Uncovering the Cells and Circuits of Touch in Normal and

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Review Uncovering the Cells and Circuits of Touch in Normal and Pathological Settings Francie Moehring,1 Priyabrata Halder,2,3 Rebecca P. Seal,2,3,4 and Cheryl L. Stucky1,* 1Department

of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA of Neurobiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA 3Center for the Neural Basis of Cognition, Pittsburgh, PA 15213, USA 4Pittsburgh Center for Pain Research, Pittsburgh, PA 15213, USA *Correspondence: [email protected] https://doi.org/10.1016/j.neuron.2018.10.019 2Department

The sense of touch is fundamental as it provides vital, moment-to-moment information about the nature of our physical environment. Primary sensory neurons provide the basis for this sensation in the periphery; however, recent work demonstrates that touch transduction mechanisms also occur upstream of the sensory neurons via non-neuronal cells such as Merkel cells and keratinocytes. Within the spinal cord, deep dorsal horn circuits transmit innocuous touch centrally and also transform touch into pain in the setting of injury. Here non-neuronal cells play a key role in the induction and maintenance of persistent mechanical pain. This review highlights recent advances in our understanding of mechanosensation, including a growing appreciation for the role of non-neuronal cells in both touch and pain. Introduction Our ability to sense touch is indispensable for daily activities throughout our lives. Gentle touch is critical for the healthy development of a newborn baby (Ardiel and Rankin, 2010; Hopper and Pinneau, 1957). In humans, tactual acuity and postural stability decrease with age, which leads to difficulties with tasks that require fine manipulations and an increase in falls due to reduced tactile sensation in the feet (Dannenbaum and Jones, 1993; Kenshalo, 1986; Tanaka et al., 1996; Tremblay et al., 2003). Despite the vital importance of touch in our lives, our understanding of exactly how mechanical stimuli are detected in the skin and converted into action potentials is still in nascent stages. Central dogma indicates that sensory neurons are the sole and initial responders to mechanical stimuli in the skin. Contradicting this, recent work indicates that initial detection involves nonneuronal cells in the skin, which in turn communicate with nearby sensory neurons. Touch input from the periphery is then transmitted centrally to the spinal cord and ultimately relayed to the somatosensory cortex through a series of neuronal circuits that conserve body somatotopy. While some forms of touch can feel pleasant, touch can also become painful after injury. The transformation of touch into pain results from peripheral nerve or tissue damage and involves changes in key dorsal horn circuits that stem from aberrant primary afferent input and altered descending supraspinal input. The transformation to painful touch is also intimately tied to the actions of non-neuronal cells (e.g., microglia, astrocytes, and oligodendrocytes) in the dorsal horn that also serve to maintain the persistent pain state. Here we review recent advances in our understanding of the molecular and cellular underpinnings of mechanosensation both in normal healthy conditions and in chronic pain specifically related to (1) advances in the field of mammalian mechanotransducers, (2) the critical role of non-neuronal cells in mechanotransduction, (3) peripheral and central pathological changes that lead to painful touch, and (4) the contribution of non-neuronal cells in the periph-

ery and spinal cord to painful touch. Overall, work discussed in this review highlights a paradigm shift in the field by revealing that sensory neurons do not act alone in detecting and conveying mechanical stimuli, but instead collaborate with various skin cells to detect and relay tactile information to the spinal cord and brain. Additionally, new studies are delineating the dorsal horn circuits that convey persistent pain, and illuminating the contributions of various glial cells to these circuits. Continued efforts to investigate the generation of touch, allodynia, and pain and the role of nonneuronal cells will reveal potential new therapeutic targets. Conventional View—Peripheral Circuitry Leading to Touch Sensation The feeling of a gentle stroke across the arm, a painful pinch, or the cool evening breeze on a hot summer night requires that sensory stimuli at the periphery are transformed into complex percepts at the level of the brain. Because changes in the external environmental often require rapid reactions, touch is processed on an extremely rapid timescale. The conventional view is that primary afferents are the first to participate in this signal transduction cascade. These neurons have a unique pseudounipolar morphology, with a single process that bifurcates into two branches: a distal branch, which can be up to a meter long, that innervates peripheral tissues and a shorter branch that terminates centrally. For the trigeminal system, which covers the head and face, the somata of peripheral sensory neurons reside within the trigeminal ganglia and terminate in the medulla. For the rest of the body, the somata reside within the dorsal root ganglia (DRG) and terminate in the spinal dorsal horn or dorsal column nuclei in the medulla (Figure 1A). The predominant class of fibers responsible for sensing light touch are Ab-fibers, which have thickly myelinated axons and thus have fast conduction velocities and low activation thresholds. Additionally, a subpopulation of C-fibers (C tactile afferents) has been shown to be crucial for gentle touch and to respond to light forces similar to Ab-fibers Neuron 100, October 24, 2018 ª 2018 Published by Elsevier Inc. 349

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(Liljencrantz and Olausson, 2014; Vallbo et al., 1999). In humans, these C tactile afferents preferentially respond to gentle slow brushing or stroking with a finger (Liljencrantz and Olausson, 2014; Vallbo et al., 1999). Because of their preferential response to gentle stroke, they are thought to be part of the limbic system that detects affective touch (Olausson et al., 2002) and are hypothesized to be important in social interactions and for skin-toskin contact. These neurons may also have a role in suppressing or augmenting pain in the setting of injury (Delfini et al., 2013; Franc¸ois et al., 2015; Kambrun et al., 2018). Specific hair follicles are innervated by low-threshold Ad-fiber lanceolate endings (D-hairs), which are precisely tuned to detect the deflection of body hairs in the tail to head direction (Brown and Iggo, 1967; Burgess et al., 1968; Rutlin et al., 2014). Similar low-threshold Ad-fiber responses have been observed in humans, but it remains to be determined if these fibers also influence touch perception (Adriaensen et al., 1983). The perception of acute noxious or painful touch is typically initiated by high-threshold unmyelinated C-fibers and thinly myelinated Ad-fibers.

