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Research Report
Psychotropic effects of Lactobacillus plantarum PS128
in early life-stressed and naïve adult mice
Yen-Wenn Liu
a
, Wei-Hsien Liu
a
, Chien-Chen Wu
a,b
, Yi-Chen Juan
a
,
Yu-Chen Wu
a
, Huei-Ping Tsai
a
, Sabrina Wang
c,
n
, Ying-Chieh Tsai
a,b,
nn
a
Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan
b
Probiotic Research Center, National Yang-Ming University, Taipei 11221, Taiwan
c
Institute of Anatomy and Cell Biology, National Yang-Ming University, 155, Section 2, Linong Street, Taipei 11221,
Taiwan
article info
Article history:
Accepted 12 November 2015
Available online 24 November 2015
Keywords:
Dopamine
Early life stress
Hypothalamic–pituitary–adrenal
axis
PS128
Psychobiotics
Serotonin
abstract
Ingestion of specific probiotics, namely “psychobiotics”, produces psychotropic effects on
behavior and affects the hypothalamic–pituitary–adrenal axis and neurochemicals in the
brain. We examined the psychotropic effects of a potential psychobiotic bacterium,
Lactobacillus plantarum strain PS128 (PS128), on mice subjected to early life stress (ELS)
and on naïve adult mice. Behavioral tests revealed that chronic ingestion of PS128
increased the locomotor activities in both ELS and naïve adult mice in the open field test.
In the elevated plus maze, PS128 significantly reduced the anxiety-like behaviors in naïve
adult mice but not in the ELS mice; whereas the depression-like behaviors were reduced in
ELS mice but not in naïve mice in forced swimming test and sucrose preference test. PS128
administration also reduced ELS-induced elevation of serum corticosterone under both
basal and stressed states but had no effect on naïve mice. In addition, PS128 reduced
inflammatory cytokine levels and increased anti-inflammatory cytokine level in the serum
of ELS mice. Furthermore, the dopamine level in the prefrontal cortex (PFC) was
significantly increased in PS128 treated ELS and naïve adult mice whereas serotonin (5-
HT) level was increased only in the naïve adult mice. These results suggest that chronic
ingestion of PS128 could ameliorate anxiety- and depression-like behaviors and modulate
neurochemicals related to affective disorders. Thus PS128 shows psychotropic properties
and has great potential for improving stress-related symptoms.
&2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC
BY license (http://creativecommons.org/licenses/by/4.0/).
http://dx.doi.org/10.1016/j.brainres.2015.11.018
0006-8993/&2015 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
Abbreviations: CFU, colony-forming unit; CORT, corticosterone; ELS, early life stress; EPM, elevated plus maze; FST, forced
swimming test; HPA axis, hypothalamus–pituitary–adrenal axis; HPLC-ECD, high-performance liquid chromatography-
electrochemical detection; IL, interleukin; LAB, lactic acid bacteria; MRS, de Man Rogosa and Sharpe; MS, maternal separation;
NELS, non-ELS; OFT, open filed test; PD, postnatal day; SPT, sucrose preference test; TNF, tumor necrosis factor
n
Corresponding author.
nn
Corresponding author at: Institute of Biochemistry and Molecular Biology, National Yang-Ming University, 155, Section 2, Linong
Street, Taipei 11221, Taiwan.
E-mail addresses: sabrina@ym.edu.tw (S. Wang), tsaiyc@ym.edu.tw (Y.-C. Tsai).
brain research 1631 (2016) 1–12
1. Introduction
Probiotics are living microorganisms known to have benefi-
cial effects on the host when ingested in adequate amounts
(FAO/WHO, 2001). Numerous studies have demonstrated the
diverse benefits of probiotics (Vyas and Ranganathan, 2012),
including anti-inflammatory effects (Liu et al., 2011), gut
protection (Zareie et al., 2006), and the attenuation of meta-
bolic dysfunctions (Barrett et al., 2012;Huang et al., 2013). In
recent years, the influences of probiotics on the central
nervous system (CNS) and behaviors via the microbiome–
gut–brain-axis have been uncovered (Collins et al., 2012). In
particular, there is an association between the gut microbiota
and stress response, and the effects of probiotics on CNS
could be mediated by humoral, immune, neural, and meta-
bolic pathways (Moloney et al., 2014;Sudo et al., 2004). Based
on the roles of probiotics in regulating stress responses and
maintaining CNS homeostasis, we think there is great poten-
tial for utilizing beneficial bacteria to improve CNS-related
abnormalities.
Several probiotics have been shown to have effects on
normalizing stress-induced abnormal behaviors and regulat-
ing the hypothalamus–pituitary–adrenal axis (HPA axis) and
the inflammatory responses in animal models (Ait-Belgnaoui
et al., 2012;Arseneault-Breard et al., 2012;Bercik et al., 2011;
Desbonnet et al., 2010). For example, pretreatment with
probiotics in immune-deficient mice has been shown to
normalize immune-mediated deficits in intestinal physiology
and to affect CNS functions (Smith et al., 2014). Administra-
tion of Lactobacillus helveticus–containing probiotics reduces
anxiety-like behaviors in rodents (Messaoudi et al., 2011;
Ohland et al., 2013). Both Bifidobacterium longum 1714 and
Bifidobacterium breve 1205 reduce anxiety-like behavior in an
anxious mouse strain (Savignac et al., 2014). It has also been
reported that the administration of probiotics restores mono-
amine levels in key brain regions of rats in a maternal
separation (MS) model of depression (Bercik et al., 2011;
Desbonnet et al., 2010). Also, probiotics have been shown to
alter behavior and CNS function in naïve adult animals
(Moloney et al., 2014). These findings not only reveal the
complexity of gut–brain interactions upon stress challenge
but also suggest that probiotics could be used to ameliorate
stress-induced disorders.
