The Vglut3Icre;Rosa26Ai14 Mouse Model as a Tool for Studying TRPM8+ Sensory Neurons

Abstract

Despite their importance to survival, cold-sensing TRPM8+ neurons represent an understudied sensory neuron population, comprising only 8-10% of all trigeminal (TG) and dorsal root ganglion (DRG) neurons. However, available transgenic mouse lines that label TRPM8+ sensory neurons have important caveats that hinder their use in functional studies. Thus, a mouse model that eases identification of these neurons would facilitate investigation of their unique physiological properties. To this end, we have found a novel use for the Vglut3iCre;Rosa26Ai14 mouse line in rapidly identifying menthol-sensitive sensory neurons in electrophysiological studies. In this mouse line, Vglut3 lineage neurons express cytoplasmic tdTomato and are easily identifiable without immunolabeling. Menthol is a potent activator of TRPM8 channels and thus a good proxy for identifying cold-sending neurons in vitro. Calcium microfluorimetry experiments on cultured Vglut3iCre;Rosa26Ai14 DRG neurons from adult mice (<24 h in culture) revealed 100% of neurons responding to menthol (100 μM) were small-diameter and of the Vglut3 lineage (tdTomato+.) These neurons did not respond to capsaicin (1 μM), a TRPV1 channel agonist, or chloroquine (1 μM), which activates TRPA1 signaling, consistent with the TRPM8+ sensory neuron phenotype. Furthermore, in current-clamp recordings, menthol evoked a train of action potentials in 100% of labeled neurons with a capacitance of < 15 pF (n = 5). Action potentials were tetrodotoxin-sensitive, confirming these neurons were not nociceptors. Thus, by targeting small (<15 pF), tdTomato+ DRG neurons in our electrophysiological recordings, we were able to increase the probability of identifying and recording from TRPM8+ neurons by 10-fold. In conclusion, the Vglut3iCre;Rosa26Ai14 is a useful model for rapidly identifying TRPM8+ sensory neurons and could facilitate electrophysiological studies of cold-responding sensory neurons.