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Behavioural Brain Research 253 (2013) 266– 273

Contents lists available at ScienceDirect

Behavioural Brain Research

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esearch report

ethylphenidate restores novel object recognition in DARPP-32nockout mice

harles J. Heysera,b,∗, Caitlyn H. McNaughtonb, Donna Vishnevetskyb, Allen A. Fienbergc

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

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.

r t i c l e i n f o

rticle history:eceived 30 April 2013eceived in revised form 18 July 2013ccepted 20 July 2013vailable online 29 July 2013

eywords:ARPP-32ethylphenidate

italinovel object recognitionemory

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 operantreversal-learning task. The present study was conducted to examine adult male and female DARPP-32knockout mice and wild-type controls in a novel object recognition test. Wild-type and knockout miceexhibited comparable behavior during the initial exploration trials. As expected, wild-type mice exhib-ited preferential exploration of the novel object during the substitution test, demonstrating recognitionmemory. In contrast, knockout mice did not show preferential exploration of the novel object, insteadexhibiting an increase in exploration of all objects during the test trial. Given that the removal of DARPP-32 is an intracellular manipulation, it seemed possible to pharmacologically restore some cellular activityand behavior by stimulating dopamine receptors. Therefore, a second experiment was conducted exam-ining the effect of methylphenidate. The results show that methylphenidate increased horizontal activityin 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, pre-treatment with methylphenidate restored the behavior of DARPP-32 knockout mice to that observed inwild-type mice given saline. These results provide additional evidence for a functional role of DARPP-32 inthe mediation of processes underlying learning and memory. These results also indicate that the behav-ioral deficits in DARPP-32 knockout mice may be restored by the administration of methylphenidate.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Over the past 30 years, using a variety of molecular, cellu-ar and behavioral approaches, dopamine- and cAMP-regulatedhosphoprotein of 32 kDa (DARPP-32) has been established as a

∗ Corresponding author at: University of California, San Diego, Department ofeuroscience, 9500 Gilman Drive, #0608, La Jolla, CA 92093-0608, USA.el.: +1 858 534 1615; fax: +1 858 534 1615.

E-mail address: cheyser@ucsd.edu (C.J. Heyser).

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.07.031

critical mediator of the biochemical, electrophysiological, tran-scriptional and behavioral effects of dopamine [18,49,53,58].However, the specific role of DARPP-32 in the processes of learn-ing and memory is still poorly understood. DARPP-32 is presentat high levels in striatal medium spiny neurons and at muchlower levels in other neuronal populations [38,39,55,56]. Activationof D1-like receptors by dopamine results in the phosphoryla-

tion of DARPP-32 by cAMP-dependent protein kinase (PKA) atthe threonine 34 (Thr34) site and converts DARPP-32 into apotent inhibitor of protein phosphatase-1 (PP1) [27]. Stimula-tion of D2-like receptors by dopamine results in the activation of

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alcium/calmodulin-dependent protein phosphatase (calcineurin)nd the dephosphorylation of DARPP-32 [25]. By inhibiting PP1,ARPP-32 controls the state of phosphorylation and physiologicalctivity of many neuronal phosphoproteins, including neurotrans-itter receptors, ion channels, ion pumps and transcription factors

for review see [10,49,53,58]).Without the aid of selective pharmacological ligands to study

he behavioral significance of DARPP-32, knockout mice were gen-rated [19]. In the initial paper, Fienberg et al. (1998) reportedhat DARPP-32 knockout mice exhibited profound deficits inheir molecular, electrophysiological, and behavioral responseso dopamine including an attenuated response to cocaine,mphetamine, and methamphetamine challenge compared toild-type controls. Previous research in our laboratory has shown

quivalent acquisition by DARPP-32 knockout mice and wild-typeice in a discriminated operant task for food reinforcement [29].owever reversal learning was impaired in the DARPP-32 knock-ut mice, who required significantly more trials to reach criterionhan wild-type mice [29]. Using a similar operant task, Stipanovicht al. [52] has shown that in response to physiological rewards,ARPP-32 accumulates in the nucleus. This effect is mediated by D1

eceptor stimulation and appears necessary for behavioral rewardearning [52]. In addition, both nuclear accumulation induced by1 stimulation and behavioral reward learning were eliminated

n DARPP32-Ser97-Ala mutant mice [52]. Changes in DARPP-32xpression have also been observed following the acquisition of annhibitory avoidance task [43]. More recently, it has been reportedhat c-Fos and DARPP-32 immunoreactivity in the accumbens shellas significantly increased on the first day of fixed-ratio-5 (FR5)

raining for food, while c-Fos and DARPP-32 expression in theore significantly increased on the second day of FR5 training [48].urthermore, D1 receptor modulation of memory retrieval per-ormance in a novel object recognition test was reported to bessociated with change in DARPP-32 in rat prefrontal cortex [31].n humans, The DARPP-32 gene has been associated with striatalopamine function and was predictive of probabilistic learning20]. Taken together, these results provide evidence for a functionalole of DARPP-32 in the mediation of processes underlying learningnd memory.