Early life stress (ELS) is known to convey negative effects
on brain development and cause behavioral changes in
adulthood (Lupien et al., 2009). In rodents, MS is the most
commonly used method to create ELS for the study of the
physiological and psychological impacts of ELS (O'Mahony
et al., 2011;Sanchez et al., 2001). The affected animals show
long-lasting behavioral and physiological phenotypes, includ-
ing enhanced stress responses, anxiety-like and depression-
like behaviors, HPA axis hyperactivity, and abnormal neuro-
chemical changes (Cryan and Holmes, 2005). In addition, MS
also increases the immune response, as indicated by
enhanced IL-6 release following concanavalin A (ConA) sti-
mulation (Desbonnet et al., 2010). In humans, ELS is also
associated with immune dysregulation (Fagundes et al.,
2013). Furthermore, ELS is a well-known risk factor for
psychopathology, including major depressive disorder and
anxiety disorders (Green et al., 2010;McCrory et al., 2010;
Tyrka et al., 2013). Moreover, in animal studies MS has been
shown to affect the development of serotonergic and dopa-
minergic systems and the HPA axis in the brain (Bravo et al.,
2014;Rentesi et al., 2010). Thus, an animal model of MS is
ideal for investigating the psychopathology of stress-related
disorders and for evaluating the psychotropic potentials of
probiotics (O'Mahony et al., 2009).
Although several probiotics showing psychotropic effects
in animal models have been classified as psychobiotics,
which are defined as probiotics that produce health benefits
in patients suffering from psychiatric illnesses, the field is
still in its infancy (Dinan et al., 2013). In an attempt to screen
for potential psychobiotics in our lactic acid bacteria (LAB)
bank, we administered several LAB strains by oral gavage to
mice that underwent ELS and evaluated their psychotropic
potentials according to their ability to reverse depression-like
behaviors. Among the strains tested, we found that Lactoba-
cillus plantarum strain PS128 (PS128), isolated from sponta-
neously fermented mustard greens in Taiwan, shows the
highest potential. To our knowledge, the ability of this strain
to normalize stress-induced depression-like behaviors has
not been reported previously. Therefore, in the current study
we further characterize its psychotropic effects on the HPA
axis, immune responses, and neurochemical changes in the
brain. In addition, we also administered PS128 to naïve adult
mice to evaluate its effects on unstressed normal mice.
2. Results
2.1. PS128 causes distinct alterations in behavior in ELS
and naïve adult mice
We administered PS128 to ELS and naïve adult mice for
4 weeks and then started the behavioral tests to evaluate
the behavioral effects of chronic PS128 ingestion. The beha-
vioral tests were conducted starting from the least stressful
to the most stressful test in the following order: SPT, OFT,
EPM, and FST. In the SPT, the ELS mice showed a clear
reduction in sucrose preference compared to NELS litter-
mates; chronic ingestion of PS128 could revert this reduction
(Fig. 1A). However, chronic ingestion of PS128 had no effect on
sucrose preference in the naïve mice (Fig. 1B). In the OFT
there was no difference in the total distance traveled by the
ELS and NELS mice. However, the ELS mice showed a reduc-
tion in the time spent in the center (Fig. 2A and B). Chronic
PS128 treatment in ELS mice significantly increased the total
distance traveled by the ELS mice but did not reverse the time
spent in the center by the mice (Fig. 2A and B); however, this
treatment significantly increased the total distance traveled
and the time spent in the center by the naïve mice (Fig. 2C
and D).
From the EPM test we observed that ELS mice spent
significantly more time in the closed arms than the NELS
group, indicating more anxiety-like behaviors in the ELS mice
(Fig. 3A). Although we did not observe any change in the
PS128-treated ELS mice, this treatment significantly reduced
the closed arm time and increased open arm time of the
naïve mice (Fig. 3A and B). In contrast, in the FST, PS128
brain research 1631 (2016) 1–122
treatment significantly reduced the immobile time in the ELS
mice, reverting the immobility to a level similar to that of the
NELS group, whereas such treatment had no effect on the
naïve mice (Fig. 4A and B). Taken together, chronic PS128
treatment increased locomotor activity in both ELS mice and
naïve rats (Fig. 2). For anxiety-like behavior evaluated by the
center time of OFT and open arm time of the EPM, PS128 had
a significant effect on naïve mice but not ELS mice (Figs. 2 and
3). Conversely, PS128 significantly reduced depression-like
behaviors in ELS mice but had no effect on naïve mice
(Figs. 1 and 4).
2.2. PS128 normalizes ELS-induced exaggerated
corticosterone release
Maternal neglect is known to increase stress reactivity in
offspring (Meaney, 2001). Thus, we measured the serum
corticosterone levels to access the HPA axis reactivity. During
the basal unstressed condition, the corticosterone level of the
ELS mice was significantly higher than that of the NELS mice
(Fig. 5A). Chronic PS128 treatment significantly reduced the
baseline corticosterone level in the ELS mice to a degree
similar to that of NELS mice (Fig. 5A). Under stressed condi-
tions, in this case 30 min after forced swimming, ELS mice
also showed significantly higher corticosterone levels than
did the NELS mice (Fig. 5A). Again, chronic PS128 treatment
could significantly reduce the elevated corticosterone level in
stressed ELS mice (Fig. 5A). In the naïve mice, chronic
ingestion of PS128 had no effect on the serum corticosterone
level either at baseline or under stressed conditions (Fig. 5B).
These results indicated that the HPA axis in the ELS mice was
dysregulated and administration of PS128 could normalize
the elevated corticosterone level (Fig. 5A).
Fig. 1 –PS128 administration reversed the reduced sucrose preference in the ELS mice but has no effect on naïve mice. (A) ELS
mice show a clear reduction in sucrose preference compared to NELS mice, and chronic PS128 ingestion reverts this reduction
to a level similar to that of NELS mice. (B) Chronic PS128 ingestion has no effect on sucrose preference in naïve mice. ELS, early
life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data in the
ELS mice experiments were analyzed by one-way ANOVA; data in the naïve mice experiments were analyzed by t-test.
