Download The dopamine reuptake inhibitor MRZ

International Journal of Neuropsychopharmacology (2014), 17, 2045–2056.
doi:10.1017/S1461145714000996
© CINP 2014
ARTICLE
The dopamine reuptake inhibitor MRZ-9547
increases progressive ratio responding in rats
S. Sommer1, W. Danysz2, H. Russ2, B. Valastro2, G. Flik3 and W. Hauber1
1
Institute of Biology, Department of Animal Physiology, University of Stuttgart, D-70550 Stuttgart, Germany
Merz Pharmaceuticals GmbH, D-60318, Frankfurt am Main, Germany
3
Brains On-Line B.V., De Mudden 16, 9747 AW Groningen, The Netherlands
2
Abstract
Received 11 March 2014; Reviewed 17 April 2014; Revised 15 May 2014; Accepted 20 May 2014;
First published online 25 June 2014
Key words: Dopamine transporter inhibition, Fatigue, Progressive ratio responding, Rat.
Introduction
Motivational dysfunctions related to effort expenditure
such as fatigue, apathy, anergia and psychomotor slowing are common symptoms in neuropsychiatric and
neurological disorders, e.g. major depression, schizophrenia, multiple sclerosis or inflammatory disease
(Friedman et al., 2011; Salamone and Correa, 2012). For
instance, in a test of effort-related choice, patients with
major depressive disorders were less willing to expend
effort for rewards than controls (Treadway et al., 2012).
Fatigue and anergia are also present in Parkinson’s disease and may be even more deleterious to quality of
life than motor dysfunction (Friedman et al., 2011). Yet,
the available treatment options rarely cause significant
improvements in fatigue in neuropsychiatric and neurological disorders (Harrington, 2012; Kluger et al., 2013).
There is evidence that tests of effort-related decision
Address for correspondence: Dr W. Hauber, Universität Stuttgart,
Biologisches Institut, Abteilung Tierphysiologie, Pfaffenwaldring 57,
D-70550 Stuttgart, Germany.
Tel.: ++49 711 685 65003 Fax: ++49 711 685 55003
Email: [email protected]
making in rodents could be useful for modeling some
motivational dysfunctions in humans and identifying potential drugs for treating effort-related symptoms
(Salamone and Correa, 2012). For instance, in a progressive ratio (PR)/chow feeding concurrent choice task in
which animals can lever press for preferred food pellets
under a PR schedule or approach and consume freely
available but less preferred lab chow, a selective adenosine A2A receptor antagonist inclined intact rats to select
the increasingly more effortful type of food-seeking behavior (Randall et al., 2012). Moreover, blockade of dopamine (DA) transmission shifted effort-based choice
behavior towards the low-cost response option, an effect
that can be reversed by co-administration of a selective
adenosine A2A receptor antagonist (Randall et al.,
2012). In view of these findings, adenosine A2A receptor
antagonists have been suggested to be potentially useful
for treating energy-related symptoms such as fatigue
(Farrar et al., 2007).
Here, we tested the effects of MRZ-9547 (d-(2(2-Oxo-4(R)-phenylpyrrolidin-1-yl)-acetamide), and its
l-enantiomer MRZ-9546 on effort-related decision making
in rats. The racemic form of these compounds referred
to as phenotropil has been shown to stimulate motor
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Drugs that are able to shift effort-related decision making in intact rats towards high-effort response options
are largely unknown. Here, we examined the effects of two candidate drugs, MRZ-9547 and its l-enantiomer
MRZ-9546 on progressive ratio (PR) responding using two different tasks, a standard PR task that involves
increasing ratio requirements and a PR/chow feeding choice task in which animals can lever press for preferred
food pellets under a PR schedule or approach freely available less preferred lab chow. Furthermore, we assessed
the mechanisms of action of both drugs using in vitro-assay methods and in vivo-microdialysis. Results reveal that
MRZ-9547 is a selective dopamine transporter (DAT) inhibitor that moderately stimulated striatal dopamine
release. MRZ-9546 was a much less potent DAT inhibitor. Furthermore, MRZ-9547 dose dependently increased
the tendency to work for food reinforcement both in the standard PR task and the PR/chow feeding choice task,
MRZ-9546 was considerably less active. Relative to MRZ-9547, other DAT-interfering drugs had only moderate
(methylphenidate) or marginal (modafinil, d-amphetamine) stimulant effects on PR responding in either task.
Collectively, our data demonstrate that the DAT inhibitor MRZ-9547 can markedly stimulate PR responding
and shift effort-related decision making in intact rats towards high-effort response options. An analysis of
effort-related decision making in rodents could provide an animal model for motivational dysfunctions related
to effort expenditure such as fatigue, e.g. in Parkinson’s disease or major depression. Our findings suggest
that DAT inhibitors such as MRZ-9547 could be potentially useful for treating energy-related symptoms in
neurological or neuropsychiatric disorders.
2046 S. Sommer et al.
Methods
All animal experiments were conducted according to the
German law of animal protection and approved by the
proper authorities.
MRZ-9546 and MRZ-9547: binding experiments
For binding experiments, human recombinant dopamine
transporter (DAT) was expressed in CHO-K1 cells. Cell
membranes were incubated with [125I] RTI-55 (0.15 nM)
for 3 h at 4 °C in 50 mM Tris-HCl, 100 mM NaCl, 1 μM leupeptin, 10 μM PMSF pH 7.4. Maximum binding was 0.047
pmol/mg protein. Non-specific binding was determined
with 10 μM nomifensine. For functional assays, human
recombinant DAT was expressed in CHO-K1 cells. Cells
were incubated with [3H]-DA for 10 min at 25 °C in 5
mM Tris-HCl, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl,
1.2 mM CaCl2, 1.2 mM MgSO4, 5 mM D-glucose, 1 mM
ascorbic acid, pH 7.1. DA taken up was determined
by scintillation counting. MRZ-9546 and MRZ-9547
were dissolved in 0.4% DMSO.
