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The triple reuptake inhibitor DOV 216,303 induces long-lasting enhancement of brain reward activity as measured by intracranial self-stimulation in rats
European Journal of Pharmacology 693 (2012) 51–56
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
The triple reuptake inhibitor DOV 216,303 induces long-lasting enhancement of brain reward activity as measured by intracranial self-stimulation in rats Jolanda Prins a,n, Paul J. Kenny b, Ivo Doomernik a, Rudy Schreiber c, Berend Olivier a,d, S. Mechiel Korte a a
Utrecht Institute for Pharmaceutical Sciences (UIPS) and Rudolf Magnus Institute of Neuroscience (RMI), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands b Laboratory of Behavioral and Molecular Neuroscience, Department of Molecular Therapeutics, The Scripps Research Institute-Scripps Florida, Jupiter, Florida, USA. c Sepracor Inc., 84 Waterford Drive, Marlborough, MA 01752, USA d Yale University School of Medicine, New Haven, CT, USA
a r t i c l e i n f o
abstract
Article history: Received 14 June 2012 Received in revised form 9 July 2012 Accepted 27 July 2012 Available online 21 August 2012
Triple reuptake inhibitors (TRIs) are potential new antidepressants, which not only enhance brain serotonin and norepinephrine concentrations but also increase dopamine levels. Therefore TRIs are believed to have faster therapeutic onset than SSRIs, and may be particularly useful for the treatment of anhedonia (i.e. inability to experience pleasure), one of the core symptoms of major depression. The current study aimed at getting better insight into the rewarding properties of DOV 216,303, which is a TRI, regarding its possible use to treat anhedonia. It is known that psychostimulant drugs lower intracranial self-stimulation (ICSS) reward thresholds, reflecting enhanced brain reward activity, whereas withdrawal from those compounds mostly results in increased ICSS thresholds. Therefore we assessed the effects of DOV 216,303 on ICSS thresholds in rats. Animals were trained in the discretetrial current-threshold procedure and after stable ICSS reward thresholds were established, animals received one injection per day of DOV 216,303 (20 mg/kg) or amphetamine (5 mg/kg) for four consecutive days. ICSS thresholds were assessed 3, 6, and 23 h after each injection. DOV 216,303 decreased ICSS thresholds up to 6 h after drug treatment. To our knowledge this is the first time that a triple reuptake inhibitor, DOV 216,303, induces relatively long-lasting enhancement of brain reward activity. Elevated ICSS thresholds were found after amphetamine administration, which is consistent with previously reported reward deficits induced after amphetamine-withdrawal. & 2012 Elsevier B.V. All rights reserved.
Keywords: Triple reuptake inhibitor TRI DOV 216,303 DOV 21,947 EB-1010 Major depression Anhedonia Reward Dopamine Monoamines
1. Introduction Pharmacological treatment of Major Depressive Disorder is mainly based on increasing serotonin and/or norepinephrine levels by selective serotonin reuptake inhibitors (SSRIs) and norepinephrine reuptake inhibitors (NRIs) or dual acting drugs, acting on both serotonin and norepinephrine (SNRIs). Nevertheless, it takes a relatively long time before the therapeutic effects of SSRIs are established. Moreover, anhedonia (one of the core symptoms of Major Depression), which is defined as the inability to experience pleasure, is often not alleviated with currently available antidepressants. A role for dopamine in the pathophysiology of depression has been postulated and extensively reviewed (Dunlop and Nemeroff, 2007; Kapur and Mann, 1992; Naranjo et al., 2001; Nestler and Carlezon, 2006). Evidence for disturbed dopaminergic reward
n
Corresponding author. Tel.: þ31 6 16375438; fax: þ31 30 253 7900. E-mail address: [email protected] (J. Prins).
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.07.047
function in depression comes from preclinical research in which the dopamine response after palatable food is blunted in the chronic mild stress animal model for depression (Di Chiara et al., 1999). Moreover, clinical research also demonstrated a role for a dysfunctional reward system in depression (Tremblay et al., 2002, 2005). Co-targeting of the dopaminergic system has been proven to be effective in antidepressant treatment. Bupropion, which is a dopamine reuptake inhibitor (DRI) and NRI, augments antidepressant treatment with SSRIs (Trivedi et al., 2006). Moreover, bupropion alone shows antidepressant effects (Dhillon et al., 2008) and even enhances brain reward function in rats (Cryan et al., 2003a). Furthermore, the D2/D3 receptor agonist pramipexole has antidepressant-like effects in an animal model of depression (Breuer et al., 2009) and can also augment the effects of SSRIbased antidepressants (Goldberg et al., 2004; Gupta et al., 2006). These insights have led to the development of triple reuptake inhibitors (TRIs). TRIs are considered as a new class of antidepressants (Guiard et al., 2009; Marks et al., 2008b; Millan, 2009; Skolnick and Basile, 2007). DOV 216,303 is such a TRI, which
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enhances brain serotonin, norepinephrinergic and dopaminergic neurotransmission (Prins et al., 2010; 2011; Skolnick et al., 2006). By impacting dopaminergic neurotransmission, TRIs are believed to have faster therapeutic onset than SSRIs (Willner, 1997) and may be particularly useful for the treatment of anhedonia (Marks et al., 2008a). The current study aimed at getting better insight into the rewarding properties of DOV 216,303. In this study only one dose of DOV 216,303 (20 mg/kg, p.o.) was used which had shown to have a clear monoamine release profile, including an increase in dopaminergic neurotransmission, even after repeated administration (Prins et al., 2010, 2011). A similar monoamine release profile has been observed after DOV 216,303 (20 mg/kg, i.p.). To assess the potential of DOV 216,303 to activate brain reward systems we examined the effects of acute, sub-chronic and after cessation of treatment with DOV 216,303 and amphetamine on intracranial self stimulation (ICSS) thresholds in rats. ICSS is considered as a direct measure of brain reward (Carlezon and Chartoff, 2007; Kenny, 2007; Markou and Koob, 1992). The hypothesis of the current study is that DOV 216,303, which is also known to increase dopaminergic neurotransmission, increases brain reward functioning as reflected by decreasing intracranial self stimulation (ICSS) thresholds.
