Feasibility and effectiveness of transcutaneous auricular vagus nerve stimulation (taVNS) in awake mice

Abstract Aims Transcutaneous auricular vagus nerve stimulation (taVNS) is widely used to treat a variety of disorders because it is noninvasive, safe, and well tolerated by awake patients. However, long‐term and repetitive taVNS is difficult to achieve in awake mice. Therefore, developing a new taVNS method that fully mimics the method used in clinical settings and is well‐tolerated by awake mice is greatly important for generalizing research findings related to the effects of taVNS. The study aimed to develop a new taVNS device for use in awake mice and to test its reliability and effectiveness. Methods We demonstrated the reliability of this taVNS device through retrograde neurotropic pseudorabies virus (PRV) tracing and evaluated its effectiveness through morphological analysis. After 3 weeks of taVNS application, the open field test (OFT) and elevated plus maze (EPM) were used to evaluate anxiety‐like behaviors, and the Y‐maze test and novel object recognition test (NORT) were used to evaluate recognition memory behaviors, respectively. Results We found that repetitive taVNS was well tolerated by awake mice, had no effect on anxiety‐like behaviors, and significantly improved memory. Conclusion Our findings suggest that this new taVNS device for repetitive stimulation of awake mice is safe, tolerable, and effective.


| INTRODUC TI ON
2][3] The concept of taVNS was proposed in 2000, and more than 20 years of research has revealed that this strategy can treat over 30 diseases and disorders. 4,55][26] taVNS has promising application value for clinical disease prevention due to its safety, tolerability, and effectiveness.
Research on the mechanism of action of taVNS has gradually progressed to include animal experiments, but there is still a significant gap between the stimulation methods used in animal research and clinical settings.Two of the important differences are the use of anesthesia and the stimulation intensity.In many clinical studies, taVNS is repeatedly administered for a long period to awake patients. 13,18,270][31][32] When an appropriate stimulation intensity is used, taVNS is well tolerated by patients even when repeated over a long period.Unfortunately, the high stimulation intensity tolerated by mice under general anesthesia was intolerable to conscious mice.Obviously, the design of a new stimulation method that completely mimics the technique used in clinical settings and can be well tolerated by awake mice is extremely necessary for generalizing research findings related to the effects of taVNS.The development of this method in animal studies avoids the interference of repeated general anesthesia to the experiment itself, which enables closer integration of basic and clinical research.
In this study, we utilized a taVNS device that could be reliably immobilized in mice to investigate the effects of repeated stimulation at an appropriate stimulation intensity in awake mice.

| Animals
Male C57BL/6J mice (at least 8 weeks old) were obtained from the Laboratory Animal Center at Xuzhou Medical University.The mice were housed in single cages at a stable temperature (22 ± 1 °C) and humidity (30%-50%) on a 12-h light/dark cycle (starting at 7:00 a.m.) with ad libitum access to food and water.The care and use of animals

| Electrode implantation
Mice were anesthetized with 1% sodium pentobarbital (40 mg/ kg, intraperitoneal injection, i.p.) and placed on a heating pad to maintain their body temperature at 35-37°C, and then, their eyes were coated with ophthalmic ointment to prevent drying.
To minimize impedance, the fur was removed from the electrode implantation site with a mild depilatory cream.Immediately, two 0.3-mm-diameter platinum wire electrodes were placed in the cymba conchae, which is innervated by the auricular branch of the vagus nerve (ABVN).The skin of the ear was penetrated with the platinum wires to form a loop (total length of approximately 1 cm, approximately 0.5 cm on the inner and outer sides of the ear, respectively) around the ear skin, and the platinum wire on the dorsal side of the ear and connected to a stimulator (Figure 1A).

| Transcutaneous auricular vagus nerve stimulation (taVNS)
The electrodes were connected to a stimulator (Kedoubc, Suzhou, China) by wires (Figure 1A).The stimulation parameters were as follows: 0.8 mA, 20 Hz, the total length of 30 min, and a 300 μs pulse width.

| Stereotaxic surgery and virus injection
To verify the distribution of the vagus nerve at the electrode site and its connection with the brain nucleus, we anesthetized the mice with 1% pentobarbital (40 mg/kg, i.p.) and then fixed them on a stereotaxic apparatus (RWD, Shenzhen, China).Then, their eyes were lubricated with ophthalmic ointment.A total of 300-500 nL volume of PRV-CAG-EGFP (BrainVTA, Wuhan, China) was injected into the helix of the ear at a rate of 0.1 μL/min (Figure 2D).After injection, the needle was slowly retracted to minimize backflow.
The mice were subsequently placed in a cage on a heating pad and returned to their home cages when they were fully awake.After allowing 5 days for virus expression, histological verification of the viral injection site and infection of target brain regions was performed (Figure 2A-C,E).

