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Aggressive experience alters place preference in green anole lizards, Anolis carolinensis
ANIMAL BEHAVIOUR, 2006, 71, 1155–1164 doi:10.1016/j.anbehav.2005.10.006
Aggressive experience alters place preference in green anole lizards, Anolis carolinensis WILL IAM J. F ARRELL & WALTER WILCZYNSKI
Department of Psychology and Institute for Neuroscience, The University of Texas at Austin (Received 29 June 2004; initial acceptance 20 September 2004; final acceptance 10 October 2005; published online 20 March 2006; MS. number: A9924R)
Males of many species establish territories from which they exclude conspecifics, and experience can influence the defence and stability of territorial boundaries. We used a conditioned place preference (CPP) procedure to determine whether engaging in a form of behaviour associated with territorial defence, aggressive behaviour, was rewarding for male green anoles and whether aggressive experience could alter the spatial/contextual preferences of these territorial lizards. When approached by a male conspecific, or when exposed to their reflection in a mirror, many male anoles show species-typical aggressive behaviours. We repeatedly exposed male anoles to a mirror or the nonreflective back of the mirror in opposing sides of a CPP apparatus that consisted of two contextually distinct chambers connected by a central tunnel. Control animals (N ¼ 10) were exposed to the back of the mirror in both sides of the apparatus. Following conditioning, animals that showed aggressive behaviour (N ¼ 13) when exposed to the mirror during conditioning increased the time that they spent in the side of the apparatus paired with the mirror relative to baseline. In contrast, the preferences of nonaggressive (N ¼ 8) and control animals were unchanged. These findings suggest that engaging in aggressive behaviour (and not simply seeing a conspecific) is rewarding for male green anoles and that aggression-related reward and associative learning could influence the formation and/or maintenance of stable territories. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animals of many species and taxa form territories from which they exclude conspecifics. The maintenance of these territories provides occupants with access to resources that are often crucial for survival and reproduction. Some of the resources that are defended include food, mates, nesting sites and physical aspects of the environment required to maintain homeostasis and avoid predators (Emlen & Oring 1977; Waser & Wiley 1979; Grant 1993; Maher & Lott 2000). Field studies indicate that, once established, territorial boundaries often remain quite stable (e.g. Knapton & Krebs 1974; Hirons 1985; Janes 1985; Price et al. 1986; Jenssen et al. 1995). One factor that contributes at least in part to this stability is the experience gained by residents within their territories. Territory residents typically have a competitive advantage over intruders and repel or
Correspondence and present address: W. J. Farrell, Queens College, Psychology Department, 65-30 Kissena Boulevard, Flushing, NY 11367, U.S.A. (email: [email protected]). W. Wilczynski is now at the Department of Psychology, and Center for Behavioral Neuroscience, Georgia State University, P.O. Box 3966, Atlanta, GA 30302-3966, U.S.A. 0003–3472/06/$30.00/0
defeat them (Waser & Wiley 1979; Archer 1988; Bradbury & Vehrencamp 1998). Furthermore, the results of displacement experiments indicate that the magnitude of the resident advantage is often related to the duration of residency (Krebs 1982; Beletsky & Orians 1989; Johnsson & Forser 2002). For example, Beletsky & Orians (1989) removed resident red-winged blackbirds, Agelaius phoenicius, from their territories for 6–7 days. Once released, the majority of residents (73%) were able to reclaim their territories from replacement males that had occupied them for 2 days or less, but the converse was true (only 16% reclaimed territories) when replacements had occupied the territory for at least 6 days. Learning that occurs while occupying an area may have a variety of influences on the formation and maintenance of stable territories. For example, knowledge gained about the topography of a region and the distribution and quality of resources may increase the perceived value of a territory and thereby enhance territorial defence (reviewed in Shutler & Weatherhead 1992; Stamps & Krishnan 1999). Furthermore, site-specific motor learning may render animals more capable of exploiting the physical dimensions of a territory (Stamps 1995). Finally, associations learned while engaging in social interactions such
1155 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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as aggression may also play a role. Organisms can learn associations regarding prior aggressive interactions and these associations can influence subsequent behaviour. For example, studies conducted with fish such as Betta splendens, blue gouramis, Trichogaster tichopterus, and sticklebacks, Gasterosteus aculeatus, indicate that many aspects of species-typical aggressive behaviour can be classically conditioned by pairing a conditioned stimulus (CS) with subsequent visual access to a conspecific (e.g. Thompson & Sturm 1965; Hollis 1984; Hollis et al. 1995; Jenkins & Rowland 1996). Furthermore, this type of training can confer an advantage upon the animals that were trained. When the same CS later predicts actual physical access to a conspecific, conditioned animals are typically more aggressive and win more contests than do fish for whom the contest has not been cued (Hollis 1984; Hollis et al. 1995). The present study focused on the possibility that engaging in aggression might also serve as a rewarding stimulus that, when associated with a particular spatial environment, could increase an animal’s attachment or preference for that environment. Evidence indicates that engaging in aggressive behaviour that ends with the defeat or experimental withdrawal of a conspecific can be rewarding. For example, mice will work or perform operant tasks for the opportunity to engage in aggressive behaviour (Legrand 1970; Connor 1974; Fish et al. 2002). Likewise, male mice (Martinez et al. 1995) and female hamsters (Meisel & Joppa 1994) form conditioned place preferences for contexts that were previously paired with the introduction of a conspecific that they attacked. Analogous findings from studies of operant conditioning (Thompson 1969; Hollis & Overmier 1982) and place preference (Bronstein et al. 1988) have also been reported for fish. The majority of studies examining aggression-related reinforcement have been restricted to laboratory-bred rodents or domesticated strains of birds and fish (but see Bronstein et al. 1988). In the present study, we tested whether engaging in aggressive behaviour might be rewarding for a wild-caught, territorial reptile, the green anole lizard, and whether such reward could alter spatial preferences. Green anoles are arboreal lizards found throughout a large region of the southeastern United States. During the breeding season, male anoles establish territories from which they exclude other males (Jenssen et al. 1995; Jenssen & Nunez 1998). While establishing and maintaining breeding territories, male anoles engage in aggressive interactions with male conspecifics, raising the possibility that reinforcement gained and associations learned during these interactions may contribute to the establishment and maintenance of stable territories. The territorial nature of male green anoles makes them an ideal organism for the study of aggression-related reinforcement/reward. Furthermore, males of this species readily display species-typical aggressive behaviours in the laboratory. Lateral compression of the body along the sagittal axis is the signature aggressive behaviour in this species (Greenberg & Noble 1944; Greenberg & Crews 1983) and is frequently accompanied by pushup displays (repetitive raising and lowering of the front half of the body), and rapid
extensions of the red dewlap beneath the throat. Skin colour can also change during aggressive interactions. A patch of skin behind the eye, the postorbital eyespot, turns black during heightened aggressive interactions and body colour can vary between light green and dark brown. Body colour darkens (range ¼ light green to dark brown) following stressful experiences such as social subordination (Greenberg et al. 1984; Greenberg & Crews 1990; Summers & Greenberg 1994), and blackening of the postorbital eyespot is directly related to elevated plasma catecholamine titres (Kleinholz 1938; Hadley & Goldman 1969; Greenberg & Crews 1983; Summers & Greenberg 1994; Summers 2001). The aggressive behaviours of green anoles are distinct and easy to quantify. Moreover, these behaviours can be elicited by exposing a male anole to his own reflection (e.g. Korzan et al. 2000a; Baxter et al. 2001). To examine whether engaging in aggressive behaviour is rewarding for male green anoles, and to determine whether associations learned during these interactions can alter the spatial preferences of combatants, we developed an apparatus and methodology to examine conditioned place preference (CPP) in this species. CPP procedures have been extensively used with rodents to examine the rewarding properties of drugs of abuse (for review see Bardo & Bevins 2000), and less frequently to determine whether social stimuli such as sexual and aggressive interactions are rewarding (e.g. Oldenburger et al. 1992; Meisel & Joppa 1994; Martinez et al. 1995). The underlying assumption of CPP procedures is that animals will increase the time that they spend in a distinct environmental context (the CS) after having associated this context with a rewarding stimulus (the unconditioned stimulus, US). The terms ‘preference’ and ‘reward’ are operationally defined in the CPP paradigm in reference to the observed behaviour of the organism, and neither term implies a conscious cognitive state. We chose to use a CPP paradigm in part because we thought that place-preference learning could be of particular ethological relevance for a territorial species such as A. carolinensis. We also chose this paradigm because wild-caught anoles show individual differences in aggressiveness, and a CPP paradigm allowed us to examine the responses of both aggressive and nonaggressive animals. The primary goals of the present study were to determine whether engaging in aggression was rewarding for male green anoles and whether they showed an increased preference for a context previously associated with aggressive interactions. However, one difficulty with examining aggression-related reward is the possibility that social exposure to a conspecific alone, rather than engaging in aggression per se, may serve as a rewarding stimulus. We examined both possibilities by quantifying the changes in place preference shown by both aggressive and nonaggressive animals following simulated social interactions. We repeatedly exposed male anoles to a mirror or the back of the mirror (a nonreflective neutral stimulus) in opposing sides of a CPP apparatus. If engaging in aggressive behaviour is rewarding for male anoles, animals that respond aggressively to their reflection during conditioning should prefer the side of the apparatus paired with the
FARRELL & WILCZYNSKI: AGGRESSION AND PREFERENCE
reflective mirror following conditioning, and nonaggressive animals should not show this change in preference. If, however, social contact alone is rewarding for male anoles, then both groups of animals should show a similar change in preference regardless of their behavioural response to the mirror.
