Peripheral gating of pain by glial endozepine.

We report that diazepam binding inhibitor (DBI) is a glial messenger mediating satellite glia-sensory neuron crosstalk in the dorsal root ganglion (DRG). DBI is highly and specifically expressed in satellite glia cells (SGCs) of mice, rat and human, but not in sensory neurons or other DRG-resident cells. Knockdown of DBI results in a robust mechanical hypersensitivity without significant effects on other sensory modalities. In vivo overexpression of DBI in SGCs reduces sensitivity to mechanical stimulation and alleviates mechanical allodynia in neuropathic and inflammatory pain models. We further show that DBI acts as a partial agonist and positive allosteric modulator at the neuronal GABAA receptors, particularly strongly effecting those with a high-affinity benzodiazepine binding site. Such receptors are selectively expressed by a subpopulation of mechanosensitive DRG neurons and these are also more enwrapped with DBI-expressing glia, as compared to other DRG neurons, suggesting a mechanism for specific effect of DBI on mechanosensation. These findings identified a new, peripheral neuron-glia communication mechanism modulating pain signalling, which can be targeted therapeutically.

Role of TMEM100 in mechanically insensitive nociceptor un-silencing.

Mechanically silent nociceptors are sensory afferents that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli during inflammation. Using RNA-sequencing and quantitative RT-PCR we demonstrate that inflammation upregulates the expression of the transmembrane protein TMEM100 in silent nociceptors and electrophysiology revealed that over-expression of TMEM100 is required and sufficient to un-silence silent nociceptors in mice. Moreover, we show that mice lacking TMEM100 do not develop secondary mechanical hypersensitivity-i.e., pain hypersensitivity that spreads beyond the site of inflammation-during knee joint inflammation and that AAV-mediated overexpression of TMEM100 in articular afferents in the absence of inflammation is sufficient to induce mechanical hypersensitivity in remote skin regions without causing knee joint pain. Thus, our work identifies TMEM100 as a key regulator of silent nociceptor un-silencing and reveals a physiological role for this hitherto enigmatic afferent subclass in triggering spatially remote secondary mechanical hypersensitivity during inflammation.

TTX-Resistant Sodium Channels Functionally Separate Silent From Polymodal C-nociceptors.

Pronounced activity-dependent slowing of conduction has been used to characterize mechano-insensitive, "silent" nociceptors and might be due to high expression of NaV1.8 and could, therefore, be characterized by their tetrodotoxin-resistance (TTX-r). Nociceptor-class specific differences in action potential characteristics were studied by: (i) in vitro calcium imaging in single porcine nerve growth factor (NGF)-responsive neurites; (ii) in vivo extracellular recordings in functionally identified porcine silent nociceptors; and (iii) in vitro patch-clamp recordings from murine silent nociceptors, genetically defined by nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) expression. Porcine TTX-r neurites (n = 26) in vitro had more than twice as high calcium transients per action potential as compared to TTX-s neurites (n = 18). In pig skin, silent nociceptors (n = 14) characterized by pronounced activity-dependent slowing of conduction were found to be TTX-r, whereas polymodal nociceptors were TTX-s (n = 12) and had only moderate slowing. Mechano-insensitive cold nociceptors were also TTX-r but showed less activity-dependent slowing than polymodal nociceptors. Action potentials in murine silent nociceptors differed from putative polymodal nociceptors by longer duration and higher peak amplitudes. Longer duration AP in silent murine nociceptors linked to increased sodium load would be compatible with a pronounced activity-dependent slowing in pig silent nociceptors and longer AP durations could be in line with increased calcium transients per action potential observed in vitro in TTX-resistant NGF responsive porcine neurites. Even though there is no direct link between slowing and TTX-resistant channels, the results indicate that axons of silent nociceptors not only differ in their receptive but also in their axonal properties.

Structure-guided examination of the mechanogating mechanism of PIEZO2.

Piezo channels are mechanically activated ion channels that confer mechanosensitivity to a variety of different cell types. Piezos oligomerize as propeller-shaped homotrimers that are thought to locally curve the membrane into spherical domes that project into the cell. While several studies have identified domains and amino acids that control important properties such as ion permeability and selectivity as well as inactivation kinetics and voltage sensitivity, only little is known about intraprotein interactions that govern mechanosensitivity-the most unique feature of PIEZOs. Here we used site-directed mutagenesis and patch-clamp recordings to investigate the mechanogating mechanism of PIEZO2. We demonstrate that charged amino acids at the interface between the beam domain-i.e., a long α-helix that protrudes from the intracellular side of the "propeller" blade toward the inner vestibule of the channel-and the C-terminal domain (CTD) as well as hydrophobic interactions between the highly conserved Y2807 of the CTD and pore-lining helices are required to ensure normal mechanosensitivity of PIEZO2. Moreover, single-channel recordings indicate that a previously unrecognized intrinsically disordered domain located adjacent to the beam acts as a cytosolic plug that limits ion permeation possibly by clogging the inner vestibule of both PIEZO1 and PIEZO2. Thus, we have identified several intraprotein domain interfaces that control the mechanical activation of PIEZO1 and PIEZO2 and which might thus serve as promising targets for drugs that modulate the mechanosensitivity of Piezo channels.

