brain research 1490 (2013) 193201

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Research Report

Interleukin-1b inhibits the differentiation of hippocampal

neural precursor cells into serotonergic neurons

Kun Zhanga, Haiyun Xua,b, Longlong Caoa, Kangsheng Lic, Qingjun Huanga,n
a
Mental Health Center, Shantou University Medical College, Shantou 515065, PR China
b
Department of Anatomy, Shantou University Medical College, Shantou 515041, PR China
c
Department of Microbiology and Immunology, Shantou University Medical College, Shantou 515041, PR China

art i cle i nfo ab st rac t

Article history: Interleukin-1 beta (IL-1b) is one of pro-inflammatory cytokines. Recent studies have shown that
Accepted 12 October 2012 IL-1b impairs hippocampal neurogenesis, mediates proliferation and differentiation of multi-
Available online 22 October 2012 potent neural precursor cells (NPCs), and exerts effects of anti-proliferation, anti-neurogenesis,

Keywords: and pro-gliogenesis on embryonic hippocampal NPCs. The aim of this study was to examine

IL-1b the effect of IL-1b on the differentiation of hippocampal NPCs into functional serotonergic

Neural precursor cell neurons, which play important roles in the pathophysiology and treatment of depression.

Differentiation Hippocampal NPCs were prepared from the hippocampus of neonatal rats (within 24 h after

Serotonin birth). After three passages and phenotyping, hippocampal NPCs were cultured in a differ-

Hippocampus entiating medium with various concentrations (5, 10, and 20 ng/mL) of IL-1b for 7 days. At the
endpoint, the serotonergic differentiation of hippocampal NPCs in IL-1b-treated cultures
decreased in a dose-dependent manner and this effect was blocked by IL-1ra, an IL-1 receptor
antagonist capable of blocking the effects of IL-1 by binding to the same receptor (IL-1R1)
without triggering signaling; serotonin in the lysate of the differentiated hippocampal NPCs
decreased in IL-1b-treated cultures; and levels of Bcl-2 and phosphorylated extracellular-
regulated kinase (pERK) were also lower in differentiated hippocampal NPCs with IL-1b
treatment. These results support the hypothesis that IL-1b is an important factor in the
stress-associated neuropathology and psychopathology and has relevance to the treatment of
depressive symptoms in patients with depression.
& 2012 Elsevier B.V. All rights reserved.

1. Introduction cells in the periphery as well as glia and neurons within the
brain (Dinarello, 1996). The action of IL-1 involves various
Interleukin-1 (IL-1) is a prototypal cytokine with pleotropic ligands and receptors: IL-1a and IL-1b activate the IL-1
effects. It comes from many types of cells including immune receptor type I (IL-1RI) (Sims et al., 1993); whereas IL-1

Abbreviations: 5-HT, serotonin; bFGF, basic fibroblast growth factor; DIV, days in vitro; DMEM, Dulbeccos modified eagles
medium; DMSO, dimethyl sulphoxide; EGF, epidermal growth factor; ERK, extracellular-regulated kinase; FBS, fetal bovine serum;
GFAP, glial fibrillary acidic protein; HPA, hypothalamic pituitary-adrenal axis; IL-1, interleukin-1; IL-1b, interleukin-1 beta;
IL-1RI, IL-1 receptor type I; IL-1ra, IL-1 receptor antagonist; MAPK, mitogen-activated protein kinase; NFkB, nuclear factor kappa B;
NPC, neural precursor cell; pERK, phosphorylated ERK
n
Corresponding author. Fax: 86 754 88900728.
E-mail address: huangqj@stumhc.cn (Q. Huang).

