SEARCH

SEARCH BY CITATION

Keywords:

  • dopamine;
  • laminin;
  • methamphetamine;
  • plasmin;
  • sensitization;
  • tissue plasminogen activator

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We have previously demonstrated that repeated, but not acute, methamphetamine (METH) treatment increases tissue plasminogen activator (tPA) activity in the brain, which is associated with the development of behavioral sensitization to METH. In this study, we investigated whether the tPA-plasmin system is involved in the development of sensitization in METH-induced dopamine release in the nucleus accumbens (NAc). There was no difference in acute METH-induced increase in extracellular dopamine levels in the NAc between wild-type and tPA-deficient (tPA−/−) mice. Repeated METH treatment resulted in a significant enhancement of METH- induced dopamine release in wild-type mice, but not tPA−/− mice. Microinjection of exogenous tPA or plasmin into the NAc of wild-type mice significantly potentiated acute METH- induced dopamine release. Degradation of laminin was evident in brain tissues incubated with tPA plus plasminogen or plasmin in vitro although tPA or plasminogen alone had no effect. Immunohistochemical analysis revealed that microinjection of plasmin into the NAc reduced laminin immunoreactivity without neuronal damage. Our findings suggest that the tPA-plasmin system participates in the development of behavioral sensitization induced by repeated METH treatment, by regulating the processes underlying the sensitization of METH-induced dopamine release in the NAc, in which degradation of laminin by plasmin may play a role.

Abbreviations used
aCSF

artificial cerebrospinal fluid

BDNF

brain-derived neurotrophic factor

DAT

dopamine transporter

ECM

extracellular matrix

LTP

long-term potentiation

METH

methamphetamine

NAc

nucleus accumbens

PAR-1

protease-activated receptor-1

PAR-1−/−

PAR-1-deficient

tPA

tissue plasminogen activator

tPA−/−

tPA-deficient

VTA

ventral tegmental area

The mesolimbic dopaminergic system from the ventral tegmental area to the nucleus accumbens (NAc) plays a crucial role in drug dependence (Wise 1996; Koob et al. 1998; Mizoguchi et al. 2004; Nestler 2005). Methamphetamine (METH), one of the most abused drugs in the world, increases extracellular dopamine levels in the NAc by reversing the dopamine transporter (DAT), which is associated with the reinforcing effect of METH (Seiden et al. 1993; Giros et al. 1996; Nakajima et al. 2004). In rodents, augmentation of behavioral responses to METH is observed during and after repeated administration. This form of behavioral plasticity, called behavioral sensitization, is regarded as an animal model for the intensification of drug craving in human addicts (Robinson and Berridge 2003) and for METH-induced psychosis (Sato et al. 1983, 1992). In parallel with behavioral sensitization, repeated METH administration enhances the dopamine release-stimulating effect of the drug in the NAc (O’Dell et al. 1991; Suzuki et al. 1997; Narita et al. 2004).

Tissue plasminogen activator (tPA), a serine protease which catalyzes the conversion of plasminogen to plasmin, plays an important role in the central nervous system. Accumulating evidence has demonstrated that tPA is involved in synaptic plasticity and remodeling, directly by itself or indirectly through plasmin. For instance, tPA is directly involved in long-term potentiation (LTP) by acting on low-density lipoprotein receptor-related proteins (Zhuo et al. 2000) and NMDA receptors (Nicole et al. 2001). On the other hand, neurite outgrowth (Krystosek and Seeds 1981), cell migration (Moonen et al. 1982; Seeds et al. 1999) and amyloid-β degradation induced by tPA (Tucker et al. 2000; Melchor et al. 2003) are mediated by plasmin. In addition, recent studies have demonstrated the role of tPA in the regulation of neurotransmitter release. Thus, depolarization-evoked dopamine release in the NAc (Ito et al. 2006) as well as norepinephrine release from hearts (Schaefer et al. 2006) are diminished in tPA-deficient (tPA−/−) mice compared with wild-type mice. We have demonstrated that tPA plays an important role in the rewarding effects of abused drugs, including METH, morphine and nicotine, through the activation of plasminogen to plasmin, and that plasmin modulates morphine- and nicotine-induced dopamine release in the NAc (Nagai et al. 2004, 2005a,b, 2006).

Regarding the molecular targets of the tPA-plasmin system, protease-activated receptor-1 (PAR-1) is activated by plasmin (Kuliopulos et al. 1999) and plasmin-induced migration requires signaling through PAR-1 (Majumdar et al. 2004). We have demonstrated that PAR-1 is involved in the enhancement of nicotine-induced dopamine release by the tPA-plasmin system (Nagai et al. 2006) as well as morphine-induced dopamine release and hyperlocomotion (Ito et al. 2007). Alternatively, the degradation of laminin, one of the major components of extracellular matrix (ECM), by plasmin is important in the maintenance of LTP in organotypic hippocampal cultures (Nakagami et al. 2000) and excitotoxin-induced neuronal cell death in the hippocampus (Chen and Strickland 1997).

