Serotonergic response to social stress and artificial social sign stimuli during paired interactions between male Anolis carolinensis

Neuroscience 123 (2004) 835– 845

SEROTONERGIC RESPONSE TO SOCIAL STRESS AND ARTIFICIAL SOCIAL SIGN STIMULI DURING PAIRED INTERACTIONS BETWEEN MALE ANOLIS CAROLINENSIS W. J. KORZAN AND C. H. SUMMERS*

Greenberg and Noble, 1944), and those that exhibit more aggression usually become socially dominant (Summers, 2002). Social stress caused by agonistic interactions between two males results in serotonergic activity with distinct temporal characteristics for dominant and subordinate animals (Summers, 2001, 2002; Summers et al., 2003a). During agonistic encounters between two males, sympathetically activated darkening of postorbital skin (eyespot formation) in both males acts as a potent social sign stimulus, influencing aggressive behavior, central serotonergic activity and the outcome of the interaction (Korzan et al., 2000a, 2002; Summers, 2002). Male A. carolinensis viewing an opponent with darkened eyespots exhibit fewer aggressive acts (Korzan et al., 2000a,b, 2001, 2002; Larson and Summers, 2001), and individuals with the most rapid eyespot formation become dominant in 95% of paired interactions (Korzan et al., 2002; Larson and Summers, 2001; Summers, 2002; Summers and Greenberg, 1994). In addition, artificially darkening the eyespot region of one male in a combative pair facilitates achievement of dominant status for that individual and intensifies aggressive behavior (Korzan et al., 2000a, 2002). In previous experiments, male A. carolinensis viewing a mirror image of an opponent with eyespots had serotonergic patterns similar to a less aggressive individual and had elevated serotonergic activity in raphe´, locus ceruleus and substantia nigra/ventral tegmental area (Korzan et al., 2001). In contrast, animals viewing a mirror image opponent without eyespots exhibited more aggression and elevated levels of serotonergic activity in limbic nuclei such as hippocampus and amygdala (Korzan et al., 2000b). Previous experiments with un-manipulated pairs showed subordinate animals had an increase in serotonergic activity in the hippocampus, nucleus accumbens and locus ceruleus at 10 min. Conversely, un-manipulated dominant males exhibited an increase in serotonergic activity in the medial amygdala. In the experiments reported here we retested the effect of manipulation of the eyespot color on serotonergic response, but this time in interacting pairs capable of completing social rank distinctions. Serotonergic activity in many brain nuclei (especially the limbic system) is thought to mediate responses to both stress and aggression in vertebrate species (Coccaro, 1992; Lopez et al., 1999; Pucilowski and Kostowski, 1983; Summers et al., 2000, 2003b). Recent work investigating serotonergic activity of dominant and subordinate individuals at multiple time points reveals changes in serotonergic activity do occur in both social ranks, but these are temporally distinguished such that dominant animals in-

Biology and Neuroscience, University of South Dakota, 414 East Clark Street, Vermillion, SD 57069-2390, USA

Abstract—Serotonergic activity is influenced by social status and manipulation of social signals. In the lizard Anolis carolinensis, eyespot formation, i.e. darkening of postorbital skin from green to black, appears during stressful and agonistic situations, forming first in males that become dominant. To assess the effect of eyespots on central serotonergic activity during social interaction, males were paired by weight and painted postorbitally with green or black paint. Manipulation of eyespot color influenced social interactions and status. All males that viewed an opponent with black painted eyespots became subordinate. In these subordinate animals, serotonergic activity was elevated in hippocampus, striatum, nucleus accumbens and locus ceruleus. In contrast, males that viewed opponents with hidden eyespots (painted green) and became dominant had increased serotonergic activity in hypothalamus, medial amygdala and raphe´. Pre-painted eyespots produced results that distinguish dominant and subordinate relationships based on serotonergic activity not previously seen in unmanipulated pairs. Results from experiments using pairs are similar to those using mirrors for medial amygdala and locus ceruleus, but not hippocampus, nucleus accumbens or raphe´. Decreased hypothalamic serotonin was associated with increased aggressive behavior. These results, when compared with previous studies, suggest some flexibility in central serotonergic systems, which may shape dominant and subordinate rank acquisition, and appear to be influenced by the completion of social role formation. Furthermore, social status and central serotonergic activity was influenced by a visual cue, the presence or absence of postorbital eyespots on an opponent. © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: aggression, Anolis carolinensis, dominant, lizard, serotonin, subordinate.

When males compete for dominant social rank, serotonergic activity can be distinguished according to the social standing achieved (Blanchard et al., 1991; Øverli et al., 1999; Winberg et al., 1992). Male Anolis carolinensis utilize aggressive interactions to establish territories during their reproductive season (Crews, 1975; Evans, 1936; *Corresponding author. Tel: ⫹1-605-677-6177; fax: ⫹1-605-677-6557. E-mail address: [email protected] (C. H. Summers). Abbreviations: CRF, corticotropin releasing factor; HPLC, high pressure liquid chromatography; LC, locus ceruleus; PVN, paraventricular nucleus; SN/VTA, substantia nigra and ventral tegmental area taken together; VLH, ventral lateral hypothalamus; 5-HIAA, 5-hydroxyindoleacetic acid; 5-HT, serotonin or 5-hydroxytryptamine.

