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Repeated agonistic encounters in hamsters modulate AVP V1a receptor binding
Hormones and Behavior 48 (2005) 545 – 551 www.elsevier.com/locate/yhbeh
Repeated agonistic encounters in hamsters modulate AVP V1a receptor binding Matthew A. Coopera,*, Mary Karomb, Kim L. Huhmana, H. Elliott Albersa,b a
Department of Psychology, Center for Behavioral Neuroscience, Georgia State University, P.O. Box 3966, Atlanta, GA 30302-3966, USA b Department of Biology, Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30302, USA Received 18 January 2005; revised 26 April 2005; accepted 30 April 2005 Available online 1 June 2005
Abstract Arginine vasopressin (AVP) regulates aggression in male Syrian hamsters. In this study, we used radioligand receptor autoradiography to examine whether changes in agonistic behavior following acute and repeated social defeat are accompanied by changes in AVP V1a receptor binding. Social defeat produced high levels of submissive behavior and a loss of territorial aggression when hamsters were subsequently tested with a novel intruder, and repeated agonistic encounters produced similar behavioral changes in subordinates. AVP V1a receptor binding was not reduced by acute social defeat but was affected by repeated agonistic encounters. Dominants had significantly more AVP V1a receptor binding in lateral portions of the ventromedial hypothalamus (VMHL) than did their subordinate opponents, but subordinates were no different from controls. In contrast, receptor binding did not differ in most other brain regions examined. The changes in receptor binding appear to be independent of testosterone levels, as testosterone levels did not differ among dominants, subordinates, and controls. Our results suggest that changes in AVP V1a receptors do not account for the changes in agonistic behavior produced by acute social defeat but AVP V1a binding in the VMHL correlates with, and may modulate, the behavioral changes that occur following repeated experiences of victory. D 2005 Elsevier Inc. All rights reserved. Keywords: Aggression; Dominant; Subordinate; Conditioned defeat; Vasopressin; Testosterone
Introduction Social defeat is a powerful stressor for laboratory rodents (Koolhaas et al., 1997). In male Syrian hamsters, losers of agonistic encounters exhibit a hormonal stress response characterized by elevated plasma adrenocortropin, h-endorphin, h-lipotropin, cortisol, and corticosterone (Huhman et al., 1991). In contrast, winners do not exhibit a hormonal stress response. Additionally, social defeat produces a striking change in future agonistic behavior that is characterized by an increase in submissive and defensive behavior and a complete loss of territorial aggression (Huhman et al., 2003; Potegal et al., 1993). The increased submissive behavior displayed following acute social defeat is regulated
* Corresponding author. Fax: +1 404 651 3929. E-mail address: [email protected] (M.A. Cooper). 0018-506X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2005.04.012
in part by CRF receptors in the bed nucleus of the stria terminalis (Cooper and Huhman, in press) and by NMDA receptors in the basolateral amygdala (Jasnow et al., 2004). However, much less is known about the neurobiological mechanisms regulating the loss of territorial aggression. In rodents, arginine vasopressin (AVP) is involved in the control of various types of social behavior including affiliation (Winslow et al., 1993), scent marking (Ferris et al., 1984, 1985), and aggression (Ferris et al., 1997; Koolhaas et al., 1990). In male Syrian hamsters, AVP activity in lateral regions of the anterior hypothalamus (AH) and lateral regions of the ventromedial hypothalamus (VMHL) have been implicated in aggression (Delville et al., 1996a, 2000; Ferris et al., 1997). Injection of a selective AVP V1a receptor antagonist into the AH inhibits inter-male aggression (Ferris and Potegal, 1988; Potegal and Ferris, 1990). Likewise, AVP injected into the AH increases offensive aggression (Ferris et al., 1997). Establishment of
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a dominance relationship in hamsters also alters components of the AVP system. Subordinates have lower levels of AVP immunoreactivity in the AH, fewer AVP immunoreactive fibers in the AH, and fewer AVP immunoreactive cell bodies in the nucleus circularis compared to dominants and socially isolated controls (Ferris et al., 1989). Thus, the loss of territorial aggression following social defeat may be due to alterations of the AVP system. In many laboratory rodents, testosterone modulates aggression in resident –intruder paradigms (Albert et al., 1986; Payne, 1973; van Oortmessen et al., 1987). In Syrian hamsters, testosterone appears to stimulate aggression in part by increasing AVP V1a receptor binding (Delville et al., 1996b). Likewise, reductions in testosterone decrease AVP V1a receptor binding in brain regions related to aggression (Caldwell and Albers, 2003; Young et al., 2000). Also, losers show reduced plasma testosterone after repeated social defeats, but not after a single social defeat (Huhman et al., 1991). Consequently, the behavioral responses of subordinates following repeated agonistic encounters may be mediated by reduced testosterone and decreased activity of the AVP system. In the present study, we investigated the effects of single and repeated agonistic encounters on AVP V1a receptor binding. In Experiment 1, we tested the hypothesis that an acute social defeat, which dramatically alters future agonistic behavior, would reduce AVP V1a receptor binding in specific brain regions known to modulate aggression in hamsters. In Experiment 2, we tested the hypothesis that repeated agonistic encounters would reduce AVP V1a receptor binding in specific brain regions as well as plasma testosterone in subordinates as compared to dominants and controls.
