Brain polarization of parietal cortex augments training-induced improvement of visual exploratory and attentional skills

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Brain polarization of parietal cortex augments training-induced improvement of visual exploratory and attentional skills Nadia Bolognini a,b,⁎, Felipe Fregni c , Carlotta Casati a,b , Elena Olgiati a , Giuseppe Vallar a,b a

Department of Psychology, University of Milano-Bicocca, Milano, Italy Neuropsychological Laboratory, IRCCS Istituto Auxologico Italiano, Milano, Italy c Department of Neurology, Berenson-Allen Center for Noninvasive Brain Stimulation, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA b

A R T I C LE I N FO

AB S T R A C T

Article history:

Recent evidence suggests that behavioural gains induced by behavioural training are

Resea

Accepted 22 June 2010

maximized when combined with techniques of cortical neuromodulation, such as

pariet

Available online 1 July 2010

transcranial Direct Current Stimulation (tDCS). Here we address the validity of this appealing approach by investigating the effect of coupling a multisensory visual field exploration training with tDCS of the posterior parietal cortex (PPC). The multisensory visual field exploration training consisted in the practice of visual search through the systematic audio-visual stimulation of the visual field. Neurologically unimpaired participants performed a bimodal exploration training for 30 min, while simultaneously receiving anodal-excitatory PPC tDCS or sham tDCS. In two different experiments, the left and the right hemisphere were stimulated. Outcome measures included visual exploration speed at different time intervals during the training, and the post-training effects on tests assessing visual scanning and visuo-spatial orienting. Results show that PPC tDCS applied to the right, but not to the left, hemisphere increases the training-induced behavioural improvement of visual exploration, as compared to sham tDCS. In addition, right PPC tDCS brings about an improvement of covert visual orienting, in a task different from the visual search practice. In an additional experiment, we confirm that right parietal tDCS by itself, even without the associated training, can lead to enhancement of visual search. Overall, anodal PPC tDCS is a promising technique to enhance visuo-spatial abilities, when combined to a visual field exploration training task. © 2010 Elsevier B.V. All rights reserved.

1.

Introduction

Transcranial Direct Current Stimulation (tDCS) has been investigated as a mean of modulating cortical excitability, in

order to reveal causal relationships between brain regions and sensorimotor and cognitive functions (Boggio et al., 2009; Priori, 2003; Vines et al., 2006), and of facilitating skill acquisition, learning, and neural plasticity (Floel et al., 2008;

⁎ Corresponding author. Department of Psychology, University of Milano-Bicocca, Viale dell'Innovazione 10, 20126 Milano, Italy. Fax: + 39 02 64483788. E-mail address: [email protected] (N. Bolognini). 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.06.053

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Fregni and Pascual-Leone, 2007; Ragert et al., 2008). tDCS essentially consists of delivering for minutes a weak direct current (below the perceptual threshold) over the scalp: the resulting constant electric field penetrates the skull. tDCS is thought to induce alterations in cortical excitability through hyperpolarizing and depolarizing shifts of the resting membrane potential (Liebetanz et al., 2002; Nitsche and Paulus, 2000; Priori, 2003). In general, whereas cathodal tDCS has a suppressive effect, anodal tDCS increases the function of the underlying cortical areas (Liebetanz et al., 2002; Nitsche and Paulus, 2000; Priori, 2003). These changes in cortical excitability induced by tDCS (increase or decrease) lead to correspondent changes in cortical function and activation. The neuromodulatory effects of tDCS on cognitive processing may also depend upon the tasks used, and the stimulation parameters applied (Monti et al., 2008; Sparing et al., 2009). Recently, it has been proposed that tDCS could be used to prime cortical excitability for a subsequent training or stimulation, in order to enhance learning processes (Bolognini et al., 2009b; Fregni and Pascual-Leone, 2007). The rationale is that learning and tDCS may share similar mechanisms of action for inducing neuroplastic cortical changes (i.e., cortical excitability shifts, changes of synaptic efficacy) (Liebetanz et al., 2002; Rioult-Pedotti et al., 1998). In addition, tDCS, by modulating the neuronal membrane threshold (for instance, with anodal stimulation inducing a local depolarization), might lead to an increased efficiency of the signal processing induced by the behavioural training. Therefore, cortical excitability changes induced by tDCS could interact with the ongoing learning processes, leading to more remarkable behavioural gains (Bolognini et al., 2009b; Fregni and Pascual-Leone, 2007). Here we were interested in determining whether adjuvant tDCS of the right parietal posterior cortex (PPC) can augment the behavioural gain induced by a visual field exploration training. As a training task, we chose a previously developed paradigm consisting in the multisensory audio-visual stimulation of the entire visual field (Bolognini et al., 2005b). Previous studies have indeed provided much evidence concerning the benefits of multisensory integration for orienting behaviour, such as the enhancement of oculomotor responses and visual sensitivity (Bolognini et al., 2005a; Driver and Spence, 1998; Frassinetti et al., 2002, 2005; Harrington and Peck, 1998; Hughes et al., 1998; McDonald et al., 2000; Spence and Driver, 2000). Accordingly, current methods for rehabilitation of unimodal sensory disorders in brain-damaged patients (e.g., hemianopia) focus on the activation of spared multisensory cerebral structures by using multisensory stimulation (Bolognini et al., 2005b). This multisensory stimulation approach appears more effective than a purely visual practice (Passamonti et al., 2009). In this study we investigated the possibility of enhancing the behavioural effects of a multisensory visual field training (see Fig. 1) through the brain polarization of the PPC. This study may provide baseline evidence for future therapeutic application in cognitive rehabilitation, proving to be useful to boost impaired visual function by an efficient combining of multisensory behavioural approaches and tDCS of relevant areas. To this aim, we conducted two double-blind, randomized, shamcontrolled experiments in neurologically unimpaired volun-

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Fig. 1 – Overview of the bimodal apparatus used for training. Participants sat at a distance of 120 cm from the board (see text for details).

teers with a two-fold aim: (1) to determine the effect of a singlesession of PPC tDCS, combined with a bimodal exploration training (Bolognini et al., 2005b), on visual search and visuospatial attention; (2) to investigate putative differences related to the hemispheric side of the PPC stimulation. One important issue is the site of stimulation: the PPC, which is a critical area for bimodal exploration training. Firstly, spatial awareness for perception and action is an important function of the “dorsal-ventral” stream of cortical pathways originating in the primary visual cortex and projecting to the PPC (Rizzolatti and Matelli, 2003; Rozzi et al., 2006). In particular, in humans the right PPC critically contributes to visual search, covert visual orienting and visual selection, by exerting top-down influences on visual processing (Corbetta et al., 2008). Secondly, it has long been known that the right hemisphere is specialized for visuospatial functions (Mesulam, 2002), being able to orient attention towards both sides of space (Pardo et al., 1991; Proverbio et al., 1994). Recent studies with tDCS also indicate that this area might be relevant for modulating visuo-spatial orienting (Ko et al., 2008; Schweid et al., 2008; Sparing et al., 2009). Thirdly, the PPC is a heteromodal region of sensory convergence, which contains unisensory and multisensory neurons responding to visual and auditory stimuli (Andersen, 1997; Bolognini et al., 2009a; Bushara et al., 1999), thus contributing to supramodal spatial attention and awareness (Macaluso and Driver, 2003). Targeting this area by anodal tDCS has been shown to be effective for enhancing audiovisual interactions (Bolognini et al., 2010). Therefore the PPC, particularly in the right cerebral hemisphere, is the most appropriate site to be targeted by tDCS. Accordingly, we first assessed the effects of stimulating the right PPC (Experiment 1) and, second, the effects of left-hemisphere PPC stimulation (Experiment 2). For PPC stimulation the excitability enhancing anodal electrode was placed over P4 (Experiment 1) or over P3 (Experiment 2), according to the international 10-20 system for EEG electrode placement; an extra-cephalic montage was used for the placement of the cathodal electrode.

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During the bimodal training (lasting for 30 min), participants received either PPC tDCS (2 mA) or sham stimulation (Gandiga et al., 2006). The effects induced by the intervention were measured by considering: (1) the improvement of the participants’ performance during the bimodal training; (2) the improvement of the participants’ skills in different visuospatial tasks (namely, visual field exploration and visuospatial orienting), by comparing their performance before and after each training session. This allowed us to monitor the effects of the intervention not only during the training, but also on visuo-spatial orienting, which, as discussed earlier, critically relies on the activity of the right PPC, but was not specifically trained. Additional control tasks were used to assess the specificity of the after-effects induced by the intervention. Finally, we further explored the effects of parietal tDCS by itself, without the training, on visual search (Experiment 3).

2.

Results

2.1. Effects of parietal tDCS combined with multisensory visual field exploration In both Experiment 1 (right PPC tDCS) and Experiment 2 (left PPC tDCS), the effects induced by the training were analyzed separately for each test by analyses of variance (ANOVAs). When appropriate, post hoc multiple comparisons were performed, using Newman–Keuls tests.

2.1.1.

