A daily single dose of a novel modafinil analogue CE-123 improves memory acquisition and memory retrieval

Article abstract of DOI:10.1016/j.bbr.2018.01.032

Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhydrylsulfinyl)methyl) thiazole (CE-123), was selec

Full text of DOI:10.1016/j.bbr.2018.01.032

Behavioural Brain Research 343 (2018) 83–94
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Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
A daily single dose of a novel modafinil analogue CE-123 improves memory
acquisition and memory retrieval
Martina Kristofovaa,1, Yogesh D. Aherb,1, Marija Ilica,1, Bojana Radoman , Predrag Kalaba ,
b
a
a
a
a
b
a
Vladimir Dragacevic , Nilima Y. Aher , Johann Leban , Volker Korz , Lisa Zanon ,
Winfried Neuhaus , Marcus Wieder , Thierry Langer , Ernst Urban , Harald H. Sitte ,
c
a
a
a
d
e
f,⁎
f,⁎
Harald Hoeger , Gert Lubec , Jana Aradska
a
Department of Pharmaceutical Chemistry, Faculty of Life Sciences, University of Vienna, Vienna, Austria
Department of Pediatrics, Medical University of Vienna, Vienna, Austria
b
c
d
e
f
Competence Unit Molecular Diagnostics, Competence Center Health and Bioresources, Austrian Institute of Technology (AIT) GmbH, Vienna, Austria
Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, Vienna, Austria
Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, Himberg, Austria
Department of Neuroproteomics, Paracelsus Medical University, Salzburg, Austria
A R T I C L E I N F O
A B S T R A C T
Keywords:
DAT inhibitor
Modafinil
Dopamine
Memory
Dopamine reuptake inhibitors have been shown to improve cognitive parameters in various tasks and animal
models. We recently reported a series of modafinil analogues, of which the most promising, 5-((benzhy-
drylsulfinyl)methyl) thiazole (CE-123), was selected for further development. The present study aims to char-
acterize pharmacological properties of CE-123 and to investigate the potential to enhance memory performance
in a rat model. In vitro transporter assays were performed in cells expressing human transporters. CE-123 blocked
uptake of [3H] dopamine (IC50 = 4.606 μM) while effects on serotonin (SERT) and the norepinephrine trans-
porter (NET) were negligible. Blood-brain barrier and pharmacokinetic studies showed that the compound
reached the brain and lower elimination than R-modafinil. The Pro-cognitive effect was evaluated in a spatial
hole-board task in male Sprague-Dawley rats and CE-123 enhances memory acquisition and memory retrieval,
represented by significantly increased reference memory indices and shortened latency. Since DAT blockers can
be considered as indirect dopamine receptor agonists, western blotting was used to quantify protein levels of
dopamine receptors D1R, D2R and D5R and DAT in the synaptosomal fraction of hippocampal subregions CA1,
CA3 and dentate gyrus (DG). CE-123 administration in rats increased total DAT levels and D1R protein levels
were significantly increased in CA1 and CA3 in treated/trained groups. The increase of D5R was observed in DG
only. Dopamine receptors, particularly D1R, seem to play a role in mediating CE-123-induced memory en-
hancement. Dopamine reuptake inhibition by CE-123 may represent a novel and improved stimulant therapeutic
for impairments of cognitive functions.
Hippocampus
Dopamine receptor
1. Introduction
affinity for multiple monoamine transporters [7].
Cocaine, unlike amphetamine, acts as a simple inhibitor that binds
The DA transporter (DAT), which governs the spatial and temporal
to the DAT and inhibits its transport activity without stimulating re-
verse transport activity [8]. Although a multitude of DAT inhibitors that
share cocaine-like behavioral effects exists, a number of atypical DAT
inhibitors (e.g., analogs of benztropine, RTI-371, GBR12909, modafinil)
exhibit different behavioral properties with a minimal abuse liability
[9–14]. Moreover, benztropine and modafinil have been reported to
antagonize the effect of cocaine [15,16], suggesting a unique ther-
apeutic potential for treatment of stimulant dependence. Several of
dynamics of dopamine neurotransmission by driving reuptake from the
extracellular space into the presynaptic neurons, is a major target for
several psychostimulant drugs including cocaine, amphetamine and
methylphenidate, likely mediating their abuse-related effects [1–4].
Most classical psychostimulants exhibit a dose-dependent biphasic ef-
fect on cognitive functions, learning and memory [5,6]. However, their
behavioral profile is also influenced by their action on and different
⁎
Corresponding authors.
E-mail addresses: gert.lubec@lubeclab.com (G. Lubec), jana.aradska@lubeclab.com (J. Aradska).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.bbr.2018.01.032
Received 12 December 2017; Received in revised form 28 January 2018; Accepted 29 January 2018
Available online 01 February 2018
0166-4328/ © 2018 Elsevier B.V. All rights reserved.

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
2. Materials and methods
2
2
1
.1. Synthesis of 5-((benzhydrylsulfinyl)methyl)thiazole (CE-123)
.1.1. Synthesis of S-di-phenylmethyl-isothiuronium hydrobromide
Diphenylmethanol (26.0 g, 140 mmol) and thiourea (13.0 g,
70 mmol) were mixed in a 1 L two-neck round bottomed flask. Water
(
65 mL) was added and the mixture was heated to 95 °C (an emulsion
was obtained) and 52 g of 48% HBr (644 mol, 4.6 equivalents) was then
added dropwise during 0.5 h. The mixture was refluxed for additional
Fig. 1. Molecular formulae of CE-123 and parent compound modafinil.
0.5 h and cooled to room temperature. The product was precipitated at
them show pro-cognitive effects similar to those of low-dose psychos-
timulants. These agents, however, are only moderately selective in-
hibitors of DAT. The fact that a locomotor stimulant effect and addic-
tiveness are not a common property of all DAT inhibitors, demonstrates
that DAT may possess some of the ligand-specific pleiotropic attributes
inherent to G-protein–coupled receptors [17,18].
A parent compound of CE-123, modafinil (Fig. 1) is a wake-pro-
moting drug approved for the treatment of narcolepsy, sleep apnea and
shift-work sleep disorders [19]. Modafinil has gained increasing interest
for its cognitive enhancing properties in healthy individuals [20,21]
and individuals with some cognitive dysfunctions, including attention
deficit hyperactivity disorder (ADHD) [22] and schizophrenia [23].
Despite a weak binding potential for DAT, modafinil occupies more
than 50% of DAT at therapeutic doses [24], that makes modafinil a
candidate for off-prescription use for cognitive enhancement.
0 °C. Subsequently, products were filtered off and washed with cold
water, dried under reduced pressure to yield 19.24 g of a white solid
(yield: 74%).
2.1.2. Synthesis of 5-(chloromethyl)thiazole hydrochloride
In a round bottom flask 10 g (86.8 mmol) of 5-(hydroxymethyl)
thiazole were dissolved in 100 mL of dichloromethane and cooled to
0 °C. Then 6.3 mL (1 equivalent, 86.8 mmol) of thionyl chloride was
added dropwise to the reaction mixture and stirred overnight at room
temperature. The product was then concentrated at reduced pressure
and dried in high vacuum for one hour to yield 9.1 g of a solid orange
material (yield: 78.5%).
2.1.3. Synthesis of 5-((benzhydrylthio)methyl)thiazole
In a round bottomed flask 13.73 g (42 mmol) of S-di-phenylmethyl-
isothiuronium hydrobromide was dissolved in 150 mL of methanol.
Afterwards, 7.09 g (42 mmol) of 5-(chloromethyl)-thiazole hydro-
chloride and 29.31 g (5 equivalents, 210 mmol) of potassium carbonate
were added to the mixture. The mixture was stirred for 2 days at room
temperature. Methanol was removed under reduced pressure and
150 mL of water were added. The reaction products were extracted with
ethylacetate, dried over Na SO , filtered off and the products were
Although the mechanism of action of modafinil is not well under-
stood, multiple neurotransmitter systems have been implicated in its
activity [25]. A major molecular target of modafinil is the DAT, how-
ever, several studies reported significant occupancy of NET by mod-
afinil [26]. In vitro (in cells expressing human transporters), modafinil
inhibits DAT activity (IC50 6.4–13 μM) and with weaker potency NET
(
IC50 35.6–182 μM) [27–29]. Given the important role of dopamine for
2
4
executive functions, attention and cognition, acting on dopaminergic
system may be a key action that promotes pro-cognitive effects.
Therefore, the design of improved selective dopamine reuptake in-
hibitors for cognitive enhancement represents the basic strategy for
own modafinil analogue development.
concentrated. The crude product was purified via flash column chro-
matography on silica gel (5% methanol in dichloromethane) to yield
17.9 g of white crystals (yield: 72.71%).
2.1.4. Synthesis of 5-((benzhydrylsulfinyl)methyl)thiazole
We recently reported a series of modafinil analogues [30–33] dis-
playing improved pharmacological properties compared to the parent
compound with pro-cognition action. We have recently demonstrated
that CE-123 enhances cognitive flexibility without producing impulsive
responding [34] and therefore CE-123 was selected for further testing
of its cognition enhancing properties.
17.9 g (60.25 mmol) of 5-((benzhydrylthio)methyl)thiazole were
dissolved in 50 mL of glacial acetic acid and 6.95 mL (60.25 mmol) of
30% H O2 was dropped into the solution and stirred for 12 h. The re-
2
action mixture was neutralized with cold 5% sodium bicarbonate so-
lution. Reaction products were extracted with ethyl acetate, dried over
Na SO , filtered off and ethyl acetate was removed under reduced
2
4
In the present study, animals were tested in a hole-board task, a
food-rewarded test of spatial learning and memory. The efficiency of
DAT inhibitors is stimulus-dependent and is therefore limited by the
tone of presynaptic activity [7]. Novelty or reward-related stimuli in-
crease activity of DA neurons in the VTA [35,36] and stimulate DA
release in hippocampus and prefrontal cortex [37,38]. The effect of DA
receptor stimulation on learning and memory that is predominantly
mediated by prefrontal cortex and hippocampus is well-documented.
