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

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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.
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Behavioural Processes 96 (2013) 27–35
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Behavioural Processes
journal homepage: www.elsevier.com/locate/behavproc
Ingestion of Mycobacterium vaccae decreases anxiety-related behavior
and improves learning in mice
Dorothy M. Matthewsa,, Susan M. Jenksb
aDepartment of Biology, The Sage Colleges, Troy, NY 12208, USA
bDepartments of Biology & Psychology, The Sage Colleges, Troy, NY 12208, USA
article info
Article history:
Received 5 January 2013
Accepted 18 February 2013
Keywords:
Mycobacterium vaccae
Serotonin
Learning
Memory
Anxiety-related behaviors
Old friends hypothesis
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.
© 2013 Elsevier B.V. All rights reserved.
Coevolution of microbes, macrobiotic organisms and their
animal hosts over the past 500 million years has resulted in
the development of some symbiotic relationships that enable
animals to adapt to the ambient environment and protect them-
selves against pathogens (Strachan, 1989; Chakrabarty, 2003;
Tlaskalova-Hogenova et al., 2011; Rook, 2012). Such relation-
ships involve bidirectional signaling between the gastrointestinal
tract and the brain via neural, hormonal and immune interactions
(Grenham et al., 2011). Recent work on communication between
the brain–gut–microbiota axis using rodents (Berick et al., 2011;
Grenham et al., 2011; Bravo et al., 2012), monkeys (Bailey et al.,
2004), pigs (Barnes et al., 2012) and humans (Knowles et al., 2008;
Khani et al., 2012) has deepened our understanding of how such
symbiotic relationships can influence animal behavior. Studies with
germ-free animals allow evaluation of the effects of the micro-
biota on the CNS; antibiotic studies provide insight on how use of
Corresponding author at: Department of Biology, The Sage Colleges, 63 First
Street, Troy, NY 12208, USA. Tel.: +1 518 925 4958; fax: +1 518 244 3174.
E-mail addresses: matthd@sage.edu,
dorothymatthews1@gmail.com (D.M. Matthews).
broad-spectrum antibiotics can modulate the microbiome and
affect behavior; infection studies show that enteric pathogens
can induce anxiety-like behaviors in animals; probiotic studies
show beneficial effects on the intestinal tract and improved behav-
iors associated with anxiety-related conditions (see Bravo et al.,
2012). For example, Li and colleagues (2009) reported that alter-
ations in the diversity of enteric bacteria influence memory and
learning in mice, Clarke et al. (2012) found sex-specific regu-
lation of hippocampal serotonin associated with anxiety using
germ-free mice and Bravo et al. (2011) further demonstrated that
Lactobacillus rhamnosus influences emotional behavior in mice
through the GI tract with involvement of the vagus nerve and
gamma-amminobutyric acid (GABA) system. This research pro-
vides evidence about how changes in the gut microbiota can lead
to modification in CNS function with ramifications for behavior.
Homeostatic function and behavior, however, can be influenced
not only by normal and disrupted enteric microbiota associations,
but by organisms present in the ambient environment as well. Rook
and Brunet (2002) have proposed and championed the “old friends”
hypothesis as a way to explain the explosion of allergic, chronic
inflammatory and autoimmune disorders present among people
living in developed nations. They suggest that contemporary urban
0376-6357/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.beproc.2013.02.007
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28 D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35
lifestyles have disrupted long established relationships during pre-
natal, neonatal and adulthood with coevolved organisms such as
helminths, soil and water microbes, farm animals and pets that are
typically recognized as harmless by the innate immune system and
induce an anti-inflammatory response. Since allergies are medi-
ated by T helper (TH2) lymphocytes, and autoimmunity is mediated
by T helper (TH1/TH17) lymphocytes, the immune dysregulation
caused by lack of exposure to “old friends” likely involves disrup-
tions not only in innate immunity but to the adaptive immune
system as well. Such dysregulation of immunoregulatory circuits
of the immune system may also potentially affect mood, cogni-
tive function and behavior (Rook et al., 2003, 2012; Raison et al.,
2010; Rook, 2012). These same homeostatic processes are likely
important to the behavioral ecology of all mammals.
A microbe that has been the subject of several studies inves-
tigating the hypothesis that extant nonpathogenic organisms can
improve behavioral health outcomes is Mycobacterium vaccae.M.
vaccae is an aerobic bacterium found in temperate environments
and animals are likely exposed to it through contact with water,
soil and vegetation (Sneath et al., 1986; Gomez et al., 2001; Kazda
et al., 2009). As an aerobe, it cannot colonize the anaerobic GI tract
of animals and is thought of as a transient commensal (Rook and
Brunet, 2005). M. vaccae was used in clinical trials in which termi-
nal lung cancer patients were inoculated with heat-killed M. vaccae.
Treated patients showed improved emotional health and general
cognitive function (O’Brien et al., 2004). These findings led to specu-
lation that an immune response to M. vaccae antigens might involve
a ubiquitous neurotransmitter such as serotonin that plays a role
in mood, arousal and learning (Leussis and Bolivar, 2006; Cools
et al., 2007; Hohmann et al., 2007; Cifariello et al., 2008). Thus, an
immune response to this microbe might positively impact behavior
influenced by emotionality.
Examining this idea in a mouse model, Lowry et al. (2007) tested
the hypothesis that peripheral exposure to M. vaccae antigen causes
a T helper cell response that activates brain serotonergic systems
in mice. Their research demonstrated that mice injected with heat-
killed M. vaccae antigen experienced (1) a TH1 and T regulatory
cell biased immune activation of a subset of serotonergic neurons
located in the dorsal raphae nucleus (DRI) of the brainstem and
that project to the hippocampus and other forebrain regions, (2)
elevated serotonin metabolism in the ventromedial prefrontal cor-
tex, and (3) a reduction in stress-related emotional behavior in the
forced swim test. Prior to this, Hunt et al. (2005) showed that heat-
killed M. vaccae could influence immunocompetence through GI
tract interaction in mice after administration by gavage.
Several researchers document the effect of immunomodula-
tion on cognition and psychiatric disorders (Brynskikh et al., 2008;
Miller, 2010; Yirmiya and Goshen, 2011). Integration of Lowry
et al.’s (2007) findings and recent research on the nature of
brain–gut–enteric microbiota interactions encouraged us to ask:
Could ingestion of M. vaccae alter anxiety behavior and influence
learning in mice? We hypothesized that if M. vaccae decreases
stress response through an immune system activation of serotonin
pathways, then mice that ingest M. vaccae may show superior com-
plex maze performance and fewer anxiety-related behaviors than
control mice.
1. General methods: all experiments
1.1. 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 con-
sistency with the mice used by Lowry et al. (2007). Each mouse
was placed in an individual polycarbonate cage with a wire bar lid
used to hold the water bottle and feed. Carefresh Natural Premium
pet bedding, obtained from Carolina Biological Supply Company,
was placed directly into the cage allowing the absorption of urine
and the animal to burrow and/or den. To allow the mice to become
acclimated to their new setting, the experiments were started when
mice were 52 days old, and weighted approximately 21–25 g.
1.2. Ethical note
All animal experiments were conducted in accordance with the
2010 US Animal Welfare Act under animal use protocols (#01-2010
and #01-2011) and animal husbandry standard operating proce-
dures approved by the Sage Colleges Institutional Animal Care and
Use Committee. All efforts were made to minimize the number of
animals used and their suffering. At the completion of each study
animals were humanely euthanized using CO2.
