Anodal transcranial direct current stimulation

increases brain intracellular pH and

modulates bioenergetics
Caroline D. Rae
1,2
, Vincent H.-C. Lee
1,2
, Roger J. Ordidge
1,3
, Angelo Alonzo
4
and Colleen Loo
4
1
Neuroscience Research Australia, Randwick, NSW, Australia
2
School of Medical Sciences, The University of New South Wales, Sydney, NSW Australia
3
Centre for Neuroscience, The University of Melbourne, Parkville, Vic, Australia
4
The Black Dog Institute, School of Psychiatry, The University of New South Wales, Sydney, NSW, Australia
Abstract
Transcranial direct current stimulation is an emerging treatment for brain disorders but its mode of action
is not well understood. We applied 10 min 1 mA anodal transcranial direct current stimulation (tDCS)
inside the bore of a 3 T MRI scanner to the left dorsolateral prefrontal cortex of 13 healthy volunteers
(aged 1928 yr) in a blinded, sham-controlled, cross-over design. Brain bioenergetics were measured
from the left temporo-frontal region using
31
P magnetic resonance spectroscopy before, during and
for 20 min following tDCS. Brain pH rose during tDCS and remained elevated afterwards.
Phosphomonoesters were signicantly decreased while inorganic phosphate (Pi) also fell. Partial-least
squares discriminant analysis of the data revealed two signicantly different subject groups: one where
phosphocreatine (PCr), ATP and Pi fell along with a larger increase in pH and one where PCr and ATP
increased along with a smaller increase in pH and a slower and more sustained decrease in Pi. Group mem-
bership was predicted by baseline pH and ATP. We interpreted the effects of tDCS as driving two bio-
chemical processes: cellular consumption of ATP causing hydrolysis of PCr via the creatine kinase
reaction driving the increase in pH; synthesis of ATP and PCr by mitochondria with concomitant drop
in Pi and phosphomonoester levels.
Received 8 October 2012; Reviewed 7 December 2012; Revised 15 January 2013; Accepted 21 January 2013
Key words: Depression, inorganic phosphate, pH, phosphomonoesters, polarization, transcranial direct
current stimulation.
Introduction
Transcranial direct current stimulation (tDCS) is a
safe method for modulating cortical excitability
and is of current interest as a possible treatment for
psychiatric conditions as well as neurological disorders
(Stagg and Nitsche, 2011). Electrodes are applied to
the scalp and low currents applied via cathodal and
anodal electrodes. Depending on the site of appli-
cation, a range of effects have been demonstrated,
including motor, somatosensory, cognitive, visual
and affective (Utz et al., 2010).
Application of currents alters blood ow, with
widespread increases in the vicinity of the anode and
decreased blood ow near the cathode, with effects
lasting for at least 50 min (10 min tDCS, 1 mA; Lang
et al., 2005). Increased oxygen delivery in the vicinity
of the anode has been shown using near-infrared spec-
troscopy, with effects lasting for several minutes after
stimulation had ceased (Merzagora et al., 2010). tDCS
alters cortical excitability, with sustained increases of
up to 150% lasting for up to 90 min, provided the
period of tDCS application exceeded 9 min in duration.
These effects may be mediated by NMDA receptors,
dopaminergic activity and/or by the serotonin system
(Stagg and Nitsche, 2011).
Current thinking on tDCS is that it acts by altering
the polarity of membranes without causing action
potentials, although polarity is dependent on the orien-
tation of axons and dendrites in the induced eld
Address for correspondence: Professor C. Rae, Neuroscience Research
Australia, Barker St, Randwick, NSW 2031, Australia.
