The use of ball pits and playpens in laboratory Lister Hooded male rats induces ultrasonic vocalisations indicating a more positive affective state and can reduce the welfare impacts of aversive procedures

The advancement and quality of science rely on research that is robust and unbiased in its experimental design, execution, analysis, and reproducibility. In preclinical research, a better understanding of animal emotions and refinement of their husbandry, housing, and handling are important goals in providing good animal welfare in a laboratory setting which underpins rigorous research quality. Induction of positive emotional state in animals is a key component of their well-being, and one approach is to increase their environmental complexity using, for example, ball pits or playpens in rats. In this study, we recorded 50 kHz ultrasonic vocalisations (USVs) during animals’ exposure to the ball pit and playpen. We have previously shown that 50 kHz USVs provide a graded and quantifiable measure of an animal’s emotional state, and here find that access to the ball pit and playpen increases 50 kHz USVs, indicative of a more positive affective state. Using our affective bias test (ABT) we next quantified the animals’ emotional response to an aversive intervention and whether this could be attenuated by access to a playpen. The playpen exposure completely mitigated the negative affective state induced by an anxiogenic drug when compared with animals who experienced the drug in the home cage. Together, these findings suggest ball pits and playpens provide a simple and effective method to improve the welfare of laboratory rats and reduce the cumulative suffering they experience from their housing conditions and minor, aversive procedures

Fecal glucocorticoid metabolites as biomarkers in equids : assay choice matters

Free ranging animals are exposed to environmental, demographic, and ecological challenges over time, which can affect their health and fitness. Non‐invasive biomarkers can provide insight into how animals cope with these challenges and assess the effectiveness of conservation management strategies. We evaluated how free ranging ponies (Equus ferus caballus) on the Carneddau Mountain range, North Wales respond to 2 stimuli: an acute stressor of an annual roundup event in November 2014, and spatial and temporal variation in ecological factors in 2018. We evaluated fecal glucocorticoid metabolites using 2 enzyme immunoassays (EIAs): an 11‐oxoetiocholanolone EIA (measuring 11,17‐dioxoandrostanes [11,17‐DOAs]) and a corticosterone EIA. The former assay has been validated in equids, whereas there is limited evidence for the suitability of the latter. We used an additional parent testosterone EIA to measure fecal androgen metabolites in response to the ecological challenges. Following the roundup, the metabolite concentrations measured by the 2 glucocorticoid EIAs were not correlated. The 11,17‐DOAs were elevated from the second day following the roundup and then slowly returned to pre‐round levels over the next 2 weeks. In contrast, the metabolites measured by the corticosterone assay showed no response to the roundup. For the ecological data, all 3 assays detected a positive correlation between metabolites and social group size in males but not in females. The metabolite concentrations measured by the testosterone and corticosterone assays were highly correlated and were temporally associated with the onset of the breeding season, whereas the 11,17‐DOAs were not. The co‐variance of metabolites measured by the corticosterone and testosterone assays, and the lack of an acute response in the corticosterone assay to the roundup, suggests that metabolites detected by the corticosterone assay were not primarily associated with increased glucocorticoid production. We recommend using well‐validated fecal biomarker assays of hypothalamus‐pituitary‐adrenal axis activity to evaluate and compare the effect of different management interventions and environmental change. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society

Rapid-acting antidepressant drugs modulate affective bias in rats

How rapid-acting antidepressants (RAADs), such as ketamine, induce immediate and sustained improvements in mood in patients with major depressive disorder (MDD) is poorly understood. A core feature of MDD is the prevalence of cognitive processing biases associated with negative affective states, and the alleviation of negative affective biases may be an index of response to drug treatment. Here, we used an affective bias behavioral test in rats, based on an associative learning task, to investigate the effects of RAADs. To generate an affective bias, animals learned to associate two different digging substrates with a food reward in the presence or absence of an affective state manipulation. A choice between the two reward-associated digging substrates was used to quantify the affective bias generated. Acute treatment with the RAADs ketamine, scopolamine, or psilocybin selectively attenuated a negative affective bias in the affective bias test. Low, but not high, doses of ketamine and psilocybin reversed the valence of the negative affective bias 24 hours after RAAD treatment. Only treatment with psilocybin, but not ketamine or scopolamine, led to a positive affective bias that was dependent on new learning and memory formation. The relearning effects of ketamine were dependent on protein synthesis localized to the rat medial prefrontal cortex and could be modulated by cue reactivation, consistent with experience-dependent neural plasticity. These findings suggest a neuropsychological mechanism that may explain both the acute and sustained effects of RAADs, potentially linking their effects on neural plasticity with affective bias modulation in a rodent model

