Transcranial magnetic stimulation promotes the proliferation of dopaminergic neuronal cells in vitro

Transcranial magnetic stimulation (TMS) is a safe and non-invasive treatment for neurological disorders. TMS has been approved as a treatment for major depressive disorders by the US Food and Drug Administration (FDA) in 2008. Due to the phenomenon of electromagnetic induction, a time-varying magnetic field induces an electric field in the conductive tissues in the brain, TMS has the ability to activate neurons in vivo. However, the effects of the magnetic fields on neurons in cell culture have not been investigated adequately. The magnetic fields affect the neurons when the potential across the neuronal membrane exceeds the threshold which in turn causes an action potential. Based on these theories, we investigated the effects of the magnetic fields generated by a monophasic stimulator with a 70 mm double coil on rat dopaminergic neuronal cell lines (N27). The directions of the magnetic fields in each coil of the double coil oppose each other. The effects of changing the direction of the magnetic field on N27 neurons was also investigated. The results of the experiments showed that both of the fields perpendicular to the coil surface promoted the proliferation of N27 dopaminergic neurons. In order to investigate the gene expression and protein expression affected by TMS, quantitative Polymerase Chain Reaction (qPCR) was used. Here we report changes in glial cell line-derived neurotrophic factor (GDNF) in dopaminergic neuronal cells (N27) after TMS treatment.


I. INTRODUCTION
Transcranial magnetic stimulation (TMS) is a neuromodulation technique capable of activating neurons in the brain non-invasively.2][3][4] The US Food and Drug Administration has approved TMS as a treatment for major depressive disorder in 2008.
In TMS, the major principle is electromagnetic induction.The time-varying magnetic field generated by the TMS stimulator induces an electric field in conductive brain tissues which activates neurons by depolarization. 5o far, limited studies have reported the effects of TMS on the proliferation of cells, especially neuronal cells in vitro.Abbasnia et al. has reported that repetitive TMS increased the proliferation and neuronal differentiation of the neural stem and progenitor cells from adult male mice. 6uo et al. has reported that repetitive TMS significantly increased the proliferation of adult neural stem cells after focal cerebral ischemia in the subventricular zone. 7To the best of our knowledge, the effects of TMS on the proliferation of dopaminergic neuronal cells (N27) and whether it is related to the directions of the magnetic fields generated by a TMS stimulator has not been investigated.
In this paper, our study aimed to investigate how TMS affects the proliferation of dopaminergic neuronal cells (N27) and if the effects are altered when applying magnetic fields with opposite directions.Magnetic fields used in the experiments were measurable while the induced electric fields were not with our current experimental setup.So we focused on the aspects of magnetic field such as direction and intensity.

A. TMS stimulator and figure-8 coil
TMS stimulators provide pulsed electric current to a coil to generate time-varying magnetic fields.In our experiments, the Magstim 200 2 monophasic stimulator with the Magstim D70 2 double 70 mm figure-8 coil was used.
In the experiments, N27 cells were divided into three groups, two treatment groups and one control group.Two treatment groups were stimulated with magnetic fields opposing each other generated by TMS stimulator and figure-8 coil.During stimulation, the coil was held horizontally and the cells in each treatment group were placed below the coil without gap.The distance between the coil surface and the N27 cells is approximately 5 mm.
The actual magnetic fields generated by TMS stimulator and the figure-8 coil were measured using a gaussmeter and a Hall probe.The origin position of measurement was the center of the figure-8 coil between two coil windings, and the measurement was collected along a straight line across the center of each winding.The measured magnetic field intensities were quantified every 10 mm at 5 mm above the coil to be consistent with the distance between cells and the coil surface during stimulation.When the power level was 100%, the maximum upward (pointing out) and downward (pointing in) magnetic fields were 1.4189 MA/m and 1.3045 MA/m respectively and the minimum was 0.193 MA/m.When the power level was set to 70%, the maximum values were correspondingly 1.0345 MA/m and 0.9743 MA/m and the minimum is 0.0528 MA/m.The maximum values of the measured magnetic fields occurred at the locations of 40 mm away from the center between the two coil windings, where minimum values occurred.

C. TMS treatment on N27 cells
On Day 0, for each group, an equal amount of N27 cells (5K) were seeded into 16 wells (4 by 4) on 96-well plates.We used 16 wells on each plate because the area that could be stimulated at the peak field intensity is limited.The two treatment groups were distinguished by the direction of magnetic fields and called "up" group and "down" group.
On Day 1, magnetic stimulation was performed on both treatment groups.The centers of the 16-wells were positioned as close to the centers of the coil windings as possible, where the maximum field occurred.The control group was placed in the same environment but at least 2 meters away from the TMS stimulator and coil to avoid extraneous influences.
The pulses used to treat each group lasted for 12 minutes with intervals of 4 seconds.The minimum discharge and recharge time for the capacitor is 4 seconds at the power level of "100%".The duration is supposed to be no longer than 12 minutes as the temperature of the figure-8 coil increases over time and would exceed the upper limit of the operation temperature of 40 • C. When the temperature of the coil exceeds 40 • C, the stimulator is put into its safe inactive default condition. 9fter a few trials, 12 minute treatment was deemed appropriate.
After 12 minute treatment, the 96-well plates of all the groups were placed back into the incubator at 37 • C with a humidified atmosphere of 5% CO 2 .
On Day 2, 24 hours after the TMS treatment, cell counting was performed for each group using MTS cell viability cell counting method.
In order to make further efforts to evaluate the effects of magnetic fields on the proliferation of N27 cells, we decreased the power level of the TMS stimulator to 70% and repeated the experiments with same procedure.The minimum discharge and recharge time for the capacitor was 3 seconds at the power level of 70%.We still used a 4 seconds interval between each pulse to ensure consistency.

