Inchworming: A Novel Motor Stereotypy in the BTBR T+ Itpr3tf/J Mouse Model of Autism

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental disorder characterized by decreased reciprocal social interaction, abnormal communication, and repetitive behaviors with restricted interest. As diagnosis is based on clinical criteria, any potentially relevant rodent models of this heterogeneous disorder should ideally recapitulate these diverse behavioral traits. The BTBR T⁺ Itpr3^tf/J (BTBR) mouse is an established animal model of ASD, displaying repetitive behaviors such as increased grooming, as well as cognitive inflexibility. With respect to social interaction and interest, the juvenile play test has been employed in multiple rodent models of ASD. Here, we show that when BTBR mice are tested in a juvenile social interaction enclosure containing sawdust bedding, they display a repetitive synchronous digging motion. This repetitive motor behavior, referred to as "inchworming," was named because of the stereotypic nature of the movements exhibited by the mice while moving horizontally across the floor. Inchworming mice must use their fore- and hind-limbs in synchrony to displace the bedding, performing a minimum of one inward and one outward motion. Although both BTBR and C56BL/6J (B6) mice exhibit this behavior, BTBR mice demonstrate a significantly higher duration and frequency of inchworming and a decreased latency to initiate inchworming when placed in a bedded enclosure. We conclude that this newly described behavior provides a measure of a repetitive motor stereotypy that can be easily measured in animal models of ASD.     

Applying Stereotactic Injection Technique to Study Genetic Effects on Animal Behaviors

Stereotactic injection is a useful technique to deliver high titer lentiviruses to targeted brain areas in mice. Lentiviruses can either overexpress or knockdown gene expression in a relatively focused region without significant damage to the brain tissue. After recovery, the injected mouse can be tested on various behavioral tasks such as the Open Field Test (OFT) and the Forced Swim Test (FST). The OFT is designed to assess locomotion and the anxious phenotype in mice by measuring the amount of time that a mouse spends in the center of a novel open field. A more anxious mouse will spend significantly less time in the center of the novel field compared to controls. The FST assesses the anti-depressive phenotype by quantifying the amount of time that mice spend immobile when placed into a bucket of water. A mouse with an anti-depressive phenotype will spend significantly less time immobile compared to control animals. The goal of this protocol is to use the stereotactic injection of a lentivirus in conjunction with behavioral tests to assess how genetic factors modulate animal behaviors.     

Combined prime-boost vaccination against tick-borne encephalitis (TBE) using a recombinant vaccinia virus and a bacterial plasmid both expressing TBE virus non-structural NS1 protein

Background  

Heterologous prime-boost immunization protocols using different gene expression systems have proven to be successful tools in protecting against various diseases in experimental animal models. The main reason for using this approach is to exploit the ability of expression cassettes to prime or boost the immune system in different ways during vaccination procedures. The purpose of the project was to study the ability of recombinant vaccinia virus (VV) and bacterial plasmid, both carrying the NS1 gene from tick-borne encephalitis (TBE) virus under the control of different promoters, to protect mice against lethal challenge using a heterologous prime-boost vaccination protocol.     

Home-cage activity in heterogeneous stock (HS) mice as a model of baseline activity

Behavioral genetic work in humans indicates that clinical hyperactivity is best viewed as the extreme end of activity levels in the population. However, current animal models of hyperactivity are not studied as quantitative traits as they are either knockout models or inbred strains. Furthermore, these animal models generally demonstrate elevated locomotion in novel environments, but not in their home-cages. This is the opposite of the symptoms seen in the human condition where childhood hyperactivity is generally more pronounced in constant, unstimulating situations. In this study we filmed an outbred population of 44 heterogeneous stock (HS) mice under red light during their active phase, to assess the reliability of individual differences in home-cage behavior and extract an index of home-cage activity (HCA) level. We then compared this measure to locomotor behavior in a novel environment — the open-field. Reliable individual differences in home-cage behaviors such as running, swinging on bars, and burrowing were found, and principal component factor analysis yielded a general activity factor, which accounted for 32% of the variance and correlated 0.90 with a subjective impression of activity level. The correlation between HCA and locomotor activity in the open-field was 0.23, which was non-significant. However, the association with HCA level appeared to increase over the five minutes of the open-field, presumably as the mice habituated. Furthermore, although mice displaying particularly high and low HCA were indistinguishable early in the open-field task, they became significantly differentiated over time. We conclude that home-cage behaviors and the open-field, after habituation, display good face and construct validity, and may provide a good model of baseline activity for quantitative trait loci (QTL) discovery and functional genomics in the HS mice.     

