JOURNAL OF NEUROCHEMISTRY | 2015 | 134 | 693703
13157
*Shire, Wayne, Pennsylvania, USA
Brains On-Line BV, Groningen, the Netherlands
Abstract
Attention decit hyperactivity disorder (ADHD) is a neurode-
velopmental disorder characterized by poor attention, impulse
control and hyperactivity. A signicant proportion of ADHD
patients are also co-morbid for other psychiatric problems
including mood disorders and these patients may be managed
with a combination of psychostimulants and anti-depressants.
While it is generally accepted that enhanced catecholamine
signalling via the action of psychostimulants is likely respon-
sible for the cognitive improvement in ADHD, other neuro-
transmitters including acetylcholine and histamine may be
involved. In the present study, we have examined the effect of
lisdexamfetamine dimesylate (LDX), an amphetamine pro-
drug that is approved for the treatment of ADHD on acetyl-
Attention decit hyperactivity disorder (ADHD) is a neuro-
developmental disorder that may present in children as young
as 5 years old and continue through adolescence and into
adulthood. ADHD is characterized clinically by three
symptom clusters, impulsivity, hyperactivity and inattention,
and is managed pharmacologically by the use of psycho-
stimulants such as methylphenidate and D-amphetamine, and
non-stimulants such as the noradrenergic reuptake inhibitor
atomoxetine and the alpha-2 adrenoceptor agonist guanfa-
cine. The aetiology of ADHD is not fully understood, but
studies in animals and the use of psychostimulants suggest
that dopamine and noradrenaline signalling play a signicant
role in the cognitive and hyperactivity components of the
disorder (Pliszka 2005; Arnsten and Pliszka 2011).
A signicant proportion of patients with ADHD are also co-
morbid for other psychiatric disorders including mood
(Pliszka 2003; Daviss 2008; Biederman et al. 2010), anxiety
(Pliszka et al. 2003, Biederman et al. 2010; Vance et al.
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
doi: 10.1111/jnc.
choline and histamine efux in pre-frontal cortex and hippo-
campus alone and in combination with the anti-depressant s-
citalopram. LDX increased cortical acetylcholine efux, an
effect that was not signicantly altered by co-administration of
s-citalopram. Cortical and hippocampal histamine were mark-
edly increased by LDX, an effect that was attenuated in the
hippocampus but not in pre-frontal cortex when co-adminis-
tered with s-citalopram. Taken together, these results suggest
that efux of acetylcholine and histamine may be involved in
the therapeutic effects of LDX and are differentially inuenced
by the co-administration of s-citalopram.
Keywords: acetylcholine, attention-decit/hyperactivity
disorder, histamine, lisdexamfetamine, s-citalopram.
J. Neurochem. (2015) 134, 693703.
2013) and bipolar disorder (Pliszka et al. 2003, Skirrow et al.
2012). The pharmacological management of many ADHD
patients who are also co-morbid for mood disorders relies on
the co-administration of either psychostimulants, including
methylphenidate, D-amphetamine, or non-stimulant medica-
tions, such as atomoxetine and guanfacine, in combination
with anti-depressants, including the selective serotonin reup-
take inhibitors (SSRIs) (Betts et al. 2014).
Received November 24, 2014; revised manuscript received March 27,
2015; accepted April 29, 2015.
Address correspondence and reprint requests to Peter H. Hutson,
Shire, 725 Chesterbrook Blvd, Wayne, PA 19087, USA. E-mail:
phutson@shire.com
Abbreviations used: aCSF, articial cerebrospinal uid; ADHD,
attention-decit/hyperactivity disorder; AP, anteriorposterior; AUC,
area under the curve; DV, dorsalventral; LDX, lisdexamfetamine
dimesylate; ML, medial-lateral; MRM, multiple-reaction-monitoring;
MS, mass spectrometry; SSRI, selective serotonin reuptake inhibitor.
