Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Animals were bred and housed in accordance with the United Kingdom Animals (Scientific Procedures) Act (1986).

NOTE: For the generation of the pCAGGs-hM3D(Gq)-IRES-RFP construct, a SalI-StuI fragment, containing the hM3D(Gq) sequence, has been isolated from plasmid 50463 (Addgene), and inserted into the expression vector pCAGGs-RFP (gift from F. Guillemot) digested with XhoI-EcoRV.

1. Preparation of Mouse Embryo Cortical Slices

1. Sterilize lab equipment (e.g., stereo microscopes) and surfaces (e.g., benches) with an appropriate detergent solvent and 70% ethanol (EtOH) solution in water (H₂O).
2. Use dissection tools that have been autoclaved and keep them all the time in 100% EtOH.
3. Prepare three 20 mL aliquots of 4% low-gelling temperature agarose in 1x phosphate buffer solution (PBS), in 50 mL tubes, and keep them at 55 °C.
4. Sacrifice pregnant mice by cervical dislocation at 13.5 or 14.5 days of gestation (embryonic day E13.5-E14.5).
5. With a pair of dissection scissors, open the abdomen of the pregnant mice, remove the uterine horns (with the embryos) and place them in ice-cold Krebs solution (Table 1), in a 90 mm Petri dish.
6. With a pair of straight student fine forceps, open the amniotic sacs, remove the embryos and transfer them in fresh ice-cold Krebs solution, in a new 90 mm Petri dish. Dissect the mouse embryo brain (forebrain, midbrain, hindbrain) from the rest of the embryo body.
7. Holding the dissected brains from the hindbrain, transfer them in ice-cold Krebs solution in a new 90 mm Petri dish, and keep them on ice.
8. With a waterproof pen, draw a straight line at the outside surface, in the middle of the bottom part of six 35 mm Petri dishes.
9. Place one 4% low-gelling agarose/PBS 20 mL aliquot at 37 °C for 5 min. Immediately afterwards poor 10 mL in two 35 mm Petri dishes and embed the dissected brains.
   NOTE: The olfactory bulbs should face downwards (bottom of the Petri dish). Embed 3-4 brains per Petri dish, align them in the straight line previously drawn. Leave 3−5 mm space between each two brains.
10. Repeat steps 1.8 and 1.9 until all dissected brains have been embedded.
11. Place the embedded brains at 4 °C, in order for the agarose to solidify, and subsequently carve the three brains into a single block of appropriate size and orientation.
    NOTE: Leave approximately 3 mm around the edges of the brain samples. Change the orientation of the brains, with the olfactory bulbs on the top.
12. Glue the block on the surface of a microtome base and using a surgical blade cut all the way through the bottom of the block, between each two brains, in order to create 3 independent blocks (each mini block containing one brain).
13. Section the blocks, in ice-cold Krebs solution, into 250 μm thick slices, using a vibrating blade microtome.
14. With a bended flat-ended micro-spatula collect only the slices containing the medial or caudal ganglionic eminences (MGE or CGE; Figure 1) and individually transfer them onto filter membranes (13 μm diameter, 8.0 µm pore size), floating on minimal essential medium (MEM, Table 1) in polystyrene center-well organ culture dishes (60 mm x 15 mm).
15. Place the dishes in a CO₂ tissue culture incubator, at 37 °C, for 1 h, and prepare for the focal electroporation.

