Intermittent Theta-Burst Transcranial Magnetic Stimulation Alters Electrical Properties of Fast-Spiking Neocortical Interneurons in an Age-Dependent Fashion

Abstract

Modulation of human cortical excitability by repetitive transcranial magnetic stimulation (rTMS) appears to be in part related to changed activity of inhibitory systems. Our own studies showed that intermittent theta-burst stimulation (iTBS) applied via rTMS to rat cortex primarily affects the parvalbumin-expressing (PV) fast-spiking interneurons (FSIs), evident via a strongly reduced PV expression. We further found the iTBS effect on PV to be age-dependent since no reduction in PV could be induced before the perineuronal nets (PNNs) of FSIs start to grow around postnatal day (PD) 30. To elucidate possible iTBS-induced changes in the electrical properties of FSIs and cortical network activity during cortical critical period, we performed ex vivo-in vitro whole-cell patch clamp recordings from pre-labeled FSIs in the current study. FSIs of verum iTBS-treated rats displayed a higher excitability than sham-treated controls at PD29-38, evident as higher rates of induced action potential firing at low current injections (100-200 pA) and a more depolarized resting membrane potential. This effect was absent in younger (PD26-28) and older animals (PD40-62). Slices of verum iTBS-treated rats further showed higher rates of spontaneous excitatory postsynaptic currents (sEPSCs). Based on these and previous findings we conclude that FSIs are particularly sensitive to TBS during early cortical development, when FSIs show an activity-driven step of maturation which is paralleled by intense growth of the PNNs and subsequent closure of the cortical critical period. Although to be proven further, rTMS may be a possible early intervention to compensate for hypo-activity related mal-development of cortical neuronal circuits.

Keywords: calcium-binding protein; cortical fast spiking interneurons; development; parvalbumin; repetitive transcranial magnetic stimulation.

Figure 1

Ex vivo—in vitro whole-cell patch recordings of Cy3-Wisteria floribunda agglutinin (WFA)-labeled fast-spiking interneurons (FSIs) after transcranial magnetic stimulation (TMS). (A) Drawing showing placement and orientation of the figure-of-eight coil used to apply the intermittent theta-burst (iTBS) protocol (see B) to the rat brain. At the orientation shown, a mediolaterally oriented electric field is induced within the brain, optimally suited to depolarize axons of the corpus callosum. (C) Red fluorescent Cy3-WFA-labeled perineuronal net of a cortical FSIs revealed by fluorescence microscopy. (D) The same neuron shown in infrared phase contrast illumination. (E) Same as in (D) but with the patch pipette attached to the cell. (F) Cell filled with Alexa488.

Figure 2

Histochemical staining of recorded FSIs for Alexa488, parvalbumin (PV) and Cy3-WFA. Photomicrographs of 30 μm-thick sections obtained from the 290 μm thick brain slices after recordings show Alexa488 stain (green), parvalbumin (PV, Alexa647, blue) and WFA (perineuronal nets, Cy3, red) of two different FSIs (A–D) at different magnification (20× in (A) and (C), 63× in (B) and (D)). Merged images to the left (A–D), individual stains to the right (A₁–D₃). The red arrows indicate labeling in the images of lower magnification. Notice, that the cell indicated by an arrow in (A) was PV-immunoreactive whereas the marked cell in (C) was PV-immunonegative.

Figure 3

Quantitative analysis of verum vs. sham intermittent theta-burst stimulation (iTBS) induced spike-firing for FSI tested at different age. The diagrams show mean rate of spike firing (spikes/s ± SEM) of FSIs induced by current injections between 100 and 500 pA for animals of different age (postnatal days, PD) which had received either sham (gray lines) or verum (black lines) iTBS. *p < 0.05 (t-test, 2-sided). Number of analyzed cells per age group (in ascending order of age): sham 6, 8, 6, 6, 11, 6; verum 9, 13, 8, 10, 18, 13.

Figure 4

Changes in cell membrane characteristics of sham and verum iTBS-treated rats of different age. Example voltage traces obtained with −50 to +115 pA current injections to determine membrane resting potential (MRP) and input resistance are shown to the upper left. Other membrane parameters were determined in voltage clamp mode, testing −100 mV to −10 mV at steps of 10 mV. Diagrams show mean values (± SEM) of MRP, input resistance, time constant, capacitance and action potential threshold plotted vs. age for sham (gray) and verum (black) iTBS-treated animals. *p < 0.05, **p < 0.01 (t-test, 2-sided). Number of analyzed cells per age group (in ascending order of age): sham 8, 8, 6, 7, 12, 6; verum 11, 15, 9, 10, 25, 15.

Figure 5

Frequency and amplitudes of spontaneous postsynaptic currents (sPSCs) in FSIs of sham or verum iTBS-treated rats of different age. (A) Example traces showing sPSC recordings in FSIs of sham (gray) and verum (black) iTBS-treated rats at PD35. (B–D) Bar diagrams show mean frequency of sPSCs (± SEM) for sham (gray) or verum (black) iTBS-treated rats of three different age ranges. Total sPSCs including excitatory (sEPSCs) and inhibitory (sIPSCs) currents were recorded with the GABA_A receptor blocker picrotoxin (PTX) not added to the bath solution (ACSF, left columns). sEPSCs were selected by blocking sIPSCs with PTX (right columns) which resulted in little reduction in sPSC rate. (E–G) Cumulative plots of sEPSC amplitudes for animals of different age having received either sham (gray) or verum (black) iTBS. Verum iTBS causes a shift towards larger sEPSCs primarily in the older animals (PD40–42). (H) Cumulative plot of sEPSC amplitudes for the sham-control animals of different age. sEPSC amplitudes increased between PD34–36 and PD40–42. *p < 0.05 (t-test, 2-sided), ^#p < 0.001 (Wilcoxon rank-sum test).