1.3. M. vaccae
M. vaccae (15,483) was purchased from the American Tissue Cell
Culture (ATCC) and stored at 5 C until reconstituted. M. vaccae
was grown in nutrient broth for four days at 37 C and stored in
a refrigerator until needed. Aliquots of 0.1 mL, containing approx-
imately 4.5×106CFU/mL (determined by a standard plate count)
was applied to the food vehicle of treatment mice, as appropriate.
1.4. Food vehicles
All mice were denied food for 24 h before administration of food
vehicles. The food vehicle given the experimental mice consisted
of a piece of white Wonder bread (produced by Hostess Brands),
approximately 1 cm ×1 cm, onto which 0.1 mL of M. vaccae was
aseptically pipetted. The bread was coated on the same side with
a thin layer of store brand creamy peanut butter to increase pal-
atability. Control mice received a food vehicle like that given the
experimental animals (1 cm ×1 cm square of white bread coated
with peanut butter), but which lacked M. vaccae. Treatment mice
in experiments 2 and 3 received a food vehicle identical to that
given the control mice, i.e. it lacked M. vaccae.
2. Experiments 1–3: Complex maze experiments
2.1. Methods
2.1.1. 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.
2.1.2. 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 learn-
ing 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 bedding or other
materials. The maze was a square Plexiglas box (14 cm high,
45 cm ×45 cm) consisting of five rows, 9 cm wide, with five door
openings, 8 cm wide. The start box was 9 cm ×13cm in size. Eight
turns are required to reach the end point of the unobstructed maze.
Three levels of maze difficulty were used in experiment 1. Each
successive level involved additional turns and openings and longer
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D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35 29
Fig. 1. Illustration of the maze layout. Mice were placed in the maze at the start box,
and rewards were placed at the finish of level 1, level 2 or level 3 as appropriate.
(Level 3 finish is shown here.)
maze run length. This was accomplished by closing off pertinent
door openings with Plexiglas barriers that were attached to the top
of the maze walls with small clips. Level 1 total maze run distance
was 54 cm, level 2 was 64cm and level 3 was 136 cm.
For level 1, barriers blocked off the two door openings in the
second row of the maze. This required that a minimum of two turns
to be made to reach the food reward. For level 2, barriers were
removed from the second row of the maze that established level 1,
and a barrier was instead placed in the door opening of the third row
of the maze. This required that a minimum of four turns be made
to reach the food reward. For level 3, all barriers were removed and
the maze was completely unobstructed, requiring a minimum of
eight turns to reach the food reward. Level 3 of the maze was used
in all testing in experiments 2 and 3. After each mouse was tested,
the maze was sanitized with 70% alcohol, rinsed with water and
allowed to dry completely.
2.1.3. Anxiety-related behaviors
Mice demonstrate a variety of anxiety-related behaviors
(Blanchard et al., 2001, 2003; Leussis and Bolivar, 2006; Bailey and
Crawley, 2009; Gould, 2010; Smolinsky et al., 2010). Seven separate
anxiety-related behaviors (Table 1) were observed and scored.
2.1.4. Analysis
Maze trials were videotaped (SONY Handicam DCR-HC85). An
experienced observer blind to the treatment for each mouse assign-
ment scored the anxiety-related behaviors from the tapes. All
statistical analyses used IBM Statistical package for the Social Sci-
ences (SPSS), version 20, and all reported values are means and
Table 1
Ethogram of anxiety-related behaviors.
Behavior pattern Description
1. Defecation Number of fecal boli released per trial
2. Elongation Mouse moves with a low-back, stretched posture or
movement while keeping hind feet stationary; number
of events per trial counted
3. Grooming Mouse uses front paws to rub face and whiskers;
number of events per trial counted
4. Immobilization Mouse remains motionless for three or more seconds;
number of events per trial counted
5. Latency to start Number of seconds mouse spends in start box after
initial placement until hindquarters cross the start box
boundary to the maze
6. Return to start Mouse moves from the start box to other area of the
maze and then returns to start box and remains for at
least a second; number of events per trial counted
7. Wall climbing Mouse puts both front paws on maze wall while on
hind legs; all events within 15 s period were scored as
one event
standard errors of the means (S.E.M.). Comparisons of two inde-
pendent means were made using a two-tailed t-test (P< 0.05).
Comparisons among means in experimental designs with multiple
between subjects factors were analyzed using analysis of variance
(ANOVA, P< 0.05) followed, when appropriate, by post hoc analysis
using pairwise comparisons with Bonferroni corrections. Compar-
isons of within-subjects factors were performed using repeated
measures analysis of variance (P< 0.05) followed, where appropri-
ate, by post hoc pairwise comparisons using Bonferroni corrections.
For experiments 1–3, a different observer rescored 8% of the tests
for an interobserver reliability estimate of 92% for the scored
anxiety-related behaviors. Maze running errors were scored as the
total number of three types of errors: wrong turns moving for-
ward; backtracking; and direct return to start box (Winocur and
Moscovitch, 1990; Devan et al., 2006).
3. Details of individual experiments
3.1. Experiment 1: Effect of M. vaccae
3.1.1. 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 1. Control mice were given
a food vehicle on days 21 and 7 as well, but it lacked M. vaccae.
Likewise, the food rewards at the finish of the maze runs of control
mice lacked M. vaccae.
Maze testing was a repeated measures design at three levels of
maze difficulty. All mice were tested during each trial and all test-
ing occurred on Sunday, Tuesday and Thursday. Four trials were
conducted at level 1, one each on Sunday, Tuesday, Thursday, and
Sunday. Likewise, four trials were conducted at level 2, one each on
Tuesday, Thursday, Sunday and Tuesday. Finally, four trials were
conducted at level 3 of the unobstructed maze, one each on Thurs-
day, Sunday, Tuesday and Thursday. This resulted in 12 trials over
a 4-week period.
Start time was recorded when all four paws of the mouse
touched the floor of the maze. Completion time for the maze was
scored when the mouse ate the food reward for a full three seconds
to ensure commitment to ingestion. The order in which individuals
from the experimental and control groups were tested was alter-
nated each time a mouse was tested. Time to finish the maze was
recorded and demonstrated anxiety-related behaviors were scored.
3.1.2. Results
3.1.2.1. Maze run time. Twelve trials of testing at three maze diffi-
culty levels revealed that mice that ingested M. vaccae completed
the maze twice as fast (X= 55.2 ±10.6 s, N= 10) as control mice
(X= 116.8 ±11.8 s, N= 8). A repeated measures ANOVA showed that
ingestion of M. vaccae had a significant effect on the time it took
for experimental and control mice to complete the maze (ANOVA:
F1,16 = 15.08, P=0.001). A main effect by maze level was observed
as well. Mauchly’s test indicated that the assumption of sphericity
had been violated, 2= 16.3, P< 0.05, therefore degrees of freedom
were corrected using Greenhouse–Geisser estimates of sphericity
(ε= 0.6), (ANOVA: F1.2, 19.2 = 30.26, P= 0.0001). Bonferroni post hoc
tests showed that performance differed at levels 1 and 2 of the
maze (Mdiff = 91.65, 95% CI [49.79, 133.52]), and at levels 1 and 3
of the maze (Mdiff = 95.93, 95% CI [49.79, 142.1.]), but not at levels
2 and 3 of the maze (Fig. 2). The group treatment by maze level
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30 D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35
Fig. 2. Comparison of the effect of ingestion of M. vaccae on maze run time at each
level of the complex maze in experiment 1. Experimental mice completed the maze
faster than control mice at each level of the maze with the largest difference in
performance seen at level 1.
interaction was also significant (ANOVA: F1.2,19.2 = 6.96, P= 0.013).
Post hoc comparison of means and confidence interval between the
two groups at each level revealed that while experimental mice
performed better than the control mice at all levels of the maze,
their performance at level 3 was not different than their perfor-
mance at level 2 (Table 2).
