This work was supported by CIHR MOP-142209 to TJS.

All experiments and protocols using laboratory animals were approved by the University of Manitoba animal ethics committee and were compliant with the guidelines of the Canadian Council on Animal Care.
1. Preparation of Instruments, Buffers and Solutions
<ol>
	<li>Sterilize by autoclaving all dissection equipment, glass Pasteur pipettes, pipette tips, filter apparatus, and deionized water.</li>
	<li>Prepare a 20% (w/v; 1.1 M) stock glucose solution in deionized water and filter-sterilize. Store at 4 &#176;C.</li>
	<li>Prepare a 100 mM sodium pyruvate solution in deionized water and filter-sterilize. Store at 4 &#176;C.</li>
	<li>Prepare calcium- and magnesium-free Hank&#39;s Balanced Salt Solution (CMF-HBSS) by making a solution of 10% HBSS (10x), 10 mM HEPES, and 100 U/mL penicillin-streptomycin in deionized water. Store at 4 &#176;C for no more than 1 to 2 months.</li>
	<li>Prepare 10 mg/mL deoxyribonuclease I (DNase) in CMF-HBSS, and then filter-sterilize and store at -20 &#176;C.</li>
	<li>Prepare glial medium by making a solution of 30 mM glucose (from the sterile solution), 95 U/mL penicillin-streptomycin, and 10-15% bovine growth serum (BGS) in Minimum Essential Medium (MEM). 
	NOTE: As indicated in the protocol, a solution of 5% BGS can be used if required to slow cell proliferation. Horse Serum may be used instead of BGS, but note that each batch must be tested before use due to inter-batch variation. Store the prepared solution at 4 &#176;C for no more than 1 to 2 months. Though many laboratories use FBS, we consistently observed that glial growth in BGS is as robust as in FBS. BGS is derived from fetal calf serum which is supplemented with chemically defined components. In addition, BGS is substantially more economical than FBS.</li>
	<li>Prepare the neuron growth and maintenance medium (neurobasal-B27, NBG) by making a solution of 1x B27 supplement and 0.5 mM L-Glutamine in 500 mL Neurobasal Medium. Store at 4 &#176;C for no more than 1 to 2 months. L-Glutamine can be substituted with GlutaMAX, which has been suggested to be more stable in the growth medium.</li>
	<li>Prepare the neuron plating medium by making a solution of 30 mM glucose (from the sterile solution), 1 mM sodium pyruvate (from the sterile solution), and 10% BGS in MEM. Store at 4 &#176;C for no more than 1 to 2 months.</li>
	<li>Prepare a solution of 1 mM Cytarabine (Ara-C) and filter-sterilize. Aliquot into 1 mL portions and store at -20 &#176;C.</li>
	<li>Prepare a solution of 100 mM APV in 100 mN filter-sterilized NaOH. Store at -20 &#176;C.</li>
	<li>Dissolve 1 mg/mL poly-L-lysine in borate buffer, pH 8.5 (50 mM boric acid and 12 mM borax) and filter-sterilize. Aliquot and store at -20 &#176;C. Instead of poly-L-lysine, one may use poly-L-ornithine which may be less toxic to cells.</li>
</ol>
2. Glia Culture Preparation
<ol>
	<li>Prepare cortical astroglia cell culture
	<ol>
		<li>Obtain rat pups within 24 h of birth. Place the pups on ice to anaesthetize them.</li>
		<li>Spray the pups with 70% ethanol and decapitate them.</li>
		<li>Place the heads in a 100 mm dish on ice containing 15 mL CMF-HBSS.</li>
		<li>Dissect out each brain by cutting the scalp, opening the skull, severing the brainstem, and then transferring the brain to 60 mm Petri dishes on ice containing 3 mL CMF-HBSS.</li>
		<li>Separate the cerebral hemispheres from the hippocampi and then strip away the meninges surrounding them. Exercise adequate precaution to ensure minimal contamination from meningeal fibroblasts. Meningeal fibroblasts in the culture can compete with glia for growth and be detrimental for neuron health. Collect all the cerebral hemispheres in a 60 mm Petri dish on ice containing 5 mL CMF-HBSS.</li>
		<li>Remove the CMF-HBSS and then mince the collected cortical tissue as finely as possible using micro-dissecting spring-scissors.</li>
		<li>Add sufficient CMF-HBSS to the chopped tissue. Using a glass Pasteur pipette, transfer the tissue pieces to a 15 mL tube. Bring the total volume to 12 mL by adding CMF-HBSS.</li>
		<li>Add 1.5 mL each of 2.5% trypsin and 10 mg/mL DNase solution. Incubate the resulting mixture at 37 &#176;C in a water bath for 12 min.</li>
		<li>Triturate the tissue 10-15 times using a glass Pasteur pipette, and further incubate it in a 37 &#176;C water bath for 3 min.</li>
		<li>Filter the supernatant through lens paper or a filter mesh into a 50 mL tube containing 5 mL of BGS. This is done to remove chunks of undissociated tissue. Alternatively, use commercially available cell strainers.</li>
		<li>Add 13.5 mL of CMF-HBSS and 1.5 mL of 2.5% trypsin to the tube containing the remaining tissue pieces and further incubate it at 37 &#176;C in a water bath for an additional 10 min.</li>
		<li>Triturate the remaining tissue once more, and then filter the suspension again into the 50 mL tube containing the initial filtrate. Next, rinse the lens paper with 3 mL of BGS. The final volume should be approximately 38 mL.</li>
		<li>Centrifuge the suspension containing the dissociated glial cells for 6 min at 130 x g. Carefully discard the supernatant using a glass Pasteur pipette. Ensure that the pelleted cells at the bottom are not disturbed. The bottom of the pellet appears slightly reddish containing blood components.</li>
		<li>Transfer the glial cells in 1 mL of glial medium into a fresh tube, taking care not to disturb the reddish portion of the pellet.</li>
