Transcranial Focal Stimulation modifies the genetic expression in the cerebral cortex and the hippocampus of healthy rats

doi:10.21203/rs.3.rs-1511681/v1

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Transcranial Focal Stimulation modifies the genetic expression in the cerebral cortex and the hippocampus of healthy rats

https://doi.org/10.21203/rs.3.rs-1511681/v1

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Transcranial Electrical Stimulation (TES) has emerged as an alternative therapy for cognitive and neuropsychiatric diseases. Transcranial Focal Stimulation (TFS) is an alternating current stimulation TES technique with a wide therapeutic potential. Nevertheless, the biological mechanisms behind the TFS effects are unknown.

Objective: The present study aims to describe the gene expression profile of the cerebral cortex and hippocampus after a short course of TFS.

Methodology: Using microarray analysis, the cerebral cortex and hippocampus of rats receiving TFS for 5 minutes were evaluated. These results were compared to those obtained from sham-stimulated rats. Relevant target genes were selected based on their biological processes, molecular function, and cellular localization. Relevant target genes were further normalized to visualize differential gene expression among experimental groups.

Results: The cerebral cortex samples found a clear differential expression profile. A total of differentially expressed genes of 102 in the cerebral cortex and 2 in the hippocampus were found. TFS increased the expression of Nsf (120%, p = 0.0004) and decreased the expression of Sema3b (57.6%, p = 0.0004) in the cerebral cortex. While in the hippocampus, TFS increased the expression of Acsm5 (200%, p = 0.001) and Cml3 (208%, p = 0.001).

Conclusion: A short course of TFS modifies the gene expression profile of the brain. The effects were more pronounced in the cerebral cortex rather than in the hippocampus. Further research is needed to validate these findings and to evaluate the long-term effects of TFS.

Transcranial Electrical Stimulation

Noninvasive neurostimulation

Microarrays

Gene expression

Transcranial Electrical Stimulation (TES) has emerged as a non-invasive therapeutic alternative for several diseases and as a potential enhancer of cognitive traits in healthy subjects (Clark et al., 2021; Cox et al., 2020; Morya et al., 2019; Schneider et al., 2021). TES techniques modify the neurophysiological properties of the brain in humans, non-human primates, and rodents (Fertonani & Miniussi, 2017; Liu et al., 2018). They also have been shown to induce plasticity-like events or neurotransmitter modulation in the human cerebral cortex (Bunai et al., 2021; Frase et al., 2021). Although, the mechanisms or biological pathways associated with these effects are unknown.

Gene expression profiling is a useful technique in toxicity screening studies (Mitchell & Mirnics, 2012; Rao et al., 2019), but it also could help us identify relevant biological effects of TES. For example, transcranial direct current stimulation (tDCS) modifies the cortical gene expression in pathways related to neuroplasticity and immune mediating effects (Holmes et al., 2016; Rabenstein et al., 2019). These effects have also been observed using different techniques, such as genetic knockdown or genetic studies (Chhabra et al., 2015; Kim et al., 2017). One of the main disadvantages of tDCS is its low special resolution and low current uniformity (van Oosterom & Strackee, 1983; Wiley & Webster, 1982).

Transcranial focal stimulation (TFS) is a TES technique that uses tripolar concentric ring electrodes (TCREs) (Besio et al., 2007). TFS has a greater current uniformity and a more focalized electric field compared to TES using other types of electrodes (T. Chen et al., 2009; Datta et al., 2008). This neuromodulation technique has shown anticonvulsant and neuroprotective effects in experimental animal models (Makeyev et al., 2013; Santana-Gómez et al., 2021; Valdés-Cruz et al., 2019). Nevertheless, the mechanisms behind these effects are unknown.

