The horizontal ladder test (HLT) protocol: a novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus.

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. To address this knowledge gap, an apparatus modeled after a horizontally placed ladder and where the height of the rungs could be adjusted was developed. The protocol that was employed within the apparatus mimicked the walk and turn test of the human standardized field sobriety test. Here, five Sinclair miniature pigs were trained to cross the horizontally placed ladder, starting at a rung height of six inches and decreasing to three inches in one-inch increments. It was demonstrated that pigs can reliably learn to cross the ladder, with few errors, under baseline/unimpaired conditions. These animals were then involved in a voluntary consumption of ethanol study where animals were longitudinally evaluated for motor coordination changes at baseline, 2.5, 5, 7.5, and 10% ethanol concentrations subsequently to consuming ethanol. Consistent with our predictions, relative to baseline performance, motor incoordination increased as voluntary consumption of escalating concentrations of ethanol increased. Together these data highlight that the horizontal ladder test (HLT) test protocol is a novel, optimized and reliable test for evaluating motor coordination as well as changes in motor coordination in pigs.

ATP1A1-linked diseases require a malfunctioning protein product from one allele.

Heterozygous germline variants in ATP1A1, the gene encoding the α1 subunit of the Na+/K+-ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αß) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na+ and K+ that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1+/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1+/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1-related diseases despite carrying a plasma-membrane protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

ATP1A1 -linked diseases require a malfunctioning protein product from one allele.

Heterozygous germline variants in ATP1A1 , the gene encoding the α1 subunit of the Na + /K + -ATPase (NKA), have been linked to diseases including primary hyperaldosteronism and the peripheral neuropathy Charcot-Marie-Tooth disease (CMT). ATP1A1 variants that cause CMT induce loss-of-function of NKA. This heterodimeric (αß) enzyme hydrolyzes ATP to establish transmembrane electrochemical gradients of Na + and K + that are essential for electrical signaling and cell survival. Of the 4 catalytic subunit isoforms, α1 is ubiquitously expressed and is the predominant paralog in peripheral axons. Human population sequencing datasets indicate strong negative selection against both missense and protein-null ATP1A1 variants. To test whether haploinsufficiency generated by heterozygous protein-null alleles are sufficient to cause disease, we tested the neuromuscular characteristics of heterozygous Atp1a1 +/- knockout mice and their wildtype littermates, while also evaluating if exercise increased CMT penetrance. We found that Atp1a1 +/- mice were phenotypically normal up to 18 months of age. Consistent with the observations in mice, we report clinical phenotyping of a healthy adult human who lacks any clinical features of known ATP1A1 -related diseases despite carrying a protein-null early truncation variant, p.Y148*. Taken together, these results suggest that a malfunctioning gene product is required for disease induction by ATP1A1 variants and that if any pathology is associated with protein-null variants, they may display low penetrance or high age of onset.

The Horizontal Ladder Test (HLT) protocol: A novel, optimized, and reliable means of assessing motor coordination in Sus scrofa domesticus.

Pigs can be an important model for preclinical biological research, including neurological diseases such as Alcohol Use Disorder. Such research often involves longitudinal assessment of changes in motor coordination as the disease or disorder progresses. Current motor coordination tests in pigs are derived from behavioral assessments in rodents and lack critical aspects of face and construct validity. While such tests may permit for the comparison of experimental results to rodents, a lack of validation studies of such tests in the pig itself may preclude the drawing of meaningful conclusions. Here, we present a novel, optimized, and reliable horizontal ladder test (HLT) test protocol for evaluating motor coordination in pigs and an initial validation of its construct validity using voluntary alcohol consumption as an experimental manipulation.

Effects of weaning age and housing conditions on phenotypic differences in mice.

Poor reproducibility is considered a serious problem in laboratory animal research, with important scientific, economic, and ethical implications. One possible source of conflicting findings in laboratory animal research are environmental differences between animal facilities combined with rigorous environmental standardization within studies. Due to phenotypic plasticity, study-specific differences in environmental conditions during development can induce differences in the animals' responsiveness to experimental treatments, thereby contributing to poor reproducibility of experimental results. Here, we studied how variation in weaning age (14-30 days) and housing conditions (single versus group housing) affects the phenotype of SWISS mice as measured by a range of behavioral and physiological outcome variables. Weaning age, housing conditions, and their interaction had little effect on the development of stereotypies, as well as on body weight, glucocorticoid metabolite concentrations, and behavior in the elevated plus-maze and open field test. These results are surprising and partly in conflict with previously published findings, especially with respect to the effects of early weaning. Our results thus question the external validity of previous findings and call for further research to identify the sources of variation between replicate studies and study designs that produce robust and reproducible experimental results.

