title: A Cell Culture System to Investigate the Presynaptic Control of Subsynaptic Membrane Differentiation at the Neuromuscular Junction.
abstract: For decades the neuromuscular junction (NMJ) has been a favorite preparation to investigate basic mechanisms of synaptic function and development. As its function is to transmit action potentials in a 1:1 ratio from motor neurons to muscle fibers, the NMJ shows little or no functional plasticity, a property that makes it poorly suited to investigate mechanisms of use-dependent adaptations of synaptic function, which are thought to underlie learning and memory formation in the brain. On the other hand, the NMJ is unique in that the differentiation of the subsynaptic membrane is regulated by one major factor secreted from motor neurons, agrin. As a consequence, myotubes grown on a laminin substrate that is focally impregnated with recombinant neural agrin closely resemble the situation in vivo, where agrin secreted from motor neurons binds to the basal lamina of the NMJ's synaptic cleft to induce and maintain the subsynaptic muscle membrane. We provide here a detailed protocol through which acetylcholine receptor clusters are induced in cultured myotubes contacting laminin-attached agrin, enabling molecular, biochemical and cell biological analyses including high resolution microscopy in 4D. This preparation is ideally suited to investigate the mechanisms involved in the assembly of the postsynaptic muscle membrane, providing distinct advantages over inducing AChR clusters using soluble agrin.
authors.affiliation: Department of Physiology II, Albert-Ludwigs University, 79104, Freiburg, Germany.
authors.forename: Nadine
authors.initials: N
authors.lastname: Schmidt
authors.affiliation: Department of Cell Biology and Genetics, Erasmus MC, 3000 CA, Rotterdam, The Netherlands.
authors.forename: Sreya
authors.initials: S
authors.lastname: Basu
authors.affiliation: Department of Physiological Genomics, Ludwig-Maximilians-University, 80336, Munich, Germany.
authors.forename: Stephan
authors.initials: S
authors.lastname: Kröger
authors.affiliation: Department of Biomedicine, University of Basel, Klingelbergstrasse 50, 4051, Basel, Switzerland. hans-rudolf.brenner@unibas.ch.
authors.forename: Hans Rudolf
authors.initials: HR
authors.lastname: Brenner
mesh_terms: D000818:Animals; D015415:Biomarkers; D019556:COS Cells; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002522:Chlorocebus aethiops; D051379:Mice; D008853:Microscopy; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism; D032446:Myoblasts / Q000166:cytology / Q000378:metabolism; D009469:Neuromuscular Junction / Q000166:cytology* / Q000502:physiology*; D017729:Presynaptic Terminals / Q000502:physiology*; D061251:Primary Cell Culture; D013570:Synaptic Membranes / Q000502:physiology*
chemical_list: D015415:Biomarkers
keywords: High resolution microscopy; Muscle; Neuromuscular junction; Synapse development