However, not all forms of learning are affected by manipula-ions of DARPP-32. For example, DARPP-32 knockout mice acquire

discriminated operant behavior [29] and were unimpaired in theater maze [15]. Therefore, the present study was conducted to

urther characterize the role of DARPP-32 in learning and mem-ry. More specifically, male and female DARPP-32 knockout micend wild-type controls were tested for novel object recognitionsing an object discrimination procedure (adapted from [6,17,46])hat is routinely used in our laboratory [28,30]. The advantages ofhe novel object recognition task are that there is no explicit needor food or water restriction and several behavioral endpoints cane rapidly obtained, including general activity, reactivity to nov-lty, and learning [9,16]. The novel object recognition task haseen shown to be sensitive to dopaminergic manipulations. Forxample, Besheer and colleagues [8] showed that systemic injec-ion of the dopamine D1 receptor antagonist SCH23390 beforehe retention test impaired the performance of rats in detec-ing the novel object. Object recognition was improved at a 4-hrelay with systemic injections of the D1 agonist SKF 81297 andpatial memory was disrupted by the selective D1 antagonistCH 23390 [31]. The improvement in recognition and tempo-al order memory performance at a 4-hr delay was associatedith increased phosphorylation of both CREB and DARPP-32 in

he PFC of rats treated with the D1 agonist SKF 81297, whereashe impairing effect of SCH 23390 was associated with decreasedhosophylation of CREB and DARPP-32 in the prefrontal cortex31]. Object recognition memory was impaired following bilateral

esearch 253 (2013) 266– 273 267

microinjections of the dopamine antagonist SCH 23390 into the PFC[36]. Further evidence for the involvement of dopamine in novelobject recognition comes from a recent study showing that theperformance of mice was systematically related to dopamine lev-els in the core region of the nucleus accumbens, but not the shell[37].

In the present study, the results of Experiment 1 show that maleand female DARPP-32 knockout mice exhibited impaired perfor-mance, with these mice exhibiting equal exploration of the noveland familiar object during the substitution test. Given that theremoval of DARPP-32 in the knockout mouse effectively altersthe intracellular circuit of these mice while leaving dopaminereceptors levels intact [19], it might be possible to restore neu-ronal performance by increasing the activational state of dopaminereceptors. In support of this hypothesis, it has been reported thatThr34-Ala DARPP-32 mutant mice require more time to acquirecocaine self-administration [60]. However, once acquired thesemice administered more cocaine at lower doses compared to wild-type mice [60]. The authors suggest that when stimuli are strong,as in the case with higher doses of cocaine, phosphorylation byPKA is sufficient to compensate for a deficiency of the DARPP-32 pathway. However, when stimuli are weaker, as in the casewith the lower doses of cocaine, mice with an impaired DARPP-32 pathway need to increase responding for cocaine in order toget the same amount of reinforcement. Therefore, Experiment2 was conducted to examine the effect of methyphenidate onnovel object recognition memory in male and female DARPP-32 knockout mice and wild-type controls. The psychostimulantmethylphenidate was selected for use in this study for severalreasons. First, it is a stimulant widely used in the treatment ofADHD [50]. Second, the pharmacodynamics of methylphenidateare similar to amphetamine and cocaine [51,54]. The administra-tion of methylphenidate increases synaptic levels of dopamine andnorepinephrine in several brain regions including the hippocam-pus and the prefrontal cortex by binding to and blocking dopamineand norepinephrine reuptake transporters [22,40]. Third, althoughthere are studies looking at cocaine and amphetamine challengein DARPP-32 knockout mice [19,52,59,60], there are no publishedreports on the effects of methylphenidate in these knockout mice.All of these studies [19,52,59,60] reported a blunted response topsychostimulant challenge in DARPP-32 knockout mice. And lastly,it has been reported that methylphenidate increased DARPP-32Thr34 phosphorylation and decreased Thr75 phosphorylation inslices from adult mice [21]. The dose of methylphenidate wasselected based on past research [23,30] and a report that indicatedthe peak drug response occurs at 20 min and remains elevatedto 80 min after an i.p. injection [24]. The duration of the novelobject recognition procedure used in the present study was 42 min,thus all behavioral observations were made during the peak drugresponse.

