We thank Kazumi Kamiyama and Manami Kawada for assistance with experiments. This work was supported by JSPS KAKENHI (Grant Number 19K08010 to N.K.) and Japan Agency for Medical Research and Development (AMED) (Grant Number JP19bk0104077 and JP22bm0804024 to T.S.).

1. Plate coating
<ol>
	<li>Coat a 96-well microplate with poly-L-lysine (1 mg/mL, diluted in 0.1 M borate buffer [pH: 8.5]; 100 &#181;L/well) and incubate overnight at 37 &#176;C. 
	NOTE: Only coat the wells that need to be used. In the experiments performed here, the middle 60 wells are used. The borate buffer is prepared by mixing 50 mM boric acid and 12 mM borate in sterilized water.</li>
	<li>Wash the plate twice with sterilized water (250 &#181;L/well).</li>
	<li>Wash the plate once with fresh culture medium without supplements (250 &#181;L/well).</li>
	<li>Dry the plate on a clean bench for 20 min.</li>
	<li>Wrap the plate with aluminum foil and keep it at 4 &#176;C until use (valid for 1 month).</li>
</ol>
2. Cell seeding
<ol>
	<li>Add 50 &#181;L/well of the culture medium to the coated plate and keep it in a 37 &#176;C, 5% CO2 incubator for 30 min to 1 h. Fill the peripheral wells with sterilized water (200 &#181;L/well). 
	NOTE: The culture medium is prepared by adding 50x B-27, 400x Glutamax, and 100 U/mL of penicillin/streptomycin to the neurobasal medium (refer to the Table of Materials for details).</li>
	<li>Remove the neuron cryovial from the liquid nitrogen tank. The neurons used here were DMSO-cryopreserved neurons11.</li>
	<li>Immerse the cryovial in a 37 &#176;C heat block for up to 3 min and partially thaw the contents. Do not warm up the cryovial for too long. Transfer the contents to a 50 mL tube as soon as it is thawed.</li>
	<li>Slowly transfer the neuron cryovial contents to a sterile 50 mL tube drop-wise (50 &#181;L/s) using a 1 mL pipette with a wide-pore tip.</li>
	<li>Rinse the empty cryovial with 1 mL of the culture medium (room temperature; RT). Transfer this 1 mL of the culture medium from the cryovial drop-wise (50 &#181;L/s) to the 50 mL tube containing the cell suspension.</li>
	<li>Add 9 mL of the culture medium (RT) to the 50 mL tube drop-wise (0.5 mL/s) and make up the volume to 11 mL. Do not repeat pipetting, but mix the cell suspension slowly.</li>
	<li>Count the cell number (use a cell counter or a hemocytometer).</li>
	<li>Transfer all the cell suspension to a reservoir and dispense the cell suspension to the 96-well plate using a multichannel pipette with wide-pore tips (1.0 x 104 cells/well). To reduce the culture medium evaporation, fill the peripheral wells with sterilized water (step 2.1). 
	NOTE: This study confirms that the culture medium evaporation is small for a 3-week culture without medium exchange. The reduction rate of the medium is 3.6% (n = 120 wells). Thus, the osmolality change will not be drastic during the 3-week incubation period.</li>
	<li>Incubate the neurons for 1-2 h in a 37 &#176;C, 5% CO2 incubator.</li>
	<li>Replace the culture medium with 100 &#181;L of preheated culture medium (37 &#176;C) per well and put it back in a 37 &#176;C, 5% CO2 incubator (no medium change is required during culture).</li>
</ol>
3. Ara-C treatment
<ol>
	<li>At 4 days in vitro (DIV), add cytosine &#946;-D-arabino-furanoside (Ara-C) to a final concentration of 0.2 &#181;M per well to reduce the growth of glial cells.</li>
</ol>
4. Drug treatments
<ol>
	<li>At 21 days in vitro, treat the cells with the drugs of interest.</li>
	<li>Keep the temperature of the plate at 37 &#176;C during drug treatments.</li>
	<li>For a positive control, treat the cells with 100 &#181;M glutamate (per well for final concentration) for 10 min before fixation.</li>
</ol>
5. Fixation
<ol>
	<li>For the fixation, use 4% paraformaldehyde in 0.1 M phosphate buffer (100 &#181;L/well).</li>
	<li>After ~20 min of fixation, wash the wells with phosphate-buffered saline (PBS; 250 &#181;L/well) 2x for 5 min each.</li>
</ol>
6. Immunocytochemistry
<ol>
	<li>Wash the cells with PBS (250 &#181;L/well) 1x for 5 min.</li>
	<li>Permeabilize the cells with 0.1% Triton X-100 (100 &#181;L/well) in PBS for 5 min.</li>
	<li>Wash the cells with PBS (250 &#181;L/well) 3x for 5 min each.</li>
	<li>For blocking, use 3% bovine serum albumin in PBS (PBSA; 100 &#181;L/well) for 1 h at RT.</li>
