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

Summary

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.

Abstract

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.

Introduction

Synapse formation is one of critical steps during development of neural circuits^1,2,3. 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 receptors^1,2. 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 diseases^4,5. 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 neurons^6,7. 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 bodies^9,10. 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.

Protocol

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 modifications^9,10. We also adopted several procedures from the Banker method for dissociated culture¹⁴.

1. Confirming the followings before starting the dissection
   1. Prepare all the required solutions and sterilize them by autoclave/filtration in advance.
   2. Keep ready all the instruments and materials to be used in each steps of this cortical neuron culture.
   3. Spray and wipe the laminar air flow cabinet, dissection table, stage plate of stereomicroscope, scissors, and forceps with 70% ethanol.
2. Euthanize the mouse upon application of CO₂ and dissect the abdomen to obtain E16 embryos.
3. 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 µM phenol red.
4. 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.
5. Cut the cortices into small pieces with microdissecting spring scissors in a laminar flow cell culture hood.
6. 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 °C.
   NOTE: This trypsinization time is critical for efficient neuron culture as the increasing time (> 4.5 min) leads to much more dead neurons.
7. Transfer the cell aggregates to a new 15 mL tube containing 10 mL of HBSS by a sterile transfer pipette, and incubate at 37 °C for 5 min. Repeat this step one more time.
8. 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.
9. 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.
10. For preparing neuron balls, take the above cell suspension and adjust the cell density to 1 x 10⁶ cells/mL using NGB medium.
11. Culture the cortical neurons as hanging drops containing 10,000 cells/drop (1 drop is 10 µL) inside the upper lids of 10 cm culture dishes.
12. 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 °C with 5% CO₂ under humidified condition to allow for neuron ball formation.

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.

1. Soak the coverslips in 1/20 diluted neutral non-phosphorous detergent in ceramic racks for 1-3 overnights.
2. Wash the coverslips 8 times in ultrapure water, and then sterilize in an oven at 200 °C for 12 h.
   NOTE: All the steps from here must be done in a laminar air flow cell culture hood.
3. 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.
4. (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 °C. Dip a Pasteur pipette into the paraffin, then rapidly touch it to make three spots near the edge of a coverslip.
5. Coat PLL (MW > 300,000) onto the paraffin-beaded glass coverslips in 60-mm dishes using PLL solution (15 μg/mL in borate buffer), and keep them for at least 1 h in a CO₂ incubator.
6. After washing 4 times with PBS, transfer the PLL-coated coverslips to a 4-well plate containing 350 µL of NGB medium in each well and cytosine β-D-arabinofuranoside hydrochloride (AraC, 3 μM) to the media to kill dividing cells.
7. 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 °C before transferring neuron balls.
8. At DIV 3 when “neuron balls” are formed very well, transfer them onto PLL-coated coverslips inside the 4-well plate (5 neuron balls/well) kept in the CO₂ incubator.
9. 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 °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.
10. 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.

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’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 –conjugated Protein A beads that are used for prototype LRRTM2 system⁸.

1. Preparation of biotinylated LRRTM2 beads
   1. 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.
   2. 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 °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.
   3. Take 20 µL from the suspension of streptavidin-coated magnetic particles (diameter: 4-5 µm) to a microcentrifuge tube. Immobilize the beads to a handmade apparatus attached with neodymium permanent magnets and wash three times with 100 µL 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.
   4. After removing completely PBS-MCBC from the beads, add predetermined volume of LRRTM2 stock (usually 500-1,000 µL, see Note 3.1.2) to the washed beads. Incubate the mixture using rotator at 4 °C for 1-2 h.
   5. Wash the beads twice with 100 µL of PBS-MCBC, and later with 100 µL of NGB medium.
   6. Resuspend the LRRTM2 beads in 50 µL of NGB medium for application to the neuron ball culture.
   7. Use same procedures for preparing the control beads (negative control) in another microcentrifuge tube except adding biotinylated-LRRTM2 proteins.
2. Removing cell bodies from neuron balls at DIV 11-12 and applying LRRTM2 beads
   1. Label the wells of a 4-well plate as “Cell body (+)” and “Cell body (-)”.
   2. Cut the ending of a yellow tip at 45° angle with a razor blade previously sprayed with 70% ethanol under stereomicroscope (Figure 1B).
   3. Put the yellow tip end on the cell body area of a neuron ball and remove the cell bodies by suction (Figure 1B).
   4. To identify each specified condition of the experiment, label the wells again as “LRRTM2 beads” and “Control beads”.
   5. 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.
   6. 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.
   7. 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.

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.

1. Fixing and staining neurons in neuron ball culture after presynapse formation with LRRTM2 beads
   1. Remove NGB medium and fix the neuron balls with 4% PFA in PBS (250 µL/well) for 20 min at room temperature, and then wash with 500 µL of PBS 4-times each 5 min.
   2. 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.
   3. Permeabilize the cells/axons of neuron ball culture with TBS containing 0.3% Triton X-100 for 5 min.
   4. Keep the cells 1 h for blocking with blocking buffer (0.1% Triton X-100 and 5% NGS (normal goat serum) in TBS).
   5. 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 °C.
   6. 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).
   7. Stain the nuclei of the cell bodies of neurons for 5 min with 4′,6-diamidino-2-phenylindole (DAPI, 1 µg/mL) in PBS.
   8. 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.
   9. Store the glass slides in a refrigerator at -20 °C until microscopy. Fluorescent signals of the glass slides are detectable for at least 1 year when the slides are stored at -20 °C.
2. Taking images under fluorescence microscope
   1. 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.
   2. Measure the IF intensity in presynapses in axon using following formula; IF intensities of Region of Interest on beads (ROI) – Off beads region intensity/Axonal intensity along 20 µm from beads – background intensity. This ratio intensity provides protein accumulation index (Figure 4A). Accomplish the measurements on original 16-bit images using Image J software.
   3. 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.
   4. For accurate measurement, choose 5-different axonal field (similar distance from cell bodies)/coverslip.
   5. Keep identical imaging conditions at different day and in between experiments

Results

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 ...

Discussion

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 chamber^20,21,22. One of advantages of this method is LRRTM2-beads. While LRRTM2 interacts with neurexin to form excitatory presynapses specifically^11,

Materials

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