10 1016@j Brs 2019 10 022

Journal Pre-proof
Transcranial alternating current stimulation of α but not β frequency sharpens multiple

visual functions
Hisato Nakazono, Katsuya Ogata, Akinori Takeda, Emi Yamada, Takahiro Kimura,
Shozo Tobimatsu
PII: S1935-861X(19)30431-0
DOI: https://doi.org/10.1016/j.brs.2019.10.022
Reference: BRS 1597
To appear in: Brain Stimulation
Received Date: 15 July 2019

Revised Date: 23 September 2019
Accepted Date: 31 October 2019
Please cite this article as: Nakazono H, Ogata K, Takeda A, Yamada E, Kimura T, Tobimatsu S,
Transcranial alternating current stimulation of α but not β frequency sharpens multiple visual functions,
Brain Stimulation, https://doi.org/10.1016/j.brs.2019.10.022.
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Nakazono et al. - 1
3 Transcranial alternating current stimulation of α but not β
4 frequency sharpens multiple visual functions
8 Hisato Nakazono1, 2, Katsuya Ogata1, 3, Akinori Takeda1, Emi Yamada1, Takahiro Kimura4,
9 Shozo Tobimatsu1
10
11
1
12 Department of Clinical Neurophysiology, Neurological Institute, Faculty of Medicine,
13 Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan
2
14 Department of Occupational Therapy, Faculty of Medical Science, Fukuoka International
15 University of Health and Welfare, Fukuoka 814-0001, Japan
3
16 Department of Speech and Hearing Sciences, School of Health Sciences at Fukuoka,
17 International University of Health and Welfare, Fukuoka 831-8501, Japan
4
18 Institute of Liberal Arts and Science, Kanazawa University, Kanazawa 920-1192, Japan
19
Nakazono et al. - 2
20 Address Correspondence to Hisato Nakazono, PhD:
21 Department of Occupational Therapy, Faculty of Medical Science, Fukuoka International
22 University of Health and Welfare, Fukuoka 814-0001, Japan
23 E-mail: nakazono@med.kyushu-u.ac.jp
24 Phone: +81-92-832-1200
25 Fax: +81-92-832-1167
26
Nakazono et al. - 3
27 Abstract
28 Background: Transcranial alternating current stimulation (tACS) can entrain and enhance
29 cortical oscillatory activity in a frequency-dependent manner. In our previous study
30 (Nakazono et al.2016), 20 Hz (β) tACS significantly increased excitability of primary
31 motor cortex compared with 10 Hz (α) tACS. α oscillations are a prominent feature of the
32 primary visual cortex (V1) in a resting electroencephalogram. Hence, we investigated
33 whether α and β tACS can differentially influence multiple visual functions.
34 Methods: Firstly, we evaluated the after-effects of α and β tACS on pattern-reversal (PR)
35 and focal-flash (FF) visual evoked potentials (VEPs). Secondly, we determined the
36 relationship between resting α oscillations and PR-VEPs modulated by tACS. Thirdly, the
37 behavioral effects of tACS were assessed by contrast sensitivity.
38 Results: α tACS modulated the amplitudes of PR-VEPs, compared with β tACS, but did
39 not modulate the FF-VEPs. Time-frequency analysis revealed that α tACS facilitated
40 event-related α phase synchronizations without increasing power, which consequently
41 increased the PR-VEP amplitudes. There was a significant positive correlation between
42 PR-VEP amplitudes and resting α oscillations. These findings suggested that α tACS
43 modulated α oscillations, and affected visual functions of contrast and spatial frequency.
44 Indeed, α tACS also improved subjects’ contrast sensitivity at the behavioral level.
45 Conversely, β tACS increased posterior α activity, but did not change VEP amplitudes.
Nakazono et al. - 4
46 Conclusions: α tACS can influence different neuronal populations from those influenced
47 by β tACS. Thus, our results provide evidence that α tACS sharpens multiple visual
48 functions by modulating α oscillations in V1.
49
Nakazono et al. - 5
50 Keywords: α and β tACS, α oscillations, contrast sensitivity, visual cortex, visual evoked
51 potentials
52
53
54 Highlights
55 We studied whether α and β tACS can differentially influence the occipital cortex.
56 Effects of α and β tACS on VEPs, resting EEG and contrast sensitivity were assessed.
57 α tACS but not β tACS was effective for sharpening multiple visual functions.
58 Effects of tACS on visual cortex can be a frequency-dependent manner.
59
60 Abbreviations:
61 tACS, transcranial alternating current stimulation; VEP, visual evoked potential; PR-VEP,
62 pattern-reversal visual evoked potential; FF-VEP, focal-flash visual evoked potential; EEG,
63 electroencephalography; ERP, event-related potential; V1, primary visual cortex; V2,
64 secondary visual cortex; ERSP, event-related spectral perturbation; ITC, inter-trial
65 coherence; cpd, cycle per degree; LMM, linear mixed-effects modeling

