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tDCS-induced modulation of GABA concentration and dopamine release in the human brain: A combination study of magnetic resonance spectroscopy and positron emission tomography

Open AccessPublished:December 23, 2020DOI:https://doi.org/10.1016/j.brs.2020.12.010

      Highlights

      • tDCS to the DLPFC increases GABA and dopamine in the striatum.
      • Reductions in GABA in the left DLPFC correlated with GABA and dopamine concentrations in the striatum.
      • tDCS to the DLPFC may modulate dopamine-GABA functions in the basal ganglia-cortical circuit.

      Abstract

      Background

      Transcranial direct current stimulation (tDCS) to the dorsolateral prefrontal cortex (DLPFC) hypothetically modulates cognitive functions by facilitating or inhibiting neuronal activities chiefly in the cerebral cortex. The effect of tDCS in the deeper brain region, the basal ganglia-cortical circuit, remains unknown.

      Objective

      To investigate the interaction between γ-aminobutyric acid (GABA) concentrations and dopamine release following tDCS.

      Method

      This study used a randomized, placebo-controlled, double-blind, crossover design. Seventeen healthy male subjects underwent active and sham tDCS (13 min twice at an interval of 20 min) with the anode placed at the left DLPFC and the cathode at the right DLPFC, followed by examinations with [11C]-raclopride positron emission topography (PET) and GABA-magnetic resonance spectroscopy (MRS). MRS voxels were set in the left DLPFC and bilateral striata. Paired t-tests and regression analyses were performed for PET and MRS parameters.

      Results

      MRS data analyses showed elevations in GABA in the left striatum along with moderate reductions in the right striatum and the left DLPFC after active tDCS. PET data analyses showed that reductions in [11C]-raclopride binding potentials (increase in dopamine release) in the right striatum were inversely correlated with those in the left striatum after active tDCS. GABA reductions in the left DLPFC positively correlated with elevations in GABA in the left striatum and with increases in right striatal dopamine release and negatively correlated with increases in left striatal dopamine release.

      Conclusion

      The present results suggest that tDCS to the DLPFC modulates dopamine-GABA functions in the basal ganglia-cortical circuit.

