A novel heterocyclic compound targeting the dopamine transporter improves performance in the radial arm maze and modulates dopamine receptors D1-D3

Article abstract of DOI:10.1016/j.bbr.2016.06.011

A series of compounds targeting the dopamine transporter (DAT) haS been shown to improve memory performance most probably by re-uptake inhibition. Although specific DAT inhibitors are available, there is limited information about specificity, mechanism an

Full text of DOI:10.1016/j.bbr.2016.06.011

Behavioural Brain Research 312 (2016) 127–137
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Behavioural Brain Research
journal homepage: www.elsevier.com/locate/bbr
Research report
A novel heterocyclic compound targeting the dopamine transporter
improves performance in the radial arm maze and modulates
dopamine receptors D1-D3
a,f,1
, Yogesh D. Ahera,b,e,1, Predrag Kalaba , Nilima Y. Aher ,
b,1
b
Sivaprakasam R. Saroja
b
b
a
a
b
Martin Zehl , Volker Korz , Saraswathi Subramaniyan , Andras G. Miklosi , Lisa Zanon ,
b,c,d
e
b
b
b
Winfried Neuhaus
Gert Lubec
, Harald Höger , Thierry Langer , Ernst Urban , Johann Leban ,
b,∗
a
Department of Pediatrics, Medical University of Vienna, 1090 Vienna, Austria
b
c
d
e
f
Department of Pharmaceutical Chemistry, University of Vienna, 1090 Vienna, Austria
Department of Anaesthesia and Critical Care, University Hospital Wuerzburg, 97080 Wuerzburg, Germany
Institute of Medical Genetics, Medical University of Vienna, 1090 Vienna, Austria
Core Unit of Biomedical Research, Division of Laboratory Animal Science and Genetics, Medical University of Vienna, A-2325 Himberg, Austria
Department of Cell Biology, Duke University School of Medicine, NC 27710, USA
h i g h l i g h t s
•
•
•
•
CE-111 specifically targets DAT and inhibits dopamine reuptake.
CE-111 improves spatial memory performance in radial arm maze.
Blood-brain barrier in vitro model shows CE-111 permeates similar to modafinil.
CE-111 modulates pre- and extra- synaptic but not postsynaptic dopamine receptors.
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 27 May 2016
Received in revised form 6 June 2016
Accepted 7 June 2016
Available online 8 June 2016
A series of compounds targeting the dopamine transporter (DAT) haS been shown to improve memory
performance most probably by re-uptake inhibition. Although specific DAT inhibitors are available, there
is limited information about specificity, mechanism and in particular the effect on dopamine receptors.
It was therefore the aim of the study to test the DAT inhibitor 4-(diphenyl-methanesulfinylmethyl)-
2
-methyl-thiazole (code: CE-111), synthetized in our laboratory for the specificity to target DAT, for
the effects upon spatial memory and for induced dopamine receptor modulation. Re-uptake inhibition
was tested for DAT (IC50 = 3.2 ␮M), serotonin transporter, SERT (IC50 = 272291 ␮M) and noradrenaline
transporter, NET (IC50 = 174 ␮M). Spatial memory was studied in the radial arm maze (RAM) in male
Sprague-Dawley rats that were intraperitoneally injected with CE-111 (1 or 10 mg/kg body weight). Per-
formance in the RAM was improved using 1 and 10 mg/kg body weight of CE-111. Training and treatment
effects on presynaptic, postsynaptic and extrasynaptic D1 and D2- receptors and dopamine receptor con-
taining complexes as well as on activated DAT were observed. CE-111 was crossing the blood-brain barrier
comparable to modafinil and was identified as effective to improve memory performance in the RAM.
Dopamine re-uptake inhibition along with modulations in dopamine receptors are proposed as potential
underlying mechanisms.
Keywords:
Hippocampus
Presynaptic
Extrasynaptic
Dopamine receptor
Radial arm maze
Dopamine reuptake inhibitor
©
2016 Published by Elsevier B.V.
1. Introduction
Both D1-like and D2-like dopamine receptors play important
roles in learning and memory, functions of the prefrontal cortex
and hippocampus [1–3]. Dopamine autoreceptors that are located
presynaptically regulate firing rate and neurotransmitter synthe-
sis or release in response to extracellular dopamine levels [2].
∗ Corresponding author at: University of Vienna, Department of Pharmaceutical
Chemistry, Althanstrasse 14, 1090 Vienna, Austria.
E-mail address: gert.lubec@univie.ac.at (G. Lubec).
1
These authors contributed equally to this work.
http://dx.doi.org/10.1016/j.bbr.2016.06.011
0
166-4328/© 2016 Published by Elsevier B.V.

1
28
S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
Presynaptic transduction and expression of D1R/D5R facilitates
long-term potentiation (LTP) at the CA1 region of the hippocampus
to subicular burst-spiking cell synapses through activation of the
cAMP-PKA cascade [4]. Activation of D2-like autoreceptors (presy-
naptic receptors) leads to a decrease in midbrain dopamine neuron
firing, dopamine synthesis and dopamine release, reduced locomo-
tor activity and enhanced motivation for food-seeking behavior,
whereas activation of postsynaptic D2-like receptors increases
to address the specific effects of memory training/CE-treatments
on the hippocampal dopaminergic system.
