J Neural Transm DOI 10.1007/s00702-013-1104-z
TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
Evidence for metaplasticity in the human visual cortex Tommaso Bocci • Matteo Caleo • Silvia Tognazzi • Nikita Francini • Lucia Briscese • Lamberto Maffei • Simone Rossi • Alberto Priori • Ferdinando Sartucci
Received: 17 June 2013 / Accepted: 14 October 2013 Ó Springer-Verlag Wien 2013
Abstract The threshold and direction of excitability changes induced by low- and high-frequency repetitive transcranial magnetic stimulation (rTMS) in the primary motor cortex can be effectively reverted by a preceding session of transcranial direct current stimulation (tDCS), a phenomenon referred to as ‘‘metaplasticity’’. Here, we used a combined tDCS–rTMS protocol and visual evoked potentials (VEPs) in healthy subjects to provide direct electrophysiological evidence for metaplasticity in the human visual cortex. Specifically, we evaluated changes in VEPs at two different contrasts (90 and 20 %) before and at different time points after the application of anodal or T. Bocci N. Francini L. Briscese F. Sartucci (&) Unit of Neurology, Department of Clinical and Experimental Medicine, Pisa University Medical School, Pisa, Italy e-mail: [email protected] URL: http://www.unipi.it T. Bocci S. Rossi Neurology and Clinical Neurophysiology Section, Department of Neurological and Neurosensorial Sciences, Azienda Ospedaliera Universitaria Senese, Siena, Italy T. Bocci A. Priori Department of Neurological Sciences, Fondazione IRCCS Ospedale Maggiore Policlinico, University of Milan, Milan, Italy M. Caleo L. Maffei F. Sartucci CNR Neuroscience Institute, Pisa, Italy S. Tognazzi F. Sartucci Cisanello Neurology Unit, Department of Clinical and Experimental Medicine, Pisa University Medical School, Pisa, Italy Present Address: F. Sartucci Unit of Neurology, Cisanello Hospital, Via Paradisa, n. 2, 56124 Pisa, Italy
cathodal tDCS to occipital cortex (i.e., priming), followed by an additional conditioning with low- or high-frequency rTMS. Anodal tDCS increased the amplitude of VEPs and this effect was paradoxically reverted by applying highfrequency (5 Hz), conventionally excitatory rTMS (p \ 0.0001). Similarly, cathodal tDCS led to a decrease in VEPs amplitude, which was reverted by a subsequent application of conventionally inhibitory, 1 Hz rTMS (p \ 0.0001). Similar changes were observed for both the N1 and P1 component of the VEP. There were no significant changes in resting motor threshold values (p [ 0.5), confirming the spatial selectivity of our conditioning protocol. Our findings show that preconditioning primary visual area excitability with tDCS can modulate the direction and strength of plasticity induced by subsequent application of 1 or 5 Hz rTMS. These data indicate the presence of mechanisms of metaplasticity that keep synaptic strengths within a functional dynamic range in the human visual cortex. Keywords Metaplasticity Visual system Transcranial direct current stimulation Repetitive transcranial magnetic stimulation Visual evoked potentials
Introduction The ability of cortical networks to keep neuronal activity within a functional dynamic range has been given considerable attention recently (Abraham 2008; Turrigiano 2011). Because of the positive feedback nature of activity-driven, Hebbian synaptic plasticity, a continuous and unidirectional increase in network excitability would destabilize a neural system. Three candidate mechanisms have been
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proposed for maintaining network activity within a useful range: synaptic scaling, the Bienenstock–Cooper–Munro (BCM) synapse and regulation of intrinsic excitability (Bienenstock et al. 1982; Turrigiano and Nelson 2000, 2004; Desai 2003). In particular, the BCM rule posits that stabilization of neuronal activity is ensured through a dynamic adaptation of the modification threshold for longterm potentiation (LTP) and long-term depression (LTD), depending on the preexisting excitability state. The polarity and strength of activity-dependent synaptic plasticity can be continuously modified as a function of the prior history of cortical activation (Abraham 2008; Cho and Bear 2010). For instance, a high level of previous activity ‘‘slides’’ to the right the modification threshold for LTP, favoring the induction of LTD, while a sustained reduction in postsynaptic activity has opposite effects, promoting the induction of LTP. This sliding of the modification threshold for LTP and LTD, depending on the previous history of neural activity, is referred to as metaplasticity (Abraham and Bear 1996; Desai 2003; Abraham 2008). Excitability changes in human cerebral cortex can be induced and investigated by noninvasive brain stimulation (NIBS) techniques such as transcranial direct current stimulation (tDCS) and single pulse (Kupers et al. 2006; Ptito et al. 2008) or repetitive transcranial magnetic stimulation (rTMS; Rossini and Rossi 2007). Previous studies showed that cathodal tDCS and low-frequency rTMS can effectively dampen neuronal excitability in motor and sensory cortices, including the visual one (Wassermann and Lisanby 2001; Fumal et al. 2003; Bocci et al. 2011), probably by adding neural noise to the perceptual process (Ruzzoli et al. 2010; Fertonani et al. 2011). Conversely, anodal tDCS and high-frequency rTMS increase cortical excitability (Bohotin et al. 2002; Palermo et al. 2011). In line with BCM rules of metaplasticity, the magnitude and direction of excitability changes that are triggered by lowand high-frequency rTMS in the primary motor cortex (M1) can be effectively reverted in healthy humans by a preceding session of tDCS (Lang et al. 2004; Siebner et al. 2004). Specifically, a conditioning session of cathodal tDCS renders excitatory a normally inhibitory subsequent session of low-frequency rTMS (Siebner et al. 2004). Whether similar phenomena can take place in visual pathways is still a matter of debate. A study based on determination of phosphene threshold (PT) in humans demonstrated modest and short-lasting metaplastic effects by applying combined tDCS and rTMS protocols (Lang et al. 2007). These differences between motor and visual cortices may be due to differences in ontogenesis, histological texture and neurotransmitters distribution among different cortical areas (Zilles et al. 2004); moreover, the location of primary visual area (V1) in the depth of interehemispheric fissure could theoretically represent an
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additional factor explaining the weak response to preconditioning tDCS. Other studies have provided support for a mechanism of metaplasticity in human visual cortex (Pasley et al. 2009). In particular, short-term light deprivation results in an increased activation of occipital cortical areas to incoming visual stimuli (Boroojerdi et al. 2000), consistent with a leftward shift of the modification threshold (Bienenstock et al. 1982; Law and Cooper 1994) due to low cortical activity during light deprivation. Accordingly, a normally inhibitory, low-frequency (1 Hz) rTMS protocol induces a significant prolongation of the cortical hyperexcitability state induced by dark rearing (Fierro et al. 2005). In addition, monocular deprivation in adult humans enhances the apparent contrast of stimuli presented to the deprived eye, indicating up-regulation of cortical gaincontrol mechanisms (Lunghi et al. 2011). In this scenario, we aimed to evaluate whether a mechanism resembling BCM-like metaplasticity also operates in the intact human primary visual cortex by recording visual evoked potentials (VEPs) triggered by gratings of various contrasts. VEPs represent the sum of post-synaptic potentials occurring in the visual cortex, time-locked to patterned stimulation, and reflecting the activation of different generators (Ghilardi et al. 1991; Tobimatsu and Celesia 2006). In this way, excitability changes induced by combining tDCS with low- and highfrequency rTMS could be objectively evaluated.
Materials and methods Ethical approval Written informed consent was obtained from all subjects prior to participation in the study. Experiments were approved by the local ethical committee and followed the tenets of Helsinki. Subjects were screened before rTMS with a standard questionnaire (Rossi et al. 2009, 2011). Subjects We enrolled a total of ten right-handed healthy volunteers (5 males and 5 females: mean age ±1 SD 26.9 ± 4.7 years, range 22–34), all with normal or corrected-to-normal vision and no history of neurological or psychiatric disorders (Chen et al. 1997); we also discarded migraineurs, who have excitability thresholds lower than healthy subjects (Schoenen 1996) and might react paradoxically to low-frequency rTMS (Brighina et al. 2002). To avoid interference with cortical excitability changes due to hormonal variations during the ovarian cycle, in relation to GABAergic and glutamatergic effects of progesterone metabolites and estradiol, respectively, females were
Effects of tDCS and rTMS on visual evoked responses
recorded at mid-cycle, i.e., 12–16 days after the 1st day of menses (Smith et al. 1999). All the participants were blinded about the polarity of conditioning stimulation. No subject was taking medications at the time of, or 1 month before, inclusion in the study and all had suspended alcohol or caffeine consumption at least 48 h before.
