Combining supervised and unsupervised analyses to quantify behavioral phenotypes and validate therapeutic efficacy in a triple transgenic mouse model of Alzheimer's disease.

Behavioral testing is an essential tool for evaluating cognitive function and dysfunction in preclinical research models. This is of special importance in the study of neurological disorders such as Alzheimer's disease. However, the reproducibility of classic behavioral assays is frequently compromised by interstudy variation, leading to ambiguous conclusions about the behavioral markers characterizing the disease. Here, we identify age- and genotype-driven differences between 3xTg-AD and non-transgenic control mice using a low-cost, highly customizable behavioral assay that requires little human intervention. Through behavioral phenotyping combining both supervised and unsupervised behavioral classification methods, we are able to validate the preventative effects of the immunosuppressant cyclosporine A in a rodent model of Alzheimer's disease, as well as the partially ameliorating effects of candidate drugs nebivolol and cabozantinib.

Behavioral effects of visual stimuli in adult zebrafish using a novel eight-tank imaging system.

Introduction: Animals respond to various environmental cues. Animal behavior is complex, and behavior analysis can greatly help to understand brain function. Most of the available behavioral imaging setups are expensive, provide limited options for customization, and allow for behavioral imaging of one animal at a time. Methods: The current study takes advantage of adult zebrafish as a model organism to study behavior in a novel behavioral setup allowing one to concurrently image 8 adult zebrafish. Results: Our results indicate that adult zebrafish show a unique behavioral profile in response to visual stimuli such as moving lines. In the presence of moving lines, the fish spent more time exploring the tank and spent more time toward the edges of the tanks. In addition, the fish moved and oriented themselves against the direction of the moving lines, indicating a negative optomotor response (OMR). With repeated exposure to moving lines, we observed a reduced optomotor response in adult zebrafish. Discussion: Our behavioral setup is relatively inexpensive, provides flexibility in the presentation of various animated visual stimuli, and offers improved throughput for analyzing behavior in adult zebrafish. This behavioral setup shows promising potential to quantify various behavioral measures and opens new avenues to understand complex behaviors.

Increasing transposase abundance with ocean depth correlates with a particle-associated lifestyle.

Transposases are mobile genetic elements that move within and between genomes, promoting genomic plasticity in microorganisms. In marine microbial communities, the abundance of transposases increases with depth, but the reasons behind this trend remain unclear. Our analysis of metagenomes from the Tara Oceans and Malaspina Expeditions suggests that a particle-associated lifestyle is the main covariate for the high occurrence of transposases in the deep ocean, and this trend holds true for individual genomes as well as in a community-wide sense. We observed a strong and depth-independent correlation between transposase abundance and the presence of biofilm-associated genes, as well as the prevalence of secretory enzymes. This suggests that mobile genetic elements readily propagate among microbial communities within crowded biofilms. Furthermore, we show that particle association positively correlates with larger genome size, which is in turn associated with higher transposase abundance. Cassette sequences associated with transposons are enriched with genes related to defense mechanisms, which are more highly expressed in the deep sea. Thus, while transposons spread at the expense of their microbial hosts, they also introduce novel genes and potentially benefit the hosts in helping to compete for limited resources. Overall, our results suggest a new understanding of deep ocean particles as highways for gene sharing among defensively oriented microbial genomes.IMPORTANCEGenes can move within and between microbial genomes via mobile genetic elements, which include transposases and transposons. In the oceans, there is a puzzling increase in transposase abundance in microbial genomes as depth increases. To gain insight into this trend, we conducted an extensive analysis of marine microbial metagenomes and metatranscriptomes. We found a significant correlation between transposase abundance and a particle-associated lifestyle among marine microbes at both the metagenome and genome-resolved levels. We also observed a link between transposase abundance and genes related to defense mechanisms. These results suggest that as microbes become densely packed into crowded particles, mobile genes are more likely to spread and carry genetic material that provides a competitive advantage in crowded habitats. This may enable deep sea microbes to effectively compete in such environments.

Drug repurposing for neurodegenerative diseases using Zebrafish behavioral profiles.

Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.

An 8-cage imaging system for automated analyses of mouse behavior.

The analysis of mouse behavior is used in biomedical research to study brain function in health and disease. Well-established rapid assays allow for high-throughput analyses of behavior but have several drawbacks, including measurements of daytime behaviors in nocturnal animals, effects of animal handling, and the lack of an acclimation period in the testing apparatus. We developed a novel 8-cage imaging system, with animated visual stimuli, for automated analyses of mouse behavior in 22-h overnight recordings. Software for image analysis was developed in two open-source programs, ImageJ and DeepLabCut. The imaging system was tested using 4-5 month-old female wild-type mice and 3xTg-AD mice, a widely-used model to study Alzheimer's disease (AD). The overnight recordings provided measurements of multiple behaviors including acclimation to the novel cage environment, day and nighttime activity, stretch-attend postures, location in various cage areas, and habituation to animated visual stimuli. The behavioral profiles were different in wild-type and 3xTg-AD mice. AD-model mice displayed reduced acclimation to the novel cage environment, were hyperactive during the first hour of darkness, and spent less time at home in comparison to wild-type mice. We propose that the imaging system may be used to study various neurological and neurodegenerative disorders, including Alzheimer's disease.

Zebrafish Larvae Position Tracker (Z-LaP Tracker): a high-throughput deep-learning behavioral approach for the identification of calcineurin pathway-modulating drugs using zebrafish larvae.

Brain function studies greatly depend on quantification and analysis of behavior. While behavior can be imaged efficiently, the quantification of specific aspects of behavior is labor-intensive and may introduce individual biases. Recent advances in deep learning and artificial intelligence-based tools have made it possible to precisely track individual features of freely moving animals in diverse environments without any markers. In the current study, we developed Zebrafish Larvae Position Tracker (Z-LaP Tracker), a modification of the markerless position estimation software DeepLabCut, to quantify zebrafish larval behavior in a high-throughput 384-well setting. We utilized the high-contrast features of our model animal, zebrafish larvae, including the eyes and the yolk for our behavioral analysis. Using this experimental setup, we quantified relevant behaviors with similar accuracy to the analysis performed by humans. The changes in behavior were organized in behavioral profiles, which were examined by K-means and hierarchical cluster analysis. Calcineurin inhibitors exhibited a distinct behavioral profile characterized by increased activity, acoustic hyperexcitability, reduced visually guided behaviors, and reduced habituation to acoustic stimuli. The developed methodologies were used to identify 'CsA-type' drugs that might be promising candidates for the prevention and treatment of neurological disorders.

Novel use of FDA-approved drugs identified by cluster analysis of behavioral profiles.

Repurposing FDA-approved drugs is an efficient and cost-effective approach in the development of therapeutics for a broad range of diseases. However, prediction of function can be challenging, especially in the brain. We screened a small-molecule library with FDA-approved drugs for effects on behavior. The studies were carried out using zebrafish larvae, imaged in a 384-well format. We found that various drugs affect activity, habituation, startle responses, excitability, and optomotor responses. The changes in behavior were organized in behavioral profiles, which were examined by hierarchical cluster analysis. One of the identified clusters includes the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506), which are immunosuppressants and potential therapeutics in the prevention of Alzheimer's disease. The calcineurin inhibitors form a functional cluster with seemingly unrelated drugs, including bromocriptine, tetrabenazine, rosiglitazone, nebivolol, sorafenib, cabozantinib, tamoxifen, meclizine, and salmeterol. We propose that drugs with 'CsA-type' behavioral profiles are promising candidates for the prevention and treatment of Alzheimer's disease.