Optical control of zebrafish behavior with halorhodopsin

Abstract

Expression of halorhodopsin (NpHR), a light-driven microbial chloride pump, enables optical control of membrane potential and reversible silencing of targeted neurons. We generated transgenic zebrafish expressing enhanced NpHR under control of the Gal4/ UAS system. Electrophysiological recordings showed that eNpHR stimulation effectively suppressed spiking of single neurons in vivo. Applying light through thin optic fibers positioned above the head of a semi-restrained zebrafish larva enabled us to target groups of neurons and to simultaneously test the effect of their silencing on behavior. The photostimulated volume of the zebrafish brain could be marked by subsequent photoconversion of co-expressed Kaede or Dendra. These techniques were used to localize swim command circuitry to a small hindbrain region, just rostral to the commissura infima Halleri. The kinetics of the hindbrain-generated swim command was investigated by combined and separate photo-activation of NpHR and Channelrhodopsin-2 (ChR2), a light-gated cation channel, in the same neurons. Together this ''optogenetic toolkit'' allows loss-of-function and gain-of-function analyses of neural circuitry at high spatial and temporal resolution in a behaving vertebrate.echnology for the inactivation of specific neurons within an otherwise intact circuit promises to accelerate research into the neural basis of behavior (1-8). The light-gated chloride pump halorhodopsin (NpHR) from the archaebacterium Natronomonas pharaonis has recently been introduced into neuroscience along with enhanced derivatives (9-14) and enables superior temporal and spatial control. Other light-controlled silencing methods are being developed (15-17), but require covalent attachment of a photo-switchable affinity label. NpHR silencing has been demonstrated electrophysiologically (10, 12) and has been used to reversibly paralyze Caenorhabditis elegans expressing NpHR in motor peripheries (12). Despite its promise, however, NpHR has so far found only limited applications for circuit analysis in vivo. In this study, we have devised a versatile and cost-effective optical stimulation strategy for manipulation of animal behavior with this tool. These advances were made possible by our choice of zebrafish as the experimental system.Zebrafish are ideal models for testing and applying lightcontrolled channels and pumps in vertebrates, since they are translucent and display a number of quantifiable behaviors during the first 2 weeks of larval development (18-21). Accordingly, Wyart et al. (22) used a re-engineered, light-gated glutamate receptor (LiGluR), to induce swimming by photostimulation of a rare type of spinal neuron. Douglass et al. (23) succeeded in triggering escape responses by activating ChR2 in single zebrafish mechanosensory cells. The adaptation of the Gal4/UAS method from Drosophila melanogaster (24) to zebrafish enables targeting transgene expression to specific brain areas and cell types (25-29) and will further contribute to the refinement of an optogenetic toolkit in thi...

Principles of designing interpretable optogenetic behavior experiments

“…The type of fluorescent protein used can modulate opsin function. For example, in zebrafish, the unaltered N. pharaonis halorhodopsin appeared to clump when expressed with the fluorophore mCherry, more than it did when fused to the fluorophore YFP (Arrenberg et al 2009). This differential effect may have persisted even after appending trafficking sequences (e.g., compare Fig.…”

Optogenetic Activation of Excitatory Premotor Interneurons Is Sufficient to Generate Coordinated Locomotor Activity in Larval Zebrafish

“…This line has been obtained from an enhancer trap screen (Scott et al, 2007) and drives the expression of Channelrhodopsin-2(H134R;ChR2) when crossed with a UAS: ChR2(H134R)-mCherry line (Arrenberg et al, 2009;Baier and Scott, 2009;Wyart et al, 2009;Del Bene and Wyart, 2012). We first investigated whether the Gal4 expression was selective to V2a interneurons by crossing the Gal4 s1011t with UAS:GFP or UAS: Kaede.…”

Optogenetic Localization and Genetic Perturbation of Saccade-Generating Neurons in Zebrafish

“…While the pathways generating eye movements converge on a shared set of oculomotor neurons in all vertebrates, it is not known whether different types of saccades are produced by the same premotor circuits and whether teleosts possess a region that is homologous to the mammalian burst generator. To gain insight into this issue, we have chosen here a functional approach in zebrafish (Arrenberg et al, 2009). We generated transgenic zebrafish larvae expressing halorhodopsin (NpHR) in the entire CNS.…”

“…These experiments help to localize an important brain function in zebrafish larvae, invite further work on homologies between teleost and mammalian hindbrain circuits, and show that optogenetic intervention can be successfully used to reverse a neurological defect in an animal model. , were discovered in a chemical mutagenesis screen using the OKR as a screening assay (Muto et al, 2005 (Scott et al, 2007;Arrenberg et al, 2009). Tg(UAS: ChR2(H134R)-mCherry)s1986t was newly generated for this project by established procedures.…”