PDE10A inhibitors stimulate or suppress motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways

Abstract

The enzyme phosphodiesterase 10A (PDE10A) regulates the activity of striatal, medium spiny neurons (MSNs), which are divided into a behaviorally stimulating, Gs-coupled D₁ receptor-expressing “direct” pathway and a behaviorally suppressant, Gi-coupled D₂ receptor-expressing “indirect” pathway. Activating both pathways, PDE10A inhibitors (PDE10AIs) combine functional characteristics of D₂ antagonists and D₁ agonists. While the effects of PDE10AIs on spontaneous and stimulated behavior have been extensively reported, the present study investigates their effects on suppressed behavior under various conditions of reduced dopaminergic neurotransmission: blockade of D₁ receptors with SCH-23390, blockade of D₂ receptors with haloperidol, or depletion of dopamine with RO-4-1284 or reserpine. In rats, PDE10AIs displayed relatively low cataleptic activity per se. After blocking D₁ receptors, however, they induced pronounced catalepsy at low doses close to those required for inhibition of apomorphine-induced behavior; slightly higher doses resulted in behavioral stimulant effects, counteracting the catalepsy. PDE10AIs also counteracted catalepsy and related behaviors induced by D₂ receptor blockade or dopamine depletion; catalepsy was replaced by behavioral stimulant effects under the latter but not the former condition. Similar interactions were observed at the level of locomotion in mice. At doses close to those inhibiting d-amphetamine-induced hyperlocomotion, PDE10AIs reversed hypolocomotion induced by D₁ receptor blockade or dopamine depletion but not hypolocomotion induced by D₂ receptor blockade. It is concluded that PDE10AIs stimulate or inhibit motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways.

Introduction

The enzyme phosphodiesterase 10A (PDE10A) modulates signal transduction by catabolizing the intracellular second messengers cAMP and cGMP. PDE10A has higher affinity for cAMP and is more efficient with cAMP as substrate (Fujishige et al. 1999; Soderling et al. 1999). PDE10A is expressed primarily in the brain, particularly in the striatum, nucleus accumbens, and olfactory tubercle, regulating the activity of dopamine-sensitive striatal medium spiny neurons (MSNs) (Loughney et al. 1999; Seeger et al. 2003; Coskran et al. 2006; Sano et al. 2008).

Striatal MSNs are organized into a dopamine D₁ receptor-expressing “direct” (striatonigral) output pathway and a dopamine D₂ receptor-expressing “indirect” (striatopallidal) output pathway (Graybiel 1990, 2000). Action selection and execution takes place via the direct pathway, whereas action suppression is mediated via the indirect pathway (Delong and Wichmann 2007; Wilson 2014). Both pathways integrate behaviorally relevant information received via cortical and thalamic glutamatergic projections with input regarding the salience of this information from mid-brain dopaminergic neurons (Surmeier et al. 2007). Processed information is ultimately fed back to the frontal cortex via both pathways (Delong and Wichmann 2007; Wilson 2014), which act like the brake and accelerator in a car (Graybiel 2000).

All currently available antipsychotics block D₂ receptors and, by reducing dopamine-evoked cAMP decrease via Gi-coupled D₂ receptors in the indirect pathway, they activate only the indirect pathway (Kapur and Mamo 2003; Agid et al. 2008). By increasing cAMP, PDE10A inhibitors (PDEA10Is) activate both the Gi-coupled D₂ receptor-expressing indirect pathway and the Gs-coupled D₁ receptor-expressing direct pathway (Nishi et al. 2008; Strick et al. 2010), thereby combining functional characteristics of D₂ antagonists and D₁ agonists. Accordingly, PDE10AIs have been proposed as a novel type of antipsychotics with a broader therapeutic profile and reduced side effects (Siuciak et al. 2006; Kehler et al. 2007; Menniti et al. 2007; Siuciak 2008). However, PDE10AIs may have effects beyond D₁- and D₂-mediated neurotransmission, also affecting striatal glutamate–NO–guanylyl cyclase signaling (Loughney et al. 1999; Seeger et al. 2003; Coskran et al. 2006; Sano et al. 2008; Grauer et al. 2009).

In order to explore their antipsychotic potential and side-effect liability, PDE10AIs have been studied predominantly for effects on stimulated or spontaneous behaviors. In line with results suggesting that PDE10AIs activate indirect pathway neurons to a greater extent than direct pathway neurons (Threlfell et al. 2009; Nishi et al. 2011), PDE10AIs produce similar effects as D₂ receptor blockers, such as inhibition of spontaneous or stimulant-induced behavior, inhibition of conditioned avoidance behavior and reversal of stimulant-induced sensory gating deficits (Menniti et al. 2007; Schmidt et al. 2008; Grauer et al. 2009; Kehler and Nielsen 2011; Gresack et al. 2013; Megens et al. 2014). Concomitant direct pathway activation explains the relatively low cataleptogenic activity (Schmidt et al. 2008; Grauer et al. 2009; Megens et al. 2014), reduced efficiency against behavioral stimulants in case of concomitant D₁ receptor stimulation (Menniti et al. 2007; Sotty et al. 2009; Gresack et al. 2013; Megens et al. 2014), preferential activity against apomorphine-induced climbing versus apomorphine-induced stereotypy (Grauer et al. 2009), cognition-enhancing effects (Rodefer et al. 2005; Grauer et al. 2009), and socializing effects (Grauer et al. 2009).

The present study investigates effects of PDE10AIs on suppressed behaviors (catalepsy in rats; hypolocomotion in mice) under various conditions of decreased dopaminergic neurotransmission, viz. blockade of D₁ receptors with SCH-23390 (Bourne 2001), blockade of D₂ receptors with haloperidol, or dopamine depletion with RO-4-1284 (Colzi et al. 1993; Filinger 1994) or reserpine. Inhibition of apomorphine-induced behavior in rats and antagonism of d-amphetamine-induced hyperlocomotion in mice are included as measures of reduced D₂ receptor signaling. The following PDE10AIs are tested: JNJ-42314415 (Megens et al. 2014), PQ-10 (Kehler and Kilburn 2009), TP-10 (Schmidt et al. 2008; Kehler and Kilburn 2009), and MP-10 (Grauer et al. 2009; Kehler and Kilburn 2009). TP-10 is not included in all assays because of limited availability. In some assays, the D₁ agonist SKF-82958 (Wang and McGinty 1997) and the D₂ antagonist haloperidol are also included for comparison. Part of the data have been presented elsewhere (Vanhoof et al. 2012; Megens et al. 2014). The present results indicate that PDE10AIs stimulate or suppress motor behavior dependent on the relative activation state of the direct and indirect striatal output pathways.

Materials and Methods

Materials and sources

Company codes refer to the following chemical structures: JNJ-42314415: 3-[6-(2-methoxyethyl)pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine; MP-10: 2-{[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)phenoxy]methyl}quinoline; PQ-10: 6,7-dimethoxy-4-[3-(quinoxalin-2-yloxy)pyrrolidin-1-yl]quinazoline); RO-4-1284: 2-ethyl-9,10-dimethoxy-3-(2-methylpropyl)-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-ol; SCH-23390: 8-chloro-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-ol; SKF-82958: 6-chloro-1-phenyl-3-prop-2-en-1-yl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol; TP-10: 2-({4-[4-pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxy}methyl)quinoline. All compounds were synthesized in our own laboratories, except apomorphine, d-amphetamine (Certa SA, Eigenbrakel, Belgium), reserpine, and SKF-82958 (Sigma-Aldrich-Fluka, Diegem, Belgium).