The literature shows that TFS has no impact on memory formation or hippocampal and cortical neuronal survival in healthy rats (Luby et al., 2014; Mucio-Ramirez et al., 2011; Mucio-Ramírez & Makeyev, 2017; Rogel-Salazar et al., 2013). However, the application of electric current in the brain is not innocuous (Antal et al., 2017) and TFS’ biological effects must be addressed carefully for long-term and clinical use. In the present study, it was proposed to identify if a short curse of TFS modifies the genetic expression in the cerebral cortex or hippocampus of healthy rats.

Animals and Habituation

Adult Wistar rats weighing 250–300 g at the beginning of the experimental manipulation were used. The rats were housed under controlled conditions (12 h light/dark cycles,22ºC) in groups of 5 before any experimental manipulation. All animals received water and food ad libitum. The manipulations and sacrifice of the animals were authorized by an internal ethics committee at the Center for Research and Advanced Studies (approval code CICUAL 125 − 15) and approved by the IMSS national committee for health research (R-2018-785-057). All the procedures were carried out following national (NOM-062-ZOO-1999) and international (National Institutes of Health Guide for the Care and Use of Laboratory Animals) laboratory animals care guidelines. None of the animals showed signs of stress or pain during or after the TFS application.

The habituation of the animals consisted of a daily administration for 7 days of saline solution (SS, NaCl 0.9%, 1ml/kg, i.p.) and 5 minutes of handling with a TCRE (10 mm diameter) on the scalp.

Transcranial Focal Stimulation

TFS consisted of the application of alternating current through the TCRE in the shaved head of the rats. The alternating current parameters were as follows: square biphasic pulses of 200 µs, at a frequency of 300 Hz, with an intensity of 50 mA, for 5 minutes. The electrical current was applied between the outer ring and the central disc of the TCRE with the middle ring electrode left floating. Before the application of TFS, the head of the rat was shaved and prepared. The rat’s head preparation consisted of the use of NuPrep (Weaver and Company, Aurora, CO), a mild abrasive, to eliminate dead skin and oils, and the use of Ten20 paste (Weaver and Company, Aurora, CO) for impedance matching between the electrode and the rat’s skin.

Experimental Groups

TFS (n = 3). One day after the last day of habituation, these animals received TFS. Three and a half hours after TFS administration, the animals were sacrificed by decapitation. The bilateral cerebral cortex (frontal, parietal, and temporal) and hippocampus were rapidly dissected under cold conditions and the structures were stored at -70°C until further use.

SS (n = 3). These animals were manipulated as those in the TFS group except that these rats did not receive TFS.

Ribonucleic Acid extraction, purification, and microarrays

Ribonucleic Acid (RNA) extraction was carried out using the TRIZOL (Cat. No. 15596026, Thermo Fisher Scientific, Massachusetts, USA) while RNA purification was carried out using magnetic pearls (Cat. No. A63987, Agencourt RNAClean XP, Indiana, USA). RNA concentration and purity were quantified using Nanodrop (Thermo Fisher Scientific, Massachusetts, USA). Rat’s transcriptome was evaluated using the Clariom S rat Assay microarrays (Cat. No. 902935, Thermo Fisher Scientific, Massachusetts, USA) and Clariom D rat Assay microarrays (Cat. No. 902631, Thermo Fisher Scientific, Massachusetts, USA). Microarray chips were processed in GeneChip Fluidics Station 450 (Thermo Fisher Scientific, Massachusetts, USA), hybridized in GeneChip Hybridization Oven 645 (Thermo Fisher Scientific, Massachusetts, USA), and scanned in GeneChip Scanner Affymetrix 3000 7G (Thermo Fisher Scientific, Massachusetts, USA). All the microarray processing was as recommended by the manufacturer (GeneChip WT Plus Reagent Kit, Cat. No. 902280, Applied Biosystems, Massachusetts, USA). CEL file-type files were used for further analysis.

Microarray data analysis

Raw microarray data (GSE198994 and GSE199183) were normalized using the robust multichip average (RMA) normalization method. General linear models and empirical Bayes statistics were used for differential gene expression. An absolute logarithmic fold change in expression greater than 1 and an unadjusted P-value lower than 0.05 were thresholds used for gene selection.

