Research articleHeat-killed Enterococcus fecalis (EC-12) supplement alters the expression of neurotransmitter receptor genes in the prefrontal cortex and alleviates anxiety-like behavior in mice
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.
References (35)
- et al.
Assessing gonadal hormone contributions to affective psychopathologies across humans and animal models
Psychoneuroendocrinology
(2014) - et al.
Transferring the blues: depression-associated gut microbiota induces neurobehavioural changes in the rat
J. Psychiatr. Res.
(2016) - et al.
Administration of Lactobacillus helveticus NS8 improves behavioral, cognitive, and biochemical aberrations caused by chronic restraint stress
Neuroscience.
(2015) - et al.
Bifidobacteria modulate cognitive processes in an anxious mouse strain
Behav. Brain Res.
(2015) - et al.
Effects of the probiotic Bifidobacterium infantis in the maternal separation model of depression
Neuroscience.
(2010) - et al.
Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse
Biol. Psychiatry
(2007) - et al.
Evidence that the anxiolytic-like effects of the beta3 receptor agonist Amibegron involve serotoninergic receptor activity
Pharmacol. Biochem. Behav.
(2013) - et al.
Effects of the beta3-adrenoceptor (Adrb3) agonist SR58611A (amibegron) on serotonergic and noradrenergic transmission in the rodent: relevance to its antidepressant/anxiolytic-like profile
Neuroscience.
(2008) - et al.
Sex differences in the neuro-immune consequences of stress: Focus on depression and anxiety
Brain Behav. Immun.
(2018) - et al.
Dysregulated diurnal cortisol pattern is associated with glucocorticoid resistance in women with major depressive disorder
Biol. Psychol.
(2013)
Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse
Biol. Psychiatry
Treatment gap for anxiety disorders is global: results of the World Mental Health Surveys in 21 countries
Depress. Anxiety
From inflammation to sickness and depression: when the immune system subjugates the brain
Nat. Rev. Neurosci.
The psychobiology of depression and resilience to stress: implications for prevention and treatment
Annu. Rev. Clin. Psychol.
The neuroactive potential of the human gut microbiota in quality of life and depression
Nat. Microbiol.
The role of inflammation and the gut microbiome in depression and anxiety
J. Neurosci. Res.
Normal gut microbiota modulates brain development and behavior
Proc. Natl. Acad. Sci. U S A.
Cited by (24)
Scope of bacterial surface effector molecules beyond probiotics
2023, Food BioscienceRole of PPAR-Allopregnanolone Signaling in Behavioral and Inflammatory Gut-Brain Axis Communications
2023, Biological PsychiatryXanthine-induced deficits in hippocampal behavior and abnormal expression of hemoglobin genes
2023, Behavioural Brain ResearchUnderstanding the Connection between Gut Homeostasis and Psychological Stress
2023, Journal of NutritionThe antidepressant potential of lactobacillus casei in the postpartum depression rat model mediated by the microbiota-gut-brain axis
2022, Neuroscience LettersCitation Excerpt :These findings indicate that Lactobacillus casei supplementation deserves further study as a potential treatment strategy for PPD. The HPA axis abnormalities are widely observed in patients with severe mental illness and play an important role in the etiology of depression and anxiety [2324]. Moreover, several previous studies suggest the association between the HPA axis and the gut microbiota.