Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 81–90

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Progress in Neuro-Psychopharmacology & BiologicalPsychiatry

Effects of atomoxetine on attention and impulsivity in the five-choiceserial reaction time task in rats with lesions of dorsal noradrenergicascending bundle

Yia-Ping Liu a,b,⁎, Teng-Shun Huang a, Che-Se Tung c, Chen-Cheng Lin a

a Department of Physiology and Biophysics, National Defense Medical Center, Taipei, Taiwan, ROCb Department of Psychiatry, Tri-Service General Hospital, Taipei, Taiwan, ROCc Division of Medical Research & Education, Cheng Hsin General Hospital, Taipei, Taiwan, ROC

Abbreviations: 5-CSRTT, five-choice serial reaction timhyperactivity disorder; BNST, bed nucleus of the schloroethyl)-N-ethyl-2-bromobenzylamine; DNAB, dobundle; ITI, inter-trial interval; LC, locus coeruleus; NAT,noradrenaline reuptake inhibitor; VNAB, ventral noradren⁎ Corresponding author at: Department of Physiology a

Medical Center, 161 Minchuan East Rd., Sec. 6, Taipei 187923100x18614; fax: +88 62 87923153.

E-mail address: yiaping@ms75.hinet.net (Y.-P. Liu).

http://dx.doi.org/10.1016/j.pnpbp.2014.08.0070278-5846/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 May 2014Received in revised form 4 August 2014Accepted 16 August 2014Available online 21 August 2014

Keywords:AtomoxetineDorsal noradrenergic ascending bundleFive-choice serial reaction time taskImpulsivityMicrodialysis

Atomoxetine, a noradrenaline reuptake inhibitor (NRI), which is a non-stimulating medicine that is used for thetreatment of patients with attention deficit hyperactivity disorder (ADHD), has been found to be effective inreducing behavioral impulsivity in rodents, but its efficacy in a dorsal noradrenergic ascending bundle(DNAB)-lesioned condition has not been examined. The present study aimed to investigate the effects ofDNAB lesions on attention and impulsive control in the five-choice serial reaction time task (5-CSRTT) in ratstreated with atomoxetine. The drug-induced changes in noradrenaline efflux in the medial prefrontal cortexwere also measured. 5-CSRTT-trained rats were included in one of the following groups: N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4)/Atomoxetine, Sham/Atomoxetine, DSP-4/Saline, or Sham/Saline. Acuteatomoxetine (0.3 mg/kg) was administered 14 days after the DSP-4 regime. The behavioral testing includedma-nipulations of the inter-trial interval (ITI), stimulation duration and food satiety. In vivomicrodialysis of the nor-adrenaline efflux in the medial prefrontal cortex and the expression of the noradrenaline transporter (NAT) inthe DNAB areas were examined. Atomoxetine reduced impulsivity and perseveration in the long-ITI conditionwith no effects on any other variables. This phenomenon was not influenced by DSP-4 pre-treatment. TheDNAB-lesioned rats had lower noradrenaline efflux in the medial prefrontal cortex. DSP-4 caused no change inNAT expression in the DNAB areas. These findings suggested that noradrenaline reuptakemay not be exclusivelyresponsible for the atomoxetine effects in adjusting impulsivity. The role of DNAB should also be considered,particularly in conditions requiring greater behavioral inhibition.

© 2014 Elsevier Inc. All rights reserved.

1. Introduction

The use of atomoxetine in treating patients with attention deficit hy-peractivity disorder (ADHD) highlights the pharmacological therapeuticsof the disease may be achieved through the manipulations of involvedneurotransmitters. Specifically, the central effects of atomoxetine pri-marily operate through an increase of cortical norepinephrine via themechanism of noradrenaline transporter (NAT) inhibitor (Bymasteret al., 2002), although with a secondary effect of elevating the corticaldopamine efflux through non-selective effects of NAT (Bymaster et al.,

e task; ADHD, attention deficittria terminalis; DSP-4, N-(2-rsal noradrenergic ascendingnoradrenaline transporter; NRI,ergic ascending bundle.nd Biophysics, National Defense14, Taiwan, ROC. Tel.: +88 62

2002; Carboni and Silvagni, 2004), and with inhibitory effects on sero-tonin reuptake transporters (Ding et al., 2014) and glutamate receptors(Ludolph et al., 2010). As atomoxetine effectively adjusts the electro-physiological activities of locus coeruleus (LC) (Bari and Aston-Jones,2013), the projection areas of LC are highly responsible for thenoradrenaline-relevant behavioral tasks (Arnsten, 2011), particularlyfor those targeting the core symptoms of ADHD, namely hyperactivity,inattention and impulsivity (Wietecha et al., 2009).

In rodents, atomoxetine has been demonstrated to affect perfor-mance on the five-choice serial reaction time task (5-CSRTT), a be-havioral test that concomitantly examining animal attention andimpulsivity. While atomoxetine has no influence on attentional accura-cy in the 5-CSRTT, it decreases premature responding in baseline condi-tions and situations with altered cognitive demand (Fernando et al.,2012; Robinson, 2012; Robinson et al., 2008). The evidence impliesthat strengthened central noradrenaline transmission results in bettercontrol of behavioral impulsivity. However, it appears paradoxical toextend this hypothesis to lesioned conditions in which central nor-adrenaline function is impaired but the animal's impulse control in the

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5-CSRTT is preserved (Carli et al., 1983) or improved (Cole and Robbins,1992). It is necessary to revisit the noradrenaline hypothesis of impul-sivity in terms of the integrity of noradrenaline neurons, particularlyin that noradrenaline neurons involve in the selective processing ofsensory stimuli (Coull, 1994), which serves an organic viewpoint forconsidering ADHD as a minimal brain dysfunction (Gilger and Kaplan,2001). Accordingly, the atomoxetine effects are worth investigatingunder intact or lesioned state of the noradrenaline pathway.

