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Ethanol Modulates Cortical Activity: Direct Evidence with Combined TMS and EEG
NeuroImage 14, 322–328 (2001) doi:10.1006/nimg.2001.0849, available online at http://www.idealibrary.com on
Ethanol Modulates Cortical Activity: Direct Evidence with Combined TMS and EEG S. Ka¨hko¨nen,* ,† M. Kesa¨niemi,* V. V. Nikouline,* J. Karhu,* M. Ollikainen,* M. Holi,* and R. J. Ilmoniemi* *BioMag Laboratory, Medical Engineering Centre, Helsinki University Central Hospital, FIN-00029 Helsinki, Finland; and †Cognitive Brain Research Unit, Department of Psychology, University of Helsinki, Helsinki, Finland Received July 10, 2000
The motor cortex of 10 healthy subjects was stimulated by transcranial magnetic stimulation (TMS) before and after ethanol challenge (0.8 g/kg resulting in blood concentration of 0.77 ⴞ 0.14 ml/liter). The electrical brain activity resulting from the brief electromagnetic pulse was recorded with high-resolution electroencephalography (EEG) and located using inversion algorithms. Focal magnetic pulses to the left motor cortex were delivered with a figure-of-eight coil at the random interstimulus interval of 1.5–2.5 s. The stimulation intensity was adjusted to the motor threshold of abductor digiti minimi. Two conditions before and after ethanol ingestion (30 min) were applied: (1) real TMS, with the coil pressed against the scalp; and (2) control condition, with the coil separated from the scalp by a 2-cm-thick piece of plastic. A separate EMG control recording of one subject during TMS was made with two bipolar platinum needle electrodes inserted to the left temporal muscle. In each condition, 120 pulses were delivered. The EEG was recorded from 60 scalp electrodes. A peak in the EEG signals was observed at 43 ms after the TMS pulse in the real-TMS condition but not in the control condition or in the control scalp EMG. Potential maps before and after ethanol ingestion were significantly different from each other (P ⴝ 0.01), but no differences were found in the control condition. Ethanol changed the TMS-evoked potentials over right frontal and left parietal areas, the underlying effect appearing to be largest in the right prefrontal area. Our findings suggest that ethanol may have changed the functional connectivity between prefrontal and motor cortices. This new noninvasive method provides direct evidence about the modulation of cortical connectivity after ethanol challenge. © 2001 Academic Press Key Words: EEG; electroencephalography; ethanol; minimum-norm estimate; motor cortex; prefrontal cortex; TMS; transcranial magnetic stimulation.
INTRODUCTION Acute ethanol ingestion has a widespread effect on brain function, but the mechanisms involved are poorly understood. Studies examining the effects of moderate 1053-8119/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
doses of alcohol on spontaneous electroencephalography (EEG) have generally reported an increase in ␣ rhythm amplitude and slowing of the dominant ␣ frequency (Lukas et al., 1986). With larger amounts, an increase in the lower frequencies is observed (Lehtinen et al., 1978, 1985). Quantitative EEG data indicate that low doses may produce alterations in (4 –7 Hz) and in slow ␣ activity (8 –9 Hz), while effects at higher frequencies tend to depend on individual factors such as drinking history and predrug EEG reading (Ehlers et al., 1989). Ethanol has been observed to attenuate different event-related potentials (ERP), but this attenuation is different for different ERP components. Ethanol has been found to attenuate consistently the amplitude of the auditory N1 deflection, even with blood alcohol concentration (BAC) below 0.05% (Hari et al., 1979; Ja¨a¨skela¨inen et al., 1996). P2 is suppressed as well, but not as readily as N1 (Pfefferbaum et al., 1979; Hari et al., 1979; Ja¨a¨skela¨inen et al., 1996). P3 latency is increased and its scalp topography is changed (Teo and Ferguson, 1986; Daruna et al., 1987; Krein et al., 1987). However, the limited number of electrodes in these studies did not allow conclusions about the source locations. Transcranial magnetic stimulation (TMS) provides a painless, noninvasive method for the study of the human central motor system (Barker, 1991). An intense current pulse through a stimulator coil placed above the head generates a time-varying magnetic field, which induces a flow of current in the brain. Modern TMS devices can focus the submillisecond pulse on an area of less than 30 mm in diameter. TMS has been used as a tool to explore motor-evoked potential (MEP) responses to psychopharmacological drugs, giving indirect evidence of cortical excitability. The paired-pulse technique applied to the motor cortex has shown that different drugs affecting the dopaminergic system affect cortical excitability differently. The dopamine D 2receptor antagonist haloperidol reduces intracortical inhibition and increases intracortical facilitation while the effects of the dopamine D 2-receptor agonist bro-
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mocriptine is the opposite (Ziemann et al., 1997). The effects of drugs have been limited to studies based on evaluating the peripheral responses, which give an indirect evidence of cortical excitability. High-resolution EEG (HR-EEG) measures cerebral activation in the millisecond range and with a spatial accuracy of about 10 mm when the number of sources is small (Scherg et al., 1990). The combination of TMS and EEG provides the means to observe directly how the stimulation of superficial cortex results in electrophysiological responses, providing information about cortical excitability in different conditions (Ilmoniemi et al., 1997). The current study was designed to investigate whether brain changes that occur during motor cortex magnetic stimulation after ingestion of moderate doses of ethanol are reflected in EEG recordings. MATERIAL AND METHODS
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mined by moving the coil in 5-mm steps over the presumed area of the left motor cortex. The coil was oriented so that the induced current was in the posterior–anterior direction during the rising phase of the biphasic 320-s pulse. The optimal position was defined as the site of stimulation which consistently yielded the largest MEP at a suprathreshold stimulation intensity. The threshold intensity was approached from above by reducing the intensity with 1% steps and was defined as the stimulus intensity which produced a MEP of ⬎50 V in electromyography (EMG) in 5 subsequent trials from 10. The experimental protocol consisted of two conditions before and after ethanol ingestion: (1) real TMS (n ⫽ 10), with the coil pressed against the scalp; and (2) control condition (n ⫽ 6) with the coil 2 cm from the scalp, but with a plastic piece connecting the head and the coil. In each condition, 120 pulses were delivered.
