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Hippocampal neurogenesis dysfunction linked to depressive-like behaviors in a neuroinflammation induced model of depression
Hippocampal neurogenesis dysfunction linked to depressive-like behaviors in a neuroinflammation induced model of depression Ming-ming Tang, Wen-juan Lin, Yu-qin Pan, Xi-ting Guan, Ying-cong Li PII: DOI: Reference:
S0031-9384(16)30177-9 doi: 10.1016/j.physbeh.2016.04.034 PHB 11310
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
4 January 2016 13 April 2016 15 April 2016
Please cite this article as: Tang Ming-ming, Lin Wen-juan, Pan Yu-qin, Guan Xi-ting, Li Ying-cong, Hippocampal neurogenesis dysfunction linked to depressive-like behaviors in a neuroinflammation induced model of depression, Physiology & Behavior (2016), doi: 10.1016/j.physbeh.2016.04.034
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ACCEPTED MANUSCRIPT Hippocampal
neurogenesis
dysfunction
linked
to
depressive-like
behaviours
in
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neuroinflammation induced model of depression
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Ming-ming Tang a,c，Wen-juan Lin a,b,*, Yu-qin Pan a, b , Xi-ting Guan a,c , Ying-cong Li a
Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing
100101, China
Brain-Behavior Research Center, Institute of Psychology, Chinese Academy of Sciences, Beijing
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b
100101, China
University of Chinese Academy of Sciences, Beijing, China
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c
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* Corresponding author at: 16 Lincui Road, Beijing 100101, China. Tel: 86-10-64853723, Fax:
Abstract
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86-10-64872070, Email: [email protected].
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Our previous work found that triple central lipopolysaccharide (LPS) administration could induce depressive-like behaviours and increased central pro-inflammatory cytokines mRNA, hippocampal cytokine mRNA in particular. Since several neruoinflammation-associated conditions have been reported to impair neurogenesis, in this study, we further investigated whether the neruoinflammation induced depression would be associated with hippocampal neurogenesis dysfunction. An animal model of depression induced by triple central lipopolysaccharide (LPS) administration was used. In the hippocampus, the neuroinflammatory state evoked by LPS was marked by an increased production of pro-inflammatory cytokines, including interleukin-1β, interleukin- 6, and tumor necrosis factor-α. It was found that rats in the neuroinflammatory state exhibited depressive-like behaviors, including reduced saccharin preference and locomotor activity as well as increased immobility time in the tail suspension test and latency to feed in the novelty suppressed feeding test. Adult hippocampal neurogenesis was
ACCEPTED MANUSCRIPT concomitantly inhibited, including decreased cell proliferation and newborn cell survival. We also demonstrated that the decreased hippocampal neurogenesis in cell proliferation was significantly correlated with the depressive-like phenotypes of decreased saccharine preference and distance
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traveled, the core and characteristic symptoms of depression, under neuro inflammation state. These findings provide the first evidence that hippocampal neurogenesis dysfunction is
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correlated with neuroinflammation-induced depression, which suggests that hippocampal neurogenesis might be one of biological mechanisms underlying depression induced by
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neruoinflammation.
Key words
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Depressive-like behavior; Neuroinflammation; Neurogenesis; Pro-inflammatory cytokine;
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Lipopolysaccharide;
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1. Introduction
It has been proposed that the actions of inflammatory processes, and of pro-inflammatory
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cytokines in particular, on the brain may account for the pathogenesis of depressive disorders, and this hypothesis has been referred to as the “cytokine or inflammatory hypothesis of depression” [1-5]. Although this hypothesis has been an interesting research focus for more than
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two decades, how neuronal-immune interactions modulate the depressive phenotype is still not fully understood. Inflammatory processes may affect more than one pathway, which is thought to be important in the pathogenesis of depression, such as monoamine alterations, glutamate neurotransmission, and glucocorticoid receptor resistance [6-8]. Novel theory suggests that adult hippocampal neurogenesis, as a novel pathway, may also be involved in depressive disorders caused by inflammation [9, 10]. Neurogenesis is the complex process of generating new neurons from neural stem or progenitor cells that occurs in the brains of many animal species, including humans [11]. Adult neurogenesis primarily occurs in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus [12]. The process of adult neurogenesis involves several steps, including proliferation, specification of the fate of neural progenitors, neuronal migration, neuronal maturation and synaptic integration of the young
ACCEPTED MANUSCRIPT neurons into the existing neuronal circuitry. It has been reported that adult hippocampal neurogenesis is related to synaptic plasticity, cognitive functioning, and psychiatric diseases [13, 14]. Neurogenesis in the dentate gyrus of the hippocampus is negatively regulated by stressful
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experiences [15] and positively regulated by chronic antidepressant treatment [16]. Recent evidence indicates that neurogenesis is influenced by inflammatory processes [17-19]. Recently,
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we found that triple central lipopolysaccharide (LPS) administration induced depressive-like behaviours and increased central pro-inflammatory cytokines mRNA, hippocampal cytokine mRNA in particular. We also found that the increased hippocampal TNF-α mRNA expression was
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the common affected factor in both chronic stress induced and LPS induced models of depression [20]. These results suggest that hippocampal inflammation may have a pivotal role in depression.
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However, it is unknown how hippocampal neurogenesis is affected by triple central LPS administration and whether the altered hippocampal neurogenesis contributes to the regulation
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of depression induced by neuroinflammation.
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Thus, the purpose of the present study was to determine the relationship between hippocampal neurogenesis and the depressive-like behavior induced by inflammation. In this
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study, we first examined the neuroinflammatory state in the hippocampus induced by triple central LPS administration by determining the protein expression of IL-1β, IL-6 and TNF-α. We then examined depressive-like behaviors and adult hippocampal neurogenesis under
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neuroinflammatory state and evaluated the correlation between the degree of depressive-like behaviors and the level of altered neurogenesis.
2. Materials and methods 2.1. Animals Male Sprague-Dawley rats that weighed 220-240 g were obtained from Vital River Laboratories (Beijing, China). All of the rats were individually housed in standard stainless steel cages in a temperature- and humidity-controlled room (22±1℃; 40-60%) with a 12:12 dark/light cycle (lights on at 07:00 am; off at 7:00 pm). The rats were given free access to food and water throughout the experiment except for the saccharin preference test time and novelty suppressed feeding test time. The rats were given 5 min of daily handling for 7 days prior to experimental use to minimize stress responses to the experimental manipulation. The experimental procedures
ACCEPTED MANUSCRIPT were approved by the Institutional Review Board of the Institute of Psychology, Chinese Academy of Sciences and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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2.2. Surgical procedures
After acclimation, the rats were anesthetized with sodium pentobarbital (1%, 35 mg/Kg;
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Merck, Darmstadt, Germany) via an intraperitoneal injection, and guide cannulas were implanted (RWD Life Science Co., Ltd, Shenzhen, China) into the lateral ventricle (AP: - 0.9 mm from bregma; LM: 1.1 mm from the sagittal suture; DV: 3.2 mm in depth relative to the skull). The top of the
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animal’s head was shaved, and the head was fixed in a stereotaxic instrument (Stoelting, Wood Dale, IL, USA), with the incisor bar set at 3.3 mm below the interaural line. Body temperature was
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maintained at 37 ℃ using a rectal thermometer and a feedback-controlled heating pad (RWD, Shenzhen, China). A 1 mm hole was drilled, and the cannula (outer diameter: 0.64 mm; inner
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diameter: 0.45 mm) was unilaterally implanted above the lateral ventricle. The guide cannula was
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fixed to the skull via acrylic resin and two stainless steel screws. At the end of the surgery, a stylet with the same length as the guide cannula was inserted to prevent obstruction. The rats were
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allowed to recover for ten days in their home cages. 2.3. Drug infusion
All of the injections were performed in freely moving animals using a 5 µl microsyringe
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attached to a micro infusion pump (RWD, Shenzhen, China). The pro-inflammatory cytokine-inducer LPS (derived from E. coli serotype 0111:B4, No: L-2880, Sigma, St Louis, MO, USA) was diluted to 100 ng/µl with sterile saline and was infused intracerebroventricularly (i.c.v.) at a dose of 100 ng/rat (flow rate: 0.5 µl/min). This dose was chosen because it has previously been shown to significantly decrease social exploration and locomotor activity and increase immobility in rats [21, 22]. In accordance with our previous studies [20, 23], the animals received infusions of LPS or saline once every two days for five days (i.e., on days 1, 3, and 5). For the labelling of S-phase mitotic cells, the thymidine analogue bromodeoxyuridine (BrdU) (Sigma-Aldrich, St Louis, MO, USA) was diluted to 100 µg/µl in sterile saline and was infused i.c.v. at a dose of 200 µg/rat (flow rate: 1 µl/min). This dose could effectively label the newborn cells [24, 25]. The animals received two infusions of BrdU at a 6 h interval in one day.