Methods Animals Male Syrian hamsters were purchased from Charles River Laboratories. Experimental animals were 3– 4 months old and weighed 120 – 140 g at the start of the study. The animals were individually housed 10 – 12 days prior to the start of the experiment in a temperature controlled (20 T 2-C) colony room and were maintained on a 14:10-h light– dark cycle. Animals were handled daily to habituate them, in part, to the training and testing procedures. In Experiment 1, older animals (>6 months) that weighed 160 –180 g were housed individually and used as resident aggressors during training, and younger animals (2 months) that weighed 100 – 110 g were group housed (5 animals per cage) and used as non-aggressive intruders during testing. All animals were housed in polycarbonate cages (20 40 20 cm) with corncob and cotton bedding materials and wire mesh tops. Food and water were available continuously. The cages of experimental animals and resident aggressors were
not changed for 1 week prior to testing. The Georgia State University Animal Care and Use Committee approved all procedures used in this study. Behavioral testing Prior to each experiment animals were matched by weight and randomly assigned to groups. All training and testing sessions were performed during the first 3 h of the dark phase of the light– dark cycle. In Experiment 1, training consisted of one 15-min exposure to a resident aggressor in the aggressor’s home cage (defeated animals, N = 7), one 15-min exposure to a non-aggressive intruder in the subject’s home cage (nondefeated animals, N = 6), or one 15-min exposure to an empty cage (handled controls, N = 6). The resident aggressors repeatedly attacked their opponents, and all of the defeated animals lost the fight. The duration of social defeat experienced by the subjects depended in part on the latency to attack by the resident aggressor. To reduce variation in the duration of social defeat experienced, we started the 15-min exposure after the first attack by the resident aggressor. The latency to attack is typically less than 30 s, but in one case the first attack occurred after 2.5 min. The non-aggressive intruders did not attack the nondefeated animals. The non-defeated animals displayed mainly social behavior, but some of them attacked the non-aggressive intruders. Twenty-four hours after training, all animals and controls were tested in their home cage for 5 min with a non-aggressive intruder. Testing sessions were recorded and later scored by an observer blind to the experimental conditions using behavioral scoring software from Noldus Observer (Noldus Information Technology, Wageningen, Netherlands). We recorded the total duration of four classes of behavior during testing: (1) social behavior: attend, approach, investigate, sniff, nose touching, and flank marking; (2) non-social behavior: locomotion, exploration, self-groom, nesting, feeding, and sleeping; (3) submissive and defensive behavior: flee, avoid, tail up, upright and side defense, full submissive posture, stretchattend, head flag, attempt to escape from cage; and (4) aggressive behavior: upright and side offensive posture, chase, and attack (including bite). Our behavioral definitions followed (Albers et al., 2002). A subset of testing sessions was scored twice and intra-observer reliability on the total duration of each behavior category was 92%. In Experiment 2, animals were weight matched and paired for 11 daily, 15-min encounters. One animal from each pair was randomly selected as resident for all encounters. Animals were categorized as dominant or subordinate based on the reliable display of agonistic behavior. Seven pairs established a dominance relationship during the first encounter and that relationship remained stable. One pair reversed their dominance relationship midway through training and this pair was dropped from analyses. Handled controls (N = 6) were exposed to a novel
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male’s empty cage for 15 min each training day. On the 12th day, all animals and controls were tested in their own home cage for 5 min with a non-aggressive intruder. Testing sessions were scored as in Experiment 1. Radioligand receptor autoradiography Immediately following testing, all animals were rapidly decapitated and their brains were extracted, frozen on dry ice, and stored at 80-C until use. Brain sections were sliced at 20 Am on a cryostat and thaw-mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA). Alternate sections were placed on separate slides in order to perform autoradiography and cresyl violet staining on adjacent sections. Brains were sliced from the rostral lateral septum to caudal regions of the lateral ventromedial hypothalamus. The sections were stored at 80-C until use. One set of slide-mounted sections was processed for AVP V1a receptor autoradiography using a 125I linear vasopressin V1a receptor antagonist (PerkinElmer Life and Analytical Sciences, Boston, MA) as previously described (Young et al., 2000). Briefly, the sections were fixed with 0.1% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 2 min at room temperature and were then prewashed twice for 10 min each in 50 mM Tris buffer (pH 7.4). Sections were next incubated for 60 min at room temperature with 50 pM 125I V1a receptor antagonist in 50 mM Tris buffer with 10 mM MgCl2, 0.1% bovine serum albumin, and 0.05% bacitracin. Unbound ligand was removed with two 5-min washes and one 35-min wash in 50 mM Tris buffer with 10 mM MgCl2. Next, slides were dipped into cold, distilled water and dried. The sections were exposed to BioMax MR film for 72 h (Kodak, Rochester, NY, USA) along with autoradiographic 125I microscales (Amersham Biosciences, Piscataway, NJ, USA).