A Systematic Review and Meta-Analysis of the Relationship Between Social Dominance Status and Common Behavioral Phenotypes in Male Laboratory Mice.

Background: Social dominance status (e.g., dominant or subordinate) is often associated with individual differences in behavior and physiology but is largely neglected in experimental designs and statistical analysis plans in biomedical animal research. In fact, the extent to which social dominance status affects common experimental outcomes is virtually unknown. Given the pervasive use of laboratory mice and culminating evidence of issues with reproducibility, understanding the role of social dominance status on common behavioral measures used in research may be of paramount importance. Methods: To determine whether social dominance status-one facet of the social environment-contributes in a systematic way to standard measures of behavior in biomedical science, we conducted a systematic review of the existing literature searching the databases of PubMed, Embase, and Web of Science. Experiments were divided into several domains of behavior: exploration, anxiety, learned helplessness, cognition, social, and sensory behavior. Meta-analyses between experiments were conducted for the open field, elevated plus-maze, and Porsolt forced swim test. Results: Of the 696 publications identified, a total of 55 experiments from 20 published studies met our pre-specified criteria. Study characteristics and reported results were highly heterogeneous across studies. A systematic review and meta-analyses, where possible, with these studies revealed little evidence for systematic phenotypic differences between dominant and subordinate male mice. Conclusion: This finding contradicts the notion that social dominance status impacts behavior in significant ways, although the lack of an observed relationship may be attributable to study heterogeneity concerning strain, group-size, age, housing and husbandry conditions, and dominance assessment method. Therefore, further research considering these secondary sources of variation may be necessary to determine if social dominance generally impacts treatment effects in substantive ways.

Social dominance hierarchy type and rank contribute to phenotypic variation within cages of laboratory mice.

A tacit assumption in laboratory animal research is that animals housed within the same cage or pen are phenotypically more similar than animals from different cages or pens, due to their shared housing environment. This assumption drives experimental design, randomization schemes, and statistical analysis plans, while neglecting social context. Here, we examined whether a domain of social context-social dominance-accounted for more phenotypic variation in mice than cage-identity. First, we determined that cages of mice could be categorized into one of three dominance hierarchies with varying degrees of dominance behavior between cage-mates, and low levels of agonistic behavior in the home-cage. Most groups formed dynamic hierarchies with unclear ranks, contrasting with recent accounts of stable transitive hierarchies in groups of mice. Next, we measured some phenotypic traits, and found that social dominance (i.e. dominance hierarchy type and degree of dominance behavior) consistently accounted for some phenotypic variation in all outcome measures, while cage-identity accounted for phenotypic variation in some measures but virtually no variation in others. These findings highlight the importance of considering biologically relevant factors, such as social dominance, in experimental designs and statistical plans.

Freestanding bipedal posture and coordinated bimanual manipulation significantly influence lateralized hand use in rhesus monkeys (Macaca mulatta) and chimpanzees (pan troglodytes).

Investigations of behavioral lateralization in nonhuman primates yield important insights into brain-behavior relationships. In turn, they provide clues about both proximal and distal factors that shape the development and expression of association between motor asymmetries and underlying neural substrates. Nonhuman primates afford unique comparative opportunities to evaluate potential routes for the evolution of handedness, as well as to uncover relationships between behavioral lateralization and underlying neural, genetic, and physiological correlates. We examined hand preference in 22 rhesus monkeys and 79 chimpanzees using unimanual reaching tasks varying in postural stability and in a coordinated bimanual task. The majority of rhesus monkeys and chimpanzees showed significant lateral biases when reaching from a freestanding posture and when engaged in a coordinated bimanual task. Population-level directional bias was not evident for any task for rhesus monkeys and was observed only in the bimanual task for chimpanzees. We did not find consistent relationships between an individual's hand preference for different types of tasks. Both freestanding bipedal posture and coordinated bimanual hand use elicited significantly stronger lateral biases in reaching when compared with quadrupedal reaching. These data support the hypothesis that both degrees of postural instability and complex manipulation, such as bimanual coordination, may influence the expression of behavioral asymmetries in primates. These results demonstrate robust lateralization occurs at the individual level. Our results also highlight the need for greater consideration of task type and descriptive data in studies aimed at evaluating brain-behavior relationships and individual differences associated with hand preference. (PsycINFO Database Record (c) 2019 APA, all rights reserved).

Effects of Cage Enrichment on Behavior, Welfare and Outcome Variability in Female Mice.

