Brain Research Bulletin 120 (2016) 1–13

Contents lists available at ScienceDirect

Brain Research Bulletin

journal homepage: www.elsevier.com/locate/brainresbull

Research report

Developmental minocycline treatment reverses the effects of neonatal

immune activation on anxiety- and depression-like behaviors,
hippocampal inflammation, and HPA axis activity in adult mice
Jafar Majidi a , Morteza Kosari-Nasab a , Ali-Akbar Salari a,b,∗
a
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
b
Laboratory of Neuropsychopharmacology and Psychoneuroimmunology, Hayyan Research Institute, University of Tabriz, Tabriz, Iran

a r t i c l e i n f o a b s t r a c t

Article history: Neonatal infection is associated with increased lifetime risk for neuropsychiatric disorders including anx-
Received 28 August 2015 iety and depression, with evidence showing that dysregulation of the hypothalamic–pituitary–adrenal-
Received in revised form 22 October 2015 (HPA)-axis system may be partly responsible. Preclinical and clinical studies demonstrate that
Accepted 23 October 2015
minocycline exhibits antidepressant effects through inhibition of microglial activation and anti-
Available online 28 October 2015
inflammatory actions, and of interest is that recent studies suggest that minocycline alleviates the
behavioral abnormalities induced by early-life insults. The current study was designed to determine
Keywords:
if developmental minocycline treatment attenuates the neonatal immune activation-induced anxiety-
Neonatal infection
Brain development
and depression-like symptoms and HPA-axis-dysregulation later in life. To this end, neonatal mice were
Affective disorders treated to either lipopolysaccharide or saline on postnatal days (PND) 3–5, then dams during lacta-
Hippocampal inflammation tion (PND 6–20) and male offspring during adolescence (PND 21–40) received oral administration of
HPA-axis activity minocycline or water via regular drinking bottles. Anxiety- and depression-like behaviors, HPA-axis-
Proinflammatory cytokines reactivity (corticosterone), and hippocampal inflammation (TNF-␣ and IL-1␤) after exposure to stress
were evaluated. The results indicated that neonatal immune activation resulted in increased anxiety and
depression-like symptoms, HPA-axis-hyperactivity, and elevated the levels of TNF-␣ and IL-1␤ in the
hippocampus in response to stress in adulthood. Interestingly, developmental minocycline treatment
significantly reduced the abnormalities induced by neonatal inflammation in adult mice. In addition,
minocycline, regardless of postnatal inflammation, did not have any detrimental effects on the above
measured parameters. Considering that minocycline is currently under exploration as an alternative
or adjunctive therapy for reducing the symptoms of neurological disorders, our findings suggest that
minocycline during development can decrease the behavioral abnormalities induced by early life inflam-
mation in adulthood.
© 2015 Elsevier Inc. All rights reserved.

1. Introduction Toneatto and Nguyen, 2007). There is significant overlap between

these two psychological problems which negatively affect daily
Extensive research indicates that major depression and anxiety activities and quality of life influencing other types of psychi-
are among the most common psychiatric disorders which fre- atric disorders (Huppert et al., 2001; Mendlowicz and Stein, 2000).
quently cause significant functional impairment in humans (Fried Accumulating evidence suggest that neurological diseases such as
and Nesse, 2015; Löwe et al., 2008; Ravindran and Stein, 2010; autism, schizophrenia, and affective disorders can be programmed
by events such as stress and infection in early life (Boksa, 2010;
Cottrell and Seckl, 2009; Depino, 2015; Dong et al., 2015; Enayati
et al., 2012; Kinney et al., 2008). Although, the etiology of anxiety
Abbreviations: LPS, lipopolysaccharide; PND, postnatal day; HPA,
hypothalamic–pituitary–adrenal axis; IL, interleukin; TNF-␣, tumor necrosis
and depression remains unclear, recent studies by our group have
factor-alpha; CORT, corticosterone; ANOVA, analysis of variance; SEM, standard provided evidence that infection/inflammation during the early
error of the mean. stages of brain development including prenatal and early postna-
∗ Corresponding author at: Drug Applied Research Center, Tabriz University
tal periods may be significantly involved in programming of these
of Medical Sciences, P.O. Box 51656−65811, Tabriz, Iran. Tel.: +98 919 4099673;
disorders in later life (Babri et al., 2014a,b; Doosti et al., 2013;
fax: +98 411 3368208.
E-mail address: [email protected] (A.-A. Salari).
Enayati et al., 2012; Majidi-Zolbanin et al., 2013, 2014, 2015). For

http://dx.doi.org/10.1016/j.brainresbull.2015.10.009
0361-9230/© 2015 Elsevier Inc. All rights reserved.
2 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13

instance, we and others have developed an animal model of neona- anti-immobility effect of desipramine in the forced swim test
tal inflammation through lipopolysaccharide (LPS) administration in animals showing an antidepressant effect (Molina-Hernández
in postnatal days (PND) 3 and 5 in mice (Doosti et al., 2013; Majidi- et al., 2008). Moreover, minocycline reduces the expression of pro-
Zolbanin et al., 2013) and rats (Sominsky et al., 2012; Walker et al., inflammatory cytokines and depression-like behavior induced by
2004, 2012) leading to anxiety- and depressive-like behaviors, and LPS in mice (O’connor et al., 2009). Minocycline has been used to
persistent abnormalities in hypothalamic–pituitary–adrenal axis prevent or ameliorate white matter damage after inflammation
(HPA) axis function later in life. Dysregulation of HPA axis has a and hypoxic-ischemic injury in both adult and neonatal rats (Cai
crucial role in the etiology of depression and anxiety-related dis- et al., 2006; Fan et al., 2005a). Fan et al. (2005a,b) demonstrated
orders, both hypo and hyperactivity of the HPA-axis have indeed that minocycline treatment attenuates lipopolysaccharide-induced
been found to be associated with higher risk of depression (Stetler white matter injury in the neonatal rat brain on PND 5, proba-
and Miller, 2011; Tsigos and Chrousos, 2002). bly through inhibition of microglial activation. In other studies,
Neonatal LPS administration has been shown to activate minocycline treatment during lactation in Fmr1 KO mice, a mouse
microglia cells and increase pro-inflammatory cytokines in the model of Fragile X Syndrome, alleviated anxiety-like behavior in
brain (Fan et al., 2005a,b). With regard to the relationship between young mice (Bilousova et al., 2009; Dansie et al., 2013). Overall, the
neonatal immune activation and microglial activation, Sominsky neuroprotective effects of minocycline are assumed to be exerted
et al. (2012) demonstrated that LPS administration in PNDs 3 and through antioxidant free-radical scavenging, inhibition of caspase
5 results in increased anxiety-like behavior and microglial activa- expression and mitogen-activated protein kinases, and the sup-
tion in the hippocampus of adult rats. In support of these findings, pression of microglial and astroglial activation and proliferation
Walker et al. (2004, 2012) reported that immune activation with leading to reduced neuroinflammation and inhibition of apoptotic
LPS on PND 3 and 5 increases anxiety-like symptoms and levels neuronal loss (Cheng et al., 2015; Soczynska et al., 2012). Minocy-
of tumor necrosis factor-alpha (TNF-␣) and interleukin (IL)-1␤ in cline is clinically well tolerated and almost completely absorbed
the hippocampus of adult rats in response to stress (Walker et al., when taken orally and which is also able to pass from the mother
2010). Microglial activation and brain inflammatory cytokines have to the offspring through the breast milk (Lee et al., 2006; Lin et al.,
been implicated in mediating the effects of stress and both of them 2005; Luzi et al., 2009; Miyaoka et al., 2012). Since inflammation
are known as major triggers for depression (Kreisel et al., 2014; is thought to be one of the important player in the development
Miller et al., 2009; Raison et al., 2006). In addition, previous studies of psychiatric diseases, and developmental drug treatment is cur-
have shown a relationship between elevated TNF-␣ and IL-1␤ and rently receiving attention as a potential strategy for the treatment
pathophysiology of anxiety and major depression (Bayramgürler of neuropsychiatric disorders, we thought that minocycline could
et al., 2013; Dowlati et al., 2010; Goshen et al., 2008; Kaster et al., be considered a pharmacological candidate for the prevention of
2012; Krügel et al., 2013; Miller et al., 2009; Raison et al., 2006; affective-like behaviors induced by neonatal immune activation.
Rossi et al., 2012; Simen et al., 2006). Given that hippocampus plays
an important role in mediating anxiety- and depression-related 2. Materials and methods
behaviors in humans and rodents, this evidence suggests that the
neuropathology of anxiety and depression induced by neonatal 2.1. Subjects and ethics
immune activation might be closely associated with hippocampal
inflammation in adulthood. Adult male and female NMRI mice (10–11 weeks old) were
Emerging evidence from preclinical and clinical studies also obtained from the animal house of Razi Institute. Animals were
shows that the development of the therapeutic strategies against maintained under standard laboratory conditions on a 12:12 h
neuropsychiatric disorders with neurodevelopmental origin is an light/dark cycle (lights on at 08:00 AM) and controlled temperature
interesting topic for researchers. For example we reported that (23 ± 1 ◦ C). Food and water were also available ad libitum. All pro-
adolescent fluoxetine treatment can reduce the effects of early cedures were approved by the Research and Ethics Committee of
postnatal inflammation induced by LPS on anxiety and depression- Tabriz University of Medical Sciences, and conducted in accordance
like behaviors in offspring during adulthood (Doosti et al., 2013; with guidelines from the National Institutes of Health.
Majidi-Zolbanin et al., 2013). While the majority of studies in this
area of research originally tend to address how antidepressant
and antipsychotic drugs at different windows of brain develop- 2.2. Newborn mice
ment such as prenatal, neonatal and adolescent periods can reduce
psychiatric symptoms by regulating different neurotransmitter Following a 2-week period of acclimatization to the new ani-
systems in the brain (Dickerson et al., 2012; Doosti et al., 2013; mal housing room, to facilitate the mating, male and female mice
Ishiwata et al., 2005; Majidi-Zolbanin et al., 2013; Nagano et al., were kept together one-by-one in a cage. Female mice were visu-
2012; Rayen et al., 2011; Richtand et al., 2012), a few studies ally monitored daily for confirmation of pregnancy, when it was
have recently considered the possibilities and ideas about early confirmed the female mice were removed from the breeding cages
pharmacological intervention by minocycline, a second-generation and housed individually in standard cages. All pregnant animals
tetracycline, for reducing brain inflammatory processes (Fan et al., were allowed to have normal delivery and the first day of birth was
2005a,b Zhu et al., 2014a,b). In this context, a series of experi- considered as PND 0. One day after the birth, all litters were culled
ments indicate that minocycline can be a potential therapeutic drug to 4 male pups per mother. On the day 21, litters were weaned
for various neurological disorders, including major depression, by removal of the mother and then were housed based on the
anxiety, schizophrenia, Huntington’s disease, Parkinson’s disease, treatment condition. In order to prevent the possible confound-
ischemia (Garrido-Mesa et al., 2013; Soczynska et al., 2012). Find- ing factors of isolation housing, the offspring were kept in groups
ings from animal and human studies support the hypothesis that of 2 animals in the cages. Only one mouse per litter was used for
minocycline can be a potential multi-target agent for the treat- each of the experiments to avoid the litter-effect.
ment of major depression and anxiety-related disorders through
anti-inflammatory and microglial activities, and neuroprotective 2.3. Neonatal immune activation
actions including neurogenesis and anti-glutamate excitotoxicity
(Garrido-Mesa et al., 2013; Soczynska et al., 2012). It has been A timeline diagram of the experiments is shown in Fig. 1. The
shown that minocycline decreases immobility and enhances the dams were removed from their pups for approximately 5 min and
 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13 3

