Elsevier

Behavioural Processes

Volume 96, June 2013, Pages 27-35
Behavioural Processes

Ingestion of Mycobacterium vaccae decreases anxiety-related behavior and improves learning in mice

https://doi.org/10.1016/j.beproc.2013.02.007Get rights and content

Abstract

Coevolution of microbes and their hosts has resulted in the formation of symbiotic relationships that enable animals to adapt to their environments and protect themselves against pathogens. Recent studies show that contact with tolerogenic microbes is important for the proper functioning of immunoregulatory circuits affecting behavior, emotionality and health. Few studies have examined the potential influence of ambient bacteria, such as Mycobacterium vaccae on the gut–brain–microbiota axis. In this preliminary research, we show that mice fed live M. vaccae prior to and during a maze learning task demonstrated a reduction in anxiety-related behaviors and maze completion time, when tested at three maze difficulty levels over 12 trials for four weeks. Treated mice given M. vaccae in their reward completed the maze twice as fast as controls, and with reduced anxiety-related behaviors. In a consecutive set of 12 maze trials without M. vaccae exposure, treated mice continued to run the maze faster for the first three trials, and with fewer errors overall, suggesting a treatment persistence of about one week. Following a three-week hiatus, a final maze run revealed no differences between the experimentals and controls. Additionally, M. vaccae-treated mice showed more exploratory head-dip behavior in a zero maze, and M. vaccae treatment did not appear to affect overall activity levels as measured by activity wheel usage. Collectively, our results suggest a beneficial effect of naturally delivered, live M. vaccae on anxiety-related behaviors and maze performance, supporting a positive role for ambient microbes in the immunomodulation of animal behavior.

Highlights

Mycobacterium vaccae-fed mice express fewer anxiety behaviors and faster complex maze run time. ► The effects of M. vaccae treatment in the complex maze lasted 8 weeks. ► M. vaccae treated mice showed more exploratory head dip behaviors in elevated zero maze. ► M. vaccae did not affect running wheel activity. ► Transient commensal bacteria may affect animal behavior via immunomodulatory mechanisms.

Section snippets

Animals

For all experiments, male, BALB/c specific pathogen free mice were obtained from Charles River Laboratories when they were about 38 days old, housed individually in an isolated animal room under a 12 h light/dark cycle and at a constant 25 °C temperature, and fed Carolina Biological Supply Company Mazuri rodent pellets (5663) (ad libitum). This mouse strain was used to maintain consistency with the mice used by Lowry et al. (2007). Each mouse was placed in an individual polycarbonate cage with a

Sample

In experiments 1–3, ten mice constituted the treatment group and eight mice constituted the control group. The same mice were used through the progression from experiments 1 to 3.

Complex maze

A Hebb–Williams style complex maze was used in this study (Fig. 1). This type of maze is widely used in measuring spatial learning tasks and working memory with rodents (Shore et al., 2001, Parle et al., 2006). This maze operates on appetitive rather than aversive principles.

The mice were tested in a maze free of

Methods

To determine whether mice that ingested live M. vaccae perform differently in a maze than control mice, experimental mice (N = 10) were immunologically primed by placing a food vehicle on the wire bar lid of their cages on two occasions: 21 days and 7 days prior to the start of maze testing in experiment 1. Since M. vaccae was incorporated into the food reward of the experimental mice at the finish of each maze run, those mice received additional M. vaccae during the 12 maze trials of experiment

Methods

To determine what would happen to complex maze performance and anxiety-related behaviors when M. vaccae was no longer administered, both experimental and control mice were tested only at level 3 of the maze without M. vaccae in the food reward. To maintain the same maze testing schedule that was used in experiment 1, experiment 2 began three days following the last test day of experiment 1. All mice were subsequently tested three times a week for four weeks, yielding a total of 12 trials. Time

Methods

To determine how well mice remembered the maze pattern, all mice were rested for three weeks and one final maze test was conducted at level 3, seven weeks after the experimental mice had last been exposed to M. vaccae. No M. vaccae was administered in the food reward at this time. Time to finish the maze and demonstrated anxiety-related behaviors were recorded as for experiments 1 and 2.

