Bryce C. Ryan

Intrauterine position effects.

A review of the literature suggests that individual variability in sex-related traits may be influenced by variations in hormonal exposure during fetal development. In litter-bearing mammals, fetuses develop in utero and may be subjected to differing hormonal environments based upon the sex of neighboring fetuses. Female fetuses developing between two males tend to show masculinized anatomical, physiological and behavioral traits as adults. Female fetuses developing without adjacent males, on the other hand, tend to show more feminized traits as adults. These traits include permanently altered hormone levels, reproductive organs, aggressive behaviors, secondary sex ratios and susceptibility to endocrine disruption. This intrauterine effect is due to the transfer of testosterone from male fetuses to adjacent fetuses. While these effects have been most clearly demonstrated in mice, other rodents and swine also show intrauterine position (IUP) effects. Some of these effects are similar to the influence of prenatal stress on adult phenotypes. A few reports on human twins suggest that variability in some masculine and feminine traits may be due to intrauterine hormonal signals. IUP effects may impact a number of scientific fields of research such as endocrine disruption, toxicology, population biology, animal production and health.

Developmental exposure to environmental estrogens alters anxiety and spatial memory in female mice

Humans and wildlife are exposed to numerous anthropogenic drugs and pollutants. Many of these compounds are hormonally active, and recent evidence suggests that the presence of these endocrine disruptors permanently alters normal development and physiology in a variety of vertebrate species. Here, we report on the effects of developmental exposure to two common estrogenic pollutants, bisphenol A and ethinyl estradiol on sexually dimorphic, non-reproductive behavior. Mice (Mus musculus domesticus) were exposed to environmentally relevant levels of these chemicals (2 and 200 microg/kg/day for bisphenol A and 5 microg/kg/day for ethinyl estradiol) throughout prenatal and early postnatal development. As adults, the animals were observed in a variety of tests measuring sexually dimorphic behaviors including short-term spatial memory (in a radial-arm maze and a Barnes maze) and anxiety (in an elevated-plus maze and a light/dark preference chamber). Developmental exposure to ethinyl estradiol was found to masculinize behavior in all of the assays used. Bisphenol A increased anxious behavior in a dose-dependent fashion but had no effect on spatial memory. These results indicate that non-reproductive, sexually dimorphic behavior is sensitive to endocrine disruption. In addition, these experiments suggest that both humans and wildlife are being exposed to levels of these endocrine disrupting compounds that are sufficient to disrupt the development of the nervous system and that may have permanent consequences on sexually dimorphic behaviors.

Social Deficits, Stereotypy, and Early Emergence of Repetitive Behavior in the C58/J Inbred Mouse Strain

Mouse lines with behavioral phenotypes relevant to symptoms in neurodevelopmental disorders may provide models to test hypotheses about disease etiology and to evaluate potential treatments. The present studies were designed to confirm and expand earlier work on the intriguing behavioral profile of the C58/J inbred strain, including low social approach and aberrant repetitive movements. Additional tests were selected to reflect aspects of autism, a severe neurodevelopmental disorder characterized by emergence of symptoms early in life, higher prevalence in males, social deficits and abnormal repetitive behavior. Mice from the C57BL/6J inbred strain, which has a similar genetic lineage and physical appearance to C58/J, served as a comparison group. Our results revealed that C58/J mice display elevated activity levels by postnatal day 6, which persist into adulthood. Despite normal olfactory ability, young adult male C58/J mice showed deficits in social approach in the three-chambered choice assay and failed to demonstrate social transmission of food preference. In contrast, female C58/J mice performed similarly to female C57BL/6J mice in both social tests. C58/J mice of both sexes demonstrated abnormal repetitive behaviors, displaying excessive jumping and back flipping in both social and non-social situations. These stereotypies were clearly evident in C58/J pups by postnatal days 20-21, and were also observed in C58/J dams during a test for maternal behavior. Overall, the strain profile for C58/J, including spontaneously developing motor stereotypies emerging early in the developmental trajectory, and social deficits primarily in males, models multiple components of the autism phenotype.

