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Neuropharmacology 64 (2013) 321e328

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Neuropharmacology

journal homepage: www.elsevier .com/locate/neuropharm

A selective dopamine reuptake inhibitor improves prefrontal cortex-dependentcognitive function: Potential relevance to attention deficit hyperactivity disorder

Brooke E. Schmeichel a, Frank P. Zemlan b, Craig W. Berridge a,*

a Psychology Department, University of Wisconsin, 1202 W. Johnson St., Madison, WI 53706, USAb P2D Biosciences, Cincinnati, OH 45219, USA

a r t i c l e i n f o

Article history:Received 15 February 2012Received in revised form28 June 2012Accepted 3 July 2012

Keywords:ADHDPrefrontal cortexCognitionPsychostimulantsNorepinephrineDopamine

* Corresponding author. Tel.: þ1 608 265 5938; faxE-mail address: berridge@wisc.edu (C.W. Berridge

0028-3908/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.neuropharm.2012.07.005

a b s t r a c t

Drugs used to treat attention deficit hyperactivity disorder (ADHD) improve prefrontal cortex (PFC)-dependent cognitive function. The majority of ADHD-related treatments act either as dual norepi-nephrine (NE) and dopamine (DA) reuptake inhibitors (psychostimulants) or selective NE reuptakeinhibitors (SNRIs). Certain benztropine analogs act as highly selective DA reuptake inhibitors whilelacking the reinforcing actions, and thus abuse potential, of psychostimulants. To assess the potential useof these compounds in the treatment of ADHD, we examined the effects of a well-characterized benz-tropine analog, AHN 2-005, on performance of rats in a PFC-dependent delayed-alternation task ofspatial working memory. Similar to that seen with all drugs currently approved for ADHD, AHN 2-005dose-dependently improved performance in this task. Clinically-relevant doses of psychostimulants andSNRIs elevate NE and DA preferentially in the PFC. Despite the selectivity of this compound for the DAtransporter, additional microdialysis studies demonstrated that a cognition-enhancing dose of AHN 2-005 that lacked locomotor activating effects increased extracellular levels of both DA and NE in the PFC.AHN 2-005 produced a larger increase in extracellular DA in the nucleus accumbens, although themagnitude of this was well below that seen with motor activating doses of psychostimulants. Collec-tively, these observations suggest that benztropine analogs may be efficacious in the treatment of ADHDor other disorders associated with PFC dysfunction. These studies provide a strong rationale for futureresearch focused on the neural mechanisms contributing to the cognition-enhancing actions and thepotential clinical utility of AHN 2-005 and related compounds.

This article is part of a Special Issue entitled ‘Cognitive Enhancers’.� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Attention-deficit hyperactivity disorder (ADHD) is conserva-tively estimated to affect 3%e5% of children and adults (Solanto,2001; Wilens et al., 2004). Psychostimulants are currently themost effective treatment for ADHD (Greenhill, 2001). However, theabuse potential of these drugs raises significant concerns abouttheir widespread use. Thus, there is a need for new drug treatmentsfor ADHD that display comparable efficacy while lacking the abusepotential of psychostimulants.

Extensive studies demonstrate that ADHD-approved medica-tions improve cognitive processes dependent on the prefrontalcortex (PFC), including working memory, planning, response inhi-bition and the regulation of impulsivity (Chamberlain et al., 2007;Diamond, 2005; Mehta et al., 2001; Turner et al., 2005). These

: þ1 608 262 4029.).

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observations are consistentwith imagingdata demonstratingADHDis associatedwith PFC dysfunction (Castellanos and Tannock, 2002).Importantly, the cognition-enhancing actions of ADHD-relateddrugs are not limited to ADHD, with similar effects observed in bothnormal human and animal subjects (Arnsten and Dudley, 2005;Berridge et al., 2006;Devilbiss andBerridge, 2008; Elliott et al.,1997;Gamo et al., 2010; Mehta et al., 2001; Rapoport and Inoff-Germain,2002). Collectively, these observations suggest that the clinicalefficacy of drugs used in the treatment of ADHD involves, at least inpart, an ability to improve PFC-dependent function.

Psychostimulants used in the treatment of ADHD (i.e. methyl-phenidate, amphetamine) act as non-selective catecholaminereuptake inhibitors (Berridge and Devilbiss, 2011). Additionally,selective norepinephrine reuptake inhibitors (SNRIs) are effectivein the treatment of ADHD, though these drugs are typically viewedas less efficacious than psychostimulants (Berridge and Devilbiss,2011). To date, selective DA reuptake inhibitors (SDRIs) have notbeen utilized in ADHD, largely due to a limited number ofcompounds that display selectivity for the DA transporter (DAT)

B.E. Schmeichel et al. / Neuropharmacology 64 (2013) 321e328322

while lacking the abuse potential of psychostimulants. However,a series of benztropine analogs has been described that display highselectivity and affinity for the DAT while lacking reinforcing effectsin rodents and monkeys (Hiranita et al., 2009; Li et al., 2005;Woolverton et al., 2001, 2000). The behavioral and pharmacologicalprofiles of these compounds suggest they may be efficacious in thetreatment of ADHD while lacking significant abuse potential.