0006-8993/$ - see front matter & 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.brainres.2012.10.025
194 brain research 1490 (2013) 193201
receptor antagonist (IL-1ra) serves to block the effects of IL-1
by binding to the same receptor (IL-1R1) without triggering
signaling (Seckinger et al., 1987). IL-1 signaling is mediated by
multiple pathways including the mitogen-activated protein
kinase (MAPK) and nuclear factor kappa B (NFkB) cascades,
translocation of transcription factors into the nucleus, and
transcription of immediate-early genes like c-jun and c-fos
(McCulloch and Downey, 2006).
IL-1 is the first cytokine that has been associated with
modulation of neuroendocrine systems, particularly the
hypothalamic pituitary-adrenal axis (HPA) (Berkenbosch
et al., 1987; Bernton et al., 1987; Sapolsky et al., 1987).
Production of brain IL-1 is an important link in stress-
induced activation of the HPA axis and secretion of gluco-
corticoids, which mediate the effects of stress on memory
functioning and neural plasticity. Moreover, IL-1 signaling
and the resultant glucocorticoid secretion mediate the devel-
opment of depressive symptoms associated with exposure to
acute and chronic stressors, at least partly via suppressing
hippocampal neurogenesis (Goshen and Yirmiya, 2009).
These previous findings suggest that IL-1 is a critical med-
iator of adaptive stress responses as well as stress-associated
neuropathology and psychopathology.
There is an accumulating array of data linking IL-1b up
with the stress-associated neuropathology and psycho-
pathology. First, IL-1b increased following various stressors
in animals and humans (Goshen and Yirmiya, 2009); second,
depressed mood was associated with serum elevations of
IL-1b in subjects without a psychiatric history (van den
Biggelaar et al., 2007); third, higher concentrations of IL-1b
inhibited learning, memory and long-term potentiation
(Depino et al., 2004; Nolan et al., 2005; Yirmiya et al., 2002);
fourth, levels of depression, anxiety, and memory impair-
ment in human volunteers were positively correlated with
serum IL-1b (Yirmiya et al., 2000). Moreover, hippocampal
IL-1b-mediated cognitive impairment was associated with
Fig. 1 Neural precursor cells in neurospheres. (A) Typical neurospheres at passage 2. (B) and (C) Immunofluorescence
images of neurospheres showing III-Tubulin (B) and GFAP positive (C) staining. (D) and (E) A neurosphere showing Nestin
positive staining (D) and nuclei of all cells identified by DAPI (E). (F) The merged image of (D) and (E).
stress (Goshen and Yirmiya, 2009; Vereker et al., 2001) and
depression (Goshen et al., 2007).
The hippocampus has been linked with some psychiatric
disorders including clinical depression. Stem cells have been
shown to reside in this brain region and to differentiate under
a variety of conditions into neural cells. Recent studies have
shown that high levels of IL-1b impairs hippocampal neuro-
genesis in aged mice (Kuzumaki et al., 2010), mediates
proliferation and differentiation of multipotent neural pre-
cursor cells (NPCs) (Wang et al., 2007), and exerts effects of
anti-proliferation, anti-neurogenesis and pro-gliogenesis on
embryonic hippocampal NPCs (Green et al., 2012).
Although effects of IL-1b on the proliferation and differentia-
tion of hippocampal NPCs have been studied, little is known
about the influence of IL-1b on embryonic hippocampal NPC
lineage restriction and differentiation. The aim of this study was
to examine the effect of this pro-inflammatory cytokine on the
differentiation of embryonic hippocampal NPCs into functional
serotonergic neurons, which play important roles in the patho-
physiology and treatment of depression (Jans et al., 2007; Mann,
1999). IL-1b in the present study was shown to inhibit the
differentiation of embryonic hippocampal NPCs into serotonergic
neurons in a dose-dependent manner and this effect was
blocked by IL-1ra. In the differentiated hippocampal NPCs,
IL-1b treatment decreased levels of serotonin (5-HT), which
was accompanied with lower Bcl-2 and phosphorylated
extracellular-regulated kinase (pERK).
2. Results
2.1. IL-1b inhibits cell proliferation and decreases the
survival rate of hippocampal NPCs
IL-1b exposure has been shown to decrease the total cells of
cultured NPCs, inhibit cell proliferation and reduce viability
 brain research 1490 (2013) 193201 195

120
100

(% of controls)
*

Total cells
80 **
**
60
40 y = -1.9543x + 95.6

20
0
0 5 10 15 20
IL-1 (ng/mL)

120 120
BrdU-positive cells

Cell viability (% of
100 100
(% of controls)

* * *
** *

controls)
80 80
y = -0.8179x + 96.863
60 y = -1.0901x + 97.231 60
40 40
20 20
0 0
0 5 10 15 20 0 5 10 15 20
IL-1 (ng/mL) IL-1 (ng/mL)

Fig. 2 Effects of IL-1b on cell proliferation and viability of hippocampal NPCs. (A) IL- IL-1b decreased the total cells in
hippocampal NPCs in a dose dependant manner. (B) IL- IL-1b reduced the BrdU incorporation rate of hippocampal NPCs. (C)
IL- IL-1b reduced the cell viability of hippocampal NPCs. Data were normalized by taking the values of controls in the absence
of IL-1b as the standards (100%) and transferring those of the IL-1b treated cultures into the percentages of the standards.
Data were expressed as mean7S.E.M. npo0.05, nnpo0.01, compared to controls.

