Differential intensity-dependent effects of magnetic stimulation on the longest neurites and shorter dendrites in neuroscreen-1 cells

Abstract

Objective: Magnetic stimulation (MS) is a potential treatment for neuropsychiatric disorders. This study investigates whether MS-regulated neuronal activity can translate to specific changes in neuronal arborization and thus regulate synaptic activity and function.

Approach: To test our hypotheses, we examined the effects of MS on neurite growth of neuroscreen-1 (NS-1) cells over the pulse frequencies of 1, 5 and 10 Hz at field intensities controlled via machine output (MO). Cells were treated with either 30% or 40% MO. Due to the nature of circular MS coils, the center region of the gridded coverslip (zone 1) received minimal (∼5%) electromagnetic current density while the remaining area (zone 2) received maximal (∼95%) current density. Plated NS-1 cells were exposed to MS twice per day for three days and then evaluated for length and number of neurites and expression of brain-derived neurotrophic factor (BDNF).

Main results: We show that MS dramatically affects the growth of the longest neurites (axon-like) but does not significantly affect the growth of shorter neurites (dendrite-like). Also, MS-induced changes in the longest neurite growth were most evident in zone 1, but not in zone 2. MS effects were intensity-dependent and were most evident in bolstering longest neurite outgrowth, best seen in the 10 Hz MS group. Furthermore, we found that MS-increased BDNF expression and secretion was also frequency-dependent. Taken together, our results show that MS exerts distinct effects when different frequencies and intensities are applied to the neuritic compartments (longest neurite versus shorter dendrite(s)) of NS-1 cells.

Significance: These findings support the concept that MS increases BDNF expression and signaling, which sculpts longest neurite arborization and connectivity by which neuronal activity is regulated. Understanding the mechanisms underlying MS is crucial for efficiently incorporating its use into potential therapeutic strategies.

Figure 1

MS-induced current density on coverslips containing cultured NS-1 cells and the MS protocol. (A) A 3-D model of the experimental setup was constructed using COMSOL Multiphysics^® CAD software, including the geometries and relative positions of both the MS coil and coverslip during stimulations, defined by the electric current per unit area of a cross section. (B) The color bar represents the induced current density (A/mm²) distribution in the coverslip, where maximum and minimum measurements are colored red and blue, respectively. (C) For all MS frequency groups, MS cycles consisted of fifteen 2-sec stimulation trains followed by 2-sec intertrain intervals for a MS cycle of 1-min total. The pulse distribution per group is illustrated by each red line in the stimulation time frame. (D) MS treatment consisted of two MS cycles, as illustrated in (C), separated by a 5-min intercycle interval.

Figure 2

Neurite length and number were regulated by MS. NS-1 cells were cultured onto a collagen-coated coverslip. MS (1, 5 or 10 Hz) at 30% MO was applied starting from the second day, twice per day, for the next 3 days. The NS-1 cells were then fixed and stained for HCS CellMask Red. (A) NS-1 cells extended longer neurites, as compared to the sham group, after 3 days of treatment with 10 Hz MS, but not 1 Hz or 5 Hz MS. (B) The average number of neurites per NS-1 cell was significantly increased by 10 Hz MS. Bars show mean ± SEM values. * p<0.05, *** p<0.005 vs. sham MS group; ^## p <0.01 vs. 1 Hz MS group; ^♮♮p <0.01, ^♮♮♮p<0.005 vs. 10 Hz MS group; Two-way ANOVA with Bonferroni post-hoc tests.

Figure 3

Neurite growth was regulated by both MS frequency and intensity. Representative images show that neurite lengths and counts per NS-1 cell were regulated by MS, as compared to the sham MS group (G). NS-1 cells treated with 1 Hz MS (A, D) expressed shorter neurites, whereas 5 Hz MS (B, E) had negligible effects. 10 Hz MS (C, F), however, consistently expressed longer neurites and higher counts per cell. The graphs illustrate average neurite lengths (H) and neurite counts (I) for NS-1 cells treated with sham MS (sham) and all MS treatment groups (1, 5, or 10 Hz) at 30% MO or 40% MO. Bars showed mean ± SEM values. * p<0.05, ** p <0.01, *** p<0.005 vs. sham MS group; ^# p<0.05, ^## p <0.01 vs. 1 Hz MS group; ^Ω p<0.05 vs. 5 Hz MS group; ^♮ p<0.05 vs. 10 Hz MS group; Two-way ANOVA with Bonferroni post-hoc tests.

