Development and Validation of Animal Models and/or Outcome Measures

ABSTRACT Research Component (RC) 2 includes the development and validation of novel animal models that model a specific pain type, and/or the development and validation of novel outcome measures. Dr. Scherrer has participated in a consensus meeting of the Preclinical Pain Research Consortium for Investigating Safety and

Efficacy (PPRECISE) Working Group, sponsored by the Analgesic, Anesthetic, and Addiction Clinical Trial Translations, Innovations, Opportunities, and Networks (ACTTION), a public-private partnership with the U.S. Food and Drug Administration. This working group was tasked with making recommendations to improve

experimental design and reporting transparency and minimize conscious and unconscious experimental bias to increase scientific rigor, reproducibility, and translatability in the pain field. Based on the conclusions of this working group, we propose to develop and validate three novel outcome measures that bring innovative, state-

of-the-art machine learning, optical recording of neural activity in behaving mice, and analytic methods from the systems neuroscience field into the field of pain drug discovery. These novel outcome measures will be used in RC5 in vivo efficacy studies to elevate the scientific rigor and translatability of our antinociceptive NTSR1 asset

discoveries. In Aim 1, we implement machine learning–based outcome measures to standardize and automate in vivo efficacy studies of NTSR1 assets. Manual scoring of rodent nocifensive behaviors by humans is inherently subjective and varies considerably between experimenters, weakening data robustness and leading to

reproducibility issues. Another limitation of manual scoring is the low throughput of the method. We will develop and validate the use of DeepEthogram, a machine learning pipeline for supervised behavior classification from raw pixels, to reproducibly and automatically score reflexive and affective-motivational pain behaviors to evaluate

the antinociceptive efficacy of drugs. In Aim 2, we develop Ca2+ imaging methods to simultaneously test the effect of NTSR1 assets on pain behaviors and on amygdalar activity. Because a number of factors can contribute to reducing nocifensive motor responses following administration of an asset, relying exclusively on behavioral

outcome measures in rodent efficacy studies may not suffice to predict translatability. Here, we will develop and validate methods to use the miniature microscope (miniscope) technology and express the Ca2+ indicator GCaMP8f in NTSR1-expressing amygdalar neurons to simultaneously record both the activity of these neurons

and pain behaviors in response to antinociceptive drugs. In Aim 3, to further test the effect of NTSR1 assets against pain experience, we will develop and validate the use of the “Crystal Skull” technology to image the effect of antinociceptive drugs on pain representation in neocortex, including areas implicated in the sensory-

discriminative and affective-motivational aspects of pain, such as the primary somatosensory cortex and cingulate cortex.