This study was supported by an NIH/NIMH grant to S.Q. (R00MH087628).

All experimental procedures involving mice were approved by the Institutional Animal Care and Use Committee of the University of Arizona, and conformed to NIH guidelines.
1. Tissue Source for Hippocampal Neuron Culture
<ol>
	<li>To generate prenatal mouse pups for hippocampal neuron culture, use time-pregnant mice (C57Bl6/J) that are bred in house17. The day with vaginal plug detection is designated as E0.5. The planned harvest time for culture is E16.5-E17.5. 
	Note: This pr...

<ol>
	<li>Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cultureing Nerve Cells. , 339-370 (1998).</li><li>Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+hippocampal+neurons.">Culturing hippocampal neurons.</a> Nat Protoc. 1, 2406-2415 (2006).</li><li>Petersen, J. D., Kaech, S., Banker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+microtubule-based+transport+of+dendritic+membrane+proteins+arises+in+concert+with+axon+specification.">Selective microtubule-based transport of dendritic membrane proteins arises in concert with axon specification.</a> J Neurosci. 34, 4135-4147 (2014).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+hippocampal+neurons+from+prenatal+mice.">Isolation and culture of hippocampal neurons from prenatal mice.</a> J Vis Exp. , (2012).</li><li>Shi, Y., Ethell, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrins+control+dendritic+spine+plasticity+in+hippocampal+neurons+through+NMDA+receptor+and+Ca2+/calmodulin-dependent+protein+kinase+II-mediated+actin+reorganization.">Integrins control dendritic spine plasticity in hippocampal neurons through NMDA receptor and Ca2+/calmodulin-dependent protein kinase II-mediated actin reorganization.</a> J Neurosci. 26, 1813-1822 (2006).</li><li>Viesselmann, C., Ballweg, J., Lumbard, D., Dent, E. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleofection+and+primary+culture+of+embryonic+mouse+hippocampal+and+cortical+neurons.">Nucleofection and primary culture of embryonic mouse hippocampal and cortical neurons.</a> J Vis Exp. , (2011).</li><li>Biffi, E., Regalia, G., Menegon, A., Ferrigno, G., Pedrocchi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+neuronal+density+and+maturation+on+network+activity+of+hippocampal+cell+cultures:+a+methodological+study.">The influence of neuronal density and maturation on network activity of hippocampal cell cultures: a methodological study.</a> PLoS One. 8, e83899 (2013).</li><li>Crump, F. T., Dillman, K. S., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cAMP-dependent+protein+kinase+mediates+activity-regulated+synaptic+targeting+of+NMDA+receptors.">cAMP-dependent protein kinase mediates activity-regulated synaptic targeting of NMDA receptors.</a> J Neurosci. 21, 5079-5088 (2001).</li><li>Leal, G., Afonso, P. M., Duarte, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+induces+synaptic+delivery+of+hnRNP+A2/B1+by+a+BDNF-dependent+mechanism+in+cultured+hippocampal+neurons.">Neuronal activity induces synaptic delivery of hnRNP A2/B1 by a BDNF-dependent mechanism in cultured hippocampal neurons.</a> PLoS One. 9, e108175 (2014).</li><li>Clarke, L. E., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+astrocytes+in+neural+circuit+development.">Emerging roles of astrocytes in neural circuit development.</a> Nat Rev Neurosci. 14, 311-321 (2013).</li><li>Kaneko, A., Sankai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+culture+of+rat+hippocampal+neurons+at+low+density+in+serum-free+medium:+combination+of+the+sandwich+culture+technique+with+the+three-dimensional+nanofibrous+hydrogel+PuraMatrix.">Long-term culture of rat hippocampal neurons at low density in serum-free medium: combination of the sandwich culture technique with the three-dimensional nanofibrous hydrogel PuraMatrix.</a> PLoS One. 9, e102703 (2014).</li><li>Millet, L. J., Bora, A., Sweedler, J. V., Gillette, M. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+cellular+peptidomics+of+supraoptic+magnocellular+and+hippocampal+neurons+in+low-density+co-cultures.">Direct cellular peptidomics of supraoptic magnocellular and hippocampal neurons in low-density co-cultures.</a> ACS Chem Neurosci. 1, 36-48 (2010).</li><li>Salama-Cohen, P., Arevalo, M. A., Meier, J., Grantyn, R., Rodriguez-Tebar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NGF+controls+dendrite+development+in+hippocampal+neurons+by+binding+to+p75NTR+and+modulating+the+cellular+targets+of+Notch.">NGF controls dendrite development in hippocampal neurons by binding to p75NTR and modulating the cellular targets of Notch.</a> Mol Biol Cell. 16, 339-347 (2005).</li><li>Xu, S. Y., Wu, Y. M., Ji, Z., Gao, X. Y., Pan, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+technique+for+culturing+primary+fetal+rat+cortical+neurons.">A modified technique for culturing primary fetal rat cortical neurons.</a> J Biomed Biotechnol. 2012, 803930 (2012).</li><li>McCarthy, K. D., de Vellis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+separate+astroglial+and+oligodendroglial+cell+cultures+from+rat+cerebral+tissue.">Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue.</a> J Cell Biol. 85, 890-902 (1980).</li><li>Qiu, S., Weeber, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reelin+signaling+facilitates+maturation+of+CA1+glutamatergic+synapses.">Reelin signaling facilitates maturation of CA1 glutamatergic synapses.</a> J Neurophysiol. 97, 2312-2321 (2007).</li><li>Qiu, S., Lu, Z., Levitt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MET+receptor+tyrosine+kinase+controls+dendritic+complexity,+spine+morphogenesis,+and+glutamatergic+synapse+maturation+in+the+hippocampus.">MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus.</a> J Neurosci. 34, 16166-16179 (2014).</li></ol>

