Effects of estrus cycle stage on defensive behavior in female Long-Evans hooded rats.

This study investigated the influence of the estrus cycle in mediating cat odor-induced unconditioned and conditioned defensive behaviors in female Long-Evans hooded rats. Unconditioned defensive behaviors were assessed during predatory cue exposure; conditioned defensive behaviors were examined 24 h after threat exposure. Estrus phases were determined by microscopic examination of vaginal smears within 10 min of completing the behavioral tests. Compared to no-odor controls, female rats exposed to cat odor exhibited both unconditioned and conditioned defensive behaviors, including elevated levels of freezing, risk assessment and avoidance. Rats in proestrus and estrus exhibited reduced levels of defensive behavior during the unconditioned test trial compared to subjects in diestrus and metestrus. Specifically, estrus stages characterized by high levels of circulating estrogens and progesterone were associated with reduced immobility (i.e. freezing) and enhanced active defense (i.e. risk assessment), profiles that may enable mate seeking and subsequent reproduction in potentially dangerous or novel environments. These results suggest a specific role for ovarian hormone fluctuations in mediating unconditioned fear- and anxiety-like defensive behaviors during exposure to predatory odors.

Neuronal correlates of asocial behavior in a BTBR T (+) Itpr3(tf)/J mouse model of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized, in part, by an inability to adequately respond to social cues. Patients diagnosed with ASD are often devoid of empathy and impaired in understanding other people's emotional perspective. The neuronal correlates of this impairment are not fully understood. Replicating such a behavioral phenotype in a mouse model of autism would allow us insight into the neuronal background of the problem. Here we tested BTBR T(+)Itpr3(tf)/J (BTBR) and c57BL/6J (B6) mice in two behavioral paradigms: the Transfer of Emotional Information test and the Social Proximity test. In both tests BTBR mice displayed asocial behavior. We analyzed c-Fos protein expression in several brain regions after each of these tests, and found that, unlike B6 mice, BTBR mice react to a stressed cagemate exposure in the Transfer of Emotional Information test with no increase of c-Fos expression in either the prefrontal cortex or the amygdala. However, after Social Proximity exposure we observed a strong increase in c-Fos expression in the CA3 field of the hippocampus and two hypothalamic regions of BTBR brains. This response was accompanied by a strong activation of periaqueductal regions related to defensiveness, which suggests that BTBR mice find unavoidable social interaction highly aversive.

Central oxytocin regulates social familiarity and scent marking behavior that involves amicable odor signals between male mice.

The effect of oxytocin on social behavior and odor communication was investigated in male C57BL/6J mice. In three-male colonies, in visible burrow systems, icv oxytocin (OT) infusion before colony formation substantially increased huddling together over the initial 8 h of grouping, accompanied by decreased expression of a number of social approaches associated with conspecific aggression and defense. OT antagonist infusion had little impact on expression of social approaches but decreased time engaging in social components including huddle over the initial 8 h. These results demonstrate a linkage of social familiarity to OT availability in the brain. In a scent marking paradigm central infusion of OT reduced territorial marking towards male conspecifics, and this in turn reduced the scent marking of untreated stimulus males to OT-infused subjects. Infusion of an OT antagonist into stimulus mice who were confronted with OT-infused subjects prevented the reduction/suppression of scent marking that was normally seen following exposure of social odors released from OT-injected mice. Odor of pair-housed mice also induced a suppression of territorial scent marking in odor recipients, but OT antagonist administration into pair-housed mice blocked this suppressive effect of odor cue. These results indicate that central OT modulates release as well as detection of amicable signals facilitating/maintaining familiar relationships and suppressing territorial behavior between male mice. Overall, these findings suggest that OT plays a significant role in regulating social familiarity via changing qualities of conspecific odor cues.

Applying the ethoexperimental approach to neurodevelopmental syndrome research reveals exaggerated defensive behavior in Mecp2 mutant mice.

