This protocol was originally published by Dr.Gary Bunker and his colleagues, and they allow for the long density and the high purity culture of embryonic loading hippocampal neurons, by suspending the neurons over a glial fitter layer. This result is at the pan dangers of our other hippocampus crucial results, because it enables neurons to grow at low densities with few contaminating glial cells. This result is therefore ideal for high resolution imaging and functioning asset of typography and mature neurons in culture.

The post-maturation of this protocol should expedite the adaption by new life because there are several key states during the procedure that are difficult to describe, so it takes you along. This protocol consists of three independent populations that are required for culturing high quality, primary neurons. These processes include glial cell culture, creating and coating of cover slips, and the preparation of hippocampal nerve cells.

To begin, early post-natal rat cortical tissue should be obtained as described in the written protocol. While not directly shown here, note that the dissection step shown later in this protocol are nearly identical to those used for the cortical dissection. For this reason, relevant steps will be highlighted during the hippocampal dissection procedure shown later in this video.

Beginning with Step Six of the written protocol, remove the HBSS from the collected cortical tissue, and then mince the tissue as finely as possible using scissors. Add enough HBSS to the chopped tissue to transfer the tissue pieces to a 50 milliliter conical tube, and then bring the total volume to 12 milliliters by adding HBSS. Add 1.5 milliliters each of 2.5%trypsin, and 10 milligram per milliliter DNA solution, invert to mix, then incubate the resulting mixture in a 37 degree Celsius water bath for 12 minutes.

Triturate the tissue 10 to 15 times using a glass Pasteur pipette, and then incubate it in the water bath for another three minutes. Ensure that any tissue pieces have settled to the bottom of the tube, then filter the supernatant into a 50 milliliter conical tube containing 5 milliliters of BGS. Add 13.5 milliliters of HBSS, and 1.5 milliliters of 2.5&trypsin to the tube containing the remaining pieces of undigested tissue, invert to mix, then incubate it in the water bath for an additional 10 minutes.

After incubation, triturate the renaming tissue 10 to 15 times, then filter it into the tube containing the initial filtrate. Next, rinse the filter by passing through it an additional 3 milliliters of BGS. At this point, the filtrate can be divided into multiple tubes to simplify both balancing and re-suspension of the pellet it cells.

Centrifuge the suspensions for six minutes at 130 G.After centrifugation, note that the bottom of the pellet appears slightly reddish, due to the presence of blood components. Remove supernatants, then add 1 milliliter of glial medium to each tube, taking care not to disturb the pellet. Transfer the portions of each pellet that do no have this reddish appearance to a fresh 50 milliliter conical tube.

Add up to 2 milliliters of glial medium per pup to re-suspend the combined tissue, and transfer each 2 milliliters of the suspension into 18 milliliters of pre-warmed glial medium in a 75 centimeter squared culture flask. Incubate the flask in a 37 degrees Celsius, 5%CO2 incubator. The day after seeding the cells, change the medium in each flask to fresh glial medium.

Note that at this time, the culture will be a mix of astrocytes and other non-target cell types. To remove non-astrocyte cell types, flasks must be forcefully agitated approximately four days after being seeded. To do this, rinse the flasks with HBSS, then add 10 milliliters of fresh HBSS to each flask and shake each vigorously five to 10 times.

Aspirate off the HBSS, and rinse twice more with HBSS. Then add 20 milliliters glial medium, and return the flasks to the incubator as before. Note that while this is vigorous, the astrocytes desired for culture will remain strongly adhered to the flask surface.

To maintain the astrocyte cultures, change the media to fresh glial medium twice a week until they are confluent. Once confluent, cells can be frozen for future use as described in the written protocol. Glia must be plated into dishes at least 14 days prior to the neuron culture initiation as described in the written protocol.

Submerge cover slips in concentrated nitric acid and sonicate them for 30 minutes at room temperature. Be careful, as the concentrated nitric acid is extremely caustic. Note also that some labs prefer to soak the cover slips in nitric acid overnight using ceramic staining racks.

Carefully discard the nitric acid in the chemical fume hood as per institutional guidelines, then rinse the cover slips three times with deionized water. Submerge the cover slips in water, and sonicate them again for 30 minutes. Next, remove the water and rinse the cover slips another three times.

Submerge the cover slips in 50 milliliters of water, and then autoclave them. In a sterile laminar flow hood, remove the water, and add 20 to 30 milliliters of ethanol to rinse. Add another 20 to 30 milliliters of ethanol, and then transfer the cover slips and ethanol to a 100 millimeter Petri dish.

Using blunt tweezers, transfer the color slips to 60 milliliter Petri dishes, four to five per dish, then allow them to air dry by leaning the cover slips against the edge of the dish. Once the ethanol has evaporated, return the cover slips to horizontal positions. Melt 10 grams of paraffin in a heat-resistant glass bottle, and maintain it at about 150 to 200 degrees Celsius.

The ideal temperature may take some tweaking, and incorrect temperatures can lead to difficulty producing the correct size of paraffin feet. Dip a Pasteur pipette into the paraffin, then quickly touch it to three spots near the edge of each cover slip. Paraffin drops will solidify into beads approximately 3 to 5 millimeters high, and 1 to 2 millimeters in diameter, and will function to provide a space between the neurons on the cover slip and the glial-mono layer below.

