Visualization of active neural circuitry in the spinal cord of intact zebrafish

1. One of the major obstacles in studying vertebrate neural networks is the difficulty in simultaneously monitoring activity in a population of neurons. To take advantage of the transparency of larval zebrafish, we used confocal microscopy to look into the spinal cord of immobilized fish to monitor neural responses during an escape behavior. 2. Populations of identified neurons were labeled with a calcium indicator and neural activity was monitored on a millisecond time scale. The calcium dependent nature of the fluorescent signals was confirmed by monitoring the accumulation, diffusion, and removal of calcium that was introduced by electrical and sensory stimulation. 3. Zebrafish, like most swimming vertebrates, have two major classes of motoneurons: large primary motoneurons thought to be used primarily for rapid movements and smaller secondary motoneurons implicated in slower movements. Our optical approach allowed us to ask how these groups of primary and secondary motoneurons respond during the escape behavior--one of the fastest and most forceful motor behaviors produced by vertebrates. 4. We demonstrate a previously unknown synchrony in the response of populations of primary and secondary motoneurons. This synchrony can account for the massive activation of the axial musculature during powerful escapes. Detection of this synchrony depended on the rapid in vivo imaging of activity in this neuronal population. This optical approach will allow functional studies of neuronal populations in the brain and spinal cord of normal and mutant lines of zebrafish.