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Figure 1. Sensory Neurons and Nonneuronal Cells Important for Touch Transduction in Hairy and Glabrous Skin (A) Cell bodies of the dorsal root ganglia (DRG) neurons are found near the spinal cord, where they extend a centrally projecting axon to the dorsal horn and a distal projecting, longer axon to the hairy and glabrous skin (or deeper muscle, bone, or tendon tissue; not shown). Ab-fibers are thickly myelinated, fast conducting, and generally have large-diameter cell bodies; Ad-fibers are thinly myelinated, conduct at medium speed, and generally have medium-diameter cell bodies, Non-myelinated C-fibers conduct much more slowly and generally have smaller cell bodies. (B) Glabrous skin innervation shows the diversity of somatosensory neurons that terminate within the thick layer of the epidermis (between keratinocytes) and the underlying dermis. Keratinocytes can communicate with the distal terminals of noxious-detecting peptidergic (deeper) and nonpeptidergic C-fibers (more superficial), as well as with the terminals of Ad-fibers and Ab-fibers. Ab-fiber endings innervate Merkel cell complexes and aid in touch sensation by helping shape our detection of two-point discrimination. (C) The mammalian hairy skin is comprised of a thinner layer of the epidermis and contains three different type of hairs, where each afferent type can be identified by its innervation pattern of the hair follicle as well as its morphology. For example, Guard hairs are the least abundant and longest hair and are innervated by Ab-fibers, which have circumferential endings. Zigzag hairs, on the other hand, are the most abundant, have a distinct zigzag shape, and are innervated by C-fibers or Ad-fibers that have circumferential endings. All mammals except humans are equipped with these highly differentiated touch organs.

In order for primary sensory neurons to respond to mechanical stimuli and initiate action potentials, they need specific molecular transducers that can be directly activated by physical energy. Elegant studies using Caenorhabditis elegans (C. elegans), Drosophila melanogaster, and even bacteria have identified a number of intrinsically mechanically gated ion channels; however, the identification of mammalian mechanotransduction channels has lagged behind (Lumpkin and Caterina, 2007; Nakatani et al., 2015). Mammalian homologs of the channels identified in C. elegans and Drosophila, such as members of the ENaC family, have been investigated; however, there is still no evidence that they act as bona fide mechanotransducers in mammalian tissues (Arnado´ttir and Chalfie, 2010; Chatzigeorgiou et al., 2010). A challenge for the field has been to determine whether a putative mechanotransducer forms the actual pore of the channel or is a required associated protein that acts to modify the mechanically gated channel. For this reason, typical methods for functional verification such as reconstitution or gene knockout are difficult to interpret. Piezo Channels Are Touch Transducers In 2010, two novel mechanically gated cation channels (Piezo1 and Piezo2) were discovered in mice (Coste et al., 2010). The

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Review channels are between 2,500 and 2,800 amino acids in length, share approximately 42% homology (Coste et al., 2010), and have the highest number of transmembrane domains of any mammalian protein (26) (Volkers et al., 2015). Despite the large sequence homology between the two channels, more is known about the structure and gating of Piezo1. A high-resolution cryoelectron microscopy (cryo-EM) structure of this channel reveals a trimer with a three-blade-propeller-shaped architecture (Coste et al., 2015; Ge et al., 2015; Saotome et al., 2018; Zhao et al., 2016). Piezo1 was shown to form an ion pore that is directly gated by membrane stretch as shown by its response to lipid bilayer tension in the absence of cytoskeleton and by its reconstitution in lipid droplets (Cox et al., 2016; Syeda et al., 2016). However, direct gating of Piezo2 by membrane tension has not yet been demonstrated. In addition, Piezo 1 is modulated by voltage, showing an increased open probability at positive voltages (Moroni et al., 2018). Both Piezo ion channels are expressed by many nonneuronal tissues, such as bladder, colon, and lung, that functionally rely on mechanotransduction (Coste et al., 2010). For example, constitutive knockout of either Piezo isoform in mice is embryonic lethal because these channels are essential for lung and vasculature function (Li et al., 2014; Ranade et al., 2014; Woo et al., 2014). Several mutations in both Piezo1 and Piezo2 have been identified in humans and present as severe physiological phenotypes. In general, Piezo1 (gain or loss of function) mutations affect the lymphatic system and red blood cells (Fotiou et al., 2015; Martin-Almedina et al., 2018). Patients with Piezo2 mutations suffer from profound mechanosensory and proprioceptive deficits (Chesler et al., 2016; Delle Vedove et al., 2016; Mahmud et al., 2017). The importance of Piezo2 in proprioception was recently confirmed in mice, where deletion of the channel selectively from proprioceptive neurons led to a profound impairment in limb motor coordination (Woo et al., 2015). Additionally, Chesler and colleagues identified two patients with null mutations in Piezo2 that led to nonsense-mediated decay and a severe reduction in the level of PIEZO2 transcripts. The patients exhibited diminished sensitivity to light touch, vibration, and two-point touch discrimination in the glabrous skin while acute mechanical pain sensation was unaltered (Chesler et al., 2016). Recent mouse and human studies indicate that Piezo2 expression in primary afferents is required for dynamic mechanical allodynia (Murthy et al., 2018b; Szczot et al., 2018). Taken together, the data indicate that normal Piezo channel activity is required for baseline tactile, dynamic mechanical allodynia, and proprioceptive responses in rodents and humans. Interestingly, it was recently discovered that Piezo2 has at least 16 alternative splice variants in sensory neurons, whereas non-neuronal tissues express only a single form (Szczot et al., 2017). The neuronal splice variants show significant differences in ion permeability, sensitivity to calcium modulation, and rates of channel inactivation (Szczot et al., 2017). This finding raises intriguing questions about how the different Piezo2 splice variants may contribute to the detection of mechanical stimuli in different subtypes of mechanosensory neurons, and whether the splice variants are differently modulated by factors in the environment, tissue injury, or disease.