0306-4522/04$30.00⫹0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2003.11.005

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crease and decrease serotonergic activity faster than subordinates (Øverli et al., 1999; Summers, 2001, 2002; Summers et al., 2003a). The intensity of aggression exhibited by A. carolinensis appears to affect serotonergic activity in specific brain nuclei (Korzan et al., 2000b, 2001; Summers, 2002), suggesting a reciprocal interaction between serotonergic activity and behavioral expression. These studies suggest that changes of central serotonergic activity are influenced by both the intensity of behavior and social status, and these factors modulate specific brain nuclei (Summers, 2001, 2002). The purpose of this experiment was to investigate the effect of artificially darkened or hidden eyespots on central serotonergic activity during aggressive interactions between pairs of male A. carolinensis that complete social acquisition of dominant and subordinate social status. The relationships among eyespots, aggression, social rank, and central serotonergic activity were considered. Those mechanisms that underlie aggression and eyespot formation during agonistic interactions are modulated by serotonergic activity (Larson and Summers, 2001). We hypothesized that manipulating the eyespot signal would influence regional serotonergic response after 10 min of paired interactions with pre-painted eyespots and completed dominant and subordinate status, similar to the effects of aggression in mirror experiments. For example, we expected that aggressive, dominant males would exhibit an increase in serotonergic activity in limbic brain regions and a decrease in raphe´ (Korzan et al., 2000b, 2001). In contrast, we hypothesized that non-aggressive subordinate males, would exhibit serotonergic activity that is increased in raphe´, but decreased in nucleus accumbens when confronted with an opponent (Korzan et al., 2000b, 2001).

EXPERIMENTAL PROCEDURES Animals Adult male (⬎60 mm snout-vent length) A. carolinensis were obtained from a commercial supplier (Marabella’s, Gonzales, LA, USA). Each was weighed, measured and housed individually in a (25 cm)3 terrarium with a wooden perch (Korzan et al., 2000a). Room lights, temperature (14L 32 °C:10D 20 °C; light:dark) and relative humidity (70 – 80%) were regulated to maintain gonadal activity (Licht, 1971). Lizards were fed live crickets and watered ad libitum. All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), under approved protocol by University of South Dakota IACUC. In addition, all efforts were made to minimize the number of animals used and their suffering. Following 1 week of acclimation, males were tested for reproductive condition. Only those responding with courtship behavior when presented with a female (Greenberg and Crews, 1990) were included in the study. The eyespot regions of each lizard were manipulated by covering them with non-toxic paints (Accent Acrylic Paints, Bloomsbury, NJ, USA). Postorbital skin was painted black (to darken the eyespot region) or green to hide the formation of natural eyespots, with N⫽10 for each group (Korzan et al., 2000a,b, 2001, 2002). The paint was applied 24 h before testing. Animals were paired, such that one had eyespots painted green (hidden) and the other had eyespots painted black (darkened), and were separated from each other by an opaque divider prior to behavioral interaction. Animals with their own

eyespots darkened, but viewing opponents with hidden eyespots (masked with green paint) achieved dominance by performing stereotypical dominant behavior such as displacing the opponent and hereafter will be referred to as “dominant” (Korzan et al., 2002). Males with hidden eyespots, but viewing opponents with eyespots darkened (marked with black paint), became subordinate by exhibiting stereotypical submissive behavior such as retreating and hereafter will be referred to as “subordinate” (Korzan et al., 2002). One group of males was left unpainted, but brushed with water (controls) and never exposed to an opponent, and was therefore isolated from behavioral interaction. There were no significant differences in mean initial weights or snout-vent lengths between pairs or treatment groups. After 1 week of acclimation to the divided enclosure the divider separating opponents was removed and behaviors such as stereotyped aggressive displays (Greenberg, 1977), perch site, body color, and chromatic patterns were observed and recorded for 10 min (Korzan et al., 2000a).

Aggressive behavior All paired males interacted aggressively. Aggressive displays have been well described previously (DeCourcy and Jenssen, 1994; Greenberg, 1977). The number of displays, which consisted of full dewlap extension with head nods performed in assertion or challenge context (DeCourcy and Jenssen, 1994; Greenberg, 1977) were counted during the 10 min trial (Korzan et al., 2000a). Other recorded behavior included the number of approaches (toward the opponent), bites, retreats and tail whipping. Maximally aggressive intent is indicated by sagittal expansion, which includes a combination of lateral compression of the rib cage, nuchal crest expansion, partial dewlap extension and lateral orientation to the opponent. Approaching often accompanies aggressive displays, and always precedes bites and tail whipping.

