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Elevated locomotor activity without altered striatal dopamine contents in Nurr1 heterozygous mice after acute exposure to methamphetamine

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Elevated locomotor activity without altered striatal dopamine contents in Nurr1 heterozygous mice after acute exposure to methamphetamine
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  Behavioural Brain Research 143 (2003) 95–100 Research report Elevated locomotor activity without altered striatal dopamine contents inNurr1 heterozygous mice after acute exposure to methamphetamine Cristina Bäckman a , ∗ , Zhi-Bing You b , Thomas Perlmann c , Barry J. Hoffer a a Cellular Neurobiology Branch, National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, MD 21221, USA b  Behavioral Neuroscience Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA c  Department of Cell and Molecular Biology, Ludwig Institute for Cancer Research, Karolinska Institute, Box 240, Stockholm S-171 77, Sweden Received 18 November 2002; accepted 8 January 2003 Abstract Genetargetingexperiments,inwhichbothallelesoftheNurr1geneweredeleted,haveshownthatthismoleculeplaysanessentialroleinthedevelopmentofmidbraindopaminergicneurons,asshownbythelossofdopaminergicmarkersandtheneurotransmitterdopamine(DA)intheventralmesencephalonofNurr1nullmutantmice.Nurr1-deficientmicediewithinafewhoursofbirth.Herein,weinvestigatedwhetheradult mice (12–15-month-old), heterozygous for the Nurr1 mutation (  Nurr1 + / − ), show alterations in locomotor function and in the nigros-triataldopaminergicsystemafteracuteexposuretomethamphetamine.Wefirstevaluatedspontaneousandamphetamine-induced(5mg/kg)locomotor response of >12-month-old wildtype (  Nurr1 + / + ) and  Nurr1 + / − mice. Both, spontaneous and methamphetamine-induced lo-comotor behavior was significantly increased in the  Nurr1 + / − animals as compared to  Nurr1 + / + mice. Striatal DA and DA metabolitelevels were measured in untreated animals and methamphetamine-treated animals. No significant differences in striatal dopamine levels orits metabolites DOPAC and HVA were found in the  Nurr1 + / − as compared to  Nurr1 + / + mice in untreated or methamphetamine-treatedanimals. These data show that deletion of a single allele of the Nurr1 gene alters the locomotor activity of 12–15-month-old  Nurr1 + / − animals. While total dopamine levels were not altered in the striatum of   Nurr1 + / − mice, future studies will be necessary to determine if processes involved with the dynamics of DA release/clearance within the nigrostriatal system may be altered in  Nurr1 + / − mutant mice.© 2003 Elsevier Science B.V. All rights reserved. Keywords:  Nurr1; Dopamine; Methamphetamine; Behavior 1. Introduction Nurr1 is a member of the nuclear hormone receptor fam-ily of ligand-inducible transcription factors. The murine or-phan nuclear receptor, Nurr1, is predominately expressed inthe CNS in limbic areas and the ventral midbrain, includingdopamine (DA) neurons [1,14,15,17]. Nurr1 expression inthe ventral midbrain occurs on embryonic day 10.5 (E10.5), just before the appearance of the dopaminergic marker en-zyme tyrosine hydroxylase (TH) at E11.5. [4,13,16]. Thisobservation led to the hypothesis that Nurr1 plays a rolein the development of midbrain dopaminergic cells. Also,as Nurr1 expression continues during adulthood, this tran-scription factor may also be required for normal functionof the mature dopaminergic system. To determine the phys-iological function of Nurr1, knockout mice were generated ∗ Corresponding author. Tel.:  + 1-410-550-6870x19.  E-mail address:  cbackman@intra.nida.nih.gov (C. Bäckman). by homologous recombination [4,13,16]. The phenotype of these mice demonstrated that Nurr1 plays an essential rolefor the generation of DA neurons. Moreover, analysis of homozygous Nurr1 (  Nurr1 − / − ) brain extracts revealed theloss of the neurotransmitter DA. This was consistent withabsent TH,  l -aromatic amino acid decarboxylase, and otherDA neuron phenotypic markers in the ventral midbrain.  Nurr1 − / − mutant mice can be distinguished after birth byhypoactivity and the lack of milk in their stomachs, anddie within hours of birth.  