The behavioral and neurochemical actions of the benztropineanalog, N-allyl-3a[bis(4fluorophenyl)methoxy]tropane (AHN 2-005), have been well-characterized. Prior work demonstrates thatthis compound displays high selectivity for the DAT relative toother transporters and receptors and lacks reinforcing effects asmeasured in conditioned place preference and self-administrationparadigms at doses that produce robust increases in extracellularDA concentrations (Hiranita et al., 2009; Katz et al., 1999, 2004; Rajeet al., 2005). To assess the potential use of AHN 2-005 in ADHD, wefirst examined the degree to which this compound improves PFC-dependent function of rats as measured in a delayed-responsetask of working memory. Importantly, the pharmacology ofperformance in this task aligns closely with the pharmacology ofADHD: all major classes of drugs used to treat ADHD (psychosti-mulants, SNRIs, a2-agonists) improve performance in this task(Arnsten, 2009; Berridge and Devilbiss, 2011). Thus this task isa useful preclinical screen for ADHD-related compounds. In thecurrent studies, AHN 2-005 dose-dependently improved perfor-mance in this task, comparable to that seen with ADHD-relateddrugs.

Available evidence indicates that clinically-relevant, cognition-enhancing doses of psychostimulants and SNRIs simultaneouslyand preferentially elevate extracellular NE and DA within the PFC(Berridge et al., 2006; Bymaster et al., 2002). This has been positedto reflect, in part, a prominent role of the NET in the clearance of DAwithin the PFC (Berridge and Devilbiss, 2011; Carboni et al., 2006;Yamamoto and Novotney, 1998). These and other observationsindicate a pivotal role of PFC catecholamines in the cognition-enhancing/therapeutic actions of ADHD-related drugs (Arnsten,2009; Arnsten and Dudley, 2005; Devilbiss and Berridge, 2008;Spencer et al., 2012). If NE and DA binding at the NE transporter(NET) in the PFC is competitive, elevations in extracellular DA areexpected to elevate extracellular NE levels. To test whether thisoccurs with AHN 2-005, additional microdialysis studies examinedthe degree to which a cognition-enhancing dose of AHN 2-005(10 mg/kg) simultaneously impacts extracellular DA and NE withinthe PFC, the nucleus accumbens and the medial septal area. Similarto that seen with clinically-relevant doses of psychostimulants andSNRIs, AHN 2-005 elicited moderate increases in extracellularlevels of both DA and NE within the PFC and medial septal area, andmodestly higher increases in extracellular levels of DA in thenucleus accumbens. These neurochemical effects were observed inthe absence of locomotor activating effects, consistent with resultsof earlier studies (Li et al., 2005).

Collectively, these preclinical observations suggest that AHN 2-005 and other benztropine analogs may have utility in the treat-ment of ADHD and other conditions associated with PFCdysfunction.

2. Methods and material

2.1. Animals and surgery

Male SpragueeDawley rats (260e280 g, Charles River, Wilmington, MA) werehoused in pairs with ad lib access to food and water on an 11:13 h light:dark cycle(lights on 7:00 AM). For microdialysis studies, probes were surgically implantedunder isoflurane anesthesia, as previously described (Berridge et al., 2006). Allprocedures were in accordance with NIH guidelines and were approved by theUniversity of Wisconsin Institutional Animal Care and Use Committee.

2.2. Spatial delayed alternation/working memory testing

Training and testing were similar to that used previously (Berridge et al.,2006; Devilbiss and Berridge, 2008). Briefly, animals were pair housed andplaced on a restricted feeding schedule in which they were allowed to eat15e25 g of standard chow immediately after each training/testing session. Thequantity of food/chow was titrated for each animal to maintain motivation forfood rewards (chocolate chips) while avoiding weight loss. The testing apparatuswas a T-maze consisting of a runway (91 cm), and two arms (66 cm) perpen-dicular to the runway and placed at the end of the runway farthest away from theexperimenter. The runway and arms were 10 cm in height and width. 20 cm fromthe end of the runway closest to the experimenter was an 18 cm tall removablegate that, when in position, created a start-box from which the animal could notenter the rest of the maze.

For this task, animals were rewarded (chocolate chip) when they entered thearm of the maze not chosen on the previous trial (10 trials per session, 1 session perday). Following each trial, the animal was placed in the start box for a delay period.Inter-trial delays were titrated for each animal to elicit performance accuracy in therange of 60e80%. If an animal exceeded this range, the delay was lengthened on thefollowing testing day and baseline testing resumed. Stable performance was definedas two consecutive days in which performance did not differ by more than 10%.Accuracy of performance increases over time, necessitating periodic increases indelays to maintain performance level in the target range. Given the need fordemonstrating stable baseline and the fact delays are periodically adjusted, animalsreceived a treatment on average of once every twoweeks. To ensure prominent PFC-dependency, delays were limited to 120-s. We have previously observed that withdelays up to 80e120-s in length, temporary inactivation of the medial PFC of ratsreduces performance to chance levels (unpublished observations, Spencer et al.,2012). Thus, even at 120-s delays this task is highly PFC-dependent. This range ofdelays is identical that that used in our previous studies documenting cognition-enhancing effects of methylphenidate in this task (Berridge et al., 2006). Given the120-s cut-off for delays, not every animal received all treatments prior to reachingthe 120-s cut-off.

