The large number of neurons and neural interconnection makes the nervous system a highly dense and complex network. Understanding the functionality of such a network requires not only high-throughput recording of neural activities with cellular resolution but also a non-invasive and precisely defined interaction with the neurons. This work demonstrates two pivotal steps towards these aims:
(i) Developing a fluorescence microscope based on Bessel light-sheet illumination to record neural activities by means of calcium imaging. Chapter II describes the successful development and construction of an in vivo light-sheet fluorescence microscope (in vivo LSFM) based on conventional digitally laser scanning light-sheet microscopy (DSLM) with interchangeable Gaussian and Bessel illumination modalities to compare the performance of both methods. The Bessel illumination modality in comparison to the Gaussian one reveals not only a two-fold improvement in axial resolution but also a reduction by the factor of 4 in the shadowing artifact and consequently, 35 times improvement in detecting the correct neural activities from the calcium signals.
(ii) Providing a suitable transgenic zebrafish model for optogenetic manipulation; Optogenetics apply light to facilitate the interaction with a genetically-engineered cell and/or populations of cells. ... mehrChapter III describes the establishment of a transgenic zebrafish expressing a red-shifted channelrhodopsin, Chlamydomonas channelrhodopsin1 fused to Volvox channelrhodopsin1 (C1V1), under the elavl3 promotor making almost the entire nervous system of larval zebrafish accessible for optical manipulation. To the best of our knowledge, this is the first demonstration of a transgenic zebrafish with C1V1 pan-neuronal expression. The establishment of the stable transgenic line is in progress.
In future, crossing this line with an already established line expressing a calcium indicator protein will provide the essential animal model for simultaneous optogenetic manipulation and high-fidelity neural activity recording in the intact nervous system.