right somatosensory cortices in emotion recognition has been argued to occur irrespective of modality and of specific emotion [25]. Recent evidence from TMS studies shows that right somatosensoric areas, like the fronto-parietal operculum, may be specifically involved in evaluating withdrawal emotions [23,26]. Not only the right fronto-parietal operculum but also the right anterior superior temporal gyrus seems to play a role in the perception of emotional prosody [3,12,21]. The anterior part of the superior temporal gyrus has been found to be a voice-selective area [27]. Regions along the anterior superior temporal gyrus show greater response to human voices as compared to non-vocal sounds [27]. This region has also been shown to be involved in both the production and perception of singing [28]. Although a large number of studies have tried to elucidate the brain regions involved in emotional prosody perception (resulting in discrepant data), only a few studies have looked at the time pattern of emotional prosody processing. To help in reconciling the contro- versial data from previous patient and imaging studies, we focused on both right fronto-parietal operculum and right anterior superior temporal gyrus in order to investigate their potentially different roles in the process of emotional prosody perception. Indeed, both right fronto-parietal operculum and right anterior superior temporal gyrus might be critical for the processing of emotional prosody, albeit possibly not for the same functional reasons. The multi-step process of emotional prosody perception has been described by Schirmer and colleagues, comprising the following mental processes in temporal order: 1) analyzing the acoustic elements of vocalizations, 2) deriving emotional significance from a set of acoustic cues, 3) applying emotional significance to higher order cognition [29]. A fourth component could be the integration of emotional prosody in language processing. Sack and colleagues introduced a procedure to map time points at which different brain regions are critical for information processing [30]. This procedure allowed us to shed light on the precise time course of the contribution of the right anterior superior temporal gyrus and the right fronto-parietal operculum in emotional prosody perception. We expected to find the following time course of involvement of the two areas that were focused on in the present study. The right anterior superior temporal gyrus has been assumed to have a prominent role in the extraction of slow acoustic elements [4,19,27,29,31]. This area will be involved at the beginning of the time-path in step 1 of the multi-step process described above. Later the perceived emotion is represented in the somatosensory right fronto-parietal operculum where simulation provides information about the specific emotion [3,32], part of step 2. Aim of the present study was to analyze the effect of triple-pulse TMS over the right anterior superior temporal gyrus and the right fronto-parietal operculum at different time intervals from stimulus onset on the perception of emotional prosody. An additional active control TMS condition was included to test whether the effect is specific to the targeted region. Results Reaction times and percentages correct were analyzed by repeated measures ANOVA, in which the factors were Site of stimulation (anterior superior temporal gyrus (STG), fronto- parietal operculum (FPO), POz), Time (6 points, varying from 400 to 1900ms, with 300 ms in between) and Emotion (fear, sad, happy). Only reaction times for correct trials were included in the analyses. Due to a high percentage correct over all subjects (84%) the results are based on the majority of data collected. Post hoc analyses were performed using pair-wise comparisons. Effects were tested at a significance level of p,.05. At the beginning of each Time Course Emotional Prosody transcranial magnetic stimulation (TMS) session subjects were asked to complete the positive and negative affect schedule (PANAS) to evaluate their current affective state [33]. This was done to be able to correct for potentially different affective states that could influence the performance on an emotion task. A 362 repeated measures ANOVA with Site of stimulation (aSTG, FPO, POz) and scores on the PANAS scale (positive scale, negative scale) revealed no significant differences between the three TMS sessions, F (2,18 ) = 1.22, p = .32. This means that we can assume that there was no significant effect of mood on the reaction times [33]. Differences in reaction times during TMS conditions were not attributable to differences in mood state between the TMS conditions. Gender was added as a between subjects factor in the repeated measures ANOVA, but no significant differences were found across gender groups, F (1, 8) = .47, p = .51. Reaction times during triple-pulse rTMS at the fronto- parietal operculum and the anterior superior temporal gyrus A 26663 repeated measures ANOVA with Site of stimulation (aSTG, FPO), Time (6 points, from 400 to 1900ms) and Emotion (fear, sad, happy) as independent variables, showed that there was no significant difference in reaction times between the two sites of stimulation, F (1,13) = .79, p = .39. There was a main effect of Time, F (5, 65) = 31.3. p,.001. In addition, the quadratic term was highly significant, F (1, 13) = 69.4, p,.001. The quadratic trend reflects a peak in reaction times at a certain time point. Post hoc analysis revealed most strong interference of the triplet of TMS pulses at 1300 ms from onset of the stimulus. At this onset time the mean reaction time was significantly larger than at all other time points, mean differences varied from 114 ms to 346 ms, (p,.05). The analysis also revealed a main effect of Emotion F (2, 26) = 7.3, p,.010. Longest reaction times were found for the detection of the emotion fear. Contrasts revealed that the difference between reaction times for correctly classifying ‘fear’ sentences compared to ‘happy’ sentences was significant F (1,13) = 26.04, (p,.05). Subjects were faster in the detection of happiness from intonations than in the detection of fear. The difference between performance on ‘fear’ sentences and ‘sad’ was also significant F (1,13) = 5.20, (p ,.05). Subjects responded faster on ‘sad’ sentences than on ‘fear’ sentences. Furthermore there was a significant interaction between Emotion and Time F (10,130) = 6.1, p,.001. This indicates that the time of delivering the triplet of TMS pulses had a different effect depending on which emotion was expressed. To explain this interaction, reaction times per emotion over the 6 time-points were analyzed. Time of stimulation significantly affected reaction times for the Emotions ‘fear’ and ‘sad’, F (5, 65) = 12.7, p,.001 and F ( 5, 65) = 28.4, p,.001 respectively. For the Emotion ‘happy’ Time of stimulation was also significant but with a larger p-value, F (5, 65) = 2.6, p,.05. Whereas the strongest interference for ‘fear’ and ‘sad’ stimuli was observed at 1300 ms, this was not apparent for happy (see figure 1). Reaction times during triple pulse rTMS with the inclusion of active control condition POz To test whether the results described in the above section are a real TMS effect, an additional active control condition over the occipital cortex (POz electrode location) was included. We can assume that this brain area is not related to emotional prosody. Inclusion of condition POz (10 participants), revealed a main effect of Site of stimulation F( 2,18) = 4.7, p,.05. Contrasts revealed that both right anterior superior temporal gyrus and right fronto- parietal operculum differed significantly from POz, F (1, 9) = 6.5, PLoS ONE | www.plosone.org 2 May 2008 | Volume 3 | Issue 5 | e2244