Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

This protocol describes a simple and reproducible method for purifying and culturing specific types of primary neuron. These cultures are suitable for electrophysiological, morphological and survival analysis. Cultures of purified neurons can be used to study fundamentals of neuronal physiology, formation of synapses as well as how neuronal networks develop.

While we focus on cortical glutamatergic principal cells and GABAergic interneurons, this procedure can be easily modified to study any neuronal lines expressing fluorescent proteins. To prepare glass coverslips, first thaw a five milliliter aliquot of 200 micrograms per milliliter PLL solution. Dilute this stock solution to 20 micrograms per milliliter by adding 45 milliliters of pure injection grade water.

Then filter sterilize the solution into a new 50 milliliter conical tube and label this tube as PLL sterile. Next, place 100 sterile round 12 millimeter glass coverslips in the sterile PLL solution. Agitate the tube every five to 10 minutes for two to three minutes to ensure even coating.

After 40 minutes of PLL coating, take two pieces of tissue paper and lay them flat in the flow cabinet. Sterilize the paper using 70%ethanol then flatten to remove creases and leave to dry. After coating the glass coverslips for one hour with PLL, remove excess PLL solution and add sterile injection grade water.

Gently agitate the coverslips for two to three seconds to remove excess PLL. Repeat this rinsing step two more times. Remove excess water and then transfer the coverslips to the sterile tissue paper.

Once dry, transfer the coverslips to a 24 well culture plate. To prepare the cell culture solutions, measure out 12 milliliters of cell culture buffer to a 15 milliliter conical tube and label as BSA. Measure out five milliliters of cell culture buffer to a different 15 milliliter tube and label as papain.

Subsequently, incubate both tubes for 15 minutes at 37 degrees Celsius. Add 120 milligrams of BSA to the tube labeled BSA. Then invert the tube to help dissolve the solution.

Then add seven milligrams of papain to the tube labeled papain. Return both tubes to the water bath for 15 minutes. Filter sterilize the BSA solution into a fresh conical tube.

Divide the sterile BSA solution into three tubes and label each tube as BSA sterile and either one, two, or three. Now filter sterilize the papain tube and label the tube as papain sterile. Return all the tubes back to the water bath and continue to incubate at 37 degrees Celsius until use.

To prepare for tissue dissection, lay out the scalpel, scissors, forceps, and spatula required for the dissection of the hippocampus and cortex. Place two 35 millimeter Petri dishes and a 100 millimeter Petri dish containing sterile filter paper in the flow cabinet. Collect transgenic NexCre;Ai9 or vesicular GABA transporter Venus mouse pups that are to be dissected using a fluorescent lamp with appropriate excitation and emission filters to discriminate fluorescent pups from wild type liter mates.

Immediately before dissecting the animals, fill in each Petri dish with chilled sterile cell culture buffer. After dissection, carefully transfer the transgenic pup's brain to a sterile filter paper. First, dissect away the cerebellum and separate the two hemispheres.

Then carefully separate the hippocampus and cortex from each hemisphere. Transfer the dissected tissue to a 35 millimeter Petri dish containing chilled cell culture buffer. Then transfer the dissected hippocampus and cortex to the lid of another 35 millimeter Petri dish.

Using the flat edge of a scalpel blade, carefully chop the tissue in a crisscross motion until only small pieces remain. Next, transfer the chopped tissue with a small amount of papain solution from the Petri dish lid to the sterile papain tube. Incubate the tissue at 37 degrees Celsius for 25 minutes.

After papain incubation, transfer the sterile papain tube and sterile BSA tubes to the flow cabinet. Then use a one milliliter Pasteur pipette to transfer only the corticohippocampal tissue from the papain tube into the BSA tube one. In order to break up any large clumps of tissue, triturate the tissue several times using a one milliliter Pasteur pipette.

Following this, triturate the tissue seven times using a fine tip Pasteur pipette. After 30 seconds, transfer one milliliter of the lower solution and tissue from BSA tube one to BSA tube two. Triturate the tissue in BSA tube two several times using a fine tip Pasteur pipette.

After trituration, transfer one milliliter of the lower tissue and solution from BSA tube one to BSA tube three. Triturate the tissue in BSA tube three several times. After trituration, transfer all solution and tissue from tubes two and three into BSA tube one.

Triturate two to three more times and centrifuge at 3, 000 times g for three minutes. Following centrifugation, carefully remove the supernatant from the pelleted tissue and resuspend the cells using a P1000 pipette in two milliliters of complete hibernate A low fluorescence medium. Then triturate the tissue 20 times to ensure complete resuspension of the tissue solution.

Subsequently, filer the cell suspension through a 30 micrometer cell sieve into a polystyrene sample tube. Prepare cell sorting collection tubes by pipetting 300 microliters of complete hibernate A media to the required number of polypropylene tubes. For each fluorescent cell type to be sorted, choose the appropriate excitation and emission filters.

Excite Venus protein using 488 nanometer excitation wavelength and detect the emitted light through 530/40 emission filter set. Excite TdTomato protein using 531 nanometer excitation wavelength and detect the emitted light through 575/30 emission filter set. For high purity, sort brightly labeled fluorescent cells.

Following cell sorting, transfer the collected cells to two milliliter round bottom centrifuge tubes. Then centrifuge the cells at 3, 000 times g for three minutes to form a cell pellet. Resuspend the cell pellet in the required amount of pre-warmed complete NBA medium to achieve a cell density of 1, 000 cells per microliter.

To confirm the presence of dissociated cells, check the cell solution under a microscope using a 4X or a 10X objective lens. Before plating the cells, vortex at a medium speed for two to three seconds to ensure an even cell suspension. Following vortexing, quickly pipette 10 microliters of the cell suspension to the center of each coverslip.

After one hour, feed the cells with 500 microliters of pre-warmed complete NBA medium and return to the incubator at 37 degrees Celsius. To co-culture the purified neurons and glial cells, passage glial cells to the required number of cell culture inserts. This is done by plating 40, 000 glial cells in a 500 microliter droplet of complete NBA medium.

After one hour, transfer glial cell culture inserts to purified neurons. Remove excess media from the culture inserts and return the plates to the incubator for culturing. Following successful sorting, plated neurons should appear round in shape with a smooth membrane and should be seen to spare up neuroids after approximately one hour in vitro.

By seven days in vitro, although some cell death may be apparent, viable cells should be present in all culture conditions. Analysis of purified cultures reveals that both glutamatergic and GABAergic neurons were able to extend axons and dendrites from their cell bodies and retain the ability to generate action potentials in response to super threshold depolarizing current injections. Notably following purification, only GABAergic neurons received significant amount of spontaneous synaptic transmission and glutamatergic neurons received very little synaptic transmission in the absence of glial cells.

Importantly, culturing purified neurons with glial cells improves neuron growth and survival as well as promoting synaptic transmission in glutamatergic cultures. Sufficient polarizing coating of the coverslips and maintaining the physiological pH of the culture media throughout the procedure are key to ensuring good quality cell cultures. Following this protocol, it should be possible to perform RNA sequencing and protein mass spectroscopy on purified cultures allowing translational and transcriptional processes to be investigated in specific neuronal types.