Effects of central noradrenaline system lesions can be approacheddifferently in terms of the ventral or dorsal noradrenergic ascendingbundles (abbreviated VNAB or DNAB, respectively) (Kostowski et al.,1978). The VNAB primarily deals with aversive sensations (Aston-Joneset al., 1999). It originates from the lateral tegmentum and targets theseptum, hypothalamus, and bed nucleus of the stria terminalis (BNST)(Moore and Card, 1984). However, the DNAB originates from thelocus coeruleus (LC) and innervates areas including the amygdala, hip-pocampus and frontal cortex (Dahlstroem and Fuxe, 1964). This path-way plays a crucial role in mood regulation and impulse control withits extensive limbic-cortical innervations. Previously, we demonstratedthat reboxetine, another selective NRI, decreases the activity of thenoradrenaline transporter (NAT) in the LC but preserves it along theterminal areas of the DNAB, such as the hippocampus and cortex (Kuet al., 2012). This implies that the interpretation of the mechanismsunderlying NRI-improved impulse control may not be limited to NATfunction only, and approach reflects more dynamic features of nor-adrenaline transmission, such asmeasuring noradrenaline effluxwithinthe lesioned DNAB projection areas, which is necessary.

N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) is aβ-chloroalkylamine.When systemically administered, it passes throughthe blood–brain barrier and causes a preferential neurotoxic effect onnoradrenaline nerve terminal projections that originate from the LC(Jonsson et al., 1981) through both transmitter-depleting and neurode-generative mechanisms (Fritschy and Grzanna, 1991b). Due to the dif-ferential sensitivity to DSP-4 of DNAB and VNAB, the peripheral DSP-4regime specifically damages the DNAB (Fritschy and Grzanna, 1991a,1991b). Its use is thus appropriate in investigating the functional roleof DNAB. Previous evidence has demonstrated that DSP-4 causes mar-ginal memory impairment (Sontag et al., 2008) yet with no effects onthe impulsivity of rats in a spatial memory performance test (Sontaget al., 2011).

In the present study, we compared the effects of atomoxetine on theperformance of rats with or without DNAB lesions in the 5-CSRTT to ex-amine whether the DNAB impairments alter the atomoxetine effects.Furthermore, noradrenaline efflux in the medial prefrontal cortex wasmeasured with in vivo microdialysis, which might provide additionalinformation for interpreting our behavioral data in termsof thedynamicreactivity of extracellular noradrenaline to atomoxetine.

2. Materials and methods

2.1. Animals

Male Sprague–Dawley (SD) rats (BioLASCO Taiwan Co., Ltd., Taipei,Taiwan, R.O.C.) were used. All rats were housed in groups of 3 and ina temperature and humidity-controlled holding facility with 12-hlight/dark cycles (lights on from 07:00 to 19:00). All animals receivedwater ad libitum. However, food was restricted so that it could beearned during the test sessions [maximum of 100 × 45-mg purified ro-dent pellets (Labdiet, St. Louis, MO, USA)] and at the end of the test[20 g/rat of standard rodent chow (BioLASCO Taiwan Co., Ltd.)]. Ratswere trained to learn the 5-CSRTT at 12 weeks of age. They reachedthe criteria for the 5-CSRTT at 16 weeks of age and body weights of300–350 g. Behavioral testing took place between 08:00 and 12:00with all rats tested at the same time every daywhen possible. All exper-imental procedures were evaluated and approved by the animalcare committee of the National Defense Medical Center (AAALAC full

accreditation). All efforts were made to reduce the number of animalsused and to minimize animal suffering during the experiments.

2.2. Training procedure for the 5-CSRTT

The training programwas identical to that of a previously describedprocedure (Liu et al., 2009, 2011). Each session beganwith the illumina-tion of the 5-CSRTT chamber (25 × 31 × 33 cm3, TSE Systems GmbH,Bad Homburg, Germany) with a house light. The rats had to nose pokethe magazine in order to initiate a trial. After a fixed inter-trial interval(ITI) of 5 s, the light at the rear of one of the response apertures wasbriefly illuminated. A response in this aperture within a limited timefrom illumination of the hole (limited hold period) was recorded as acorrect response and was rewarded by delivery of a food pellet to themagazine. Response in a non-illuminated holewas recorded as an incor-rect response and was punished by a 5 s timeout period, during whichthe house light was extinguished. Trials in which the rat failed to re-spond within the 5 s limited hold period were recorded as omissions,and the rat was punished with a 5 s timeout period. Responses in anyone of the apertures prior to the illumination (i.e. during the ITI) werenot punished by a timeout, thus needed to be recorded separately asprematures (the initial response) and perseverations (responses after-ward in the same aperture). For assessing animals' waiting capacity,the ITI was adjusted to 3 or 7 s in separated testing sessions. A sessionwas either terminated after a maximum of 100 completed trials or30 min, depending on which came first.

2.3. Experimental design

Thirty-two 5-CSRTT-trained rats were randomly assigned to one ofthe following groups: Sham/Saline, DSP-4/Saline, Sham/Atomoxetine,or DSP-4/Atomoxetine (8 rats in each group). Rats in the sham groupreceived saline vehicle instead of DSP-4. Behavioral testing with orwithout atomoxetine was performed 14 days later and was finishedwithin 30 days of the DSP-4 or vehicle injection. The behavioral testingincluded manipulations of ITI, stimulation duration, and food satiety.The brains of the rats were immediately removed after the last testfor western blotting (to examine NAT levels). Ten separate 5-CSRTT-trained rats were used for the dialysis experiment. Rats were randomlyassigned to the DSP-4 or sham group (5 rats in each group). These ratswere running the 5-CSRTT routinely, and the dialysis experiment wasconducted 30 days after the DSP-4 or vehicle injection.

