Fyn Is Required for Haloperidol-induced Catalepsy in Mice
2006; Elsevier BV; Volume: 281; Issue: 11 Linguagem: Inglês
10.1074/jbc.m511608200
ISSN1083-351X
AutoresKotaro Hattori, Shigeo Uchino, Tomoko Isosaka, Mamiko Maekawa, Masaomi Iyo, Toshio Sato, Shinichi Kohsaka, Takeshi Yagi, Shigeki Yuasa,
Tópico(s)Epilepsy research and treatment
ResumoFyn-mediated tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D2 receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D2 receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D2 receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response. Fyn-mediated tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits has been implicated in various brain functions, including ethanol tolerance, learning, and seizure susceptibility. In this study, we explored the role of Fyn in haloperidol-induced catalepsy, an animal model of the extrapyramidal side effects of antipsychotics. Haloperidol induced catalepsy and muscle rigidity in the control mice, but these responses were significantly reduced in Fyn-deficient mice. Expression of the striatal dopamine D2 receptor, the main site of haloperidol action, did not differ between the two genotypes. Fyn activation and enhanced tyrosine phosphorylation of the NMDA receptor NR2B subunit, as measured by Western blotting, were induced after haloperidol injection of the control mice, but both responses were significantly reduced in Fyn-deficient mice. Dopamine D2 receptor blockade was shown to increase both NR2B phosphorylation and the NMDA-induced calcium responses in control cultured striatal neurons but not in Fyn-deficient neurons. Based on these findings, we proposed a new molecular mechanism underlying haloperidol-induced catalepsy, in which the dopamine D2 receptor antagonist induces striatal Fyn activation and the subsequent tyrosine phosphorylation of NR2B alters striatal neuronal activity, thereby inducing the behavioral changes that are manifested as a cataleptic response. Typical antipsychotic agents, such as haloperidol and chlorpromazine, have extrapyramidal side effects (EPS) 2The abbreviations used are: EPS, extrapyramidal side effects; BSS, balanced salt solution; D2-R, dopamine D2-receptor; HAL, Haloperidol; NMDA, N-methyl-d-aspartate; NMDA-R, N-methyl-d-aspartate receptor; PKA, protein kinase A; PKC, protein kinase C; SFKs, Src family kinases; TBS, Tris-buffered saline; TH, tyrosine hydroxylase. that resemble Parkinson disease. Drug-induced catalepsy, the impairment of movement initiation, in rodents is an animal model of EPS and is mainly caused by blockade of the dopamine D2 receptor (D2-R) (1Crocker A.D. Hemsley K.M. Prog. Neuropsychopharmacol. Biol. Psychiatry. 2001; 25: 573-590Crossref PubMed Scopus (46) Google Scholar, 2Wadenberg M.L. Soliman A. VanderSpek S.C. Kapur S. Neuropsychopharmacology. 2001; 25: 633-641Crossref PubMed Scopus (221) Google Scholar). Haloperidol-induced responses are also dependent on N-methyl-d-aspartate receptor (NMDA-R) activity, because prior administration of the NMDA-R antagonist MK-801 attenuates haloperidol-induced catalepsy (3Moore N.A. Blackman A. Awere S. Leander J.D. Eur. J. Pharmacol. 1993; 237: 1-7Crossref PubMed Scopus (46) Google Scholar, 4Chartoff E.H. Ward R.P. Dorsa D.M. J. Pharmacol. Exp. Ther. 1999; 291: 531-537PubMed Google Scholar). D2-R and NMDA-R are co-expressed in close proximity along the dendrites of medium spiny neurons in the striatum, and they are functionally coupled in terms of controlling extrapyramidal functions (5Chase T.N. Parkinsonism Relat. Disord. 2004; 10: 305-313Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). The NMDA-Rs are hetero-oligomeric ligand-gated ion channels composed of a single NR1 subunit and one type of NR2 (A–D) subunit (6Sucher N.J. Awobuluyi M. Choi Y.B. Lipton S.A. Trends Pharmacol. Sci. 1996; 17: 348-355Abstract Full Text PDF PubMed Scopus (253) Google Scholar). The most abundant receptor subunits in the striatum are NR1, NR2A, and NR2B (7Standaert D.G. Testa C.M. Young A.B. Penney Jr., J.B. J. Comp. Neurol. 