Tel.: +61 293 991211 Fax: +61 293 991026
Email: c.rae@unsw.edu.au
International Journal of Neuropsychopharmacology, Page 1 of 12. CINP 2013
doi:10.1017/S1461145713000084
ARTI CLE
(Zaghi et al., 2010). Application of electric currents to
brain cortical tissue increases metabolism (McIlwain,
1953), possibly through membrane polarization
causing subsequent activation of NMDA receptors
(Nitsche et al., 2003; Fritsch et al., 2010) with bursts
of lactate production noted following stimulation
indicative of increased metabolism of glucose
(McIlwain, 1955). Periods of anodal stimulation
exceeding 5 min result in prolonged (up to 5 h with
direct cortical stimulation in rats) increases in excit-
ability (Creutzfeldt et al., 1962; Bindman et al., 1964).
Electric pulses have a larger proportionate effect on
metabolism in white matter than in grey (Kurokawa,
1960) due to the larger spread of the pulse through
this medium. Early work where electrical pulses
were applied to tissue slices showed breakdown of
phosphocreatine (PCr) and increased inorganic phos-
phate (Pi), which recovered to baseline levels following
cessation of the pulses (Heald, 1954). This effect was
independent of, and occurred on a faster time-scale
to, changes induced by K
+
depolarization.
In humans, application of tDCS has shown
mixed outcomes. Rango et al. (2008), who stimulated
the right motor cortex (1.5 mA for 15 min), reported
increases in myoinositol 30 min after tDCS but no
effect on any other metabolite measured. Stagg et al.
(2009) stimulated the left sensorimotor cortex (1 mA
for 10 min) and reported reduced GABA with no sig-
nicant change in glutamate/glutamine 1520 min
post stimulus, while a 30 min 2 mA stimulus increased
glu/gln 30 min post stimulus (Clark et al., 2011).
Anodal tDCS applied to the primary motor cortex for
20 min at 1 mA resulted in decreased bioenergetic
ratios [adenosine triphosphate (ATP)/Pi and PCr/Pi]
90 min after commencement of tDCS, which correlated
with cerebral glucose uptake rates (Binkofski et al.,
2011).
Brain bioenergetics are known to be sensitive to
brain workload (Kato et al., 1996; Rango et al., 1997)
and are known to be altered in some psychiatric dis-
orders, e.g. (Volz et al., 1998). We wished to determine
whether brain bioenergetics changed during tDCS and
to delineate what these changes, if any, were.
Method
Participants
Altogether, 13 healthy subjects (age range 1928 yr,
median 22 yr; ve males) were recruited with informed
consent from the student population of the University
of New South Wales (NSW, Australia). Subjects were
considered ineligible if there was any current or past
psychiatric illness, general systemic or neurological ill-
ness (epilepsy, seizures or head trauma). Also excluded
were those taking or recently taking psychotropic
medications, including benzodiazepines, those with a
history of alcohol abuse [2001 Australian National
Health and Medical Research Council (NHMRC)
guidelines], recreational drug users, excessive caffeine
consumption (NSW Health guidelines), magnetic res-
onance imaging (MRI) contraindications or those
with any possibility of being pregnant. Potential sub-
jects were screened for the above using a 22-item ques-
tionnaire, adapted from the Transcranial Magnetic
Stimulation Adult Safety Screen (Keel et al., 2001),
including additional questions about past and present
neurological and psychiatric disorder, drug use and
general medical illness. A positive response on any
item was further assessed by a psychiatrist (C.L.).
This study was conducted within the parameters
for human research as specied by the Australian
NHMRC and approved by the University of NSW
Human Research Ethics Committee.
Procedure
Study design followed a double-blind, cross-over
design with subjects receiving either 10 min of active
or sham tDCS (see below), where the subject and the
researcher analysing the results were blind to treat-
ment condition. Four subjects received active treatment
prior to sham and nine received sham treatment rst.
Subjects were studied twice at similar times of day,
with at least 2 d (or 1 wk if active tDCS preceded
sham tDCS) between visits. At the commencement
and completion of each session, subjects completed a
visual analogue scale, a self-rating questionnaire
evaluating their tDCS experience and any psychologi-
cal changes or after effects they experienced
during or after tDCS. This included ratings of concen-
tration/alertness, tension/anxiety, fatigue/inertia,
depression/dejection, vigour/activity, anger/hostility,
confusion/bewilderment and sleepiness on a scale
fromminus to plus 3, where zero represented no change
compared to baseline, 3=much worse, +3=much better.
