This work is partly supported by JSPS Grant-in-Aid for Scientific Research (KAKENHI) (C) (No. 22500336, 25430068, 16K07061) (Y. Sasaki). We thank Dr. Terukazu Nogi and Ms. Makiko Neyazaki (Yokohama City University) for kindly providing biotinylated LRRTM2 protein. We also thank Honami Uechi and Rie Ishii for technical assistance.

The experiments described in this manuscript were performed according to the guidelines outlined in the Institutional Animal Care and Use Committee of the Yokohama City University.
1. Preparation of neuron balls as hanging drop culture (Days in vitro (DIV) 0-3)
NOTE: The procedures described here for the preparation of neuron ball culture are based on the method previously reported by the Sasaki group with some modifications9,10. We also adopted several procedures from the Banker method for dissociated culture14.
<ol>
	<li>Confirming the followings before starting the dissection
	<ol>
		<li>Prepare all the required solutions and sterilize them by autoclave/filtration in advance.</li>
		<li>Keep ready all the instruments and materials to be used in each steps of this cortical neuron culture.</li>
		<li>Spray and wipe the laminar air flow cabinet, dissection table, stage plate of stereomicroscope, scissors, and forceps with 70% ethanol.</li>
	</ol>
	</li>
	<li>Euthanize the mouse upon application of CO2 and dissect the abdomen to obtain E16 embryos.</li>
	<li>Remove the brains from embryos carefully with the help of fine tips of forceps and transfer them into 60 mm cell culture dishes containing 4 mL of HEPES Buffered Salt Solution (HBSS). 
	NOTE: The dissection medium HBSS contains 10 mM HEPES (pH 7.4), 140 mM NaCl, 5.4 mM KCl, 1.09 mM Na2HPO4, 1.1 mM KH2PO4, 5.6 mM D-glucose, and 5.64 &#181;M phenol red.</li>
	<li>Remove the scalp, cut the olfactory bulb, separate cortices from each cerebral hemisphere using the fine tips of forceps under stereomicroscope, and transfer to another 60 mm dishes containing fresh HBSS. Use at least 3-5 embryos for each separate neuron ball culture.</li>
	<li>Cut the cortices into small pieces with microdissecting spring scissors in a laminar flow cell culture hood.</li>
	<li>Transfer minced cortices to a 15 mL tube and trypsinize the minced cortices in 4 mL of 0.125% trypsin in HBSS for 4.5 min in a water bath at 37 &#176;C. 
	NOTE: This trypsinization time is critical for efficient neuron culture as the increasing time (&#62; 4.5 min) leads to much more dead neurons.</li>
	<li>Transfer the cell aggregates to a new 15 mL tube containing 10 mL of HBSS by a sterile transfer pipette, and incubate at 37 &#176;C for 5 min. Repeat this step one more time.</li>
	<li>Transfer the cell aggregates to a new 15 mL tube containing 2 mL of Neurobasal media containing GlutaMax, B27 supplement (NGB medium), 0.01% DNase I and 10% horse serum.</li>
	<li>Triturate the trypsinized cortices by repeatedly pipetting them up and down (3-5 times) using fire-polished fine glass Pasteur pipette. 
	NOTE: Diameter of fire-polished fine glass Pasteur pipette is very important as described in the Banker method paper14. If the pipette is too narrow to pass cortices, prepare pipettes possessing 2-3 different sizes, and try from larger pipette.</li>
	<li>For preparing neuron balls, take the above cell suspension and adjust the cell density to 1 x 106 cells/mL using NGB medium.</li>
	<li>Culture the cortical neurons as hanging drops containing 10,000 cells/drop (1 drop is 10 &#181;L) inside the upper lids of 10 cm culture dishes.</li>
	<li>Add 7 mL of phosphate buffered saline (PBS) to the bottom part of culture dishes, then keep in an incubator for 3 days at 37 &#176;C with 5% CO2 under humidified condition to allow for neuron ball formation.</li>
</ol>
2. Placing neuron balls on PLL-coated glass coverslips and culture maintenance (DIV 3-11)
NOTE: Before coating of glass coverslips with poly-L-lysine (PLL), washing the coverslips using detergent is important. Glass coverslips are sometimes not so clean for neuronal culture and uniform coating with PLL. Non-uniform PLL coating may result in uneven axonal extension of neuron balls.
<ol>
	<li>Soak the coverslips in 1/20 diluted neutral non-phosphorous detergent in ceramic racks for 1-3 overnights.</li>
	<li>Wash the coverslips 8 times in ultrapure water, and then sterilize in an oven at 200 &#176;C for 12 h. 
	NOTE: All the steps from here must be done in a laminar air flow cell culture hood.</li>
	<li>Transfer baked coverslips to 100-mm dishes. After sealing the dish by parafilm between a lid and a bottom dish, baked coverslips can be kept for long-term storage.</li>