***po0.001.
Fig. 2 –Open field test indicates that chronic PS128 ingestion increases locomotor activities in both ELS and naïve mice and
reduces anxiety-like behaviors in naïve mice. (A) In the open field test the total travel distance is increased in ELS mice after
PS128 administration. (B) The time spent in the center area is significantly reduced in ELS mice, and PS128 administration had
no effect on the center time of ELS mice. (C) The total travel distance in naïve mice is also increased following PS128
administration. (D) Time spent in the center area is significantly increased in naïve mice after chronic PS128 administration.
ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128.
Data in the ELS mice experiments were analyzed by one-way ANOVA; data in the naïve mice experiments were analyzed by t-
test. *po0.05; ***po0.001.
brain research 1631 (2016) 1–12 3
2.3. PS128 decreases ELS-induced inflammation
To evaluate the effects of PS128 on the immune system of ELS
mice, we measured the levels of the pro-inflammatory
cytokines TNF-αand IL-6 and the anti-inflammatory cytokine
IL-10 in serum and in mitogen-stimulated splenocytes. The
ELS mice showed a significant reduction in serum TNF-α,
whereas the IL-6 level was increased compared with that of
NELS mice (Fig. 6A and B). The IL-10 concentration of ELS
mice seemed lower than that of the NELS mice; however, this
did not reach statistical significance (Fig. 6C). After chronic
administration of PS128, the ELS mice showed no significant
change in TNF-αlevel (Fig. 6A). However, this treatment
significantly reduced the IL-6 level and increased the IL-10
level in the ELS mice (Fig. 6B and C). In the cultured
splenocytes, which were obtained from NELS, ELS, and
PS128-treated ELS mice, we collected culture medium and
measured the aforementioned cytokines. We found that in
the unstimulated culture there were no differences in TNF-α,
IL-6, and IL-10 concentrations among different groups
(Fig. 6D, E and F). When we stimulated the culture with ConA,
we found that splenocytes from ELS mice produced signifi-
cantly more TNF-αthan splenocytes from NELS mice (Fig. 6D).
In splenocytes from PS128-treated ELS mice, this enhance-
ment was attenuated (Fig. 6D). The level of IL-6 in LPS-
stimulated splenocytes from ELS mice was not different from
that of NELS mice; however, the IL-6 response of splenocytes
from PS128-treated ELS mice was significantly higher than
that of the other two groups (Fig. 6E). The levels of IL-10 in
LPS-stimulated splenocytes from ELS mice showed a trend of
reduction; however, the difference was not statistically sig-
nificant. The IL-10 levels of splenocytes from the PS128-
treated ELS group were significantly higher than those of
the ELS mice (Fig. 6F). These results showed that PS128
modulated immune responses in ELS mice. However, PS128
treatment showed no clear effects on cytokine levels in
Fig. 3 –Elevated plus maze test shows PS128 administration decreases anxiety-like behaviors in naïve mice but not ELS mice.
(A) Time spent in closed arm is significantly increased in ELS mice and PS128 administration has no effect on ELS mice. (B) In
naïve mice, PS128 administration significantly reduced the time spent in the closed arm and increased the time spent in the
open arm. ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered
PS128. Data of closed or open arm in the ELS mice were analyzed separately by one-way ANOVA; data of closed or open arm
in the naïve mice experiments were analyzed separately by t-test. *po0.05; ***po0.001.
Fig. 4 –Forced swim test indicates PS128 administration reduces depression-like behaviors in ELS mice but not in naïve mice.
(A) The immobile time is significantly increased in ELS mice, and PS128 administration reduces the immobile time to the level
similar to that of NELS mice. (B) PS128 administration has no effect on the immobile time of naïve mice. ELS, early life stress;
ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data in the ELS mice
experiments were analyzed by one-way ANOVA; data in the naïve mice experiments were analyzed by t-test. ***po0.001.
brain research 1631 (2016) 1–124
serum or stimulated splenocytes from naïve mice (data not
shown).
2.4. PS128 altered the serotonin and dopamine systems in
both ELS and naïve adult mice
Because ELS has been reported to increase serotonergic and
dopaminergic activities in the PFC, and to increase dopami-
nergic activity in the striatum (Rentesi et al., 2013), we also
investigated whether PS128 could alter the ELS-induced
changes in the serotonin and dopamine systems. We ana-
lyzed levels of 5-HT, DA, and their metabolites in the PFC and
striatum by HPLC-ECD. In the PFC, ELS mice showed signifi-
cantly reduced 5-HT levels and had no change in their 5-HIAA
concentration. As a result, the ratio of 5-HIAA to 5-HT was
significantly increased in ELS mice compared to that of NELS
mice, indicating an increase in serotonergic activity (Table 1).
Chronic PS128 ingestion did not increase the reduced 5-HT in
ELS mice, but instead reduced the 5-HIAA concentration and
consequently reduced the ratio of 5-HIAA to 5-HT to the level
similar to that of NELS mice (Table 1). However, in the naïve
adult mice PS128 increased 5-HT concentration and
decreased both the 5-HIAA concentration and the ratio of 5-
HIAA to 5-HT (Table 2). The serotonin system in the striatum
was not significantly different in ELS, NELS, PS128-treated ELS
mice, or naïve mice (Table 1).
In the dopaminergic system, the DA concentration of PFC
tended to decrease in ELS mice compared with NELS mice;
however, this difference was not statistically significant
(Table 1). The concentration of DA metabolite DOPAC was
not changed in ELS mice compared to NELS mice (Table 1).