Subjects
EEG
Ten healthy right-handed nonsmoking male volunteers (24 ⫾ 3.7 years; range 19 –38 years; weight 73.6 ⫾ 7.0 kg) with no personal history of drug/ethanol abuse participated in the study. The subjects reported having no systemic, neurological, or psychiatric disorders, and using no medications. They were instructed to abstain from food for at least 3 h and from ethanol for at least 48 h prior to participation. The experimental procedures were approved by the local ethical committee. All subjects gave their written informed consent.
The EEG was recorded from 60 scalp electrodes referred to the forehead. The average potential from all recording electrodes was used as the reference for constructing the potential maps. To avoid artifacts in the EEG caused by the TMS pulses, an EEG amplifier designed for TMS was used (Virtanen et al., 1999). Low-conductivity small Ag/AgCl-pellet electrodes eliminated possible heating effects. The saturation of the EEG amplifiers during the TMS pulse was avoided by using a sample-and-hold circuit that pinned the amplifier output to a constant level during the pulse. The amplifiers recovered in 5 ms after the end of the magnetic pulse. The data were sampled at the rate of 1450 Hz and digitally low-pass filtered at 250 Hz. After the rejection of noisy epochs, at least 100 responses were averaged. A separate EMG control recording of one subject was made to ensure that the observed EEG activity was not contaminated by muscle activity. Two bipolar platinum needle electrodes were inserted to the main muscle mass of the left temporal muscle 3 cm apart. Stimulation, recording and averaging procedures were the same as for all the other recordings, with the exception that for the control we used two TMS coil orientations: orthogonal and parallel to the presumed orientation of temporal muscle fibers.
Experimental Design and Ethanol Challenge Subjects received the conditions in the same order: the baseline TMS-EEG recording was followed by ethanol ingestion. Subjects had 30 min to drink the ethanol (0.8 g/kg) dissolved in 10% lemon-flavored solution. After an additional 30-min waiting period, the repeated TMS-EEG experiment was performed. The BAC was estimated using the SD-2 breath analyzer (Lion Laboratories, Barry, UK) immediately prior to and 30 min after ethanol ingestion. Magnetic Stimulation The subject was seated in a comfortable reclining chair. Studies were performed while the subject had his eyes open; earplugs were used to avoid possible adverse effects of the stimulation click. Magnetic pulses to the left motor cortex were delivered at the random interstimulus interval (ISI) of 1.5–2.5 s. The stimulator coil consisted of two coplanar circular 40mm-diameter wings, each made of 15 turns of copper wire. The figure-of-eight coil was placed against the scalp. For the measurement of motor threshold, the optimal position of coil for eliciting a MEP in the resting right abductor digiti minimi (ADM) was first deter-
Analysis Because of possible artifacts from scalp muscle activation or from the auditory response to the coil click, the time range from 20 to 50 ms after magnetic stimulation was selected for further analysis. Those 41 electrodes that remained reliably attached to the head during the whole recording in all subjects were used for data analysis. Common average reference was used for statistical analysis. The global mean field amplitude (GMFA) was calculated by the formula
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冑
N ¥ i⫽1 (v i ⫺v ) 2
N
,
where v i is the potential of the electrode i, v is the average potential of electrodes, and N is the number of electrodes. Statistical analysis was carried out with a permutation test to evaluate differences between potential maps (Karniski et al., 1994). For the estimation of ethanol effects, subtraction potentials were calculated (before and after alcohol ingestion). The distribution of neuronal activity was estimated by computing the primary current distribution J p(r) that had the smallest overall amplitude (minimum norm),
冑兰兩J p共r兲兩 2dv among those current distributions that would give rise to the data (Ha¨ma¨la¨inen and Ilmoniemi, 1994). A spherical head model was used in the calculations. RESULTS The baseline BAC was 0.0 ml/liter in all subjects. Thirty minutes after the consumption of ethanol, the BAC was 0.77 ⫾ 0.14 ml/liter (range 0.6 –1 ml/liter). In the four subjects tested after the TMS session, it was 0.78 ⫾ 0.13 ml/liter. The grand-averaged HR-EEG responses evoked by TMS before and after ethanol ingestion are shown in Fig. 1. The GMFA of the EEG peaked 43 ms after the TMS pulse before and after ethanol ingestion. No such peak was observed in the control conditions or in the control scalp EMG (Figs. 2 and 3). GMFA differed significantly in control