A multi-lab experimental assessment reveals that replicability can be improved by using empirical estimates of genotype-by-lab interaction.

The utility of mouse and rat studies critically depends on their replicability in other laboratories. A widely advocated approach to improving replicability is through the rigorous control of predefined animal or experimental conditions, known as standardization. However, this approach limits the generalizability of the findings to only to the standardized conditions and is a potential cause rather than solution to what has been called a replicability crisis. Alternative strategies include estimating the heterogeneity of effects across laboratories, either through designs that vary testing conditions, or by direct statistical analysis of laboratory variation. We previously evaluated our statistical approach for estimating the interlaboratory replicability of a single laboratory discovery. Those results, however, were from a well-coordinated, multi-lab phenotyping study and did not extend to the more realistic setting in which laboratories are operating independently of each other. Here, we sought to test our statistical approach as a realistic prospective experiment, in mice, using 152 results from 5 independent published studies deposited in the Mouse Phenome Database (MPD). In independent replication experiments at 3 laboratories, we found that 53 of the results were replicable, so the other 99 were considered non-replicable. Of the 99 non-replicable results, 59 were statistically significant (at 0.05) in their original single-lab analysis, putting the probability that a single-lab statistical discovery was made even though it is non-replicable, at 59.6%. We then introduced the dimensionless "Genotype-by-Laboratory" (GxL) factor-the ratio between the standard deviations of the GxL interaction and the standard deviation within groups. Using the GxL factor reduced the number of single-lab statistical discoveries and alongside reduced the probability of a non-replicable result to be discovered in the single lab to 12.1%. Such reduction naturally leads to reduced power to make replicable discoveries, but this reduction was small (from 87% to 66%), indicating the small price paid for the large improvement in replicability. Tools and data needed for the above GxL adjustment are publicly available at the MPD and will become increasingly useful as the range of assays and testing conditions in this resource increases.

Exploration in the Presence of Mother in Typically and Non-typically Developing Pre-walking Human Infants.

In previous phenotyping studies of mouse and rat exploratory behavior we developed a computational exploratory data analysis methodology including videotaping, tracking, preparatory methods for customized data analysis, a methodology for improving the replicability of results across laboratories, and algorithmic design for exposing the natural reference places (origins) used by animals during exploration. We then measured the animals' paths in reference to these origins, revealing robust, highly replicable modules termed excursions, which are performed from the origin into the environment and back to the origin. Origin-related exploration has been claimed to be phylogenetically conserved across the vertebrates. In the current study we use the same methodology to examine whether origin-related exploration has also been conserved in human pre-walking typically developing (TD) and a group of non-typically developing (NTD) infants in the presence of their stationary mother. The NTDs had been referred to a center for the early treatment of autism in infancy by pediatric neurologists and clinicians. The TDs established a reference place (origin) at mother's place and exhibited a modular partitioning of their path into excursions performed in reference to mother, visiting her often, and reaching closely. In contrast, the NTDs did not establish a distinct origin at the mother's place, or any other place, and did not partition the exploratory path into excursions. Once this difference is validated, the differences between the human infant groups may serve as an early referral tool for child development specialists. The absence of distinct modularity in human infants at risk of autism spectrum disorder can guide the search for animal models for this disorder in translational research.

Vertical exploration and dimensional modularity in mice.

Exploration is a central component of animal behaviour studied extensively in rodents. Previous tests of free exploration limited vertical movement to rearing and jumping. Here, we attach a wire mesh to the arena wall, allowing vertical exploration. This provides an opportunity to study the morphogenesis of behaviour along the vertical dimension, and examine the context in which it is performed. In the current set-up, the mice first use the doorway as a point reference for establishing a borderline linear path along the circumference of the arena floor, and then use this path as a linear reference for performing horizontal forays towards the centre (incursions) and vertical forays on the wire mesh (ascents). Vertical movement starts with rearing on the wall, and commences with straight vertical ascents that increase in extent and complexity. The mice first reach the top of the wall, then mill about within circumscribed horizontal sections, and then progress horizontally for increasingly longer distances on the upper edge of the wire mesh. Examination of the sequence of borderline segments, incursions and ascents reveals dimensional modularity: an initial series (bout) of borderline segments precedes alternating bouts of incursions and bouts of ascents, thus exhibiting sustained attention to each dimension separately. The exhibited separate growth in extent and in complexity of movement and the sustained attention to each of the three dimensions disclose the mice's modular perception of this environment and validate all three as natural kinds.

