Aberrant responses in social interaction of dopamine transporter knockout mice

Behavioural Brain Research 148 (2004) 185–198

Research report

Aberrant responses in social interaction of dopamine transporter knockout mice Ramona M. Rodriguiz a , Richard Chu a , Marc G. Caron b , William C. Wetsel a,∗ a

Departments of Psychiatry, Behavioral Sciences, Cell Biology, and Medicine, Mouse Behavioral and Neuroendocrine Analysis Core Facility, Duke University Medical Center, Durham, NC 27710, USA b Departments of Cell Biology and Medicine, Duke University Medical Center, Howard Hughes Medical Institute, Durham, NC 27710, USA Received 25 January 2003; received in revised form 19 May 2003; accepted 20 May 2003

Abstract The dopamine (DA) transporter (DAT) controls the temporal and spatial resolution of dopaminergic neurotransmission. Disruption of the Dat1 gene in mice leads to increased extracellular DA concentrations and reduced expression of D1- and D2-like receptors in striatum. The mutants are hyperactive in the open field and they display deficits in learning and memory. In humans, dopaminergic dysfunction has been associated with a number of different psychiatric disorders and some of these conditions are accompanied by abnormal social responses. To determine whether social responses were also impaired in DAT knockout (KO) mice, behaviors of group- and isolation-housed animals were compared. All group-housed animals readily established hierarchies. However, the social organizations of the mutants were changed over time. Under both group- and isolation-housed conditions, mutants exhibited increased rates of reactivity and aggression following mild social contact. In isolation, exposure to a novel environment exacerbated these abnormal responses. Regardless of housing context, stereotyped and perseverative patterns of social responses were a common feature of the KO repertoire. In fact, many abnormal behaviors were due to the emergence and predominance of these inflexible behaviors. These data suggest that KO mice may serve as a useful animal model for understanding not only how DA dysfunction contributes to social abnormalities, but also how behavioral inflexibility distorts their social responses. © 2003 Elsevier B.V. All rights reserved. Keywords: Dopamine; Dopamine transporter; Mice; Psychiatric disorder; Social interaction

1. Introduction Dopamine (DA) is a catecholamine that is present in both the central and peripheral nervous systems and it has been shown to regulate a variety of behaviors [35]. Aberrations in DA neurotransmission are an important factor contributing to a broad spectrum of psychiatric illnesses that are characterized by abnormalities in mood, motor activity, and the fragmentation of thought processes [14,45]. These clinical disturbances can include schizophrenia or the schizotypical disorders, problems related to substance abuse, affective disorders, and pediatric conditions including attention-deficit hyperactivity disorder (ADHD) and related disruptive behavior disorders [1]. Although each condition represents a discrete etiology, nearly all of these disorders include inappropriate social interactions that can lead to a combative ∗

Corresponding author. Tel.: +1-919-684-4574; fax: +1-919-684-3071. E-mail address: [email protected] (W.C. Wetsel).

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behaviors or heightened states of aggression [6,28,50,54]. While little is known about the precise nature of the neural mechanisms that may underlie the abnormal social responses in these patients, amelioration of violent outbursts and agonistic behaviors has been achieved with a number of agents that affect DA neurotransmission [41,45]. These findings suggest that some features of social behavior are regulated by the DA system, and that abnormalities in DA functioning can influence the propensity of an individual to exhibit an aggressive or combative phenotype. Through homologous recombination, a strain of mice has been produced in which the gene encoding the DA transporter (DAT) has been disrupted [24]. The DAT knockout (KO) mice present a behavioral phenotype that represents an exaggerated version of a condition achieved in animals and humans that are chronically exposed to high levels of psychostimulants [20,24]. The KO animals exhibit a marked hyperactivity when placed into a novel environment, as well as heightened rates of stereotypic and perseverative behaviors

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[20]. Besides these behavioral anomalies, there are also a number of different neurochemical and biochemical changes that characterize this mutant. For instance, while tissue concentrations of DA are decreased 20-fold, synthesis rates are increased [24]. Extracellular levels of striatal DA are increased at least 5-fold and the clearance of catecholamine is greatly prolonged compared to that of wild type (WT) controls. Besides the hyperdopaminergia, expression of D1- and D2-like receptors is suppressed [24,29] and the presynaptic D2 autoreceptors are desensitized and down-regulated [31]. Recently, changes in the sensitivity and density of DA receptors have been shown to be important regulators in the expression of aggressive behavior in mice [5,22,23,47]. Chronic administration of psychostimulants also produces high rates of aggression in mice, as well as stereotypic motor routines and fragmented behavioral interactions [19,42]. The relationship between increased DA transmission and aggressive behaviors suggest that social interactions may be perturbed in the KO mice. Since the KO mice present a number of different behaviors and biochemical changes that might be anticipated to contribute to altered social interactions, behaviors of these animals and their WT controls were evaluated in four different social paradigms. A test of social organization in group-reared animals was used to determine whether KO mice can form social hierarchies and whether these hierarchies are stable across time. The social-hierarchy rankings were further confirmed by tube-dominance testing. Isolation-induced aggression was examined both in the home cage and in a novel environment. Finally, the appearance and rates of repetitive and stereotypic responses were studied across all of the testing contexts.

2. Materials and methods 2.1. Animals The WT and KO littermates were generated from C57BL/6J–129/SvJ hybrid DAT heterozygotes that had been intercrossed for more than 15 generations as previously described [24]. Animals were weaned at 21–30 days of age and housed in groups of four segregated by sex and genotype. Between 10 and 16 weeks of age, mice were divided into four groups. The first group was used for hierarchy and tube-dominance testing and housed four per cage. A second group was assigned to urine and lemon/almond olfactory discrimination testing; animals were housed individually and tested approximately 2 weeks later. A third set of mice was evaluated for social transmission of food preferences and was housed two per cage. A final group was placed in isolation and tested at least 2 weeks later in the resident–intruder test; the same mice were tested 7–10 days later in the dyadic paradigm. Male C3H/HeJ mice (Jackson Laboratories, Bar Harbor, ME) were used as test partners in the resident–intruder

