Maintenance of acetylcholine receptor number by neuregulins at the neuromuscular junction in vivo

Science

Volumn 276, Issue 5312, 1997, Pages 599-603

  Sandrock Jr , Alfred W ^a,b   Dryer, Stuart E ^a,d   Rosen, Kenneth M ^a   Gozani, Shai N ^a   Kramer, Rainer ^c   Theill, Lars E ^c   Fischbach, Gerald D ^a  

^a HARVARD MEDICAL SCHOOL   (United States)

^b MASSACHUSETTS GENERAL HOSPITAL   (United States)

^c AMGEN INC   (United States)

^d FLORIDA STATE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CHOLINERGIC RECEPTOR; IMMUNOGLOBULIN; NEU DIFFERENTIATION FACTOR;

AMPLITUDE MODULATION; ANIMAL CELL; ARTICLE; MOUSE; NEUROMUSCULAR FUNCTION; NONHUMAN; POSTSYNAPTIC MEMBRANE; PRIORITY JOURNAL; PROTEIN PURIFICATION; RECEPTOR BINDING; RECEPTOR DENSITY; SYNAPTIC TRANSMISSION;

EID: 1842338111     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5312.599     Document Type: Article

References (57)

1. Z. W. Hall and J. R. Sanes, Cell 72 / Neuron 10 (suppl.), 99 (1993).
2. Z. W. Hall and J. R. Sanes, Cell 72 / Neuron 10 (suppl.), 99 (1993).
3. U. J. McMahan, Cold Spring Harbor Symp. Quant. Biol. 55, 407 (1990); M. Gautam et al., Cell 85, 525 (1996).
4. U. J. McMahan, Cold Spring Harbor Symp. Quant. Biol. 55, 407 (1990); M. Gautam et al., Cell 85, 525 (1996).
5. L. W. Role, V. R. Matossian, R. J. O'Brien, G. D. Fischbach, J. Neurosci. 5, 2197 (1985).
6. J. P. Merlie and J. R. Sanes, Nature 317, 66 (1985); D. Goldman and J. Staple, Neuron 3, 219 (1989); J. R. Sanes et al., Development 113, 1181 (1991); A. M. Simon, P. Hoppe, S. J. Burden, ibid. 114, 545 (1992).
7. J. P. Merlie and J. R. Sanes, Nature 317, 66 (1985); D. Goldman and J. Staple, Neuron 3, 219 (1989); J. R. Sanes et al., Development 113, 1181 (1991); A. M. Simon, P. Hoppe, S. J. Burden, ibid. 114, 545 (1992).
8. J. P. Merlie and J. R. Sanes, Nature 317, 66 (1985); D. Goldman and J. Staple, Neuron 3, 219 (1989); J. R. Sanes et al., Development 113, 1181 (1991); A. M. Simon, P. Hoppe, S. J. Burden, ibid. 114, 545 (1992).
9. J. P. Merlie and J. R. Sanes, Nature 317, 66 (1985); D. Goldman and J. Staple, Neuron 3, 219 (1989); J. R. Sanes et al., Development 113, 1181 (1991); A. M. Simon, P. Hoppe, S. J. Burden, ibid. 114, 545 (1992).
10. H. R. Brenner, A. Herczeg, C. R. Slater, Development 116, 41 (1992).
11. M. Gautam et al., Nature 377, 232 (1995).
12. T. M. Jessell, R. E. Siegel, G. D. Fischbach, Proc. Natl. Acad. Sci. U.S.A. 76, 5397 (1979); M. H. BucCaron, P. Nystrom, G. D. Fischbach, Dev. Biol. 95, 378 (1983); T. B. Usdin and G. D. Fischbach, J. Cell Biol. 103, 493 (1986); G. D. Fischbach and K. M. Rosen, Annu. Rev. Neurosci., in press.
13. T. M. Jessell, R. E. Siegel, G. D. Fischbach, Proc. Natl. Acad. Sci. U.S.A. 76, 5397 (1979); M. H. BucCaron, P. Nystrom, G. D. Fischbach, Dev. Biol. 95, 378 (1983); T. B. Usdin and G. D. Fischbach, J. Cell Biol. 103, 493 (1986); G. D. Fischbach and K. M. Rosen, Annu. Rev. Neurosci., in press.
14. T. M. Jessell, R. E. Siegel, G. D. Fischbach, Proc. Natl. Acad. Sci. U.S.A. 76, 5397 (1979); M. H. BucCaron, P. Nystrom, G. D. Fischbach, Dev. Biol. 95, 378 (1983); T. B. Usdin and G. D. Fischbach, J. Cell Biol. 103, 493 (1986); G. D. Fischbach and K. M. Rosen, Annu. Rev. Neurosci., in press.