Environmental bias? Effects of housing conditions,laboratory environment and experimenter on behavioral tests

Behavioral testing does not always yield similar results when replicated in different laboratories, and it usually remains unclear whether the variability in results is caused by different laboratory environments or different experimenters conducting the tests. In our study, we applied a systematic variation of housing conditions, laboratories and experimenters in order to test the influence of these variables on the outcome of behavioral tests. We wanted to know whether known effects of different housing conditions on behavior can be demonstrated regardless of the respective laboratory and experimenters. In this study, we compared the behavior of mice kept under enriched housing conditions with mice kept in unstructured cages regarding their exploratory, locomotor and anxiety-related behavior in the barrier test, in the open-field test and in the elevated plus-maze test. Experiments were conducted by six different persons in two different laboratories. In spite of an extensive protocol standardizing laboratory environment, animal maintenance and testing procedures, significant differences in absolute values between different laboratories as well as between different experimenters were noticed in the barrier test and in the elevated plus-maze test but not in the open-field test. However, with regard to the differences between enriched and unstructured housing conditions, overall consistent results were achieved by different experimenters in both laboratories. We conclude that the reliability of behavioral phenotyping is not challenged seriously by experimenter and laboratory environment as long as appropriate standardizations are met and suitable controls are involved.     

The Use of the Puzzle Box as a Means of Assessing the Efficacy of Environmental Enrichment

Environmental enrichment can dramatically influence the development and function of neural circuits. Further, enrichment has been shown to successfully delay the onset of symptoms in models of Huntington’s disease ^1-4, suggesting environmental factors can evoke a neuroprotective effect against the progressive, cellular level damage observed in neurodegenerative disorders. The ways in which an animal can be environmentally enriched, however, can vary considerably. Further, there is no straightforward manner in which the effects of environmental enrichment can be assessed: most methods require either fairly complicated behavioral paradigms and/or postmortem anatomical/physiological analyses. This protocol describes the use of a simple and inexpensive behavioral assay, the Puzzle Box ^5-7 as a robust means of determining the efficacy of increased social, sensory and motor stimulation on mice compared to cohorts raised in standard laboratory conditions. This simple problem solving task takes advantage of a rodent’s innate desire to avoid open enclosures by seeking shelter. Cognitive ability is assessed by adding increasingly complex impediments to the shelter’s entrance. The time a given subject takes to successfully remove the obstructions and enter the shelter serves as the primary metric for task performance. This method could provide a reliable means of rapidly assessing the efficacy of different enrichment protocols on cognitive function, thus paving the way for systematically determining the role specific environmental factors play in delaying the onset of neurodevelopmental and neurodegenerative disease.     

Morris Water Maze Test: Optimization for Mouse Strain and Testing Environment

The Morris water maze (MWM) is a commonly used task to assess hippocampal-dependent spatial learning and memory in transgenic mouse models of disease, including neurocognitive disorders such as Alzheimer’s disease. However, the background strain of the mouse model used can have a substantial effect on the observed behavioral phenotype, with some strains exhibiting superior learning ability relative to others. To ensure differences between transgene negative and transgene positive mice can be detected, identification of a training procedure sensitive to the background strain is essential. Failure to tailor the MWM protocol to the background strain of the mouse model may lead to under- or over- training, thereby masking group differences in probe trials. Here, a MWM protocol tailored for use with the F1 FVB/N x 129S6 background is described. This is a frequently used background strain to study the age-dependent effects of mutant P301L tau (rTg(TauP301L)4510 mice) on the memory deficits associated with Alzheimer’s disease. Also described is a strategy to re-optimize, as dictated by the particular testing environment utilized.     