and social dyadic testing (see [23]). These animals were housed in groups of four to five and were between 9 and 12 weeks of age at the time of testing. Visual acuity of the C3H/HeJ mice was assessed 24 h before testing (see [48]). Briefly, mice were placed on a pedestal in the middle of a 20 cm × 18 cm × 20 cm circular arena and were presented with a black and white patterned back of an infant spoon. The spoon was passed through the mouse’s visual field six times in a left–right counterbalanced order. C3H/HeJ mice that oriented to the spoon on at least five of six trials were used for social testing. Standard laboratory chow and water were available ad libitum. Animals were housed in a humidity- and temperaturecontrolled room with a 14 h/10 h light/dark cycle (lights on at 07:00 h). All studies were conducted with an approved protocol from the Duke University Institutional Animal Care and Use Committee and were in accordance with the NIH guidelines for the Care and Use of Laboratory Animals. 2.2. Genotyping Mice were genotyped by PCR using sense WT (5 -CCCGTCTACCCATGAGTAAAA-3 ), sense KO (5 -TGACCGCTTCCTCGTGC-3 ), and a common antisense primer (5 -CTCCACCTTCCTAGCACTAAC-3 ). Reactions were run with purified DNA at 94 ◦ C for 30 s, 62 ◦ C for 60 s, and 72 ◦ C for 60 s at 35 cycles. The PCR products were 550 and 850 bp for the respective WT and KO alleles. 2.3. Olfactory discrimination tests Olfaction is an important component of social behaviors in mice [34]. Odorant discrimination tests were run with mice that had been housed individually for at least 2 weeks. Animals were compared for their abilities to discriminate olfactants from pooled urine samples [4]. The comparisons included urine of unfamiliar group-housed C3H males to saline, urine from estrus females to saline, and male vs. female urine. Fifty microliters of urine was pipetted onto 1 cm × 1 cm cotton wadding and was placed into Tissue-Tek cassettes (3 cm × 4 cm × 0.4 cm; VWR Scientific, Bridgeport, NJ) mounted on opposite walls of the home cage, 1 cm above the floor. The time spent with each cassette was coded for 5 min using the Noldus Observer (Noldus Information Technology, Leesburg, VA). Selection was defined as the mouse sniffing or manipulating the cassette, or being within 1 body length of the cassette and oriented towards it. One week later, an olfactory place preference test was run where animals had to discriminate between lemon and almond scents (Simply Organic, Boulder, CO) (see [26]). The mouse 3-chambered place preference apparatus (Med-Associates, St. Albans, VT) was equipped with photocells to monitor activity and location of the mouse; ground 5015 diet (PMI Nutrition International LLC, Brentwood, MO) was the reinforcement. Mice were maintained

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at 95% body weights and were acclimated to the apparatus for 30 min over 5 days to insure that no chamber preferences developed before conditioning. Thereafter, mice were randomly assigned to groups where on alternate days lemon scent was paired with chow and almond was paired with no food for 16 days; or vice versa. A 100 ␮l aliquot of the odor was placed in the center of a tray below the floor. The location of the reinforced chamber was randomized across animals. On the day following conditioning, mice were tested for place preference without food for 30 min. Preference for the conditioning odor was calculated as the time spent in the chamber previously paired with the food relative to the total time spent in both chambers. Social transmission of food preferences was used as a third discrimination test (see [44]). Males of the same genotype and weight were housed two per cage and were deprived of food for 16 h prior to test. One mouse was designated as a “demonstrator” and the other as “tester”. Flavored diets were prepared by mixing 50 g of crushed rodent chow (5001 diet) with 0.1 ml almond or lemon flavoring (Simply Organic, Boulder, CO), 1 g of granulated sugar, and 50 ml of water to produce a mash. Testing began with placing the demonstrator into a standard mouse cage with two bowls each containing either 2 g of lemon or almond-flavored chow with the choice of flavoring block-randomized across genotype. The demonstrator ate for 30 min and was immediately returned to its home cage. Social exchanges between the demonstrator and tester were observed for 20 min. Subsequently, the tester was placed into a clean cage containing one dish each of almond- and lemon-scented food. Mice were allowed to freely feed for 60 min. Following testing, the amount of food remaining in each dish was weighed and any spillage was noted. Preference for a given flavored diet was determined by calculating the differences between the amounts of each flavored diet consumed relative to the total amount consumed during the test. No preference for either diet produced preference scores equal to or approaching zero; scores approaching or equal to 1 indicated a preference for one diet over the other. 2.4. Social-hierarchy testing Mice from each genotype were ranked according to weight and assigned to groups of four animals each with similar weights that had never been previously housed together. Two hours (09:00 h) after onset of the light cycle the next day, mice were housed with their newly assigned group and observed for 20 min under standard lighting conditions. Social interactions were coded for 10 min at 2-h interval until the onset of the dark cycle (21:00 h), producing a total of 80 min of observations for the first day of testing. Behavioral observations were continued between 10:00 and 21:00 h for 5 additional days in the same fashion. A continuous coding method was used where the sequence of events between mice was coded beginning with the initiator of the social exchange, the type of behavior observed, and

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the response of the recipient(s) [3]. Subsequent responses were continuously recorded between animals until the end of the social exchange. Behaviors for each mouse were summed over each day for mild social investigation (MSI), stationary reactivity (SR), locomotor reactivity (LR), and aggressive behaviors expressed as rate/h. Dominance matrices were constructed based upon a time lag-1 transitional frequency matrix [3] where the behaviors of each target mouse (i.e. each member of the social group) towards its cage-mates are represented as the rate of behavioral events that were either initiated or received by that target. A 4 × 4 matrix was constructed for each cage representing the initiator or recipient status for animals on each test day and behavioral category. The conditional probability for each mouse was calculated as the total number of initiations by that animal for a particular behavioral category minus the number of behavioral receipts by the same mouse within the same category. This difference was divided by the total number of social interactions for that animal [(initiations − receipts)/total interactions]. Each animal was ranked within its social group based upon the conditional probability scores for aggression (i.e. a composite of agonistic, threatening behaviors, and attacks). Individual mice with high scores for aggression (i.e. animals that initiated more attacks than it received) were ranked as dominant (rank 1). The remaining animals were ranked as either active (rank 2) or passive (rank 3) subordinate, or submissive (rank 4). Active-subordinate mice (rank 2) initiated more or equal numbers of aggressive acts relative to those received; however, their scores were lower than the dominant animal. Passive-subordinate mice (rank 3) were ranked higher than submissive animals and these animals initiated fewer instances of aggressive behavior than active-subordinate males. Individual mice with the lowest aggression scores were ranked as submissive (rank 4). In certain cases, animals from the same cage were assigned the same rank-position within the hierarchy (i.e. ties). In situations where no aggressive behavior was evident, all animals were given their rank from the previous day. From the social rankings, changes in the hierarchy positions for each mouse were calculated by time-series analysis [3]. Briefly, the cumulative differences in the social ranks between adjacent days were calculated for each animal. A zero score indicated no change in ranking, whereas positive scores were indicative of an increase and negative scores a reduction in social status. In addition, the type of social hierarchy formed within each social group was based upon the individual rankings of the mice and determined to comprise either a 4-, 3-, or 2-stage hierarchy (see [52]). A 4-rank hierarchy involved dominant, active-subordinate, passive-subordinate, or submissive animals. A 3-rank system included a dominant mouse with either active or passive subordinates, but no submissive animals. Two-rank hierarchies included those cages with a single dominant mouse and the remaining positions comprised of either submissive or passive-subordinate animals.

