Valerenic Acid Protects Against Physical and Psychological Stress by Reducing the Turnover of Serotonin and Norepinephrine in Mouse Hippocampus-Amygdala Region

Abstract

In a previous study, we demonstrated that a Valeriana officinalis extract could attenuate increases in serum corticosterone levels in a mouse model of physical and psychological stress. In addition, our results showed that the extract could modulate serotonin (5-HT) and norepinephrine (NE) turnover in the hippocampus and amygdala region. In this study, we intended to investigate the effects of valerenic acid (VA), the main component of V. officinalis extract, on corticosterone levels in serum in normal mice and monoamine turnover in hippocampus-amygdala homogenates in a mouse model of physical and psychological stress. To determine the minimum dose of VA for antianxiety effect, eight-week-old ICR mice were orally administered VA (0.2, 0.5, and 1.0 mg/kg/0.3 mL) once daily for 3 weeks to probe for immobility time and serum corticosterone levels. At a VA dose of 0.5 and 1.0 mg/kg, animals showed a decrease in the duration of immobility time and serum corticosterone levels. To confirm the antianxiety effect of VA, eight-week-old ICR mice received VA at a dose of 0.5 mg/kg, orally, once daily for 3 weeks, before being subjected to physical or psychological stress for 3 days, in a specially designed communication box, followed by estimation of levels of monoamines and their metabolites in the hippocampus-amygdala region. In conclusion, VA administration at 0.5 mg/kg can mitigate the physical and psychological stress response by decreasing the turnover of 5-HT to 5-hydroxyindoleacetic acid and NE to 3-methoxy-4-hydroxyphenylethyleneglycol sulfate in the hippocampus and amygdala.

Introduction

Fear and/or anxiety are one of the most common mental disorders in human beings and are mediated through the ventral hippocampus and amygdala,^1,2 two closely connected regions of the brain.^3,4

Monoamine neurotransmitters such as serotonin (5-hydroxytryptamine [5-HT]), norepinephrine (NE), and dopamine are some of the important modulators of cognition, mood, emotion, and depression in the brain.⁵ Commercially available antidepressants target one or more of these monoamines by blocking their neuronal reuptake.⁶ The benzodiazepine class of drugs is believed to have utility as first-line agents for most anxiety disorders.⁷ However, these are known to cause undesirable side effects such as cognitive decline in the elderly and reduction of response in some humans.⁸

Extracts from the roots of valerian (Valeriana officinalis L., Valerianaceae) have been widely used in alternative medicine for the treatment of anxiety, epilepsy, and sleep disorders.⁹ The genus Valeriana has been widely studied with a special focus on its anxiolytic, sedative properties and as an herbal cure for insomnia.¹⁰ In addition, valerian extracts have also been shown to have several effects against neurological disorders, including Parkinson's, Alzheimer's disease, ischemia^11–13, and anxiety.^10,14 In previous studies, we have shown valerian extract to reduce serum corticosterone levels in normal and aged animals,¹⁵ and in animals subjected to physical and psychological stress (PS and PCS, respectively).¹⁴ In addition, we demonstrated that valerian extract modulates the turnover of 5-HT and NE in the hippocampus and amygdala of animals, following PS and PCS.¹⁴

Valerenic acid (VA), a main constituent of V. officinalis, has been used as a nonsedative anxiolytic and anticonvulsant¹⁶ because it has antianxiety effects in mice via the interaction with GABA receptors comprising β_2/3-subunits and enhancement of transmembrane chloride currents of GABA receptors.¹⁷ The goal of this study was to determine the minimum effective dose of VA for antianxiety properties in a mouse model through behavioral (forced swim test [FST]) and biochemical (serum corticosterone levels) evaluations. In addition, we also sought to ascertain the turnover of monoamines, 5-HT and NE, in tissue homogenates of the hippocampus-amygdala region of mice that had been pretreated with VA before being subject to PS or PCS.

Materials and Methods

Study animals and treatment regimen

Six-week-old male ICR mice were purchased from OrientBio, Inc. (Seongnam, South Korea) and housed at 23°C with 60% humidity and a 12-h light/dark cycle, with free access to food and tap water. Animal handling and care conformed to guidelines compliant with current international laws and policies (NIH Guide for the Care and Use of Laboratory Animals, NIH Publication No. 85-23, 1985, revised 1996) and were approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-120103-10). All experiments and procedures were designed to minimize the number of animals used and suffering caused.

For determination of minimum effective dose of VA, following a 2-week acclimation period, animals were divided into five groups (n=7): vehicle (physiological saline) control, and VA in 0.3 mL vehicle at three concentrations (0.2, 0.5, and 1.0 mg/kg, respectively). Animals were administered assigned treatments orally, once daily, for 3 weeks. Subsequent to this, animals were subject to a forced swimming test followed by estimation of serum corticosterone levels (Fig. 1).

