Methylphenidate restores novel object recognition in DARPP-32 knockout mice

Behavioural Brain Research 253 (2013) 266–273

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Methylphenidate restores novel object recognition in DARPP-32 knockout mice Charles J. Heyser a,b,∗ , Caitlyn H. McNaughton b , Donna Vishnevetsky b , Allen A. Fienberg c a b c

Department of Neuroscience, University of California, San Diego, La Jolla, CA 92093, USA Department of Psychology, Franklin & Marshall College, Lancaster, PA 17603, USA Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY 10021, USA

h i g h l i g h t s • • • • •

Male and female DARPP-32 knockout mice were impaired in novel object recognition. The effect of methylphenidate on locomotor activity was blunted in DARPP-32 knockout mice. Discriminative performance of knockout mice during the test trial was restored by the administration of methylphenidate. The administration of methylphenidate disrupted novel object recognition in wild-type mice. These data provide further evidence for the involvement of DARPP-32 in learning and memory.

a r t i c l e

i n f o

Article history: Received 30 April 2013 Received in revised form 18 July 2013 Accepted 20 July 2013 Available online 29 July 2013 Keywords: DARPP-32 Methylphenidate Ritalin Novel object recognition Memory

a b s t r a c t Previously, we have shown that Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) knockout mice required significantly more trials to reach criterion than wild-type mice in an operant reversal-learning task. The present study was conducted to examine adult male and female DARPP-32 knockout mice and wild-type controls in a novel object recognition test. Wild-type and knockout mice exhibited comparable behavior during the initial exploration trials. As expected, wild-type mice exhibited preferential exploration of the novel object during the substitution test, demonstrating recognition memory. In contrast, knockout mice did not show preferential exploration of the novel object, instead exhibiting an increase in exploration of all objects during the test trial. Given that the removal of DARPP32 is an intracellular manipulation, it seemed possible to pharmacologically restore some cellular activity and behavior by stimulating dopamine receptors. Therefore, a second experiment was conducted examining the effect of methylphenidate. The results show that methylphenidate increased horizontal activity in both wild-type and knockout mice, though this increase was blunted in knockout mice. Pretreatment with methylphenidate significantly impaired novel object recognition in wild-type mice. In contrast, pretreatment with methylphenidate restored the behavior of DARPP-32 knockout mice to that observed in wild-type mice given saline. These results provide additional evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory. These results also indicate that the behavioral deficits in DARPP-32 knockout mice may be restored by the administration of methylphenidate. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Over the past 30 years, using a variety of molecular, cellular and behavioral approaches, dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) has been established as a

∗ Corresponding author at: University of California, San Diego, Department of Neuroscience, 9500 Gilman Drive, #0608, La Jolla, CA 92093-0608, USA. Tel.: +1 858 534 1615; fax: +1 858 534 1615. E-mail address: [email protected] (C.J. Heyser). 0166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbr.2013.07.031

critical mediator of the biochemical, electrophysiological, transcriptional and behavioral effects of dopamine [18,49,53,58]. However, the specific role of DARPP-32 in the processes of learning and memory is still poorly understood. DARPP-32 is present at high levels in striatal medium spiny neurons and at much lower levels in other neuronal populations [38,39,55,56]. Activation of D1-like receptors by dopamine results in the phosphorylation of DARPP-32 by cAMP-dependent protein kinase (PKA) at the threonine 34 (Thr34) site and converts DARPP-32 into a potent inhibitor of protein phosphatase-1 (PP1) [27]. Stimulation of D2-like receptors by dopamine results in the activation of

