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Transcranial alternating current stimulation rescues motor deficits in a mouse model of Parkinson's disease via the production of glial cell line-derived neurotrophic factor

  • Hong Ju Lee
    Affiliations
    Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 50612, Republic of Korea

    Graduate Training Program of Korean Medical Therapeutics for Healthy Aging, Pusan National University, Yangsan, 50612, Republic of Korea
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  • Da Hee Jung
    Affiliations
    Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 50612, Republic of Korea

    Graduate Training Program of Korean Medical Therapeutics for Healthy Aging, Pusan National University, Yangsan, 50612, Republic of Korea
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  • Young Jin Jung
    Affiliations
    School of Healthcare and Biomedical Engineering, Chonnam National University, Yeosu, 59626, Republic of Korea
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  • Hwa Kyoung Shin
    Correspondence
    Corresponding authors. Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 50612, Republic of Korea.
    Affiliations
    Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 50612, Republic of Korea

    Graduate Training Program of Korean Medical Therapeutics for Healthy Aging, Pusan National University, Yangsan, 50612, Republic of Korea
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  • Byung Tae Choi
    Correspondence
    Corresponding authors. Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 50612, Republic of Korea.
    Affiliations
    Department of Korean Medical Science, School of Korean Medicine, Pusan National University, Yangsan, 50612, Republic of Korea

    Graduate Training Program of Korean Medical Therapeutics for Healthy Aging, Pusan National University, Yangsan, 50612, Republic of Korea
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Open AccessPublished:April 13, 2022DOI:https://doi.org/10.1016/j.brs.2022.04.002

      Highlights

      • HD-tACS application alleviates motor dysfunction in PD mouse model.
      • Effects of beta-frequency 20 Hz HD-tACS is more prominent than other frequency.
      • HD-tACS with 20 Hz promotes the survival of dopaminergic neurons in the substantia nigra.
      • HD-tACS with 20 Hz upregulates GDNF production in the striatum.

      Abstract

      Background

      Therapeutic effects of transcranial alternating current stimulation (tACS) for treating Parkinson's disease (PD) are limited to modulating abnormally synchronized oscillations; however, long-lasting tACS effects may involve non-neuronal mechanisms like the regulation of neurotrophic factors.

      Objectives/Hypothesis

      We investigated whether tACS exerts neuroprotective effects on dopaminergic neurons in a mouse model of PD by regulating endogenous glial cell line-derived neurotrophic factor (GDNF).

      Methods

      Repeated high-definition tACS (HD-tACS, 20 min, 89.1 μA/mm2) was administered over the primary motor cortex of C57BL/6J 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mice. Behavioral tests assessing motor function, immunohistochemistry, western blots, enzyme-linked immunosorbent assays, and flow cytometric analyses were performed to examine suitable tACS conditions and its underlying mechanisms.

      Results

      Stimulation at representative frequencies (theta to gamma; 20-Hz beta frequency, in particular) attenuated motor dysfunction and protected the dopaminergic neurons with increased GDNF production. Beta-frequency (20 Hz) tACS application significantly attenuated motor deficits to levels comparable with those of levodopa treatment. Moreover, beta-frequency tACS induced the survival of dopaminergic neurons in the substantia nigra with upregulated production of endogenous GDNF in striatal parvalbumin-positive interneurons. An inhibitor of the GDNF receptor-associated rearranged during transfection (RET) kinase suppressed most aspects of the tACS-induced behavioral recovery, dopaminergic cell survival, and GDNF production. Beta-frequency tACS activated RET-related survival signaling for dopaminergic neurons in the substantia nigra.

      Conclusions

      Application of tACS over the primary motor cortex may exert protective effects on dopaminergic neurons in the substantia nigra via activation of endogenous GDNF production by striatal parvalbumin-positive interneurons and its survival signaling.

