Generating NTRK3-FlpO Mice for Genetically Intersecting A-beta Nociceptors

PROJECT SUMMARY The dorsal root ganglion (DRG) contains diverse types of primary sensory afferents to mediate versatile somatosensation, including pain. Nociceptive DRG neurons (nociceptors) can be divided into different types, including C, Aδ, and Aβ nociceptors, based on their conduction velocities from slow to fast. Though multiple Cre lines targeting mouse C and Aδ nociceptors have been generated, no genetic line is currently available for specifically accessing and manipulating Aβ nociceptors, an important population of nociceptive afferents particularly in humans. Intersectional genetic mapping is a powerful approach for interrogating molecularly defined populations of neurons that can't be specifically manipulated using a single Cre line. Results from our lab suggest the existence of an evolutionarily conserved Aβ nociceptors expressing NTRK3 and neuromedin B (NMB) in both mice and humans. Interestingly, these neurons barely express PIEZO2, suggesting that they may use a different molecular mechanism for sensing mechanical pain. The Luo lab has possessed a Nmb-Cre mouse line, so we propose to generate and characterize a NTRK3-FlpO knockin mouse line in this R21 application. With this new mouse line, we can utilize an intersectional genetic strategy to specifically interrogate mouse Aβ nociceptors for the first time. In Aim 1, we will generate new Ntrk3-FlpO founder mouse lines using the Penn CRISPR/Cas9 Mouse Targeting Core Facility and characterize their recombination pattern by crossing to Flp dependent reporter mouse lines. In aim 2, we will breed the Ntrk3-FlpO line with correct combination patterns to Nmb-Cre and examine the intersectional recombination using Cre and Flp double dependent reporter lines. We will quantify the number/percentage of the double positive DRG neurons, characterize co-expression of known molecular markers of different types of mouse DRG neurons, examine their central and peripheral projections, and determine their physiological properties. Taken together, we anticipate generating a new mouse line, which would allow the field to genetically label and manipulate mouse Aβ nociceptors. This would pave a new avenue to discover molecular mechanisms underlying mechanical nociception and determine functions of Aβ nociceptors in chronic pain. This new Ntrk3-FlpO mouse line will also be an invaluable tool for other investigators to study Aβ low-threshold mechanoreceptors, proprioceptors, or any other cell types expressing Ntrk3.