tDCS, the current was turned on for only 5 s at the beginning of the sham session and then turned off in a ramp-shaped fashion. That induces initial skin sensations indistinguishable from real tDCS. Subjects were not able to discriminate between the applied anodal, cathodal or sham tDCS. Repetitive transcranial magnetic stimulation (rTMS)
Visual evoked potentials (VEPs) Transient VEPs were recorded with an AgCl electrode placed on the Oz position of the 10–20 International EEG System. The reference electrode was positioned over Cz and the ground was located on the forehead (Harding et al. 1996; Porciatti and Sartucci 1999). VEPs were recorded in response to abrupt reversal (1 Hz) of a horizontal square wave grating (spatial frequency 2 c/°, variable contrast), generated by computer on a display (Samsung SyncMaster 1100DF 2100 CRT monitor, Ridgefield Park, NJ, US; refresh rate 60 Hz; subtending 20° 9 15° of visual angle) by a VSG card (Cambridge Research Systems, Rochester, Kent, UK). Subjects maintained stable fixation on a central dot (diameter, 0.2°) throughout stimulus presentation. The display was centered on the vertical meridian (central stimulation). Stimulation was always monocular (one eye patched). A monocular stimulation was preferred to avoid a binocular summation of the amplitudes of evoked responses. Signals were amplified, band pass filtered (0.1–100 Hz) and fed to a computer for storage and analysis (Caleo et al. 2007). At least two series of 50 events (total: 100 traces) were averaged with the stimulus contrast reversal. Transcranial direct current stimulation (tDCS) We applied tDCS using a battery-driven constant current stimulator (HDCStim, Newronika, Italy) and a pair of electrodes in two 5 9 7-cm water-soaked synthetic sponges. For cathodal stimulation, the cathode was placed on Oz (according to the 10–20 international EEG system) and the anode on the right shoulder. For anodal stimulation, the current flow was reversed. In the real tDCS conditions, direct current was transcranially applied for 20 min with an intensity of 1.5 mA, and constant current flow was measured by an ampere meter. The intensity and duration of stimulation were comparable to those used in previous studies (Priori et al. 1998; Antal et al. 2004, 2006; Lang et al. 2007; Antal and Paulus 2008; Feurra et al. 2011). We applied current at a density of 0.43 mA/cm2 and delivered a total charge of 57.5 mC/cm2. These values are below the threshold for tissue damage (Nitsche et al. 2003a). Apart from occasional, transient and short-lasting tingling and burning sensations below the electrodes, direct current stimulation strength remained below the conscious sensory threshold throughout the experimental session. For a sham
A Magstim Super Rapid Transcranial Magnetic Stimulator (Magstim Company, Dyfed, UK, 2.2 T maximum field output) connected to a standard eight-shaped focal coil with wing diameters of 70 mm was used. The coil was placed with its handle pointing upward, so as to induce a current flowing in a craniocaudal direction (Fierro et al. 2005), centered on the Oz point on the skull and kept in a constant position by a tripod. The magnetic stimuli induced biphasic pulses with a mean duration of 200 ls and a rise time of 100 ls. Both low-frequency rTMS (1 Hz over a period of 20 min, for a total of 600 pulses) and highfrequency rTMS (5 Hz for 60 s) were tested following preconditioning with tDCS. We always employed subthreshold intensities, i.e., 85 % of individual resting motor threshold (RMT) prior determined for each subject (Rossi et al. 2009). Experimental protocol The protocol was structured in two main experiments and in a control experiment (modified from Lang et al. 2007). All subjects were enrolled in each experimental session. Figure 1a illustrates the time line of the experimental procedures. The first main experiment consisted of three sessions. In separate sessions, we applied different interventional protocols: anodal tDCS followed by real 1 Hz rTMS or cathodal tDCS followed by real 1 Hz rTMS or sham tDCS followed by real 1 Hz rTMS. Different sessions on the same participant were separated by at least 1 week, to avoid possible carryover effects, and the order of interventional protocols was pseudo-randomized and balanced across subjects. The same subjects joined in the second main experiment. As with the main experiment, we applied three different interventional procedures in separate sessions: anodal tDCS followed by real 5 Hz rTMS or cathodal tDCS followed by real 5 Hz rTMS or sham tDCS followed by real 5 Hz rTMS. In these two main experiments, rTMS was applied 15–20 min following the completion of the tDCS session. Finally, in the control experiment the participants underwent anodal or cathodal 20 min tDCS followed by sham rTMS. Throughout the three experiments, VEPs were recorded at two different contrasts (K90 and K20 %) and evaluated basally (T0), after tDCS (T1), immediately after (T2) and
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Fig. 1 a Summary of the experimental protocol. A 20 min session of anodal, cathodal or sham tDCS was given to the primary visual area (V1) on Oz. For anodal tDCS, the anode was placed over the V1, and the cathode over the right shoulder. Polarity was reversed for cathodal tDCS. VEPs were recorded at two different contrasts (90 and 20 %) and evaluated basally (T0), after tDCS (T1), immediately after (T2) and 30 min (T3) and 60 min (T4) after the completion of either inhibitory (1 Hz for 20 min) or excitatory (5 Hz for 60 s) rTMS. To control for nonspecific effects of rTMS, real rTMS was replaced by
sham rTMS using a specifically designed placebo coil. b Representative VEPs recorded in the same subject that underwent anodal (top traces) and cathodal (bottom traces) tDCS, followed respectively by high-frequency and low-frequency rTMS. Different sessions were separated by a week. Note the surprisingly robust decrease in VEP amplitude following high-frequency (normally excitatory) rTMS preceded by anodal tDCS (top traces). A conventionally inhibitory (1 Hz) rTMS preceded by cathodal tDCS results instead in a significant potentiation of VEPs amplitudes (bottom traces)
30 min (T3) and 60 min (T4) following the completion of rTMS. Modifications in VEPs latency and amplitude were considered at different times (see Fig. 1a). We also tested the RMT at T0, T1, T2, T3 and T4 (before recording VEPs) to assess whether rTMS effects were regionally specific for the visual networks or spread to motor areas. RMT is defined as the minimum stimulator output that induces motor evoked potentials (MEPs) of more than 50 lV in at least five out of ten trials when the first digital interosseus (FDI) muscle is completely relaxed (Ni et al. 2007). The whole protocol had a duration of about 120 min, with each session of VEPs recording lasting for about 10 min. All participants were naı¨ve to VEP recordings and admitted into the experimental room only before the first session of recordings (i.e., T0). During the time intervals between VEPs testing, they were seated on a chair in a lighted room (ambient light of about 200 lux).
Data analysis
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A two-way repeated measures analysis of variance (ANOVA; STATISTICA 5.5, StatSoft Inc.) was used to compare peak-to-peak amplitude and peak latency values at different times and between the type of intervention (anodal tDCS, cathodal tDCS or sham tDCS; 1 Hz rTMS, 5 Hz rTMS or sham rTMS) and contrast of visual stimuli (K90 or K20 %). All individual values were normalized at baseline (i.e., T0). In the main experiments, normalized values were entered into a two-way ANOVA with stimulation (three levels: anodal tDCS ? low or high-frequency rTMS, cathodal tDCS ? low or high-frequency rTMS sham tDCS ? low- or high-frequency rTMS) and time (four levels: T1, T2, T3 and T4) as factors. Similarly, a twoway repeated measures ANOVA was used for control experiments, with stimulation (two levels: anodal tDCS ? sham rTMS and cathodal tDCS ? sham rTMS)
Effects of tDCS and rTMS on visual evoked responses
and time (four levels) as factors. To isolate the group or groups that differ from each other, we used a multiple comparison procedure (post hoc analysis, Holm–Sidak method). One-way ANOVA was used to compare the variations in VEP amplitude over time for each specific experimental condition. All data were analyzed in terms of both latency and peak-to-peak or baseline-to-peak amplitude, as concerns N1 and P1 VEPs components, respectively. All parameters were evaluated statistically using a B0.05 level of significance. Data are given as mean ± SEM.