		<li>Add up to 2 mL of glial medium per pup to resuspend the combined dissociated glial cells. For example, if 10 pups were used, and then the total resuspension volume would be 20 mL.</li>
		<li>Prepare one 75 cm2 cell culture flask per pup. Add 18 mL pre-warmed glial medium to each flask.</li>
		<li>Transfer 2 mL of cell suspension to each flask and incubate in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>After one day, replace with 20 mL glial medium. At this stage, the culture is a mix of glial and other unwanted cell-types.</li>
		<li>Four days after seeding the cells, vigorously shake the flasks to dislodge non-astrocyte cells. To do this, rinse the flasks once with CMF-HBSS, and then add 10 mL fresh CMF-HBSS to each flask. Shake the flasks vigorously 5-10 times, and then aspirate off the CMF-HBSS. Rinse twice with CMF-HBSS, and then add 20 mL glial medium. 
		NOTE: This step is crucial to ensure the removal of non-target cell types, and should not result in the loss of the desired astrocytes.</li>
		<li>Replace with fresh glial medium twice a week until the cells are confluent (Figure 1).</li>
	</ol>
	</li>
	<li>Freezing glia
	<ol>
		<li>Once the cultures are confluent, aspirate off the medium and wash the cell monolayer with 10 mL CMF-HBSS.</li>
		<li>Add 10 mL of 0.25% trypsin/EDTA to each flask and incubate at 37 &#176;C for 3 min.</li>
		<li>Tap flasks gently to dislodge the cells. Add 1 mL BGS to each flask and transfer the cell suspensions into 15 mL tubes.</li>
		<li>Centrifuge the tubes at 130 x g for 6 min. Discard the resulting supernatant.</li>
		<li>Resuspend the cell pellet in 2 mL glial medium.</li>
		<li>Prepare the glial freezing medium (1-part dimethyl sulfoxide and 4-parts BGS). Add 0.5 mL of this solution to each of 4 cryogenic vials per flask.</li>
		<li>Transfer 0.5 mL of cell suspension to each vial, and then freeze the cells slowly by placing the vials in an insulated container in a -80 &#176;C freezer overnight. After one day, transfer the vials to a liquid nitrogen freezer for long-term storage.</li>
	</ol>
	</li>
	<li>Plating revived glia
	<ol>
		<li>Plate glia 1-2 weeks before the neuron culture day.</li>
		<li>Prepare a 50 mL tube with 10 mL warmed glial medium.</li>
		<li>Thaw 1 frozen vial of glia in a 37 &#176;C water bath for 2-3 min.</li>
		<li>Transfer the contents of the vial into the 50 mL tube containing 10 mL glial medium.</li>
		<li>Centrifuge the tube for 5 min at 130 x g and aspirate off the supernatant.</li>
		<li>Resuspend the pellet in 20 mL glial medium.</li>
		<li>Add 3 mL fresh glial medium to each of the culture dishes (60 mm), and then transfer 1 mL of the cell suspension to each dish. Incubate the cultures in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>Feed the cells twice a week. If the cell density reaches 50% confluence well before the neuron culture day, switch to glial medium containing 5% BGS to slow the glial growth rate. The desired confluence of the glial monolayer for neuron culture is 50% - 70%.</li>
		<li>Replace the glial medium with NBG medium (6 mL/dish) 1-2 days before the neuron culture.</li>
	</ol>
	</li>
</ol>
3. Coverslip Preparation
<ol>
	<li>Cleaning and Preparation of Paraffin Beads
	<ol>
		<li>Submerge coverslips in concentrated nitric acid (70% wt/wt; CAUTION: highly corrosive) and sonicate them for 30 min at room temperature. Other laboratories have found it beneficial to incubate the coverslips in 70% nitric acid in ceramic racks for 12-24 h at room temperature instead.</li>
		<li>Carefully discard the nitric acid in a designated chemical fume hood in accordance to institutional guidelines. Rinse the coverslips 3-5 times with deionized water.</li>
		<li>Submerge the coverslips in deionized water and sonicate them again for 30 min (skip if following the alternative method).</li>
		<li>Remove the deionized water and rinse the coverslips another three times.</li>
		<li>Submerge the coverslips in 50 mL deionized water and sterilize them by autoclaving. Alternatively, sterilize the coverslips at 225 &#176;C in an oven for 6 h. If using this alternative approach, skip to step 3.1.9.</li>
		<li>Once autoclaved, transfer the coverslips to a sterile laminar flow hood for the following steps.</li>
		<li>Remove the deionized water and rinse with 20-30 mL of 100% ethanol.</li>
		<li>Add 20-30 mL of 100% ethanol and transfer the coverslips and ethanol to a 100 mm Petri dish.</li>
		<li>Use blunt tweezers to transfer coverslips to 60 mm Petri dishes, 4-5 coverslips per dish, and then allow them to air-dry by leaning the coverslips against the edge of the dish. Once the ethanol has evaporated, lay the coverslips flat on the surface.</li>
		<li>Transfer 10 g paraffin to a suitable heat-resistant glass bottle. Melt the paraffin and maintain it at about 150-200 &#176;C on a hot plate for at least 1 h. The ideal temperature to which the paraffin should be heated may take some tweaking. Non-ideal temperatures can lead to difficulty producing the correct size of the paraffin beads. The waiting period after melting helps to ensure consistent temperature throughout the liquid.</li>
		<li>Dip a Pasteur pipette into the paraffin, and then quickly touch it to three spots near the edge of each coverslip (each spot being approximately 120&#176; apart). The drops will solidify into beads approximately 0.3-0.5 mm high and 1.0-2.0 mm in diameter and will function to provide a space between the neurons on the coverslip and the glial monolayer below (Figure 2).</li>
