Behavioral and neurochemical characterization of mice deficient in the phosphodiesterase-1B (PDE1B) enzyme

Neuropharmacology 53 (2007) 113e124 www.elsevier.com/locate/neuropharm

Behavioral and neurochemical characterization of mice deficient in the phosphodiesterase-1B (PDE1B) enzyme J.A. Siuciak a,*, S.A. McCarthy a, D.S. Chapin a, T.M. Reed b, C.V. Vorhees c, D.R. Repaske d a

CNS Discovery Research, Pfizer Global Research & Development, Pfizer Inc., Eastern Point Road, Groton, CT 06340, USA b Department of Biology, College of Mount Saint Joseph, Cincinnati, OH, USA c Division of Neurology, Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA d Division of Endocrinology, Cincinnati Children’s Hospital Medical Center and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA Received 30 December 2006; received in revised form 13 April 2007; accepted 16 April 2007

Abstract PDE1B is a calcium-dependent cyclic nucleotide phosphodiesterase that is highly expressed in the striatum. In order to investigate the physiological role of PDE1B in the central nervous system, PDE1B knockout mice (C57BL/6N background) were assessed in behavioral tests and their brains were assayed for monoamine content. In a variety of well-characterized behavioral tasks, including the elevated plus maze (anxietylike behavior), forced swim test (depression-like behavior), hot plate (nociception) and two cognition models (passive avoidance and acquisition of conditioned avoidance responding), PDE1B knockout mice performed similarly to wild-type mice. PDE1B knockout mice showed increased baseline exploratory activity when compared to wild-type mice. When challenged with amphetamine (AMPH) and methamphetamine (METH), male and female PDE1B knockout mice showed an exaggerated locomotor response. Male PDE1B knockout mice also showed increased locomotor responses to higher doses of phencyclidine (PCP) and MK-801; however, this effect was not consistently observed in female knockout mice. In the striatum, increased dopamine turnover (DOPAC/DA and HVA/DA ratios) was found in both male and female PDE1B knockout mice. Striatal serotonin (5-HT) levels were also decreased in PDE1B knockout mice, although levels of the metabolite, 5HIAA, were unchanged. The present studies demonstrate increased striatal dopamine turnover in PDE1B knockout mice associated with increased baseline motor activity and an exaggerated locomotor response to dopaminergic stimulants such as methamphetamine and amphetamine. These data further support a role for PDE1B in striatal function. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Phosphodiesterase; Knockout; Mouse; Dopamine; Serotonin; Striatum

1. Introduction The cyclic nucleotides cAMP and cGMP are second messengers mediating intracellular signal transduction. A key element in regulation of these signaling pathways is metabolic

Abbreviations: DA, dopamine; DOPAC, dihydroxyphenlyacetic acid; 5HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin; HVA, homovanillic acid; PDE1B, phosphodiesterase 1B; PDEs, phosphodiesterases. * Corresponding author. Tel.: þ1 860 715 2120; fax: þ1 860 686 0013. E-mail addresses: [email protected], judith.siuciak@gmail. com (J.A. Siuciak). 0028-3908/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2007.04.009

inactivation of cyclic nucleotides by cyclic nucleotide phosphodiesterases (PDEs) (for reviews, Soderling and Beavo, 2000; Bender and Beavo, 2006). In mammals, PDEs comprise a superfamily of enzymes divided into 11 families that are differentially localized throughout the organism. The ability to selectively regulate cyclic nucleotide signaling through pharmacological manipulation of these enzymes may offer unique therapeutic opportunities. Type 1 cyclic nucleotide phosphodiesterases (PDE1) are highly enriched in brain and are characterized by Ca2þ-dependent stimulation via the Ca2þ-binding protein calmodulin (CaM). Three PDE1 isoforms have been identified (PDE1A,

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PDE1B, and PDE1C) and all are expressed within the central nervous system. PDE1A is expressed throughout the brain, with high levels in the hippocampus, cerebellum and low levels in the striatum (Borisy et al., 1992; Yan et al., 1994). In situ hybridization and immunocytochemistry demonstrate high levels of PDE1B mRNA and/or protein in the caudate putamen, nucleus accumbens, olfactory tubercle and dentate gyrus, moderate levels in the olfactory bulb and cortex and lower levels in various other brain areas including the globus pallidus, substantia nigra, and hypothalamus (Polli and Kincaid, 1992, 1994; Repaske et al., 1993; Yan et al., 1994). The expression pattern of PDE1B observed within the caudate putamen is similar to that found for the D1 dopamine receptor and correlates strongly with brain areas that are richest in dopaminergic innervation, suggesting an important role in antagonism of cAMP-regulated signaling in dopaminoceptive neurons. PDE1C is expressed primarily in olfactory epithelium, cerebellum and striatum (Yan et al., 1995, 1996). Previous studies have reported the generation and initial characterization of the PDE1B knockout mouse (Reed et al., 2002). In this initial study, PDE1B knockout mice maintained on a C57BL6/129svj genetic background demonstrated altered spontaneous locomotor activity and Morris water maze behavior. Gender differences in locomotor activity were also reported for PDE1B knockout mice. The aim of the present experiments was to backcross PDE1B knockout mice onto a congenic C57BL/6N genetic background. Congenic male and female PDE1B wild-type and knockout mice would then be assessed in a variety of well-established behavioral tests including spontaneous locomotor activity, hot plate (analgesia), forced swim test (depression-like behavior), elevated plus maze (anxiety-like behavior), passive avoidance (cognition) and conditioned avoidance responding (acquisition and response to the suppressant effects of an antipsychotic drug). High levels of PDE1B are expressed within the striatal medium spiny neurons (Repaske et al., 1993; Furuyama et al., 1994), the activity of which are regulated by both cortical/thalamic glutamatergic inputs and midbrain dopaminergic projections. Previous studies have reported that PDE1B knockout mice demonstrate an exaggerated response to a single dose of METH (Reed et al., 2002; Ehrman et al., 2006). In order to investigate the interaction of PDE1B with both dopaminergic and glutamatergic systems, the locomotor response of PDE1B knockout mice to multiple doses of DA-releasing agents (METH and AMPH) and NMDA receptor antagonists (MK-801 and PCP) was also examined. Finally, in order to further explore a potential association between PDE1B and dopamine, we have examined the regional brain content of monoamines and their respective metabolites in these mice. 2. Methods