Analysis of monoamines Brains were obtained by rapidly decapitating subjects; within 5 s of capture at the end of the 10 min interaction, and were immediately frozen on dry ice. Frozen brains were sliced coronally in 300 ␮m sections and nuclei brain regions were identified using a stereotaxic atlas (Greenberg, 1982) and map of central catecholamines (Lopez et al., 1992) for A. carolinensis, and were microdissected using a 300 mm punch (Summers et al., 2003a). Regions of the brain analyzed were chosen based on neurochemical responsiveness to aggressive social stress (Summers et al., 1998, 2003a), physical stress (Emerson et al., 2000), stress hormones (Summers et al., 2000), behavioral significance (Greenberg et al., 1979, 1984, 1988; Greenberg, 1983, 2003), homologies to the mammalian forebrain limbic system (Bruce and Neary, 1995) including the subiculum (inclusive range as measured from the center of the parietal eye: anterior 0.6 mm–posterior 1.2 mm, lateral 0.3–1.0 mm, deep 0.6 – 0.9 mm), hippocampus (a 0.4 mm–p 1.4 mm, l 0.1– 0.6 mm, d 0.6 –1.4 mm), lateral amygdala (a 0.4 mm–p 0.4 mm, l 0.1–1.4 mm, d 0.9 –1.3 mm), paleostriatum (a 0.4 mm–p 0.4 mm, l 0.3–1.0 mm, d 1.4 –2.0 mm), nucleus accumbens (a 0.2 mm–p 0.4 mm, l 0.1– 0.4 mm, d 1.6 –2.0 mm), septum (ap 0.0 mm–p 0.8 mm, l 0.0 – 0.4 mm, d 1.4 –2.0 mm), hypothalamus (p 0.2–1.8 mm, l 0.1– 0.9 mm, d 1.7–3.4 mm), medial amygdala (p 0.6 –1.2 mm, l 0.7–1.4 mm, d 1.5–2.3 mm), periaqueductal gray (p 2.3–2.5 mm, l 0.0 – 0.5 mm, d 1.5–2.2 mm), serotonin (5-HT) cell bodies of the raphe´ nuclei (p 2.6 –3.8 mm, l 0.0 – 0.2 mm, d 2.5–3.0 mm), and the other monoaminergic producing nuclei, including the substantia nigra (SN; p 2.4 – 2.8 mm, l 0.3– 0.5 mm, d 2.5–2.7 mm) and ventral tegmental area (VTA; p 2.3–2.5 mm, l 0.5– 0.9 mm, d 2.5–2.7 mm) analyzed together (SN/VTA), and locus ceruleus (LC; p 2.4 – 3.0 mm, l 0.3– 0.6 mm, d 2.0 –2.4 mm).

W. J. Korzan and C. H. Summers / Neuroscience 123 (2004) 835– 845 Serotonin (5-HT) and its catabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured (dopaminergic and noradrenergic parameters were also measured, but will be reported elsewhere) using high pressure liquid chromatography (HPLC) with electrochemical detection (Summers et al., 1998). Briefly, microdissected samples were expelled into 60 ␮l of a sodium acetate buffer (pH 5.1) containing an internal standard (␣-methyl dopamine; Sigma, St. Louis, MO, USA), freeze-thawed and centrifuged at 15,000⫻g for 2 min. Following centrifugation, 2 ␮l of a 1 mg/ml ascorbate oxidase solution (Sigma) was added to each sample. The supernatant was removed and 40 ␮l was injected into a chromatographic system (Waters Associates, Inc.; Milford, MA, USA) and analyzed with an ESA 5200 Coulochem II liquid chromatography system with electrochemical detection (ESA, Bedford, MA, USA) using two electrodes at reducing, then oxidizing potentials of ⫺40 and ⫹320 mV (Matter et al., 1998). The sodium phosphate –10% acetonitrile mobile phase was brought to a final pH of 2.9. Separation of the monoamines was achieved with a 10 cm⫻4.6 mm reverse phase, 3 ␮m particle size, Hypersil ODS column (Keystone Scientific, Bellefonte, PA, USA), and mobile phase flow rate maintained at 1 ml/min with a Waters 515 HPLC pump. Sample peak areas were quantified by comparison to standard solutions of known concentrations (5-HT and 5-HIAA; Sigma) and corrected for recovery of the internal standard using interpretation software (Kontron D450, Softron, Basel, Switzerland). Figures presented depict 5-HT, 5-HIAA concentrations and ratios of catabolite to transmitter (i.e. 5-HIAA/5-HT) as an estimate of monoaminergic turnover and activity. Monoaminergic activity is often approximated by the ratio of the catabolite to transmitter, such as 5-HIAA/ 5-HT. This is especially important for analyzing serotonergic activity in studies of behavior or stress (Summers, 2001), as accessible 5-HT is often greater than demand for individual or even multiple behavioral events, hence 5-HT levels usually remain unchanged. Constant 5-HT levels may also occur when synthesis is rapidly elevated in response to stress. Behaviorally or stressinduced changes in the catabolite 5-HIAA are often seen along with changes in 5-HIAA/5-HT (Winberg and Nilsson, 1993). However, the ratio is a more direct index of serotonergic activity than catabolite levels per se, because variance related to tissue sampling, and to total levels of 5-HT and 5-HIAA, are reduced (Shannon et al., 1986). Although, changes in ratio are often a result of changes in 5-HIAA levels, experiments that immediately follow behavioral interaction may discern a reduction in 5-HT concentrations reflecting recent release, and have more effect than 5-HIAA levels on the ratio.