      Graphical abstract

      Keywords

      1. Introduction

      Parkinson's disease (PD) is a progressive disorder characterized by classic diagnostic motor symptoms associated with degeneration of the nigrostriatal dopaminergic neurons [,
      • Nasrolahi A.
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      • Akbarzadeh A.
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      • Sadigh-Eteghad S.
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      Neurotrophic factors hold promise for the future of Parkinson's disease treatment: is there a light at the end of the tunnel?.
      ]. Abnormally synchronized oscillations at specific frequency bands within or between brain areas also contribute to the pathophysiology of motor symptoms in PD, including tremor and bradykinesia [
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      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Guerra A.
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      • Paparella G.
      • Fabbrini G.
      • et al.
      Driving motor cortex oscillations modulates bradykinesia in Parkinson's disease.
      ]; these aberrant neuronal oscillations can be suppressed by dopaminergic medication, resulting in the improvement of motor symptoms [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ]. Transcranial alternating current stimulation (tACS), a noninvasive brain stimulation technique, comprises application of a weak sinusoidal alternating electric current at a specified frequency over the scalp to modulate cortical neural activity and brain oscillations [
      • Brittain J.S.
      • Probert-Smith P.
      • Aziz T.Z.
      • Brown P.
      Tremor suppression by rhythmic transcranial current stimulation.
      ,
      • Khatoun A.
      • Asamoah B.
      • Mc Laughlin M.
      Simultaneously excitatory and inhibitory effects of transcranial alternating current stimulation revealed using selective pulse-train stimulation in the rat motor cortex.
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      • Henin S.
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      Immediate neurophysiological effects of transcranial electrical stimulation.
      ]. However, new therapeutic tools for PD first require testing on common rodent models. These include neurotoxin-induced models such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) models [
      • Chia S.J.
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      tACS has gained popularity for modulating brain oscillations, for its ability to synchronize intrinsic neural activities at a relevant frequency, and for enhancing the power of entrained endogenous oscillations via rhythmic stimulation [
      • Guerra A.
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      • Giangrosso M.
      • Cannavacciuolo A.
      • Paparella G.
      • Fabbrini G.
      • et al.
      Driving motor cortex oscillations modulates bradykinesia in Parkinson's disease.
      ,
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      • Hampshire A.
      • et al.
      Externally induced frontoparietal synchronization modulates network dynamics and enhances working memory performance.
      ,
      • Fritsch B.
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      • Martinowich K.
      • Schambra H.M.
      • Ji Y.
      • Cohen L.G.
      • et al.
      Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning.
      ]. Externally enforced rhythms generate activity and facilitate connectivity in brain networks by synchronizing brain regions, and these oscillations in the brain impact task-specific behaviors [
      • Guerra A.
      • Colella D.
      • Giangrosso M.
      • Cannavacciuolo A.
      • Paparella G.
      • Fabbrini G.
      • et al.
      Driving motor cortex oscillations modulates bradykinesia in Parkinson's disease.
      ,
      • Guerra A.
      • Pogosyan A.
      • Nowak M.
      • Tan H.
      • Ferreri F.
      • Di Lazzaro V.
      • et al.
      Phase dependency of the human primary motor cortex and cholinergic inhibition cancelation during beta tACS.
      ]. Certain frequencies of tACS application have functional consequences [
      • Brittain J.S.
      • Probert-Smith P.
      • Aziz T.Z.
      • Brown P.
      Tremor suppression by rhythmic transcranial current stimulation.
      ,
      • Joundi R.A.
      • Jenkinson N.
      • Brittain J.S.
      • Aziz T.Z.
      • Brown P.
      Driving oscillatory activity in the human cortex enhances motor performance.
      ,
      • Asamoah B.
      • Khatoun A.
      • Mc Laughlin M.
      tACS motor system effects can be caused by transcutaneous stimulation of peripheral nerves.
      ], and individualized tACS in patients with PD improves motor performance [
      • Brittain J.S.
      • Probert-Smith P.
      • Aziz T.Z.
      • Brown P.
      Tremor suppression by rhythmic transcranial current stimulation.
      ,
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      • et al.
      Personalized transcranial alternating current stimulation (tACS) and physical therapy to treat motor and cognitive symptoms in Parkinson's disease: a randomized cross-over trial.
      ].
      The underlying mechanisms of tACS are controversial and under-investigated [
      • Liu A.
      • Voroslakos M.
      • Kronberg G.
      • Henin S.
      • Krause M.R.
      • Huang Y.
      • et al.
      Immediate neurophysiological effects of transcranial electrical stimulation.
      ]. The current therapeutic use of tACS is mainly limited to modulating abnormally synchronized oscillations of the brain. Unlike tACS, transcranial direct current stimulation applies directional non-oscillatory voltage components and mediates the activation of N-methyl-d-aspartate receptors and voltage-gated calcium channels, resulting in increased intracellular calcium levels. The subsequent downstream molecular cascades produce long-lasting effects [
      • Liebetanz D.
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      • Tsao M.C.
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      • et al.
      Repeated transcranial direct current stimulation improves cognitive dysfunction and synaptic plasticity deficit in the prefrontal cortex of streptozotocin-induced diabetic rats.
      ], and transcranial electrical stimulation can indirectly impact behaviors, such as motor learning, cognition, and motor function, via non-neuronal neurotrophic factors [
      • Frohlich F.
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      Endogenous electric fields may guide neocortical network activity.
      ,
      • Jung D.H.
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      • Jung Y.J.
      • Kim K.B.
      • et al.
      Therapeutic effects of anodal transcranial direct current stimulation in a rat model of ADHD.
      ,
      • Ahn S.M.
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      • Shin Y.I.
      • et al.
      Contralesional application of transcranial direct current stimulation on functional improvement in ischemic stroke mice.
      ].
      There is no treatment available to slow or stop the neurodegenerative process in PD, and current therapies are only partially able to alleviate disease signs and symptoms [,
      • Nasrolahi A.
      • Mahmoudi J.
      • Akbarzadeh A.
      • Karimipour M.
      • Sadigh-Eteghad S.
      • Salehi R.
      • et al.
      Neurotrophic factors hold promise for the future of Parkinson's disease treatment: is there a light at the end of the tunnel?.
      ]. Glial cell line-derived neurotrophic factor (GDNF) shows positive effects on the survival of dopaminergic neurons, making it a suitable candidate for the prevention of degenerative cell loss in PD [
      • Nasrolahi A.
      • Mahmoudi J.
      • Akbarzadeh A.
      • Karimipour M.
      • Sadigh-Eteghad S.
      • Salehi R.
      • et al.
      Neurotrophic factors hold promise for the future of Parkinson's disease treatment: is there a light at the end of the tunnel?.
      ,
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ]. Exogenous GDNF treatment for PD has serious limitations, including requiring surgery and intracranial delivery as restorative therapy [
      • Enterria-Morales D.
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      • Blesa J.
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      • Gallet S.
      • Prevot V.
      • et al.
      Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons.
      ]. Therefore, the parvalbumin-positive interneurons are a relevant treatment target for PD, as these cells are the main source of striatal GDNF [
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ,
      • Enterria-Morales D.
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      • Blesa J.
      • Lopez-Lopez I.
      • Gallet S.
      • Prevot V.
      • et al.
      Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons.
      ].
      The effects of tACS on cortical excitability are highly complex, which is likely due to the underlying balance between dominant cortical excitation and inhibition [
      • Khatoun A.
      • Asamoah B.
      • Mc Laughlin M.
      Simultaneously excitatory and inhibitory effects of transcranial alternating current stimulation revealed using selective pulse-train stimulation in the rat motor cortex.
      ]. However, we hypothesized that administration of tACS may enhance neural activity, which may then induce non-neuronal changes associated with the production of endogenous neurotrophic factors, such as GDNF. In this study, we employed high-definition tACS with multiple small ring-based electrodes instead of conventional saline sponge-based rectangular pads to improve the ease of stimulation and the ability to reach the cerebral cortex. We applied high-definition tACS (HD-tACS) at various frequencies and evaluated the therapeutic effects using behavioral assessments of motor function, as well as biochemical assays. Molecular investigations of the GDNF expression patterns were also performed to shed light on the underlying mechanisms.