in previous studies (Green et al., 2012; Wang et al., 2007). To
compare the contributions of the latter two to the decrease in
total cells, we normalized all three sets of data in relative
numbers (percentages of controls without exposure to IL-1b)
as shown in Fig. 2. Of all three sets of data, the total cells
decreased most significantly with IL-1b increase. Accordingly,
the linear model of this data set had a biggest slope (in
absolute value) (Fig. 2A). This decrease in total cells was due
to the inhibiting effects of IL-1b on cell proliferation and
viability of hippocampal NPCs indicated by the observation
that these two indices changed as functions of IL-1b concen-
tration. More specifically, the slope of linear model of cell
proliferation data (Fig. 2B) had a bigger absolute value
compared to that of the cell viability data (Fig. 2C), suggesting
that the former made a bigger contribution to the total cell
decrease than the latter did.
Then the percentage composition of MAP-2/DAPI cells, of
5-HT/DAPI cells, and of 5-HT/MAP-2 cells were calculated.
One-way ANOVA was performed with these quantitative data
followed by post-hoc comparisons. A significant effect of IL-1b
on the percentage composition of MAP-2/DAPI positive cells
(F(3, 44) 12.65, po0.001; n 12/condition) was found. Post-hoc
comparisons showed that both 10 and 20 ng/mL IL-1b sig-
nificantly decreased the percentage composition of MAP-2/
DAPI positive cells compared to that in the control group
without IL-1b (Fig. 4A). A similar inhibiting effect of IL-1b on
the percentage composition of 5-HT/DAPI positive cells was
also found (Fig. 4B). More significantly, the percentage com-
position of 5-HT/MAP-2 positive cells was decreased by IL-1b
treatment. The linear model of this data set showed a biggest
slope in its absolute value (Fig. 4C) compared to those of the
other two linear models (Fig. 4A and B).

2.2. IL-1b inhibits the differentiation of hippocampal 2.3. IL-1ra blocks the inhibiting effect of IL-1b on the
NPCs into 5-HT neurons differentiation of hippocampal NPCs into 5-HT neurons

IL-1b at various concentrations (5, 10, 20 ng/mL) was applied to
hippocampal NPCs in serum free differentiating medium for 7
DIV. At the end, cultured cells were stained by means of double
immunofluorescence staining with primary antibodies to MAP-2
and 5-HT. All cells were counterstained with DAPI solution to
identify the nuclei of cells. As shown in Fig. 3, cultures treated
with IL-1b displayed fewer MAP-2 and 5-HT positive neurons. In
addition, 10 and 20 ng/mL IL-1b seemed to cause apoptosis in the
cultured cells indicated by the condensed nuclei and cell
clumping.
To quantify the inhibiting effect of IL-1b on the neuronal
differentiation of hippocampal NPCs, MAP-2 and 5-HT posi-
tive cells, as well as the number of all nuclei, were counted.
To confirm the effect of IL-1b on the differentiation of hippo-
campal NPCs, IL-1ra was applied, at the concentrations of 5, 10,
and 20 ng/mL, to the cultures in the presence of 10 ng/mL IL-1b.
IL-1ra is known to block the effects of IL-1 by binding to the same
IL-1R1 receptor without triggering signaling (Seckinger et al.,
1987). As expected, IL-1ra blocked the inhibiting effect of IL-1b on
the differentiation of hippocampal NPCs into 5-HT positive
neurons. One-way ANOVA with the quantitative data of the
groups of 5, 10, and 20 ng/mL IL-1ra, as well as the group treated
with IL-1b (10 ng/mL) but without IL-1ra, indicated a significant
effect of IL-1ra on the effect of IL-1b (F(3, 44) 11.49, po0.001;
n 12/condition). post-hoc comparisons showed that both 10 and
20 ng/mL IL-1ra significantly blocked the inhibiting effect of IL-1b
196 brain research 1490 (2013) 193201

Fig. 3 Double immunofluorescence staining of differentiated hippocampal NPCs in the absence or presence of various
concentrations of IL-1b. The first row shows 5-HT and MAP-2 positive neurons, nuclei of all cells identified by DAPI, and the
merged image of them in the absence of IL-1b. The second row shows 5-HT and MAP-2 positive neurons, nuclei of all cells
identified by DAPI, and the merged image of them in the presence of 5 ng/mL IL-1b. The third row shows 5-HT and MAP-2
positive neurons, nuclei of all cells identified by DAPI, and the merged image of them in the presence of 10 ng/mL IL-1b. The
fourth row shows 5-HT and MAP-2 positive neurons, nuclei of all cells identified by DAPI, and the merged image of them in
the presence of 20 ng/mL IL-1b. The bars in all images are equal to 50 lm.

