Genetic and optical targeting of neural circuits and behavior--zebrafish in the spotlight

Abstract

Methods to label neurons and to monitor their activity with genetically encoded fluorescent reporters have been a staple of neuroscience research for several years. The recent introduction of photoswitchable ion channels and pumps, such as channelrhodopsin (ChR2), halorhodopsin (NpHR), and light-gated glutamate receptor (LiGluR), is enabling remote optical manipulation of neuronal activity. The translucent brains of zebrafish offer superior experimental conditions for optogenetic approaches in vivo. Enhancer and gene trapping approaches have generated hundreds of Gal4 driver lines in which the expression of UAS-linked effectors can be targeted to subpopulations of neurons. Local photoactivation of genetically targeted LiGluR, ChR2, or NpHR has uncovered novel functions for specific areas and cell types in zebrafish behavior. Because the manipulation is restricted to times and places where genetics (cell types) and optics (beams of light) intersect, this method affords excellent resolving power for the functional analysis of neural circuitry.

Figure 1. Examples of Gal4-VP16 driver lines labeling subsets of neurons in the zebrafish brain

Panels show twenty-four examples of UAS:Kaede expression patterns picked up in a recent ET screen, with the Gal4-VP16 line number indicated. All are dorsal images of 5 or 6 dpf live larvae, mounted in agarose. Scale bar is 200 m. Reprinted from [•16]

Figure 2. Cellular architecture of the zebrafish brain revealed by Kaede photoconversion

a–c, Combining Kaede and BGUG. Observing single cells in the context of a broader expression pattern requires three transgenes, 1) a Gal4 line, in this case the tectum-specific ET line s1038t, 2) UAS:Kaede (or a red-fluorescent protein), and 3) the BGUG transgene, which leads to highly variegated expression of GFP in Gal4-positive neurons (a). GFP-positive cells are made visible by red-conversion of Kaede (b). A tectal periventricular interneuron with laminated dendritic arbor is revealed using this approach (c). d, e, Labeling long-range projections. ET line s1019t drives expression in habenular neurons (d), with projections to the interpeduncular nucleus (IPN, box in d). Targeted photoconversion of the right habenula (inset, e) labels right axons red and leaves left axons green, showing that each habenula targets different parts of the IPN (e). Scale bars are 50μm (c) and 200μm (d).

Figure 3. Photostimulation of LiGluR-expressing subsets of spinal neurons elicits distinct motor responses

The Gal4 ET lines indicated on the left were crossed to UAS:iGluR(M439C). Each line drives the UAS-linked transgene to a different, occasionally overlapping subset of spinal neurons (side views of spinal cord, four to five segments shown). MAG was added to the bath. The fish larvae were then mounted in agarose, with their tails free to move. At least three distinct types of tail movements were observed (superimposed high-speed video frames on the right) in response to application of UV light (circular illumination area). a, Stimulation of RB cells in Gal4^s1102t evoked a “C bend”, indicative of an attempted escape. b, Combined stimulation of motor neurons and KA cells in Gal4^s1020t evoked tail beats similar to swimming. c, Stimulation of the KA cell population alone in Gal4^s1003t also evoked swimming. d, Stimulation of motor neurons alone in Gal4^s1041t evoked a twitch.

Figure 4. Options for the spatially confined delivery of light into the brain of a zebrafish larva

a, Side view of a 6 dpf zebrafish larva, carrying the pan-neuronal Gal4s1101t driver and UAS:Kaede. b–e, Four methods for the local excitation of fluorophores and photoswitches. The images show the zebrafish head with a schematic depiction of illumination volumes (red) for each method.