Nakazono et al. - 6
66 Introduction
67 Transcranial alternating current stimulation (tACS) is a non-invasive technique
68 known to modulate brain oscillations. Interestingly, tACS affects brain functions or
69 behavior in a frequency-dependent manner [1–5]. The effective stimulation frequency of
70 tACS is regarded as matching the targeted brain oscillations [4,6]. Indeed,
71 electroencephalographic (EEG) studies have revealed that tACS at α frequency (~10 Hz)
72 over the visual cortex enhances resting α activity in the parieto-occipital area during [7] and
73 after stimulation [8–11]. Posterior α oscillations are a prominent feature in resting EEG,
74 and are thought to have an association with a broad range of visual neurocognitive
75 functions. However, the role of these oscillations in visual system function remains unclear
76 [12]. Recently, tACS has been applied to investigate a causal link between α oscillations
77 and visual processing [7,13–15]. Nevertheless, frequency-dependent effects of α tACS
78 were not observed in some studies [16,17]. Therefore, the effects of α tACS on the visual
79 cortex may be complex and depend on task demands.
80 In this study, we recorded visual evoked potentials (VEPs) to determine the effects
81 of α tACS on the visual system. The human visual system processes different visual
82 information via multiple, parallel pathways or channels, and VEPs can be used to assess
83 such pathways [18]. For example, in pattern-reversal VEPs (PR-VEPs), the small
84 checkerboard pattern stimulates the fovea and activates mostly contrast channels [19],
Nakazono et al. - 7
85 whereas flash stimulation is processed by luminance channels [20]. Several earlier studies
86 have postulated that VEPs are related to α oscillations [21–23]. In such a model, the phases
87 of the background α oscillations are partially aligned with the visual stimulus and, in turn,
88 the oscillations contribute to the generation of VEP components. However, this relationship
89 remains controversial [24–26]. Therefore, we hypothesized that if there is a causal
90 relationship between background α oscillations and visual system processes, then α tACS
91 should modulate not only resting α activity but also visual system functions such as VEPs
92 and visual task performance. To test this hypothesis, we first investigated whether α (10
93 Hz) tACS modulated PR- and focal-flash (FF-) VEP amplitudes after stimulation, as
94 compared with β (20 Hz) tACS, in Experiment 1. Next, we assessed the event-related α
95 phase concentration or power dynamics in VEPs modulated by α and β tACS using
96 time-frequency analysis. We also investigated resting α power modulations after tACS in
97 Experiment 2. Finally, we measured the behavioral effects of α and β tACS on contrast
98 sensitivity in Experiment 3 because PR-VEP amplitudes depend on pattern spatial
99 frequency [27]. Since there may be different spatial frequency preferences in the separate
100 visual cortical areas (e.g., primary (V1) and secondary (V2) visual cortices) [28], we used
101 three different spatial frequencies for the contrast sensitivity assessments.
102
103 Material and methods

Nakazono et al. - 8
104 Participants
105 A total of 33 healthy subjects (17 females; mean ± SD, 26.8 ± 7.8 years old) took
106 part. None of the subjects had any history of neurological, psychiatric or other medical
107 problems. All subjects had normal or corrected-to-normal visual acuity. The right eye was
108 dominant in 30 subjects. All participants gave written informed consent in accordance with
109 the Declaration of Helsinki. The study was approved by the Ethics Committee of Kyushu
110 University. Thirteen subjects (7 females; 26.1 ± 7.4 years old) joined in Experiment 1. The
111 sample size was chosen based on previous studies of tACS targeted to the visual cortex [8–
112 11,13]; this number of subjects showed sufficient statistical power (1 − β = 0.9).
113 Subsequently, sample sizes of N ≥ 15 were determined to be necessary for Experiments 2
114 and 3, to achieve statistical power of 0.8, on the basis of the effect size in Experiment 1.
115 Therefore, 17 subjects (7 females; 28.4 ± 8.3 years old; 5 had also participated in
116 Experiment 1) and 15 subjects (8 females; 29.5 ± 9.1 years old; 7 had also participated in
117 Experiments 1 and/or 2) took part in Experiments 2 and 3, respectively.
118
119 Experimental designs
120 This research was designed as a randomized, crossover and single-blinded study.
121 Sessions with the different tACS frequencies (i.e., Experiment 1: α and β tACS,
122 Experiments 2 and 3: α and β tACS and sham) were separated by at least 2 days.
Nakazono et al. - 9
123 In Experiment 1, PR- and FF-VEPs were recorded before, and every 10 min after,
124 tACS, for up to 30 min (Fig. 1A). The order of VEP types was randomized across subjects.
125 The sham condition was not used in Experiment 1 because we were primarily concerned
126 with the difference in VEP alteration between FF- and PR-VEPs following α and β tACS.
127 In Experiment 2, resting EEG and VEP recordings were completed before, and up to 10
128 min after, stimulation (Fig. 1B). In the VEP measurement, PR-VEPs were recorded because
129 α tACS was found to be effective only on PR-VEPs in Experiment 1. Resting EEG was
130 always recorded before PR-VEP recordings to minimize the influence of VEP stimuli on
131 the resting α oscillations. In Experiment 3, contrast sensitivity was measured at baseline
132 and up to 10 min after tACS (Fig. 1C). Each session started with a training run to
133 familiarize the subject with the contrast sensitivity task. The sham condition was employed
134 in Experiments 2 and 3, to confirm the tACS effects.
135
136 tACS
137 Participants sat on a comfortable chair in a quiet room with dim lighting during
138 experiments. tACS was delivered by a battery-driven current stimulator (DC
139 Stimulator-Plus, NeuroConn GmbH, Ilmenau, Germany) through two self-adhesive
140 electrodes (PALS electrodes, Axelgaard Manufacturing Co., Ltd., Fallbrook, CA). tACS
141 was applied at 1 mA (peak-to-peak) for 20 min (the impedance was kept below 5 kΩ), with
Nakazono et al. - 10
142 5 s ramping up and down. The target tACS electrode (3.5 × 3.5 cm) was placed at Oz’ (2.5
143 cm above the inion), and the reference electrode (7 × 5 cm) was positioned at Cz
144 (International 10–20 system). The tACS electrodes were affixed using a support bandage.
145 These electrode positions were chosen to affect the occipital cortex [29,30]. For sham
146 stimulation, α tACS was applied for only 30 s at the beginning of the 20 min period. After
147 stimulation, all subjects were asked to report whether they experienced any tACS-induced
148 sensations.
149
150 Recordings of VEPs and resting EEG
151 VEPs were recorded from the electrode at Oz’ with the reference electrode at FCz
152 (forehead) [31,32]. For PR-VEP recordings, a checkerboard pattern with 15-min checks
153 was used. For FF-VEP recordings, a uniform white field was presented for 200 ms and then
154 replaced by a black field for 800 ms. For both types of VEPs, the stimuli subtended 6° of
155 visual angle from the vertical and horizontal midline in the lower visual field (Fig. 1A) [32].
156 The resting EEG was recorded from Oz’ and Pz (International 10–20 system) [9,33] with
157 the reference electrode at FCz. To measure background brain oscillations, subjects’ resting
158 EEG responses were recorded for 60 s. Participants were instructed to fixate on a red point
159 at the center of the screen during VEP and resting EEG recordings (Fig. 1). Details of VEP
160 and EEG recordings are provided in the supplementary methods.