		<li>Sterilize the dishes under UV light for 30 min, and then cover them with aluminum foil and store at room temperature. Prepared coverslips can be stored and used for up to one month. Paraffin beads should be allowed to adhere to the coverslips for at least one week before use. This will prevent the paraffin from detaching during the remaining steps of the protocol.</li>
	</ol>
	</li>
	<li>Coating Coverslips with Poly-L-Lysine
	<ol>
		<li>Obtain Petri dishes containing previously prepared coverslips with the paraffin beads.</li>
		<li>Add 200 &#181;L of poly-L-lysine in borate buffer to the surface of each coverslip and let the solution sit on the coverslips overnight, covered, at room temperature. Coat the same surface as the one on which the paraffin beads are situated. Note that if properly cleaned, the poly-L-lysine should spread easily to cover the surface of the coverslip.</li>
		<li>After one day, wash the coverslips twice by soaking them in sterile deionized water for 2 h each time. After the final wash, replace the water with 4 mL of neuronal plating medium per dish. Note that it is important that the surface of the coverslips not be allowed to dry after having been coated with poly-L-lysine.</li>
		<li>Place the coverslip-containing dishes in a 37 &#176;C, 5% CO2 incubator. The coated coverslips should be incubated for at least 24 h before the neuron culture. This is done to prime the medium for neuron culture.</li>
	</ol>
	</li>
</ol>
4. Dissection of Hippocampus and Plating Neurons
<ol>
	<li>Obtain a pregnant rat (embryonic day 18-19, as measured from the day of vaginal plug discovery) and euthanize by isoflurane-anesthetized decapitation or other approved method.</li>
	<li>Spray the underside of the rat with 70% ethanol, and then make an 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.</li>
	<li>Rinse the dissection instruments again with 70% ethanol, and then cut through the abdominal wall to expose the uterus. Remove the two uterine horns and place them in a sterile 100 mm Petri dish.</li>
	<li>Perform the remaining steps in a laminar flow hood. Remove the uterine membrane surrounding the fetuses. Decapitate them and place the heads in a 100 mm Petri dish containing 20 mL CMF-HBSS.</li>
	<li>Dissect out the brains and immediately place them in a 60 mm Petri dish containing 2 mL CMF-HBSS on ice. Collect only 2-3 brains per dish to allow sufficient space for the next step.</li>
	<li>Under a dissecting microscope strip away the meninges from the midline of the cerebral hemispheres, and then dissect out the hippocampi and place them in a 60 mm Petri dish containing 4.5 mL CMF-HBSS. 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.</li>
	<li>Transfer the collected hippocampal tissue and CMF-HBSS to a 15 mL tube. Add 0.5 mL of 2.5% trypsin, and then incubate at 37 &#176;C for 10 min. Papain may be used instead of trypsin because it is gentler and may increase yield.</li>
	<li>Gently remove the trypsin solution by Pasteur pipette, leaving the hippocampal tissue undisturbed at the bottom of the tube.</li>
	<li>Gently wash the tissue twice with 5 mL CMF-HBSS while incubating it at 37 &#176;C for 5 min. After the second wash, bring the total volume to at least 5 mL by adding a sufficient volume of CMF-HBSS to the tissue. If there are hippocampi from more than 10 pups, up to 8 mL CMF-HBSS can be added.</li>
	<li>Prepare two variants of fire-polished pipettes for tissue dissociation. The tip of one variant is of the normal diameter of the pipette but with fire-polished smoothened edges. The other variant is one with the tip diameter reduced by half. Briefly expose the tip of a glass Pasteur pipette diagonally to a flame source, and then examine the tips of the pipettes.
	<ol>
		<li>Repeat this step until the tip edge had been smoothened or the diameter has been reduced. 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.</li>
	</ol>
	</li>
	<li>Triturate the hippocampal tissue first by about 10 passes through a smooth-edged normal glass Pasteur pipette, and then a further 5 to 10 passes through the pipette with the reduced diameter. The goal is to obtain a homogenous solution of cells with no or very few tissue clumps.</li>
	<li>Determine the cell density using a hemocytometer or an automated cell counter. Typically, 0.8 to 1.0 million cells per pup are obtained when hippocampi from the two hemispheres are combined.</li>
	<li>Transfer a sufficient volume of the cell suspension to dishes containing poly-L-lysine-coated coverslips in 4 mL plating medium to obtain a plating density of 300,000 cells per 60 mm dish. The density of the cells can vary from 100,000 to 500,000, depending upon the intended experiment.</li>
	<li>After 3-4 h, transfer the coverslips with neurons attached to dishes containing glial cells in 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 4-5 coverslips per glia dish.</li>
	<li>Two or three days after plating, add the Ara-C solution to a final concentration of 5 &#181;M per dish to arrest glial growth.</li>
	<li>Feed the cells once a week by replacing 2 mL of the old medium with 2.5 mL 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 (Figure 3). Neuron survival can be improved by adding APV to the culture medium to a final concentration of 100 &#181;M. APV is an NMDA receptor antagonist and promotes survival by reducing glutamate excitotoxicity.</li>
</ol>

