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Investigating the effects of LI-rMS on single neurons at a range of different frequencies: anatomical and molecular changes

Chapter 1 outlines the increasing scientific base of the biological importance of low intensity magnetic stimulation effects. In particular, because initial studies indicate that LI-rTMS has therapeutic benefits and low intensity stimulation forms a peri-focal by-field of human rTMS, the effects of LI-rTMS merit systematic investigation. To understand the long-term effects of LI-rTMS at a brain systems level (such as cortical excitability), it is necessary to perform fundamental investigation of effects at the cellular and molecular level in CNS tissue. A small number of studies have started to investigate these effects; however comparison between studies is difficult due to high variability of experimental models, in particular the stimulation parameters used. A systematic investigation of the effects of different frequencies on a range of biological parameters at the single cell level, after both single or multiple stimulation sessions, has not been carried out.
Here we investigate the effects of LI-rMS on anatomical and molecular changes in single neurons at a range of different frequencies. We applied different LI-rMS to primary cortical cultures in order to systematically investigate the effects of single and multiple LI-rMS, with the help of a small custom-build magnetic stimulation device that was tailored for the specific in vitro set-up, to deliver a defined magnetic field in the tissue of interest.
We applied four consecutive stimulation sessions per frequency and quantified survival of different cell populations and changes in neuronal morphology. We then sought insight into the underlying mechanisms involved in these long-term effects, and investigated modulation of intracellular calcium in real time during stimulation.
Consistent with a wide range of studies, both in high and low intensity literature, we showed that intracellular calcium was increased by LI-rMS. We also applied pharmacological agents to demonstrate for the first time that the source of intracellular calcium changes resulted from intracellular stores. We showed that a single session of LI-rMS induced changes in the expression of genes involved in downstream calcium signalling cascades and that these changes were stimulation-specific and consistent with the changes in cellular viability observed after repeated stimulation.
Article 1
Grehl, S., Viola, H. M., Fuller-Carter, P. I., Carter, K. W., Dunlop, S. A., Hool, L. C., Sherrard, R.M., Rodger, J. (2015). Cellular and Molecular Changes to Cortical Neurons Following Low Intensity Repetitive Magnetic Stimulation at Different Frequencies. Brain Stimulation, 8 (1), 114-123. (Appendix E)
Cellular and molecular changes to cortical neurons following low intensity repetitive magnetic stimulation at different frequencies


Repetitive transcranial magnetic stimulation (rTMS) is used in clinical treatment to non-invasively stimulate the brain and promote long-term plastic change in neural circuit function (Pell et al., 2011; Thickbroom, 2007), with benefits for a wide range of neurological disorders (Adeyemo, Simis, Macea, & Fregni, 2012; Daskalakis, 2014; George, Taylor, & Short, 2013). In addition, there is increasing evidence that low intensity magnetic stimulation (LI-rTMS) may also be therapeutic, particularly in mood regulation and analgesia (Di Lazzaro et al., 2013; Martiny et al., 2010; Robertson et al., 2010). Nonetheless, clinical outcomes of rTMS and LI-rTMS are variable (Wassermann & Zimmermann, 2012) and greater knowledge of the mechanisms underlying different stimulation regimens is needed in order to optimise these treatments.
Investigating the mechanisms of both high and low intensity rTMS is important because most human rTMS protocols deliver a range of stimulation intensities across and within the brain. Human rTMS is most commonly delivered using butterfly figure-of-eight shaped coils (Fatemi-Ardekani, 2008; Thielscher & Kammer, 2004) to produce focal high-intensity fields that depolarise neurons in a small region of the cortex underlying the intersection of the 2 loops (Fatemi-Ardekani, 2008; Thielscher & Kammer, 2004), which in turn can modulate activity in down-stream neural centres (Aydin-Abidin, Trippe, Funke, Eysel, & Benali, 2008; Valero-Cabré, Payne, Rushmore, Lomber, & Pascual-Leone, 2005). However, this stimulation focus is surrounded by a weaker magnetic field such that a large volume of adjacent cortical and sub-cortical tissue is also stimulated, albeit at a lower intensity that is below activation threshold (Cohen et al., 1990; Deng et al., 2013). While the functional importance of this para-focal low-intensity stimulation in the context of human rTMS is unclear, low-intensity magnetic stimulation on its own modifies cortical function (Capone et al., 2009; Robertson et al., 2010) and brain oscillations (Cook et al., 2004). Moreover, animal and in vitro studies demonstrate that low-intensity stimulation alters calcium signalling (Pessina et al., 2001; Piacentini et al., 2008), gene expression (Mattsson & Simkó, 2012), neuroprotection (Yang et al., 2012) and the structure and function of neural circuits (Makowiecki, Harvey, Sherrard, & Rodger, 2014; Rodger et al., 2012). However, the mechanisms underlying outcomes of low-intensity magnetic stimulation, particularly in conjunction with different stimulation frequencies, have not been investigated.
To address this, we undertook a systematic investigation of the fundamental morphological and molecular effects of five repetitive low intensity magnetic stimulation (LI-rMS) protocols in a simple in vitro system with defined magnetic field parameters. We show for the first time that LI-rMS induces calcium release from intracellular stores. Moreover, we show specific effects of different stimulation protocols on neuronal survival and morphology and associated changes in expression of genes mediating apoptosis and neurite outgrowth. Taken together, our data demonstrate that even low intensity magnetic stimulation induces long-term modifications to neuronal structure, which might have implications for understanding the effects of high-intensity human rTMS in the whole brain.
C57Bl/6j mice pups were sourced from the Animal Resources Centre (Canning Vale, WA, Australia). Experimental procedures were approved by the UWA Animal Ethics Committee (03/100/957).
Tissue culture
To investigate changes in neuron biology following LI-rMS stimulation, we used neuronal enriched cultures from postnatal day 1 mouse cortex. Pups were euthanased by pentobarbitone sodium (150 mg/kg i.p.), decapitated and both cortices removed. Pooled cortical tissue was dissociated and prepared following standard procedures (Meloni, Majda, & Knuckey, 2001). Cells were suspended in NB media (Neurobasal-A, 2% B27 (Gibco®), 0.6 mg/ml creatine, 0.5 mM L-glutamine, 1 % Penicillin/Streptomycin, and 5 mM Hepes) and plated on round poly-D-lysine coated coverslips at a density of 75000 cells/well (day 0 in vitro; DIV 0). On DIV 3, half the culture medium was removed and replaced with fresh media containing cytosine arabinofuranoside (6 μM; Sigma) to inhibit glial proliferation. Cells were grown at 37°C in an incubator (5% CO2 + 95% air) for 10 days and half the medium was replaced on DIV 6 and 9. To ensure that any experimental effects were not due to either different litters or culture sessions, plated coverslips from each litter were randomly allocated to stimulation groups. The whole culture-stimulation procedure was repeated 3 times.