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lished from 125I microscales. Specific labeling was calculated by taking the measurements from each brain region and subtracting non-specific labeling, which was measured in adjacent brain regions devoid of signal. Using cresyl violet-stained sections, we identified the lateral septum (LS), lateral portions of the bed nucleus of the stria terminalis (BNST), medial preoptic area (MPOA), medial preoptic nucleus (MPN), lateral portions of the anterior hypothalamus (AH), lateral hypothalamus (LH), central amygdala (CeA), Ca1 region of the hippocampus (Ca1), and lateral portions of the ventromedial hypothalamus (VMHL) as shown in Fig. 1. The VMHL includes the lateral portions of the ventromedial hypothalamus and the medial aspects of the medial tuberal nucleus and has also been called the ventrolateral hypothalamus (DeLeon et al., 2002; Delville et al., 2000). We selected these brain regions based on their putative roles in aggressive behavior as well as the presence of V1a receptors (DeLeon et al.,
Testosterone assay In Experiment 2, testosterone levels were measured by radioimmunoassay (Active DSL 4000 Testosterone RIA, Diagnostic Systems Laboratories, Webster, TX, USA). Immediately following testing, trunk blood was collected by rapid decapitation, samples were centrifuged, and plasma was stored at 20-C until assayed. The assay was performed without extraction and validated for hamster serum by linearity of dilution and recovery methods. Intraassay reliability was 95.6% and all samples were run in the same assay. Analysis AVP V1a receptor binding density was analyzed using Scion Image software (an NIH image program available on the Internet at http://rsb.info.nih.gov/nih-image/). Optical densities were converted to disintegrations per minute/ milligram tissue equivalent using a standard curve estab-
Fig. 1. Diagrams showing the location of regions selected for quantification of AVP V1a binding (shaded areas). Diagrams indicate the distance from bregma and were modified from the hamster atlas of Morin and Wood (2001). Lateral septum (LS); bed nucleus of the stria terminalis (BNST); medial preoptic area (MPOA); medial preoptic nucleus (MPN); anterior hypothalamus (AH); lateral hypothalamus (LH); central amygdala (CeA); hippocampus (Ca1); lateral ventromedial hypothalamus (VMHL).
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2002; Delville et al., 2000; Young et al., 2000). For each brain region, optical densities were quantified using a box that was 0.35 mm 0.35 mm. Densities were recorded in three brain sections 60 Am apart and averaged per animal. A second scorer made independent measurements that produced the same statistically significant results found by the primary scorer. Treatment effects were analyzed using one-way ANOVAs for each brain region and Tukey tests were used for post hoc analysis when necessary. In Experiment 2, dominant – subordinate pairs were treated as paired samples and the data were analyzed using a series of paired-sample t tests. Comparisons are considered significant at P < 0.05.
Results Experiment 1 Acutely defeated animals showed more submissive/ defensive behavior (128.4 s, SE = 24.1) in the presence of a non-aggressive intruder than did non-defeated animals (49.0 s, SE = 16.0) (t(11) = 2.75, P = 0.02). Also, none of the seven defeated animals directed aggression against the non-aggressive intruder, whereas three of six non-defeated animals did. High levels of AVP V1a receptor binding were observed in the LS, BNST, CeA, and VMHL and low to moderate receptor binding was observed in the MPOA, MPN, AH, LH, and Ca1 region of the hippocampus. Acutely defeated animals, non-defeated animals, and handled controls did not differ significantly in the amount of AVP V1a receptor binding observed in any of the brain regions examined ( P > 0.05) (Fig. 2). Also, we found no indication that the three non-defeated animals that were aggressive differed in AVP V1a receptor binding in any of the brain regions examined compared their non-aggressive counterparts.
Experiment 2 As expected, previously dominant animals showed significantly less submissive/defensive behavior (0.0 s) and more aggressive behavior (177.8 s, SE = 29.6) in response to a non-aggressive intruder than did previously subordinate animals (88.8 s, SE = 26.0; 48.2 s, SE = 32.1, respectively) (t(5) = 3.42, P = 0.02; t(5) = 2.51, P = 0.05, respectively). However, plasma testosterone levels did not significantly differ between dominants (6.6 ng/ml, SE = 0.8), subordinates (6.7 ng/ml, SE = 0.4), and controls (8.0 ng/ml, SE = 0.9) ( P > 0.05). Representative film autoradiographs of hamster brain illustrating AVP V1a-selective radioligand binding in the VMHL are shown in Fig. 3. Dominant animals had greater AVP V1a receptor binding in the VMHL than did subordinate animals (t paired(5) = 3.95, P = 0.01; Fig. 4). A similar trend was observed in the AH (t paired(5) = 2.22, P = 0.077; Fig. 4). Dominants appeared to have slightly greater AVP V1a receptor binding than did subordinates in the BNST, but the apparent difference was non-significant. V1a receptor binding in control animals was often in between that of dominants and subordinates (except in the VMHL and MPN) and was not significantly different from either dominants or subordinates in any brain region examined.
Discussion AVP V1a receptor binding in control animals was similar to that previously described for adolescent and adult male hamster brain (DeLeon et al., 2002; Johnson et al., 1995; Young et al., 2000). We found that an acute social defeat produced pronounced changes in future agonistic behavior but did not alter AVP V1a receptor binding. In contrast, after repeated agonistic interactions AVP V1a receptor binding in the VMHL was correlated with dominance status. A similar, but non-significant, relationship was observed in the AH.