2.4. DNAB lesions

TheDNAB lesionswere conducted based onmethods described else-where (Cryan et al., 2002; Ku et al., 2012). In brief, ratswere given an in-traperitoneal (i.p.) injection of DSP-4 (50 mg/kg) 14 days prior to thestart of behavioral testing in order to allow for recovery of the peripher-al noradrenergic system (Fritschy and Grzanna, 1991a). Due to the dif-ferential sensitivity to DSP-4 of DNAB and VNAB, the peripheral DSP-4regime specifically damages the DNAB only, thus leaves the VNAB inte-gral (Fritschy and Grzanna, 1991a,1991b). Zimeldine (10 mg/kg, i.p.)was injected 30 min prior to the DSP-4 administration in order toprotect the serotonergic nerve terminals (Heal et al., 1993). The degreeof noradrenaline depletion was determined by the noradrenalinelevels, which were determined with the high performance liquidchromatography (HPLC) method that has been employed elsewhere(Lu et al., 1992).

2.5. Microdialysis

Rats were anesthetized and mounted in a stereotaxic apparatus(David Kopf Instruments, Tujunga, CA, USA) with their body tempera-ture maintained at 37 °C. A microdialysis probe with 2 mmmembranelength (MAB, Microbiotech/se AB, Stockholm, Sweden) was implanted

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in the medial prefrontal cortex (AP: +3.2 mm, Lat: 0.5 mm, depth:4 mm) based on the coordinate of Paxinos and Watson (1986). AKrebs-Ringer solution (pH 7.4) was made up with Milli-Q deionizedwater containing (in mM): NaCl 145, KCl 2.7, CaCl2 1.2, MgCl2 1, andNaH2PO4 2. This solution was passed through the probe at 1 μL/minwith a syringe pump (CMA-100; CMA Microdialysis AB, Kista, Sweden).Samples were collected from the outflow tube of the probe into500-mL Eppendorf Tubes containing 1.5 μL of 10% acetic acid. Dialysatesamples were collected 1 h later, and the sampling was ended 300 minafter the commencement of sampling for a total of 11 dialysate samplescollected for analysis (3 baseline and 8 post-drug samples at 30-min in-tervals). Noradrenaline was quantified with HPLC with an electrochemi-cal detector with a VT-03 cell (Antec, Zoeterwoude, Netherlands). Themobile phase was made up of (in mM) NaH2PO4 100, sodium octanesulfonate 0.74, EDTA 0.02, KCl 2, and 10% methanol. The column wasC18, 3m, 100 mm × 2.1 mm (GRACE). The detection sensitivity ofnoradrenaline was 10–15 pM.

2.6. Western blot

Brain tissues were homogenized and then centrifuged at 16,000 gfor 30 min at 4 °C. The supernatant was denatured by heating at 95 °Cfor 10 min. Proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis in an 8% gel, transferred ontoa polyvinylidene fluoride membrane (EMD Millipore Corporation,Billerica,MA, USA) and immediately blockedwith 5% bovine serumalbu-min (Sigma-Aldrich Co. LLC, St. Louis, MO, USA) at room temperature for1 h. The membranes were incubated overnight at 4 °C with a polyclonalrabbit anti-rat NAT antibody (1:500; EMD Millipore Corporation), anda monoclonal mouse anti-β-actin antibody (1:5000; Sigma-AldrichCo. LLC) was used as the loading control. The membrane was then incu-bated with the corresponding horseradish peroxidase-conjugated goatanti-rabbit IgG (Jackson ImmunoResearch Laboratories, Inc., WestGrove, PA) and goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.,Santa Cruz, CA, USA) secondary antibodies for 1 h at room temperature.Phosphate-buffered saline with Tween 20 was used as the washingagent for each step. After reaction in the ECL solution (GE HealthcareBio-Sciences, Piscataway, NJ, USA), bound antibody was visualized witha chemiluminescence imaging system (Syngene, Cambridge, UK). Theoptical density of each specific band was measured with a computer-assisted imaging analysis system (Gene ToolsMatch software, Syngene).

2.7. Drugs

Zimeldine (Sigma-Aldrich Co. LLC) was dissolved in distilled water.DSP-4 (Sigma-Aldrich Co. LLC) and atomoxetine (Tocris Bioscience,Bristol, UK) were dissolved in 0.9% saline. The drugs were freshly pre-pared prior to their use, and all injections were made i.p. Atomoxetinewas given 40 min before the 5-CSRTT. The dosages for Zimeldine(10 mg/kg) and DSP-4 (50 mg/kg) were based on a previous DNAB le-sion study (Ku et al., 2012). The dosage thatwas chosen for atomoxetinewas 0.3 mg/kg because that dose has been shown to effectively reducepremature responses in similar 5-CSRTT studies (Robinson, 2012).

2.8. Measures and statistics

Accuracy was measured by the percentage of correct responses[correct responses / (correct responses + incorrect responses)]. Thepercentage of omissions was calculated by the formula [(times the ratfailed to respond sufficiently soon after stimulus onset / the total trialnumber) × 100]. ITI responses were scored if the rat responded in anyhole during the ITI. The first was defined as a premature response, andthose that occurred afterward were defined as perseverative responses.The correct latency wasmeasured as the time that elapsed between theoccurrence of the visual stimulus and a nose poke in the correct hole.Collection latency was measured as the time that elapsed between a

nose poke in the correct hole and the retrieval of the reward from themagazine. A multi-way ANOVA was conducted with LESION andDRUG as the between-subject factors. The within-subject factors wereITI length, stimulus duration, and satiety condition (for different5-CSRTT manipulations) and TIME (for dialysis experiments). Furthermultiple comparisons were performed by Student's t-test or Dunnettt-test (for dialysis data), or Tukey's method (for behavioral data).p values less than 0.05 were considered statistically significant.

