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Restorative Neurology and Neuroscience 31 (2013) 275–285 DOI 10.3233/RNN-120297 IOS Press

275

Both the cutaneous sensation and phosphene perception are modulated in a frequency-specific manner during transcranial alternating current stimulation

Zs. Turia,∗, G.G. Ambrusa,b, K. Janacseka,c, K. Emmerta, L. Hahna, W. Paulusa and A. Antala aDepartment of Clinical Neurophysiology, Georg-August University of Göttingen, Göttingen, Germany bDepartment of Medical Psychology and Medical Sociology, Georg-August University of Göttingen, Göttingen, Germany cDepartment of Clinical Psychology and Addiction, Institute of Psychology, Eötvos Loránd University, Budapest, Hungary

Abstract. Purpose: Transcranial alternating current stimulation (tACS) is a non-invasive stimulation technique for shaping neuroplastic processes and possibly entraining ongoing neural oscillations in humans. Despite the growing number of studies using tACS, we know little about the procedural sensations caused by stimulation. In order to fill this gap, we explored the cutaneous sensation and phosphene perception during tACS. Methods: Twenty healthy participants took part in a randomized, single-blinded, sham-controlled study, where volunteers received short duration stimulation at 1.0 mA intensity between 2 to 250 Hz using the standard left motor cortex – contralateral supraorbital montage. We recorded the perception onset latency and the strength of the sensations assessed by visual rating scale as dependent variables. Results: We found that tACS evoked both cutaneous sensation and phosphene perception in a frequency-dependent manner. Our results show that the most perceptible procedural sensations were induced in the beta and gamma frequency range, especially at 20 Hz, whereas minimal procedural sensations were indicated in the ripple range (140 and 250 Hz). Conclusions: We believe that our results provide a relevant insight into the procedural sensations caused by oscillatory currents, and will offer a basis for developing more sophisticated stimulation protocols and study designs for future investigations.

Keywords: Oscillatory current, transcranial alternating current stimulation (tACS), procedural sensations, cutaneous sensation, phosphene perception, motor cortex

∗Corresponding author: Zsolt Turi, Department of Clinical Neu- rophysiology, Georg-August University of Göttingen, Robert Koch Straße 40, 37075 Göttingen, Germany. Tel.: +49 551 3919265; Fax: +49 551 398126; E-mail: [email protected].

1. Introduction

Transcranial alternating current stimulation (tACS) is a relatively new technique for inducing neuroplas- tic changes in humans non-invasively (Antal et al., 2008). It can be used to effectively change a wide range

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of behavioral measures in both the motor and cogni- tive domains in a frequency-dependent manner (Antal et al., 2008; Chaieb, Antal, & Paulus, 2011; Kanai, Paulus, & Walsh, 2010; Pogosyan, Gaynor, Eusebio, & Brown, 2009; Sela, Kilim, & Lavidor, 2012; Zaghi, Acar, Hultgren, Boggio, & Fregni, 2010).

Alternating currents are generated by a battery- driven stimulator and delivered to the head via a pair of rubber conductive electrodes, similar to other types of transcranial electrical stimulation methods, such as transcranial direct current stimulation (tDCS) and transcranial random noise stimulation (tRNS). These electrodes are typically inserted into a sponge saturated with saline solution to minimize procedural sensations (i.e. cutaneous sensation) caused by stimulation. Even so, cutaneous sensations and phosphenes, which are flashing sensations in the visual field, can be perceived during tACS (Antal et al., 2008).

Similar to tACS, skin-related procedural sensations and adverse-effects also occur during and after tDCS (Poreisz, Boros, Antal, & Paulus, 2007). The most common cutaneous sensations associated with tDCS were mild itching, tingling and burning sensations. Interestingly, Ambrus and colleagues found that tDCS had a markedly lower detection threshold (0.4 mA) than transcranial random noise stimulation (tRNS) (1.2 mA), the latter transmitting randomly distributed currents between 0.1–600 Hz (Ambrus, Paulus & Antal, 2010).