Repetitive Magnetic Stimulation

LI-rMS stimulation was delivered to cells in the incubator with a custom built round coil (8 mm inside diameter, 16.2 mm outside diameter, 10 mm thickness, 0.25 mm copper wire, 6.1 Ω resistance, 462 turns) placed 3 mm from the coverslip (Fig 2.1A) and driven by a 12 V magnetic pulse generator: a simple resistor-inductor circuit under control of a programmable (C-based code) micro-controller card (CardLogix, USA). The non-sinusoidal monophasic pulse (Peterchev et al., 2011) had a measured 320µs rise time and generated an intensity of 13 mT as measured at the target cells by hall effect (ss94a2d, Honeywell, USA) and assessed by computational modelling using Matlab (Mathworks, USA; Fig 2.1B,C). Coil temperature did not rise above 37oC, ruling out confounding effects of temperature change. Vibration from the bench surface (background) and the top surface of the coil were measured at 10 Hz stimulation using a single-point-vibrometer (Polytec, USA); coil vibration was within vibration amplitude of background (Fig 2.1D).
Stimulation was delivered for 10 continuous minutes per day at 1 of 5 frequencies: 1 Hz, which reduces, or 10 Hz which increases, cortical excitability in human rTMS (Fitzgerald et al., 2006; Hoogendam et al., 2010); we also used 100 Hz, consistent with very low intensity pulsed magnetic field stimulation (Ash et al., 2009; Di Lazzaro et al., 2013), continuous theta burst stimulation (cTBS: 3 pulses at 50 Hz repeated at 5 Hz) showing inhibitory effects on cortical excitability post-stimulation in human rTMS (Gamboa et al., 2011; Huang et al., 2005) or biomimetic high frequency stimulation (BHFS: 62.6 ms trains of 20 pulses, repeated at 9.75 Hz). The BHFS pattern was designed on electro-biomimetic principles (Martiny et al., 2010), based on the main parameter from our previous studies (Makowiecki et al., 2014; Rodger et al., 2012) which was modelled on endogenous patterns of electrical fields around activated nerves during exercise (patent PCT/AU2007/000454, Global Energy Medicine). The total number of pulses delivered for each stimulation paradigm is shown in Table 1. We chose a standard duration of stimulation of 10 minutes (rather than a standard number of pulses) because studies of brain plasticity reveal that 10 minutes of physical training or LI-rTMS is sufficient to induce functional and structural plasticity (Angelov et al., 2007; Makowiecki et al., 2014; Rodger et al., 2012). For all experiments, controls were treated identically but the coils were not activated. An overview of experiments and summary of experimental design is shown in Fig 2.1E.