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2.5. Tube-dominance testing Following assessment of the social hierarchy, dominance was further evaluated in a tube-dominance test. This apparatus consisted of clear PVS tubing (3 cm in diameter and 45 cm long) that was anchored to a solid wooden base. All testing was performed at <40 lx between 10:00 and 16:00 h. In order to relate the social-hierarchy rankings to the tube-dominance performance, each mouse was paired with each of its cage-mates for three trials. All tests within a social group were randomized across cage-mates so that a given mouse did not encounter the same animal twice in succession. Social behaviors were continuously coded for each animal until one of the mice exited the tube or 120 s elapsed. The submissive mouse was identified as that animal which first withdrew from the tube [37]. If no animal exited the tube, the trial was coded as a “tie” for each mouse. The frequencies of behaviors initiated in the tube test by each mouse were analyzed by genotype as a function of the animal’s social status in the home cage on the last day of social-hierarchy testing. These behaviors included agonistic behaviors and pushing. Agonistic behaviors included all instances of biting, clawing, tail-rattling, forcing the test partner to retreat, or chasing the opponent from the tube. Mice were also ranked by the number of times they forced their cage-mates from the tube. In the case of ties, animals were each assigned a mid-point between the two tied ranking positions. 2.6. Resident–intruder and dyadic testing WT and KO mice were housed individually for at least 2 weeks before the experiments and bedding was not changed during this time. Animals were matched by weight to an unfamiliar group-housed C3H/HeJ male. C3H/HeJ animals were selected as social partners due to the high amounts of social investigation these mice provide without initiating unprovoked agonistic or attack behaviors [23]. Social tests were conducted at least 2 h following the onset of the dark cycle in dim (<20 lx), indirect lighting. Tests were terminated when a mouse escaped from the test chamber, when attacks continued for more than 60 s, or when an animal was injured. All social interchanges were filmed and coded live by trained observers who were blind to the genotypes of the animals. Mice were first tested in the intruder assay where a C3H/ HeJ male was introduced into the home cage (29 cm × 18 cm × 13 cm) of a WT or KO mouse where the food, water, and wire-bar lid were removed from the cage and only the filter top was replaced to reduce the amount of upright and unsupported rearing. Following introduction of the intruder, all behavioral interchanges were coded in 5-s blocks over 5 min. At the end of this period, the C3H/HeJ intruder was removed to a clean cage. All WT and KO cages were cleaned after testing and were left undisturbed until dyadic testing 7–10 days later.

In the dyadic test, each WT and KO mouse was paired with an unfamiliar C3H/HeJ animal in a novel observation chamber. Chambers were cleaned prior to and between each test with Anlage detergent (Quip Laboratories, Wilmington, DE) and dried. Clean irradiated 1/8 in. cob bedding (Andersen Inc., Maumee, OH) was placed into the 48.3 cm × 26.8 cm × 20.3 cm cage (Allentown Caging, Allentown, NJ) before testing. Mice were placed at opposite ends of the cage and separated by a solid partition. Following 5 min of acclimatization, the barrier was removed and the animals were allowed to freely interact for 5 min. All behavior was coded as described above. Since a small proportion of the tests ended with the partner escaping from the dyadic test chamber, all behaviors are calculated as a rate per 5 min [(no. of incidences/total test time in s) × 300 s]. For both intruder and dyadic tests, genotypic differences were analyzed for each category of social behaviors (see below). 2.7. Behavioral coding for social interactions Inter-observer (Cohen’s κ = 0.90–0.92) and intra-observer agreement (Cohen’s κ = 0.95–0.98) were determined for the 18 behaviors coded in the social-hierarchy, resident–intruder, and dyadic tests, and the six behaviors reported in tube-dominance testing. Social interaction events were coded where a single social interaction was defined as beginning with contact of the test partner by either the WT or KO mouse and continuing until an animal disengaged from the exchange (see [3]). Disengagement was defined as when an animal turns-away or leaves the proximity of its partner. Social behaviors were scored by specifying the initiator of the social interaction, behavior performed, and response of the partner [11,23,27]. For all the experiments except tube-dominance testing, 18 behaviors were coded and organized into five behavioral domains representing MSI, SR, LR, threatening postures (TP), and aggression/attack behaviors. MSI involved three non-aggressive interchanges that included approaching, sniffing, or nosing the anogenital region of the other mouse. SR comprised five interactions that included boxing (upright posture with the front paws held-out defensively, but not in contact with the other mouse), holding (a posture similar to boxing, but the front paws are in contact with other mouse), vocalizations, kicking, and eye closing. LR involved jumping or rapid escaping following social investigation by the C3H partner. While mice will perform these behaviors as defensive acts immediately following an attack, both forms of reactivity for the purposes of this study were limited to instances of SR and LR that occurred following social investigation by the partner mouse. TP included all instances of climbing on the opponent mouse that were met with five different reactive or defensive postures: feinting (a rapid approach that appears to be the initiation of an attack, but terminates prior to physical contact between mice), clawing, lunging or chasing, and tail-rattling. Agonistic behaviors comprised three responses: pinning the opponent to the floor of the

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chamber, biting, and attacking (rapid lunge which results in knocking-down, biting, and clawing) the partner. For social-hierarchy testing, the aggressive behavior included all instances of threatening or agonistic behaviors, and attacks. An additional category of behaviors included rapid retreating responses with a hunched or contracted body posture without kicking or threatening the partner, lowering the head to a level below that of the approaching mouse, or turning away with the head down in response to social investigation by a cage-mate. 2.8. Repetitive social behaviors Social behaviors were re-examined for the appearance of repetitive behaviors in the social-hierarchy test where mice were group-housed and in the resident–intruder and dyadic tests with isolated animals. Repetitive behaviors were defined as any single social behavior that was executed two or more times in succession without an intervening behavior from a testing partner. All non-social stereotyped behaviors (e.g. face-wiping, digging, isolated jumping, etc.) were excluded. Besides single social behaviors, animals’ responses were also examined for evidence of repetitive sequences or patterns of social behaviors in the different test conditions. All responses were expressed as a frequency of stereotyped or perseverative behaviors over time. 2.9. Statistics All data are presented as the mean ± S.E.M., except for the hierarchy results that are expressed as the total percent of cages that exemplified a particular organization on a given test day. All analyses were performed with the Statistical Package for the Social Sciences 9 (Chicago, IL). For analyses of olfactory responses, differences in the preference scores between WT and KO tester mice were analyzed by an analysis of variance (ANOVA) test. A posteriori analyses were run with a Bonferroni test where a P < 0.05 was considered significant. Strength of preferences for particular odorants within each genotype was examined using a t-test for independent measures. Changes in social rank were analyzed with multivariate analyses of variance (MANOVA). Individual changes in hierarchy status across the 6 test days were analyzed with repeated measures ANOVAs within each genotype. The individual cage ranks for each animal on day 1 served as the independent/predictor variable for the analyses. A change in the type of hierarchies constructed across test days was analyzed with χ2 analyses. To determine whether a positive relationship existed between the tube-dominance behaviors and social rank of the mouse within the home cage, the day 6 rankings for each mouse in the hierarchy were recoded so that the most dominant animal was ranked as ‘1’, subordinates as ‘2’, and submissive mice as ‘3’. ANOVAs were used for analyzing the effects of genotype and cage rank on the total as-