	<li>(Optional) Apply paraffin dots to the coverslips. The dots make space to prevent neuron balls detaching from the coverslips during immunostaining by direct contact of neuron balls to plastic dishes. Melt paraffin in a suitable bottle in a hot water bath at about 90 &#176;C. Dip a Pasteur pipette into the paraffin, then rapidly touch it to make three spots near the edge of a coverslip.</li>
	<li>Coat PLL (MW &#62; 300,000) onto the paraffin-beaded glass coverslips in 60-mm dishes using PLL solution (15 &#956;g/mL in borate buffer), and keep them for at least 1 h in a CO2 incubator.</li>
	<li>After washing 4 times with PBS, transfer the PLL-coated coverslips to a 4-well plate containing 350 &#181;L of NGB medium in each well and cytosine &#946;-D-arabinofuranoside hydrochloride (AraC, 3 &#956;M) to the media to kill dividing cells.</li>
	<li>Keep this 4-well plate containing the PLL-coated coverslips in the CO2 incubator at least for 20 min to ensure the temperature of the medium reach 37 &#176;C before transferring neuron balls.</li>
	<li>At DIV 3 when &#8220;neuron balls&#8221; are formed very well, transfer them onto PLL-coated coverslips inside the 4-well plate (5 neuron balls/well) kept in the CO2 incubator.</li>
	<li>After 48 h, replace the neuron ball culture medium with fresh AraC-free NGB medium. Use a hot plate in a laminar air flow cell culture hood whose temperature is kept ready at 37 &#176;C immediately before this procedure. 
	NOTE: It is necessary to perform the medium changing as rapidly as possible on a hot plate to reduce the time that the cultures are at the outside of the CO2 incubator.</li>
	<li>Keep this neuron ball culture in the CO2 incubator for up to DIV 11. 
	NOTE: At DIV 11-12, neuron balls extending neurites up to 1-2 mm are used for experiments.</li>
</ol>
3. Applying LRRTM2 beads on neuron ball culture with or without cell bodies (DIV 11-12)
NOTE: Before application of LRRTM2 beads on neuron ball culture, it is recommended to remove cell bodies. Therefore, prepare LRRTM2 beads at first, then remove the cell bodies and later apply LRRTM2 beads to culture as early as possible. Biotinylated LRRTM2 is provided by the Nogi&#8217;s group (Yokohama City University) as conditioned medium. They use an expression vector including biotin acceptor sequence and biotin ligase from E. coli (BirA)15,16 to attach biotin to LRRTM2, and the expression vector is transfected to Expi293F cells included in the Expi293 Expression System. The vector information is available in Supplementary Figure 1. Biotinylated LRRTM2-conjugated streptavidin beads reduced background of immunostaining greatly compared to LRRTM2-Fc &#8211;conjugated Protein A beads that are used for prototype LRRTM2 system8.
<ol>
	<li>Preparation of biotinylated LRRTM2 beads
	<ol>
		<li>To remove excess biotin from conditioned medium of Expi293F cells expressing biotinylated LRRTM2, apply 1.7 mL of the conditioned medium mixed with 0.8 mL of PBS (total 2.5 mL) to PD-10 gel filtration column. PD-10 column is pre-equilibrated with 25 mL of ultrapure water and 25 mL of PBS.</li>
		<li>Elute with 3.5 mL of PBS and collect the flow-through as a LRRTM2 stock. 
		NOTE: This LRRTM2 stock can be dispensed to aliquots and stored at -80 &#176;C for long-term. Expression levels of biotinylated LRRTM2 sometimes vary from lot to lot. Thus, proper volume of LRRTM2 stock to conjugate to the beads should be determined to form presynapses enough on neuron balls, when new lot of LRRTM2 stock is used at first time.</li>
		<li>Take 20 &#181;L from the suspension of streptavidin-coated magnetic particles (diameter: 4-5 &#181;m) to a microcentrifuge tube. Immobilize the beads to a handmade apparatus attached with neodymium permanent magnets and wash three times with 100 &#181;L of PBS-MCBC in 1.5 mL microcentrifuge tubes. 
		NOTE: PBS-MCBC contains PBS including 5 mM MgCl2, 3 mM CaCl2, 0.1% BSA, and 0.1% Complete EDTA-free.</li>
		<li>After removing completely PBS-MCBC from the beads, add predetermined volume of LRRTM2 stock (usually 500-1,000 &#181;L, see Note 3.1.2) to the washed beads. Incubate the mixture using rotator at 4 &#176;C for 1-2 h.</li>
		<li>Wash the beads twice with 100 &#181;L of PBS-MCBC, and later with 100 &#181;L of NGB medium.</li>
		<li>Resuspend the LRRTM2 beads in 50 &#181;L of NGB medium for application to the neuron ball culture.</li>
		<li>Use same procedures for preparing the control beads (negative control) in another microcentrifuge tube except adding biotinylated-LRRTM2 proteins.</li>
	</ol>
	</li>
	<li>Removing cell bodies from neuron balls at DIV 11-12 and applying LRRTM2 beads
	<ol>
		<li>Label the wells of a 4-well plate as &#8220;Cell body (+)&#8221; and &#8220;Cell body (-)&#8221;.</li>
		<li>Cut the ending of a yellow tip at 45&#176; angle with a razor blade previously sprayed with 70% ethanol under stereomicroscope (Figure 1B).</li>
