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2 Clinical Neurophysiology 121 (10) Contents lists available at ScienceDirect Clinical Neurophysiology journal homepage: Phosphene thresholds evoked with single and double TMS pulses Thomas Kammer *, Lisa W. Baumann Department of Psychiatry, University of Ulm, Leimgrubenweg 12, D Ulm, Germany article info abstract Article history: Accepted 6 December 09 Available online 15 January 10 Keywords: Magnetic stimulation Occipital cortex Phosphene thresholds Double pulses Fusion time Objective: To evaluate the quantitative advantage of double pulses vs. single pulses in TMS phosphenes evoked from the occipital cortex. Methods: In 10 healthy subjects single pulse thresholds were compared with thresholds from double pulses of equal strength at a stimulus onset asynchrony (SOA) of 2, 5, 10, and ms, both with and pulse forms. In a second experiment fusion time, i.e. the double pulse SOA where the percept passes from one into two phosphenes was determined. Results: Thresholds obtained with double pulses did not depend on SOA. They were lowered to about 90% of single pulse thresholds. Biphasic pulses yielded lower thresholds (89%) than pulses. Fusion time was about 45 ms but highly varied inter-individually and did not depend on stimulation intensity. Conclusions: Although double pulses are more efficient compared to single pulses the advantage is rather small. Previous recommendations to apply double pulses in phosphene studies cannot be confirmed, at least for SOAs up to ms. The independence of fusion time to stimulus intensity indicates a non-linear relation between network activity and the percept of phosphene persistence. Significance: Phosphene threshold studies do not gain advantages by the application of double pulses. Ó 09 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. 1. Introduction Transcranial magnetic stimulation (TMS) over the occipital pole is able to evoke elementary visual phenomena called phosphenes (Barker et al., 1985; Meyer et al., 1991; Marg and Rudiak, 1994; Kammer, 1999). However, several authors reported that many subjects failed to perceive phosphenes with a single TMS pulse (Amassian et al., 1989; Beckers and Hömberg, 1991; Aurora et al., 1998; Kamitani and Shimojo, 1999; Corthout et al., 00). Following the recommendations of Ray et al. (1998) in some phosphene studies double pulses have been used instead of single pulses (e.g. Boroojerdi et al., 00; Brighina et al., 02; Fierro et al., 05) in order to increase the amount of subjects perceiving phosphenes. So far, in three studies the effects single pulses and double pulses to evoke phosphenes have been compared in a systematic approach. In the first report on the effects of double-pulse TMS on phosphene perception, Ray et al. (1998) reported a reduction of phosphene thresholds down to 0.9 of single pulse thresholds using double pulses with a stimulus onset asynchrony (SOA) of ms. They were able to evoke phosphenes even with single pulses in all healthy subjects investigated. They furthermore reported that repetitive TMS trains of 5 Hz reduced phosphene thresholds down to 0.6 of single pulse thresholds. Gerwig et al. * Corresponding author. Tel.: ; fax: address: thomas.kammer@uni-ulm.de (T. Kammer). (05) compared single and double-pulse TMS in healthy subjects and in patients suffering from migraine. They were able to elicit phosphenes in all the subjects with single pulses, too. With double pulses at a SOA of ms phosphene thresholds decreased to 0.7 of single pulse thresholds both in healthy subjects and in migraine patients. Sparing et al. (05) investigated phosphene detection rates with double pulses of different intensities at SOAs between 2 and 12 ms. They searched for an equivalent of short-interval intracortical inhibition (SICI) known from the motor system (Kujirai et al., 1993; Ilic et al., 02) expecting an increase in phosphene threshold with double pulses in the sequence sub threshold supra threshold stimulation intensity. They did not identify a double pulse condition reducing detection rates compared to the corresponding single pulse condition, but rather no change or even an increase in detection rates depending on stimulation intensity, i.e. a facilitation comparable to Ray et al. (1998) and Gerwig et al. (05). From electrophysiological studies (Moliadze et al., 03) we know that a single pulse already evokes a complex pattern of excitation and inhibition in the visual cortex. Double pulses increase the complexity of network reaction (Moliadze et al., 05). In the present study we set out to quantify the advantage of double pulses compared to single pulses in generating phosphenes. To that aim we measured phosphene thresholds using single pulses and double pulses with several SOAs. We included the TMS pulse form ( and pulses) as independent factor in order to /$36.00 Ó 09 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved. doi: /j.clinph

