Proteomic analysis of rat hippocampus after repeated psychosocial stress

Neuroscience 137 (2006) 1237–1246

PROTEOMIC ANALYSIS OF RAT HIPPOCAMPUS AFTER REPEATED PSYCHOSOCIAL STRESS L. CARBONI,a* C. PIUBELLI,b,e C. POZZATO,c H. ASTNER,d R. ARBAN,a P. G. RIGHETTI,e1 M. HAMDANd AND E. DOMENICIb

for the first time in relation to stress. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: social defeat, 2-D electrophoresis, mass spectrometry, behavioral model, rat brain.

a Department of Behavioural Neuroscience, Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Via A. Fleming 4, 37135 Verona, Italy b Department of Molecular Psychiatry, Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Via A. Fleming 4, 37135 Verona, Italy

Stressful life events are often thought to be responsible for triggering the onset of major depression episodes, although a genetic predisposition is probably required (Brown, 1998; Kendler et al., 1999). Moreover, an abnormal stress response mediated by the hypothalamic–pituitary–adrenal axis is frequently observed in patients affected by mood disorders (Plotsky et al., 1998). As a consequence, several animal models of depression have been developed, based on the induction of a variety of stressful conditions which bring about depression-like symptoms in the rodent (Vollmayr and Henn, 2003; Blanchard et al., 2001; Dugovic et al., 1999). In the social defeat model, the experimental subject is exposed to a dominant and aggressive conspecific animal. Animals exposed to the defeat show signs of anhedonia and neuroendocrine responses recalling those of depressive patients (Fuchs and Flügge, 2002; Kudryavtseva et al., 1991; Koolhaas et al., 1995). Moreover, some of the behavioral and endocrine effects caused by the psychosocial stress can be reversed by treatment with antidepressants (Czeh et al., 2001; von Frijtag et al., 2002; Kramer et al., 1999). The behavioral and endocrine changes induced in the experimental animal by exposure to this paradigm are often maintained for a long time after the end of the stressful events (Koolhaas et al., 1997; Meerlo et al., 2002; von Frijtag et al., 2000). The molecular modifications induced in the brain which are responsible for causing and sustaining the behavioral and neuroendocrine alterations in the model are unknown. Thus, this study was carried out to gain information about the molecular changes induced in the limbic system by chronic and acute exposure to psychosocial stress in rats. We examined the hippocampus, since the molecular changes brought about by stress hormones in this brain region are considered key in mediating behavioral responses (McEwen, 1999; Kim and Yoon, 1998; Fuchs and Flügge, 1998; Kim and Diamond, 2002). In addition, there is a large body of evidence that shows the impact of stress on hippocampal function and plasticity, as documented by electrophysiological, neuroanatomical and cellular physiology studies (de Kloet et al., 2005). A proteomic technique was used to search for novel proteins or signaling pathways involved in the induction of the stress response.

c

Department of Neuropsychopharmacology, Psychiatry Centre of Excellence for Drug Discovery, GlaxoSmithKline, Via A. Fleming 4, 37135 Verona, Italy

d Department of Computational, Analytical and Structural Science, Discovery Research, GlaxoSmithKline, Via A. Fleming 4, 37135 Verona, Italy e Department of Agricultural and Industrial Biotechnologies, University of Verona, Strada Le Grazie 15, 37134 Verona, Italy

Abstract—Since stress plays a role in the onset and physiopathology of psychiatric diseases, animal models of chronic stress may offer insights into pathways operating in mood disorders. The aim of this study was to identify the molecular changes induced in rat hippocampus by repeated exposure to psychosocial stress with a proteomic technique. In the social defeat model, the experimental animal was defeated by a dominant male eight times. Additional groups of rats were submitted to a single defeat or placed in an empty cage (controls). The open field test was carried out on parallel animal groups. The day after the last exposure, levels of hippocampal proteins were compared between groups after separation by 2-D gel electrophoresis and image analysis. Spots showing significantly altered levels were submitted to peptide fingerprinting mass spectrometry for protein identification. The intensity of 69 spots was significantly modified by repeated stress and 21 proteins were unambiguously identified, belonging to different cellular functions, including protein folding, signal transduction, synaptic plasticity, cytoskeleton regulation and energy metabolism. This work identified molecular changes in protein levels caused by exposure to repeated psychosocial stress. The pattern of changes induced by repeated stress was quantitatively and qualitatively different from that observed after a single exposure. Several changed proteins have already been associated with stress-related responses; some of them are here described 1

Present address: Department of Chemistry, Materials and Chemical Engineering “Giulio Natta,” Politecnico di Milano, Via Mancinelli 7, Milano 20131, Italy. *Corresponding author. Tel: ⫹39-045-821-8294; fax: ⫹39-045-821-8047. E-mail address: [email protected] (L. Carboni). Abbreviations: DRP-2, dihydropyrimidinase-related protein-2; GFAP, glial fibrillary acidic protein; GRP75, stress-70 protein; GRP78, 78-kDa glucose-regulated protein; HSC71, heat-shock cognate 71 kDa protein; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; pI, isoelectric point; PPIA, peptidyl-prolyl cis-trans isomerase A.