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sertive, agonistic, and pushing behaviors performed in the tube-dominance test. Post hoc Bonferroni comparisons were used to examine the effects of cage rankings on the performance of these behaviors. Differences between WT and KO mice in the resident– intruder and dyadic tests were examined with univariate ANOVA for MSI, SR, LR, TP, attacks, and the total number of social initiations. Interactions between the main effects of testing context and genotype were explored with a two-way multifactor ANOVA. To control for the possibility that dissimilarities between WT and KO mice were contingent upon the frequency of behaviors executed by the test animal, an analysis of covariance (ANCOVA) was performed. For these analyses, the frequency of total behavioral initiations was designated as the covariate and genotype as a fixed factor. As a complementary analysis to the ANCOVA, linear regression analyses were used where behavioral initiations and genotype were entered as the coefficients of the model in a step-wise order. Changes in r2 were reported for the addition of each coefficient into the model.

3. Results 3.1. Olfactory discrimination test Pheromone and other odorants are powerful regulators of social interaction [16,34,36]. Both WT and KO males spent significantly more time investigating the male urine [F(1, 35) = 178.09, P < 0.001] or female urine [F(1, 35) = 390.8, P < 0.001] than saline (Fig. 1A). However, the WT controls engaged the female urine approximately 17% longer than mutants [t(1, 17) = 2.56, P < 0.02]. Despite this fact, both WT and KO males preferred urine from females than males. These data show that both WT and KO males can discriminate olfactory stimuli. To be meaningful within the context of social interactions, animals must be capable of making an association between the olfactory cue and a particular event [34]. To test this ability, olfaction was evaluated in a place preference paradigm. During the acclimatization period, no chamber preference was noted for either genotype (Fig. 1B). Following conditioning, both WT and KO mice preferred the odor that had been previously paired with food and no genotypic differences were discerned in the strength of this preference. These data support the idea that KO males can make olfactory discriminations and that they are able to associate these stimuli with particular events. Since social interaction and aggressive behavior in mice are contingent upon olfactory recognition [34,36], WT and KO mice were housed in pairs and tested for social transmission of food preferences. Results from pilot studies revealed that both lemon- and almond-flavored diets were equally consumed by both genotypes. When exposed to flavored chow after 16 h of food restriction, the demonstrators rapidly

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3.2. Social-hierarchy testing Differences in social interactions between WT and KO males were evaluated under group-housed conditions. During the first 20 min after placement with unfamiliar animals, both WT and KO animals showed equally high rates of social investigation (WT: 13.4 ± 2.8, n = 8; KO: 12.0 ± 2.9, n = 8), low rates of reactivity (WT: 1.6 ± 0.9, n = 28; KO: 2.5 ± 1.8, n = 28), TP and aggressive behaviors (WT: 2.9 ± 1.9, n = 28; KO: 4.1 ± 2.4, n = 28). When summed across all observations on the first test day, no genotype differences in MSI were noted (Fig. 2A). Additionally, both genotypes showed the same magnitude of reductions of MSI on day 2. Although the mutants maintained these attenuated MSI rates across subsequent test days, the WT males exhibited an increase instance of these behaviors on days 3–5 [MANOVA; Fday 3 (1, 50) = 5.61, P = 0.03; Fday 4 = 7.23, P = 0.02; Fday 5 = 12.94, P = 0.003]. These high rates were not sustained such that by the sixth day of testing, MSI for the WT males were reduced to mutant levels.

Fig. 1. Analyses of olfactory responses in WT and KO mice. (A) Percent of time spent investigating urine from group-housed C3H males vs. saline, urine from estrus females vs. saline, and female vs. male urine. WT, open bars; KO, filled bars. ∗ P < 0.05 from WT mice. (B) Olfactory place preference discrimination by WT and KO mice. Differences in time spent between the chambers during the pre-conditioning (WT, open bar; KO, gray bar) and test phases are displayed (WT, striped bar; KO, gray striped bar). ∗ P < 0.05 from pre-conditioning phase. (C) Preferences for the familiar- or novel-scented food in the transmission of food preference test. WT, open bar; KO, filled bar.

approached the diets (WT: 5.6 ± 2.1 s; KO: 6.5 ± 2.4 s), and consumed similar amounts of chow (WT: 0.539 ± 0.09 g, n = 8; KO 0.420 ± 0.12 g, n = 8) regardless of flavor. During 20 min of interaction with the demonstrator in the home cage, WT and KO test mice were equally likely to approach and sniff the demonstrator’s mouth (data not shown). When the test mouse was given a choice between the “familiar-” and a novel-flavored diet immediately after the social interaction, both WT and KO mice exhibited strong preferences for one of the diets (Fig. 1C). The WT animals preferred the familiar diet (WT: 0.62 ± 0.10, n = 8); mutants preferred the novel-flavored diet (KO: −0.64 ± 0.04, n = 8). There were no differences in the total amounts of food consumed by either genotype. These findings demonstrate that both WT and KO can recognize different odors; however, the KO mice prefer the novel- rather than familiar-flavored food.

Fig. 2. Rates of social investigation, reactivity, and aggression in the home cage. (A) Incidences of MSI between group-reared male mice as a function of genotype across 6 days of testing. (B) Reactivity rates (LR and SR) in response to social investigation by WT and KO cage-mates. (C) TP and attack behavior for WT and KO males. All results are presented as the mean rate per cage per day. WT mice are designated by an open circle, and KO males by a closed triangle (n = 28 males per genotype). ∗ P < 0.05 for WT mice.