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calcium/calmodulin-dependent protein phosphatase (calcineurin) and the dephosphorylation of DARPP-32 [25]. By inhibiting PP1, DARPP-32 controls the state of phosphorylation and physiological activity of many neuronal phosphoproteins, including neurotransmitter receptors, ion channels, ion pumps and transcription factors (for review see [10,49,53,58]). Without the aid of selective pharmacological ligands to study the behavioral significance of DARPP-32, knockout mice were generated [19]. In the initial paper, Fienberg et al. (1998) reported that DARPP-32 knockout mice exhibited profound deficits in their molecular, electrophysiological, and behavioral responses to dopamine including an attenuated response to cocaine, amphetamine, and methamphetamine challenge compared to wild-type controls. Previous research in our laboratory has shown equivalent acquisition by DARPP-32 knockout mice and wild-type mice in a discriminated operant task for food reinforcement [29]. However reversal learning was impaired in the DARPP-32 knockout mice, who required significantly more trials to reach criterion than wild-type mice [29]. Using a similar operant task, Stipanovich et al. [52] has shown that in response to physiological rewards, DARPP-32 accumulates in the nucleus. This effect is mediated by D1 receptor stimulation and appears necessary for behavioral reward learning [52]. In addition, both nuclear accumulation induced by D1 stimulation and behavioral reward learning were eliminated in DARPP32-Ser97-Ala mutant mice [52]. Changes in DARPP-32 expression have also been observed following the acquisition of an inhibitory avoidance task [43]. More recently, it has been reported that c-Fos and DARPP-32 immunoreactivity in the accumbens shell was significantly increased on the first day of fixed-ratio-5 (FR5) training for food, while c-Fos and DARPP-32 expression in the core significantly increased on the second day of FR5 training [48]. Furthermore, D1 receptor modulation of memory retrieval performance in a novel object recognition test was reported to be associated with change in DARPP-32 in rat prefrontal cortex [31]. In humans, The DARPP-32 gene has been associated with striatal dopamine function and was predictive of probabilistic learning [20]. Taken together, these results provide evidence for a functional role of DARPP-32 in the mediation of processes underlying learning and memory. However, not all forms of learning are affected by manipulations of DARPP-32. For example, DARPP-32 knockout mice acquire a discriminated operant behavior [29] and were unimpaired in the water maze [15]. Therefore, the present study was conducted to further characterize the role of DARPP-32 in learning and memory. More specifically, male and female DARPP-32 knockout mice and wild-type controls were tested for novel object recognition using an object discrimination procedure (adapted from [6,17,46]) that is routinely used in our laboratory [28,30]. The advantages of the novel object recognition task are that there is no explicit need for food or water restriction and several behavioral endpoints can be rapidly obtained, including general activity, reactivity to novelty, and learning [9,16]. The novel object recognition task has been shown to be sensitive to dopaminergic manipulations. For example, Besheer and colleagues [8] showed that systemic injection of the dopamine D1 receptor antagonist SCH23390 before the retention test impaired the performance of rats in detecting the novel object. Object recognition was improved at a 4-hr delay with systemic injections of the D1 agonist SKF 81297 and spatial memory was disrupted by the selective D1 antagonist SCH 23390 [31]. The improvement in recognition and temporal order memory performance at a 4-hr delay was associated with increased phosphorylation of both CREB and DARPP-32 in the PFC of rats treated with the D1 agonist SKF 81297, whereas the impairing effect of SCH 23390 was associated with decreased phosophylation of CREB and DARPP-32 in the prefrontal cortex [31]. Object recognition memory was impaired following bilateral

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microinjections of the dopamine antagonist SCH 23390 into the PFC [36]. Further evidence for the involvement of dopamine in novel object recognition comes from a recent study showing that the performance of mice was systematically related to dopamine levels in the core region of the nucleus accumbens, but not the shell [37]. In the present study, the results of Experiment 1 show that male and female DARPP-32 knockout mice exhibited impaired performance, with these mice exhibiting equal exploration of the novel and familiar object during the substitution test. Given that the removal of DARPP-32 in the knockout mouse effectively alters the intracellular circuit of these mice while leaving dopamine receptors levels intact [19], it might be possible to restore neuronal performance by increasing the activational state of dopamine receptors. In support of this hypothesis, it has been reported that Thr34-Ala DARPP-32 mutant mice require more time to acquire cocaine self-administration [60]. However, once acquired these mice administered more cocaine at lower doses compared to wildtype mice [60]. The authors suggest that when stimuli are strong, as in the case with higher doses of cocaine, phosphorylation by PKA is sufficient to compensate for a deficiency of the DARPP32 pathway. However, when stimuli are weaker, as in the case with the lower doses of cocaine, mice with an impaired DARPP32 pathway need to increase responding for cocaine in order to get the same amount of reinforcement. Therefore, Experiment 2 was conducted to examine the effect of methyphenidate on novel object recognition memory in male and female DARPP32 knockout mice and wild-type controls. The psychostimulant methylphenidate was selected for use in this study for several reasons. First, it is a stimulant widely used in the treatment of ADHD [50]. Second, the pharmacodynamics of methylphenidate are similar to amphetamine and cocaine [51,54]. The administration of methylphenidate increases synaptic levels of dopamine and norepinephrine in several brain regions including the hippocampus and the prefrontal cortex by binding to and blocking dopamine and norepinephrine reuptake transporters [22,40]. Third, although there are studies looking at cocaine and amphetamine challenge in DARPP-32 knockout mice [19,52,59,60], there are no published reports on the effects of methylphenidate in these knockout mice. All of these studies [19,52,59,60] reported a blunted response to psychostimulant challenge in DARPP-32 knockout mice. And lastly, it has been reported that methylphenidate increased DARPP-32 Thr34 phosphorylation and decreased Thr75 phosphorylation in slices from adult mice [21]. The dose of methylphenidate was selected based on past research [23,30] and a report that indicated the peak drug response occurs at 20 min and remains elevated to 80 min after an i.p. injection [24]. The duration of the novel object recognition procedure used in the present study was 42 min, thus all behavioral observations were made during the peak drug response.