However, the ratio of DOPAC to DA was significantly higher
than that of the NELS mice (Table 1). Although the level of
HVA was significantly reduced in ELS mice, the HVA-to-DA
ratio was not significantly different from that of the NELS
mice (Table 1). Following chronic PS128 ingestion, the treated
ELS mice had a significantly elevated DA level compared to
untreated ELS mice (Table 1). The DOPAC and HVA concen-
trations of PS128-treated ELS mice were also significantly
higher than those of the untreated ELS mice. As a result, the
ratio of DOPAC to DA and HVA to DA were both significantly
lower than that of the untreated ELS mice (Table 1). In
particular, the ratio of HVA to DA in PS128-treated mice
was even lower than that of the NELS mice (Table 1).
In naïve adult mice, PS128 treatment significantly
increased the concentration of DA, DOPAC, and HVA
(Table 2). However, the DA turnover rate was not different
from that of the saline-treated mice (Table 2). In addition,
there was no change in the dopaminergic activity in the
striatum in ELS, PS128-treated ELS, and naïve mice groups
(Tables 1 and 2).
3. Discussion
In the present study we demonstrated the psychotropic
effects of PS128 on ELS and naïve adult mice. We used MS
as the adverse event to create ELS. MS is a well-established
paradigm used in rat models of ELS and has been shown to
result in prolonged and consistent dysfunctions in the gut–
brain-axis (O'Mahony et al., 2011). Hence, it proved to be a
useful platform for screening psychobiotics (Dinan et al.,
2013). For that reason, we had adapted the rat MS procedure
with some modifications because our mice had ELS. It has
been reviewed that MS in mice is difficult to work with
because it produces unreliable phenotypes that may be
caused by either different stressor protocols or mice strains
(Millstein and Holmes, 2007;Savignac et al., 2011). However,
we have consistently found that mice that underwent our
modified MS procedure developed reliable abnormal beha-
viors (Fig. 1). In addition to behavioral changes, we also found
dysfunctions in the HPA axis, immune response, and neuro-
transmitters in the ELS mice. These findings suggest that the
modified MS procedure could be used in mice for under-
standing the adverse effects of ELS and for screening
psychobiotis.
Our ELS mice showed both anxiety-like and depression-like
phenotypes because they had reduced center time in OFT,
increased closed arm time in EPM, increased immobile time in
the FST, and reduced preference for sucrose (Figs. 1–4). Most
Fig. 5 –Chronic PS128 administration reduces corticosterone levels in ELS mice but not naïve mice. (A) ELS treatment
significantly elevates blood corticosterone levels under both basal and stressed states. PS128 administration effectively
reduces the corticosterone levels in the ELS mice to a level similar to that of NELS mice under both states. (B) PS128
administration has no effect on the corticosterone levels in naïve mice in both basal and stressed states. ELS, early life stress;
ELSþ128, ELS mice administered PS128; NELS, non-ELS; Naïveþ128, naïve mice administered PS128. Data of basal or stressed
state in the ELS mice experiments were analyzed separately by one-way ANOVA; data of basal or stressed state in the naïve
mice experiments were analyzed separately by t-test. *po0.05; **po0.01; ***po0.001.
brain research 1631 (2016) 1–12 5
studies using MS show a single trait consisting of either
anxiety-like or depression-like behavior (as reviewed in
Moloney et al., 2014). In addition, the serum corticosterone
levels in our ELS mice were significantly higher in both basal
andstressedstates(Fig. 5). However, most of the MS animals in
previous studies showed increased corticosterone levels only
during the stressed state (Rees et al., 2006). These results
suggest that our ELS procedure produces a more robust
behavioral phenotype with severely dysregulated stress
response.
In our ELS mice we found a significant increase in serum
IL-6 levels accompanied by decreased serum IL-10 levels
(Fig. 6). These changes have not been previously described
in MS rats (Desbonnet et al., 2010;O'Mahony et al., 2009).
Fig. 6 –Effects of PS128 administration on serum cytokines and cytokines released from stimulated splenocytes from ELS and
naïve mice. (A) ELS treatment reduces serum TNF-αconcentration, and PS128 administration has no effect on the TNF-αlevel
of ELS mice. (B) Serum IL-6 level is significantly elevated in ELS mice, and chronic PS128 administration reversed this
elevation. (C) Chronic PS128 administration increases serum levels of anti-inflammatory cytokine IL-10 in ELS mice. (D) ConA
stimulation significantly induced TNF-αrelease in cultured splenocytes from ELS mice. Splenocytes from PS128-treated ELS
mice show a significant TNF-αrelease. (E) IL-6 release is significantly elevated in splenocytes from PS128-treated ELS mice
following LPS stimulation. (F) Splenocytes from ELS mice show reduced IL-10 release following LPS stimulation, whereas the
IL-10 release in splenocytes from PS128-treated ELS mice is similar to that of NELS mice. ConA, concanavalin A; LPS,
lipopolysaccharide; ELS, early life stress; ELSþ128, ELS mice administered PS128; NELS, non-ELS. Data of serum cytokine
levels in the ELS mice experiments were analyzed by one-way ANOVA; data of medium or ConA/LPS stimulated splenocyte
experiments were analyzed separately by one-way ANOVA. *po0.05; **po0.01; ***po0.001.
brain research 1631 (2016) 1–126
Elevation of serum IL-6 is associated with stress-related
disorders in humans who had early adverse experiences
during childhood (Coelho et al., 2014). It has been shown that
adults with childhood maltreatment and patients with
depressive disorders have increased serum IL-6 levels
(Carpenter et al., 2010;Pace et al., 2006). In addition, a clinical
study has demonstrated a higher ratio of serum IL-6 to IL-10
and a lower serum IL-10 level in patients with major depres-
sion compared to patients without major depression
(Dhabhar et al., 2009). Therefore, our data suggest that the
ELS in mice could induce an immune alternation that is
similar to changes found in clinical studies. Furthermore, the
HPA axis is also known to directly interact with the immune
system (Moloney et al., 2014). Increased secretion of circulat-
ing glucocorticoids, including corticosterone, could inhibit
both innate and adaptive immune responses (Sorrells and
Sapolsky, 2007). Thus, there may be a complex cross-
regulation between the HPA axis and the immune system,
and this cross-regulation may lead to the modulation of
animal behavior. Sustained elevation of corticosterone might
also result in the reduced serum TNF-αobserved in the ELS
mice (Fig. 6A).
