ubject Number : S-16-NM-0098
Support Type : 機器利用
Title (English) : In vitro 3D cortical column formation by guided proliferation and differentiation of neuronal cells, induced by electro-magnetic oscillation
Username (English) : Agrawal Lokesh
Affiliation (English) :University of Tsukuba, Graduate School of Pure and Applied Sciences

1. Summary
We want to study the effect of Electromagnetic waves with different frequencies and time latencies, what happens with the membrane channels when applied in the neuronal culture. Each protein is made up with the special arrangement of alpha helix and beta pleated sheets. This arrangement of beta sheets provide the mechanical barrier for energy transmission and creates a barrier between two isolated groups of alpha helices. This separation creates an imaginary cavity architecture, which resonates and transmit information on a particular frequency with which it comes in the harmony. When we apply low voltage electromagnetic pulses with kilohertz to megahertz, frequencies it may initiate the firing in neurons because of increased activity of microtubule bundles and ion channels.

2. Experimental
Facility: Cell culture facility, Laser conforcal fluorescent microscope.
1. Experimental Setup:
1. Standard Protocol to culture Rat Brain
Cortex Primary Neuronal Cells (Lonza) :
Required chemicals:
R-Cx-500 Rat Brain Cortex (Cx) ≥ 1 ml cell suspension
Primary Neuron Basal Medium (200 ml), SingleQuotsTM: NSF-1, 4 ml; L-Glutamine, 2 ml; GA, 0.2 ml; Calcium staining dye Flura4.
Required Apparatus:-
35mm glass bottom Petri dish, Pipette man
,Incubator, Centrifuge, 0.2mm syringe filter,
Time-lapse microscope and AC function
generator.
1. Thawing of Cells / Initiation of Culture Process :-
Note: Doing a trypan blue viability count upon thaw is not recommended as live cells will also uptake the dye.
1. DAY 1: Remove a vial of cells from liquid nitrogen and place in a water bath preheated to 37°C. IMPORTANT: Do not centrifuge or vortex the cells. Keep the time between removing the vial from the liquid nitrogen tank and placing into a pre-heated water bath as short as possible.
2. After 2½ minutes, remove vial and disinfect the outside of the vial by wiping with 70% ethanol. Place in a laminar flow hood. Proceed with the next step immediately after thawing.
3. Gently transfer 1 ml cells into a 15 ml centrifuge tube and immediately add prewarmed medium drop-wise onto the cells, while rotating the tube by hand. This should take approximately 2 minutes.
IMPORTANT: Do not add the whole volume of medium at once to the cells. This may result in osmotic shock. If one vial of cells is to be used for several different experiments at one time, mix the cells first by pipetting slowly up and down once, then aliquot the cells into the appropriate vessels.
4. Mix cell suspension by inverting the tube carefully, twice. IMPORTANT: Do not vortex the cells.
5. Transfer cell suspension to appropriate flasks, petri dishes or well plates. See chart below for recommended volumes of medium.
6. Incubate the cells for 4 hours in a 37°C, 5% CO 2 incubator.
7. Remove the medium from the cells leaving a small volume to ensure the cells do not dry out and add fresh, pre-warmed medium.
8. Incubate the cells at 37°C with 5% Co2.

Cell death will be observed; Cultivation of the cells should be continued.
9. DAY 5: Change the medium.
10. For a longer period of cultivation, replace50% of the media with fresh, pre-warmed media every 3 to 4 days.

Volume of Medium Plating Format
1. 9 ml for 1 ml cells suspension
2. 200 μl/well 96-well plate
3. 1 ml/well 24-well plate

Maintenance
1. After initial media change on day 5, replace 50% of the growth media every 3 to 4 days.
2. Warm an appropriate amount of medium to 37°C in a sterile container. Remove 50% of the medium from the cell culture. Replace with the warmed, fresh medium and return the cells to the incubator.
3. Avoid repeated warming and cooling of the medium. If the entire contents are not needed for a single procedure, transfer only the required volume to a sterile secondary container.
4. Compensation for media loss due to evaporation should be taken into consideration. Add additional media whenever necessary.

Fig1: Double stained fixed cultured neuron Primary Antigen Chicken Anti-Map2 (1/400) and Mouse Anti-GAD65 (1/160), Secondary Antigen 1.AF 594 Anti chick IgG (1/500) and AF488 Anti-mouse IgG (1/500). Florescence imaging with confocal microscope (40X).

