Adaptation of nanocrystals for in vivo use in tracking of single molecules at the neuromuscular junction

The project aims at developing nanocrystals or Quantum dot (QD) technology for in vivo use in general, and for tracking single molecules in particular. Quantum dots emit fluorescence and have emission spectra that are orders of magnitude more intense tha n conventional dyes. An antibody is usually needed to ensure specific binding of the QD. To date however, the use of QDs in vivo has been limited since the conjugation of an antibody to the QD makes it relatively large. Project 1: Is aimed at the adaptati on of QDs. QDs conjugated to [a]-bungarotoxin to label acetylcholine receptors (AChRs) we will use the technology to track single molecules in the synapse, in order to elucidate basic mechanism for synapse formation, synapse maintenance, and synaptic comp etition. Project 2: Rapsyn is a synapse protein that binds AChRs and it is essential for synapse formation. We plan to clone dominant negative forms of rapsyn into plasmids and overexpress them in muscle cells by single cell injection of the plasmids. We will use modified QDs to track the fate of rapsyn and AChRs after transfection to investigate the effect of dominant negative forms of rapsyn at the adult NMJ. Project 3: Competition between neural inputs is one of the fundamental problems in neurobiology . This competition between active and less active synapses be recapitulated adult animals by the formation of ectopic synapses or by the partial blockade of single synapses. In both cases a "winning" and a "losing"synapse can be studied, and the receptor dynamics investigated using QDs. This will allow us to study early events in clusters in membrane regions where the ultimate fate of the clusters will be known.