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Elevated locomotor activity without altered striatal dopamine contents in Nurr1 heterozygous mice after acute exposure to methamphetamine
Behavioural Brain Research 143 (2003) 95–100
Research report
Elevated locomotor activity without altered striatal dopamine contents in Nurr1 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 Behavioral Neuroscience Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA Department of Cell and Molecular Biology, Ludwig Institute for Cancer Research, Karolinska Institute, Box 240, Stockholm S-171 77, Sweden b
c
Received 18 November 2002; accepted 8 January 2003
Abstract Gene targeting experiments, in which both alleles of the Nurr1 gene were deleted, have shown that this molecule plays an essential role in the development of midbrain dopaminergic neurons, as shown by the loss of dopaminergic markers and the neurotransmitter dopamine (DA) in the ventral mesencephalon of Nurr1 null mutant mice. Nurr1-deficient mice die within a few hours of birth. Herein, we investigated whether adult mice (12–15-month-old), heterozygous for the Nurr1 mutation (Nurr1+/− ), show alterations in locomotor function and in the nigrostriatal dopaminergic system after acute exposure to methamphetamine. We first evaluated spontaneous and amphetamine-induced (5 mg/kg) locomotor response of >12-month-old wildtype (Nurr1+/+ ) and Nurr1+/− mice. Both, spontaneous and methamphetamine-induced locomotor behavior was significantly increased in the Nurr1+/− animals as compared to Nurr1+/+ mice. Striatal DA and DA metabolite levels were measured in untreated animals and methamphetamine-treated animals. No significant differences in striatal dopamine levels or its metabolites DOPAC and HVA were found in the Nurr1+/− as compared to Nurr1+/+ mice in untreated or methamphetamine-treated animals. 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 family of ligand-inducible transcription factors. The murine orphan nuclear receptor, Nurr1, is predominately expressed in the CNS in limbic areas and the ventral midbrain, including dopamine (DA) neurons [1,14,15,17]. Nurr1 expression in the ventral midbrain occurs on embryonic day 10.5 (E10.5), just before the appearance of the dopaminergic marker enzyme tyrosine hydroxylase (TH) at E11.5. [4,13,16]. This observation led to the hypothesis that Nurr1 plays a role in the development of midbrain dopaminergic cells. Also, as Nurr1 expression continues during adulthood, this transcription factor may also be required for normal function of the mature dopaminergic system. To determine the physiological function of Nurr1, knockout mice were generated
∗
Corresponding author. Tel.: +1-410-550-6870x19. E-mail address: [email protected] (C. Bäckman).
by homologous recombination [4,13,16]. The phenotype of these mice demonstrated that Nurr1 plays an essential role for the generation of DA neurons. Moreover, analysis of homozygous Nurr1 (Nurr1−/− ) brain extracts revealed the loss of the neurotransmitter DA. This was consistent with absent TH, l-aromatic amino acid decarboxylase, and other DA neuron phenotypic markers in the ventral midbrain. Nurr1−/− mutant mice can be distinguished after birth by hypoactivity and the lack of milk in their stomachs, and die within hours of birth. Nurr1+/− mice have moderately lower levels of Nurr1 protein than Nurr1+/+ mice [9], which is consistent with Nurr1 mRNA expression [13]. The newborn Nurr1+/− mice have decreased TH expression in SN and ventral tegmental area and significantly lower levels of DA in striatum [16]. Zetterström et al. [16] also reported that adult heterozygotes display apparently no histological and behavioral abnormalites despite reduced striatal DA levels. In contrast, Le et al. [9] found that striatal DA levels in young adult Nurr1+/− mice are not significantly different from Nurr1+/+ mice.