Fig. 2. AVP V1a receptor binding in defeated animals (n = 7), non-defeated animals (n = 6), and handled controls (n = 6) (mean T SEM). No treatment effects were found in any of the brain regions shown ( P > 0.05).
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Fig. 3. Film autoradiographs of V1a-selective radioligand binding in representative dominant (a), subordinate (b), and control hamsters (c). Arrows indicate AVP V1a receptor binding in the lateral ventromedial hypothalamus, VMHL.
Our results suggest that AVP V1a receptor binding correlates with and may modulate behavioral responses to repeated, but not single, agonistic encounters. These results
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are consistent with previous research implicating AVP activity in the AH and VMHL in the control of offensive aggression in male Syrian hamsters (Delville et al., 1996a,b; Ferris et al., 1989, 1997). We expected that behavioral changes to repeated agonistic encounters would correlate with changes in AVP V1a receptor binding and plasma testosterone levels. AVP injected into the VMHL and lateral regions of the AH facilitates offensive aggression in male Syrian hamsters (Delville et al., 1996a; Ferris et al., 1997), and aggression is reduced by injection of a selective V1a receptor antagonist into the AH (Ferris and Potegal, 1988; Potegal and Ferris, 1990). Moreover, AVP V1a receptor binding is testosterone dependent in specific brain regions, such as the VMHL, AH, MPOA, MPN, and BNST (Caldwell and Albers, 2003; Delville and Ferris, 1995; Johnson et al., 1995; Young et al., 2000). In the present study, the formation of stable dominance relationships altered AVP V1a receptor binding in the VMHL, but we found no difference in plasma testosterone levels between dominates and subordinates. Thus, the changes observed in V1a receptor binding appear to have occurred independent of testosterone. We expected to find reduced testosterone in subordinates because repeated social defeats have been shown to reduce plasma testosterone in male Syrian hamsters (Huhman et al., 1991). The Huhman et al. (1991) study tested losers after they were repeatedly defeated by different aggressors, whereas we tested subordinates after multiple encounters with the same opponent. It seems possible that in our study testosterone levels could have decreased initially in subordinates and returned to baseline after the establishment of stable dominance relationships. Indeed, testosterone levels might be expected to reflect dominance status only when dominance relationships are unstable (Sapolsky, 1993). The VMHL and AH are not the only brain regions involved in the regulation of agonistic behavior. In various species, the activity of neurons in brain regions such as the
Fig. 4. AVP V1a receptor binding in dominants (n = 6), subordinates (n = 6), and controls (n = 6) (mean T SEM). Dominants showed significantly greater receptor binding in the lateral ventromedial hypothalamus (VMHL) compared to subordinates (**P = 0.01), and a similar trend was observed in the anterior hypothalamus (AH) (*P = 0.077). No changes were observed in the other brain regions examined.
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BNST, LS, and medial amygdala has also been implicated in the control of agonistic behavior (Delville et al., 2000; Han et al., 1996a,b; Kollack-Walker et al., 1997; Koolhaas et al., 1990; Potegal et al., 1996a,b). In hamsters, flank marking, which is a form of scent marking, is regulated mainly by AVP in the MPOA-AH (Albers and Bamshad, 1998; Ferris et al., 1990), and dominant hamsters flank mark more than subordinates (Ferris et al., 1986, 1987). The MPOA-AH is reciprocally connected with the LS, BNST, and the periaqueductal gray (Albers et al., 1992; Ferris et al., 1990), and these sites, as well as the CeA, modulate flank marking (Bamshad and Albers, 1996; Bamshad et al., 1996; Hennessey et al., 1992; Irvin et al., 1990). In our study, AVP V1a receptor binding was not significantly different between dominants and subordinates in the brain regions which compose the neural circuit for flank marking (i.e., MPOA-AH, LS, BNST, CeA). The finding that significant differences in AVP V1a receptor binding were restricted to the VMHL suggests that the changes in AVP V1a receptor binding that occur following the establishment of dominance relationships are most likely related to aggression and not other aspects of agonistic behavior (e.g., submission or flank marking). Acute social defeat produced pronounced changes in agonistic behavior but did not alter AVP V1a receptor binding. This result suggests that changes in AVP V1a receptor populations are not required for the increased submission and decreased aggression that occurs following acute social defeat. Repeated encounters also produced changes in agonistic behavior, and dominants had greater AVP V1a receptor binding in the VMHL than did subordinates. In the VMHL, differences in AVP V1a receptor binding appeared to be due to up-regulation in dominants, while subordinates did not differ from controls. These results are consistent with a role for V1a receptors in the VMHL in the control of aggressive behavior but indicate that agonistic behavior can change dramatically before changes in V1a receptor binding occur. In sum, the present data suggest that the behavioral changes that occur following acute social defeat do not require AVP V1a receptor binding changes, but they suggest a role for V1a receptors in modulating behavioral responses to repeated experiences of victory.
Acknowledgments We thank Heather Caldwell for advice on autoradiography and AVP V1a binding patterns. We are also grateful to Jeris Israel and Lauren Zelinski for technical assistance. This work was supported by grant MH62044 to KLH, grant MH62641 to HEA, and grant NRSA MH072085 to MAC. Also, this material is based upon work supported in part by The Center for Behavioral Neuroscience, an STC Program of the National Science Foundation under agreement No. IBN-9876754.