Slots for mirrors Doors Tan rear and side walls
Black rear wall
Smooth plastic floor
Textured black floor
METHODS
Subjects Wild-caught male and female green anoles were obtained from Charles Sullivan Inc. (Nashville, Tennessee, U.S.A.) during April, July and September. Subjects were adult male anoles, which ranged in size from 55 to 69 mm (snout-to-vent length) SVL. All subjects were assumed to be sexually mature adults based on the ratings scale of Jenssen et al. (2001), which classifies males with a SVL of 50 mm or greater as adults. Each male anole was housed with a female in a glass terrarium measuring 17.8 30.5 45.7 cm for a minimum of 13 days and for a maximum of 265 days (X SE ¼ 161 12:4 days) before the experiment. The front and back of each terrarium were clear glass and the side walls were constructed of opaque (white) Plexiglas, which visually isolated adjacent male/female pairs. The bottom of each cage was covered by about 2.5 cm of sphagnum peat moss and was outfitted with a plastic water bowl. A single wooden dowel (1.6 cm diameter) placed diagonally between the bottom-front and rear-top corners of the cage was provided as a perch. Animals were housed under conditions designed to simulate the breeding season and promote gonadal activity (Licht 1967, 1971). The animal room was maintained on a 14:10 h light:dark cycle (lights on at 0700 hours) and the ambient temperature varied from 30 C during the day to 23 C at night. Incandescent lights (60 W) positioned above each terrarium served as an additional source of heat, and full-spectrum fluorescent lights (Vitalite, Duro-Test, Philadelphia, Pennsylvania, U.S.A.) provided additional illumination. Half-grown crickets were provided three times a week as food, and water was provided continuously in bowls and by periodic misting of the cage walls. All subjects were observed courting the female with whom they had been paired within 3 days before the beginning of the experiment to ensure that they were socially active.
Apparatus Conditioning and testing were conducted in a CPP apparatus (Fig. 1) that consisted of two side chambers (8.0 15.5 13.0 cm) joined by a central tunnel (11.5 8.5 8.0 cm). The dimensions of the side chambers were chosen to provide the animals enough room to display aggressively while ensuring that they saw the mirror inserted into these chambers during the appropriate conditioning sessions (see below). The central tunnel was deliberately designed to be small to facilitate alternations between the sides of the apparatus when
Orange ceiling
Tunnel (blue)
Blue ceiling
Figure 1. Schematic representation of the conditioned placepreference apparatus.
appropriate. Both pilot observations and observations conducted during the experiment indicated that male anoles were capable of turning around in the central tunnel and did so frequently. The two side chambers of the apparatus and the central tunnel were designed to be contextually distinct both from each other and from any habitat that the subjects had previously experienced. The left chamber had a clear plastic front wall, beige rear and side walls, an orange ceiling and a smooth plastic floor. The right chamber had clear front and rear walls, a black side wall, a light blue ceiling and a textured black floor (plastic needlepoint backing). A narrow slot was cut into the ceiling of both side chambers, allowing a mirror (12.7 10.2 cm) to be lowered into the chamber along the wall directly opposite to the opening of the central tunnel. The floor and walls of the central tunnel were dark blue plastic and the ceiling was clear Plexiglas. Guillotine doors were at the left and right ends of the central tunnel and could be raised or lowered as necessary to allow subjects access to the various segments of the apparatus. The right door was black and the left door was white. Conditioning and testing sessions were videotaped from the front of the apparatus. A camcorder (RCA, CC437) served as a camera and was connected to a time-lapse VCR (Gyyr, TLC1824). Place-preference tests were recorded at 1/6 normal tape speed, allowing videotapes to be coded rapidly (6 normal speed) when reviewed using a standard VHS player. Conditioning sessions were recorded at standard tape speed to ensure the accurate quantification of rapidly occurring aggressive behaviours.
Procedure CPP conditioning and testing were conducted over 8 days in a room maintained at 30 C. The procedure was divided into baseline, conditioning and testing phases.
Baseline (day1) On the first day of the experiment, animals were tested for initial (baseline) spatial preferences in the CPP apparatus. At the beginning of the baseline session, a single male lizard was removed from his home cage and confined to the central tunnel of the CPP apparatus for
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5 min. All anoles were placed into the central tunnel head first with their nose facing the rear of the apparatus. However, animals frequently turned around several times during the 5-min period in the central tunnel, rendering it impossible to control the direction of orientation during this period. Following the 5-min period in the central tunnel, both guillotine doors were raised simultaneously, allowing the subject free access to all portions of the apparatus for 2 h. Videotaped recordings obtained during the baseline session were then reviewed and the time spent in the left and right side of the apparatus was quantified. Subjects were deemed to have entered one side of the apparatus when their entire head had exited the central tunnel and to have exited that side when their entire body (excluding the tail) was once again within the central tunnel. The side in which an individual animal spent the most time during the baseline session was designated as that subject’s preferred side of the apparatus and the opposing side was designated as the less preferred side. Animals that did not enter all areas of the apparatus were excluded from the experiment (N ¼ 6).
Conditioning (days 2–7) Conditioning commenced 1 day after the baseline preference test and continued for a total of 6 days (days 2–7). Animals underwent six 45-min conditioning sessions, one per day. During three sessions, subjects were confined to their less preferred side of the apparatus and were exposed to a mirror. During the remaining sessions, on alternate days, animals were confined to their preferred side of the apparatus and were exposed to the nonreflective back of the mirror. We paired the nonreflective back of the mirror with the side of the apparatus that was preferred during the baseline to hold the physical act of inserting a piece of glass into the side chambers constant across the previously preferred and less preferred chambers. The nonreflective back of the mirror does not elicit aggressive displays from male green anoles. We used a 6-day-long conditioning regimen (with three mirror exposures) because the results of previous studies indicated that aggressive behaviour shown during daily visual exposure to conspecifics (videotaped or real) increases over the first 3 days and subsequently declines (Yang et al. 2001; Yang & Wilczynski 2002). Control animals underwent similar procedures but were exposed to the back of the mirror in both sides of the apparatus. The control group was included to ensure that repeated exposure to the various sides of the apparatus did not systematically alter preferences. Each conditioning session began with a 5-min habituation period before the mirror was inserted. Following this habituation period, an experimenter lowered the mirror (or the back of the mirror) into the apparatus through the slot in the roof of the chamber. Behaviour was videotaped during the next 10 min and the mirror was then removed. Subjects remained in the apparatus for 30 min following removal of the mirror (or the back of the mirror) and were then returned to their home cage. We exposed 21 animals to the mirror during conditioning and used 10 additional animals in the control group. Conditioning began
in the preferred side of the apparatus for five control animals and 10 animals exposed to the mirror.
Testing (day 8) One day after the final conditioning session, subjects were retested for place preference using the baseline testing procedure.
Behavioural Quantification and Data Analysis Video recordings and/or real-time observations obtained during conditioning sessions (mirror only) were used to determine the latency required to perform the lateral compression (LC) display and show a darkened eyespot. Animals that never showed either LC or a darkened eyespot were assigned latencies equivalent to the duration of an individual mirror exposure (600 s). Pilot observations and real-time observations during conditioning trials indicated that subjects did not display aggressive behaviour when exposed to the nonreflective back of the mirror. Therefore, behaviour during these trials was not quantified. Animals were classified as aggressive if they became laterally compressed during any of the conditioning sessions in which they were exposed to the mirror and were classified as nonaggressive if they never displayed this response. Video recordings obtained during conditioning sessions were subsequently used to quantify the frequency of pushups (PU) and dewlap extensions (DE) displayed during mirror exposure. LC was used to classify animals as aggressive, rather than PU or DE, because both PU and DE can occur in the absence of aggression (e.g. spontaneous advertisement or during courtship) and are also major components of aggressive display. We assessed changes in place preference by comparing the amount of time spent in the less preferred side of the apparatus (paired with the mirror during conditioning) during the baseline and postconditioning test sessions. One-way ANOVA with Tukey’s HSD test was used for between-subjects comparisons of mean preference change (test minus baseline). Paired t tests and repeated measures ANOVA were used to examine within-subject differences. Two correlations were also performed to examine the relationships between the magnitude of individual animals’ preference changes and either the number of pushups or the mean latency to become laterally compressed during conditioning. Results are expressed as mean SE. The results of statistical tests were considered significant if P < 0.05 (two-tailed). RESULTS
Response to Mirror Of the 21 animals exposed to the mirror during conditioning, 13 displayed LC and were classified as aggressive. The remaining eight animals never became compressed and were classified as nonaggressive. The results of two between-subjects ANOVAs indicated that mean SVL (F2,28 ¼ 1.32, P ¼ 0.28) and number of days in captivity (F2,28 ¼ 1.11, P ¼ 0.35) did not differ
FARRELL & WILCZYNSKI: AGGRESSION AND PREFERENCE
significantly between the 13 aggressive animals, eight nonaggressive animals and the 10 control animals. Ten of the 13 animals classified as aggressive showed LC during all three of the conditioning sessions in which they were exposed to the mirror. The remaining three aggressive animals displayed LC during trial 1 (N ¼ 2) or 2 (N ¼ 1). The mean latency required for aggressive animals to show LC was 179.0 32.4 s and did not change significantly across the three conditioning trials in which animals were exposed to the mirror (repeated measures ANOVA: F2,24 ¼ 0.34, P ¼ 0.715). Aggressive animals also showed darkening of their postorbital eyespot during 82.1% of the conditioning trials in which they saw the mirror. Eyespot darkening occurred with an average latency of 231.1 34.0 s and did not change significantly across the three exposures to the mirror (repeated measures ANOVA: F2,24 ¼ 1.28, P ¼ 0.298). Nonaggressive animals never displayed a darkened postorbital eyespot when exposed to the mirror. Aggressive animals also performed DE and PU. DEs were most common at the onset of aggressive interactions, while PUs often continued throughout. The mean number of DEs and PUs displayed by aggressive animals during each 10-min mirror exposure were 2.9 0.6 and 113.7 16.0, respectively. The mean frequency with which aggressive animals displayed DE and PU did not differ significantly across the three conditioning trials in which they were exposed to the mirror (repeated measures ANOVA: DE: F2,24 ¼ 3.15, P ¼ 0.06; PU: F2,24 ¼ 1.94, P ¼ 0.17) but the frequency of DE tended to decline progressively across trials. While nonaggressive animals never showed LC or darkening of the postorbital eyespot when exposed to the mirror, they did occasionally (16.7% of trials) perform DEs and/or PUs (X SE ¼ 0:5 0:3 DEs and 8.1 6.9 PUs per trial). These behaviours were only performed by 50% of the nonaggressive animals and were never expressed during more than one trial per animal.