161
162 Measurement of contrast sensitivity
163 Contrast sensitivity was measured with single Gabor patches (visual angle, 10°;
164 vertical orientation) in binocular vision (Fig. 1C). Contrast sensitivity was measured at
165 three different spatial frequencies (0.5, 3 and 9 cycle per degree (cpd)), and the test
166 consisted of 30 trials (10 trials per spatial frequency, in random order). For further details,
167 see supplementary methods.
168
169 Data Analyses
170 Data analyses were performed using functions implemented in the MNE-Python
171 software [34] and custom-programmed tools in Python.
172
173 Analysis of VEP amplitudes
174 The artifact-free VEP data from Oz’ were averaged offline. We measured the major
175 components of FF- and PR-VEPs: peak-to-peak amplitudes of PR-VEPs (N75, P100 and
176 N145) and FF-VEPs (N1, P1 and N2) (Fig. 2A) [32]. The two VEPs recordings from before
177 tACS were averaged, and used as the baseline (pre-tACS). The VEP amplitudes in trials
178 after tACS (post-tACS) were normalized relative to those of the pre-tACS.
179
180 Time-frequency analysis for VEP data
181 In the time-frequency analysis, the event-related spectral perturbation (ERSP) and
182 inter-trial coherence (ITC) were calculated. The epochs of 2000 ms (with 1000 ms before
183 pattern-reversal) were extracted from the PR-VEP data recorded at electrode Oz’ in
184 Experiment 2. Trial-by-trial time-frequency analysis was performed using Morlet wavelet
185 transforms in frequencies between 4 and 40 Hz, in steps of 1 Hz, with wavelet cycles
186 increased from 2 to 20. ERSP represents mean power changes relative to the prestimulus
187 period (-300 to 0 ms) across epochs, and was calculated as the log-ratio [log10
188 (poststimulus period/prestimulus period)]. ERSP measures event-related changes in spectral
189 power over time [35]. Meanwhile, the ITC is a measure of the phase synchronization of the
190 EEG activity to the time-locking events [36,37]. The ITC values range from 0 to 1, with
191 ITC values close to 1 reflecting almost perfect phase coincidence across epochs.
192 To evaluate the ERSP and ITC related to VEP responses, we defined the analysis
193 time windows from 50 to 200 ms after pattern-reversal (Supplementary Fig. S2). Moreover,
194 frequency bands of interest were selected in the α (10 ± 1 Hz) and β (20 ± 1 Hz) ranges,
195 taking into account the tACS frequencies. The ERSP and ITC values of the analysis time
196 windows in the α or β ranges were averaged for each subject. To measure the after-effects
197 of tACS, two VEP recordings before tACS were pooled as the pre-tACS, and then
198 subtracted from the post-tACS for each subject.