ACCEPTED MANUSCRIPT 2.4. Experimental procedures Experiment 1. The effects of central LPS treatment on the protein expression of pro-inflammatory cytokines in the hippocampus.
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After the rats recovered from surgery, 18 rats were randomly assigned to two groups (9 rats per group) and administered central LPS or saline treatments, 1 h after light on once every two
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days (on days 1, 3, and 5) to examine the effects of the treatment on the protein expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the hippocampus (Fig. 1A). The rats were sacrificed 24 h after the last LPS infusion (on day 6). To examine and conform the
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neuroinflammation state, inflammatory cytokines were determined by two technical methods, i.e. samples from five rats in each group were used for western blotting, and samples from four rats
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in each group were used for immunohistochemistry.
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proliferation in the hippocampus.
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Experiment 2. The effects of central LPS treatment on depressive-like behaviors and cell
The same central LPS treatments were used as in Experiment 1. Twelve rats were randomly
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assigned to two groups (6 rats per group). The timeline was shown in Fig. 2A. To label the cell proliferation after LPS treatment, all rats were given BrdU infusions on day 6. Behavioral measurements were obtained on day 7. The behavioral tests were conducted between
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8:00-14:00. The order of testing was balanced by group: one rat from each group in sequence. Brain samples were dissected after the termination of behavioural measurements.
Experiment 3. The effects of central LPS treatment on depressive-like behaviors and newborn cell survival in the hippocampus. In this experiment, the same central LPS treatments were used as in Experiment 1. Twelve rats were randomly divided into two groups (6 rats per group). The timeline was shown in Fig. 3A. To label the newborn cell survival after LPS treatment, all rats were given BrdU infusions on day 0 before the LPS treatment. Behavioral measurements were obtained on day 6. Brain samples were dissected after the termination of behavioural measurements.
ACCEPTED MANUSCRIPT 2.5. Behavioral measurements Saccharin preference test (SPT) Three days of habituation occurred prior to the test. The rats were provided a bottle that
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contained water and a second bottle with a 0.5% saccharin solution during a 4 h window (8:00-12:00 a.m.) and were deprived of liquid at other times (20 h). On the test day, each rat was
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given the two bottles (saccharin and water) for 1 h between 8:00-9:00 a.m. after a 20 h deprivation of liquid. The intake amount of each liquid was determined by weighing the bottles before and after the 1 h window. The saccharin preference was calculated as the ratio of
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saccharin intake to the total intake (saccharin solution plus water intake). Intake of a reduced proportion of sweet solution has been used as a measurement of anhedonia, which is a core
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symptom of depression [26]. The positions of the bottles on the cages were changed after 30 min.
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After the preference test, the rats were given free access to water.
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Open field test (OFT)
Reduction of locomotor activity is one of characteristic symptoms of major depression [27],
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and is linked to reductions in energy generation in the depressive episode of mood disorder [28]. After the saccharin preference test, locomotor activity was measured for 5 min in an open field test [22, 29]. The apparatus was a circular arena, 180 cm in diameter with a 50 cm wall, in a
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room with a dim illumination (40 W). The rat was placed in the center of the field, and the variables of distance traveled and the number of rearing were recorded and analyzed as our previous work by a computer-based system (Ethovision software, Noldus Information Technology, Wageningen, The Netherlands) [8]. After each trial, the apparatus was cleaned with a 30% ethanol solution. The order of testing was balanced by group.
Tail suspension test (TST) The TST is a standardized test of depressive-like behavior in which depression is inferred from an increased duration of immobility [30, 31]. The rats were suspended from a steel beam (1500 cm in height with an 80 cm pothook) using adhesive tape at 4-5 cm from the tip of the tail. The rats’ behavior was videotaped for 5 min and scored by a highly trained observer blind to the treatment. Immobility was defined as a complete lack of movement in the four limbs and trunk.
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Novelty suppressed feeding test (NSFT) The NSFT is a conflict test that elicits competing motivations: the drive to eat and the fear of
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venturing into the center of the brightly lit arena [32]. Food was removed from the cage 24 h prior to testing. The rats were weighed just before they were deprived of food and again before
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testing to assess body weight loss. For testing, the food-deprived rats were placed in an open field (60 cm × 60 cm × 20 cm) with a small amount of food in the center. The amount of time to take the first bite was recorded as the latency to feed. The behaviors were videotaped, and the
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latency for each rat was scored offline by an experimenter blind to the condition of each rat. Home cage food intake during a 5 min period was measured immediately after the test as a
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2.6. Immunohistochemistry
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differences in hunger or motivation.
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control value to test whether feeding differences in the novel environment were because of
The animals were deeply anesthetized via an intraperitoneal infusion of a 10% chloral
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hydrate solution and were cardiac perfused with 200 ml of phosphate-buffered saline (PBS), followed by 200 ml of 4% paraformaldehyde (PFA). The brains were excised, and tissues from bregma -2 mm to bregma -5 mm were harvested and post-fixed in 4% PFA at 4 ℃ overnight.
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The brains were embedded in paraffin and cut into 5-μm-thick sections. The sections were dewaxed in xylenes and rehydrated via immersion in a series of graded alcohols (100%, 95%, 85%, and 75%). To unmask antigens, the sections were heated in 10 mM citrate buffer (pH 6.0) in a microwave oven at 80-85 ℃ for 15 min and were then cooled down for 2 h at room temperature (RT). For cytokine fluorescence staining, the procedures used were as previously described [33]. After antigen unmasking, the sections were washed in PBS and then incubated with a 5% normal goat serum solution for 1 h at RT. The sections were incubated with a rabbit anti-IL-1β antibody (1:1000; ab9787, Abcam, Cambridge, UK), a rabbit anti-IL-6 antibody (1:800; ab6672, Abcam) and a rabbit anti-TNF-α antibody (1:2000; ab66579, Abcam) overnight at 4 ℃. The sections were subsequently washed in PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100; Jackson, West Grove, USA) for 1 h at RT. Counterstaining was
ACCEPTED MANUSCRIPT performed with 4’,6-diamidino-2-phenylindole (DAPI). For BrdU labelling, the procedures used were as previously described [32, 34]. The slices were pre-treated with 2 N HCl at 37 ℃ for 30 min and neutralized with 0.1 M borate buffer (pH
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8.5) for 10 min. For diaminobenzidine (DAB) staining, endogenous peroxidases were blocked
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using a solution that contained 0.3% hydrogen peroxide for 15 min at 37 ℃ away from light.
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Serum blocking was performed with a 5% normal rabbit serum solution for 1 h at RT. The sections were incubated with a mouse anti-BrdU (1:100; BM0201, Boster, Wuhan, China) antibody overnight at 4 ℃. Then, the sections were washed and incubated with peroxidase-conjugated
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rabbit anti-mouse IgG (1:500; ZSGB-BIO, Beijing, China) for 1 h at RT, followed by 10 min of DAB reaction at RT. The DAB-stained sections were dehydrated in graded alcohols and xylenes and
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covered with neutral balsam. The fluorescent stained sections were covered with mounting medium. All of the sections were imaged using a fluorescence microscope (Leica Microsystems,
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Wetzlar, Germany), and the number of positive cells was counted using Image Pro-plus software
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(Media Cybernetics, Bethesda, MD, USA). Cells were counted within defined regions of interest in the dentate gyrus (DG, containing the subgranular zone, where adult neurogenesis occurs), which
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was delineated in each section according to the stereotaxic brain atlas (Paxinos and Watson, 2006). In total, ten 5-μm coronal sections were examined per animal, from bregma -2.92 mm to bregma -3.96 mm for the DG (plates 57-63, Paxinos & Watson, 2006). BrdU cell counting was
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carried out as previously described [35, 36]. To obtain the total number of BrdU cells for the given regions, the numbers from all of the sections counted were tallied and multiplied by 20, in accordance with the section sampling interval.
