anatomical determination was needed. Talairach coordinates for this electrode location were approximately (0, 295, 16) [44]. At the beginning of the TMS sessions the right fronto-parietal operculum and the right anterior superior temporal gyrus were targeted and marked on an EEG-cap [45]. Mean distance between right fronto-parietal operculum and right anterior superior temporal gyrus measured on the EEG cap was 28 mm (SD 4.9 mm). Triple-pulse TMS (tpTMS) was applied using a Medtronic MagPro X100 stimulator (Medtronic, Skovlunde, Denmark) with a figure-of-eight coil (diameter 75 mm), with a dynamic cooling device (Cool-B65). The coil was maintained in a constant position with the Medronic coil arm. The orientation of the coil was set with the handle pointing downwards, making an angle of 90u with the midline of the head. TMS Procedure At the beginning of each TMS session, subjects completed the positive and negative affect schedule (PANAS), to assess their current affective state [33]. This self-assessment questionnaire consists of 10 positive (interested, excited, strong, enthusiastic, etc.) and 10 negative affects (distressed, upset, guilty, etc). All items have to be rated on a scale from 1 to 5, based on the strength of emotion where 1 = ‘‘ very slightly or not at all,’’ and 5 = ‘‘extremely’’. Participants were seated in a comfortable chair, in front of a computer screen and fitted with an EEG cap to allow marking of stimulation sites. First, individual motor thresholds (MT) of the (preferable) left hemisphere were determined using the thumb (abductor pollicis brevis) movement procedure [46]. Stimulation intensity was set at 90% MT (mean 45.6 (SD 6.9)). First, participants completed a training session of 6 sentences. The entire procedure was completed in three sessions on separate days, around the same hour. Order of the experimental blocks and the two brain areas of interest (right fronto-parietal operculum and right anterior superior temporal gyrus) were counterbalanced over subjects. The extra active control condition was included later (in 10 subjects), this area was always stimulated in the last session. A triplet of TMS pulses was given at various onset times per stimulus to identify the time points at which right anterior superior temporal gyrus and right fronto-parietal operculum are critical for processing emotional prosody. In every tpTMS run, the TMS coil was placed over the area of interest while the prosodic discrimination of emotion task was completed. During each trial, a triplet of three single TMS pulses was applied with an inter- pulse-interval of 100 ms, based on a recent TMS study [30]. In an event-related potential study, a peak was found around 360 ms, under voluntary processing of emotional prosody in words [36]. In a pilot study mean reaction time for detection of emotions was 2389 ms (SD 884) without TMS. Duration of response prepara- tion is 6400 ms. Based on these data, the onset times of tpTMS in the present study varied between 400 and 1900 ms, in steps of 300 ms. There was a total of 8 experimental blocks of the same length during which tpTMS was applied at different pseudo- References 1. Mitchell RL, Crow TJ (2005) Right hemisphere language functions and schizophrenia: the forgotten hemisphere? Brain 128: 963–978. 2. Morris JS, Scott SK, Dolan RJ (1999) Saying it with feeling: neural responses to emotional vocalizations. Neuropsychologia 37: 1155–1163. 3. Adolphs R, Damasio H, Tranel D (2002) Neural systems for recognition of emotional prosody: a 3-D lesion study. Emotion 2: 23–51. 4. Wildgruber D, Pihan H, Ackermann H, Erb M, Grodd W (2002) Dynamic brain activation during processing of emotional intonation: influence of acoustic parameters, emotional valence, and sex. NeuroImage 15: 856–869. Time Course Emotional Prosody randomized time points on the scalp positions. The number of sentences per emotion per time-point was 8. Every ten seconds a new sentence was presented. Minimum time between triplets of pulses was 8.5 seconds, enough to avoid carry-over effects [22]. The procedure for the right fronto-parietal operculum and right anterior superior temporal gyrus TMS sessions took 90 minutes at maximum. The same design was used in an extra session, for the active control condition, i.e. real TMS was performed to this area but we did not expect effects on the task, because this area is not related to emotion in language. During this condition, stimulation was applied with the centre of the coil at the POz electrode site, the duration was 60 minutes. This is 30 minutes less than in the other two TMS conditions, because no neuronavigation was needed. Emotional prosody task Sentences of neutral content pronounced in an emotional (anxious, sad or happy) tone of voice by male and female actors were used as stimuli. The task was developed and presented using Eprime software [47]. These were selected from three different validated prosody tests [48–50]. All sentences were Dutch, examples are, ‘‘The old car drives through the streets of the capital’’ and ‘‘Jan has been to the hairdresser’’. The sentences were tested by ten separate subjects. The digitized stimuli were of approximately equal length and presented through two computer sound boxes (mean duration 2042 ms (SD 755)). During listening, the names of the emotions to be discriminated were presented on the computer screen. The visual presentation of the answer choices was included to aid subjects as to which categories they were to choose from. Without the visual presentation, subjects would have to memorize the different categories, and the task would have a stronger working memory component. Participants were instruct- ed to use the index finger of their right hand to make a ‘fear’ response on a keypad, the middle finger to make a ‘sad’ response or the thumb for a ‘happy’ response as soon as they identified the emotion expressed in the tone of voice. Speed and accuracy were stressed as objectives. Acknowledgments The authors are grateful to Prof. J. Vroomen, Prof. G. Vingerhoets and dr. E. Bos for allowing us to make use of their stimuli. The authors are also grateful to dr. R.Renken who helped us with experimental setup and to dr. R. Kortekaas for editorial aid. Furthermore the authors thank the participants of this study. Author Contributions Conceived and designed the experiments: AA MH. Performed the experiments: MH LB. Analyzed the data: MH. Contributed reagents/ materials/analysis tools: LB. Wrote the paper: MH. Other: Critically read the manuscript and gave helpful comments to improve the text: RK. Contributed to the text of the paper and designed the study: MH AA. Read the manuscript and gave suggestions from improvement: LB. Made the tables and figures: LB. 5. Kotz SA, Meyer M, Alter K, Besson M, von Cramon DY, et al. (2003) On the lateralization of emotional prosody: An event-related functional MR investiga- tion. Brain and Language 86: 366–376. 6. Hornak J, Bramham J, Rolls ET, Morris RG, O’Doherty J, et al. (2003) Changes in emotion after circumscribed surgical lesions of the orbitofrontal and cingulate cortices. Brain 126: 1691–1712. 7. Wildgruber D, Hertrich I, Riecker A, Erb M, Anders S, et al. (2004) Distinct Frontal Regions Subserve Evaluation of Linguistic and Emotional Aspects of Speech Intonation. Cerebral Cortex 14: 1384–1389. PLoS ONE | www.plosone.org 6 May 2008 | Volume 3 | Issue 5 | e2244