      Keywords

      Introduction

      Transcranial direct current stimulation (tDCS) to the dorsolateral prefrontal cortex (DLPFC) has attracted attention as a new treatment for neuropsychiatric disorders, and clinical improvement in Alzheimer’s disease [
      • Khedr E.M.
      • El Gamal N.F.
      • El-Fetoh N.A.
      • Khalifa H.
      • Ahmed E.M.
      • Ali A.M.
      • et al.
      A double-blind randomized clinical trial on the efficacy of cortical direct current stimulation for the treatment of Alzheimer’s disease.
      ] and depression [
      • Brunoni A.R.
      • Moffa A.H.
      • Sampaio B.
      • Borrione L.
      • Moreno M.L.
      • Fernandes R.A.
      • et al.
      Trial of electrical direct-current therapy versus escitalopram for depression.
      ] has been reported. The mechanism of tDCS is thought to be through electrophysiological modulation of neurotransmitters such as glutamate and γ-aminobutyric acid (GABA) at the site of stimulation [
      • Filmer H.L.
      • Dux P.E.
      • Mattingley J.B.
      Applications of transcranial direct current stimulation for understanding brain function.
      ]. Recently, tDCS to the DLPFC has been shown to increases dopamine release in the striatum [
      • Fonteneau C.
      • Redoute J.
      • Haesebaert F.
      • Le Bars D.
      • Costes N.
      • Suaud-Chagny M.F.
      • et al.
      Frontal transcranial direct current stimulation induces dopamine release in the ventral striatum in human.
      ], and we showed a clear correlation between improvements in cognitive function and dopamine release in the right striatum [
      • Fukai M.
      • Bunai T.
      • Hirosawa T.
      • Kikuchi M.
      • Ito S.
      • Minabe Y.
      • et al.
      Endogenous dopamine release under transcranial direct-current stimulation governs enhanced attention: a study with positron emission tomography.
      ]. Since the currently accepted mechanism of tDCS relates to the occurrence of polarity-specific changes in neuronal excitability, i.e., increments or decrements [
      • Creutzfeldt O.D.
      • Fromm G.H.
      • Kapp H.
      Influence of transcortical d-c currents on cortical neuronal activity.
      ], these findings help understand a tDCS-related positive effect on cognitive functions in a variety of situations, i.e., a contribution of the dopaminergic system beyond the neuronal excitability theory itself. Although an online effect of tDCS reportedly failed to alter brain GABA levels [
      • Hone-Blanchet A.
      • Edden R.A.
      • Fecteau S.
      Online effects of transcranial direct current stimulation in real time on human prefrontal and striatal metabolites.
      ], there has been no study about its offline effect on GABA, which is the most conventional application in a repeated/intermittent stimulation manner. Because tDCS was reported to alter dopamine in the striatum as mentioned, it can be speculated that tDCS modulates neurotransmitters in the basal ganglia-cortical circuit. This speculation (stimulation-induced dopamine release) may be further supported by a previous non-invasive brain stimulation study with transcranial magnetic stimulation (TMS) showing that the stimulation to the left DLPFC enhanced dopamine release in the striatum [
      • Strafella A.P.
      • Paus T.
      • Barrett J.
      • Dagher A.
      Repetitive transcranial magnetic stimulation of the human prefrontal cortex induces dopamine release in the caudate nucleus.
      ]. However, it remains unclear how tDCS modulates GABA within the basal ganglia-cortical circuit in relation to the dopaminergic response.
      The basal ganglia-cortical circuit is not only an anatomical system but also a functional system that is always working properly during motor, emotional and cognitive performances [
      • Alexander G.
      Parallel organization of functionally segregated circuits linking basal ganglia and cortex.
      ,
      • Middleton F.A.
      Basal-ganglia “projections” to the prefrontal cortex of the primate.
      ,
      • Haber S.N.
      The primate basal ganglia: parallel and integrative networks.
      ]. Glutamate, dopamine and GABA are key players as neurotransmitters engaging in basal ganglia-cortical circuit regulation, which has been closely linked with various cognitive functions in humans [
      • Braskie M.N.
      • Wilcox C.E.
      • Landau S.M.
      • O’Neil J.P.
      • Baker S.L.
      • Madison C.M.
      • et al.
      Relationship of striatal dopamine synthesis capacity to age and cognition.
      ,
      • Landau S.M.
      • Lal R.
      • O’Neil J.P.
      • Baker S.
      • Jagust W.J.
      Striatal dopamine and working memory.
      ,
      • Edden R.A.E.
      • Muthukumaraswamy S.D.
      • Freeman T.C.A.
      • Singh K.D.
      Orientation discrimination performance is predicted by GABA concentration and gamma oscillation frequency in human primary visual cortex.
      ,
      • Sumner P.
      • Edden R.A.E.
      • Bompas A.
      • Evans C.J.
      • Singh K.D.
      More GABA, less distraction: a neurochemical predictor of motor decision speed.
      ]. Among these neurotransmitters, dopamine may have two functional roles: excitatory and inhibitory [
      • Gao W.J.
      • Goldman-Rakic P.S.
      Selective modulation of excitatory and inhibitory microcircuits by dopamine.
      ]. A previous functional connectivity study showed that l-DOPA administration to healthy subjects increased neuronal variability in various somatosensory and motor regions [
      • Cole D.M.
      • Beckmann C.F.
      • Oei N.Y.L.
      • Both S.
      • van Gerven J.M.A.
      • Rombouts S.A.R.B.
      Differential and distributed effects of dopamine neuromodulations on resting-state network connectivity.
      ], suggesting that dopamine signaling increased intranetwork functional connectivity and tissue activity within the somatosensory region. In this respect, dopamine works as an excitatory molecule, unlike GABA, which functions in an inhibitory manner. Considering neuropsychiatric disorders such as Parkinson’s disease (PD) and schizophrenia, therapeutic targets are mostly dopamine and GABA systems within this circuit. In addition, recent reports about these neuropsychiatric disorders with tDCS ameliorating dopamine-related or GABA-involved symptoms [
      • McLaren M.E.
      • Nissim N.R.
      • Woods A.J.
      The effects of medication use in transcranial direct current stimulation: a brief review.
      ] would suggest that there is some interaction or modification in these two neurotransmitter systems under tDCS. Indeed, most striatal medium spiny neurons contain GABA, and anodal stimulation that would theoretically depolarize neurons under glutamatergic activation may increase GABA concentrations in the striatum.
      Many mechanistic studies of tDCS have likely focused on the neurons and glia residing in the cerebral cortical regions, not in a deeper brain region such as the basal ganglia. Here, we investigated tDCS-induced changes in dopamine and GABA levels in the basal ganglia-cortical circuit region using [11C]-raclopride positron emission topography (PET) and GABA-MRS to explore the relationship between these two neurotransmitters following tDCS to the DLPFC.

