Early life experience alters behavior during social defeat: Focus on serotonergic systems

Neuroscience 136 (2005) 181–191

EARLY LIFE EXPERIENCE ALTERS BEHAVIOR DURING SOCIAL DEFEAT: FOCUS ON SEROTONERGIC SYSTEMS K. L. GARDNER,a K. V. THRIVIKRAMAN,b S. L. LIGHTMAN,a P. M. PLOTSKYb AND C. A. LOWRYa*

Key words: maternal separation, social defeat, serotonin, dorsal raphe nucleus, anxiety, c-Fos.

a

Henry Wellcome Laboratories for Integrative Neuroscience and Endocrinology, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK

Previous human and animal studies have shown that early life experience can alter neuroendocrine, autonomic, and behavioral responses to stress throughout adult life (Heim and Nemeroff, 2001). Maternal separation is one model that has been used extensively to characterize the effects of early life experience on subsequent responses to stress in adulthood (Plotsky and Meaney, 1993). This model is based on separation of neonatal rat pups from their mothers during a critical period of development. Rats exposed to long periods of maternal separation during the first 2 weeks of life (MS180 rats) have augmented stress-induced hypothalamic–pituitary–adrenal (HPA) axis and behavioral responses in adulthood, including increased anxiety-like behaviors, anhedonia, increased preference for ethanol, and impairment of male sexual behavior (Plotsky and Meaney, 1993; Ladd et al., 1996; Wigger and Neumann, 1999; Huot et al., 2001; Rhees et al., 2001; Kalinichev et al., 2002). In contrast, studies have shown that animals exposed to short periods of maternal separation during the first 2 weeks of life (MS15 rats) have a decreased HPA axis response to stress and decreased anxiety in adulthood (Meaney et al., 1996; Ladd et al., 2000). One mechanism through which early life experience may influence neuroendocrine and behavioral changes seen in adults is through actions on neuromodulatory systems, such as serotonergic systems. Studies have shown that early life experience has long-term effects on 5-hydroxytryptamine1B (5-HT1B) receptor gene expression, 5-HT2C receptor density, and 5-HT neurotransmission (Smythe et al., 1994; Neumaier et al., 2002; Gartside et al., 2003; Daniels et al., 2004; Van Riel et al., 2004). Social defeat, which is the result of intraspecific confrontation between male rats, is a powerful, biologically relevant, psychosocial stress which impacts on serotonergic systems and behavior. Animals that have experienced defeat display changes in neuroendocrine, autonomic, and behavioral function (Raab et al., 1986; Herbert, 1987). Social defeat has been shown to increase the expression of the protein product of the immediate-early gene c-fos in the dorsal raphe nucleus (DR) (Martinez et al., 1998), although it has not been determined if the increased c-Fos expression in the DR is localized to serotonergic or nonserotonergic neurons. Furthermore, animals exposed to social defeat display a down-regulation of 5-HT1A receptors in the DR and an increase in anxiety-like behaviors (Short, 2003). Social defeat is therefore a useful tool to

b

Department of Psychiatry and Behavioral Sciences, Emory School of Medicine, 1639 Pierce Drive, Atlanta, GA, 30322, USA

Abstract—Early life experience can have prolonged effects on neuroendocrine, autonomic, and behavioral responses to stress. The objective of this study was to investigate the effects of early life experience on behavior during social defeat, as well as on associated functional cellular responses in serotonergic and non-serotonergic neurons within the dorsal raphe nucleus, a structure which plays an important role in modulation of stress-related physiology and behavior. Male Long Evans rat pups were exposed to either normal animal facility rearing or 15 min or 180 min of maternal separation from postnatal days 2–14. As adults, these rats were exposed to a social defeat protocol. Differences in behavior were seen among the early life treatment groups during social defeat; rats exposed to 180 min of maternal separation from postnatal days 2–14 displayed more passive–submissive behaviors and less proactive coping behaviors. Analysis of the distribution of tryptophan hydroxylase and c-Fos-like immunoreactivity in control rats exposed to a novel cage and rats exposed to social defeat revealed that, independent of the early life experience, rats exposed to social defeat showed an increase in the number of c-Fos-like immunoreactive nuclei in serotonergic neurons in the middle and caudal parts of the dorsal dorsal raphe nucleus and caudal part of the ventral dorsal raphe nucleus, regions known to contain serotonergic neurons projecting to central autonomic and emotional motor control systems. This is the first study to show that the dorsomedial part of the mid-rostrocaudal dorsal raphe nucleus is engaged by a naturalistic stressor and supports the hypothesis that early life experience alters behavioral coping strategies during social conflict; furthermore, this study is consistent with the hypothesis that topographically organized subpopulations of serotonergic neurons principally within the mid-rostrocaudal and caudal part of the dorsal dorsal raphe nucleus modulate stress-related physiological and behavioral responses. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹44-117-331-3119; fax: ⫹44-117-331-3120. E-mail address: [email protected] (C. A. Lowry). Abbreviations: AFR, animal facility rearing; ANOVA, analysis of variance; BNST, bed nucleus of the stria terminalis; DR, dorsal raphe nucleus; DRD, dorsal raphe nucleus, dorsal part; DRV, dorsal raphe nucleus, ventral part; HPA, hypothalamic–pituitary–adrenal; MANOVA, multivariate analysis of variance; MS15, 15 min of maternal separation from postnatal days 2–14; MS180, 180 min of maternal separation from postnatal days 2–14; PB, 0.1 M sodium phosphate buffer; PBS, 0.05 M phosphate-buffered saline; PBST, 0.3% Triton X-100 in 0.05 M phosphate-buffered saline; PND, postnatal day; TrpOH, tryptophan hydroxylase; 5-HT, 5-hydroxytryptamine.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.07.042

181

182

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

help elucidate changes in serotonergic systems and stress-responsivity following early life experience. This study aimed to test the hypothesis that early life adverse experience alters serotonergic activity within the DR and associated behavioral responses during social defeat. In addition, as previous studies have demonstrated that stress-related stimuli have selective actions on subpopulations of serotonergic neurons within the mid-rostrocaudal and caudal parts of the DR (Abrams et al., 2005; Grahn et al., 1999; Staub et al., 2005), we hypothesized that social defeat increases the neuronal activity of serotonergic neurons within these specific regions.

