This method allows the visualization of Zebrafish brain in later larval stages to allow the observation of the neuronal architecture in a highly detailed fashion in vivo that has previously not been possible. Pigment cells, which emerged during later larval development and prevents the brain from clear imaging, are no problem with this method because they are simply removed. With the method, it should be possible to study processes that occur at later stages during larval development of the brain, processes that impact on synaptic plasticity, degeneration of neurons or their regeneration.
Before beginning the experiment use a micropipette puller to prepare sharp, thin, glass needles from glass capillaries. Next, use a plastic Pasteur pipette to collect larva into a 90 millimeter diameter Petri dish containing the appropriate solution. Transfer the selected larva to a 35 millimeter diameter Petri dish containing ACSF and place a 24 by 24 millimeter, square, glass cover slip into the Petri dish lid.
Then aliquot the volume of ACSF needed for the experiment into an appropriate phial for oxygenation with carbogen. To embed the larva, use a Pasteur pipette to transfer the anesthetized larva to a mounting chamber under a stereomicroscope. If any larva are still able to move, do not use the fish for the experiment until the fish are completely unable to move.
When the larva are immobile, carefully remove the excess medium and immediately add at least one milliliter of low melting agarose onto the larva. Orient the Zebrafish with the dorsal region facing up as close to the surface of the agarose as possible. If the larva will be imaged using an inverted microscope, after a solidifying, trim the agarose containing the larva into a small cuboid block.
To expose the brain for imaging, trim away the excess agarose over the brain region of interest as necessary and use a glass needle to make a small incision through the skin near, but not over, the region of interest without penetrating too deeply into the tissue. Barely moving the needle just under the skin surface, continue to carefully make very small cuts around the region of interest until the skin over the region of interest can be removed or pushed aside. When the tissue has been removed from all the embryos, add a small drop of low melting agarose to the previously prepared imaging chamber of an inverted microscope and use a small spatula to flip the cuboid agarose block 180 degrees onto the agarose drop in the imaging chamber.
When the agarose has solidified, fill the imaging chamber with fresh ACSF and begin imaging the larva. Here, an intact 14 days post-fertilization transgenic larva with the skull still intact can be observed. As shown, the pigment cells within the overlaying skin are distributed all over the head, interfering with the fluorescent signal in the region of interest.
After open skull surgery, the area of interest becomes freely accessible for detailed high resolution imaging. The skin, skull, and or blood brain barrier also hinder the penetration of substances into the brain, as illustrated by the robust nuclear dye staining observed in these brain cells only after open skull surgery. These advantages are also observed at 30 days post-fertilization.
In transgenic fish containing a fluorescent brain vasculature due to the expression of the red fluorescent protein, mCherry, in endothelial cells, color-coding at the depth values of the image stack demonstrates that even blood vessels deeply buried within the brain to nearly 250 microns are still clearly visible and traceable. When performing this procedure always keep in mind that this is a surgery in a living animal. Therefore, it requires respect and a focused mind.
After this procedure, recording of the neuronal morphology, including synaptic structures, physiological and intracellular transport events can be performed in larvae older than seven days post-fertilization. With this technique, we hope to provide an approach that allows to study also cellular processes in the brain that occur at later larval stages, and that are involved in establishing, for example, such complex behaviors as social behavior or decision-making.