Bimodal exploration training

In order to assess the progressive improvement of performance during the bimodal training, we analyzed mean reaction times (RTs) to visual–auditory stimuli by repeatedmeasures ANOVAs with Condition (Real PPC tDCS, sham tDCS), Block (mean reaction times, ms, after 8 min of training, Block 1; after 16 min, Block 2; after 24 min, Block 3; after 32 min, Block 4), and Side (left-sided, right-sided stimuli) as main factors. During training, each participant detected the visual targets, regardless of their spatial location, without making any false alarm or omission error. Therefore, accuracy scores were not analysed.

2.1.2.

Visual exploration

Visual scanning performances (response latencies, ms; accuracy, percent correct) in the EF Test, in the Triangles Test, and in the Visual Scanning Test—TAP [i.e., “Test Battery for Attentional Performance” (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994)] were analyzed by repeated measures one-way ANOVAs in the following conditions: baseline, post PPC tDCS, post sham tDCS.

2.1.3.

Visuo-spatial orienting

For the Unimodal Visual Test and the Covert Visual Orienting Test from the TAP, only response latencies were analysed, since participants were 100% accurate. For the Unimodal Visual Test, mean RTs were analysed by repeated measures ANOVAs, with Condition (baseline, post PPC tDCS, post sham tDCS), and Side (left-sided and right-sided stimuli) as main

factors. For the Covert Visual Orienting Test the main factors were Condition, Side, and Cue (valid, invalid).

2.1.4.

Control tests

For the Working Memory and the Alertness test from the TAP, repeated measures one-way ANOVAs were performed on mean RTs in the following conditions: baseline, post PPC tDCS, post sham tDCS. Accuracy scores were not analysed since participants' performance was 100% correct.

2.2.

Experiment 1: right hemisphere stimulation

2.2.1.

Bimodal exploration training

As shown by Fig. 2A, the participants' latencies diminished across blocks, with the decrease being steeper between Block 1 and Block 2 during Real PPC tDCS. The main effects of Block (F3,27 = 11.69, p < 0.001, pη2 = 0.56), and of Side (F1,9 = 10.60, p < 0.01, pη2 = 0.54) were significant. As for the Side effect, responses were faster to right-sided stimuli (262 ms, Standard Error, i.e., SEM = 24), as compared to left-sided stimuli (271 ms, SEM = 26); this side difference is not shown in Fig. 2A, as it did not interact with the other main factors. The main effect of Condition was not significant (F1,9 = 2.26, p = 0.17, pη2 = 0.2). The Condition by Block interaction was significant (F3,27 = 3.69, p < 0.02, pη2 = 0.29). The Condition by Side (F1,9 = 0.92, p = 0.36, pη2 = 0.09), Block by Side (F3,27 = 0.47, p = 0.7, pη2 = 0.05), and Condition by Block by Side (F3,27 = 0.95, p = 0.4, pη2 = 0.1) interactions were not significant. As for the significant Condition by Block interaction, after Real PPC tDCS latencies decreased between Block 1 and Block 2 (p < 0.001); latencies in Block 1 differed also from latencies in Block 3, and in Block 4 (p < 0.001). No other difference was significant, indicating that after Block 2 up to the end of the training the participants' performance remained stable. Conversely, during sham tDCS, a decrease of latencies emerged only at the end of the training, as shown by the only significant difference between Block 1 and Block 4 (p < 0.05).

2.2.2.

Visual exploration

2.2.2.1. E-F test.

Fig. 2B shows that visual exploration improved in the two post-training evaluations, as compared to the baseline, with the reduction of latencies being greater after PPC tDCS. The effect of Condition was significant (F2,18 = 12.3, p < 0.001, pη2 = 0.58). The differences between the baseline, and both post PPC tDCS and post sham tDCS (p < 0.001), and the difference between post PPC tDCS and post sham tDCS were significant (p < 0.05). As for accuracy, the ANOVA showed a significant effect of Condition (F2,18 = 3.82, p < 0.05, pη2 = 0.6): participants scored 92% (SEM= 3) in the baseline, 95% (SEM= 2) post PPC tDCS, and 91% (SEM = 4) post sham tDCS. The differences between post PPC tDCS, and both the baseline and post sham tDCS were significant (p < 0.05); the difference between the two latter conditions was not significant (p = 0.6).

2.2.2.2. Triangles test. As shown in Fig. 2B, latencies were lower after PPC tDCS, as compared with the baseline and the sham stimulation. The effect of condition was significant (F2,18 = 21.42, p < 0.0001, pη2 = 0.7). Responses were faster after PPC tDCS, and sham tDCS, as compared to the baseline

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Fig. 2 – Experiment 1: right hemisphere stimulation. (A) Bimodal exploration training: mean RTs (SEM) by block (1–4, after 8, 16, 24, and 32 min of training), and condition (sham, PPC tDCS). (B) Visual exploration: mean RTs (SEM) by condition (baseline, post sham tDCS, post right PPC tDCS) in the EF, Triangles, and Visual Scanning-TAP Tests. (C) Visuo-spatial orienting: mean RTs (SEM) by condition (as in B) in the Unimodal Visual and Covert Visual Orienting Tests.

(p < 0.001); the difference between post PPC tDCS and post sham tDCS was also significant (p < 0.05). As for accuracy, participants scored 78% (SEM = 4) in the baseline, 91% (SEM = 2) post PPC tDCS, and 88% (SEM = 3) post sham tDCS. The effect of Condition was significant (F2,18 = 12.18, p < 0.001, pη2 = 0.58). The differences between the baseline and both post PPC tDCS conditions were significant (p < 0.001). The difference between the two post-training evaluations was not significant (p = 0.4).

2.2.2.3. Visual Scanning Test—TAP. Fig. 2B shows faster response latencies after training. The effect of Condition was significant (F2,18 = 6.15, p < 0.01, pη2 = 0.4). The differences between the baseline, and both the post PPC tDCS (p < 0.001) and the post sham tDCS (p < 0.05) were significant. The difference between the post PPC and the post sham tDCS conditions approached significance (p = 0.06). As for accuracy, the participants' scores were 84% (SEM = 3) correct in the baseline, 90% (SEM = 3) after PPC tDCS, and 87% (SEM = 3) after sham tDCS. The effect of Condition (F2,18 = 4.18, p < 0.05, pη2 = 0.34) was

significant. The difference between post PPC tDCS and the baseline (p < 0.05) was significant, with no other difference attaining the significance level.

2.2.3.

Visuo-spatial orienting

2.2.3.1. Unimodal Visual Test. Fig. 2C shows lower latencies in the post PPC tDCS session, as compared to both the baseline, and post sham tDCS. The main effects of Condition (F2,18 = 6.92, p < 0.01, pη2 = 0.43), and Side (F1,9 = 9.28, p < 0.05, pη2 = 0.5) were significant, while the Condition by Side interaction was not significant (F2,18 = 0.4, p = 0.9, pη2 = 0.01). Responses to right-sided stimuli [244 ms (SEM = 19)] were faster than those to left-sided stimuli [253 ms (SEM = 18)]. This side difference is not shown in Fig. 2C, as it did not interact with the other main factor. Latencies decreased in the post PPC tDCS evaluation, as compared to both the baseline, and the post sham tDCS (p < 0.01), with no significant difference between the two latter conditions (p = 0.8).

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2.2.3.2. Covert Visual Orienting—TAP. Fig. 2C shows overall lower latencies after PPC tDCS, for both valid and invalid cues, and an overall validity effect. The main effects of Cue (F1,9 = 86.6, p < 0.001, pη2 = 0.9), and of Condition (F2,18 = 6.28, p < 0.01, pη2 = 0.4) were significant. The main effect of Side (F1,9 = 3.73, p = 0.09, pη2 = 0.29), not shown in Fig. 2C, was not significant. The Condition by Side (F2,18 = 2.14, p = 0.15, pη2 = 0.2), Condition by Cue (F2,18 = 1.34, p = 0.29, pη2 = 0.1), Side by Cue (F2,18 = 4.15, p = 0.08, pη2 = 0.3), and Condition by Side by Cue (F2,18 = 0.96, p = 0.4, pη2 = 0.1) interactions were not significant. The differences between the post PPC tDCS [247 ms (SEM= 11)], and both the baseline [280 ms (SEM= 9)] (p < 0.05) and the post sham tDCS [291 ms, (SEM= 15)] (p < 0.01) were significant. No other effect reached the significance level. 2.2.4.

Control tests

In the Working Memory Test the participants' latencies were 560 ms (SEM= 22) in the baseline, 578 ms (SEM= 51) in the post PPC tDCS, and 550 ms (SEM = 39) in the post sham tDCS. No significant difference among conditions was found (F2,18 = 0.2, p = 0.8, pη2 = 0.02).

In the Alertness Test the participants' latencies were 248 ms (SEM= 12) in the baseline, 246 ms (SEM= 16) in the post PPC tDCS, and 242 ms (SE = 13) in the post sham tDCS. No significant difference among conditions was found (F2,18 = 0.33, p = 0.7, pη2 = 0.04).

2.3.

Experiment 2: left hemisphere stimulation

2.3.1.