	<li>Incubate the cells with anti-drebrin (1:1) and anti-Microtubule Associated Protein 2 (MAP2) (1:000) antibodies (60 &#181;L/well) at 4 &#176;C overnight.</li>
	<li>Wash the cells with PBS (250 &#181;L/well) 4x for 5 min each.</li>
	<li>Incubate the cells with appropriate secondary antibodies and 4&#8242;,6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:1000) in PBSA (60 &#181;L/well) for 2 h at RT.</li>
	<li>Wash the cells with PBS (250 &#181;L/well) 4x for 5 min each.</li>
	<li>Store the cells in PBS containing 0.1% sodium azide (150 &#181;L/well).</li>
</ol>
7. Image acquisition and analysis
<ol>
	<li>To acquire the images, use an appropriate microscope.</li>
	<li>To identify the cell bodies of neurons, use both MAP2-positive and DAPI-positive regions.</li>
	<li>To identify the dendrites of neurons, use MAP2-positive signals without cell bodies.</li>
	<li>To identify drebrin clusters, use drebrin-positive signals along MAP2 positive dendrites.</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+DMSO-based+method+for+cryopreservation+of+primary+hippocampal+and+cortical+neurons.">A simple DMSO-based method for cryopreservation of primary hippocampal and cortical neurons.</a> Journal of Neuroscience Methods. 333, 108578 (2020).</li><li>Pischedda, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreservation+of+primary+mouse+neurons:+The+benefit+of+neurostore+cryoprotective+medium.">Cryopreservation of primary mouse neurons: The benefit of neurostore cryoprotective medium.</a> Frontiers in Cellular Neuroscience. 12, 81 (2018).</li><li>Kobayashi, Y., et al. <a target="_blank" 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href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor-dependent+and+-independent+effects+of+natural+compounds+and+crude+drugs+on+synaptic+states+as+revealed+by+drebrin+imaging+analysis.">NMDA receptor-dependent and -independent effects of natural compounds and crude drugs on synaptic states as revealed by drebrin imaging analysis.</a> The European Journal of Neuroscience. 53 (11), 3548-3560 (2021).</li><li>Mizui, T., Takahashi, H., Sekino, Y., Shirao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+drebrin+A+in+immature+neurons+induces+the+accumulation+of+F-actin+and+PSD-95+into+dendritic+filopodia,+and+the+formation+of+large+abnormal+protrusions.">Overexpression of drebrin A in immature neurons induces the accumulation of F-actin and PSD-95 into dendritic filopodia, and the formation of large abnormal protrusions.</a> Molecular and Cellular Neurosciences. 30 (1), 149-157 (2005).</li><li>Sekino, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+N-methyl-D-aspartate+receptor+induces+a+shift+of+drebrin+distribution:+disappearance+from+dendritic+spines+and+appearance+in+dendritic+shafts.">Activation of N-methyl-D-aspartate receptor induces a shift of drebrin distribution: disappearance from dendritic spines and appearance in dendritic shafts.</a> Molecular and Cellular Neurosciences. 31 (3), 493-504 (2006).</li><li>Takahashi, H., Yamazaki, H., Hanamura, K., Sekino, Y., Shirao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+the+AMPA+receptor+regulates+drebrin+stabilization+in+dendritic+spine+morphogenesis.">Activity of the AMPA receptor regulates drebrin stabilization in dendritic spine morphogenesis.</a> Journal of Cell Science. 122, 1211-1219 (2009).</li><li>Aoki, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drebrin+A+is+a+postsynaptic+protein+that+localizes+in+vivo+to+the+submembranous+surface+of+dendritic+sites+forming+excitatory+synapses.">Drebrin A is a postsynaptic protein that localizes in vivo to the submembranous surface of dendritic sites forming excitatory synapses.</a> The Journal of Comparative Neurology. 483 (4), 383-402 (2005).</li><li>Koganezawa, N., Hanamura, K., Sekino, Y., Shirao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+drebrin+in+dendritic+spines.">The role of drebrin in dendritic spines.</a> Molecular and Cellular Neurosciences. 84, 85-92 (2017).</li><li>Shirao, T., et al. <a target="_blank" 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Tomoaki Shirao is the CEO of AlzMed, Inc. The study was funded by AlzMed, Inc. (500,000 JPY to NK for the project entitled "High-throughput analysis of synaptic function").

