Neuropharmacology 89 (2015) 225e231

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Low dose pramipexole causes D3 receptor-independent reduction of locomotion and responding for a conditioned reinforcer P.N. McCormick a, *, P.J. Fletcher a, b, V.S. Wilson a, J.D.C. Browne a, J.N. Nobrega a, b, c, d, G.J. Remington a, d a

Centre for Addiction and Mental Health, Toronto, ON, Canada Department of Psychology, University of Toronto, Toronto, ON, Canada Department of Pharmacology and Toxicology, University of Toronto, ON, Canada d Department of Psychiatry, University of Toronto, Toronto, ON, Canada b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 May 2014 Received in revised form 15 September 2014 Accepted 23 September 2014 Available online 13 October 2014

Pramipexole is a clinically important dopamine receptor agonist with reported selectivity for dopamine D3 receptors over other dopaminergic and non-dopaminergic sites. Many of its behavioural effects are therefore attributed to D3 receptor activity. Here we relate pramipexole's ex vivo D2 and D3 receptor binding (measured using [3H]-(þ)-PHNO binding experiments) to its effects on locomotion and operant responding for primary and conditioned reinforcers. We show that pramipexole has inhibitory behavioural effects on all three behaviours at doses that occupy D3 but not D2 receptor. However, these effects are 1) not inhibited by a D3 selective dose of the antagonist SB-277011-A, and 2) present in D3 receptor knockout mice. These results suggest that a pharmacological mechanism other than D3 receptor activity must be responsible for these behavioural effects. Finally, our receptor binding results also suggest that these behavioural effects are independent of D2 receptor activity. However, firmer conclusions regarding D2 involvement would be aided by further pharmacological or receptor knock-out experiments. The implications of our findings for the understanding of pramipexole's behavioural and clinical effects are discussed. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Pramipexole Dopamine D3 receptor Dopamine D2 receptor [3H]-(þ)-PHNO Locomotion Operant responding

1. Introduction Pramipexole is a dopaminergic agonist with interesting pharmacological and clinical profiles. Pharmacologically, pramipexole displays approximately 100-fold in vitro selectivity for dopamine D3 versus D2 receptors (Mierau et al., 1995; Millan et al., 2002) and greater levels of selectivity relative to other dopamine receptors (D4, D1, D5) and non-dopaminergic sites including serotonergic and adrenergic receptors (Millan et al., 2002). Pramipexole is the only clinically-approved dopaminergic agonist displaying this level of D3 receptor selectivity, and is used primarily to treat Parkinson's disease (Guttman, 1997; Shannon et al., 1997), restless leg syndrome (Montplaisir et al., 1999) and fibromyalgia (Holman and Myers, 2005), and is currently under investigation for treatment

* Corresponding author. Centre for Addiction and Mental Health, 250 College St., Room 320, Toronto, ON M5T 1R8, Canada. Tel.: þ1 416 535 8501x34719; fax: þ1 416 979 4292. E-mail addresses: [email protected], [email protected] (P.N. McCormick). http://dx.doi.org/10.1016/j.neuropharm.2014.09.026 0028-3908/© 2014 Elsevier Ltd. All rights reserved.

of depression (Corrigan et al., 2000), bipolar disorder (Goldberg et al., 2004) and schizophrenia (Kelleher et al., 2012). Pramipexole exhibits several side effects including excessive daytime sleepiness (Schlesinger and Ravin, 2003), sleep attacks (Frucht et al., 1999) and a range of more unusual behavioural disturbances collectively referred to as impulse control disorder (Aiken, 2007; Holman, 2009). Associated with chronic pramipexole treatment, impulse control disorder consists of sudden, drastic behavioural changes such as development of problem gambling (Kolla et al., 2010), exercise dependence (Vitale et al., 2010), and changes in sexual behaviour (Munhoz et al., 2009). The therapeutic and side effects of pramipexole are thought to result from its dopamine D3 receptor activity (Piercey, 1998). Several of pramipexole's behavioural effects in animals have also been attributed to this receptor (Collins et al., 2012, 2007, 2005). Furthermore, the interest in pramipexole as a psychiatric treatment stems in large part from the presumed benefits of D3 receptor stimulation (Aiken, 2007; Corrigan et al., 2000; Kelleher et al., 2012).