conditions from the one measured before and after ethanol ingestion (t ⫽ 3.15, P ⫽ 0.002; t ⫽ 2.89, P ⫽ 0.03, respectively) in the real-TMS condition. The potential maps in the real-TMS condition differed significantly before and after ethanol ingestion (permutation test; P ⫽ 0.01), but no differences were found in the control condition (permutation test; P ⬎ 0.05). The subtraction potentials showed that left parietal and right frontal areas were the most reactive to ethanol (Fig. 4). The P-map showed most pronounced differences before and after ethanol ingestion in frontal and left parietal areas (Fig. 5). Figure 4 displays the differences between the current density distributions obtained before and after ethanol ingestion. These data show that ethanol changed mostly the activation of the right prefrontal area. DISCUSSION This study indicates that ethanol changes TMSevoked EEG responses. Only males were included in
FIG. 1. Grand-averaged TMS-evoked HR-EEG responses with the signals from electrodes enlarged before (dotted lines) and after ethanol (solid) challenge. Artifacted electrodes were removed.
this study to avoid sex-related differences in the ethanol challenge. Women show a higher level of BAC after the same dose of ethanol compared to men (Marshall et al., 1983). Previous studies have shown that TMS evokes complex EEG responses, which can contain an auditory and possibly also a somatosensory component (Nikouline et al., 1999; Tiitinen et al., 1999). TMS pulses are accompanied by a click of up to 130 dB (Starck et al., 1996). Thus, a reliable interpretation of physiological effects caused by TMS requires an evaluation of acoustical effects. Because part of the auditory response is due to bone-conducted sound (Nikouline et al., 1999), we used a piece of plastic between the magnetic coil and the scalp to maintain bone conduction in the control condition. In addition to this, an analysis was set up in the time range to 50 ms only after magnetic stimulation to exclude possible auditory artifacts. Another possible source of confounding artifacts is TMSevoked activity in the scalp muscle fibers or somatosensory-evoked potentials arising from the area of stimulation. In the direct control recordings from the scalp muscle, we observed no averaged EMG activity due to repetitive TMS in the analyzed latency range or in the latencies subject to trigeminal or scalp evoked reflex activity (Bennett et al., 1987). Ethanol changed TMS-evoked potentials over right frontal and left parietal areas. The physiological significance of these changes is difficult to interpret. The sequence of activation after TMS is not well under-
ETHANOL MODULATES CORTICAL ACTIVITY
FIG. 2. Global mean field amplitude in the real-TMS condition (A and A⬘) and the control condition (B and B⬘) before (dotted lines) and after (solid lines) alcohol challenge.
stood. However, there are experimental data on the effect of a brief electrical pulse applied to the surface of feline motor cortex using intracellular recordings (Rosenthal et al., 1967). The study showed that the electrical pulse causes either direct discharge or early excitatory postsynaptic potentials (EPSP) and discharges in both output neurons and interneurons in the motor cortex, followed by prolonged inhibitory postsynaptic potentials (IPSP). The effect of electrical pulse on motor cortex is probably similar to the effect of a magnetically induced electric field. The IPSPs are probably GABA-mediated; ethanol has been shown to potentiate this inhibition (Krnjevic et al., 1966; Givens and Breese, 1990; Ticku, 1990). Heterotopical transcallosal anatomical projections from motor areas to prefrontal cortex have been described in animals (Luttenberg, 1974; Cavada and Reinoso-Suarez, 1981). This finding together with high reactivity of prefrontal cortex to ethanol suggests that functional connectivity between the motor cortex (which we stimulated) and prefrontal cortex may have changed after ethanol ingestion. The grand-average differential activation map of Fig. 4 indicates that the effect of ethanol was small on sensorimotor activation but quite pronounced on right frontal cortex activation. However, we used the uncontrolled study design. In future studies the placebo control will allow the possible confounding factors to be taken into consideration. Ziemann et al. (1996), measuring peripheral motor responses, were the first to use TMS in the study of ethanol effects. Their results showed indirectly that ethanol may enhance intracortical inhibition and can suppress intracortical facilitation in a paired-pulse paradigm. Their subjects were challenged to nearly the same amount of ethanol (BAC 0.7 ml/liter) as our subjects. Simultaneous EEG and TMS allow one to detect the direct effect of magnetic stimulation on brain function.