2. Material and methods 2.1. Animals Twenty-four male Wistar rats (Harlan, Horst, the Netherlands) weighing between 290–350 g at the time of surgery were socially housed, four per cage on a 12 h light-dark cycle with lights on at 6:00 h and off at 18:00 h. Food and water were available ad libitum, except for the first 3 days of ICSS training where animals were given 20 g food per animals per day in order to have a more optimized training of the animals (Carr, 1990). All animal experimental procedures were carried out in accordance with the government guidelines and approved by the Ethical Committee for Animal Research at Utrecht University, the Netherlands. 2.2. Surgery Animals were anesthetized by inhalation of isoflurane gas (2–3%), mixed with nitrous oxide and oxygen and placed in a stereotaxic instrument (Kopf, David Kopf Instruments). Lidocaine hydrochloride (2%) þadrenaline (0.001%) were applied in the incision as local anesthetic. Bipolar ICSS electrodes (cut to a length of 11 mm) were implanted into the lateral hypothalamus (LH). The coordinates of the LH were AP: 0.5 mm from bregma; ML: 71.7 mm from bregma; DV: 8.3 mm from dura. The incisor bar was adjusted to 5 mm above the interaural line (Pellegrino et al., 1979). Electrodes were anchored with four screws and dental acrylic on the skull. All animals received Rimadyl (5 mg/kg, subcutaneously) for pain relief twice daily, up to a total of four injections.
a computer running MED-PC IV (Med Associates, Inc.) controlling all stimulation settings, programs and recording of data. 2.4. ICSS procedure In order for the animals to make the association that turning the wheel results in electrical stimulation, all animals were initially trained to turn the wheel manipulandum on a fixed ratio 1 schedule of reinforcement. In this training phase each quarter turn of the wheel resulted in an electrical stimulus with train duration of 500 ms. After several successful training sessions (more than 1000 turnings in 30 min), the rats were trained on a discrete-trial current-threshold procedure according to the procedure described by Markou and Koob (1992). At the start of a trial rats received a free, non-contingent stimulus and had 7.5 s to react and turn the wheel a quarter turn to obtain a second, contingent stimulus (positive response) of the same current intensity and duration (100 ms). In case no response occurred during the 7.5 s period, a negative response was recorded. The 7.5 s period in which a positive or negative response occurred was followed by an inter trial interval (ITI) with a duration range from 7.5 to 12.5 s. Responses during the ITI resulted in a delay of onset of the next trial of 12.5 s. Turning the wheel in the 2 s after a positive response did not have further consequences. Responses during this period often reflect the force with which the animal turned the wheel, because a powerful pull will result in more than a quarter turn of the wheel. Animals were subjected to alternating descending and ascending series of current intensities starting with a descending series. The stimulus intensity of the first series was set 40 mA above each animal’s own baseline. Current levels were presented in sets of five trials of the same current intensity and altered by steps of 5 mA. 2.5. Parameters 2.5.1. ICSS thresholds The current threshold for a series was defined as the midpoint between two consecutive current intensities for which animals responded in at least three of the five trials and two consecutive current intensities for which animals did not respond in three or more of the five trials. The overall threshold of the session was defined as the mean of the thresholds of the four alternating descending and ascending series. Stable thresholds were defined as less than 10% change in threshold over three consecutive days after at least 10 days of testing. 2.5.2. Response latencies The response latency is the time between the presentation of the non-contingent stimulus and the turning of the wheel by the animal. The overall response latencies were defined as the mean response latency of all trials during which a positive response occurred. 2.6. Drugs
2.3. ICSS apparatus All ICSS experiments were performed in eight soundattenuating operant chambers (30.5 30 17 cm) with a grid floor and a wheel manipulandum in one of the sides. The implanted electrode was connected to the electrical stimulator through a swivel and a bipolar connector cable (Plastics One), ensuring unrestrained movement throughout the ICSS procedure. The electrical stimulations were delivered by a constant current stimulator (Med Associates Inc.). The stimulator was connected to
Animals were divided into three treatment groups. Treatment groups consisted of vehicle (n ¼4, sterile water, administered p.o. by oral gavage and n ¼3, 0.9% saline, administered i.p.), 5 mg/kg Damphetamine (dissolved in 0.9% saline administered i.p. in a volume of 1 ml/kg, n ¼6), or 20 mg/kg DOV 216,303 [(þ/ )-1(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride) synthesized by Sepracor Inc., Marlborough, USA], (dissolved in sterile water and administered p.o. in a volume of 2 ml/kg by oral gavage, n¼7).
J. Prins et al. / European Journal of Pharmacology 693 (2012) 51–56
2.7. Experimental design An overview of the experimental set-up is pictured in Fig. 1. After stable ICSS thresholds were established (less than 10% change in threshold over three consecutive days), animals were randomly assigned to their treatment groups. Either vehicle, D-amphetamine or DOV 216,303 was administered and ICSS thresholds were measured 3, 6 and 23 h after drug treatment. At 24 h, the second drug treatment was given and again ICSS thresholds were measured at 3, 6 and 23 h after administration. In total four injections were given on four consecutive days and 3, 6 and 23 h after each drug treatment ICSS thresholds were measured. Furthermore ICSS thresholds were regularly measured from day three up to 10 days after the last drug treatment.
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data. Response latencies were expressed as absolute values (s). ICSS current-thresholds and response latencies were analyzed by repeated measures ANOVA with time (three levels: 3, 6 and 23 h) and day (four levels: day 1–4) as within subject factors and treatment (vehicle, D-amphetamine and DOV 216,303) as between subject factor. In case of significant overall effects or interaction effects, effects of treatment individual time points were analyzed with a one-way ANOVA following post-hoc Bonferroni comparisons. When the assumption of sphericity was violated, reported results were corrected by the Greenhouse– Geisser correction factor.
3. Results
2.8. Statistical analysis
3.1. Baseline ICSS thresholds
Mean absolute baseline thresholds and response latencies ( 7S.E.M.) were measured for each experimental group. All ICSS current-thresholds were expressed as a percentage of the baseline
Mean ( 7S.E.M.) baseline ICSS thresholds were calculated by taking an average from thresholds 10 days prior to the start of treatment. Mean absolute baseline ICSS thresholds were 104.1712.7 mA, 108.6 712.2 mA and 93.678.4 mA for the control, amphetamine and DOV 216,303 treated groups respectively. As expected, no significant difference could be found between the two different vehicle treatments [F(1, 5) ¼0.302], therefore the water and saline groups were taken together and in further analysis considered as one vehicle group. 3.2. ICSS thresholds after treatment
Fig. 1. Experimental design of the study. After ICSS surgery, training and until stable ICSS thresholds were established (not shown), animals received one injection per day of D-amphetamine, DOV216,303 or vehicle for four consecutive days. ICSS thresholds were assessed 3, 6, and 23 h after each injection.