| Immunohistochemistry
Mice were sacrificed in a CO 2 chamber and then subjected to cardiac perfusion with 20 mL of phosphate-buffered saline (PBS) followed by 20 mL of 4% paraformaldehyde (PFA).The mouse brains were removed, post-fixed in PFA for 4-6 h, and subsequently dehydrated with 30% sucrose at 4°C until the tissue sank to the bottom.
Coronal brain sections (30 mm thick) were prepared with a freezing microtome (VT1000S, Leica Microsystems).Confocal images were acquired using a confocal microscope (LSM 880, Carl Zeiss).To obtain images of entire sections, image tiles were stitched together using Zeiss software.The images were further processed using ZEN software (version 2.3, blue edition; Zeiss, Germany).

| Open field test (OFT)
Mice were placed in a white plastic open field (40 cm × 40 cm × 50 cm), which was divided into a 3 × 3 central area, four corner areas, and four side areas.The trajectories of the mice over 5 min were recorded by the SMART tracking system (Panlab, Spain) to assess anxiety-related behaviors.

| Elevated plus maze (EPM) test
The EPM test is used to assess anxiety levels. 33,34The maze was elevated 50 cm above the ground and consisted of two open arms (30 cm × 5 cm), two closed arms (30 cm × 5 cm, 20 cm tall walls) with open roofs, and a central platform (5 cm × 5 cm).The mice were placed on the central platform facing an open arm.They were allowed to freely explore the maze for 5 min, and their behavior was recorded and analyzed with EthoVision XT 14.0 software.

| Novel object recognition test (NORT)
Mice were placed in a square arena ( the arena for 10 min.After 2 h of rest in their home cages, the mice were returned to the arena after one object was randomly replaced by a new object.The time spent exploring each object was recorded over 5 min to assess the memory of the mice.

| Y-maze test
The Y-maze test was performed with a Y-shaped device consisting of 3 white, closed arms at an angle of 120° to each other (A, B, and C), with each arm being 30 cm long, 6 cm wide, and 15 cm high.After closing arm C with a white plastic sheet, the mice were placed in arm A and allowed to freely explore arms A and B. After 2 h of rest in their home cages, the mice were returned to the arena.During the test, arm C was opened, and the exploratory behavior of the mice in the three arms over 5 minutes was analyzed.

| Statistical analysis
The data are presented as the mean ± SD.All statistical analyses were performed with GraphPad Prism 7.0 (GraphPad Software, Inc., USA).Two-way ANOVA and Bonferroni post hoc analyses were used in analyses with multiple comparisons.Paired two-tailed t-tests or unpaired two-tailed t-tests were used for comparison between two groups.p < 0.05 indicated statistical significance.