3.1.2.2. Anxiety-related behaviors. A mixed measures ANOVA with
Bonferroni corrections was performed for each of the anxiety
behaviors, with the group (experimental [N= 10] versus control
[N= 8]) as the between-subjects independent variable, the maze
level (level 1 versus level 2 versus level 3) as the within-subjects
variable, and the duration or count or the anxiety behaviors as
the dependent variables. For all of the behaviors except return to
start, Mauchly’s tests of sphericity were violated and the degrees
of freedom were corrected using Greenhouse–Geisser estimates of
sphericity (ε). Table 3 presents the main effects. The main effect of
maze level was observed for all seven behaviors. Four behaviors
exhibited treatment (group) effects: elongation, immobilization,
Table 2
Summary of repeated measures ANOVA multiple comparisons of M. vaccae treat-
ment by maze level for maze run time in experiment 1.
Maze Level 1 Level 2 Level 3
X(SEM)
95% CI
X(SEM)
95% CI
X(SEM)
95% CI
Exp 87.9a(23.5) 44.0b(7.4) 33.6b(8.6)
[38.05,
137.7]
[28.25,
59.65]
[15.47,
51.78]
Control 209.1c
(26.3)
69.7a(8.3) 71.5a(9.6)
[153.39,
264.8]
[52.16,
87.27]
[51.17,
91.76]
Note. CI =confidence interval. Different subscript letters indicate statistically signif-
icant differences P< 0.05.
grooming and latency to start. A maze level by treatment inter-
action was shown for three behaviors: immobilization, grooming
and latency to start.
Anxiety-related behaviors for both experimentals and controls
decreased from maze level 1 to maze level 3. Post hoc comparisons
are reported only for the behaviors which had both a significant
group effect and maze level effect. The most common pattern was
a decrease in anxiety-related behaviors between levels 1 and 2,
with no significant differences between levels 2 and 3. Bonferroni
post hoc tests showed that both the experimentals and the con-
trols exhibited significantly fewer immobilizations between maze
level 1 and maze level 2 (Xdiff = 0.70, 95% CI [0.214,1.17], P=0.004),
and maze level 3 (Xdiff = 0.63, 95% CI [0.110,1.153], P= 0.015), which
were not significantly different from each other. Likewise, for
latency to start, both the experimentals and the controls exhib-
ited significantly fewer immobilizations between maze level 1 and
maze level 2 (Xdiff = 63.22, 95% CI [29.90, 96.55], P= 0.0001), which
were not significantly different from each other. This pattern was
the same for the grooming behavior (levels 1–2: Xdiff = 0.38, 95% CI
[0.056, 0.71], P= 0.019; levels 1–3: Xdiff = 0.49, 95% CI [0.07, 0.90],
P= 0.019). For elongation a different pattern emerged: elongations
at level 1 and 3 (Xdiff = 2.92, 95% CI [2.04, 3.81], P= 0.0001) and at
level 2 and 3 (Xdiff = 2.10 95% CI [0.95, 3.25], P= 0.0001) were signif-
icantly different, but not between levels 1 and 2. For return to start,
wall climbing and defecation, the pattern of decline was mixed for
both experimentals and controls in terms of whether a significant
difference from level 1 was observed at level 2 first or not until level
3, and the group effect was not significant for these behaviors.
Table 3
Summary of main effects and interaction effects for anxiety behaviors in experiment 1.
Behavior Main effects Interaction
Level Group
Immobilization F1.17, 18.7 = 11.88 F1,16 = 12.99 F1.17,18.7 = 12.96
P= 0.002 P= 0.002 P= 0.03
Grooming F1.26, 20.11 = 8.99 F1,16 = 11.02 F1.26,20.11 = 6.02
P= 0.005 P= 0.004 P= 0.018
Latency to start F1.01,16.19 = 23.7 F1,16 = 12.99 F1.01,16.19 = 7.49
P= 0.001 P= 0.002 P= 0.014
Elongation F1.51, 24.11 = 22.61 F1,16 = 5.84
P= 0.001 P= 0.03
Return to start F2,32 = 9.38
P= 0.001
Wall climbing F1.48,23.70 = 5.50
P= 0.02
Defecation F1.39,22.31 = 8.6
P= 0.004
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D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35 31
Table 4
Summary of repeated measures ANOVA multiple comparisons of M. vaccae treatment by maze level for anxiety behaviors in experiment 1.
Maze Level 1 Level 2 Level 3
Behavior X(SEM) 95% CI X(SEM) 95% CI X(SEM) 95% CI
Immobilization
Exp 0.2a(0.3) 0.0b(0.0) 0.03b(0.1)
[0.30, 0.75] [0.8, 0.8] [0.15, 0.20]
Control 1.3c(0.3) 0.2a(0.0) 0.3a(0.1)
[0.73, 1.9] [0.6, 0.25] [0.6, 0.44]
Grooming
Exp 0.2a,d (0.2) 0.1a,d (0.1) 0.1a(0.1)
[0.27, 0.57] [0.11, 0.31] [0.12, 0.22]
Control 1.1b(0.2) 0.4c(0.1) 0.3a,c,d (0.1)
[0.70, 1.60] [0.18, 0.64] [0.07, 0.44]
Latency to start
Exp 31.5a(17.5) 3.8b, e (2.0) 3.5b(1.8)
[5.52, 68.57] [0.34, 8.0] [0.33, 7.33]
Control 108.2c(19.5) 9.5d(2.2) 8.1d,e(2.0)
[66.80, 149.64] [4.82, 14.15] [3.78, 12.35]
Note. CI =confidence interval. Different subscript letters indicate statistically significant differences P< 0.05.
The treatment by maze level interaction was significant for three
of the behaviors: immobilization, grooming and latency to start
(Table 4). These three behaviors show the pattern of performance
in which controls at maze levels 2 and 3 approximate that of exper-
imentals at maze level 1. The significant interaction indicates that
the difference between the groups in these anxiety-related behav-
iors due to treatment was present at maze level 1 but not at levels 2
or 3 of the maze. Although controls exhibited more returns to start,
wall climbing and defecation than experimentals at each maze
level, these differences were not significant in interaction across
the maze levels. By the third level of the maze, the controls were
showing fewer anxiety-related behaviors, approximating the lower
levels of anxiety behaviors expressed by the experimentals at the
first two levels. The measure of latency to start appeared to be the
best indicator of initial anxiety for the experimentals and the con-
trols, with a large mean differences at maze level 1 compared to
maze levels 2 and 3. While experimentals showed significantly
less hesitation to start than controls at each level, this was the
only behavior in which the controls at both levels 2 and 3 exhib-
ited less of the particular anxiety behavior the experimentals at
level 1.
The patterns of reduced run time and reduced demonstration of
anxiety-related behaviors were similar for both experimentals and
controls across the three levels of the maze. This suggests that as
anxiety-related behaviors decline, running speed improves.
3.1.2.3. Errors. Mice treated with M. vaccae demonstrated fewer
errors (X= 3.7 ±0.6, N= 10) across all three levels during maze runs
than the control mice (X= 4.6 ±0.7, N= 8), but these differences
were not statistically significant.
4. Experiment 2: M. vaccae removal
4.1. Methods
To determine what would happen to complex maze perfor-
mance 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 experi-
ment 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 to finish the
maze and demonstrated anxiety-related behaviors were recorded
as for experiment 1.