backcross onto a C57BL/6N (CRL) background. Mice generated for the present studies were bred using a knockout  knockout and wild-type  wild-type breeding strategy. Adult mice were housed in groups of 5e10 at ambient temperatures of 20e22  C, and under a 12:12-h light/dark cycle (lights on at 06:00 h). Mice were allowed food and water ad libitum. All studies were conducted during the light cycle. With the exception of the conditioned avoidance responding test, which used only male mice, all other behavioral tests utilized both male and female mice. All procedures relating to animal care and treatment were conducted according to the guidelines of the Institutional Animal Care and Use Committee at Pfizer and the National Institutes of Health guidelines.

2.2. Observational battery Mice were examined according to the protocols of the observational neurological test battery described elsewhere (Irwin, 1968). While placed in an open field, mice were assessed for transfer arousal, involuntary movements (tremors, convulsions, etc.), gait, gait abnormalities, mobility, arousal, respiration, stereotypy, unusual behavior, rears (defined as any time both front paws leave the floor), defecation, urination, diarrhea, polyuria, piloerection, response to stimuli to assess reactivity (approach with blunt object, tail pinch). Corneal reflex, righting reflex and inverted grid test are also assessed.

2.3. Locomotor activity Locomotor activity was performed as previously described (Siuciak and Fujiwara, 2004). Briefly, animals were placed into the automated locomotor activity boxes (Accuscan Instruments, Columbus, OH, 20  20  25 cm) and data were collected for 2 h. Data were reported as total horizontal activity representing the number of infrared beam breaks (mean  S.E.M.). During pharmacologically stimulated locomotor activity experiments, animals were initially habituated to the boxes for 2 h. After habituation animals were briefly removed from the boxes, injected with drug (phencyclidine (PCP), MK-801, amphetamine (AMPH), or methamphetamine (METH)) and immediately returned to the same chamber. Separate groups of mice were used for all stimulant-induced locomotor studies. Data were collected for an additional 2 h post-drug administration. Statistical analysis was performed using an ANOVA followed by Fisher’s test with significance set at p < 0.05.

2.4. Elevated plus maze The elevated plus maze was performed as previously described (MacQueen et al., 2001) and used commercially available equipment (HamiltonKinder, Poway, CA). Subjects were scored for a 5 min period by an automated infrared photo-beam system that measured entries into arms, time spent in arms, and total distance traveled. Statistical analysis was performed using an ANOVA followed by Fisher’s test with significance set at p < 0.05.

2.5. Forced swim test The forced swim test was performed as previously described (Siuciak and Fujiwara, 2004). Mice were placed into 1-L beakers (KIMAX #14005) filled with 800 ml of water (20e22  C) for a 7 min swim period which consisted of a 2 min acclimation period and a 5 min scoring period. During the 5 min scoring period, ten 30 s intervals were scored for a total possible score of 0e10 for each mouse. Animals were manually scored by a single experimenter during the last 5 min of the test and assigned either a ‘‘0’’ if actively swimming for the majority of the 30 s interval or ‘‘1’’ if immobile, except for small movements needed to keep afloat. Data were analyzed with a KruskaleWallis test, followed by ManneWhitney U-test with significance set at p < 0.05.

2.1. Animals 2.6. Hot plate Breeding pairs of PDE1B wild-type and knockout mice were obtained from Dr. David Repaske (Cincinnati, OH) and a colony was established and maintained at Charles River Laboratories (CRL, Wilmington, MA). All PDE1B wild-type and knockout mice were selected from the tenth generation

The hot plate was performed as previously described (Siuciak et al., 1994). Briefly, the test was conducted with a commercially available hot plate (Columbus Instruments, Columbus, OH) set at 55  C. Mice were individually

J.A. Siuciak et al. / Neuropharmacology 53 (2007) 113e124 placed on to the hot plate surface inside a Plexiglas cylinder and allowed to remain there until either lifting and licking a hindpaw or attempting escape (four paw jump). This time was recorded and the animal was removed from the apparatus. To prevent possible tissue damage, in the absence of a response, tests were terminated after 60 s and animals were assigned a time of 60 s. No animals reached the 60 s cut-off time in these experiments, and parametric statistics were used. Statistical analysis was performed using an ANOVA followed by Fisher’s test with significance set at p < 0.05.

2.7. Passive avoidance The passive avoidance test was performed as previously described (MacQueen et al., 2001). On day 1 (training day), mice were placed into commercially available shuttleboxes (Coulbourn Instruments). Mice were allowed a 60 s acclimation period during which the house lights were on. At the end of the acclimation period, the house light on the occupied compartment was lit, the guillotine door was opened, and the latency to enter the dark side of the shuttlebox was recorded. After the mouse entered the dark side, the door separating the two sides of the shuttlebox was closed and a 2 s, 0.6 mA constant current, continuous scrambled footshock was administered through the grid floor. The mouse was allowed to remain in the dark compartment for 10 s prior to being returned to the home cage. On the next day (24 h later, test day), mice were again placed into the apparatus and retested for latency to cross to the dark chamber. The test day latency was used as the measure of retention for the association between the dark compartment and foot-shock. If a mouse did not enter the dark side within 300 s, it was removed from the lit chamber, returned to its home cage and given a score of 300 for the day. Statistical analysis was performed using a Kruskal-Wallis test followed by a ManneWhitney U-test with significance set at p < 0.05.