		<li>Put the yellow tip end on the cell body area of a neuron ball and remove the cell bodies by suction (Figure 1B).</li>
		<li>To identify each specified condition of the experiment, label the wells again as &#8220;LRRTM2 beads&#8221; and &#8220;Control beads&#8221;.</li>
		<li>Apply the LRRTM2 and control beads on neuron ball culture, and submerge to the bottom of the plates for 1 min using ferrite magnets to start presynapse formation. This procedure ensures to touchdown all beads at the same time. Especially, it would be effective for short time incubation (e.g., 30 min and 1 h) with LRRTM2 beads synchronously.</li>
		<li>To perform time-course experiments, label each separate well as 0 min, 30 min, 1 h, 2 h, 4 h and 18 h and apply LRRTM2 beads at the indicated time intervals.</li>
		<li>After the addition of LRRTM2 beads, incubate the neuron ball culture with the beads for specified time (0 min to 18 h) to form presynapses.</li>
	</ol>
	</li>
</ol>
4. Immunostaining and microscopy
NOTE: Fix the cells for 4 h via incubation with LRRTM2 beads in the experimental conditions with and without cell bodies, as the axons tend gradually to die in absence of cell bodies after 4 h. In case of time course with LRRTM2 beads, fix the cells at the indicated specified time.
<ol>
	<li>Fixing and staining neurons in neuron ball culture after presynapse formation with LRRTM2 beads
	<ol>
		<li>Remove NGB medium and fix the neuron balls with 4% PFA in PBS (250 &#181;L/well) for 20 min at room temperature, and then wash with 500 &#181;L of PBS 4-times each 5 min.</li>
		<li>Wash the fixed cells with TBS (Tris-buffered saline: 50 mM Tris-HCl (pH 7.3) and 150 mM NaCl) for more than 5 min.</li>
		<li>Permeabilize the cells/axons of neuron ball culture with TBS containing 0.3% Triton X-100 for 5 min.</li>
		<li>Keep the cells 1 h for blocking with blocking buffer (0.1% Triton X-100 and 5% NGS (normal goat serum) in TBS).</li>
		<li>Incubate the cells with primary antibodies; anti-rabbit-vGlut1 (vesicular glutamate transporter 1 (1:4000)) and anti-mouse-Munc18-1 (1:300) diluted with antibody diluent for overnight at 4 &#176;C.</li>
		<li>Wash the coverslips 4 times with immunofluorescence (IF) buffer (0.1% Triton X-100 and 2% BSA in TBS) and incubate for 30 min with fluorophore (Alexa dye)-conjugated 2nd antibodies; anti-mouse-IgG-Alexa 555 (1:500), anti-rabbit-IgG-Alexa 488 (1:1000).</li>
		<li>Stain the nuclei of the cell bodies of neurons for 5 min with 4&#8242;,6-diamidino-2-phenylindole (DAPI, 1 &#181;g/mL) in PBS.</li>
		<li>Wash the coverslips three times with PBS and then mount on glass slides using mounting media containing 167 mg/mL poly (vinyl) alcohol and 6 mg/mL N-propyl gallate.</li>
		<li>Store the glass slides in a refrigerator at -20 &#176;C until microscopy. Fluorescent signals of the glass slides are detectable for at least 1 year when the slides are stored at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Taking images under fluorescence microscope
	<ol>
		<li>Capture differential interference contrast and IF images under an inverted fluorescent microscope with a cooled CCD camera using 60X oil immersion lens. For image acquisition software, use a software installed microscope system. Use Image J as image analysis software.</li>
		<li>Measure the IF intensity in presynapses in axon using following formula; IF intensities of Region of Interest on beads (ROI) &#8211; Off beads region intensity/Axonal intensity along 20 &#181;m from beads &#8211; background intensity. This ratio intensity provides protein accumulation index (Figure 4A). Accomplish the measurements on original 16-bit images using Image J software.</li>
		<li>To quantify the accumulation level of particular protein in presynapse induced with LRRTM2 beads, always select the area away from 2-field of view or more apart from the cell body with microscope (60X). 
		NOTE: The selection of area in neuron ball for imaging is crucial as dense axons are near the cell body and periphery of neuron ball can provide single axon.</li>
		<li>For accurate measurement, choose 5-different axonal field (similar distance from cell bodies)/coverslip.</li>
		<li>Keep identical imaging conditions at different day and in between experiments</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We developed novel method to examine presynapse formation stimulated with LRRTM2-beads using neuron ball culture. Currently, most of presynapse formation assay includes poly-D-lysine (PDL)-coated beads and dissociated culture/microfluidic chamber20,21,22. One of advantages of this method is LRRTM2-beads. While LRRTM2 interacts with neurexin to form excitatory presynapses specifically11,<sup cla...