1994; 343: 1-16Crossref PubMed Scopus (331) Google Scholar, 8Kosinski C.M. Standaert D.G. Counihan T.J. Scherzer C.R. Kerner J.A. Daggett L.P. Velicelebi G. Penney J.B. Young A.B. Landwehrmeyer G.B. J. Comp. Neurol. 1998; 390: 63-74Crossref PubMed Scopus (57) Google Scholar). These three subunits are involved in extrapyramidal functions (5Chase T.N. Parkinsonism Relat. Disord. 2004; 10: 305-313Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), and we have found that an NR2B-selective antagonist attenuates haloperidol-induced catalepsy (9Yanahashi S. Hashimoto K. Hattori K. Yuasa S. Iyo M. Brain Res. 2004; 1011: 84-93Crossref PubMed Scopus (23) Google Scholar). Phosphorylation of tyrosine residues on the NMDA-R has been reported to modulate its channel characteristics (10Wang Y.T. Salter M.W. Nature. 1994; 369: 233-235Crossref PubMed Scopus (602) Google Scholar, 11Tingley W.G. Ehlers M.D. Kameyama K. Doherty C. Ptak J.B. Riley C.T. Huganir R.L. J. Biol. Chem. 1997; 272: 5157-5166Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Depriving the striatum of dopaminergic input increases the tyrosine phosphorylation of the striatal NMDA-R and the motor response (12Menegoz M. Lau L.F. Herve D. Huganir R.L. Girault J.A. Neuroreport. 1995; 7: 125-128PubMed Google Scholar, 13Oh J.D. Russell D.S. Vaughan C.L. Chase T.N. Russell D. Brain Res. 1998; 813: 150-159Crossref PubMed Scopus (153) Google Scholar), but infusing the striatum with a tyrosine kinase inhibitor, genistein, attenuates both the tyrosine phosphorylation and the motor response induced by dopaminergic deprivation (13Oh J.D. Russell D.S. Vaughan C.L. Chase T.N. Russell D. Brain Res. 1998; 813: 150-159Crossref PubMed Scopus (153) Google Scholar). Fyn is a member of the Src family kinases (SFKs) and is associated with the NMDA-R at postsynaptic densities. Fyn phosphorylates NMDA-R subunits and modifies their channel activity (14Salter M.W. Kalia L.V. Nat. Rev. Neurosci. 2004; 5: 317-328Crossref PubMed Scopus (650) Google Scholar). One of the NMDA-R subunits, NR2B, is preferentially phosphorylated by Fyn, and its phosphorylation has been implicated in several brain functions, including ethanol tolerance, long term potentiation, and seizure susceptibility (15Miyakawa T. Yagi T. Kitazawa H. Yasuda M. Kawai N. Tsuboi K. Niki H. Science. 1997; 278: 698-701Crossref PubMed Scopus (267) Google Scholar, 16Yaka R. Phamluong K. Ron D. J. Neurosci. 2003; 23: 3623-3632Crossref PubMed Google Scholar, 17Nakazawa T. Komai S. Tezuka T. Hisatsune C. Umemori H. Semba K. Mishina M. Manabe T. Yamamoto T. J. Biol. Chem. 2001; 276: 693-699Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 18Kojima N. Ishibashi H. Obata K. Kandel E.R. Learn. Mem. 1998; 5: 429-445PubMed Google Scholar). The Tyr-1472 of NR2B is a particularly key site for Fyn-mediated phosphorylation (17Nakazawa T. Komai S. Tezuka T. Hisatsune C. Umemori H. Semba K. Mishina M. Manabe T. Yamamoto T. J. Biol. Chem. 2001; 276: 693-699Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 19Cheung H.H. Gurd J.W. J. Neurochem. 2001; 78: 524-534Crossref PubMed Scopus (75) Google Scholar). To investigate the role of Fyn in the cataleptic behavior induced by haloperidol, we studied these haloperidol effects in Fyn-deficient mice and biochemically analyzed the Fyn-mediated signal transduction initiated by haloperidol. Based on our results, we discuss the significance of Fyn activation in haloperidol-induced catalepsy within the scope of signal transduction from D2-R inhibition to modulation of the extrapyramidal system. Animals—Fyn tyrosine kinase-deficient mice were generated by inserting the β-galactosidase gene (lacZ) into the reading frame of the fyn gene as described previously (20Yagi T. Shigetani Y. Okado N. Tokunaga T. Ikawa Y. Aizawa S. Oncogene. 1993; 8: 3343-3351PubMed Google Scholar). Because the lacZ introduced is expressed in both heterozygous (+/fynZ) and homozygous (fynZ/fynZ) mice, heterozygous mice were mainly used as the controls instead of wild-type mice to compensate for the possible effect of lacZ expression as a foreign gene. The background of this mutant's strain is C57BL/6J. Genotypes were analyzed by the PCR. All animals were maintained under standard laboratory conditions as described previously (15Miyakawa T. Yagi T. Kitazawa H. Yasuda M. Kawai N. Tsuboi K. Niki H. Science. 