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