EXPERIMENTAL PROCEDURES Maternal separation All animal care was conducted in accordance with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Emory University Institutional Animal Care and Use Committee. Particular care was taken in order to minimize the number of animals used and their suffering. Timed-pregnant Long Evans Crl:(LE)BR rats (Charles River, Portage, MI, USA) arrived at the Emory University vivarium on gestational day 12. Upon arrival, dams were individually housed and maintained on a 12-h light/dark cycle (lights on at 07:00 h) with food and water available ad libitum. The animal care and maternal separation procedures used have been described in detail previously (Plotsky and Meaney, 1993; Huot et al., 2001). The day of birth was designated as postnatal day 0 (PND0). Male rat pups available for this study (72 rat pups, 36 rat pups of which were used in this study) were pooled and standardized to foster litters consisting of eight rat pups each (nine foster litters in total). The nine foster litters were randomly assigned to one of three rearing conditions: (1) animal facility rearing (AFR)— handling of pups twice a week during routine cage changes, (2) MS15— daily 15-min period of maternal separation from PND2-14 inclusive, (3) MS180 — daily 180-min period of maternal separation from PND2-14 inclusive. Protocols involving manipulation of the pups took place between 08:00 and 12:00 h daily. During separation, each dam was removed from its maternity cage and placed into an adjacent identical cage until the end of the separation period. Pups were then removed as complete foster litters from the nest, placed into an empty cage and transferred to an incubator in an adjacent room. The incubator was maintained at 32⫾0.5 °C from PND2– PND5 and 30⫾0.5 °C from PND6 –PND14. At the end of the separation period, foster litters were returned to their maternity cages, followed by reunion with the dams. Bedding in the transfer cages and incubator was never changed. During PND4 –PND14, half of the bedding in the maternity cages was changed once a week while the pups and dams were out of the cage. During PND15–PND18 foster litters were not disturbed. Beginning on PND18, bedding was completely changed twice a week. Pups were weaned on PND21, housed with their foster littermates until PND30, then, using a completely randomized design, pair housed throughout adulthood with a member of the same early life treatment group. Of the 12 pairs of rats available for each early life experience treatment group, six pairs of rats were randomly selected for this experiment. Two days prior to the experiment adult rats were separated and individually housed.

Social defeat For the social defeat protocol, half of the individually housed rats from each early life treatment group were randomly assigned to the novel cage control group; the other half were assigned to the

social defeat group. The social defeat paradigm consisted of both a pre-defeat phase and a defeat phase, each lasting 10 min (Martinez et al., 1998). Resident males were heavier and older than the male test subjects (intruders). The average weight of the intruders was 393⫾10.6 g and the average weight of the residents was 574⫾48.7 g; the residents were always at least 57 g heavier than the intruders. Bedding in the resident’s cage was left unchanged the week prior to the experiment. The resident female was removed from the resident’s cage 15 min prior to the experiment. The resident’s cage (59.4 cm length⫻30.8 cm width⫻ 22.9 cm height) was fitted with a perforated Plexiglas plate to divide the length of the cage into a small (1/3) and a large (2/3) compartment with the resident in the large compartment, which prevented physical contact during the pre-defeat phase but allowed the rats to see and smell each other. Immediately following the initial 10 min pre-defeat phase, the Plexiglas divide was removed and the rats were allowed to interact for 10 min. Behavior during both phases of the social defeat was recorded using a video camera mounted on a tripod at the side of the cage. Control rats were placed in a clean novel cage (1042 cm3 floor space, identical to the home cage) for 20 min in the experimental room, out of visual sight of the resident’s cage, during the social defeat. Experiments were completed within the first 6 h of the light phase.

Behavior Behavior of the intruder was analyzed using EthoLog© 2.2.5 (Eduardo B. Ottoni, São Paulo, SP, Brazil), a computer-based ethological observation tool. Behaviors were coded by an observer blind to the early life treatment group. During the pre-defeat phase eight behaviors were scored using criteria based on a previous study (Martinez et al., 1998; Table 1). During the defeat phase all of the pre-defeat behaviors (except for sniffing resident, close to partition, and digging) and seven additional behaviors were measured (Table 2).

Statistical analysis of behavior Pre-defeat behavior and defeat behavior were analyzed separately. To determine if rats in different early life treatment groups showed different patterns of behavior during the pre-defeat phase, multivariate analysis of variance MANOVA was performed separately for the frequency and duration of specific behaviors, using all behavioral scores in each analysis. In the presence of significant treatment effects using MANOVA, post hoc single ANOVAs were conducted for each behavioral category to determine if the frequency or duration of these categories of behavior was different among the three treatment groups. Table 1. Behaviors of the intruder measured during the pre-defeat phase of the social defeat protocol Behavior

Definition

Sniffing resident Close to partition

Sniffing through the holes of the partition All other behavior conducted in the proximity of the partition, including sniffing, pacing, rearing and general locomotion close to partition Walking around the cage Sniffing the substrate without locomotion Moving sawdust with forelimbs Bipedal posture Licking or scratching coat Intruder is completely motionless except for movement associated with respiration

Locomotion Sniffing bedding Digging Rearing Self-grooming Freezing

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191 Table 2. Additional behaviors of the intruder measured during the defeat phase of the social defeat protocol Behavior

Definition

Upright defensive behavior Passive genital sniff Full submission

Rearing while facing the resident Being sniffed The intruder lies on its back with its belly exposed to the resident The intruder crouches below the resident and turns to expose part of its belly Fleeing from the resident Biting the resident Sniffing the resident’s genitals

Sideways submission

Escape Attack Genital sniff

For statistical analysis of defeat behavior, the behavioral measurements were divided into categories: passive–submissive, proactive coping, and neutral (Table 3). This model is based on previous studies identifying different behavioral coping strategies in male rodents, e.g. passive coping (reactive coping) and active coping (proactive coping) where aggressive males appear to have a more proactive behavioral response and non-aggressive or reactive males appear to be more adaptable in their behavioral strategies, responding only when absolutely necessary (Koolhaas et al., 1999; De Boer and Koolhaas 2003; Miczek et al., 2004). Therefore, we would predict that individual scores for the duration of passive–submissive and proactive coping to be inversely correlated. This was in fact the case (r⫽⫺0.572, P⫽0.013). Separate MANOVAs were used to analyze the frequency and duration of behaviors to determine if rats in different early life treatment groups showed different patterns of behavior during the defeat phase. In the presence of significant treatment effects using MANOVA, post hoc single ANOVAs were conducted using 1) the frequency or duration of passive–submissive behaviors, 2) the frequency or duration of proactive coping behaviors, and 3) the ratio of the frequency or duration of passive–submissive to proactive coping behaviors to determine if they were different among the three treatment groups. MANOVA analysis was conducted using JMP statistical software package (version 4.0.4; SAS Institute Inc., Cary, NC, USA) using the IDENTITY model for pre-defeat behavior and the SUM model for defeat behavior contrasting passive–submissive and proactive coping behaviors; in all cases neutral behaviors were excluded to avoid linearity in the model (i.e. all scores summing to the maximum value of 600 s). When appropriate, orthogonal contrasts were conducted to compare individual treatment groups. Subsequent post hoc ANOVAs and when appropriate Fisher’s protected least significant difference tests were conducted using SYSTAT 5.02 for Windows (SYSTAT Inc., Evanston, IL, USA).

