mGluR1 in cerebellar Purkinje cells essential for long-term depression, synapse elimination, and motor coordination

Science

Volumn 288, Issue 5472, 2000, Pages 1832-1835

  Ichise, Taeko ^a   Kano, Masanobu ^b   Hashimoto, Kouichi ^b   Yanagihara, Dai ^c,d   Nakao, Kazuki ^a   Shigemoto, Ryuichi ^e   Katsuki, Motoya ^a   Aiba, Atsu ^a  

^a UNIVERSITY OF TOKYO   (Japan)

^b KANAZAWA UNIVERSITY   (Japan)

^c TOYOHASHI UNIVERSITY OF TECHNOLOGY   (Japan)

^d RIKEN BRAIN SCIENCE INSTITUTE   (Japan)

^e NATIONAL INSTITUTE FOR PHYSIOLOGICAL SCIENCES   (Japan)

Author keywords

[No Author keywords available]

Indexed keywords

METABOTROPIC RECEPTOR;

ANIMAL EXPERIMENT; ARTICLE; CEREBELLUM; DEPRESSION; MOTOR COORDINATION; MOUSE; NERVE CELL DIFFERENTIATION; NERVE CELL PLASTICITY; NERVE GROWTH; NONHUMAN; PRIORITY JOURNAL; PURKINJE CELL; TRANSGENE;

ANIMALS; EXCITATORY POSTSYNAPTIC POTENTIALS; GENE TARGETING; LOCOMOTION; MICE; MICE, TRANSGENIC; MOTOR ACTIVITY; NERVE FIBERS; NEURONAL PLASTICITY; PSYCHOMOTOR PERFORMANCE; PURKINJE CELLS; RECEPTORS, METABOTROPIC GLUTAMATE; SYNAPSES; TRANSGENES;

ANIMALIA;

EID: 0034625542     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.288.5472.1832     Document Type: Article

