Auricular Transcutaneous Vagus Nerve Stimulation Acutely Modulates Brain Connectivity in Mice

Abstract

Brain electrical stimulation techniques take advantage of the intrinsic plasticity of the nervous system, opening a wide range of therapeutic applications. Vagus nerve stimulation (VNS) is an approved adjuvant for drug-resistant epilepsy and depression. Its non-invasive form, auricular transcutaneous VNS (atVNS), is under investigation for applications, including cognitive improvement. We aimed to study the effects of atVNS on brain connectivity, under conditions that improved memory persistence in CD-1 male mice. Acute atVNS in the cymba conchae of the left ear was performed using a standard stimulation protocol under light isoflurane anesthesia, immediately or 3 h after the training/familiarization phase of the novel object-recognition memory test (NORT). Another cohort of mice was used for bilateral c-Fos analysis after atVNS administration. Spearman correlation of c-Fos density between each pair of the thirty brain regions analyzed allowed obtaining the network of significant functional connections in stimulated and non-stimulated control brains. NORT performance was enhanced when atVNS was delivered just after, but not 3 h after, the familiarization phase of the task. No alterations in c-Fos density were associated with electrostimulation, but a significant effect of atVNS was observed on c-Fos-based functional connectivity. atVNS induced a clear reorganization of the network, increasing the inter-hemisphere connections and the connectivity of locus coeruleus. Our results provide new insights into the effects of atVNS on memory performance and brain connectivity extending our knowledge of the biological mechanisms of bioelectronics in medicine.

Keywords: auricular transcutaneous vagus nerve stimulation; brain connectivity; c-Fos functional networks; electrostimulation; memory persistence.

Figure 1

(A) atVNS improves object-recognition memory persistence in naïve mice when administered immediately after the familiarization phase of the novel object-recognition test (NORT). Discrimination index and total exploration time in NORT for atVNS (0 h), atVNS (3 h), and No stimulation conditions in naïve CD-1 mice [atVNS (0 h) condition, n = 11; atVNS (3 h) condition, n = 12; No stimulation condition, n = 12]. *p < 0.05 by the one-way ANOVA. (B) c-Fos density in No stimulation and atVNS (0 h) conditions, separating contralateral (right, R) and ipsilateral (left, L) sides according to the site of the stimulation. The brain regions analyzed are organized from frontal to caudal and grouped in the cortical (orange), hippocampal (purple), amygdalar (red), thalamic (green), and brainstem (blue) groups.

Figure 2

(A) Effects of atVNS on global network connectivity. Total connectivity presented as z-score, comparing No stimulation and atVNS (0 h) conditions. ***p < 0.001 by the Kruskal–Wallis test. (B) Network connectivity graphs displaying only the significant correlations (p < 0.05). Connecting lines represent Spearman correlation (positive correlation in blue, negative correlation in red). Strongest significant correlations are highlighted in orange (p < 0.01) and yellow (p < 0.001). Regions are presented from frontal to caudal and separating left (light gray) and right (light yellow) sides. Regions are grouped into the cortical (orange), hippocampal (purple), amygdalar (red), thalamic (green), and brainstem (blue) groups. (C) Network connectivity Kamada–Kawai plots displaying only positive significant z-score for No stimulation and atVNS (0 h) conditions. Colors represent the cortical (orange), hippocampal (purple), amygdalar (red), thalamic (green), and brainstem (blue) groups. Regions are grouped based on the connectivity strength between them.

Figure 3

(A) Brain regions ranked in descending order based on nodal strength and nodal degree coefficients for No stimulation and atVNS (0 h) conditions. Regions are displayed with color coding: cortex (orange), hippocampus (purple), amygdala (red), thalamus (green), and brainstem (blue). (B) Difference in the total connectivity of right and left locus coeruleus (LC) regions with the rest of the brain areas analyzed, presented as z-score and comparing No stimulation and atVNS (0 h) conditions. **p < 0.01; ***p < 0.001 by the Kruskal–Wallis test.