4.2. Results
4.2.1. Maze run time
In the consecutive set of 12 trials at level 3 of the complex maze
without M. vaccae in the food reward, experimental mice contin-
ued to complete the maze twice as fast (X= 21.6 ±10.1 s, N= 10)
as the control mice (X= 47.0 ±11.3 s, N= 8). A repeated measures
ANOVA over the course of the 12 trials, however, revealed that these
differences were not statistically significant. Further analysis of dif-
ferences in the maze performance of experimental (X= 18.8 ±6.3 s,
N= 10) and control mice (X= 50.1 ±7.1 s, N= 8) at trials 1 and 2
(ANOVA: F1= 15.33, P= 0.001) and experimental (X= 26.0 ±6.3 s,
N= 10) and control mice (X= 49.5 ±7.0 s, N= 8) at trials 1, 2 and
3 (ANOVA: F1,2 = 6.92, P= 0.018) revealed that experimental mice
completed the maze faster than control mice and that these differ-
ences were statistically significant. At trials 4–12 of maze testing,
however, statistically significant differences in the maze run time
of the two groups were not observed.
4.2.2. Anxiety-related behaviors
Repeated measures ANOVA for each of the seven anxiety-related
behaviors indicated that experimental (X= 0.1 ±0.04, N= 10) and
control mice (X= 0.3 ±0.05, N= 8) differed significantly from one
another in only one behavior, grooming (F1,16 = 10.73, P= 0.005).
4.2.3. Errors
Experimental mice demonstrated fewer errors (X= 2.2 ±0.6,
N= 10) than control mice (X= 4.1 ±0.7, N=8) during the 12 trials of
maze testing. Analysis of these results show that mice who previ-
ously ingested M. vaccae displayed significantly fewer errors than
control mice (ANOVA: F1,16 = 4.53, P= 0.049). There was no main
effect of trial number, or group by trial interaction. This indicates
that even though experimental mice were not running faster than
control mice over the course of the 12 trials of testing at level 3,
they were making less errors in the maze than the control mice.
5. Experiment 3: Strength of memory
5.1. Methods
To determine how well mice remembered the maze pattern, all
mice were rested for three weeks and one final maze test was con-
ducted at level 3, seven weeks after the experimental mice had last
been exposed to M. vaccae.NoM. vaccae was administered in the
food reward at this time. Time to finish the maze and demonstrated
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32 D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35
anxiety-related behaviors were recorded as for experiments 1 and
2.
5.2. 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 with fewer anxiety-related behaviors (X= 0.8 ±0.2, N= 10)
than control mice (X= 1.1 ±0.3, N= 8). However, these differences
were not statistically significant. Similarly, the experimental mice
demonstrated fewer errors (X= 1.9 ±0.6, N= 10) during maze runs
than the control mice (X= 2.4 ±0.6, N= 8), but the differences in
errors were not statistically significant.
6. Experiment 4: Elevated Zero Maze
6.1. 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).
6.2. Subjects
Forty-one mice were divided into a control group (N= 11) who
did not receive M. vaccae in their food vehicle and three treat-
ment groups (N= 30) of 10 mice each. Mice in the treatment groups
all received M. vaccae in their food vehicle but differed from one
another in the time of testing in the EZM following their last M.
vaccae treatment: 12 h (N=10), 18 h (N= 10) or 24h (N= 10).
6.3. Elevated zero maze
The maze (Med Associates, St. Albans, VT) consisted of a circular
platform (7.0 cm wide with a 45.5cm inner diameter) that was ele-
vated 64.5 cm above the floor. It was equally divided into two closed
quadrants and two open quadrants. The two closed areas had walls
on both sides that were 20.5 cm in height. The open areas lacked
walls, but were bordered by a narrow lip of clear plastic (0.5 cm
high) to diminish the likelihood of mice falling onto the floor. The
activity of the mice was monitored by an overhead camera and
scored by visual observation of behaviors.
6.4. M. vaccae exposure
Experimental mice were exposed to M. vaccae using the
immunological priming schedule utilized in experiment 1. Experi-
mental mice were given a food vehicle on the wire lid of their cages
which contained M. vaccae on three occasions: 3 weeks before maze
testing, 1 week before maze testing and on the day before EZM test-
ing. Control mice were given a food vehicle on the same schedule
as experimental mice, but which lacked M. vaccae.
6.5. EZM testing protocol
EZM testing occurred in a room separate from the home colony
and under dim light (approximately 70 lux) using a protocol
described by Walf and Frye (2007). Each mouse was transported
to the testing room and placed within the closed area of the EZM
at the boundary to the open area, facing inward. EZM testing lasted
for 5 min. Placement in each of the two closed areas of the maze
was alternated between subjects. After each mouse was tested,
the maze was sanitized with 70% alcohol, and allowed to dry com-
pletely.
We scored three behaviors from the EZM trials: number of
entries into the open maze area, time spent in the open area, and
the number of head dips from the closed area and from the open
area. Head dips are a standard ethological measurement used in
EZM testing indicative of motivation to explore and risk assess-
ment (Shepherd et al., 1994; Bourin et al., 2007; Walf and Frye,
2007). Entry into the open area of the maze was recorded when
all four paws were in the open area. Time spent in the open area
was scored as the percentage of time that mice spent with all four
paws in the open area. Head dips are defined as the mouse looking
over the edge of the maze by arching the neck and pointing the
nose down toward the floor. Head dip from the closed area was
scored when one or more paws remained in the closed area while
the mouse looks over the edge. Head dip from open area was scored
when the mouse had four paws in the open area while looking over
the edge of the maze.
6.6. Analysis
Experiment 4 zero maze trials were videotaped using the same
equipment as in experiments 1–3. An experienced observer who
was blind to the treatment assignment scored the entries into the
open area, time spent in the open area and head dipping behaviors
from the tapes.
6.7. Results
There were no statistically significant differences between
experimentals and controls in the time spent in the open area and in
the number of entries into the open area from the closed area. Mice
did differ in the number of combined head dips (combined open
and closed area head dips) (ANOVA: F3,41 = 3.01, P= 0.042). Post hoc
pairwise comparisons revealed that there was a significant differ-
ence in the head dipping behavior of the 12 h group (X= 13.0 ±1.55,
CI [9.86,16.14]) and the control group (X= 8.50 ±1.62, CI [5.21,
11.79]), but no significant differences between the 18 h and 24 h
groups.
7. Experiment 5: activity testing
7.1. Methods
7.1.1. 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).
7.1.2. 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.
7.1.3. Activity testing protocol
Mice were acclimated for two weeks within the activity cages
with the wheels immobilized. On the day following the first expo-
sure of experimental mice to M. vaccae, the wheels were released.
Running activity was collected from the counters daily for the
next 23 days, and the mean km distance traveled/day was calcu-
lated.
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D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35 33
7.1.4. Results
The mean daily distance traveled by the experimental mice
(X= 6.3 ±1.4 km, N= 8) did not significantly differ from the control
mice (X= 6.8 ±1.7 km, N= 7) over the 23 days of activity testing.
8. 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 dif-
ferent in the M. vaccae treated group as compared to the control
group (Table 3). These effects do not appear to be due to differ-
ences in generalized activity levels related to treatment with M.
vaccae. Experiment 5 did not show differences in wheel running
activity due to treatment with M. vaccae. Additional evaluation of
anxiety using a standard anxiety-testing maze, EZM, revealed only
differences in one measure: head dipping.
In complex maze experiment 1, mice given M. vaccae showed
superior performance compared to controls with both faster
maze run time and reduced expression of anxiety-like behaviors.
Although maze level effects were significant overall, the primary
difference was seen at level 1 for both running time and anxi-
ety behaviors. The difference in performance of the two groups
of mice was greatest during early exposure to the maze when
the novelty of the task might have been most anxiety-provoking
to these animals. The behavior latency to start reveals hesita-
tion to exit the start box and enter the maze. The experimentals
entered the maze more readily than the controls at every level.
