Rapid and reversible effects of activity on acetylcholine receptor density at the neuromuscular junction in vivo

Science

Volumn 286, Issue 5439, 1999, Pages 503-507

  Akaaboune, Mohammed ^a   Culican, Susan M ^a   Turney, Stephen G ^a   Lichtman, Jeff W ^a  

^a WASHINGTON UNIVERSITY SCHOOL OF MEDICINE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BUNGAROTOXIN; CHOLINERGIC RECEPTOR;

ANIMAL TISSUE; ARTICLE; CONTROLLED STUDY; HALF LIFE TIME; MOUSE; NEUROMUSCULAR BLOCKING; NEUROMUSCULAR SYNAPSE; NEUROTRANSMISSION; NONHUMAN; PRIORITY JOURNAL; RECEPTOR DENSITY;

ANIMALS; BUNGAROTOXINS; CELL MEMBRANE; CURARE; DIFFUSION; ELECTRIC STIMULATION; FLUORESCENT DYES; HALF-LIFE; MICE; MUSCLE CONTRACTION; MUSCLE DENERVATION; NEUROMUSCULAR BLOCKADE; NEUROMUSCULAR BLOCKING AGENTS; NEUROMUSCULAR JUNCTION; RECEPTOR AGGREGATION; RECEPTORS, CHOLINERGIC; RHODAMINES; SYNAPTIC TRANSMISSION;

ANIMALIA;

EID: 0033569388     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.286.5439.503     Document Type: Article

References (42)