To investigate the influence of different stimulation frequencies at the cellular level, we used immunohistochemistry to examine neuronal survival and the prevalence of different cell types. Cells plated on glass coverslips were grown in 12 spatially separated wells of a 24 well plate to ensure no overlap of magnetic field. Wells were stimulated for 10 minutes daily from DIV 6-9 and cells were fixed with 4% paraformaldehyde 24 hours after the last stimulation. Mouse anti-active Caspase-3 (1:50, Abcam) and TUNEL (DeadEnd™ Fluorometric TUNEL System, Promega) double labelling were carried out to identify apoptosis. Glia and neurons were labelled with rabbit anti-GFAP (1:500, Dako) or mouse anti-βIII Tubulin (1:500, Covance). Subpopulations of neurons were identified, using rabbit anti-calbindin D-28K (inhibitory and small excitatory neurons; 1:500, Chemicon (Brederode van, Helliesen, & Hendrickson, 1991)) or mouse anti-SMI-32 (excitatory neurons; 1:2000, Covance (DeFelipe, 1997)). Antibody binding was visualised using fluorescently labelled secondary antibodies (Alexa Fluor 546 and Alexa Fluor 488; Invitrogen). Cell nuclei were labelled with either Hoechst (1:1000, Sigma Aldrich) or Dapi (DeadEnd™). Coverslips were mounted with Fluoromount-G.

Histological Analysis

For each experimental group, histological analyses were performed blind to stimulation paradigm on 12-18 images containing cultured cells from 2-3 different litters. Five semi-randomly distributed images per immunostained coverslip were taken from locations underneath the desired magnetic field (13 mT), in order to analyse cells that had received similar stimulation intensity.
We counted cells labelled with the following antibody combinations: βIII Tubulin or GFAP (neurons/glia), Caspase-3 and TUNEL (apoptotic cells), or Calbindin or SMI-32 (inhibitory/excitatory neurons). Raw counts were normalized to the total cells numbers (Hoechst or DAPI labelled) in the analysed field (FA). Cells that were not immunolabelled for either marker were identified as ‘other’ and included in the total cell count.
Morphometric analysis was undertaken on individually visualized neurons. Calbindin labelled neurons had weakly labelled processes thus neurite morphology could not be reliably distinguished. Thus, only SMI-32 positive cells were analysed. For every cell, the longest neurite was traced and its total length calculated with Image J. To estimate neuronal morphology, fast Sholl analysis (Gutierrez & Davies, 2007) was performed, using an Image-Pro®Plus (Media Cybernetics, Inc.) based macro (M. Doulazmi, UPMC).
Calcium Imaging
To assess the mechanisms underlying LI-rMS effects, we measured real-time changes in intracellular calcium during stimulation. On DIV 6-10, cells were incubated in 1 µM Fura-2AM (Molecular Probes) supplemented media at 37°C for 90-120 min. Immediately prior to experimentation, cells were transferred to Fura-2AM supplemented imaging solution containing: 140 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, 0.5 mM MgCl2, 10 mM Glucose and 10 mM Hepes (pH 7.4). Intracellular calcium was assessed at 37°C as described previously (Viola, Arthur, & Hool, 2007) to evaluate ratiometric change and estimate intracellular concentration [Ca2+]i (nM). Fura-2 340/380 nm ratiometric fluorescence was captured using a Hamamatsu Orca ER digital camera attached to an inverted Nikon TE2000-U microscope (ex 340/380 nm, em 510 nm) and analysed by manually tracing cells in MetaMorph® 6.3 (Molecular Devices).
Ratiometric fluorescent values were recorded from 5 min pre-stimulation to 5 min post-stimulation (or control) at 1 minute time intervals. Analyses were made off-line such that the experimenter was blind to stimulation group. Ratiometric fluorescent values were averaged over the last 3 min of stimulation (µMain : minutes 8-10), and normalised to the pre-stimulation baseline (µPre: minutes 3-5). Percentage Fura-2 ratiometric signal change (Y%change) was calculated (Y%change = ((µPre – µMain )/ µMain ) * 100) (Fig 2.1E). Pilot experiments showed that there was no effect of culture age (DIV) on calcium responses, thus data were pooled across days.
Following each experiment, cells were fixed with 4% paraformaldehyde and immunostained for GFAP/βIII Tubulin/Hoechst, as described above, to confirm that imaged cells were neurons. Only data from βIII tubulin-positive neurons were included in the analysis.
To investigate the source of increased intracellular calcium, we assessed alterations in intracellular calcium when neurons were either placed in calcium-free imaging solution, or exposed to thapsigargin (3 μM, SIGMA) to deplete intracellular calcium stores. For calcium-free studies, cells were placed in calcium-free imaging solution (140 mM NaCl, 5 mM KCl, 0.5 mM MgCl2, 10 mM HEPES, 10 mM Glucose, 3 mM EGTA, pH 7.4) immediately prior to experimentation. To confirm that results were not based on changes in ion concentration, in some experiments we compensated for the drop in Ca2+ with an equi-molar replacement of Mg2+ to keep a constant surface charge effect across the membrane. Results showed no difference between these 2 solutions.
Thapsigargin studies (supplemented 10 min prior to experimentation) were performed in normal 2.5 mM calcium containing imaging solution. All imaging solutions were supplemented with 1 µM Fura-2AM. Cell viability was confirmed with propidium iodide (PI) post-stimulation and cells that were permeable to PI were excluded from subsequent analysis (Appendix A) .
PCR Array
To investigate the molecular events triggered by different frequency stimulation, changes in gene expression were examined in a separate series of cultures following a single stimulation at DIV 6. For each group (3 frequencies plus control), three replicates of ten wells underwent one stimulation session. Five hours after the end of stimulation, total RNA was extracted with Trizol (Life Technologies) followed by purification on RNeasy kit columns (Qiagen). cDNA was transcribed from 200 ng of total RNA using the RT² Easy First Strand cDNA Synthesis Kit (Qiagen). For each sample, 250 ng of cDNA was applied to the Mouse cAMP / Ca2+ Signaling Pathway Finder PCR Array and amplified on a Rotorgene 6000. Results were analysed by a researcher (K Carter), who did not know the tissue groupings, on the Qiagen RT2 Profiler PCR array data analysis (v3.5) using the geometric mean of housekeeping genes glyceraldehyde-3-phosphate dehydrogenase and glucuronidase beta. Normalized mean expression levels (log2(2-ΔCt)) were used to determine differentially expressed genes between each group and control. Changes in gene expression were analysed further in R 3.0.1 using the NMF package (Gaujoux & Seoighe, 2010), Ingenuity Pathways Analysis (IPA) and WebGestalt (Zhang, Kirov, & Snoddy, 2005).