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An increasing body of evidence implicates the transcription factor Nurr1 as an important regulator of dopamine function in the nigrostriatal system in the adult animal. A recent study has shown that Nurr1 and dopamine transporter (DAT) gene expression are markedly reduced in the ventral mesensephalon of human cocaine abusers [2], suggesting that Nurr1 may play a role in vivo in adaptation to repeated exposure to cocaine. Another study has shown parallel decreases in Nurr1- and TH-immunofluorescence as a function of age, indicating that age-related decline of DA phenotypic markers is associated with down-regulation of Nurr1 expression in the SN [5]. Le et al. [9] reported that reduction of Nurr1 expression in Nurr1+/− mice, confers an increased vulnerability to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) when compared to Nurr1+/+ mice. Nurr1 has also been shown to enhance the transcriptional activity of human dopamine transporter gene constructs transiently transfected into a cell line that expresses a dopaminergic phenotype [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 a response element within a region of the tyrosine hydroxylase promoter necessary for midbrain-specific expression. Given the previously demonstrated effects of Nurr1 in adult and aged midbrain dopaminergic neurons, we were interested in examining if nigrostriatal dopaminergic function could be altered in >12-month-old heterozygous mice with the deletion of a single Nurr1 gene. To answer these questions, wildtype controls and heterozygous Nurr1 mutant mice at 12–15 months of age were subjected to a behavioral test which provides an index of nigrostriatal dopaminergic function; spontaneous and methamphetamine-induced (5 mg/kg) locomotor activity. In addition, striatal DA and metabolite concentrations were measured under basal conditions and in mice receiving methamphetamine.
2. Materials and methods 2.1. Animals Nurr1 null mutant mice were obtained from Dr. Thomas Perlmann at the Karolinska Institute, Sweden. The generation of Nurr1 null mutant mice has been described in detail elsewhere [16]. Mutant mice were generated from >10 generations 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 were genotyped by means of polymerase chain reactions using gene specific primers. Aged male animals (12–15-month-old) were kept in groups of three to four in plastic cages in the same room and maintained on a 12 h/12 h light/dark cycle with food and water available ad libitum. All animal procedures were approved by the NIDA Animal Care and Use Committee.
2.2. Locomotor activity To assess spontaneous locomotor activity in a novel environment, mice were placed in an Omnitech Animal Activity Monitor for a 30-min period (day 1). To assess methamphetamine-stimulated locomotor activity, methamphetamine (5 mg/kg) was injected intraperitoneally immediately prior to placement of the animal within the activity monitor chamber for a 60-min test period. All the animals treated with methamphetamine received daily intraperitoneal (i.p.) saline injections and were introduced to the activity monitor chambers for 3 days prior to drug treatment to minimize responses induced by exposure to a novel environment, handling and/or pain induced by the injection. In the present study, different types of behaviors are reported: total distance, horizontal activity, vertical activity, and stereotypic movements. The total scores obtained over the entire testing period were used in the analyses of spontaneous activity. Scores obtained during the methamphetamine-stimulated test are presented as two blocks of 30 min each. 2.3. Quantification of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) Methamphetamine-treated animals were sacrificed 2 h after drug treatment and brain tissue from untreated and methamphetamine-treated animals was extracted to analyze striatal dopamine and dopamine metabolite contents by HPLC. Methamphetamine-treated animals were sacrificed 2 h after drug treatment. Strital tissue was analyzed for content of DA, and its metabolites DOPAC and HVA. After euthanasia by CO2 saturation, the brain was removed, placed in a mouse brain coronal matrix immersed in an ice-cold saline solution and cut into 1 mm sections. Bilateral punches from the striatum were collected from one 1-mm section between bregma 1.60 and 0.22. Samples were immediately frozen on dry ice and stored at −80 ◦ C until processed. Each tissue sample was homogenized with 0.01 M perchloric acid using a glass homogenizer. Immediately following the homogenization, the samples were transferred to Eppendof tubes and place at 4 ◦ C for 2 h to allow sufficient extraction, and then centrifuged for 20 min 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 measured with an HPLC coupled to an ESA Coulochem II Detector (model 5200) with a dual-electrode microdialysis cell, and an ESA model 501 data station (ESA, Inc., Chelmsford, MA). Sample injection onto the column (3 m particle size, 3 mm × 150 mm, Analytical MD-150, ESA, Inc.) was made automatically through remote control of the autosampler. The mobile phase for catecholamine separation consists of 75 mM NaH2 PO4 , 1.5 mM OSA, 10 M EDTA, and 7% acetonitrile (pH 3.0 adjusted with H3 PO4 ). Dopamine and its metabolites were quantified on both reducing (−250 mV)
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and oxidizing electrodes (350 mV) and then calculated as pmole/mg wet tissue. 2.4. Statistical analysis The data given represent mean ± S.E.M. values. Body weights and spontaneous behavior scores were compared between Nurr1+/+ and Nurr1+/− mice using an unpaired t-test. For group analysis and statistical comparison for behavior scores after methamphetamine treatment and DA/metabolite concentration determinations, an ordinary ANOVA with Fisher’s a posteriori analysis was used. For all analyses, the threshold for statistical significance was P < 0.05.