Mechanically Sensitive Potassium Channels Two-Pore Potassium Channels Other ion channels that are inherently sensitive to mechanical stimuli in mammals include the two-pore-domain potassium channels (K2P channels): TREK-1 (TWIK-1-related K+ channel), TREK-2, and TRAAK (TWIK-related arachidonic acid-stimulated K+ channel) (Bang et al., 2000; Fink et al., 1996, 1998; Lesage et al., 2000). These force-sensitive channels are activated by asymmetric tension in the lipid membrane that is induced by membrane curvature (Brohawn et al., 2014; Sun et al., 2016). Whereas Piezo1 increases membrane excitability upon mechanical stimulation, activation of K2P channels suppresses neuronal excitability, acting as a brake on transmission. Therefore, the role of these K2P channels may be to counteract or balance the activity of other mechanotransduction channels in the membrane. In support of this hypothesis, TREK-1, TREK-2, and TRAAK knockout mice show increased sensitivity to punctate €l et al., 2009; Pereira mechanical stimuli (Alloui et al., 2006; Noe et al., 2014). Voltage-Gated Potassium Channels Although most studies on mechanically sensitive potassium channels have focused on the K2P family, there is evidence that voltage-gated potassium channels also respond to mechanical force (Gu et al., 2001; Hao et al., 2013; Laitko et al., 2006). For example, membrane probing of HEK293 cells expressing Kv1.1 oligomers leads to mechanically activated currents (Hao et al., 2013). Additionally, mice expressing a dominant-negative Kv 1.1 channel in sensory neurons display a decreased paw withdrawal threshold (Hao et al., 2013), demonstrating that in the absence of Kv1.1, animals develop mechanical allodynia. These data suggest that both K2P and Kv 1.1 channels act as brakes to dampen the effects of non-selective mechanically sensitive cation channels in the membrane. Other Potential Mechanosensors and Recent Advances OSCA/TMEM63 Channels In 2014, Yuan and colleagues used a hyperosmolarity screen to identify ion channels that sense changes in osmolarity and allow calcium influx. Examining Arabidopsis mutants, the authors identified a channel that they termed hyperosmolality-induced [Ca2+]i increase 1 (OSCA1) (Yuan et al., 2014). Expression of OSCA1.1 and OSCA3.1 in heterologous cells conferred the response to stretch (Zhang et al., 2018) and reconstitution of OSCA1.2 in liposomes allowed for the induction of large stretch-activated currents (Murthy et al., 2018a). In addition, fruit fly, mouse, and human TMEM63, a homolog of OSCA, was also shown exhibit large inward currents in response to membrane stretch in heterologous cells (Murthy et al., 2018a). Together, these data suggest that OSCA/TMEM63 proteins are inherently mechanically activated ion channels. TMEM150C TMEM150C was originally hypothesized to be a mechanosensitive ion channel with six predicted transmembrane domains that underlies slowly adapting type currents (Hong et al., 2016). However, two more recent studies suggest that TMEM150C is instead a modulator of other mechanically sensitive ion channels (Anderson et al., 2018; Dubin et al., 2017). The most likely explanation for the differences is that Hong and colleagues

