Neural and endocrine responses to social stress differ during actual and virtual aggressive interactions or physiological sign stimuli

Abstract

Neural and endocrine responses provide quantitative measures that can be used for discriminating behavioral output analyses. Experimental design differences often make it difficult to compare results with respect to the mechanisms producing behavioral actions. We hypothesize that comparisons of distinctive behavioral paradigms or modification of social signals can aid in teasing apart the subtle differences in animal responses to social stress. Eyespots are a unique sympathetically activated sign stimulus of the lizard Anolis carolinensis that influence aggression and social dominance. Eyespot formation along with measurements of central and plasma monoamines enable comparison of paired male aggressive interactions with those provoked by a mirror image. The results suggest that experiments employing artificial application of sign stimuli in dyadic interactions amplify behavioral, neural and endocrine responses, and foreshorten behavioral interactions compared to those that develop among pairs naturally. While the use of mirrors to induce aggressive behavior, produces simulated interactions that appear normal, some behavioral, neural and endocrine responses are amplified in these experiments as well. In contrast, mirror image interactions also limit the level of certain behavioral and neuroendocrine responses, as true social communication does not occur during interaction with mirror images, rank relationships can never be established. Multiple experimental approaches, such as combining naturalistic social interactions with virtual exchanges and/or manipulation of sign stimuli, can often provide added depth to understanding the motivation, context, and mechanisms that produces specific behaviors. The addition of endocrine and neural measurements helps identify the contributions of specific behavioral elements to the social processes proceeding.

1. Introduction

Molecularly, genetically and/or chemically oriented experiments that elucidate physiologically significant mechanisms often require analysis of behavioral output to confirm relevance for animals in nature (Campos, Rojas, and Wilczynski 2020; Nikonov and Maruska 2019; Butler et al. 2018). However, experimental designs for behavioral analyses can hold hidden complications and the outward expression or lack of behavioral responses can unfortunately be misinterpreted. A number of classic and elegant experimental protocols have been designed to elucidate the intentions for and motivations behind social behaviors (Clark and Utez 1991; Craig 1918; Lissmann 1932; Lorenz and Tinbergen 1938; ter Pelkwijk and Tinbergen 1937; Tinbergen and Perdeck 1950), but as the neural mechanisms promoting those behaviors are not entirely known, it is not always simple to compare results from different classic approaches (Morgan 1894). The purpose of this article is to compare the outcomes of three classic behavioral approaches, using a sympathetically derived sign stimulus (Kleinholz 1938; Tinbergen 1939; Korzan, Summers, Ronan, et al. 2000) and analysis of neural and endocrine responsiveness.

Experiments on socially derived aggressive behavior elucidate the mechanisms for determining social stress and status (Carpenter and Summers 2009; Smith et al. 2014; Smith et al. 2016; Forster et al. 2005; Korzan et al. 2007; Korzan and Summers 2007; Summers, Forster, et al. 2005; Korzan, Overli, and Summers 2006). Agonistic social interaction can be achieved via multiple designs such as introducing animals in pairs or groups, or by using a mirror or monitor to allow an individual to “interact” with its own reflection or videotaped presentation. The quality and frequency of aggressive display produced may be very similar, independent of ethological method employed (see Figure 2). However, the interpretation of the results produced, relative to the impact of the social stressor (with synergistic behavioral signals increasing neuroendocrine responses), by various approaches may be quite different.

Figure 2.

A) Pairing males or simulating an opponent with a mirror elicits aggressive behavior in all A. carolinensis males. During aggressive encounters, darkened eyespots (either naturally or artificially) always limit aggressive behavior. Frequency of aggressive behavior (including displays, head nods, dewlap extensions, bites, jaw gapes, chases and approaches; taken together per minute ± SEM) is significantly greater (‡) for unpainted males becoming dominant (gray bars) than for subordinate males (clear bars). Black-eyespot-painted naïve males (gray bar) always became dominant and behaved more aggressively than those with hidden eyespots (green paint; clear bars). Artificially darkened eyespots (clear bar) inhibited aggression in animals reacting to their own mirror image, but males that had eyespots hidden by green paint (gray bar) displayed significantly elevated aggression (‡). B) Mean plasma norepinephrine (NE ± SEM) concentrations during agonistic interactions. C) Mean serotonergic activity (estimated by 5-HIAA/5-HT ± SEM) in the medial cortex (hippocampus). Males that experienced two or more of these factors: 1. performing aggression, 2. viewing aggression, and 3. viewing blackened eyespots had a significantly (*) elevated B) NE response, and C) serotonergic activity compared to control (hatched bars). Viewing 4. darkened body skin on an opponent limited NE and 5-HIAA/5-HT. Among paired animals or treatments (clear bars: subordinate/unaggressive males versus gray bars for dominant/aggressive males), ‡ denotes significant increases in NE or serotonergic response compared within treatment sets (i.e. sub vs dom, or low vs high aggression). Among dominant and/or aggressive males (gray bars), unmodified lizards had significantly greater plasma NE than paired lizards with painted eyespots (♦). Painted anoles viewing their own mirror image with eyespots hidden had higher NE (†) and serotonergic activity (♦; though not significant to natural dominant males) than either type of paired dominant male. The green painted male (eyespots hidden; clear bar) in a pair became less aggressive and subordinate with significantly elevated NE (*) and serotonergic activity (*) compared to opponents painted/darkened eyespots. Green-painted males in a pair, also had more NE (§) and serotonergic activity (§ also higher in natural subordinates) than males who were unaggressive toward their own mirror image (compare clear bars) in response to dark-painted eyespots.

