Scratching after Stroking and Poking: A Spinal Circuit Underlying Mechanical Itch

Neuron

Previews the authors have presented us with a powerful new framework capturing the dominant factors controlling dendritic protein distributions, which will serve as a foundation upon which future models must be built. Its impact will likely be amplified by the development of the smart webtool (http://www. tchumatchenko.de/Visualisation.html) to allow experimentalists to plug in parameter values for their protein of choice. In summary, like all good models, the Fonkeu et al. (2019) framework gives us both an insight into the mechanisms underlying a complicated system and a useful machine for making quantitative, testable predictions for future experiments.

REFERENCES Abbott, L.F., Farhi, E., and Gutmann, S. (1991). The path integral for dendritic trees. Biol. Cybern. 66, 49–60. Alon, U. (2006). An Introduction to Systems Biology: Design Principles of Biological Circuits (Chapman and Hall/CRC). Bressloff, P.C., and Earnshaw, B.A. (2007). Diffusion-trapping model of receptor trafficking in dendrites. Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 75, 041915. Crank, J. (1979). The Mathematics of Diffusion (Oxford University Press). Fonkeu, Y., Kraynyukova, N., Hafner, A.-S., Kochen, L., Sartori, F., Schuman, E.M., and Tchumatchenko, T. (2019). How mRNA localization and protein synthesis sites influence dendritic protein distribution and dynamics. Neuron 103, this issue, 1109–1122.

Kosik, K.S. (2016). Life at low copy number: how dendrites manage with so few mRNAs. Neuron 92, 1168–1180. Mahmutovic, A., Fange, D., Berg, O.G., and Elf, J. (2012). Lost in presumption: stochastic reactions in spatial models. Nat. Methods 9, 1163–1166. Santamaria, F., Wils, S., De Schutter, E., and Augustine, G.J. (2006). Anomalous diffusion in Purkinje cell dendrites caused by spines. Neuron 52, 635–648. Segev, I., Rinzel, J., and Shepherd, G.M. (1995). The Theoretical Foundation of Dendritic Function: Selected Papers of Wilfrid Rall with Commentaries (MIT Press). Williams, A.H., O’Donnell, C., Sejnowski, T.J., and O’Leary, T. (2016). Dendritic trafficking faces physiologically critical speed-precision tradeoffs. eLife 5, e20556.

Scratching after Stroking and Poking: A Spinal Circuit Underlying Mechanical Itch Zilong Wang,1 Christopher R. Donnelly,1 and Ru-Rong Ji1,2,3,* 1Center

for Translational Pain Medicine, Department of Anesthesiology, Duke University Medical Center, Durham, NC 27710, USA of Neurobiology, Duke University Medical Center, Durham, NC 27710, USA 3Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2019.09.009 2Department

Mechanical itch is a desire to scratch due to light mechanical stimuli. In this issue of Neuron, Pan et al. (2019) identify a feedforward inhibition circuit in the spinal cord dorsal horn that processes mechanical itch as well as spontaneous itch. How do you feel when your skin is gently grazed by a hair or when an insect is climbing on your skin? Most people will have an instinctual urge to scratch their skin in response to such stimuli. This sensation is defined as mechanical itch, a distinct sensory entity from chemical itch elicited by chemical pruritogens. Under physiological conditions, mechanical itch is a warning sign, serving a protective role to alert an organism to possible environmental hazards. Unfortunately, hypersensitivity of mechanical itch is also a symptom in chronic itch (Ikoma et al., 2006). Distinct cellular mechanisms may underlie mechanical and chemical itch (Bourane et al., 2015;

Feng et al., 2018). It remains unknown which population(s) of peripheral (primary) sensory neurons mediates mechanical itch, nor do we understand which population of neurons in the spinal cord transmits mechanical itch. In this issue of Neuron, Pan et al. (2019) identified a unique subpopulation of spinal cord excitatory interneurons (INs) that are required for mechanical itch. These spinal INs express the neuropeptide Ucn3 in inner lamina II and lamina III. Strikingly, using an intersectional genetic strategy to ablate >97% of Ucn3+ INs, Pan et al. (2019) demonstrate that mechanical itch elicited by gentle touch is completely abolished without affecting

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chemical pruritogen-induced itch. Further experiments using DREADD-based chemogenetic approaches demonstrate that silencing spinal Ucn3+ INs attenuates mechanical itch, while acute activation of Ucn3+ neurons is sufficient to evoke spontaneous scratching. Which neurons provide afferent synaptic inputs onto Ucn3+ INs? Further analysis reveals that Ucn3+ INs receive inputs from Ab low-threshold mechanoreceptors (Ab-LTMRs). Interestingly, monosynaptic tracing discovered that 89% of Ucn3-innervating DRG sensory neurons co-express Toll-like receptor 5 (TLR5). Thus, TLR5+ LTMRs make synaptic connections to spinal Ucn3+

Neuron

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ΘΙ NPY ?