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The current study is part of a larger research program investigating D3 receptor mechanisms in operant behaviour, with the broader goal of assessing D3 receptor stimulation as a potential treatment for “motivational” impairments in psychiatric disorders. To these ends we examined pramipexole's effects on operant responding for a primary reinforcer (water) and a conditioned reinforcer (a water-predictive cue), behaviours representing basic operant processes. We related these behavioural effects to pramipexole's interaction with dopamine D2 and D3 receptors, measured using ex vivo [3H]-(þ)-PHNO binding experiments. The results of this investigation are discussed in the context of pramipexole's behavioural pharmacology as well its clinical mechanism of action. 2. Methods 2.1. Subjects The experiments were conducted with approval of the local Animal Care Committee and in accordance with the guidelines of the Canadian Council on Animal Care. Subjects were male SpragueeDawley rats, male wild-type C57BL/6J mice, or male D3 knockout (KO) mice from the C57BL/6J strain. Rats were purchased from Charles River Laboratories (Senneville, QC, Canada) and weighed 300e400 g during behavioural testing. Rats were pair housed under reversed lighting (lights on 9 pme9 am), and given unlimited access to food and water. During a one week acclimation period the animals were exposed repeatedly to handling by the experimenters. To reduce injection-related stress animals were given several mock injections and one actual injection of saline. Rats in the operant responding experiments were switched to single housing conditions several days prior to the first training session to prevent competition for water during water restriction. D3 mutant mice, weighing 24e30g, were bred and genotyped in house and were descendent from Jackson Laboratories stock (Bar Harbor, ME, USA). A mutant breeding colony was maintained by backcrossing to C57BL/6J wild-type mice. Homozygous D3 KO mice were obtained from heterozygous mating pairs. Offspring were pair housed with littermates post-weaning. 2.2. Pharmacological agents [3H]-(þ)-PHNO (64 Ci/mmol, 16 mM) was synthesized by Moravek Biochemicals (Brea, USA). Pramipexole and SB-277011-A were purchased from Toronto Research Chemicals (Toronto, Canada) and Abcam Biochemicals (Cambridge, UK) respectively. Saline was used as a vehicle solution, except for in experiments involving SB277011-A, in which 30% b-cyclodextrin (SigmaeAldrich) was used. Drugs were administered i.p. in a volume of 1 mL/kg (rat) or 5 mL/kg (mouse). Drugs were coinjected in experiments examining the effect of one drug on the behavioural effects of another. 2.3. Determination of drug occupancy of D2 and D3 receptors Full description of the ex vivo [3H]-(þ)-PHNO binding methodology can be found in McCormick et al., 2013. Briefly, animals were first injected i.p. with the experimental drug (one of various doses of pramipexole or SB-277011-A). One hour later animals were injected i.v. via a tail vein with a tracer dose of [3H]-(þ)-PHNO (0.1 nmol given as a 0.3 mL bolus in saline). One hour later the animals were sacrificed by decapitation and samples of dorsal striatum (STR), nucleus accumbens (NACC) and cerebellar lobes 9 and 10 (LOB) were excised into weighed plastic vials. These regions were chosen based on the pharmacology of their [3H]-(þ)-PHNO specific binding sites; D2 receptors in STR, a mixture of D2 and D3 receptors in NACC, and D3 receptors in LOB (McCormick et al., 2010, 2013). Additionally a sample of cerebellar cortex (CER) was collected to serve as a reference region devoid of either D2/D3 receptor expression (Martres et al., 1985; Stanwood et al., 2000). Five mL of 0.6 M NaOH was added and the tissue left to dissolve for 24 h at room temperature. Ten mL of Bio-Safe II scintillation fluid was then added and the samples were mixed and left in the dark for 24 h. Their 3H content was then determined by liquid scintillation spectrometry. The 3H content was first expressed as disintegrations per min per mg of tissue (DPM/mg). The specific binding ratio (SBR), representing the ratio of specific to nondisplaceable radiotracer binding, was then calculated as [(DPM/mg ROI) e (DPM/mg CER)] ÷ (DPM/mg CER). The DPM/mg in CER was also independently examined to assess drug effects on non-displaceably bound radiotracer (which can indicate changes in haemodynamic and/or metabolic factors). 2.4. Behavioural experiments In the operant behavioural experiments animals were restricted to 2 h of daily water access (3e5 pm) so as to render water and saccharin solution effective reinforcers. In the locomotion experiments animals were allowed unlimited access to food and water. In order to reduce animal numbers a within-subject, Latin squares design was used. Group sizes ranged from n ¼ 8 in locomotion experiments to n ¼ 16 in the operant experiments. Exact group sizes are given in the corresponding figures.

