Axon morphology and intrinsic cellular properties determine repetitive transcranial magnetic stimulation threshold for plasticity

Abstract

Introduction: Repetitive transcranial magnetic stimulation (rTMS) is a widely used therapeutic tool in neurology and psychiatry, but its cellular and molecular mechanisms are not fully understood. Standardizing stimulus parameters, specifically electric field strength, is crucial in experimental and clinical settings. It enables meaningful comparisons across studies and facilitates the translation of findings into clinical practice. However, the impact of biophysical properties inherent to the stimulated neurons and networks on the outcome of rTMS protocols remains not well understood. Consequently, achieving standardization of biological effects across different brain regions and subjects poses a significant challenge.

Methods: This study compared the effects of 10 Hz repetitive magnetic stimulation (rMS) in entorhino-hippocampal tissue cultures from mice and rats, providing insights into the impact of the same stimulation protocol on similar neuronal networks under standardized conditions.

Results: We observed the previously described plastic changes in excitatory and inhibitory synaptic strength of CA1 pyramidal neurons in both mouse and rat tissue cultures, but a higher stimulation intensity was required for the induction of rMS-induced synaptic plasticity in rat tissue cultures. Through systematic comparison of neuronal structural and functional properties and computational modeling, we found that morphological parameters of CA1 pyramidal neurons alone are insufficient to explain the observed differences between the groups. Although morphologies of mouse and rat CA1 neurons showed no significant differences, simulations confirmed that axon morphologies significantly influence individual cell activation thresholds. Notably, differences in intrinsic cellular properties were sufficient to account for the 10% higher intensity required for the induction of synaptic plasticity in the rat tissue cultures.

Conclusion: These findings demonstrate the critical importance of axon morphology and intrinsic cellular properties in predicting the plasticity effects of rTMS, carrying valuable implications for the development of computer models aimed at predicting and standardizing the biological effects of rTMS.

Keywords: axons; excitation; inhibition; morphology; organotypic tissue cultures; synaptic plasticity; whole-cell patch-clamp recordings.

Figure 1

10 Hz repetitive magnetic stimulation (rMS) induces synaptic plasticity in mouse CA1 pyramidal neurons. (A) Schematic illustration of the experimental setting. Organotypic tissue cultures are stimulated in a standard 35 mm petri dish filled with extracellular solution using a 70 mm figure-of-eight coil (900 pulses, 10 Hz, at 50% maximum stimulator output). (B) Overview of an organotypic tissue culture. Visualization of cytoarchitecture with DAPI. DG, Dentate gyrus; EC, entorhinal cortex; CA1 and CA3, Cornu Ammonis areas 1 and 3. Scale bar, 500 μm. (C) Patched CA1 pyramidal neurons filled with biocytin and identified post hoc with streptavidin-A488. Scale bar, 50 μm. (D,E) Sample traces and group data of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from mouse CA1 pyramidal neurons in sham-(control) and rMS-stimulated cultures 2–4 h after stimulation (control, n = 31 cells; rMS, n = 28 cells; Mann–Whitney test). (F,G) Sample traces and group data of GABA receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from mouse CA1 pyramidal neurons in sham-(control) and rMS-stimulated cultures 2–4 h after stimulation (control, n = 14 cells; rMS, n = 14 cells; Mann–Whitney test). Individual data points are indicated in this and the following figures by gray dots. Data are mean ± SEM. NS, not significant. *p < 0.05. **p < 0.01.

Figure 2

10 Hz repetitive magnetic stimulation (rMS) at 50% maximum stimulator output fails to induce synaptic plasticity in rat CA1 pyramidal neurons. (A) Overview images of a mouse and rat organotypic tissue culture. DG, Dentate gyrus; EC, entorhinal cortex; CA1 and CA3, Cornu Ammonis areas 1 and 3. Scale bar, 1500 μm. (B) Patched rat CA1 pyramidal neuron filled with biocytin and identified post hoc with streptavidin-A488. Scale bar, 50 μm. (C,D) Sample traces and group data of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from rat CA1 pyramidal neurons in sham-(control) and rMS-stimulated cultures 2–4 h after stimulation (control, n = 38 cells; rMS, n = 71 cells; Mann–Whitney test). (E,F) Sample traces and group data of GABA receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) recorded from rat CA1 pyramidal neurons in sham-(control) and rMS-stimulated cultures 2–4 h after stimulation (control, n = 12 cells; rMS, n = 9 cells; Mann–Whitney test). Data are mean ± SEM. NS, Not significant.

