Understanding novel neuromodulation pathways in tDCS: brain stem recordings in rats during trigeminal nerve direct current stimulation

Abstract

tDCS is widely assumed to cause neuromodulation via the electric field in the cortex acting directly on cortical neurons. However, recent evidence suggests that tDCS may indirectly influence brain activity through cranial nerve pathways, notably the trigeminal nerve, but these neuromodulatory pathways remain unexplored. To investigate the first stages in this potential pathway we developed an animal model to study the effect of trigeminal nerve direct current stimulation (TN-DCS) on neuronal activity in the principal sensory nucleus (NVsnpr) and the mesencephalic nucleus of the trigeminal nerve (MeV). We conducted experiments on twenty-four male Sprague Dawley rats (n = 10 NVsnpr, n = 10 MeV during anodic stimulation, and n = 4 MeV during cathodic stimulation). DC stimulation, ranging from 0.5 to 3 mA, targeted the trigeminal nerve’s marginal branch. Concurrently, single-unit electrophysiological recordings were obtained using a 32-channel silicon probe, encompassing three 1-min intervals: pre, during, and post-stimulation. Xylocaine trigeminal nerve blockage served as a control. TN-DCS increased neuronal spiking activity in both NVsnpr and MeV, returning to baseline during the post-stimulation phase. The 3 mA DC stimulation of the blocked trigeminal nerve failed to induce increased spiking activity in the trigeminal nuclei. These findings provide empirical support for trigeminal nuclei modulation via TN-DCS, suggesting the cranial nerve pathways could play a role in mediating the tDCS effects in humans.

Introduction

Transcranial direct current stimulation (tDCS) is a non-invasive electrical stimulation method, in which electrodes are placed on the scalp to administer a weak direct electrical current (1 to 2 mA) [1]. tDCS has significant potential as a treatment option due to its affordability, portability, safety, and user-friendly nature [2]. However, understanding the underlying mechanisms of action remains a primary challenge in tDCS research [3].

One widely recognized effect of tDCS is its ability to alter membrane polarity in cortical neurons, thereby influencing the generation of action potentials [4, 5]. However, the accepted view that tDCS acts via the currents that pass through the scalp, skull, and cerebrospinal fluid, to directly affect cortical neurons, has recently been challenged [6–8]. An alternative, somewhat controversial, explanation is that the impact of tDCS on neural circuits may also be indirect, acting through cranial/peripheral nerves such as the trigeminal and the greater occipital nerves [3, 6–8]. Note, that in one of the most common human tDCS montages one electrode is placed in a supraorbital location where it will stimulate the trigeminal nerve, while the other is placed over the motor cortex where it will stimulate the occipital nerve. Evidence for the stimulation of the trigeminal nerve during tDCS is supported by two recent studies. The first study [9] found that patients with trigeminal neuralgia exhibited a significant reduction of over 40% in fractional anisotropy (indicative of demyelination and microstructural changes) in the ipsilateral trigeminal root entry zone after tDCS treatment. The second study reported a notable increase in motor evoked potentials (MEPs) amplitude after applying tDCS to the motor cortex. Importantly, this increase was not replicated when a topical anesthetic was applied to the skin under the electrodes, suggesting that the effects of tDCS might involve peripheral and/or cranial nerve stimulation alongside direct brain activation [10]. Thus, while there is some limited experimental evidence to support the hypothesis that tDCS neuromodulation is mediated via trigeminal nerve stimulation, a clear understanding of the neuromodulatory pathways involved is currently absent.

The trigeminal nerve has extensive connections to the brainstem and various other brain structures. It projects to the trigeminal nuclei, e.g., principal sensory nucleus (NVsnpr) and mesencephalic nucleus (MeV), which reciprocally project to locus coeruleus (LC), raphe nuclei (RN), and NTS [11, 12]. The LC and RN are rich in noradrenergic and serotoninergic innervation, respectively [11]. Additionally, the NTS is linked to glutamatergic and GABAergic systems [13]. Through these projections and connections, an increase in the activity of trigeminal nuclei can have several behavioral and physiological consequences [14] including enhanced vigilance [15, 16], accelerated mental information processing [17], reduced response delays [18], augmentation of the oxygen-dependent signal in cerebral blood flow, and mydriasis in response to tasks [19].

Similarly, recent evidence indicates that non-invasive transcutaneous direct current (DC) electrical stimulation of the greater occipital nerve establishes a ‘bottom-up’ communication pathway from the periphery via the brainstem (nucleus tractus solitarius (NTS)) and then the reticular formation to the relevant subcortical and cortical regions [6, 7].

The NVsnpr is composed of neurons that are believed to be either sensory relay neurons, firing in proportion to stimulus intensity, or neurons that transform depolarization into bursts to alter sensory signals [20, 21]. Evidence also indicates that neurons within this nucleus can adjust their response stability based on specific stimulus attributes, such as duration and frequency [22]. Besides, it has been reported that trigeminal nerve stimulation elicits diverse activity patterns in MeV neurons [23].

Little is known about the electrophysiological effects of trigeminal nerve DC stimulation (TN-DCS) on neuronal activity in the NVsnpr and MeV. In this study, we aimed to develop a rat model that isolates the trigeminal nerve stimulation component of human tDCS and use this model to investigate its effects on neuronal activity within the NVsnpr and MeV.

Materials and methods

Animals

For all experiments, we used a total of 24 adult male Sprague Dawley rats (Charles River Laboratories, Germany), divided as follows: 10 rats for NVsnpr (with 5 of these used for the xylocaine condition), 10 rats for MeV during both anodic TN-DCS and xylocaine conditions and 4 rats for MeV during cathodic TN-DCS and tDCS conditions. The rats weighed between 250-450 g at the start of each experiment. The number of animals was chosen based on previous studies in the field. No randomization or blinding was performed in this study. Before surgery, the rats were housed in pairs per cage. The animals were housed in cages in a temperature- and humidity-controlled room, maintaining a diurnal light-dark cycle with ad libitum access to food and water.

Anesthesia and surgery

During the experiment days, the rats were anesthetized via an intraperitoneal (i.p.) injection of a 1.5 g/kg body weight dose of urethane (ethyl carbamate; Sigma-Aldrich, U2500) [24]. Subsequently, they were positioned in a stereotaxic frame (World Precision Instruments Ltd., Stevenage, UK) on a heated pad, while the core temperature was monitored using a metal rectal probe. The depth of anesthesia was regularly assessed by observing the toe-pinch reflex. Approximately 100 µL of urethane was administered via i.p. injection as needed to maintain a consistent anesthesia level. Once the scalp was removed, the skull was carefully adjusted to achieve a level of 0 degrees on both the anteroposterior and mediolateral planes. This adjustment ensured that the lambda-bregma and lateral ridges differences were <200 μm. At the end of each experiment, the animal was euthanized using an overdose i.p. injection of sodium pentobarbital (Dolethal ND, Vetoquinol, France) at 200 mg/kg [25].