In a previous study, we have demonstrated that repeated METH treatment dose-dependently induced tPA mRNA expression as well as enzyme activity in the NAc, whereas acute METH treatment had no effect. Although there was no difference in acute METH-induced hyperlocomotion between wild-type and tPA−/− mice, METH-induced conditioned place preference and behavioral sensitization after repeated METH treatment were significantly reduced in tPA−/− mice compared with wild-type mice. The defect of behavioral sensitization in tPA−/− mice was reversed by microinjection of exogenous tPA into the NAc (Nagai et al. 2005b; Yamada et al. 2005). These results suggest that tPA plays a role in the development of behavioral sensitization induced by repeated METH treatment, but the underlying mechanism remains to be determined.

In the present study, we investigated whether the tPA-plasmin system participates in the sensitization of METH-induced dopamine release in the NAc of mice after repeated treatment. Furthermore, we examined whether PAR-1 activation or laminin degradation is involved in modulation by the tPA-plasmin system of dopamine release in the NAc after repeated METH treatment.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Animals

Male ICR mice (7-weeks old) were obtained from Japan SLC Inc. (Shizuoka, Japan). Wild-type (C57BL/6J), tPA−/− (stock number 002508, Carmeliet et al. 1994) and PAR-1-deficient (PAR-1−/−) (stock number 002862, Connolly et al. 1996) mice were provided by the Jackson Laboratory (Bar Harbor, ME, USA), and the presence or absence of either PAR-1, tPA, or the neomycin cassette was verified according to the manufacturer’s instructions. When comparing the wild-type and knock-out forms, only congenic animals were used. The animals were housed in plastic cages and kept in a regulated environment (23 ± 1°C, 50 ± 5% humidity), with a 12/12 h light-dark cycle (lights on at 9:00 am). Food (CA1; Clea Japan Inc., Tokyo, Japan) and tap water were available ad libitum.

All animal care and use were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of Kanazawa University.

METH treatment

METH hydrochloride (Dainihon Pharmaceutical Co. Ltd., Osaka, Japan) was dissolved in physiological saline. For acute METH treatment, mice were given a subcutaneous injection of METH (1 mg/kg). For repeated METH treatment, animals were subjected to a 5-day regimen in which METH (1 mg/kg) was injected subcutaneously once a day for 5 days. Control animals were given the same volume of saline under the same injection schedule as used for acute and repeated administrations of METH.

In vivo microdialysis

Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and a guide cannula (MI-AG-6, Eicom Corp., Kyoto, Japan) was implanted in the NAc (+1.5 mm anteroposterior, +0.8 mm mediolateral from the bregma, −4.0 mm dorsoventral from the skull) according to the mouse brain atlas (Franklin and Paxinos 1997). For repeated METH treatment (Fig. 1), mice were given a subcutaneous injection of METH (1 mg/kg) or saline for 4 days 1 or 2 days after the operation. One day after the last injection of repeated METH or saline, microdialysis was performed. For acute METH treatment (Figs 2 and 3), microdialysis was performed 1 or 2 days after the operation. A dialysis probe equipped with a microinjection tube (MIA-6-1; 1 mm membrane length, Eicom Corp.) was inserted through the guide cannula, and perfused with artificial cerebrospinal fluid (147 mmol/L NaCl, 4 mmol/L KCl, and 2.3 mmol/L CaCl2) at a flow rate of 1.0 μL/min (Nagai et al. 2004). The microdialysis probes were constructed of three stainless tubes, two silicatubes (inlet and outlet) for microdialysis with a 75 μm outer diameter and a microinjection silica tube with a 75 μm outer diameter. The microinjection tube was place in parallel with the tubes for microdialysis. The microinjection tube was half the length of the dialysis membrane. These three silica tubes were sealed together with epoxy resin, and each was secured with stainless steel tubing at the top of the probe.

image

Figure 1.  Acute and repeated METH-induced changes in extracellular dopamine levels in the NAc of wild-type and tPA−/− mice. METH was administered at a dose of 1 mg/kg for 5 days in the repeated METH groups. Values indicate the means ± SEM (= 7 for acute METH-treated wild-type mice; = 7 for repeated METH-treated wild-type mice; = 8 for acute METH-treated tPA−/− mice; = 7 for repeated METH-treated tPA−/− mice). Basal levels of dopamine (pg/20 min/20 μL) were 1.2 ± 0.2 in acute METH-treated wild-type mice, 1.2 ± 0.2 in repeated METH-treated wild-type mice, 1.7 ± 0.4 in acute METH-treated tPA−/− mice and 1.3 ± 0.4 in repeated METH-treated tPA−/− mice. One-way anova with repeated measures indicated significant effects of group [F(3, 25) = 4.841, < 0.01] and time [F(5, 125) = 34.961, < 0.0001], but not the interaction of group with time [F(15, 125) = 1.621, > 0.05]. *< 0.05 versus acute METH-treated wild-type mice. #< 0.05 versus repeated METH-treated wild-type mice.

Download figure to PowerPoint

image

Figure 2.  Effect of microinjections of either tPA (a) or plasmin (b) into the NAc on acute METH-induced dopamine release in ICR mice. (a) tPA (100 ng) was microinjected during a 10-min period into the NAc at a volume of 1 μL. Ten min after the microinjection, METH (1 mg/kg) was administrated s.c. Values indicate the means ± SEM (= 8 for vehicle-treated mice, = 10 for tPA-treated mice). One-way anova with repeated measures revealed significant effects of group [F(1, 16) = 5.382, < 0.05] and time [F(5, 80) = 6.181, < 0.0001], but not the interaction of group with time [F(5, 80) = 0.914, > 0.05]. *< 0.05 versus vehicle-treated group. (b) Plasmin (100 ng) was microinjected during a 10-min period into the NAc in a volume of 1 μL. Ten min after the microinjection, METH (1 mg/kg) was administrated s.c. Values indicate the means ± SEM (= 6 for vehicle-treated mice; = 9 for plasmin-treated mice). One-way anova with repeated measures revealed significant effects of group [F(1, 13) = 4.891, < 0.05] and time [F(5, 65) = 7.463, < 0.0001], but not the interaction of group with time [F(5, 65) = 1.331, > 0.05]. *< 0.05 versus vehicle-treated group.

