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Impaired in vivo dopamine release in parkin knockout mice
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Research Report
Impaired in vivo dopamine release in parkin knockout mice Genko Oyama a,b , Kenji Yoshimi b,c , Shihoko Natori a,b , Yoko Chikaoka a , Yong-Ri Ren a , Manabu Funayama a , Yasushi Shimo a , Ryosuke Takahashi c,d , Taizo Nakazato b,c , Shigeru Kitazawa b,c , Nobutaka Hattori a,c,⁎ a
Department of Neurology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan Department of Neurophysiology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan c CREST, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi-shi, Saitama 305-8575, Japan d Department of Neurology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
parkin is the most frequent causative gene among familial Parkinson's disease (PD).
Accepted 25 June 2010
Although parkin deficiency induces autosomal recessive juvenile parkinsonism (AR-JP,
Available online 8 July 2010
PARK2) in humans, parkin knockout (PKO) mice consistently show few signs of dopaminergic degeneration. We aimed to directly measure evoked extracellular dopamine (DA) overflow
Keywords:
in the striatum with in vivo voltammetry. The amplitude of evoked DA overflow was low in
parkin knockout mice
PKO mice. The half-life time of evoked DA overflow was long in PKO mice suggesting lower
Dopamine release
release and uptake of dopamine. Facilitation of DA overflow by repetitive stimulation
In vivo voltammetry
enhanced in the older PKO mice. Decreased dopamine release and uptake in young PKO
Medial forebrain bundle
mice suggest early pre-symptomatic changes in dopamine neurotransmission, while the enhanced facilitation in the older PKO mice may reflect a compensatory adaptation in dopamine function during the late pre-symptomatic phase of Parkinson's disease. Our results showed parkin deficiency may affect DA release in PKO mice, although it does not cause massive nigral degeneration or parkinsonian symptoms as in humans. © 2010 Elsevier B.V. All rights reserved.
1.
Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The main pathology in PD is the loss of dopaminergic neurons in the substantia nigra pars compacta. Although most PD cases are sporadic, to date, 16 subtypes of familial forms of parkinsonism and 8 causative genes have been identified (Mizuno et al., 2006; Satake et al., 2009; Thomas and Beal, 2007; Vila and
Przedborski, 2004). Recent evidence suggests that the interaction between genetic and environmental factors could contribute to the pathogenesis of PD (Hattori and Mizuno, 2004). Thus, the pathogenesis of familial forms of PD foreshadows a common pathway of nigral degeneration in not only various types of familial forms of PD but also the sporadic form. parkin is the gene responsible for PARK2, an autosomal recessive type of a familial form of parkinsonism (Kitada et al., 1998). This form is the most frequent among young-onset PD, and is
⁎ Corresponding author. Department of Neurology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan. Fax: +81 3 5684 0476. E-mail address: [email protected] (N. Hattori). Abbreviations: AR-JP, autosomal recessive early-onset parkinsonism; DA, dopamine; MFB, medial forebrain bundle; PKO, parkin knockout; WT, wild type 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.06.065
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characterized by L-DOPA responsive dystonia and parkinsonism (Hattori and Mizuno, 2004). parkin deficiency is expected to be associated with loss-offunction effect based on the recessive mode of inheritance. However, the parkin knockout (PKO) mouse lacks extensive nigral degeneration or marked parkinsonism-like motor deficits (Goldberg et al., 2003; Itier et al., 2003; Perez and Palmiter, 2005; Sato et al., 2006; Von Coelln et al., 2004). On the other hand, these mice show mild changes in extracellular dopamine (DA) (Goldberg et al., 2003; Itier et al., 2003) and upregulation of DA receptor binding in the striatum (Sato et al., 2006). Other studies also reported non-motor manifestations of PD (Zhu et al., 2007) and certain cognitive changes in PKO mice (Goldberg et al., 2003; Itier et al., 2003; Zhu et al., 2007). In spite of previous failures detecting PD-like changes in PKO mice in vivo, physiological dysfunction of dopamine neurotransmission remains to be evaluated. In mouse species, lack of motor deficit does not suggest intact dopaminergic functions (Jackson-Lewis and Przedborski, 2007). In this study, we aimed to measure evoked extracellular DA overflow in the striatum in vivo by a high-speed electrochemical technique using carbon-fiber microelectrodes (Nakazato and Akiyama, 1999; Natori et al., 2009; Suzuki et al., 2007; Yavich et al., 2004).
2.
Results
2.1.
General growth and behavioral tests of PKO mice
In agreement with previous studies (Goldberg et al., 2003; Itier et al., 2003; Perez and Palmiter, 2005; Sato et al., 2006; Von Coelln et al., 2004), PKO mice developed normally except for their smaller body weight (WT; 34.3 ± 1.0 g, PKO; 30.0 ± 0.7 g), the main effects of gene and age were significant (F(1,62) = 16.3, P < 0.001; F(3,62) = 23.3, P < 0.001), but the general behavior seemed to be normal. The pole and accelerating rotarod tests were used to evaluate motor function prior to voltammetry tests. In the
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pole test, neither age nor genotype had significant effects on the time for turning on the pole (TTurn) (F(3,55) = 0.32, P = 0.821; F(1,62) = 2.38, P = 0.149, Fig. 1A). With regard to the time required for landing (TLand), the main effect of age was significant (F(3,55) = 12.74, P < 0.001) but the effect of genotype was not (F(1,62) = 2.174, P = 0.146, Fig. 1B). The effect of age on the time for landing could be due to the difference in body weight, because heavier mice went down the pole faster (correlation between TLand and bodyweight, r = −0.49, P < 0.01). In the accelerating rotarod test, the main effect of age on the falling latency was significant (F(3,55) = 9.54, P < 0.001, Fig. 1C), but the main effect of genotype was not (F(1,62 = 0.36, P = 0.55). The above results are consistent with previous studies (Goldberg et al., 2003; Itier et al., 2003; Perez and Palmiter, 2005; Sato et al., 2006; Von Coelln et al., 2004) and indicate that the genotype does not significantly affect general motor function in PKO mice.
2.2.
Evoked DA overflow
During the 2-s stimulation of the nigrostriatal fibers in the medial forebrain bundle (MFB), a fast and short-lasting increase in electrochemical current was observed (Fig. 2A). The maximum amplitude of the evoked dopamine overflow (Δ[DA]max) was lower in PKO than in WT (Fig. 2C). The main genotype effect was significant (F(1,62) = 11.4, P = 0.0016), but that of age was not (F(3,55) = 0.843, P = 0.48). Administration of nomifensine, a competitive DA uptake inhibitor, increased the maximum amplitude (Fig. 2B,D). The genotype effect was again significant after nomifensine (F(1,59) =18.23, P < 0.001), but that of age was not (F(3,52) = 0.52, P = 0.67). There were significant differences (P < 0.05) in Δ[DA]max before nomifensine between WT and PKO at 3 and 6 months of age (Fig. 2C), and in Δ[DA]max after nomifensine at 3, 6 and 9 months of age (Fig. 2D). However, there were no differences either before or after nomifensine at 12 months of age. In the half-life time (T1/2) of the evoked DA overflow, the main genotype effect was significant before nomifensine (F(1,62) = 6.89, P = 0.01) but that of age was not (F(3,55) = 0.634, P = 0.597) (Fig. 2E). The administration of nomifensine significantly increased T1/2 both in WT
Fig. 1 – Behavioral tests performed before voltammetric measurement. (A) The latency before the mouse turned toward the ground (TTurn) and (B) the time from turn to landing (TLand) in the pole test. (C) Accelerating rotarod test (3-cm diameter), in which the rod accelerated from 3 to 35 rpm over 5 min. Data are mean ± SEM of 8 to 10 mice.