Olfactory cues are sufficient to elicit social approach behaviors but not social transmission of food preference in C57BL/6J mice

Mouse models for the study of autistic-like behaviors are increasingly needed to test hypotheses about the causes of autism, and to evaluate potential treatments. Both the automated three-chambered social approach test and social transmission of food preference have been proposed as mouse behavioral assays with face validity to diagnostic symptoms of autism, including aberrant reciprocal social interactions and impaired communication. Both assays measure aspects of normal social behavior in the mouse. However, little is known regarding the salient cues present in each assay that elicit normal social approach and communication. To deconstruct the critical components, we focused on delivering discrete social and non-social olfactory and visual cues within the context of each assay. Results indicate that social olfactory cues were sufficient to elicit normal sociability in the three-chambered social approach test. On social transmission of food preference, isolated social olfactory cues were sufficient to induce social investigation, but not sufficient to induce food preference. These findings indicate that olfactory cues are important in mouse social interaction, but that additional sensory cues are necessary in certain situations. The present evidence that both the three-chambered social approach assay and the social transmission of food preference assay require socially relevant cues to elicit normal behavior supports the use of these two assays to investigate autism-related behavioral phenotypes in mice.

Rebuttal of “Flawed Experimental Design Reveals the Need for Guidelines Requiring Appropriate Positive Controls in Endocrine Disruption Research” by vom Saal

That the developmental effects of the potent environment estrogen ethinyl estradiol (EE2) address important agency research needs and that EE2 is not just a ‘‘positive control.’’ Why the Long Evans (LE) rat and several other rat strains are excellent animal models for the study of the potential adverse effects of xenoestrogens. Why the argument that LE rats and other rat strains are ‘‘insensitive strains’’ is not consistent with (a) the pharmacological and toxicological data showing that humans and these rat strains display similar sensitivities to EE2 and (b) the cellular and molecular biology of the action of estrogens on different target tissues How the effects of leaching of bisphenol A (BPA) from polycarbonate cages have been grossly exaggerated. That governmental regulatory agencies have concluded that studies like ours (Ryan et al., 2010) are useful for risk assessment, whereas many of the ‘‘positive’’ low-dose studies were ‘‘inadequate’’ or the results have not been replicated. THE DEVELOPMENTAL EFFECTS OF THE POTENT ENVIRONMENT ESTROGEN EE2 ADDRESS IMPORTANT AGENCY RESEARCH NEEDS AND EE2 IS NOT JUST A POSITIVE CONTROL

Novel object exploration in the C58/J mouse model of autistic-like behavior

Mouse models of autistic like behaviors are a valuable tool to use when studying the causes, symptoms, and potential treatments for autism. The inbred C58/J strain is a strain of interest for this model and has previously been shown to possess face validity for some of the core traits of autism, including low social behavior and elevated motor stereotypies. Higher order repetitive behaviors have not been extensively studied in this strain, or in mice in general. In this study, we looked for evidence of higher-order repetitive behaviors in the C58/J strain using a novel object assay. This assay utilized a mouses natural exploratory behavior among unfamiliar objects to identify potential sequencing patterns in motor activity. The motor stereotypies displayed by the C58/J strain during testing were consistent with past studies. The C58/J strain also displayed a high preference for a single object in the round arena assays and the females demonstrating elevated sequencing patterns in the round arena. Although the C58/J strain did not show pervasive evidence of higher-order repetitive behaviors across all measures, there was evidence of higher order repetitive behaviors in certain situations. This study further demonstrates the potential of the C58/J mouse strains as a model for lower-order and potentially, higher-order repetitive behaviors. This study also demonstrates that the shape of the novel object arena can change the behavior displayed by the test animals. Further studies utilizing the C58/J strain and further validation of the novel object assay are warranted.

Novel Object Exploration as a Potential Assay for Higher Order Repetitive Behaviors in Mice

Restricted, repetitive behaviors (RRBs) are a core feature of autism spectrum disorder (ASD) and disrupt the lives of affected individuals. RRBs are commonly split into lower-order and higher-order components, with lower order RRBs consisting of motor stereotypies and higher order RRBs consisting of perseverative and sequencing behaviors. Higher order RRBs are challenging to model in mice. Current assays for RRBs in mice focus primarily on the lower order components, making basic biomedical research into potential treatments or interventions for higher-order RRBs difficult. Here we describe a new assay, novel object exploration. This assay uses a basic open-field arena with four novel objects placed around the perimeter. The test mouse is allowed to freely explore the arena and the order in which the mouse investigates the novel objects is recorded. From these data, patterned sequences of exploration can be identified, as can the most preferred object for each mouse. The representative data shared here and past results using the novel object exploration assay illustrate that inbred mouse strains do demonstrate different behavior in this assay and that strains with elevated lower order RRBs also show elevated patterned behavior. As such, the novel object exploration assay appears to possess good face validity for higher order RRBs in humans and may be a valuable assay for future studies investigating novel therapeutics for ASD.