Review ArticleMemory and Cognition-Related NeuroplasticityEnhancement by Transcranial Direct Current Stimulation inRodents: A Systematic Review

Carla Cavaleiro,1,2,3 João Martins,1,2,3,4 Joana Gonçalves ,1,2

and Miguel Castelo-Branco 1,2,3,4

1Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, Coimbra, Portugal2Institute of Nuclear Sciences Applied to Health (ICNAS), University of Coimbra, Coimbra, Portugal3CNC.IBILI Consortium, University of Coimbra, Coimbra, Portugal4Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Correspondence should be addressed to Joana Gonçalves; jgoncalves@fmed.uc.ptand Miguel Castelo-Branco; mcbranco@fmed.uc.pt

Received 6 December 2019; Revised 27 January 2020; Accepted 6 February 2020; Published 26 February 2020

Guest Editor: Luca Marsili

Copyright © 2020 Carla Cavaleiro et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Brain stimulation techniques, including transcranial direct current stimulation (tDCS), were identified as promising therapeutictools to modulate synaptic plasticity abnormalities and minimize memory and learning deficits in many neuropsychiatricdiseases. Here, we revised the effect of tDCS on the modulation of neuroplasticity and cognition in several animal diseasemodels of brain diseases affecting plasticity and cognition. Studies included in this review were searched following the terms(“transcranial direct current stimulation”) AND (mice OR mouse OR animal) and according to the PRISMA statementrequirements. Overall, the studies collected suggest that tDCS was able to modulate brain plasticity due to synaptic modificationswithin the stimulated area. Changes in plasticity-related mechanisms were achieved through induction of long-term potentiation(LTP) and upregulation of neuroplasticity-related proteins, such as c-fos, brain-derived neurotrophic factor (BDNF), or N-methyl-D-aspartate receptors (NMDARs). Taken into account all revised studies, tDCS is a safe, easy, and noninvasive brain stimulationtechnique, therapeutically reliable, and with promising potential to promote cognitive enhancement and neuroplasticity. Sincethe use of tDCS has increased as a novel therapeutic approach in humans, animal studies are important to better understand itsmechanisms as well as to help improve the stimulation protocols and their potential role in different neuropathologies.

1. Introduction

Transcranial direct current stimulation (tDCS) is a noninva-sive brain stimulation technique that promotes transientpolarity-dependent changes in spontaneous neuronal activ-ity. This effect is mediated by the application of constantlow-amplitude electrical currents using epicranially posi-tioned electrodes above a specific brain region of interest[1–4]. The therapeutic use of low-amplitude electrical cur-rents has a long historical track. Accordingly, both Greeksand Romans used electric torpedo fishes for migraine treat-ment, and in the 11th century, a similar therapeutic proce-dure was attempted to handle epilepsy [5]. In the 19th

century, the application of galvanic currents was attemptedto heal melancholia [6]. Over the years, the scientific commu-nity interest in brain stimulation grew, and several noninva-sive brain stimulation techniques were developed such astDCS, deep brain stimulation, or transcranial magnetic stim-ulation. The epicranial application of direct currents pro-motes a weak electric field force and produces neuronalmembrane potential changes [7, 8]. These alterations occurthrough sodium and calcium currents [1] modulating spon-taneous neuronal activity [2]. The consequent regional neu-ronal inhibition or excitation depends on the applied currentpolarity [9, 10]. So, it was overall observed that cathodal cur-rents produce inhibitory effects, and thus hyperpolarization,

HindawiNeural PlasticityVolume 2020, Article ID 4795267, 23 pageshttps://doi.org/10.1155/2020/4795267

whereas anodal currents increase excitability in the form ofdepolarization [2, 11] (Figure 1).

There is nowadays an ongoing discussion regarding thefactors that interfere with tDCS outcomes. The initial brainresting state of each subject [12], his/her baseline perfor-mance [13], specific individual variations in brain tissuemorphology [14], or even more particular details from theexperimental design or stimulation protocol used [15]influence these outcomes. In vivo and in vitro studies areconsensual to demonstrate that tDCS-modulated corticalexcitability depends on several stimulation parameters, suchas duration and frequency of stimulation [16]; polarity,intensity, and density of the applied current [17, 18]; andelectrode size and position in the scalp [18–20]. Despite that,beneficial effects of tDCS in several brain disorders, such asPD [21, 22], depression [23], stroke [24, 25], or autism [26,27], have been documented, and there is growing evidenceproposing tDCS application in multiple other disease condi-tions affecting cognition and neuroplasticity mechanisms.

Both preclinical and clinical studies have demonstratedtherapeutic effects of tDCS. Indeed, in human studies, anodaltDCS applied intermittently in the prefrontal cortex (PFC)during slow-wave sleep period, improved recall of declarativememories (word pairs). The authors correlated these findingswith enhancement of slow oscillatory electroencephalogram(EEG) activity (<3Hz, delta (δ) waves), responsible forneuronal plasticity facilitation [28]. Also, anodal tDCS overdorsolateral prefrontal cortex (DLPFC) improved workingmemory in PD patients and in major depression patients byboosting cortical excitability [21, 23]. Accordingly, preclini-cal animal studies reported that cortical anodal tDCSimproved spatial memory in both wild type (WT) [29] andthe AD rat model [30]. Beneficial effects were also found dur-ing the early stage of traumatic brain injury (TBI) [31] andfollowing a pilocarpine-induced status epilepticus in normalrats [32]. Moreover, improvements were also reported con-cerning short-term memory in an animal model of attentiondeficit hyperactivity disorder (ADHD) [33].

The molecular mechanisms underlying the tDCS-mediated cognitive improvements and neuroplasticity pro-cesses have become the focus of recent interest. Accordingly,tDCS modulation over several cognition-related plasticitygenes and their signaling pathways has been studied. In thisreview, we provide a state of the art on the application ofdifferent protocols of tDCS in animal models highlightingits effectiveness on neuroplasticity mechanisms and, conse-quently, their related learning and memory processes. Sincethe published systematic reviews focused on human applica-tion of tDCS, here, we provide a comprehensive revision ofthe effect of tDCS in in vivo rodent models of normal andpathological brain functioning.

2. Methods

2.1. Data Sources and Search. Studies included in this reviewwere identified by searching PubMed. The search was rununtil 31 October 2019. The search terms were (“transcranialdirect current stimulation”) AND (mice OR mouse OR ani-mal). Articles were firstly assessed based on their abstracts

and titles, aiming to include studies that reported applyingtDCS to cognitive impairment in animal models. Simulta-neously, the following exclusion criteria were adopted toreject studies: (1) not written in English; (2) performingreviews; (3) in human subjects; (4) in vitro models; (5)employing other brain stimulation techniques (e.g., transcra-nial magnetic stimulation (TMS), deep brain stimulation(DBS), or transcranial alternating current stimulation(tACS)); and (6) not explicitly describing the tDCS protocol(stimulation area, number of sessions, frequency, intensity,and pattern).

2.2. Data Extraction. A data extraction sheet was developedseeking to retrieve relevant information from each study,namely, study design, sample size, animal model, whetheradditional therapy was performed, details of the tDCS proto-col, outcome measures, and behavioral results.

2.3. Study Selection. The database search was elaboratedaccording to the PRISMA statement requirements [34]. 404records were found, which underwent a preliminary screen-ing (of titles and abstracts), with 314 records being excludedbecause they did not meet the eligibility criteria. After thefull-text analysis of each of the 90 individual articles, 44rodent studies focusing on tDCS effects over cognition andneuroplasticity in both healthy and neuropathological animalmodels were selected (Figure 2).