Statistical analysis

Data from all groups were explored for outliers and normal distribution with SPSS Statistics 20 (IBM). For cell survival and calcium imaging experiments, effects of frequency were analysed with One-way Analysis of Variance (Kruskal-Wallis; H) and Mann-Whitney (U) pairwise comparisons with Bonferroni-Dunn correction where appropriate. Neurite length data was analysed with Univariate ANOVA (F). Gene expression levels from the PCR array were compared by two sample t-test. All values are expressed as mean ± SEM and considered significant at p < 0.05.


We delivered multiple sessions of LI-rMS (DIV6-9) to see the cumulative effects on the survival and morphology of single neurons. We then examined potential mechanisms underpinning these cellular effects by identifying acute changes induced by a single session of LI-rMS: (1) intracellular calcium during stimulation; (2) gene expression 5 hrs after a single stimulation session, an interval necessary for transcriptional changes to have occurred. The experimental design is summarized in Fig 2.1E.

Table of contents :

1.1 Stimulating the brain
1.1.1 A History
1.2 Electromagnetic principles
1.3 Stimulation parameters
1.3.1 The stimulator output
1.3.2 Coil design
1.3.3 Stimulation intensity
1.3.4 Temporal pulse spacing (frequency)
1.4 TMS application
1.5 Underlying biological mechanism of rTMS
1.5.1 Mathematical modelling
1.5.2 rTMS in animal models
1.5.3 Cortical excitability – Neuroplasticity
1.5.4 LTP-LTD
1.5.5 Calcium hypothesis
1.5.6 State dependent effects
1.5.7 Involvement of neuronal subclasses
1.5.8 Frequency effects on further signalling pathways Downstream activation – converging pathways Early response gene activation BDNF regulation
1.6 Low intensity (LI) magnetic stimulation
1.6.1 Frequency-specific effects – underlying mechanisms Neuroplasticity – Calcium signalling Intracellular signalling
1.6.2 In conclusion
1.7 AIMS
1.7.1 Advantages of in vitro systems
1.7.2 Rationale of aims and experimental models
A. The effects of LI-rMS on the individual neuron
B. The effects of LI-rMS on neuronal circuit repair
B.1 The cerebellum, cerebellar cortex and olivary nucleus circuit
B.2 Olivo-cerebellar development
B.3 Olivo-cerebellar injury
B.4 Molecules involved in reinnervation
B.5 Cerebellar rTMS
C. Custom tailoring LI-rMS delivery to the in vitro set-up
Article 1
Article 2
Article 3
5.1 What’s new
5.2 Theory of mechanism
5.2.1 Calcium hypothesis Applied calcium hypothesis Mechanism of stimulation-specific effects: cellular death Neural circuit-specific effects
5.2.2 Stimulation load
5.3 Stimulation delivery
5.3.1 Stimulation delivery in our specific in vitro set-ups
5.4 Why does it all matter?


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