MRZ-9547 and MRZ-9546: brain extracellular
concentrations
Animals
Male Sprague–Dawley rats (Elevage-Janvier, France)
weighing 250–300 g were kept up to four per cage
(55 × 39 × 27 cm, Tecniplast, Italy) in a room with controlled temperature (21 ± 1 °C), humidity (60 ± 3%) under
a 12:12 h light–dark cycle (lights on at 06:00 hours).
Standard laboratory food (Altromin, Germany) and
water were available ad libitum. At least 6 d after arrival
they were subjected to the stereotaxic surgery. After surgery, animals were housed individually in cages (Ehret,
Germany, 26 × 18 × 42 cm) with the grid lid elevated by
a 5 cm frame.
Surgery
Using standard stereotaxic procedures, siliconized guide
cannula (MAB 6/14) (MAB, Sweden) were implanted
unilaterally in anesthetized animals aiming the caudateputamen (CPu)(AP: +0.1 mm, L: +2.6 or −2.6 mm, DV:
−3.2 mm relative to bregma) with the incisor bar set
3.3 mm under the interaural line according to the atlas
of (Paxinos and Watson, 1986). For induction of anesthesia 50 mg/kg pentobarbital i.p. was used followed by
1 ml/kg Metacam™ 10% 1.0 ml. Each rat was given at
least 2 d to recover from surgery before starting microdialysis experiments using standard microdialysis techniques and analytical procedures (see Supplementary
Methods and Materials).
Drugs
MRZ-9547 (Merz Pharmaceuticals, Germany) was dissolved fresh in distilled water for each test day and administered at a volume up to 4 ml/kg i.p. (10, 50, 100 mg/kg,
n = 4–5 per dose).
Data analysis
Pharmacokinetic variables (e.g. area under curve) given
were calculated using Excel add on ‘PK-Solver’ (Zhang
et al., 2010).
MRZ-9547: striatal DA and DOPAC release
Animals
Male Sprague–Dawley rats weighing about 300 g (Harlan,
The Netherlands) were housed in plastic cages (60 × 40 ×
20 cm; max. 5 rats per cage) for 7 d until surgery.
Thereafter, animals were housed individually in cages
(30 × 30 × 40 cm), food and water were available ad libitum
in a room with controlled temperature (20–24 °C), humidity (45–70%) under a 12:12 h light–dark cycle (lights
on at 07:00 hours). Post-surgery recovery was at least 48 h.
Surgery
Rats were anesthetized using isoflurane (2%, 800 ml/min
O2). Bupivacain/epinephrine were used for local anesthesia. Flunixin (1 ml/kg, s.c.) was used to induce pre-/
peri-operative analgesia. The animals were placed
in a stereotaxic frame (Kopf instruments, USA), and
I-shaped probes (Hospal AN 69 membrane, 3 mm exposed surface; Brainlink, The Netherlands) were inserted
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activity in rats (Zvejniece et al., 2011) and enhance physical capacity and cognition in humans (Malykh and
Sadaie, 2010). We examined the effects of MRZ-9547
and MRZ-9546 on PR responding using two different
tasks, a standard PR task that involves increasing ratio
requirements per reinforcer until an animal ceases lever
pressing and a PR/chow feeding choice task in which animals can lever press for preferred food pellets under a PR
schedule or approach and consume freely available but
less preferred lab chow (Schweimer and Hauber, 2005).
Furthermore, we assessed mechanisms of action of
MRZ-9547 and MRZ-9546 using in vitro-assay methods
and in vivo-microdialysis. For comparison, we included
methylphenidate, modafinil and d-amphetamine which,
in standard PR tasks, enhanced breakpoints (Poncelet
et al., 1983; Mayorga et al., 2000; Young and Geyer,
2010), a putative measure of how much effort an animal
is willing to invest to gain access to a reinforcer
(Stewart, 1975). However, it is yet unknown in which
ways these drugs influence effort-related choice behavior.
We hypothesize that MRZ-9547 and its l-enantiomer as
well as methylphenidate, modafinil and d-amphetamine
are not only able to invigorate PR responding in the
standard PR task but also to increase selection of the higheffort over the low-effort response option in the PR/chow
feeding choice task.
Dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats 2047
into the CPu. Coordinates for the tips of the probes were:
posterior (AP)=+0.9 mm to bregma, lateral (L)=+3.0 mm
to midline and ventral (V)=−6.5 mm to dura (Paxinos
and Watson, 1986). Each rat was given at least 2 d to recover from surgery before starting microdialysis experiments using standard microdialysis techniques and
analytical procedures (see Supplementary Materials and
Methods). MRZ-9547 was given i.p. at 100 mg/kg.
Statistical analysis
Statistical analyses were performed on relative data
(per cent change from the baseline) using a two-way
analysis of variance (ANOVA) for repeated measurements with treatment and time as main factors followed
by a Student–Newman–Keuls post-hoc test (SigmaPlot
for Windows v. 12, SPSS Corporation, 2012).