The manner in which laboratory rodents are housed is driven by economics (minimal use of space and resources), ergonomics (ease of handling and visibility of animals), hygiene, and standardization (reduction of variation). This has resulted in housing conditions that lack sensory and motor stimulation and restrict the expression of species-typical behavior. In mice, such housing conditions have been associated with indicators of impaired welfare, including abnormal repetitive behavior (stereotypies, compulsive behavior), enhanced anxiety and stress reactivity, and thermal stress. However, due to concerns that more complex environmental conditions might increase variation in experimental results, there has been considerable resistance to the implementation of environmental enrichment beyond the provision of nesting material. Here, using 96 C57BL/6 and SWISS female mice, respectively, we systematically varied environmental enrichment across four levels spanning the range of common enrichment strategies: (1) bedding alone; (2) bedding + nesting material; (3) deeper bedding + nesting material + shelter + increased vertical space; and (4) semi-naturalistic conditions, including weekly changes of enrichment items. We studied how these different forms of environmental enrichment affected measures of animal welfare, including home-cage behavior (time-budget and stereotypic behavior), anxiety (open field behavior, elevated plus-maze behavior), growth (food and water intake, body mass), stress physiology (glucocorticoid metabolites in fecal boluses and adrenal mass), brain function (recurrent perseveration in a two-choice guessing task) and emotional valence (judgment bias). Our results highlight the difficulty in making general recommendations across common strains of mice and for selecting enrichment strategies within specific strains. Overall, the greatest benefit was observed in animals housed with the greatest degree of enrichment. Thus, in the super-enriched housing condition, stereotypic behavior, behavioral measures of anxiety, growth and stress physiology varied in a manner consistent with improved animal welfare compared to the other housing conditions with less enrichment. Similar to other studies, we found no evidence, in the measures assessed here, that environmental enrichment increased variation in experimental results.

Phenotypic variability between Social Dominance Ranks in laboratory mice.

The laboratory mouse is the most prevalent animal used in experimental procedures in the biomedical and behavioural sciences. Yet, many scientists fail to consider the animals' social context. Within a cage, mice may differ in their behaviour and physiology depending on their dominance relationships. Therefore, dominance relationships may be a confounding factor in animal experiments. The current study housed male and female C57BL/6ByJ mice in same-sex groups of 5 in standard laboratory conditions and investigated whether dominance hierarchies were present and stable across three weeks, and whether mice of different dominance ranks varied consistently in behaviour and physiology. We found that dominance ranks of most mice changed with time, but were most stable between the 2nd and 3rd week of testing. Phenotypic measures were also highly variable, and we found no relation between dominance rank and phenotype. Further, we found limited evidence that variation in measures of phenotype was associated with cage assignment for either males or females. Taken together, these findings do not lend support to the general assumption that individual variation among mice is larger between cages than within cages.

A cross-species judgement bias task: integrating active trial initiation into a spatial Go/No-go task.

Judgement bias tasks are promising tools to assess emotional valence in animals, however current designs are often time-consuming and lack aspects of validity. This study aimed to establish an improved design that addresses these issues and can be used across species. Horses, rats, and mice were trained on a spatial Go/No-go task where animals could initiate each trial. The location of an open goal-box, at either end of a row of five goal-boxes, signalled either reward (positive trial) or non-reward (negative trial). Animals first learned to approach the goal-box in positive trials (Go) and to re-initiate/not approach in negative trials (No-go). Animals were then tested for responses to ambiguous trials where goal-boxes at intermediate locations were opened. The Go:No-go response ratio was used as a measure of judgement bias. Most animals quickly learned the Go/No-go discrimination and performed trials at a high rate compared to previous studies. Subjects of all species reliably discriminated between reference cues and ambiguous cues, demonstrating a monotonic graded response across the different cue locations, with no evidence of learning about the outcome of ambiguous trials. This novel test protocol is an important step towards a practical task for comparative studies on judgement biases in animals.

Evaluation of the effects of space allowance on measures of animal welfare in laboratory mice.

We studied how space allowance affects measures of animal welfare in mice by systematically varying group size and cage type across three levels each in both males and females of two strains of mice (C57BL/6ByJ and BALB/cByJ; n = 216 cages, a total of 1152 mice). This allowed us to disentangle the effects of total floor area, group size, stocking density, and individual space allocation on a broad range of measures of welfare, including growth (food and water intake, body mass); stress physiology (glucocorticoid metabolites in faecal boli); emotionality (open field behaviour); brain function (recurrent perseveration in a two-choice guessing task); and home-cage behaviour (activity, stereotypic behaviour). While increasing group size was associated with a decrease in food and water intake in general, and more specifically with increased attrition due to escalated aggression in male BALB mice, no other consistent effects of any aspect of space allowance were found with respect to the measures studied here. Our results indicate that within the range of conditions commonly found in laboratory mouse housing, space allowance as such has little impact on measures of welfare, except for group size which may be a risk factor for escalating aggression in males of some strains.