2. Methods 2.1. Animals DARPP-32 knockout (−/−) and wild-type control (+/+) male and female mice were generated and bred as previously described [19]. The mice used in the present study were selected from offspring of heterozygous × heterozygous breeding pairs. A total of 15 knockout and 15 wild-type adult (4- to 5-month old) male and female mice were used in Experiment 1. A separate group of 22 knockout and 22 wild-type adult (3- to 4-month old) male and female mice were used in Experiment 2. Mice were housed 3–4 in Plexiglas cages (28 cm × 17 cm × 11.5 cm) with sawdust bedding changed weekly. All mice were kept under conditions of a regular light/dark cycle (lights on 8:00AM, lights off 8:00PM) in a colony room maintained at a temperature of 24 ◦ C. Food and water were available ad libitum throughout training. All studies were conducted in accordance with the Guide for the Care and Use of Laboratory Animals [1] and were approved by the Institutional Animal Care and Use Committee of Franklin & Marshall College.

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2.2. Apparatus The apparatus consisted of a circular open field (120 cm in diameter). The floor was made of wood and was painted dark green. The walls (30 cm in height) were made of aluminum and were painted flat black. The objects used in the experiments were a yellow smooth plastic square (6 cm × 6 cm × 6 cm), an opaque pink smooth rectangular plastic bottle on its side (5 cm × 14 cm × 5 cm), a white smooth cardboard box (7.5 cm × 8 cm × 3 cm), a clear grooved glass dome (8 cm in diameter, 4 cm in height), and a gray smooth plastic rectangle with a hole in the center (6 cm × 12 cm × 6 cm). All objects were of sufficient weight such that they could not be moved by the animal. The open field was illuminated by diffuse incandescent lighting (15 lux). A video camera was located directly over the center of the open field and was connected to a VCR and monitor located in the room immediately outside the testing room to minimize noise and the presence of the experimenter. 2.3. Procedure 2.3.1. Novel object exploration: Experiment 1 Prior to the start of testing all mice were handled for 5 min per day for five days. Testing consisted of five 6-min trials, with a 3-min intertrial interval between each trial. During the intertrial interval the mouse was placed in a holding cage, which remained inside the testing room. In the first trial (Pre-Exposure), each mouse was placed individually into the center of the otherwise empty open field for 6 min. For the next three trials (Sample Trials 1–3) four different objects were placed into the open field. In all cases the configuration of the objects was such that the objects were equidistant from each other and from the wall (i.e., forming a square). In the last trial (Test), one of the objects was replaced with a novel object. Objects and their placement into the open field were varied across mice to avoid positional biases. To control for possible odor cues the objects were cleaned with a 10% ethanol solution at the end of each trial and the floor of the open field wiped down to eliminate possible scent/trail markers. During the test phase, the novel object was also wiped down prior to testing so that the objects would all have the same odor. In each trial, duration of contact with each object was recorded using a stopwatch. Exploration was defined as direct contact of the nose or front paws with the object. In addition, the number of outer and inner line crosses was recorded for each trial. In order to score line crosses, the open field was divided into 28 sections: 16 outer sections and 12 inner sections. To record a line cross, the mouse must have three limbs cross into and out of the section. All behaviors were scored from videotape to ensure accuracy. All observers were blind to sex and genotype and were trained to a scoring criterion of greater than 95% inter-rater reliability. 2.3.2. Novel object exploration – methylphenidate challenge: Experiment 2 Adult male and female WT and KO mice were randomly assigned to either the saline or methylphenidate group. The same apparatus and procedure described in Experiment 1 was used in this challenge. Each animal was given an intraperitoneal injection (i.p.) of either saline or 5 mg/kg methylphenidate (Sigma–Aldrich) 20-min prior to the start of the pre-exposure trial. Injections were given in a volume of 1 ml/100 g body weight and each animal was tested only once. 2.4. Data analysis The data were analyzed using analysis of variance (ANOVA) and t-tests; the specific details are included in the results section. Tukey’s tests and simple main effects analyses [33] were used to determine the locus of significant main effects and interactions. A significance level of p < 0.05 was used for all statistical analyses.