For tACS, however, the stimulation-related cuta- neous sensations have not yet been explored experi- mentally. Generally, in non-invasive brain stimulation (NiBS) studies, the presence of stimulation-related cutaneous sensations was considered as a negligible, non-relevant factor for a long time and relatively few studies addressed this issue previously (Ambrus et al., 2010, 2012; Gandiga, Hummel, & Cohen, 2006). The studies investigating phosphene perception (Kanai, Chaieb, Antal, Walsh, & Paulus, 2008; Kar & Krekel- berg, 2012; Paulus, 2010; Schutter & Hortensius, 2010) concentrated on the origin of the phosphene, rather than on the effect of stimulation frequency in evok- ing the phosphene perception itself. Here, we aim to explore and clarify the effect of stimulation frequency on (1) cutaneous sensation, and (2) on phosphene per- ception evoked by tACS, giving us methodical insights into the frequency-dependent effects of these phenom- ena. This information might be useful for developing and optimizing stimulation protocols specifically for future studies using oscillating currents, by taking

the frequency-dependent procedural sensations into account.

2. Methods

2.1. Participants

Twenty healthy volunteers (7 male; age range: 21–29 years; mean age: 24.9 years) took part in the experiment. Participants had no previous history of neurological or psychiatric disorders, and they had no metal implants in the head or neck area. Partici- pants gave written and verbal informed consent before participating. The experiment was in accordance with the guidelines of Declaration of Helsinki, and it was approved by the ethics committee of the University of Göttingen.

2.2. Stimulation

tACS was generated by a battery-driven electrical stimulator (NeuroConn GmbH, Illmenau) through a pair of conductive rubber electrodes with a current intensity of 1.0 mA (peak-to-peak). One electrode was placed over the left motor cortex, whereas the other electrode was placed over the contralateral supraor- bital region. The size of the stimulating electrodes was 3 cm × 3.5 cm, which were inserted in electrode sponge wrappers (5 cm × 7 cm) (Fig. 1, A). Twenty two stimulation frequencies between 2 and 250 Hz were applied and compared to seven no-stimulation trials in order to assess most accurately the minimum and maxi- mum points of the frequency range in which phosphene perception could be evoked (Fig. 1, B). The selection of the stimulation frequencies was predominantly based on previous tACS studies using stimulation frequen- cies at theta (5–6.5 Hz by Feurra, Bianco, Santarnecchi, Del Testa, Rossi & Rossi, 2011; Sela et al., 2012), alpha (10 Hz by Wach, Krause, Moliadze, Paulus, Schnitzler & Pollok, 2012), beta (20–30 Hz by Antal et al., 2008) gamma (50–80 Hz by Feurra, Paulus, Walsh & Kanai, 2011) and at the ripple range (140 Hz by Moliadze, Atalay, Antal & Paulus, 2012). The duration of the stimulation was 31 seconds for each trial.

2.3. Procedure

The experimental procedure was identical to that used in a previous study: see Ambrus and colleagues

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Fig. 1. A) The stimulation montage and the stimulation conditions. B) The non-linear arrangement of stimulation frequencies between 2 and 250 Hz. C) The stimulation protocol for the verum stimulation trials. The stimulation intensity gradually increased from zero to 1.0 mA in the fade-in phase and was decreased to zero after 15 seconds of stimulation with maximal intensity.

(2010). Briefly, participants were seated in a dimly illu- minated room and in a reclining chair placed in front of the investigators in order to minimize the observer- expectancy effect. The entire experiment consisted of one session lasting approximately 90 minutes. The experimental session contained 29 trials, which were divided into 22 verum and 7 no-stimulation trials (sim- ilar to Ambrus et al., 2010). Thus, participants received each verum stimulation trial only once, whereas they received the no-stimulation trial 7 times. The order of the verum and no stimulation trials were individually randomized.

Participants started each trial by pressing the SPACE button on the keyboard. Each verum stimulation trial consisted of a fade-in period (8 sec), stimulation period with maximum intensity (15 sec) and fade-out period (8 sec). In the fade-in period, the stimulation intensity was progressively ramped-up from zero to 1 mA inten- sity. In the fade-out period the stimulation intensity was gradually ramped-down from 1.0 mA to zero again

(Fig. 1, C). In the case of the no-stimulation trials, the stimulator was unplugged and disconnected from the trigger cable, and no stimulation was applied. All the other parameters were identical to those in the verum stimulation condition.

