Cerebral activation by the signals ascen

PII: S - 2 ( ) 0 - Neuroscience Vol 113, No 2, pp 375^386, 2002 ß 2002 IBRO Published by Elsevier Science Ltd All rights reserved Printed in Great Britain 0306-4522 / 02 $22.00+0.00 www.neuroscience-ibro.com CEREBRAL ACTIVATION BY THE SIGNALS ASCENDING THROUGH UNMYELINATED C-FIBERS IN HUMANS: A MAGNETOENCEPHALOGRAPHIC STUDY T D TRAN,a;bà K INUI,a M HOSHIYAMA,a;c K LAM,a Y QIUa and R KAKIGIa a Department of Integrative Physiology, National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan b c Department of Pediatrics, Faculty of Medicine, The University of Tokyo, Tokyo, Japan Department of Health of Sciences, Faculty of Medicine, Nagoya University, Nagoya, Japan AbstractöCerebral processing of ¢rst pain, associated with AN-¢bers, has been studied intensively, but the cerebral processing associated with unmyelinated C-¢bers, relating to second pain, remains to be investigated This is the ¢rst study to clarify the primary cortical processing of second pain by magnetoencephalography, through the selective activation of C-¢bers, by the stimulation of a tiny area of skin with a CO2 laser In the hemisphere contralateral to the side stimulated, a one-source generator in the upper bank of the Sylvian ¢ssure (secondary somatosensory cortex, SII) or two-source generators in SII and the hand area of the primary somatosensory cortex (SI) were the optimal con¢gurations for the ¢rst component 1M The onset and peak latency of the two sources in SI and SII were not signi¢cantly di¡erent In the hemisphere ipsilateral to the stimulation, only one source was estimated in SII, and its peak latency was signi¢cantly (approximately 18 ms on average) longer than that of the SII source in the contralateral hemisphere From our ¢ndings we suggest that parallel activation of SI and SII contralateral to the stimulation represents the ¢rst step in the cortical processing of C-¢ber-related activities, probably related to second pain ß 2002 IBRO Published by Elsevier Science Ltd All rights reserved Key words: CO2 laser, tiny skin surface areas, second pain, BESA, primary somatosensory cortex (SI), secondary somatosensory cortex (SII) into the cortical representation of pain has focused on the signals ascending through thin myelinated AN-¢bers In contrast, the cortical representation of the activation of unmyelinated C-¢bers has been neglected although the signals ascending through these ¢bers would play an important role in pain perception, and consequently, the temporal and spatial processing of the activation in human brain is not well understood The main reason for this is a lack of appropriate methods to activate C-¢bers selectively Activating C-¢bers selectively by conduction blockade of AN-¢bers (Bromm and Treede, 1987), utilizing low intensity stimulation (Towell et al., 1996) or feedback-controlled laser heat stimulation (Magerl et al., 1999), was di⁄cult to perform Recently, a non-invasive and simple method for the selective activation of C a¡erent sensory terminals in the skin by CO2 laser stimulation of a tiny surface area has been reported (Bragard et al., 1996) The physiological background of this method is that the C a¡erent sensory terminals in the skin have a higher density and lower activation threshold than the AN-terminals (Schmidt et al., 1994; Treede et al., 1994) Ultralate laser-evoked potentials (LEPs), suggested as being associated with the activation of C-¢bers, were obtained directly con¢rming the applicability of this method to eliciting a C-¢ber response (Bragard et al., 1996; Opsommer et al., 1999, 2001) Recently, we also recorded ultralate LEPs by modifying the method and reported Primary a¡erent ¢bers, which selectively respond to stimuli that threaten to cause damage, are classi¢ed as nociceptors There are two systems for nociceptive perception, one ascending through AN-nociceptors and AN-¢bers and related to ¢rst or sharp pain, and one involving C-nociceptors and C-¢bers and related to second or burning pain (Konietzny et al., 1981; Ochoa and Torebjork, 1989) The use of a CO2 laser to activate speci¢cally nociceptors, is ideal for the study of pain (Kenton et al., 1980; Bromm, 1984) The stimulus activates both AN-¢bers and C-¢bers However, the activation of AN-¢bers suppresses that of C-¢bers (Kenton et al., 1980; Bromm, 1984) Therefore, most of the research *Corresponding author Tel.: +81-564-557769; fax: +81-564527913 E-mail address: dieptuan@nips.ac.jp (T D Tran) Abbreviations : 1M, the ¢rst magnetic response; BESA, brain electric source analysis ; ECD, equivalent current