Compound administration

Reserpine was dissolved in 10% hydroxypropyl-β-cyclodextrin containing ascorbic acid (4:1 ratio relative to reserpine; w:w). The other compounds were dissolved in water (apomorphine, d-amphetamine), acidified with tartaric acid if required (haloperidol, JNJ-42314415, SCH-23390), or in 10% or 20% hydroxypropyl-β-cyclodextrin acidified with tartaric acid (MP-10, PQ-10, SKF-82958, TP-10). The test formulations were stored at room temperature in closed containers protected from light and subcutaneously (s.c.) administered (10 mL/kg).

Animals (species, weight, and sex)

Male Wistar rats (Crl:WI; 220 ± 50 g) and male NMRI mice (Crl:NMRI(Han); 22 ± 5 g) were obtained from Charles River Breeding Laboratories (Sulzfeld, Germany for rats; L’Arbresle, France for mice) and housed under standard laboratory conditions (21 ± 2°C; 45–65% relative humidity; light–dark cycle set at 12 h). According to standard procedures, all animals were fasted overnight (tap water remaining available ad libitum), allowing oral dosing on an empty stomach (not applicable to the present study). The Institutional Ethics Committee on Animal Experimentation approved the experimental protocols, in compliance with Belgian law (Royal Decree on the protection of laboratory animals 6 April 2010).

Test descriptions

Apomorphine-induced agitation in rats

Apomorphine (1.0 mg/kg, i.v., intravenous)-induced agitation was scored (0, 1, 2, or 3) every 5 min over the first hour after injection of apomorphine. Criteria for drug-induced inhibition of agitation were: less than six times score 3 (1.5% false positives), less than six times score ≥2 (0.0% false positives), or less than seven times score ≥1 (0.0% false positives).

Observation test in rats

Catalepsy was scored (0, 1, 2 or 3) by two independent observers at 1, 2, 3, and 4 h after administration of test compound or solvent. The catalepsy scores from the two observers were summed for further evaluation. Criteria for drug-induced catalepsy: score >2 for slight catalepsy (occurrence in 1800 control observations: 0.0%; 450 rats and four observations per rat) and score 6 for pronounced catalepsy (not observed in controls). Other abnormalities were considered drug-induced, if occurring in a dose-dependent way. The interactions with SCH-23390 (0.63 mg/kg, s.c.), haloperidol (0.63 mg/kg, s.c.), and RO-4-1284 (10 mg/kg, s.c.) were evaluated by coadministering these agents simultaneously with the test formulations.

Reserpine-induced behavior in rats

Reserpine (10 mg/kg, s.c.) induces miosis, palpebral ptosis, and sedation and blocks the tail-pinch response (the biting and grasping response when an artery clamp is put on the rat’s tail, 1 cm from the distal end). The pupil diameter (of the right eye; using a microscopic micrometer; expressed in 1/24 mm units) and the latency of the tail-pinch response (s; cutoff time: 120 sec) were measured and palpebral opening (score 0, 1, 2, 3, 4, or 5) and sedation (hypomotility; score 0, 1, 2, 3) were scored 1 h after the reserpine challenge. To assess intrinsic effects of the test compounds, the pupil diameter and the tail-pinch response were also measured just before the reserpine injection. Test compound or solvent was administered 1 h before the reserpine challenge. Based on frequency distributions obtained in a historical control population of solvent-pretreated rats (n = 400), the following all-or-none criteria were selected to assess drug-induced effects: (1) before reserpine injection – induction of miosis: pupil diameter <11 units (4.3% false positive controls; n > 400); induction of mydriasis: pupil diameter >25 units (5.2% false positives); inhibition and blockade of tail-pinch response: latency >45 sec and >120 sec, respectively (5.2% and 0.2% false positives, respectively); (2) after reserpine injection – reversal of the tail-pinch response blockade: latency <120 sec (4.1% false positives); reversal of miosis: pupil diameter >8 units (1.1% false positives); reversal of palpebral ptosis: score for palpebral opening >1 (3.2% false positives); and reversal of sedation: score <3 (1.1% false positives).

Locomotor activity in mice

The procedure room was only sparsely lit (3–30 lx) to provide better contrast for the video tracking. Each locomotor activity arena (gray PVC cylinder; height: 40 cm; diameter: 22.5 cm) was placed on an infrared LED (8 × 8 LEDs) lit box (white PVC squared box; 40 × 40 cm²; height 12.5 cm). An infrared-sensitive tube camera and a white light source (in arena: 4–7 lx) were mounted to the ceiling above the arena to track the animal. Distance travelled was recorded and analyzed using the Noldus Ethovision XT Video Tracking System (Version 3.1; Noldus, Wageningen, the Netherlands). Locomotion was measured over a 30-min period by introducing the mouse into the motor activity arena 30 min after pretreatment with test compound or solvent. The arena was cleaned after each trial. In order to stimulate or suppress locomotion, following treatments were used: d-amphetamine (5.0 mg/kg, s.c.; −0.5 h), SCH-23390 (0.08 mg/kg, i.v.; 0 h), RO-4-1284 (0.63 mg/kg, s.c.; −0.5 h), or haloperidol (0.31 mg/kg, i.v.; 0 h).

General procedure and statistics

General procedure

All experiments were performed by unbiased trained technicians using coded solutions. Doses were selected from the geometrical series 0.01–0.02–0.04…40.0–80.0–160 mg/kg. Each dose group consisted of at least five animals which were tested in separate daily experimental sessions in order to account for day-to-day variability and to minimize systematic errors. Control injections of solvent were included in each experimental session. In Figures 4–6, dose 0 mg/kg refers to results obtained in solvent-treated control animals that were tested concurrently with the test compounds.

Figure 4.

Reversal of Ro-4-1284 (10 mg/kg, s.c.)-induced catalepsy in rats that were cotreated (at the same time) with the PDE10AIs JNJ-42314415, PQ-10, or MP-10 or the dopamine D₁ agonist SKF-82958. Shown are individual and median catalepsy scores (symbols and stripes, respectively) per dose group measured 2, 3, and 4 h after dosing. The dotted horizontal lines represent the critical levels adopted for drug-induced reversal of catalepsy (score <2 at 2 h; score <5 at 3 and 4 h) that were used for the determination of the ED₅₀ values listed in Tables 6–8, 10. The PDE10AIs dose dependently reduced catalepsy scores to below the critical level. The D₁ agonist was effective at the 2-h interval and, to a lesser extent, at the 3-h time interval. PDE10AI, phosphodiesterase 10A inhibitor.

Figure 6.

Dose-dependent reversal of reserpine-induced sedation and replacement by excitation. Shown are individual and median scores (symbols and stripes, respectively) for sedation and excitation (measured 1 h post reserpine) in rats pretreated with the PDE10AIs JNJ-42314415, PQ-10, MP-10, and the D₁ agonist SKF-82958 1 h before the reserpine challenge. The dotted horizontal lines represent the critical score adopted for reversal of sedation (score <3). In case of excitation, score >0 was used for the determination of ED₅₀ values. The PDE10AIs dose dependently reversed the reserpine-induced sedation, which was replaced by excitation. The D₁ agonist also reversed the reserpine-induced sedation but without resulting in excitation. PDE10A, phosphodiesterase 10A inhibitor.

ED₅₀ determination

All-or-none criteria for drug-induced effects were defined by analyzing a frequency distribution of a series of historical control data, aiming for less than 5% responders in the control population. The fraction of animals responding to these criteria in animals pretreated with test compound was determined per dose level (n ≥ 5 in the relevant dose range; at least three doses). ED₅₀s (the dose inducing the defined effect in 50% of the tested animals) and corresponding 95% confidence limits were determined according to the modified Spearman-Karber estimate, using theoretical probabilities instead of empirical ones (Tsutakawa 1982). This modification allows estimation of the ED₅₀ and its confidence interval as a function of the slope of the log dose–response curve (Lewi et al. 1977).