Results None of the participants reported any adverse effects during or after the experiment. Neither tDCS nor rTMS induced phosphenes. Figure 1b shows representative VEPs of a subject who underwent anodal and cathodal tDCS, at an interval of 1 week, followed in either case by 1 Hz rTMS. Figures 2 and 3 illustrate the effects of tDCS and rTMS on mean P1 and N1 amplitudes, respectively, at maximal contrast (K90 %); Fig. 4 shows the effects of tDCS and rTMS on P1 and N1 amplitudes at lower contrast (K20 %). Control experiment In line with previous reports targeting various neocortical areas (Nitsche and Paulus 2001; Antal et al. 2004; Lang et al. 2007; Feurra et al. 2011), anodal tDCS induced a long-lasting increase in cortical excitability, whereas cathodal tDCS resulted in opposite effects, both on P1 and N1 components (Figs. 2a, 3a). Polarity-specific aftereffects on cortical excitability account for a significant interaction ‘‘time by intervention’’ at high (P1: F(9, 27) = 14.42, p \ 0.0001; N1: F(9, 27) = 4.07, p = 0.002, two-way repeated measures ANOVA followed by Holm–Sidak test) and low contrasts (P1 and N1 with p \ 0.001). These results were confirmed by evaluating changes from baseline for anodal (F = 2.7, p = 0.04 for P1; F = 28.6, p \ 0.0001 for N1, one-way ANOVA) and cathodal stimulation (F = 22.8, p \ 0.0001 for P1; F = 76.2, p \ 0.0001 for N1). While tDCS conditioning caused a polarity-specific modulation of VEPs amplitudes, we found that mean latencies for N1 and P1 were unaffected during the experiment (one-way ANOVA, p [ 0.1). As reported elsewhere (Antal et al. 2004), the effects of tDCS are more pronounced for N1 than P1 (anodal tDCS:
Fig. 2 Stimulation-induced changes in P1 amplitude at high contrast (90 %). a Control experiment: as expected, P1 amplitude is increased by anodal tDCS and dampened by cathodal tDCS, both followed by sham rTMS (one-way ANOVA followed by Holm–Sidak method, p \ 0.05, compared to baseline). b, c Aftereffects of rTMS are critically dependent on the previous priming with tDCS. Conventionally inhibitory, 1 Hz rTMS leads to a paradoxical and highly significant increase in P1 amplitude if preceded by cathodal tDCS (b), while 5 Hz rTMS dampens VEPs amplitude if preceded by anodal tDCS (c). 1 and 5 Hz rTMS, when preceded by sham tDCS, produce a slight but significant modification of baseline VEP amplitudes (oneway ANOVA followed by Holm–Sidak method, p \ 0.05). Values are expressed as percentage of baseline ±1 SD
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Fig. 4 Results of the first main experiment (anodal, cathodal or sham tDCS followed by 1 Hz rTMS) with low-contrast visual stimuli (K20 %). a, b The amplitude variations for P1 (a) and N1 (b) are similar to those reported at higher contrast. In particular, a sequence of two conventionally inhibitory stimuli (cathodal tDCS and lowfrequency rTMS) leads to a paradoxical increase of VEPs amplitude (p \ 0.0001). The normally inhibitory effect of 1 Hz rTMS is greatly enhanced when rTMS is performed after a session of anodal tDCS to increase cortical excitability (p \ 0.0001). Values are expressed as percentage of baseline ±1 SD
Fig. 3 Stimulation induced changes in N1 amplitude at high contrast (90 %). a Control experiment: N1 amplitude is increased by anodal tDCS and dampened by cathodal tDCS, both followed by sham rTMS (one-way ANOVA followed by Holm–Sidak method, p \ 0.05, compared to baseline). b, c Cathodal, inhibitory tDCS followed by 1 Hz rTMS lead to a significant increase in N1 amplitude (p \ 0.01), while 5 Hz rTMS dampens N1 amplitude if preceded by anodal tDCS (p \ 0.01). All the results are in line with those previously showed for P1 component. Values are expressed as percentage of baseline ±1 SD
F(9, 27) = 4.19, p = 0.0018; cathodal tDCS: F(9, 27) = 9.37, p \ 0.001, two-way repeated measures ANOVA with ‘‘time’’ and ‘‘VEPs component’’ as factors) and for cathodal stimulation compared with anodal one. This analysis was
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performed by evaluating the respective percentage changes from baseline (P1 and N1: p \ 0.01, two-way repeated measures ANOVA with ‘‘time’’ and ‘‘stimulation’’ as factors). We looked for eventual gender differences, but we did not disclose significant modifications in VEPs amplitude during control experiments between males and females (p [ 0.1). Main experiments In the first main experiment (Figs. 2b, 3b), the preconditioning tDCS had a profound impact on the effects induced by subsequent intervention with 1 Hz rTMS. When the excitability of the V1 had been increased by 20 min of anodal tDCS, a subsequent period of 1 Hz rTMS robustly decreased VEP responses for at least 60 min after the completion of rTMS (Figs. 2b, 3b, open squares). The
Effects of tDCS and rTMS on visual evoked responses
effect was observed for both P1 (K90 %: F = 57.16, p \ 0.0001: Fig. 2, panel b; K20 %: F = 155.5, p \ 0.0001: Fig. 4, panel a, one-way ANOVA with ‘‘time’’ as factor) and N1 components (K90 %: F = 41.82, p \ 0.0001: Fig. 3, panel b; K20 %: F = 72.48, p \ 0.0001: Fig. 4, panel b). This reduction was consistently greater than that induced by either cathodal tDCS alone (p \ 0.05) or 1 Hz rTMS alone, preceded by sham tDCS (p \ 0.0001 for P1; p \ 0.05 for N1, one-way repeated measures ANOVA with ‘‘time’’ as factor; p = 0.011, two-way repeated measures ANOVA, with ‘‘time’’ and ‘‘stimulation’’ as factors). Surprisingly, when cathodal tDCS was used to reduce V1 excitability, the same 1 Hz rTMS increased paradoxically visual cortex responses at both high (K90 %: F(18, 54) = 4.61, p \ 0.0001 for P1: Fig. 2, panel b; F(18, 54) = 12.12, p \ 0.0001 for N1: Fig. 3, panel b, twoway ANOVA with ‘‘stimulation’’ and ‘‘time’’ as factors) and low contrasts (K20 %: F(18, 54) = 5.34, p \ 0.0001 for P1: Fig. 4, panel a; F(18, 54) = 3.97, p \ 0.0001 for N1: Fig. 4, panel b). The results were confirmed by evaluating changes from baseline in this experimental condition (cathodal tDCS ? low-frequency rTMS) at high (p \ 0.0001 for P1; p = 0.01 for N1, one-way ANOVA) and low contrasts (p \ 0.0001 for P1; p \ 0.0001 for N1). This paradoxical increase in excitability was stable throughout the whole observation time, both at high (P1 and N1: T1: p \ 0.0001; T2: p \ 0.0001; T3: p \ 0.0001; T4: p \ 0.0001, one-way repeated measures ANOVA) and low contrasts (P1 and N1 with p \ 0.0001 for all comparisons). Analysis of the data from individual subjects, as previously reported for the motor cortex (Siebner et al. 2004), showed that there was a significant inverse correlation between the magnitude of effects immediately after tDCS and the subsequent aftereffect of rTMS. Subjects who had the largest increase in excitability after anodal tDCS showed the greatest depression after 1 Hz rTMS (Pearson’s correlation; p \ 0.006). Similarly, subjects with the largest reduction in excitability after cathodal tDCS had the largest increase after 1 Hz rTMS (Pearson’s correlation; p \ 0.03). When the excitability of the V1 was dampened by 20 min of cathodal tDCS, a subsequent period of 5 Hz rTMS increased VEP responses for at least 20 min after the completion of rTMS (Figs. 2c, 3c). The effect was observed for both P1 (K90 %: F = 28.3, p \ 0.0001, oneway ANOVA with ‘‘time’’ as factor) and N1 components (K90 %: F = 12.0, p \ 0.0001). 5 Hz rTMS preceded by sham tDCS also slightly increased P1 (p = 0.013, one-way repeated measures ANOVA) and N1 amplitude (p = 0.003). This increase was less robust than that induced by cathodal tDCS followed by 5 Hz rTMS (K90 %: F = 54.3, p \ 0.0001 for P1; F = 160.7, p \ 0.0001 for N1).
Importantly, a paradoxical depression of VEPs amplitude was induced by anodal tDCS followed by 5 Hz rTMS (second main experiment; Figs. 2c, 3c). The effect was consistent for both the main components of VEPs (K90 %: F(18, 54) = 7.40, p \ 0.0001 for P1; F(18, 54) = 4.73, p \ 0.0001 for N1; two-way ANOVA with ‘‘stimulation’’ and ‘‘time’’ as factors). To rule out a global modification of cortical excitability, we checked the regional specificity of our low-frequency rTMS protocol by assessing the RMT in the right motor cortex to rTMS. We found no significant changes in RMT at T0, T1, T2, T3 or T4, confirming the spatial selectivity of our protocol (one-way ANOVA on ranks, p [ 0.1). Similarly, the latency of the major VEPs components, both at high and low contrasts, did not change; mean latency of N1 and P1 was around 75 and 107 ms, respectively, and remained unaltered at T0, T1, T2, T3 and T4 (one-way ANOVA on ranks, p [ 0.1, for all the experimental conditions tested). Comparison between responses to high and low-contrast stimuli In the first main experiment, the effects of both anodal and cathodal tDCS were more pronounced at low- (K20 %) compared with high-contrast (K90 %) stimuli. This result was statistically significant when anodal stimulation preceded low-frequency rTMS for both the two main components of visual responses (N1: two-way ANOVA F(18, 54) = 4.51, p = 0.012, followed by Holm–Sidak test; P1: two-way ANOVA F(18, 54) = 3.7, p = 0.021). When cathodal tDCS preceded rTMS, this effect was statistically significant only for P1, but not for the first component (N1: two-way ANOVA F(18, 54) = 1.27, p = 0.32; P1: two-way ANOVA F(18, 54) = 3.16, p = 0.043, Holm–Sidak test, p \ 0.05).