Repeated measures ANOVA with time and day as within factors and treatment as between subjects factor revealed a significant effect of treatment [F(2, 17) ¼5.850, p ¼0.012]. Furthermore, an interaction between time and the three different treatments [F(4, 34)¼ 7.997 p o0.001] was found. One-way ANOVA per time point revealed significant differences between
Fig. 2. Percentage change in ICSS thresholds after treatment. A. ICSS thresholds 3, 6, and 23 h after each injection for four consecutive days. B. Average ICSS thresholds per time point. #P o 0.05 compared to other treatment group (DOV 216,303 or D-amphetamine) nPo 0.05 compared to vehicle group. Bars represent mean 7S.E.M.
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treatment groups. Although a large variation in individual data points 3 h after amphetamine treatment was observed (see also Fig. 4). Post-hoc bonferroni comparisons between amphetaminetreated and DOV 216,303-treated groups revealed significant differences at time point t3, t6 and t23 of day 1, and differences at t6 and t23 of day 2, 3 and 4 (Fig. 2A). No effect of day [F(2, 31) ¼1.967, e ¼0.607] or day x treatment interaction [F(4, 31) ¼1.646 e ¼0.607] could be observed. Therefore, the 3, 6 and 23 h measurements of all days were averaged and visualized in Fig. 2B. One way ANOVA on these averaged data revealed significant effects at time point t3 [F(2, 19) ¼3.818, p¼0.043], t6 [F(2, 19) ¼7.671, p ¼0.004] and t23 [F(2, 19) ¼8.812, p ¼0.002]. Post-hoc bonferroni comparisons revealed a significant decrease in thresholds 6 and 23 h after DOV216,303 administration compared to the same time points after amphetamine administration. Compared to the vehicle group, post-hoc bonferroni comparisons showed significant elevations in ICSS thresholds at t23 after amphetamine administration, reflecting decreased reward values of the stimulation. Significant decreases in ICSS thresholds were observed until 6 h after DOV 216,303 administration when compared to vehicle. Since ICSS thresholds showed no significant differences between the treatment groups from day three up to 10 days after the last injection, these data are not presented. 3.3. ICSS thresholds vehicle group As can be seen in Fig. 2A and B, ICSS thresholds of the vehicle group increase during the day, where t23 is in the morning, at the beginning of the light cycle and t6 is in the afternoon, 2 h before the lights went off. One way ANOVA revealed significant differences between time points at day 2 [F(2, 20)¼7.219, p o0.01] and day 3 [F(2, 20)¼4.810, po0.05]. Post-hoc bonferroni comparisons revealed a trend towards an increased threshold at t3 compared to t23 at day 2 (p ¼0.055) and day 3 (p ¼0.053) and significant increases in thresholds at t6 compared to t23 at both days. 3.4. Response latencies Mean ( 7S.E.M.) baseline response latencies were calculated by taking an average from response latencies 10 days prior to the start of treatment. Mean absolute baseline ICSS response latencies were 2.86 70.10 s, 2.73 70.17 s and 2.4470.16 s for the control, amphetamine and DOV 216,303 treated groups respectively. Repeated measures ANOVA with time and day as within factors and treatment as between subjects factor revealed only a
significant main effect of day [F(2, 28) ¼4.289 p ¼0.031] (Fig. 3). No significant difference could be observed between treatment groups [F(1, 17) ¼0.718] (see Fig. 3).
4. Discussion The most important finding of the present study is that the triple reuptake inhibitor (TRI) DOV 216,303 leads to long-lasting enhancement of brain reward activity, reflected by long-lasting decreased intracranial self-stimulation (ICSS) thresholds. Such long-lasting hedonic effects were not observed in the amphetamine-treated animals. Moreover, amphetamine-treated animals showed withdrawal-like reward deficits (elevated ICSS thresholds) 23 h after administration. No significant differences between response latencies of the different treatment groups were observed (see Fig. 3), indicating that a possible hyperactivity or behavioral impairment due to drug treatment do not attribute to the observed changes in ICSS thresholds (see Fig. 2). ICSS thresholds are considered as a direct measure of brain reward function in rats and mice (Carlezon and Chartoff, 2007; Kenny, 2007; Markou and Koob, 1992). Many psychostimulant drugs (e.g. amphetamine, cocaine, and heroin) amplify reward signals in the brain reflected in lowering of ICSS thresholds (Kenny, 2007). Drug-induced lowering of ICSS thresholds can be explained by increased reward signaling resulting in potentiating the reward perceived by ICSS. On the contrary, withdrawalinduced elevations of ICSS thresholds can be explained by decreased activity of reward circuitry and desensitization of the rewarding effects of ICSS (Kenny, 2007; Kornetsky and Esposito, 1979). Previously, it was shown that rats increased their levels of heroin self-administration to avoid this withdrawal-associated state of negative reward, suggesting that withdrawal-induced reward dysfunction serves as a substrate for negative reinforcement that also contributes to the development of habitual drug use (Kenny et al., 2006). The relatively long-lasting stimulatory effects (up to 6 h) on brain reward by DOV 216,303 observed in the current study are quite different from the transient effects of psychostimulant drugs like amphetamine (Leith and Barrett, 1976; Lin et al., 2000) and cocaine (Kenny et al., 2003), which only decrease ICSS thresholds for a relatively short period (from 15 min up to 3 h). In the present study we focused especially on long-lasting drug effects on brain reward systems and we therefore might have missed the early short-term hedonic effects of amphetamine. Although many amphetamine-treated animals did show decreases in ICSS
Fig. 3. Absolute response latencies (s) 3, 6, and 23 h after each injection for four consecutive days. B¼ baseline (average response latencies 10 days prior to the start of treatment).