15. T. M. Jessell, R. E. Siegel, G. D. Fischbach, Proc. Natl. Acad. Sci. U.S.A. 76, 5397 (1979); M. H. BucCaron, P. Nystrom, G. D. Fischbach, Dev. Biol. 95, 378 (1983); T. B. Usdin and G. D. Fischbach, J. Cell Biol. 103, 493 (1986); G. D. Fischbach and K. M. Rosen, Annu. Rev. Neurosci., in press.
16. P. L. Falls et al., Cell 72, 801 (1993).
17. W. E. Holmes et al., Science 256, 1205 (1992); D. Wen et al., Cell 69, 559 (1992).
18. W. E. Holmes et al., Science 256, 1205 (1992); D. Wen et al., Cell 69, 559 (1992).
19. M. A. Marchionni et al., Nature 362, 312 (1993).
20. K. M. Rosen et al., Cold Spring Harbor Symp. Quant. Biol., in press.
21. A. D. J. Goodearl et al., J. Cell Biol. 130, 1423 (1995).
22. G. Corfas et al., Neuron 14, 103 (1995).
23. N. Altiok, J. L. Bessereau, J. P. Changeux, EMBO J. 14, 4258 (1995); X. J. Zhu, C. Lai, S. Thomas, S. J. Burden, ibid., p. 5842.
24. N. Altiok, J. L. Bessereau, J. P. Changeux, EMBO J. 14, 4258 (1995); X. J. Zhu, C. Lai, S. Thomas, S. J. Burden, ibid., p. 5842.
25. S. A. Jo, X. Zhu, M. A. Marchionni, S. J. Burden, Nature 373, 158 (1995).
26. L. M. Moscoso et al., Dev. Biol. 172, 158 (1995).
27. G. C. Chu, L. M. Moscoso, M. X. Sliwkowski, J. P. Merlie, Neuron 14, 329 (1995).
28. A. W. Sandrock et al., J. Neurosci. 15, 6124 (1995).
29. J. C. Martinou, D. L. Falls, G. D. Fischbach, J. P. Merlie, Proc. Natl. Acad. Sci. U.S.A. 88, 7669 (1991).
30. H. R. Brenner and B. Sakmann, Nature 271, 366 (1978); J. Physiol. (London) 337, 159 (1983); H. R. Brenner, V. Witzemann, B. Sakmann, Nature 344, 544 (1990).
31. H. R. Brenner and B. Sakmann, Nature 271, 366 (1978); J. Physiol. (London) 337, 159 (1983); H. R. Brenner, V. Witzemann, B. Sakmann, Nature 344, 544 (1990).
32. H. R. Brenner and B. Sakmann, Nature 271, 366 (1978); J. Physiol. (London) 337, 159 (1983); H. R. Brenner, V. Witzemann, B. Sakmann, Nature 344, 544 (1990).
33. D. Meyer and C. Birchmeier, ibid. 378, 386 (1995).
34. R. Kramer et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4833 (1996).
35. K. Lee et al., Nature 378, 394 (1995).
36. M. Gassman et al., Nature 378, 390 (1995).
37. M. J. Dennis, L. Ziskind-Conhaim, A. J. Harris, Dev. Biol. 81, 266 (1981).
38. Y. Kuo, X. Yang, L. Role, Soc. Neurosci. Abstr. 20, 452.18 (1994); W.-H. Ho et al., J. Biol. Chem. 270, 14523 (1995).
39. Y. Kuo, X. Yang, L. Role, Soc. Neurosci. Abstr. 20, 452.18 (1994); W.-H. Ho et al., J. Biol. Chem. 270, 14523 (1995).
40. J. A. Loeb and G. D. Fischbach, J. Cell Biol. 130, 127 (1995).
41. S. J. Burden, P. B. Sargent, U. J. McMahan, ibid. 82, 412 (1979).
42. D. Goldman, B. M. Carlson, J. Staple, Neuron 7, 649 (1991).
43. 4] containing S1 nuclease (350 U/ml) (Pharmacia, Piscataway, NJ), and the reactions were incubated for 1 hour at 37°C. The products were separated by electrophoresis on DNA sequencing gels and visualized with a phosphorimager screen (Molecular Dynamics, Sunnyvale, CA) exposed for 24 to 72 hours. The density of equivalent areas was determined, and values for relative neuregulin levels were expressed as the ratio to the signal for GAPDH.
44. A. Orr-Urteger et al., Proc. Natl. Acad. Sci. U.S.A. 90,1867 (1993); M. S. Chen et al., J. Comp. Neurol. 349, 389 (1994); D. Meyer and C. Birchmeier, Proc. Natl. Acad. Sci. U.S.A. 91, 1064 (1994).
45. A. Orr-Urteger et al., Proc. Natl. Acad. Sci. U.S.A. 90,1867 (1993); M. S. Chen et al., J. Comp. Neurol. 349, 389 (1994); D. Meyer and C. Birchmeier, Proc. Natl. Acad. Sci. U.S.A. 91, 1064 (1994).
