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Behavioural Brain Research 243 (2013) 165– 170

Contents lists available at SciVerse ScienceDirect

Behavioural Brain Research

j ourna l ho mepage: www.elsev ier .com/ locate /bbr

esearch report

-actin may play an important role in IL-1�-stimulated hippocampal neurons

ueping Zhanga,b,1, Zhongxiang Caia,1, Gaohua Wanga,∗, Huiling Wanga, Zhongchun Liua, Xin Guoa,an Yanga, Xiaoping Wanga, Hesheng Wanga, Chang Shua, Ling Xiaoa

Department of Psychiatry, Renmin Hospital, Wuhan University, Jiefang Road 238#, Wuhan 430060, ChinaAdult Education College, Ningxia Medical University, Shengli South Street 1190#, Yinchuan, 750004, China

i g h l i g h t s

IL-1� increased viability at low doses for hippocampal neurons.High doses IL-1� blunted increases effection of low doses for hippocampal neurons.IL-1� increased the expression of F-actin at low doses in hippocampal neurons.IL-1� decreased the expression of F-actin at high doses in hippocampal neurons.

r t i c l e i n f o

rticle history:eceived 26 November 2012eceived in revised form9 December 2012ccepted 3 January 2013vailable online 11 January 2013

eywords:

a b s t r a c t

Objectives: The aim of this study was to investigate whether IL-1� treatment affects F-actin in hippocam-pal neurons.Materials and methods: Primary culture of hippocampal neurons were prepared from rat embryos, andthe effect of different concentrations of IL-1� on the cell phenotype was investigated. Cell viability wasmonitored by CCK-8 activity analysis. Western blotting and immunofluorescence were performed toanalyze protein levels and morphological changes in cells treated with IL-1�.Results: IL-1� increased viability at low doses and these increases were severely blunted at higher con-

-actinL-1�ippocampal neurons

centrations. Similarly, the expression of F-actin was increased at low concentrations and decreased athigh concentrations.Conclusions: IL-1� has a critical role in regulating hippocampal neuron viability by stimulating F-actinexpression. These data suggest that IL-1� secretion at low doses is beneficial for the expression of F-actin, which can improve the survival of hippocampal neurons during stress. However,these effect wereseverely blunted at higher concentrations.

. Introduction

Major depressive disorder (MDD), which is a devastating psy-hiatric disorder, is among the most common and life-threateningllnesses, and it has a 12-month prevalence of 6.6% [1]. MDD has aomplex etiology, with biological, psychological, and environmen-al components, and it is associated with the onset of depressionnd with a loss in hippocampal volume [2,4–7]. Many studiesave demonstrated hippocampal atrophy in depressed patients andhown that antidepressant treatment increases hippocampal neu-

ogenesis [2–5]. Although a number of researchers have investedheir efforts in studying the underlying mechanism of this finding,he complete regulatory mechanisms are still not understood. Thus,

∗ Corresponding author. Tel.: +86 27 88041911 8320; fax: +86 27 88072022.E-mail address: wangghwh@tom.com (G. Wang).

1 Both authors contributed equally to this work.

166-4328/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.bbr.2013.01.001

© 2013 Elsevier B.V. All rights reserved.

elucidating new mechanisms is essential for discovering noveleffective therapeutic targets for treating depression.

In this respect, there has been increasing interest in the puta-tive involvement of the immune system in psychiatric disorders[8]. Interleukin-1 beta (IL-1�) is a proinflammatory cytokine withimportant roles in innate immunity, as well as in normal tissuehomeostasis. In particular, levels of IL-1� have been shown tobe elevated in depressed patients, both in the cerebrospinal fluidand in the blood; moreover, the levels appear to be higher inpatients with more severe depressive symptoms [9,10]. And IL-1� was detected not only in damaged brain tissue but also inbrain tissue undergoing repair. Animal studies have demonstratedthat exposure to stress increases IL-1� in the hippocampus, andthe exogenous administration of IL-1� to rats has been found

to produce depressive-like symptoms, but these depressive-likebehavioral changes were induced by blockade of the IL-1RI receptor[11]. However, in brain slices, IL-1� had the capacity to stimulateneurite growth.

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F-actin is an important cytoskeletal protein that maintains theormal intricate architecture of the hippocampus, and it can behanged by pathological extracellular stimuli through signalingascades [12–14]. Many studies have investigated the relationshipetween F-actin and IL-1� [15]. In particular, some studies investi-ated the change in F-actin after treatment with IL-1� in astrocytes16,17]. However, no studies have investigated the relationshipetween IL-1� and F-actin in hippocampal neurons.

