Activity-dependent silencing reveals functionally distinct itch-generating sensory neurons

​Authors: This work is the collaboration between Alex Binshtok's group in the Department of Medical  Neurobiology, Institute of Medical Research Israel-Canada, Faculty of Medicine,  The Edmond and Lily Safra Center for Brain Sciences, Hebrew University and Clifford Woolf's group in the Boston Childers Hospital and Harvard Medical School.Corresponding Authors: Alex Binshtok and Clifford Woolf. Student contributors: David Roberson from Harvard Medical School, Sagi Gudes from the Hebrew University, Jared Sprague from Harvard Medical School, Felix Blasl from the Hebrew UniversityShort description of the findings: Itch is a complex unpleasant cutaneous sensation that in some respects resembles pain, yet is different in terms of its intrinsic sensory quality and the urge to scratch. Although some types of the itch like urticaria (hives) could be effectively treated with anti-histaminergic agents, itch accompanying most chronic pruritic diseases, including atopic dermatitis (eczema), allergic itch and dry skin itch, is not predominantly mediated by histamine. The understanding of the molecular and cellular mechanisms underlying the sensation of itch is essential for the development of effective and selective treatment of itch, which in some cases could become a devastating condition.The signaling machinery  responsive to histaminergic and non-histaminergic  itch-generating ligands are well described and distinct, although at a cellular level, there is overlapping responsiveness of peripheral neurons  to itch-producing pruritogens and even to pain-producing algogens. It remains controversial, therefore, if there are separate peripheral neurons  that mediate histamine itch and non-histamine itch. This distinction is clinically important since therapies targeting histaminergic itch fibers might be ineffective for treating non-histaminergic itch if the neurons mediating the two itches are functionally distinct in the adult.To study if histaminergic and non-histaminergic itch are functionally distinct we adapted a method originally designed for achieving a pain-specific peripheral nerve block  to selectively silence the peripheral terminals of different subsets of itch - and noxious stimuli-responsive primary afferents in an activity-dependent manner. To do this we targeted the charged, membrane-impermeable lidocaine derivative QX-314-a sodium channel blocker through large pore ion channels activated specifically by different algogens and pruritogens. Thus we were able to selectively  inhibit  pain- and itch-related Recordings of  calcium signals from cultured peripheral neurons  demonstrated that histamine responsive neurons are a small subset of neurons expressing noxious heat detecting TRPV1 channel and that non-histaminergic pruritogens such as chloroquine activate  small subset of the peripheral neurons expressing TRPA1 channel – the protein that detects noxious chemical stimuli.  We showed by electrophysiological recording that activation of these large pore channels by histamine and chloroquine enables sufficient permeation of QX-314 into trigeminal neurons to block sodium currents, and that this block effect is specific; only the pruritogen-activated neurons are blocked. This has enabled us to exploit the silencing of different afferents to tease out their functional sensitivity to defined stimuli. This approach differs from interventions that only block a particular receptor or channel in that it targets action potential generation and conduction of the activated axon, and differs from genetic targeted ablation of different sensory neuronal subtypes in that it is temporary with no and will not lead to any compensatory changes.Utilizing this selective silencing strategy by monitoring the scratching behaviors of mice following injection of histamine and non-histaminergic pruritogens we reveal that the fibers that mediate histamine and non-histamine itch are functionally separable. Moreover we demonstrate that activation of these itch-generating fibers is not required for eliciting normal behavioral responses to acute mechanical and thermal pain, cold or tactile stimuli. The fact that both pain-sensing and itch-generating neurons express same transducer channels TRPV1 and TRPA1 implies that noxious heat or mechanical stimuli will produce to itch in addition to pain.  But normally we will withdraw the arm from hot kettle and will rub it but not scratch it. Using selective silencing strategy of TRPV1 and TRPA1 expressing neurons we showed that  certain peripheral neurons may normally indirectly inhibit noxious stimuli from eliciting itch.Our findings suggest that primary afferent itch-generating neurons encode functionally distinct histamine and non-histaminergic itch pathways. In addition to revealing the modality specificity and functional specialization of somatosensory afferents, our findings could also help direct development of new treatments for itch.