Statistical analyses Comparisons of frequency of aggression between eyespot painted groups were performed by paired t-test. Monoamines and monoamine ratios comparisons between eyespot painted groups were performed by one-way analysis of variance and paired t-test and comparisons between controls and eyespot painted groups were performed by independent t-test.

RESULTS Manipulation of the eyespots had a substantial influence on behavior, 5-HT, its catabolite 5-HIAA and serotonergic activity (5-HIAA/5-HT) in various brain nuclei. Subordinate animals exhibited increased serotonergic activity in hippocampus, striatum, LC and nucleus accumbens and a decrease in substantia nigra/ventral tegmental area. In comparison, dominant animals had increased serotonergic activity in medial amygdala, lateral amygdala, hypothalamus, LC and raphe´ when compared with controls. However, serotonergic activity was not significantly (P⬎0.05)

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affected in septum, subiculum and periaqueductal gray nuclei, suggesting that generalized activation or inhibition was not occurring throughout the entire brain. Behavior In each paired interaction both males initially responded aggressively to one another. Agonistic behavior (such as displays and total aggression) was frequently exhibited by individuals that became dominant (Korzan et al., 2002). Individuals with eyespots darkened by black paint achieved dominant status over their hidden eyespots opponents in 100% of interactions, as previously reported (Korzan et al., 2002). Total aggression (Fig. 1A), including displays, head nods, bites, jaw gapes and approaches, was significantly (t⫽3.95, P⬍0.0075) higher from dominant individuals. Attack latency (Fig. 1B) was significantly (t⫽2.90, P⬍0.0186) longer in subordinate individuals. Hippocampus In the hippocampus, the pattern of serotonergic activity (estimated by 5-HIAA/5-HT ratio) was similar to that seen in most of the limbic structures. Levels of 5-HT in the hippocampus (Fig. 2, Top a) were significantly (t⫽2.87, P⬍0.013) lower in subordinate individuals compared with isolated controls, but dominant animals showed no difference compared with controls or compared with subordinates. There was no significant difference in 5-HT catabolite 5-HIAA (Fig. 2, Top b) among the three test groups, but a trend toward increase for dominant and subordinate animals. Hippocampal serotonergic activity was significantly (t⫽4.20, P⬍0.001; t⫽3.13, P⬍0.026) elevated in subordinate individuals when compared with isolated and dominant males (Fig. 2, Top c). Striatum (paleostriatum) 5-HT levels in striatum located ventrally in Anolis brain (Fig. 2, Middle a) were significantly lower in subordinate males compared with dominant (t⫽2.80, P⬍0.031) and control individuals (t⫽2.49, P⬍0.026). There was no significant difference in 5-HT catabolite 5-HIAA (Fig. 2, Middle b) among the three test groups. However, serotonergic activity was significantly elevated in subordinate individuals when compared with control (t⫽4.24, P⬍0.001) or dominant males (t⫽4.34, P⬍0.005; Fig. 2, Middle c). Nucleus accumbens In nucleus accumbens, 5-HT levels were significantly (t⫽3.03, P⬍0.019) elevated in dominant individuals compared with subordinate males (Fig. 2, Bottom a). Levels of 5-HT catabolite 5-HIAA levels were similar for all test groups in nucleus accumbens (Fig. 2, Bottom b). Serotonergic activity in nucleus accumbens exhibited a similar pattern as the previous brain nuclei, with subordinate animals having significantly (t⫽3.64, P⬍0.015) elevated activity compared with dominant males (Fig. 2, Bottom c).

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compared with subordinate (t⫽8.02, P⬍0.001) or control individuals (t⫽2.54, P⬍0.03; Fig. 3, Top c). This pattern of serotonergic activity in medial amygdala was opposite to the serotonergic pattern in hippocampus, striatum and nucleus accumbens.

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Similar to medial amygdala, 5-HT concentrations in lateral amygdala (the ventrolateral portion of the anterior dorsal ventricular ridge) were significantly elevated in subordinate animals compared with dominant (t⫽3.22, P⬍0.018) and control males (t⫽2.64, P⬍0.02; Fig. 3, Middle a). Similar to hippocampus, medial amygdala, striatum and nucleus accumbens, 5-HIAA concentrations in lateral amygdala were not significantly different for the three test groups (Fig. 3, Middle b). However, serotonergic activity in lateral amygdala was significantly elevated in dominant males compared with subordinate (t⫽2.81, P⬍0.031) or control individuals (t⫽2.43, P⬍0.029; Fig. 3, Middle c). This pattern is similar to that shown in the medial amygdala but the opposite of the pattern in hippocampus, striatum and nucleus accumbens.