2.7. Western blot The rats were decapitated, and the brains were rapidly removed on ice for determining the protein levels of cytokine. The brain was placed in a stainless steel brain matrix, and the dorsal hippocampus were dissected bilaterally according to the atlas of Paxinos and Watson [37]. All of the tissues were placed into liquid nitrogen to be frozen. The tissue samples were homogenized in 20 volumes of buffer (pH 7.5, containing 50 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 0.05 mM okadaic acid, 1 µM sodium orthovanadate, 5 µg/ml pepstatin A and 0.5% Nonidet P-40). The protein content of the lysates was determined using BCA assay kits (Thermo, MA, USA). The
ACCEPTED MANUSCRIPT lysates were mixed with 5×loading buffer to prepare sample solutions of a certain concentration. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels, and then were moved to a 0.2 µm nitrocellulose (NC)
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filter (Sigma-Aldrich, St. Louis, MO, USA) by electrophoretic transfer. The NC filters were incubated in blocking buffer (5% nonfat dry milk powder in Tris-buffered saline containing 0.05%
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Tween-20, TBST) overnight at 4℃ and then washed for 10 min×3 in TBST. The NC filters were incubated with primary antibodies for IL-1β, IL-6 or TNF-α(1:2500, 1:1000, and 1:1000, respectively; Abcam, UK) and β-tubulin (1:1000; Santa, Germany) for 2 h at RT and then washed
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for 10 min×3 in TBST. The NC filters were incubated with the peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000; ZSGB-BIO, China) for 1 h at RT, then washed and
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treated with enhanced chemiluminescence (ECL) reagents (Pierce Biotechnology, Rockford, USA). The blots were exposed to film and analyzed using Quantity One 1-D analysis software (UVP,
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Upland, CA, USA). The relative protein levels were calculated from the ratio of the absorbance of
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the cytokines relative to the absorbance of the corresponding β-tubulin to correct for small
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difference in protein loading.
2.8. Statistical analysis
The values are presented as the mean ± the standard error of the mean (SEM). The data
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were analyzed using t test. The correlations were analyzed using Pearson’s correlation. The level of significance was set at p ≤ 0.05.
3. Results 3.1. Central LPS treatment induces an inflammatory state in the hippocampus The effects of LPS treatment on the protein levels of IL-1β, IL-6, and TNF-α in the hippocampus are shown in Fig. 1. IL-1β-positive cells were identified primarily in the polymorphic layer of the dentate gyrus (PoDG) and the CA3 pyramidal layer closest to the DG. IL-6 and TNF-α had similar labelling patterns in the hippocampus (Fig. 1B). The rats exposed to the LPS treatments showed an increased production of IL-1β (t = 2.47, p < 0.05), IL-6 (t = 4.25, p < 0.01), and TNF-α (t = 3.78, p < 0.01) in the dorsal hippocampus compared with the saline control group (Fig. 1C-D). The results obtained from both western blotting and immunohistochemistry
ACCEPTED MANUSCRIPT demonstrated that the central LPS treatments induced a significant neuroinflammatory state in the hippocampus.
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Fig.1. near here. 1.5 column.
3.2. Central LPS treatment inhibits cell proliferation in the hippocampus and induces
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depressive-like behaviors
To determine the effects of neuroinflammation on adult hippocampal neurogenesis, cell proliferation was examined. Representative BrdU-stained sections are shown in Fig. 2B&C. The
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quantitative analyses of BrdU-positive cell numbers are shown in Fig. 2D. Compared with the Saline group, the rats of LPS group showed a decreased number of BrdU-positive cells in the
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dentate gyrus (t = 7.05, p < 0.001), indicating inhibition of cell proliferation. The depressive-like behaviors measured after LPS treatment are shown in Fig. 2E-I. In SPT,
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compared with the saline control group, the rats exposed to LPS exhibited a significant decrease
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in saccharin preference (t = 5.21, p < 0.001), a symptom of anhedonia. The total liquid intake in SPT did not differ between the groups (Saline: 24.82±3.47 g; LPS: 20.33±4.52 g) (t = 0.79, p >
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0.05), indicating LPS treatment did not influence physical function of drinking. In TST, compared with the saline control group, the rats exposed to LPS exhibited a significant increase in immobility time (t = 4.08, p < 0.01), indicating that LPS treatment induced the hopelessness. In
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OFT, compared with the saline control group, the rats exposed to LPS exhibited a significant decrease in distance travelled (t = 2.37, p < 0.05) and in rearing (t = 3.47, p < 0.01), indicating that LPS treatment induced reduction in locomotor activities and exploration. In NSFT, compared with the saline control group, the rats exposed to LPS exhibited a significant increase in latency to feed (t = 3.17, p < 0.05), indicating low motivation to feed in a novel environment. However, Home cage food intake in NSFT did not differ between the groups (Saline: 7.12±1.55 g; LPS: 7.23±1.32 g) (t = 0.06, p > 0.05), indicating LPS treatment did not influence physical function of eating.
Fig.2. near here. 2 column.
ACCEPTED MANUSCRIPT 3.3. Central LPS treatment inhibits newborn cell survival in the hippocampus and induces depressive-like behaviors To further examine the effects of neuroinflammation on adult hippocampal neurogenesis,
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newborn cell survival was examined. Representative BrdU-stained sections are shown in Fig. 3B&C. The quantitative analyses of BrdU-positive cell numbers are shown in Fig. 3D. In the LPS
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group, the number of BrdU-positive cells in the dentate gyrus was less than the Saline group (t = 5.96, p < 0.001), indicating inhibition of newborn cell survival in the neuroinflammatory state. The depressive-like behaviors measured after LPS treatment are shown in Fig. 3E-I. In SPT,
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compared with the saline control group, the rats exposed to LPS exhibited a significant decrease in saccharin preference (t = 6.28, p < 0.001), whereas the total liquid intake in SPT did not differ
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between the groups (Saline: 24.43±2.86 g; LPS: 17.68±4.07 g) (t = 0.55, p > 0.05), indicating LPS treatment induced anhedonia but not physical function of drinking. In TST, compared with the
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saline control group, the rats exposed to LPS exhibited a significant increase in immobility time (t
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= 3.87, p < 0.01), indicating that LPS treatment induced the hopelessness. In OFT, compared with the saline control group, the rats exposed to LPS exhibited a significant decrease in distance
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travelled (t = 3.01, p < 0.05) and in number of rearing (t = 2.36, p < 0.05), indicating that LPS treatment induced reduction in locomotor activities and exploration. In NSFT, compared with the saline control group, the rats exposed to LPS exhibited a significant increase in latency to feed (t =
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4.24, p < 0.01), but home cage food intake in NSFT did not differ between the groups (Saline: 6.58 ±1.29 g; LPS: 7.07±1.84 g) (t = 0.22, p > 0.05), indicating LPS treatment induced low motivation to feed in a novel environment, which was not because of the differences in hunger. All the results in this experiment were similar to the results from Experiment 2.
Fig.3 near here. 2 column. 3.4. Depressive-like behaviors correlate with neurogenesis in the hippocampus To examine whether the function of hippocampal neurogenesis was related to the behavioral performance, Pearson’s correlation analysis was performed by using the data from both the control and LPS group as illustrated by Silverman, et al. [29]. The analysis indicated that most of the behavioral parameters correlated with the number of proliferating cells and the number of survival new born cells. For cell proliferation, as shown in Fig. 4A-E, the BrdU-positive
ACCEPTED MANUSCRIPT cell number in proliferation groups were positively correlated with the ratio of saccharin intake (r = 0.85, p < 0.001) and the distance travelled in the open field (r = 0.74, p < 0.01) and were negatively correlated with the immobility time in the TST (r = -0.77, p < 0.01) and the latency to
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feed in the NSFT (r = -0.61, p < 0.05). The BrdU-positive cell number in proliferation were not significantly correlated with the rearing times in the open field (r = -0.51, p > 0.05). For newborn
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cell survival, as shown in Fig. 4F-J, the BrdU-positive cell number in survival groups were positively correlated with the ratio of saccharin intake (r = 0.81, p < 0.01) and the distance travelled (r = 0.66, p < 0.05) and the rearing times (r = 0.61, p < 0.05) in the open field and
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negatively correlated with the immobility time in the TST (r = -0.70, p < 0.05) and the latency to
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feed in the NSFT (r = -0.71, p < 0.01).
Fig.4. near here. 2 column.
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To make sure whether the function of hippocampal neurogenesis was specifically correlated
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to the neuroinflamation induced depressive-like behaviors, Pearson’s correlation analysis was further performed by using the data separately from the LPS group or the control group (n=6 for
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each group). For LPS group, the analysis showed that the decreased hippocampal neurogenesis in cell proliferation was significantly correlated with the depressive-like phenotypes of decreased saccharine preference (r = 0.81, p < 0.05) and distance traveled (r = 0.81, p < 0.05), indicating the
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core and characteristic symptoms of major depression were linked to hippocampal neurogenesis. There exist no significant correlations between behavioral parameters and the parameters of hippocampal neurogenesis in the control group.