Stereotaxic coordinates and electrode placement

The Paxinos and Watson rat brain atlas was used to locate the NVsnpr relative to Bregma [26]. To this end, a burr hole craniotomy was performed using the 78001 Microdrill (RWD Life Science Co., Shenzhen, China) at the following coordinate AP: −9.0 mm, ML: ±2.9 mm, and DV:-7.5-9 mm. The MeV was located using the criteria described by Totah et al. [24],. A cranial window was made at AP: −4 to −5 mm and ML: ±1.2 mm relative to true lambda [26]. The dura was retracted and the electrode was lowered at a 15° angle to the horizontal plane to avoid the transverse sinus overlaying the target area until reaching 5-7 mm below the cortical surface [24]. The electrode location in the MeV was confirmed by the audible presence of jaw movement-responsive cells [24].

Stimulation protocol

To stimulate the trigeminal nerve’s marginal branch, a rectangular metal electrode (1 cm²) was positioned at the lower lip of the rat serving as an anode. Another identical electrode was attached to the tail to serve as a cathode. The contact surfaces of the electrodes with the rat were covered with conductive gel (Signa Gel, Parker Labs, New Jersey). To compare the effects of TN-DCS with a more standard animal tDCS montage we added a group of animals with an epicranial metal electrode (0.2 cm²) placed directly on the skull over the lambdoid suture (ML: ± 2 mm) on the recording side serving as an anode and a rectangular metal electrode (1 cm²) attached to the tail serving as a cathode. Thus, in this control tDCS group an electric field would be generated in the brain without stimulating the trigeminal nerve, while in the TN-DCS group, the trigeminal nerve would be stimulated without (see xylocaine control experiment below used to confirm this) generating an effective electric field in the brain.

DC electrical stimulation was provided by an AM 2200 analog current source (AM Systems, Sequim, WA) connected to stimulation electrodes. The current source received an analog voltage waveform generated from a data acquisition card (NI USB-6216, National Instruments, Austin, TX) operating at a sampling rate of 30 kHz. Dedicated MATLAB software (MathWorks, Natick, MA, USA) controlled the data acquisition card. In humans, tDCS is delivered continuously with stimulation lasting a few minutes. Therefore, our stimulation protocol consisted of the following sequence: 1 min without any stimulation (pre-time epoch), followed by 1 min of continuous DC stimulation (during-time epoch), and finally another 1 min without any stimulation (post-time epoch).

To effectively stimulate peripheral/cranial nerves in both humans and animals currents of a few milliamperes are needed [8, 27]. In human tDCS, currents of 1 or 2 mA are routinely used and these are enough to stimulate the trigeminal nerve [9]. Therefore, for the TN-DCS experiments, DC stimulation was delivered once (no repetitions) for each amplitude at +0.5, +1, +2, and +3 mA as well as −0.5, −1, −2, and −3 mA in the NVsnpr, and +1, +2, and +3 mA as well as −1, −2, and −3 mA in the MeV. When a current of 1 mA is delivered to a tDCS scalp electrode in humans, it generates an electric field of below 1 V/m in the cortex [28, 29]. The much thinner skull in rats means that to deliver an equivalent electric field (that is representative of human tDCS) we must use much lower amplitudes. Therefore, in the tDCS control experiments, the same stimulation protocol was used (1-min pre, 1 min continuous DC stimulation, 1-min post) but with lower amplitudes known to generate electric field strengths (0.13 to 14.3 mA/cm²) that are the best possible approximations of those typically observed in human tDCS (0.029-0.080 mA/cm²): +0.1, +0.2, and +0.3 mA as well as −0.1, −0.2, and −0.3 mA. While there are differences in electric field strengths, these selected amplitudes were chosen to closely mimic the effects of human tDCS, allowing for a relevant comparison of neurophysiological outcomes in the rat model [30, 31]. A 3-min washout period was used between each stimulation in all experiments.

In a further control experiment, we wanted to be sure that the effects seen in the TN-DCS condition were caused by trigeminal nerve stimulation and not the electric field in the brain generated via volume conduction. Therefore, the trigeminal nerve was blocked by injecting 1 ml xylocaine 2% (Xyl-M; VMD, Belgium) unilaterally into the nerve and its branches on the face. This allowed us to test whether brain stem nuclei responses were due to activation of the trigeminal nerve or the result of volume conduction.

Recording setup

We recorded neurons using a 50 μm thick silicone probe with 32 channels spanning across 1550 µm (Atlas Neuro, Leuven, Belgium, Model: E32 + R-50-S1M-L20 NT). The output of the channels was amplified (×192) and digitized using a 16-bit, 30 kHz acquisition board and 32-channel recording headstage (RHD 32 by Intan Technologies, Los Angeles, CA, USA). The probe data were monitored and recorded throughout the experiment on a PC hard drive at a sampling rate of 30 kHz via the Open Ephys GUI (https://open-ephys.org/).

Data analysis

DC stimulation will create an electrical stimulation artifact in the recorded neuronal data. The 32-channel Intan amplifier we used was AC coupled. This meant that the DC stimulation artifact was visible as an onset and offset artifact lasting ~10 ms when stimulation was switched on or off. To remove these artifacts we identified the time-points just before and after the artifact and used linear interpolation to remove it. Effectively, this means that we lost around 17 ms of data when DC stimulation was switched on or off. Next, we performed spike sorting using SpyKING CIRCUS 1.0.1 [32]. Briefly, the data first underwent several preprocessing steps. SpyKING CIRCUS filtered all the signals with an auto-defined cut-off frequency for a high-pass at 300 Hz (template width of 3 ms and a spatial radius of 250 µm for the templates). Then, spikes were automatically detected considering λ = 6 in the spike threshold (θ_k) calculation formula. Accordingly, spatial whitening was done to eliminate erroneous spatial correlations among adjacent recording electrodes. Afterward, the dimensionality of the temporal waveforms was decreased by basis estimation (principal component analysis). Following the preprocessing steps, the data were clustered, and template matching and automated merging (spike trains binning) were performed [32]. The clustering was subsequently manually examined and adjusted using phy a GUI-based software for manual curation (https://github.com/cortex-lab/phy) and only distinct single-unit clusters were selected for further analysis. Our assessment of waveform quality involved detecting potential action potentials, and only clusters that showed recognizable action potential waveforms and had a V-shaped autocorrelogram were labeled as “good” before being exported to MATLAB. Following this, we proceeded to refine the data by checking for interspike interval (ISI) violations, which occurred when a putative unit fired more than twice within a 2 millisecond interval. Clusters that exhibited over 3% ISI violations were discarded from the analysis. Following the manual clustering phase, we further examined the distinct neuronal units in MATLAB. In MATLAB the mean spike rate in the 1 min pre, 1 min during, and 1-min post-time epochs was calculated for further statistical analysis. Additionally, a spike rate over time waveform was calculated by taking the mean spike rate over 1-s bins (see next section for more details).

