In vivo imaging of the developing neuromuscular junction in neonatal mice

Although fluorescently labeled structures can be analyzed more easily at high resolution in fixed-tissue preparations than in living animals, some biological questions can only be answered by time-lapse imaging. Changes in nervous system wiring during development cannot be determined reliably by taking tissue from different animals at staggered time points. Rather, the same cells and connections must be viewed repeatedly. To study developmental synapse elimination, we image muscles in transgenic mice that express fluorescent proteins in motor neurons and follow the same neuromuscular junctions (NMJs) over multiple days. This protocol describes the use of confocal microscopy for in vivo imaging of developing NMJs in transgenic neonatal mice expressing cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP). The sternomastoid, a flat, accessible neck muscle with large junctions, is imaged. A principal advantage of confocal microscopy is the ability to acquire multiple fluorescence channels simultaneously. If the channels are acquired sequentially, there is inevitably misalignment because of movement. Moreover, the total imaging time scales linearly with the number of channels. With simultaneous acquisition, only a single scan may be required. With perfect alignment between channels, irrespective of movement that might occur during a scan, color differences can be used to study interactions between axons over time. A limitation of this technique is that axons must be brightly labeled and at the muscle surface. NMJs that are more than one muscle fiber deep may be difficult to scan because of index of refraction changes that cause image blurring.