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Review used HEK293 cells that endogenously express Piezo1 and TMEM150C likely potentiated the endogenous Piezo1 currents in their study (Dubin et al., 2017). Indeed, Anderson and colleagues used HEK293 cells with endogenous Piezo1 deleted and showed that TMEM150C potentiates the currents of the mechanically gated channels Piezo1, Piezo2, and TREK1 and that TMEM150C co-immunoprecipitates with TREK1 and Piezo2 (Anderson et al., 2018). These data suggest that TMEM150C directly interacts with these mechanically gated ion channels to potentiate their currents via an unknown mechanism. Taken together, the results point to the identity of new mechanically relevant ion channels and modulators and the likelihood that more channels and modulators are waiting to be discovered. The data also show that molecular touch mechanisms are complex and it is probable that multiple touch-relevant channels work together to drive the activity of touch- and pain-sensing fibers. Roles for Non-neuronal Cells in Mechanotransduction Non-neuronal cells are known to have an important role in sensory transduction processes underlying taste and hearing (Chaudhari and Roper, 2010; Fettiplace and Kim, 2014). In the somatosensory field, however, the canonical view has been that the sensory neurons alone are sufficient to initiate mechanotransduction. This concept is somewhat surprising given that most mechanosensory neurons innervate specialized end organs in the skin (Figures 1B and 1C) that are known to have key roles: (1) Merkel cells respond to sustained touch and pressure and aid in two-point discrimination; (2) Ruffini’s end organs sense stretching of skin around objects and over joints; (3) Pacinian corpuscles sense fast vibrations and deep pressure; (4) Meissner’s corpuscles sense slow vibrations and changes in texture; and (5) hair follicles detect hair movement in response to very light touch, clothing, and air currents (Delmas et al., 2011; Fleming and Luo, 2013). Thus, numerous specialized non-neuronal end organs in the skin sense different features of mechanical stimuli. How touch is tuned at each neurite complex is likely due to a variety of factors including the distinct expression patterns and density of mechanotransduction channels in sensory endings that innervated the end organ or the non-neuronal cells that comprise the structural ending. The response also depends on other factors such as the depth of the end organs in the skin (Delmas et al., 2011; Fleming and Luo, 2013), the extent of terminal branching, and the types of surrounding non-neuronal cells. Merkel Cells Merkel cells are touch-sensitive cells that are specifically enriched in areas of high tactile acuity (fingertips, whisker hair follicles, and touch domes) (Merkel, 1875) (Figure 1). Located in the deep basal epidermal cell layer, they form complexes with SAI (slowly adapting type I) afferents, where they detect sustained pressure and encode textures and object features (Iggo and Muir, 1969; Maksimovic et al., 2014). Pressure applied to a Merkel cell elicits long-lasting and irregular action potential firing characteristic of the SAI afferents (Iggo and Muir, 1969). These data suggest that Merkel cells are indispensable for normal touch sensation. Indeed, mice lacking Merkel cells due to an Atoh1specific knockout display a complete loss of SAI firing patterns and female Keratin14 Atoh1 conditional knockout mice display a lack of texture discrimination (Maricich et al., 2009, 2012)

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Merkel cells express Piezo2, which has been documented to contribute to two-point tactile discrimination. Epidermal-specific Piezo2 knockout mice lack the touch-evoked currents in Merkel cells and afferents that innervate Merkel cells in the mice exhibit truncated action potential firing (Maksimovic et al., 2014; Woo et al., 2014). Signaling from Merkel cells to SA1 afferents is still not entirely resolved, but has been reported to rely on serotonin (Chang et al., 2016). A two-receptor-site model was put forth suggesting that Merkel cells and sensory afferents work in concert to transduce touch stimuli (Fagan and Cahusac, 2001; Nakatani et al., 2015; Yamashita and Ogawa, 1991). However, computational models have shown that Merkel cell-neurite complexes and neurites cannot solely be responsible for the sustained action potential firing of SAI afferents and that a third player responsible for ultra-slowly adapting currents is also required (Gerling et al., 2018). This ultra-slowly adapting current is necessary for the slow adaptation observed in SAI afferent firing and may be initiated by other skin cells (such as keratinocytes) or the neurite itself. Interestingly, in addition to mediating baseline touch sensations, new research indicates that Piezo2 in Merkel cells is also required for the modulation and dampening of touch-evoked itch sensation, as animals with Piezo2 deleted from epidermal cells show increased alloknesis (itchy skin) in response to light mechanical stimulation (Feng et al., 2018). These data suggest that epidermal cells may play a more prominent role in the detection and transmission of sensory information than previously recognized. Keratinocytes Merkel cells constitute only a small percentage (roughly 3%–6%) of the cells in the epidermis (Figures 1B and 1C) (Fradette et al., 2003; Halata et al., 2003; Moll et al., 1986). Other cells include melanocytes, Langerhans (dendritic) cells, and keratinocytes. Keratinocytes are the predominant cell type, making up 95% of the epidermis and forming the main barrier to the external environment (Fuchs, 1995). All primary afferent subtypes terminate within or in close proximity to the keratinocyte layers within the skin (Figures 1B and 1C) (Abraira and Ginty, 2013; Kruger et al., 1981; Li and Ginty, 2014; Owens and Lumpkin, 2014; Pare´ et al., 2001). ‘‘Synapse-like’’ cell-cell membrane contacts have been observed between sensory nerve endings and keratinocytes (Chaˆteau and Misery, 2004; Chaˆteau et al., 2007; Hilliges et al., 1995; Talagas et al., 2018). Thus, keratinocytes are wellpositioned to act as an intermediary between the external environment and sensory neuron terminals. Recent work has shown that optogenetic inhibition of epidermal cells (including keratinocytes) dampens both innocuous and noxious behavioral responses to mechanical stimuli, indicating that keratinocytes are a key player in how animals sense touch (Moehring et al., 2018). Furthermore, keratinocytes shape the responses of both nociceptive and non-nociceptive afferents (Baumbauer et al., 2015; Pang et al., 2015). Mechanical stimulation of keratinocytes has been shown to directly depolarize the keratinocyte membrane (Moehring et al., 2018) and to elicit inward currents in adjacent sensory neurons in co-cultures of keratinocytes and sensory neurons (Klusch et al., 2013). Mechanistically, keratinocytes signal to sensory nerve terminals by releasing ATP, which activates sensory neuron P2X4 receptors (Moehring et al., 2018). However, the mechanotransducer