4. Flura4 Staining of calcium channels:
Chemical preparation:-
Preparation of 250 mM Stock Solution of probnecid :-
Add 1ml of Fluo-4 Direct calcium assay buffer to each 77mg vial of water soluble probenecid.
• Vortex until dissolved.
• Store at <= -20°C (Up to 6 months).

Preparation Of 2x-Loading Solution:-
• Add 10 ml fluo-4-calcium assay buffer + 200 μl probenecid stock solution to one bottle of Fluo-4-direct calcium reagent(Comp. A).
• Vortex and allow it to completely dissolve.
Loading of culture cells with 2X-fluo-4-
Calcium reagent:
• Add loading solution to petri dish amount should be exactly equivalent to culture medium in petri dish.
• Incubate the culture dish for at least 30-60 min at 37°C.
• Remove the solution containing dye and wash the dish with HBSS (Ca and Mg free) for three times.
• Add 2 ml pre-warmed live cell imaging solution.
• Observe under the Time-lapse microscope for the excitation 494nm and emission at 516nm.

2. Times lapse Recording set up (494 – 516 Green fluorescence):

Fig2: Experimental setup for time-lapse recording

We made very special arrangements to perform this experiment, as picturized in the above figures. Maintaining the environment of time-lapse chamber equipped with Co2 Cylinder and humidifier is very essential to keep the neuron culture in healthy condition for the live imaging of neuron firing.
Study under the subthreshold level of stimulus:-
1. Connect the source voltage with gold electrode.
2. Set the frequency of AC signal from Hz to MHz according to your interest.
3. Dip the both gold electrodes in the culture dish containing the medium.
4. Switch on the voltage source and apply the minimum bias required to stimulate the firing.
5. Observe under the time-lapse microscope.
6. Captured the images continuously for at least 20 min to observe the change in fluorescence with activation and inactivation of calcium channels.
7. Change the bias to activate and deactivate the number of calcium channels.

3. Results and Discussion
We could observe firing activities under the application of different voltage bias and frequencies. We are trying to repeat the results so we could get a clear video showing the protein activation induced firing.

4. Others
References:
1.Abbott, L.F. Lapique’s introduction of the integrate-and-fire model neuron (1907). Brain Research Bulletin 50, 303–304 (1999).
2. Hodgkin AL, Huxley AF, Katz B Measurements of current-voltage relations in the membrane of the giant axon of Loligo. Journal of Physiology 116, 424–448 (1952).
3. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ. Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflügers Archiv European Journal of Physiology 391, 85–100 (1981).
4. Izhikevich E. M., Edelman G. M., Large-Scale Model of Mammalian Thalamocortical Systems PNAS 105, 3593-3598 (2008).
5. Tecuci, G. Artificial intelligence. Wiley Interdisciplinary Reviews: Computational Statistics, 4, 168–180 (2012).
6. Can Neural Activity Propagate by Endogenous Electrical Field? Chen Qiu, Rajat S. Shivacharan, Mingming Zhang and Dominique M. Durand, Journal of Neuroscience 2 December 2015, 35 (48) 15800-15811
7. Spatial mapping of juxtacrine axo-glial interactions identifies novel molecules in peripheral myelination, Y. Poitelon, et al, Nature Communications 6, Article number: 8303 (2015)
8. Ion specific effects in bundling and depolymerization of taxol-stabilized microtubules, Daniel J. Needleman et al; Faraday Discuss., 2013,166, 31-45
9. Xu, K., Zhong, G., Zhuang, X., Actin, spectrin and associated proteins form a periodic cytoskeleton structure in axons, Science 339, 452-456 (2013).

Acknowledgement:
We would like to show our sincere gratitude to all faculties at Nanotechnology Platform in National Institute for material sciences, for their tremendous support and on for sharing their pearls of wisdom with us during the course of this research , specially Hattori san and Xianglan Li San who provide me hands on training on animal cell culture , Confocal microscope and Time lapse microscope. We really appreciate their cooperation during the course of experiment.
5. Publication/Presentation
1. Subrata Ghosh , Satyajit Sahu , Lokesh Agrawal , Takashi Shiga and Anirban Bandyopadhyay, “Inventing a co-axial atomic resolution patch clamp to study a single resonating protein complex and ultra-low power communication deep inside a living neuron cell ”, Journal of Integrative Neuroscience, Vol. 15, No. 3 (2016) 1–31
World Scientific Publishing Europe Ltd, DOI: 10.1142/S0219635216500321.