The Role of Kv7/M Potassium Channels in Controlling Ectopic Firing in Nociceptors

Peripheral nociceptive neurons encode and convey injury-inducing stimuli toward the central nervous system. In normal conditions, tight control of nociceptive resting potential prevents their spontaneous activation. However, in many pathological conditions the control of membrane potential is disrupted, leading to ectopic, stimulus-unrelated firing of nociceptive neurons, which is correlated to spontaneous pain. We have investigated the role of KV7/M channels in stabilizing membrane potential and impeding spontaneous firing of nociceptive neurons. These channels generate low voltage-activating, noninactivating M-type K+ currents (M-current, IM), which control neuronal excitability. Using perforated-patch recordings from cultured, rat nociceptor-like dorsal root ganglion neurons, we show that inhibition of M-current leads to depolarization of nociceptive neurons and generation of repetitive firing. To assess to what extent the M-current, acting at the nociceptive terminals, is able to stabilize terminals' membrane potential, thus preventing their ectopic activation, in normal and pathological conditions, we built a multi-compartment computational model of a pseudo-unipolar unmyelinated nociceptive neuron with a realistic terminal tree. The modeled terminal tree was based on the in vivo structure of nociceptive peripheral terminal, which we assessed by in vivo multiphoton imaging of GFP-expressing nociceptive neuronal terminals innervating mice hind paw. By modifying the conductance of the KV7/M channels at the modeled terminal tree (terminal gKV7/M) we have found that 40% of the terminal gKV7/M conductance is sufficient to prevent spontaneous firing, while ~75% of terminal gKV7/M is sufficient to inhibit stimulus induced activation of nociceptive neurons. Moreover, we showed that terminal M-current reduces susceptibility of nociceptive neurons to a small fluctuations of membrane potentials. Furthermore, we simulated how the interaction between terminal persistent sodium current and M-current affects the excitability of the neurons. We demonstrated that terminal M-current in nociceptive neurons impeded spontaneous firing even when terminal Na(V)1.9 channels conductance was substantially increased. On the other hand, when terminal gKV7/M was decreased, nociceptive neurons fire spontaneously after slight increase in terminal Na(V)1.9 conductance. Our results emphasize the pivotal role of M-current in stabilizing membrane potential and hereby in controlling nociceptive spontaneous firing, in normal and pathological conditions

Multispectral labeling technique to map many neighboring axonal projections in the same tissue

We describe a method to map the location of axonal arbors of many individual neurons simultaneously based on the spectral properties of retrogradely transported dyelabeled vesicles. We inject overlapping regions of an axon target area with three or more different colored retrograde tracers. Based on the combinations and intensities of the colors in the individual vesicles transported to neuronal somata we calculate the projection sites of each neuron’s axon.This neuronal positioning system (NPS) enables mapping of many axons in a simple automated way. NPS combined with spectral (Brainbow) labeling of the input to autonomic ganglion cells show that the locations of ganglion cell projections to a mouse salivary gland relate to the identities of their preganglionic axonal innervation. We also show that NPS can delineate projections of many axons simultaneously in the mouse CNS.Molecular and Cellular Biolog

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

The peripheral terminals of primary sensory neurons detect histamine and non-histamine itch-provoking ligands through molecularly distinct transduction mechanisms. It remains unclear, however, whether these distinct pruritogens activate the same or different afferent fibers. We utilized a strategy of reversibly silencing specific subsets of murine pruritogen-sensitive sensory axons by targeted delivery of a charged sodium-channel blocker and found that functional blockade of histamine itch did not affect the itch evoked by chloroquine or SLIGRL-NH2, and vice versa. Notably, blocking itch-generating fibers did not reduce pain-associated behavior. However, silencing TRPV1+ or TRPA1+ neurons allowed AITC or capsaicin respectively to evoke itch, implying that certain peripheral afferents may normally indirectly inhibit algogens from eliciting itch. These findings support the presence of functionally distinct sets of itch-generating neurons and suggest that targeted silencing of activated sensory fibers may represent a clinically useful anti-pruritic therapeutic approach for histaminergic and non-histaminergic pruritus

Age-dependent decrease in inhibitory drive on the excitatory superficial spinal dorsal horn neurons

The excitatory and inhibitory interneurons of superficial laminae I-II of the spinal dorsal horn (SDH) receive and process pain-related information from the primary afferents and transmit it to the brain via the projection neurons. Thus, the interaction between excitatory and inhibitory SDH interneurons is crucial in determining the output from the spinal cord network. Disruption of this interaction in pathological conditions leads to increased SDH output to the higher brain centers, which could underlie pathological pain. Here, we examined whether the changes in the intrinsic SDH connectivity also occur with age, possibly underlying age-related increase in pain sensitivity. Using Vgat;tdTomato transgenic mouse line, we compared the spontaneous inhibitory postsynaptic currents (sIPSCs) in inhibitory tdTomato+ and excitatory tdTomato− interneurons between adult (3–5 m.o.) and aged (12–13 m.o.) mice. We demonstrate that in adult mice, the amplitude and frequency of the sIPSCs on the excitatory interneurons were significantly higher than on inhibitory interneurons. These differences were annulled in aged mice. Further, we show that in aged mice, excitatory neurons receive less inhibition than in adult mice. This could lead to overall disinhibition of the SDH network, which might underlie increased pain perception among the aged population