Activation of D1-like receptors enhance synaptic plasticity, in particular
long-term potentiation [39,40], one of the cellular mechanisms of
learning and memory [41]. D1-like receptors seem to be key mediators
of downstream effects subsequent to DAT inhibition. However, effects
of DAT inhibitors on the hippocampus were so far neglected.
To generate a profile of subregion-specific protein level changes of
dopamine receptors and the DAT following drug treatment, three major
hippocampal subregions were examined individually.
pressure. The solid product (8.8 g) was purified by flash column chro-
matography on silica gel (5% methanol in dichloromethane), con-
centrated and dried in high vacuum to yield 6.6 g of the white crys-
talline product (yield: 50.2%) The C-18 HPLC - determined purity was
99.7%
(Supplementary
data).
[M+H+] = 314.0667,
[M
+
+N1a ] = 336.0486 (Supplementary data).
H NMR (500 MHz, CDCl3-d, 23 °C): δ = 8.86 (s, 1H, CH, thiazole-
2), 7.67 (s, 1H, CH, thiazol-4), 7.39 (2x CH-2,6, phenyls), 7.45 (CH-3,5,
phenyl), 7.39 (CH-3,5, phenyl), 7.36 (2x CH-4, phenyls), 4.65 (s, 1H,
CH), δ = 4.13, 3.89 (AB, 2H, CH2) (Supplementary data).
13
C[42]NMR (125.75 MHz, CDCl3-d, 23 °C): δ = 46.64 (CH2),
69.95 (CH), 128.54 (C-4, phenyl), 128.77 (C-4, phenyl), 129.33 (2x C-
3,5, phenyl), 129.52 (2x C-3,5, phenyls), 128.66 (C-2,6, phenyl),
128.81 (C-2,6, phenyl), 134.05 (Cq-1, phenyl), 134.74 (Cq-1, phenyl),
124.89 (C-2, thiazole), 144.18 (C-4, thiazole), 154.97 (C-5, thiazole).
(Supplementary data).
Herein we report a novel modafinil analogue CE-123 with superior
pharmacological properties compared to the parent compound with
respect to DAT, SERT and NET, specifically inhibits DAT in vitro, pre-
sumably penetrates the BBB, shows slower elimination from blood, CSF
and brain and exerts no adverse effects and is significantly increasing
performance in a spatial memory task.
Details on formulae, separation and information on purity are given
in Supplementary data.
2.2. Molecular docking
The protein homology model was kindly provided by the group of
84

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
Gerhard Ecker from the University of Vienna. The provided homology
model was derived from the drosophila DAT structure as described in
Saha et al. [43]. The DAT structure was in complex with nortriptyline
2.5. Transport across blood-brain barrier in vitro model
The transport studies were accomplished with Transwell models
using mouse cell line cerebEND which was a kind gift from Prof. Carola
Förster [48]. CerebEND cells are able to form tight cell layers similar to
primary mouse brain endothelial cells and have been characterized as
blood-brain barrier in vitro model in a comprehensive manner pre-
viously [48,49]. Experiments were conducted on collagen IV coated
Transwell inserts as described recently [31,49]. Compound solutions
were added to the apical compartment, and inserts were transferred at
37 °C into DMEM (+0.2% DMSO) filled wells after five, 15, 30 and
60 min. Transendothelial electrical resistance (TEER) values before and
after the studies confirmed that cell layer integrity was not compro-
mised during experiments. Diazepam and CE-123 (100 mM) were dis-
solved in DMSO, compounds were diluted in DMEM to a test con-
centration of 100 μM (test solution). Diazepam was added to CE-123 in
DMEM as internal standard to account for cell layer’s variability.
Samples of the test solution, the basolateral and apical compartments
after the transport study were collected and prepared for HPLC analysis
as recently described in detail [31,50]. Cleared volume versus (vs.) time
curves and permeability coefficients were calculated using peak areas
of HPLC analysis following the clearance principle according to Nova-
kova et al. [50]. Permeability coefficient PCall [μm/min] describes the
transport across the cell layer and the membrane support. PCcell values
[μm/min] mean the permeability across only the cell layer (after sub-
straction of transport results across blank inserts without cells).
[
44].
Molecular docking was performed using Autodock Vina [45]. The
ligands were prepared using the Maestro Modelling suite as shown in
Fig. 3. The ligands were prepared with flexible bonds using the auto-
matic detection of Autodock Vina. For the molecular docking run
standard parameters were used, protein flexibility was not regarded and
the exhaustiveness level was set to 16. Waters were removed and the
binding site was defined as the center of mass of nortriptyline. The
search space was restricted to the protein structure. Docking analysis
was performed using compound R/S-CE-123 and cocaine.
The interaction pattern between the best docked poses and the
amino acids in the binding pocket were visualized using the Maestro
Modelling suite and the pharmacophore models were generated using
LigandScout [46].
2.3. Reuptake inhibition assays
Dulbecco’s modified Eagle’s medium (DMEM), trypsin and fetal
bovine serum (FBS) were purchased from Sigma-Aldrich Handels GmbH
3
3
(
Austria). [ H]5-HT (Hydroxytryptamine; 5-[1,2- H[N]]; 27,8 Ci/
3
mmol), [ H]DA (Dihydroxyphenylethylamine; 3,4-[ring-2,5,6-3[H]]-
Dopamine; 36,6 Ci/mmol) and [ H]MPP+ (Methyl-4-phenylpyr-
idinium iodide; 1-[methyl- H]; 80 Ci/mmol) were purchased from
3
3
Perkin Elmer, Boston, MA.
Effects of CE-123 on reuptake of their respective substrates were
analyzed as described by [30,47]. Briefly, HEK293 cells stably expres-
sing human isoforms of the dopamine transporter (DAT), the nor-
epinephrine transporter (NET) and the serotonin transporter (SERT)
2.6. Plasma, cerebrospinal fluid (CSF) and brain levels of CE-123
Pharmacokinetic analysis was performed by Pharmacelsus
(Saarbrücken, Germany). Sprague Dawley rats were treated with CE-
123 or R-modafinil at a dose of 10 mg/kg via intraperitoneal adminis-
tration. Blood, CSF and brains were collected from rats at 15 min, 1 and
7 h after dosing. The plasma was prepared within 45 min after sampling
and was kept at −20 °C until being assayed. Rats were sacrificed by
inhalation of an overdose of isoflurane for collection of CSF and brain.
Rats were placed in a stereotaxic device and aliquots of 20 μL CSF were
obtained by puncture of the cisterna magna. The sample was frozen on
dry ice within 1–2 min of sampling. Subsequently, brains were dissected
and frozen on dry ice within 1–2 min of sampling.
were used for reuptake inhibition assays. All cell lines were seeded on
4
9
2
6-well plates pre-coated with poly-D-lysine (PDL) (5 × 10 cells/well)
4 h prior to the experiment. Each well was washed with 100 μL of
Krebs-HEPES buffer (KHB; 10 mM HEPES, 120 mM NaCl, 3 mM KCl,
2
mM CaCl2·2H2O, 2 mM MgCl2·6H2O, 5 mM D-(+)-Glucose mono-
hydrate, pH 7.3). Cells were pre-incubated 5 min in KHB containing
different dilutions (0.001 μM, 0.01 μM, 0.1 μM, 1 μM, 10 μM, 0.1 mM
and 1 mM) of CE-123. CE-123 was dissolved first in 99.9% dimethyl
sulfoxide (DMSO) and subsequently diluted in KHB. Afterwards, cells
were incubated in KHB containing the same dilutions of CE-123 with
Plasma, CSF and brain levels of CE-123 were measured by HPLC–MS
(Thermo TSQ Quantum Discovery Max, Thermo Fisher Scientific, USA)
by standard validated protocols defined by the contractor
(Pharmacelsus, Germany).
3
3
addition of 0.2 μM [ H]-dopamine (for HEK-DAT), 0.05 μM [ H]MPP+
3
(
for HEK-NET) and 0.4 μM [ H]5-HT (for HEK-SERT). Incubation times
were 1 min for HEK-DAT and HEK-SERT and 3 min for HEK-NET. For
determination of non-specific uptake in HEK-DAT and HEK-NET 10 μM
mazindole was used and 10 μM paroxetine was used for HEK-SERT.
After incubation at room temperature, reactions were stopped by the
addition of 100 μL of ice-cold KHB. Finally, cells were lysed with 300 μL
of 1% SDS and released radioactivity was measured by a liquid scin-
tillation counter (Tri-carb-2300TR, Perkin Elmer).
2.7. Animals
Forty-eight male Sprague Dawley rats, age between 12 and 14
weeks, divided in 4 groups were used for the experiments. They were
bred and maintained in cages made of Makrolon filled with autoclaved
woodchips in the Core Unit of Biomedical Research, Division of
Laboratory Animal Science and Genetics, Medical University of Vienna.
Food and water in bottles were available ad libitum and reared under
normal animal facility conditions (temperature: 22 ± 2 °C; humidity:
55 ± 5%; 12 h artificial light/12 h dark cycle: light on at 7:00 a.m.).
All procedures were carried out according to the guidelines of the Ethics
committee, Medical University of Vienna, and were approved by
Federal Ministry of Education, Science and Culture, Austria (BMWFW-
66.009/0114-WF/II/3b/2014). All efforts were made to minimize an-
imal suffering and to reduce the number of animals used.
