Vagus nerve stimulation -induced anxiolytic and neurotrophic effect in healthy rats

Abstract

Memory, learning, anxiety, and depression have concerned science for years, increasingly leading to research into new therapeutic targets. The study investigated the effect of acupuncture stimulation of the auricular branch of the vagus nerve (ABVN) on memory, anxiety-like and depression-like behaviors in healthy rats. Healthy rats were divided into groups: (1) control, without treatment; (2) acupuncture in the left ABVN; and (3) acupuncture in the right ABVN. Tests were conducted to evaluate memory (inhibitory avoidance and object recognition), anxiety-like (open field, elevated plus maze, and light/dark box), and depressive-like (sucrose preference and forced swimming) behaviors. In addition, the concentration of brain-derived neurotrophic factor (BDNF) in the hippocampus of the rats was evaluated. The control group did not show any changes in the behavioral tests. The animals that received acupuncture (ABVN-R and ABVN-L) remained longer on the light side of the light/dark box test compared to the control group. Acupuncture in the ABVN-R also increased the concentration of BDNF in the hippocampus of the animals. Accordingly, acupuncture stimulation of the ABVN did not show an antidepressant effect or improve memory in healthy animals; however, it did show an anxiolytic effect and increased neurotrophic levels in the hippocampus.

Key words
vagus nerve stimulation; auriculotherapy; acupuncture; ear

INTRODUCTION

Cognitive processes play a key role in stress-related neuropsychiatric disorders, including emotional disorders such as anxiety and depression, and memory problems. Clinical and basic research evidence, using animals’ models, has demonstrated several interactions between different behaviors, such as memory-anxiety and memory-depression. Indeed, there is a traditional dichotomy between “emotional” domains (such as anxiety and depression) and “cognitive” domains (such as memory and learning) in behavioral neuroscience. Although relatively artificial, these boundaries seem to favor the segmentation of research with these different behaviors (Paylor et al. 2006, Kalueff & Murphy 2007).

Several substances are related to these changes, with the brain-derived neurotrophic factor (BDNF) standing out due to its high level of expression in the brain and its potent effects on synapses. Indeed, numerous studies have firmly established that BDNF plays a critical role in terms of learning and memory. Furthermore, evidence strongly suggests that deficits in terms of signaling BDNF contribute to the pathogenesis of several major diseases and disorders, such as depression and anxiety (Lu et al. 2014). Therefore, the study of cognitive and emotional disorders, in an associated way, is crucial for a better understanding of these problems, besides contributing to the prevention or treatment of these alterations and the quality of life of the affected individuals.

Given the lack of pharmacological treatments that modify the course of cognitive and emotional impairments, there has been an increasing clinical and research focus on non-pharmacological interventions for these disorders. Many treatment approaches, such as acupuncture and cognitive training, aim to slow cognitive decline and alleviate the signs and symptoms of emotional disturbances, offering potential benefits for mood and quality of life (Horr et al. 2015, Farah et al. 2016).

In this context, there is growing evidence for the use of acupuncture on the auricular branch of the vagus nerve (ABVN) in cognitive and emotional disorders (Li et al. 2020, Farmer et al. 2021, Zhang et al. 2022). The vagus nerve is widely distributed in our body and plays an important role in homeostasis (Browning 2015). Recent studies suggest that the vagus nerve provides an important route of information in the nervous system and its projections are associated with different brain functions (Busch et al. 2013, Chung et al. 2011, Lin & Hsieh 2014). Considering the foregoing, the purpose of this study was to evaluate the effects of manual acupuncture stimulation of the ABVN on learning, memory, anxiety, and depression- like behaviors in healthy rats.

MATERIALS AND METHODS

Animals

The study sample consisted of adult Wistar rats (Rattus novergicus), healthy males, aged between 45-60 days and weighing around 220g, obtained from the Central Animal Facility of the Federal University of Santa Catarina (Universidade Federal de Santa Catarina/UFSC) and the Regional University of Blumenau (Fundação Universidade Regional de Blumenau/FURB). All rats were maintained in the Laboratory of Neurobiology of Pain and Inflammation (Laboratório de Neurobiologia da Dor e Inflamação/LANDI/UFSC) or Laboratory of Biochemistry (FURB), under standard laboratory conditions, with temperature of 22 ± 2°C, on a 12-hour light/dark cycle, and with free access to water and food. The experiments were conducted in the LANDI (inhibitory avoidance and object recognition) or in the Biochemistry Laboratory (elevated plus maze, light-dark box, open field, sucrose preference, and forced swimming), in accordance with international standards for the study of laboratory animals. The experimental procedures were approved by the Regional University of Blumenau (FURB) Ethics Committee under protocol number 037/19. A total of 126 animals were used in this study, divided into three experimental groups for each behavioral test. Biochemical tests were performed on the animals after the memory tests. At the end of the experiments, all the animals were euthanized by an overdose of anesthetic (xylazine and ketamine) followed by decapitation.

