This protocol shows that it's possible to generate novel neurons directly in the brain from resident glia and that these reprogrammed cells mature into subtype-specific neurons. The main advantage is the cre-dependent AAV virus that enables specific targeting of the NG2-glia and the GFP reporter that labels the reprogrammed neurons for analysis. Generating novel neurons in the brain may lead to future development for cell replacement therapies in the brain.
In our protocol we generate the parvalbumin-positive interneurons that have implications for psychiatric disorders. In vivo reprogramming can be applied to other brain areas and circuits depending on what neuronal phenotype and neurological condition you want to target. Demonstrating the procedure will be Jenny Johansson:a technician, Maria Pereira:a former PhD student, and Marcella Birtele:a PhD student in our lab.
To produce AAV5 viral vector, seed HEK293T cells with standard culture medium in five T175 flasks. When the cells reach 50 to 70%confluency, prepare the following mix for transvection. In a 50 milliliter centrifuge tube, add equal molar amounts of vector plasmid, and pDG series helper plasmid.
Add Tris-EDTA buffer to a final volume of 144 microliters then add ultrapure water to result in a total volume of 1296 microliters and mix. Next, add 144 microliters of 2.5 molar calcium chloride and mix. After that, add 1.92 milliliters of HBS to the DNA solution and vortex immediately.
Incubate at room temperature for exactly 60 seconds. Subsequently, transfer the solution to 28 milliliters of fresh cell culture medium and mix. Replace the medium in the flasks with the medium containing transvection mix.
Wait for three days, and transfer the media to waste. Add five milliliters of harvest buffer to each flask, then add another four milliliters of DPBS to each flask to rinse the remaining cells and pool with the cell solution. Centrifuge the harvested cells at 1, 000 x g for five minutes at four degrees Celsius.
After centrifugation, remove the supernatant and dissolve the pellet in 15 milliliters of lysis buffer. Freeze the 50 milliliter centrifuge tube on dry ice for 15 minutes and store in a minus 20 degrees Celsius freezer. Before use, thaw the harvested cell lysate in a water bath at 37 degrees Celsius.
In this procedure, perform AAV purification by iodixanol gradient ultracentrifugation and use ultracentrifuge ceiling tubes with centrifugation at 350, 000 x g for one hour and 45 minutes at room temperature. To extract the AAV containing phase, insert a 10 milliliter syringe with an 18 gauge needle at about two millimeters below the 40 to 60%phase border with the bevel facing upwards and withdraw. Make sure to stop before reaching the protein band after five to six milliliters of the viral vector has been extracted.
Then, purify and concentrate the diluted iodixanol gradient through an anion exchange filter, by pushing it through at a rate no faster than one drop per second. Subsequently, push three milliliters of IE buffer slowly through the filter to wash it. Next, elute the mixture into a centrifugal filter unit with one to two milliliters of elution buffer.
Add DPBS to the device to a final volume of four milliliters. Centrifuge at 2, 000 x g at room temperature until less than 0.5 milliliters of DPBS is left in the filter. Afterward, remove liquid from the bottom of the tube, refill with four milliliters of DPBS, and centrifuge again.
Repeat this step two more times, make sure that the volume of concentrated vector on the filter is about 200 microliters after the last centrifugation. Remove the 200 microliter concentrated vector using a pipette, and push it through a 0.22 micrometer filter to sterilize. Next, aliquot 200 microliters into a glass vial with interlocked insert.
To inject reprogramming factors, place an anesthetized mouse in the stereotaxic frame. Administer appropriate analgesia at the beginning of the surgery, then bring the tip of the glass capillary of the injection needle just above bregma. Make sure the capillary tip is perfectly straight in both A-P and M-L planes.
Reset both the M-L and A-P values to 0.0 on the digital coordinate counter. To make sure the head of the animal is in a perfectly flat position, use the digital coordinate counter to measure the D-V coordinate value when the A-P arm is at plus 2.0 and minus 2.0 as well as when the M-L arm is at plus 2.0 and minus 2.0. Adjust the height of the tooth bar and ear bars accordingly.
Afterward, raise the syringe slightly and drill a hole using a dental drill at the injection coordinates. Start to drill at the site, working in a circular and gentle manner. Then, place a piece of cotton gauze over the open incision and flush the syringe with saline solution.
After flushing, draw up an air bubble and then one microliter of solution containing the viral vector. Make sure that the viral solution can be easily visualized below the air bubble. Next, lower the syringe, progressing slowly to the desired depth, and be sure that the trajectory is clear of bone fragments.
Subsequently, inject one microliter of the viral solution at a rate of 0.4 microliters per minute. When the injection is done, allow two minutes for diffusion before syringe withdrawal. After diffusion, slowly retract the syringe until the tip of the capillary is completely withdrawn from the brain, then carefully suture the incision and remove the animal from the stereotaxic frame.
Monitor the animal in a postoperative station until consciousness is regained. Animals are kept for up to 12 weeks after virus injection to allow resident glia to reprogram into mature neurons. To prepare brain slices for electrophysiology using a vibratome, section the brain from the most rostral part down to the striatal level at high speed.
Then section the striatum coronally at 275 micrometers at 0.1 millimeters per second. After each section, carefully remove the non-injected striatal side and transfer the injected side into a vial with a bottom net and oxygenized CREB sense slide in the water bath at room temperature. Keep the vial at room temperature until all the sections are cut.
Afterward, slowly increase the temperature of the water bath to 37 degrees Celsius and leave it for 30 minutes, then turn off the heater and let it cool down to room temperature. After transferring one tissue section to a recording chamber for electrophysiology, mount the glass pipette on the recording electrode and lower it into the solution. Double-check the resistance of the electrode.
Then, slowly approach the reprogrammed cell with the pipette, keeping a slight positive pressure in the electrode to avoid plugging the tip, and check that the cell is GFP positive before patching. When the cell is patched, maintain the cell in current clamp from minus 60 to minus 70 millivolts, and inject 500 millisecond currents from minus 20 to plus 90 picoamperes with 10 picoampere increments to induce action potentials. This is indicative of a neuronal maturation and successful reprogramming.
Subsequently, switch to voltage clamp and measure the inward sodium and delayed rectifying potassium currents at depolarizing steps of 10 millivolts. Here is a post recorded biocytin filled reprogrammed neuron which shows mature neuronal morphology and the dendritic spines. Here, electrophysiological recordings of the reprogrammed neurons show the presence of postsynaptic functional connections with spontaneous activity measures.
Traces show the inhibitory activity that is blocked with picrotoxin, a GABAA receptor antagonist and the excitatory activity that is blocked with CNQX, an AMPA receptor antagonist. The patched neurons already show postsynaptic activity at five weeks post-injection, and continue at eight and 12 weeks post-injection. The number of neurons with current induced action potentials also increases over time.
A more detailed analysis revealed several distinct firing patterns where the majority of cells show fast spiking activity similar to parvalbumin interneurons. Immunohistochemical analysis at 12 weeks further showed co-expression of the reporter GFP and the parvalbumin. Following this procedure you can examine the gene expression with patch seek technique or assess the three-dimensional synaptic connectivity with monosynaptic tracing and iDISCO.
Now that it's possible to reprogram resident glia into parvalbumin expressing interneurons, we have started to investigate whether these are authentic and can be used as a therapeutic tool. Remember to follow the established protocols and guidelines when handling animals and using AAV virus.