2. Methods

2.1. Animals

DARPP-32 knockout (−/−) and wild-type control (+/+) male and female micewere generated and bred as previously described [19]. The mice used in the presentstudy 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 femalemice were used in Experiment 1. A separate group of 22 knockout and 22 wild-typeadult (3- to 4-month old) male and female mice were used in Experiment 2. Micewere housed 3–4 in Plexiglas cages (28 cm × 17 cm × 11.5 cm) with sawdust beddingchanged 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 temperatureof 24 ◦C. Food and water were available ad libitum throughout training. All studieswere conducted in accordance with the Guide for the Care and Use of LaboratoryAnimals [1] and were approved by the Institutional Animal Care and Use Committeeof Franklin & Marshall College.

2 rain Research 253 (2013) 266– 273

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Fig. 1. Mean (±SEM) duration of contact (s) across the three sample trials for maleand female wild-type and knockout mice (top). All mice exhibited a decrease inobject exploration across trials (i.e., habituation). No differences were observedbetween the groups. Mean (± SEM) duration of contact (s) for each object during theTest Trial (when a familiar object was replaced with a novel object) (bottom). Male

68 C.J. Heyser et al. / Behavioural B

.2. Apparatus

The apparatus consisted of a circular open field (120 cm in diameter). The flooras made of wood and was painted dark green. The walls (30 cm in height) wereade of aluminum and were painted flat black. The objects used in the experi-ents were a yellow smooth plastic square (6 cm × 6 cm × 6 cm), an opaque pink

mooth rectangular plastic bottle on its side (5 cm × 14 cm × 5 cm), a white smoothardboard box (7.5 cm × 8 cm × 3 cm), a clear grooved glass dome (8 cm in diame-er, 4 cm in height), and a gray smooth plastic rectangle with a hole in the center6 cm × 12 cm × 6 cm). All objects were of sufficient weight such that they could note moved by the animal. The open field was illuminated by diffuse incandescent

ighting (15 lux). A video camera was located directly over the center of the openeld and was connected to a VCR and monitor located in the room immediatelyutside the testing room to minimize noise and the presence of the experimenter.

.3. Procedure

.3.1. Novel object exploration: Experiment 1Prior to the start of testing all mice were handled for 5 min per day for five days.

esting consisted of five 6-min trials, with a 3-min intertrial interval between eachrial. During the intertrial interval the mouse was placed in a holding cage, whichemained inside the testing room. In the first trial (Pre-Exposure), each mouse waslaced individually into the center of the otherwise empty open field for 6 min. Forhe next three trials (Sample Trials 1–3) four different objects were placed into thepen field. In all cases the configuration of the objects was such that the objectsere equidistant from each other and from the wall (i.e., forming a square). In the

ast trial (Test), one of the objects was replaced with a novel object. Objects and theirlacement into the open field were varied across mice to avoid positional biases. Toontrol for possible odor cues the objects were cleaned with a 10% ethanol solutiont the end of each trial and the floor of the open field wiped down to eliminateossible scent/trail markers. During the test phase, the novel object was also wipedown 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 stop-atch. Exploration was defined as direct contact of the nose or front paws with the

bject. In addition, the number of outer and inner line crosses was recorded for eachrial. In order to score line crosses, the open field was divided into 28 sections: 16uter sections and 12 inner sections. To record a line cross, the mouse must havehree limbs cross into and out of the section. All behaviors were scored from video-ape to ensure accuracy. All observers were blind to sex and genotype and wererained to a scoring criterion of greater than 95% inter-rater reliability.

.3.2. Novel object exploration – methylphenidate challenge: Experiment 2Adult male and female WT and KO mice were randomly assigned to either the

aline or methylphenidate group. The same apparatus and procedure described inxperiment 1 was used in this challenge. Each animal was given an intraperitonealnjection (i.p.) of either saline or 5 mg/kg methylphenidate (Sigma–Aldrich) 20-minrior to the start of the pre-exposure trial. Injections were given in a volume of

ml/100 g body weight and each animal was tested only once.

.4. Data analysis

The data were analyzed using analysis of variance (ANOVA) and t-tests; thepecific details are included in the results section. Tukey’s tests and simple mainffects analyses [33] were used to determine the locus of significant main effectsnd interactions. A significance level of p < 0.05 was used for all statistical analyses.