2. Materials and methods
2.1. Synthesis of CE-111
(
4-(benzhydrylsulfinylmethyl)-2-methyl-thiazole)
2.1.1. Synthesis of benzhydryl carbamimidothioate
Diphenylmethanol (13 g, 0.07 mol) and thiourea (6.5 g,
locomotor activity [5,6]. The presynaptic D2R plays an impor-
tant role in hippocampal long-term synaptic plasticity and spatial
learning [7]. D1R and D2R regulate synthesis and degradation of
the dopamine transporter (DAT) which removes dopamine from
the synaptic cleft to terminate dopamine-mediated signalling [8].
However, kinases like PKA or PKC phosphorylate DAT, that is either
internalized to cease the transport or operate in reverse; as such,
the phosphorylated DAT may release dopamine into the synapse
0
.085 mol) are added in 0.5 l flask and 32.5 ml of water was
◦
added. The mixture is then heated to 95 C (an emulsion is
obtained) and 26 g of 48% HBr (0.322 mol, 4.6 equivalents) is then
gradually added during 0.5 h. The mixture is heated under reflux
◦ ◦
106–107 C) for 0.5 h and cooled to 80–85 C. The mixture is then
(
cooled in ice and precipitated crystals formed. After filtration and
washing with water, a colorless crystalline substance is obtained.
The product is then dried in the high vacuum. About 9.62 g of the
product was obtained as a white crystalline solid (yield: 74%).
9]. The phosphorylation of DAT at the Thr53 site is important
[
for amphetamine- induced DAT-mediated dopamine release [10].
Inhibition of DAT has been shown to enhance LTP in the hippocam-
2.1.2. Synthesis of 4-(benzhydrylthiomethyl)-2-methyl-thiazole
4
-Chloromethyl-2-methyl-thiazole
hydrochloride
was
pal CA1- region mediated by the D3R [11]. In general, dopamine
levels at the synaptic cleft or at the extrasynaptic region deter-
mine activity and function of dopamine receptors. Low dopamine
concentrations (<500 nM) have been shown to enhance inhibitory
postsynaptic currents (IPSCs) via D1 receptors whereas at high
concentrations IPSCs are decreased via activation of D2R [12].
GABAergic inhibitory currents in CA1 pyramidal neurons are inhib-
ited by activation of D3R and a D3-selective agonist, PD128907
enhances LTP in the CA1 stratum radiatum but not in stratum oriens
obtained from Acros Organics (New Jersey, USA; Geel, Belgium).
In a round bottom flask 3.51 g (10.86 mmol) of benzhydryl car-
bamimidothioate is dissolved in 75 ml of methanol. Afterwards, 2 g
(
10.86 mmol) of 4-chloromethyl-2-methyl-1,3-thiazole and 7.5 g (5
equivalents) of potassium carbonate are added to the mixture. The
mixture is left to stir overnight at room temperature. Methanol
is evaporated and water is added. The solution is then extracted
3x) with 100 ml of ethylacetate. Organic phases are collected, com-
bined, dried with Na SO and then filtered. Ethylacetate is removed
by rotary evaporation and then semi-solid product is obtained.
Crude product is purified via the flash column chromatography on
silica gel. Methanol of 5% in dichloromethane is used as the mobile
phase. About 2.6 g of the crude solid product is obtained (yield:
9.07%).
H NMR (200 MHz, DMSO-d ): 2.63 (3H, s, CH ), 3.62 (2H, s,
(
2
4
[
13].
Some psychostimulants inhibit DAT-mediated dopamine reup-
take to elevate extracellular dopamine levels. As such, increased
dopamine levels are associated with cognitive improvements [14].
Selective dopamine reuptake inhibitors such as modafinil and
the benztropine analog AHN 2-005 improve both, hippocampal-
and prefrontal cortex- dependent cognitive functions [15–18].
In search for highly selective DAT inhibitors, we synthesized a
heterocyclic compound, CE-111 and tested its blocking efficacy
in HEK293-expressing DAT, NET or SERT. The effect of CE-111
5
6
3
CH ), 5.33 (1H, s, CH), 7.15 (1H, s, CHthiazole), 7.21–7.46 (10H, m,
2
CHaromatic).
2
.1.3. Synthesis of
4-(benzhydrylsulfinylmethyl)-2-methyl-thiazole (CE-111)
on memory performance was assessed in a radial arm maze
In
a
round bottom flask, 1.44 g (4.62 mmol) of 4-
(
RAM). Hippocampal dopamine receptors/transporter complexes
(benzhydrylthiomethyl)-2-methylthiazole is dissolved in 10 ml
of glacial acetic acid (174.82 mmol). 0.55 ml (4.85 mmol) of 30%
hydrogen peroxide is dropped into the solution and stirred for
12 h. Acid is neutralized with 5% sodium bicarbonate and ice.
were analysed by blue-native electrophoresis after CE-111 treat-
ments in RAM training. Moreover, presynaptic, postsynaptic and
extrasynaptic dopamine receptors/transporter were also studied

S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
129
Fig. 1. (a) Structure of CE-111 (4-(diphenyl-methanesulfinylmethyl)-2-methyl-thiazole). (b) Evaluation of CE-111 activity on HEK293 cells stably expressing human isoforms
of DAT, SERT and NET. It specifically inhibited DAT-mediated dopamine uptake (n = 3). (c) Substrate-efflux assay was performed to examine whether CE-111 has any ability
to release the neurotransmitter from inside the cells, like amphetamine does. Figure depicts that CE-111 is not behaving as an amphetamine like compound. (d) Behavioural
experiments on RAM show that both 10mg and 1mg groups improved memory performance on 8th and 9th day, respectively (F (9, 269) = 4.660, P < 0.0001). The data was
analyzed using repeated measurements ANOVA with Bonferroni post hoc test. **P < 0.01, ***P < 0.001. Values are expressed as mean ± s.e.m, n = 8–10.