3. Results 3.1. Body weights Nurr1+/− mice developed normally with no obvious behavioral abnormalities. The body weights (mean in grams ± S.E.M.) of the Nurr1+/− (48.3±1.06) mice were not significantly different from those of the Nurr1+/+ mice (49.9 ± 0.89). 3.2. Locomotor activity Spontaneous and amphetamine-induced locomotor activity was assessed in Nurr1+/+ (n = 33) versus Nurr1+/− (n = 43) mice by using Omnitech activity chambers. Nurr1+/+ and Nurr1+/− mice exhibited significant differences in spontaneous and methamphetamine-induced (5 mg/kg) behaviors (Fig. 1). Horizontal activity and total distance scores were significantly higher in Nurr1+/− mice as compared to Nurr1+/+ during exposure to a novel environment (Fig. 1). Significant differences between Nurr1+/+ versus Nurr1+/− mice were observed for all methamphetamine-induced measured activities: horizontal activity, total distance, vertical activity, and stereotypic activity. Methamphetamine injection led to a dramatic increase in locomotion in both Nurr1+/+ and Nurr1+/− mice (Figs. 1 and 2). The level of locomotor activation was significantly higher in Nurr1+/− mice as compared to Nurr1+/+ (Fig. 2). The proportion of Nurr1+/− mice showing scores greater than their respective Nurr1+/+ controls were consistently higher during both testing periods (1–30 min versus 30–60 min) for horizontal activity and total distance. Vertical and stereotypic activity scores were significantly increased in the Nurr1+/− group during the second testing period (30–60 min), 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 metabolites DOPAC and HVA were measured in the striatum of
Fig. 1. Exposure to a novel environment increases spontaneous activity in Nurr1+/− animals as compared to Nurr1+/+ . Animals were tested for 30 min 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).
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Fig. 2. Effects of acute methamphetamine (5 mg/kg) on horizontal activity (A), total distance (B), vertical activity (C), and stereotypic activity (D), as assessed in an Omnitech Activity Monitor. Motor activity was examined directly after methamphetamine exposure, and is presented in two blocks of 30 min each. Methamphetamine stimulation significantly increased all behaviors in the Nurr1+/− animals as compared to Nurr1+/+ (ordinary ANOVA + Fisher’s a posteriori test; ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS, not significant).
Nurr1+/+ and Nurr1+/− untreated mice (n = 11 and 14, respectively) and 2 h after methamphetamine exposure (n = 10 and 21, respectively; Fig. 3). DA and HVA levels were increased in both heterozygous and wildtype mice 2 h after methamphetamine exposure (Fig. 3). DOPAC levels were decreased after methamphetamine exposure (Fig. 3). However, no significant differences in DA, DOPAC, or HVA levels were found between Nurr1+/+ and Nurr1+/− animals during control conditions or after methamphetamine treatment (Fig. 3).
4. Discussion The implication of Nurr1 as a transcription factor that regulates nigrostriatal DA function together with the observation that Nurr1+/− neonatal and young adult mice exhibit deficits in striatal dopamine content or ni-
grostriatal dopamine functions suggest that this neurotransmitter system may be further compromised in aged Nurr1+/− mice. Accordingly, the question of whether locomotor 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. Our present data indicates that Nurr1+/− mice exhibit a significant increase in horizontal activity and total distance traveled recorded during spontaneous behavior. This behavioral difference was augmented when the Nurr1+/− mice were given a single injection of methamphetamine (5 mg/kg). Methamphetamine-treated Nurr1+/− mice exhibited a significant increase in horizontal and vertical activity, total distance traveled, and stereotypic activity as compared to Nurr1+/+ controls. Interestingly, analysis of dopamine, DOPAC, and HVA contents in the striatum revealed no alterations in Nurr1+/− mice suggesting that augmentation of locomotor activity in these animals is not
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Fig. 3. Striatal dopamine (DA) and dopamine metabolites, DOPAC and HVA, concentrations for untreated and methamphetamine-treated (5 mg/kg) Nurr1+/− and Nurr1+/+ mice. No significant differences were observed between the groups for either DA, DOPAC, or HVA levels in the untreated or methamphetamine-treated groups (ordinary ANOVA + Fisher’s a posteriori test; NS, not significant).