1997; 278: 698-701Crossref PubMed Scopus (267) Google Scholar). All experimental procedures were in accordance with the 1996 National Institutes of Health guidelines and were approved by the Animal Care Committee of the Chiba University Graduate School of Medicine, Osaka University, and the National Institute of Neuroscience, National Center of Neurology and Psychiatry. Pharmacological Agents—Haloperidol, the dopamine D2-R-selective antagonist L-741,626 (21Kulagowski J.J. Broughton H.B. Curtis N.R. Mawer I.M. Ridgill M.P. Baker R. Emms F. Freedman S.B. Marwood R. Patel S. Ragan C.I. Leeson P.D. J. Med. Chem. 1996; 39: 1941-1942Crossref PubMed Scopus (253) Google Scholar), and the D2-R-selective agonist (–)-quinpirole were purchased from Sigma. The drugs were administered by intraperitoneal injection in a volume of 10 μl/g body weight. All solutions were prepared immediately prior to the experiments. To exclude the effect of drug tolerance, no animals were used more than once in the pharmacological experiments. Antibodies—Goat polyclonal anti-D2-R antibody (N19) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal anti-tyrosine hydroxylase (TH) antibody was obtained from Chemicon (Temecula, CA). A mouse monoclonal anti-phosphotyrosine antibody (Tyr(P)-100) was purchased from Cell Signaling Technology (Beverly, MA). Phosphorylation site-specific rabbit polyclonal antibody against p-Src (Y418) and p-Src (Y529) was obtained from BIO-SOURCE (Camarillo, CA) and against p-NR2B (Y1472) was from Sigma. Rabbit polyclonal anti-NR2B antibody was a gift from Dr. Masahiko Watanabe (22Watanabe M. Fukaya M. Sakimura K. Manabe T. Mishina M. Inoue Y. Eur. J. Neurosci. 1998; 10: 478-487Crossref PubMed Scopus (266) Google Scholar). Anti-Src mouse monoclonal antibody (GD11) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-Fyn rat monoclonal antibody (γC3) was raised by Dr. Masahiro Yasuda (23Yasunaga M. Yagi T. Hanzawa N. Yasuda M. Yamanashi Y. Yamamoto T. Aizawa S. Miyauchi Y. Nishikawa S. J. Cell Biol. 1996; 132: 91-99Crossref PubMed Scopus (37) Google Scholar). The anti-βIII tubulin mouse monoclonal antibody was purchased from Promega (Madison, WI). Assessment of Catalepsy—Catalepsy was measured by a bar test (24Boulay D. Depoortere R. Oblin A. Sanger D.J. Schoemaker H. Perrault G. Eur. J. Pharmacol. 2000; 391: 63-73Crossref PubMed Scopus (56) Google Scholar). The test was carried out 1 h after intraperitoneal injection of haloperidol (0–1.0 mg/kg) or L-741,626 (0–10 mg/kg). A 3-mm-diameter wooden bar was fixed horizontally 4 cm above the floor of a Plexiglas cage. The animals were placed inside the test cage and allowed to acclimatize for 5 min prior to performing the bar test. Both forepaws were then gently placed on the bar, and the length of time during which each mouse maintained the initial position was measured (maximum cut-off time, 180 s). Analysis of Rigidity—Muscle rigidity after haloperidol administration was assessed by a mechanographic technique using a modified device designed for rat experiments (25Lorenc-Koci E. Wolfarth S. Ossowska K. Exp. Brain Res. 1996; 109: 268-276Crossref PubMed Scopus (80) Google Scholar). The mouse was placed in a narrow, well ventilated plastic tube to restrict body movement, and one hind leg was bound to a force sensor (AD4937-5N, A & D Co. Ltd., Tokyo, Japan) that records linear reciprocating motion (15-mm distance, 15 cycles/min) via a crank and motor. The raw data from the force sensor were analyzed on a Macintosh computer connected to an A/D converter and software (PowerLab 4s, chart version 3.6 ADInstruments, Mountain View, CA), and the resistance of the flexor and extensor muscles to forced extension and flexion of the knee and ankle joint was measured. The mice were attached to the above device, and the difference in muscle resistance before and after the administration of vehicle or haloperidol (1.0 mg/kg) was recorded. The mean amplitude of 10 consecutive waves at each time point was calculated. Spikes that