Statistical analysis
For the PR task, breaking points (i.e. the maximum ratio
achieved), number of lever presses per minute, response
latency (i.e. the latency to enter the food magazine after
completion of a ratio) and perseverative lever presses
(i.e. extra lever presses after completion of a ratio that
had no programmed consequences) were recorded from
both PR test days with drug administration. Means across
both test days were calculated and analyzed using an
ANOVA followed by Dunnett’s post-hoc test. For the PR
choice task, breaking points and the chow intake (in g),
for the consumption test, the pellet intake was measured
and analyzed as above. All statistical computations were
carried out with Statistica™ (StatSoft®, USA) using the
level of statistical significance of p < 0.05.
Experiment 1: effects of MRZ-9547 on PR responding
Animals
Experiment 2: effects of MRZ-9546 on PR responding
Male Sprague–Dawley rats (Janvier, France) weighing between 210 and 225 g at the beginning of the experiments
were used. They were housed in groups of four animals
in transparent plastic cages (55 × 39 × 27 cm, Ferplast,
Germany) and kept under a 12:12 h light–dark cycle (lights
on at 07:00 hours). Throughout the experiments water was
available ad libitum and standard laboratory maintenance
chow (Altromin, Germany) was restricted to 15 g per animal and day to maintain them at ∼85% of their freefeeding weight. Temperature (22 ± 2 °C) and humidity
(50 ± 10%) were kept constant in the animal house.
Unless otherwise noted, the same procedures as in experiment 1 were used. Two treatment groups were included that received either vehicle (n = 10) or MRZ-9546
(100 mg/kg, n = 10). The day after completion of behavioral experiments, animals assigned to the MRZ-9546
treatment group during behavioral testing received a
final drug injection and, 120 min later, a terminal heart
function under isoflurane anesthesia. Collected blood
samples were analyzed for MRZ-9546 plasma concentrations (see Supplementary Materials and Methods).
Drugs
MRZ-9547 (Merz Pharmaceuticals, Germany) was dissolved in distilled water fresh for each test day and administered 30 min before behavioral test onset at a volume
up to 5 ml/kg i.p. Four treatment groups were included
that received either vehicle (n =10) or MRZ-9547 (25 mg/kg,
n = 10; 50 mg/kg, n = 10, 100 mg/kg, n = 10).
Behavioral procedures and apparatus
Three tests were performed in a consecutive manner on
subsequent days, a ‘standard’ PR task, a PR/chow feeding
choice task followed by a consumption test. The standard
PR task involved increasing ratio requirements per reinforcer until an animal ceased lever pressing (Schweimer
and Hauber, 2005), in the PR/chow feeding choice task
animals could lever press for preferred food pellets
under a PR schedule or approach and consume freely
available but less preferred lab chow (Schweimer and
Hauber, 2005). In the consumption test the amount of
pellets (used in the PR tasks as reward) freely available
in a dish ingested under drug/vehicle was measured
Experiment 3: effects of MRZ-9547 and modafinil
on PR responding
Unless otherwise noted, the same procedures as in
experiment 1 were used. MRZ-9547 was dissolved in distilled water. Modafinil ((2-(diphenylmethyl)sulfinyl)
acetamide) purchased from Sequoia (UK) was dissolved
in 1% w/v methylcellulose (Sigma, Germany) in 0.9%
NaCl water fresh for each test day. Drugs administered
30 min before behavioral test onset at a volume of
2 ml/kg i.p. IP injections of 1% w/v methylcellulose
(Sigma, Germany) in 0.9% NaCl water (2 ml/kg) served
as controls. Five treatment groups were included
that received either vehicle (n = 11), MRZ-9547 (50 mg/kg,
n = 11; 100 mg/kg, n = 10) or modafinil (32 mg/kg, n = 10;
64 mg/kg, n = 10). The day after completion of behavioral
experiments, animals assigned to treatment groups during behavioral testing received a final injection of
MRZ-9547 or modafinil and, 120 min later, a terminal
heart function under isoflurane anesthesia. Collected
blood samples were analyzed for MRZ-9547 and modafinil plasma concentrations (see Supplementary Materials
and Methods).
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Behavioral experiments
(for details of behavioral testing see Supplementary
Materials and Methods).
2048 S. Sommer et al.
Experiment 4: effects of MRZ-9547 and methylphenidate
on PR responding
Experiment 5: effects of MRZ-9547 and d-amphetamine
on PR responding
Unless otherwise noted, the same procedures as in experiment 1 were used. MRZ-9547 and d-amphetamine
(Sigma, Germany) was dissolved in distilled water fresh
for each test day and administered 30 min before behavioral test onset at a volume of 2 ml/kg. Injections of
distilled water served as controls. Four treatment groups
were included that received either vehicle (n = 10),
MRZ-9547 (50 mg/kg, n = 10; 100 mg/kg, n = 10) or
d-amphetamine (1 mg/kg, n = 10).
Results
MRZ-9547 and MRZ-9546: mechanisms of action
MRZ-9547 turned out to be a DAT inhibitor as shown by
displacement of binding of [125I] RTI-55 (IC50 = 4.82 ±
0.05 μM, n = 3) to human recombinant DAT expressed
in CHO-K1 cells and inhibition of DA uptake (IC50 = 14.5
± 1.6 μM, n = 2) in functional assays in the same cells. It
inhibited norepinephrine transporter (NET) with an IC50
of 182 μM (one experiment in duplicate). The potencies for
the l-enantiomer MRZ-9546 were as follows: DAT binding
(Ki = 34.8 ± 14.8 μM, n = 3), DAT function (IC50 = 65.5 ± 8.3 μM,
n = 2) and NET function (IC50 = 667 μM, one experiment performed in duplicate). (Binding to other targets, see
Supplementary Results).