      2. Materials and methods

      2.1 Experimental procedures

      First, to examine the efficacy of tACS (20 min, 89.1 μA/mm2) for PD motor dysfunction and to choose the frequency appropriate for improving motor function, we performed tACS at four different frequency bands, i.e., theta (θ, 6 Hz), alpha (α, 10 Hz), beta (β, 20 Hz), and gamma (γ, 60 Hz) in MPTP-induced PD mice and compared them with the untreated PD mice (Experiment 1). Next, we applied the frequency that proved more effective in improving motor function to investigate the mechanisms underlying the observed tACS effects. The mice were randomly divided into five groups: control, MPTP (MPTP-injected mice with sham stimulation), MPTP + tACSβ (MPTP-injected mice treated with beta-20 Hz tACS), MPTP + tACSβ+PP1 (MPTP-injected mice treated with beta-20 Hz tACS and protein phosphatase 1 [PP1], a GDNF receptor-associated RET kinase inhibitor), and MPTP + L-Dopa (MPTP-injected mice treated with levodopa [l-dopa]) (Experiment 2). All mouse experiments were performed on the indicated day (Fig. S1). A detailed description of the materials and methods used in this study is provided in the online-only Data Supplement (Supplementary Material and Methods).

      3. Results

      3.1 Behavioral motor deficits and regulation of dopaminergic neurons according to the frequency band of tACS applied to the MPTP-induced Parkinson's model

      Following the application of tACS on the primary motor cortex of the PD mice model, we examined the survival of the dopaminergic neurons and GDNF production corresponding to the tACS frequency, since motor dysfunctions in PD are mainly caused by degeneration of nigrostriatal dopaminergic neurons and changes in GDNF expression. Our results suggested that administration of tACS over the primary motor cortex, especially beta-frequency tACS at 20 Hz, attenuated motor deficits and had a neuroprotective effect on dopaminergic neurons (Figs. S2 and S3). A detailed description of the results of this study is provided in the online-only Data Supplement (Supplementary Results).

      3.2 Effects of beta-frequency tACS on improvement of motor deficits in the MPTP-induced Parkinson's model

      The application of tACS at 20 Hz, which was a more effective frequency than the others for improving motor function and upregulating GDNF, was used in subsequent experiments to determine whether it was effective for motor deficits and regulation of GDNF production in the PD mouse model. Moreover, we employed the l-dopa-treated group as the positive control, and the PP1-treated group as the GDNF signaling-inhibiting group. The MPTP, MPTP + tACSβ, MPTP + L-dopa, and MPTP + tACSβ+PP1 groups showed no significant change in body weight compared with the control mice (Fig. S4A) (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Jung D.H.
      • Ahn S.M.
      • Pak M.E.
      • Lee H.J.
      • Jung Y.J.
      • Kim K.B.
      • et al.
      Therapeutic effects of anodal transcranial direct current stimulation in a rat model of ADHD.
      ] = 2.771 P = 0.056). All behavioral tests for motor functions were significantly changed in the MPTP group compared with the control group (rotarod test: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 21.509 P < 0.001, P < 0.001; pole test: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 19.196 P < 0.001, P < 0.001; open-field test, mean speed: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 55.529 P < 0.001, P < 0.001; total distance: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 59.342 P < 0.001, P < 0.001; activity time: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 11.193 P < 0.001, P < 0.001; average speed (CatWalk): F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 11.915 P < 0.001, P < 0.001; CatWalk, number of front paw steps: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 36.480 P<0.001, P < 0.001; number of hind paw steps: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 47.485 P < 0.001, P < 0.001).
      Latency to fall time in the rotarod test was significantly longer in the MPTP + tACSβ and MPTP + L-dopa groups than in the MPTP group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 21.509 P < 0.001, tACSβ: P=0.008, l-dopa: P=0.01) (Fig. 1A), and the T-turn time in the pole test was also more significantly decreased in both groups than in the MPTP group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 19.196 P < 0.001, tACSβ: P<0.001, l-dopa: P=0.01) (Fig. 1B). In the open-field test, all measured parameters were markedly increased in the MPTP + tACSβ and MPTP + L-dopa groups than in the MPTP group (mean speed: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 55.529 P < 0.001, tACSβ: P<0.001, l-dopa: P<0.001; total distance: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 59.342 P < 0.001, tACSβ: P=0.025, l-dopa: P=0.028; activity time: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 11.193 P < 0.001, tACSβ: P=0.006, l-dopa: P=0.012) (Fig. 1C−F). In the CatWalk test, the ability to walk on a runway, including average speed and number of steps, was markedly improved in the MPTP + tACSβ and MPTP + L-dopa groups than in the MPTP group (average speed: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 11.915 P < 0.001, tACSβ: P=0.015, l-dopa: P=0.014; number of front paw steps: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 36.480 P<0.001, tACSβ: P<0.001, l-dopa: P<0.001; number of hind paw steps: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 47.485 P < 0.001, tACSβ: P<0.001, l-dopa: P<0.001) (Fig. 1G−I). The stride length and step cycle of the MPTP + tACSβ and MPTP + L-dopa groups were markedly increased compared to those of the MPTP group, suggesting similar effects of tACSβ and l-dopa on bradykinesia and rigidity (stride length: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 15.556 P < 0.001, tACSβ: P=0.024, l-dopa: P=0.001; right front paw step cycle: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 18.272 P < 0.001, tACSβ: P=0.018, l-dopa: P=0.005; left front paw step cycle: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 10.962 P < 0.001, tACSβ: P=n.s, l-dopa: P=0.046) (Figs. S4C and D). The parameter mean intensity, reflecting bradykinesia and rigidity severity, was significantly changed only in the MPTP + L-dopa group (right front paw mean intensity: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 14.413 P < 0.001, P=0.049; left front paw intensity: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 14.242 P < 0.001, P=0.008) (Fig. S4B). PP1 prevented the recovery of most behavioral functions in the CatWalk test, apart from the mean intensity (Fig. 1, Fig. S4B−D). In addition, the control group tended to prefer alternate step patterns with a secondary inclination for cruciate-type sequences in the step sequence of the CatWalk test (Aa: 29.9%, Ab: 30.6%, Ca: 9%, Cb: 30.5%), whereas the MPTP group showed a preference toward cruciate step patterns and a reduced preference for alternate step patterns (Aa: 26.5%, Ab: 17.9%, Ca: 27.8%, Cb: 27.8%). The MPTP + tACSβ and MPTP + L-Dopa groups showed an increased rate of alternate step patterns and a decreased rate of cruciate step patterns (tACSβ: Aa: 25.3%, Ab: 29.6%, Ca: 21.2%, Cb: 23.9%; l-dopa: Aa: 27.9%, Ab: 28%, Ca: 17.1%, Cb: 27%) (Fig. S4E). These behavioral results indicate that beta-frequency 20 Hz tACS over the primary motor cortex may attenuate motor deficits in the PD mouse model.
      Fig. 1
      Fig. 1Effects of beta-frequency 20 Hz tACS on the improvement of motor function in the MPTP-induced Parkinson's model. Quantification of motor function results of the (A) rotarod test, (B) pole test, (C–F) open-field test, and (G–I) CatWalk test. The beta-frequency 20 Hz tACS showed beneficial effects similar to those of l-dopa treatment in attenuating motor dysfunction in MPTP-induced mice. All behavioral test results for motor function were significantly changed in the beta-frequency 20 Hz tACS-treated group compared to those in the MPTP group. All tACSβ effects in the behavioral tests were inhibited by PP1, an inhibitor of the receptor tyrosine kinase RET. n = 7. All data are expressed as the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. each group.