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Repeated agonistic encounters in hamsters modulate AVP V1a receptor binding Matthew A. Coopera,*, Mary Karomb, Kim L. Huhmana, H. Elliott Albersa,b a
Department of Psychology, Center for Behavioral Neuroscience, Georgia State University, P.O. Box 3966, Atlanta, GA 30302-3966, USA b Department of Biology, Center for Behavioral Neuroscience, Georgia State University, Atlanta, GA 30302, USA Received 18 January 2005; revised 26 April 2005; accepted 30 April 2005 Available online 1 June 2005
Abstract Arginine vasopressin (AVP) regulates aggression in male Syrian hamsters. In this study, we used radioligand receptor autoradiography to examine whether changes in agonistic behavior following acute and repeated social defeat are accompanied by changes in AVP V1a receptor binding. Social defeat produced high levels of submissive behavior and a loss of territorial aggression when hamsters were subsequently tested with a novel intruder, and repeated agonistic encounters produced similar behavioral changes in subordinates. AVP V1a receptor binding was not reduced by acute social defeat but was affected by repeated agonistic encounters. Dominants had significantly more AVP V1a receptor binding in lateral portions of the ventromedial hypothalamus (VMHL) than did their subordinate opponents, but subordinates were no different from controls. In contrast, receptor binding did not differ in most other brain regions examined. The changes in receptor binding appear to be independent of testosterone levels, as testosterone levels did not differ among dominants, subordinates, and controls. Our results suggest that changes in AVP V1a receptors do not account for the changes in agonistic behavior produced by acute social defeat but AVP V1a binding in the VMHL correlates with, and may modulate, the behavioral changes that occur following repeated experiences of victory. D 2005 Elsevier Inc. All rights reserved. Keywords: Aggression; Dominant; Subordinate; Conditioned defeat; Vasopressin; Testosterone
Introduction Social defeat is a powerful stressor for laboratory rodents (Koolhaas et al., 1997). In male Syrian hamsters, losers of agonistic encounters exhibit a hormonal stress response characterized by elevated plasma adrenocortropin, h-endorphin, h-lipotropin, cortisol, and corticosterone (Huhman et al., 1991). In contrast, winners do not exhibit a hormonal stress response. Additionally, social defeat produces a striking change in future agonistic behavior that is characterized by an increase in submissive and defensive behavior and a complete loss of territorial aggression (Huhman et al., 2003; Potegal et al., 1993). The increased submissive behavior displayed following acute social defeat is regulated
* Corresponding author. Fax: +1 404 651 3929. E-mail address: [email protected] (M.A. Cooper). 0018-506X/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2005.04.012
in part by CRF receptors in the bed nucleus of the stria terminalis (Cooper and Huhman, in press) and by NMDA receptors in the basolateral amygdala (Jasnow et al., 2004). However, much less is known about the neurobiological mechanisms regulating the loss of territorial aggression. In rodents, arginine vasopressin (AVP) is involved in the control of various types of social behavior including affiliation (Winslow et al., 1993), scent marking (Ferris et al., 1984, 1985), and aggression (Ferris et al., 1997; Koolhaas et al., 1990). In male Syrian hamsters, AVP activity in lateral regions of the anterior hypothalamus (AH) and lateral regions of the ventromedial hypothalamus (VMHL) have been implicated in aggression (Delville et al., 1996a, 2000; Ferris et al., 1997). Injection of a selective AVP V1a receptor antagonist into the AH inhibits inter-male aggression (Ferris and Potegal, 1988; Potegal and Ferris, 1990). Likewise, AVP injected into the AH increases offensive aggression (Ferris et al., 1997). Establishment of
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a dominance relationship in hamsters also alters components of the AVP system. Subordinates have lower levels of AVP immunoreactivity in the AH, fewer AVP immunoreactive fibers in the AH, and fewer AVP immunoreactive cell bodies in the nucleus circularis compared to dominants and socially isolated controls (Ferris et al., 1989). Thus, the loss of territorial aggression following social defeat may be due to alterations of the AVP system. In many laboratory rodents, testosterone modulates aggression in resident –intruder paradigms (Albert et al., 1986; Payne, 1973; van Oortmessen et al., 1987). In Syrian hamsters, testosterone appears to stimulate aggression in part by increasing AVP V1a receptor binding (Delville et al., 1996b). Likewise, reductions in testosterone decrease AVP V1a receptor binding in brain regions related to aggression (Caldwell and Albers, 2003; Young et al., 2000). Also, losers show reduced plasma testosterone after repeated social defeats, but not after a single social defeat (Huhman et al., 1991). Consequently, the behavioral responses of subordinates following repeated agonistic encounters may be mediated by reduced testosterone and decreased activity of the AVP system. In the present study, we investigated the effects of single and repeated agonistic encounters on AVP V1a receptor binding. In Experiment 1, we tested the hypothesis that an acute social defeat, which dramatically alters future agonistic behavior, would reduce AVP V1a receptor binding in specific brain regions known to modulate aggression in hamsters. In Experiment 2, we tested the hypothesis that repeated agonistic encounters would reduce AVP V1a receptor binding in specific brain regions as well as plasma testosterone in subordinates as compared to dominants and controls.