A critical step in this method is to thaw the cell suspension. Transfer of the cell suspension before it becomes too warm is very important. To avoid the rapid change of osmolality however, do not transfer the cell suspension to a large volume of the medium at once. Drop-wise addition of the culture medium is also crucial to avoid sudden changes in osmotic pressure.
The neurons can be cultured in other culture vessels: 24-well plates, 8-well chambers, 60 mm dishes, or MED probes. In those cases, however, the final concentration and the timing of the addition of Ara-C need to be adjusted. In addition, the density of neurons needs to be optimized in different types of experiments. For example, high-density culture is required for electrophysiological experiments, and in that case, the media exchange is needed twice a week (Figure 6). Thus, a low-density culture requires fewer steps than a high-density culture.
Low-density neuronal culture often requires advance techniques; however, using ready-to-use frozen stock solves this problem. The described method does not depend on the skill of an experimenter. The quality of frozen stock is stable and can be stably cultured as long as they are stored in liquid nitrogen and avoid temperature changes for up to 4 years.
Culturing the cells for 3 weeks without medium exchange raises the question on whether there are significant osmolality changes or culture media evaporation. However, we have confirmed that the culture media evaporation is small (reduction rate of 3.6%). The localization of synaptic proteins and the morphology of the neurons appear normal after 3 weeks. Therefore, 3-week culture without media exchange does not cause large osmolality changes affecting the conditions of the cultured neurons. Keeping the plate in an incubator after Ara-C treatment is also an important point that minimizes evaporation.
There is no limitation regarding the use of the frozen stock neurons. However, there are some limitations of the low-density culture method. We confirmed that the low-density culture could be applied for morphological observation of neurons, evaluation of synaptic function, and GFP transfection. However, we have not examined live cell imaging. In addition, as mentioned above, high-density culture is required to perform electrophysiology.
Synapse maturation typically takes 3 weeks7, and we cannot confirm that the cultured neurons have proper synapses until the end. If the synapse maturation is not good after 3 weeks, we would have to culture again. By knowing the quality of the neurons before starting the experiments, we can save these 3 weeks. Therefore, to perform experiments efficiently, it is best to check the quality of the neurons in advance. Frozen stocks make it possible to check the quality of the neurons beforehand. Each batch of frozen stocks is generated from one litter of rats, and we can use one of the stocks from each batch for a quality check. Drebrin is a good marker for the quality check of the neurons. As described, drebrin accumulates in the spine head in mature neurons, and it reacts to synaptic stimulation. Thus, we can check the quality of the neurons in frozen stocks by using drebrin as a marker.
This method can be applied to evaluate the effect of drugs on the synaptic state. The drebrin exodus from dendritic spines occurs during the initial stages of synaptic plasticity22. Therefore, the detection of drebrin cluster reduction elicited by drug treatment shows that the drug stimulates the synapse and causes synaptic plasticity. Furthermore, to identify whether the reduction is NMDAR dependent, an experiment using 2-amino-5-phosphonovaleric acid (APV, an NMDAR antagonist) is useful. Using drebrin as a marker, even an NMDAR-dependency is clearly determined10,15. The method described is useful in drug screenings, safety pharmacological studies, and evaluation of synaptic function.