Subject preparation
Electrode position was determined by the International
10/20 System for EEG Electrodes and used the montage
recommended for treatment of depression. Subjects
head measurements were taken and the F3 and F8
locations were marked using a 10/20 cap. Direct cur-
rents were applied through a pair of saline-soaked, sur-
face sponge electrodes (75 cm). For both active and
sham tDCS, the anode electrode was placed over F3
2 C. D. Rae et al.
(left dorsolateral prefrontal cortex) and the cathode
electrode on the contralateral side over F8, a montage
recently successfully used in the treatment of
depression (Loo et al., 2012). Electrodes were xed
using a rubber head band around the head. Subjects
were then positioned in the scanner and lay supine
for the duration of the experiment.
A 10 cm diameter
31
P surface coil (Pulseteq Ltd, UK)
was placed next to the head, centred over the left
inferior frontal/left temporal lobe proximate to the
anodal electrode and secured using Velcro strips (for
anatomical location, see Fig. 1). The coil was not placed
directly over the electrode as: (1) surface coils of this
design have maximum ux orthogonal to the magnetic
eld and zero ux along the magnetic eld; (2) there
may be safety concerns with placing a loop surface
coil directly over a working electrode; (3) computer
modelling suggests that cerebral effects are diffuse
and not limited to the area under the electrode when
the electrodes are widely spaced (Sadleir et al., 2010).
MRI protocol
All spectra were acquired using a Philips 3T Achieva
TX MRI (Philips, The Netherlands) using the 10 cm
diameter
31
P circular surface coil. Each spectrum was
acquired using a pulse-acquire sequence (TR=2 s,
bandwidth 3500 Hz) with an adiabatic (sech) pulse.
Coil localization was veried using scout images to
locate the residual signal from the ducial marker
phantom in the centre of the coil. This phantom
was lled with a solution of 1 M phenylphosphonate
(=16 ppm) as an intensity marker. Following
volume shimming a scout
31
P spectrum was collected
and the transmitter frequency set 300 Hz to high fre-
quency of the resonance from -ATP (16 ppm) in
order to optimize and standardize the frequency
response window of the adiabatic pulse.
All spectra represented the sum of 16 transients.
Spectra were acquired in blocks of ve (prior to treat-
ment), 17 (during treatment) or 41 (following treat-
ment). In each case, the rst spectrum was used to
establish steady-state magnetization and was dis-
carded. Typical spectra from each phase of data collec-
tion are shown in Fig. 1.
Anodal transcranial direct current stimulation
A battery-driven, constant-current stimulator (Eldith
DC-Stimulator; MR NeuroConn GmbH, Germany)
delivered 1 mA (current density of 0.03 A/cm
2
) to
the brain via the electrodes. For sham stimulation,
the current was turned on for 30 s, using a procedure
previously found to result in adequate subject blinding
(Loo et al., 2012). The quality of data collected during
the stimulation period was good (see spectrum in
Fig. 1) with no frequency spikes and with no signi-
cant difference in measured noise (residuals of
AMARES t) between sham or control or between
data collected in the before, during or after stimulation
blocks.
Analysis of data
Spectra were processed using jMRUI (http://sermn02.
uab.es/mrui/mrui_Overview.shtml; V3.0). Quanti-
cation of the reconstructed signals was performed
in the time-domain. The AMARES algorithm
(Vanhamme et al., 1997) was used to t decaying sinu-
soids, corresponding to Lorentzian line shapes in the
frequency domain, to the resonances from the phan-
tom, phosphomonoesters (PMEs), Pi, phosphodiesters
(PDEs), PCr and the three resonances of ATP (,
and ). The line-width of Pi was constrained to not
exceed that of PCr, while that of the PDE peak was
constrained not to exceed 100 Hz. All other resonances,
frequencies and phases were unconstrained. The rst
20 points of the free induction decay were multiplied
by a quarter sine wave (weighting) to minimize the
large anisotropic signal underlying the spectrum.