3. Results

3.1. Role of Anodal tDCS in Cognition Processing in HealthyAnimals. In healthy animals, studies demonstrated memoryimprovement in association with induction of synapticplasticity mechanisms. In fact, tDCS to prefrontal corteximproved monkey’s performance on an associative learningtask by altering low-frequency oscillations and functionalconnectivity, both locally and between distant brain areas[35]. Regarding rodent models, data are controversial regard-ing fear condition. Right frontal anodal tDCS administered24 h before behavioral task did not alter contextual and audi-tory learning and memory [36]. Additionally, another studydescribed that while the anodal stimulation did not affect fearretrieval, posttraining cathodal stimulation improved fearmemory retrieval [37, 38]. However, left prefrontal anodaland cathodal tDCS impaired the acquisition of both contex-tual and cued fear memory, which could be explained byactivity modulation of deep structures such as the amygdalaand hippocampus [39].

Concerning learning and memory, de Souza Custódioand colleagues [29] reported better spatial working memoryperformance following administration of anodal currents tothe medial prefrontal cortex (mPFC). In agreement, it wasdescribed that administration of hippocampal anodal tDCSimproves learning and memory in the Morris water mazeand novel object recognition tests [40]. Moreover, memoryperformance in the passive avoidance learning task wasenhanced by anodal stimulation [41]. Also, cortical cathodalstimulation together with visuospatial memory training ledto cognitive improvement [42].

2 Neural Plasticity

The revised in vivo animal model studies regarding tDCSeffects in memory and cognition of healthy animals are listedbelow in Table 1.

3.2. Beneficial Role of tDCS in Brain Diseases.Overall, reportsusing animal models of brain diseases described a beneficialrole of tDCS in the mitigation of memory symptoms of neu-rologic conditions such as Alzheimer’s disease (AD) or trau-matic brain injury (TBI). More recent studies demonstrated

that tDCS rescued AD-related cognitive symptoms, namely,spatial memory and motor skills [30, 43, 44]. The repetitivestimulation with anodal tDCS in the AD-like dementia ratmodel reduced the time interval animals needed to reacha food pellet and also decreased the number of errors inthe attempt [43]. The same research group showed laterthat the abovementioned protocol rescued spatial learningand memory in a Aβ1-40-lesioned AD rat model [30].Moreover, the impact of tDCS on cognitive performance

Electric currentinto the brain

Electric currentinto the brain

Referenceelectrode

Activeelectrode

Direct currentstimulator

Anodal stimulation

Depolarized soma

Excitation

Cathodal stimulation

Hyperpolarized soma

Inhibition

−

+

−

+

Figure 1: Illustration of transcranial direct current stimulation in the mice. Anodal stimulation depolarizes the neuronal membrane andenhances excitability. On the other hand, cathodal stimulation hyperpolarizes the neuronal membrane and decreases excitability.

# of records identified throughPubmed database searching: 404

# of records screened: 404

# of full-text articlesassessed for eligibility: 90

# of rodent studies includedin the qualitative synthesis: 44

# of records excluded: 314220 reviews and methods(i)

(ii)(iii)(iv)

8 in vitro studies36 human studies50 studies regarding otherbrain stimulation techniques(e.g., TMS, DB and tACS)

Figure 2: Search flow diagram (in accordance with PRISMA statement). Abbreviations: DB: deep brain stimulation; tACS: transcranialalternating current stimulation; TMS: transcranial magnetic stimulation.

3Neural Plasticity

Table1:Effectof

transcranialdirectcurrentstim

ulationon

mem

oryandlearning

ofhealthyanim

als.

Autho

rYear

Animal

mod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(m2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Dockery

etal.[42]

2011

NDM

Long-

Evans

rats;

males

41a-tD

CS

vs.

c-tD

CS

Fron

talcortex

(leftor

right

hemisph

ere)

0.2

0.035

3057.1

Back

10.5

NY(3

days)

↑Visuo

spatialw

orking

mem

ory(c-tDCS)

deSouza

Custódio

etal.[29]

2013

NDM

Wistar

rats;m

ales

23a-tD

CS

LeftmPFC

0.4

0.25

11N/A

Neck

1N

Y(5

days)

↑Spatialw

orking

mem

ory

(1h,

4h,

and10

hpo

ststim

ulation)

Faraji

etal.[96]

2013

NDM

Long-

Evans

rats;

males

24a-tD

CS

Somatosensory

cortex

(bilateral)

0.065

N/A

10N/A

Backof

skull

N/A

NY

↑Corticaln

eurald

ensity

↑Motor

learning

(a-tDCSappliedbilaterally

orinto

pawpreferredto

reaching

contralateral

hemisph

ere)

Pod

daetal.[40]

2016

NDM

C57BL/6

mice;

males

16a-tD

CS

vs.

c-tD

CS

Leftparietal

cortex

(dorsalto

hipp

ocam

pal

form

ation)

0.35

0.06

20N/A

Ventral

thorax

5.2

NN

(single

session)

↑Spatiallearningand

mem

ory

(a-tDCS;benefits

observableon

eweekafter)

↑BDNFexpression

sin

the

hippocam

pus

CREB/CBPpathway

activation

Manteghi

etal.[36]

2017

NDM

NMRI

mice;

males

64a-tD

CS

Right

fron

tal

cortex

0.2

0.04

20N/A

Chest

9.5

N/A

N(single

session)

↓Freezing

time%

and↑

latencyto

thefreezing

(tDCSfollowing0.1mg/kg

ACPAinjection)

Nasehi

etal.[37]

2017

NDM

NMRI

mice;

males

128

a-tD

CS

vs.

c-tD

CS

Right

fron

tal

cortex

0.2

0.04

20N/A

Ventral

thorax

9.5

NY(2

sessions)

↑Fear

mem

ory

retrieval/freezing

time

(a-tDCSandprop

rano

lol

injectionbefore

cond

itioning)

Nasehi

etal.[38]

2017

NDM

NMRI

mice;

males

128

a-tD

CS

vs.

c-tD

CS

Leftfron

tal

cortex

0.2

0.04

20N/A

Ventral

thorax

9.5

NY(2

sessions)

↑Con

textualfearmem

ory

acqu

isition

(a-tDCSbefore

pre-

orpo

sttraining)

Abbasi

etal.[39]

2017

NDM

NMRI

mice;

males

41a-tD

CS

vs.

c-tD

CS

LeftPFC

0.2

0.04

20or

30N/A

Chest

9.5

NN

(single

session)

↓Con

textualand

cued

fear

mem

ory

4 Neural Plasticity

Table1:Con

tinu

ed.

Autho

rYear

Animal

mod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(m2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Martins

etal.[97]

2019

NDM

Male

Wistar

rats;m

ales

50a-tD

CS

LeftmPFC

0.4

N/A

13N/A

N/A

N/A

N/A

Y(5

days)

↑Spatialw

orking

mem

ory

↑GAP-43(extinctby

AMPARantagonistPRP)

Yuetal.

[41]

2019

NDM

Sprague

Daw

ley

rats;m

ales

224

a-tD

CS

SCdo

rsalto

the

hippocam

pus

0.25

0.25

30N/A

Anterior

chest

N/A

(EEG

electrod

e)Y

N(single

session)

↑Mem

ory

(passive

avoidance

mem

oryretention)

↑LT

Pin

CA1hippocam

pus

(blocked

byTrkB

antagonist)

↑BDNFin

CA1

hippocam

pus

Abbreviations:rtD

CS:repetitive

transcranialdirectcurrentstimulation;a-tD

CS:anod

altranscranialdirectcurrentstimulation;c-tD

CS:cathod

altranscranialdirectcurrentstimulation;SC

:stereotaxiccoordinates;

C57BL/6:

mou

sestrain;N

MRI:Naval

Medical

ResearchInstituteou

tbredmice;NBM:n

odiseasemod

el;SHR:spo

ntaneous

hypertensive

rats;W

KY:W

istarKyoto

rats;P

FC:p

refron

talcortex;m

PFC

:medial

prefrontal

cortex;ITC:inferotempo

ralcortex;CAI:comuam

mon

is1region

inthehipp

ocam

pus;PRP:perampanel;ACPA:anticitrullin

ated

proteinantibody

(selective

cann

abinoidCBIreceptor

agon

ist);

AMPAR:α

-amino-3-hydroxy-5-methyl-4-isoxazolepropion

icacid

receptor;T

rk:tropo

myosinreceptor

kinase

receptor;C

REB/CBP:cAMPrespon

seelem

entbind

ingprotein;

BDNF:

brain-derivedneurotroph

icfactor;G

AP-43:

grow

th-associatedprotein43;L

TP:lon

g-term

potentiation

;EEG:electroenceph

alograph

y;A/m

2 :am

pere

persquare

meter;m

A:m

illiampere;cm

2 :square

centim

eter;m

m:m

illim

eter;h

:hou

r;min:m

inute;vs.:versus;Y

:yes;N

:no;N/A

:not

available.