3. Results

3.1. Effects of DNAB lesions on noradrenaline and NAT levels

TheHPLCmethod confirmed the reduction in noradrenaline levels inthe DNAB (medial prefrontal cortex: 75% and LC: 68%) of the DSP-4-treated rats. Western blotting demonstrated no group differences forNAT levels between the sham control and DSP-4-treated rats (Fig. 1).

3.2. Effects of DNAB lesions and atomoxetine on noradrenaline efflux in themedial prefrontal cortex

Rats with DNAB lesions had equal serotonin efflux to the sham con-trol group in themedial prefrontal cortex [68 and 66 fmol/sample for le-sioned and non-lesioned rats, respectively], indicating the efficacy of theserotonergic protection by Zimeldine. In the baseline condition, le-sioned rats had a lower noradrenaline efflux than the sham controlgroup [df=8, t=4.7, p b 0.001] (left panel, Fig. 2). TIME had an overalleffect in increasing the noradrenaline efflux following the atomoxetineinjection [F(5,40) = 3.52, p b 0.01, by calculating 6 samples after theinjection in order to highlight the duration of the atomoxetine effectsfor the time when the 5-CSRTT was performed]. The atomoxetine-increased noradrenaline efflux was more pronounced in the shamgroup [tD(3,12) = 3.3, p b 0.01, comparisons from sample 3 to 5,i.e., repeated measurements] but not in the DSP-4-treated rats (rightpanel, Fig. 2).

3.3. Effects of DNAB lesions and atomoxetine on 5-CSRTT with different ITIlengths

It was ensured that rats exhibited a similar performance for all thesix variables (i.e., no group difference reaching statistically significantfor correct response, omissions, prematures, perseverations, correct la-tency, and collect latency) before moving to the pharmacological ma-nipulations (i.e., pseudorandomly assigned to Sham/Saline, DSP-4/Saline, Sham/Atomoxetine, or DSP-4/Atomoxetine). For these 4 groups,The behavioral data of the 5-CSRTTwere as the following: for correct re-sponses (%): 82.2±6.1, 79.5±4.8, 84.0±5.2, and 79.8±6.5; for omis-sions (%): 7.5 ± 0.8, 8.9 ± 1.1, 7.1 ± 0.9, and 6.6 ± 0.5; for prematures(count): 22.5 ± 3.1, 24.0 ± 5.7, 21.8 ± 2.9, and 23.6 ± 3.5; for persev-erations (count): 3.1 ± 0.7, 3.4 ± 0.9, 2.8 ± 0.5, and 3.3 ± 0.7; forcorrect latency (ms): 810 ± 62, 783 ± 77, 882 ± 69, and 847 ± 75;for collect latency (ms): 2332 ± 148, 2178 ± 204, 2491 ± 189, and2245 ± 163, for Sham/Saline, DSP-4/Saline, Sham/Atomoxetine, orDSP-4/Atomoxetine, respectively.

For behavioral performance after pharmacological manipulations,all rats performed at a similar level with respect to correct responses,omissions (in percent), correct latency, and collection latency (in ms)with different ITI lengths. There was a significant overall effect of ITIlength (repeated measurements, i.e. 3, 5 and 7 s) on prematuresand perseverations [F(2,56) = 10.1, p b 0.01 and F(2,56) = 24.4,p b 0.001, respectively], and a post-hoc analysis confirmed that theprematures and perseverations at 3, 5, and 7 s were different fromeach other.

For premature responding, atomoxetine had a main effect in re-ducing this variable in the long ITI condition (i.e. 7 s) [F(1,28) = 10.9,p b 0.01] with no interactions with LESION. Further analysis by Tukey

Fig. 1. Western blot analysis of noradrenaline transporter (NAT) of SHAM or DSP-4 treated rats (for each group, N = 4) in the (A) medial prefrontal cortex (mPFC), (B) hippocampus(HIPPO), (C) hypothalamus (HYP) and (D) locus coeruleus (LC). Values are presented as mean + SEM (mPFC medial prefrontal cortex, HIPPO hippocampus, HYP hypothalamus, LClocus coeruleus).

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method revealed that the main effect of atomoxetine was mostly con-tributed by the difference between Sham/Saline and Sham/Atomoxetine. Perseverations were reduced in the long ITI condition byboth DSP-4 and atomoxetine [F(1,28) = 18.8, p b 0.01 and F(1,28) =14.3, p b 0.01, main effects of LESION and DRUG, respectively] with nointeraction between these two factors. Further analysis by Tukey meth-od revealed that these effects were mainly contributed by the differ-ences between Sham/Saline and Sham/Atomoxetine, DSP-4/Saline andSham/Saline, and DSP-4/Saline and DSP-4/Atomoxetine (Fig. 3C and D).

3.4. Effects of DNAB lesions and atomoxetine on 5-CSRTT with a shortenedstimulus duration

A shorter stimulus duration (i.e. 0.5 s) led to a decrease in correct re-sponses [F(1,28)=7.9, p b 0.01] (Fig. 5A) and an increase in prematuresand perseverations [F(1,28) = 14.6, p b 0.001 and F(1,28) = 15.8,p b 0.001, respectively] in all groups (Fig. 4C and D). Omissions, correctlatency and collection latency were not affected by the manipulation ofthe stimulus duration.

Fig. 2. The effects ofDNAB-lesions (A, in arbitrarymagnitude) and atomoxetine (0.3 mg/kg) (B, in percent of baseline values) on extracellular noradrenaline in themedial prefrontal cortex(for each group, N = 5). A total of 11 dialysate samples were collected for analysis (3 baseline and 8 post-drug samples at 30-min intervals). Values are presented as mean + SEM.***p b 0.001, compared with the SHAM group; *p b 0.05, comparison between sample 3 and 5 of the sham group.