Obtain dishes containing previously prepared cover slips with paraffin feet. Add 200 microliters of poly-l-lysine and borate buffer to the surface of each cover slip, and let the solution sit on the cover slips overnight, covered at room temperature. The surface being coated should be the same one on which the paraffin beads are situated.

If properly cleaned, the poly-l-lysine should spread easily to cover the surface of the cover slip. The next morning, wash the cover slips twice by soaking them in sterile water for two hours each time. After the final wash, replace the water with 4 milliliters of plating medium per dish.

Note that it is important that the surface of the cover slips not be allowed to dry after having been coated with poly-l-lysine. Place the cover slip dishes in a 37 degree Celsius incubator. The coated cover slip should be incubated for at least 24 hours before the initiation of the neuron culture.

Obtain an embryonic day 18 or 19 pregnant rat, and euthanize by an approved method. Spray the underside of the rat with 70%ethanol, then make a midline incision from the pubic region through to the end of the abdominal cavity. To avoid contamination, take care to cut only through the skin at this step.

Sterilize the dissection instruments using 70%ethanol, then cut through the abdominal wall to expose the uterus. Remove the two uterine horns, and place them in a sterile dish. In a sterile laminar flow hood, remove the uterine membranes surrounding the fetuses, then decapitate them and place the heads in a 100 millimeter Petri dish containing 20 milliliters of HBSS.

Dissect out the brains, and immediately place them in a 60 millimeter Petri dish containing 2 milliliters of HBSS on ice. Collect only two to three brains per dish to allow sufficient space for the next step. Under a dissecting microscope, strip away the meninges from the midline of the cerebral hemispheres.

Next, dissect out the hippocampi. Note that the hippocampus can be distinguished as a crescent shaped structure in the medial surface of the cortical hemisphere, and is easy to remove as it is bordered laterally by a ventricle. Once freed, place the hippocampi in a 60 millimeter Petri dish containing 4.5 milliliters of HBSS and keep on ice.

Note that the steps taken up to this point also hold for the cortical dissection, except in that the cortices are kept as opposed to hippocampi. For the cortical dissection, the next step would be to separate the cortex from the rest of the brain, as shown here, and then collect the cortices and HBSS as shown previously. Once all hippocampal tissue has been collected in HBSS, transfer it to a 15 milliliter conical tube.

Add 5 milliliters of 2.5%trypsin, then incubate it at 37 degrees Celsius for 10 minutes. Gently remove the trypsin solution by Pasteur pipette, leaving the hippocampal tissue undisturbed at the bottom of the tube. Wash the tissue twice by gently adding 5 milliliters of HBSS, incubating at 37 degrees Celsius for five minutes, and then removing it again by Pasteur pipette, ensuring not to disturb the tissue at the bottom.

While waiting for the previously shown incubation step, prepare two variants of flame-polished pipettes for tissue dissociation. To do this, briefly expose the tip of the glass Pasteur pipette to a flame, then examine the tip of the pipette. For the first variant, repeat this step until the edges of the pipette tip are smoothened, but the diameter remains unchanged.

For the second, repeat until the edges are smoothed and the tip diameter has been reduced by about half. It is important to practice to find the ideal diameter to ensure that the tissue is dissociated to yield single cells without damaging these cells in the process. After the second wash, bring the total volume to 5 milliliters by adding a sufficient volume of HBSS to the tissue.

Triturate the hippocampal tissue first by about 10 passes through the smooth-edged normal pipette, then with a further five to 10 passes through the reduced tip diameter pipette. The goal here is to obtain a homogenous solution of cells with no, or very few, tissue clumps. Determine the cell density using a hemocytometer.

Typical yields are about 800, 000 to one million cells per pup. Transfer a sufficient volume of the cell suspension to dishes containing poly-l-lysine coated cover slips in 4 milliliters of plating medium, to obtain a plating density of between 100, 000 to 500, 000 cells per 60 millimeter dish, depending on the intended experiment. Place the dishes in a 37 degree Celsius 5%CO2 incubator.

After three to four hours, transfer the cover slips with neurons attached to dishes containing glial cells and NBG medium. This should be done such that the side on which the neurons were plated is facing down towards the glial cell layer. Add a total of four or five cover slips per glia dish.

Two or three days after plating, add the aerocele solution to a final concentration of five micromolar per dish to arrest glial cell growth. Feed cells once a week by replacing 2 milliliters of the old medium with 2.5 milliliters of fresh medium at each feeding. The extra medium added is to compensate for evaporation over time.

And only a portion of the medium is changed to ensure consistent conditioning of the medium by the glial cells. These cultures are typically healthy for at least one month. Neurons were fixed with 4%paraformaldehyde, then incubated in primary antibodies for fluorescence imaging.

As seen by the Tau and MAP2 immuno-stains, these neurons develop extensive arborization with well-developed axons and dendrites. Mature neurons also develop synaptic spines as identified by the selective localization of Drebrin One, that are closely opposed to pre-synaptic specializations as identified by synapse in one immuno-stating. These primary neurons are well-suited for functional and structural studies of neurons and synapses, including studies of signal transduction, neuronal polarity, subcellular trafficking, and synapse development and plasticity, among others.

This video should assist in the understanding and accurate execution of several key steps several to this protocol. Proper adherence to the method described should result in healthy, long density neuron culture with extensive arborization and morphologically and functionally well developed synapses.