The development of an in vitro experimental system that can assess synaptic functions involved in learning and memory is important. Neuronal culture is a valuable system for evaluating synaptic functions in vitro. The neuronal culture technique was first used in the 1980s, and in the 1990s, low-density culture of primary hippocampal neurons was developed1,2,3 for the study of individual neurons in terms of subcellular localization of protein components, protein trafficking, neuronal polarity, spine morphology, synapse development, and plasticity4,5,6,7,8. However, there are many steps involved in this technique: mating animals, dissecting embryos, preparing culture vessels, and culturing cells for 3 weeks with media changes once a week. In addition, it requires advance techniques3.
We have developed frozen stocks of dissociated hippocampal neurons from rat embryos9,10. The frozen stocks of neurons are ready-to-use, and no advance techniques are required for culturing the cells11,12. In other words, culturing the neurons from frozen stocks does not depend on the technique of an experimenter. It eliminates the need for animal experiments (e.g., permission for animal experiments, arranging timed pregnant animals, and dissecting rat embryos), thereby reducing the number of animals used. Recently, high-quality, ready-to-use frozen stocks of neurons have become commercially available. Here, we used commercially available frozen stocks from the embryonic day (E) 18 rat hippocampus13,14,15. Culturing the neurons from a frozen stock does not require glia-conditioned media or co-culture with glial cells. Ordinary primary culture media without additional serum can be used to culture the cells; hence we can acquire reproducible data. Furthermore, there is no need for media exchange for 3 weeks after the cell seeding as the growth of glial cells is reduced (Figure 1).
Dendritic spines are the postsynaptic compartment of most excitatory synapses. They contain receptor proteins, postsynaptic scaffold proteins, and actin cytoskeletal proteins. We focused on an actin-binding protein drebrin5,6,7,16,17,18. Drebrin accumulates at the spine head in mature neurons19, and we reported drebrin as a marker for the synaptic state15,17,20,21,22,23. By conducting a high-content analysis using drebrin as readout, we have recently reported the inhibitory effects of phencyclidine analogs on N-methyl-D-aspartic acid-type glutamate receptors (NMDARs)10 and the NMDAR-dependent effects of natural compounds and crude drugs on synaptic states15.
Here, we detail how to culture frozen stocks of neurons at low density. In addition, we show a drebrin imaging-based evaluation of the synaptic state using 96-well plates.

