Reproductive toxicology. Triethylene glycol diacetate.

Our previous studies on scalp-recorded event-related potentials (ERPs) showed that somatosensory N140 evoked by a tactile vibration in working memory tasks was enhanced when human subjects expected a coming visual stimulus that had been paired with the tactile stimulus. The results suggested that such enhancement represented the cortical activities involved in tactile-visual crossmodal association. In the present study, we further hypothesized that the enhancement represented the neural activities in somatosensory and frontal cortices in the crossmodal association. By applying independent component analysis (ICA) to the ERP data, we found independent components (ICs) located in the medial prefrontal cortex (around the anterior cingulate cortex, ACC) and the primary somatosensory cortex (SI). The activity represented by the IC in SI cortex showed enhancement in expectation of the visual stimulus. Such differential activity thus suggested the participation of SI cortex in the task-related crossmodal association. Further, the coherence analysis and the Granger causality spectral analysis of the ICs showed that SI cortex appeared to cooperate with ACC in attention and perception of the tactile stimulus in crossmodal association. The results of our study support with new evidence an important idea in cortical neurophysiology: higher cognitive operations develop from the modality-specific sensory cortices (in the present study, SI cortex) that are involved in sensation and perception of various stimuli.


INTRODUCTION
Recent monkey studies have shown evidence that cells in primary somatosensory cortex (SI) and secondary somatosensory cortex (SII) change their firing correlated with tactile unimodal working memory [1][2][3][4]. In a recent human study [5], it was shown that SI cortex retained a memory trace of the tactile stimulus for a short period. Further, cells in the somatosensory cortex of monkeys were shown to respond to task-related stimuli of more than one sensory modality in working memory tasks [6][7][8]. Crossmodal effects have also been observed in studies on neural mechanisms of attention in monkeys, in which firing changes in cells of somatosensory cortex were found in the crossmodal attention switch [9,10], and in attention studies of humans, in which changes in early modalityspecific sensory (visual, auditory, and somatosensory) ERP (eventrelated potential) components were detected [11,12]. The above observations suggest that crossmodal links affect sensory-perceptual processes within modality-specific cortical regions [11].
In behavioral studies, it has been shown that viewing the stimulated body part can improve tactile discrimination at the stimulated site [13][14][15]. The visual-tactile improvement may be linked to modulations of neural activities in SI [15,16] through the higher-level multimodal associative cortex [16][17][18][19], suggesting the involvement of both sensory and associative cortical areas in visual-tactile crossmodal associations.
In our previous study [20], we found that the amplitude of the ERP component N140 evoked by the tactile stimulus was increased when the subject expected a coming visual stimulus that had been paired with the tactile stimulus in comparison to this component evoked by the same tactile stimulus without crossmodal expectation. It has been suggested that the somatosensory N140 is generated by sources in multiple cortical areas, including frontal cortex and SII cortex [21][22][23]. By applying independent component analysis (ICA) in the present study to the EEG (electroencephalogram) data recorded in the unimodal and crossmodal tasks, we explored independent components (ICs) that represented neural activities in cortical areas. We found that the crossmodal modulation of the N140 represented the neural activities in somatosensory (SI, and possibly SII as well) and frontal cortical areas that cooperated with each other in crossmodal association in the tasks.