Spatial cues were minimized by black plastic draping that surrounded the maze.All training and testing were conducted by a single individual. Themazewas cleanedwith 5% ethanol between animals. For a given animal, fecal boli and urine wereremoved/absorbed by a dry tissue prior to the start of the next trial. Intraperitonealtreatments were counter-balanced within and across animals and were adminis-tered 20-min prior to testing.

2.3. Microdialysis studies

On the day prior to testing, a microdialysis probewas inserted into one or two ofthe following regions: PFC (Aþ3.2; L0.8; V-5.2), the nucleus accumbens (Aþ1.7; L1.4;V-7.85), or the medial septal area (Aþ0.25; L1.05; V-6.5 at 6� from vertical) asdescribed previously (Berridge et al., 2006; Berridge and Stalnaker, 2002). The last0.5e1.0 mm of a dialysis probe contained an epoxy plug (corresponding to theventral-most portion of the probe when implanted). The length of functionalmembrane was 4 mm for PFC, 3 mm for the medial septal area and 2 mm for thenucleus accumbens. This active membrane began immediately above the epoxyplug. Animals were housed in a Plexiglas testing chamber (32 � 32 � 40 cm) con-tained within a ventilated, sound-attenuating outer chamber for 1e2 days (seebelow). Artificial extracellular fluid (AECF: 147 mM NaCl, 1.3 mM CaCl2, 0.9 mMMgCl2, 2.5 mM KCl; pH 7.4) was perfused through the dialysis probe.

DA and NE were measured in dialysate samples using HPLC with electro-chemical detection as previously described (Berridge and Stalnaker, 2002). Briefly,AECF was delivered at a rate of 1.5 ml/min through dialysis membrane (MW cut-off13,000, o.d. 250 mm; Spectrum Labs, Rancho Dominguez, CA). 30-min samples werecollected prior to and following vehicle or AHN 2-005 treatment. For the PFC andmedial septal area, samples were split and analyzed for both DA and NE. 20 mlaliquots were injected onto an HPLC-EC system consisting of an ESA Model 582pump set at 0.6 ml/min and an ESA 5100A Coulochem II detector with 2 electrodes inseries: �.025V, þ.220V (ESA Inc. Boston, MA). For DA, samples were injected ontoa Velosep C18 100 � 3.2 mm column with a mobile phase consisting of: 200 mMsodium phosphate (pH 3.0e4.5), 0.1 mM EDTA, 0.3 mM sodium octyl-sulfate, and 5%v/v methanol. For NE, samples were injected onto an ion exchange column (ESA,MD-16, #70e7277) and the mobile phase consisted of 150 mM ammonium acetate(pH 6.0), 0.14 mM EDTA, 15% v/v methanol, and 5% acetonitrile. The quantitationlimit for NE and DA (using a criterion of 3 times background noise) was approxi-mately 0.3 pg. NE levels display robust elevations during quiet waking relative tosleep (Berridge and Stalnaker, 2002). To avoid potential arousal-state relatedincreases in NE release, baseline samples were collected during periods when theanimal was awake a majority of the time (this occasionally required gentle tappingon the chamber and/or leaving the outer chamber door ajar). An average baselinevalue was calculated from three 30-min baseline samples displaying no greater than10% variation from the average value. The mean baseline concentration of NE persample was 1.50 � 0.12 pg within the PFC (n ¼ 14) and 1.15 � 0.15 pg within themedial septal area (n ¼ 8). The mean baseline concentration of DA was

B.E. Schmeichel et al. / Neuropharmacology 64 (2013) 321e328 323

0.83 � 0.06 pg within the PFC (n ¼ 14), 6.9 � 0.60 pg within the nucleus accumbens(n ¼ 15), and 1.06 � 0.19 pg within the MSA (n ¼ 8).

Following collection of at least three baseline samples, animals received anintraperitoneal (IP) injection of vehicle or a dose of AHN 2-005 demonstrated toimprove PFC-dependent working memory performance (10 mg/kg; see Fig. 1). IPinjections were performed without picking up the animal by gently lifting a back legwhen the animal faced away from the experimenter (which is typical), minimizingthe stress/arousal associated with injection.

2.4. Measures of locomotor activity, feeding, drinking and sleep/arousal

To better compare the behavioral actions of cognition-enhancing doses of AHN2-005 to clinically-relevant doses of psychostimulants and other ADHD-relateddrugs (see Berridge et al., 2006; Devilbiss and Berridge, 2008), spontaneousbehavior was scored fromvideotaped records in a subset of microdialysis animals, asdescribed previously (see Berridge and Foote, 1996). For these analyses, behaviorwas scored in the one 30-min epoch immediately preceding and two 30-min epochsimmediately following treatment. The following behaviors were scored: 1) thenumber of quadrant entries (a measure of horizontal locomotion defined by hindlegs crossing into a new quadrant of the testing chamber); 2) the number of rears(both free and wall); 3) time spent eating; 4) time spent drinking; 5) time spentasleep (body resting on floor, head resting on floor); 6) time spent in quiet waking(head raised off of floor, body resting on floor); 7) time spent in active waking (allwaking behavior other than quiet waking). In earlier studies we demonstrated thatthese behavioral measures of sleep-wake state align closely with EEG/EMGmeasures of sleep-wake state (Berridge and Foote, 1996; Berridge et al., 1999;Berridge and Wifler, 2000).