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3.5. Effects of DNAB lesions and atomoxetine on 5-CSRTT in satiety

Satiety led to a decrease in correct responses [F(1,28) = 6.6,p b 0.05] (Fig. 5A) and an increase in collection latency [F(1,28) = 8.3,p b 0.01] (Fig. 5F) in all groups. Under satiety conditions, atomoxetineincreased the collection latency in lesioned rats [F(1,28) = 10.1,p b 0.01 and F(1,28) = 5.3, p b 0.05, for LESION × DRUG and a simplemain effect of DRUG, respectively] (Fig. 5F). Omissions, prematures, per-severations, and correct latency were not affected by the manipulationof satiety.

4. Discussion

In the present study, DNAB lesions and atomoxetine treatment weretwo pharmacological manipulations with distinct impacts on the nor-adrenaline system, and they both led to reduced impulsiveness in the5-CSRTT. DNAB lesioning results in a long-lasting weakening of nor-adrenaline transmission, whereas the atomoxetine causes an acuteincrement of synaptic noradrenaline, possibly referring to tonic andphasic control, respectively (Devilbiss and Berridge, 2006; Howellset al., 2012). In the present study, the DSP-4 treated rats exhibited areduced baseline noradrenaline efflux with a sluggish response toatomoxetine, demonstrating that the impaired noradrenergic tonicitymay further affect its phasic reactivity. The mitigation of impulsivenessby atomoxetine that is seen in the 5-CSRTT might be due to drug-induced temporarily disabled utility of the NAT, as it is the key site ofdrug action (Robinson, 2012). This drug-induced effect appears to bepreserved under DNAB-lesioned conditions because atomoxetine trig-gered a similar surge pattern of synaptic noradrenaline in both lesionedand sham-control rats. Given the evidence that NAT function is notaffected substantively by DNAB lesions (in the present study and inKu et al., 2012), the impulsivity-regulating mechanisms underlyingatomoxetine and DSP-4 may operate independently.

DSP-4 results in a long-term decrease of DNAB tonicity (Fritschy andGrzanna, 1991a,1991b), as demonstrated by the reduced baseline nor-adrenaline efflux seen in the medial prefrontal cortex of DNAB rats.This is possibly related to the reduced noradrenaline tonicity of the LC,in which the activation threshold is reset to a lower level (Meffordand Potter, 1989), thus changing the animal's behavior in processing in-coming information. This observation, together with the preserved NATutility, implies two possible interpretations. The first is that the DSP-4did not render an adequate impact on the given areas of the DNAB.Given the facts that the NAT is not the only substrate for DSP-4 [DSP-4exerts its neurotoxicity impacts through, at least, NAT and dopamine βhydroxylase (DBH) (Wang et al., 2014)], we thus preferred the secondpossible interpretation that the reduction of baseline synthesis and

release rather than the reuptake, of noradrenaline, might be more rele-vant to the mechanism responsible for the lower perseverative re-sponses in DNAB-lesioned rats; this is somewhat in contrast to theprevious negative view of DNAB lesions (Mason and Iversen, 1979).Our data also indicated that some apparatus other than the presynapticmechanisms is likely to be involved in the DNAB-lesioned conditions.For example, DSP-4 at the same dose also induces an up-regulation ofpostsynaptic β-adrenergic receptors in the cortex (Dooley et al., 1983;Harro et al., 1999; Theron et al., 1993).

Pre-treatment with DSP-4 has been reported to potentiatemethylphenidate-increased impulsivity, as indexed by an increase inthe number of inspections of holes in a spatial memory performancetest (Sontag et al., 2011). However, the increase of inspection numberis qualitatively different compared to the concept of the prematureresponding thatwas used in the present study, with the latter providinga superior description of how rats prepare for actions and withholdmotor impulsivity while waiting for prompt information. Our DSP-4data generally replicated the findings that rats with DNAB lesionsmade fewer premature and perseverative actions than sham controlswhen the stimuli-waiting period was longer than expectation (Coleand Robbins, 1992). Note that in the present study the ITI was notpresented in a variable and unpredictable manner. Thus, the animals'arousal level remained at a necessary level for operating the automaticorienting response was less effortful (Cole and Robbins, 1992; Schneiderand Shiffrin, 1977), which possibly explained that DSP-4 had only aminor influence on visual discrimination, consistent with the effects oflocal DNAB lesions with 6-OHDA (Carli et al., 1983; Cole and Robbins,1992) or more selectively, with anti-dopamine-beta hydroxylase(DβH)–saporin (Milstein et al., 2007).

In the long-ITI condition, atomoxetine significantly reduced prema-ture and perseverative responses at a dose of 0.3 mg/kg, which wasthe same dose used by Robinson (2012), but it did not lead to a con-comitant increase in omissions, correct latency, or collection latency,indicating that the atomoxetine-improved impulse control was lessconfounded by non-specific effects. In addition, our finding thatthe collection latency was reduced by a solitary effect of DSP-4 oratomoxetine in satiated rats indicates that the eagerness of retrievingfood pellets is also controlled by a surge of synaptic noradrenalineor under low-noradrenaline tonicity, which is possibly related to theclinical evidence that atomoxetine alters appetite in attention-deficitpatients (Durell et al., 2013).

The novelty and significance of the present study were that wedemonstrated that impulsive perseverations in the circumstance oflong-ITI condition, i.e., requiring greater withholding effort or moretop-down regulation (Arnsten, 2011; Bari and Aston-Jones, 2013), arecontrolled by both the integrity of central noradrenergic function and

Fig. 3. Effects of DNAB lesions and atomoxetine treatment in different ITI conditions (3, 5, 7 s) on the performance of the 5-CSRTT in rats (for each group, N= 8) in the aspects of correctresponses (A), omissions (B), prematures (C), perseverations (D), correct latency (E) and collection latency (F). Values are presented asmean+SEM. **p b 0.01, comparedwith the SHAMgroup; #p b 0.05, compared with the SAL control group (ATO atomoxetine, SAL saline).