Independent components (ICs)
Thirty different independent components were found through ICA from each task of each subject. In comparison among them, 2 independent components across 2 tasks and 10 subjects showed consistent temporal activities and topographies of their coefficients of spatial projection to scalp electrodes. We defined those 2 ICs as IC-F (F: frontal) and IC-RS (RS: right somatosensory).
Topographies The IC-F appeared to be active in prefrontal areas, and the IC-RS appeared to be active in right somatosensory areas. The topography of the IC-F is shown in Figure 2, and of the IC-RS is shown in Figure 3. Individual topographic maps were normalized by root mean square, and made the same polarities [24]. Topographies of both IC-F and IC-RS were apparently consistent across 10 subjects and the two tasks. Topographies of IC-F and IC-RS were averaged respectively across subjects and tasks (Figure 4), and the grand mean of the topographies of each IC was then submitted to BESA2000 to obtain the location of the IC-related dipole in the brain ( Figure 4). The IC-F-dipole location was found to be around the medial prefrontal areas, anterior part of the midline of the brain (Talairach coordinates [25]: 0. 5, 19.5, 43.4). This location was estimated to be in anterior cingulate cortex (ACC, area 32). IC-RS-dipole was estimated around the right primary somatosensory area (Talairach coordinates: 33.0,222.5, 41.6, area 3).
Temporal activities Temporal activities of two independent components, IC-F and IC-RS were analyzed. Back-projections of the IC-F showed waveforms with peaks similar to the original ERP components, P100 and N140 ( Figure 5). A four-way repeated measures MANOVA (see Materials and Methods) showed no significant difference in latency of those two components between the IC-F back-projections and the original ERPs (P100: F = 3.3, df (Effect) = 1, df (Error) = 9, p = 0.103; N140: F = 0.2, df (Effect) = 1, df (Error) = 9, p = 0.691). Results of the amplitude analysis showed that substantial proportions of the original P100 and N140 were contributed by the IC-F. No significant difference was observed between modalities in IC-F contributions to the N140, although the significant difference between modalities in this component was shown in the original ERPs.
Back-projections of the IC-RS showed a component similar to the original ERP component P45 observed at three electrodes on the right side, contralateral to the tactile stimulus ( Figure 6). No significant difference was observed between the IC-RS component and the ERP P45 in latency among the 9 electrodes. This IC-RS component was significantly affected among these 9 electrodes by AP (F = 12.1, df (Effect) = 2, df (Error) = 8, p,0.005) and LR (F = 33.6, df (Effect) = 2, df (Error) = 8, p,0.0005), but not by Modality (F = 4.3, df (Effect) = 1, df (Error) = 9, p = 0.07) although Post hoc test (Tukey HSD) showed that this component was significantly higher in the crossmodal task at several electrodes (figure 7).
Time-frequency representation (TFR), coherence, and Granger causality spectra Power spectra for IC-F and the original ERPs at FCz, and for IC-RS and the original ERPs at C4 were analyzed across all subjects ( Figure 8). At these two electrode sites, back-projections from IC-F and IC-RS showed highest amplitude respectively. Results indicated that in the time range of 100,300 ms, independent components and ERPs showed activities mainly with frequency in the theta band (3)(4)(5)(6)(7).
Coherence between IC-F and IC-RS is indicated in Figure 9 (left side). A three-way repeated measures MANOVA (see Materials and Methods) showed no significant effects of any main factor (Modality, Duration, Frequency-Band). Post hoc test (Tukey HSD), however, showed in the crossmodal task the significantly stronger coherence in the theta band during 100,200 ms after the onset of S-1 compared with the baseline (2100,0 ms).
Granger causality spectra were obtained (see Materials and Methods) to test the direction of the connectivity between IC-F and IC-RS in the crossmodal task since significant coherence was observed between these ICs in the task. Results showed trends that the connectivity after the onset of the stimulus (0,300 ms) was stronger than before the stimulus (2100,0 ms) in the task (Fig. 10). The causality index (CI) was significantly affected by Frequency-Band (F = 10.5, df (Effect) = 3, df (Error) = 7, p,0.01), but not by other two factors (Modulation, Duration). The interaction between Modulation and Frequency-Band was marginal (F = 4.2, df (Effect) = 3, df (Error) = 7, p = 0.05). Post hoc analysis showed that, in general, the pre-stimulus CI was the smallest. For crossmodal bottom-up modulation (Fig. 10), CI in the theta band in the duration from 100 ms to 200 ms after the onset of S-1 was significantly (p,0.001) larger than that before the S-1 (2100,0 ms).