Discussion The present study shows that preconditioning V1 excitability with tDCS can modulate the direction and strength of plasticity induced by subsequent application of rTMS, irrespective of whether the train of stimulation was ‘‘conventionally’’ inhibitory (1 Hz) or excitatory (5 Hz). No significant changes in RMT values over time are reported, confirming the spatial selectivity of our combined tDCS/ rTMS protocol. Our results provide evidence in favor of the existence of metaplasticity-like phenomena in the intact human visual cortex. Indeed, preconditioning with tDCS effectively shifts the threshold for synaptic potentiation and depression. Specifically, a conditioning session of cathodal tDCS renders excitatory a normally inhibitory subsequent
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session of low-frequency rTMS, while anodal tDCS renders inhibitory a normally excitatory session of high-frequency rTMS. Thus, the polarity of rTMS-induced effects depends on the previous network activity. Importantly, also the strength of rTMS effects is impacted by the recent history of network activity, as the normal depression induced by 1 Hz rTMS is much increased when it is preceded by anodal tDCS to increase visual cortex excitability. Similarly, the potentiating effects of 5 Hz rTMS are magnified by a previous dampening of visual cortex responses with cathodal tDCS. We obtained similar results for both N1 and P1 components of VEPs; since they appear to be generated in striate and extrastriate cortex, respectively (Di Russo et al. 2001, 2005), these findings point to the existence of BCMlike mechanisms in both primary and higher-order visual areas. Remarkably, a similar trend was disclosed at high (K90 %) and low contrasts (K20 %), which could suggest an involvement of both parvo- and magnocellular streams in the BCM-like regulation of visual responses. However, these effects are more pronounced for N1 than P1, probably due to different sources of activation, and for cathodal stimulation compared with anodal one. This is in agreement with the results of Antal et al. (2004), confirming that the nervous system is more prone to response depression (i.e., NIBS-induced down-regulation of cortical excitability) than to potentiation (i.e., NIBS-induced up-regulation of cortical excitability) (Creutzfeldt et al. 1962; Bindman et al. 1964; Froc et al. 2000). Another interesting finding is that we observed a greater variation of VEPs amplitudes at lower contrasts, following either cathodal or anodal tDCS. This is possibly due to a ceiling effect: in fact, high contrasts (90 %) activate visual areas maximally and the relative weak modulation induced by tDCS may not produce a similar prominent effect (Antal et al. 2004). In this context, we preferred to use monocular viewing conditions to avoid binocular summation of the evoked responses (Bobak et al. 1988). In the present work, we have not included a full sham condition (i.e., sham tDCS ? sham rTMS) to measure spontaneous fluctuations in VEPs amplitude and latency across recording sessions. However, previous work from our group (Bocci et al. 2011; Fig. 5) has demonstrated no longitudinal changes in VEP parameters over time in control conditions. Also in the present experiments, VEP amplitudes remained quite stable over time. In particular, VEP responses remained unaltered for 60 min following tDCS alone, despite repeated exposure to gratings (Figs. 2a, 3a). We are thus confident that there is no significant drift in the recordings across experimental sessions. A previous study based on the evaluation of phosphene threshold (Lang et al. 2007) has shown only modest and short-lasting metaplastic-like changes in the intact human
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visual cortex. Here, we tackled this issue using VEPs amplitudes, which is a more sensitive and objective method than the psychophysical assessment of phosphene threshold (PT). The evaluation of changes in VEPs amplitude and latency offers a unique opportunity to study the whole stimuli processing along visual pathways: N1 and P1 reflect the activation of parallel rather than sequential anatomical pathways, as N1 is mostly a foveal contrast-dependent component, while P1 probably represents luminance parafoveal processing (Ghilardi et al. 1991). Conversely, PT is a psychophysical index and, therefore, an indirect measure of visual cortex excitability. Some studies have recently highlighted a possible retinal origin of phosphenes (Schutter and Hortensius 2010): both volumeconduction effects and subtle rhythmic muscle contraction in eyes make it difficult to separate cortical from retinal sources of activation (Schutter and Hortensius 2010). Moreover, transcranial alternating current stimulation (tACS) and rTMS applied either to the posterior parietal cortex or frontal eye field can reduce phosphene threshold and exert a direct, top-down modulation over extrastriate visual areas (Silvanto et al. 2006; Taylor et al. 2010). Altogether, these findings strengthen the hypothesis that phosphene perception arises from a wider network than previously expected, ranging from occipital to central and parietal cortex (Lamme and Roelfsema 2000; Lamme et al. 2000; Pascual-Leone and Walsh 2001; Taylor et al. 2010). The stimulation protocol that we employed is also quite different from that used by Lang et al.; as current density distribution strictly depends both on the position of the electrodes and the geometry of tissue compartments (Holdefer et al. 2006; Miranda et al. 2006), we preferred to use a classical monopolar montage. This approach reduces interference between anodal and cathodal effects and decreases cutaneous shunt through the scalp, rising at the same time the current density in the depth of primary visual cortex. Finally, it is worth remembering that in the study by Lang et al., the effects on PT of low-frequency rTMS following priming tDCS was not explored. The most likely interpretation of our results is that a tDCS-induced modulation in cell excitability of cortical neurons alters the modification threshold for synaptic plasticity induced by rTMS, ultimately affecting the synaptic potentials responsible for the generation of VEPs. Other studies in the literature have provided evidence in favor of similar BCM-like mechanisms in the visual cortex of human subjects. In particular, Boroojerdi et al. (2000) and Fierro et al. (2005) examined the effects of light deprivation on PT. A reduced PT was detected as soon as 45 min after the onset of light deprivation, and PT returned to baseline levels following re-exposure to light (Boroojerdi et al. 2000; Fierro et al. 2005). Of note, 1 Hz rTMS applied during the last 15 min of light deprivation
Effects of tDCS and rTMS on visual evoked responses
significantly prolonged the time needed to recover baseline PT values following light re-exposure (Fierro et al. 2005). Thus, conventionally inhibitory rTMS following light deprivation produces a paradoxical effect, tending to maintain higher cortical excitability (i.e., reduced phosphene threshold). Boroojerdi et al. (2000) also examined visual stimulus-dependent activation of cortical areas by functional magnetic resonance imaging and found increased visual cortex activation after 60 min of light deprivation. Altogether, these data are consistent with enhanced excitability of visual cortex after a period of reduced network activity. Recently, Lunghi et al. (2011) have shown that brief periods of monocular deprivation (patching of one eye) disrupt ocular balance in human adult visual cortex, with the deprived eye prevailing in conscious perception twice as much as the non-deprived eye. The apparent contrast of stimuli presented to the deprived eye was also increased, suggesting that visual deprivation may paradoxically enhance signal strength of the deprived eye (Lunghi et al. 2011). It is important to note that animal studies have provided important support for BCM-like synapses in the visual cortex. A key study (Kirkwood et al. 1996) compared visual cortical slices from naı¨ve rats and rats reared in the dark to reduce afferent input activity to the cortex. The authors found that the threshold for synaptic potentiation/ depression (i.e., LTP/LTD) was significantly shifted to the left in the slices from the dark-reared animals (Kirkwood et al. 1996). Thus, when neuronal activity levels are reduced by visual deprivation, the LTD/LTP plasticity threshold is lowered such that stimulation is more likely to trigger synaptic potentiation (LTP) and less likely to cause synaptic weakening (LTD). These data provide direct evidence that the threshold for LTP/LTD is not fixed, but can slide according to the previous levels of activity. This mechanism of sliding is important since it allows keeping synaptic strengths within a functional dynamic range (Abraham 2008). Previous evidence in animal models has suggested a role for different molecules and signaling pathways in the regulation of metaplastic and homeostatic mechanisms (Abraham 2008; Turrigiano 2008, 2011). No pharmacological data are currently available to explain metaplastic regulation of neuronal excitability for the human cerebral cortex; therefore further studies are needed to reveal the molecular bases of this kind of plasticity. There are other questions raised by the present study that remain to be addressed in future investigations. First, it will be important to dissect the contribution of pre- and post-synaptic mechanisms to the induction of metaplastic phenomena in the cortex. Second, it will be interesting to disclose eventual differences among cortical layers in the activation of BCM-like mechanisms.
How tDCS may prime new plasticity responses to rTMS remains open to speculation. Previous studies on the visual cortex in animal models (Philpot et al. 2001, 2003) have shown that metaplasticity is potently regulated by Nmethyl-D-aspartate (NMDA) receptor function, through modifications both in the number of NMDA receptors and in the lengthening of NMDA-mediated excitatory postsynaptic current (EPSCs). Thus, the aftereffects induced by tDCS may be mainly dependent on NMDA receptors (Liebetanz et al. 2002; Nitsche et al. 2003b). However, it is possible that other neurotransmitters could account for tDCS-induced changes in responsiveness of the visual cortex, as suggested by the evidence that amantadine, a low-affinity, non-competitive NMDA receptor antagonist, is not effective in modulating VEPs amplitude (Bandini et al. 2002). Another possibility is that tDCS modulates metaplasticity changes through modifications in GABAergic synaptic transmission (Nitsche et al. 2004), as BCMlike plasticity also depends on GABA receptor function (Kalantzis and Shouval 2009).
Conclusions Discovery of BCM-like regulation of visual responses in humans is important since homeostatic phenomena and metaplasticity are believed to underlie the ability of cortical networks to maintain stable function in the face of developmental or learning-induced changes in drive. Metaplasticity could play a role in circuit rearrangements triggered in the visual cortex by alterations in sensory input, as suggested by previous animal studies (Ranson et al. 2012). Metaplasticity could also be crucial for the process of plasticity in the adult, healthy visual system; it is known that adult visual cortex retains a surprisingly high degree of plasticity fundamental in reaction to sensory loss (Sabel 2008; Lunghi et al. 2011). Similarly, our results might be helpful in evaluating dynamic changes in visual responsiveness following stroke or other brain lesions, in adults as well as in childhood. In this context, it is interesting to note that neuromodulation by NIBS techniques as rTMS is increasingly being used in a series of pathologies to inactivate/stimulate cortical networks, and in the treatment of visual dysfunctions such as amblyopia (Thompson et al. 2008). Our data indicate that the magnitude and direction of plasticity induced by rTMS depends on the preexisting, functional state of the cortex, pointing to possible interindividual differences in the response to inhibitory/excitatory rTMS. Acknowledgments We gratefully acknowledge the participation of all subjects and Mr. C. Orsini and Ms. M. Naraci for their excellent technical assistance. The paper was supported in part by the Italian
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T. Bocci et al. operating and development MIUR PRIN grant year 2006, n. 2006062332_002. Alberto Priori is founder and shareholder of Newronika srl, Milan, Italy.