dipole; EEG, electroencephalography; fMRI, functional magnetic resonance imaging; GOF, goodness of ¢t; LEF, laser-evoked magnetic ¢eld; LEP, laser-evoked potential ; MEG, magnetoencephalography; MRI, magnetic resonance imaging ; N1^P1, the ¢rst negative and positive electric response; PCA, principal component analysis ; PET, positron emission tomography; RV, residual variance ; SI, primary somatosensory cortex; SII, secondary somatosensory cortex; VPI, ventroposterior inferior thalamic nucleus; VPL, ventroposterior lateral thalamic nucleus 375 NSC 5640 8-7-02 Cyaan Magenta Geel Zwart 376 T D Tran et al that the conduction velocity of the peripheral nerve following this speci¢c stimulation was approximately 0.8^ 1.5 m/s, which is within the range for C-¢bers (Tran et al., 2001, 2002) The advantage of this method is that it is not only nociceptive-speci¢c but also selective for C-¢bers Magnetoencephalography (MEG) has the theoretical advantage of localizing cortical activity as there is less of an e¡ect of cerebrospinal £uid and skull MEG and electroencephalography (EEG) have a very high temporal resolution, in the order of millisecond, as compared with functional magnetic resonance imaging (fMRI) and positron emission tomography (PET), and MEG has a much higher spatial resolution than EEG, in the order of millimeter Therefore, MEG is considered a good method for elucidating the spatial and temporal relationship in early cortical processing The objective of this study was to determine how the signals ascending through C-¢bers are processed in the cerebral cortex in humans by analyzing MEG, that is, ultralate laserevoked magnetic ¢elds (LEFs) To activate C-¢bers selectively, we adopted the aforementioned method of laser stimulation of a tiny area of skin EXPERIMENTAL PROCEDURES than AN-¢ber receptors (Treede et al., 1994) Accordingly, the selectivity for C-¢bers with this method may result from a higher probability to hit C-¢ber than AN-¢ber terminals To avoid magnetic noise caused by the stimulator, the stimulator was located outside the shielded room, and the laser beam was carried through specially developed optical ¢bers approximately 3.5 m in length, which penetrated the wall of the shielded room The laser power was attenuated to approximately 40% of the original power by passage through the ¢bers The stimulus intensity reaching the skin was 5^12 W with a 20-ms duration, which caused pressure, touch, or slight burning pain (or second pain) We used the smallest intensity that generated a clear ultralate LEP in each subject in our previous study (Tran et al., 2001, 2002) A higher intensity may evoke an AN-¢ber response, which would suppress the response of C-¢bers, as suggested by the gating theory for C- and AN-¢bers (Price et al., 1997; Bjerring and Arendt-Nielsen, 1988) To avoid habituation, the irradiated points were shifted slightly for each stimulus on a 25U25 mm area of skin corresponding to the area of the array of holes and the stimulus interval was set randomly from to 20 s (Chen et al., 1998) Stimulation was given at between the ¢rst and second metacarpal bones of the dorsum of the left hand The room temperature was kept at approximately 22^25‡C, and sound and light were regulated The subjects were calm, vigilant, attentive and relaxed, with eyes opened Their eyes were protected from the CO2 laser beam by goggles In our previous studies, we found that 10 trials were su⁄cient to elicit clear LEP responses (Tran et al., 2001, 2002; Qiu et al., 2001) More than 10 trials would attenuate the responses due to habituation Thus, 10 trials were averaged in each session and eight artifact-free sessions were grand-averaged Reproducibility was con¢rmed from these di¡erent sessions Subjects Fifteen healthy volunteers participated in this study (11 males and four females) However, we only analyzed results obtained from nine subjects (seven males and two females), because we could not clearly identify the ultralate LEF component due to artifacts or no consistent response in six subjects Their ages ranged from 27 to 42 (mean ỵ S.D.: 32.7 ỵ 4.1) years All the subjects gave informed consent The ethical committee at our institute approved the study None of the subjects su¡ered from diseases that might a¡ect normal somatosensory perception All subjects were sta¡ in our department and were well trained as subjects in MEG study CO2 laser stimulation of the skin A special CO2 laser stimulator was designed by Nippon Infrared Industries Co Ltd (Kawasaki, Japan) to elicit LEPs and LEFs Its maximum power was 12.5 W and the laser wavelength was 10.6 Wm The diameter of the