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noradrenergic reuptake utility. These twomechanisms are, in away, notmutually exclusive because both atomoxetine and DSP-4 lesion tendto reduce the tonicity of LC (Bari and Aston-Jones, 2013; Fritschyand Grzanna, 1991a,1991b) and in the present study the decreased per-severations in DSP-4 rats can be further reduced by atomoxetine. Theperseverative impulsiveness appeared unable to be explained solelyby noradrenaline efflux in the medial prefrontal cortex, as the reducedperseverations occur in lesioned-rats (Cole and Robbins, 1992) with a

lower noradrenaline efflux, yet also occur in atomoxetine rats with asurged noradrenaline efflux. It is possible that, under the conditionthat the DNAB is impaired, a VNAB-relevant compensatory effect mayoccur to boost the atomoxetine-exerted behavioral inhibition. The sim-ilar phenomenon was previously reported in which rats of DNAB lesionreduced their immobility in a forced swim test under the treatmentof another NRI, reboxetine (Ku et al., 2012). On the other hand, com-pensatory changes in cortical postsynaptic receptors might be also

Fig. 4. Effects of DNAB lesions and atomoxetine treatment in different stimulus durations (1, 0.5 s) on the performance of the 5-CSRTT in rats (for each group, N = 8) in the aspects ofcorrect responses (A), omissions (B), prematures (C), perseverations (D), correct latency (E) and collection latency (F). Values are presented asmean+SEM(ATO atomoxetine, SAL saline).

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responsible, given the evidence that noradrenaline depletionmay inducean increase in the binding profile of postsynaptic beta-adrenoceptors(Levin and Biegon, 1984).

In the present study, the occurrence of nose pokes during the ITI didnot terminate the ongoing trial (i.e. without time-out punishment) (Liuet al., 2009, 2011), explaining that the amounts of baseline ITI responseswere greater than those in the time-out punishing context. This may ina way provide a utility against possible bottom effects, and thus helpfulfor examining the drug effects to reduce impulsiveness (rather than

increase impulsiveness). For the same reason that the ITI respondingis unpunished,multiple prematures during the ITI in fact reflect a deficitcombining both premature and perseverative characteristics. By sepa-rating these two variables, our study demonstrated that, while theywere adjusted in the same direction, perseveration decreased more inrats with DNAB lesions or atomoxetine treatment, suggesting that theperseverative activity adjusted rapidly to the change in noradrenalinefunction, and this was similar to that of the perseverative panel pushes(Cole and Robbins, 1992). Note that the occurrence of perseveration

Fig. 5. Effects of DNAB lesions and atomoxetine treatment in different satiety conditions (food restricted versus satiated) on the performance of the 5-CSRTT in rats (for each group, N=8)in the aspects of correct responses (A), omissions (B), prematures (C), perseverations (D), correct latency (E) and collection latency (F). Values are presented as mean + SEM (ATOatomoxetine, SAL saline).

88 Y.-P. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 81–90

was greatly increased in the long-ITI condition as there was more timeavailable for multiple nose pokes. This may also indicate that persever-ation can be activated in an unfamiliar condition in which the expectedstimulus is delayed (Cole and Robbins, 1992).

Inattention and impulsiveness are two independent symptoms inclinical ADHD, as in the rat study of the 5-CSRTT in which the correctand premature responses are not necessarily associated with eachother (Cole and Robbins, 1989; Godoi et al., 2005; Robbins et al.,1998). However, in certain lesioned or challenged conditions, higherpremature responses may occur concurrently with impaired accuracy(Baarendse and Vanderschuren, 2012; Rogers et al., 2001), suggesting

a response vigor that co-occurswith a decline in discriminative accuracyas rats encounter a greater attention workload. This proposal can bepartly supported by the negative relationship between attention andimpulsivity in our study, in which the shortened stimulus duration ledto a drop in correct responses and a concurrent increase in prematureand perseverative actions. This negative relationship was less to dowith DNAB integrity but was manipulation-specific because it wasonly exhibited when changes were made for the stimulus duration,rather than the ITI or the satiety state.

The selectivity of atomoxetine in noradrenaline-related attentionaltasks depends largely on the drug dose and the sensitivity of the test

89Y.-P. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 81–90

(Newman et al., 2008). In the present study, atomoxetine at a doseof 0.3 mg/kg reduced perseverative responses in both lesioned andnon-lesioned rats. Thus, the use of a single dose of atomoxetine limitsthe interpretation of the behavioral and the neurochemical data, partic-ularly that higher doses of atomoxetine (for example, 1 and 3 mg/kg)were reported more effective in reducing the premature responding(Fernando et al., 2012) and producing longer lasting increases of nor-adrenaline efflux in the prefrontal cortex (Bymaster et al., 2002;Swanson et al., 2006). Future studies of DNAB effects on the perfor-mance of the 5-CSRTT following long-term and multiple doses ofatomoxetine are suggested. Another limitation is that the behavioraleffects in the present study cannot be explained by noradrenalineonly, and, at least, the involvement of dopamine should be considered,as atomoxetine at a dose of 0.3 mg/kg increases the extracellularconcentrations of both noradrenaline and dopamine in the prefrontalcortex (Bymaster et al., 2002),where these two systems share their pro-jection terminals (Devoto et al., 2005; Gresch et al., 1995).

In conclusion, the present study suggests that the impulsivity-regulating mechanisms underlying atomoxetine and DSP-4 may oper-ate independently. As the noradrenaline reuptakemaynot be exclusive-ly responsible for the atomoxetine effects in adjusting impulsivity,the role of DNAB should also be considered, particularly in conditionsrequiring greater behavioral inhibition.

Acknowledgments

This study was supported by grants from the National DefenseMedical Center (I-20 and MAB101-39) at Taipei, Taiwan. The experi-ments of the present study complied with the current laws of Taiwan.

References

Arnsten AF. Catecholamine influences on dorsolateral prefrontal cortical networks. BiolPsychiatry 2011;69(12):89–99.