3 T. Kammer, L.W. Baumann / Clinical Neurophysiology 121 (10) quantify the advantage of pulses (Kammer et al., 07). Furthermore, we quantified the fusion time for the perception of one versus two distinct phosphenes. 2. Methods 2.1. Subjects and apparatus Ten healthy subjects (age between 24 and 31, 5 females, 5 males) participated in the study after giving written informed consent. They were paid for participation. The study was approved by the local internal review board of the Medical Faculty, University of Ulm. Subjects were stimulated with a Medtronic Magpro stimulator X100 (Skovlunde, Denmark), using a figure-of-eight coil, MC-B70 (for physical details see Thielscher and Kammer (04)), fixed on a tripod. Double pulses with various SOAs were triggered by the PC with a precision of 100 ls using the digital output port of the graphics board VSG 2/3 (Cambridge Research Systems, Rochester, UK). In two training sessions subjects were familiarized with the occipital stimulation and the observation of phosphenes. Subjects were sitting in a comfortable chair and fixated a spot on a white screen in a slightly illuminated room. Phosphene perception had to fulfill the following three criteria (Kammer et al., 05): (a) dependence on the stimulated hemisphere, i.e. perception in the left visual field with stimulation at the right occipital pole and vice versa (Meyer et al., 1991); (b) visibility of phosphenes with eyes both open and closed; (c) dependence of phosphene location within the visual field on gaze direction. After familiarization subjects were trained to perceive phosphenes at threshold by at least two runs of threshold measurement Experimental procedure Phosphene perception thresholds from the left occipital pole were measured following a previously established protocol (Kammer et al., 01b). First, a stimulation site was determined by moving the coil in steps of about 5 mm over the left occipital pole while the subject was stimulated with a suprathreshold intensity known from the training sessions until he or she observed a sharply delineated phosphene clearly restricted to the right visual field ( hot spot ). Then, for each stimulation condition 7 magnetic stimuli were delivered at 6 8 different stimulator output intensities each in steps of 3%, resulting in stimuli per measurement. All stimuli were randomly intermixed (method of constant stimuli). A computer program presented the actual stimulation intensity on a screen and the experimenter transferred it to the stimulator manually. After the subject released the magnetic stimulation with a key press, he or she observed the white screen and reported the presence or the absence of a phosphene after each stimulus ( yes no ) by pressing one out of two response keys. After the response, the delay time of 00 ms started and the experimenter transferred the next stimulation intensity to the stimulator. An acoustical signal indicated the end of the delay time allowing the subject to release the next magnetic stimulus. Thus, stimulation frequency did not exceed 0.2 Hz (decision time + 00 ms + reaction time to the acoustical signal). In the first protocol phosphene thresholds were measured with single pulse TMS and with double-pulse TMS at SOAs of 2, 5, 10, and ms. Every session started with a single-pulse measurement. Then the four SOAs were measured in random order. Finally, a second single-pulse measurement was performed, resulting in 6 measurements per session. Two sessions were performed, one with and the other with TMS pulse forms. The handle of the coil was oriented horizontally, and the first upstroke of the induced current was oriented latero-medially for pulses and medio-laterally for pulses (Kammer et al., 01b, 07). In the second protocol fusion time was determined by the application of two TMS pulses with different SOAs (method of constant stimuli). After each stimulus with two pulses subjects had to report the percept of one or two distinct phosphenes by pressing a key. At least six SOAs were presented seven times randomly intermixed. In each subject the shortest and the longest SOA in the protocol were determined individually before by test stimuli revealing the percept of one or two phosphenes, respectively. Due to large interindividual differences the SOAs ranged from 2 to 17 ms as a time window tested in the fastest subject, and ms as a time window tested in the slowest subject. Three fusion time measurements were performed with stimulation intensities of 110% 1%, and 1% of individual single pulse phosphene threshold measured as first run in the session. The protocol was measured with mono- and TMS pulse forms. The experiments were performed in four sessions on four different days. The