indicated spontaneous movements of the mice were excluded from the count. In Situ Hybridization Histochemistry—The distribution of D2-R gene expression in the striatum of the control and Fyn-deficient mice was compared. The probe was prepared as follows. A cDNA fragment encoding the sequence of mouse D2-R (1.3 kbp, a gift from Dr. T. Kaneko, Kyoto University) was cloned into the pBluescript II/KS–vector, and the clone was digested and used as a DNA template to synthesize an antisense or sense digoxigenin-labeled cRNA probe. The probe was prepared with T7 or T3 RNA polymerase and a digoxigenin RNA labeling kit (Roche Applied Science). Staining was performed as reported previously (26Yuasa S. Anat. Embryol. 1996; 194: 223-234Crossref PubMed Scopus (77) Google Scholar). The sense cRNA probe was employed as the control, and no signals in the brain were detected with it. Immunoblot Analysis—One hour after administration of the vehicle, haloperidol (1.0 mg/kg), or L-741,626 (5 mg/kg), the striatum was immediately dissected and frozen in liquid nitrogen. The striatum was placed in buffer containing 10% sucrose, 3% SDS, 10 mm Tris-HCl, pH 6.8, 1 mm sodium orthovanadate, and 1 mm phenylmethylsulfonyl fluoride and homogenized with a Polytron homogenizer (Kinematica AG, Lucerne, Switzerland). Samples were spun down (20,000 × g, 15 min) to remove insoluble material, and the protein concentration was determined with the BCA protein assay reagent (Pierce). After the addition of 40 mm dithiothreitol, the samples were boiled for 5 min, and an equal amount of protein (40 μg per lane) from each sample was separated by electrophoresis on 10% polyacrylamide gels. The gels were transferred onto Immobilon membranes (Millipore, Bedford, MA). The membranes were blocked with 1% bovine serum albumin in Tris-buffered saline (TBS; pH 7.5) containing 0.1% Tween 20 (TBS-T) or 10% skim milk in TBS-T for 1 h and probed with primary antibodies (1:750 dilution for TH, 1:4000 for anti-βIII tubulin, and 1:1000 for other antibodies). After washing three times with TBS-T, the membranes were incubated with horseradish peroxidase-labeled secondary antibodies (anti-goat, anti-rabbit, anti-rat, or anti-mouse IgG, 1:20,000 dilution, all purchased from The Jackson Laboratories, West Grove, PA). After washing three times, the signals were detected with ECL Plus (Amersham Biosciences) and ATTO Cool Saver (ATTO Corp., Tokyo, Japan). The membranes were then incubated with stripping buffer (100 mm 2-mercaptoethanol, 2% SDS, 62.5 mm Tris-HCl, pH 6.7) at 50 °C for 30 min, washed, blocked, and reprobed with other antibodies. Immunoprecipitation and Western Blotting—The procedures for immunoprecipitation were as described previously (15Miyakawa T. Yagi T. Kitazawa H. Yasuda M. Kawai N. Tsuboi K. Niki H. Science. 1997; 278: 698-701Crossref PubMed Scopus (267) Google Scholar). Striatum obtained 1 h after vehicle or haloperidol (1.0 mg/kg) administration was placed in lysis buffer (10 mm Tris-HCl, pH 7.4, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 0.15 m NaCl, 1 mm EDTA, and 1 mm sodium orthovanadate) and homogenized with a Polytron homogenizer. Samples were spun down to remove insoluble material, and the protein concentration was determined. Equal amounts of protein (500 μg) were then used for immunoprecipitation. Samples were precleared with protein G-Sepharose (Amersham Biosciences), incubated for 1 h at 4 °C with 1 μg of the anti-Fyn or anti-Src antibody, and then incubated for 1 h at 4°C with 10 μl of protein G-Sepharose. After three washes with lysis buffer, the pelleted protein G-Sepharose was boiled for 5 min in 30 μl of SDS sample buffer, and 15 μl of the supernatant was subjected to SDS-PAGE. The separated proteins were subsequently blotted onto Immobilon, probed with each antibody, and visualized as described above. Primary Cultures of Striatal Neurons—Primary cultures of striatal neurons were prepared from the fetal striata of wild-type and Fyn-deficient mice at embryonic day 17. Striata from 6 to 8 fetal brains were dissected and placed in Hanks' balanced salt solution (Invitrogen) and then were transferred into a