two-photon imaging of zebrafish forebrain 

Behavior Tracking and Neural Activity Pattern in the Zebrafish Forebrain

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.

Author Spotlight: Advancements in Adult Zebrafish Brain Research 

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

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   

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.

two-photon-imaging-of-zebrafish-forebrain

Research

JoVE Journal

Neuroscience

252 visualizações.  Academia Sinica. Ligue o laser e aguarde 30 minutos para que a energia se estabilize. Ajuste o comprimento de onda para 920 nanômetros e a potência para aproximadamente 50 miliwatts na amostra. Coloque a câmara de gravação com o peixe-zebra contido na cabeça na plataforma experimental.Posicione a lente objetiva próxima à superfície do prosencéfalo. Para gravação de comportamento, ligue as luzes infravermelhas de 810 nanômetros em ambos os lados do tanque. Selecione a fonte de entrada para ...

Vídeo: Two-Photon Imaging of Zebrafish Forebrain   

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 ...

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.

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.

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Developmental Biology

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Biology

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...