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Since less than 5% of the observed social interactions included jumping or escape behaviors (i.e. LR), all instances of SR and LR were collapsed and analyzed as a single variable (Fig. 2B). Although reactivity was initially higher for the mutants on day 1 relative to WT controls [F(1, 50) = 3.52, P = 0.05], no differences were discerned on day 2. While reactivity was attenuated and maintained from test day 3 onwards, KO mice consistently demonstrated higher rates of vocalization, holding, boxing, and eye-closing compared to WT controls [Fday 3 = 14.41, P = 0.002; Fday 4 = 3.81, P = 0.05; Fday 5 = 3.162, P = 0.05; Fday 6 = 8.91, P = 0.01]. The KO animals were significantly more aggressive following the formation of new social groups on day 1 than the WT males [Fig. 2C; F(1, 50) = 3.62, P = 0.05]. After this time, aggression between cage-mates was reduced for both genotypes over the next two days. By the fourth day, mutants consistently showed higher levels of agonistic behaviors than the WT controls [Fday 4 (1, 50) = 3.65, P < 0.05; Fday 5 = 4.210, P < 0.04; Fday 6 = 6.10, P < 0.03]. Together, these data show that group-housed WT male engage primarily in MSI, while KO animals show reactivity and aggression. 3.3. Establishment and stability of the social hierarchy In group-housed conditions, mutants rarely engaged in mild social exchanges after the first encounter and were more likely to continue initiating reactive and aggressive behaviors with their cage-mates than WT controls. These observations suggest that establishment and maintenance of social hierar-

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chies may be perturbed with KO animals. To address this issue, the data were re-analyzed with respect to the amount of aggression initiated by a mouse towards its cage-mates relative to the agonistic responses it received across a given day. From these observations, animals were assigned daily ranks and were classified as dominant, active subordinate, passive subordinate, or submissive (see Section 2 for definitions). All WT and KO mice established a dominance hierarchy within the first day (Fig. 3A and B). Over time, the hierarchy between group-reared WT males remained stable with most of cages displaying 4-ranked organizations (Fig. 3A). In striking contrast, although 100% of the KO cages established a 4-rank hierarchy on day 1, the rankings varied across days with this arrangement being displaced with a 2to 3-rank hierarchy, and the 2-rank hierarchy predominating over time (Fig. 3B). Hence, by the end of testing, approximately only 25% of the mutants’ cages had a 3- to 4-rank system, whereas the remaining KO cages exhibited a more stratified rank organization consisting of a single despot animal (χ2 = 84.65, P < 0.001). Collectively, these data suggest that although group-reared mutants can initially establish a well-defined social organization, these hierarchies are unstable and can transform rapidly over time. By comparison, the WT males establish and maintain a 4- or 3-rank hierarchy system. To investigate individual changes within the dominance hierarchy, the average rank shifts for the WT and KO mice were plotted daily relative to each animals’ cage rank on day 1. Although few shifts in social dominance were observed among the WT males (Fig. 3C), significant changes

Fig. 3. Analyses of social hierarchies for group-reared WT and KO mice. (A) The percentage of WT mice housed in social groups characterized by one of three hierarchal systems across 6 test days. Four-rank systems are shown with a black bar, 3-ranked systems as light gray bars, and 2-ranked systems as white bars. The data are displayed as the percent of individual animals that have organized their dominance hierarchy by rankings of 2, 3, or 4 compared to the total number of mice observed. (B) The percentage of KO males in social groups characterized by the three hierarchal systems as a function of test day. (C) The daily shifts in dominance rank of WT males (open symbols) as a function of their day 1 cage-rank. (D) The daily rank shifts of KO males (filled symbols). On day 6, dominant mice are shown as diamonds, active subordinates as triangles, passive subordinates as squares, and submissives as circles.

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in daily rankings were found for the mutants [Fig. 3D; Frepeated (3, 51) = 3.75, P < 0.016] and these changes were predictable based upon the animal’s ranking on day 1 [Frepeated (3, 28) = 8.45, P < 0.001]. Notably, dominant males were more likely to experience a decline in social status, such that they became subordinate at the end of testing. Concurrently, subordinate KO males on day 1 typically achieved dominance status within the group by day 4. Although KO mice are more likely to experience significant changes in their rankings within the hierarchy across time, these data show that the changes are not random but are predictable based upon their initial status within the social group. 3.4. Tube-dominance testing Since mice can recognize dominant and subordinate animals across different testing contexts [34], both genotypes were tested in a tube-dominance test following completion of social-hierarchy testing. Testing consisted of introducing two cage-mates simultaneously into opposite ends of a clear tube and monitoring all instances of interactions between the two animals. A mouse was considered to “win” if it was the last to leave the tube. The hierarchy rankings in group-housing on day 6 were used as a basis of comparison for dominant or submissive behaviors in the tube-dominance test. The propensity of the mice to exhibit assertive behaviors in the tube-dominance test was found to be dependent upon both genotype and its position in the hierarchy on day 6 of testing (Fig. 4). Overall, WT males initiated more assertive responses than KO animals during tube testing [Fig. 4A; F(1, 55) = 16.57, P < 0.001]. These included clawing, tail-rattling, biting, chasing, pushing, or forcing their cage-mate to retreat from the tube. However, when the rates of assertive behaviors were examined as a function of the animal’s hierarchy status in the home cage, only the mutants demonstrated significant differences in the occurrences of these behaviors as a function of cage rank [F(1, 55) = 6.65, P < 0.003]. Post hoc analyses revealed that dominant KO males displayed more assertive responses than the subordinate (P < 0.003) and submissive (P < 0.004) animals. Although there is a tendency of the WT males to show a similar relationship between dominance status and assertive behaviors, the differences did not reach statistical significance. Finally, both subordinate and submissive WT males showed higher frequencies of these behaviors relative to KO males of the same rankings (P < 0.02). To examine the basis of differences in assertive behaviors, responses were divided into agonistic (e.g. clawing, tail-rattling, biting, and chasing) and pushing behaviors (Fig. 4B and C). With respect to agonistic behaviors, WT animals had a stronger tendency to initiate these responses than KO mice [F(1, 55) = 3.33, P < 0.06]. Cage ranks on the sixth day of hierarchy testing were found to be significant predictors of aggression for both genotypes in the tube-dominance test [F(1, 55) = 7.69, P < 0.001]. When

Fig. 4. Social behaviors in the tube-dominance test for group-reared WT and KO males. (A) Total assertive behaviors (composite of agonistic and pushing behaviors) committed by WT and KO mice. (B) The frequency of agonistic behaviors by WT and KO animals. (C) The average rate of pushing behaviors by WT and KO males. All data are shown as the mean rates initiated per mouse over testing as a function of dominant, subordinate, and submissive social status on day 6 of testing in the home cage. WT males are shown as open bars and KO animals as closed bars. ∗ P < 0.05 from dominant KO mice.

data from WT and KO males were examined in separate univariate ANOVAs with cage rank as the fixed factor, only the mutants showed changes in aggressive behavior [F(1, 27) = 6.32, P < 0.006]. The dominant KO animals exhibited the highest levels of agonistic behaviors relative to both subordinate (P < 0.04) and submissive (P < 0.02) mice with subordinates initiating more agonistic responses than submissives (P < 0.03). With respect to pushing, WT mice engaged in similar amounts of this behavior regardless of social rank (Fig. 4C). By contrast, a strong relationship was observed among KO animals with respect to pushing and to their home-cage rank [F(1, 27) = 3.45, P < 0.05]. Here, submissive animals displayed the lowest rates of this behavior (P < 0.05). These results demonstrate that expression of agonistic and pushing behaviors in the tube-test for KO mice is highly dependent upon the social rank of the animal within its home cage; this same relationship only applies to agonistic responses for WT animals.

