In recent years, there has been a resurgence of enthusiasm surrounding tDCS, as it emerged as a promising instrument for modulating cortical function in the human brain and addressing neuropsychiatric disorders [28]. The appeal of tDCS lies in its capacity to non-invasively alter cortical activity and modulate excitability through undamaged cranial structures [28]. Numerous investigations have been undertaken in recent years to examine the physiological impacts and mechanisms of action of tDCS [29, 30]. 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) [30, 31]. 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 [32, 33]. The majority of the electrical current is attenuated by the scalp and skull, leaving only a small portion to reach the brain. Recent investigations propose that as much as 75% of the administered current does not effectively reach its target within the brain [34]. One potential rationale lies in the notion that tDCS might also impact neural circuits through an indirect route, namely, using peripheral nerves [5, 8].
Extensive empirical support substantiates the potential viability of trigeminal nerve stimulation (TNS) as a viable substitute for conventional therapeutic options in addressing neuropsychiatric disorders [35–39]. The clinical efficacy of TNS is well-established [40, 41]. However, the neurobiological mechanisms by which this efficacy is asserted remain largely unexplored [42].
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. With cathodic stimulation, the mean spike rate remained significantly high up to one minute after discontinuation of stimulation. However, this was not the case with anodic stimulation. Otherwise, we found no difference in the mean spike rate in the NVsnpr between the anodic and cathodic stimulations. Interestingly, these effects disappeared after administration of xylocaine to the marginal branch of TN where the stimulation electrode was positioned, suggesting that the increase in the mean spike rate in the NVsnpr was caused by direct stimulation of the trigeminal nerve and not any current spread to the nuclei under investigation.
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. 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 [43]. In humans, it has been shown that the application of acute, cyclic, 20-minute 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 [44].
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 1mA condition and from 15.72 ± 1.63 Hz to 26.63 ± 2.68 Hz in 3 mA condition. 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 [45].
An increase in the activity of trigeminal nuclei can have several behavioral and physiological consequences [12–17]. There are several pathway mechanisms via which TNS has the potential to produce these effects, with one example pathway being vitae the LC. LC is a pivotal hub within the 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 [46–48]. Furthermore, trigeminal nerve stimulation could also reach the LC by traveling through the nucleus of the NTS and the reticular formation (RF) [49, 50]. These projections do not end in the LC core but in the periphery where the LC dendrites are located and thereby can influence the electrical coupling of LC neurons [51]. Interestingly, the 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 [52]. LC, a major noradrenergic area in the brain, can cause a desynchronization of neuronal activity, resulting in heightened levels of arousal and improved attention. This suggests that the LC plays a significant role in influencing these cognitive processes by modulating the electrical patterns in the brain. Furthermore, LC has the potential to impact an individual's mood [17]. 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 [53].
The trigeminal nerve, via its brain stim nuclei, also reaches NTS. Subsequently, this nucleus innervates various brain areas including LC, RN, and last but not least amygdala and hippocampus [54]. 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 [55–57].
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 [58].
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 DC-TNS. 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-minute 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. 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.