After self-initiating the stimulation trial by the but- ton press, participants were required to indicate the presence of (1) cutaneous sensations, (2) phosphenes, or (3) the occurrence of both percepts via one of the three possible response buttons. The selected option(s) and the perception onset latency between the start of the stimulation trial and the response were stored for off-line analysis. Subsequently, participants were pre- sented with a visual rating scale for indicating the intensity of the perceived procedural sensations (i.e., for cutaneous sensations, for phosphenes, or for both). Participants were instructed to indicate the subjective intensity of the perceived sensation by changing the position of the mouse along a continuous line any- where between the two endpoints. They were told

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that the left point represents the lowest point at which the procedural sensation can be detected, whereas the right endpoint indicates the maximum intensity beyond which the sensation is no longer bearable. The cho- sen intensity was then also stored for later analysis. In the next step, participants were asked whether they felt (1) a tingling sensation, (2) an itching sensation, and/or (3) a burning sensation during the stimulation, and they were also requested to provide the degree of the perceived intensity in a numerical rating scale between 0 and 100 in the case of confirmatory answers (Williamson & Hoggart, 2005). In order to reduce the possible carry-over effects between the experi- mental trials, the next trial was only started when the participants clearly indicated the end of the per- ceived procedural sensations arising from the previous trial.

2.4. Questionnaire

After the end of the experiment, participants filled out a post-experimental questionnaire taken, in a mod- ified form, from Poreisz and colleagues (2007). The questionnaire assessed whether participants experi- enced fatigue, nervousness, anxiety and headache before and after the entire experiment using a numer- ical rating scale from 0 to 100, where 0 represented the absence of the sensation. Participants completed the questionnaire under the supervision of the experi- menter.

2.5. Statistical analysis

The calculation of perception onset latency is described in a previous paper by Ambrus and col- leagues (2010). In brief, cumulative perception onset latency for each stimulation condition was computed, which was then averaged to the number of positive answers.

The assessment of the normality of data was performed using the Shapiro-Wilk test. Since the data significantly deviated from a normal distribu- tion (p < 0.05), non-parametric tests were used for the analysis. Wilcoxon’s matched pairs test was used to compare the intensity of the cutaneous sensation between each verum stimulation trial (22 trials) and the average of the 7 no-stimulation trials (no-stimulation condition). To test whether the proportion of the reported procedural sensations (i.e. positive answers for each stimulation frequency) was different from

the value reported in the no-stimulation condition, a binominal test was used. The proportion value for the binominal test was set according to the averaged reported value in the no-stimulation condition (9.29 for cutaneous sensation and 4.29 for phosphene per- ception). In the case of multiple comparisons, we used the Bonferroni correction (Bender & Lange, 2001). Null hypotheses below the alpha-value of 0.0022 were rejected. Trends were considered above the alpha- value of 0.0022 and below of 0.1.

In the case of the side of phosphene perception, we analyzed data from 16 participants due to the failure in data collection for the first 4 participants.

3. Results

In order to explore the procedural sensations during tACS between 2–250 Hz, we measured participants’ perception onset latency and intensity of the sensations by using a visual rating scale. All participants toler- ated the stimulation; no serious adverse-effects were reported by the volunteers.

3.1. Cutaneous sensation

Between 30% (at 250 Hz) to 70% (at 20 Hz) of the participants reported cutaneous sensation in the verum stimulation condition and ∼10% of them in the no-stimulation condition (Fig. 2). A binominal test was used to determine whether the proportion of the positive answers for cutaneous sensations in the verum stimulation frequencies was different from 9.29%, the average reported value correspond to the no-stimulation condition. We found a significant dif- ference in all stimulation trials (ps ≤ 0.0015) except for 250 Hz, where the alpha-value reached only a trend (p = 0.008). Thus, at all stimulation frequencies with the exception of 250 Hz, participants reported cuta- neous sensations significantly more frequently than in the no-stimulation condition.

The intensity of the cutaneous sensation assessed using the 100-point visual rating scale ranged between 2 and 19.4 points, where the minimum was at 250 Hz and the maximum at 20 Hz (Fig. 3). The Wilcoxon’s matched pairs test revealed that the intensity for cutaneous sensation was significantly different from the no-stimulation condition (median = 0) at 7 Hz (median = 4.5), 20 Hz (median = 7), 30 Hz (median = 11) and 73 Hz (median = 3.5) (all ps ≤ 0.0018).