. Results

.1.1. Experiment 1: novel object exploration

The number of outer line crosses and inner line crosses werenalyzed by a 2 (group: wild-type vs. knockout) × 2 (sex) × 5trial) mixed analysis of variance (ANOVA), with trial as a within-ubjects factor. As can be seen in Table 1, the number of outerine crosses decreased across trials for all mice: main effect of trial(4,104) = 59.33, p = 0.000. Tukey’s tests indicated that the micexhibited significantly more outer line crosses on the pre-exposurerial than on first trial with objects (sample trial 1), which was sig-ificantly greater than sample trials 2, 3 and the test trial, whichere not different from each other. No other statistical differencesere obtained. The analysis on the number of inner line crosses

ndicated a main effect of trial F(4,104) = 8.122, p = 0.001, along with significant sex × trial interaction F(4,104) = 3.35, p = 0.013. As caneen in Table 1, no differences were observed among the mice dur-ng the pre-exposure trial. Male mice made significantly more inner

and female wild-type mice showed preferential exploration for the novel object inthe test trial. Male and female DARPP-32 knockout mice explored all objects forsimilar durations during the test trial.

line crosses on sample trials 1–3 and the test trial than on the pre-exposure trial. Female wild-type and knockout mice made a similarnumber of inner line crosses during all trials. No statistical differ-ences were observed between wild-type and knockout mice on anymeasure of horizontal activity (see Table 1).

The duration of exploration across the three sample trials (Sam-ple Trials 1–3) was analyzed by a 2 (group) × 2 (sex) × 3 (trial)mixed ANOVA, with trial as a within-subjects factor. Neither sex norgenotype significantly affected object exploration during the sam-ple phase (see Fig. 1, top). Male and female wild-type and knockoutmice showed a significant decrease in object exploration acrosstrials (i.e., habituation). This was confirmed by a significant maineffect of trial F(2,52) = 21.57, p = 0.000. Tukey’s test indicated thatmice explored the objects significantly longer in sample trial 1 andsample trial 2, than during sample trial 3.

The duration of exploration during the Test trial (where a famil-iar object was replaced with a novel object) was analyzed by a2 (group) × 2 (sex) × 4 (object) mixed ANOVA, with object as awithin-subjects factor. This analysis revealed a significant maineffect of object F(3,78) = 8.43, p = 0.000, along with a significantgroup × object interaction F(3,78) = 3.91, p = 0.012. Male and femalewild-type mice explored the novel object significantly more thanthe familiar objects (see Fig. 1, bottom). In contrast, male andfemale knockout mice explored all objects for a similar duration.

A discrimination index was calculated for the test trial using thefollowing formula: discrimination index = (novel object time/totalexploration time for all four objects) × 100. The discriminationindex was analyzed by a one-way ANOVA and by single sample

C.J. Heyser et al. / Behavioural Brain Research 253 (2013) 266– 273 269

Table 1Mean (±SE) number of line crosses for wild-type and knockout mice.

Type Gender Pre-exposure Trial 1 Trial 2 Trial 3 Test

Outer line crossesWT Male 103.9 (10.9) 88.3 (8.9) 57.7 (5.7) 46.0 (8.9) 40.4 (5.9)

Female 139.4 (20.3) 110.4 (16.9) 79.5 (14.4) 58.6 (10.5) 53.9 (7.2)KO Male 112.7 (13.9) 93.9 (11.5) 59.25 (7.7) 47.9 (3.5) 53.4 (3.6)

Female 118.8 (9.4) 107.1 (16.4) 62.1 (9.4) 53.3 (10.9) 60.0 (7.5)Inner line crossesWT Male 38.7 (10.3) 78.7 (6.3) 72.7 (10.6) 57.0 (8.9) 55.6 (10.7)

Female 47.1 (8.5) 56.0 (11.8) 40.2 (9.2) 40.0 (7.4) 42.2 (10.4))

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-tests comparing each group to chance performance (25% in thistudy). These analyses showed that wild-type mice performed sig-ificantly above chance performance: male t(6) = 4.14, p = 0.006;

emale t(7) = 2.52, p = 0.040. The performance of the knockoutice did not differ from chance: male t(7) = 1.58, p = 0.158; female

(6) = 1.12, p = 0.306. Directly comparing the groups revealed thatild-type mice had a significantly greater discrimination index

46.60 ± 4.73%) than knockout mice (29.86 ± 2.50%), F(1,26) = 9.62, = 0.005. Therefore, only wild-type mice exhibited preferentialxploration of the novel object (i.e., object recognition memory).