Based on this knowledge, the goal of this study was to investi-ate the effect of different concentrations of IL-1� on F-actin inhe hippocampus in vitro and to further define the relationshipetween IL-1� and F-actin.

. Materials and methods

.1. Animals

All of the experiments involving animals were performed in accordance withhe Chinese Health Guide for the Care and Use of Laboratory Animals and werepproved by the Institutional Animal Care and Use Committee of Wuhan University.dult male and female Wistar rats (240–270 g) were purchased from the Wuhanniversity Center for Animal Experiments. Wistar rats were housed under stan-ard conditions (22 ± 1 ◦C, 60% relative humidity) in a facility with a 12-h/12-h

ight/dark cycle (lights on from 06:00 to 18:00 h) and had free access to food andater. All efforts were undertaken to minimize the number of rats used and their

uffering.

.2. Cell culture

Primary hippocampal neurons were isolated from 16 to 18-day-old Wis-ar rat embryos as described previously [18]. Briefly, the brains were removednd dissociated from the meninges, and the hippocampi were isolated. The hip-ocampi were washed three times in Ca2+/Mg2+-free Hank’s Balanced Salt SolutionHBSS), followed by trituration, and were digested for 10 min at 37 ◦C and 5%O2 in Dulbecco’s modified Eagle’s medium/high glucose (DMEM/HG, GNM12800,ibco, California, American), containing 2 mg/ml papain, in the presence of 10nits/ml DNase I (EN0521, Fermentas, Vilnius, Lithuania). Subsequently, the diges-ion was terminated by adding 10% fetal bovine serum. The hippocampal tissueas washed three times in DMEM/HG and dissociated with a flame-polished Pas-

eur pipette. The cells were counted, resuspended in plating medium (DMEM/HG),lated on poly-l-lysine-coated coverslips (0.1 mg/ml) at a density of 1 × 105 cellser coverslip for most experiments, and plated at a density of 2 × 105/cm2 inoly-l-lysine-coated 6-well plates for biochemical experiments. Four hours afterlating, the medium was replaced with neuronal culture medium (21103-49,ibco, California, American; 2% B-27, 1% glutamine, 100 units/ml penicillin andtreptomycin), and AraC (5 �M) was added to the cultures after 2 days in vitro.ippocampal neurons were used for experiments after a culturing period of 7–9ays.

.3. Drug administration

To analyze the effect of IL-1� on hippocampal neurons, IL-1� was purchasedrom Peprotech (400-01B, Peprotech, New Jersey, American) and dissolved inimethylsulfoxide, and aliquots were stored at −80 ◦C. Subsequently, aliquots wereissolved in fresh PBS before use. After the cells were maintained for 8 days, IL-1�as administered at final concentrations of 0 (PBS was added to control neurons at

final concentration of 0.1%), 0.01, 0.1, 1, 5, 10, 20, 50, and 100 ng/ml. After 12 h ofreatment, the cells were harvested for use in other experiments.

To further confirm the effects of IL-1� on hippocampal neurons, we appliedL-1ra (1545-RA/CF, R&D Systems, Minnesota, American) at a concentration that

as 100-fold higher than that of IL-1� in the culture solution and treated theells with IL-1� for another 12 h. The cells were then harvested for use in otherxperiments.

.4. Immunocytochemistry

After treatment, the neuronal identity and morphology of the cultures were con-rmed by immunocytochemical labeling for neuron-specific enolase (NSE) (BA0535,oster, Hubei, China) [19]. Hippocampal neurons plated on 6-well plates at a den-ity of 0.7–1 × 106 cells/ml were rinsed with 0.01 M PBST 3 times each for 5 minnd fixed with 4% paraformaldehyde for 20 min at 4 ◦C. The cells were then washedith 0.01 M PBST 3 times each for 5 min at room temperature. The cells were sub-

equently permeabilized with 0.1% Triton X-100 for 20 min in 0.01 M PBST and 3%

2O2 for 15 min at room temperature. The cells were blocked with 5% goat serum

or 30 min in a wet box at 37 ◦C. Cells were incubated overnight at 4 ◦C with theabbit anti-NSE (1:100) primary antibody. After washing three times with 0.01 MBST, goat secondary antibody (sc-2091, Santa Cruz Biotechnology) was applied for0 min at room temperature. Subsequently, cells were stained with DAB for 20 min

esearch 243 (2013) 165– 170

and hematoxylin for 1 min. After dehydration with a gradient of ethanol, the cellswere mounted and observed under a microscope.