While field experiments yield invaluable information, it is notoriously difficult to design experiments for the field to test specific questions about factors that induce or inhibit behavior. It is possible to relate broadly comparable results between lab and field, for example, aggressive social interaction between a tethered intruder and a territorially dominant male Sceloporus jarrovi, stimulates a very rapid increase in serotonergic activity in the telencephalon of the territorial male (Matter, Ronan, and Summers 1998), similar to that seen in captive A. carolinensis dyads (Summers, Summers, et al. 2003).

Numerous protocols have been developed to examine aggression, social status, social memory, and the neural and physiological mechanisms that underlie these behavioral states (Yang and Wilczynski 2002; Korzan and Summers 2007; Summers et al. 2017). Most of these have been carried out using physical characteristics (Matched by body length and weight) to create size-matched pairs, including those on the lizard A. carolinensis (Summers and Greenberg 1994, 1995; Summers, Korzan, et al. 2005; Summers et al. 1998; Summers, Watt, et al. 2005; Summers, Summers, et al. 2003; Greenberg 1977; Greenberg, Chen, and Crews 1984; Greenberg and Crews 1990). Another classic experimental paradigm makes use of mirrors (Lissmann 1932; Tinbergen 1948) for individuals of species that do not recognize their own image; and has been used for freshwater fish (Lissmann 1932; Desjardins and Fernald 2010), marine fish (Oliveira, Carneiro, and Canario 2005), anadromous fish (Tinbergen 1948), and the lizard A. carolinensis (Baxter 2001; Baxter, Ackermann, et al. 2001; Baxter, Clark, et al. 2001; Watt et al. 2007; Korzan, Summers, Ronan, et al. 2000; Korzan et al. 2001; Korzan, Summers, and Summers 2000). Similarly, video images may provoke behavioral responses in marine (Goncalves et al. 2000) as well as freshwater fish (Gerlai, Fernandes, and Pereira 2009; Luca and Gerlai 2012; Chouinard-Thuly et al. 2017; Qin et al. 2014) and the lizard A. carolinensis (Plavicki, Yang, and Wilczynski 2004; Yang et al. 2001; Yang and Wilczynski 2002, 2003); as long as the individual does not recognize that the image on the playback screen is not socially interactive. These paradigms may provoke aggressive behaviors that appear quite similar.

For some species, sign stimuli (Tinbergen 1939; Tinbergen and Perdeck 1950), may also modify social stress/aggressive reactivity(Korzan and Summers 2007; Summers, Forster, et al. 2005). For some of these species, such as veiled chameleons Chamaeleo calyptratus (Ligon and McGraw 2013), the cichlid Astatotilapia (Haplochromis) burtoni (Muske and Fernald, 1987b) and swordtail Xiphophorus birchmanni and X. multilineatus fish (Muske and Fernald, 1987a), and the lizard A. carolinensis, the sign stimulus is a dynamic physiological response (Hutton et al. 2015), which may dramatically summon or limit aggressive responses (Summers and Greenberg 1994; Ligon and McGraw 2018). The lizard A. carolinensis has postorbital skin chromatophores that darken due to adrenal catecholamines acting on β₂-adrenergic receptors (Ligon and McCartney 2016; Goldman and Hadley 1969; Hadley and Goldman 1969), referred to as eyespots. Darkened eyespots inhibit aggression in observers, promote social dominance of the wearer, and influence stress-related hormones and neurotransmitters (Korzan, Summers, and Summers 2002; Korzan and Summers 2004; Korzan et al. 2001; Korzan, Summers, Ronan, et al. 2000; Korzan, Summers, and Summers 2000). The timing of Anolis eyespot darkening is influenced by the monoamine neurotransmitters serotonin (5-HT) (Larson and Summers, 2001) and dopamine (DA) (Höglund et al., 2004). The latency to eyespot darkening is important, because it is the relative timing of that event that designates social status; dominant males have faster eyespot darkening than subordinate males (Larson and Summers, 2001; Summers and Greenberg, 1994). Social status plays an important role in determining neuroplasticity and function (Fernald and Maruska 2012; Maruska, Carpenter, and Fernald 2012; Meyer et al. 2004). While influencing the temporal resolution of eyespot formation, the 5-HT reuptake inhibitor sertraline also inhibited aggression and reversed social dominance relationships (Larson and Summers, 2001). Artificial manipulation of these sign stimuli also modifies social stress responsiveness (Korzan and Summers 2004; Korzan, Summers, Miner, et al. 2000; Korzan et al. 2001; Korzan, Summers, Ronan, et al. 2000; Korzan, Summers, and Summers 2000, 2002). Viewing eyespot color, modified by paint (Tinbergen and Perdeck, 1950) matching darkened postorbital skin pigmentation, limits aggressive head nods, chases, and attacks in male A. carolinensis, in dyads or mirror images (Figure 1). As artificially darkened eyespots produces social dominance for that male in naïve pairs, this manipulation influences both the aggression producing social stress, and social rank (Korzan, Summers, and Summers 2002; Muske and Fernald 1987).

Figure 1.

The experimental design begins with 7 days of acclimation to cages for 60 male A. carolinensis. At the end of day 7, 40 of those males have their eyespot regions covered with either green paint (N=20) to hide the natural eyespot darkening in response to plasma NE during aggressive interaction, or with black paint (N=20) to mimic instantaneous eyespot darkening. The remaining 20 lizards are left unpainted, to allow natural eyespot darkening during aggression. Additional lizards (N=20; not shown) are painted with water as controls. All together, there are 7 groups defined by paint color or by dominant vs. subordinate social status: 1. Water-painted controls, 2. Natural (unpainted) males who become dominant, 3. Natural males who become subordinate, 4. Black-painted males of a dyad (these males become dominant) 5. Green-painted males of a dyad (become subordinate) 6. Black painted males facing a mirror (eyespot lowers aggression, cannot resolve social status), and 7. Green painted males facing a mirror (lack of eyespot escalate aggressive behavior, cannot resolve social status). On day 8, the dyads and males paired with a mirror are allowed to interact with their opponent or mirror (an opaque divider is pulled to reveal the opponent or mirror) for 10 minutes. In dyads social rank is determined. At the end of 10 min of aggression, the lizards are rapidly (within 15 s) caught and killed.