Intracellular pH was calculated by jMRUI using the
chemical shift of the Pi peak relative to that of the
PCr peak at =0.000 ppm (Iotti et al., 2000).
Statistical analyses using the IBM SPSS Statistics
program (version 20) tested the effect of tDCS on six
brain bioenergetic measures (phosphomonesters, Pi,
PDEs, PCr, -ATP and pH). In order to measure the
effects of tDCS over time, planned contrasts compared
the during and after time-points to baseline, follow-
ing normalization of the data to the baseline state. A
multivariate analysis of variance (MANOVA) was
conducted with two within-groups factors: tDCS con-
dition (active or sham); time (before, during and after
tDCS). The MANOVA was followed up by separate
analyses of variance (ANOVAs) for each brain bioener-
getic measure with simple effects testing the direction
of any signicant interactions. Following the results
of a partial least-squares discriminant analysis
(PLS-DA), where participants were divided into two
distinct bioenergetic groups (described below), further
ANOVAs adding a between-groups factor (i.e. tDCS
conditiontimegroup) were also conducted.
Multivariate pattern recognition and data reduction
tools are capable of taking into account several predic-
tive variables simultaneously. They are especially use-
ful for analysis of the type of metabolic data presented
here as they can objectively distil the major response
Brain bioenergetics and tDCS 3
Phantom
PME
PDE
Pi
PCr
ATP After
Before
20 10
Chemical shift (, ppm)
(a) (b)
0.0 10 20
During
Fig. 1. Typical 51.73 MHz
31
P spectra. Shown are example spectra from before, during and after conditions (n=13, active
transcranial direct current stimulation). Spectra were acquired with a 10 cm surface coil positioned on the left temporal lobe
(a, coronal and b, axial view) using a pulse and collect sequence with a sech pulse centred 300 Hz to high frequency of the
4 C. D. Rae et al.
variables to a few controlled factors, termed latent vari-
ables (Wold, 1994). Multivariate data analysis was per-
formed using the program Simca P+ (v11.5; Umetrics,
Sweden). Data were imported for each variable as the
change in value compared to the baseline mean such
that there were two values for each metabolite (during
tDCS cf. baseline and after tDCS cf. baseline) in each of
the two conditions (sham and active). Data were uni-
variance scaled to standardize variance between the
high and low concentration metabolites (Wold et al.,
1998) to ensure equal contribution to the model of
high and low value variables. A simple principal com-
ponent analysis model was generated initially that was
used objectively to classify subjects into one of two
groups (Fig. S1). Data were then subjected to PLS-DA
to further test for within-group variability. This
approach is useful where data reduction is required
and discrimination is the goal (Barker and Rayens,
2003). Variable importance in the projection (VIP)
scores were used to determine which variables were
discriminatory and which were not (Quintas et al.,
2012) and the model was recalculated using only dis-
criminatory variables. Model robustness and predic-
tive strength was veried by cross-validation (Q
2
).
Results
The quality of spectra obtained before, during and
after electrical stimulation in the magnet was similar
in each phase of the experiment and in active and
sham conditions (Fig. 1), with no change in signal:
noise and no signicant difference in the noise levels
as measured by the residuals following tting in
jMRUI. Careful placement of the external resistor box
as close to the MRI room entry point as possible was
found in pilot experiments to be adequate to eliminate
frequency spikes.