Rett syndrome is a Pervasive Developmental Disorder (PDD) associated with de novo mutations of the methyl CpG-binding protein 2 (MECP2) gene. Mecp2 functions as a transcription factor that regulates the expression of hundreds of genes. Identification of the role of Mecp2 in specific neurodevelopmental symptoms remains an important research aim. We previously demonstrated that male mice possessing a truncation mutation in Mecp2 are hyper-social. We predicted that reduced fear or anxiety might underlie this enhanced affiliation. In order to probe risk assessment and anxiety-like behavior, we compared Mecp2 truncation mutants to their wild-type littermates in the elevated plus maze and elevated zero maze. Additionally, subjects were administered the mouse defense test battery to evaluate unconditioned fear- and panic-like behavior to a graded set of threat scenarios and a predator stimulus. Mutant mice showed no significant changes in anxiety-like behavior. Yet, they displayed hyper-reactive escape and defensive behaviors to an animate predatory threat stimulus. Notably, mutant mice engaged in exaggerated active defense responding to threat stimuli at nearly all phases of the fear battery. These results reveal abnormalities in emotion regulation in Mecp2 mutants particularly in response to ecologically relevant threats. This hyper-responsivity suggests that transcriptional targets of Mecp2 are critical to emotion regulation. Moreover, we suggest that detailed analysis of defensive behavior and aggression with ethologically relevant tasks provides an avenue to interrogate gene-behavior mechanisms of neurodevelopmental and other psychiatric conditions.

Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice.

Corticotropin-releasing factor (CRF) plays an essential role in coordinating the autonomic, endocrine and behavioral responses to stressors. In this study, we investigated the role of CRF within the medial prefrontal cortex (mPFC) in modulating unconditioned defensive behaviors, by examining the effects of microinfusing cortagine a selective type-1 CRF receptor (CRF1) agonist, or acidic-astressin a preferential CRF1 antagonist, into the mPFC in male CD-1 mice exposed to a live predator (rat exposure test--RET). Cortagine microinfusions significantly reduced several indices of defense, including avoidance and freezing, suggesting a specific role for CRF1 within the infralimbic and prelimbic regions of the mPFC in modulating unconditioned behavioral responsivity to a predator. In contrast, microinfusions of acidic-astressin failed to alter defensive behaviors during predator exposure in the RET. Cortagine microinfusions also reduced Fos protein production in the medial, central and basomedial, but not basolateral subnuclei of the amygdala in mice exposed to the rat predatory threat stimulus. These results suggest that CRF1 activation within the mPFC attenuates predator-induced unconditioned anxiety-like defensive behaviors, likely via inhibition of specific amygdalar nuclei. Furthermore, the present findings suggest that the mPFC represents a unique neural region whereby activation of CRF1 produces behavioral effects that contrast with those elicited following systemic administration of CRF1 agonists.

The role of behavior in translational models for psychopathology: functionality and dysfunctional behaviors.

The history of science has frequently included a problem-based impetus toward research that can be translated expeditiously into solutions. A current problem is that psychopathologies, typically chronic, contribute hugely to the economic and social burden of medical care, especially in the United States. For behavioral neuroscientists a psychopathology-aimed translational research emphasis particularly involves animal models to facilitate the experimental and invasive work necessary to an understanding of the biology of normal and aberrant behavior. When the etiology of a particular psychopathology is unknown, and there are no specific biomarkers, behavioral parallels between the focal disorder and its putative models become crucial elements in assessing model validity. Evaluation of these parallels is frequently neglected, reflecting in part the lack of a systematic conceptualization of the organization of behavior and how this may be conserved across species. Recent work specifically attempting to bridge this gap suggests that analysis of behaviors that are functional - adaptive in crucial situations such as danger or social contexts - can facilitate an understanding of the parallels between behaviors of human and nonhuman species, including the dysfunctional behaviors of psycho pathologies. As research with animal models comes to provide a more systematic analysis of particular behaviors and their adaptive functions, cross-talk between model and focal psychopathology may be advantageous to understanding both.

Heparan sulfate deficiency in autistic postmortem brain tissue from the subventricular zone of the lateral ventricles.

Abnormal cellular growth and organization have been characterized in postmortem tissue from brains of autistic individuals, suggestive of pathology in a critical neurogenic niche, the subventricular zone (SVZ) of the brain lateral ventricles (LV). We examined cellular organization, cell proliferation, and constituents of the extracellular matrix such as N-sulfated heparan sulfate (HS) and laminin (LAM) in postmortem brain tissue from the LV-SVZ of young to elderly individuals with autism (n=4) and age-matched typically developing (TD) individuals (n=4) using immunofluorescence techniques. Strong and systematic reductions in HS immunofluorescence were observed in the LV-SVZ of the TD individuals with increasing age. For young through mature, but not elderly, autistic pair members, HS was reduced compared to their matched TDs. Cellular proliferation (Ki67+) was higher in the autistic individual of the youngest age-matched pair. These preliminary data suggesting that HS may be reduced in young to mature autistic individuals are in agreement with previous findings from the BTBR T+tf/J mouse, an animal model of autism; from mice with genetic modifications reducing HS; and with genetic variants in HS-related genes in autism. They suggest that aberrant extracellular matrix glycosaminoglycan function localized to the subventricular zone of the lateral ventricles may be a biomarker for autism, and potentially involved in the etiology of the disorder.