We propose that behavioral, endocrine and transmitter interactions that regulate aggression and social status relationships in A. carolinensis, can be used to evaluate the relative value and meaning of experiments stimulating aggressive interactions using mirrors or pairs of animals, and manipulation of behavior via eyespot with black paint. We hypothesize that variations on four stress-related signals (1. Being highly aggressive, 2. Viewing aggressiveness in an opponent, 3. Viewing darkened eyespots on an opponent, and 4. Viewing brown skin on an opponent) produced by these manipulations, result in synergistic reactions, such that combinations of 1, 2, and/or 3 increase neuroendocrine stress responsiveness, and the addition of 4 reduces it.

2. Methods

2.1. Animals

All experiments were conducted with wild adult (> 60 mm snout-vent length), size matched (mean weight varied by < 6 mg, and mean snout-vent length varied by < 2 mm) to minimize the influence of body size on behavioral interactions (Tokarz 1985) male A. carolinensis lizards (purchased from Thibodaux Live Supplies, Thibodaux, LA). Animals were housed in one half of a terrarium of 17,550 cm³ divided by a removable opaque partition that obscures either a mirror or another male. Lights, temperature (14L32°C:10D20°C; lights on at 0600) and relative humidity (70 - 80%) were regulated to maintain gonadal activity (Licht 1971; Summers 1984, 1988). Lizards were fed live crickets every other day and watered ad libitum. Following one week of acclimation, males were tested for reproductive condition. Only males responding with courtship behavior when presented with a female (Greenberg and Crews 1990) were included in the study. Reproductive state and androgen levels may be associated with aggressive capacity (Greenberg and Crews 1990; Lovern, McNabb, and Jenssen 2001).

2.2. Experimental design.

The data presented herein are derived from three experiments conducted (Figure 2)with male A. carolinensis paired to determine social rank, or with single males interacting with their mirror image (Korzan, Summers, and Summers 2002; Summers, Summers, et al. 2003; Korzan and Summers 2004; Korzan, Summers, Ronan, et al. 2000; Korzan, Summers, and Summers 2000). Paired lizards were completely naïve to each other prior to experimental social interaction, and social dominance was determined during the aggressive bouts, and not beforehand. The postorbital skin (eyespot region) of each lizard (chosen at random) in two of the experiments, was manipulated by covering with non-toxic paint from a commercial supplier 24 hours before behavioral testing (Accent Acrylic Paints, Bloomsbury, NJ).

In one experiment size-matched pairs were used, and one of each pair had eyespot regions painted black (N = 10; the male lizards became dominant due to their eyespots being painted black) or green, (N = 10; lizards which became subordinates because their eyespots were hidden by green paint). In addition, there were 10 controls painted with water. All of these animals were assigned to paint color groups randomly before interactions occurred, and before social status was determined.

In another experiment, single lizards confronted mirror images. Again, eyespots were pained either green or black (N = 10 males with eyespots painted black, 10 with eyespots hidden by green paint, and 10 water painted controls).

In experiments with natural pairs the eyespots were left unmanipulated (N = 10 dominant males, 10 subordinate males, and 10 isolated controls). Lizards with natural eyespots interacted in pairs, whereas painted males interacted with a mirrored reflection (viewing the same eyespot on the opponent) or with a live opponent with the opposite eyespot paint color (males with eyespots hidden by green paint faced males with eyespots darkened by black paint). Behavior was manually recorded for ten minutes (Summers 2002) following removal of an opaque divider, either between two animals or hiding the mirror. Observations were made with room lights off and cage lights on (Korzan, Summers, Ronan, et al. 2000). Darkened room, cage illumination and distance of observers from cages (1.5 m) minimized observer effects on lizard behavior (Sugerman 1990). All behavioral observations were performed in three days between 12:30 pm and 3:00 pm central time. This window of observation was selected because room humidity and temperature were consistent at 32°C. All animal experiments were conducted in a manner that minimized suffering and the number of animals used, in compliance with APA ethical standards for research with animals, and 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.

2.3. Aggressive behavior

All paired male A. carolinensis interacted aggressively in the stereotyped manner that has been previously described (Crews 1975, 1979; DeCourcy and Jenssen 1994; Greenberg 1977; Greenberg and Noble 1944; Evans 1936). All displays performed in assertion or challenge context (DeCourcy and Jenssen 1994; Greenberg 1977) were counted during a ten minute period (Korzan, Summers, Ronan, et al. 2000). Behavioral records included approaches, bites, chases, jaw gapes, and extension of dewlap with head nods. Maximally aggressive display behavior (sagittal expansion) includes a combination of lateral compression of the rib cage, nuchal crest expansion, dewlap extension, sagittal spatial positioning, and head nods. Animals were scored with an approach every time they slowly decreased the distance between themselves and their opponent and the opponent did not move away. Aggressive approach often precedes display, and always precedes biting behavior. Winning an antagonistic encounter is accomplished by a greater frequency of and shorter latency to aggressive acts (such as biting) by a given male, and by chasing and displacing the opponent. Fight-winning males were confirmed to be socially dominant by realization of the most superior perching position (the highest position directly under the overhead light), continued displacement of the subordinate individual from the superior perch position and from other locations within the cage, and by lighter body coloration.

