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Alteration of methamphetamine-induced striatal dopamine release in mint-1 knockout mice
Neuroscience Research 43 (2002) 251 /257 www.elsevier.com/locate/neures
Alteration of methamphetamine-induced striatal dopamine release in mint-1 knockout mice Atsushi Mori a,*, Keiji Okuyama b, Masato Horie a, Yoshihiro Taniguchi b, Takashi Wadatsu b, Naoki Nishino b, Yoshikazu Shimada b, Norihiro Miyazawa b, Satoshi Takeda b, Masashi Niimi b, Hiroyuki Kyushiki b, Mari Kondo b, Yasuhide Mitsumoto a a
Second Institute of New Drug Research, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno Kawauchi-cho, Tokushima 771-0192, Japan b Otsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno Kawauchi-cho, Tokushima 771-0192, Japan Received 15 November 2001; accepted 22 March 2002
Abstract Mint-1, which is also called as X11 or mammalian Lin10, protein has been implicated in the synaptic vesicle exocytosis and the targeting and localization of synaptic membrane proteins. Here, we established mint-1 gene knockout (mint-1 KO) mice and investigated vesicular and transporter-mediated dopamine (DA) release evoked by high K and methamphetamine (METH), respectively. Compared with wild-type control, high K -evoked striatal DA release was attenuated, but not significantly, in the KO mice as measured by microdialysis method. The METH-induced DA release was significantly attenuated in the KO mice. In addition, METH-induced stereotypy was also significantly attenuated in the KO mice. Mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object as compared with wild-type mice. These results suggest that mint-1 protein is involved in transporter-mediated DA release induced by METH. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Mint-1; Knockout mouse; Dopamine release; Microdialysis; Stereotypy; Dopamine transporter
1. Introduction Mint-1, which is also called as X11 or mammalian Lin10, is a neuronal protein that interacts with munc181, an essential component of synaptic vesicle exocytosis. The implication of mint-1 in synaptic neurotransmitter release has been reported genetically or biochemically (Nguyen and Sudhof, 1997; Okamoto and Sudhof, 1997; Butz et al., 1998; Maximov et al., 1999; Biederer and Sudhof, 2000). Furthermore, mint-1 protein is thought to play an important role in the targeting and localization of synaptic membrane proteins, such as N -methyl-D aspartate (NMDA) receptor 2B (NR2B) subunit (Setou et al., 2000).
* Corresponding author. Tel.: /81-88-665-2126; fax: /81-88-6656106 E-mail address: [email protected] (A. Mori).
Neurotransmitters, such as catecholamines (CAs), can be released through two mechanisms (Raiteri et al., 1979): exocytotic release (vesicular release), which is calcium- and impulse-dependent, and transportermediated release (non-vesicular release), which is not impulse-dependent and has little calcium-dependence (Hurd and Ungerstedt, 1989; Pierce and Kalivas, 1997). In the impulse-dependent exocytotic release, the resting potential of the nerve cell is dependent largely upon the K gradient across the cell membrane (Alberts et al., 1989). The depolarization-induced synaptic vesicle exocytotic release of CAs to the synaptic cleft is dependent upon ATP hydrolysis, and is initiated by Ca2 influx (Winkler, 1988). In the transporter-mediated release, on the other hand, the release of CAs can be induced by reversing the Na gradient across the membrane. Speaking restrictedly to dopamine (DA) release, a methamphetamine (METH) or amphetamine (AMPH) administration induces this type of release. This process
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has been called reverse transport (Sulzer et al., 1995). In addition, a systemic administration of these drugs induces stereotypy, an animal model of a positive symptom in schizophrenia, by excessive dopaminergic neurotransmission through both DA releases (Randrup and Munkvad, 1965; Ellinwood et al., 1973) and DA uptake inhibitory action (Liang and Rutledge, 1982; Schmidt and Gibb, 1985; O’Dell et al., 1991; Seiden et al., 1993; Cubells et al., 1994) in rodents. Recently, we established mint-1 gene knockout (mint1 KO) mice by gene targeting in embryonic stem cells. If mint-1 protein is an essential component for exocytosis of synaptic vesicles and/or targeting and localization of neurotransmitter transporter proteins, alteration of neurotransmitter release through synaptic vesicles and/ or transporter might occur in mint-1 KO mice. Therefore, the main objective in this study was to clarify the precise role of mint-1 protein in the neurotransmitter release in vivo using mint-1 KO mice. Especially, high K condition and METH were employed to evoke DA release through vesicular and transporter-mediated release, respectively.
2. Experimental procedures 2.1. Animals and housing All procedures were conducted in accordance with ‘The Guidelines for Animal Experimentation in Otsuka Pharmaceutical Co., Ltd.’ Mint-1 KO mice were generated according to the methods described by Hogan et al. (Hogan et al., 1994). The targeting vector was constructed by replacing a 1.0-kb fragment containing the first methionine of the mint-1 gene with the pMC1-neopolyA cassette (Stratagene, USA, Fig. 1A). The linearized construct was electroporated into CCE28 ES cells derived from 129/Sv mouse, then G418-resistant ES clones were screened by probing Spe I-digested genomic DNAs with 5? and 3? external probes to obtain homologous recombinant (Fig. 1A). Chimeras were mated with C57BL/6J mice to obtain the heterozygous mutants. Mint-1 KO mice and wild-type mice used in the experiments were obtained by intercross of the heterozygous mutants, of which genetic background was hybrid of 129/Sv and C57BL/6J. The genotype of the mice was determined by Southern blot analysis or by PCR analysis of the genomic DNA prepared from the tails. In Northern blot analysis, total RNA from the mouse brain was hybridized with 5? end fragment (808 bp) of open reading frame of the mouse mint-1 cDNA. Anti-mint-1 chicken antibody, which was raised against N-terminal portion (residues 17 /229) of mouse mint-1 recombinant protein expressed in Escherichia coli , was used for Western blot analysis.