Statistical analyses

The data processing and statistical analyses were conducted using MATLAB 2022a (MathWorks, Natick, MA, USA). Following the recommendations of [33], we employed a linear mixed-effects (LME) model to assess the impact of stimulation amplitude and time epoch (pre, during, and post) on spike rate for both NVsnpr and MeV data. We treated stimulation amplitude, time epoch, and their interaction as fixed effects while considering rats and neurons as random effects. Here, fixed effects provide information about the overall impact of predictors on the response variable (spike rate), while random effects account for variability within groups or clusters in our data [33].

The model codes in MATLAB’s fitlme.m were as follows:

1. Spike Rate ~ Amplitude + Time epoch + Amplitude × Time epoch + (1|Neuron) + (1|Rat) to test the effect of amplitude and time epochs on spike rate,

2. Spike Rate ~ Amplitude + Time epoch + Polarity + Amplitude × Time epoch × Polarity + (1|Neuron) + (1|Rat) to test the effect of polarity on spike rate, and

3. Spike Rate ~ 1 + Xylo × Time epoch + (1 | Neuron) + (1 | Rat) to test the effect of xylocaine blockage on spike rate.

A two-way ANOVA was then performed to test for the significance of the model’s fixed effects and interactions. To conduct post-hoc testing, we employed a two-sided Wilcoxon signed rank test (spike rate was not normally distributed) with a strict Bonferroni correction to account for all possible comparisons. For the NVsnpr dataset, this involved 4 stimulation amplitudes and 3 time epochs, resulting in 12 comparisons. We had 3 stimulation amplitudes and 3 time epochs for the MeV dataset, leading to 9 comparisons. Cohen’s d was computed for pairwise comparisons between specific time epochs: pre vs. dur, pre vs. post, and dur vs. post. For each comparison, we used the means and standard deviations from the respective conditions, using the formula

$$d=\frac{M_1-M_2}{{\mathit{SD}}_{\mathit{pooled}}},$$

where M₁ and M₂ represent the means of the two conditions being compared, and SD _pooled is the pooled standard deviation. For each pairwise comparison—pre-dur, pre-post, and dur-post—we computed the means and standard deviations of the respective conditions to derive the effect sizes.

Additionally, we calculated z-scores for each condition based on their means and standard deviations derived from the 1-min pre-stimulation period as well as during stimulation and post-stimulation periods. This was performed to evaluate how many standard deviations each time epoch’s mean deviated from a known population mean. The z-score formula is given by

$$z=\frac{M-\mu }{\sigma /\sqrt{n}}$$

where M is the sample mean, μ is the population mean, σ is the population standard deviation, and n denotes the sample size.

To test whether spike shape changed during stimulation as compared to the pre-stimulation time epoch we extracted action potential amplitude and action potential duration. We then used a t-test (action potential amplitude and duration normally distributed) to examine if action potential amplitude or duration were significantly different in the pre-stimulation or the during-stimulation time epochs.

To calculate the spike rate over time, and to perform individual neuron statistics, for each neuron, we binned spikes into 1-s intervals and calculated the mean spike rate within that 1-s bin. We then used a t-test to compare the spike-rate distribution in the pre-time epoch and the during-time epoch. This allowed us to determine if each neuron showed: a significant increase in spike rate in response to stimulation; a significant decrease in response to stimulation; or no significant change. In all comparisons, a significance level of 0.05 was considered.

Results

Single unit response to TN-DCS in NVsnpr and MeV nuclei

Figure 1A depicts a schematic diagram of the Sprague Dawley rat brain sagittal plane showing the relative position of stimulating and recording electrodes and the positional relationship of the NVsnpr, MeV, and trigeminal nerve. It also shows an example spike train from the NVsnpr (Fig. 1B) and MeV (Fig. 1C). TN-DCS is turned on at the 60-s time point and there is a noticeable increase in spike rate in both of the trigeminal nuclei in these examples. Individual spikes as identified after spike sorting are indicated with a red dot. Figure 2 illustrates single unit responses in the NVsnpr (upper panel) and the MeV (lower panel) to three different stimulation amplitudes; represented by yellow, red, and blue lines for 3, 2, and 1 mA, respectively. This figure shows an example recording of the pre-during-post time epochs during TN-DCS. Note that in the example shown, while the spike rate increases, the spike shape does not appear to be affected by the stimulation time epochs. A statistical analysis on all spikes found no significant differences between the action potential duration in the pre and during time epochs (p = 0.177, t-stat = −1.521) nor in amplitude (p = 0.785, t-stat = 0.273) for spikes in the NVsnpr. This was also applied to the MeV spikes, where neither the action potential duration (p = 0.3173, t-stat = 1.009) nor amplitude (p = 0.1771, t-stat = 1.367) showed a significant difference between the pre and during time epochs.

Fig. 1. Representation of the electrode and recorded nuclei, and individual neuronal firing response to trigeminal nerve direct current stimulation (TN-DCS) in the Sprague Dawley Rat Brain.

A Schematic diagram showing stimulation and recording electrodes and the relative positional relationship of the recorded nuclei in the sagittal section of the Sprague Dawley rat brain. B Example of spike trains showing typical fast irregular firing of neurons in (B) the principle sensory nucleus (NVsnpr) and (C) the mesencephalic nucleus of the trigeminal nerve (MeV) before and after stimulation onset at 3 mA. A sharp increase in the number of spikes is observed upon the initiation of trigeminal nerve direct current stimulation (TN-DCS). Red dots represent spikes detected by the spike sorting process. Figure 1A is created with BioRender.com.

Fig. 2. The effects of trigeminal nerve direct current stimulation (TN-DCS) on spike rate at individual neuron level.