2.4. Plasma catecholamine assay

Within 5 seconds of completion of the experiment animals were decapitated and blood was taken immediately from the body and head into capillary tubes. The tubes were then centrifuged to separate blood cells and plasma. Determination of catecholamine concentration from small volumes of plasma (25-100 μl) was achieved by HPLC (Lin et al. 1984). An internal standard, DHBA (100 ng/ml), was added along with 25-100 μl of plasma into a 1.5 ml syringe-filter cartridge (EG&G WALLAC/ AKRON) with 50 mg of acid-washed aluminum oxide (BAS). Immediately upon the addition of 1 ml Tris buffer (1.86 M, pH 8.65) samples were vortexed and capped, then rotated for 10 min and vortexed again. The supernatant was aspirated through the filter and the alumina was then washed and vortexed 4 times with 1 ml H2O to which a small volume of pH 7 buffer was added and aspirated to near dryness each time. A 200 μl microcentrifuge tube was placed on the cartridge as a receiver tube and centrifuged to remove any residual fluid. A new receiver tube was placed on the cartridge and 100 μl 0.1 N perchloric acid (HClO₄) was added to the sample. At this time, the samples were vortexed for 30 seconds, allowed to stand for 3-5 min and vortexed again. The cartridge and receiver tube were centrifuged until no fluid remained in the cartridge. Sixty μl of HClO₄ extract was injected directly into an HPLC system (Waters) and analyzed electrochemically with an LC-4B potentiostat (BAS). The electrode potential was set at +0.6 V with respect to an Ag/AgCl reference electrode. Mobile phase consisted of 14 g citric acid, 8.6 g sodium acetate, 110 mg 1-octanesulfonic acid (sodium salt), 150 mg EDTA disodium salt, and 100 ml methanol in 1 L of deionized water. Flow rate was maintained at 1.0 ml/min.

2.5. Analysis of central monoamines

Brains were obtained within five seconds of capture at the end of ten minutes of interaction, and were immediately frozen on dry ice. Frozen brains were sliced coronally in 300 μm sections and medical cortices containing the hippocampal granule and pyramidal cells 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, Summers, et al. 2003).

Serotonin (5-HT) and its catabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured (dopaminergic and noradrenergic parameters were also measured, but reported elsewhere) using HPLC with electrochemical detection (Summers et al. 1998). Briefly, microdissected samples were expelled into 60 μl of a sodium acetate buffer (pH 5.1) containing an internal standard (α-methyl dopamine; Sigma, St. Louis, MO), freeze-thawed and centrifuged at 15,000 x g for 2 min. Following centrifugation, 2 μl of a 1 mg/ml ascorbate oxidase solution (Sigma, St. Louis, MO) was added to each sample. The supernatant was removed and 40 μl 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) using two electrodes at reducing, then oxidizing potentials of - 40 and +320 mV (Matter, Ronan, and Summers 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 x 4.6 mm reverse phase, 3 μm particle size, Hypersil ODS column (Keystone Scientific, Bellefonte, PA), 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, St. Louis, MO) and corrected for recovery of the internal standard α-methyl dopamine using interpretation software (Kontron D450, Softron, Basel Switzerland). Transmitter and catabolite concentrations are then normalized to the quantity of protein (Bradford 1976) contained in punches, and levels are determined as pg/μg protein (Summers et al. 1998). Results presented depict, concentrations and ratios of catabolite (5-HIAA) to transmitter (5-HT) as an estimate of monoaminergic turnover and activity (i.e., 5-HIAA/5-HT).

2.6. Interpretation of data

Although neurotransmitter levels, expressed as pg amine/μg protein, are uncomplicated, understanding relative changes in activity of monoamine systems often requires some interpretation. The timing of acquisition of samples is critical for interpretation of results. If there has been sufficient time following behavioral or environmental stimulus, the data may be presented as ratios of catabolite to transmitter (i.e., 5-HIAA/5-HT), which is an estimate of monoaminergic turnover. As such, the activity of a given monoamine system can be said to increase as the ratio increases. This kind of monoaminergic activity, approximated by the ratio of the catabolite to transmitter, presumes that transmitter levels (i.e., 5-HT) decrease (or remain constant) as they are secreted and converted to catabolite (5-HIAA) and thereby catabolite concentrations increase. The ratio is especially useful for analyzing monoaminergic activity in studies of behavior or stress (Summers 2001). Behaviorally- or stress-induced changes in the catabolite are often seen along with changes in the ratio (Winberg and Nilsson 1993). The ratio is often a more direct index of monoaminergic activity than catabolite levels per se, because variance related to tissue sampling, and to total levels of transmitter and catabolite, are reduced (Shannon, Gunnet, and Moore 1986). Accessible transmitter concentration is often greater than demand for individual or even multiple behavioral events; hence monoamine levels often remain unchanged. Constant transmitter concentrations may also occur when synthesis is rapidly elevated in response to stimulus or stress, as production may offset release. Behaviorally significant events are usually followed by changes in monoaminergic ratio. Our data are derived from experiments in which the 5-HIAA/5-HT ratio appears to accurately estimate serotonergic activity, including increased release, production and catabolism of 5-HT.

2.7. Statistical Analyses

Comparison across experimental designs (natural pairs X pairs with eyespots manipulated X mirror interactions) was accomplished by two-way analysis of variance (ANOVA), contrasting treatment effects with dominant social status or highly aggressive behavior (a hallmark of dominant social rank), but excluding isolated controls. Interaction between treatment and rank were derived from this two-way analysis. Comparison of the effects of 1) dominant status or aggressive behavior, or 2) subordinate rank or unaggressive behavior with isolated controls by treatment was accomplished by one-way ANOVA, followed by Duncan's multiple range tests. When comparing dominant with subordinate members of a paired interaction, a paired t-test (t_p) was utilized, and independent t-test (t) for comparison of eyespot color in mirrored interactions.

3. Results

Natural or manipulated social interactions had a substantial influence on behavior, plasma catecholamines, and serotonergic activity (5-HIAA/5-HT) in a context dependent manner. Eyespot manipulation influenced behavioral expression and thus altered social status and the underlying mechanisms that influence behavior such as plasma catecholamines and central serotonergic activity.

