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note
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A mouse GDNF cDNA was cloned by the reverse transcription polymerase chain reaction (RT-PCR) using total RNA from embryonic (E13) head as template. A 700-bp Bam HI fragment, containing the full-length cDNA for mouse GDNF, was cloned into the plasmid cassette pMyo-hGH, containing a 1.6-kb Hind III-Kpn I fragment, corresponding to myogenin promoter (10-11), and a 2.1-kb Bam HI-Hind III fragment, containing human growth hormone gene including a polyadenylation signal. The resulting plasmid pMyo-GDNF was digested with Not I and Xho I; the 4.4-kb transgene fragment was eluted from an agarose gel and used for pronuclear injection (B6/CBA strain). Integration of the transgene into the mouse genome was determined by PCR on genomic DNA from mouse tail using the 5′-oligo (5′-TGATGTGGTAGTGGTAGGTCT-3′) and the 3′-oligo (5′-CAGGCATATTGGAGTCACTGG-5′). Genotype was confirmed by Southern (DNA) blot analysis as described (11) using a 0.7-kb digoxigenin-labeled PCR product, corresponding to the mouse GDNF cDNA as a probe.
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1 offspring from different transgenic lines was assessed by in situ hybridization on cryostat sections (16 μm) of P1 mice cut in the transverse plane at the level of the forelimbs using riboprobes to GDNF, as described (7)
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1 offspring from different transgenic lines was assessed by in situ hybridization on cryostat sections (16 μm) of P1 mice cut in the transverse plane at the level of the forelimbs using riboprobes to GDNF, as described (7).
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17
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7144263314
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max immunoassay kit (Promega), following the manufacturer's instructions using whole muscles (upper hind limbs) dissected from PS and adult animals
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max immunoassay kit (Promega), following the manufacturer's instructions using whole muscles (upper hind limbs) dissected from PS and adult animals.
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19
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7144232091
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note
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Mice were anesthetized and a ventral midline incision was made to expose sternomastoid muscles. Tetramethyl rhodamine isothiocyanate α-bungarotoxin (TRITC-αBTX, 5 μg/ml; Molecular Probes) was applied to the muscles (20 min) to label postsynaptic acetylcholine receptors (AChRs). After transcardial perfusion with 2% paraformaldehyde, whole sternomastoid muscles were dissected and pinned on a Sylgard-lined dish. Nerve terminals and axons were labeled with antibodies to neurofilament (SMI312, Sternberger) and synaptic vesicles (G95, P. Greengard). NMJs doubly labeled for nerve terminals and AChRs were viewed with high-numerical-aperture objectives with confocal optics using a real-time scanner (Noran, Odyssey). The number of incoming axons could be accurately defined by carefully focusing through the region of the NMJ where the nerves enter.
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Whole diaphragm muscles with an intact portion of the phrenic nerve were dissected from P9-P10 mice in oxygenated culture medium (Dulbecco's). Electrophysiological recordings of numbers of axons innervating single muscle fibers were made using intracellular recording as described [P. A. Redfern, J. Physiol. (London) 209, 701 (1970)] in the presence of 1 to 3 μM curare to prevent muscle contraction.
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Redfern, P.A.1
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0026529469
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P6-P8 mice were anesthetized and a ventral midline incision was made to expose the left sternomastoid muscle. Motor neurons that innervate the sternomastoid muscle were retrogradely labeled with Fluoro-Gold (Fluorochrome) as described [D. M. Rotto-Percelay et al., Brain Res. 574, 291 (1992)]. Briefly, three separate injections (200 to 400 nl) of 4% Fluoro-Gold in saline were made into the left sternomastoid muscle. The wound was sutured closed and the mouse pups were returned to their mother. At P11 (3 to 5 days later), the pups were reanesthetized; brainstem and spinal cords were processed for motor neuronal cell counting with the following changes: Brainstem and spinal cords were cut in the horizontal plane at 12 μm, mounted serially on glass slides, counterstained with ethidium bromide [L. C. Schmued, L. W. Swanson, P. E. Sawchenko, J. Histochem. Cytochem. 30, 123 (1982)], and cover-slipped in glycerin. Fluoro-Gold-labeled neurons were examined with a fluorescence microscope with an ultraviolet filter set (Leica) and imaged with a silicon-intensified camera. As a precaution to prevent errors associated with double counting, only cells in which a nucleus could be seen were counted and adjacent sections were compared [physical dissector; R. E. Coggeshall, Trends Neurosci. 15, 9 (1992)].