FIG. 1.

Experimental protocol and floor plan scheme of the communication box used in the study.

For studying the effect of VA treatment on monoamine-to-metabolite turnover in mice subject to PS or PCS, following a 2-week acclimation period, animals were divided into five groups (n=8): control, vehicle control followed by PS (V-PS), VA-treatment followed by PS (VA-PS), vehicle control followed by PCS (V-PCS), and VA-treatment followed by PCS (VA-PCS). The dose of VA chosen for these experiments was 0.5 mg/kg, orally, and treatment duration was 3 weeks (Fig. 1). After 3 weeks, animals were subject to PS or PCS before estimation of monoamine and metabolite levels, as described below.

Forced swim test

Two hours after the last administration of the assigned treatment, animals were placed inside a 25 cm glass cylinder (with a 14 cm diameter) containing 20 cm of water that was maintained at 24°C±2°C and were forced to swim for 5 min. The immobility time for each animal was recorded by using the video-based Ethovision System during the last 3 min of the 5-min test.

Estimation of serum corticosterone level

One hour after the FST, five mice from each group were anesthetized with 100 mg/kg Zoletil 50^® (Virbac, Carros, France), and blood sample from each was collected by cardiac puncture by using a 1-mL syringe. Samples were allowed to clot and then centrifuged for 30 min at 1000 g to separate serum. Corticosterone levels in serum were measured by using a commercial enzyme-linked immunosorbent assay kit (IBL, Hamburg, Germany) as per the manufacturer's instructions. The absorbance was read at 450 nm.

Exposure to PS and PCS

Mouse models of PS and PCS were developed using a method that employed a communication box and has been previously described by Ogawa and Kuwabara.¹⁸ Briefly, the communication box was divided into rooms A and B by using a transparent acrylic box (16×16×64 cm). Room A included eight small rooms with plastic flooring while room B had eight small rooms with a floor made of exposed metal grid for electric conduction (Fig. 1). To mimic PS, mice in room B were given an electrical shock (0.3 mA for 10 s, followed by rest period of 2 min) for 60 min through the floor to evoke nociceptive responses such as jumping up, defecating, and vocalization. On the other hand, mice in room A were exposed to the responses of mice in room B to induce PCS in these animals. Mice were subjected to PS and PCS for 60 min every morning (11:00–11:30) for 3 days before being sacrificed for evaluation. At the end of exposure on the third day, the mice were kept in the cages for 1 h before they were euthanized.

Tissue harvesting and processing

One hour after the final PS or PCS exposure, mice were anesthetized with 100 mg/kg Zoletil 50 (Virbac), and their brains were processed to measure the concentrations of 5-HT, NE, and their respective metabolites (5-hydroxyindoleacetic acid [5-HIAA] and 3-methoxy-4-hydroxyphenylethyleneglycol sulfate [MHPG-SO₄]) therein. In brief, the brain was removed from the braincase, the hippocampus-amygdala region was isolated on ice, and the samples were frozen in liquid nitrogen. The frozen tissue was fractured in 0.2 M perchloric acid containing 0.1 mM disodium ethylenediaminetetraacetic acid (EDTA) and isoproterenol as an internal standard. The homogenate was then centrifuged at 20,000 g for 15 min. The supernatant was separated; its pH was adjusted to 3.0 by using 1 M sodium acetate and finally passed through a 0.2-μm regenerated cellulose filter. An aliquot of each filtrate was used for determining the brain levels of monoamine and their metabolites.

Estimation of levels of monoamines and their metabolites in hippocampus and amygdala homogenates

The concentrations of 5-HT, NE, 5-HIAA, and MHPG-SO₄ in the hippocampus-amygdala region were ascertained by high-performance liquid chromatography (HPLC) as described by Nadaoka et al.¹⁹ An aliquot of the processed homogenate was injected onto a C₁₈ reverse-phase column (250×4.6 mm, 5 μm; Agilent Technologies, Santa Clara, CA, USA) in a HPLC system (Agilent 1100 series, Agilent Technologies) equipped with an electrochemical detector. The mobile phase used (0.1 M acetate-citrate buffer with 17% methanol) allowed for the separation of the two major monoamines 5-HT and NE and their respective metabolites, 5-HIAA and MHPG-SO₄.²⁰ Sodium octyl sulfate (190 mg/L) was added as an ion-pairing agent, and EDTA (5 mg/L) was added as an antioxidant. Each peak area was normalized to isoproterenol concentration. The level of 5-HT, NE, and their metabolites were detected by using a Waters 474 scanning fluorescence detector (Waters, Milford, MA, USA). All detected compounds were quantified by comparing the area under the peaks with the area of reference standards by using specific HPLC software (Chromatography Station for Windows). The turnover ratio of 5-HIAA to 5-HT is considered the index of cell activity resulting in release of 5-HT, reuptake, and metabolism to 5-HIAA.