Synapse formation is one of critical steps during development of neural circuits1,2,3. Formation of synapses that are specialized compartments composed of pre- and post-synapses is a complex and multistep process involving appropriate recognition of axons and dendrites, formation of active zone and postsynaptic density, and proper alignment of ion channels and neurotransmitter receptors1,2. In each process, many kinds of synaptic proteins accumulate to these specialized compartments in proper timing by transporting synaptic proteins from cell bodies and/or by local translation in the compartments. These synaptic proteins are considered to be arranged in organized manner to form functional synapses. Dysfunction of some synaptic proteins involving synapse formation results in neurological diseases4,5. However, it remains unclear how synaptic proteins accumulate in proper timing.
To investigate how synaptic proteins accumulate in organized manner, it is necessary to examine accumulation of synaptic proteins in chronological order. Some reports demonstrated live imaging to observe synapse formation in dissociated culture of neurons6,7. However, it is time-consuming to find neurons which just start synapse formation under microscopy. To observe accumulation of synaptic proteins efficiently, synapse formation must start at the time when researchers want to induce the formation. The second challenge is to distinguish accumulation of synaptic proteins due to transport from cell bodies or local translation in synapses. For that purpose, translation level is necessary to be measured under the condition that does not allow transport of synaptic proteins from cell bodies.
We developed novel presynapse formation assay using combination of neuron ball culture with beads to induce presynapse formation8. Neuron ball culture is developed to examine axonal phenotype, due to the formation of axonal sheets surrounding cell bodies9,10. We used magnetic beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2) that is a presynaptic organizer to induce excitatory presynapses (Figure 1A)11,12,13. By using the LRRTM2 beads, presynapse formation start at the time when the beads are applied. This means that presynapse formation starts in thousands of axons of a neuron ball at same times, thus it allows to examine precise time course of accumulation of synaptic proteins efficiently. In addition, neuron ball culture is easy to block transport synaptic proteins from soma by removing cell bodies (Figure 1B)8. We have already confirmed that axons without cell bodies can survive and are healthy at least 4 h after removal of cell bodies. Thus, this protocol is suitable to investigate from where synaptic proteins are derived (cell body or axon), and how synaptic proteins accumulate in organized manner.