irradiation beam was mm, which cannot be adjusted To selectively activate C-¢bers by stimulation of a tiny skin area, we used a thin (0.1 mm in depth) aluminum plate (40 mm in length and 60 mm in width) In a 25U25 mm square on this plate, parallel lines were drawn every mm, giving 26U26 intersections A total of 676 (26U26) tiny holes were drilled at these intersections, each with a diameter of 0.4 mm, corresponding to an area of 0.125 mm2 for each hole This thin plate was used as a spatial ¢lter and attached to the skin at the site to be stimulated The array of holes allowed the 2-mm laser beam to pass through 1^4 holes to reach the skin The principle of this method was based on those of previous studies (Bragard et al., 1996; Opsommer et al., 1999, 2001), but was modi¢ed by using a thin plate with many small holes (Tran et al., 2001, 2002; Qiu et al., 2001) The physiological background for this method is that skin has a higher innervation density of C-¢ber terminals In humans, they are 3^4 times more numerous than AN-¢ber terminals (Ochoa and Mair, 1969; Schmidt et al., 1994) In rats, the absolute density distribution is estimated at 2^8 terminals/mm2 (Lynn and Baranowski, 1987) In addition, C-¢ber receptors have a lower activation threshold for laser thermal stimuli NSC 5640 8-7-02 MEG recordings The ultralate LEFs were measured with a dual 37-channel biomagnetometer (Magnes, Biomagnetic Technologies, San Diego, CA, USA) The detection coils of the biomagnetometer were arranged in a uniformly distributed array in concentric circles over a spherically concave surface The device was 144 mm in the diameter and 122 mm in the radius of its curvature The outer coils were 72.5‡ apart Each coil was 20 mm in diameter, and the centers of the coils were 22 mm apart Each coil was connected to a superconducting quantum interference device (SQUID) When the left and right hemispheres were recorded simultaneously, the two probes were centered at C3 and C4 (International 10^20 System) in each subject The C3 and C4 positions covered the primary and secondary somatosensory cortices, SI and SII, of the left and right hemisphere, respectively (Fig 1a) When the centro-parietal region was examined, one probe was centered on the Cz (vertex) position (International 10^20 System) The magnetic ¢elds were recorded with a 0.1^50-Hz bandpass ¢lter, and digitized at a sampling rate of 1041.7 Hz The analysis window was s after the stimuli, and a pre-stimulus period of 300 ms was used as the dc baseline The bandpass ¢lter was further set at 0.1^20 Hz at the analyzing step There were a total of 10 sessions, with for each session (10 trials) and 1-min intervals between sessions So, the total experiment lasted for about 30 The subject’s head was ¢xed to the lower device by tape to avoid head movement The head location was checked after the experiment, and it did not show signi¢cant change EEG recordings The ultralate LEP was recorded simultaneously with the LEF The placement of electrodes was based on the International 10^ 20 System Three exploring electrodes were placed at Cz (vertex), C3 (around the hand area of SI in the left hemisphere), and C4 (around the hand area of SI in the right hemisphere) referred to linked earlobes (A1+A2), since LEP is maximum in amplitude at Cz (Opsommer et al., 2001), and SI activity could be Cyaan Magenta Geel Zwart MEG responses following C-¢ber stimulation recorded at C3 and C4 Impedance was maintained below k6 The ground electrode was placed on the forehead The same ¢lters, sampling rate, and analysis window were used as for the LEF recordings The potentials were recorded with a sensitivity of 10 WV/cm Electrooculographs were also simultaneously recorded for monitoring eye movements, though those contam- 377 inated with large artifacts caused by eye movements were not averaged When the LEF recordings at positions C3 and C4 were made, it was di⁄cult to place the electrode around the lower temporal area due to muscle artifacts caused by the subject’s position, magnetic noise caused by the EEG electrode just beneath the biomagnetometer, and pain in the scalp area caused by the pressure of the biomagnetometer and EEG electrode Therefore, only for the LEF recording at the Cz position, additional exploring electrodes at T4T6 (midway between T4 and T6; over the temporal area in the right hemisphere) and T3T5 (midway between T3 and T5; over the temporal area in the left hemisphere) were placed and referred to Fz, in an attempt to record the early component (corresponding to the early N component in the late LEP of AN-¢ber activation These were