Drugs were administered i.p. 30 min prior to testing and at least 48 h were allowed for drug washout between sessions. 2.4.1. Responding for a conditioned reinforcer in rats These experiments took place in standard rat operant conditioning chambers (Med Associates Inc., St. Albans, USA) equipped with a white house light, two retractable levers, two red stimulus lights, a small speaker for delivery of auditory stimuli, and a liquid drop dispenser which delivered water into a recessed magazine. The first phase of the experiment, conducted with the levers retracted, consisted of 10 Pavlovian conditioning sessions in which an auditory-visual stimulus (the conditioned stimulus, CS) was associated with the delivery of ~50 mL of water (the unconditioned stimulus, US). The CS was a 5 s interval in which the house light was turned off and the red stimulus lights were illuminated, terminating in an auditory pulse. The US was delivered immediately following each CS presentation. The CS and US were paired 30 times per session on a random time 60 s (RT60) schedule. In the testing phase of the experiment CS presentation was used to reinforce lever pressing, i.e. it acted as a conditioned reinforcer (CR). Both levers were extended; pressing the left lever resulted in CR presentation, while pressing the right lever had no programmed consequence. The CR was identical to the CS in the conditioning phase, but was never followed by delivery of water. Prior to testing, the animals were given a lever habituation session in which responses on the active lever were reinforced on a random ratio 2 (RR2) schedule. This session ended either after 10 active lever responses or when 40 min had elapsed. Four 40 min testing sessions followed in which active lever responses were reinforced on an RR2 schedule, whereas inactive lever presses had no programmed consequence. Test sessions were preceded by drug or vehicle treatment. 2.4.2. Responding for a conditioned reinforcer in mice These experiments took place in mouse operant conditioning chambers (Med Associates Inc., St. Albans, USA) equipped with a white house light, two retractable levers, two red stimulus lights, a small speaker for delivery of auditory stimuli, and a liquid dipper dispenser which raised 20 ml of liquid into a recessed magazine. Saccharin solution (0.2%) served as the US. Mice were given a 30 min habituation session in order that they learn to acquire saccharin from the dipper; the dipper was raised to present saccharin and descended 5s after the animal's head entered the magazine, allowing up to 60 presentations on an RT30 schedule. Ten 40 min Pavlovian conditioning sessions followed in which 40 CS-US pairings were presented on an RT60 schedule. The CS consisted of a 5 s period in which the houselight was turned off and stimulus lights were illuminated. The US, which immediately followed the CS, consisted of 8 s of access to saccharin solution. The illumination conditions during the US were identical to those during the CS (i.e. houselight off, stimulus lights illuminated). Four 40 min CR sessions were then conducted, with both levers extended, preceded by drug or vehicle treatment. Pressing the left lever resulted in delivery of the CR on an RR2 schedule, whereas there was no consequence to pressing the right lever. The CR consisted of a 5s period in which the houselight was turned off and stimulus lights on, and terminated in 2 s access to the elevated, empty dipper. 2.4.3. Responding for water in rats The animals in this experiment were the same subjects used in the previous experiment examining responding for a conditioned reinforcer. Only the right lever was extended. Animals were first trained to lever press on a fixed ratio 1 (FR1) schedule in which each response resulted in the delivery of ~50 mL of water. After two 30 min FR1 sessions the schedule was changed to RR2. Four 30 min RR2 sessions followed to allow responding levels to stabilize. Following these stabilization sessions were four 30 min drug/vehicle testing sessions in which the animals responded for water on an RR2 schedule. 2.4.4. Measurement of locomotion in rats Experiments measuring locomotion were conducted in infrared actimeter boxes. These were transparent plexiglass cages with a floor area measuring 21  43 cm, equipped with 12 horizontal infrared beams. One 30 min habituation session was followed by four 1 h testing sessions preceded by drug or vehicle treatment. Locomotion was quantified as the total infrared beam breaks per session. 2.5. Statistical analysis Statistical analyses were conducted using SPSS version 15. Radiotracer binding data were analysed using one factor ANOVA followed by post hoc Dunnett's test. Behavioural data were analysed using repeated measures ANOVA. In the experiments examining responding for a conditioned reinforcer two within subject factors (treatment and lever) were included, whereas in experiments examining locomotion or responding for water there was only a single within subject factor (treatment). Post hoc pairwise comparisons were done using Bonferroni multiplecomparison-corrected t tests. In the experiments examining responding for the conditioned reinforcer, where a significant treatment  lever interaction was found, post hoc comparisons were done separately on left and right lever data. Means were considered significantly different when p < 0.05.

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3. Results 3.1. Determination of drug occupancy of D2 and D3 receptors Fig. 1 shows the effect of various doses of pramipexole and SB277011-A on regional [3H]-(þ)-PHNO binding in brain. There was a significant effect of treatment (pramipexole dose) on [3H]-(þ)-PHNO SBR in STR (F(3,36) ¼ 59.87, p < 0.0001), NACC (F(3,36) ¼ 31.18, p < 0.0001) and LOB (F(3,36) ¼ 23.08, p < 0.0001), as well as on [3H]-(þ)-PHNO DPM/mg in CER (F(3,36) ¼ 6.49, p ¼ 0.0013). Pairwise comparisons indicated that pramipexole inhibited [3H]-(þ)-PHNO SBR in LOB at all three doses tested (Fig. 1, C), whereas only the highest dose was able to inhibit [3H](þ)-PHNO SBR in STR and NACC (Fig. 1, A and B). At the two highest doses pramipexole also reduced [3H]-(þ)-PHNO DPM/mg in CER (Fig. 1, D). There was a significant effect of SB-277011-A on [3H]-(þ)-PHNO only in LOB (F(3,27) ¼ 35.13, p < 0.0001) and NACC (F(3,27) ¼ 5.77, p ¼ 0.0035). Pairwise comparisons indicated that SB-277011-A inhibited [3H]-(þ)-PHNO SBR in LOB at all doses (Fig. 1, G), but inhibited [3H]-(þ)-PHNO SBR in NACC only at the highest dose (Fig. 1, NACC) and failed to inhibit [3H]-(þ)-PHNO SBR in STR (Fig. E). SB-277011-A had no effect on [3H]-(þ)-PHNO DPM/mg in CER (Fig. 1, H).

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versus inactive; F(1,27) ¼ 53.63, p < 0.001) as well as a significant treatment by lever interaction (F(3,81) ¼ 9.18, p < 0.001). Pairwise comparisons showed that the lowest dose of pramipexole (0.01 mg/ kg) had no effect on responding, whereas the 0.1 and 1 mg/kg doses reduced responding on both active and inactive levers (Fig. 2 A). SB-277011-A had no effect on lever responding at any administered dose (Fig. 3 A) (F(3,45) ¼ 1.13, p ¼ 0.348), while a significant main effect of lever was found (F(1,15) ¼ 53.95, p < 0.001). In the pramipexole plus SB-277011-A co-administration experiment there was a significant main effect of treatment (F(2,23) ¼ 17.09, p < 0.001) and lever (F(1,14) ¼ 37.11, p < 0.001) and a significant treatment by lever interaction (F(2,28) ¼ 15.08, p < 0.001). Pairwise comparisons confirmed that the reduction of responding induced by 0.1 mg/kg pramipexole was not affected by co-administration of 10 mg/kg SB-277011-A (Fig. 3 B). Additionally, in both wild type and D3 receptor knockout mice (Fig. 4) there was a significant main effect of treatment (wild type, F(3,33) ¼ 5.93, p ¼ 0.002; knockout, F(3,33) ¼ 31.88, p < 0.001) and lever (wild type, F(1,11) ¼ 6.76, p ¼ 0.025; knockout, F(1,11) ¼ 7.60, p < 0.019), and a significant treatment by lever interaction (wild type, F(3,33) ¼ 4.32, p ¼ 0.011; knockout, F(3,33) ¼ 4.26, p < 0.012). Pairwise comparisons indicated that in wild type mice the reduction of responding reached significance at 1 mg/kg whereas in the knockout mice both the 0.1 and 1 mg/kg doses caused significant reduction in responding (Fig. 4).

