The overall goal of this experiment is to visualize genetically modified cortical GABAergic interneurons during tangential migration in the most naturalistic environment possible. This method can help answer key questions in developmental neurosciences by screening the impact of the loss of function of specific genes of proteins during brain development. The main advantage of this technique is that it is low cost and relatively easy to perform compared to other similar methods such as in utero electroporation.
So this technique can ultimately improve diagnosis for patients with genetic neurodevelopmental disorders such as autism and epilepsy by clarifying the pathogenecity of specific mutations identified in patients as well as the roles of novel disease genes during brain development. Begin by setting up the biosafety cabinet with all the instruments needed for dissection and culture. Generously spray all instruments in the biosafety cabinet with 70%ethanol and sterilize the instruments and cabinet interior with UV light for 15 to 20 minutes.
When ready to collect embryos, generously spray the abdomen of the euthanized dam with 70%ethanol. Pull the abdominal skin up with a pair of sterilized forceps and with the other hand, use sterilized surgical scissors to cut the skin from the abdomen. With a second pair of sterilized forceps and scissors, pull the abdominal fascia up and cut it while carefully avoiding the uterus.
Using a third pair of sterilized forceps and scissors, hold the uterine horns and cut them out of the pelvic cavity. Place the dissected uterine horns in a sterile 60 millimeter petridish filled with neural based lebovitz culture medium supplemented with amino acids, vitamins and inorganic salts. In the sterile biosafety cabinet, use two pairs of fine tweezers to dissect the embryos out of the placenta and isolate the heads by decapitation.
Bevel cut the tip of a sterile three milliliter plastic transfer pipette and use this to aspirate the heads and transfer them into a new sterile 60 millimeter petri dish filled with the same supplemented neural based culture medium to wash out the blood. Transfer them again into a third petri dish layered with solidified black wax and filled with neural based supplemented culture medium. Place the 16 millimeter petri dish layered with black wax and containing the decapitated heads in neural based supplemented culture medium under the dissecting microscope in the biosafety cabinet.
Use fine tweezers held in the left hand to stabilize the head with the rostral part facing right. Then use the nano injector in the right hand to inject one to two microliters of the dye spiked plasmid solution into the right lateral ventricle. Electroporate the injected brain by placing the head between the electrodes with the negative electrode positioned dorsally and parallel to the head and the positive electrode toward the ventral side of the head to target the MGE.
Once the electrodes are well positioned, deliver foursquare pulses of 40 volts for 50 milliseconds at 500 millisecond interpulse intervals. While still working in the sterile environment of the biosafety cabinet, start dissecting the brain out of the skull. First, stabilize the head on the layer of black wax by inserting a needle into each eye while carefully avoiding the brain.
Then use a pair of fine tweezers to hold the left side of the neck and a second pair of fine tweezers to tear the skin from the skull from back to front. While holding the head laterally with tweezers in one hand use another pair of tweezers in the other hand to carefully cut the skull at the level of the brainstem and pull the skull up. With each tweezer, cut the skull in the sagittal plane towards the front and then incise laterally to liberate the skull fragments.
Then lift the brainstem and carefully cut the meninges and cranial nerves until the brain is completely out of the skull. Fill a 35 millimeter petri dish with molten agarose solution kept liquid at 42 degrees celsius. Then quickly transfer an electroporated brain to the agarose filled dish using the previously cut transfer pipette.
Stir the agarose with a metal stick to keep the brain in the middle of the well to prevent sinking and position the brain in a rostral caudal plane parallel to the dish. Stop stirring when the agarose starts to solidify to avoid any damage to the brain. While waiting for the agarose embedding to solidify, transfer 750 microliters of N2 supplemented culture medium in each well of the six well culture plate according to the number of brains to process.
And place a 30 millimeter membrane insert in each medium filled well. Use a razor blade to cut the agarose surrounding the brain to form a rectangular block leaving a margin of one to two millimeters around the brain. Ensure that the rostral part of the brain is perpendicular to the anterior limit of the block to facilitate orientation for the subsequent sectioning steps.
Glue the agarose block on the vibratome platform rostral edge facing down and ventral edge facing the user. Cut the brain in coronal sections to obtain 250 micron thick sections at four degrees celsius. With sterilized spatulas, collect two to three sections containing the MGE and place all brain sections from one animal on a single 30 millimeter membrane insert.
While carefully avoiding overlap between sections. Place the culture plate in a ventilated sterile incubator at 37 degrees celsius with 60%humidity and five percent CO2 for 48 or 72 hours. After the desired incubation time, transfer the sections of interest to an eight chambered coverslip filled with three to five microliters of culture medium.
Place the coverslip in an environmental chamber connect it to an inverted spinning disk confocal equipped with a computer-assisted acquisition software to set up the time-lapse imaging session. Interneurons electroporated at E 13.5 with an experimental plasmid expressing the TD tomato cassette and and SH RNA targeting a gene of interest are seen in tangential migration in an organo-typic brain slice obtained from a DLX5 6Cre RCEE GFP mouse embryo after 48 hours of culture. Interneurons were visualized using time-lapse live imaging for eight hours.
In this example, the same electroporated interneuron is seen in the process of nucleokinesis. While migrating tangentially in the cortex parallel to the peel surface. The neuron initially displays an elongated cell body in the process of nucleokinesis.
As well as a trailing process and a branched leading process. After three hours of live imaging, the nucleokinesis is completed. The trailing process has retracted and the leading process is extending two long branches.
After five hours and 30 minutes, one of the leading process branches has retracted and the neuron cell body has moved forward about 10 microns. Finally, after eight hours of imaging, migration has paused, the neuron has extended its leading process again and a trailing process has appeared. But the nucleus has not yet moved forward.
Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it's important to keep a sterile environment at all possible times. So following this procedures, other methods like MG electroporations followed by explants can be performed to address additional questions for answers to elucidate the dynamic cytoskeletal processes occurring during neuronal migration at the ultracellular level.