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TRANSLATIONAL NEUROSCIENCES - ORIGINAL ARTICLE
Evidence for metaplasticity in the human visual cortex Tommaso Bocci • Matteo Caleo • Silvia Tognazzi • Nikita Francini • Lucia Briscese • Lamberto Maffei • Simone Rossi • Alberto Priori • Ferdinando Sartucci
Received: 17 June 2013 / Accepted: 14 October 2013 Ó Springer-Verlag Wien 2013
Abstract The threshold and direction of excitability changes induced by low- and high-frequency repetitive transcranial magnetic stimulation (rTMS) in the primary motor cortex can be effectively reverted by a preceding session of transcranial direct current stimulation (tDCS), a phenomenon referred to as ‘‘metaplasticity’’. Here, we used a combined tDCS–rTMS protocol and visual evoked potentials (VEPs) in healthy subjects to provide direct electrophysiological evidence for metaplasticity in the human visual cortex. Specifically, we evaluated changes in VEPs at two different contrasts (90 and 20 %) before and at different time points after the application of anodal or T. Bocci N. Francini L. Briscese F. Sartucci (&) Unit of Neurology, Department of Clinical and Experimental Medicine, Pisa University Medical School, Pisa, Italy e-mail: [email protected] URL: http://www.unipi.it T. Bocci S. Rossi Neurology and Clinical Neurophysiology Section, Department of Neurological and Neurosensorial Sciences, Azienda Ospedaliera Universitaria Senese, Siena, Italy T. Bocci A. Priori Department of Neurological Sciences, Fondazione IRCCS Ospedale Maggiore Policlinico, University of Milan, Milan, Italy M. Caleo L. Maffei F. Sartucci CNR Neuroscience Institute, Pisa, Italy S. Tognazzi F. Sartucci Cisanello Neurology Unit, Department of Clinical and Experimental Medicine, Pisa University Medical School, Pisa, Italy Present Address: F. Sartucci Unit of Neurology, Cisanello Hospital, Via Paradisa, n. 2, 56124 Pisa, Italy
cathodal tDCS to occipital cortex (i.e., priming), followed by an additional conditioning with low- or high-frequency rTMS. Anodal tDCS increased the amplitude of VEPs and this effect was paradoxically reverted by applying highfrequency (5 Hz), conventionally excitatory rTMS (p \ 0.0001). Similarly, cathodal tDCS led to a decrease in VEPs amplitude, which was reverted by a subsequent application of conventionally inhibitory, 1 Hz rTMS (p \ 0.0001). Similar changes were observed for both the N1 and P1 component of the VEP. There were no significant changes in resting motor threshold values (p [ 0.5), confirming the spatial selectivity of our conditioning protocol. Our findings show that preconditioning primary visual area excitability with tDCS can modulate the direction and strength of plasticity induced by subsequent application of 1 or 5 Hz rTMS. These data indicate the presence of mechanisms of metaplasticity that keep synaptic strengths within a functional dynamic range in the human visual cortex. Keywords Metaplasticity Visual system Transcranial direct current stimulation Repetitive transcranial magnetic stimulation Visual evoked potentials
Introduction The ability of cortical networks to keep neuronal activity within a functional dynamic range has been given considerable attention recently (Abraham 2008; Turrigiano 2011). Because of the positive feedback nature of activity-driven, Hebbian synaptic plasticity, a continuous and unidirectional increase in network excitability would destabilize a neural system. Three candidate mechanisms have been
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proposed for maintaining network activity within a useful range: synaptic scaling, the Bienenstock–Cooper–Munro (BCM) synapse and regulation of intrinsic excitability (Bienenstock et al. 1982; Turrigiano and Nelson 2000, 2004; Desai 2003). In particular, the BCM rule posits that stabilization of neuronal activity is ensured through a dynamic adaptation of the modification threshold for longterm potentiation (LTP) and long-term depression (LTD), depending on the preexisting excitability state. The polarity and strength of activity-dependent synaptic plasticity can be continuously modified as a function of the prior history of cortical activation (Abraham 2008; Cho and Bear 2010). For instance, a high level of previous activity ‘‘slides’’ to the right the modification threshold for LTP, favoring the induction of LTD, while a sustained reduction in postsynaptic activity has opposite effects, promoting the induction of LTP. This sliding of the modification threshold for LTP and LTD, depending on the previous history of neural activity, is referred to as metaplasticity (Abraham and Bear 1996; Desai 2003; Abraham 2008). Excitability changes in human cerebral cortex can be induced and investigated by noninvasive brain stimulation (NIBS) techniques such as transcranial direct current stimulation (tDCS) and single pulse (Kupers et al. 2006; Ptito et al. 2008) or repetitive transcranial magnetic stimulation (rTMS; Rossini and Rossi 2007). Previous studies showed that cathodal tDCS and low-frequency rTMS can effectively dampen neuronal excitability in motor and sensory cortices, including the visual one (Wassermann and Lisanby 2001; Fumal et al. 2003; Bocci et al. 2011), probably by adding neural noise to the perceptual process (Ruzzoli et al. 2010; Fertonani et al. 2011). Conversely, anodal tDCS and high-frequency rTMS increase cortical excitability (Bohotin et al. 2002; Palermo et al. 2011). In line with BCM rules of metaplasticity, the magnitude and direction of excitability changes that are triggered by lowand high-frequency rTMS in the primary motor cortex (M1) can be effectively reverted in healthy humans by a preceding session of tDCS (Lang et al. 2004; Siebner et al. 2004). Specifically, a conditioning session of cathodal tDCS renders excitatory a normally inhibitory subsequent session of low-frequency rTMS (Siebner et al. 2004). Whether similar phenomena can take place in visual pathways is still a matter of debate. A study based on determination of phosphene threshold (PT) in humans demonstrated modest and short-lasting metaplastic effects by applying combined tDCS and rTMS protocols (Lang et al. 2007). These differences between motor and visual cortices may be due to differences in ontogenesis, histological texture and neurotransmitters distribution among different cortical areas (Zilles et al. 2004); moreover, the location of primary visual area (V1) in the depth of interehemispheric fissure could theoretically represent an
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additional factor explaining the weak response to preconditioning tDCS. Other studies have provided support for a mechanism of metaplasticity in human visual cortex (Pasley et al. 2009). In particular, short-term light deprivation results in an increased activation of occipital cortical areas to incoming visual stimuli (Boroojerdi et al. 2000), consistent with a leftward shift of the modification threshold (Bienenstock et al. 1982; Law and Cooper 1994) due to low cortical activity during light deprivation. Accordingly, a normally inhibitory, low-frequency (1 Hz) rTMS protocol induces a significant prolongation of the cortical hyperexcitability state induced by dark rearing (Fierro et al. 2005). In addition, monocular deprivation in adult humans enhances the apparent contrast of stimuli presented to the deprived eye, indicating up-regulation of cortical gaincontrol mechanisms (Lunghi et al. 2011). In this scenario, we aimed to evaluate whether a mechanism resembling BCM-like metaplasticity also operates in the intact human primary visual cortex by recording visual evoked potentials (VEPs) triggered by gratings of various contrasts. VEPs represent the sum of post-synaptic potentials occurring in the visual cortex, time-locked to patterned stimulation, and reflecting the activation of different generators (Ghilardi et al. 1991; Tobimatsu and Celesia 2006). In this way, excitability changes induced by combining tDCS with low- and highfrequency rTMS could be objectively evaluated.
Materials and methods Ethical approval Written informed consent was obtained from all subjects prior to participation in the study. Experiments were approved by the local ethical committee and followed the tenets of Helsinki. Subjects were screened before rTMS with a standard questionnaire (Rossi et al. 2009, 2011). Subjects We enrolled a total of ten right-handed healthy volunteers (5 males and 5 females: mean age ±1 SD 26.9 ± 4.7 years, range 22–34), all with normal or corrected-to-normal vision and no history of neurological or psychiatric disorders (Chen et al. 1997); we also discarded migraineurs, who have excitability thresholds lower than healthy subjects (Schoenen 1996) and might react paradoxically to low-frequency rTMS (Brighina et al. 2002). To avoid interference with cortical excitability changes due to hormonal variations during the ovarian cycle, in relation to GABAergic and glutamatergic effects of progesterone metabolites and estradiol, respectively, females were
Effects of tDCS and rTMS on visual evoked responses
recorded at mid-cycle, i.e., 12–16 days after the 1st day of menses (Smith et al. 1999). All the participants were blinded about the polarity of conditioning stimulation. No subject was taking medications at the time of, or 1 month before, inclusion in the study and all had suspended alcohol or caffeine consumption at least 48 h before.