The BTBR T+ tf/J mouse model for autism spectrum disorders-in search of biomarkers.

Autism spectrum disorders (ASD) form a common group of neurodevelopmental disorders appearing to be under polygenic control, but also strongly influenced by multiple environmental factors. The brain mechanisms responsible for ASD are not understood and animal models paralleling related emotional and cognitive impairments may prove helpful in unraveling them. BTBR T+ tf/J (BTBR) mice display behaviors consistent with the three diagnostic categories for ASD. They show impaired social interaction and communication as well as increased repetitive behaviors. This review covers much of the data available to date on BTBR behavior, neuroanatomy and physiology in search for candidate biomarkers, which could both serve as diagnostic tools and help to design effective treatments for the behavioral symptoms of ASD.

Absence of social conditioned place preference in BTBR T+tf/J mice: relevance for social motivation testing in rodent models of autism.

A major goal of translation research in autism is to characterize the physiological and psychological processes underlying behavioral abnormalities. Since autism reflects impairments in social motivation, we modified the mouse three-chamber social approach apparatus for use as a social conditioned place preference arena. We paired one of two unique contexts with social interactions in juvenile mice for five or ten conditioning sessions in BTBR T+tf/J mice and a control strain with normal approach behaviors (C57BL/6J) since the BTBR T+tf/J inbred mouse strain displays a variety of behavioral alterations analogous to symptoms of autism spectrum disorders. While C57BL/6J mice formed a conditioned place preference to the context associated with social interactions, particularly those receiving ten days of conditioning, BTBR T+tf/J mice did not. Neither absence of social proximity nor avoidance due to high rates of autogrooming appeared to underlie the impaired positive incentive value of the unconditioned social stimulus in the BTBR T+tf/J strain. These data contribute to a growing body of evidence suggesting that the BTBR T+tf/J strain shows impairments in all diagnostic domains of autism including social motivation. Additionally, social conditioning testing might provide an important social motivation measure in other rodent models of neuropsychiatric disorders characterized by social abnormalities.

Reduced sulfate plasma concentrations in the BTBR T+tf/J mouse model of autism.

Clinical studies have shown that children diagnosed with autism show abnormal sulfate chemistry, which is critical for cellular and metabolic processes. To determine if the inbred BTBR T+tf/J mouse shows autism-relevant aberrations in sulfate chemistry, the present study examined plasma sulfate concentrations in BTBR T+tf/J, inbred C57BL/6J, and outbred CD-1 mice. Results showed that the BTBR T+tf/J mouse exhibits significantly lower plasma sulfate concentrations in comparison to both C57BL/6J and CD-1 mice. These results suggest that the BTBR mouse shows autism-relevant abnormalities in sulfate chemistry and may serve additional utility in examining the role of sulfate and sulfate-dependent systems in relation to autism-relevant behavioral aberrations.

Facial expressions of mice in aggressive and fearful contexts.

Some animals display a variety of context dependent facial expressions. Previous studies have shown that rodents display a facial grimace while in pain. To determine if the facial expressions of mice extend beyond pain, facial expressions were analyzed in the presence of non-social, social and predator stimuli. In a vibrissae contact test, the whiskers of mice were stroked by the bristles of a brush. In a social proximity test, two mice were placed together in a small chamber where contact was virtually unavoidable. In a resident-intruder test of aggression, an unknown mouse was placed into the homecage of another mouse. In a cat odor exposure test and in a live rat exposure test, mice were presented with the respective stimuli. Results from this study indicated that mice showed two patterns of expression, either a full display of changes in the measured facial components, characterized by tightened eyes, flattened ears, nose swells and cheek swells; or a more limited display of these facial changes. The full display of changes occurred in the vibrissae contact test, the social proximity test, and in resident mice in the resident-intruder test. The more limited display of facial changes occurred in the cat odor exposure test, the rat exposure test and in intruder mice in the resident-intruder test. The differential display of facial changes across conditions indicated that mice showed tightened eyes and flattened ears in situations that provided the immediate potential for contact, suggesting that such changes are involved in protection of sensitive and/or vulnerable body parts. Furthermore, the display of facial expressions by mice indicates that these expressions are widely distributed across evolution.