0306-4522/06$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.10.045

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EXPERIMENTAL PROCEDURES Study design A proteomic analysis of rat hippocampus was carried out on animals subjected to repeated- or single-stress paradigms in order to identify molecular changes brought about by psychosocial stress. In the repeated-stress paradigm, a group of animals (n⫽6) was exposed eight times to the social stress protocol. The day after the last exposure, hippocampi were dissected and analyzed by 2-D gel electrophoresis. Each sample was prepared from a single animal and analyzed on two replicate gels. Samples belonging to a corresponding control, non-stressed group (n⫽6), were analyzed at the same time under the same procedure. Behavioral data in the open field test were collected on parallel groups of stressed and control animals (n⫽8 –10) to determine whether an anxiety-related behavior was induced by the social defeat procedure. In the single-stress paradigm, a group of rats (n⫽5) was exposed only once to the social stress protocol; the following day hippocampi were dissected and analyzed by 2-D electrophoresis together with a group of control animals (n⫽5). Again, behavioral data were collected in parallel groups of rats (n⫽9 –11).

Animals Long Evans rats (400 – 450 g) were purchased from Harlan, Portage, MI, USA; Sprague–Dawley female (250 –300 g) and male rats (300 –350 g at the beginning of the experiment) were from Charles River, Calco, Lecco, Italy. All animals were housed under standard laboratory conditions (22⫾1 °C), on a 12-h light/dark cycle with lights on at 6:00 a.m. Food and water were available ad libitum. All procedures were carried out in accordance with the Italian law (art. 7, Legislative Decree no. 116, 27 January 1992), which acknowledges the European Directive 86/609/EEC, and were fully compliant with GlaxoSmithKline policy on the care and use of laboratory animals and related codes of practice. This policy aims to minimize the number of animals utilized and ensures minimal discomfort throughout laboratory procedures.

Social defeat procedure The resident–intruder protocol described by Meerlo et al. (1996) was followed. Long Evans rats were used as dominant male rats. They were co-housed with Sprague–Dawley females sterilized by tubal ligation in cages of 60⫻35⫻20 cm for two weeks to induce territorial dominance and then trained to be aggressive against intruding male rats for the following week. After this period only responsive animals were used for the stress procedure and latency to the first attack was measured to select dominants (inclusion criterion: latency to first attack⬍30 s). Sprague–Dawley male rats (300 –350 g at the beginning of the experiment, Charles River), housed individually for seven days before the experiment, were used as intruder subjects. In this stress procedure, intruder rats were introduced into the dominant resident cage after removal of the female. Following the first attack, intruders were physically isolated using a Plexiglas cylinder (diameter: 19 cm; height: 24 cm) and left in visual, auditory and olfactory contact with the dominant for the following 30 min. In repeated exposures, the intruder was exposed to randomly selected different dominants. Control rats were introduced in a novel empty cage, with odor of another male Sprague–Dawley rat, using the same protocol as for the intruder. Social encounters were carried out between 9:00 a.m. and 12:00 p.m.

Open field test Open field test was carried out under red dim light conditions (2 lux) 30 h after the last social defeat stress. Animals were exposed

for 5 min to an unfamiliar test arena (100 cm⫻100 cm), divided into 16 white squares (12.5⫻12.5 cm) to allow assessment of lines crossing as an index of locomotor activity. Animals were placed in the center of the arena, videotaped and analyzed blindly. A decrease in spontaneous exploratory behavior, assessed as reduction in line crossings, was taken as an index of anxiety related behavior. Data were analyzed with Student’s t-test (GB-STAT 7.0 statistical software, Dynamic Microsystems) and were considered significant if P⬍0.05.