The overall MANOVA revealed no main effect of
tDCS condition (F
6,7
=2.469, p=0.131; Wilks =0.321)
but a signicant main effect of time (F
12,38
=3.248, p=
0.003; Wilks =0.244) and a signicant tDCS con-
ditiontime interaction (F
12,38
=2.141, p=0.037; Wilks
=0.356). The follow-up ANOVAs found a signicant
tDCS conditiontime interaction for the measures
of PMEs with tDCS condition having an effect
during (F
1,11
=25.802, p<0.001) and after (F
1,11
=7.674,
p=0.018) stimulation. Simple effects showed that
PME levels decreased during active tDCS (F
1,12
=8.94,
p=0.011) but not during sham tDCS (F
1,12
=1.04, p=
0.329) and remained decreased after active tDCS
(F
1,12
=10.04, p=0.008) but not after sham tDCS (F
1,12
=0.93, p=0.354). There was also a signicant tDCS
conditiontime interaction for pH levels, indicating
an effect of tDCS during stimulation (F
1,11
=5.978, p
=0.033) but only a trend level effect after stimulation
(F
1,11
=3.616, p=0.084). Simple effects showed that
pH levels increased during active tDCS (F
1,12
=20.03,
p=0.001) but not during sham tDCS (F
1,12
=0.02, p=
0.879) and remained increased after active tDCS
(F
1,12
=10.06, p=0.008) but not after sham tDCS (F
1,12
=0.60, p=0.455). There were also tDCS condition
time interaction trends that suggested decreases in
PDE levels during active tDCS (F
1,11
=3.260, p=0.098)
and in Pi after active tDCS (F
1,11
=3.315, p=0.096)
but these did not reach signicance. There were no
signicant tDCS conditiontime interactions for PCr
or -ATP (Fig. 2).
Simple conservation of mass implies that
decreases in PMEs and Pi should be accompanied
by alterations in other phosphate moieties, but these
were not apparent at the group level. Given that
there seemed to be some variability in individual
responses to tDCS, particularly in PCr and ATP,
which appeared directed rather than random, we
undertook an objective, within-group data discrimi-
nation process, PLS-DA, to examine the group data
for within group variability.
An initial principal components model of all the
data was conducted to look for variance in the data
and to identify any meaningful eigenvectors related
to PCr and ATP. This analysis generated a three com-
ponent model accounting for 70% of the variance in
the data (Fig. S1). This model was poorly cross-
validated (Q
2
=39%) but it was used to classify subjects
into one of two groups. Group membership was
then used as a dummy variable in a PLS-DA model,
which accounted for 88% of the variance in the data,
and showed good cross-validation (Q
2
=64%). VIP
scores for all variables from this model were inspected
and those variables that did not contribute signi-
cantly to the model were removed (Fig. S2). Finally, a
new PLS-DA model was generated using only discri-
minating variables. This was a two component model
accounting for 88% of the variance with a cross-
validation score (Q
2
) of 74% (Fig. 3). This model
showed clear separation of the 13 subjects into two
resonance from -adenosine triphosphate (ATP), with TR=2 s, bandwidth 3500 Hz. The ducial marker, located in the centre
of the coil, can be seen on the left side of the images. Spectra in this diagram have been Fourier transformed with 8 Hz
exponential multiplication. PME, Phosphomonoesters; Pi, inorganic phosphate; PDE, phosphodiesters; PCr, phosphocreatine.
Brain bioenergetics and tDCS 5
groups, where the major discriminating variables were
changes in ATP and PCr (Fig. 3). Changes in levels of
PMEs during sham stimulation were the only sham
condition variables to be included.
Based on this classication, we subjected the data to
a further ANOVA analysis with classication group as
a factor in addition to time and tDCS condition. This
showed signicant three-way interactions in ATP
(F
1,11
=6.803, p=0.024) and PCr levels (F
1,11
=14.608, p
=0.003), with ATP and PCr levels higher during
tDCS than in the sham condition in the black group
and ATP and PCr levels lower during tDCS than in
the sham condition in the red group. These groupwise
data are shown in Fig. 4.