2.4. DAT-release assay
Monensin sodium salt and the D-amphetamine hemisulfate salt were
purchased from Sigma-Aldrich Co. The substrate/efflux experiments
were performed as described before [47]. Briefly, HEK-DAT cells were
grown in 5 mm diameter PDL-coated coverslips. Cells were incubated
with 0.05 μM [3H]MPP+ at 37 °C for 20 min. The coverslips were
transferred onto superfusion chambers (0.2 mL) and excess radio-
activity was washed out with KHB for 40 min (0.7 mL/min) at 25 °C to
obtain stable baselines. During the experiment the buffer was ex-
changed either to monensin or remained at control buffer after the
collection of three baseline fractions for another four fractions. Subse-
quently, CE-123 or D-amphetamine was added for another five fractions
as indicated in Fig. 1B. Finally, the remaining radioactivity was col-
lected by treatment with 1% SDS.
2.8. Hole-board apparatus and procedure
Hippocampus-dependent spatial learning and memory was eval-
uated by a hole-board task as previously described with a small mod-
ification [51,52]. The hole-board setup consisted of a black plastic
85

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
board (1 m × 1 m) with 16 regularly arranged holes (7 cm in diameter
and 7 cm deep) and was surrounded by plexiglass walls 30 cm in height
kg drug or vehicle (DMSO) administered intraperitoneally in doses of 1
and 10 mg/kg body weight. Drug treatment did not impair general
health condition such as movement, gait, salivation, sedation, tremor,
fur condition, convulsion, diarrhoea, etc. (Supplementary data).
(
from BiObserve, St. Augustin, Germany). The walls were marked with
different black and white cues at each side. Four out of 16 holes were
baited in a fixed pattern with standard food pellets (dustless precision
pellets, 45 mg, Bio-Serv, Frenchtown, NJ, USA). Beneath the hole-board
there was a second board on which food pellets were scattered ran-
domly to mask olfactory cues. The apparatus was kept at an elevation of
2.10. Behavioral tests
Behavioral tests including Open Field [54], Elevated Plus Maze
[54], Forced Swim Test [55,56] and Rota Rod [54,55] were used to
determine potential effect of CE-123 on anxiety-, exploratory behavior,
anti-depressant effect and motor function. A neurological observational
battery [54] was applied to reveal defects in gait or posture, changes in
muscle tone, grip strength, visual acuity and temperature. All proce-
dures are described in details in Supplementary data.
80 cm above the floor in a room with indirect illumination by floor
positioned lamps directed to the ceiling providing equal light intensities
in all corners. A camera fixed to the ceiling viewing the experimental
area monitored trials and videos were stored digitally.
Rats were kept in their home cages within the testing room
throughout the experiments. They were handled for 15 min each day for
adaptation 3 consecutive days before habituation. The body weight of
the animals was recorded from first day of handling throughout the
experiment. The animals were food-deprived (receiving 5–6 g food
2.11. Synaptosome isolation
(
ssniff Spezialdiäten GmbH) per day and ad libitum water) to decrease
Hippocampal subregions were homogenized with pipette tips in a
Syn-PER synaptic protein extraction reagent (Thermo Scientific,
Rockford, IL, USA) containing a Protease Inhibitor Cocktail (PIC, Roche
the body weight to 80–85% of free feeding weight and maintained the
same throughout the experiment.
Prior to the start of the training on the fourth and fifth day of food
deprivation, rats were given two habituation sessions in order to fa-
miliarize them to the hole-board setup and to the reward system. On the
first day of habituation, pellets were placed all over the board as well as
in the holes and rats were allowed to explore the apparatus and eat the
food for fifteen minutes, whereas on the second day, pellets were placed
only in the holes and exploration/eating time was reduced to five
minutes. After free exploration of the apparatus the animals were
carefully picked up and taken back to the home cage.
Molecular Biochemicals) and a Phosphatase Inhibitor Cocktail
(PhosStop, Roche Molecular Biochemicals). The homogenate was cen-
trifuged at 1200 × g for 10 min to remove cell debris. The resulting
supernatant was centrifuged at 15,000 × g for 30 min. The synapto-
some pellets were resuspended in SDS-buffer (50 mM TrisHCl pH 8.0,
150 mM NaCl, 1% SDS, 1x PhosStop, 1x PIC). Protein concentration was
estimated using a bicinchoninic (BCA) assay kit (Pierce, Rockford, IL,
USA) according to manufacturer’s instruction.
During three days of training and testing (five trials on day 1, four
trials on day 2, and the retention trial on day 3) a fixed pattern of baited
holes (4 out of 16) with a 20 min inter-trial interval was maintained
throughout the experiment for all rats to assess working memory, while
the other arms were always left un-baited in order to test reference
memory. The training began at 9:30 a.m. and the retention trial (day 3)
began 24 h after the last trial on day 2 (10:30 a.m.). Each trial started by
placing the rat at one of the corners of the hole-board and the start
position changed subsequently in the following trials. A trial was
stopped after 2 min or when an animal had found all 4 pellets. The
2.12. Immunoblotting
Protein levels of D1R, D2R, D5R, DAT, DATp were determined using
a standard western blotting procedure [32]. Polyclonal rabbit anti-D1R
(1:5000, GeneScript, USA), rabbit anti-D2R (1:5000, GeneScript),
rabbit anti-D5R (1:3000, ab181623, Abcam), rabbit anti-DAT (1:3000,
GeneScript) and rabbit anti-DATp (1:3000, ab183486, Abcam) anti-
bodies were used. Specific peptide sequences were used for im-
munization and custom-production of the affinity-purified rabbit
polyclonal
antibodies
D1R
(-TSTMDEAGLPAERD-),
D2R
®
board was cleaned with 1% Incidin after each trial in order to remove
(-NWSRPFNGSEGKAD-) and DAT (-TNSTLINPPQTPVEAQERETW-)
(GenScript, Piscataway, NJ, USA) and specificity of antibodies was
determined [31]. Five to thirty micrograms of each sample were in-
cubated at 37 °C for 30 min prior to separation on 10% SDS-PAGE. For
immunoblotting, proteins were transferred to PVDF membranes (GE
Healthcare), blocked for one hour at 22 °C with 5% non-fat milk in TBS
containing 0.1% Tween-20 (0.1% TBST), and incubated with primary
antibodies for 16 h at 4 °C. All antibodies were incubated in 5% non-fat
dry milk in 0.1% TBST. Membranes were then washed six times for
10 min in 0.1% TBST. An HRP-conjugated anti-rabbit secondary anti-
body (1:10,000, ab191866, Abcam) was used and blots were visualised
with ECL solution (#170-5061, BIO–RAD). Immunoreactive bands were
imaged on films, digitized at a resolution of 600 d.p.i., and quantitated
using ImageJ software. As loading control membranes were stained
with PhastGel BlueR (GE Healthcare) according to the manufacturer’s
instruction. Immunoblot data were normalized to corresponding whole-
lane densitometric volumes of total protein-stained membranes as
previously described [57].
any olfactory cues. The time needed to find all of the pellets (latency),
the working-memory errors (i.e., visiting a hole that was baited but had
already been visited, with the pellet picked up during a specific trial)
and the reference-memory errors (i.e., visiting an unbaited hole) were
counted. The memory index was calculated for working (first entry into
baited holes/total visits of baited holes)(WMI) and reference memory
(
total visits of baited holes/total visits of all holes)(RMI). Thus a value
of 1 represents no error reflecting best performance whereas zero errors
combined with no hole visits indicate the index as zero. On the last day,
one hour after the retention trial, animals were deeply anaesthetized
2
with CO and sacrificed by cervical dislocation. Brain tissues were
quickly removed, hippocampi were rapidly dissected on a cold plate set
at 4–6 °C and CA1, CA3 and DG were micro-dissected from left and right
hippocampi under a stereoscope as described previously [53]. The tis-
sues were stored separately at −80 °C for further biochemical analysis.
Untrained/yoked rats underwent similar handling, habituation and
food restriction regimen and spent the same amount of time in the hole-
board as their trained counterpart rats except there was no food during
the trials, thus minimizing the likelihood of learning to associate the
cues with the reward.
2.13. Statistical analysis
Data were analyzed by unpaired Student's t-test when comparing
only two groups and with two-way ANOVA followed by Dunnett's or
Bonferroni's post hoc test (as indicated in each figure legend), when
comparing more than two groups (GraphPad Software, San Diego CA,
USA). Non-linear regression analysis was carried out for reuptake and
release assays. The probability level of P < .05 was considered as
2
.9. Drug administration
CE-123 was freshly dissolved in 100% DMSO and administered via
intraperitoneal injection everyday 30 min before the start of the beha-
vioral experiment throughout the training sessions. Rats received 1 mL/
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M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
3
3
3
Fig. 2. (A) Reuptake inhibition of [3H]monoamines by CE-123. Inhibition of [ H]DA, [ H]MPP+ and [ H]5-HT reuptake by increasing concentrations of CE-123 in HEK293 cells stably
expressing human isoforms of DAT, NET and SERT. Unspecific uptake was determined by using 10 μM mazindole for HEK-DAT and HEK-NET and 10 μM paroxetine for HEK-SERT. The
percentage of maximum uptake was determined by using 1% DMSO in KHB. (B) DAT-release assay. The release assay was performed in HEK-DAT cells. Cells were grown on PDL-coated
3
coverslips, treated with 0.05 μM [ H]MPP+ at 37 °C for 20 min and washed with KHB for 40 min in superfusion chambers. The first three (baseline) and next four fractions were without
any compounds and with 10 μM monensin, respectively. Final five fractions were with either 10 μM CE-123 or 10 μM D-amphetamine. Non-linear regression analysis was carried out by
using GraphPad Prism 5. Values are presented as mean ± SD.
statistically significant. Details are described in the corresponding sec-
tions.
