Toward a Smart Sensing System to Monitor Small Animal's Physical State via Multi-Frequency Resonator Array.

This article presents a highly scalable and rack-mountable wireless sensing system for long-term monitoring (i.e., sense and estimate) of small animal/s' physical state (SAPS), such as changes in location and posture within standard cages. The conventional tracking systems may lack one or more features such as scalability, cost efficiency, rack-mount ability, and light condition insensitivity to work 24/7 on a large scale. The proposed sensing mechanism relies on relative changes of multiple resonance frequencies due to the animal's presence over the sensor unit. The sensor unit can track SAPS changes based on changes in electrical properties in the sensors near fields, appearing in the resonance frequencies, i.e., an Electromagnetic (EM) Signature, within the 200 MHz-300 MHz frequency range. The sensing unit is located underneath a standard mouse cage and consists of thin layers of a reading coil and six resonators tuned at six distinct frequencies. ANSYS HFSS software is used to model and optimize the proposed sensor unit and calculate the Specific Absorption Rate (SAR) obtained under 0.05 W/kg. Multiple prototypes have been implemented to test, validate, and characterize the performance of the design by conducting in vitro and in vivo experiments on Mice. The in-vitro test results have shown a 15 mm spatial resolution in detecting the mouse's location over the sensor array having maximum frequency shifts of 832 kHz and posture detection with under 30° resolution. The in-vivo experiment on mouse displacement resulted in frequency shifts of up to 790 kHz, indicating the SAPS's capability to detect the Mice's physical state.

Zebrafish automatic monitoring system for conditioning and behavioral analysis.

Studies using zebrafish (Danio rerio) in neuro-behavioural research are growing. Measuring fish behavior by computational methods is one of the most efficient ways to avoid human bias in experimental analyses, extending them to various approaches. Sometimes, thorough analyses are difficult to do, as fish can behave unpredictably during an experimental strategy. However, the analyses can be implemented in an automated way, using an online strategy and video processing for a complete assessment of the zebrafish behavior, based on the detection and tracking of fish during an activity. Here, a fully automatic conditioning and detailed analysis of zebrafish behavior is presented. Microcontrolled components were used to control the delivery of visual and sound stimuli, in addition to the concise amounts of food after conditioned stimuli for adult zebrafish groups in a conventional tank. The images were captured and processed for automatic detection of the fish, and the training of the fish was done in two evaluation strategies: simple and complex. In simple conditioning, the zebrafish showed significant responses from the second attempt, learning that the conditioned stimulus was a predictor of food presentation in a specific space of the tank, where the food was dumped. When the fish were subjected to two stimuli for decision-making in the food reward, the zebrafish obtained better responses to red light stimuli in relation to vibration. The behavior change was clear in stimulated fish in relation to the control group, thus, the distances traveled and the speed were greater, while the polarization was lower in stimulated fish. This automated system allows for the conditioning and assessment of zebrafish behavior online, with greater stability in experiments, and in the analysis of the behavior of individual fish or fish schools, including learning and memory studies.

The ZE-Tunnel: An Affordable, Easy-to-Assemble, and User-Friendly Benchtop Zebrafish Swim Tunnel.

The popularity of zebrafish in both basic biological and biomedical research has led to an increased need for understanding their behavior. Locomotor behavior is an important outcome of different factors, such as specific genotypes or external stimuli that influence the nervous and musculoskeletal system. Locomotion can be studied by forced swimming in a swim tunnel, a device capable of generating a laminar water flow at different speeds in a chamber where zebrafish can be placed. However, commercially available swim tunnels are relatively expensive and in-house built systems are mostly presented without clear building instructions or proper validation procedures. In this study, we developed an alternative, cheap (<250 euro), and user-friendly, but customizable benchtop swim tunnel, called the "Zebrafish exercise-tunnel" (ZE-Tunnel). Detailed step-by-step instructions on how to construct the tunnel components, including the frame, mechanical, and electric components are given. The ZE-Tunnel was reliably used to exercise fish for prolonged periods and its performance was successfully validated by replicating previously published experiments on critical speed testing in zebrafish. Finally, implementation of behavioral video analysis using freely available motion-tracking software showed differences in swimming dynamics in the Chihuahua skeletal zebrafish mutant.

