Time Lapse in Vivo Visualization of Developmental Stabilization of Synaptic Receptors at Neuromuscular Junctions Received for publication, July 27, 2010, and in revised form, August 30, 2010 Published, JBC Papers in Press, September 2, 2010, DOI 10.1074/jbc.M110.168880 Pessah Yampolsky ‡ , Pier Giorgio Pacifici ‡ , Lukas Lomb § , Gu ¨ nter Giese ¶ , Ru ¨ diger Rudolf  , Ira V. Ro ¨ der  , and Veit Witzemann ‡1 From the Departments of ‡ Molecular Neurobiology, § Biomolecular Mechanisms, and ¶ Biomedical Optics, Max Planck Institute for Medical Research, 69120 Heidelberg, Germany and the  Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, Karlsruhe 76131, Germany The lifetime of nicotinic acetylcholine receptors (AChRs) in neuromuscular junctions (NMJs) is increased from <1 day to >1 week during early postnatal development. However, the exact timing of AChR stabilization is not known, and its correlation to the concurrent embryonic to adult AChR channel conversion, NMJ remodeling, and neuromuscular diseases is unclear. Using a novel time lapse in vivo imaging technology we show that replacement of the entire receptor population of an individual NMJ occurs end plate-specifically within hours. This makes it possible to follow directly in live animals changing sta- bilities of end plate receptors. In three different, genetically modified mouse models we demonstrate that the metabolic half-life values of synaptic AChRs increase from a few hours to several days after postnatal day 6. Developmental stabilization is independent of receptor subtype and apparently regulated by an intrinsic muscle-specific maturation program. Myosin Va, an F-actin-dependent motor protein, is also accumulated synapti- cally during postnatal development and thus could mediate the stabilization of end plate AChR. In developing and adult muscle, acetylcholine receptors (AChRs) 2 at the neuromuscular junction (NMJ) undergo dra- matic changes in their metabolic stability. In newly formed syn- apses, AChRs become clustered and stabilized at a relatively rapid turnover rate of t1 ⁄2  1 day (1). The initial clustering requires the muscle-specific kinase MuSK (2), associated sig- naling components (3), as well as the receptor-aggregating pro- tein, rapsyn (4). Rapsyn, which interacts directly with AChR (5), contributes to receptor stability (6). In the adult synapses the AChRs turnover is rather slow (t1 ⁄2  10 days) (1, 7–10). Recent investigation of AChR has revealed that stabilization is not a static integration process, but results from AChR recycling that is regulated by muscle activity (11). There are indications that the actin cytoskeleton is involved in regulating synaptic targeting and stabilization of AChRs. Lately, class V myosin motor proteins have been identified to play important roles in synaptic plasticity. Although myosin Va (12) or myosin Vb (13) seems to be crucial for recycling of AMPA receptors of central synapses, proper turnover of AChR in adult neuromuscular synapses is dependent on myosin Va (14). The mechanism that localizes and stabilizes the AChRs within a postsynaptic scaffold and the operational and organi- zational sequence of postnatal stabilization processes including the developmentally occurring AChR channel conversion, however, remains unknown. The temporal resolution of AChR stabilization as well as the direct observation of the rapid dynamics of channel conversion during early postnatal development has not been accomplished and appeared inaccessible. In the current study we imple- mented in vivo time lapse imaging techniques for direct, con- tinuous visualization of surface AChR trafficking in vivo. This way we narrowed down the analysis time frame to the period between postnatal days 3 and 8 in mice, when major remodeling and maturation processes of the NMJs are initiated. We track AChR channel conversion at real time resolution, and we observe directly AChR stabilization events at single NMJs. The determination of the metabolic stability of end plate receptors is crucial for the understanding of the regulation of AChR turn- over and activity-dependent plasticity of NMJs. Furthermore, in neuromuscular diseases, including myasthenic or dystrophic disorders, pathological changes could affect AChR stability. The direct visualization of receptor stability will be an impor- tant tool to investigate motor function deficits or genetic disorders. EXPERIMENTAL PROCEDURES Animals and Genotyping—The mouse line AChR -GFP/-GFP was generated by homologous recombination fusing GFP into the  subunit gene as described previously (15, 31). GFP-labeled  subunits,  -GFP subunits, are assembled into AChR -GFP complexes that substitute the wild type  subunit-containing AChR. Dilute and wild type mice of the DLS/LeJ and the C57BL/6J strains, respectively, were purchased from Jackson Laboratories and then maintained in the local animal facility. All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (Publication No. 85-23, revised 1996) and the European Community guidelines for the use of experimental animals. 1 To whom correspondence should be addressed: Jahnstrasse 29, 69120 Heidelberg, Germany. Fax: 49-6221-486498; E-mail: witzeman@ mpimf-heidelberg.mpg.de. 2 The abbreviations used are: AChR, acetylcholine receptor; NMJ, neuromus- cular junction; r-bgt, rhodamine-labeled -bungarotoxin; P, postnatal day. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 45, pp. 34589 –34596, November 5, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. NOVEMBER 5, 2010 • VOLUME 285 • NUMBER 45 JOURNAL OF BIOLOGICAL CHEMISTRY 34589 by guest on June 3, 2020 http://www.jbc.org/ Downloaded from