5Neural Plasticity

of streptozotocin-induced diabetic rats has been evaluated.Both anodal and cathodal stimulations in the prefrontalcortex restored memory impairment [45, 46] together withrestoration of LTP [45]. Other authors evaluated the poten-tial therapeutic effects of tDCS in memory impairment inan animal model of ADHD. It was found that this neuro-modulation technique was able to improve short- andlong-term memory deficits in the spontaneous hypertensiverats (SHR) but not in their control, Wistar Kyoto rats [33,47]. In addition, no changes were detected in workingmemory of these control rats following administration oftDCS [47].

Anodal tDCS also ameliorated behavioral and spatialmemory function in the early phase after TBI when it wasdelivered two weeks postinjury. However, earlier stimulationonly improved spatial memory [31]. In a later phase of TBI, itwas possible to observe motor recovery as well as spatialmemory improvement following repeated anodal tDCS[48]. A growing number of studies has been reporting prom-ising effects of neurostimulation in models of addictive disor-ders, by reducing craving and maladaptive pervasive learning[49]. In fact, repeated anodal stimulation in mouse frontalcortex decreased nicotine-induced conditioned place prefer-ence and further improved working memory [50]. Samepolarity currents also could prevent cocaine-induced loco-motor hyperactivity and place preference conditioning [51].In addition, it has been reported that cathodal stimulationhas an anticonvulsive effect [16, 32, 52–54]. Indeed, theadministration of hippocampal tDCS rescued cognitiveimpairment by reducing hippocampal neural death andsupragranular and CA3 mossy fiber sprouting in a lithium-pilocarpine-induced status epilepticus rat [32]. Other neuro-plastic effects were evidenced in the reversion of motorsymptoms in PD by tDCS administration. The applicationof anodal currents enhanced graft survival and dopaminergicre-innervation of the surrounding striatal tissue and pro-nounced behavioral recovery [55].

Despite the fact that many studies reported recovery frommemory deficits following tDCS stimulation, there are someopposing reports in animal models of disease affecting cogni-tion. In a recent study from Gondard and collaborators usinga triple transgenic (3xTg) mouse model of AD, it was evi-denced that a neurostimulation was not able to amelioratememory symptoms [56]. To reconcile this discrepancy, pre-vious authors have suggested the importance of choosingan optimal current intensity in order to modulate corticalexcitability since LTP alterations were dependent on currentintensity [57].

The reports regarding tDCS effects in cognition andmemory in animal models of brain disease are listed inTables 2 and 3.

3.3. Effect of tDCS on Cellular and Molecular NeuroplasticityMechanisms. Neuronal network reorganization underliesneuroplasticity processes like developmental synaptogenesis,or neurogenesis and synaptic turnover later on, which ulti-mately contributes to optimal brain development and aging,as well as functional recovery upon trauma [58]. Interest-ingly, several reports using genetic engineered animals, phar-

macologically induced animal models of disease, or in vitrotechniques enlightened the potential of direct current stimu-lation (DCS) to interact with a myriad of neuroplasticity-related processes such as neuroinflammation [59, 60], neuralstem cell migration [59], neurite growth [61], or neurogen-esis [62]. Moreover, both human and in vivo animal studiesevidenced a tDCS-induced effect on memory and learning[28, 35, 63]. However, the underlying cellular and molecularmechanisms remain to be elucidated.

3.3.1. Modulation of the Excitatory/Inhibitory Network. Todate, animal experimental evidence highlighted tDCS influ-ences on synaptic plasticity, through alterations in thefunctional connectivity of cognition-related areas [35] andby modulation of excitatory/inhibitory network tonus[64], which may involve both the GABAergic and gluta-matergic systems. Accordingly, a study conducted witholder adults remarked an anodal stimulation effect ingamma-aminobutyric acid (GABA) levels [65]. Similarly, inhuman healthy volunteers, an anodal tDCS effect in motorlearning was correlated with a decrease in GABA levels, anoutcome known to be a determinant factor in the promotionof long-term potentiation- (LTP-) dependent plasticity andtherefore learning [66, 67].

Several preclinical studies probed LTP enhancement fol-lowing direct current stimulation. Anodal DCS enhancedLTP in both mouse cortex [68] and rat hippocampal slices[69, 70]. Further, this neurostimulation method increasedlocal field potential (LFPs) amplitudes in primary somato-sensory cortex of rabbits [63]. Also, other works demon-strated that neurostimulation-enhanced hippocampal LTPwas associated with better spatial memory performancealong with an increase in brain-derived neurotrophic factor(BDNF) expression levels [40]. An opposite effect on LTPand LFPs was obtained with administration of cathodal cur-rents. In agreement, a report from Sun et al. [71] evidencedthat cathodal currents applied in mouse neocortical slicesinduced field excitatory postsynaptic potential depression.This type of LTD was smothered by application of anmGluR5 negative allosteric modulator [72]. These findingssupport a possible modulatory effect of tDCS on mGluR5-mTOR signaling [72]; these molecular pathways are recog-nized to disturb cognition-related synaptic plasticity.

Further evidence supporting tDCS effect on LTP-likemechanisms was recently brought to light by Stafford et al.[73]. These authors observed that a single anodal tDCSincreased both the phosphorylation at the S831 of GluA1subunit and the translocation of α-amino-3-hydroxy5-methyl-4-isoxazole propionic acid receptors (AMPARs)from cytosolic to synaptic fractions in the hippocampus.These data could be favoring learning enhancement, as thistranslocation has been associated with hippocampal LTPinduction [72]. Accordingly, others reported a spatial work-ing memory enhancement after anodal stimulation over leftmedial PFC that was lost with the administration of theAMPAR antagonist perampanel (PRP). In contrast tocathodal currents, anodal currents enhanced intracellularcalcium (Ca2+) intake in cell cultures including astrocytes[74–76], a process associated with AMPAR phosphorylation

6 Neural Plasticity

Table2:Im

pactof

transcranialdirectcurrentstim

ulationon

mem

oryandlearning

inanim

almod

elsof

braindisorders.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Kam

ida

etal.[32]

2011

Lithium-

pilocarpine

hydrochloride

(60mg/kg)SC

injectionat

P20-21

Wistar

rats;m

ales

18c-tD

CS

Hippo

campu

s0.2

0.035

30N/A

Backof

neck

N/A

NY(dailyfor2

weeks)

↓SE

-ind

uced

hipp

ocam

palcellloss

inCA3region

Yuetal.

[43]

2014

Scop

olam

ine

IPinjection

Sprague

Daw

ley

rats;both

gend

ers

16N/A

Parietalcortex

(hippo

campu

s)0.1

N/A

20N/A

N/A

N/A

N/A

Y(2x/daydu

ring

5days

aweek,

for4weeks)

↓Tim

eand↓

numberof

errors

toreachfood

pellets

Significant

differencesin

ACh

content(day

14and

day28)

↑Motor

function

Pedron

etal.[49]

2014

NicotineIP

injection

(1mh/kg

2x/day

for14

days)

Swiss

mice;

females

152

a-tD

CS

Leftfron

tal

cortex

0.2

0.035

2×20

N/A

Ventral

thorax

9.5

NY(5

days)

↑Working

mem

ory

↑Nicotine-indu

ced

placepreference

cond

itioning

(3weeks

poststim

ulation)

Yuetal.