Immunohistochemistry Following behavioral testing, rats were returned to their home cages and transferred back into the housing room for 60 min. Rats

183

were then anesthetized with sodium pentobarbital (Fatal Plus, Vortech Pharmaceuticals, Dearborn, MI, USA), transferred to a procedure room and perfused via the ascending aorta with ice cold 0.9% saline followed by 4% paraformaldehyde in 0.1 M sodium phosphate buffer (PB), pH 7.4. Brains were post-fixed for 24 h in the same fixative, rinsed twice for 12 h in 0.05 M phosphate-buffered saline (PBS; pH 7.4) and immersed in 20% sucrose solution in PBS for cryoprotection. Brains were blocked at approximately ⫺6.0 mm bregma using a rat brain matrix (RBM-4000C, ASI Instruments, Warren, MI, USA), rapidly frozen in liquid nitrogen cooled isopentane and stored at ⫺80 °C. Six alternate sets of 30 ␮m cryostat sections were prepared and placed in cryoprotectant (27% ethylene glycol, 16% glycerol in PB; pH 7.4) in 24-well polystyrene tissue culture plates. One set of sections was used for double-immunostaining for c-Fos and tryptophan hydroxylase (TrpOH). Sections were washed in PBS then incubated with freshly prepared 1% H2O2 in PBS for 10 min, rinsed with PBS, then PBS containing 0.3% Triton X-100 (PBST), followed by incubation with rabbit polyclonal anti-c-Fos antiserum (Cat. No. ab-5, Lot #D14854; Oncogene Sciences, Calbiochem, Nottingham, UK) diluted 1:10,000 in PBST for 16 h. Sections were washed using PBST then incubated with biotinylated swine anti-rabbit antiserum (1:200; Cat. No. E0353, DAKO, Cambridgeshire, UK) for 80 min. Sections were washed again using PBST then incubated with ABC reagent (1:200; Cat. No. PK-6101, Vector Laboratories, Burlingame, CA, USA) for 80 min. After washing with PBST, then PBS, sections were incubated with substrate (Vector SG peroxidase substrate, Cat. No. SK-4700) for 20 min. Sections were rinsed thoroughly with PBS then incubated with an affinity-purified sheep anti-TrpOH antibody (1:12,000; Cat. No. 9260-2505, Lot #21040310, Biogenesis, Ltd., Poole, UK) in PBST for 16 h. Sections were washed using PBST then incubated with biotinylated rabbit anti-sheep antiserum (1:200; Cat. No. PK6106, Vector Laboratories) for 80 min. Sections were washed using PBST then incubated with PBS containing substrate (0.05% 3,3=-diaminobenzidine tetrahydrochloride, Cat. No. D-5637, Sigma) and 0.012% H2O2 for 35 min. Sections were rinsed briefly in distilled water then transferred to glass slides and coverslipped using DPX mounting medium (BDH Laboratory Supplies, Poole, UK).

Data quantification and statistical analysis of cell counts Determination of c-Fos-, TrpOH- and double-immunostained (c-Fos and TrpOH) cell counts within subdivisions of the DR was aided by comparing the stained sections with illustrations in a rat brain stereotaxic atlas (Paxinos and Watson, 1998). For quantitative analysis of cells positive for c-Fos and TrpOH, one section from each of four rostrocaudal levels (⫺7.30, ⫺8.00, ⫺8.18, ⫺8.54 mm bregma) in each rat was selected based on topographical features of TrpOH immunostaining observed at 10⫻ magnification (Abrams et al., 2004). In each section, the numbers of c-Fos-positive, TrpOHpositive, and double-stained cells were counted within subdivisions of the DR (Paxinos and Watson, 1998) at 400⫻ magnification by an observer blind to the early life and stress treatments.

Table 3. Categories of behavioral measurements used to analyze defeat behavior Category

Description

Behaviors included

Passive–submissive

Behaviors with vigilance, anxiety-related, fear-related, or risk assessment components as well as submissive behaviors Confrontational behaviors and behaviors with exploration or escape components

Passive genital sniff, genital sniff, sniffing bedding, freezing, sideways submission, and full submission

Proactive coping Neutral a

(E.g. explorative escape as defined by De Boer and Koolhaas, 2003).

Attack, upright defensive behavior, rearing,a and escape Self-grooming and locomotion

184

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

To determine the effects of and potential interactions among early life experience, social defeat, and brain region a single multifactorial analysis of variance (ANOVA) with repeated measures was used for each dependent variable (c-Fos-ir/TrpOHimmunonegative cells, TrpOH-ir cells, and double-immunostained cells), with early life experience and social defeat as between subjects factors and brain region as a within subjects factor. Prior to the multifactorial ANOVA with repeated measures, missing values (3% of the 1155 total data points) were replaced using the Petersen method (Petersen, 1985). These replacement values were used for the multifactorial ANOVA with repeated measures only. If the multifactorial ANOVA with repeated measures indicated an effect of early life experience, social defeat, or interactions among these factors and brain region, the topographical distribution of the effect was determined using a two-factor ANOVA for each brain region. All statistics were performed using SYSTAT 5.02 for Windows.

RESULTS Pre-defeat behavior Analysis of the frequencies of behaviors during the predefeat phase using MANOVA revealed different patterns of behavior among the three early life event treatment groups (Wilks’ ␭(12,18)⬇2.396; P⫽0.0457; Table 4). Post hoc analysis using orthogonal contrasts revealed that MS180 rats had a different pattern of behavior compared with AFR rats (F(6,9)⫽4.309; P⫽0.0252). The comparison of the patterns of behavior of MS180 rats and MS15 rats approached statistical significance (F(6,9)⫽3.145; P⫽0.060), while there was no difference in the patterns of behavior of MS15 and MS180 rats (F(6,9)⫽1.112; P⫽0.425). Further analysis of the frequencies of behaviors during the pre-defeat phase using post hoc ANOVA and pairwise comparisons revealed that rats with different early life experience responded with different frequencies of rearing (F(2,14)⫽6.801; P⫽0.009). MS180 and MS15 rats reTable 4. Frequency and duration of the intruders’ behaviors during the pre-defeat phase of the social defeat protocol Behavior