Differential modulation of voltage-gated sodium channels by nerve growth factor in three major subsets of TrkA-expressing nociceptors.

Nerve growth factor is an inflammatory mediator that induces long-lasting hyperalgesia, which can partially be attributed to nerve growth factor-induced sensitization of primary afferent nociceptors. It was shown that nerve growth factor increases the excitability of polymodal C-fibre nociceptors by modulating tetrodotoxin-sensitive and tetrodotoxin-resistant voltage-gated sodium channels, but hitherto only little is known about the effects of nerve growth factor on sodium currents in other nociceptor subtypes that express the nerve growth factor receptor TrkA. We previously characterized two reporter mouse lines that allow the unequivocal identification of two important subclasses of TrkA-expressing nociceptors - i.e. neuropeptide Y receptor type 2 (NPY2R+ ) Aδ-fibre nociceptors that mediate pinprick pain and nicotinic acetylcholine receptor alpha-3 subunit (CHRNA3+ ) silent nociceptors, which are the most abundant TrkA+ nociceptors in visceral organs and deep somatic tissues. Here, we utilized these mouse lines to investigate the expression patterns and the possible nerve growth factor-dependent modulation of sodium channels in these neurons using whole-cell patch-clamp recordings and quantitative real-time polymerase chain reaction. We demonstrate that NPY2R+ nociceptors, CHRNA3+ 'silent' nociceptors and polymodal C-fibre nociceptors express different combinations of sodium channel α- and ß-subunits and accordingly exhibit functionally different sodium currents. Moreover, we demonstrate that nerve growth factor produces robust hyperpolarizing shifts in the half-activation voltage of tetrodotoxin-resistant currents in NPY2R+ nociceptors and polymodal C-fibre nociceptors and also shifts the half-activation of tetrodotoxin-sensitive currents in polymodal C-fibre nociceptors. In silent nociceptors, however, nerve growth factor solely increases the current density of the tetrodotoxin-resistant current but does not alter other sodium channel properties. Considering the different peripheral target tissues and the previously reported roles in different forms of pain of the nociceptor subpopulations that were examined here, our results suggest that nerve growth factor differentially contributes to the development visceral and cutaneous pain hypersensitivity and highlights the importance of developing different therapeutic strategies for different forms of pain.

Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation.

Touch Receptor-Derived Sensory Information Alleviates Acute Pain Signaling and Fine-Tunes Nociceptive Reflex Coordination.

Painful mechanical stimuli activate multiple peripheral sensory afferent subtypes simultaneously, including nociceptors and low-threshold mechanoreceptors (LTMRs). Using an optogenetic approach, we demonstrate that LTMRs do not solely serve as touch receptors but also play an important role in acute pain signaling. We show that selective activation of neuropeptide Y receptor-2-expressing (Npy2r) myelinated A-fiber nociceptors evokes abnormally exacerbated pain, which is alleviated by concurrent activation of LTMRs in a frequency-dependent manner. We further show that spatial summation of single action potentials from multiple NPY2R-positive afferents is sufficient to trigger nocifensive paw withdrawal, but additional simultaneous sensory input from LTMRs is required for normal well-coordinated execution of this reflex. Thus, our results show that combinatorial coding of noxious and tactile sensory input is required for normal acute mechanical pain signaling. Additionally, we established a causal link between precisely defined neural activity in functionally identified sensory neuron subpopulations and nocifensive behavior and pain.

PIEZO2 is required for mechanotransduction in human stem cell-derived touch receptors.

Human sensory neurons are inaccessible for functional examination, and thus little is known about the mechanisms mediating touch sensation in humans. Here we demonstrate that the mechanosensitivity of human embryonic stem (hES) cell-derived touch receptors depends on PIEZO2. To recapitulate sensory neuron development in vitro, we established a multistep differentiation protocol and generated sensory neurons via the intermediate production of neural crest cells derived from hES cells or human induced pluripotent stem (hiPS) cells. The generated neurons express a distinct set of touch receptor-specific genes and convert mechanical stimuli into electrical signals, their most salient characteristic in vivo. Strikingly, mechanosensitivity is lost after CRISPR/Cas9-mediated PIEZO2 gene deletion. Our work establishes a model system that resembles human touch receptors, which may facilitate mechanistic analysis of other sensory subtypes and provide insight into developmental programs underlying sensory neuron diversity.