Physiological mechanisms underlying transcranial direct-current stimulation effects on mice somatosensory and cerebellar cortices

Laburpena

Transcranial direct-current stimulation (tDCS) is one of the most commonly used non-invasive brain stimulation techniques consisting in the application of weak electrical currents through the scalp. The technique began to gain popularity about 20 years ago when its neuromodulatory effects were demonstrated during the administration of tDCS and, perhaps most importantly, lasting several minutes after stimulus cessation. It is known from in vitro studies, that immediate observed neuronal modulation is caused by the polarization of the different neuronal compartments (soma, dendrites, axon) caused by the displacement and redistribution of charges due to the electric field. On the other hand, long-term effects have been related with membrane receptors changes, protein modifications and glial activity. However, there are still huge gaps of knowledge about the physiological mechanisms underlying its effects. For this reason, the main objective of the present Doctoral Thesis was to examine and characterize the effects and mechanisms behind the neuromodulation of tDCS in somatosensory (S1) and cerebellar (CrusI-II) cortices in the alert mice. First, the strength of the electric field generated by tDCS was assessed, observing a decay with distance from the electrode in a logarithmic manner when applied over S1 (S1-tDCS) or lateral cerebellum (Cb-tDCS). In addition, the actual electric field generated in somatosensory cortex when tDCS was applied over lateral cerebellum was two orders of magnitude lower than the electric field generated by tDCS directly applied over S1. After that, tDCS effects on somatosensory and cerebellar cortices excitability were characterized by means of electrophysiological and immunohistochemical measures. For S1-tDCS, there was a polarity and intensity-dependent modulation of sensory evoked potentials (SEPs) during the application of transcranial currents with anodal increasing and cathodal decreasing S1 excitability. Nonetheless, after tDCS cessation, just cathodal stimulation maintained a decreased excitability for up to one hour. This result was in accordance with an increase in GAD65-67 levels observed in the stimulated hemisphere after 20 minutes of cathodal tDCS. With respect to Cb-tDCS, there was also an immediate increase with anodal and decrease with cathodal Cb-tDCS of the cerebellar SEPs during the application of the current. Nevertheless, no long-term effects were observed after tDCS cessation for both electrophysiological nor GAD65-67 or vGlut1 levels. After that, the impact of Cb-tDCS on interconnected distant regions (S1) was evaluated, showing a decrease in S1 excitability during anodal and an increase during cathodal Cb-tDCS. Interestingly, after the instant decrease in S1 excitability observed during the first minutes of anodal Cb-tDCS, there was a return to control levels in the last minutes of stimulation and, intriguingly, an increase in excitability appeared just after anodal Cb-tDCS was switched off. No changes were observed after cathodal Cb-tDCS, and no changes were observed for GAD65-67 or vGlut1 levels in S1 after anodal nor cathodal Cb-tDCS. Lastly, a more detailed analysis of tDCS effects on neuronal excitability was explored by single-cell recordings during Cb-tDCS in awake mice, showing a polarity and intensity-dependent modulation of ongoing firing activity of Purkinje and non-Purkinje cells in a heterogeneous manner. To unravel the causes of this heterogeneous behaviour, juxtacellular labelling of the recorded neurons in anesthetized mice was performed. The observed results indicate that the somatodendritic axis orientation with respect to the tDCS-generated electric field was the main factor determining the modulation of the Purkinje cells. In conclusion, present results show direct evidence of the different effects that tDCS may have on different stimulated regions, providing evidence of the importance of potential distant effects induced by this neuromodulatory technique. Finally, the data presented in this thesis constitutes the first in vivo experimental evidence of the fundamental role of the somatodendritic axis orientation on tDCS polarizing effects.