<table>
	<tbody>
		<tr>
			<td>Antibody diluent</td>
			<td>DAKO</td>
			<td>S2022</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 594 AffiniPure Donkey Anti-Mouse IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>715-585-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure Donkey Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse anti-Munc18-1</td>
			<td>BD Biosciences</td>
			<td>610336</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Alubumin (BSA)</td>
			<td>Nacalai Tesque</td>
			<td>01863-48</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-Culture Treated Multidishes (4 well dish)</td>
			<td>Nunc</td>
			<td>176740</td>
			<td></td>
		</tr>
		<tr>
			<td>Complete EDTA-free</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>cooled CCD camera</td>
			<td>Andor Technology</td>
			<td>iXON3</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Matsunami</td>
			<td>C015001</td>
			<td>Size: 15 mm, Thickness: 0.13-0.17 mm</td>
		</tr>
		<tr>
			<td>Cytosine &#946;-D-arabinofuranoside (AraC)</td>
			<td>Sigma-Aldrich</td>
			<td>C1768</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;,6-Diamidino-2-phenylindole Dihydrochloride (DAPI)</td>
			<td>Nacalai Tesque</td>
			<td>11034-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclease 1 (DNase I)</td>
			<td>Wako pure chemicals</td>
			<td>047-26771</td>
			<td></td>
		</tr>
		<tr>
			<td>Expi293 Expression System</td>
			<td>Thermo Fisher Scientific</td>
			<td>A14635</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Sigma-Aldrich</td>
			<td>H1270</td>
			<td></td>
		</tr>
		<tr>
			<td>image acquisition software</td>
			<td>Nikon</td>
			<td>NIS-element AR</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>NIH</td>
			<td>Image J</td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
		<tr>
			<td>Inverted fluorecent microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal media</td>
			<td>Thermo Fisher Scientific</td>
			<td>#21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum (NGS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>#143-06561</td>
			<td></td>
		</tr>
		<tr>
			<td>N-propyl gallate</td>
			<td>Nacalai Tesque</td>
			<td>29303-92</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Nacalai Tesque</td>
			<td>26126-25</td>
			<td></td>
		</tr>
		<tr>
			<td> 
			Paraplast Plus</td>
			<td>Sigma-Aldrich</td>
			<td>P3558</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Hydrobromide (MW &#62; 300,000)</td>
			<td>Nacalai Tesque</td>
			<td>28359-54</td>
			<td></td>
		</tr>
		<tr>
			<td>poly (vinyl alcohol)</td>
			<td>Sigma</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr>
			<td>Prepacked Disposable PD-10 Columns</td>
			<td>GE healthcare</td>
			<td>17085101</td>
			<td></td>
		</tr>
		<tr>
			<td>rabbit anti-vesicular glutamate transporter 1</td>
			<td>Synaptic Systems</td>
			<td>135-302</td>
			<td></td>
		</tr>
		<tr>
			<td>SCAT 20X-N (neutral non-phosphorous detergent)</td>
			<td>Nacalai Tesque</td>
			<td>41506-04</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin-coated magnetic particles</td>
			<td>Spherotech Inc</td>
			<td>SVM-40-10</td>
			<td>diameter: 4-5 &#181;m</td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Nacalai Tesque</td>
			<td>35501-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Nacalai Tesque</td>
			<td>18172-94</td>
			<td></td>
		</tr>
	</tbody>
</table>