Fig. 1. Experimental design: There were four main experiments in this study, the experiments show the effects of neonatal immune activation alone or in combination with
minocycline treatment on anxiety- and depression-like behaviors, HPA axis function and stress-induced inflammatory cytokines (TNF-␣ and IL-1␤) in the hippocampus in
male mice in adulthood.

the pups were weighed and received subcutaneously (in the inter- 2.5. Behavioral testing
scapular region) injection of LPS (Escherichia coli 0111:B4, Sigma
Co., USA; 50 ␮g/kg) or vehicle solution (1 ml/kg) on the PNDs 3 and Behavioral assessments began at PND 70. The observers blind
5. Both dose and time of LPS treatment in newborn mice were cho- to the treatment recorded all parameters for each of the behavioral
sen based on our previous studies, where it has been shown to tests by using a stopwatch. In addition, all behavioral tests were
induce anxiety- and depression-like behaviors in adulthood (Doosti conducted in a quiet room during the light period (between 13:00
et al., 2013; Majidi-Zolbanin et al., 2013). LPS was dissolved in and 18:00 h) under illumination of 75 lux and the mice were kept
saline (0.9% NaCl) and injections were performed between 12:00 in the room for at least 1 h before the assessment. At the end of each
and 13:00 P.M. Each injection was performed through a needle test session, the arena was carefully cleaned with 70% ethanol and
(30-gauge) connected by polyethylene tubing to a 10-␮l Hamilton after test the cage was transported back to the colony room.
syringe. Neonate mice were returned to their housing immedi-
ately following saline or LPS administration. In order to perform
the behavioral tests, mice from both neonatal treatment conditions 2.5.1. Open field
were divided into 2 experiments (each experiment was only used The open field was conducted as previously described by our
for three behavioral tests, with a 10-days interval between each group (Amani et al., 2013). The open field apparatus consisted of a
test; N = 9/group). white wooden box (40 × 40 × 20 cm) with 16 squares (10 × 10 cm;
12 outer and 4 inner) which was directly illuminated by a 100 W
bulb placed 90 cm above the center of the apparatus floor. The test
period was initiated when a single mouse was placed in the middle
2.4. Minocycline treatment of the apparatus and allowed to move freely for 5 min. The inner
zone time in and entries (an entry was defined as all four paws)
Minocycline (30 mg/kg/day) was administered via drinking were recorded as indices of anxiety-like behavior.
water (the bottles were opaque to protect the drug from light) to the
nursing mothers (PND 6–20) or adolescent offspring (PND 21–40).
We chose this mode of delivery instead of invasive and stressful 2.5.2. Light-dark box
procedures resulting from repeated injections or oral gavages, to The light–dark box apparatus consisted of a white-black wooden
minimize the effect of repeated stress. This method of minocy- rectangular box (length 46 cm, width 27 cm, and height 30 cm),
cline administration has been previously reported to result in high which was divided into two compartments (light and large:
(detectable) concentrations of the drug in the blood of adult mice 27 cm × 27 cm, dark and small: 18 cm × 27 cm) by a partition. These
and in the breast milk of lactating dams (Lin et al., 2005; Luzi et al., areas were connected by a small central open door (7.5 cm × 7.5 cm)
2009). Before the experiment began, we recorded the average water located in the center of the partition at floor level. The large com-
consumption according to the body weight per cage per day for each partment was open at the top, illuminated by a 100 W bulb located
mouse in the laboratory. In addition, minocycline was dissolved in 90 cm above the apparatus. The small compartment had a remov-
the drinking water and its concentration was calculated at three- able black lid at the top. To start the test, each mouse was placed
day intervals according to the average liquid consumption and body in the center of the light compartment, facing away from the door.
weight per cage. The mice did not receive any source of water The animal was allowed to freely explore both compartments for
except for the drinking water containing minocycline solutions. 5 min. The following parameters were recorded: light compartment
Thus, they were motivated by thirst to drink the drug solutions. time, light compartment entries, and latency of entry into the light
To examine possible effects of the chronic minocycline treatment, compartment after the first entry into the dark division. A decrease
the liquid consumption of the control mice was measured every in the amount of time spent and numbers of entries into the light
three days which showed no significant change in the liquid intake compartment are indicative of anxiety-like behavior (Amani et al.,
compared with the drug-receiving mice. 2013).
4 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13