DISCUSSION
ICA is a technique that has been successfully applied to human EEG studies in the last decade [24,[26][27][28][29][30][31]. ICA completely decomposes single-trial (or continuous) EEG data, separating the data into distinct information sources. By using this technique in data analysis, the multi-channel EEG can be decomposed into spatially fixed, temporally maximally independent components, and the scalp maps associated with some of these ICs resembled the scalp projections of synchronous activity inside the brain in a cognitive task [27]. Thus, when subjects perform behavioral tasks the ICs likely represent neural activities in those brain areas where they are located [24,26,27,31,32]. In the present study, the use of the ICA technique enabled us to find from the original EEG data two ICs (IC-F and IC-RS) that represented neural activities correlated with tactile working memory tasks, unimodal or crossmodal. This finding strongly suggests that cortical locations of those two ICs, medial prefrontal cortex and SI are involved in perception of the tactile stimulus and crossmodal associations in the task, and it may therefore provide us with a better understanding of the neural mechanism underlying the crossmodal working memory. The results of our study showed the beneficial application of the ICA technique that leads us to valuable findings that would not have been possible with the traditional ERP analysis. Studies have shown that P100 and N140 are enhanced when attention is directed to the somatosensory stimuli, and are modulated by endogenous spatial attention as well [22,[33][34][35][36][37][38][39][40]. In the present study, the subject's attention was directed to the tactile stimulus (S-1) to detect the frequency of vibration. The level of attention was essentially the same in both tasks. Backprojections of IC-F showed its substantial non-differential (similar in both tasks) contributions to P100 and N140, indicating that medial prefrontal cortex, most likely ACC, was one of the major sources of these two ERP components, which represented neural activities of ACC in attention to the same tactile stimulus (S-1) in the tasks. This finding showed that the ACC was involved in attention on the tactile stimulus as early as 100 ms after the onset of the stimulus. The present finding is consistent with the findings of other studies showing that the ACC plays an important role in attention of various stimuli [23,[41][42][43][44][45][46][47][48].
The back-projection analysis showed that another independent component found in this study, IC-RS, was the main generator for the ERP component P45 that typically represents the neural activity in SI cortex evoked by the contralateral somatosensory stimulus [21,35,36,49]. This suggested that changes in backprojection from the IC-RS represented neural activities of SI cortex in the task. The location of the IC-RS also supported the notion that the dynamic changes in IC-RS activity represented the changes in SI activity. Significant differences in IC-RS backprojection between the unimodal task and the crossmodal task were observed after the onset of the tactile stimulus, apparently because of the enhancement of IC-RS activity in the crossmodal task. The enhancement and the location of IC-RS strongly suggested that the crossmodal association between tactile and visual stimuli involved activities in the SI cortex as early as 100 ms after the onset of the tactile stimulus, or even earlier since P45 of the back-projection from IC-RS also showed trends toward differential reaction between the tasks. This new finding in the present study agrees with the findings in other studies that show participation of SI cortex in crossmodal association in monkeys [6][7][8], and in humans [15,16].
In our previous study [20], we argued that the enhancement of N140 in the crossmodal task was unlikely to be due to attention, movement, or load of the task, but rather was related to crossmodal transfer of information between the tactile and the visual modalities in the task. The IC-F found in our present study had a sizeable contribution to the N140, but its contribution did not show any significant difference between the tasks. Although we were not able to locate an IC that was consistent across subjects and tasks in SII area because of the limitations of the ICA technique, it is a reasonable assumption that the significant difference in N140 between the tasks likely resulted from the activity in SII since SII has been shown to be another major  source, in addition to the prefrontal source, to generate somatosensory N140 [22,[50][51][52][53]. Nonetheless, the possibility that other prefrontal areas contributed to the difference in N140 cannot be completely eliminated. It has also been suggested that P100-generators are located in the SII cortex [51,[54][55][56][57]. Therefore, the crossmodal modulation of both P100 and N140 generated by the subject's expectation of visual stimuli in the task may involve the change in neural activities in the SII cortex. The results of our study suggest that the crossmodal association may not only occur in association cortical regions, such as frontal cortex and posterior parietal cortex, but also occur in tactile modalityspecific cortical regions, such as SI and SII cortex.
Studies have shown that the ACC is involved in attentional modulation of sensory processing in primary visual and primary auditory cortices (e.g., [47] ). The theta oscillation in ACC may play an important role in the attentional modulation [58][59][60]. Our coherence analysis indicated that in the crossmodal task, compared with the baseline period the coherence in theta range during the period of 100,200 ms after the onset of the tactile stimulus was significantly increased between IC-F and IC-RS, showing that the activity in SI cortex may be synchronized with the activity in ACC in crossmodal association. This coherence between two areas suggested that ACC cooperated with SI cortex in attention and perception of the tactile stimulus under the influence of the crossmodal association. The Granger causality analysis of the coherence indicates that activities of ACC may be affected by SI cortex (bottom-up) as early as 100,200 ms after the onset of the tactile stimulus.
In conclusion, modulation was observed in the present study on activities of somatosensory cortex in the crossmodal task. Although how tactile crossmodal association is processed in the somatosensory system is still not well understood, our study clearly shows that SI cortex (presumably SII cortex as well) participates in the taskrelated crossmodal association that has been suggested by previous monkey and human studies (e.g., [7,16] ). In the process of crossmodal association, somatosensory cortex appeared also to cooperate with the higher level association cortex, the medial prefrontal cortex, in attention and perception of the tactile stimulus. Taken together, the results of our study support the idea with new evidence that higher cognitive operations develop from the modality-specific sensory cortices that are involved in sensation and perception of various stimuli [1,[61][62][63][64][65][66][67][68], and fit the concept of the perception-action cycle [69,70] that describes the cortical neural dynamics of sensory-motor behaviors.