on the percentage composition of 5-HT/MAP-2 positive cells.
20 ng/mL IL-1ra almost completely blocked the effect of IL-1b
(Fig. 4D). However, IL-1ra alone had no effect on the differentia-
tion of hippocampal NPCs as no difference was found when
various concentrations (5, 10, 20, 40, and 80 ng/mL) of this ligand
were applied to cultured NPCs for 7 DIV under differentiation
conditions (data not shown).
2.4. IL-1b decreases levels of 5-HT in differentiated
hippocampal NPCs
Hippocampal NPCs-derived neurons have been shown to
release 5-HT (Chen et al., 2007). This previous finding encour-
aged us to measure 5-HT concentrations in the differentiated
hippocampal NPCs and to examine the effect of IL-1b on
levels of this neurotransmitter in differentiated cells.
Enzyme-linked immunosorbent assay (ELISA) was performed
with the lysate of the differentiated hippocampal NPCs. The
results showed that IL-1b effectively decreased levels of 5-HT
in the lysate of the differentiated hippocampal NPCs. One-
way ANOVA showed a significant effect of IL-1b (F(3, 12) 4.57,
po0.01; the experiment with four groups was repeated four
times) on levels of 5-HT. post-hoc comparisons showed that
both 10 and 20 ng/mL IL-1b caused moderate but significant
decreases in 5-HT of cultures (Fig. 5A). These moderate
decreases in 5-HT likely resulted from the reduced number
of 5-HT neurons in the IL-1b treated cultures. To prove
this claim, we normalized the data of 5-HT and 5-HT
positive neurons and compared the two curves in a same
figure (Fig. 5B).
2.5. IL-1b decreases levels of Bcl-2 and pERK in
differentiated hippocampal NPCs
In a recent study, the differentiation of neural stem cells into
serotonergic neurons was associated with the expression of
 brain research 1490 (2013) 193201 197

50 20

MAP-2/DAPI cells (%)

5-HT/DAPI cells (%)

40
15
30 * *
10
20
y = -0.5886x + 31.4 5 *
10 *
y = -0.5371x + 12.2
0 0
0 5 10 15 20 0 5 10 15 20
IL-1 (ng/mL) IL-1 (ng/mL)

50 50

5-HT/MAP-2 cells (%)

**
HT/MAP5-2 cells (%)

40 * 40
*
30 30
20 ** ** 20 *

10 y = -1.4171x + 39.4
10
0 0
0 5 10 15 20 0 5 10 15 20
IL-1 (ng/mL) IL-1ra (ng/mL)

Fig. 4 The inhibiting effects of IL-1b on neuronal differentiation of hippocampal NPCs. (A) IL-1b decreases the ratio of MAP-
2/DAPI cells in differentiated hippocampal NPCs. (B) IL-1b decreases the ratio of 5-HT/DAPI cells in differentiated
hippocampal NPCs. (C) IL-1b decreases the ratio of 5-HT/MAP-2 cells in differentiated hippocampal NPCs. (D) IL-1ra blocks
the inhibiting effect of IL-1b on the serotonergic differentiation of hippocampal NPCs. Data were expressed as mean7S.E.M.
n
po0.05, nnpo0.01, compared to control groups.

130 120
neurons (% of controls)

*
Changes in 5-HT &

110 * 100
numbers of 5-HT
5-HT (pg/mL)

5-HT levels
90 80 y = -0.7081x + 99.565

70
60
50
40 Numbers of
30 y = -2.4585x + 79.07
5-HT
10 20
neurons
0 5 10 15 20 0
IL-1 (ng/mL) 0 5 10 15 20
IL-1 (ng/mL)

Fig. 5 IL-1b decreases levels of 5-HT in differentiated hippocampal NPCs. 5-HT levels in differentiated hippocampal NPCs
were measured by means of ELISA. (A) Levels of 5-HT in the lysate of the differentiated hippocampal NPCs treated with 10
and 20 ng/mL IL-1b were decreased. (B) The IL-1b induced decrease in 5-HT levels was not parallel to the decrease in
numbers of 5-HT positive neurons. Data were expressed as mean7S.E.M. npo0.05, compared to the controls in the absence
of IL-1b.

