Accepted

Mechanical allodynia is a cardinal feature of pathological pain. Recent work has demonstrated the necessity of A[graphic1] low-threshold mechanoreceptors (Aβ-LTMRs) for mechanical allodynia-like behaviors in mice, but it remains unclear whether these neurons are sufficient to produce pain under pathological conditions. Thus, we generated a transgenic mouse in which channelrhodopsin-2 (ChR2) is conditionally expressed in Vesicular Glutamate Transporter 1 (Vglut1) sensory neurons (Vglut1-ChR2), which is a heterogeneous population of large-sized sensory neurons with features consistent with (A[graphic3]-LTMRs). In naive male Vglut1-ChR2 mice, transdermal hindpaw photostimulation evoked withdrawal behaviors in an intensity- and frequency-dependent manner, which were abolished by local anesthetic and also selective A-fiber blockade. Surprisingly, male Vglut1-ChR2 mice did not show significant differences in light-evoked behaviors or real-time aversion after nerve injury, despite marked hypersensitivity to punctate mechanical stimuli. Thus, we conclude that optogenetic activation of cutaneous Vglut1-ChR2 neurons alone is not sufficient to produce pain-like behaviors in neuropathic mice. Mechanical allodynia, wherein innocuous touch is perceived as pain, is a common feature of pathological pain. To test the contribution of low-threshold mechanoreceptors to nerve injury-induced mechanical allodynia, we generated and characterized a new transgenic mouse (Vglut1-ChR2) to optogenetically activate cutaneous Vglut1-positive LTMRs. Using this mouse, we found that light-evoked behaviors were unchanged by nerve injury, which suggests that activation of Vglut1-positive LTMRs alone is not sufficient to produce pain. The Vglut1-ChR2 mouse will be broadly useful for the study of touch, pain and itch.

The cold and menthol sensitive Transient Receptor Potential Melastatin 8 (TRPM8) channel is important for both physiological temperature detection and cold allodynia. Activation of G-protein coupled receptors (GPCRs) by pro-inflammatory mediators inhibits these channels. It was proposed that this inhibition proceeds via direct binding of G to the channel. TRPM8 requires the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P or PIP] for activity. It was claimed however that a decrease in cellular levels of this lipid upon receptor activation does not contribute to channel inhibition. Here we show that supplementing the whole cell patch pipette with PI(4,5)P reduced inhibition of TRPM8 by activation of G-coupled receptors in mouse dorsal root ganglion (DRG) neurons isolated from both sexes. Stimulating the same receptors activated Phospholipase C (PLC) and decreased plasma membrane PI(4,5)P levels in these neurons. PI(4,5)P also reduced inhibition of TRPM8 by activation of heterologously expressed G-coupled muscarinic M1 receptors. Co-expression of a constitutively active G protein that does not couple to PLC inhibited TRPM8 activity, and in cells expressing this protein decreasing PI(4,5)P levels using a voltage sensitive 5'-phosphatase induced a stronger inhibition of TRPM8 activity than in control cells. Our data indicate that upon GPCR activation, G binding reduces the apparent affinity of TRPM8 for PI(4,5)P and thus sensitizes the channel to inhibition induced by decreasing PI(4,5)P levels.Increased sensitivity to heat in inflammation is partially mediated by inhibition of the cold- and menthol sensitive TRPM8 ion channels. Most inflammatory mediators act via GPCR-s that activate the Phospholipase C pathway leading to the hydrolysis of PI(4,5)P How receptor activation by inflammatory mediators leads to TRPM8 inhibition is not well understood. Here we propose that direct binding of G both reduces TRPM8 activity, and sensitizes the channel to inhibition by decreased levels of its cofactor PI(4,5)P Our data demonstrate the convergence of two downstream effectors of receptor activation G and PI(4,5)P hydrolysis in regulation of TRPM8.