Aqueous mixture is extracted (3x) with 50 ml of ethyl acetate.
plemented with 5 mM d-glucose). To determine unspecific uptake
in HEK-DAT and HEK-NET 10 ␮M of mazindole were used while
10 ␮M of paroxetine were used for HEK-SERT. The tritiated sub-
strates used for HEK-DAT, HEK-SERT and HEK-NET were 0.2 ␮M
Organic phases are collected, combined, dried with Na SO4 then
2
filtered and ethyl acetate is concentrated on rotary evaporator.
Semi solid product is purified via flash column chromatography
on silica gel. 5% methanol in dichloromethane is used as a mobile
phase. Product is dried in a high vacuum. By this procedure 1.12 g
of the final pure product was obtained (yield: 73.98%) (Fig. 1a and
Supplementary Fig. 1).
3
3
3
+
H-Dop, 0.4 ␮M H-5HT and 0.05 ␮M H MPP , respectively. Cells
were washed once with KHB buffer and incubated with compounds
either 5 min for HEK-DAT and HEK-SERT cells or 8 min for HEK-NET
cells. Subsequently, substrates were added and the reactions were
stopped with ice-cold KHB buffer after either 1 min for HEK-DAT
and HEK-SERT cells or 3 min for HEK-NET cells. Cells were lysed
with 1% SDS and released radioactivity was measured by a liquid
scintillation counter (Tri-carb-2300TR, Perkin Elmer).
+
+
MS. [M+H ] = 328.0824 (MW is 327.08), [M+Na ] = 350.0644.
H NMR (200 MHz, DMSO-d ): 2.65 (3H, s, CH ), 3.73–3.99 (2H,
6
3
dd, CH ), 5.43 (1H, s, CH), 7.32–7.58 (11H, aromatic CH).
2
The substrate/efflux experiments were performed as described
before [21]. Briefly, HEK-DAT cells were grown in 5 mm diameter
2
.2. Uptake and release assays
3
+
PDL-coated coverslips. Cells were incubated with 0.1 ␮M H MPP
Dulbecco’s modified Eagle’s medium (DMEM) and trypsin were
◦
at 37 C for 20 min. The coverslips were transferred onto superfu-
sion chambers (0.2 ml) and excess radioactivity was washed out
purchased from PAA Laboratories GmbH (Pasching, Austria). Fetal
calf serum was purchased from Invitrogen. [3H]5-HT ([3H]5-
hydroxytryptamine; [3H]serotonin; 28.3 ␮Ci/mmol) and [3H]DA
◦
with KHB buffer for 40 min (0.7 ml/min) at 25 C to obtain stable
baselines. The experiment was started with the collection of frac-
tions (2 min). After collection of first three baseline fractions, either
CE-111 or D-Amphetamine was added as depicted in Fig. 1C. Finally,
the remaining radioactivity was collected by treating with 1% SDS.
(
[3H]dihydroxyphenylethylamine, [3H]dopamine; 46 ␮Ci/mmol)
were purchased from Perkin Elmer, Boston, MA. [3H]1-Methyl-
-phenylpyridinium ([3H]MPP+; 85 ␮Ci/mmol) was supplied by
4
American Radiolabeled Chemicals (St. Louis, MO). Paroxetine was
purchased from Santa Cruz Biotechnology, USA, while mazindole
and D-amphetamine were purchased from Sigma–Aldrich Co.
For uptake experiments, the human isoforms of DAT, SERT and
NET were expressed in HEK293 (HEK-DAT, HEK-SERT and HEK-
NET) cells. CE-111-mediated monoamine transporter effects on
substrate uptake were analysed as described previously [19,20].
In brief, cells were grown in poly-d-lysine (PDL) coated 96-well
plates. CE-111 was dissolved in DMSO and subsequently diluted
in Krebs–Ringer–HEPES buffer (KHB; 25 mM HEPES.NaOH, pH 7.4,
2.3. Transport studies across blood-brain barrier in vitro model
The used Transwell model was based on mouse cell line
cerebEND which was a kind gift from Prof. Carola Förster [22].
CerebEND cells form a tight barrier similar to primary mouse
brain endothelial cells and have been characterized as blood-
brain barrier in vitro model in a comprehensive manner previously
[22,23]. CerebEND cells were cultivated on collagen IV coated Tran-
swell inserts (1 ␮m pore size, BDL353103, Becton & Dickenson)
1
20 mM NaCl, 5 mM KCl, 1.2 mM CaCl , and 1.2 mM MgSO4 sup-
2

1
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S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
as described in Neuhaus et al. [23]. On day 13 after cell seed-
ing transport studies were accomplished according to Novakova
et al. [24]. In brief, 12-well plates prefilled with DMEM (Dulbecco’s
modified Eagle medium) and substance solutions were prewarmed
first. Arms were baited with a small pellet only once, 3 cm from the
end. Re-entry into a baited arm was counted as a working mem-
ory error (WME), whereas any entry into an un-baited arm was
recorded as a reference memory error (RME). Untrained animals
(yoked) were also treated same as trained animals in terms of han-
dling, habituation and food restriction and exposed to the maze for
the same amount of time as their trained counterparts but without
food rewards in the arms. Thus, they do not learn and form any
memory. The training sessions were recorded with a computerized
tracking video camcorder: 1/3 SSAM HR EX VIEW HAD. Six hours
after the l of the tenth training session, the animals were deeply
◦
at 37 C. After medium exchange to pure DMEM, cell layers were
◦
equilibrated at 37 C for 30 min. The transport study started after
addition of compound solutions to the apical compartment, and
inserts were transferred into a new, prewarmed and DMEM filled
◦
well in the incubator at 37 C after five, 15, 30 and 60 min. Mea-
surement of transendothelial electrical resistance (TEER) before
and after the studies confirmed cell layer integrity throughout
the total experiments. Stock solutions of Diazepam, CE-111 and
Modafinil were 100 mM in DMSO, tested compound concentra-
tion was 100 ␮M in DMEM (test solution). Diazepam was added
to CE-111 or Modafinil in DMEM as internal standard to account
for cell layer’s variability. Basolateral samples, apical solution after
the transport study and test solutions were collected and prepared
for following HPLC analysis by precipitation with acetonitril in a
anaesthetized with CO and killed by neck dislocation to study the
2
protein synthesis-dependent dopamine receptors and transporter.
