Two-Photon Calcium Imaging of Forebrain Activity in Behaving Adult Zebrafish

Here, we present a protocol to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish.

Adult zebrafish (Danio rerio) exhibit a rich repertoire of behaviors for studying cognitive functions. They also have a miniature brain that can be used for measuring activities across brain regions through optical imaging methods. However, reports on the recording of brain activity in behaving adult zebrafish have been scarce. The present study describes procedures to perform two-photon calcium imaging in the dorsal forebrain of adult zebrafish. We focus on steps to restrain adult zebrafish from moving their heads, which provides stability that enables laser scanning imaging of the brain activity. The head-restrained animals can freely move their body parts and breathe without aids. The procedure aims to shorten the time of head restraint surgery, minimize brain motion, and maximize the number of neurons recorded. A setup for presenting an immersive visual environment during calcium imaging is also described here, which can be used to study neural correlates underlying visually triggered behaviors.

Calcium fluorescence imaging with genetically encoded indicators or synthetic dyes has been a powerful method of measuring neuronal activity in behaving animals, including non-human primates, rodents, birds, and insects¹. The activity of hundreds of cells, up to approximately 800 µm below the brain surface, can be measured simultaneously using multi-photon imaging^2,3. The activity of specific cell types can also be measured by expressing calcium indicators in genetically defined neuronal populations. Application of the imaging method for small vertebrate models opens up new possibi....

All animal procedures were approved and carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Academia Sinica. Details of the research tools can be found in the Table of Materials.

1. Preparation of recording chamber

1. Prepare a semi-hexagonal tank, a base plate, and a head stage (Figure 1A; Supplementary Files 1-3). The head stage consists of two metal posts.......

The protocol consists of two parts: head restraint surgery and two-photon calcium imaging of neuronal activities in the forebrain. The success of surgery is defined by the survival of the animal and the stability of the head restraint. The survival rate can be greatly improved by frequent perfusion of 0.01% TMS solution through the mouth during surgery. Fish should recover from anesthesia and breathe actively within 1-2 min after being immersed into fish tank water. Two-photon calcium imaging enables activity recording o.......

Here, we describe a detailed protocol to restrain the head of adult zebrafish for two-photon calcium imaging. There are two critical steps to achieve a head restraint that is stable enough for laser scanning imaging. First, the head bar has to be glued to the specific attachment sites of the skulls. Other parts of the skull are often too thin to provide mechanical stability and may even be fractured during strong body movements. Second, the skin above the attachment sites has to be thoroughly removed. Residual water.......

This work was supported by the Institute of Molecular Biology, Academia Sinica, and National Science and Technology Council, Taiwan. The Machine Shop at the Institute of Physics, Academia Sinica helped to fabricate custom-designed parts. We also want to thank P. Argast (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for the design of the quick-lock mechanism of the head stage.