Nurr1 + / − mice have moderatelylower levels of Nurr1 protein than  Nurr1 + / + mice [9],which is consistent with Nurr1 mRNA expression [13]. The newborn  Nurr1 + / − mice have decreased TH expression inSN and ventral tegmental area and significantly lower levelsof DA in striatum [16]. Zetterström et al. [16] also reported that adult heterozygotes display apparently no histologicaland behavioral abnormalites despite reduced striatal DAlevels. In contrast, Le et al. [9] f ound that striatal DA levels in young adult  Nurr1 + / − mice are not significantly differentfrom  Nurr1 + / + mice. 0166-4328/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved.doi:10.1016/S0166-4328(03)00029-9  96  C. Bäckman et al./Behavioural Brain Research 143 (2003) 95–100 An increasing body of evidence implicates the transcrip-tion factor Nurr1 as an important regulator of dopaminefunction in the nigrostriatal system in the adult animal. Arecent study has shown that Nurr1 and dopamine trans-porter (DAT) gene expression are markedly reduced in theventral mesensephalon of human cocaine abusers [2], sug- gesting that Nurr1 may play a role in vivo in adaptationto repeated exposure to cocaine. Another study has shownparallel decreases in Nurr1- and TH-immunofluorescenceas a function of age, indicating that age-related decline of DA phenotypic markers is associated with down-regulationof Nurr1 expression in the SN [5]. Le et al. [9] reported that reduction of Nurr1 expression in  Nurr1 + / − mice, confersan increased vulnerability to the selective dopaminergicneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP) when compared to  Nurr1 + / + mice. Nurr1 hasalso been shown to enhance the transcriptional activity of human dopamine transporter gene constructs transientlytransfected into a cell line that expresses a dopaminergicphenotype [11]. In gain-of-function experiments, Sakurada et al. [12] have demonstrated that Nurr1 is able to activate transcription of the tyrosine hydroxylase gene by binding aresponse element within a region of the tyrosine hydroxy-lase promoter necessary for midbrain-specific expression.Given the previously demonstrated effects of Nurr1 inadult and aged midbrain dopaminergic neurons, we wereinterested in examining if nigrostriatal dopaminergic func-tion could be altered in >12-month-old heterozygous micewith the deletion of a single Nurr1 gene. To answer thesequestions, wildtype controls and heterozygous Nurr1 mutantmice at 12–15 months of age were subjected to a behav-ioral test which provides an index of nigrostriatal dopamin-ergic function; spontaneous and methamphetamine-induced(5mg/kg) locomotor activity. In addition, striatal DA andmetabolite concentrations were measured under basal con-ditions and in mice receiving methamphetamine. 2. Materials and methods 2.1. Animals Nurr1 null mutant mice were obtained from Dr. ThomasPerlmann at the Karolinska Institute, Sweden. The genera-tion of Nurr1 null mutant mice has been described in detailelsewhere [16]. Mutant mice were generated from >10 gen- erations of backcrosses onto a C57BL6 inbred mouse strain.  Nurr1 + / − mice were mated to produce the  Nurr1 + / + and  Nurr1 + / − offspring used in the present study. Mice weregenotyped by means of polymerase chain reactions usinggene specific primers.Aged male animals (12–15-month-old) were kept ingroups of three to four in plastic cages in the same roomand maintained on a 12h/12h light/dark cycle with foodand water available ad libitum. All animal procedures wereapproved by the NIDA Animal Care and Use Committee. 2.2. Locomotor activity To assess spontaneous locomotor activity in a novelenvironment, mice were placed in an Omnitech AnimalActivity Monitor for a 30-min period (day 1). To assessmethamphetamine-stimulated locomotor activity, metham-phetamine (5mg/kg) was injected intraperitoneally imme-diately prior to placement of the animal within the activitymonitor chamber for a 60-min test period. All the animalstreated with methamphetamine received daily intraperi-toneal (i.p.) saline injections and were introduced to the ac-tivity monitor chambers for 3 days prior to drug treatment tominimize responses induced by exposure to a novel environ-ment, handling and/or pain induced by the injection. In thepresent study, different types of behaviors are reported: totaldistance, horizontal activity, vertical activity, and stereotypicmovements. The total scores obtained over the entire testingperiod were used in the analyses of spontaneous activity.Scores obtained during the methamphetamine-stimulatedtest are presented as two blocks of 30min each. 2.3. Quantification of dopamine (DA),3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) Methamphetamine-treated animals were sacrificed 2hafter drug treatment and brain tissue from untreated andmethamphetamine-treated animals was extracted to analyzestriatal dopamine and dopamine metabolite contents byHPLC. Methamphetamine-treated animals were sacrificed2h