Test groups comprised male mint-1 KO mice and wild-type controls at 3 /10 months of age. All comparisons between two groups were made in mice of equivalent age. They had free access to food and water, and were maintained in a 12-h light/dark cycle. 2.2. In vivo microdialysis To examine DA release, an in vivo microdialysis method was used. Under pentobarbital anesthesia (50 mg/kg i.p.), a guide cannula for a microdialysis probe (EICOM, Japan) was stereotaxically implanted in the striatum (2 mm lateral to the right from the bregma and 1.5 mm deep from the skull surface). The next day, a microdialysis probe with 2 mm active membrane (EICOM) was inserted into the guide cannula and perfused continuously with artificial cerebrospinal fluid (aCSF, composition in mM: NaCl 147, KCl 4, CaCl2 2.3) at a flow rate of 2 ml/min, using a microinfusion pump (CMA/100, BAS, Japan). The dialysate was collected every 20 min (40 ml) and was immediately analyzed for DA by a high performance liquid chromatography (HPLC). After a 3-h equilibration period (9 fractions), the mice received a KCl pulse (100 mM for 30 min) delivered locally through the dialysis probe, and 10 fractions were analyzed. Then, METH (10 mg/kg, Dainippon Pharmaceutical Co., Ltd., Japan) was administered subcutaneously, and 9 fractions were analyzed. The HPLC system was equipped with an electrochemical detector (ECD-300, EICOM) on a separation column (Eicompak CA-ODS, 15 cm /2.1 mm I.D., EICOM). The mobile phase, pH 6.0, consisted of a 0.1 M phosphate buffer containing 500 mg/l sodium-1-octane-sulfonate, 50 mg/l ethylenediaminetetraacetic acid, disodium salt, and 20% (v/v) methanol. The flow rate was set at 0.25 ml/min. The electrode potential was set at /0.4 V against Ag/AgCl reference electrode potential. The DA content in each fraction was expressed as a percentage of the basal value, which was the DA content just before the KCl pulse (the value of DA content in fraction number 9). The summed effects of the local perfusion of the dialysis membrane with KCl or those of the METH injection over the course of the experiment were measured by determining the area under the curve (AUC). 2.3. Behavioral tests Basic motor skills were assessed using the subjects of tests described by Irwin (Irwin, 1968). The measurement of locomotor activity was performed as previously described (Kikuchi et al., 1995) with minor modifications. Mice were individually placed in plastic columnar chambers (30 cm in diameter and 30 cm high), and locomotor activity was immediately measured for 1 h, as described below. A pivot was fixed at the center of the
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Fig. 1. Targeted disruption of the mouse mint-1 gene. (A) Maps of the wild-type mint-1 locus, the targeting vector and mutated mint-1 locus. The first exon is shown by closed boxes. The location of the 5? external probe (5? probe) and the 3? external probe (3? probe) is shown. The 5? probe hybridized to a 12-kb Spe I fragment from the wild-type mint-1 locus and a 3.4-kb Spe I fragment from the mutated mint-1 locus, and 3? probe hybridized to an 12-kb Spe I fragment from the wild-type mint-1 locus and a 8.5-kb fragment from the mutated mint-1 locus. The regions used for PCR analysis for the genotyping of the mice are shown. ATG, initiation methionine; Neo, the neomycin resistance gene driven by the MC1 promoter; DT-A, the diphtheria toxin A-fragment gene driven by the MC1 promoter; S, Spe I; P, Pst I; H, Hind III; N, Not I; X, Xho I. (B) Southern blot analysis of the mouse genomic DNA. Spe I-digested genomic DNAs were hybridized with the 5? probe, as shown in A. (///), wild-type; (///), heterozygous mutant; (///), homozygous mutant. (C) Northern blot analysis for the mint-1 mRNA expression. Total RNA from the mouse brain was hybridized with the mouse mint-1 cDNA fragment. (D) Western blot analysis for the mint-1 protein expression. The mouse brain homogenate was analyzed using the anti-mouse mint-1 antibody.
