Multiple blocks of intermittent and continuous theta-burst stimulation applied via transcranial magnetic stimulation differently affect sensory responses in rat barrel cortex

Abstract

Key points: Theta-burst stimulation (TBS) applied via transcranial magnetic stimulation is able to modulate human cortical excitability. Here we investigated in a rat model how two different forms of TBS, intermittent (iTBS) and continuous (cTBS), affect sensory responses in rat barrel cortex. We found that iTBS but less cTBS promoted late (>18 ms) sensory response components while not affecting the earliest response (8-18 ms). The effect increased with each of the five iTBS blocks applied. cTBS somewhat reduced the early response component after the first block but had a similar effect as iTBS after four to five blocks. We conclude that iTBS primarly modulates the activity of (inhibitory) cortical interneurons while cTBS may first reduce general neuronal excitability with a single block but reverse to iTBS-like effects with application of several blocks.

Abstract: Cortical sensory processing varies with cortical state and the balance of inhibition to excitation. Repetitive transcranial magnetic stimulation (rTMS) has been shown to modulate human cortical excitability. In a rat model, we recently showed that intermittent theta-burst stimulation (iTBS) applied to the corpus callosum, to activate primarily supragranular cortical pyramidal cells but fewer subcortical neurons, strongly reduced the cortical expression of parvalbumin (PV), indicating reduced activity of fast-spiking interneurons. Here, we used the well-studied rodent barrel cortex system to test how iTBS and continuous TBS (cTBS) modulate sensory responses evoked by either single or double stimuli applied to the principal (PW) and/or adjacent whisker (AW) in urethane-anaesthetized rats. Compared to sham stimulation, iTBS but not cTBS particularly enhanced late (>18 ms) response components of multi-unit spiking and local field potential responses in layer 4 but not the very early response (<18 ms). Similarly, only iTBS diminished the suppression of the second response evoked by paired PW or AW-PW stimulation at 20 ms intervals. The effects increased with each of the five iTBS blocks applied. With cTBS a mild effect similar to that of iTBS was first evident after 4-5 stimulation blocks. Enhanced cortical c-Fos and zif268 expression but reduced PV and GAD67 expression was found only after iTBS, indicating increased cortical activity due to lowered inhibition. We conclude that iTBS but less cTBS may primarily weaken a late recurrent-type cortical inhibition mediated via a subset of PV+ interneurons, enabling stronger late response components believed to contribute to the perception of sensory events.

Figure 1. Study design

The first block ‘pre-tests’ included recordings with a single metal electrode for finding upper layer 4 of the cortical barrel corresponding to the principal whisker (PW) and repeated recordings via the triple electrode bundle placed at the same site until spiking activity stabilized. Then, three blocks of measurements were performed (pre) using PW deflections at five different velocities (light grey), testing paired PW deflections at either 20 or 100 ms intervals (middle grey) and combined AW–PW stimulation also at intervals of 20 or 100 ms intervals (dark grey), mixed with single PW and AW deflections in a quasi-random order, before the first iTBS, cTBS or sham stimulation block had been applied (TMS). Identical measurement blocks were repeated after each TMS block (TMS1 to TMS5) and a further 60 and 120 min after the last TMS block (post1, post2). iTBS and cTBS were applied according to Huang et al. (2005). For further description of TMS, see Methods.