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Fig. 2 – Evoked dopamine overflow. (A and B): Representative records of evoked dopamine by a single medial forebrain bundle (MFB) stimulation (50 Hz × 100 pulses, gray lines) in 3-month-old PKO and WT mice before (A) and after (B) administration of nomifensine. (C and D): The maximal amplitude of evoked DA (Δ[DA]max ) before (C) and after (D) nomifensine. The half-life time of evoked DA (T1/2 ) before (E) and after (F) nomifensine. Data are mean ± SEM. *P < 0.05 (Bonferroni's correction).
and PKO (F(1,52) = 51.5, P < 0.001). After nomifensine, the genotype effect was again significant (F(1, 59) = 6.70, P = 0.01), but that of age was not (F(3,52) = 1.26, P = 0.30) (Fig. 2F).
2.3.
DA dynamics under repeated stimulations
After evaluating the amplitude of 2-s stimulation, dopamine dynamics induced by repeated stimulation were evaluated. Consistent with previous studies (Kita et al., 2007; Yavich et al.,
2004), both depression between block and facilitation within block were observed in all mice (Fig. 3A). Between-block depression was not different between WT and PKO mice and there was no significant age effect (Fig. 3B). The between-block depression was similar after administration of nomifensine except at 9 months of age (F(1,13) = 5.97, P = 0.03). On the other hand, facilitation was reduced by age in both WT and PKO mice. The effect of genotype was significant in 9- (F(1,13) = 7.47, P = 0.017) and 12-month-old groups (F(1,14) = 7.35, P = 0.017).
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Fig. 3 – Repeated train stimulations. (A) Representative records of WT and KO from 3-month-age group, before and after nomifensine. Every burst-block consisted of six 2-s stimulations with 5-s intervals and each burst-block was repeated three times every 90 s. Each peak-amplitude was measured by subtracting the baseline before the first pulse from the peak. (B) Depression of the first stimulation among the blocks. The amplitude of the first stimulation of each block is expressed relative to the amplitude of the first stimulation of the first train. (C) Facilitation within the first burst block. The peak amplitude to each 2-s stimulation is expressed relative to the amplitude on the first stimulation. Data are mean ± SEM of 7–8 mice. *P < 0.05 (Repeated ANOVA/Bonferroni's correction).
This facilitation disappeared after administration of nomifensine (Fig. 3C). To evaluate the effect of inter-stimulation interval, three 2-s stimulations were applied at various intervals (interval test, Fig. 4). The net increase in amplitude was expressed as a percentage of the first burst's amplitude. Before the administration of nomifensine, a slight depression was observed at long intervals of 40 and 20 s, but it changed to facilitation at short intervals of less than 10 s, in both WT and PKO mice. There were no significant differences between WT and PKO, except for the 12month-old groups (2nd: F(1,14) =7.94, P=0.014, 3rd: F(1,14) =5.52, P=0.0033). There was a significant age effect in the second (F(3,27) =3.59, P=0.027) and third pulses (F(3,27) =3.49, P=0.029) of WT, whereas PKO did not show any significant change with age.
Nomifensine administration resulted in the disappearance of facilitation and appearance of depression at short intervals less than 10 s, which was considered due to paired pulse depression by D2 autoregulation (Benoit-Marand et al., 2001; Schmitz et al., 2002; Schmitz et al., 2003). There were no significant differences in the effects of genotype and age.
2.4.
Neurochemical analysis
In order to confirm the density of DA projection to the striatum, the striatal tissue contents of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5Hydroxyindoleacetic acid (5-HIAA) were measured by highperformance liquid chromatography (HPLC, Table 1). The
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Fig. 4 – The interval test. (A) Representative records of WT and KO from 3-month-age group. Three 2-s stimulations were applied at different intervals of 40, 20, 10, 7, 5, and 3 s before (gray lines) and after (black lines) nomifensine. The net increase of amplitude was measured by subtracting the baseline of each pulse from each peak. The amplitudes of the second and third pulse relative to the first pulse were plotted against stimulation interval before (B) and after (C) nomifensine. Data are mean±SEM of 7–8 mice.
contents of DA and monoamine metabolites did not show significant genotype effects (DA: F(1,62) = 0.06, P = 0.81; DOPAC: F(1,62) = 0, P = 1.00, HVA: F(1,62) = 0.27, P = 0.61, 5-HIAA; F(1,62) = 0.10,
P = 0.75). However, there was a significant age effect on DOPAC concentration (F(3,55) = 5.14, P = 0.003), mainly due to low concentration in 3-month-old mice.
Table 1 – Neurochemical analysis.
3 months 6 months 9 months 12 months
WT (n = 7) KO (n = 8) WT (n = 7) KO (n = 7) WT (n = 7) KO (n = 6) WT (n = 8) KO (n = 8)
DA
DOPAC
HVA
5-HIAA
93.9 ± 6.8 94.2 ± 4.4 89.8 ± 7.6 88.4 ± 8.3 70.2 ± 8.8 80.4 ± 14.1 95.8 ± 5.2 91.7 ± 8.2
13.6 ± 1.6 12.9 ± 0.8 22.4 ± 2.1 18.5 ± 1.2 11.8 ± 2.1 19.4 ± 5.8 23.2 ± 2.5 20.2 ± 3.2
30.8 ± 4.0 29.2 ± 1.6 31.7 ± 3.6 31.5 ± 2.5 21.9 ± 3.4 28.0 ± 1.8 32.4 ± 1.7 30.3 ± 1.6
5.3 ± 0.6 5.1 ± 0.2 5.8 ± 0.6 5.6 ± 0.4 4.7 ± 0.8 5.3 ± 0.5 5.5 ± 0.3 4.7 ± 0.2
Striatal contents in pmol/mg of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) measured by HPLC, after electrochemical tests. There are no significant genotype effects. However, there was a significant age effect on DOPAC due to 3-month age group (Uni-ANOVA/Bonferroni's correction). Data are mean ± SEM. P < 0.05.
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2.5.
Histology
In the majority of cases, the electrode tip was located within the TH-positive fiber bundles. Deviation of the electrode tip from the center of TH-positive fibers was not different between WT (0.19 ±0.03 mm) and PKO mice (0.15 ± 0.02) (F(1,55) = 1.20, P = 0.28).
3.