693
694 P. H. Hutson et al.
Studies in animals and patients with mood disorders have
indicated that enhancement of monoamine dysfunction plays
a central, although not exclusive, role in the management of
mood symptoms (Hamon and Blier 2013). Similarly, it is
generally thought that enhanced cortical and hippocampal
monoamine signalling following the administration of psy-
chostimulants such as D-amphetamine is most likely respon-
sible for the efcacy across the cognitive symptom domains
in ADHD. However, these are not the only neurochemical
substrates that play a role in cognitive processing. Both the
cholinergic (Wilens et al. 1999; Degroot and Parent 2000;
Kay 2000; Potter et al. 2006; Day et al. 2007; Alvarez 2009;
Brioni et al. 2011; Savage 2012) and histaminergic (Kay
2000; Day et al. 2007; Alvarez 2009; Brioni et al. 2011;
Kohler et al. 2011) systems have been implicated in
learning, memory, attention, arousal and vigilance and,
consequently, may be involved in the cognitive decits in
ADHD. Consistent with this, histamine H3 receptor antag-
onists, which increase histamine efux via an action on
inhibitory H3 autoreceptors, improve attention and impul-
sivity (Witkin and Nelson 2004; Day et al. 2007). In this
regard, it is well documented that D-amphetamine, in addition
to enhancing catecholamine function, also increased the
extracellular concentration of acetylcholine in several brain
regions including the hippocampus (Day and Fibiger 1992;
Imperato et al. 1993) and cortex (Day and Fibiger 1992;
Arnold et al. 2001; Zmarowski et al. 2007). However, these
ndings (with the exception of Zmarowski et al. (2007)
should be interpreted with caution as they all included an
acetylcholinesterase inhibitor in the perfusate, which could
inuence cholinergic autoreceptor regulation of acetylcholine
efux and the effect of drugs that modulate acetylcholine
efux. In contrast, the effect of D-amphetamine on histamine
efux is less well documented, although increased brain
histamine efux has been observed following methamphet-
amine (Ito et al. 1996) and also atomoxetine and methyl-
phenidate, drugs that increase catecholamine efux in the
CNS and are used to treat ADHD (Horner et al. 2007; Liu
et al. 2008).
Lisdexamfetamine dimesylate (LDX) is a D-amphetamine
pro-drug, currently approved for use in ADHD patients
6 years and older. LDX comprises the naturally occurring
amino acid L-lysine, linked via an amide bond to D-
amphetamine, and is pharmacologically inert in vitro, lacking
afnity for a wide range of molecular targets including g-
protein-coupled receptors, ion channels, transporters and
enzymes (Hutson et al. 2014). Following its absorption into
the blood stream, LDX is metabolized by a peptidase
associated with red blood cells to generate D-amphetamine
and L-lysine (Pennick 2010). The pharmacology of LDXs
metabolite, D-amphetamine, is well documented (Heal et al.
2013) and the primary consequence of its multiple molecular
actions is to increase the synaptic availability of dopamine
and noradrenaline in the brain. As might be expected, LDX
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
has also been shown to increase extracellular dopamine, but
commensurate with its pro-drug activity this occurs at a
smaller magnitude over a more prolonged time course and
consequently causes less locomotor stimulant activity than an
equi-molar dose of D-amphetamine (Rowley et al. 2012).
Therefore, given the proposed role for both acetylcholine
and histamine in cognitive function and that a signicant
proportion of ADHD patients are co-morbid for mood
disorder, the aim of the current studies was to determine the
effect on acetylcholine and histamine efux in the ventral
hippocampus and pre-frontal cortex of LDX alone and in
combination with the SSRI antidepressant s-citalopram, two
medications that may be co-administered when managing
ADHD patients with co-morbid mood symptoms (Pliszka
2003; Betts et al. 2014).
Materials and methods
Animals
Twenty-ve adult male SpragueDawley rats (Harlan, Horst, the
Netherlands) were used. Experiments were conducted in strict
accordance with EU directive 2010/63/EU and approved by a local
Institutional Animal Care and Use Committee. Animals were
housed, in a temperature (22  2C) and humidity (55  15%)
controlled environment on a 12-h light cycle (07:0019:00).
Standard diet (RMH-B 2181; AB Diets (Woerden, the Netherlands))
and water were available ad libitum prior to experimentation.
Surgery
Rats were anaesthetized using isourane (2% and 500 mL/min O2).
For analgesia, Finadyne (1 mg/kg, s.c.) was administered, topical
anaesthesia was applied on the skull using a mixture of bupivacaine
and epinephrine. Microdialysis probes (polyacrylonitrile membrane;
Brainlink B.V. (Groningen, the Netherlands) were implanted into
the pre-frontal cortex (4 mm exposed membrane surface; coordi-
nates: anteriorposterior (AP) = +3.4 mm (from bregma); medial-
lateral (ML): +0.8 mm (from midline); dorsalventral (DV):
6.0 mm (from skull) and into the hippocampus (4 mm exposed
membrane surface; coordinates: AP = 5.3 mm (from bregma);
ML: +4.8 mm (from midline); DV: 9.0 mm (from skull), the
incisor bar was set at 3.3 mm (Paxinos and Watson 2008). The
probes were attached to the skull with stainless steel screws and
dental cement.