Brain tissues were quickly removed and hippocampi were rapidly
◦
◦
dissected on a cold plate set at 4–6 C and stored at −80 C.
2.5. Sample preparation
◦
ratio of 1:1 at 4 C for at least 30 min. After centrifugation super-
Hippocampal total membrane fraction was prepared as
natants were subjected to HPLC analysis which was accomplished
described previously [25]. Briefly, homogenized hippocampi in
ice-cold homogenization buffer [10 mM HEPES, pH 7.5, 300 mM
sucrose, one complete protease inhibitor tablet (Roche Molecular
Biochemicals) per 50 ml] were centrifuged for 10 min at 1000g and
the nuclei pellet was discarded. The supernatant was centrifuged at
as recently published [24] applying 50:50 CH CN:potassium phos-
3
phate buffer (10 mM, pH = 3) as elution buffer (retention time for
Modafinil: 3.9 min; CE-111 8.7 min; Diazepam: 10.5 min) and UV-
detection at 220/254 nm. Peak areas were used to calculate cleared
volume versus (vs.) time curves and permeability coefficients fol-
lowing the clearance principle according to Novakova et al. [24].
PKall accords to the permeability coefficient in ␮m/min across the
cell layer and the membrane support. For PKcell values [␮m/min]
the permeability across blank inserts without cells was subtracted
resulting in the permeability of the compounds only across the cell
layer.
5
0, 000g for 30 min. The resulting pellet was resuspended in wash
buffer (homogenization buffer without sucrose), incubated on ice
for 30 min and centrifuged at 50, 000g for 30 min to obtain a mem-
brane fraction. Membrane protein was extracted with extraction
buffer (1.5 M 6-aminocaproic acid, 300 mM Bis–Tris, pH 7.0, 1% n-
Dodecyl b-d-maltoside) and the protein content was measured by
using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL,
USA).
2.4. Radial arm maze (RAM)
Presynaptic and postsynaptic membrane proteins were pre-
pared as described in [26] with some modifications. All the steps
Twelve to fourteen weeks old male Sprague Dawley rats (n = 72)
◦
were carried out at 4 C unless it is indicated. Hippocampi were
were used in all experiments. All procedures were carried out
according to the guidelines of the European Communities Council
Directive of 24 November 1986 (86/609/EEC) and the Ethics com-
mittee, Medical University of Vienna, and were approved by Federal
Ministry of Education, Science and Culture, Austria (BMWFW-
homogenized (0.5 mM CaCl , 10 mM MgCl , 320 mM sucrose and
2
2
protease inhibitor) and brought to a final sucrose concentration of
.25 M. The homogenate containing 1.25 M of sucrose was over-
1
laid by 1 M sucrose and centrifuged at 100, 000g for 3 h. Interfaced
synaptosomal fraction was collected, mixed with 0.1 mM CaCl and
2
6
6.009/0114-WF/II/3b/2014). All efforts were made to minimize
centrifuged at 15, 000g for 30 min to obtain a synaptosomal pellet.
The pellet was resuspended with 20 mM Tris buffer pH-6 contain-
animal suffering and to reduce the number of animals used. The
maintenance of the animals, details about the apparatus and the
performance of the 12 arms containing RAM experiment was
described previously [19,20].
ing 0.25 mM CaCl and 1% Triton X-100, mixed/incubated for 30 min
2
and then centrifuged at 50, 000g for 30 min. The supernatant con-
taining the extrasynaptic membrane proteins was collected and the
pellet was treated with 20 mM Tris buffer pH-8 containing 0.25 mM
CaCl2 and 1% Triton X-100 and after mixed/incubated for 1 h, the
mixture was centrifuged at 50, 000g for 30 min. The supernatant
containing presynaptic proteins was collected; the pellet contain-
ing PSD proteins was extracted with 0.25 mM tris buffer pH-7.4
containing 3% of SDS. Presynaptic and extrasynaptic proteins were
In brief; rats were handled for 30 min/day for 5 days to adapt to
the experimenter and to the surrounding conditions. The amount
of food was restricted prior to the actual experiment to reduce the
body weight to ∼80–85% for motivation. The food restriction was
maintained during handling and subsequent training days. Water
was provided ad libitum during the handling, habituation and train-
ing. Twelve arms RAM was used to train the animals and access the
memory formation. Two days before the training period, rats were
allowed to explore the maze (habituation) for 5 min and eat the food
pellets which were scattered all over the maze. During the 10 train-
ing days (1 trial/day), eight arms were baited with food to assess
working memory and four remained un-baited to access reference
memory. The pattern of baited and un-baited arms was consistent
throughout the training for each rat but differed among rats. CE-111
was freshly dissolved in 100% DMSO and injected intraperitoneally
in doses of 1 and 10 mg/kg body weight, 1 ml/kg everyday 30 min
before the start of the training sessions. Pure DMSO was used as
vehicle.