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thresholds (see Fig. 4), this is not reflected in the mean value because some individuals already show strong withdrawal effects, i.e. increased ICSS thresholds, 3 h after amphetamine treatment. This explanation is in agreement with earlier findings in which it was shown that after acute amphetamine administration shortlasting decreased ICSS thresholds were observed followed in time by an increase in ICSS thresholds, reflecting drug withdrawal effects (Cryan et al., 2003b; Leith and Barrett, 1976; Kornetsky and Esposito, 1979; Lin et al., 1999, 2000 ). Remarkably, no such withdrawal effects were detected after DOV 216,303 treatment. Even 23 h after DOV 216,303 treatment, no withdrawal-associated reward deficits were present, since no elevated ICSS thresholds were observed. However, to be absolutely sure that no withdrawal effects take place, in the future ICSS thresholds also have to be measured for more time-points (between 24 and 48 h) after DOV 216,303 treatment. Our data with DOV 216,303 are in accordance with other studies in which monoamine reuptake inhibitors have an effect on brain reward sensitivity. The norepinephrine/dopamine reuptake inhibitor (NDRI) bupropion dose-dependently enhanced brain reward function 30 min after treatment. But this effect was relatively short-term, because 24 h after treatment ICSS thresholds were back to baseline levels (Cryan et al., 2003a). Data points in between were not reported, so nothing is known about the time-course of this effect. Furthermore, the NRI desipramine lowered ICSS thresholds 30 min after administration, but had no effect after chronic treatment (Paterson et al., 2008). Neither acute nor chronic treatment nor withdrawal from the SSRI fluoxetine altered ICSS thresholds. Although chronic fluoxetine did not have an effect themselves on brain reward systems, they did alter the ability of amphetamine to increase ICSS thresholds (Lin et al., 1999). However, other studies showed opposite effects in that acute as well as chronic treatment with fluoxetine elevated reward thresholds (Lee and Kornetsky, 1998). Moreover, another study showed that chronic blockade of the serotonin transporter by fluoxetine, or a deletion of this transporter in SERT knockout rats, results in reduced responding for natural food reward (Sanders et al., 2007), suggesting that the serotonin system is involved in reward-related processes (Kranz et al., 2010). So, our data showed an increased sensitivity of brain reward systems by DOV 216,303 for a longer period than reported in these studies. The combination of increasing all three monoamines at the same time might be a possible explanation for this enhancement of brain reward function, which might be a key therapeutic advantage in the treatment of anhedonia.
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Since DOV 216,303 enhances dopaminergic neurotransmission, its possible use as an antidepressant drug raises concerns related to its abuse potential. Cocaine (which is like DOV 216,303 also a TRI) or amphetamine also increase dopamine neurotransmission, and are known for their intrinsic abuse potential and reinforcing effects (Kuhar et al., 1991). It is known that rats titrate their patterns of self-administration of intravenous cocaine (Kenny et al., 2003), heroin (Kenny et al., 2006) or nicotine (Kenny and Markou, 2006) at a level that achieves maximal drug-induced lowering of ICSS thresholds. Thus, the stimulatory effects of these drugs on brain reward systems represent an important source of positive reinforcement that motivates habitual consumption (Kenny, 2007). Before it can be concluded that DOV 216,303 does not have abuse potential, similar selfadministration experiments as described above have to be performed. Recently, a preclinical study assessing the potential abuse liability of DOV 216,303 showed that the compound only partially substituted cocaine in a drug-discrimination assay in rats and produced locomotor sensitization in mice at doses that are at least six times higher than the minimally effective dose in antidepressant tests (Caldarone et al., 2010). Furthermore, a clinical trial did not mention adverse effects as abuse potential of DOV 216,303 (Beer et al., 2004; Skolnick et al., 2006). Moreover, a recent study showed that Amitifadine (DOV 21,947 or EB-1010, the active enantiomer of DOV 216,303) significantly improved symptoms (including anhedonia), in patients with major depressive disorder without observing adverse side effects (Tran et al., 2012). Nevertheless, more experiments have to be performed before it can be concluded that DOV 216,303 has no addictive properties at all. In conclusion, DOV 216,303 (20 mg/kg, p.o.), which has previously shown to have an antidepressant-like action in the forced swim test in rats and mice (Caldarone et al., 2010), can activate brain reward systems for a relatively long period. Therefore it is hypothesized that TRIs, especially due to their dopaminergic component, can be used to treat anhedonia. Enhancement of dopaminergic neurotransmission in antidepressant treatment, however, should be carefully investigated with regards to abuse potential.
Acknowledgments The authors would like to thank Koen Westphal, Gerdien Korte-Bouws and Hans Sturkenboom for their excellent technical assistance and animal upkeep.
References
Fig. 4. Individual data points of percentage change in ICSS thresholds 3 h after amphetamine treatment on day 1, 2, 3 and 4. The horizontal dashed line represents the 0% baseline ICSS threshold.