Reproducibility and replicability of rodent phenotyping in preclinical studies.

The scientific community is increasingly concerned with the proportion of published "discoveries" that are not replicated in subsequent studies. The field of rodent behavioral phenotyping was one of the first to raise this concern, and to relate it to other methodological issues: the complex interaction between genotype and environment; the definitions of behavioral constructs; and the use of laboratory mice and rats as model species for investigating human health and disease mechanisms. In January 2015, researchers from various disciplines gathered at Tel Aviv University to discuss these issues. The general consensus was that the issue is prevalent and of concern, and should be addressed at the statistical, methodological and policy levels, but is not so severe as to call into question the validity and the usefulness of model organisms as a whole. Well-organized community efforts, coupled with improved data and metadata sharing, have a key role in identifying specific problems and promoting effective solutions. Replicability is closely related to validity, may affect generalizability and translation of findings, and has important ethical implications.

Generative rules of Drosophila locomotor behavior as a candidate homology across phyla.

The discovery of shared behavioral processes across phyla is a significant step in the establishment of a comparative study of behavior. We use immobility as an origin and reference for the measurement of fly locomotor behavior; speed, walking direction and trunk orientation as the degrees of freedom shaping this behavior; and cocaine as the parameter inducing progressive transitions in and out of immobility. We characterize and quantify the generative rules that shape Drosophila locomotor behavior, bringing about a gradual buildup of kinematic degrees of freedom during the transition from immobility to normal behavior, and the opposite narrowing down into immobility. Transitions into immobility unfold via sequential enhancement and then elimination of translation, curvature and finally rotation. Transitions out of immobility unfold by progressive addition of these degrees of freedom in the opposite order. The same generative rules have been found in vertebrate locomotor behavior in several contexts (pharmacological manipulations, ontogeny, social interactions) involving transitions in-and-out of immobility. Recent claims for deep homology between arthropod central complex and vertebrate basal ganglia provide an opportunity to examine whether the rules we report also share common descent. Our approach prompts the discovery of behavioral homologies, contributing to the elusive problem of behavioral evolution.

Coping with Space Neophobia in Drosophila melanogaster: The Asymmetric Dynamics of Crossing a Doorway to the Untrodden.

We discover and examine within a wide phylogenetic perspective spatial neophobia, avoidance of untrodden terrain, in fruit flies, in an experimental setup that reduces the gap between the field and the laboratory. In our setup, fruit flies use a natal fruit as their origin, freely exploring for days their surroundings, which consists of a mixture of trodden and untrodden terrain. The interface between trodden and untrodden is, however, reduced in our setup to a wide doorway, opened within a surrounding wall. Crossing this doorway, characterized by a sharp contrast interface between trodden and untrodden, generates a behavior whose dynamics betrays the flies' space neophobia. The moment-by-moment dynamics of crossing is remarkably similar to that reported in mouse models of anxiety. This means that neophobic behavior is either homologous across arthropods and vertebrates or, not less interesting, convergent, whereby the same behavior is mediated in the two phyla by two completely different schemata.

Display of individuality in avoidance behavior and risk assessment of inbred mice.

Factors determining individuality are still poorly understood. Rodents are excellent model organisms to study individuality, due to a rich behavioral repertoire and the availability of well-characterized isogenic populations. However, most current behavioral assays for rodents have short test duration in novel test environments and require human interference, which introduce coercion, thereby limiting the assessment of naturally occurring individuality. Thus, we developed an automated behavior system to longitudinally monitor conditioned fear for assessing PTSD-like behavior in individual mice. The system consists of a safe home compartment connected to a risk-prone test compartment (TC). Entry and exploration of the TC is solely based on deliberate choice determined by individual fear responsiveness and fear extinction. In this novel ethological assay, C57BL/6J mice show homogeneous responses after shock exposure (innate fear), but striking variation in long-lasting fear responses based on avoidance and risk assessment (learned fear), including automated stretch-attend posture quantification. TC entry (retention) latencies after foot shock differed >24 h and the re-explored TC area differed >50% among inbred mice. Next, we compared two closely related C57BL/6 substrains. Despite substantial individual differences, previously observed higher fear of C57BL/6N vs. C57BL/6J mice was reconfirmed, whereas fear extinction was fast and did not differ. The observed variation in fear expression in isogenic mice suggests individual differences in coping style with PTSD-like avoidance. Investigating the assumed epigenetic mechanisms, with reduced interpretational ambiguity and enhanced translational value in this assay, may help improve understanding of personality type-dependent susceptibility and resilience to neuropsychiatric disorders such as PTSD.

