High frequency repetitive Transcranial Magnetic Stimulation promotes long lasting phrenic motoneuron excitability via GABAergic networks

Abstract

Repetitive transcranial magnetic stimulation (rTMS) is a promising, innovative, and non-invasive therapy used clinically. Efficacy of rTMS has been demonstrated to ameliorate psychiatric disorders and neuropathic pain through neuromodulation of affected neural circuits. However, little is known about the mechanisms and the specific neural circuits via which rTMS facilitates these functional effects. The aim of this study was to begin revealing the mechanisms by which rTMS may tap into existing neural circuits, by using a well characterized spinal motor circuit - the phrenic circuit. Here we hypothesized that rTMS can be used to enhance phrenic motoneuron excitability in anesthetized Sprague Dawley rats. Multiple acute rTMS protocols were used revealing 10 Hz rTMS protocol induced a robust, long-lasting increase in phrenic motoneuron excitability, functionally evaluated by diaphragm motor evoked potentials (59.1 ± 21.1 % of increase compared to baseline 60 min after 10 Hz protocol against 6.0 ± 5.8 % (p = 0.007) for Time Control, -5.8 ± 7.4 % (p < 0.001) for 3 Hz, and 5.2 ± 12.5 % (p = 0.008) for 30 Hz protocols). A deeper analyze allowed to discriminate "responder" and "non-responder" subgroups among 10 Hz rTMS treated animals. Intravenous injections of GABA_A and GABA_B receptor agonists prior to 10 Hz rTMS treatment, abolished the enhanced phrenic motoneuron excitability, suggesting GABAergic input plays a mechanistic role in rTMS-induced phrenic excitability. These data demonstrate that a single high frequency rTMS protocol at 10 Hz increases phrenic motoneuron excitability, mediated by a local GABAergic "disinhibition". By understanding how rTMS can be used to affect neural circuits non-invasively we can begin to harness the therapeutic potential of this neuromodulatory strategy to promote recovery after disease or injury to the central nervous system.

Keywords: GABAergic modulation; Motoneuron excitability; Preclinical model; rTMS.

Fig. 1.. Effect of different rTMS protocols on phrenic motoneuron excitability.

(A) Schematic of magnetic coil used for Transcranial Magnetic Stimulation (TMS) positioned over the rat’s head. The proposed descending pathways that are stimulated are represented with action potential volleys. Pseudorabies virus is applied to the diaphragm for retrograde labeling of phrenic motor network. (B) Representative diaphragmatic motor evoked potentials (MEPdia) recordings before and 60 min after 3 Hz, 10 Hz and 30 Hz rTMS or Sham rTMS. The white arrow shows the MEPdia at baseline, the black arrow indicates the increase in MEPdia response, corresponding to an increase in phrenic motoneuron excitability. (C) Time course showing the evolution of the percentage of MEPdia compared to baseline values following Sham, 3 Hz, 10 Hz or 30 Hz rTMS treatment. * p < 0.01 compared to Sham rTMS, 3 Hz and 30 Hz groups; a p < 0.05 compared to 1, 10 and 20 min post-rTMS; b p < 0.05 compared to 1 min post-rTMS (Two-way ANOVA, Fisher LSD post-hoc test).

Fig. 2.. Meta-analysis of 10 Hz rTMS treated animals resulted in statistical separation of “responder” and “non-responder” subpopulations.

(A) Diaphragm motor evoked potential (MEPdia) amplitude before and 60 min following Sham and 10 Hz rTMS treatment groups. (B) Exponential regression of 10 Hz rTMS treated group statistically separated into 2 subpopulations (“Responder” and “Non-responder”) based on the Mean + Standard Deviation (SD) values of the Sham rTMS group. Each dot represents the percent of animals considered as “Responder” for Mean + given SD. The threshold for discriminating the two populations was set at Sham rTMS Mean + 2SD. (C) Time-course of percent change in MEPdia amplitude following indicated protocols compared to baseline, with 10 Hz rTMS separated into Responder and Non-responder groups. (D) Percent change from baseline in MEPdia amplitude at 60 min post-rTMS for Sham, Non-responder and Responder 10 Hz rTMS groups. # p < 0.001 compared to baseline (Wilcoxon t-test); * p < 0.05 compared to Sham rTMS and Non-responder groups; a p < 0.01 compared to 1, 10, 20 and 40 min post-rTMS; b p < 0.005 compared to 1 min post-rTMS (Two-way ANOVA, Fisher LSD post-hoc test); ** p < 0.05 compared to Sham rTMS and Non-responder groups (Kruskal-Wallis One-way ANOVA (Dunn’s Method)).

Fig. 3.. Experimental timeline and GABA agonists dosage.

(A) GABAergic agonists were delivered prior to Sham or rTMS treatment. The phrenic motoneuronal excitability was assessed 7 times (MEPdia). (B) Plasma concentration of GABA_A agonist (Clonazepam) and (C) GABA_B agonist (Baclofen) immediately after drug injection (Baseline) and 60 min post-rTMS. Paired t-test, * p < 0.001 compared to baseline.

Fig. 4.. Phrenic motoneuron excitability and mean arterial pressure following delivery of GABA agonist.

(A) Phrenic motoneuron excitability evaluated by MEPdia amplitude before and 5 min following GABA_A agonist injection (Clonazepam). (B) Phrenic motoneuron excitability evaluated by MEPdia amplitude before and 5 min following GABA_B agonist injection (Baclofen). (C) Mean arterial pressure before, 5 min and 60 min after 10 Hz or sham rTMS protocol with Clonazepam injection. (D) Mean arterial pressure before, 5 min and 60 min after 10 Hz or sham rTMS protocol with Baclofen injection. Paired t-test, # p < 0.01 compared to before GABA agonist injection.

Fig. 5.. 10 Hz rTMS protocol and phrenic motoneuronal excitability with GABA agonists.

(A) Representative MEPdia traces prior to and 60 min post-10 Hz rTMS, Sham rTMS + GABA_A agonist (Clonazepam), Sham rTMS + GABA_B agonist (Baclofen), 10 Hz rTMS + Clonazepam and 10 Hz rTMS + Baclofen protocols. The white arrow shows the MEPdia at baseline, the black arrows show the MEPdia increase following 10 Hz rTMS protocol. (B) Time-course of percent change in MEPdia amplitude following indicated protocols compared to baseline. # p < 0.05 compared to Sham rTMS + GABA_A agonist and 10 Hz rTMS + GABA_A agonist groups; * p < 0.009 compared to Sham rTMS + GABA_A agonist, Sham rTMS + GABA_B agonist, 10 Hz rTMS + GABA_A agonist and 10 Hz + GABA_B agonist; a p < 0.05 compared to 1, 10 and 20 min post-rTMS; b p < 0.05 compared to 1 min post-rTMS (Two-way ANOVA, Fisher LSD post-hoc test).

Fig. 6.. Anatomical localization of GABA_A and GABA_B on phrenic motor network identified using transsynaptic pseudorabies virus (PRV).

When applied onto the diaphragm, PRV infects phrenic motoneurons (A) (located in the cervical spinal cord level 3–5 in the rat). PRV is then retrogradely transported and trans-synaptically labels phrenic interneurons (B, E). Immunohistochemistry for GABA_A (C) and GABA_B (F) receptors used to demonstrate examples of phrenic GABAergic receptor expression (D, G). Orthogonal projections of overlays are demonstrated in D and G. White asterisk (*) in D and G denote high-magnification (40x) insets. CC: central canal. Scale bars: 50 μm in A, D, G; 2 μm in insets.