the anti-apoptotic protein Bcl-2 via ERK pathway (Chiou et al., of phosphorylated ERK (pERK) in the differentiated hippo-
2006). Inspired by this significant finding, we examined campal NPCs (Fig. 6B).
effects of IL-1b on the expression of Bcl-2 protein in differ-
entiated hippocampal NPCs and on the phosphorylation of
ERK in these cells by performing ELISA. As shown in Fig. 6, IL- 3. Discussion
1b decreased levels of Bcl-2 in differentiated hippocampal
NPCs in a dose-dependent manner (Fig. 6A); one-way ANOVA In accordance with previous studies (Green et al., 2012; Wang
indicated a significant effect of IL-1b on levels of Bcl-2 (F(3, 12) et al., 2007), IL-1b treatments applied in the present study
7.37, po0.01) and post-hoc comparisons revealed that both significantly decreased the number of total cells in cultured
10 and 20 ng/mL IL-1b significantly decreased levels of Bcl-2 hippocampal NPCs (Fig. 2A). Furthermore, we compared the
in differentiated hippocampal NPCs compared to the controls contributions of decreased cell proliferation and cell viability
in the absence of IL-1b. Similarly, this cytokine reduced levels to the decrease in total cells. The results suggest that the
198 brain research 1490 (2013) 193201
120
*
100
Bcl-2 (pg/mL)
80
60
40
20
0 0
0 5 10 15
IL-1 (ng/mL)
Fig. 6 IL-1b decreases levels of Bcl-2 and pERK in differentiated hippocampal NPCs. Levels of Bcl-2 and pERK in
differentiated hippocampal NPCs were measured by means of ELISA. (A) 10 and 20 ng/mL IL-1b decreased levels of Bcl-2
in differentiated hippocampal NPCsr. (B) All IL-1b treatments decreased levels of pERK in differentiated hippocampal NPCs.
Data were expressed as mean7S.E.M. npo0.05, nnpo0.01, compared to the controls in the absence of IL-1b.
inhibiting effect of IL-1b on cell proliferation (Fig. 2B) made a
bigger contribution to the total cell decrease than its toxic
effect on cell viability (Fig. 2C) did. Indeed, all IL-1b treat-
ments significantly inhibited the cell proliferation of the
cultured cells; whereas only 10 and 20 ng/mL IL-1b decreased
the cell viability. These results are in line with the morpho-
logical observation on the immunoflurescence staining of
cultured cells, in which condensed nuclei were more fre-
quently seen in cultures treated with 10 and 20 ng/mL IL-1b
(Fig. 3), suggesting higher percentages of apoptosis. Similar to
our finding, apoptosis of NPCs was induced by IL-1b in a
previous study (Wang et al., 2007).
IL-1b exposure for 7 days inhibited the differentiation of
hippocampal NPCs into MAP-2 positive neurons (Fig. 3 and
Fig. 4A). This finding is in accordance with a previous report
that a significant reduction in the percentage composition of
newly-born neurons and post-mitotic neurons was observed
when embryonic rat hippocampal NPCs differentiated in the
presence of IL-1b for 7 days (Green et al., 2012). Furthermore,
the present study for the first time found that the IL-1b
exposure more severely inhibited the differentiation of hip-
pocampal NPCs into 5-HT neurons (Fig. 3 and Fig. 4C),
suggesting that serotonergic differentiation from hippocam-
pal NPCs is more vulnerable to the IL-1b treatment than the
differentiation of hippocampal NPCs into MAP-2 neurons. An
inference from this relatively selective action of IL-1b on
serotonergic differentiation from hippocampal NPCs is: high
levels of IL-1b under stressful conditions may predominantly
inhibit the in vivo differentiation of hippocampal NPCs into
serotonergic neurons. Via this action, increased IL-1b in the
brain may decrease the number of serotonergic neurons in
the hippocampus thus play an important role in the stress-
associated neuropathology and psychopathology.
Proliferating and differentiated hippocampal NPCs have
been demonstrated to express IL-1R1 (Green et al., 2012;
Wang et al., 2007). Therefore, we assumed that the inhibiting
effect of IL-1b on serotonergic differentiation of hippocampal
NPCs shown in the present study resulted from the specific
action of this ligand on IL-1R1. This assumption was
approved true by our findings that IL-1ra, another ligand
capable of binding the same IL-1R1 receptor without triggering
signaling (Seckinger et al., 1987), significantly blocked the
inhibiting effect of IL-1b on the percentage composition of
160
140
p-ERK (pg/mL)
* 120
*
100 **
80 **
60
40
20
20 0 5 10 15 20
IL-1 (ng/mL)
5-HT/MAP-2 positive cells (Fig. 4D); whereas no significant effect