<ol><li>Banker, G. A., Cowan, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rat+hippocampal+neurons+in+dispersed+cell+culture%5BTitle%5D)+AND+%22Brain+Res%22%5BJournal%5D)">Rat hippocampal neurons in dispersed cell culture.</a> Brain Res. 126, 397-425 (1977).</li>
<li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Culturing+hippocampal+neurons%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li>
<li>Adamantidis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optogenetics%3A+10+years+after+ChR2+in+neurons--views+from+the+community%5BTitle%5D)+AND+%22Nat+Neurosci%22%5BJournal%5D)">Optogenetics: 10 years after ChR2 in neurons--views from the community.</a> Nat Neurosci. 18, 1202-1212 (2015).</li>
<li>Ho, V. M., Lee, J. A., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+cell+biology+of+synaptic+plasticity%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">The cell biology of synaptic plasticity.</a> Science. 334, 623-628 (2011).</li>
<li>Huganir, R. L., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPARs+and+synaptic+plasticity%3A+the+last+25+years%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">AMPARs and synaptic plasticity: the last 25 years.</a> Neuron. 80, 704-717 (2013).</li>
<li>Luscher, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+AMPA+receptor+cycling+in+synaptic+transmission+and+plasticity%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Role of AMPA receptor cycling in synaptic transmission and plasticity.</a> Neuron. 24, 649-658 (1999).</li>
<li>Varoqueaux, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuroligins+determine+synapse+maturation+and+function%5BTitle%5D)+AND+%22Neuron%22%5BJournal%5D)">Neuroligins determine synapse maturation and function.</a> Neuron. 51, 741-754 (2006).</li>
<li>Huettner, J. E., Baughman, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+culture+of+identified+neurons+from+the+visual+cortex+of+postnatal+rats%5BTitle%5D)+AND+%22J+Neurosci%22%5BJournal%5D)">Primary culture of identified neurons from the visual cortex of postnatal rats.</a> J Neurosci. 6, 3044-3060 (1986).</li>
<li>Lester, R. A., Quarum, M. L., Parker, J. D., Weber, E., Jahr, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Interaction+of+6-cyano-7-nitroquinoxaline-2%2C3-dione+with+the+N-methyl-D-aspartate+receptor-associated+glycine+binding+site%5BTitle%5D)+AND+%22Mol+Pharmacol%22%5BJournal%5D)">Interaction of 6-cyano-7-nitroquinoxaline-2,3-dione with the N-methyl-D-aspartate receptor-associated glycine binding site.</a> Mol Pharmacol. 35, 565-570 (1989).</li>
<li>Brewer, G. J., Torricelli, J. R., Evege, E. K., Price, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimized+survival+of+hippocampal+neurons+in+B27-supplemented+Neurobasal%2C+a+new+serum-free+medium+combination%5BTitle%5D)+AND+%22J+Neurosci+Res%22%5BJournal%5D)">Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination.</a> J Neurosci Res. 35, 567-576 (1993).</li>
<li>Banker, G., Goslin, K. Rat hippocampal neurons in low-density culture. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aGoslin%2C+K+%22Culturing+nerve+cells%22">Culturing nerve cells</a>. 2, 339-370 (1998).</li>
<li>Hanisch, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Microglia+as+a+source+and+target+of+cytokines%5BTitle%5D)+AND+%22Glia%22%5BJournal%5D)">Microglia as a source and target of cytokines.</a> Glia. 40, 140-155 (2002).</li>
</ol>