199
200 Analysis of resting EEG
201 Resting EEG data from the electrodes at Oz’ and Pz were each split into 2 s epochs.
202 A fast Fourier transform was applied to each epoch, to calculate power spectra of 0.5–30 Hz,
203 then log-transformed spectra were averaged [38]. To evaluate power changes induced by α
204 and β tACS, the mean power in the α (10 ± 1 Hz) or β (20 ± 1 Hz) range was calculated. To
205 measure the after-effects of tACS, the pre-tACS power values were subtracted from the
206 post-tACS data (Δlog10 power = log10 power (post) − log10 power (pre)). Moreover, we
207 measured α and β peak frequencies in resting EEG data of pre-tACS (Supplementary Table
208 S1, Supplementary Fig. S3).
209
210 Analysis of contrast sensitivity
211 We excluded trials without a button press, then calculated the average contrast
212 sensitivity for each spatial frequency and each subject separately as a baseline (pre-tACS).
213 The individual contrast sensitivity values (i.e., 10 trials of each spatial frequency) in
214 post-tACS were normalized to the pre-tACS baseline value in each subject.
215
216 Statistical analyses
217 The tACS after-effects on each measurement were evaluated using a linear
218 mixed-effects modeling (LMM) with subjects as a random effect, and time (Experiment 1:
219 post 0–30 min; Experiment 2 and Experiment 3: post 0–10 min) and tACS (α and β tACS
220 or sham) as the fixed effects for all experiments. When a main effect of tACS was observed,
221 we used pairwise comparisons for a post-hoc analysis, and p values were adjusted using
222 Bonferroni correction for multiple comparisons. To evaluate the relationship between VEP
223 amplitudes and resting α power in Experiment 2, the after-effect at time points post 0-10
224 min on each measurement were averaged together as a total post-tACS, and then Pearson’s
225 correlation coefficients were calculated. To test the stability for pre-tACS in each
226 evaluation, LMM was performed with tACS as a factor. The data are presented as means ±
227 standard error of the mean. The significance level for all tests was set at p < 0.05. Statistical
228 analysis was carried out with SPSS (version 17.0 for Windows, IBM, Armonk, NY).
229
230 Results
231 Regarding the stability of pre-tACS measurements in each experiment
232 (Experiment 1: amplitudes of PR- and FF-VEPs; Experiment 2: N75-P100 amplitude of
233 PR-VEPs, and α or β range values (ERSP, ITC and resting EEG power); Experiment 3:
234 contrast sensitivities). There were no significant differences between tACS sessions in any
235 of the measurements (p ≥ 0.098).
236
237 Experiment 1
238 α tACS enhanced PR-VEPs but not FF-VEPs
239 α tACS increased PR-VEP N75-P100 amplitudes compared with β tACS (Fig. 2B
240 upper left). LMM revealed a significant effect of tACS, but no significant effects of time or
241 tACS × time interaction were found (Table 1). The PR-VEP P100-N145 amplitudes did not
242 differ significantly between the α and β tACS conditions (Table 1, Fig. 2B upper right). In
243 addition, there were no significant effects of time, tACS or their interaction on FF-VEP
244 N1-P1 amplitudes and P1-N2 amplitudes (Table 1, Fig. 2B lower).
245
246 Experiment 2
247 α tACS modulated event-related α phase synchronization without power change
248 We replicated the finding from Experiment 1 that α tACS increased N75-P100
249 amplitudes. In brief, LMM revealed a significant effect of tACS, but there was no
250 significant effect of time or time × tACS interaction (Table 1). Post-hoc analysis revealed
251 that N75-P100 amplitudes were significantly enhanced after α tACS, compared with sham,
252 and that there was a tendency toward higher amplitudes for α tACS compared with β tACS
253 (see Fig. 3 and figure legend).
254 Based on the time-frequency analysis, Figure 4A shows the changes from
255 pre-tACS in averaged ERSP in the α (10 ± 1 Hz) and β (20 ± 1 Hz) ranges. For the α ERSP,
256 LMM revealed a significant effect of tACS without significant effects of time or time ×
257 tACS interaction (Table 1). Post-hoc analysis revealed that β tACS decreased the α ERSP
258 compared with α tACS, but not compared with the sham condition (Fig. 4A left). In the β
259 ERSP, there were no significant effects of tACS, time, or time × tACS interaction (Table 1,
260 Fig. 4A right).
261 Figure 4B shows the after-effects of tACS on α and β ITC. LMM revealed a
262 significant effect of tACS without significant effects of time or time × tACS interaction
263 (Table 1). Post-hoc analysis showed that α tACS enhanced α ITC compared with that of β
264 tACS and sham (Fig. 4B left). In β ITC, there was a significant effect of tACS, without a
265 significant effect of time or time × tACS interaction (Table 1). Pairwise comparisons
266 revealed that β ITC was increased for α tACS compared with β tACS, but did not differ
267 from sham (Fig. 4B right).
268
269 α and β tACS augmented resting α activity
270 Figure 5A shows the grand-averaged power spectra across all subjects at Pz. α
271 tACS increased the resting α power, while β tACS increased both of resting α and β power.
272 In the α power, LMM revealed significant effects of tACS and time, but no significant time
273 × tACS interaction was found (Table 1). Both α and β tACS increased α power compared
274 with sham (Fig. 5B left). In β power, there was a significant effect of tACS without a
275 significant effect of time or time × tACS interaction (Table 1). β tACS increased β power
276 compared with both α tACS and sham (Fig. 5B right). Examination of the after-effects on α
277 and β power at electrode Oz’, showed that the power modulation by tACS was less marked
278 than that at electrode Pz (Supplementary Fig. S4).
279 tACS-induced changes in resting α power at Oz’ or Pz were positively correlated
280 with changes in PR-VEP amplitudes in the α tACS condition only (Fig. 6). We also
281 confirmed that the mean peak frequencies in the α and β ranges at electrodes Oz’ and Pz
282 were very close to the tACS frequencies (Supplementary Table S1). When subjects’ peak
283 frequencies in the α and β ranges were compared between Oz’ and Pz, both peak
284 frequencies at Pz were higher than those at Oz’ (Supplementary Fig. S3).
285
286 Experiment 3
287 α tACS improved contrast sensitivity
288 For each of the three spatial frequencies (i.e., 0.5-9 cpd), LMM revealed
289 significant effects of tACS, but no significant effects of time or time × tACS interaction
290 were found (Table 1). In comparison with the sham stimulation, α tACS improved the
291 contrast sensitivities at all three spatial frequencies (p ≤ 0.032, corrected), whereas β tACS
292 only increased the contrast sensitivity at 3 cpd (p = 0.018, corrected) (Fig. 7).
293
294 No effects of tACS-induced flickering sensation
295 In each of the three experiments, about half of the subjects reported a slight
296 flickering sensation during β tACS (see more details in Supplementary results). To
297 investigate the possible influence of tACS-induced flickering perception, we classified our
298 subjects into two groups: a group of those with the sensation (flickering group) and a group
299 of those without it (non-flickering group). We compared the total post-tACS between the
300 two groups, but found no significant differences (p ≥ 0.52, unpaired t-test) (Supplementary
301 Fig. S5). Therefore, our findings cannot be explained simply by flickering sensations.
302
303 Discussion
304 Our aim was to elucidate the effects of α tACS on the visual system. In
305 Experiment 1, α tACS increased the amplitudes of only the early components of PR-VEPs,
306 and this effect lasted for at least 30 min after stimulation. In Experiment 2, α tACS
307 increased resting α oscillatory activity, and a significant positive correlation between
308 PR-VEP amplitudes and resting α oscillatory activity was observed. α tACS also increased
309 the α phase synchronization in time-frequency analysis. Experiment 2 also demonstrated
310 that β tACS enhanced resting α activity, but did not affect VEP amplitudes. In Experiment
311 3, α tACS improved contrast sensitivity. Taken together, α tACS is more effective for
312 modulating PR-VEPs and contrast sensitivity than is β tACS. Possible neural mechanisms
313 and physiological implications are discussed below.
314 α tACS modulates early PR-VEP components and contrast sensitivity
315 In general, VEPs represent the activation of neuronal populations specific to the
316 nature of the visual stimulation. PR-VEP stimuli with an optimal check size preferentially
317 stimulate the fovea, and activate the contrast and spatial frequency channels of V1 [19].
318 Flash stimuli are processed by luminance channels [20]. Therefore, we assume that α tACS
319 affects specific channels of V1, and consequently increases the N75-P100 amplitudes of
320 PR-VEPs. Magnetoencephalographic studies have revealed that the N75 and P100
321 components have sources around the calcarine fissure of striate cortex (V1) whereas the
322 N145 source is localized to striate or extrastriate cortex [18]. Additionally, in a study by
323 Plant et al. (1983) early components of PR-VEP amplitudes were modulated by the spatial
324 frequency of visual stimuli, whereas those of late components were not [39]. In an animal
325 study, the optimal spatial frequency of cells in V1 ranged from 0.5 to 8 cpd, and cells in V2
326 responded to lower spatial frequencies than did cells in V1 [40]. Indeed, our finding
327 revealed that α tACS improved contrast sensitivity for the spatial frequencies between
328 0.5-9 cpd. Collectively, these findings support our assumption that α tACS activates the
329 contrast and spatial frequency channels of V1.
330
331 The interaction between posterior α oscillations and VEP amplitudes