dissociation medium containing Hanks' balanced salt solution, 0.05% DNase I, and 1% trypsin/EDTA and incubated at 37 °C for 7 min. After sedimentation, the supernatant was removed, and the pellet was washed three times with Hanks' balanced salt solution containing 1% penicillin/streptomycin. The tissue was gently placed in Hanks' balanced salt solution containing 0.05% DNase I and triturated with a plastic pipette until a homogeneous suspension was obtained. After centrifugation at 130 × g for 8 min, the cell pellet was resuspended in Neurobasal/B27 medium (Invitrogen) containing 0.5 mm l-glutamine and penicillin/streptomycin (100 units/ml). The cell cultures were seeded at a density of 3 × 105 cells/cm2 on 0.1% polyethyleneimine-coated cover glasses in 1.9 cm2/well dishes (Nunclon, Nunc). Cells were maintained at 37 °C under a humidified 5% CO2 atmosphere. The cultured striatal neurons were identified immunocytochemically with anti-GAD65 antibody (Chemicon) and anti-MAP2 antibody (Sigma). More than 95% of both the wild-type and Fyn-deficient neurons were double-labeled by anti-GAD65 and anti-MAP2 (supplemental Fig. S1). Calcium Imaging—Calcium imaging was carried out as described previously (27Uchino S. Watanabe W. Nakamura T. Shuto S. Kazuta Y. Matsuda A. Nakajima-Iijima S. Kudo Y. Kohsaka S. Mishina M. FEBS Lett. 2001; 506: 117-122Crossref PubMed Scopus (9) Google Scholar). Briefly, striatal primary cells were incubated with 10 μm fura-2/AM (Dojindo) for 1 h at 30°C in balanced salt solution (BSS) consisting of (in mm) NaCl 130, KCl 5.4, glucose 5.5, HEPES 10, and CaCl2 2, and adjusted to pH 7.4 with NaOH. After washing, the cover glasses that contained cultured neurons were mounted on the stage of an inverted fluorescence microscope (IX50; Olympus) and perfused with BSS at a flow rate of 1.8 ml/min. The perfusion medium was prewarmed and maintained at 32.6 ± 1.1 °C in the measurement dish. Fluorescence images obtained by alternate excitation with 340 and 380 nm light through the ×20 objective lens and CCD camera (C2400-8; Hamamatsu Photonics, Hamamatsu, Japan) were fed into an image processor (Argus 50, Hamamatsu) for ratiometric analysis. The effect of the D2-R antagonist L-741,626 on the channel activity of NMDA receptors was investigated in the presence of the selective D2-R agonist quinpirole in the perfusion medium. As shown in supplemental Fig. S2, quinpirole alone had a dose-dependent inhibitory effect on the channel activity of the NMDA receptors of the striatal primary neurons of the control mice consistent with its inhibitory effect reported in the striatal slice culture (28Cepeda C. Buchwald N.A. Levine M.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9576-9580Crossref PubMed Scopus (478) Google Scholar). Quinpirole was observed to have almost the same degree of the inhibitory effect on Fyn-deficient neurons (49% decrease at 50 μm). Preparation of Protein Samples from the Cultured Neurons—Striatal neurons were cultured in 1.9 cm2/well dishes (Nunclon, Nunc) as described above. Each solution used in the following experiments was pre-warmed to 37 °C in a water bath. Culture dishes were warmed on a heat block to 37 °C. The culture medium was removed, and the cultured cells were incubated with BSS for at least 5 min. The cells were then incubated with the following: 1) BSS for 7 min followed by incubation in quinpirole (50 μm) in BSS for 7 min, or 2) in quinpirole (50 μm) in BSS for 7 min followed by a mixture of quinpirole (50 μm) and L-741,626 (10 μm) in BSS for 7 min. The solution was removed, and the cells were immediately lysed in 150 μl of SDS sample buffer. Statistical Analyses—The results of the catalepsy assessment and calcium imaging were evaluated by the Kruskal-Wallis test followed by the Mann-Whitney U test. The results of the muscle rigidity analysis were evaluated by a two-way repeated measure ANOVA. The results of Western blotting were evaluated by one-way ANOVA followed by Bartlett's test. All data are expressed as the mean ± S.E. Haloperidol induced catalepsy in the +/fynZ mice, and the duration of the catalepsy increased in a dose-dependent manner (Fig. 