The authors have nothing to disclose.

While the &#34;sandwich&#34; method of culturing neurons has been well-described elsewhere 2,11, there are several steps throughout the protocol that are quite difficult to describe in text alone, which can lead to frustration for investigators who wish to adopt it.
The method can be divided into three broad workflows: glial culture, coverslip preparation and neuron culture and maintenance. Each of the three preparations are critical f...

The brain is organized into intricate networks of neurons. The contribution of individual neurons to network activity and brain function can be studied by selective alteration of their molecular composition and perturbance of their physiological properties. Genetic and chemical manipulation of individual neurons is arguably easier in cultured neurons than in intact brain tissue, unencumbered by the latter&#39;s cellular heterogeneity and complexity. Neurons in culture develop well-defined axonal and dendritic arbors and form extensive synaptic connections with each other.
While neuron culture from adult animals or from other regions of the nervous system is possible, embryonic hippocampal cultures are frequently preferred due to their defined pyramidal cell population and relatively low glial density 1,2. Hippocampal neurons grown at low density in culture are particularly amenable to the study of subcellular localization, protein trafficking, neuronal polarity and synapse development. Neurons in culture have also been extensively employed in studying molecular processes in synaptic plasticity 3,4,5,6. Neuron culture preparations from mice with global genetic deletions that do not survive postnatally have been especially useful in studying cellular and synaptic roles of certain genes 7.
As in the brain, cultured hippocampal neurons are dependent on trophic support from glial cells. This complicates their culture, and has led to the development of several different methods by which this support is supplied. One commonly used method involves plating neurons directly onto a monolayer of glial cells 8, or allowing contaminant glial cells from the acquired hippocampal tissue to proliferate and form a monolayer beneath the neurons 9. While this method has found some success, the impurity of the resulting neuronal culture is disadvantageous for imaging experiments. Another commonly used method of neuron culture is to leave-out the glial feeder layer altogether, and instead provide trophic support in the form of a defined growth medium 10.
Here, we describe the &#34;sandwich&#34; or &#34;Banker&#34; method of neuron culture 2,11. This method involves plating the hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells separated by paraffin wax beads. This facilitates long-term culture of a homogenous population of neurons without contaminating glia while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays.