Even though at level 1 both experimentals and controls exhibited
much more hesitation than they did at levels 2 and 3, experimen-
tals were approximately three times less hesitant than controls at
each maze level (Table 4). These results indicate that M. vaccae
treatment may have abated anxiety in the experimentals which
affected both motivation to run through the maze and expression
of anxiety-related behaviors. The other three behaviors for which
there was a significant group effect (immobilization, grooming and
elongation) showed a similar pattern. The experimentals and con-
trols did not differ in errors at each level, and the run time of
the two groups was similar by level 3 despite the fact that the
maze difficulty increased at each maze level. Therefore the per-
formance differences across the levels within each group may be
primarily due to increasing familiarity with the maze. The main
outcome of this experiment is that M. vaccae treated mice showed
superior maze run time and diminished anxiety compared to the
controls, and that difference was most pronounced in early maze
trials.
The reduction of anxiety-related behaviors resulting from M.
vaccae ingestion may have allowed more rapid complex maze
investigation, resulting in reduced run time once the maze is
learned at each maze level. If we consider these results in the con-
text of the findings of Lowry et al. (2007) who demonstrated that
injected M. vaccae antigen stimulates brain serotonin production
and decreases stress-related behaviors in mice for a short period
of time in a forced swim test, then it seems feasible that inges-
tion of live M. vaccae may stimulate serotonin production through
immunological mechanisms. This suggests that ingested M. vac-
cae may upregulate DRI serotonergic neurons that modulate stress
responsive behaviors. Plasticity in the stress response influencing
cognitive behavior can be modulated through a T cell response.
While many studies (Grenham et al., 2011; Bravo et al., 2012; Clarke
et al., 2012) show that microbiota can activate innate and adap-
tive immune mechanisms that influence anxiety and behavior, our
research indicates that ingestion of an ambient bacterium which is
not part of the enteric microbiome may have this same beneficial
effect.
In a consecutive set of 12 trials without M. vaccae treatment
at level 3 of the maze (experiment 2), experimental mice contin-
ued to run the maze faster and with less anxiety-related behaviors
than control mice. This pattern was only statistically significant,
however, during the first three trials of maze testing in this exper-
iment. It appears that while M. vaccae ingestion had an effect on
maze performance of experimental mice for about a week after
M. vaccae removal, this effect was not long lasting. Among the
test behaviors, a significant group difference was only observed
for grooming. This indicates that anxiety-behaviors were no longer
influencing maze performance. However, there were significant
differences in the number of maze navigational errors with con-
trol mice demonstrating two times as many errors as experimental
mice. The experimental mice may have remembered the complex
maze pattern better than the controls for the 4-week duration of
experiment 2. Lastly, following a three-week rest period (experi-
ment 3), a final trial revealed no statistically significant differences
in run time, anxiety-related behaviors or errors between the two
groups. The similarities in maze performance and demonstrated
anxiety-related behaviors may have occurred because, after 25
maze trials, both groups of mice knew the maze pattern equally
well.
Although our study did not aim to elucidate the underlying
neuroimmunological mechanisms potentially responsible for the
results we observed, those mechanisms must interact with several
higher order behavioral systems. M. vaccae treatment may have
increased motivation to run the maze due to a facilitatory interac-
tion with rewarding properties inherent in maze exploration itself,
the action of running, and/or to an acutely rewarding feature of the
M. vaccae in the food vehicle given at the end of each maze trial
in experiment 1. Acquisition of conditioned cues may have been
heightened by the treatment resulting in increased performance
times and anxiety reduction which would then have facilitated
familiarization with the maze. In experiment 1, after every four tri-
als, the maze complexity and distance to complete the maze were
increased. Both the experimental mice and control mice exhibited
fewer anxiety-related behaviors with each subsequent level of the
maze despite increasing complexity. However, the experimentals
continued to exhibit fewer anxiety-related behaviors than the con-
trol mice at each new level of the maze. A further consideration
is the level of stress reactivity exhibited by various mouse strains.
BALB/c is considered to be a stress reactive mouse strain (Palumbo
et al., 2009). M. vaccae treatment might produce a different outcome
relative to the stress profile of the strain.
In order to examine the effect of ingested M. vaccae on anx-
iety behavior in a non-cognitive task, we observed performance
in an elevated zero maze (experiment 4). The experimental and
control groups did not differ in their willingness to enter the
open areas of the maze whether tested 12, 18 or 24 h after the
last exposure to ingested M. vaccae or the placebo. However, the
experimental and control groups did differ in head dip behavior
in the 12 h test. As reviewed in Shepherd et al. (1994), includ-
ing ethological measures of behavior indicative of risk assessment
and exploration add clarity and reliability to the standard meas-
ures of open arm entries and time spent, elucidating anxiolytic
and anxiogenic drug effects. In experiment 4, we observed signif-
icant differences in the exploratory risk assessment behavior of
head dipping. Head dipping in elevated mazes is commonly con-
sidered to be an exploratory movement (Bourin et al., 2007) with
increased head dips being indicative of decreased anxiety (Braun
et al., 2011). Head dipping in the EZM may be related to explo-
ration and risk assessment involved in “looking for an escape route”
behavior. In this way it may be related to behaviors required for
exploring, spatially navigating, and learning a complex maze such
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34 D.M. Matthews, S.M. Jenks / Behavioural Processes 96 (2013) 27–35
as that used in experiments 1–3. Thus, M. vaccae treatment may
affect the motivational system involved in exploration of novel
and potentially threatening environments, and enhance spatial
memory. Moreover, the results of experiment 5 indicate that the
behavioral differences observed in mice treated with M. vaccae
versus controls in experiments 1–4 were likely not due to effects
on basic activity level. It should be noted, however, that the small
sample size used in this experiment may not have provided enough
power to detect a treatment effect of M. vaccae exposure on activ-
ity. Future research should be conducted to answer this question
more fully.
Raison et al. (2010) speculate that the mammalian microbiome
plays a critical role in the development of the immune system and
the maintenance of human health. Saprophytic mycobacteria are
common in the environment (Kazda et al., 2009) and while they do
not replicate in the gut, were likely to have always been present in
the gastrointestinal tract of our ancestors due to contact with mud
and water (Rook, 2010). Repeated, long-term exposure to M. vac-
cae could serve as an adaptive mechanism for the development of
tolerance responses to stressful situations. Further, such a scenario
dovetails with the old friends hypothesis (Raison et al., 2010). Thus,
by upsetting the long established relationship of our immune sys-
tem to ambient bacteria such as M. vaccae, complex behaviors such
as learning could be negatively affected through the dysregulation
of immunoregulatory responses coupled to the neuromodulation
of emotionality.
While recent research has shed light on the ability of the micro-
biota to influence behavior via neural, hormonal and immune
interactions (Li et al., 2009; Bravo et al., 2011; Clarke et al., 2012), it
is surprising to think that a common ambient microbe may modu-
late anxiety behaviors. Our research provides initial data suggesting
that ingestion of live M. vaccae can reduce anxiety behaviors related
to exploration of novel environments, and exert a previously unre-
ported influence on learning in mice. This effect on behavior was
fast acting, observed by the first maze run trial, but the effect dimin-
ished with M. vaccae removal. This suggests that M. vaccae may
act as a kind of pharmabiotic, inducing short-term physiological
changes affecting behavior.