Arsenic Speciation in Bituminous Coal Fly Ash and Transformations in Response to Redox Conditions.

The risk of the mobilization of coal ash into the environment has highlighted the need for the assessment of the environmental behavior of coal ash, particularly with respect to toxic trace elements such as arsenic (As). Here, we examined As speciation in coal fly ash samples and transformations in response to aquatic redox conditions. X-ray absorption spectroscopy indicated that 92-97% of total As occurred as As(V), with the remainder present as As(III). Major As-bearing hosts in unamended ashes were glass, iron (oxyhydr)oxides, and calcium arsenate. Oxic leaching resulted in immediate As mobilization to the aqueous phase, reprecipitation of As-iron ferrihydrite, and As adsorption to mineral surfaces. Under anoxic conditions, the (reductive) dissolution of As-bearing phases such as iron ferrihydrite resulted in increased dissolved As compared to oxic conditions and reprecipitation of iron arsenate. Overall, As in coal ash is not environmentally stable and can participate in local biogeochemical cycles.

Maternal separation followed by isolation-housing differentially affects prepulse inhibition of the acoustic startle response in C57BL/6 mice.

Exposure to chronic stress is associated with an increased incidence of neuropsychiatric dysfunction. The current study evaluated two competing hypotheses, the cumulative stress and the match/mismatch hypothesis of neuropsychiatric dysfunction, using two paradigms relating to exposure to "stress": pre-weaning maternal separation and post-weaning isolation-housing. C57BL/6 offspring were reared under four conditions: typical animal facility rearing (AFR, control), early handling (EH, daily 15 min separation from dam), maternal separation (MS, daily 4 hr separation from dam), and maternal and peer separation (MPS, daily 4 hr separation from dam and from littermates). After weaning, mice were either housed socially (2-3/cage) or in isolation (1/cage) and then tested for prepulse inhibition in adulthood. Isolation-housed MPS subjects displayed greater deficits in prepulse inhibition relative to socially-housed MPS subjects while socially-housed AFR subjects displayed greater deficits in prepulse inhibition relative to isolation-housed AFR subjects. The results indicate that these treatment conditions represent a potentially valuable model for evaluating the match/mismatch hypothesis in regards to neuropsychiatric dysfunction.

Effect of Cage-Induced Stereotypies on Measures of Affective State and Recurrent Perseveration in CD-1 and C57BL/6 Mice.

Stereotypies are abnormal repetitive behaviour patterns that are highly prevalent in laboratory mice and are thought to reflect impaired welfare. Thus, they are associated with impaired behavioural inhibition and may also reflect negative affective states. However, in mice the relationship between stereotypies and behavioural inhibition is inconclusive, and reliable measures of affective valence are lacking. Here we used an exploration based task to assess cognitive bias as a measure of affective valence and a two-choice guessing task to assess recurrent perseveration as a measure of impaired behavioural inhibition to test mice with different forms and expression levels of stereotypic behaviour. We trained 44 CD-1 and 40 C57BL/6 female mice to discriminate between positively and negatively cued arms in a radial maze and tested their responses to previously inaccessible ambiguous arms. In CD-1 mice (i) mice with higher stereotypy levels displayed a negative cognitive bias and this was influenced by the form of stereotypy performed, (ii) negative cognitive bias was evident in back-flipping mice, and (iii) no such effect was found in mice displaying bar-mouthing or cage-top twirling. In C57BL/6 mice neither route-tracing nor bar-mouthing was associated with cognitive bias, indicating that in this strain these stereotypies may not reflect negative affective states. Conversely, while we found no relation of stereotypy to recurrent perseveration in CD-1 mice, C57BL/6 mice with higher levels of route-tracing, but not bar-mouthing, made more repetitive responses in the guessing task. Our findings confirm previous research indicating that the implications of stereotypies for animal welfare may strongly depend on the species and strain of animal as well as on the form and expression level of the stereotypy. Furthermore, they indicate that variation in stereotypic behaviour may represent an important source of variation in many animal experiments.

An Exploration Based Cognitive Bias Test for Mice: Effects of Handling Method and Stereotypic Behaviour.