The authors declare that they have no competing financial interests.

We present a detailed protocol for long-term culture of ultra-low density hippocampal glutamatergic neurons under serum free conditions. At ~2000 neurons/cm2, the density is at least two fold lower than most &#39;low density&#39; culture preparations with or without glia support reported by the existing literature2,3,11,13,14. In addition to being ultra-low density, this protocol is novel and significant in two more ways. First, no glia feeder layer is needed as the low density neurons obtain trophi...

Growing hippocampal neurons under in vitro conditions enables observation and experimental manipulation of these neurons that are otherwise not possible in vivo. This experimental approach is widely used to reveal neuronal mechanisms of growth, polarity, neurite specification, trafficking and subcellular localization of proteins, synapse formation and functional maturation1. These in vitro cultured hippocampal neurons, when harvested from late embryonic stages, are relatively pure (&#62;90%) glutamatergic cells of pyramidal morphology2. Because neurons were grown in a 2-D surface under in vitro conditions, this method allows easy observation, such as live imaging or immunocytochemistry (ICC) staining through a single focal plane3; or manipulations, such as drug treatment and transfections3-6. When grown at high density, neurons tend to have high rates of survival because of higher concentrations of secreted growth factors in addition to alimentary support from the growth media, and also because of neurite contact-dependent mechanisms7. However, low density hippocampal neurons are desirable for morphological studies, where an individual neuron can be imaged in its entirety or stained for ICC analysis. Low density neurons are hard to maintain in culture due to lack of paracrine support and thus often require trophic support from a glial (typically cortical astrocyte) feeder layer, which has to be prepared prior to neuron culture2. When co-cultured with a glial cell feeder layer, low density neurons are grown on coverslips, and then flipped on top of the glia layer so that the low density neurons and glia are facing each other. A small confined space between glia and neurons is created by placing paraffin wax dots on the corners of the coverslips, therefore creating a &#39;sandwich&#39; layout2,8,9. The low density neurons will grow within the confined space between glia and the coverslip, which creates a permissive microenvironment with concentrated factors secreted by neurons and glia. This approach yields low-density, fully developed neurons that are spaced reasonably apart, therefore facilitating ICC labeling or live imaging studies.
An apparent drawback of neuron-glia co-culture, aside from being time-consuming and laborious, is that it prevents study of neuron-specific, or cell-autonomous, mechanisms. Although this system is far less complex than in vivo neural tissue, glia impact on neuron development through secreted, not-yet-fully-defined factors can confound the experiments10. Therefore, in experiments that require the investigation of neuron specific mechanisms, defined culture conditions that remove serum and glial support layer are necessary. A previous study has succeeded in culturing low concentrations of neurons (~9,000 cells/cm2) using a three dimensional hydrogel matrix11. Since a relatively pure neuronal population can be cultured at high density under serum free conditions without glial support, we hypothesize that ultra-low density of hippocampal neurons can be grown in serum free defined culture medium by co-culturing them with high density neurons, in a way that is analogous to the conventionally adopted neuron-glia co-culture. Indeed, high density hippocampal neuron cultures in a &#39;sandwich&#39; configuration have been recently used to support a small number of specialized magnocellular endocrine neurons12.
Therefore, co-culture with high density neurons may allow low density neurons to receive trophic factors support that is sufficient to enable long term survival. This protocol of culturing ultra-low density neurons was thus formulated and validated. The protocol can be implemented within one single experiment, by preparing high density (~250,000 cells/ml) dissociated hippocampal neurons first, and then making a dilution to yield a density ~10,000 neurons/mL (~3,000 neurons/coverslip, or ~2,000 neurons/cm2), which is much lower than most reported low density cultures2,3,9,11,13. This culture condition is loosely referred to as &#39;ultra-low density&#39; culture and used with &#39;low-density&#39; inter-changeably. The high density neurons are plated on the poly-D-lysine coated 24-well plates; while the low density neurons are seeded on poly-D-lysine coated 12-mm glass coverslips that are placed inside another 24-well plate. The coverslips with adhering low density neurons are flipped on top of the high density