Sample preparation for 2D electrophoresis The day after the last social defeat procedure the animals were killed and their brains removed. Hippocampi were rapidly dissected, frozen on dry ice and stored at ⫺80 °C. Each left hippocampus was homogenized with a glass/Teflon homogenizer at a concentration of 10% (w/v) in a solubilizing solution containing: 7 M urea (Sigma-Aldrich, St. Louis, MO, USA), 2 M thiourea (Fluka, Buchs, Switzerland), 40 mM Tris (Sigma-Aldrich), 3 mM tributylphosphine (Fluka), 2% (w/v) 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate, CHAPS (Fluka), 1% Pharmalytes 3.5–10 (Amersham Biosciences, Uppsala, Sweden), Complete™ protease inhibitor (Roche, Basel, Switzerland). Samples were sonicated three times for 10 seconds on ice with an ultrasonic processor with probe (Ultrasonic 2000, Dynatech Laboratories Inc., Chantilly, VA, USA). The extract was centrifuged at 1000⫻g and the pellet discarded. Proteins in the supernatant were separated by 2-D electrophoresis. An aliquot of this supernatant was dialyzed against 1% sodium dodecyl sulfate (SigmaAldrich) in water and used to measure protein concentration by the BCA method (Pierce, Rockford, IL, USA).

2-D electrophoresis Isoelectric focusing was carried out on 17 cm immobilized pH gradient strips (3–10 non-linear pH gradient, Bio-Rad, Hercules, CA, USA). The strips were rehydrated for 16 h with 0.4 mg protein in 450 ␮L of the solubilizing solution already described, with the addition of 10 mM iodoacetamide (Sigma-Aldrich) as alkylating agent (Herbert et al., 2001). Focusing was carried out at 20 °C for 75,000 Vh at a maximum of 10,000 V in a Protean IEF Cell (Bio-Rad). Immobilized pH gradient strips were then incubated with gentle shaking in an equilibration solution (6 M urea, 2% sodium dodecyl sulfate, 375 mM Tris pH 8.8, 4 mM tributylphosphine) for 25 min. The strips were then laid on top of homemade polyacrylamide slab gels (8 –18% T gradient, 20⫻20.5 cm; acrylamide from Bio-Rad) in the presence of 0.5% agarose (SigmaAldrich) prepared in running buffer and stained with Bromophenol Blue (Sigma-Aldrich). An aliquot of Precision™ mass markers (Bio-Rad) was loaded in parallel on each gel. Gels were run in the Protean plus Dodeca Cell apparatus (Bio-Rad) at 18 °C and 12 mA/gel overnight in 125 mM Tris, 960 mM glycine (SigmaAldrich), 0.5% sodium dodecyl sulfate. After 1 h washing in 10% v/v ethanol, 7% (v/v) acetic acid in water, staining was carried out overnight with Sypro Ruby fluorescent stain (Bio-Rad). Gels were destained 1 h in a 10% v/v ethanol and 7% (v/v) acetic acid solution and maintained in water. Images were acquired with a CCD camera on the VersaDoc imaging system (Bio-Rad). Image analysis was carried out with the PDQuest software (Bio-Rad). Protein levels were evaluated as volumes (spot area⫻optical density) for the protein spots matched among gels. Spot volume was normalized for each gel on total density in valid spots. Data were log transformed and analyzed with Student’s t-test with the statistics tools included in the PDQuest software. Spots which gave significant results (P⬍0.05) were verified visually to exclude artifacts. Volume values of satisfactory spots were re-checked by Student’s t-test on the Prism software (GraphPad Software Inc., San Diego, CA, USA).

L. Carboni et al. / Neuroscience 137 (2006) 1237–1246

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Table 1. Identification of 21 proteins which showed a significantly different level after repeated social defeats SSP

MW [Da]