Bimodal exploration training

Fig. 3A shows a gradual decrease of response latencies throughout the four blocks of training. The main effects of Block (F3,27 = 5.18, p < 0.001, pη2 = 0.37), and of Side (F1,9 = 8.14, p < 0.05, pη2 = 0.47) were significant. A decrease of latencies took place only at the end of the training during both left PPC tDCS and sham tDCS. The difference between Block 4 [249 ms (SEM = 24)] and Block 1 [291 ms (SEM = 20)] was significant (p < 0.05), with no other difference attaining the significance level. Latencies to right-sided stimuli [267 ms (SEM= 22)] were faster than those to left-sided stimuli [275 ms (SEM= 24)] (not shown in Fig. 3A). The main effect of Condition (F1,9 = 1.74, p = 0.2, pη2 = 0.16), and the Condition by Block (F1,9 = 0.37, p = 0.8,

Fig. 3 – Experiment 2: Left hemisphere stimulation. A/B/C, see legend to Fig. 2.

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pη2 = 0.04), Condition by Side (F1,9 = 0.05, p = 0.8, pη2 = 0.01), Block by Side (F1,9 = 0.35, p = 0.8, pη2 = 0.04), and Condition by Block by Side (F 1,9 = 0.33, p = 0.8, pη2 = 0.03) interactions were not significant.

2.3.2.

Visual exploration

2.3.2.1. E-F test. Fig. 3B shows a faster visual search after training, in both the post PPC and the post sham tDCS conditions. The effect of Condition was significant (F2,18 = 4.4, p < 0.05, pη2 = 0.33). The differences between the baseline and both the post PPC tDCS and the post sham tDCS conditions were significant (p < 0.05); RTs in the two latter conditions were comparable (p = 0.08). As for accuracy, the participants scores were 90% (SEM = 6) in the baseline, 92% (SEM = 5) in the post PPC tDCS, and 88% (SEM = 10) in the post sham tDCS. No differences among the three conditions were found (F2,18 = 2.29, p = 0.5, pη2 = 0.2). 2.3.2.2. Triangles test. Fig. 3B shows lower latencies after training in both post tDCS conditions. The effect of Condition was significant (F2,18 = 3.74, p < 0.05, pη2 = 0.29). The differences between the baseline, and both post PPC tDCS, and post sham tDCS were significant (p < 0.05), with no difference between the two post-training evaluations (p = 0.7). The participants' accuracy scores were 90% (SEM = 4) correct in the baseline, 95% (SEM = 3) after PPC tDCS, and 91% (SEM = 4) after sham tDCS. The effect of Condition (F2,18 = 5.31, p < 0.02, pη2 = 0.37) was significant. The differences between the post PPC tDCS condition, and both the baseline (p < 0.01) and the post sham tDCS (p < 0.05) conditions were significant.

2.3.4.

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Control tests

In the Working Memory Test the participants' latencies were 531 ms (SEM = 29) in the baseline, 512 ms (SEM= 39) post PPC tDCS, and 520 ms (SEM = 43) post sham tDCS. No significant difference among conditions (F2,18 = 0.04, p = 0.9, pη2 = 0.01) was found. In the Alertness Test the participants' latencies were 207 ms (SEM= 9) in the baseline, 223 ms (SEM= 12) post PPC tDCS, and 225 ms (SEM= 13) post sham tDCS. No significant difference among conditions (F2,18 = 0.23, p = 0.8, pη2 = 0.17) was found.

2.4. Hemispheric asymmetry: comparing the effect of right versus left PPC tDCS The results reported above suggest that stimulation of the right PPC has specific effects on bimodal exploration training, as well as on visual exploration and visuo-spatial orienting. Left PPC tDCS has no specific effects, with the exception of visual scanning (with a 4% accuracy increase in the Triangles Test). We assessed this possible hemispheric asymmetry by comparing directly the mean benefit induced by right and left PPC tDCS. To this aim, we compared the differences in latencies (Δ reaction times: ΔRTs) between sham and real tDCS (ΔRTs = RTs sham tDCS − RTs PPC tDCS) found for right (Experiment 1) and left (Experiment 2) hemisphere stimulation.

2.4.1.

Bimodal training

2.3.2.3. Visual Scanning Test—TAP. Fig. 3B shows a decrease of latencies after training. The effect of Condition (F2,18 = 9.98, p < 0.001, pη2 = 0.5) was significant. The differences between the baseline, and both the post PPC (p < 0.001), and the post sham (p < 0.05) tDCS conditions were significant, with no other difference attaining the significance level. The accuracy scores were 88% (SEM= 2) correct in the baseline, 90% (SEM= 3) in the post PPC, and 92% (SEM= 2) in the post sham conditions. The effect of Condition (F2,18 = 1.59, p = 0.2, pη2 = 0.18) was not significant.

Fig. 4A shows that right PPC tDCS induced a greater benefit (overall 32 ms, averaged across the four blocks), than left PPC tDCS (14 ms). Mean ΔRTs (sham–real PPC tDCS) to bimodal stimuli throughout the training were analysed by a repeated measures ANOVA with a between-subjects main factor (Hemisphere: right PPC tDCS, left PPC tDCS), and two withinsubjects main factors (Block: 1–2–3–4, and Side: left-sided, right-sided stimuli). The effect of the Hemisphere main factor was significant (F1,18 = 4.82 p < 0.05, pη2 = 0.44). The main effects of Block (F3,54 = 2.59 p = 0.07, pη2 = 0.13), and of Side (F1,18 = 1.56, p = 0.2, pη2 = 0.07), as well as the Hemisphere by Block (F3,54 = 1.17, p = 0.33, pη2 = 0.06), Hemisphere by Side (F1,18 = 1.87, p = 0.18, pη2 = 0.09), Block by Side (F3,54 = 1.92, p = 0.14, pη2 = 0.08), and Hemisphere by Block by Side (F3,54 = 0.48, p = 0.7, pη2 = 0.1) interactions were not significant.

2.3.3.

2.4.2.

Visuo-spatial orienting

2.3.3.1. Unimodal Visual Test. Fig. 3C shows the participants' latencies. The main factors of Condition (F2,18 = 0.79, p = 0.5, pη2 = 0.1), and of Side (F1,9 = 3.84, p = 0.08, pη2 = 0.3) (not shown in Fig. 3C), as well as the Condition by Side (F2,18 = 0.32, p = 0.7, pη2 = 0.03) interaction were not significant. 2.3.3.2. Covert Visual Orienting—TAP. Fig. 3C shows lower latencies for valid cues. The main effect of Cue (F1,9 = 72.95, p < 0.001, pη2 = 0.89) was significant. The main effects of Condition (F2,18 = 1.84, p = 0.2, pη2 = 0.17), and Side (F1,9 = 4.18, p = 0.08, pη2 = 0.32) (not shown in Fig. 3C) were not significant. The Condition by Side (F2,18 = 0.1, p = 0.9, pη2 = 0.1), Condition by Cue (F2,18 = 0.02, p = 0.9, pη2 = 0.01), Side by Cue (F1,9 = 1.75, p = 0.1, pη2 = 0.2), and Condition by Side by Cue (F2,18 = 0.36, p = 0.7, pη2 = 0.04) interactions were not significant.

Visual exploration

The mean ΔRTs (sham–real PPC tDCS) after right and left PPC tDCS are shown in Fig. 4B. The mean ΔRTs after right and left PPC tDCS for the EF Test, the Triangles Test, and the Visual Scanning Test (TAP) were compared by unpaired t-tests. Significant differences were found for the EF test (t18 = 2.67, p < 0.05), and for the Triangles Test (t18 = 2.51, p < 0.05), with right PPC tDCS bringing about a greater benefit. In the Visual Scanning Test (TAP) no significant differences were found (t18 = 1.03, p = 0.3).

2.4.3.

Visuo-spatial orienting

Fig. 4C shows the average ΔRTs for right and left PPC tDCS, with a greater reduction of latencies after right PPC tDCS with respect to its sham condition, for both the Unimodal Visual and the Covert Visual Orienting tests. For the Unimodal Visual Test, mean ΔRTs were analysed by a repeated measures ANOVA with a between-subjects main

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pη2 = 0.07), Side by Cue (F1,18 = 0.81, p = 0.38, pη2 = 0.08), and Hemisphere by Side by Cue (F1,18 = 0.35, p = 0.56, pη2 = 0.02) interactions. Finally, the difference between right- and left-hemisphere stimulation was also assessed by comparing with a paired t-test the average size effects (i.e., partial Eta Squared, pη2) found in the two experiments for the main effect of tDCS Condition in the bimodal training and the following tests: EF test, Triangles test, Unimodal Visual Test, Covert Visual Orienting Test. The pη2 measures the degree of association between an effect and the dependent variable, namely the proportion of the total variance that is attributable to a main factor (Cohen, 1973). For the right hemisphere (Experiment 1), the mean pη2 was 0.5 (range 0.2–0.7), for the left hemisphere (Experiment 2) the mean pη2 was 0.2 (range 0.1–0.5). The difference was significant (t(4) = 3.45, p < 0.05), indicating a greater overall effect of right PPC tDCS.