<table><tbody><tr><td>96 well plate</td><td>Zeon Corporation</td><td></td><td>Gifted</td></tr><tr><td>96 well plate</td><td>greiner</td><td>655986</td><td></td></tr><tr><td>Anti-drebrin antibody (M2F6)</td><td>MBL</td><td>D029-3</td><td>Mouse monoclonal (dilution 1:1)</td></tr><tr><td>Anti-MAP2 antibody</td><td>Millipore</td><td>AB5622</td><td>Rabbit&amp;nbsp; (dilution 1:1000)</td></tr><tr><td>Anti-mouse Alexa Fluor 568</td><td>Thermo Fisher Scientific</td><td>A11031</td><td>Dilution 1: 500</td></tr><tr><td>Anti-rabbit Alexa Fluor&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>A11008</td><td>Dilution 1: 500</td></tr><tr><td>B-27</td><td>Gibco</td><td>17504-044</td><td>2 v/v% for MEA plates; 50x for normal plates</td></tr><tr><td>Borax</td><td>Sigma</td><td>B-9876</td><td>Final concentration 12 mM</td></tr><tr><td>Boric acid</td><td>WAKO</td><td>021-02195</td><td>Final concentration 50 mM</td></tr><tr><td>Bovine serum albumin</td><td>Millipore</td><td>12659-100G</td><td>Final concentration: 3% in PBS</td></tr><tr><td>Confocal quantitative image cytometer&lt;br/&gt; CellVoyager CQ1</td><td>YOKOGAWA</td><td></td><td>Phase contrast images</td></tr><tr><td>Cytosine &amp;beta;-D-arabino-furanoside (Ara-C)</td><td>Sigma</td><td>C-6645</td><td>Diluted in dH&lt;sub&gt;2&lt;/sub&gt;O (final concentration: 0.2 &amp;micro;M)</td></tr><tr><td>DAPI</td><td>FUJIFILM</td><td>340-07971</td><td>Dilution 1:1000</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-061</td><td>&amp;nbsp;2.5 mM for MEA plates; 400x for normal plates</td></tr><tr><td>In Cell Analyzer 2200</td><td>Cytiva</td><td></td><td>Fluorescence images</td></tr><tr><td>Laminin</td><td>Sigma</td><td>114956-81-9</td><td>Final concentration: 20 &amp;micro;g/mL</td></tr><tr><td>Maestro</td><td>Axion Biosystems</td><td></td><td>MEA recordings</td></tr><tr><td>MEA plate</td><td>Axion Biosystems</td><td>M768-tMEA-48W</td><td></td></tr><tr><td>Neurobasal</td><td>Gibco</td><td>21103-049</td><td></td></tr><tr><td>Paraformaldehyde</td><td>nacalai tesque</td><td>26126-25</td><td>Final concentration: 4% in PBS</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140-122</td><td>&amp;nbsp;100 U/mL for normal plates</td></tr><tr><td>Penicillin/Streptomycin</td><td>nacalai tesque</td><td>26253-84</td><td>100 &amp;micro;g/mL for MEA plates</td></tr><tr><td>polyethyleimine</td><td>Sigma</td><td>9002-98-6</td><td>Final concentration: 0.1%</td></tr><tr><td>Poly-L-lysine</td><td>Sigma</td><td>P2636</td><td>Diluted in the borate buffer (final concentration: 1 mg/mL)</td></tr><tr><td>SKY Neuron</td><td>AlzMed , Inc.</td><td>ARH001</td><td>1.0 x 10&lt;sup&gt;6&lt;/sup&gt; cells/tube</td></tr><tr><td>Sodium azide</td><td>FUJIFILM</td><td>195-11092</td><td>0.1%</td></tr><tr><td>SodiumL(+)-Glutamate monohydrate</td><td>WAKO</td><td>194-02032</td><td>Diluted in dH&lt;sub&gt;2&lt;/sub&gt;O (final concentrations: 1 &amp;micro;M, 3 &amp;micro;M, 10 &amp;micro;M, 30 &amp;micro;M, 100 &amp;micro;M)</td></tr></tbody></table>

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.