pl

MOWSEscore

8402

44423

8

1.65 E9

6214

28701

7.2

8.72 E6

4407 7503 7608 5308 3602

46985 57687 85358 36323 79443

6.5 7 8.3 6.5 6.1

1.75 2.57 1.20 1.97 3.09

7615 7703

67644 17743

2509 3606 2604 5215

MASCOT score

Z-score

Protein name

Accession number

% Sequence coverage

No. of peptides

Regulation

%

P

96

2.25

P16617

37.5

17

Up

20

0.0402

96

1.75

O9DBJ1

40.32

12

Up

20

0.0401

E11 E16 E8 E6 E8

151 218 91 92 106

2.38 2.39 2.34 2.28 2.38

P04764 P11980 P20004 P14152 P15690

44.3 48.1 18 26 18

16 28 14 13 15

Up Up Up Down Down

40 50 130 60 40

0.0155 0.0368 0.0041 0.005 0.0078

7.6 8.6

6.49 E14 1.08 E7

165 79

2.4 2.17

P50137 P10111

33.1 54

20 11

Down Up

20 20

0.0255 0.0204

70871 73860 72347 28575

5.4 6.2 5.1 6.6

1.18 E15 1.16 E10 8.4 E12 1.00 E6

173 114 98 100

2.41 2.28 2.35 1.92

P08109 P48721 P06761 P52555

44 30 32.3 38

24 18 18 10

Up Down Down Down

160 40 50 40

0.0031 0.0038 0.0088 0.038

6108

24408

7

2.23 E5

99

2.16

P62828

37

10

Up

40

0.0226

5706

68268

5.8

2.03 E9

64

2.26

P50516

26.7

15

Down

70

0.0224

2415 2508 3215 3521

49943 56115 30609 62278

5.4 5.3 6.3 6.3

6.47 E8 7.52 E18 1.73 E13

122 347 94 135

2.37 2.38 2.04 2.23

P47819 P23565 P47756 P47942

36 61.8 30 40.2

17 35 10 18

Down Up Down Down

30 40 60 20

0.0227 0.0175 0.0052 0.0113

5206

31776

6.3

9.07 E6

147

2.36

P16446

43

12

Up

20

0.0193

1001

16696

4.1

4.50 E5

96

2.32

Phosphoglycerate kinase Phosphoglycerate mutase 1 Alpha enolase Pyruvate kinase Aconitate hydratase Malate dehydrogenase NADH-ubiquinone oxidoreductase Transketolase Peptidyl-prolyl isomerase A HSC71 GRP75 GRP78 Endoplasmic reticulum protein ERp29 GTP-binding nuclear protein Ran Vacuolar ATP synthase GFAP Alpha-internexin F-actin capping protein Dihydropyrimidinase related protein-2 Phosphatidylinositol transfer protein Calmodulin

P02593

51

9

Up

30

0.0256

SSP is the identification number of the selected spot assigned by the image analysis software. Theoretical values of molecular mass (MW) and pl are shown in columns 2 and 3. Probability scores of the identification are taken from the three database search software types, they are described in the Experimental Procedures section. SwissProt protein names and accession numbers are provided in columns 7 and 8, respectively. The number of protein peptides that matches the theoretical peptides from the database entry and the sequence coverage percentage are indicated in columns 10 and 9 respectively. The regulation refers to the stressed group as compared to the control group and the percentage of variation is shown. The P value was determined using Student’s t test.

Protein identification with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry Each selected spot was carefully cut with a spot cutter (Bio-Rad) and destained with two 10 min-washing steps in 50% acetonitrile (Sigma-Aldrich) (v/v), 50% of 5 mM Tris, pH 8.5, followed by a third wash with 5 mM Tris pH 8.5 for 10 min. The spots were dried in a Speedvac sc110A device (Thermo Savant, NY, USA) for 1 h at room temperature and then covered with 15 ␮L of sequencing grade modified trypsin (Sigma-Aldrich) (0.02 mg/mL) in NH4HCO3 buffer (40 mM, pH 8.5) and left at 37 °C overnight. The spots were then crushed and peptides were extracted twice in 50 ␮L 50% acetonitrile, 50% H2O with 1% formic acid (v/v) and a third time with 50 ␮L acetonitrile. The extractions were conducted in an ultrasonic bath for 15 min. The three extraction solutions were mixed and evaporated to dryness in the Speedvac device and the residues dissolved in 10 ␮L H2O with 0.1% trifluoroacetic acid. For an additional purification, the samples were cleaned by using ZIP-TIP C18 (Millipore, Bedford, MA, USA). Two microliters of the resulting solution were mixed with an equivalent volume of matrix solution, prepared fresh every day by dissolving 10 mg/mL ␣-cyano4-hydroxycinnamic acid (Sigma-Aldrich) in acetonitrile:ethanol (1:1, v/v). One microliter of the resulting mixture was loaded onto the MALDI sample plate and allowed to dry. Measurements were performed using a TofSpec 2E MALDI-TOF instrument (Micro-

mass, Manchester, UK), operated in reflector mode, with an accelerating voltage of 20 kV. Measured peptide masses were analyzed by two different software tools, which allowed a double confirmation of protein identification: MASCOT software (Matrix Science, London, UK) (Perkins et al., 1999), which incorporates a probability-based scoring, was used to search Swiss-Prot, TrEMBL and NCBI nonredundant databases with Mammalia (mammals) as taxonomic category. ProFound (V4.10.5) searches were performed using the same databases with the same taxonomic category. Database interrogation was carried out using monoisotopic peptide masses, 150 ppm mass tolerance, and 1 as the maximum number of missed tryptic cleavages. The molecular masses of the intact proteins and associated isoelectric (pI) points were taken from the 2-D maps. The interrogation also included possible modifications such as oxidation of Met and the alkylation of Cys residues by iodoacetamide. The criteria for positive identification of proteins were set as follow: protein scores were considered significant when the probability of random events was less than 1 in 20 (P ⬍0.05). For MASCOT analyses, this means that proteins with a probability-based MOWSE score (the reported MASCOT score in the identification Tables 1 and 2) greater than the threshold calculated by the tool algorithm were considered significant identifications. For ProFound it roughly corresponds to a Z-score greater than 1.65.