Time–activity relationship

ED₅₀s were plotted versus time after dosing using GraphPad Prism® (software version 5.00 for Windows, GraphPad Software, San Diego, CA; http://www.graphpad.com). The ED₅₀ at time of peak effect and the onset and duration of action (at four times the peak-effect dose) were estimated from the curve of best fit through these data points as generated by nonlinear regression of the polynomial second order function y = a + bx + cx². Goodness of fit was determined by calculating R² and S_y.x.

Results

Apomorphine-induced agitation in rats

Inhibition of apomorphine-induced behavior relates to the D₂ receptor blocking activity of antipsychotics (Leysen and Niemegeers 1985; Leysen 2000). Apomorphine antagonism was used in the present study as sensitive index for the functional D₂ antagonism of PDE10AIs in rats, being measured at doses close to the ED₅₀ for striatal PDE10A occupancy (Megens et al. 2014).

The ED₅₀s of JNJ-42314415, PQ-10, TP-10, and MP-10 obtained for inhibition of apomorphine-induced behavior at various time intervals after s.c. injection are listed in Table 1 and graphically presented in Figure 1 (solid circles; the other data are discussed below). The PDE10AIs showed a fast onset of action (<0.5 h) and reached their peak effect within 0.5–1 h (nonlinear regression estimates in Table 2). JNJ-42314415, MP-10, and TP-10 were about equipotent and about three to four times more potent than PQ-10 in terms of peak-effect dose. MP-10 showed a somewhat shorter duration (2.8 h) than the other compounds (4.3–4.8 h).

Table 1.

ED₅₀s (and 95% confidence limits; mg/kg) of the test compounds for inhibition of apomorphine-induced agitation in rats as a function of time after s.c. injection

	ED50s (95% confidence limits; mg/kg, s.c.)
Time (h)	JNJ-42314415	PQ-10	TP-10	MP-10
0.5	1.02 (0.75–1.38)	3.6 (2.88–4.4)	1.17 (0.86–1.58)	1.17 (0.86–1.58)
1	1.02 (0.75–1.38)	4.1 (3.0–5.5)	1.02 (0.75–1.38)	0.67 (0.45–1.01)
2	1.77 (1.44–2.18)	6.2 (4.1–9.2)	1.77 (1.18–2.65)	2.69 (1.99–3.6)
4	4.1 (3.0–5.5)	7.1 (4.7–10.6)	4.1 (3.0–5.5)	8.1 (6.0–11.0)
8	8.2 (5.5–12.2)	43 (28.7–64)	12.3 (9.1–16.7)

Figure 1.

ED_50s (95% confidence limits; mg/kg) of PDE10AIs for various effects in rats cotreated with the D₁ antagonist SCH-23390 (0.63 mg/kg, s.c., 0 h) in comparison with the ED₅₀ for antagonism of apomorphine-induced behavior in rats not cotreated with SCH-23390. The ED₅₀s have been plotted as a function of time after s.c. injection of the compounds. In the rats cotreated with the D₁ antagonist, PDE10AIs induced (slight and pronounced) catalepsy at doses similar to those required for apomorphine antagonism. At 10-fold higher doses, catalepsy disappeared, being replaced by agitation. The various effects followed a parallel time course. For TP-10 only ED₅₀s for apomorphine antagonism are shown. PDE10AI, phosphodiesterase 10A inhibitor.

Table 2.

Inhibition of apomorphine-induced agitation in rats: nonlinear regression estimates for the time and dose of peak effect, and for the onset and duration of action after s.c. dosing

	Peak effect
Compound	Time (h)	ED50 (mg/kg)	Onset1 (h)	Duration1 (h)	R2	Sy.x
JNJ-42314415	0.5	0.95	<0.5	4.3	0.985	0.068
PQ-10	0.83	3.5	<0.5	4.8	0.930	0.163
TP-10	0.68	1.07	<0.5	4.3	0.995	0.044
MP-10	0.77	0.88	<0.5	2.8	0.929	0.217

The values were interpolated from the nonlinear regression lines in Figure 1. Values for R² and S_y.x show the goodness of fit of the nonlinear regression lines.

¹Onset and duration of action were assessed at four times the peak-effect dose.

Observation tests in rats

Interaction with the D₁ antagonist SCH-23390

In order to study involvement of dopamine D₁ receptors, catalepsy was studied after JNJ-42314415, PQ-10, and MP-10 in rats cotreated or not cotreated with the D₁ antagonist SCH-23390 (0.63 mg/kg, s.c., 0 h). In rats not cotreated with SCH-23390 (Fig. 2: upper panel for each compound; open circles), the PDE10AIs failed to induce catalepsy in a dose-dependent and consistent manner. MP-10 tended to induce slight catalepsy (score >2) around 1.25–2.5 mg/kg (maximum 60–80% responders) but only incidentally induced pronounced catalepsy (score 6). In rats cotreated with SCH-23390 (Fig. 2: bottom panel for each compound; closed circles), however, the PDE10AIs consistently induced pronounced catalepsy (score 6). The dose–response curves were bell-shaped: catalepsy decreased again at higher dose levels, being replaced by stereotyped sniffing and rearing. The ED₅₀s for the various effects in rats cotreated with SCH-23390 (Table 3) have been plotted as a function of time in Figure 1 in comparison with the ED₅₀s for apomorphine antagonism in rats not cotreated with SCH-23390. The time course for the effects in the rats cotreated with SCH-23390 was found to proceed more or less in parallel with the apomorphine antagonism in the absence of SCH-23390. In the rats cotreated with SCH-23390, the PDE10AIs already induced catalepsy at low doses comparable to those for apomorphine antagonism (Table 4), reflecting a very small safety margin under these conditions. SCH-23390 (0.63 mg/kg, s.c.) alone did not induce catalepsy (score >2 was observed only once in 35 rats tested at four time intervals).

Figure 2.

Potentiation of PDE10AI-induced catalepsy by cotreatment with the D₁ antagonist SCH-23390. Individual (circles) and median scores (stripes) for catalepsy obtained per dose level 1, 2, 3, and 4 h after s.c. injection of JNJ-42314415, PQ-10, and MP-10 in rats cotreated with SCH-23390 (0.63 mg/kg, s.c., 0 h; bottom panel for each compound; closed circles) or not cotreated (upper panel for each compound; open circles). Median catalepsy scores of successive dose groups have been interconnected. The horizontal dotted lines represent the critical levels adopted for slight catalepsy (score >2) and pronounced catalepsy (maximum score 6). The PDE10AIs consistently induced pronounced catalepsy with bell-shaped dose–response curves in the rats cotreated with the D₁ antagonist, whereas they induced almost no or only slight catalepsy in the rats not cotreated with the D₁ antagonist. PDE10AI, phosphodiesterase 10A inhibitor.

Table 3.

ED₅₀s (and 95% confidence limits) for induction of slight and pronounced catalepsy, the subsequent disappearance of catalepsy, and the induction of agitation in rats cotreated with SCH-23390 (0.63 mg/kg, s.c.)