332 For many years, researchers have debated whether VEPs are modulated by
333 ongoing α activity. There are two hypothetical models that explain the relationship between
334 VEPs and ongoing α activity: the evoked model and the phase-reset model. The former
335 assumes that VEPs are independent of background oscillatory activity [24–26]. The latter
336 supposes that ongoing α rhythm is partially phase reset by a sensory input, and part of the
337 VEP is generated by a reorganization of ongoing α oscillations [22,23,41]. So far, the
338 debate has not been conclusively settled.
339 Our time-frequency analysis revealed that α tACS increased the α ITC but not the
340 α ERSP, along with enhancing PR-VEP amplitudes. In the phase-reset model, it is predicted
341 that the event-related phase reorganization occurs independent of the event-related power
342 modulation [35,42,43]. In other words, the phase resetting of background α oscillations can
343 lead to increased α phase concentration (i.e., α ITC) without event-related α power (i.e.,
344 α ERSP) modulation. Thus, our α tACS after-effects agree well with predictions of the
345 phase-reset model. Furthermore, α tACS increased resting α activity, and there was a
346 positive correlation between PR-VEP amplitudes and resting α activity modulation, which
347 also supports the phase-reset model [41]. Conversely, β tACS was found to increase resting
348 α activity, whereas it did not change VEP amplitudes or α ITC. These β tACS effects
349 suggest that background α enhancement does not necessarily result in VEP modulation,
350 which is in line with the evoked model. Interestingly, β tACS decreased the α ERSP
351 compared with α tACS, but not compared with the sham condition. This effect suggests that
352 α and β tACS both increase resting α activity but induce different poststimulus α dynamics.
353 Thus, our findings indicate that the phase-reset model cannot completely explain our results,
354 suggesting that the two different models could coexist in parieto-occipital cortex. Indeed,
355 several studies have proposed the coexistence of these two models, according to which,
356 both partial phase-reset and partial evoked models contribute to the dynamics to VEP
357 generation [35,44].
358 Although α oscillations have been extensively investigated, their functional role is
359 not fully understood. One influential hypothesis is that α oscillations reflect the functional
360 inhibition of task-irrelevant brain regions [45,46]. In fact, previous studies revealed that
361 strong posterior α activity has a negative impact on visual behavioral performance [47,48].
362 However, other studies have demonstrated positive correlations of α activity with
363 behavioral performance [49–51], and it has been suggested that the functional role of α
364 oscillations may vary along the cortical hierarchy [52,53]. Thus, we speculate that α and β
365 tACS could modulate different neuronal networks or sources that are partially involved in
366 the generation of α oscillations, and consequently would induce different effects on
367 PR-VEP amplitudes.
368
369 tACS for spike timing-dependent plasticity