3.1. Behavior

In all experiments, males responded aggressively toward their reflected or real opponent. Although isolated (control) males will spontaneously exhibit stereotyped socially aggressive displays (Yang et al. 2001), in these experiments they never did. Therefore, social behavior of experimental males was not statistically compared with that of isolated controls. Social interaction affected the number of aggressive behaviors per minute by male A. carolinensis (Figure 1), significant across a two-way design (F_5,54 = 4.4, P < 0.002). There was a significant effect of social rank (F_1,54 = 20.5, P < 0.0001), but no effect of treatment (natural pairs versus painted pairs or mirror; F_2,54 = 0.55, P > 0.58), nor interaction between treatment and rank (F_2,54 = 0.07, P > 0.93). That is, changes in aggressive behavior (including displays, head nods, bites, jaw gapes and approaches) per minute were dependent on social rank or eyespot color alone. There were no significant differences among subordinate or unaggressive males (one way: F_2,25 = 0.60, P > 0.56), nor were there among dominant aggressive males (one way: F_2,24 = 0.22, P > 0.81) separated by whether they were in natural pairs, painted pairs, or interacting with a mirror. However, there was significantly more aggressive behavior expressed by dominant compared to subordinate males (t_p = 6.46, P < 0.0001, Figure 1 see ‡) in natural pairs, and by males that became dominant after their eyespots were artificially darkened (t_p = 2.92, P < 0.02, Figure 2A see ‡) compared to subordinate males (with eyespots hidden). Aggression was also significantly (t = 2.33, P < 0.031, Fig. 2A see ‡) elevated for males that interacted with their own mirror image when their eyespots were hidden compared to those with darkened eyespots (Korzan, Summers, Ronan, et al. 2000; Korzan, Summers, and Summers 2002). In addition, all males with eyespots painted black became socially dominant when faced with an opponent with eyespots hidden by green paint (Korzan, Summers, and Summers 2002).

3.2. Plasma catecholamines

Aggressive interactions affected plasma catecholamines (Figure 2B). The effect was significant across a two-way design (excluding controls; F_5,40 = 3.0, P < 0.023), and showed an interaction effect between social rank and eyespot manipulation (F_2,40 = 6.9, P < 0.003). That is, changes in NE concentrations were dependent on the level of aggression, eyespot production (or presence) and perception of the eyespot. Across subordinate or unaggressive males, there was a significant (one way: F_3,37 = 5.82, P < 0.003) rise in plasma norepinephrine compared with isolated controls only among paired males (Figure 2B see *), and not in those viewing a black eyespot on themselves in a mirror. Subordinate paired males also secreted more plasma NE than less aggressive males interacting with a mirror, when the eyespots of these groups of males were painted green (Figure 2B, see §). Across dominant aggressive males, plasma NE concentrations were not elevated in males painted with black eyespots aggressively interacting with a male whose eyespots were hidden. However, in natural dominant males there was a significant (one way: F_3,37 = 15.74, P < 0.0001) rise in plasma NE compared to controls (Figure 2B see *) and dominant males painted black (Figure 2 see ♦), exceeded by that of males acting aggressively against a mirror of themselves without (hidden) eyespots (Figure 2B see †). When controlled experiments are considered separately, plasma concentrations of NE were elevated significantly in one or both of the animals by all three treatment regimes. However, there was no significant (t_p = 0.66, P > 0.53) difference between males that became dominant or subordinate when the animals were paired without manipulation, both rose significantly. By contrast, plasma NE concentrations only rose significantly (t_p = 2.6, P < 0.04, Fig. 2B see ‡) in the subordinate less aggressive male when eyespots were manipulated to produce a winner with blackened eyespots (Korzan, Summers, and Summers 2002). A third configuration of NE secretion was evident for interactions with mirror images, in which NE was also significantly (t = 2.68, P < 0.023, Figure 2B see ‡) elevated when males interacted with an image of an opponent without eyespots present as compared to males viewing themselves with darkened eyespots (Korzan, Summers, Ronan, et al. 2000).

3.3. Hippocampal serotonergic activity

In the hippocampus, the pattern of serotonergic activity (estimated by 5-HIAA/5-HT ratio) stimulated by aggressive interactions was similar to that seen in other limbic structures (Summers and Winberg 2006). Hippocampal serotonergic activity was significantly stimulated by aggression and eyespots (Figure 2C) across a two-way design (excluding controls; F_5,49 = 3.65, P < 0.008), and showed an interaction effect between social rank and eyespot manipulation (F_2,49 = 6.49, P < 0.003). That is, like NE, changes in hippocampal 5-HIAA/5-HT levels were dependent on the level of aggression, eyespot production (or presence) and perception of the eyespot. Across subordinate or unaggressive males, there was a significant (one way: F_3,42 = 16.4, P < 0.0001) rise in hippocampal serotonergic activity among paired males compared with isolated controls (Figure 2C, see *), but not in those viewing a mirror image of themselves with black eyespots. Paired subordinates also expressed greater hippocampal 5-HIAA/5-HT levels than less aggressive males interacting with a mirror (Figure 2C, see §). Across dominant aggressive males, hippocampal 5-HIAA/5-HT levels were not elevated in males painted with black eyespots aggressively interacting with a male whose eyespots were hidden. However, in natural (unmanipulated) dominant males there was a significant (one way: F_3,44 = 7.0, P < 0.001, see *) rise in hippocampal serotonergic activity compared to controls, similar to that of males acting aggressively against a mirror of themselves without eyespots (hidden), which was also significantly greater than the painted dominant male of a pair (Figure 2C, see ♦). When controlled experiments are considered separately, hippocampal serotonergic activity was significantly elevated in one or both of the animals by all three treatment regimes. However, there was no significant (t_p = 0.53, P > 0.62) difference in 5-HIAA/5-HT between males that became dominant or subordinate when the animals were paired without manipulation, hippocampal serotonergic activity rose significantly both dominant and subordinate males in natural pairs. Hippocampal serotonergic activity did rise significantly (t_p = 2.3, P < 0.05) in painted- subordinate with less aggressive males (Figure 2C, see ‡), compared to the winner with blackened eyespots (Korzan, Summers, and Summers 2002). Also in interactions with mirror images, hippocampal serotonergic activity was also significantly (t = 2.64, P < 0.017; also see ‡) elevated when males interacted with an image of an opponent without eyespots present as compared with aggressive males with blackened eyespots (Korzan, Summers, Ronan, et al. 2000).