Intervention

The animals were previously anesthetized with 1-2% isoflurane (Cristália, São Paulo, Brazil), through a nasal cone. After verifying the animal’s state of consciousness, by pinching the tail and posterior interdigital folds, two acupuncture needles (Dong Bang@, 0.18 X 8 mm) were implanted in the upper (center of the cymba shell) and inferior (center of the concha cava) regions of the ear. This intervention was performed in different groups: the first one received acupuncture stimulation on the left ABVN (ABVN-L); while the second group received the intervention on the right ABVN (ABVN-R). Needles were implanted at a depth of 1-2 mm from the surface of the skin, in the upper and lower regions of the pinna. They were stimulated manually by means of two half-turns per second, for 30 seconds in each needle. After this procedure, the needles were removed and two stainless steels micro-needles, sterile, specific for auricular acupuncture (Dong Bang@, 0.22 mm X 1.6 mm), were implanted in the same regions of the previous needling, and fixed with micropore microporous tape (3M™ Micropore™) (Neves et al. 2022). Immediately, the animals were removed from anesthesia and taken to the experimentation room. To minimize biases, the animals were randomized between the groups and were performed by an evaluator blinded to the treatments. Behavioral tests were performed approximately 30 minutes after this procedure, and the animals still had microneedles in their ears.

Inhibitory avoidance test (IAT)

To perform the test, the animal received a single training session and two test sessions. The first test session was conducted one and a half hours after training to assess short-term memory (STM). A second test session was held twenty-four hours later to evaluate long-term memory retention (LTM). In both phases, the latency of the animals to step off the platform was measured. The apparatus used for training in the inhibitory avoidance task consists of an acrylic box whose floor is formed by parallel bars. On the left side of the box is a platform on which the animals were placed. During training, the descent latency of the apparatus platform was recorded, and when the animal stepped down (with all four paws), it received a 0.3 mA shock for 2 seconds (Moretti et al. 2014). After 1.5 hours and 24 hours, the procedure was the same as that used in the training session, except that, when stepping down from the platform, the animal did not receive a shock. The retention test was assessed by measuring the latency to descend from the platform, which served as an index of memory performance. On re-exposure, the animals remained in the apparatus for a maximum of three minutes (one hundred and eighty seconds) (Arteni et al. 2002). The apparatus was cleaned with 70% alcohol between one animal and another, to eliminate possible odors that could affect the behavioral response of the animals.

Open field test (OFT)

The apparatus consists of a wooden arena covered with waterproof Formica (100 x 100 cm), entirely painted white. The floor of the apparatus was divided into 25 squares of 20 x 20cm (black dividing lines), 9 squares form the central area and 16 from the peripheral area; and the walls are 40 cm high. The luminosity used in the apparatus during the experiments was set at 10 lux. Rats were placed individually in the center of the open field test (OFT), and the number of squares crossed with all four paws was recorded over a 5-minute period. A reduction in the number of crossings compared to the control group was interpreted as a decrease in locomotor activity. The increase in time spent in the central area, or decreased latency to enter the central area were considered indicators of reduced anxiety (Prut & Belzung 2003).

Novel object recognition (NOR) test

The NOR test was performed according to Leger et al. (2013) and Prado Lima et al. (2018), with modifications. The open field apparatus is used for the test. The experiment consisted of two stages: habituation and testing. After the OFT, the animals were again placed in the same apparatus for 5 minutes, now with the presence of two equal objects (colored cubes). In the following steps, 1.5 h and 24 h later, the rats were re-exposed to the apparatus for 5 minutes, however, one of the objects was replaced by another one of a different color and size (1.5h: plastic parts; 24h: cubes of wood). The following parameters were considered as exploration: smelling, touching, or looking at the object less than 2 cm away. The discrimination index was represented in percentage, using the following formula (where t represents the time in seconds and 3 represents the new object): Discrimination index = t (s) 3 / t (s) 2 + t (s) 3 x 100%.

Elevated plus maze (EPM)

Anxious-like behavior was assessed using the EPM (Pellow et al. 1985). The EPM consists of an apparatus with two open arms (50 cm × 10 cm) and two closed arms (50 cm × 10 cm × 40 cm), in opposition, which extend from a central platform (10 × 10 cm) and elevated 50 cm from the floor. The animals were placed in the center of the apparatus, with their faces turned towards one of the closed arms, being allowed to freely explore the environment for 5 minutes, without any previous habituation. To characterize the EPM animal behavior, some parameters were manually measured, such as the number of entries into the open (EOA) and closed (ECA) arms, time in the open (TOA) and closed (TCA) arms, in seconds. The percentage of TOA and TCA were calculated according to the formula: [TOA / (TOA) + TCA)] x 100. An anxiogenic-like effect was considered when there was a reduction in the time spent exploring the open arms (EOA and TOA). Furthermore, the ECA was considered a representative measure of the spontaneous locomotor activity of the animal in the apparatus.