Genotype-specific environmental impact on the variance of blood values in inbred and F1 hybrid mice

Mice are important models for biomedical research because of the possibility of standardizing genetic background and environmental conditions, which both affect phenotypic variability. Inbred mouse strains as well as F₁ hybrid mice are routinely used as genetically defined animal models; however, only a few studies investigated the variance of phenotypic parameters in inbred versus F₁ hybrid mice and the potential interference of the genetic background with different housing conditions. Thus, we analyzed the ranges of clinical chemical and hematologic parameters in C3H and C57BL/6 inbred mice and their reciprocal F₁ hybrids (B6C3F1, C3B6F1) in two different mouse facilities. Two thirds of the blood parameters examined in the same strain differed between the facilities for both the inbred strains and the F₁ hybrid lines. The relation of the values between inbred and F₁ hybrid mice was also affected by the facility. The variance of blood parameters in F₁ hybrid mice compared with their parental inbred strains was inconsistent in one facility but generally smaller in the other facility. A subsequent study of F₁ hybrid animals derived from the parental strains C3H and BALB/c, which was done in the latter housing unit, detected no general difference in the variance of blood parameters between F₁ hybrid and inbred mice. Our study clearly demonstrates the possibility of major interactions between genotype and environment regarding the variance of clinical chemical and hematologic parameters.     

Social Communication in Mice – Are There Optimal Cage Conditions?

Social communication is heavily affected in patients with neuropsychiatric disorders. Accordingly, mouse models designed to study the mechanisms leading to these disorders are tested for this phenotypic trait. Test conditions vary between different models, and the effect of these test conditions on the quantity and quality of social interactions and ultrasonic communication is unknown. The present study examines to which extent the habituation time to the test cage as well as the shape / size of the cage influence social communication in freely interacting mice. We tested 8 pairs of male mice in free dyadic social interactions, with two habituation times (20 min and 30 min) and three cage formats (rectangle, round, square). We tested the effect of these conditions on the different types of social contacts, approach-escape sequences, follow behavior, and the time each animal spent in the vision field of the other one, as well as on the emission of ultrasonic vocalizations and their contexts of emission. We provide for the first time an integrated analysis of the social interaction behavior and ultrasonic vocalizations. Surprisingly, we did not highlight any significant effect of habituation time and cage shape / size on the behavioral events examined. There was only a slight increase of social interactions with the longer habituation time in the round cage. Remarkably, we also showed that vocalizations were emitted during specific behavioral sequences especially during close contact or approach behaviors. The present study provides a protocol reliably eliciting social contacts and ultrasonic vocalizations in adult male mice. This protocol is therefore well adapted for standardized investigation of social interactions in mouse models of neuropsychiatric disorders.     

Marble Burying and Nestlet Shredding as Tests of Repetitive,Compulsive-like Behaviors in Mice

Obsessive-compulsive disorder (OCD) and autism spectrum disorders (ASD) are serious and debilitating psychiatric conditions and each constitutes a significant public health concern, particularly in children. Both of these conditions are highlighted by the repeated expression of meaningless behaviors. Individuals with OCD often show checking, frequent hand washing, and counting. Children with ASDs also engage in repetitive tapping, arm or hand flapping, and rocking. These behaviors can vary widely in intensity and frequency of expression. More intense forms of repetitive behaviors can even result in injury (e.g. excessive grooming, hand washing, and self-stimulation). These behaviors are therefore very disruptive and make normal social discourse difficult. Treatment options for repetitive behaviors in OCD and ASDs are somewhat limited and there is great interest in developing more effective therapies for each condition. Numerous animal models for evaluating compulsive-like behaviors have been developed over the past three decades. Perhaps the animal models with the greatest validity and ease of use are the marble burying test and the nestlet shredding test. Both tests take advantage of the fact that the target behaviors occur spontaneously in mice. In the marble burying test, 20 marbles are arrayed on the surface of clean bedding. The number of marbles buried in a 30 min session is scored by investigators blind to the treatment or status of the subjects. In the nestlet shredding test, a nestlet comprised of pulped cotton fiber is preweighed and placed on top of cage bedding and the amount of the nestlet remaining intact after a 30 min test session is determined. Presently, we describe protocols for and show movie documentation of marble burying and nestlet shredding. Both tests are easily and accurately scored and each is sensitive to small changes in the expression of compulsive-like behaviors that result from genetic manipulations, disease, or head injury.     