sequence of protocols was randomized in the following way: In the first session both the protocol (phosphene threshold or fusion time) and the pulse form ( or ) was chosen at random. In the second session the alternate protocol was measured with the same pulse form as in the first session. In the third and fourth session the protocols from the first and second session were measured changing the pulse form with respect to the first two sessions Data analysis For each run a sigmoidal function was fitted to the reported responses over stimulation intensity (logistic function using psignifit, cf. Wichmann and Hill (01)). The stimulation intensity at % yes -responses was taken as the phosphene threshold (cf. Kammer et al., 01b). Thus, calculated phosphene thresholds could reach non-integer values. Similarly, fusion time was determined in each run at % between one - and two -responses after fitting a sigmoidal function to the reported responses. The inference statistics was calculated with Statistica (V 7.1, StatSoft, Tulsa, OK, USA). 3. Results After the two training sessions all 10 subjects were able to perceive phosphenes and to report the absence or presence of a phosphene with stimulation intensities at threshold Single and double pulses Phosphene thresholds with single pulses measured three times in each subject were reproducible (no significant differences in repeated measure ANOVAs) and therefore were collapsed to one datum ( 35.5 ± 6.3%, mean ± SEM, 31.7 ± 6.1%). Double pulse phosphene thresholds did not change with different SOAs ( F 3,27 = 0.77, p = 0.52; F 3,27 = 1.48, p = 0.24, Fig. 1a) and therefore were collapsed to one datum. In a 2-way repeated measure ANOVA Factor PULSE FORM showed significant differences (F 1,9 = 7.57, p < 0.05). Biphasic thresholds were lower compared to thresholds (.0 ± 5.7% vs ± 6.1%). Factor NUMBER OF PULSES was significant too (F 1,9 = 9.61, p < 0.05). Thresholds with double pulses were lower compared to thresholds with single pulses (.4 ± 5.8% vs ± 6.0%). For pulses, double pulses lowered threshold to 0.89 of single pulse threshold; for pulses, the factor

4 378 T. Kammer, L.W. Baumann / Clinical Neurophysiology 121 (10) a 60 Phosphene threshold (%) Fusion time (ms) b Phosphene threshold (%) was 0.94 (Fig. 1b). The interaction PULSE FORM NUMBER OF PULSES was not significant (F 1,9 = 1., p = 0.28, Fig. 1b) Fusion time Fusion time was numerically lower with stimulation (42.1 ± 7.3 ms) compared to stimulation (45.0 ± 7.5 ms): The 2-way repeated measure ANOVA did not reveal significant differences: neither with factor INTENSITY (F 2,18 = 0.88, p = 0.43) nor with factor PULSE FORM (F 1,9 = 0.71, p = 0.42). The interaction was not significant, too (F 2,18 = 0.04, p = 0.96, Fig. 2). 4. Discussion single double SOA (ms) single Pulse double Fig. 1. Phosphene thresholds in percent of maximum stimulator output with single and double pulses. Data from 10 subjects. (a) Thresholds for double pulses at SOAs of 2, 5, 10, and ms compared with single pulse. No significant difference was found for the four SOAs tested. (b) Mean thresholds for double pulses a four SOAs compared with single pulse thresholds. See text for results of ANOVA. Filled squares: pulse form, open circles: pulse form. Error bars indicate SEM. Phosphene thresholds measured with double pulses are lower compared to single pulses. However, the numerical difference is marginal: a relative decrease of about 0.9. The difference is in the same range as the difference between and stimuli, and it is independent of SOA. Fusion time, i.e. the SOA for the transition between the percept of one versus two distinct phosphenes highly varies between subjects but does not vary with stimulation intensity TMS intensity (% of phosphene threshold) Fig. 2. Fusion time, i.e. the SOA in ms of double pulses where subjects observed two distinct phosphenes or one fused phosphene with equal probability, in dependence of stimulus intensity, normalized to single pulse phosphene threshold. Data from 10 subjects. See text for results of ANOVA. Filled squares: pulse form, open circles: pulse form. Error bars indicate SEM. The present data confirm previous observations that the use of double pulses lower phosphene thresholds compared to single pulses, but the effects are rather small. The decrease is in the same range as observed by Ray et al. (1998), and similar to our data they did not observe an influence of SOA on threshold decrease. Using the same stimulator and the same coil as in the present study Gerwig et al. (05) found in healthy subjects a reduction of phosphene thresholds comparing single and double pulses from 64.4% down to 44.6%, i.e. a factor of They only applied pulses, and SOA was ms for double pulses. The longest SOA we investigated was ms. We observed in the mode a reduction of phosphene thresholds