| RE SULTS
First, we evaluated the reliability of the placement of the taVNS device in the mouse ear.The impedance of the stimulating electrodes stabilized 8 days after implantation and was maintained throughout the 3-week taVNS procedure (Figure 1B), suggesting that the attachment between the stimulating electrodes and the cymba conchae was stable.The auricular branch of the vagus nerve (ABVN) is a branch of the vagus nerve that forms a cutaneous receptive field over the auricle 35,36 and projects somatosensory afferents to the nucleus tractus solitarius (NTS) and spinal trigeminal nucleus (Sp5). 16 utilizing the retrograde neurotropic pseudorabies virus (PRV) tracing strategy, [37][38][39][40] we confirmed the connection of the ABVN with the NTS and Sp5, providing an anatomical basis for the reliable application of taVNS.PRV-CAG-EGFP was unilaterally injected into the helix of the mouse ear (Figure 2D).Five days later, we sacrificed the mice and observed EGFP-labeled neurons in the NTS and Sp5 (Figure 2B,C).
We subsequently confirmed the effectiveness of this taVNS strategy.We assessed changes in the excitability of the NTS and Sp5 after taVNS by evaluating the expression of c-Fos, a molecular proxy of neural activity. 41After 3 weeks of taVNS, the expression levels of c-Fos in the NTS and Sp5 were dramatically higher than those in the control group (c-Fos in the NTS: control = 103.56± 30.59, taVNS = 342.78± 46.05, p < 0.0001; c-Fos in the Sp5: control = 101.67± 17.75, taVNS = 238.33 ± 29.51, p < 0.0001) (Figure 3).While taVNS was administered under anesthesia in previous studies, 2,12,28,42,43 4).Compared with the control group mice, the taVNS group mice exhibited no significant differences in body weight (Figure S1).Consistent with previous findings, 25 compared with the control group, the taVNS group showed a significant improvement in object recognition memory after 3 weeks of stimulation, which provides convincing evidence for the effect of taVNS on improving memory behaviors (novel object recognition test (NORT): control = 0.54 ± 0.33, taVNS = 1.62 ± 0.34, p < 0.0001; Y-maze: control = 6.64 ± 1.87, taVNS = 9.77 ± 2.09, p = 0.0008) (Figure 5).We report a new method for applying taVNS in awake mice that is safe, noninvasive, and well tolerated, and based on anatomical validation and improvements in memory, we confirm that this new stimulation modality is effective.
In a large number of previous clinical studies, taVNS was applied persistently and repetitively, often in awake patients, for safety and maneuverability reasons. 13,44In contrast, in many animal experiments, mice are given general anesthesia when receiving taVNS due to technical and equipment limitations. 2,3,12,25,28,42,43Repeated anesthesia may have marked impacts on the brain, such as neurotoxicity and cognitive impairment. 7In the present study, taVNS was administered to awake mice, allowing basic research findings to be generalized to clinical situations and providing a technique for treating various diseases with taVNS in animals.
Platinum wires were used as the electrode for taVNS due to their excellent histocompatibility and corrosion resistance.The impedance of the stimulating electrodes remained stable approximately 1 week after implantation, which indicated that the device could be reliably fixed and had stable performance.Neuroanatomical studies have shown that the only branch of the vagus nerve on the surface of the body is the ABVN, and the cymba conchae is innervated exclusively by the ABVN. 12,25,45The NTS and Sp5, as the brainstem nuclei, in which vagus nerve afferent fibers terminate, are considered relay stations for visceral information, and they receive signals from the ear and regulate body functions. 3,35The ABVN-innervated region of the ear, known as the cymba conchae, was found to be clearly connected to the NTS and Sp5 in the brainstem through retrograde transsynaptic PRV tracing.This work further confirmed that the electrode implantation site was located in a dense region of the vagus nerve in the ear, providing an anatomical basis for the effects of taVNS.
Notably, the stimulus intensity used for taVNS was effective in freely moving awake mice, as evidenced by elevated c-Fos expression in the NTS and Sp5.In the preliminary experiment, we monitored the behavior of mice receiving taVNS at different stimulus intensities and determined that 0.8 mA was the highest stimulus intensity that did not interfere with free movement in awake mice under the current experimental conditions.Consistent with the findings of previous studies, our present study showed that taVNS improved memory but did not induce anxiety in mice. 3,25All mice tolerated taVNS without adverse events during the experiment (see Data S2).
Inevitably, there are certain limitations to this method.Because of the platinum wires connection between the mice's ears and the stimulator, some interference may occur if behavioral tests need to be performed at the same time as the taVNS.However, in general, tried to consider wireless stimulation to address this limitation.For example, wireless stimulation is performed by embedding an induction coil in the mouse, but in this case, the stability of the stimulation is difficult to ensure as the mouse position changes.A previous study applied wireless optoelectronic devices to vagus nerve stimulation in mice, but there were many drawbacks, such as invasiveness, limited efficiency, and large individual differences. 46Taken together, our current method is applicable to most studies.

| CON CLUS ION
This study developed a new taVNS method that utilizes a device that can be fully immobilized, fully mimics the taVNS technique used in clinical settings, and is well tolerated by freely moving awake mice.In addition, administration of taVNS to awake mice for 3 consecutive weeks significantly improved memory but had no effect on anxiety-like behaviors.This new taVNS method has promising applications in research on awake animals and has the potential to improve the generalizability of basic research findings in clinical settings.
F I G U R E 5 Application of taVNS for 3 weeks improves memory in freely moving mice.(A) Experimental scheme showing the application of taVNS and behavioral tests.(B) The exploration time ratio for the novel and familiar objects in the NORT was significantly higher in the taVNS group than in the control group (control = 0.54 ± 0.33, taVNS = 1.62 ± 0.34, ****p < 0.0001).n = 12 mice/group.(C) Representative heat maps showed the time spent exploring each object of control and taVNS mice.(D) There was a significant increase in the number of spontaneous alternations in the Y-maze test in the taVNS group compared to the control group (control = 6.64 ± 1.87, taVNS = 9.77 ± 2.09, ***p = 0.0008).n = 12 mice/group.(E) Representative heat maps showed the exploratory behavior in the three arms of control and taVNS mice.The data were analyzed by unpaired two-tailed t-test.The data are presented as the mean ± SD.NORT, novel object recognition test.
and the experimental protocols used in this study were approved by the Institutional Animal Care and Use Committee and the Office of Laboratory Animal Resources of Xuzhou Medical University under the Regulations for the Administration of Affairs Concerning Experimental Animals (1988) in China.