◦
precipitated overnight with 3 fold of acetone at −20 C. Precipi-
tated proteins were solubilized with 0.25 mM Tris buffer (pH-7.4)
containing 3% of SDS.
2.6. Electrophoresis and western blotting
Blue-native electrophoresis and immunoblotting were
closely performed as described before [25]. Specific peptide
sequences were used to produce D1R (-TSTMDEAGLPAERD-
),
D2R (-NWSRPFNGSEGKAD-), D3R (-LRHPSLEGGAGMPS-),
DAT (-TNSTLINPPQTPVEAQERETW-) and DATph (-
TNSTLINPPQpTPVEAQERETW-) antibodies from GenScript.
Antibodies were tested in SDS-PAGE and BN-PAGE (Supplementary
Figs. 2 and 3).
Each trial began by placing the rat in the central platform, after
0 s the cylinder was slowly lifted. The session lasted for 8 min or
until all the baited arms (n = 8) were entered-whatever occurred
1

S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
131
Fig. 2. Permeability of CE-111 (a) and modafinil (b) across the 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 PKall [␮m/min] revealed almost no difference between the transport of CE-111 and modafinil. Subtraction of the blank values led to permeability coefficient PKcell
[␮m/min] values reflecting the permeability of the compounds only across the cell layer. Normalization to internal control diazepam showed almost no difference between
the permeability of CE-111 and modafinil. The data are presented as mean ± SD, n = 3–5.
2.7. Immunohistochemistry
3. Results
Rats were anesthetized and transcardially perfused with PBS
3.1. CE-111 specifically blocked dopamine uptake and is not a
substrate of DAT
and then with 4% paraformaldehyde. Excised brains were post-
fixed with 4% paraformaldehyde for 1 h and 100 ␮m thick sections
were made by using Vibratome. Finely powdered DIL dye was ran-
domly placed on hippocampal sub regions followed by overnight
incubation in PBS at the room temperature. Following 30 min of
The effect of CE-111 on monoamine transporters, DAT-, NET-
3
3
and SERT- mediated respective substrate uptake ( H-DA, H-5HT
3
and H MPP+) was assessed using HEK293 cells which expressed
blocking with 10% donkey serum, either D1R or D2R antibody
the corresponding transporters. CE-111 strongly blocks DAT-
mediated substrate uptake (IC50 = 3.25 ± 0.23SD ␮M) while weakly
blocks NET (IC50 = 174 ± 15.5SD ␮M) and negligible effects on SERT
(IC50 = 272291 ± 3453SD ␮M) (Fig. 1b). We then sought whether
CE-111 specifically blocks DAT or it also behaves like a substrates
of DAT to promote DAT-mediated release to increase the extracel-
lular dopamine levels. We therefore performed a release assay to
determine if CE-111 acts as a substrate to induce release of triti-
ated MPP+ from HEK-DAT cells. CE-111 treatment did not induce
DAT-mediated release while D-Amphetamine as a positive control
did (Fig. 1c). The observations clearly indicate that CE-111 specif-
ically blocks DAT without acting as a substrate of DAT to increase
the extracellular dopamine levels.
◦
(
1:200) was added and incubated for 24 h at 4 C. Tissue was then
washed with PBS for 30 min and incubated with secondary anti-
body (anti-rabbit IgG Alexa Fluor 647, 1:1000; Cell Signaling) for
1
h. After washing with PBS for 1hr, tissue was counterstained with
DAPI (3 ␮g/ml). Then again the tissue was briefly washed with PBS
and the tissue slide was mounted (DAKO; fluorescence mounting
medium). The tissue was imaged with Zeiss LSM 700 using 63X
oil immersion objective at 1 Air Unit and the 3D-image was con-
structed by using imageJ.
2.8. Statistics
3.2. CE-111 permeated across the blood-brain barrier in vitro
similar to modafinil
Non-linear regression analysis was carried out to determine
the IC50 values of CE-111 on DAT, NET and SERT. For behavioural
data analysis, a repeated measurement of two way ANOVA with
the factors, CE-111 and the time were used. A pairwise multiple
comparison was done using the Bonferroni post hoc test. The den-
sitometry values of western blot quantification were compared
using two-way ANOVA with Bonferroni post hoc test for multiple
comparisons to reveal the differences among groups. The proba-
bility level of p < 0.05 was considered as statistically significant.
Statistics was done using GraphPad Prism 6 (version 6.00, GraphPad
Software, San Diego CA, USA).
Before conducting the in vivo experiments, the ability of CE-111
to cross the blood-brain barrier was assessed in vitro. Transport
studies across cerebEND cell layers revealed that CE-111 permeated
in a similar manner compared to modafinil (Fig. 2). Normaliza-
tion of permeability data to internal control diazepam resulted
in ratios of 0.79 ± 0.07 for CE-111 and 0.73 ± 0.10 for Modafinil,
respectively. Thus, data suggested significant blood-brain barrier
permeability of CE-111 since modafinil is known as a well perme-
ating compound. Based on these findings following in vivo studies
were accomplished.

