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.
Highlights
When delivered by Sindbis virus, the same recombinant gene, allatostatin www.frontiersin.org receptor (AlstR):internal ribosomal entry sequence (IRES):eGFP, produced robust fluorescence (Figure 1C), demonstrating that expression levels achieved by Sindbis infection are higher than those obtained by conventional transgenesis
Our results demonstrate that spatial and temporal control of gene expression in zebrafish can be achieved by viral gene delivery and the Tet system
Whereas viruses are fast to generate and easy to apply, the Tet system allows for the conditional, non-invasive expression of transgenes in defined cell types
Summary
Encoded tools to visualize and manipulate defined neurons (Zhang et al, 2007; Luo et al, 2008) open exciting new perspectives for the mechanistic analysis of neuronal circuits (Adamantidis et al, 2007; Zhang and Oertner, 2007; Douglass et al, 2008; Huber et al, 2008; Arrenberg et al, 2009; Gradinaru et al, 2009; Petreanu et al, 2009; Wyart et al, 2009). Exploiting the full potential of these tools would be greatly facilitated by small animal models that permit optical access to many individual neurons in vivo. Advanced genetic methods are required to express transgenes at high levels in specific cell types. We capitalized on experience in different species to establish gene expression systems in zebrafish that achieve high-level, conditional expression of transgenes in small and specific neuronal subsets. We first explored whether transgenes can be introduced into zebrafish neurons by viral gene delivery. This approach is well-established in mammals (Luo et al, 2008) it has, to our knowledge, not been explored systematically in the zebrafish brain, despite some encouraging reports (Wagle et al, 2004)
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