      Material and methods

      Subjects

      We recruited 22 healthy 20–26-year-old men using poster advertisements from a neighboring university. Five subjects were excluded for the following reasons: 2 experienced claustrophobia in the MRI scanner and 3 showed signal artifacts induced by motion. Therefore, data from 17 subjects were analyzed in this study (Fig. 1A). All subjects were right-handed native Japanese with an intelligence quotient (IQ) higher than 80 (mean [SD] IQ, 109.2 [6.7]; IQ range of 97–120) on the Japanese reading test (JART) [
      • Matsuoka K.
      • Uno M.
      • Kasai K.
      • Koyama K.
      • Kim Y.
      Estimation of premorbid IQ in individuals with Alzheimer’s disease using Japanese ideographic script (Kanji) compound words: Japanese version of National Adult Reading Test.
      ]. Subjects with a history of traumatic brain injury, organic brain diseases, psychiatric disorder, seizure, chronic headache, or an episode of unconsciousness lasting more than 5 min and subjects who could not undergo an MRI scan were excluded from the study. Written informed consent was obtained before enrollment. The Ethics Committees of Hamamatsu University School of Medicine and Hamamatsu Medical Photonics Foundation approved the methods and procedures used for this study, which were applied in accordance with the Declaration of Helsinki. The study was registered with the University Hospital Medical Information Network Clinical Trials Registry (no. UMIN000020583).
      Fig. 1
      Fig. 1Experimental design
      Consolidated Standards of Reporting Trials (CONSORT) flow chart (A) and placement of volumes of interest (B).

      Experimental design

      This study used a randomized, sham-controlled, double-blind, crossover design. The participants completed two sessions at least 1 month apart to control for carry-over effects. The participants then underwent 26 min of tDCS (either active or sham) to the DLPFC, after which PET, magnetic resonance imaging (MRI) and MRS were performed. PET was performed at 50 min after initiation of tDCS, followed by MRS measurement at 140 min post-stimulation (Supplementary Fig. 1).

      Intervention: tDCS

      Two soaked (NaCl 0.9%) anodal and cathodal electrodes (35 cm2) using DC-STIMULATOR PLUS (neuroCare Group GmbH, Ilmenau, Germany) were placed at the left frontal areas and right frontal areas, respectively. Thirteen minutes of tDCS with a current intensity of 2 mA was applied twice at an interval of 20 min. After more than a month, sham stimulation was applied. With the same electrodes placed as in the true stimulation condition, the sham stimulation was switched off 30 s after the stimulation started. The stimulation type (active or sham) was randomized across sessions and crossovered across participants. The interventions with tDCS were almost identical to those used in a recent study [
      • Fukai M.
      • Bunai T.
      • Hirosawa T.
      • Kikuchi M.
      • Ito S.
      • Minabe Y.
      • et al.
      Endogenous dopamine release under transcranial direct-current stimulation governs enhanced attention: a study with positron emission tomography.
      ]. No side effects were found during and after active and sham tDCS in all participants.

      MRI scanning and MRS measurements

      Scanning was performed with a 3T MRI machine (Ingenia; Philips Healthcare, Best, The Netherlands). MRI was applied to ascertain the areas of concern for establishing the volumes of interest (VOIs) with the following acquisition parameters: three-dimensional mode sampling, TR shortest (6 ms) and TE shortest (2.7 ms), 8° flip angle, 0.9 × 0.9 × 0.9 mm3 voxel size, 210 slices).
      Spectroscopy measurements were acquired just after each PET scan. We obtained spectroscopy measurements using the MEGA-PRESS sequence for detecting GABA and other brain metabolites. VOIs for MRS were established in the left DLPFC and bilateral striata (Fig. 1B). The MEGA-PRESS spectra were acquired from 45 × 30 × 20 mm3 voxels (left DLPFC) and 30 × 30 × 30 mm3 voxels (bilateral striata). We used the following spectroscopy parameters: TR/TE = 2000/68 ms, 320 averages, 10-min scan. We analyzed GABA and NAA concentrations using LCModel software (Version 6.3-1L; Stephen Provencher Inc., Oakville, Ontario, Canada).