The angular interval between the direction of progression and body orientation in normal, alcohol- and cocaine treated fruit flies.

In this study we characterize the coordination between the direction a fruit-fly walks and the direction it faces, as well as offer a methodology for isolating and validating key variables with which we phenotype fly locomotor behavior. Our fundamental finding is that the angular interval between the direction a fly walks and the direction it faces is actively managed in intact animals and modulated in a patterned way with drugs. This interval is small in intact flies, larger with alcohol and much larger with cocaine. The dynamics of this interval generates six coordinative modes that flow smoothly into each other. Under alcohol and much more so under cocaine, straight path modes dwindle and modes involving rotation proliferate. To obtain these results we perform high content analysis of video-tracked open field locomotor behavior. Presently there is a gap between the quality of descriptions of insect behaviors that unfold in circumscribed situations, and descriptions that unfold in extended time and space. While the first describe the coordination between low-level kinematic variables, the second quantify cumulative measures and subjectively defined behavior patterns. Here we reduce this gap by phenotyping extended locomotor behavior in terms of the coordination between low-level kinematic variables, which we quantify, combining into a single field two disparate fields, that of high content phenotyping and that of locomotor coordination. This will allow the study of the genes/brain/locomotor coordination interface in genetically engineered and pharmacologically manipulated animal models of human diseases.

Measuring behavior of animal models: faults and remedies.

Widely used behavioral assays need re-evaluation and validation against their intended use. We focus here on measures of chronic anxiety in mouse models and posit that widely used assays such as the open-field test are performed at the wrong time, for inadequate durations and using inappropriate mouse strains. We propose that behavioral assays be screened for usefulness on the basis of their replicability across laboratories.

Short and long term measures of anxiety exhibit opposite results.

Animal models of human diseases of the central nervous system, generalized anxiety disorder included, are essential for the study of the brain-behavior interface and obligatory for drug development; yet, these models fail to yield new insights and efficacious drugs. By increasing testing duration hundredfold and arena size tenfold, and comparing the behavior of the common animal model to that of wild mice, we raise concerns that chronic anxiety might have been measured at the wrong time, for the wrong duration, and in the wrong animal. Furthermore, the mice start the experimental session with a short period of transient adaptation to the novel environment (habituation period) and a long period reflecting the respective trait of the mice. Using common measures of anxiety reveals that mice exhibit opposite results during these periods suggesting that chronic anxiety should be measured during the post-habituation period. We recommend tools for measuring the transient period, and provide suggestions for characterizing the post habituation period.

The developmental dynamics of behavioral growth processes in rodent egocentric and allocentric space.

In this review I focus on how three methodological principles advocated by Philip Teitelbaum influenced my work to this day: that similar principles of organization should be looked for in ontogeny and recovery of function; that the order of emergence of behavioral components provides a view on the organization of that behavior; and that the components of behavior should be exhibited by the animal itself in relatively pure form. I start by showing how these principles influenced our common work on the developmental dynamics of rodent egocentric space, and then proceed to describe how these principles affected my work with Yoav Benjamini and others on the developmental dynamics of rodent allocentric space. We analyze issues traditionally addressed by physiological psychologists with methods borrowed from ethology, EW (Eshkol-Wachman) movement notation, dynamical systems and exploratory data analysis. Then we show how the natural origins of axes embodied by the behavior of the organism itself, are used by us as the origins of axes for the measurement of the developmental moment-by-moment dynamics of behavior. Using this methodology we expose similar principles of organization across situations, species and preparations, provide a developmental view on the organization of behavior, expose the natural components of behavior in relatively pure form, and reveal how low level primitives generate higher level constructs. Advances in tracking technology should allow us to study how movements in egocentric and allocentric spaces interlace. Tracking of multi-limb coordination, progress in online recording of neural activity in freely moving animals, and the unprecedented accumulation of genetically engineered mouse preparations makes the behavioral ground plan exposed in this review essential for a systematic study of the brain/behavior interface.

Validation of the dimensionality emergence assay for the measurement of innate anxiety in laboratory mice.