Methods Animals Male Syrian hamsters were purchased from Charles River Laboratories. Experimental animals were 3– 4 months old and weighed 120 – 140 g at the start of the study. The animals were individually housed 10 – 12 days prior to the start of the experiment in a temperature controlled (20 T 2-C) colony room and were maintained on a 14:10-h light– dark cycle. Animals were handled daily to habituate them, in part, to the training and testing procedures. In Experiment 1, older animals (>6 months) that weighed 160 –180 g were housed individually and used as resident aggressors during training, and younger animals (2 months) that weighed 100 – 110 g were group housed (5 animals per cage) and used as non-aggressive intruders during testing. All animals were housed in polycarbonate cages (20 40 20 cm) with corncob and cotton bedding materials and wire mesh tops. Food and water were available continuously. The cages of experimental animals and resident aggressors were
not changed for 1 week prior to testing. The Georgia State University Animal Care and Use Committee approved all procedures used in this study. Behavioral testing Prior to each experiment animals were matched by weight and randomly assigned to groups. All training and testing sessions were performed during the first 3 h of the dark phase of the light– dark cycle. In Experiment 1, training consisted of one 15-min exposure to a resident aggressor in the aggressor’s home cage (defeated animals, N = 7), one 15-min exposure to a non-aggressive intruder in the subject’s home cage (nondefeated animals, N = 6), or one 15-min exposure to an empty cage (handled controls, N = 6). The resident aggressors repeatedly attacked their opponents, and all of the defeated animals lost the fight. The duration of social defeat experienced by the subjects depended in part on the latency to attack by the resident aggressor. To reduce variation in the duration of social defeat experienced, we started the 15-min exposure after the first attack by the resident aggressor. The latency to attack is typically less than 30 s, but in one case the first attack occurred after 2.5 min. The non-aggressive intruders did not attack the nondefeated animals. The non-defeated animals displayed mainly social behavior, but some of them attacked the non-aggressive intruders. Twenty-four hours after training, all animals and controls were tested in their home cage for 5 min with a non-aggressive intruder. Testing sessions were recorded and later scored by an observer blind to the experimental conditions using behavioral scoring software from Noldus Observer (Noldus Information Technology, Wageningen, Netherlands). We recorded the total duration of four classes of behavior during testing: (1) social behavior: attend, approach, investigate, sniff, nose touching, and flank marking; (2) non-social behavior: locomotion, exploration, self-groom, nesting, feeding, and sleeping; (3) submissive and defensive behavior: flee, avoid, tail up, upright and side defense, full submissive posture, stretchattend, head flag, attempt to escape from cage; and (4) aggressive behavior: upright and side offensive posture, chase, and attack (including bite). Our behavioral definitions followed (Albers et al., 2002). A subset of testing sessions was scored twice and intra-observer reliability on the total duration of each behavior category was 92%. In Experiment 2, animals were weight matched and paired for 11 daily, 15-min encounters. One animal from each pair was randomly selected as resident for all encounters. Animals were categorized as dominant or subordinate based on the reliable display of agonistic behavior. Seven pairs established a dominance relationship during the first encounter and that relationship remained stable. One pair reversed their dominance relationship midway through training and this pair was dropped from analyses. Handled controls (N = 6) were exposed to a novel
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male’s empty cage for 15 min each training day. On the 12th day, all animals and controls were tested in their own home cage for 5 min with a non-aggressive intruder. Testing sessions were scored as in Experiment 1. Radioligand receptor autoradiography Immediately following testing, all animals were rapidly decapitated and their brains were extracted, frozen on dry ice, and stored at 80-C until use. Brain sections were sliced at 20 Am on a cryostat and thaw-mounted on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA, USA). Alternate sections were placed on separate slides in order to perform autoradiography and cresyl violet staining on adjacent sections. Brains were sliced from the rostral lateral septum to caudal regions of the lateral ventromedial hypothalamus. The sections were stored at 80-C until use. One set of slide-mounted sections was processed for AVP V1a receptor autoradiography using a 125I linear vasopressin V1a receptor antagonist (PerkinElmer Life and Analytical Sciences, Boston, MA) as previously described (Young et al., 2000). Briefly, the sections were fixed with 0.1% paraformaldehyde in phosphate-buffered saline (pH 7.4) for 2 min at room temperature and were then prewashed twice for 10 min each in 50 mM Tris buffer (pH 7.4). Sections were next incubated for 60 min at room temperature with 50 pM 125I V1a receptor antagonist in 50 mM Tris buffer with 10 mM MgCl2, 0.1% bovine serum albumin, and 0.05% bacitracin. Unbound ligand was removed with two 5-min washes and one 35-min wash in 50 mM Tris buffer with 10 mM MgCl2. Next, slides were dipped into cold, distilled water and dried. The sections were exposed to BioMax MR film for 72 h (Kodak, Rochester, NY, USA) along with autoradiographic 125I microscales (Amersham Biosciences, Piscataway, NJ, USA).