bottom of each chamber, where the animals were placed, and six microswitches were fitted beneath the bottom. The locomotor movement of the animals generated a pulse by turning on the microswitches and electrically charging a magnetic counter, and the counts of the movement for initial 10 min and 1 h were calculated using a computer. In the elevated plus maze test, a plus-maze was elevated to a height of 40 cm above the floor. It was comprised of two open arms (5 /25 cm) and two enclosed arms (5 /25 /15 cm) that extended from a common central platform (5 /5 cm). The maze floor was made of black Plexiglas, while the side and end walls of the enclosed arms were made of clear Plexiglas. Each mouse was placed in the middle of an open arm facing the central platform. The test was started when the mouse entered the platform. During a 5-min test
period, the number of entries into the open and enclosed arms, and the time spent there was recorded. In the observation, all four paws had to be within an arm for an entry to be measured. As indices of anxiety, the percentages of open arm entries and time spent in the open arms were calculated. In the tail suspension test, the apparatus was a 4channel device incorporating an electric balance, an A/D converter, a testing box (30 /25 /25 cm), and a personal computer, in which BIBU96 software was installed (Yamashita Giken, Japan). The immobility state was determined by two indices (1.5 V of The Lowest Activity Value and 1 s of Rest Delay Time) in the software. A mouse was suspended from a hook in the testing box by an attachment, which was applied to the tail at 15 /20 mm from its tip. The duration of immobility was measured using the computer for a
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period of 6 min following the start of suspension. The data was acquired for the period from 2 to 6 min. The experiments were carried out in a soundproof room. In the METH-induced stereotypy, METH was administered subcutaneously at a dose of 10 mg/kg to mice, and the mice were placed individually in a glass beaker. Stereotypy was observed for 1 min at 10-min intervals from 20 to 40 min after the METH injection. The stereotypy induced by METH was graded 0 /3 according to the following criteria described by Puech et al. (Puech et al., 1981): 0 /absence of stereotypy or any abnormal movement; 1 /slight stereotyped head movements and intermittent sniffing; 2 /intense head movements, mild licking interspersed with sniffing; 3 /intense licking and/or gnawing. The total scores for three observations were then calculated.
3. Results 3.1. Generation of mint-1 KO mice Mint-1 KO mice were obtained with mating the heterozygous mutants. Although the mint-1 KO mice were just lighter in body weight than wild-type mice, they did not have abnormal physical features. And histological analysis of the brain in the mint-1 KO mice did not reveal any gross developmental abnormalities (data not shown). Southern blot analysis indicated that the mint-1 gene was homozygously disrupted in mint-1 KO mice (Fig. 1B). Northern blot analysis using the mouse mint-1 cDNA fragment as a probe showed that its mRNA was absent in the brain of mint-1 KO mice (Fig. 1C). Western blot analysis using anti-mouse mint-1 antibody showed that the mint-1 protein was not detectable in the brain of mint-1 KO mice (Fig. 1D). The expression of both mint-2, which is another neuronal isoform of mint-1 and has similar domain structures, mRNA and protein were not affected by the mint-1 mutation (data not shown).
Fig. 2. Effects of high K and methamphetamine on the extracellular levels of DA in the striatum of wild-type (k) and mint-1 KO mice (m). KCl was added to the perfusate at a concentration of 100 mM during 30 min marked by a horizontal bar, and methamphetamine was administered subcutaneously at a dose of 10 mg/kg at an arrow point (fraction no. 19). The data are expressed as percentages of fraction no. 9 and indicate the means9/S.E.M. values of six determinations. There is a significant difference (P B/0.05) between the wild-type control group and mint-1 KO group in the area of fraction nos. 19 /28 (Student’s t -test).
was similar in both genotypes (259/7 nM for wild-type and 289/2 nM for mint-1 KO mice in 40 ml of fraction No. 9). However, mint-1 KO mice showed lower, although not significant, DA release by the KCl pulse (64% of wild-type in AUC) than wild-type mice. Significant suppression of DA release by the METH injection (41% of wild-type in AUC) was observed in mint-1 KO mice. These data showed that the neurotransmitter release in mint-1 KO mice was suppressed only when the release was stimulated by a high K or METH injection. In addition, as a pharmacological behavioral analysis, METH-induced stereotypy was tested (Fig. 3). The expression of stereotypy in mint-1 KO mice was significantly attenuated as compared with that of wildtype mice and thus the suppression of METH-induced
3.2. Effects of mint-1 deficiency on DA release To investigate the effects of mint-1 on neurotransmitter release, extracellular DA levels were measured using in vivo microdialysis in the striatum of freely moving mice (Fig. 2). DA release was evoked by a KCl pulse through a dialysis probe or a systemic injection of METH to cause vesicular or transporter-mediated release, respectively. The basal value of DA content
Fig. 3. Performance in methamphetamine-induced stereotypy of mint1 mutant mice. Methamphetamine was administered subcutaneously at a dose of 10 mg/kg, and stereotypy was graded according to a criteria described by Puech et al. (Puech et al., 1981). The data are expressed as the total scores of three observations, and columns indicate the means9/S.E.M. values of six animals. * P B/0.05 (Wilcoxon’s rank sum test).
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DA release was also demonstrated behaviorally as well as neurochemically.
3.3. Effects of mint-1 deficiency on behavior If the deficiency of mint-1 causes the abnormal neurotransmission through the dysfunction of exocytosis of synaptic vesicles and/or of transporter proteins, behavioral difference could be seen between wild-type and mint-1 KO mice. Therefore, we evaluated normal reflexes in standard neurological tests, horizontal movement (ambulation), and neuropsychological events (Table 1). Firstly, we performed behavioral observations according to Irwin’s method. Although mint-1 KO mice did not have tremor or ataxia, nor display an abnormal stance or posture, they showed more sensitive and prominent behavioral response to an approaching object than wild-type mice by touch escape test described by Irwin (Irwin, 1968). Only seven of 79 wild-type mice (8.8%) showed the slight escape response when the sides and body of the animal was stroked. On the other hand, 63 of 100 mint-1 KO mice (63%) showed the vigorous and rapid escape response. Locomotor activity was assessed in activity cages by recording every 10 min for 1 h. Although mint-1 KO mice showed a higher locomotor activity than wild-type mice both in the initial 10-min and 1-h measurement, there were no significant differences between the two genotypes. The elevated plus maze and tail suspension tests were performed as neuropsychological tests. Statistical analysis of the indices in these tests did not reveal any significant differences in effects between the genotypes. Thus, both anxiety and depression levels seem to be similar between the wild-type and mint-1 KO mice. Table 1 Performance of the mint-1 gene knockout and wild-type mice on the multiple behavioral task Tests
N
Locomotor activity (counts) 10 min 10 60 min 10
Wild-type
N
Mint-1 KO
151.89/32.2 335.69/90.3
9 9
184.79/48.1 403.89/112.6
Elevated plus maze % of Time % of Entry
10 10
23.49/4.5 37.79/8.3
9 9
27.09/9.0 34.39/8.5
Tail suspension Immobility time (s)
7
186.89/10.6
9
206.19/9.7
Each test was performed as described in Section 2. % of Time and % of Entry in elevated plus maze represents percentage of time spent in the open arms and percentage of open arm entries, respectively. Immobility time in Tail suspension represents the data acquired for the period from 2 to 6 min. The data are the means9/S.E.M.