2.5. Drug treatment

One goal of the current study was to compare the neurochemical and behavioralactions of AHN 2-005 with previously described actions of psychostimulants andselective NE reuptake inhibitors. Given virtually all previous work with these drugsin rats has been conducted during the light phase of the circadian cycle, the currentstudies tested animals between the hours of 0900 and 1800. Additionally, a majorityof work with psychostimulants and selective NE reuptake blockers in animals usedIP administration. Moreover, we previously demonstrated that methylphenidateexerts similar cognition-enhancing and neurochemical actions when administeredIP and orally, provided dose is adjusted to yield similar peak and clinically-relevantplasma concentrations (Berridge et al., 2006; Devilbiss and Berridge, 2008). Thus, forthese studies animals received IP treatment with vehicle (0.9% saline) or AHN 2-005dissolved in vehicle.

2.6. Statistical analyses

Given increasing delays are needed to maintain a set performance level and welimited delay length to 120 s, it was not possible that every animal receive everydose of AHN 2-005. Thus, the dose-dependent effects of AHN 2-005 on workingmemory performance were analyzed with a between-subject one-way ANOVA.Post-hoc analyses were conducted by the Dunnett’s test, comparing drug-treatmentwith vehicle-treatment. The neurochemical effects of 10 mg/kg AHN 2-005 wereanalyzed using a mixed-design two-way ANOVA with treatment as a between-

Fig. 1. AHN 2-005 improves PFC-dependent working memory performance. Shown arethe mean (�SEM) percent change from baseline in animals treated with vehicle orvarying doses of AHN 2-005. AHN 2-005 produced a dose-dependent improvement inperformance in this task with significant improvement occurring at the 10 mg/kg dose.The magnitude of AHN 2-005-induced improvement in performance is comparable tothat seen with systemic administration of clinically-relevant doses of psychostimu-lants. *P < 0.05 vs. vehicle-treatment.

subjects (2 levels) and time as a within-subjects factor (11 levels). In the case ofmedial septal area, effects of 10 mg/kg AHN 2-005 were analyzed using a one-wayANOVA with time as a within-subjects factor (11 levels). For the neurochemicaldata, matched-pair t-tests were used to determine whether, within a given treat-ment group, post-treatment measures differed significantly from the baseline epochthat immediately preceded AHN 2-005 administration. Effects of 10 mg/kg AHN 2-005 on spontaneous behavior (locomotor, eating, drinking and sleep-wake) duringthe first two 30-min post-treatment epochs were analyzed using a mixed-designtwo-way ANOVA with treatment as a between-subjects and time as a within-subjects factor. When statistical significance (P < 0.05) was indicated, post-hocanalyses were conducted using independent t-tests.

2.7. Histological analyses and data selection

Placement of microdialysis probes was verified in 40-mm thick coronal sectionsstained with Neutral Red dye. Neurochemical data were included only whenhistological analyses verified accurate placement of microdialysis probes and NE orDA concentrations were stable (<10% variability) throughout baseline.

3. Results

3.1. Effects of AHN 2-005 on working memory performance

To assess the effects of AHN-2005 on working memory perfor-mance, animals were treated with vehicle (n¼ 8), 1.0 mg/kg AHN 2-005 (n¼ 7) or 10.0mg/kg AHN 2-005 (n¼ 8) 20-min prior to testingin the T-maze. This dose range was based on: 1) published obser-vations indicating behavioral and neurochemical actions of AHN 2-005 (Hiranita et al., 2009; Li et al., 2005; Raje et al., 2005); 2) limitedpilot studies; 3) limited observations indicating that at 30 mg/kglocomotor-activating effects may begin to emerge (unpublishedobservations, Frank Zemlan, Ph.D.). The delay interval was adjustedto produce moderate baseline performance levels. For this study,mean baseline accuracy was: vehicle, 76% � 2% (SEM); 1.0 mg/kgAHN 2-005, 78% � 1%; 10 mg/kg AHN 2-005, 76% � 2%. Delaysranged between 10 and 120-s with an average delay of 82 � 9 s. Asshown in Fig. 1, AHN 2-005 dose-dependently improved perfor-mance from baseline, with a significant improvement at 10 mg/kg(F2,20 ¼ 5.81, P ¼ 0.01). The magnitude of this improvement issimilar to that seen previously in our laboratory with clinically-relevant doses of methylphenidate tested under identical condi-tions (Berridge et al., 2006; Devilbiss and Berridge, 2008).

3.2. Effects of a cognition-enhancing dose of AHN 2-005 on NE and/or DA efflux in the PFC, nucleus accumbens, and medial septal area

To better understand the neurochemical actions that maycontribute to the cognition-enhancing effects of AHN 2-005, addi-tional studies examined the effects of a cognition-enhancing doseof AHN 2-005 (10 mg/kg) on NE and DA efflux in the PFC, nucleusaccumbens (DA only), and the medial septal area, a subcorticalregion that, like the PFC, receives only a moderate innervation ofboth DA and NE (see Figs. 2e4). Additionally, in a subset ofmicrodialysis animals we examined the locomotor, wake-promoting, feeding and drinking effects of this dose of AHN 2-005.