Figure 4

Degree of MS influence over longest neurite and shorter dendrite lengths. Neurite outgrowth measurements were broken down into longest neurites and shorter dendrites for further analysis. Graphs show either longest neurite (A) or shorter dendrite (C) lengths measured for NS-1 cells fixed with sham MS treatment (sham) or MS (1, 5 or 10 Hz) at 30% or 40% MO. (B) Further illustrates any trends in the percentage of longest neurites with specified greater length ranges for all test groups. Bars show mean ± SEM values. * p<0.05, ** p <0.01, *** p<0.005 vs. sham MS group; ^# p<0.05, ^## p <0.01, ^### p<0.005 vs. 1 Hz MS group; ^Ω p<0.05, ^ΩΩ p <0.01 vs. 5 Hz MS group; ^♮p<0.05, ^♮♮♮ p<0.005 vs. 10 Hz MS group; Two-way ANOVA with Bonferroni post-hoc tests.

Figure 5

MS intensity differentially increased longest neurite growth. The following results were derived from three representative experiments measuring neurite length and number in zones 1 and 2 of gridded coverslips after MS (30% MO) to either 1, 5, or 10 Hz. (A) Schematic of the gridded coverslip divided into Z1 and Z2, where NS-1 cells received minimal (Z1) and maximal (Z2) MS-induced current densities. Z1 was further segmented into Z1 inner (Z1_i) and Z1 outer (Z1_o) in order to test for effects from detailed changes in induced current density. Z1 and Z2 regions were established to distinguish cells either in or out of r_dz and their respective electric field distributions in our experimental model. If we consider 100% induced current density as maximal MS efficiency, Z1_i was subject to <~5% efficiency, Z1_o between ~10–30%, and Z2 primarily ~95% efficiency. Zonal differential data between Z1 and Z2 are illustrated via shorter dendrite (B) and longest neurite (C) lengths and percentage of longest neurites within specified greater lengths range groups (E). Similarly, Z1_i and Z1_o differential neurite lengths are compared in (D). Bars show mean ± SEM values. * p<0.05, ** p <0.01, *** p<0.005 vs. sham MS group; ^# p<0.05, ^### p<0.005 vs. 1 Hz MS group; ^Ω p<0.05 vs. 5 Hz MS group; ^♮ p<0.05, ^♮♮♮p<0.005 vs. 10 Hz MS group; Two-way ANOVA with Bonferroni post-hoc tests.

Figure 6

MS treatment increased BDNF levels in NS-1 cells. NS-1 cells were treated with MS at 1, 5 or 10 Hz and harvested after 3 days for ELISA analysis of BDNF protein content: proBDNF and mBDNF in cell lysates (A) and mBDNF in culture media (B). Bars show mean ± SEM values; n=4; * p<0.05, ** p <0.01, *** p<0.005 vs. sham MS group; ^# p<0.05, ^### p<0.005 vs. 1 Hz MS group; ^Ω p<0.05, ^ΩΩ p <0.01 vs. 5 Hz MS group; ^♮ p<0.05, ^♮♮♮ p<0.005 vs. 10 Hz MS group; Two-way ANOVA with Bonferroni post-hoc tests.

Figure 7

Proposed illustration of the regulation of neurite growth by MS. (A) Stimulation with MS under physiological conditions leads to the neurite branching and longest neurite elongation. A calcium gradient exists under physiological conditions where higher concentrations of calcium are near dendrites and the lowest calcium concentrations are in axons. (B) Overexcitation, such as treatment with high frequency/intensity MS, leads to sustained elevation of intracellular calcium concentrations overwhelming mechanisms to maintain the calcium gradient, thus inhibiting the neurite branching and longest neurite elongation.