the optimal sites for recording this early N component (Kunde and Treede, 1993)) Simultaneous EEG recordings are not used to explore source locations, but simply used for reference and a comparison of latency with MEG responses Data analysis Single-dipole source analysis A source analysis based on a single moving equivalent current dipole (ECD) model was applied to the magnetic ¢eld distribution (Sarvas, 1987) The location, orientation and amplitude of a single ECD were estimated at the latency of peak root mean square (a measure of the magnetic signal strength of the collected data) The origin of the head-based coordinate system was the midpoint between the pre-auricular points The x-axis indicated the coronal plane with a positive value toward the anterior direction, the y-axis indicated the mid-sagittal plane with a positive value toward the left pre-auricular point, and the z-axis lay on the transverse plane perpendicular to the x^y plane with a positive value toward the upper side (Fig 1b) The correlations between the theoretical ¢eld generated by the model and the observed ¢eld were calculated We used our standard criteria for single-dipole model analysis First, during the response period, 10 ms before and after the peak of each component, we ascertained that the dipole location determined with the ECD model must remain stationary (within cm of each coordinate), ensuring the consistency of the source location Second, the correlation coe⁄cient must be above 0.95 Multi-dipole source analysis In the case that the singledipole modeling failed to localize the ultralate LEF generators, we used the brain electric source analysis (BESA) software package made by Scherg (BESA 2000, NeuroScan, McLean, VA, USA) for the computation of theoretical source generators in a three-layer spherical head model The BESA was modi¢ed for the use of our 2U37-channel magnetometers, one each placed on the two hemispheres This method allows the spatio-temporal modeling of multiple simultaneous sources over de¢ned intervals Before starting the modeling, a principal component analysis (PCA) was applied to identify how many principal Fig Placement of MEG sensors and the head-based coordinate system (a) Placement of MEG sensors of one probe centered at C4 in the right hemisphere It covers the primary and secondary somatosensory cortices, SI and SII, of the right hemisphere ; another probe is centered at C3 (b) The origin is the point exactly halfway between the pre-auricular points (PAs) The x-axis indicates a line extending through the origin and the nasion, with positive x coming out of the head at the nasion The z-axis is a line extending through the origin and the top of the head, with positive values toward the upper side This axis is perpendicular to the plane formed by the left and right PAs and nasion The y-axis is a line perpendicular to the x-axis and z-axis extending through the origin and the sides of the head, with positive values toward the left PA (modi¢ed from Nihashi et al., 2001) NSC 5640 8-7-02 Cyaan Magenta Geel Zwart 378 T D Tran et al Fig LEFs and LEPs in three subjects Superimposed waveforms recorded from 37 channels at positions C4 and C3, corresponding to the hemisphere contralateral and ipsilateral to the hand stimulation in three subjects, and simultaneously recorded LEPs at the Cz electrode, are shown The peak latency of 1M recorded from the contralateral hemisphere was signi¢cantly shorter than that of 1M from the ipsilateral hemisphere and of the N1^P1 components (also see Table 1) Triangles point at the peak latencies of 1M and N1^P1 components Calibration: 200 fT for MEG recordings, and 10 WV for EEG recordings components explain the signi¢cant variance The number of components su⁄ce to explain more than 95% of the power was determined as the number of dipoles The location and orientation of the dipoles were calculated by an iterative leastsquares ¢t The residual variance (%RV) indicated the percentage of data, which could not be explained by the model The goodness of ¢t (GOF) was expressed as (1003%RV) A GOF larger than 90% is considered to be a good multiple-dipole NSC 5640 8-7-02 model For comparison, BESA was also conducted when the single-ECD model successfully estimated the source generator The di¡erences in latency between components of the contralateral and ipsilateral hemispheres, between components of MEG recordings and EEG recordings, and between dipoles in BESA analyses were analyzed using two-tailed Wilcoxon pairedsample test, where P-values less than 0.05 were considered to be signi¢cant in all the analyses Cyaan Magenta