neurons 2 hr later after the neurons are descended and attached to the coverslips. In addition, instead of using paraffin wax dots to elevate the coverslips above the high density neuron layer, an 18 G syringe needle was used to etch the bottom of the 24 well plates with two parallel stripes. The resulting displaced, adjoining plastics provide an elevated support for the glass coverslips. This space is consistently measured at 150-200 &#956;m, which allows sufficient oxygen and culture medium exchange while providing a microenvironment with concentrated trophic factors. Under this condition, the low density neurons grow extensively, and can survive beyond three months in culture. When these neurons are transfected with GFP plasmid after three weeks in culture, the dendrites are profusely studded with dendritic spines. As a proof of principle, data are presented to show that this co-culture system supports ultra-low density cultures of hippocampal neurons seeded on poly-D-Lysine &#39;micro-islands&#39;, where neurons form autaptic connections that may facilitate investigation of cell-autonomous, network-independent mechanisms.

<table border="0" cellpadding="0" cellspacing="0" width="713">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Neurobasal medium</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">21103-049</td>
			<td style="width:200px;">Protect from light</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">B27 supplement</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">17504-044</td>
			<td>aliquot, store in 0.6ml size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">GlutaMAX-I</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">35050-061</td>
			<td>dilute 100X&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">antibiotic-antimycotic (AA)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:144px;">15240-096</td>
			<td>dilute 100X&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Complete Culture medium</td>
			<td style="width:135px;"></td>
			<td style="width:144px;"></td>
			<td>Neurobasal medium with 1X B27, 1X AA, 1X GlutaMAX-I</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Wash medium</td>
			<td style="width:135px;"></td>
			<td style="width:144px;"></td>
			<td>same as &#39;Neurobasal medium&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Feed medium</td>
			<td style="width:135px;"></td>
			<td style="width:144px;"></td>
			<td>Neurobasal with 1X B27 supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">DNAse I</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:144px;">D5025</td>
			<td>prepare 100X stock at 0.6mg/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">poly-D-lysine</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:144px;">P6407</td>
			<td>M.W. 70000-150000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">borate buffer</td>
			<td style="width:135px;">Sigma-Aldrich</td>
			<td style="width:144px;">B6768 (boric acid); 71997(borax)</td>
			<td>1.24g boric acid &#38; 1.9g borax in 400ml H2O, pH to 8.5 use HCl</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">12-mm round glass coverslips</td>
			<td style="width:135px;">Glasswarenfabrik Karl Hecht GmbH</td>
			<td style="width:144px;">1001/12</td>
			<td>No. 1 glass, purchase from Carolina Biological Supply</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">proFection transfection kit</td>
			<td style="width:135px;">Promega</td>
			<td style="width:144px;">E1200</td>
			<td>see&#160; protocol for details</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">2X HEPES buffered saline (HBS)</td>
			<td style="width:135px;">Promega</td>
			<td style="width:144px;">E1200</td>
			<td>see&#160; protocol for details</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Syringe filter</td>
			<td style="width:135px;">Pall Corporation</td>
			<td style="width:144px;">4192</td>
			<td>0.2um pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Endofree plasmid prep kit</td>
			<td style="width:135px;">Qiagen</td>
			<td style="width:144px;">12362</td>
			<td>for preparation of transfection grade plasmid DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-MAP2 antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">MAB3418</td>
			<td>mouse antibody, clone AP20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-p-Tau antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">AB10417</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-NR1 antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">MAB1586</td>
			<td>mouse antibody, clone R1JHL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">anti-GluR1 antibody</td>
			<td style="width:135px;">Millipore</td>
			<td style="width:144px;">AB1504</td>
			<td>rabbit polyclonal antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Hank&#39;s balanced salt solution</td>
			<td style="width:135px;">ThermoFisher</td>
			<td style="width:144px;">14025092</td>
			<td>500ml size</td>
		</tr>
	</tbody>
</table>