Computational fluid dynamics applied to the ventilation of small-animal laboratory cages.

Several studies based on in vivo or in vitro models have found promising results for the noble gas argon in neuroprotection against ischaemic pathologies. The development of argon as a medicinal product includes the requirement for toxicity testing through non-clinical studies. The long exposure period of animals (rats) during several days results in technical and logistic challenges related to the gas administration. In particular, a minimum of 10 air changes per hour (ACH) to maintain animal welfare results in extremely large volumes of experimental gas required if the gas is not recirculated. The difficulty with handling the many cylinders prompted the development of such a recirculation-based design. To distribute the recirculating gas to individually ventilated cages and monitor them properly was deemed more difficult than constructing a single large enclosure that will hold several open cages. To address these concerns, a computational fluid dynamics (CFD) analysis of the preliminary design was performed. A purpose-made exposure chamber was designed based on the CFD simulations. Comparisons of the simulation results to measurements of gas concentration at two cage positions while filling show that the CFD results compare well to these limited experiments. Thus, we believe that the CFD results are representative of the gas distribution throughout the enclosure. The CFD shows that the design provides better gas distribution (i.e. a higher effective air change rate) than predicted by 10 ACH.

MouseBytes, an open-access high-throughput pipeline and database for rodent touchscreen-based cognitive assessment.

Open Science has changed research by making data accessible and shareable, contributing to replicability to accelerate and disseminate knowledge. However, for rodent cognitive studies the availability of tools to share and disseminate data is scarce. Automated touchscreen-based tests enable systematic cognitive assessment with easily standardised outputs that can facilitate data dissemination. Here we present an integration of touchscreen cognitive testing with an open-access database public repository (mousebytes.ca), as well as a Web platform for knowledge dissemination (https://touchscreencognition.org). We complement these resources with the largest dataset of age-dependent high-level cognitive assessment of mouse models of Alzheimer's disease, expanding knowledge of affected cognitive domains from male and female mice of three strains. We envision that these new platforms will enhance sharing of protocols, data availability and transparency, allowing meta-analysis and reuse of mouse cognitive data to increase the replicability/reproducibility of datasets.

Digging deeper: methodologies for high-content phenotyping in Caenorhabditis elegans.

Deep phenotyping is an emerging conceptual paradigm and experimental approach aimed at measuring and linking many aspects of a phenotype to understand its underlying biology. To date, deep phenotyping has been applied mostly in cultured cells and used less in multicellular organisms. However, in the past decade, it has increasingly been recognized that deep phenotyping could lead to a better understanding of how genetics, environment and stochasticity affect the development, physiology and behavior of an organism. The nematode Caenorhabditis elegans is an invaluable model system for studying how genes affect a phenotypic trait, and new technologies have taken advantage of the worm's physical attributes to increase the throughput and informational content of experiments. Coupling of these technical advancements with computational and analytical tools has enabled a boom in deep-phenotyping studies of C. elegans. In this Review, we highlight how these new technologies and tools are digging into the biological origins of complex, multidimensional phenotypes.

Continuous measurement of locomotor activity during convalescence and acclimation in group-housed rats.

Locomotor activity is affected by a range of factors in addition to experimental treatment, including the breeding environment. Appropriate convalescence and acclimation are important for animal experiments, because environmental changes and physical burden can result from surgery, transportation, and cage exchange. However, the duration that locomotor activity is affected by these factors is currently unclear, because it has traditionally been difficult to measure locomotor activity in multiple group-housed animals in any location other than the analysis room. In the present study, we analyzed the locomotor activity of group-housed rats using a nano tag® after surgery, transportation, and cage exchange. The nano tag®, a new device for analyzing activity, can measure locomotor activity in laboratory animals with no limitation on the number of animals in same cage. Any type of cage can be used for analysis, at any time of day, and in any location. Nano tags® were subcutaneously implanted in male rats (F344/NSlc, 6 weeks of age) and locomotor activity was continuously measured after surgery, transportation, and cage exchange. Significant activity changes were observed in rats after transportation and cage exchange, 9 days and 3 h after the event, respectively. The results suggest that continuous measurement of locomotor activity with nano tags® can be used to monitor changes in activity induced by environmental changes, and will be helpful for designing animal experiments analyzing locomotor activity.