associated with whole tissue contents of DA and metabolite levels. The mechanism by which deletion of a Nurr1 allele results in an increase in locomotor behavior after acute exposure to methamphetamine is not immediately apparent. Methamphetamine treatment increases extracellular concentrations of DA in the striatum, in part, by reversing the function of the dopamine transporter and facilitating cytoplasmic DA release, as well as by releasing vesicular stores of dopamine [6,10]. Most parameters involving the nigrostriatal system, including DA content, tyrosine hydroxylase, and DA uptake have all been shown to be non-functional after Nurr1 ablation [4,13,16]. The observation made here that Nurr1+/− mice respond to methamphetamine with a significant increase in locomotor behavior suggests that transmission within the nigrostriatal dopaminergic pathway has been altered. However, DA and DA metabolite levels mea-
sured in the striatum of 12–15-month-old either untreated or methamphetamine-treated Nurr1+/− animals were not altered as compared to Nurr1+/+ . Since reduced DA levels have been reported in newborn heterozygous animals and young adults [16], compensatory mechanisms may have developed in aged Nurr1+/− animals to maintain normal levels of DA in the striatum. Compensatory mechanisms in the dopaminergic system of aging animals have previously been described by Friedemann and Gerhardt [7]. In this study, aged Fischer 344 rats did not show age-related differences in striatal DA levels, DA metabolite levels, and turnover indices as compared to young animals. However, DA overflow and clearance rates were decreased in aged animals. Interestingly, our preliminary in vivo electrochemistry results (data not published) suggest that the dynamics of dopamine release/uptake may be affected in Nurr1+/− animals. Correlated with this, recent biochemical studies have shown that
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Nurr1 may regulate the expression of other dopaminergic cell components such as the dopamine transporter [11]; thus, the behavioral effects observed here may be mediated by cell mechanisms other than DA synthesis. In addition, it should also be considered that Nurr1 is present in other motor neuronal systems [14,15,17] that are known to be affected by methamphetamine exposure, such as the motor cortex [3,8]. Therefore, neuronal alterations in brain areas other than the ventral mesencephalon may mediate the altered locomotor responses observed in heterozygote animals. In conclusion, this study shows that deletion of one allele of the Nurr1 gene alters the locomotor activity of 12–15-month-old Nurr1+/− animals. While dopamine levels were not altered in the striatum of the Nurr1+/− mice, the dynamics of DA release and uptake within the nigrostriatal system may be altered in these heterozygotes. Further studies will be necessary to determine the changes in dopaminergic neurons caused by partial inactivation of Nurr1 gene activity, which underlie changes in motor behavior.
Acknowledgements This work was supported by the Intramural Research Program, NIDA, NIH.
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References [14] [1] Bäckman C, Perlmann T, Wallen A, Hoffer BJ, Morales M. A selective group of dopaminergic neurons express Nurr1 in the adult mouse brain. Brain Res 1999;18:125–32. [2] Bannon MJ, Pruetz B, Manning-Bog AB, Whitty CJ, Michelhaugh SK, Sacchetti P, et al. Decreased expression of the transcription factor NURR1 in dopamine neurons of cocaine abusers. Proc Natl Acad Sci USA 2002;99:6382–5. [3] Burrows KB, Meshul CK. Methamphetamine alters presynaptic glutamate immunoreactivity in the caudate nucleus and motor cortex. Synapse 1997;27:133–44. [4] Castillo SO, Baffi J, Palkovits M, Goldstein DS, Kopin IJ, Witta J, et al. Dopamine biosynthesis is selectively abolished in substantia nigra/ventral tegmental area but not in hypothalamic neurons in