3.2. Behavioural experiments 3.2.1. Responding for a conditioned reinforcer The results of the conditioned reinforcement experiments are shown in Figs. 2e4. There was a significant main effect of treatment (pramipexole dose; F(3,81) ¼ 14.43, p < 0.001) and lever (active

3.2.2. Responding for water The effect of pramipexole on lever responding for water reinforcement can be seen in Fig. 2 B. There was a significant effect of pramipexole dose on lever responses (F(3,33) ¼ 26.21, p < 0.001).

Fig. 1. Inhibition of regional [3H]-(þ)-PHNO binding by pramipexole (AeD) and SB-277011-A (EeH). For pramipexole group sizes were: VEH and 0.01 mg/kg, n ¼ 14; 0.1 and 1 mg/ kg, n ¼ 6. For SB-277011-A group sizes were n ¼ 8 per dose including VEH. Abbreviations: STR, striatum; NACC, nucleus accumbens; LOB, cerebellar lobes 9 and 10; CER, cerebellar cortex; REF, reference region; SBR, specific binding ratio; DPM/mg, disintegrations per minute per mg of tissue; VEH, vehicle. Statistical symbols indicate comparison to the VEH group: *, p < 0.05; **, p < 0.01.

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Fig. 2. Behavioural effects of pramipexole in rat: A) responding for a conditioned reinforcer (n ¼ 28, two cohorts of n ¼ 12 and 16); B) responding for water (n ¼ 12); C) locomotion (n ¼ 16). Abbreviations: VEH, vehicle. Active and Inactive refer to the levers associated with presentation of the conditioned reinforcer or no programmed consequence, respectively. Statistical symbols indicate comparison to the VEH group: ***, p < 0.001.

Consideration of pairwise comparisons indicated that only the two highest doses of pramipexole significantly affected lever responses.

Fig. 3. Behavioural effects of SB-277011-A: A) lack of effect of SB-277011-A on responding for a conditioned reinforcer (n ¼ 16); B) lack of effect of SB-277011-A on pramipexole-induced reduction in responding for a conditioned reinforcer (n ¼ 16); C) lack of effect of SB-277011-A on locomotion and pramipexole-induced inhibition of locomotion (n ¼ 8). In addition to the statistical results shown symbolically it should be noted that no differences were found between PRAM and PRAM þ SB group in active lever responding, inactive lever responding or locomotion. Abbreviations: VEH vehicle; PRAM, 0.1 mg/kg pramipexole; SB, 10 mg/kg SB-277011-A. Active and Inactive refer to the levers associated with presentation of the conditioned reinforcer or no programmed consequence, respectively. Statistical symbols indicate comparison to the VEH group: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

4. Discussion 3.2.3. Locomotion The effect of pramipexole on locomotion can be seen in Fig. 2 C. There was a significant effect of dose (F(3,45) ¼ 32.92, p < 0.001), and pairwise comparisons indicated that the highest two doses significantly affected locomotion. Pramipexole's effects on locomotion were bidirectional, inhibiting locomotion at the medium dose while increasing locomotion at the highest dose to levels greater than those seen in the vehicle treated group. In the pramipexole plus SB-277011-A co-administration experiment (Fig. 3 C) there was a significant main effect of treatment (F(3,21) ¼ 7.71, p < 0.001). Pairwise comparisons indicated that SB-277011-A alone had no effect on locomotion, nor did it affect inhibition of locomotion induced by pramipexole.

The interpretation that will be advanced here is that pramipexole, when administered at D3 receptor-occupying doses, has behavioural effects that are pharmacologically independent of the D3 receptor. This interpretation rests on several key findings. Our radiotracer binding results demonstrate that there are behaviourally active doses of pramipexole that occupy D3 but not D2 receptors suggesting, firstly, that D2 is not a direct site of action at these doses. Secondly, our experiments utilizing the D3 selective antagonist SB-277011-A and D3 knockout mice indicate that the behavioural effects of pramipexole are also independent of D3 receptor activity. These findings and their implications are discussed below.

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Fig. 4. Effect of pramipexole on responding for a conditioned reinforcer in A) wild type mice (n ¼ 12), and B) D3 receptor knockout mice (n ¼ 12). Abbreviations: VEH, vehicle. Active and Inactive refer to the levers associated with presentation of the conditioned reinforcer or no programmed consequence, respectively. Statistical symbols indicate comparison to the VEH group: *, p < 0.05; ***, p < 0.001.