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Table 2. Identification of eight proteins which showed a significantly different level after a single social defeat SSP

MW [Da]

pl

5102 2110 6306

24687 21784 39153

5.8 5.5 7.1

7601

81428

4109 1102 2409 4205

21371 14504 41700 29820

MOWSEscore

MASCOT score

Z-score

Protein name

Accession number

% Sequence coverage

No. of peptides

Regulation

%

P

1.15 E4 1.30 E5 1.06 E9

96 81 109

2.31 2.2 2.22

O35244 P35704 P09117

48 36 41

12 8 12

Up Up Up

170 90 50

0.0077 0.0321 0.0166

8.3

2.22 E10

123

2.19

P10860

28

16

Up

140

0.0321

11.3 4.5 5.3 5.7

2.07 E5 66 5.24 E08 2.7 E7

Peroxiredoxin 6 Peroxiredoxin 2 Fructose-bisphosphate aldolase C Glutamate dehydrogenase Myelin basic protein Beta-synuclein Beta-actin Prohibitin

P02688 Q63754 P60711 P24142

40.7 34 36 39

10 7 12 10

Up Up Up Down

90 120 80 10

0.0343 0.0001 0.0098 0.0244

80 1.13 E4 69 98

1.74 2.3 2.42 2.19

SSP is the identification number of the selected spot assigned by the image analysis software. Theoretical values of molecular mass (MW) and pI are listed in columns 2 and 3. Probability scores of the identification are taken from the three database search software types; they are described in the Experimental Procedures section. SwissProt protein names and accession numbers are given in columns 7 and 8, respectively. The number of protein peptides that matches the theoretical peptides from the database entry and the sequence coverage percentage are indicated in columns 10 and 9 respectively. The regulation refers to the stressed group as compared to the control group and the percentage of variation is shown. The P value was determined using Student’s t test.

last exposure. In the open field test, animals belonging to the stressed group showed a significantly reduced number of line crossings as compared with the undefeated controls (defeated group: 67.6⫾16.1, mean⫾S.E.M.; control group: 105.4⫾7.8; P⬍0.05; Fig. 1, panel A). This reduction suggests an increase in anxiety-related behaviors. The proteomic analyses were carried out on two parallel, independent groups of rats (repeated stress and controls) which were not submitted to behavioral tests. Hippocampal proteins were extracted and separated by 2-D electrophoresis, thus obtaining maps in which 896⫾121 spots were detectable (mean⫾S.D.). For proteins that were unambiguously matched among the gels, levels were evaluated as volumes (spot area⫻optical density) with the image analysis software. The comparison between groups with image analysis followed by statistical testing revealed that 69

A third tool, ProteinProbe (V3.4, BioLynx; Micromass), was also used as an additional confirmation of the identified proteins and the resulting MOWSE score was considered significant if there was a difference of two orders of magnitude in the score value between the first and the second hit. The result of a database search was significant if the protein was ranked as best hit and there was sequence coverage of at least 20%. Positive identification of the protein was assigned only if the mass deviation of the matched peptides was constant over the whole mass range. Matched peptide masses were evenly distributed throughout the complete protein amino acid sequence, and the identified protein molecular mass and pI values corresponded to the measured values, with few exceptions.

RESULTS In the repeated-stress paradigm (see “study design” section), rats were exposed to social defeat stress for eight times and behavioral tests were conducted 30 h after the

B

A

120

100

*

Line crossings (n)

Line crossings (n)

120

80 60 40 20 0 Control

* p<0.05

100 80 60 40 20 0

Defeat

Control n=8-10

Defeat

n=10-11

Fig. 1. Results of the open field test performed 30 h after the last defeat procedure. Panel A shows the total number of line crossings of the animals after the repeated stress procedure (eight defeat session). * P⬍0.05 Student’s t-test. Panel B shows the number of line crossings of the animals which received a single exposure to the dominant rat.

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Fig. 2. Hippocampal proteins separated by 2-D electrophoresis. The pH range of 3–10 is non linear. The acrylamide gradient is 8 –18.5% T. Molecular mass markers from top: 250, 150, 100, 75, 50, 37, 25, 15, 10 kDa. The gel was stained with Sypro Ruby fluorescent stain. The spot numbers refer to identified proteins that showed modified levels after repeated exposure to social defeat, which are reported in Table 1. All gels were run together in the same apparatus at the same time.