370 It has been suggested that tACS entrains ongoing oscillations in a
371 frequency-dependent manner [4]. Many studies have shown that α tACS over the occipital
372 cortex enhances α power in the parieto-occipital area during or after stimulation [7–11]. In
373 this study, we replicated the finding that α tACS increased the resting α power, and further
374 demonstrated that β tACS enhanced not only resting β power but also α power in the
375 parieto-occipital cortex. Meanwhile, all tACS conditions, including sham, increased resting
376 α power over time (post 0 vs. post 10) in Experiment 2. Increased resting α activity in a
377 sham condition was also reported in a previous tACS study as natural α increase [11], and it
378 may explain the effect of time revealed by LMM in Experiment 2. Recently, network
379 modeling studies revealed that when the stimulation frequency of tACS was a harmonic of
380 the endogenous oscillations, the endogenous rhythmic activity was increased as a
381 subharmonic resonance [54,55]. These resonance or entrainment effects are in good
382 accordance with our results. However, the entrainment evidence regarding tACS has been
383 supported during (i.e., online to) stimulation [7,56], and the mechanisms of after-effects
384 (offline effects) are unclear. A recent study showed that the entrainment effects of tACS did
385 not last after stimulation [10], and spike timing–dependent plasticity (STDP) was proposed
386 as a plausible candidate to explain after-effects [8,10,57]. According to the STDP
387 hypothesis, α tACS can selectively strengthen the synaptic weights of neuronal circuits
388 (recurrent loops) with a reverberation frequency at 10 Hz, and lead to the increased α
389 oscillatory activity, after stimulation, as a synaptic plasticity effect [8,10]. In this model,
390 online entrainment of tACS can contribute frequency-specific synaptic strengthening by
391 STDP [57]. Therefore, we speculate that β tACS can affect the neuronal circuits that
392 reverberate at 10 Hz and at 20 Hz, related to the above-mentioned subharmonic resonance
393 mechanisms, and result in enhancement of power in both the α and β ranges after
394 stimulation.
395 It is interesting to note that the modulation of resting α and β oscillations after
396 tACS was more pronounced at Pz than at Oz’ (Fig. 5 and Supplementary Fig. S4). In the
397 resting state, posterior α oscillations have a widespread origin in the parieto-occipital areas
398 [58] and may reflect a complex product of both thalamo-cortical and cortico-cortical
399 interactions in the visual cortex [12]. Indeed, subjects’ α or β peak frequencies in resting
400 EEG in pre-tACS differed somewhat between the Oz’ and Pz electrodes (Supplementary
401 Fig. S3). This may imply that posterior α and β oscillations reflect summation of several
402 local network rhythms. However, it is beyond the scope of our study to discriminate the
403 cortical sources modulated by tACS in the present experiments. This point should be
404 clarified in the future studies.
405
406 Limitations
407 Although tACS was applied at a constant amplitude (i.e., 1 mA), about half of
408 subjects noticed a slight flickering sensation during β tACS. Indeed, a previous study
409 revealed that the tACS-induced retinal flickering threshold was lowest for β tACS [59]. To
410 avoid the perception of flicker, different intensities are necessary for α and β tACS.
411 However, current intensity is an important factor influencing tACS effects [55,60], and thus
412 we used the same intensity at α and β tACS in this study. Moreover, although we
413 investigated the influences of tACS-induced flickering, these perceptions could not explain
414 the after-effects of α and β tACS on PR-VEPs and resting α power modulation.
415
416 Conclusions
417 Our findings revealed that α tACS over occipital cortex increased PR-VEP
418 amplitude and resting α oscillatory activity. However, β tACS induced the enhancement of
419 resting α power without VEP amplitude modulation. These results suggest that the
420 generators of VEPs and posterior α oscillations partially share the same neuronal networks,
421 and that tACS can affect such networks in a frequency-dependent manner.
422
423 Acknowledgments:
424 We are grateful to Dr. Junji Kishimoto for the constructive comments and support for
425 statistical analysis. We thank Claire Barnes, PhD, from Edanz Group
426 (www.edanzediting.com/ac) for editing a draft of this manuscript.

427
428 Author contributions
429 H.N., K.O. and S.T. designed the study; H.N. and K.O. collected the data; T.K., A.T. and
430 E.Y. supervised data collection; H.N., A.T. and K.O. analyzed the data; H.N., K.O. and S.T.
431 wrote the paper; T.K. commented on the paper.
432
433 Funding
434 This work was supported in part by the Japan Society for the Promotion of Science a
435 Grant-in-Aid for Scientific Research (JSPS KAKENHI 15J03164 to HN) and Grant-in-Aid
436 for Early-Career Scientists (JSPS KAKENHI 18K17720 to HN) and Grant-in-Aid for
437 Scientific Research on Innovative Areas (MEXT KAKENHI 15H05875 to ST).
438
439 Competing Interests:
440 The authors declare no conflict of interest.