using double pulses of only An explanation for this striking numerical difference could be that we trained our subjects prior to the threshold measurements. The training might sensitize subjects to observe phosphenes, consistent with the lower absolute threshold values we observed (32% and 29% vs. 64% and 45% in case of Gerwig et al. (05)). Another difference is the SOA used for double pulses. ms used by Gerwig et al. (05) is above the mean fusion time we determined: From that we would expect that the majority of our subjects would have observed two distinct phosphenes at an SOA of ms. One might speculate whether the appearance of two distinct phosphenes might lower the perceptual threshold. Our threshold results are consistent with the single cell recordings in cats by Moliadze et al. (05). They showed that neurons responding with an increase in activity due to a single TMS pulse further increase their activity with two pulses of equal strength in a non-linear manner. This kind of summation also seems to take place using trains of stimuli, as demonstrated by Ray et al. (1998). The influence of pulse form on phosphene thresholds has previously been demonstrated for single pulses (Kammer et al., 07). Similar to the motor cortex (e.g. Kammer et al., 01a) pulses are more efficient compared to pulses. The replication of this observation in the visual cortex confirms a general neurophysiologic principle that holds for peripheral nerve preparations (Maccabee et al., 1998) as well as for cortical networks. However, the underlying mechanism is not clarified. The polarity change might cause a sequence of hyperpolarization and depolarization recruiting more sodium channels compared to a pure depolarization (Maccabee et al., 1998). Alternatively, optimal neuronal responses might depend on the duration of the induced current in its optimal orientation (Maccabee et al., 1998; Davey and Epstein, 00). Fusion time does not increase with stronger stimulation. This negative finding demonstrates that the persistence of a phosphene

5 T. Kammer, L.W. Baumann / Clinical Neurophysiology 121 (10) does not depend on stimulation intensity. By means of single cell recordings in striate cortex in cats it has been shown that stronger pulses result in both a longer excitation response as well as a longer inhibitory phase (Moliadze et al., 03). From that our finding suggests that the electrophysiological equivalent of the percept does not depend on the activity of single neurons but rather reflects certain network activity that does not change within the range of stimulation intensities tested. The observation demonstrates one more physiological difference between the reaction of motor cortex and visual cortex to magnetic stimulation, since in the motor system with increasing stimulation intensity the number of evoked I-waves and thus the duration of the cortical responses increase. It is in line with the observation that the visual system does not generate short interval intracortical inhibition (SICI) applying paired-pulse stimuli in the intensity sequence subthreshold suprathreshold, as has been shown both in humans (Sparing et al., 05) and cats (Moliadze et al., 05). The present study is the seventh on phosphenes from our laboratory. In the present study as well as in five preceding studies (Kammer, 1999; Kammer and Beck, 02; Kammer et al., 01b, 03, 05) all subjects included into these studies were able to perceive phosphenes immediately or after a short-interval of training. Only in Kammer et al. (07) one of the 15 subjects failed to reach the established criteria (Kammer et al., 05, see Section 2) and was therefore excluded from the study. We cannot explain the discrepancy between our experience and other studies failing to evoke phosphenes in almost every subject. We only can speculate that there might be a kind of learning process required in some subjects in order to perceive phosphenes, much as with some gestalt processes (Kammer et al., 05). Since the differences between single and double pulses we observed have been so small in our view there is no need to apply double pulses in phosphene studies unless the particular neurophysiologic constellation of summation is the goal of the investigation. However, we would recommend to train the subjects in order to reach stable perception situations. Acknowledgement The authors would like to thank Anne-Katharina Fladung for helpful discussions. References Amassian VE, Cracco RQ, Maccabee PJ. Focal stimulation of human cerebral cortex with the magnetic coil: a comparison with electrical stimulation. Electroenceph Clin Neurophysiol 1989;74:1 16. Aurora SK, Ahmad BK, Welch KMA, Bhardhwaj P, Ramadan NM. Transcranial magnetic stimulation confirms hyperexcitability of occipital cortex in migraine. 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