Voltage-sensitive dye imaging of transcranial magnetic stimulation-induced intracortical dynamics

Abstract

Transcranial magnetic stimulation (TMS) is widely used in clinical interventions and basic neuroscience. Additionally, it has become a powerful tool to drive plastic changes in neuronal networks. However, highly resolved recordings of the immediate TMS effects have remained scarce, because existing recording techniques are limited in spatial or temporal resolution or are interfered with by the strong TMS-induced electric field. To circumvent these constraints, we performed optical imaging with voltage-sensitive dye (VSD) in an animal experimental setting using anaesthetized cats. The dye signals reflect gradual changes in the cells' membrane potential across several square millimeters of cortical tissue, thus enabling direct visualization of TMS-induced neuronal population dynamics. After application of a single TMS pulse across visual cortex, brief focal activation was immediately followed by synchronous suppression of a large pool of neurons. With consecutive magnetic pulses (10 Hz), widespread activity within this "basin of suppression" increased stepwise to suprathreshold levels and spontaneous activity was enhanced. Visual stimulation after repetitive TMS revealed long-term potentiation of evoked activity. Furthermore, loss of the "deceleration-acceleration" notch during the rising phase of the response, as a signature of fast intracortical inhibition detectable with VSD imaging, indicated weakened inhibition as an important driving force of increasing cortical excitability. In summary, our data show that high-frequency TMS changes the balance between excitation and inhibition in favor of an excitatory cortical state. VSD imaging may thus be a promising technique to trace TMS-induced changes in excitability and resulting plastic processes across cortical maps with high spatial and temporal resolutions.

Keywords: excitation–inhibition balance; plasticity; primary visual cortex.

Fig. 1.

Visualization of TMS-induced cortical activity using VSD imaging in a single experimental session. (A) Sketch of relative coil position (35-mm coil casing, blue), recording chamber (border outlined in gray, outer/inner diameter = 32/24 mm), and vascular pattern of the imaged region (cat V1, A, anterior; L , lateral). (Scale bar, 1 mm.) (Lower) Zoom in from Upper: The coil center was 9 mm medial and 8 mm posterior with respect to the center of the recording chamber (Materials and Methods). Yellow arrow indicates the first hemicycle current direction of the biphasic pulse. Measurements of cortical activity were achieved through metal-free optical and stereotactic settings (Materials and Methods). (B) Magnetic field strength and its timing measured separately in air (Materials and Methods shows estimates of induced electric field). (C) Spatiotemporal activity patterns induced by a single TMS pulse (Left trace) and 10 Hz TMS (Right trace, five pulses); green and red arrows mark time of stimulation. Activity was averaged over 40 repetitions. Colorbar indicates activity levels expressed as fractional change in fluorescence relative to blank condition (∆F/F). (D) Time courses of spatial averages across recording frames. Stippled black trace was recorded after application of TTX (10 Hz, 30 repetitions) to verify that no artifacts were present. Gray area depicts confidence levels (95%) of baseline activity. Ten hertz rTMS produced gradual buildup of activity after initial suppression present in both TMS conditions (green arrow overlaps first red).

Fig. 2.

Buildup of an excitatory cortical state through high-frequency rTMS (nine different experiments). (A) Initial TMS pulses induced strong suppression followed by rebound of small amplitude in the case of single-pulse stimulation (green). Contrastingly, in the case of 10 Hz (red) each consecutive TMS pulse gave rise to a stepwise increase in cortical activity. Arrows mark TMS pulse time (first red arrow is covered by the green). Color shadings depict SEM. When calculated from the suppressive baseline, amplitudes of activity following 10 Hz TMS were in the same range (right bar in B) and highly correlated with visually evoked responses (r = 0.81, pairwise comparisons). Note that for low-frequency stimulation, the first-pulse excitatory peak appeared less pronounced than that for 10 Hz, because its onset was scattered across experiments, possibly reflecting reduced excitability. (B) TMS-induced amplitudes of activity during early phase (white bar, mean across five time frames with lowest responses) and late phase (gray bar, mean between 500 ms and 800 ms; compare gray area in A) compared with visually evoked responses (black contour). Error bars depict SEM. (C) Spontaneous activity after 10 Hz rTMS (red) revealed monotonous increase (8 × 10⁻⁴ units/s, dashed line shows linear regression) not present after low-frequency stimulation (green).

Fig. 3.

Induction of LTP-like facilitation. After application of 10 Hz rTMS (red data points), visually evoked activity was enhanced for 30–90 min (n = 13 experiments) compared with pre-TMS times (blue). This effect was counteracted by subsequent application of 1 Hz TMS, resulting in LTD-like suppression (green, n = 4), but also declined to baseline levels without further interventions (n = 5). Gray bars at bottom mark TMS times; *P < 0.05, **P < 0.01, two-sample two-tailed t test; vertical gray lines show SEM.

Fig. 4.

Weakened inhibition after high-frequency rTMS. Traces depict evolution of visually evoked activity over time (spatial averages across image frames). Before application of TMS, a notch during the rising phase (blue curves) was evident, indicating inhibition (main text). In A–E, individual experiments are shown with the notch encircled. After 10 Hz rTMS notches were almost completely abolished (pink curves), suggesting weakened inhibition, leading to higher amplitudes and shorter response latencies (compare blue and pink curves). Controls with subsequent application of 1 Hz rTMS (D and E, green curves) showed reduction of response amplitudes and recurrence of the notch, signifying regained inhibition. In 4 of 13 experiments, the notch was absent from the start (example in F); the opposing effects of single-pulse vs. 10 Hz TMS on response amplitudes were, however, clearly present. (G, Left) Mean across 9 different experiments (colored areas show SEM). Pre-TMS (blue) activity during the notch phase was significantly lower (with maximal effect 75 ms after stimulus onset, **P < 0.01, two-tailed t test) than after 10 Hz rTMS (pink). Amplitudes at 75 ms in pre-TMS conditions were on average 2.6 ± 0.4 SEM, ×10⁻⁴ ∆F/F compared with 5.4 ± 0.7 SEM, ×10⁻⁴ ∆F/F after rTMS. (Lower Right) Time courses between −200 ms and 800 ms, stimulus onset at zero. Arrows point to differences in amplitudes. Square outlines zoom-in shown at Left. (Upper Right) Pre- and post-TMS (10 Hz) differences in visually evoked amplitudes (time averages across the gray-shaded timespan in Lower plot) for the individual experiments (n = 13).