      3.3 Effects of beta-frequency tACS on dopaminergic neuron survival and GDNF production in the MPTP-induced Parkinson's model

      When considering the dopaminergic neurons in the PD model, the integral optical density (IOD) in the striatum and the number of TH-positive cells in the substantia nigra were significantly decreased in the MPTP-induced mice than in the control mice (striatum: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 63.893 P < 0.001, P < 0.001; substantia nigra: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 44.005 P < 0.001, P < 0.001). The MPTP + tACSβ and MPTP + L-dopa groups showed markedly increased staining of the dopaminergic neurons than the MPTP group, and these effects were reversed in the MPTP + tACSβ+PP1 group (striatum: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 63.893 P < 0.001, tACSβ: P=0.009, PP1: P=0.005, l-dopa: P=0.002; substantia nigra: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 44.005 P < 0.001, tACSβ: P<0.001, PP1: P<0.001, l-dopa: P<0.001) (Fig. 2A−C). Likewise, the number of TH/cleaved caspase 3 double-positive cells in the substantia nigra was significantly increased in the MPTP mice, though significantly decreased in the MPTP + tACSβ and MPTP + L-dopa groups. Moreover, the mean number of these double-positive cells was higher in the MPTP + tACSβ+PP1 group than in the MPTP + tACSβ group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 19.812 P < 0.001, MPTP: P<0.001, tACSβ: P=0.031, PP1: P<0.001, l-dopa: P=0.016) (Fig. 2D−F, Figs. S5–1A).
      Fig. 2
      Fig. 2Effects of beta-frequency 20 Hz tACS on dopaminergic neurons in the MPTP-induced Parkinson's model. (A) Photomicrographs showing TH-positive cells in the striatum and substantia nigra and (B and C) the corresponding bar charts. The beta-frequency 20 Hz tACS- and l-dopa-treated groups exhibited increase in the integral optical density (IOD) of TH in the striatum and in the number of TH-positive cells in the substantia nigra compared to those in the MPTP group. However, the PP1-treated 20-Hz tACS group showed a decreased IOD in the striatum and a lower number of TH-positive cells in the substantia nigra. (D and E) Photomicrographs showing TH/cleaved caspase 3 (cCaspase 3) double-positive cells in the substantia nigra (the images in E are magnifications of the areas indicated by dashed lines in D) and (F) the corresponding bar chart. n = 7. The number of TH/cCaspase 3 double-positive cells was significantly lower in the beta-frequency 20-Hz tACS-treated group than in the MPTP group, whereas the cell number in the PP1-treated 20-Hz tACS group was markedly higher than that in the 20-Hz tACS-treated group. All data are expressed as the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. control group. Scale bars, A and D = 100 μm; E = 20 μm.
      To examine the regulation of GDNF production by beta-frequency tACS, we evaluated the neurotrophic factor GDNF using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and flow cytometric assays. All measured parameters of these assays showed significantly decreased levels in the MPTP group than in the control mice (IOD: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Brown P.
      Oscillatory nature of human basal ganglia activity: relationship to the pathophysiology of Parkinson's disease.
      ] = 48.316 P < 0.001, P<0.001; GDNF ELISA: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 25.168 P < 0.001, P<0.001; tyrosine hydroxylase (TH) ELISA: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 10.009 P < 0.001, P < 0.001). The IOD of GDNF in the striatum was markedly higher in the MPTP + tACSβ group than in the MPTP group, but the IOD in the MPTP + tACSβ+PP1 group was similar to that of the MPTP mice (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Brown P.
      Oscillatory nature of human basal ganglia activity: relationship to the pathophysiology of Parkinson's disease.
      ] = 48.316 P < 0.001, tACSβ: P=0.017, PP1: P=0.02) (Fig. 3A and B). The levels of GDNF and TH in the dorsal striatum were significantly increased in both MPTP + tACSβ and MPTP + L-dopa groups, but decreased in the MPTP + tACSβ+PP1 group (GDNF: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 25.168 P < 0.001, tACSβ: P<0.001, PP1: P<0.001, l-dopa: P=0.007; TH: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 10.009 P < 0.001, tACSβ: P=0.049, PP1: P=0.003, l-dopa: P=0.003) (Fig. 3C and D). Next, we examined the parvalbumin-positive interneurons to identify whether it was related to GDNF production in the striatum of adult mice (Fig. 3E−G, Figs. S5–1B). The cell percentage of the Q2 area, showing high fluoresce intensity to both GDNF and parvalbumin, in the dorsal striatum was significantly decreased in the MPTP mice (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 13.732 P < 0.001, P < 0.001). In comparison to the MPTP group, the cell percentage was substantially increased in the MPTP + tACSβ and MPTP + L-dopa groups, although only the former was significantly different. In addition, this cell percentage was markedly decreased in the MPTP + tACSβ+PP1 group than in the MPTP + tACSβ group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 13.732 P < 0.001, tACSβ: P = 0.033, PP1: P = 0.045). These results suggest that in the PD mouse model, beta-frequency 20 Hz tACS may protect dopaminergic neurons via upregulation of endogenous GDNF production in the striatum by activation of parvalbumin interneurons, the main source of striatal GDNF.
      Fig. 3
      Fig. 3Effects of beta-frequency 20-Hz tACS on the dopaminergic neurons and GDNF secreting cells in the MPTP-induced Parkinson's model. (A) Photomicrographs showing the integral optical density (IOD) of GDNF in the striatum and (B) the corresponding bar chart. (C and D) The levels of GDNF and TH in the dorsal striatum measured by ELISA assays. n = 7. For GDNF, the striatal IOD was markedly higher in the beta-frequency 20 Hz tACS-treated group than in the MPTP group. The 20-Hz tACS group showed significant increases in GDNF and TH levels in the dorsal striatum, whereas the PP1-treated 20-Hz tACS group exhibited decreased levels. (E) Representative flow cytometry plots, (G) the corresponding bar chart, and (F) photomicrographs showing GDNF/parvalbumin double-positive cells in the dorsal striatum. n = 6. The cell percentage of the Q2 area was significantly increased in the 20-Hz tACS-treated group than in the MPTP group. The PP1-treated 20-Hz tACS group showed a decreased percentage of these cells. GDNF expression was localized to the parvalbumin-positive interneurons. All data are expressed as the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. each group. Scale bars, A = 100 μm, F = 20 μm.