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3.5. Resident–intruder and dyadic testing Aside from examining animals under group-housing conditions, social behaviors can be studied in isolated mice in the resident–intruder paradigm that makes use of the familiar environment and the dyadic test that utilizes a novel test context. Here, mice are housed individually for at least 2 weeks prior to the tests. The mutants showed at least a 2-fold increase in the initiation of single social behaviors towards the test partner compared to WT males [F(1, 57) = 135.76, P < 0.001]. Although the initiation of social behaviors was increased by 18% for WT animals in the dyadic test (Fig. 5A), rates for mutants were augmented by 25% yielding a significant genotype and test-context interaction [F(1, 57) = 4.39, P < 0.04]. Once the WT or KO animals made social contact in the intruder or dyadic tests, mutants displayed at least twice as many behaviors per interaction compared to the WT controls [F(1, 57) = 33.23, P < 0.001, data not shown]. These data show that the KO mice not only engage in more social behaviors towards unfamiliar part-

Fig. 5. Social behaviors by isolate-housed WT and KO males in the resident–intruder and dyadic paradigms. (A) The total social behaviors initiated by either WT or KO males comprising of MSI, SR, LR, TP, and attacks. (B) MSI initiated by the WT and mutant animals. (C) SR rates initiated by WT and KO animals in response to investigations by the C3H partner. (D) LR by WT and KO mice. (E) TP by WT and KO animals. (F) Total attacks by the WT and KO males. Responses of WT males are shown as open bars and mutants as closed bars. ∗ P < 0.05 from WT controls.

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ners, but they also execute higher rates of these behaviors during each social encounter relative to their WT controls. In the resident–intruder assay, WT and KO display similar levels of MSI (Fig. 5B). By comparison, KO males exhibited higher levels of SR [F(1, 27) = 5.92, P < 0.01], LR [F(1, 27) = 7.89, P < 0.01], and TP [F(1, 27) = 6.53, P < 0.01] than WT controls (Fig. 5C–E). No genotypic differences, however, were discerned for the rates of attacks against the intruder (Fig. 5F, left). Overall, the results from the resident–intruder test demonstrate that in a familiar environment the KO mice develop a socially reactive disposition. Nevertheless, despite increased levels of reactivity, the probability of attack by the mutants was no greater than that observed for the WT controls. To determine whether the same social interactions in the familiar environment were also evident under novel circumstances, behaviors of WT and KO mice were assessed in the dyadic test 7–10 days later. WT and KO animals showed moderate rates MSIs (Fig. 5B). Although SR occurred infrequently for WT controls, a 2-fold increase in these behaviors was observed in the KO mice [Fig. 5C; F(1, 27) = 6.11, P < 0.01]. By comparison, no significant differences in LR were discerned between genotypes (Fig. 5D). On the other hand, mutants displayed higher frequencies of TP than WT mice [Fig. 5E; F(1, 27) = 5.03, P < 0.01]. This propensity to threaten the test partner was accompanied by a 5-fold increase in attack rates by mutants relative to WT controls [Fig. 5F; F(1, 27) = 6.31, P < 0.01]. Together, these findings demonstrate that while the social behaviors of WT controls are little affected by test context, mutants become more aggressive under novel conditions. An analysis of context effects revealed that MSI and SR were not significantly influenced by genotype assignment. A significant genotype by test-context interaction was obtained for LR [Fgenotype×context (1, 57) = 7.83, P < 0.007] and a posteriori analyses demonstrated that LR was augmented for mutants in the intruder relative to the dyadic test (P < 0.01); no changes were ascribed to WT males. The genotype by test-context interaction for TP did not reach significance [Fgenotype×context (1, 57) = 3.28, P < 0.07]. By comparison, a significant genotype by context interaction was obtained when the frequency of attack behaviors was considered [Fgenotype×context (1, 57) = 20.33, P < 0.001]. This result can be attributed to the 3-fold enhancement (P < 0.001) in attacks when mutants were exposed to the novel environment. These findings show that the KO animals are highly reactive to novelty and this condition enhances the appearance of attack behaviors. 3.6. Stereotyped and perseverative social behaviors KO mice display increased stereotypy in the open field and they engage in high rates of perseverative responses in the radial arm maze [20]. To determine whether the appearance of repetitive behaviors was a generalized feature of KO mice, social behaviors were re-examined first

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Fig. 6. Rates of stereotyped and perseverative social behaviors in group- or isolation-housed WT and KO mice. (A) Average daily rates of single repetitive behaviors by group-housed following a single contact by a cage-mate. WT males are shown as open circles and mutants as closed circles. ∗ P < 0.05 from WT animals. (B) The rate of single repetitive behaviors (MSI, SR, LR, TP, and attack) by isolated WT and KO males in the resident–intruder and social dyadic tests. ∗ P < 0.05 from group-housed mice. (C) A comparison of perseverative social behaviors (MSI, reactivity, aggression) in the groupand isolate-housed (composite resident–intruder and dyadic test) conditions. (D) The incidence of perseverative patterns of behavior (MSI, SR, LR, TP, and attack) following a single social contact in the intruder and dyadic tests. Note that the WT males show no instances of these responses. Responses for the WT males are shown as open bars; mutants as closed bars. ∗ P < 0.05 from WT controls.

in the group-housed mice in the social-hierarchy test and then in the isolate-housed mice in the resident–intruder and dyadic tests. Among group-housed animals, mutants exhibited significantly higher rates of repetitive behaviors on each day of testing compared to WT males [Fig. 6A; Fdays1–6 (1, 51) = 9.76–52.67, P < 0.001]. Similar genotype differences were also observed with isolated males in the resident–intruder intruder [Fig. 6B; F(1, 57) = 8.47, P < 0.01] and dyadic tests [F(1, 57) = 10.81, P < 0.001]. Parenthetically, it should be emphasized that these repetitions, regardless of context, were not specific to any behavioral category (i.e. MSI, LR, SR, TP, or attack), but were unique to each animal. Since isolation rearing can induce reactivity and aggression in mice [10,33], the frequencies of repetitive behaviors were compared between group- and isolation-housed mice. Because frequencies of behaviors in the former tests were originally expressed as a group, their rates of repeated behaviors were recalculated as individual rates/5 min so that they could be compared directly to the combined intruder and dyadic results (Fig. 6C). The appearance of repetitive behaviors was enhanced by isolation for both genotypes [F(1, 85) = 179.2, P < 0.0001]. While the effect was significant for WT animals (P < 0.05), it was especially apparent for KO mice (P < 0.001). This result suggests that the high frequencies of stereotyped and perseverative behaviors characteristic of the mutants can be modulated through housing conditions. Besides analyzing single repetitive responses, behavioral records were also examined for evidence of repetitive sequences or patterns. Complex behavioral patterns were very difficult to identify under group-housing conditions because an individual animal often interacted with several animals