Individual neuron examples of the effect of 3 different amplitudes (blue, red, and yellow representing 1, 2, and 3 mA, respectively) of trigeminal nerve direct current stimulation (TN-DCS) on spike rate and shape in principle sensory nucleus (NVsnpr; A) and mesencephalic nucleus of trigeminal nerve (MeV; B) neurons. Application of 3 mA TN-DCS increases spike rate during stimulation in both nuclei without affecting spike shape. The y-axis represents the spike rate per second and the x-axis depicts time in seconds.

Neuronal responses in NVsnpr to TN-DCS: Effect of stimulation amplitude, time epoch, and polarity

A total of 132 single units were isolated from the NVsnpr. Figure 3 presents data for all these neurons grouped, illustrating their responses to TN-DCS under various stimulation amplitude ranges and polarities (Fig. 3A, B for anodic and cathodic amplitudes, respectively) across the pre, during, and post-time epochs. The LME model was used to analyze the data.

Fig. 3. Effect of different amplitudes and time epochs of trigeminal nerve direct current stimulation (TN-DCS) on neuronal spike rate in the principal sensory nucleus of the trigeminal nerve (NVsnpr).

A Anodic TN-DCS: The mean neuronal spike rate increased with increasing amplitude. The gray shadows and the blue lines (top) represent the 95% confidence intervals (CIs) and the average spike rate over time for all neurons. The bar graphs and error bars (bottom) display the mean spike rate and standard deviation (SD) for all pre, during, and post-TN-DCS time epochs across different amplitude levels. A horizontal line denotes a significant increase in mean neuronal spike rate when switching from the pre-time epoch to the during-time epoch or from the during-time epoch to the post-time epoch. Each grey dot (bottom) represents a single unit. B cathodic TN-DCS: The mean neuronal spike rate increased with increasing amplitude and remained significantly higher up to 1 min after stimulation discontinuation. All figures in (B) follow the same style convention as in (A). Data were analyzed using a linear mixed-effect model followed by a two-sided Wilcoxon signed rank test with a strict Bonferroni correction. C Pie chart representing the relative number of neurons based on their response to TN-DCS. The signs “0”, “−“, and “+” indicate no change, decrease, or increase in the spike rate in response to stimulation, respectively.

Anodic stimulation

For the anodic stimulation, we found a significant impact of amplitude on spike rate (F_{(1, 717)} = 7.787, p = 0.005) and an interaction between amplitude and time epoch (F_{(2, 717)} = 7.951, p < 0.001). However, we found no significant effect of the time epoch (F_{(2, 717)} = 1.073, p = 0.342) on the spike rate. This indicates that higher amplitudes of TN-DCS were associated with higher spike rates in the NVsnpr during stimulation (p < 0.001 for pre- vs. during stimulation time epochs across all amplitudes, see Table 1 for full details of all post-hoc testing). Accordingly, higher anodic amplitudes were linked to higher Cohen’s d values indicating higher effect sizes (Table 1).

Table 1.

Size Post-hoc testing metrics for the impact of direct current trigeminal nerve stimulation (TN-DCS) on the spike rat in the principal sensory nucleus of the trigeminal nerve (NVsnpr) in rats.

Effect size measure	Anodic stimulation of NVsnpr
	0.5 mA			1 mA			2 mA			3 mA
	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post
P value	<0.001	1	<0.001	<0.001	1	<0.001	<0.001	0.141	0.114	<0.001	<0.001	0.060
Z value	-6.334	-1.022	-4.422	-7.672	-0.943	-4.470	-6.735	-2.517	-2.591	-6.154	-4.792	-2.804
Cohen’s d	0.273	0.029	0.231	0.264	0.186	0.070	0.420	0.253	0.168	0.678	0.489	0.191
Effect size measure	Cathodic stimulation of NVsnpr
	-0.5 mA			-1 mA			-2 mA			-3 mA
	Pre-During	During- Post	Pre-Post	Pre-During	During-Post	Pre-Post	Pre-During	During-Post	Pre-Post	Pre-During	During-Post	Pre-Post
P value	<0.001	1	<0.001	<0.001	0.271	<0.001	<0.001	<0.001	<0.001	<0.001	0.036	<0.001
Z value	-7.062	-0.533	-4.375	-8.054	-2.280	-4.183	-7.623	-4.826	-6.313	-6.791	-2.962	-4.557
Cohen’s d	0.176	0.009	0.168	0.368	0.191	0.162	0.491	0.364	0.117	0.605	0.301	0.297

This table reports the effect size measures (Cohen’s d) along with p-values and Z-values (deviations from the mean) for different anodic and cathodic amplitudes of TN-DCS in rats under three conditions: pre- vs. during, post- vs. during, and pre- vs. post-stimulation.

Cathodic stimulation

When analyzing the cathodic stimulation, we observed a significant effect of amplitude (F_{(1, 864)} = 4.064, p = 0.044) and an interaction between amplitude and time epoch (F_{(2, 864)} = 4.058, p = 0.017), but there was no significant effect of time epoch alone (F_{(2, 864)} = 0.461, p = 0.630) on the spike rate. These results suggested that higher amplitudes of TN-DCS were linked to higher spike rates in the NVsnpr in the during- compared to the pre-time epoch and interestingly, also in the post compared to the pre-stimulation time epoch (p < 0.001 pre- vs. during and pre- vs. post-stimulation time epochs across all amplitudes, Table 1). Similar to the anodic time epoch, we found between increased cathodic stimulation amplitudes higher Cohen’s d values, indicating a larger effect size (see Table 1).

Anodic vs. cathodic stimulation

We combined the datasets and added polarity as a fixed effect to test if there was a difference in the spike rate response to anodic and cathodic stimulation. Our analysis revealed no statistically significant effect of polarity on spike rates in the NVsnpr (F_{(1, 1581)} = 1.532, p = 0.215). With cathodic stimulation, the mean spike rate remained persistently high up to 1 min after discontinuation of stimulation. However, this was not the case with anodic stimulation (Fig. 3). It was also found that the effects of amplitude (F_{(1, 1581)} = 9.108, p = 0.002), the interaction between amplitude and polarity (F_{(1, 1581)} = 13.53, p < 0.001), the interaction between amplitude and time epoch (F_{(2, 1581)} = 5.967, p = 0.002), and the interaction between amplitude, polarity, and time epoch (F_{(2, 1581)} = 9.587, p < 0.001) on the spike rate in the NVsnpr were statistically significant. The effects of the time epoch and the interaction between the time epoch and polarity on the spike rate in the NVsnpr were not significant (p > 0.5 for all comparisons).