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However, the single-pulse technique does not allow the separation of cortical inhibition and facilitation. According to the minimum-norm estimate, ethanol appeared to change mostly the activation of the right prefrontal area. Imaging studies using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have suggested that the right prefrontal area may be involved in several human emotional responses. In the SPECT study of Tiihonen et al. (1994) during orgasm and in the PET study of London et al. (1990) in cocaine challenge, right prefrontal areas were activated. Our results correspond to the cerebral blood flow (CBF) data obtained by Tiihonen et al. (1994) using SPECT and Volkow et al. (1989) using PET, which indicate right prefrontal activation. In contrast to Volkow et al. (1989), we did not see the activation of temporal areas. In addition to these, the PET study of de Wit et al. (1990) was devoted to cerebral glucose utilization during alcohol intoxication. Results of this study showed that 0.8 but not 0.5 g/kg of ethanol can decrease whole-brain glucose utilization. Schwartz et al. (1993), studying cerebral blood flow with SPECT, showed that not only alcohol but its main metabolite acetate contributes to the changes in
FIG. 3. (A) An averaged EMG to the motor threshold TMS pulses as recorded with the bipolar platinum electrodes in one subject. The amplifier was gated for 5 ms. A segment from 8 to 70 ms was detrended and potential from 5 to 8 ms was forced to be zero in order to compensate for the very abrupt (rectangular-like) shift after the gating period and slowly recovering potential (⬎100 ms), which is likely related to the specificity of EMG electrodes. (B) An example of EMG during the teeth-clenching activation (recorded from the same pair of electrodes).
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FIG. 4. Difference between the potentials before and after ethanol ingestion at 43 ms after magnetic stimulation of the left motor cortex on the left. The red color indicates a positive change in the potential; blue indicates negative change. Electrode locations are shown by the green dots. Cortical activation map based on potential map on the right. The minimum-norm estimate of the grand average of the change in the cortical activity is shown color coded on the brain surface constructed from a magnetic resonance images. CURRY software (Philips GmbH, Germany) was used for the illustration. The TMS coil position is indicated with a cross.
CBF during alcohol intoxication. Recently, fMRI has showed a reduction of the amplitude of visual cortical activation in response to photic stimulation by approximately 33% following ethanol administration (Cohen
FIG. 5. Topographic distribution of the differences between before and after ethanol ingestion revealed by paired t test. The electrodes with the black perimeter showed the strongest difference. Topmost positions, frontal electrodes; bottommost, occipital electrodes. Artifacted electrodes, with red color, were removed before analysis.
et al., 1998). These results also suggest that the baseline right-hemispheric predominance of activation in response to photic stimulation may be reduced following ethanol, suggesting a greater effect on the right hemisphere. Discrepancies in different studies may be linked to different methodologies of measurements, different doses of ethanol challenge, and different timing of recording. PET/SPECT scanning is able to locate brain areas with an accuracy of a few millimeters, but temporal resolution is on the order of several seconds. It is possible that PET/SPECT scanning does not detect fast changes of neural activity following ethanol ingestion. The interpretation of rCBF data in these studies is complicated by the fact that ethanol has both vasodilatory and vasoconstrictive effects, which might change blood flow directly. In the studies of Volkow et al. (1989) and Tiihonen et al. (1994), the same range of doses (0.5–1.0 ml/kg) was used as in the present study, but the delivery of ethanol took only 10 min. The effect of ethanol on brain function depends on the time course of its concentration in plasma (Schwarz et al., 1981). A different timing of measurement could lead to different patterns of brain activation. In conclusion, we were able to detect effects of ethanol on TMS-evoked EEG responses. Pronounced changes in right prefrontal electrodes suggest changes in functional connectivity between the stimulated sensorimotor cortex and contralateral prefrontal cortex. In the future, different sites of cerebral cortex could be stimulated after the administration of ethanol and
ETHANOL MODULATES CORTICAL ACTIVITY
psychopharmacological agents: immediate and remote effects can be monitored with EEG. This provides direct information about cortical reactivity and neuronal connections of different brain areas after drug challenge.
ACKNOWLEDGMENTS This work was supported by Helsinki University Central Hospital Research Funds and Yrjo¨ Jahnsson’s Foundation. We thank Perttu Sipila¨, M.Sc., Montreal Neurological Institute, McGill University, Montreal, Canada, and Mr. Anssu Ranta-aho, BioMag Laboratory, Helsinki University Central Hospital, Helsinki, Finland, for helping in preparing illustrations.