2.5.3. Elevated zero maze 2.6. HPA axis reactivity to stress

The elevated zero maze was a ring-shaped apparatus, con-
structed from wood, elevated 40 cm from the floor. This apparatus We assessed HPA axis reactivity in adult male offspring (PND
consisted of a circular platform (outer diameter 46 cm, width 90) 25 min after the forced swim test. The blood was collected via
5.5 cm) divided into four quadrants of equal lengths with two tail vein puncture and circulating level of corticosterone (CORT;
open opposite (with 1 cm high curbs to prevent falls), and two Bio-Medical Assay Company, China) in the serum was measured as
equal closed (surrounded by a 20 cm wall from the surface of the previously described by using CORT specific quantitative sandwich
maze) quadrants with an open roof. The room was illuminated by ELISA kit according to the manufacturer’s instructions. All samples
a 60-W bulb 1.5 m above the apparatus. Each animal was indi- and standards were assayed in duplicate.
vidually placed into one of the two closed quadrants at the start
of their 5 min sessions. The following behavioral parameters were 2.7. Stress protocol, hippocampal collection and cytokine
recorded: open quadrant time, open quadrant entries, the number measurements
of head dips in the open region; the number of stretch attend pos-
tures (defined as the animal having their front two paws out of the We evaluated the impacts of neonatal immune activation and
closed arm and their hind paws within the closed arm) in the closed minocycline treatment on IL-1␤ and TNF-␣ proteins in the hip-
quadrants (Salari et al., 2015). pocampus following acute restraint stress (mice were individually
placed in plastic transparent cylinders, 4 cm diameter and 10 cm
long, under a single 60-W light bulb for 60 min) based on a
2.5.4. Sucrose preference recent study where LPS administration on PNDs 3 and 5 resulted
The sucrose preference test was performed as described pre- in increased TNF-␣ and IL-1␤ in the hippocampus in response
viously by Monteggia et al. (2007), over a 48-h period between of stress (Walker et al., 2010). Immediately after stress expo-
PND 70 and 72. No previous food or water deprivation was applied sure, the offspring were deeply anaesthetized by an i.p. injection
before the test. After three days of acclimation to the two-bottle of ketamine hydrochloride (50 mg/kg; Alfasan, Woerden-Holland)
choice paradigm (two identical water bottles, both containing tap plus Xylazine (5 mg/kg; Alfasan, Woerden-Holland). To prevent
water, were placed on the cages during acclimation, PND 67–70), contamination of blood cytokine in the brain, mice were perfused
each offspring was given two bottles, one containing a 2% sucrose with ice-cold pyrogen-free saline before they were killed. Then,
solution (A) and the other containing tap water (B). The position of whole brains were rapidly removed, placed on an ice-cold sur-
the bottles A and B was changed every 12 h to avoid a “side” bias. face in a Petri dish filled with saline, and the hippocampus was
The amount of the sucrose solution or water consumed was mea- dissected. The tissues were snap frozen in liquid nitrogen, placed
sured by weighing the bottles immediately before and after the test. into microcentrifuge tubes and stored at −80 ◦ C until processing.
The sucrose preference was calculated as the percentage of sucrose Hippocampal tissue was homogenized in 500 ␮l buffer (TBS plus
solution ingested relative to the total amount of liquid consumed. 0.2% Triton X-100, 2 mM EDTA, PBS 1 mM PMSF, and protease
After the test, offspring were given free access to water. Anhedonia, inhibitor cocktail) and centrifuged at 15,000 × g for 15 min at 4 ◦ C.
a core symptom of depression, was defined as a decrease in sucrose The supernatants were collected and total protein was determined
preference. by Micro BCA Protein Assay Kit. The levels of IL-1␤ and TNF-␣ in
the hippocampus was detected using ELISA kits (R&D systems, USA)
according to kit instructions. The concentration of cytokine protein
2.5.5. Tail suspension test is presented as pg per mg protein.
The tail suspension test was performed as previously described
(Salari et al., 2015). At the beginning of the experiment, each mouse 2.8. Statistics
was individually suspended by the tail using a clamp, 2 cm from
the end, in a grey wooden enclosure (40 cm high, 30 cm wide and All data were analyzed using the statistical package of SPSS
20 cm deep) such that the head of mouse was about 25 cm above the (IBM-Version-21). In order to evaluate the effects of neonatal
floor. The total duration of immobility was recorded (in seconds) immune activation in offspring, the data were analyzed using
during the 5 min test period. Any animal that climbed their tails one-way analysis of variance (ANOVA). The interactions between
were removed from the experimental group, and were not used neonatal immune activation and minocycline were analyzed using
in the analysis. Immobility was defined as the lack of motion of two-way ANOVA. The data between drug (minocycline) and control
the whole body, whereas mobility was defined as movement of the (water) subgroups in LPS/saline-treated groups were also analyzed
hind legs. separately by using one-way ANOVA. All data are presented as the
mean ± standard error of the mean (S.E.M). A P-value less than 0.05
was considered statistically significant.
2.5.6. Forced swim test
The forced swim test was performed as described elsewhere 3. Results
(Salari et al., 2015). To apply this behavioral model to mice, the fol-
lowing procedure was adopted: Mice were individually placed into 3.1. Effect of minocycline on neonatal immune
the transparent glass cylinders (Height: 25 cm, Diameter: 10 cm), activation-induced anxiety-like behavior in adult mice
filled with water to a height of 15 cm and maintained at 25 ± 1 ◦ C.
The water was replaced by fresh water between each test. The 3.1.1. Open field
total duration of immobility was recorded during the last 4 min of Male offspring treated with minocycline were tested on PND
the 6 min testing period. At the end of the swimming session, the 70 in the open field. There were no interactions between neona-
animals were removed from the cylinder, dried with towels, and tal immune activation × minocycline treatment in the inner zone
placed gently near an electric heater for 15–30 min. Each mouse time and entries. As shown in Fig. 2, neonatal immune activation
was judged to be immobile when it ceased struggling and remained with LPS significantly decreased time spent [F1,16 = 6.49, P = 0.021]
floating motionless in the water and making only those movements and number of entries [F1,16 = 6.29, P = 0.023] in the inner zone of
necessary to keep its head above water. The lack of struggling was the open field, indicating higher levels of anxiety-like behaviors
considered as indicative of a state of behavioral despair. compared to the saline group. Moreover, the results of the open
 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13 5

that minocycline treatment during lactation and adolescence sig-

nificantly elevated amount of time spent [F1,16 = 5.22, P = 0.036]
into the light compartment and reduced the latency [F1,16 = 9.6,
P = 0.007] in the LPS offspring relative to LPS/Water offspring. These
results confirm the above findings and therefore it can be concluded
that minocycline treatment during lactation and adolescence can
reverse anxiety-like behavior induced by neonatal immune activa-
tion in mice, as a result, we can consider this as a beneficial effect of
minocycline for the treatment of anxiety with neurodevelopmental
origin in mice.

3.1.3. Elevated zero-maze

After light-dark box paradigm, the offspring were placed in the
elevated zero maze on PND 90 and their behavior was recorded for
5 min. Two-way ANOVA revealed a main effect of neonatal immune
activation and minocycline treatment on open quadrant time
([F1,32 = 15.48, P < 0.001]; [F1,32 = 7.36, P = 0.011], resp.), open quad-
rant entries (neonatal immune activation: [F1,32 = 9.74, P = 0.004]),
and stretch attend postures ([F1,32 = 10.82, P = 0.002]; [F1,32 = 21.21,
P < 0.001], resp.) in closed quadrants. A significant interaction
existed between neonatal immune activation and minocycline
treatment [F1,32 = 6.25, P = 0.018] for stretch attend postures and
there were no interactions for the other parameters. No significant
change in the head dips was observed.
In addition, one-way ANOVA revealed that early postnatal expo-
sure to LPS significantly reduced open quadrant time [F1,16 = 14.58,
P = 0.002] and open quadrant entries [F1,16 = 11.69, P = 0.004], and
increased stretch attend postures [F1,16 = 17.76, P = 0.001], which
reflects higher levels of anxiety in LPS-treated offspring rela-
tive to the saline-treated group (Fig. 4). On the other hand, as
illustrated in Fig. 4, minocycline treatment during development
significantly increased open quadrant time [F1,16 = 9.25, P = 0.008]
and open quadrant entries [F1,16 = 6.63, P = 0.02], and decreased
stretch attend postures [F1,16 = 28.87, P < 0.001], showing lower lev-
els of anxiety-like behavior in LPS offspring in comparison with
LPS + water animals. Therefore, these results indicate that minocy-
Fig. 2. Effects of neonatal immune activation alone or in combination with minocy- cline acts as an anxiolytic agent during development and is able to
cline treatment on anxiety-like behavior in the open field in adult male mice, PND prevent anxiety-like behavior induced by neonatal inflammation
70. Values are presented as mean ± S.E.M. (N = 9) inner zone time (A) and inner zone
in adult mice.
entries (B). Significant differences: *P < 0.05, compared to the saline-treated mice;
$
P<0.05, compared to the LPS-treated mice.