2.5. Experiment 3: effect of parietal tDCS alone on visual search A control experiment was run, in order to explore whether PPC tDCS by itself, without any behavioural training, was capable of modulating visual search. To this aim we used the EF test. Fig. 5 shows the average latencies and accuracy scores of the participants' performances in the four assessed conditions of the EF Test (baseline, post sham tDCS, post right PPC tDCS, and post left PPC tDCS). Right PPC tDCS brought about a reduction of latencies. Latency and accuracy scores were analysed by two repeated measures one-way ANOVAs. The effect of Condition was significant (F3,33 = 3.23, p < 0.05, pη2 = 0.37). The participants' performance in the anodal post Right PPC tDCS differed from the other conditions (p < 0.05), with no other comparison being significant. As for the accuracy scores, no differences among conditions (F3,33 = 0.81, p = 0.49, pη2 = 0.2) were found.

3. Fig. 4 – Hemispheric asymmetries of PPC tDCS. Mean differences in RTs (SEM) between sham and real tDCS (ΔRTs: sham—PPC tDCS), by hemispheric side of stimulation (left, right) in the bimodal exploration training (A), Visual exploration Test (B), and Visuo-spatial orienting Test (C). factor (Hemisphere: right PPC tDCS, left PPC tDCS), and one within-subjects main factor (Side: left-sided, right-sided stimuli). The main effect of Hemisphere was significant (F 1,18 = 7.37, p < 0.01, pη2 = 0.52). The main effect of Side (F1,18 = 0.09, p = 0.8), and the Hemisphere by Side Interaction (F1,18 = 0.05, p = 0.8, pη2 = 0.02) were not significant (not shown in Fig. 3C). For the Covert Visual Orienting Test of the TAP, mean ΔRTs were analysed by a repeated measures ANOVA with the Hemisphere and Side main factors already used in the previous analysis, and the Cue main factor (valid, invalid). The main effect of Hemisphere was significant (F1,18 = 8.22, p < 0.01, pη2 = 0.6). The main effects of Side (F1,18 = 1.56, p = 0.2, pη2 = 0.09), and of Cue (F1,18 = 1.54, p = 0.2, pη2 = 0.06) were not significant, as well as the Hemisphere by Side (F1,18 = 2.04, p = 0.17, pη2 = 0.1), Hemisphere by Cue (F1,18 = 0.95, p = 0.34,

Discussion

The brain polarization of the PPC of the right hemisphere, combined with intensive multisensory stimulation, improves

Fig. 5 – Experiment 3: effect of PPC tDCS alone on visual search in the EF test. Mean RTs (SEM) by condition (baseline, post sham tDCS, post right PPC tDCS, post left PPC tDCS).

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performance during a bimodal audio-visual exploratory task. So far, non-invasive cortical stimulation during visuo-spatial tasks has been conducted predominantly with TMS (Bolognini et al., 2009a; Fierro et al., 2000, 2006; Muggleton et al., 2008; Ruff et al., 2008, 2009). This is the first demonstration that anodalexcitatory tDCS delivered over the right PPC improves visual search in neurologically unimpaired participants. The strategy used here is that of facilitating directly visual exploration during practice by stimulating the right PPC, which is known to be involved in visual search and visuo-spatial functions (Corbetta et al., 2008; Halligan et al., 2003; Vallar, 2001). In particular, a first finding concerns the time course of the tDCS effects during training. A speed up of responses to audiovisual stimuli during training emerges early, after the first 8 min of right PPC stimulation. This effect is critically dependent on the side of stimulation, being specific for the right PPC. Indeed, when the left PPC is targeted by tDCS, as well as during the sham tDCS stimulation, performance improves later in training, only at its end, after 30 min of practice. This improvement of performance during the bimodal stimulation of the visual field is in line with previous findings that this procedure improves visual exploration in brain-damaged patients with visual halffield deficits (Bolognini et al., 2005b). Importantly, combining anodal tDCS over the right PPC during practice boosts responses to bimodal stimuli during the first minutes of training. This finding is in line with previous evidence from our lab showing that anodal parietal tDCS can enhance spatial orienting to both unisensory (visual and auditory) and crossmodal (audiovisual) stimuli (Bolognini et al., 2010). However, since we did not directly monitor the strategy used by our participants to search for the bimodal targets during the training (for instance, by recording eye-movements), our current conclusion is that an improvement of multisensoryguided visual field scanning takes place, due to the combined use of multisensory stimulation and parietal tDCS. Additionally, such an improvement is not merely trainingspecific: it extends across all the tests assessing visual exploration with unimodal stimulus presentation, where the additional benefit induced by right PPC tDCS is again found. This effect is further confirmed by Experiment 3, where we show a modulation of visual search by anodal tDCS delivered to the right PPC, also when given alone, without any behavioural training. In line with the present evidence, previous fMRI and TMS results (Corbetta et al., 1995; Ellison et al., 2003; Leonards et al., 2000; Muggleton et al., 2008; Ruff et al., 2008, 2009; Shulman et al., 2001) show that a neural network comprising the PPC supports visual search, with a main contribution from the right cerebral hemisphere. Our results indicate that coupling anodal tDCS with a visual search training can enhance the response of a neural network–in this study the attentional system (Corbetta et al., 2008; Halligan et al., 2003; Posner, 1980)–to a kind of stimulation (the bimodal training), by simultaneously priming it with a different form of stimulation (tDCS). In line with the present findings, in one study (Antal et al., 2004), the excitability of MT+/V5 and M1 was increased or decreased by anodal or cathodal tDCS, while participants were learning a visuallyguided manual tracking task. Accuracy of tracking movements was increased by anodal stimulation, with cathodal stimulation being ineffective. Interestingly, the positive effect of anodal tDCS was restricted to the learning phase, suggesting a specific

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effect of the stimulation. Similar results for language learning are on record (Floel et al., 2008). The second finding is that modulating the activity of the right PPC by tDCS has the additional effect of improving visuo-spatial orienting. The effect is likely to be more closely related to tDCS alone, as no specific repeated training sessions for this behaviour were given. Indeed, an improvement of visuo-spatial orienting, as assessed at the end of the intervention (Unimodal Visual Test; Covert Visual Orienting Test from the TAP, Zimmerman and Fimm, 1992; Zoccolotti et al., 1994) was selectively induced by right PPC tDCS, with no effects of the bimodal exploration training only with sham tDCS or left PPC tDCS. In particular, enhancing the excitability of the right PPC speeds up covert orienting to visual stimuli (Unimodal Visual Test). The improvement involves both visual half-fields, contralateral and ipsilateral to the side of the stimulation. This result is in line with the view that the right hemisphere (particularly, the right PPC) can direct attention to both the left and the right sides of space, whereas the left hemisphere directs attention to the right side only. Neuroimaging studies have shown increased right posterior parietal activations when participants attend to both the contralateral left, and the ipsilateral right visual halffields (Corbetta et al., 1993, 2000). Conversely, right braindamaged patients with left unilateral spatial neglect show an overall slowness of responses to visual targets presented in both half-fields, with reaction times in the left visual field being slowest overall (Bartolomeo and Chokron, 1999). Moreover, right parietal tDCS improves also performance in the Covert Visual Orienting Test from the TAP (Zoccolotti et al., 1994). In this paradigm, while keeping central fixation, participants have to respond to the appearance of peripheral targets, that can be preceded by a cue, i.e. a central arrow pointing to the side where the target stimulus is going to appear (“valid” trial), or to the opposite location (“invalid” trial). Therefore, for invalid targets, participants must first disengage attention from the cued location, then move it, and finally engage attention at the uncued location, where the target appears. With this paradigm, studies in right-brain-damaged patients have highlighted the key role of the PPC in the covert orienting of attention: lesions involving the right posterior-superior parietal cortex (Posner et al., 1984), and the right temporo-parietal junction (Friedrich et al., 1998) disrupt the ability to disengage attention from ipsilesional stimuli. The finding that the right PPC tDCS affects responses to both valid and invalid trials in both visual half-fields suggests that the right PPC is important for the visual orientation of attention, for all of its component processes (disengaging, orienting, and engaging) (Posner et al., 1984). Noteworthy, neither enhancing the activity of left PPC by tDCS, nor the bimodal exploration training alone, improves orienting of visual attention. These results can be explained by a more specific effect of right PPC stimulation on spatial orienting, which occurs independent of the visual search practice. In this regard, one recent tDCS study has shown that unilateral stimulation of the PPC modulates bidirectionally the performance of neurologically unimpaired participants in a visual dot detection task, depending on both the side of stimulation and current polarity (Schweid et al., 2008; Sparing et al., 2009). Particularly, anodal tDCS improves the detection of contralateral stimuli, while cathodal tDSC ameliorates the