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Ethanol Modulates Cortical Activity: Direct Evidence with Combined TMS and EEG S. Ka¨hko¨nen,* ,† M. Kesa¨niemi,* V. V. Nikouline,* J. Karhu,* M. Ollikainen,* M. Holi,* and R. J. Ilmoniemi* *BioMag Laboratory, Medical Engineering Centre, Helsinki University Central Hospital, FIN-00029 Helsinki, Finland; and †Cognitive Brain Research Unit, Department of Psychology, University of Helsinki, Helsinki, Finland Received July 10, 2000
The motor cortex of 10 healthy subjects was stimulated by transcranial magnetic stimulation (TMS) before and after ethanol challenge (0.8 g/kg resulting in blood concentration of 0.77 ⴞ 0.14 ml/liter). The electrical brain activity resulting from the brief electromagnetic pulse was recorded with high-resolution electroencephalography (EEG) and located using inversion algorithms. Focal magnetic pulses to the left motor cortex were delivered with a figure-of-eight coil at the random interstimulus interval of 1.5–2.5 s. The stimulation intensity was adjusted to the motor threshold of abductor digiti minimi. Two conditions before and after ethanol ingestion (30 min) were applied: (1) real TMS, with the coil pressed against the scalp; and (2) control condition, with the coil separated from the scalp by a 2-cm-thick piece of plastic. A separate EMG control recording of one subject during TMS was made with two bipolar platinum needle electrodes inserted to the left temporal muscle. In each condition, 120 pulses were delivered. The EEG was recorded from 60 scalp electrodes. A peak in the EEG signals was observed at 43 ms after the TMS pulse in the real-TMS condition but not in the control condition or in the control scalp EMG. Potential maps before and after ethanol ingestion were significantly different from each other (P ⴝ 0.01), but no differences were found in the control condition. Ethanol changed the TMS-evoked potentials over right frontal and left parietal areas, the underlying effect appearing to be largest in the right prefrontal area. Our findings suggest that ethanol may have changed the functional connectivity between prefrontal and motor cortices. This new noninvasive method provides direct evidence about the modulation of cortical connectivity after ethanol challenge. © 2001 Academic Press Key Words: EEG; electroencephalography; ethanol; minimum-norm estimate; motor cortex; prefrontal cortex; TMS; transcranial magnetic stimulation.
INTRODUCTION Acute ethanol ingestion has a widespread effect on brain function, but the mechanisms involved are poorly understood. Studies examining the effects of moderate 1053-8119/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
doses of alcohol on spontaneous electroencephalography (EEG) have generally reported an increase in ␣ rhythm amplitude and slowing of the dominant ␣ frequency (Lukas et al., 1986). With larger amounts, an increase in the lower frequencies is observed (Lehtinen et al., 1978, 1985). Quantitative EEG data indicate that low doses may produce alterations in (4 –7 Hz) and in slow ␣ activity (8 –9 Hz), while effects at higher frequencies tend to depend on individual factors such as drinking history and predrug EEG reading (Ehlers et al., 1989). Ethanol has been observed to attenuate different event-related potentials (ERP), but this attenuation is different for different ERP components. Ethanol has been found to attenuate consistently the amplitude of the auditory N1 deflection, even with blood alcohol concentration (BAC) below 0.05% (Hari et al., 1979; Ja¨a¨skela¨inen et al., 1996). P2 is suppressed as well, but not as readily as N1 (Pfefferbaum et al., 1979; Hari et al., 1979; Ja¨a¨skela¨inen et al., 1996). P3 latency is increased and its scalp topography is changed (Teo and Ferguson, 1986; Daruna et al., 1987; Krein et al., 1987). However, the limited number of electrodes in these studies did not allow conclusions about the source locations. Transcranial magnetic stimulation (TMS) provides a painless, noninvasive method for the study of the human central motor system (Barker, 1991). An intense current pulse through a stimulator coil placed above the head generates a time-varying magnetic field, which induces a flow of current in the brain. Modern TMS devices can focus the submillisecond pulse on an area of less than 30 mm in diameter. TMS has been used as a tool to explore motor-evoked potential (MEP) responses to psychopharmacological drugs, giving indirect evidence of cortical excitability. The paired-pulse technique applied to the motor cortex has shown that different drugs affecting the dopaminergic system affect cortical excitability differently. The dopamine D 2receptor antagonist haloperidol reduces intracortical inhibition and increases intracortical facilitation while the effects of the dopamine D 2-receptor agonist bro-
322
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mocriptine is the opposite (Ziemann et al., 1997). The effects of drugs have been limited to studies based on evaluating the peripheral responses, which give an indirect evidence of cortical excitability. High-resolution EEG (HR-EEG) measures cerebral activation in the millisecond range and with a spatial accuracy of about 10 mm when the number of sources is small (Scherg et al., 1990). The combination of TMS and EEG provides the means to observe directly how the stimulation of superficial cortex results in electrophysiological responses, providing information about cortical excitability in different conditions (Ilmoniemi et al., 1997). The current study was designed to investigate whether brain changes that occur during motor cortex magnetic stimulation after ingestion of moderate doses of ethanol are reflected in EEG recordings. MATERIAL AND METHODS
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mined by moving the coil in 5-mm steps over the presumed area of the left motor cortex. The coil was oriented so that the induced current was in the posterior–anterior direction during the rising phase of the biphasic 320-s pulse. The optimal position was defined as the site of stimulation which consistently yielded the largest MEP at a suprathreshold stimulation intensity. The threshold intensity was approached from above by reducing the intensity with 1% steps and was defined as the stimulus intensity which produced a MEP of ⬎50 V in electromyography (EMG) in 5 subsequent trials from 10. The experimental protocol consisted of two conditions before and after ethanol ingestion: (1) real TMS (n ⫽ 10), with the coil pressed against the scalp; and (2) control condition (n ⫽ 6) with the coil 2 cm from the scalp, but with a plastic piece connecting the head and the coil. In each condition, 120 pulses were delivered.