field assessment showed that minocycline treatment during devel- 3.2. Effect of minocycline on neonatal immune
opment significantly resulted in decreased the number of entries activation-induced depression-like behavior in adult mice
[F1,16 = 6.41, P = 0.022] into the inner zone in LPS-treated offspring
relative to the LPS + water group. 3.2.1. Sucrose preference
To examine whether minocycline treatment during develop-
3.1.2. Light–dark box ment exerts a significant effect on depressive-like behavior, we
The light–dark box test was performed ten days after the open first tested the offspring (PND 70) for sucrose preference to
field on PND 80 and its results are presented in Fig. 3. Two-way assess offspring’s response to a natural reward. It has been sug-
ANOVA revealed a main effect of neonatal immune activation gested that a loss of sensitivity to reward is an indicator of
for light compartment time [F1,32 = 5.23, P < 0.029], light com- anhedonia-like behavior, one important feature of human major
partment entries [F1,32 = 9.53, P = 0.004], and latency [F1 ,32 = 7.66, depressive disorder. Two-way ANOVA revealed a main effect of
P = 0.009], and minocycline treatment for the latency [F1,32 = 6.07, neonatal immune activation [F1,32 = 8.07, P = 0.008] and minocy-
P < 0.019] in the light-dark box test. There was also a significant cline treatment [F1,32 = 4.2, P = 0.05] on sucrose preference test.
interaction between neonatal immune activation × minocycline However, there was no significant interaction between neonatal
treatment [F1,32 = 8.52, P = 0.006] for the latency. However, there immune activation × minocycline treatment for sucrose prefer-
were no significant interactions between neonatal immune acti- ence. As illustrated in Fig. 5A, mice neonatally treated with LPS
vation × minocycline treatment for the other parameters in the showed significantly less preference [F1,16 = 7.4, P = 0.015] for the
test. Additional statistical analysis revealed that neonatal treat- sucrose solution than saline-treated mice, consistent with an anhe-
ment with LPS significantly reduced the light compartment donic response. On the other side, minocycline treatment reversed
time [F1,16 = 9.7, P = 0.007] and entries [F1,16 = 5.06, P = 0.039], and the decrease in sucrose preference [F1,16 = 4.61, P = 0.047] in LPS
increased latency [F1,16 = 9.79, P = 0.006]. These findings are indica- offspring, showing higher preference for the sucrose solution and
tive for higher levels of anxiety in LPS-treated offspring in lower depressive-like behavior in LPS offspring in comparison with
comparison with the saline-treated animals. Fig. 3 also shows LPS + water group.
6 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13

Fig. 3. Effects of neonatal immune activation alone or in combination with minocycline treatment on anxiety-like behavior in the light–dark box in adult male mice, PND80.
Values are presented as mean ± S.E.M. (N = 9) light compartment time (A), light compartment entries (B), or latency of entry into the light compartment (C). Significant
differences: *P < 0.05 and **P < 0.01, compared to the saline-treated mice; $ P < 0.05 and $$ P < 0.01, compared to the LPS-treated mice.

3.2.2. Tail suspension test treatment in the tail suspension test. Early neonatal exposure to LPS
The tail suspension test was performed ten days after sucrose increased the total duration of immobility [F1,16 = 8.74, P = 0.009]
preference test on PND 80 and its data is shown in Fig. 5B. Two- in offspring, which is indicative for higher levels of depression
way ANOVA revealed a main effect of neonatal immune activation in LPS-treated males compared to the saline-treated group. On
[F1,32 = 7.09, P = 0.012] and minocycline treatment [F1,32 = 4.53, the other hand, minocycline treatment during lactation and ado-
P = 0.041] on the immobility time. There was no significant lescence resulted in decreased the immobility time [F1,16 = 4.53,
interaction between neonatal immune activation × minocycline
 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13 7

Fig. 4. Effects of neonatal immune activation alone or in combination with minocycline treatment on anxiety-like behavior in the zero maze in adult male mice, PND 90.
Values are presented as mean ± S.E.M. (N = 9) open quadrant time (A), open quadrant entries (B), the number of the stretch attend postures (C), or the number of head dips
(D). Significant differences: **P < 0.01, compared to the saline-treated mice; $ P < 0.05, $$ P < 0.01 and $$$ P < 0.001, compared to the LPS-treated mice.

P = 0.049], which reflects lower levels of depressive-like behavior In addition, Fig. 6A shows that neonatal immune activation
in LPS offspring relative to the LPS + Water offspring. increased the corticosterone levels [F1,16 = 13.97, P = 0.002] in LPS
males relative to the saline-treated group. This finding reflects
3.2.3. Forced swim test higher levels of HPA axis reactivity to stress in LPS offspring than
The offspring were tested in forced swim test on PND 90, two- saline offspring. One-way ANOVA also indicated that minocycline
way ANOVA revealed a main effect of neonatal immune activation treatment during development significantly decreased stress-
[F1,32 = 8.22, P = 0.007] and minocycline treatment [F1,32 = 13.40, induced corticosterone levels in adult male mice [F1,16 = 9.28,
P = 0.001] on the immobility time. There was no significant P = 0.008] in response to stress in comparison with the LPS + water
interaction between neonatal immune activation × minocycline group. These findings demonstrate that minocycline exposure dur-
treatment in the forced swim test. As shown in Fig. 5C, neona- ing brain development can normalize HPA axis reactivity to stress
tal immune activation increased the total duration of immobility following neonatal immune activation.
[F1,16 = 12.21, P = 0.003] in offspring, which reflects higher levels
of depression in LPS offspring, as compared to the saline-treated 3.4. Effect of minocycline following neonatal immune activation
group. On the other side, minocycline treatment during devel- on stress-induced hippocampal IL-1ˇ and TNF-˛ in adult mice
opment gave rise to reduced the immobility time [F1,16 = 10.91,
P = 0.004], showing lower levels of depressive-like behavior in LPS 3.4.1. IL-1ˇ in the hippocampus
offspring in comparison with LPS + Water offspring. These findings As illustrated in Fig. 6B, the levels of IL-1␤ protein in the hip-
confirm the above data showing that minocycline treatment dur- pocampus were measured after stress exposure in adult mice.
ing lactation and adolescence can reverse depressive-like behavior Two-way ANOVA revealed a main effect of neonatal immune
induced by neonatal immune activation in mice in adulthood. activation [F1,32 = 44.68, P < 0.001] and minocycline treatment
[F1,32 = 23.8, P < 0.001]. A significant interaction existed between
3.3. Effect of minocycline following neonatal immune activation neonatal immune activation and minocycline treatment [F1,32 = 6.7,
on HPA axis reactivity to stress in adult mice P = 0.014]. One way ANOVA indicated that postnatal immune
activation elevated the hippocampal IL-1␤ levels [F1,16 = 30.26,
Stress-induced corticosterone levels were measured in response P < 0.001] in LPS-treated mice, as compared to the saline-treated
to the elevated plus maze (Fig. 6A) in adult mice. Two-way ANOVA mice. The levels of IL-1␤ in the hippocampus of the saline-
revealed a main effect of neonatal immune activation [F1,32 = 22, minocycline-treated mice were significantly lower [F1,16 = 4.93,
P < 0.001] and minocycline treatment [F1,32 = 7.59, P = 0.01]. How- P = 0.041] than those saline-water-treated mice. Furthermore,
ever, there was no significant interaction effect for neonatal Fig. 6B shows that developmental minocycline exposure markedly
immune activation × minocycline treatment [F1,32 = 3.75, P = 0.62]. reduced the levels of IL-1␤ protein in the hippocampus of the
8 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13

Fig. 5. Effects of neonatal immune activation alone or in combination with minocycline treatment on depression-like behaviors (sucrose preference, PND 70: A; tail suspension
test, PND80: B; forced swim test, PND90: C) in adult male mice. Values are presented as mean ± S.E.M. (N = 9) percentage of sucrose preference, or of total duration of immobility.
Significant differences: *P < 0.05 and **P < 0.01, compared to the saline-treated mice; $ P < 0.05 and $$ P < 0.01, compared to the LPS-treated mice.

mice that neonatally were exposed to LPS [F1,16 = 18.97, P < 0.001]. development of anxiety- and depression-like behaviors, HPA axis
These data clearly demonstrate that minocycline treatment follow- hyperactivity and hippocampal inflammation in adulthood (Doosti
ing neonatal immune activation during perinatal and adolescent et al., 2013; Majidi-Zolbanin et al., 2013; Walker et al., 2004, 2010,
periods can decrease stress-induced IL-1␤ in the hippocampus. 2012). Given that we have previously discussed in detail the influ-
ence of neonatal immune activation on HPA axis development and
3.4.2. TNF-˛ in the hippocampus its relevance to anxiety- and depression-like behaviors elsewhere
We also measured the levels of TNF-␣ protein in the hip- (Doosti et al., 2013; Majidi-Zolbanin et al., 2013), here we further
pocampus after stress exposure in adult mice. Two-way ANOVA focus on the role of hippocampal inflammation and minocycline
revealed a main effect of neonatal immune activation [F1,32 = 44.1, effects on these affective disorders. Consistent with our findings it
P < 0.001] and minocycline treatment [F1,32 = 20.97, P < 0.001]. How- has been shown that neonatal infection increases the expression
ever, there was no significant interaction for neonatal immune of glial cell marker and IL-1␤-related genes in the hippocampus
activation and minocycline treatment. As shown in Fig. 6C, the of neonate and adult rats in response to LPS administration (Bilbo
data analysis revealed that neonatal immune activation resulted et al., 2005a). Besides, Walker et al. (2004, 2012) reported that early
in an increase in TNF-␣ protein in the hippocampus [F1,16 = 27.31, neonatal LPS exposure in rats significantly elevates anxiety-like
P < 0.001] in mice. We also found a significant lower level of behavior and hippocampal IL-1␤ and TNF-␣ levels after exposure
TNF-␣ protein [F1,16 = 10.44, P = 0.005] in the hippocampus of the to stress in adulthood (Walker et al., 2010). Interestingly, this group
saline-minocycline-treated mice in comparison with the saline- later showed that postnatal LPS administration results in increased
water-treated mice. Moreover, minocycline treatment following inflammation and microglial activation in the hippocampus in adult
neonatal immune activation significantly decreased the hippocam- rats that were co-incident with an elevation in levels of anxiety-like
pal TNF-␣ levels [F1,16 = 11.31, P = 0.004] in adult mice after stress behavior (Sominsky et al., 2012). In light of this evidence, a wide
exposure. These findings indicate developmental minocycline spectrum of studies suggest that dysregulation of inflammatory
treatment following neonatal immune activation can reduce stress- processes need to be carefully considered as a major pathophys-
induced TNF-␣ in the hippocampus. iological mechanism of major depression (Dowlati et al., 2010;
Miller et al., 2009; Raison et al., 2006). From a neuroimmunological
4. Discussion point of view, pro-inflammatory agents such as IL-1␤ and TNF-␣
are thought to be among the most important players in the patho-
Our results (Fig. 7) confirm and extend the earlier findings, sug- genesis of major depression and anxiety-related disorders, through
gesting that neonatal immune activation can be a risk factor for the their direct effects on neural cells within CNS and alterations in
 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13 9