4. Discussion The
results
demonstrated
that
triple
repeated
central
LPS
challenge
evoked
neuroinflammation in the hippocampus of rats that was marked by elevated protein levels of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. This neuroinflammatory state induced significant depressive-like behaviors in the rats. These neuroinflammatory responses also inhibited adult hippocampal neurogenesis, which was marked by decreased BrdU positive cell number. The degree of inhibition in adult hippocampal neurogenesis was closely correlated with the level of depressive-like behaviors under neuroinflammation state. The present findings
ACCEPTED MANUSCRIPT provide the first evidence that hippocampal neurogenesis dysfunction is closely related to neuroinflammation-induced depression.
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Consistent with the results of our previous studies [20, 22, 23], the results of the present study confirmed that repeated central infusion of LPS induced significant depressive-like
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behaviors, including decreased locomotor activity in the open field, decreased saccharin preference, increased immobility time in the TST, and increased latency to food in the NSFT. Although LPS is frequently used in studies of depression [7, 38], the depressive-like behaviour
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induced by a single injection of LPS, regardless of peripheral or central administration, usually lasts a few hours, seldom longer than 24 h [8, 21, 23], whereas repeated peripheral LPS injections
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induce immune and behavioural tolerance [39-41]. The altered behavior within 24h post-LPS are often argued as being sickness behavior or proposed to be a mixture of both sickness behavior
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and depressive-like behavior [2, 30]. It has been demonstrated that the acute sickness behavioral
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effects (2-4 h), at least in mice, have been dissociated from longer lasting depressive-like behavioral effects (24-28 h) following LPS injection [7, 30]. In the present study, the altered
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behaviors was observed 24h after central LPS treatment and thus was regarded as depressive-like behavior. Importantly, the present study demonstrated the induction of a neuroinflammatory state by central LPS challenge, by showing that hippocampal production of
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pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) was significantly increased 24 h post-LPS. Positive cells for IL-1β, IL-6, and TNF-α were identified primarily in the polymorphic layer of the dentate gyrus (PoDG) and the CA3 pyramidal layer closest to the DG. The present results, along with the results of our previous studies, make repeated central LPS challenge a reliable and useful animal model of neuroinflammation-induced depression.
With the advantage of this new animal model of depression, we further examined the changes in adult hippocampal neurogenesis under the neuroinflammatory state and found that both cell proliferation and new born cell survival were inhibited by central LPS challenge. The observation that brain LPS infusion impairs the survival of new born cells is in agreement with previous findings in which a role for neuroinflammation in regulating the survival stage of the neurogenic process was demonstrated [42]. It had also been reported that an intraperitoneal
ACCEPTED MANUSCRIPT infection of LPS significantly decreased survival of new born cells and immature neurons [19]. However, these initial works and later replication reports are only limited to the effect of inflammation on the survival stage of neurogenesis [19, 42, 43]. Notably, our data demonstrate
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that repeated central LPS infusions affect not only survival of new cells but also cell proliferation, indicating the effect of neuroinflammation on hippocampal neurogenesis is involved in
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decreasing both survival and proliferation components.
To elucidate whether the impairment of hippocampal neurogenesis was related to the
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behavioral performances, we plotted the behavioral performance parameters against the BrdU-positive cell numbers for each animal. Pearson’s correlation identified a strong correlation
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between the depressive-like behaviors and the inhibition of neurogenesis. Notably, a further Pearson’s correlation analysis by using the data independently from the LPS group indicated that
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the decreased hippocampal neurogenesis in cell proliferation was significantly correlated with
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the decreased saccharine preference and the distance traveled, core and characteristic phenotypes of depression. In contrast, the reports of previous studies regarding the correlation
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between the inhibition of neurogenesis and depression in animal stress models have been inconsistent. Several previous studies have suggested that decreased hippocampal neurogenesis was associated with depressive-like phenotypes induced by stress [44, 45]. However, other
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studies have demonstrated that a stress-induced decrease in neurogenesis did not correlate with an anhedonic phenotype [46, 47]. One explanation for these conflicting data is that the relationship between behavioral performance and neurogenesis may be dependent on the method of assessment, timing, or extent of neurogenesis disruption, namely, the nature and effects of the stressors. Indeed, it has been reported that several stressors fail to reduce adult hippocampal neurogenesis [48]. Interestingly, our finding is in accord with the findings reported by Süß et al. in which severe peripheral inflammation did not induce neuroinflammation and impairment of hippocampal neurogenesis, and also failed to induce depressive-like behavior, but locomotor deficits [49]. Both work suggested, in opposite ways, that the hippocampal neurogenesis might be one of biological mechanisms underlying depression induced by neuroinflammation. Further research as to whether blockade of inhibition of neurogenesis could reverse neuroinflammation-induced depression is currently in progress.
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In conclusion, the present study demonstrated that neuroinflammation evoked by triple repeated central LPS challenge induced depressive-like behaviors and inhibited adult
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hippocampal neurogenesis. The depressive-like behavioral symptoms were correlated with changes in adult hippocampal neurogenesis. These findings provide the first demonstration that
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hippocampal neurogenesis dysfunction is associated with neuroinflammation-induced depression, which suggested that the dysfunction of hippocampal neurogenesis might be one of biological
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mechanisms underlying depression induced by neuroinflammation.
Acknowledgements
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This study was supported by the grants from the National Natural Science Foundation of China (31170987, 31440045, and 30770718) and the key laboratory of Mental Health of Chinese
Conflict of Interest
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Academy of Sciences (KLMH20142G01).
References
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The authors have no conflicts of interest to declare.
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Figure legends
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Fig. 1. Central LPS challenge induced an inflammatory state in the hippocampus. (A) Rats were given triple LPS infusions in the lateral ventricle, and brain samples were collected 24 h after the LPS infusions. (B) Immunohistochemistry indicated that increased pro-inflammatory
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cytokine proteins are present in the PoDG and CA3 after LPS administration. IL-1β, IL-6 and TNF-α (green); DAPI (blue). (C&D) Western blots indicated the production of dorsal hippocampal IL-1β, IL-6 and TNF-α increased after exposure to LPS (n = 5 per group). * p < 0.05; ** p < 0.01. The data are presented as the mean ± SEM. IL, interleukin; TNF, tumor necrosis factor; LPS, lipopolysaccharide;
PoDG,
polymorphic
layer
of
the
dentate
gyrus;
DAPI,
4’,6-diamidino-2-phenylindole.
Fig. 2. Effects of central LPS challenge on cell proliferation in the hippocampus and depressive-like behavior. (A) To examine the effect of central LPS challenge on cell proliferation, BrdU infusions were given after LPS infusions. (B) Representative BrdU-stained sections of Saline group. (C) Representative BrdU-stained sections of LPS group. (D) BrdU-positive cell number in
ACCEPTED MANUSCRIPT dentate gyrus of hippocampus was lower in LPS rats. (E) Saccharin preference. (F) Immobility in the TST. (G) Distance travelled during 5 min in an open field. (H) Rearing behavior in open field. (I) Latency to feed in the NSFT. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are presented as
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the mean ± SEM. n = 6 per group. LPS, lipopolysaccharide; BrdU, Bromodeoxyuridine; SPT, saccharin preference test; OFT, open field test; TST, tail suspension test; NSFT, novelty suppressed
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feeding test.
Fig. 3. Effects of central LPS challenge on and newborn cell survival in the hippocampus
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and depressive-like behavior. (A) To examine the effect of central LPS challenge on newborn cell survival, BrdU infusions were given before LPS challenge. (B) Representative BrdU-stained
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sections of Saline group. (C) Representative BrdU-stained sections of LPS group. (D) BrdU-positive cell number in dentate gyrus of hippocampus was lower in LPS rats. (E) Saccharin preference. (F)
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Immobility in the TST. (G) Distance travelled during 5 min in an open field. (H) Rearing behavior in
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open field. (I) Latency to feed in the NSFT. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are presented as the mean ± SEM. n = 6 per group. LPS, lipopolysaccharide; BrdU,
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Bromodeoxyuridine; SPT, saccharin preference test; OFT, open field test; TST, tail suspension test; NSFT, novelty suppressed feeding test.
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Fig. 4. Correlation between behavioral performances and neurogenesis in the dentate gyrus. (A-E) Behavioral performance parameters correlated with the cell proliferation indicated by BrdU-positive cell number in the dentate gyrus. (F-J) Behavioral performance parameters correlated with the cell survival indicated by BrdU-positive cell number in the dentate gyrus. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are presented as the mean ± SEM. BrdU, Bromodeoxyuridine; LPS, lipopolysaccharide.