In-vivo experiments
Experiments started after 1 day of recovery. The microdialysis
probes were connected with exible PEEK tubing (PK005-020;
Western Analytical Products Inc., Lake Elsinore, CA, USA) to a
microperfusion pump (Harvard, Holliston, MA, USA) and perfused
with articial cerebrospinal uid (aCSF) perfusate containing
147 mM NaCl, 3.0 mM KCl, 1.2 mM CaCl2 and 1.2 mM MgCl2,
at a ow rate of 1.5 lL/min. An acetylcholinesterase inhibitor was
not included in the aCSF in any experiment. After collection of three
basal samples, LDX or vehicle was administered (t = 20 min) and
subsequently s-citalopram or vehicle at (t = 0 min = start collection
of rst post-dosing sample), samples were collected at 30-min
intervals for 4 h post-administration while the microdialysis probes

were perfused continuously. The 20-min LDX pre-treatment time
was chosen based on previous studies (Hutson et al. 2014). Samples
were collected into mini-vials (4001029; Microbiotech/se AB,
Stockholm, Sweden) already containing 15 lL of 0.04% ascorbic
acid with 20 mM formic acid using an automated fraction collector
(UV 8301501, TSE, Univentor, Zejtun, Malta). All samples were
split into two fractions, one for acetylcholine and one for histamine
analysis. The fractions were stored at 80C until analyses were
performed.
After the experiments, the animals were euthanized by an intra-
cardial overdose of pentobarbital under isourane anaesthesia (as
described above). The brains were removed from the skull and
stored in 4% paraformaldehyde for at least 3 days. The brains were
then sectioned by hand to verify probe positioning. All probes were
found to be targeted to the brain area of interest.
Detection and analysis of acetylcholine
Analysis was performed essentially as described by Giorgetti et al.
(2010). Internal standard solution was added to an aliquot of each
sample. Samples (5 lL) were injected onto an ion exchange
(150 9 2.1 mm, 5 lm) analytical column (Thermo Scientic
BioBasic SCX, Keystone, CO, USA) by an automated sample
injector (SIL-10 ADvp; Shimadzu, Tokyo, Japan). Analytes were
separated using a gradient of ammonium acetate, ammonium
formate and acetic acid in acetonitrile:ultrapuried H2O (80 : 20
volume/volume) containing 0.1% formic acid, at a temperature of
30C. The MS analyses were performed using an API 3000 MS/
MS system consisting of an API 3000 MS/MS detector and a
Turbo Ion Spray interface (Applied Biosystems, Bleiswijk, the
Netherlands). The acquisitions on API 3000 were performed in
positive ionization mode. The instrument was operated in
multiple-reaction-monitoring (MRM) mode for detection of the
compound (transitions m/z 146.0 and 87.1) and its internal
standard (transitions m/z 155.3 and 87).
Detection and analysis of histamine
Analysis was performed using a derivatization agent SymDAQTM,
Brainlink B.V. (Groningen, the Netherlands). By interaction with
primary amines SymDAQTM facilitates liquid chromatography and
analysis of a range of monoamine and amino acid neurotrans-
mitters. The method used was essentially described by Jacobsen
et al.(2012) and Hoand et al. (2012). Internal standard solution
was added to an aliquot of each sample. This mixture was
derivatized with SymDAQTM automatically in the autosampler.
After a pre-dened reaction period, the sample (45 lL) was
injected. Samples were injected onto a reversed phase
(2.0 9 100 mm, particle size: 2.5 lm) analytical column (Phe-
nomenex, Utrecht, the Netherlands) by an automated sample
injector (SIL-30AC; Shimadzu). Analytes were separated on a
gradient of acetonitrile and methanol in ammonium formate
10 mM + 0.1% formic acid in ultrapure water at a temperature of
40C. The MS analyses were performed using an API 4000 MS/
MS system consisting of an API 4000 MS/MS detector and a
Turbo Ion Spray interface (Applied Biosystems). The acquisitions
on API 4000 were performed in positive ionization mode. The
instrument was operated in MRM mode for detection of the
derivatized histamine (transitions m/z 357.2 and 314.0) and its
internal standard (transitions m/z 361.2 and 318.0). Using the
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
LDX and s-citalopram effects on Ach and histamine 695
response ratio of histamine and the internal standard suitable
calibration curves were tted using weighted (1/x) regression, and
the sample concentrations were determined using these calibration
curves. Accuracy was veried by quality control samples after
each sample series. Concentrations were calculated with AnalystTM
data system (version 1.5.2; Applied Biosystems).
Drugs
LDX was provided by Shire Pharmaceuticals LLC (Hampshire
International Business Park Chineham, Basingstoke, Hampshire,
UK) and was dissolved in deionized water. S-citalopram was
obtained from Sigma UK Ltd, Dorset, England and was dissolved in
saline. LDX was administered orally (1.5 mg/kg, p.o.) 20 min prior
to the administration of s-citalopram at time = 0 by intraperitoneal
injection (5 mg/kg, i.p.). Doses of LDX (Rowley et al. 2012;
Hutson et al. 2014) and s-citalopram (Marcus et al. 2012) were
selected from previously published studies with LDX and from the
literature, respectively. Both drugs were made up each day within
2 h of dosing and given using a dose volume of 2 mL/kg body
weight. The dose of s-citalopram is expressed as mg/kg base; the
LDX dose is expressed in terms of D-amphetamine base (salt/base
correction factor = 3.37).