2.8. Conditioned avoidance responding (CAR) test The CAR test was performed as previously described (Siuciak et al., 2006) on a separate group of na€ıve PDE1B wild-type and knockout mice. Briefly, testing was performed during the lights on period of the light/dark cycle, between 07:00 and 12:00 h using commercially available shuttleboxes (Coulbourn Instruments). The shuttle boxes were divided by a guillotine door into two sides, and enclosed in sound attenuating chambers. The shuttleboxes were fitted with metal grid floors equipped with scrambled/constant current shockers. Training consisted of repeated pairings of a conditioned stimulus (activation of house lights, cue lights, and the opening of the guillotine door), followed 5 s later by a 0.6 mA constant current, continuous scrambled footshock. The shock was terminated when the animal crossed to the other side of the shuttlebox, or after 10 s. Thirty trials were completed per session, and the number of avoidances (animal crossed before receiving shock, maximum 30), escapes (animal crossed after receiving shock, maximum 30), escape failures (animal failed to cross, maximum 30), latency to avoid (maximum 5 s), latency to escape (maximum 10 s), and adaptation crossovers (number of crossovers for a 5 min period before the onset of trials with chamber lights off) were recorded by the computer program. Inter-trial intervals were 30 s and were conducted with the guillotine door closed. Training was continued until a shock avoidance criterion of 80% avoidance was achieved, at which point animals were used for drug studies. Statistical analysis was performed using a KruskaleWallis test followed by a ManneWhitney U-test with significance set at p < 0.05.

2.9. HPLC analysis of brain monoamines HPLC analysis was performed as previously described (Siuciak et al., 2006) on a separate group of behaviorally na€ıve PDE1B wild-type and knockout mice. Mice were decapitated, the brains were removed and the striatum and hippocampus were dissected over ice. Each region was pooled bilaterally and stored at 70  C for subsequent analysis of dopamine (DA), serotonin (5-HT), norepinephrine (NE) and metabolite levels (DOPAC, HVA, 5-HIAA) by high-pressure liquid chromatography with electrochemical detection (HPLC-EC) (Coulochem II system, ESA Inc., Chelmsford, MA). Tissue samples were homogenized in a buffer (0.1 M phosphate-citrate buffer, pH 2.5,

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containing 15% methanol), centrifuged, and supernatants were injected onto the HPLC-EC system and were eluted with a mobile phase (MD-TM, ESA Inc.) at a flow rate of 1 ml/min through a reverse phase C-18 column (model HR-80, 3 mm column, ESA Inc.). Detector conditions (model 5011 high sensitivity cell, ESA Inc.) were 400 mV for the conditioning cell, 40 mV for electrode 1 and 320 mV for electrode 2 with sensitivity set at 1 mA for striatal tissue analysis and 200 nA for hippocampal tissue analysis. A t-test was used to assess statistical significance between wild-type and knockout mice.

2.10. Drugs Phencyclidine hydrochloride (PCP), MK-801 maleate, D-amphetamine sulfate, D-methamphetamine hydrochloride, and clozapine were all purchased from SigmaeAldrich (St. Louis, MO). Vehicles used included saline (MK801, AMPH, METH) and dH2O (PCP), and 0.3% tartaric acid for clozapine. All compounds were dosed and reported as the free base. All compounds were administered subcutaneously (s.c.), with the exception of MK-801 which was dosed via the intraperitoneal (i.p.) route, in a volume of 10 ml/kg.

3. Results 3.1. Behavioral observation No gross abnormalities were detected in PDE1B knockout mice. All mice appeared healthy, active and well groomed. No differences were observed between wild-type and knockout mice in a functional observational battery. Gross alterations of locomotor activity and reactivity to auditory and tactile stimulation were not apparent in the mutant mice. No tremors or convulsions were observed. All reflexes appeared normal. Body weight for male and female PDE1B wild-type and knockout mice was assessed over an eleven week period (n ¼ 44e48/group, data not shown). A two-way repeated measures ANOVA indicated no effect of genotype (F1,72 ¼ 0.99, not significant (n.s.)). There was a significant effect of gender (F1,72 ¼ 573, p < 0.0001) and post hoc analysis indicated female mice of both genotypes weighed less than the male mice. There was a significant effect of age, such that animals gained weight over time (F9,648 ¼ 64, p < 0.0001). 3.2. Spontaneous locomotor activity Fig. 1 shows a time course of horizontal locomotor activity in male and female PDE1B wild-type and knockout mice (n ¼ 44e48 mice/group). A two-way repeated measures ANOVA of the time-course data revealed a significant effect of genotype (F1,180 ¼ 24.2, p < 0.0001) such that PDE1B knockout mice showed increased locomotor activity when placed in a novel environment as compared to wild-type mice. There was also a significant effect of gender (F1,180 ¼ 9.4, p < 0.03) with the female mice generally showing higher levels of locomotor activity relative to males. There was no interaction (F1,180 ¼ 0.05, n.s.). The activity of both wild-type and knockout mice decreased over the 120 min test period as reflected by a significant effect of time (F23,4140 ¼ 268.9, p < 0.0001). Post hoc analysis of 120 min total activity indicated a significant difference between male wild-type and knockout mice (25% increase,

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Fig. 1. Baseline locomotor activity in male and female PDE1B wild-type (WT) and knockout (KO) mice. Data shown are horizontal activity (mean  S.E.M.) over 120 min test session for n ¼ 44e48 mice /group.

p ¼ 0.0003) and female wild-type and knockout mice (20% increase, p ¼ 0.001) (Table 1).

was no significant interaction between genotype and gender (F1,36 ¼ 3.0, n.s.).

3.3. Hot plate

3.4. Elevated plus maze

Male and female PDE1B wild-type and knockout mice responded similarly in the hot-plate assay (see Table 1, n ¼ 10 mice/group). There were no significant effects of either gender (F1,36 ¼ 0.002, n.s.) or genotype (F1,36 ¼ 0.02, n.s.). There

There were no significant effects of either gender or genotype for any of the elevated plus maze behaviors measured (See Table 1, n ¼ 10 mice/group) including percent time spent in open arm (gender, F1,77 ¼ 0.51, n.s.; genotype, F1,77 ¼ 0.003, n.s.; interaction, F1,77 ¼ 0.008, n.s.), percent time spent in closed arm (gender, F1,77 ¼ 0.008, n.s.; genotype, F1,77 ¼ 0.113, n.s.; interaction, F1,77 ¼ 0.156, n.s.), percent open arm entries (gender, F1,77 ¼ 1.62, n.s.; genotype, F1,77 ¼ 1.59, n.s.; interaction, F1,77 ¼ 0.34, n.s.) and percent closed arm entries (gender, F1,77 ¼ 1.22, n.s.; genotype, F1,77 ¼ 0.001, n.s.; interaction, F1,77 ¼ 0.001, n.s.).