[30]

2015

Bilateral

hippocam

pus

Aβ1-40

injection

Sprague

Daw

ley

rats;

females

36a-tD

CS

Right

fron

tal

cortex

0.02;0.06;

0.1;0.2

0.0314

20N/A

Ventral

thorax

10N

Y(10sessions

in2weeks)

↑Spatiallearning

performance

(best

with0.1mAand

0.2mAstim

ulation)

↓GFA

Pexpression

inCA1hippocam

pus

region

andDG(best

with0.1mA

stim

ulation)

Yoon

etal.[31]

2016

Lateralfl

uid

percussion

metho

d

Sprague

Daw

ley

rats;m

ales

36a-tD

CS

Hippo

campu

s0.2

0.0225

2028.2

Chest

48(corset)

YY(dailyfor5

days)

↑Perilesion

alarea

BDNFexpression

(tDCS2weeks

post-TBI)

↑Cho

line/creatinine

ratios

(tDCS1week

post-TBI)

Motor

performance

recovery

(2weeks

oftD

CS)

7Neural Plasticity

Table2:Con

tinu

ed.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Leffa

etal.[33]

2016

SHR

SHRrats

andWKT

rats;m

ales

48a-tD

CS

Fron

talcortex

0.5

1.5

2033.4

Between

ears

1.5

NY(8

consecutive

days)

↑DAlevelsin

STRin

both

ratstrainsand

inthehipp

ocam

pus

followingtD

CS

treatm

entin

WKY

↑BDNFlevelsin

WKYrats

Short-term

mem

ory

improvem

ent

Wuetal.

[45]

2017

STZ-ind

uced

diabeticrats

Sprague

Daw

ley

rats;m

ales

130

a-tD

CS

dPFC

0.2

0.0314

30N/A

Anterior

chest

0.25

YY

↑Spatialw

orking

mem

oryandmPFC

LTPrestoring

Pedron

etal.[50]

2017

Cocaine

injections

Swiss

mice;

females

165

a-tD

CS

Leftfron

tal

cortex

0.2

0.035

2×20

N/A

Ventral

thorax

9.5

N

Y(5

days

stim

ulation,

twiceaday;5h

interstimulation

interval)

↓Cocaine-ind

uced

locomotor

activity

Nococaine-indu

ced

placepreference

(5mg/kg

and

25mg/kg)

↑Zif2

68basal

expression

underthe

electrod

earea,inthe

STRandcortex

Leffa

etal.[55]

2018

ADHD

SHRand

WKYrats;

males

30a-tD

CS

(bicephalic)

Fron

talcortex

(sup

raorbital

area)

0.5

1.5(ECG

electrod

e)20

33.3

Neck

1.5(ECG

electrod

e)N/A

Y(8

days)

↓Inflam

matory

cytokinesand

reversionof

long-

term

mem

orydeficits

inSH

Rrats

Bragina

etal.[47]

2018

CCI

Mice;N/A

40a-tD

CS

Parietal

somatosensory

cortex

0.1

N/A

15N/A

Ventral

thorax

N/A

N/A

Y(dailyfor4

days,over4

weeks

and3days

interval)

Motor

coordination

recovery

Spatialm

emoryand

learning

performance

improvem

ent

↑CBFbilaterally

(regionalarteriolar

dilatation

and

hypo

xiaredu

ction)

Roostaei

etal.[44]

2019

STZ-ind

uced

diabeticrats

Wistar

rats;m

ales

64a-tD

CSvs.

c-tD

CS

Leftfron

tal

cortex

0.2

3.5

20N/A

Ventral

thorax

9.5

NY(twiceaday

over

2days)

Restoration

ofST

Z-

indu

cedam

nesia

(bothpo

larities)

8 Neural Plasticity

Table2:Con

tinu

ed.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Gon

dard

etal.[55]

2019

Animalmod

elof

AD-triple

transgenic

(3xT

g)mice

Triple

transgenic

(3xT

g)mice;

males

27c-tD

CSand

a-tD

CS

Second

ary

motor

cortex

(m2)

(c-TDCS)

anddo

rsal

tempo

ral

hippocam

pus

(a-tDCS)

0.05

0.0325

20N/A

N/A

0.0325

N/A

Y(5

days/week

for3weeks)

Notreatm

enteffect

onmem

oryou

tcom

eor

AD

neurop

atho

logical

biom

arkers

Abbreviations:rtD

CS:repetitive

transcranialdirectcurrentstimulation;a-tD

CS:anod

altranscranialdirectcurrentstimulation;c-tD

CS:cathod

altranscranialdirectcurrentstimulation;TBI:traumaticbraininjury;

ADHD:attention

deficith

yperactivitydisorder;SHR:spo

ntaneous

hypertensive

rats;W

KY:W

istarKyoto

rats;C

BF:cerebralbloodflow

;PFC

:prefron

talcortex;mPFC

:medialprefron

talcortex;dP

FC:dorsolateral

prefrontalcortex;D

G:dentategyrus;ST

R:striatum;M

2:second

arymotor

cortex;ITC:inferotem

poralcortex;CA3:cornuam

mon

is3region

inthehippocam

pus,ST

Z:streptozotocin;AD:A

lzheim

er’sdisease;Aβ1-

40:am

yloidbeta

peptide1–40;ACh:

acetylcholine;BDNF:

brain-derivedneurotroph

icfactor;DA:do

pamine;GFA

Pglialfibrillaryacidic

protein;

Zif2

68:zinc

finger

transcriptionfactor

268;

LTP:long-term

potentiation

;IP:intraperiton

eal;SC

:subcutaneous;SE

:status

epilepticus;ECG:electrocardiograph

y;mg:

milligram;kg:kilogram

;A/m

2 :am

pere

persquare

meter;mA:milliampere;cm

2 :square

centim

eter;

mm:m

illim

eter;h

:hou

r;min:m

inute;vs.:versus;Y

:yes;N

:no;N/A

:not

available.

9Neural Plasticity

Table3:Roleof

transcranialdirectcurrentstim

ulationon

neurop

lasticitywithafocuson

anim

almod

elsof

neurotraum

a.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Nekhend

zyetal.[97]

2004

Inflam

matory

nociceptionmod

el

Sprague

Daw

ley

rats;m

ales

31c-tD

CS

Fron

talcortex

2.25

N/A

45N/A

Bim

astoid

N/A

YY(8

days)

↓Nociceptive

respon

se(effectslasted

upto

50min

poststim

ulation)

Taibetal.

[98]

2009

Left

hemicerebellectom

yWistar

rats;m

ales

9a-tD

CS

Right

orleft

motor

cortex

0.4

0.071

2056.3

Supraorbital

region

0.0064

N/A

Y↑Tcorticom

uscular

respon

seam

plitud

es

Kim

etal.

[99]

2010

MCAO

Sprague

Daw

ley

rats;both

gend

ers

61c-tD

CSvs.

a-tD

CS

Leftprim

ary

motor

cortex

(M1)

0.1

0.785

30N/A

Trunk

9Y

Y(2

weeks)

Neuroprotection

over

whitematterand

ischem

icsize

↓(a-tDCS)

↑Motor

function

Jiangetal.

[95]

2012

MCAO

Sprague

Daw

ley

rats;both

gend

ers

90a-tD

CSand

c-tD

CS

Motor

cortex

0.1

0.785

301.27

Trunk

9Y

Y(daily,for

3,7,or

14days

postlesion

)

↑Motor

function

(7and14

days

poststroke)

↑Dendriticspine

density

↓PX1expression

(7th

and14

hday

poststroke)

Yoonetal.