<table><tbody><tr><td>&lt;strong&gt;Dissection Instruments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro Dissecting Scissors</td><td>Roboz</td><td>RS-5910</td><td></td></tr><tr><td>Micro Dissecting Spring Scissors</td><td>Roboz</td><td>RS-5650</td><td></td></tr><tr><td>Micro Dissecting Spring Scissors</td><td>Roboz</td><td>RS-5605</td><td></td></tr><tr><td>Dumont Forceps (#5)</td><td>Roboz</td><td>RS-5045</td><td></td></tr><tr><td>Dumont Forceps (#PP)</td><td>Roboz</td><td>RS-4950</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tissue Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trypsin (2.5%)</td><td>Gibco</td><td>15-090-046</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Gibco</td><td>25-200-072</td><td></td></tr><tr><td>Swinnex Filter Holder, 25&amp;nbsp;mm</td><td>EMD Millipore</td><td>SX0002500</td><td>Used as cell strainer. Assemble first with filter and autoclave</td></tr><tr><td>Isoflorane</td><td>Pharmaceutical Partners of Canada Inc.</td><td>CP0406V2</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific</td><td>1492</td><td></td></tr><tr><td>Grade 105 Lens Cleaning Tissue</td><td>GE Healthcare</td><td>2105-841</td><td>Used as cell strainer. Assemble first in filter holder and autoclave</td></tr><tr><td>Glass Pasteur pipettes with cotton filter</td><td>VWR</td><td>14672-412</td><td></td></tr><tr><td>HEPES (1 M)</td><td>Gibco</td><td>15-630-080</td><td></td></tr><tr><td>Hank&#039;s Balanced Salt Solution without Calcium, Magnesium, Phenol Red (HBSS, 10x)</td><td>Gibco</td><td>14-185-052</td><td></td></tr><tr><td>Glass Pasteur pipettes</td><td>VWR</td><td>14672-380</td><td></td></tr><tr><td>Deoxyribonuclease I from bovine pancreas (Dnase)</td><td>Sigma-Aldrich</td><td>DN25-100mg</td><td></td></tr><tr><td>Butane bunsen burner</td><td>Wall-Lenk Mfg. Co.</td><td>Model 65</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5810R</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Tissue Culture&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15-140-122</td><td></td></tr><tr><td>Petri Dish (100 mm)</td><td>Fisher</td><td>FB0875712</td><td></td></tr><tr><td>Petri Dish (60 mm)</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>Horse Serum</td><td>Gibco</td><td>16050-122</td><td>Can be used in place of BGS, but each lot must be tested due to inter-lot variation. Heat-inactivation of serum is recommended.</td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>S318-1</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma-Aldrich</td><td>P2256</td><td></td></tr><tr><td>Minimum Essential Medium (MEM)</td><td>Gibco</td><td>11-095-080</td><td></td></tr><tr><td>Neurobasal Medium</td><td>Gibco</td><td>21-103-049</td><td></td></tr><tr><td>L-Glutamine (200 mM)</td><td>Gibco</td><td>25-030-081</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Gibco</td><td>35050061</td><td>Can be used in place of L-Glutamine in the NBG medium</td></tr><tr><td>Culture Dish (60 mm)</td><td>Corning, Inc</td><td>353002</td><td></td></tr><tr><td>Culture Flasks (75 cm^2)</td><td>Greiner Bio-One</td><td>658170</td><td></td></tr><tr><td>Cytarabine (Ara-C)</td><td>Sigma-Aldrich</td><td>C3350000</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>Bovine Growth Serum (BGS)</td><td>HyClone</td><td>SH3054103</td><td>Heat-inactivation is recommended before use.</td></tr><tr><td>B27 Supplement (50x)</td><td>Gibco</td><td>17-504-044</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>D8418-100ml</td><td></td></tr><tr><td>Cryogenic Vials</td><td>VWR</td><td>89094-806</td><td></td></tr><tr><td>DL-2-Amino-5-phosphonopentanoic acid (APV)</td><td>Sigma-Aldrich</td><td>A5282</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Coverslip Preparation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sodium tetraborate decahydrate (borax)</td><td>Sigma-Aldrich</td><td>B9876-1KG</td><td></td></tr><tr><td>Poly-L-Lysine Hydrobromide</td><td>Sigma-Aldrich</td><td>P2636</td><td></td></tr><tr><td>Histoplast Paraffin Wax</td><td>Fisher</td><td>22-900-700</td><td></td></tr><tr><td>Gravity Convection Oven</td><td>VWR</td><td>89511-404</td><td>Used for alternative coverslip cleaning method discussed in protocol</td></tr><tr><td>Ultrasonic Bath (Sonicator)</td><td>Fisher Scientific</td><td>15337400</td><td></td></tr><tr><td>Nitric Acid</td><td>Anachemia</td><td>62786-460</td><td></td></tr><tr><td>Ceramic Staining Racks</td><td>Thomas Scientific</td><td>8542E40</td><td>Used for alternative coverslip cleaning method discussed in protocol</td></tr><tr><td>Coverslips</td><td>Glaswarenfabrik Karl Hecht&amp;nbsp;GmbH</td><td>1001/18</td><td>Manufacturer is very important, as neurons do not adhere well to lower quality glass&amp;nbsp;</td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B0252</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Miscellaneous&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Sterile Syringe Filters</td><td>VWR</td><td>28145-477</td><td>Used with BD syringe for filter-sterilization</td></tr><tr><td>Syringe&amp;nbsp;</td><td>BD</td><td>302832</td><td>Used with VWR sterile syringe filters for fliter-sterilization</td></tr><tr><td>Water Bath</td><td>Fisher Scientific</td><td>15-460-16Q</td><td></td></tr><tr><td>Inverted Microscope</td><td>Olympus</td><td>CKX41</td><td></td></tr><tr><td>15 mL Conical Sterile Centrifuge Tubes</td><td>ThermoScientific</td><td>339650</td><td></td></tr><tr><td>50 mL Conical Sterile Centrifuge Tubes</td><td>ThermoScientific</td><td>339652</td><td></td></tr></tbody></table>

low-density primary hippocampal neuron culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

In this &#34;sandwich&#34; method of primary nerve cell culture, hippocampal neurons (Figure 3) grow on a bed of glial cells (Figure 1) separated by paraffin beads (Figure 2). This ensures that neurons selectively grow on glass coverslips with minimal glial cell contamination but receive adequate trophic support from glia growing on the tissue culture dish. Typically, neurons can be maintained in culture for &#62;3 weeks and develop exten...