3. Results 3.1.1. Experiment 1: novel object exploration The number of outer line crosses and inner line crosses were analyzed by a 2 (group: wild-type vs. knockout) × 2 (sex) × 5 (trial) mixed analysis of variance (ANOVA), with trial as a withinsubjects factor. As can be seen in Table 1, the number of outer line crosses decreased across trials for all mice: main effect of trial F(4,104) = 59.33, p = 0.000. Tukey’s tests indicated that the mice exhibited significantly more outer line crosses on the pre-exposure trial than on first trial with objects (sample trial 1), which was significantly greater than sample trials 2, 3 and the test trial, which were not different from each other. No other statistical differences were obtained. The analysis on the number of inner line crosses indicated a main effect of trial F(4,104) = 8.122, p = 0.001, along with a significant sex × trial interaction F(4,104) = 3.35, p = 0.013. As can seen in Table 1, no differences were observed among the mice during the pre-exposure trial. Male mice made significantly more inner

Fig. 1. Mean (±SEM) duration of contact (s) across the three sample trials for male and female wild-type and knockout mice (top). All mice exhibited a decrease in object exploration across trials (i.e., habituation). No differences were observed between the groups. Mean (± SEM) duration of contact (s) for each object during the Test Trial (when a familiar object was replaced with a novel object) (bottom). Male and female wild-type mice showed preferential exploration for the novel object in the test trial. Male and female DARPP-32 knockout mice explored all objects for similar durations during the test trial.

line crosses on sample trials 1–3 and the test trial than on the preexposure trial. Female wild-type and knockout mice made a similar number of inner line crosses during all trials. No statistical differences were observed between wild-type and knockout mice on any measure of horizontal activity (see Table 1). The duration of exploration across the three sample trials (Sample Trials 1–3) was analyzed by a 2 (group) × 2 (sex) × 3 (trial) mixed ANOVA, with trial as a within-subjects factor. Neither sex nor genotype significantly affected object exploration during the sample phase (see Fig. 1, top). Male and female wild-type and knockout mice showed a significant decrease in object exploration across trials (i.e., habituation). This was confirmed by a significant main effect of trial F(2,52) = 21.57, p = 0.000. Tukey’s test indicated that mice explored the objects significantly longer in sample trial 1 and sample trial 2, than during sample trial 3. The duration of exploration during the Test trial (where a familiar object was replaced with a novel object) was analyzed by a 2 (group) × 2 (sex) × 4 (object) mixed ANOVA, with object as a within-subjects factor. This analysis revealed a significant main effect of object F(3,78) = 8.43, p = 0.000, along with a significant group × object interaction F(3,78) = 3.91, p = 0.012. Male and female wild-type mice explored the novel object significantly more than the familiar objects (see Fig. 1, bottom). In contrast, male and female knockout mice explored all objects for a similar duration. A discrimination index was calculated for the test trial using the following formula: discrimination index = (novel object time/total exploration time for all four objects) × 100. The discrimination index was analyzed by a one-way ANOVA and by single sample

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Table 1 Mean (±SE) number of line crosses for wild-type and knockout mice. Type Outer line crosses WT KO Inner line crosses WT KO

Gender

Pre-exposure

Trial 1

Trial 2

Trial 3

Test

Male Female Male Female

103.9 (10.9) 139.4 (20.3) 112.7 (13.9) 118.8 (9.4)

88.3 (8.9) 110.4 (16.9) 93.9 (11.5) 107.1 (16.4)

57.7 (5.7) 79.5 (14.4) 59.25 (7.7) 62.1 (9.4)

46.0 (8.9) 58.6 (10.5) 47.9 (3.5) 53.3 (10.9)

40.4 (5.9) 53.9 (7.2) 53.4 (3.6) 60.0 (7.5)

Male Female Male Female

38.7 (10.3) 47.1 (8.5) 39.5 (7.2) 36.0 (3.8)

78.7 (6.3) 56.0 (11.8) 80.7 (9.9) 49.0 (16.4)

72.7 (10.6) 40.2 (9.2) 63.2 (8.0) 47.7 (14.1)

57.0 (8.9) 40.0 (7.4) 61.9 (8.2) 37.7 (12.2)

55.6 (10.7) 42.2 (10.4) 58.9 (8.4) 52.3 (10.5)

t-tests comparing each group to chance performance (25% in this study). These analyses showed that wild-type mice performed significantly above chance performance: male t(6) = 4.14, p = 0.006; female t(7) = 2.52, p = 0.040. The performance of the knockout mice did not differ from chance: male t(7) = 1.58, p = 0.158; female t(6) = 1.12, p = 0.306. Directly comparing the groups revealed that wild-type mice had a significantly greater discrimination index (46.60 ± 4.73%) than knockout mice (29.86 ± 2.50%), F(1,26) = 9.62, p = 0.005. Therefore, only wild-type mice exhibited preferential exploration of the novel object (i.e., object recognition memory). Upon closer examination of Fig. 1 (bottom), it appeared that DARPP-32 knockout mice were increasing their exploration to all objects during the substitution test. To quantify this, a difference score was calculated for familiar and novel object [DS = duration of exploration during the test − duration of exploration during the last sample trial]. The difference score was analyzed by a 2 (group) × 2 (sex) × 2 (familiar vs. novel object) mixed ANOVA, with object as a within-subjects factor. The ANOVA revealed a significant main effect of object F(1,26) = 12.20, p < 0.01, along with a significant group × object interaction F(1,26) = 8.40, p < 0.01. As can be seen in Fig. 2, exploration of the familiar objects was unchanged and an increase exploration of the novel object was observed in male and female wild-type mice. In contrast, male and female DARPP32 knockout mice increased exploration to both the familiar and novel objects. Therefore, DARPP-32 knockout mice do respond to the change made during the test (substitution of a novel object), however their response is not directed at the novel object. 3.1.2. Experiment 2: methylphenidate challenge The number of outer line crosses and inner line crosses were analyzed by a 2 (group: wild-type vs. knockout) × 2 (sex) × 2

