The present study allows us to describe an overall differential expression in two brain regions induced by a short course of TFS (5 minutes, 10 mm diameter). This differential expression was more evident in the cerebral cortex compared to the hippocampus of the rats. Also, it revealed the differential expression of cortical and hippocampal genes that could be relevant for understanding the basic mechanism and possible biological effects of TFS.
This study demonstrates that a short course of TFS modifies the gene expression in the cerebral cortex and hippocampus of healthy rats. These results partially agree with previous studies demonstrating changes in the expression of neuroplasticity and immune-mediating genes induced by tDCS in healthy animals (Holmes et al., 2016; Rabenstein et al., 2019). Overexpression of immune-mediating genes such as major histocompatibility complex I (MHC-I) could be associated with an inflammatory response after the application of TES (Pikhovych et al., 2016; Rabenstein et al., 2019; Rueger et al., 2012). In contrast to those studies, TFS does not modify immune-mediating genes, but it does modify genes related to neuroplasticity. This difference in effects could be a consequence of a difference in the duration of administration of the TES. In the studies using tDCS, the researchers administered the stimulation for 20 minutes while TFS was applied for only 5 minutes in this study. Preclinical evidence indicates that TES effects could depend on the duration of the stimulation (Liu et al., 2018; Wen et al., 2017). Then, a longer or repetitive application of TFS may also show a modified expression of immune-mediated genes.
Another interesting finding was the difference between the amount of differentially expressed genes between the cerebral cortex (102 genes) and the hippocampus (2 genes). This could be a consequence of a difference in the distance between the electrode and the stimulated brain region. Computational models infer a decay in the electric field strength as the distance from the stimulating electrode increases (Besio et al., 2011; Datta et al., 2008). Also, tDCS displays this characteristic (Huang et al., 2017). Although previous studies have shown that the electric field strength in deeper structures may not induce changes in the neuronal spiking rate (Anastassiou et al., 2011), recent evidence shows that TES using alternating current induces changes in deeper structures in the brain (Louviot et al., 2021). In consequence, TFS parameters could be optimized depending on the desired target, for example, the hippocampus.
In this study, Ppia was selected for gene normalization. This gene encodes for an enzyme related to transductional processes, it may play a role in cyclosporin A-mediated immunosuppression, and it is not modified by TFS application. These characteristics allowed us to reliably analyze target genes. Previous evidence recommends the use of metabolic (Glyceraldehyde-3-phosphate dehydrogenase), structural (actin β or β2 microglobulin), or unassigned (Ppia) genes for normalization depending on the experimental intervention (Chapman & Waldenström, 2015; Radonić et al., 2004). Preceding experiments evaluating gene expression in brain samples after tDCS used the ribosomal protein L13A (Kim et al., 2017). In the present study, most of the selected constitutive genes showed wide variations across experimental groups and regions between groups (data not shown). Then, the metabolic or structural constitutive genes could not be adequate for normalization in the context of TES or TFS.
The current study describes a significantly lower expression of Sema3b and a significantly higher expression of Nsf in the TFS as compared with the Sham group in the cerebral cortex. Sema3b belongs to the semaphorins family, this family of proteins is widely known as axon repulsion (Bron et al., 2007; Chédotal et al., 1998). Although Sema3b has shown both attractive and repulsive properties for axonal guidance (Julien et al., 2005), the diminished expression induced by TFS could be an early signal for axonal growth and rewiring in the cerebral cortex facilitating neuroplasticity. Conversely, Nsf encodes for a regulatory protein involved in receptor trafficking (Whiteheart et al., 1994) and, it is essential for adequate neurotransmitter receptor trafficking, including the γ-aminobutyric acid (GABA) (Chou et al., 2010; Goto et al., 2005), metabotropic glutamate receptor 2 (GluR2) (Beretta et al., 2005; Joels & Lamprecht, 2010), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (Steinberg et al., 2004), and dopamine receptor (S. Chen & Liu, 2010). Five minutes of TFS increased the expression of Nsf, then increased neurotransmitter receptor expression. Previous evidence showed TFS increases GABA and decreases glutamate release in normal conditions (Santana-Gomez et al., 2015). Thus, TFS could be modifying neurotransmitter release and its receptor concentration.
A short course of TFS induces the overexpression of Acsm5 and Cml3 in the hippocampus. Acsm5 is an enzyme that belongs to a family of coenzyme A synthetases, and this family participates in the metabolism of fatty acids (Watkins et al., 2007). This gene is widely expressed in the liver with almost no expression in other parts of the body (Yu et al., 2014). On the other hand, the brain expression of Cml3 increases with the age of the rat (Yu et al., 2014). Although studies have shown that tDCS increases glucose metabolic consumption (Jeong et al., 2021; Kraus et al., 2020), there is no information about the effects of TES techniques on fatty acid metabolism. Interestingly, this protein has been related to alteration in the development of the zebrafish and the Xenopus laevis (Karmodiya et al., 2014; Popsueva et al., 2001). The information on Cml3 and Acsm5 physiological and biological implications is scarce, and this prevents us from making conclusions about their relevance for TFS therapy.
Among the limitations of the present study are the lower specificity of the microarray analysis and a relatively short administration time of TFS (compared to other TES techniques). Microarray experiments could be considered a screening method and their results must be validated using more specific techniques (Chuaqui et al., 2002). The length for TFS administration was selected based on previous experiments and the animal comfort for noninvasive manipulations. Microarray experiments are expensive and these results should be used to extract the most relevant hypotheses in validation experiments.