Discussion
In the present study, although there were no differences between PKO and WT mice in the behavioral test, PKO mice showed lower amplitudes and longer half-lives of evoked DA than WT mice did (Fig. 2). This may suggest early presymptomatic changes in dopaminergic transmission in PKO mice. In addition, the facilitation was reduced by age in general, but remained high in the older PKO mice compare to WT mice (Fig. 3C). This facilitation may suggest a compensatory adaptation during the late pre-symptomatic phase or possibly during the symptomatic phase. The amplitude of evoked DA overflow is a consequence of competing mechanisms of synaptic release and uptake (Schmitz et al., 2002; Schmitz et al., 2003). On the other hand, the falling phase of evoked DA depends on the DA uptake by DA transporter (DAT). Therefore the increase of half-life observed in PKO mice suggests a reduced DA uptake (Fig. 2), which rather increases the amplitude of dopamine overflow. These results suggest that the decrease of evoked DA amplitude is due to reduced DA vesicular release. Itier et al. (2003) showed low amphetamine- or methamphetamineinduced DA efflux in PKO mice, but this may reflect reverse transport of dopamine through DAT (Riddle et al., 2006). A recent study showed impaired DA release in acute striatal slices of PKO mice (Kitada et al., 2009a). Interestingly, Kitada et al. (2009a) did not detect differences in 3H labeled DA uptake in PKO compared to WT, whereas Itier et al. (2003) showed functional reduction in DA uptake. The reduction in DA uptake can be a secondary change to compensate the decreased DA release. However the opposite is also possible, that is, decreased DA release could be a secondary change to compensate for reduced uptake. These hypotheses require confirmation in future studies. In the brain, PARKIN protein is found in the presynaptic vesicles (Kubo et al., 2001; Mouatt-Prigent et al., 2004). Although exactly how parkin deficiency alters DA transmission at the presynapse remains obscure, we speculate three possibilities. First, since PARKIN is an ubiquitin-E3 ligase, disruption of proteolysis of functional synaptic proteins may impact synaptic function, as another synapse-localized E3 ligase, SCRAPPER may do (Yao et al., 2007). Second, parkin deficiency may cause changes in mitochondrial function for energy metabolism (Darios et al., 2003; Periquet et al., 2005), so that the highly energy-dependent processes of exocytosis may be influenced. Third, mitochondrial dysfunction can lead to higher H2O2 level, which is a potent suppressor of DA release (Avshalumov and Rice, 2003). More studies are needed to confirm these hypotheses. The within-block facilitation has been explained as modification of D2 receptor auto-regulation (Kita et al., 2007),
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which suppresses the subsequent DA release in repeated pulse stimuli (Benoit-Marand et al., 2001; Schmitz et al., 2002, 2003). However, the within-block facilitation in our study completely disappeared in the presence of nomifensine (Fig. 3C), which suggests that facilitation also reflects DAT function. Indeed, D2 auto-receptor and DAT interact with each other (Baik et al., 1995; Benoit-Marand et al., 2001; Dickinson et al., 1999; Schmitz et al., 2002, 2003). The lack of changes in the interval tests after suppression of DAT may suggest a relatively intact pre-synaptic D2 receptor function (Fig. 4C). However, there is a possibility that slowed DAT increases basal DA levels and consequently alters autoreceptor activation. Furthermore, nomifensine is a competitive inhibitor and does not block DAT completely. More direct approach would be needed to assess the contribution of autoreceptors. Although PARK2 is young-onset, the changes of dopaminergic neurotransmission related to PD are expected to progress by age. In this study, the age effect for evoked DA amplitude was not significant, and less evident in older animals (Fig. 2). Considering the parkin mRNA and protein show marked increase in expression levels during midgestational development in the central nervous system followed by a steady increase until adulthood (Kuhn et al., 2004), we speculate that parkin deficiency can influence DA release in the early developmental phase in PKO mice, followed by some possible secondary changes to compensate for it. On the other hand, enhancement of facilitation was evident in matured (9–12-month-old) mice, suggesting progressive changes by aging (Fig. 3C). These dysfunctions may be subtle and may not impact motor function under physiological condition in young mice. In addition, the life span of mice may be too short, even in aged mice with deficits of parkin and other recessive genes (DJ1−, PINK1) (Kitada et al., 2009b), to confirm the degenerative changes seen in parkin−/− human. In conclusion, we presented direct evidence for changes in dopaminergic neurotransmission in PKO mice in vivo. When combined with the in situ study of PKO mice (Kitada et al., 2009a), our results indicate that genetic deficits in parkin may lead to early-onset physiological dysfunctions of dopaminergic neurons.
4.
Experimental procedures
4.1.
Animals
The parkin−/− mouse (male, 3–12 months old), which carries a chromosomal replacement of exon 3 of the parkin gene (Kitao et al., 2007) was used. The mice were backcrossed into the C57BL/6 J mice for 10 generations, then both +/+ wildtype (WT) and −/− (PKO) littermates were sub-strained and maintained in parallel in the same animal facility at Juntendo University, Tokyo. WT and PKO mice of close birth dates were subjected to behavioral tests on the same day in a shuffled sequence. DNA samples from each animal were confirmed after all experiments through genotyping. Three-, six-, nine- and twelve-month-old mice were subjected to the accelerating rotarod and the pole tests on the same day and then evaluated with in vivo voltammetry
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within three weeks in a random sequence. Mice that died during the surgeries (two in 6 months, two in 9 months, and three in 12 months) and the recordings (two in 6 months and one in 9 months) were not included in the data analysis. Two to six mice were housed per cage, fed solid food with water available ad libitum and kept under controlled conditions maintained on a 12-h light–dark cycle at constant temperature and humidity. The procedures followed in using laboratory animals were in accordance with the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan and all experiments were approved by the Ethics Review Committee for Animal Experimentation of Juntendo University School of Medicine. All efforts were made to minimize the number of animals used and their suffering.
4.2.
Behavioral tests
Behavioral tests were performed during the light cycle and conducted by the same investigator. The behavioral tests were performed in the same room housing the animals. Accelerating rotarod test. Mice were placed on the 3-cm diameter rotating rod covered by rubber (Ugo Basile, Acceler Rotarod 7650 for mice, Jones and Roberts, Italy). The rotation was accelerated, starting at 3 rpm and constantly accelerated to 35 rpm over 5 min, and the fall latency was recorded. The test was repeated three times with a 15-min inter-test interval. The first and second trials were considered acclimation trials, mice were placed on the rod immediately after falling, up to 5 min. The first fall latency of the third trial was analyzed. Pole test. Mice were placed on the top of a 30-cm-tall, clothwrapped, 1.5-cm-diameter pole. The latency before the mouse turned towards the ground (Tturn) and time from the turn to landing (Tland) were recorded. The test was repeated three times with a 15-min inter-test interval. The first and second trials were acclimation trials and data from the third trial was collected and analyzed.
4.3.