Upon closer examination of Fig. 1 (bottom), it appeared thatARPP-32 knockout mice were increasing their exploration to allbjects during the substitution test. To quantify this, a differencecore was calculated for familiar and novel object [DS = duration ofxploration during the test − duration of exploration during the lastample trial]. The difference score was analyzed by a 2 (group) × 2sex) × 2 (familiar vs. novel object) mixed ANOVA, with object as

within-subjects factor. The ANOVA revealed a significant mainffect of object F(1,26) = 12.20, p < 0.01, along with a significantroup × object interaction F(1,26) = 8.40, p < 0.01. As can be seenn Fig. 2, exploration of the familiar objects was unchanged andn increase exploration of the novel object was observed in malend female wild-type mice. In contrast, male and female DARPP-2 knockout mice increased exploration to both the familiar andovel objects. Therefore, DARPP-32 knockout mice do respond tohe change made during the test (substitution of a novel object),owever their response is not directed at the novel object.

.1.2. Experiment 2: methylphenidate challenge

The number of outer line crosses and inner line crosses werenalyzed by a 2 (group: wild-type vs. knockout) × 2 (sex) × 2

ig. 2. Mean (±SEM) difference score (s) [duration of exploration during theest − duration of exploration during the last sample trial]. Male and female wild-ype mice showed renewed exploration of the novel object in the test trial. Inontrast, male and female DARPP-32 knockout mice increased exploration of allbjects during the test trial.

63.2 (8.0) 61.9 (8.2) 58.9 (8.4)47.7 (14.1) 37.7 (12.2) 52.3 (10.5)

(drug: saline vs. methylphenidate) × 5 (trial) mixed analysis of vari-ance (ANOVA), with trial as a within-subjects factor. The ANOVAconducted on the number of outer line crosses revealed a sig-nificant main effect of trial F(4,144) = 45.79, p = 0.000 and drugF(1,36) = 83.26, p = 0.000, along with a significant group × druginteraction 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 allmice. In order to deconstruct the interaction, it is important tonote that there were no differences in line crosses between wild-type and knockout mice given saline injections. Both wild-type andknockout mice pretreated with methylphenidate exhibited a sig-nificant increase in the number of outer line crosses comparedto their saline controls. However, the methylphenidate-inducedincrease in outer line crosses was significantly greater in wild-typemice compared to knockout mice across all trials (see Table 2).The analysis on the number of inner line crosses indicated agroup × drug interaction F(1,36) = 7.36, p = 0.010 and a significantgroup × drug × trial interaction F(4,144) = 8.75, p = 0.000. The num-ber of inner line crosses did not differ between wild-type andknockout mice given saline during any of the five trials. Wild-typemice given methylphenidate made significantly more inner linecrosses during the pre-exposure trial than wild-type mice givensaline. Deconstruction of the interaction using simple main effectsrevealed that methylphenidate-treated wild-type mice exhibitedsignificantly fewer inner line crosses on sample trial 1–3 and duringthe test compared to the pre-exposure trial. In contrast, knock-out mice pre-treated with methylphenidate significantly increasedinner line crosses on all trials compared with the pre-exposure trial(see Table 2).

As can be seen in Fig. 3 (top), methylphenidate selectivelyaffected object exploration during the sample phase. A 2 (group) × 2(sex) × 2 (drug: saline vs. methylphenidate) × 3 (trial) mixedANOVA conducted on the duration of object contact across thethree sample trials revealed a significant main effect of trialF(2,72) = 8.20, p = 0.001, along with a significant group × trial inter-action F(2,72) = 3.73, p = 0.029, a significant drug × trial interactionF(2,72) = 4.16, p = 0.019, and a group × drug × trial interactionF(2,72) = 7.07, p = 0.002. In order to deconstruct the interaction,it is important to note that there were no differences in objectexploration between wild-type and knockout mice given salineinjections or knockout mice pretreated with methylphenidate. Ascan be seen in Fig. 3 (top), these three groups (saline-treated wild-type and knockout mice and methylphenidate-treated knockoutmice) displayed a decrease in object exploration across trials (i.e.,habituation). Tukey’s test indicated that mice explored the objectssignificantly longer in sample trials 1 and 2 than during sample trial3. In contrast, methylphenidate-treated wild-type mice exploredthe objects significantly less than saline-treated wild-type mice on

sample trial 1 and 2. In fact, the pattern of object exploration inmethylphenidate-treated mice was opposite to that observed in allother mice; these mice significantly increased object explorationacross trials (see Fig. 3, top).

