Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of
live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescentlymarked
plasma membranes can be scanned in a confocal laser scanning microscope
in order to determine the volume of muscle tissue and the number of muscle fibers
present. Efficient approaches for the measurement of cell number and size in live
animals over time are described and validated against more arduous segmentation
methods. Methods are described that permit the control of muscle electrical, and
thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced
muscle growth. In larvae, a protocol is described that allows reintroduction of
patterned electrical-evoked contractile activity. The described methods minimize the
effect of inter-individual variability and will permit analysis of the effect of electrical,
genetic, drug, or environmental stimuli on a variety of cellular and physiological
growth parameters in the context of the living organism. Long-term follow-up of the
measured effects of a defined early-life intervention on individuals can subsequently
be performed.