In addition to these findings, concentrations of neuro-
transmitters DA and 5-HT were significantly reduced in the
PFC of ELS mice, but there was an increase in the turnover
rate of both transmitters (Tables 1 and 2). The increased DA
Table 1 –Alteration of 5-HT and DA neurochemicals in brain regions of ELS mice.
Prefrontal cortex Hippocampus Striatum
NELS ELS ELSþPS128 NELS ELS ELSþPS128 NELS ELS ELSþPS128
Monoamines and metabolites
5-HT 88.5745 42.3714
***
50.1730
*
69.774.7 52.7757.6724 5647109 6037156 6547120
5-HIAA 21.377.2 25.879.0 12.574.7
**,###
62.5721 51.9718 39.7714 77.1743 98.7750 88.8757
DA 1687114 66.1733
**
1907133
##
36.0714 29.8724 33.0712 406971176 39317938 39997939
DOPAC 50.4711 59.9729 71.2728
*
46.3721 36.279.0 34.8714 417795 4667224 333765
HVA 158751 129746 167771 72.7722 57.4710 59.0739 4597225 6877242 5197104
Turnover ratio
5-HIAA:5-HT 0.30170.38 0.61570.29
**
0.22970.12
###
0.93070.29 1.0870.55 0.73470.42 0.092470.020 0.18070.13 0.13070.058
DOPAC:DA 0.46870.28 0.83070.36
**
0.41670.18
###
1.0670.51 1.0470.36 0.93070.29 0.092370.0069 0.12370.064 0.085870.019
HVA:DA 1.6071.1 2.3171.2 0.97770.72
##
1.8170.71 1.6870.54 1.4570.35 0.16170.016 0.17970.057 0.13370.028
Concentrations of monoamines, metabolites and turnovers (ng/g wet tissue) are expressed as mean7SEM. DA, dopamine; DOPAC, 3,4-
dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HT, serotonin or 5-hydroxytryptamine; 5-HIAA, 5-hydroxyindoleacetic acid. Data were
analyzed by one-way ANOVA. Statistically significant values are highlighted in gray.
*
po0.05;
**
po0.01;
***
po0.001 vs. NELS;
##
po0.01;
###
po0.001 vs. ELS.
Table 2 –Alterations of 5-HT and DA neurochemicals in brain regions of normal adult mice.
Prefrontal cortex Hippocampus Striatum
Saline PS128 Saline PS128 Saline PS128
Monoamines and metabolites
5-HT 47.8719 95.6735
**
78.4718 99.6748 8297146 8687217
5-HIAA 25.3716 17.676.3 N/D N/D 65.3773 39.3750
DA 142766 3017157
*
50.2720 62.8749 808071070 736471883
DOPAC 95.8739 121751 57.0712 65.0718 316748 351789
HVA 123737 147742 47.177.1 54.6720 521776 469793
Turnover ratio
5-HIAA:5-HT 0.63870.64 0.16070.037 N/D N/D 0.077070.040 0.078070.044
DOPAC:DA 0.69270.34 0.54070.22 1.3170.60 1.4770.67 0.039670.0077 0.045770.017
HVA:DA 0.82370.38 0.67970.28 1.0770.40 0.98170.53 0.064770.0084 0.066710.0051
Concentrations of monoamines, metabolites and turnovers (ng/g wet tissue) are expressed as mean7SEM. DA, dopamine; DOPAC, 3,4-
dihydroxyphenylacetic acid; HVA, homovanillic acid; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, 5-hydroxytryptamine or serotonin. Data were
analyzed by t-test. Statistically significant values are highlighted in gray. N/D, not detected.
*
po0.05;
**
po0.01 vs. Saline.
brain research 1631 (2016) 1–12 7
and 5-HT turnover rate has also been reported in the
maternal deprivation model in rats (Rentesi et al., 2013).
The serotonergic system has long been linked to anxiety,
and insufficient 5-HT concentration may lead to hyper-
excitability in the amygdala, a condition that is associated
with adulthood anxiety (Etkin and Wager, 2007). Thus,
decreased 5-HT levels in the PFC may result in dysregulated
excitability in the amygdala and consequently produce
anxiety-like behaviors in ELS mice (Fig. 3). Taken together,
our ELS procedure produced mice with multiple deficits,
including abnormal behaviors, HPA dysregulation, immune
alternation, and changes in neurochemicals in the PFC.
Following the daily gavage of PS128 for 4 weeks, we found
that chronic administration of PS128 reduced the ELS-
induced depression-like behaviors, normalized the HPA axis
and immune systems, and modulated the changes in the DA
and 5-HT system in the PFC. From previous studies, ELS is
known to modulate gut microbiota and disrupt the integrity
of the gut barrier, thus resulting in increased inflammation
and abnormal behaviors (O'Mahony et al., 2011), both of
which can be restored by the ingestion of beneficial bacteria.
Reports have shown that consumption of a commercially
available probiotic combination of Lactobacillus rhamnosus
R0011 and Lactobacillus helveticus R0052 increases the amount
of gut lactobacilli, reduces permeability in the colon, and
reduces the elevated serum corticosterone induced by MS in
rat pups (Gareau et al., 2007). A recent study also shows that
administration of the commensal bacterium Bacteroides fragi-
lis improves gut barrier integrity and normalizes anxiety-like
behaviors in a mouse model known to display features of
autism spectrum disorder (Hsiao et al., 2013). Also, adminis-
tration of Lactobacillus farciminis has been shown to attenuate
gut hyperpermeability and the abnormal activation of the
HPA axis induced by psychological stress (Ait-Belgnaoui et al.,
2012). These results suggest that beneficial bacteria in the gut
contribute to the improvement of the stress response. Thus,
it is possible that PS128 normalizes the MS-induced dysregu-
lation of the HPA axis via modulating the gut microbiota and
strengthening the gut barrier. We did not directly examine
the gut integrity of PS128-treated mice; however, judging by
the lack of effect on levels of corticosterone in PS128-treated
naïve mice, which would have normal gut microbiota and gut
barrier functions, it is likely that the effect of PS128 on the
HPA axis of ELS mice involves the improvement of gut
integrity.