Location and plasticity of the sodium spike initiation zone in nociceptive terminals in vivo

Referred to by: Sharon R. Ha, Matthew N. Rasband. The SIZ of Pain. Neuron, Volume 102, Issue 4, 22 May 2019, Pages 709-711Nociceptive terminals possess the elements for detecting, transmitting, and modulating noxious signals, thus being pivotal for pain sensation. Despite this, a functional description of the transduction process by the terminals, in physiological conditions, has not been fully achieved. Here, we studied how nociceptive terminals in vivo convert noxious stimuli into propagating signals. By monitoring noxious-stimulus-induced Ca2+ dynamics from mouse corneal terminals, we found that initiation of Na+ channel (Nav)-dependent propagating signals takes place away from the terminal and that the starting point for Nav-mediated propagation depends on Nav functional availability. Acute treatment with the proinflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) resulted in a shift of the location of Nav involvement toward the terminal, thus increasing nociceptive excitability. Moreover, a shift of Nav involvement toward the terminal occurs in corneal hyperalgesia resulting from acute photokeratitis. This dynamic change in the location of Nav-mediated propagation initiation could underlie pathological pain hypersensitivity.Support is gratefully acknowledged from the Israel Science Foundation under grant agreement 1470/17 (R.H.G., O.B., B. K., S.L., and A.M.B.); the Deutsch-Israelische Projectkooperation program of the Deutsche Forschungsgemeinschaft (DIP) under grant agreement BI 1665/1-1ZI1172/12-1 (R.H.G., O.B., B. K., S.L., and A.M.B.); the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC grant agreement 260914 (R.H.G., O.B., B. K., S.L., and A.M.B.); the Jacob and Lena Joels Chair for Excellence in Life and Medical Sciences (A.M.B.); the Hoffman Leadership Program (R.H.G. and O.B.); and grant SAF2017-83674-C2-2-R from Agencia Estatal de Investigación, Spain, and ERDF, European Union (A.I.-P.).Peer reviewe

Optical assessment of nociceptive TRP channel function at the peripheral nerve terminal

This article belongs to the Special Issue TRP Channels.Free nerve endings are key structures in sensory transduction of noxious stimuli. In spite of this, little is known about their functional organization. Transient receptor potential (TRP) channels have emerged as key molecular identities in the sensory transduction of pain-producing stimuli, yet the vast majority of our knowledge about sensory TRP channel function is limited to data obtained from in vitro models which do not necessarily reflect physiological conditions. In recent years, the development of novel optical methods such as genetically encoded calcium indicators and photo-modulation of ion channel activity by pharmacological tools has provided an invaluable opportunity to directly assess nociceptive TRP channel function at the nerve terminal.This work was funded by The Spanish Agencia Estatal de Investigación and the European Regional Development Fund grants RTI2018-100994-AI00 and SAF2017-83674-C2-2-R and -1-R, The Spanish Ministerio de Universidades Fellowship FPU17/00483, The Generalitat Valenciana Excellence Project Program grant PROMETEO/2018/114, The Israeli Science Foundation, grant agreement 1470/17, Canadian Institutes of Health Research (CIHR), the International Development Research Centre (IDRC), the Israel Science Foundation (ISF) and the Azrieli Foundation, grant agreement 2545/18, and the Deutsch-Israelische Projectkooperation Program of the Deutsche Forschungsgemeinschaft (DIP), grant agreement BI 1665/1-1ZI1172/12-1.Peer reviewe

Abnormal Reinnervation of Denervated Areas Following Nerve Injury Facilitates Neuropathic Pain

An injury to peripheral nerves leads to skin denervation, which often is followed by increased pain sensitivity of the denervated areas and the development of neuropathic pain. Changes in innervation patterns during the reinnervation process of the denervated skin could contribute to the development of neuropathic pain. Here, we examined the changes in the innervation pattern during reinnervation and correlated them with the symptoms of neuropathic pain. Using a multispectral labeling technique—PainBow, which we developed, we characterized dorsal root ganglion (DRG) neurons innervating distinct areas of the rats’ paw. We then used spared nerve injury, causing partial denervation of the paw, and examined the changes in innervation patterns of the denervated areas during the development of allodynia and hyperalgesia. We found that, differently from normal conditions, during the development of neuropathic pain, these areas were mainly innervated by large, non-nociceptive neurons. Moreover, we found that the development of neuropathic pain is correlated with an overall decrease in the number of DRG neurons innervating these areas. Importantly, treatment with ouabain facilitated reinnervation and alleviated neuropathic pain. Our results suggest that local changes in peripheral innervation following denervation contribute to neuropathic pain development. The reversal of these changes decreases neuropathic pain