With this method, we can study the effects of intrinsic activity on the maturation of cortical interneuron precursors. This technique provides an efficient way for targeting and manipulating the cellular properties of cortical interneuron progenitors that is accessible to the majority of the scientific community. This protocol involves the meticulous handling of samples and equipment, and it can best be appreciated by visual demonstration.
Using a waterproof pen, draw a straight line across the middle of the bottom part of the bottom surface of six 35-millimeter Petri dishes. Add 10 milliliters of liquid 4%low gelling agarose in PBS into two of the 35-millimeter Petri dishes and embed three to four dissected brains in the agarose with the olfactory bulbs facing down per dish across the straight line, leaving three to five millimeters of space between each sample. When all of the brains have been embedded, place the dishes at four degrees Celsius to allow the agarose to solidify and carve the three brains into a single block of agarose, leaving approximately three millimeters of gel around the edges of the samples.
Glue the block on the surface of a microtome base and use a surgical blade to cut all the way through the bottom of the block between each tissue sample to obtain three independent blocks. Next, use a vibrating blade microtome to section the blocks in ice cold Krebs solution into 250-micrometer thick slices, using a bent flat microspatula to collect only the slices containing the medial or caudal ganglionic eminences. Then, place each section onto individual 13-micrometer diameter, eight-micrometer pore size filter membranes floating on minimal essential medium in individual polystyrene center well organ culture dishes.
When all of the sections have been obtained, place the dishes in a carbon dioxide tissue culture incubator at 37 degrees Celsius for one hour. Before the focal DNA injection, place one-millimeter diameter, 10-millimeter long agarose columns punched with a glass 225-millimeter long, two-milliliter capacity glass pipette into ice cold Krebs solution. Using a surgical blade, cut one smaller piece of agarose to fit on the surface of the electroporation electrode and one bigger piece to be used as a base for the focal DNA injections.
Then, place the agarose blocks into ice cold Krebs solution. For the injection, mix the expression and control vector DNA at a one-microgram per microliter concentration for each vector and add fast green stock solution at a one to 10th dilution. Fill a pulled 0.5-millimeter inner diameter, one-milliliter outer diameter glass micropipette with 10 microliters of the DNA mixture and load the micropipette into a pneumatic PicoPump injector.
Place the bigger block of agarose under a stereo microscope and place the slice to be injected onto the piece of agarose. Then, inject 25 to 50-nanoliter volumes into the selected medial ganglionic or caudal ganglionic eminence region of the slice. Immediately after the injection, place the small agarose block on the Petri dish electrode and use a flat ended microspatula to attach the agarose column to the mobile cover electrode.
Transfer the injected slice with its supporting membrane onto the agarose block and place the top electrode with the agarose column on top of the selected region of the slice. Then, electroporate the region with two five-millisecond pulses of 125 volts, 500 milliseconds apart. After the electroporation, place the slice with its supporting membrane back into its holding dish and return the dish to the tissue culture incubator.
After one hour, replace the minimal essential medium with a basic medium appropriate for primary neuronal cultures and return the slices to the incubator overnight. In these electroporation experiments, approximately 50%of the GFP positive neurons co-expressed the RFP positive injected protein and therefore, the GFP positive RFP negative population served as an internal control for the effect of the injected DNA ligands. Clozapine and oxide administration selectively increases the activity of transfected RFP positive cells, as demonstrated by the expression of the activity-dependent protein, c-Fos, in these newborn coronal tissue sections.
Clozapine and oxide treatment also results in an increase in the proportion of GFP-positive RFP-positive cells relative to the number of GFP-positive RFP-negative cells compared to vehicle administered littermates. This procedure can be used to study the effects of activity-modulated grafted interneurons on circulatory plasticity and brain function of the host.