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Highlights Repeated central lipopolysaccharide infusions cause neuroinflammation in hippocampus; Neuroinflammation induces depressive-like behavior; Neuroinflammation impairs adult hippocampal neurogenesis; Depressive-like behavior correlates with hippocampal neurogenesis.
S0031-9384(16)30177-9 doi: 10.1016/j.physbeh.2016.04.034 PHB 11310
To appear in:
Physiology & Behavior
Received date: Revised date: Accepted date:
4 January 2016 13 April 2016 15 April 2016
Please cite this article as: Tang Ming-ming, Lin Wen-juan, Pan Yu-qin, Guan Xi-ting, Li Ying-cong, Hippocampal neurogenesis dysfunction linked to depressive-like behaviors in a neuroinflammation induced model of depression, Physiology & Behavior (2016), doi: 10.1016/j.physbeh.2016.04.034
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ACCEPTED MANUSCRIPT Hippocampal
neurogenesis
dysfunction
linked
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neuroinflammation induced model of depression
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Ming-ming Tang a,c，Wen-juan Lin a,b,*, Yu-qin Pan a, b , Xi-ting Guan a,c , Ying-cong Li a
Key Laboratory of Mental Health, Institute of Psychology, Chinese Academy of Sciences, Beijing
100101, China
Brain-Behavior Research Center, Institute of Psychology, Chinese Academy of Sciences, Beijing
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b
100101, China
University of Chinese Academy of Sciences, Beijing, China
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* Corresponding author at: 16 Lincui Road, Beijing 100101, China. Tel: 86-10-64853723, Fax:
Abstract
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86-10-64872070, Email: [email protected].
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Our previous work found that triple central lipopolysaccharide (LPS) administration could induce depressive-like behaviours and increased central pro-inflammatory cytokines mRNA, hippocampal cytokine mRNA in particular. Since several neruoinflammation-associated conditions have been reported to impair neurogenesis, in this study, we further investigated whether the neruoinflammation induced depression would be associated with hippocampal neurogenesis dysfunction. An animal model of depression induced by triple central lipopolysaccharide (LPS) administration was used. In the hippocampus, the neuroinflammatory state evoked by LPS was marked by an increased production of pro-inflammatory cytokines, including interleukin-1β, interleukin- 6, and tumor necrosis factor-α. It was found that rats in the neuroinflammatory state exhibited depressive-like behaviors, including reduced saccharin preference and locomotor activity as well as increased immobility time in the tail suspension test and latency to feed in the novelty suppressed feeding test. Adult hippocampal neurogenesis was
ACCEPTED MANUSCRIPT concomitantly inhibited, including decreased cell proliferation and newborn cell survival. We also demonstrated that the decreased hippocampal neurogenesis in cell proliferation was significantly correlated with the depressive-like phenotypes of decreased saccharine preference and distance
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traveled, the core and characteristic symptoms of depression, under neuro inflammation state. These findings provide the first evidence that hippocampal neurogenesis dysfunction is
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correlated with neuroinflammation-induced depression, which suggests that hippocampal neurogenesis might be one of biological mechanisms underlying depression induced by
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neruoinflammation.
Key words
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Depressive-like behavior; Neuroinflammation; Neurogenesis; Pro-inflammatory cytokine;
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Lipopolysaccharide;
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1. Introduction
It has been proposed that the actions of inflammatory processes, and of pro-inflammatory
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cytokines in particular, on the brain may account for the pathogenesis of depressive disorders, and this hypothesis has been referred to as the “cytokine or inflammatory hypothesis of depression” [1-5]. Although this hypothesis has been an interesting research focus for more than
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two decades, how neuronal-immune interactions modulate the depressive phenotype is still not fully understood. Inflammatory processes may affect more than one pathway, which is thought to be important in the pathogenesis of depression, such as monoamine alterations, glutamate neurotransmission, and glucocorticoid receptor resistance [6-8]. Novel theory suggests that adult hippocampal neurogenesis, as a novel pathway, may also be involved in depressive disorders caused by inflammation [9, 10]. Neurogenesis is the complex process of generating new neurons from neural stem or progenitor cells that occurs in the brains of many animal species, including humans [11]. Adult neurogenesis primarily occurs in the subventricular zone (SVZ) of the lateral ventricles and in the subgranular zone (SGZ) of the dentate gyrus in the hippocampus [12]. The process of adult neurogenesis involves several steps, including proliferation, specification of the fate of neural progenitors, neuronal migration, neuronal maturation and synaptic integration of the young
ACCEPTED MANUSCRIPT neurons into the existing neuronal circuitry. It has been reported that adult hippocampal neurogenesis is related to synaptic plasticity, cognitive functioning, and psychiatric diseases [13, 14]. Neurogenesis in the dentate gyrus of the hippocampus is negatively regulated by stressful
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experiences [15] and positively regulated by chronic antidepressant treatment [16]. Recent evidence indicates that neurogenesis is influenced by inflammatory processes [17-19]. Recently,
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we found that triple central lipopolysaccharide (LPS) administration induced depressive-like behaviours and increased central pro-inflammatory cytokines mRNA, hippocampal cytokine mRNA in particular. We also found that the increased hippocampal TNF-α mRNA expression was
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the common affected factor in both chronic stress induced and LPS induced models of depression [20]. These results suggest that hippocampal inflammation may have a pivotal role in depression.
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However, it is unknown how hippocampal neurogenesis is affected by triple central LPS administration and whether the altered hippocampal neurogenesis contributes to the regulation
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of depression induced by neuroinflammation.
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Thus, the purpose of the present study was to determine the relationship between hippocampal neurogenesis and the depressive-like behavior induced by inflammation. In this
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study, we first examined the neuroinflammatory state in the hippocampus induced by triple central LPS administration by determining the protein expression of IL-1β, IL-6 and TNF-α. We then examined depressive-like behaviors and adult hippocampal neurogenesis under
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neuroinflammatory state and evaluated the correlation between the degree of depressive-like behaviors and the level of altered neurogenesis.
2. Materials and methods 2.1. Animals Male Sprague-Dawley rats that weighed 220-240 g were obtained from Vital River Laboratories (Beijing, China). All of the rats were individually housed in standard stainless steel cages in a temperature- and humidity-controlled room (22±1℃; 40-60%) with a 12:12 dark/light cycle (lights on at 07:00 am; off at 7:00 pm). The rats were given free access to food and water throughout the experiment except for the saccharin preference test time and novelty suppressed feeding test time. The rats were given 5 min of daily handling for 7 days prior to experimental use to minimize stress responses to the experimental manipulation. The experimental procedures
ACCEPTED MANUSCRIPT were approved by the Institutional Review Board of the Institute of Psychology, Chinese Academy of Sciences and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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2.2. Surgical procedures
After acclimation, the rats were anesthetized with sodium pentobarbital (1%, 35 mg/Kg;
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Merck, Darmstadt, Germany) via an intraperitoneal injection, and guide cannulas were implanted (RWD Life Science Co., Ltd, Shenzhen, China) into the lateral ventricle (AP: - 0.9 mm from bregma; LM: 1.1 mm from the sagittal suture; DV: 3.2 mm in depth relative to the skull). The top of the
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animal’s head was shaved, and the head was fixed in a stereotaxic instrument (Stoelting, Wood Dale, IL, USA), with the incisor bar set at 3.3 mm below the interaural line. Body temperature was
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maintained at 37 ℃ using a rectal thermometer and a feedback-controlled heating pad (RWD, Shenzhen, China). A 1 mm hole was drilled, and the cannula (outer diameter: 0.64 mm; inner
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diameter: 0.45 mm) was unilaterally implanted above the lateral ventricle. The guide cannula was
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fixed to the skull via acrylic resin and two stainless steel screws. At the end of the surgery, a stylet with the same length as the guide cannula was inserted to prevent obstruction. The rats were
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allowed to recover for ten days in their home cages. 2.3. Drug infusion
All of the injections were performed in freely moving animals using a 5 µl microsyringe
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attached to a micro infusion pump (RWD, Shenzhen, China). The pro-inflammatory cytokine-inducer LPS (derived from E. coli serotype 0111:B4, No: L-2880, Sigma, St Louis, MO, USA) was diluted to 100 ng/µl with sterile saline and was infused intracerebroventricularly (i.c.v.) at a dose of 100 ng/rat (flow rate: 0.5 µl/min). This dose was chosen because it has previously been shown to significantly decrease social exploration and locomotor activity and increase immobility in rats [21, 22]. In accordance with our previous studies [20, 23], the animals received infusions of LPS or saline once every two days for five days (i.e., on days 1, 3, and 5). For the labelling of S-phase mitotic cells, the thymidine analogue bromodeoxyuridine (BrdU) (Sigma-Aldrich, St Louis, MO, USA) was diluted to 100 µg/µl in sterile saline and was infused i.c.v. at a dose of 200 µg/rat (flow rate: 1 µl/min). This dose could effectively label the newborn cells [24, 25]. The animals received two infusions of BrdU at a 6 h interval in one day.
