Comprehensive ethological analysis of fear expression in rats using DeepLabCut and SimBA machine learning model.

Introduction: Defensive responses to threat-associated cues are commonly evaluated using conditioned freezing or suppression of operant responding. However, rats display a broad range of behaviors and shift their defensive behaviors based on immediacy of threats and context. This study aimed to systematically quantify the defensive behaviors that are triggered in response to threat-associated cues and assess whether they can accurately be identified using DeepLabCut in conjunction with SimBA. Methods: We evaluated behavioral responses to fear using the auditory fear conditioning paradigm. Observable behaviors triggered by threat-associated cues were manually scored using Ethovision XT. Subsequently, we investigated the effects of diazepam (0, 0.3, or 1 mg/kg), administered intraperitoneally before fear memory testing, to assess its anxiolytic impact on these behaviors. We then developed a DeepLabCut + SimBA workflow for ethological analysis employing a series of machine learning models. The accuracy of behavior classifications generated by this pipeline was evaluated by comparing its output scores to the manually annotated scores. Results: Our findings show that, besides conditioned suppression and freezing, rats exhibit heightened risk assessment behaviors, including sniffing, rearing, free-air whisking, and head scanning. We observed that diazepam dose-dependently mitigates these risk-assessment behaviors in both sexes, suggesting a good predictive validity of our readouts. With adequate amount of training data (approximately > 30,000 frames containing such behavior), DeepLabCut + SimBA workflow yields high accuracy with a reasonable transferability to classify well-represented behaviors in a different experimental condition. We also found that maintaining the same condition between training and evaluation data sets is recommended while developing DeepLabCut + SimBA workflow to achieve the highest accuracy. Discussion: Our findings suggest that an ethological analysis can be used to assess fear learning. With the application of DeepLabCut and SimBA, this approach provides an alternative method to decode ongoing defensive behaviors in both male and female rats for further investigation of fear-related neurobiological underpinnings.

Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells.

Single-strand breaks (SSBs) represent the major form of DNA damage, yet techniques to map these lesions genome-wide with nucleotide-level precision are limited. Here, we present a method, termed SSiNGLe, and demonstrate its utility to explore the distribution and dynamic changes in genome-wide SSBs in response to different biological and environmental stimuli. We validate SSiNGLe using two very distinct sequencing techniques and apply it to derive global profiles of SSBs in different biological states. Strikingly, we show that patterns of SSBs in the genome are non-random, specific to different biological states, enriched in regulatory elements, exons, introns, specific types of repeats and exhibit differential preference for the template strand between exons and introns. Furthermore, we show that breaks likely contribute to naturally occurring sequence variants. Finally, we demonstrate strong links between SSB patterns and age. Overall, SSiNGLe provides access to unexplored realms of cellular biology, not obtainable with current approaches.