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.

The protocol described here enables successful ultra-low density, long-term culture of pure glutamatergic neurons without the need of glia cells serving as a feeder layer. The protocol is diagramed in Figure 1, which involves preparation of high density (on poly-D-lysine coated 24 wells) and low-density neurons (on poly-D-lysine coated glass coverslips) separately, and subsequent co-culture that can be maintained up to three months.
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Watch this Scientific Journal Video about A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture at JoVE.com

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

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 ...

The overall goal of this procedure is to establish healthy longterm cultures of primary mouse hippocampal neurons at ultra-low density, in order to facilitate immunocytochemistry labeling, and the study of neuronal cell autonomous mechanisms. This study can help understand some key questions in the field of neuroscience research such as the studies on the specificity of protein in neuromorphology of function of development. The main advantage of this technique is that it allows neurons to grow at ultra-low density on a cover slip without the support of a glial feeder layer.Demonstrating the procedure will be Mariel Piechowicz, a student from my laboratory. Prepare two high-density and two low-density 24-well plates. Using a 28 gauge needle, etch the bottom of 24 well plates, so that the displaced plastics will act as a support for the cover slips with low-density neurons growing on them.Transfer the sterile glass cover slips into the two 24-well plates designated for low-density cultures. Coat all four plates with 300 microliters of 0.1 milligram per milliliter Poly-D-lysine diluted in borate buffer, and return them to the incubator for overnight incubation coating. Before tissue collection, prepare two 10 centimeter petri dishes filled with ice-cold sterile HBSS.Under the microscope, hold the embryo by the neck with the forceps, and make the first cut right on the midline, below the cerebellum level. Subsequently, make a cut on the left side of the base of the skull. Next, make another cut on the right side of the base of the skull.Leave a small part of the skull bone and skin tissue at the rostral end uncut so that the whole brain can be flipped aside for extraction easily. Now transfer the brain into the HBSS solution. Inspect the brain to make sure that it is intact without gross cut off regions.Then, repeat the procedures to collect the rest of the embryo brains in a new petri dish containing 20 milliliters of cold HBSS buffer. Position each brain with the ventral side up. While holding down the brain with forceps at the caudal end, make a cut diagonally between the middle rostral point and the caudal temporal region with a sharp tweezer.Repeat this for the other hemisphere. Afterward, separate the two hemispheres with intact hippocampus from the rest of the brain's stem tissues. Repeat the procedures for all the brains and pool all the dissected hemispheres together.Then, remove the meninges using two pairs of tweezers. Identify the developing hippocampus as a curved structure that is folded inside the temporal lobe and separate it from the adjoining cortical tissues. Collect and pool all the hippocampi tissues together.In this procedure, transfer all the dissected hippocampi into the enzymatic digestion solution in a 15 milliliter conical tube. Next, place the tube inside a water bath and incubate at 37 degrees Celsius for 25 minutes. After 25 minutes, carefully remove the enzyme solution.Then, add five milliliters of warm wash medium to bring the total volume to 10 milliliters. Gently wash the tissue by slowly inverting the tube five times. Remove the wash medium and add 10 milliliters of wash medium again, for an additional wash.After that, remove the wash medium and add three milliliters of fresh warm wash medium again. Shake the tube by hand rigorously 15 times to dislodge the digested neurons from the tissue. The medium should become turbid once the cells are separated from the tissue into the wash medium.Then, collect the cell suspension in a new 15-milliliter conical tube, while leaving the remaining tissue in the tube. After that, add another two milliliters of wash medium to the tissue, and