Fig. 6. Effects of neonatal immune activation alone or in combination with minocycline treatment on HPA axis reactivity to stress (corticosterone levels, PND90) and stress-
induced inflammatory cytokines (IL-1␤: B; and TNF-␣: C; PND 100) in the hippocampus in adult male mice. Values are presented as mean ± S.E.M. (N = 9) corticosterone
concentration, or cytokine protein levels. Significant differences: # P < 0.05 and ## P < 0.01, compared to the saline-treated mice; **P < 0.01 and ***P < 0.001, compared to the
saline-treated mice; $$ P < 0.01 and $$$ P < 0.001, compared to the LPS-treated mice.

neurotransmitters and neuropeptides levels (Dowlati et al., 2010; regulation of depression (Bremner et al., 2000) and anxiety (Engin
Loftis et al., 2010; Miller et al., 2009; Raison et al., 2006; Salim and Treit, 2007) behaviors and HPA axis activity (Herman et al.,
et al., 2012). This view, in agreement to our findings, is supported 2005). While there is a strong relationship between inflammation
by clinical studies where IL-1␤ and TNF-␣ appear to be signifi- in the brain and the etiology and pathophysiology of major depres-
cantly higher in patients with major depression, and of interest is sion and anxiety-related disorders, little attention has been paid
that antidepressant drugs can reverse these altered levels (Dowlati to where depression is characterized by increased inflammation in
et al., 2010; Hannestad et al., 2011; Owen et al., 2001; Song et al., the context of HPA axis hyperactivity and glucocorticoid resistance
2009; Tuglu et al., 2003). Preclinical studies also support these find- (Pariante and Lightman, 2008). According to the fact that increased
ings, in recent years, it has repeatedly been reported that these two CORT level during inflammation may result from stimulation of the
cytokines have a direct role in the regulation of depression and HPA axis by pro-inflammatory cytokines such as IL-1␤ and TNF-␣
anxiety-like symptoms in animal models (Babri et al., 2014b; Chen (Munck et al., 1984), it can be concluded that elevated levels of IL-
et al., 2013; Goshen et al., 2008; Kaster et al., 2012; Rossi et al., 1␤ and TNF-␣ in the hippocampus and HPA axis dysregulation as
2012). Further confirmations come from previous literature where a result of neonatal immune activation in mice likely contribute
treatment with anti-TNF and -IL-1␤ agents or the deletion of related to alterations in anxiety- and depression-like phenotypes in adult-
genes was associated with decreased depressive and anxiety symp- hood.
toms (Bayramgürler et al., 2013; Goshen et al., 2008; Karson et al., In the second part of this study, we show that the adverse
2013; Koo and Duman, 2009; Krügel et al., 2013; Murray et al., consequences of neonatal immune activation on anxiety- and
2013; Simen et al., 2006). Considering that the major source of depression-like behaviors, and hippocampal inflammation were
TNF and IL-1␤ in the CNS is activated microglia and astrocytes, reversed by developmental minocycline treatment in adult male
and the fact these glia cells and cytokines are involved in inflam- mice. In addition, developmental minocycline exposure, regardless
mation, neuronal death and neurogenesis (Ben-Hur et al., 2003; of postnatal inflammation, did not have any detrimental effects
Ekdahl et al., 2009; Goshen et al., 2008; Iosif et al., 2006; Kohman on the above measured parameters. Recent studies have demon-
and Rhodes, 2013; Zunszain et al., 2012), in this study, their poten- strated the neuroprotective effects of minocycline on anxiety and
tial effects on the structure and function of hippocampal neurons depression-related disorders in humans and animals (Soczynska
might have influenced the behavior in mice. The point which needs et al., 2012). For instance, minocycline has shown to be beneficial in
to be taken into consideration is that hippocampus is involved in the reducing the immobility time, an indicator of antidepressant effect,
10 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13

Fig. 7. This figure indicates the summary of results. ↑ = Increase and ↓ = Decrease.

in the forced swim test (Molina-Hernández et al., 2008). Further- cued these abnormalities (Zhu et al., 2014a). Consistent with these
more, minocycline was able to attenuate LPS-induced expression findings, previous literature supports the idea that impaired neu-
of pro-inflammatory cytokines and prevent LPS- and interferon- roprotection like decreased neuronal survival and neurogenesis in
alpha-induced depressive-like behavior in mice (O’connor et al., the hippocampus is strongly involved in the pathogenesis of major
2009; Zheng et al., 2015). In line with this, it has been shown that depression disorder, thus these actions can be considered as poten-
minocycline attenuates LPS-induced neuroinflammation, sickness tial pathophysiological factors and therapeutic targets for patients
behavior, and anhedonia in adult mice through reduced microglial with major depression (Pae et al., 2008). On the other side, it has
activation and decreased mRNA levels of IL-1␤ in the hippocam- been shown that neonatal E. coli administration reduces both the
pus (Henry et al., 2008). Another study found that minocycline survival and neurogenic potential of hippocampal progenitors and
affects the maturation of dendritic spines in the developing hip- impairs adult performance in hippocampus-dependent memory
pocampal neurons, resulting in anxiolytic behavior in young Fmr1 tasks, which is attributed to decreased hippocampal neurogenesis
KO mice (Bilousova et al., 2009). Interestingly, in agreement with (Bilbo et al., 2005b; Bland et al., 2010). Moreover, neonatal exposure
our findings, a recent study demonstrated that adolescent minocy- to LPS has been shown to induce an acute expansion of the hip-
cline treatment attenuated the behavioral deficits and inhibited pocampal microglia population, resulting in reduced neurogenesis
the activated microglia in the brain of offspring that prenatally in the hippocampus of rats (Smith et al., 2014). Although the find-
exposed to inflammation (Zhu et al., 2014b). Further evidence ings from the present study do not provide direct evidence about
comes from the experiments where minocycline has been used to the effect of neonatal immune activation on the neuronal survival
prevent or ameliorate white matter damage induced by neonatal and neurogenesis, our results may reflect the ability of inflamma-
exposure to LPS, the protective effect of minocycline was associ- tion to modulate depression and anxiety-like behaviors through
ated with suppressed microglial activation and decreased the levels inhibition of neurogenesis in the hippocampus. This conclusion
of IL-1␤ and TNF-␣ proteins in the neonatal rat brain (Fan et al., comes from previous studies in which LPS and TNF-␣ have shown to
2005a). Along with these finding, Zhu et al. (2014a,b) reported that be the known agents impairing newborn and adult neurogenesis in
neonatal intra-hippocampal injection of LPS in rats leads to behav- different brain regions like hippocampus (Borsini et al., 2015; Cacci
ioral alterations such as deficits in social interaction, novel object et al., 2005; Das and Basu, 2008; Järlestedt et al., 2013; Monje et al.,
recognition and prepulse inhibition, and increased the number of 2003). In support of this argument we recently demonstrated that
activated microglial cells in the hippocampus, which application of early neonatal TNF-␣ treatment in mice results in increased anxi-
either minocycline, risperidone or both of them significantly res- ety and depression-like behavior in adulthood (Babri et al., 2014b).
 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13 11