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Fig. 2. The percentages of the participants who reported cutaneous sensation during the various stimulation trials. The horizontal axis represents the stimulation frequencies (Hz). During the no-stimulation condition, there was no current flowing through the stimulation electrodes. Asterisk represents significant differences in the cutaneous sensation reported during the verum stimulation trials compared to the proportion value (9.29%) measured in the no-stimulation condition (ps ≤ 0.0015) (binominal test), whereas ‘+’ shows a trend toward significance (p = 0.008).

Fig. 3. The intensity of the reported cutaneous sensations during the stimulation trials assessed using a visual rating scale. The horizontal axis represents the stimulation frequencies (Hz). During the no-stimulation condition, there was no current flowing through the stimulation electrodes. Error bars indicate standard error of mean. Asterisk represents significant differences (p ≤ 0.0018) compared to the no-stimulation condition (Wilcoxon’s matched pairs test), whereas ‘ + ’ shows a trend toward significance (0.0028 ≥ ps ≤ 0.0406).

All the remaining stimulation trials (except for 250 Hz) showed a trend towards significance (0.0028 ≥ ps ≤ 0.0406). In other words, participants indicated significantly more intense cutaneous sensa- tions when they received tACS at 7, 20, 30 and 73 Hz.

On the other hand, at 250 Hz the reported intensity was statistically not different from the no-stimulation condition. In the case of the remaining frequencies the participants have demonstrated a trend towards reporting more intense cutaneous sensations.

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The intensity of itching, tingling and burning sen- sations was assessed by using a 100-point numerical rating scale. Volunteers reported itching sensations with a maximum intensity of 15.7 points (at 30 Hz) and a minimum of 2.5 points (at 250 Hz). Wilcoxon’s matched pairs test between the no-stimulation condi- tion (median = 0) and verum stimulation trials revealed only one significant difference at 30 Hz (median = 8) (Z = −3,059, p < 0.0022). At 3, 4, 5, 7, 9, 10, 20, 40, 60, and between 70 and 80 Hz, the analysis revealed a trend toward significance (0.0022 ≥ ps ≤ 0.0506).

Tingling sensations were rated between 8.4 points (at 7 Hz) and 1 point (at 250 Hz). Wilcoxon’s matched pairs test revealed no significant difference between any of the verum stimulation trials and the no- stimulation condition (all ps ≥ 0.01). At 2, 3, 4, 5, 6, 7, 8, 9, 20, 30, 40, 60, 70, 71, 72, 73, 74, 80 and at 140 Hz, we found a trend toward significance (0.01 ≥ ps ≤ 0.092).

The maximum value for burning sensations was 8.7 points (at 9 Hz) and the minimum value was 1.7 points at 40 Hz. No burning sensations were reported at 250 Hz. Wilcoxon’s matched pairs test showed no significant difference between any of the verum stim- ulation trials and the no-stimulation condition (all ps > 0.041). At 5, 7, 9, 20, 60, 73, 80 and at 140 Hz, the test showed a trend (0.041 ≥ ps ≤ 0.067). Thus, these three observations indicate that the reported intensity

of the outcome measures (itching, tingling and burn- ing sensations) was in no cases significantly different between the no-stimulation and verum stimulation tri- als (except one single case at 30 Hz in the itching sensation).

3.2. Phosphene perception

More than 50% of the participants indicated phosphene perception during stimulation trials between 7 and 60 Hz. At 20 Hz, 95% of the partic- ipants experienced a flashing light sensation, which was the highest percentage reported. Interestingly, 140 Hz and 250 Hz did not induce phosphenes in any of our volunteers (Fig. 4). Participants reported phosphene perception in 4.3% of the no-stimulation condition trials.

A binominal test was used to determine, whether the proportion of the positive answers indicated in the verum stimulation trials was different from that value indicated in the no-stimulation condition (4.29%). We found a significant difference between 2 and 75 Hz (ps ≤ 0.0013) and a trend at 80 Hz (p = 0.0095). In other words, participants reported significantly more frequently phosphene perceptions between 2 and 75 Hz than in the no-stimulation condition. This difference at 80 Hz was reduced to a trend.

Fig. 4. The percentages of participants who reported phosphene perception during the stimulation trials. The horizontal axis represents the stimulation frequencies (Hz). During the no-stimulation condition, there was no current flowing through the stimulation electrodes. Asterisk represents significant differences in phosphene detection reported during the verum stimulation trials compared to the proportion value (4.29%) measured in the no-stimulation condition (ps ≤ 0.0013) (binominal test), whereas + shows a trend toward significance (p = 0.0095).