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4. Discussion Direct evidence to clarify the effects of mint-1 deficiency on DA release was acquired from the data of in vivo microdialysis and METH-induced stereotypy (Figs. 2 and 3). In microdialysis, although basal value of DA content was the same in the two genotypes, the striatal DA release induced by a KCl pulse and METH injection in mint-1 KO mice was suppressed to 64 and 41%, respectively, as compared with the wild-type controls. These results indicate that mint-1 deficiency causes an alteration of the neurotransmitter release through vesicular or transporter-mediated release. As a reason for the suppression of the former DA release, at first, the disruption of mint-1/munc18-1/syntaxin1 complex (Okamoto and Sudhof, 1997) was considered from the results of high K stimulation. As seen in a previous study with Doc2a, which is another neuronal protein interacted with both munc18 and munc13 (Orita et al., 1997; Verhage et al., 1997), KO mice (Sakaguchi et al., 1999), DA release was usually normal in mint-1 KO mice. Although the detailed action mechanisms of Doc2a are not clear, Doc2a is reported not to be essential for Ca2-dependent neurotransmitter release but may modulate neurotransmitter release during repetitive synaptic activation. Therefore, the suppression of high K -evoked DA release in mint-1 KO mice may not be caused by the attenuation of munc18-related exocytosis of synaptic vesicles. However, further investigation will be needed to clarify how mint-1 protein is involved in the high K -evoked DA release. DA release induced by a METH injection is nonvesicular and transporter-mediated release (Liang and Rutledge, 1982), and it was significantly suppressed to 41% in mint-1 KO mice as measured by microdialysis method. In addition, METH-induced stereotypy was significantly attenuated in the KO mice. These results from both neurochemical and behavioral analysis suggest that DA release via dopamine transporters (DAT) was clearly inhibited by mint-1 deficiency. Setou et al. reported that NR2B subunit in NMDA receptor was transported along microtubules by KIF17, a neuronspecific molecular motor in neuronal dendrites, and the transport was accomplished by direct interaction of the KIF17 tail with a PDZ domain of mint-1, which was a constituent of a protein complex including CASK, velis, and NR2 subunit (Setou et al., 2000). More recently, it has been suggested that PICK1, which is a member of PDZ domain-containing protein, participates in the localization and expression of monoamine transporters (Torres et al., 2001). Taken together, the results presented here suggest that mint-1 may be involved in the targeting and localization of DAT proteins. In the observation of neurological and neuropsychological behavior, mint-1 KO mice were indistinguishable from their wild-type in gross behavior. And there were
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no significant behavioral differences between mint-1 KO mice and wild-type control in neuropsychological tests such as the elevated plus maze test for anxiety and the tail suspension test for depression (Table 1). Compared with wild-type controls, however, mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object. On the other hand, the locomotor activity for the initial 10 min was measured as an activity index in a novel environment. Mint-1 KO mice showed slightly higher, although not significant, than wild-type controls in this index (Table 1). Another study of ours demonstrated that 18-month-old mint-1 KO mice showed a significantly higher locomotor activity for the initial 10 min than age-matched wildtype controls (unpublished data). From these behavioral observations, only when the mice were stimulated by an approaching object or a novel environment, they showed sensitive responses. Interestingly, like mint-1 KO mice, DAT KO mice also showed hyperactivity in a novel environment, and it was considered that an excess of DA in the synaptic cleft caused the hyperactivity (Ralph et al., 2001). Even in mint-1 KO mice, the same event might occur by the suppressed DA release, which was caused by the disappearance of DAT proteins from pre-synaptic membranes. In summary, we demonstrated the alterations of dopamine release in mint-1 KO mice in vivo and the presumably concomitant behavioral changes. The neurochemical and behavioral changes could be due to the impairment of transporter-mediated DA release. These findings indicate that mint-1 proteins may have an important role in the targeting and localization of DAT proteins. Various psychiatric disorders, such as schizophrenia, drug abuse and attention deficit-hyperactivity disorder (ADHD), believed to have hyperdopaminergic activity have been associated with DAT dysfunction. Therefore, functional regulation of DAT proteins by mint-1 molecule represents a new avenue of future research to understand the pathogenesis of these disorders.
Acknowledgements We thank Drs Akira Tanigami, Toyoki Mori, Hisashi Kitagawa, Masami Nakai, and Mr Tetsuro Kikuchi for their helpful comments and support.