Variability of response to TN-DCS among neurons and rats

To examine the variability within responses we report the LME model estimate of the covariance of the random effects, i.e. rat and neuron. The model showed a lower covariance estimate for rats (std = 5.428 (cathodic) and std = 4.732 (anodic)) than for neurons (std = 9.047 (cathodic) and std = 9.874 (anodic)), indicating that the variability among neurons was much higher than the variability among rats.

Sixty-two percent of the NVsnpr neurons exhibited no significant response to stimulation at the lowest amplitude of TN-DCS. Conversely, 33% and 5%, respectively, indicated a significant increase and significant decrease in the spike rate after stimulation. After 3 and −3 mA stimulation, the percentage of neurons exhibiting a significant spike rate rise increased significantly to 65% and 70%, respectively (Fig. 3C).

Neuronal responses in MeV to TN-DCS: Impact of amplitude and time epoch, and their interaction

Anodic stimulation

A total of 74 single units were isolated from the MeV during anodic stimulation (n = 10). Figure 4A depicts the data for these neurons, grouped to illustrate their responses to TN-DCS across various amplitude ranges in the pre, during, and post-time epochs. Our findings revealed a significant effect of amplitude (F_{(1, 462)} = 6.050, p = 0.014) and the interaction between amplitude and time epoch (F_{(2, 462)} = 3.367, p = 0.035) on the spike rate. However, there was no significant effect of the time epoch alone (F_{(2, 462)} = 0.402, p = 0.668) on the spike rate. This meant that higher amplitudes were associated with higher spike rates in the MeV in the during-time epoch but not in the pre- or post-time epochs (p < 0.001 for pre- vs. during stimulation time epochs across all amplitudes, Table 2). The mean neuronal spike rate increased with increasing amplitude and remained significantly higher up to 1 min after stimulation discontinuation (p < 0.001 for all comparisons). Similar to the NVsnpr, we found higher Cohen’s d values with higher anodic amplitudes, i.e., larger effect sizes (see Table 2 for full details).

Fig. 4. Effect of different amplitudes, time epochs, and polarities of trigeminal nerve direct current stimulation (TN-DCS) on neuronal spike rate in the mesencephalic nucleus (MeV) of the trigeminal nerve.

A Anodic TN-DCS (n = 10): The mean neuronal spike rate increased with amplitude. B Cathodic TN-DCS (n = 4): The mean neuronal spike rate increased with amplitude. The gray shadows and the blue lines represent the 95% confidence intervals (CIs) and the average spike rate over time for all neurons. The bar graphs and error bars display the mean spike rate and standard deviation (SD) for all pre, during, and post-TN-DCS time epochs across different amplitude levels and polarities. A horizontal line denotes a significant increase in mean neuronal spike rate when switching from the pre-time epoch to the during-time epoch or from the during-time epoch to the post-time epoch. Each grey dot represents a single unit. Data were analyzed using a linear mixed-effect model followed by a two-sided Wilcoxon signed rank test with a strict Bonferroni correction. C Pie chart representing the relative number of neurons based on their response to TN-DCS. The signs “0”, “−“, and “+” indicate no change, decrease, or increase in the spike rate in each amplitude, respectively.

Table 2.

Effect size measures for the impact of anodic and cathodic direct current trigeminal nerve stimulation (TN-DCS) on the spike rat in the principal sensory nucleus of the mesencephalic nucleus (MeV) in rats.

Effect size measure	Anodic stimulation of MeV
	1 mA			2 mA			3 mA
	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post
P value	<0.001	1	<0.001	<0.001	0.028	<0.001	<0.001	0.016	0.001
Z value	-5.776	-1.610	-3.769	-6.509	-3.034	-4.200	-6.509	-3.205	-3.915
Cohen’s d	0.245	0.050	0.186	0.487	0.216	0.271	0.693	0.428	0.279
Effect size measure	Cathodic stimulation of MeV
	-1 mA			-2 mA			-3 mA
	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post
P value	1	0.445	1	0.017	<0.001	0.475	0.005	0.004	1
Z value	-1.354	2.084	0.709	-4.736	6.657	2.057	-5.439	5.471	-0.349
Cohen’s d	0.095	0.020	0.066	0.181	0.401	-0.204	0.614	0.611	0.038

This table includes Cohen’s d values, p-values, and Z-values for various anodic amplitudes of TN-DCS in three conditions: pre- vs. during, post- vs. during, and pre- vs. post-stimulation. Higher absolute Z-values denote greater deviation from the mean, while larger Cohen’s d values signify more substantial effects.

Cathodic stimulation

During cathodic stimulation, 62 single units (n = 4) were separated from the MeV. The data for these neurons are shown in Fig. 4B. The data are grouped to show the neurons’ responses to TN-DCS in the pre-, during-, and post-time epochs across different amplitude ranges. Our study showed that the amplitude had a significant impact on the spike rate (F_{(1, 462)} = 12.644, p < 0.001), as did the interaction between the amplitude and time epoch (F_{(2, 462)} = 5.300, p = 0.005). On the other hand, the time epoch by itself did not significantly affect the spike rate (F_{(2, 462)} = 0.952, p = 0.386). This means that higher stimulation amplitudes were linked to higher spike rates in the MeV in the during-time epoch but not in the pre- or post-time epochs. As the amplitude increased, the mean neuronal spike rate also increased but returned to baseline after the stimulation was stopped (p < 0.001 across all comparisons). For higher cathodic amplitudes, we observed bigger effect sizes, with higher Cohen’s d values, similar to the NVsnpr (see Table 2 for complete details).

Anodic vs. cathodic stimulation

To see if the spike rate response to anodic and cathodic stimulation differed, we merged the datasets and included polarity as a fixed effect. Polarity had no statistically significant impact on spike rates in the MeV, according to our study (F(_{1, 924)} = 0.244, p = 0.621). The increase in the mean spike rate persisted for a minute after the stimulation was stopped with anodic stimulation. With cathodic stimulation, however, this was not the case (Fig. 3). It was also found that amplitude (F_{(1, 924)} = 4.624, p = 0.031) had a significant effect on the spike rate in the MeV. The effects of the interaction between amplitude and polarity, the interaction between amplitude and time epoch, the interaction between the time epoch and polarity, and the interaction between amplitude, polarity, and time epoch on the spike rate in the NVsnpr were not significant (p > 0.5 for all comparisons).