MS15

AFR

Frequency Sniffing resident Close to partition Locomotion Sniffing bedding Digging Rearing Self-grooming Freezing

13.5⫾2.9 45.3⫾2.2 38⫾2.5 20.3⫾3.2 3⫾3.0 20.6⫾1.3* 4.8⫾1.3 3.2⫾1.8

9.8⫾2.5 46.4⫾4.8 39⫾2.8 25.4⫾3.5 3.6⫾2.2 27⫾2.8 2.6⫾0.8 3.4⫾1.0

Duration Sniffing resident Close to partition Locomotion Sniffing bedding Digging Rearing Self-grooming Freezing

49.6⫾11.6 217.3⫾9.2 77.9⫾6.5 116.2⫾18.7 5.3⫾5.3 72.4⫾7.5 23.5⫾8.0 11.6⫾7.2

21.1⫾3.1 240.3⫾29.4 74.9⫾6.7 149.5⫾22.4 4.7⫾3.3 98.5⫾16.9 12.8⫾4.0 5.6⫾1.7

MS180

8.2⫾2.3 42.8⫾3.6 32⫾2.7 25.7⫾1.4 1.3⫾0.6 16.2⫾2.0** 4⫾1.0 5.2⫾2.0 29.5⫾7.2 204.0⫾12.1 75.3⫾8.8 164.6⫾18.3 2.7⫾1.2 65.7⫾10.6 12.8⫾4.2 16.2⫾8.9

Values are presented as mean⫾S.E.M. * P⬍0.05 compared to AFR; ** P⬍0.01 compared to AFR.

Table 5. Frequency and duration of the intruders’ behaviors during the defeat phase of the social defeat protocol Behavior Frequency Locomotion Sniffing bedding Rearing Self-grooming Freezing Upright defensive behavior Passive genital sniff Full submission Sideways submission Escape Attack Genital sniff Duration Locomotion Sniffing bedding Rearing Self-grooming Freezing Upright defensive behavior Passive genital sniff Full submission Sideways submission Escape Attack Genital sniff

MS15

AFR

MS180

48.3⫾9.1 13.2⫾3.0 13.5⫾4.8 1.3⫾0.3 26.7⫾4.4 15⫾3.6

36⫾6.3 12.7⫾3.2 17.2⫾6.0 0.3⫾0.2 21.5⫾3.2 12.0⫾1.9

48.7⫾9.0 16.7⫾3.6 8.3⫾2.6 1.2⫾1.0 34.2⫾6.4 8.8⫾3.6

8.2⫾2.5 3.8⫾1.2 2.7⫾1.5 4.8⫾2.1 3.0⫾1.7 16.0⫾4.3

6.8⫾2.8 5.8⫾1.6 2.3⫾1.5 4.5⫾1.5 4.0⫾1.5 12.8⫾5.6

13.2⫾3.5 4.8⫾0.9 4.7⫾1.9 3.8⫾1.6 0.7⫾0.3 14.8⫾5.8

136.6⫾19.9 50.2⫾11.3 52.9⫾19.4 5.2⫾2.4 149.0⫾28.0 80.7⫾23.5

87.6⫾16.7 55.6⫾14.9 66.4⫾23.6 2.1⫾1.3 162.3⫾44.4 53.7⫾5.5

131.1⫾26.4 71.8⫾18.0 35.0⫾13.6 4.7⫾3.0 168.6⫾36.4 30.7⫾12.8

28.5⫾10.2 23.9⫾10.3 7.7⫾3.4 4.9⫾2.2 4.5⫾1.9 45.1⫾8.9

39.5⫾22.3 50.2⫾16.0 4.5⫾3.3 5.8⫾1.6 4.7⫾2.1 45.8⫾24.0

53.9⫾17.9 41.6⫾14.8 12.6⫾7.8 5.5⫾1.8 0.3⫾0.2 50.4⫾21.2

Values are presented as mean⫾S.E.M.

sponded with a lower incidence of rearing behavior compared with AFR rats (Table 4). There were no effects of early life experience on the frequency of any other behavior. Analysis of the durations, as opposed to the frequencies, of behaviors, during the pre-defeat phase using MANOVA revealed that there were no differences in the patterns of the durations of behaviors among the three early life event treatment groups (Wilks’ ␭(12,18)⬇1.001; P⫽0.4848; Table 4). Defeat behavior The means and S.E.M.s of the frequencies and durations of all behaviors during the defeat phase are listed in Table 5. During the defeat phase, the resident male rat always attacked the intruder and the intruder always displayed submissive behavior during the 10 min period (Table 5). Rats often showed different behavioral strategies to demonstrate submissive status. For example four rats (two MS15 rats, one AFR rat, and one MS180 rat) never showed sideways submission but did show full submission, while one rat (AFR) never showed full submission but did show sideways submission. None of the intruders defeated the resident male rat. Analysis of the frequencies of behaviors during the defeat phase using MANOVA revealed different patterns of behavior among the three early life event treatment groups

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

185

Fig. 1. Frequency of behaviors during the defeat phase of the social defeat protocol including (a), passive–submissive behaviors, (b), proactive coping behaviors, and (c), neutral behaviors. (d) Ratio of the frequency of passive–submissive behaviors versus the frequency of proactive coping behaviors. * P⬍0.05 compared with MS15; † P⬍0.05 compared with AFR. Results are presented as the mean⫾S.E.M.

Fig. 2. Duration of behaviors during the defeat phase of the social defeat protocol, including (a), passive–submissive behaviors, (b), proactive coping behaviors, and (c), neutral behaviors. (d) Ratio of the duration of passive–submissive behaviors versus the duration of proactive coping behaviors. * P⬍0.05 compared with MS15; † P⬍0.05 compared with AFR. Results are presented as the mean⫾S.E.M.

(F(2,15)⫽6.636; P⫽0.0086; Fig. 1). Post hoc analysis using orthogonal contrasts revealed that MS180 rats had a different pattern of behavior compared with either MS15 rats (F(1,15)⫽12.113; P⫽0.0034) or AFR rats (F(1,15)⫽7.140; P⫽0.0174). There were no differences in the patterns of behavior of MS15 and AFR rats (F(1,15)⫽0.654; P⫽0.432). Further analysis of the frequencies of behaviors during the defeat phase using post hoc ANOVA and pairwise comparisons revealed that early life experience altered the ratio of passive–submissive versus proactive coping behaviors (F(2,15)⫽6.017; P⫽0.012). MS180 rats displayed a higher ratio of passive–submissive versus proactive coping behaviors compared with either MS15 or AFR rats (Fig. 1d).