Figure 2. Changes in the dynamics of sensory MUA after theta-burst TMS

Peri-stimulus time histograms (PSTHs) show grand average MUA of sensory responses for experiments with either iTBS application (left column, mean of 28 recording sites), cTBS (middle column, 34 recording sites) or sham stimulation (right column, 23 recording sites). Black areas always show pre-TMS activity while grey areas show responses after one block of stimulation (upper panels, ‘1 × iTBS, cTBS or sham’), after five blocks of stimulation (middle panels, ‘5× …’) or 60–120 min after the last TMS block had been applied (lower panels, ‘post …’; post1 and post2 recording sessions averaged). A, responses obtained with a single deflection of the principal whisker (PW); B, responses obtained with 2-fold stimulation of the PW at 20 ms interval (PW–PW20ms). The grey-shaded rectangles indicate the time windows used for quantification of the early (8–18 ms) and late (19–39 ms) components of the sensory responses. Note that for PW–PW20ms the two analysis time windows refer to the early and late responses evoked by the second PW deflection. PW deflection was always performed with a velocity of 800 deg s⁻¹. The ordinate of the PSTHs (spikes per 30 stimuli per bin) refers to the number of spikes accumulated within each 1 ms bin with repetition of 30 identical stimuli. The arrows point to the artefacts resulting from the voltage step needed to move the piezoelectric actuator.

Figure 3. Statistics of iTBS and cTBS effects on sensory responses evoked by single PW or AW deflection

Diagrams show mean sensory response amplitudes (±SEM) before (pre), after one to five TMS blocks (TMS1, TMS2 … TMS5) and 60 and 120 min after the fifth TMS block (post1, post2) separately for the early (left column) and late (right column) response component. Spikes evoked by 30 stimuli of the same kind were summed within 8–18 ms after whisker deflection (800 deg s⁻¹) in the case of the early response and between 19 and 39 ms in the case of the late response. The number of spikes was then divided by the number of integrated bins (1 ms) to achieve the same scaling as used for the PSTHs in Fig.2 (spikes per 30 stimuli per bin). Data from sham stimulation experiments are shown in blue, iTBS in red and cTBS in green. Asterisks (*P < 0.05, **P < 0.01) indicate statistically significant differences from the ‘pre’ condition for each experimental condition (post hoc Dunnett's test, note colour code); hash symbols (#P < 0.05, ##P < 0.01) indicate statistically significant differences between iTBS and sham (red) or cTBS and sham (green) (post hoc Tukey test); and dollar symbols ($P < 0.05, $$P < 0.01) indicate statistically significant differences between iTBS and cTBS (post hoc Tukey test).

Figure 4. No correlation between changes in spectral power of the EEG and sensory response rates

A, means (±SEM) of the ratio of the power of the theta and delta frequency band of the EEG (θ/δ ratio) for the different time points (pre to post2) of the three experimental conditions. B–D, comparisons of changes in EEG power ratio with changes of sensory response spike rates (PW early response) for the different experimental conditions (B, sham; C, iTBS; D, cTBS). The dotted horizontal line gives a reference to the ‘pre’ θ/δ power ratio of the EEG. Statistically significant differences (P < 0.05) are shown between cTBS and sham condition (*) or between cTBS and iTBS (#, post hoc Tukey test).

Figure 5. No TMS effect on response gain

For each of the three experimental conditions (sham, iTBS, cTBS) the diagrams show mean early (continuous lines) and late (dashed lines) response rates evoked by five different whisker deflection velocities during the pre (black), TMS1 (dark grey) and TMS5 (light grey) conditions. The error bars were excluded here for better readability of the diagrams. Both iTBS and cTBS mainly induced vertical shifts of the curves without a change in slope. While early response components showed a clear stimulus–response relationship, late responses did not except when strongly increased after the fifth iTBS block. Only at deflection velocities of 800–1000 deg s⁻¹ did varying response saturation change the slope of the curves.

Figure 6. Changes in the amplitude of the second response with paired whisker stimulation following theta-burst TMS

Early and late response components of the second response evoked by paired whisker stimulation, as compared to Fig.3 (A, PW twice at 20 ms interval; B, AW–PW stimulation at 20 ms interval; C, simultaneous AW–PW stimulation). As in Fig.3, sham experiments are shown in blue, iTBS experiments in red and cTBS experiments in green. Asterisks (*P < 0.05, **P < 0.01) indicate statistically significant differences from the pre condition for each experimental condition (post hoc Dunnett's test, note colour code); hash symbols (#P < 0.05, ##P < 0.01) indicate statistically significant differences between iTBS and sham (red) or cTBS and sham (green) (post hoc Tukey test); and dollar symbols ($P < 0.05, $$P < 0.01) indicate statistically significant differences between iTBS and cTBS (post hoc Tukey test).