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mice with targeted disruption of the Nurr1 gene. Mol Cell Neurosci 1998;11:36–46. Chu Y, Kompoliti K, Cochran EJ, Mufson EJ, Kordower JH. Age-related decreases in Nurr1 immunoreactivity in the human substantia nigra. J Comp Neurol 2002;450:203–14. Cubells JF, Rayport S, Rajendran G, Sulzer D. Methamphetamine neurotoxicity involves vacuolation of endocytic organelles and dopamine-dependent intracellular oxidative stress. J Neurosci 1994;14:2260–71. Friedemann MN, Gerhardt GA. Regional effects of aging on dopaminergic function in the Fischer-344 rat. Neurobiol Aging 1992;13:325–32. Gluck MR, Moy LY, Jayatilleke E, Hogan KA, Manzino L, Sonsalla PK. Parallel increases in lipid and protein oxidative markers in several mouse brain regions after methamphetamine treatment. J Neurochem 2001;79:152–60. Le W, Conneely OM, He Y, Jankovic J, Appel SH. Reduced Nurr1 expression increases the vulnerability of mesencephalic dopamine neurons to MPTP-induced injury. J Neurochem 1999;73:2218–21. O’Dell SJ, Weihmuller FB, Marshall JF. Multiple methamphetamine injections induce marked increases in extracellular striatal dopamine which correlate with subsequent neurotoxicity. Brain Res 1991;564:256–60. Sacchetti P, Mitchell TR, Granneman JG, Bannon MJ. Nurr1 enhances transcription of the human dopamine transporter gene through a novel mechanism. J Neurochem 2001;76:1565–72. Sakurada K, Ohshima-Sakurada M, Palmer TD, Gage FH. Nurr1 an orphan nuclear receptor, is a transcriptional activator of endogenous tyrosine hydroxylase in neural progenitor cells derived from the adult brain. Development 1999;126:4017–26. Saucedo-Cardenas O, Quintana-Hau JD, Le W-D, Smidt MP, Cox JJ, DeMayo F, et al. Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc Natl Acad Sci USA 1998;95:4013–8. Saucedo-Cardenas O, Conneel OM. Comparative distribution of Nurr1 and Nurr77 nuclear receptors in the mouse central nervous system. J Mol Neurosci 1996;7:51–63. Xiao Q, Castillo SO, Nikodem VM. Distribution of messenger RNAs for the orphan nuclear receptors Nurr1 and Nur77 (NGFI-B) in adult rat brain using in situ hybridization. Neuroscience 1996;75:221–30. Zetterström RH, Solomin L, Jansson L, Hoffer BJ, Olson L, Perlmann T. Dopamine neuron agenesis in Nurr1-deficient mice. Science 1997;276:248–50. Zetterström RH, Williams R, Perlmann T, Olson L. Cellular expression of the immediate early transcription factors Nurr1 and NGFI-B suggests a gene regulatory role in several brain regions including the nigrostriatal dopamine system. Brain Res Mol Brain Res 1996;41:111–20.
Research report
Elevated locomotor activity without altered striatal dopamine contents in Nurr1 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 Behavioral Neuroscience Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD 21224, USA Department of Cell and Molecular Biology, Ludwig Institute for Cancer Research, Karolinska Institute, Box 240, Stockholm S-171 77, Sweden b
c
Received 18 November 2002; accepted 8 January 2003
Abstract Gene targeting experiments, in which both alleles of the Nurr1 gene were deleted, have shown that this molecule plays an essential role in the development of midbrain dopaminergic neurons, as shown by the loss of dopaminergic markers and the neurotransmitter dopamine (DA) in the ventral mesencephalon of Nurr1 null mutant mice. Nurr1-deficient mice die within a few hours of birth. Herein, we investigated whether adult mice (12–15-month-old), heterozygous for the Nurr1 mutation (Nurr1+/− ), show alterations in locomotor function and in the nigrostriatal dopaminergic system after acute exposure to methamphetamine. We first evaluated spontaneous and amphetamine-induced (5 mg/kg) locomotor response of >12-month-old wildtype (Nurr1+/+ ) and Nurr1+/− mice. Both, spontaneous and methamphetamine-induced locomotor behavior was significantly increased in the Nurr1+/− animals as compared to Nurr1+/+ mice. Striatal DA and DA metabolite levels were measured in untreated animals and methamphetamine-treated animals. No significant differences in striatal dopamine levels or its metabolites DOPAC and HVA were found in the Nurr1+/− as compared to Nurr1+/+ mice in untreated or methamphetamine-treated animals. 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 family of ligand-inducible transcription factors. The murine orphan nuclear receptor, Nurr1, is predominately expressed in the CNS in limbic areas and the ventral midbrain, including dopamine (DA) neurons [1,14,15,17]. Nurr1 expression in the ventral midbrain occurs on embryonic day 10.5 (E10.5), just before the appearance of the dopaminergic marker enzyme tyrosine hydroxylase (TH) at E11.5. [4,13,16]. This observation led to the hypothesis that Nurr1 plays a role in the development of midbrain dopaminergic cells. Also, as Nurr1 expression continues during adulthood, this transcription factor may also be required for normal function of the mature dopaminergic system. To determine the physiological function of Nurr1, knockout mice were generated
∗
Corresponding author. Tel.: +1-410-550-6870x19. E-mail address: [email protected] (C. Bäckman).