Subjects
EEG
Ten healthy right-handed nonsmoking male volunteers (24 ⫾ 3.7 years; range 19 –38 years; weight 73.6 ⫾ 7.0 kg) with no personal history of drug/ethanol abuse participated in the study. The subjects reported having no systemic, neurological, or psychiatric disorders, and using no medications. They were instructed to abstain from food for at least 3 h and from ethanol for at least 48 h prior to participation. The experimental procedures were approved by the local ethical committee. All subjects gave their written informed consent.
The EEG was recorded from 60 scalp electrodes referred to the forehead. The average potential from all recording electrodes was used as the reference for constructing the potential maps. To avoid artifacts in the EEG caused by the TMS pulses, an EEG amplifier designed for TMS was used (Virtanen et al., 1999). Low-conductivity small Ag/AgCl-pellet electrodes eliminated possible heating effects. The saturation of the EEG amplifiers during the TMS pulse was avoided by using a sample-and-hold circuit that pinned the amplifier output to a constant level during the pulse. The amplifiers recovered in 5 ms after the end of the magnetic pulse. The data were sampled at the rate of 1450 Hz and digitally low-pass filtered at 250 Hz. After the rejection of noisy epochs, at least 100 responses were averaged. A separate EMG control recording of one subject was made to ensure that the observed EEG activity was not contaminated by muscle activity. Two bipolar platinum needle electrodes were inserted to the main muscle mass of the left temporal muscle 3 cm apart. Stimulation, recording and averaging procedures were the same as for all the other recordings, with the exception that for the control we used two TMS coil orientations: orthogonal and parallel to the presumed orientation of temporal muscle fibers.
Experimental Design and Ethanol Challenge Subjects received the conditions in the same order: the baseline TMS-EEG recording was followed by ethanol ingestion. Subjects had 30 min to drink the ethanol (0.8 g/kg) dissolved in 10% lemon-flavored solution. After an additional 30-min waiting period, the repeated TMS-EEG experiment was performed. The BAC was estimated using the SD-2 breath analyzer (Lion Laboratories, Barry, UK) immediately prior to and 30 min after ethanol ingestion. Magnetic Stimulation The subject was seated in a comfortable reclining chair. Studies were performed while the subject had his eyes open; earplugs were used to avoid possible adverse effects of the stimulation click. Magnetic pulses to the left motor cortex were delivered at the random interstimulus interval (ISI) of 1.5–2.5 s. The stimulator coil consisted of two coplanar circular 40mm-diameter wings, each made of 15 turns of copper wire. The figure-of-eight coil was placed against the scalp. For the measurement of motor threshold, the optimal position of coil for eliciting a MEP in the resting right abductor digiti minimi (ADM) was first deter-
Analysis Because of possible artifacts from scalp muscle activation or from the auditory response to the coil click, the time range from 20 to 50 ms after magnetic stimulation was selected for further analysis. Those 41 electrodes that remained reliably attached to the head during the whole recording in all subjects were used for data analysis. Common average reference was used for statistical analysis. The global mean field amplitude (GMFA) was calculated by the formula
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冑
N ¥ i⫽1 (v i ⫺v ) 2
N
,
where v i is the potential of the electrode i, v is the average potential of electrodes, and N is the number of electrodes. Statistical analysis was carried out with a permutation test to evaluate differences between potential maps (Karniski et al., 1994). For the estimation of ethanol effects, subtraction potentials were calculated (before and after alcohol ingestion). The distribution of neuronal activity was estimated by computing the primary current distribution J p(r) that had the smallest overall amplitude (minimum norm),
冑兰兩J p共r兲兩 2dv among those current distributions that would give rise to the data (Ha¨ma¨la¨inen and Ilmoniemi, 1994). A spherical head model was used in the calculations. RESULTS The baseline BAC was 0.0 ml/liter in all subjects. Thirty minutes after the consumption of ethanol, the BAC was 0.77 ⫾ 0.14 ml/liter (range 0.6 –1 ml/liter). In the four subjects tested after the TMS session, it was 0.78 ⫾ 0.13 ml/liter. The grand-averaged HR-EEG responses evoked by TMS before and after ethanol ingestion are shown in Fig. 1. The GMFA of the EEG peaked 43 ms after the TMS pulse before and after ethanol ingestion. No such peak was observed in the control conditions or in the control scalp EMG (Figs. 2 and 3). GMFA differed significantly in control