Geel Zwart MEG responses following C-¢ber stimulation Table Peak latency of the 1M (LEF) and N1^P1 (LEP) components following stimulation of the dorsum of the left hand LEF: 1M (contralateral) 1M (ipsilateral) LEP: N1 P1 Mean ỵ S.D (ms) Range (ms) 746 ỵ 64a 764 ỵ 64a 586^808 601^820 782 ỵ 66b 963 ỵ 77 639^827 786^1058 a Peaks of 1M contralateral were signi¢cantly shorter than those of 1M ipsilateral by 10^31 ms (18 ỵ ms) (two-tailed Wilcoxon paired-sample test, P = 0.005, n = 9) b The peak latency of N1 was signi¢cantly longer than that of 1M in the contralateral hemisphere and ipsilateral hemisphere (twotailed Wilcoxon paired-sample test, P = 0.02, n = 7) Magnetic resonance imaging (MRI) overlaying MRI scans (Shimadzu Magnes 150 XT 1.5 T, Kyoto, Japan) were obtained for all subjects T1-weighted coronal, axial and sagittal images with continuous slices 1.5 mm in thickness were used for overlays with ECD sources detected by MEG The same anatomical landmarks used to create the MEG headbased three-dimensional (3D) coordinate system (the nasion and bilateral pre-auricular points) were visualized in the MRI images by a⁄xing to these points high-contrast cod liver oil capsules (3 mm in diameter), whose short relaxation time provides a high-intensity signal in T1-weighted images The common MEG and MRI anatomical landmarks allowed easy transformation of the head-based 3D coordinate system used for the MEG source analysis in MRI The head coordinate system in BESA is almost the same, rotated by 90‡, as the BTi system, and therefore can be used to overlay individual MRI with a minor adjustment RESULTS LEF waveforms In six out of 15 subjects, we could not clearly identify the ultralate LEF component due to artifacts or because no consistent response was recognized Therefore, we only analyzed results obtained from nine subjects At positions C3 and C4, the ¢rst magnetic component, 1M, was clearly identi¢ed in both hemispheres ipsilateral and contralateral to the side stimulated (Fig 2) In the contralateral hemisphere, the onset and peak latencies of the 1M ranged from 517 to 742 ms (mean ỵ S.D.: 674 ỵ 71 ms) and from 586 to 808 ms (746 ỵ 64 ms), respectively While in the ipsilateral hemisphere, the onset and peak latencies of the 1M ranged from 548 to 757 ms (710 ỵ 64 ms) and from 601 to 820 ms (764 ỵ 64 ms), respectively (Table 1) The 1M recorded from the ipsilateral hemisphere was signi¢cantly longer in latency in all the subjects, and the inter-hemispheric di¡erence in latency ranged from 10 to 31 ms (18 þ ms) (P = 0.005, n = 9) (Table 1, Fig 2) MEG recordings at the Cz position did not yield consistent LEF responses, although a clear LEP in the simultaneous EEG recordings at Cz were identi¢ed NSC 5640 8-7-02 379 LEP waveforms In the simultaneous LEP recordings, we could clearly identify a major positive component, P1, in all nine subjects A small negative component, N1, preceding the P1 component was identi¢ed in seven subjects The N1 and P1 were consistent and maximal at the Cz (vertex) electrode (Fig 2) No component earlier than the N1 was identi¢ed at any electrode The peak latency of the N1 and P1 component ranged from 639 to 827 ms (782 ỵ 66 ms) and from 786 to 1058 ms (963 ỵ 77 ms), respectively The peak of the 1M of LEF was before the onset (three subjects) or on the ascending slope of the N1 component (Fig 2) The peak latency of the N1 component (in seven subjects) of LEP was always and signi¢cantly later than that of the 1M component of LEF recorded from the contralateral hemisphere, 36 ms on average (P = 0.02, n = 7), and from the ipsilateral hemisphere, 21 ms on average (P = 0.02, n = 7) Source localization of the 1M component Contralateral hemisphere First, we analyzed the ECD location for the 1M component in all nine subjects using a single-dipole model In ¢ve subjects, the isocontour map showed a one-dipole pattern (Fig 3a), and the ECD of 1M was estimated to lie in the upper bank of the Sylvian ¢ssure with a high correlation value (0.98 ỵ 0.01), corresponding to SII (Fig 3b) In the other four subjects, the isocontour map showed a complicated magnetic ¢eld rather than a typical single-dipole pattern (Figs 4a and 5a), which probably indicated multiple sources In these subjects, we could not reliably estimate the location of the dipole using the single-dipole model, that is, the correlation value was less than 95% or the location was estimated in a strange area such as deep white matter We applied a multi-dipole model (BESA) for such subjects In all four subjects, a PCA showed two principal components, and the two-dipole model was