Using light-sheet microscopy to study spontaneous activity in the developing lateral-line system

Abstract

Hair cells are the sensory receptors in the auditory and vestibular systems of all vertebrates, and in the lateral-line system of aquatic vertebrates. During development, spontaneous activity in hair cells shapes the formation of these sensory systems. In auditory hair cells of mice, coordinated waves of spontaneous activity can be triggered by concomitant activity in nearby supporting cells. But in mammals, developing auditory and vestibular hair cells can also autonomously generate spontaneous events independent of supporting cell activity. To date, significant progress has been made studying spontaneous activity in the auditory and vestibular systems of mammals, in isolated cultures. The purpose of this work is to explore the zebrafish lateral-line system as a model to study and understand spontaneous activity in vivo. Our work applies genetically encoded calcium indicators along with light-sheet fluorescence microscopy to visualize spontaneous calcium activity in the developing lateral-line system. Consistent with our previous work, we show that spontaneous calcium activity is present in developing lateral-line hair cells. We now show that supporting cells that surround hair cells, and cholinergic efferent terminals that directly contact hair cells are also spontaneously active. Using two-color functional imaging we demonstrate that spontaneous activity in hair cells does not correlate with activity in either supporting cells or cholinergic terminals. We find that during lateral-line development, hair cells autonomously generate spontaneous events. Using localized calcium indicators, we show that within hair cells, spontaneous calcium activity occurs in two distinct domains-the mechanosensory bundle and the presynapse. Further, spontaneous activity in the mechanosensory bundle ultimately drives spontaneous calcium influx at the presynapse. Comprehensively, our results indicate that in developing lateral-line hair cells, autonomously generated spontaneous activity originates with spontaneous mechanosensory events. Overall, with robust spontaneous activity three different cell types, the developing lateral line is a rich model to study these activities in an intact sensory organ. Future work studying this model may further our understanding of these activities and their role in sensory system formation, function and regeneration.