conditions from the one measured before and after ethanol ingestion (t ⫽ 3.15, P ⫽ 0.002; t ⫽ 2.89, P ⫽ 0.03, respectively) in the real-TMS condition. The potential maps in the real-TMS condition differed significantly before and after ethanol ingestion (permutation test; P ⫽ 0.01), but no differences were found in the control condition (permutation test; P ⬎ 0.05). The subtraction potentials showed that left parietal and right frontal areas were the most reactive to ethanol (Fig. 4). The P-map showed most pronounced differences before and after ethanol ingestion in frontal and left parietal areas (Fig. 5). Figure 4 displays the differences between the current density distributions obtained before and after ethanol ingestion. These data show that ethanol changed mostly the activation of the right prefrontal area. DISCUSSION This study indicates that ethanol changes TMSevoked EEG responses. Only males were included in
FIG. 1. Grand-averaged TMS-evoked HR-EEG responses with the signals from electrodes enlarged before (dotted lines) and after ethanol (solid) challenge. Artifacted electrodes were removed.
this study to avoid sex-related differences in the ethanol challenge. Women show a higher level of BAC after the same dose of ethanol compared to men (Marshall et al., 1983). Previous studies have shown that TMS evokes complex EEG responses, which can contain an auditory and possibly also a somatosensory component (Nikouline et al., 1999; Tiitinen et al., 1999). TMS pulses are accompanied by a click of up to 130 dB (Starck et al., 1996). Thus, a reliable interpretation of physiological effects caused by TMS requires an evaluation of acoustical effects. Because part of the auditory response is due to bone-conducted sound (Nikouline et al., 1999), we used a piece of plastic between the magnetic coil and the scalp to maintain bone conduction in the control condition. In addition to this, an analysis was set up in the time range to 50 ms only after magnetic stimulation to exclude possible auditory artifacts. Another possible source of confounding artifacts is TMSevoked activity in the scalp muscle fibers or somatosensory-evoked potentials arising from the area of stimulation. In the direct control recordings from the scalp muscle, we observed no averaged EMG activity due to repetitive TMS in the analyzed latency range or in the latencies subject to trigeminal or scalp evoked reflex activity (Bennett et al., 1987). Ethanol changed TMS-evoked potentials over right frontal and left parietal areas. The physiological significance of these changes is difficult to interpret. The sequence of activation after TMS is not well under-
ETHANOL MODULATES CORTICAL ACTIVITY
FIG. 2. Global mean field amplitude in the real-TMS condition (A and A⬘) and the control condition (B and B⬘) before (dotted lines) and after (solid lines) alcohol challenge.
stood. However, there are experimental data on the effect of a brief electrical pulse applied to the surface of feline motor cortex using intracellular recordings (Rosenthal et al., 1967). The study showed that the electrical pulse causes either direct discharge or early excitatory postsynaptic potentials (EPSP) and discharges in both output neurons and interneurons in the motor cortex, followed by prolonged inhibitory postsynaptic potentials (IPSP). The effect of electrical pulse on motor cortex is probably similar to the effect of a magnetically induced electric field. The IPSPs are probably GABA-mediated; ethanol has been shown to potentiate this inhibition (Krnjevic et al., 1966; Givens and Breese, 1990; Ticku, 1990). Heterotopical transcallosal anatomical projections from motor areas to prefrontal cortex have been described in animals (Luttenberg, 1974; Cavada and Reinoso-Suarez, 1981). This finding together with high reactivity of prefrontal cortex to ethanol suggests that functional connectivity between the motor cortex (which we stimulated) and prefrontal cortex may have changed after ethanol ingestion. The grand-average differential activation map of Fig. 4 indicates that the effect of ethanol was small on sensorimotor activation but quite pronounced on right frontal cortex activation. However, we used the uncontrolled study design. In future studies the placebo control will allow the possible confounding factors to be taken into consideration. Ziemann et al. (1996), measuring peripheral motor responses, were the first to use TMS in the study of ethanol effects. Their results showed indirectly that ethanol may enhance intracortical inhibition and can suppress intracortical facilitation in a paired-pulse paradigm. Their subjects were challenged to nearly the same amount of ethanol (BAC 0.7 ml/liter) as our subjects. Simultaneous EEG and TMS allow one to detect the direct effect of magnetic stimulation on brain function.