Our receptor binding experiments indicate that both pramipexole and SB-277011-A are selective for dopamine D3 over D2 receptors, as indicated by the higher occupancy seen in the D3rich cerebellum lobes 9 & 10 compared to the D2-rich striatum (Fig. 1). Moreover, for both drugs a range of doses was found that resulted in substantial occupancy of D3 receptors (i.e reduced [3H]-(þ)-PHNO binding in cerebellum lobes 9 & 10) but no measureable D2 receptor occupancy (reduced striatal binding) (Fig. 1). For pramipexole this range had an upper boundary between 0.1 and 1 mg/kg (the lowest dose to produce D2 occupancy). While not meant to precisely quantify selectivity, these results suggest a D3 over D2 selectivity of between 10- and 100fold; in general agreement with in vitro reports (Mierau et al., 1995; Millan et al., 2002). For SB-277011-A no D2 occupancy was found at doses as high as 15 mg/kg, the highest dose administered. This is in agreement with a recent paper in which doses up to 30 mg/kg resulted in D3 but not D2 receptor occupancy (Barth et al., 2013). Interestingly, the lowest dose of pramipexole (0.01 mg/kg) caused an increase in [3H]-(þ)-PHNO binding to striatal D2 receptors. This could potentially be explained by an autoreceptor action of pramipexole; i.e., by lowering extracellular dopamine concentration pramipexole reduces competition between dopamine and [3H]-(þ)-PHNO, resulting in increased radiotracer binding. This speculative interpretation is supported by data demonstrating that pramipexole decreases striatal extracellular dopamine concentration measured by in vivo microdialysis (Carter and Muller, 1991). While determining the pharmacological identity of this autoreceptor site was not a goal of this study, our binding data would more strongly implicate D3 than D2 receptors in this regard. It is also unclear from our data whether the increase in [3H](þ)-PHNO binding seen after pramipexole treatment is related to the behavioural effects we observe. At all doses tested pramipexole reduced operant responding (see Fig. 2). This does not appear to be the result of direct D2 receptor stimulation since D2 occupancy was seen only at the higher of the two behaviourally active doses. In the locomotion experiments pramipexole also had an inhibitory effect at a dose (0.1 mg/ kg) which did not result in D2 occupancy. These results suggest that the reduced operant responding and reduced locomotion induced by pramipexole at low doses are independent of direct D2 receptor stimulation. Li et al. have attempted to clarify in mice the role of D2 receptors in the reduction of locomotion caused by several D2/D3 agonists (Li et al., 2010). They found, firstly, that this effect could not be blocked by the D2 antagonist L-74126, a difficult finding to interpret given that L-74126 itself has an inhibitory effect on locomotion which may have been conflated with any effects on agonist-induced activity. Secondly, they found that agonist-induced reduction of

locomotion appeared absent in D2 receptor knockout mice. This does not indicate, however, whether the D2 receptor is the binding site of these drugs at the doses in question or whether its role is, alternatively, in events downstream of drugereceptor interactions. With respect to the current data we have found no evidence for occupancy of D2 receptors at doses that inhibit both locomotion and operant responding. We have not, however, directly tested whether the behavioural effects of this dose are attributable to D2 occupancy below the limit of detection of the current method. This might result, for example, from a higher affinity of pramipexole for one splice variant of the D2 receptor or for presynaptic versus postsynaptic D2 receptors. We consider it unlikely that consideration of splice variants is important for interpretation of our results given that pramipexole's has very similar affinity for D2 splice variants, at least in human cloned D2 receptors (Millan et al., 2002). Consideration of presynaptic versus postsynaptic D2 receptors would be necessary only in the event that pramipexole and [3H]-(þ)-PHNO had very different relative affinities for presynaptic versus postsynaptic receptors, i.e. that they bind to different populations of D2 receptors, making interpretation of displacement/competition data difficult. We know of no published results that could shed light on this point. An additional point worth considering is the pharmacokinetics of [3H]-(þ)-PHNO. After bolus injection [3H]-(þ)-PHNO brain (and plasma) concentrations peak in less than 5 min and then fall for the remainder of the experiment (Wilson et al, 2005). Under these conditions [3H](þ)-PHNO SBR would be relatively insensitive to decreases in pramipexole receptor occupancy occurring over the course of the binding experiment since there is no net brain influx to fuel the corresponding increase in [3H]-(þ)-PHNO binding signal that would be required for detection of this occupancy change. Thus it is unlikely that our results represent an underestimation of D2 (or D3) receptor occupancy; indeed, the levels of occupancy reported likely represent the highest to occur during our binding experiment. Finally, from an explanatory point of view involvement of the D2 receptors in the behvaioural effects of low dose pramipexole would require postulation of a biphasic effect of D2 receptor stimulation on locomotion, with reduced activity seen at doses producing low occupancy (ie < 0.1 mg/kg) and increased activity at doses producing high occupancy (>0.1 mg/kg). This represents a less parsimonious and therefore less favourable account of the data presented here. The D3 antagonist SB-277011-A had no effect on operant responding or locomotion at doses that resulted in large D3 receptor occupancy. The lack of effect on locomotion is consistent with published reports (Reavill et al., 2000; Ross et al., 2007). It is less clear how our conditioned reinforcement results relate to published reports. In general it has been found that SB-277011 and other D3 antagonists disrupt the ability of drugs of abuse and