D. MTS cell viability cell counting method
Promega's CellTiter 96 ® AQ ueous One Solution Cell Proliferation Assay was the method we used for cell counting in the experiments.
After 20 µl MTS reagent (CellTiter 96 ® AQ ueous One Solution Reagent) was added to each well, the plate was incubated at 37 • C in a humidified, 5% CO 2 atmosphere for 90 minutes.Then the absorbance at 490 nm with a 96-well plate reader was read and recorded. 10

E. Real-time quantitative polymerase chain reaction (qPCR) analysis
In order to further investigate the change in the N27 cells as a result of the applied magnetic fields, we performed real-time quantitative polymerase chain reaction (qPCR) to analyze Glial cell linederived neurotrophic factor (GDNF).GDNF is known to support growth and survival of dopaminergic neurons. 11,12eal-time quantitative polymerase chain reaction (qPCR) provides a simple and elegant method for the detection and quantification of DNA or RNA. 13,14

A. Difference between cell numbers in each group after TMS treatment at the power level of 100%
After TMS treatment, the numbers of viable cells in each well were counted by the MTS cell counting method.There were significant differences in the numbers of cells between the control group and treatment groups.This means the proliferation rate has been increased by the use of TMS treatment at a power level of 100% regardless of the direction of the magnetic field, upward or downward.Statistical analysis was performed using GraphPad Prism.The numbers of N27 cells in each well were the same on the first day and the wells were independent with each other.The results are shown in Figure 1 below.There are significant differences in the cell numbers between treatment groups and control group.

B. GNDF mRNA analysis
In order to find what caused the difference in cell growth in N27 cells, we analyzed the glial cell line-derived neurotrophic factor (GDNF) for both the treatment groups and the control group by doing qPCR analysis.The results of GDNF mRNA level are given in Fig. 2.
The results show that the down group significantly increased their GDNF mRNA levels and the up group slightly increased their GDNF mRNA levels.The GDNF mRNA levels show a good matching with the cell numbers shown in Fig. 1.

C. Difference between cell numbers in each group after TMS treatment at the power level of 70%
In order to further evaluate the effects of magnetic fields, we decreased the power level to 70% and did the same experiment following the exact same steps as before.The results are shown in Figure 3 below.
There are significant differences of the numbers of N27 cells between the control group and treatment groups and there is not much difference in the effects on N27 cell growth with opposite magnetic fields.Given the fact that the induced electric field amplitude is a minimum in the center of each coil winding 15 and the direction of the magnetic field minimally influences cell growth, another experiment was conducted.The goal of which was to see the effects of magnetic stimulation when the N27 cells were placed in the center of a coil between two windings, where the induced electric field amplitude is a maximum.
The results are shown in Figure 4 below that there is a significant difference of cell numbers between treatment group and control group.

IV. CONCLUSION
Our experiments investigated the effects of TMS on the proliferation of N27 cells and whether the effects are related to the direction of magnetic field generated by the TMS stimulator.
All the results showed there is a significant difference of the cell numbers between treatment groups and control group no matter whether the cells are placed under each coil winding separately or under the center between two windings.It can be concluded that TMS promotes the proliferation of dopaminergic neuronal cells (N27).The conclusion is supported by the analysis of GDNF mRNA levels with TMS treatment at the power level of 100%.Whereas, there is no evidence to prove that the direction of magnetic fields affects the positive results of TMS on the proliferation of N27 cells.

V. DISCUSSION
In our experiments, the conclusions are the same when putting the cells in the center of each coil winding or the center between two coil windings given the amplitudes of induced electric fields on these points are different.So far it is not clear how the positive effect of TMS on cell growth relates to the field intensity.This will be further investigated in future.
Our results of the experiments lead to the conclusion that TMS promotes the proliferation of dopaminergic neuronal cells (N27).But the mechanism behind these results has not been identified.The brain-derived neurotrophic factor (BDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF) and early growth response protein 1 (EGR1) will be of interest in the future study.

FIG. 1 .
FIG.1.Comparison of the numbers of N27 cells between control group and treatment groups after 12 minutes TMS treatment at a power level of 100%.("Down" and "Up" refer to the direction of magnetic fields).The statistically significant differences are indicated by asterisks as follows: * p<0.05, ** p<0.01, *** p<0.001.