Statistical analysis
Three pre-administration samples with less than 50% variation were
taken at baseline. Their mean was set at 100%. Data are expressed as
percentage of basal level (mean  SEM), within the same subject.
Prior to performing inter-group analyses, outlier analyses were
performed. Outliers are dened as samples where relative increase at
a given time point is outside the 95% condence interval (2 SDs) at
that time point within the same treatment group. Only these specic
relative levels were excluded from graphing, statistical analysis and
AUC calculation. Compound effects were expressed as area under
the curve (AUC0240 min) of relative levels using the linear
trapezoidal method. Missing data were replaced with the average
relative level at the relevant time point within the same treatment
group. Statistical analyses were performed on the relative data using
SigmaPlot for Windows version 12 (SPSS Corporation, San Jose,
CA, USA). Treatment and time effects were compared, using two-
way ANOVA for repeated measurements followed by a Student
NewmanKeuls post hoc test. The effects of the treatment on
acetylcholine and histamine concentrations, expressed as AUCs,
were compared using one-way ANOVA followed by a Student
NewmanKeuls post hoc test. Table 1 shows F and p values of the
statistical analysis, results of the post hoc tests are given in the
results section. The level of statistical signicance was dened a
priori at p < 0.05 for all tests.
Results
Effects of LDX and s-citalopram on cortical and
hippocampal acetylcholine efflux
The time course and AUC0240 min values for the change of
acetylcholine efux following administration of LDX
(1.5 mg/kg, p.o) or vehicle at t = 20 min and s-citalopram
(5 mg/kg freebase, i.p.) or vehicle at t = 0 min are shown
in Fig. 1(a, b, upper panel) and (c, d, lower panel) for
696 P. H. Hutson et al.

Table 1 Summary table of ANOVA results for pre-

ANOVA Source of variation F p
sented data
Acetylcholine Two-way RM Treatment 9 time F24,166 = 8.64 < 0.001
Levels in PFC
Acetylcholine One way Between groups F3,21 = 7.80 0.001
AUC0-240 in
PFC
Acetylcholine Two-way RM Treatment 9 time F24,160 = 5.46 < 0.001
Levels in vHipp
Acetylcholine One way Between groups F3,20 = 6.37 0.003
AUC0-240 in vHipp
Histamine Two-way RM Treatment 9 time F24,167 = 4.35 < 0.001
Levels in PFC
Histamine One way Between groups F3,21 = 5.23 0.007
AUC0-240 in PFC
Histamine Two-way RM Treatment 9 time F24,160 = 6.23 < 0.001
Levels in vHipp
Histamine One way Between groups F3,20 = 9.76 < 0.001
AUC0-240 in vHipp

For all analysis all groups were initially compared in a one-way or two-way repeated measures
ANOVA. Based on the outcome, the specic interactions were determined using a Student
NewmanKeuls post hoc test. Results of the post hoc analysis are described in more detail in
the results section.

pre-frontal cortex and the ventral hippocampus, respectively.
Basal acetylcholine concentrations are given in Table 2.
Administration of vehicle caused a transient and non-
signicant increase of pre-frontal cortex acetylcholine efux
to approximately 155% of baseline values at t = 30 min and
which returned to normal levels at t = 60 min (Fig. 1a). S-
citalopram (5 mg/kg freebase, i.p.) caused a small, transient
increase of acetylcholine efux at t = 30 min which was
comparable to that observed in the vehicle-treated animals
(Fig. 1a). When compared using the AUC0240 min values,
there was no signicant difference between s-citalopram and
vehicle-treated rats (Fig. 1b). LDX caused a time-related
increase of acetylcholine efux which was signicantly
(p < 0.05) greater than vehicle-treated animals between
t = 60 and 210 min. A maximum value of 208% of basal
was reached at t = 60 min after which values declined to
basal values at t = 240 min (Fig. 1a). This increase in
acetylcholine efux was reected in the AUC0240 min values
which were signicantly (p = 0.021) higher than vehicle-
treated animals (Fig. 1b).
In animals treated with LDX and s-citalopram, acetylcho-
line efux increased along a similar time course to that
shown by the LDX/vehicle group with the exception that
efux values did not decline at t = 240 min; in fact,
acetylcholine efux was highest at this time point (230%
of basal) and was signicantly (p < 0.05) greater than
vehicle-treated rats between t = 60 and 240 min (Fig. 1a).
This extended effect on acetylcholine efux was reected in
the mean AUC0240 min value, which was numerically higher
but not signicantly different from LDX/vehicle-treated
animals (Fig. 1b). As with the LDX/vehicle group, mean
AUC0240 min acetylcholine efux in the LDX/s-citalopram
group was signicantly different (p = 0.006) from vehicle/
vehicle-treated rats (Fig. 1b).