Results

The experimental mice completed the maze faster (X = 12.9 ± 3.0 s, N = 10) than control mice (X = 20.0 ± 4.6 s, N = 8), and

Methods

To evaluate the effects of M. vaccae treatment on anxiety-related behaviors in addition to those measured during a complex maze learning task, an elevated zero maze was employed. The elevated zero maze (EZM) examines anxiety behaviors based on the premise that mice have an aversion for open, more illuminated spaces (Jonas et al., 2010) and allows for exploration uninterrupted by a central space, such as in the elevated plus maze (Shepherd et al., 1994, Walf and Frye, 2007, Braun et al., 2011).

Subjects

Subjects

Fifteen mice (8 = treatment, 7 = control) were individually housed in home cages with standard running wheels. Rotation of the wheels was recorded by magnetically activated counters (Mini Mitter, Respironics Company).

M. vaccae exposure

Experimental mice were exposed to M. vaccae at three different times: on the day before the wheels were released (day zero), on day 14 and on day 21.

Activity testing protocol

Mice were acclimated for two weeks within the activity cages with the wheels immobilized. On the day following the first exposure of

Discussion

This research shows that ingestion of live M. vaccae prior to and during a complex maze learning task (experiments 1–3) reduced maze run time and anxiety-related behaviors in BALB/c mice. Four of the seven measured anxiety-related behaviors, immobilization, grooming, latency to start and elongation, were significantly different in the M. vaccae treated group as compared to the control group (Table 3). These effects do not appear to be due to differences in generalized activity levels related to

Conflict of interest statement

All authors declare that there are no conflicts of interest.

Acknowledgements

This research was supported by a Sage Colleges Faculty Research Grant. The authors gratefully acknowledge Charles Rivers Laboratories for supplying the animals used in this study. We thank the anonymous reviewers for their careful and constructive review of the manuscript. We also thank T.H. Reynolds for sharing equipment, K. Light and V. Bolivar for helpful comments on the research, B. Elder, M. Grubb, M.J. Matthews and L. Drickamer for their thoughtful review of versions of the manuscript, K.

References (51)

  • C.A. Lowry et al.

    Identification of an immune-responsive mesolimbocortical serotonergic system: potential role in regulation of emotional behaviour

    Neuroscience

    (2007)
  • A.H. Miller

    Depression and immunity: a role for T cells?

    Brain Behav. Immun.

    (2010)
  • M.E. O’Brien et al.

    SRL172 (killed Mycobacterium vaccae) in addition to standard chemotherapy improves quality of life without affecting survival, in patients with advanced non-small-cell lung cancer: phase III results

    Ann. Oncol.

    (2004)
  • G.A.W. Rook et al.

    Can we vaccinate against depression?

    Drug Discovery Today

    (2012)
  • R. Yirmiya et al.

    Immune modulation of learning, memory, neural plasticity and neurogenesis

    Brain Behav. Immun.

    (2011)
  • K.R. Bailey et al.

    Anxiety-related behaviors in mice

  • M.T. Bailey et al.

    Prenatal stress alters bacterial colonization of the gut in infant monkeys

    J. Pediat. Gastroenterol. Nutr.

    (2004)
  • J.L. Barnes et al.

    Intestinal adaptation is stimulated by partial enteral nutrition supplemented with the prebiotic short-chair fructooligosaccharide in a neonatal intestinal failure piglet model

    J. Parent. Enteral Nutr.

    (2012)
  • P. Berick et al.

    The intestinal microbiota affects central levels of brain-derived neurotropic factor and behavior in mice

    Gastroenterology

    (2011)
  • J.A. Bravo et al.

    Ingestion of a Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve

    Proc. Natl. Acad. Sci. U.S.A.

    (2011)
  • M. Bourin et al.

    Animal models of anxiety in mice

    Fundam. Clin. Pharmacol.

    (2007)
  • A.M. Chakrabarty

    Microorganisms and cancer: quest for therapy

    J. Bacteriol.

    (2003)
  • G. Clarke et al.

    The microbiome–gut–brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner

    Mol. Psychiatry

    (2012)
  • R. Cools et al.

    Serotonergic regulation of emotion and behavioural control responses

    Trends Cogn. Sci.

    (2007)
  • B.D. Devan et al.

    Phosphodiesterase inhibition by sildenafil citrate attenuates a maze learning impairment in rats induced by nitric oxide synthase inhibition

    Psychopharmacology (Berl)

    (2006)
  • Cited by (0)

    View full text