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Alteration of methamphetamine-induced striatal dopamine release in mint-1 knockout mice Atsushi Mori a,*, Keiji Okuyama b, Masato Horie a, Yoshihiro Taniguchi b, Takashi Wadatsu b, Naoki Nishino b, Yoshikazu Shimada b, Norihiro Miyazawa b, Satoshi Takeda b, Masashi Niimi b, Hiroyuki Kyushiki b, Mari Kondo b, Yasuhide Mitsumoto a a
Second Institute of New Drug Research, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno Kawauchi-cho, Tokushima 771-0192, Japan b Otsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno Kawauchi-cho, Tokushima 771-0192, Japan Received 15 November 2001; accepted 22 March 2002
Abstract Mint-1, which is also called as X11 or mammalian Lin10, protein has been implicated in the synaptic vesicle exocytosis and the targeting and localization of synaptic membrane proteins. Here, we established mint-1 gene knockout (mint-1 KO) mice and investigated vesicular and transporter-mediated dopamine (DA) release evoked by high K and methamphetamine (METH), respectively. Compared with wild-type control, high K -evoked striatal DA release was attenuated, but not significantly, in the KO mice as measured by microdialysis method. The METH-induced DA release was significantly attenuated in the KO mice. In addition, METH-induced stereotypy was also significantly attenuated in the KO mice. Mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object as compared with wild-type mice. These results suggest that mint-1 protein is involved in transporter-mediated DA release induced by METH. # 2002 Elsevier Science Ireland Ltd and the Japan Neuroscience Society. All rights reserved. Keywords: Mint-1; Knockout mouse; Dopamine release; Microdialysis; Stereotypy; Dopamine transporter
1. Introduction Mint-1, which is also called as X11 or mammalian Lin10, is a neuronal protein that interacts with munc181, an essential component of synaptic vesicle exocytosis. The implication of mint-1 in synaptic neurotransmitter release has been reported genetically or biochemically (Nguyen and Sudhof, 1997; Okamoto and Sudhof, 1997; Butz et al., 1998; Maximov et al., 1999; Biederer and Sudhof, 2000). Furthermore, mint-1 protein is thought to play an important role in the targeting and localization of synaptic membrane proteins, such as N -methyl-D aspartate (NMDA) receptor 2B (NR2B) subunit (Setou et al., 2000).
* Corresponding author. Tel.: /81-88-665-2126; fax: /81-88-6656106 E-mail address: [email protected] (A. Mori).
Neurotransmitters, such as catecholamines (CAs), can be released through two mechanisms (Raiteri et al., 1979): exocytotic release (vesicular release), which is calcium- and impulse-dependent, and transportermediated release (non-vesicular release), which is not impulse-dependent and has little calcium-dependence (Hurd and Ungerstedt, 1989; Pierce and Kalivas, 1997). In the impulse-dependent exocytotic release, the resting potential of the nerve cell is dependent largely upon the K gradient across the cell membrane (Alberts et al., 1989). The depolarization-induced synaptic vesicle exocytotic release of CAs to the synaptic cleft is dependent upon ATP hydrolysis, and is initiated by Ca2 influx (Winkler, 1988). In the transporter-mediated release, on the other hand, the release of CAs can be induced by reversing the Na gradient across the membrane. Speaking restrictedly to dopamine (DA) release, a methamphetamine (METH) or amphetamine (AMPH) administration induces this type of release. This process
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has been called reverse transport (Sulzer et al., 1995). In addition, a systemic administration of these drugs induces stereotypy, an animal model of a positive symptom in schizophrenia, by excessive dopaminergic neurotransmission through both DA releases (Randrup and Munkvad, 1965; Ellinwood et al., 1973) and DA uptake inhibitory action (Liang and Rutledge, 1982; Schmidt and Gibb, 1985; O’Dell et al., 1991; Seiden et al., 1993; Cubells et al., 1994) in rodents. Recently, we established mint-1 gene knockout (mint1 KO) mice by gene targeting in embryonic stem cells. If mint-1 protein is an essential component for exocytosis of synaptic vesicles and/or targeting and localization of neurotransmitter transporter proteins, alteration of neurotransmitter release through synaptic vesicles and/ or transporter might occur in mint-1 KO mice. Therefore, the main objective in this study was to clarify the precise role of mint-1 protein in the neurotransmitter release in vivo using mint-1 KO mice. Especially, high K condition and METH were employed to evoke DA release through vesicular and transporter-mediated release, respectively.
2. Experimental procedures 2.1. Animals and housing All procedures were conducted in accordance with ‘The Guidelines for Animal Experimentation in Otsuka Pharmaceutical Co., Ltd.’ Mint-1 KO mice were generated according to the methods described by Hogan et al. (Hogan et al., 1994). The targeting vector was constructed by replacing a 1.0-kb fragment containing the first methionine of the mint-1 gene with the pMC1-neopolyA cassette (Stratagene, USA, Fig. 1A). The linearized construct was electroporated into CCE28 ES cells derived from 129/Sv mouse, then G418-resistant ES clones were screened by probing Spe I-digested genomic DNAs with 5? and 3? external probes to obtain homologous recombinant (Fig. 1A). Chimeras were mated with C57BL/6J mice to obtain the heterozygous mutants. Mint-1 KO mice and wild-type mice used in the experiments were obtained by intercross of the heterozygous mutants, of which genetic background was hybrid of 129/Sv and C57BL/6J. The genotype of the mice was determined by Southern blot analysis or by PCR analysis of the genomic DNA prepared from the tails. In Northern blot analysis, total RNA from the mouse brain was hybridized with 5? end fragment (808 bp) of open reading frame of the mouse mint-1 cDNA. Anti-mint-1 chicken antibody, which was raised against N-terminal portion (residues 17 /229) of mouse mint-1 recombinant protein expressed in Escherichia coli , was used for Western blot analysis.
