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,</su...

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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 ÃƒÂ¢Ã¢â€šÂ¬Ã…â€œNeuron BallÃƒÂ¢Ã¢â€šÂ¬Ã‚Â 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.

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 ...

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