Table 1 Behavioral responses of PDE1B wild-type and knockout mice Assay

Wild-type

Knockout

Spontaneous locomotor activity (120 min total horizontal locomotor activity) Male Female

19846  967 23007  872

24789  1060** 27538  910**

3.5. Forced swim test Hot plate (s) Male Female Elevated plus maze % Time open arm Male Female % Time closed arm Male Female % Entries open arm Male Female % Entries closed arm Male Female

17.3  1.5 22.3  1.7

22.0  5.4 16.7  1.4

12.6  2.6 15.5  4.9

12.4  4.4 16.1  5.6

80.0  2.8 78.5  4.8

76.5  5.2 78.8  5.3

8.3  1.0 11.4  1.7

7.2  2.3 8.3  1.2

42.3  0.9 40.2  1.5

42.3  2.2 40.1  2.2

*p < 0.05, **p < 0.01 vs. gender-matched wild-type mice.

No effects of genotype or gender were observed on immobility in the forced swim test (male PDE1B wild-type mice ¼ 9.3  0.3, knockout mice ¼ 9.4  0.2; U ¼ 195, n.s., n ¼ 20/group). Similar results were found in female mice (female PDE1B wild-type mice ¼ 9.6  0.2, knockout mice ¼ 9.3  0.2; U ¼ 127, n.s., n ¼ 16e20/group). 3.6. Passive avoidance On the training day, no significant overall effect was observed by KruskaleWallis ( p ¼ 0.84) between male and female PDE1B wild-type and knockout mice (see Fig. 2, n ¼ 10 mice/group). Cross-over latency at 24 h, a measure of retention, showed an overall significant effect by KruskaleWallis

J.A. Siuciak et al. / Neuropharmacology 53 (2007) 113e124 30 TRAIN LATENCY 250

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Fig. 2. Response of male and female PDE1B wild-type (WT) and knockout (KO) mice in the passive avoidance test. Data shown are latency (mean  S.E.M.) to enter the dark side of the shuttlebox chamber on both the training day, and 24 h later (test day) for n ¼ 10 mice/group.

test ( p ¼ 0.02). Post hoc analysis indicated no significant differences between male PDE1B wild-type and knockout mice (U ¼ 40, n.s.) or between female PDE1B wild-type and knockout mice (U ¼ 37, n.s.). However, overall female mice had significantly shorter test latencies than their male counterparts (wild-type mice, male vs. female, U ¼ 22, p ¼ 0.03; knockout mice, male vs. female, U ¼ 25, p ¼ 0.03). These data suggest that both knockout mice and controls successfully suppressed their preference for dark areas by avoiding the compartment within which they were previously shocked. 3.7. Conditioned avoidance responding Male PDE1B wild-type and knockout mice rapidly learned to avoid a shock by responding to the guillotine door opening and crossing into the non-shock chamber (Fig. 3, n ¼ 16 mice/ group). No significant differences were found between wildtype and knockout mice at any time point. By the fourth training day, the average percentage of successful avoidance responses had reached a plateau of >80% per session for both wild-type and knockout mice (wild-type vs. knockout mice, day 4, U ¼ 149, n.s.). Suppression of conditioned avoidance responding is a widely used model for the identification of potential antipsychotic drugs (for review see Wadenberg and Hicks, 1999). In order to verify that PDE1B wild-type and knockout mice were not only able to acquire the task but respond normally to antipsychotic drugs, the ability of clozapine to suppress CAR was assessed after training. No significant differences were found when comparing the effects of clozapine in wild-type and knockout mice in CAR. In wild-type mice, clozapine administration resulted in a dose-dependent inhibition of CAR with an ED50 value 1.34 mg/kg (95% CI ¼ 1.04e 1.73) without producing significant response failures (data not shown). Similar results were obtained in PDE1B knockout mice (ED50 value ¼ 1.60 mg/kg; 95% CI ¼ 1.30e1.96, no response failures).

WT

AVOIDANCES (MEAN +/- SEM)

MEAN (+/- SEM) SECONDS TO CROSS OVERS

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Fig. 3. Acquisition of conditioned avoidance task in male PDE1B wild-type (WT) and knockout (KO) mice. Data shown are for avoidance responses (mean  S.E.M.) for n ¼ 16 mice/group.

3.8. AMPH- and METH-stimulated locomotor activity Mice were allowed to habituate to the activity chambers for a 2 h session in order to allow the significant differences in baseline activity between the two groups to diminish. Following this habituation period, separate groups of mice received either vehicle, METH (0.56, 1.0, or 1.78 mg/kg), or AMPH (1.0, 1.78 or 3.2 mg/kg). Fig. 4 shows the effects of METH administration in male and female PDE1B wild-type and knockout mice (n ¼ 6e18 mice/group). After the 2 h habituation period, vehicle-treated wild-type and knockout mice showed similar locomotor responses (females, p ¼ 0.36, n.s.; males, p ¼ 0.79, n.s.). Both male and female PDE1B wild-type and knockout mice showed a dose-dependent increase in horizontal locomotor activity in response to METH (dose: F3,164 ¼ 230.9, p < 0.0001). Statistical analysis revealed there was a significant effect of genotype (F1,164 ¼ 119.6, p < 0.0001) and gender (F1,164 ¼ 38.6, p < 0.0001), as well as a significant interaction between genotype and dose (F3,164 ¼ 19.3, p < 0.0001). Post hoc analysis revealed that PDE1B knockout mice treated with METH showed a significantly increased locomotor response to METH administration compared to PDE1B wild-type mice at all doses tested. The female knockout mice showed increases of 75% (0.56 mg/kg), 104% (1.0 mg/kg), and 43% (1.78 mg/kg) compared to female wild-type mice. The male knockout mice showed increases of 90% (0.56 mg/kg), 84% (1.0 mg/kg), and 50% (1.78 mg/kg) compared to male wildtype mice. Fig. 5 shows the effects of AMPH administration in male and female PDE1B wild-type and knockout mice (n ¼ 6e20 mice/group). After a two hour habituation period, vehicletreated wild-type and knockout mice showed similar locomotor responses (females, p ¼ 0.59, n.s.; males, p ¼ 0.53, n.s.). Both male and female PDE1B wild-type and knockout mice showed a dose-dependent increase in horizontal locomotor activity in response to AMPH (dose: F3,182 ¼ 246.5, p < 0.0001). Statistical analysis revealed there was

