In vivo imaging

There has been a long-standing motivation to follow the cellular and molecular events of synapse formation and remodeling directly in vivo. When biological processes are exclusively studied using timed sequences of fixed preparations, transition states may easily be missed and the temporal order of cellular events might not be determined adequately. Furthermore, versatile genetic tools typical for Drosophila allow dissecting the cellular mechanisms responsible for localization and turnover of proteins in a straightforward manner.

Due to the transparent nature of Drosophila larvae, the glutamatergic synapses forming at the developing larval NMJ are ideally suited for in vivo imaging in an intact organism. Because non-anesthetized Drosophila larvae move strongly, in vivo imaging on the level of individual synapses demands stable anesthetization in order to allow confocal imaging. We developed an anesthetization method that allowed us to anesthetize and image animals repetitively without affecting their viability or developmental speed. Thereby the development of identified NMJs can be followed in GluR-IIA^GFP expressing larvae for extended periods. Confocal stacks covering a full NMJ are taken every few hours. In all experiments, an individual imaging session lasted only a few minutes. Between the imaging sessions, the animals moved freely in their "natural environment" (food).

Refs: Nature Protocols (2007); 2 (12): 3285-3298. Nature Neuroscience (2005); 8(7): 898-905.