Variability of response to TN-DCS among neurons and rats

Once more for MeV, we present the LME model estimate of the covariance of the random effects, i.e., rat and neuron, to investigate the variability within responses. The model revealed that the variability between individual neurons was larger than the variability between different rats, with the rat covariance estimate (std = 3.988 for cathodic and 2.839 for anodic) being lower than the neuron covariance estimate (std = 10.661 for cathodic and 10.976 for anodic).

With the lowest amplitude of TN-DC, 70% of the MeV neurons showed no response to stimulation. On the other hand, 20% showed an increase, and 10% showed a decrease in the spike rate following stimulation. The proportion of neurons with a rise in the spike rate significantly increased to 62% following 3 mA and to 70% following −3 mA stimulation (Fig. 4C).

Neuronal responses in NVsnpr to TN-DCS: Effect of TN blockage via xylocaine

In the experiment with the xylocaine blocker, a group of 20 individual neurons (in a total of 5 animals) were isolated from the NVsnpr. The grouped data for these neurons are presented in Fig. 5, illustrating their responses to TN-DCS with and without xylocaine blocker. The results of our investigation revealed a significant effect of xylocaine on the spike rate in the NVsnpr (F_{(1, 162)} = 10.609, p = 0.001). Moreover, we observed significant effects of the time epoch (F_{(2, 162)} = 12.855, p < 0.001) and interaction between xylocaine and the time epoch (F_{(2, 162)} = 3.521, p = 0.031) on the spike rate. This result indicates that TN blockage via xylocaine effectively inhibited the spike-rate effect of TN-DCS in the NVsnpr (p < 0.001 for pre vs. during time epoch before xylocaine injection vs. p > 0.05 for pre vs. during time epoch after xylocaine injection). See Table 3 for full post-hoc results.

Fig. 5. Effect of trigeminal nerve direct current stimulation (TN-DCS) at 3 mA amplitude on neurons’ spike rate in the trigeminal nerve’s principal sensory nucleus (NVsnpr) with and without xylocaine injection.

A significant decrease in neuronal spike rate is observed after xylocaine injection (right panel). The gray shadows and the blue lines (top) represent the 95% confidence intervals (CIs) and the average spike rate over time for all neurons. The bar graphs and error bars (bottom) display the mean spike rate and standard deviation (SD) for all pre, during, and post-TN-DCS time epochs across different amplitude levels. A horizontal line denotes a significant increase in mean neuronal spike rate when switching from the pre-time epoch to the during-time epoch or from the during- to the post-time epoch. Each grey dot (bottom) represents a single unit. Data were analyzed using a linear mixed-effect model followed by a two-sided Wilcoxon signed rank test with a strict Bonferroni correction.

Table 3.

Effect size measures for the impact of trigeminal nerve direct current stimulation (TN-DCS) on the spike rat in the principal sensory nucleus of the trigeminal nerve (NVsnpr) before and after unilateral xylocaine blocker injection to multiple branches of the trigeminal nerve in rats.

Effect size measure	Anodic stimulation of NVsnpr
	3 mA without xylocaine blocker			3 mA with xylocaine blocker
	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post
P value	<0.001	1	<0.001	1	1	1
Z value	-3.825	-4.053	-0.910	NA	NA	NA
Cohen’s d	0.750	0.596	0.163	NA	NA	NA

This table includes Cohen’s d values, p-values, and Z-values for 3 mA amplitude before and after blocker injection in three time epochs: pre- vs. during, post- vs. during, and pre- vs. post-stimulation. Higher absolute Z-values denote greater deviation from the mean, while larger Cohen’s d values signify more substantial effects. NA, not applicable – no post-hoc testing was performed as there was no main effect.

MeV neural responses to TN-DCS: Impact of xylocaine-induced TN blocking

Using the xylocaine blocker, 52 distinct neurons (from n = 4 rats) were isolated from the MeV in the experiment. Figure 6 displays the aggregated data for these neurons, showing how they responded to TN-DCS both with and without a xylocaine blocker. Our study’s findings showed that xylocaine significantly affected the MeV’s spike rate (F_{(1, 291)} = 39.821, p < 0.001). Additionally, we found that the time epoch had a substantial impact on the spike rate (F_{(2, 291)} = 29.055, p < 0.001), as did the interaction between xylocaine and the time epoch (F_{(2, 291)} = 12.634, p < 0.001). The acquired data suggests that the increase in the spike rate caused by TN-DCS in the MeV was successfully suppressed by unilateral TN blocking via xylocaine (p < 0.001 for pre vs. during time epoch before xylocaine injection vs. p > 0.05 for pre vs. during time epoch after xylocaine injection). Complete post-hoc findings are shown in Table 4.

Fig. 6. Effect of trigeminal nerve direct current stimulation (TN-DCS) at 3 mA amplitude on neurons’ spike rate in the trigeminal nerve’s mesencephalic nucleus (MeV) with and without xylocaine injection. Following xylocaine administration, a significant reduction in neuronal spike rate is seen (right panel).

The average spike rate over time for all neurons is shown by the blue lines at the top and the gray shadows on the 95% confidence intervals (CIs). The mean spike rate and standard deviation (SD) for each pre-, during-, and post-TN-DCS time epoch across various amplitude levels are shown in the bar graphs and error bars (bottom). A move from the pre-time to the during-time or from the during- to the post-time epoch results in a considerable rise in mean neuronal spike rate, as indicated by a horizontal line. Each grey dot (bottom) represents a single unit. Data were analyzed using a linear mixed-effect model followed by a two-sided Wilcoxon signed rank test with a strict Bonferroni correction.

Table 4.

Effect size measures for the impact of trigeminal nerve direct current stimulation (TN-DCS) on the spike rat in the mesencephalic nucleus of the trigeminal nerve (MeV) before and after unilateral xylocaine blocker injection to multiple branches of the trigeminal nerve in rats.

Effect size measure	Anodic stimulation of MeV
	3 mA without xylocaine blocker			3 mA with xylocaine blocker
	Pre-during	During-post	Pre-post	Pre-during	During-post	Pre-post
P value	<0.001	<0.001	1	0.150	1	0.066
Z value	-5.439	-5.470	-0.349	-1.957	0.100	-2.285
Cohen’s d	0.614	0.611	0.038	0.136	0.017	0.126

This table includes Cohen’s d values, p-values, and Z-values for 3 mA amplitude before and after blocker injection in three time epochs: pre- vs. during, post- vs. during, and pre- vs. post-stimulation. Higher absolute Z-values denote greater deviation from the mean, while larger Cohen’s d values signify more substantial effects. NA, not applicable – no post-hoc testing was performed as there was no main effect.

