Excitability Of Sensory Neurons Research Articles

HCN3 ion channels: roles in sensory neuronal excitability and pain.

HCN ion channels conducting the Ih current control the frequency of firing in peripheral sensory neurons signalling pain. Previous studies have demonstrated a major role for the HCN2 subunit in chronic pain but the potential involvement of HCN3 in pain has not been investigated. HCN3 was found to be widely expressed in all classes of sensory neurons (small, medium, large) where it contributes to Ih . HCN3 deletion increased the firing rate of medium but not small, sensory neurons. Pain sensitivity both acutely and following neuropathic injury was largely unaffected by HCN3 deletion, with the exception of a small decrease of mechanical hyperalgesia in response to a pinprick. We conclude that HCN3 plays little role in either acute or chronic pain sensation. HCN ion channels govern the firing rate of action potentials in the pacemaker region of the heart and in pain-sensitive (nociceptive) nerve fibres. Intracellular cAMP promotes activation of the HCN4 and HCN2 isoforms, whereas HCN1 and HCN3 are relatively insensitive to cAMP. HCN2 modulates action potential firing rate in nociceptive neurons and plays a critical role in all modes of inflammatory and neuropathic pain, although the role of HCN3 in nociceptive excitability and pain is less studied. Using antibody staining, we found that HCN3 is expressed in all classes of somatosensory neurons. In small nociceptive neurons, genetic deletion of HCN2 abolished the voltage shift of the Ih current carried by HCN isoforms following cAMP elevation, whereas the voltage shift was retained following deletion of HCN3, consistent with the sensitivity of HCN2 but not HCN3 to cAMP. Deletion of HCN3 had little effect on the evoked firing frequency in small neurons but enhanced the firing of medium-sized neurons, showing that HCN3 makes a significant contribution to the input resistance only in medium-sized neurons. Genetic deletion of HCN3 had no effect on acute thresholds to heat or mechanical stimuli in vivo and did not affect inflammatory pain measured with the formalin test. Nerve-injured HCN3 knockout mice exhibited similar levels of mechanical allodynia and thermal hyperalgesia to wild-type mice but reduced mechanical hyperalgesia in response to a pinprick. These results show that HCN3 makes some contribution to excitability, particularly in medium-sized neurons, although it has no major influence on acute or neuropathic pain processing.

The contributions and mechanisms of changes in excitability during simple forms of learning in Aplysia.

Learning and memory have long been thought to involve changes in synaptic connections between neurons. However, in many cases learning-related plasticity also involves changes in the excitability of neurons. These findings have raised questions about the relative importance of these two types of mechanisms to behavioral learning, and also about the extent to which they involve shared or unique molecular mechanisms. We have taken a reductionist approach to these questions by addressing them in a simple model organism, Aplysia californica. Studies of a semi-intact Aplysia siphon withdrawal preparation suggest that classical conditioning involves an increase in the evoked firing of sensory neurons (SNs) as well as facilitation of the monosynaptic PSP to motor neurons (MNs). Furthermore, these two mechanisms may act cooperatively at the cellular level: increased SN firing produces more PSPs, each of which is facilitated, leading to a multiplicative increase in depolarization of the MN and siphon withdrawal. The changes in SN firing and the monosynaptic PSP also share several mechanisms at the molecular level, suggesting that they may both be due in part to a decrease in K+ current that causes an increase in SN excitability as well as an increase in SN spike width and thus increased transmitter release. However, changes in the monosynaptic PSP also involve additional mechanisms that are not shared and may affect different aspects of synaptic transmission as well. Studies of operant conditioning of feeding suggest that it involves similar mechanisms as classical conditioning of siphon withdrawal. In particular, for both types of associative learning adenylyl cyclase appears to serve as a molecular coincidence detector that leads to increased activation of PKA and changes in excitability of key neurons in the neural circuit. Furthermore, in both cases those changes in excitability make an important contribution to the behavioral learning.

Tetrodotoxin-Sensitive Sodium Channels Mediate Action Potential Firing and Excitability in Menthol-Sensitive Vglut3-Lineage Sensory Neurons.

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3lineage) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3lineage neurons. Whole-cell recordings showed that small-diameter Vglut3lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (NaV) currents in menthol-sensitive Vglut3lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive NaV transcripts between TRPM8-positive and -negative Vglut3lineage neurons; however, NaV1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of NaV subunits, with NaV1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3lineage neurons rely primarily on TTX-resistant NaV channels. Additionally, when NaV1.1 channels were blocked, the remaining NaV current readily entered into slow inactivation in menthol-sensitive Vglut3lineage neurons. Thus, these data demonstrate that TTX-sensitive NaVs drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability.SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of NaV1.1 as a therapeutic target for sensory disorders.