The open field test is a common tool to measure innate anxiety in rodents. In the usual configuration of this test the animal is forced to explore the open arena and its behavior includes both anxiety and non-anxiety responses. However, the open arena is generally small and allows only limited expression of exploratory behavior. The recently developed dimensionality emergence assay in which an animal is housed in a home cage with free access to a large circular arena elicits graded exploration and promises to serve as a more ethological test of anxiety. Here we examined the predictive validity of this assay for anxiety-related measures in mice. First, we compared their behavior in the presence or absence of access to the home cage and found that mice with access to the home cage exhibited a gradual build-up in exploration of the arena while those without did not. Then we identified behavioral measures that responded to treatment with the anxiolytic drug diazepam. Diazepam altered several classical measures of innate anxiety, such as distance traveled and thigmotaxis, but also led to a dose-dependent acceleration of the build-up as reflected in a significantly reduced latency to attain several exploratory landmarks. Finally, we tested the utility of the dimensionality emergence assay in assessing alterations in innate anxiety reported in mice carrying a knockout allele for the serotonin 1A receptor (Htr1a). Our findings support the validity of the dimensionality emergence assay as a method to extract an expanded repertoire of behavioral measures for the assessment of anxiety in laboratory mice.

Quantifying the buildup in extent and complexity of free exploration in mice.

To obtain a perspective on an animal's own functional world, we study its behavior in situations that allow the animal to regulate the growth rate of its behavior and provide us with the opportunity to quantify its moment-by-moment developmental dynamics. Thus, we are able to show that mouse exploratory behavior consists of sequences of repeated motion: iterative processes that increase in extent and complexity, whose presumed function is a systematic active management of input acquired during the exploration of a novel environment. We use this study to demonstrate our approach to quantifying behavior: targeting aspects of behavior that are shown to be actively managed by the animal, and using measures that are discriminative across strains and treatments and replicable across laboratories.

Ten ways to improve the quality of descriptions of whole-animal movement.

The demand for replicability of behavioral results across laboratories is viewed as a burden in behavior genetics. We demonstrate how it can become an asset offering a quantitative criterion that guides the design of better ways to describe behavior. Passing the high benchmark dictated by the replicability demand requires less stressful and less restraining experimental setups, less noisy data, individually customized cutoff points between the building blocks of movement, and less variable yet discriminative dynamic representations that would capture more faithfully the nature of the behavior, unmasking similarities and differences and revealing novel animal-centered measures. Here we review ten tools that enhance replicability without compromising discrimination. While we demonstrate the usefulness of these tools in the context of inbred mouse exploratory behavior they can readily be used in any study involving a high-resolution analysis of spatial behavior. Viewing replicability as a design concept and using the ten methodological improvements may prove useful in many fields not necessarily related to spatial behavior.

Knots: attractive places with high path tortuosity in mouse open field exploration.

When introduced into a novel environment, mammals establish in it a preferred place marked by the highest number of visits and highest cumulative time spent in it. Examination of exploratory behavior in reference to this "home base" highlights important features of its organization. It might therefore be fruitful to search for other types of marked places in mouse exploratory behavior and examine their influence on overall behavior.Examination of path curvatures of mice exploring a large empty arena revealed the presence of circumscribed locales marked by the performance of tortuous paths full of twists and turns. We term these places knots, and the behavior performed in them-knot-scribbling. There is typically no more than one knot per session; it has distinct boundaries and it is maintained both within and across sessions. Knots are mostly situated in the place of introduction into the arena, here away from walls. Knots are not characterized by the features of a home base, except for a high speed during inbound and a low speed during outbound paths. The establishment of knots is enhanced by injecting the mouse with saline and placing it in an exposed portion of the arena, suggesting that stress and the arousal associated with it consolidate a long-term contingency between a particular locale and knot-scribbling.In an environment devoid of proximal cues mice mark a locale associated with arousal by twisting and turning in it. This creates a self-generated, often centrally located landmark. The tortuosity of the path traced during the behavior implies almost concurrent multiple views of the environment. Knot-scribbling could therefore function as a way to obtain an overview of the entire environment, allowing re-calibration of the mouse's locale map and compass directions. The rich vestibular input generated by scribbling could improve the interpretation of the visual scene.

Freedom of movement and the stability of its unfolding in free exploration of mice.