[56]

2012

MCAO

Sprague

Daw

ley

rats;m

ales

30a-tD

CS

Leftprim

ary

motor

cortex

(M1)

0.2

N/A

2028.2

Anterior

chest

48.0

Y

Y(5

days

stim

ulation1

weekvs.1

day

postischem

ia)

↑Motor

function

and

Barnesmaze

performance

(a-tDCS

applied1weekafter

ischem

icinjury)

↑MAP-2

andGAP-43

expression

arou

ndthe

perilesion

alarea

(a-tDCSapplied1

weekpo

stischem

icinjury)

Lasteetal.

[100]

2012

CFA

injection/chronic

inflam

mation

indu

ction

Wistar

rats;m

ales

18a-tD

CS

Parietal

cortex

0.5

1.5(ECG

electrod

e)20

33.4

Supraorbital

area

1.5(ECG

electrod

e)N

Y(8

days)

Significant

differences

inno

ciceptive

respon

se(immediately

afterand24

hpo

ststim

ulation)

Adachi

etal.[101]

2012

CRS

Wistar

rats;m

ales

48a-tD

CS

Parietal

cortex

0.5

1.5(ECG

electrod

e)20

3.3

Supraorbital

area

1.5(ECG

electrod

e)N

Y(8

days)

↓Nociceptive

respon

sein

chronicstress

cond

ition

↓TNFexpression

inthehippocam

pus

10 Neural Plasticity

Table3:Con

tinu

ed.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Peruzzotti-

Jametti

etal.[60]

2013

MCAO

C57BL/6

mice;

males

137

c-tD

CSvs.

a-tD

CS

Leftparietal

cortex

0.25

0.0144

4055

Ventral

thorax

5.2

N/A

N(single

session)

↑Infarctv

olum

eand↑

BBBleakage(a-tDCS

ipsilesion

alhemisph

ere)

CorticalG

luactivity

↓,function

al↑and

ischem

icdamage↓

(c-tDCSipsilesion

alhemisph

ere)

Notturno

etal.[102]

2014

MCAO

Sprague

Daw

ley

rats;both

gend

ers

53c-tD

CS

Leftmotor

cortex

0.2

0.07

120

28.6

Ventral

thorax

10.5

YN

(single

session)

Ischem

iavolume↓

Luetal.

[103]

2015

MPTPinjection

C57bl

mice;

males

36a-tD

CS

Leftfron

tal

cortex

0.2

0.035

1057

Between

shou

lders

9N/A

Y(dailyfor3

weeks)

↑Motor

coordination

(until21

days

poststim

ulation)

↑TH

andDA

expression

↓Oxidative

stress

Spezia

Adachi

etal.[104]

2015

Restraint

stress

mod

elWistar

rats;m

ales

78a-tD

CS

Parietal

cortex

(midlin

e)0.5

1.5(ECG

electrod

e)20

N/A

Supraorbital

area

1.5(ECG

electrod

e)N

Y(dailyfor8

days)

↓Stress-ind

uced

nociceptiverespon

se↑Painthreshold

↓BDNFlevels(spinal

cord

andbrainstem)in

unstressed

anim

als

Yoonetal.

[31]

2016

Lateralfl

uid

percussion

metho

d

Sprague

Daw

ley

rats;m

ales

36a-tD

CS

Hippo

campu

s0.2

0.0225

2028.2

Chest

48(corset)

YY(dailyfor5

days)

↑Perilesion

alarea

BDNFexpression

(tDCS2weeks

post-TBI)

↑Cho

line/creatinine

ratios

(tDCS1week

post-TBI)

Motor

performance

recovery

(2weeks

oftD

CS)

Leffaetal.

[33]

2016

SHR

SHRrats

andWKT

rats;m

ales

48a-tD

CS

Fron

talcortex

0.5

1.5

2033.4

Betweenears

1.5

NY(8

consecutive

days)

↑DAlevelsin

STRin

both

ratstrainsandin

thehipp

ocam

pus

followingtD

CS

treatm

entin

WKY

↑BDNFlevelsin

WKY

rats

11Neural Plasticity

Table3:Con

tinu

ed.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Liuetal.

[105]

2016

PTI

Sprague

Daw

ley

rats;N

/A58

c-tD

CS

Right

S1FL

2N/A

2020.37

Venter

N/A

N/A

N(single

session)

Ischem

iaexpansion

inhibition

(c-tDCS

immediately

postischem

iaindu

ction)

↓NeuN

expression

(c-tDCS+

PSS

grou

p)

Braun

etal.

[106]

2016

MCAO

Wistar

rats;m

ales

41c-tD

CSvs.

a-tD

CS

Leftprim

ary

motor

cortex

(M1)

0.5

0.035

15N/A

Ventral

thorax

N/A

Y

Y(10-day

stim

ulation;

5days

with

2-day

interval)

Gaitrecoveryatday16

poststroke

(both

polarities)

Faster

recovery

oflim

bstrength

(fully

recoveredstrength

atday10

andgaitat

day

14withc-tD

CS)

↑Microgliaand

neuroblastsin

lesion

ipsilateralcortex

Cioatoetal.

[107]

2016

Sciaticnerve

chronic

constriction

Wistar

rats;m

ales

84a-tD

CSand

c-tD

CS

Parietal

cortex

(bicephalic)

0.5

1.5(ECG

electrod

e)20

N/A

Supraorbital

area

1.5(ECG

electrod

e)N

Y(8

days)

Nociceptive

relieve

(for

upto

7days

poststim

ulation)

Reversion

ofTIL-1þ

levels(48hand7days

poststim

ulation)

Filhoetal.

[108]

2016

Partialsciaticnerve

compression

Wistar

rats;m

ales

144

a-tD

CS

Parietal

cortex

0.5

1.5(ECG

electrod

e)20

N/A

Supraorbital

area

1.5(ECG

electrod

e)N

Y(8

days)

↓BDNFexpression

(48hpo

ststim

ulation)

Reversion

ofbehavioralalteration

s(analgesicand

anxiolytic)associated

withneurop

athicpain

Moreira

etal.[109]

2016

Painand

menop

ause

(ovariectomised

anim

als)

Wistar

rats;

females

45C-tDCS

Parietal

cortex

0.5

1.5(ECG

electrod

e)20

N/A

Supraorbital

area

1.5(ECG

electrod

e)N

Y(8

days)

↓Hypothalamic

BDNFlevelsandT

serum

BDNFin

ovariectom

ised

anim

als

Dim

ovetal.

[87]

2016

N/A

Wistar

rats;m

ales

25c-tD

CS

Leftprim

ary

motor

cortex

(M1)

0.25

0.0227

15N/A

Ventral

thorax

N/A

NN

(single

session)

Bilateral↓

Egr-1

expression

inthePAG

SpinalENK

immun

oreactivity↓in

theDHSC

12 Neural Plasticity

a-

Table3:Con

tinu

ed.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Liuetal.

[110]

2017

PTI

Sprague

Daw

ley

rats;m

ales

58c-tD

CS

S1FL

2N/A

20N/A

3mm

lateral

tolambd

aN/A

N/A

N(single

session)

Preventionof

ischem

iainjury

expansion

during

hyperacute

phaseof

ischem

ia(c-tDCS+

PSS)

Kim

&Han

[111]

2017

Mod

ified

Tang’s

metho

d[128]

Sprague

Daw

ley

rats;N

/A31

a-tD

CS

Leftmotor

cortex

0.2

130

0.26

Ventral

thorax

9Y

N

Earlyrecovery

ofconsciou

snessand

MEPandSE

Pprolon

gedlatency

(tDCSappliedright

afterTBI)

↓AstroglialG

FAP

immun

oreactivity

Winkler

etal.[112]

2017

Striatal6-OHDA

injection

Sprague

Daw

ley

rats;

females

24a-tD

CSand

c-tD

CS

Leftmotor

cortex

N/A

0.16

208

Chest

3N

Y(dailyfor

14days)

Graftsurvival,striatal

dopaminergic

reinnervationand

motor

recovery

(a-tDCS)

deSouza

etal.[113]

2017

PSN

LSw

iss

mice;

males

N/A

a-tD

CSand

c-tD

CS

Parietal

cortex

(bicephalic)

0.5

N/A

(EEG

electrod

e)5;10;15;20

N/A

Supraorbital

area

N/A

(EEG

electrod

e)N

N(single

session)

Antiallodyniceffect

(seen4h

poststim

ulationof

15min

and20

min)

Leffaetal.