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Marks, D.M., Pae, C.U., Patkar, A.A., 2008a. Triple reuptake inhibitors: a premise and promise. Psychiatry Investig. 5, 142–147. Marks, D.M., Pae, C.U., Patkar, A.A., 2008b. Triple reuptake inhibitors: the next generation of antidepressants. Curr. Neuropharmacol. 6, 338–343. Millan, M.J., 2009. Dual- and triple-acting agents for treating core and co-morbid symptoms of major depression: novel concepts, new drugs. Neurotherapeutics 6, 53–77. Naranjo, C.A., Tremblay, L.K., Busto, U.E., 2001. The role of the brain reward system in depression. Prog. Neuropsychopharmacol. Biol. Psychiatry 25, 781–823. Nestler, E.J., Carlezon Jr., W.A., 2006. The mesolimbic dopamine reward circuit in depression. Biol. Psychiatry 59, 1151–1159. Paterson, N.E., Semenova, S., Markou, A., 2008. The effects of chronic versus acute desipramine on nicotine withdrawal and nicotine self-administration in the rat. Psychopharmacology (Berl) 198, 351–362. Pellegrino, L., Pellegrino, A., Cushman, A., 1979. A stereotaxic Atlas of the Rat Brain. Plenum Press, New York. Prins, J., Denys, D.A., Westphal, K.G., Korte-Bouws, G.A., Quinton, M.S., Schreiber, R., Groenink, L., Olivier, B., Korte, S.M., 2010. The putative antidepressant DOV 216,303, a triple reuptake inhibitor, increases monoamine release in the prefrontal cortex of olfactory bulbectomized rats. Eur. J. Pharmacol. 633, 55–61. Prins, J., Westphal, K.G., Korte-Bouws, G.A., Quinton, M.S., Schreiber, R., Olivier, B., Korte, S.M., 2011. The potential and limitations of DOV 216,303 as a triple reuptake inhibitor for the treatment of major depression: A microdialyis study in olfactory bulbectomized rats. Pharmacol. Biochem. Behav. 97, 444–452. Sanders, A.C., Hussain, A.J., Hen, R., Zhuang, X., 2007. Chronic blockade or constitutive deletion of the serotonin transporter reduces operant responding for food reward. Neuropsychopharmacology 32, 2321–2329. Skolnick, P., Basile, A.S., 2007. Triple reuptake inhibitors (‘‘broad spectrum’’ antidepressants). CNS Neurol. Disord. Drug Targets 6, 141–149. Skolnick, P., Krieter, P., Tizzano, J., Basile, A., Popik, P., Czobor, P., Lippa, A., 2006. Preclinical and clinical pharmacology of DOV 216,303, a ‘‘triple’’ reuptake inhibitor. CNS Drug Rev. 12, 123–134. Tran, P., Skolnick, P., Czobor, P., Huang, N.Y., Bradshaw, M., McKinney, A., Fava, M., 2012. Efficacy and tolerability of the novel triple reuptake inhibitor amitifadine in the treatment of patients with major depressive disorder: a randomized, double-blind, placebo-controlled trial. J. Psychiatr. Res. 46, 64–71. Tremblay, L.K., Naranjo, C.A., Cardenas, L., Herrmann, N., Busto, U.E., 2002. Probing brain reward system function in major depressive disorder: altered response to dextroamphetamine. Arch. Gen. Psychiatry 59, 409–416. Tremblay, L.K., Naranjo, C.A., Graham, S.J., Herrmann, N., Mayberg, H.S., Hevenor, S., Busto, U.E., 2005. Functional neuroanatomical substrates of altered reward processing in major depressive disorder revealed by a dopaminergic probe. Arch. Gen. Psychiatry 62, 1228–1236. Trivedi, M.H., Fava, M., Wisniewski, S.R., Thase, M.E., Quitkin, F., Warden, D., Ritz, L., Nierenberg, A.A., Lebowitz, B.D., Biggs, M.M., Luther, J.F., Shores-Wilson, K., Rush, A.J., 2006. Medication augmentation after the failure of SSRIs for depression. N. Engl. J. Med. 354, 1243–1252. Willner, P., 1997. The mesolimbic dopamine system as a target for rapid antidepressant action. Int. Clin. Psychopharmacol. 12 (Suppl 3), S7–14.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Behavioural pharmacology
The triple reuptake inhibitor DOV 216,303 induces long-lasting enhancement of brain reward activity as measured by intracranial self-stimulation in rats Jolanda Prins a,n, Paul J. Kenny b, Ivo Doomernik a, Rudy Schreiber c, Berend Olivier a,d, S. Mechiel Korte a a
Utrecht Institute for Pharmaceutical Sciences (UIPS) and Rudolf Magnus Institute of Neuroscience (RMI), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands b Laboratory of Behavioral and Molecular Neuroscience, Department of Molecular Therapeutics, The Scripps Research Institute-Scripps Florida, Jupiter, Florida, USA. c Sepracor Inc., 84 Waterford Drive, Marlborough, MA 01752, USA d Yale University School of Medicine, New Haven, CT, USA
a r t i c l e i n f o
abstract
Article history: Received 14 June 2012 Received in revised form 9 July 2012 Accepted 27 July 2012 Available online 21 August 2012
Triple reuptake inhibitors (TRIs) are potential new antidepressants, which not only enhance brain serotonin and norepinephrine concentrations but also increase dopamine levels. Therefore TRIs are believed to have faster therapeutic onset than SSRIs, and may be particularly useful for the treatment of anhedonia (i.e. inability to experience pleasure), one of the core symptoms of major depression. The current study aimed at getting better insight into the rewarding properties of DOV 216,303, which is a TRI, regarding its possible use to treat anhedonia. It is known that psychostimulant drugs lower intracranial self-stimulation (ICSS) reward thresholds, reflecting enhanced brain reward activity, whereas withdrawal from those compounds mostly results in increased ICSS thresholds. Therefore we assessed the effects of DOV 216,303 on ICSS thresholds in rats. Animals were trained in the discretetrial current-threshold procedure and after stable ICSS reward thresholds were established, animals received one injection per day of DOV 216,303 (20 mg/kg) or amphetamine (5 mg/kg) for four consecutive days. ICSS thresholds were assessed 3, 6, and 23 h after each injection. DOV 216,303 decreased ICSS thresholds up to 6 h after drug treatment. To our knowledge this is the first time that a triple reuptake inhibitor, DOV 216,303, induces relatively long-lasting enhancement of brain reward activity. Elevated ICSS thresholds were found after amphetamine administration, which is consistent with previously reported reward deficits induced after amphetamine-withdrawal. & 2012 Elsevier B.V. All rights reserved.
Keywords: Triple reuptake inhibitor TRI DOV 216,303 DOV 21,947 EB-1010 Major depression Anhedonia Reward Dopamine Monoamines
1. Introduction Pharmacological treatment of Major Depressive Disorder is mainly based on increasing serotonin and/or norepinephrine levels by selective serotonin reuptake inhibitors (SSRIs) and norepinephrine reuptake inhibitors (NRIs) or dual acting drugs, acting on both serotonin and norepinephrine (SNRIs). Nevertheless, it takes a relatively long time before the therapeutic effects of SSRIs are established. Moreover, anhedonia (one of the core symptoms of Major Depression), which is defined as the inability to experience pleasure, is often not alleviated with currently available antidepressants. A role for dopamine in the pathophysiology of depression has been postulated and extensively reviewed (Dunlop and Nemeroff, 2007; Kapur and Mann, 1992; Naranjo et al., 2001; Nestler and Carlezon, 2006). Evidence for disturbed dopaminergic reward
n
Corresponding author. Tel.: þ31 6 16375438; fax: þ31 30 253 7900. E-mail address: [email protected] (J. Prins).