1
32
S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
Fig. 3. BN-PAGE western blot of 10 mg/kg body weight group. (a) Hippocampal membrane proteins (native) were separated by blue-native electrophoresis and immunoblotted
with indicatedantibodies(n = 8–10). Molecularweightrangewas between720 kDand 480 kD. (b–f) Quantifiedresultsof D1R(F(1, 28) = 24.87, P < 0.0001), D2R(F(1, 29) = 18.76,
P = 0.0002), D3R (F (1, 27) = 44.33, P < 0.0001), DAT (F (1, 29) = 2.229, P = 0.1462) and DATph (DAT Thr ) (F (1, 31) = 6.709, P = 0.0145). The data was analyzed by two-way ANOVA
with Bonferroni post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Values are expressed as mean ± s.e.m, n = 8–10.
53
3
.3. CE-111 improved memory performance
images of dopamine receptors and transporters from 10 mg/kg
group. As seen in Fig. 3b, the D1R complex levels were reduced
in CE-111 trained and vehicle yoked groups compared to vehicle
trained group. Furthermore, they were also reduced in vehi-
cle yoked compared to CE-111 yoked group (F (1, 28) = 24.87,
P < 0.0001). Whereas, the D2R complex was reduced in CE-111
yoked group compared to CE-111 trained and vehicle yoked groups
and in vehicle trained group compared to CE-111 trained group
(F (1, 29) = 18.76, P = 0.0002) (Fig. 3c). The D3R complex lev-
els were reduced in CE-111 trained and vehicle yoked groups
compared to vehicle trained group while it was significantly
increased in CE-111 yoked group compared to vehicle yoked
group (F (1, 27) = 44.33, P < 0.0001) (Fig. 3d). No significant changes
were observed in DAT-containing complexes (F (1, 29) = 2.229,
Memory performance of rats from the 1 mg, 10 mg and con-
trol groups was progressively improved over the training days. The
memory index (MI) was calculated by dividing the number of first
entries into the baited arms by total entries into the baited arms. In
the control group, memory index was gradually increased until 6th
day and remained stable during the further training days; however,
continuous improvement was observed in 1 mg and 10 mg CE-111
treated groups. Thus, the significant memory improvement at the
8
th and 9th day was respectively observed in the 10 mg and 1 mg
group compared to the control group (F (9, 269) = 4.660, P < 0.0001)
Fig. 1d).
(
ph
53
P = 0.1462) (Fig. 3e). However, DAT (Thr )-containing complexes
were significantly reduced in CE-111 yoked, CE-111 trained and
vehicle trained groups compared to vehicle yoked group (F (1,
3.4. Memory training and CE-111 treatments modulated
dopamine receptors and transporter complexes
3
1) = 6.709, P = 0.0145) (Fig. 3f). Like 10 mg/kg group, significant
changes of dopamine receptors or transporter were observed in the
mg/kg body weight CE-111 treated group (Supplementary Fig. 5).
Hippocampal dopamine receptors and transporter complexes
were separated by BN-PAGE and subsequently immunoblotted
with specified antibodies as mentioned earlier. The Coomassie R-
1
3
50 stained PVDF membrane was used as the loading control [27]
(
Supplementary Fig. 4). Fig. 3a shows blue native-western blot

S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
133
Fig. 4. Presynaptic and postsynaptic dopaminergic system of 1 mg/kg body weight group. (a) Postsynaptic marker PSD-95 was enriched in postsynaptic fraction, while
presynaptic marker synaptophysin was enriched in presynaptic fraction, shows the quality of our separation methods. (b) SDS-PAGE western blotting of presynaptic and
postsynaptic fraction with indicated antibodies. (c–g) Quantified results of D1R (F (1, 30) = 4.372, P = 0.0451) and D2 R (F (1, 30) = 14.29, P = 0.0007) from presynaptic fraction
and D1R (F (1, 32) = 1.081, P = 0.3064), D2R (F (1, 31) = 0.4490, P = 0.5078) and D3R (F (1, 31) = 0.02589, P = 0.8732) from postsynaptic fraction. The data was analyzed by two
way-ANOVA with Bonferroni post hoc test. *P < 0.05, ***P < 0.001. Values are expressed as mean ± s.e.m, n = 8–9.
3
.5. CE-111 modulated presynaptic and extrasynaptic dopamine
111 trained and vehicle yoked groups (F (1, 30) = 14.29, P = 0.0007)
(Fig. 4d). Surprisingly, no significant changes in D1R, D2R and D3R
complex levels were observed in the PSD fractions (D1R (F (1,
32) = 1.081, P = 0.3064), D2R (F (1, 31) = 0.4490, P = 0.5078) and D3R
(F (1, 31) = 0.02589, P = 0.8732)) (Fig. 4e–g). However, the extrasy-
naptic D1R levels were significantly increased in CE-111 trained
group compared to CE-111 yoked and vehicle trained groups (F
(1, 28) = 24.86, P < 0.0001) (Fig. 5b). The D2R complex levels were
significantly increased in CE-111 trained group when compared to
CE-111 yoked group (F (1, 31) = 9.794, P = 0.0038) (Fig. 5c). Although
the extrasynaptic DAT was comparable between the groups (F
(1, 31) = 1.884, P = 0.1798), DAT Thr53 was significantly reduced in
vehicle yoked group compared to CE-111 yoked and vehicle trained
groups (F (1, 32) = 5.559, P = 0.0247) (Fig. 5d and e).