MRZ-9547: striatal DA/DOPAC concentrations
MRZ-9547 (100 mg/kg i.p., n = 6) moderately, but significantly increased striatal DA concentration (Fig. 1b).
When DA reached peak levels, there was a parallel, strong
decrease in DOPAC levels which persisted even after
DA levels returned to baseline (Supplementary Fig. S1).
Experiment 1: MRZ-9547 effects on PR responding
PR task
MRZ-9547 produced a dose-dependent increase of
breaking points (Fig. 2a). In line with this description,
an ANOVA revealed a significant effect of treatment
(F(3, 36) = 19.57, p < 0.001). Post-hoc comparisons of drug
vs. vehicle treatment groups indicated a significant increase of breaking points in animals treated with
MRZ-9547 across all doses relative to vehicle controls
(p < 0.01, respectively). (For results for lever presses per
minute, perseverative lever presses and response latencies
see Supplementary Results.)
PR choice task
The breaking points for pressing the lever for preferred
pellets differed between treatment groups (F(3, 36) =
29.43, p < 0.001)(Fig. 2b). Post-hoc comparisons revealed a
significant increase of breaking points in animals treated
with MRZ-9547 across all doses relative to vehicle controls (p < 0.001, respectively). Furthermore, the intake of
freely available, less preferred lab chow differed significantly between treatment groups (F(3, 36) = 5.0, p < 0.01)
(Fig. 2c). Post-hoc comparisons showed a significant decrease of lab chow intake in animals treated with
MRZ-9547 50 mg/kg (p = 0.05) or 100 mg/kg (p < 0.01) relative to vehicle controls.
Consumption test
As shown in Fig. 2d, the amount of pellets ingested differed between treatment groups (F(3, 36) = 7.40, p < 0.001)
and post-hoc comparisons demonstrated a significant
decrease of pellet intake in animals treated with
MRZ-9547 50 and 100 mg/kg relative to vehicle controls
(p < 0.001, respectively).
Experiment 2: MRZ-9546 effects on PR responding
MRZ-9547 and MRZ-9546: brain extracellular
concentrations
After i.p. administration, MRZ-9547 (10, 50, 100 mg/kg,
n = 4–5 per dose) displayed a gradual, delayed and dosedependent increase in brain concentration (Fig. 1a).
The dose of 50 mg/kg resulted in brain concentrations
of 22 μM, i.e. slightly above affinity for DAT (see above),
PR task
MRZ-9546 (100 mg/kg) moderately enhanced breaking
points (Supplementary Fig. S2A). Accordingly, an
ANOVA revealed a significant effect of treatment
(F(1, 18) = 5.22, p < 0.05). Similarly, MRZ-9546 elicited an
increase of lever presses per minutes (F(1, 18) = 4.83,
p < 0.05) but did not alter the number of perseverative
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Unless otherwise noted, the same procedures as in experiment 1 were used. MRZ-9547 and methylphenidate
(Sigma, Germany) was dissolved in distilled water fresh
for each test day and administered 30 min before behavioral test onset at a volume of 2 ml/kg. Injections of distilled water served as controls. Four treatment groups
were included that received either vehicle (n = 11),
MRZ-9547 (50 mg/kg, n = 10; 100 mg/kg, n = 10) or methylphenidate (5 mg/kg., n = 10; 10 mg/kg, n = 10). The day after
completion of behavioral experiments, animals assigned
to treatment groups during behavioral testing received
a final injection of MRZ-9547 or methylphenidate and,
120 min later, a terminal heart function under isoflurane
anesthesia. Collected blood samples were analyzed for
MRZ-9547 and methylphenidate plasma concentrations
(see Supplementary Materials and Methods).
whereas at 10 mg/kg concentrations were below this
threshold.
Dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats 2049
(a)
10 mg/kg
t1/2
Tmax
Cmax
AUC 0-t
min
min
nmol/L
nmol/L *min
50 mg/kg
152,2
80,0
5149,0
869620,9
t1/2
Tmax
Cmax
AUC 0-t
100 mg/kg
min
234,9
min
120,0
nmol/L
21453,7
nmol/L *min 3898477,1
t1/2
Tmax
Cmax
AUC 0-t
min
205,9
min
60,0
nmol/L
52806,0
nmol/L *min 9735003,1
MRZ-9547 (10 mg/kg)
MRZ-9547 (50 mg/kg)
MRZ-9547 (100 mg/kg)
60000
Concentration (nM)
50000
40000
30000
Administration
20000
10000
0
(b)
0
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60
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*
DA levels (% of baseline)
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–60 –40 –20 0
20 40 60 80 100 120 140 160 180
240 260 280 300
Time (min)
Fig. 1. The graph shows MRZ-9547 brain extracellular fluid concentrations (a) after i.p. administration (10, 50, 100 mg/kg) and
changes in striatal dopamine (DA) concentrations (b) after i.p. administration of MRZ-9547 (100 mg/kg). AUC: area under curve;
Cmax: maximum concentration; T1/2: half-life time; Tmax: time point of maximum concentration; values are mean±S.E.M. *p < 0.05
vs. basal values, Student–Newman–Keuls test.
lever presses (F(1, 18) = 0.10, n.s.) or response latencies
(F(1, 18) = 0.09, n.s.) (data not shown).
PR choice task
The breaking points for pressing the lever for preferred
pellets were higher in animals that received MRZ-9546
(F(1, 18) = 6.81, p < 0.05)(Supplementary Fig. S2B), whereas
their intake of lab chow was moderately lower, an
effect that missed significance (F(1, 18) = 1.92, n.s.)
(Supplementary Fig. S2C).