0014-2999/$ - see front matter & 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2012.07.047
function in depression comes from preclinical research in which the dopamine response after palatable food is blunted in the chronic mild stress animal model for depression (Di Chiara et al., 1999). Moreover, clinical research also demonstrated a role for a dysfunctional reward system in depression (Tremblay et al., 2002, 2005). Co-targeting of the dopaminergic system has been proven to be effective in antidepressant treatment. Bupropion, which is a dopamine reuptake inhibitor (DRI) and NRI, augments antidepressant treatment with SSRIs (Trivedi et al., 2006). Moreover, bupropion alone shows antidepressant effects (Dhillon et al., 2008) and even enhances brain reward function in rats (Cryan et al., 2003a). Furthermore, the D2/D3 receptor agonist pramipexole has antidepressant-like effects in an animal model of depression (Breuer et al., 2009) and can also augment the effects of SSRIbased antidepressants (Goldberg et al., 2004; Gupta et al., 2006). These insights have led to the development of triple reuptake inhibitors (TRIs). TRIs are considered as a new class of antidepressants (Guiard et al., 2009; Marks et al., 2008b; Millan, 2009; Skolnick and Basile, 2007). DOV 216,303 is such a TRI, which
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enhances brain serotonin, norepinephrinergic and dopaminergic neurotransmission (Prins et al., 2010; 2011; Skolnick et al., 2006). By impacting dopaminergic neurotransmission, TRIs are believed to have faster therapeutic onset than SSRIs (Willner, 1997) and may be particularly useful for the treatment of anhedonia (Marks et al., 2008a). The current study aimed at getting better insight into the rewarding properties of DOV 216,303. In this study only one dose of DOV 216,303 (20 mg/kg, p.o.) was used which had shown to have a clear monoamine release profile, including an increase in dopaminergic neurotransmission, even after repeated administration (Prins et al., 2010, 2011). A similar monoamine release profile has been observed after DOV 216,303 (20 mg/kg, i.p.). To assess the potential of DOV 216,303 to activate brain reward systems we examined the effects of acute, sub-chronic and after cessation of treatment with DOV 216,303 and amphetamine on intracranial self stimulation (ICSS) thresholds in rats. ICSS is considered as a direct measure of brain reward (Carlezon and Chartoff, 2007; Kenny, 2007; Markou and Koob, 1992). The hypothesis of the current study is that DOV 216,303, which is also known to increase dopaminergic neurotransmission, increases brain reward functioning as reflected by decreasing intracranial self stimulation (ICSS) thresholds.
2. Material and methods 2.1. Animals Twenty-four male Wistar rats (Harlan, Horst, the Netherlands) weighing between 290–350 g at the time of surgery were socially housed, four per cage on a 12 h light-dark cycle with lights on at 6:00 h and off at 18:00 h. Food and water were available ad libitum, except for the first 3 days of ICSS training where animals were given 20 g food per animals per day in order to have a more optimized training of the animals (Carr, 1990). All animal experimental procedures were carried out in accordance with the government guidelines and approved by the Ethical Committee for Animal Research at Utrecht University, the Netherlands. 2.2. Surgery Animals were anesthetized by inhalation of isoflurane gas (2–3%), mixed with nitrous oxide and oxygen and placed in a stereotaxic instrument (Kopf, David Kopf Instruments). Lidocaine hydrochloride (2%) þadrenaline (0.001%) were applied in the incision as local anesthetic. Bipolar ICSS electrodes (cut to a length of 11 mm) were implanted into the lateral hypothalamus (LH). The coordinates of the LH were AP: 0.5 mm from bregma; ML: 71.7 mm from bregma; DV: 8.3 mm from dura. The incisor bar was adjusted to 5 mm above the interaural line (Pellegrino et al., 1979). Electrodes were anchored with four screws and dental acrylic on the skull. All animals received Rimadyl (5 mg/kg, subcutaneously) for pain relief twice daily, up to a total of four injections.
a computer running MED-PC IV (Med Associates, Inc.) controlling all stimulation settings, programs and recording of data. 2.4. ICSS procedure In order for the animals to make the association that turning the wheel results in electrical stimulation, all animals were initially trained to turn the wheel manipulandum on a fixed ratio 1 schedule of reinforcement. In this training phase each quarter turn of the wheel resulted in an electrical stimulus with train duration of 500 ms. After several successful training sessions (more than 1000 turnings in 30 min), the rats were trained on a discrete-trial current-threshold procedure according to the procedure described by Markou and Koob (1992). At the start of a trial rats received a free, non-contingent stimulus and had 7.5 s to react and turn the wheel a quarter turn to obtain a second, contingent stimulus (positive response) of the same current intensity and duration (100 ms). In case no response occurred during the 7.5 s period, a negative response was recorded. The 7.5 s period in which a positive or negative response occurred was followed by an inter trial interval (ITI) with a duration range from 7.5 to 12.5 s. Responses during the ITI resulted in a delay of onset of the next trial of 12.5 s. Turning the wheel in the 2 s after a positive response did not have further consequences. Responses during this period often reflect the force with which the animal turned the wheel, because a powerful pull will result in more than a quarter turn of the wheel. Animals were subjected to alternating descending and ascending series of current intensities starting with a descending series. The stimulus intensity of the first series was set 40 mA above each animal’s own baseline. Current levels were presented in sets of five trials of the same current intensity and altered by steps of 5 mA. 2.5. Parameters 2.5.1. ICSS thresholds The current threshold for a series was defined as the midpoint between two consecutive current intensities for which animals responded in at least three of the five trials and two consecutive current intensities for which animals did not respond in three or more of the five trials. The overall threshold of the session was defined as the mean of the thresholds of the four alternating descending and ascending series. Stable thresholds were defined as less than 10% change in threshold over three consecutive days after at least 10 days of testing. 2.5.2. Response latencies The response latency is the time between the presentation of the non-contingent stimulus and the turning of the wheel by the animal. The overall response latencies were defined as the mean response latency of all trials during which a positive response occurred. 2.6. Drugs
2.3. ICSS apparatus All ICSS experiments were performed in eight soundattenuating operant chambers (30.5 30 17 cm) with a grid floor and a wheel manipulandum in one of the sides. The implanted electrode was connected to the electrical stimulator through a swivel and a bipolar connector cable (Plastics One), ensuring unrestrained movement throughout the ICSS procedure. The electrical stimulations were delivered by a constant current stimulator (Med Associates Inc.). The stimulator was connected to
Animals were divided into three treatment groups. Treatment groups consisted of vehicle (n ¼4, sterile water, administered p.o. by oral gavage and n ¼3, 0.9% saline, administered i.p.), 5 mg/kg Damphetamine (dissolved in 0.9% saline administered i.p. in a volume of 1 ml/kg, n ¼6), or 20 mg/kg DOV 216,303 [(þ/ )-1(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride) synthesized by Sepracor Inc., Marlborough, USA], (dissolved in sterile water and administered p.o. in a volume of 2 ml/kg by oral gavage, n¼7).