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detection of ipsilateral stimuli, and worsens the detection of contralateral stimuli in a bilateral stimulation condition, with an extinction-like pattern. Importantly, however, no hemispheric asymmetries were found, at variance with the present study. Additionally, in right-brain-damaged patients with left visuo-spatial neglect, both cathodal inhibitory tDCS applied over the undamaged left PPC, and anodal facilitatory tDCS applied over the damaged right PPC, reduce the patients' rightward bias in line bisection. Consistent with these findings in humans, cathodal tDCS applied to the right visuo-parietal cortex (the posterior middle suprasylvian cortex) of the cat, is able to reduce detection as well as orienting responses to static visual targets, presented in the contralateral left side of space; the study did not include however cathodal tDCS to the left hemisphere (Schweid et al., 2008). The finding that right PPC tDCS was able to modulate the participants' performance in two different types of tasks (i.e., visual search, covert visual orienting) leads to consider the possibility that neurofunctional mechanisms shared by both tasks were affected. Noteworthy, the PPC (and, in particular, the inferior parietal lobule) is involved in salience mapping, namely the generation of a search template of task-relevant objects and locations (Buschman and Miller, 2007; Egner et al., 2008; Rafal, 2006). Indeed, a general function of the PPC is the selection of sensory information for action in extra-personal space, and their transformation into representations to be used by the motor systems (Rafal, 2006). The suggestion has been made that the PPC mediates a top-down search template entailing a combination of spatial and feature-based information about a searched target (Corbetta et al., 1995, 2000, 2008; Egner et al., 2008). This role fits also well with the view that the PPC integrates multimodal information, in order to construct representations of the locations of motivationally relevant stimuli in extrapersonal space (Andersen, 1997; Macaluso and Driver, 2003). PPC-based mechanisms of this sort may support both visual search and visual orienting. Finally, the effects of tDCS were absent for tasks assessing functions such as Alertness and Working Memory, which are closely linked to neither the ongoing practice nor to the stimulated PPC. Working memory, which in the present study was assessed by a task from the TAP (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994), involves a wide cortical neural circuitry: the prefrontal cortex plays a major role in verbal working memory, while the PPC is more involved in visuospatial working memory (Knudsen, 2007). Alertness, also assessed by a subtest from the TAP (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994), involves a widespread set of regions, including major nodes in the frontal lobes, thalamic and brain-stem structures (Posner, 2008; Sturm and Willmes, 2001). Overall, the present study shows that combining a noninvasive technique of neuro-modulation, such as tDCS, with behavioural interventions, represents a powerful method to augment the response of the targeted system to the behavioural practice (Bolognini et al., 2009b; Fregni and PascualLeone, 2007). The basic idea of this approach is that the ongoing state of the cortex at the time of the behavioural training bears a relationship with its effectiveness: namely, cortical stimulation may maximize the effects induced by practice. Anodal tDCS cortex can elicit prolonged cortical

excitability increases (Nitsche and Paulus, 2000, 2001), by modulating the resting membrane threshold; these effects facilitate (in the case of anodal stimulation) processing in the stimulated area. The acquisition of new skills is also accompanied by changes in neuronal activity and excitability (Muellbacher et al., 2002; Karni et al., 1998), which, in turn, may reflect modifications in synaptic strength (Hess and Donoghue, 1996; Rioult-Pedotti et al., 1998). In sum, anodal tDCS appears to have the potential to improve learning. The present findings are encouraging for future intervention in brain-damaged patients with sensory and spatial disorders. The uninjured nervous tissue may be particularly receptive to modulation by various external manipulations, including behavioural training and neuromodulation approaches, such as non-invasive brain stimulation. Both strategies, behavioural therapy and cortical stimulation, have some similarities in their mechanisms of action, as both induce similar changes in cortical excitability, associated with long-lasting after-effects (Bolognini et al., 2009b). Accordingly, their combination might be more beneficial than their use alone. In addition, it appears that PPC tDCS might jointly affect other functions (i.e., visuo-spatial orienting), not directly trained, but dependent on the tDCS modulation of the stimulated area. In sum, the present results widen the possibilities for treating cognitive deficits by using DC polarization. Also possible limitations of the present study should be taken into consideration. A first matter of concern may be the running of two separate experiments on the right and on the left hemisphere in different participants. However, we used a bimodal training paradigm, and we wished to avoid submitting neurologically unimpaired participants to multiple training sessions, in order to prevent possible ceiling effects. A second note of caution concerns the size of the participants’ sample, that might have been larger, as in other studies using tDCS to assess cognitive function (de Vries et al., 2009; Floel et al., 2008; Sparing et al., 2009). The observed hemispheric (i.e., left vs. right) effects, however, are quite consistent, notwithstanding the comparatively small number of participants, as also shown by the analysis on the size effects. Finally, this line of research may be developed by comparing the effects of cathodal vs. anodal tDCS, in order to assess which of these stimulations is more effective for improving visual search and visuo-spatial orienting.

4.

Experimental procedures

4.1. Experiments 1 and 2: effects of parietal tDCS combined with the multisensory visual field exploration 4.1.1.

Study design

We conducted two double-blind, randomized, sham-controlled experiments in healthy volunteers to determine the effect of a single-session of PPC tDCS, combined with a bimodal exploration training (Bolognini et al., 2005b), on visual search and visuospatial attention. The study conformed to the ethical standards of the Declaration of Helsinki (BMJ 1991; 302: 1194), and was approved by the Ethical Committee of the Italian Auxological Institute, Milan, Italy. We first assessed the effects of

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stimulating the right PPC (Experiment 1), and, second, the effects of left hemisphere PPC stimulation (Experiment 2).

4.1.2.

Participants

Twenty neurologically unimpaired right-handed participants took part in the study: ten in Experiment 1 (8 females; mean age: 24, range: 21–26), and ten in Experiment 2 (8 females; mean age: 23, range 20–25). Handedness was assessed by the Edinburgh Inventory (Oldfield, 1971). All participants were undergraduate students; they were naïve as to the experimental procedure, and the purpose of the study. The study coordinator explained the risk/benefits of the study and screened interested individuals for eligibility, according to standard international guidelines (Poreisz et al., 2007). Participants entered the study if they fulfilled the following criteria: (1) no history or clinical evidence of disease, including neurological and psychiatric disorders; (2) no history of substance abuse or dependence; (3) no use of central nervous system-effective medication. All participants gave their written informed consent to participate in the study. The study was performed in the Neuropsychological Laboratory of the Italian Auxological Institute, Milan, Italy.

4.1.3.

Bimodal exploration training

The bimodal visual field training was performed on a 2 m × 2 m training board. The board comprised a central 1 m × 2 m panel, and two 50 cm × 2 m wings that could be moved inwards (see Fig. 1). The right and the left hand-side wings of the board were positioned 45° inwards, as shown in Fig. 1. In the board, 48 red light-emitting diodes (LED, diameter 1 cm, luminance 90 cd m2) were distributed in six horizontal rows, with eight lights per row. Forty-eight piezoelectric loudspeakers (0.4 W, 8Ω) were located below each light, with the sound being generated by a PC controlled white-noise generator (80 dB, duration 100 ms). LED were separated by 25 cm. Participants sat at a 1.2 m distance from the central part of the board, so that their visual fields were filled out by the board. During the training, cross-modal visual–auditory stimuli were presented, namely, a sound coupled to the visual target, originating from one out of the 48 positions (separated by 12° of visual angle) in the board. The sound was always presented simultaneously to the light, and in the same position of the visual stimulus. Previous studies have shown that the maximal cross-modal interaction effects, enhancing visual responses, are induced by spatially and temporally coincident audio-visual stimuli (Bolognini et al., 2005a). During training, participants were required to look at the fixation point, a yellow star (2°) located in the centre of the apparatus, and to explore the visual field, in order to detect the presence of the visual stimulus, which was shown for 100 ms. The inter-stimulus interval varied randomly between 1 and 3 s. The participants' task was to report each presented stimulus by pressing with their index finger the right button of the PC wireless mouse. False positive responses were recorded. The execution of oculomotor exploration by each participant was monitored visually by the experimenter, standing near the apparatus. The training included four blocks of 96 visual–auditory trials (two trials for each spatial location), each lasting about 7 min, separated by 3–4 min breaks. The duration of the

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training session was about 30 min. No participant committed false positive or omission errors. Stimuli presentation and responses recording were under computer control (E-prime Software, Psychology Software Tools Inc., www.psychotoolbox.org) (see Fig. 1).

4.1.4.

Transcranial Direct Current Stimulation (tDCS)

During the bimodal training, participants received either sham or PPC tDCS. A pair of surface sponge electrodes (7 × 5: 35 cm2) was soaked in saline and placed at target areas of stimulation. Rubber bandages were used to hold the electrodes in place for the duration of stimulation. For PPC stimulation the excitability enhancing anodal electrode was placed over P4 (Experiment 1), or over P3 (Experiment 2), according to the international 10-20 system for EEG electrode placement; the cathodal electrode was placed over the contralateral deltoid muscle. The advantage of the extracephalic montage is to avoid the confounding effect of the reference electrode (Nitsche et al., 2003). Positioning the reference electrode on the body might result in brain stem stimulation. However, the direct functional effects of tDCS are restricted to the area under the electrode, since the electrical field strength is fairly homogeneous under the electrode, but decreases very rapidly at a distance from it (Miranda et al., 2006; Wagner et al., 2007). This montage has been successfully used in previous tDCS studies (Ferrucci et al., 2008; Monti et al., 2008; Priori et al., 2008; Stone and Tesche, 2009). Stimulation intensity was set at 2 mA, lasting for 30 min. This duration of stimulation was adequate to induce a lasting effect: with only 13 min of 1 mA tDCS stimulation, the effects on neural excitability outlast the period of stimulation by up to 90 min (Nitsche and Paulus, 2001). For sham stimulation, the electrodes were placed in the same positions as PPC tDCS, but the stimulator was turned off after 30 s of stimulation. This ensured that participants felt the initial itching sensation at the beginning of tDCS, prevented any effective modulation of cortical excitability by tDCS, and allowed for a successful blinding of participants for the real vs. sham stimulation condition (Gandiga et al., 2006). Additionally, the device used was particularly reliable for double-blind studies (http://www.eldith.de/products/stimulator): during the sham stimulation, a switch was prearranged to interrupt the electrical current while maintaining the “ON” display, and showing the stimulation parameters throughout the procedure to both the experimenter and the participant. Additionally, after the completion of the study, participants were asked to identify in which session they had received the “real” cortical stimulation (tDCS). The instructions given to the participants were identical for the two sessions (“real” and “sham” tDCS). The experienced discomfort, mostly due to the headband, was negligible in all participants, who, as specifically required at the end of each session, were unable to distinguish between anodal tDCS and sham.