Download figure to PowerPoint

image

Figure 3.  Effect of microinjection of plasmin into the NAc on METH-induced doamine release in PAR-1−/− mice. Plasmin (100 ng) was microinjected during a 10-min period into the NAc in a volume of 1 μL. Ten min after the microinjection, METH (1 mg/kg) was administrated s.c.. Values indicate the means ± SEM (= 5 for vehicle-treated wild-type mice, = 8 for plasmin-treated wild-type mice, = 5 for vehicle-treated PAR-1−/− mice, = 5 for plasmin-treated PAR-1−/− mice). One-way anova with repeated measures indicated significant effects of group [F(3, 19) = 3.392, < 0.05] and time [F(5, 95) = 9.440, < 0.01], but not the interaction of group with time [F(15, 95) = 1.033, > 0.05] There was no difference in METH-induced dopamine release between wild-type and PAR-1−/− mice in the presence or absence of pretreatment with plasmin.

Download figure to PowerPoint

Outflow fractions were collected every 20 min. Following the collection of three baseline fractions, human recombinant tPA (100 ng, provided by Eisai Co. Ltd., Tokyo, Japan) or human plasmin (100 ng, Chromogenix, Molndal, Sweden) dissolved in 1 μL of artificial cerebrospinal fluid solution was injected during a 10-min period through the microinjection tube into the NAc (Nagai et al. 2005a). Ten min after the microinjection, METH (1 mg/kg, s.c.) was administrated. Dopamine levels in the dialysates were analyzed using an HPLC system equipped with an electrochemical detector (Nagai et al. 2004, 2006).

Measurement of locomotor activity

Animals were treated with saline on day 1 and METH (1 mg/kg, s.c.) once daily for 7 days (from day 2 to day 8). Mice were habituated to a transparent acrylic cage (25 cm × 25 cm × 20 cm) for 120 min before the measurement of locomotor activity at 10-min intervals for 60 min using digital counters with an infrared sensor on days 1, 2, and 8 (Brainscience Idea Inc., Osaka, Japan) (Nagai et al. 2005b).

Western blotting

Striatal tissues containing NAc were homogenized in 0.1 mol/L Tris–HCl (pH 7.2) containing 0.1% Triton X-100 and centrifuged at 10 000g at 4°C for 20 min. Supernatants was incubated with either 0.3 μmol/L tPA and 0.5 μmol/L plasminogen (Chromogenix) or 1 μmol/L plasmin at 37°C for 30 min. Samples were subjected to 6% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under reducing conditions, followed by transfer onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk in Tris-buffered saline-Tween 20 (10 mmol/L Tris–HCl (pH. 7.5), 100 mmol/L NaCl, and 1% Tween-20), and incubated with rabbit anti-laminin antibody (1 : 2000, Sigma, St. Louis, MO, USA) at 4°C overnight. After incubation with a horseradish peroxidase-conjugated donkey anti-rabbit IgG antibody (1 : 5000, GE Healthcare Bio-Science Corp., Piscataway, NJ, USA) at 25°C for 1 h, the immune complex was detected using ECL plus western blotting detection reagents (GE Healthcare Bio-Science Corp.).

Immunohistochemistry

Thirty min after the injection of plasmin into the NAc, the animals were anesthetized with ether, and transcardially perfused with isotonic 0.1 mol/L phosphate buffer (pH 7.4) followed by isotonic 4% paraformaldehyde. The brains were removed, post-fixed in 4% paraformaldehyde for 2 h and then cryoprotected in 30% sucrose in 0.1 mol/L phosphate buffer. The brains were embedded in Tissue-Tek O.C.T. compound (Sakura Finetech) and stored at −80°C. Briefly, sections (14 μm) were fixed with 4% paraformaldehyde, and washed with 0.3% Triton X-100 in phosphate-buffered saline. They were blocked with 10% normal goat serum and 1% bovine serum albumin in phosphate-buffered saline for 30 min, and incubated in rabbit anti-laminin IgG (1 : 30) at 4°C overnight. Sections were then incubated in Alexa Fluor 488-conjugated goat anti-rabbit IgG (1 : 200, Invitrogen Corp.) for 1 h. Samples were observed with a confocal microscope (model LSM510, Carl Zeiss, Oberkochen, Germany).

Nissl stain

Mice were anesthetized with ether, and transcardially perfused with isotonic 0.1 mol/L phosphate buffer (pH 7.4) followed by isotonic 4% paraformaldehyde. The brains were removed, post-fixed in 4% paraformaldehyde for 2 h and then cryoprotected in 30% sucrose in 0.1 mol/L phosphate buffer. Sections (14 μm) were stained with 1% Cresyl violet. Samples were observed with a microscope (model Axioskop, Carl Zeiss).