Consumption test
As shown in Supplementary Fig. S2D, the amount of
pellets ingested was lower in animals that received
MRZ-9546 (F(1, 18) = 4.56, p < 0.05).
Plasma concentrations
The plasma concentrations of MRZ-9546 120 min after
administration are: 83.9 ± 3.1; 172.0 ± 4.6; 343.3 ± 11.6 and
673.6 ± 44.9 μM for 25; 50; 100 and 200 mg/kg respectively.
Experiment 3: MRZ-9547 and modafinil effects
on PR responding
PR task
MRZ-9547 but not modafinil increased breaking points
(Fig. 3a). In line with this description, an ANOVA showed
a significant effect of treatment (F(4, 47) = 19.27, p < 0.001).
Post-hoc comparisons revealed a significant increase of
breaking points in animals treated with MRZ-9547 50
or 100 mg/kg relative to vehicle controls (p < 0.01,
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–20
2050 S. Sommer et al.
(a)
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PR test: last ratio achieved
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Veh
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Choice test: chow ingested (g)
(d)
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Choice test: chow ingested (g)
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MRZ-9547 Modafinil
Fig. 2. Effects of MRZ-9547 (25, 50, 100 mg/kg, i.p.): (a) on
lever pressing in a progressive ratio (PR) task, (b) on lever
pressing, (c) chow consumption in a PR chow feeding
choice task and (d) on pellet intake in a consumption test.
(Means ± S.E.M, *p < 0.05; **p < 0.01, different from vehicle
controls).
Fig. 3. Effects of MRZ-9547 (50, 100 mg/kg, i.p.) and modafinil
(32, 64 mg/kg, i.p.): (a) on lever pressing in a progressive ratio
(PR) task, (b) on lever pressing, (c) chow consumption (c) in
a PR chow feeding choice task and (d) on pellet intake in a
consumption test. (Means ± S.E.M, (*)p = 0.054, *p < 0.05, **p < 0.01,
different from vehicle controls, Dunnett test).
respectively) but not in those treated with modafinil 32
or 64 mg/kg (p > 0.05, respectively). (For additional results
see Supplementary Results.)
F(4, 47) = 34.02, p < 0.0001 (Fig. 3b). Post-hoc comparisons
revealed a significant increase of breaking points in animals treated with MRZ-9547 across both doses relative
to vehicle controls (p < 0.001, respectively) but not in animals treated with modafinil (32, 64 mg/kg; p > 0.05, respectively). Furthermore, the intake of lab chow differed
significantly between treatment groups F(4, 47) = 8.42,
p < 0.001 (Fig. 3c). Accordingly, post-hoc comparisons
PR choice task
The breaking points for pressing the lever for preferred
pellets clearly differed between treatment groups
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140
(b)
Choice test: last ratio achieved
0
Choice test: last ratio achieved
(b)
Dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats 2051
Plasma levels
Experiment 4: MRZ-9547 and methylphenidate effects
on PR responding
PR task
MRZ-9547 and, to a lesser extent, methylphenidate
increased breaking points (Fig. 4a). Accordingly, an
ANOVA revealed a significant effect of treatment
(F(4, 46) = 49.40, p < 0.0001) and post-hoc comparisons
revealed a significant increase of breaking points in animals treated with MRZ-9547 50 or 100 mg/kg as well
as methylphenidate 10 mg/kg relative to vehicle controls
(p < 0.01, respectively). (For results for lever presses per
minute, perseverative lever presses and response latencies
see Supplementary Results.)
PR choice task
The breaking points for pressing the lever for pellets
differed between treatment groups (F(4, 46) = 36.05,
p < 0.0001) (Fig. 4b). Post-hoc comparisons revealed a significant increase of breaking points in animals treated
with MRZ-9547 (50, 100 mg/kg) and methylphenidate
(10 mg/kg) (p < 0.001, respectively). Furthermore, the intake of lab chow differed between treatment groups.
However, compared to other experiments, the lab chow
intake in vehicle controls was, for unknown reasons,
low and the there was only a trend for a significant
treatment effect (F(4, 46) = 2.27, p = 0.07) (Fig. 4c).
Consumption test
As shown in Fig. 4d, the amount of pellets ingested differed between treatment groups (F(4, 46) = 4.63, p < 0.01)
PR test: last ratio achieved
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Methylphenidate
Fig. 4. Effects of MRZ-9547 (50, 100 mg/kg, i.p.) and
methylphenidate (5, 10 mg/kg, i.p.): (a) on lever pressing in
a progressive ratio (PR) task, (b) on lever pressing, (c) chow
consumption in a PR chow feeding choice task and (d) on
pellet intake in a consumption test. (Means ± S.E.M, *p < 0.05,
**p < 0.01, different from vehicle controls, Dunnett test).
and post-hoc comparisons demonstrated a significant
decrease of pellet intake in animals treated with MRZ9547 50 mg/kg (p < 0.05) and 100 mg/kg (p < 0.001) as
well as methylphenidate 5 and 10 mg/kg (p < 0.05,
respectively). Plasma concentrations are given in Supplementary Results.
Downloaded from http://ijnp.oxfordjournals.org/ by guest on October 26, 2016
MRZ-9547 given at 50 mg/kg was detected in plasma at
the concentrations of 245.7 ± 21.2 and 194.6 ± 10.5 μM 30
and 120 min after treatment. For 100 mg/g corresponding
values were 329.6 ± 54.1 and 353.8 ± 28.9 μM. Modafinil at
the dose of 32 mg/kg was present in the plasma at the
concentrations of 50.6 ± 9.4 and 6.85 ± 1.45 μM for 30 and
120 min, respectively. For 64 mg/kg the respective values
were 90.06 ± 24.45 and 14.23 ± 1.14 μM.