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Ucn3

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Mechanical itch Mechanical allodynia

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TLR5+ AE-LTMR Type I

TLR5

TLR5+ AE-LTMR Type II

Figure 1. Schematic of Spinal Cord Mechanical Itch Pathway Mediated by TLR5+ Ab-LTMR and Ucn3+ Ins Notably, a different subgroup of TLR5+ Ab-LTMR may mediate mechanical allodynia.

neurons (Figure 1). This is consistent with a previous study demonstrating TLR5 expression primarily in Ab-LTMRs (Xu et al., 2015). The previous study also established a method of inhibiting Ab-LTMRs: activation of TLR5+ A-fibers with flagellin, a bacterial ligand of TLR5, allows cellular entry of QX-314 (cell-impermeable lidocaine derivative), leading to a functional blockade of A-fibers and subsequent blockade of mechanical allodynia after chemotherapy and nerve injury (Xu et al., 2015). In contrast, co-application of QX-314 and flagellin does not affect C-fibers, which are blocked by co-application of capsaicin and QX-314 (Binshtok et al., 2007). Using this blockade method, Pan et al. (2019) confirmed that silencing TLR5+ neurons by QX-314/flagellin blocked mechanical allodynia after spared nerve injury. Furthermore, they demonstrated that intradermal co-application of QX314 and flagellin abolished mechanical

itch induced by light punctate stimuli. Calcium imaging revealed that activation of TLR5 by flagellin also activates A-fiber neurons in vitro. Importantly, intradermal injection of flagellin alone could induce spontaneous itch, and this pruritus requires both TLR5 and Ucn3+ neurons. Deep sequencing of DRG neurons reveals that Ab rapidly adapting LTMRs (RA-LTMRs) have the highest expression of Tlr5, while Ab slowly adapting LTMRs (SA-LTMRs) and Ad LTMRs also show significant Tlr5 expression (Zheng et al., 2019). Pan et al. (2019) also conducted an elegant electrophysiological characterization of synaptic transmission between TLR5+ LTMRs and Ucn3+ INs in spinal cord slices. They showed that QX-314 and flagellin co-application abolished Ab-evoked excitatory postsynaptic currents (EPSCs) in 30 out of 31 recorded Ucn3+ INs, without affecting Ad-evoked EPSCs, indicating a selectivity of TLR5 for

Ab-LTMRs. Pan et al. (2019) also recorded Ab-eEPSCs in Ucn3 INs in laminae IIi-III and found that QX-314/flagellin abolished Ab-eEPSCs in 32.0% of the recorded neurons. Thus, there are at least two subtypes of TLR5+ LTMRs: type I neurons for mechanical allodynia, as previously demonstrated (Xu et al., 2015), and type II neurons, which innervate Ucn3+ INs to transmit mechanical itch (Figure 1). Pan et al. (2019) also demonstrated that Ucn3+ INs receive feedforward inhibition from INs. NPY+ inhibitory INs were previously shown to gate mechanical itch (Bourane et al., 2015). The authors further showed that most Ucn3+ INs received inhibitory synaptic inputs from NPY+ INs, and optogenetic activation of NPY+ INs could evoke IPSCs in Ucn3+ neurons. Pan et al. (2019) also demonstrated that Ucn3+ INs receive GABAergic and/or glycinergic inhibition from NPY+ inhibitory INs. Furthermore, chemogenetic activation of the NPY+ INs attenuated mechanical itch, while ablation of these neurons could augment mechanical itch. Thus, NPY+ inhibitory INs suppress mechanical itch through Ucn3+ INs. Almost simultaneously, Acton et al. (2019) reported that neuropeptide Y receptor Y1 (Y1R) expressing neurons are also essential for generating mechanical itch, but not chemical itch. Ablation or silencing of Y1R+ INs attenuated mechanical itch. In contrast, activation of Y1R+ INs increased mechanical itch and induced spontaneous scratch. Acton et al. (2019) also demonstrated that NPY+ INs form functional inhibitory synapses with Y1R+ INs in the spinal dorsal horn to gate mechanical itch through the NPY-Y1 pathway. Notably, ablation of NPY+ neurons results in spontaneous itch, which is completely abolished by ablation of Y1R+ INs. As both Ucn3+ and Y1R+ INs are implicated in mechanical itch, it is reasonable to postulate that these two populations of excitatory INs may overlap. However, Pan et al. (2019) showed that only 14% of Ucn3+ neurons co-express Y1R in the dorsal horn. It is also significant that the distribution patterns of Y1R+ neurons in these two studies are different, and more Y1R+ neurons were detected in Acton’s study, likely owing to differences in the sensitivity of the detection tools. Future studies are warranted to Neuron 103, September 25, 2019 953