Exploration is a central component of human and animal behavior that has been studied in rodents for almost a century. The measures used by neuroscientists to characterize full-blown exploration are limited in exposing the dynamics of the exploratory process, leaving the morphogenesis of its structure and meaning hidden. By unfettering exploration from constraints imposed by hunger, thirst, coercion, and the confines of small cage and short session, using advanced computational tools, we reveal its meaning in the operational world of the mouse. Exploration consists of reiterated roundtrips of increasing amplitude and freedom, involving an increase in the number of independent dimensions along which the mouse moves (macro degrees of freedom). This measurable gradient can serve as a standard reference scale for the developmental dynamics of some aspects of the mouse's emotional-cognitive state and for the study of the interface between behavior and the neurophysiologic and genetic processes mediating it.

Mouse cognition-related behavior in the open-field: emergence of places of attraction.

Spatial memory is often studied in the Morris Water Maze, where the animal's spatial orientation has been shown to be mainly shaped by distal visual cues. Cognition-related behavior has also been described along "well-trodden paths"--spatial habits established by animals in the wild and in captivity reflecting a form of spatial memory. In the present study we combine the study of Open Field behavior with the study of behavior on well-trodden paths, revealing a form of locational memory that appears to correlate with spatial memory. The tracked path of the mouse is used to examine the dynamics of visiting behavior to locations. A visit is defined as either progressing through a location or stopping there, where progressing and stopping are computationally defined. We then estimate the probability of stopping at a location as a function of the number of previous visits to that location, i.e., we measure the effect of visiting history to a location on stopping in it. This can be regarded as an estimate of the familiarity of the mouse with locations. The recently wild-derived inbred strain CZECHII shows the highest effect of visiting history on stopping, C57 inbred mice show a lower effect, and DBA mice show no effect. We employ a rarely used, bottom-to-top computational approach, starting from simple kinematics of movement and gradually building our way up until we end with (emergent) locational memory. The effect of visiting history to a location on stopping in it can be regarded as an estimate of the familiarity of the mouse with locations, implying memory of these locations. We show that the magnitude of this estimate is strain-specific, implying a genetic influence. The dynamics of this process reveal that locations along the mouse's trodden path gradually become places of attraction, where the mouse stops habitually.

Analysis of the trajectory of Drosophila melanogaster in a circular open field arena.

BACKGROUND: Obtaining a complete phenotypic characterization of a freely moving organism is a difficult task, yet such a description is desired in many neuroethological studies. Many metrics currently used in the literature to describe locomotor and exploratory behavior are typically based on average quantities or subjectively chosen spatial and temporal thresholds. All of these measures are relatively coarse-grained in the time domain. It is advantageous, however, to employ metrics based on the entire trajectory that an organism takes while exploring its environment. METHODOLOGY/PRINCIPAL FINDINGS: To characterize the locomotor behavior of Drosophila melanogaster, we used a video tracking system to record the trajectory of a single fly walking in a circular open field arena. The fly was tracked for two hours. Here, we present techniques with which to analyze the motion of the fly in this paradigm, and we discuss the methods of calculation. The measures we introduce are based on spatial and temporal probability distributions and utilize the entire time-series trajectory of the fly, thus emphasizing the dynamic nature of locomotor behavior. Marginal and joint probability distributions of speed, position, segment duration, path curvature, and reorientation angle are examined and related to the observed behavior. CONCLUSIONS/SIGNIFICANCE: The measures discussed in this paper provide a detailed profile of the behavior of a single fly and highlight the interaction of the fly with the environment. Such measures may serve as useful tools in any behavioral study in which the movement of a fly is an important variable and can be incorporated easily into many setups, facilitating high-throughput phenotypic characterization.

Coordination of steering in a free-trotting quadruped.

Typically, locomotion has been studied by restricting the animal's path and/or speed, focusing on stride and step kinematics. Here we incorporate measurements of the legs and trunk in the support and swing phases, during trotting with various speeds and curvatures. This paradigm releases the animal from the confines of the treadmill and runway into the open space. The diagonal step, a new unit of locomotion, is defined by regarding the line between the two supporting diagonal legs as a frame of reference for the description of the dynamics of the virtual line between the two swinging diagonal legs. This analysis reveals that during free trotting the mouse uses three types of steps: fixating, opening, and closing steps. During progression along a straight path, the mouse uses fixating steps, in which the swinging diagonal maintains a fixed direction, landing on the supporting foreleg; during progression along a curved path the mouse uses opening and closing steps alternately. If two steps of the same type are performed sequentially, they engender an abrupt change of direction. Our results reveal how steering with the swinging diagonal, while using a virtually bipedal gait, engenders the whole repertoire of free-trotting behavior.