Statistical analysis

All data are expressed as the mean ± SEM. In analysis of the time course of microdialysis, anova with repeated measures was used and followed by the Student–Newman-Keuls test when F ratios were significant (< 0.05). Statistical differences in the analysis of laminin protein levels were determined using one-way anova, followed by the Student Newman–Keuls test when F ratios were significant (< 0.05).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Changes in METH-induced dopamine release in tPA−/− mice

First, to investigate whether the tPA-plasmin system is involved in the development of sensitization in METH-induced dopamine release, we measured the levels of extracellular dopamine in the NAc of wild-type and tPA−/− mice after acute or repeated METH treatment, using in vivo microdialysis (Fig. 1). Acute METH treatment at 1 mg/kg increased extracellular dopamine levels to 253 ± 32% and 235 ± 44% of the basal levels 40 min after treatment in wild-type and tPA−/− mice, respectively. There was no difference in acute METH-induced dopamine release between the two genotypes. Repeated METH treatment potentiated METH-induced dopamine release in wild-type mice (412 ± 39% of the basal levels), but not in tPA−/− mice (280 ± 45% of the basal levels). One-way anova with repeated measures indicated significant effects of group [F(3, 25) = 4.841, < 0.01] and time [F(5, 125) = 34.961, < 0.0001], but not their interaction [F(15, 125) = 1.621, > 0.05]. Post hoc test indicated a significant difference between acute and repeated METH treatment in wild-type mice (< 0.01) whereas no difference was seen in tPA−/− mice. Furthermore, dopamine responses in tPA−/− mice were significantly diminished compared with those in wild-type mice after repeated METH treatment (< 0.05). These results suggest that tPA is involved in the development of sensitization on METH-induced dopamine release after repeated treatment.

Effect of tPA and plasmin on acute METH-induced dopamine release in the NAc of ICR mice

We have previously demonstrated that repeated, but not acute, treatment with METH increased the enzyme activity of tPA in the NAc (Nagai et al. 2005b). Accordingly, to clarify the tPA-dependent mechanism underlying the development of sensitization in METH-induced dopamine release, we studied the effects of microinjections of exogenous tPA or plasmin into the NAc on acute METH-induced dopamine release in the NAc of ICR mice (Fig. 2). Acute METH-induced dopamine release (226 ± 29% of the basal levels at 40 min in the vehicle-treated group) was significantly potentiated by a prior microinjection of recombinant tPA into the NAc (344 ± 55% of the basal levels at 40 min in the tPA-treated group) as the response observed after repeated METH treatment (Fig. 2a). One-way anova with repeated measures revealed significant effects of group [F(1, 16) = 5.382, < 0.05] and time [F(5, 80) = 6.181, < 0.0001], but not their interaction [F(5, 80) = 0.914, > 0.05]. Thus, it is possible that an increase in tPA expression in the NAc after repeated METH treatment may contribute to the development of sensitization in METH-induced dopamine release. Similarly, microinjection of plasmin (100 ng) into the NAc enhanced acute METH-induced dopamine release (394 ± 60% of the basal levels) (Fig. 2b). One-way anova with repeated measures revealed significant effects of group [F(1, 13) = 4.891, < 0.05] and time[F(5, 65) = 7.463, < 0.0001], but not the interaction of group with time [F(5, 65) = 1.331, > 0.05]. These results suggest that tPA potentiates METH-induced dopamine release in the NAc, through the conversion of plasminogen to plasmin.

PAR-1 is not involved in METH-induced dopamine release

In previous studies, we demonstrated that the tPA-plasmin system potentiates nicotine- and morphine-induced dopamine release in the NAc by activating PAR-1 expressed on dopaminergic nerve terminals (Nagai et al. 2006; Ito et al. 2007). Therefore, we examined whether PAR-1 is involved in the potentiation of METH-induced dopamine release by plasmin, using PAR-1−/− mice. Microinjection of plasmin into the NAc significantly potentiated acute METH-induced dopamine release both in wild-type and PAR-1−/− mice (Fig. 3). There was no difference between wild-type and PAR-1−/− mice in METH-induced dopamine release in the presence or absence of pre-treatment with plasmin. One-way anova with repeated measures indicated significant effects of group [F(3, 19) = 3.392, < 0.05] and time [F(5, 95) = 9.440, < 0.01], but not their interaction [F(15, 95) = 1.033, > 0.05].

We also compared repeated METH-induced behavioral sensitization in PAR-1−/− mice with that in wild-type mice. As shown in Table 1, there was no difference between wild-type and PAR-1−/− mice in either acute METH-induced hyperlocomotion or repeated METH-induced behavioral sensitization. Collectively, it is unlikely that PAR-1 plays a significant role in the development of sensitization in dopamine release and hyperlocomotion induced by repeated METH treatment.

Table 1.   Behavioral sensitization induced by repeated METH treatment in wild-type and PAR-1−/− mice
GenotypesTreatment
Saline (day 1)METH (day 2)METH (day 8)
  1. Animals were treated with saline on day 1 and METH (1 mg/kg, s.c.) once daily for 7 days (day 2–8). Locomotor activity was measured for 1 h after treatment on days 1, 2, and 8. Values indicate the means ± SE (= 9–10). One-way anova revealed significant effects of METH treatment [F(5, 51) = 12.66, < 0.0001]. *< 0.05 versus respective saline treatment (day 1). #< 0.05 versus respective acute METH treatment (day 2).