was observed when various concentrations of this ligand alone
were applied to cultured NPCs for 7 days under differentiation
conditions (data not shown).
The moderate decreases in 5-HT by 10 and 20 ng/mL IL-1b
are believed to result from the reduced number of 5-HT
neurons in the IL-1b treated cultures. This claim was proved
true by the comparison of the two sets of data of the
decreases in 5-HT levels and in numbers of 5-HT positive
neurons as shown in Fig. 5B. The liner model of 5-HT neurons
showed a bigger slope (in absolute value) than that of 5-HT
levels, suggesting the existence of a compensating mechan-
ism in the existing 5-HT neurons for the decrease in 5-HT
positive neurons in the cultures treated with IL-1b. This
phenomenon ruled out the possibility that IL-1b inhibited
the synthesis of 5-HT in the existing 5-HT neurons in the
cultures. Further studies should measure the activities of
tryptophan hydroxylase, a rate limiting biosynthetic enzyme
in the serotonin pathway and regulating levels of serotonin.
The wider implications of the finding that IL-1b exposure
for 7 days decreased levels of 5-HT in the differentiated cells
should not be ignored given that 5-HT is a central neuro-
transmitter and plays important roles in the pathophysiology
and treatment of depression. Indeed, IL-1b was elevated in
subjects with major depressive disorders; antidepressant
treatments reduced levels of IL-1b while it improved depres-
sive symptoms (Hannestad et al., 2011; Himmerich et al.,
2010). More relevantly, IL-1b caused fluoxetine resistance in
an animal model of depression (Pineda et al., 2012); the same
antidepressant treatment stimulated serotonergic differen-
tiation of neural stem cells in vitro (Chen et al., 2007).
Exposure to 10 and 20 ng/mL IL-1b for 7 days decreased
levels of Bcl-2 in differentiated hippocampal NPCs (Fig. 6A).
This decrease in Bcl-2 was accompanied with the lower cell
densities in IL-1b treated cultures, suggesting an association
between the two indices. One possibility is that fewer cells
produce less Bcl-2; another possibility is that lower levels of
Bcl-2 lead to lower density of viable cells. Although further
studies are necessary to determine a certain relationship
between lower levels of Bcl-2 and fewer cells in IL-1b treated
cultures, the existence of this association reminded us of the
observation that Bcl-2 triggered the differentiation of the
embryonic stem cells into mature neurons (Rolletschek
 brain research 1490 (2013) 193201
et al., 2001). In addition, another member of the Bcl-2 family,
Bcl-xL stimulated the differentiation of the embryonic cells of
a mouse into both the dopaminergic and serotonergic neu-
rons (Shim et al., 2004). Together, all these data suggest an
important role of Bcl-2 in the neuronal differentiation of
embryonic stem cells and hippocampal NPCs.
While inhibiting the serotonergic differentiation and redu-
cing Bcl-2 levels, exposure to IL-1b for 7 days also decreased
pERK in differentiated hippocampal NPCs (Fig. 6B). This
finding is in line with the report showing that IL-1b increase
was coupled with down-regulation of ERK in cortical tissue
prepared from aged rats (Maher et al., 2004). More relevantly,
moclobemide, an antidepressant drug with the capacity of
increasing levels of 5-HT and norepinephrine, induced neural
stem cell differentiation into serotonergic neurons through
activating the phosphorylation of ERK and increasing Bcl-2
expression (Chiou et al., 2006; Choi et al., 2003; Hayley et al.,
2005). Although future studies are necessary to specify the
target cells in which IL-1b inhibits the phosphorylation of
ERK, our data and results of the abovementioned previous
studies suggest an interpretation: increased IL-1b inhibits
phosphorylation of ERK in the cells and/or down-regulates
Bcl-2 expression, which, in turn, decreases serotonergic
differentiation of hippocampal NPCs. This explanation is of
help in understanding roles of IL-1b in stress-associated
neuropathology and psychopathology. Specifically, under
stressful conditions, enhanced IL-1b, via the abovementioned
pathway, may finally impair the functions of serotonergic
system in the brain thus inducing or aggravating depressive
symptoms in human subjects. Following this theory, normal-
izing IL-1b levels should be one of principles in treating
stress-associated neuropathology and psychopathology.
Indeed, a recent human study showed that antidepressant
