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 imaging2,3. 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 possibilities in the field of neuronal computation across brain regions.
Zebrafish are a widely used model system in neuroscience research. Larval zebrafish at around 6 days post-fertilization have been used for calcium imaging due to their miniature brain and transparent body4. Juvenile zebrafish (3-4 weeks old) are also used for studying the neural mechanisms underlying sensorimotor pathways5,6. However, the maximal performance level for complex behaviors, including associative learning and social behaviors, is reached at an older age7,8. Thus, a reliable protocol is required to study multiple cognitive functions in the brains of adult zebrafish using imaging methods. While zebrafish larva and juvenile zebrafish can be embedded in agarose for in vivo imaging, adult zebrafish at 2 months or older suffer from hypoxia in such conditions and are physically too strong to be restrained by agarose. Therefore, a surgical procedure is required to stabilize the brain and enable the animal to breathe freely through the gills.
Here, we describe a head restraint protocol that involves a novel design of a single head bar. The reduced surgery time of 25 min is twice as fast as the previous method9. We also describe the design of the recording chamber (semi-hexagonal tank), the head stage and a quick-lock mechanism to combine the two parts9. Finally, the setup to present an immersive visual stimulus to study visually triggered brain activity and behaviors is also described. Overall, the procedures described here can be used to perform two-photon calcium imaging in genetically defined cell populations in a head-restrained adult zebrafish, enabling the investigation of brain activities during various behavioral paradigms.
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
Figure 1: Instruments required for head restraint surgery. (A) The quick-lock mechanism between the circular plate of the head stage and the base plate inside the semi-hexagonal tank. Computer-aided design (CAD) files of the custom-made parts can be found in Supplementary Files 1–4. (B) Ω-shaped head bar for the head restraint. (C) The three-axis micromanipulator used to position the head bar to the attachment site. Inset: orientation of the head bar in the clay. (D) Cannon to hold the fish during surgery. Inset: orientation of fish within the cannon. (E) Fish loading module and the micromanipulator used to load the fish onto the head stage. Inset: orientation of fish within the module. Please click here to view a larger version of this figure.
2. Head restraint surgery
Figure 2: Key steps during head restraint surgery. (A) Composition of the capsule inside the cannon. (B) Attachment sites on the skull (red). Red arrows specify blood vessel sites. (C) Top: head bar attached to the fish skull. Bottom: fish loaded onto the head stage. (D) Cuts needed to remove the skin above the forebrain. Numbers denote the cutting sequence. Avoid skin removal at the marked site (arrowhead) to prevent bleeding of the animal. Please click here to view a larger version of this figure.
3. Two-photon imaging
Figure 3: Setup to perform calcium imaging, behavior recording, and visual stimulus display. (A) Three projectors present a visual stimulus onto the walls of the semi-hexagonal tank. IR lights on the side are used to illuminate the body of the zebrafish. (B) Positioning of the objective lens. Left: front view. Right: side view. The distance between the 16x objective lens and the targeted brain region is around 2.5 mm. (C) Example two-photon image. Left: maximum projection of the entire dorsal forebrain in Tg[neuroD:GCaMP6f]. Right: zoomed-in image to reveal neurons across multiple brain regions. Inset: a higher magnification from a different brain region. Images are averages of 10 s of data recorded at 5Hz. Please click here to view a larger version of this figure.
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 of individual neurons in the dorsal forebrain at a depth up to 200 µm from the brain surface through intact skulls (~40 µm in thickness). This imaging range covers multiple zones of the dorsal telencephalon (D), including the medial zone (Dm), the rostral part of the central zone (rDc), the caudal part of the central zone (cDc), and the lateral zone (Dl). Together they make up 30% of the telencephalon in adult zebrafish (Figure 3C). With volumetric imaging using the piezo actuator, we typically record the activity of 150 neurons in Dl or cDc, and 300 neurons in Dm and rDc in Tg[neuroD:GCaMP6f]10. Simultaneous behavioral recording is performed during brain imaging, which enables the identification of neuronal correlates of motor outputs (Figure 4).
