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>

<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" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairment+of+ciliary+dynamics+in+an+APP+knock-in+mouse+model+of+Alzheimer's+disease.">Impairment of ciliary dynamics in an APP knock-in mouse model of Alzheimer's disease.</a> Biochemical and Biophysical Research Communications. 610, 85-91 (2022).</li><li>Kobayashi, 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=Properties+of+primary+cilia+in+melanin-concentrating+hormone+receptor+1-bearing+hippocampal+neurons+in+vivo+and+in+vitro.">Properties of primary cilia in melanin-concentrating hormone receptor 1-bearing hippocampal neurons in vivo and in vitro.</a> Neurochemistry International. 142, 104902 (2021).</li><li>Koganezawa, N., 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=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" 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+neurons.">The role of drebrin in neurons.</a> Journal of Neurochemistry. 141 (6), 819-834 (2017).</li><li>Sekino, Y., Koganezawa, N., Mizui, T., 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=Role+of+drebrin+in+synaptic+plasticity.">Role of drebrin in synaptic plasticity.</a> Advances in Experimental Medicine and Biology. 1006, 183-201 (2017).</li><li>Togo, K., 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=Postsynaptic+structure+formation+of+human+iPS+cell-derived+neurons+takes+longer+than+presynaptic+formation+during+neural+differentiation+in+vitro.">Postsynaptic structure formation of human iPS cell-derived neurons takes longer than presynaptic formation during neural differentiation in vitro.</a> Molecular Brain. 14 (1), 149 (2021).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160; (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&#160;</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 
			CellVoyager CQ1</td>
			<td>YOKOGAWA</td>
			<td></td>
			<td>Phase contrast images</td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabino-furanoside (Ara-C)</td>
			<td>Sigma</td>
			<td>C-6645</td>
			<td>Diluted in dH2O (final concentration: 0.2 &#181;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>&#160;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 &#181;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>&#160;100 U/mL for normal plates</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>nacalai tesque</td>
			<td>26253-84</td>
			<td>100 &#181;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 106 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 dH2O (final concentrations: 1 &#181;M, 3 &#181;M, 10 &#181;M, 30 &#181;M, 100 &#181;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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JoVE Journal

Neuroscience

2.2K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

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 ...

Bilaminar Co-culture of Primary Rat Cortical Neurons and Glia

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or co-culture model. This system is suitable for a variety of experimental needs requiring either a glass or plastic growth substrate and can also be used for culture of other types of neurons.
Rat cortical neurons obtained from the late embryonic stage (E17) are plated on glass coverslips or tissue culture dishes facing a feeder layer of glia grown on dishes or plastic coverslips (known as Thermanox), respectively. The choice between the two configurations depends on the specific experimental technique used, which may require, or not, that neurons are grown on glass (e.g. calcium imaging versus Western blot). The glial feeder layer, an astroglia-enriched secondary culture of mixed glia, is separately prepared from the cortices of newborn rat pups (P2-4) prior to the neuronal dissection.
A major advantage of this culture system as compared to a culture of neurons only is the support of neuronal growth, survival, and differentiation provided by trophic factors secreted from the glial feeder layer, which more accurately resembles the brain environment in vivo. Furthermore, the co-culture can be used to study neuronal-glial interactions1.
At the same time, glia contamination in the neuronal layer is prevented by different means (low density culture, addition of mitotic inhibitors, lack of serum and use of optimized culture medium) leading to a virtually pure neuronal layer, comparable to other established methods1-3. Neurons can be easily separated from the glial layer at any time during culture and used for different experimental applications ranging from electrophysiology4, cellular and molecular biology5-8, biochemistry5, imaging and microscopy4,6,7,9,10. The primary neurons extend axons and dendrites to form functional synapses11, a process which is not observed in neuronal cell lines, although some cell lines do extend processes.
A detailed protocol of culturing rat hippocampal neurons using this co-culture system has been described previously4,12,13. Here we detail a modified protocol suited for cortical neurons. As approximately 20x106 cells are recovered from each rat embryo, this method is particularly useful for experiments requiring large numbers of neurons (but not concerned about a highly homogenous neuronal population). The preparation of neurons and glia needs to be planned in a time-specific manner. We will provide the step-by-step protocol for culturing rat cortical neurons as well as culturing glial cells to support the neurons.

Here we provide a protocol for culturing rat cortical neurons in the presence of a glial feeder layer. The cultured neurons establish polarity and create synapses, and can be separated from the glia for use in various applications, such as electrophysiology, calcium imaging, cell survival assays, immunocytochemistry, and RNA/DNA/protein isolation.

This video will guide you through the process of culturing rat cortical neurons in the presence of a glial feeder layer, a system known as a bilaminar or ...

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 ...

Assaying DNA Damage in Hippocampal Neurons Using the Comet Assay

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique.
	The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell 1,2. Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.

The comet assay is an efficient way of detecting single- and double-strand breaks, including alkali-labile sites and DNA-DNA/DNA-protein cross-links on the DNA in all cells including hippocampal neurons. The method takes advantage of the differential migration of DNA in an electric field due to differences in amount of DNA damage.

A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been ...

Preparation of Primary Neurons for Visualizing Neurites in a Frozen-hydrated State Using Cryo-Electron Tomography

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the structure of neurites is critical to understanding how these processes move materials and signals that support synaptic communication. Electron microscopy (EM) has been traditionally used to assess the ultrastructural features within neurites; however, the exposure to organic solvent during dehydration and resin embedding can distort structures. An important unmet goal is the formulation of procedures that allow for structural evaluations not impacted by such artifacts.
Here, we have established a detailed and reproducible protocol for growing and flash-freezing whole neurites of different primary neurons on electron microscopy grids followed by their examination with cryo-electron tomography (cryo-ET). This technique allows for 3-D visualization of frozen, hydrated neurites at nanometer resolution, facilitating assessment of their morphological differences. Our protocol yields an unprecedented view of dorsal root ganglion (DRG) neurites, and a visualization of hippocampal neurites in their near-native state. As such, these methods create a foundation for future studies on neurites of both normal neurons and those impacted by neurological disorders.

To preserve neuronal processes for ultrastructural analysis, we describe a protocol for plating of primary neurons on electron microscopy grids followed by flash freezing, yielding samples suspended in a layer of vitreous ice. These samples can be examined with a cryo-electron microscope to visualize structures at the nanometer scale.

Neurites, both dendrites and axons, are neuronal cellular processes that enable the conduction of electrical impulses between neurons. Defining the ...

Calcium Phosphate Transfection of Primary Hippocampal Neurons

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this method on hard-to-transfect cells like primary neurons. Here we describe our detailed protocol for calcium phosphate transfection of hippocampal neurons cocultured with astroglial cells.

Calcium phosphate precipitation is a convenient and economical method for transfection of cultured cells. With optimization, it is possible to use this ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

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 ...