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16. We generated transgenic mice (L7-mGluR1) that expressed mGluR1α under the control of the PC-specific L7 promoter [J. Oberdick, R. J. Smeyne, J. R. Mann, S. Zackson, J. I. Morgan, Science 248, 223 (1990); R. J. Smeyne et al., Science 254, 719 (1991); R. J. Smeyne et al., Mol. Cell. Neurosci. 6, 230 (1995)]. A 3.7-kb Sac II-Fsp I fragment of rat mGluR1α cDNA containing a 26 base pair (bp) 5′ untranslated region (UTR), a 3597-bp coding region, and a 36-bp 3′ UTR was introduced into exon 4 of the L7 gene cassette. We obtained eight independent L7-mGluR1 transgenic founder mice by microinjecting the transgene into the pronuclei of fertilized mGluR1 (+/-) eggs. The mGluR1-rescue mice were obtained by breeding mGluR1 (+/-) with transgenic mice. The cerebellum-restricted expression of the transgene was examined by Western blotting with polydonal antibodies to rat mGluR1 (Upstate Biotechnology, Lake Placid, NY). One line expressed the L7- mGluR1α transgene in the cerebellum but not in the cerebral cortex.
17. We generated transgenic mice (L7-mGluR1) that expressed mGluR1α under the control of the PC-specific L7 promoter [J. Oberdick, R. J. Smeyne, J. R. Mann, S. Zackson, J. I. Morgan, Science 248, 223 (1990); R. J. Smeyne et al., Science 254, 719 (1991); R. J. Smeyne et al., Mol. Cell. Neurosci. 6, 230 (1995)]. A 3.7-kb Sac II-Fsp I fragment of rat mGluR1α cDNA containing a 26 base pair (bp) 5′ untranslated region (UTR), a 3597-bp coding region, and a 36-bp 3′ UTR was introduced into exon 4 of the L7 gene cassette. We obtained eight independent L7-mGluR1 transgenic founder mice by microinjecting the transgene into the pronuclei of fertilized mGluR1 (+/-) eggs. The mGluR1-rescue mice were obtained by breeding mGluR1 (+/-) with transgenic mice. The cerebellum-restricted expression of the transgene was examined by Western blotting with polydonal antibodies to rat mGluR1 (Upstate Biotechnology, Lake Placid, NY). One line expressed the L7-mGluR1α transgene in the cerebellum but not in the cerebral cortex.
18. Adult wild-type, mGluR1 (-/-). and mGluR1-rescue (Tg/+) mice were deeply anesthetized with pentobarbital (100 mg/kg of body weight) and were perfused transcardially with 3.5% paraformaldehyde, 0.05% glutaraldehyde, and 1% picric acid in 0.1 M sodium phosphate buffer (pH 7.3). The brains were cryoprotected with 25% (w/w) sucrose in 0.1 M phosphate buffer overnight at 4°C and cut on a freezing microtome or cryostat into 40-μm-thick parasagittal sections. The free-floating sections were incubated overnight at room temperature with antibodies to mGluR1α (1.0 μg/ml) [R. Shigemoto et al., Nature 381, 523 (1996)], 0.1% Triton X-100, and 0.25% carrageenan. These sections were then washed with phosphate buffered saline (PBS), incubated with biotinylated antibody to guinea pig immunoglobulin G, washed again, and reacted with avidin-biotin-peroxidase complex (ABC Kit, Vector. Burtingame, CA). Finally, the sections were reacted with 0.02% dlaminobenzidine tetrahydrochloride and 0.002% hydrogen peroxide in 50 mM tris-HCl (pH 7.6).
19. 2. Bicuculline (10 μM) was included to block spontaneous inhibitory postsynaptic currents. Membrane currents were recorded with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA). Glass pipettes filled with standard extracellular solution were used to stimulate CFs in the granule cell layer. The PULSE software (version 8.2, HEKA, Lambrecht, Germany) was used for stimulation and data acquisition. The signals were filtered at 3 kHz and digitized at 20 kHz.
20. 2. Bicuculline (10 μM) was included to block spontaneous inhibitory postsynaptic currents. Membrane currents were recorded with an Axopatch-1D amplifier (Axon Instruments, Foster City, CA). Glass pipettes filled with standard extracellular solution were used to stimulate CFs in the granule cell layer. The PULSE software (version 8.2, HEKA, Lambrecht, Germany) was used for stimulation and data acquisition. The signals were filtered at 3 kHz and digitized at 20 kHz.
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28. G. N. Orlovsky, F. V. Severin, M. L Shik, Biofizika 11, 509 (1966); J. A. Gruner, J. Altman, N. Spivack, Exp. Brain Res. 40, 361 (1980); M. Udo, K. Matsukawa, H. Kamei, Y. Oda, J. Neurophysiol. 44, 119 (1980); P. A. Fortier, A. M. Smith, S. Rossignol, Exp. Brain Res. 66, 271 (1987); N. Kashiwabuchi et al., Cell 81, 245 (1995); L. Chen, S. Bao, J. M. Lockard, J. K. Kim, R. F. Thompson, J. Neurosci. 16, 2829 (1996).
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31. Mice were placed under pentobarbital anaesthesia (40 to 50 mg/kg of body weight) before surgical operation. A platform for fixation of the head to a stereotaxic frame was built in aseptic conditions. After making an incision in the skin, four small screws around the central long bolt were mounted on the parietal cranium, and were sealed by dental cement. Cinematographic recordings were started 2 to 4 days after the surgery. During recordings, a mouse was mounted on the treadmill with its head fixed, but its body and limbs were not restrained. Locomotion was induced by moving the belts at moderate velocities (12, 14, and 16 cm/s). Movement of the mice was filmed with a video camera (SONY DXC-107A, Tokyo, Japan) equipped with a shutter operating at 60 fields per second. A field-by-field analysis (16.7-ms time resolution) of the videotapes revealed the temporal measurements.
32. Animals were housed at 21° ± 1°C with free access to food and water and tested at 12 to 15 weeks. Experiments were conducted in accordance with ethical guidelines of the Institute of Medical Science, University of Tokyo. In the open-field test, each mouse was placed in the middle of a 75-cm-diameter enclosure, and the walking route of the mouse was traced with a behavioral tracing analyzer (Muromachi Kikai, Tokyo). The total walking distance was recorded every 30 min over a 2-hour period.
33. The Rota-Rod Treadmill (Muromachi Kikai) consists of a gritted plastic rod (3 cm in diameter, 10 cm long) flanked by two large round plates (50 cm in diameter). The time the mouse remained on the rod was measured. A maximum of 120 s was allowed to test each animal.
34. We thank S. Nakanishi for rat mGluR1α cDNA. Supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (to M.K. and A.A.); the Grant-in-Aid for Special Scientific Research on Agriculture, Forestry, and Fisheries (to A.A); the Human Frontier Science Program (to M.K.); and by the Special Coordination Funds for Promoting Science and Technology from Science and Technology Agency of Japan (to M.K.). Language assistance was provided by M. Ohara.