Place Preference During the baseline test for spatial preferences, animals (13 aggressive, 8 nonaggressive and 10 control animals) spent an average of 3787.1 258.8 s and 2895.2 246.4 s in the right and left sides of the apparatus, respectively. There was no significant difference between baseline preference for the left or right side (paired t test: t30 ¼ 1.85, P ¼ 0.07). Individual animals did show significant baseline preferences for one side of the apparatus or the other. Animals spent a mean SE of 4501.9 175.8 s in their preferred side of the apparatus and 2180.4 142.2 s in their less preferred side (paired t test: t30 ¼ 8.21, P < 0.001). Table 1 summarizes the time that subjects spent in their preferred and less preferred sides of the apparatus during the baseline and postconditioning preference tests. Table 2 summarizes the number of alternations between right and left sides of the apparatus during both baseline and postconditioning tests. Preference changes following conditioning indicated that mirror exposure alone (without taking into account animals’ behavioural responses to the mirror) was
insufficient to elicit an enhanced preference for the side of the CPP apparatus paired with the mirror during conditioning. Animals exposed to the mirror during conditioning (13 aggressive and 8 nonaggressive animals) increased the time that they spent in the less preferred side of the apparatus (paired with mirror) by an average of 1246.0 468.4 s relative to baseline, while control animals showed a concurrent decrease (X SE ¼ 194:7 688:6 s), but these values did not differ significantly (ANOVA: F1,29 ¼ 3.02, P ¼ 0.09). However, there was a relationship between engaging in aggressive behaviour during conditioning and an enhanced preference for the side of the apparatus paired with the mirror (i.e. postconditioning minus baseline). For the 21 anoles exposed to the mirror (N ¼ 13 aggressive and 8 nonaggressive animals), there was a significant positive relationship between the number of PUs during conditioning and preference change (Pearson correlation: r19 ¼ 0.52, N ¼ 21, P < 0.02) and a significant negative relationship between mean latency to display LC and preference change (r19 ¼ 0.50, N ¼ 21, P ¼ 0.02; Fig. 2). Mean preference changes for the less preferred (paired with mirror) side of the apparatus differed by group (aggressive, nonaggressive, control: ANOVA: F2,28 ¼ 5.71, P < 0.01; Fig. 3a). Post hoc analyses indicated that aggressive animals showed a significantly larger preference change than did both nonaggressive (Tukey’s HSD test: P < 0.03) and control animals (Tukey’s HSD: P < 0.02). The changes in preference by nonaggressive and control animals were not significantly different (Tukey’s HSD test: P ¼ 0.99). The mean time that aggressive animals spent in the side of the apparatus paired with the mirror also increased significantly relative to their own baselines (paired t test: t12 ¼ 4.19, P < 0.01; Fig. 3b). In contrast, the preferences of nonaggressive (paired t test: t7 ¼ 0.41, P ¼ 0.69) and control animals (paired t test: t9 ¼ 0.28, P ¼ 0.78) did not change. Of the animals classified as aggressive, 84.6% increased the time that they spent in the side of the apparatus paired with the mirror following conditioning. In contrast, only 37.5% and 30.0% of nonaggressive and control animals showed increases.
DISCUSSION The results suggest that engaging in an aggressive interaction can be rewarding for male green anoles and that aggressive experience can result in an enhanced preference for the site of previous aggression in this species. Animals that responded aggressively towards their reflection during conditioning subsequently increased the time they spent in the side of the apparatus paired with the mirror. In contrast, control animals that were never exposed to the mirror and nonaggressive animals that never displayed aggressive responses failed to show similar increases. With respect to aggression-related reward/reinforcement, our findings are consistent with those previously reported for mammals (Azrin et al. 1965; Tellegen et al. 1969; Legrand 1970; Connor 1974; Meisel & Joppa 1994; Martinez et al. 1995; Fish et al. 2002), birds (Cherek
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Table 1. Time distribution during baseline and postconditioning place-preference tests Preferred side
Mirror (N¼21) Aggressive (N¼13) Nonaggressive (N¼8) Control (N¼10)
Less preferred side
Baseline
Postconditioning
Baseline
Postconditioning
4453.2234.6 4243.2327.4 4794.4295.7 4604.2246.5
3464.4500.6 2575.9585.3 4908.3666.2 4702.8702.4
2123.1181.9 2226.5241.0 1955.1282.6 2300.7228.3
3369.1517.2 4395.6593.3 1701.0619.5 2106.0689.7
Values are mean SE times (s). The mirror group comprised both the aggressive and nonaggressive animals.
Table 2. Number of alternations between the left and right sides of the conditioned place-preference apparatus during baseline and postconditioning place-preference tests Group Mirror (N¼21) Aggressive (N¼13) Nonaggressive (N¼8) Control (N¼10)
Baseline 5.290.87 5.081.21 5.631.27 4.201.19
Postconditioning 5.521.45 4.541.26 7.133.29 3.401.14
Values are mean SE numbers. The mirror group comprised both the aggressive and nonaggressive animals.
It is unclear why some male anoles responded aggressively towards their reflection while others did not. It is unlikely that the nonaggressive animals failed to see their reflection. The size of the mirror used and its position 6000 (a) 5000 Change in preference (s)
et al. 1973) and fish (Thompson 1969; Hollis & Overmier 1982; Bronstein et al. 1988). To our knowledge, the present results are the first demonstration of aggressionrelated reward in a reptile. Interestingly, the place preferences of aggressive anoles changed rapidly (three trials with mirror), suggesting that territorial males of this species may be particularly adept at forming aggressionrelated contextual associations and supporting the notion that aggression-related reward and associative learning might contribute to the formation and/or maintenance of stable territories. However, the CPP apparatus and procedures that we used in the present study did not mimic the natural conditions under which green anoles form territories. Additional research under more natural conditions will be required to examine further the contribution of aggression-related reward and associative learning to territory formation. The data obtained from nonaggressive animals are of particular interest because they suggest that engaging in aggressive behaviour was a prerequisite for enhanced place preference in our paradigm. Kelsey & Cassidy (1976) found that isolated male mice preferentially entered the arm of a T-maze where they had previously fought and that the formation of this preference could be blocked by allowing social interaction (through a wire mesh) with a conspecific in the opposing arm of the maze. These findings suggest that, at least under some circumstances, reward gained during aggressive interactions may be derived from social contact rather than engaging in aggression. It is unlikely, however, that reward obtained simply from any form of social interaction can explain our results. The preferences of nonaggressive anoles were unchanged by the conditioning procedure, even though these animals were exposed to their reflection for the same amount of time as the aggressive animals. Only animals that reacted aggressively to the mirror developed a CPP.
4000 3000 2000 1000 0 –1000
r = +0.52
–2000 –3000
P = 0.017 0
200
400
600
800
1000
Number of pushups 6000 (b)
5000 Change in preference (s)
1160
4000 3000 2000 1000 0 –1000 r = –0.50
–2000 –3000
P = 0.020 0
200
400
600
Mean lateral compression latency (s) Figure 2. Relationships between change in preference (postconditioning minus baseline) for the side of the conditioned placepreference apparatus paired with the mirror during conditioning and (a) the total number of pushups performed during mirror exposure and (b) the mean latency to display lateral compression when exposed to the mirror.