Wild-type mice1120 ± 2081844 ± 249*2705 ± 235*,#
PAR-1−/− mice1223 ± 1521965 ± 166*2620 ± 101*,#

Effect of plasmin on laminin degradation in vitro and in vivo

To explore the possible mechanism underlying the plasmin-induced potentiation of acute METH-induced dopamine release in the NAc, we examined the changes in laminin contents in brain tissue after treatment with plasmin in vitro. Treatment of striatal tissues with either tPA or plasminogen alone had no effect, but the combination significantly reduced laminin levels [laminin α1 subunit; F(3, 12) = 27.199, < 0.01 (Fig. 4a and b), laminin β1γ1 subunits; F(3, 12) = 34.719, < 0.01 (Fig. 4a and c)]. Similarly, incubation of brain tissues with plasmin decreased laminin levels in a concentration-dependent manner [laminin α1 subunit; F(2, 9) = 68.48, < 0.01 (Fig. 4d and e), laminin β1γ1subunits; F(2, 9) = 54.706, < 0.01 (Fig. 4d and f)]. These results suggest that plasmin produced by tPA from plasminogen can degrade laminin in brain tissue in vitro.

image

Figure 4.  Effects of tPA, plasminogen and plasmin on laminin degradation in vitro. (a and d) Representative photograph of western blotting for laminin after treatment with tPA, plasminogen (a) and plasmin (d). (b, c, d and f) Densitometric analysis of laminin α1 (b and e) and β1γ1 (c and f) chain. (a–c) The homogenate of striatum including the NAc was incubated with recombinant human tPA (300 nmol/L) with or without human plasminogen (500 nmol/L) for 30 min at 37°C. (b) anova [F(3, 12) = 27.199, < 0.01]. (c) anova [F(3, 12) = 34.719, < 0.01]. (d–f) The homogenate of striatum was incubated with recombinant human plasmin (10 or 100 nmol/L) for 30 min at 37°C. (e) anova [F(2, 9) = 68.48, < 0.01]. (f) anova [F(2, 9) = 54.706, < 0.01]. **< 0.01 versus control.

Download figure to PowerPoint

Lastly, we examined the degradation of laminin in the NAc after plasmin treatment in vivo. Immunohistochemical analysis indicated that laminin-like immunoreactivity in the NAc was markedly reduced 30 min after the microinjection of plasmin into the NAc (Fig. 5a) compared with the vehicle-injected control group (Fig. 5b). Nissl staining indicated no apparent cell damage after plasmin treatment (Fig. 5c and d).

image

Figure 5.  Immunohistochemical detection of laminin in the NAc after microinjection of plasmin. (a and b) Representative photographs of laminin immunoreactivity in the NAc of vehicle- (a) or plasmin-microinjected mice (b). (c and d) Representative photographs of Nissl-stained sections in the NAc of vehicle- (c) or plasmin-microinjected mice (d). Scale bar indicates 100 μm in Fig. 5a and c and 50 μm in Fig. 5a and c insert. aca: anterior commissure anterior part.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

In the present study, we demonstrated that repeated METH treatment in wild-type mice resulted in the development of sensitization in METH-induced dopamine release in the NAc whereas such sensitization of dopamine release was not evident in tPA−/− mice. Since there was no difference in acute METH-induced dopamine release between wild-type and tPA−/− mice, it is unlikely that there is a mechanism defect of METH-induced DAT-mediated dopamine release in the NAc (Seiden et al. 1993; Giros et al. 1996; Nakajima et al. 2004). Rather, it is likely that tPA plays a role in the development of sensitization in METH-induced dopamine release. The present findings are consistent with our previous findings that behavioral sensitization after repeated METH treatment was attenuated in tPA−/− mice, although there was no difference in acute METH-induced hyperlocomotion between wild-type and tPA−/− mice (Nagai et al. 2005b). It should be noted that the levels of tPA mRNA and its enzyme activity in the NAc were markedly increased after repeated METH treatment, although acute METH treatment had no effect (Nagai et al. 2005b). Taken together, it is suggested that the induction of tPA in the NAc after repeated METH treatment plays a role in the development of sensitization to METH.

In contrast to acute METH-induced dopamine release, we have demonstrated that acute morphine- (Nagai et al. 2004) and nicotine-induced dopamine release (Nagai et al. 2006) as well as potassium depolarization-evoked dopamine release (Ito et al. 2006) are all diminished in tPA−/− mice compared with wild-type mice. One possible explanation for this discrepancy is that METH-induced dopamine release is a DAT-mediated Ca2+-independent process (Nakajima et al. 2004), whereas dopamine release induced by morphine and nicotine is Ca2+-dependent (Harsing et al. 1992; Keren et al. 1997). However, the fact that microinjection of exogenous tPA and plasmin into the NAc can enhance METH-induced dopamine release does not support the aforementioned hypothesis. Rather, it is likely that the tPA-plasmin system can potentiate METH-induced, DAT-mediated and Ca2+-independent dopamine release as it does in morphine-, nicotine and depolarization-induced dopamine release. An alternative explanation is that because acute METH treatment has no effect on tPA release and expression in the brain (Nagai et al. 2005b), acute METH-induced dopamine release is not affected by the deficiency of tPA. On the other hand, since acute treatment with morphine (Ito et al. 2007), nicotine (Nagai et al. 2006) and potassium depolarization (Gualandris et al. 1996) induces tPA expression and increases extracellular tPA activity in the brain, dopamine release is diminished in tPA−/− mice compared with wild-type mice.