1). By contrast, the duration of the catalepsy in the fynZ/fynZ mice was significantly shorter (Fig. 1). At the 1.0 mg/kg dose, there was no difference in the cataleptic response between the +/fynZ mice and the wild-type mice (154.5 ± 47.1 s). The D2-R-selective antagonist L-741,626 was confirmed to induce catalepsy in the control mice (supplemental Fig. S3), as reported previously in rats (29Millan M.J. Dekeyne A. Rivet J.M. Dubuffet T. Lavielle G. Brocco M. J. Pharmacol. Exp. Ther. 2000; 293: 1063-1073PubMed Google Scholar), but the duration of the catalepsy was significantly reduced in fynZ/fynZ mice (supplemental Fig. S3). Because there was no significant difference in the temporal patterns of the locomotor activity between +/fynZ mice and fynZ/fynZ mice (30Miyakawa T. Yagi T. Kagiyama A. Niki H. Brain Res. Mol. Brain Res. 1996; 37: 145-150Crossref PubMed Scopus (61) Google Scholar), the altered cataleptic response in the fynZ/fynZ mice was concluded not to be due to a locomotion defect. Because Fyn-deficient mice are more fearful than control mice (31Miyakawa T. Yagi T. Watanabe S. Niki H. Brain Res. Mol. Brain Res. 1994; 27: 179-182Crossref PubMed Scopus (78) Google Scholar), we suspected that they might avoid a procedure like the "bar test" and that the duration of catalepsy would be misleadingly short as a result. We therefore also measured muscular rigidity to minimize any such emotional influence on the response to haloperidol. Haloperidol induced a marked increase in hind limb muscle rigidity in the +/fynZ mice that was detectable as early as 45 min after administration (1.0 mg/kg) and persisted for more than 2 h, but no increase in muscle rigidity was detected in the fynZ/fynZ mice (Fig. 2 and supplemental Fig. S4). To exclude the possibility that the failure to respond to haloperidol was because of a difference in D2-R expression, in situ hybridization of D2-R mRNA and Western blotting of D2-R protein were performed on the striatum of +/fynZ and fynZ/fynZ mice. As shown in Fig. 3, no clear difference was observed in either striatal D2-R gene expression (Fig. 3A) or the protein level (Fig. 3B). Western blotting analysis of striatal TH was also performed to determine whether there was any difference between the two genotypes in the abundance of the rate-limiting enzyme in dopamine biosynthesis, but little difference in the amount of TH protein was found (Fig. 3C). The effect of haloperidol on protein tyrosine phosphorylation in the striatum of the +/fynZ and fynZ/fynZ mice was compared by Western blotting. One hour after haloperidol administration (1.0 mg/kg), a marked increase in tyrosine phosphorylation of several proteins, including 60-, 110-, and 180-kDa proteins, was observed in the striatum of the +/fynZ mice but not of the fynZ/fynZ mice (Fig. 4A). Because the 60-kDa protein corresponds in size to SFKs, we measured tyrosine phosphorylation of the activation-related Tyr-418 residue on SFKs. As shown in Fig. 4B, phospho-Tyr-418 increased after haloperidol injection of the +/fynZ mice, but no such effect was observed in the fynZ/fynZ mice. Basal Tyr(P)-418 immunoreactivity was much lower in the fynZ/fynZ mice. Because the anti-pY418 antibody recognizes both Fyn and Src, we immunoprecipitated Fyn and Src, and we examined the phosphorylation of the activation-related residue, Tyr-418, and of the inhibition-related residue, Tyr(P)-529, by Western blotting in the +/fynZ mice. As shown in Fig. 4C, Fyn but not Src was activated at Tyr-418 by haloperidol, and no change was observed in the phosphorylation at Tyr-529. Because the 180-kDa protein corresponds in size to the NR2B subunit, we also measured the phosphorylation of the Tyr-1472 of NR2B, the key phosphorylation site, by Fyn. The results showed that phospho-Tyr-1472 increased in the +/fynZ mice but not in the fynZ/fynZ mice (Fig. 4D). The basal level of Tyr(P)-1472 in the fynZ/fynZ mice was not significantly different from the basal level in the +/fynZ mice. Marked increases in tyrosine phosphorylation of the 60-, 110-, and 180-kDa proteins and up-regulation of Tyr(P)-418 and Tyr(P)-1472 were also observed following