presynapse formation assay using presynapse organizer beads and &ldquo;neuron ball&rdquo; culture

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in appropriate order by transport from cell bodies and/or local translation in immature synapses. However, it is not fully understood how synaptic proteins accumulate in synapses in proper order. Here, we present a novel method to analyze presynaptic formation by using the combination of neuron ball culture with beads to induce presynapse formation. Neuron balls that is neuronal cell aggregates provide axonal sheets far from cell bodies and dendrites, so that weak fluorescent signals of presynapses can be detected by avoiding overwhelming signals of cell bodies. As beads to trigger presynapse formation, we use beads conjugated with leucine-rich repeat transmembrane neuronal 2 (LRRTM2), an excitatory presynaptic organizer. Using this method, we demonstrated that vesicular glutamate transporter 1 (vGlut1), a synaptic vesicle protein, accumulated in presynapses faster than Munc18-1, an active zone protein. Munc18-1 accumulated translation-dependently in presynapse even after removing cell bodies. This finding indicates the Munc18-1 accumulation by local translation in axons, not transport from cell bodies. In conclusion, this method is suitable to analyze accumulation of synaptic proteins in presynapses and source of synaptic proteins. As neuron ball culture is simple and it is not necessary to use special apparatus, this method could be applicable to other experimental platforms.

Here, we show representative results of accumulation of presynaptic proteins in LRRTM2-induced presynapses of axonal sheets of neuron ball culture. As presynaptic proteins, we analyzed the excitatory synaptic vesicle protein vGlut1 and the active zone protein Munc18-1. We also examined time course of accumulation of vGlut1 and Munc18-1 in presynapses, and obtained results indicating source of Munc18-1 in presynapses using axons removing cell bodies and a protein synthesis inhibitor. Recently, we have investigated a role ...

Watch this Scientific Journal Video about Presynapse Formation Assay Using Presynapse Organizer Beads and Ã¢â‚¬Å“Neuron BallÃ¢â‚¬Â Culture at JoVE.com

Presynapse Formation Assay Using Presynapse Organizer Beads and &ldquo;Neuron Ball&rdquo; Culture

Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of &#8220;neuron ball&#8221; culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

During neuronal development, synapse formation is an important step to establish neural circuits. To form synapses, synaptic proteins must be supplied in ...

presynapse-formation-assay-using-presynapse-organizer-beads-neuron

Research

JoVE Journal

Neuroscience

8.1K Views.  Yokohama City University Graduate School of Medical Life Science. Presynapse formation is a dynamic process including accumulation of synaptic proteins in proper order. In this method, presynapse formation is triggered by beads conjugated with a presynapse organizer protein on axonal sheets of “neuron ball” culture, so that accumulation of synaptic proteins is easy to be analyzed during presynapse formation.