In ventral hippocampus, vehicle administration caused a
transient and non-signicant increase in acetylcholine efux
to approximately 117% of baseline values at t = 30 min
and which returned to normal levels at t = 60 min
(Fig. 1c). S-citalopram also transiently increased (125%
of basal) acetylcholine efux at t = 0 min but then values
declined below vehicle control values and remained at
these levels for the duration of the study. The decrease was
signicantly lower (p < 0.05) than vehicle controls at
t = 90 min (Fig. 1c). This change was reected in the
mean AUC0240 min value which was numerically lower but
not signicantly different from the mean AUC0240 min
value for vehicle-treated rats (Fig. 1d). In contrast to the
effects in the pre-frontal cortex, LDX caused a small
increase of hippocampal acetylcholine efux reaching a
maximum of ~ 143% of basal values at t = 90 min which
was sustained for 210 min and then returned to basal
values at t = 240 min (Fig. 1c). This effect of LDX was
only signicantly different (p < 0.05) from vehicle-treated
animals at t = 180 min. The overall effect of LDX on
hippocampal acetylcholine efux was reected in the
AUC0240 min value which was not signicantly different
from vehicle-treated animals (Fig. 1d). In rats administered
LDX and s-citalopram, acetylcholine efux was signi-
cantly (p < 0.05) greater than vehicle-treated rats between
t = 120 and 240 min. A maximal value of ~ 205% of basal

2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703


(a)
(c)
Fig. 1 Effect of vehicle or lisdexamfetamine dimesylate (LDX)
(1.5 mg/kg, p.o.) given at t = 20 min and vehicle or s-citalopram
(5 mg/kg freebase, i.p.) given at t = 0 min on the acetylcholine efux
time course (a and c) and area under the curve (AUC0240 min) (b and
d) in the pre-frontal cortex (a and b) and ventral hippocampus (c and d).
Time course data are expressed as mean  SEM (half error bars are
shown for clarity), % of baseline values, (n = 6 per group, except for
Table 2 Basal extracellular concentrations of acetylcholine and hista-
mine in the pre-frontal cortex and hippocampus
Pre-frontal cortex Hippocampus
Concentration (nM) (n = 25) (n = 24)
Acetylcholine 2.71  0.26 1.15  0.08
Histamine 3.88  0.41 2.16  0.26
was reached at t = 180 min where it was sustained for the
duration of the study (Fig. 1c). The overall effect of LDX
in combination with s-citalopram on hippocampal acetyl-
choline efux was reected in the AUC0240 min value
which was signicantly (p = 0.046) different from vehicle-
treated animals and was numerically higher but not
signicantly different when compared with LDX-treated
rats (Fig. 1d).
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
LDX and s-citalopram effects on Ach and histamine 697
(b)
(d)
PFC data from vehicle + LDX group where n = 7). AUC0240 min data
are expressed as mean  SEM (n = 6 per group, except for PFC data
from vehicle + LDX group where n = 7). *p < 0.05 LDX + s-citalopram
compared with vehicle + vehicle, p < 0.05 LDX + s-citalopram
compared with vehicle + LDX. Arrows indicate time(s) of administra-
tion.
Effects of LDX and s-citalopram on cortical and
hippocampal histamine efflux
The time course and AUC0240 min values for the change of
histamine efux following administration of LDX (1.5 mg/
kg, p.o.) or vehicle at t = 20 min and s-citalopram (5 mg/
kg freebase, i.p.) or vehicle at t = 0 min are shown in
Fig. 2(a, b, upper panel) and (c, d, lower panel) for
pre-frontal cortex and the ventral hippocampus respectively.
Basal concentrations of histamine efux are given in
Table 2.
Administration of vehicle transiently and non-signicantly
increased histamine efux to 151% of baseline values at
t = 30 min which returned to control values at t = 150 min
(Fig. 2a). S-citalopram (5 mg/kg freebase, i.p.) transiently
and non-signicantly increased histamine efux reaching a
maximal value of 197% of basal, at t = 30 min and which
698 P. H. Hutson et al.
(a)
(c)
Fig. 2 Effect of vehicle or lisdexamfetamine dimesylate (LDX)
(1.5 mg/kg, p.o.) given at t = 20 min and vehicle or s-citalopram
(5 mg/kg freebase, i.p.) given at t = 0 min on the histamine efux time
course (a and c) and area under the curve (AUC0240 min) (b and d) in
the pre-frontal cortex (a and b) and ventral hippocampus (c and d).
Time course data are expressed as mean  SEM (half error bars are
shown for clarity), % of baseline values, (n = 6 per group, except for
then declined to control values at t = 90 min (Fig. 2a).