The question now is how minocycline treatment can reverse the the paper. All authors read and approved the final version of the
effects of neonatal immune activation in adulthood, in other words, manuscript.
what are the potential mechanisms? Given that neuroinflamma-
tion is mediated by microglia and astrocytes in the CNS, we think Acknowledgment
that the neuroprotective mechanism of minocycline appears to be
directly related to its anti-inflammatory effects. It has clearly been This study was supported by a grant from the Drug Applied
documented that minocycline suppresses microglial and astroglial Research Center at Tabriz University of Medical Sciences (GN-90-
activation, inhibits the increased production of pro-inflammatory 32).
cytokines, like TNF-␣ and IL-1␤, enhance neuroplasticity and neu-
rogenesis in the hippocampus (Cheng et al., 2015; Garrido-Mesa References
et al., 2013; Jiang et al., 2015; Soczynska et al., 2012). Since brain is
very vulnerable and sensitive to inflammation during early neona- Amani, M., Samadi, H., Doosti, M.-H., Azarfarin, M., Bakhtiari, A., Majidi-Zolbanin,
N., Mirza-Rahimi, M., Salari, A.-A., 2013. Neonatal NMDA receptor blockade
tal development, an age when murine developmental neurogenesis
alters anxiety- and depression-related behaviors in a sex-dependent manner
is highly active (Smith et al., 2014), it is very likely that the nega- in mice. Neuropharmacology 73, 87–97.
tive effect of LPS on structure and function of hippocampal neurons Babri, S., Doosti, M.-H., Salari, A.-A., 2014a. Strain-dependent effects of prenatal
might have been enhanced by increased levels of TNF-␣ and IL-1␤ maternal immune activation on anxiety- and depression-like behaviors in
offspring. Brain Behav. Immun. 37, 164–176.
in adulthood. It is obvious that the hippocampus is an impor- Babri, S., Doosti, M.-H., Salari, A.-A., 2014b. Tumor necrosis factor-alpha during
tant limbic structure which plays a crucial role in anxiety and neonatal brain development affects anxiety- and depression-related behaviors
depression, and HPA axis system (Bremner et al., 2000; Engin and in adult male and female mice. Behav. Brain Res. 261, 305–314.
Bayramgürler, D., Karson, A., Özer, C., Utkan, T., 2013. Effects of long-term
Treit, 2007; Herman et al., 2005). Previous literature has unequiv- etanercept treatment on anxiety- and depression-like neurobehaviors in rats.
ocally linked the existence of structural and functional disorders Physiol. Behav. 119, 145–148.
in the hippocampus, such as a reduction in the total number of Ben-Hur, T., Ben-Menachem, O., Furer, V., Einstein, O., Mizrachi-Kol, R., Grigoriadis,
N., 2003. Effects of proinflammatory cytokines on the growth, fate, and motility
neurons, to major depression (Bremner et al., 2000) which the of multipotential neural precursor cells. Mol. Cell. Neurosci. 24, 623–631.
effectiveness of antidepressants such as fluoxetine appear to be Bilbo, S.D., Biedenkapp, J.C., Der-Avakian, A., Watkins, L.R., Rudy, J.W., Maier, S.F.,
dependent on induction of hippocampal neurogenesis (Santarelli 2005a. Neonatal infection-induced memory impairment after
lipopolysaccharide in adulthood is prevented via caspase-1 inhibition. J.
et al., 2003). Similarly, administration of minocycline markedly
Neurosci. 25, 8000–8009.
increases hippocampal neurogenesis and reduces the levels of pro- Bilbo, S.D., Levkoff, L.H., Mahoney, J.H., Watkins, L.R., Rudy, J.W., Maier, S.F., 2005b.
inflammatory cytokines in the brain (Garrido-Mesa et al., 2013; Neonatal infection induces memory impairments following an immune
challenge in adulthood. Behav. Neurosci. 119, 293.
Jiang et al., 2015; Soczynska et al., 2012). From a therapeutic point
Bilousova, T., Dansie, L., Ngo, M., Aye, J., Charles, J.R., Ethell, D.W., Ethell, I.M., 2009.
of view, based on the above evidence from experimental and clin- Minocycline promotes dendritic spine maturation and improves behavioural
ical studies with minocycline, we postulate that minocycline may performance in the fragile X mouse model. J. Med. Genet. 46, 94–102.
treat neonatal immune activation-induced anxiety and depression- Bland, S.T., Beckley, J.T., Young, S., Tsang, V., Watkins, L.R., Maier, S.F., Bilbo, S.D.,
2010. Enduring consequences of early-life infection on glial and neural cell
like behaviors, and HPA axis dysregulation through the inhibition genesis within cognitive regions of the brain. Brain Behav. Immun. 24,
of microglial and/or astroglial activation, anti-inflammatory activ- 329–338.
ity and upregulating hippocampal neurogenesis. Considering that Boksa, P., 2010. Effects of prenatal infection on brain development and behavior: a
review of findings from animal models. Brain Behav. Immun. 24, 881–897.
neonatal infection can have profound effects on human’s long- Borsini, A., Zunszain, P.A., Thuret, S., Pariante, C.M., 2015. The role of inflammatory
term health during adulthood. In this study, we used the neonatal cytokines as key modulators of neurogenesis. Trends Neurosci. 38, 145–157.
immune activation model for inducing anxiety- and depression- Bremner, J.D., Narayan, M., Anderson, E.R., Staib, L.H., Miller, H.L., Charney, D.S.,
2000. Hippocampal volume reduction in major depression. Am. J. Psychiatry
related behaviors in mice, which is based on the well-documented 157 (1), 115–118.
association between early life exposure to infection and increased Cacci, E., Claasen, J.H., Kokaia, Z., 2005. Microgliaderived tumor necrosis factor-␣
risk of neuropsychiatric disorders including depression and anxi- exaggerates death of newborn hippocampal progenitor cells in vitro. J.
Neurosci. Res. 80, 789–797.
ety in adulthood. Using animal models like this one can be of great
Cai, Z., Lin, S., Fan, L.-W., Pang, Y., Rhodes, P., 2006. Minocycline alleviates
value, to help understand and uncover the etiologies of affective hypoxic–ischemic injury to developing oligodendrocytes in the neonatal rat
disorders which are multifactorial, resulting from a complex inter- brain. Neuroscience 137, 425–435.
Chen, J., Song, Y., Yang, J., Zhang, Y., Zhao, P., Zhu, X.-J., Su, H.-c., 2013. The
play between genetic and environmental factors. Given that the
contribution of TNF-␣ in the amygdala to anxiety in mice with persistent
neuroprotective effects of minocycline are currently under explo- inflammatory pain. Neurosci. Lett. 541, 275–280.
ration as an alternative or adjunctive therapy for alleviating the Cheng, S., Hou, J., Zhang, C., Xu, C., Wang, L., Zou, X., Yu, H., Shi, Y., Yin, Z., Chen, G.,
symptoms of neuropsychiatric disorders, finding potential ther- 2015. Minocycline reduces neuroinflammation but does not ameliorate neuron
loss in a mouse model of neurodegeneration. Sci. Rep. 5, 10535.
apeutic efficacy of minocycline in neurodevelopmental disorders Cottrell, E.C., Seckl, J.R., 2009. Prenatal stress, glucocorticoids and the
makes it an attractive drug for further investigation in humans. programming of adult disease. Front. Behav. Neurosci. 3.
Much more work is also needed to understand the benefits and Dansie, L., Phommahaxay, K., Okusanya, A., Uwadia, J., Huang, M., Rotschafer, S.,
Razak, K., Ethell, D., Ethell, I., 2013. Long-lasting effects of minocycline on
risks of developmental minocycline treatment against psychiatric behavior in young but not adult Fragile X mice. Neuroscience 246, 186–198.
disorders induced by common early-life insults including infection Das, S., Basu, A., 2008. Inflammation: a new candidate in modulating adult
and stress. neurogenesis. J. Neurosci. Res. 86, 1199–1208.
Depino, A., 2015. Early prenatal exposure to LPS results in anxiety-and
depression-related behaviors in adulthood. Neuroscience 299, 56–65.
Dickerson, D.D., Restieaux, A.M., Bilkey, D.K., 2012. Clozapine administration
Conflict of interest ameliorates disrupted long-range synchrony in a neurodevelopmental animal
model of schizophrenia. Schizophr. Res. 135, 112–115.
Dong, E., Dzitoyeva, S.G., Matrisciano, F., Tueting, P., Grayson, D.R., Guidotti, A.,
None. 2015. Brain-derived neurotrophic factor epigenetic modifications associated
with schizophrenia-like phenotype induced by prenatal stress in mice. Biol.
Psychiatry 77, 589–596.
Doosti, M.-H., Bakhtiari, A., Zare, P., Amani, M., Majidi-Zolbanin, N., Babri, S., Salari,
Authors’ contributions A.-A., 2013. Impacts of early intervention with fluoxetine following early
neonatal immune activation on depression-like behaviors and body weight in
Ali-Akbar Salari conceived and designed the study; Jafar Majidi, mice. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 43, 55–65.
Dowlati, Y., Herrmann, N., Swardfager, W., Liu, H., Sham, L., Reim, E.K., Lanctôt, K.L.,
Morteza Kosari-Nasab and Ali-Akbar Salari performed research, 2010. A meta-analysis of cytokines in major depression. Biol. Psychiatry 67,
Ali-Akbar Salari analyzed and interpreted the data, and wrote 446–457.
12 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13