Watch this Scientific Journal Video about Low-Density Primary Hippocampal Neuron Culture at JoVE.com

Low-Density Primary Hippocampal Neuron Culture

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

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Research

JoVE Journal

Neuroscience

20.7K Views.  University of Manitoba. This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

Video: Low-Density Primary Hippocampal Neuron Culture

Nucleofection and Primary Culture of Embryonic Mouse Hippocampal and Cortical Neurons

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and synapse formation and function. An advantage of culturing these neurons is that they readily polarize, forming distinctive axons and dendrites, on a two dimensional substrate at very low densities. This property has made them extremely useful for determining many aspects of neuronal development. Furthermore, by providing glial conditioning for these neurons they will continue to develop, forming functional synaptic connections and surviving for several months in culture. In this protocol we outline a technique to dissect, culture and transfect embryonic mouse hippocampal and cortical neurons. Transfection is accomplished by electroporating DNA into the neurons before plating via nucleofection. This protocol has the advantage of expressing fluorescently-tagged fusion proteins early in development (~4-8hrs after plating) to study the dynamics and function of proteins during polarization, axon outgrowth and branching. We have also discovered that this single transfection before plating maintains fluorescently-tagged fusion protein expression at levels appropriate for imaging throughout the lifetime of the neuron (&gt; 2 months in culture). Thus, this methodology is useful for studying protein localization and function throughout CNS development with little or no disruption of neuronal function.

This protocol outlines the steps required to dissect, transfect via electroporation and culture mouse hippocampal and cortical neurons. Short-term cultures may be used for studies of axon outgrowth and guidance, while long-term cultures can be used for studies of synaptogenesis and dendritic spine analysis.

Hippocampal and cortical neurons have been used extensively to study central nervous system (CNS) neuronal polarization, axon/dendrite outgrowth, and ...

Primary Culture of Hippocampal Neurons from P0 Newborn Rats

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and memory. Primary hippocampal cell culturing allows neuroscientists to examine the activity and properties of neurons at the individual cell and single synapse level. In this video, we will demonstrate how to isolate and grow primary hippocampal cells from newborn rats. The hippocampus may be isolated from each newborn animal in as short as 2 to 3 minutes, and the cultures can be maintained for up to two weeks. We will also briefly demonstrate how to use these hippocampal neurons for ratiometric calcium imaging. While this protocol describes the process for the hippocampus, with little to no modification, it can be applied to other regions of the brain.

The dissection and growth of cells from an individual brain area facilitates investigation of cellular and physiological parameters. We describe a method for primary cell culturing that produces neuron-enriched cultures in a serum-free environment.

The physiological properties of hippocampal neurons are commonly investigated, especially because of the involvement of the hippocampus in learning and ...

Biology

Isolation and Culture of Hippocampal Neurons from Prenatal Mice

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual neurons, researchers are able to analyze properties related to cellular trafficking, cellular structure and individual protein localization using a variety of biochemical techniques. Results from such experiments are critical for testing theories addressing the neural basis of memory and learning. However, unambiguous results from these forms of experiments are predicated on the ability to grow neuronal cultures with minimum contamination by other brain cell types. In this protocol, we use specific media designed for neuron growth and careful dissection of embryonic hippocampal tissue to optimize growth of healthy neurons while minimizing contaminating cell types (i.e. astrocytes). Embryonic mouse hippocampal tissue can be more difficult to isolate than similar rodent tissue due to the size of the sample for dissection. We show detailed dissection techniques of hippocampus from embryonic day 19 (E19) mouse pups. Once hippocampal tissue is isolated, gentle dissociation of neuronal cells is achieved with a dilute concentration of trypsin and mechanical disruption designed to separate cells from connective tissue while providing minimum damage to individual cells. A detailed description of how to prepare pipettes to be used in the disruption is included. Optimal plating densities are provided for immuno-fluorescence protocols to maximize successful cell culture. The protocol provides a fast (approximately 2 hr) and efficient technique for the culture of neuronal cells from mouse hippocampal tissue.

We provide a protocol for the culture of highly purified hippocampal neurons from prenatal mouse brains without the use of a feeder glial cell layer.

Primary cultures of rat and murine hippocampal neurons are widely used to reveal cellular mechanisms in neurobiology. By isolating and growing individual ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis of primary cultures of neurons. We describe a set of procedures for: labeling newborn mice to be genotyped, rapid genotyping, and establishing low-density cultures of brain neurons from these mice. Individual mice are labeled by tattooing, which allows for long-term identification lasting into adulthood. Genotyping by the described protocol is fast and efficient, and allows for automated extraction of nucleic acid with good reliability. This is useful under circumstances where sufficient time for conventional genotyping is not available, e.g., in mice that suffer from neonatal lethality. Primary neuronal cultures are generated at low density, which enables imaging experiments at high spatial resolution. This culture method requires the preparation of glial feeder layers prior to neuronal plating. The protocol is applied in its entirety to a mouse model of the movement disorder DYT1 dystonia (&#916;E-torsinA knock-in mice), and neuronal cultures are prepared from the hippocampus, cerebral cortex and striatum of these mice. This protocol can be applied to mice with other genetic mutations, as well as to animals of other species. Furthermore, individual components of the protocol can be used for isolated sub-projects. Thus this protocol will have wide applications, not only in neuroscience but also in other fields of biological and medical sciences.