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Fig. 5. The intensity of the reported phosphene perception during the stimulation trials assessed using a visual rating scale. The horizontal axis represents the stimulation frequencies (Hz). During the no-stimulation condition, there was no current flowing through the stimulation electrodes. Error bars indicate standard error of mean. Asterisk represents significant differences (ps ≤ 0.0012) compared to the no-stimulation condition (Wilcoxon’s matched pairs test), + shows a trend toward significance (0.0028 ≥ ps ≤ 0.0463).

The reported intensity for phosphene perception was assessed using a 100-point visual rating scale. Between 8 and 60 Hz, the indicated intensity was over 10 points, with a maximum intensity of 44.75 points at 20 Hz (Fig. 5). Wilcoxon’s matched pairs test demon- strated that the intensity for phosphene perception was significantly different (all ps ≤ 0.0012) from the no- stimulation condition (median = 0) at 8 Hz (median = 6.5), 9 Hz (median = 12.5), 10 Hz (median = 18), 20 Hz (median = 39.5), 30 Hz (median = 16), 40 Hz (median = 30.5) and 60 Hz (median = 3). At 6, 7 and between 70 and 75 Hz, the test revealed a trend toward significance (0.0028 ≥ ps ≤ 0.0463). Thus, partici- pants indicated significantly more intense phosphene perception between 8 and 60 Hz compared to the no- stimulation condition. Above 75 Hz, the intensity for phosphene sensation was statistically indistinguish- able from the no-stimulation condition.

In the case of phosphene perception, the right or both eyes were indicated in 59.1% and 38.6% of the trials. Participants observed phosphenes in the left eye only in 2.3% of the cases. Wilcoxon’s matched pairs test showed a significant difference between the right and left eye (Z = −3.443, p = 0.001), between both eyes and the left eye (Z = −3.047, p = 0.002), but not between the right and both eyes (Bonferroni corrected alpha- value = 0.016). Thus, participants perceived more

phosphenes on the right eye alone, or on both eyes but not on the left eye alone. It appears to be that the evoked phosphene perception heavily depends on the position of the electrodes. Volunteers tend to report more phosphene perceptions for the electrode that is closer to the retina (i.e., right supraorbital region) than for the electrode that is on the motor cortex (left side).

3.3. Perception onset latency

At 5 and 8 Hz, all responding participants reported cutaneous sensations during the fade-in phase of the stimulation (Fig. 6). In the case of phosphene percep- tion, participants indicated phosphenes in the fade-in phase at 20 and 30 Hz. If the response was less than 8 seconds, volunteers perceived the procedural sen- sations even in the fade-in period, where the current intensity has not yet reached the maximum value (which is 1.0 mA).

3.4. Questionnaire

After the experiment, participants filled out a questionnaire with regards to nervousness, fatigue, headache and anxiety. Before the experiment, 30% of the participants reported nervousness with an average intensity of 3.2 out of 100 (3.2/100), 60% experienced

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Fig. 6. The averaged perception onset latency for the positive answers for cutaneous sensations (A) and for phosphene perception (B). The vertical axis represents the stimulation frequencies (Hz). During the no-stimulation condition, there was no current flowing through the stimulation electrodes. The horizontal axis represents the perception onset latency in seconds for each stimulation trial. The maximum time for the stimulation trials is 31 seconds, which includes 8 seconds of fade-in phase, 15 seconds short stimulation with maximum intensity of 1.0 mA (peak-to-peak) and 8 seconds if fade-out phase. Error bars indicate standard error of mean.

fatigue (17.7/100), 30% headache (12.6/100) and none of them had reported feelings of anxiety. After the experiment, none of them reported nervousness, 85% of the participants indicated fatigue (25.7/100) and 45% reported headache (11.6/100).

The Wilcoxon’s matched pairs test revealed that before and after the experiment the difference in the indicated intensity for nervousness (Z = −2.207, p = 0.024), fatigue (Z = −2.253, p = 0.027) and headache (Z = −0.564, p = 0.573) was not significant (Bonferroni corrected alpha-value = 0.016). Thus, although none of the participants indicated nervous- ness at the end of the experiment, the decrease has not reached a level of significance. The intensities for fatigue and headache were unchanged from before to after the experiment.