      PET data acquisition and image data analyses

      As recently described in detail [
      • Fukai M.
      • Bunai T.
      • Hirosawa T.
      • Kikuchi M.
      • Ito S.
      • Minabe Y.
      • et al.
      Endogenous dopamine release under transcranial direct-current stimulation governs enhanced attention: a study with positron emission tomography.
      ], positron emission tomography was performed using a high-resolution brain PET scanner (SHR12000; Hamamatsu Photonics K.K., Hamamatsu, Japan), and the [11C]-raclopride binding potential (BPND) in each region was estimated based on a noninvasive Logan plot analysis using software (PMOD 3.5; PMOD Technologies LLC, Zurich, Switzerland). Regions of interest (ROIs) were located bilaterally in the striatum, in which tDCS has been shown to increase the level of endogenous dopamine release.

      Statistical analyses

      Data were analyzed using software (Statistical Package for Social Sciences ver. 23; SPSS Inc., Chicago, IL, USA). Paired t tests were applied to compare the GABA:NAA ratios in the respective brain regions between active and sham stimulation conditions. Pearson correlations were used to assess the correlations between differences ([active]-[sham]) in the GABA:NAA ratio in each region (left DLPFC and bilateral striata) and %reduction ({[sham]-[active]}/[sham]) of [11C]-raclopride BPND in the bilateral striata after active and sham stimulations. For all results, statistical significance was inferred for p < 0.05 after correction for multiple comparisons.

      Results

      Post-tDCS changes in the GABA:NAA ratio and correlations

      Paired t tests showed that the GABA:NAA ratio in the left striatum was higher after active stimulation than after sham stimulation, but correction for multiple comparisons prevented our data from reaching significance (P = 0.021). In contrast, the mean value of the difference in the GABA:NAA ratio in the right striatum and left DLPFC after active tDCS was below zero (Table 1, Fig. 2A). A significant negative correlation was found between changes in the GABA:NAA ratios in the left striatum and those in the left DLPFC (r = −0.575, p = 0.016) (Fig. 2B), indicating that the higher the GABA:NAA ratio in the left striatum became, the lower the value in the left DLPFC. There was no correlation between the right striatum and the left DLPFC (Fig. 2C).
      Table 1Levels of GABA/NAA after active and sham stimulation.
      StimulationStriatumDLPFC
      LeftRightLeft
      Active0.38 ± 0.05∗0.31 ± 0.050.23 ± 0.06
      Sham0.34 ± 0.060.33 ± 0.050.25 ± 0.05
      Data are presented as mean ± SD. ∗p < 0.05 vs sham.
      DLPFC: dorsolateral prefrontal cortex.
      Fig. 2
      Fig. 2Changes in GABA:NAA ratios in the cortico-striatal regions
      The GABA:NAA ratio was higher in the left striatum after active stimulation than sham stimulation (∗p < 0.05). The ordinate denotes %change. B and C shows correlations with the GABA:NAA ratio.

      Correlations of [11C]-raclopride binding between the bilateral striata

      The %reduction in [11C]-raclopride BPND (an index of dopamine release) was higher in the right striatum (Fig. 3A), as reported previously [
      • Fukai M.
      • Bunai T.
      • Hirosawa T.
      • Kikuchi M.
      • Ito S.
      • Minabe Y.
      • et al.
      Endogenous dopamine release under transcranial direct-current stimulation governs enhanced attention: a study with positron emission tomography.
      ]. There was a significantly negative correlation between %reduction in [11C]-raclopride BPND in the right and left striata (r = −0.542, p = 0.025) (Fig. 3B). This [11C]-raclopride PET result indicated that dopamine release in the bilateral striatal regions after prefrontal tDCS varied in opposite directions.
      Fig. 3
      Fig. 3Changes in [11C]-raclopride BPND in the striatum
      Significant increases in [11C]-raclopride BPND in the right striatum (A). Correlation of changes in [11C]-raclopride BPND between the bilateral striata (B).