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Social Status Fig. 1. In pairs of interacting A. carolinensis, A. males that become dominant exhibit more aggressive behavior than males that become subordinate (paired t-test, t⫽3.95, P⬍0.0075) B. and males that become dominant have shorter latency to attack when compared with animals that receive subordinate status (paired t-test, t⫽2.9, P⬍0.0186). Gray bars represent dominant males and black bars represent subordinate males. Animals that became dominant viewed an opponent with eyespots hidden with green paint. Whereas, animals that became subordinate viewed an opponent with eyespots darkened with black paint.

Medial amygdala In contrast to hippocampus, striatum and nucleus accumbens, 5-HT levels in the medial amygdala were significantly elevated in subordinate animals compared with dominant (t⫽2.73, P⬍0.041) and control males (t⫽2.92, P⬍0.02; Fig. 3, Top a). Similar to hippocampus, striatum and nucleus accumbens, medial amygdala 5-HIAA levels were not significantly different for the three test groups (Fig. 3, Top b). However, serotonergic activity in medial amygdala was significantly elevated in dominant males

Levels of 5-HT in hypothalamus were significantly lower in dominant males when compared with subordinate (t⫽3.07, P⬍0.028) and control males (t⫽2.72, P⬍0.019; Fig. 3, Bottom a). Similar to other forebrain limbic regions (hippocampus, striatum, nucleus accumbens, medial and lateral amygdala), 5-HIAA levels in hypothalamus were unaffected by eyespot presence or status (Fig. 3, Bottom b). Serotonergic activity in hypothalamus was significantly elevated in dominant individuals compared with subordinates (t⫽2.80, P⬍0.049) and controls (t⫽2.32, P⬍0.039; Fig. 3, Bottom c). This pattern was similar to medial amygdala and lateral amygdala but opposite of the other forebrain limbic areas. Raphe´ 5-HT levels in the raphe´ were not significantly different for all three groups (Fig. 4, Top a). In raphe´ 5-HIAA levels were significantly elevated in dominant animals compared with subordinate (t⫽3.22, P⬍0.018) and control males (t⫽3.28, P⬍0.006; Fig. 4, Top b). Serotonergic activity in the raphe´ was significantly elevated in dominant individuals compared with subordinate (t⫽3.25, P⬍0.017) and control animals (t⫽2.97, P⬍0.012; Fig. 4, Top c). SN/VTA In the SN/VTA 5-HT concentrations of subordinate individuals were significantly (t⫽2.77, P⬍0.017) elevated compared with controls, but not different from dominant males (Fig. 4, Middle a). Levels of 5-HIAA were significantly increased in dominant animals compared with subordinate (t⫽3.02, P⬍0.024) and control males (t⫽3.60, P⬍0.003; Fig. 4, Middle b). Serotonergic activity in SN/VTA was significantly lower in subordinate animals compared with

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S o c i a l S ta t u s Fig. 2. Mean concentrations⫾S.E.M. of 5-HT (a), 5-HIAA (b) and serotonergic activity (the ratio of 5-HIAA to 5-HT; c) are reported for the following nuclei (top) hippocampus, (middle) striatum (paleostriatum) and (bottom) nucleus accumbens. Black bars represent subordinate males, white represent control animals and gray bars represent dominant males. Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (P⬍0.05). Animals that became dominant viewed an opponent with eyespots hidden with green paint. Whereas, animals that became subordinate viewed an opponent with eyespots darkened with black paint.

dominant (t⫽4.86, P⬍0.003) and control individuals (t⫽4.82, P⬍0.0001; Fig. 4, Middle c). LC Levels of 5-HT in LC were significantly lower in subordinate animals compared with dominant (t⫽3.47, P⬍0.013) and control males (t⫽2.41, P⬍0.032; Fig. 4, Bottom a). Levels of 5-HIAA in LC were significantly (t⫽2.22, P⬍0.05), increased in subordinate animals compared with control males (Fig. 4, Bottom b). Serotonergic activity in LC was significantly elevated in subordinate (t⫽2.44,

P⬍0.033) and dominant (t⫽2.81, P⬍0.016) individuals compared with control males (Fig. 4, Bottom c).