Ekdahl, C., Kokaia, Z., Lindvall, O., 2009. Brain inflammation and adult Luzi, P., Abraham, R.M., Rafi, M.A., Curtis, M., Hooper, D.C., Wenger, D.A., 2009.
neurogenesis: the dual role of microglia. Neuroscience 158, 1021–1029. Effects of treatments on inflammatory and apoptotic markers in the CNS of
Enayati, M., Solati, J., Hosseini, M.-H., Shahi, H.-R., Saki, G., Salari, A.-A., 2012. mice with globoid cell leukodystrophy. Brain Res. 1300, 146–158.
Maternal infection during late pregnancy increases anxiety-and Majidi-Zolbanin, J., Azarfarin, M., Samadi, H., Enayati, M., Salari, A.-A., 2013.
depression-like behaviors with increasing age in male offspring. Brain Res. Adolescent fluoxetine treatment decreases the effects of neonatal immune
Bull. 87, 295–302. activation on anxiety-like behavior in mice. Behav. Brain Res. 250, 123–132.
Engin, E., Treit, D., 2007. The role of hippocampus in anxiety: intracerebral infusion Majidi-Zolbanin, J., Doosti, M.-H., Baradaran, B., Amani, M., Azarfarin, M., Salari,
studies. Behav. Pharmacol. 18, 365–374. A.-A., 2014. Neonatal immune activation during early and late postnatal brain
Fan, L.-W., Pang, Y., Lin, S., Rhodes, P., Cai, Z., 2005a. Minocycline attenuates development differently influences depression-related behaviors in adolescent
lipopolysaccharide-induced white matter injury in the neonatal rat brain. and adult C57BL/6 mice. Neuroimmunol. Neuroinflamm. 1, 35.
Neuroscience 133, 159–168. Majidi-Zolbanin, J., Doosti, M.-H., Kosari-Nasab, M., Salari, A.-A., 2015. Prenatal
Fan, L.W., Pang, Y., Lin, S., Tien, L.T., Ma, T., Rhodes, P.G., Cai, Z., 2005b. Minocycline maternal immune activation increases anxiety-and depressive-like behaviors
reduces lipopolysaccharideinduced neurological dysfunction and brain injury in offspring with experimental autoimmune encephalomyelitis. Neuroscience
in the neonatal rat. J. Neurosci. Res. 82, 71–82. 294, 69–81.
Fried, E.I., Nesse, R.M., 2015. Depression sum-scores don’t add up: why analyzing Mendlowicz, M.V., Stein, M.B., 2000. Quality of life in individuals with anxiety
specific depression symptoms is essential. BMC Med. 13, 72. disorders. Am. J. Psychiatry 157 (May (5)), 669–682.
Garrido-Mesa, N., Zarzuelo, A., Galvez, J., 2013. What is behind the non-antibiotic Miller, A.H., Maletic, V., Raison, C.L., 2009. Inflammation and its discontents: the
properties of minocycline? Pharmacol. Res. 67, 18–30. role of cytokines in the pathophysiology of major depression. Biol. Psychiatry
Goshen, I., Kreisel, T., Ben-Menachem-Zidon, O., Licht, T., Weidenfeld, J., Ben-Hur, 65, 732–741.
T., Yirmiya, R., 2008. Brain interleukin-1 mediates chronic stress-induced Miyaoka, T., Wake, R., Furuya, M., Liaury, K., Ieda, M., Kawakami, K., Tsuchie, K.,
depression in mice via adrenocortical activation and hippocampal Taki, M., Ishihara, K., Araki, T., 2012. Minocycline as adjunctive therapy for
neurogenesis suppression. Mol. Psychiatry 13, 717–728. patients with unipolar psychotic depression: an open-label study. Prog.
Hannestad, J., DellaGioia, N., Bloch, M., 2011. The effect of antidepressant Neuro-Psychopharmacol. Biol. Psychiatry 37, 222–226.
medication treatment on serum levels of inflammatory cytokines: a Molina-Hernández, M., Tellez-Alcántara, N.P., Pérez-García, J., Olivera-Lopez, J.I.,
meta-analysis. Neuropsychopharmacology 36, 2452–2459. Jaramillo-Jaimes, M.T., 2008. Antidepressant-like actions of minocycline
Henry, C.J., Huang, Y., Wynne, A., Hanke, M., Himler, J., Bailey, M.T., Sheridan, J.F., combined with several glutamate antagonists. Prog. Neuro-Psychopharmacol.
Godbout, J.P., 2008. Minocycline attenuates lipopolysaccharide (LPS)-induced Biol. Psychiatry 32, 380–386.
neuroinflammation, sickness behavior, and anhedonia. J. Neuroinflamm. 5, Monje, M.L., Toda, H., Palmer, T.D., 2003. Inflammatory blockade restores adult
2094–2095. hippocampal neurogenesis. Science 302, 1760–1765.
Herman, J.P., Ostrander, M.M., Mueller, N.K., Figueiredo, H., 2005. Limbic system Monteggia, L.M., Luikart, B., Barrot, M., Theobold, D., Malkovska, I., Nef, S., Parada,
mechanisms of stress regulation: hypothalamo–pituitary–adrenocortical axis. L.F., Nestler, E.J., 2007. Brain-derived neurotrophic factor conditional
Prog. Neuro-Psychopharmacol. Biol. Psychiatry 29, 1201–1213. knockouts show gender differences in depression-related behaviors. Biol.
Huppert, J.D., Weiss, K.A., Lim, R., Pratt, S., Smith, T.E., 2001. Quality of life in Psychiatry 61, 187–197.
schizophrenia: contributions of anxiety and depression. Schizophr. Res. 51, Munck, A., Guyre, P., Holbrook, N., 1984. Physiological functions of glucocorticoids
171–180. in stress and their relation to pharmacological actions*. Endocr. Rev. 5, 25–44.
Iosif, R.E., Ekdahl, C.T., Ahlenius, H., Pronk, C.J., Bonde, S., Kokaia, Z., Jacobsen, Murray, C.L., Obiang, P., Bannerman, D., Cunningham, C., 2013. Endogenous IL-1 in
S.-E.W., Lindvall, O., 2006. Tumor necrosis factor receptor 1 is a negative cognitive function and anxiety: a study in IL-1RI−/− mice. PLoS One 8, e78385.
regulator of progenitor proliferation in adult hippocampal neurogenesis. J. Nagano, M., Liu, M., Inagaki, H., Kawada, T., Suzuki, H., 2012. Early intervention with
Neurosci. 26, 9703–9712. fluoxetine reverses abnormalities in the serotonergic system and behavior of
Ishiwata, H., Shiga, T., Okado, N., 2005. Selective serotonin reuptake inhibitor rats exposed prenatally to dexamethasone. Neuropharmacology 63, 292–300.
treatment of early postnatal mice reverses their prenatal stress-induced brain O’connor, J., Lawson, M., Andre, C., Moreau, M., Lestage, J., Castanon, N., Kelley, K.,
dysfunction. Neuroscience 133, 893–901. Dantzer, R., 2009. Lipopolysaccharide-induced depressive-like behavior is
Järlestedt, K., Naylor, A., Dean, J., Hagberg, H., Mallard, C., 2013. Decreased survival mediated by indoleamine 2, 3-dioxygenase activation in mice. Mol. Psychiatry
of newborn neurons in the dorsal hippocampus after neonatal LPS exposure in 14, 511–522.
mice. Neuroscience 253, 21–28. Owen, B.M., Eccleston, D., Ferrier, I.N., Young, A.H., 2001. Raised levels of plasma
Jiang, Y., Liu, Y., Zhu, C., Ma, X., Ma, L., Zhou, L., Huang, Q., Cen, L., Pi, R., Chen, X., interleukin-1beta in major and postviral depression. Acta Psychiatr. Scand.
2015. Minocycline enhances hippocampal memory, neuroplasticity and 103, 226–228.
synapse-associated proteins in aged C57 BL/6 mice. Neurobiol. Learn. Mem. Pae, C.-U., Marks, D.M., Han, C., Patkar, A.A., 2008. Does minocycline have
121, 20–29. antidepressant effect? Biomed. Pharmacother. 62, 308–311.
Karson, A., Demirtaş, T., Bayramgürler, D., Balcı, F., Utkan, T., 2013. Chronic Pariante, C.M., Lightman, S.L., 2008. The HPA axis in major depression: classical
administration of infliximab (TNF␣ Inhibitor) decreases depression and theories and new developments. Trends Neurosci. 31, 464–468.
anxietylike behaviour in rat model of chronic mild stress. Basic Clin. Raison, C.L., Capuron, L., Miller, A.H., 2006. Cytokines sing the blues: inflammation
Pharmacol. Toxicol. 112, 335–340. and the pathogenesis of depression. Trends Immunol. 27, 24–31.
Kaster, M.P., Gadotti, V.M., Calixto, J.B., Santos, A.R., Rodrigues, A.L.S., 2012. Ravindran, L., Stein, M., 2010. The pharmacologic treatment of anxiety disorders: a
Depressive-like behavior induced by tumor necrosis factor-␣ in mice. review of progress. J. Clin. Psychiatry 71, 839.
Neuropharmacology 62, 419–426. Rayen, I., Van Den Hove, D.L., Prickaerts, J., Steinbusch, H.W., Pawluski, J.L., 2011.
Kinney, D.K., Munir, K.M., Crowley, D.J., Miller, A.M., 2008. Prenatal stress and risk Fluoxetine during development reverses the effects of prenatal stress on
for autism. Neurosci. Biobehav. Rev. 32, 1519–1532. depressive-like behavior and hippocampal neurogenesis in adolescence. PLoS
Kohman, R.A., Rhodes, J.S., 2013. Neurogenesis, inflammation and behavior. Brain One 6, e24003.
Behav. Immun. 27, 22–32. Richtand, N.M., Ahlbrand, R., Horn, P., Tambyraja, R., Grainger, M., Bronson, S.L.,
Koo, J.W., Duman, R.S., 2009. Interleukin-1 receptor null mutant mice show McNamara, R.K., 2012. Fluoxetine and aripiprazole treatment following
decreased anxiety-like behavior and enhanced fear memory. Neurosci. Lett. prenatal immune activation exert longstanding effects on rat locomotor
456, 39–43. response. Physiol. Behav. 106, 171–177.
Kreisel, T., Frank, M., Licht, T., Reshef, R., Ben-Menachem-Zidon, O., Baratta, M., Rossi, S., Sacchetti, L., Napolitano, F., De Chiara, V., Motta, C., Studer, V., Musella, A.,
Maier, S., Yirmiya, R., 2014. Dynamic microglial alterations underlie Barbieri, F., Bari, M., Bernardi, G., 2012. Interleukin-1␤ causes anxiety by
stress-induced depressive-like behavior and suppressed neurogenesis. Mol. interacting with the endocannabinoid system. J. Neurosci. 32, 13896–13905.
Psychiatry 19, 699–709. Salari, A.-A., Bakhtiari, A., Homberg, J.R., 2015. Activation of GABA-A receptors
Krügel, U., Fischer, J., Radicke, S., Sack, U., Himmerich, H., 2013. Antidepressant during postnatal brain development increases anxiety-and depression-related
effects of TNF-␣ blockade in an animal model of depression. J. Psychiatr. Res. behaviors in a time-and dose-dependent manner in adult mice. Eur.
47, 611–616. Neuropsychopharmacol. 25, 1260–1274.
Lee, C.Z., Yao, J.S., Huang, Y., Zhai, W., Liu, W., Guglielmo, B.J., Lin, E., Yang, G.-Y., Salim, S., Chugh, G., Asghar, M., 2012. Inflammation in anxiety. Adv. Protein Chem.
Young, W.L., 2006. Dose–response effect of tetracyclines on cerebral matrix Struct. Biol. 88, 1–25.
metalloproteinase-9 after vascular endothelial growth factor Santarelli, L., Saxe, M., Gross, C., Surget, A., Battaglia, F., Dulawa, S., Weisstaub, N.,
hyperstimulation. J. Cereb. Blood Flow Metab. 26, 1157–1164. Lee, J., Duman, R., Arancio, O., 2003. Requirement of hippocampal neurogenesis
Lin, S., Wei, X., Bales, K.R., Paul, A.B., Ma, Z., Yan, G., Paul, S.M., Du, Y., 2005. for the behavioral effects of antidepressants. Science 301, 805–809.
Minocycline blocks bilirubin neurotoxicity and prevents Simen, B.B., Duman, C.H., Simen, A.A., Duman, R.S., 2006. TNF␣ signaling in
hyperbilirubinemiainduced cerebellar hypoplasia in the Gunn rat. Eur. J. depression and anxiety: behavioral consequences of individual receptor
Neurosci. 22, 21–27. targeting. Biol. Psychiatry 59, 775–785.
Loftis, J.M., Huckans, M., Morasco, B.J., 2010. Neuroimmune mechanisms of Smith, P.L., Hagberg, H., Naylor, A.S., Mallard, C., 2014. Neonatal peripheral
cytokine-induced depression: current theories and novel treatment strategies. immune challenge activates microglia and inhibits neurogenesis in the
Neurobiol. Dis. 37, 519–533. developing murine hippocampus. Dev. Neurosci. 36, 119–131.
Löwe, B., Spitzer, R.L., Williams, J.B., Mussell, M., Schellberg, D., Kroenke, K., 2008. Soczynska, J.K., Mansur, R.B., Brietzke, E., Swardfager, W., Kennedy, S.H.,
Depression, anxiety and somatization in primary care: syndrome overlap and Woldeyohannes, H.O., Powell, A.M., Manierka, M.S., McIntyre, R.S., 2012. Novel
functional impairment. Gen. Hosp. Psychiatry 30, 191–199. therapeutic targets in depression: minocycline as a candidate treatment.
Behav. Brain Res. 235, 302–317.
 J. Majidi et al. / Brain Research Bulletin 120 (2016) 1–13 13