Fig. 2. Mean (±SEM) difference score (s) [duration of exploration during the test − duration of exploration during the last sample trial]. Male and female wildtype mice showed renewed exploration of the novel object in the test trial. In contrast, male and female DARPP-32 knockout mice increased exploration of all objects during the test trial.

(drug: saline vs. methylphenidate) × 5 (trial) mixed analysis of variance (ANOVA), with trial as a within-subjects factor. The ANOVA conducted on the number of outer line crosses revealed a significant main effect of trial F(4,144) = 45.79, p = 0.000 and drug F(1,36) = 83.26, p = 0.000, along with a significant group × drug interaction F(1,36) = 17.87, p = 0.000. As can be seen in Table 2, the number of outer line crosses decreased across trials for all mice. In order to deconstruct the interaction, it is important to note that there were no differences in line crosses between wildtype and knockout mice given saline injections. Both wild-type and knockout mice pretreated with methylphenidate exhibited a significant increase in the number of outer line crosses compared to their saline controls. However, the methylphenidate-induced increase in outer line crosses was significantly greater in wild-type mice compared to knockout mice across all trials (see Table 2). The analysis on the number of inner line crosses indicated a group × drug interaction F(1,36) = 7.36, p = 0.010 and a significant group × drug × trial interaction F(4,144) = 8.75, p = 0.000. The number of inner line crosses did not differ between wild-type and knockout mice given saline during any of the five trials. Wild-type mice given methylphenidate made significantly more inner line crosses during the pre-exposure trial than wild-type mice given saline. Deconstruction of the interaction using simple main effects revealed that methylphenidate-treated wild-type mice exhibited significantly fewer inner line crosses on sample trial 1–3 and during the test compared to the pre-exposure trial. In contrast, knockout mice pre-treated with methylphenidate significantly increased inner line crosses on all trials compared with the pre-exposure trial (see Table 2). As can be seen in Fig. 3 (top), methylphenidate selectively affected object exploration during the sample phase. A 2 (group) × 2 (sex) × 2 (drug: saline vs. methylphenidate) × 3 (trial) mixed ANOVA conducted on the duration of object contact across the three sample trials revealed a significant main effect of trial F(2,72) = 8.20, p = 0.001, along with a significant group × trial interaction F(2,72) = 3.73, p = 0.029, a significant drug × trial interaction F(2,72) = 4.16, p = 0.019, and a group × drug × trial interaction F(2,72) = 7.07, p = 0.002. In order to deconstruct the interaction, it is important to note that there were no differences in object exploration between wild-type and knockout mice given saline injections or knockout mice pretreated with methylphenidate. As can be seen in Fig. 3 (top), these three groups (saline-treated wildtype and knockout mice and methylphenidate-treated knockout mice) displayed a decrease in object exploration across trials (i.e., habituation). Tukey’s test indicated that mice explored the objects significantly longer in sample trials 1 and 2 than during sample trial 3. In contrast, methylphenidate-treated wild-type mice explored the objects significantly less than saline-treated wild-type mice on sample trial 1 and 2. In fact, the pattern of object exploration in methylphenidate-treated mice was opposite to that observed in all other mice; these mice significantly increased object exploration across trials (see Fig. 3, top).

