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, et al. Cortisol plasma levels in social anxiety disorder patients correlate with serotonin-1A receptor binding in limbic brain regions. Int J Neuropsychopharmacol 2010: 1-15. 14. Akimova E, Lanzenberger R, Kasper S. The serotonin-1A receptor in anxiety disorders. Biol Psychiatry 2009; 66: 627-35.
Pages to are hidden for
"SUPPLEMENTARY INFORMATION"Please download to view full document