DISCUSSION A conspecific social interaction for access to resources is a common feature in vertebrate and invertebrate taxa, and this competition often results in aggressive, stressful situations. For male A. carolinensis in this study, viewing an opponent with darkened eyespots diminished total aggression, increased attack latency and elevated

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Medial Amygdala 4

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S o c i a l S ta t u s Fig. 3. Mean concentrations⫾S.E.M. of 5-HT (a), 5-HIAA (b) and serotonergic activity (ratio, c) for (top) medial amygdala, (middle) lateral amygdala (anterior dorsal ventricular ridge) and (bottom) hypothalamus. Black bars represent subordinate males, white represent control animals and gray bars represent dominant males. Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (P⬍0.05). Animals that became dominant viewed an opponent with eyespots hidden with green paint. Whereas, animals that became subordinate viewed an opponent with eyespots darkened with black paint.

serotonergic activity in hippocampus, striatum, nucleus accumbens and LC (Figs. 1, 2, 4). Conversely, males viewing an opponent with eyespot regions masked with paint exhibited elevated aggressive behavior (Fig. 1) and higher serotonergic activity in medial amygdala, lateral amygdala, hypothalamus and raphe´ (Figs. 3, 4). Serotonergic activity was unaffected in septum, subiculum and periaqueductal gray, suggesting that generalized activation or inhibition did not occur throughout the entire brain. The combined results of this and previous studies suggest that social signals, such as darkened

eyespots, influence agonistic behavior in paired interactions and are correlated with activation or inhibition of serotonergic activity in nuclei known to be specifically associated with aggression and/or stress. Previous studies have demonstrated that stressful, aggressive encounters influence monoamine patterns in specific brain nuclei in a context dependent manner (Korzan et al., 2000b; Summers et al., 1998). Many vertebrates exhibit central serotonergic changes following stressful social interactions (Blanchard et al., 1993; Kudryavtseva, 2000; Øverli et al., 1999; Summers, 2002; Win-

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B

10 8

A

AB

b. 5-HIAA (pg/ µ g protein)

B

5

0.8 0.7

A

4

A

c. 5-HIAA/5-HT

B

B

0.6

A

0.5

6

3

0.4

4

2

0.3

2

1

0

0

0.2

Control Dom

S ub

Locus Ceruleus

a. 5-HT (pg/ µ g protein)

9 8

B

9

0.1 Control Dom

b. 5-HIAA (pg/ µ g protein)

8

B

B

7

A

6

A

5

0.0

1.6

0.6

3

3

2

2

0.4

1

1

0.2

0

0

S ub

B

1.0

A

5

0.8

Control Dom

S ub

c. 5-HIAA/5-HT

1.4

4

4

Control Dom

1.2

7 6

S ub

Control Dom

S ub

0.0

B A

Control Dom

S ub

S o c i a l S t a t us Fig. 4. Mean concentrations⫾S.E.M. of 5-HT (a), 5-HIAA (b) and serotonergic activity (ratio, c) for (top) raphe´, (middle) substantia nigra/ventral tegmental area (SN/VTA) and (bottom) LC. Black bars represent subordinate males, white represent control animals and gray bars represent dominant males. Means that share any superscript letter are not significantly different, and those with no common superscript letter are significantly different (P⬍0.05). Animals that became dominant viewed an opponent with eyespots hidden with green paint. Whereas, animals that became subordinate viewed an opponent with eyespots darkened with black paint.

berg and Nilsson, 1993). Thus, changes in serotonergic activity can be modulated by behavior, visual sign stimuli, and social status (Korzan et al., 2000b, 2001). Behavior and visual stimuli modify serotonergic activity in subordinate males Regional serotonergic activity recorded for paired males differed from that measured in aggressive interactions with mirror images with respect to eyespot color and aggression. In the currently reported study only in the medial amygdala (Fig. 3, top) was increased serotonergic activity

expressed along with increased aggressive behavior for animals viewing an opponent with hidden eyespots, as in previous experiments utilizing mirrors (reflected opponents; Korzan et al., 2000b, 2001). In those studies utilizing a mirror image to evoke aggressive response, single males were introduced to a reflected image of themselves and therefore social rank could not be established. During paired interactions males that viewed an opponent with the visual sign stimulus (darkened eyespots) became subordinate (Korzan et al., 2002) and exhibited slower onset and lower levels of aggression, plus higher levels of serotoner-

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gic activity in many brain nuclei. Males that became subordinate viewed a highly aggressive opponent that also exhibited the visual signal of darkened eyespots (Korzan et al., 2002). Presence of the visual signal may exacerbate the stress response in individuals that view the signal and become subordinate (Korzan et al., 2002). The combination of these two social signals (i.e. viewing aggression and eyespots) may be the cause of changes in serotonergic activity associated with subordinate status. Previous studies of unmanipulated pairs have shown that subordinate male A. carolinensis have increased serotonergic activity at 10 min in many central nuclei including hippocampus, medial amygdala, nucleus accumbens and LC (Summers et al., 2003a). Similarly in this study the subordinate males expressed increased serotonergic activity in hippocampus, striatum, nucleus accumbens and LC and also decreased levels in substantia nigra/ventral tegmental area (Fig. 4). In unmanipulated pairs, both dominant and subordinate males have elevated serotonergic activity in hippocampus, medial amygdala and nucleus accumbens (Summers et al., 2003a). While social status was clearly determined, the outcome of the aggressive interaction was not conclusive for at least 4 – 8 min (Summers, 2002), and elevated serotonergic activity at 10 min could reflect social stress during that period. That interpretation of the data is supported by experiments using mirrors, in which no social rank acquisition was possible, nor was completion of the interaction. During interaction with a mirrored reflection, the most aggressive males, similar in behavior to dominant males, have the most elevated limbic serotonergic activity, and also the most aggression directed toward them (Korzan et al., 2000b), suggesting that an incomplete and highly aggressive interaction produces increased serotonergic activity in the limbic brain. That is, unresolved aggressive interactions are stressful, and produce active serotonergic reactions in limbic regions regulating stress and aggressive responsiveness. However, for subordinate animals, even after the social acquisition of status is complete, being confined within the behavioral theater of the dominant animal is also stressful, and produces elevated limbic serotonergic activity at 10 min (Fig. 2), and chronic serotonergic activity in the amygdala (Summers et al., 1998). Behavior and visual stimuli modify serotonergic activity in dominant males Rapid activation of serotonergic systems promote or is associated with dominant status in unstable social situations (Edwards and Kravitz, 1997; Raleigh et al., 1991; Summers et al., 2003a) and activation of dominant social/ territorial display (Baxter et al., 2001; Baxter, 2001). In A. carolinensis, males with eyespots painted dark, viewing an opponent with the eyespot regions covered with green paint, achieve dominant status (Korzan et al., 2002) and show elevated amygdalar, hypothalamic and brainstem serotonergic activity (Figs. 3, 4). Males that became dominant viewed an opponent exhibiting low amounts of aggression and without the presence of the eyespot stimulus. When eyespots are manipulated during paired interactions

