Using G-CaMP 1.6 to Monitor Visually-Evoked Synaptic Activity in Tectal Neurons in vivo

Abstract

To investigate the visual system of the zebrafish, different techniques were used to express genetically-encoded Ca2+ indicators and test their functionality in vivo. XFPs, G-CaMP 1.6 and troponeons were expressed in a mosaic pattern and in single neurons under different promoters in the whole central nervous system of larval zebrafish. Light-evoked responses to a flickering LED in G-CaMP1.6 expressing tectal neurons were recorded with single photon excitation. Furthermore, changes of intracellular [Ca2+] were measured in separate dendritic structures. For the characterization of expression patterns, repeated two-photon imaging of the whole tectum opticum was performed, allowing the monitoring of single cells in high resolution over time.The analysis of the expression of green fluorescent protein (GFP) showed that nearly all cell types present in the adult, are found as early as 7 dpf (days post fertilisation) in the zebrafish larvae, which was unknown previously. Equally, at 7dpf, the lamination of the tectum opticum was found to be similar to that of the adult. Furthermore, the onset of development of the tectal laminar structure seems to coincidence with the first arborization of retinal axons onto their tectal target (84 hpf).Linking of these morphological aspects, with functional properties of tectal neurons in the larva, gives valuable information about different players in the visual processing in the tectum. The system developed in the course of this study presents an experimental tool for the investigation of the cytoarchitecture of neural circuits with a quasi non-invasive monitoring of network activity throughout early development in an in vivo preparation, which has direct application to complex questions of systems level neuroscience.