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Fig. 4. Response of male and female PDE1B wild-type (WT) and knockout (KO) mice to methamphetamine (METH). Data shown are total horizontal activity over 120 min test session in male (left panel) and female (right panel) WT and KO mice following METH administration (0.56, 1.0 and 1.78 mg/kg, s.c.) for n ¼ 6e18 mice/group. *p < 0.05, **p < 0.01 versus VEH-treated WT or KO mice, respectively. #p < 0.05, ##p < 0.01 versus WT mice.

a significant effect of genotype (F1,182 ¼ 16.3, p < 0.0001) and gender (F1,182 ¼ 28.6, p < 0.0001), as well as a significant interaction between genotype and dose (F3,182 ¼ 5.5, p < 0.001). Post hoc analysis revealed male PDE1B knockout mice treated with AMPH showed a significant increase in the locomotor response to AMPH administration compared to PDE1B wild-type mice at the two highest doses (42% (1.78 mg/kg) and 15% (3.2 mg/kg)). Female PDE1B

knockout receiving AMPH showed a significant increase compared to wild-type mice only at a single dose (62% (1.78 mg/kg)). At the lower dose, there was a trend toward an increase in the locomotor activity of the female knockout mice ( p ¼ 0.08). In contrast, the female PDE1B knockout mice treated with the highest dose (3.2 mg/kg) showed a slightly decreased response compared to the female wildtype mice ( p ¼ 0.08).

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Fig. 5. Response of male and female PDE1B wild-type (WT) and knockout (KO) mice to amphetamine (AMPH). Data shown are total horizontal activity over 120 min test session in male (left panel) and female (right panel) WT and KO mice following AMPH administration (1.0, 1.78 and 3.2 mg/kg, s.c.) for n ¼ 6e20 mice/group. *p < 0.05, **p < 0.01 versus VEH-treated WT or KO mice, respectively. #p < 0.05, ##p < 0.01, #p ¼ 0.08 versus WT mice.

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3.9. MK-801 and PCP-stimulated locomotor activity Mice were allowed to habituate to the activity chambers for a 2 h session in order to allow the significant differences in baseline activity between the two groups to diminish as before. Following this habituation period, separate groups of mice received either vehicle, MK-801 (0.1, 0.178, or 0.32 mg/kg), or PCP (1.0, 3.2, or 5.6 mg/kg). Fig. 6 shows the effects of MK-801 administration in male and female PDE1B wild-type and knockout mice (n ¼ 10e24 mice/group). After a 2 h habituation period, vehicle-treated wild-type and knockout mice showed similar locomotor responses (females, p ¼ 0.42, n.s.; males, p ¼ 0.16, n.s.). Both male and female PDE1B wild-type and knockout mice showed a dose-dependent increase in horizontal locomotor activity in response to MK-801 (dose: F3,168 ¼ 105.9, p < 0.0001). Statistical analysis revealed there was a significant effect of genotype (F1,168 ¼ 29.7, p < 0.0001) and a significant interaction between genotype and dose (F3,168 ¼ 4.1, p < 0.008). There was no effect of gender (F1,168 ¼ 0.01, n.s.). Post hoc analysis revealed male PDE1B knockout mice treated with MK-801 showed a significantly increased locomotor response compared to wild-type mice at the two highest doses (0.178 and 0.32 mg/kg). Female PDE1B knockout mice receiving MK801 showed a significant increase compared to wild-type mice only at a single dose (0.178 mg/kg). Fig. 7 shows the effects of PCP administration in male and female PDE1B wild-type and knockout mice (n ¼ 10e12 mice/group). After a 2 h habituation period, vehicle-treated wild-type and knockout mice showed similar locomotor responses (females, p ¼ 0.82, n.s.; males, p ¼ 0.55, n.s.). Both male and female PDE1B wild-type and knockout mice showed a dose-dependent increase in horizontal locomotor activity in **

##

HORIZONTAL LOCOMOTOR ACTIVITY (MEAN +/- SEM)

50000

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response to PCP (dose: F3,167 ¼ 75.6, p < 0.0001). Statistical analysis revealed there was a significant effect of genotype (F1,167 ¼ 3.9, p ¼ 0.05) and a trend towards an interaction between genotype and dose (F3,167 ¼ 2.4, p < 0.07). There was no effect of gender (F1,167 ¼ 2.1, n.s.). Post hoc analysis revealed male PDE1B knockout mice treated with PCP showed a significantly increased (59%) locomotor response compared to wild-type mice at the highest dose (5.6 mg/kg). In contrast, female PDE1B wild-type and knockout receiving PCP showed similar locomotor activity at all doses. 3.10. DA, DOPAC and HVA levels The concentrations of DA, DOPAC, and HVA, as well as the ratios of DOPAC/DA and HVA/DA from the striatum of PDE1B wild-type and knockout mice are shown in Table 2. In male PDE1B knockout mice, the turnover rates, reflected in the DOPAC/DA ratio and the HVA/DA ratio were increased (21% and 17%, respectively), compared to male wild-type mice. DOPAC levels were significantly increased (21%), and there was a trend towards an increase in HVA content ( p ¼ 0.08), while DA levels were unchanged. In female PDE1B knockout mice, DOPAC and the DOPAC/DA ratio were elevated (21% and 44%, respectively), while DA levels were decreased (21%), compared to female wild-type mice. There was a trend towards an increase in DOPAC levels ( p ¼ 0.07). The concentrations of DA, DOPAC, and HVA, as well as the ratios of DOPAC/DA and HVA/DA from the hippocampus of PDE1B wild-type and knockout mice are shown in Table 2. In male PDE1B knockout mice, levels of DA were increased (22%) and the HVA/DA ratio was decreased (24%) compared to male wild-type mice. No significant changes were observed