Expanding our scientific horizons: utilization of unique model organisms in biological research

During the past century, research studies using animal models have contributed to numerous scientific discoveries and have been vital for the understanding of numerous biological processes, including disease. Over the past decades, the scientific community has defined a small number of model organisms that includes a few mammals, fish (mainly zebrafish), birds (mainly chicken), frogs, flies, and nematodes. Rodents are by far the most commonly employed laboratory animals in biomedical research. Mice share many biological similarities to humans and can be genetically manipulated to express mutations linked to human diseases. Mice and rats reproduce relatively quickly and have a short life span, which allows scientists to study progressive disorders, including aging. A large range of inbred mice strains enables accurate and reproducible experiments by decreasing the variability often associated with animal models and biological systems in general. Finally, mice are cost‐effective, small, and relatively easy to handle, transport, and house. All of these advantages combine to make mice the major species for recapitulating and studying human diseases. However, focusing exclusively on one or very few animal models may lead researchers to lose sight of other species with vastly different biology that might inform and affect our understanding of disease pathogenesis.     

Excessive D1 Dopamine Receptor Activation in the Dorsal Striatum Promotes Autistic-Like Behaviors

The dopamine system has been characterized in motor function, goal-directed behaviors, and rewards. Recent studies recognize various dopamine system genes as being associated with autism spectrum disorder (ASD). However, how dopamine system dysfunction induces ASD pathophysiology remains unknown. In the present study, we demonstrated that mice with increased dopamine functions in the dorsal striatum via the suppression of dopamine transporter expression in substantia nigra neurons or the optogenetic stimulation of the nigro-striatal circuitry exhibited sociability deficits and repetitive behaviors relevant to ASD pathology in animal models, while these behavioral changes were blocked by a D1 receptor antagonist. Pharmacological activation of D1 dopamine receptors in normal mice or the genetic knockout (KO) of D2 dopamine receptors also produced typical autistic-like behaviors. Moreover, the siRNA-mediated inhibition of D2 dopamine receptors in the dorsal striatum was sufficient to replicate autistic-like phenotypes in D2 KO mice. Intervention of D1 dopamine receptor functions or the signaling pathways-related D1 receptors in D2 KO mice produced anti-autistic effects. Together, our results indicate that increased dopamine function in the dorsal striatum promotes autistic-like behaviors and that the dorsal striatum is the neural correlate of ASD core symptoms.     

Dissociation of Learned Helplessness and Fear Conditioning in Mice: A Mouse Model of Depression

The state of being helpless is regarded as a central aspect of depression, and therefore the learned helplessness paradigm in rodents is commonly used as an animal model of depression. The term ‘learned helplessness’ refers to a deficit in escaping from an aversive situation after an animal is exposed to uncontrollable stress specifically, with a control/comparison group having been exposed to an equivalent amount of controllable stress. A key feature of learned helplessness is the transferability of helplessness to different situations, a phenomenon called ‘trans-situationality’. However, most studies in mice use learned helplessness protocols in which training and testing occur in the same environment and with the same type of stressor. Consequently, failures to escape may reflect conditioned fear of a particular environment, not a general change of the helpless state of an animal. For mice, there is no established learned helplessness protocol that includes the trans-situationality feature. Here we describe a simple and reliable learned helplessness protocol for mice, in which training and testing are carried out in different environments and with different types of stressors. We show that with our protocol approximately 50% of mice develop learned helplessness that is not attributable to fear conditioning.     