120
20
Choice test: last ratio achieved
As shown in Fig. 3d, the amount of pellets ingested differed between treatment groups (F(4, 47) = 4.49, p < 0.01)
and post-hoc comparisons demonstrated a significant or
near significant decrease of pellet intake in animals treated with MRZ-9547 100 mg/kg (p < 0.001) and modafinil
64 mg/kg (p = 0.054).
Choice test: chow ingested (g)
Consumption test
(a) 140
Consumption test: pellets ingested (g)
showed a significant decrease of lab chow intake in
animals treated with MRZ-9547 50 mg/kg (p < 0.05) or
100 mg/kg (p < 0.01), whereas no significant differences
were detected in animals treated with modafinil
(32, 64 mg/kg; p > 0.05, respectively).
2052 S. Sommer et al.
PR choice task
Consumption test
As shown in Fig. 5d, the amount of pellets ingested differed between treatment groups (F(3, 36) = 8.67, p < 0.001)
and post-hoc comparisons demonstrated a significant decrease of pellet intake in animals treated across drug/
doses relative to vehicle control (p < 0.01, respectively).
Discussion
The main finding in the present study was that MRZ-9547
turned out to be a selective DAT inhibitor that, relative to
other DAT interfering drugs including MRZ-9546, markedly stimulated PR responding and shifted effort-related
decision making in intact rats towards high effort
response options.
MRZ-9547 and MRZ-9546: mechanisms of action
MRZ-9547 was found to be an effective DAT inhibitor,
whereas activity of MRZ-9546 was about 4-fold weaker.
Moreover, MRZ-9547 seems to be relatively selective
as other targets tested were affected only at 30-fold higher
concentrations except NET (over 10-fold difference). After
i.p. administration, plasma and brain extracellular fluid
(ECF) concentrations of MRZ-9547 displayed a dosedependent and sustained increase up to 120 min.
Notably, plasma levels of MRZ-9546 remained elevated
for 120 min after i.p. injection. Furthermore, MRZ-9547
at 100 mg/kg only moderately enhanced striatal DA levels
PR test: last ratio achieved
100
80
60
40
20
(b)
0
140
(c)
0
**
**
120
100
80
60
40
20
5
(d)
0
16
4
3
**
2
1
**
14
12
**
10
**
8
**
6
4
2
0
Veh
50
100
MRZ-9547
1
Amphetamine
Fig. 5. Effects of MRZ-9547 (50, 100 mg/kg, i.p.) and
d-amphetamine (1 mg/kg, i.p.): (a) on lever pressing in a
progressive ratio (PR) task, (b) on lever pressing, (c) chow
consumption (c) in a PR chow feeding choice task and (d) on
pellet intake in a consumption test. (Means ± S.E.M, *p < 0.05,
**p < 0.01, different from vehicle controls; in Fig. 1b:
d-amphetamine vs. vehicle control, p = 0.06; in Fig. 1c:
d-amphetamine vs. vehicle control, p = 0.08, Dunnett test).
relative to baseline (130%), an increase that is comparable to
some other DAT inhibitors such GBR12921 (Hurd and
Ungerstedt, 1989) or mazindol (Nakachi et al., 1995) but
markedly lower than the increase (400%) produced by the
DAT inhibitor methylphenidate (10 mg/kg) (Hurd and
Ungerstedt, 1989).
Downloaded from http://ijnp.oxfordjournals.org/ by guest on October 26, 2016
The breaking points for pressing the lever for pellets
differed between treatment groups (F(3, 36) = 35.98,
p < 0.0001) (Fig. 5b). Post-hoc comparisons revealed a significant increase of breaking points in animals treated with
MRZ-9547 (50, 100 mg/kg) and a trend for a significant effect in animals treated with d-amphetamine (p = 0.066).
Furthermore, the intake of lab chow differed between
treatment groups (F(3, 36) = 13.86, p < 0.001) (Fig. 5c).
Accordingly, post-hoc comparisons showed a significant
decrease of lab chow intake in animals treated with either
dose of MRZ-9547 (p < 0.01, respectively) relative to vehicle
controls, whereas there was only a trend for a significant
effect in animals treated with d-amphetamine (p = 0.08).
**
120
Choice test: last ratio achieved
MRZ-9547 but not d-amphetamine increased breaking
points (Fig. 5a). Accordingly, an ANOVA revealed a significant effect of treatment (F(3, 36) = 15.22, p < 0.0001) and
post-hoc comparisons revealed a significant increase of
breaking points in animals treated with MRZ-9547 50 or
100 mg/kg relative to vehicle controls (p < 0.01, respectively). (For additional results see Supplementary Results.)
**
140
Choice test: chow ingested (g)
PR task
(a) 160
Consumption test: pellets ingested (g)
Experiment 5: effects of MRZ-9547 and d-amphetamine
on PR responding
Dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats 2053
Drug effects in the PR task
Drug effects in the PR/chow feeding choice task
Across experiments, MRZ-9547 dose-dependently and
consistently inclined rats to select the increasingly effortful type of food-seeking behavior in presence of a low
Downloaded from http://ijnp.oxfordjournals.org/ by guest on October 26, 2016
Across experiments, MRZ-9547 consistently and dosedependently increased the last ratio achieved. Relative
to MRZ-9547, the ability of MRZ-9546 to increase the
maximal ratio was considerably less pronounced.