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lished from 125I microscales. Specific labeling was calculated by taking the measurements from each brain region and subtracting non-specific labeling, which was measured in adjacent brain regions devoid of signal. Using cresyl violet-stained sections, we identified the lateral septum (LS), lateral portions of the bed nucleus of the stria terminalis (BNST), medial preoptic area (MPOA), medial preoptic nucleus (MPN), lateral portions of the anterior hypothalamus (AH), lateral hypothalamus (LH), central amygdala (CeA), Ca1 region of the hippocampus (Ca1), and lateral portions of the ventromedial hypothalamus (VMHL) as shown in Fig. 1. The VMHL includes the lateral portions of the ventromedial hypothalamus and the medial aspects of the medial tuberal nucleus and has also been called the ventrolateral hypothalamus (DeLeon et al., 2002; Delville et al., 2000). We selected these brain regions based on their putative roles in aggressive behavior as well as the presence of V1a receptors (DeLeon et al.,
Testosterone assay In Experiment 2, testosterone levels were measured by radioimmunoassay (Active DSL 4000 Testosterone RIA, Diagnostic Systems Laboratories, Webster, TX, USA). Immediately following testing, trunk blood was collected by rapid decapitation, samples were centrifuged, and plasma was stored at 20-C until assayed. The assay was performed without extraction and validated for hamster serum by linearity of dilution and recovery methods. Intraassay reliability was 95.6% and all samples were run in the same assay. Analysis AVP V1a receptor binding density was analyzed using Scion Image software (an NIH image program available on the Internet at http://rsb.info.nih.gov/nih-image/). Optical densities were converted to disintegrations per minute/ milligram tissue equivalent using a standard curve estab-
Fig. 1. Diagrams showing the location of regions selected for quantification of AVP V1a binding (shaded areas). Diagrams indicate the distance from bregma and were modified from the hamster atlas of Morin and Wood (2001). Lateral septum (LS); bed nucleus of the stria terminalis (BNST); medial preoptic area (MPOA); medial preoptic nucleus (MPN); anterior hypothalamus (AH); lateral hypothalamus (LH); central amygdala (CeA); hippocampus (Ca1); lateral ventromedial hypothalamus (VMHL).
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2002; Delville et al., 2000; Young et al., 2000). For each brain region, optical densities were quantified using a box that was 0.35 mm 0.35 mm. Densities were recorded in three brain sections 60 Am apart and averaged per animal. A second scorer made independent measurements that produced the same statistically significant results found by the primary scorer. Treatment effects were analyzed using one-way ANOVAs for each brain region and Tukey tests were used for post hoc analysis when necessary. In Experiment 2, dominant – subordinate pairs were treated as paired samples and the data were analyzed using a series of paired-sample t tests. Comparisons are considered significant at P < 0.05.
Results Experiment 1 Acutely defeated animals showed more submissive/ defensive behavior (128.4 s, SE = 24.1) in the presence of a non-aggressive intruder than did non-defeated animals (49.0 s, SE = 16.0) (t(11) = 2.75, P = 0.02). Also, none of the seven defeated animals directed aggression against the non-aggressive intruder, whereas three of six non-defeated animals did. High levels of AVP V1a receptor binding were observed in the LS, BNST, CeA, and VMHL and low to moderate receptor binding was observed in the MPOA, MPN, AH, LH, and Ca1 region of the hippocampus. Acutely defeated animals, non-defeated animals, and handled controls did not differ significantly in the amount of AVP V1a receptor binding observed in any of the brain regions examined ( P > 0.05) (Fig. 2). Also, we found no indication that the three non-defeated animals that were aggressive differed in AVP V1a receptor binding in any of the brain regions examined compared their non-aggressive counterparts.
Experiment 2 As expected, previously dominant animals showed significantly less submissive/defensive behavior (0.0 s) and more aggressive behavior (177.8 s, SE = 29.6) in response to a non-aggressive intruder than did previously subordinate animals (88.8 s, SE = 26.0; 48.2 s, SE = 32.1, respectively) (t(5) = 3.42, P = 0.02; t(5) = 2.51, P = 0.05, respectively). However, plasma testosterone levels did not significantly differ between dominants (6.6 ng/ml, SE = 0.8), subordinates (6.7 ng/ml, SE = 0.4), and controls (8.0 ng/ml, SE = 0.9) ( P > 0.05). Representative film autoradiographs of hamster brain illustrating AVP V1a-selective radioligand binding in the VMHL are shown in Fig. 3. Dominant animals had greater AVP V1a receptor binding in the VMHL than did subordinate animals (t paired(5) = 3.95, P = 0.01; Fig. 4). A similar trend was observed in the AH (t paired(5) = 2.22, P = 0.077; Fig. 4). Dominants appeared to have slightly greater AVP V1a receptor binding than did subordinates in the BNST, but the apparent difference was non-significant. V1a receptor binding in control animals was often in between that of dominants and subordinates (except in the VMHL and MPN) and was not significantly different from either dominants or subordinates in any brain region examined.
Discussion AVP V1a receptor binding in control animals was similar to that previously described for adolescent and adult male hamster brain (DeLeon et al., 2002; Johnson et al., 1995; Young et al., 2000). We found that an acute social defeat produced pronounced changes in future agonistic behavior but did not alter AVP V1a receptor binding. In contrast, after repeated agonistic interactions AVP V1a receptor binding in the VMHL was correlated with dominance status. A similar, but non-significant, relationship was observed in the AH.