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Review that enables keratinocytes to respond to mechanical probing has not yet been identified. A prime candidate for this transducer is Piezo1, which is highly expressed in mouse skin (Coste et al., 2010). Collectively, these data indicate that keratinocytes are intimately involved in communicating information about somatosensory stimuli that impact the skin to the nervous system Oral Epithelium Akin to skin, the epithelial cells in the oral cavity are regularly subjected to mechanical forces that occur during speaking, eating, and swallowing. It has been suggested that our tongue is tactually more sensitive than our fingertips (Aktar et al., 2015). Recent evidence demonstrates that the specialized end organs of the oral epithelium called end bulbs of Krause express Piezo2 (Moayedi et al., 2018). This suggests that the end bulbs of Krause, which are innervated by sensory neuron fibers, may also participate in mechanical detection and encoding that occurs in the oral cavity. This type of cellular communication may contribute significantly to complex fine motor skills such as speech or the ability to sense food textures. Together these data reveal a paradigm shift that changes our view of somatosensation from a point where sensory neurons were thought to act alone in transduction of stimuli, to a perspective that supporting cells may play a much greater role in baseline mechanotransduction mechanisms than originally thought. Peripheral Non-neuronal Cells in Mechanical Allodynia While the ability to detect acute painful stimuli is essential for the organism’s survival, the perception of pain in response to normally non-painful stimuli (i.e., allodynia) is a maladaptive hallmark of many diseases. Because mechanical allodynia is evoked by non-painful stimuli, it is hypothesized that low-threshold primary afferent fibers convey touch-evoked pain. Blocking A-fibers decreases dynamic mechanical allodynia evoked by slow brushing across the skin (Kilo et al., 1994; Koltzenburg et al., 1992; Ochoa and Yarnitsky, 1993; Torebjo¨rk et al., 1992). Static allodynia, evoked by indentation of the skin, however, is not blocked by this paradigm, suggesting that unmyelinated fibers may be important for conveying this type of allodynia (Kilo et al., 1994; Koltzenburg et al., 1992; Ochoa and Yarnitsky, 1993; Torebjo¨rk et al., 1992). Research has also shown that a class of C-fibers that are normally mechanically insensitive (‘‘silent’’) respond to mechanical stimuli after injury (Feng and €bler Gebhart, 2011; Gebhart, 1999; Gold and Gebhart, 2010; Ha et al., 1990; Michaelis et al., 1996; Prato et al., 2017; Schaible and Schmidt, 1988). This type of C-fiber was recently characterized in viscera and deep somatic tissues as expressing the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) (Prato et al., 2017). The identity of the analogous cutaneous C-fiber is not yet known. To date, most research on mechanical allodynia has focused on how disease or injury affects neuronally expressed ion channels. Several studies have shown that ion channels expressed on sensory nerve terminals become sensitized to mechanical stimuli after injury (reviewed in Basbaum et al., 2009; Basso and Altier, 2017; Ellis and Bennett, 2013; Gold and Gebhart, 2010; Julius, 2013; Lolignier et al., 2015; Luo et al., 2015; Stein et al., 2009; Stucky et al., 2009; Woolf and Ma, 2007). However, the precise

mechanisms through which diseases and/or injuries alter the mechanical sensitivity of ion channels leading to the perception of pain instead of touch are unknown. It may involve various pathways such as (1) PKA and PKC signaling that lead to sensitization of ion channels (Brackley et al., 2017; Dubin et al., 2012; Fan et al., 2009; Fischer et al., 2010; Meents et al., 2017; Schmidt et al., 2009); (2) alterations in the composition of the plasma membrane lipid bilayer, and subsequent alterations in gating of a mechanically-gated ion channel (Barabas et al., 2014; Narayanan et al., 2018); or (3) novel channel interactions such as the coupling of an ion channel normally not involved in mechanosensation to a mechanically gated ion channel or the coupling of a mechanically sensitive ion channel to an accessory protein that enhances its function (e.g., TMEM150C) (Anderson et al., 2018; Dubin et al., 2017). Ultimately, more research is needed to determine how mechanically gated ion channels become sensitized in pathological states associated with painful touch. Keratinocytes Despite the fact that non-neuronal cells such as keratinocytes are often the primary site of injury and, therefore, are in an ideal location to modify sensory neuron signaling in disease states, little is known about their role in these processes. Altered keratinocyte function and signaling is a hallmark of skin disorders such as dermatitis and psoriasis (Martin et al., 2015). During the development of psoriasis, keratinocytes release chemokines that allow them to communicate with and recruit immune cells to the lesion site, thereby leading to the skin inflammation that is typical of psoriatic plaques (Ayala-Fonta´nez et al., 2016). In another skin disorder, Hidradenitis Suppurativa, keratinocytes have an intrinsic defect that enhances chronic inflammation of the skin through the release of pro-inflammatory molecules (Hotz et al., 2016; Lima et al., 2016), and this produces painful lumps in the armpit and groin areas. Additionally, keratinocytes from patients with mechanical allodynia show changes in the gene expression profiles of ion channels that are known to be involved in pain conditions. For example, hairy skin and keratinocytes from patients with complex regional pain syndrome and post-herpetic neuralgia exhibit increased expression of various voltage-gated sodium channels (Nav1.1, Nav1.2, Nav1.6, Nav1.7, and Nav1.8) (Zhao et al., 2008). Thus, pathological alterations in keratinocytes contribute to the development of cutaneous pain and itch, symptoms frequently experienced by patients with skin disorders. In addition to resident skin cells in disease pathology, mobile peripheral immune cells recruited to the site of injury have also been shown to play a crucial role in pain modulation (reviewed in Ji et al., 2016; Marchand et al., 2005; Pinho-Ribeiro et al., 2017). With rapid progress being made on non-neuronal cell communication mechanisms at baseline, it is likely that these mechanisms will be further investigated in disease states, opening the possibility of identifying novel peripheral pharmaceutical targets. Anatomy of Touch Pathways The complex percepts of touch are made up of both discriminative and affective features that are signaled through multiple pathways from the periphery to the brain. The first pathway consists of large, myelinated low-threshold mechanosensory afferents that project through the dorsal columns, terminate in