2. Acute Brain Slice Electroporation

1. Before beginning the electroporation procedure, prepare 50 mL of 1% agarose gel in a 100 mm Petri dish. Leave the agarose gel to solidify at room temperature (RT) for approximately 30 min.
2. Prepare tiny agarose columns (1 mm diameter and 10 mm length), punched with a glass pipette (225 mm length; 2 mL capacity) and transfer them in ice-cold Krebs solutions.
   NOTE: A rubber dropper bulb suitable for 2 mL pipettes is attached to the pipette, and by pressing it the column can be released from the glass pipette to the Krebs solution.
3. With a surgical blade, cut small agarose blocks of two sizes: a small one that will fit on the surface of the electrode (see below) and a bigger one, which will be used as a base for performing the focal DNA injections into the brain slices. Transfer the agarose blocks in ice-cold Krebs solution as well.
4. Prepare the set-up for the focal injections and the acute electroporation (Figure 2).
   NOTE: For the injections, the following equipment is needed: 1) a bright field dissection stereo-microscope, 2) a pneumatic pico-pump injector, 3) a micromanipulator, 4) a magnetic stand, and 5) a steel base plate. For the acute slice electroporation, the following equipment is needed: 1) one square platinum 10 mm Petri dish electrode, 2) one square platinum 10 mm cover electrode, 3) a micromanipulator, and 4) an electroporator.
5. Focal DNA injections
   1. Prepare a DNA mixture of expression vectors: pCAGGs-IRES-GFP (control vector) + pCAGGs-hM3D(Gq)-IRES-RFP, at a concentration of 1 µg/µL for each vector and add fast green solution (stock 25 mg/mL) in a 1/10 dilution.
   2. Fill a pulled glass micropipette (0.5 mm inner diameter and 1 mm outer diameter) with 10 µL of the DNA mixture and inject small amounts (on the 25-50 nL range) into the selected region (MGE/CGE) of the slice (Figure 1 and Figure 2).
6. Acute electroporation
   NOTE: The electroporation should be performed immediately after the focal DNA injection.
   1. Place the small agarose block on the Petri dish electrode and attach the agarose column to the mobile cover electrode with the help of a flat-ended micro-spatula.
   2. Transfer the slice with its supporting membrane onto the agarose block and place the top electrode with the agarose column on top of the selected region (MGE/CGE) of the slice.
      NOTE: Charging voltages of 125 V (two pulses of 5 ms each, interval 500 ms) will yield a successful electroporation (Figure 3).
7. After the electroporation, place the slice with its supporting membrane into the dish and transfer the dish in a CO₂ tissue culture incubator, at 37 °C.
8. After 1 h, exchange the MEM medium for a basic medium appropriate for primary neuronal cultures (neuron basic medium; Table 1) and incubate the slices overnight, for approximately 18-24 h.

3. Preparation of Cell-grafts

1. Check the efficiency of the electroporation in all slices. Select only the slices where an acceptable number of fluorescent cells is observed (Figure 3).
   NOTE: Electroporate approximately 30 slices/per experiment in order to acquire the appropriate number of cells for grafting (see below).
2. Dissect the selected region (MGE/CGE) from each slice and cut the tissue into small pieces in ice cold Krebs solution, under a fluorescent dissection stereo-microscope.
3. Meanwhile, place a 1.5 mL tube with 900 µL neuron basic medium in a water bath at 37 °C.
4. Transfer the tissue pieces with a P1000 micropipettor to a 1.5 mL tube containing 500 µL of L15/DNase medium prepared by adding 100 µL of 1 mg/mL DNase stock in DMEM/F12 medium into 900 µL of L15 medium. Wash the tissue pieces by tapping.
5. Add 100 µL of DNase (stock 1 mg/mL in DMEM/F12 medium) into the 900 µL neuron basic medium.
6. Discard the L15/DNase medium and resuspend the tissue pieces in 200 µL neuron basic/DNase medium prepared in step 3.5.
7. Set a P200 micropipettor to 180 µL and mechanically dissociate the tissue pieces by pipetting up and down gently, 20-30 times, until a smooth and "creamy" suspension is obtained.
8. Add 200 µL of neuron basic/DNase medium (final total volume 400 µL) and resuspend.
9. Take a 4 µL aliquot of cells, dilute appropriately and mount on a haemocytometer.
10. Under a bright field microscope, check the efficiency of the dissociation and count the number of cells.
    NOTE: If the dissociation is successful, bright (alive) single cells and not cell-aggregates will be observed.
11. Centrifuge cell suspension from step 3.8, at 1,000 rpm, at RT, for 5 min, and subsequently, remove the supernatant from the tube and add the appropriate L15/DNase medium (usually 5-7 µL) so that the final concentration of cells will be 8 x 10⁵-1.2 x 10⁶ cells/µL.
    NOTE: During the resuspension, it is extremely important to avoid air bubbles.
12. Place the cell aliquot on ice and have additional L15/DNase medium for the injections.