	ED50s (95% confidence limits; mg/kg, s.c.)
	Induction of catalepsy
Time (h)	Slight (score >2)	Pronounced (score = 6)	Disappearance of slight catalepsy (score ≤2)	Induction of agitation
JNJ-42314415
1	1.54 (1.14–2.09)	1.54 (1.14–2.09)	10.8 (8.0–14.6)	10.8 (8.0–14.6)
2	1.54 (1.14–2.09)	1.77 (1.44–2.18)	10.8 (8.0–14.6)	12.4 (9.1–16.7)
3	2.03 (1.50–2.75)	2.04 (1.51–2.76)	16.3 (12.0–22.1)	21.5 (13.3–35)
4	3.5 (2.87–4.4)	3.6 (2.88–4.4)	16.3 (12.0–22.1)	>40
PQ-10
1	3.6 (2.37–5.3)	4.7 (3.4–6.3)	32 (21.7–49)	>40
2	5.4 (4.0–7.3)	5.4 (4.0–7.3)	43 (–)	>40
3	8.2 (6.0–11)	10.8 (8.0–14.6)	43 (–)	>40
4	18.7 (13.8–25.3)	21.5 (15.9–29.1)	>40	>40
MP-10
1	0.294 (0.182–0.48)	0.44 (0.297–0.67)	4.1 (2.73–6.1)	5.4 (3.6–8.1)
2	0.44 (0.276–0.72)	0.51 (0.32–0.82)	7.1 (4.7–10.6)	7.1 (4.7–10.6)
3	0.77 (0.57–1.05)	1.78 (1.18–2.65)	14.2 (11.5–17.4)	21 (–)
4	2.69 (1.80–4.0)	3.1 (2.28–4.2)	18.7 (13.8–25.3)	>20

Table 4.

ED₅₀s of the PDE10AIs at time of peak-effect for induction of slight and pronounced catalepsy, the subsequent disappearance of catalepsy, and the induction of agitation in rats cotreated with SCH-23390 (0.63 mg/kg, s.c.) in relation to the ED₅₀ for inhibition of apomorphine-induced agitation

	ED50 (mg/kg, s.c.) at time of peak effect1
Effects	JNJ-42314415	PQ-10	MP-10
Apomorphine antagonism	0.95 [1.0]	3.5 [1.0]	0.88 [1.0]
Induction of slight catalepsy2 (score >2)	1.35 [1.4]	3.6 [1.0]	0.30 [0.34]
Induction of pronounced catalepsy2 (score 6)	1.46 [1.5]	4.3 [1.2]	0.43 [0.49]
Disappearance of slight catalepsy2 (score ≤2)	10.6 [11]	32 [9.1]	4.0 [4.5]
Induction of agitation2	9.2 [9.6]	>40 [>11]	5.2 [5.9]

PDE10AI, phosphodiesterase 10A inhibitor.

¹ED₅₀s at time of peak effect were interpolated from the nonlinear regression lines plotted in Figure 1.

²In animals cotreated with SCH-23390 (0.63 mg/kg, s.c). Figures between square brackets represent the ratio of the ED₅₀ over the ED₅₀ for apomorphine antagonism.

As the functional D₁ agonism of PDE10AIs apparently counteracts the catalepsy that may be expected from their functional D₂ antagonism, it is studied below whether this activity component may also counteract catalepsy and related behaviors induced by other mechanisms, viz. D₂ receptor blockade with haloperidol or dopamine depletion using Ro-4-1284 or reserpine.

Interaction with the D₂ antagonist haloperidol

Figure 3 illustrates the dose-dependent reversal of haloperidol (0.63 mg/kg, s.c.)-induced catalepsy after cotreatment with JNJ-42314415, TP-10, MP-10, and the D₁ agonist SKF-82958. Shown are individual data and median scores per dose group obtained 2, 3, and 4 h after dosing. In solvent-cotreated rats (n = 321), haloperidol consistently induced catalepsy, which became more pronounced at later time intervals. Catalepsy scores <2 at 2 h and <5 at 3 h and 4 h occurred in only 6.2%, 4.7%, and 3.1% of these control rats, respectively, and were adopted as critical levels for reversal of haloperidol-induced catalepsy (dotted horizontal lines in Fig. 3). The three PDE10AIs dose dependently reversed the haloperidol-induced catalepsy (Fig. 3, Table 5). The lowest ED₅₀ obtained over the three time intervals was 4.7 mg/kg for JNJ-42314415, 2.69 mg/kg for TP-10, and 6.2 mg/kg for MP-10, corresponding to 4.9, 2.5, and 7.0 times, respectively, the ED₅₀ for apomorphine antagonism at time of peak effect (Table 2). SKF-82958 reversed haloperidol-induced catalepsy at 1 h with a bell-shaped dose–response curve (estimated ED₅₀: 1.02 mg/kg) but was hardly effective at the later time intervals.

Figure 3.

Reversal of haloperidol (0.63 mg/kg, s.c.)-induced catalepsy in rats that were cotreated (at the same time) with the PDE10AIs JNJ-42314415, TP-10, or MP-10 or the dopamine D₁ agonist SKF-82958. Shown are individual and median catalepsy scores (symbols and stripes, respectively) per dose group measured 2, 3, and 4 h after dosing. The dotted horizontal lines represent the critical levels adopted for drug-induced reversal of catalepsy (score <2 for 2 h; score <5 for 3 and 4 h) that were used for the determination of the ED₅₀ values listed in Table 5. The PDE10AIs dose dependently reduced catalepsy scores to below the critical level. The D₁ agonist showed less consistent reversal of catalepsy. PDE10AI, phosphodiesterase 10A inhibitor.

Table 5.

Dose–response relations and corresponding ED₅₀s for reversal of haloperidol (0.63 mg/kg, s.c.)-induced catalepsy in rats that received test compound simultaneously with haloperidol

Effect		Dose (mg/kg, s.c.)						ED50
Time (h)	Criterion	0.31	0.63	1.25	2.5	5.0	10	(95% CL; mg/kg)
JNJ-42314415
2	Score <2				0	1	5	6.2 (4.6–8.3)
3	Score <5				0	3	4	5.4 (3.6–8.0)
4	Score <5				1	4	4	4.7 (3.1–7.0)
TP-10
2	Score <2		0	0	3	4	5	2.69 (1.80–4.0)
3	Score <5		0	0	2	4	5	4.1 (3.0–5.5)
4	Score <5		0	0	3	4	4	3.1 (1.92–5.0)
MP-10
2	Score <2			0	0	1	4	7.1 (4.7–10.6)
3	Score <5			0	0	2	2	≥10
4	Score <5			0	0	2	4	6.2 (4.1–9.2)
SKF-82958
2	Score <2	2	2	4	3	3	2	1.02 (–)
3	Score <5	0	2	1	1	3	0	∼5.0
4	Score <5	0	0	0	0	2	0	>10

The number of rats responding to the listed criterion is listed per dose level (n = 5 per dose). Values representing more than 50% responders are formatted bold.

Interaction with the monoamines-depleting agent RO-4-1284

RO-4-1284 (10 mg/kg, s.c.) consistently induced catalepsy and sedation in solvent-cotreated rats (n = 50). JNJ-42314415, PQ-10, and MP-10 dose dependently reversed the RO-4-1284-induced sedation and catalepsy, which were replaced by behavioral excitation (Table 8; Fig. 4). The effects occurred as doses close to the ED₅₀ for apomorphine antagonism (Table 9). The D₁ agonist SKF-82958 tended to reverse the RO-4-1284-induced catalepsy at 2.5 mg/kg without affecting sedation. At this dose, however, the combination with RO-4-1284 resulted in convulsions, spasms and body twitches in two of the five rats tested (one of them died and was not included in Table 10).

Table 8.