FARRELL & WILCZYNSKI: AGGRESSION AND PREFERENCE
3500 3000
(a)
*
Change in preference (s)
2500 2000 1500 1000 500 0 –500 –1000 –1500
Nonaggressive
Aggressive
Control
6000 Time spent in less preferred side (s)
(b)
**
5000
Baseline Test
4000 3000 2000 1000 0
Nonaggressive
Aggressive
Control
Figure 3. (a) Mean SE change in preference (postconditioning minus baseline) and (b) mean SE time spent in the less preferred side (paired with mirror) of the conditioned place-preference apparatus (*P < 0.05; **P < 0.01, two tailed).
within the apparatus were chosen to preclude this possibility. Furthermore, real-time and videorecorded observations during conditioning sessions indicated that nonaggressive animals showed eye, head and body position changes consistent with seeing their reflection. Nonaggressive animals also did not respond to the mirror with changes in body colour (darkening of the skin) typical of animals that have been rendered submissive and that are highly stressed (Greenberg et al. 1984; Greenberg & Crews 1990; Summers & Greenberg 1994). All of the nonaggressive subjects were either completely or partially green at the beginning and end of each mirror exposure. Perhaps the novelty of the CPP apparatus or some aspect of their experience before capture contributed to this phenomenon. There is evidence indicating that contextual familiarity influences aggressive behaviour in this species (Leuk 1995) as well as in other anoles (Stamps & Krishnan1994a, b). The past experience of wild-caught animals in our study is unknown. Finally, the extensive handling of animals in our CPP paradigm (twice per day through training, conditioning and testing) may have resulted in elevated stress
levels, which also may have made some of the animals less aggressive. This seems unlikely, however, since all of the anoles were either completely or partially green immediately before mirror exposure during conditioning trials. In contrast, highly stressed animals typically adopt a dark brown body colour. In our paradigm, subjects were exposed to a mirror for 10 min and remained in the apparatus for 30 min following removal of the mirror. We chose to use a mirror to elicit aggressive behaviour because pilot observations indicated that mirror exposure was a more potent stimulus than were videorecorded images of another male. The use of a mirror also ensured that subjects would perceive a well-matched opponent but would be unlikely to perceive the experience as a defeat, an event that probably would not be perceived as rewarding and that could cause animals to become submissive. We allowed animals to remain in the apparatus for 30 min after removing the mirror to increase the likelihood that the interaction would more closely resemble a victory followed by the withdrawal of the opposing combatant. The results suggest that male green anoles experienced some type of aggression-related reward in our paradigm. The exact source of this reinforcement, however, is unknown. One possibility is that neural and/or endocrine changes that occur during the aggressive interaction make the experience rewarding. A second, not mutually exclusive, possibility is that physiological changes that follow the aggressive interaction made the experience rewarding. Both short- and long-term processes may be involved in aggression-related reward. The results of microdialysis studies examining dopamine release in the nucleus accumbens of resident male rats both during and following 10-min aggressive interactions indicate that extracellular dopamine levels rise during aggressive interactions, peak 20 min following the encounter and decline progressively over the next 90 min (van Erp & Miczek 2000; Ferrari et al. 2003). Similar changes in accumbal dopamine have been reported following exposure to rewarding stimuli (Hernandez & Hoebel 1988; Pfaus et al. 1990; Yoshida et al. 1992), raising the possibility that at least a portion of this dopamine increase may be related to aggressionrelated reward. Similar changes in dopamine levels in rats occurred in the absence of aggressive behaviour when measurements were taken at the same time of day and in the same context as 10 previous aggressive encounters (Ferrari et al. 2003). This finding is also consistent with a reward-based interpretation of these data. However, results from A. carolinensis differ from those reported for rats. Dopaminergic activity as estimated from the ratio of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) to dopamine in accumbal tissue samples obtained immediately following a 10-min aggressive interaction with a mirror was inversely related to the amount of aggressive behaviour displayed (Korzan et al. 2000b), raising the possibility that aggression-related reward was derived from events that occurred following the aggressive interaction in our paradigm. Unfortunately, the pattern of dopamine turnover and/or release during the minutes and hours following aggressive interactions has yet to be described in anoles.
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Changes in the plasma levels of hormones such as testosterone (T) following aggressive interactions represent potential postaggression sources of aggression-related reward. A potential for T as a primary reinforcer for aggressive behaviour is intriguing for two reasons. First, in birds (Wingfield & Wada 1989; Wingfield & Hahn 1994), plasma levels of T can become elevated following aggressive encounters, and in A. carolinensis (Greenberg & Crews 1990), winners of staged aggressive interactions had higher androgen titres than both losers and control animals 1 h after the initiation of the interaction. The androgen levels of the winners subsequently declined and were not significantly different from those of control animals when examined both 1 day and 1 week later. Second, there is evidence indicating that T administration can act as a rewarding stimulus. Rats will form conditioned preferences for contexts associated with T injection (e.g. Alexander et al. 1994; Packard et al. 1998). Furthermore, the rewarding effect of T is rapid; conditioning occurs within 30 min of injection. This rapid effect suggests that a nongenomic action underlies the rewarding effect of this compound and increases the likelihood that postaggression surges in T could have served as a rewarding stimulus in our paradigm. Exogenous testosterone administration also yields a rapid increase in striatal dopamine levels in male jackey dragons, Amphibolurur muricatus, another territorial lizard species (Watt et al. 2002). Furthermore, Yang & Wilczynski (2002) found that the relationship between aggression and plasma androgens could best be explained by a model in which expressing aggressive displays mediated plasma androgen levels rather than vice versa. Involvement of a postaggression surge of T in aggressionrelated reward could explain why, in our paradigm, we observed CPP only in those animals that acted aggressively towards the mirror. This discussion has been based, at least in part, on the assumption that the animals that responded aggressively towards their reflection during conditioning perceived the outcome of the interaction as a victory following the disappearance of the mirror and thus the perceived opponent. It is also possible, however, that the male anoles may have perceived the outcome of the interaction as a draw since neither the aggressive animal nor its reflection behaved or changed skin colour in ways that are indicative of a submissive animal. If the anoles perceived the conflict as ending in a draw, the opportunity to resolve the conflict during subsequent encounters may have been the rewarding stimulus that caused the aggressive anoles to increase the time that they spent in the side of the apparatus paired with the mirror. Such an explanation might explain the apparent contradiction between our finding that engaging in aggressive interactions may be rewarding for male anoles and prior reports in this species indicating opponent recognition and reduced aggression during subsequent interactions with the same individual once a social hierarchy has been established (e.g. Forster et al. 2005). However, the results of particular dyadic interactions can alter the subsequent behaviour of both the winner and the looser. Therefore, reductions in aggressive behaviour across successive interactions between the same two combatants might be mediated more by the
submissive or avoidance behaviour of the initial loser rather than by a propensity by the prior winner to become less aggressive to a familiar combatant. Furthermore, the ‘mirror opponent’ to which the nonaggressive animals were exposed also disappeared when the mirror was removed during the conditioning sessions. Therefore, the nonaggressive anoles also experienced an unresolved interaction during the conditioning sessions, but they did not subsequently show an enhanced preference for the side of the CPP apparatus paired with the mirror during conditioning. The results suggest that engaging in an aggressive interaction can be rewarding for green anoles. Yang et al. (2001) showed that experience with aggressive interactions yielded increased aggression to novel challengers. Our results suggest that aggression-related reward may underlie that phenomenon. We also found that prior experience gained during simulated aggressive encounters can lead to the establishment of a place preference for the context and location where the previous aggressive encounter occurred. Based on these findings, aggression-related reward and reinforcement may thus relate to territoriality in two ways: (1) by increasing aggression towards future challengers and (2) by establishing site preferences for the territory itself or locations within it. Future experiments manipulating androgens and/or the dopamine system can address the physiological and neural mechanisms of this process by elucidating the influences of endogenous testosterone and behaviourally triggered testosterone or dopamine surges following aggressive interactions. The results of such studies, combined with additional experiments examining the specific behavioral changes that occur between pairs of anoles during successive aggressive interactions in both the field and laboratory settings, should help to further our understanding of the behavioural and physiological regulation of aggressive behaviour and of its consequences regarding territory formation. Acknowledgments Support for this research was contributed by National Institutes of Health F32MH067292-01 to W. J. Farrell and National Science Foundation IBN 0090739 to W. Wilczynski. References Alexander, G. M., Packard, M. G. & Hines, M. 1994. Testosterone has rewarding affective properties in male rats: implications for the biological basis of sexual motivation. Behavioral Neuroscience, 108, 424–428. Archer, J. 1988. The Behavioural Biology of Aggression. New York: Cambridge University Press. Azrin, N. H., Hutchinson, R. R. & McLaughlin, R. 1965. The opportunity for aggression as an operant reinforcer during aversive stimulation. Journal of the Experimental Analysis of Behavior, 8, 171–180. Bardo, M. T. & Bevins, R. A. 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology, 153, 31–43. Baxter, L. R., Ackermann, R. F., Clark, E. C. & Baxter, J. E. 2001. Brain mediation of anolis social dominance displays. I. Differential basal ganglia activation. Brain, Behavior and Evolution, 57, 169–183.