Figure 3

Modeling of electric fields in mouse and rat tissue cultures. (A) Visualization of the macroscopic electric field simulations from magnetic stimulation in vitro. (B) Three-dimensional mesh models of mouse and rat tissue cultures and the electric fields generated by a single magnetic pulse, respectively. (C) Comparison of the maximum electric field generated at distinct stimulation intensities in mouse and rat tissue cultures. The electric field generated in mouse slice cultures at 50% maximum stimulator output is attained with 53% maximum stimulator output in rat tissue cultures. (D) Group data of AMPA receptor-mediated mEPSCs recorded 2–4 h after stimulation from rat CA1 pyramidal neurons in sham-(control) and rMS-stimulated cultures; stimulation at 53% maximum stimulator output (control, n = 12 cells; rMS, n = 12 cells; Mann–Whitney test). Data are mean ± SEM. NS, not significant.

Figure 4

No significant differences in baseline network activity in mouse and rat tissue cultures. (A,B) Overview images of mouse and rat tissue culture on high-density microelectrode array chips. DG, dentate gyrus; EC, entorhinal cortex; CA1 and CA3, Cornu Ammonis areas 1 and 3. (C) Raster plots of spikes during a 10 min recording period in mouse and rat tissue cultures. (D–F) Group data of mean firing rate and mean field potential rate from mouse and rat tissue cultures (mouse, n = 4 cultures; rat, n = 5 cultures; Mann–Whitney test). Data are mean ± SEM. NS, not significant.

Figure 5

No significant morphological differences of CA1 pyramidal neurons in mouse and rat tissue cultures. (A) Examples of patched and biocytin-filled rat CA1 pyramidal neurons identified post hoc with streptavidin-A488, Scale bar, 100 μm. (B) Examples of three-dimensional neuronal reconstructions of mouse and rat CA1 pyramidal neurons. (C–H) Group data of mouse and rat apical and basal dendrites (mouse, n = 11 cells; rat, n = 11 cells; statistical comparisons for panels (C,D,G,H) were performed with Mann–Whitney test; statistical comparisons for panels (E,F) were performed with 2-way ANOVA). (I) Rat CA1 pyramidal neuron patched and filled with biocytin, identified post hoc with streptavidin-A488, and used for comprehensive neuronal reconstruction, encompassing dendritic and axonal neuronal structures. Scale bar, 50 μm. (J–L) Group data of mouse and rat axons [mouse, n = 6 cells; rat, n = 6 cells; statistical comparisons for panels (J,L) were performed with Mann–Whitney test; statistical comparisons for panel (K) were performed with 2-way ANOVA].

Figure 6

Multiscale single-cell modeling of electromagnetic stimulation. (A) Changes in membrane voltage, to electromagnetic stimulation were modeled in realistic dendritic and axonal morphologies from reconstructed mouse and rat CA1 pyramidal neurons. (B) Group data of realistic dendritic morphologies with a standardized artificial axon (mouse, n = 6 cells; rat, n = 6 cells; Mann–Whitney test). (C) Group data of simulations with realistic dendritic and axonal morphologies (mouse, n = 6 cells; rat, n = 6 cells; Mann–Whitney test). (D) Group data for mouse and rat CA1 pyramidal neurons, categorizing those with axons exhibiting lowest (left) and highest (right) rMS depolarization thresholds (mouse, n = 6 cells; rat, n = 6 cells; Kruskal-Wallis test). Data are mean ± SEM. NS, not significant. *p < 0.01.

Figure 7

Rat CA1 pyramidal neurons exhibit lower excitability in comparison to mice. (A) Sample traces from input–output recordings of CA1 pyramidal neurons of mouse and rat tissue cultures. (B,C) Group data of resting membrane potentials and input resistances from mouse and rat CA1 pyramidal neurons (mouse, n = 44 cells; rat, n = 56 cells; Mann–Whitney test). (D,E) Group data of action potential (AP) amplitude and threshold from mouse and rat CA1 pyramidal neurons (mouse, n = 44 cells; rat, n = 56 cells; Mann–Whitney test). (F) Current/frequency curve of CA1 pyramidal neurons of mouse and rat tissue cultures (mouse, n = 52 cells; rat, n = 63 cells; 2-way ANOVA). Data are mean ± SEM. NS, not significant. **p < 0.01. ***p < 0.001.

Figure 8

10 Hz repetitive magnetic stimulation (rMS) at 60% MSO induces synaptic plasticity in rat CA1 pyramidal neurons. (A) Group data of AMPA receptor-mediated miniature excitatory postsynaptic currents (mEPSCs) recorded from rat CA1 pyramidal neurons from sham-(control) and rMS-stimulated cultures (control, n = 34 cells; rMS, n = 16 cells; Mann–Whitney test). (B) Sample traces and group data of miniature inhibitory postsynaptic currents (mIPSCs) recorded from rat CA1 pyramidal neurons from sham- (control) and rMS- stimulated cultures (control, n = 14 cells; rMS, n = 17 cells; Mann–Whitney test. One data point outside of axis limits in mIPSC amplitude and frequency respectively). Data are mean ± SEM. NS, not significant. *p < 0.05. ***p < 0.001.