3.2.1. PFC DA and NEWithin the PFC, 10 mg/kg AHN 2-005 modestly elevated extra-

cellular DA levels, with increases of 70%e85% above baseline levelsbetween the 2nd and 5th 30-min post-treatment sample(Figs. 2e4; vehicle n ¼ 7; AHN 2-005 n ¼ 8; treatment F1,13 ¼ 22.6,P < 0.001, time F10,130 ¼ 8.5, P < 0.001, treatment � timeF10,130 ¼ 5.4, P < 0.001). AHN 2-005 also significantly elevatedextracellular NE levels in the PFC by 75%e90% in post-treatmentsamples 2e5, comparable to that seen with PFC DA (Figs. 2e4;vehicle n ¼ 6; AHN 2-005 n ¼ 8; treatment F1,12 ¼ 12.5, P < 0.001,time F10,120 ¼ 3.8, P < 0.001, treatment � time F10,120 ¼ 4.5,P < 0.001). The sustained neurochemical action of AHN 2-005 is

Fig. 2. Photomicrographs depicting placement of dialysis probes within the prefrontal cortex (PFC, Panel A), nucleus accumbens (NAc, Panel B), and the medial septal area (MSA,Panel C). For PFC, probes were placed within the medial subdivision of this region. In Panel B, dashed line indicates approximate boundary of the nucleus accumbens. For MSA,probe placement spanned the medial septum and diagonal band of Broca. Dashed line in Panel C indicates dorsal border of the medial septum. 40-mm coronal sections were stainedwith Neutral Red. Arrows indicate probe track. AC ¼ anterior commissure; CC ¼ corpus callosum; LV ¼ lateral ventricle; M ¼ midline.

B.E. Schmeichel et al. / Neuropharmacology 64 (2013) 321e328324

consistent with the relatively long half-life of this compound (Rajeet al., 2005).

The magnitude of the AHN 2-005-induced increase in PFC DA iscomparable to that seen with clinically-relevant doses of methyl-phenidate and the SNRI, atomoxetine (Berridge et al., 2006;

Fig. 3. Chromatograms of AHN 2-005-induced increases in DA and NE levels. Shownare chromatograms from a pre-treatment (PRE) and post-treatment (POST) sample forPFC DA (Top Panel), PFC NE (Middle Panel) and nucleus accumbens DA (NAc DA,Bottom Panel). Numbers adjacent to peaks indicate retention time and quantity (pg),respectively. 10 mg/kg AHN 2-005 increased the height of the NE and DA peaks (scale isthe same in PRE vs. POST).

Bymaster et al., 2002). The magnitude of the AHN 2-005-inducedincrease in PFC NE is comparable to that seen with atomoxetineand somewhat less than that seen with methylphenidate (Berridgeet al., 2006; Bymaster et al., 2002).

Fig. 4. Effects of a cognition-enhancing dose of AHN 2-005 on extracellular levels ofNE and DA within the PFC (Panel A), nucleus accumbens (NAc, DA only, Panel B), andmedial septal area (MSA, Panel C). Shown are the mean (�SEM) DA and NE levels (per20 ml sample) expressed as a percentage of baseline in 30-min samples collected priorto (negative numbers) and following (positive numbers) injection of vehicle or 10 mg/kg AHN 2-005. AHN 2-005 elicited significant though relatively restrained increases inextracellular DA and NE in the PFC and MSA (w75%e100% above baseline) and largerincreases in extracellular DA levels in the nucleus accumbens (w200% above baseline).þP < 0.05, þþP < 0.01 compared to sample immediately preceding drug administration(Sample e 1); *P < 0.05, **P < 0.01 compared to vehicle-treated animals.

B.E. Schmeichel et al. / Neuropharmacology 64 (2013) 321e328 325

3.2.2. Nucleus accumbens DACompared to the PFC, AHN 2-005 produced a more robust

increase in extracellular levels of DA in the nucleus accumbens,consistent with the high density of DA fibers and DAT in this region.Specifically, in all samples collected after the first 30-min post-treatment, we observed an increase in accumbens DA that rangedbetween approximately 170%e200% above baseline levelsfollowing AHN 2-005 (Figs. 2e4; vehicle n ¼ 6; AHN 2-005 n ¼ 8;treatment F1,12 ¼ 140.2, P < 0.001, time F10,120 ¼ 11.7, P < 0.001,treatment � time F10,120 ¼ 11.0, P < 0.001). There were no obviousdifferences between baseline DA levels or drug-induced alterationsin DA levels when probes were estimated to have been locatedwithin the core subregion vs. the shell subregion of the nucleusaccumbens. Thus, averaging across post-treatment samples 2e5,AHN 2-005 increased DA levels in the shell accumbens by175%� 41% and increased core accumbens DA levels by 197%� 68%.