Light/dark box (LDB) test

This test was performed according to Kumar et al. (2013), with minor adaptations. For the test, we used an acrylic box unevenly divided into two chambers (2/3 light illuminated by a 60-Watt lamp above the box and 1/3 dark compartment with black walls and covered by a black lid). The luminosity used in the apparatus during the experiments was 40 lux. The animals were placed individually in the center of the illuminated box and observed for 5 minutes. Manually, the parameters (i) time spent in the clear space, and (ii) number of transitions in each compartment were recorded as a measure of anxiogenic behavior.

Sucrose preference test (SPT)

The SPT was used to assess the behavior of anhedonia or loss of pleasure. The SPT was carried out in the animals’ home box, using a trough filled with 250ml of water and another, of equal volume, with a 3% sucrose solution, positioned side by side in the containment grid. The test lasted 2 hours, and 1 hour after the start, the position of the bottles was changed to avoid bias in preference for the place. SPT was calculated using the following formula: %SP= [sucrose solution intake (ml) / (sucrose solution intake (ml) + water intake (ml)] x 100 (Riaz et al. 2015). Both SP and total fluid intake (water intake + sucrose intake) were calculated for all experimental groups.

Forced swimming test (FST)

The forced swim test is a widely used test to study depressive-like behavior in rodents and assess hopelessness behavior (Porsolt et al. 1977). The rat was gently lowered into an opaque plastic cylinder (60 cm x 15 cm), filled with water to a depth of 30 cm at 25°C. The animals were trained on-site for 15 minutes, and the test was performed 24 hours later. During the test, the time spent immobile, swimming, and climbing, as well as the frequency of climbing, are recorded over a 5-minute period. Immobility is defined as the absence of any movement except for those necessary to keep the head above water. Swimming is characterized by sudden movements of the front paws (beyond what is required to keep the head above water) aimed at moving the body through the water. Climbing is noted when the animal uses its front paws to contact the walls of the cylinder (Mezadri et al. 2011).

Enzyme-linked immunoabsorbent assay

The animals that passed the memory tests were euthanized by an overdose of anesthetic (xylazine and ketamine) followed by decapitation, and the hippocampus were collected for further quantification of brain-derived neurotrophic factor (BDNF). The samples were homogenized with a PBS (phosphate buffered saline) solution containing Tween 20 (0.05%), phenylmethylsulfonyl fluoride (PMSF, 0.1 mM), ethylenediaminetetraacetic acid (EDTA, 10 mM), Aprotinin (2 ng/ml) and benzethonium chloride (0.1 mM). After transferring the homogenates to Eppendorf tubes, they were centrifuged at 3000 r.p.m (10 min, 4°C) and the supernatants were collected and stored at -80°C for further analysis. The total protein content of the supernatant was measured using the Bradford method (Bradford 1976). BDNF levels were measured according to the rat ELISA kit data sheets (R&D Systems, Minneapolis, MN, USA; DY248). Measurements (pg/mg of protein) were performed in an ELISA plate reader (Multi Reader Infinite M200 TECAN) at 450 nm.

Statistical analysis

All data were analyzed for normality using the Shapiro-Wilk test. One or two-way analysis of variance (ANOVA) with Tukey’s post-test was used for parametric data. In addition, for the object recognition test and for the BDNF analysis, the Student’s t test was used, against a hypothetical value of 50%. Results are presented as the mean and standard error of the mean (S.E.M.) for each group. All analyses and graphs were performed using the GraphPad Prism 8.0 program (GraphPad Software, Inc., San Diego, CA). P values <0.05 were considered indicative of significance.

RESULTS

Manual acupuncture of the ABVN preserves memory function in healthy rats

Rats in the control group did not change the latency time to step off the platform, at times 1.5 h and 24 h, when compared to the training phase (baseline), as shown in Figure 1. We can observe there was no statistical difference between the control group and the experimental groups (Figure 1). The Inhibitory Avoidance Test (IAT) was employed to evaluate both short-term (1.5 hours) and long-term (24 hours) aversive memory by measuring the latency of animals to step off the platform in both tests. The latency to descend from the platform was used as an index of memory performance. Consequently, the results indicate that stimulation of the auricular branch of the vagus nerve (ABVN-L and ABVN-R) did not affect latency, suggesting no alterations in short-term or long-term memory.