High‐throughput analysis of vocalizations reveals sex‐specific changes in Fmr1 mutant pups

There have been several reports that individuals with Fragile X syndrome (FXS) and animal models of FXS have communication deficits. The present study utilized two different call classification taxonomies to examine the sex‐specificity of ultrasonic vocalization (USV) production on postnatal day (PD8) in the FVB strain of Fmr1 knockout (KO) mice. One classification protocol requires the investigator to score each call by hand, while the other protocol uses an automated algorithm. Results using the hand‐scoring protocol indicated that male Fmr1 KO mice exhibited longer calls (P = .03) than wild types on PD8. Male KOs also produced fewer complex, composite, downward, short and two‐syllable call‐types, as well as more frequency steps and chevron call‐types. Female heterozygotes exhibited no significant changes in acoustic or temporal aspects of calls, yet showed significant changes in call‐type production proportions across two different classification taxonomies (P < .001). They exhibited increased production of harmonic and frequency steps calls, as well as fewer chevron, downward and short calls. According to the second high‐throughput analysis, female heterozygotes produced significantly fewer single‐type and more multiple‐type syllables, unlike male KOs that showed no changes in these aspects of syllable production. Finally, we correlated both scoring methods and found a high level of correlation between the two methods. These results contribute further knowledge of sex differences in USV calling behavior for Fmr1 heterozygote and KO mice and provide a foundation for the use of high‐throughput analysis of neonatal USVs.     

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association between environmental cues and aversive experiences. In this test, mice are placed into a conditioning chamber and are given parings of a conditioned stimulus (an auditory cue) and an aversive unconditioned stimulus (an electric footshock). After a delay time, the mice are exposed to the same conditioning chamber and a differently shaped chamber with presentation of the auditory cue. Freezing behavior during the test is measured as an index of fear memory. To analyze the behavior automatically, we have developed a video analyzing system using the ImageFZ application software program, which is available as a free download at http://www.mouse-phenotype.org/. Here, to show the details of our protocol, we demonstrate our procedure for the contextual and cued fear conditioning test in C57BL/6J mice using the ImageFZ system. In addition, we validated our protocol and the video analyzing system performance by comparing freezing time measured by the ImageFZ system or a photobeam-based computer measurement system with that scored by a human observer. As shown in our representative results, the data obtained by ImageFZ were similar to those analyzed by a human observer, indicating that the behavioral analysis using the ImageFZ system is highly reliable. The present movie article provides detailed information regarding the test procedures and will promote understanding of the experimental situation.     

Variability in empathic fear response among 11 inbred strains of mice

Empathy is an important emotional process that involves the ability to recognize and share emotions with others. We have previously developed an observational fear learning (OFL) behavioral assay to measure empathic fear in mice. In the OFL task, a mouse is conditioned for context‐dependent fear when it observes a conspecific demonstrator receiving aversive stimuli. In the present study, by comparing 11 different inbred mouse strains that are commonly used in the laboratory, we found that empathic fear response was highly variable between different strains. Five strains – C57BL/6J, C57BL/6NTac, 129S1/SvImJ, 129S4/SvJae and BTBR T⁺ Itpr3^tf/J – showed observational fear (OF) responses, whereas AKR/J, BALB/cByJ, C3H/HeJ, DBA/2J, FVB/NJ and NOD/ShiLtJ mice exhibited low empathic fear response. Importantly, day 2 OF memory was significantly correlated with contextual memory in the classical fear conditioning among the 11 strains. Innate differences in anxiety, locomotor activity, sociability and preference for social novelty were not significantly correlated with OFL. Interestingly, early adolescent C57BL/6J mice exhibited an increase in acquisition of OF. The level of OFL in C57BL/6J strain was not affected by sex or strains of the demonstrator. Taken together, these data strongly suggest that there are naturally occurring OFL‐specific genetic variations modulating empathic fear behaviors in mice. The identification of causal genes may uncover novel genetic pathways and underlying neural mechanisms that modulate empathic fear and, ultimately, provide new targets for therapeutic intervention in human mental disorders associated with impaired empathy.     