Chronic treatment of PS128 also alleviated the increased
serum IL-6 concentration in ELS mice. The serum concentra-
tion of IL-6 is positively correlated with depression severity in
patients (Pace et al., 2006). Preclinical animal studies have
shown that reduction of IL-6 is associated with reducing
depression-like behaviors. For example, previous studies
show that the administration of Bifidobacterium infantis 35624
to MS rats attenuated IL-6 release and reduced immobility in
FST (Desbonnet et al., 2010). Similarly, we found that chronic
PS128 administration also reduced serum IL-6 and improved
depression-like behaviors in ELS mice (Figs. 4A and 6B). We
also found that administration of PS128 significantly
increases IL-6 production in LPS-stimulated splenocytes,
suggesting PS128 treatment could increase the function of
the immune system against foreign pathogens (Fig. 6E).
The effects of PS128 on ELS mice are clearly different from
that of the naïve mice. We found PS128 could reduce anxiety-
like behaviors in naïve mice but not ELS mice (Fig. 3). How-
ever, the depression-like behaviors were reduced in PS128-
treated ELS mice but not naïve mice (Fig. 4). In addition, PS128
treatment reduced serum corticosterone levels under both
basal and stressed conditions in ELS mice but had no effect
on naïve mice (Fig. 5). Furthermore, PS128 treatment
increased the 5-HT concentration in PFC only in naïve mice
(Table 2). Because the corticosterone response of the HPA axis
in naïve mice was not changed, the reduction of anxiety-like
behaviors in naïve mice cannot be explained by regulating
the HPA axis response. Instead, it might be related to the
increased 5-HT level in the PFC for the reason mentioned
previously, that is, that 5-HT could modulate the activity of
amygdala, which is associated with anxiety-like behavior
(Etkin and Wager, 2007). However, in ELS mice the reduced
5-HT concentration in PFC cannot be reversed by adminis-
tration of PS128, and we did not see a reduction in anxiety-
like behavior (Fig. 3 and Table 1).
Administration of PS128 increased locomotor activity in
both ELS mice and naïve mice. Previous studies have shown
that the administration of DA or its analog into specific brain
regions such as the striatum or the nucleus accumbens
stimulates locomotion in animals (Mabrouk et al., 2014;
Woodruff et al., 1976). Because we did not observe changes
in the striatal dopaminergic activity (data not shown), this
locomotor enhancement probably did not involve the stria-
tum. However, a previous study has shown that the admin-
istration of Bifidobacterium infantis 35624 or citalopram to MS
rats modulated the level of noradrenaline in the brain stem
and normalized depression-like behavior (Desbonnet et al.,
2010). Changes in the noradrenaline system could also
potentially modulate the locomotor activity and should be
further explored in future studies of PS128.
The most interesting finding in this study is that PS128
significantly reduced the hyperactive HPA axis response and
the depression-like behaviors in ELS mice (Figs. 1,4and 5).
We also found an increased DA concentration and DA turn-
over rate in the PFC of PS128-treated ELS mice (Table 1). PFC is
known to be a highly evolved brain region that is responsible
for regulating working memory, decision-making, and emo-
tions, including stress response (Shansky and Lipps, 2013).
Normal PFC function, especially regarding the reward/avoid-
ance emotional responses, requires an adequate DA concen-
tration (Arnsten, 2009). In addition, the mesocortical DA
system may have a direct influence on the HPA axis
(Feenstra et al., 1992). Thus, the hyperactive HPA axis and
depression-like behaviors seen in ELS mice might be directly
associated with their altered DA function in the PFC (Table 1).
Interestingly, following PS128 administration, the DA system
of ELS mice was restored to the control level, and so were the
HPA axis responses and depression-like behaviors (Figs. 4,5
and Table 1). Hence, PS128 might suppress the exaggerated
release of corticosterone and normalized depression-like
behavior through increasing dopaminergic activity in the PFC.
In summary, we assessed the psychotropic effects of
PS128 in both ELS and naïve adult mice in an attempt to
identify novel psychobiotic strains. We demonstrate that
PS128 could normalize ELS-induced HPA axis hyperactivity
brain research 1631 (2016) 1–128
and depression-like behaviors, and that in naïve mice it
reduced anxiety-like behaviors. Some of the ELS-induced
serum cytokine and PFC neurochemical changes are also
restored by chronic PS128 administration. The psychotropic
effects of PS128 on ELS and naïve adult mice suggest great
potential for PS128 to be used to improve affective behaviors
under both normal and diseased conditions. In addition, to
our knowledge, PS128 is the first psychobiotic that increases
locomotor activity and modulates both serotonergic and
dopaminergic systems, which further expands its possible
application in the treatment of psychiatric and neurological
disorders.
4. Experimental procedure
4.1. Preparation of PS128
PS128 was isolated from fermented mustard greens, which is
a traditional Hakka ethnic food product. It was identified as a
novel strain by phylogenetic classification of its 16S rDNA
sequence. The isolated PS128 was inoculated in Man Rogosa
Sharpe broth (MRS; BD Difco, Becton-Dickinson, Sparks, MD
MD, USA), cultured at 37 1C for 18 h, and then harvested by
centrifugation at 6000 gfor 10 min. The pellet was re-
suspended in MRS plus 12.5% glycerol to a final concentration
of 5 10
9
colony-forming units per milliliter. The re-
suspended solution was then aliquoted in freezer tubes and
stored at 20 1C until use. When in use the aliquot was pre-
warmed to 37 1C for 1 h and re-suspended in saline before
being administered to mice.