male anoles that became dominant show increased serotonergic activity in nuclei such as medial amygdala, lateral amygdala, hypothalamus, LC and raphe´ at 10 min of agonistic interaction. Previous experiments utilizing unmanipulated pairs indicate that the medial amygdala in dominant males shows a slower secondary serotonergic response when eyespots form naturally (Summers et al., 1998), as compared with manipulated eyespot experiments. These results suggest that the presence or absence of eyespots is influencing the temporal component of serotonergic activity in the medial amygdala. Therefore, the formation of social rank and changes in serotonergic activity resulting from this may be facilitated through manipulation of the eyespot social signal. Comparison between ranks and previous studies Results from this and previous experiments, especially comparing serotonergic activity from unmanipulated pairs (Summers et al., 2003a) and individuals interacting with a mirror image (Korzan et al., 2000b), suggest that the presence or absence of the visual signal immediately upon initiation of social interaction may exaggerate the stress response stimulated by aggressive interactions (Korzan et al., 2002). For example, serotonergic activity in hippocampus is influenced by physical stress (Emerson et al., 2000), elevated by glucocorticoid stress hormones (Summers et al., 2000, 2003b), increased in both dominant and subordinate males of unmanipulated pairs (Summers et al., 2003a), but only elevated in more aggressive males (viewing hidden eyespots) interacting with a mirror image (Korzan et al., 2000b). By contrast, we report here (Fig. 2) that elevated serotonergic activity in hippocampus occurs only in subordinate males. These subordinate males are experiencing a stressful situation accompanied by elevated plasma corticosterone (Summers et al., 2003a). Aggressive behavior is being directed toward them (as was the case in the mirror experiment), and that probably accounts for some stimulation of hippocampal serotonergic activity and elevated corticosterone. However, the opponent they face has a darkened eyespot, as opposed to an opponent without an eyespot, as was the case that produced elevated hippocampal serotonergic activity among males interacting with a mirror. In a comparison of the medial amygdala, again stress hormones like corticosterone stimulate elevate the 5-HIAA/5-HT ratio, and when male A. carolinensis are tested by interactions with a mirror image and in painted pairs (Fig. 3), enhanced serotonergic activity occurs in the amygdala of more aggressive males (dominant in pairs), viewing an opponent without eyespots (hidden by green paint). In both of these limbic regions elevated serotonergic activity is correlated with aggressive activity directed toward them or their opponent and darkened eyespot color, but only when taken in combinations. The results taken together, suggest that heightened stressful experiences result in elevated serotonergic activity in hippocampus and amygdala, but appear to be differentiated by social rank, and the experiences that are unique to dominant and subordinate roles.