      3.4 Effects of beta-frequency tACS on cell signaling regulating the survival of dopaminergic neurons in the substantia nigra of MPTP-induced mice

      GDNF-related signaling may be involved in the survival of the dopaminergic neurons in the substantia nigra, as suggested by the results obtained with PP1, the RET inhibitor interfering with GDNF receptor signaling. Therefore, we examined the expression of RET and the downstream markers since activation of RET-signaling by GDNF requires the GDNF family receptor-α (GFRα) for cell survival via subsequent downstream signaling.
      According to the western blot analyses, the ratio of pRET/RET was not different between the control group and MPTP-treated groups, with the exception of the MPTP + tACSβ+PP1 group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Brown P.
      Oscillatory nature of human basal ganglia activity: relationship to the pathophysiology of Parkinson's disease.
      ] = 4.801 P = 0.005, P = 0.003). The ratio of phosphorylated AKT (pAKT)/AKT, but not of phosphorylated extracellular signal-regulated kinase (pERK)/ERK, was significantly lower in the MPTP group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 5.376 P = 0.003, P = 0.006). Both ratios were significantly increased in the MPTP + tACSβ group than in the MPTP group (pAKT: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 5.376 P = 0.003, P = 0.041; pERK: F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 3.665 P = 0.018, P = 0.037) (Fig. 4A−D). Furthermore, immunohistochemical assays confirmed pRET and pAKT expression in dopaminergic neurons in the substantia nigra. The mean number of TH/pRET double-positive cells was markedly decreased in MPTP mice than in the control mice (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 14.882 P < 0.001, P=0.001). Although the MPTP + tACSβ and MPTP + L-dopa groups had more TH/pRET double-positive cells than the MPTP group, these differences were not significant. However, the MPTP + tACSβ+PP1 group had significantly fewer TH/pRET double-positive cells than the MPTP + tACSβ group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 14.882 P < 0.001, P<0.001). The mean number of TH/pAKT double-positive cells was significantly decreased in the MPTP group than in the control group, but markedly increased in the MPTP + tACSβ group than in the MPTP group (F [
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ] = 6.006 P = 0.001, MPTP: P=0.002, tACSβ: P=0.028) (Fig. 4E−G, Figs. S5–2). These findings indicate that beta-frequency 20 Hz tACS may protect dopaminergic neurons in the substantia nigra via GDNF-related survival signaling in this mouse model of PD.
      Fig. 4
      Fig. 4Effects of beta-frequency 20 Hz tACS on GDNF-related survival signaling in the substantia nigra of MPTP-induced mice. (A) Western blot and (B–D) the corresponding densitometry results for RET, AKT, and ERK expression in the substantia nigra. n = 6. The beta-frequency 20 Hz tACS-treated group showed significantly higher pAKT and pERK expression levels than the MPTP group. The PP1-treated 20 Hz tACS group exhibited lower pRET expression levels than the 20-Hz tACS-treated group. β-actin was used as a loading control for the western blot analyses. (E) Photomicrographs showing TH/pRET and TH/pAKT double-positive cells in the substantia nigra (images presented below are magnifications of the areas indicated by dashed lines). (F and G) the corresponding bar charts. n = 7. The number of pAKT-expressing dopaminergic neurons was significantly increased in the 20-Hz tACS-treated group than in the MPTP group. The PP1-treated 20-Hz tACS group exhibited significantly lower pRET expression than the 20-Hz tACS-treated group. All data are expressed as the mean ± SEM; ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 vs. each group. Scale bars, E = 100 μm; magnified images = 20 μm.