Sominsky, L., Walker, A., Ong, L., Tynan, R., Walker, F., Hodgson, D., 2012. Increased Walker, A.K., Hawkins, G., Sominsky, L., Hodgson, D.M., 2012. Transgenerational
microglial activation in the rat brain following neonatal exposure to a bacterial transmission of anxiety induced by neonatal exposure to lipopolysaccharide:
mimetic. Behav. Brain Res. 226, 351–356. implications for male and female germ lines. Psychoneuroendocrinology 37,
Song, C., Halbreich, U., Han, C., Leonard, B., Luo, H., 2009. Imbalance between 1320–1335.
pro-and anti-inflammatory cytokines, and between Th1 and Th2 cytokines in Walker, F.R., March, J., Hodgson, D.M., 2004. Endotoxin exposure in early life alters
depressed patients: the effect of electroacupuncture or fluoxetine treatment. the development of anxiety-like behaviour in the Fischer 344 rat. Behav. Brain
Pharmacopsychiatry 42, 182–188. Res. 154, 63–69.
Stetler, C., Miller, G.E., 2011. Depression and hypothalamic–pituitary–adrenal Zheng, L.-S., Kaneko, N., Sawamoto, K., 2015. Minocycline treatment ameliorates
activation: a quantitative summary of four decades of research. Psychosom. interferon-alpha-induced neurogenic defects and depression-like behaviors in
Med. 73, 114–126. mice. Front. Cell. Neurosci. 9.
Toneatto, T., Nguyen, L., 2007. Does mindfulness meditation improve anxiety and Zhu, F., Zheng, Y., Ding, Y., Liu, Y., Zhang, X., Wu, R., Guo, X., Zhao, J., 2014a.
mood symptoms? a review of the controlled research. Can. J. Psychiatry 52, Minocycline and risperidone prevent microglia activation and rescue
260. behavioral deficits induced by neonatal intrahippocampal injection of
Tsigos, C., Chrousos, G.P., 2002. Hypothalamic–pituitary–adrenal axis, lipopolysaccharide in rats. PLoS One 9, e93966.
neuroendocrine factors and stress. J. Psychosom. Res. 53, Zhu, F., Zheng, Y., Liu, Y., Zhang, X., Zhao, J., 2014b. Minocycline alleviates
865–871. behavioral deficits and inhibits microglial activation in the offspring of
Tuglu, C., Kara, S.H., Caliyurt, O., Vardar, E., Abay, E., 2003. Increased serum tumor pregnant mice after administration of polyriboinosinic–polyribocytidilic acid.
necrosis factor-alpha levels and treatment response in major depressive Psychiatry Res. 219, 680–686.
disorder. Psychopharmacology 170, 429–433. Zunszain, P.A., Anacker, C., Cattaneo, A., Choudhury, S., Musaelyan, K., Myint, A.M.,
Walker, A., Nakamura, T., Hodgson, D., 2010. Neonatal lipopolysaccharide exposure Thuret, S., Price, J., Pariante, C.M., 2012. Interleukin-1␤: a new regulator of the
alters central cytokine responses to stress in adulthood in Wistar rats. Stress kynurenine pathway affecting human hippocampal neurogenesis.
13, 506–515. Neuropsychopharmacology 37, 939–949.