4. Intracranial Injections

NOTE: The following procedures take place in a procedure room within the Animal House Facility. Since the cells will be injected directly to the brain of newborn pups without exposing the brain, aseptic conditions are kept by sterilizing the working space with 70% EtOH solution and using autoclaved glass needles. The following equipment is needed for the intracranial injections: 1) a bright field dissection stereo-microscope, 2) a micro-injector, and 3) a heating pad for mouse recovery.

1. Prepare a glass needle by pulling needles according to the manufacturer’s recommendations. Glass needles with an 80 µm outer diameter, 40 µm inner diameter, and a 30° bevel are used in this protocol.
   NOTE: As mentioned above, needles should be autoclaved before use.
2. Backfill manually the needle with biologically inert oil using a 30 G, 2 inch needle and a syringe.
3. Assemble and insert the needle to the injector unit according to the manufacturer's instructions.
4. Determine the injection settings at maximum volume (69 nL) and a relative slow rate (23 nL/s).
5. Empty the needle until the plunger is fully extended.
6. Fill the needle.
   1. Cut a small piece from a grafting tape and place it under a bright field dissection stereo-microscope. With a P10 micropipette, transfer 5 µL from the sample (cell aliquot) onto the tape, so that a spherical drop is formed.
   2. Place the tip of the needle into the sample and fill the needle (the plunger retracts and draws the sample with it).
      NOTE: Since the sample should be quite viscous, fill the needle in small steps so that the sample will equilibrate, and at a slow rate that prevents bubbles from forming. The sample should be smooth and homogeneous within the needle. If the sample is too viscous and it is not possible to fill the needle, add as much L15/DNase medium as it is required to obtain the correct viscosity. Nevertheless, this will change the concentration of the sample, and ideally should be avoided.
7. Anaesthetize new born pups (postnatal day 0-2 [P0-P2]) on ice for 2-5 min.
   NOTE: Make sure that the pup is not moving.
8. Place the anaesthetized pup under the bright field dissection stereo-microscope.
9. Perform 3-4 injections of 69 nL each in each hemisphere.
   NOTE: The injection sites are located approximately 1 mm lateral to the midline, and between 1 mm caudal to bregma and 1 mm rostral to the interaural line. The tip of the needle should be placed approximately 1 mm deep to the pial surface. After each injection, the needle is left in place for approximately 30 s and withdrawn in periods.
10. Immediately after the injections, place the pup on a heating pad with the heating at its lowest setting (37 °C). When the pup recovers transfer it to the cage with its mother.
    NOTE: Never leave the mother with no pups in the cage. The whole procedure (from removing the pup from its littermates, until it is returned) should last less than 10 min.

5. Clozapine-N-oxide Injections

1. Prepare the DREADD ligand, CNO stock solution by diluting 1 mg of CNO in 50 µL of dimethyl sulfoxide (DMSO) until the solution is translucent. Top-up to 10 mL with saline so that the final concentration of CNO is 0.1 mg/mL.
   NOTE: DMSO is toxic. Avoid using it to a concentration higher than 0.1%. Use as a control a saline solution containing the same DMSO concentration as the CNO containing solution.
2. From postnatal day 14 and for 3 constitutively days (P14-P16), in each mouse perform two intraperitoneal (I.P.) injections of CNO (CNO concentration: 1 mg/kg) or 0.05% DMSO in saline, per day, 12 h apart.
3. On the last day (P17), perform a single injection and sacrifice mice by cervical dislocation within a time window of 30 min-1 h.