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MK-801 DOSE (mg/kg) FEMALES WT

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Fig. 6. Response of male and female PDE1B wild-type (WT) and knockout (KO) mice to MK-801. Data shown are total horizontal activity over 120 min test session in male (left panel) and female (right panel) WT and KO mice following MK-801 administration (0.1, 0.178 and 0.32 mg/kg, i.p.) for n ¼ 10e24 mice/group. *p < 0.05, **p < 0.01 versus VEH-treated WT or KO mice, respectively. #p < 0.05, ##p < 0.01 versus WT mice.

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Fig. 7. Response of male and female PDE1B wild-type (WT) and knockout (KO) mice to PCP. Data shown are total horizontal activity over 120 min test session in male (left panel) and female (right panel) WT and KO mice following PCP administration (1, 3.2 and 5.6 mg/kg, s.c.) for n ¼ 10e12 mice/group. *p < 0.05, **p < 0.01 versus VEH-treated WT or KO mice, respectively. #p < 0.05, ##p < 0.01 versus WT mice.

in levels of DOPAC, HVA or in the DOPAC/DA ratio. In female wild-type and knockout mice, no significant differences were observed in DA or metabolite levels or turnover ratios. 3.11. 5-HT and 5-HIAA levels The levels of 5-HT and 5-HIAA, and the ratio of 5-HIAA/5HT from the striatum of PDE1B wild-type and knockout mice are shown in Table 3. Levels of 5-HT were decreased in both male (15%) and female (22%) knockout mice compared to their respective wild-type controls. The levels of 5-HIAA and the 5-HIAA/5-HT ratio were similar between wild-type and knockout mice. The levels of 5-HT and 5-HIAA and the ratio of 5-HIAA/5HT from the hippocampus of PDE1B wild-type and knockout mice are shown in Table 3. No significant differences were observed between wild-type and knockout mice for 5-HT or 5HIAA content or for the 5-HIAA/5-HT ratio.

3.12. Norepinephrine levels Levels of NE in the striatum were below the limits of detection. Hippocampal NE content was similar in wild-type and knockout mice (male wild-type ¼ 0.41  0.01 vs. male knockout ¼ 0.44  0.01 ng/mg tissue, t ¼ 1.28, n.s.; female wildtype ¼ 0.38  0.02 vs. knockout ¼ 0.36  0.01 ng/mg tissue, t ¼ 0.77, n.s.; n ¼ 10 mice/group). 4. Discussion Previous studies have reported the generation and initial behavioral characterization of PDE1B knockout mice (Reed et al., 2002). Although the present studies are an extension of this previous work, one significant difference between these two studies is the genetic background of the knockout mouse. In the initial study, PDE1B mice were backcrossed to C57Bl/ 6N (CRL) for three generations and then maintained on

Table 2 Levels of DA, DOPAC and HVA in selected brain regions in PDE1B wild-type and knockout mice DA

DOPAC

Striatum Wild-type Knockout Wild-type Knockout

Female Female Male Male

10.59  0.38 8.36  0.44** 10.82  0.42 10.64  0.70

0.79  0.03 0.90  0.04 0.90  0.03 1.09  0.07*

Hippocampus Wild-type Knockout Wild-type Knockout

Female Female Male Male

0.020  0.001 0.021  0.001 0.023  0.001 0.028  0.002*

0.009  0.004 0.009  0.001 0.009  0.001 0.010  0.001

DOPAC/DA 0.075  0.002 0.108  0.003** 0.083  0.003 0.103  0.002**

0.43  0.02 0.44  0.04 0.41  0.02 0.35  0.02

Data shown are ng/mg tissue for n ¼ 10 mice/group. *p < 0.05, **p < 0.01 vs. gender-matched wild-type mice.

HVA

HVA/DA

0.98  0.04 0.96  0.05 0.95  0.03 1.09  0.07

0.092  0.002 0.115  0.005** 0.088  0.002 0.103  0.004**

0.020  0.001 0.019  0.001 0.019  0.001 0.018  0.001

0.98  0.05 0.93  0.11 0.84  0.05 0.64  0.04**

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121

Table 3 Levels of 5-HT and 5-HIAA in selected brain regions in PDE1B wild-type and knockout mice 5-HT

5-HIAA

5-HIAA/5-HT

Striatum Wild-type Knockout Wild-type Knockout

Female Female Male Male

0.46  0.02 0.36  0.03** 0.55  0.02 0.47  0.03*

0.31  0.01 0.27  0.02 0.29  0.02 0.26  0.01

0.68  0.03 0.77  0.05 0.53  0.02 0.57  0.02

Hippocampus Wild-type Knockout Wild-type Knockout

Female Female Male Male

0.63  0.02 0.64  0.02 0.61  0.02 0.65  0.02

0.43  0.02 0.40  0.02 0.33  0.02 0.37  0.01

0.68  0.03 0.62  0.02 0.54  0.02 0.57  0.02

Data shown are ng/mg tissue for n ¼ 10 mice/group. *p < 0.05.