There were no statistically signicant differences
in scores on the self-rating questionnaire between
sham and active states. Example mean rankings (S.D.)
for sham and active conditions respectively for
depression/dejection were 0.0 (0.0) and 0.23 (0.73), for
tension/anxiety 0.20 (0.42) and 0.23 (1.17) and for con-
centration/alertness were 0.00 (0.47) and 0.08 (0.95).
When asked to rate their experience on a scale of 15,
where 1=mildly enjoyable and 5=extremely unplea-
sant, subjects undergoing sham treatment reported a
mean score (S.D.) of 1.90 (0.57) compared to the active
condition 2.08 (0.49).
Finally, we inspected the baseline
31
P data in order
to determine whether initial raw
31
P levels bore any
relationship to group membership outcomes. There
were no signicant between-group differences in
baseline
31
P data. We built a PLS-DA model containing
only the initial baseline data (ratio of PMEs, Pi, PDE,
PCr and ATP to the phantom, plus pH). We found
that using initial pH and ATP levels only, we could
classify the subjects correctly to red or black groups
in all but two cases and these latter were only just out-
side the classication line (Fig. S3). Membership of the
black group was associated with lower initial pH and
lower initial [ATP]. Initial levels of PCr, PMEs and
PDEs were found not to signicantly contribute to
classication.
Discussion
Here, we show for the rst time a signicant increase
in brain intracellular pH in humans during tDCS,
coupled with a signicant decrease in PMEs. At the
group level, these effects of tDCS were without signi-
cant concomitant changes in brain high-energy
phosphates.
Why should applied direct current produce an
elevation in pH?
Brain pH is known to increase in response to acti-
vation. The accepted mechanism for this is via acti-
vation of the creatine kinase system according to the
reaction:
Creatine +ATP PCr +ADP +H
+
. . . . (1)
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31
P metabolite levels before, during and after transcranial direct current stimulation for all 13 subjects. Measurements
are normalized to each mean baseline measurement (100) made relative to the ducial marker resonances, phenylphosphonate
at =15.8 ppm. Active condition is shown in hatched bars and sham condition in white bars. Error bars are S.E.M. and
are N=4 (before), N=16 (during) and N=40 (after) repeated measures on 13 subjects. PCr, Phosphocreatine; ATP, adenosine
triphosphate.
6 C. D. Rae et al.
4
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Fig. 3. Partial-least squares discriminant analysis (PLS-DA) plot of the effects of transcranial direct current stimulation (tDCS)
on all 13 subjects. (a) The plot shows the results of a PLS-DA model where the difference between baseline adenosine
Brain bioenergetics and tDCS 7
Hydrolysis of PCr requires a proton (H
+
), resulting
in net alkalinization of the milieu. Under conditions
of induced workload in the brain, such as that induced
by photic stimulation of the visual cortex using a ash-
ing chequerboard, the resultant activity causes a rapid
reduction in PCr and a rapid (seconds scale) rise in
brain pH (Sappey-Marinier et al., 1992; Kato et al.,
1996; Rango et al., 1997). It is generally accepted that
ATP levels are not altered by this workload, being
strongly buffered via the creatine kinase-catalysed
reaction and through increased synthesis of ATP,
although the time-scale on which this occurs is not
fully explored.
In practice, if increased ATP hydrolysis is the
only demanding process going on, the increase in
pH should also be accompanied by a net drop in
PCr accompanied by either stability of ATP levels
or a small drop (due to demand outstripping
re-supply). We see at the whole group level no net
change in levels of PCr or ATP but we do see a drop
in PMEs and possibly Pi. Direct current is known to
alter membrane potential (Gross et al., 1986), which
will activate the Na+K+-ATPase (Mata et al., 1980).
Direct current is also reported to alter Ca
2+
inux
(Onuma and Hui, 1985) with concomitant activation
of the Ca
2+
ATPase.
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B
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D
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106
104
102
100
98
96
B
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p
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p
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(
%

c
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)
p
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(
%

c
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r
o
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)
P
C
r

(
%

c
o
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r
o
l
)
Fig. 4.