Surgical preparation
Carbon-fiber microelectrodes were stereotaxically implanted in the right striatum and a pair of stimulating electrodes placed in the MFB on the same side, as described previously (Natori et al., 2009; Suzuki et al., 2007; Yavich et al., 2004). Mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and placed on a stereotaxic frame (Narishige, Tokyo). Anesthesia was maintained with 1% isoflurane inhalation with 30% O2 and 70% N2O. Rectal temperature was maintained at 37 ± 1.0 °C using a heating lamp and a body warmer. The carbonfiber (HTA-7, Toho Tenax Co., Tokyo; 7 μm in diameter) was cut to a length of 500 μm from the end of the pulled capillary glass, and insulated with epoxy glue, and inserted into the striatum (electrode tip position at 0.5 mm anteriorly (A), 2 mm laterally (L), and 3.2 mm ventrally (V) relative to the bregma) (insertion angle: 15° relative to the coronal plane and actually inserted from A1.5 mm at dura and proceeded 3.3 mm) according to a mouse brain atlas (Franklin and Paxinos, 1997). A pair of stainless steel bipolar stimulating electrodes (Unique Medical Co., Japan; distance between the poles: 1 mm, the stainless needle was insulated except for the 0.3-
mm tip) were implanted in the MFB (AP, −2.0 mm; L, 1.1 mm; V, −4.75 to −5.5 mm). The dorso-ventral placement of the stimulating electrode was adjusted to obtain the maximal DA overflow. The stimulation electrode was advanced by the steps 0.125 mm every 120 s, until the recorded evoked response's amplitude was equivalent to the preceding step and there was no further advancement (typically, 4.75 to 5.0 mm from dura). Two silver/silver chloride wires (reference and auxiliary electrodes), were combined in a glass tube filled with 3% agar made of phosphate buffered saline (pH 7.4, 100 mM phosphate, 0.9% NaCl, PBS) and placed onto the dural surface of the contralateral side.
4.4.
In vivo voltammetry
Stimulated DA overflow was measured by chronoamperometry (differential pulse voltammetry with constant holding potential) as described previously (Nakazato and Akiyama, 1999), with minor modifications. In short, four pairs of squarewave pulses of 20 ms duration and 200 mV amplitude were applied with progressive 100 mV shifts (−100 to 100, 0 to 200, 100 to 300 and 200 to 400 mV) and the current at the last 14 ms was measured and differentiated into high and low potential pairs. One cycle of recording lasted 240 ms and the holding potential between the recording pulses was −200 mV. In addition, biphasic activation pulses (1285 mV, 10 ms) were applied at the beginning of every cycle, to improve the endurance and stability of the carbon-fibers (Nakazato and Akiyama, 1999). Voltage controls, current recordings, and stimulation triggers were controlled automatically with A/D, D/A, timing boards (PCI-3165, PCI-3310 and PCI-6103, respectively, Interface Inc., Hiroshima, Japan), and a three-electrode potentiostat (Rainbow Science Co., Japan). The peak electrochemical current of DA was obtained at 0 to 200-mV pulse in vitro. The peak potential of evoked response in vivo was observed at 200- and 300-mV pulses. Electrochemical currents at 200-mV pulses were regarded as DA responses. After the experiments, the working electrode and the reference electrodes were calibrated using 1.0-μM DA in vitro in PBS (pH 7.4).
4.5. Electrical stimulation of MFB and stimulation protocols Biphasic constant current pulses were delivered to the MFB for 2-s (50 Hz; 100 biphasic 200 μA constant-current pulses; 2 ms each) through the stimulating electrodes from an isolated stimulator (SS-202J connected to SEN-7203, Nihon-Koden, Co., Japan). Stimulation pulses were automatically triggered from our recording system. After adjusting the depth of MFB stimulating electrode to yield the maximal evoked response, the DA current in response to 2-s stimulation was recorded more than three times. The maximum amplitude of the evoked DA overflow (Δ[DA]max) was expressed in molar concentrations (μmol per liter) based on post-calibration data. The half-life time (T1/2) of the evoked DA overflow was defined as the period of time (in seconds) for Δ[DA]max decreasing by half. To evaluate the dynamic regulation of DA release, we delivered different sequences of pulse trains to the MFB. In the first test, six blocks of three 2-s stimulation pulses were applied
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with different inter-pulse intervals of 40, 20, 10, 7, 5, and 3 s in this order (interval test). Each three-pulse block was followed by a break of 100 s, and a break of 200 s was placed before starting the next six-block sequence. In the second test, the dynamic modulation of release was evaluated using the same procedure used in the previous study (Yavich et al., 2004). In this test three burst blocks, each consisting of six 2-s stimulation pulses with 5-s intervals, were delivered with a 90-s break after each block. After completion of these measurements, nomifensine maleate (Sigma, St. Louis, MO, 7 mg/kg, subcutaneously), an uptake inhibitor of the DAT, was administered. DA responses to 2-s MFB stimulation were continuously recorded every 200 s to test the effect of the drug. Twenty minutes after the administration of nomifensine, the same sequences mentioned above were repeated.
4.6.
Neurochemical analysis
The striatal contents of DA and monoamine metabolites were measured by HPLC. After completion of the electrochemical tests, mice were deeply anesthetized with pentobarbital and perfused transcardially with 10 mM cold PBS. The brain tissue samples were cut immediately at the level of bregma with the aid of a mouse brain matrix (RBM-2000C; Activational System, Warren, MI). The anterior striatal tissues were dissected out, frozen on dry ice, and stored at −80 °C until use. The caudal part of the brain was fixed with ice-cold 4% buffered (pH 7.4) formaldehyde solution and used for histological identification of the MFB electrode position. Dissected striatal tissues were sonicated in ×20 volume of ice-cold 0.1 M perchloric acid (PCA) and centrifuged at 20,000 × g for 10 min at 4 °C. The resulting supernatant samples were applied to the HPLC system, which was equipped with a reverse-phase C18 column (150 × 4.6 mm; ODS-100s, Tosoh, Tokyo) and an eight-electrode coulometric electrochemical detection system (ESA-400, ESA, Inc., Chelmsford, MA). The concentration of DA and monoamine metabolites was expressed as picomoles per gram (pmol/mg) of tissue weight.
4.7.
Histological staining and examination
The location of the stimulation electrodes was verified histologically at the end of the study. After the voltammetric recording, the position of the stimulating electrode was marked with a DC current (40 μA DC for 20 s). The harvested brains were paraffin-embedded and sliced into 5-μm-thick sections and MFB was immunostained with tyrosine hydroxylase (TH).
4.8.
Statistical analysis
The effects of the two genotypes and four ages were analyzed by UNI-ANOVA (SPSS, Chicago, IL). When the measurements were repeated on the same mouse, repeated measurements (four training days, four pre- and post-administration periods, three blocks, six stimulations or six intervals) were treated as within-subject factors and the genotype as a between-subject factor. If a major effect was identified, multiple comparisons were performed using Bonferroni's correction. All values are
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expressed as mean ± SEM. A P value less than 5% denoted the presence of a statistically significant difference between the groups.