a mixed C57BL6/129svj genetic background for behavioral evaluation. In the present studies, the knockout mice have been backcrossed onto a C57BL/6N background for ten generations. The genetic background of knockout mice has been reported to play an important role in the phenotype (Groenink et al., 2003). Similar to the previous studies using a C57BL6/ 129svj genetic background, disruption of the PDE1B gene in mice maintained on a C57BL/6N background did not lead to any gross alteration in behavior. PDE1B knockout mice appeared healthy and behaved normally in a functional observational battery. Previous studies using PDE1B knockout mice have reported impaired spatial learning in the Morris water maze (Reed et al., 2002; Ehrman et al., 2006). We have extended these studies to include the assessment of PDE1B knockout mice in two additional cognitive tests, passive avoidance and acquisition of conditioned avoidance responding. We saw no effects of genetic deletion of PDE1B on cognitive performance using these two models employing shock avoidance. Thus, the previous deficit observed with the Morris water maze may represent a specific deficit in hippocampal-dependent spatial learning. Consistent with the previous reports (Reed et al., 2002; Ehrman et al., 2006), PDE1B knockout mice show increased spontaneous locomotor activity. Both male and female knockout mice show similar increases in locomotor activity compared to their wild-type counterparts (approximately 25% increase over 120 min test period). PDE1B mRNA has been localized within the caudate putamen with an expression pattern similar to the D1 dopamine receptor and correlating strongly with brain areas rich in dopaminergic innervation, suggesting an important role in modulation of cAMP-regulated signaling in dopamine-receptive neurons (Reed et al., 2002). Previous studies have demonstrated that PDE1B knockout mice show an increased response to the locomotor stimulant METH, which has dopamine releasing properties. In these studies (Reed et al., 2002; Ehrman et al., 2006), a single dose level of METH was used (1 mg/kg and 0.8 mg/kg, respectively). In addition, mice were habituated for a shorter duration (1 h). The present studies confirm and extend these previous reports to show that both male and female

PDE1B knockout mice show an exaggerated response to METH (44 -104% increase) across the spectrum of doses employed (0.56, 1.0 and 1.78 mg/kg). We have also assessed the responses of PDE1B knockout mice to an additional dopaminergic locomotor stimulant, AMPH. It was of interest to note that the response to AMPH, although still enhanced, was less robust than that observed for METH. Male PDE1B knockout mice showed an increased responsiveness to AMPH, but only at the two higher doses tested (1.78 mg/kg, 42% increase and 3.2 mg/kg, 15% increase). The lowest doses of METH (0.56 mg/kg) and AMPH (1.0 mg/kg) produced equivalent locomotor responses in male wild-type mice (approximately 13,000 counts/120 min test). In contrast, these same doses in knockout mice produced different results (METH(0.56) ¼ 26,367 counts vs. AMPH(1.0) ¼ 16,765 counts), suggesting a differential responsiveness to the two stimulants in PDE1B knockout mice. In female mice the response to AMPH was even less pronounced, with the only significant increase observed at the 1.78 mg/kg dose (62%). AMPH and the N-methylated analogue, METH, are both phenylethylamines, which share several pharmacokinetic and pharmacodynamic properties (Melega et al., 1995). Despite these similarities, recent studies have demonstrated neurochemical differences between METH and AMPH. Microdialysis studies have shown that both METH and AMPH effect DA and 5-HT systems (Kuczenski et al., 1995; Shoblock et al., 2003, 2004), both of which are altered in the striatum of PDE1B knockout mice in the present studies. METH is more effective at releasing serotonin than AMPH (Kuczenski et al., 1995). In contrast, AMPH is more effective than METH at raising DA levels in the prefrontal cortex and striatum (Kuczenski et al., 1995; Shoblock et al., 2003). It is possible that the observed increased response of PDE1B knockout mice to METH compared to AMPH is due to a differential modulation of serotonin versus dopamine. This is further supported by data that PDE1B knockout mice demonstrated an exaggerated locomotor response to the 5-HT releasing drug p-chloroamphetamine (Ehrman and Vorhees, unpublished observations). However, METH and AMPH also exhibit different interactions with biogenic amine transporters which may be responsible for some of their observed neurochemical

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differences (Shoblock et al., 2004). It is also possible that the present results are attributable to knockout-induced alterations in monoamine transporters, although future studies are needed to assess this possibility. The striatal medium spiny neurons, which express high levels of PDE1B, are also regulated by cortical/thalamic glutamatergic inputs. In order to investigate the interaction of PDE1B with glutamatergic systems, we assessed the locomotor response of PDE1B knockout mice to the NMDA receptor antagonists, MK-801 and PCP. Following MK-801 administration, male PDE1B knockout mice showed a significant increase in locomotor response compared to wild-type mice at the two highest doses (0.178 and 0.32 mg/kg). Female PDE1B knockouts receiving MK-801 showed a significant increase compared to PDE1B wild-type mice only at a single dose (0.178 mg/kg). PCP produced less robust effects, with the increased response in male knockout mice limited to the highest dose (5.6 mg/kg, 59%), and no differences in the female knockout mice. One possible explanation is that PDE1B knockout mice have a generalized increase in responsivity to a broad range of locomotor stimulants, particularly since they demonstrate baseline hyperactivity. However, the differential response across stimulants and genders argues against such a conclusion. Male and female knockout mice showed similar increases in baseline locomotor activity, yet showed significantly different responses to stimulants, particularly to the NMDA antagonists. In the present studies, we demonstrate that PDE1B knockout mice show increased striatal dopamine turnover as well as an increased locomotor response to dopamine releasing agents such as AMPH and METH. It is well established that dopamine and glutamate interact extensively within the striatum (for review see Missale et al., 2006). The D1 receptor and the NMDA receptor are both expressed within the medium spiny neurons of the striatum and are co-localized within the postsynaptic density of the spine heads of these neurons. In addition, both these receptors appear to co-regulate receptor translocation in response to individual receptor stimulation and may act and be expressed as a complex (Missale et al., 2006). NMDA antagonists have been reported to increase dopamine turnover in various brain regions (Rao et al., 1990; Maj et al., 1991) and increase the firing rate of dopaminergic neurons (French and Ceci, 1990; Murase et al., 1993). Thus, it is interesting to speculate that the administration of MK-801 or PCP to PDE1B knockout mice, which already have altered baseline dopaminergic activity, results in the observed increased hyperlocomotor responsivity of these NMDA antagonists. Previous experiments have demonstrated that PDE1B-dependent hyperlocomotor responses are due to interactions with DARPP-32-dependent (dopamine and cAMP regulated phosphoprotein 32 kDa) pathways which respond to both D1 and NMDA receptor activation (Reed et al., 2002; Ehrman et al., 2006). However, the exact mechanisms by which PDE1B is involved in these receptor-activated DARPP-32 pathways is unknown at this time. We have also assessed the levels of monoamines and metabolites within the striatum and hippocampus of PDE1B