31
P metabolite levels before, during and after transcranial direct current stimulation for each of the groups identied
by partial-least squares discriminant analysis. Measurements are normalized to each mean baseline measurement (100) made
relative to the ducial marker resonances, phenylphosphonate at =15.8 ppm. The two subgroups are coloured as in Fig. 3
(red or black) with the respective sham stimulation shown in white bars with red or black outline. Error bars are S.E.M. and
are N=4 (before), N=16 (during) and N=40 (after) repeated measures on 13 subjects. PCr, Phosphocreatine; ATP, adenosine
triphosphate.
triphosphate (ATP), phosphocreatine (PCr) and pH in the during and after states in the active condition were used as inputs,
along with phosphomonoesters (PME) in the active state during tDCS and in the sham state in the during and after
conditions. The gure shows clear separation of the two groups of subjects based on their high energy phosphate response to
tDCS. The large outer ellipse represents the 95% condence interval (Hotelling score). The model described a two component
model accounting for 88% of the variance in the data with a cross-validation value (Q
2
) of 74%. (i) and (ii) show the loadings
for each variable for each component.
8 C. D. Rae et al.
Another possible explanation for increase in pH is
via electrolysis of water. A number of authors have
reported an increase in medium pH after application
of direct currents to biological samples (Song et al.,
2007), an alkalization that increases with increased
applied current and that is also time dependent.
Direct current application is a form of electrophoresis
and may be expected to induce movement of proteins
and other charged species, including phospholipids
(Jaffe, 1977), although it is difcult to speculate
what the energy cost of protein and phospholipid
physical movement to the cell would be as there is a
paucity of published data on the subject. Data showing
non-synaptic effects of cathodal stimulation support
the physical effects argument (Ardolino et al., 2005)
with the authors predicting increased pH as one
outcome.
In this work, we also saw a drop in the levels of
PME during tDCS and, to a lesser extent, of Pi.
Under steady-state conditions, the concentration of Pi
within the brain is related to the properties of Na
+-linked Pi inux and passive Pi efux and is in equi-
librium with the cellular redox states (NAD
+
/NADH)
and the substrates and products of the reaction cata-
lysed by glyceraldehyde-3-phosphate dehydrogenase
(Masuda et al., 1990). The amount of Pi is therefore
related to its use in the synthesis of ATP and
glyceraldehyde-3-phosphate (the latter being relatively
small compared to that of ATP), its production through
hydrolysis of ATP and its inux and efux from the
cell (Rae et al., 2003). The largest driver of decrease
in Pi in this instance is likely to be through increased
net synthesis of ATP.
The PME peak is composed of a mix of moieties,
including phosphocholine, phosphoethanolamine,
AMP and phosphorylated glycolytic intermediates,
such as glucose-6-phosphate (G6P) and 3-phosphogly-
cerate (3PG). It has been shown to change on the time-
scale of this experiment in response to various chal-
lenges, such as alanine infusion (which stimulates glu-
coneogenesis and increases the PME resonance by
increasing G6P and 3PG levels), by activation of phos-
pholipases, or by stimulation of release of phos-
phoethanolamine. It is not possible here to say which
species are contributing to the loss of PME signal.
Movement of protein and phospholipids by electro-
phoresis with subsequent reorganization of cell mem-
branes may create a demand for the membrane
precursors phosphocholine and phosphoethanola-
mine. There are no reports of loss of the choline signal
in
1
H spectroscopy studies (Rango et al., 2008; Rae
et al., 2009; Clark et al., 2011). This resonance is derived
from both phosphorylated and unphosphorylated
forms of choline and would be expected to
decrease if signicant membrane incorporation were
occurring due to subsequent signicant shortening of
relaxation properties with resultant increased signal
decay. So along with the evidence of a trend towards
decreased Pi levels following tDCS, the loss of
PME is suggestive of a general demand elsewhere for
phosphate moieties.