F I G U R E 1 | 3 of 8 YU
The diagram of electrode implantation of the taVNS device in awake mice.(A) Schematic representation of the method used to implant the taVNS device in the mouse auricle, which is the distribution area of the ABVN.(B) The electrode impedance data showed that the impedance was stable 8 days after electrode implantation.***p < 0.001, ****p < 0.0001; ns, no significant difference (p > 0.05).The data were analyzed by paired two-tailed t-tests.The data are presented as the mean ± SD.ABVN, auricular branch of the vagus nerve; taVNS, transcutaneous auricular vagus nerve stimulation.et al.
25 cm × 25 cm × 30 cm) with two identical objects in adjacent corners and allowed to freely explore F I G U R E 2 Schematic of validation of the reliability of the taVNS method by utilizing the retrograde neurotropic PRV tracing strategy.(A) Schematic representation of the location of brainstem nuclei NTS and Sp5.(B) Representative confocal image showing the EGFP-positive neurons in the NTS.(C) Representative confocal image showing the EGFP-positive neurons in the Sp5.(D) Schematic representation of injection of the retrograde PRV-CAG-EGFP virus into the helix of the mouse ear.(E) Representative confocal image showing the virus injection site in the ear.Scale bar: 200 μm.NTS, nucleus tractus solitarius; PRV, pseudorabies virus; Sp5, spinal trigeminal nucleus.
electrical stimulation was applied in awake mice in the present study; consequently, it was necessary to monitor anxiety-like behaviors and body weight to exclude the possibility of negative emotions caused by an improper stimulus intensity.The time spent in the center or open area in the open field test (OFT) or elevated plus maze (EPM), respectively, did not significantly differ between the taVNS group and control group (OFT: control = 3.84 ± 1.44, taVNS = 4.83 ± 1.24, p = 0.0842; EPM: control = 20.48 ± 4.37, taVNS = 23.43 ± 4.18, p = 0.1051) (Figure

F I G U R E 3
taVNS effectively activates the NTS and Sp5 in freely moving awake mice.(A) Representative images of c-Fos-positive neurons in the NTS in control and taVNS group mice.n = 9 sections from three mice per group.Scale bar: 200 μm.(B) The expression levels of c-Fos in the NTS were significantly higher than those in the control group (control = 103.56± 30.59, taVNS = 342.78± 46.05, ****p < 0.0001).(C) Representative images of c-Fos-positive neurons in the Sp5 in control and taVNS group mice.n = 9 sections from three mice per group.Scale bar: 200 μm.(D) The expression levels of c-Fos in the Sp5 were significantly higher than those in the control group (control = 101.67± 17.75, taVNS = 238.33 ± 29.51, ****p < 0.0001).The data were analyzed by unpaired two-tailed ttest.The data are presented as the mean ± SD.AP, area postrema.
this type of connection has little effect on mice because behavioral tests are rarely required at the time of stimulation.Of course, we F I G U R E 4 Application of taVNS for 3 weeks has no effect on anxietylike behaviors in freely moving mice.(A) Experimental scheme showing the application of taVNS and behavioral tests.(B) There was no significant difference between control and taVNS mice in the percentage of time spent in the central area in the OFT (control = 3.84 ± 1.44, taVNS = 4.83 ± 1.24, p = 0.0842).n = 12 mice/group.(C) Representative heat maps showed the time spent in the open field chamber of control and taVNS mice.(D) There was no significant difference between control and taVNS mice in the percentage of time spent in the open area in the EPM test (control = 20.48 ± 4.37, taVNS = 23.43 ± 4.18, p = 0.1051).n = 12 mice/group.(E) Representative heat maps showed the time spent in open arms of control and taVNS mice.ns, no significant difference (p > 0.05).The data were analyzed by unpaired two-tailed t-test.The data are presented as the mean ± SD.EPM, elevated plus maze; OFT, open field test.