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peripheral or intra-accumbens injection of a-Flupenthixol. Hormones and Behavior, 34, 39–47. Pfaus, J. G., Damsma, G., Nomikos, G. G., Wenkstern, D. G., Blaha, C. D., Phillips, A. G. & Fibiger, H. C. 1990. Sexual behavior enhances central dopamine transmission in the male rat. Brain Research, 530, 345–348. Price, K., Broughton, K., Boutin, S. & Sinclair, A. R. E. 1986. Territory size and ownership in red squirrels: response to removals. Canadian Journal of Zoology, 64, 1144–1147. Shutler, D. & Weatherhead, P. J. 1992. Surplus territory contenders in male red-winged blackbirds: where are the desperados? Behavioral Ecology and Sociobiology, 31, 97–106. Stamps, J. A. 1995. Motor learning and the value of familiar space. American Naturalist, 146, 41–58. Stamps, J. A. & Krishnan, V. V. 1994a. Territory acquisition in lizards I. First encounters. Animal Behaviour, 47, 1375–1385. Stamps, J. A. & Krishnan, V. V. 1994b. Territory acquisition in lizards II. Establishing social and spatial relationships. Animal Behaviour, 47, 1387–1400. Stamps, J. A. & Krishnan, V. V. 1999. A learning-based model of territory establishment. Quarterly Review of Biology, 74, 291–318. Summers, C. H. 2001. Mechanisms for quick and variable responses. Brain, Behavior and Evolution, 57, 283–292. Summers, C. H. & Greenberg, N. 1994. Somatic correlates of adrenergic activity during aggression in the lizard, Anolis carolinensis. Hormones and Behavior, 28, 29–40. Tellegen, A., Horn, J. M. & Legrand, R. G. 1969. Opportunity for aggression as a reinforcer in mice. Psychonomic Science, 14, 104–105. Thompson, T. 1969. Aggressive behaviour of siamese fighting fish: analysis and synthesis of conditioned and unconditioned
components. In: Aggressive Behaviour (Ed. by S. Garattini & E. B. Sigg), pp. 15–31. New York: J. Wiley. Thompson, T. & Sturm, T. 1965. Classical conditioning of aggressive display in Siamese fighting fish. Journal of the Experimental Analysis of Behavior, 8, 397–403. Waser, P. W. & Wiley, R. H. 1979. Mechanisms and evolution of spacing in animals. In: Handbook of Behavioral Neurobiology (Ed. by P. Marler & J. G. Vandenbergh), pp. 159–223. New York: Plenum. Watt, M. J., Forster, G. L., Joss, J. M. P., Blaha, C. D. & Summers, C. H. 2002. Rapid testosterone modulation of striatal dopamine release. Society for Neuroscience Abstracts, 28, 863. 11. Wingfield, J. C. & Hahn, T. P. 1994. Testosterone and territorial behaviour in sedentary and migratory sparrows. Animal Behaviour, 47, 77–89. Wingfield, J. C. & Wada, M. 1989. Changes in plasma level of testosterone during male–male interactions in the song sparrow Melospiza melodia: time course and specificity of response. Journal of Comparative Physiology, 166, 189–194. Yang, E.-J. & Wilczynski, W. 2002. Relationships between hormones and aggressive behavior in green anole lizards: an analysis using structural equation modeling. Hormones and Behavior, 42, 192–205. Yang, E.-J., Phelps, S. M., Crews, D. & Wilczynski, W. 2001. The effects of social experience on aggressive behavior in the green anole lizard (Anolis carolinensis). Ethology, 107, 777–793. Yoshida, M., Yokoo, H., Mizoguchi, K., Kawahara, H., Tsuda, A., Nishikawa, T. & Tanaka, M. 1992. Eating and drinking cause increased dopamine release in the nucleus accumbens and the ventral tegmental area in the rat: measurement by in vivo microdialysis. Neuroscience Letters, 139, 73–76.
Aggressive experience alters place preference in green anole lizards, Anolis carolinensis WILL IAM J. F ARRELL & WALTER WILCZYNSKI
Department of Psychology and Institute for Neuroscience, The University of Texas at Austin (Received 29 June 2004; initial acceptance 20 September 2004; final acceptance 10 October 2005; published online 20 March 2006; MS. number: A9924R)
Males of many species establish territories from which they exclude conspecifics, and experience can influence the defence and stability of territorial boundaries. We used a conditioned place preference (CPP) procedure to determine whether engaging in a form of behaviour associated with territorial defence, aggressive behaviour, was rewarding for male green anoles and whether aggressive experience could alter the spatial/contextual preferences of these territorial lizards. When approached by a male conspecific, or when exposed to their reflection in a mirror, many male anoles show species-typical aggressive behaviours. We repeatedly exposed male anoles to a mirror or the nonreflective back of the mirror in opposing sides of a CPP apparatus that consisted of two contextually distinct chambers connected by a central tunnel. Control animals (N ¼ 10) were exposed to the back of the mirror in both sides of the apparatus. Following conditioning, animals that showed aggressive behaviour (N ¼ 13) when exposed to the mirror during conditioning increased the time that they spent in the side of the apparatus paired with the mirror relative to baseline. In contrast, the preferences of nonaggressive (N ¼ 8) and control animals were unchanged. These findings suggest that engaging in aggressive behaviour (and not simply seeing a conspecific) is rewarding for male green anoles and that aggression-related reward and associative learning could influence the formation and/or maintenance of stable territories. Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
Animals of many species and taxa form territories from which they exclude conspecifics. The maintenance of these territories provides occupants with access to resources that are often crucial for survival and reproduction. Some of the resources that are defended include food, mates, nesting sites and physical aspects of the environment required to maintain homeostasis and avoid predators (Emlen & Oring 1977; Waser & Wiley 1979; Grant 1993; Maher & Lott 2000). Field studies indicate that, once established, territorial boundaries often remain quite stable (e.g. Knapton & Krebs 1974; Hirons 1985; Janes 1985; Price et al. 1986; Jenssen et al. 1995). One factor that contributes at least in part to this stability is the experience gained by residents within their territories. Territory residents typically have a competitive advantage over intruders and repel or
Correspondence and present address: W. J. Farrell, Queens College, Psychology Department, 65-30 Kissena Boulevard, Flushing, NY 11367, U.S.A. (email: [email protected]). W. Wilczynski is now at the Department of Psychology, and Center for Behavioral Neuroscience, Georgia State University, P.O. Box 3966, Atlanta, GA 30302-3966, U.S.A. 0003–3472/06/$30.00/0
defeat them (Waser & Wiley 1979; Archer 1988; Bradbury & Vehrencamp 1998). Furthermore, the results of displacement experiments indicate that the magnitude of the resident advantage is often related to the duration of residency (Krebs 1982; Beletsky & Orians 1989; Johnsson & Forser 2002). For example, Beletsky & Orians (1989) removed resident red-winged blackbirds, Agelaius phoenicius, from their territories for 6–7 days. Once released, the majority of residents (73%) were able to reclaim their territories from replacement males that had occupied them for 2 days or less, but the converse was true (only 16% reclaimed territories) when replacements had occupied the territory for at least 6 days. Learning that occurs while occupying an area may have a variety of influences on the formation and maintenance of stable territories. For example, knowledge gained about the topography of a region and the distribution and quality of resources may increase the perceived value of a territory and thereby enhance territorial defence (reviewed in Shutler & Weatherhead 1992; Stamps & Krishnan 1999). Furthermore, site-specific motor learning may render animals more capable of exploiting the physical dimensions of a territory (Stamps 1995). Finally, associations learned while engaging in social interactions such
1155 Ó 2006 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights reserved.
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as aggression may also play a role. Organisms can learn associations regarding prior aggressive interactions and these associations can influence subsequent behaviour. For example, studies conducted with fish such as Betta splendens, blue gouramis, Trichogaster tichopterus, and sticklebacks, Gasterosteus aculeatus, indicate that many aspects of species-typical aggressive behaviour can be classically conditioned by pairing a conditioned stimulus (CS) with subsequent visual access to a conspecific (e.g. Thompson & Sturm 1965; Hollis 1984; Hollis et al. 1995; Jenkins & Rowland 1996). Furthermore, this type of training can confer an advantage upon the animals that were trained. When the same CS later predicts actual physical access to a conspecific, conditioned animals are typically more aggressive and win more contests than do fish for whom the contest has not been cued (Hollis 1984; Hollis et al. 1995). The present study focused on the possibility that engaging in aggression might also serve as a rewarding stimulus that, when associated with a particular spatial environment, could increase an animal’s attachment or preference for that environment. Evidence indicates that engaging in aggressive behaviour that ends with the defeat or experimental withdrawal of a conspecific can be rewarding. For example, mice will work or perform operant tasks for the opportunity to engage in aggressive behaviour (Legrand 1970; Connor 1974; Fish et al. 2002). Likewise, male mice (Martinez et al. 1995) and female hamsters (Meisel & Joppa 1994) form conditioned place preferences for contexts that were previously paired with the introduction of a conspecific that they attacked. Analogous findings from studies of operant conditioning (Thompson 1969; Hollis & Overmier 1982) and place preference (Bronstein et al. 1988) have also been reported for fish. The majority of studies examining aggression-related reinforcement have been restricted to laboratory-bred rodents or domesticated strains of birds and fish (but see Bronstein et al. 1988). In the present study, we tested whether engaging in aggressive behaviour might be rewarding for a wild-caught, territorial reptile, the green anole lizard, and whether such reward could alter spatial preferences. Green anoles are arboreal lizards found throughout a large region of the southeastern United States. During the breeding season, male anoles establish territories from which they exclude other males (Jenssen et al. 1995; Jenssen & Nunez 1998). While establishing and maintaining breeding territories, male anoles engage in aggressive interactions with male conspecifics, raising the possibility that reinforcement gained and associations learned during these interactions may contribute to the establishment and maintenance of stable territories. The territorial nature of male green anoles makes them an ideal organism for the study of aggression-related reinforcement/reward. Furthermore, males of this species readily display species-typical aggressive behaviours in the laboratory. Lateral compression of the body along the sagittal axis is the signature aggressive behaviour in this species (Greenberg & Noble 1944; Greenberg & Crews 1983) and is frequently accompanied by pushup displays (repetitive raising and lowering of the front half of the body), and rapid
extensions of the red dewlap beneath the throat. Skin colour can also change during aggressive interactions. A patch of skin behind the eye, the postorbital eyespot, turns black during heightened aggressive interactions and body colour can vary between light green and dark brown. Body colour darkens (range ¼ light green to dark brown) following stressful experiences such as social subordination (Greenberg et al. 1984; Greenberg & Crews 1990; Summers & Greenberg 1994), and blackening of the postorbital eyespot is directly related to elevated plasma catecholamine titres (Kleinholz 1938; Hadley & Goldman 1969; Greenberg & Crews 1983; Summers & Greenberg 1994; Summers 2001). The aggressive behaviours of green anoles are distinct and easy to quantify. Moreover, these behaviours can be elicited by exposing a male anole to his own reflection (e.g. Korzan et al. 2000a; Baxter et al. 2001). To examine whether engaging in aggressive behaviour is rewarding for male green anoles, and to determine whether associations learned during these interactions can alter the spatial preferences of combatants, we developed an apparatus and methodology to examine conditioned place preference (CPP) in this species. CPP procedures have been extensively used with rodents to examine the rewarding properties of drugs of abuse (for review see Bardo & Bevins 2000), and less frequently to determine whether social stimuli such as sexual and aggressive interactions are rewarding (e.g. Oldenburger et al. 1992; Meisel & Joppa 1994; Martinez et al. 1995). The underlying assumption of CPP procedures is that animals will increase the time that they spend in a distinct environmental context (the CS) after having associated this context with a rewarding stimulus (the unconditioned stimulus, US). The terms ‘preference’ and ‘reward’ are operationally defined in the CPP paradigm in reference to the observed behaviour of the organism, and neither term implies a conscious cognitive state. We chose to use a CPP paradigm in part because we thought that place-preference learning could be of particular ethological relevance for a territorial species such as A. carolinensis. We also chose this paradigm because wild-caught anoles show individual differences in aggressiveness, and a CPP paradigm allowed us to examine the responses of both aggressive and nonaggressive animals. The primary goals of the present study were to determine whether engaging in aggression was rewarding for male green anoles and whether they showed an increased preference for a context previously associated with aggressive interactions. However, one difficulty with examining aggression-related reward is the possibility that social exposure to a conspecific alone, rather than engaging in aggression per se, may serve as a rewarding stimulus. We examined both possibilities by quantifying the changes in place preference shown by both aggressive and nonaggressive animals following simulated social interactions. We repeatedly exposed male anoles to a mirror or the back of the mirror (a nonreflective neutral stimulus) in opposing sides of a CPP apparatus. If engaging in aggressive behaviour is rewarding for male anoles, animals that respond aggressively to their reflection during conditioning should prefer the side of the apparatus paired with the
FARRELL & WILCZYNSKI: AGGRESSION AND PREFERENCE
reflective mirror following conditioning, and nonaggressive animals should not show this change in preference. If, however, social contact alone is rewarding for male anoles, then both groups of animals should show a similar change in preference regardless of their behavioural response to the mirror.