by homologous recombination [4,13,16]. The phenotype of these mice demonstrated that Nurr1 plays an essential role for the generation of DA neurons. Moreover, analysis of homozygous Nurr1 (Nurr1−/− ) brain extracts revealed the loss of the neurotransmitter DA. This was consistent with absent TH, l-aromatic amino acid decarboxylase, and other DA neuron phenotypic markers in the ventral midbrain. Nurr1−/− mutant mice can be distinguished after birth by hypoactivity and the lack of milk in their stomachs, and die within hours of birth. Nurr1+/− mice have moderately lower levels of Nurr1 protein than Nurr1+/+ mice [9], which is consistent with Nurr1 mRNA expression [13]. The newborn Nurr1+/− mice have decreased TH expression in SN and ventral tegmental area and significantly lower levels of DA in striatum [16]. Zetterström et al. [16] also reported that adult heterozygotes display apparently no histological and behavioral abnormalites despite reduced striatal DA levels. In contrast, Le et al. [9] found that striatal DA levels in young adult Nurr1+/− mice are not significantly different from Nurr1+/+ mice.
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An increasing body of evidence implicates the transcription factor Nurr1 as an important regulator of dopamine function in the nigrostriatal system in the adult animal. A recent study has shown that Nurr1 and dopamine transporter (DAT) gene expression are markedly reduced in the ventral mesensephalon of human cocaine abusers [2], suggesting that Nurr1 may play a role in vivo in adaptation to repeated exposure to cocaine. Another study has shown parallel decreases in Nurr1- and TH-immunofluorescence as a function of age, indicating that age-related decline of DA phenotypic markers is associated with down-regulation of Nurr1 expression in the SN [5]. Le et al. [9] reported that reduction of Nurr1 expression in Nurr1+/− mice, confers an increased vulnerability to the selective dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) when compared to Nurr1+/+ mice. Nurr1 has also been shown to enhance the transcriptional activity of human dopamine transporter gene constructs transiently transfected into a cell line that expresses a dopaminergic phenotype [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 a response element within a region of the tyrosine hydroxylase promoter necessary for midbrain-specific expression. Given the previously demonstrated effects of Nurr1 in adult and aged midbrain dopaminergic neurons, we were interested in examining if nigrostriatal dopaminergic function could be altered in >12-month-old heterozygous mice with the deletion of a single Nurr1 gene. To answer these questions, wildtype controls and heterozygous Nurr1 mutant mice at 12–15 months of age were subjected to a behavioral test which provides an index of nigrostriatal dopaminergic function; spontaneous and methamphetamine-induced (5 mg/kg) locomotor activity. In addition, striatal DA and metabolite concentrations were measured under basal conditions and in mice receiving methamphetamine.
2. Materials and methods 2.1. Animals Nurr1 null mutant mice were obtained from Dr. Thomas Perlmann at the Karolinska Institute, Sweden. The generation of Nurr1 null mutant mice has been described in detail elsewhere [16]. Mutant mice were generated from >10 generations 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 were genotyped by means of polymerase chain reactions using gene specific primers. Aged male animals (12–15-month-old) were kept in groups of three to four in plastic cages in the same room and maintained on a 12 h/12 h light/dark cycle with food and water available ad libitum. All animal procedures were approved by the NIDA Animal Care and Use Committee.