spots (8% of spots) were significantly altered in the stress group as compared with control group. Spots were cut and submitted to peptide fingerprinting mass spectrometry and database search to identify the proteins. The 21 spots which were unambiguously matched to known proteins in the databases are listed in Table 1 and shown in Fig. 2. The repeated stress paradigm was able to modulate proteins possessing a chaperone function such as stress-70 protein (GRP75), 78-kDa glucose-regulated protein (GRP78), heatshock cognate 71 kDa protein (HSC71), peptidyl-prolyl cistrans isomerase A (PPIA), ERp29. Proteins mediating signal transduction and synaptic plasticity were also modified after exposure to repeated social stress (dihydropyrimidinase-related protein-2 [DRP-2], calmodulin, phosphatidylinositol transfer protein, vacuolar ATP synthase). Proteins involved in cytoskeleton regulation, such as F-actin capping protein, ␣-internexin, Ran GTPase and glial fibrillary acidic protein (GFAP) were also modified by stress. Other varied proteins are involved in energy metabolism pathways. Phosphoglycerate kinase, phosphoglycerate mutase, ␣-enolase, pyruvate kinase belong to the glycolytic pathway of glucose metabolism. Aconitate hydratase and malate dehydrogenase belong to the Krebs cycle; subunit 75 of complex I is part of the mitochondrial oxidative phosphorylation and transketolase is involved in the pentose phosphate pathway. In the single-stress paradigm, rats were exposed to a single social stress procedure and the open field test was

carried out to evaluate whether the procedure modified anxiety-like behavior. Animals exposed to a single defeat reduced the exploratory behavior when exposed to the arena 6 h after the social defeat (Marini et al., 2005). In this study, when experimental animals were tested 30 h after the exposure to the social defeat, no statistical difference in anxiety-related behavior could be detected between stressed and control groups (68.8⫾11.3 line crossings vs. 42.1⫾11.3, P⫽0.1123) (Fig. 1, panel B), suggesting that normal behavior may be reinstated. In order to evaluate the change induced by a single defeat session as compared by repeated stress, a proteomic analysis of hippocampal proteins was carried out at the same time point of 30 h after the stress. Stained maps displayed 669⫾80 proteins per gel (mean⫾S.D.), a reduced number when compared with the results obtained in the repeated stress group, that we ascribed to technical variability usually observed between separate runs and staining procedures. In comparing stressed versus control groups, 30 spots (4% of spots) displayed a statistically significant difference in their level in maps prepared from the hippocampi of defeated animals. All differently expressed spots were cut and peptide fingerprinting mass spectrometry was applied to identify the corresponding proteins. Peroxiredoxin 6, peroxiredoxin 2, fructose bis-phosphate aldolase, glutamate dehydrogenase, myelin basic protein, ␤-synuclein and ␤-actin were upregulated in the stressed group; prohibitin was downregulated. Additional data on the identified proteins

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Fig. 3. Hippocampal proteins separated by 2-D electrophoresis in the same conditions as in Fig. 2. The spot numbers refer to identified proteins whose levels were modified after exposure to a single social defeat, which are reported in Table 2. All gels were run together in the same apparatus at the same time.

are listed in Table 2 and shown in Fig. 3. The other identifications were unsuccessful due to insufficient amount of material or to absent database matching.

DISCUSSION This study reports a proteomic analysis of molecular changes in rat hippocampus induced by repeated and single exposure to psychosocial stress. The unbiased proteomics approach was able to reveal new proteins or signaling pathways involved in the induction of the stress response along with previously characterized mechanisms contributing to the stress response. Overall, the level of expression modulation is below two-fold, in line with previous large scale expression profiling data in the CNS, indicating that mRNA and protein level changes in the brain are rarely high in the absence of a pharmacological challenge (Voshol et al., 2003). Due to the characteristics of 2-D electrophoresis, the results are somewhat biased toward abundant, soluble cytosolic proteins; therefore it is likely that many other changes went undetected. It is also important to keep in mind that the differences that were detected could be due to a modification in the total amount of the changed proteins as well as to differences in posttranslational modifications, which may result in separate spots on the maps. In spite of these considerations, the study showed that, in the repeated-stress paradigm, where increase in anxiety behaviors persisted 30 h after the end of the last stress,

overall a larger percentage of molecular modifications was detected after repeated defeats as compared with the single exposure paradigm (69 versus 30 modified spots per group, respectively, which translates in 8% versus 4% when corrected for the total number of detected spots). Interestingly, no overlap was detected between the molecular changes induced by the repeated and single stress paradigms, although these findings should be interpreted with some caution. Indeed, a large number of false negatives can occur using this technique (due to the limited sensitivity of spot detection in 2-D gels), and subtle changes in low abundance proteins might have escaped the detection. Therefore we cannot rule out the possible existence of changes in common between the acute and chronic paradigm that the proteomic approach was unable to identify. However, our findings are consistent with the literature suggesting that chronic and acute stress procedures have a different impact on behavioral and endocrine parameters that are reflected also at the molecular and cellular level (Koolhaas et al., 1997), in particular as far as cellular plasticity mechanisms are concerned (de Kloet et al., 2005). It is indeed worth noting that a number of modulated proteins induced by repeated exposure to stress are associated with different cellular functions, including protein folding, signal transduction, synaptic plasticity, cytoskeleton regulation and energy metabolism, which do not seem to be activated after a single stress exposure.