[46]

2018

ADHD

SHRand

WKYrats;

males

30a-tD

CS

(bicephalic)

Fron

talcortex

(sup

raorbital

area)

0.5

1.5(ECG

electrod

e)20

33.3

Neck

1.5(ECG

electrod

e)N/A

Y(8

days)

↓Inflam

matory

cytokinesand

reversionof

long-term

mem

orydeficitsin

SHRrats

Paciello

etal.[114]

2018

NIH

LWistar

rats;m

ales

124

a-tD

CS

Tem

poral

lobe

(aud

itory

cortex)

0.35

0.0625

2056

Ventral

thorax

12N

Y(2

days)

↑Dendriticspines

density(layer

2/3

pyramidalneuron

sof

theauditory

cortex)

↑BDNFand

synaptop

hysin

expression

inauditory

cortex

(24h

poststim

ulation)

Fregni

etal.

[115]

2018

N/A

Wistar

rats;m

ales

32N/A

N/A

(bicephalic)

N/A

N/A

20N/A

N/A

N/A

N/A

Y(8

days)

tDCSpriorto

stress

expo

sure

prevented

thermalhyperalgesia

13Neural Plasticity

Table3:Con

tinu

ed.

Autho

rYear

Animalmod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Leeetal.

[116]

2019

MPTPinjection

C57bl

mice;male

60a-tD

CS

Primary

motor

cortex

(M1)

N/A

N/A

30N/A

Between

shou

lders

N/A

N/A

Y(dailyfor5

days)

↑Motor

coordination

Rescueof

MTPT-

indu

cedmitocho

ndrial

dysfun

ction(Ç

ATP

andGDH

and$Drp1

levels)

Callaietal.

[117]

2019

CCI-IO

NWistar

rats,m

ales

151

a-tD

CS

Parietal

cortex

(bicephalic)

0.5

1.5(ECG

electrod

e)20

N/A

Supraorbital

area

1.5(ECG

electrod

e)N

Y(8

days)

↓Mechanical

hyperalgesia

↓TNF-aexpression

(7days

poststim

ulation)

↓IL-10(7

days

poststim

ulation)

↓LD

Hserum

levels

Scarabelot

etal.[118]

2019

CFA

injection/chronic

inflam

mation

indu

ction

Sprague

Daw

ley

rats;m

ales

104

a-tD

CS

Parietal

cortex

(bicephalic)

0.5

1.5(ECG

electrod

e)20

N/A

Supraorbital

area

1.5(ECG

electrod

e)N/A

Y(8

days)

↓Therm

aland

mechanical

hyperalgesia

↑IL-6

(inbrainstem

24hpo

ststim

ulation)

↓IL-10(7

days

poststim

ulation)

Normalizationof

BDNFlevels(24h

poststim

ulation)

Abbreviations:rtD

CS:repetitive

transcranialdirect

currentstim

ulation;

a-tD

CS:anod

altranscranialdirect

currentstim

ulation;

c-tD

CS:cathod

altranscranialdirect

currentstim

ulation;

C57BL/6:mou

sestrain;

SHR:spon

taneou

shypertensive

rats;WKY:WistarKyoto

rats;ADHD:attentiondeficithyperactivitydisorder;6-OHDA:6-hydroxydop

amine;

MPTP:1-methyl-4-ph

enyl-1,2,3,6-tetrahydrop

yridine;

TBI:

traumatic

braininjury;PTI:ph

otothrom

bicischem

ia;MCAO:middlecerebral

artery

occlusion;

PSS:periph

eral

sensorystim

ulation;

NIH

L:no

ise-indu

cedhearingloss;CC-ION:chronicconstriction

ofthe

infraorbital

nerve(painmod

el);PSN

L:partialsciaticnerveligation(painmod

el);CRS:

chronicrestraintstress

(painmod

el);CFA

:com

pleteFreund

’sadjuvant

(painmod

el);BBB:b

lood

–brain

barrier;S1FL

:forelim

bregion

oftheprim

arysomatosensory

cortex;M1:

prim

arymotor

area;P

AG:periaquedu

ctal

grey;D

HSC

:dorsalho

rnof

thespinal

cord;M

EP:m

otor-evokedpo

tentials;S

EP:som

atosensory

evoked

potentials;G

FAP:g

lialfibrillaryacidic

protein;

BDNF:

brain-derivedneurotroph

icfactor;Glu:g

lutamateNeuN:neuron

almarker;PX1:

pann

exin

1;TH:thyroxinehydroxylase;Egr-1:earlygrow

threspon

seprotein1;

TNF:

tumor

necrosisfactor;IL-1þ:interleuk

in1beta;IL-6:

interleukin6;

IL-10:

interleukin10;L

DH:lactate

dehydrogenaseenzyme;ATP:adeno

sine

tripho

sphate;G

DH:glutamatedehydrogenase;

Drp1:

dynamin-related

protein;

MAP-2:microtubu

leassociated

protein

2;GAP-43:

grow

thassociated

protein

43;ENK:earlyem

bryo

specificNK;DA:do

pamine;

ECG:electrocardiograph

y;EEG:

electroencephalogram

;A/m

2 :am

pere

persquare

meter;m

A:m

illiampere;cm

2 :square

centim

eter;m

in:m

inute;vs.:versus;Y

:yes;N

:no;N/A

:not

available.

14 Neural Plasticity

nd trafficking to postsynaptic density [77] and ultimately,allowing LTP facilitation, a cellular correlate of learningand memory.

3.3.2. Activation of Neuroplasticity-Associated GeneExpression. Neurostimulation could have long-lasting effectsin memory as data from different studies evidenced [40].Authors have been argued that tDCS cognition modulationis associated with neuroplasticity-associated gene expressionalterations [78]. One of the neuroplasticity-associated genes,known to be essential for hippocampal LTP, is BDNF [79].Several studies elucidated the role of BDNF in memory mod-ulation by tDCS. In fact, it was reported that anodal currentscould increase BDNF expression [68], and its activation viatropomyosin receptor kinase (Trk) receptors [80], triggeringNMDAR opening, and inducing a later phase LTP (L-LTP)facilitation [81]. Accordingly, Yu et al. [41] found that theadministration the Trk inhibitor ANA-12 prevented theanodal tDCS-induced hippocampal CA1 LTP increase. Otherstudies, using the same polarity currents, revealed a linkbetween the upregulation of BDNF and cAMP response ele-ment binding protein/CREB-binding protein (CREB/CBP)[40] involved in LTP and memory formation [82]. Also, theapplication of cortical anodal currents in frontal cortex wasable to upregulate BDNF together with striatal dopamine[33]. The upregulation of BDNF following neurostimulationwas associated with the augmentation of expression levels ofimmediate early genes (IEGs), such as c-fos and zif268 [69].Moreover, Kim et al. [78] confirmed that repetitive anodaltDCS in right sensorimotor cortex of healthy rats promoteda significant increase of mRNA levels of plasticity-associated genes, namely, BDNF, cAMP response elementbinding protein (CREB), synapsin I, Ca2+/calmodulin-dependent protein kinase II (CaMKII), activity-regulatedcytoskeleton-associated protein (Arc), and c-fos. It was alsodemonstrated that sensory evoked cortical responses wereboosted after tDCS via alpha-1 adrenergic receptor-mediated astrocytic Ca2+/IP3 signaling, thus involving alsoglia and the adrenergic system [75]. Anodal tDCS actionsin glia were further confirmed by Mishima et al. [76]. Usinga mouse model lacking Ca2+ uptake in astrocytes, the inositoltrisphosphate receptor type 2 (IP3R2) knockout (KO) mouseand also an adrenergic receptor antagonist, they confirmeddecreased microglia motility along with soma enlargementin tDCS stimulated animals [76].

