3.1. Salivary testosterone
The mean endogenous testosterone level in men (155.61!4.47pg/ml) was significantly higher than in women(95.35!3.49pg/ml,t(232)=10.76,p<0.001). 3.2. Valenceand arousalratings(testosterone- independentanalyses)
Valence ratings differed significantly across valence cate- gories (men: mean valence ratings for negative pictures 1.31!0.27, mean valence ratings for positive pictures 2.72!0.18, mean valence ratings for neutral pictures 2.10!0.16; F(2,94)=984.44, p<0.001; women: mean valence ratings for negative pictures 1.17!0.14, mean
valence ratings for positive pictures 2.76!0.19, mean valence ratings for neutral pictures 2.10!0.17; F(2,136)=3162.50,p<0.001).
Arousalratingsforthedifferentvalencecategoriesofthe pictures were significantly different, with highest arousal ratings fornegative pictures and lowest arousalratings for neutralpictures(men:meanarousalratingnegativepictures 2.23!0.03,meanarousalratingpositivepictures1.85!0.04, mean arousal rating neutral pictures 1.34!0.02; F(2,190)=413.47, p<0.001; women: mean arousal rating negative pictures 2.40!0.03, mean arousal ratingpositive pictures 1.95!0.03, mean arousal rating neutral pictures 1.41!0.03; F(2,274)=734.50,p<0.001).Menandwomen differed significantly in the arousal ratings of all valences (arousal ratings for positive pictures: F(1,232)=4.14, p<0.05; arousal ratings for negative pictures: F(1,232)= 14.30, p<0.001; arousal ratings for neutral pictures: F(1,232)=3.95,p<0.05).
Inmenaswellasinwomen,arousalratingsweresignifi- cantly higher for recalled than for non-recalled pictures (women:meanarousalrecalledpictures2.05,meanarousal non-recalled pictures 1.83; t(1,137)=14.15, p<0.001; men: mean arousal recalled pictures 1.97, mean arousal non-recalledpictures1.71;t(1,95)=14.63,p<0.001). 3.3. Brainactivationduringpictureencoding (testosterone-independent)
Encodingofpicturesofeithervalenceascomparedtobase- line (i.e.scrambledpictures)activated alarge networkof neocortical and limbic brain regions including the frontal lobe, the temporal lobe, medial temporal lobe structures (hippocampus, parahippocampal gyrus and amygdala), the subthalamicnucleus,thecingulategyrus,theinsularcortex, thecuneus,aswellasparietalandoccipitalregions(Tables
S2—S7andFig.S2A—C).
A very similar activation pattern, including amygdala, hippocampusandinsularcortexwasfoundwhencomparing theencodingofemotionalpictures(negativeandpositive)to theencodingofneutralpictures(TablesS8—S11andFig.S3A andB).
Whencomparing negativetopositivepictureswe found activationinfrontal,temporalandoccipitalregions.Results forthepositivevs.negativecontrastrevealedactivationsin thefrontal,temporal,parietallobesandintheinsula(Tables
S12andS13).
All of these activation differences were very robustat significancelevelsofp<0.05FWEcorrectedforwholebrain. 3.4. Brainactivationrelatedto endogenous testosteronelevels
Wefoundapositivecorrelationbetweentestosteronelevels and amygdala activation during encoding of neutral vs. scrambledpicturesinmenonly(contrast: neutralpictures >scrambledpictures;Fig.1A,leftamygdala,["25"3"16], 29voxels,t(1,94)=4.17,r=0.39,p(SVC)=0.002(FWE)).Inan exploratory whole-brainanalysis nosuprathreshold clusters survivedthesignificancelevelofp<0.05family-wiseerror corrected in men and women. The amygdala was robustly (p(FWE)<0.05) activated during processing of neutral vs.
scrambledpicturesinthetestosterone-independentanalysis bothinmenandwomen(Fig.S2C).Therewerenosuprathres- hold activations with decreasing endogenous testosterone levels during processing of neutral vs. scrambled pictures neitherinmennorinwomen.
During encoding of negative vs. scrambled pictures no suprathresholdclusterswithincreasingnordecreasingendo- genouslevelsoftestosteronewerefoundneitherinmennor inwomen.Duringencodingofpositivevs.scrambledpictures thesamepatternemerged.
Weadditionallyanalyzedthecontrastsnegativepictures vs.neutralpicturesandpositivepicturesvs.neutralpictures
inrelation to both increasing anddecreasing testosterone levels.Weonlyfoundasignificantactivationinthecontrast positivepicturesvs.neutralpictureswithdecreasingtestos- terone levels (left amygdala, [!25!3!16], 3 voxels, t(1,94)=4.17,r=0.35,p(SVC)=0.010(FWE)),whichlikely reflectstheactivationfoundinthecontrastneutralpictures vs.scrambled pictures with increasing testosterone levels (samecoordinates).
