The overall goal of this procedure is to micro inject morph oligonucleotides into the cerebral ventricle of the adult zebrafish brain and to knock down the activity of genes in the radio glial cells. This is accomplished by first anesthetizing the zebra fish. Next, a slit is generated in the skull, overlaying the optic tum using a barbed dent needle.
Then the morpho solution is injected through the incision site into the cerebral ventricular fluid. Finally, the injection efficiency and accuracy are checked under the fluorescence microscope. Ultimately, results can be obtained the too efficient and widespread blockage of protein production from the genes of interest in the radial glial cells of the adult zebrafish brain through morpho mediated gene knockdown, which can be assayed by histological sections and immunohistochemical stainings.
The main advantage of this technique over the existing methods, like transgenic approaches or electro operation based delivery methods, is that CVMI is less time consuming, more efficient in targeting the whole population of radio glial cells. Those adjustable and many genes can be knocked down at the same time. To prepare the injection mixture begin by making a 500 Micromolar Morpho stock solution.
Using PBS for 10 injection, prepare 10 microliters of injection mix by combining nine microliters of diluted or undiluted morpho solution with one microliter of a fluorescent tracking dye. Store it room temperature. Use a needle puller to prepare glass injection capillaries according to the following parameters.
Turn on the pressure source and adjust the pressure settings to 50 PSI or 3.5 bars. Adjust the micro injector settings as follows. Hold pressure 20 PSI eject pressure 10 PSI period value 2.5 and 100 milliseconds.
Range of gating. Turn on the ring illuminator and locate the ring over the microscope stage. Place the capillary holder at an appropriate position next to the microscope.
Insert the glass capillary into the holder and adjust the injection angle to 45 degrees. Using find end forceps, snap off the tip of the glass capillary and calibrate the pressure output from the orifice. By immersing the tip of the needle into the fish water and applying a continuous pressure pulse, the air bubbles should form a single row indicating optimum pressure or orifice size.
Load the glass capillary with the injection solution. Avoiding air bubbles apply pressure to remove the air between the tip of the glass capillary and the loaded injection solution. After preparing a 0.1%stalk solution of the anesthetic meab, transfer the desired number of fish into a transport container with fish water.
Prepare a working solution of anesthetic by mixing 200 milliliters of fish water with five milliliters of MISP stock solution. Fill a Petri dish halfway with the anesthetic to use for injections. Incubate a fish in the anesthetic until it stops moving.
Then use a net to transfer it into the Petri dish of anesthetic. To facilitate incision, use the forceps to gently hold the fish and orient it with its head tilted slightly downwards. Using the tip of a 30 gauge barbed in needle, make a small slit on the skull without penetrating too deeply as this will cause damage to the brain tissue.
While continuing to hold the fish, insert the tip of the glass capillary through the incision site about half a millimeter, and while avoiding contact with the brain tissue, orient it towards the cephalon. Inject the solution, it should disperse immediately. Transfer the fish into fresh fish water and allow it to recover.
About 30 minutes later, re anesthetize the fish and check it under a fluorescence microscope to look for widespread distribution of the liquid. Based on red fluorescence. Return the fish to its tank and place it back into circulation.
Until use for behavioral analyses or for preparing tissue specimens as seen here, an accurate injection leads to dispersion of the injected solution throughout the brain and efficient targeting of the cells close to the ventricular surface. However, injections where the glass capillary impales, the brain tissue will have a dense fluorescent signal at the point of injection. And if the injected amount is not enough, a weak fluorescent signal will be observed.
This figure demonstrates that vivo morphos can penetrate a few cell diameters inside the ventricular surface, and hence can efficiently target the ventricular cells as compared to control.Morpho. Injection of morpho against PCNA efficiently knocks down gene expression in ventricular cells. In these immunofluorescence images, BRDU and QCD immuno staining after A-B-R-D-U pulse chase experiment demonstrate that knockdown of PCNA with antisense morphoses leads to a significant reduction in newborn neurons, suggesting that CVMI can be used to knock down endogenous genes that subsequently gives rise to functional consequences.
So after its development, this technique paved the way for the researchers in the field of neuroscience to explore the molecular mechanisms involved in the regenerator neurogenesis response of the adult S of fish brain.