A Review of Freely Available, Open-Source Software for the Automated Analysis of the Behavior of Adult Zebrafish.

The analysis of behavior in animal models is an important objective in many research fields, including neuroscience, psychology, toxicology, and neuropsychopharmacology. Animal models have been used for many years, and several behavioral paradigms, such as locomotor activity, social interactions, and cognitive behavior, have been studied in animal models to correlate the behaviors with pharmacological or environmental interventions and with molecular, biochemical, and physiological findings. We reviewed the literature looking for open-source, freely available software to analyze animal behavior and found 12 freely available programs: ToxTrack, EthoWatcher, Mouse Behavior Tracker, Mouse Move, JAABA, wrMTrck, AnimalTracker, idTracker, Ctrax, Mousetracker, VideoHacking, and Cowlog, which were developed with different programs, work on different platforms, and have particular types of inputs and outputs and analysis capabilities. We reviewed some examples of their use, tested some of them, and provided several recommendations for the future development of programs for the automated analysis of behavior in animal models. In conclusion, we show freely available software for the automated analysis of behavior in animal models such as adult zebrafish and provide information for researchers and students looking for quick, easy-to-implement, and inexpensive behavior analysis alternatives.

Quantified water intake in laboratory cats from still, free-falling and circulating water bowls, and its effects on selected urinary parameters.

OBJECTIVES: The study objectives were to determine if the method of water presentation (still [S], circulating [C] or free-falling [FF] bowl systems) influences daily water consumption in cats in a controlled environment, and whether differences in water intake affect urine relative super saturation (RSS) for calcium oxalate and struvite, urine specific gravity (USG), urine osmolality (Uosmol) and urine volume. METHODS: Sixteen healthy laboratory cats fed a dry diet were individually housed with urine collection systems. Each cat underwent a randomized 2 week crossover period with all bowl systems, allowing a 1 week acclimation period between each crossover. Water intake was measured daily by bowl weight, accounting for spillage and evaporation. USG and urine volume were measured daily, whereas other urinary parameters were measured at various time points throughout each 14 day crossover period. RESULTS: Fourteen cats completed the study. Average daily water intake (ml/kg/day), urine volume, USG and urine RSS for struvite and calcium oxalate were not significantly different between water bowls. Uosmol was significantly higher in C compared with S and FF bowl systems (P = 0.009 for both). Three individual cats demonstrated a significant water bowl preference (Cat 4: C >S, P = 0.039; Cat 10: FF >C, P = 0.005; Cat 11: S >C, P = 0.037). CONCLUSIONS AND RELEVANCE: Overall, water bowl type had no appreciable effect on water intake. Uosmol was the only urinary parameter found to be significantly different, and was higher for the C bowl. The implication of this is unknown, considering water intake did not differ significantly between bowls. Alternative methods to increase water intake should be implemented beyond providing unique water bowls in patients where augmented water intake would be beneficial for disease management.

Designing and Testing of Self-Cleaning Recirculating Zebrafish Tanks.

Maintenance of large number of zebrafish in captive conditions is a daunting task. This can be eased by the use of recirculating racks with self-cleaning zebrafish tanks. Commercially available systems are costly, and compatibility of intercompany products has never been investigated. Although various cost-effective designs and methods of construction of custom-made recirculating zebrafish racks are available in literature, the design of self-cleaning zebrafish tanks is still not available. In this study, we report the design and method of construction of the self-cleaning unit, which can be fitted in any zebrafish tank. We validated the design by investigating sediment cleaning process in rectangular and cylindrical tank geometries using time lapse imaging. Our results suggest that for both tank geometries, the tanks fitted with self-cleaning unit provided superior sediment cleaning than the tanks fitted with overflow-drain unit. Although the self-cleaning unit could clean the sediment completely from both geometries over prolonged period, the cleaning of sediments was faster in the cylindrical tank than the rectangular tank. In conclusion, cost and efforts of zebrafish maintenance could be significantly reduced through the installation of our self-cleaning unit in any custom-made zebrafish tank.