These differences are most likely due to small coordinate deviations
between the two structures and do not reflect an independent and
different binding mode. A common structural feature of CE-123 and
modafinil is the lack of ionic interaction with negatively charged
ASP79, an interaction demonstrated in cocaine binding. No direct dif-
ferences are seen due to this interaction in the docked poses of S/R-CE-
3. Results
3.1. CE-123 strongly inhibits DAT-mediated dopamine reuptake without
123 and cocaine, however.
acting as a substrate
CE-123 was characterized in HEK293 cells expressing cloned human
transporters. A reuptake inhibition assay was used to determine the
efficacy of CE-123 to block the uptake of substrates by different
3
.3. CE-123 permeates across the blood-brain barrier in vitro
3
3
+
The cerebEND cell line possesses principal features of the blood-
monoamine transporters DAT, NET and SERT ([ H]DA, [ H]MPP and
3
brain barrier (BBB) in vivo. In the present study cerebEND cell layers
were used as an in vitro model to study the ability of CE-123 to cross the
BBB. Transport studies across cerebEND cell layers revealed a perme-
ability coefficient PCcell for CE-123 of 30.31 ± 6.94 μm/min (Fig. 4).
Normalization of permeability data to internal control Diazepam re-
sulted in a ratio of 0.87 ± 0.04 for CE-123. Thus, significant BBB
permeability could be expected for CE-123, since Diazepam is known as
a well permeating compound. Based on these findings following in vivo
studies were accomplished.
[
H]5-HT, respectively). CE-123 strongly inhibits DAT-mediated do-
pamine reuptake (IC50 = 4.606 μM ± 0.140) whilst inhibition of NET
and SERT is negligible (Fig. S2A, Supplementary data).
The DA-release assay was performed to examine whether CE-123
3
acts as a substrate and induces release of [ H]MPP+ from HEK-DAT
cells. 10 μM of CE-123 was used while 10 μM D-amphetamine serving as
a positive control was assayed under the same conditions. Fig. 2B shows
that CE-123 only negligibly induces DAT-mediated substrate efflux
(
Supplementary data). To facilitate reversal of normal transporter flux
and unambiguously distinguish inhibitor from releaser [58,59], cells
were pre-treated with 10 μM monensin. Monensin pretreatment did not
3
3.4. Plasma, CSF and brain levels of CE-123
increase CE-123-induced efflux of [ H]MPP + , whereas efflux induced
by DAT substrate D-amphetamine was significantly enhanced (Fig. 2B).
The observation from HEK-DAT cells clearly indicates that CE-123
specifically blocks DAT without acting as a substrate.
Next, we examined whether the data obtained in the in vitro model
also holds true in an animal experiment; hence, we tested whether CE-
23 can cross the BBB in the rat. We assessed in a pharmacokinetic
1
study, plasma, CSF and brain concentrations of CE-123 or R-modafinil
at 15 min, 1 and 7 h after the dose. Table 1 shows plasma, CSF and brain
levels of drugs after a single intraperitoneal administration of CE-123
and R-modafinil at 10 mg/kg. For brain, the levels of CE-123 and R-
modafinil were 4.4 ± 0.5 and 1.1 ± 0.3 μg/g, respectively, at 15 min,
and 2.0 ± 0.4 and 0.1 ± 0.0 μg/g at 1 h; for CSF the respective values
were 0.3 ± 0.1 and 0.5 ± 0.2, and 0.3 ± 0.04 and 0.1 ± 0.03 μg/
mL; for plasma the respective values were 7.7 ± 1.1 and 2.3 ± 1.0,
and 3.0 ± 0.9 and 0.2 ± 0.1 μg/mL. 7 h after the injection CE-123
was still detected in plasma, CSF and brain (Table 1). The brain level of
3
.2. Docking of CE-123 into DAT homology model
To characterize the CE-123 binding site in the DAT, we carried out a
docking study of the two enantiomers with a DAT homology model
derived from drosophila [43,60] (Fig. 3). A binding site of CE-123 en-
antiomers overlaps with the substrate-binding pocket in the center of
DAT that is also the binding site for cocaine [44], modafinil [28] and
benztropine analogues [61]. The highest scored ligand positions are
shown in Fig. 3. The top-ranked binding poses revealed significant si-
milarity between the binding modes of the enantiomers, although there
are differences in the interacting amino acids: e.g. ASP476 is only
present for S-CE-123 and ALA81 is only present as an interaction
partner for R-CE-123. A closer look at the pharmacophore features re-
vealed that the interactions are unspecific hydrophobic interactions.
4.4 μg/g at 15 min indicates rapid brain entry for our compound.
Moreover, the different plasma, CSF and brain levels of CE-123 and
modafinil at the 15-min and 1-h time point demonstrates that CE-123
has a higher elimination rate constant than R-modafinil.
87

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
Fig. 3. Docking of R/S-CE-123 and cocaine into DAT homology model. The highest scored pose of R-CE-123, S-CE-123 and cocaine are shown in the upper panels as well as the ligand
interaction diagram showing the interactions between the ligands and the surrounding amino acids in the middle panels. In the ligand interaction diagram the residues are represented as
colored spheres (green = hydrophobic, cyan = polar, red = negatively charged, purple = positively charged) labeled with the residue name and number. Pharmacophore features are
shown in the bottom panels indicating that – with the exception of an ionic interaction (shown in blue on cocaine) – only hydrophobic interactions (shown as yellow regions) are present.
(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3
.5. Spatial hole-board training
treatment effect (F2,24 = 2.177, P > .05) at day 1. At day 2 a sig-
nificant trial effect (F3,36 = 10.35, P < .0001) and treatment effect
(F2,24 = 4.683, P = .0192), but no significant trial x treatment effect
(F6,72 = 1.765, P > .05) was observed. The Dunnett's post hoc test re-
vealed a significant difference between the CE-123 at 1 mg/kg and
vehicle treated groups at day 2 (P = .0294). A one-way ANOVA at day
3 (consolidation phase) resulted in a significant treatment effect
(F2,36 = 12.38, p < .0001). Trial-by-trial analysis revealed that re-
ference memory indices were significantly higher on day 1, trial 3 at
10 mg/kg (P = .0386), trial 4 (P = .0068 at 10 mg/kg and P = .0003 at
1 mg/kg), at the end of the acquisition phase (day 2, trial 7, P = .0147,
The effect of CE-123 on spatial learning and memory was evaluated
using a hole-board procedure with intraperitoneal administration of
drug (1 mg/kg and 10 mg/kg) or vehicle. Working memory indices
(
WMI) and reference memory indices (RMI) were calculated for all
training trials and used for analysis. The analysis of RMI and latency
was performed for each day separately as they represent different
phases of training [52]. A two-way ANOVA for repeated measures on
RMI revealed a significant trial effect (F4,48 = 17.88, P < .0001) and
trial x treatment effect (F8,96 = 2.915, P < .0059), but no significant
88

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
3.6. CE-123 does not affect coordinative function and locomotion
Potential neurotoxic effects of the CE-123 were examined in the
open field (OF), the elevated plus maze (EPM), the forced swim test
FST) and the rota rod test. After 3 days of handling, animals were
administrated CE-123 at a dose of 10 mg/kg or vehicle for the following
0 days. From the 5th day of drug administration behavioral tests were
(
1
conducted in the following order: OF, EPM, observation battery, rota
rod and FST. The motor coordinative function was evaluated through
the rota rod test. Administration of CE-123 at 10 mg/kg dose did not
produce any significant effect on motor coordination and locomotion
(
OF) (Supplementary data). Behavioral tests assessing anxiety-like be-
havior (OF and EPM) and a test used for evaluation of anti-depressant
effects (FST) did not show significant changes between the drug- and
vehicle-treated groups (Supplementary data).
3.7. Training and drug administration modulates synaptic dopamine
receptor levels in hippocampal subregions
Because DA receptors are important regulators of DAT function, as
well as downstream processes involved in memory function, we decided
to examine dopamine receptor level changes in synaptosomal fractions
of three major hippocampal subregions after treatment with the selec-
tive DAT inhibitor CE-123 (1 mg/kg). D1R protein levels were sig-
nificantly increased in CA1 and CA3 hippocampal subregions following
CE-123 treatment and training in the hole-board task. A simple ad-
ministration of drug (no training) led to significant increase of D1R
protein levels in CA1 and DG, whereas D1R protein levels in CA3 were
significantly decreased (Fig. 6).
D2R protein levels were significantly decreased in CA1 in the
trained group (P = .0232), whereas no significant changes in D2R
protein levels were observed in other hippocampal subregions.
In DG D5R protein levels were significantly increased (P = .0375 for
trained groups, P = .0325 for yoked groups) in the drug-treated group
compared to the vehicle-treated group, neither in CA1, nor in CA3
significant changes in D5R protein levels were shown.
Fig. 4. Permeability of CE-123 across a blood-brain barrier in vitro model. Differences of
cleared volume versus (vs.) time curves between the transport across the blank inserts and
inserts with cultivated cerebEND cells confirmed the significant barrier formed by the
blood-brain barrier in vitro model. Resulting permeability coefficients PCall [μm/min]
revealed similar transport properties of CE-123 in comparison to Diazepam. Substraction
of the blank values led to permeability coefficient PCcell [μm/min] values reflecting the
permeability of the compounds only across the cell layer. The data are presented as
mean ± SD, n = 5.
trial 9, P = .021 at 1 mg/kg) and during retrieval (day 3, trial 10,
P < .0001 at 1mg/kg) in CE-123 treated groups compared to vehicle
treated group (Fig. 5A).