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Chaperone proteins GRP75 and GRP78 displayed a reduced level in hippocampi from long-term stressed animals, whereas the opposite trend was shown for HSC71 and PPIA. HSC71, GRP75 and GRP78 belong to the 70 kDa heat-shock proteins, which in ordinary conditions play a role in helping protein folding and assembly. In the presence of cellular stress, their expression is usually induced and they exert a protective effect, helping to manage insults and to prevent apoptosis (Welch, 1992; Parcellier et al., 2003). The increase in HSC71 is in line with previous results since it is known that other stresses are able to induce its expression in brain: restraint-water immersion stress increases HSC71 mRNA in rat hippocampus and cerebral cortex (Fukudo et al., 1999, 1995). Similarly, a sub-chronic immobilization stress or cocaine administration can increase HSC71 immunoreactivity in the rat hippocampus (Hayase et al., 2003). It is conceivable that the increase in HSC71 is an attempt to reduce the negative consequences of stress. PPIA (also known as rotamase A or cyclophilin A), although widely expressed in many other tissues, is present in the highest concentration in brain. It has been hypothesized that PPIA plays a role in the maturation and folding of neurone-specific membrane proteins, especially ion channels (Helekar and Patrick, 1997). Modifications in the expression levels of this protein are not easily induced, so that its mRNA is often used as an internal control to normalize the levels of other mRNAs. Nevertheless, since a reduced level of PPIA was observed during plastic reorganization of brain cortex, a role was suggested for this protein as an inhibitor of cortical plasticity (Arckens et al., 2003). In rat hippocampus, limbic seizures are able to increase PPIA mRNA levels (Yount et al., 1992). HSC71 and PPIA have a cytoplasmic localization, whereas GRP75 and GRP78 are contained mainly in the mitochondrion and endoplasmic reticulum, respectively, where they exert a role as chaperones. Data in the literature indicate that a modification of GRP78 levels is induced in mental disorders (Katayama et al., 1999; Brown et al., 2000). It should also be kept in mind that the reduction in GRP78 levels discovered in this study may also be due to a specific decrease of a posttranslationally modified form, rather than the standard form of this protein. Glycosylated and phosphorylated forms exist for GRP78 which appear as separate bands on isoelectric-focusing (Laitusis et al., 1999), due to differences in the pI. ERp29 is an endoplasmic reticulum protein which was found in association with GRP78, suggesting a function of chaperone in this intracellular compartment specifically involving secretory proteins. It is highly expressed in brain, with the highest levels in cerebellum (Hubbard, 2002). Törönen et al. (2002) found that an acute treatment with MK801, an N-methylD-aspartate glutamate receptor antagonist, increased ERp29 mRNA in entorhinal cortex and the authors speculated that this protein may be involved in the secretion of brain-derived neurotrophic factor. If their hypothesis is

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correct, a reduction of ERp29 in repeatedly stressed animals may be related to the reduction of hippocampal brain-derived neurotrophic factor after stress (Smith et al., 1995). Proteins involved in synaptic functions Vacuolar ATP synthase belongs to a family of enzymes responsible for the acidification of intracellular compartments, mediating endocytosis and intracellular transport (Nishi and Forgac, 2002). In brain, they are involved in the re-uptake of neurotransmitters into synaptic vesicles (Maycox et al., 1990). Based on the findings obtained after a block of vacuolar ATPase rat hippocampal slices (Zhou et al., 2000), it may be hypothesized that the consequence of the reduced level of vacuolar ATP synthase found in repeatedly stressed rats would be a depression of GABAergic and glutamatergic transmission. A modification of neuronal activity is also mediated by proteins implicated in second messenger signaling. Among them, phosphatidyl inositol transfer protein is involved in phosphoinositide synthesis and in vesicle traffic, including exocytotic release (Liscovitch and Cantley, 1995); therefore the observed increase in its levels may impact on several cellular processes regulated by polyphosphoinositides (Pacheco and Jope, 1996). Similarly involved in second-messenger pathways, calmodulin is a Ca2⫹ binding protein that works as an intracellular receptor for calcium and, in its bound form, modulates the activity of several calcium-responsive target proteins. Calcium and calcium-regulated proteins, especially Ca2⫹/calmodulin kinase II, are suggested to modulate the synaptic plasticity induced by stress in the hippocampus (Kim and Yoon, 1998). A regulation of Ca2⫹/calmodulin kinase II has also been hypothesized in the mechanism of action of antidepressive agents (Popoli et al., 2001). DRP-2 (also known as CRMP-2 or TOAD64) shows reduced levels in the hippocampus of repeatedly stressed rats in this study. In developing hippocampal neurones, DRP-2 is involved in the regulation of axon formation, helping to establish and maintain neuronal polarity (Inagaki et al., 2001) by inducing microtubule assembly (Fukata et al., 2002). In addition to the function carried out during neuronal development, it is likely that this protein plays other roles in the adult. Yoshimura et al. (2002) reported that DRP-2 was identified in the post-synaptic density of adult rat brain as a substrate of Ca2⫹/calmodulin kinase II, thus suggesting that the protein plays a role in synaptic transmission and plasticity. It is therefore possible that the reduction of DRP-2 after repeated defeats is due to its involvement in the well-known modifications of plasticity and transmission observed in hippocampus after stress (Kim and Yoon, 1998; McEwen, 1999; Kim and Diamond, 2002). Cytoskeleton proteins F-actin capping protein regulates the organization of actin filaments by binding to the “fast-growing end” with high affinity (Yamashita et al., 2003). Its reduced level may