      4. Discussion

      The therapeutic effects of tACS for neurological or psychiatric disorders are considered to be primarily associated with modulation of brain oscillations, since the brains of patients with such disorders show abnormally synchronized oscillations. Modulation of relevant brain oscillations is also applied when treating motor symptoms of PD [
      • Lalo E.
      • Thobois S.
      • Sharott A.
      • Polo G.
      • Mertens P.
      • Pogosyan A.
      • et al.
      Patterns of bidirectional communication between cortex and basal ganglia during movement in patients with Parkinson disease.
      ,
      • Tinkhauser G.
      • Pogosyan A.
      • Tan H.
      • Herz D.M.
      • Kuhn A.A.
      • Brown P.
      Beta burst dynamics in Parkinson's disease OFF and ON dopaminergic medication.
      ,
      • Guerra A.
      • Colella D.
      • Giangrosso M.
      • Cannavacciuolo A.
      • Paparella G.
      • Fabbrini G.
      • et al.
      Driving motor cortex oscillations modulates bradykinesia in Parkinson's disease.
      ]. However, we investigated the therapeutic effects of successive tACS on the recovery of motor symptoms in PD in association with endogenous GDNF production. The main findings of our study included: 1) high-definition tACS applied over the primary motor cortex ameliorated motor dysfunction in the MPTP-induced mouse model of PD by facilitating the survival of dopaminergic neurons and upregulating GDNF production; 2) the therapeutic effects of beta-frequency 20 Hz tACS were more prominent than those of other frequency bands used; and 3) the application of beta-frequency 20 Hz tACS promoted the survival of dopaminergic neurons in the substantia nigra via upregulation of endogenous GDNF production in striatal parvalbumin interneurons. These results suggest that the application of beta-frequency tACS ameliorates motor impairments in PD and is a potential adjuvant therapy for patients with PD.
      Mapping of oscillatory frequencies provides a personalized neurotherapeutic target and can guide noninvasive brain stimulation. Although it remains unresolved as to whether brain oscillations refiect a fundamental mechanism in cortical information processing, frequency-specific tACS is widely employed to induce rhythmic current flows that interact with entrained spontaneous oscillations [
      • Guerra A.
      • Colella D.
      • Giangrosso M.
      • Cannavacciuolo A.
      • Paparella G.
      • Fabbrini G.
      • et al.
      Driving motor cortex oscillations modulates bradykinesia in Parkinson's disease.
      ,
      • Fritsch B.
      • Reis J.
      • Martinowich K.
      • Schambra H.M.
      • Ji Y.
      • Cohen L.G.
      • et al.
      Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning.
      ]. The motor system is not typically targeted by tACS, which can also modulate other task-specific functions, such as hearing, cognition, and memory [
      • Asamoah B.
      • Khatoun A.
      • Mc Laughlin M.
      tACS motor system effects can be caused by transcutaneous stimulation of peripheral nerves.
      ]. Rhythmic oscillations can be categorized into low- and high-frequency bands according to the main states of the brain at sleep and during awake rest, respectively [
      • Feurra M.
      • Bianco G.
      • Santarnecchi E.
      • Del Testa M.
      • Rossi A.
      • Rossi S.
      Frequency-dependent tuning of the human motor system induced by transcranial oscillatory potentials.
      ]. Moreover, individualized tACS improves motor performance and reduces classic symptoms, such as tremors in patients with PD [
      • Brittain J.S.
      • Probert-Smith P.
      • Aziz T.Z.
      • Brown P.
      Tremor suppression by rhythmic transcranial current stimulation.
      ,
      • Del Felice A.
      • Castiglia L.
      • Formaggio E.
      • Cattelan M.
      • Scarpa B.
      • Manganotti P.
      • et al.
      Personalized transcranial alternating current stimulation (tACS) and physical therapy to treat motor and cognitive symptoms in Parkinson's disease: a randomized cross-over trial.
      ].
      Each frequency band of tACS has been associated with specific functions, e.g., beta- and gamma-bands are antikinetic and prokinetic, respectively [
      • Guerra A.
      • Colella D.
      • Giangrosso M.
      • Cannavacciuolo A.
      • Paparella G.
      • Fabbrini G.
      • et al.
      Driving motor cortex oscillations modulates bradykinesia in Parkinson's disease.
      ,
      • Joundi R.A.
      • Jenkinson N.
      • Brittain J.S.
      • Aziz T.Z.
      • Brown P.
      Driving oscillatory activity in the human cortex enhances motor performance.
      ,
      • Brown P.
      Oscillatory nature of human basal ganglia activity: relationship to the pathophysiology of Parkinson's disease.
      ]. In this study, we first applied tACS at representative frequency bands from theta to gamma in a PD mouse model to compare the therapeutic effects and to determine the most effective frequency for restoring motor function impaired by PD. We applied tACS over the primary motor cortex, centralized over the large caudal forelimb area [
      • Tennant K.A.
      • Adkins D.L.
      • Donlan N.A.
      • Asay A.L.
      • Thomas N.
      • Kleim J.A.
      • et al.
      The organization of the forelimb representation of the C57BL/6 mouse motor cortex as defined by intracortical microstimulation and cytoarchitecture.
      ], to determine any beneficial effects on motor function. All tACS frequencies, with the exception of the gamma frequency, significantly alleviated motor deficits in the MPTP-induced PD model; beta-frequency tACS had the most beneficial effects on recovery from motor dysfunction, producing improvements in all behavioral tests.
      Diagnostic motor symptoms in patients with PD result from the degeneration of nigrostriatal dopaminergic neurons [,
      • Nasrolahi A.
      • Mahmoudi J.
      • Akbarzadeh A.
      • Karimipour M.
      • Sadigh-Eteghad S.
      • Salehi R.
      • et al.
      Neurotrophic factors hold promise for the future of Parkinson's disease treatment: is there a light at the end of the tunnel?.
      ], and it is known that GDNF, a potent trophic factor, promotes the survival of these dopaminergic neurons [
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ,
      • Enterria-Morales D.
      • Del Rey N.L.
      • Blesa J.
      • Lopez-Lopez I.
      • Gallet S.
      • Prevot V.
      • et al.
      Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons.
      ]. In this study, the death of dopaminergic neurons and the production of GDNF were examined in relation to the applied tACS frequency. All the frequencies used increased the number of dopamine cells in the substantia nigra and promoted their survival compared to the untreated PD mouse model. However, alpha- and beta-frequency tACS showed better effects on the number of dopamine cells in the substantia nigra with increased levels of TH and GDNF in the dorsal striatum. Taking the results of the behavioral tests and dopamine cell counts into consideration, it seems that tACS application may protect dopaminergic neurons by regulating GDNF production in the PD mouse model and that beta-frequency 20 Hz tACS is more effective at preventing damaged characteristics than other frequency bands.