Consistent with these findings, MRZ-9547 (5–50 mg/kg,
i.p.) enhanced locomotor activity to a markedly higher
degree than MRZ-9546 (5–50 mg/kg, i.p.) (Zvejniece
et al., 2011). Of note, perseverative lever presses and response latencies were not altered by MRZ-9547 and
MRZ-9546 suggesting that animals were sensitive to the
ratio requirements of the operant schedule and that
drugs did not induce motor effects interfering with reward approach. By contrast, modafinil did not alter the
last ratio achieved and other performance measures.
Modafinil at doses used here produced a large variety
of neurochemical and behavioral effects (Eagle et al.,
2007), e.g. a reduced food intake (Nicolaidis and De
Saint Hilaire, 1993). Consistent with latter observation,
in the consumption test (Experiment 4) modafinil
(64 mg/kg) reduced food intake suggesting that the dose
was behaviorally active. In other words, inappropriate
dosing might not account for the lack of effect of modafinil on the last ratio achieved. Moreover, methylphenidate
at 10 mg/kg enhanced the last ratio achieved confirming
earlier evidence that methylphenidate at a similar dose
enhanced breakpoints (Mayorga et al., 2000) By contrast,
d-amphetamine (1 mg/kg) had only very subtle effects on
the last ratio achieved. This finding is difficult to compare
with the literature as earlier studies reported mixed
effects in PR tasks, i.e. increased (Poncelet et al., 1983)
and decreased (Mobini et al., 2000) breakpoints.
Overall, our data are largely consistent with earlier
reports (Poncelet et al., 1983) and demonstrate that
DAT-interacting drugs can increase in PR tasks the tendency to work for reinforcement to variable degrees.
Importantly, breakpoints in PR tasks are not simply a
measure of ‘reward’, but predominantly a measure of
how much effort an animal will invest to gain access to
a reinforcer (Stewart, 1975). DA-related mechanisms, in
particular in the nucleus accumbens, are crucial to mediate the tendency to work for reinforcement in PR
tasks (Zhang et al., 2003). For instance, DA-related signal
transduction activity in the nucleus accumbens, i.e.
pDARPP-32(Thr34) expression, was greater in rats with
high vs. low lever pressing in a PR task suggesting that
accumbens signal transduction activity is related to individual differences in work output (Randall et al., 2012).
These findings indicate that a stimulation of mesoaccumbens DA systems may be one likely neurochemical
substrate of MRZ-9547 to amplify PR responding.
effort response option. In vehicle controls across all
experiments, the maximum ratio achieved in the PR/
chow feeding choice task was considerably lower than
the one achieved in the PR task, probably because less
preferred lab chow is freely available. In other words,
choosing how much effort to exert under a PR schedule
is influenced by the presence or absence of a low effort response option. This effect was also observed in animals
treated with 25 and 50 mg/kg MRZ-9547, but not in animals treated with 100 mg/kg MRZ-9547. In other words,
cost/benefit-related decision making in animals subjected
to low and intermediate doses of MRZ-9547 was sensitive
to the presence of a low-cost response option. As pellet intake under MRZ-9547 at 50 and 100 mg/kg was reduced
in the consumption test, the enhanced preference for the
effortful response option under a PR schedule may not
be related to a drug-induced increase of the palatability
or incentive value of the pellets. Reduced pellet intake
in the consumption test suggests that MRZ-9547 may
have appetite suppressant effects as observed with other
DAT-interfering drugs, e.g. amphetamine (Holtzman
and Jewett, 1971), methylphenidate (Bello and Hajnal,
2006) or modafinil (Nicolaidis and De Saint Hilaire,
1993). In a related PR/chow feeding choice task, a putative
appetitive suppressant drug, i.e. the cannabinoid CB1 antagonist/inverse agonist AM251, as well as pre-feeding
decreased both pellet reinforced lever pressing and lab
chow consumption (see Randall et al., 2012 for a detailed
discussion). Thus, the effects of appetite suppressant
manipulations and DAT interfering drugs with appetite
suppressant activity differ markedly in this task suggesting that other factors than appetitive suppression have a
major influence on choice behavior. Of note, in the choice
test, the total gram amount of pellets and lab chow
ingested by rats treated with the highest doses of
MRZ-9547, modafinil, methylphenidate or amphetamine
did not differ significantly from respective vehicle controls, but in MRZ-9547- and d-amphetamine-treated rats
the relative amount of pellets ingested was significantly
higher (data not shown). Furthermore, it should be
pointed out that, in vehicle controls of Experiment 5,
food intake in the choice and consumption test, for unknown reasons, was only moderate. However, as in our
other experiments, the last ratio achieved in the choice
test was markedly lower than in the PR test, thus reduced
chow intake in vehicle controls of Experiment 5 might not
have altered their choice behavior.
MRZ-9546 was markedly less active than MRZ-9547 as
measured by the maximal ratio achieved in the choice
test, whereas modafinil was not effective. By contrast,
methylphenidate increased this measure, but seemingly
did not reduce the amount of lab chow ingested, an artifact that mainly reflects reduced lab chow ingestion in respective vehicle controls (see also above). Consistent with
earlier findings in an effort-related choice task (Cousins
et al., 1994), d-amphetamine at the dose tested only
moderately increased the last ratio achieved.