4.1.5.

Tasks

Before and immediately after the training, the participants underwent an examination of visual exploration and visuospatial attention by using different outcomes (see below). In addition, two control tests were administered, in order to

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assess whether PPC tDCS was associated with non-specific cognitive changes, unrelated to the bimodal training.

The scores were the number of correct counts for the 20 arrays (range 0–20) and the searching times (latencies, ms).

4.1.5.1. Visual exploration.

4.1.5.3. Visual Scanning Test—TAP. Visual scanning was assessed by a subtest from the TAP (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994). The target pattern, a square open on the upper side, had to be detected in a 5 × 5 arrangement of squares with openings on other sides, presented on a computer PC monitor (IBM ThinkVision L190 19″). The participants’ task was to scan the pattern, and to press on a response box a right-sided response key with the right index finger, when the target was present, and a left-sided response key with the left index finger when no target was detected. Participants were required to respond as fast and as accurately as possible. One hundred trials were given in a random fixed order, with the target being present in 50 trials. Correct responses and time for visual scanning were recorded.

Three different tests were used to evaluate the participants' ability to scan the visual field, searching for a target presented among distracters. In these tests, participants were required to explore the visual field by using eye movements.

4.1.5.2. Visual search test. This test included two subtests: the E-F Test and the Triangles Test (Bolognini et al., 2005b). In both subtests, the visual search tasks were shown on slides with a projector (SONY-VPL-ES4 Projector). The stimulus arrays were displayed at a distance of 1.2 m from the participants’ eyes and subtended 35° ×28° of visual angle. Stimuli and responses were generated and measured by E-Prime software, running on an IBM compatible Pentium PC. Each trial began with a fixation red cross (1 s), which was followed by the presentation of the search array. During both subtests, participants were required to scan the visual field, searching for targets embedded among distracters (targets and distracters had the same size, i.e., 5 ×5 cm). The search array was shown on the screen until participants made a “present” or “absent” response by means of a key press on the keyboard, using their right index finger. No time limits were given, with participants being instructed to reply as accurately and as faster as possible. Following a response, a blank screen was presented for 1 s prior to the start of the next trial. 4.1.5.2.1. E-F test. Each array included 21 stimuli, distributed at random over it. The stimuli were green letters, projected on a black background. Twenty trials were presented: 16 trials in which the target was present, and four in which the target was absent. Participants were instructed to fixate the red cross located in the centre of the slide (i.e., the fixation point, shown for 1 s), and to search, after the disappearance of the cross, for a single target (i.e., the green letter “F”) intermingled among distracters (i.e., the green letters “E”). Participants had received instructions to report the presence of the target. Using their right index finger, participants pressed a “yes” key response of the keyboard (the leftward arrow) if the target was present, and a “no” key response (the rightward arrow) if it was absent. The score was the number of correctly reported targets (range 0–16). Search times (latencies, ms) were recorded. False alarms, namely the erroneous report of a target in an array where it was absent, were also recorded. 4.1.5.2.2. Triangles test. Each array included 21 stimuli, distributed at random on a black background. Different shapes of the same size (4 cm × 4 cm) were used as stimuli: yellow squares as distracters and yellow triangles as targets. The number of targets presented in the stimulus array ranged from 0 to 13 (Target/Distracters Ratio: 0–60%). As the number of targets increased, the number of distracters decreased, so that each stimulus array always included 21 stimuli. Twenty trials were given. Participants were instructed to fixate the red cross presented in the centre of the slide, namely the fixation point shown for 1 s. After the disappearance of the cross, participants visually scanned the stimulus array, searching for the total number of targets. Participants had received instructions to press the space bar of the keyboard when they had finished to inspect the stimulus array. Immediately afterwards they were asked to report verbally the number of detected targets.

4.1.5.4. Visuo-spatial orienting. Two tests assessed the orientation of visuo spatial attention. The tasks differed from the visual scanning tests (Visual Search Tests: E-F and Triangles; Visual Scanning Test – TAP) in that only single targets were presented, without distracters. 4.1.5.4.1. Unimodal visual test. The same apparatus employed for the training procedure (see above, Bimodal Exploration Training) was used. A visual target was presented for 100 ms in one of 48 different spatial positions (12 spatial locations in each quadrant of the visual field). For each of the 48 spatial positions, two trials were given, for a total of 24 trials in each of the four quadrants. Each target stimulus was shown for 100 ms. In 24 catch trials no visual stimulus was given, in order to assess false positive responses. The total number of trials amounted to 120 (96 with a target stimulus, and 24 catch). Participants received instructions to look at the central fixation point (a cross located at the centre of the apparatus), and to press, using the right index finger, the right button of the PC wireless mouse, when they detected a target. The scores were the number of correctly detected targets and the latencies, by the left- and the right-sided position of the target, with respect to the mid-sagittal plane of the participants’ body. 4.1.5.4.2. Covert visual orienting test—TAP. This task was a subtest of the TAP (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994), assessing the covert orientation of attention (Posner, 1980). The covert shift of attention is considered to be a process preliminary to eye movements, by which a new visual target is selected. The test consisted in a simple reaction time task with a preceding cue–i.e., an arrow in the centre of the screen–pointing with high probability to the side where the target stimulus was going to appear (valid cues 80%, invalid cues 20% of the trials). Stimuli were displayed on a computer screen (IBM ThinkVision L190 19"). Participants were asked to respond to the target stimulus by pressing a response key with their right index finger, as quickly as possible. The test included 48 trials for each half-field. Latencies to valid and invalid cues were recorded separately for the left and the right visual half-fields. 4.1.5.5. Control tests 4.1.5.5.1. Working memory test—TAP. This task from the TAP (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994)

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required a continuous control of the flow of information through temporary memory. Numbers (2 s of presentation) were presented centrally on a computer screen (IBM ThinkVision L190 19″), and had to be compared with previously exposed numbers. When a number was repeated within a short (1 s) interval, participants had to press a key on a response box, using their right hand. Sixty trials were presented. The scores were the response latencies (ms), and the number of correct responses (range 0–60).

4.1.5.5.2. Alertness test—TAP. This test from the TAP (Zimmerman and Fimm, 1992; Zoccolotti et al., 1994) was a simple reaction time task with a centrally presented visual test stimulus, displayed on a computer screen (IBM ThinkVision L190 19″). Simple reaction times provide a measure of tonic alertness (van Zomeren and Brouwer, 1994). Participants were instructed to respond as quickly as possible, by pressing a response key with their right index finger, to the appearance of the centrally presented target. The test included 40 trials. Latencies (ms) were recorded.

4.1.6.

Procedure

Participants underwent two training sessions, counterbalanced across participants, and conducted over different days, with the inter-sessions interval being at least 1 week. In each Experiment (1 and 2, either real or sham tDCS), participants underwent approximately 30 min of bimodal training, combined with 30 min of sham or real tDCS stimulation. To evaluate the effects induced by the training associated with tDCS (real vs. sham), all participants underwent a pretraining (baseline), and a post-training evaluation of visual search and visuo-spatial attention; both evaluations were conducted in each experimental session (PPC and sham tDCS sessions). Two pre-training evaluations for each session allowed controlling for the occurrence of learning effects between consecutive evaluations. The pre-training evaluation was performed the day before the experimental session, the post-training evaluation immediately after the end of the training. Tests were given in a randomized order across participants. Each experimental session lasted about 2 h: pre-training evaluation (40 min), training with real/sham tDCS (30 min), post-training evaluation (40 min).

4.2. Experiment 3: effect of parietal tDCS alone on visual search This experiment aimed at exploring the effects of PPC tDCS alone on visual search, as assessed through the E-F Test (see above, for details). Twelve healthy participants, different from those involved in the previous experiments, were recruited for this study (10 females, all right-handed, mean age: 26, range: 22– 27). Participants underwent four counterbalanced experimental sessions: a baseline, during which no stimulation was given, and three tDCS sessions, in which the task was administered after the delivery of 15 min anodal-excitatory tDCS (2 mA) over the right PPC (i.e., right PPC tDCS), the left PPC (i.e., left PPC tDCS), or after the delivery of the placebo stimulation (i.e., sham tDCS, given over the right hemisphere in six participants, and over the left hemisphere in six participants). Stimulation sessions were separated by at least 2 h, in order to avoid carry-over effects and

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to guarantee a sufficient washout of the effects of the previous run (Boggio et al., 2009; Ragert et al., 2008; Sparing et al., 2009). The tDCS procedure was the same as described for the main experiment (see above, Transcranial Direct Current Stimulation (tDCS) for details).