The touchscreen operant platform for testing learning and memory in rats and mice.

An increasingly popular method of assessing cognitive functions in rodents is the automated touchscreen platform, on which a number of different cognitive tests can be run in a manner very similar to touchscreen methods currently used to test human subjects. This methodology is low stress (using appetitive rather than aversive reinforcement), has high translational potential and lends itself to a high degree of standardization and throughput. Applications include the study of cognition in rodent models of psychiatric and neurodegenerative diseases (e.g., Alzheimer's disease, schizophrenia, Huntington's disease, frontotemporal dementia), as well as the characterization of the role of select brain regions, neurotransmitter systems and genes in rodents. This protocol describes how to perform four touchscreen assays of learning and memory: visual discrimination, object-location paired-associates learning, visuomotor conditional learning and autoshaping. It is accompanied by two further protocols (also published in this issue) that use the touchscreen platform to assess executive function, working memory and pattern separation.

The touchscreen operant platform for testing working memory and pattern separation in rats and mice.

The automated touchscreen operant chamber for rats and mice allows for the assessment of multiple cognitive domains within the same testing environment. This protocol presents the location discrimination (LD) task and the trial-unique delayed nonmatching-to-location (TUNL) task, which both assess memory for location. During these tasks, animals are trained to a predefined criterion during ∼20-40 daily sessions. In LD sessions, touching the same location on the screen is rewarded on consecutive trials, followed by a reversal of location-reward contingencies. TUNL, a working memory task, requires animals to 'nonmatch' to a sample location after a delay. In both the LD and TUNL tasks, spatial similarity can be varied, allowing assessment of pattern separation ability, a function that is thought to be performed by the dentate gyrus (DG). These tasks are therefore particularly useful in animal models of hippocampal, and specifically DG, function, but they additionally permit discernment of changes in pattern separation from those in working memory.

The touchscreen operant platform for assessing executive function in rats and mice.

This protocol details a subset of assays developed within the touchscreen platform to measure various aspects of executive function in rodents. Three main procedures are included: extinction, measuring the rate and extent of curtailing a response that was previously, but is no longer, associated with reward; reversal learning, measuring the rate and extent of switching a response toward a visual stimulus that was previously not, but has become, associated with reward (and away from a visual stimulus that was previously, but is no longer, rewarded); and the 5-choice serial reaction time (5-CSRT) task, gauging the ability to selectively detect and appropriately respond to briefly presented, spatially unpredictable visual stimuli. These protocols were designed to assess both complementary and overlapping constructs including selective and divided visual attention, inhibitory control, flexibility, impulsivity and compulsivity. The procedures comprise part of a wider touchscreen test battery assessing cognition in rodents with high potential for translation to human studies.

A novel approach to scavenging anesthetic gases in rodent surgery.

Laboratory animal procedures using gas anesthetics may amass elevated waste gas concentrations in operating rooms if controls are not implemented for capturing and removing the vapors. Area sampling using an infrared analyzer indicated isoflurane concentrations likely to exceed occupational exposure guidelines. Our study showed environmental concentrations of oxygen as high as 40% and isoflurane concentrations >100 ppm when no controls or merely passive controls were utilized. These extraneous isoflurane emissions were determined to be originating from the pre-procedural induction process as well as the gas delivery nose cone. A novel waste gas collection cylinder was designed to enclose the gas delivery nose cone and animal head during the administration of anesthetic gases. The vented cylinder utilized a house vacuum to remove the waste anesthetic gases from the surgical field. A commercially available induction chamber designed to be actively and externally exhausted was used to lower concentrations during the induction process. With implementation of local exhaust ventilation controls, waste anesthetic gas concentrations decreased to below recommended occupational exposure levels. In vitro (sham) testing compared favorably to in vivo measurements validating the reduction capability of active ventilation during rodent anesthetic administration. In vivo isoflurane reductions for the induction chamber emissions, the operating room, and the surgeon's breathing zone were 95%, 60%, and 53%, respectively. The same measurements for an in vitro procedure were 98%, 84%, and 87%, respectively.