Kinesin family member 2A gates nociception.

Nociceptive axons undergo remodeling as they innervate their targets during development and in response to environmental insults and pathological conditions. How is nociceptive morphogenesis regulated? Here, we show that the microtubule destabilizer kinesin family member 2A (Kif2a) is a key regulator of nociceptive terminal structures and pain sensitivity. Ablation of Kif2a in sensory neurons causes hyperinnervation and hypersensitivity to noxious stimuli in young adult mice, whereas touch sensitivity and proprioception remain unaffected. Computational modeling predicts that structural remodeling is sufficient to explain the phenotypes. Furthermore, Kif2a deficiency triggers a transcriptional response comprising sustained upregulation of injury-related genes and homeostatic downregulation of highly specific channels and receptors at the late stage. The latter effect can be predicted to relieve the hyperexcitability of nociceptive neurons, despite persisting morphological aberrations, and indeed correlates with the resolution of pain hypersensitivity. Overall, we reveal a critical control node defining nociceptive terminal structure, which is regulating nociception.

Encoding of inflammatory hyperalgesia in mouse spinal cord.

ABSTRACT: Inflammation modifies the input-output properties of peripheral nociceptive neurons such that the same stimulus produces enhanced nociceptive firing. This increased nociceptive output enters the superficial dorsal spinal cord (SDH), an intricate neuronal network composed largely of excitatory and inhibitory interneurons and a small percentage of projection neurons. The SDH network comprises the first central nervous system network integrating noxious information. Using in vivo calcium imaging and a computational approach, we characterized the responsiveness of the SDH network in mice to noxious stimuli in normal conditions and investigated the changes in SDH response patterns after acute burn injury-induced inflammation. We show that the application of noxious heat stimuli to the hind paw of naïve mice results in an overall increase in SDH network activity. Single-cell response analysis reveals that 70% of recorded neurons increase or suppress their activity, while ∼30% of neurons remain nonresponsive. After acute burn injury and the development of inflammatory hyperalgesia, application of the same noxious heat stimuli leads to the activation of previously nonresponding neurons and desuppression of suppressed neurons. We further demonstrate that an increase in afferent activity mimics the response of the SDH network to noxious heat stimuli under inflammatory conditions. Using a computational model of the SDH network, we predict that the changes in SDH network activity result in overall increased activity of excitatory neurons, amplifying the output from SDH to higher brain centers. We suggest that during acute local peripheral inflammation, the SDH network undergoes dynamic changes promoting hyperalgesia.

mTORC2 mediates structural plasticity in distal nociceptive endings that contributes to pain hypersensitivity following inflammation.

The encoding of noxious stimuli into action potential firing is largely mediated by nociceptive free nerve endings. Tissue inflammation, by changing the intrinsic properties of the nociceptive endings, leads to nociceptive hyperexcitability and thus to the development of inflammatory pain. Here, we showed that tissue inflammation-induced activation of the mammalian target of rapamycin complex 2 (mTORC2) triggers changes in the architecture of nociceptive terminals and leads to inflammatory pain. Pharmacological activation of mTORC2 induced elongation and branching of nociceptor peripheral endings and caused long-lasting pain hypersensitivity. Conversely, nociceptor-specific deletion of the mTORC2 regulatory protein rapamycin-insensitive companion of mTOR (Rictor) prevented inflammation-induced elongation and branching of cutaneous nociceptive fibers and attenuated inflammatory pain hypersensitivity. Computational modeling demonstrated that mTORC2-mediated structural changes in the nociceptive terminal tree are sufficient to increase the excitability of nociceptors. Targeting mTORC2 using a single injection of antisense oligonucleotide against Rictor provided long-lasting alleviation of inflammatory pain hypersensitivity. Collectively, we showed that tissue inflammation-induced activation of mTORC2 causes structural plasticity of nociceptive free nerve endings in the epidermis and inflammatory hyperalgesia, representing a therapeutic target for inflammatory pain.

The Input-Output Relation of Primary Nociceptive Neurons is Determined by the Morphology of the Peripheral Nociceptive Terminals.

The output from the peripheral terminals of primary nociceptive neurons, which detect and encode the information regarding noxious stimuli, is crucial in determining pain sensation. The nociceptive terminal endings are morphologically complex structures assembled from multiple branches of different geometry, which converge in a variety of forms to create the terminal tree. The output of a single terminal is defined by the properties of the transducer channels producing the generation potentials and voltage-gated channels, translating the generation potentials into action potential (AP) firing. However, in the majority of cases, noxious stimuli activate multiple terminals; thus, the output of the nociceptive neuron is defined by the integration and computation of the inputs of the individual terminals. Here, we used a computational model of nociceptive terminal tree to study how the architecture of the terminal tree affects the input-output relation of the primary nociceptive neurons. We show that the input-output properties of the nociceptive neurons depend on the length, the axial resistance (Ra), and location of individual terminals. Moreover, we show that activation of multiple terminals by a capsaicin-like current allows summation of the responses from individual terminals, thus leading to increased nociceptive output. Stimulation of the terminals in simulated models of inflammatory or neuropathic hyperexcitability led to a change in the temporal pattern of AP firing, emphasizing the role of temporal code in conveying key information about changes in nociceptive output in pathologic conditions, leading to pain hypersensitivity.SIGNIFICANCE STATEMENT Noxious stimuli are detected by terminal endings of primary nociceptive neurons, which are organized into morphologically complex terminal trees. The information from multiple terminals is integrated along the terminal tree, computing the neuronal output, which propagates toward the CNS, thus shaping the pain sensation. Here, we revealed that the structure of the nociceptive terminal tree determines the output of nociceptive neurons. We show that the integration of noxious information depends on the morphology of the terminal trees and how this integration and, consequently, the neuronal output change under pathologic conditions. Our findings help to predict how nociceptive neurons encode noxious stimuli and how this encoding changes in pathologic conditions, leading to pain.