MATERIALS AND METHODS
Details of experimental procedures for behavioral tasks and EEG recording have been described previously [71].

Participants
Twelve paid normal adult volunteers were recruited for the present study (10 men, 2 women, aged 19-47 years). Two participants were excluded because of excessive blinking or excessive muscle artifacts. Thus data from 10 subjects were collected and analyzed in the study. The data of 8 out of those 10 subjects were also used previously [20]. All participants signed informed consent. The protocols of the experiments were approved by the IRB of the Johns Hopkins School of Medicine.

Stimuli and EEG recording
Experiments were carried out in a quiet, dimly lit room. Participants sat in a comfortable chair, facing a light-emitting Electroencephalograms (EEG) were recorded by an EEG recording system (SynAmp, Neuroscan, Ltd Corp). Thirty-two Ag-AgCl scalp electrodes (Quick-Caps, Neuroscan) were arranged in standard locations (Guideline thirteen, American Clinical Neurophysiology Society, 2003). EEG signals from all of these electrodes were referenced to linked earlobes. The impedance of each electrode was kept below 5 kV. The electro-oculogram (EOG) was recorded for horizontal eye movements and for vertical eye movements. Signals of EEG (30 electrodes) and EOG (2 electrodes) were filtered (0.1-100 Hz band-pass), amplified, digitized (500 Hz sampling rate), and saved for off-line analysis.

Behavioral tasks
The scalp-ERPs were recorded when participants performed a tactile-tactile delayed matching-to-sample task (unimodal task) or a tactile-visual delayed matching-to-sample task (crossmodal task). The subjects were instructed to focus on the LED throughout a recording session to avoid eye movement and eye blinking within any trial of the task. Trials that showed eye-blinks, excessive eye movements, or muscle artifacts were excluded from data analysis.  In the unimodal task, a complete trial contained a sequence of events (Figure 1 upper). A trial started with stimulus-1 (S-1), a 100ms tactile vibration of either high (150 Hz) or low (80 Hz) frequency. After a delay of 1,500 ms, stimulus-2 (S-2), a 100-ms tactile vibration again (150 Hz or 80 Hz) was presented. During the delay, the subject was instructed to memorize the vibration frequency of S-1, and to expect S-2 that would match the frequency of S-1. The subject indicated at the end of the trial whether S-2 matched S-1 by pressing one of two buttons (e.g., left for match, right for nonmatch). The frequency of S-1 or S-2 was presented randomly from trial to trial to prevent the subject from getting any clue for performance. The intertrial interval between trials was chosen randomly in a range of 4-5 seconds. The subject's response time to S-2 was recorded.