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the gracile and cuneate nuclei of the brainstem, and encode fine, discriminative touch (Johnson and Hsiao, 1992) (Figure 2A). The second major touch relay from the periphery projects to laminae IIi–IV of the spinal dorsal horn, also known as the low-threshold mechanosensor-recipient zone (LTMR-RZ) (Abraira et al., 2017). All three classes of low-threshold mechanosensory afferents (Ab, Ad, and C-LTMRs) send projections that terminate in this region either exclusively or as collaterals from dorsal column projections. These inputs transmit crude touch information as well as affective aspects of touch and, after injury, are recruited into nociceptive circuits to convey light touch as painful. Cellular Organization of the Spinal Cord Dorsal Horn Classically, the dorsal horn is organized as Rexed laminae based on the pattern of nissl-stained cell bodies in the dorsoventral orientation (Rexed, 1952). The most dorsal laminae (I and II) receive predominantly nociceptive and thermal afferent input, while more ventral laminae (III–VI) receive input from lowthreshold mechanoreceptors and proprioceptors. The dorsal horn itself is composed of a large number of molecularly and morphologically diverse excitatory and inhibitory interneurons as well as distinct sets of projection neurons (Figure 2B). Wide dynamic range projection neurons in lamina I and V respond to lowthreshold input and send information to the brain through the anterior spinothalamic tract. Within the LTMR-RZ, postsynaptic

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Figure 2. Spinal Cord Circuits Important for Touch and Pain Processing (A) High-threshold (HT) C- and Ad-fibers that transmit pain, itch, and temperature project to the dorsal laminae I and II. Low-threshold (LT) C- and Ad-fibers that transmit aspects of touch project to lamina II inner and III. A-fibers that transmit touch information project directly through the dorsal columns to the brainstem as well as send collaterals into the deeper dorsal horn laminae Iii–IV. This region is known as the low-threshold mechanosensor-recipient zone (LTMR-RZ). Descending cortical projections modulate touch and innervate specifically the LTMR-RZ. (B) LTMRs innervate neurons in the LTMR-RZ, including excitatory vertical cells (Ver), VGLUT3 interneurons, RORa interneurons, and post-synaptic dorsal column (PSDC) neurons as well as inhibitory dynorphin (Dyn), parvalbumin (PV), and islet cells. Vertical cells receive inputs from all types of afferents as well as a large number of intrinsic neuronal populations. VGLUT3 neurons directly or indirectly innervate the Cr and PKCg populations; however, a cell population in this pathway remains uncharacterized. PSDC neurons ascend through the postsynaptic dorsal column tract of the dorsal horn. RORa neurons receive inputs from descending corticospinal tract, and connect to premotor (PMN) and motor neurons (MN) in the ventral horn. These dorsal horn neurons modulate motor function in concert with somatosensory inputs.

dorsal column (PSDC) neurons transmit touch information to the brainstem dorsal column nuclei. These neurons receive most of their input from local spinal neurons (Abraira et al., 2017). Interestingly, PSDC neurons in the deep dorsal horn also have been implicated in visceral pain (Al-Chaer et al., 1996). The cellular and functional organization of dorsal horn interneurons is still not well understood. Recent efforts to solve this issue have emphasized histological and molecular approaches. Using histological methods, both excitatory (gastrin-releasing peptide [GRP], preprotachykinin B, and neurotensin) and inhibitory (galanin, neuropeptide Y [NPY], parvalbumin [PV], and neuronal nitric oxide synthase) interneuron populations that do not overlap were identified (Gutierrez-Mecinas et al., 2016; Polga´r et al., 2013). The impetus to organize the dorsal horn into non-overlapping populations is driven by the idea that distinct somatosensory functions are defined by population codes. Studies to date indicate emerging roles for the GRP neurons in promoting chemical itch (Fleming et al., 2012; Sun and Chen, 2007), NPY neurons in preventing mechanical itch (Bourane et al., 2015a), galanin neurons in preventing chemical itch (Kardon et al., 2014; Sardella et al., 2011), and PV neurons in preventing mechanical allodynia (Hughes et al., 2012; Petitjean et al., 2015). It is likely, however, that these heterogeneous populations will have roles in more than one modality. Molecular efforts to define functional populations within the dorsal horn have focused most recently on the use of singlecell transcriptome approaches. In these studies, cell populations are defined by parsing individual neurons into groups based on