Acknowledgments The authors thank Dr. Akitane Akiyama for his help with the experimental equipment and Mr. Taka-aki Yanase for technical assistance. This work was supported in part by the Strategic Research Program for Brain Sciences (SRPBS) and a Grant-in-Aid for Scientific Research on Priority Areas “Emergence of Adaptive Motor Function through Interaction between Body, Brain and Environment” from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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Research Report
Impaired in vivo dopamine release in parkin knockout mice Genko Oyama a,b , Kenji Yoshimi b,c , Shihoko Natori a,b , Yoko Chikaoka a , Yong-Ri Ren a , Manabu Funayama a , Yasushi Shimo a , Ryosuke Takahashi c,d , Taizo Nakazato b,c , Shigeru Kitazawa b,c , Nobutaka Hattori a,c,⁎ a
Department of Neurology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan Department of Neurophysiology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan c CREST, Japan Science and Technology Agency, Honcho 4-1-8, Kawaguchi-shi, Saitama 305-8575, Japan d Department of Neurology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawaharacho, Sakyoku, Kyoto 606-8507, Japan b
A R T I C LE I N FO
AB S T R A C T
Article history:
parkin is the most frequent causative gene among familial Parkinson's disease (PD).
Accepted 25 June 2010
Although parkin deficiency induces autosomal recessive juvenile parkinsonism (AR-JP,
Available online 8 July 2010
PARK2) in humans, parkin knockout (PKO) mice consistently show few signs of dopaminergic degeneration. We aimed to directly measure evoked extracellular dopamine (DA) overflow
Keywords:
in the striatum with in vivo voltammetry. The amplitude of evoked DA overflow was low in
parkin knockout mice
PKO mice. The half-life time of evoked DA overflow was long in PKO mice suggesting lower
Dopamine release
release and uptake of dopamine. Facilitation of DA overflow by repetitive stimulation
In vivo voltammetry
enhanced in the older PKO mice. Decreased dopamine release and uptake in young PKO
Medial forebrain bundle
mice suggest early pre-symptomatic changes in dopamine neurotransmission, while the enhanced facilitation in the older PKO mice may reflect a compensatory adaptation in dopamine function during the late pre-symptomatic phase of Parkinson's disease. Our results showed parkin deficiency may affect DA release in PKO mice, although it does not cause massive nigral degeneration or parkinsonian symptoms as in humans. © 2010 Elsevier B.V. All rights reserved.
1.
Introduction
Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The main pathology in PD is the loss of dopaminergic neurons in the substantia nigra pars compacta. Although most PD cases are sporadic, to date, 16 subtypes of familial forms of parkinsonism and 8 causative genes have been identified (Mizuno et al., 2006; Satake et al., 2009; Thomas and Beal, 2007; Vila and
Przedborski, 2004). Recent evidence suggests that the interaction between genetic and environmental factors could contribute to the pathogenesis of PD (Hattori and Mizuno, 2004). Thus, the pathogenesis of familial forms of PD foreshadows a common pathway of nigral degeneration in not only various types of familial forms of PD but also the sporadic form. parkin is the gene responsible for PARK2, an autosomal recessive type of a familial form of parkinsonism (Kitada et al., 1998). This form is the most frequent among young-onset PD, and is
⁎ Corresponding author. Department of Neurology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan. Fax: +81 3 5684 0476. E-mail address: [email protected] (N. Hattori). Abbreviations: AR-JP, autosomal recessive early-onset parkinsonism; DA, dopamine; MFB, medial forebrain bundle; PKO, parkin knockout; WT, wild type 0006-8993/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.06.065
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characterized by L-DOPA responsive dystonia and parkinsonism (Hattori and Mizuno, 2004). parkin deficiency is expected to be associated with loss-offunction effect based on the recessive mode of inheritance. However, the parkin knockout (PKO) mouse lacks extensive nigral degeneration or marked parkinsonism-like motor deficits (Goldberg et al., 2003; Itier et al., 2003; Perez and Palmiter, 2005; Sato et al., 2006; Von Coelln et al., 2004). On the other hand, these mice show mild changes in extracellular dopamine (DA) (Goldberg et al., 2003; Itier et al., 2003) and upregulation of DA receptor binding in the striatum (Sato et al., 2006). Other studies also reported non-motor manifestations of PD (Zhu et al., 2007) and certain cognitive changes in PKO mice (Goldberg et al., 2003; Itier et al., 2003; Zhu et al., 2007). In spite of previous failures detecting PD-like changes in PKO mice in vivo, physiological dysfunction of dopamine neurotransmission remains to be evaluated. In mouse species, lack of motor deficit does not suggest intact dopaminergic functions (Jackson-Lewis and Przedborski, 2007). In this study, we aimed to measure evoked extracellular DA overflow in the striatum in vivo by a high-speed electrochemical technique using carbon-fiber microelectrodes (Nakazato and Akiyama, 1999; Natori et al., 2009; Suzuki et al., 2007; Yavich et al., 2004).
2.
Results
2.1.
General growth and behavioral tests of PKO mice
In agreement with previous studies (Goldberg et al., 2003; Itier et al., 2003; Perez and Palmiter, 2005; Sato et al., 2006; Von Coelln et al., 2004), PKO mice developed normally except for their smaller body weight (WT; 34.3 ± 1.0 g, PKO; 30.0 ± 0.7 g), the main effects of gene and age were significant (F(1,62) = 16.3, P < 0.001; F(3,62) = 23.3, P < 0.001), but the general behavior seemed to be normal. The pole and accelerating rotarod tests were used to evaluate motor function prior to voltammetry tests. In the
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pole test, neither age nor genotype had significant effects on the time for turning on the pole (TTurn) (F(3,55) = 0.32, P = 0.821; F(1,62) = 2.38, P = 0.149, Fig. 1A). With regard to the time required for landing (TLand), the main effect of age was significant (F(3,55) = 12.74, P < 0.001) but the effect of genotype was not (F(1,62) = 2.174, P = 0.146, Fig. 1B). The effect of age on the time for landing could be due to the difference in body weight, because heavier mice went down the pole faster (correlation between TLand and bodyweight, r = −0.49, P < 0.01). In the accelerating rotarod test, the main effect of age on the falling latency was significant (F(3,55) = 9.54, P < 0.001, Fig. 1C), but the main effect of genotype was not (F(1,62 = 0.36, P = 0.55). The above results are consistent with previous studies (Goldberg et al., 2003; Itier et al., 2003; Perez and Palmiter, 2005; Sato et al., 2006; Von Coelln et al., 2004) and indicate that the genotype does not significantly affect general motor function in PKO mice.
2.2.
Evoked DA overflow
During the 2-s stimulation of the nigrostriatal fibers in the medial forebrain bundle (MFB), a fast and short-lasting increase in electrochemical current was observed (Fig. 2A). The maximum amplitude of the evoked dopamine overflow (Δ[DA]max) was lower in PKO than in WT (Fig. 2C). The main genotype effect was significant (F(1,62) = 11.4, P = 0.0016), but that of age was not (F(3,55) = 0.843, P = 0.48). Administration of nomifensine, a competitive DA uptake inhibitor, increased the maximum amplitude (Fig. 2B,D). The genotype effect was again significant after nomifensine (F(1,59) =18.23, P < 0.001), but that of age was not (F(3,52) = 0.52, P = 0.67). There were significant differences (P < 0.05) in Δ[DA]max before nomifensine between WT and PKO at 3 and 6 months of age (Fig. 2C), and in Δ[DA]max after nomifensine at 3, 6 and 9 months of age (Fig. 2D). However, there were no differences either before or after nomifensine at 12 months of age. In the half-life time (T1/2) of the evoked DA overflow, the main genotype effect was significant before nomifensine (F(1,62) = 6.89, P = 0.01) but that of age was not (F(3,55) = 0.634, P = 0.597) (Fig. 2E). The administration of nomifensine significantly increased T1/2 both in WT
Fig. 1 – Behavioral tests performed before voltammetric measurement. (A) The latency before the mouse turned toward the ground (TTurn) and (B) the time from turn to landing (TLand) in the pole test. (C) Accelerating rotarod test (3-cm diameter), in which the rod accelerated from 3 to 35 rpm over 5 min. Data are mean ± SEM of 8 to 10 mice.