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Table 2 Mean (±SE) number of line crosses for wild-type and knockout mice given saline (SAL) or 5 mg/kg methylphenidate (MPH). Type

Gender

Outer line crosses SAL WT MPH KO SAL MPH Inner line crosses SAL WT MPH KO SAL MPH

Pre-exposure

Trial 1

Trial 2

Trial 3

Test

134.0 (20.4) 276.8 (32.0) 141.9 (19.9) 201.0 (22.3)

103.0 (13.8) 229.4 (30.5) 123.72 (13.5) 159.8 (13.9)

79.7 (8.9) 181.5 (21.2) 86.2 (8.4) 128.0 (18.5)

73.9 (13.2) 179.7 (18.9) 75.8 (10.8) 111.6 (18.0)

61.8 (9.8) 173.25 (26.7) 68.6 (10.9) 110.0 (17.3)

40.5 (5.5) 30.3 (6.1) 39.4 (10.6) 58.8 (6.3)

54.0 (7.3) 27.5 (5.8) 39.2 (9.7) 59.6 (6.0)

37.0 (4.3) 50.0 (5.4) 38.1 (3.3) 35.6 (5.3)

42.8 (7.2) 25.8 (3.5) 32.5 (9.3) 57.3 (4.9)

A 2 (group) × 2 (sex) × 2 (drug: saline vs. methylphenidate) × 4 (object) mixed ANOVA conducted on the duration of object contact during the substitution test revealed a significant main effect of object F(3,108) = 13.53, p = 0.000, along with a significant group × drug × object interaction F(3,108) = 6.91, p = 0.000. Male and female wild-type mice given saline explored the novel object significantly more than the familiar objects (see Fig. 3, bottom). In contrast, male and female knockout mice pretreated with saline explored all objects equally. Methylphenidate-treated wild-type mice explored all objects for a similar duration. In

Fig. 3. Mean (±SEM) duration of contact (s) across the three sample trials for wild-type and knockout mice given saline or 5.0 mg/kg methylphenidate (top). Wild-type mice given saline and knockout mice given either saline or methylphenidate exhibited a decrease in object exploration across trials (i.e., habituation). Object exploration was significantly reduced in wild-type mice pretreated with methylphenidate on sample trial 1 and 2 compared to all other groups. Mean (±SEM) duration of contact (s) for each object during the Test Trial (when a familiar object was replaced with a novel object (bottom). Wild-type mice given saline exhibited preferential exploration of the novel object during the test trial. Wild-type mice pretreated with methylphenidate explored all objects for a similar duration. Salinetreated knockout mice explored all objects equally during the test trial, whereas knockout mice pretreated with methylphenidate exhibited preferential exploration for the novel object in the test trial.

49.6 (5.6) 27.1 (5.2) 41.5 (8.9) 52.7 (4.8)

contrast, knockout mice receiving methylphenidate explored the novel object significantly longer than the familiar objects. The analyses conducted on the discrimination index showed that wildtype mice given saline (DI = 43.46 ± 2.73%) performed significantly above chance performance (25% for this study) t(10) = 6.74, p = 0.00, whereas those treated with methylphenidate (DI = 31.03 ± 3.59%) did not differ from chance t(10) = 1.69, p = 0.121. The performance of the knockout mice treated with saline (DI = 30.62 ± 3.79%) did not differ from chance t(10) = 1.48, p = 0.168, whereas those treated with methylphenidate (DI = 43.06 ± 3.92%) were significantly above chance performance t(10) = 4.60, p = 0.001. Therefore, wild-type mice treated with saline exhibited preferential exploration of the novel object (i.e., recognition memory) and methylphenidate disrupted this performance. An opposite pattern was observed in knockout mice, with preferential exploration of the novel object observed only in mice pretreated with methylphenidate. As can be seen in Fig. 4, DARPP-32 knockout mice treated with methylphenidate show a pattern of responding similar to that observed in saline-treated wild-type mice using the difference score [DS = duration of exploration during the test − duration of exploration during the last sample trial]. Specifically, exploration of the familiar objects was unchanged and an increase exploration of the novel object was observed in these mice. Wild-type mice receiving methylphenidate exhibited impaired performance, with no changes to either familiar or novel objects. As was observed in Experiment 1, saline-treated DARPP-32 knockout mice increased exploration to both the familiar and novel objects. These observations were confirmed by ANOVA: main effect of object F(1,36) = 6.36, p < 0.05 and a significant group × drug × object interaction F(1,36) = 5.69, p < 0.05. Therefore, methylphenidate

Fig. 4. Mean (±SEM) difference score (s) [duration of exploration during the test − duration of exploration during the last sample trial]. Wild-type mice given saline showed renewed exploration of the novel object during the test trial. Wildtype mice pretreated with methylphenidate exhibited no change in responding for either object during the test trial. Saline-treated knockout mice increased exploration of all objects during the test trial, whereas knockout mice pretreated with methylphenidate exhibited renewed exploration of the novel object during the test trial.

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improved performance in DARPP-32 knockout mice and impaired performance in wild-type mice at this dose.