Statistical analyses

Data are presented as mean±SEM (standard error of mean) values for each experiment. The statistical significance of observed differences between the groups in immobilization time, corticosterone levels in normal mice, turnover of 5-HT, NE after PS and PCS, and the differences between the means were analyzed by using one-way analysis of variance with Bonferroni's post-hoc test.

Results

Minimum concentration of VA for antianxiety effect in mice subjected to FST

The duration of immobilization time in animals in the vehicle control group was 117.1±25.4 s. In comparison, animals receiving 0.2 mg/kg VA showed a slight decrease in immobilization time, down to 91.1% of the vehicle-treated group. However, the most striking results were seen in groups receiving 0.5 and 1.0 mg/kg VA and in these animals the average immobilized time had decreased to 63.5% and 68.5%, respectively (Fig. 2).

FIG. 2.

Effects of valerenic acid (VA) on immobilized activity in the forced swimming test and serum corticosterone level of vehicle-, 0.2, 0.5, and 1.0 mg/kg VA-treated group (n=7 per group; ^aP<.05 indicating a significant difference as compared to the vehicle group). Error bars indicate standard error of mean (SEM).

A similar pattern was observed in serum corticosterone levels. Animals in control group had serum corticosterone level 104.0±22.7 ng/mL and these values were similar in animals treated with 0.2 mg/kg VA. However, as seen before, in the 0.5 and 1.0 mg/kg VA-treated groups, serum corticosterone level was significantly decreased to 74.3% and 74.7%, respectively, of the vehicle-treated group (Fig. 2).

Effects of VA on turnover of NE following PS or PCS

In the control group, NE and MHPG-SO₄ levels were 480.9±68.90 and 74.86±10.29 ng/g, respectively, in the hippocampus-amygdala homogenates, while the ratio of NE to MHPG-SO₄ was 0.1582±0.0029. In the V-PS group, NE level in the homogenates was notably decreased to 52.4% of control group, while MHPG-SO₄ levels were dramatically elevated to 267.4% of the control group. Consequently, the ratio of MHPG-SO₄: NE in the V-PS group had prominently increased to 474.3% of the control group. In the VA-PS group, NE levels had increased to 132.6% of the V-PS group, whereas we did not observe any noteworthy change in MHPG-SO₄ levels between V-PS and VA-PS groups. However, the ratio of MHPG-SO₄: NE in the VA-PS group was substantially lower at 67.1% of the V-PS group.

In the V-PCS group, NE levels in the hippocampus-amygdala homogenates were decreased to 57.7% of control group and was similar to that in the V-PS group. In addition, MHPG-SO₄ levels in the V-PCS group were elevated to 238.9% of the control group. However, we did not observe any significant difference in MHPG-SO₄ levels between V-PS and V-PCS although the MHPG-SO₄ levels were low in V-PCS group. The ratio of MHPG-SO₄: NE in the VA-PS group was also drastically increased to 415.5% of the control group, but we did not observe any significant differences in the ratio of MHPG-SO₄: NE between V-PS and V-PCS group. In the VA-PCS group, NE levels had increased to 140.2% of the V-PCS group, but they were lower than that in the control group. In the VA-PCS group, we did not observe any significant difference in MHPG-SO₄ levels from V-PCS group. However, the ratio of MHPG-SO₄: NE in the VA-PCS group was significantly lower, at 62.1% of the V-PCS group (Fig. 3).

FIG. 3.

Effect of VA on levels of norepinephrine (NE), its metabolite (3-methoxy-4-hydroxyphenylethyleneglycol sulfate [MHPG-SO₄]) and ratio (MHPG-SO₄/NE) in the control, vehicle control followed by physical stress (V-PS), VA-treatment followed by PS (VA-PS), vehicle control followed by PCS (V-PCS), and VA-treatment followed by PCS (VA-PCS) (n=8 per group; ^aP<.05, indicating a significant difference as compared to the control group; ^bP<.05, significantly different from the V-PS vs. VA-PS or V-PCS vs. VA-PCS group). Error bars indicate SEM.

Effects of VA on turnover of 5-HT following PS or PCS

The levels of 5-HT and 5-HIAA in hippocampus-amygdala homogenates from the control group were 333.7±39.57 and 291.4±34.84 ng/g, respectively, whereas the 5-HT:5-HIAA ratio was 0.8735±0.02297. Levels of 5-HIAA in the homogenates from the V-PS and V-PCS were notably higher in comparison to those from the control group. However, we did not observe any significant changes in 5-HT levels in homogenates from animals subjected to PS or PCS, irrespective of VA treatment. Additionally, we did not observe any perceptible differences in 5-HIAA levels between the V-PS and VA-PS groups. Levels of 5-HIAA in the VA-PCS group were lower at, 76.1% of the control group. Also, the ratio of 5-HIAA: 5-HT was markedly higher in V-PS and V-PCS groups in comparison to the control group. In the VA-PCS group, the ratio of 5-HIAA: 5-HT was found to have decreased to 75.7% of the control group (Fig. 4).