ACCEPTED MANUSCRIPT 2.4. Experimental procedures Experiment 1. The effects of central LPS treatment on the protein expression of pro-inflammatory cytokines in the hippocampus.
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After the rats recovered from surgery, 18 rats were randomly assigned to two groups (9 rats per group) and administered central LPS or saline treatments, 1 h after light on once every two
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days (on days 1, 3, and 5) to examine the effects of the treatment on the protein expression of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) in the hippocampus (Fig. 1A). The rats were sacrificed 24 h after the last LPS infusion (on day 6). To examine and conform the
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neuroinflammation state, inflammatory cytokines were determined by two technical methods, i.e. samples from five rats in each group were used for western blotting, and samples from four rats
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in each group were used for immunohistochemistry.
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proliferation in the hippocampus.
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Experiment 2. The effects of central LPS treatment on depressive-like behaviors and cell
The same central LPS treatments were used as in Experiment 1. Twelve rats were randomly
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assigned to two groups (6 rats per group). The timeline was shown in Fig. 2A. To label the cell proliferation after LPS treatment, all rats were given BrdU infusions on day 6. Behavioral measurements were obtained on day 7. The behavioral tests were conducted between
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8:00-14:00. The order of testing was balanced by group: one rat from each group in sequence. Brain samples were dissected after the termination of behavioural measurements.
Experiment 3. The effects of central LPS treatment on depressive-like behaviors and newborn cell survival in the hippocampus. In this experiment, the same central LPS treatments were used as in Experiment 1. Twelve rats were randomly divided into two groups (6 rats per group). The timeline was shown in Fig. 3A. To label the newborn cell survival after LPS treatment, all rats were given BrdU infusions on day 0 before the LPS treatment. Behavioral measurements were obtained on day 6. Brain samples were dissected after the termination of behavioural measurements.
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contained water and a second bottle with a 0.5% saccharin solution during a 4 h window (8:00-12:00 a.m.) and were deprived of liquid at other times (20 h). On the test day, each rat was
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given the two bottles (saccharin and water) for 1 h between 8:00-9:00 a.m. after a 20 h deprivation of liquid. The intake amount of each liquid was determined by weighing the bottles before and after the 1 h window. The saccharin preference was calculated as the ratio of
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saccharin intake to the total intake (saccharin solution plus water intake). Intake of a reduced proportion of sweet solution has been used as a measurement of anhedonia, which is a core
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symptom of depression [26]. The positions of the bottles on the cages were changed after 30 min.
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After the preference test, the rats were given free access to water.
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Open field test (OFT)
Reduction of locomotor activity is one of characteristic symptoms of major depression [27],
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and is linked to reductions in energy generation in the depressive episode of mood disorder [28]. After the saccharin preference test, locomotor activity was measured for 5 min in an open field test [22, 29]. The apparatus was a circular arena, 180 cm in diameter with a 50 cm wall, in a
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room with a dim illumination (40 W). The rat was placed in the center of the field, and the variables of distance traveled and the number of rearing were recorded and analyzed as our previous work by a computer-based system (Ethovision software, Noldus Information Technology, Wageningen, The Netherlands) [8]. After each trial, the apparatus was cleaned with a 30% ethanol solution. The order of testing was balanced by group.
Tail suspension test (TST) The TST is a standardized test of depressive-like behavior in which depression is inferred from an increased duration of immobility [30, 31]. The rats were suspended from a steel beam (1500 cm in height with an 80 cm pothook) using adhesive tape at 4-5 cm from the tip of the tail. The rats’ behavior was videotaped for 5 min and scored by a highly trained observer blind to the treatment. Immobility was defined as a complete lack of movement in the four limbs and trunk.
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Novelty suppressed feeding test (NSFT) The NSFT is a conflict test that elicits competing motivations: the drive to eat and the fear of
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venturing into the center of the brightly lit arena [32]. Food was removed from the cage 24 h prior to testing. The rats were weighed just before they were deprived of food and again before
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testing to assess body weight loss. For testing, the food-deprived rats were placed in an open field (60 cm × 60 cm × 20 cm) with a small amount of food in the center. The amount of time to take the first bite was recorded as the latency to feed. The behaviors were videotaped, and the
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latency for each rat was scored offline by an experimenter blind to the condition of each rat. Home cage food intake during a 5 min period was measured immediately after the test as a
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2.6. Immunohistochemistry
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differences in hunger or motivation.
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control value to test whether feeding differences in the novel environment were because of
The animals were deeply anesthetized via an intraperitoneal infusion of a 10% chloral
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hydrate solution and were cardiac perfused with 200 ml of phosphate-buffered saline (PBS), followed by 200 ml of 4% paraformaldehyde (PFA). The brains were excised, and tissues from bregma -2 mm to bregma -5 mm were harvested and post-fixed in 4% PFA at 4 ℃ overnight.
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The brains were embedded in paraffin and cut into 5-μm-thick sections. The sections were dewaxed in xylenes and rehydrated via immersion in a series of graded alcohols (100%, 95%, 85%, and 75%). To unmask antigens, the sections were heated in 10 mM citrate buffer (pH 6.0) in a microwave oven at 80-85 ℃ for 15 min and were then cooled down for 2 h at room temperature (RT). For cytokine fluorescence staining, the procedures used were as previously described [33]. After antigen unmasking, the sections were washed in PBS and then incubated with a 5% normal goat serum solution for 1 h at RT. The sections were incubated with a rabbit anti-IL-1β antibody (1:1000; ab9787, Abcam, Cambridge, UK), a rabbit anti-IL-6 antibody (1:800; ab6672, Abcam) and a rabbit anti-TNF-α antibody (1:2000; ab66579, Abcam) overnight at 4 ℃. The sections were subsequently washed in PBS and incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG (1:100; Jackson, West Grove, USA) for 1 h at RT. Counterstaining was
ACCEPTED MANUSCRIPT performed with 4’,6-diamidino-2-phenylindole (DAPI). For BrdU labelling, the procedures used were as previously described [32, 34]. The slices were pre-treated with 2 N HCl at 37 ℃ for 30 min and neutralized with 0.1 M borate buffer (pH
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8.5) for 10 min. For diaminobenzidine (DAB) staining, endogenous peroxidases were blocked
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using a solution that contained 0.3% hydrogen peroxide for 15 min at 37 ℃ away from light.
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Serum blocking was performed with a 5% normal rabbit serum solution for 1 h at RT. The sections were incubated with a mouse anti-BrdU (1:100; BM0201, Boster, Wuhan, China) antibody overnight at 4 ℃. Then, the sections were washed and incubated with peroxidase-conjugated
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rabbit anti-mouse IgG (1:500; ZSGB-BIO, Beijing, China) for 1 h at RT, followed by 10 min of DAB reaction at RT. The DAB-stained sections were dehydrated in graded alcohols and xylenes and
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covered with neutral balsam. The fluorescent stained sections were covered with mounting medium. All of the sections were imaged using a fluorescence microscope (Leica Microsystems,
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Wetzlar, Germany), and the number of positive cells was counted using Image Pro-plus software
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(Media Cybernetics, Bethesda, MD, USA). Cells were counted within defined regions of interest in the dentate gyrus (DG, containing the subgranular zone, where adult neurogenesis occurs), which
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was delineated in each section according to the stereotaxic brain atlas (Paxinos and Watson, 2006). In total, ten 5-μm coronal sections were examined per animal, from bregma -2.92 mm to bregma -3.96 mm for the DG (plates 57-63, Paxinos & Watson, 2006). BrdU cell counting was
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carried out as previously described [35, 36]. To obtain the total number of BrdU cells for the given regions, the numbers from all of the sections counted were tallied and multiplied by 20, in accordance with the section sampling interval.