4. Discussion The results of Experiment 1 show that male and female DARPP-32 knockout mice are impaired in novel object recognition. Knockout mice did not exhibit preferential exploration of the novel object in the test trial, but instead increased exploration to all objects. In Experiment 2, the discriminative performance of DARPP-32 knockout mice during the test trial was restored by the administration of methylphenidate to that observed in salinetreated wild-type mice. This was at a dose that disrupted novel object recognition in wild-type mice. In addition, the administration of methylphenidate significantly increased locomotor activity in both wild-type and knockout mice, however this increase was blunted in DARPP-32 knockout mice. These data are consistent with other reports showing less reactivity to psychostimulant challenge in DARPP-32 knockout mice [19,52,59,60] and provide further evidence for the involvement of DARPP-32 in learning and memory [29,31,43,48,52]. The present results show that DARPP-32 knockout mice did not discriminate between a familiar object and a novel one in the novel object recognition test. Locomotor activity, exploration of the sample objects and habituation of the exploration response were similar for male and female knockout and wild-type mice across all trials. Therefore, it seems unlikely that the deficits in object recognition are due to alteration in locomotor activity or exploration during the sample phase. What is particularly interesting about the behavior of the knockout mice is that these mice did respond to the object change during the test trial. Unlike wild-type mice, which showed the expected preferential exploration of the novel object during the substitution test, DARPP-32 knockout mice increased exploration to all objects. Thus, although DARPP-32 knockout mice are responding to the change in the environment, it is not directed at the specific feature that produced the change. It is important to remember that the novel object recognition test relies on the behavioral observation that animals will seek out and explore novelty [7,17,32] and that repeated exposure to the novel feature(s) will result in habituation of the exploration response [6]. This aspect of the task appears unchanged in DARPP-32 knockout mice, as all mice showed habituation across the sample trials. Once the animal is habituated, changes in the environment (e.g., the replacement of a familiar object with a novel object) will result in a renewed exploration that is directed at the novel object [6,17]. This is the foundation by which this test is used as a method for the study of recognition memory. In many ways the behavior of the knockout mice is analogous to a dishabituated response to the whole environment as opposed to a discriminated pattern of responding to a particular feature that has changed. Methyphenidate improved discrimination performance of male and female DARPP-32 knockout mice in the novel object recognition test at a dose that disrupts novel object recognition in wild-type mice. The disrupting effect of methylphenidate on the performance of wild-type mice in the novel object recognition test is consistent with previous reports [13,30]. These data are also in agreement with evidence that the positive effects of psychostimulants on cognitive performance are seen in subjects with deficits [34], whereas negative (disruptive) effects are observed in healthy populations [57]. The administration of methylphenidate significantly increased locomotor activity in both knockout and wild-type mice compared to saline-treated mice, though this increase was significantly blunted in DARPP-32 mice. This is consistent with previous reports showing an attenuated locomotor response to cocaine, amphetamine, and methamphetamine challenge

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in DARPP-32 mice compared to wild-type controls [19,52,60]. Although methylphenidate increased locomotor activity in all mice, a significant disruption in object exploration during the sample trials was observed only in wild-type mice. Clearly, exaggerated locomotor activity can interfere with object exploration in general and object exploration during the sample phase (Trials 1–3) is critical for object recognition during the test [2]. Of course, it is also possible that methylphenidate decreases preference for novelty [14] or possibly increases neophobia. Regarding the performance of DARPP-32 knockout mice, it is clear that knockout mice seek out and respond to novelty as evidenced by equivalent object exploration during the sample phase comparable to wild-type mice. Knockout mice also respond to the novelty change during the test trial by increasing exploration of all objects. We hypothesize that the effect of methylphenidate may be to increase the saliency of the specific novel object and/or direct attention to the specific change that has occurred during the object substitution test trial. These results are in support of the notion that dopamine and norepinephrine are involved in the process of allocating responses in various situations [41,44] and it is this process that appears disrupted in DARPP-32 knockout mice. This behavioral profile is reminiscent of the performance of DARPP-32 knockout mice in the discriminated operant task [29]. More specifically, no differences were observed between DARPP-32 knockout mice and wild-type mice during acquisition of this task. Significant impairments in DARPP-32 knockout mice were observed only when the parameters of the task were changed during the reversal procedure [29]. It would be interesting in future studies to see if methylphenidate improves reversal learning in DARPP-32 knockout mice. It has been hypothesized that aspects of memory rely, in part, on optimal levels of synaptic dopamine and norepinephrine and that stimulant dugs work to restore cognitive function in individuals by increasing suboptimal catecholamine activity [4]. As noted previously, the benefits of methylphenidate for cognitive processes have been attributed to both the DA- and NE-enhancing properties of the drug [3]. Although is not possible to identify the specific action of methylphenidate responsible for the beneficial effects observed in DARPP-32 knockout mice in the present study, previous research has shown that the novel object recognition task is sensitive to dopaminergic manipulations [8,31,35–37]. For example, object recognition was improved following an injection of the D1 agonist SKF 81297 [31], whereas impairments were observed following and injection of the D1 receptor antagonist SCH 233390 [8]. In addition, the improvement in recognition memory performance was associated with increased phosphorylation of both CREB and DARPP-32 in the PFC of rats treated with the D1 agonist SKF 81297, whereas the impairing effect of SCH 23390 was associated with decreased phosophylation of CREB and DARPP-32 in the prefontal cortex [31]. Currently the involvement of NE in novel object recognition memory has received limited attention and the pattern of results is much less clear. For example, administration of the ␣2 -adrenoceptor antagonists dexefaroxan immediately after the sample phase has been shown to improve memory in the object recognition task in rats [12]. It has also been reported that post-sample infusion of NE into the basolateral amygdala improved recognition memory when tested 24 later [42]. In contrast, no effects on recognition memory were observed in DSP-4-treated animals [47] or following a 28-day treatment regime of the selective norepinephrine reuptake inhibitor venlafaxine [11]. Recently, it has been reported that the activation of ␤1 -adrenoceptors induced a rapid and transient increase in DARPP-32 phosphorylation and the activation of ␣2 -adrenoceptors also induced a rapid and transient increase in DARPP-32 phosphorylation, which subsequently decreased below basal levels [26]. In that study, the authors also report that the activation of ␣2 -adrenoceptors attenuated dopamine D1 and adenonsine A2A receptor/DARPP-32 signaling, whereas blockade of

