Optogenetic dissection of neuronal circuits in zebrafish using viral gene transfer and the Tet system

Abstract

The conditional expression of transgenes at high levels in sparse and specific populations of neurons is important for high-resolution optogenetic analyses of neuronal circuits. We explored two complementary methods, viral gene delivery and the iTet-Off system, to express transgenes in the brain of zebrafish. High-level gene expression in neurons was achieved by Sindbis and Rabies viruses. The Tet system produced strong and specific gene expression that could be modulated conveniently by doxycycline. Moreover, transgenic lines showed expression in distinct, sparse and stable populations of neurons that appeared to be subsets of the neurons targeted by the promoter driving the Tet-activator. The Tet system therefore provides the opportunity to generate libraries of diverse expression patterns similar to gene trap approaches or the thy-1 promoter in mice, but with the additional possibility to pre-select cell types of interest. In transgenic lines expressing channelrhodopsin-2, action potential firing could be precisely controlled by two-photon stimulation at low laser power, presumably because the expression levels of the Tet-controlled genes were high even in adults. In channelrhodopsin-2-expressing larvae, optical stimulation with a single blue LED evoked distinct swimming behaviors including backward swimming. These approaches provide new opportunities for the optogenetic dissection of neuronal circuit structure and function.

Dopaminergic Modulation of Mitral Cells and Odor Responses in the Zebrafish Olfactory Bulb

“…Expression of Chr2YFP in this line recapitulates the expression of GFP in the OB of a dlx4/6:GFP transgenic line described previously (Zerucha et al, 2000;Zhu et al, 2009). In the adult OB, the dlx4/6 promoter/enhancer elements drive expression in periglomerular and/or shortaxon cells, which are likely to include DAINs (Zhu et al, 2009).…”

“…In the OB of adult fish, this line expresses the fluorescent marker predominantly or exclusively in MCs (Li et al, 2005;Yaksi and Friedrich, 2006). Optogenetic experiments were performed using a transgenic line that expresses channelrhodopsin-2 fused to yellow fluorescent protein (Chr2YFP) under the control of dlx4/6 promoter/enhancer elements from zebrafish (Zerucha et al, 2000) and the tet-off system [Dlx4/6:itTA/Ptet:Chr2YFP Line 01 (Zhu et al, 2009)]. In the adult OB, this line expresses Chr2YFP at high levels in interneurons of the glomerular layer (Zhu et al, 2009).…”

“…To examine the mode and kinetics of dopaminergic transmission, we optically stimulated DA-INs using a transgenic fish line expressing channelrhodopsin-2 fused to YFP (Chr2YFP) (Nagel et al, 2003;Boyden et al, 2005) under the control of dlx4/6 promoter/enhancer elements and the Tet system [Dlx4/6:itTA/Ptet:Chr2YFP Line 01 (Zhu et al, 2009)]. Expression of Chr2YFP in this line recapitulates the expression of GFP in the OB of a dlx4/6:GFP transgenic line described previously (Zerucha et al, 2000;Zhu et al, 2009).…”

Principles of designing interpretable optogenetic behavior experiments

“…Of course, transgenic animals with cassettes bearing opsins under specific promoters can also be directly generated, e.g., in C. elegans ). Other expression systems-for instance the Tet off system (Zhu et al 2009;Bundschuh et al 2012), the Cre/lox (see above) or FLP/FRT systems (Schmitt et al 2012;Fenno et al 2014)-can also be used in many different species.…”

Generation and Characterization of a Transgenic Zebrafish Expressing the Reverse Tetracycline Transactivator

“…This transgenic line is very useful for the study of gene function during heart development and disorders, but lacks clear background in terms of rtTA gene integration. The Tet-on system is also used for conditional control of transgene expression in zebrafish brain and for structural and functional dissection of neuronal circuits (Zhu et al, 2009). In addition, two dually inducible Tet-on systems are developed for reversible and tightly controlled gene expression in zebrafish (Knopf et al, 2010).…”