Oxytocin receptor and Mecp2 308/Y knockout mice exhibit altered expression of autism-related social behaviors.

The development of tasks measuring behaviors specific to the three major symptom categories for autism makes it possible to differentiate mouse models of autism spectrum disorders (ASD) in terms of changes in these specific categories. Prior studies indicate that BTBR T+tf/J mice, the strain that has been evaluated most extensively, show autism-relevant changes in all three symptom categories; reciprocal social interactions; communication; and repetitive, ritualized behaviors. This report reviews the behaviors of oxytocin receptor (Oxtr) and Mecp2(308/Y) wild-type (WT) and knockout (KO) mice, in a number of tests specifically designed to provide information on behaviors that may show functional parallels to the core symptoms of ASD. Oxtr KO mice show robust decreases in reciprocal social interactions, and reduced levels of communication, but no changes in repetitive, ritualized behaviors; whereas Mecp2(308/Y) KO mice show a slight but consistent enhancement of social behavior and communication, and no changes in repetitive, ritualized behaviors. This data base, although small, strongly indicates that mouse models can sort the diagnostic symptoms of autism, and suggests that biological and physiological analyses of these strains may be capable of providing differential information on the brain systems involved in particular symptoms of this disorder. Profiles of behavioral changes in other mouse models of ASD should provide additional specificity in the search for biomarkers associated with particular ASD symptoms and symptom clusters.

BTBR T+tf/J mice: autism-relevant behaviors and reduced fractone-associated heparan sulfate.

BTBR T+tf/J (BTBR) mice have emerged as strong candidates to serve as models of a range of autism-relevant behaviors, showing deficiencies in social behaviors; reduced or unusual ultrasonic vocalizations in conspecific situations; and enhanced, repetitive self-grooming. Recent studies have described their behaviors in a seminatural visible burrow system (VBS); a Social Proximity Test in which avoidance of a conspecific is impossible; and in an object approach and investigation test evaluating attention to specific objects and potential stereotypies in the order of approaching/investigating objects. VBS results confirmed strong BTBR avoidance of conspecifics and in the Social Proximity Test, BTBR showed dramatic differences in several close-in behaviors, including specific avoidance of a nose-to-nose contact that may potentially be related to gaze-avoidance. Diazepam normalized social avoidance by BTBRs in a Three-Chamber Test, and some additional behaviors - but not nose to nose avoidance - in the Social Proximity Test. BTBR also showed higher levels of preference for particular objects, and higher levels of sequences investigating 3- or 4-objects in the same order. Heparan sulfate (HS) associated with fractal structures in the subventricular zone of the lateral ventricles was severely reduced in BTBR. HS may modulate the functions of a range of growth and guidance factors during development, and HS abnormalities are associated with relevant brain (callosal agenesis) and behavioral (reductions in sociality) changes; suggesting the value of examination of the dynamics of the HS system in the context of autism.

Oxytocin receptor knockout mice display deficits in the expression of autism-related behaviors.

A wealth of studies has implicated oxytocin (Oxt) and its receptors (Oxtr) in the mediation of social behaviors and social memory in rodents. It has been suggested that failures in this system contribute to deficits in social interaction that characterize autism spectrum disorders (ASD). In the current analyses, we investigated the expression of autism-related behaviors in mice that lack the ability to synthesize the oxytocin receptor itself, Oxtr knockout (KO) mice, as compared to their wild-type (WT) littermates. In the visible burrow system, Oxtr KO mice showed robust reductions in frontal approach, huddling, allo-grooming, and flight, with more time spent alone, and in self-grooming, as compared to WT. These results were corroborated in the three-chambered test: unlike WT, Oxtr KO mice failed to spend more time in the side of the test box containing an unfamiliar CD-1 mouse. In the social proximity test, Oxtr KO mice showed clear reductions in nose to nose and anogenital sniff behaviors oriented to an unfamiliar C57BL/6J (B6) mouse. In addition, our study revealed no differences between Oxtr WT and KO genotypes in the occurrence of motor and cognitive stereotyped behaviors. A significant genotype effect was found in the scent marking analysis, with Oxtr KO mice showing a decreased number of scent marks, as compared to WT. Overall, the present data indicate that the profile for Oxtr KO mice, including consistent social deficits, and reduced levels of communication, models multiple components of the ASD phenotype. This article is part of a Special Issue entitled Oxytocin, Vasopressin, and Social Behavior.