3.2.3. Medial septal area DA and NETo assess the degree towhich AHN 2-005 alters NE and DA efflux

in a subcortical region that, like the PFC, receives a moderateinnervation by both NE and DA fibers, additional studies examinedthe effects of AHN 2-005 on DA (n ¼ 8) and NE (n ¼ 8) efflux withinthe medial septal area (Figs. 3 and 4). Vehicle treatment hadminimal effects on extracellular DA and NE in the PFC andaccumbens in the current studies as well as our previously pub-lished studies measuring NE within the medial septal area(Berridge, 2006). Thus, we did not include vehicle-treated controlsin the studies examining the effects of AHN 2-005 on medial septalNE and DA. As in the PFC, AHN 2-005 significantly increasedextracellular levels of both DA and NE in themedial septal area (DA:time F10,70 ¼ 6.4, P < 0.001; NE: time F10,70 ¼ 11.1, P < 0.001). Themagnitude of AHN 2-005-induced increases in NE and DA abovebaseline in this regionwas comparable to that seen in the PFC (posttreatment samples 2e5, DA ¼ 55%e82%; NE ¼ 50%e100%).

3.3. Effects of AHN 2-005 on locomotor activity, eating/drinking andsleep-wake

To better compare thebehavioral effects of a cognition-enhancingdose AHN 2-005 to the well-characterized behavioral effects ofpsychostimulants, in a subset of randomly selected microdialysisanimals, a broad array of behavioral effects of AHN 2-005 (n¼ 7) andvehicle treatment (n¼ 7)were examinedduring thefirst two 30-minpost-treatment epochs. AHN 2-005 lacked pronounced locomotor-activating actions (Table 1; Quadrant Entries, treatment F1,12 ¼ 1.6,P ¼ 0.24, time F1,12 ¼ 5.2, P ¼ 0.04, treatment � time F1,12 ¼ 2.8,P¼0.12;Rears, treatment F1,12¼2.0,P¼0.18, time F1,12¼2.8,P¼0.12,treatment � time F1,12 ¼ 0.3, P ¼ 0.63). The minimal level of loco-motor activity observed following AHN 2-005 and vehicle treatmentis comparable to that seen in spontaneous waking (Berridge and

Table 1Behavioral actions of AHN 2-005.

Quad entries Rears Eat Dr

Post1Veh 4 � 2 2 � 1 72 � 68 0AHN 16 � 8 1 � 1 0 � 0 6Post2Veh 2 � 2 1 � 1 95 � 95 11AHN 1 � 1 0 � 0 9 � 9 0

Shown are the effects of a cognition-enhancing dose of AHN 2-005 (AHN; 10 mg/kg) on lotime (seconds) spent eating (Eat), drinking (Drink), asleep (Sleep), in quiet waking (Quietfor vehicle-treated (Veh) and AHN 2-005-treated animals for the two 30-min epochs immon any of these behavioral measures relative to vehicle treatment, except for a modest intime spent in quiet waking during this epoch in AHN 2-005-treated animals represents

Foote, 1996; Berridge and O’Neill, 2001) and well below that seenwith moderate doses of psychostimulants (Kuczenski et al., 1997).Similarly, AHN 2-005 lacked significant effects on time spent eatingand drinking (Eating, treatment F1,12¼ 0.6, P¼ 0.47, time F1,12¼ 0.01,P ¼ 0.93, treatment� time F1,12 ¼ 0.3, P ¼ 0.87; Drinking, treatmentF1,12 ¼ 0.2, P ¼ 0.69, time F1,12 ¼ 0.2, P ¼ 0.69, treatment � timeF1,12 ¼ 1.8, P ¼ 0.20). Finally, AHN 2-005 lacked robust wake-promoting effects, particularly after the first post-injection 30-minepoch (Sleep, treatment F1,12 ¼ 1.0, P ¼ 0.34, time F1,12 ¼ 5.4,P ¼ 0.04, treatment � time F1,12 ¼ 1.2, P ¼ 0.30; Quiet Wake, treat-ment F1,12 ¼ 9.5, P ¼ 0.01, time F1,12 ¼ 8.08, P ¼ 0.015,treatment � time F1,12 ¼ 3.8, P ¼ 0.07; Active Wake, treatmentF1,12 ¼ 0.03, P ¼ 0.86, time F1,12 ¼ 3.3, P ¼ 0.10, treatment � timeF1,12 ¼ 0.5, P ¼ 0.51).

4. Discussion

Drugs used to treat ADHDhave been demonstrated to improve anarray of PFC-dependent processes (Chamberlain et al., 2007;Diamond, 2005; Mehta et al., 2001; Turner et al., 2005). The currentstudies demonstrate that the selective DA reuptake inhibitor, AHN 2-005, improves PFC-dependent cognitive function asmeasured in thisworkingmemory taskwhile lacking locomotor-activatingorarousal-promoting actions. This behavioral profile is similar to all drugscurrently approved for use in ADHD, including psychostimulants,SNRIs and a2-agonists (see Arnsten, 2009; Berridge and Devilbiss,2011). Moreover, at a cognition-enhancing dose AHN 2-005modestly elevated extracellular levels of DA and NE within the PFC(e.g. 75%e100%) similar to that seenwith cognition-enhancing dosesof psychostimulants and SNRIs (Berridge et al., 2006; Bymaster et al.,2002). These latter observations are consistent with recent studiesdemonstrating that methylphenidate acts directly within themedialPFC to improveworkingmemory performance (Spencer et al., 2012).Collectively, these observations suggest a potential use of this orrelated benztropine analogs in the treatment of ADHD and otherconditions associated with prefrontal dysfunction.