270 C.J. Heyser et al. / Behavioural Brain Research 253 (2013) 266– 273

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

Type Gender Pre-exposure Trial 1 Trial 2 Trial 3 Test

Outer line crossesWT SAL 134.0 (20.4) 103.0 (13.8) 79.7 (8.9) 73.9 (13.2) 61.8 (9.8)

MPH 276.8 (32.0) 229.4 (30.5) 181.5 (21.2) 179.7 (18.9) 173.25 (26.7)KO SAL 141.9 (19.9) 123.72 (13.5) 86.2 (8.4) 75.8 (10.8) 68.6 (10.9)

MPH 201.0 (22.3) 159.8 (13.9) 128.0 (18.5) 111.6 (18.0) 110.0 (17.3)Inner line crossesWT SAL 37.0 (4.3) 42.8 (7.2) 40.5 (5.5) 54.0 (7.3) 49.6 (5.6)

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MPH 50.0 (5.4) 25.8 (3.5)KO SAL 38.1 (3.3) 32.5 (9.3)

MPH 35.6 (5.3) 57.3 (4.9)

A 2 (group) × 2 (sex) × 2 (drug: saline vs. methylphenidate) × 4object) mixed ANOVA conducted on the duration of object con-act during the substitution test revealed a significant mainffect of object F(3,108) = 13.53, p = 0.000, along with a signif-cant group × drug × object interaction F(3,108) = 6.91, p = 0.000.

ale and female wild-type mice given saline explored the novel

bject significantly more than the familiar objects (see Fig. 3,ottom). In contrast, male and female knockout mice pretreatedith saline explored all objects equally. Methylphenidate-treatedild-type mice explored all objects for a similar duration. In

ig. 3. Mean (±SEM) duration of contact (s) across the three sample trialsor wild-type and knockout mice given saline or 5.0 mg/kg methylphenidatetop). Wild-type mice given saline and knockout mice given either saline or

ethylphenidate exhibited a decrease in object exploration across trials (i.e., habit-ation). Object exploration was significantly reduced in wild-type mice pretreatedith 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 familiarbject was replaced with a novel object (bottom). Wild-type mice given saline exhib-ted preferential exploration of the novel object during the test trial. Wild-type miceretreated with methylphenidate explored all objects for a similar duration. Saline-reated knockout mice explored all objects equally during the test trial, whereasnockout mice pretreated with methylphenidate exhibited preferential explorationor the novel object in the test trial.

30.3 (6.1) 27.5 (5.8) 27.1 (5.2)39.4 (10.6) 39.2 (9.7) 41.5 (8.9)58.8 (6.3) 59.6 (6.0) 52.7 (4.8)

contrast, knockout mice receiving methylphenidate explored thenovel object significantly longer than the familiar objects. Theanalyses conducted on the discrimination index showed that wild-type mice given saline (DI = 43.46 ± 2.73%) performed significantlyabove 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 performanceof the knockout mice treated with saline (DI = 30.62 ± 3.79%) didnot differ from chance t(10) = 1.48, p = 0.168, whereas those treatedwith methylphenidate (DI = 43.06 ± 3.92%) were significantly abovechance performance t(10) = 4.60, p = 0.001. Therefore, wild-typemice treated with saline exhibited preferential exploration of thenovel object (i.e., recognition memory) and methylphenidate dis-rupted this performance. An opposite pattern was observed inknockout mice, with preferential exploration of the novel objectobserved only in mice pretreated with methylphenidate.

As can be seen in Fig. 4, DARPP-32 knockout mice treatedwith methylphenidate show a pattern of responding similar tothat observed in saline-treated wild-type mice using the differ-ence score [DS = duration of exploration during the test − durationof exploration during the last sample trial]. Specifically, explo-ration of the familiar objects was unchanged and an increaseexploration of the novel object was observed in these mice.Wild-type mice receiving methylphenidate exhibited impairedperformance, with no changes to either familiar or novel objects. Aswas 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 ofobject F(1,36) = 6.36, p < 0.05 and a significant group × drug × objectinteraction F(1,36) = 5.69, p < 0.05. Therefore, methylphenidate

Fig. 4. Mean (±SEM) difference score (s) [duration of exploration during thetest − duration of exploration during the last sample trial]. Wild-type mice givensaline showed renewed exploration of the novel object during the test trial. Wild-type mice pretreated with methylphenidate exhibited no change in responding foreither object during the test trial. Saline-treated knockout mice increased explo-ration of all objects during the test trial, whereas knockout mice pretreated withmethylphenidate exhibited renewed exploration of the novel object during the testtrial.

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mproved performance in DARPP-32 knockout mice and impairederformance in wild-type mice at this dose.