Analysis of the durations, as opposed to the frequencies, of behaviors during the defeat phase using MANOVA revealed different patterns of behavior among the three early life event treatment groups (F(2,15)⫽5.581; P⫽ 0.0154). Post hoc analysis using orthogonal contrasts revealed that MS180 rats had a different pattern of behavior compared with MS15 rats (F(1,15)⫽11.008; P⫽0.0047). MS180 rats responded with more passive–submissive behavior and less proactive coping behavior compared with MS15 rats (Fig. 2). The comparison of the patterns of behavior of MS180 and AFR rats approached statistical significance (F(1,15)⫽3.996; P⫽0.064). There were no differences in the patterns of behavior of MS15 and AFR rats (F(1,15)⫽1.739; P⫽0.207).

Abbreviations used in the figures Aq B9 BL Ce CLi Con CRF DRI Dtgx IPA IPVL ml mlf

cerebral aqueduct supralemniscal serotonergic cell group basolateral amygdaloid nucleus central amygdaloid nucleus caudal linear nucleus control corticotropin-releasing factor dorsal raphe nucleus, interfascicular part dorsal tegmental decussation interpeduncular nucleus, apical subdivision interpeduncular nucleus, ventrolateral subdivision medial lemniscus medial longitudinal fasciculus

MnR PAG PnR RMg ROb RPa scp SD VLPAG vgtx 3

median raphe nucleus periaqueductal gray pontine raphe nucleus raphe magnus nucleus raphe obscurus nucleus raphe pallidus nucleus superior cerebellar peduncle social defeat ventrolateral periaqueductal gray ventral tegmental decussation oculomotor nucleus

186

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

Further analysis of the durations of behaviors during the defeat phase using post hoc ANOVA and pairwise comparisons revealed that early life events altered the total duration of passive–submissive behavior (F(2,15)⫽3.865; P⫽0.044; Fig. 2a), proactive coping behavior (F(2,15)⫽ 4.468; P⫽0.03; Fig. 2b) and the ratio of passive–submissive versus proactive coping behaviors (F(2,15)⫽4.358; P⫽0.032; Fig. 2d). There were no differences among early life experience treatment groups in the duration of neutral behaviors (e.g. self-grooming and locomotion; Fig. 2c). Post hoc pairwise comparisons revealed that the MS180 rats showed more passive–submissive behavior compared with MS15 rats (Fig. 2a) and less proactive coping behavior compared with MS15 and AFR rats (Fig. 2b). MS180 rats also showed a higher ratio of passive–submissive versus proactive coping behaviors compared with both AFR and MS15 rats (Fig. 2d).

Double-immunostained neurons Social defeat altered the number of c-Fos/TrpOH doubleimmunostained neurons in the DR (social defeat main effect; F(1,29)⫽5.58; Pⱕ0.05). Post hoc analysis revealed that the effects of social defeat were restricted to specific subdivisions of the DR. Post hoc two-factor ANOVA within each brain region revealed that the effects of social defeat on c-Fos expression in TrpOH-immunoreactive neurons were restricted to the mid-rostrocaudal dorsal raphe nucleus, dorsal part (DRD) (⫺8.00 mm bregma; F(1,27)⫽ 14.43, Pⱕ0.001), the caudal DRD (⫺8.54 mm bregma; F(1,28)⫽5.38, Pⱕ0.05), and the caudal dorsal raphe nucleus, ventral part (DRV) (⫺8.54 mm bregma; F(1,28)⫽ 4.35, Pⱕ0.05) (Fig. 3). Effects of social defeat approached statistical significance in the rostral DRD (social defeat main effect; ⫺7.30 mm bregma; F(2,27)⫽2.73, P⫽0.083) and the mid-rostrocaudal DRV (social defeat main effect; ⫺8.00 mm bregma; F(2,27)⫽2.88, P⫽0.073).

Fig. 3. Effects of early life experience and social defeat on the number of cells positive for both c-Fos and TrpOH immunoreactivity (doubleimmunoreactive). (a) Graphs illustrating the effects of early life events and social defeat on the number of c-Fos/TrpOH-immunoreactive neurons in different subdivisions of the DR. The bregma levels of the four levels of the DR studied are listed above each column of graphs. * Main effect of social defeat based on two factor ANOVA within each brain region, P⬍0.05. Results are presented as the mean⫾S.E.M. (b) Photomicrographs showing the four rostrocaudal levels with overlays of drawings of the DR subdivisions analyzed. Scale bar⫽500 ␮m.

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

187

Fig. 4. Effects of early life experience and social defeat on the number of TrpOH-immunonegative, c-Fos-immunoreactive cells. (a) Graphs illustrating the effects of early life experience and social defeat on the number of c-Fos-immunoreactive neurons in specific subdivisions of the DR. The bregma levels of the four levels of the DR studied are listed above each column of graphs. * Main effect of social defeat based on two factor ANOVA within each brain region, P⬍0.05. Results are presented as the mean⫾S.E.M. (b) Photomicrographs showing the four rostrocaudal levels with overlays of drawings of the DR subdivisions analyzed. Scale bar⫽500 ␮m.

c-Fos single-immunostained neurons Social defeat altered c-Fos immunoreactivity in TrpOHimmunonegative cells in the DR in a regionally dependent manner (social defeat⫻brain region interaction; F(10,290)⫽ 4.55; P⬍0.0001). Post hoc two-factor ANOVA within each brain region revealed that the effects of social defeat on c-Fos expression were restricted to the mid-rostrocaudal DRD (early life event⫻social defeat interaction; ⫺8.00 mm bregma; F(2,27)⫽3.44, Pⱕ0.05), the DR, ventrolateral part (social defeat main effect; ⫺8.18 mm bregma; F(1,29)⫽

6.66, Pⱕ0.05), and the caudal DRV (social defeat main effect; ⫺8.54 mm bregma; F(1,28)⫽10.36, Pⱕ0.01) (Fig. 4). Effects of social defeat approached statistical significance in the rostral DRD (social defeat main effect; ⫺7.30 mm bregma; F(1,27)⫽3.81, P⫽0.061) and the caudal DRD (⫺8.54 mm bregma; F(2,28)⫽2.74, P⫽0.082). TrpOH-immunostained neurons Statistical analysis revealed there were no effects of social defeat or early life experience on the total number of

188

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

Table 6. Total number of TrpOH immunoreactive neurons ELE

MS15 MS15 AFR AFR MS180 MS180

Social defeat

Control Social defeat Control Social defeat Control Social defeat

⫺7.30

⫺8.00

DRD

DRV

DRD

DRV

DRVL

36.0⫾4.7 31.0⫾4.7 37.5⫾6.4 22.2⫾3.4 35.7⫾4.3 36.8⫾3.8

24.6⫾3.6 25.2⫾4.7 27.2⫾3.0 16.8⫾1.5 24.7⫾4.8 29.0⫾3.6

53.7⫾5.9 53.6⫾3.9 57.8⫾5.3 86.3⫾3.6 70.8⫾5.5 69.0⫾7.9

112.0⫾14 145.7⫾5.9 123.0⫾8.7 110.3⫾7.5 120.7⫾5.9 113.7⫾5.0

66.8⫾7.0 77.5⫾8.2 78.4⫾7.0 57.3⫾3.4 69.2⫾5.5 87.3⫾9.5

ELE, early life event.