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Review €ring et al., 2018; the similarity of their gene expression profiles (Ha Sathyamurthy et al., 2018). Interestingly, in a number of cases, neurons that belong to a genetically defined population are clustered within a single lamina. This clustering is consistent with the concept that functional output is intimately tied to laminar organization. Another study, focusing specifically on the LTMR-RZ, used the Allen Brain Institute Spinal Cord Atlas to identify eleven largely non-overlapping populations of excitatory and inhibitory neurons that also showed distinct morphological and electrophysiological properties (Abraira et al., 2017). Examination of the connectivity of these populations to local, peripheral, and supraspinal neurons identified several overarching principles for how cutaneous light touch is wired. In terms of peripheral connections, the study showed that each identified interneuron population is connected to at least two types of LTMRs, but that not all LTMRs form monosynaptic connections with each identified interneuron population. With respect to local connections, neurons within the LTMR-RZ were found to be highly interconnected, consistent with previous reports determined using paired electrophysiological recordings (Abraira et al., 2017; Schneider, 2008). The authors also reported that descending cortical projections span almost exclusively the LTMR-RZ and form contacts with each of the eleven identified populations, indicating widespread direct cortical modulation of the circuitry for light touch. Since multiple areas of the cortex intimately collaborate to generate touch perception, understanding the precise effects on the spinal circuitry will require a more detailed characterization of the connectivity made by each region. In this respect, direct descending inputs from somatosensory cortex were shown to amplify low-threshold primary afferent input onto cholecystokinin-expressing excitatory neurons in lamina III–IV, and this impacted both light touch sensitivity and mechanical allodynia (Liu et al., 2018). In a separate study, excitatory neurons in lamina IIi–IV that express the retinoid receptor-related orphan receptor (ROR)a were shown to be innervated by motor areas including cortex. Loss of these neurons produced defects in light touch and in a corrective foot response measured as foot slips (Bourane et al., 2015b). The work suggested a role for these neurons in the integration of touch and motor control. Thus, a new framework for understanding the functional organization of the dorsal horn is emerging from recent studies of its cellular organization. A concerted effort to reconcile disparities among the newly defined populations will further enhance its utility. The identification of key excitatory populations and their connections to the larger spinal/CNS network is rapidly accelerating due to the rapid expansion of tools that permit the functional manipulation of select cell types, for example with optogenetics and chemogenetics and Cre recombinase-expressing mouse lines (Daigle et al., 2018; Harris et al., 2014; Madisen et al., 2015). Using these approaches, three additional excitatory interneuron populations important for mechanical allodynia have been identified: vesicular glutamate transporter (VGLUT) 3 neurons in laminae III-IV, calretinin interneurons in lamina II, and somatostatin interneurons in laminae II–III (Duan et al., 2014; Peirs et al., 2015) (Figure 2B). Studies of these neuronal populations revealed a few key features of the functional organization of the mechanical allodynia circuitry. In the study of calretinin interneurons, together with

excitatory interneurons expressing the gamma subunit of protein kinase C (PKCg), it was suggested, from an analysis of cFos, a marker of neuronal activity, that although both inflammatory and neuropathic peripheral insults produce mechanical allodynia, the allodynia may be transmitted by different aspects of the dorsal horn circuitry (Peirs et al., 2015). Specifically, mechanical allodynia induced by inflammatory injuries engages calretinin interneurons, whereas that induced by neuropathic injuries engages PKCg interneurons. The differential pattern of spinal neuron recruitment likely reflects different primary afferents affected by each type of injury. Consistent with this idea, it was recently reported that primary afferents expressing CGRPa are important for conveying mechanical allodynia induced by a neuropathic, but not an inflammatory, injury (Cowie et al., 2018). Investigation into the role of excitatory somatostatin interneurons and an inhibitory population expressing dynorphin (Dyn) highlighted a fundamental mechanistic principle for the mechanical allodynia circuitry in the dorsal horn (Duan et al., 2014). Specifically, the study provided further support for the idea that touch becomes painful when there is a decrease in inhibitory tone that allows LTMRs access to nociceptive dorsal horn circuits (Torsney and MacDermott, 2006). In addition to Dyn interneurons, PV interneurons in lamina III are also important for preventing LTMRmediated activation of nociceptive circuits under non-injury conditions (Petitjean et al., 2015). Given that a decrease in inhibitory tone from either of these populations is sufficient to generate the allodynic state, it is of interest to understand the degree to which injury causes weakened inhibition by these populations and if the impairment of each population depends on the type of injury (e.g., inflammatory, mononeuropathic, polyneuropathic). Together, these findings call for a systematic interrogation of the relevant circuitry from periphery to brain as a function injury type. Since mechanical allodynia is a common attribute of many types of chronic pain, such information will be highly valuable for the design of properly targeted pain therapeutics. Non-neuronal Cells in Persistent Mechanical Pain in the Dorsal Horn Work to understand the functional role of the dorsal horn in touch and pain processing also includes interactions with glial cells. Glial players include astrocytes, microglia, and oligodendrocytes, which are each essential for many aspects of dorsal horn function and have roles in the generation of mechanical allodynia following injury. Oligodendrocytes Of the three cell types, our understanding of the role of oligodendrocytes in persistent forms of pain such as mechanical allodynia is the least well understood. Oligodendrocytes are responsible for myelination in the CNS, and have functions related to the integrity of neurons (Nave and Werner, 2014). Death or damage as occurs in demyelinating diseases like multiple sclerosis often gives rise to neuropathic pain. Though still unclear, pain arising from this condition likely stems from changes in axonal integrity during the earliest stages of demyelination (Oluich et al., 2012). In the dorsal horn, ablation of oligodendrocytes damages interneurons as well as the spinothalamic tract (Gritsch et al., 2014). Damage to the spinothalamic tract has been shown to cause changes in thalamic activity similar to what is observed