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2.7. Experimental design An overview of the experimental set-up is pictured in Fig. 1. After stable ICSS thresholds were established (less than 10% change in threshold over three consecutive days), animals were randomly assigned to their treatment groups. Either vehicle, D-amphetamine or DOV 216,303 was administered and ICSS thresholds were measured 3, 6 and 23 h after drug treatment. At 24 h, the second drug treatment was given and again ICSS thresholds were measured at 3, 6 and 23 h after administration. In total four injections were given on four consecutive days and 3, 6 and 23 h after each drug treatment ICSS thresholds were measured. Furthermore ICSS thresholds were regularly measured from day three up to 10 days after the last drug treatment.
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data. Response latencies were expressed as absolute values (s). ICSS current-thresholds and response latencies were analyzed by repeated measures ANOVA with time (three levels: 3, 6 and 23 h) and day (four levels: day 1–4) as within subject factors and treatment (vehicle, D-amphetamine and DOV 216,303) as between subject factor. In case of significant overall effects or interaction effects, effects of treatment individual time points were analyzed with a one-way ANOVA following post-hoc Bonferroni comparisons. When the assumption of sphericity was violated, reported results were corrected by the Greenhouse– Geisser correction factor.
3. Results
2.8. Statistical analysis
3.1. Baseline ICSS thresholds
Mean absolute baseline thresholds and response latencies ( 7S.E.M.) were measured for each experimental group. All ICSS current-thresholds were expressed as a percentage of the baseline
Mean ( 7S.E.M.) baseline ICSS thresholds were calculated by taking an average from thresholds 10 days prior to the start of treatment. Mean absolute baseline ICSS thresholds were 104.1712.7 mA, 108.6 712.2 mA and 93.678.4 mA for the control, amphetamine and DOV 216,303 treated groups respectively. As expected, no significant difference could be found between the two different vehicle treatments [F(1, 5) ¼0.302], therefore the water and saline groups were taken together and in further analysis considered as one vehicle group. 3.2. ICSS thresholds after treatment
Fig. 1. Experimental design of the study. After ICSS surgery, training and until stable ICSS thresholds were established (not shown), animals received one injection per day of D-amphetamine, DOV216,303 or vehicle for four consecutive days. ICSS thresholds were assessed 3, 6, and 23 h after each injection.
Repeated measures ANOVA with time and day as within factors and treatment as between subjects factor revealed a significant effect of treatment [F(2, 17) ¼5.850, p ¼0.012]. Furthermore, an interaction between time and the three different treatments [F(4, 34)¼ 7.997 p o0.001] was found. One-way ANOVA per time point revealed significant differences between
Fig. 2. Percentage change in ICSS thresholds after treatment. A. ICSS thresholds 3, 6, and 23 h after each injection for four consecutive days. B. Average ICSS thresholds per time point. #P o 0.05 compared to other treatment group (DOV 216,303 or D-amphetamine) nPo 0.05 compared to vehicle group. Bars represent mean 7S.E.M.
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treatment groups. Although a large variation in individual data points 3 h after amphetamine treatment was observed (see also Fig. 4). Post-hoc bonferroni comparisons between amphetaminetreated and DOV 216,303-treated groups revealed significant differences at time point t3, t6 and t23 of day 1, and differences at t6 and t23 of day 2, 3 and 4 (Fig. 2A). No effect of day [F(2, 31) ¼1.967, e ¼0.607] or day x treatment interaction [F(4, 31) ¼1.646 e ¼0.607] could be observed. Therefore, the 3, 6 and 23 h measurements of all days were averaged and visualized in Fig. 2B. One way ANOVA on these averaged data revealed significant effects at time point t3 [F(2, 19) ¼3.818, p¼0.043], t6 [F(2, 19) ¼7.671, p ¼0.004] and t23 [F(2, 19) ¼8.812, p ¼0.002]. Post-hoc bonferroni comparisons revealed a significant decrease in thresholds 6 and 23 h after DOV216,303 administration compared to the same time points after amphetamine administration. Compared to the vehicle group, post-hoc bonferroni comparisons showed significant elevations in ICSS thresholds at t23 after amphetamine administration, reflecting decreased reward values of the stimulation. Significant decreases in ICSS thresholds were observed until 6 h after DOV 216,303 administration when compared to vehicle. Since ICSS thresholds showed no significant differences between the treatment groups from day three up to 10 days after the last injection, these data are not presented. 3.3. ICSS thresholds vehicle group As can be seen in Fig. 2A and B, ICSS thresholds of the vehicle group increase during the day, where t23 is in the morning, at the beginning of the light cycle and t6 is in the afternoon, 2 h before the lights went off. One way ANOVA revealed significant differences between time points at day 2 [F(2, 20)¼7.219, p o0.01] and day 3 [F(2, 20)¼4.810, po0.05]. Post-hoc bonferroni comparisons revealed a trend towards an increased threshold at t3 compared to t23 at day 2 (p ¼0.055) and day 3 (p ¼0.053) and significant increases in thresholds at t6 compared to t23 at both days. 3.4. Response latencies Mean ( 7S.E.M.) baseline response latencies were calculated by taking an average from response latencies 10 days prior to the start of treatment. Mean absolute baseline ICSS response latencies were 2.86 70.10 s, 2.73 70.17 s and 2.4470.16 s for the control, amphetamine and DOV 216,303 treated groups respectively. Repeated measures ANOVA with time and day as within factors and treatment as between subjects factor revealed only a
significant main effect of day [F(2, 28) ¼4.289 p ¼0.031] (Fig. 3). No significant difference could be observed between treatment groups [F(1, 17) ¼0.718] (see Fig. 3).