Fz
In the crossmodal task (Figure 1 upper), the task sequence was identical to the unimodal task except that in this task S-2 was a visual cue (100 ms), a green or red LED associated with the tactile vibration. Associations between the tactile stimuli (S-1) and the visual stimuli (S-2) were assigned before the subject started performing the task (e.g., green associated with high frequency; red with low frequency) and counterbalanced across subjects. At the end of each trial, the subject indicated by pressing a button whether S-2 (LED) was associated with S-1.

EEG data analysis
Original EEG data from which trials with eye-blinks, excessive eye movements, or muscle artifacts had been excluded were filtered with a digital zero-phase filter (Finite Impulse Response filter, pass band 2 to 30 Hz). Amplitude of an ERP component was calculated as the difference between its peak and the baseline (200 ms preceding the onset of S-1) mean value. Its latency was measured from S-1 onset to the peak. A three-way repeated measures MANOVA was performed for comparisons of amplitude and latency of the original ERP components. The within-subject factors of the analysis were left-right electrode locations (LR) (left, center, right-corresponding to electrode locations of 3, z, and 4), anterior-posterior electrode locations (AP) (frontal, frontocentral, central, centroparietal, parietal levels-electrode locations of F, FC, C, CP, and P), and Modality (crossmodal and unimodal).

Independent component analysis
Analysis of EEG data recorded from 30 electrodes was performed by using Matlab 7.0 (Math Works, Natick, MA) and EEGLAB4.51(Swartz Center for Computational Neurosciences, La Jolla, CA; http://www.sccn.ucsd.edu/eeglab), a freely available open source software toolbox. BESA2000 (MEGIS, Graefeling, Germany) was also used to localize dipoles of independent components (ICs).
The filtered-EEG data (2,30 Hz) that preserved theta, alpha, and beta band information [31] were used for the ICA study. The onset of S-1 in each trial was used as the task-event marker to separate a trial into a period before the onset and a period after the onset. In each trial, filtered-EEG of 1,500 ms (500 ms before the onset of S-1 and 1,000 ms after it) were extracted from the continuous EEG to form a data epoch. The mean value of EEG amplitude in the first 100 ms of the epoch was calculated from all trials of each task in each individual subject. To obtain the EEG data epoch for further processing, this mean value was then subtracted from each corresponding data epoch to reduce the influence of EEG variance across trials.
All data epochs were put together and submitted to infomax ICA [72,73] that comes from the ICA families performing blind source separation. A 30630 unmixing square matrix was found by using Infomax ICA. When this matrix was multiplied by the EEG data epochs, maximally, temporally independent activities were obtained. In this calculation of the independent activities, a weight change of 10e-6 together with iterations ,800 were set as the stop criterion [24].
Let X denote the EEG data and M denote the unmixing square matrix. Then independent activities (S) are: S = MX. We can change the formula into X = M 21 S. In this formula, one row of the matrix S represents the temporal activity of one IC, and the corresponding column of the matrix M 21 represents this IC's spatial pattern at the scalp electrodes. The back-projection of an IC at one electrode is obtained by multiplying the temporal activity of this IC with its coefficient of the corresponding spatial pattern at this electrode. EEG at one electrode can be considered as the sum of back-projections of all ICs at this electrode. The temporal independent activity and its corresponding spatial pattern together characterize an IC that may correlate with the activity of a neuronal clique. In the present study, we screened activities of ICs to determine potentially common temporal patterns of those ICs across all trials of each task and subjects, and we also visually screened topographies of ICs to assess their potentially common spatial patterns across tasks and subjects. ICs showing event (onset of S-1)-related activities consistently across trials of each task and subjects, and spatial topographies consistently across tasks and subjects, were selected in the screening. The spatial topographies may reflect the dipole activity, presumably caused by partially synchronous activities within certain cortical source patches that produce far-field potentials through volume conduction. The above process of selection resulted in identification of ICs for each subject and each task, The grand average topography across subjects and tasks of a selected IC was submitted to BESA2000 that uses a standard four-shell spherical head model (i.e., brain, cerebrospinal fluid, bone, and scalp) to find the location of the IC-related dipole (source model) in the brain. The dipole was derived in BESA2000 by fitting it iteratively to the averaged IC topography parameters until minimal residual variance was reached. In the present study, the values of residual variance lower than 10% were used as the threshold [74].