Fractone-associated N-sulfated heparan sulfate shows reduced quantity in BTBR T+tf/J mice: a strong model of autism.

BTBR T+tf/J (BTBR) mice show abnormal social, communicatory, and repetitive/stereotyped behaviors paralleling many of the symptoms of autism spectrum disorders. BTBR also show agenesis of the corpus callosum (CC) suggesting major perturbations of growth or guidance factors in the dorsal forebrain [1]. Heparan sulfate (HS) is a polysaccaride found in the brain and other animal tissues. It binds to a wide variety of ligands and through these ligands modulates a number of biological processes, including cell proliferation and differentiation, migration and guidance. It is aggregated on fractal-like structures (fractones) in the subventricular zone (SVZ), that may be visualized by laminin immunoreactivity (LAM-ir), as well as by HS immunoreactivity (HS-ir). We report that the lateral ventricles of BTBR mice were drastically reduced in area compared to C57BL/6J (B6) mice while the BTBR SVZ was significantly shorter than that of B6. In addition to much smaller fractones for BTBR, both HS and LAM-ir associated with fractones were significantly reduced in BTBR, and their anterior-posterior distributions were also altered. Finally, the ratio of HS to LAM in individual fractones was significantly higher in BTBR than in B6 mice. These data, in agreement with other findings linking HS to callosal development, suggest that variations in the quantity and distribution of HS in the SVZ of the lateral ventricles may be important modulators of the brain structural abnormalities of BTBR mice, and, potentially, contribute to the behavioral pathologies of these animals.

General and social anxiety in the BTBR T+ tf/J mouse strain.

BTBR T+ tf/J (BTBR) is an inbred mouse strain that shows behavioral traits with analogies to the three diagnostic symptoms of autism spectrum disorder (ASD); deficits in social interaction, impaired communication, and repetitive behaviors with restricted interests. Previous findings reveal that when compared to C57BL/6J (B6) and other inbred strains, BTBR exhibit normal to low anxiety-like traits in paradigms designed to assess anxiety-related behaviors. The current study assessed the generality of these anxiety findings. In experiment 1, B6 and BTBR mice were tested in the elevated plus maze (EPM), mouse defense test battery (MDTB) and elevated zero-maze. BTBR mice exhibited an anxiogenic profile in the EPM, with a reduction in open arm time and an increase in risk assessment behaviors, as compared to B6. In the MDTB, BTBR showed enhanced vocalization to the predator, and significantly less locomotor activity than B6 in the pre-threat situation, but significantly more locomotion than B6 following exposure to a predator threat, suggesting enhanced defensiveness to the predator. In the zero-maze, BTBR mice showed a significantly higher number of entries and time spent in the open segments of the apparatus, when compared to B6. In experiment 2, a three-chambered social preference test was used to evaluate effects of the systemic administration of an anxiolytic compound, diazepam, on B6 and BTBR social approach. Diazepam consistently increased time in the compartment containing the social stimulus, for both B6 and BTBR mice. However, in the vehicle treated groups, B6 mice spent significantly more time while BTBR mice spent significantly less time in the social stimulus compartment; after diazepam administration both B6 and BTBR strains significantly preferred the social stimulus chamber. These results suggest that while the anxiety responses of BTBR mice to novel situations (EPM and zero-maze) are inconsistent, BTBR mice appear to be more defensive to animate threat stimuli (predator or another mouse). Reduction of anxiety by diazepam normalized the social preference of BTBR for a mouse stimulus in the three-chambered test.

Involvement of dorsal raphe nucleus and dorsal periaqueductal gray 5-HT receptors in the modulation of mouse defensive behaviors.