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Role of the hypothalamus Stress-induced adjustments in, or control by, the hypothalamus have been the focus of many studies investigating the control and regulation of the stress response. One particularly important area of the hypothalamus involved with the stress response is the paraventricular nucleus (PVN). The PVN is highly innervated with serotonergic fibers and terminals, and the majority of hormonal corticotropin releasing factor (CRF) cell bodies are also located in this region (Dinan, 1996). Serotonin directly stimulates CRF release from the PVN (Chaouloff, 1993; Dinan, 1996), and CRF release from the PVN initiates the HPA stress response, resulting in increased corticosterone in the bloodstream (Dinan, 1996). At the conclusion of a 10 min interaction between non-manipulated pairs of anoles, both dominant and subordinate males exhibit increased corticosterone levels (Summers et al., 2003a). These results support the concept that agonistic interactions in vertebrates are stressful for both dominant (winning) and subordinate (losing) males (Øverli et al., 1999; Sapolsky, 1982; Summers, 2001, 2002). However, previous studies have not demonstrated neurochemical changes in the hypothalamus of A. carolinensis males following social interaction. Our results may suggest that early in the course of the socially aggressive interaction, the hormonal stress response has been activated in the hypothalamus only in dominant males (Fig. 3). Due to size constraints with anole brains we had to sample the entire hypothalamus, and therefore our results do not only reflect changes in the PVN. We found that dominant (aggressive) males exhibited a significant decrease in 5-HT, and increased serotonergic activity (5-HIAA/5-HT) compared with controls and subordinate males (Fig. 3). This effect may be exaggerated in this experiment by artificially darkened eyespots, however, because in unmanipulated pairs, both dominant and subordinate males have elevated plasma corticosterone and limbic serotonergic activity by 10 min (Summers et al., 2003a). Shortly after the 10 min sampling period, the stress response of dominant males appears to be diminishing. Recent experimental results demonstrate that although dominant males have elevated plasma corticosterone after 10 min of aggressive interaction, by 20 min plasma corticosterone in dominant males has returned to baseline, and remains unaffected thereafter (Summers et al., 2003a). In addition to the widely known role of the hypothalamus in the stress response, it is also involved in initiating and modulating aggressive behavior (Halasz et al., 2002; Kruk et al., 1998; Siegel et al., 1999). Regions such as the anterior and ventral lateral hypothalamus (VLH) have been identified as key sites of control of aggressive behavior (Delville et al., 2000; Ferris et al., 1999). Anterior hypothalamus and VLH possess high 5-HT1B binding sites and are highly innervated by 5-HT fibers and terminals in hamsters (Delville et al., 1996; Ferris and Delville, 1994). Administration of 5-HT reuptake inhibitor fluoxetine in the VLH blocks offensive aggressive behavior in golden hamsters (Delville et al., 1996). Similarly, in the entire hypothalamus,

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more aggressive dominant male A. carolinensis exhibited a significant decrease in 5-HT compared with controls and subordinate males, although serotonergic activity (estimated by 5-HIAA/5-HT) is elevated (Fig. 3). This finding, combined with previous work, suggests that a decrease in the available 5-HT in the hypothalamus facilitates aggressive behavior. However, although chronically elevated 5-HT inhibits aggression in Anolis (Larson and Summers, 2001), these results (Figs. 2– 4) do not suggest that 5-HT has generalized inhibitory effects on aggression regardless of brain area, as this and other studies have shown elevated serotonergic activity in limbic regions of more aggressive dominant males (Korzan et al., 2000b; Summers et al., 2003a). Aggression and social status correlated with negative feedback in the raphe´ Vertebrate limbic systems are highly innervated by serotonergic cells of the raphe´ (Chaouloff, 1993; Dinan, 1996; Smeets and Steinbusch, 1988). Serotonergic activity in the raphe´ is often opposite to that in many limbic regions (Emerson et al., 2000; Korzan et al., 2001; Summers et al., 2003a). Inhibiting the release of 5-HT to limbic structures via lesions of the raphe´ increases defensive aggression in rats (Yamamoto and Ueki, 1977). In this study we observed that heightened aggressive behavior by dominant animals did not result in a change of activity in most limbic areas, but that serotonergic activity in hypothalamus, amygdala and raphe´ was increased. However, less aggressive subordinate males exhibited increased forebrain limbic serotonergic activity and no change in raphe´ serotonergic activity. These results suggest that the change in serotonergic activity associated with aggression and social status may be facilitated through feedback mechanisms such as activation of 5-HT1A receptors by 5-HT in the raphe´, which decrease 5-HT release at the limbic structures. Inhibitors of 5-HT1A receptors impede aggression in Anolis (Deckel and Fuqua, 1998), perhaps working via feedback mechanisms in the raphe´ or hypothalamus. Summary Social status, behavior and visual perception (of social sign stimulus and aggression) influence serotonergic profiles in paired male A. carolinensis. Individuals receive and produce multiple socially relevant visual stimuli during an interaction, such as eyespots, viewing behavior and body color. All of these factors influence behavior and serotonergic activity of the viewer. During social interactions decreased hypothalamic 5-HT was associated with increased aggressive behavior. Patterns of serotonergic activity in the raphe´ and limbic system suggest serotonergic activity is modulated through feedback mechanisms in the raphe´ during aggressive interactions. This work suggests the combination of social signals, rank and behavior, but not any single factor, may be the impetus for the changes in serotonergic activity associated with subordinate and dominant profiles.

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Acknowledgements—We thank Kenneth Renner for technical assistance. We also thank Gina Forster, Michael Watt, Tangi R. Summers, Øyvind Øverli and Ken Renner for critically reading of this manuscript. We would also like to thank Dr. Yuhlong Lio, Professor of Mathematics for statistical advice. Supported by NIH grant 1 F31 MH64983-01, Sigma Xi grants in aid and NSF EPSCoR graduate fellowship granted to W.J.K.; and by NIH grant P20 RR15567.

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(Accepted 5 November 2003)