Assessment of environmental enrichment for different primate species under low budget: A case study

The 2011 European Union Zoo Inquiry presented evidence that most zoos fail to achieve adequate levels of nonhuman animal welfare. Appropriate environmental enrichment (EE) can play a role in the promotion of welfare. However, financial and staff constraints frequently make it challenging to implement EE on a daily basis. The aim of this study was to test how individuals of three different nonhuman primate species at the Maia Zoo in Portugal (white-handed gibbons, Hylobates lar; Mona monkeys, Cercopithecus mona; and brown lemurs, Eulemur fulvus) reacted to EE devices. Another aim was to investigate whether simple, inexpensive devices can have positive outcomes for animal welfare. The results showed that all individuals reacted to the EE intervention, but individual responses to the EE devices differed. EE devices had consistently positive effects for all three species as demonstrated by an increase in the expression of natural behaviors and a general decrease in abnormal behaviors (46% compared with baseline). This study demonstrates that it is important to simultaneously test multiple individuals from different species in different situations to evaluate the effectiveness of EE.     

Developmental exposure to low-dose estrogenic endocrine disruptors alters sex differences in exploration and emotional responses in mice

Estrogenic endocrine disruptors (EEDs) are naturally occurring or man-made compounds present in the environment that are able to bind to estrogen receptors and interfere with normal cellular development in target organs and tissues. There is mounting evidence that EEDs can interfere with the processes of sexual differentiation of brain and behavior in different animal models. We investigated the effects of maternal exposure to EEDs, at concentrations within the range of human exposure and not patently teratogenic, on behavioral responses of male and female house mice (Mus musculus domesticus) before and after puberty. Pregnant dams were trained to spontaneously drink daily doses of corn oil with or without the estrogenic plastic derivative, bisphenol A (BPA 10 microg/kg), or the estrogenic insecticide methoxychlor (MXC 20 microg/kg) from gestation day 11 to postpartum day 8. Their male and female offspring were examined at different ages to examine several components of explorative and emotional behaviors in 3 experimental paradigms: a novelty test before puberty and, as adults, a free-exploratory open-field test and the elevated plus maze test. The main results are sex differences in control mice on a number of behavioral responses at both ages and in all experimental paradigms, while perinatal exposure to BPA or MXC decreased or eliminated such sex differences. The present findings are evidence of long-term consequences of developmental exposure to BPA and MXC on neurobehavioral development and suggest a differential effect of low-dose exposure to these estrogenic chemicals in males and females.     

Behavioral phenotyping of transgenic and knockout mice: practical concerns and potential pitfalls

New technologies in molecular genetics have dramatically increased the number of targeted gene mutations available to the biomedical research community. Many mutant mouse lines have been generated to provide animal models for human genetic disorders, offering insights into anatomical, neurochemical, and behavioral effects of aberrant gene expression. A variety of assays have been developed to identify and characterize phenotypic changes. In the behavioral domain, our phenotyping strategy involves a comprehensive standardized methodological approach that assesses general health, reflexes, sensory abilities, and motor functions. This assessment is followed by a series of complementary tasks in the specific behavioral domain(s) hypothesized to reveal the function(s) of the gene. Our multitiered approach minimizes intersubject variability by standardizing the experimental history for all animals, improves interlaboratory reliability by providing a clearly defined experimental protocol, and minimizes artifactual interpretations of behavioral data by careful preliminary assessments of basic behaviors, followed by multiple tests within the behavioral domain of interest. Despite meticulous attention to experimental protocol, attention to environmental factors is essential. Differences in noise, light, home cage environment, handling, and diet can dramatically alter behavior. Baseline differences in the behaviors of inbred strains used to generate targeted mutant mouse lines can directly influence the behavioral phenotype of the mutant line. Strategies aimed at minimizing environmental variability and contributions of background genes will enhance the robustness of mouse behavioral phenotyping assays.