Slots for mirrors Doors Tan rear and side walls
Black rear wall
Smooth plastic floor
Textured black floor
METHODS
Subjects Wild-caught male and female green anoles were obtained from Charles Sullivan Inc. (Nashville, Tennessee, U.S.A.) during April, July and September. Subjects were adult male anoles, which ranged in size from 55 to 69 mm (snout-to-vent length) SVL. All subjects were assumed to be sexually mature adults based on the ratings scale of Jenssen et al. (2001), which classifies males with a SVL of 50 mm or greater as adults. Each male anole was housed with a female in a glass terrarium measuring 17.8 30.5 45.7 cm for a minimum of 13 days and for a maximum of 265 days (X SE ¼ 161 12:4 days) before the experiment. The front and back of each terrarium were clear glass and the side walls were constructed of opaque (white) Plexiglas, which visually isolated adjacent male/female pairs. The bottom of each cage was covered by about 2.5 cm of sphagnum peat moss and was outfitted with a plastic water bowl. A single wooden dowel (1.6 cm diameter) placed diagonally between the bottom-front and rear-top corners of the cage was provided as a perch. Animals were housed under conditions designed to simulate the breeding season and promote gonadal activity (Licht 1967, 1971). The animal room was maintained on a 14:10 h light:dark cycle (lights on at 0700 hours) and the ambient temperature varied from 30 C during the day to 23 C at night. Incandescent lights (60 W) positioned above each terrarium served as an additional source of heat, and full-spectrum fluorescent lights (Vitalite, Duro-Test, Philadelphia, Pennsylvania, U.S.A.) provided additional illumination. Half-grown crickets were provided three times a week as food, and water was provided continuously in bowls and by periodic misting of the cage walls. All subjects were observed courting the female with whom they had been paired within 3 days before the beginning of the experiment to ensure that they were socially active.
Apparatus Conditioning and testing were conducted in a CPP apparatus (Fig. 1) that consisted of two side chambers (8.0 15.5 13.0 cm) joined by a central tunnel (11.5 8.5 8.0 cm). The dimensions of the side chambers were chosen to provide the animals enough room to display aggressively while ensuring that they saw the mirror inserted into these chambers during the appropriate conditioning sessions (see below). The central tunnel was deliberately designed to be small to facilitate alternations between the sides of the apparatus when
Orange ceiling
Tunnel (blue)
Blue ceiling
Figure 1. Schematic representation of the conditioned placepreference apparatus.
appropriate. Both pilot observations and observations conducted during the experiment indicated that male anoles were capable of turning around in the central tunnel and did so frequently. The two side chambers of the apparatus and the central tunnel were designed to be contextually distinct both from each other and from any habitat that the subjects had previously experienced. The left chamber had a clear plastic front wall, beige rear and side walls, an orange ceiling and a smooth plastic floor. The right chamber had clear front and rear walls, a black side wall, a light blue ceiling and a textured black floor (plastic needlepoint backing). A narrow slot was cut into the ceiling of both side chambers, allowing a mirror (12.7 10.2 cm) to be lowered into the chamber along the wall directly opposite to the opening of the central tunnel. The floor and walls of the central tunnel were dark blue plastic and the ceiling was clear Plexiglas. Guillotine doors were at the left and right ends of the central tunnel and could be raised or lowered as necessary to allow subjects access to the various segments of the apparatus. The right door was black and the left door was white. Conditioning and testing sessions were videotaped from the front of the apparatus. A camcorder (RCA, CC437) served as a camera and was connected to a time-lapse VCR (Gyyr, TLC1824). Place-preference tests were recorded at 1/6 normal tape speed, allowing videotapes to be coded rapidly (6 normal speed) when reviewed using a standard VHS player. Conditioning sessions were recorded at standard tape speed to ensure the accurate quantification of rapidly occurring aggressive behaviours.
Procedure CPP conditioning and testing were conducted over 8 days in a room maintained at 30 C. The procedure was divided into baseline, conditioning and testing phases.
Baseline (day1) On the first day of the experiment, animals were tested for initial (baseline) spatial preferences in the CPP apparatus. At the beginning of the baseline session, a single male lizard was removed from his home cage and confined to the central tunnel of the CPP apparatus for
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5 min. All anoles were placed into the central tunnel head first with their nose facing the rear of the apparatus. However, animals frequently turned around several times during the 5-min period in the central tunnel, rendering it impossible to control the direction of orientation during this period. Following the 5-min period in the central tunnel, both guillotine doors were raised simultaneously, allowing the subject free access to all portions of the apparatus for 2 h. Videotaped recordings obtained during the baseline session were then reviewed and the time spent in the left and right side of the apparatus was quantified. Subjects were deemed to have entered one side of the apparatus when their entire head had exited the central tunnel and to have exited that side when their entire body (excluding the tail) was once again within the central tunnel. The side in which an individual animal spent the most time during the baseline session was designated as that subject’s preferred side of the apparatus and the opposing side was designated as the less preferred side. Animals that did not enter all areas of the apparatus were excluded from the experiment (N ¼ 6).
Conditioning (days 2–7) Conditioning commenced 1 day after the baseline preference test and continued for a total of 6 days (days 2–7). Animals underwent six 45-min conditioning sessions, one per day. During three sessions, subjects were confined to their less preferred side of the apparatus and were exposed to a mirror. During the remaining sessions, on alternate days, animals were confined to their preferred side of the apparatus and were exposed to the nonreflective back of the mirror. We paired the nonreflective back of the mirror with the side of the apparatus that was preferred during the baseline to hold the physical act of inserting a piece of glass into the side chambers constant across the previously preferred and less preferred chambers. The nonreflective back of the mirror does not elicit aggressive displays from male green anoles. We used a 6-day-long conditioning regimen (with three mirror exposures) because the results of previous studies indicated that aggressive behaviour shown during daily visual exposure to conspecifics (videotaped or real) increases over the first 3 days and subsequently declines (Yang et al. 2001; Yang & Wilczynski 2002). Control animals underwent similar procedures but were exposed to the back of the mirror in both sides of the apparatus. The control group was included to ensure that repeated exposure to the various sides of the apparatus did not systematically alter preferences. Each conditioning session began with a 5-min habituation period before the mirror was inserted. Following this habituation period, an experimenter lowered the mirror (or the back of the mirror) into the apparatus through the slot in the roof of the chamber. Behaviour was videotaped during the next 10 min and the mirror was then removed. Subjects remained in the apparatus for 30 min following removal of the mirror (or the back of the mirror) and were then returned to their home cage. We exposed 21 animals to the mirror during conditioning and used 10 additional animals in the control group. Conditioning began
in the preferred side of the apparatus for five control animals and 10 animals exposed to the mirror.
Testing (day 8) One day after the final conditioning session, subjects were retested for place preference using the baseline testing procedure.
