Synopsis

Keywords: Spectroscopy, Neuroscience

The neurochemical substrate of the measures observed with transcranial magnetic stimulation remains unclear. MR spectroscopy may provide valuable insights into understanding the synaptic GABAergic and glutamatergic activity, however studies comparing these methods haven’t provided an adequate explanation. We aimed to compare GABA and glutamate concentrations with short-interval afferent inhibition measured with TMS. We found positive correlation between the amount of inhibition and tonic GABA levels, however this finding needs to be carefully scrutinized.

Introduction

Short-interval afferent inhibition (SAI) is an electrophysiology protocol that uses transcranial magnetic stimulation (TMS) to assess the inhibition in the motor cortex provoked by the sensory afferent input. In fact, TMS over the primary motor cortex (M1) allows to measure the synaptic GABAergic and glutamatergic activity indirectly¹. MR spectroscopy (MRS) remains the only method capable of quantifying GABA and Glutamate (Glu) in vivo². However, a proper understanding of the link between GABA and Glu concentration and SAI has not been yet investigated. We aimed to compare the tonic levels of GABA and Glu acquired with edited-MRS with the maximum amount of inhibition identified in the SAI curve. Moreover, we evaluated the possible link of single steps of SAI (peripheral stimulation and TMS over M1) with GABA and Glu.

Methods

7 healthy, right-handed subjects (19-33 years old) were scanned on a Philips Achieva 7T scanner with dual-transmit 32-channel receive array head coil. Small (15x15cm²) in-house made dielectric pad was placed over the motor cortex to improve the local B₁⁺ field. Spectroscopic voxel (2x2.5x2.5 cm³) was planned on a structural image and placed in the left M1. MRS was acquired using MEGA-sLASER with GOIA refocusing pulses³, interleaved outer volume suppression and VAPOR water suppression and following parameters: TR/TE=4000/74ms; 6ms gaussian editing pulses at 1.9ppm and 7.46ppm (edit-ON and edit-OFF respectively); 96 transients; 4kHz spectral bandwidth. Data were frequency and phase corrected, aligned and subtracted using custom-made script and FID-A⁴ functions to form a difference spectrum (DIFF). Edit-OFF and DIFF spectra were quantified with LCModel 6.3⁵. Basis sets were generated using custom-built fully localized density matrix simulations⁶. Quantification parameters for DIFF spectra were adapted from Zollner et al⁷. GABA (from DIFF-spectrum) and Glu (from OFF-spectrum) were referenced to total Cr (Cr+PCr) from OFF-spectrum.
In order to proceed with the SAI protocol, firstly we identified the perception threshold for the median nerve stimulation, which is the minimum stimulation that the subject can clearly perceive after the delivery of the electrical pulse (200us duration) on the wrist. Then, after identifying the motor hotspot, we measured the individual motor threshold at rest, which is the minimum TMS intensity capable of evoking motor evoked potentials (MEPs) higher or equal to 50 uV in 5 out of 10 consecutive stimuli⁸. Finally, SAI was performed and the maximum level of inhibition was selected on a temporal curve obtained after applying TMS (120% of resting motor threshold) for all time points between 20 and 30 milliseconds after the median nerve stimulation (90% of motor threshold) in steps of 1 ms.

Results

Spectra showed overall good spectral quality with mean CRLBs[%] of 5 (GABA) and 5.7 (Glutamate) (Fig. 1). Mean SNR/Linewidth was 40/7.9Hz and 54/8.8Hz for the DIFF-spectra and the OFF-spectra respectively as reported by LCModel. No significant correlation was found between GABA/tCr and motor threshold (r = 0.239, p-value = 0.27) or perception threshold (r = 0.001, p-value = 0.96), however a significant positive correlation was found between GABA/tCr and maximum inhibition from SAI protocol (r = 0.58, p-value = 0.05). There was no correlation between Glu/tCr and motor threshold (r = 0.001, p-value = 0.95), perception threshold (r = 0.042, p-value = 0.66) or maximum inhibition (r = 0.17, p-value = 0.36).

Discussion

In this study we show that while perception levels and motor threshold are not linked to the excitatory and inhibitory neurotrasmitters’ concentration in the sensory-motor cortex, the maximum level of inhibition after afferent input is related to the amount of Glu and GABA.
Previous pharmacological studies have shown that SAI is likely mediated by GABA and more precisely it showed reduction when benzodiazepines, which are GABA_A receptor agonist, were administered^9,10. Therefore, our finding is in line by showing that higher levels of GABA induce higher level of inhibition. On the other hand, previous studies did not show a convincing relationship between GABAergic inhibitory TMS protocols and concentrations of Glu and GABA^11,12 This might be due to methodological differences between our study and the previous ones or to the different mechanisms involved when the inhibition is evaluated after the direct transcranial stimulation or after the afferent input. Our results are preliminary and more datasets will need to be acquired to support our findings.

Acknowledgements

Supported by Lundbeck Fonden grant number R346-2020-1822.

References

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