most appropriate; source in SII and source in the hand areas of SI (GOF: 92.9 ỵ 0.7%) (Figs 4b and 5b), these source locations overlapped on MRI (Figs 4c and 5c) The onset and peak latency of sources and were obtained from the source waveforms (Figs 4b and 5b), and there was no signi¢cant di¡erence between them (P = 0.50, n = 4) The current strength of dipole and was 24.2 ỵ 9.3 and 13.4 ỵ 7.8 nA m, respectively For comparison, BESA was also conducted in subjects in whom the single-ECD model could successfully estimate the source generators In all ¢ve subjects, PCA showed only one principal component, and source located in SII showed very strong activity but source in SI showed no or slight activity (Fig 3c) Ipsilateral hemisphere In all nine subjects, the isocontour map was a simple one-dipole type (Figs 3a, 4a and 5a) and the ECD was estimated to lie in SII using the single-dipole model (correlation = 0.97 ỵ 0.01) The Cyaan Magenta Geel Zwart 380 T D Tran et al Fig Source generator analysis in subject (a) Isocontour maps at the peak latency of 1M in both hemispheres (b) Single-dipole model estimates of the dipole location of 1M in the upper bank of the Sylvian ¢ssure (SII) in the hemisphere contralateral (correlation = 0.98) and ipsilateral (correlation = 0.95) to the stimulation on the subject’s MRI scan (c) For comparison (see Figs 4b and 5b), multiple-source analysis using BESA also showing only one dipole with GOF = 93.6% This supported the result obtained with the single-dipole model Notice that the dipole location in BESA is shown in a spherical model, it does not really re£ect the exact coordinates of each individual As can be seen, source seems to be located in the temporal area, but its real location superimposed on the subject’s MRI scan was in the inferior part of the parietal lobe (see b) source location overlapped on MRI as shown in Figs 3b, 4c and 5c BESA was also conducted and showed only one principal component, the source of which was located in SII (Figs 4b and 5b) NSC 5640 8-7-02 Vertex (Cz) position We also recorded LEF by centering the MEG device at Cz in all nine subjects However, no signi¢cant or consistent response was identi¢ed at this position Cyaan Magenta Geel Zwart MEG responses following C-¢ber stimulation 381 Fig Source generator analysis in subject (a) The isocontour map of the contralateral hemisphere shows a complicated magnetic ¢eld, which implied multiple sources, whereas that of the ipsilateral hemisphere shows a typical one-dipole pattern (b) Using BESA, the two-dipole model was the most appropriate in the contralateral hemisphere, with one dipole (red) located in the upper bank of the Sylvian ¢ssure (SII) and one dipole (blue) located in the upper part of the postcentral gyrus (SI) In the ipsilateral hemisphere, the activity in SII was very strong but that in SI was not identi¢ed (c) MRI overlay of source generators in the contralateral (estimated by BESA, without showing orientation) and ipsilateral (estimated by singledipole model, showing orientation) hemisphere There were two dipoles in the SII (red) and SI (blue) contralateral hemisphere, and only one dipole in the SII ipsilateral hemisphere (red with orientation) DISCUSSION This is the ¢rst study to record C-¢ber-speci¢c cortical activity in humans, by using a novel method for selectively activating C-¢bers and MEG (LEF) with high temporal and spatial resolution Simultaneous EEG (LEP) was useful not only for identifying LEF components but also for comparing the response latency and source generators of LEP and LEF We found the ¢rst LEF component, 1M, with very long peak latency at approximately 750 ms This was slightly shorter than NSC 5640 8-7-02 that of the major N1^P1 components of LEP, peaking at approximately 780 and 960 ms, respectively Since no earlier component was identi¢ed, we believe that 1M represents the ¢rst cortical activity after the signals ascending through C-¢bers are received The main source, of 1M, was consistently estimated to lie in the bilateral SII A previous study relating to the activation of C-¢bers using high-resolution EEG following CO2 laser stimulation (Opsommer et al., 2001) also reported activation in the bilateral SII By contrast, two PET studies of capsaicin-induced pain, which directly Cyaan Magenta Geel Zwart 382 T D Tran et al Fig (Continued) activates C-¢bers, yielded a very weak signal (Andersson et al., 1997) or did not produce a signi¢cant activation of SII (Iadarola et al., 1998) The reason for this discrepancy is unclear, but may reside in the di¡erent stimulus characteristics and recording techniques