2.5. Neuron viability assays

The viability assay [20] assesses the ability of cells to maintain or recover theirviability after treatment. Neuron viability assays were performed in three indepen-dent experiments. Hippocampal neurons were plated in quintuplicate onto 96-wellplates at a density of 1 × 105 cells/well. Hippocampal neuron growth was mea-sured using the Cell Counting Kit-8 (CCK-8) following the manufacturer’s protocol(C0038, Beyotime, Shanghai, China). A total of 10 �l of CCK-8 reagent was added toeach well and allowed to incubate for 4 h. The amount of CCK-8 reagent reducedto formazan by cellular dehydrogenase indicates cell viability and was measuredby reading the absorbance at 450 nm in a 96-well plate reader. The absorbancereading was subtracted from the background control and was averaged over threemeasurements.

2.6. Immunofluorescence

The morphology of F-actin in hippocampal neurons after treatment with dif-ferent concentrations of IL-1� was observed using immunofluorescence [21]. Cellson coverslips were stained for F-actin after fixation with ice-cold 4% paraformalde-hyde for 30 min. The cells were then incubated with phalloidin (0.01 mg/ml, P5282,Sigma, Missouri, America) for 60 min at room temperature. After washing with0.01 M PBS 3 times, the stained cells were mounted using anti-fade mediumwith DAPI and were examined using a fluorescence microscope (CKX41, Olympus,Japan).

2.7. Western blotting

To analyze the effect of treatment with different concentrations of IL-1� onF-actin protein levels in hippocampal neurons, Western blotting was performed.Cultured hippocampal neurons were homogenized in ice-cold lysis buffer containingPMSF, NaF, Na3VO4, complete PhoStop, and EDTA. The cells were homogenized withan ultrasound wave cell disruption instrument (KS-600, Kesheng, Zhejiang, China)and centrifuged at 12,000 × g for 15 min at 4 ◦C. The supernatants were collected andstored at −80 ◦C. The protein concentrations were measured using the Bradford Pro-tein Assay Kit (301003, BestBio, Shanghai, China) according to the manufacturer’sprotocol. The tissue or cell extracts were analyzed by 4–10% SDS/PAGE and blottedonto PVDF membranes. The membranes were first blocked in 5% non-fat dry milk inTris–buffered saline containing Tween 20 (TBST) for 2 h at room temperature. Afterwashing three times for 5 min each in TBST buffer, the membranes were incubatedwith primary antibodies (ab205, Abcam, Massachusetts, American) overnight at 4 ◦Cwith gentle rocking. The secondary antibodies were added and incubated with gen-tle rocking for 45 min at room temperature before the membranes were washed asdescribed above. To analyze relative protein quantity, GAPDH was used as a loadingcontrol.

2.8. Statistical methods

Data comparisons were performed using a one-way analysis of variance(ANOVA) followed by a post hoc LSD test or Tamhane’s T2 test. These testswere used to evaluate significant differences between different groups. Analyseswere performed using the Statistic software SPSS 13.0. Data were acquired from3 separate experiments. A value of P < 0.05 was considered statistically signifi-cant.

3. Results

3.1. Successful cell culture

To verify that the cells were growing normally, we observedthem under the microscope. The cultured cells on cover slips weregrown for 1–9 days and observed under an inverted microscope(Fig. 1A). Protrusions were visibly growing from the cell body. Aftera culture period of 7 days, the cell edges were large and smoothand exhibited strong refraction. Additionally, cell synapses wereconnected with each other to form a network.

We utilized two approaches to further verify that the primaryhippocampal neuron culture was successful. In the first approach,we used immunocytochemistry to assess the purity of the hip-

pocampal neurons (Fig. 1B). Hippocampal neurons were stainedwith positive markers. It was observed that the positive cells wereconnected to the surrounding cells. Positive cells were presentthroughout the entire field of view, and the culture was enriched in

X. Zhang et al. / Behavioural Brain Research 243 (2013) 165– 170 167

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ig. 1. Successful cell culture. (A) Hippocampal neuron culture at 1, 2, 3, 4, 5, 6,ippocampal neurons at 8 days. (C) Neuron viability was detected by the CCK-8 ass

eurons (>95%). In the other approach, neuron viability was eval-ated by the CCK-8 assay at 1, 3, 5, 7, 9, and 12 days. The viabilityas markedly increased in the cell culture from 5 to 12 days, and

he highest viability was observed at 9 days (Fig. 1C).