4.2. Animals and housing
Timed-pregnant female and naïve adult male C57BL/6J mice
were purchased from the National Laboratory Animal Center
(Taipei, Taiwan). On arrival, the mice were accommodated in
the specific pathogen-free room at the Laboratory Animal
Center of National Yang-Ming University. The room was kept
at 22721C, 50–60% humidity, and under 12 h light/dark cycle.
The mice were provided with water and chow ad libitum
(LabDiet Autoclavable Rodent Diet 5010; PMI Nutrition Inter-
national, Brentwood, MO, USA). After 1 week of acclimation
the experiments were started. All animal experimental pro-
cedures were reviewed and approved by the Institutional
Animal Care and Use Committee, National Yang-Ming Uni-
versity (IACUC No. 1001102).
4.3. Maternal separation stress
We adapted MS procedures from a rat model of ELS described
previously and made some modifications (Desbonnet et al.,
2010). Briefly, between postnatal day (PD) 2 and PD 14, male
and female neonates in the stress group were separated from
their mothers and littermates and placed in a small glass
bottle (5 cm in diameter) for 3 h per day (11:00–14:00) at room
temperature without a heating pad (Supplementary Fig 1).
The no MS group was left with their mother undisturbed. For
the next 2 weeks, all the mice were left undisturbed except
for the routine bedding change and were weaned at PD 28. On
PD 29, only males pups were selected and were randomly
assigned to the ELS experimental group (ELSþPS128, n¼10)
and ELS control group (ELS, n¼12). Mice without MS were
regarded as the no ELS control group (NELS; n¼10).
4.4. Experimental design and sample collection
To evaluate the psychotropic effects of PS128, we administered
PS128 to the ELS experimental group and to a separate naïve
adult male group (naïve). The ELS and naïve experimental
groups were given saline re-suspended PS128 daily (10
9
CFU/
mouse/day) by gavage for 4 weeks from PD 29 and from 8 weeks
old, respectively, whereas the ELS and naïve control groups were
given saline by gavage during the same period. At the end of the
PS128 treatment period, when ELS group mice were 8 weeks old
andnaïvegroupmicewere12weeksold,themiceunderwenta
battery of behavioral tests. The tests were given in sequence
from the least stressful to the most stressful in the following
order: sucrose preference test (SPT); open field test (OFT);
elevated plus maze (EPM); and forced swimming test (FST). After
the 4-day SPT the mice were subjected to OFT on the next day.
EPM test were conducted immediately after OFT on the same
test day. On the next day the first session of FST began. All the
tests were conducted during the light phase.
On the last day of FST, before the swim start, the mice were
subjected to orbital sinus blood collection to obtain the basal
state samples. Then, the mice underwent a 6-min forced swim
and were returned to their home cage. After 30 min, blood
samples were collected again to obtain the stressed state
samples. All blood sampling was conducted within the same
3-hperiodinanefforttominimize the effect of circadian rhythm
on corticosterone release. The collected whole blood was cen-
trifuged at 3000 gfor 10 min at 4 1C, and then the serum was
stored at 80 1C until use. Following blood collection, the mice
were sacrificed by cervical dislocation. The brains were quickly
removed and placed on dry ice. The ice-cold brain was dissected
on a filter paper placed on a glass dish on ice. The prefrontal
cortex (PFC), hippocampus, and striatum samples were dissected
out and immediately preserved in ice-cold 0.6% perchloric acid
and stored at 80 1C until use. Spleen tissues were also
collected, temporarily stored in ice-cold RPMI 1640 medium
(Invitrogen, Carlsbad, CA, USA), and then processed in spleno-
cyte experiments (see mitogen-stimulated splenocyte section).
4.5. Sucrose preference test
SPT was modified from a previous study by Yu et al. (2012).To
habituate the mice to drinking sucrose-containing solution,
on the first day of the test the mice were given two bottles of
1% sucrose solution (g/mL) at 18:00 for 24 h. On the second
day, one of the sucrose bottles was replaced with tap water
and left for 24 h. On the third day, the mice were water-
deprived for 18 h from 18:00 until 12:00 next day. On the
fourth day, immediately after water deprivation, the mice
were given one bottle of 1% sucrose and one bottle of tap
water for 5 h. The position of the bottles was switched to
avoid location preference. The sucrose and water consumed
during the 5-h test were measured. The sucrose preference
(%) was calculated as follows: (weight of sucrose consumed/
total weight of sucrose and water consumed) 100.
brain research 1631 (2016) 1–12 9
4.6. Open field test
Locomotor activity of the mice was examined by the OFT. In
this test the mouse was placed in the open field activity
chamber for 10 min (Tru Scan Activity System; Coulbourn
Instruments, Whitehall, PA, USA). The square activity cham-
ber (25.4 25.4 38 cm
3
) is made of Plexiglas walls with two
photobeam sensor bars on each side. The box was cleaned
with 70% ethanol after each test. The activities were auto-
matically recorded and quantified with the Tru Scan 2.2 soft-
ware (Coulbourn Instruments). The total distance traveled,
moving time, and center distance and time were measured by
the Tru Scan Activity System. The center area was defined as
a region in the center measuring 12.5 12.5 cm
2
.
4.7. Elevated plus maze test
The elevated plus maze is composed of two closed arms and two
open arms (height, 45 cm; full arm length, 66 cm; arm width,
10 cm; wall height of closed arm, 30 cm). This test was used to
assess anxiety-like behavior of the mice. The mouse was placed
in the center arm crossing area (10 10 cm
2
)andallowedfree
exploration in the maze for 10 min. The maze was cleaned with
70% ethanol after each test. The mouse activity was recorded by
a video camera mounted on the ceiling of the maze center. The
recorded activity was later analyzed by EthoVision video tracking
software (Noldus Information Technology, Wageningen, the
Netherlands). The total travel distance and duration spent in
the open and closed arms were quantified.