after drug treatment. Strital tissue was analyzed for con-tent of DA, and its metabolites DOPAC and HVA. After eu-thanasia by CO 2  saturation, the brain was removed, placedin a mouse brain coronal matrix immersed in an ice-coldsaline solution and cut into 1mm sections. Bilateral punchesfrom the striatum were collected from one 1-mm sectionbetween bregma 1.60 and 0.22. Samples were immediatelyfrozen on dry ice and stored at − 80 ◦ C until processed.Each tissue sample was homogenized with 0.01M per-chloric acid using a glass homogenizer. Immediately fol-lowing the homogenization, the samples were transferred toEppendof tubes and place at 4 ◦ C for 2h to allow sufficientextraction, and then centrifuged for 20min at 20,000 rpm.The supernatants were then taken out from the Eppendof tubes and place in a refrigerated autosampler (CMA 280).DA and its metabolites, DOPAC and HVA, were measuredwith an HPLC coupled to an ESA Coulochem II Detec-tor (model 5200) with a dual-electrode microdialysis cell,and an ESA model 501 data station (ESA, Inc., Chelms-ford, MA). Sample injection onto the column (3  m particlesize, 3mm × 150mm, Analytical MD-150, ESA, Inc.) wasmade automatically through remote control of the autosam-pler. The mobile phase for catecholamine separation consistsof 75mM NaH 2 PO 4 , 1.5mM OSA, 10  M EDTA, and 7%acetonitrile (pH 3.0 adjusted with H 3 PO 4 ). Dopamine andits metabolites were quantified on both reducing ( − 250mV)  C. Bäckman et al./Behavioural Brain Research 143 (2003) 95–100  97 and oxidizing electrodes (350mV) and then calculated aspmole/mg wet tissue. 2.4. Statistical analysis The data given represent mean  ± S . E . M .  values. Bodyweights and spontaneous behavior scores were comparedbetween  Nurr1 + / + and  Nurr1 + / − mice using an unpaired t  -test. For group analysis and statistical comparison forbehavior scores after methamphetamine treatment andDA/metabolite concentration determinations, an ordinaryANOVA with Fisher’s a posteriori analysis was used. Forall analyses, the threshold for statistical significance was P <  0 . 05. 3. Results 3.1. Body weights Nurr1 + / − mice developed normally with no obvious be-havioral abnormalities. The body weights (mean in grams ± S . E . M . ) of the  Nurr1 + / − (48 . 3 ± 1 . 06) mice were not signif-icantly different from those of the  Nurr1 + / + mice (49 . 9 ± 0 . 89). 3.2. Locomotor activity Spontaneous and amphetamine-induced locomotor activ-ity was assessed in  Nurr1 + / + ( n  =  33) versus  Nurr1 + / − ( n  =  43) mice by using Omnitech activity chambers.  Nurr1 + / + and  Nurr1 + / − mice exhibited significant dif-ferences in spontaneous and methamphetamine-induced(5mg/kg) behaviors (Fig. 1). Horizontal activity and to- tal distance scores were significantly higher in  Nurr1 + / − mice as compared to  Nurr1 + / + during exposure to anovel environment (Fig. 1). Significant differences between  Nurr1 + / + versus  Nurr1 + / − mice were observed for allmethamphetamine-induced measured activities: horizon-tal activity, total distance, vertical activity, and stereotypicactivity. Methamphetamine injection led to a dramatic in-crease in locomotion in both  Nurr1 + / + and  Nurr1 + / − mice(Figs. 1 and 2). The level of locomotor activation was signif-icantly higher in  Nurr1 + / − mice as compared to  Nurr1 + / + (Fig. 2). The proportion of   Nurr1 + / − mice showing scoresgreater than their respective  Nurr1 + / + controls were con-sistently higher during both testing periods (1–30min ver-sus 30–60min) for horizontal activity and total distance.Vertical and stereotypic activity scores were significantlyincreased in the  Nurr1 + / − group during the second testingperiod (30–60min), as compared to wildtypes. 3.3. DA, DOPAC, and HVA levels in the striatum of  Nurr1 + / + versus Nurr1 + / − mice Postmortem tissue concentrations of DA and its metabo-lites DOPAC and HVA were measured in the striatum of  Fig. 1. Exposure to a novel environment increases spontaneous activityin  Nurr1 + / − animals as compared to  Nurr1 + / + . Animals were tested for30min after being placed for the first time in an Omnitech chamber.  Nurr1 + / − animals showed a significant increase in (A) horizontal activity( t  -test;  ∗∗ P <  0 . 01) and (B) total distance traveled ( t  -test;  ∗∗∗ P <  0 . 001).There was no difference between the groups in (C) vertical activity scores( t  -test; NS, not significant).  