We have reported that METH-induced locomotor sensitization is significantly attenuated in tPA−/− mice although there is no difference in acute METH-induced hyperlocomotion between wild-type and tPA−/− mice (Nagai et al. 2005b). Furthermore, dopamine release in the NAc is thought to play a crucial role in the locomotor-stimulating effects of drugs of abuse (Koob et al. 1998; Ito et al. 2007). Thus, it is suggested that tPA-plasmin system plays a role in the development of behavioral sensitization induced by repeated METH treatment through the regulation of processes underlying the sensitization of dopamine release in the NAc. We propose that the induction of tPA in the NAc following repeated METH treatment is a critical step for the development of sensitization in METH-induced dopamine release, leading to behavioral sensitization.

There are several potential targets of the tPA-plasmin system in the brain. For example, tPA enhances NMDA receptor signaling by cleaving the NR1 subunit at the arginine 260 of the amino-terminal domain (Nicole et al. 2001; Fernández-Monreal et al. 2004). Thus, the NR1 subunit of NMDA receptors may be a possible target for the tPA-plasmin system to potentiate METH-induced dopamine release in the NAc. This assumption is consistent with observations that NMDA receptor antagonists prevent the development of locomotor sensitization to amphetamines (Karler et al. 1991; Stewart and Druhan 1993; Wolf and Jeziorski 1993). However, since plasmin leads to complete degradation of the amino-terminal domain of NR1, the proteolytic pattern being different from that of tPA (Fernández-Monreal et al. 2004), it is unlikely that NMDA receptor activation caused by cleavage of the NR1 subunit is involved in the sensitization of METH-induced dopamine release by the tPA-plasmin system.

Alternatively, it has been demonstrated that tPA, by activating plasmin, converts the precursor pro-brain-derived neurotrophic factor (BDNF) to mature BDNF, and that this conversion is critical for the expression of late-phase LTP (Pang et al. 2004). Furthermore, BDNF is implicated in METH-induced dopamine release and behavior effects (Narita et al. 2003). Thus, it is possible that BDNF is involved in the sensitization of METH-induced dopamine release by the tPA-plasmin system.

Regarding a target molecule for the tPA-plasmin system, we have demonstrated that plasmin activates PAR-1 expressed on the nerve terminals of dopaminergic neurons in the NAc. In addition, we indicated that nicotine-induced dopamine release and reward was diminished in PAR-1−/− mice and that tyrTRAP7, a PAR-1 antagonist peptide, blocked the ameliorating effect of plasmin on the defect of nicotine-induced dopamine release in tPA−/− mice (Nagai et al. 2006). Moreover, the PAR-1 antagonist blocked the ameliorating effect of plasmin on the defect of morphine-induced dopamine release in tPA−/− mice (Ito et al. 2007). Accordingly, we examined METH-induced dopamine release and locomotor sensitization in PAR-1−/− mice. As there was no difference in METH-induced dopamine release and locomotor sensitization, it is unlikely that PAR-1 is a target molecular for the tPA-plasmin system to potentiate METH-induced dopamine release.

We therefore focused on the role of laminin, an ECM protein susceptible to plasmin since previous studies indicated that laminin degradation by plasmin is involved in the maintenance of LTP in the hippocampus (Nakagami et al. 2000) and excitotoxin-induced cell death in the hippocampus (Chen and Strickland 1997). Laminin levels in brain tissue containing the NAc were reduced by treatment with tPA plus plasminogen or plasmin in vitro. Furthermore, microinjection of plasmin into the NAc resulted in a marked decrease in laminin immunoreactivity without any apparent cell damage. These findings suggest that degradation of laminin may be involved in the potentiation of METH-induced dopamine release by the tPA-plasmin system. Although further studies are necessary to substantiate this hypothesis, our previous study that the microinjection of purified matrix metalloprotease-2, an enzyme known to cleave ECM such as laminin (Yong et al. 2001), into the NAc enhances METH-induced dopamine release without affecting basal dopamine levels (Mizoguchi et al. 2007) may support the hypothesis.

On the other hand, repeated treatment with drugs of abuse produces structural adaptations. Exposure to amphetamine produces a long-lasting increase in the length of dendrites and the number of branched spines on medium spiny neurons (Robinson and Kolb 1997). Development of sensitization in METH-induced dopamine release in the NAc and locomotor activity was significantly attenuated in tPA−/− mice compared with wild-type mice. Thus, it is possible that tPA may play a role in repeated METH-induced structural changes through the degradation of laminin, which may underlie the behavioral and neurochemical sensitization to the methamphetamine.