      Thus, we applied 20 Hz tACS and compared its effects with those of the clinical drug l-dopa to elucidate the underlying GDNF-related mechanisms in the PD mouse model. GDNF preferentially binds to GFRα, and this trophic factor affects the protection and regeneration of dopaminergic neurons via subsequent activation of the receptor tyrosine kinase RET [
      • Drinkut A.
      • Tillack K.
      • Meka D.P.
      • Schulz J.B.
      • Kugler S.
      • Kramer E.R.
      Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model.
      ]. RET does not directly interact with GDNF and requires GFRα as a co-receptor [
      • Mulligan L.M.
      RET revisited: expanding the oncogenic portfolio.
      ]. GDNF/RET signaling may be a potential target for neuroregenerative therapy in PD because the absence of RET completely abolishes the neuroprotective and regenerative effects of GDNF [
      • Bespalov M.M.
      • Saarma M.
      GDNF family receptor complexes are emerging drug targets.
      ]. Therefore, we employed the RET kinase inhibitor PP1 in tACS-treated mice to examine the effects of GDNF-signaling inhibition [
      • Carlomagno F.
      • Vitagliano D.
      • Guida T.
      • Napolitano M.
      • Vecchio G.
      • Fusco A.
      • et al.
      The kinase inhibitor PP1 blocks tumorigenesis induced by RET oncogenes.
      ]. Beta-frequency tACS ameliorated motor dysfunction in the PD mouse model to an extent that was comparable with that of l-dopa treatment and showed neuroprotective effects on the dopaminergic neurons with upregulated endogenous GDNF production in the PD mouse model. However, treatment with the RET kinase inhibitor PP1 abrogated the tACS-induced recovery of motor deficits, suggesting that GDNF/RET kinase-related signaling is involved in the therapeutic effects of tACS.
      GDNF is expressed by neurons of the substantia nigra including the striatum in the normal rodent brain [
      • Trupp M.
      • Belluardo N.
      • Funakoshi H.
      • Ibanez C.F.
      Complementary and overlapping expression of glial cell line-derived neurotrophic factor (GDNF), c-ret proto-oncogene, and GDNF receptor-alpha indicates multiple mechanisms of trophic actions in the adult rat CNS.
      ]; however, the GABAergic parvalbumin-positive interneurons, which are distributed uniformly over the striatum, are the main source of striatal GDNF, with more than 95% of these cells expressing GDNF [
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ,
      • Enterria-Morales D.
      • Del Rey N.L.
      • Blesa J.
      • Lopez-Lopez I.
      • Gallet S.
      • Prevot V.
      • et al.
      Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons.
      ]. Parvalbumin-positive interneurons are a relevant treatment target for PD because they represent the only cell type that synthesizes GDNF in the striatum, and they are densely innervated by nigrostriatal dopaminergic nerve endings [
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ,
      • Enterria-Morales D.
      • Del Rey N.L.
      • Blesa J.
      • Lopez-Lopez I.
      • Gallet S.
      • Prevot V.
      • et al.
      Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons.
      ]. GDNF is transported from the axon terminals to the cell soma of substantia nigra neurons by long-range retrograde signaling of mesencephalic dopaminergic neurons via the nigrostriatal pathway [
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ,
      • Ibanez C.F.
      Message in a bottle: long-range retrograde signaling in the nervous system.
      ].
      GDNF/RET signaling leads to the survival of dopaminergic neurons via activation of downstream signaling cascades, including mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/AKT [
      • Drinkut A.
      • Tillack K.
      • Meka D.P.
      • Schulz J.B.
      • Kugler S.
      • Kramer E.R.
      Ret is essential to mediate GDNF's neuroprotective and neuroregenerative effect in a Parkinson disease mouse model.
      ,
      • Airaksinen M.S.
      • Saarma M.
      The GDNF family: signalling, biological functions and therapeutic value.
      ]. Thus, we examined the expression of striatal GDNF and parvalbumin-positive interneurons expressing GDNF. Beta-frequency 20 Hz tACS had a neuroprotective effect on the dopaminergic neurons via upregulated endogenous GDNF production in striatal parvalbumin interneurons. Beta-frequency 20 Hz tACS and l-dopa treatment produced similar results; however, l-dopa treatment did not cause significant changes in the mean IOD of GDNF in the striatum and in the GDNF expression of parvalbumin interneurons. The proportion of activated RET was significantly downregulated by treatment with PP1, whereas activated ERK and AKT were significantly increased by beta-frequency 20 Hz tACS, though not l-dopa treatment. Our results suggest that the application of tACS, especially beta-frequency 20 Hz tACS, activates the release of endogenous GDNF from striatal parvalbumin interneurons at the nerve terminals of the dopaminergic neurons in the substantia nigra. Retrograde signaling of mesencephalic neurons induced survival signaling of the dopaminergic neurons and subsequently restored damaged motor dysfunction in the PD mouse model (Fig. 5).
      Fig. 5
      Fig. 5Simplified schematic showing the proposed therapeutic mechanism of tACS application in the PD mouse model. Striatal parvalbumin-positive interneurons may be activated by successive applications of tACS and released GDNF at the nerve terminals as retrograde signaling to the dopaminergic neurons in the substantia nigra. This, in turn, induces survival signaling in these dopaminergic neurons, thereby protecting the dopaminergic neurons in the substantia nigra, and subsequently alleviating motor dysfunctions.
      tACS involves a relatively weak current, producing a modest effect on cortical excitability, but is able to provoke neural spikes and ultimately modify multiunit activity [
      • Fritsch B.
      • Reis J.
      • Martinowich K.
      • Schambra H.M.
      • Ji Y.
      • Cohen L.G.
      • et al.
      Direct current stimulation promotes BDNF-dependent synaptic plasticity: potential implications for motor learning.
      ,
      • Zaghi S.
      • Acar M.
      • Hultgren B.
      • Boggio P.S.
      • Fregni F.
      Noninvasive brain stimulation with low-intensity electrical currents: putative mechanisms of action for direct and alternating current stimulation.
      ]. Low-frequency alternating current stimulation is able to produce excitability of somata that mimics anodal and cathodal direct current stimulation; however, it reduces neuronal polarization much faster than the neuronal membrane time constant of alternating current frequency [
      • Liu A.
      • Voroslakos M.
      • Kronberg G.
      • Henin S.
      • Krause M.R.
      • Huang Y.
      • et al.
      Immediate neurophysiological effects of transcranial electrical stimulation.
      ,
      • Voroslakos M.
      • Takeuchi Y.
      • Brinyiczki K.
      • Zombori T.
      • Oliva A.
      • Fernandez-Ruiz A.
      • et al.
      Direct effects of transcranial electric stimulation on brain circuits in rats and humans.
      ]. Synchronous components of the membrane potential signals the activation of cell assemblies and enables firing to occur [
