Analyzing the Prey-Evoked Neuronal Activity in a Transgenic Zebrafish Larva

Abstract

Source: Muto, A. et al., Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae. J. Vis. Exp. (2018)

This video demonstrates the method for studying visual prey detection in transgenic zebrafish larvae using calcium indicator-based fluoresce imaging with their neurons expressing fluorescent calcium indicator complexes.

Protocol

1. Ablation of a Subpopulation of Neurons Using a Two-photon Laser Microscope

NOTE: If users plan on performing Ca imaging following ablation, use the UAShspzGCaMP6s line. If users plan on performing behavioral recording following ablation, use the UAS:EGFP line, as the ablation of EGFP-positive cells is easier to perform than of GCaMP6s-expressing cells.

1. Begin by setting up the mating of a Gal4 line that labels specific neurons to be studied and UAS:EGFP or UAShspzGCaMP6s. Be sure to use the nacre background for both parents so that nacre homozygotes can be obtained.
   

Results

Figure 1. Zebrafish larvae. (A) Five-day old (5-day post-fertilization (5-dpf)) zebrafish larva and its prey, a paramecium. (B) 4-dpf nacre larva embedded in 2% low melting-point agarose. Note that the nacre strain lacks black pigments o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NuSieve GTG Agarose	Lonza	Cat.#50080	low-melting temperature agarose
6 cm petri dish	FALCON	Product#:351007
dissecting needle	AS ONE Corporation	Cat. No. 2-013-01	https://keystone-lab.com/en/item/detail/404142
LSM7MP	Carl Zeiss		two-photon laser scanning microscope
W Plan-Apochromat 63x/1.0	Carl Zeiss		63X objective lens
Imager.Z1	Carl Zeiss		an epi-fluorescence microscope
ZEN	Carl Zeiss		Image acquisition software for confocal microscopes
Secure-Seal Hybridization Chamber Gasket, 8 chambers, 9 mm diameter x 0.8 mm depth	Molecular Probes	Catalogue # S-24732	Used as a recording chamber in Ca imaging
Surgical knife	MANI	Ophthalmic knife MST15
ORCA-Flash4.0	Hamamatsu Photonics	model:C11440-22CU	A scientific CMOS camera
HCImage	Hamamatsu Photonics		Image acuisition software
Hard Disk Recording module	Hamamatsu Photonics		An software module that enables saving the movie files onto a hard disc drive in a short time
Point Grey Grasshopper3 4.1 MP Mono USB3 Visio	FLIR Systems, Inc.	Product No. GS3-U3-41C6NIR-C	CMOS camera
XIMEA xiQ camera	XIMEA	Product No. MQ042RG-CM	CMOS camera
A ring LED light	CCS	Model: LDR2-100SW2-LA	White LED
Nylon mesh 32µm	Tokyo Screen	N-No.380T	http://www.tokyo-screen.com/cms/sta20347/
Nylon mesh 13µm	Tokyo Screen	N-No. 508T-K	http://www.tokyo-screen.com/cms/sta20347/
Metal seive 150 micron aperture	Tokyo Screen		http://www.tokyo-screen.com/cms/sta20341/#ami
Metal seive 75 micron aperture	Tokyo Screen		http://www.tokyo-screen.com/cms/sta20341/#ami
EBIOS	Asahi Food & Healthcare, Co. Ltd.		Dry beer yeast
LabVIEW	National Instruments		An integrated development environment for programming
Mai-Tai HP	Spectra Physics		Two-photon laser