In conclusion, we have demonstrated that the tPA-plasmin system participates in the development of behavioral sensitization induced by repeated METH treatment by regulating the processes underlying the sensitization of METH-induced dopamine release in the NAc, in which degradation of laminin by plasmin may play a role.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported in part by a Grant-in-Aid for Scientific Research (18790052, 19790053 and 19390062) and by the 21st Century COE program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and grants from the Smoking Research Foundation, Japan, Suzuken Memorial Foundation, Uehara Memorial Foundation, Kobayashi Magobe Memorial Foundation, and JSPS and KOSEF under the Japan-Korea Basic Scientific Cooperation Program. We thank Eizai Co. Ltd. for providing human recombinant tPA.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  • Carmeliet P., Schoonjans L., Kieckens L. et al. (1994) Physiological consequences of loss of plasminogen activator gene function in mice. Nature 368, 419424.
  • Chen Z. L. and Strickland S. (1997) Neuronal death in the hippocampus is promoted by plasmin-catalyzed degradation of laminin. Cell 91, 917925.
  • Connolly A. J., Ishihara H., Kahn M. L., Farese R. V. Jr and Coughlin S. R. (1996) Role of the thrombin receptor in development and evidence for a second receptor. Nature 381, 516519.
  • Fernández-Monreal M., López-Atalaya J. P., Benchenane K. et al. (2004) Arginine 260 of the amino-terminal domain of NR1 subunit is critical for tissue-type plasminogen activator-mediated enhancement of N-methyl-D-aspartate receptor signaling. J. Biol. Chem. 279, 5085050856.
  • Franklin J. B. J. and Paxinos G. T. (1997) The mouse brain in stereotaxic coordinates. Academic, New York.
  • Giros B., Jaber M., Jones S. R., Wightman R. M. and Caron M. G. (1996) Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379, 606612.
  • Gualandris A., Jones T. E., Strickland S. and Tsirka S. E. (1996) Membrane depolarization induces calcium-dependent secretion of tissue plasminogen activator. J. Neurosci. 16, 22202225.
  • Harsing L. G. Jr, Sershen H., Vizi S. E. and Lajtha A. (1992) N-type calcium channels are involved in the dopamine releasing effect of nicotine. Neurochem. Res. 17, 729734.
  • Ito M., Nagai T., Kamei H., Nakamichi N., Nabeshima T., Takuma K. and Yamada K. (2006) Involvement of tissue plasminogen activator-plasmin system in depolarization-evoked dopamine release in the nucleus accumbens of mice. Mol. Pharmacol. 70, 17201725.
  • Ito M., Nagai T., Mizoguchi H., Fukakusa A., Nakanishi Y., Kamei H., Nabeshima T., Takuma K. and Yamada K. (2007) Possible involvement of protease-activated receptor-1 in the regulation of morphine-induced dopamine release and hyperlocomotion by the tissue plasminogen activator-plasmin system. J. Neurochem. 101, 13921399.
  • Karler R., Calder L. D. and Turkanis S. A. (1991) DNQX blockade of amphetamine behavioral sensitization. Brain Res. 552, 295300.
  • Keren O., Gafni M. and Sarne Y. (1997) Opioids potentiate transmitter release from SK-N-SH human neuroblastoma cells by modulating N-type calcium channels. Brain Res. 764, 277282.
  • Koob G. F., Sanna P. P. and Bloom F. E. (1998) Neuroscience of addiction. Neuron 21, 467476.
  • Krystosek A. and Seeds N. W. (1981) Plasminogen activator release at the neuronal growth cone. Science 213, 15321534.
  • Kuliopulos A., Covic L., Seeley S. K., Sheridan P. J., Helin J. and Costello C. E. (1999) Plasmin desensitization of the PAR1 thrombin receptor: kinetics, sites of truncation, and implications for thrombolytic therapy. Biochemistry 38, 45724585.
  • Majumdar M., Tarui T., Shi B., Akakura N., Ruf W. and Takada Y. (2004) Plasmin-induced migration requires signaling through protease-activated receptor 1 and integrin α 9β 1. J. Biol. Chem. 279, 3752837534.
  • Melchor J. P., Pawlak R. and Strickland S. (2003) The tissue plasminogen activator-plasminogen proteolytic cascade accelerates amyloid-beta (Aβ) degradation and inhibits Aβ-induced neurodegeneration. J. Neurosci. 23, 88678871.
  • Mizoguchi H., Yamada K., Mizuno M., Mizuno T., Nitta A., Noda Y. and Nabeshima T. (2004) Regulations of methamphetamine reward by extracellular signal-regulated kinase 1/2/ets-like gene-1 signaling pathway via the activation of dopamine receptors. Mol. Pharmacol. 65, 12931301.
  • Mizoguchi H., Yamada K., Niwa M. et al. (2007) Reduction of methamphetamine-induced sensitization and reward in matrix metalloproteinase-2 and -9-deficient mice. J. Neurochem. 100, 15791588.
  • Moonen G., Grau-Wagemans M. P. and Selak I. (1982) Plasminogen activator-plasmin system and neuronal migration. Nature 298, 753755.
  • Nagai T., Yamada K., Yoshimura M., Ishikawa K., Miyamoto Y., Hashimoto K., Noda Y., Nitta A. and Nabeshima T. (2004) The tissue plasminogen activator-plasmin system participates in the rewarding effect of morphine by regulating dopamine release. Proc. Natl. Acad. Sci. USA 101, 36503655.