.2. Effects of IL-1 ̌ on hippocampal neuron viability

To determine the effect of IL-1� on rat hippocampal neurons, wexposed neurons that were cultured for 8 days to different concen-rations of IL-1� and determined their viability. Initially, viabilityas increased at 0.01 ng/ml, and significant differences in viabilityere observed between the neurons cultured under this concen-

ration and those in the control group. When the concentration wasncreased to 0.1 ng/ml, the hippocampal neurons exhibited a moreignificant increase in viability after 12 h of treatment, and the high-st viability was observed at 0.1 ng/ml. Subsequently, the viabilityas significantly reduced at increasingly higher concentrations of

L-1� (Fig. 2A).Taken together, these data confirmed that IL-1� increased neu-

onal survival in a dose-dependent manner at low doses and lessffective increased neuronal survival in a dose-dependent man-er at high doses. The increase in cell survival was significant at.1 ng/ml (P < 0.01). In contrast, the effects described above werebolished when the cells were treated with IL-1ra, and no sig-ificant changes in CCK-8 activity were observed between the

roups (Fig. 3A). For example, when IL-1ra was used to inhibit theffect of IL-1�, the CCK-8 activity levels decreased from 1.14 ± 0.04o 0.93 ± 0.02 at 0.01 ng/ml and increased from 0.88 ± 0.03 to.96 ± 0.07 at 20 ng/ml.

nd 9 days. (B) Immunocytochemistry was performed to detect the purity of the, 3, 5, 7, 9, and 12 days.

3.3. Effects of IL-1 ̌ on F-actin in hippocampal neurons

One of the specific objectives of this study was to investigatethe role of different concentrations of IL-1� on hippocampal neu-rons. We investigated the protein expression and morphology ofF-actin by performing Western blotting, immunofluorescence andimmunocytochemistry. Western blotting analysis demonstratedthat protein expression was higher in the 0.01 ng/ml group thanin the control group. Similarly, protein expression was higher inthe 0.1 ng/ml group than in the 0.01 ng/ml group. However, theseincreases were severely blunted at higher concentrations. In par-ticular, the level of F-actin was diminished at 50 and 100 ng/ml(Fig. 2B).

In addition, immunofluorescence (Fig. 2C) and immunocyto-chemistry (Fig. 2D) experiments revealed the same tendency. At the0.01 and 0.1 ng/ml concentrations, the number of protrusions wasgreater than that in the control group. With respect to morphologyand number, the protrusions were larger and greater in number atthe 0.1 ng/ml concentration than at the 0.01 ng/ml concentration.However, with increasingly higher concentrations, the protrusionedges were no longer smooth, and the protrusions became shorterand fewer in number. In contrast, the effects described above wereabolished when the cells were treated with IL-1ra, and no signif-icant changes in morphology were observed between the groups(Fig. 3B).

Taken together, these results support the conclusion thatdifferent concentrations of IL-1� mediate different effects onF-actin expression in hippocampal neurons. IL-1� increasedthe expression of F-actin at low doses in a dose-dependent

168 X. Zhang et al. / Behavioural Brain Research 243 (2013) 165– 170

Fig. 2. The effects of IL-1� on hippocampal neurons. (A) Neuron viability was detected by the CCK-8 assay after treatment with IL-1� at concentrations of 0, 0.01, 0.1, 10, 20,50, and 100 ng/ml. (B) Top: quantitative results of the protein levels of F-actin in different groups by Western blotting analysis. The data used in the analysis are the average of3 .01 ngW tions

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anner and abolished it at higher doses in a dose-dependentanner.

. Discussion

As a member of the pluripotent cytokine family that is involvedn various inflammatory pathways, IL-1� may also protect againstr aggravate central nervous system insults [9–11]. Neuronal struc-ural integrity is deeply dependent on the dynamic balance of

/ml IL-1� group. †P < 0.05 versus the 0.1 ng/ml IL-1� group. Bottom: representativeof IL-1�. GAPDH was used as a loading control. (C) The changes in F-actin expressionF-actin by immunocytochemistry analysis at different concentrations of IL-1�.

the cytoskeletal system. F-actin, an important component of thecytoskeleton, plays a role in hippocampal neuron function [12–14].In the present study, we investigated the role of F-actin in hip-pocampal neurons using different concentrations of IL-1�. Ourresults show that IL-1� could both increase the expression of F-

actin and hippocampal neuronal activity at low concentrationsand produce neurotoxic effects at high concentrations, consistentwith previous reports [11,22]. These effects could be inhibitedby IL-1ra [11,23]. The results indicate that the effect of IL-1� on