wild-type and knockout mice. The current studies demonstrate gender- and region-specific alterations in dopaminergic and serotonergic activity predominantly within the striatum of PDE1B knockout mice. The observed changes were primarily alterations in DA turnover, reflected as increases in the DOPAC/DA and HVA/DA ratios and were found in both male and female knockout mice. Other changes were also observed in male mice (increased striatal DOPAC, decreased hippocampal HVA/DA ratio, increased hippocampal DA) and may be responsible for some of the gender differences observed in response to stimulant administration. Within the 5HT system, the predominant effect was a decrease in striatal 5-HT content found in both male and female knockout mice. A previous report in PDE1B knockout mice (Ehrman et al., 2006) found no differences in monoamine levels in these same brain areas. However, the predominant changes in the present study were found in striatal dopamine turnover (DOPAC/DA and HVA/DA ratios), a variable not reported in this previous study. Furthermore, this previous study performed neurochemical analyses in animals used in behavioral studies, while the present studies utilized na€ıve mice. This, along with other methodological differences (i.e. background strain, housing, handling and/or HPLC methods), may reflect the discrepancies in the results. At present, it is unclear how genetic deletion of PDE1B leads to changes in dopamine turnover. Changes in the expression of dopamine receptors, alterations in receptor signal transduction or modification of the receptor phosphorylation may play a role. Studies by Yamashita et al. (1997) report that the nonselective PDE1 inhibitor vinpocetine caused dopamine depletion in primary cultured rat mesencephalic neurons, suggesting PDE1 regulates the release of dopamine or other neurotransmitters. Removing PDE1B cyclic nucleotide hydrolyzing ability presumably increases the magnitude and prolongs the duration of the D1 receptor generated increase in cyclic nucleotides and their downstream phosphorylation signaling. Studies using striatal slices from PDE1B knockout mice (Reed et al., 2002) reported increased phosphorylation of DARPP-32 (dopamine and cAMP regulated phosphoprotein 32 kDa). Additional studies will be required to further understand the interactions between PDE1B and dopamine within the striatum. In light of the findings discussed above, it is interesting to compare the behavioral phenotype of the PDE1B knockout mice with that of the PDE10A knockout mice that we have recently generated and characterized in similar behavioral tests and neurochemical assays (Siuciak et al., 2006). PDE10A is also highly enriched in the striatal medium spiny neurons and is similarly a dual substrate enzyme (for review see Siuciak and Strick, 2006). In contrast to the PDE1B knockouts, PDE10A knockout mice are hypoactive in baseline locomotor tests and exhibit a blunted locomotor response to the NMDA antagonists MK-801 and PCP (Siuciak et al., 2006). Furthermore, PDE10A knockout mice show normal responses to METH and AMPH and have no alterations in brain levels of monoamines compared to wild-type mice. The unique behavioral phenotype resulting from the elimination

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of PDE1B in comparison to PDE10A, despite their similar substrate specificity, suggests that each is differentially compartmentalized within the medium spiny neurons and that they regulate distinct cyclic nucleotide pools. These two striatal PDEs may regulate unique signaling pathways or may work in opposition within the same pathways. Additional studies with PDE1B knockout mice using wellcharacterized behavioral tasks, including the elevated plus maze (anxiety-like behavior), forced swim test (depressionlike behavior), hot-plate (nociception) and two learning and memory models (passive avoidance and acquisition of CAR) demonstrated that PDE1B knockout mice performed similarly to wild-type mice. However, the aim of the present studies was a broad behavioral screen and as such, does not represent an exhaustive exploration of these mice in all available models targeting these disease areas. Future experiments in these knockout mice, using modifications of the present models, additional animal models or employing drug challenges, may reveal additional abnormal phenotypes. For example, studies have demonstrated that water temperature is an important determinant of degree of immobility in the forced swim test (Cryan et al., 2005), and assessment of pain sensitivity can be dependent upon the temperature of the hotplate, and therefore additional experiments manipulating water or plate temperature could be performed to determine the generality of the effects reported herein. In conclusion, the present studies demonstrate increased striatal DA turnover in PDE1B knockout mice associated with increased baseline motor activity and an exaggerated locomotor response to dopaminergic stimulants such as methamphetamine and amphetamine. These data further support a role for PDE1B in striatal function. Acknowledgments Portions of this work were presented at the Society for Neuroscience, San Diego, CA, November 2001. The authors would like to thank the Genetically Modified Mouse breeding group for assistance with these studies. References Bender, A.T., Beavo, J.A., 2006. Cyclic nucleotide phosphodiesterases: molecular regulation to clinical use. Pharmacol. Rev. 58, 488e520. Borisy, F.F., Ronnett, G.V., Cunningham, A.M., Juilfs, D., Beavo, J., Snyder, S.H., 1992. Calcium/calmodulin-activated phosphodiesterase expressed in olfactory receptor neurons. J. Neurosci. 12, 915e923. Cryan, J.F., Valentino, R.J., Lucki, I., 2005. Assessing substrates underlying the behavioral effects of antidepressants using the modified rat forced swimming test. Neurosci. Biobehav. Rev. 29, 547e569. Ehrman, L.A., Williams, M.T., Schaefer, T.L., Gudelsky, G.A., Reed, T.M., Fienberg, A.A., Greengard, P., Vorhees, C.V., 2006. Phosphodiesterase 1B differentially modulates the effects of methamphetamine on locomotor activity and spatial learning through DARPP32-dependent pathways: evidence from PDE1B-DARP32 double-knockout mice. Genes Brain Behav. 5, 540e551. French, E.D., Ceci, A., 1990. Non-competitive N-methyl-D-aspartate antagonists are potent activators of ventral tegmental A10 dopamine neurons. Neurosci. Lett. 119, 159e162.

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