During two-photon imaging, tail movements should not induce a visible motion artifact in the image. A small (<1 µm) and transient motion can be observed during extreme struggling. These motions are typically reversible, so imaging can be continued afterward. We also observe a slow drift (<1 µm min-1) in the lateral and axial directions9. To prevent the loss of neurons due to axial sample drift, we typically restrict our imaging session to 10 min. Photobleaching of the calcium indicator should not be observed after a 10 min imaging session under the laser power specified.
Figure 4: Behavior tracking and neural activity pattern in adult zebrafish. (A) An example frame of camera recording of behavior (ventral view). (B) Activity of forebrain neurons (ΔF/F, black) and tail movement intensity (blue). The intensity of tail movement was quantified by the mean of the absolute pixel-wise difference between successive video frames. Please click here to view a larger version of this figure.
Supplementary File 1: Design of the base plate. Please click here to download this File.
Supplementary File 2: Design of the circular plate. Please click here to download this File.
Supplementary File 3: Design of the semi-hexagonal tank. Please click here to download this File.
Supplementary File 4: Design of the head bar. Please click here to download this File.
Supplementary Table 1: Troubleshooting details. Please click here to download this File.
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 should also be dried thoroughly. This allows the skull to tightly bind to the tissue glue. A table that lists potential problems, their causes, and the solutions is provided (Supplementary Table 1).
Due to the curved geometry of the zebrafish forebrain, the excitation beam and resultant emission travel through different thicknesses of the brain tissue across the imaging plane. Thus, the fluorescence of neurons imaged within an optical section vary based on the neuron's distance from the brain boundary. This leads to low data yield within a recording session. The problem is particularly severe when imaging the Dl and cDc (Figure 3C). Here, we use a piezo actuator to perform recordings across multiple axial positions, which compromise the frame rate but increase the number of neurons recorded. Importantly, as neuronal activities at multiple axial positions are recorded within a single behavioral session, the data has a higher statistical power in comparison to activity data recorded from multiple behavioral sessions. Alternatively, three-photon imaging2 and adaptive optics11 can record the neuronal activities in deep tissues and relax the high curvature problem. However, the repetition rate of three-photon lasers and the heterogenous structure of skulls should be considered.
In comparison to previous methods9,12 that use multiple metal parts to stabilize the head, the current approach made a significant improvement by using a single piece of an Ω-shaped head bar. This simplified design reduces the surgery time by 50%. The protocol is also easier to learn and typically can be mastered within 2 weeks of training. Although the surgery time is largely reduced in comparison to the previous protocol9, we did not observe obvious differences in survival rate nor behavior under the head restraint. For example, the duration of time during which the animal remains active under the microscope is similar (3-8 h, depending on the animal). The amount of time required for the animal to wake up from anesthesia after the surgery is also similar (1-2 min). The transgenic line10 used in this protocol involves a single transgene that encodes the calcium indicator in the forebrain from the larval stage to adulthood, similar to the expression timeline of the triple transgenic line used in a previous study12.
Currently, the protocol has the following limitations. First, neuronal activity can be imaged through the skull at a depth up to 200 µm from the brain surface using a two-photon microscope. The image quality at various depths has been previously quantified9,12. These imaging depths allow access to the dorsal forebrain areas Dm, Dc, and Dl, but potentially exclude the ventral forebrain areas Vv, Vs, Vp, and Vd in adult zebrafish. In addition, optical access to the majority of the midbrain and the hindbrain is obstructed by the presence of the head bar and dental cement. Additional modifications to the protocol are required to perform imaging in these brain regions. Furthermore, we typically restrict an imaging session to 10 min to prevent significant sample drift. The potential cause of the drift is the weakening of the bonding between the skull and the tissue glue after vigorous tail movements. If longer recording sessions are needed, volumetric imaging with small axial steps followed by 3D image registration can be performed.