Accordingly, there was no signicant difference in mean
AUC0240 min values between s-citalopram and vehicle-
treated rats (Fig. 2b). In contrast, LDX caused a time-related
increase of histamine efux which was signicantly
(p < 0.05) greater than vehicle-treated animals between
t = 60 and 240 min and reached a maximum of ~ 250% of
basal at t = 90 min after which it declined towards basal
values at t = 240 min (Fig. 2a). The increase above vehicle
controls was reected in the mean AUC0240 min value which
was signicantly (p < 0.014) higher than vehicle-treated
animals (Fig. 2b). In animals treated with LDX and
s-citalopram, histamine efux increased over the 240 min
time course and was signicantly higher than vehicle
controls between t = 180 and 210 min; however, the max-
imal value attained at 120 min was only ~ 190% of basal
(Fig. 2a). The overall effect of LDX/s-citalopram adminis-
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
(b)
(d)
PFC data from vehicle + LDX group where n = 7). AUC0240 min data
are expressed as mean  SEM (n = 6 per group, except for PFC data
from vehicle + LDX group where n = 7). *p < 0.05 LDX + s-citalopram
compared with vehicle + vehicle, p < 0.05 LDX + s-citalopram
compared with vehicle + LDX. Arrows indicate time(s) of administra-
tion.
tration on pre-frontal cortex histamine efux was reected in
the mean AUC0240 min value which was not signicantly
different from the vehicle/vehicle group and was numerically
lower but not signicantly different from LDX/vehicle-
treated animals (Fig. 2b).
Similar to that seen in the pre-frontal cortex, vehicle
administration caused a small (~ 150% of basal), non-
signicant and transient increase of ventral hippocampus
histamine efux which was maximal 30 min after adminis-
tration and returned to control values at t = 150 min
(Fig. 2c). S-citalopram transiently and non-signicantly
increased histamine efux, reaching a maximal value of
183% of basal at t = 30 min and which then declined to
control values at t = 90 min (Fig. 2c). The lack of effect of
s-citalopram compared with vehicle-treated rats on
hippocampal acetylcholine efux was reected in the mean
AUC0240 min values (Fig. 2d). LDX caused a time-related

increase of histamine efux which was signicantly
(p < 0.05) greater than vehicle-treated animals between
t = 60 and 240 min and reached a maximum of 271% of
basal at t = 150 min after which it declined towards basal
values at t = 240 min (Fig. 2c). The overall effect of
LDX on hippocampal histamine efux was reected in the
AUC0240 min value which was signicantly (p < 0.001)
different from vehicle/vehicle-treated animals (Fig. 2d). In
rats administered LDX/s-citalopram, histamine efux
increased over the 240 min time course and was signicantly
higher than vehicle controls between t = 120 and 240 min;
however, the maximal value attained at 120 min was only
190% of basal which was sustained for the duration of the
study (Fig. 2c). The overall smaller effect of LDX/s-citalop-
ram administration on hippocampal histamine efux was
reected in the mean AUC0240 min value which was not
signicantly different from the vehicle/vehicle group but was
signicantly different (p = 0.019) from LDX/vehicle-treated
animals (Fig. 2d).
Discussion
Results in the present study demonstrate that LDX (1.5 mg/
kg) increased acetylcholine efux in the cortex but not in the
hippocampus. The present experiments were conducted
without the inclusion of an acetylcholinesterase inhibitor in
the aCSF which has been used previously to articially raise
the extracellular acetylcholine concentration. However, this
may inuence cholinergic auto- and heteroreceptor function
and hence drug-induced effects on acetylcholine efux, as
was shown in the striatum where amphetamine failed to
increase acetylcholine efux when a low but not a higher
concentration of neostigmine was used in the aCSF (Acquas
and Fibiger 1998). In the case of cortical or hippocampal
acetylcholine efux, there are no similar systematic studies to
investigate the effect of acetylcholinesterase inhibition on
drug-induced responses. However, the effect of similar doses
of D-amphetamine and LDX (present study) on cortical
acetylcholine efux were comparable and appeared not to be
affected by the presence (Arnold et al. 2000) or absence
(Zmarowski et al. 2007) of an acetylcholinesterase inhibitor.
It is unclear how LDX (or D-amphetamine)-induced
increases of acetylcholine efux in cortex and hippocampus
are mediated, and additional studies would be required to be
conducted under similar conditions (i.e. absence of an
acetylcholinesterase inhibitor). Interestingly, the potential
role of noradrenaline in mediating the amphetamine-induced
increase of acetylcholine efux has been less extensively
explored, although Moroni et al. (1983) showed that
noradrenaline decreased acetylcholine efux in the cortex,
so it seems unlikely that noradrenaline release by LDX
would contribute to this effect, at least in cortex.
D-Amphetamine has previously been demonstrated to
increase hippocampal acetylcholine efux (Day and Fibiger
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
LDX and s-citalopram effects on Ach and histamine 699
1992, 1994; Imperato et al. 1993), an effect that was blocked
by the D1 receptor antagonist SCH23390 (Day and Fibiger
1994), so it was surprising that LDX failed to do so.