4.1. What PFC-dependent processes are targeted by AHN 2-005?

These preclinical studies were designed to initially assess thepotential use of benztropine analogs in the treatment of ADHD.Pharmacological and lesion studies demonstrate that the delayed-response test of spatial working memory used in these studies ishighly dependent on the PFC (Kesner et al., 1989; Spencer et al.,2012). Additionally, the pharmacological sensitivity of perfor-mance in this task closely aligns with the pharmacology of ADHD(Arnsten, 2009; Berridge et al., 2012; Gamo et al., 2010). This is incontrast with other animal-based tests of PFC-dependent function,including sustained attention and attentional set shifting, whichdisplay a pharmacological sensitivity distinct from both delayed

ink Sleep Quiet wake Active wake

� 0 1213 � 219 121 � 31 213 � 114� 6 812 � 194 377 � 60* 490 � 136

� 11 1448 � 224 82 � 22 61 � 57� 0 1450 � 130 167 � 70 73 � 52

comotor activity as measured by the number of quadrant entries and rears as well asWake) and in active waking (Active Wake). Data are expressed as the mean (�SEM)ediately following treatment (Post1 and Post2). AHN 2-005 had no significant effectscrease in quiet waking during the first 30-min post-treatment epoch. The amount ofonly 20% of this epoch. *P < 0.01 vs. vehicle-treatment.

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alternation testing and ADHD. Specifically, working memoryperformance is improved by stimulation of PFC a2-receptors andimpaired by a1-receptor activation, while attention set shifting andsustainedattentionare improvedbya1-receptorsand, at leastwhereexamined, are insensitive to a2-receptor activation (Berridge et al.,2012; Lapiz and Morilak, 2006). Moreover, although workingmemory performance is maximally improved by doses of methyl-phenidate that produce clinically-relevant plasma concentrations,attention set shifting and sustained attention are maximallyimprovedbydoses that yieldplasmaconcentrations above the rangetypically associated with clinical efficacy (Berridge et al., 2012,2006). The close alignment between the pharmacology of perfor-mance in this delayed-alternation task of spatial working memoryand ADHD may reflect the fact that performance in this task issimultaneously dependent on a variety of cognitive and behavioralprocesses affected in ADHD, including attention, planning, resis-tance to distractors, and working memory. The multiplicity ofbehavioral and cognitive processes involved in performance of thistask currently precludes definitive identification of the precisesubset of cognitive processes affected by AHN 2-005.

Nonetheless, in both human and animal subjects, psychosti-mulants and other drugs used to treat ADHD have been demon-strated to facilitate a variety of PFC-dependent processes, includingplanning, sustained attention, working memory, response inhibi-tion, and the regulation of impulsivity (Berridge et al., 2012, 2006;Mehta et al., 2004; Mehta et al., 2001; Robbins and Arnsten, 2009).These observations are consistent with imaging data demon-strating ADHD-associated hypofrontality is reversed by clinically-relevant doses of psychostimulants (Bush et al., 2008; Vaidyaet al., 1998). Based on these and other observations, it has beenproposed that the treatment of ADHD involves, at least in part,drug-induced general improvement in PFC-dependent functionwhich is manifested across an array of PFC-dependent tasks(Arnsten and Pliszka, 2011). From this perspective, the ability ofAHN 2-005 to improve performance in this well-validated test ofPFC-dependent function suggests it would likely facilitate perfor-mance on other tests of PFC-dependent function.

4.2. Potential neurocircuitry and receptor mechanisms underlyingthe cognition-enhancing actions of AHN 2-005

Currently, the receptor mechanisms involved in the cognition-enhancing effects of AHN 2-005 are unknown. However, post-synaptic DA D1 and NE a2 receptors located within the PFC havebeen documented to facilitate PFC-dependent function asmeasuredin delayed-response tasks of working memory (Arnsten, 2007).Evidence further suggests a prominent role of these receptors in thecognition-enhancing actions of drugs used to treat ADHD. Thisincludes the fact that clinically-relevant doses of psychostimulantsand SNRIs elevate extracellular NE and DA preferentially within thePFC (Berridge et al., 2006; Bymaster et al., 2002), while systemic a2and D1 receptor antagonists block the cognition-enhancing actionsof these drugs (Arnsten and Dudley, 2005; Gamo et al., 2010).Moreover, recent studies demonstrate that psychostimulant infu-sion directly into the PFC of rats improves working memoryperformance (Spencer et al., 2012) similar to that seen with intra-PFC infusion of clinically efficacious a2-agonists (Arnsten, 2009).These observations suggest the hypothesis that the cognition-enhancing actionsof AHN2-005 involvePFCD1and/ora2-receptors.

Of course this hypothesis does not imply that the cognitiveeffects of AHN 2-005 (or drugs used to treat ADHD) are solelydependent on actions within the PFC. For example, substantialevidence implicates frontostriatal circuitry in ADHD (Castellanosand Tannock, 2002). AHN 2-005 and ADHD-related treatmentsmay alter frontostriatal signaling through actions within the PFC.