Video: Presynapse Formation Assay Using Presynapse Organizer Beads and “Neuron Ball” Culture

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. The existence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS) using a novel serum&#8208;free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs from different regions of the embryonic CNS. These in vitro expanded NSCs are multipotent and can give rise to the three major cell types of the CNS. While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable and consistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue and provides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brain microdissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in NSA culture. After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of the NSCs for future experiments.

This video protocol demonstrates the application of the neurosphere assay for the isolation and expansion of neural stem cells from the ganglionic eminences of embryonic day 14-mouse brain.

In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs during the life ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

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 ...

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (&#947;-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. 
During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. 
To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts.

Inhibitory Synapse Formation in a Co-culture Model Incorporating GABAergic Medium Spiny Neurons and HEK293 Cells Stably Expressing GABAA Receptors

The molecular mechanisms that co-ordinate the formation of inhibitory GABAergic synapses during ontogeny are largely unknown. To study these processes,we have developed a co-culture model system which incorporates embryonic medium spiny GABAergic neurons cultured together with stably transfected human embryonic kidney 293 (HEK293) cells expressing functional GABAA receptors.

Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA ...

Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.
Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.
Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.
On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena ...

Live-cell Measurement of Odorant Receptor Activation Using a Real-time cAMP Assay

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. To efficiently and accurately deorphanize ORs, we combine the use of a heterologous cell line to express mammalian ORs and a genetically modified biosensor plasmid to measure cAMP production downstream of OR activation in real time. This assay can be used to screen odorants against ORs and vice versa. Positive odorant-receptor interactions from the screens can be subsequently confirmed by testing against various odor concentrations, generating concentration-response curves. Here we used this method to perform a high-throughput screening of an odorous compound against a human OR library expressed in Hana3A cells and confirmed that the positively-responding receptor is the cognate receptor for the compound of interest. We found this high-throughput detection method to be efficient and reliable in assessing OR activation and our data provide an example of its potential use in OR functional studies.

Characterizing the function of odorant receptors serves an indispensable part in the deorphanization process. We describe a method to measure the activation of odorant receptors in real time using a cAMP assay.

The enormous sizes of the mammalian odorant receptor (OR) families present difficulties to find their cognate ligands among numerous volatile chemicals. ...

Live Imaging of Primary Cerebral Cortex Cells Using a 2D Culture System

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or postmitotic neurons. Later, some progenitors switch to a gliogenic fate, adding to the astrocyte and oligodendrocyte populations. Using time-lapse video-microscopy of primary cerebral cortex cell cultures, it is possible to study the cellular and molecular mechanisms controlling the mode of cell division and cell cycle parameters of progenitor cells. Similarly, the fate of postmitotic cells can be examined using cell-specific fluorescent reporter proteins or post-imaging immunocytochemistry. More importantly, all these features can be analyzed at the single-cell level, allowing the identification of progenitors committed to the generation of specific cell types. Manipulation of gene expression can also be performed using viral-mediated transfection, allowing the study of cell-autonomous and non-cell-autonomous phenomena. Finally, the use of fusion fluorescent proteins allows the study of symmetric and asymmetric distribution of selected proteins during division and the correlation with daughter cells fate. Here, we describe the time-lapse video-microscopy method to image primary cerebral cortex murine cells for up to several days and analyze the mode of cell division, cell cycle length and fate of newly generated cells. We also describe a simple method to transfect progenitor cells, which can be applied to manipulate genes of interest or simply label cells with reporter proteins.

Live imaging is a powerful tool to study cellular behaviors in real time. Here, we describe a protocol for time-lapse video-microscopy of primary cerebral cortex cells that allows a detailed examination of the phases enacted during the lineage progression from primary neural stem cells to differentiated neurons and glia.

During cerebral cortex development, progenitor cells undergo several rounds of symmetric and asymmetric cell divisions to generate new progenitors or ...

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.