  • Nagai T., Kamei H., Ito M., Hashimoto K., Takuma K., Nabeshima T. and Yamada K. (2005a) Modification by the tissue plasminogen activator-plasmin system of morphine-induced dopamine release and hyperlocomotion, but not anti-nociceptive effect in mice. J. Neurochem. 93, 12721279.
  • Nagai T., Noda Y., Ishikawa K. et al. (2005b) The role of tissue plasminogen activator in methamphetamine-related reward and sensitization. J. Neurochem. 92, 660667.
  • Nagai T., Ito M., Nakamichi N., Mizoguchi H., Kamei H., Fukakusa A., Nabeshima T., Takuma K. and Yamada K. (2006) The rewards of nicotine: regulation by tissue plasminogen activator-plasmin system through protease activated receptor-1. J. Neurosci. 26, 1237412383.
  • Nakagami Y., Abe K., Nishiyama N. and Matsuki N. (2000) Laminin degradation by plasmin regulates long-term potentiation. J. Neurosci. 20, 20032010.
  • Nakajima A., Yamada K., Nagai T. et al. (2004) Role of tumor necrosis factor-alpha in methamphetamine-induced drug dependence and neurotoxicity. J. Neurosci. 24, 22122225.
  • Narita M., Aoki K., Takagi M., Yajima Y. and Suzuki T. (2003) Implication of brain-derived neurotrophic factor in the release of dopamine and dopamine-related behaviors induced by methamphetamine. Neuroscience 119, 767775.
  • Narita M., Akai H., Nagumo Y., Sunagawa N., Hasebe K., Nagase H., Kita T., Hara C. and Suzuki T. (2004) Implications of protein kinase C in the nucleus accumbens in the development of sensitization to methamphetamine in rats. Neuroscience 127, 941948.
  • Nestler E. J. (2005) Is there a common molecular pathway for addiction? Nat. Neurosci. 8, 14451449.
  • Nicole O., Docagne F., Ali C., Margaill I., Carmeliet P., MacKenzie E. T., Vivien D. and Buisson A. (2001) The proteolytic activity of tissue-plasminogen activator enhances NMDA receptor-mediated signaling. Nat. Med. 7, 5964.
  • O’Dell S. J., Weihmuller F. B. and Marshall J. F. (1991) Multiple methamphetamine injections induce marked increases in extracellular striatal dopamine which correlate with subsequent neurotoxicity. Brain Res. 564, 256260.
  • Pang P. T., Teng H. K., Zaitsev E. et al. (2004) Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity. Science 306, 487491.
  • Robinson T. E. and Berridge K. C. (2003) Addiction. Annu. Rev. Psychol. 54, 2553.
  • Robinson T. E. and Kolb B. (1997) Persistent structural modifications in nucleus accumbens and prefrontal cortex neurons produced by previous experience with amphetamine. J. Neurosci. 17, 84918497.
  • Sato M., Chen C. C., Akiyama K. and Otsuki S. (1983) Acute exacerbation of paranoid psychotic state after long-term abstinence in patients with previous methamphetamine psychosis. Biol. Psychiatry 18, 429440.
  • Sato M., Numachi Y. and Hamamura T. (1992) Relapse of paranoid psychotic state in methamphetamine model of schizophrenia. Schizophr. Bull. 18, 115122.
  • Schaefer U., Machida T., Vorlova S., Strickland S. and Levi R. (2006) The plasminogen activator system modulates sympathetic nerve function. J. Exp. Med. 203, 21912200.
  • Seeds N. W., Basham M. E. and Haffke S. P. (1999) Neuronal migration is retarded in mice lacking the tissue plasminogen activator gene. Proc. Natl. Acad. Sci. USA 96, 1411814123.
  • Seiden L. S., Sabol K. E. and Ricaurte G. A. (1993) Amphetamine: effects on catecholamine systems and behavior. Annu. Rev. Pharmacol. Toxicol. 33, 639677.
  • Stewart J. and Druhan J. P. (1993) Development of both conditioning and sensitization of the behavioral activating effects of amphetamine is blocked by the non-competitive NMDA receptor antagonist, MK-801. Psychopharmacology 110, 125132.
  • Suzuki H., Shishido T., Watanabe Y., Abe H., Shiragata M., Honda K., Horikoshi R. and Niwa S. (1997) Changes of behavior and monoamine metabolites in the rat brain after repeated methamphetamine administration: effects of duration of repeated administration. Prog. Neuropsychopharmacol. Biol. Psychiatry 21, 359369.
  • Tucker H. M., Kihiko M., Caldwell J. N. et al. (2000) The plasmin system is induced by and degrades amyloid-beta aggregates. J. Neurosci. 20, 39373946.
  • Wise R. A. (1996) Neurobiology of addiction. Curr. Opin. Neurobiol. 6, 243251.
  • Wolf M. E. and Jeziorski M. (1993) Coadministration of MK-801 with amphetamine, cocaine or morphine prevents rather than transiently masks the development of behavioral sensitization. Brain Res. 613, 291294.
  • Yamada K., Nagai T. and Nabeshima T. (2005) Drug dependence, synaptic plasticity, and tissue plasminogen activator. J. Pharmacol. Sci. 97, 157161.
  • Yong V. W., Power C., Forsyth P. and Edwards D. R. (2001) Metalloproteinases in biology and pathology of the nervous system. Nat. Rev. Neurosci. 2, 502511.
  • Zhuo M., Holtzman D. M., Li Y., Osaka H., DeMaro J., Jacquin M. and Bu G. (2000) Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation. J. Neurosci. 20, 542549.