The overall goal of this procedure is to examine adult neurogenesis using a neurosphere assay derived from the adult zebrafish brain, and to manipulate gene expression in zebrafish neurospheres. So, this method can help to answer a key question in neurogenesis, and, in particular, the key mechanism that drive neural stem cell self-renewal and the determination of this cell. The main advantage of this technique is that it's fairly simple, it's reliable, and it will help to obtain a neural stem progenitor cell from the zebrafish brain, and to understand their property in vitro.
So we first add the idea for this method, when we were investigating epigenetic regulation in the zebrafish developing brain, and using our previous experts, we adapted mouse neurosphere protocol to zebrafish. To begin, prepare a dissection bed by filling a Petri dish with gel packs. Then, cover the dish with its corresponding lid, and incubate it at 20 degrees Celsius.
Once the gel is frozen, remove the dish from the freezer, and place a clean square of filter paper on top of its lid. Proceed to wrap both the paper and dish with plastic film. Next, treat all micro-dissection instruments with 70%ethanol.
Place the sterilized tools next to a dissecting microscope, and position the completed dissection bed under the microscope with optical fiber illumination. Immediately place a previously-prepared head specimen on top of the dissection bed and orient it so that its dorsal side is facing down. Then, use scissors to make a longitudinal cut through the soft tissue from the back of the head to the mouth.
Afterwards, expose the base of the skull with forceps. And remove all of the adjacent tissue. Next, cut one of the lateral walls of the skull, starting at the back of the head, and moving towards the tectum region of the brain.
Repeat this process for the contralateral side. Proceed to cut the optic nerve. And then remove the two lateral-most sides of the skull at the level of the tectum.
Finally, turn the head ventral side up, and with forceps, peel off the most apical part of the skull to expose the brain. Afterwards, transfer the brain and any remaining parts of the skull to a dish containing dissection medium, which is composed of DMEM/F12 supplemented with penicillin streptomycin. With the plastic handle of a micro-knife, under the microscope, clean the brain tissue, being careful not to damage any neural structures.
Use up to two zebrafish brains to generate whole brain-derived neurospheres. Move this dish containing the tissue into a bio-safety cabinet, then transfer the brain to a sterile 1.5 milliliter tube containing 800 microliters of dissection medium. Remove the media from the tube by pipetting, being careful not to touch the tissue.
Proceed to digest the brain by adding 500 microliters of previously prepared papain solution. And incubating it at 37 degrees Celsius for 10 minutes. Following incubation, transfer the papain solution and digested brain into a 15 milliliter conical tube using a cut 1, 000 microliter pipette tip.
Using the same tip, gently pipette up and down 10 times to dissociate the tissue, avoiding the formation of air bubbles. Incubate the conical again at 37 degrees Celsius for 10 minutes, and afterwards, dissociate the brain tissue further by pipetting up and down approximately 10 times, using an uncut 1, 000 microliter tip. To stop the enzymatic reaction, add two milliliters of previously prepared washing solution to the tube, and centrifuge it at 800 x g for five minutes at room temperature.
Carefully decant the cell suspension, pour off the supernatant, and then vigorously tap the conical. To finish cell resuspension, pipette up and down very carefully the cells in the remaining solution with a regular 1, 000 milliliter tip. Then, add two milliliters of washing solution, and check the cells under a microscope.
If a single cell suspension has been attained, proceed to centrifuge the tube using the conditions previously described. Remove the supernatant after centrifugation, and resuspend the pellet in one milliliter in fresh Z-condition medium. Proceed to stain the cells with Trypan blue, and then count them using a hemocytometer.
To prepare for neurosphere generation, fill each well of a 24-well plate with 300 microliters of fresh Z-condition medium. Then, add 200 microliters of the cell suspension to each well, seeding them at a density of approximately 500 cells per microliter. Afterwards, incubate the plate at 30 degrees Celsius and 5%carbon dioxide.
After one day of culture, view the cells in the 24-well plate under a microscope. Expect to observe single cell suspensions at this stage. If any debris is seen to have collected in the center of a well, remove it by pipetting out approximately 100 microliters of medium, and replace this liquid by adding 100 microliters of fresh Z-condition medium.
Once all debris has been removed, incubate the plate under the conditions described previously. After an additional day of culture, transfer 250 microliters of cell suspension from a single well into a new, empty well. Repeat this step for all 24 wells.
Then, add 250 microliters of fresh Z-condition medium to each well, and homogenize the suspensions by gently pipetting up and down. Incubate the plates again under the same conditions. And repeat this expansion procedure after an additional day of culture.
Expect to observe a progressive increase in the size of neurospheres on the third, and fourth days of this period. To begin passaging, after four days, remove 250 microliters of medium, but no neurospheres, from each well. Then, use a one milliliter pipette to mechanically dissociate the neurospheres in the remaining medium volume.
Proceed to pool the cell suspension from each dissociated well. Then, count the cells with a hemocytometer, and distribute 250 microliters of primary culture supernatant, containing 800 cells per microliter, into each well of a new 24-well plate. Continue by adding 250 microliters of Z-condition medium to each well.
and then incubating the plate as previously described. Zebrafish primary neurospheres are capable of self-renewal, as demonstrated by the formation of secondary neurospheres at passage one, and tertiary structures at passage two, which suggests a pool of stem, or progenitor, cells. These stem or progenitor cells can also be differentiated, as supported by the observation that neurospheres cultured for four days under differentiation conditions give rise to glia and cells with axonal-like projections, indicated by arrows here.
Differentiation experiments can also be performed on transfected neurospheres. Here, neurospheres were treated with either a scrambled control, or an antagonist of micro R-107, a small non-coding RNA, and differentiated as before. Antagonist transfected neurospheres demonstrate cells with abnormal axonal processes, which were significantly thicker than processes in controls.
RT-PCR analysis reveals that inhibition of micro R-107 leads to a significant increase in the expression of certain neuroblast or axon-specific molecules compared to controls, but does not significantly affect glial markers. In contrast, neurospheres that were treated with the micro R-107 mimic, resulting in the gain of micro R-107 expression, and subsequently differentiated, demonstrate significantly decreased levels of neuroblasts or axon markers compared to controls. These results suggest that micro R-107 acts as a regulator of neural differentiation in zebrafish.
Once mastered, this technique is fairly fast, it can be performed in about two hours, and it's a technology that will help scientists to advance other methods, such as neural regeneration in adults and zebrafish brain, in particular to understand the property of neural stem cell activation. So after its development, this technique paves way for researchers in the field of development and neuroscience to investigate neurogenesis in zebrafish. After watching this video, you should have a good understanding on how to perform a neurosphere assay using the adult zebrafish brain, and apply it to investigate biological questions in neurogenesis research as our stem and progenitor cells differentiate.