Figure 1
Effect of ABVN stimulation on the left and right pinnae on inhibitory avoidance and on object recognition in male rats. Inhibitory avoidance (a) training; (b) short-term memory (1.5 h after the test) and (c) long-term memory (24 h after the test) were evaluated. Object recognition (d) training, (e) short-term memory (1.5 h after the test) and (f) long-term memory (24 h after the test) were also evaluated. Results represent the mean of 6 animals per group. Data normality was analyzed using the Shapiro-Wilk test. The results are presented as mean and standard error of the mean (SEM) for each group, which were statistically evaluated by two-way ANOVA analysis of variance with repeated measures, followed by Tukey’s pos-hoc (a, b and c) or by Student’s t test against a hypothetical value of 50% (d, e and f).

Regarding the results of the NOR test, the animals in the control group, when exposed to two identical objects, remained approximately half-time in each of them (Figure 1d), demonstrating no preference for the objects. Figure 1e represents the test phase, performed 1.5 h after training, where one of the objects was replaced by another of different color and size. Figure 1f represents the test 24 h after training, where one of the objects was replaced by another one with a different color and size again. The data show no difference, showing that the rats did not learn the task. These data suggest that manual acupuncture in the ABVN did not alter animals’ short- and long-term memory parameters.

Manual acupuncture of the auricular branch of the vagus nerve (ABVN) demonstrated an anxiolytic effect in healthy rats

Considering the EPM test, there was no difference between the groups in the percentage of time spend in the open or closed arms (Figures 2a, b) and in the number of entries in the open and closed arm (Figure 2c, d), suggesting that this test was not sensitive enough to detect potential behavioral change.

Figure 2
Effect of ABVN stimulation on the left and right pinna on the EPM in male rats. (a) the percentage of time in the open arm, (b) percentage of time in the closed arm, (c) number of entries in the open arm and (d) number of entries in the closed arm. Results represent the average of 6 animals per group. Data normality was analyzed using the Shapiro-Wilk test. The results are presented as mean and standard error of mean (SEM) for each group, which were statistically evaluated by one-way ANOVA analysis of variance, followed by Tukey’s post-hoc.

On the other hand, it was observed that in the light/dark box (LDB) test, the animals in the control group spent less time in the light environment compared to the animals in the experimental groups, as shown in Figure 3a. In addition, the number of transitions made by the animals to each side of the apparatus was not statistically different, indicating that was not a locomotor bias (Figure 3b). Therefore, we can suggest that animals in the ABVN acupuncture stimulation groups (left and right) exhibited reduced anxious-like behavior compared to the control group in this test.

Figure 3
Effect of ABVN stimulation on the left and right pinna on the LDB and the OPT in male rats. (a) time (s) in the light compartment and (b) number of transitions from light to dark in LDB. (c) time (s) in the center and (d) number of crossings in OPT. Results represent the average of 6 animals per group (LDB) or 6 animals per group (OTP). Data normality was analyzed using the Shapiro-Wilk test. The results are presented as mean and standard error of mean (SEM) for each group, which were statistically evaluated by one-way ANOVA analysis of variance, followed by Tukey’s post-hoc. P values < 0.05 were considered as indicative of significance in relation to the control group. *p<0.05 and **p < 0.01.

Finally, in the open field test, the animals treated with stimulation by acupuncture in the ABVN did not show a statistical difference from the control group, both in terms of length of stay in the center and the number of entries into the center of the apparatus. Therefore, we can observe that there was no statistical difference between the groups in any of the analyzed parameters (Figures 3c, d). These results reinforce that ABVN stimulation did not alter spontaneous locomotion (number of crossings) and did not induce anxiogenic-like behavior (time spent in the center of the apparatus).

Manual acupuncture of the auricular branch of the vagus nerve (ABVN) did not alter depressive-like behaviors in healthy rats

The results of the sucrose preference test showed no differences between the experimental groups, as sucrose consumption was similar across all groups (Figure 4a). This suggests that manual acupuncture of the auricular branch of the vagus nerve (ABVN) did not induce anhedonic behavior. Similarly, there were no significant differences between groups in immobility time (Figure 4b), swimming time (Figure 4c), climbing time (Figure 4d), or the number of climbs (Figure 4e). Therefore, we can infer that ABVN acupuncture stimulation did not affect depressive-like behavior in healthy mice.

Figure 4
Effect of ABVN stimulation in the left and right pinna on SP and on FST in male rats. The percentage of consumed sucrose was evaluated in SP (a). (b) immobility time (s), (c) swimming time (s), (d) climbing time (s) and (e) number of climbs were evaluated in FST. Results represent the average of 6 animals per group (SP) or average of 6 animals per group (FST). Data normality was analyzed using the Shapiro-Wilk test. The results are presented as mean and standard error of mean (SEM) for each group, which were statistically evaluated by one-way ANOVA analysis of variance, followed by Tukey’s post-hoc.

