Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium c... more Selective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N-ethyl bromide (QX-314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX-314. We investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch-clamp recordings, and cellular QX-314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed. Of the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red-sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX-314 permeated through TRPA1 channels and accumulated intracellularly after activation of these channels. Upon sciatic injections, QX-314 markedly prolonged bupivacaine's nociceptive block and also extended (to a lesser degree) its motor block. Bupivacaine's blockade of C-, but not A-fibre, CAPs in sciatic nerves was extended by co-application of QX-314. Surprisingly, however, this action was the same in wild-type, TRPA1-knockout and TRPV1/TRPA1-double knockout mice, suggesting a TRP-channel independent entry pathway. Consistent with this, high doses of bupivacaine promoted a non-selective, cellular uptake of QX-314. Bupivacaine, combined with QX-314, produced a long-lasting sensory nerve block. This did not require QX-314 permeation through TRPA1, although bupivacaine activated these channels. Regardless of entry pathway, the greatly extended duration of block produced by QX-314 and bupivacaine may be clinically useful.
ABSTRACTDevelopment of new non-addictive analgesics requires advanced strategies to differentiate... more ABSTRACTDevelopment of new non-addictive analgesics requires advanced strategies to differentiate human pluripotent stem cells (hPSCs) into relevant cell types amenable for translational research. Here, we developed a highly efficient and reproducible method that differentiates hPSCs into peptidergic and non-peptidergic nociceptors. By modulating specific cell signaling pathways, hPSCs were first converted into SOX10+ neural crest cells, followed by differentiation into sensory neurons with an in vivo-like pseudo-unipolar morphology. Detailed characterization confirmed that the hPSC-derived nociceptors displayed molecular and cellular features comparable to native dorsal root ganglion (DRG) neurons, and expressed high-threshold primary sensory neuron markers, transcription factors, neuropeptides, and over 150 ion channels and receptors, including critical pain-relevant drug targets (e.g., TRPV1, TAC1, CALCA, NAV1.7, NAV1.8). Moreover, after confirming robust functional activities an...
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The hallmark of many bacterial infections is pain. The underlying mechanisms of pain during live ... more The hallmark of many bacterial infections is pain. The underlying mechanisms of pain during live pathogen invasion are not well understood. Here, we elucidate key molecular mechanisms of pain produced during live methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that spontaneous pain is dependent on the virulence determinant agr and bacterial pore-forming toxins (PFTs). The cation channel, TRPV1, mediated heat hyperalgesia as a distinct pain modality. Three classes of PFTs-alpha-hemolysin (Hla), phenol-soluble modulins (PSMs), and the leukocidin HlgAB-directly induced neuronal firing and produced spontaneous pain. From these mechanisms, we hypothesized that pores formed in neurons would allow entry of the membrane-impermeable sodium channel blocker QX-314 into nociceptors to silence pain during infection. QX-314 induced immediate and long-lasting blockade of pain caused by MRSA infection, significantly more than lidocaine or ibuprofen, two widely used clinical an...
Potentially harmful stimuli are detected at the skin by nociceptor sensory neurons that drive rap... more Potentially harmful stimuli are detected at the skin by nociceptor sensory neurons that drive rapid protective withdrawal reflexes and pain. We set out to define, at a millisecond timescale, the relationship between the activity of these sensory neurons and the resultant behavioral output. Brief optogenetic activation of cutaneous nociceptors was found to activate only a single action potential in each fiber. This minimal input was used to determine high-speed behavioral responses in freely behaving mice. The localized stimulus generated widespread dynamic repositioning and alerting sub-second behaviors whose nature and timing depended on the context of the animal and its position, activity, and alertness. Our findings show that the primary response to injurious stimuli is not limited, fixed, or localized, but is dynamic, and that it involves recruitment and gating of multiple circuits distributed throughout the central nervous system at a sub-second timescale to effectively both al...
Pain is elicited by cold, and a major feature of many neuropathic pain states is that normally in... more Pain is elicited by cold, and a major feature of many neuropathic pain states is that normally innocuous cool stimuli begin to produce pain (cold allodynia). To expand our understanding of cold induced pain states we have studied cold pain behaviors over a range of temperatures in several animal models of chronic pain. We demonstrate that a Peltier-cooled cold plate with +/- 1 degrees C sensitivity enables quantitative measurement of a detection withdrawal response to cold stimuli in unrestrained rats. In naïve rats the threshold for eliciting cold pain behavior is 5 degrees C. The withdrawal threshold for cold allodynia is 15 degrees C in both the spared nerve injury and spinal nerve ligation models of neuropathic pain. Cold hyperalgesia is present in the spared nerve injury model animals, manifesting as a reduced latency of withdrawal response threshold at temperatures that elicit cold pain in naïve rats. We also show that following the peripheral inflammation produced by intrapla...