Dose–response relations and corresponding ED₅₀s for various effects observed over a 4-h period after challenge with RO-4-1284 (10 mg/kg, s.c.) in rats that received test compound simultaneously with RO-4-1284

Effect		Dose of MP-10 (mg/kg, s.c.)							ED50
Time (h)	Criterion	0.63	1.25	2.5	5.0	10	20	40	(95% CL; mg/kg)
Reversal of sedation/passivity
1	Absence (6.0%)1	2	5	5	5				0.68 (0.50–0.91)
2	Absence (4.0%)1	2	5	5	5				0.68 (0.50–0.91)
3	Absence (2.0%)1	2	5	5	5				0.68 (0.50–0.91)
4	Absence (16%)1	1	2	5	5				1.17 (0.78–1.76)
Reversal of catalepsy
2	Score <2 (0.0%)1	1	5	5	5				0.78 (0.57–1.05)
3	Score <5 (2.0%)1	1	4	5	5				0.89 (0.59–1.33)
4	Score <5 (8.0%)1	0	2	5	5				1.35 (1.00–1.82)
Induction of excitation
1	Presence (0.0%)1	0	1	0	3				4.7 (–)
2	Presence (0.0%)1	0	0	1	4				3.5 (2.36–5.3)
3	Presence (0.0%)1	0	0	1	4				3.5 (2.36–5.3)
4	Presence (0.0%)1	0	0	0	2				≥5.0

The number of rats responding to the listed criterion is listed per dose level (n = 5 per dose group). Values representing more than 50% responders are formatted bold.

¹Percentage of false positives (solvent-pretreated rats responding to the criterion [n = 50]).

Table 9.

ED₅₀s (95% confidence limits; mg/kg) of the PDE10AIs for interactions with RO-4-1284-induced behavior related to their ED₅₀ for inhibition of apomorphine-induced behavior

	JNJ-42314415		PQ-10		MP-10
Compound	ED50 (95% CL)	Ratio1	ED50 (95% CL)	Ratio1	ED50 (95% CL)	Ratio1
Apomorphine test
Inhibition of agitation	0.95	[1.0]	3.5	[1.0]	0.88	[1.0]
RO-4-1284 interaction
Reversal of sedation	0.59 (0.44–0.80)	[0.62]	3.6 (2.89–4.3)	[1.0]	0.68 (0.50–0.91)	[0.77]
Reversal of catalepsy	1.03 (0.69–1.54)	[1.1]	4.7 (2.91–7.6)	[1.3]	0.78 (0.57–1.05)	[0.89]
Induction of excitation	2.05 (1.37–3.1)	[2.2]	14.2 (8.8–22.8)	[4.0]	3.1 (1.91–5.0)	[3.5]

Listed are ED₅₀s obtained at time of peak effect together with the ratio over the ED₅₀ for apomorphine antagonism. PDE10AI, phosphodiesterase 10A inhibitor

¹The ratio over the corresponding ED₅₀ for apomorphine antagonism is listed between square brackets.

Table 10.

Dose–response relations and corresponding ED₅₀s for various effects observed over a 4-h period after challenge with RO-4-1284 (10 mg/kg, s.c.) in rats that received test compound simultaneously with RO-4-1284

Effect		Dose of SKF-82958 (mg/kg, s.c.)							ED50
Time (h)	Criterion	0.04	0.08	0.16	0.31	0.63	1.25	2.51	(95% CL; mg/kg)
Reversal of sedation/passivity
1	Absence (6.0%)2	2	1	0	2	3	0	0/4	>2.5
2	Absence (4.0%)2	1	0	0	2	3	0	0/4	>2.5
3	Absence (2.0%)2	1	0	0	1	2	0	0/4	>2.5
4	Absence (16%)2	0	0	0	0	1	0	1/4	>2.5
Reversal of catalepsy
2	Score <2 (0.0%)2	0	1	0	1	1	0	3/4	2.1 (–)
3	Score <5 (2.0%)2	1	1	1	1	2	1	2/4	2.5 (–)
4	Score <5 (8.0%)2	0	1	0	1	1	1	1/4
Induction of excitation
1	Presence (0.0%)2	0	0	0	0	0	0	0/4	>2.5
2	Presence (0.0%)2	0	0	0	0	0	0	0/4	>2.5
3	Presence (0.0%)2	0	0	0	0	0	0	0/4	>2.5
4	Presence (0.0%)2	0	0	0	0	0	0	0/4	>2.5

The number of rats responding to the listed criterion is listed per dose level (n = 5 per dose group). Values representing more than 50% responders are formatted bold.

¹One of the five rats tested at 2.5 mg/kg died and was not included.

²Percentage of false positives (solvent-pretreated rats responding to the criterion [n = 50]).

Table 6.

Dose–response relations and corresponding ED₅₀s for various effects observed over a 4-h period after challenge with RO-4-1284 (10 mg/kg, s.c.) in rats that received test compound simultaneously with RO-4-1284

Effect		Dose of JNJ-42314415 (mg/kg, s.c.)							ED50
Time (h)	Criterion	0.63	1.25	2.5	5.0	10	20	40	(95% CL; mg/kg)
Reversal of sedation/passivity
1	Absence (6.0%)1	3	5	5	5	5	5	5	0.59 (0.44–0.80)2
2	Absence (4.0%)1	2	2	5	5	5	5	5	1.03 (0.69–1.54)
3	Absence (2.0%)1	2	2	4	4	5	5	5	1.36 (0.79–2.33)
4	Absence (16%)1	1	3	2	4	5	5	5	1.79 (1.04–3.1)
Reversal of catalepsy
2	Score <2 (0.0%)1	1	0	4	5	5	5	5	2.05 (1.52–2.78)
3	Score <5 (2.0%)1	1	3	5	5	5	5	5	1.03 (0.69–1.54)
4	Score <5 (8.0%)1	0	2	4	5	5	5	5	1.56 (1.04–2.33)
Induction of excitation
1	Presence (0.0%)1	0	0	2	4	4	4	1	4.1 (2.38–7.0)
2	Presence (0.0%)1	0	0	4	2	4	5	3	3.6 (2.20–5.7)
3	Presence (0.0%)1	0	0	2	2	4	5	2	4.1 (2.53–6.6)
4	Presence (0.0%)1	0	0	1	2	4	4	5	6.2 (3.6–10.6)

The number of rats responding to the listed criterion is listed per dose level (n = 5 per dose group). Values representing more than 50% responders are formatted bold.

¹Percentage of false positives (solvent-pretreated rats responding to the criterion [n = 50]).

²ED₅₀ estimated assuming no responders at the nontested dose of 0.31 mg/kg.

Table 7.

Dose–response relations and corresponding ED₅₀s for various effects observed over a 4-h period after challenge with RO-4-1284 (10 mg/kg, s.c.) in rats that received test compound simultaneously with RO-4-1284

Effect		Dose of PQ-10 (mg/kg, s.c.)							ED50
Time (h)	Criterion	0.63	1.25	2.5	5.0	10	20	40	(95% CL; mg/kg)
Reversal of sedation/passivity
1	Absence (6.0%)1		0	0	5	4	5		3.6 (2.89–4.3)
2	Absence (4.0%)1		0	2	3	4	4		4.7 (2.38–7.0)
3	Absence (2.0%)1		1	2	3	2	2		7.1 (–)
4	Absence (16%)1		0	2	0	1	3		16.2 (–)
Reversal of catalepsy
2	Score <2 (0.0%)1		0	0	1	3	5		8.2 (5.4–12.2)
3	Score <5 (2.0%)1		0	3	2	3	5		4.7 (2.91–7.6)
4	Score <5 (8.0%)1		0	1	0	3	4		10.7 (7.2–16.1)
Induction of excitation
1	Presence (0.0%)1		0	0	0	0	3		19 (–)
2	Presence (0.0%)1		0	0	0	0	3		19 (–)
3	Presence (0.0%)1		0	0	1	0	3		19 (–)
4	Presence (0.0%)1		0	0	0	0	0		>20 (–)

The number of rats responding to the listed criterion is listed per dose level (n = 5 per dose group). Values representing more than 50% responders are formatted bold.

¹Percentage of false positives (solvent-pretreated rats responding to the criterion [n = 50]).

