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					Lanzenberger et al. 2011, Supplementary Information                         1/13




             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
Lanzenberger et al. 2011, Supplementary Information   2/13


Supplementary figure (see legend at page 3/14)
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 5-

HT1A 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                                         4/13


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

               Injected dose (MBq/kg body weight)                     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



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                              Limit of sensitivity          Interassay CV

         Cortisol                                      0.4   ng/mL                6    %

         Progesterone                               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                                        5/13

                                        2
Region                       Partial R for   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                                       6/13

                                        2
Region                       Partial R for   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                                            7/13



                                        2
Region                       Partial R 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



 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                                 8/13



                     3
    ROI volumes (cm )                     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                                 9/13


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        11
                                                        C 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 (three-

dimensional mode) started simultaneously with bolus injection of [carbonyl-11C] WAY-

100635 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
Lanzenberger et al. 2011, Supplementary Information                              10/13

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 hormone-

induced 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                                 11/13

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                            12/13

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                                        13/13


Supplementary References

1.     Wagner CK. The many faces of progesterone: a role in adult and developing male
       brain. Front Neuroendocrinol 2006; 27: 340-59.
2.     Castren M, Patchev VK, Almeida OF, Holsboer F, Trapp T, Castren E. Regulation of
       rat mineralocorticoid receptor expression in neurons by progesterone. Endocrinology
       1995; 136: 3800-6.
3.     Nishi M, Ogawa H, Ito T, Matsuda KI, Kawata M. Dynamic changes in subcellular
       localization of mineralocorticoid receptor in living cells: in comparison with
       glucocorticoid receptor using dual-color labeling with green fluorescent protein
       spectral variants. Mol Endocrinol 2001; 15: 1077-92.
4.     Nishi M, Tanaka M, Matsuda K, Sunaguchi M, Kawata M. Visualization of
       glucocorticoid receptor and mineralocorticoid receptor interactions in living cells with
       GFP-based fluorescence resonance energy transfer. J Neurosci 2004; 24: 4918-27.
5.     Fejes-Toth G, Pearce D, Naray-Fejes-Toth A. Subcellular localization of
       mineralocorticoid receptors in living cells: effects of receptor agonists and antagonists.
       Proc Natl Acad Sci U S A 1998; 95: 2973-8.
6.     Carey MP, de Kloet ER. Interaction of progesterone with the hippocampal
       mineralocorticoid receptor. Ann N Y Acad Sci 1994; 746: 434-7.
7.     Lanzenberger RR, Mitterhauser M, Spindelegger C, Wadsak W, Klein N, Mien LK, et
       al. Reduced Serotonin-1A Receptor Binding in Social Anxiety Disorder. Biol
       Psychiatry 2007; 61: 1081-9.
8.     Wadsak W, Mien LK, Ettlinger D, Lanzenberger R, Haeusler D, Dudczak R, et al.
       Simple and fully automated preparation of [carbonyl-11C]WAY-100635.
       Radiochimica Acta 2007; 95: 417-22.
9.     Spindelegger C, Lanzenberger R, Wadsak W, Mien LK, Stein P, Mitterhauser M, et al.
       Influence of escitalopram treatment on 5-HT 1A receptor binding in limbic regions in
       patients with anxiety disorders. Mol Psychiatry 2009; 14: 1040-50.
10.    Holm S. A simple sequentially rejective multiple test procedure. Scandinavian Journal
       of Statistics 1979; 6: 65–70.
11.    Witte AV, Floel A, Stein P, Savli M, Mien LK, Wadsak W, et al. Aggression is related
       to frontal serotonin-1A receptor distribution as revealed by PET in healthy subjects.
       Hum Brain Mapp 2009; 30: 2558-70.
12.    Moser U, Wadsak W, Spindelegger C, Mitterhauser M, Mien LK, Bieglmayer C, et al.
       Hypothalamic serotonin-1A receptor binding measured by PET predicts the plasma
       level of dehydroepiandrosterone sulfate in healthy women. Neurosci Lett 2010; 476:
       161-5.
13.    Lanzenberger R, Wadsak W, Spindelegger C, Mitterhauser M, Akimova E, Mien LK,
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14.    Akimova E, Lanzenberger R, Kasper S. The serotonin-1A receptor in anxiety
       disorders. Biol Psychiatry 2009; 66: 627-35.

				
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