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0020357721
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P6-P8 mice were anesthetized and a ventral midline incision was made to expose the left sternomastoid muscle. Motor neurons that innervate the sternomastoid muscle were retrogradely labeled with Fluoro-Gold (Fluorochrome) as described [D. M. Rotto-Percelay et al., Brain Res. 574, 291 (1992)]. Briefly, three separate injections (200 to 400 nl) of 4% Fluoro-Gold in saline were made into the left sternomastoid muscle. The wound was sutured closed and the mouse pups were returned to their mother. At P11 (3 to 5 days later), the pups were reanesthetized; brainstem and spinal cords were processed for motor neuronal cell counting with the following changes: Brainstem and spinal cords were cut in the horizontal plane at 12 μm, mounted serially on glass slides, counterstained with ethidium bromide [L. C. Schmued, L. W. Swanson, P. E. Sawchenko, J. Histochem. Cytochem. 30, 123 (1982)], and cover-slipped in glycerin. Fluoro-Gold-labeled neurons were examined with a fluorescence microscope with an ultraviolet filter set (Leica) and imaged with a silicon-intensified camera. As a precaution to prevent errors associated with double counting, only cells in which a nucleus could be seen were counted and adjacent sections were compared [physical dissector; R. E. Coggeshall, Trends Neurosci. 15, 9 (1992)].
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P6-P8 mice were anesthetized and a ventral midline incision was made to expose the left sternomastoid muscle. Motor neurons that innervate the sternomastoid muscle were retrogradely labeled with Fluoro-Gold (Fluorochrome) as described [D. M. Rotto-Percelay et al., Brain Res. 574, 291 (1992)]. Briefly, three separate injections (200 to 400 nl) of 4% Fluoro-Gold in saline were made into the left sternomastoid muscle. The wound was sutured closed and the mouse pups were returned to their mother. At P11 (3 to 5 days later), the pups were reanesthetized; brainstem and spinal cords were processed for motor neuronal cell counting with the following changes: Brainstem and spinal cords were cut in the horizontal plane at 12 μm, mounted serially on glass slides, counterstained with ethidium bromide [L. C. Schmued, L. W. Swanson, P. E. Sawchenko, J. Histochem. Cytochem. 30, 123 (1982)], and cover-slipped in glycerin. Fluoro-Gold-labeled neurons were examined with a fluorescence microscope with an ultraviolet filter set (Leica) and imaged with a silicon-intensified camera. As a precaution to prevent errors associated with double counting, only cells in which a nucleus could be seen were counted and adjacent sections were compared [physical dissector; R. E. Coggeshall, Trends Neurosci. 15, 9 (1992)].
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7144222319
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note
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P9-P10 mice were perfused transcardially with 2% paraformaldehyde. The nerve to the sternomastoid muscle was dissected and stored in a 2% paraformaldehyde-2% glutaraldehyde solution for 5 to 6 hours. After an overnight rinse in 5% sucrose in phosphate-buffered saline, nerves were stained in 1% osmium tetroxide. Plastic sections (1 μm) were serially mounted and stained with 1% toluidine blue-1% sodium borate. Cross sections of the nerve were photographed under a dissecting scope (50x) and the number of myelinated axons (of all diameters) counted.
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0018651915
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Whole sternomastoid muscles with an intact portion of the incoming nerve were dissected from P10 mice in oxygenated culture medium (Dulbecco's). Motor unit twitch tension recordings using a force transducer (Cambridge Neuroscience) were performed as described [W. J. Betz, J. H. Calwell, R. R. Ribchester, J. Physiol. (London) 297, 463 (1979)].
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We thank L. Worley for excellent technical assistance. Supported by grants from NIH and the Muscular Dystrophy Association
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We thank L. Worley for excellent technical assistance. Supported by grants from NIH and the Muscular Dystrophy Association.
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