2.2. Locomotor activity To assess spontaneous locomotor activity in a novel environment, mice were placed in an Omnitech Animal Activity Monitor for a 30-min period (day 1). To assess methamphetamine-stimulated locomotor activity, methamphetamine (5 mg/kg) was injected intraperitoneally immediately prior to placement of the animal within the activity monitor chamber for a 60-min test period. All the animals treated with methamphetamine received daily intraperitoneal (i.p.) saline injections and were introduced to the activity monitor chambers for 3 days prior to drug treatment to minimize responses induced by exposure to a novel environment, handling and/or pain induced by the injection. In the present study, different types of behaviors are reported: total distance, horizontal activity, vertical activity, and stereotypic movements. The total scores obtained over the entire testing period were used in the analyses of spontaneous activity. Scores obtained during the methamphetamine-stimulated test are presented as two blocks of 30 min each. 2.3. Quantification of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) Methamphetamine-treated animals were sacrificed 2 h after drug treatment and brain tissue from untreated and methamphetamine-treated animals was extracted to analyze striatal dopamine and dopamine metabolite contents by HPLC. Methamphetamine-treated animals were sacrificed 2 h after drug treatment. Strital tissue was analyzed for content of DA, and its metabolites DOPAC and HVA. After euthanasia by CO2 saturation, the brain was removed, placed in a mouse brain coronal matrix immersed in an ice-cold saline solution and cut into 1 mm sections. Bilateral punches from the striatum were collected from one 1-mm section between bregma 1.60 and 0.22. Samples were immediately frozen on dry ice and stored at −80 ◦ C until processed. Each tissue sample was homogenized with 0.01 M perchloric acid using a glass homogenizer. Immediately following the homogenization, the samples were transferred to Eppendof tubes and place at 4 ◦ C for 2 h to allow sufficient extraction, and then centrifuged for 20 min 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 measured with an HPLC coupled to an ESA Coulochem II Detector (model 5200) with a dual-electrode microdialysis cell, and an ESA model 501 data station (ESA, Inc., Chelmsford, MA). Sample injection onto the column (3 m particle size, 3 mm × 150 mm, Analytical MD-150, ESA, Inc.) was made automatically through remote control of the autosampler. The mobile phase for catecholamine separation consists of 75 mM NaH2 PO4 , 1.5 mM OSA, 10 M EDTA, and 7% acetonitrile (pH 3.0 adjusted with H3 PO4 ). Dopamine and its metabolites were quantified on both reducing (−250 mV)
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and oxidizing electrodes (350 mV) and then calculated as pmole/mg wet tissue. 2.4. Statistical analysis The data given represent mean ± S.E.M. values. Body weights and spontaneous behavior scores were compared between Nurr1+/+ and Nurr1+/− mice using an unpaired t-test. For group analysis and statistical comparison for behavior scores after methamphetamine treatment and DA/metabolite concentration determinations, an ordinary ANOVA with Fisher’s a posteriori analysis was used. For all analyses, the threshold for statistical significance was P < 0.05.
3. Results 3.1. Body weights Nurr1+/− mice developed normally with no obvious behavioral abnormalities. The body weights (mean in grams ± S.E.M.) of the Nurr1+/− (48.3±1.06) mice were not significantly different from those of the Nurr1+/+ mice (49.9 ± 0.89). 3.2. Locomotor activity Spontaneous and amphetamine-induced locomotor activity was assessed in Nurr1+/+ (n = 33) versus Nurr1+/− (n = 43) mice by using Omnitech activity chambers. Nurr1+/+ and Nurr1+/− mice exhibited significant differences in spontaneous and methamphetamine-induced (5 mg/kg) behaviors (Fig. 1). Horizontal activity and total distance scores were significantly higher in Nurr1+/− mice as compared to Nurr1+/+ during exposure to a novel environment (Fig. 1). Significant differences between Nurr1+/+ versus Nurr1+/− mice were observed for all methamphetamine-induced measured activities: horizontal activity, total distance, vertical activity, and stereotypic activity. Methamphetamine injection led to a dramatic increase in locomotion in both Nurr1+/+ and Nurr1+/− mice (Figs. 1 and 2). The level of locomotor activation was significantly higher in Nurr1+/− mice as compared to Nurr1+/+ (Fig. 2). The proportion of Nurr1+/− mice showing scores greater than their respective Nurr1+/+ controls were consistently higher during both testing periods (1–30 min versus 30–60 min) for horizontal activity and total distance. Vertical and stereotypic activity scores were significantly increased in the Nurr1+/− group during the second testing period (30–60 min), 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 metabolites DOPAC and HVA were measured in the striatum of
Fig. 1. Exposure to a novel environment increases spontaneous activity in Nurr1+/− animals as compared to Nurr1+/+ . Animals were tested for 30 min 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).
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Fig. 2. Effects of acute methamphetamine (5 mg/kg) on horizontal activity (A), total distance (B), vertical activity (C), and stereotypic activity (D), as assessed in an Omnitech Activity Monitor. Motor activity was examined directly after methamphetamine exposure, and is presented in two blocks of 30 min each. Methamphetamine stimulation significantly increased all behaviors in the Nurr1+/− animals as compared to Nurr1+/+ (ordinary ANOVA + Fisher’s a posteriori test; ∗∗ P < 0.01, ∗∗∗ P < 0.001, NS, not significant).