This head restraint protocol opens up several future applications. First, two-photon calcium imaging, optogenetic manipulations, electrophysiology, or even ultrasound imaging can be performed in vivo. With three-photon imaging, neuronal activity across the entire forebrain can also be recorded2. In addition, a mechanical device can be constructed to reversibly grab and release the two ends of the head bar, which enables longitudinal investigation of neural activity and circuit morphology across days. Finally, the visual display setup we described provides a 180° horizontal field of view to the animal. The setup can be used to construct an immersive virtual reality environment that interacts with head-restrained fish9. Overall, this protocol enables activity measurement of neuronal populations across the pallium of behaving adult zebrafish.
The authors have nothing to disclose.
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.
Acquisition card | MBF Bioscience | Vidrio vDAQ | Microscope |
Back-projection film | Kimoto | Diland screen – GSK | present visual stimulus |
Band-pass filter (510/80 nm) | Chroma | ET510/80m | Microscope |
Base plate for the semi-hexagonal tank | custom made | see supplemental files | recording chamber |
Camera filter (<875 nm) | Edmund optics | #86-106 | Behavior recording |
Camera filter (>700 nm) | Edmund optics | #43-949 | Behavior recording |
Camera lens | Thorlabs | MVL50M23 | Behavior recording |
Chameleon Vision-S | Coherent | Vision-S | Laser |
Circular plate for the head stage | custom made | see supplemental files | recording chamber |
Controller for piezo actuator | Physik Instrumente | E-665. CR | Microscope |
Current amplifier | Thorlabs | TIA60 | Microscope |
Elitedent Q-6 | Rolence Enterprise | Q-6 | Surgery: UV lamp |
Emission Filter 510/80 nm | Chroma | ET510/80m | Microscope |
Head bar | custom made | see supplemental files | recording chamber |
Infrared light | Thorlabs | M810L3 | Behavior recording |
LED projector | AAXA | P2B LED Pico Projector | present visual stimulus |
Moist paper tissue (Kimwipe) | Kimtech Science | 34155 | Surgery: moist paper tissue |
Motorized XY sample stage | Zaber | X-LRM050 | Microscope |
Neutral Density Filters (50% Transmission) | Thorlabs | NE203B | present visual stimulus |
Ø1/2" Post Holder | ThorLabs | PH1.5V | Surgery: hollow tube for cannon |
Ø1/2" Stainless Steel Optical Post | ThorLabs | TR150/M | Surgery: fish loading module |
Objective lens 16x, 0.8NA | Nikon | CF175 | Microscope |
Oil-based modeling clay | Ly Hsin Clay | C4086 | Surgery: head bar holder |
Optical adhesive | Norland Products | NOA68 | Surgery: UV curable glue |
Photomultiplier tube | Hamamatsu | H11706P-40 | Microscope |
Piezo actuator | Physik Instrumente | P-725.4CA PIFOC | Microscope |
Pockels Cell | Conoptics | M350-80-LA-BK-02 | Microscope |
Red Wratten filter (> 600 nm) | Edmund optics | #53-699 | present visual stimulus |
Resonant-Galvo Scan System | INSS | RGE-02 | Microscope |
Right-Angle Clamp for Ø1/2" Post | ThorLabs | RA90/M | Surgery: fish loading module |
Rotating Clamp for Ø1/2" Post | ThorLabs | SWC/M | Surgery: fish loading module |
ScanImage | MBF Bioscience | Basic version | Microscope |
Semi-hexagonal tank | custom made | see supplemental files | recording chamber |
Super-Bond C&B Kit | Sun Medical Co. | Super-Bond C&B | Surgery: dental cement |
Tricaine methanesulfonate | Sigma Aldrich | E10521 | Surgery: anesthetic |
USB Camera | FLIR | BFS-U3-13Y3M-C | Behavior recording |
Vetbond | 3M | 1469SB | Surgery: tissue glue |