Elsevier

Neuroscience Letters

Volume 720, 16 February 2020, 134753
Neuroscience Letters

Research article
Heat-killed Enterococcus fecalis (EC-12) supplement alters the expression of neurotransmitter receptor genes in the prefrontal cortex and alleviates anxiety-like behavior in mice

https://doi.org/10.1016/j.neulet.2020.134753Get rights and content

Highlights

  • Gut microbiota promotes mental health by combating depression and anxiety.

  • EC-12 supplementation exhibited an anti-depressive trend in mice.

  • EC-12 supplement significantly enhances the expression of Adrb3, Avpr1a, and Drd5.

  • EC-12 supplement enhances Butyricicoccus and Enterococcus in the gut.

  • Heat-killed EC-12, a novel probiotic to promote mental health.

Abstract

Gut microbiota plays a crucial role in the maintenance of mental health and influences mental disorders such as depression and anxiety. Several studies have reported the beneficial affects of probiotics in mental health. Heat-killed Enterococcus faecalis strain EC-12 (EC-12), a lactic acid bacterium induces activation of the immune system. However, little is known about the effect of EC-12 on mental health. In the present study, the anti-anxiety effect of EC-12 was elucidated in vivo. Male mice fed on diet supplemented with EC-12 showed decreased anxiety-like behavior in open-field and elevated plus-mazetest. In addition, EC-12 supplementation exhibited an anti-depressive trend in mice subjected to forced swim test. The expression of neurotransmitter receptor genes: Adrb3 and Avpr1a were significantly enhanced in EC-12 supplemented mice compared to that of the control mice. In mice, analyses of gut microbiota composition by next generation sequencing revealed significant increase in Butyricicoccus and Enterococcus with EC-12 supplementation. Significant difference was not detected in the expression of neurotransmitter receptor genes in the prefrontal cortex with the administration of sodium butyrate compared to that of the control group. The mechanism associated with EC-12 mediated reduced anxiety-like behavior and altered gene expression in the brain needs to be further elucidated. Taken together, the present study is the first to report the possibility of exploiting the anti-anxiety effect of heat-killed EC-12 as a novel probiotic to promote mental health.

Introduction

Mental health is a growing concern worldwide as people affected with depression and anxiety is on the rise globally. A survey conducted by World Health Organization reported 10 % of respondents with symptoms of depression and anxiety [1]. The cause of depression and anxiety has been hypothesized to be associated with stress, hormonal dysfunction, and inflammation [[2], [3], [4]]. Recent research focusing on the role of gut microbiota on human health has received a great deal of attention. Evidences gathered from improved molecular tools, metagenomic studies, and animal models have indicated a strong link between the gut microbiota composition and mental health (gut microbiota-brain axis) [5,6]. Lower richness and diversity of gut microbiota was seen in patients with depression compared to that of healthy individuals matched for gender, age, and ethnicity [7]. Studies in germ-free (GF) mice have reported reduced depression- and anxiety-like behaviors [8,9]. Transplantation of fecal microbiota from depressed patients to microbiota-depleted rodents induced behavioral and physiological features of depression- and anxiety-like behaviors in the recipient animals [7,9].

Probiotics are bacteria that are intended to confer health benefits when consumed. Several human and animal studies have proved the ability of lactic acid bacteria such as Lactobacillus and Bifidobacterium in alleviating the symptoms of depression and anxiety. In stress induced anxious animal models, administration of L. rhamnosus and L. helveticus significantly decreased depression- and anxiety-like behaviors [10,11]. In a mouse model with heightened anxiety, intake of B. longum and B. breve markedly reduced depression- and anxiety-like behaviors [12,13]. Studies related to neonatal stress indicate significant decrease in depressive-like symptoms in probiotic B. infantism treated adult rats that experienced maternal separation in the neonatal period [14]. In healthy humans, consumption of a mixture of L. helveticus and B. longum as a probiotic supplement had beneficial psychological effects [15].

Enterococcus faecalis strain EC-12 (EC-12), a lactic acid bacteria has been reported to possess several beneficial effects in humans and animals. Consumption of EC-12 activates immune cells and modulates intestinal immune response, cytokines, and IgA secretion [[16], [17], [18]]. In addition, EC-12 alters the metabolic ingredients and composition of the microbiota in the feces. However, the effect of heat-killed EC-12 is attributed to its immunomodulatory mechanism rather than the ability to alter the intestinal microbiota. Several studies have focused on the beneficial use of lactic acid bacteria as probiotics for the treatment of depression and anxiety. However, the effect of heat-killed EC-12 on mental health still needs to be elucidated.

In the present study, we investigated the in vivo effects of EC-12 on depression and anxiety in mice. EC-12 supplementation significantly reduced anxiety-like behavior and altered the expression of neurotransmitters receptor genes in the prefrontal cortex. Microbiota composition analyses by next generation sequencing revealed increased number of Butyricicoccus and Enterococcus. Further, tissues obtained from the prefrontal cortex of mice administered with butyrate were subjected to gene expression analyses. However significant difference was not detected between the butyrate and control groups.

Section snippets

Animals

Male C57BL/6 J mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). Animals were maintained at 23 ± 3 °C, 14 h/10 h light/dark cycle, with free access to food and tap water. Male 8-week-old mice were randomly assigned into the control (n = 8) and EC-12 (n = 8) groups. The control group was fed on AIN-93 M basal diet (Oriental Yeast CO. LTD., Tokyo, Japan) and the EC-12 group was fed on AIN-93 M diet with heat-killed EC-12 at a concentration of 0.125 %. After 4 weeks of treatment,

EC-12 supplementation induced in vivo behavioral modifications in mice

The effect of EC-12 supplementation on depressive- and anxiety-like behavior in mice was determined using OFT, EPM, and FST. In the OFT, time spent by the mice in the center area was significantly increased in the EC-12 group compared to that of the control group (Fig. 1A). However, the overall distance remained same in both the EC-12 and control groups suggesting that the general activity of mice was not affected. In the EPM test, time spent in the open arms was slightly increased in the EC-12

Discussion

Growing evidence has proved the prominent role of gut microbiota in regulating the activity of the nervous system. GF mice exhibit reduced anxiety-like behavior compared to that of mice harboring the conventional microbiota [8]. GF mice when exposed to gut microbiota early in life show similar characteristics as that of the conventional mice, signifying the role of gut microbiota in the regulation of anxiety-like behavior. Administration of exogenous probiotic microbes, such as Bifidobacterium

Conclusion

Recent study reports that EF-2001 (another heat-killed Enterococcus faecalis strain) prevents IBD-like symptoms and depressive-like behavior in DSS-treated mice [35]. Here, we also report the important finding that the heat-killed EC-12 contribute to the positive effect seen on anxiety related behavior in normal condition. Further studies are required to comprehend the exact mechanism of the effect of the heat-killed bacteria on brain.

CRediT authorship contribution statement

Jun Kambe: Conceptualization, Investigation, Writing - original draft. Sovijit Watcharin: Methodology. Yuko Makioka-Itaya: Resources. Ryo Inoue: Investigation. Gen Watanabe: Formal analysis. Hirohito Yamaguchi: Writing - review & editing. Kentaro Nagaoka: Supervision, Writing - review & editing, Funding acquisition.

Declaration of Competing Interest

This work was supported by Combi Corporation.

Acknowledgements

We would like to thank Editage (www.editage.com) for English language editing.

Funding

This work was supported by Combi Corporation.

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