TrpOH-immunoreactive neurons sampled and no interactions among social defeat, early life experience, or brain region (Table 6).

DISCUSSION Consistent with our hypothesis, exposure of rat pups to different early life experiences altered their pattern of behavior during social conflict in adulthood. MS180 rats responded to a social defeat encounter with more passive– submissive behaviors and less proactive coping behaviors compared with either AFR or MS15 rats. These findings suggest that early life experience is an important determinant of intra-specific conflict behavior during adulthood. In contrast to our hypothesis, different early life experience did not alter c-Fos expression within DR serotonergic neurons. However, we found that, independent of early life experience, social defeat increased c-Fos expression in subpopulations of serotonergic and non-serotonergic neurons within the DR, consistent with the hypothesis that subpopulations of serotonergic neurons, principally within the mid-rostrocaudal and caudal part of the dorsal DR, modulate stress-related physiological and behavioral responses. Effects of early life experience on behavior during the pre-defeat period MS180 and MS15 rats responded with a lower frequency of rearing behavior compared with AFR rats. This finding is difficult to interpret, however, as rearing scores included rearing that occurred away from the partition but not rearing that may have occurred close to the partition. Nonetheless, this is consistent with the finding that MS180 rats responded with less proactive coping behavior (which included rearing behavior) compared with AFR rats during social defeat (see below). Effects of early life experience on behavior during social defeat and the relationship to serotonergic activity Exposure of rat pups to different early life experience altered the pattern of behavior during social conflict in adulthood. These findings are consistent with previous studies demonstrating that maternal separation of rat pups of several rat strains results in increases in anxiety-like behavior in adulthood in a variety of tests including the

open field, elevated plus-maze, auditory startle, and novelty-induced suppression of feeding tests compared with neonatally handled rats (McIntosh et al., 1999; Wigger and Neumann, 1999; Caldji et al., 2000; Huot et al., 2001; Kalinichev et al., 2002) or AFR rats (Ogawa et al., 1994; Caldji et al., 2000). This is the first study in rats to suggest that early life experience alters proactive coping behaviors, including escape and aggressive behaviors, during adult social encounters. Our study using c-Fos expression as an indicator of functional cellular responses in serotonergic neurons does not support a role for early life events in modulating the excitability of serotonergic systems following exposure of adults to a social defeat paradigm. This is in contrast to our expectations based on previous studies which have described effects of early life events on other aspects of serotonergic function in adult rats including changes in 5-HT1A receptor-mediated autoinhibition (Gartside et al., 2003), 5-HT1B receptor expression within the DR (Neumaier et al., 2002), and postsynaptic 5-HT-mediated hyperpolarization in hippocampal CA1 pyramidal neurons (Van Riel et al., 2004). Measurement of c-Fos immunoreactivity can provide information regarding the number of neurons with stimulus-induced functional cellular responses but does not provide information regarding the threshold for neuronal activation or the duration of neuronal activation. Therefore it is possible that early life experience altered the threshold for or duration of activation of serotonergic neurons in the present study but that, since all rats were defeated, the c-Fos responses represent a maximal response in all treatment groups. Alternatively, it is possible that early life experience did not alter the threshold for or duration of activation of serotonergic neurons in the present study, but other aspects of serotonergic function that may be independent of the excitability of serotonergic neurons were altered (e.g. the availability of tryptophan (the amino acid precursor of 5-HT), the expression or phosphorylation state of TrpOH (the rate-limiting enzyme for the biosynthesis of 5-HT), or postsynaptic responses to 5-HT receptor activation). Future studies will be required to distinguish between these different possibilities. Effects of social defeat on c-Fos expression in serotonergic neurons in the DR An important finding in the present study is that social defeat increased the excitability of subpopulations of se-

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

189

Table 6. Continued ⫺8.18

⫺8.54

DRD

DRV

DRVL

DRD

DRV

DRI

50.0⫾4.8 60.3⫾5.8 55.2⫾5.1 53.6⫾6.5 53.7⫾4.8 54.2⫾4.3

81.3⫾13.6 78.7⫾5.1 76.3⫾0.1 82.6⫾6.0 70.3⫾6.9 77.5⫾5.9

69.8⫾3.4 88.2⫾7.6 75.0⫾10.1 63.8⫾4.9 68.8⫾3.6 66.0⫾11.2

53.7⫾5.8 54.2⫾7.5 61.5⫾5.8 42.8⫾3.9 47.5⫾6.9 43.7⫾4.8

25.7⫾4.1 34.7⫾2.7 30.6⫾4.1 27.8⫾6.8 27.3⫾3.2 28.3⫾4.0

40.8⫾6.0 49.0⫾4.1 42.5⫾2.9 44.3⫾5.5 47.2⫾7.3 44.5⫾3.6

rotonergic neurons within the DR, specifically in the dorsal part of the mid-rostrocaudal and caudal DR but not in the rostral DR or any other subdivision of the DR studied. This finding is consistent with previous studies examining the effects of social defeat on c-Fos expression in the DR of adult male rats (Martinez et al., 1998; Chung et al., 2000) and hamsters (Kollack-Walker et al., 1997), although these previous studies did not verify whether the increases in c-Fos expression were in subdivisions of the DR or in serotonergic neurons. A role for serotonergic systems in behavioral responses to social defeat is supported by the finding that lesions of the serotonergic system prevent adaptive responses that are normally observed following daily repeated social defeat, such as increased freezing during the defeat phase (Chung et al., 2000). Consistent with this hypothesis, previous studies have shown that in a variety of vertebrate species, subordinate animals have increases in serotonergic function (Blanchard et al., 1991; Summers et al., 2003; Walletschek and Raab, 1982). Since all rats, regardless of early life experience, showed elements of both passive–submissive and proactive coping behaviors during the defeat phase of the social defeat protocol, these specific subpopulations of serotonergic neurons may be involved in modulating behavioral responses during social defeat, e.g. anxiety- or fear-related behavior, escape, or aggression, specific motor patterns associated with these behaviors, or physiological correlates of these behaviors. Although the neural mechanisms underlying this anatomical specificity are not clear, retrograde tracing studies have revealed that the dorsomedian part of the DR, compared with other areas of the DR, receives unique afferent input from the bed nucleus of the stria terminalis (BNST; Peyron et al., 1998), an area important in mediating anxiety-related behavior (Davis, 1998; Gewirtz et al., 1998; Levita et al., 2004; Walker et al., 2003). The finding that social defeat increased c-Fos expression in serotonergic neurons within the dorsal part of the middle and caudal DR complements recent studies showing that uncontrollable stress, the anxiogenic drugs caffeine, n-methyl-beta-carboline-3-carboxamide, and m-chlorophenyl piperazine and the anxiety-related neuropeptide, urocortin 2 (Ucn 2) increase c-Fos expression in the middle and caudal DR, particularly the dorsal part (Grahn et al., 1999; Abrams et al., 2005; Staub et al., 2005). The convergent effects of social defeat and uncontrollable stress on serotonergic neurons within the middle and caudal DR suggest that