Interaction with the monoamines-depleting agent reserpine

Reserpine (10 mg/kg, s.c.) induced palpebral ptosis, sedation, and blocked the tail-pinch response (the grasping and biting response when an artery clamp is put on the distal end of the rat’s tail; latency >120 sec). Before the reserpine challenge, the PDE10AIs inhibited the tail-pinch response per se (latency >45 sec) at doses only 1.2–3.2 times the ED₅₀ for apomorphine antagonism (Fig. 5, Table 11). The dose–response relations for the inhibition of the tail-pinch response tended to be bell-shaped, except for PQ-10 (tested in a small dose range only). While inhibiting the tail-pinch response before reserpine, the PDE10AIs in contrast dose dependently reversed the reserpine-induced blockade of the tail-pinch response 1 h after reserpine. This effect occurred at 3.3–8.5 times the ED₅₀ for apomorphine antagonism. The PDE10AIs even restored normal response latencies (<45 sec) at slightly higher doses (5.0–11 times the ED₅₀ for apomorphine antagonism). Reversal of the reserpine-induced tail-pinch response blockade was thus observed at similar doses as those inhibiting the tail-pinch response before reserpine (Table 11). The PDE10AIs also reversed the reserpine-induced sedation (at 0.54–3.2 times the ED₅₀ for apomorphine antagonism; Fig. 6), which was replaced by behavioral stimulation (sniffing and rearing) at 1.9–4.9 times the ED₅₀ for apomorphine antagonism. The PDE10AIs also tended to reverse the reserpine-induced palpebral ptosis (at 0.96–3.2 times the ED₅₀ for apomorphine antagonism) but did not affect pupil diameter up to the highest doses tested (data not shown).

Figure 5.

Dose-dependent inhibition of the tail-pinch response measured immediately before reserpine challenge in rats pretreated s.c. with the PDE10AIs JNJ-42314415, PQ-10, or MP-10 or the dopamine D₁ agonist SKF-82958 1 h before the reserpine challenge (left panel) and dose-dependent reversal of the blockade of the tail-pinch response measured in the same rats 1 h after reserpine (right panel). Shown are individual and median response latencies (symbols and stripes, respectively) per dose group measured immediately before and 1 h after the reserpine challenge. The dotted and solid horizontal lines represent the critical latencies adopted for inhibition and blockade of the tail-pinch response (>45 sec and >120 sec, respectively) that were used for the determination of ED₅₀ values listed in Table 11. The PDE10AIs inhibited the tail-pinch response before the reserpine challenge with an apparently bell-shaped dose–response relation, whereas they reversed the reserpine-induced blockade of the tail-pinch response 1 h after the reserpine challenge. The D₁ agonist only tended to inhibit the tail-pinch response before reserpine but efficiently reversed the reserpine-induced blockade of the tail-pinch response 1 h after the reserpine challenge. PDE10AI, phosphodiesterase 10A inhibitor.

Table 11.

ED₅₀s (95% CL; mg/kg, s.c.) for inhibition and blockade of the tail-pinch response before reserpine challenge and for reversal of reserpine-induced phenomena (sedation, palpebral ptosis, blockade of the tail-pinch response) 1 h after reserpine challenge in rats pretreated s.c. with the test compounds 1 h before reserpine challenge

	ED50s (95% confidence limits; mg/kg, s.c.)
	Before reserpine			After reserpine
	Tail-pinch response				Tail-pinch response
Compound	Apomorphine antagonism	Inhibition (>45 sec)	Blockade (>120 sec)	Reversal sedation	Reversal ptosis	Reversal blockade (<120 sec)	Normalization (<45 sec)	Induction excitation
JNJ-42314415	0.95	1.35 (0.90–2.02)	2.701 (–)	1.17 (0.78–1.75)	3.1 (1.62–5.9)	4.1 (3.0–5.5)	8.2 (5.0–13.2)	2.35 (1.37–4.0)
PQ-10	3.5	6.2 (4.6–8.4)	8.2 (6.0–11.0)	7.1 (5.8–8.7)	8.2 (5.4–12.2)	14.2 (9.4–21.2)	≥20	10.7 (7.2–16.1)
MP-10	0.88	1.341 (–)	60% at 2.5 mg/kg	0.51 (0.32–0.82)	1.78 (0.89–3.6)	3.1 (1.71–5.6)	4.7 (2.73–8.1)	1.78 (0.89–3.6)
SKF-82958	–	0.443 (40–60%)	>10	0.097 (0.060–0.157)	40% at 0.31–10 mg/kg	0.112 (0.062–0.203)	0.51 (40–80%)	>2.52

For comparison, ED₅₀s for inhibition of apomorphine-induced behavior (at time of peak effect) have been listed in the first column.

¹Estimated ED₅₀ (bell-shaped dose–response curve).

²SKF-82958 induced excitation before reserpine (ED₅₀: 0.44 [0.36–0.55] mg/kg) but not after reserpine.

³Estimated ED50 (maximum 40–60% responders).

Before the reserpine challenge, the D₁ agonist SKF-82958 inhibited the tail-pinch response in some rats (from 0.16 mg/kg onward; maximum 40% responders), presumably related to behavioral stimulant effects (sniffing, rearing; ED₅₀: 0.44 [0.36–0.55] mg/kg). After the reserpine challenge, SKF-82958 reversed the reserpine-induced sedation (ED₅₀: 0.097 mg/kg; Fig. 6) and the blockade of the tail-pinch response (ED₅₀: 0.112 mg/kg; Fig. 5, Table 11), and tended to normalize the tail-pinch response (estimated ED₅₀: 0.51 mg/kg;) and to reverse the ptosis (from 0.31 mg/kg onward; data not shown) without affecting miosis (ED₅₀: >10 mg/kg; data not shown). The overt behavioral stimulation seen with SKF-82958 before the reserpine challenge was not seen after the reserpine challenge (Table 11). In contrast, the PDE10AIs showed behavioral stimulant effects after but not preceding the reserpine challenge.

Locomotor activity assays in mice

In addition to the above studies at the level of catalepsy in rats, similar studies were performed at the level of locomotion in mice. First, inhibition of d-amphetamine-induced hyperlocomotion was tested as index of functional D₂ antagonism. Subsequently, the ability of PDE10AIs to reverse the hypolocomotion induced by the D₁ antagonist SCH-23390, the monoamines-depleting agent, Ro-4-1284, or the D₂ antagonist haloperidol was evaluated. Total distance travelled in solvent-pretreated control mice receiving no challenge was 4167 ± 1135 cm (mean ± SD; n = 1444).

Inhibition of hyperlocomotion induced by the dopamine-releasing agent d-amphetamine

In solvent-pretreated control mice, d-amphetamine (5.0 mg/kg, s.c.;−0.5 h) increased distance travelled to 12,764 ± 4278 cm (mean ± SD; n = 462). Only 3.0% of these control mice travelled <5500 cm. The PDE10AIs and the D₂ receptor blocker haloperidol reduced the d-amphetamine-induced hyperlocomotion in a dose-dependent manner to normal values <5500 cm (Fig. 7, first column; ED₅₀s in Table 12, first column).

Figure 7.

Activity profile in locomotor activity tests in mice. Dose–response relations for inhibition of d-amphetamine-induced hyperlocomotion and reversal of hypolocomotion induced by the D₁ antagonist SCH-23390, the monoamines-depleting agent RO-4-1284 or the D₂ antagonist haloperidol 0.5 h after s.c. injection. The solid horizontal line represents the averaged distance obtained in solvent-pretreated control mice, the gray area represents the activity range obtained in challenged solvent-treated control mice, and the area between the two dotted horizontal lines represents the locomotor activity level obtained in nonchallenged control mice. The PDE10AIs dose dependently inhibited d-amphetamine-induced hyperlocomotion and reversed the hypolocomotion induced by the D₁ antagonist SCH-23390 or the monoamines-depleting agent RO-4-1284 but hardly affected the hypolocomotion induced by the D₂ antagonist haloperidol. PDE10A, phosphodiesterase 10A inhibitor.