Test groups comprised male mint-1 KO mice and wild-type controls at 3 /10 months of age. All comparisons between two groups were made in mice of equivalent age. They had free access to food and water, and were maintained in a 12-h light/dark cycle. 2.2. In vivo microdialysis To examine DA release, an in vivo microdialysis method was used. Under pentobarbital anesthesia (50 mg/kg i.p.), a guide cannula for a microdialysis probe (EICOM, Japan) was stereotaxically implanted in the striatum (2 mm lateral to the right from the bregma and 1.5 mm deep from the skull surface). The next day, a microdialysis probe with 2 mm active membrane (EICOM) was inserted into the guide cannula and perfused continuously with artificial cerebrospinal fluid (aCSF, composition in mM: NaCl 147, KCl 4, CaCl2 2.3) at a flow rate of 2 ml/min, using a microinfusion pump (CMA/100, BAS, Japan). The dialysate was collected every 20 min (40 ml) and was immediately analyzed for DA by a high performance liquid chromatography (HPLC). After a 3-h equilibration period (9 fractions), the mice received a KCl pulse (100 mM for 30 min) delivered locally through the dialysis probe, and 10 fractions were analyzed. Then, METH (10 mg/kg, Dainippon Pharmaceutical Co., Ltd., Japan) was administered subcutaneously, and 9 fractions were analyzed. The HPLC system was equipped with an electrochemical detector (ECD-300, EICOM) on a separation column (Eicompak CA-ODS, 15 cm /2.1 mm I.D., EICOM). The mobile phase, pH 6.0, consisted of a 0.1 M phosphate buffer containing 500 mg/l sodium-1-octane-sulfonate, 50 mg/l ethylenediaminetetraacetic acid, disodium salt, and 20% (v/v) methanol. The flow rate was set at 0.25 ml/min. The electrode potential was set at /0.4 V against Ag/AgCl reference electrode potential. The DA content in each fraction was expressed as a percentage of the basal value, which was the DA content just before the KCl pulse (the value of DA content in fraction number 9). The summed effects of the local perfusion of the dialysis membrane with KCl or those of the METH injection over the course of the experiment were measured by determining the area under the curve (AUC). 2.3. Behavioral tests Basic motor skills were assessed using the subjects of tests described by Irwin (Irwin, 1968). The measurement of locomotor activity was performed as previously described (Kikuchi et al., 1995) with minor modifications. Mice were individually placed in plastic columnar chambers (30 cm in diameter and 30 cm high), and locomotor activity was immediately measured for 1 h, as described below. A pivot was fixed at the center of the
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Fig. 1. Targeted disruption of the mouse mint-1 gene. (A) Maps of the wild-type mint-1 locus, the targeting vector and mutated mint-1 locus. The first exon is shown by closed boxes. The location of the 5? external probe (5? probe) and the 3? external probe (3? probe) is shown. The 5? probe hybridized to a 12-kb Spe I fragment from the wild-type mint-1 locus and a 3.4-kb Spe I fragment from the mutated mint-1 locus, and 3? probe hybridized to an 12-kb Spe I fragment from the wild-type mint-1 locus and a 8.5-kb fragment from the mutated mint-1 locus. The regions used for PCR analysis for the genotyping of the mice are shown. ATG, initiation methionine; Neo, the neomycin resistance gene driven by the MC1 promoter; DT-A, the diphtheria toxin A-fragment gene driven by the MC1 promoter; S, Spe I; P, Pst I; H, Hind III; N, Not I; X, Xho I. (B) Southern blot analysis of the mouse genomic DNA. Spe I-digested genomic DNAs were hybridized with the 5? probe, as shown in A. (///), wild-type; (///), heterozygous mutant; (///), homozygous mutant. (C) Northern blot analysis for the mint-1 mRNA expression. Total RNA from the mouse brain was hybridized with the mouse mint-1 cDNA fragment. (D) Western blot analysis for the mint-1 protein expression. The mouse brain homogenate was analyzed using the anti-mouse mint-1 antibody.