Manual acupuncture of the auricular branch of the vagus nerve (ABVN) increased BDNF levels in the hippocampus of healthy rats

According to the results shown in Figure 5, we observed that treatment with acupuncture in the right ABVN was able to increase BDNF levels in the hippocampus of healthy rats (t (df = 10) = 1.817, p = 0.0496) when compared to the control group.

Figure 5
Effect of ABVN stimulation on the BDNF concentration in the cortex of male rats. Data normality was analyzed using the Shapiro-Wilk test. The results represent the mean of 6 animals per group and the vertical lines indicate the standard error of the mean (SEM). Asterisks show the level of statistical significance in relation to the control group (Student’s t test), *p < 0.05.

DISCUSSION

Although several studies have investigated the effects of manual acupuncture on the auricular branch of the vagus nerve (ABVN) (Farmer et al. 2021, Li et al. 2014a, b, Noble et al. 2019a, b), there is a notable lack of research examining the bilateral effects of this stimulation (left and right ear), particularly in acute settings. The lack of standardization has contributed to the divergence of results in the literature (Farmer et al. 2021). Therefore, this study aimed to investigate the effects of manual acupuncture on the ABVN of both the right and left ears in rats subjected to behavioral tests for anxiety-like and depressive-like behaviors, as well as memory improvement. The results sought to evaluate, within a controlled environment, the natural anxious and depressive behaviors of rats exposed to specific conditions, such as height and water. The study analyzed the behaviors of different rat groups to assess whether bilateral ABVN stimulation provides benefits in treating depression, anxiety, and memory.

Considering that motor impairments can affect the assessment of animals in behavioral tests for anxiety, depression, and memory, the typical exploratory behavior of animals was evaluated using the Open Field Test (OFT), and the number of crossings in the EPM and LDB tests. Statistical analysis showed no significant difference in exploratory behavior between the treated groups and the control group. We also assessed the effect of acupuncture stimulation of the auricular branch of the vagus nerve (ABVN) on anxiety-like behavior (measured by the EPM and OFT) and found no significant differences between groups. In contrast, Noble et al. (2019a) demonstrated that rats treated with auricular nerve stimulation spent more time in the open arms of the EPM test, indicating an anxiolytic effect. However, stimulation can also reduce the conditioned fear response, potentially affecting the anxiety response in tests (Noble et al. 2019a, b). These findings are consistent with studies by Li et al. (2020) and Farmer et al. (2021), which emphasize the importance of considering stimulation parameters, such as frequency and duration, in relation to clinical efficacy, and highlight the need for standardization in testing methods.

On the other hand, in the LDB test, an experiment that also verifies anxious-like behavior, we verified an anxiolytic effect in the groups treated with stimulation in the ABVN. The animals in the treated groups spent more time on the light side of the box, regardless of whether the stimulation was applied to the left or right ear, compared to the control animals. However, when we evaluated the alternation of animals in the light and dark compartments, we did not verify any statistical difference with the control group. In fact, clinical research with humans has shown that patients with treatment-resistant anxiety disorders generally tolerated vague nerve stimulation intervention, in addition to showing acute and long-term improvement (George et al. 2008).

According to the experiments by Noble et al. (2019a), where male rats with surgical implants around the vagus nerve were used, ABVN stimulation demonstrated an anxiety reduction. The role of the peripheral parasympathetic system in the anxiolytic effect of vagus nerve stimulation was examined by blocking peripheral muscarinic receptors with intraperitoneal administration of methyl scopolamine. These results indicate that the anxiolytic effect of ABVN stimulation involves parasympathetic activity, and they support the hypothesis that stimulation can reduce the conditioned fear response by interfering with the anxiety response. Consistent with this hypothesis, chronic ABVN stimulation reduces anxiety in both rats and humans (George et al. 2008, Furmaga et al. 2012, Shah et al. 2016).

Subsequently, we investigated the depressive-like behavior of rats using SPT and FST tests. We found that the animals submitted to ABVN stimulation did not show a decrease in depressive behavior compared to the control group. Although the behavior of the rats has not been affected by the stimulation tested in the present research, the chronic treatment with vague stimulation has been experimentally studied and presents interesting results. The study employed transcutaneous stimulation of the auricular vagus nerve at three different frequencies (5 Hz, 20 Hz, and 100 Hz), administered for 30 minutes once a day over 28 days, in rats subjected to mild stress. The authors concluded that the different frequencies of treatment, rather than the cumulative effect of chronic stimulation, were responsible for the observed antidepressant-like effects. Specifically, rats exhibited increased sucrose preference and longer swimming times in the Forced Swim Test at a frequency of 20 Hz. In contrast, frequencies of 5 Hz and 100 Hz did not produce antidepressant effects (Li et al. 2020). In fact, non-invasive transcutaneous stimulation of the ABVN in humans has shown that left-sided stimulation facilitates invigoration for food rewards. Thus, the estimation of ABVN can improve the search for rewards, opening the way for new research for the treatment of motivational deficiencies (Neuser et al. 2020).