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Fig. 2 – Evoked dopamine overflow. (A and B): Representative records of evoked dopamine by a single medial forebrain bundle (MFB) stimulation (50 Hz × 100 pulses, gray lines) in 3-month-old PKO and WT mice before (A) and after (B) administration of nomifensine. (C and D): The maximal amplitude of evoked DA (Δ[DA]max ) before (C) and after (D) nomifensine. The half-life time of evoked DA (T1/2 ) before (E) and after (F) nomifensine. Data are mean ± SEM. *P < 0.05 (Bonferroni's correction).
and PKO (F(1,52) = 51.5, P < 0.001). After nomifensine, the genotype effect was again significant (F(1, 59) = 6.70, P = 0.01), but that of age was not (F(3,52) = 1.26, P = 0.30) (Fig. 2F).
2.3.
DA dynamics under repeated stimulations
After evaluating the amplitude of 2-s stimulation, dopamine dynamics induced by repeated stimulation were evaluated. Consistent with previous studies (Kita et al., 2007; Yavich et al.,
2004), both depression between block and facilitation within block were observed in all mice (Fig. 3A). Between-block depression was not different between WT and PKO mice and there was no significant age effect (Fig. 3B). The between-block depression was similar after administration of nomifensine except at 9 months of age (F(1,13) = 5.97, P = 0.03). On the other hand, facilitation was reduced by age in both WT and PKO mice. The effect of genotype was significant in 9- (F(1,13) = 7.47, P = 0.017) and 12-month-old groups (F(1,14) = 7.35, P = 0.017).
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Fig. 3 – Repeated train stimulations. (A) Representative records of WT and KO from 3-month-age group, before and after nomifensine. Every burst-block consisted of six 2-s stimulations with 5-s intervals and each burst-block was repeated three times every 90 s. Each peak-amplitude was measured by subtracting the baseline before the first pulse from the peak. (B) Depression of the first stimulation among the blocks. The amplitude of the first stimulation of each block is expressed relative to the amplitude of the first stimulation of the first train. (C) Facilitation within the first burst block. The peak amplitude to each 2-s stimulation is expressed relative to the amplitude on the first stimulation. Data are mean ± SEM of 7–8 mice. *P < 0.05 (Repeated ANOVA/Bonferroni's correction).
This facilitation disappeared after administration of nomifensine (Fig. 3C). To evaluate the effect of inter-stimulation interval, three 2-s stimulations were applied at various intervals (interval test, Fig. 4). The net increase in amplitude was expressed as a percentage of the first burst's amplitude. Before the administration of nomifensine, a slight depression was observed at long intervals of 40 and 20 s, but it changed to facilitation at short intervals of less than 10 s, in both WT and PKO mice. There were no significant differences between WT and PKO, except for the 12month-old groups (2nd: F(1,14) =7.94, P=0.014, 3rd: F(1,14) =5.52, P=0.0033). There was a significant age effect in the second (F(3,27) =3.59, P=0.027) and third pulses (F(3,27) =3.49, P=0.029) of WT, whereas PKO did not show any significant change with age.
Nomifensine administration resulted in the disappearance of facilitation and appearance of depression at short intervals less than 10 s, which was considered due to paired pulse depression by D2 autoregulation (Benoit-Marand et al., 2001; Schmitz et al., 2002; Schmitz et al., 2003). There were no significant differences in the effects of genotype and age.
2.4.
Neurochemical analysis
In order to confirm the density of DA projection to the striatum, the striatal tissue contents of DA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5Hydroxyindoleacetic acid (5-HIAA) were measured by highperformance liquid chromatography (HPLC, Table 1). The
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Fig. 4 – The interval test. (A) Representative records of WT and KO from 3-month-age group. Three 2-s stimulations were applied at different intervals of 40, 20, 10, 7, 5, and 3 s before (gray lines) and after (black lines) nomifensine. The net increase of amplitude was measured by subtracting the baseline of each pulse from each peak. The amplitudes of the second and third pulse relative to the first pulse were plotted against stimulation interval before (B) and after (C) nomifensine. Data are mean±SEM of 7–8 mice.
contents of DA and monoamine metabolites did not show significant genotype effects (DA: F(1,62) = 0.06, P = 0.81; DOPAC: F(1,62) = 0, P = 1.00, HVA: F(1,62) = 0.27, P = 0.61, 5-HIAA; F(1,62) = 0.10,
P = 0.75). However, there was a significant age effect on DOPAC concentration (F(3,55) = 5.14, P = 0.003), mainly due to low concentration in 3-month-old mice.
Table 1 – Neurochemical analysis.
3 months 6 months 9 months 12 months
WT (n = 7) KO (n = 8) WT (n = 7) KO (n = 7) WT (n = 7) KO (n = 6) WT (n = 8) KO (n = 8)
DA
DOPAC
HVA
5-HIAA
93.9 ± 6.8 94.2 ± 4.4 89.8 ± 7.6 88.4 ± 8.3 70.2 ± 8.8 80.4 ± 14.1 95.8 ± 5.2 91.7 ± 8.2
13.6 ± 1.6 12.9 ± 0.8 22.4 ± 2.1 18.5 ± 1.2 11.8 ± 2.1 19.4 ± 5.8 23.2 ± 2.5 20.2 ± 3.2
30.8 ± 4.0 29.2 ± 1.6 31.7 ± 3.6 31.5 ± 2.5 21.9 ± 3.4 28.0 ± 1.8 32.4 ± 1.7 30.3 ± 1.6
5.3 ± 0.6 5.1 ± 0.2 5.8 ± 0.6 5.6 ± 0.4 4.7 ± 0.8 5.3 ± 0.5 5.5 ± 0.3 4.7 ± 0.2
Striatal contents in pmol/mg of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindoleacetic acid (5-HIAA) measured by HPLC, after electrochemical tests. There are no significant genotype effects. However, there was a significant age effect on DOPAC due to 3-month age group (Uni-ANOVA/Bonferroni's correction). Data are mean ± SEM. P < 0.05.
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2.5.
Histology
In the majority of cases, the electrode tip was located within the TH-positive fiber bundles. Deviation of the electrode tip from the center of TH-positive fibers was not different between WT (0.19 ±0.03 mm) and PKO mice (0.15 ± 0.02) (F(1,55) = 1.20, P = 0.28).
3.