However, given both AHN 2-005 and psychostimulants increase DAsignaling within the striatum (Berridge et al., 2006), the cognition-enhancing effects of these compounds may also involve drug actionwithin the striatum. Such amechanismwould differ from the SNRIs,which haveminimal effects on extracellular DAwithin the striatum/core nucleus accumbens (Bymaster et al., 2002). Given psychosti-mulants are viewed asmore effective in the treatment of ADHD thanSNRIs (Greenhill, 2001), elevations in striatal DA signaling may benecessary to achieve maximal efficacy. Nonetheless, in recentstudies we observed that, in contrast to that seen with intra-PFCinfusions, methylphenidate infusion into the dorsomedial striatumhad no effect on working memory performance even thoughperformance in this task is highly dependent on this region (Spenceret al., 2012). This latter observation suggests the AHN 2-005 likelydoes not act within the dorsomedial striatum to facilitate frontos-triatal function as measured by working memory performance.

Finally, AHN 2-005 also produced moderate elevations inextracellular NE and DA outside the PFC and striatum (i.e. withinthe medial septal area) similar to that seen with cognition-enhancing doses of psychostimulants (see Results 3.2.3; Berridgeet al., 2006). Thus, actions on NE/DA signaling outside frontos-triatal circuitry may contribute to the cognition-enhancing actionsof AHN 2-005 and ADHD-related drugs. Indeed, the medial septumis implicated in delayed-response tasks of spatial working memory(Pang et al., 2011; Smith and Pang, 2005). Future studies will needto identify the neurocircuitry underlying the cognition-enhancingeffects of AHN 2-005 and ADHD-related drugs.

4.3. Non-psychostimulant-like behavioral actions of AHN 2-005

AHN 2-005 lacked prominent wake-promoting, locomotor-acti-vating and feeding/drinking effects. This behavioral profile is similarto clinically-relevant doses of psychostimulants and other drugsused in the treatment of ADHD (Berridge et al., 2006). In part, thisappears to reflect relatively mild increases in extracellular NE/DAelicited by AHN 2-005, including in regions associated withpsychostimulant-induced arousal (e.g. medial septal area; Berridgeet al., 1999). Previous studies demonstrated AHN 2-005 andrelated benztropine analogs lack reinforcing effects (Hiranita et al.,2009; Li et al., 2005; Woolverton et al., 2001, 2000). This is unex-pected, given the effects of this compound on extracellular DA in thenucleus accumbens observed here and elsewhere (see above andRaje et al., 2005). However, it should be noted that although AHN 2-005 increased accumbens DA in the current studies, the magnitudeof this (200%) was substantially less than that typically associatedwithmotor-activating doses of commonly abused psychostimulants,including amphetamine, methamphetamine and cocaine (600%e<1000%; Florin et al., 1994, 1995; Kuczenski et al., 1995). Thereasons for the reduced action of AHN 2-005 on locomotion, rein-forcement and extracellular DA are not fully understood. However,available evidence suggests that benztropine analogs physicallyinteract with the DAT differently than cocaine-like DAT inhibitors,leading to a relatively slow DAT association rate (Desai et al., 2005;Loland et al., 2008). This is posited to result in behavioral andneurochemical profiles that are distinct from the cocaine and otherpsychostimulants. Despite our incomplete understanding of thepharmacology of AHN 2-005, the available evidence indicates thatits ability to selectively target the DAT is not associated with rein-forcing actions and thus is not a contraindicator for clinical use.

4.4. Potential mechanisms involved in the simultaneous elevation ofDA and NE levels within the PFC

Earlier work demonstrates that blockade of the NET leads to anincrease in extracellular DA in the PFC and other regions that

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display moderate NE and DA innervations and moderate DATdensity (Carboni et al., 2006, 1990; Sesack et al., 1998; Yamamotoand Novotney, 1998). Our neurochemical observations furtherdemonstrate a close relationship between extracellular DA and NElevels, with a highly selective DAT inhibitor (AHN 2-005) elevatingboth DA and NE in the PFC and medial septal area (see Results 3.2).Given extracellular concentrations of DA and NE are similar in theseregions (see Methods 2.3 and Results 3.2), the ability of AHN 2-005to increase extracellular NE levels may involve drug-inducedincreases in competition between DA and NE at the NET. Alterna-tively or additionally, AHN 2-005-induced increases in DA signalingmay directly or indirectly activate noradrenergic neurons thatproject to the PFC (Foote et al., 1983) andmedial septal area (Españaand Berridge, 2006).

4.5. Summary

These studies demonstrate that the selective dopamine reup-take inhibitor, AHN 2-005, improves PFC-dependent cognitivefunction while simultaneously elevating extracellular NE and DA inthe PFC. These actions are similar to those observed with low-dosepsychostimulants and SNRIs used in the treatment of ADHD, sug-gesting that AHN 2-005 or other benztropine analogs may be usefulin treating this disorder.

Financial disclosures

Dr. Berridge has received consulting fees from Phase 2Discovery. P2D Bioscience is developing AHN 2-005 for the treat-ment of attention deficit/hyperactivity disorder. FPZ is a full timeemployee of P2D Bioscience. Ms. Schmeichel has no financialdisclosures to report.

Acknowledgments

This work was supported by PHS grants, MH081843, DA000389,and MH08138, the Wisconsin Institutes of Discovery and theUniversity of Wisconsin Graduate School.

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