To confirm the previous observations, we highlight the studies with non-invasive transcutaneous stimulation of ABVN developed by Ferstl et al. (2022), where the stimulation improved mood after a prolonged period of exertion and the acute motivational effects are partially dependent on the mood states. These results showed that transcutaneous ABVN stimulation can help improve the effect, suggesting that non-invasive transcutaneous ABVN stimulation could be a useful tool to complement current behavioral therapies. In addition to behavioral studies, experiments on inhibitory avoidance and object recognition were carried out to analyze memory. The results showed that the animals submitted to ABVN stimulation of the right and left ear did not present changes in short or long-term memory compared to the control. Given the results presented, we can see that the stimulation did not cause improvement in memory or learning. We did not find similar works with animals. However, Sun et al. (2021) demonstrated that transcutaneous stimulation of the auricular vagus nerve increased performance in working memory tasks in healthy people.

Depression and anxiety are commonly treated with similar therapeutics, almost all classes of compounds used to treat depression also reduce anxiety. Vagus nerve stimulation treatment in rats showed antidepressant-like and anxiolytic-like effects. An inhibitor of Tropomyosin receptor kinase B (TrkB), K252a, blocked the anxiolytic-like effect of chronic treatment and the antidepressant-like effect of acute vagus nerve stimulation. TrkB and its primary ligand BDNF are expressed in neurons and other cells. In the work, researchers used Sprague-Dawley rats, evaluated in the novelty suppressed feeding test and forced swim test, for anxiety and depression, respectively (Shah et al. 2016). Vagus nerve stimulation rapidly phosphorylates tyrosine 515 in the TrkB receptor, and this effect persists over time. This effect is distinct from those phosphorylated by standard antidepressant medications (Furmaga et al. 2011) and may explain the increase in BDNF observed in the present study.

CONCLUSIONS

In conclusion, our study investigated the effects of manual acupuncture stimulation of the auricular branch of the vagus nerve (ABVN) on behavioral aspects in healthy rats, focusing on anxiety-like behaviors, depressive-like behaviors, and memory. While the treatment did not produce significant changes in depressive-like behaviors or memory performance, it did demonstrate an anxiolytic effect, as evidenced by increased time spent in the light compartment of the Light/Dark Box test. Additionally, stimulation of the right ear resulted in elevated levels of neurotrophic factors in the hippocampus. These findings highlight the potential of manual acupuncture in modulating anxiety but suggest that further research is needed to explore its effects on other behavioral aspects and to establish its efficacy in clinical settings. This study contributes to the growing body of research on non-pharmacological interventions for neuropsychiatric conditions and underscores the importance of continued investigation into optimal stimulation parameters and their impact on various behavioral outcomes.

ACKNOWLEDGMENTS

This article is dedicated to the memory of Prof. Dr. Adair Roberto Soares dos Santos. We are immensely grateful to Prof. Adair, great friend, and instructor, who contributed intensely to the present work (concept, research project, provision of facilities and supervision). However, he passed away before the work was completed. We also thank Programa de Pós-Graduação em Neurociências da Universidade Federal de Santa Catarina (UFSC), and Laboratório Multiusuário de Estudos Biológicos (LAMEB/UFSC). This study was financed in part by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