Platelet-derived growth factor activates nociceptive neurons by inhibiting M-current and contributes to inflammatory pain.

Endogenous inflammatory mediators contribute to the pathogenesis of pain by acting on nociceptors, specialized sensory neurons that detect noxious stimuli. Here, we describe a new factor mediating inflammatory pain. We show that platelet-derived growth factor (PDGF)-BB applied in vitro causes repetitive firing of dissociated nociceptor-like rat dorsal root ganglion neurons and decreased their threshold for action potential generation. Injection of PDGF-BB into the paw produced nocifensive behavior in rats and led to thermal and mechanical pain hypersensitivity. We further detailed the biophysical mechanisms of these PDGF-BB effects and show that PDGF receptor-induced inhibition of nociceptive M-current underlies PDGF-BB-mediated nociceptive hyperexcitability. Moreover, in vivo sequestration of PDGF or inhibition of the PDGF receptor attenuates acute formalin-induced inflammatory pain. Our discovery of a new pain-facilitating proinflammatory mediator, which by inhibiting M-current activates nociceptive neurons and thus contributes to inflammatory pain, improves our understanding of inflammatory pain pathophysiology and may have important clinical implications for pain treatment.

Location and Plasticity of the Sodium Spike Initiation Zone in Nociceptive Terminals In Vivo.

Nociceptive 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 1ß (IL-1ß) 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.

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.

KV 7/M channels as targets for lipopolysaccharide-induced inflammatory neuronal hyperexcitability.

KEY POINTS: Neuroinflammation associated with CNS insults leads to neuronal hyperexcitability, which may culminate in epileptiform discharges. Application of the endotoxin lipopolysaccharide (LPS) to brain tissue initiates a neuroinflammatory cascade, providing an experimental model to study the mechanisms of neuroinflammatory neuronal hyperexcitability. Here we show that LPS application to hippocampal slices markedly enhances the excitability of CA1 pyramidal cells by inhibiting a specific potassium current, the M-current, generated by KV 7/M channels, which controls the excitability of almost every neuron in the CNS. The LPS-induced M-current inhibition is triggered by sequential activation of microglia, astrocytes and pyramidal cells, mediated by metabotropic purinergic and glutamatergic transmission, leading to blockade of KV 7/M channels by calcium released from intracellular stores. The identification of the downstream molecular target of neuroinflammation, namely the KV 7/M channel, potentially has far reaching implications for the understanding and treatment of many acute and chronic brain disorders. ABSTRACT: Acute brain insults and many chronic brain diseases manifest an innate inflammatory response. The hallmark of this response is glia activation, which promotes repair of damaged tissue, but also induces structural and functional changes that may lead to an increase in neuronal excitability. We have investigated the mechanisms involved in the modulation of neuronal activity by acute inflammation. Initiating inflammatory responses in hippocampal tissue rapidly led to neuronal depolarization and repetitive firing even in the absence of active synaptic transmission. This action was mediated by a complex metabotropic purinergic and glutamatergic glia-to-neuron signalling cascade, leading to the blockade of neuronal KV 7/M channels by Ca2+ released from internal stores. These channels generate the low voltage-activating, non-inactivating M-type K+ current (M-current) that controls intrinsic neuronal excitability, and its inhibition was the predominant cause of the inflammation-induced hyperexcitability. Our discovery that the ubiquitous KV 7/M channels are the downstream target of the inflammation-induced cascade, has far reaching implications for the understanding and treatment of many acute and chronic brain disorders.

The role of slow and persistent TTX-resistant sodium currents in acute tumor necrosis factor-α-mediated increase in nociceptors excitability.

Tetrodotoxin-resistant (TTX-r) sodium channels are key players in determining the input-output properties of peripheral nociceptive neurons. Changes in gating kinetics or in expression levels of these channels by proinflammatory mediators are likely to cause the hyperexcitability of nociceptive neurons and pain hypersensitivity observed during inflammation. Proinflammatory mediator, tumor necrosis factor-α (TNF-α), is secreted during inflammation and is associated with the early onset, as well as long-lasting, inflammation-mediated increase in excitability of peripheral nociceptive neurons. Here we studied the underlying mechanisms of the rapid component of TNF-α-mediated nociceptive hyperexcitability and acute pain hypersensitivity. We showed that TNF-α leads to rapid onset, cyclooxygenase-independent pain hypersensitivity in adult rats. Furthermore, TNF-α rapidly and substantially increases nociceptive excitability in vitro, by decreasing action potential threshold, increasing neuronal gain and decreasing accommodation. We extended on previous studies entailing p38 MAPK-dependent increase in TTX-r sodium currents by showing that TNF-α via p38 MAPK leads to increased availability of TTX-r sodium channels by partial relief of voltage dependence of their slow inactivation, thereby contributing to increase in neuronal gain. Moreover, we showed that TNF-α also in a p38 MAPK-dependent manner increases persistent TTX-r current by shifting the voltage dependence of activation to a hyperpolarized direction, thus producing an increase in inward current at functionally critical subthreshold voltages. Our results suggest that rapid modulation of the gating of TTX-r sodium channels plays a major role in the mediated nociceptive hyperexcitability of TNF-α during acute inflammation and may lead to development of effective treatments for inflammatory pain, without modulating the inflammation-induced healing processes.