bottom of each chamber, where the animals were placed, and six microswitches were fitted beneath the bottom. The locomotor movement of the animals generated a pulse by turning on the microswitches and electrically charging a magnetic counter, and the counts of the movement for initial 10 min and 1 h were calculated using a computer. In the elevated plus maze test, a plus-maze was elevated to a height of 40 cm above the floor. It was comprised of two open arms (5 /25 cm) and two enclosed arms (5 /25 /15 cm) that extended from a common central platform (5 /5 cm). The maze floor was made of black Plexiglas, while the side and end walls of the enclosed arms were made of clear Plexiglas. Each mouse was placed in the middle of an open arm facing the central platform. The test was started when the mouse entered the platform. During a 5-min test
period, the number of entries into the open and enclosed arms, and the time spent there was recorded. In the observation, all four paws had to be within an arm for an entry to be measured. As indices of anxiety, the percentages of open arm entries and time spent in the open arms were calculated. In the tail suspension test, the apparatus was a 4channel device incorporating an electric balance, an A/D converter, a testing box (30 /25 /25 cm), and a personal computer, in which BIBU96 software was installed (Yamashita Giken, Japan). The immobility state was determined by two indices (1.5 V of The Lowest Activity Value and 1 s of Rest Delay Time) in the software. A mouse was suspended from a hook in the testing box by an attachment, which was applied to the tail at 15 /20 mm from its tip. The duration of immobility was measured using the computer for a
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period of 6 min following the start of suspension. The data was acquired for the period from 2 to 6 min. The experiments were carried out in a soundproof room. In the METH-induced stereotypy, METH was administered subcutaneously at a dose of 10 mg/kg to mice, and the mice were placed individually in a glass beaker. Stereotypy was observed for 1 min at 10-min intervals from 20 to 40 min after the METH injection. The stereotypy induced by METH was graded 0 /3 according to the following criteria described by Puech et al. (Puech et al., 1981): 0 /absence of stereotypy or any abnormal movement; 1 /slight stereotyped head movements and intermittent sniffing; 2 /intense head movements, mild licking interspersed with sniffing; 3 /intense licking and/or gnawing. The total scores for three observations were then calculated.
3. Results 3.1. Generation of mint-1 KO mice Mint-1 KO mice were obtained with mating the heterozygous mutants. Although the mint-1 KO mice were just lighter in body weight than wild-type mice, they did not have abnormal physical features. And histological analysis of the brain in the mint-1 KO mice did not reveal any gross developmental abnormalities (data not shown). Southern blot analysis indicated that the mint-1 gene was homozygously disrupted in mint-1 KO mice (Fig. 1B). Northern blot analysis using the mouse mint-1 cDNA fragment as a probe showed that its mRNA was absent in the brain of mint-1 KO mice (Fig. 1C). Western blot analysis using anti-mouse mint-1 antibody showed that the mint-1 protein was not detectable in the brain of mint-1 KO mice (Fig. 1D). The expression of both mint-2, which is another neuronal isoform of mint-1 and has similar domain structures, mRNA and protein were not affected by the mint-1 mutation (data not shown).
Fig. 2. Effects of high K and methamphetamine on the extracellular levels of DA in the striatum of wild-type (k) and mint-1 KO mice (m). KCl was added to the perfusate at a concentration of 100 mM during 30 min marked by a horizontal bar, and methamphetamine was administered subcutaneously at a dose of 10 mg/kg at an arrow point (fraction no. 19). The data are expressed as percentages of fraction no. 9 and indicate the means9/S.E.M. values of six determinations. There is a significant difference (P B/0.05) between the wild-type control group and mint-1 KO group in the area of fraction nos. 19 /28 (Student’s t -test).
was similar in both genotypes (259/7 nM for wild-type and 289/2 nM for mint-1 KO mice in 40 ml of fraction No. 9). However, mint-1 KO mice showed lower, although not significant, DA release by the KCl pulse (64% of wild-type in AUC) than wild-type mice. Significant suppression of DA release by the METH injection (41% of wild-type in AUC) was observed in mint-1 KO mice. These data showed that the neurotransmitter release in mint-1 KO mice was suppressed only when the release was stimulated by a high K or METH injection. In addition, as a pharmacological behavioral analysis, METH-induced stereotypy was tested (Fig. 3). The expression of stereotypy in mint-1 KO mice was significantly attenuated as compared with that of wildtype mice and thus the suppression of METH-induced
3.2. Effects of mint-1 deficiency on DA release To investigate the effects of mint-1 on neurotransmitter release, extracellular DA levels were measured using in vivo microdialysis in the striatum of freely moving mice (Fig. 2). DA release was evoked by a KCl pulse through a dialysis probe or a systemic injection of METH to cause vesicular or transporter-mediated release, respectively. The basal value of DA content
Fig. 3. Performance in methamphetamine-induced stereotypy of mint1 mutant mice. Methamphetamine was administered subcutaneously at a dose of 10 mg/kg, and stereotypy was graded according to a criteria described by Puech et al. (Puech et al., 1981). The data are expressed as the total scores of three observations, and columns indicate the means9/S.E.M. values of six animals. * P B/0.05 (Wilcoxon’s rank sum test).
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DA release was also demonstrated behaviorally as well as neurochemically.
3.3. Effects of mint-1 deficiency on behavior If the deficiency of mint-1 causes the abnormal neurotransmission through the dysfunction of exocytosis of synaptic vesicles and/or of transporter proteins, behavioral difference could be seen between wild-type and mint-1 KO mice. Therefore, we evaluated normal reflexes in standard neurological tests, horizontal movement (ambulation), and neuropsychological events (Table 1). Firstly, we performed behavioral observations according to Irwin’s method. Although mint-1 KO mice did not have tremor or ataxia, nor display an abnormal stance or posture, they showed more sensitive and prominent behavioral response to an approaching object than wild-type mice by touch escape test described by Irwin (Irwin, 1968). Only seven of 79 wild-type mice (8.8%) showed the slight escape response when the sides and body of the animal was stroked. On the other hand, 63 of 100 mint-1 KO mice (63%) showed the vigorous and rapid escape response. Locomotor activity was assessed in activity cages by recording every 10 min for 1 h. Although mint-1 KO mice showed a higher locomotor activity than wild-type mice both in the initial 10-min and 1-h measurement, there were no significant differences between the two genotypes. The elevated plus maze and tail suspension tests were performed as neuropsychological tests. Statistical analysis of the indices in these tests did not reveal any significant differences in effects between the genotypes. Thus, both anxiety and depression levels seem to be similar between the wild-type and mint-1 KO mice. Table 1 Performance of the mint-1 gene knockout and wild-type mice on the multiple behavioral task Tests
N
Locomotor activity (counts) 10 min 10 60 min 10
Wild-type
N
Mint-1 KO
151.89/32.2 335.69/90.3
9 9
184.79/48.1 403.89/112.6
Elevated plus maze % of Time % of Entry
10 10
23.49/4.5 37.79/8.3
9 9
27.09/9.0 34.39/8.5
Tail suspension Immobility time (s)
7
186.89/10.6
9
206.19/9.7
Each test was performed as described in Section 2. % of Time and % of Entry in elevated plus maze represents percentage of time spent in the open arms and percentage of open arm entries, respectively. Immobility time in Tail suspension represents the data acquired for the period from 2 to 6 min. The data are the means9/S.E.M.