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Previews further characterize the co-localization of Ucn3+ and NPY1R+ INs in greater detail. It is also important to identify neurotransmitters or neuromodulators that can trigger mechanical itch in the circuits. Is the neuropeptide Urocortin-3 an itch mediator? Notably, the TLR5+-Ucn3+ circuit may also converge with other itch pathways, including spontaneous itch and chronic itch. Pan et al. (2019) demonstrated that mechanical itch in histamineinduced alloknesis and several models of chronic itch are largely abolished in Ucn3+ IN-ablated mice. Spontaneous itch in chronic models was also greatly attenuated after Ucn3+ IN ablation. Furthermore, inhibition of TLR5+-LTMR with intradermal flagellin/QX-314 greatly attenuated both histamine and calcipotriol induced alloknesis. Given a critical role of GRPR+ INs in chemical itch, chronic itch, and spontaneous itch (Sun et al., 2009), it is important to know whether Ucn3+ INs synapse on GRPR+ neurons to mediate spontaneous itch. However, GRPR+ neurons are not required for Y1R+ IN-mediated mechanical itch (Acton et al., 2019). Taken together, the mechanical itch pathway identified by Pan et al. (2019) provides a critical step forward in our understanding of the microcircuits respon-

sible for distinct forms of itch. TLR5+ LTMRs appear to mediate both mechanical allodynia (type I) and mechanical itch (type II) (Figure 1). It remains to be investigated whether NPY+ and Y1+ INs also receive inputs from TLR5+ LTMRs. Merkel cells are touch receptors and regulate mechanical itch, and notably, alloknesis in aging and dry skin is associated with a loss of Merkel cells (Feng et al., 2018). It will be interesting to investigate the possibility of a functional link between Merkel cells and TLR5+ Ab-LTMRs in mechanical itch. Recently, Sakai and Akiyama (2019) reported that silencing TLR5+ Ab fibers with co-injection of flagellin and QX-314 could provoke mechanical itch. It is likely that Ab-LTMRs could have both positive and negative regulations of mechanical itch, depending on whether the spinal cord inhibitory gate is open under the different physiological or pathological conditions. REFERENCES Acton, D., Ren, X., Di Costanzo, S., Dalet, A., Bourane, S., Bertocchi, I., Eva, C., and Goulding, M. (2019). Spinal neuropeptide Y1 receptorexpressing neurons form an essential excitatory pathway for mechanical itch. Cell Rep. 28, 625–639.e6. Binshtok, A.M., Bean, B.P., and Woolf, C.J. (2007). Inhibition of nociceptors by TRPV1-mediated entry

of impermeant sodium channel blockers. Nature 449, 607–610. Bourane, S., Grossmann, K.S., Britz, O., Dalet, A., Del Barrio, M.G., Stam, F.J., Garcia-Campmany, L., Koch, S., and Goulding, M. (2015). Identification of a spinal circuit for light touch and fine motor control. Cell 160, 503–515. Feng, J., Luo, J., Yang, P., Du, J., Kim, B.S., and Hu, H. (2018). Piezo2 channel-Merkel cell signaling modulates the conversion of touch to itch. Science 360, 530–533. €nder, S., Yosipovitch, Ikoma, A., Steinhoff, M., Sta G., and Schmelz, M. (2006). The neurobiology of itch. Nat. Rev. Neurosci. 7, 535–547. Pan, H., Fatima, M., Li, A., Lee, H., Cai, W., Horwitz, L., Hor, C.C., Zaher, N., Cin, M., Slade, H., et al. (2019). Identification of a spinal circuit for mechanical and persistent spontaneous itch. Neuron 103, this issue, 1135–1149. Sakai, K., and Akiyama, T. (2019). Disinhibition of touch-evoked itch in a mouse model of psoriasis. J. Invest. Dermatol. 139, 1407–1410. Sun, Y.G., Zhao, Z.Q., Meng, X.L., Yin, J., Liu, X.Y., and Chen, Z.F. (2009). Cellular basis of itch sensation. Science 325, 1531–1534. Xu, Z.Z., Kim, Y.H., Bang, S., Zhang, Y., Berta, T., Wang, F., Oh, S.B., and Ji, R.R. (2015). Inhibition of mechanical allodynia in neuropathic pain by TLR5-mediated A-fiber blockade. Nat. Med. 21, 1326–1331. Zheng, Y., Liu, P., Bai, L., Trimmer, J.S., Bean, B.P., and Ginty, D.D. (2019). Deep sequencing of somatosensory neurons reveals molecular determinants of intrinsic physiological properties. Neuron 103, 598–616.e7.

Are You There, Cortex? It’s Me, Acetylcholine Kevin J. Monk1,2 and Marshall G. Hussain Shuler1,2,* 1The Solomon H. Snyder Department of Neuroscience, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA 2Kavli Neuroscience Discovery Institute, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205, USA *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2019.08.039

It is not well understood how associations between two temporally removed stimuli form. In this issue of Neuron, Guo et al. (2019) implicate basal forebrain cholinergic neurons as providing a link between auditory cues and the aversive outcomes they predict. We decide between courses of action based on their expected outcomes. Yet, how we come to expect future events given predictive cues is not well under-

stood; especially challenging to neuroscientists is how the brain connects two stimuli when they are temporally removed from each other. What areas are involved

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in learning these contingencies? Who is learning from whom? What are the resultant changes? In this issue of Neuron, Guo et al. (2019) uncover a means by