therapy decreased IL-1b serum levels while improved the
psychopathological status of patients suffering from a
depressive episode (Himmerich et al., 2010). In a more recent
study IL-1b caused fluoxetine resistance in an animal model
of depression (Pineda et al., 2012).
In sum, IL-1b exposure for 7 days reduced cell proliferation
and cell viability, inhibited serotonergic differentiation of
embryonic hippocampal NPCs, which effect was blocked by
IL-1ra. The levels of 5-HT in hippocampal NPCs were also
decreased by the same treatment. In the meanwhile, IL-1b
decreased levels of Bcl-2 and pERK in differentiated hippo-
campal NPCs. These results support the hypothesis that IL-1b
is an important factor in the stress-associated neuropathology
and psychopathology and has relevance to the treatment of
depressive symptoms in patients with depression.
4. Experimental procedures
4.1. Preparation and treatment of rat hippocampal NPC
cultures
All animal procedures applied in this study were in accor-
dance with the guideline set up by the Animal Care and Use
Committee of Shantou University Medical College and
approved by the committee. To save animals and rule out
possible influence of sex hormones on hippocampal NPCs,
199
neonatal, rather than adult rats, were used in this study. The
protocols for the preparation and culture of rat hippocampal
NPCs by Wan et al. (2006) were followed. Briefly, neonatal
(Sprague-Dawley) rats (within 24 h after birth) were decapi-
tated and bilateral hippocampi were dissected out. The
hippocampal tissue was then dissociated by gentle mechan-
ical pipetting through fire-polished Pasteur pipettes. The
resultant suspensions were filtered through a nylon mesh
of 70 mm followed by the incubation in DMEM/F12 (prolifera-
tion) medium supplemented with 10% fetal bovine serum
(FBS) (all from Gibco) for 30 min at 37 1C, 5% CO2. The
suspension of the cultures was collected and the cells in it
were seeded at 106/ml into uncoated culture flasks (75 cm2)
with the same DMEM/F12 medium and incubated for one day
(1 DIV). Then the most of suspension was discarded and the
same volume of DMEM/F12 medium with 10% FBS was added.
In this proliferation medium, the incubation continued for
another 3 days at 37 1C, 5% CO2. At the end of this culturing
period, typical neurospheres were found in the culture under
a microscopy (Fig. 1A). After a centrifugation of 1000 rpm/min
for 5 min at 22 1C and discarding the suspension, the cultured
cells were rinsed with PBS (pH 7.4; 2  5 min), then the neural
stem cell culture medium was added, in which DMEM/F12
was supplemented with 2% B27 (Gibco), basic fibroblast
growth factor (bFGF, 20 ng/mL, Peprotech), and human
recombinant epidermal growth factor (EGF, 20 ng/mL, Pepro-
tech). During the following 7 DIV, an half of the medium was
replaced with fresh one each 3- or 4 DIV, and for each 7 DIV
cultured cells were passaged by dissociating neurospheres
and transferring dissociated cells into fresh medium in new
flasks at 1  106/ml. At the end, the neurospheres showed bIII-
Tubulin- (Fig. 1B) and GFAP-positive (Fig. 1C) staining. Of all
cells in the neurospheres, more than 90% are nestin positive
(Fig. 1DF).
After cultured for 21 DIV as described above, the neuro-
spheres were incubated in DMEM/F12 with trypsin (0.25%)
and EDTA (0.02%) for 35 min at 37 1C. Then the dissociated
cells were seeded at 106/ml into poly-D-lysine-coated 48-well
plates and cultured for 7 DIV in the differentiating medium of
DMEM/F122% B27 (without FBS, bFGF, and EGF) with various
concentrations (5, 10, 20 ng/mL) of IL-1b. These doses of IL-1b
were in the range of that applied in previous studies (Green
et al., 2012; Wang et al., 2007), which showed significant
effects on brain NPCs of neonatal rats. During this period the
medium was replaced on third and fifth day; into the fresh
medium the same concentrations of IL-1b were added. At the
end of this culture period, the effects of IL-1b on the
differentiation of hippocampal NPCs and levels of 5-HT, Bcl-2
and pERK were examined as described later. To examine the
blocking effect of IL-1ra on actions of IL-1b, IL-1ra was applied
at the final concentrations of 5, 10, and 20 ng/mL in the
presence of IL-1b.
4.2. Analysis of cell proliferation
Hippocampal NPCs were seeded at a density of 4  105 in
poly-D-lysine-coated 6-well plates with the proliferation
medium and allowed to proliferate for 3 DIV. IL-1b was added
