The overall goal of this procedure is to obtain electrical recordings from parametal neurons in organotypic slices of neocortex. This is accomplished by first cutting acute slices from rats at postnatal ages P eight to P 10. The second step of the procedure is to affect Bic transfection of CD NA.The third step of the procedure is to culture the slices for three to seven days.
The final step of the procedure is to obtain electrical recordings from transfected cells using current or voltage clamp. Ultimately, results can be obtained that show parametal cells in organotypic slices have similar properties to those from acute slices of the same age. Transfection of GFP does not alter these properties, allowing the study of neurons after genetic manipulations of ion channels through whole cell current or voltage clamp recordings.
The chief advantage of this technique is as opposed to say acute slices, is that cells can be maintained in a normal environment for long enough to be able to genetically manipulate ion channels. The method can help answer key questions in the ion channel field, such as the functional roles of specific ion channel types, Prepare the laminar flow hood, vacuum flow hood, surgical supplies and solutions as specified in the accompanying manuscript. Wear gloves for all subsequent steps after filtering the prepared 250 milliliters cutting solution aliquot 40 milliliters of it into a 50 milliliter beaker.
Place the cutting chamber, 40 milliliter aliquot, and the remaining 210 milliliters of the cutting solution in the freezer at minus 20 degrees Celsius under the laminar flow hood. Place 1.1 milliliters of culture media into three diagonal wells of a six well plate. Repeat this for a second.
Six well plate. Place the six well plates into an incubator at 37 degrees Celsius for at least one hour. Next, use sterile scissors to shorten four transfer pipettes to be used for transferring slices.
Another three pipettes are kept intact when the solutions have become slushy mixtures. Remove the 40 milliliter aliquot of cutting solution from the freezer and put it under the vacuum hood. Bubble it with 95%oxygen, 5%carbon dioxide.
Then remove the 210 milliliters of cutting solution from the freezer and put it under the laminar flow hood bubble. It also with 95%oxygen, 5%carbon dioxide. Next, remove the wash media from the refrigerator and bubble it with 100%oxygen under the laminar flow hood.
Place two milliliters of culture media in a Petri dish. Place the remaining culture media in the refrigerator for changing media during the week to prepare for the slicing of the brain. Pour about 10 milliliters of cold cutting solution from the 50 milliliter beaker into a 25 milliliter beaker under the vacuum hood.
Following anesthesia and decapitation, remove the brain of a postnatal day, eight to 10 rat. Wash the brain in the beaker containing 30 milliliters of cold cutting solution for about 30 seconds. Then carefully transfer the brain to the 25 milliliter beaker.
Change gloves and saturate the gloves with ethanol. Then carry the brain in the cold cutting solution to the laminar flow hood. The next step is to orient and block the brain by removing the cerebellum and the frontal pole and making a partial mid sagittal cut glue the blocked brain on the stage with the frontal cortex up.
Make additional scalpel cuts as needed to restrict the size of the slices. Then place the stage with the brain into the sterilized metal slicing chamber. Attach to the vibrating tissue slicer.
Fill the bath with ice cold bubbling cutting solution. Pour 30 milliliters of the remaining cutting solution into a 50 milliliter beaker and bubble with 95%oxygen, 5%carbon dioxide. This beaker will be used to collect the slices.
Cut 250 micron coronal slices of the frontal parietal cortex. Place at least six slices into the 50 milliliter peaker containing cutting solution. Next place about two milliliters of wash media in a 35 millimeter Petri dish.
Using a cut pipette, transfer the slices from the cutting solution to the Petri dish containing wash media. Wash the slices three times using fresh pipettes to add media and to aspirate the waste solutions after washing, transfer the slices to the 35 millimeter Petri dish containing culture media. Next, remove the tops from the packaging for the mesh inserts, but leave the inserts in the packages.
Then use forceps to lift one mesh insert from its package using a cut transfer pipette. Transfer one slice from the culture media to the mesh. Insert and position the slice in the middle of the membrane using an intact transfer pipette.
Remove any excess culture media.Now. Place the mesh insert with its slice in one of the fluid-filled wells of the six well plate. Be careful to prevent formation of air bubbles under the mesh after placing one slice in each of the fluid filled wells.
Place both six well plates back in the incubator and incubate at 37 degrees Celsius for about one hour. In the meantime, clean the work area under the laminar flow hood in preparation for transfection. The next step is to load the BioRad Helios gene gun, leaving the first position free.
Load a cartridge containing CD NA coated bullets into every other position of the cartridge holder of the gene gun. Then place the cartridge holder into the gene gun. First, clear the gun by firing a barrel with no bullets.
Then advance the barrel to a cartridge containing CDNA.Bullets. Take the six well plates from the incubator and place them under the laminar flow hood holding the gun vertically just above the top of the six well plate shoot a slice at 100 PSI then advance the barrel and clear it again by firing a blank. Shoot the remaining slices with one shot per slice.
After shooting, return the six well plates to the incubator and incubate the slices at 37 degrees Celsius for two to seven days with 5%carbon dioxide during the incubation period. Replace the media every other day as follows, working under the laminar flow hood, add new media to the three unoccupied wells. Then replace the six well plate in the incubator for at least one hour after an hour return the six well plate to the laminar hood and use ethanol cleaned curved forceps to move each mesh insert and its slice to a new well aspirate the old media and return the plate to the incubator.
After several days of incubation, the slices are now ready for whole cell recordings. Remove a six well plate from the incubator under the laminar flow hood. Use ethanol cleaned curved forceps to remove one mesh insert and its slice.
Then replace the six well plate in the incubator. Next, transfer the Petri dish with a mesh insert and slice to the recording area, applying tension with forceps or a flexible piece of metal. Use a number 11 scalpel to cut the mesh away from around the slice if necessary, finish the final cuts with the scissors.
Then use forceps to transfer the slice and the attached mesh to the recording chamber. On the stage of an upright microscope, bathe the slice in carbogen related artificial cerebral spinal fluid under IR DIC optics. Visualize the parametal neurons in layers two, three, or five under epi fluorescence and the fitz filter.
Locate the green transfected cells under IR DIC microscopy. Look for the CDNA bullets, which show up as small black dots inside the cells. Select a transfected cell for whole cell recording.
Next, advance a micro electrode to the selected cell and use gentle suction to attain a giga ohm seal. Then apply additional suction to break into the cell for whole cell.Recording. The cell is now ready for either current or voltage clamp recordings.
This figure shows a fluorescent image of cells transfected with CDNA for GFP. The following recordings are taken from cells transfected with CDNA for GFP. Shown here is an action potential in a layer three parametal cell in response to a supra threshold 10 millisecond current injection.
This figure shows repetitive firing from a layer five parametal neuron in response to a two second 400 pico amp current injection. This figure shows a voltage clamp record from a layer five parametal neuron. Tetrodatoxin was used to block the voltage gated sodium current.
The traces represent outward potassium currents elicited by a family of 500 millisecond voltage steps from a holding potential of minus 70 millivolts. Once master, the slight preparation and transfection can be done in about two hours, including preparation time. When attempting this Procedure.
It's important to maintain sterile conditions and to restrict the thickness and dimensions of the slice to prevent drying. Thanks for watching. Good luck with your experiment.
Thank you.