4. Discussion

The main purpose of this exploratory study was to provide basic information about the cutaneous sensation and phosphene perception evoked by tACS

at different stimulation frequencies between 2 and 250 Hz. By keeping the stimulation intensity con- stant, we found that the procedural sensations were modulated in a frequency-dependent manner, but in different ways: We found a clear and consistent pattern of frequency-dependency for phosphene perception in both outcome measures, while in the case of cutaneous sensations, these differences were less pronounced, but still present in terms of perceived intensity.

Our research was also motivated by the implications of the well-known placebo/nocebo effect, which was explored with regard to tDCS by several previous stud- ies (Ambrus et al., 2010, 2012; Antal, Terney, Kühnl, & Paulus, 2010). The placebo/nocebo effect derives from highly active psychological mechanisms such as expectation and conditioning (de la Fuente-Fernández et al., 2001), which can induce a significant bias in the response to a therapy or intervention (Enck, Benedetti, & Schedlowski, 2008). So far, only limited information was available concerning the tACS-evoked procedu- ral sensations, information which might be crucial in experiments using a placebo-controlled, double- blinded study-design.

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The presence of cutaneous sensations have often been regarded as a relatively non-influential and neg- ligible procedural concomitants for NiBS methods, such as for tDCS (Gandiga, Hummel, & Cohen, 2006; Nitsche et al., 2008). However, experimental evidence demonstrated that the tDCS-mediated neuroplastic changes were surprisingly task-specific, possibly mod- ulated -at least partly- by the attentional involvement of the participants (e.g. Antal, Terney, Poreisz, & Paulus, 2007). In this current experiment, 30 to 70% of the participants reported perceiving cutaneous sensations in all of the stimulation trials. These values were sig- nificantly higher at each stimulation frequency (except for 250 Hz) compared to the value reported in the no-stimulation condition. It might be argued that the cutaneous sensations evoked by tACS modify the alert- ness of the volunteers and are possibly capable of altering attentional processes as well. Therefore, the role of the skin sensations in tACS have to be taken into consideration in future studies. Future studies might also study the location of the cutaneous sensation on the scalp and whether or not the location is modulated by the different frequencies.

The indicated intensity for cutaneous sensation was frequency-dependent and generally low. We found significant differences mainly at the beta band fre- quencies (20 and 30 Hz), whereas cutaneous sensations in a high oscillating frequency (250 Hz) were almost unnoticeable. Recently, Jelinek and McIntyre (2010) have also found a modulatory effect of the stimula- tion frequency of electrocutaneous stimulation on the perceived magnitude of cutaneous sensation. By using squared-wave, low-intensity electrocutaneous stimula- tion over the anterior forearm between 20 and 200 Hz at suprathreshold intensity (10% above the detec- tion threshold), Jelinek and McIntyre showed that the higher the stimulation frequency was, the higher the reported magnitude of sensation was indicated, with a plateau at 120 Hz. Importantly, we also found an effect of stimulation frequency on the magnitude of the perceived sensation, however, in a different way. Our results showed that the perceived intensity of cutaneous sensations gradually increased as a func- tion of stimulation frequency up to the beta frequency range, and did not show a trend for further increase beyond the beta range. On the contrary, the lowest cutaneous sensation was indicated at the highest inten- sity. It is difficult to explain the discrepancy between the two studies, but the different waveform (sinusoid vs. squared-wave), electrode placement (head vs. fore-

arm), current intensity (predetermined at 1.0 mA vs. individually adjusted) may all contribute to the diver- gent findings.

Phosphene perception evoked by tACS has recently received much attention with NIBS research. The main debate was on the possibility that tACS is capable of entraining ongoing brain oscillatory mechanisms and evoke phosphene perception in a frequency-dependent manner (Kanai et al., 2008), or the possibility that the phosphene perception could be partially explained by retinal contribution due to the volume-conduction effect (Kar & Krekelberg, 2012; Schutter & Horten- sius, 2010).

Similar to the cutaneous sensation results, the most frequently reported and the most intense phosphene perception was also observed at 20 Hz, but in gen- eral, the phosphene perception reached the significance level at each stimulation frequency between 2 and 75 Hz. The strength of the phosphene perception also differed significantly from the no-stimulation condi- tion between 8 and 60 Hz. In other words, we found a bell-shaped relationship between the stimulation frequency and the evoked magnitude of phosphene per- ception. Frequencies beyond 8 Hz produced increased phosphene perception up to the maximum at 20 Hz while perception declined dramatically from 60 Hz onward.

