UNCORRECTED PROOF Neuroscience Letters xxx (xxxx) xxx-xxx Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com 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 Jun Kambe a , Sovijit Watcharin a , Yuko Makioka-Itaya b , Ryo Inoue c , Gen Watanabe a , Hirohito Yamaguchi d , Kentaro Nagaoka a, a Laboratory of Veterinary Physiology, Tokyo University of Agriculture and Technology, Tokyo, Japan b Functional Foods, Combi Corporation, Saitama, Japan c Laboratory of Animal Science, Kyoto Prefectural University, Kyoto, Japan d Cancer Research Center, Qatar Biomedical Research Institute, Hamad Bin Khalifa University, Qatar ARTICLE INFO Keywords: Gut microbiota Probiotics EC-12 Anxiety Depression ABSTRACT Gut microbiota plays a crucial role in the maintenance of mental health and inUuences mental disorders such as depression and anxiety. Several studies have reported the beneTcial 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 anx- iety-like behavior in open-Teld and elevated plus-maze tests. 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 in- crease 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 anx- iety-like behavior and altered gene expression in the brain needs to be further elucidated. Taken together, the present study is the Trst to report the possibility of exploiting the anti-anxiety effect of heat-killed EC-12 as a novel probiotic to promote mental health. 1. Introduction Mental health is a growing concern worldwide as people affected with depression and anxiety is on the rise globally. A survey con- ducted by World Health Organization reported 10 % of respondents with symptoms of depression and anxiety [1]. The cause of depres- sion and anxiety has been hypothesized to be associated with stress, hormonal dysfunction, and inUammation [2 4]. Recent research fo- cusing on the role of gut microbiota on human health has received a great deal of attention. Evidences gathered from improved molec- ular 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]. Stud- ies in germ-free (GF) mice have reported reduced depression- and anx- iety-like behaviors [8,9]. Transplantation of fecal microbiota from de- pressed 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 beneTts when consumed. Several human and animal studies have proved the ability of lactic acid bacteria such as Lactobacillus and Bi>dobacterium in alleviating the symptoms of depression and anxiety. In stress in- duced anxious animal models, administration of L. rhamnosus and L. Corresponding author. Email address: nagaokak@cc.tuat.ac.jp (K. Nagaoka) https://doi.org/10.1016/j.neulet.2020.134753 Received 7 November 2019; Received in revised form 28 December 2019; Accepted 9 January 2020 Available online xxx 0304-3940/ © 2020. Research article