However, all of the previous studies employed the use of an
acetylcholinesterase inhibitor in the aCSF and hence direct
comparison with our own studies with LDX in the absence of
an acetylcholinesterase inhibitor is difcult for the reasons
outlined above. To our knowledge, there are no studies that
have systematically examined the effect of amphetamine on
cortical and hippocampal acetylcholine efux with and
without an acetylcholinesterase inhibitor in the aCSF. The
reasons for the lack of effect of LDX in the hippocampus are
not clear at present.
In the current study, s-citalopram had no effect on acetyl-
choline efux in the cortex and slightly reduced efux in the
hippocampus. As with the effects of D-amphetamine, it is
difcult to compare the effects of s-citalopram on acetylcho-
line efux in cortex and hippocampus in the current study with
previous reports where different SSRIs and acetylcholinester-
ase inhibitors were used. Furthermore, there appear to be no
systematic investigations of how acetylcholinesterase inhibi-
tion may inuence SSRI-induced changes of acetylcholine
efux in these brain regions. Notwithstanding these caveats,
results in the current study with s-citalopram appear to be
consistent with previous observations showing a lack of effect
on hippocampal or cortical acetylcholine efux following the
systemic administration of the SSRIs uoxetine (Hirano et al.
1995; Degroot and Nomikos 2005) and citalopram (Egashira
et al. 2006).
The lack of effect of SSRIs on acetylcholine efux is of
interest as these agents show limited effects in treating
cognitive symptoms in major depression (Jeon et al. 2014;
Keefe et al. 2014) despite their pronounced effects on
enhancing forebrain monoamine availability (Owen and
Whitton 2006; Fernandez-Pastor et al. 2013; Kaminska
et al. 2013; Ortega et al. 2013). Clearly, enhancing forebrain
monoamine function alone by SSRIs appears to be insuf-
cient to broadly improve cognitive dysfunction in major
depression. More recently, Mork et al. (2013) found that
vortioxetine, a clinically effective anti-depressant, also
improved episodic memory and increased cortical acetyl-
choline efux; however, this compound has broad pharma-
cological activity in addition to serotonin reuptake inhibition,
which may explain its neurochemical and behavioural effects
(Pehrson et al. 2013), and acetylcholine efux was deter-
mined in the presence of an acetylcholinesterase inhibitor,
thereby articially elevating the extracellular concentration
of acetylcholine.
The effects of combining LDX and s-citalopram on
acetylcholine efux in the cortex essentially reected the
effect of LDX alone with the exception of a marked decrease
of acetylcholine concentration at 240 min in the LDX/
vehicle group, which was not observed in the LDX+s-
citalopram group. While this represents a statistically signif-
700 P. H. Hutson et al.
icant difference between the groups at this time point, the
reasons for which are uncertain, it does not affect the overall
drug effect when compared using the AUC data. In the
hippocampus, the combination of s-citalopram and LDX
signicantly increased acetylcholine efux at specic time
points (120240 min) and overall (AUC) when compared
with vehicle controls, although this effect was not signi-
cantly greater than LDX alone when examined over
the whole time course, largely because the increase in
acetylcholine only occurred 2 h after administration. It is
conceivable that with a greater number of animals per
treatment group, more pronounced interaction effects would
have been observed. Importantly, the effect of combining
these two drugs did not diminish the effects of LDX alone,
suggesting that cortical and hippocampal acetylcholine efux
may be enhanced in patients treated with both drugs.
In the present study, LDX caused a large, sustained
increase in cortical and hippocampal histamine efux, which
is consistent with previous studies of methamphetamine in
the striatum (Ito et al. 1996) methylphenidate and atomoxe-
tine in the cortex (Horner et al. 2007; Liu et al. 2008). The
effect of methamphetamine on striatal histamine efux
appears to be mediated via an indirect action of dopamine
on D2 receptors (Ito et al. 1996), and while a similar dose of
LDX increased both dopamine and noradrenaline efux in
the cortex (Rowley et al. 2014), it is unknown at this time if
the effect of LDX on histamine efux in these regions is
indirectly mediated by dopamine and/or noradrenaline.
Unlike vortioxetine, which increased cortical histamine
efux (Mork et al. 2013), s-citalopram had no effect on
histamine efux beyond that of vehicle administration in
either brain region. It is possible that the effects of
vortioxetine on histamine efux are mediated by pharmacol-
ogy other than serotonin reuptake inhibition (Pehrson et al.
2013), although this is currently unknown and studies with
other SSRIs appear to be lacking. The lack of effect of s-
citalopram on histamine efux may contribute to its limited
efcacy in ameliorating cognitive dysfunction in mood
disorders (Jeon et al. 2014).