      Correlations between the cortical MRS and striatal PET data

      Correlation analysis showed a significant positive correlation between %reduction of [11C]-raclopride BPND (dopamine release index) in the left striatum and a difference in the GABA:NAA ratio in the left DLPFC (r = 0.564, p = 0.018) (Fig. 4A) and a negative correlation between %reduction of [11C]-raclopride BPND in the right striatum and a difference in the GABA:NAA ratio in the left DLPFC (r = −0.686, p = 0.002) (Fig. 4B), indicating that as %reduction of [11C]-raclopride BPND (dopamine release index) in the left striatum decreased and that in the right striatum increased, the GABA:NAA ratio in the left DLPFC decreased.
      Fig. 4
      Fig. 4Correlations between the cortical MRS and striatal PET data.
      A positive correlation is present between the left DLPFC MRS data and the left striatal PET data, and a negative correlation is present between the left DLPFC MRS data and the right striatal PET data (p < 0.05).

      Discussion

      The present study showed that tDCS under this particular electrode montage (an anode over the left DLPFC and a cathode over the right DLPFC) caused significant increases in dopamine release in the right striatum and GABA concentrations in the left striatum. The GABA concentrations in the left DLPFC under the anode tended to be lower, in line with a previous report [
      • Filmer H.L.
      • Dux P.E.
      • Mattingley J.B.
      Applications of transcranial direct current stimulation for understanding brain function.
      ] that stimulation of the anode suppressed GABA transmission. Positive correlations of cortical GABA change with ipsilateral striatal GABA changes and with contralateral striatal dopamine release after DLPFC anodal tDCS suggest that tDCS could modulate the monoaminergic systems in the deep brain structures, which would go beyond the theory of a tDCS mechanism based on glutamatergic and GABAergic activities changing within the cortical region.
      In the present double-blind, crossover design study, the participants received 13 min of stimulation twice at an interval of 20 min. It has been reported that tDCS induces after-effects lasting for more than 24 h with a single stimulation [
      • McIntire L.K.
      • McKinley R.A.
      • Goodyear C.
      • McIntire J.P.
      The effects of anodal transcranial direct current stimulation on sleep time and efficiency.
      ,
      • Nelson J.T.
      • McKinley R.A.
      • Golob E.J.
      • Warm J.S.
      • Parasuraman R.
      Enhancing vigilance in operators with prefrontal cortex transcranial direct current stimulation (tDCS).
      ] or repeated stimulations within short intervals [
      • Monte-Silva K.
      • Kuo M.F.
      • Hessenthaler S.
      • Fresnoza S.
      • Liebetanz D.
      • Paulus W.
      • et al.
      Induction of late LTP-like plasticity in the human motor cortex by repeated non-invasive brain stimulation.
      ]; hence, our study was sufficient to evaluate after-effects using PET and MRS after the stimulations. Regarding the tDCS montage, stimulation of the motor cortex is well known to induce changes in motor performance and learning [
      • Morya E.
      • Monte-Silva K.
      • Bikson M.
      • Esmaeilpour Z.
      • Biazoli C.E.
      • Fonseca A.
      • et al.
      Beyond the target area: an integrative view of tDCS-induced motor cortex modulation in patients and athletes.
      ]. In contrast to motor cortex stimulation, stimulation of the DLPFC has been shown to strongly correlate with cognitive function and caused higher electric field peaks in the striatum than stimulation with other montages [
      • Gomez-Tames J.
      • Asai A.
      • Hirata A.
      Significant group-level hotspots found in deep brain regions during transcranial direct current stimulation (tDCS): a computational analysis of electric fields.
      ]. Similarly, our previous study focused on attention enhancement and dopamine release in the striatum [
      • Fukai M.
      • Bunai T.
      • Hirosawa T.
      • Kikuchi M.
      • Ito S.
      • Minabe Y.
      • et al.
      Endogenous dopamine release under transcranial direct-current stimulation governs enhanced attention: a study with positron emission tomography.
      ]. There has been only one study of the striatal GABA system in which GABA concentrations did not significantly change under online stimulation: 1 mA of continuous stimulation for 30 min [
      • Hone-Blanchet A.
      • Edden R.A.
      • Fecteau S.
      Online effects of transcranial direct current stimulation in real time on human prefrontal and striatal metabolites.
      ]. The discrepancy with our results could be explained by different protocols, such as the timing of the stimulation, the current intensity (1 mA vs. 2 mA) and interstimulation intervals.
      In the present study, GABA in the left striatum and dopamine in the right striatum were significantly increased after active tDCS. The left DLPFC and right striatal GABA levels tended to decrease. There was a positive correlation between reduced GABA in the left DLPFC and increased GABA in the left striatum and dopamine in the right striatum and a negative correlation between reduced GABA in the left DLPFC and increased dopamine in the left striatum. Fig. 5 is a schematic summary of the present study, illustrating that DLPFC anodal tDCS upregulates ipsilateral striatal GABA concentrations and contralateral striatal dopamine release. Previous animal experiments with neurochemical substances have shown that prefrontal cortical dopamine and GABA cooperate to modulate the release of dopamine in the striatum [
      • Del Arco A.
      • Mora F.
      Glutamate-dopamine in vivo interaction in the prefrontal cortex modulates the release of dopamine and acetylcholine in the nucleus accumbens of the awake rat.
      ] and that stimulation of GABA systems decreased dopamine release in the striatum [
      • Pitman K.A.
      • Puil E.
      • Borgland S.L.
      GABAB modulation of dopamine release in the nucleus accumbens core.
      ,
      • Balla A.
      • Nattini M.E.
      • Sershen H.
      • Lajtha A.