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However, the single-pulse technique does not allow the separation of cortical inhibition and facilitation. According to the minimum-norm estimate, ethanol appeared to change mostly the activation of the right prefrontal area. Imaging studies using positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have suggested that the right prefrontal area may be involved in several human emotional responses. In the SPECT study of Tiihonen et al. (1994) during orgasm and in the PET study of London et al. (1990) in cocaine challenge, right prefrontal areas were activated. Our results correspond to the cerebral blood flow (CBF) data obtained by Tiihonen et al. (1994) using SPECT and Volkow et al. (1989) using PET, which indicate right prefrontal activation. In contrast to Volkow et al. (1989), we did not see the activation of temporal areas. In addition to these, the PET study of de Wit et al. (1990) was devoted to cerebral glucose utilization during alcohol intoxication. Results of this study showed that 0.8 but not 0.5 g/kg of ethanol can decrease whole-brain glucose utilization. Schwartz et al. (1993), studying cerebral blood flow with SPECT, showed that not only alcohol but its main metabolite acetate contributes to the changes in
FIG. 3. (A) An averaged EMG to the motor threshold TMS pulses as recorded with the bipolar platinum electrodes in one subject. The amplifier was gated for 5 ms. A segment from 8 to 70 ms was detrended and potential from 5 to 8 ms was forced to be zero in order to compensate for the very abrupt (rectangular-like) shift after the gating period and slowly recovering potential (⬎100 ms), which is likely related to the specificity of EMG electrodes. (B) An example of EMG during the teeth-clenching activation (recorded from the same pair of electrodes).
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FIG. 4. Difference between the potentials before and after ethanol ingestion at 43 ms after magnetic stimulation of the left motor cortex on the left. The red color indicates a positive change in the potential; blue indicates negative change. Electrode locations are shown by the green dots. Cortical activation map based on potential map on the right. The minimum-norm estimate of the grand average of the change in the cortical activity is shown color coded on the brain surface constructed from a magnetic resonance images. CURRY software (Philips GmbH, Germany) was used for the illustration. The TMS coil position is indicated with a cross.
CBF during alcohol intoxication. Recently, fMRI has showed a reduction of the amplitude of visual cortical activation in response to photic stimulation by approximately 33% following ethanol administration (Cohen
FIG. 5. Topographic distribution of the differences between before and after ethanol ingestion revealed by paired t test. The electrodes with the black perimeter showed the strongest difference. Topmost positions, frontal electrodes; bottommost, occipital electrodes. Artifacted electrodes, with red color, were removed before analysis.
et al., 1998). These results also suggest that the baseline right-hemispheric predominance of activation in response to photic stimulation may be reduced following ethanol, suggesting a greater effect on the right hemisphere. Discrepancies in different studies may be linked to different methodologies of measurements, different doses of ethanol challenge, and different timing of recording. PET/SPECT scanning is able to locate brain areas with an accuracy of a few millimeters, but temporal resolution is on the order of several seconds. It is possible that PET/SPECT scanning does not detect fast changes of neural activity following ethanol ingestion. The interpretation of rCBF data in these studies is complicated by the fact that ethanol has both vasodilatory and vasoconstrictive effects, which might change blood flow directly. In the studies of Volkow et al. (1989) and Tiihonen et al. (1994), the same range of doses (0.5–1.0 ml/kg) was used as in the present study, but the delivery of ethanol took only 10 min. The effect of ethanol on brain function depends on the time course of its concentration in plasma (Schwarz et al., 1981). A different timing of measurement could lead to different patterns of brain activation. In conclusion, we were able to detect effects of ethanol on TMS-evoked EEG responses. Pronounced changes in right prefrontal electrodes suggest changes in functional connectivity between the stimulated sensorimotor cortex and contralateral prefrontal cortex. In the future, different sites of cerebral cortex could be stimulated after the administration of ethanol and
ETHANOL MODULATES CORTICAL ACTIVITY
psychopharmacological agents: immediate and remote effects can be monitored with EEG. This provides direct information about cortical reactivity and neuronal connections of different brain areas after drug challenge.
ACKNOWLEDGMENTS This work was supported by Helsinki University Central Hospital Research Funds and Yrjo¨ Jahnsson’s Foundation. We thank Perttu Sipila¨, M.Sc., Montreal Neurological Institute, McGill University, Montreal, Canada, and Mr. Anssu Ranta-aho, BioMag Laboratory, Helsinki University Central Hospital, Helsinki, Finland, for helping in preparing illustrations.
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