Nurr1+/+ and Nurr1+/− untreated mice (n = 11 and 14, respectively) and 2 h after methamphetamine exposure (n = 10 and 21, respectively; Fig. 3). DA and HVA levels were increased in both heterozygous and wildtype mice 2 h after methamphetamine exposure (Fig. 3). DOPAC levels were decreased after methamphetamine exposure (Fig. 3). However, no significant differences in DA, DOPAC, or HVA levels were found between Nurr1+/+ and Nurr1+/− animals during control conditions or after methamphetamine treatment (Fig. 3).
4. Discussion The implication of Nurr1 as a transcription factor that regulates nigrostriatal DA function together with the observation that Nurr1+/− neonatal and young adult mice exhibit deficits in striatal dopamine content or ni-
grostriatal dopamine functions suggest that this neurotransmitter system may be further compromised in aged Nurr1+/− mice. Accordingly, the question of whether locomotor 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. Our present data indicates that Nurr1+/− mice exhibit a significant increase in horizontal activity and total distance traveled recorded during spontaneous behavior. This behavioral difference was augmented when the Nurr1+/− mice were given a single injection of methamphetamine (5 mg/kg). Methamphetamine-treated Nurr1+/− mice exhibited a significant increase in horizontal and vertical activity, total distance traveled, and stereotypic activity as compared to Nurr1+/+ controls. Interestingly, analysis of dopamine, DOPAC, and HVA contents in the striatum revealed no alterations in Nurr1+/− mice suggesting that augmentation of locomotor activity in these animals is not
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Fig. 3. Striatal dopamine (DA) and dopamine metabolites, DOPAC and HVA, concentrations for untreated and methamphetamine-treated (5 mg/kg) Nurr1+/− and Nurr1+/+ mice. No significant differences were observed between the groups for either DA, DOPAC, or HVA levels in the untreated or methamphetamine-treated groups (ordinary ANOVA + Fisher’s a posteriori test; NS, not significant).
associated with whole tissue contents of DA and metabolite levels. The mechanism by which deletion of a Nurr1 allele results in an increase in locomotor behavior after acute exposure to methamphetamine is not immediately apparent. Methamphetamine treatment increases extracellular concentrations of DA in the striatum, in part, by reversing the function of the dopamine transporter and facilitating cytoplasmic DA release, as well as by releasing vesicular stores of dopamine [6,10]. Most parameters involving the nigrostriatal system, including DA content, tyrosine hydroxylase, and DA uptake have all been shown to be non-functional after Nurr1 ablation [4,13,16]. The observation made here that Nurr1+/− mice respond to methamphetamine with a significant increase in locomotor behavior suggests that transmission within the nigrostriatal dopaminergic pathway has been altered. However, DA and DA metabolite levels mea-
sured in the striatum of 12–15-month-old either untreated or methamphetamine-treated Nurr1+/− animals were not altered as compared to Nurr1+/+ . Since reduced DA levels have been reported in newborn heterozygous animals and young adults [16], compensatory mechanisms may have developed in aged Nurr1+/− animals to maintain normal levels of DA in the striatum. Compensatory mechanisms in the dopaminergic system of aging animals have previously been described by Friedemann and Gerhardt [7]. In this study, aged Fischer 344 rats did not show age-related differences in striatal DA levels, DA metabolite levels, and turnover indices as compared to young animals. However, DA overflow and clearance rates were decreased in aged animals. Interestingly, our preliminary in vivo electrochemistry results (data not published) suggest that the dynamics of dopamine release/uptake may be affected in Nurr1+/− animals. Correlated with this, recent biochemical studies have shown that
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Nurr1 may regulate the expression of other dopaminergic cell components such as the dopamine transporter [11]; thus, the behavioral effects observed here may be mediated by cell mechanisms other than DA synthesis. In addition, it should also be considered that Nurr1 is present in other motor neuronal systems [14,15,17] that are known to be affected by methamphetamine exposure, such as the motor cortex [3,8]. Therefore, neuronal alterations in brain areas other than the ventral mesencephalon may mediate the altered locomotor responses observed in heterozygote animals. In conclusion, this study shows that deletion of one allele of the Nurr1 gene alters the locomotor activity of 12–15-month-old Nurr1+/− animals. While dopamine levels were not altered in the striatum of the Nurr1+/− mice, the dynamics of DA release and uptake within the nigrostriatal system may be altered in these heterozygotes. Further studies will be necessary to determine the changes in dopaminergic neurons caused by partial inactivation of Nurr1 gene activity, which underlie changes in motor behavior.
Acknowledgements This work was supported by the Intramural Research Program, NIDA, NIH.
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