tDCS, the current was turned on for only 5 s at the beginning of the sham session and then turned off in a ramp-shaped fashion. That induces initial skin sensations indistinguishable from real tDCS. Subjects were not able to discriminate between the applied anodal, cathodal or sham tDCS. Repetitive transcranial magnetic stimulation (rTMS)
Visual evoked potentials (VEPs) Transient VEPs were recorded with an AgCl electrode placed on the Oz position of the 10–20 International EEG System. The reference electrode was positioned over Cz and the ground was located on the forehead (Harding et al. 1996; Porciatti and Sartucci 1999). VEPs were recorded in response to abrupt reversal (1 Hz) of a horizontal square wave grating (spatial frequency 2 c/°, variable contrast), generated by computer on a display (Samsung SyncMaster 1100DF 2100 CRT monitor, Ridgefield Park, NJ, US; refresh rate 60 Hz; subtending 20° 9 15° of visual angle) by a VSG card (Cambridge Research Systems, Rochester, Kent, UK). Subjects maintained stable fixation on a central dot (diameter, 0.2°) throughout stimulus presentation. The display was centered on the vertical meridian (central stimulation). Stimulation was always monocular (one eye patched). A monocular stimulation was preferred to avoid a binocular summation of the amplitudes of evoked responses. Signals were amplified, band pass filtered (0.1–100 Hz) and fed to a computer for storage and analysis (Caleo et al. 2007). At least two series of 50 events (total: 100 traces) were averaged with the stimulus contrast reversal. Transcranial direct current stimulation (tDCS) We applied tDCS using a battery-driven constant current stimulator (HDCStim, Newronika, Italy) and a pair of electrodes in two 5 9 7-cm water-soaked synthetic sponges. For cathodal stimulation, the cathode was placed on Oz (according to the 10–20 international EEG system) and the anode on the right shoulder. For anodal stimulation, the current flow was reversed. In the real tDCS conditions, direct current was transcranially applied for 20 min with an intensity of 1.5 mA, and constant current flow was measured by an ampere meter. The intensity and duration of stimulation were comparable to those used in previous studies (Priori et al. 1998; Antal et al. 2004, 2006; Lang et al. 2007; Antal and Paulus 2008; Feurra et al. 2011). We applied current at a density of 0.43 mA/cm2 and delivered a total charge of 57.5 mC/cm2. These values are below the threshold for tissue damage (Nitsche et al. 2003a). Apart from occasional, transient and short-lasting tingling and burning sensations below the electrodes, direct current stimulation strength remained below the conscious sensory threshold throughout the experimental session. For a sham
A Magstim Super Rapid Transcranial Magnetic Stimulator (Magstim Company, Dyfed, UK, 2.2 T maximum field output) connected to a standard eight-shaped focal coil with wing diameters of 70 mm was used. The coil was placed with its handle pointing upward, so as to induce a current flowing in a craniocaudal direction (Fierro et al. 2005), centered on the Oz point on the skull and kept in a constant position by a tripod. The magnetic stimuli induced biphasic pulses with a mean duration of 200 ls and a rise time of 100 ls. Both low-frequency rTMS (1 Hz over a period of 20 min, for a total of 600 pulses) and highfrequency rTMS (5 Hz for 60 s) were tested following preconditioning with tDCS. We always employed subthreshold intensities, i.e., 85 % of individual resting motor threshold (RMT) prior determined for each subject (Rossi et al. 2009). Experimental protocol The protocol was structured in two main experiments and in a control experiment (modified from Lang et al. 2007). All subjects were enrolled in each experimental session. Figure 1a illustrates the time line of the experimental procedures. The first main experiment consisted of three sessions. In separate sessions, we applied different interventional protocols: anodal tDCS followed by real 1 Hz rTMS or cathodal tDCS followed by real 1 Hz rTMS or sham tDCS followed by real 1 Hz rTMS. Different sessions on the same participant were separated by at least 1 week, to avoid possible carryover effects, and the order of interventional protocols was pseudo-randomized and balanced across subjects. The same subjects joined in the second main experiment. As with the main experiment, we applied three different interventional procedures in separate sessions: anodal tDCS followed by real 5 Hz rTMS or cathodal tDCS followed by real 5 Hz rTMS or sham tDCS followed by real 5 Hz rTMS. In these two main experiments, rTMS was applied 15–20 min following the completion of the tDCS session. Finally, in the control experiment the participants underwent anodal or cathodal 20 min tDCS followed by sham rTMS. Throughout the three experiments, VEPs were recorded at two different contrasts (K90 and K20 %) and evaluated basally (T0), after tDCS (T1), immediately after (T2) and
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Fig. 1 a Summary of the experimental protocol. A 20 min session of anodal, cathodal or sham tDCS was given to the primary visual area (V1) on Oz. For anodal tDCS, the anode was placed over the V1, and the cathode over the right shoulder. Polarity was reversed for cathodal tDCS. VEPs were recorded at two different contrasts (90 and 20 %) and evaluated basally (T0), after tDCS (T1), immediately after (T2) and 30 min (T3) and 60 min (T4) after the completion of either inhibitory (1 Hz for 20 min) or excitatory (5 Hz for 60 s) rTMS. To control for nonspecific effects of rTMS, real rTMS was replaced by
sham rTMS using a specifically designed placebo coil. b Representative VEPs recorded in the same subject that underwent anodal (top traces) and cathodal (bottom traces) tDCS, followed respectively by high-frequency and low-frequency rTMS. Different sessions were separated by a week. Note the surprisingly robust decrease in VEP amplitude following high-frequency (normally excitatory) rTMS preceded by anodal tDCS (top traces). A conventionally inhibitory (1 Hz) rTMS preceded by cathodal tDCS results instead in a significant potentiation of VEPs amplitudes (bottom traces)
30 min (T3) and 60 min (T4) following the completion of rTMS. Modifications in VEPs latency and amplitude were considered at different times (see Fig. 1a). We also tested the RMT at T0, T1, T2, T3 and T4 (before recording VEPs) to assess whether rTMS effects were regionally specific for the visual networks or spread to motor areas. RMT is defined as the minimum stimulator output that induces motor evoked potentials (MEPs) of more than 50 lV in at least five out of ten trials when the first digital interosseus (FDI) muscle is completely relaxed (Ni et al. 2007). The whole protocol had a duration of about 120 min, with each session of VEPs recording lasting for about 10 min. All participants were naı¨ve to VEP recordings and admitted into the experimental room only before the first session of recordings (i.e., T0). During the time intervals between VEPs testing, they were seated on a chair in a lighted room (ambient light of about 200 lux).
Data analysis
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A two-way repeated measures analysis of variance (ANOVA; STATISTICA 5.5, StatSoft Inc.) was used to compare peak-to-peak amplitude and peak latency values at different times and between the type of intervention (anodal tDCS, cathodal tDCS or sham tDCS; 1 Hz rTMS, 5 Hz rTMS or sham rTMS) and contrast of visual stimuli (K90 or K20 %). All individual values were normalized at baseline (i.e., T0). In the main experiments, normalized values were entered into a two-way ANOVA with stimulation (three levels: anodal tDCS ? low or high-frequency rTMS, cathodal tDCS ? low or high-frequency rTMS sham tDCS ? low- or high-frequency rTMS) and time (four levels: T1, T2, T3 and T4) as factors. Similarly, a twoway repeated measures ANOVA was used for control experiments, with stimulation (two levels: anodal tDCS ? sham rTMS and cathodal tDCS ? sham rTMS)
Effects of tDCS and rTMS on visual evoked responses
and time (four levels) as factors. To isolate the group or groups that differ from each other, we used a multiple comparison procedure (post hoc analysis, Holm–Sidak method). One-way ANOVA was used to compare the variations in VEP amplitude over time for each specific experimental condition. All data were analyzed in terms of both latency and peak-to-peak or baseline-to-peak amplitude, as concerns N1 and P1 VEPs components, respectively. All parameters were evaluated statistically using a B0.05 level of significance. Data are given as mean ± SEM.