2.7. Western blot The rats were decapitated, and the brains were rapidly removed on ice for determining the protein levels of cytokine. The brain was placed in a stainless steel brain matrix, and the dorsal hippocampus were dissected bilaterally according to the atlas of Paxinos and Watson [37]. All of the tissues were placed into liquid nitrogen to be frozen. The tissue samples were homogenized in 20 volumes of buffer (pH 7.5, containing 50 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 0.05 mM okadaic acid, 1 µM sodium orthovanadate, 5 µg/ml pepstatin A and 0.5% Nonidet P-40). The protein content of the lysates was determined using BCA assay kits (Thermo, MA, USA). The
ACCEPTED MANUSCRIPT lysates were mixed with 5×loading buffer to prepare sample solutions of a certain concentration. The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% polyacrylamide gels, and then were moved to a 0.2 µm nitrocellulose (NC)
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filter (Sigma-Aldrich, St. Louis, MO, USA) by electrophoretic transfer. The NC filters were incubated in blocking buffer (5% nonfat dry milk powder in Tris-buffered saline containing 0.05%
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Tween-20, TBST) overnight at 4℃ and then washed for 10 min×3 in TBST. The NC filters were incubated with primary antibodies for IL-1β, IL-6 or TNF-α(1:2500, 1:1000, and 1:1000, respectively; Abcam, UK) and β-tubulin (1:1000; Santa, Germany) for 2 h at RT and then washed
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for 10 min×3 in TBST. The NC filters were incubated with the peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5000; ZSGB-BIO, China) for 1 h at RT, then washed and
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treated with enhanced chemiluminescence (ECL) reagents (Pierce Biotechnology, Rockford, USA). The blots were exposed to film and analyzed using Quantity One 1-D analysis software (UVP,
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Upland, CA, USA). The relative protein levels were calculated from the ratio of the absorbance of
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the cytokines relative to the absorbance of the corresponding β-tubulin to correct for small
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difference in protein loading.
2.8. Statistical analysis
The values are presented as the mean ± the standard error of the mean (SEM). The data
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were analyzed using t test. The correlations were analyzed using Pearson’s correlation. The level of significance was set at p ≤ 0.05.
3. Results 3.1. Central LPS treatment induces an inflammatory state in the hippocampus The effects of LPS treatment on the protein levels of IL-1β, IL-6, and TNF-α in the hippocampus are shown in Fig. 1. IL-1β-positive cells were identified primarily in the polymorphic layer of the dentate gyrus (PoDG) and the CA3 pyramidal layer closest to the DG. IL-6 and TNF-α had similar labelling patterns in the hippocampus (Fig. 1B). The rats exposed to the LPS treatments showed an increased production of IL-1β (t = 2.47, p < 0.05), IL-6 (t = 4.25, p < 0.01), and TNF-α (t = 3.78, p < 0.01) in the dorsal hippocampus compared with the saline control group (Fig. 1C-D). The results obtained from both western blotting and immunohistochemistry
ACCEPTED MANUSCRIPT demonstrated that the central LPS treatments induced a significant neuroinflammatory state in the hippocampus.
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Fig.1. near here. 1.5 column.
3.2. Central LPS treatment inhibits cell proliferation in the hippocampus and induces
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depressive-like behaviors
To determine the effects of neuroinflammation on adult hippocampal neurogenesis, cell proliferation was examined. Representative BrdU-stained sections are shown in Fig. 2B&C. The
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quantitative analyses of BrdU-positive cell numbers are shown in Fig. 2D. Compared with the Saline group, the rats of LPS group showed a decreased number of BrdU-positive cells in the
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dentate gyrus (t = 7.05, p < 0.001), indicating inhibition of cell proliferation. The depressive-like behaviors measured after LPS treatment are shown in Fig. 2E-I. In SPT,
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compared with the saline control group, the rats exposed to LPS exhibited a significant decrease
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in saccharin preference (t = 5.21, p < 0.001), a symptom of anhedonia. The total liquid intake in SPT did not differ between the groups (Saline: 24.82±3.47 g; LPS: 20.33±4.52 g) (t = 0.79, p >
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0.05), indicating LPS treatment did not influence physical function of drinking. In TST, compared with the saline control group, the rats exposed to LPS exhibited a significant increase in immobility time (t = 4.08, p < 0.01), indicating that LPS treatment induced the hopelessness. In
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OFT, compared with the saline control group, the rats exposed to LPS exhibited a significant decrease in distance travelled (t = 2.37, p < 0.05) and in rearing (t = 3.47, p < 0.01), indicating that LPS treatment induced reduction in locomotor activities and exploration. In NSFT, compared with the saline control group, the rats exposed to LPS exhibited a significant increase in latency to feed (t = 3.17, p < 0.05), indicating low motivation to feed in a novel environment. However, Home cage food intake in NSFT did not differ between the groups (Saline: 7.12±1.55 g; LPS: 7.23±1.32 g) (t = 0.06, p > 0.05), indicating LPS treatment did not influence physical function of eating.
Fig.2. near here. 2 column.
ACCEPTED MANUSCRIPT 3.3. Central LPS treatment inhibits newborn cell survival in the hippocampus and induces depressive-like behaviors To further examine the effects of neuroinflammation on adult hippocampal neurogenesis,
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newborn cell survival was examined. Representative BrdU-stained sections are shown in Fig. 3B&C. The quantitative analyses of BrdU-positive cell numbers are shown in Fig. 3D. In the LPS
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group, the number of BrdU-positive cells in the dentate gyrus was less than the Saline group (t = 5.96, p < 0.001), indicating inhibition of newborn cell survival in the neuroinflammatory state. The depressive-like behaviors measured after LPS treatment are shown in Fig. 3E-I. In SPT,
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compared with the saline control group, the rats exposed to LPS exhibited a significant decrease in saccharin preference (t = 6.28, p < 0.001), whereas the total liquid intake in SPT did not differ
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between the groups (Saline: 24.43±2.86 g; LPS: 17.68±4.07 g) (t = 0.55, p > 0.05), indicating LPS treatment induced anhedonia but not physical function of drinking. In TST, compared with the
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saline control group, the rats exposed to LPS exhibited a significant increase in immobility time (t
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= 3.87, p < 0.01), indicating that LPS treatment induced the hopelessness. In OFT, compared with the saline control group, the rats exposed to LPS exhibited a significant decrease in distance
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travelled (t = 3.01, p < 0.05) and in number of rearing (t = 2.36, p < 0.05), indicating that LPS treatment induced reduction in locomotor activities and exploration. In NSFT, compared with the saline control group, the rats exposed to LPS exhibited a significant increase in latency to feed (t =
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4.24, p < 0.01), but home cage food intake in NSFT did not differ between the groups (Saline: 6.58 ±1.29 g; LPS: 7.07±1.84 g) (t = 0.22, p > 0.05), indicating LPS treatment induced low motivation to feed in a novel environment, which was not because of the differences in hunger. All the results in this experiment were similar to the results from Experiment 2.
Fig.3 near here. 2 column. 3.4. Depressive-like behaviors correlate with neurogenesis in the hippocampus To examine whether the function of hippocampal neurogenesis was related to the behavioral performance, Pearson’s correlation analysis was performed by using the data from both the control and LPS group as illustrated by Silverman, et al. [29]. The analysis indicated that most of the behavioral parameters correlated with the number of proliferating cells and the number of survival new born cells. For cell proliferation, as shown in Fig. 4A-E, the BrdU-positive
ACCEPTED MANUSCRIPT cell number in proliferation groups were positively correlated with the ratio of saccharin intake (r = 0.85, p < 0.001) and the distance travelled in the open field (r = 0.74, p < 0.01) and were negatively correlated with the immobility time in the TST (r = -0.77, p < 0.01) and the latency to
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feed in the NSFT (r = -0.61, p < 0.05). The BrdU-positive cell number in proliferation were not significantly correlated with the rearing times in the open field (r = -0.51, p > 0.05). For newborn
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cell survival, as shown in Fig. 4F-J, the BrdU-positive cell number in survival groups were positively correlated with the ratio of saccharin intake (r = 0.81, p < 0.01) and the distance travelled (r = 0.66, p < 0.05) and the rearing times (r = 0.61, p < 0.05) in the open field and
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negatively correlated with the immobility time in the TST (r = -0.70, p < 0.05) and the latency to
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feed in the NSFT (r = -0.71, p < 0.01).
Fig.4. near here. 2 column.
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To make sure whether the function of hippocampal neurogenesis was specifically correlated
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to the neuroinflamation induced depressive-like behaviors, Pearson’s correlation analysis was further performed by using the data separately from the LPS group or the control group (n=6 for
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each group). For LPS group, the analysis showed that the decreased hippocampal neurogenesis in cell proliferation was significantly correlated with the depressive-like phenotypes of decreased saccharine preference (r = 0.81, p < 0.05) and distance traveled (r = 0.81, p < 0.05), indicating the
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core and characteristic symptoms of major depression were linked to hippocampal neurogenesis. There exist no significant correlations between behavioral parameters and the parameters of hippocampal neurogenesis in the control group.