Neuronal responses in MeV to standard tDCS: Effect of stimulation amplitude, time epoch, and polarity

During anodic and cathodic tDCS, we identified 19 neurons from MeV (from n = 4 rats). Upon conducting anodic stimulation, we found that amplitude, polarity, time epoch, and their combination did not significantly affect spike rate (p > 0.05) (Fig, 7A and B). Additionally, we discovered that whilst 80% of the isolated MeV neurons exhibited no response to tDCS, 12% and 8% of them respectively displayed an significant increase and a significant reduction in spiking during tDCS. The proportion of respondents remained constant as the amplitude increased (Fig. 7C).

Fig. 7. Effect of different amplitudes, time epochs, and polarities of a standard transcranial direct current stimulation (tDCS) on neuronal spike rate in the mesencephalic nucleus (MeV) of the trigeminal nerve.

A Anodic tDCS (n = 4): As amplitude increased, the mean neuronal spike rate remained unchanged. B Cathodic tDCS (n = 4): As amplitude increased, the mean neuronal spike rate remained unchanged. The average spike rate over time for all neurons is shown by the blue lines and the gray shadows, respectively, and the 95% confidence intervals (CIs). For each before, during, and post-tDCS time epochs across various amplitude levels and polarities, the mean spike rate and standard deviation (SD) are shown in bar graphs and error bars. Every gray dot stands for a single unit. Data were analyzed using a linear mixed-effect model followed by a two-sided Wilcoxon signed rank test with a strict Bonferroni correction .C Pie chart representing the relative number of neurons based on their response to TN-DCS. The signs “0”, “−”, and “+” indicate no change, decrease, or increase in the spike rate in each amplitude, respectively.

Discussion

In recent years, there has been a resurgence of enthusiasm surrounding tDCS, as it emerged as a promising technique for modulating cortical function in the human brain and addressing neuropsychiatric disorders [34]. The appeal of tDCS lies in its capacity to non-invasively alter cortical activity and modulate excitability through undamaged cranial structures [34]. Numerous investigations have been undertaken in recent years to examine the physiological impacts and mechanisms of action of tDCS [35, 36]. There exists a consensus that the main mechanism by which DC stimulation influences the cerebral cortex is a slight alteration of the resting membrane potential of neurons (subthreshold depolarization or hyperpolarization depending on the polarity, duration, and strength of stimulation) [36, 37]. Recent research involving animals claims that tDCS, administered at intensities between 1 and 4.16 A/m², can exert a direct influence on subcortical structures, including the medial longitudinal fascicle and the red nucleus [38, 39]. The majority of the electrical current is attenuated by the scalp and skull, leaving only a small portion to reach the brain. Lately, it has been proposed that as much as 75% of the administered current does not effectively reach its target within the brain [40]. Our results were in line with these findings showing no effects of standard tDCS (see Fig. 7) on the neuronal spike rate in the MeV when cranial/peripheral nerves are not activated. Besides, a significant number of neurons remained non-responder to tDCS even at the highest implemented amplitude. Although it should be noted that one could still expect to see effects from standard tDCS in a cortical area located closer to the electrode and with a stronger electrical field.

One potential explanation for tDCS effects observed in humans lies in the notion that tDCS might also impact neural circuits through an indirect route, namely, using cranial/peripheral nerves [5, 10]. The results from a recent study revealed a significant increase in MEPs after tDCS over the motor cortex, suggesting a modulatory effect on corticospinal neurotransmission. The absence of significant modulation with a local anesthetic indicates that tDCS effects may involve peripheral and/or cranial nerve stimulation [10]. Extensive empirical support substantiates the potential viability of trigeminal nerve stimulation (TNS) as a viable substitute for conventional therapeutic options in addressing neuropsychiatric disorders [41–45]. The clinical efficacy of TNS is well-established [46, 47]. However, the neurobiological mechanisms by which this efficacy is asserted remain largely unexplored [48].

The results of our study showed that TN-DCS stimulation of the trigeminal nerve with direct current significantly increased the mean spike rate in the NVsnpr during stimulation. In this study, the mean basic spike rate in the NVsnpr was found to be between 14.81 ± 2.26 Hz (mean ± standard deviation) (3 mA) and 18.01 ± 1.56 Hz (0.5 mA) which increased to 31.58 ± 3.43 Hz in 3 mA and 21.45 ± 1.69 in 0.5 mA, respectively. Additionally, when the amplitude of TN-DCS rose, the number of respondents with a spike rate increase increased significantly. This is in line with the findings of Laturnus et al., who reported an increase in the basal median spike rate in the NVsnpr to 17.26 Hz [29.28 interquartile range (IQR)] after the introduction of the rat’s whiskers to 100 trials of seven-second white noise stimuli [49]. In humans, it has been shown that the application of acute, cyclic, 20 min TNS leads to a noteworthy alteration in the activity of the bilateral polysynaptic pathway that involves areas such as the trigeminal nuclei in the brain stem [50].

We observed the same pattern in the MeV where the basic mean firing rate increased from 16.98 ± 2.10 Hz to 20.82 ± 2.33 Hz in the 1 mA amplitude and from 15.72 ± 1.63 Hz to 26.63 ± 2.68 Hz in 3 mA amplitude. Besides, the number of responders with an increase in their spike rate significantly increased with an increase in the amplitude of TN-DCS. In an indirect relevance to this line of evidence, studies suggest that TNS via macrovibrissae movement causes an abrupt increase (with a very short latency of 1.3 ± 0.2 ms) in the neuronal discharge in the MeV which immediately returns to the basic level upon the termination of stimulation [51].

In the NVsnpr and with cathodic (and not anodic) stimulation, the mean spike rate remained significantly high up to 1 min after discontinuation of stimulation. Surprisingly, in the MeV and with anodic (and not cathodic) stimulation, the mean spike rate remained significantly high up to 1 min after discontinuation of stimulation. Otherwise, we found no difference in the mean spike rate in the NVsnpr and MeV between the anodic and cathodic stimulations. Evidence shows that axonal stimulation may depend, among other factors, on the axonal orientation relative to the electrode. Axons parallel to the electrode respond more favorably to cathodic stimulation, while those perpendicular to the electrode respond more favorably to anodic stimulation. The consequences of polarity changes are more complex in deep brain structures, as they can stimulate neurons with different orientations while inhibiting other neuronal populations [52–54]. Our study demonstrated that the administration of xylocaine to the marginal branch of TN, where the stimulation electrode was positioned, eliminated the enhancing effects of TN-DCS on neuronal activity in the NVsnpr and MeV. This suggests that the increase in mean spike rate in the NVsnpr and MeV was caused by direct stimulation of the trigeminal nerve and not by any current spreading to the nuclei under investigation.