Following the protocol, the neurons were cultured in a 96-well plate for 21 days, and then treated with glutamate (Figure 1). The neurons developed normally without an exchange of the culture medium for 3 weeks (Figure 2). We treated the cells with several concentrations of glutamate (1 &#181;M, 3 &#181;M, 10 &#181;M, 30 &#181;M, and 100 &#181;M diluted in sterilized water) for 10 min and fixed them. Immunocytochemistry was performed, and fluorescence images of drebrin and MAP2 were acquired using an automated fluorescence microscope with an sCMOS camera. As shown in Figure 3, drebrin-positive dendritic spines are clearly observed along MAP2-positive dendrites. It has been shown that glutamate stimulation elicits Ca2+ influx through NMDAR, which causes drebrin exodus from dendritic spines resulting in a reduction of drebrin cluster densities5,17. Accordingly, we observed the dose-dependent reduction of drebrin cluster densities against glutamate stimulation10 (Figure 4). As shown in Figure 5, this method is highly reproducible if drebrin is used as a marker for synaptic states.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64872/64872fig01.jpg" /> 
Figure 1: Scheme of the method. The neurons were cultured in a 96-well plate for 21 days, and then treated with glutamate. <a href="https://www.jove.com/files/ftp_upload/64872/64872fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64872/64872fig02.jpg" /> 
Figure 2: Bright-field images of cultured neurons using a 96-well plate. Phase contrast images were obtained from each developmental stage (DIV 1, 7, 14, 21) using a confocal quantitative image cytometer. Scale bar: 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64872/64872fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64872/64872fig03.jpg" /> 
Figure 3: Representative images of immunostained cultured neurons. (Left) Merged fluorescence images of drebrin (green) and MAP2 (red). Each fluorescence image of drebrin and MAP2 showed in the middle and right panels, respectively. White rectangles show the area magnified below. Scale bars; upper panels: 50 &#181;m, lower panels: 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64872/64872fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64872/64872fig04.jpg" /> 
Figure 4: Glutamate-dependent dose-response changes in normalized drebrin cluster density. (A) Representative fluorescence images immunostained using drebrin (green) and MAP2 (red) from the well that is treated with 0 &#181;M, 10 &#181;M, and 100 &#181;M glutamate (from left to right). Scale bar: 50 &#181;m. (B) Drebrin cluster density was normalized by the average of control (0 &#181;M). 0 &#181;M, N = 58 wells; 1 &#181;M, N = 46; 3 &#181;M, N = 54; 10 &#181;M, N = 45; 30 &#181;M, N = 54; 100 &#181;M, N = 55, from 13 experiments using different lots. ** P &#60; 0.01 versus control (0 &#181;M) by Dunnett&#39;s multiple comparisons test following ANOVA. <a href="https://www.jove.com/files/ftp_upload/64872/64872fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64872/64872fig05.jpg" /> 
Figure 5: Glutamate-dependent dose-response changes in drebrin cluster density. The raw data from six experiments using different lots. N = 4 wells for each concentration (0 &#181;M, 3 &#181;M, 10 &#181;M, 30 &#181;M, and 100 &#181;M). Values are expressed as mean &#177; SEM. <a href="https://www.jove.com/files/ftp_upload/64872/64872fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/64872/64872fig06.jpg" /> 
Figure 6: Application of the frozen stocks of neurons to electrophysiological experiments. (A) A protocol for electrophysiological experiments using microelectrode array (MEA) plates. Coating: One day before plating the cells, each 48-well MEA plate was pre-coated with a polyethyleneimine (PEI: 0.1%) solution and incubated for 1 h at 37&#8239;&#176;C. The MEA plate was then washed 3x with sterilized water and dried for 1 h. Then, the MEA plate was kept at 4&#8239;&#176;C overnight. High-density culture: 50,000 cells/well of the neurons were plated onto 48-well MEA plates. The cell seeding step was performed as described in section 2 of the above-described protocol. Laminin (20&#8239;&#181;g/mL) added culture medium (add 2 v/v% B-27, 2.5 mM Glutamax, and 100 &#181;g/mL of penicillin/streptomycin to the neurobasal medium) was used to plate the neurons. Thereafter, the neurons were cultured at 37&#8239;&#176;C, 5% CO2 in the culture medium. The media was fully exchanged on DIV 1 with the culture medium up to DIV 3. Ara-C was added at DIV 4 (final 0.2 &#181;M). From DIV 5 onward and 2 times a week, 50% of the media was changed with the culture medium. The activity of the neurons on each well of the MEA plate was recorded with an MEA system. (B) Spontaneous neuronal activity was acquired at 37 &#176;C under a 5% CO2 atmosphere using an MEA system at a sampling rate of 12.5 kHz/channel at DIV 21. Recordings from 4 channels out of the 16 channels within a well are shown. For all the recordings, a Butterworth band-pass filter (200-3,000 Hz) was applied. Arrowheads show the timing of synchronized burst firing. <a href="https://www.jove.com/files/ftp_upload/64872/64872fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Easy and Reproducible Low-Density Primary Culture using Frozen Stock of Embryonic Hippocampal Neurons at JoVE.com

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

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 ...

easy-reproducible-low-density-primary-culture-using-frozen-stock

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Neuroscience

2.3K Views.  Gunma University. 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.