Table 12.

ED₅₀s (95% confidence limits) for inhibition of d-amphetamine-induced hyperlocomotion and for reversal of hypolocomotion induced by the D₁ antagonist SCH-23390, the monoamines-depleting agent RO-4-1284, or the D₂ antagonist haloperidol measured in mice 0.5 h after s.c. injection

	ED50s (95% confidence limits; mg/kg, s.c., −0.5 h)
Compound	d-Amphetamine (<5500 cm)	SCH-23390 (>2500 cm)	RO-4-1284 (>2500 cm)	Haloperidol (>2500 cm)
JNJ-42314415	8.1 (5.4–12.2)	7.1 (4.8–10.7)	2.69 (1.99–3.6)	>401
PQ-10	12.4 (9.1–16.7)	16.3 (10.1–26.3)	5.4 (–)	>40
MP-10	1.78 (1.10–2.87)	1.17 (0.86–1.58)	0.58 (0.34–1.01)	>40
TP-10	1.78 (1.19–2.66)	1.78 (1.18–2.65)	No stock	>10
Haloperidol	0.112 (0.075–0.168)	–	–	–
SKF-82958	–	0.148 (0.109–0.200)	0.294 (0.218–0.40)	>10

¹60% responders at 10 mg/kg.

Reversal of hypolocomotion induced by the D₁ antagonist SCH-23390

In solvent-pretreated control mice, SCH-23390 (0.08 mg/kg, i.v., 0 h) decreased distance travelled to 1408 ± 513 cm (mean ± SD; n = 279). Only 1.4% of these SCH-23390-challenged control mice travelled a distance >2500 cm. The PDE10AIs and the D₁ agonist SKF-82958 reversed the SCH-23390-induced hypolocomotion to normal values (>2500 cm; Fig. 7; second column). The ED₅₀s of the PDE10AIs for this effect were similar to those required for inhibition of d-amphetamine-induced hyperlocomotion (Table 12).

Reversal of hypolocomotion induced by the monoamines-depleting agent RO-4-1284

In solvent-pretreated control mice, dopamine depletion with RO-4-1284 (0.63 mg/kg, s.c., −0.5 h) induced pronounced hypolocomotion (total distance [mean ± SD]: 772 ± 411 cm; n = 218). Values >2500 cm were rarely measured (in only 0.4% of the control mice). The tested PDE10AIs and the D₁ agonist SKF-82958 reversed the RO-4-1284-induced hypolocomotion to normal values (>2500 cm; Fig. 7; third column). The dose–response curves of JNJ-42311445 and PQ-10 tended to be bell-shaped and effect size tended to be higher for MP-10 than for JNJ-42311445 and PQ-10. The PDE10AIs were somewhat (two to three times) more potent against Ro-4-1284 than against d-amphetamine or SCH-23390 while SKF-82958 was equipotent in both assays (Table 12).

Reversal of hypolocomotion induced by the D₂ antagonist haloperidol

In solvent-pretreated mice, the D₂ antagonist haloperidol (0.31 mg/kg, i.v., 0 h) induced hypolocomotion (total distance [mean ± SD]: 1257 ± 1370 cm; n = 1833). Values >2500 cm occurred in only 4.0% of these control mice. The PDE10AIs and the D₁ agonist SKF-82958 were not able to reverse the haloperidol-induced hypolocomotion to values >2500 cm, except for a tendency in the lower dose range of the PDE10AIs (maximum percentage responders: 60% at 10 mg/kg for JNJ-42314415; 40% at 10 mg/kg for PQ-10, 40% at 0.63 mg/kg for MP-10, and 0% for TP-10).

Discussion

PDE10AIs resemble D₂ receptor blockers by, for example, inhibiting spontaneous and stimulant-induced behavior, inhibiting conditioned behavior, and reversing stimulant-induced sensory gating deficits (see Introduction). These effects are mediated by indirect pathway activation, partially counteracted by direct pathway activation when D₁ receptors are concomitantly stimulated (Menniti et al. 2007; Gresack et al. 2013; Megens et al. 2014). By stimulating dopamine D₂ receptors, nonselective dopamine agonists such as apomorphine or endogenous dopamine (e.g., after release by d-amphetamine) deactivate the indirect pathway, thereby reducing behavioral inhibition. By concomitantly stimulating D₁ receptors, such compounds also activate the direct pathway, favoring behavioral stimulation. PDE10AIs inhibit the effect of dopamine agonists on the indirect pathway but facilitate their effect on the direct pathway. Acting together, both counteracting effects provide an explanation for the relatively reduced efficacy of PDE10AIs against nonselective dopamine agonists such as apomorphine and dopamine-releasing agents such as amphetamine as compared with their pronounced effects against nondopaminergic stimulants such as NMDA antagonists and antimuscarinics or D₂-selective dopamine agonists such as quinpirole (Menniti et al. 2007; Sotty et al. 2009; Gresack et al. 2013; Megens et al. 2014). Both counteracting activity components may also explain the failure of MP-10 against acute exacerbations of schizophrenia (DeMartinis et al. 2012). The present study investigated the effects of PDE10AIs on suppressed behavior induced by reducing dopaminergic neurotransmission rather than on stimulated or spontaneous behavior. The results show that PDE10AIs can reverse suppressed behavior and even stimulate behavior depending on the relative activation state of the direct and indirect pathways.

Under basal conditions, PDE10AIs have relatively low cataleptogenic activity, due to concomitant direct pathway activation (Kehler et al. 2007; Schmidt et al. 2008; Megens et al. 2014). After D₁ receptor blockade with SCH-23390, however, PDE10AIs induced pronounced catalepsy at low doses comparable to those required for inhibition of apomorphine-induced behavior. Similar levels of catalepsy occur with D₂ receptor blockers only at >10-fold higher dose increments (Langlois et al. 2012). The potentiation of PDE10AI-induced catalepsy by a D₁ antagonist confirms that PDE10AIs suppress central D₂ receptor-mediated neurotransmission very efficiently when concomitant direct pathway activation is prevented by D₁ receptor blockade and implies a risk of increased EPS when combining a PDE10AI with a neuroleptic with associated D₁ antagonism (e.g., clozapine or olanzapine; Schotte and Leysen 1998).

The time course for catalepsy in animals cotreated with SCH-23390 was found to proceed more or less in parallel with inhibition of apomorphine-induced behavior in the absence of SCH-23390. This confirms previously reported data on the time course of (mild) catalepsy after PDE10AIs alone (Kehler et al. 2007; Schmidt et al. 2008). However, catalepsy after D₂ receptor blockers intensifies over time with retesting of the animals (Stanley and Glick 1976; Hillegaart et al. 1987), this is apparently not the case after PDE10AIs. The reversal of haloperidol-induced catalepsy by PDE10AIs in the present study apparently conflicts with the reported potentiation of haloperidol-induced catalepsy after the PDE10AI papaverine (Siuciak et al. 2006). When a lower dose of haloperidol was used, however, we also showed PDE10AI-induced potentiation of catalepsy but only at low doses with a bell-shaped dose–response relation (Vanhoof et al. 2012).