at once. For this reason, only behaviors from isolated mice were scored. These complex behaviors were comprised of rapid, multiple executions of several individual behaviors in succession or by the continuous repetition of a unique pattern of three or more behaviors. Typically, these patterns followed behavioral initiation by the C3H partner. Although the WT males never exhibited these behaviors, they were clearly evident in KO mice (Fig. 6D). Since mutants display higher rates of social behaviors than WT controls (recall Fig. 5A–F), these findings suggest that some of the observed increases in SR, LR, TP, and attacks in KO mice may be due to their perseverative responses. To investigate whether the mutant’s repetitive behaviors contributed to their higher rates of abnormal social responses, an ANCOVA was run where the rate of social repetitions was controlled. It was predicted that if repeated or stereotyped responses were an important modulator of the behavioral repertoire in KO mice, genotype effects would be reduced. Only those behaviors where significant genotype effects had been found in the intruder and dyadic tests were used in this analysis. When the rates of social repetitions were controlled, differences between WT and KO males for SR, LR, and TP in the resident–intruder test did not achieve significance. With regards to dyadic testing, no genotypic differences were discerned for TP and attacks. Interestingly, the observed differences in the rates of MSI and SR between WT controls and mutants were preserved with the ANCOVA tests [F s(1, 28) = 10.3–10.7, Ps < 0.004]. To examine the ANCOVA results in greater detail, the data were re-examined with linear regression models where the rate of repeated social initiations and the genotype of the mice were entered in a step-wise order. In the resident– intruder test, no significant genotype changes in r2 were

R.M. Rodriguiz et al. / Behavioural Brain Research 148 (2004) 185–198 Table 1 Step-wise regression parameters for MSI, SR, LR, TP, and attack behaviors Test Intrudera SR LR TP Dyadicb MSI SR TP Attack

Parameter

r2 change

Repeated behaviors genotype Repeated behaviors genotype Repeated behaviors genotype

0.386 0.062 0.672 0.015 0.657 0.037

Repeated genotype Repeated genotype Repeated genotype Repeated genotype

0.317 0.123 0.415 0.165 0.644 0.003 0.851 0.001

behaviors behaviors behaviors behaviors

F score change 16.99 2.98 55.32 0.015 51.66 3.14 12.53 4.66 19.20 10.25 48.78 0.19 154.01 0.22

Significance

0.001 ns 0.001 ns 0.001 ns 0.001 0.05 0.001 0.001 0.001 ns 0.001 ns

a Since no main effects of genotype by ANOVA were obtained for MSI and attack behaviors in the resident–intruder paradigm, regression analyses were not performed. b Since no WT and KO significant differences were obtained by ANOVA for LR in the dyadic test, no regression analyses were run.

found for SR, LR, and TP (Table 1). Similarly, in the dyadic test, no alterations in r2 for genotype were noted for TP and attacks. These data are consistent with the ANCOVA findings by demonstrating that abnormal social behaviors of KO mice are due to their stereotyped behaviors. By comparison, evaluation of SR and MSI in the dyadic model revealed that while genotype differences were robust, the repetition of behavior significantly influenced the magnitude of their responses. Together, these data support the hypothesis that behavioral differences between the two genotypes are directly related to the higher rate of stereotyped and perseverative responses of the mutants, albeit these effects appear to be contingent upon the environmental contexts in which the mice are tested. Hence, exposure to a novel environment can preserve the higher rates of MSI and SR for the KO animals.

4. Discussion In the present investigations, we show that the WT and KO mice are equally capable of discriminating olfactory stimuli. Hence, any impairment in social behaviors cannot be attributed to deficiencies in olfaction. Group-housed mutants are more reactive and display more aggressive behaviors than WT controls. Although mice from both genotypes form dominance hierarchies, WT mice form 3- to 4-ranked hierarchies whereas those from KO mice are 2-ranked and their hierarchies are less stable over time. Confirmation of dominance hierarchy status in the tube-dominance test supported the ethological results. However, the appearance of assertive, agonistic, and attack behaviors is more rigidly

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maintained in KO animals by their ranking positions in the home cage. Isolation-housing exacerbates the reactivity and aggression of KO mice and novelty further augments the appearance of threatening and attack behaviors. During the scoring of behaviors, it was observed that the KO animals often engage in stereotyped and perseverative social behaviors and these behaviors become more robust over time. When these behaviors were statistically controlled, the appearance of many KO abnormal social behaviors was dramatically reduced. These findings suggest that stereotypy and perseveration may distort the social interactions of KO mice such that their behavioral repertoires become restricted and inflexible. Social interaction was examined in WT and KO animals under group- and isolation-rearing conditions. Although it has been suggested that isolation rearing is similar to conditions encountered by young male mice in naturalistic settings [7], males will also develop social organizations in group-housed conditions in the laboratory that are analogous to those observed in the wild [15,39,52]. In group-housing, KO males displayed more changes in social organization than WT animals. In most cases, there was a complete inversion of the hierarchy with the emergence of a single despot animal. This same social organization was preserved and maintained in another test of hierarchy, the tube-dominance test. When the assertive behaviors were analyzed relative to the cage status of the animals, it was found that all mutant behaviors in the tube-test were significantly regulated by their status in the social groups, whereas pushing behaviors in the WT males were not. These findings suggest that in KO males the formation of a despot hierarchy not only constrains behaviors within the cage among animals, but it also preserves it outside the home cage. These data are relevant to KO mice because these mutants are hyperdopaminergic and pharmacological induction of this state by amphetamine in normal rats and mice alters agonistic behaviors such that the emergence of abnormal social responses is highly dependent upon the pre-existing dominant status of the test animal [51]. Chronic amphetamine treatment in primates also exacerbates the hierarchy such that dominant males assert their status more aggressively, whereas subordinates become more subservient in their social interactions [25]. Interestingly, amphetamine-treated submissive animals also become more vigilant and are hyper-reactive to social contact [21,38]. Hence, states of hyperdopaminergia serve not only to promote certain social behaviors, but they also exaggerate the expression of these behaviors in different social contexts. In concert with these findings, our own results demonstrate that disruptions in the DA system can distort social interactions and lead not only to increased aggression, but also to heightened reactivity and subordinate postures. While group-housed of KO mice displayed enhanced reactivity and agonistic responses, social isolation further exacerbated these responses. In the resident–intruder and dyadic paradigms, SR, LR, and TP occurred at higher frequencies in mutant than WT mice. It should be noted that the incidence of TP was even more enhanced when KO