receptors and transporter
We then sought to study the presynaptic, postsynaptic and
extrasynaptic dopamine receptors as well as transporter, as it is
unknown how their distribution is affected either during the mem-
ory formation or in treatment with the psychostimulants. The
presynaptic, postsynaptic and extrasynaptic proteins were pre-
pared as described in Section 2. The postsynaptic marker, PSD-95
and the presynaptic marker, synaptophysin, respectively observed
at PSD and presynaptic fractions demonstrate the efficiency of sam-
ple preparation method (Fig. 4a). Western blot images from 1 mg/kg
are shown in Fig. 4b. The presynaptic D1R complex levels were sig-
nificantly decreased from CE-111 yoked to CE-111 trained group (F
(
1, 30) = 4.372, P = 0.0451) (Fig. 4c). The presynaptic D2R complex
levels were significantly reduced in CE-111 yoked compared to CE-

1
34
S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
Fig. 5. Extrasynaptic dopaminergic system of 1 mg/kg body weight group. (a) SDS-PAGE western blotting of extrasynaptic fraction with indicated antibodies. (b–e) Quantified
results of D1R (F (1, 28) = 24.86, P < 0.0001), D2R (F (1, 31) = 9.794, P = 0.0038), DAT (F (1, 31) = 1.884, P = 0.1798) and DATph (DAT Thr ) (F (1, 32) = 5.559, P = 0.0247). The data
was analyzed by two-way ANOVA with Bonferroni post hoc test. **P < 0.05, **P < 0.01, ****P < 0.0001. Values are expressed as mean ± s.e.m, n = 8–9.
53
3.6. Distribution of D1R and D2R
only at the 10 mg/kg dose, but not at the 1 mg/kg concentration
16], we observed a continuous memory improvement using CE-
[
In order to elucidate the distribution of D1R and D2R on the
111 during the last three days of training at both, 10 mg/kg and
1 mg/kg dosages. Moreover, modafinil is effective at a comparable
dose of approximately 1–3 mg/kg body weight in medical use but
it is less specifically acting at the dopamine transporter.
hippocampal pyramidal cells, we stained neurons with DIL dye
and co-localized the dopamine receptors. D1R is mainly expressed
around the soma but also expressed on dendritic spines and axonal
segments of CA1 pyramidal cells (Fig. 6a). Like D1R, D2Rs are
expressed both, around the soma and dendritic spines (Fig. 6b).
This observation suggests that both D1R and D2R may have dif-
ferent functional properties on neuronal plasticity based on their
cellular distribution.
4
.2. Effect of CE-111 on D1R-, D2R-, D3R-, DAT- and DATph
containing complexes
-
The D1R are exclusively excitatory, while D2R are inhibitory,
and D3R either inhibitory or excitatory. D1R is important for hip-
pocampal spatial long-term memory formation [31,32], while mice
lacking D2R and D3R showed impaired spatial working memory
[33]. D1R complex levels were reduced in the vehicle yoked group
compared to the CE-111 yoked group suggesting an influence of
CE-111 on D1R-containing complexes, which were abundant in
the vehicle trained group compared to both vehicle yoked groups
and the CE-111 trained group. However, the level of D1R-complex
was similar between CE-111 yoked and CE-111 trained groups.
This result suggests that CE-111 has the same effect on D1R in
trained and control groups while D1R was differentially modulated
during training in vehicle groups. Interestingly, we observed com-
pletely opposite effects on D2R-containing complexes. The D2R
complex level was reduced in CE-111 yoked group compared to
vehicle yoked group, but increased in CE-111 trained group com-
pared to vehicle trained group. It suggests that CE-111 modulates
D2R during memory training while control groups had no effect
on D2R. Though D1R and D2R belong to different subfamilies of
4
. Discussion
4.1. CE-111 is a specific DAT inhibitor and improves memory
performance
Psychostimulants can induce diverse behavioral effects and
might lead to either cognitive improvements or deficits and behav-
ioral adverse effects [28]. These effects are usually determined by
the dosages or target specificity of the compound; higher doses
or multiple targets usually exerts adverse side effects [29]. Cocaine
inhibits NET, DAT and SERT, as an example [30]. Here, CE-111 selec-
tively inhibited DAT-mediated rather than NET- or SERT-mediated
uptake suggesting that it might serve as a potential specific psy-
chostimulant. Prior to in vivo studies, in vitro transport across the
blood-brain barrier in vitro model showed that CE-111 permeated
similar such as modafinil. In contrast to the benztropine analog,
AHN 2-005 that induced improvement in cognitive performance

S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
135
Fig. 6. Distribution of hippocampal D1R and D2R. (a) Immunohistochemistry with D1R and DIL labelling. White spots on the soma and dendritic spines are co-localized
points. (b) Upper panel shows D2R around the soma with DAPI in blue. Lower panel with DIL and D2R are shown white co-localized points.
dopamine receptors, either co-expression or co-activation of D1R-
D2R elevates intracellular calcium levels, suggesting that D1R and
D2R could also show similar functional properties [34]. Unlike to
either D1R or D2R, both, training and CE-treatments differentially
modulated D3R-containing complexes. D3R levels were higher in
the CE-111 yoked group compared to the vehicle yoked group and
the CE-111 trained group. This suggests that CE-111 facilitates the
expression of plasma membrane-associated D3R levels, but the
memory training reduces to the level of yoked control. On the
other hand, D3R levels were abundant in the vehicle trained group
compared to CE-111 trained and vehicle yoked groups, suggest-
ing that training without CE-111 promotes more D3R-contaning
complexes on the plasma membrane. Taken together, CE-111 and
memory training modulate D1R-, D2R- and D3R-contaning com-
plexes, but in a different manner. Interestingly, the total level of
thesis and release. D2R knockout mice (D2KO) showed impaired
hippocampus-dependent LTP, LTD and spatial memory. However,
DATcre
when the presynaptic D2R (D2
) was selectively deleted, the
hippocampal LTD was impaired and LTP remained intact in the CA1,
whereas spatial memory was equally impaired as in D2KO mice [7].
