two-photon imaging of zebrafish forebrain 

ゼブラフィッシュの前脳における行動追跡と神経活動パターン

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 &#181;m 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.

著者スポットライト:成体ゼブラフィッシュの脳研究の進歩 

ゼブラフィッシュ成魚の行動性における前脳活動の2光子カルシウムイメージング

The fish fore-brain hosts areas suspected to be homologous to mammalian brain regions involved in complex behavior. These include the hippocampus, the cortex, and the amygdala. We are currently trying to understand the effect of sensory information on the activity of these brain regions.Current research in zebra fish neuroscience uses two photon calcium imaging of immobilized larvae. Combined with virtual reality based behavior essays, to study sensory motor processing in the brain. Most research on the zebra fish brain is done on larvae, however some complex behaviors in fish do not fully develop until adulthood.Our protocol allows the study of brain activity in the adult during behavior. This protocol reduced surgery times by fifty percent compared to the previous method. It's a more efficient approach for restraining the head of adult zebra fish for a better image.This head restraint protocol opened an opportunity to perform an in vivo experiment on the adult zebra fish brain. In addition to the two-photon calcium image, head restraint potentially allows the opto-genetic manipulation, electro-physiology, and even ultra-sound imaging of live fish.

Watch this Scientific Journal Video about Two-Photon Imaging of Zebrafish Forebrain at JoVE.com

Two-Photon Imaging of Zebrafish Forebrain   

ゼブラフィッシュ前脳の2光子イメージング 

レーザーをオンにし、電源が安定するまで30分間待ちます。サンプルの波長を 920 ナノメートル、出力を約 50 ミリワットに設定します。頭部を拘束したゼブラフィッシュの入った記録チャンバーを実験台に置きます。対物レンズを前脳表面の近くに配置します。行動を記録するには、タンクの両側にある810ナノメートルの赤外線ライトをオンにします。VR表示用の入力ソースを選択し、データの記録を開始します。次に、レーザーシャッターを開き、フォトマルチプライヤチューブを有効にします。背側前脳が現れるまで、対物レンズを徐々に上げます。ピエゾアクチュエータを使用して複数の深度で画像を取得し、記録されるニューロンの数を増やします。2光子カルシウムイメージングは、無傷の頭蓋骨を通して最大200ミクロンの深さの背側前脳の個々のニューロン活動を記録します。イメージング範囲には、古領域、Dm、cDc、rDc、およびゼブラフィッシュの成体の前脳の30%をカバーするDlの一部が含まれます。脳イメージング中に同時に行われる行動記録により、運動出力の神経相関を特定することができます。イメージング中、尾の動きは目に見えるモーションアーチファクトを引き起こしてはなりません。

two-photon-imaging-of-zebrafish-forebrain

Research

JoVE Journal

Neuroscience

Two-Photon Imaging of Zebrafish Forebrain 

250 ビュー.  Academia Sinica. レーザーをオンにし、電源が安定するまで30分間待ちます。サンプルの波長を 920 ナノメートル、出力を約 50 ミリワットに設定します。頭部を拘束したゼブラフィッシュの入った記録チャンバーを実験台に置きます。対物レンズを前脳表面の近くに配置します。行動を記録するには、タンクの両側にある810ナノメートルの赤外線ライトをオンにします。VR表示用の入...

ビデオ: Two-Photon Imaging of Zebrafish Forebrain   

Two-Photon Calcium Imaging of Forebrain Activity in Behaving 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.

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 ...

神経科学

Preparation of Recording Chamber for Zebrafish Imaging

To begin, prepare a semi-hexagonal tank, a base plate, and a head stage. Place the semi-hexagonal tank on the experimental platform of the microscope. Aim the 810 nanometer infrared light and the camera to the head stage for behavioral recording.Then, set up an 875 nanometer short-pass filter, and a 700 nanometer long-pass filter in front of the camera to prevent the two-photon laser and the visual stimulus from interfering with behavioral recording. To present the visual stimulus, attach back projection films to the inner side of the three walls of the semi-hexagonal tank. Aim the three projectors at the three surfaces of the tank.

ゼブラフィッシュイメージングのための記録チャンバーの作製

Head-Fixation Surgery in Zebrafish

To prepare an omega-shaped head bar for the zebrafish head restraint, attach a piece of oil-based modeling clay to a three-axis micro manipulator. Then insert an arm of the head bar into the clay and orient the head bar horizontally. Prepare the cannon for holding the fish's body in place during surgery.To remove excess skin and water from the skull's attachment sites, prepare four tissue swabs by cutting a paper towel into a three centimeter square. Then fold the paper squares along their diagonal to produce a tube-like structure and twist the ends of the tube into a fine point. After anesthetizing the fish with 0.03%TMS, perform syringe perfusion with 0.01%TMS to maintain sedation throughout the procedure.Wrap the fish in moist paper tissue starting from the tip of the tail to around one millimeter caudal to the gill. Then wrap a soft plastic tubing with a longitudinal slit around the paper tissue covering the fish from the end of the tail to around two millimeter caudal to the gill. Wrap the paper towel around the tubing.Then wrap a soft plastic tube cut from the bulb of a plastic transfer pipette around the paper towel. Load the wrapped fish into the cannon. Next, using a scalpel, remove the skin covering the attachment sites, two triangular areas of the skull rostrolateral to the cerebellum and above the gills.Then use tissue swabs to dry the attachment sites and clear any remaining skin. Using a 10 microliter pipette tip, apply a drop of tissue glue at the center of each attachment site. Place the fish body in an upright position and position the head bar slightly above and caudal to the attachment sites.Next, prepare dental cement by mixing one small spoon of polymer with four drops of quick monomer and one drop of catalyst five. Stir the mixture evenly for 15 seconds. Immediately after mixing, use a micropipette and a 10 microliter pipette tip to apply the cement onto the attachment sites and the region between the sites.Then using a micro manipulator, gently press the head bar onto the cement. Cover the head bar with the remaining cement and wait for 12 minutes to allow the curing of the cement. Next, to improve optical access to the forebrain for calcium imaging, use a scalpel to remove the skin above the telencephalon.After curing, remove the clay from the head bar. Next, prepare a fish loading module for the micro manipulator. Then transfer the entire capsule from the cannon to the pitch adjuster in the fish loading module.Using the micro manipulator, position the fish with the head bar placed on top of the metal posts of the head stage. Slightly increase the pitch angle of the fish. Apply ultraviolet curable glue to secure the head bar to the metal posts.Then pull out the fish from the capsule and lock the head stage onto the base plate within the semi hexagonal tank. After surgery, immerse the animal in fish tank water to recover from anesthesia.

ゼブラフィッシュの頭部固定手術

Turn on the laser and wait for 30 minutes for the power to stabilize. Set the wavelength to 920 nanometers and power to approximately 50 milliwatts at the sample. Place the recording chamber with the head-restrained zebrafish on the experimental platform.Position the objective lens close to the forebrain surface. For behavior recording, turn on the 810 nanometer infrared lights on both sides of the tank. Select the input source for VR display and start recording data.Then, open the laser shutter and enable the photo multiplier tube. Gradually elevate the objective lens until the dorsal forebrain is revealed. Use a piezo actuator to acquire images at multiple depths to increase the number of neurons recorded.Two photon calcium imaging records individual neuron activity in the dorsal forebrain up to 200 microns deep through intact skulls. The imaging range includes the paleo areas, Dm, cDc, rDc, and part of the Dl covering 30%of the adult zebrafish forebrain. Simultaneous behavioral recording performed during brain imaging enables the identification of neuronal correlates of motor outputs.During imaging, tail movements should not cause visible motion artifacts.