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␣2 -adrenoceptors enhanced signaling [26]. Clearly, the improved performance in methylphenidate-treated mice cannot be due to the NE-DARPP-32 as this pathway is not present in DARPP-32 knockout mice. However, NE transmission in general has not been studied in DARPP-32 knockout mice. Therefore, future studies should examine specific DA and NE ligands in these mice to identity the underlying neurobiology of the performance restoring effects of methylphenidate in knockout mice. The interpretation of these results must be viewed with caution. First, the male and female DARPP-32 knockout mice used in this study have a complete genetic removal of DARPP-32. As mentioned in the introduction, phosphorylation of DARPP-32 at the Thr34 site by PKA converts DARPP-32 into a potent inhibitor of PP-1 [10,53,55]. However, there are 3 additional binding sites on DARPP-32: Thr75, Ser97 and Ser130 [53]. Therefore it is not possible to identify the specific pathway in these knockout mice that is responsible for the alterations in behavior observed. Second, there is always the possibility that some form of developmental compensation has occurred in these mice that we have not yet identified, as this genetic deletion is present from conception. And lastly, DARPP32s effects are dependent on neuronal localization. Bateup et al. [5] reported using regionally selective conditional knockout mice that the loss of DARPP-32 in striatonigral neurons decreased basal and cocaine-induced locomotion and abolished dyskinetic behaviors in response to the Parkinson’s disease drug L-DOPA. Conversely, the loss of DARPP-32 in striatopallidal neurons produced a robust increase in locomotor activity and a strongly reduced cataleptic response to antipsychotic drug haloperidol. In conclusion, the results of the present study provide further evidence for the role of DARPP-32 in learning and memory using the novel object recognition procedure and that disrupted performance in these mice was restored by methylphenidate. These results may be due to alterations in behavioral flexibility (i.e., the ability to adapt to changes in the environment) and may reflect underlying alterations in attention and/or motivational systems in DARPP-32 knockout mice. These results may have implications for a variety of neurological and psychiatric disorders, given the role of dopamine in these disorders [3,4,45], particularly given that post-mortem studies in humans suggest possible alterations of DARPP-32 levels in schizophrenia and bipolar disorder [58]. Conflict of interest The authors declare no conflict of interest. Acknowledgements The authors wish to thank Dr. Paul Greengard at the Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, NY for the generous donation of the DARPP-32 knockout mice. References [1] Guide for the care and use of laboratory animals. Washington, DC: National Academy Press; 1996. [2] Ainge JA, Heron-Maxwell C, Theofilas P, Wright P, de Hoz L, Wood ER. The role of the hippocampus in object recognition in rats: examination of the influence of task parameters and lesion size. Behavioural Brain Research 2006;167:183–95. [3] Arnsten AF. Modulation of prefrontal cortical–striatal circuits: relevance to therapeutic treatments for Tourette syndrome and attention-deficit hyperactivity disorder. Advances in Neurology 2001;85:333–41. [4] Arnsten AF, Li BM. Neurobiology of executive functions: catecholamine influences on prefrontal cortical functions. Biological Psychiatry 2005;57:1377–84. [5] Bateup HS, Santini E, Shen W, Birnbaum S, Valjent E, Surmeier DJ, et al. Distinct subclasses of medium spiny neurons differentially regulate striatal motor behaviors. Proceedings of the National Academy of Sciences of the United States of America 2010;107:14845–50.

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