on the day of seeding at various concentrations (5, 10, and
20 ng/mL). 5-bromo-20 -deoxyuridine (BrdU, 0.2 uM) (Sigma)
200 brain research 1490 (2013) 193201
was added after 3 DIV for the final 24 h of culture. At the end,
BrdU positive cells were labeled immunocytochemically
using a primary mono-clonal antibody (Chemicon; 1:800)
and a FITC conjugated secondary antibody (Jackson Immuno
Research Lab; 1:50). Cell nuclei were identified with DAPI.
4.3. Immunofluorescence staining
Cultured cells were washed with PBS and fixed with 4%
paraformaldehyde in 0.01 M PBS (pH 7.4) for 30 min at room
temperature. After washing with PBS (3  5 min) and 0.3%
Triton X-100 in PBS (PBS-T; 10 min), cells were incubated with
the mouse anti-nestin (1:100; Millipore), rabbit anti-MAP-2
(1:50; Cell Signal), mouse anti-5-HT (1:50; Abcam), mouse
anti-bIII-tubulin (1:200; Promega), or rabbit anti-GFAP (1:200;
Dako) in PBS containing 1% BSA overnight at 4 1C. Then cells
were washed in PBS (3  5 min) and incubated for 1 h at room
temperature with the secondary antibodies fluorescein-
conjugated goat anti-rabbit (FITC;1:100; Jackson Immuno
Research Lab) and rhodamine-conjugated goat anti-mouse
(RITC; 1:50; Jackson Immuno Research Lab). Cells were coun-
terstained with DAPI solution to identify the nuclei. Primary
antiserum omission controls and normal mouse and goat
serum controls were used to confirm the specificity of the
immunofluorescence labeling.
4.4. Cells counts and analysis
For each treatment condition, fluorescent images of immu-
nopositive cells were viewed under a fluorescence micro-
scope (Nikon Elipse 80i; Japan) equipped with a CCD/Nikon
digital camera DXM1200F and the software ACT-1 (Nikon
ACT-1, version 2.70), with which photographs were taken at
 40 or  20 magnification and saved in a HP computer. The
number of total cells, and the number of each cell phenotype
were counted in 5 randomly-chosen fields of view per cover-
slip. For each condition, cells from 4 cover-slips were stained
and counted, and each experiment was repeated 3 times.
4.5. MTT assay
After 6 DIV under proliferation conditions, in the presence of
various concentrations (5, 10, and 20 ng/mL) of IL-1b, neuro-
spheres were dissociated and cells of them were seeded at
105 cells in a 96-well plate, in the proliferation medium. One h
later, cells were incubated with 0.5 mg/mL Thiazolyl Blue
Tetrazolium Bromide (Sigma) on phenol-red free DMEM at
37 1C for 3 h under a humidified atmosphere containing 5%
CO2. After removing the solution, the cells were lysed in
dimethyl sulphoxide (DMSO; Sigma) and the lysates were
transferred to a fresh 96-well plate for the final absorbance
reading on a Tecan Sunrise spectrophotometer at an absor-
bance wavelength of 540 nm and a reference wavelength of
690 nm. Each experiment was repeated three times.
4.6. Enzyme-linked immunosorbent assay (ELISA)
The differentiated cells were treated with the radio immuno-
precipitation assay solution and the supernatant was collected.
Samples and standards (50 ml) at different concentrations were
added into the wells of the levels of an ELISA plate (R&D), and
incubated for 1 h at room temperature. After the plate was
washed three time with the wash buffer (5% Tween 20 in PBS),
primary antibody (100 ml) to 5-HT, Bcl-2, or pERK was added at a
certain concentration to all wells and incubated for 1 h at room
temperature. After three washes in wash buffer, peroxidase-
conjugated polyclonal secondary antibody (1: 5001000; Sigma)
was added and incubated for 1 h at room temperature. Once
again, wells were washed three times, then the chromogen
containing tetramethyl-benzidine (TMB, Sigma) and subsrate
(100 mM sodium accetae, 3 mM citric acid, 0.1% TMB in DMSO,
0.04% H2O2) was added and incubated at 37 1C for 15 min,
followed by addition of 10% H2SO4 to stop the reaction. The
plate was read at 450 nm using a Bio-Tek EL808 Ultra Microplate
Reader. Each individual sample was analyzed in triplicate.
4.7. Statistical analysis
The results were presented as the mean7S.E.M. Statistical
analysis was performed using the one-way analysis of var-
iance test followed by least significant difference test.
A difference was considered to be statistically significant
when po0.05.
Acknowledgments
This work was supported by a Grant (30770771) from the
National Science Foundation of PR China (QJH).
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