4.3.

Statistical analyses

Statistical analyses were performed using Statistica for Windows (release 6.0, StatSoft). In Experiment 1 (right PPC tDCS plus multisensory training), Experiment 2 (left PPC tDCS plus multisensory training), and Experiment 3 (PPC tDCS alone, without multisensory training), for each test the effects induced by the training were analyzed by repeated measures analyses of variance (ANOVAs) performed on the reaction times (RTs), converted in logarithm (e.g. Ratcliff and Murdock, 1976). Since different participants were recruited in Experiments 1 and 2, the data were analysed separately for each experiment, in order to control for inter-participant variability. When appropriate, multiple comparisons were computed, using the Newman– Keuls test. In addition size effects were computed, by calculating the partial Eta Squared (pη2), which measures the degree of association between an effect and the dependent variable, i.e. the proportion of the total variance that is attributable to a main factor or to an interaction (Cohen, 1973). In each test used in Experiments 1–2, no significant differences between the two baseline conditions (i.e., the two pre-training evaluations, performed before real and sham tDCS sessions), as assessed by t-tests, were found (p > 0.09 for each comparison). Accordingly, the data from the two pretraining evaluations were averaged. The lack of differences between the two baseline conditions ruled out the possibility that a learning effect took place between the two baseline sessions. In sum, we considered three time points (baseline, after real tDCS, and after sham tDCS).

Acknowledgments This research was supported in part by PRIN grants from the Ministero Italiano dell'Università e della Ricerca Scientifica (MIUR) to GV and NB, and from the IRCCS Istituto Auxologico Italiano to GV (Ricerca Corrente). We thank Rocco De Marco, Carlo Toneatto, Silvia Convento, Paola Fortis and Valeria Velardo for assistance.

REFERENCES

Andersen, R.A., 1997. Multimodal integration for the representation of space in the posterior parietal cortex. Philos. Trans. R. Soc. Lond. B Biol. Sci. 352, 1421–1428. Antal, A., Nitsche, M.A., Kincses, T.Z., Kruse, W., Hoffmann, K.P., Paulus, W., 2004. Facilitation of visuo-motor learning by transcranial direct current stimulation of the motor and extrastriate visual areas in humans. Eur. J. Neurosci. 19, 2888–2892. Bartolomeo, P., Chokron, S., 1999. Left unilateral neglect or right hyperattention? Neurology 53, 2023–2027. Boggio, P.S., Fregni, F., Valasek, C., Ellwood, S., Chi, R., Gallate, J., Pascual-Leone, A., Snyder, A., 2009. Temporal lobe cortical

88

BR A IN RE S EA RCH 1 3 49 ( 20 1 0 ) 7 6 –89

electrical stimulation during the encoding and retrieval phase reduces false memories. PLoS ONE 4, e4959. Bolognini, N., Frassinetti, F., Serino, A., Ladavas, E., 2005a. “Acoustical vision” of below threshold stimuli: interaction among spatially converging audiovisual inputs. Exp. Brain Res. 160, 273–282. Bolognini, N., Rasi, F., Coccia, M., Ladavas, E., 2005b. Visual search improvement in hemianopic patients after audio-visual stimulation. Brain 128, 2830–2842. Bolognini, N., Miniussi, C., Savazzi, S., Bricolo, E., Maravita, A., 2009a. TMS modulation of visual and auditory processing in the posterior parietal cortex. Exp. Brain Res. 195, 509–517. Bolognini, N., Pascual-Leone, A., Fregni, F., 2009b. Using non-invasive brain stimulation to augment motor training-induced plasticity. J. Neuroeng. Rehabil. 6, 8. Bolognini, N., Olgiati, E., Rossetti, A., Maravita, A., 2010. Enhancing multisensory spatial orienting by brain polarization of the parietal cortex. Eur. J. Neurosci. 31, 1800–1806. Buschman, T.J., Miller, E.K., 2007. Top-down versus bottom-up control of attention in the prefrontal and posterior parietal cortices. Science 315, 1860–1862. Bushara, K.O., Weeks, R.A., Ishii, K., Catalan, M.J., Tian, B., Rauschecker, J.P., Hallett, M., 1999. Modality-specific frontal and parietal areas for auditory and visual spatial localization in humans. Nat. Neurosci. 2, 759–766. Cohen, J., 1973. Eta-squared and partial Eta-squared in fixed factor anova designs. Educ. Psychol. Meas. 33, 107–112. Corbetta, M., Miezin, F.M., Shulman, G.L., Petersen, S.E., 1993. A PET study of visuospatial attention. J. Neurosci. 13, 1202–1226. Corbetta, M., Shulman, G., Miezin, F., Petersen, S., 1995. Superior parietal cortex activation during spatial attention shifts and visual feature conjunction. Science 270, 802–805. Corbetta, M., Kincade, J.M., Ollinger, J.M., McAvoy, M.P., Shulman, G.L., 2000. Voluntary orienting is dissociated from target detection in human posterior parietal cortex. Nat. Neurosci. 3, 292–297. Corbetta, M., Patel, G., Shulman, G.L., 2008. The reorienting system of the human brain: from environment to theory of mind. Neuron 58, 306–324. de Vries, M.H., Barth, A.C., Maiworm, S., Knecht, S., Zwitserlood, P., Floel, A., 2009. Electrical stimulation of Broca's area enhances implicit learning of an artificial grammar. J. Cogn. Neurosci. Driver, J., Spence, C., 1998. Crossmodal attention. Curr. Opin. Neurobiol. 8, 245–253. Egner, T., Monti, J.M., Trittschuh, E.H., Wieneke, C.A., Hirsch, J., Mesulam, M.M., 2008. Neural integration of top-down spatial and feature-based information in visual search. J. Neurosci. 28, 6141–6151. Ellison, A., Rushworth, M., Walsh, V., 2003. The parietal cortex in visual search: a visuomotor hypothesis. Suppl. Clin. Neurophysiol. 56, 321–330. Ferrucci, R., Mameli, F., Guidi, I., Mrakic-Sposta, S., Vergari, M., Marceglia, S., Cogiamanian, F., Barbieri, S., Scarpini, E., Priori, A., 2008. Transcranial direct current stimulation improves recognition memory in Alzheimer disease. Neurology 71, 493–498. Fierro, B., Brighina, F., Oliveri, M., Piazza, A., La Bua, V., Buffa, D., Bisiach, E., 2000. Contralateral neglect induced by right posterior parietal rTMS in healthy subjects. NeuroReport 11, 1519–1521. Fierro, B., Brighina, F., Giglia, G., Palermo, A., Francolini, M., Scalia, S., 2006. Paired pulse TMS over the right posterior parietal cortex modulates visuospatial perception. J. Neurol. Sci. 247, 144–148. Floel, A., Rosser, N., Michka, O., Knecht, S., Breitenstein, C., 2008. Noninvasive brain stimulation improves language learning. J. Cogn. Neurosci. 20, 1415–1422.