A two-way ANOVA for repeated measures on latency revealed a
significant trial effect (F4,48 = 5.333, P = .0012) and treatment effect
(F
(F
(F
2,24 = 4.009, P = .0315), but no significant trial × treatment effect
8,96 = 1.147, P > .05) at day 1. At day 2 a significant trial effect
3,36 = 16.35, P < .0001) and treatment effect (F2,24 = 4.131,
3.8. CE-123 modulates synaptic dopamine transporter levels in
hippocampal subregions
P = .0287), but no significant trial x treatment effect (F6,72 = 0.7827,
P > .05) was observed. The Dunnett's post hoc test revealed significant
differences between vehicle and CE-123 for both 1 mg/kg (P = .0402)
and 10 mg/kg (P = .0477) doses at day 1, and between vehicle and CE-
We compared total DAT protein levels and phosphorylation of DAT
at Thr53 (DATp) in the hippocampal subregions of drug-treated and
vehicle-treated animals. Results have revealed altered dopamine
transporter levels in hippocampal subregions following drug adminis-
tration.
123 at 1 mg/kg (P = .0193) at day 2 (Fig. 5B). A one-way ANOVA at
day resulted in significant treatment effect (F2,36 = 8.707,
3
a
DA transporter reuptake inhibition by CE-123 in CA1 leads to in-
creased phosphorylation of DAT at Thr53 in the trained group, whereas
total DAT protein levels were not changed (Fig. 7; trained groups,
P = .0002; yoked groups, P = .976).
In DG of trained animals total DAT protein levels were significantly
increased in the drug-treated group as compared to vehicle-treated
group (P < .0001).
P = .0008). The time to find all pellets was significantly decreased on
day 1 (trial 2; trial 3; trial 4 and trial 5), on day 2 (trial 7 and trial 9)
and during retrieval (day 3, trial 10, P = .0006) when compared drug-
treated groups to the vehicle-treated group.
A two-way ANOVA analysis on WMI did not show significant dif-
ferences between CE-123 treated groups and vehicle treated group
(
Fig. 5C). However, this may be due to a ceiling effect.
In CA3 DATp protein levels were significantly decreased in the drug-
treated group compared to vehicle-treated group in the yoked groups
Table 1
Plasma, CSF and brain levels of CE-123 and R-modafinil in Sprague Dawley rats after a single intraperitoneal administration of 10 mg/kg.
Time (min)
Plasma (ng/ml)
CE-123
CSF (ng/ml)
CE-123
Brain (ng/g)
CE-123
R-modafinil
R-modafinil
R-modafinil
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
Mean
SD
1
6
4
5
0
20
7701.9
2972.6
64.1
1097.4
859.3
43.4
2277.7
199.5
0.0
1030.8
86.8
0.0
606.9
279.2
4.9
52.6
35.6
3.0
524.5
113.5
1.2
197.0
32.4
2.0
4398.6
1987.7
20.9
506.9
354.3
21.7
1088.8
117.7
0.0
259.1
4.9
0.0
89

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
Fig. 5. (A) The performance was significantly better
with improved RMI in the drug treated groups
compared to vehicle during acquisition phase on day
1
and day 2 and during retrieval on day 3 of the hole-
board testing (B). Latency to complete the task was
significantly decreased in the drug treated groups
compared to vehicle treated group during acquisition
phase on day 1 at both doses and on day 2 and day 3
at dose of 1 mg/kg. (C) Working memory indices.
Mean and SEM are shown in the graph for every trial.
The data were analyzed using two-way ANOVA with
Bonferroni's or Dunnett's post hoc analysis for day 1
and 2 and one-way ANOVA for day 3. (*P < .05;
**P < .01; ***P < .001; ****P < .0001; n = 13).
(
P = .0205); whereas DATp protein levels in trained groups were
brain levels higher than for R-modafinil, moreover, CE-123 was de-
tected in the brain at 1 h post-administration in levels almost 20 times
higher than those achieved following administration of R-modafinil.
These data along with a memory enhancing effect observed in the hole-
board test indicate that CE-123 presumably permeates across the blood-
brain barrier and approaches its sites of action in vivo.
Over the years modafinil has gained popularity for its reported pro-
cognitive effects, which were extensively evaluated in healthy in-
dividuals and psychiatric disorders [20,21]. Studies involving animals
reported variable effects on different neurocognitive domains including
attention, executive functions, learning and memory. Working memory-
enhancing effect of modafinil has been demonstrated by employing
distinct spatial memory tasks [29,70,71]. Effects of modafinil on long-
term memory after chronic or subacute administration has been re-
ported as well [52,72,73].
The memory-enhancing potential of CE-123 was evaluated in a
spatial hole-board task in male Sprague-Dawley rats. Animals learned
the hole-board task as shown by the gradually decreasing number of
errors and latency during training trials. CE-123 shows a unique en-
hancing effect on memory acquisition on day 1 after single adminis-
tration prior to training at both drug concentrations. A second dose on
day 2 prior to training shows an enhancing effect only at 1 mg/kg, same
as for the retrieval phase. A dose-dependent effect on cognition,
learning and memory consolidation was reported previously for clas-
sical psychostimulants [6,74], as well as for modafinil [71,73].
Moreover, significantly decreased latency in a treated group may
not only refer to enhanced cognitive performance, but may also indicate
increased motivation to perform a given task [75], consistent with
previous reports on motivation-enhancing effect of DAT inhibitors
comparable.
4
. Discussion
In comparison to other psychostimulants including the lead com-
pound modafinil, CE-123 with respect to NET, DAT and SERT is an
apparently very selective DAT inhibitor with negligible binding to NET
and SERT. The very selective inhibition of the DAT may underlie the
fact that no neurological adverse side effects as e.g. coordination dis-
turbances were observed after drug treatment in contrast to modafinil
(
racemate), which displayed impairment in motor coordination on the
rota rod (data not shown). These effects are usually determined by ki-
netic characteristics and target specificity of the compound; higher
doses or multiple targets usually exert adverse drug reactions [62].
The lack of cocaine-like locomotor stimulation could be also at-
tributed to a DAT binding mode that is distinct from that shown by
cocaine and cocaine-like compounds. Examples are modafinil and
benztropine-like ligands, known to interact with an inward-facing
conformational state [28,63–65]. Moreover, several atypical DAT in-
hibitors have been reported to attenuate cocaine-induced behavioural
effects in vivo [66–69], suggesting a potential application of our com-
pound as an anti-relapse medication. The docking experiments from the
current study revealed that CE-123 lacks ionic interaction with nega-
tively charged ASP79, an interaction demonstrated in cocaine binding.
We could not show binding to inward-facing DAT conformation, how-
ever.
In order to reach its target in the CNS, CE-123 has to overcome the
blood-brain barrier. Before in vivo studies were conducted, results of in
vitro transport studies suggested that CE-123 could permeate in a
manner similar to diazepam and in comparison to a previous study also
similar to modafinil [31].
The structural modification in the design of CE-123 includes sub-
stitution of carboxyl-amide moiety of modafinil with a thiazole at-
tached on position 5 via a methylene bridge to the sulfoxide moiety
[
76,77]. However, the biological relevance of this behavioral result has
to be proven in further studies.
It has to be mentioned that the observed memory enhancing effect
can be also attributed to a rescuing effect. DMSO, a commonly used
drug delivery vehicle, has demonstrable pharmacological and patho-
logical effects on the central nervous system [78,79]. We have recently
reported that DMSO reduced spatial memory performance and LTP
magnitude, when compared to saline control. R-modafinil rescued
spatial memory and dentate gyrus synaptic plasticity from impairing
(
Fig. 1). A substitution of the carboxyl-amide group with the hetero-
cycle (thiazole) could result in the increased metabolic stability of the
compound. Pharmacokinetic analysis indicated rapid brain uptake with
90

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
Fig. 6. CE-123-induced synaptosomal changes in dopamine receptor protein levels in hippocampal subregions CA1, CA3 and DG. Hippocampal synaptosomal proteins were separated by
SDS-PAGE and immunoblotted with indicated antibodies. Apparent molecular weights were about 100 kDa for D1R, 50 kDa for D2R and for D5R. The data were analyzed by unpaired
Student's t-test. *P < .05, **P < .01, ****P < .0001. Values are expressed as mean ± SD; n = 11–13.
effects of DMSO [52].
information encoding and storage by gating hippocampal long term
plasticity and may drive the qualitative processing of information being
stored by the hippocampus [87]. Moreover, D1-like receptors seem to
be key mediators of downstream effects subsequent to DAT inhibition.
Several reports support this hypothesis. It has been shown that D1R is
required for the majority of modafinil-induced effects on exploration
[1], which is supported by the observation that D1/D5 antagonists at-
tenuate modafinil-induced increases in activity and exploratory beha-
viour [1]. Modafinil and GBR-12909 induce an increase in motivation
in a D1R expression-dependent manner [76]. With respect to spatial
memory, D1/D5 antagonism impairs long-term spatial memory in rats
[88] whereas D1/D5 agonism enhances spatial memory [88].
The current study revealed that CE-123 administration induces re-
gion- and training-specific changes in DA receptors protein levels. Drug
treatment led to increase of D1R protein levels in CA1 and DG, how-
ever, in CA3 D1R protein level changes seems to be dependent on
training and not simply reflect drug administration. In the DG drug
CE-123 had no effect on working memory performance, even
though it was expected, probably due to a ceiling effect. To determine a
full working memory-enhancing effects of CE-123, a more complex task
would be required.
The hippocampus is a functionally complex brain region that plays a
role in spatial navigation and memory. Each hippocampal subregion
represents a distinct unit at the cellular, molecular and functional level
and contributes differently to hippocampal information processing
[
80,81]. Dopamine neurotransmission affects hippocampal synaptic
plasticity [82–84] and influences hippocampal-associated functions
85,86].