Previous findings point to the involvement of the dorsal raphe nucleus (DRN) and dorsal periaqueductal gray (dPAG) serotonergic receptors in the mediation of defensive responses that are associated with specific subtypes of anxiety disorders. These studies have mostly been conducted with rats tested in the elevated T-maze, an experimental model of anxiety that was developed to allow the measurement, in the same animal, of two behaviors mentioned: inhibitory avoidance and one-way escape. Such behavioral responses have been respectively related to generalized anxiety disorder (GAD) and panic disorder (PD). In order to assess the generality of these findings, in the current study we investigated the effects of the injection of 5-HT-related drugs into the DRN and dPAG of another rodent species, mouse, on the mouse defense test battery (MDTB), a test of a range of defensive behaviors to an unconditioned threat, a predator. Male CD-1 mice were tested in the MDTB after intra-DRN administration of the 5-HT(1A) receptor antagonist WAY-100635 or after intra-dPAG injection of two serotonergic agonists, the 5-HT(1A) receptor agonist 8-OH-DPAT and the 5-HT(2A/2C) receptor agonist DOI. Intra-DRN injection of WAY-100635 did not change behavioral responses of mice confronted with a rat in the MDTB. In the dPAG, both 8-OH-DPAT and DOI consistently impaired mouse escape behavior assessed in the MDTB. Intra-dPAG infusion of 8-OH-DPAT also decreased measures of mouse risk assessment in the rat exposure test. In conclusion, the current findings are in partial agreement with previous results obtained with rats tested in the elevated T-maze. Although there is a high level of similarity between the behavioral effects obtained in rats (elevated T-maze) and mice (MDTB and RET) with the infusion of 5-HT agonists into the dPAG, the same is not true regarding the effects of blockade of DRN 5-HT(1A) receptors in these rodent species. These data suggest that there may be differences between mice and rats regarding the involvement of the DRN in the mediation of defensive behaviors.

Risk assessment as an evolved threat detection and analysis process.

Risk assessment is a pattern of activities involved in detection and analysis of threat stimuli and the situations in which the threat is encountered. It is a core process in the choice of specific defenses, such as flight, freezing, defensive threat and defensive attack, that counter the threat and minimize the danger it poses. This highly adaptive process takes into account important characteristics, such as type and location (including distance from the subject) of the threat, as well as those (e.g. presence of an escape route or hiding place) of the situation, combining them to predict which specific defense is optimal with that particular combination of threat and situation. Risk assessment is particularly associated with ambiguity either of the threat stimulus or of the outcome of available defensive behaviors. It is also crucial in determining that threat is no longer present, permitting a return to normal, nondefensive behavior. Although risk assessment has been described in detail in rodents, it is also a feature of human defensive behavior, particularly in association with ambiguity. Rumination may be a specifically human form of risk assessment, more often expressed by women, and highly associated with anxiety. Risk assessment behaviors respond to drugs effective against generalized anxiety disorder; however, flight, a dominant specific defense in many common situations, shows a pharmacological response profile closer to that of panic disorder. Risk assessment and flight also appear to show some consistent differences in terms of brain regional activation patterns, suggesting a potential biological differentiation of anxiety and fear/panic systems. An especially intriguing possibility is that mirror neurons may respond to some of the same types of situational differences that are analyzed during risk assessment, suggesting an additional functional role for these neurons.

A novel social proximity test suggests patterns of social avoidance and gaze aversion-like behavior in BTBR T+ tf/J mice.

The BTBR T+ tf/J (BTBR) inbred mouse strain displays a low sociability phenotype relevant to the first diagnostic symptom of autism, deficits in reciprocal social interactions. Previous studies have shown that BTBR mice exhibit reduced social approach, juvenile play, and interactive behaviors. The present study evaluated the behavior of the BTBR and C57BL/6J (B6) strains in social proximity. Subjects were closely confined and tested in four experimental conditions: same strain male pairs (Experiment 1); different strain male pairs (Experiment 2); same strain male pairs and female pairs (Experiment 3); same strain male pairs treated with an anxiolytic (Experiment 4). Results showed that BTBR mice displayed decreased nose tip-to-nose tip, nose-to-head and upright behaviors and increased nose-to-anogenital, crawl under and crawl over behaviors. These results demonstrated avoidance of reciprocal frontal orientations in the BTBR, providing a parallel to gaze aversion, a fundamental predictor of autism. For comparative purposes, Experiment 3 assessed male and female mice in a three-chamber social approach test and in the social proximity test. Results from the three-chamber test showed that male B6 and female BTBR displayed a preference for the sex and strain matched conspecific stimulus, while female B6 and male BTBR did not. Although there was no significant interaction between sex and strain in the social proximity test, a significant main effect of sex indicated that female mice displayed higher levels of nose tip-to-nose tip contacts and lower levels of anogenital investigation (nose-to-anogenital) in comparison to male mice, all together suggesting different motivations for sociability in males and females. Systemic administration of the anxiolytic, diazepam, decreased the frequency of two behaviors associated with anxiety and defensiveness, upright and jump escape, as well as crawl under behavior. This result suggests that crawl under behavior, observed at high levels in BTBR mice, is elicited by the aversiveness of social proximity, and possibly serves to avoid reciprocal frontal orientations with other mice.