X. Zhang et al. / Behavioural Brain Re

Fig. 3. The effects of IL-1� on hippocampal neurons could be inhibited by IL-1ra.(A) Neuron viability was detected by the CCK-8 assay at different concentrations ofIL-1� and IL-1ra. (B) The changes in F-actin by immunocytochemistry analysis atd

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depression. Neuroscience Letters 2011;503(1):15–9.[3] Yang C, Wang G, Wang H, Liu Z, Wang X. Cytoskeletal alterations in rat

ifferent concentrations of IL-1� and IL-1ra.

ippocampal neurons could be due to changes in F-actin expres-ion.

IL-1� is expressed as a 17.4-kD precursor, which is encodedy a distinct gene and is produced, primarily by monocytes, inhe periphery. In the central nervous system, IL-1� is producedot only by microglia and invading monocytes/macrophages butlso by neurons and astrocytes, as suggested by various in vivond in vitro studies that showed that IL-1� was detrimental toippocampal neurons [2,3]. We found that IL-1� exerted negativeffects on hippocampal neurons when administered at high con-entrations (often ≥1 ng/ml) in cell culture, which is in agreementith previous studies [24,25]. However, few studies have been con-ucted regarding the effects of IL-1� at lower concentrations. We

sed low IL-1� concentrations of 0.01, 0.1 and 1 ng/ml to stimu-

ate hippocampal neurons and found that viability was significantncreased after 12 h.

search 243 (2013) 165– 170 169

Cytoskeletal proteins are downstream of various signaling path-ways. Our data are in agreement with previous in vivo observationsof cytoskeletal alterations in the hippocampus following stress[2,3]. F-actin is one of the major components of the cellular scaf-folding that serves to maintain cell shape, and F-actin is the mostprominent cytoskeletal protein at synapses. F-actin can interlink toa variety of cytoskeletal networks through actin-binding proteins.Previous studies have demonstrated that inflammatory cytokinescan induce F-actin polymerization and reorganization in other cells[16,17]. For example, some studies have shown that IL-1� inducesrearrangement of F-actin in astrocytes. However, other studies haveshown through the immunostaining of astroglial cells that therewere no remarkable changes in F-actin induced by IL-1� treatment.Data available in the literature suggest a complex and uncertaincorrelation between IL-1� and F-actin in other cells. Therefore, webegan this study by investigating the effect of different concentra-tions of IL-1� on F-actin.

In the present study, the observed increases in F-actin wereinduced by low concentrations of IL-1�. In contrast, F-actin exhib-ited a lower expression at high concentrations of IL-1�. Thesechanges could all be reversed by IL-1ra. Surprisingly, the changesin F-actin expression were similar to the changes in hippocampalneuron viability, as mentioned before. And large numbers of stud-ies show that F-actin is closely related with the cells activity andmigration, especially in the researches of tumor cell [26–30]. Usingsome methods that perturb individual aspects of F-actin polymer-ization and reconstruction, they demonstrated that the dynamicsof tumor cells were restrained [26,27]. So our findings suggest thatthe potent IL-1� effects on neuronal activity in primary cell cul-ture may occur directly or indirectly through effects on F-actin. Toour knowledge, these data provide the first direct evidence that theregulation of F-actin is crucial to the role of IL-1� in hippocampalneurons and may be a useful new intervention target for MDD.

In summary, our results demonstrate that IL-1� has a criticalrole in regulating hippocampal neuron viability by stimulating F-actin. This study shows that the effects of IL-1� on hippocampalneurons are dependent on its concentration and that F-actin iscapable of enhancing hippocampal neuron viability in cell culture.It is possible that IL-1� secretion at low doses is beneficial for theexpression of F-actin, which can improve the survival of hippocam-pal neurons during stress. However, high doses have the oppositeeffect. These findings further elucidate the actions of IL-1� stimu-lation on F-actin expression in hippocampal neurons and could beused to identify new targets for antidepressant drugs.

Acknowledgements

Our research was supported by two grants from Key Technol-ogy Research of Major Mental Illness Prevention and Treatmentfor the Barriers to the Recognition and Prevention of Depres-sion and Anxiety in General Hospital (Grant No. 2012BAIO1B00),and the 985 Project of Cognitive and Neural Information Science,Wuhan University, PR China (Grant No. 904273258). The authorsalso acknowledge technical supports from the HuBei Provincial KeyLibrary of Digestive Disease and the Central Laboratory of RenminHospital of Wuhan University.

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