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bring about an impaired cytoskeletal reorganization. ␣-Internexin belongs to the class IV intermediate filament family proteins and it is widely expressed in the adult brain. Although neurofilament abnormalities lead to deleterious effects and even to cell death, the function of this class is not precisely known; possibly it is related to neurite elongation (Lariviere and Julien, 2004). GFAP is the major intermediate filament protein in astrocytes. In agreement with the results obtained in our rat model, a reduction in GFAP immunoreactivity was observed in human hippocampus both in depressive patients and in patients treated with corticosteroids (Muller et al., 2001). Ran GTPase is involved in nuclear transport, mitotic spindle assembly and nuclear envelope assembly (Quimby and Dasso, 2003).

CONCLUSION In conclusion, this work showed that changes in protein levels could be detected in the hippocampus of rats subjected to a repeated or single paradigm of social defeat. The single stress paradigm induced a lower number of changes and they were different from the repeated stress pattern. Most modulated spots are ascribed to the pool of soluble and abundant cytoplasmic proteins, which are more easily detected in 2-D electrophoresis experiments (Fountoulakis, 2004). Several changed proteins have already been associated with stress-related responses; some of them are here described for the first time in relation to stress. The above findings may open new opportunities for further investigations on the modulation induced in the brain by stress at the molecular level.

Energy metabolism Enzymes belonging to glycolytic pathway displayed an up-regulation, which was not matched by Krebs cycle enzymes or by the respiratory chain. Since the glycolytic pathway is mainly carried out in astrocytes (Tsacoupoulos and Magistretti, 1996), it is conceivable that chronic stress may increase lactate production, as was described after other stresses (Schasfoort et al., 1988; Elekes et al., 1996). The decrease in the 75 kDa subunit of the NADH-ubiquinone oxidoreductase may impair the function of complex I, which was also detected in other models of chronic stress (Madrigal et al., 2001), thus contributing to the generation of damaging reactive oxygen species (Liu et al., 2002). The present results show that in the single-stress paradigm a difference can be detected at the molecular level when no overt stress-induced behavior is apparent. It is known that long-term effects are induced even after a single exposure to social defeat (Koolhaas et al., 1997; Meerlo et al., 2002; von Frijtag et al., 2000), in which protein changes may be involved. Among the proteins modulated by the exposure to a single social defeat, peroxiredoxin 2 and peroxiredoxin 6 belong to a family of enzymes which are believed to function as antioxidants (Phelan, 1999). ␤-Synuclein is hypothesized to play a role in pre-synaptic terminal function, including plasticity and memory (Clayton and George, 1999), therefore suggesting a possible involvement in plasticity induced by stress. Glutamate dehydrogenase, the enzyme that controls the main catabolic pathway of glutamate in astrocytes, is increased after a single exposure to psychosocial stress. This enzyme is known to be induced by glucocorticoids (Hardin-Pouzet et al., 1996), suggesting an explanation for the higher levels observed after the exposure to a stressing condition. The glycolytic enzyme fructose bisphosphate aldolase C, which is increased after single social defeat, is also increased in brains of patients affected by mood disorders or schizophrenia (Johnston-Wilson et al., 2000). Also, structural proteins such as actin and myelin basic protein are changed by the exposure to the single paradigm of social stress.

Acknowledgments—We thank Federico Faggioni for technical support, Dr. Fabio Bordi for helpful suggestions on the animal model and Dr. Fabrizio Caldara for assistance on bioinformatic tools.

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(Accepted 17 October 2005) (Available online 7 December 2005)