A previous study using
31
P MRS after imposition
of a longer stimulation than that used here (20 min
1 mA anodal stimulation) reported an alteration in
the ratio of ATP/Pi and PCr/Pi but did not indicate
the degree to which each of these metabolites
were responsible for the change (Binkofski et al.,
2011). Additional clues about the workload induced
by tDCS may come from work subjecting rat skin to
an electric current (Cheng et al., 1982). Cheng et al.
showed increased ATP production, increased
protein synthesis and increased amino acid uptake
with applied anodal current up to 1000 A. tDCS
induces an electric potential across a cell; this would
have an effect directly on the proton-motive force
in the mitochondria (which is the sum of the
electrical potential difference and the chemical poten-
tial difference; Mitchell, 1976) and would drive the
ATP-synthase accordingly. Indeed, it has recently
been shown that the F
0
F
1
-ATPase is sensitive to
applied current with this altering the production rate
of ATP with a maximum stimulation around 0.5 mA
(Lohrasebi et al., 2008). ATP generated in the mito-
chondrion equilibrates with mitochondria PCr via
the mitochondrial creatine kinase. This reaction takes
place in a relatively small compartment and therefore
is unlikely to inuence intracellular pH measurements
made by MRS.
It is at this point that we turn to the PLS-DA
analysis of the data to dig further into whether
or not there are bioenergetic responses to tDCS.
Here, we were able to show division of our 13 subjects
into two distinct populations: one where ATP pro-
duction did not keep pace with ATP loss, resulting in
net decreases in ATP and PCr and with a smaller
increase in pH (the designated red group, Figs 3
and 4); one where ATP production exceeded ATP
loss (the black group) and where the pH increase
was larger. This suggests that two different biochemi-
cal processes were in play: hydrolysis of ATP and acti-
vation of the cytosolic creatine kinase system and
net synthesis of ATP, which acts to restore ATP
levels and also those of PCr.
Group membership was also predicted by baseline
levels of ATP and baseline pH. Lower levels of ATP
and pH are associated with slower speeds of
Brain bioenergetics and tDCS 9
processing, including performance at the symbol-digit
modalities test (Rae et al., 2003), which this electrode
montage and anodal tDCS has been shown to improve
(Loo et al., 2012).
Although preliminary, it would seem that a more
positive mood change score was associated with a
higher bioenergetic response. Altered brain bioener-
getic levels have been reported in both depression
and schizophrenia (Pettegrew et al., 1991; Volz et al.,
1998) and bioenergetic levels are known to be related
to normal brain function (Vloz et al., 1998; Rae et al.,
2003). It remains to be seen whether similar
31
P MRS
outcomes are seen following anodal tDCS in subjects
with psychiatric disorders.
In summary, we nd that tDCS is associated with an
induced metabolic workload and with an induction in
ATP synthesis and an increase in brain pH. The brain
pH increase was contributed to by changes in the cre-
atine kinase steady-state equilibrium, created by
hydrolysis of PCr due to demand for ATP. The degree
of pH change was mediated by the ability to synthesize
ATP in mitochondria, with this being inuenced by the
ability to supply phosphate units from PMEs and
PDEs. We provide evidence for individual differences
in response to applied direct current and a potential
set of biomarkers, baseline pH and ATP, for measuring
response to tDCS administered to non-motor areas of
the brain.
Supplementary material
For supplementary material accompanying this paper,
visit http://dx.doi.org/10.1017/S1461145713000084.
Acknowledgements
The authors thank Dan Moran and Ken Rayner of
Diagnostic Medical Services for expert radiography.
All scanning was performed at the DMS imaging
facility, located at Neuroscience Research Australia.
This work was supported by the Australian National
Health and Medical Research Council (grants to CR
#630516 & CL#510142). The MRUI software package
was kindly provided by the participants of the EU
Network programmes: Human Capital and Mobility,
[CHRX-CT94-0432] and Training and Mobility of
Researchers, [ERB-FMRX-CT970160].
Statement of Interest
None.
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