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

An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are reprogrammed from human fibroblasts using episomal vectors. Colonies of iPSCs can be observed 30 days after initiation of fibroblast reprogramming. Pluripotent colonies are manually picked and grown in neural induction medium to permit differentiation into neural progenitor cells (NPCs). iPSCs rapidly convert into neuroepithelial cells within 1 week and retain the capability to self-renew when maintained at a high culture density. Primary mouse NPCs are differentiated into astrocytes by exposure to a serum-containing medium for 7 days and form a monolayer upon which embryonic day 18 (E18) rat cortical neurons (transfected with channelrhodopsin-2 (ChR2)) are added. Human NPCs tagged with the fluorescent protein, tandem dimer Tomato (tdTomato), are then seeded onto the astrocyte/cortical neuron culture the following day and allowed to differentiate for 28 to 35 days. We demonstrate that this system forms synaptic connections between iPSC-derived neurons and cortical neurons, evident from an increase in the frequency of synaptic currents upon photostimulation of the cortical neurons. This co-culture system provides a novel platform for evaluating the ability of iPSC-derived neurons to create synaptic connections with other neuronal populations.

A protocol to generate a co-culture system consisting of neurons derived from induced pluripotent stem cells (iPSCs), primary cortical neurons and astrocytes is described. This co-culture system allows detection of the formation of synaptic contacts and circuits between new, iPSC-derived neurons and pre-existing cortical neurons expressing channelrhodopsin-2.

Here we describe a protocol to generate a co-culture consisting of 2 different neuronal populations. Induced pluripotent stem cells (iPSCs) are ...

Developmental Biology

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 ...

Isolation and Culture of Rat Embryonic Neural Cells: A Quick Protocol

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied successfully to the isolation of mouse and human primary neurons and neural progenitors. Dissociated neurons are maintained in serum-free medium up to several weeks. These cultures can be used for nucleofection, immunocytochemistry, nucleic acids preparation, as well as electrophysiology. Older neuronal cultures can also be transfected with a good efficiency rate by lentiviral transduction and, less efficiently, with calcium phosphate or lipid-based methods such as lipofectamine.

We describe a rapid methodology to isolate and culture hippocampal and cortical neurons from rodent embryos. This protocol allows us to perform experiments in which nearly pure neuronal cultures are required.

We are describing a quick method to dissociate and culture hippocampal or cortical neurons from E15-17 rat embryos. The procedure can be applied ...

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 ...

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 ...

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.

Presented here is a protocol for the spontaneous generation of neurospheres enriched in neural progenitor cells from high density plated neurons. During the same experiment, when neurons are plated at a lower density, the protocol also results in prolonged primary rat neuron cultures.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have ...

Use of Primary Cultured Hippocampal Neurons to Study the Assembly of Axon Initial Segments

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, assembly and activity-dependent plasticity. Giant ankyrin-G, the master organizer of AIS, directly associates with membrane-spanning voltage gated sodium (VSVG) and potassium channels (KCNQ2/3), as well as 186 kDa neurofascin, a L1CAM cell adhesion molecule. Giant ankyrin-G also binds to and recruits cytoplasmic AIS molecules including beta-4-spectrin, and the microtubule-binding proteins, EB1/EB3 and Ndel1. Giant ankyrin-G is sufficient to rescue AIS formation in ankyrin-G deficient neurons. Ankyrin-G also includes a smaller 190 kDa isoform located at dendritic spines instead of the AIS, which is incapable of targeting to the AIS or rescuing the AIS in ankyrin-G-deficient neurons. Here, we described a protocol using cultured hippocampal neurons from ANK3-E22/23-flox mice, which, when transfected with Cre-BFP exhibit loss of all isoform of ankyrin-G and impair the formation of AIS. Combined a modified Banker glia/neuron co-culture system, we developed a method to transfect ankyrin-G null neurons with a 480 kDa ankyrin-G-GFP plasmid, which is sufficient to rescue the formation of AIS. We further employ a quantification method, developed by Salzer and colleagues to deal with variation in AIS distance from the neuronal cell bodies that occurs in hippocampal neuron cultures. This protocol allows quantitative studies of the de novo assembly and dynamic behavior of AIS.

Here, we described a protocol to quantitatively study the assembly and structure of the axon initial segments (AIS) of hippocampal neurons that lack pre-assembled AIS due to the absence of a giant ankyrin-G.

Neuronal axon initial segments (AIS) are sites of initiation of action potentials and have been extensively studied for their molecular structure, ...

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 ...