. Discussion

The results of Experiment 1 show that male and femaleARPP-32 knockout mice are impaired in novel object recogni-

ion. Knockout mice did not exhibit preferential exploration of theovel object in the test trial, but instead increased explorationo all objects. In Experiment 2, the discriminative performancef DARPP-32 knockout mice during the test trial was restored byhe administration of methylphenidate to that observed in saline-reated wild-type mice. This was at a dose that disrupted novelbject recognition in wild-type mice. In addition, the administra-ion of methylphenidate significantly increased locomotor activityn both wild-type and knockout mice, however this increase waslunted in DARPP-32 knockout mice. These data are consistent withther reports showing less reactivity to psychostimulant challengen DARPP-32 knockout mice [19,52,59,60] and provide further evi-ence for the involvement of DARPP-32 in learning and memory29,31,43,48,52].

The present results show that DARPP-32 knockout mice didot discriminate between a familiar object and a novel one in theovel object recognition test. Locomotor activity, exploration of theample objects and habituation of the exploration response wereimilar for male and female knockout and wild-type mice across allrials. Therefore, it seems unlikely that the deficits in object recog-ition are due to alteration in locomotor activity or explorationuring the sample phase. What is particularly interesting about theehavior of the knockout mice is that these mice did respond to thebject change during the test trial. Unlike wild-type mice, whichhowed the expected preferential exploration of the novel objecturing the substitution test, DARPP-32 knockout mice increasedxploration to all objects. Thus, although DARPP-32 knockout micere responding to the change in the environment, it is not directedt the specific feature that produced the change. It is importanto remember that the novel object recognition test relies on theehavioral observation that animals will seek out and explore nov-lty [7,17,32] and that repeated exposure to the novel feature(s)ill result in habituation of the exploration response [6]. This aspect

f the task appears unchanged in DARPP-32 knockout mice, as allice showed habituation across the sample trials. Once the animal

s habituated, changes in the environment (e.g., the replacementf a familiar object with a novel object) will result in a renewedxploration that is directed at the novel object [6,17]. This is theoundation by which this test is used as a method for the study ofecognition memory. In many ways the behavior of the knockoutice is analogous to a dishabituated response to the whole envi-

onment as opposed to a discriminated pattern of responding to aarticular feature that has changed.

Methyphenidate improved discrimination performance ofale and female DARPP-32 knockout mice in the novel object

ecognition test at a dose that disrupts novel object recognition inild-type mice. The disrupting effect of methylphenidate on theerformance of wild-type mice in the novel object recognition test

s consistent with previous reports [13,30]. These data are also ingreement with evidence that the positive effects of psychostim-lants on cognitive performance are seen in subjects with deficits34], whereas negative (disruptive) effects are observed in healthyopulations [57]. The administration of methylphenidate signifi-antly increased locomotor activity in both knockout and wild-type

ice compared to saline-treated mice, though this increase was

ignificantly blunted in DARPP-32 mice. This is consistent withrevious reports showing an attenuated locomotor responseo cocaine, amphetamine, and methamphetamine challenge

esearch 253 (2013) 266– 273 271

in DARPP-32 mice compared to wild-type controls [19,52,60].Although methylphenidate increased locomotor activity in allmice, a significant disruption in object exploration during the sam-ple trials was observed only in wild-type mice. Clearly, exaggeratedlocomotor activity can interfere with object exploration in generaland object exploration during the sample phase (Trials 1–3) iscritical for object recognition during the test [2]. Of course, it is alsopossible that methylphenidate decreases preference for novelty[14] or possibly increases neophobia. Regarding the performanceof DARPP-32 knockout mice, it is clear that knockout mice seekout and respond to novelty as evidenced by equivalent objectexploration during the sample phase comparable to wild-typemice. Knockout mice also respond to the novelty change during thetest trial by increasing exploration of all objects. We hypothesizethat the effect of methylphenidate may be to increase the saliencyof the specific novel object and/or direct attention to the specificchange that has occurred during the object substitution test trial.These results are in support of the notion that dopamine andnorepinephrine are involved in the process of allocating responsesin various situations [41,44] and it is this process that appearsdisrupted in DARPP-32 knockout mice. This behavioral profile isreminiscent of the performance of DARPP-32 knockout mice in thediscriminated operant task [29]. More specifically, no differenceswere observed between DARPP-32 knockout mice and wild-typemice during acquisition of this task. Significant impairments inDARPP-32 knockout mice were observed only when the parame-ters of the task were changed during the reversal procedure [29].It would be interesting in future studies to see if methylphenidateimproves reversal learning in DARPP-32 knockout mice.