Behavioural Quantification and Data Analysis Video recordings and/or real-time observations obtained during conditioning sessions (mirror only) were used to determine the latency required to perform the lateral compression (LC) display and show a darkened eyespot. Animals that never showed either LC or a darkened eyespot were assigned latencies equivalent to the duration of an individual mirror exposure (600 s). Pilot observations and real-time observations during conditioning trials indicated that subjects did not display aggressive behaviour when exposed to the nonreflective back of the mirror. Therefore, behaviour during these trials was not quantified. Animals were classified as aggressive if they became laterally compressed during any of the conditioning sessions in which they were exposed to the mirror and were classified as nonaggressive if they never displayed this response. Video recordings obtained during conditioning sessions were subsequently used to quantify the frequency of pushups (PU) and dewlap extensions (DE) displayed during mirror exposure. LC was used to classify animals as aggressive, rather than PU or DE, because both PU and DE can occur in the absence of aggression (e.g. spontaneous advertisement or during courtship) and are also major components of aggressive display. We assessed changes in place preference by comparing the amount of time spent in the less preferred side of the apparatus (paired with the mirror during conditioning) during the baseline and postconditioning test sessions. One-way ANOVA with Tukey’s HSD test was used for between-subjects comparisons of mean preference change (test minus baseline). Paired t tests and repeated measures ANOVA were used to examine within-subject differences. Two correlations were also performed to examine the relationships between the magnitude of individual animals’ preference changes and either the number of pushups or the mean latency to become laterally compressed during conditioning. Results are expressed as mean SE. The results of statistical tests were considered significant if P < 0.05 (two-tailed). RESULTS
Response to Mirror Of the 21 animals exposed to the mirror during conditioning, 13 displayed LC and were classified as aggressive. The remaining eight animals never became compressed and were classified as nonaggressive. The results of two between-subjects ANOVAs indicated that mean SVL (F2,28 ¼ 1.32, P ¼ 0.28) and number of days in captivity (F2,28 ¼ 1.11, P ¼ 0.35) did not differ
FARRELL & WILCZYNSKI: AGGRESSION AND PREFERENCE
significantly between the 13 aggressive animals, eight nonaggressive animals and the 10 control animals. Ten of the 13 animals classified as aggressive showed LC during all three of the conditioning sessions in which they were exposed to the mirror. The remaining three aggressive animals displayed LC during trial 1 (N ¼ 2) or 2 (N ¼ 1). The mean latency required for aggressive animals to show LC was 179.0 32.4 s and did not change significantly across the three conditioning trials in which animals were exposed to the mirror (repeated measures ANOVA: F2,24 ¼ 0.34, P ¼ 0.715). Aggressive animals also showed darkening of their postorbital eyespot during 82.1% of the conditioning trials in which they saw the mirror. Eyespot darkening occurred with an average latency of 231.1 34.0 s and did not change significantly across the three exposures to the mirror (repeated measures ANOVA: F2,24 ¼ 1.28, P ¼ 0.298). Nonaggressive animals never displayed a darkened postorbital eyespot when exposed to the mirror. Aggressive animals also performed DE and PU. DEs were most common at the onset of aggressive interactions, while PUs often continued throughout. The mean number of DEs and PUs displayed by aggressive animals during each 10-min mirror exposure were 2.9 0.6 and 113.7 16.0, respectively. The mean frequency with which aggressive animals displayed DE and PU did not differ significantly across the three conditioning trials in which they were exposed to the mirror (repeated measures ANOVA: DE: F2,24 ¼ 3.15, P ¼ 0.06; PU: F2,24 ¼ 1.94, P ¼ 0.17) but the frequency of DE tended to decline progressively across trials. While nonaggressive animals never showed LC or darkening of the postorbital eyespot when exposed to the mirror, they did occasionally (16.7% of trials) perform DEs and/or PUs (X SE ¼ 0:5 0:3 DEs and 8.1 6.9 PUs per trial). These behaviours were only performed by 50% of the nonaggressive animals and were never expressed during more than one trial per animal.
Place Preference During the baseline test for spatial preferences, animals (13 aggressive, 8 nonaggressive and 10 control animals) spent an average of 3787.1 258.8 s and 2895.2 246.4 s in the right and left sides of the apparatus, respectively. There was no significant difference between baseline preference for the left or right side (paired t test: t30 ¼ 1.85, P ¼ 0.07). Individual animals did show significant baseline preferences for one side of the apparatus or the other. Animals spent a mean SE of 4501.9 175.8 s in their preferred side of the apparatus and 2180.4 142.2 s in their less preferred side (paired t test: t30 ¼ 8.21, P < 0.001). Table 1 summarizes the time that subjects spent in their preferred and less preferred sides of the apparatus during the baseline and postconditioning preference tests. Table 2 summarizes the number of alternations between right and left sides of the apparatus during both baseline and postconditioning tests. Preference changes following conditioning indicated that mirror exposure alone (without taking into account animals’ behavioural responses to the mirror) was
insufficient to elicit an enhanced preference for the side of the CPP apparatus paired with the mirror during conditioning. Animals exposed to the mirror during conditioning (13 aggressive and 8 nonaggressive animals) increased the time that they spent in the less preferred side of the apparatus (paired with mirror) by an average of 1246.0 468.4 s relative to baseline, while control animals showed a concurrent decrease (X SE ¼ 194:7 688:6 s), but these values did not differ significantly (ANOVA: F1,29 ¼ 3.02, P ¼ 0.09). However, there was a relationship between engaging in aggressive behaviour during conditioning and an enhanced preference for the side of the apparatus paired with the mirror (i.e. postconditioning minus baseline). For the 21 anoles exposed to the mirror (N ¼ 13 aggressive and 8 nonaggressive animals), there was a significant positive relationship between the number of PUs during conditioning and preference change (Pearson correlation: r19 ¼ 0.52, N ¼ 21, P < 0.02) and a significant negative relationship between mean latency to display LC and preference change (r19 ¼ 0.50, N ¼ 21, P ¼ 0.02; Fig. 2). Mean preference changes for the less preferred (paired with mirror) side of the apparatus differed by group (aggressive, nonaggressive, control: ANOVA: F2,28 ¼ 5.71, P < 0.01; Fig. 3a). Post hoc analyses indicated that aggressive animals showed a significantly larger preference change than did both nonaggressive (Tukey’s HSD test: P < 0.03) and control animals (Tukey’s HSD: P < 0.02). The changes in preference by nonaggressive and control animals were not significantly different (Tukey’s HSD test: P ¼ 0.99). The mean time that aggressive animals spent in the side of the apparatus paired with the mirror also increased significantly relative to their own baselines (paired t test: t12 ¼ 4.19, P < 0.01; Fig. 3b). In contrast, the preferences of nonaggressive (paired t test: t7 ¼ 0.41, P ¼ 0.69) and control animals (paired t test: t9 ¼ 0.28, P ¼ 0.78) did not change. Of the animals classified as aggressive, 84.6% increased the time that they spent in the side of the apparatus paired with the mirror following conditioning. In contrast, only 37.5% and 30.0% of nonaggressive and control animals showed increases.
DISCUSSION The results suggest that engaging in an aggressive interaction can be rewarding for male green anoles and that aggressive experience can result in an enhanced preference for the site of previous aggression in this species. Animals that responded aggressively towards their reflection during conditioning subsequently increased the time they spent in the side of the apparatus paired with the mirror. In contrast, control animals that were never exposed to the mirror and nonaggressive animals that never displayed aggressive responses failed to show similar increases. With respect to aggression-related reward/reinforcement, our findings are consistent with those previously reported for mammals (Azrin et al. 1965; Tellegen et al. 1969; Legrand 1970; Connor 1974; Meisel & Joppa 1994; Martinez et al. 1995; Fish et al. 2002), birds (Cherek
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Table 1. Time distribution during baseline and postconditioning place-preference tests Preferred side
Mirror (N¼21) Aggressive (N¼13) Nonaggressive (N¼8) Control (N¼10)
Less preferred side
Baseline
Postconditioning
Baseline
Postconditioning
4453.2234.6 4243.2327.4 4794.4295.7 4604.2246.5
3464.4500.6 2575.9585.3 4908.3666.2 4702.8702.4
2123.1181.9 2226.5241.0 1955.1282.6 2300.7228.3
3369.1517.2 4395.6593.3 1701.0619.5 2106.0689.7
Values are mean SE times (s). The mirror group comprised both the aggressive and nonaggressive animals.
Table 2. Number of alternations between the left and right sides of the conditioned place-preference apparatus during baseline and postconditioning place-preference tests Group Mirror (N¼21) Aggressive (N¼13) Nonaggressive (N¼8) Control (N¼10)
Baseline 5.290.87 5.081.21 5.631.27 4.201.19
Postconditioning 5.521.45 4.541.26 7.133.29 3.401.14
Values are mean SE numbers. The mirror group comprised both the aggressive and nonaggressive animals.
It is unclear why some male anoles responded aggressively towards their reflection while others did not. It is unlikely that the nonaggressive animals failed to see their reflection. The size of the mirror used and its position 6000 (a) 5000 Change in preference (s)
et al. 1973) and fish (Thompson 1969; Hollis & Overmier 1982; Bronstein et al. 1988). To our knowledge, the present results are the first demonstration of aggressionrelated reward in a reptile. Interestingly, the place preferences of aggressive anoles changed rapidly (three trials with mirror), suggesting that territorial males of this species may be particularly adept at forming aggressionrelated contextual associations and supporting the notion that aggression-related reward and associative learning might contribute to the formation and/or maintenance of stable territories. However, the CPP apparatus and procedures that we used in the present study did not mimic the natural conditions under which green anoles form territories. Additional research under more natural conditions will be required to examine further the contribution of aggression-related reward and associative learning to territory formation. The data obtained from nonaggressive animals are of particular interest because they suggest that engaging in aggressive behaviour was a prerequisite for enhanced place preference in our paradigm. Kelsey & Cassidy (1976) found that isolated male mice preferentially entered the arm of a T-maze where they had previously fought and that the formation of this preference could be blocked by allowing social interaction (through a wire mesh) with a conspecific in the opposing arm of the maze. These findings suggest that, at least under some circumstances, reward gained during aggressive interactions may be derived from social contact rather than engaging in aggression. It is unlikely, however, that reward obtained simply from any form of social interaction can explain our results. The preferences of nonaggressive anoles were unchanged by the conditioning procedure, even though these animals were exposed to their reflection for the same amount of time as the aggressive animals. Only animals that reacted aggressively to the mirror developed a CPP.