The impact of exposure to microbes on animal behavior in natu-
ral environments is unknown. We could hypothesize, for example,
that differential exposure to microbes such as M. vaccae may influ-
ence the expression of behavioral phenotypes related to being a
“wanderer” or a “resident” (as in prairie voles, Microtus ochrogaster,
Getz et al., 1993; Solomon and Jacquot, 2002; Ophir et al., 2008)
with implications for spatial exploration related to differential
reproduction. Research that incorporates a behavioral ecologi-
cal perspective on brain–gut–microbe interactions is necessary to
understand the underlying mechanisms that shape the evolution of
those interactions. Our results contribute preliminary evidence of
an adaptively significant behavioral response of mice to M. vaccae
ingestion that could have arisen from the coevolution of mam-
malian neuroimmunological systems and ambient microbes.
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 Labora-
tories 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 thought-
ful review of versions of the manuscript, K. Jones, S. Statham and J.
Dahlgren for graphical assistance, and R. Spica, J. Spear, A. Mathews,
M.J. Matthews and J. Bonaccorso for technical support.
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... Although a small number of soil microorganisms are pathogenic, evidence increasingly suggests that exposure to diverse soil microorganisms can help train the immune system, reduce inflammation, and improve mental health. [113,114] Similar results can be found in children living with pets and/or siblings. [115] Accumulating evidence suggests that such microbial exposures have the potential to influence neurological processes and emotions through modulation of the HPA axis and immune pathways. ...
... [116] For example, exposing mice to the common soil bacterium Mycobacterium vaccae shows a reduction in anxiety-related behaviors and improvement in cognitive tasks. [114] Nevertheless, such fledgling empirical work needs further confirmatory and higher-quality longitudinal studies to drive public policy towards both achieving planetary health [20,24,117] and implementing nature-based interventions that make use of the preventive and therapeutic potential of green space. [118] Furthermore, the inclusion of new materials and the design of built environments based on bio-architecture might be interesting and innovative interventions that could be detached from the current hypotheses. ...
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The complexity of the human mind and its interaction with the environment is one of the main epistemological debates throughout history. Recent ideas, framed as the 4E perspective to cognition, highlight that human experience depends causally on both cerebral and extracranial processes, but also is embedded in a particular sociomaterial context and is a product of historical accumulation of trajectory changes throughout life. Accordingly, the human microbiome is one of the most intriguing actors modulating brain function and physiology. Here, we present the 4E approach to the Human Microbiome for understanding mental processes from a broader perspective, encompassing one's body physiology and environment throughout their lifespan, interconnected by microbiome community structure and dynamics. We review evidence supporting the approach theoretically and motivates the study of the global set of microbial ecosystem networks encountered by a person across their lifetime (from skin to gut to natural and built environments). We furthermore trace future empirical implementation of the approach. We finally discuss novel research opportunities and clinical interventions aimed toward developing low‐cost/high‐benefit integrative and personalized bio‐psycho‐socio‐environmental treatments for mental health and including the brain‐gut‐microbiome axis. The 4E approach to the Human Microbiome understands mental processes broadly, encompassing body physiology, microbiome dynamics, and environment throughout lifespan. We pursue mechanistic network models in the form of embedded gut‐brain‐behavior interactomes, reaching convergence of methods and analyses and public policy and novel evidence‐based clinical interventions coupled with patients’ lifestyle.
... In summary, although i.n.-administered M. vaccae NCTC 11659 prevents the aggravating effects of stress on DSS-induced colitis when administered during chronic psychosocial stress exposure and shows at least mild stress protective effects when administered prior to stressor exposure [32], future studies are required to elucidate the exact underlying mechanisms. [43,58], pulmonary allergic inflammation in mice [75], and (using M. vaccae ATCC 15483 T ) anxiety-related behavior in mice [315]. TB participants receiving standard drug therapy in addition to daily p.o. administration with M. vaccae NCTC 11659 for one month showed an ameliorated TB-associated weight loss and inflammation, reduced hepatotoxicity of TB drugs, and an improved clearance of sputum from M. tuberculosis [43]. ...
... The cytokine environment in the BAL showed a bias toward increased IL-10 production, suggesting for the first time an involvement of Tregs following i.g.-administered M. vaccae NCTC 11659, with potentially beneficial consequences for the treatment of allergy. Noteworthy, mice treated p.o. with M. vaccae ATCC 15483 T via food pellets (4.5 × 10 6 CFU/mL per food pellet) on days -21 and -7 before behavioral testing in the Hebb-Williams-style complex maze or the elevated zero-maze (EZM) [315] showed a faster maze run time and reduced expression of anxiety-related behavior. Although the neurobiological mechanisms were not elucidated, the authors speculate that the effects of M. vaccae ATCC 15483 T might be due to its influence on the serotonergic system in the midbrain and pontine raphe nuclei, as shown previously [21]. ...
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Inflammatory diseases and stressor-related psychiatric disorders, for which inflammation is a risk factor, are increasing in modern Western societies. Recent studies suggest that immunoregulatory approaches are a promising tool in reducing the risk of suffering from such disorders. Specifically, the environmental saprophyte Mycobacterium vaccae National Collection of Type Cultures (NCTC) 11659 has recently gained attention for the prevention and treatment of stress-related psychiatric disorders. However, effective use requires a sophisticated understanding of the effects of M. vaccae NCTC 11659 and related rapidly growing mycobacteria (RGMs) on microbiome–gut–immune–brain interactions. This historical narrative review is intended as a first step in exploring these mechanisms and provides an overview of preclinical and clinical studies on M. vaccae NCTC 11659 and related RGMs. The overall objective of this review article is to increase the comprehension of, and interest in, the mechanisms through which M. vaccae NCTC 11659 and related RGMs promote stress resilience, with the intention of fostering novel clinical strategies for the prevention and treatment of stressor-related disorders.
... Although there are undoubtedly differences in the immune system of humans and mice or rats, experiments have shown, that there are potential connections between environmental bacteria and important physiological functions of vertebrates. The administration of living bacteria of the species Mycobacterium vaccae (a non-disease-causing soil bacterium) to mice, reduced, similar to K. alysoides, fear-like behaviors and improved their ability to learn (Matthews and Jenks, 2013). Even heat-killed bacteria of this type retained their immunomodulatory effects and and fear-like behavior in mice (Reber et al. 2016, Loupy et al. 2019. ...
Chapter
Microbes are essential for life on earth. The recently proposed “one health” concept emphasizes the importance of the microbiome and its diversity on ecosystem and human health. In this chapter, losses in microbial diversity including both soils and human (intestine) and potential consequences on the functioning of the respective systems and interlinkages are depicted. In particular, the use of plant protection agents and antimicrobials in human medicine as well as resistance in soils is discussed in the prospect of the declining biodiversity. The development of the human microbiome and how environmental microbes can proliferate over large distances and impact human health and diseases are outlined. Sustainable agricultural management is of crucial importance for terrestrial ecosystem functioning in the face of changing climate. We discuss potential steps for the latter, in combination with a human lifestyle that is re-connecting to a microbial rich and diverse nature and show how essential this is to maintain and retrieve human health.
... Notably, exposure to M. vaccae in immune system activation and serotonin pathways could influence behavioral and emotional responses (Brevik et al., 2020). Some research demonstrated that injection with heat-killed M. vaccae to mice (Mus musculus) could influence immunocompetence through gastrointestinal tract interaction, immune activation of serotonergic neurons located in related parts of the brain, upregulation of serotonin metabolism in the prefrontal cortex, and the production of metabolites such as lipids (Foxx et al., 2021;Matthews and Jenks, 2013). However, a limited number of VOCs and metabolism that are derived from M. vaccae studies have been reported (McNerney et al., 2012;Nawrath et al., 2012). ...