FIG. 4.

Effect of VA on levels of serotonin (5-hydroxytryptamine [5-HT]), its metabolite (5-hydroxyindoleacetic acid [5-HIAA]), and ratio (5-HIAA/5-HT) in the control, vehicle control followed by physical stress (V-PS), VA-treatment followed by PS (VA-PS), vehicle control followed by PCS (V-PCS), and VA-treatment followed by PCS (VA-PCS) (n=8 per group; ^aP<.05, indicating a significant difference as compared to the control group; ^bP<.05, significantly different from the V-PS vs. VA-PS or V-PCS vs. VA-PCS group). Error bars indicate SEM.

Discussion

In a previous study we observed that animals treated with a V. officinalis extract display a reduced immobility time and an abrogated rise in corticosterone levels on exposure to PS or PCS. Our postulated mechanism for this ameliorative effect is that the Valerian extract modulates the turnover of 5-HT and NE in the hippocampus and amygdala. In addition, it has been reported that a dichloromethane extract of Valeriana wallichii²¹ and a supercritical CO₂ extract of Valeriana glechomifolia²² display antidepressant-like effect that is mediated through noradrenergic and dopaminergic neurotransmission, with no significant dependence on serotonergic neurotransmission.

VA has the ability to permeate the blood-brain barrier²³ and the bioavailability and half-life of VA after oral administration is 33.7% and 2.7–5 h, respectively.²⁴ In this study, our first goal was to estimate the minimum dose of VA required for antianxiety effect in normal mice that had been subjected to stress through FST and the readouts for this were immobilization time and serum corticosterone levels in the animals. We observed that animals that had been treated with 0.5 and 1.0 mg/kg of VA before FST showed a marked decrease in duration of immobilization and serum levels of corticosterone in comparison with control animals. This result coincides with the findings of our previous study, which showed substantial reductions in serum corticosterone levels in normal mice treated with 100 mg/kg V. officinalis extract containing 3.4 mg/g of VA.¹⁵ In addition, studies have also demonstrated the anxiolytic action of VA in rats¹⁰ and mice.^17,25–27

Since the hippocampus and amygdala regions of the brain are closely associated with fear and anxiety¹, our next goal was to observe the effects of VA on the neurotransmitters that regulate these regions, 5-HT and NE, and their metabolites, following animal exposure to PS or PCS. Lesions in the hippocampus have been shown to decrease plasma corticosterone levels following exposure to stress.²⁸ In addition, NE-immunoreactive fibers are abundantly expressed in hippocampus and amygdala and are synthesized in the locus coeruleus and lateral tegmental areas.²⁹ 5-HT closely regulates various sensory, motor, and cortical functions through activation of multiple 5-HT receptor subtypes,³⁰ and abnormalities in these receptor systems are associated with psychiatric disorders such as anxiety and depression.³¹

Under stressful conditions monoamines are rapidly degraded in the synaptic cleft, and hence, monoamine-to-metabolite ratio is a suitable indicator for assessment of stress-response in the central nervous system.^32,33 In this study, PS or PCS significantly increased the ratio of 5-HIAA: 5-HT or MHPG-SO₄: NE in the hippocampus-amygdala homogenates. This observation is corroborated by the results of previous studies that reveal an increased turnover of NE and 5-HT in the brain under stress-generating conditions.³⁴ However, in our study, animals that had been treated with VA displayed a substantial attenuation of PS- or PCS-induced increase in NE turnover in the hippocampus-amygdala region. Interestingly, we observed a suppression of stress-induced increases in 5-HIAA: 5-HT ratio only in VA-fed animals that were subjected to PCS. Focusing on the monoamine-to-metabolites ratio as a marker of anxiety and depression, this result suggests that VA could be potentially used to treat PCS, but not PS. There are studies that report VA to possess anxiolytic activity in mouse^17,25–27 and rat models,¹⁰ but these studies focused on the effect of VA on gamma-aminobutyric acid receptors. In this study, we demonstrated the effects of VA on monoamine-to-metabolite ratio in PS and PCS models by using communication box.

In conclusion, VA is one of possible active compounds in valerian extracts that ameliorate the detrimental effects of physical and psychological stress by attenuating the monoamine-to-metabolite turnover in the hippocampus-amygdala region.

Acknowledgments

This research was supported by High Value-added Food Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea (111118-032-HD210). This was also partially supported by the Research Institute for Veterinary Science, Seoul National University.

Author Disclosure Statement

No competing financial interests exist.