Aston-Jones G, Delfs JM, Druhan J, Zhu Y. The bed nucleus of the stria terminalis. A targetsite for noradrenergic actions in opiate withdrawal. Ann N Y Acad Sci 1999;877:486–98.

Baarendse PJ, Vanderschuren LJ. Dissociable effects of monoamine reuptake inhibitors ondistinct forms of impulsive behaviour in rats. Psychopharmacology (Berl) 2012;219(2):313–26.

Bari A, Aston-JonesG. Atomoxetinemodulates spontaneous and sensory-evoked dischargeof locus coeruleus noradrenergic neurons. Neuropharmacology 2013;64:53–64.

Bymaster FP, Katner JS, Nelson DL, Hemrick-Luecke SK, Threlkeld PG, Heiligenstein JH,et al. Atomoxetine increases extracellular levels of norepinephrine and dopaminein prefrontal cortex of rat: a potential mechanism for efficacy in attention deficit/hyperactivity disorder. Neuropsychopharmacology 2002;27:699–711.

Carboni E, Silvagni A. Dopamine reuptake by norepinephrine neurons: exception or rule?Crit Rev Neurobiol 2004;16(1–2):121–8.

Carli M, Robbins TW, Evenden JL, Everitt BJ. Effects of lesions to ascending noradrenergicneurones on performance of a 5-choice serial reaction task in rats; implications fortheories of dorsal noradrenergic bundle function based on selective attention andarousal. Behav Brain Res 1983;9(3):361–80.

Cole BJ, Robbins TW. Effects of 6-hydroxydopamine lesions of the nucleus accumbenssepti on performance of a 5-choice serial reaction time task in rats: implications fortheories of selective attention and arousal. Behav Brain Res 1989;33:165–79.

Cole BJ, Robbins TW. Forebrain norepinephrine: role in controlled information processingin the rat. Neuropsychopharmacology 1992;7(2):129–42.

Coull JT. Pharmacological manipulations of the alpha 2-noradrenergic system. Effects oncognition. Drugs Aging 1994;5(2):116–26.

Cryan JF, Page ME, Lucki I. Noradrenergic lesions differentially alter the antidepressant-like effects of reboxetine in a modified forced swim test. Eur J Pharmacol 2002;436(3):197–205.

Dahlstroem A, Fuxe K. Evidence for the existence of monoamine neurons in the centralnervous system: I. Demonstration of monoamines in the cell bodies of brainstemneurons. Acta Physiol Scand Suppl 1964;232:1–55.

Devilbiss DM, Berridge CW. Low-dose methylphenidate actions on tonic and phasic locuscoeruleus discharge. J Pharmacol Exp Ther 2006;319(3):1327–35.

Devoto P, Flore G, Saba P, Fa M, Gessa GL. Stimulation of the locus coeruleus elicitsnoradrenaline and dopamine release in the medial prefrontal and parietal cortex.J Neurochem 2005;92:368–74.

Ding YS, Naganawa M, Gallezot JD, Nabulsi N, Lin SF, Ropchan J, et al. Clinical dosesof atomoxetine significantly occupy both norepinephrine and serotonin transports:implications on treatment of depression and ADHD. Neuroimage 2014;86:164–71.

Dooley DJ, Bittiger H, Hauser KL, Bischoff SF, Waldmeier PC. Alteration of central alpha2- and beta-adrenergic receptors in the rat after DSP-4, a selective noradrenergicneurotoxin. Neuroscience 1983;9(4):889–98.

Durell TM, Adler LA, Williams DW, Deldar A, McGough JJ, Glaser PE, et al. Atomoxetinetreatment of attention-deficit/hyperactivity disorder in young adults with assess-ment of functional outcomes: a randomized, double-blind, placebo-controlled clinicaltrial. J Clin Psychopharmacol 2013;33(1):45–54.

Fernando AB, Economidou D, Theobald DE, Zou MF, Newman AH, Spoelder M, et al.Modulation of high impulsivity and attentional performance in rats by selectivedirect and indirect dopaminergic and noradrenergic receptor agonists. Psychophar-macology (Berl) 2012;219(2):341–52.

Fritschy JM, Grzanna R. Experimentally-induced neuron loss in the locus coeruleus ofadult rats. Exp Neurol 1991a;111(1):123–7.

Fritschy JM, Grzanna R. Selective effects of DSP-4 on locus coeruleus axons: are therepharmacologically different types of noradrenergic axons in the central nervoussystem? Prog Brain Res 1991b;88:257–68.

Gilger JW, Kaplan BJ. Atypical brain development: a conceptual framework forunderstanding developmental learning disabilities. Dev Neuropsychol 2001;20(2):465–81.

Godoi FR, Oliveira MG, Tufik S. Effects of paradoxical sleep deprivation on the perfor-mance of rats in a model of visual attention. Behav Brain Res 2005;165:138–45.

Gresch PJ, Sved AF, ZigmondMJ, Finlay JM. Local influence of endogenous norepinephrineon extracellular dopamine in rat medial prefrontal cortex. J Neurochem 1995;65:111–6.

Harro J, Pähkla R, Modiri A-R, Harro M, Kask A, Oreland L. Dose-dependent effects ofnoradrenergic denervation by DSP-4 treatment on forced swimming and beta-adrenoceptor binding in the rat. J Neural Transm 1999;106(7–8):619–29.

Heal DJ, Butler SA, Prow MR, Buckett WR. Quantification of presynaptic alpha 2-adrenoceptors in rat brain after short-term DSP-4 lesioning. Eur J Pharmacol 1993;249:37–41.

Howells FM, Stein DJ, Russell VA. Synergistic tonic and phasic activity of the locuscoeruleus norepinephrine (LC-NE) arousal system is required for optimal attentionalperformance. Metab Brain Dis 2012;27(3):267–74.