      • Dunlop D.S.
      • Javitt D.C.
      GABAB/NMDA receptor interaction in the regulation of extracellular dopamine levels in rodent prefrontal cortex and striatum.
      ]. Consistently, we found in the present study that the GABA reductions in the left DLPFC were associated with striatal dopamine release, the GABA increases in the left striatum reduced dopamine release, and the GABA reductions in the right striatum coincided with dopamine release. A previous MRS study showed that the lower left DLPFC GABA was associated with greater performance of working memory [
      • Yoon J.H.
      • Grandelis A.
      • Maddock R.J.
      Dorsolateral prefrontal cortex GABA concentration in humans predicts working memory load processing capacity.
      ]. In line with this finding, we reported that improvement of attention correlates with striatal dopamine release [
      • Fukai M.
      • Bunai T.
      • Hirosawa T.
      • Kikuchi M.
      • Ito S.
      • Minabe Y.
      • et al.
      Endogenous dopamine release under transcranial direct-current stimulation governs enhanced attention: a study with positron emission tomography.
      ] in the tDCS experiment, in which the GABA content in the DLPFC under the anodal stimulation was lower as shown in the current study. The functional connectivity study on the cortico-striatal circuit showed that anodal tDCS on the motor cortex (albeit not DLPFC) augmented functional coupling between the striatum and parietal cortex on the same hemispheric side [
      • Polanía R.
      • Paulus W.
      • Nitsche M.A.
      Modulating cortico-striatal and thalamo-cortical functional connectivity with transcranial direct current stimulation.
      ], suggesting that the striatum and cerebral cortex were functionally connected possibly using information of neurotransmitters such as GABA and dopamine as found in this study. Thus, anodal tDCS to the DLPFC may exert a modulatory effect on behavior by regulating neurotransmitters possibly to make functional connectivity optimally tuned within the cortico-striatal circuit.
      Fig. 5
      Fig. 5Illustration of the prefrontal tDCS effect on the GABA and dopamine systems
      Cortical tDCS differentially affects the monoaminergic systems in deep brain regions.
      The present study showing tDCS-induced dopamine and GABA changes in the striatum may be a clinical proof of concept, which allows us to apply this method to patients with monoaminergic dysfunction. Indeed, tDCS to the DLPFC may be useful in Parkinson’s disease (PD) because it has been reported that PD patients manifest dopamine deficiency and a decrease in GABA in the basal ganglia as assessed by MRS [
      • Gong T.
      • Xiang Y.
      • Saleh M.G.
      • Gao F.
      • Chen W.
      • Edden R.A.E.
      • et al.
      Inhibitory motor dysfunction in Parkinson’s disease subtypes.
      ]. In many studies of tDCS applied to PD patients, the motor cortex is a main target, and the effects have varied depending on the study [
      • Simpson M.W.
      • Mak M.
      The effect of transcranial direct current stimulation on upper limb motor performance in Parkinson’s disease: a systematic review.
      ]. Considering the present result, i.e., the significant effect of tDCS to the DLPFC on dopamine and GABA in the basal ganglia, tDCS to the DLPFC may be promising for treating PD patients. Furthermore, as shown in patients with AD, whose cognitive functions were improved by stimulation of the temporal cortex [
      • Cai M.
      • Guo Z.
      • Xing G.
      • Peng H.
      • Zhou L.
      • Chen H.
      • et al.
      Transcranial direct current stimulation improves cognitive function in mild to moderate Alzheimer disease: a meta-analysis.
      ], DLPFC stimulation may be effective in modulating affective changes in the course of the disease. The lack of studies examining the cholinergic system after tDCS may propel researchers to investigate whether tDCS to the DLPFC can exert an influence on the cholinergic system or accumulation of misfolded proteins such as amyloid and tau not only in the cortex but also in the deep brain regions in patients with AD.
      There are some limitations to be noted in the present study. First, since PET and MRS were not simultaneously measured, interpretation of a direct relation between GABA and dopamine should be cautiously made. However, consecutive examinations within a day may be sufficient for discussing effects across the different modalities (PET and MRS). Simultaneous PET/MRI experiments may solve this problem. Second, a lack of MRS data from the right DLPFC due to time constraints did not allow us to further discuss the contribution of cathodal tDCS to changes in GABA. This needs future study. Third, although the effects of tDCS may vary based on individual factors, e.g., brain morphometry and BDNF gene polymorphisms [
      • Bouchard A.E.
      • Dickler M.
      • Renauld E.
      • Lenglos C.
      • Ferland F.
      • Edden R.A.
      • et al.
      The impact of brain morphometry on tDCS effects on GABA levels.
      ,
      • Antal A.
      • Chaieb L.
      • Moliadze V.
      • Monte-Silva K.
      • Poreisz C.
      • Thirugnanasambandam N.
      • et al.
      Brain-derived neurotrophic factor (BDNF) gene polymorphisms shape cortical plasticity in humans.
      ], it is unlikely that the rather homogeneous population, young Japanese male subjects, would show significant differences in parameters that confound the present results. Fourth, despite no emotional or behavioral changes apparent from self-reported data of spending one month between measurements (active and sham tDCS), an intra-individual fluctuation in the brain was not measurable in this study. As such, we have to assume different brain states because there are too many influencing factors and our brain is an enormously complex system. Lastly, because of the limited number of regions selected, a strict correction for multiple tests prevented our data (i.e., increase in GABA in the left striatum) from reaching significance in this study. Therefore, further exploration of the cortico-striatal region with a larger sample size is needed.