In poststroke recovery, it was found that anodal currentssignificantly increased the GAP-43 and the microtubule-associated protein 2 (MAP-2) expression around the infarctarea [56]. These neuronal growth-promoting proteins areoverexpressed during dendritic remodeling and axonalregrowth throughout the acute phase of stroke [83, 84].Anodal stimulation also modulated pannexin-1 (PX1) hemi-channel levels [85, 86] and, following an ischemic insult, neu-rostimulation decreased rat PX1 mRNA and, consequently,augmented dendritic spine density in the surrounding areasof cerebral infarction; these cellular outcomes were associ-ated with the improvement of motor function [85]. Someauthors proposed that tDCS-induced improvement of stro-ke/TBI symptoms might be due to increase of BDNF expres-

sion and associated with choline/creatine ratios in theperilesional cortex [31].

Overall, tDCS methodology was able to modulate molec-ular pathways involved in the regulation of cognition-relatedsynaptic plasticity mechanisms (Figure 3). The revised in vivoanimal studies regarding tDCS-induced effects in the cellularand molecular mechanisms of memory and learning arelisted in Table 4.

4. Discussion

This systematic review collected several studies that confirmthe potential effects of tDCS on neuronal activity and synap-tic plasticity. Here, we documented a variable combination ofstimulation protocols, stimulation areas, and healthy and dis-ease animal models. Most of the existent literature is focusedon human application of tDCS. The comprehensive revisionof the effect of tDCS on rodent models of normal and patho-logical brain functioning does therefore provide a novel con-tribution to the field. Overall, the revised studies indicatedthat tDCS was able to modulate synaptic plasticity and, con-sequently, learning and memory processes [87, 88].

Memory formation and consolidation are recognized torely on activity-dependent modifications, such as LTD andLTP [89], both dependent on the activation of calcium-dependent kinases (e.g., CaMKs), which in turn control thetrafficking of NMDARs and AMPARs [90]. Despite the wideset of stimulation protocols, tDCS-induced modulation ofNMDAR signaling and synaptic protein upregulation result-ing in LTP and cognitive enhancement have been consis-tently reported in animal studies. Anodal tDCS increasedAMPAR synapse translocation [73, 89] and induced spatialmemory improvement by involving both CREB and BDNFexpression alterations [53]. Also, an increase in hippocampaland cortical mRNA levels of c-fos, synapsin, CaMKII, andArc was observed poststimulation [78].

Similar results highlighting tDCS effects in neuroplasti-city were obtained with in vitro studies. Accordingly, Ranieriand coworkers [69] probed that anodal currents increasedNMDAR-dependent LTP in hippocampal CA3-CA1 synap-ses [69], in part, due to production of BDNF [68]. In addi-tion, it was demonstrated that tDCS-induced hippocampalBDNF release is dependent on histone acetylation of BDNFgene promoters [40]. Overall, the abovementioned worksprovide positive evidence for the effect of tDCS on cognitivefunction enhancement.

Although tDCS impaired the acquisition of both contextualand cued fear memory [39], there are no studies on possiblecascades/proteins involved in tDCS-induced neuroplasticityalterations following fear memory changes. Nevertheless, avery recent paper demonstrated chronic repetitive TMS ofthe ventromedial prefrontal cortex reversed stress-inducedbehavior impairments with an increase of c-fos activity [91].

Cortical anodal currents have been shown to be mostlyexcitatory and support memory enhancement and neuro-plasticity. The literature is also consistent with the notionthat the stimulation over the cortical region functionallyinvolved in a certain cognitive task increases performancein that specific task. Marshall et al. demonstrated that anodal

15Neural Plasticity

currents over the PFC, a region involved in memory encod-ing, during slow wave sleep improved declarative memory[28]. However, it was described that cortical cathodal stimu-lation simultaneously with training task was able to increasevisuospatial working memory, in spite of the fact that it wasassociated with decreased excitability [42]. This suggests thatmodulatory effects of tDCS were influenced by the polarity-dependent electrical dynamics established between the stim-ulated area and its related neuronal networks. In agreement,a recent report observed an inhibitory effect in motor learn-ing tasks following anodal currents in the cerebellum; theanodal excitatory effect over the Purkinje cell activity ledto an overall inhibition of downstream structures, reducingas a result the vestibulo-ocular reflex gain [90]. Similar par-adoxical results have been observed in humans. Recently,Moliadze and collaborators [92] reported that tDCS-induced neural modulation depended on several parame-ters, namely, the age. In fact, an excitatory effect was seenin young subjects, but not in the older participants.

Nowadays, TMS, another important noninvasive brainstimulation technique, is useful for evaluating excitability inthe primary motor cortex (M1) and conductivity along thecortical-spinal tract. This technique has been amply used inrehabilitation of stroke patients [93] and in neuropsychiatric

disorders, namely, depression [94]. tDCS and TMS areundergoing the most active investigation and share a capacityto modulate regional cortical excitability, and both are well-tolerated by children and adults [95]. However, TMS hasbeen already approved for clinical use and tDCS is stillundergoing investigation as a plausible therapy for a rangeof neuropsychiatric disorders [95]. The rational, in part, forthis is because data on the efficacy and safety of tDCS aresparse and employ heterogeneous stimulation protocols.Indeed, there is a paucity of strictly conducted randomized,sham controlled clinical trials, and case considerable follow-up periods, which makes it difficult to use these results toinform clinical practice concerning the putative beneficial roleof tDCS. Moreover, tDCS effects seem to be clearly dependenton structure, connectivity, and function of the target brainregion. Importantly, these outcomes were intrinsically corre-lated with GABAergic neurotransmission which raises theissue that one has to take into account that during develop-ment GABA can act as an excitatory neurotransmitter [96].

5. Conclusions

There is growing evidence that tDCSmodulates brain activityand, consequently, enhances synaptic plasticity and cognitive

Postsynaptic neuron

Early genesBdnf

mTOR

Protein kinasesP

AMPA receptorNMDA receptor

BDNFTrkB receptor

Anodal tDCS

Synaptic cle� Behavioral changes

LTP facilitatation

Formation of new protein (BDNF)

GSK3Ca2+

Ca2+

Figure 3: Schematic illustration of molecular mechanisms underlying the effect of anodal transcranial direct current stimulation (tDCS) onneuronal physiology. The neurostimulation in the target cortical area depolarizes neuronal membrane and glutamate released in presynapticneuron and binds in NMDA and AMPA receptors (see book chapter Rozisky et al., 2015). Consequently, there is intracellular Ca2+

upregulation in the postsynaptic neuron, which can activate protein kinases that in turn modulate numerous neuronal signaling pathways(such as the mTOR pathway) leading to transcriptional changes. The tDCS also activates molecular cascades to promote BDNFproduction. As a long-term mechanism, gene transcription is modulated leading to the formation of new proteins that in turn lead tofacilitation of LTP and improvement of cognition. Abbreviations: AMPA: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; BDNF:brain-derived neurotrophic factor; CBP: CREB-binding protein; CREB: cAMP response element binding protein; GSK3: glycogen synthasekinase 3; LTP: long-term potentiation; mTOR: mammalian target of rapamycin; NMDA: N-methyl-D-aspartate; TrkB: tropomyosinreceptor kinase B.