To study a possible mechanism of action, western blot analysis was
[
used to quantify changes at the protein levels of DAT and dopamine
receptors (D1R, D2R and D5R) in the synaptosomal fraction of the three
major hippocampal subregions. Based on current knowledge, it is evi-
dent that D1-like receptors play a causal role in the establishment of
91

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
Fig. 7. CE-123-induced synaptosomal changes in DAT and DATp (Thr53) protein levels in hippocampal subregions CA1, CA3 and DG. Hippocampal synaptosomal proteins were separated
by SDS-PAGE and immunoblotted with indicated antibodies. The apparent molecular weights were about 115 kDa for DAT and 75 kDa for DATp. Data were analysed by unpaired
Student's t-test. *P < .05, ****P < .0001. Values are expressed as mean ± SD; n = 10–13.
administration leads to a significant increase of D5R protein levels that
was training-independent. D1 and D5 receptors show differences in
structural features, regional, cellular and subcellular distribution,
probably predicting differences in receptor signalling as well as biolo-
gical function [89]. Therefore, also different hippocampal subregions
exerting distinct functions in information processing may be differently
modulated by the activation of D1 and/or D5 receptors. We can only
speculate that the cognition enhancing effect of DAT inhibitors, such as
CE-123, may be due to drug-induced adaptations in the cellular me-
chanisms involving hippocampal D1/D5 receptor activation that in turn
underlie the formation of a persistent memory trace in the hippocampus
cell-surface DAT and enhance DAT function [95,98]. Own results show
that CE-123-induced down-regulation of total D2R protein levels is
training-dependent and occurs only in CA1. However, differentiation
between the short and long D2R splicing variant would be required as
they seem to have different neuronal distributions and functions.
Future studies with CE-123 need to address further behavioral,
pharmacokinetic and molecular questions in several brain regions, in-
cluding examination of interaction with a large array of different re-
ceptors and transporters, to unveil the specific and detailed molecular
and cellular mechanism of action. Selectivity and the pharmacological
profile may be improved by enantiomeric separation as follow up stu-
dies.
[
90].
As already shown above, CE-123 directly modulates DAT function,
however, dynamic regulation of surface density representing transport
capacity may be involved as well [91]. Many psychostimulants influ-
ence DAT trafficking. Amphetamine increases internalization of DAT
leading to a decrease of surface density [92]; in contrast, cocaine and
methylphenidate increase DAT surface density [93,94]. Regulation of
DAT surface expression is driven by ERK-dependent processes [95,96],
which involve ERK-mediated phosphorylation of Thr53 regulating DAT
insertion into the cell membrane. The membrane-proximal residue
Thr53 which precedes Pro54 as the DAT ERK phosphorylation site [97]
is the site playing a major role in the transport kinetic mechanism. DAT
inhibitors such as nomifensine, modafinil and its analogues, appear to
behave similarly in this mechanism as cocaine. We observed an increase
of surface-associated functional DAT in CA1 in the treated/trained
group. The findings indicate the presence of a rapid feedback me-
chanism associated with the phosphorylation state that regulates DA
clearance by modulating DAT surface density. Therefore our compound
may not only simply inhibit dopamine reuptake, but also prevent in-
ternalization of this transporter itself.
Declarations of interest
None.
Acknowledgements
We are very grateful to Prof. Carola Förster from the University
Hospital Wuerzburg (Germany) for providing the cell line cerebEND.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at https://doi.org/10.1016/j.bbr.2018.01.032.
References
Previous studies have suggested that activation of the short D2R
variant on presynaptic site initiates a signalling cascade to upregulate
[1] J.W. Young, K. Kooistra, M.A. Geyer, Dopamine receptor mediation of the ex-
ploratory/hyperactivity effects of modafinil, Neuropsychopharmacology 36 (2011)
92

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
1
385–1396.
(2015) 215.
[
[
[
[
[
2] J. Zhu, M.E. Reith, Role of the dopamine transporter in the action of psychosti-
[30] Y.D. Aher, S. Subramaniyan, B. Shanmugasundaram, A. Sase, S.R. Saroja, M. Holy,
et al., A novel heterocyclic compound CE-104 enhances spatial working memory in
the radial arm maze in rats and modulates the dopaminergic system, Front. Behav.
Neurosci. 10 (2016) 20.
[31] S.R. Saroja, Y.D. Aher, P. Kalaba, N.Y. Aher, M. Zehl, V. Korz, et al., A novel het-
erocyclic compound targeting the dopamine transporter improves performance in
the radial arm maze and modulates dopamine receptors D1-D3, Behav. Brain Res.
312 (2016) 127–137.
[32] A. Sase, Y.D. Aher, S.R. Saroja, M.K. Ganesan, S. Sase, M. Holy, et al., A heterocyclic
compound CE-103 inhibits dopamine reuptake and modulates dopamine trans-
porter and dopamine D1-D3 containing receptor complexes, Neuropharmacology
102 (2016) 186–196.
[33] A.M. Hussein, Y.D. Aher, P. Kalaba, N.Y. Aher, V. Dragacevic, B. Radoman, et al., A
novel heterocyclic compound improves working memory in the radial arm maze
and modulates the dopamine receptor D1R in frontal cortex of the Sprague-Dawley
rat, Behav. Brain Res. 332 (2017) 308–315.
mulants, nicotine, and other drugs of abuse, CNS Neurol. Disord.: Drug Targets 7
(2008) 393–409.
3] J.L. Katz, S. Izenwasser, P. Terry, Relationships among dopamine transporter affi-
nities and cocaine-like discriminative-stimulus effects, Psychopharmacology 148
(2000) 90–98.
4] M.C. Ritz, R.J. Lamb, S.R. Goldberg, M.J. Kuhar, Cocaine receptors on dopamine
transporters are related to self-administration of cocaine, Science 237 (1987)
1
219–1223.
5] S. Wood, J.R. Sage, T. Shuman, S.G. Anagnostaras, Psychostimulants and cognition:
a continuum of behavioral and cognitive activation, Pharmacol. Rev. 66 (2014)
1
93–221.
6] T.X. Xu, Q. Ma, R.D. Spealman, W.D. Yao, Amphetamine modulation of long-term
potentiation in the prefrontal cortex: dose dependency, monoaminergic contribu-
tions, and paradoxical rescue in hyperdopaminergic mutant, J. Neurochem. 115
(2010) 1643–1654.
[
[
[
7] L.L. Howell, S.S. Negus, Monoamine transporter inhibitors and substrates as treat-
[34] A. Nikiforuk, P. Kalaba, M. Ilic, V. Korz, V. Dragacevic, J. Wackerlig, et al., A novel
dopamine transporter inhibitor CE-123 improves cognitive flexibility and maintains
impulsivity in healthy male rats, Front. Behav. Neurosci. 11 (2017) 222.
[35] H.M. Bayer, P.W. Glimcher, Midbrain dopamine neurons encode a quantitative
reward prediction error signal, Neuron 47 (2005) 129–141.
[36] J.Y. Cohen, S. Haesler, L. Vong, B.B. Lowell, N. Uchida, Neuron-type-specific signals
for reward and punishment in the ventral tegmental area, Nature 482 (2012) 85–88.
[37] J.A. Ihalainen, P. Riekkinen Jr., M.G. Feenstra, Comparison of dopamine and nor-
adrenaline release in mouse prefrontal cortex, striatum and hippocampus using
microdialysis, Neurosci. Lett. 277 (1999) 71–74.
ments for stimulant abuse, Adv. Pharmacol. 69 (2014) 129–176.
8] H.H. Sitte, M. Freissmuth, Amphetamines, new psychoactive drugs and the mono-
amine transporter cycle, Trends Pharmacol. Sci. 36 (2015) 41–50.
9] G. Tanda, A.H. Newman, J.L. Katz, Discovery of drugs to treat cocaine dependence:
behavioral and neurochemical effects of atypical dopamine transport inhibitors,
Adv. Pharmacol. 57 (2009) 253–289.
[
10] T. Hiranita, D.S. Wilkinson, W.C. Hong, M.F. Zou, T.A. Kopajtic, P.L. Soto, et al., 2-
isoxazol-3-phenyltropane derivatives of cocaine: molecular and atypical system
effects at the dopamine transporter, J. Pharmacol. Exp. Therap. 349 (2014)
2
97–309.
[38] D. De Bundel, T. Femenia, C.M. DuPont, A. Konradsson-Geuken, K. Feltmann,
B. Schilstrom, et al., Hippocampal and prefrontal dopamine D1/5 receptor in-
volvement in the memory-enhancing effect of reboxetine, Int. J.
[
[
[
11] H.Y. Zhang, G.H. Bi, H.J. Yang, Y. He, G. Xue, J. Cao, et al., The novel Modafinil
Analog, JJC8-016, as a potential cocaine abuse pharmacotherapeutic,
Neuropsychopharmacology 42 (2017) 1871–1883.
Neuropsychopharmacol. 16 (2013) 2041–2051.
12] M.E. Reith, B.E. Blough, W.C. Hong, K.T. Jones, K.C. Schmitt, M.H. Baumann, et al.,
Behavioral, biological, and chemical perspectives on atypical agents targeting the
dopamine transporter, Drug Alcohol. Depend. 147 (2015) 1–19.
13] S.M. Li, T.A. Kopajtic, M.J. O’Callaghan, G.E. Agoston, J. Cao, A.H. Newman, et al.,
N-substituted benztropine analogs: selective dopamine transporter ligands with a
fast onset of action and minimal cocaine-like behavioral effects, J. Pharmacol. Exp.