      • Stern E.A.
      • Jaeger D.
      • Wilson C.J.
      Membrane potential synchrony of simultaneously recorded striatal spiny neurons in vivo.
      ]. Moreover, tACS induces a mechanism called “stochastic resonance,” which results in neurons that are more likely to fire in response to other neurons during the depolarization state [
      • McDonnell M.D.
      • Abbott D.
      What is stochastic resonance? Definitions, misconceptions, debates, and its relevance to biology.
      ]. These resonate according to the spike activity by tACS and should be considered a hypothetical mechanism for the long-lasting therapeutic tACS effects caused by non-neuronal neurotrophic factors.
      Our study has some limitations that curb its ability to clearly define the mechanisms underlying the beneficial effects of tACS for patients with PD. First, we could not explore if and how tACS directly promoted the production of endogenous GDNF via striatal parvalbumin interneurons. Alternating current stimulation over the primary motor cortex may activate the downstream corticostriatal neurons via the subcortically projecting pyramidal neurons [
      • Apicella A.J.
      • Wickersham I.R.
      • Seung H.S.
      • Shepherd G.M.
      Laminarly orthogonal excitation of fast-spiking and low-threshold-spiking interneurons in mouse motor cortex.
      ] and influence corticostriatal activity in PD animal models, possibly reflecting glutamatergic neurotransmission [
      • Guerra A.
      • Suppa A.
      • D'Onofrio V.
      • Di Stasio F.
      • Asci F.
      • Fabbrini G.
      • et al.
      Abnormal cortical facilitation and L-dopa-induced dyskinesia in Parkinson's disease.
      ]. Parvalbumin-positive interneurons are interconnected by electrical synapses, i.e., dendro-dendritic gap junctions, and constitute a unique neuronal network, causing the whole population to fire almost synchronously [
      • Sharott A.
      • Moll C.K.
      • Engler G.
      • Denker M.
      • Grun S.
      • Engel A.K.
      Different subtypes of striatal neurons are selectively modulated by cortical oscillations.
      ,
      • d'Anglemont de Tassigny X.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF-based therapies, GDNF-producing interneurons, and trophic support of the dopaminergic nigrostriatal pathway. Implications for Parkinson's disease.
      ] and exhibit voltage-dependent membrane potential oscillations [
      • Beatty J.A.
      • Song S.C.
      • Wilson C.J.
      Cell-type-specific resonances shape the responses of striatal neurons to synaptic input.
      ]. Thus, tACS-induced spikes may activate the network of parvalbumin-positive interneurons, which are the primary target in patients with PD, via glutamatergic neurotransmission and voltage-dependent membrane potential.
      Second, the electric fields produced by tACS can directly affect the neurons in the targeted cortex as shown in our previous analysis of a finite element model simulation [
      • Ahn S.M.
      • Jung D.H.
      • Lee H.J.
      • Pak M.E.
      • Jung Y.J.
      • Shin Y.I.
      • et al.
      Contralesional application of transcranial direct current stimulation on functional improvement in ischemic stroke mice.
      ]. The 20 Hz beta frequency is the natural frequency of the motor regions [
      • Feurra M.
      • Bianco G.
      • Santarnecchi E.
      • Del Testa M.
      • Rossi A.
      • Rossi S.
      Frequency-dependent tuning of the human motor system induced by transcranial oscillatory potentials.
      ], and it is not yet understood why this frequency induces more GDNF production than other frequency bands. Previous studies suggest that the tACS frequency usually applied approximates the natural resonance frequency of local neural circuits [
      • Guerra A.
      • Pogosyan A.
      • Nowak M.
      • Tan H.
      • Ferreri F.
      • Di Lazzaro V.
      • et al.
      Phase dependency of the human primary motor cortex and cholinergic inhibition cancelation during beta tACS.
      ], and short treatment of tACS at 20 Hz results in selectively increased motor cortex excitability [
      • Feurra M.
      • Bianco G.
      • Santarnecchi E.
      • Del Testa M.
      • Rossi A.
      • Rossi S.
      Frequency-dependent tuning of the human motor system induced by transcranial oscillatory potentials.
      ]. It remains unclear how tACS, and beta-frequency tACS in particular, affects neural activity and promotes endogenous GDNF release from striatal parvalbumin interneurons. This aspect requires further investigation.
      It has been shown that the production of endogenous GDNF prevents degeneration of dopaminergic neurons in patients with PD [
      • Nasrolahi A.
      • Mahmoudi J.
      • Akbarzadeh A.
      • Karimipour M.
      • Sadigh-Eteghad S.
      • Salehi R.
      • et al.
      Neurotrophic factors hold promise for the future of Parkinson's disease treatment: is there a light at the end of the tunnel?.
      ,
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ], and activation of GDNF-producing striatal parvalbumin interneurons (e.g., by pharmacological, electric, or magnetic means) could have therapeutic effects for patients with PD [
      • Hidalgo-Figueroa M.
      • Bonilla S.
      • Gutierrez F.
      • Pascual A.
      • Lopez-Barneo J.
      GDNF is predominantly expressed in the PV+ neostriatal interneuronal ensemble in normal mouse and after injury of the nigrostriatal pathway.
      ]. The current study demonstrates that the application of tACS over the primary motor cortex produces therapeutic effects in the PD mouse model; we suggest that this occurs via the activation of endogenous GDNF production by striatal parvalbumin interneurons and its survival signaling for dopaminergic neurons, thereby improving motor function. We propose that tACS, especially beta-frequency 20 Hz tACS, represents a viable adjuvant treatment option for patients with PD to ameliorate motor impairments.

      Funding

      This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government ( MSIT ) ( 2021R1A2C1003646 ). This research was also supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government ( MSIT ) ( 2021M3A9E4081781 ).

      Declaration of competing interest

      None.

      Financial disclosure

      No authors have received any funding from any institution, including personal relationships, interests, grants, employment, affiliations, patents, inventions, honoraria, consultancies, royalties, stock options/ownership, or expert testimony for the last 12 months.

      Data and materials availability

      All data required to assess the manuscript's conclusions are present in the paper and/or Supplementary Materials.

      CRediT authorship contribution statement

      Hong Ju Lee: conceived and designed the experiments, analyzed the data. performed the experiments, wrote the manuscript. Da Hee Jung: performed the experiments. Hwa Kyoung Shin: analyzed the data. Byung Tae Choi: conceived and designed the experiments. analyzed the data. wrote the manuscript, analyzed the data, wrote the manuscript, All authors contributed extensively to this work.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

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