involved in the process of emotional prosody perception at the same time. A functional network of brain areas involved in phonological processes has been described in a recent meta-analysis on left hemisphere language areas [38]. In this study an area that corresponds to the fronto-parietal operculum, the rolandic operculum, has been described as part of the audio-motor loop. In this audio-motor loop the rolandic area is in charge of a motor representation of auditory stimuli to guide comprehension [38]. The fronto-parietal operculum forms part of the somatosensory cortex comprising representations of the lips, jaw and tongue. In emotional prosody perception these representations may be evoked to simulate the perceived auditory emotion. This simulation guides the comprehension of the acoustic properties of the human voice, that are processed in the anterior superior temporal gyrus at the same time. A recent imaging study provides evidence that the fronto-parietal operculum acts in an audio- motor loop to guide comprehension of emotional prosody. This study demonstrated that passive listening to nonverbal emotional vocalizations activates a network of premotor cortical regions involved in the control of facial movement [39]. This is consistent with Damasio’s conceptualization of an ‘‘as-if body loop’’, i.e. a neural internal simulation that uses the brain’s body maps, but bypasses the actual body [40]. Another explanation could be inadvertent stimulation of the non-targeted area due to proximity of the right anterior superior temporal gyrus and the right fronto-parietal operculum. The mean distance between the two sites of interest was however 28 mm with a small SD of 4.9 mm, which is large enough to assume that stimulating one area does not directly affect the other [41]. Finally, it should be taken into account that the clicking sound of the TMS coil may have distracted the subject from the task and that this lowered performance. We cannot rule out this possibility, but there is enough reason to believe that there was (also) a real TMS effect. The results of our study provide evidence that the clicking alone cannot completely explain the delays in reaction times. Although the anterior superior temporal gyrus is closer to the ear, reaction times to stimulation of the right fronto-parietal operculum were even slower than with stimulation to the anterior superior temporal gyrus. A possible effect of the clicking sound can not explain this sufficiently. Moreover, the time pattern was not found for all emotions, if slower performances would be a result of acoustic interference of the clicking sound, we may have expected generalized effects. Van Rijn and colleagues also stimulated the right fronto- parietal operculum and observed a difference in emotional prosody recognition between sham stimulation (no magnetic stimulation but with the characteristic ‘click’) and real stimulation [23]. This suggests that the TMS-effect is distinct from a mere effect of the click-sound. The inclusion of the active control condition also suggests that the effect we found should be attributed to the direct magnetic stimulation of the brain. Reaction times in the active control condition were significantly shorter than in two areas that have been related to emotional prosody processing. Stimulation of right anterior superior temporal gyrus and right fronto-parietal operculum resulted in longer reaction times than stimulation of an area that has not been associated with emotional prosody. The present study confirms that both the right anterior superior temporal gyrus and the right fronto-parietal operculum are involved in the process of emotional prosody perception. Moreover, the results reveal a relatively late critical time of involvement of both regions for recognizing emotional prosody in entire sentences. This may reflect that emotional cues can still be ambiguous at the beginning of sentences but become more apparent half-way through the sentence. Our study forms the first step in disentangling the Time Course Emotional Prosody temporal involvement of different brain areas in the process of emotional prosody recognition in natural speech. Materials and Methods Subjects Fourteen healthy subjects (aged 19 to 41, 7 females) participated in the first two conditions (right anterior superior temporal gyrus and right fronto-parietal operculum). Ten (aged 19 to 29, 6 females) of these 14 also participated in the third active control condition (midline parietal-occipital junction EEG site, POz). All were right-handed, measured with the Edinburgh Handedness Inventory and had an adequate understanding of Dutch (comprehension and reading). Participants were checked for TMS and MRI exclusion criteria. They were given extensive written and oral explanation of the procedures and signed an informed consent. The experiment was conducted in accordance to the Declaration of Helsinki and with local ethics committee approval (University Medical Centre Groningen). Experimental setup A neural navigator (NeNa) was used to locate the intended stimulation areas of each participant [42]. This frameless stereotaxy device uses a structural MRI scan of a person’s head to guide a TMS coil to the proper region on the skull. Anatomical MRI scans were made on a Philips Intera 3 Tesla MR-system at the BCN-Neuroimaging Center, Groningen. Regions of interest were drawn in MRIcro a few days before the TMS experiment by the primary investigator and checked by another researcher until the two reached consensus (see figure 2). Number of voxels per region of interest was ca. 120, 1 to 2 cm2, in accordance with the size of the region TMS affects [43]. The fronto-parietal operculum was defined as the inferior peri-central sulcus area, at the border of the frontal and parietal lobes, marked by the central sulcus, situated at the bottom of the central sulcus (covering both precentral as well as postcentral regions) above the lateral sulcus, in accordance with the study of van Rijn [3,23]. Talairach coordinates were ca. (53, 26, 14) [44]. The right anterior superior temporal gyrus was defined as approximately 1.5 cm posterior to the temporal pole, under the precentral sulcus, bordered by the lateral sulcus and superior temporal sulcus, based on imaging studies [5,19], Talairach coordinates ca. (52, 24, 24) [44]. The additional TMS condition, EEG location POz could be marked on an EEG cap using the international 10–20 system, therefore no Figure 2. This figure shows the two TMS locations that are targeted in the present study drawn in MRIcro, the fronto- parietal operculum and the anterior superior temporal gyrus. doi:10.1371/journal.pone.0002244.g002 PLoS ONE | www.plosone.org 5 May 2008 | Volume 3 | Issue 5 | e2244