46. A. Orr-Urteger et al., Proc. Natl. Acad. Sci. U.S.A. 90,1867 (1993); M. S. Chen et al., J. Comp. Neurol. 349, 389 (1994); D. Meyer and C. Birchmeier, Proc. Natl. Acad. Sci. U.S.A. 91, 1064 (1994).
47. 3/min. Two 26G monopolar needle electrodes (Nicolet, Madison, WI) were inserted into the anterior axillary region in order to supramaximally stimulate the brachial plexus with 5-to 10-mA constant current pulses of 200 μs duration (WPI A360, Sarasota, FL). Trains of 10 stimuli were delivered at frequencies of 3 or 30 Hz. CMAPs were recorded with a 24G monopolar needle electrode (Nicolet) inserted into the flexor muscle compartment of the ipsilateral forelimb and were amplified with a low noise differential amplifier (WPI Isodam, Sarasota, FL). A small disposable surface electrode (Nicolet) attached to the skin of the forepaw served as the reference electrode for the differential measurement. Another disposable surface electrode was attached to the tail to serve as the ground. For systemic curare infusions, a 27G butterfly catheter was inserted into the peritoneal cavity through a small skin incision. Incremental doses (35, 70, 140, 280, 560, and 1120 nmol/kg) of d-tubocurarine chloride (Sigma) in 0.5 to 1.0 ml of 0.9% NaCI were injected through the catheter every 10 min (the catheter was flushed with 1 ml saline after each injection). During the timed curare infusions, trains of 10 repetitive nerve stimulations (3 Hz alternating with 30 Hz) were performed at 1-min intervals, and the ratio of amplitudes of the second to sixth CMAPs was calculated online.
48. 2+ in the bath solution was selected with this in mind. Because most endplate responses were less than 5 mV, no corrections were made for nonlinear summation of synaptic potentials.
49. 125I-α-bungarotoxin (Amersham; specific activity = 2000 Ci/mmol) in Puck's saline solution containing 1 mg of bovine serum albumin (BSA) per milliliter. The diaphragms were then rinsed four times (15 min each) with PBS and fixed for 15 min at room temperature with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). After being rinsed in L15 medium (Gibco) containing BSA (1 mg/ml), the diaphragms were divided midsagittally into hemidiaphragms. In some experiments, endplate-rich strips (measuring approximately 20 × 1.5 mm) were prepared from each hemidiaphragm by cutting along either side of the main intramuscular branch of the phrenic nerve. These strips were immersed in 0.5 ml of PBS, and the radioactivity was determined in a gamma counter. Specific α-bungarotoxin binding was calculated by subtracting counts per minute found in endplate-rich strips of diaphragms that had been incubated with excess unlabeled α-bungarotoxin. In other experiments, hemidiaphragms were immersed in 30% sucrose in PBS overnight at 4°C before being placed on microscope slides (SuperFrost Plus, Fisher Scientific) where they were teased into fascicles of fewer than five muscle fibers with a pair of fine forceps. After drying, the teased fibers were rinsed with distilled water and the slides were dipped in a 1:1 mixture of NT-3B emulsion (Kodak) with water. The slides were placed in a desiccated, light-tight slide box and stored at 4°C for 8 hours before being developed, fixed, dehydrated with ethanol and xylene, and cover-slipped in Cytoseal (VWR Scientific Products, Bridgeport, NJ). For the photomicrographs of Fig. 3C, endplates were photographed on 35-mm color slide