98  C. Bäckman et al./Behavioural Brain Research 143 (2003) 95–100 Fig. 2. Effects of acute methamphetamine (5mg/kg) on horizontal activity (A), total distance (B), vertical activity (C), and stereotypic activity (D), asassessed in an Omnitech Activity Monitor. Motor activity was examined directly after methamphetamine exposure, and is presented in two blocks of 30mineach. Methamphetamine stimulation significantly increased all behaviors in the  Nurr1 + / − animals as compared to  Nurr1 + / + (ordinary ANOVA + Fisher’sa posteriori test;  ∗∗ P <  0 . 01,  ∗∗∗ P <  0 . 001, NS, not significant).  Nurr1 + / + and  Nurr1 + / − untreated mice ( n  =  11 and 14,respectively) and 2h after methamphetamine exposure ( n = 10 and 21, respectively; Fig. 3). DA and HVA levels were increased in both heterozygous and wildtype mice 2h aftermethamphetamine exposure (Fig. 3). DOPAC levels were decreased after methamphetamine exposure (Fig. 3). How- ever, no significant differences in DA, DOPAC, or HVA lev-els were found between  Nurr1 + / + and  Nurr1 + / − animalsduring control conditions or after methamphetamine treat-ment (Fig. 3). 4. Discussion The implication of Nurr1 as a transcription factorthat regulates nigrostriatal DA function together withthe observation that  Nurr1 + / − neonatal and young adultmice exhibit deficits in striatal dopamine content or ni-grostriatal dopamine functions suggest that this neuro-transmitter system may be further compromised in aged  Nurr1 + / − mice. Accordingly, the question of whether lo-comotor performance (related to nigrostriatal function)and its correlation with striatal DA levels, is affected in>12-month-old  Nurr1 + / − , was the aim of this study. Ourpresent data indicates that  Nurr1 + / − mice exhibit a sig-nificant increase in horizontal activity and total distancetraveled recorded during spontaneous behavior. This be-havioral difference was augmented when the  Nurr1 + / − mice were given a single injection of methamphetamine(5mg/kg). Methamphetamine-treated  Nurr1 + / − mice ex-hibited a significant increase in horizontal and verticalactivity, total distance traveled, and stereotypic activityas compared to  Nurr1 + / + controls. Interestingly, analysisof dopamine, DOPAC, and HVA contents in the striatumrevealed no alterations in  Nurr1 + / − mice suggesting thataugmentation of locomotor activity in these animals is not  C. Bäckman et al./Behavioural Brain Research 143 (2003) 95–100  99Fig. 3. Striatal dopamine (DA) and dopamine metabolites, DOPAC and HVA, concentrations for untreated and methamphetamine-treated (5mg/kg)  Nurr1 + / − and  Nurr1 + / + mice. No significant differences were observed between the groups for either DA, DOPAC, or HVA levels in the untreated ormethamphetamine-treated groups (ordinary ANOVA + Fisher’s a posteriori test; NS, not significant). associated with whole tissue contents of DA and metabolitelevels.The mechanism by which deletion of a Nurr1 alleleresults in an increase in locomotor behavior after acute ex-posure to methamphetamine is not immediately apparent.Methamphetamine treatment increases extracellular con-centrations of DA in the striatum, in part, by reversing thefunction of the dopamine transporter and facilitating cyto-plasmic DA release, as well as by releasing vesicular storesof dopamine [6,10]. Most parameters involving the nigros- triatal system, including DA content, tyrosine hydroxylase,and DA uptake have all been shown to be non-functional af-ter Nurr1 ablation [4,13,16]. The observation made here that  Nurr1 + / − mice respond to methamphetamine with a sig-nificant increase in locomotor behavior suggests that trans-mission within the nigrostriatal dopaminergic pathway hasbeen altered. However, DA and DA metabolite levels mea-sured in the striatum of 12–15-month-old either untreatedor methamphetamine-treated  Nurr1 + / − animals were notaltered as compared to  Nurr1 + / + . Since reduced DA levelshave been reported in newborn heterozygous animals andyoung adults [16], compensatory mechanisms may have de- veloped in aged  Nurr1 + / − animals to maintain normal lev-els of DA in the striatum. Compensatory mechanisms in thedopaminergic system of aging animals have previously beendescribed by Friedemann and Gerhardt [7]. In this study, aged Fischer 344 rats did not show age-related differencesin striatal DA levels, DA metabolite levels, and turnover in-dices as compared to young animals. However, DA overflowand clearance rates were decreased in aged animals. Inter-estingly, our preliminary in vivo electrochemistry results(data not published) suggest that the dynamics of dopaminerelease/uptake may be affected in  Nurr1 + / − animals. Corre-lated with this, recent biochemical studies have shown that
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