Frassinetti, F., Bolognini, N., Ladavas, E., 2002. Enhancement of visual perception by crossmodal visuo-auditory interaction. Exp. Brain Res. 147, 332–343. Frassinetti, F., Bolognini, N., Bottari, D., Bonora, A., Ladavas, E., 2005. Audiovisual integration in patients with visual deficit. J. Cogn. Neurosci. 17, 1442–1452. Fregni, F., Pascual-Leone, A., 2007. Technology insight: noninvasive brain stimulation in neurology-perspectives on the therapeutic potential of rTMS and tDCS. Nat. Clin. Pract. Neurol. 3, 383–393. Friedrich, F.J., Egly, R., Rafal, R.D., Beck, D., 1998. Spatial attention deficits in humans: a comparison of superior parietal and temporal–parietal junction lesions. Neuropsychology 12, 193–207. Gandiga, P.C., Hummel, F.C., Cohen, L.G., 2006. Transcranial DC stimulation (tDCS): a tool for double-blind sham-controlled clinical studies in brain stimulation. Clin. Neurophysiol. 117, 845–850. Halligan, P.W., Fink, G.R., Marshall, J.C., Vallar, G., 2003. Spatial cognition: evidence from visual neglect. Trends Cogn. Sci. 7, 125–133. Harrington, L.K., Peck, C.K., 1998. Spatial disparity affects visual–auditory interactions in human sensorimotor processing. Exp. Brain Res. 122, 247–252. Hess, G., Donoghue, J.P., 1996. Long-term potentiation and long-term depression of horizontal connections in rat motor cortex. Acta Neurobiol. Exp. (Wars) 56, 397–405. Hughes, H.C., Nelson, M.D., Aronchick, D.M., 1998. Spatial characteristics of visual–auditory summation in human saccades. Vis. Res. 38, 3955–3963. Karni, A., Meyer, G., Rey-Hipolito, C., Jezzard, P., Adams, M.M., Turner, R., Ungerleider, L.G., 1998. The acquisition of skilled motor performance: fast and slow experience-driven changes in primary motor cortex. Proc. Natl. Acad. Sci. USA. 95, 861–868. Knudsen, E.I., 2007. Fundamental components of attention. Annu. Rev. Neurosci. 30, 57–78. Ko, M.H., Han, S.H., Park, S.H., Seo, J.H., Kim, Y.H., 2008. Improvement of visual scanning after DC brain polarization of parietal cortex in stroke patients with spatial neglect. Neurosci. Lett. 448, 171–174. Leonards, U., Sunaert, S., Van Hecke, P., Orban, G., 2000. Attention mechanisms in visual search—an fMRI study. J. Cogn. Neurosci. 12, 61–75. Liebetanz, D., Nitsche, M.A., Tergau, F., Paulus, W., 2002. Pharmacological approach to the mechanisms of transcranial DC-stimulation-induced after-effects of human motor cortex excitability. Brain 125, 2238–2247. Macaluso, E., Driver, J., 2003. Multimodal spatial representations in the human parietal cortex: evidence from functional imaging. Adv. Neurol. 93, 219–233. McDonald, J.J., Teder-Salejarvi, W.A., Hillyard, S.A., 2000. Involuntary orienting to sound improves visual perception. Nature 407, 906–908. Mesulam, M.M., 2002. Functional anatomy of attention and neglect: from neurons to networks. In: Karnath, H.O., Milner, A.D., Vallar, G. (Eds.), The cognitive and neural bases of spatial neglect. Oxford University Press, Oxford, pp. 33–45. Miranda, P.C., Lomarev, M., Hallett, M., 2006. Modeling the current distribution during transcranial direct current stimulation. Clin. Neurophysiol. 117, 1623–1629. Monti, A., Cogiamanian, F., Marceglia, S., Ferrucci, R., Mameli, F., Mrakic-Sposta, S., Vergari, M., Zago, S., Priori, A., 2008. Improved naming after transcranial direct current stimulation in aphasia. J. Neurol. Neurosurg. Psychiatry 79, 451–453. Muellbacher, W., Richards, C., Ziemann, U., Wittenberg, G., Weltz, D., Boroojerdi, B., Cohen, L., Hallett, M., 2002. Improving hand function in chronic stroke. Arch. Neurol. 59, 1278–1282.

BR A IN RE S E A RCH 1 3 49 ( 20 1 0 ) 7 6 –8 9

Muggleton, N.G., Cowey, A., Walsh, V., 2008. The role of the angular gyrus in visual conjunction search investigated using signal detection analysis and transcranial magnetic stimulation. Neuropsychologia 46, 2198–2202. Nitsche, M.A., Paulus, W., 2000. Excitability changes induced in the human motor cortex by weak transcranial direct current stimulation. J. Physiol. 527 (Pt 3), 633–639. Nitsche, M.A., Paulus, W., 2001. Sustained excitability elevations induced by transcranial DC motor cortex stimulation in humans. Neurology 57, 1899–1901. Nitsche, M.A., Liebetanz, D., Antal, A., Lang, N., Tergau, F., Paulus, W., 2003. Modulation of cortical excitability by weak direct current stimulation—technical, safety and functional aspects. Suppl. Clin. Neurophysiol. 56, 255–276. Oldfield, R.C., 1971. The assessment and analysis of handedness: the Edinburgh inventory. Neuropsychologia 9, 97–113. Pardo, J.V., Fox, P.T., Raichle, M.E., 1991. Localization of a human system for sustained attention by positron emission tomography. Nature 349, 61–64. Passamonti, C., Bertini, C., Ladavas, E., 2009. Audio-visual stimulation improves oculomotor patterns in patients with hemianopia. Neuropsychologia 47, 546–555. Poreisz, C., Boros, K., Antal, A., Paulus, W., 2007. Safety aspects of transcranial direct current stimulation concerning healthy subjects and patients. Brain Res. Bull. 72, 208–214. Posner, M.I., 1980. Orienting of attention. Q. J. Exp. Psychol. 32, 3–25. Posner, M.I., 2008. Measuring alertness. Ann. NY Acad. Sci. 1129, 193–199. Posner, M.I., Walker, J.A., Friedrich, F.J., Rafal, R.D., 1984. Effects of parietal injury on covert orienting of attention. J. Neurosci. 4, 1863–1874. Priori, A., 2003. Brain polarization in humans: a reappraisal of an old tool for prolonged non-invasive modulation of brain excitability. Clin. Neurophysiol. 114, 589–595. Priori, A., Mameli, F., Cogiamanian, F., Marceglia, S., Tiriticco, M., Mrakic-Sposta, S., Ferrucci, R., Zago, S., Polezzi, D., Sartori, G., 2008. Lie-specific involvement of dorsolateral prefrontal cortex in deception. Cereb. Cortex 18, 451–455. Proverbio, A.M., Zani, A., Gazzaniga, M.S., Mangun, G.R., 1994. ERP and RT signs of a rightward bias for spatial orienting in a split-brain patient. NeuroReport 5, 2457–2461. Rafal, R.D., 2006. Oculomotor functions of the parietal lobe: effects of chronic lesions in humans. Cortex 42, 730–739. Ragert, P., Vandermeeren, Y., Camus, M., Cohen, L.G., 2008. Improvement of spatial tactile acuity by transcranial direct current stimulation. Clin. Neurophysiol. 119, 805–811. Ratcliff, R., Murdock, B.B., 1976. Retrieval processes in recognition memory. Psychol. Rev. 83, 190–214. Rioult-Pedotti, M.S., Friedman, D., Hess, G., Donoghue, J.P., 1998. Strengthening of horizontal cortical connections following skill learning. Nat. Neurosci. 1, 230–234.

89

Rizzolatti, G., Matelli, M., 2003. Two different streams form the dorsal visual system: anatomy and functions. Exp. Brain Res. 153, 146–157. Rozzi, S., Calzavara, R., Belmalih, A., Borra, E., Gregoriou, G.G., Matelli, M., Luppino, G., 2006. Cortical connections of the inferior parietal cortical convexity of the macaque monkey. Cereb. Cortex 16, 1389–1417. Ruff, C.C., Bestmann, S., Blankenburg, F., Bjoertomt, O., Josephs, O., Weiskopf, N., Deichmann, R., Driver, J., 2008. Distinct causal influences of parietal versus frontal areas on human visual cortex: evidence from concurrent TMS-fMRI. Cereb. Cortex 18, 817–827. Ruff, C.C., Blankenburg, F., Bjoertomt, O., Bestmann, S., Weiskopf, N., Driver, J., 2009. Hemispheric differences in frontal and parietal influences on human occipital cortex: direct confirmation with concurrent TMS-fMRI. J. Cogn. Neurosci. 21, 1146–1161. Schweid, L., Rushmore, R.J., Valero-Cabre, A., 2008. Cathodal transcranial direct current stimulation on posterior parietal cortex disrupts visuo-spatial processing in the contralateral visual field. Exp. Brain Res. 186, 409–417. Shulman, G.L., Ollinger, J.M., Linenweber, M., Petersen, S.E., Corbetta, M., 2001. Multiple neural correlates of detection in the human brain. Proc. Natl. Acad. Sci. U. S. A. 98, 313–318. Sparing, R., Thimm, M., Hesse, M.D., Kust, J., Karbe, H., Fink, G.R., 2009. Bidirectional alterations of interhemispheric parietal balance by non-invasive cortical stimulation. Brain 132, 3011–3020. Spence, C., Driver, J., 2000. Attracting attention to the illusory location of a sound: reflexive crossmodal orienting and ventriloquism. NeuroReport 11, 2057–2061. Stone, D.B., Tesche, C.D., 2009. Transcranial direct current stimulation modulates shifts in global/local attention NeuroReport 20, 1115–1119. Sturm, W., Willmes, K., 2001. On the functional neuroanatomy of intrinsic and phasic alertness. Neuroimage 14, S76–S84. Vallar, G., 2001. Extrapersonal visual unilateral spatial neglect and its neuroanatomy. Neuroimage 14, S52–S58. van Zomeren, A., Brouwer, W., 1994. Clinical neuropsychology of attention. Oxford University Press, New York. Vines, B.W., Schnider, N.M., Schlaug, G., 2006. Testing for causality with transcranial direct current stimulation: pitch memory and the left supramarginal gyrus. NeuroReport 17, 1047–1050. Wagner, T., Fregni, F., Fecteau, S., Grodzinsky, A., Zahn, M., Pascual-Leone, A., 2007. Transcranial direct current stimulation: a computer-based human model study. Neuroimage 35, 1113–1124. Zimmerman, P., Fimm, B., 1992. Testbatterie zur Aufmerksamkeitsprufung (TAP). Psytest, Würselen. Zoccolotti, P., Pizzamiglio, L., Pittau, P.A., Galati, G., 1994. Batteria di Test per l'esame dell'Attenzione. Psytest, Roma.