Behavioural tests to assess affective states are widely used in human research and have recently been extended to animals. These tests assume that affective state influences cognitive processing, and that animals in a negative affective state interpret ambiguous information as expecting a negative outcome (displaying a negative cognitive bias). Most of these tests however, require long discrimination training. The aim of the study was to validate an exploration based cognitive bias test, using two different handling methods, as previous studies have shown that standard tail handling of mice increases physiological and behavioural measures of anxiety compared to cupped handling. Therefore, we hypothesised that tail handled mice would display a negative cognitive bias. We handled 28 female CD-1 mice for 16 weeks using either tail handling or cupped handling. The mice were then trained in an eight arm radial maze, where two adjacent arms predicted a positive outcome (darkness and food), while the two opposite arms predicted a negative outcome (no food, white noise and light). After six days of training, the mice were also given access to the four previously unavailable intermediate ambiguous arms of the radial maze and tested for cognitive bias. We were unable to validate this test, as mice from both handling groups displayed a similar pattern of exploration. Furthermore, we examined whether maze exploration is affected by the expression of stereotypic behaviour in the home cage. Mice with higher levels of stereotypic behaviour spent more time in positive arms and avoided ambiguous arms, displaying a negative cognitive bias. While this test needs further validation, our results indicate that it may allow the assessment of affective state in mice with minimal training-a major confound in current cognitive bias paradigms.

Refinement of experimental design and conduct in laboratory animal research.

The scientific literature of laboratory animal research is replete with papers reporting poor reproducibility of results as well as failure to translate results to clinical trials in humans. This may stem in part from poor experimental design and conduct of animal experiments. Despite widespread recognition of these problems and implementation of guidelines to attenuate them, a review of the literature suggests that experimental design and conduct of laboratory animal research are still in need of refinement. This paper will review and discuss possible sources of biases, highlight advantages and limitations of strategies proposed to alleviate them, and provide a conceptual framework for improving the reproducibility of laboratory animal research.

Experimenter effects on behavioral test scores of eight inbred mouse strains under the influence of ethanol.

Eight standard inbred mouse strains were evaluated for ethanol effects on a refined battery of behavioral tests in a study that was originally designed to assess the influence of rat odors in the colony on mouse behaviors. As part of the design of the study, two experimenters conducted the tests, and the study was carefully balanced so that equal numbers of mice in all groups and times of day were tested by each experimenter. A defect in airflow in the facility compromised the odor manipulation, and in fact the different odor exposure groups did not differ in their behaviors. The two experimenters, however, obtained markedly different results for three of the tests. Certain of the experimenter effects arose from the way they judged behaviors that were not automated and had to be rated by the experimenter, such as slips on the balance beam. Others were not evident prior to ethanol injection but had a major influence after the injection. For several measures, the experimenter effects were notably different for different inbred strains. Methods to evaluate and reduce the impact of experimenter effects in future research are discussed.

Brief and long periods of maternal separation affect maternal behavior and offspring behavioral development in C57BL/6 mice.

For rats, maternal mediation of brief and longer term dam-pup separations were thought to account for pup differences in adult "emotionality." In this study, early handling (EH), maternal separation (MS), and maternal peer separation (MPS) groups were compared to an animal facility reared (AFR) group for maternal behavior and offspring adult open-field behavior in C57BL/6 mice. Although MS and MPS dams displayed higher levels of maternal behavior upon reunion, these group differences did not predict offspring open-field behavior. However, when offspring behavior was analyzed as a function of specific aspects of maternal behavior, irrespective of treatment group, pups that received high levels of quiescent nursing and activity, but not licking, were less "emotional." Individual differences in maternal licking of pups predicted variability of "emotional" behavior for AFR and EH pups. Thus, for this strain of mouse, individual and not treatment differences in maternal care predict offspring "emotional" development.

The precision of video and photocell tracking systems and the elimination of tracking errors with infrared backlighting.

Automated tracking offers a number of advantages over both manual and photocell tracking methodologies, including increased reliability, validity, and flexibility of application. Despite the advantages that video offers, our experience has been that video systems cannot track a mouse consistently when its coat color is in low contrast with the background. Furthermore, the local lab lighting can influence how well results are quantified. To test the effect of lighting, we built devices that provide a known path length for any given trial duration, at a velocity close to the average speed of a mouse in the open-field and the circular water maze. We found that the validity of results from two commercial video tracking systems (ANY-maze and EthoVision XT) depends greatly on the level of contrast and the quality of the lighting. A photocell detection system was immune to lighting problems but yielded a path length that deviated from the true length. Excellent precision was achieved consistently, however, with video tracking using infrared backlighting in both the open field and water maze. A high correlation (r=0.98) between the two software systems was observed when infrared backlighting was used with live mice.