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623 Figure legends
624
625 Figure 1.
626 Experimental design. (A) Time course and stimuli in Experiment 1. The visual stimuli for
627 the pattern-reversal VEPs (PR-VEPs) and focal-flash VEPs (FF-VEPs) were presented in
628 the lower hemi-field, and subtended 6° in radius. Both types of VEPs were recorded before
629 (pre-tACS) and after (post-tACS) tACS. VEPs were measured for up to 30 min after tACS.
630 (B) Time course and stimuli in Experiment 2. (C) Time course and stimuli in Experiment 3.
631 Measurements were performed before, immediately after, and 10 min after tACS in
632 Experiments 2 and 3.
633
634 Figure 2.
635 The after-effects of tACS on FF-and PR-VEPs in Experiment 1. (A) The waveforms of PR-
636 VEPs (upper) and FF-VEPs (lower) in a representative subject pre-tACS. Waveforms of
637 PR-VEPs consisted of N75, P100 and N145 components, while those of FF-VEPs consisted
638 of N1, P1 and N2 components. (B) Time courses of the PR-VEP amplitudes (upper) and
639 FF-VEP amplitudes (lower) following α or β tACS. VEP amplitudes in post-tACS were
640 normalized to those in pre-tACS. α tACS increased only N75-P100 amplitudes compared
641 with β tACS (upper left). Values are expressed as mean ± SEM. ** p < 0.001.
642 Figure 3.
643 The after-effects of tACS on PR-VEP amplitudes in Experiment 2. Time course of the
644 normalized N75-P100 amplitudes after α tACS, β tACS and sham is shown. α tACS
645 enhanced N75-P100 amplitudes of PR-VEP compared with β tACS or sham (α tACS vs. β
646 tACS: p = 0.085; α tACS vs. sham: p = 0.017; β tACS vs. sham: p = 1.0, corrected).
647 * p < 0.05.
648
649 Figure 4.
650 The after-effects of tACS on event-related spectral perturbation (ERSP) (A) and inter-trial
651 coherence (ITC) (B) in the α (10 ± 1 Hz) and β (20 ± 1 Hz) ranges. ∆ ERSP and ∆ ITC
652 were calculated by subtracting ERSP or ITC of the pre-tACS from those of the post-tACS.
653 (A) β tACS decreased the α ERSP compared with α tACS (α tACS vs. β tACS: p = 0.017;
654 α tACS vs. sham: p = 0.462; β tACS vs. sham: p = 0.496, corrected), but there were no
655 significant differences between tACS and sham in the α and β ERSP (α range (left); 10 ± 1
656 Hz, β range (right); 20 ± 1 Hz). (B) α tACS increased the α ITC compared with β tACS
657 and sham (α tACS vs. β tACS: p < 0.001; α tACS vs. sham: p = 0.048; β tACS vs. sham: p
658 = 0.664, corrected). Meanwhile, α tACS increased the β ITC compared with β tACS,
659 although there were no significant differences between tACS and sham (α tACS vs. β
660 tACS: p = 0.011; α tACS vs. sham: p = 0.342; β tACS vs. sham: p = 0.513, corrected).
661 * p < 0.05, ** p < 0.001.
662
663 Figure 5.
664 The after-effects of tACS on resting power at Pz. (A) Grand-averaged resting EEG activity
665 for tACS sessions (left; α tACS, middle; β tACS, right; sham) in pre- and post-tACS.
666 Shaded areas indicate the standard error of the mean. Red dashed lines indicate the α (10 ±
667 1 Hz) and β (20 ± 1 Hz) ranges. (B) The after-effects of tACS on resting EEG (α range
668 (left); 10 ± 1 Hz, β range (right); 20 ± 1 Hz). ∆ log10 power was calculated by subtracting
669 log10 power of the pre-tACS from those of the post-tACS (Δlog10 power = log10 power
670 (post) − log10 power (pre)). α and β tACS increased resting α activity compared with sham
671 (α tACS vs. β tACS: p = 1.0; α tACS vs. sham: p = 0.029; β tACS vs. sham: p = 0.002,
672 corrected), while β tACS also increased β power compared with α tACS or sham (10 Hz
673 tACS vs. 20 Hz tACS: p = 0.025; 10 Hz tACS vs. sham: p = 1.0; 20 Hz tACS vs. sham: p =
674 0.044, corrected). * p < 0.05, ** p < 0.01.
675
676 Figure 6.
677 Correlations between the resting α power at the Oz’ (left) and Pz (right) electrodes and
678 N75-P100 amplitudes of PR-VEPs. The normalized data on resting α power and N75-P100
679 amplitudes for the two post-tACS times (i.e., post 0, 10 min) were collapsed for each
680 subject and each electrode, to calculate the ∆ resting α power and N75-P100 ratio,
681 respectively. Each circle represents the after-effects of resting α power at Oz’ or Pz vs.
682 N75-P100 amplitudes in the α tACS, β tACS or sham conditions. In both electrodes, a
683 positive correlation was found in the α tACS condition but no such relationship was
684 obtained in the β tACS and sham conditions (α tACS, Pz: r = 0.523, p = 0.031; Oz’: r =
685 0.533, p = 0.028; other tACS conditions, p ≥ 0.236, Pearson correlation). Lines are the
686 regression lines for each correlation. * p < 0.05.
687
688 Figure 7.
689 The after-effects of tACS on contrast sensitivity. These figures show the mean contrast
690 sensitivity values at post-tACS normalized by the ones at pre-tACS (0.5 cpd: left; 3 cpd:
691 middle; 9 cpd: right). α tACS improved the contrast sensitivity for 0.5–9 cpd stimuli,
692 compared with sham, whereas β tACS only improved contrast sensitivity for the 3 cpd
693 stimulus. * p < 0.05, ** p < 0.001.

Table 1. Results of a linear mixed-effects model for the three experiments
tACS Time Interaction