Jonsson G, Hallman H, Ponzio F, Ross S. DSP4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine)—a useful denervation tool for central and peripheral noradren-aline neurons. Eur J Pharmacol 1981;72(2–3):173–88.

Kostowski W, Jerlicz M, Bidzin' ski A, Hauptmann M. Evidence for existence of twoopposite noradrenergic brain systems controlling behavior. Psychopharmacology(Berl) 1978;59:11–312.

Ku YC, Tsai YJ, Tung CS, Fang TH, Lo SM, Liu YP. Different involvement of ventraland dorsal norepinephrine pathways on norepinephrine reuptake inhibitor-inducedlocomotion and antidepressant-like effects in rats. Neurosci Lett 2012;514(2):179–84.

Levin BE, Biegon A. Reserpine and the role of axonal transport in the independent regula-tion of pre- and postsynaptic beta-adrenoreceptors. Brain Res 1984;311(1):39–50.

Liu YP, Lin YL, Chuang CH, Kao YC, Chang ST, Tung CS. Alpha adrenergic modulationon effects of norepinephrine transporter inhibitor reboxetine in five-choice serial re-action time task. J Biomed Sci 2009;16:72–83.

Liu YP, Tung CS, Lin YL, Chuang CH. Wake-promoting agent modafinil worsenedattentional performance following REM sleep deprivation in a young-adult ratmodel of 5-choice serial reaction time task. Psychopharmacology (Berl) 2011;213:155–66.

Lu CC, Tseng CJ, Wan FJ, Yin TH, Tung CS. Role of locus coeruleus and serotonergic drugactions on schedule-induced polydipsia. Pharmacol Biochem Behav 1992;43(1):255–61.

Ludolph AG, Udvardi PT, Schaz U, Henes C, Adolph O,Weigt HU, et al. Atomoxetine acts asan NMDA receptor blocker in clinically relevant concentrations. Br J Pharmacol 2010;160(2):283–91.

Mason ST, Iversen SD. Theories of the dorsal bundle extinction effect. Brain Res 1979;180(1):107–37.

Mefford IN, Potter WZ. A neuroanatomical and biochemical basis for attention deficit dis-order with hyperactivity in children: a defect in tonic adrenaline mediated inhibitionof locus coeruleus stimulation. Med Hypotheses 1989;29(1):33–42.

Milstein JA, Lehmann O, Theobald DE, Dalley JW, Robbins TW. Selective depletion of cor-tical noradrenaline by anti-dopamine beta-hydroxylase-saporin impairs attentionalfunction and enhances the effects of guanfacine in the rat. Psychopharmacology(Berl) 2007;190(1):51–63.

Moore RY, Card JP. Noradrenaline-containing neuron systems. In: Bjorklund A, Hokfelt T,editors. Handbook of chemical neuroanatomy. Classical Transmitters in theCNSAmsterdam: Elsevier; 1984. p. 123–56.

Newman LA, Darling J, McGaughy J. Atomoxetine reverses attentional deficits producedby noradrenergic deafferentation of medial prefrontal cortex. Psychopharmacology(Berl) 2008;200(1):39–50.

Paxinos G,Watson C. The rat brain in stereotaxic coordinates. 2nd ed. NewYork: AcademicPress; 1986.

Robbins TW, Granon S, Muir JL, Durantou F, Harrison A, Everitt BJ. Neural systems under-lying arousal and attention. Implications for drug abuse. Ann N Y Acad Sci 1998;846:222–37.

Robinson ES. Blockade of noradrenaline re-uptake sites improves accuracy and impulsecontrol in rats performingafive-choice serial reaction time tasks. Psychopharmacology(Berl) 2012;219(2):303–12.

Robinson ES, Eagle DM, Mar AC, Bari A, Banerjee G, Jiang X, et al. Similar effects of theselective noradrenaline reuptake inhibitor atomoxetine on three distinct forms ofimpulsivity in the rat. Neuropsychopharmacology 2008;33(5):1028–37.

Rogers RD, Baunez C, Everitt BJ, Robbins TW. Lesions of the medial and lateral striatum inthe rat produce differential deficits in attentional performance. Behav Neurosci 2001;115(4):799–811.

Schneider W, Shiffrin RM. Controlled and automatic information processing: 1. Detection,search and attention. Psychol Rev 1977;84:1–66.

90 Y.-P. Liu et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 56 (2015) 81–90

Sontag TA, Hauser J, Kaunzinger I, Gerlach M, Tucha O, Lange KW. Effects of the noradren-ergic neurotoxin DSP4 on spatial memory in the rat. J Neural Transm 2008;115(2):299–303.

Sontag TA, Hauser J, Tucha O, Lange KW. Effects of DSP4 and methylphenidate on spatialmemory performance in rats. Atten Defic Hyper Disord 2011;3(4):351–8.

Swanson CJ, Perry KW, Koch-Krueger S, Katner J, Svensson KA, Bymaster FP. Effect ofthe attention deficit/hyperactivity disorder drug atomoxetine on extracellularconcentrations of norepinephrine and dopamine in several brain regions of the rat.Neuropharmacology 2006;50(6):755–60.

Theron CN, de Villiers AS, Taljaard JJ. Effects of DSP-4 on monoamine and monoaminemetabolite levels and on beta adrenoceptor binding kinetics in rat brain at differenttimes after administration. Neurochem Res 1993;18(12):1321–7.

Wang Y, Musich PR, SerranoMA, Zou Y, Zhang J, ZhuMY. Effects of DSP4 on the noradren-ergic phenotypes and its potential molecular mechanisms in SH-SY5Y cells. NeurotoxRes 2014;25(2):193–207.

Wietecha LA,Williams DW, Herbert M,Melmed RD, GreenbaumM, Schuh K. Atomoxetinetreatment in adolescentswith attention-deficit/hyperactivity disorder. J Child AdolescPsychopharmacol 2009;19(6):719–30.