Results None of the participants reported any adverse effects during or after the experiment. Neither tDCS nor rTMS induced phosphenes. Figure 1b shows representative VEPs of a subject who underwent anodal and cathodal tDCS, at an interval of 1 week, followed in either case by 1 Hz rTMS. Figures 2 and 3 illustrate the effects of tDCS and rTMS on mean P1 and N1 amplitudes, respectively, at maximal contrast (K90 %); Fig. 4 shows the effects of tDCS and rTMS on P1 and N1 amplitudes at lower contrast (K20 %). Control experiment In line with previous reports targeting various neocortical areas (Nitsche and Paulus 2001; Antal et al. 2004; Lang et al. 2007; Feurra et al. 2011), anodal tDCS induced a long-lasting increase in cortical excitability, whereas cathodal tDCS resulted in opposite effects, both on P1 and N1 components (Figs. 2a, 3a). Polarity-specific aftereffects on cortical excitability account for a significant interaction ‘‘time by intervention’’ at high (P1: F(9, 27) = 14.42, p \ 0.0001; N1: F(9, 27) = 4.07, p = 0.002, two-way repeated measures ANOVA followed by Holm–Sidak test) and low contrasts (P1 and N1 with p \ 0.001). These results were confirmed by evaluating changes from baseline for anodal (F = 2.7, p = 0.04 for P1; F = 28.6, p \ 0.0001 for N1, one-way ANOVA) and cathodal stimulation (F = 22.8, p \ 0.0001 for P1; F = 76.2, p \ 0.0001 for N1). While tDCS conditioning caused a polarity-specific modulation of VEPs amplitudes, we found that mean latencies for N1 and P1 were unaffected during the experiment (one-way ANOVA, p [ 0.1). As reported elsewhere (Antal et al. 2004), the effects of tDCS are more pronounced for N1 than P1 (anodal tDCS:
Fig. 2 Stimulation-induced changes in P1 amplitude at high contrast (90 %). a Control experiment: as expected, P1 amplitude is increased by anodal tDCS and dampened by cathodal tDCS, both followed by sham rTMS (one-way ANOVA followed by Holm–Sidak method, p \ 0.05, compared to baseline). b, c Aftereffects of rTMS are critically dependent on the previous priming with tDCS. Conventionally inhibitory, 1 Hz rTMS leads to a paradoxical and highly significant increase in P1 amplitude if preceded by cathodal tDCS (b), while 5 Hz rTMS dampens VEPs amplitude if preceded by anodal tDCS (c). 1 and 5 Hz rTMS, when preceded by sham tDCS, produce a slight but significant modification of baseline VEP amplitudes (oneway ANOVA followed by Holm–Sidak method, p \ 0.05). Values are expressed as percentage of baseline ±1 SD
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Fig. 4 Results of the first main experiment (anodal, cathodal or sham tDCS followed by 1 Hz rTMS) with low-contrast visual stimuli (K20 %). a, b The amplitude variations for P1 (a) and N1 (b) are similar to those reported at higher contrast. In particular, a sequence of two conventionally inhibitory stimuli (cathodal tDCS and lowfrequency rTMS) leads to a paradoxical increase of VEPs amplitude (p \ 0.0001). The normally inhibitory effect of 1 Hz rTMS is greatly enhanced when rTMS is performed after a session of anodal tDCS to increase cortical excitability (p \ 0.0001). Values are expressed as percentage of baseline ±1 SD
Fig. 3 Stimulation induced changes in N1 amplitude at high contrast (90 %). a Control experiment: N1 amplitude is increased by anodal tDCS and dampened by cathodal tDCS, both followed by sham rTMS (one-way ANOVA followed by Holm–Sidak method, p \ 0.05, compared to baseline). b, c Cathodal, inhibitory tDCS followed by 1 Hz rTMS lead to a significant increase in N1 amplitude (p \ 0.01), while 5 Hz rTMS dampens N1 amplitude if preceded by anodal tDCS (p \ 0.01). All the results are in line with those previously showed for P1 component. Values are expressed as percentage of baseline ±1 SD
F(9, 27) = 4.19, p = 0.0018; cathodal tDCS: F(9, 27) = 9.37, p \ 0.001, two-way repeated measures ANOVA with ‘‘time’’ and ‘‘VEPs component’’ as factors) and for cathodal stimulation compared with anodal one. This analysis was
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performed by evaluating the respective percentage changes from baseline (P1 and N1: p \ 0.01, two-way repeated measures ANOVA with ‘‘time’’ and ‘‘stimulation’’ as factors). We looked for eventual gender differences, but we did not disclose significant modifications in VEPs amplitude during control experiments between males and females (p [ 0.1). Main experiments In the first main experiment (Figs. 2b, 3b), the preconditioning tDCS had a profound impact on the effects induced by subsequent intervention with 1 Hz rTMS. When the excitability of the V1 had been increased by 20 min of anodal tDCS, a subsequent period of 1 Hz rTMS robustly decreased VEP responses for at least 60 min after the completion of rTMS (Figs. 2b, 3b, open squares). The
Effects of tDCS and rTMS on visual evoked responses
effect was observed for both P1 (K90 %: F = 57.16, p \ 0.0001: Fig. 2, panel b; K20 %: F = 155.5, p \ 0.0001: Fig. 4, panel a, one-way ANOVA with ‘‘time’’ as factor) and N1 components (K90 %: F = 41.82, p \ 0.0001: Fig. 3, panel b; K20 %: F = 72.48, p \ 0.0001: Fig. 4, panel b). This reduction was consistently greater than that induced by either cathodal tDCS alone (p \ 0.05) or 1 Hz rTMS alone, preceded by sham tDCS (p \ 0.0001 for P1; p \ 0.05 for N1, one-way repeated measures ANOVA with ‘‘time’’ as factor; p = 0.011, two-way repeated measures ANOVA, with ‘‘time’’ and ‘‘stimulation’’ as factors). Surprisingly, when cathodal tDCS was used to reduce V1 excitability, the same 1 Hz rTMS increased paradoxically visual cortex responses at both high (K90 %: F(18, 54) = 4.61, p \ 0.0001 for P1: Fig. 2, panel b; F(18, 54) = 12.12, p \ 0.0001 for N1: Fig. 3, panel b, twoway ANOVA with ‘‘stimulation’’ and ‘‘time’’ as factors) and low contrasts (K20 %: F(18, 54) = 5.34, p \ 0.0001 for P1: Fig. 4, panel a; F(18, 54) = 3.97, p \ 0.0001 for N1: Fig. 4, panel b). The results were confirmed by evaluating changes from baseline in this experimental condition (cathodal tDCS ? low-frequency rTMS) at high (p \ 0.0001 for P1; p = 0.01 for N1, one-way ANOVA) and low contrasts (p \ 0.0001 for P1; p \ 0.0001 for N1). This paradoxical increase in excitability was stable throughout the whole observation time, both at high (P1 and N1: T1: p \ 0.0001; T2: p \ 0.0001; T3: p \ 0.0001; T4: p \ 0.0001, one-way repeated measures ANOVA) and low contrasts (P1 and N1 with p \ 0.0001 for all comparisons). Analysis of the data from individual subjects, as previously reported for the motor cortex (Siebner et al. 2004), showed that there was a significant inverse correlation between the magnitude of effects immediately after tDCS and the subsequent aftereffect of rTMS. Subjects who had the largest increase in excitability after anodal tDCS showed the greatest depression after 1 Hz rTMS (Pearson’s correlation; p \ 0.006). Similarly, subjects with the largest reduction in excitability after cathodal tDCS had the largest increase after 1 Hz rTMS (Pearson’s correlation; p \ 0.03). When the excitability of the V1 was dampened by 20 min of cathodal tDCS, a subsequent period of 5 Hz rTMS increased VEP responses for at least 20 min after the completion of rTMS (Figs. 2c, 3c). The effect was observed for both P1 (K90 %: F = 28.3, p \ 0.0001, oneway ANOVA with ‘‘time’’ as factor) and N1 components (K90 %: F = 12.0, p \ 0.0001). 5 Hz rTMS preceded by sham tDCS also slightly increased P1 (p = 0.013, one-way repeated measures ANOVA) and N1 amplitude (p = 0.003). This increase was less robust than that induced by cathodal tDCS followed by 5 Hz rTMS (K90 %: F = 54.3, p \ 0.0001 for P1; F = 160.7, p \ 0.0001 for N1).
Importantly, a paradoxical depression of VEPs amplitude was induced by anodal tDCS followed by 5 Hz rTMS (second main experiment; Figs. 2c, 3c). The effect was consistent for both the main components of VEPs (K90 %: F(18, 54) = 7.40, p \ 0.0001 for P1; F(18, 54) = 4.73, p \ 0.0001 for N1; two-way ANOVA with ‘‘stimulation’’ and ‘‘time’’ as factors). To rule out a global modification of cortical excitability, we checked the regional specificity of our low-frequency rTMS protocol by assessing the RMT in the right motor cortex to rTMS. We found no significant changes in RMT at T0, T1, T2, T3 or T4, confirming the spatial selectivity of our protocol (one-way ANOVA on ranks, p [ 0.1). Similarly, the latency of the major VEPs components, both at high and low contrasts, did not change; mean latency of N1 and P1 was around 75 and 107 ms, respectively, and remained unaltered at T0, T1, T2, T3 and T4 (one-way ANOVA on ranks, p [ 0.1, for all the experimental conditions tested). Comparison between responses to high and low-contrast stimuli In the first main experiment, the effects of both anodal and cathodal tDCS were more pronounced at low- (K20 %) compared with high-contrast (K90 %) stimuli. This result was statistically significant when anodal stimulation preceded low-frequency rTMS for both the two main components of visual responses (N1: two-way ANOVA F(18, 54) = 4.51, p = 0.012, followed by Holm–Sidak test; P1: two-way ANOVA F(18, 54) = 3.7, p = 0.021). When cathodal tDCS preceded rTMS, this effect was statistically significant only for P1, but not for the first component (N1: two-way ANOVA F(18, 54) = 1.27, p = 0.32; P1: two-way ANOVA F(18, 54) = 3.16, p = 0.043, Holm–Sidak test, p \ 0.05).