Article
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Mycobacterium vaccae is a species of nonpathogenic bacterium that lives naturally in soil. This study compared the physiological effects at a metabolomic level with autonomic nervous system responses in adults during soil-mixing activities, based on the presence or absence of M. vaccae in the soil. Twenty-nine adult participants performed soil-mixing activities for 5 minutes using sterilized soil with culture media and M. vaccae , respectively. Blood samples were drawn twice from each participant after each activity. Electroencephalograms and electrocardiograms were measured during the activity. Serum metabolites underwent metabolite profiling by gas chromatography, followed by multivariate analyses. Soil-emitted volatile organic compounds were identified using the solid-phase microextraction and gas chromatography-mass spectroscopy, followed by multivariate analyses. The volatile compound analysis revealed that the metabolites related to esters and sulfur-containing compounds are greater in soil with M. vaccae . Serum metabolomics revealed that the treatment group (soil inoculated by M. vaccae ) possesses relatively higher levels of inter-alia organic and amino acids compared with the control group (soil mixed with culture media). In the treatment group, the electroencephalogram and electrocardiogram revealed that alpha band activity of the occipital lobe increases, while heart rate decreases. This study concludes that M. vaccae soil contact can affect human metabolic and autonomic reactions.
... These taxa included DA101-a very abundant soil bacterium that is associated with grasslands [63]-and with the presence of photoheterotrophs (which use light energy as their energy source and rely on organic compounds from the environment as their carbon source, as opposed to exclusively carbon dioxide). They also included Mycobacterium, a bacterial genus that (a) contains the common non-pathogenic soil-dwelling M. vaccae which has been associated with reduced inflammatory responses [64,65], reduced anxiety-like behaviour [65,66], and prevention of stress-induced sleep impairment [67] in laboratory rodent models; and (b) has been linked to non-specific beneficial effects ...
Chapter
Humans have evolved in a microbe-rich environment and have become dependent on some of these microbes to colonise us, provide essential chemicals, and prime our immune systems. In many urbanised, western countries, there has been a loss of contact with these biodiverse environmental microbiota, which might be associated with the increased burden from diseases such as asthma, allergies, and autoimmune disorders. Here we summarise our growing understanding of the relationship between exposure to biodiverse environmental microbiota and their potential to provide health benefits. We start by covering the known range of health outcomes associated with green space exposure and then explore the possibility that these benefits are mediated by microbes. We provide evidence to support the notion that environmental microbiota influence the human microbiota and that this in turn leads to a range of health effects. The evidence is strong enough to recommend biodiverse green space exposure both as a clinical and as a public health intervention and discuss what types of environments might be most suitable to recommend. To maximise potential health benefits, we need to improve both the quantity and quality of green spaces and ensure that these are accessible to those communities that stand to benefit most.
... It has been universally accepted that the gut microbiota is a diverse and complex ecosystem predominantly dominated by bacteria, which has profound impact on the physiological processes and health maintenance of their hosts. As the research on intestinal microflora has received increasing attentions, and the relevance of symbiotic microorganisms for host health and specific behaviors has also been widely reported, its unique role has gradually been recognized (Matthews and Jenks 2013;Gould et al. 2018;Schretter et al. 2018;Jia et al. 2021). In this study, we depicted the gut microbiota of the G. courtoisi, a critically endangered passerine bird endemic to China, and revealed the microbial species and structural composition presented in its intestine. ...
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Blue-crowned laughingthrush (Garrulax courtoisi), passeriformes, is a critically endangered bird endemic to China. Gut microbiota is well known to play a pivotal role in host health and survival. Thus, the understanding of the microbial communities associated with Garrulax courtoisi could be beneficial to save this species from the brink of extinction. In this study, we used 16 s rDNA amplicon sequencing to investigate the gut community composition and microbial diversity of the Garrulax courtoisi population reared in Nanchang Zoo. The results showed that there were 31 phyla that were dominated by Firmicutes, Proteobacteria, Bacteroidetes, and Cyanobacteria in the intestine of Garrulax courtoisi. Compared with previous studies on birds, the Cyanobacteria exhibited an excessive abundance, which may be largely related to the personal lifestyle of Garrulax courtoisi. At the genus level, a total of 552 genera were identified, among which, 21 key genera constituted the core microbiome, including some culturable bacterial genera such as Lactobacillus, Acinetobacter, and Deinococcus. In the meanwhile, we found that there were remarkable intraspecific differences both in terms of microbial community structures, representative biomarkers and predicted functions between the parental generation and their offspring of the population investigated in this study. Furthermore, we also summarized their different eating behaviors and predicted its association with gut microbiota. This study provided the needed pieces of information about these extremely rare birds, Garrulax courtoisi, whose community composition and microbial diversity are hardly known. Importantly, these findings could contribute to our knowledge of the gut health of Garrulax courtoisi and advance the comprehensive conservation of this endangered bird.
... Several researchers have indicated a link between the gut microbiota and learning [83] or memory [84]. In particular, specific probiotics have been developed to improve learning and memory in mice [85,86]. In Drosophila, the gut microbiota is much simpler compared to other animals [87]. ...
Article
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Mounting evidence indicates that the gut microbiota is linked to several physiological processes and disease development in mammals; however, the underlying mechanisms remained unexplored mostly due to the complexity of the mammalian gut microbiome. The fruit fly, Drosophila melanogaster, is a valuable animal model for studying host-gut microbiota interactions in translational aspects. The availability of powerful genetic tools and resources in Drosophila allowed the scientists to unravel the mechanisms by which the gut microbes affect fitness, health, and behavior of their hosts. Drosophila models have been extensively used not only to study animal behaviors (i.e., courtship, aggression, sleep, and learning & memory), but also some human related neurodegenerative diseases (i.e., Alzheimer’s disease and Parkinson’s disease) in the past. This review comprehensively summarizes the current understanding of the gut microbiota of Drosophila and its impact on fly behavior, physiology, and neurodegenerative diseases.
... Specific bacteria have already been identified with a particular interest for their effects on cognitive functions. In rodents, Mycobacterium vaccae, a commensal bacterium, is as a modulator of cognition, acting both through the immune and serotonergic systems [70][71][72] . In the present study, this specific bacterium did not differ between the two experimental groups. ...
Article
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The influence of feed supplements on behavior and memory has been recently studied in livestock. The objectives of the study were to evaluate the effects of a synbiotic on: an episodic-like (SOR: Spontaneous Object Recognition), a working (BARR: Fence barrier task), a long-term (TMAZE: Spatial T-maze task) memory test and on gut microbiota composition. Eighteen female piglets were supplemented from 1 to 28 days of age with a synbiotic (SYN), while 17 served as control (CTL). Feces were collected on days 16, 33 and 41 for 16S rRNA gene composition analyses. In the SOR, SYN piglets interacted more quickly with the novel object than CTL piglets. In the BARR, SYN piglets had shorter distances to finish the test in trial 3. In the TMAZE, SYN piglets were quicker to succeed on specific days and tended to try the new rewarded arm earlier during the reversal stage. Difference of microbiota composition between treatments was nonexistent on D16, a tendency on D33 and significant on D41. The synbiotic supplement may confer memory advantages in different cognitive tasks, regardless of the nature of the reward and the memory request. Difference in memory abilities can potentially be explained by differences in microbiota composition.