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associated discriminative stimuli to reinstate drug self administration after extinction (Andreoli et al., 2003; Cervo et al., 2007; Di Ciano et al., 2003; Gal and Gyertyan, 2006; Gilbert et al., 2005; Heidbreder et al., 2007; Vengeliene et al., 2006; Xi et al., 2004). There is little evidence however for effects of D3 antagonism on operant responding for, or discriminative stimuli associated with, non-drug reinforcement (e.g. food or sucrose) (Cervo et al., 2007; Di Ciano et al., 2003; Gal and Gyertyan, 2006). Our results suggest that D3 receptors are not involved in operant responding under the current conditions and fit generally with the observation that D3 receptor antagonism is without effect in non-drug reinforcement. For current purposes the important finding is that SB-277011-A is behaviourally silent under the conditions described here, making it a useful probe of pramipexole's pharmacology. Co-administration of SB-277011-A did not alter pramipexole's inhibitory effects on operant responding or locomotion (see Fig. 3), suggesting that the mechanisms mediating these effects are independent of the D3 receptor. This interpretation is strongly supported by pramipexole's behavioural effects in D3 knockout mice (Fig. 4). The lack of effect of SB-277011-A also suggests that pramipexole's behavioural effects do not result from D3 receptor mediated changes in extracellular dopamine, discussed above in the context of the radiotracer binding results, since this as well would be inhibited by a D3 receptor antagonist. If the inhibitory behavioural effects of pramipexole measured here are not due to D3 or D2 receptor activity then what other mechanisms might be involved? According to in vitro reports pramipexole's next highest affinity binding sites are the dopamine D4 and adrenergic a2 receptors (Millan et al., 2002). Behaviourally, D4 is one of the least explored dopaminergic receptors and it is therefore difficult to assess the relevance of this receptor to the current results. There is, however, evidence implicating the adrenergic system in the in vivo pharmacology of pramipexole. For example, Chernoloz et al. report that pramipexole inhibits adrenergic neuronal firing rate (Chernoloz et al., 2009). Brooks and Weinstock (1991) found that pramipexole has hypotensive effects that can be blocked by the a2 antagonist rauwolscine. Currently, we are using a2 selective antagonists to further probe the behavioural pharmacology of pramipexole. Our findings have important implications for the pharmacological interpretation of studies describing pramipexole's behavioural effects at similar doses. Several recent reports are especially relevant in terms of dosing and pharmacological interpretability. Collins et al. (2012) examined pramipexole's behavioural effects in the context of cocaine self-administration and found that pramipexole substituted for cocaine in the presence of cocaine-associated cues. Furthermore, the rate of responding maintained by pramipexole peaked at a dose of 0.1 mg/kg (and declined at higher doses), suggesting that this was a D3 not D2 receptor mediated effect. However, as we have shown here, pramipexole has behavioural effects at this dose that are D3 independent. Thus further evidence is required to establish the pharmacology of pramipexole's substitution for cocaine. A second finding of the Collins et al. study was that pramipexole increased operant responding for a cocaineassociated conditioned reinforcer at doses of >0.32 mg/kg, but not at lower doses. The authors provide pharmacological evidence that this is a D2 receptor mediated effect, an interpretation supported by the current radiotracer binding data. Other studies by Collins et al. examined the effect of putative D3 selective agonists on yawning, body temperature and penile erection in rats (Collins et al., 2007, 2009, 2005). For pramipexole they found that rates of yawning and penile erection increased at D3 occupying doses (peaking at 0.1 mg/kg) whereas hypothermia was only induced at D2 occupying doses (>0.1 mg/kg). Similar patterns of response were seen for other agonists including PD-128907, 7-

OH-DPAT and quinpirole. Collins et al. further demonstrated that agonist-induced yawning and penile erection could be inhibited by SB-277011-A and that hypothermia could be inhibited by the D2 selective antagonist L-741,626, respectively, suggesting the pharmacological assignment of these effects to D3 and D2 receptors. While the pharmacological evidence supporting D2 mediation of hypothermia seems clear, two important features of their data cast doubt on the interpretation of yawning and penile erection as D3 mediated. Firstly, agonist-induced yawning was inhibited only by very high doses (32 mg/kg) of SB-277011-A which, according to our results, would result in complete saturation of D3 receptors, and not by lower more pharmacologically relevant doses. It is unclear why this should be the case if agonist-induced yawning is D3 mediated. More importantly, Collins et al. report that the inhibitory effect of SB-277011-A and other D3 antagonists on agonist-induced yawning and penile erection consists of a downward shift in the agonist doseeresponse curve without apparent change in potency (Collins et al., 2005). These are the defining features of noncompetitive inhibition, indicating an agonist site of action different from that of the inhibitor. Thus, the inhibition data of Collins et al. actually suggest that the D3 mechanism by which the antagonists inhibit agonist-induced yawning and penile erection is not the mechanism by which they were induced. That is, the data argue for a non-D3 mechanism mediating agonist-induced yawning and penile erection. Together with the current findings, these arguments warrant further exploration of the behavioural pharmacology of pramipexole and other dopaminergic agonists. Finally, the current results suggest a rethinking of pramipexole's clinical pharmacology. If the dose-occupancy data reported here translate to humans, clinical doses of pramipexole ranging up to 0.1 mg/kg (i.e. 4.5 mg in a 70 kg individual) would primarily occupy D3 as opposed to D2 receptors. However, as we have shown this does not imply that D3 receptors mediate the observed effects. While it is intuitively attractive to credit dopaminergic mechanisms for many of pramipexole's clinical benefits (e.g. dopamine mimetic effects in Parkinson's disease) and side effects (e.g. development of problem gambling and other “motivational” disturbances) the current study emphasizes that without direct pharmacological evidence such conclusion are premature. Funding and disclosure In the last three years, GJ Remington has received research support from the Canadian Diabetes Association, the Canadian Institutes of Health Research, Medicure, Neurocrine Biosciences, Novartis, Research Hospital FundeCanada Foundation for Innovation, and the Schizophrenia Society of Ontario and has served as a uticos consultant or speaker for Novartis, Laboratorios Farmace Rovi, Synchroneuron, and Roche. Acknowledgements The authors would like to thank Tamara Arenovich for statistical advice and Dr. Alan Wilson for helpful discussions regarding interpretation of the data. References Aiken, C.B., 2007. Pramipexole in psychiatry: a systematic review of the literature. J. Clin. Psychiatry 68 (8), 1230e1236. Andreoli, M., Tessari, M., Pilla, M., Valerio, E., Hagan, J.J., Heidbreder, C.A., 2003. Selective antagonism at dopamine D3 receptors prevents nicotine-triggered relapse to nicotine-seeking behavior. Neuropsychopharmacology 28 (7), 1272e1280. Barth, V., Need, A.B., Tzavara, E.T., Giros, B., Overshiner, C., Gleason, S.D., et al., 2013. In vivo occupancy of dopamine D3 receptors by antagonists produces neurochemical and behavioral effects of potential relevance to attention-deficithyperactivity disorder. J. Pharmacol. Exp. Ther. 344 (2), 501e510.