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4. Discussion Direct evidence to clarify the effects of mint-1 deficiency on DA release was acquired from the data of in vivo microdialysis and METH-induced stereotypy (Figs. 2 and 3). In microdialysis, although basal value of DA content was the same in the two genotypes, the striatal DA release induced by a KCl pulse and METH injection in mint-1 KO mice was suppressed to 64 and 41%, respectively, as compared with the wild-type controls. These results indicate that mint-1 deficiency causes an alteration of the neurotransmitter release through vesicular or transporter-mediated release. As a reason for the suppression of the former DA release, at first, the disruption of mint-1/munc18-1/syntaxin1 complex (Okamoto and Sudhof, 1997) was considered from the results of high K stimulation. As seen in a previous study with Doc2a, which is another neuronal protein interacted with both munc18 and munc13 (Orita et al., 1997; Verhage et al., 1997), KO mice (Sakaguchi et al., 1999), DA release was usually normal in mint-1 KO mice. Although the detailed action mechanisms of Doc2a are not clear, Doc2a is reported not to be essential for Ca2-dependent neurotransmitter release but may modulate neurotransmitter release during repetitive synaptic activation. Therefore, the suppression of high K -evoked DA release in mint-1 KO mice may not be caused by the attenuation of munc18-related exocytosis of synaptic vesicles. However, further investigation will be needed to clarify how mint-1 protein is involved in the high K -evoked DA release. DA release induced by a METH injection is nonvesicular and transporter-mediated release (Liang and Rutledge, 1982), and it was significantly suppressed to 41% in mint-1 KO mice as measured by microdialysis method. In addition, METH-induced stereotypy was significantly attenuated in the KO mice. These results from both neurochemical and behavioral analysis suggest that DA release via dopamine transporters (DAT) was clearly inhibited by mint-1 deficiency. Setou et al. reported that NR2B subunit in NMDA receptor was transported along microtubules by KIF17, a neuronspecific molecular motor in neuronal dendrites, and the transport was accomplished by direct interaction of the KIF17 tail with a PDZ domain of mint-1, which was a constituent of a protein complex including CASK, velis, and NR2 subunit (Setou et al., 2000). More recently, it has been suggested that PICK1, which is a member of PDZ domain-containing protein, participates in the localization and expression of monoamine transporters (Torres et al., 2001). Taken together, the results presented here suggest that mint-1 may be involved in the targeting and localization of DAT proteins. In the observation of neurological and neuropsychological behavior, mint-1 KO mice were indistinguishable from their wild-type in gross behavior. And there were
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no significant behavioral differences between mint-1 KO mice and wild-type control in neuropsychological tests such as the elevated plus maze test for anxiety and the tail suspension test for depression (Table 1). Compared with wild-type controls, however, mint-1 KO mice showed more sensitive and prominent behavioral response to an approaching object. On the other hand, the locomotor activity for the initial 10 min was measured as an activity index in a novel environment. Mint-1 KO mice showed slightly higher, although not significant, than wild-type controls in this index (Table 1). Another study of ours demonstrated that 18-month-old mint-1 KO mice showed a significantly higher locomotor activity for the initial 10 min than age-matched wildtype controls (unpublished data). From these behavioral observations, only when the mice were stimulated by an approaching object or a novel environment, they showed sensitive responses. Interestingly, like mint-1 KO mice, DAT KO mice also showed hyperactivity in a novel environment, and it was considered that an excess of DA in the synaptic cleft caused the hyperactivity (Ralph et al., 2001). Even in mint-1 KO mice, the same event might occur by the suppressed DA release, which was caused by the disappearance of DAT proteins from pre-synaptic membranes. In summary, we demonstrated the alterations of dopamine release in mint-1 KO mice in vivo and the presumably concomitant behavioral changes. The neurochemical and behavioral changes could be due to the impairment of transporter-mediated DA release. These findings indicate that mint-1 proteins may have an important role in the targeting and localization of DAT proteins. Various psychiatric disorders, such as schizophrenia, drug abuse and attention deficit-hyperactivity disorder (ADHD), believed to have hyperdopaminergic activity have been associated with DAT dysfunction. Therefore, functional regulation of DAT proteins by mint-1 molecule represents a new avenue of future research to understand the pathogenesis of these disorders.
Acknowledgements We thank Drs Akira Tanigami, Toyoki Mori, Hisashi Kitagawa, Masami Nakai, and Mr Tetsuro Kikuchi for their helpful comments and support.
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