L-741,626 administration to the control mice, but no such effects were observed in the fynZ/fynZ mice (supplemental Fig. S5). There were no sex differences in the results of either the behavioral or biochemical studies (data not shown). The same findings in regard to the haloperidol-induced enhancement of tyrosine phosphorylation were also observed in the wild-type mice, and no clear difference was detected between +/fynZ mice and wild-type mice (data not shown). To investigate whether the Fyn-mediated increase in NMDA receptor phosphorylation by D2-R blockade affects NMDA receptor activity, we prepared striatal primary cultures and assessed the channel activity of NMDA receptors by the calcium imaging method. After 4–7 days of culture, we loaded 10 μm fura-2/AM into the primary cells and measured the increase in intracellular calcium concentration ([Ca2+]i) by calcium fluorimetry. Exposure to 3 μm NMDA/10 μm glycine for 30 s induced a robust response in more than 95% of the cells analyzed, and repeated applications of NMDA/glycine at 5-min intervals evoked reproducible responses (data not shown), indicating little desensitization of the NMDA receptors under our experimental conditions. We first examined the effect of a D2-R-selective antagonist, L-741,626, on the channel activity of NMDA receptors of wild-type neurons in the presence of a D2-R-selective agonist, quinpirole (50 μm). After confirming that the responses evoked were reproducible by two successive applications of 3 μm NMDA, 10 μm glycine (control responses), we added 10 μm L-741,626 to the perfusion buffer BSS. After 5 min, we measured the 3 μm NMDA, 10 μm glycine-induced increases in [Ca2+]i and compared them with the control responses. As shown in Fig. 5A, larger responses than the control responses were detected in the presence of L-741,626, but in some neurons, almost identical responses were observed both in the presence and absence (control) of L-741,626. The distribution of changes in [Ca2+]i responses after the addition of L-741,626 is shown in Fig. 5B (open bars). After the addition of L-741,626, a large number of neurons (74.7%) showed larger responses than the control responses (mean change, 125.7 ± 34.1%). To determine whether the larger NMDA/glycine-induced responses induced by L-741,626 were mediated by Fyn, we performed the same experiments on primary cultures prepared from Fyn-deficient mice. After the addition of L-741,626, only 46.6% of the Fyn-deficient neurons showed larger responses than the control responses, and L-741,626 addition had little enhancing effect on the responses (mean change, 106.8 ± 26.9% of the control responses; see Fig. 5B, filled bars). A subset of Fyn-deficient neurons exhibited larger responses after L-741,626 addition, and their responses peaked at around 120% of the control responses. In addition, the numbers of neurons exhibiting larger responses after L-741,626 addition was lower in the presence of the Src family inhibitor PP2 (10 μm) (mean change, 99.9 ± 15.7% of the control responses). We then examined the effect of L-741,626 on Fyn activation and NMDA receptor phosphorylation in the presence of quinpirole (50 μm) by Western blot analysis, as shown in Fig. 6. Primary cells were exposed or not exposed (control) to 10 μm L-741,626 for 7 min prior to sample preparation. In the wild-type cells, immunoreactivity for anti-pY1472 antibody and anti-pY418 antibody in the L-741,626-treated cell extracts was stronger than in the control cell extracts. By contrast, when Fyn-deficient cells were used, there were no significant differences in immunoreactivity for anti-pY1472 antibody and anti-pY418 antibody between the L-741,626-exposed cell extracts and the control cell extracts. The results of this study show that Fyn is required for haloperidol-induced catalepsy. We also found that haloperidol induces Fyn activation and a Fyn-dependent increase in NR2B phosphorylation in mouse striatum. We used striatal primary neurons to verify that D2-R blockade induced Fyn activation, enhancement of NR2B phosphorylation, and potentiation of th
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