4. Discussion The most important finding of the present study is that the triple reuptake inhibitor (TRI) DOV 216,303 leads to long-lasting enhancement of brain reward activity, reflected by long-lasting decreased intracranial self-stimulation (ICSS) thresholds. Such long-lasting hedonic effects were not observed in the amphetamine-treated animals. Moreover, amphetamine-treated animals showed withdrawal-like reward deficits (elevated ICSS thresholds) 23 h after administration. No significant differences between response latencies of the different treatment groups were observed (see Fig. 3), indicating that a possible hyperactivity or behavioral impairment due to drug treatment do not attribute to the observed changes in ICSS thresholds (see Fig. 2). ICSS thresholds are considered as a direct measure of brain reward function in rats and mice (Carlezon and Chartoff, 2007; Kenny, 2007; Markou and Koob, 1992). Many psychostimulant drugs (e.g. amphetamine, cocaine, and heroin) amplify reward signals in the brain reflected in lowering of ICSS thresholds (Kenny, 2007). Drug-induced lowering of ICSS thresholds can be explained by increased reward signaling resulting in potentiating the reward perceived by ICSS. On the contrary, withdrawalinduced elevations of ICSS thresholds can be explained by decreased activity of reward circuitry and desensitization of the rewarding effects of ICSS (Kenny, 2007; Kornetsky and Esposito, 1979). Previously, it was shown that rats increased their levels of heroin self-administration to avoid this withdrawal-associated state of negative reward, suggesting that withdrawal-induced reward dysfunction serves as a substrate for negative reinforcement that also contributes to the development of habitual drug use (Kenny et al., 2006). The relatively long-lasting stimulatory effects (up to 6 h) on brain reward by DOV 216,303 observed in the current study are quite different from the transient effects of psychostimulant drugs like amphetamine (Leith and Barrett, 1976; Lin et al., 2000) and cocaine (Kenny et al., 2003), which only decrease ICSS thresholds for a relatively short period (from 15 min up to 3 h). In the present study we focused especially on long-lasting drug effects on brain reward systems and we therefore might have missed the early short-term hedonic effects of amphetamine. Although many amphetamine-treated animals did show decreases in ICSS
Fig. 3. Absolute response latencies (s) 3, 6, and 23 h after each injection for four consecutive days. B¼ baseline (average response latencies 10 days prior to the start of treatment).
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thresholds (see Fig. 4), this is not reflected in the mean value because some individuals already show strong withdrawal effects, i.e. increased ICSS thresholds, 3 h after amphetamine treatment. This explanation is in agreement with earlier findings in which it was shown that after acute amphetamine administration shortlasting decreased ICSS thresholds were observed followed in time by an increase in ICSS thresholds, reflecting drug withdrawal effects (Cryan et al., 2003b; Leith and Barrett, 1976; Kornetsky and Esposito, 1979; Lin et al., 1999, 2000 ). Remarkably, no such withdrawal effects were detected after DOV 216,303 treatment. Even 23 h after DOV 216,303 treatment, no withdrawal-associated reward deficits were present, since no elevated ICSS thresholds were observed. However, to be absolutely sure that no withdrawal effects take place, in the future ICSS thresholds also have to be measured for more time-points (between 24 and 48 h) after DOV 216,303 treatment. Our data with DOV 216,303 are in accordance with other studies in which monoamine reuptake inhibitors have an effect on brain reward sensitivity. The norepinephrine/dopamine reuptake inhibitor (NDRI) bupropion dose-dependently enhanced brain reward function 30 min after treatment. But this effect was relatively short-term, because 24 h after treatment ICSS thresholds were back to baseline levels (Cryan et al., 2003a). Data points in between were not reported, so nothing is known about the time-course of this effect. Furthermore, the NRI desipramine lowered ICSS thresholds 30 min after administration, but had no effect after chronic treatment (Paterson et al., 2008). Neither acute nor chronic treatment nor withdrawal from the SSRI fluoxetine altered ICSS thresholds. Although chronic fluoxetine did not have an effect themselves on brain reward systems, they did alter the ability of amphetamine to increase ICSS thresholds (Lin et al., 1999). However, other studies showed opposite effects in that acute as well as chronic treatment with fluoxetine elevated reward thresholds (Lee and Kornetsky, 1998). Moreover, another study showed that chronic blockade of the serotonin transporter by fluoxetine, or a deletion of this transporter in SERT knockout rats, results in reduced responding for natural food reward (Sanders et al., 2007), suggesting that the serotonin system is involved in reward-related processes (Kranz et al., 2010). So, our data showed an increased sensitivity of brain reward systems by DOV 216,303 for a longer period than reported in these studies. The combination of increasing all three monoamines at the same time might be a possible explanation for this enhancement of brain reward function, which might be a key therapeutic advantage in the treatment of anhedonia.
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Since DOV 216,303 enhances dopaminergic neurotransmission, its possible use as an antidepressant drug raises concerns related to its abuse potential. Cocaine (which is like DOV 216,303 also a TRI) or amphetamine also increase dopamine neurotransmission, and are known for their intrinsic abuse potential and reinforcing effects (Kuhar et al., 1991). It is known that rats titrate their patterns of self-administration of intravenous cocaine (Kenny et al., 2003), heroin (Kenny et al., 2006) or nicotine (Kenny and Markou, 2006) at a level that achieves maximal drug-induced lowering of ICSS thresholds. Thus, the stimulatory effects of these drugs on brain reward systems represent an important source of positive reinforcement that motivates habitual consumption (Kenny, 2007). Before it can be concluded that DOV 216,303 does not have abuse potential, similar selfadministration experiments as described above have to be performed. Recently, a preclinical study assessing the potential abuse liability of DOV 216,303 showed that the compound only partially substituted cocaine in a drug-discrimination assay in rats and produced locomotor sensitization in mice at doses that are at least six times higher than the minimally effective dose in antidepressant tests (Caldarone et al., 2010). Furthermore, a clinical trial did not mention adverse effects as abuse potential of DOV 216,303 (Beer et al., 2004; Skolnick et al., 2006). Moreover, a recent study showed that Amitifadine (DOV 21,947 or EB-1010, the active enantiomer of DOV 216,303) significantly improved symptoms (including anhedonia), in patients with major depressive disorder without observing adverse side effects (Tran et al., 2012). Nevertheless, more experiments have to be performed before it can be concluded that DOV 216,303 has no addictive properties at all. In conclusion, DOV 216,303 (20 mg/kg, p.o.), which has previously shown to have an antidepressant-like action in the forced swim test in rats and mice (Caldarone et al., 2010), can activate brain reward systems for a relatively long period. Therefore it is hypothesized that TRIs, especially due to their dopaminergic component, can be used to treat anhedonia. Enhancement of dopaminergic neurotransmission in antidepressant treatment, however, should be carefully investigated with regards to abuse potential.
Acknowledgments The authors would like to thank Koen Westphal, Gerdien Korte-Bouws and Hans Sturkenboom for their excellent technical assistance and animal upkeep.
References
Fig. 4. Individual data points of percentage change in ICSS thresholds 3 h after amphetamine treatment on day 1, 2, 3 and 4. The horizontal dashed line represents the 0% baseline ICSS threshold.
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