volume correction was applied.
Tracer Kinetic Modelling of [carbonyl-11C] WAY-100635 Emission Data:
For quantification of the 5-HT1A receptor binding potential we used the kinetic
modelling tool of the image quantification and kinetic modeling software PMOD 2.7.
(PMOD Technologies Ltd., Zurich, Switzerland, http://www.pmod.com/) (7). The
Simplified Reference Tissue Model (SRTM) based on a two-tissue compartmental
model implemented in PMOD was applied using the cerebellar cortex excluding
vermis and venous sinus as reference region, utilizing its low 5-HT1A receptor density.
Decay-corrected time activity curves (TACs) were obtained using 30 frames of the
dynamic PET data and the three-dimensional ROIs. We calculated the regional BP
and the regional relative delivery of the radioligand normalized to the cerebellum
(R1). Right and left ROIs (except for the raphe region and the medial orbitofrontal
cortex) were combined to improve signal-to-noise ratio. To exclude steroid hormoneinduced effects on the reference regions, we performed a correlation analysis on the
cerebellar TACs.
Hormone assays:
To minimize circadian effects and the influence of wake up time, the first venous
blood sampling was done in the morning hours at 8:51 a.m. ± 72.6 minutes (mean ±
SD), approximately 90 minutes after self-determined awakening. A second sample
was taken 21 ± 10.4 minutes later. Hormonal levels used for statistics were the mean
values of both samples which were taken and measured independently. Hormone
Lanzenberger et al. 2011, Supplementary Information
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levels are given as total (protein-unbound plus protein-bound) plasma levels (for
details, see supplementary table 2). Assays were performed using the E170 Module
(Roche E170 Modular Analytical System ®). Electrochemoluminescence (ECLIA)
was used for quantification of total progesterone and cortisol in plasma. The lower
limit of sensitivity was 0.2 ng/L, the interassay coefficient of variation was 4-7% for
progesterone, and 0.4 µg/L and 6% for cortisol, respectively (for details of plasma
levels, limits of sensitivity and interassay coefficients of variation, see supplementary
table 2 and 3). There was no measurement of cortisol binding globulin (transcortin)
for calculation of free cortisol.
Magnetic resonance imaging (MRI):
Structural whole-brain T1-weighted MR images (MPRAGE sequence) were acquired
on a 3 Tesla Bruker Scanner (128 axial slices, in-plane resolution 0.78 X 0.86 mm,
slice thickness 1.56 mm, matrix 256x256).
Data Analyses:
Data analysis procedures have been described in detail previously (7). In brief, the
structural MR image was coregistered to the summed PET image (frames 1-30) in
each
subject
using
the
statistical
parametric
mapping
software
SPM2
(http://www.fil.ion.ucl.ac.uk/spm/). ROIs (except for the raphe region) were defined on
individual co-registered MR images using PMOD 2.7 (for details, see (9)). Given the
lack of MR criteria for raphe boundaries, the ROI of the raphe region in the midbrain
had to be directly delineated on the PET summation image.
The amygdala was located directly anterior to the hippocampus which was found in
the medial temporal lobe on slices including the midbrain. A fixed-size cubic volume
of interest (747mm3) for the raphe nuclei was placed in the medulla oblongata on
Lanzenberger et al. 2011, Supplementary Information
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slices showing the interpeduncular cistern. The anterior cingulate cortex bound
anteriorally to the cingulate sulcus and the insula within the lateral sulcus were
delineated on axial slices visualizing the caudate nucleus and the putamen.
Statistical Analyses:
Multiple regression analyses were performed with region specific 5-HT1A BP as
dependent variable and mean progesterone plasma level, age, ROI volume and
radiochemical variables, including injected activity and specific activity, as
independent variables. The Bonferroni-Holm (10) correction was used to correct for
multiple testing because of the multiple dependent variables (ROIs). The gain in
explained variance of 5-HT1A BP which can be exclusively attributed to progesterone
was computed by comparing the adjusted R squared between the model including
progesterone and the model excluding progesterone. In a second step, cortisol
plasma levels were included into the model as additional independent variables to
examine their mediating effects. Furthermore, regression analyses using the region
specific delivery rate R1 as dependent variable were computed to exclude effects on
regional BP due to tracer delivery. Regression diagnostics did not indicate
multicollinearity among the predictors. Additionally, the residuals were normally
distributed, and there was no presence of heteroscedasticity. SPSS version 15.0 for
Windows was used for statistical computations. The two-tailed significance level was
set at 0.05.
For additional information about the association between the 5-HT1A receptor, steroid
hormones and psychiatric symptoms (e.g., aggression, anxiety and depression), see
(7, 11-14).
Lanzenberger et al. 2011, Supplementary Information
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