The present data show that PDE10AI-induced direct pathway activation also counteracts catalepsy and related behaviors induced by reducing D₂ receptor-mediated neurotransmission via other mechanisms such as blockade of D₂ receptors with haloperidol or dopamine depletion with RO-4-1284 or reserpine. The PDE10AIs were somewhat less potent against haloperidol than against RO-4-1284 or reserpine, presumably because they had to overcome the D₂ receptor blockade by haloperidol, whereas D₂ receptors are functioning normal after dopamine depletion. Moreover, a switch in receptor-mediated signal transduction mechanism to a supersensitive form of D₁-mediated neuronal plasticity has been demonstrated in the dopamine-depleted striatum (Gerfen 2000).

The opposing effects of PDE10AIs in the reserpine test are striking. PDE10AIs inhibit the tail-pinch response before reserpine at doses close to the ED₅₀ for apomorphine antagonism, presumably via indirect pathway activation. After reserpine, however, only slightly higher doses induce the opposite effect, viz. reversal of the reserpine-induced tail-pinch response blockade, presumably via direct pathway activation. In other words, indirect or direct pathway activation predominates under condition of normal and low dopaminergic neurotransmission, respectively.

The anticataleptic properties of PDE10AIs at conditions of low dopaminergic transmission may suggest therapeutic potential in Parkinson’s disease. The anticataleptic effects are mediated via direct pathway activation, an effect that is shared with D₁ agonists. D₁ agonists have therapeutic potential in Parkinson’s disease but suffer from side effects (Mailman et al. 2001; Hurley and Jenner 2006). In view of minimal expression of PDE10A in peripheral tissue (Seeger et al. 2003; Coskran et al. 2006), PDE10AIs are likely devoid of the disturbing peripheral cardiovascular side effects of direct D₁ receptor agonists (Westfall and Westfall 2005).

The results obtained in mice further substantiate the results obtained in rats. Like haloperidol, the PDE10AIs inhibited d-amphetamine-induced hyperlocomotion, attesting to their functional D₂ antagonism. At similar doses, the PDE10AIs also counteracted the hypolocomotion induced by the D₁ antagonist SCH-23390 or the monoamines-depleting agent RO-4-1284. Despite their activity against haloperidol-induced catalepsy in rats, PDE10AIs hardly affected haloperidol-induced hypolocomotion in mice. As a similar profile was obtained with the D₁ agonist SKF-82958, the effects of PDE10AIs against SCH-23390 and RO-4-1284 are most likely related to direct pathway activation.

The minor effects obtained with the D₁ agonist SKF-82958 in rats do not fully support the hypothesis that common direct pathway activation mediates the effects of PDE10AI against the sedation and catalepsy induced by haloperidol, RO-4-1284, and reserpine. SKF-82958 antagonized haloperidol-induced catalepsy only at the 2-h interval but this may be related to short duration of action (Desai et al. 2005). However, the D₁ agonist also reversed the reserpine-induced effects only partially and tended to reverse RO-4-1284-induced catalepsy only at very high doses. The effect on D₁ signaling under these conditions is apparently more efficient with a PDE10AI than that with an orthosteric D₁ agonist. This may be the case because, under dopamine depletion, D₁ receptors become supersensitive resulting from a signaling switch from induction of immediate early genes (such as c-fos) toward ERK/MAP signaling (Gerfen et al. 2002), whereas PDE10AIs induce both immediate early genes and ERK/MAP signaling (Strick et al. 2010).

Behavioral stimulant effects of PDE10AIs were observed in combination with high doses of the D₁ antagonist SCH-23390 or the dopamine-depleting agents RO-4-1284 or reserpine but not in combination with the D₂ receptor blocker haloperidol. Like D₂ receptor blockers, PDE10AIs increase striatal dopamine turnover (Schmidt et al. 2008). In contrast to D₂ receptor blockers, however, PDE10AIs do not directly prevent D₂ receptor stimulation, thereby leaving dopaminergic neurotransmission largely intact. Thus, the above behavioral stimulant effects of PDE10AIs may arise from progressively increasing activation of the direct pathway overcoming the effect of D₁ receptor blockade and resulting in behavioral stimulation as long as the indirect pathway is not disinhibited by D₂ receptor blockade. The behavioral stimulant effects are observed at doses close to those required for reversal of the cataleptic state, reflecting very efficient direct pathway activation and limited safety margin for anti-Parkinson effects. Concomitant direct pathway activation may also explain the bell-shaped dose–response for inhibition of the tail-pinch response before reserpine. Similar bell-shaped effects on glucose metabolism in the lateral habenula of mice were also attributed to increased direct pathway activation at high PDE10AI doses (Dedeurwaerdere et al. 2011).

Balance between direct and indirect pathway activation is necessary for numerous behaviors including reward processing (Lobo et al. 2010; Beutler et al. 2011). Dopamine D₁ receptors in the direct striatal pathway facilitate several dopamine-dependent functions including appetitive behaviors. Global loss of dopamine D₁ receptors demonstrates its importance for feeding and reward acquisition to the general ability to thrive (Drago et al. 1994; Xu et al. 1994; Wall et al. 2011). In this regard, direct striatal pathway activation by PDE10AIs might improve negative and depressive symptomatology in schizophrenic patients. In view of the monoamine hypothesis of depression (Hirschfeld 2000), the antidepressant potential of PDE10AIs may be supported by the reversal of the effects of monoamines depletion observed in the present study.

Together with previously reported data, the present results indicate that PDE10AIs suppress stimulant behavior via indirect pathway activation, but the effect is counteracted by D₁ receptor stimulation and corresponding activation of the direct pathway. In the absence of counteracting direct pathway activation (D₁ receptor stimulation), however, this mechanism is very efficient, resulting in a limited safety margin toward cataleptic side effects. Conversely, PDE10AIs can reverse behavioral suppression (catalepsy, hypolocomotion) via direct pathway activation, but the effect is counteracted by D₂ receptor blockade and corresponding disinhibition of the indirect pathway. In the absence of concomitant, counteracting indirect pathway activation (D₂ receptor blockade), however, this mechanism is very efficient with a limited safety margin toward behavioral stimulant side effects. In analogy, the brake and accelerator of a car also function more efficiently when used separately rather than simultaneously, yet both are indispensable to adjust speed when out of balance. Further clinical studies have to show which patients may benefit from the dual activity profile of PDE10AIs.

Glossary

AE

adverse event

cAMP

cyclic adenosine monophosphate

cGMP

cyclic guanosine monophosphate

CL

confidence limits

EPS

extrapyramidal symptoms

i.v.

intravenous

JNJ-42314415

3-[6-(2-methoxyethyl)pyridin-3-yl]-2-methyl-8-morpholin-4-ylimidazo[1,2-a]pyrazine

MP-10

2-{[4-(1-methyl-4-pyridin-4-yl-1H-pyrazol-3-yl)phenoxy]methyl}quinoline

MSNs

medium spiny neurons

PDE10AI

phosphodiesterase 10A inhibitor

PQ-10

6,7-dimethoxy-4-[3-(quinoxalin-2-yloxy)pyrrolidin-1-yl]quinazoline

RO-4-1284

2-ethyl-9,10-dimethoxy-3-(2-methylpropyl)-1,3,4,6,7,11b-hexahydro-2H-pyrido[2,1-a]isoquinolin-2-ol

PDE

phosphodiesterase

PKA

protein kinase A

SCH-23390

8-chloro-3-methyl-5-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-ol

SKF-82958

6-chloro-1-phenyl-3-prop-2-en-1-yl-2,3,4,5-tetrahydro-1H-3-benzazepine-7,8-diol

TP-10

2-({4-[4-pyridin-4-yl-1-(2,2,2-trifluoroethyl)-1H-pyrazol-3-yl]phenoxy}methyl)quinoline

s.c.

subcutaneous

Disclosure

All authors are employees of Janssen Research & Development, a division of Janssen Pharmaceutica NV. All authors had access to the study data, provided direction and comments on the manuscript, made the final decision about where to publish these data, and approved submission to the journal.