4. Discussion

Each of the paradigms: 1. Natural Dyad, 2. Eyespot Painted Dyad, and 3. Eyespot Painted Mirror Interaction, produced significant aggressiveness (Figure 2A). Not surprisingly, lizards which became dominant produced more and quicker aggression, but they also exhibited faster eyespot darkening than the subordinate male. When black eyespots were artificially added to naïve males, presence of dark eyespots reduced aggression in opponents whose eyespots were hidden by green paint. In mirror images, black eyespots reduced the level of aggression in the male wearing the artificial sign stimuli. Animals viewing individuals with artificially hidden eyespots continued to be aggressive at a high rate (Figure 2A), whether they were viewing an opponent or themselves. The neurochemical and endocrine responses, however, were not necessarily similar to the results for aggressive behavior. In natural pairs following ten minutes of aggressive interaction plasma NE (Figure 2B) and hippocampal serotonergic activity (Figure 2C) were equally elevated in dominant and subordinate individuals compared to controls. However, for pairs with artificially manipulated eyespots, only the males who became subordinate (due to eyespots being hidden by green paint) showed evidence of elevated plasma NE or hippocampal 5-HIAA/5-HT compared to controls; which was also higher than that of dominant (black painted) individuals (Figure 2B). In interactions with mirror images, those males expressing low levels of aggression, because they viewed themselves with darkened eyespots, did not stimulate elevated plasma NE or hippocampal serotonergic activity. Those viewing hidden eyespots, who also were more aggressive toward their own image, did have significantly greater plasma NE and hippocampal 5-HIAA/5-HT levels. The conundrum is that no one behavioral attribute produced elevated neuroendocrine responses. The lizard A. carolinensis is an extremely stress sensitive species, relative to both social and environmental conditions (Summers 1988; Summers, Suedkamp, and Grant 1995; Summers and Norman 1988; Summers 2001; Summers and Winberg 2006). To understand the neuroendocrine effects, we must comprehend the synergistic effects of the stressful stimuli encountered during aggressive interactions.

Naïve size matched male Anolis carolinensis lizards react very aggressively toward one another when introduced during the mating season. Female A. carolinensis also display competitive aggression toward other females, albeit for different objectives and outcomes (Andrews and Summers 1996; Summers, Suedkamp, and Grant 1995; Summers, Hunter, and Summers 1997). The level of aggression in males is initially indistinguishable between two equally matched lizards, but quickly becomes resolved as a dominant-subordinate relationship becomes established. By four or five minutes the subordinate male is significantly less aggressive (Summers 2002), and plasma corticosterone concentrations peak (Summers, Watt, et al. 2005). Early during that time, the eyespots of the dominant male have darkened first (Summers 2001, 2002; Larson and Summers 2001; Summers and Greenberg 1994; Plavicki, Yang, and Wilczynski 2004). Temperature and humidity are critical (Licht 1971; Summers and Norman 1988); as a reduction of 4-6°C of ambient temperature can create an ten-fold increase in the latency to eyespot darkening (Wilczynski et al. 2015; Korzan, Overli, and Summers 2006). Also, later during that period, subordinate males form eyespots (Summers and Greenberg 1994) and subsequently their entire body changes color from green to brown (Korzan, Summers, and Summers 2002; Wilczynski et al. 2015). Similarly, when the eyespots are painted black, the combative male viewing darkened eyespots on his opponent becomes less aggressive, whether the opponent is live or a mirrored reflection (Korzan, Summers, Ronan, et al. 2000; Korzan, Summers, and Summers 2002). Therefore, darkened eyespots inhibit aggression among rivals whether they occur naturally via heightened plasma catecholamines or artificially from black paint. In addition, darkened eyespots are equally effective for inhibiting aggression from a live opponent or when they view themselves in the mirror. However, individuals of A. carolinensis are capable of recognizing each other, and adjusting aggressive behavior to suit the social status of a remembered opponent (Forster et al. 2005). Finally, social experience is critically important, such that memory of the opponent is more potent than any effect of the eyespots (Korzan et al. 2007; Korzan and Summers 2007). Therefore, it is important to remember that the experiments presented herein, were carefully controlled and individual lizards were never used in more than one social interaction.

When the territory of male cichlid fish species (Neolamprologus pulcher, Lamprologus callipterus, Tropheus moorii, Pseudosimochromis curvifrons and Oreochromis mossambicus) is entered by conspecific intrusion there is a significant increase in the primary androgen 11-ketotestosterone (11-KT) (Hirschenhauser et al. 2004). What is more, in one of these species, O. mossambicus, simply watching territorial combat is enough to elevate both 11-KT and testosterone (T) (Oliveira et al. 2001). While O. mossambicus will avidly attack a mirror image, and while aggression mounts over time, no increases in 11-KT or T are ever generated (Oliveira, Carneiro, and Canario 2005). Oliveira and his colleagues concluded that information from the opponent of the contest is required for a hormonal response to be triggered in this type of aggressive encounter.