Time-frequency representations (TFRs) and coherence
TFRs of ICs and ERPs from the electrodes that showed the largest IC back-projections were computed on single trials in the frequency range of 2,30 Hz by using Hanning windowed short time Fourier transformation. The window had a fixed length of 250 ms, moving across every time stamp. The mean value of the windowed-period was taken away to avoid the variation of direct current. Zeros were then added after each windowed-period to make TFRs smoother across the Frequency axis. The ratio of the zero-pad to the windowed-period was 32. The TFRs were then normalized for each frequency by subtracting the baseline (200 ms before the onset of S-1) mean value, and dividing by the baseline standard deviation [75]. The coherence spectrum of the two independent components (ICs) was calculated by using: C 12~S 12 j j 2 S 1 S 2 S 1 , S 2 are the power spectra of the two ICs respectively. S 12 is the cross spectrum between the two ICs. The value of coherence (C 12 ) at frequency f ranges from 1, indicating maximum interdependence between the two ICs, down to 0, indicating no interdependence. Trials were shuffled 200 times to examine the significance of the coherence values (p,0.05).
The window length used in the coherence calculation was the same as those in calculation of the power spectrum. The ratio of the zero-pad to the windowed-period was 8. TFRs of ICs and ERPs, and coherence between ICs were also calculated with the window length of 500 ms. Results were similar to those obtained from the above analysis with the window length of 250 ms (see supplementary material, Figure S1 and Figure S2).

Granger Causality Spectral Analysis
In order to examine the directional relationship between the two ICs, Granger causality spectral analysis [76,77] was applied to evaluate the relative strength of influence. For each subject, the mean value of EEG of each trial was calculated and subtracted from the trial to get zero-mean stochastic process that is required for application of the autoregressive modeling. The multivariate autoregressive (MVAR) model was estimated with the 100-ms window for all trials in the time range from 100 ms before, to 300 ms after, the onset of S-1. The MVAR model of order m describes the data as: Where E t is a temporally uncorrelated residual error with covariance matrix D, and A k are 262 (2 ICs) coefficient matrices. Once the model coefficients A k and D are estimated, the spectral matrix can be written as: Where the asterisk denotes matrix transposition and complex is the transfer function of the system. In the present study, the optimal order for the MVAR model was determined by the Akaike Information Criterion (AIC) [78]. The order of 5 was selected because the AIC dropped monotonically with increasing model order up to 5. The Granger causality spectra were then calculated. The power at a specific frequency could be decomposed into an intrinsic part and a predicted part by other signals. The Granger causality at each frequency was thus defined by the ratio of predicted power to total power [77]. Causality Index was calculated by using: Where D 11 , D 22 , and D 12 are elements of D, S 11 ( f ) and S 22 ( f ) are the power spectra of channel 1 and channel 2 at frequency f respectively [77]. Trials were also shuffled 200 times to examine the significance. Granger causality spectra that were significant (p,0.05) were averaged across subjects. A three-way repeated-measure MAN-OVA was performed to compare causality values among time durations, and frequency bands with factors: Modulation (Topdown: direction of causality from IC-F to IC-RS; Bottom-up: IC-RS to IC-F), Duration, and Frequency Band.