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis

Fecal Glucocorticoid Metabolites as Biomarkers in Equids: Assay Choice Matters

Free ranging animals are exposed to environmental, demographic, and ecological challenges over time, which can affect their health and fitness. Non-invasive biomarkers can provide insight into how animals cope with these challenges and assess the effectiveness of conservation management strategies. We evaluated how free ranging ponies (Equus ferus caballus) on the Carneddau Mountain range, North Wales respond to 2 stimuli: an acute stressor of an annual roundup event in November 2014, and spatial and temporal variation in ecological factors in 2018. We evaluated fecal glucocorticoid metabolites using 2 enzyme immunoassays (EIAs): an 11-oxoetiocholanolone EIA (measuring 11,17-dioxoandrostanes [11,17-DOAs]) and a corticosterone EIA. The former assay has been validated in equids, whereas there is limited evidence for the suitability of the latter. We used an additional parent testosterone EIA to measure fecal androgen metabolites in response to the ecological challenges. Following the roundup, the metabolite concentrations measured by the 2 glucocorticoid EIAs were not correlated. The 11,17-DOAs were elevated from the second day following the roundup and then slowly returned to pre-round levels over the next 2 weeks. In contrast, the metabolites measured by the corticosterone assay showed no response to the roundup. For the ecological data, all 3 assays detected a positive correlation between metabolites and social group size in males but not in females. The metabolite concentrations measured by the testosterone and corticosterone assays were highly correlated and were temporally associated with the onset of the breeding season, whereas the 11,17-DOAs were not. The co-variance of metabolites measured by the corticosterone and testosterone assays, and the lack of an acute response in the corticosterone assay to the roundup, suggests that metabolites detected by the corticosterone assay were not primarily associated with increased glucocorticoid production. We recommend using well-validated fecal biomarker assays of hypothalamus-pituitary-adrenal axis activity to evaluate and compare the effect of different management interventions and environmental change. © 2021 The Authors. The Journal of Wildlife Management published by Wiley Periodicals LLC on behalf of The Wildlife Society

Evaluation of Dynamic Cell Processes and Behavior Using Video Bioinformatics Tools

Just as body language can reveal a person’s state of well-being, dynamic changes in cell behavior and morphology can be used to monitor processes in cultured cells. This chapter discusses how CL-Quant software, a commercially available video bioinformatics tool, can be used to extract quantitative data on: (1) growth/proliferation, (2) cell and colony migration, (3) reactive oxygen species (ROS) production, and (4) neural differentiation. Protocols created using CL-Quant were used to analyze both single cells and colonies. Time-lapse experiments in which different cell types were subjected to various chemical exposures were done using Nikon BioStations. Proliferation rate was measured in human embryonic stem cell colonies by quantifying colony area (pixels) and in single cells by measuring confluency (pixels). Colony and single cell migration were studied by measuring total displacement (distance between the starting and ending points) and total distance traveled by the colonies/cells. To quantify ROS production, cells were pre-loaded with MitoSOX Red™, a mitochondrial ROS (superoxide) indicator, treated with various chemicals, then total intensity of the red fluorescence was measured in each frame. Lastly, neural stem cells were incubated in differentiation medium for 12 days, and time lapse images were collected daily. Differentiation of neural stem cells was quantified using a protocol that detects young neurons. CLQuant software can be used to evaluate biological processes in living cells, and the protocols developed in this project can be applied to basic research and toxicological studies, or to monitor quality control in culture facilities