The averaged cumulative perception onset latency for phosphene perception was the lowest between 9 and 40 Hz, thus, participants responded numerically faster for the stimulation frequencies that evoked the most intense visual flickering percepts (please note that this result was not statistically verified; see Results). This result might be due to the fact that participants were required to simultaneously attend to two differ- ent procedural sensations (cutaneous sensations and phosphene perceptions), which might have increased the perception onset latency for reporting cutaneous sensations. Importantly, participants were always pro- vided with a subsequent self-paced screen with visual analog scales for both procedural sensations (i.e., irre- spectively of indicating only one of the sensations initially). Accordingly, participants had the possibil- ity to indicate the other procedural sensation for each experimental trial using the visual analog scale. There- fore, we believe that multisensory interactions or split attention processes minimally affected our main results.

In this study, none of the participants reported any serious adverse-effects during or after the stimulation.

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Nevertheless, 30% of the participants reported minimal nervousness (3.2 out of 100) before the experiment, which reduced to zero after the testing. Contrary to a previous paper investigating applications of tDCS during and after stimulation (Poreisz et al., 2007), the subjective intensity of headache and fatigue did not differ significantly before and after the experi- ment according to the self-reports of the participants in this current study. The baseline amount of fatigue and headache (with relatively low intensities in both cases), however, was relatively high. The reason for this elevated amount is unknown to us, but one specula- tive reason would be that most of the participants were university students, and these values reflect their reg- ular, daily routines. Interestingly, most of the previous studies assessed the status of the participants during or after but not before the intervention, therefore, we have no previous comparisons in this regard.

In summary, beta (13–30 Hz) and gamma (>30 Hz) frequencies induced the most intense and frequent procedural sensations during stimulation, but the pro- cedural sensations were perceivable between 7 and 80 Hz in general. The role of beta and gamma fre- quency ranges have been reported to be associated with motor and cognitive processes (Donner & Siegel, 2011; Engel & Fries, 2010), and there is already an increasing interest in the tACS literature for the beta-range (Kanai et al., 2008; Pogosyan et al., 2009; Zaghi et al., 2010). Future studies using the standard electrode montage at beta and gamma frequency ranges might consider the possibility that the magnitude and the frequency of the phosphenes and the cutaneous sensations could influ- ence the outcome of the stimulation in both motor and cognitive functions. Our data suggests that future stud- ies are needed to optimize sham stimulation protocol for tACS, especially in the beta-range.

5. Limitations

(1) One of the main limitations of our study was that only some selected frequencies were investigated within the range of 2 and 250 Hz. Thus, this study offers only limited information about the cutaneous sensations and phosphene perception at higher fre- quencies (i.e.>80 Hz). (2) Another key concern might be the short stimulation duration used in our study. Importantly, the aim of our exploratory research was to investigate a broad range of the frequency spectrum during tACS, which limited the stimulation duration

because of the length of the entire experiment. By considering the data presented in this report, future studies might aim to focus on the temporal develop- ment of the evoked procedural sensations in different frequency ranges. (3) Future studies might aim to employ repeated trials for each stimulation frequencies to increase the intra-subject validity of the reported pro- cedural sensations. (4) Our results are also montage- and intensity- specific. It is generally accepted that the stimulation-related cutaneous sensations significantly depend on the current density (Ambrus et al., 2010; Brunoni et al., 2011) and different montages possibly evoke procedural sensations differently (Poreisz et al., 2007).

6. Conclusion

In this study we have demonstrated that both cutaneous sensations and phosphene perception is evoked by tACS in a frequency-dependent manner, mostly in the beta and gamma frequencies. The bet- ter understanding of the stimulation-related procedural sensations might form a basis for developing more optimized stimulation protocols and study designs for paradigms using oscillatory currents in the future.

Acknowledgments

We would like to thank to Dr. Christina Schütte and Philipp Berens for their careful reading of the manuscript and their valuable comments. We also thank for Dr. Leila Chaieb for improving the English style of the manuscript. Thanks for Dr. Caspar Stephani for the medical screening of the participants and for our volunteers for their time. This study was funded by the Rose Foundation (ZsT, WP, GGA) and by the Hungarian-German Scientific Exchange Program (DAAD-MÖB) Grant number 29775 (2012- 2013) (KJ).

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