2054 S. Sommer et al.
DAT ligands and PR responding
Overall, these experiments demonstrate corresponding
effect sizes of DAT-interfering drugs on the tendency
to work for food reinforcement in two related tasks;
however, the influence of a given drug on task performance was strikingly different. Because of their effects on
extracellular DA levels, one might assume that DATinteracting drugs would elicit comparable behavioral
effects. Yet considerable evidence suggests that this notion is not correct for a number of reasons. First, DAT
ligands with distinct chemical structures induce specific
conformational changes in the transporter protein that
can be differentially transduced by cells, ultimately inducing a unique profile of behavioral effects (Schmitt
et al., 2013). Furthermore, depending on the dose, the pattern and magnitude of DA release induced by a given
DAT ligand in distinct DA terminal regions can differ
considerably (Koda et al., 2010) highlighting the complex
behavioral pharmacology of DAT ligands. Although it is
conceivable that MRZ-9547, and to a much lesser extent
MRZ-9546 and methylphenidate, might invigorate PR
responding by interfering with mesoaccumbens DA systems, the specific neurochemical and behavioral mechanisms underlying the particularly prominent effects of
MRZ-9547 are yet unclear. Relative to methylphenidate
(Koda et al., 2010), the impact of MRZ-9547 on striatal
extracellular DA concentrations demonstrated by microdialysis was only moderate implying that there is no simple relationship between DAT ligand evoked DA release
and PR responding.
Clinical significance
An analysis of effort-related decision making in rodents
may not only be important for an understanding of the
neural and neurochemical basis of motivational processes
but could also provide an animal model for motivational
dysfunctions related to effort expenditure (Salamone and
Correa, 2012) such as fatigue, apathy, anergia and psychomotor slowing in neuropsychiatric and neurological
disorders, e.g. major depression, schizophrenia, multiple
sclerosis or inflammatory disease (Friedman et al., 2011;
Salamone and Correa, 2012). For instance, in a test of
effort-related choice in humans, patients with major depressive disorders were less willing to expend effort for
rewards than controls (Treadway et al., 2012). Fatigue
and anergia are also common in Parkinson’s disease
and may be even more deleterious to quality of life
than motor dysfunction (Friedman et al., 2011). Current
treatment options rarely cause significant improvements
in fatigue (Kluger et al., 2013) and the few available studies on the pharmacotherapy of fatigue in Parkinson’s
disease indicate that only methylphenidate had some
beneficial effects, whereas L-DOPA had only minimal,
modafinil and amphetamine no beneficial effects
(Friedman et al., 2011). Our findings indicate that, relative
to methylphenidate, MRZ-9547 was able to markedly
invigorate progressive ratio responding in rodents
and, therefore, could be potentially useful for treating
energy-related symptoms such as fatigue or anergia in
Parkinson’s disease and possibly other neurological or
neuropsychiatric disorders discussed above.
It is well known that DAT ligands such as cocaine are
reinforcing (Ritz et al., 1987), hence, a possible concern is
their addictive liability. For instance, human imaging studies revealed a relationship between reinforcing effects
and degree of DAT blockade by cocaine and methylphenidate after intravenous injection (Volkow et al., 1997).
However, there are also DAT inhibitors that show little
or no reinforcing properties (Desai et al., 2005; Volkow
et al., 2005; Li et al., 2011). Although MRZ-9547 in the racemic form, known as phenotropil, has been examined in
human studies (Malykh and Sadaie, 2010), reports on its
addictive liability are, to the best of our knowledge, not
available. To be reinforcing in humans, DAT inhibitors
have to block >50% of DAT in less than 15 min and
clear out of the brain rapidly to enable fast repeated administration (Volkow et al., 2005). Our pharmacokinetic
data in rats suggest that, relative to cocaine extracellular
brain levels (Bradberry et al., 1993; Pettit and Pettit,
1994), MRZ-9547 extracellular brain levels after i.p. administration increased slower and remained elevated
Downloaded from http://ijnp.oxfordjournals.org/ by guest on October 26, 2016
Collectively, these data show that among the
DAT-interfering drugs tested, MRZ-9547 most potently
increased the tendency to work for reinforcement,
whereas MRZ-9546 and methylphenidate were markedly
less effective. Given that accumbens DA transmission
exerts a powerful influence over effort-related choice
behavior (Salamone et al., 2012), a stimulation of mesoaccumbens DA systems may be one likely neurochemical
substrate of MRZ-9547 to modulate cost/benefit related
decision making. Considerable evidence suggests that
adenosine A2A receptor antagonists were able to reverse
a shift from lever pressing to chow intake induced by
concomitant systemic blockade of DA receptors or DA
depletion (Salamone and Correa, 2012). Furthermore,
the antidepressant bupropion, a drug that can inhibit
catecholamine uptake, reversed a shift from lever pressing to chow intake after a tetrabenazine-induced DA
depletion (Nunes et al., 2013). Furthermore, in a similar
PR/chow choice task as used here, systemic administration of the adenosine A2A receptor antagonist MSX-3
inclined intact rats to select the increasingly more effortful
type of food-seeking behavior and decreased chow consumption, an effect that might rely in particular on functional interactions of DA and adenosine systems in the
nucleus accumbens (Randall et al., 2012). Relative to
MRZ-9547, MSX-3 appears to be less potent to increase
the last ratio achieved. However, respective comparisons
are limited, e.g. because PR schedules in respective PR/
free feeding choice tasks differ. Thus, a comparative
evaluation of drug effects in the same task is required.
Dopamine reuptake inhibitor MRZ-9547 increases progressive ratio responding in rats 2055
longer pointing to lower reinforcing effects. However,
detailed studies addressing this issue are warranted.
Supplementary material
For supplementary material accompanying this paper,
visit http://dx.doi.org/10.1017/S1461145714000996
Acknowledgments
We are grateful to I. Newar for assistance in animal
husbandry. This work was supported by Merz
Pharmaceuticals.
Statement of Interest
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