Since microarray data came from two different platforms (Clariom S and Clariom D). We used as reference the probes included in Clariom S for the normalization. Batch effects (platform) correction was performed using the ComBat method (C. Chen et al., 2011).

Differentially expressed genes were submitted to a gene ontology enrichment analysis (Ashburner et al., 2001) using the TopGO package (Alexa & Rahnenfuhrer, 2021). Gene enrichment analysis classifies genes by their cellular compartments, molecular functions, or biological processes. The genes that were statistically significantly classified in any gene ontology category were selected for a literature review. According to the gene ontology enrichment analysis and the literature review, a few genes were selected as relevant target genes for normalization. For the normalization of target genes, a set of possible constitutive genes was screened (Chapman & Waldenström, 2015; Radonić et al., 2004). The normalization consisted of the ratio of the expression value of each target gene over the expression of the constitutive gene in each of the subjects in the same experimental group and it is represented in arbitrary units (AU).

Statistical analysis and software

Data are presented as means ± standard error of the mean if their nature is quantitative. Otherwise, the data are presented as a percentage. Data normality was evaluated by the Shapiro-Wilk test. Student T or Mann-Whitney U tests were used depending on the data distribution. P-values lower than 0.05 were considered statistically significant.

All the microarray and statistical analyses were performed in R studio (version 1.4) using the R language (version 4.1, for the full list of packages to be used, go to supplementary table 1).

TFS modifies the gene expression profile of the cerebral cortex and hippocampus of healthy rats

Principal component analysis (PCA) was used to evaluate overall differences among the experimental groups. Initially, the cerebral cortex PCA data showed that the first two components account for 66.6% of the total variance (Fig. 1a). The hippocampal PCA data showed an overlapping between these two groups (Fig. 1b) with 56.9% of the total variance explained by the first two components.

The cerebral cortex samples showed a differential expression of 102 genes in the microarray analysis. Fifty-two percent of these genes were downregulated in the TFS group, while the remaining were upregulated (48%). Most of these genes (95%) are coding sequences, 3% were multiple or complex, and 2% were unassigned genes. Enrichment analysis showed that the differentially expressed genes are significantly located in nine different cellular compartments, they participate in two different molecular functions, and they are part of ten different biological processes (Table 1).

Table 1

TopGO enrichment analysis of the differentially expressed genes between TFS and Sham groups in the cerebral cortex
TopGo Category	GO.ID	Description	Participating genes (#)
Cellular compartment	GO:0043229	Intracellular organelle	50
	GO:0043226	Organelle	53
	GO:0043231	Intracellular membrane-bounded organelle	45
	GO:0031984	Organelle sub-compartment	9
	GO:0098827	Endoplasmic reticulum sub-compartment	6
	GO:0005789	Endoplasmic reticulum membrane	6
	GO:0043227	Membrane-bounded organelle	47
	GO:0042175	Nuclear outer membrane-endoplasmic reticulum membrane network	6
	GO:0000776	Kinetochore	3
Molecular Function	GO:0005506	Iron ion binding	3
Molecular Function	GO:0016705	Oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen	3
Biological Processes	GO:0048863	Stem cell differentiation	4
	GO:0098876	Vesicle-mediated transport to the plasma membrane	3
	GO:0048762	Mesenchymal cell differentiation	4
	GO:0060485	Mesenchyme development	4
	GO:0048545	Response to steroid hormone	6
	GO:0006886	Intracellular protein transport	8
	GO:0140014	Mitotic nuclear division	5
	GO:0065004	Protein-DNA complex assembly	3
	GO:0006323	DNA packaging	3
	GO:0050770	Regulation of axonogenesis	3

The hippocampal samples showed two differentially expressed genes between the TFS and Sham groups in the microarray analysis: the acyl-CoA synthetase medium-chain family member 5 (Acsm5) and the N-acetyltransferase 8 (GCN5-related) family member 3 (Cml3). These two genes are coding sequences and, they are downregulated in the TFS group. After enrichment analysis, no significant cellular locations, molecular functions, or biological processes were found.