F [df, error] p F [df, error] p F [df, error] p
Experiment 1 (n=13)
PR-VEP (N75-P100) 12.878 [1,84] <0.001 0.077 [3,84] 0.972 0.227 [3,84] 0.877
(P100-N145) 0.388 [1,84] 0.535 0.323 [3,84] 0.809 0.213 [3,84] 0.887
FF-VEP (N1-P1) 1.976 [1,84] 0.163 0.303 [3,84] 0.823 0.867 [3,84] 0.462
(P1-N2) 0.096 [1,84] 0.758 0.344 [3,84] 0.794 0.386 [3,84] 0.764
Experiment 2 (n=17)
PR-VEP (N75-P100) 4.487 [2,80] 0.014 0.407 [1,80] 0.525 0.664 [2,80] 0.518
α ERSP 4.033 [2,80] 0.021 0.108 [1,80] 0.743 0.743 [2,80] 0.483
β ERSP 0.366 [2,80] 0.694 0.806 [1,80] 0.372 1.128 [2,80] 0.329
α ITC 7.073 [2,80] <0.001 0.212 [1,80] 0.647 0.193 [2,80] 0.825
β ITC 4.445 [2,80] 0.015 0.086 [1,80] 0.77 0.322 [2,80] 0.726
resting α power 6.901 [2,80] 0.002 6.478 [1,80] 0.013 0.101 [2,80] 0.904
resting β power 4.535 [2,80] 0.014 0.002 [1,80] 0.969 0.041 [2,80] 0.959
Experiment 3 (n=15)
0.5 cpd 3.544 [2,873.019] 0.029 0.15 [1,873.013] 0.699 1.545 [2,873.026] 0.214
3 cpd 7.931 [2,875.013] <0.001 0.988 [1,875.011] 0.32 0.111 [2,875.016] 0.895
9 cpd 5.544 [2,857.069] 0.004 0.678 [1,856.992] 0.411 1.538 [2,856.999] 0.215
The bold font indicates that the differences reached statistical significance.
PR-VEP: pattern-reversal VEP; FF-VEP: focal-flash VEP; ERSP: event-related spectral
perturbation; ITC: inter-trial coherence
Highlights
We studied whether α and β tACS can differentially influence the occipital cortex.
Effects of α and β tACS on VEPs, resting EEG and contrast sensitivity were
assessed.
α tACS but not β tACS was effective for sharpening multiple visual functions.
Effects of tACS on visual cortex can be a frequency-dependent manner.

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Transcranial alternating current stimulation of α but not β frequency sharpens multiple

To appear in: Brain Stimulation

Received Date: 15 July 2019

© 2019 Elsevier Inc. All rights reserved.

3 Transcranial alternating current stimulation of α but not β

4 frequency sharpens multiple visual functions

13 Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan

15 University of Health and Welfare, Fukuoka 814-0001, Japan

17 International University of Health and Welfare, Fukuoka 831-8501, Japan

20 Address Correspondence to Hisato Nakazono, PhD:

21 Department of Occupational Therapy, Faculty of Medical Science, Fukuoka International

22 University of Health and Welfare, Fukuoka 814-0001, Japan

29 cortical oscillatory activity in a frequency-dependent manner. In our previous study

30 (Nakazono et al.2016), 20 Hz (β) tACS significantly increased excitability of primary

32 primary visual cortex (V1) in a resting electroencephalogram. Hence, we investigated

33 whether α and β tACS can differentially influence multiple visual functions.

34 Methods: Firstly, we evaluated the after-effects of α and β tACS on pattern-reversal (PR)

37 behavioral effects of tACS were assessed by contrast sensitivity.

40 event-related α phase synchronizations without increasing power, which consequently

48 functions by modulating α oscillations in V1.

58 Effects of tACS on visual cortex can be a frequency-dependent manner.

63 electroencephalography; ERP, event-related potential; V1, primary visual cortex; V2,

64 secondary visual cortex; ERSP, event-related spectral perturbation; ITC, inter-trial

65 coherence; cpd, cycle per degree; LMM, linear mixed-effects modeling

67 Transcranial alternating current stimulation (tACS) is a non-invasive technique

68 known to modulate brain oscillations. Interestingly, tACS affects brain functions or

69 behavior in a frequency-dependent manner [1–5]. The effective stimulation frequency of

70 tACS is regarded as matching the targeted brain oscillations [4,6]. Indeed,

77 and visual processing [7,13–15]. Nevertheless, frequency-dependent effects of α tACS

79 cortex may be complex and depend on task demands.

89 remains controversial [24–26]. Therefore, we hypothesized that if there is a causal

95 phase concentration or power dynamics in VEPs modulated by α and β tACS using

96 time-frequency analysis. We also investigated resting α power modulations after tACS in

97 Experiment 2. Finally, we measured the behavioral effects of α and β tACS on contrast

98 sensitivity in Experiment 3 because PR-VEP amplitudes depend on pattern spatial

103 Material and methods

113 Subsequently, sample sizes of N ≥ 15 were determined to be necessary for Experiments 2

117 Experiments 1 and/or 2) took part in Experiments 2 and 3, respectively.

119 Experimental designs

134 in Experiments 2 and 3, to confirm the tACS effects.

138 experiments. tACS was delivered by a battery-driven current stimulator (DC

139 Stimulator-Plus, NeuroConn GmbH, Ilmenau, Germany) through two self-adhesive

150 Recordings of VEPs and resting EEG

160 and EEG recordings are provided in the supplementary methods.

162 Measurement of contrast sensitivity

167 see supplementary methods.

169 Data Analyses

171 software [34] and custom-programmed tools in Python.

173 Analysis of VEP amplitudes

180 Time-frequency analysis for VEP data

188 (poststimulus period/prestimulus period)]. ERSP measures event-related changes in spectral

198 subtracted from the post-tACS for each subject.

200 Analysis of resting EEG

208 S1, Supplementary Fig. S3).

210 Analysis of contrast sensitivity

216 Statistical analyses

231 Regarding the stability of pre-tACS measurements in each experiment

232 (Experiment 1: amplitudes of PR- and FF-VEPs; Experiment 2: N75-P100 amplitude of

235 of the measurements (p ≥ 0.098).

238 α tACS enhanced PR-VEPs but not FF-VEPs

244 N1-P1 amplitudes and P1-N2 amplitudes (Table 1, Fig. 2B lower).

247 α tACS modulated event-related α phase synchronization without power change