16 Neural Plasticity

Table4:Cellularandmolecular

mechanism

sun

derlying

transcranialdirectcurrentstim

ulationeffectin

thebrain.

Autho

rYear

Animal

mod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Márqu

ez-

Ruizet.

[63]

2012

NDM

New

Zealand

White

albino

rabbits;

N/A

13a-tD

CS

and

c-tD

CS

Somatosensory

cortex

(S1)

0.5;1;1.5

and2

0.7857

103.7

Ear

35N

N(single

session)

↑LF

Pin

S1(a-tDCS)

and↓LF

PS1

(c-tDCS)

Roh

anetal.[57]

2015

NDM

Sprague

Daw

ley

rats;m

ale

34a-tD

CS

SCdo

rsalto

the

hippocam

pus

0.1or

0.25

0.25

30N/A

Between

shou

lders

8.04

NN

(single

session)

#LT

PandPPFin

the

hippocam

pus

Yoon

etal.[31]

2016

Lateral

fluid

percussion

metho

d

Sprague

Daw

ley

rats;m

ales

36a-tD

CS

Hippocampu

s0.2

0.0225

2028.2

Chest

48(corset)

YY(dailyfor

5days)

#perilesion

alarea

BDNFexpression

(tDCS2weeks

post-TBI)

#choline/creatinine

ratios

(tDCS1week

post-TBI)

Motor

performance

recovery

(2weeks

oftD

CS)

Leffa

etal.[33]

2016

SHR

SHRrats

andWKT

rats;m

ales

48a-tD

CS

Fron

talcortex

0.5

1.5

2033.4

Between

ears

1.5

NY(8

consecutive

days

#DAlevelsin

STRin

both

ratstrainsandin

thehipp

ocam

pus

followingtD

CS

treatm

entin

WKY

#BDNFlevelsin

WKY

rats

Short-term

mem

ory

improvem

ent

Pod

daetal.[40]

2016

NDM

C57BL/6

mice;

males

16a-tD

CS

vs.

c-tD

CS

Leftparietal

cortex

(dorsal

tohipp

ocam

pal

form

ation)

0.35

0.06

20N/A

Ventral

thorax

5.2

NN

(single

session)

#spatiallearningand

mem

ory(a-tDCS);

benefitsobservable

oneweekafter

#BDNFexpression

inthehippocam

pus

CREB/CBPpathway

activation

17Neural Plasticity

Table4:Con

tinu

ed.

Autho

rYear

Animal

mod

elSpecim

en;

gend

erN

Stim

ulationparameters

Mainfind

ings

Stim

ulationelectrod

eReference

electrod

e

Anesthesia

rtDCS

Polarity

Position

Stim

ulation

intensity

(mA)

Size

(cm

2 )

Stim

ulation

duration

(min)

Current

density

(A/m

2 )Position

Area

(cm

2 )

Mon

aietal.[75]

2016

NDM

G7N

G817

mice;N/A

10a-tD

CS

Primaryvisual

cortex

(VI)

0.1

0.02

10N/A

Neck

N/A

NN

(single

session)

Upto

50%

expansion

ofvisualevokeactive

area

(upto

2h

poststim

ulationeffect)

tDCS-indu

ced

plasticity

depend

son

theactivity

ofIP,R

2,andA1A

R

Kim

etal.

[78]

2017

NDM

Sprague

Daw

ley

rats;m

ales

90a-tD

CS

Right

sensorim

otor

cortex

0.25

0.071

20N/A

Right

anterior

chest

0.5

NY(7

days)

#BDNF,

CREB,

synapsin,and

CaM

KII

mRNAexpression

levels(ipsilateral

cortex)andc-fos

(hippocampu

s)

Stafford

etal.[73]

2018

NDM

Sprague

Daw

ley

rats,m

ales

16a-tD

CS

Caudalto

bregma

0.25

0.25

30N/A

Ventral

thorax

N/A

NN

(single

session)

↑AMPAR

translocationto

the

synapsein

the

hipp

ocam

pusand↑

phosph

orylationof

the

S831

siteon

GluA1

Martins

etal.[97]

2019

NDM

Male

Wistar

rats;m

ales

50a-tD

CS

LeftmPFC

0.4

N/A

13N/A

N/A

N/A

N/A

Y(5

days)

↑Spatialw

orking

mem

ory

↑GAP-43(extinctby

AMPARantagonist

PRP)

Yuetal.

[41]

2019

NDM

Sprague

Daw

ley

rats;m

ales

224

a-tD

CS

SCdo

rsalto

the

hippocam

pus

0.25

0.25

30N/A

Anterior

chest

N/A

(EEG

electrod

e)Y

N(single

session)

↑Mem

ory(passive

avoidancemem

ory

retention)

↑LT

Pin

CA1

hippocam

pus(blocked

byTrkBan

tagonist)

↑BDNFin

CA1

hippocam

pus

Abbreviations:rtD

CS:repetitive

transcranialdirectcurrentstimulation;a-tD

CS:anod

altranscranialdirectcurrentstimulation;c-tD

CS:cathod

altranscranialdirectcurrentstimulation;C57BL/6:mou

sestrain;SC:

stereotaxiccoordinates;NDM:n

odiseasemod

el;SHR:spo

ntaneous

hypertensive

rats;W

KY:W

istarKyoto

rats;P

FC:prefron

talcortex;mPFC

:medialp

refron

talcortex;dP

FC:dorsolateralp

refron

talcortex;DG:

dentategyrus;ST

R:striatum;S1:somatosensory

cortex;V

1:prim

aryvisualcortex;ITC:inferotem

poralcortex;CA1:cornuam

mon

is1region

inthehipp

ocam

pus;TBI:traumaticbraininjury;P

RP:peram

panel;

CREB:cAMPrespon

seelem

ent-bind

ingprotein(transcription

factor);CREB/CBP:cAMPrespon

seelem

entb

inding

protein;

BDNF:brain-derivedneurotroph

icfactor;D

A:dop

amine;GAP-43:grow

thassociate

protein43;C

aMKII:C

a2+/calmod

ulin-dependent

proteinkinase;m

RNA:m

essengerribonu

cleicacid;IP3R

2:inositoltripho

sphatetype

2receptor;A

1AR:adeno

sine

A2A

receptor;A

MPAR:α-amino-3-hydroxy-5-

methyl-4-isoxazolepropion

icacidreceptor;G

luA1:AMPAreceptor

subu

nitA

1;LF

P:localfieldpo

tential;LT

P:lon

g-term

potentiation

;PPF:paired

pulsefacilitation;EEG:eletroencephalography;A

/m2 :am

pereper

square

meter;m

A:m

illiampere;cm

2 :square

centim

eter;m

m:m

illim

eter;h

:hou

r;min:m

inute;vs.:versus;Y

:yes;N

:no;N/A

:not

available.

18 Neural Plasticity

performance. Overall, reports from laboratory animalresearch present tDCS as a promising noninvasive brainstimulation technique. The presented evidence is thereforeconsistent with human studies suggesting that this techniqueis useful to mitigate neurologic symptoms of several braindisorders, thus improving learning and memory. Furtherresearch is needed so that this technique can be fully trans-lated into optimal therapeutic strategies.

Conflicts of Interest

The authors declare that there is no conflict of interestregarding the publication of this paper.

Authors’ Contributions

Joana Gonçalves and Miguel Castelo-Branco share seniorauthorship.

Acknowledgments

This work was supported by grants POCI-01-0145-FEDER-016428 and CENTRO-01-0145-FEDER-000016 financed byCentro 2020 FEDER, COMPETE, FLAD Life Sciences Ed 22016, FCT/UID 4950 COMPETE, POCI-01-0145-FEDER-007440, FCT, and European Grant H2020 STIPED.

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