gently separate the remaining tissue by triturating with a plastic pipette 15 times. Let the cell suspension sit for five minutes before pooling it into a new 15-milliliter conical tube that contains the collected shake off cells.Then, count the density of neurons with a hemocytometer. Calculate the cell suspension volume needed to yield a density of 250, 000 neurons per milliliter, and add it to one of the two conical tubes containing 30 milliliters of complete culture medium, to yield a high-density neuron plating medium. Transfer 1.2 milliliters of this high-density neuron solution to another 30 milliliters of complete culture medium, to yield a concentration of 10, 000 neurons per milliliter, and use this solution to seed the low-density cover slips.In this procedure, place 24-well plates with etched bottoms for high-density neurons in the culture hood. Remove 300 microliters of the pre-conditioned medium by vacuum, then plate 0.6 milliliters of high-density cell suspension at 250, 000 neurons per milliliter. Place the other two 24-well plates with cover slips in the culture hood.Remove 300 microliters of the preconditioned medium by vacuum, before plating 0.6 milliliters of low-density cell suspension at 10, 000 neurons per milliliter on the cover slips. Afterward, return all four 24-well plates to the incubator, and incubate for two hours. After that, flip the low-density cover slips with adhering neurons to the high-density culture and make sure the neurons are facing each other.Subsequently, return the two plates with co-culture to the incubator. Feed the co-cultures by adding 300 microliters of fresh feed medium at DIV five. Following this initial feeding, add 300 microliters of fresh feed medium without cytosine arabinoside to each well every week.Should the culture be kept for more than one month, replace half of the medium with fresh feed medium every week beyond one month time point. Morphologically, both high-density and low-density neurons should look healthy up to three months. Here, immunocytochemitry labeling is used to reveal the functional glutamatergic synapses in which NMDA receptor subunit NR1 and the ApoE receptor subunit GluR1 are labeled with double staining.And the functional synapses can be readily identified and quantified using image J.Low-density neurons are also labeled with antibody against dendritic protein marker MAP2 in combination with axonal protein marker p-Tau. This double labeling allows clear distinction of dendrites and axons. Low-density cultures can also be successfully transfected with DNA plasmids using calcium phosphate methods, although the transfection rate is typically very low at later stages.This figure illustrates that EGFP transfection can reveal neuronal morphology, and render fine dendritic spine structures visible. Lastly, as a proof of concept, ultra-low density neurons can be grown under conditions that promote autapse formation. These ultra-low density autaptic neurons grown on Poly-D-lysine coated microislands, can be sustained by the high-density feeder neurons to beyond two months with this co-culture protocol.Once mastered, this technique can be done in two to three hours on the day of cell culture if it is performed properly. While attempting this procedure, it is critical to coat the glass cover slips and 24-well plate bottoms with Poly-D-lysine. A simple etch of the plate bottoms is adequate to provide mechanical and nutritional support to the low-density hippocampal neurons to grow on the cover slips.After watching this video, you should have a good understanding on how to grow ultra-low density primary mouse embryonic hippocampal neurons, in the absence of a glial feeder layer. Hopefully you can utilize this method for your own experimental purpose.

a-simplified-method-for-ultra-low-density-long-term-primary

Research

JoVE Journal

Neuroscience

15.5K Views.  University of Arizona College of Medicine-Phoenix. The overall goal of this procedure is to establish healthy longterm cultures of primary mouse hippocampal neurons at ultra-low density, in order to facilitate immunocytochemistry labeling, and the study of neuronal cell autonomous mechanisms. This study can help understand some key questions in the field of neuroscience research such as the studies on the specificity of protein in neuromorphology of function of development. The main advantage of this technique is that it allows neurons to gro...