Synaptic plasticity is fundamental to many neurobiological functions, including memory and pain. ... more Synaptic plasticity is fundamental to many neurobiological functions, including memory and pain. Central sensitization refers to the increased synaptic efficacy established in somatosensory neurons in the dorsal horn of the spinal cord following intense peripheral noxious stimuli, tissue injury or nerve damage. This heightened synaptic transmission leads to a reduction in pain threshold, an amplification of pain responses and a spread of pain sensitivity to non-injured areas. In the cortex, LTP -a long-lasting highly localized increase in synaptic strength -is a synaptic substrate for memory and learning. Analysis of the molecular mechanisms underlying the generation and maintenance of central sensitization and LTP indicates that, although there are differences between the synaptic plasticity contributing to memory and pain, there are also striking similarities.
In naive animals, there is a distinct laminar termination of primary afferent neurones in the spi... more In naive animals, there is a distinct laminar termination of primary afferent neurones in the spinal dorsal horn, such that lamina II (substantia gelatinosa; SG) receives predominantly high-threshold inputs from thinly myelinated Ad and unmyelinated C primary afferent fibres . Consequently, electrophysiological recordings from SG neurones in adult spinal cord slices reveal a characteristic distribution of EPSCs in response to dorsal root stimulation . Ab fibre-evoked polysynaptic EPSCs are observed in only a very small proportion of SG neurones, while, mono-and/or polysynaptic Ad fibremediated EPSCs are detectable in the vast majority ). Following complete peripheral nerve injury, Ab fibres sprout from their normal termination site in the deep dorsal horn into the SG . Though some controversy exists regarding the extent and pattern of such central sprouting ), novel mono-(Kohama et al. 2000) or polysynaptic ) Ab fibre-mediated EPSCs begin to be observed in SG neurones following a sciatic nerve transection. Anatomical changes compatible with A fibre sprouting have also been reported following chronic constriction injury (CCI) of the sciatic nerve , a partial nerve injury model where some intact peripheral innervation and input to the dorsal horn remains. In addition to central sprouting, peripheral nerve injury is associated with atrophic changes in central afferent terminals , which might alter excitatory synaptic transmission. Changes also occur in intrinsic dorsal horn neurones, for example, partial (spared nerve injury (SNI) and CCI), but not complete, sciatic nerve injury results in a selective loss of GABA A -
Bradykinin, an inflammatory mediator, sensitizes nociceptor peripheral terminals reducing pain th... more Bradykinin, an inflammatory mediator, sensitizes nociceptor peripheral terminals reducing pain threshold. We now show that the B2kinin receptor is expressed in rat dorsal horn neurons and that bradykinin, a B2-specific agonist, augments AMPA- and NMDA-induced, and primary afferent-evoked EPSCs, and increases the frequency and amplitude of miniature EPSCs in superficial dorsal horn neuronsin vitro. Administration of bradykinin to the spinal cordin vivoproduces, moreover, an NMDA-dependent hyperalgesia. We also demonstrate that nociceptive inputs result in the production of bradykinin in the spinal cord and that an intrathecal B2-selective antagonist suppresses behavioral manifestations of central sensitization, an activity-dependent increase in glutamatergic synaptic efficacy. Primary afferent-evoked central sensitization is, in addition, reduced in B2receptor knock-out mice. We conclude that bradykinin is released in the spinal cord in response to nociceptor inputs and acts as a syn...
Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidon... more Epoxyeicosatrienoic acids (EETs) are cytochrome P450-epoxygenase-derived metabolites of arachidonic acid that act as endogenous signaling molecules in multiple biological systems. Here we have investigated the specific contribution of 5,6-EET to transient receptor potential (TRP) channel activation in nociceptor neurons and its consequence for nociceptive processing. We found that, during capsaicin-induced nociception, 5,6-EET levels increased in dorsal root ganglia (DRGs) and the dorsal spinal cord, and 5,6-EET is released from activated sensory neuronsin vitro. 5,6-EET potently induced a calcium flux (100 nm) in cultured DRG neurons that was completely abolished when TRPA1 was deleted or inhibited. In spinal cord slices, 5,6-EET dose dependently enhanced the frequency, but not the amplitude, of spontaneous EPSCs (sEPSCs) in lamina II neurons that also responded to mustard oil (allyl isothiocyanate), indicating a presynaptic action. Furthermore, 5,6-EET-induced enhancement of sEPSC...
TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted t... more TRPA1 is a nonselective cation channel expressed by nociceptors. Although it is widely accepted that TRPA1 serves as a broad irritancy receptor for a variety of reactive chemicals, its role in cold sensation remains controversial. Here, we demonstrate that mild cooling markedly increases agonist-evoked rat TRPA1 currents. In the absence of an agonist, even noxious cold only increases current amplitude slightly. These results suggest that TRPA1 is a key mediator of cold hypersensitivity in pathological conditions in which reactive oxygen species and proinflammatory activators of the channel are present, but likely plays a comparatively minor role in acute cold sensation. Supporting this, cold hypersensitivity can be induced in wild-type but notTrpa1−/−mice by subcutaneous administration of a TRPA1 agonist. Furthermore, the selective TRPA1 antagonist HC-030031 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide] reduces cold hypersensitivity in ...