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males were exposed to a novel environment. This heightened responsivity may contribute to the augmentation in attack behaviors that are only observed in the dyadic context. In a recent report, Spielewoy et al. [49] reported that social behavior of KO mice in the resident–intruder paradigm did not differ from that of WT controls. Their methodology, however, differed from our own in several respects. First, male mice were housed with females and pups for 4 weeks prior to testing. In addition, the floor of the test chamber was covered with sawdust that had been soiled by unfamiliar mice prior to testing. Finally, social interactions were evaluated between animals of the same genotype rather than with a standardized social partner. Due to these differences, it is difficult to determine whether the animals’ social behaviors were differentially modified by conditions prior to testing, the pre-existing social dominance status of the WT or KO intruder males, or other circumstances. Despite this fact, two variables deserve special mention. First, it has been demonstrated that soiled materials from unfamiliar animals can disrupt and reduce the appearance of agonistic and exploratory behaviors in mice [32,53]. Hence, the presence of bedding materials from many male and female animals in the intruder cage may have attenuated the agonistic responses in Spielewoy et al.’s [49] investigations. Second, in the present experiment, we paired WT and KO mice with C3H males because these latter animals provide a stable social environment [23] where the behaviors of the WT and KO animals can be easily compared. However, despite these distinctions in methodology, Spielewoy et al. [49] did note that in other test contexts the KO animals were especially aroused by novelty. This same response was seen in our own dyadic tests. In the present study, KO mice exhibit increases in social contact and heightened aggressive responses towards unfamiliar males. Since KO mice are hyperactive in a novel environment [20,24,43,49], it may be the case that the high social initiation rates are the result of enhanced motor activity. In social tests, the enhanced interactions were not unregulated because the KO animals did not engage in thigmotaxic behavior as has been reported for the open field. Instead, they deliberately approached the C3H partner and interacted with it. Furthermore, the types of behaviors displayed by the mutants progressed from mild social encounter, through reactivity, to agonistic responses. As time progressed, the nature of the interactions became progressively more stereotyped and perseverative. Interestingly, these same behavioral characteristics have been reported for KO animals when they are placed into the open field [20,43] or a radial arm maze [20]. During social interactions, the mutants engaged in complex patterns of repetitive responses along with stereotypies of single behaviors. Parenthetically, KO males exhibited individualized patterns of repetitive responses that were activated upon contact with the test partner and they increased in frequency over testing. It is important to note that these stereotyped and perseverative behaviors contributed significantly to the display of abnor-

mal social behaviors in KO mice. This strong propensity may have some clinical relevance because stereotypic and ritualized behavioral patterns are observed in animal models of schizophrenia and mania, as well as in schizophrenic patients themselves [38]. Notably, the impaired social interactive responses of these patients are typically associated with the positive and negative symptoms of schizophrenia [2,6]. Interestingly, mice that have been selectively bred for long- or short-attack latencies have also been found to exhibit differences in DA functioning [5], and the augmented aggression reported for the latter group of animals has been attributed, in part, to increased stereotypic responses that limit the behavioral repertoire of the animal [5,47]. It is also well known that in animals, chronic amphetamine injections will produce perseveration [17,18] and “fragmentation” of social behavior [38,40]. Since amphetamine increases extracellular levels of DA [30,46] and because extracellular levels of this monoamine are chronically augmented in KO mice [29], social responses of mutants appear similar to those of animals given chronic amphetamine injections where behavior becomes both stereotyped and inflexible over time. The increased agonistic responses of KO mice were related to enhanced reactivity to mild social contact. This propensity was further potentiated when testing was conducted in a novel environment. One reason why novelty may increase the display of aggressive behaviors in KO males is that the animals may have a pre-existing lower tolerance for social stimulation than WT controls. It has been proposed that that reduced thresholds to environmental stimulation can promote a lower tolerance for social contact, such that, increased aggression occurs [9,12]. Changes in reactivity to social contact are also observed in amphetamine-induced animal models of mania and schizophrenia [17,38]. The recruitment of cortical and limbic DA systems in response to social stimulation has been reported to support emotional arousal and anticipatory fear, as well as the initiation and inhibition of behavioral responses [51]. If this is the case in KO mice, then differences in arousal to perceived threat under various circumstances might support a vast array of social responses through the differential recruitment of the DA system (see [8,22]). Since pharmacological manipulations of DA receptors can disrupt behavioral reactivity that can affect aggressive responses in mice [23] and because DA receptor functioning is already perturbed in the KO mice [24,29], heightened reactivity may support their increased aggression. Future manipulations of specific aspects of the DA and other interacting monoamine systems may provide further information regarding the role of reactivity in the regulation of aggressive responses in the KO animals. The KO mouse displays biochemical, neurochemical, and behavioral features consistent with an exaggerated model of chronic amphetamine stimulation (see [20,24,29,31]). However, psychostimulants alone cannot recapitulate the phenotype of KO mice. In our studies, the KO animals present a spectrum of behaviors that resemble those of patients diagnosed with variety of disorders attributed to DA dysfunction.

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Although the social behaviors of KO mice cannot be ascribed solely to a particular psychiatric disorder, many patients with DA disturbances are highly reactive to social stimuli. While the expression of this reactivity may take many forms, a common feature is that the behaviors may become stereotyped and inflexible [6,38]. In the present studies, we have shown that most of the aberrant social responses of KO mice are due to an increase in stereotyped and perseverative behaviors. These responses reduce the behavior repertoire of the mutants so that they are unable to readily adapt to changing social contingencies. It is well known that stimulation or antagonism of DA circuits can disrupt social interactions across a variety of animal species [38,51], including the human [13,45]. To this end, use of different pharmacological agents capable of modulating DA-dependent behaviors in the KO mice may provide novel insights not only into the management of their abnormal social responses, but also into the potential underlying mechanisms that control these behaviors.

Acknowledgements We wish to thank Ms. Jiechun Zhou for breeding, genotyping, and maintaining the mice under group housing conditions, Ms. Robin Stout for separating and maintaining the mice under isolation-housing and testing, and Mr. Ramon Rodriguiz for the construction of the social dyadic testing chambers and the apparatus for tube-dominance testing. These studies were supported in part by the American Psychological Association Postdoctoral Minority Fellowship in Neuroscience (R.M.R.), by Research Grant No. 12-FY99-468 from the March of Dimes Birth Defects Foundation (W.C.W.), and by a grant from the National Alliance for Research on Schizophrenia and Depression (W.C.W.).

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