serotonergic neurons in this region may play a particularly important role in modulating physiological and behavioral responses to stressors with a strong element of unpredictability, uncontrollability, or defeat. Anatomical tracing studies reveal that this part of the DR gives rise to strong projections to forebrain structures mediating emotional behavior, including the basolateral and central amygdaloid nuclei, dorsal hypothalamic area, and BNST (Petrov et al., 1992, 1994; Commons et al., 2003; Abrams et al., 2005). These strong connections between the dorsal part of the mid-rostrocaudal DR and forebrain structures associated with regulation of emotional behavior suggest that serotonergic neurons in this region may play an important role in regulating affective behavioral responses, including adaptive behavioral responses to repeated social encounters. Effects of social defeat on c-Fos expression in non-serotonergic neurons in the DR We found a significant interaction between early life experience and social defeat on c-Fos expression in non-serotonergic neurons in the mid-rostrocaudal DRD; this effect may be due to increased c-Fos expression in local GABAergic interneurons in MS15 rats relative to MS180 rats, which could lead to a differential time course of serotonergic activation in these rats, to different patterns of behavior during later stages of social defeat, and to different patterns of behavioral adaptation during subsequent social encounters. This hypothesis will need to be tested in future studies.

CONCLUSIONS This is the first study to demonstrate that early life experience in rat pups is an important determinant of behavior during social conflict in adulthood. Maternal separation increased passive–submissive behaviors and decreased proactive coping behaviors during a social defeat encounter. We have also identified the topographical distribution of subpopulations of serotonergic neurons that are associated with physiological or behavioral responses during social defeat. These findings raise new questions with respect to the relationship between early life experience and intra-specific conflict during adulthood, and provide new opportunities to characterize the molecular, cellular, and functional properties of subpopulations of serotonergic neurons associated with physiological and behavioral responses to unpredictable or uncontrollable stress.

190

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191

Acknowledgments—We gratefully acknowledge Dr. Rosemary Greenwood, Dr. Jason Osborne, and Dr. Katharine Semsar for assistance in the statistical analysis of behavior. Supported by the BBSRC (Grant BBS/B/06806 to S.L.L.), NIMH (Grant MH50113 to P.P.M.), the Center for Behavioral Neuroscience STC Program of the National Science Foundation (#IBN-9876754), and the Neuroendocrinology Charitable Trust (Grant PMS/VW-01/02-808 to C.A.L.). C.A.L. is a Wellcome Trust Research Fellow (RCDF 068558/Z/02/Z).

REFERENCES Abrams JK, Johnson PL, Hollis JH, Lowry CA (2004) Anatomical and functional topography of the dorsal raphe nucleus. In: Stress: Current neuroendocrine and genetic approaches (Pacak K, Aguilera G, Sabban E, Kvenansky R, eds). Ann N Y Acad Sci 1018:46 –57. Abrams JK, Johnson PL, Hay-Schmidt A, Mikkelsen JD, Shekhar A, Lowry CA (2005) Serotonergic systems associated with arousal and vigilance behaviors following administration of anxiogenic drugs. Neuroscience 133(4):983–997. Blanchard DC, Cholvanich P, Blanchard RJ, Clow DW, Hammer RP Jr, Rowlett JK, Bardo MT (1991) Serotonin, but not dopamine, metabolites are increased in selected brain regions of subordinate male rats in a colony environment. Brain Res 568:61– 66. Caldji C, Francis D, Sharma S, Plotsky PM, Meaney MJ (2000) The effects of early rearing environment on the development of GABAA and central benzodiazepine receptor levels and novelty-induced fearfulness in the rat. Neuropsychopharmacology 22:219 –229. Chung KKK, Martinez M, Herbert J (2000) c-Fos expression, behavioural, endocrine and autonomic responses to acute social stress in male rats after chronic restraint: modulation by serotonin. Neuroscience 95:453– 463. Commons KG, Connolley KR, Valentino RJ (2003) A neurochemically distinct dorsal raphe-limbic circuit with a potential role in affective disorders. Neuropsychopharmacology 28:206 –215. Daniels WM, Pietersen CY, Carstens ME, Stein DJ (2004) Maternal separation in rats leads to anxiety-like behavior and a blunted ACTH response and altered neurotransmitter levels in response to a subsequent stressor. Metab Brain Dis 19:3–14. Davis M (1998) Are different parts of the extended amygdala involved in fear versus anxiety? Biol Psychiatry 44:1239 –1247. De Boer SF, Koolhaas JM (2003) Defensive burying in rodents: ethology, neurobiology and psychopharmacology. Eur J Pharmacol 463(1–3):145–161. Gartside SE, Johnson DA, Leitch MM, Troakes C, Ingram CD (2003) Early life adversity programs changes in central 5-HT neuronal function in adulthood. Eur J Neurosci 17:2401–2408. Gewirtz JC, McNish KA, Davis M (1998) Lesions of the bed nucleus of the stria terminalis block sensitization of the acoustic startle reflex produced by repeated stress, but not fear-potentiated startle. Prog Neuropsychopharmacol Biol Psychiatry 22:625– 648. Grahn RE, Will MJ, Hammack SE, Maswood S, McQueen MB, Watkins LR, Maier SF (1999) Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor. Brain Res 826:35– 43. Heim C, Nemeroff CB (2001) The role of childhood trauma in the neurobiology of mood and anxiety disorders: preclinical and clinical studies. Biol Psychiatry 49:1023–1039. Herbert J (1987) Neuroendocrine responses to social stress. Baillieres Clin Endocrinol Metab 1:467– 490. Huot RL, Thrivikraman KV, Meaney MJ, Plotsky PM (2001) Development of adult ethanol preference and anxiety as a consequence of neonatal maternal separation in Long Evans rats and reversal with antidepressant treatment. Psychopharmacology (Berl) 158:366–373. Kalinichev M, Easterling KW, Plotsky PM, Holtzman SG (2002) Longlasting changes in stress-induced corticosterone response and anxiety-like behaviors as a consequence of neonatal maternal