REFERENCES

• ARTENI NS, LAVINSKY D, RODRIGUES AL, FRISON VB & NETTO CA. 2002. Agmatine facilitates memory of an inhibitory avoidance task in adult rats. Neurobiol Learn Mem 78(2): 465-469. DOI: 10.1006/nlme.2002.4076.
• BRADFORD MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.
• BROWNING KN. 2015. Role of central vagal 5-HT3 receptors in gastrointestinal physiology and pathophysiology. Front Neurosci 29(9): 413. DOI: 10.3389/fnins.2015.00413.
• BUSCH V, ZEMAN F, HECKEL A, MENNE F, ELLRICH J & EICHHAMMER P. 2013. The effect of transcutaneous vagus nerve stimulation on pain perception-an experimental study. Brain Stimul 6(2): 202-209. DOI: 10.1016/j.brs.2012.04.006.
• CHUNG WY, ZHANG HQ & ZHANG SP. 2011. Peripheral muscarinic receptors mediate the anti-inflammatory effects of auricular acupuncture. Chin Med 6: 3. DOI: 10.1186/1749-8546-6-3.
• FARAH WH ET AL. 2016. Non-pharmacological treatment of depression: a systematic review and evidence map. Evid Based Med 21(6): 214-221. DOI: 10.1136/ebmed-2016-110522.
• FARMER AD ET AL. 2021. International Consensus Based Review and Recommendations for Minimum Reporting Standards in Research on Transcutaneous Vagus Nerve Stimulation (Version 2020). Front Hum Neurosc 23(14): 568051. DOI: 10.3389/fnhum.2020.568051.
• FERSTL M, TECKENTRUP V, LIN WM, KRÄUTLEIN F, KÜHNEL A, KLAUS J, WALTER M & KROEMER NB. 2022. Non-invasive vagus nerve stimulation boosts mood recovery after effort exertion. Psychol Med 52(14): 3029-3039. DOI: 10.1017/S0033291720005073.
• FURMAGA H, CARRENO FR & FRAZER A. 2012. Vagal nerve stimulation rapidly activates brain-derived neurotrophic factor receptor TrkB in rat brain. PLoS ONE 7(5): e34844. DOI: 10.1371/journal.pone.0034844.
• FURMAGA H, SHAH A & FRAZER A. 2011. Serotonergic and noradrenergic pathways are required for the anxiolytic-like and antidepressant-like behavioral effects of repeated vagal nerve stimulation in rats. Biol Psychiatry 70(10): 937-945. DOI: 10.1016/j.biopsych.2011.07.020.
• GEORGE MS, WARD HE JR, NINAN PT, POLLACK M, NAHAS Z, ANDERSON B, KOSE S, HOWLAND RH, GOODMAN WK & BALLENGER JC. 2008. A pilot study of vagus nerve stimulation (VNS) for treatment-resistant anxiety disorders. Brain Stimul 1(2): 112-121. DOI: 10.1016/j.brs.2008.02.001.
• HORR T, MESSINGER-RAPPORT B & PILLAI JA. 2015. Systematic review of strengths and limitations of randomized controlled trials for non-pharmacological interventions in mild cognitive impairment: focus on Alzheimer’s disease. J Nutr Health Aging 19(2): 141-153. DOI: 10.1007/s12603-014-0565-6.
• KALUEFF AV & MURPHY DL. 2007. The importance of cognitive phenotypes in experimental modeling of animal anxiety and depression. Neural Plast 2007: 52087. DOI: 10.1155/2007/52087.
• KUMAR V, BHAT ZA & KUMAR D. 2013. Animal models of anxiety: a comprehensive review. J Pharmacol Toxicol Methods 68(2): 175-183. DOI: 10.1016/j.vascn.2013.05.003.
• LEGER M, QUIEDEVILLE A, BOUET V, HAELEWYN B, BOULOUARD M, SCHUMANN-BARD P & FRERET T. 2013 Object recognition test in mice. Nat Protoc 8: 2531-2537. https://doi.org/10.1038/nprot.2013.155.
  » https://doi.org/10.1038/nprot.2013.155
• LI S ET AL. 2020. Transcutaneous Auricular Vagus Nerve Stimulation at 20 Hz Improves Depression-Like Behaviors and Down-Regulates the Hyperactivity of HPA Axis in Chronic Unpredictable Mild Stress Model Rats. Front Neurosci 15(14): 680. DOI: 10.3389/fnins.2020.00680.
• LI S, ZHAI X, RONG P, MCCABE MF, WANG X, ZHAO J, BEN H & WANG S. 2014a. Therapeutic effect of vagus nerve stimulation on depressive-like behavior, hyperglycemia and insulin receptor expression in Zucker fatty rats. PLoS ONE 9(11): e112066. DOI: 10.1371/journal.pone.0112066.
• LI S, ZHAI X, RONG P, MCCABE MF, ZHAO J, BEN H, WANG X & WANG S. 2014b. Transcutaneous auricular vagus nerve stimulation triggers melatonin secretion and is antidepressive in Zucker diabetic fatty rats. PLoS ONE 9(10): e111100. DOI: 10.1371/journal.pone.0111100.