These studies suggest that presynaptic D2R but not postsynaptic
D2R plays a vital role in hippocampus-dependent spatial memory
and LTD which is as well associated with spatial memory consol-
idation and novelty detection [37,38]. In addition, the stimulation
of inhibitory D2R lowers extracellular dopamine levels in striatum
and prefrontal cortex [39]. Here, the presynaptic D2R was signifi-
cantly reduced in the CE-111 yoked group compared to the vehicle
yoked group. Furthermore, in the CE-111 trained group D2R lev-
els were comparable with those of the vehicle trained group, but
significantly higher when compared to the CE-111 yoked group.
Interestingly, the same pattern of increased D2R was observed from
presynaptic fraction and native protein studies (total membrane
fraction). Herein, these results strongly support the importance of
our studies on the presynaptic dopaminergic system.
Dopamine reduces the hippocampal and the entorhinal exci-
tatory input to subicular neurons by decreasing the glutamate
release following activation of the presynaptic D1R [40]. It
also inhibits NMDAR-mediated excitatory postsynaptic potentials
through presynaptic D1R [41]. The postsynaptic D1R physically
interacts with the GluN1 subunit of the NMDAR to avoid ligand-
induced internalization in order to facilitate both, NMDAR and
D1R-mediated signalling [42]. These statements are in line with
the view that NMDAR-mediated synaptic plasticity is respectively
facilitated and inhibited by postsynaptic D1R and presynaptic
D1R. Our results indicate that presynaptic D1R was significantly
decreased in the CE-111 trained group compared to the CE-111
vehicle group; thus our results suggests that CE-111 may prob-
ably facilitate NMDAR-mediated signalling to enhance memory
formation. It is noteworthy to mention here that the extrasynap-
tic D1R might be from extrasynaptic regions of both presynapse
and postsynapse. On the whole, we speculate that the presence
of CE-111 might have exerted a modulatory action of pre- and
ph
DAT-containing complexes was not altered while DAT (Thr53)
was increased in the vehicle yoked group compared to the other
three groups. It suggests that phosphorylation at threonine 53
is an important target site to regulate either trafficking or re-
uptake/efflux property of DAT during drug treatments and memory
formation.
4.3. CE-111 and presynaptic and extrasynaptic dopamine
receptors
GABAergic neurons in globus pallidus express presynaptic
D2R which inhibits GABA release. The presynaptic D2R synapse
becomes super-sensitized after dopamine loss. These supersensi-
tive D2Rs may compensate for the loss of dopamine in Parkinson’s
disease to induce a strong disinhibition of globus pallidus neu-
ron activity that may contribute to the motor-stimulating effects
[
35]. Dopamine release facilitates motor functions through the
activation of postsynaptic D2R. However, presynaptic D2R is acti-
vated or recruited at high levels of dopamine release through
a feedback mechanism in order to suppress the motor func-
tions [36]. In other words, postsynaptic D2R activates multiple
signalling pathways while presynaptic D2R controls dopamine syn-

1
36
S.R. Saroja et al. / Behavioural Brain Research 312 (2016) 127–137
extrasynaptic D1R; whereas it has no effect on postsynaptic D1R
as the postsynaptic D1R remained comparable between groups.
Future studies should address the functional role of presynaptic
and postsynaptic D1R in memory formation and in the treatment
of psychostimulants in more detail. Similar to the postsynaptic
D1R and D2R levels, the postsynaptic D3R levels are not different
between groups. Because we could not detect D3R in presynap-
tic and extrasynaptic fractions, we conclude that D3R is mainly
expressed in hippocampal postsynaptic regions. The activation
of hippocampal postsynaptic D3R reduces GABAergic transmis-
sion in the CA1 region by inducing PKA-dependent endocytosis of
GABA receptors [13]. Cocaine-induced synaptic plasticity of mes-
encephalic dopaminergic neurons is mediated by presynaptic D3R
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[
[
[
[
[
43].
Both DAT and DAT Thr53 were mainly detected in the extrasy-
naptic and not in presynaptic fraction, proposing that DAT is
primarily accumulated at the extrasynaptic sites. Similar to DAT-
containing complexes, the extrasynaptic DAT levels were also
comparable between the groups. However, DAT Thr53 was sig-
nificantly reduced in the vehicle yoked group compared to the
remaining groups. Even though the functional properties of DAT
[
[
Thr are not known yet, an in vitro study proposed that DAT Thr53
site plays an important role in DAT-mediated reuptake [10]. Our
IHC studies showed the presence of D1R and D2R at the somatic
sites and dendritic spines, showing that dopaminergic neurons
target both spines and soma. However, the physiological and func-
tional roles of dendritic and somatic dopaminergic receptors are
unknown. Future studies should address its distinguished proper-
ties during drug treatments, learning and memory.
53
(
2013) 321–328.
Herein, we propose that the novel compound CE-111 specifi-
cally inhibits DAT-mediated dopamine uptake as well as improves
memory performance of rats in the RAM. At the molecular level, the
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Conflict of interest
The authors declare that there is no conflict of interest
Acknowledgements
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We are indebted to Prof. Harald H. Sitte for allowing us to use
the space and instruments in his lab, and Marion Holy for her guid-
ance to perform the uptake inhibition assays. Furthermore, we are
very grateful to Prof. Carola Förster from the University Hospital
Wuerzburg (Germany) for providing us the cerebEND cell line.
10924–10938.
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