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Review for central pain syndromes. Pain under these circumstances is likely due to aberrant activity in the damaged, but still intact neurons (Wang and Thompson 2008; Wasner et al., 2008). An improved mechanistic understanding of how oligodendrocytes contribute to the generation of neuropathic pain will provide new treatment options for pain in such disorders. Microglia Microglia make major contributions to the initiation and maintenance of mechanical allodynia. The cells have highly ramified processes that continuously monitor the environment through transient contacts with the synapse (Davalos et al., 2005; Nimmerjahn et al., 2005). Their principal roles are to clear debris in response to damage and to shape neuronal activity and synaptic plasticity (Salter and Beggs, 2014). Upon peripheral nerve injury, microglia become activated through signaling cascades initiated by damage to primary sensory neurons (Guan et al., 2016; Wlaschin et al., 2018). Activation is characterized by morphological and transcriptional changes that result in the release of neurotrophic and pro-inflammatory molecules such as brain-derived neurotrophic factor, tumor necrosis factor (a), and interleukin 1-b (Ji et al., 2018). These and other microglial-derived molecules promote central sensitization and the development of mechanical allodynia by a number of different mechanisms that act to either suppress inhibitory transmission or potentiate excitatory transmission (Ji et al., 2018; Taves et al., 2013). Astrocytes Activated astrocytes also contribute to mechanical allodynia. In healthy tissue, astrocytes are important regulators of neuronal activity through their ability to influence extracellular levels of neurotransmitters and co-agonists, potassium homeostasis, and by conferring other forms of metabolic regulation. The precise mechanisms that promote astrocyte activation are still incompletely understood, but evidence points to the involvement of pro-inflammatory mediators released by activated microglia (Liddelow et al., 2017). Reactive astrocytes are typically identified by the upregulation of glial fibrillary acidic protein, but single-cell transcriptome analyses are revealing additional genes that are upregulated with activation, including complement cascade genes and neurotrophic factors (Zamanian et al., 2012). With expression of these genes, reactive astrocytes participate in both the destruction of synapses and in their repair. With respect to mechanical allodynia, mechanisms that promote central sensitization by astrocytes generally act to enhance glutamatergic transmission. For example, reactive astrocytes show reduced levels of the high-affinity glutamate transporter EAAT2/Glt-1, responsible for regulating extracellular glutamate levels and enhanced release of D-serine, a co-agonist of NMDA-type glutamate receptors (Gao and Ji, 2010; Moon et al., 2015). Reactive astrocytes, acting in concert with microglia, also release proinflammatory mediators that contribute to the pain state (Gao and Ji, 2010). Many of the neural-glial interactions identified as having a role in mechanical allodynia are understood to the extent that they provide promising new targets for therapeutic intervention. However, precisely which neurons in the dorsal horn are affected by these events is, in most cases, still not known. With rapid progress being made in elucidating the structure, cell types, and connections within the dorsal horn circuitry for persistent

356 Neuron 100, October 24, 2018

mechanical pain, a next step will be to map the neural-glial interactions onto these networks. This more comprehensive picture will further enhance the design of novel therapeutics and inspire new combinations of therapies that could prove even more effective in combating pain. Conclusion Our ability to feel active and passive tactile sensation across our entire body touches nearly every aspect of our daily life, wellbeing, communication with others, work, and play. For such a vital sensation, it is surprising that so little is still known about the underlying biological mechanisms. Recent efforts are making significant headway in revealing the processes underlying the complex and multifaceted sensation of touch, with non-neuronal cells in the periphery contributing intimately with primary sensory neurons to relaying touch signals centrally. The role of neurons and glial cells to touch in the dorsal horn has yet to be uncovered, but our understanding of the circuitry, functioning like railroad track switches, to turn touch into pain with injury or disease is becoming clearer. Many mechanisms underlying touch remain to be discovered, making this an exciting time to engage in the field of touch somatosensation. ACKNOWLEDGMENTS The authors would like to thank Neil Smith Illustration (http://www. neilsmithillustration.co.uk/) for his outstanding skills and creativity in refining and improving the scientific illustrations in Figures 1 and 2 created by the authors. The research reported in this manuscript was supported by NIH grants NS040538 and NS070711 to C.L.S. and NS104964 to R.P.S. In addition, the authors would also like to thank the Research and Education Initiative Fund, a component of the Advancing a Healthier Wisconsin Endowment at the Medical College of Wisconsin. AUTHOR CONTRIBUTIONS Conceptualization, F.M., C.L.S., and R.P.S.; Writing – Original Draft, F.M., C.L.S., and R.P.S.; Writing – Review & Editing, F.M., P.H., C.L.S., and R.P.S.; Funding Acquisition, C.L.S. and R.P.S. REFERENCES Abraira, V.E., and Ginty, D.D. (2013). The sensory neurons of touch. Neuron 79, 618–639. Abraira, V.E., Kuehn, E.D., Chirila, A.M., Springel, M.W., Toliver, A.A., Zimmerman, A.L., Orefice, L.L., Boyle, K.A., Bai, L., Song, B.J., et al. (2017). The cellular and synaptic architecture of the mechanosensory dorsal horn. Cell 168, 295–310.e19. Adriaensen, H., Gybels, J., Handwerker, H.O., and Van Hees, J. (1983). Response properties of thin myelinated (A-delta) fibers in human skin nerves. J. Neurophysiol. 49, 111–122. Aktar, T., Chen, J., Ettelaie, R., and Holmes, M. (2015). Tactile sensitivity and capability of soft-solid texture discrimination. J. Texture Stud. 46, 429–439. Al-Chaer, E.D., Lawand, N.B., Westlund, K.N., and Willis, W.D. (1996). Visceral nociceptive input into the ventral posterolateral nucleus of the thalamus: a new function for the dorsal column pathway. J. Neurophysiol. 76, 2661–2674. €l, J., Chemin, J., Guy, N., Alloui, A., Zimmermann, K., Mamet, J., Duprat, F., Noe Blondeau, N., Voilley, N., Rubat-Coudert, C., et al. (2006). TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 25, 2368–2376. Anderson, E.O., Schneider, E.R., Matson, J.D., Gracheva, E.O., and Bagriantsev, S.N. (2018). TMEM150C/Tentonin3 is a regulator of mechano-gated ion channels. Cell Rep. 23, 701–708.

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