Fig. 2. AVP V1a receptor binding in defeated animals (n = 7), non-defeated animals (n = 6), and handled controls (n = 6) (mean T SEM). No treatment effects were found in any of the brain regions shown ( P > 0.05).
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Fig. 3. Film autoradiographs of V1a-selective radioligand binding in representative dominant (a), subordinate (b), and control hamsters (c). Arrows indicate AVP V1a receptor binding in the lateral ventromedial hypothalamus, VMHL.
Our results suggest that AVP V1a receptor binding correlates with and may modulate behavioral responses to repeated, but not single, agonistic encounters. These results
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are consistent with previous research implicating AVP activity in the AH and VMHL in the control of offensive aggression in male Syrian hamsters (Delville et al., 1996a,b; Ferris et al., 1989, 1997). We expected that behavioral changes to repeated agonistic encounters would correlate with changes in AVP V1a receptor binding and plasma testosterone levels. AVP injected into the VMHL and lateral regions of the AH facilitates offensive aggression in male Syrian hamsters (Delville et al., 1996a; Ferris et al., 1997), and aggression is reduced by injection of a selective V1a receptor antagonist into the AH (Ferris and Potegal, 1988; Potegal and Ferris, 1990). Moreover, AVP V1a receptor binding is testosterone dependent in specific brain regions, such as the VMHL, AH, MPOA, MPN, and BNST (Caldwell and Albers, 2003; Delville and Ferris, 1995; Johnson et al., 1995; Young et al., 2000). In the present study, the formation of stable dominance relationships altered AVP V1a receptor binding in the VMHL, but we found no difference in plasma testosterone levels between dominates and subordinates. Thus, the changes observed in V1a receptor binding appear to have occurred independent of testosterone. We expected to find reduced testosterone in subordinates because repeated social defeats have been shown to reduce plasma testosterone in male Syrian hamsters (Huhman et al., 1991). The Huhman et al. (1991) study tested losers after they were repeatedly defeated by different aggressors, whereas we tested subordinates after multiple encounters with the same opponent. It seems possible that in our study testosterone levels could have decreased initially in subordinates and returned to baseline after the establishment of stable dominance relationships. Indeed, testosterone levels might be expected to reflect dominance status only when dominance relationships are unstable (Sapolsky, 1993). The VMHL and AH are not the only brain regions involved in the regulation of agonistic behavior. In various species, the activity of neurons in brain regions such as the
Fig. 4. AVP V1a receptor binding in dominants (n = 6), subordinates (n = 6), and controls (n = 6) (mean T SEM). Dominants showed significantly greater receptor binding in the lateral ventromedial hypothalamus (VMHL) compared to subordinates (**P = 0.01), and a similar trend was observed in the anterior hypothalamus (AH) (*P = 0.077). No changes were observed in the other brain regions examined.
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BNST, LS, and medial amygdala has also been implicated in the control of agonistic behavior (Delville et al., 2000; Han et al., 1996a,b; Kollack-Walker et al., 1997; Koolhaas et al., 1990; Potegal et al., 1996a,b). In hamsters, flank marking, which is a form of scent marking, is regulated mainly by AVP in the MPOA-AH (Albers and Bamshad, 1998; Ferris et al., 1990), and dominant hamsters flank mark more than subordinates (Ferris et al., 1986, 1987). The MPOA-AH is reciprocally connected with the LS, BNST, and the periaqueductal gray (Albers et al., 1992; Ferris et al., 1990), and these sites, as well as the CeA, modulate flank marking (Bamshad and Albers, 1996; Bamshad et al., 1996; Hennessey et al., 1992; Irvin et al., 1990). In our study, AVP V1a receptor binding was not significantly different between dominants and subordinates in the brain regions which compose the neural circuit for flank marking (i.e., MPOA-AH, LS, BNST, CeA). The finding that significant differences in AVP V1a receptor binding were restricted to the VMHL suggests that the changes in AVP V1a receptor binding that occur following the establishment of dominance relationships are most likely related to aggression and not other aspects of agonistic behavior (e.g., submission or flank marking). Acute social defeat produced pronounced changes in agonistic behavior but did not alter AVP V1a receptor binding. This result suggests that changes in AVP V1a receptor populations are not required for the increased submission and decreased aggression that occurs following acute social defeat. Repeated encounters also produced changes in agonistic behavior, and dominants had greater AVP V1a receptor binding in the VMHL than did subordinates. In the VMHL, differences in AVP V1a receptor binding appeared to be due to up-regulation in dominants, while subordinates did not differ from controls. These results are consistent with a role for V1a receptors in the VMHL in the control of aggressive behavior but indicate that agonistic behavior can change dramatically before changes in V1a receptor binding occur. In sum, the present data suggest that the behavioral changes that occur following acute social defeat do not require AVP V1a receptor binding changes, but they suggest a role for V1a receptors in modulating behavioral responses to repeated experiences of victory.
Acknowledgments We thank Heather Caldwell for advice on autoradiography and AVP V1a binding patterns. We are also grateful to Jeris Israel and Lauren Zelinski for technical assistance. This work was supported by grant MH62044 to KLH, grant MH62641 to HEA, and grant NRSA MH072085 to MAC. Also, this material is based upon work supported in part by The Center for Behavioral Neuroscience, an STC Program of the National Science Foundation under agreement No. IBN-9876754.
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