3.5. Memory performance(testosterone- independentanalyses)
Subjects recalled more emotional than neutral pictures, (men: positive pictures 10.68"0.33, negative pictures 10.73"0.33, neutral pictures 6.26"0.29; F(2,190)= 175.64,p<0.001;women:positivepictures12.73"0.25, negative pictures 11.30"0.26, neutral pictures 7.00"0.25;F(2,274)=226,24,p<0.001).Womenrecalled significantlymore positivepictures than men(F(1,232)= 25.32,p<0.001),butmenandwomendidnotdifferinthe recallofnegativepictures(F(1,232)=1.89,p=0.17).Inthe recallofneutralpicturesmenandwomendifferedmargin- ally(F(1,232)=3.77,p=0.05).
Inmen,amygdalaactivationwasrelatedtobettermem- ory performance. Brain activation in the amygdala (ROI- analysis)washigherduringencodingofsubsequentlyrecalled picturesthanduringencodingofsubsequentlynotrecalled (forgotten) pictures (contrast: recalled pictures>not recalled pictures; left amygdala: [!19, !3, !16], t(95)=5.47,p(SVC)<0.001;right amygdala:[25,3,!20], t(95)=3.89,p(SVC)=0.005).Wefoundnohigheractivation in the amygdala for forgotten pictures compared with recalledpictures.
In women, we observed a similar pattern in the left amygdala([!19, !6, !16], t(137)=5.47,p(SVC)=0.004). Therightamygdalashowedincreased activationfor subse- quentlyrecalledvs.forgottenpicturesaswell,butitdidnot survive SVC for the bilateral amygdala ([19, !6, !16], t(137)=2.89, p(SVC)=0.068). Again, we found no higher activationintheamygdalaforforgottenpicturescompared withrecalledpictures.
3.6. Testosteronelevels, memoryperformance andemotionalarousal
Endogenous testosterone levels in men were significantly correlatedwithrecallsuccessofneutralpictures(r=0.28, p=0.006; Fig. 1B), but not of positive pictures (r=0.14, p=0.18)ornegativepictures(r=0.13,p=0.22).Thecorre- lation with all pictures showed borderline significance (r=0.20,p=0.05).Wedidnotfind anysignificantcorrela- tionsbetweenendogenoustestosteronelevelsandmemory inwomen(allp#0.75).
Lookingatcorrelationsbetweenendogenoustestosterone levelsandarousalratingsinmen,wefoundapositivecorrela- tionbetweenendogenoustestosteronelevelsandthearousal ratingsforneutralpictures(r=0.22,p=0.03).Correlations betweenendogenoustestosteronelevelsandarousalratings forpositive or negativepictures didnotreach significance (arousalratingspositivepictures:r=0.16,p=0.11;arousal ratingsnegativepictures:r=!0.06,p=0.58).Wedidnotfind Figure1 Correlations of testosterone levels with amygdala
activityandmemoryperformanceinmen.(A)Positivecorrela- tion between testosterone levels at encoding and amygdala activationduringencodingofneutralpictures.Activationswere overlaidonsectionsofaT1-weightedmagneticresonanceimage ofSPM5,displayedatanuncorrectedthresholdofp<0.001and usingcolor-codedtvalues.Peakactivationintheamygdalawas at coordinate position [!25, !3, !16], pFWE small volume cor-
rected=0.002.(B) Correlationbetweentestosteronelevelsand
neutralpicturememory(numberofcorrectlyrecalledneutral pictures).
anysignificantcorrelationsbetweenendogenoustestosterone levelsandarousallevelsinwomen(allp!0.29).
An additional analysis dividing women in free-cycling women(n=50)andwomen takinghormonalcontraceptives (n=88)suggests,thatthemenstrualcycleandtheintakeof hormonalcontraceptivesdidnotaffectourfindings(formem- oryperformanceofneutral pictures: interactionmenstrual cycle"endogenous testosterone levels p=0.28, intake of hormonal contraceptives"endogenous testosterone levels p=0.57;forarousal ratingsofneutralpictures:interaction menstrualcycle"endogenous testosterone levels p=0.23, intakeof hormonal contraceptives"endogenous testoster- onelevelsp=0.36).WhenconductingseparatefMRI-analyses forwomen takinghormonalcontraceptivesandfreecycling women,theresultsstayedsimilar.
3.7. Working memorytask
We found a trend for a negative correlation betweenthe measurementfor workingmemory performance andendo- genoustestosterone levels inmen(r=#0.17, p=0.09).In women we did not find a significant correlation between testosterone levels and working memory performance (p=0.36).