film under Nomarski optics. For morphometric studies, bright-field images were captured with a charge-coupled device (CCD) camera (Hamamatsu). With a 20× objective lens, most of the grains at each endplate were in sharp focus. Monochromatic (gray-scale) digitized images only of endplates viewed en face were collected with Image-1 software (Corel). With the use of SigmaScanPro software (Jandel Scientific), a box was drawn around the endplate, and a 0 to 100% intensity scale was created for each image. The number of pixels that fell below a 50% intensity level were counted. This intensity threshold was chosen because it gave accurate grain counts. Statistical significance was estimated with the Mann-Whitney rank sums test with the use of SigmaStat (Jandel Scientific).
50. Mice were killed by an overdose of isoflurane, and diaphragms were dissected, rinsed with PBS, fixed for 15 min with 3% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4), immersed in 30% sucrose in PBS overnight at 4°C, and teased into fascicles of fewer than five muscle fibers, as described above. After drying, the slides were dipped into a reaction mixture containing acetylthiocholine iodide (0.5 mg/ml), 100 mM sodium citrate, and 30 mM cupric sulfate in 0.1 M sodium maleate buffer (pH 6.0). The reaction was activated by the addition of 10% (v/v) 5 mM potassium ferricyanide. After reacting for 20 to 30 min at room temperature, the slides were rinsed with PBS, dehydrated with ethanol and xylene, and cover-slipped in Cytoseal (VWR). In control experiments, acetylthiocholine iodide was replaced with butyrylthiocholine iodide. For the photomicrographs of Fig. 4C, AChE-stained endplates were photographed on 35-mm color slide film under bright-field optics. For morphometric studies, bright-field images were captured with a CCD camera (Hamamatsu) equipped with an intensifier. Monochromatic (gray-scale) digitized images only of endplates viewed en face were collected using Image-1 software (Corel). Using SigmaScanPro software (Jandel Scientific), a 0 to 100% intensity scale was created and the area occupied by pixels falling below a 35% intensity level was calculated. This intensity threshold accurately defined the boundaries of AChE-stained endplates. Statistical comparisons were made using SigmaStat (Jandel Scientific).
51. A. Mallart and A. R. Martin, J. Physiol. (London) 196, 593 (1968).
52. 2, so that transmitter release was not reduced. In these experiments, neuromuscular transmission was blocked postsynaptically with d-tubocurarine (1.0 to 1.5 μg/ ml) (Sigma) in order to keep evoked EPPs below the action potential threshold. Trains of stimuli were delivered at 25 Hz.
53. J. J. Plomp; G. T. van Kempen, P. C. Molenaar, J. Physiol. (London) 458, 487 (1992); ibid. 478, 125 (1994).
54. J. J. Plomp; G. T. van Kempen, P. C. Molenaar, J. Physiol. (London) 458, 487 (1992); ibid. 478, 125 (1994).
55. S. G. Cull-Candy, R. Miledi, A. Trautmann, O. D. Uchitel, ibid. 299, 621 (1980); J. J. Plomp et al., Ann. Neurol. 37, 627 (1995).
56. S. G. Cull-Candy, R. Miledi, A. Trautmann, O. D. Uchitel, ibid. 299, 621 (1980); J. J. Plomp et al., Ann. Neurol. 37, 627 (1995).
57. Supported by grants from NIH National Institute of Neurological Disorders and Stroke awarded to G.D.F. (R01-NS18458) and to A.W.S. (K08-NS01580). We thank D. Kane of Amgen for genotyping and shipping the mice.