4000 3000 2000 1000 0 –1000
r = +0.52
–2000 –3000
P = 0.017 0
200
400
600
800
1000
Number of pushups 6000 (b)
5000 Change in preference (s)
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4000 3000 2000 1000 0 –1000 r = –0.50
–2000 –3000
P = 0.020 0
200
400
600
Mean lateral compression latency (s) Figure 2. Relationships between change in preference (postconditioning minus baseline) for the side of the conditioned placepreference apparatus paired with the mirror during conditioning and (a) the total number of pushups performed during mirror exposure and (b) the mean latency to display lateral compression when exposed to the mirror.
FARRELL & WILCZYNSKI: AGGRESSION AND PREFERENCE
3500 3000
(a)
*
Change in preference (s)
2500 2000 1500 1000 500 0 –500 –1000 –1500
Nonaggressive
Aggressive
Control
6000 Time spent in less preferred side (s)
(b)
**
5000
Baseline Test
4000 3000 2000 1000 0
Nonaggressive
Aggressive
Control
Figure 3. (a) Mean SE change in preference (postconditioning minus baseline) and (b) mean SE time spent in the less preferred side (paired with mirror) of the conditioned place-preference apparatus (*P < 0.05; **P < 0.01, two tailed).
within the apparatus were chosen to preclude this possibility. Furthermore, real-time and videorecorded observations during conditioning sessions indicated that nonaggressive animals showed eye, head and body position changes consistent with seeing their reflection. Nonaggressive animals also did not respond to the mirror with changes in body colour (darkening of the skin) typical of animals that have been rendered submissive and that are highly stressed (Greenberg et al. 1984; Greenberg & Crews 1990; Summers & Greenberg 1994). All of the nonaggressive subjects were either completely or partially green at the beginning and end of each mirror exposure. Perhaps the novelty of the CPP apparatus or some aspect of their experience before capture contributed to this phenomenon. There is evidence indicating that contextual familiarity influences aggressive behaviour in this species (Leuk 1995) as well as in other anoles (Stamps & Krishnan1994a, b). The past experience of wild-caught animals in our study is unknown. Finally, the extensive handling of animals in our CPP paradigm (twice per day through training, conditioning and testing) may have resulted in elevated stress
levels, which also may have made some of the animals less aggressive. This seems unlikely, however, since all of the anoles were either completely or partially green immediately before mirror exposure during conditioning trials. In contrast, highly stressed animals typically adopt a dark brown body colour. In our paradigm, subjects were exposed to a mirror for 10 min and remained in the apparatus for 30 min following removal of the mirror. We chose to use a mirror to elicit aggressive behaviour because pilot observations indicated that mirror exposure was a more potent stimulus than were videorecorded images of another male. The use of a mirror also ensured that subjects would perceive a well-matched opponent but would be unlikely to perceive the experience as a defeat, an event that probably would not be perceived as rewarding and that could cause animals to become submissive. We allowed animals to remain in the apparatus for 30 min after removing the mirror to increase the likelihood that the interaction would more closely resemble a victory followed by the withdrawal of the opposing combatant. The results suggest that male green anoles experienced some type of aggression-related reward in our paradigm. The exact source of this reinforcement, however, is unknown. One possibility is that neural and/or endocrine changes that occur during the aggressive interaction make the experience rewarding. A second, not mutually exclusive, possibility is that physiological changes that follow the aggressive interaction made the experience rewarding. Both short- and long-term processes may be involved in aggression-related reward. The results of microdialysis studies examining dopamine release in the nucleus accumbens of resident male rats both during and following 10-min aggressive interactions indicate that extracellular dopamine levels rise during aggressive interactions, peak 20 min following the encounter and decline progressively over the next 90 min (van Erp & Miczek 2000; Ferrari et al. 2003). Similar changes in accumbal dopamine have been reported following exposure to rewarding stimuli (Hernandez & Hoebel 1988; Pfaus et al. 1990; Yoshida et al. 1992), raising the possibility that at least a portion of this dopamine increase may be related to aggressionrelated reward. Similar changes in dopamine levels in rats occurred in the absence of aggressive behaviour when measurements were taken at the same time of day and in the same context as 10 previous aggressive encounters (Ferrari et al. 2003). This finding is also consistent with a reward-based interpretation of these data. However, results from A. carolinensis differ from those reported for rats. Dopaminergic activity as estimated from the ratio of the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) to dopamine in accumbal tissue samples obtained immediately following a 10-min aggressive interaction with a mirror was inversely related to the amount of aggressive behaviour displayed (Korzan et al. 2000b), raising the possibility that aggression-related reward was derived from events that occurred following the aggressive interaction in our paradigm. Unfortunately, the pattern of dopamine turnover and/or release during the minutes and hours following aggressive interactions has yet to be described in anoles.
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Changes in the plasma levels of hormones such as testosterone (T) following aggressive interactions represent potential postaggression sources of aggression-related reward. A potential for T as a primary reinforcer for aggressive behaviour is intriguing for two reasons. First, in birds (Wingfield & Wada 1989; Wingfield & Hahn 1994), plasma levels of T can become elevated following aggressive encounters, and in A. carolinensis (Greenberg & Crews 1990), winners of staged aggressive interactions had higher androgen titres than both losers and control animals 1 h after the initiation of the interaction. The androgen levels of the winners subsequently declined and were not significantly different from those of control animals when examined both 1 day and 1 week later. Second, there is evidence indicating that T administration can act as a rewarding stimulus. Rats will form conditioned preferences for contexts associated with T injection (e.g. Alexander et al. 1994; Packard et al. 1998). Furthermore, the rewarding effect of T is rapid; conditioning occurs within 30 min of injection. This rapid effect suggests that a nongenomic action underlies the rewarding effect of this compound and increases the likelihood that postaggression surges in T could have served as a rewarding stimulus in our paradigm. Exogenous testosterone administration also yields a rapid increase in striatal dopamine levels in male jackey dragons, Amphibolurur muricatus, another territorial lizard species (Watt et al. 2002). Furthermore, Yang & Wilczynski (2002) found that the relationship between aggression and plasma androgens could best be explained by a model in which expressing aggressive displays mediated plasma androgen levels rather than vice versa. Involvement of a postaggression surge of T in aggressionrelated reward could explain why, in our paradigm, we observed CPP only in those animals that acted aggressively towards the mirror. This discussion has been based, at least in part, on the assumption that the animals that responded aggressively towards their reflection during conditioning perceived the outcome of the interaction as a victory following the disappearance of the mirror and thus the perceived opponent. It is also possible, however, that the male anoles may have perceived the outcome of the interaction as a draw since neither the aggressive animal nor its reflection behaved or changed skin colour in ways that are indicative of a submissive animal. If the anoles perceived the conflict as ending in a draw, the opportunity to resolve the conflict during subsequent encounters may have been the rewarding stimulus that caused the aggressive anoles to increase the time that they spent in the side of the apparatus paired with the mirror. Such an explanation might explain the apparent contradiction between our finding that engaging in aggressive interactions may be rewarding for male anoles and prior reports in this species indicating opponent recognition and reduced aggression during subsequent interactions with the same individual once a social hierarchy has been established (e.g. Forster et al. 2005). However, the results of particular dyadic interactions can alter the subsequent behaviour of both the winner and the looser. Therefore, reductions in aggressive behaviour across successive interactions between the same two combatants might be mediated more by the
submissive or avoidance behaviour of the initial loser rather than by a propensity by the prior winner to become less aggressive to a familiar combatant. Furthermore, the ‘mirror opponent’ to which the nonaggressive animals were exposed also disappeared when the mirror was removed during the conditioning sessions. Therefore, the nonaggressive anoles also experienced an unresolved interaction during the conditioning sessions, but they did not subsequently show an enhanced preference for the side of the CPP apparatus paired with the mirror during conditioning. The results suggest that engaging in an aggressive interaction can be rewarding for green anoles. Yang et al. (2001) showed that experience with aggressive interactions yielded increased aggression to novel challengers. Our results suggest that aggression-related reward may underlie that phenomenon. We also found that prior experience gained during simulated aggressive encounters can lead to the establishment of a place preference for the context and location where the previous aggressive encounter occurred. Based on these findings, aggression-related reward and reinforcement may thus relate to territoriality in two ways: (1) by increasing aggression towards future challengers and (2) by establishing site preferences for the territory itself or locations within it. Future experiments manipulating androgens and/or the dopamine system can address the physiological and neural mechanisms of this process by elucidating the influences of endogenous testosterone and behaviourally triggered testosterone or dopamine surges following aggressive interactions. The results of such studies, combined with additional experiments examining the specific behavioral changes that occur between pairs of anoles during successive aggressive interactions in both the field and laboratory settings, should help to further our understanding of the behavioural and physiological regulation of aggressive behaviour and of its consequences regarding territory formation. Acknowledgments Support for this research was contributed by National Institutes of Health F32MH067292-01 to W. J. Farrell and National Science Foundation IBN 0090739 to W. Wilczynski. References Alexander, G. M., Packard, M. G. & Hines, M. 1994. Testosterone has rewarding affective properties in male rats: implications for the biological basis of sexual motivation. Behavioral Neuroscience, 108, 424–428. Archer, J. 1988. The Behavioural Biology of Aggression. New York: Cambridge University Press. Azrin, N. H., Hutchinson, R. R. & McLaughlin, R. 1965. The opportunity for aggression as an operant reinforcer during aversive stimulation. Journal of the Experimental Analysis of Behavior, 8, 171–180. Bardo, M. T. & Bevins, R. A. 2000. Conditioned place preference: what does it add to our preclinical understanding of drug reward? Psychopharmacology, 153, 31–43. Baxter, L. R., Ackermann, R. F., Clark, E. C. & Baxter, J. E. 2001. Brain mediation of anolis social dominance displays. I. Differential basal ganglia activation. Brain, Behavior and Evolution, 57, 169–183.
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