Confocal Imaging of Fluorescently Labeled Proteins in the Drosophila Larval Neuromuscular Junction

The Drosophila larval neuromuscular junction (NMJ) consists of a presynaptic motor neuron terminal and a postsynaptic muscle cell that offer an accessible and popular model system for the analysis of synaptic growth and function. I describe techniques for visualizing fluorescently labeled proteins within dissected, formaldehyde-fixed second to third instar larval NMJs. In addition, I present two strategies using confocal microscopy to solve a particular problem in NMJ analysis: distinguishing fluorescence in the presynaptic nerve terminal from that in the adjacent postsynaptic muscle cell. This problem arises from the fact that the membrane of the muscle cell envelops the motor neuron terminal with a convoluted process called the subsynaptic reticulum, obscuring the boundary between muscle and nerve. A first strategy entails taking thin optical sections through synaptic boutons to capture a cross section of the nerve terminal, and a second strategy involves visualizing epitope-tagged isoforms of particular proteins that have been transgenically expressed in either the nerve or the muscle.