Proceedings of the National Academy of Sciences, 1999
Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain refle... more Inflammatory pain manifests as spontaneous pain and pain hypersensitivity. Spontaneous pain reflects direct activation of specific receptors on nociceptor terminals by inflammatory mediators. Pain hypersensitivity is the consequence of early posttranslational changes, both in the peripheral terminals of the nociceptor and in dorsal horn neurons, as well as later transcription-dependent changes in effector genes, again in primary sensory and dorsal horn neurons. This inflammatory neuroplasticity is the consequence of a combination of activity-dependent changes in the neurons and specific signal molecules initiating particular signal-transduction pathways. These pathways phosphorylate membrane proteins, changing their function, and activate transcription factors, altering gene expression. Two distinct aspects of sensory neuron function are changed as a result of these processes, basal sensitivity, or the capacity of peripheral stimuli to evoke pain, and stimulus-evoked hypersensitivit...
We tested whether it is possible to selectively block pain signals in the orofacial area by deliv... more We tested whether it is possible to selectively block pain signals in the orofacial area by delivering the permanently charged lidocaine derivative QX-314 into nociceptors via TPRV1 channels. We examined the effects of co-applied QX-314 and capsaicin on nociceptive, proprioceptive, and motor function in the rat trigeminal system. QX-314 alone failed to block voltage-gated sodium channel currents (I Na ) and action potentials (APs) in trigeminal ganglion (TG) neurons. However, co-application of QX-314 and capsaicin blocked I Na and APs in TRPV1-positive TG and dental nociceptive neurons, but not in TRPV1-negative TG neurons or in small neurons from TRPV1 knock-out mice. Immunohistochemistry revealed that TRPV1 is not expressed by trigeminal motor and trigeminal mesencephalic neurons. Capsaicin had no effect on rat trigeminal motor and proprioceptive mesencephalic neurons and therefore should not allow QX-314 to enter these cells. Co-application of QX-314 and capsaicin inhibited the jaw-opening reflex evoked by noxious electrical stimulation of the tooth pulp when applied to a sensory but not a motor nerve, and produced long-lasting analgesia in the orofacial area. These data show that selective block of pain signals can be achieved by co-application of QX-314 with TRPV1 agonists. This approach has potential utility in the trigeminal system for treating dental and facial pain.
The peripheral terminals of primary sensory neurons detect histamine and non-histamine itchprovok... more The peripheral terminals of primary sensory neurons detect histamine and non-histamine itchprovoking 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
TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by... more TRPA1, a member of the transient receptor potential (TRP) family of ion channels, is expressed by dorsal root ganglion neurons and by cells of the inner ear, where it has proposed roles in sensing sound, painful cold, and irritating chemicals. To test the in vivo roles of TRPA1, we generated a mouse in which the essential exons required for proper function of the Trpa1 gene were deleted. Knockout mice display behavioral deficits in response to mustard oil, to cold ( approximately 0 degrees C), and to punctate mechanical stimuli. These mice have a normal startle reflex to loud noise, a normal sense of balance, a normal auditory brainstem response, and normal transduction currents in vestibular hair cells. TRPA1 is apparently not essential for hair-cell transduction but contributes to the transduction of mechanical, cold, and chemical stimuli in nociceptor sensory neurons.
Background and PurposeSelective nociceptor fibre block is achieved by introducing the cell membra... more Background and PurposeSelective nociceptor fibre block is achieved by introducing the cell membrane impermeant sodium channel blocker lidocaine N‐ethyl bromide (QX‐314) through transient receptor potential V1 (TRPV1) channels into nociceptors. We screened local anaesthetics for their capacity to activate TRP channels, and characterized the nerve block obtained by combination with QX‐314.Experimental ApproachWe investigated TRP channel activation in dorsal root ganglion (DRG) neurons by calcium imaging and patch‐clamp recordings, and cellular QX‐314 uptake by MS. To characterize nerve block, compound action potential (CAP) recordings from isolated nerves and behavioural responses were analysed.Key ResultsOf the 12 compounds tested, bupivacaine was the most potent activator of ruthenium red‐sensitive calcium entry in DRG neurons and activated heterologously expressed TRPA1 channels. QX‐314 permeated through TRPA1 channels and accumulated intracellularly after activation of these chann...
Molecular mechanisms underlying C-fiber stimulation-induced ERK (extracellular signal-regulated k... more Molecular mechanisms underlying C-fiber stimulation-induced ERK (extracellular signal-regulated kinase) activation in dorsal horn neurons and its contribution to central sensitization have been investigated. In adult rat spinal slice preparations, activation of C-fiber primary afferents by a brief exposure of capsaicin produces an eightfold to 10-fold increase in ERK phosphorylation (pERK) in superficial dorsal horn neurons. The pERK induction is reduced by blockade of NMDA, AMPA/kainate, group I metabotropic glutamate receptor, neurokinin-1, and tyrosine receptor kinase receptors. The ERK activation produced by capsaicin is totally suppressed by inhibition of either protein kinase A (PKA) or PKC. PKA or PKC activators either alone or more effectively together induce pERK in superficial dorsal horn neurons. Inhibition of calcium calmodulin-dependent kinase (CaMK) has no effect, but pERK is reduced by inhibition of the tyrosine kinase Src. The induction of cAMP response element bindi...
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