Interestingly, the combination of LDX and s-citalopram
appeared to result in a non-signicant attenuation of
histamine efux in the cortex. There was a signicant
increase above vehicle controls at later time points but the
overall change as reected by AUC was not signicantly
different from either vehicle or LDX alone. It is conceivable
that with a greater number of animals per treatment group,
more pronounced interaction effects would have been
observed. A similar pattern was observed in hippocampus
with histamine efux in the LDX + s-citalopram group being
signicantly higher than vehicle controls at time points 120
240 min but the magnitude was signicantly less than that
produced by LDX alone at timepoints 60180 min. When
compared over the full time course, the AUC data revealed a
signicant attenuation of histamine efux compared with
2015 International Society for Neurochemistry, J. Neurochem. (2015) 134, 693--703
LDX alone. The reason for the smaller increase in histamine
efux following combined administration of LDX and s-
citalopram is unclear, but these data suggest that cortical and
hippocampal histamine efux following combined adminis-
tration of LDX and s-citalopram is still greater than vehicle
controls but may be attenuated when compared with LDX
alone.
The signicance of the increase in acetylcholine and
histamine following LDX in relation to its therapeutic effects
in the management of ADHD is unknown. The cell bodies of
cholinergic neurons originate in the nucleus of the diagonal
band, nucleus basalis, substantia inominata and the septum
and project to several forebrain structures, including the
cortex and hippocampus. The effects of acetylcholine release
are mediated by both g-protein-coupled muscarinic and
ionotropic nicotinic receptors of which there are multiple
subtypes that are present on pre- and post-synaptic neurons.
Acetylcholine has been shown to play a signicant role in
arousal and in learning and memory as evidenced by the
successful use of acetylcholinesterase inhibitors for the
treatment of cognitive dysfunction in Alzheimers disease
(Wilkinson et al. 2004) and in cognitive function in animal
models (Prickaerts et al. 2005). Of note, cholinesterase
inhibitors (Wilens et al. 2000; Doyle et al. 2006) and
nicotinic receptor agonists (Wilens et al. 1999; Potter et al.
2006, 2014; Day et al. 2007; Apostol et al. 2012) have been
examined for efcacy in ADHD albeit with mixed success, in
some cases probably due to the poor tolerability of the drugs
used.
Histamine-containing neurons originate from a single
group of cell bodies located in the tuberomammillary nucleus
(Panula et al. 1989) but project widely throughout the brain
innervating monoamine-containing cell bodies and forebrain
regions. As with acetylcholine, histamine is also thought to
play a signicant role in arousal and cognitive function (Kay
2000; Witkin and Nelson 2004; Day et al. 2007; Alvarez
2009; Brioni et al. 2011; Kohler et al. 2011). The effects of
histamine are mediated via three g-protein-coupled receptors
(H1, H2, H3) which are localized in many forebrain regions.
Of particular relevance, H3 receptors are pre-synaptic
autoreceptors on histamine terminals, which regulate the
release of histamine, but they also exist as heteroreceptors
regulating dopamine (Medhurst et al. 2007; Esbenshade
et al. 2012), serotonin, noradrenaline (Medhurst et al. 2007;
Dremencov et al. 2011) and acetylcholine (Bacciottini et al.
2002; Medhurst et al. 2007; Esbenshade et al. 2012) release
in several brain regions including cortex and hippocampus.
Surprisingly, and despite their relevant neurochemical and
behavioural prole, histamine H3 inverse agonists/antago-
nists have failed to show efcacy in patients with ADHD
(Herring et al. 2012; Weisler et al. 2012).
While the predominant effects of drugs that are used to
treat ADHD are monoaminergic, it is conceivable that
increased acetylcholine and or histamine transmission by the

action of psychostimulants may play a role in improving
cognitive function in ADHD (Horner et al. 2007; Liu et al.
2008) and the present study showed that LDX increased the
efux of both transmitters in cortex and or hippocampus.
Furthermore, the present study indicates that when LDX was
combined with s-citalopram (as might be the case in the
management of comorbid mood symptoms in ADHD), the
changes of acetylcholine and histamine were still present,
albeit somewhat attenuated in the case of histamine efux.
Consequently, if these transmitters contribute to the overall
improvement in cognitive function in ADHD, this should not
be markedly diminished by the co-administration of s-
citalopram in ADHD patients who are co-morbid for mood
disorders.
Acknowledgments and conflict of interest
disclosure
Complete Healthcare Communications, Inc. (CHC; Chadds Ford,
PA, USA) provided support in formatting, proofreading and
copyediting this manuscript. This study was conducted by Brains
On-Line BV (Groningen, the Netherlands), with funding provided
from Shire Development LLC (Wayne, PA). Shire Development
LLC (Wayne, PA) provided funding to Complete Healthcare
Communications, Inc. (CHC; Chadds Ford, PA) for support in
formatting, proofreading, and copyediting this manuscript. Peter H.
Hutson is an employee of Shire and holds stock and/or stock options
in Shire Development LLC. Mariette S. Heins and Joost H.A.
Folgering are employees of Brains On-Line BV, which was
contracted by Shire Development LLC to conduct this study.
All experiments were conducted in compliance with the ARRIVE
guidelines.
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