P.N. McCormick et al. / Neuropharmacology 89 (2015) 225e231 Brooks, D.P., Weinstock, J., 1991. The pharmacology of pramipexole in the spontaneously hypertensive rat. Eur. J. Pharmacol. 200 (2e3), 339e341. Carter, A.J., Muller, R.E., 1991. Pramipexole, a dopamine D2 autoreceptor agonist, decreases the extracellular concentration of dopamine in vivo. Eur. J. Pharmacol. 200 (1), 65e72. Cervo, L., Cocco, A., Petrella, C., Heidbreder, C.A., 2007. Selective antagonism at dopamine D3 receptors attenuates cocaine-seeking behaviour in the rat. Int. J. Neuropsychopharmacol. 10 (2), 167e181. Chernoloz, O., El Mansari, M., Blier, P., 2009. Sustained administration of pramipexole modifies the spontaneous firing of dopamine, norepinephrine, and serotonin neurons in the rat brain. Neuropsychopharmacology 34 (3), 651e661. Collins, G.T., Cunningham, A.R., Chen, J., Wang, S., Newman, A.H., Woods, J.H., 2012. Effects of pramipexole on the reinforcing effectiveness of stimuli that were previously paired with cocaine reinforcement in rats. Psychopharmacol. Berl. 219 (1), 123e135. Collins, G.T., Newman, A.H., Grundt, P., Rice, K.C., Husbands, S.M., Chauvignac, C., et al., 2007. Yawning and hypothermia in rats: effects of dopamine D3 and D2 agonists and antagonists. Psychopharmacol. Berl. 193 (2), 159e170. Collins, G.T., Truccone, A., Haji-Abdi, F., Newman, A.H., Grundt, P., Rice, K.C., et al., 2009. Proerectile effects of dopamine D2-like agonists are mediated by the D3 receptor in rats and mice. J. Pharmacol. Exp. Ther. 329 (1), 210e217. Collins, G.T., Witkin, J.M., Newman, A.H., Svensson, K.A., Grundt, P., Cao, J., et al., 2005. Dopamine agonist-induced yawning in rats: a dopamine D3 receptormediated behavior. J. Pharmacol. Exp. Ther. 314 (1), 310e319. Corrigan, M.H., Denahan, A.Q., Wright, C.E., Ragual, R.J., Evans, D.L., 2000. Comparison of pramipexole, fluoxetine, and placebo in patients with major depression. Depress Anxiety 11 (2), 58e65. Di Ciano, P., Underwood, R.J., Hagan, J.J., Everitt, B.J., 2003. Attenuation of cuecontrolled cocaine-seeking by a selective D3 dopamine receptor antagonist SB-277011-A. Neuropsychopharmacology 28 (2), 329e338. Frucht, S., Rogers, J.D., Greene, P.E., Gordon, M.F., Fahn, S., 1999. Falling asleep at the wheel: motor vehicle mishaps in persons taking pramipexole and ropinirole. Neurology 52 (9), 1908e1910. Gal, K., Gyertyan, I., 2006. Dopamine D3 as well as D2 receptor ligands attenuate the cue-induced cocaine-seeking in a relapse model in rats. Drug Alcohol Depend. 81 (1), 63e70. Gilbert, J.G., Newman, A.H., Gardner, E.L., Ashby Jr., C.R., Heidbreder, C.A., Pak, A.C., et al., 2005. Acute administration of SB-277011A, NGB 2904, or BP 897 inhibits cocaine cue-induced reinstatement of drug-seeking behavior in rats: role of dopamine D3 receptors. Synapse 57 (1), 17e28. Goldberg, J.F., Burdick, K.E., Endick, C.J., 2004. Preliminary randomized, doubleblind, placebo-controlled trial of pramipexole added to mood stabilizers for treatment-resistant bipolar depression. Am. J. Psychiatry 161 (3), 564e566. Guttman, M., 1997. Double-blind comparison of pramipexole and bromocriptine treatment with placebo in advanced Parkinson's disease. International Pramipexole-Bromocriptine Study Group. Neurology 49 (4), 1060e1065. Heidbreder, C.A., Andreoli, M., Marcon, C., Hutcheson, D.M., Gardner, E.L., Ashby Jr., C.R., 2007. Evidence for the role of dopamine D3 receptors in oral operant alcohol self-administration and reinstatement of alcohol-seeking behavior in mice. Addict. Biol. 12 (1), 35e50. Holman, A.J., 2009. Impulse control disorder behaviors associated with pramipexole used to treat fibromyalgia. J. Gambl. Stud. 25 (3), 425e431. Holman, A.J., Myers, R.R., 2005. A randomized, double-blind, placebo-controlled trial of pramipexole, a dopamine agonist, in patients with fibromyalgia receiving concomitant medications. Arthritis Rheum. 52 (8), 2495e2505. Kelleher, J.P., Centorrino, F., Huxley, N.A., Bates, J.A., Drake, J.K., Egli, S., et al., 2012. Pilot randomized, controlled trial of pramipexole to augment antipsychotic treatment. Eur. Neuropsychopharmacol. 22 (6), 415e418. Kolla, B.P., Mansukhani, M.P., Barraza, R., Bostwick, J.M., 2010. Impact of dopamine agonists on compulsive behaviors: a case series of pramipexole-induced pathological gambling. Psychosomatics 51 (3), 271e273.