We describe procedures for labeling and genotyping newborn mice and generating primary neuronal cultures from them. The genotyping is rapid, efficient and reliable, and allows for automated nucleic-acid extraction. This is especially useful for neonatally lethal mice and their cultures that require prior completion of genotyping.

High-resolution analysis of the morphology and function of mammalian neurons often requires the genotyping of individual animals followed by the analysis ...

A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic hippocampal neurons can often grow successfully on glass coverslips at high density under serum-free conditions, but low density cultures typically require a supply of trophic factors by co-culturing them with a glia feeder layer, preparation of which can be time-consuming and laborious. In addition, the presence of glia may confound interpretation of results and preclude studies on neuron-specific mechanisms. Here, a simplified method is presented for ultra-low density (~2,000 neurons/cm2), long-term (&#62;3 months) primary hippocampal neuron culture that is under serum free conditions and without glia cell support. Low density neurons are grown on poly-D-lysine coated coverslips, and flipped on high density neurons grown in a 24-well plate. Instead of using paraffin dots to create a space between the two neuronal layers, the experimenters can simply etch the plastic bottom of the well, on which the high density neurons reside, to create a microspace conducive to low density neuron growth. The co-culture can be easily maintained for &#62;3 months without significant loss of low density neurons, thus facilitating the morphological and physiological study of these neurons. To illustrate this successful culture condition, data are provided to show profuse synapse formation in low density cells after prolonged culture. This co-culture system also facilitates the survival of sparse individual neurons grown in islands of poly-D-lysine substrates and thus the formation of autaptic connections.

Low density cultures of primary hippocampal neurons usually require glia feeder layer to supply neurotrophic factors and sustain longevity. We describe here a simplified method to culture ultra-low density neurons on glass coverslips in the presence of a high density neuronal feeder layer, which facilitates investigation of specific neuronal-autonomous mechanisms.

Culturing primary hippocampal neurons in vitro facilitates mechanistic interrogation of many aspects of neuronal development. Dissociated embryonic ...

Super-Resolution Imaging to Study Co-Localization of Proteins and Synaptic Markers in Primary Neurons

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. Imaging studies are widely used to investigate their function and plasticity in physiological and pathological conditions. Because of their size and structure, localization studies of proteins require high-resolution imaging techniques. In this protocol, we describe a procedure to study in primary neurons the co-localization of target proteins with synaptic markers at a super-resolution level using structured illumination microscopy (SIM). SIM is a patterned-light illumination technique that doubles the spatial resolution of wide-field microscopy, reaching a detail of around 100 nm. The protocol indicates the required controls and settings for robust co-localization studies and an overview of the statistical methods to analyze the imaging data properly.

This protocol shows how to employ super-resolution microscopy to study protein co-localization in primary neuronal cultures.

Synapses are the functional elements of neurons and their defects or losses are at the basis of several neurodegenerative and neurological disorders. ...

Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal neurons allows the study of individual neurons or subcellular components. We have shown subcellular protein localization within a neuron by immunocytochemistry, neuronal polarity, synaptic morphology, and its developmental change using a low-density primary hippocampal culture. Recently, ready-to-use frozen stocks of neurons have become commercially available. These frozen stocks of neurons reduce the time needed to prepare animal experiments and also contribute to the reduction of the number of animals used. Here, we introduce a reproducible low-density primary culture method using a 96-well plate. We used a commercially available frozen stock of neurons from the rat embryonic hippocampus. The neurons can be stably cultured long-term without media changes by reducing the growth of glial cells at particular timepoints. This high-throughput assay using low-density culture allows reproducible imaging-based evaluations of synaptic plasticity.

A ready-to-use frozen stock of neurons is a powerful tool for evaluating synaptic functions. Here, we introduce an easy low-density primary culture from frozen stock using a 96-well plate.

Neuronal culture is a valuable system for evaluating synaptic functions and drug screenings. In particular, a low-density culture of primary hippocampal ...

A Technique to Culture Mouse Hippocampal Neurons

Source: Yang, R., et al. <a href="https://app.jove.com/v/61411">Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments.</a> J. Vis. Exp. (2021)
The video demonstrates a method for culturing neurons from murine hippocampi. Initially, the hippocampi undergo enzymatic digestion to break down the extracellular tissue matrix. Subsequently, the digested tissue is mechanically disrupted to isolate individual neurons. Finally, these neurons are cultured on glial feeder cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparing glia cell feeder dishes ...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Primary Neuronal Culture

Generating a Low-Density Primary Neuron Culture From Frozen Embryonic Neurons

Source: Koganezawa, N. et al., <a href="https://app.jove.com/v/64872">Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons.</a>&#160;J. Vis. Exp. (2023)
This video demonstrates the method of culturing cryopreserved embryonic neurons, involving controlled thawing, dilution with a neurobasal medium, and seeding onto a poly-L-lysine-coated plate. Subsequent incubation supports the growth and maintenance of neuronal connections.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Plate coating

	Coat a 96-well ...