Constitutive and target gene selection

The expression of neither the metabolic (Glyceraldehyde-3-phosphate dehydrogenase) nor structural (actin β or β2 microglobulin) showed a stable expression across the experimental groups. However, cortical Peptidylprolyl Isomerase A (Ppia) expression was similar in the TFS compared to the Sham group (23.8 ± 1.1 vs 23.3 ± 1.8 AU, p > 0.05). The same finding applied for hippocampal Ppia expression (26.4 ± 1 vs 24.4 ± 2.2 AU, p > 0.05). Therefore, this gene was selected for further normalization of the target genes.

From all the differentially expressed genes of the cortical samples, semaphorin 3B (Sema3b) and N-ethylmaleimide-sensitive Factor (Nsf) were selected according to the previously commented methodology. The normalized Sema3b/Ppia gene expression was downregulated in the TFS group compared to the Sham group (1.4 ± 0.1 vs 3.3 ± 0.1, p = 0.0004, Fig. 2a). Conversely, the normalized Nsf/Ppia gene expression was upregulated in the TFS group compared to the Sham group (1.1 ± 0.02 vs 0.5 ± 0.01, p = 0.0004, Fig. 2b).

In hippocampus samples, the normalized Acsm5/Ppia gene expression was upregulated in the TFS group compared to the Sham group (5.4 ± 0.4 vs 2.7 ± 0.4, p = 0.001, Fig. 3a). Similarly, the Cml3/Ppia gene expression was upregulated in the TFS group compared to the Sham group (9.6 ± 1.1 vs 4.6 ± 0.3, p = 0.001, Fig. 3b).

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.

TFS modifies the genetic expression in the brain after a short-term administration. These changes are more evident in the cerebral cortex than in the hippocampus. TFS, like other TES techniques, could induce neuroplasticity in the cerebral cortex. This modification of neuron communication can have relevant applications for cognitive science and neuropsychiatric disorders. Unlike other TES techniques, TFS displays an increased spatial resolution and uniform current density. These two characteristics make it an excellent option for clinical applications where specific targeting is necessary. Further studies need to be conducted to validate these findings and their clinical relevance.

Funding: The research in this paper was funded by the FIS/IMSS/PROT/PRIO/019/092.

Conflict of interest: The authors have no relevant financial or non-financial interests to disclose.

Author contribution: DPP: Methodology, Software, Formal Analysis, Investigation, Data Curation, Writing-Original Draft, and Visualization. IFR: Methodology, Investigation, Writing-Review & Editing, and Supervision. WB: Conceptualization, Investigation, Writing-Review & Editing, Project administration, and Funding acquisition. LR: Conceptualization, Investigation, Writing-Review & Editing, Project administration, and Funding acquisition. LBO: Methodology and Investigation. SOS: Conceptualization, Investigation, Writing-Review & Editing, Project administration, and Funding acquisition.

Data availability: All the data is freely available in the Gene Expression Omnibus (cat#: ). Any other request can be done to the corresponding author.

Ethics approval: The manipulations and sacrifice of the animals were authorized by an internal ethics committee at the Center for Research and Advanced Studies (approval code CICUAL 125-15) and approved by the IMSS national committee for health research (R-2018-785-057). All the procedures were carried out following national (NOM-062-ZOO-1999) and international (National Institutes of Health Guide for the Care and Use of Laboratory Animals) laboratory animals care guidelines.

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Transcranial Focal Stimulation modifies the genetic expression in the cerebral cortex and the hippocampus of healthy rats

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Introduction

Materials And Methods

Animals and Habituation

Transcranial Focal Stimulation

Experimental Groups

Ribonucleic Acid extraction, purification, and microarrays

Microarray data analysis

Statistical analysis and software

Results

TFS modifies the gene expression profile of the cerebral cortex and hippocampus of healthy rats

Constitutive and target gene selection

Discussion

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