separation in Long-Evans rats. Pharmacol Biochem Behav 73: 131–140. Kollack-Walker S, Watson SJ, Akil H (1997) Social stress in hamsters: defeat activates specific neurocircuits within the brain. J Neurosci 17:8842– 8855. Koolhaas JM, Korte SM, De Boer SF, Van Der Vegt BJ, Van Reenen CG, Hopster H, De Jong IC, Ruis MA, Blokhuis HJ (1999) Coping styles in animals: current status in behavior and stress-physiology. Neurosci Biobehav Rev 23(7):925–935. Ladd CO, Huot RL, Thrivikraman KV, Nemeroff CB, Meaney MJ, Plotsky PM (2000) Long-term behavioral and neuroendocrine adaptations to adverse early experience. Prog Brain Res 122: 81–103. Ladd CO, Owens MJ, Nemeroff CB (1996) Persistent changes in corticotropin-releasing factor neuronal systems induced by maternal deprivation. Endocrinology 137:1212–1218. Levita L, Hammack SE, Mania I, Li XY, Davis M, Rainnie DG (2004) 5-Hydroxytryptamine1A-like receptor activation in the bed nucleus of the stria terminalis: electrophysiological and behavioral studies. Neuroscience 128:583–596. Martinez M, Phillips PJ, Herbert J (1998) Adaptation in patterns of c-fos expression in the brain associated with exposure to either single or repeated social stress in male rats. Eur J Neurosci 10:20 –33. McIntosh J, Anisman H, Merali Z (1999) Short- and long-periods of neonatal maternal separation differentially affect anxiety and feeding in adult rats: gender-dependent effects. Dev Brain Res 113:97–106. Meaney MJ, Diorio J, Francis D, Widdowson J, LaPlante P, Caldji C, Sharma S, Seckl JR, Plotsky PM (1996) Early environmental regulation of forebrain glucocorticoid receptor gene expression: implications for adrenocortical responses to stress. Dev Neurosci 18:49 –72. Miczek KA, Covington HE 3rd, Nikulina EM Jr, Hammer RP (2004) Aggression and defeat: persistent effects on cocaine self-administration and gene expression in peptidergic and aminergic mesocorticolimbic circuits. Neurosci Biobehav Rev 27(8):787– 802. Neumaier JF, Edwards E, Plotsky PM (2002) 5-HT(1B) mRNA regulation in two animal models of altered stress reactivity. Biol Psychiatry 51:902–908. Ogawa T, Mikuni M, Kuroda Y, Muneoka K, Mori KJ, Takahashi K (1994) Periodic maternal deprivation alters stress response in adult offspring: potentiates the negative feedback regulation of restraint stress-induced adrenocortical response and reduces the frequencies of open field-induced behaviors. Pharmacol Biochem Behav 49:961–967. Paxinos G, Watson C (1998) The rat brain in stereotaxic coordinates, 4th edition. San Diego: Academic Press. Petersen R (1985) Design and analysis of experiments. New York: Marcel Dekker, Inc. Petrov T, Krukoff TL, Jhamandas JH (1992) The hypothalamic paraventricular and lateral parabrachial nuclei receive collaterals from raphe nucleus neurons: a combined double retrograde and immunocytochemical study. J Comp Neurol 318:18 –26. Petrov T, Krukoff TL, Jhamandas JH (1994) Chemically defined collateral projections from the pons to the central nucleus of the amygdala and hypothalamic paraventricular nucleus in the rat. Cell Tissue Res 277:289 –295. Peyron C, Petit JM, Rampon C, Jouvet M, Luppi PH (1998) Forebrain afferents to the rat dorsal raphe nucleus demonstrated by retrograde and anterograde tracing methods. Neuroscience 82:443– 468. Plotsky PM, Meaney MJ (1993) Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stress-induced release in adult rats. Brain Res Mol Brain Res 18:195–200. Raab A, Dantzer R, Michaud B, Mormede P, Taghzouti K, Simon H, Le Moal M (1986) Behavioural, physiological and immunological con-

K. L. Gardner et al. / Neuroscience 136 (2005) 181–191 sequences of social status and aggression in chronically coexisting resident-intruder dyads of male rats. Physiol Behav 36:223–228. Rhees RW, Lephart ED, Eliason D (2001) Effects of maternal separation during early postnatal development on male sexual behavior and female reproductive function. Behav Brain Res 123:1–10. Short K (2003) Uncontrollable social defeat, like inescapable shock, causes an anxiety increase and a down-regulation of dorsal raphe serotonin 1A receptors with the same time course; both are prevented by a 5-HT1AR antagonist. Society for Neuroscience Abstract Viewer/Itinerary Planner Program No. 713.16. Smythe JW, Rowe WB, Meaney MJ (1994) Neonatal handling alters serotonin (5-HT) turnover and 5-HT2 receptor binding in selected brain regions: relationship to the handling effect on glucocorticoid receptor expression. Brain Res Dev Brain Res 80:183–189. Staub D, Spiga F, Lowry CA (2005) Effects of urocortin 2 on behavior and c-Fos expression in topographically organized subpopulations of serotonergic neurons. Brain Res 1044(2):176 –189.

191

Summers CH, Summers TR, Moore MC, Korzan WJ, Woodley SK, Ronan PJ, Hoglund E, Watt MJ, Greenberg N (2003) Temporal patterns of limbic monoamine and plasma corticosterone response during social stress. Neuroscience 116(2):553–563. Van Riel E, Van Gemert NG, Meijer OC, Joels M (2004) Effect of early life stress on serotonin responses in the hippocampus of young adult rats. Synapse 53:11–19. Walker DL, Toufexis DJ, Davis M (2003) Role of the bed nucleus of the stria terminalis versus the amygdala in fear, stress, and anxiety. Eur J Pharmacol 463:199 –216. Walletschek H, Raab A (1982) Spontaneous activity of dorsal raphe neurons during defensive and offensive encounters in the treeshrew. Physiol Behav 28:697–705. Wigger A, Neumann ID (1999) Periodic maternal deprivation induces gender-dependent alterations in behavioral and neuroendocrine responses to emotional stress in adult rats. Physiol Behav 66:293–302.

(Accepted 20 July 2005) (Available online 21 September 2005)