Discussion
In the present study, although there were no differences between PKO and WT mice in the behavioral test, PKO mice showed lower amplitudes and longer half-lives of evoked DA than WT mice did (Fig. 2). This may suggest early presymptomatic changes in dopaminergic transmission in PKO mice. In addition, the facilitation was reduced by age in general, but remained high in the older PKO mice compare to WT mice (Fig. 3C). This facilitation may suggest a compensatory adaptation during the late pre-symptomatic phase or possibly during the symptomatic phase. The amplitude of evoked DA overflow is a consequence of competing mechanisms of synaptic release and uptake (Schmitz et al., 2002; Schmitz et al., 2003). On the other hand, the falling phase of evoked DA depends on the DA uptake by DA transporter (DAT). Therefore the increase of half-life observed in PKO mice suggests a reduced DA uptake (Fig. 2), which rather increases the amplitude of dopamine overflow. These results suggest that the decrease of evoked DA amplitude is due to reduced DA vesicular release. Itier et al. (2003) showed low amphetamine- or methamphetamineinduced DA efflux in PKO mice, but this may reflect reverse transport of dopamine through DAT (Riddle et al., 2006). A recent study showed impaired DA release in acute striatal slices of PKO mice (Kitada et al., 2009a). Interestingly, Kitada et al. (2009a) did not detect differences in 3H labeled DA uptake in PKO compared to WT, whereas Itier et al. (2003) showed functional reduction in DA uptake. The reduction in DA uptake can be a secondary change to compensate the decreased DA release. However the opposite is also possible, that is, decreased DA release could be a secondary change to compensate for reduced uptake. These hypotheses require confirmation in future studies. In the brain, PARKIN protein is found in the presynaptic vesicles (Kubo et al., 2001; Mouatt-Prigent et al., 2004). Although exactly how parkin deficiency alters DA transmission at the presynapse remains obscure, we speculate three possibilities. First, since PARKIN is an ubiquitin-E3 ligase, disruption of proteolysis of functional synaptic proteins may impact synaptic function, as another synapse-localized E3 ligase, SCRAPPER may do (Yao et al., 2007). Second, parkin deficiency may cause changes in mitochondrial function for energy metabolism (Darios et al., 2003; Periquet et al., 2005), so that the highly energy-dependent processes of exocytosis may be influenced. Third, mitochondrial dysfunction can lead to higher H2O2 level, which is a potent suppressor of DA release (Avshalumov and Rice, 2003). More studies are needed to confirm these hypotheses. The within-block facilitation has been explained as modification of D2 receptor auto-regulation (Kita et al., 2007),
219
which suppresses the subsequent DA release in repeated pulse stimuli (Benoit-Marand et al., 2001; Schmitz et al., 2002, 2003). However, the within-block facilitation in our study completely disappeared in the presence of nomifensine (Fig. 3C), which suggests that facilitation also reflects DAT function. Indeed, D2 auto-receptor and DAT interact with each other (Baik et al., 1995; Benoit-Marand et al., 2001; Dickinson et al., 1999; Schmitz et al., 2002, 2003). The lack of changes in the interval tests after suppression of DAT may suggest a relatively intact pre-synaptic D2 receptor function (Fig. 4C). However, there is a possibility that slowed DAT increases basal DA levels and consequently alters autoreceptor activation. Furthermore, nomifensine is a competitive inhibitor and does not block DAT completely. More direct approach would be needed to assess the contribution of autoreceptors. Although PARK2 is young-onset, the changes of dopaminergic neurotransmission related to PD are expected to progress by age. In this study, the age effect for evoked DA amplitude was not significant, and less evident in older animals (Fig. 2). Considering the parkin mRNA and protein show marked increase in expression levels during midgestational development in the central nervous system followed by a steady increase until adulthood (Kuhn et al., 2004), we speculate that parkin deficiency can influence DA release in the early developmental phase in PKO mice, followed by some possible secondary changes to compensate for it. On the other hand, enhancement of facilitation was evident in matured (9–12-month-old) mice, suggesting progressive changes by aging (Fig. 3C). These dysfunctions may be subtle and may not impact motor function under physiological condition in young mice. In addition, the life span of mice may be too short, even in aged mice with deficits of parkin and other recessive genes (DJ1−, PINK1) (Kitada et al., 2009b), to confirm the degenerative changes seen in parkin−/− human. In conclusion, we presented direct evidence for changes in dopaminergic neurotransmission in PKO mice in vivo. When combined with the in situ study of PKO mice (Kitada et al., 2009a), our results indicate that genetic deficits in parkin may lead to early-onset physiological dysfunctions of dopaminergic neurons.
4.
Experimental procedures
4.1.
Animals
The parkin−/− mouse (male, 3–12 months old), which carries a chromosomal replacement of exon 3 of the parkin gene (Kitao et al., 2007) was used. The mice were backcrossed into the C57BL/6 J mice for 10 generations, then both +/+ wildtype (WT) and −/− (PKO) littermates were sub-strained and maintained in parallel in the same animal facility at Juntendo University, Tokyo. WT and PKO mice of close birth dates were subjected to behavioral tests on the same day in a shuffled sequence. DNA samples from each animal were confirmed after all experiments through genotyping. Three-, six-, nine- and twelve-month-old mice were subjected to the accelerating rotarod and the pole tests on the same day and then evaluated with in vivo voltammetry
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within three weeks in a random sequence. Mice that died during the surgeries (two in 6 months, two in 9 months, and three in 12 months) and the recordings (two in 6 months and one in 9 months) were not included in the data analysis. Two to six mice were housed per cage, fed solid food with water available ad libitum and kept under controlled conditions maintained on a 12-h light–dark cycle at constant temperature and humidity. The procedures followed in using laboratory animals were in accordance with the Guidelines for Proper Conduct of Animal Experiments by the Science Council of Japan and all experiments were approved by the Ethics Review Committee for Animal Experimentation of Juntendo University School of Medicine. All efforts were made to minimize the number of animals used and their suffering.
4.2.
Behavioral tests
Behavioral tests were performed during the light cycle and conducted by the same investigator. The behavioral tests were performed in the same room housing the animals. Accelerating rotarod test. Mice were placed on the 3-cm diameter rotating rod covered by rubber (Ugo Basile, Acceler Rotarod 7650 for mice, Jones and Roberts, Italy). The rotation was accelerated, starting at 3 rpm and constantly accelerated to 35 rpm over 5 min, and the fall latency was recorded. The test was repeated three times with a 15-min inter-test interval. The first and second trials were considered acclimation trials, mice were placed on the rod immediately after falling, up to 5 min. The first fall latency of the third trial was analyzed. Pole test. Mice were placed on the top of a 30-cm-tall, clothwrapped, 1.5-cm-diameter pole. The latency before the mouse turned towards the ground (Tturn) and time from the turn to landing (Tland) were recorded. The test was repeated three times with a 15-min inter-test interval. The first and second trials were acclimation trials and data from the third trial was collected and analyzed.
4.3.
