이른 출생 후 마우스 두뇌로 Interneuron 선구자의 Homochronic 이식

The overall goal of this procedure is to harvest interneuron precursors from distinct brain regions in postnatal mouse pups, and transplant these cells into age-matched recipients to assess how environmental changes influence interneuron fate and maturation. This method can help answer key questions in the field of developmental neuroscience about how intrinsic genetic programs and environmental signals interact to sculpt the fate of neural cells. The main advantage of this technique is that alterations in the fate and maturation of interneuron precursors can be analyzed after their adoptive transfer into a new brain environment.

Visual demonstration of the cell harvesting and grafting steps is critical because small errors in the technique can lead to inefficient and unsuccessful transplant experiments. To begin, place one razor blade through the anterior frontal lobe of a postnatal day one pup brain, and additional razor blades into the slots just posterior to the first razor blade to generate two 0.5 millimeter slices. Transfer the striatal slices into a Petri dish with bubbled sucrose artificial cerebrospinal fluid or sACSF and place the remaining brain tissue into a Petri dish with sACSF on ice.

Place the slices under a dissecting microscope and use forceps to pinch the striatum from both hemispheres, placing the striatal chunks in a 50-milliliter tube of sACSF on ice as they are harvested. If the brain was harvested from a fluorescent reporter mouse, use fluorescence microscopy to distinguish the striatal td-tomato signal from the stronger fluorescent signal of the globus pallidus. To remove the hippocampus, use forceps to hemisect the brain along the midline, and place one hemisphere under a dissecting microscope medial surface up.

Use forceps to remove the ventral brain tissue and identify the sausage-shaped hippocampus spanning the anteroposterior access along the dorsal and ventricular side of the cortex. Insert the tips of the forceps in front of the anterior hippocampus, and advance the forceps posteriorly while pinching along the hippocampal cortical border to gently separate the hippocampus from the cortex. Place the isolated tissue in a 50-milliliter tube of sACSF on ice and repeat the hippocampus harvest for the contralateral hemisphere.

Then place the hemisected cortex medial side down and use the forceps to remove the most dorsal, ventral, anterior, and posterior portions of the cortex. Transfer the remaining square of medial cortex into a 50-milliliter tube of sACSF on ice. After removing the brain, pin the brain ventral side down through the cerebellum and anterior cortex or olfactory bulbs.

Using curved forceps, gently separate the posterior cortex from the underlying hippocampus and other tissue in each hemisphere. And peel the dorsal cortex forward to expose the underlying structures. Use forceps to pinch the hippocampus in one hemisphere at the midline, and remove the hippocampus by peeling it laterally away from the brain.

Place the hippocampus into a 50-milliliter tube of sACSF on ice and harvest the contralateral hippocampus in the same manner. Next gently scrape around the edges of the striatum to loosen it from the surrounding tissue. Gently pinch below the striatum to harvest the striatal tissue.

After harvesting the cortex from both hemispheres, as previously demonstrated, transfer the striatum under a fluorescent dissecting scope. Use forceps to remove any bright red td-tomato positive globus pallidus tissue from each striatum. When all of the tissues have been harvested, transfer tissue to a five-millimeter round bottom tube and replace the sACSF in each collection tube with two milliliters of freshly-prepared pronase-sACSF solution for a 20-minute incubation at room temperature with occasional flicking.

At the end of the incubation, carefully replace the pronase-sACSF with one to two milliliters of reconstitution solution, and mechanically dissociate the tissues with fire-polished Pasteur pipes. When the tissue is cloudy and no tissue chunks are visible, strain the cell solution through a 50-micron filter into new five-milliliter round bottom tubes to remove any cell clumps before fluorescence-activated cell sorting. After receiving the cells from flow cytometry, transfer the cell suspensions into 1.5-milliliter conical tubes for centrifugation.

After centrifugation, aspirate all but the last 20 microliters of supernatant from each tube, and reconstitute the pellets for counting. If necessary, dilute the cell solution with sACSF to a one to three times 10 to the 5th cells per microliter concentration. Load a fine-tipped micropipette with mineral oil.

Attach the micropipette to a nanoliter injector, and secure the nanoliter injection apparatus to a manipulator attached to a magnetic base. While the first pup is being anesthetized on ice, pipette the cell solution several times to ensure a homogenous cell distribution. Eject the mineral oil and completely fill the pipette with the single cell suspension.

After confirming a lack of response to toe pinch, place the pup on a Petri dish cover with the head resting on adhesive putty, so that the top of the head is relatively flat. Cover the head with a piece of lab tape with a diamond hole, pulling the tape taught until the skin is stretched so that the head is firmly secured and lambda is visible through the hole. Move the secured pup under the nanoliter injector and lower the micropipette so that the tip is directly above lambda.

Observe the X, Y coordinates on the manipulator and adjust the manipulator knobs until the micropipette is positioned at the appropriate coordinates along the medial, lateral, and anteroposterior axes of the head. Lower the micropipette until the tip creates a small concavity on the skin, and turn the z-axis manipulator knob firmly but gently to drive the micropipette through the skin and skull into the brain. Retract the micropipette slightly until the tip is surrounded by a cone of skin shaped like a tent.

And view the coordinates along the z-axis. Then lower the micropipette to the appropriate experimental depth and inject the cells. Wait 15 seconds after the cells have been delivered before retracting the micropipette and repeat injection at a second location if desired.

Place the pup on a heating pad and monitor until full recovery is achieved before transferring back to home cage. Grafted cells migrate throughout the targeted brain regions with variable cell survival rates ranging from dozens to several thousand. The grafted cells localize within the appropriate regions with many cells displaying interneuron morphologies and well-characterized interneuron neurochemical markers.

The donor cells survive and mature even when they are grafted into new environments with heterotopic transplantations. Electrophysiological analysis of the grafted cells reveals adult-like physiological properties and distinct firing patterns, representative of well-defined interneuron subtypes, suggesting that grafted interneurons are able to properly mature in the host environment. Transplanted cells also display spontaneous excitatory postsynaptic currents.

Recording from pyramidal cells adjacent to transplanted interneurons expressing channelrhodopsin-2 reveals postsynaptic GABAergic currents that are evoked by blue light in these endogenous pyramidal cells. While attempting this procedure, it's important to keep the cells on ice and triturate the cells gently with a Pasteur pipette to minimize cell death. Following this procedure, other methods such as single cell sequencing can be performed on the grafted cells to more fully define how environmental changes affect a cell's transcriptome.

After watching this video, you should be able to isolate interneuron precursors from different brain regions of neonate pups and transplant these cells into various brain regions of age-matched postnatal pups.