Article
This essay describes my ongoing series "Hypersymbiotics™," which began in 2012 and explores the potential ways in which our microbiome, genetics, epigenetics and even our environment could potentially be enhanced to turn us into human 'super-organisms.' The series includes performances and installations involving BioArt, as well as photographic documentation of ephemeral artworks and takes the form of a vehicle for public discussion about new healthcare technologies. The essay discusses artworks made using synthetic biology techniques including CRISPR genetic modification in bacteria and yeasts, and gene editing in plants, as well as using artificial intelligence and stem cell research. It critiques the role of the media and advertising in the promotion of complex new biomedical technologies. The "Hypersymbiotics™" series is deeply concerned with promoting public understanding of the ethical implications of new scientific developments and enabling reflection and debate. At its core the artwork is about knowledge, power, and control and where that resides.
Article
New discoveries in drugs and drug delivery systems are focused on identifying and delivering a pharmacologically effective agent, potentially targeting a specific molecular component. However, current drug discovery and therapeutic delivery approaches do not necessarily exploit the complex regulatory network of an indispensable microbiota that has been engineered through evolutionary processes in humans or has been altered by environmental exposure or diseases. The human microbiome, in all its complexity, plays an integral role in the maintenance of host functions such as metabolism and immunity. However, dysregulation in this intricate ecosystem has been linked with a variety of diseases, ranging from inflammatory bowel disease to cancer. Therapeutics and bacteria have an undeniable effect on each other and understanding the interplay between microbes and drugs could lead to new therapies, or to changes in how existing drugs are delivered. In addition, targeting the human microbiome using engineered therapeutics has the potential to address global health challenges. Here, we present the challenges and cutting-edge developments in microbiome-immune cell interactions and outline novel targeting strategies to advance drug discovery and therapeutics, which are defining a new era of personalized and precision medicine.
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Microorganisms and macroorganisms such as helminths from mud, animals, and feces play a critical role in driving immunoregulation. The term "old friends" is broader than "hygiene" to describe this hypothesis, and it implicates exposures to microbes and other organisms during critical phases of human development. Diseases and conditions of the modern era, including multiple sclerosis, type 1 diabetes, and allergies, involve disrupted immunoregulatory circuits, likely reflecting reduced exposures to "old friend" organisms with which humans coevolved. Several clinical trials are testing these concepts, determining whether renewed exposures to "old friend" organisms can help to combat these modern-era diseases.
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In microtine rodents, formation of communal groups often is associated with environmental stresses of winter or high population density. Social organization of the prairie vole (Microtus ochrogaster) consisted of communal groups formed by addition of philopatric young and unrelated adults to an original male-female pair or single-female breeding unit. Owing to high juvenile mortality during spring-early autumn, most social groups remained as male-female pairs or single females. Male-female pairs displayed behavioral traits associated with monogamy, including sharing a common nest and home range, mate guarding, dissolution of pairs by mortality, and low incidence of remating. Male-female pairs rarely involved related animals. Single-female breeding units were survivors of male-female pairs that had not formed a new pair; prevalence of single-female breeding units was not related to availability of unpaired males. When juvenile survival increased in late autumn, communal groups became the predominant social group. Formation of communal groups was unrelated to low temperatures and was not contingent upon the nonreproductive status of members. Although present at low population density, communal groups were the most common type of social group at high densities. We conclude that increased survival of juveniles in late autumn leads to the formation of communal groups and increases in population density.
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Memory, one of the most complex functions of the brain comprises of multiple components such as perception, registration, consolidation, storage, retrieval and decay. The present study was undertaken to evaluate the impact of different training sessions on the retention capacity of rats. The capacity of retention of learnt task was measured using exteroceptive behavioral models such as Hexagonal swimming pool apparatus, Hebb-Williams maze and Elevated plus-maze. A total of 150 rats divided into fifteen groups were employed in the present study. The animals were subjected to different training sessions during first three days. The ability to retain the learned task was tested after single, sub-acute, acute, sub-chronic and chronic exposure to above exteroceptive memory models in separate groups of animals. The memory score of all animals was recorded after 72 h, 192 h and 432 h of their last training trial. Rats of single exposure group did not show any effect on memory. Sub-acute training group animals showed improved memory up to 72 h only, where as in acute and sub-chronic training groups this memory improvement was extended up to 192 h. The rats, which were subjected to chronic exposures showed a significant improvement in retention capacity that lasted up to a period of eighteen days. These observations suggest that repeated rehearsals at regular intervals are probably necessary for consolidation of long-term memory. It was observed that sub-acute, acute and sub-chronic exposures, improved the retrieval ability of rats but this memory improving effect was short lived. Thus, rehearsal or training plays a crucial role in enhancing one's capacity of retaining the learnt information.
Book
Affecting approximately twenty percent of the world population, mood and anxiety disorders have been the subject of ever-increasing research. This increased research parallels a remarkable growth in the use of the laboratory mouse as a tool to understand the biological and genetic basis of mood and anxiety disorders as well as to develop improved treatments. In Mood and Anxiety Related Phenotypes in Mice: Characterization Using Behavioral Tests, world-renowned researchers provide an overview of behavioral approaches utilized in the characterization of mood and anxiety related behaviors in mice as well as commonly used behavioral assays to assess the potential antidepressant and anxiolytic efficacy of novel compounds. As a volume in the successful Neuromethods™ series, the chapters provide authoritative reviews including up-to-date detailed protocols of the most commonly used approaches in the field. Mood and Anxiety Related Phenotypes in Mice: Characterization Using Behavioral Tests is an ideal resource for scientists actively pursuing or interested in establishing behavioral protocols in their laboratories, while also serving as a reference for those students, scientists, and practitioners who have an interest in better understanding the preclinical behavioral methods used in mood and anxiety research.
Chapter
The fascinating story of the ubiquitous mycobacteria and their never-ending threat to human and animal health comes to a close with the following set of photographs. Both the breadth and depth of material on mycobacteria are illustrated with the short captions and links to relevant chapters of this book are provided. Readers should be aware that all photos were taken as working documents for laboratory and travel reports over the last three decades. This chapter therefore does not have the aim of representing issues relating to mycobacteria as a set of “artistic photos.” Rather, the intention of the authors was to summarize the story by showing at least some of environments where mycobacteria survive or thrive, often posing risks to other inhabitants of the habitat, including invertebrates, amphibians, fish or mammals, and even humans. The history of mycobacteria begins thousands of years ago, long before some of the species were recognized as pathogens and from ancient times they represent a permanent health risk to animals and humans. The scientific description of mycobacteria has a much shorter history. Two papers cited in this book appeared in 1875 and since that time thousands more have been published. The methods used in mycobacterial research have developed from simple observation, microscopy, culture, experiments on animals and case studies to the use of sophisticated instruments, applied in electron and confocal microscopy, liquid culture with automatic mycobacterial growth detection, radioisotope techniques, genetics, immunology, molecular biology, genomics, proteomics, etc.
Article
Many studies have documented intraspecific differences in the behavior of males or females. In some species, many adults are territorial while others have larger home ranges encompassing multiple territories. Although these two types of behavior have been documented, they are not well understood in mammals. Therefore, in the mono gamous prairie vole (Microtus ochrogaster) we characterized individuals that engage in these two behavior patterns as residents and wanderers. We monitored populations enclosed in 0.1-ha. enclosures at Miami University's Ecology Research Center. As many as 26% of animals were wanderers: animals captured frequently, but less than 75% of the time, at one nest. As expected, wanderers had larger home ranges than residents. Wanderers were primarily adult males (70%) but included some adult females. This behavior pattern was not fixed, since some wanderers previously had been residents and at least 31% of males and 57% of adult female wanderers became residents during the same field season. Wanderers were not in worse physical condition, as estimated by body mass, and survived for slightly longer than residents. Thus, it does not appear that wanderers are making the best of a bad situation, but analysis of parentage is critical to validate this conclusion.
Article
Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.Keywords: allergy; hygiene hypothesis; intestinal permeability; leaky gut; probiotics