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.