It has been hypothesized that aspects of memory rely, in part, onoptimal levels of synaptic dopamine and norepinephrine and thatstimulant dugs work to restore cognitive function in individuals byincreasing suboptimal catecholamine activity [4]. As noted previ-ously, the benefits of methylphenidate for cognitive processes havebeen attributed to both the DA- and NE-enhancing properties of thedrug [3]. Although is not possible to identify the specific action ofmethylphenidate responsible for the beneficial effects observed inDARPP-32 knockout mice in the present study, previous researchhas shown that the novel object recognition task is sensitiveto dopaminergic manipulations [8,31,35–37]. For example, objectrecognition was improved following an injection of the D1 agonistSKF 81297 [31], whereas impairments were observed following andinjection of the D1 receptor antagonist SCH 233390 [8]. In addition,the improvement in recognition memory performance was associ-ated with increased phosphorylation of both CREB and DARPP-32in the PFC of rats treated with the D1 agonist SKF 81297, whereasthe impairing effect of SCH 23390 was associated with decreasedphosophylation of CREB and DARPP-32 in the prefontal cortex [31].Currently the involvement of NE in novel object recognition mem-ory has received limited attention and the pattern of results is muchless clear. For example, administration of the �2-adrenoceptorantagonists dexefaroxan immediately after the sample phase hasbeen shown to improve memory in the object recognition task inrats [12]. It has also been reported that post-sample infusion ofNE into the basolateral amygdala improved recognition memorywhen tested 24 later [42]. In contrast, no effects on recognitionmemory were observed in DSP-4-treated animals [47] or follow-ing a 28-day treatment regime of the selective norepinephrinereuptake inhibitor venlafaxine [11]. Recently, it has been reportedthat the activation of �1-adrenoceptors induced a rapid and tran-sient 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 belowbasal levels [26]. In that study, the authors also report that theactivation of �2-adrenoceptors attenuated dopamine D1 and ade-nonsine A2A receptor/DARPP-32 signaling, whereas blockade of

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2-adrenoceptors enhanced signaling [26]. Clearly, the improvederformance in methylphenidate-treated mice cannot be due to theE-DARPP-32 as this pathway is not present in DARPP-32 knockoutice. However, NE transmission in general has not been stud-

ed in DARPP-32 knockout mice. Therefore, future studies shouldxamine specific DA and NE ligands in these mice to identity thenderlying neurobiology of the performance restoring effects ofethylphenidate in knockout mice.The interpretation of these results must be viewed with cau-

ion. First, the male and female DARPP-32 knockout mice used inhis study have a complete genetic removal of DARPP-32. As men-ioned in the introduction, phosphorylation of DARPP-32 at thehr34 site by PKA converts DARPP-32 into a potent inhibitor ofP-1 [10,53,55]. However, there are 3 additional binding sites onARPP-32: Thr75, Ser97 and Ser130 [53]. Therefore it is not possi-le to identify the specific pathway in these knockout mice that isesponsible for the alterations in behavior observed. Second, theres always the possibility that some form of developmental compen-ation has occurred in these mice that we have not yet identified, ashis genetic deletion is present from conception. And lastly, DARPP-2s effects are dependent on neuronal localization. Bateup et al. [5]eported using regionally selective conditional knockout mice thathe loss of DARPP-32 in striatonigral neurons decreased basal andocaine-induced locomotion and abolished dyskinetic behaviorsn response to the Parkinson’s disease drug L-DOPA. Conversely,he loss of DARPP-32 in striatopallidal neurons produced a robustncrease in locomotor activity and a strongly reduced catalepticesponse to antipsychotic drug haloperidol.

In conclusion, the results of the present study provide furthervidence for the role of DARPP-32 in learning and memory using theovel object recognition procedure and that disrupted performance

n these mice was restored by methylphenidate. These results maye due to alterations in behavioral flexibility (i.e., the ability todapt to changes in the environment) and may reflect underlyinglterations in attention and/or motivational systems in DARPP-32nockout mice. These results may have implications for a variety ofeurological and psychiatric disorders, given the role of dopamine

n these disorders [3,4,45], particularly given that post-mortemtudies in humans suggest possible alterations of DARPP-32 levelsn schizophrenia and bipolar disorder [58].

onflict of interest

The authors declare no conflict of interest.

cknowledgements

The authors wish to thank Dr. Paul Greengard at the Laboratoryf Molecular and Cellular Neuroscience, The Rockefeller University,ew York, NY for the generous donation of the DARPP-32 knockoutice.

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