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Li, S.M., Collins, G.T., Paul, N.M., Grundt, P., Newman, A.H., Xu, M., et al., 2010. Yawning and locomotor behavior induced by dopamine receptor agonists in mice and rats. Behav. Pharmacol. 21 (3), 171e181. Martres, M.P., Sales, N., Bouthenet, M.L., Schwartz, J.C., 1985. Localisation and pharmacological characterisation of D-2 dopamine receptors in rat cerebral neocortex and cerebellum using [125I]iodosulpride. Eur. J. Pharmacol. 118 (3), 211e219. McCormick, P.N., Kapur, S., Graff-Guerrero, A., Raymond, R., Nobrega, J.N., Wilson, A.A., 2010. The antipsychotics olanzapine, risperidone, clozapine, and haloperidol are D2-selective ex vivo but not in vitro. Neuropsychopharmacology 35 (8), 1826e1835. McCormick, P.N., Wilson, V.S., Wilson, A.A., Remington, G.J., 2013. Acutely administered antipsychotic drugs are highly selective for dopamine D2 over D3 receptors. Pharmacol. Res. 70 (1), 66e71. Mierau, J., Schneider, F.J., Ensinger, H.A., Chio, C.L., Lajiness, M.E., Huff, R.M., 1995. Pramipexole binding and activation of cloned and expressed dopamine D2, D3 and D4 receptors. Eur. J. Pharmacol. 290 (1), 29e36. Millan, M.J., Maiofiss, L., Cussac, D., Audinot, V., Boutin, J.A., Newman-Tancredi, A., 2002. Differential actions of antiparkinson agents at multiple classes of monoaminergic receptor. I. A multivariate analysis of the binding profiles of 14 drugs at 21 native and cloned human receptor subtypes. J. Pharmacol. Exp. Ther. 303 (2), 791e804. Montplaisir, J., Nicolas, A., Denesle, R., Gomez-Mancilla, B., 1999. Restless legs syndrome improved by pramipexole: a double-blind randomized trial. Neurology 52 (5), 938e943. Munhoz, R.P., Fabiani, G., Becker, N., Teive, H.A., 2009. Increased frequency and range of sexual behavior in a patient with Parkinson's disease after use of pramipexole: a case report. J. Sex. Med. 6 (4), 1177e1180. Piercey, M.F., 1998. Pharmacology of pramipexole, a dopamine D3-preferring agonist useful in treating Parkinson's disease. Clin. Neuropharmacol. 21 (3), 141e151. Reavill, C., Taylor, S.G., Wood, M.D., Ashmeade, T., Austin, N.E., Avenell, K.Y., et al., 2000. Pharmacological actions of a novel, high-affinity, and selective human dopamine D3 receptor antagonist, SB-277011-A. J. Pharmacol. Exp. Ther. 294 (3), 1154e1165. Ross, J.T., Corrigall, W.A., Heidbreder, C.A., LeSage, M.G., 2007. Effects of the selective dopamine D3 receptor antagonist SB-277011A on the reinforcing effects of nicotine as measured by a progressive-ratio schedule in rats. Eur. J. Pharmacol. 559 (2e3), 173e179. Schlesinger, I., Ravin, P.D., 2003. Dopamine agonists induce episodes of irresistible daytime sleepiness. Eur. Neurol. 49 (1), 30e33. Shannon, K.M., Bennett Jr., J.P., Friedman, J.H., 1997. Efficacy of pramipexole, a novel dopamine agonist, as monotherapy in mild to moderate Parkinson's disease. The Pramipexole Study Group. Neurology 49 (3), 724e728. Stanwood, G.D., Artymyshyn, R.P., Kung, M.P., Kung, H.F., Lucki, I., McGonigle, P., 2000. Quantitative autoradiographic mapping of rat brain dopamine D3 binding with [(125)I]7-OH-PIPAT: evidence for the presence of D3 receptors on dopaminergic and nondopaminergic cell bodies and terminals. J. Pharmacol. Exp. Ther. 295 (3), 1223e1231. Vengeliene, V., Leonardi-Essmann, F., Perreau-Lenz, S., Gebicke-Haerter, P., Drescher, K., Gross, G., et al., 2006. The dopamine D3 receptor plays an essential role in alcohol-seeking and relapse. Faseb J. 20 (13), 2223e2233. Vitale, C., Santangelo, G., Verde, F., Amboni, M., Sorrentino, G., Grossi, D., et al., 2010. Exercise dependence induced by pramipexole in Parkinson's Disease-a case report. Mov. Disord. 25 (16), 2893e2894. Wilson, A.A. , McCormick, P., Kapur, S., Willeit, M., Garcia, A., Hussey, D., Houle, S., Seeman, P., Ginovart, N., 2005. Radiosynthesis and evaluation of [11C]-(þ)-4propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol as a potential radiotracer for in vivo imaging of the dopamine D2 high-affinity state with positron emission tomography. J. Med. Chem. 48 (12), 4153e4160. Xi, Z.X., Gilbert, J., Campos, A.C., Kline, N., Ashby Jr., C.R., Hagan, J.J., et al., 2004. Blockade of mesolimbic dopamine D3 receptors inhibits stress-induced reinstatement of cocaine-seeking in rats. Psychopharmacol. Berl. 176 (1), 57e65.