A SUBPOPULATION OF ITCH RECEPTORS MARKED BY RET EXPRESSION

Sensory neurons are a heterogeneous group of cells that are specialized to detect stimuli acting on the skin such as touch, temperature, pain and itch. A major challenge in the sensory biology field is to isolate and characterize specific functional subsets of neurons as an exhaustive knowledge of many of them is still lacking. In my PhD project I established a protocol to analyse and sort by Fluorescently Activated Cell Sorting (FACS) peripheral sensory neurons, in order to isolate specific subsets and further perform gene expression profiling. In particular I applied these techniques to Avil-Cre::ReteGFP mice, where Ret-positive neurons express eGFP specifically in Dorsal Root Ganglia (DRG) neurons after Cre recombination. Ret is a receptor for the GDNF family expressed in at least 3 sensory neuron subsets: non-peptidergic nociceptors, Rapidly Adapting (RA) mechanoreceptors and C-fiber low-threshold mechanoreceptors. However, our immunohistochemical analysis suggested that there might be more Ret-positive subsets. I analysed Ret-positive neurons by FACS, combining the analysis of the pattern of endogenous eGFP expression with IB4-binding arrangement and I identified 5 diverse Ret-eGFP-positive subsets. I focused on two of them, Ret-eGFPLo:IB4Neg and Ret-eGFPHi:IB4Neg that did not bind to IB4 and expressed low and high levels of eGFP, respectively. Their expression profiles suggested that Ret-eGFPHi:IB4Neg neurons represent the previously described RA Mechanoreceptors, while the Ret-eGFPLo:IB4Neg subset constitutes a new Ret-positive class of sensory neurons involved in itch perception. To verify this assumption, we functionally characterized Ret-eGFPLo:IB4Neg neurons. We focused on three molecules whose receptors we found enriched in the subset: histamine, a well known pruritogen, IL-31, a cytokine that has been linked to the pathology of Atopic Dermatitis, and LY344864, a serotonin agonist whose receptors, 5-HT1f, was among the most expressed genes within the Ret-eGFPLo:IB4Neg subset. By calcium imaging we demonstrated that these substances are able to elicit a neuronal response mainly in Ret-eGFP/IB4 negative cells. Moreover, when injected in the nape of the neck of mice, they all cause scratching, substantiating a putative role of this population as itch receptors.. My data indicate that we have discovered a new Ret-positive subset of sensory neurons involved in itch perception. A more extensive characterization of these cells, for example with a Sst-ires-Cre line which we found to specifically mark Ret-eGFPLo:IB4Neg subset, will further clarify their role within the DRG in vivo.