Therap. 336 (2011) 575–585.
14] M. Darwish, M. Kirby, E.T. Hellriegel, P. Robertson Jr., Armodafinil and modafinil
have substantially different pharmacokinetic profiles despite having the same
terminal half-lives: analysis of data from three randomized, single-dose, pharma-
cokinetic studies, Clin. Drug Invest. 29 (2009) 613–623.
[39] H. Gurden, M. Takita, T.M. Jay, Essential role of D1 but not D2 receptors in the
NMDA receptor-dependent long-term potentiation at hippocampal-prefrontal cortex
synapses in vivo, J. Neurosci. 20 (2000) RC106.
[40] N. Lemon, D. Manahan-Vaughan, Dopamine D1/D5 receptors gate the acquisition
of novel information through hippocampal long-term potentiation and long-term
depression, J. Neurosci. 26 (2006) 7723–7729.
[41] S.J. Martin, P.D. Grimwood, R.G. Morris, Synaptic plasticity and memory: an eva-
luation of the hypothesis, Ann. Rev. Neurosci. 23 (2000) 649–711.
[42] G. Brackman, M. Risseeuw, S. Celen, P. Cos, L. Maes, H.J. Nelis, et al., Synthesis and
evaluation of the quorum sensing inhibitory effect of substituted triazolyldihy-
drofuranones, Bioorg. Med. Chem. 20 (2012) 4737–4743.
[
[
15] R.I. Desai, T.A. Kopajtic, M. Koffarnus, A.H. Newman, J.L. Katz, Identification of a
dopamine transporter ligand that blocks the stimulant effects of cocaine, J.
Neurosci. 25 (2005) 1889–1893.
16] S.V. Mahler, M. Hensley-Simon, P. Tahsili-Fahadan, R.T. LaLumiere, C. Thomas,
R.V. Fallon, et al., Modafinil attenuates reinstatement of cocaine seeking: role for
cystine-glutamate exchange and metabotropic glutamate receptors, Addict. Biol. 19
[43] K. Saha, J.S. Partilla, K.R. Lehner, A. Seddik, T. Stockner, M. Holy, et al., ‘Second-
generation’ mephedrone analogs, 4-MEC and 4-MePPP, differentially affect mono-
amine transporter function, Neuropsychopharmacology 40 (2015) 1321–1331.
[44] T. Beuming, J. Kniazeff, M.L. Bergmann, L. Shi, L. Gracia, K. Raniszewska, et al.,
The binding sites for cocaine and dopamine in the dopamine transporter overlap,
Nat. Neurosci. 11 (2008) 780–789.
[
(
2014) 49–60.
[45] O. Trott, A.J. Olson, AutoDock vina: improving the speed and accuracy of docking
with a new scoring function, efficient optimization, and multithreading, J.
Computational Chemistry. 31 (2010) 455–461.
[46] G. Wolber, T. Langer, LigandScout: 3-D pharmacophores derived from protein-
bound ligands and their use as virtual screening filters, J. Chem. Inform. Model. 45
(2005) 160–169.
[47] S. Sucic, S. Dallinger, B. Zdrazil, R. Weissensteiner, T.N. Jorgensen, M. Holy, et al.,
The N terminus of monoamine transporters is a lever required for the action of
amphetamines, J. Biol. Chem. 285 (2010) 10924–10938.
[48] C. Silwedel, C. Forster, Differential susceptibility of cerebral and cerebellar murine
brain microvascular endothelial cells to loss of barrier properties in response to
inflammatory stimuli, J. Neuroimmunol. 179 (2006) 37–45.
[
[
[
17] K.C. Schmitt, R.B. Rothman, M.E. Reith, Nonclassical pharmacology of the dopa-
mine transporter: atypical inhibitors, allosteric modulators, and partial substrates,
J. Pharmacol. Exp. Therap. 346 (2013) 2–10.
18] J.D. Urban, W.P. Clarke, M. von Zastrow, D.E. Nichols, B. Kobilka, H. Weinstein,
et al., Functional selectivity and classical concepts of quantitative pharmacology, J.
Pharmacol. Exp. Therap. 320 (2007) 1–13.
19] N.J. Wesensten, G. Belenky, M.A. Kautz, D.R. Thorne, R.M. Reichardt, T.J. Balkin,
Maintaining alertness and performance during sleep deprivation: Modafinil versus
caffeine, Psychopharmacology 159 (2002) 238–247.
20] M.J. Minzenberg, C.S. Carter, Modafinil: a review of neurochemical actions and
effects on cognition, Neuropsychopharmacology 33 (2008) 1477–1502.
21] R.M. Battleday, A.K. Brem, Modafinil for cognitive neuroenhancement in healthy
non-sleep-deprived subjects: a systematic review, Eur. Neuropsychopharmacol.: J.
Eur. Coll. Neuropsychopharmacol. 25 (2015) 1865–1881.
[
[
[49] W. Neuhaus, F. Gaiser, A. Mahringer, J. Franz, C. Riethmuller, C. Forster, The pi-
votal role of astrocytes in an in vitro stroke model of the blood-brain barrier, Front.
Cell. Neurosci. 8 (2014) 352.
[
[
[
22] S.M. Wang, C. Han, S.J. Lee, T.Y. Jun, A.A. Patkar, P.S. Masand, et al., Modafinil for
the treatment of attention-deficit/hyperactivity disorder: a meta-analysis, J.
Psychiatric Res. 84 (2017) 292–300.
[50] I. Novakova, E.A. Subileau, S. Toegel, D. Gruber, B. Lachmann, E. Urban, et al.,
Transport rankings of non-steroidal antiinflammatory drugs across blood-brain
barrier in vitro models, PLoS One 9 (2014) e86806.
[51] S. Subramaniyan, V. Hajali, T. Scherf, S.J. Sase, F.J. Sialana, M. Groger, et al.,
Hippocampal receptor complexes paralleling LTP reinforcement in the spatial
memory holeboard test in the rat, Behav. Brain Res. 283 (2015) 162–174.
[52] B. Shanmugasundaram, Y.D. Aher, J. Aradska, M. Ilic, D. Daba Feyissa, P. Kalaba,
et al., R-modafinil exerts weak effects on spatial memory acquisition and dentate
gyrus synaptic plasticity, PLoS One 12 (2017) e0179675.
23] L. Scoriels, P.B. Jones, B.J. Sahakian, Modafinil effects on cognition and emotion in
schizophrenia and its neurochemical modulation in the brain, Neuropharmacology.
6
4 (2013) 168–184.
24] N.D. Volkow, J.S. Fowler, J. Logan, D. Alexoff, W. Zhu, F. Telang, et al., Effects of
modafinil on dopamine and dopamine transporters in the male human brain:
clinical implications, JAMA 301 (2009) 1148–1154.
[
25] P. Gerrard, R. Malcolm, Mechanisms of modafinil: a review of current research,
Neuropsychiatr. Dis. Treat. 3 (2007) 349–364.
[53] K.W. Li, Neuroproteomics, Humana Press/Springer, New York, 2011.
[54] R. Weitzdoerfer, H. Hoeger, E. Engidawork, M. Engelmann, N. Singewald, G. Lubec,
et al., Neuronal nitric oxide synthase knock-out mice show impaired cognitive
performance, Nitric Oxide : Biol. Chem. 10 (2004) 130–140.
[55] D.D. Feyissa, Y.D. Aher, E. Engidawork, H. Hoger, G. Lubec, V. Korz, Individual
differences in male rats in a behavioral test battery: a multivariate statistical ap-
proach, Front. Behav. Neurosci. 11 (2017) 26.
[56] R.D. Porsolt, G. Anton, N. Blavet, M. Jalfre, Behavioural despair in rats: a new
model sensitive to antidepressant treatments, Eur. J. Pharmacol. 47 (1978)
379–391.
[
26] M.J. Minzenberg, A.J. Watrous, J.H. Yoon, S. Ursu, C.S. Carter, Modafinil shifts
human locus coeruleus to low-tonic, high-phasic activity during functional MRI,
Science 322 (2008) 1700–1702.
[27] B.K. Madras, Z. Xie, Z. Lin, A. Jassen, H. Panas, L. Lynch, et al., Modafinil occupies
dopamine and norepinephrine transporters in vivo and modulates the transporters
and trace amine activity in vitro, J. Pharmacol. Exp. Therap. 319 (2006) 561–569.
[28] C.J. Loland, M. Mereu, O.M. Okunola, J. Cao, T.E. Prisinzano, S. Mazier, et al., R-
Modafinil (armodafinil): a unique dopamine uptake inhibitor and potential medi-
cation for psychostimulant abuse, Biol. Psychiatry 72 (2012) 405–413.
[29] Y. Karabacak, S. Sase, Y.D. Aher, A. Sase, S.R. Saroja, A. Cicvaric, et al., The effect of
modafinil on the rat dopamine transporter and dopamine receptors D1-D3 paral-
leling cognitive enhancement in the radial arm maze, Front. Behav. Neurosci. 9
[57] C. Welinder, L. Ekblad, Coomassie staining as loading control in Western blot
analysis, J. Proteome Res. 10 (2011) 1416–1419.
[58] M.H. Baumann, J.S. Partilla, K.R. Lehner, E.B. Thorndike, A.F. Hoffman, M. Holy,
et al., Powerful cocaine-like actions of 3,4-methylenedioxypyrovalerone (MDPV), a
93

M. Kristofova et al.
Behavioural Brain Research 343 (2018) 83–94
principal constituent of psychoactive’ bath salts’ products,
Neuropharmacology 109 (2016) 223–235.
Neuropsychopharmacology 38 (2013) 552–562.