Surgical preparation
Carbon-fiber microelectrodes were stereotaxically implanted in the right striatum and a pair of stimulating electrodes placed in the MFB on the same side, as described previously (Natori et al., 2009; Suzuki et al., 2007; Yavich et al., 2004). Mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and placed on a stereotaxic frame (Narishige, Tokyo). Anesthesia was maintained with 1% isoflurane inhalation with 30% O2 and 70% N2O. Rectal temperature was maintained at 37 ± 1.0 °C using a heating lamp and a body warmer. The carbonfiber (HTA-7, Toho Tenax Co., Tokyo; 7 μm in diameter) was cut to a length of 500 μm from the end of the pulled capillary glass, and insulated with epoxy glue, and inserted into the striatum (electrode tip position at 0.5 mm anteriorly (A), 2 mm laterally (L), and 3.2 mm ventrally (V) relative to the bregma) (insertion angle: 15° relative to the coronal plane and actually inserted from A1.5 mm at dura and proceeded 3.3 mm) according to a mouse brain atlas (Franklin and Paxinos, 1997). A pair of stainless steel bipolar stimulating electrodes (Unique Medical Co., Japan; distance between the poles: 1 mm, the stainless needle was insulated except for the 0.3-
mm tip) were implanted in the MFB (AP, −2.0 mm; L, 1.1 mm; V, −4.75 to −5.5 mm). The dorso-ventral placement of the stimulating electrode was adjusted to obtain the maximal DA overflow. The stimulation electrode was advanced by the steps 0.125 mm every 120 s, until the recorded evoked response's amplitude was equivalent to the preceding step and there was no further advancement (typically, 4.75 to 5.0 mm from dura). Two silver/silver chloride wires (reference and auxiliary electrodes), were combined in a glass tube filled with 3% agar made of phosphate buffered saline (pH 7.4, 100 mM phosphate, 0.9% NaCl, PBS) and placed onto the dural surface of the contralateral side.
4.4.
In vivo voltammetry
Stimulated DA overflow was measured by chronoamperometry (differential pulse voltammetry with constant holding potential) as described previously (Nakazato and Akiyama, 1999), with minor modifications. In short, four pairs of squarewave pulses of 20 ms duration and 200 mV amplitude were applied with progressive 100 mV shifts (−100 to 100, 0 to 200, 100 to 300 and 200 to 400 mV) and the current at the last 14 ms was measured and differentiated into high and low potential pairs. One cycle of recording lasted 240 ms and the holding potential between the recording pulses was −200 mV. In addition, biphasic activation pulses (1285 mV, 10 ms) were applied at the beginning of every cycle, to improve the endurance and stability of the carbon-fibers (Nakazato and Akiyama, 1999). Voltage controls, current recordings, and stimulation triggers were controlled automatically with A/D, D/A, timing boards (PCI-3165, PCI-3310 and PCI-6103, respectively, Interface Inc., Hiroshima, Japan), and a three-electrode potentiostat (Rainbow Science Co., Japan). The peak electrochemical current of DA was obtained at 0 to 200-mV pulse in vitro. The peak potential of evoked response in vivo was observed at 200- and 300-mV pulses. Electrochemical currents at 200-mV pulses were regarded as DA responses. After the experiments, the working electrode and the reference electrodes were calibrated using 1.0-μM DA in vitro in PBS (pH 7.4).
4.5. Electrical stimulation of MFB and stimulation protocols Biphasic constant current pulses were delivered to the MFB for 2-s (50 Hz; 100 biphasic 200 μA constant-current pulses; 2 ms each) through the stimulating electrodes from an isolated stimulator (SS-202J connected to SEN-7203, Nihon-Koden, Co., Japan). Stimulation pulses were automatically triggered from our recording system. After adjusting the depth of MFB stimulating electrode to yield the maximal evoked response, the DA current in response to 2-s stimulation was recorded more than three times. The maximum amplitude of the evoked DA overflow (Δ[DA]max) was expressed in molar concentrations (μmol per liter) based on post-calibration data. The half-life time (T1/2) of the evoked DA overflow was defined as the period of time (in seconds) for Δ[DA]max decreasing by half. To evaluate the dynamic regulation of DA release, we delivered different sequences of pulse trains to the MFB. In the first test, six blocks of three 2-s stimulation pulses were applied
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with different inter-pulse intervals of 40, 20, 10, 7, 5, and 3 s in this order (interval test). Each three-pulse block was followed by a break of 100 s, and a break of 200 s was placed before starting the next six-block sequence. In the second test, the dynamic modulation of release was evaluated using the same procedure used in the previous study (Yavich et al., 2004). In this test three burst blocks, each consisting of six 2-s stimulation pulses with 5-s intervals, were delivered with a 90-s break after each block. After completion of these measurements, nomifensine maleate (Sigma, St. Louis, MO, 7 mg/kg, subcutaneously), an uptake inhibitor of the DAT, was administered. DA responses to 2-s MFB stimulation were continuously recorded every 200 s to test the effect of the drug. Twenty minutes after the administration of nomifensine, the same sequences mentioned above were repeated.
4.6.
Neurochemical analysis
The striatal contents of DA and monoamine metabolites were measured by HPLC. After completion of the electrochemical tests, mice were deeply anesthetized with pentobarbital and perfused transcardially with 10 mM cold PBS. The brain tissue samples were cut immediately at the level of bregma with the aid of a mouse brain matrix (RBM-2000C; Activational System, Warren, MI). The anterior striatal tissues were dissected out, frozen on dry ice, and stored at −80 °C until use. The caudal part of the brain was fixed with ice-cold 4% buffered (pH 7.4) formaldehyde solution and used for histological identification of the MFB electrode position. Dissected striatal tissues were sonicated in ×20 volume of ice-cold 0.1 M perchloric acid (PCA) and centrifuged at 20,000 × g for 10 min at 4 °C. The resulting supernatant samples were applied to the HPLC system, which was equipped with a reverse-phase C18 column (150 × 4.6 mm; ODS-100s, Tosoh, Tokyo) and an eight-electrode coulometric electrochemical detection system (ESA-400, ESA, Inc., Chelmsford, MA). The concentration of DA and monoamine metabolites was expressed as picomoles per gram (pmol/mg) of tissue weight.
4.7.
Histological staining and examination
The location of the stimulation electrodes was verified histologically at the end of the study. After the voltammetric recording, the position of the stimulating electrode was marked with a DC current (40 μA DC for 20 s). The harvested brains were paraffin-embedded and sliced into 5-μm-thick sections and MFB was immunostained with tyrosine hydroxylase (TH).
4.8.
Statistical analysis
The effects of the two genotypes and four ages were analyzed by UNI-ANOVA (SPSS, Chicago, IL). When the measurements were repeated on the same mouse, repeated measurements (four training days, four pre- and post-administration periods, three blocks, six stimulations or six intervals) were treated as within-subject factors and the genotype as a between-subject factor. If a major effect was identified, multiple comparisons were performed using Bonferroni's correction. All values are
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expressed as mean ± SEM. A P value less than 5% denoted the presence of a statistically significant difference between the groups.
Acknowledgments The authors thank Dr. Akitane Akiyama for his help with the experimental equipment and Mr. Taka-aki Yanase for technical assistance. This work was supported in part by the Strategic Research Program for Brain Sciences (SRPBS) and a Grant-in-Aid for Scientific Research on Priority Areas “Emergence of Adaptive Motor Function through Interaction between Body, Brain and Environment” from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
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