An increase in the activity of trigeminal nuclei can have several behavioral and physiological consequences [14–19]. There are several pathway mechanisms via which TNS has the potential to produce these effects, with one example pathway being via the LC. LC is a pivotal hub within the ascending reticular activating system (ARAS), serving as the primary origin of norepinephrine (NE) within the central nervous system [3]. There are multiple routes through which trigeminal input could reach both the ARAS and LC. It has been shown that each trigeminal nucleus, NVsnpr, and MeV included, sends projections to the LC [55–57]. Furthermore, trigeminal nerve stimulation could also reach the LC by traveling through the nucleus of the NTS and the reticular formation [58, 59]. These projections do not end in the LC core but in the pericoerulear regions where the LC dendrites are located and thereby can influence the electrical coupling of LC neurons [60]. Interestingly, a decrease in trigeminal signals can result in lower levels of neurotrophic factors for LC neurons and thereby their dysfunction. This dysfunction can extend to the glial cells due to the strong electrical connection between MeV-LC cells and nearby astrocytes [61]. The LC is a key noradrenergic region in the brain, involved in modulating arousal levels and enhancing attention. Pupil dilation, regulated by the LC-NE system, is correlated with cognitive performance in healthy individuals [62]. A recent study from our lab investigated the effects of TNS on pupil dilation in healthy subjects. Compared to sham stimulation and median nerve stimulation, TNS resulted in greater pupil dilation [27]. These findings suggest that TNS may activate NE pathways, such as the LC-noradrenergic system [14, 27, 62]. This implies that the LC, and thus TNS, may play a significant role in modulating cognitive processes by influencing NE pathways in the brain [14].

Furthermore, LC has the potential to impact an individual’s mood [19]. In addition, research has demonstrated that stimulating the LC can effectively reduce the perception of acute pain by reducing the release of neurotransmitters from nociceptive afferents. This may explain the analgesic effects rendered by TNS [63].

The trigeminal nerve, via its brain stem nuclei, also reaches NTS. Subsequently, this nucleus innervates various brain areas including LC, RN, and last but not least amygdala and hippocampus [64]. Moreover, the LC and the RN are connected through reciprocal pathways, indicating that these neuromodulatory regions are likely to interact when they are directly or indirectly activated by TNS [65–67].

These three brainstem regions i.e., LC, RN, and NTS, possess anatomical positioning that enables them to directly or indirectly impact the neurochemistry of extensive areas within the central nervous system. This may explain some of the behavioral and neurophysiological effects observed during TNS and tDCS [68].

To the best of our knowledge, this research represents a novel endeavor, being the first kind to characterize the effects of trigeminal nerve direct current stimulation on brainstem nuclei. The control of stimulation intensities and stimulation’s DC nature enabled an exploration of the dose-response relationship, providing valuable knowledge about the neural mechanisms at play during TN-DCS. Additionally, the use of extracellular recordings offered a high temporal resolution, ensuring the accurate measurement of spike rates and their dynamic changes in response to stimulation. However, several limitations should be acknowledged. First and foremost is the generalizability of the findings, as individual variations among animals may not be fully accounted for. Nevertheless, we tried to use the LME model to analyze our data and address this issue. Furthermore, the invasive nature of extracellular recordings raises concerns about potential alterations in the neural activity being studied, which may affect the reliability of the results. The limited duration of the 3-min stimulation may not capture long-term effects or chronic responses adequately. It’s important to note that this study solely focuses on electrophysiological recordings, potentially overlooking other essential aspects of neural responses, such as changes in gene expression or synaptic plasticity. Besides, the stimulation of other cranial nerves such as the facial nerve (cranial nerve VII) through muscle spindles in the region could not be sufficiently ruled out using the results of this study. However, it should be noted that this sensory information is carried to the higher cortical areas via trigeminal nuclei and dorsal and ventral trigeminothalamic tract [69]. Furthermore, it has been demonstrated that lidocaine (~xylocaine) has no impact on the conductance of proprioceptive information. Given that other cranial nerves in the stimulated region, primarily relay motor and proprioceptive information, our data using the xylocaine block condition may suggest that these effects are primarily due to stimulation of the trigeminal nerve and not other cranial nerves [70]. Lastly, the absence of behavioral assessments in the study limits our ability to gain a comprehensive understanding of the functional consequences of the observed neuronal changes. Interpreting the spike rate changes in these nuclei in response to electrical stimulation presents a complex challenge. Numerous factors can influence neuronal firing, including network effects and neurotransmitter interactions. Therefore, it is crucial to interpret the results within the broader context of neural function and connectivity.

Conclusion

Our study demonstrated that both anodic and cathodic TN-DCS significantly increased neuronal spike rates in the NVsnpr and MeV nuclei during stimulation. These effects were reversible with xylocaine administration into the trigeminal nerve in rats. However, conventional tDCS did not affect the MeV spike rate. Contrary to the prevailing view, our findings suggest that peripheral/cranial nerve stimulation may play a substantial role in mediating the effects of tDCS, challenging the conventional notion of direct transcranial neuronal stimulation as the primary mechanism. However, it’s important to acknowledge the preliminary nature of this work. Future investigations should encompass additional nuclei between the trigeminal nuclei and the target brain areas to provide a comprehensive understanding of the neural pathways involved. This study serves as a starting point, encouraging further research and potential revisions of existing neuromodulation paradigms.

Author contributions

Conceptualization, MML, and AM; resources, AM and MML; data curation, AM and MML; methodology, MML; formal analysis, AM and MML, original draft writing, AM; review and editing, BA, and MML; supervision, MML; project administration, MML, funding, MML.

Funding

This study was supported by Fonds Wetenschappelijk Onderzoek [grant number G0B4520N] and the National Institutes of Health [grant number 1R01MH123508-01].

Data availability

Any datasets and codes that are a part of this study are available from the corresponding author upon reasonable request.

Competing interests

The authors declare no competing interests.

Ethical approval

All animal experiments adhered to the ARRIVE guidelines and were in accordance with the EU Directive 2010/63/EU for animal experiments. The experimental procedures conducted in this study received approval from the KU Leuven ethics committee for laboratory experimentation (License numbers: 201/2018 and 072/2020).