[78] J.L. Hanslick, K. Lau, K.K. Noguchi, J.W. Olney, C.F. Zorumski, S. Mennerick, et al.,
Dimethyl sulfoxide (DMSO) produces widespread apoptosis in the developing
central nervous system, Neurobiol. Dis. 34 (2009) 1–10.
[79] M. Cavas, D. Beltran, J.F. Navarro, Behavioural effects of dimethyl sulfoxide
(DMSO): changes in sleep architecture in rats, Toxicol. Lett. 157 (2005) 221–232.
[80] R.P. Kesner, I. Lee, P. Gilbert, A behavioral assessment of hippocampal function
based on a subregional analysis, Rev. Neurosci. 15 (2004) 333–351.
[81] D.R. Vago, A. Bevan, R.P. Kesner, The role of the direct perforant path input to the
CA1 subregion of the dorsal hippocampus in memory retention and retrieval,
Hippocampus 17 (2007) 977–987.
[
59] P. Scholze, J. Zwach, A. Kattinger, C. Pifl, E.A. Singer, H.H. Sitte, Transporter-
mediated release: a superfusion study on human embryonic kidney cells stably
expressing the human serotonin transporter, J. Pharmacol. Exp. Therap. 293 (2000)
8
70–878.
[
60] L.G. Ferreira, R.N. Dos Santos, G. Oliva, A.D. Andricopulo, Molecular docking and
structure-based drug design strategies, Molecules 20 (2015) 13384–13421.
61] H. Bisgaard, M.A. Larsen, S. Mazier, T. Beuming, A.H. Newman, H. Weinstein, et al.,
The binding sites for benztropines and dopamine in the dopamine transporter
overlap, Neuropharmacology 60 (2011) 182–190.
[
[
62] A.S. Faqi, A Comprehensive Guide to Toxicology in Nonclinical Drug Development,
[82] A. Kulla, D. Manahan-Vaughan, Depotentiation in the dentate gyrus of freely
moving rats is modulated by D1/D5 dopamine receptors, Cereb. Cortex. 10 (2000)
614–620.
[83] S. Sajikumar, J.U. Frey, Late-associativity, synaptic tagging, and the role of dopa-
mine during LTP and LTD, Neurobiol. Learn. Mem. 82 (2004) 12–25.
[84] D. Jenson, K. Yang, A. Acevedo-Rodriguez, A. Levine, J.I. Broussard, J. Tang, et al.,
Dopamine and norepinephrine receptors participate in methylphenidate enhance-
ment of in vivo hippocampal synaptic plasticity, Neuropharmacology 90 (2015)
23–32.
[85] J.I. Broussard, K. Yang, A.T. Levine, T. Tsetsenis, D. Jenson, F. Cao, et al., Dopamine
regulates aversive contextual learning and associated in vivo synaptic plasticity in
the hippocampus, Cell. Rep. 14 (2016) 1930–1939.
[86] J.I. Rossato, L.R. Bevilaqua, I. Izquierdo, J.H. Medina, M. Cammarota, Dopamine
controls persistence of long-term memory storage, Science 325 (2009) 1017–1020.
[87] N. Hansen, D. Manahan-Vaughan, Dopamine D1/D5 receptors mediate informa-
tional saliency that promotes persistent hippocampal long-term plasticity, Cereb.
Cortex 24 (2014) 845–858.
2
nd ed.
[
63] C.J. Loland, R.I. Desai, M.F. Zou, J. Cao, P. Grundt, K. Gerstbrein, et al.,
Relationship between conformational changes in the dopamine transporter and
cocaine-like subjective effects of uptake inhibitors, Mol. Pharmacol. 73 (2008)
8
13–823.
[
64] K.C. Schmitt, J. Zhen, P. Kharkar, M. Mishra, N. Chen, A.K. Dutta, et al., Interaction
of cocaine-, benztropine-, and GBR12909-like compounds with wild-type and mu-
tant human dopamine transporters: molecular features that differentially determine
antagonist-binding properties, J. Neurochem. 107 (2008) 928–940.
65] K.C. Schmitt, M.E. Reith, The atypical stimulant and nootropic modafinil interacts
with the dopamine transporter in a different manner than classical cocaine-like
inhibitors, PLoS One 6 (2011) e25790.
[
[
66] C. Velazquez-Sanchez, A. Ferragud, J. Murga, M. Carda, J.J. Canales, The high af-
finity dopamine uptake inhibitor, JHW 007, blocks cocaine-induced reward, loco-
motor stimulation and sensitization, Eur. Neuropsychopharmacol.: J. Eur. Coll.
Neuropsychopharmacol. 20 (2010) 501–508.
[
67] P. Tahsili-Fahadan, R. Malcolm, G. Aston-Jones, Modafinil: an anti-relapse medi-
cation, Neuropsychopharmacology 35 (2010) 343–344.
[88] W.C. da Silva, C.C. Kohler, A. Radiske, M. Cammarota, D1/D5 dopamine receptors
modulate spatial memory formation, Neurobiol. Learn. Mem. 97 (2012) 271–275.
[89] A.S. Undieh, Pharmacology of signaling induced by dopamine D(1)-like receptor
activation, Pharmacol. Therap. 128 (2010) 37–60.
[90] C.M. O’Carroll, S.J. Martin, J. Sandin, B. Frenguelli, R.G. Morris, Dopaminergic
modulation of the persistence of one-trial hippocampus-dependent memory, Learn.
Mem. 13 (2006) 760–769.
[
68] H.Y. Zhang, G.H. Bi, H.J. Yang, Y. He, G. Xue, J. Cao, et al., The novel modafinil
analog, JJC8-016, as a potential cocaine abuse pharmacotherapeutic,
Neuropsychopharmacology (2017).
[
69] A.L. Anderson, M.S. Reid, S.H. Li, T. Holmes, L. Shemanski, A. Slee, et al., Modafinil
for the treatment of cocaine dependence, Drug Alcohol. Depend. 104 (2009)
1
33–139.
[91] B.D. Richardson, K. Saha, D. Krout, E. Cabrera, B. Felts, L.K. Henry, et al.,
Membrane potential shapes regulation of dopamine transporter trafficking at the
plasma membrane, Nat. Commun. 7 (2016) 10423.
[92] W.C. Hong, S.G. Amara, Differential targeting of the dopamine transporter to re-
cycling or degradative pathways during amphetamine- or PKC-regulated en-
docytosis in dopamine neurons, FASEB J. 27 (2013) 2995–3007.
[
70] H.M. Murphy, D. Ekstrand, M. Tarchick, C.H. Wideman, Modafinil as a cognitive
enhancer of spatial working memory in rats, Physiol. Behav. 142 (2015) 126–130.
71] M. Bezu, B. Shanmugasundaram, G. Lubec, V. Korz, Repeated application of
Modafinil and Levodopa reveals a drug-independent precise timing of spatial
working memory modulation, Behav. Brain Res. 312 (2016) 9–13.
[
[
[
[
[
72] H. Burgos, A. Castillo, O. Flores, G. Puentes, C. Morgan, A. Gatica, et al., Effect of
modafinil on learning performance and neocortical long-term potentiation in rats,
Brain Res. Bull. 83 (2010) 238–244.
73] T. Shuman, S.C. Wood, S.G. Anagnostaras, Modafinil and memory: effects of
modafinil on Morris water maze learning and Pavlovian fear conditioning, Behav.
Neurosci. 123 (2009) 257–266.
[93] D.C. Mash, J. Pablo, Q. Ouyang, W.L. Hearn, S. Izenwasser, Dopamine transport
function is elevated in cocaine users, J. Neurochem. 81 (2002) 292–300.
[94] R.A. Vaughan, J.D. Foster, Mechanisms of dopamine transporter regulation in
normal and disease states, Trends Pharmacol. Sci. 34 (2013) 489–496.
[95] J.A. Moron, I. Zakharova, J.V. Ferrer, G.A. Merrill, B. Hope, E.M. Lafer, et al.,
Mitogen-activated protein kinase regulates dopamine transporter surface expression
and dopamine transport capacity, J. Neurosci. 23 (2003) 8480–8488.
[96] E.A. Bolan, B. Kivell, V. Jaligam, M. Oz, L.D. Jayanthi, Y. Han, et al., D2 receptors
regulate dopamine transporter function via an extracellular signal-regulated kinases
1 and 2-dependent and phosphoinositide 3 kinase-independent mechanism, Mol.
Pharmacol. 71 (2007) 1222–1232.
[97] B.K. Gorentla, A.E. Moritz, J.D. Foster, R.A. Vaughan, Proline-directed phosphor-
ylation of the dopamine transporter N-terminal domain, Biochemistry 48 (2009)
1067–1076.
[98] S.M. Meiergerd, T.A. Patterson, J.O. Schenk, D2 receptors may modulate the
function of the striatal transporter for dopamine: kinetic evidence from studies in
vitro and in vivo, J. Neurochem. 61 (1993) 764–767.
74] E. Whiting, H.J. Chenery, J. Chalk, R. Darnell, D.A. Copland, The explicit learning
of new names for known objects is improved by dexamphetamine, Brain Lang. 104
(2008) 254–261.
75] R. Haseneder, L. Starker, J. Berkmann, K. Kellermann, B. Jungwirth, M. Blobner,
et al., Sevoflurane anesthesia improves cognitive performance in mice, but does not
influence in vitro long-term potentation in hippocampus CA1 stratum radiatum,
PLoS One 8 (2013) e64732.
[
76] J.W. Young, M.A. Geyer, Action of modafinil–increased motivation via the dopa-
mine transporter inhibition and D1 receptors? Biol. Psychiatry 67 (2010) 784–787.
77] M. Oemisch, K. Johnston, M. Pare, Methylphenidate does not enhance visual
working memory but benefits motivation in macaque monkeys,
[
94