Capsaicininduced pain is a tonic stimulus, whereas laser-evoked pain is a phasic stimulus PET records all activities for several seconds after the onset of stimulation, but MEG consistently records early cortical activities This is the biggest advantage of MEG over fMRI and PET Probably, the metabolic changes in SII with a relatively short duration are much smaller than the later activities in other areas Our ¢ndings, along with additional evidence that nociceptive neurons with large receptive ¢elds exist in the caudal part of SII (Whitsel et al., 1969) and in the neighboring areas 7b and the retroinsular cortex (Robinson and Burton, 1980), suggest that the SII region is involved in the processing of C-¢bers In addition to the bilateral SII sources, one source in the upper part of the postcentral gyrus, corresponding to SI, contralateral to the side of the stimulation was also found in four subjects (Figs 4b and 5b) Since the stimulus site was on the hand, the location of the source probably corresponded to the hand area of SI The acti- NSC 5640 8-7-02 vation of SI was also detected in the PET studies (Andersson et al., 1997; Iadarola et al., 1998), however, the temporal aspect of SI activity was not clari¢ed An intrinsic optical imaging study in monkeys using heating stimuli, which induced second pain, showed activation in area 3a (Tommerdahl et al., 1998) This supported our ¢nding of the involvement of SI in the activation of C-¢bers The lack of a SI source in the other ¢ve subjects can be explained by one or more of the following: (1) the SI activity is too weak, due to a paucity of nociceptive neurons in this area, (2) a dipole derived from SI, probably from area or 3a (Tommerdahl et al., 1998; Ploner et al., 2000; Kanda et al., 2000), induces a radial orientation, which cannot be detected by MEG, and therefore (3) the detection of SI activity also depends on the anatomical variability in the extension of the postcentral gyrus Although the second source (in BESA analysis) was not consistently recorded, whenever present, its waveform was obvious and source strength was signi¢cant These sources were estimated to lie in the SI area (Figs 4b and 5b) Our ¢ndings of the simultaneous activation of SI and SII in response to the selective stimulation of C-¢bers were well compatible with the temporal activation pattern of the ¢rst pain processing induced by stimulation of Cyaan Magenta Geel Zwart MEG responses following C-¢ber stimulation 383 Fig Source generator analysis in subject See caption to Fig AN-¢bers, but not that of tactile processing induced by stimulation of AL-¢bers In the ¢rst pain associated with AN-¢bers, SI sources were activated simultaneously with SII sources or even later (Tarkka and Treede, 1993; Ploner et al., 1999; Kanda et al., 2000) Our ¢nding in C-¢bers was consistent with this, suggesting a parallel activation of SI and SII in human pain processing This concept was supported by anatomical data obtained in monkeys, showing that nociceptive input is likely processed by distinct spinothalamocortical pathways via the ventroposterior inferior thalamic nucleus (VPI) to SII and via the ventroposterior lateral thalamic nucleus (VPL) to SI (Garraghty et al., 1991; Gingold et al., 1991; Stevens et al., 1993) In contrast, intracranial cor- NSC 5640 8-7-02 tical recordings and MEG studies of tactile processing in humans, for example following upper limb stimulation, revealed a sequential activation of SI and SII (Allison et al., 1989a, b; Hari et al., 1993; Kakigi, 1994; Schnitzler et al., 1999) Since no earlier components were identi¢ed by either EEG or MEG and there was a very slow conduction velocity (0.8^1.5 m/s) following this type of stimulation (Tran et al., 2001, 2002), the activation of SII and SI areas contralateral to the stimulation site probably represents the ¢rst steps in the cortical processing relating to the activation of C-¢bers The SII in the ipsilateral hemisphere was always activated later than the contralateral SII, by approximately 18 ms on average This delay Cyaan Magenta Geel Zwart 384 T D Tran et al Fig (Continued) between ipsilateral and contralateral activations could be due to a delay of transmission in the callosal ¢bers or in the ipsilateral pathway from the VPI nuclei (Forss et al., 1999) The activity of the cingulate cortex is very di⁄cult to detect by MEG due to its deep location, the possibility of the canceling out of the bilateral activities, and its radial orientation Therefore, it was not detected in the present study Activation of the cingulate cortex, however, was found in capsaicin-induced