1. D. M. Fambrough and H. C. Hartzell, Science 176, 189 (1972); H. C. Fertuck and M. M. Salpeter, Proc. Natl. Acad. Sci. USA 71. 1376 (1974); J. Cell Biol. 63, 144 (1976).
2. D. M. Fambrough and H. C. Hartzell, Science 176, 189 (1972); H. C. Fertuck and M. M. Salpeter, Proc. Natl. Acad. Sci. USA 71. 1376 (1974); J. Cell Biol. 63, 144 (1976).
3. D. M. Fambrough and H. C. Hartzell, Science 176, 189 (1972); H. C. Fertuck and M. M. Salpeter, Proc. Natl. Acad. Sci. USA 71. 1376 (1974); J. Cell Biol. 69, 144 (1976).
4. P. Ravdin and D. Axelrod, Anal. Biochem. 80, 585 (1977).
5. S. G. Turney, S. M. Culican, J. W. Lichtman, J. Neurosci. Methods 64, 199 (1996).
6. M. M. Rich and J. W. Lichtman, J. Neurosci. 9, 1781 (1989); R. J. Balice-Gordon and J. W. Lichtman, ibid. 13, 834 (1993); Nature 372, 519 (1994).
7. M. M. Rich and J. W. Lichtman, J. Neurosci. 9, 1781 (1989); R. J. Balice-Gordon and J. W. Lichtman, ibid. 13, 834 (1993); Nature 372, 519 (1994).
8. M. M. Rich and J. W. Lichtman, J. Neurosci. 9, 1781 (1989); R. J. Balice-Gordon and J. W. Lichtman, ibid. 13, 834 (1993); Nature 372, 519 (1994).
9. 1/2 = ∼5 months; n = 29). In addition, the results reported here show that the loss of fluorescence was related to muscle fiber activity, ruling out bleaching or unbinding as the explanation of the loss.
10. E. F. Stanley and D. B. Drachman, Science 222, 67 (1983); D. A. Ramsay, D. B. Drachman, R. J. Drachman, E. F. Stanley, Brain Res. 581, 198 (1992).
11. E. F. Stanley and D. B. Drachman, Science 222, 67 (1983); D. A. Ramsay, D. B. Drachman, R. J. Drachman, E. F. Stanley, Brain Res. 581, 198 (1992).
12. α-Bungarotoxin (15 to 20 μg/ml of Ringer's solution) or curare (2.5 mg/ml) or both were used to block neuromuscular transmission for the duration of the experiment by placing a coverslip over the exposed muscle to prevent drying and by replacing the fluid with additional blocking solution once per hour.
13. A mixture (50:50) of rhodamine-tagged and unlabeled α-bgt was used both to label the muscle and to maintain the neuromuscular blockade.
14. Based on the facts that (i) one bgt molecule inactivates an AChR, (ii) all receptors can bind two bgt molecules and (iii) binding of each α-bgt is independent, we calculated the relation between fluorescence label and percentage of receptors blocked. For example, application of r-α-bgt for 45 min labels 50% of the maximum fluorescence intensity, which means that 68% of receptors are blocked. In the experiments with a nonlocking dose of α-bgt, we blocked 20 to 40% of the receptors by labeling ∼13 to 24% of the maximum fluorescence, as this degree of blockade is well below the 70% needed to impede action potentials in the muscle [C. J. Lingle and J. H. Steinbach, Int. Anesthesiol Clin. 26, 288 (1988)].
15. The ∼14-day half-life is slightly longer than previous estimates of normal receptor turnover based on saturation with radiolabeled α-bgt (1). This difference is probably because the short period of increase AChR loss after receptor saturation (Fig. 1) was not previously noted.
16. In junctions saturated with labeled α-bgt such that all the receptors were associated with two bgt molecules, the loss of label was substantially slower on the second day after application than on the first day (Fig. 1). Moreover, when curare was applied to these muscles on the second day, the loss rate of the previously labeled receptors rapidly increased to ∼5% per hour (n = 20). Because curare cannot bind to AChRs that already have two α-bgt molecules bound [D. X. Fu and S. M. Sine, J. Biol. Chem. 269, 26152 (1994)], neuromuscular blockade rather than receptor occupation by blocking ligands induced accelerated receptor loss from the neuromuscular junction.
17. If one assumes both slowly degrading and rapidly degrading receptors at denervated junctions [S. L. Shyng and M. M. Salpeter, J. Cell Biol. 108, 647 (1989)] and a half-life of 14 days (from the observations reported here) for slowly degraded AChRs, the result is a combined degradation half-life of 2.8 days at 1 week after denervation, which is very close to the half-life of 2.7 days found here.
18. J. S. Andreose, R. Xu, T. Lomo, M. M. Salpeter, C. Fumagalli [J. Neurosci. 13, 3433 (1993)] previously showed that the acceleration in the rate of receptor loss after denervation was also blocked by electrical stimulation.
19. M. Akaaboune, S. M. Culican, S. C. Turney, J. W. Lichtman, data not shown.
20. Perijunctional receptors were imaged with 1.4 numerical aperture objectives and high-intensity laser illumination on a confocal scanning light microscope (Bio-Rad 1024). The gain was intentionally set high to allow the faint perijunctional signal to be seen (Fig. 3). These muscles were fixed in 4% paraformaldehyde, washed in saline overnight (to remove curare in the blocked preparations), and stained with r-α-bgt (5 μg/ml) for 30 min.
21. H. C. Hartzell and D. M. Fambrough, J. Gen. Physiol. 60, 248 (1972); M. Stya and D. Axelrod, J. Neurosci. 4, 70 (1984).
22. H. C. Hartzell and D. M. Fambrough, J. Gen. Physiol. 60, 248 (1972); M. Stya and D. Axelrod, J. Neurosci. 4, 70 (1984).
23. J. R. Sanes and J. W. Lichtman, Annu. Rev. Neurosci. 22, 389 (1999).
24. M. M. Salpeter and R. Harris, J. Cell-Biol. 96, 1781 (1983); M. M. Salpeter, M. Marchaterre, R. Harris, ibid. 106, 2087 (1988).
25. M. M. Salpeter and R. Harris, J. Cell-Biol. 96, 1781 (1983); M. M. Salpeter, M. Marchaterre, R. Harris, ibid. 106, 2087 (1988).
26. M. M. Salpeter and R. H. Loring, Prog. Neurobiol. 25, 297 (1985).
27. S. M. Culican et al., in preparation.
28. T. A. Levitt, R. H. Loring, M. M. Salpeter, Science 210, 550 (1980); R. S. Brett, S. C. Younkin, M. Konieczkowski, R. M. Slugg, Brain Res. 233, 133 (1982).
29. T. A. Levitt, R. H. Loring, M. M. Salpeter, Science 210, 550 (1980); R. S. Brett, S. C. Younkin, M. Konieczkowski, R. M. Slugg, Brain Res. 233, 133 (1982).
30. M. J. Dennis and R. Miledi, J. Physiol. (London) 237, 431 (1974).
31. R. A. McKinney, M. Capogna, R. Durr, B. H. Gahwiler, S. M. Thompson, Nature Neurosci. 2, 44 (1999).
32. S. Rotzler, H. Schramek, H. R. Brenner, Nature 349, 337 (1991); J. L. McManaman, J. C. Blosser, S. H. Appel, J. Neurosci. 1, 771 (1981).
33. S. Rotzler, H. Schramek, H. R. Brenner, Nature 349, 337 (1991); J. L. McManaman, J. C. Blosser, S. H. Appel, J. Neurosci. 1, 771 (1981).
34. D. J. Perkel, J. J. Petrozzino, R. A. Nicoli, J. A. Connor, Neuron 11, 817 (1993); J. A. Cummings, R. M. Mulkey, R. A. Nicoli, R. C. Malenka, ibid. 16, 825 (1996).
35. D. J. Perkel, J. J. Petrozzino, R. A. Nicoli, J. A. Connor, Neuron 11, 817 (1993); J. A. Cummings, R. M. Mulkey, R. A. Nicoli, R. C. Malenka, ibid. 16, 825 (1996).
36. T. Manabe, P. Renner, R. A. Nicoll, Nature 355, 50 (1992).
37. D. V. Lissin, R. C. Carroll, R. A. Nicoll, R. C. Malenkavon, M. Zastrow, J. Neurosci. 15, 1263 (1999);
38. R. C. Carroll, D. V. Lissin, M. V. Zastrow, R. A. Nicoll, R. C. Malenka, Nature Neurosci. 2, 454 (1999).
39. J. L. Gooch, M. R. Suchyta, J. M. Balbierz, J. H. Petajan, T. P. Clemmer, Crit. Care Med. 19, 1125 (1991); V. Segredo et al., N. Engl. J. Med. 327, 524 (1992); T. L. Kurt, J. Toxicol. Clin. Toxicol. 34, 253 (1996).
40. J. L. Gooch, M. R. Suchyta, J. M. Balbierz, J. H. Petajan, T. P. Clemmer, Crit. Care Med. 19, 1125 (1991); V. Segredo et al., N. Engl. J. Med. 327, 524 (1992); T. L. Kurt, J. Toxicol. Clin. Toxicol. 34, 253 (1996).
41. J. L. Gooch, M. R. Suchyta, J. M. Balbierz, J. H. Petajan, T. P. Clemmer, Crit. Care Med. 19, 1125 (1991); V. Segredo et al., N. Engl. J. Med. 327, 524 (1992); T. L. Kurt, J. Toxicol. Clin. Toxicol. 34, 253 (1996).
42. This work was supported by a Human Frontier Science Program Long-Term Fellowship (M.A.) and the NIH and the Muscular Dystrophy Association (J.W.L). We thank the members of our laboratory for helpful discussions about this work.