      Conclusions

      The tDCS montage including the left anodal DLPFC and right cathodal DLPFC can generate GABAergic excitation in the ipsilateral striatum concomitant with mild GABAergic reductions in the left DLPFC. Although data were obtained from different modalities (PET and MRI), the 24-h lasting after-effects of tDCS [
      • Monte-Silva K.
      • Kuo M.F.
      • Hessenthaler S.
      • Fresnoza S.
      • Liebetanz D.
      • Paulus W.
      • et al.
      Induction of late LTP-like plasticity in the human motor cortex by repeated non-invasive brain stimulation.
      ] allows us to speculate that dopamine release is responsive to the stimulation in an antagonistic manner to the GABA response. DLPFC-tDCS may modulate dopamine-GABA systems in the basal ganglia-cortical circuit at a molecular level.

      CRediT authorship contribution statement

      Tomoyasu Bunai: Methodology, Data curation, Formal analysis, Investigation, Writing - original draft. Tetsu Hirosawa: Investigation. Mitsuru Kikuchi: Project administration, Funding acquisition. Mina Fukai: Investigation. Masamichi Yokokura: Investigation. Shigeru Ito: Investigation. Yohei Takata: Software. Tatsuhiro Terada: Investigation. Yasuomi Ouchi: Conceptualization, Methodology, Writing - review & editing, Funding acquisition.

      Declaration of competing interest

      Fig. S1
      Fig. S1Design and timeline of this study.
      The type of stimulation (active or sham) was randomized in a double-blind manner. Thirteen minutes duration tDCS was applied twice with an interval of 20 min. PET was performed at 50 min after initiation of tDCS, followed by MRS measurement at 140 min. Participants underwent twice experiments in a cross over fashion at least 1 month apart.

      Acknowledgments

      This research is supported by the Center of Innovation Program from Japan Science and Technology Agency, JST , and partly by Scientific Research on Innovative Areas ( JP16H06402 ). The authors wish to thank Professor Richard A. Edden at The Johns Hopkins University (USA) for his great help with the GABA-MRS study and the staff of Hamamatsu Medical Imaging Center for their valuable support. We also thank Philips for the MRS technical support.

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