Name: a simplified method for ultra-low density, long-term primary hippocampal neuron culture
Uploaded: 2016-03-05T15:00:00
Description: Watch this Scientific Journal Video about A Simplified Method for Ultra-Low Density, Long-Term Primary Hippocampal Neuron Culture at JoVE.com

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. 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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>

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

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 ...

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps shape attention and also protects cells from over-stimulation. Adaptation within the olfactory circuit of C. elegans was first described by Colbert and Bargmann1,2. Here, the authors defined parameters of the olfactory adaptation paradigm, which they used to design a genetic screen to isolate mutants defective in their ability to adapt to volatile odors sensed by the Amphid Wing cells type C (AWC) sensory neurons. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor3 for 30 min they will adapt their responsiveness to the odor and will then ignore the adapting odor in a chemotaxis behavioral assay for ~1 hr. When wildtype C. elegans animals are exposed to an attractive AWC-sensed odor for ~1 hr they will then ignore the adapting odor in a chemotaxis behavioral assay for ~3 hr. These two phases of olfactory adaptation in C. elegans were described as short-term olfactory adaptation (induced after 30 min odor exposure), and long-term olfactory adaptation (induced after 60 min odor exposure). Later work from L'Etoile et al.,4 uncovered a Protein Kinase G (PKG) called EGL-4 that is required for both the short-term and long-term olfactory adaptation in AWC neurons. The EGL-4 protein contains a nuclear localization sequence that is necessary for long-term olfactory adaptation responses but dispensable for short-term olfactory adaptation responses in the AWC4. By tagging EGL-4 with a green fluorescent protein, it was possible to visualize the localization of EGL-4 in the AWC during prolonged odor exposure. Using this fully functional GFP-tagged EGL-4 (GFP::EGL-4) molecule we have been able to develop a molecular readout of long-term olfactory adaptation in the AWC5. Using this molecular readout of olfactory adaptation we have been able to perform both forward and reverse genetic screens to identify mutant animals that exhibit defective subcellular localization patterns of GFP::EGL-4 in the AWC6,7. Here we describe: 1) the construction of GFP::EGL-4 expressing animals; 2) the protocol for cultivation of animals for long-term odor-induced nuclear translocation assays; and 3) the scoring of the long-term odor-induced nuclear translocation event and recovery (re-sensitization) from the nuclear GFP::EGL-4 state.

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

Here we describe a molecular readout of long-term olfactory adaptation in Caenorhabditis elegans. The Protein Kinase G, EGL-4, is necessary for stable adaptation responses in the primary sensory neuron pair called AWC. During prolonged odor exposure EGL-4 translocates from the cytosol to nucleus of the AWC.

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and social interactions can be best studied by observing unrestrained, freely behaving animals. Weakly electric fish (WEF) display readily observable exploratory and social behaviors by emitting electric organ discharge (EOD). Here, we describe three effective techniques to synchronously measure the EOD, body position, and posture of a free-swimming WEF for an extended period of time. First, we describe the construction of an experimental tank inside of an isolation chamber designed to block external sources of sensory stimuli such as light, sound, and vibration. The aquarium was partitioned to accommodate four test specimens, and automated gates remotely control the animals&#39; access to the central arena. Second, we describe a precise and reliable real-time EOD timing measurement method from freely swimming WEF. Signal distortions caused by the animal&#39;s body movements are corrected by spatial averaging and temporal processing stages. Third, we describe an underwater near-infrared imaging setup to observe unperturbed nocturnal animal behaviors. Infrared light pulses were used to synchronize the timing between the video and the physiological signal over a long recording duration. Our automated tracking software measures the animal&#39;s body position and posture reliably in an aquatic scene. In combination, these techniques enable long term observation of spontaneous behavior of freely swimming weakly electric fish in a reliable and precise manner. We believe our method can be similarly applied to the study of other aquatic animals by relating their physiological signals with exploratory or social behaviors.

We describe a set of techniques for studying spontaneous behavior of freely swimming weakly electric fish over an extended period of time, by synchronously measuring the animal&#39;s electric organ discharge timing, body position and posture both accurately and reliably in a specially designed aquarium tank inside a sensory isolation chamber.

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure the activity of neurons during various behaviors. To correlate neural activity with behavior, the animal should not be immobilized but should be able to move. Many behavioral changes occur during long time scales and require recording over many hours of behavior. This also makes it necessary to culture the worms in the presence of food. How can worms be cultured and their neural activity imaged over long time scales? Agarose Microchamber Imaging (AMI) was previously developed to culture and observe small larvae and has now been adapted to study all life stages from early L1 until the adult stage of C. elegans. AMI can be performed on various life stages of C. elegans. Long-term calcium imaging is achieved without immobilizing the animals by using short externally triggered exposures combined with an electron multiplying charge-coupled device (EMCCD) camera recording. Zooming out or scanning can scale up this method to image up to 40 worms in parallel. Thus, a method is described to image behavior and neural activity over long time scales in all life stages of C. elegans.

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Imaging behavior and neural activity over long time scales without immobilization of the animal is a prerequisite to understand behavior. Agarose microfluidic chambers imaging (AMI) can be used to image neural activity and behavior for all life stages of Caenorhabditis elegans.

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

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.

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 ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of their normal in vivo interactions. Slices obtained from a variety of transgenic mouse lines or use of viral vectors for expression of fluorescently tagged proteins or reporters in wild type brain slices allow for high-resolution imaging by fluorescence microscopy. Although several methods have been developed for imaging brain slices, combining slice culture with the ability to perform repetitive high-resolution imaging of specific cells in live slices over long time periods has posed problems. This is especially true when viral vectors are used for expression of exogenous proteins since this is best done in a closed system to protect users and prevent cross contamination. Simple modifications made to the roller tube brain slice culture method that allow for repetitive high-resolution imaging of slices over many weeks in an enclosed system are reported. Culturing slices on photoetched coverslips permits the use of fiducial marks to rapidly and precisely reposition the stage to image the identical field over time before and after different treatments. Examples are shown for the use of this method combined with specific neuronal staining and expression to observe changes in hippocampal slice architecture, viral-mediated neuronal expression of fluorescent proteins, and the development of cofilin pathology, which was previously observed in the hippocampus of Alzheimer's disease (AD) in response to slice treatment with oligomers of amyloid-&#946; (A&#946;) peptide.

Presented here is a modified roller tube method for culturing and intermittent high-resolution imaging of rodent brain slices over many weeks with precise repositioning on photoetched coverslips. Neuronal viability and slice morphology are well maintained. Applications of this fully enclosed system using viruses for cell-type specific expression are provided.

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Long-term Sensory Conflict in Freely Behaving Mice

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning in mice. By permanently wearing a device fixed on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages. Therefore, this protocol readily enables the study of the visual system and multisensory interactions over an extended timeframe that would not be accessible otherwise. In addition to lowering the experimental costs of long-term sensory learning in naturally behaving mice, this approach accommodates the combination of in vivo and in vitro experiments. In the reported example, video-oculography is performed to quantify the vestibulo-ocular reflex (VOR) and optokinetic reflex (OKR) before and after learning. Mice exposed to this long-term sensory conflict between visual and vestibular inputs presented a strong VOR gain decrease but exhibited few OKR changes. Detailed steps of device assembly, animal care, and reflex measurements are hereby reported.

The presented protocol produces a persistent sensory conflict for experiments aimed at studying long-term learning. By permanently wearing a fixed device on their heads, mice are continuously exposed to a sensory mismatch between visual and vestibular inputs while freely moving in home cages.

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...