4.8. Forced swimming test
The FST was used to assess the depression-like behaviors in the
mice. Briefly, mice were put in a transparent acrylic cylinder
(height, 30 cm; internal diameter, 10 cm) containing 15 cm water
(23–25 1C) to swim for 6 min on the first day. After the swim, the
mice were dried with tissue paper and returned to their home
cage. The next day, the mice were given a second swim session
for 5 min. Only naïve adult mice were subjected to the first and
second sessions of FST. Mice in the ELS experiments underwent
only the first day session. The swim sessions were recorded by a
video camera and the behavior was analyzed by EthoVision
video tracking software. The immobile time were quantified
from the first day session of ELS groups and from the second day
session of naïve adult mice.
4.9. Serum corticosterone
The serum was diluted first, and then we used a commercial
CORT EIA kit (Cayman Chemical, Michigan, MI, USA) to
analyze corticosterone concentration. The corticosterone
concentration was interpolated using the standards provided
in the kit following the manufacturer’s instructions.
4.10. Serum cytokine
We measured cytokine levels in the serum samples collected
during the baseline period and in the culture medium of
stimulated splenocytes. Tumor necrosis factor (TNF)-α, inter-
leukin (IL)-6, and IL-10 were measured using commercial
ELISA kits (eBioscience, San Diego, CA, USA) according to
the manufacturer’s instructions.
4.11. Mitogen-stimulated splenocytes
Spleen preserved in ice-cold RPMI 1640 medium was first
squashed by a sterile piston and then filtered through a sieve
mesh cell strainer (Becton, Dickinson and Company, East
Rutherford, NJ, USA) to isolate single cells. The preparation
was centrifuged at 1000 rpm for 5 min. The pellet was
collected and treated with lysis buffer for 5 min to remove
erythrocyte. Then, the preparation was centrifuged again to
remove lysis buffer and washed in serum-free RPMI 1640
medium. After the final centrifugation to remove serum-free
medium, the splenocyte pellet was re-suspended in RPMI
1640 medium plus 10% fetal bovine serum and seeded at
210
6
cells/200 μL/well in 96-well flat-bottom plates. The
stimulated splenocytes were cultured with mitogen conca-
navalin A (ConA; 4 μg/mL) or lipopolysaccharide (LPS;
0.6 μg/mL) in the culture medium for 48 h at 37 1C (5% CO
2
).
The control splenocytes were cultured for the same period of
time without mitogens.
4.12. Quantification of monoamines and metabolites
The frozen PFCs, hippocampus, and striata were thawed and
homogenized with a micro-sonicator (Q125 sonicator; Qsonica,
Newton, CT, USA). The homogenized samples were then cen-
trifuged at 12,000 gfor 10 min at 4 1C. The supernatants were
filtered through a 0.22-mm polyvinylidene difluoride membrane
(4-mm syringe filter; Millex-GV; Millipore, Billerica, MA, USA).
Afterproperdilutionwetook20-μl samples to measure the
concentrations of monoamines and their metabolites by the
high-performance liquid chromatography-electrochemical
detection system (HPLC-ECD). The HPLC-ECD comprised a
micropump (CMA-100; CMA, Stockholm, Sweden), an online
injector (CMA-160), a Microtech LC-pump (Microtech Scientific,
Sunnyvale, CA, USA), a BAS-4C electrochemical detector (Bioa-
nalytical Systems, Inc., West Lafeyette, IN, USA), and a reversed-
phase column (Kinetex C
18
,2.6μm, 100 2.1 mm I.D.; Phenom-
enex, Torrance, CA, USA) as previously described (Cheng et al.,
2000). The potential for the glassy carbon working electrode was
set at þ650 mV with respect to an Ag/AgCl reference electrode at
room temperature (25 1C). The mobile phase containing 0.1 M
NaH
2
PO
4
, 8% methanol, 0.74 mM SOS (1-octanesulfonic acid,
sodium salt), 0.03 mM EDTA, and 2 mM KCl was adjusted to pH
3.74 with H
3
PO
4
.Dilutedfiltrates were then injected (20 μL) into
the chromatographic system at a flow rate of 0.2 mL/min.
Concentrations of DA, 3,4-dihydroxyphenylacetic acid (DOPAC),
homovanillic acid (HVA), 5-HT, and 5-hydroxyindoleacetic acid
(5-HIAA) in the samples were interpolated using standards
(Sigma-Aldrich, St. Louis, MO, USA) ranging from 1 to 100 ng/mL.
4.13. Statistical analysis
All data were expressed as mean7SEM. Differences between
groups were analyzed by one-way or two-way analysis of
variance (ANOVA) with Bonferroni’s post-test or two-tailed t-
test when appropriate.
brain research 1631 (2016) 1–1210
Contributions
WSL, YCJ, and YCT designed the study. WSL, YCJ, YCW, and
HPT performed the experiments. WSL, SW, and YCJ analyzed
and interpreted the data. YWL, SW, WSL, CCW, YCJ, and YCT
wrote the manuscript.
Disclosure
The authors have declared no conflict of interest related to
this study.
Acknowledgments
We thank Prof. Tung-Hu Tsai for providing the HPLC-ECD
system, Ko-Fan Lu and Heng-Chun Lin for contributing part
of the work, and Dr. Wang-Tso Lee and Dr. Cheng-Jee Hong
for helpful discussions. This work was supported by the
Academic Technology Development Program (101-EC-17-A-
17-S1-197) of the Ministry of Economic Affairs, Republic of
China. The funding source had no contribution to the study
design, collection, analysis, interpretation of the data, or
writing of the report for publication.
Appendix A. Supplementary material
Supplementary data associated with this article can be found
in the online version at http://dx.doi.org/10.1016/j.brainres.
2015.11.018.
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