In A. carolinensis, plasma T has also been reported to rise quickly and dramatically (Greenberg and Crews 1990), as do the stress hormones corticosterone (Summers, Watt, et al. 2005) and the catecholamines in individuals engaging in aggressive activity (Korzan, Summers, and Summers 2002). Hormonal or neural arginine vasotocin, also impacts social behavior in A. carolinensis (Campos, Rojas, and Wilczynski 2020). The results presented herein, suggest that combinations of activity and visual stressors: 1. being aggressive, 2. viewing aggression in an opponent, and 3. viewing darkened eyespots on an opponent, provoked differentially in each paradigm (Figures 1 and 2) elevate plasma catecholamines, while 4. viewing brown skin on an opponent limits catecholamine secretion. During paired interactions between unpainted anoles, norepinephrine (NE; the primary plasma catecholamine in this lizard) increases in both dominant (via stressors 1 and 2) and subordinate males (via stressors 2 and 3; Figure 2B). When eyespot color is manipulated in a pair, those with dark eyespots (viewing opponents with hidden eyespots), become very aggressive, but exhibit no change in plasma NE (limited by 4, and a lack of 2 and 3; Figure 2B). Less aggressive males, with hidden eyespots, but viewing an aggressive opponent with dark eyespots have elevated plasma NE (reacting to stressors 2 and 3; Figure 2B), as well as elevated plasma epinephrine and dopamine (Korzan, Summers, and Summers 2002). In contrast, aggression and a lack of eyespot color (painted green) promoted elevated NE in males interacting with a mirror image (stimulated by stressors 1 and 2; Figure 2B); plasma DA and Epi were also elevated (Korzan, Summers, Ronan, et al. 2000). Less aggressive males viewing a mirror image opponent with dark eyespots exhibited no change in plasma catecholamines (due to a lack of stressors 1 and 2 mitigating the effect of stressor 3).

Limbic serotonergic activity increases in response to aggressive social interaction, and this neurochemical response changes over time, with more rapid increase and return to baseline in dominant individuals (Summers and Greenberg 1995; Summers et al. 1998; Summers, Summers, et al. 2003; Summers, Korzan, et al. 2005). Subordinate response are slower; both to initiate and terminate. While social status alone does not provoke large changes in serotonergic activity, small differences in tonic anterior hypothalamic 5-HIAA/5-HT activity in the does make a difference in terms of later aggressiveness (Summers, Korzan, et al. 2005). Like plasma catecholamines and corticosterone, after ten minutes of social aggression, hippocampal serotonergic activity is equally elevated in dominant (via stressors 1 and 2) and subordinate (via stressors 2 and 3) males with naturally forming eyespots (Figure 2C). However, like plasma catecholamines, hippocampal serotonergic activity will distinguish social rank when eyespots are artificially manipulated. Males that become dominant due to artificially blackened eyespots have unchanged hippocampal 5-HIAA/5-HT, but the subordinate males that oppose them have elevated serotonergic activity. When a mirror is substituted for a live opponent, hippocampal serotonergic activity is elevated only in males fighting an opponent with no eyespots (Korzan, Summers, and Summers 2000). Similar changes are measured in other limbic regions, such as amygdala, subiculum, and nucleus accumbens, but the opposite pattern is measured from the 5-HT producing cells of the raphe in the midbrain, suggesting negative feedback there (Korzan, Summers, and Summers 2000; Korzan et al. 2001; Summers, Summers, et al. 2003).

Therefore, central serotonergic activity early during aggressive interactions does not inhibit aggression (Summers, Korzan, et al. 2005). An inverse relationship between serotonin and aggression is widely considered to be the mechanism regulating aggression in humans and animals (Coccaro 1992; Nelson and Chiavegatto 2001). Our results, and similar data from experiments in fish (Overli, Harris, and Winberg 1999), suggest that acutely increased limbic serotonin does not negatively regulate aggression while it occurs (Summers, Korzan, et al. 2005), but rather the opposite – that heightened aggressions cumulatively enhance serotonergic activity (Summers, Forster, et al. 2005). We suggest that rapidly increased serotonergic activity (Matter, Ronan, and Summers 1998; Summers, Summers, et al. 2003; Summers, Matter, et al. 2003) reflects regulation of central circuitry that modulates behavioral and neuroendocrine stress responsiveness (Summers 2001; Summers, Korzan, et al. 2005; Summers and Winberg 2006). However, longer term serotonergic activity, such as has been demonstrated for serotonergic reuptake inhibitors (Larson and Summers 2001; Deckel 1996; Deckel and Jevitts 1997; Kavoussi, Liu, and Coccaro 1994), probably does influence aggressive behavior, perhaps as a part of stress management (Summers, Korzan, et al. 2005). The level of serotonergic activity in aggression-related circuitry prior to aggressive interaction, does appear to influence the propensity to act aggressively (Summers, Korzan, et al. 2005).