pain PET studies (Andersson et al., 1997; Iadarola et al., 1998; May et al., 1998) and the EEG study (Opsommer et al., 2001) In the EEG study, the cingulate activity found at the positive component, which had a latency much longer than the 1M component (Table 1, Fig 1) Therefore, our negative ¢nding for cingulate activity did not rule out a role for the cingulate cortex in the perception of second pain In all subjects, 1M peaked before the peak latency of the N1 component of LEP, 36 ms on average In fact, in three subjects the 1M component peaked prior to the onset of N1 There was no activity in LEP corresponding to 1M identi¢ed (e.g subject 1, Fig 2) The reason why EEG could not detect any activity at the latency of the 1M component remains undetermined, although this NSC 5640 8-7-02 particular phenomenon has always been present in previous pain studies of AN-¢bers (Huttunen et al., 1986; Kakigi et al., 1995; Yamasaki et al., 1999; Inui et al., 2002) Moreover, this earliest MEG component was not a¡ected by a change in attention (distraction), although the EEG response was a¡ected (Yamasaki et al., 1999) Taken together, we suppose that the mechanisms generating the 1M of LEF and N1^P1 of LEP are probably di¡erent However, we could not neglect the possibility that the 1M of MEG and the early EEG component, N1, arose from the same generators Theoretically, EEG recordings are sensitive to both tangentially and radially oriented dipoles, and both shallowly and deeply located dipoles, but summation and/or interference of such activities usually make EEG waveforms more complicated, and may sometimes considerably reduce their amplitude Even if EEG can record volume-conducted activities, tangentially oriented dipoles with small amounts of currents located in a very restricted region may not be clearly recorded In contrast, MEG recordings are very sensitive to a tangentially oriented dipole located beneath the MEG sensors We must also consider the possibility that volume-conducted currents generated in the bilateral hemispheres may interfere and cancel out each other on Cyaan Magenta Geel Zwart MEG responses following C-¢ber stimulation EEG recordings Therefore, we speculate that activities at the period of MEG 1M peak could not be recorded by EEG due to one or a combination of the reasons described above, although we could not draw de¢nitive conclusion about this ¢nding at present To the question of whether C-¢ber responses in our study represent the second pain-related cerebral activation, we believe they There are three types of C-¢bers: (1) polymodal or mechano-heat-responsive C-¢bers, (2) warm or heat-responsive C-¢bers, and (3) silent or mechano- and heat-insensitive C-¢bers, and second pain is transmitted by (1) (Konietzny et al., 1981; Ochoa and Torebjork, 1989) In normal subjects, CO2 laser stimulation can activate (1) and (2) (Bromm and Treede, 1984) However, it is found that (2) has a higher heat threshold (48‡C) than (1) (40‡C) (Weidner et al., 1999) In the present study, we used a CO2 laser to stimulate a tiny area of skin at weak intensity This stimulation caused a sensation of pressure, touch or slight burning pain, and probably activated polymodal C-¢bers The level of activity in nociceptors may or may not be associated with pain, depending on whether the stimulus intensity is below or above the pain threshold However, there is a clear causal relationship between 385 the two (Willis, 1995) Moreover, SI and SII are mainly involved in sensory-discriminative aspects of pain (Treede et al., 1999) Therefore, when peripheral C-¢ber signals reach the SI and SII cortices, a step, which is involved in the sensory-discriminative aspect of pain, there is probably no di¡erence between the cerebral activation of C-¢ber responses in the present study and second pain-related cerebral activation In summary, by using selective C-¢ber stimuli, we for the ¢rst time performed a MEG study of C-¢ber-related cortical activation in humans, possibly corresponding to second pain Our ¢ndings suggest that the activation of SI and SII contralateral to the site of stimulation, possibly through di¡erent thalamocortical pathways, represents the ¢rst step in the cortical processing of peripheral C-¢ber pain inputs, and indicates a parallel input processing for the second pain perception In the early stage of cortical processing, the spatial and temporal aspects of the C-¢ber nociceptive processing are very similar to those of AN-¢ber nociceptive processing Investigations of the cortical processing in pain 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