4. Discussion The
results
demonstrated
that
triple
repeated
central
LPS
challenge
evoked
neuroinflammation in the hippocampus of rats that was marked by elevated protein levels of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. This neuroinflammatory state induced significant depressive-like behaviors in the rats. These neuroinflammatory responses also inhibited adult hippocampal neurogenesis, which was marked by decreased BrdU positive cell number. The degree of inhibition in adult hippocampal neurogenesis was closely correlated with the level of depressive-like behaviors under neuroinflammation state. The present findings
ACCEPTED MANUSCRIPT provide the first evidence that hippocampal neurogenesis dysfunction is closely related to neuroinflammation-induced depression.
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Consistent with the results of our previous studies [20, 22, 23], the results of the present study confirmed that repeated central infusion of LPS induced significant depressive-like
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behaviors, including decreased locomotor activity in the open field, decreased saccharin preference, increased immobility time in the TST, and increased latency to food in the NSFT. Although LPS is frequently used in studies of depression [7, 38], the depressive-like behaviour
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induced by a single injection of LPS, regardless of peripheral or central administration, usually lasts a few hours, seldom longer than 24 h [8, 21, 23], whereas repeated peripheral LPS injections
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induce immune and behavioural tolerance [39-41]. The altered behavior within 24h post-LPS are often argued as being sickness behavior or proposed to be a mixture of both sickness behavior
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and depressive-like behavior [2, 30]. It has been demonstrated that the acute sickness behavioral
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effects (2-4 h), at least in mice, have been dissociated from longer lasting depressive-like behavioral effects (24-28 h) following LPS injection [7, 30]. In the present study, the altered
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behaviors was observed 24h after central LPS treatment and thus was regarded as depressive-like behavior. Importantly, the present study demonstrated the induction of a neuroinflammatory state by central LPS challenge, by showing that hippocampal production of
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pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) was significantly increased 24 h post-LPS. Positive cells for IL-1β, IL-6, and TNF-α were identified primarily in the polymorphic layer of the dentate gyrus (PoDG) and the CA3 pyramidal layer closest to the DG. The present results, along with the results of our previous studies, make repeated central LPS challenge a reliable and useful animal model of neuroinflammation-induced depression.
With the advantage of this new animal model of depression, we further examined the changes in adult hippocampal neurogenesis under the neuroinflammatory state and found that both cell proliferation and new born cell survival were inhibited by central LPS challenge. The observation that brain LPS infusion impairs the survival of new born cells is in agreement with previous findings in which a role for neuroinflammation in regulating the survival stage of the neurogenic process was demonstrated [42]. It had also been reported that an intraperitoneal
ACCEPTED MANUSCRIPT infection of LPS significantly decreased survival of new born cells and immature neurons [19]. However, these initial works and later replication reports are only limited to the effect of inflammation on the survival stage of neurogenesis [19, 42, 43]. Notably, our data demonstrate
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that repeated central LPS infusions affect not only survival of new cells but also cell proliferation, indicating the effect of neuroinflammation on hippocampal neurogenesis is involved in
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decreasing both survival and proliferation components.
To elucidate whether the impairment of hippocampal neurogenesis was related to the
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behavioral performances, we plotted the behavioral performance parameters against the BrdU-positive cell numbers for each animal. Pearson’s correlation identified a strong correlation
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between the depressive-like behaviors and the inhibition of neurogenesis. Notably, a further Pearson’s correlation analysis by using the data independently from the LPS group indicated that
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the decreased hippocampal neurogenesis in cell proliferation was significantly correlated with
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the decreased saccharine preference and the distance traveled, core and characteristic phenotypes of depression. In contrast, the reports of previous studies regarding the correlation
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between the inhibition of neurogenesis and depression in animal stress models have been inconsistent. Several previous studies have suggested that decreased hippocampal neurogenesis was associated with depressive-like phenotypes induced by stress [44, 45]. However, other
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studies have demonstrated that a stress-induced decrease in neurogenesis did not correlate with an anhedonic phenotype [46, 47]. One explanation for these conflicting data is that the relationship between behavioral performance and neurogenesis may be dependent on the method of assessment, timing, or extent of neurogenesis disruption, namely, the nature and effects of the stressors. Indeed, it has been reported that several stressors fail to reduce adult hippocampal neurogenesis [48]. Interestingly, our finding is in accord with the findings reported by Süß et al. in which severe peripheral inflammation did not induce neuroinflammation and impairment of hippocampal neurogenesis, and also failed to induce depressive-like behavior, but locomotor deficits [49]. Both work suggested, in opposite ways, that the hippocampal neurogenesis might be one of biological mechanisms underlying depression induced by neuroinflammation. Further research as to whether blockade of inhibition of neurogenesis could reverse neuroinflammation-induced depression is currently in progress.
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In conclusion, the present study demonstrated that neuroinflammation evoked by triple repeated central LPS challenge induced depressive-like behaviors and inhibited adult
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hippocampal neurogenesis. The depressive-like behavioral symptoms were correlated with changes in adult hippocampal neurogenesis. These findings provide the first demonstration that
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hippocampal neurogenesis dysfunction is associated with neuroinflammation-induced depression, which suggested that the dysfunction of hippocampal neurogenesis might be one of biological
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mechanisms underlying depression induced by neuroinflammation.
Acknowledgements
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This study was supported by the grants from the National Natural Science Foundation of China (31170987, 31440045, and 30770718) and the key laboratory of Mental Health of Chinese
Conflict of Interest
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Academy of Sciences (KLMH20142G01).
References
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The authors have no conflicts of interest to declare.
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Figure legends
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Fig. 1. Central LPS challenge induced an inflammatory state in the hippocampus. (A) Rats were given triple LPS infusions in the lateral ventricle, and brain samples were collected 24 h after the LPS infusions. (B) Immunohistochemistry indicated that increased pro-inflammatory
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cytokine proteins are present in the PoDG and CA3 after LPS administration. IL-1β, IL-6 and TNF-α (green); DAPI (blue). (C&D) Western blots indicated the production of dorsal hippocampal IL-1β, IL-6 and TNF-α increased after exposure to LPS (n = 5 per group). * p < 0.05; ** p < 0.01. The data are presented as the mean ± SEM. IL, interleukin; TNF, tumor necrosis factor; LPS, lipopolysaccharide;
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4’,6-diamidino-2-phenylindole.
Fig. 2. Effects of central LPS challenge on cell proliferation in the hippocampus and depressive-like behavior. (A) To examine the effect of central LPS challenge on cell proliferation, BrdU infusions were given after LPS infusions. (B) Representative BrdU-stained sections of Saline group. (C) Representative BrdU-stained sections of LPS group. (D) BrdU-positive cell number in
ACCEPTED MANUSCRIPT dentate gyrus of hippocampus was lower in LPS rats. (E) Saccharin preference. (F) Immobility in the TST. (G) Distance travelled during 5 min in an open field. (H) Rearing behavior in open field. (I) Latency to feed in the NSFT. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are presented as
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the mean ± SEM. n = 6 per group. LPS, lipopolysaccharide; BrdU, Bromodeoxyuridine; SPT, saccharin preference test; OFT, open field test; TST, tail suspension test; NSFT, novelty suppressed
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Fig. 3. Effects of central LPS challenge on and newborn cell survival in the hippocampus
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and depressive-like behavior. (A) To examine the effect of central LPS challenge on newborn cell survival, BrdU infusions were given before LPS challenge. (B) Representative BrdU-stained
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sections of Saline group. (C) Representative BrdU-stained sections of LPS group. (D) BrdU-positive cell number in dentate gyrus of hippocampus was lower in LPS rats. (E) Saccharin preference. (F)
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Immobility in the TST. (G) Distance travelled during 5 min in an open field. (H) Rearing behavior in
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open field. (I) Latency to feed in the NSFT. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are presented as the mean ± SEM. n = 6 per group. LPS, lipopolysaccharide; BrdU,
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Bromodeoxyuridine; SPT, saccharin preference test; OFT, open field test; TST, tail suspension test; NSFT, novelty suppressed feeding test.
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Fig. 4. Correlation between behavioral performances and neurogenesis in the dentate gyrus. (A-E) Behavioral performance parameters correlated with the cell proliferation indicated by BrdU-positive cell number in the dentate gyrus. (F-J) Behavioral performance parameters correlated with the cell survival indicated by BrdU-positive cell number in the dentate gyrus. * p < 0.05; ** p < 0.01; *** p < 0.001. The data are presented as the mean ± SEM. BrdU, Bromodeoxyuridine; LPS, lipopolysaccharide.
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Highlights Repeated central lipopolysaccharide infusions cause neuroinflammation in hippocampus; Neuroinflammation induces depressive-like behavior; Neuroinflammation impairs adult hippocampal neurogenesis; Depressive-like behavior correlates with hippocampal neurogenesis.