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 â€œNeuron Ballâ€ 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 ...

This protocol is suitable to investigate where presynaptic proteins originate:cell bodies or axons. And how presynaptic proteins accumulate in presynapses in an organized manner during the synapse formation. This technique can induce thousands of presynapses at the same time, and does not use any special apparatus to maintain axons in culture allowing efficient analysis of the formation of many presynapses in axons.Along with graduate student, Honami, demonstrating this procedure will be Rie Ishii, an undergraduate student in my laboratory. Begin this procedure with mouse euthanasia as described in the text protocol. Dissect the abdomen to obtain E16 embryos.Remove the brains from embryos carefully with the help of fine-tipped forceps, and transfer them into 60-millimeter cell culture dishes containing four milliliters of HEPES buffered salt solution or HBSS. Remove the meninges and cut the olfactory bulb. Separate cortices from each cerebral hemisphere using the fine tips of forceps under the stereo microscope and transfer to another 60-millimeter dish containing fresh HBSS.Use at least three to five embryos for each separate neuron ball culture. Cut the cortices into small pieces with microdissecting spring scissors in a laminar flow cell culture hood. Now, transfer minced cortices to a 15-milliliter tube.Trypsinize the minced cortices in four milliliters of 0.125%trypsin in HBSS for 4.5 minutes in a water bath at 37 degrees Celsius. Transfer the cell aggregates to a new 15-milliliter tube containing 10 milliliters of HBSS by a sterile transfer pipette. Incubate at 37 degrees Celsius for five minutes before repeating this step one more time.Then transfer the cell aggregates to a new 15-milliliter tube containing two milliliters of NGB medium, 0.01%DNase I, and 10%horse serum. Triturate the trypsinized cortices by repeatedly pipetting them up and down three to five times using a fire-polished fine glass Pasteur pipette. For preparing neuron balls, adjust the cell density in the cell suspension to one million cells per milliliter using NGB medium.Add seven milliliters of PBS to the bottom part of the culture dishes. Culture the cortical neurons as 10 microliter hanging drops containing 10, 000 cells per drop inside the upper lids of 10-centimeter culture dishes. Keep the dishes in an incubator for three days at 37 degrees Celsius with 5%CO2 under humidified conditions to allow for neuron ball formation.Coat poly-L-lysine or PLL onto the paraffin-beaded glass cover slips in 60-millimeter dishes using a 15 microgram per milliliter PLL solution in borate buffer. Keep them for at least one hour in a CO2 incubator at 37 degrees Celsius. After washing four times with PBS, transfer the PLL-coated cover slips to a four-well plate containing 350 microliters of NGB medium with three micromolar AraC in each well.AraC is added to the media to kill dividing cells. Incubate the four-well plates containing the PLL-coated cover slips in the CO2 incubator for at least 20 minutes to ensure the temperature of the medium reaches 37 degrees Celsius before transferring the neuron balls. At DIV 3 when neuron balls are formed very well, transfer them onto PLL-coated cover slips.Inside the four-well plate, add five neuron balls per well. At DIV 11 transfer 20 microliters from the suspension of streptavidin-coated magnetic particles to a microcentrifuge tube. Immobilize the beads to a handmade apparatus attached with neodymium permanent magnets and wash three times with 100 microliters of PBS-MCBC in 1.5-milliliter microcentrifuge tubes.After completely removing PBS-MCBC from the beads, add a predetermined volume of LRRTM2 stock to the washed beads. Incubate the mixture using a rotator at four degrees Celsius for one to two hours. Following incubation, wash the beads twice with 100 microliters of PBS-MCBC.Then, wash the beads with 100 microliters of NGB medium. Resuspend the LRRTM2 beads in 50 microliters of NGB medium for application to the neuron ball culture. Now cut the end of a yellow tip at 45-degree angle with a razor blade under the stereo microscope.Put the yellow tip end on the cell body area of a neuron ball and remove the cell bodies by suction. Apply the LRRTM2 and control beads on the neuron ball culture. Then submerge the beads to the bottom of the plates of the neuron ball culture for one minute using ferrite magnets to start pre-synapse formation.This procedure ensures to touchdown all beads at the same time. Fix and stain the neurons in the neuron ball culture after pre-synapse formation with beads as described in the text protocol. Capture differential interference contrast and immunofluorescence images under an inverted fluorescent microscope with a cooled CCD camera using a 60X oil immersion lens.Measure the immunofluorescence intensity in pre-synapses in the axon. Use immunofluorescence intensities of the region of interest on beads. Minus the off beads region intensity divided by the axonal intensity, along 20 microns from the beads.Minus background intensity. This ratio intensity provides the protein accumulation index. To quantify the accumulation level of a particular protein in a presynapse induced with LRRTM2 beads, always select the area that is two fields of view or more apart from the cell body.For accurate measurement, choose five different axonal fields per coverslip. Shown here is the application of LRRTM2 beads into neuron ball culture at DIV 11 induced accumulation of Munc18-1 in presynapses of axons of neuron balls. The beads on axons of neuron balls are visible in the phase microscopy images.And the accumulation of presynaptic proteins are observed by the immunofluorescence images. Even in axons which are removed cell bodies, accumulation of Munc18-1 was observed under the beads, similar to axons of neuron balls with cell bodies. When the peripheral region of the axonal sheet was analyzed by high magnification objective lens, vGlut1 and Munc18-1 accumulated clearly in presynapses of axons under the beads with and without cell bodies.Time course experiments demonstrated that accumulation of vGlut1 in presynapses increased significantly at 30 minutes. On the other hand, Munc18-1 accumulation started to increase significantly at two hours and reached a plateau at four hours. These data indicate that the synaptic vesicle protein vGlut1 accumulates in presynapses earlier than the active zone protein Munc18-1.The Munc18-1 accumulation in presynapses of Fmr1-KO neurons increased 1.5 times more than those in wild type, indicating involvement of FMRP in Munc18-1 accumulation. Protein synthesis inhibitor, anisomycin, suppressed the Munc18-1 accumulation significantly in axons with and without cell bodies. This indicates that the accumulation is protein synthesis dependent.Pipetting the trypsinized cortices using a fire-polished fine glass Pasteur pipette is an important step. Experimenters prepare the fire-polished pipettes with two to three different diameters, and chose a pipette with an appropriate diameter. Using this procedure, synaptic release from presynapses induced by LRRTM2 beads is measured by live imaging.This additional method would answer whether protein synthesis in axons is involved in the regulation of synaptic release. Using this method, researchers examine how presynaptic proteins accumulate in presynapses in an organized manner, as well as the location of the source of presynaptic proteins.

Research

JoVE Journal

Neuroscience

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 ...

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 ...

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 ...

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 ...

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 ...

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

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 ...

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 ...

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. ...

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 ...