These classic behavioral tests have important differences. Perhaps the most obvious, is that during social interaction with a mirror image, no social rank or role can be established (Oliveira, Carneiro, and Canario 2005), and artificial eyespots can stimulate continuous intense (green/hidden) or limited (black) levels of self-directed aggression. Similarly, animals with synthetically manipulated eyespot-sign stimuli, may artificially inaugurate social roles and prematurely establish social rank. The responses of both sympathetic-adrenal and limbic systems suggest that aggressive social interaction is a stressful event for both dominant and subordinate males; each showing an approximately equivalent response of endocrine NE concentration (Figure 2B) and hippocampal serotonergic activity (Figure 2C) after ten minutes of combative engagement in un-manipulated pairs. However, the responses of dominant males are widely divergent from those of subordinate males at ten minutes, simply by artificially applying or hiding the sign stimulus, eyespots, with black or green paint. What is more intriguing, is that the endocrine noradrenergic and hippocampal serotonergic responses are reversed for painted dominant/aggressive males or subordinate/less aggressive males when they are facing each other compared with when they face a mirror image. That is, subordinate males (green paint hiding eyespots) have significantly higher plasma NE concentrations and hippocampal serotonergic activity than the aggressive dominant males (black paint making eyespots) they face. However, when the most aggressive males face a mirror image of themselves with eyespots hidden by green paint, plasma NE concentrations and serotonergic activity are elevated compared to less aggressive males viewing themselves with blackened eyespots. Three things account for these differences: First, when naïve pairs of males interact, those with artificially darkened eyespots become dominant (Korzan, Summers, and Summers 2002), and the interaction is foreshortened. Second, when an animal faces a mirror image, male lizards facing an image with dark eyespots give up quickly, but those facing an image of an opponent who looks like a subordinate male with no eyespots will continue aggressive behavior throughout the experiment. The lack of eyespot formation in their perceived opponent makes them expect their opponent to capitulate, but when their mirror image does not, they continue aggressive behaviors. Third, male lizards facing natural opponents, painted opponents, or mirrors, confront a separate assortment of socially stressful signals.

Male A. carolinensis lizards create or confront four social signals that act to promote or alleviate neuroendocrine stress responses. First, these animals can 1. be aggressive. An aggressive posture stimulates neural and endocrine stress responsiveness (Miczek et al. 2002; Summers, Watt, et al. 2005; Summers, Forster, et al. 2005). Second, 2. viewing aggression is equally stressful (Summers, Korzan, et al. 2005). Third, 3. viewing an opponent with blackened eyespots, which occur most rapidly on, and signal the presence of, the most aggressive dominant males, is also socially stressful (Summers, Forster, et al. 2005). Creating or confronting any one of these social signals produces elevated plasma NE concentrations and limbic serotonergic activity. Fourth, the lizard can signal social submission and subordinate status by 4. darkening the skin of its entire body from green to brown. Numerous species have visible melanin-related pigment changes that are associated with stress (Khan et al. 2016), including A. carolinensis (Greenberg, Chen, and Crews 1984; Hemer, Salas, and LaPointe 1981; Vaughan and Greenberg 1987). This darker body color appears to ameliorate the other socially stressful signals, reducing plasma NE and hippocampal serotonergic activity. In the absence of an opponent with dark skin, a combination of being aggressive, viewing an aggressive opponent, or viewing an opponent with dark eyespot maximizes the neuroendocrine stress responses (Summers, Forster, et al. 2005). Two stressors are more potent than one, and maximize the response; whereas dark skin signals the end of hostilities (Ligon 2014), and perhaps also the end to the need for neuroendocrine stress activity.

5. Conclusions

Any given behavioral paradigm is likely to distort the interpretation of experimental results, even when the experimental paradigm is essentially naturalistic. In natural settings when unusual environmental or social conditions dominate, even field results can bias the interpretation of data. We suggest that to understand the motivations, contexts and mechanisms that produce and shape behavior, caution should be used when deploying virtual interactions or artificial sign stimulus manipulations. Ideally, of course, multiple experimental approaches should be used, particularly including those that enhance distinctions and differences but remembering that one cannot be substituted for another, especially when considering neural and endocrine outcomes.

In our experiments, data from un-manipulated pairs made it clear that socially aggressive interaction was stressful for both dominant and subordinate males (Overli et al. 2004; Summers, Summers, et al. 2003; Summers, Watt, et al. 2005; Summers and Winberg 2006), but not which attributes of the interaction made it stressful for each. In Anolis carolinensis, it appears that being aggressive, viewing aggression, or viewing dark eyespots on an opponent are all components of the dynamic that produces neuroendocrine and behavioral stress responses. It appears that the stimuli are synergistic, such that one stimulus is not sufficient, but two are (Summers, Forster, et al. 2005). This idea is reminiscent of that proposed by Tinbergen such that two visual sign stimuli synergistically promote social release of behavior (Tinbergen 1948).

Therefore, we conclude that naturalistic experiments, both in the field and in the lab, should be compared to establish a baseline for the influence of social or environmental stimuli on behavior and neuroendocrine activity. However, these data may not distinguish relevant social or environmental factors, such as the mechanisms underlying social rank. Interpreting the data from unmanipulated pairs alone, might lead to the conclusion that stress hormones and transmitters have no influence on dominant-subordinate relationships. Similarly, modifying the context of social interaction by manipulating the sign stimuli available (such as painting over eyespots) or adjusting the perception of social response, by using mirrors or video displays, should always be compared to more naturalistic interactions, and then interpreted cautiously, with a view to what parameter has been limited in the design. For example, in the experiments with mirrors, no social rank is ever established, yielding very high NE levels and hippocampal 5-HIAA/5-HT activity in animals that viewed their opponent (themselves) with a green body and hidden eyespots that never seemed to acquiesce, and therefore the combat continued unabated. On the other hand, by manipulating the social sign stimulus (painting the eyespots) prior to paired social interaction, although the behavioral results were very similar to those of naturalistic pairs, the neuroendocrine results revealed that subordinate animals (even though this status was artificially induced by hiding the eyespots) were more stressed than dominant animals, even from the very beginning. This is an important result, because dominant animals in natural pairs are clearly stressed (Figure 2B and C), but the neuroendocrine machinery underlying that stress response appears to be different from that expressed by subordinate males. Manipulated social interactions have the potential for differentiating mechanisms underlying behavioral responses that are distinctly based on social or environmental conditions.

Highlights.

• Social stress behavior is similar in mirror, dyad, and altered sign stimulus trials

• Mirror, dyad, and altered sign stimulus trials change the stress type experienced

• Altered stress types change the social experience and neuroendocrine responses

• Altered stress types increase or decrease plasma norepinephrine concentrations

• Altered stress types increase or decrease hippocampal serotonergic activity