• LIN YW & HSIEH CL. 2014. Auricular electroacupuncture reduced inflammation-related epilepsy accompanied by altered TRPA1, pPKCα, pPKCε, and pERk1/2 signaling pathways in kainic acid-treated rats. Mediators Inflamm 2014: 493480. DOI: 10.1155/2014/493480.
• LU B, NAGAPPAN G & LU Y. 2014. BDNF and synaptic plasticity, cognitive function, and dysfunction. Handb Exp Pharmacol 220: 223-250. DOI: 10.1007/978-3-642-45106-5_9.
• MEZADRI TJ, BATISTA GM, PORTES AC, MARINO-NETO J & LINO-DE-OLIVEIRA C. 2011. Repeated rat-forced swim test: reducing the number of animals to evaluate gradual effects of antidepressants. J Neurosci Methods 195(2): 200-205. DOI: 10.1016/j.jneumeth.2010.12.015.
• MORETTI M, MATHEUS FC, DE OLIVEIRA PA, NEIS VB, BEN J, WALZ R, RODRIGUES AL & PREDIGER RD. 2014. Role of agmatine in neurodegenerative diseases and epilepsy. Front Biosci (Elite Ed) 6(2): 341-359. DOI: 10.2741/E710.
• NEUSER MP, TECKENTRUP V, KÜHNEL A, HALLSCHMID M, WALTER M & KROEMER NB. 2020. Vagus nerve stimulation boosts the drive to work for rewards. Nat Commun 11(1): 3555. DOI: 10.1038/s41467-020-17344-9.
• NEVES ML, KARVAT J, SIMÕES RR, SPERETTA GFF, LATARO RM, DA SILVA MD & SANTOS ARS. 2022. The antinociceptive effect of manual acupuncture in the auricular branch of the vagus nerve in visceral and somatic acute pain models and its laterality dependence. Life Sci 15(309): 121000. DOI: 10.1016/j.lfs.2022.121000.
• NOBLE LJ, CHUAH A, CALLAHAN KK, SOUZA RR & MCINTYRE CK. 2019a. Peripheral effects of vagus nerve stimulation on anxiety and extinction of conditioned fear in rats. Learn Mem 26(7): 245-251. DOI: 10.1101/lm.048447.118.
• NOBLE LJ, MERUVA VB, HAYS SA, RENNAKER RL, KILGARD MP & MCINTYRE CK. 2019b. Vagus nerve stimulation promotes generalization of conditioned fear extinction and reduces anxiety in rats. Brain Stimu 12(1): 9-18. DOI: 10.1016/j.brs.2018.09.013.b.
• PAYLOR R, SPENCER CM, YUVA-PAYLOR LA & PIEKE-DAHL S. 2006. The use of behavioral test batteries, II: effect of test interval. Physiol Behav 87(1): 95-102. DOI: 10.1016/j.physbeh.2005.09.002.
• PELLOW S, CHOPIN P, FILE SE & BRILEY M. 1985. Validation of open:closed arm entries in an elevated plus-maze as a measure of anxiety in the rat. J Neurosci Methods 14(3): 149-167. DOI: 10.1016/0165-0270(85)90031-7.
• PORSOLT RD, BERTIN A & JALFRE M. 1977. Behavioral despair in mice: a primary screening test for antidepressants. Arch Int Pharmacodyn Ther 229(2): 327-336.
• PRADO LIMA MG, SCHIMIDT HL, GARCIA A, DARÉ LR, CARPES FP, IZQUIERDO I & MELLO-CARPES PB. 2018. Environmental enrichment and exercise are better than social enrichment to reduce memory deficits in amyloid beta neurotoxicity. Proc Natl Acad Sci USA 115(10): E2403-E2409. DOI: 10.1073/pnas.1718435115.
• PRUT L & BELZUNG C. 2003. The open field as a paradigm to measure the effects of drugs on anxiety-like behaviors: a review. Eur J Pharmacol 463: 3-33.
• RIAZ MS, BOHLEN MO, GUNTER BW, QUENTIN H, STOCKMEIER CA & PAUL IA. 2015. Attenuation of social interaction-associated ultrasonic vocalizations and spatial working memory performance in rats exposed to chronic unpredictable stress. Physiol Behav 152(Pt A): 128-134. DOI: 10.1016/j.physbeh.2015.09.005.
• SHAH AP, CARRENO FR, WU H, CHUNG YA & FRAZER A. 2016. Role of TrkB in the anxiolytic-like and antidepressant-like effects of vagal nerve stimulation: Comparison with desipramine. Neuroscience 322(13): 273-286. DOI: 10.1016/j.neuroscience.2016.02.024.
• SUN JB ET AL. 2021. Transcutaneous Auricular Vagus Nerve Stimulation Improves Spatial Working Memory in Healthy Young Adults. Front Neurosci 23(15): 790793. DOI: 10.3389/fnins.2021.790793.
• ZHANG X, GUO YM, NING YP, CAO LP, RAO YH, SUN JQ, QING MJ & ZHENG W. 2022. Adjunctive vagus nerve stimulation for treatment-resistant depression: a preliminary study. Int J Psychiatry Clin Pract 26(4): 337-342. DOI: 10.1080/13651501.2021.2019789.

Publication Dates

• Publication in this collection
  21 Mar 2025
• Date of issue
  2025

History

• Received
  06 Nov 2024
• Accepted
  30 Nov 2024