Figure 7. Changes in LFP waveforms evoked by single and paired PW stimulation after TBS

The diagrams show grand average LFPs obtained from 24 recording sites for the sham experiments (top row) and 36 recording sites in the iTBS and cTBS experiments (middle and bottom row, respectively). A, LFPs evoked by single PW stimulation; B, LFPs evoked by paired PW stimulation (PW–PW20). Shown are LFPs of the pre-TMS state (black), after one iTBS or cTBS block (dark grey) and after five iTBS or cTBS blocks (light grey). Open rectangles indicate the early (8–18 ms) and late (19–39 ms) time windows chosen for statistical analysis of MUA (compare with Fig.2). All LFP measurements were performed at the same time as the MUA recordings. Horizontal bars below the diagrams indicate statistically significant differences of LFP waveforms between TMS1 and pre (black) or TMS5 and pre (grey) at P < 0.03 (non-parametric permutation-based t test, two-sided, corrected for multiple measurements by Dunnett's test).

Figure 8. Histological verification of a recording site

The top image shows serum protein staining surrounding the electrode track (arrow) within layer 3 close to layer 4 of the right hemisphere. The middle image shows the barrel layout visualized by cytochrome oxidase (COX) staining within layer 4 about 120–150 μm deeper. The bottom image shows an overlay of the top and middle images and graphical assignment of the barrels. Accordingly, the track of the bundle of the three electrodes terminated close to the upper border of barrel D2.

Figure 9. TMS-induced changes of neuronal activity markers

A, example images of histological sections through sensorimotor cortex showing cells labelled via antibodies directed to c-Fos, GAD67, parvalbumin and calbindin and subsequent diaminobenzidine staining for animals of the sham, iTBS and cTBS series. Shown are only the upper cortical layers (1–4, pial side on top) which showed the strongest effects. Scale bar = 100 μm. B, Nissl-stained frontal section at the level of sensorimotor cortex including representation of the vibrissal pad showing the location of the region of interest (ROI) for counting labelled cells (rectangle, about 1 mm² reaching from pia mater to white matter, 0.720 mm width × 1.44 mm height, at 1.2–1.8 mm from bregma). Horizontal sections through the same area of the right hemisphere (planar to cortical surface, dashed lines) were prepared to verify the electrode tracks (see Fig.8). C, bar graphs showing the mean number (+SEM) of labelled neurons counted within the ROI of four sections obtained from each animal with successful immunohistochemistry (number of animals indicated below the bar diagrams). PV, parvalbumin; CB, calbindin; GAD67, acid decarboxylase; c-Fos, zif268, immediate early gene products. *P < 0.05 relative to sham, ##P < 0.01, ###P < 0.001 iTBS vs. cTBS (Student's t test).

Figure 10. Scheme of cortical connections affected by TMS

Stimulation of the axons of the corpus callosum (lightning symbol) results not only in antidrome activation of the pyramidal cells of origin, but also in orthodrome activation of collateral synapses at both inhibitory interneurons (CB- and PV-type neurons, numbers 2–5) and other pyramidal cells (number 6). All these connections are involved in intra- and inter-areal cortical processing. Recurrent perisomatic inhibition can be either local (numbers 3 and 4) or via a long loop involving pyramidal cells of other cortical layers or areas (indicated by the dashed rectangle, connection number 5). It is assumed that different populations of PV-type interneurons exist, one receiving primarily cortical input and being affected by TMS, and another population primarily receiving thalamic input (number 1) and supposed not to be (or less) affected by TMS (see Staiger et al. for different populations of PV-type interneurons).