Discussion The present study shows that preconditioning V1 excitability with tDCS can modulate the direction and strength of plasticity induced by subsequent application of rTMS, irrespective of whether the train of stimulation was ‘‘conventionally’’ inhibitory (1 Hz) or excitatory (5 Hz). No significant changes in RMT values over time are reported, confirming the spatial selectivity of our combined tDCS/ rTMS protocol. Our results provide evidence in favor of the existence of metaplasticity-like phenomena in the intact human visual cortex. Indeed, preconditioning with tDCS effectively shifts the threshold for synaptic potentiation and depression. Specifically, a conditioning session of cathodal tDCS renders excitatory a normally inhibitory subsequent
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session of low-frequency rTMS, while anodal tDCS renders inhibitory a normally excitatory session of high-frequency rTMS. Thus, the polarity of rTMS-induced effects depends on the previous network activity. Importantly, also the strength of rTMS effects is impacted by the recent history of network activity, as the normal depression induced by 1 Hz rTMS is much increased when it is preceded by anodal tDCS to increase visual cortex excitability. Similarly, the potentiating effects of 5 Hz rTMS are magnified by a previous dampening of visual cortex responses with cathodal tDCS. We obtained similar results for both N1 and P1 components of VEPs; since they appear to be generated in striate and extrastriate cortex, respectively (Di Russo et al. 2001, 2005), these findings point to the existence of BCMlike mechanisms in both primary and higher-order visual areas. Remarkably, a similar trend was disclosed at high (K90 %) and low contrasts (K20 %), which could suggest an involvement of both parvo- and magnocellular streams in the BCM-like regulation of visual responses. However, these effects are more pronounced for N1 than P1, probably due to different sources of activation, and for cathodal stimulation compared with anodal one. This is in agreement with the results of Antal et al. (2004), confirming that the nervous system is more prone to response depression (i.e., NIBS-induced down-regulation of cortical excitability) than to potentiation (i.e., NIBS-induced up-regulation of cortical excitability) (Creutzfeldt et al. 1962; Bindman et al. 1964; Froc et al. 2000). Another interesting finding is that we observed a greater variation of VEPs amplitudes at lower contrasts, following either cathodal or anodal tDCS. This is possibly due to a ceiling effect: in fact, high contrasts (90 %) activate visual areas maximally and the relative weak modulation induced by tDCS may not produce a similar prominent effect (Antal et al. 2004). In this context, we preferred to use monocular viewing conditions to avoid binocular summation of the evoked responses (Bobak et al. 1988). In the present work, we have not included a full sham condition (i.e., sham tDCS ? sham rTMS) to measure spontaneous fluctuations in VEPs amplitude and latency across recording sessions. However, previous work from our group (Bocci et al. 2011; Fig. 5) has demonstrated no longitudinal changes in VEP parameters over time in control conditions. Also in the present experiments, VEP amplitudes remained quite stable over time. In particular, VEP responses remained unaltered for 60 min following tDCS alone, despite repeated exposure to gratings (Figs. 2a, 3a). We are thus confident that there is no significant drift in the recordings across experimental sessions. A previous study based on the evaluation of phosphene threshold (Lang et al. 2007) has shown only modest and short-lasting metaplastic-like changes in the intact human
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visual cortex. Here, we tackled this issue using VEPs amplitudes, which is a more sensitive and objective method than the psychophysical assessment of phosphene threshold (PT). The evaluation of changes in VEPs amplitude and latency offers a unique opportunity to study the whole stimuli processing along visual pathways: N1 and P1 reflect the activation of parallel rather than sequential anatomical pathways, as N1 is mostly a foveal contrast-dependent component, while P1 probably represents luminance parafoveal processing (Ghilardi et al. 1991). Conversely, PT is a psychophysical index and, therefore, an indirect measure of visual cortex excitability. Some studies have recently highlighted a possible retinal origin of phosphenes (Schutter and Hortensius 2010): both volumeconduction effects and subtle rhythmic muscle contraction in eyes make it difficult to separate cortical from retinal sources of activation (Schutter and Hortensius 2010). Moreover, transcranial alternating current stimulation (tACS) and rTMS applied either to the posterior parietal cortex or frontal eye field can reduce phosphene threshold and exert a direct, top-down modulation over extrastriate visual areas (Silvanto et al. 2006; Taylor et al. 2010). Altogether, these findings strengthen the hypothesis that phosphene perception arises from a wider network than previously expected, ranging from occipital to central and parietal cortex (Lamme and Roelfsema 2000; Lamme et al. 2000; Pascual-Leone and Walsh 2001; Taylor et al. 2010). The stimulation protocol that we employed is also quite different from that used by Lang et al.; as current density distribution strictly depends both on the position of the electrodes and the geometry of tissue compartments (Holdefer et al. 2006; Miranda et al. 2006), we preferred to use a classical monopolar montage. This approach reduces interference between anodal and cathodal effects and decreases cutaneous shunt through the scalp, rising at the same time the current density in the depth of primary visual cortex. Finally, it is worth remembering that in the study by Lang et al., the effects on PT of low-frequency rTMS following priming tDCS was not explored. The most likely interpretation of our results is that a tDCS-induced modulation in cell excitability of cortical neurons alters the modification threshold for synaptic plasticity induced by rTMS, ultimately affecting the synaptic potentials responsible for the generation of VEPs. Other studies in the literature have provided evidence in favor of similar BCM-like mechanisms in the visual cortex of human subjects. In particular, Boroojerdi et al. (2000) and Fierro et al. (2005) examined the effects of light deprivation on PT. A reduced PT was detected as soon as 45 min after the onset of light deprivation, and PT returned to baseline levels following re-exposure to light (Boroojerdi et al. 2000; Fierro et al. 2005). Of note, 1 Hz rTMS applied during the last 15 min of light deprivation
Effects of tDCS and rTMS on visual evoked responses
significantly prolonged the time needed to recover baseline PT values following light re-exposure (Fierro et al. 2005). Thus, conventionally inhibitory rTMS following light deprivation produces a paradoxical effect, tending to maintain higher cortical excitability (i.e., reduced phosphene threshold). Boroojerdi et al. (2000) also examined visual stimulus-dependent activation of cortical areas by functional magnetic resonance imaging and found increased visual cortex activation after 60 min of light deprivation. Altogether, these data are consistent with enhanced excitability of visual cortex after a period of reduced network activity. Recently, Lunghi et al. (2011) have shown that brief periods of monocular deprivation (patching of one eye) disrupt ocular balance in human adult visual cortex, with the deprived eye prevailing in conscious perception twice as much as the non-deprived eye. The apparent contrast of stimuli presented to the deprived eye was also increased, suggesting that visual deprivation may paradoxically enhance signal strength of the deprived eye (Lunghi et al. 2011). It is important to note that animal studies have provided important support for BCM-like synapses in the visual cortex. A key study (Kirkwood et al. 1996) compared visual cortical slices from naı¨ve rats and rats reared in the dark to reduce afferent input activity to the cortex. The authors found that the threshold for synaptic potentiation/ depression (i.e., LTP/LTD) was significantly shifted to the left in the slices from the dark-reared animals (Kirkwood et al. 1996). Thus, when neuronal activity levels are reduced by visual deprivation, the LTD/LTP plasticity threshold is lowered such that stimulation is more likely to trigger synaptic potentiation (LTP) and less likely to cause synaptic weakening (LTD). These data provide direct evidence that the threshold for LTP/LTD is not fixed, but can slide according to the previous levels of activity. This mechanism of sliding is important since it allows keeping synaptic strengths within a functional dynamic range (Abraham 2008). Previous evidence in animal models has suggested a role for different molecules and signaling pathways in the regulation of metaplastic and homeostatic mechanisms (Abraham 2008; Turrigiano 2008, 2011). No pharmacological data are currently available to explain metaplastic regulation of neuronal excitability for the human cerebral cortex; therefore further studies are needed to reveal the molecular bases of this kind of plasticity. There are other questions raised by the present study that remain to be addressed in future investigations. First, it will be important to dissect the contribution of pre- and post-synaptic mechanisms to the induction of metaplastic phenomena in the cortex. Second, it will be interesting to disclose eventual differences among cortical layers in the activation of BCM-like mechanisms.
How tDCS may prime new plasticity responses to rTMS remains open to speculation. Previous studies on the visual cortex in animal models (Philpot et al. 2001, 2003) have shown that metaplasticity is potently regulated by Nmethyl-D-aspartate (NMDA) receptor function, through modifications both in the number of NMDA receptors and in the lengthening of NMDA-mediated excitatory postsynaptic current (EPSCs). Thus, the aftereffects induced by tDCS may be mainly dependent on NMDA receptors (Liebetanz et al. 2002; Nitsche et al. 2003b). However, it is possible that other neurotransmitters could account for tDCS-induced changes in responsiveness of the visual cortex, as suggested by the evidence that amantadine, a low-affinity, non-competitive NMDA receptor antagonist, is not effective in modulating VEPs amplitude (Bandini et al. 2002). Another possibility is that tDCS modulates metaplasticity changes through modifications in GABAergic synaptic transmission (Nitsche et al. 2004), as BCMlike plasticity also depends on GABA receptor function (Kalantzis and Shouval 2009).
Conclusions Discovery of BCM-like regulation of visual responses in humans is important since homeostatic phenomena and metaplasticity are believed to underlie the ability of cortical networks to maintain stable function in the face of developmental or learning-induced changes in drive. Metaplasticity could play a role in circuit rearrangements triggered in the visual cortex by alterations in sensory input, as suggested by previous animal studies (Ranson et al. 2012). Metaplasticity could also be crucial for the process of plasticity in the adult, healthy visual system; it is known that adult visual cortex retains a surprisingly high degree of plasticity fundamental in reaction to sensory loss (Sabel 2008; Lunghi et al. 2011). Similarly, our results might be helpful in evaluating dynamic changes in visual responsiveness following stroke or other brain lesions, in adults as well as in childhood. In this context, it is interesting to note that neuromodulation by NIBS techniques as rTMS is increasingly being used in a series of pathologies to inactivate/stimulate cortical networks, and in the treatment of visual dysfunctions such as amblyopia (Thompson et al. 2008). Our data indicate that the magnitude and direction of plasticity induced by rTMS depends on the preexisting, functional state of the cortex, pointing to possible interindividual differences in the response to inhibitory/excitatory rTMS. Acknowledgments We gratefully acknowledge the participation of all subjects and Mr. C. Orsini and Ms. M. Naraci for their excellent technical assistance. The paper was supported in part by the Italian
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T. Bocci et al. operating and development MIUR PRIN grant year 2006, n. 2006062332_002. Alberto Priori is founder and shareholder of Newronika srl, Milan, Italy.
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