Mechanical allodynia, a widespread pain symptom that still lacks effective therapy, is associated with the activation of a dorsally directed polysynaptic circuit within the spinal dorsal horn (SDH) or medullary dorsal horn (MDH), whereby tactile inputs into deep SDH/MDH can gain access to superficial SDH/MDH, eliciting pain. Inner lamina II (II i ) interneurons expressing the ␥ isoform of protein kinase C (PKC␥ ϩ ) are key elements for allodynia circuits, but how they operate is still unclear. Combining behavioral, ex vivo electrophysiological, and morphological approaches in an adult rat model of facial inflammatory pain (complete Freund's adjuvant, CFA), we show that the mechanical allodynia observed 1 h after CFA injection is associated with the following (1) sensitization (using ERK1/2 phosphorylation as a marker) and (2) reduced dendritic arborizations and enhanced spine density in exclusively PKC␥ ϩ interneurons, but (3) depolarized resting membrane potential (RMP) in all lamina II i PKC␥ ϩ /PKC␥ Ϫ interneurons. Blocking MDH 5HT 2A receptors (5-HT 2A R) prevents facial mechanical allodynia and associated changes in the morphology of PKC␥ ϩ interneurons, but not depolarized RMP in lamina II i interneurons. Finally, activation of MDH 5-HT 2A R in naive animals is enough to reproduce the behavioral allodynia and morphological changes in PKC␥ ϩ interneurons, but not the electrophysiological changes in lamina II i interneurons, induced by facial inflammation. This suggests that inflammation-induced mechanical allodynia involves strong morphological reorganization of PKC␥ ϩ interneurons via 5-HT 2A R activation that contributes to open the gate for transmission of innocuous mechanical inputs to superficial SDH/MDH pain circuitry. Preventing 5-HT 2A R-induced structural plasticity in PKC␥ ϩ interneurons might represent new avenues for the specific treatment of inflammation-induced mechanical hypersensitivity.Inflammatory or neuropathic pain syndromes are characterized by pain hypersensitivity such as mechanical allodynia (pain induced by innocuous mechanical stimuli). It is generally assumed that mechanisms underlying mechanical allodynia, because they are rapid, must operate at only the level of functional reorganization of spinal or medullary dorsal horn (MDH) circuits. We discovered that facial inflammation-induced mechanical allodynia is associated with rapid and strong structural remodeling of specifically interneurons expressing the ␥ isoform of protein kinase C (PKC␥) within MDH inner lamina II. Moreover, we elucidated a 5-HT 2A receptor to PKC␥/ERK1/2 pathway leading to the behavioral allodynia and correlated morphological changes in PKC␥ interneurons. Therefore, descending 5-HT sensitize PKC␥ interneurons, a putative "gate" in allodynia circuits, via 5-HT 2A receptor-induced structural reorganization.

No abstract

A fundamental challenge in the optogenetic toolbox is that all opsins, regardless of their excitation spectra, are activated by blue light. We hypothesized that pairing a red-shifted channelrhodopsin with a blue light-sensitive anion channel of appropriately matching kinetics shall render neurons responsive to a red but not blue light. To achieve this, we used a semi-rational mutagenesis strategy to optimize the kinetics and light spectrum of a chloride channelrhodopsin. By pairing optimized variants of blue light-sensitive anion channel ZipACR with vfChrimson, a fast red-shifted channelrhodopsin, we created a system in which red light derives precise and faithful action potentials of high frequencies, while blue light, through shunting inhibition, nullifies the effect of the red-shifted ChR2. Additionally, by a simple switch between red and blue lights, one can effectively excite or inhibit the activity of the same neurons.