Cholecystokinin type B receptor-mediated inhibition of A-type K+ channels enhances sensory neuronal excitability through the phosphatidylinositol 3-kinase and c-Src-dependent JNK pathway

BackgroundCholecystokinin (CCK) is implicated in the regulation of nociceptive sensitivity of primary afferent neurons. Nevertheless, the underlying cellular and molecular mechanisms remain unknown.MethodsUsing patch clamp recording, western blot analysis, immunofluorescent labelling, enzyme-linked immunosorbent assays, adenovirus-mediated shRNA knockdown and animal behaviour tests, we studied the effects of CCK-8 on the sensory neuronal excitability and peripheral pain sensitivity mediated by A-type K+ channels.ResultsCCK-8 reversibly and concentration-dependently decreased A-type K+ channel (IA) in small-sized dorsal root ganglion (DRG) neurons through the activation of CCK type B receptor (CCK-BR), while the sustained delayed rectifier K+ current was unaffected. The intracellular subunit of CCK-BR coimmunoprecipitated with Gαo. Blocking G-protein signaling with pertussis toxin or by the intracellular application of anti-Gβ antibody reversed the inhibitory effects of CCK-8. Antagonism of phosphatidylinositol 3-kinase (PI3K) but not of its common downstream target Akts abolished the CCK-BR-mediated IA response. CCK-8 application significantly activated JNK mitogen-activated protein kinase. Antagonism of either JNK or c-Src prevented the CCK-BR-mediated IA decrease, whereas c-Src inhibition attenuated the CCK-8-induced p-JNK activation. Application of CCK-8 enhanced the action potential firing rate of DRG neurons and elicited mechanical and thermal pain hypersensitivity in mice. These effects were mediated by CCK-BR and were occluded by IA blockade.ConclusionOur findings indicate that CCK-8 attenuated IA through CCK-BR that is coupled to the Gβγ-dependent PI3K and c-Src-mediated JNK pathways, thereby enhancing the sensory neuronal excitability in DRG neurons and peripheral pain sensitivity in mice.

NaV 1.6 regulates excitability of mechanosensitive sensory neurons.

Voltage-gated sodium channels are critical for peripheral sensory neuron transduction and have been implicated in a number of painful and painless disorders. The β-scorpion toxin, Cn2, is selective for NaV 1.6 in dorsal root ganglion neurons. NaV 1.6 plays an essential role in peripheral sensory neurons, specifically at the distal terminals of mechanosensing fibres innervating the skin and colon. NaV 1.6 activation also leads to enhanced response to mechanical stimulus in vivo. This works highlights the use of toxins in elucidating pain pathways moreover the importance of non-peripherally restricted NaV isoforms in pain generation. Peripheral sensory neurons express multiple voltage-gated sodium channels (NaV ) critical for the initiation and propagation of action potentials and transmission of sensory input. Three pore-forming sodium channel isoforms are primarily expressed in the peripheral nervous system (PNS): NaV 1.7, NaV 1.8 and NaV 1.9. These sodium channels have been implicated in painful and painless channelopathies and there has been intense interest in them as potential therapeutic targets in human pain. Emerging evidence suggests NaV 1.6 channels are an important isoform in pain sensing. This study aimed to assess, using pharmacological approaches, the function of NaV 1.6 channels in peripheral sensory neurons. The potent and NaV 1.6 selective β-scorpion toxin Cn2 was used to assess the effect of NaV 1.6 channel activation in the PNS. The multidisciplinary approach included Ca2+ imaging, whole-cell patch-clamp recordings, skin-nerve and gut-nerve preparations and in vivo behavioural assessment of pain. Cn2 facilitates NaV 1.6 early channel opening, and increased persistent and resurgent currents in large-diameter dorsal root ganglion (DRG) neurons. This promotes enhanced excitatory drive and tonic action potential firing in these neurons. In addition, NaV 1.6 channel activation in the skin and gut leads to increased response to mechanical stimuli. Finally, intra-plantar injection of Cn2 causes mechanical but not thermal allodynia. This study confirms selectivity of Cn2 on NaV 1.6 channels in sensory neurons. Activation of NaV 1.6 channels, in terminals of the skin and viscera, leads to profound changes in neuronal responses to mechanical stimuli. In conclusion, sensory neurons expressing NaV 1.6 are important for the transduction of mechanical information in sensory afferents innervating the skin and viscera.

MiR-143 Regulated Inflammatory Pain by Target Rab1a in Spinal Dorsal Horn

Inflammation induces changes in nociceptive neurons which contribute to both peripheral and central sensitization of pain-sensitive pathways. Plastic changes in sensory neuron excitability are considered the cellular basis of persistent pain. It is well investigated that microRNA can modulate gene expression impact on pain. It has reported miR-143 was downregulated in dorsal root ganglion (DRG) in inflammatory mice. However, less information is available about contribution of miRNA in pain behavior. Here, we demonstrate that miR-143 is also downregulated in spinal dorsal horn. Local increase of miR-143 by using an miR-143 mimic can suppress established inflammatory pain. Furthermore, block miR-143 function was sufficient to cause pain-related behaviors in intact mice. miR-143 targeted the Rab1a gene, and decreased miR-143 associated with pain caused increased Rab1a protein expression, independent of messenger RNA levels. Consistently, miR-143 overexpression in spinal cord suppressed increased Rab1a expression and normalized long-lasting hyperexcitability of nociceptive neurons. These findings demonstrate miR-143 downregulation is causally involved in maintenance of inflammatory pain through regulation of Rab1a and miR-143 supplement offers a novel therapeutic strategy specific for chronic inflammatory pain.

TRESK (K2P18.1) Background Potassium Channel Is Activated by Novel-Type Protein Kinase C via Dephosphorylation.

TRESK (K2P18.1) background K+ channel is a major determinant of the excitability of primary sensory neurons. It has been reported that human TRESK is activated by the protein kinase C (PKC) activator PMA (phorbol 12-myristate 13-acetate) in Xenopus oocytes. In the present study, we investigated the mechanism of this PKC-dependent TRESK regulation. We show that TRESK is activated by coexpression of the novel-type PKC isoforms η and ε The effect of PKC is not mediated by calcineurin phosphatase, which is known to evoke the calcium-dependent TRESK activation. Mutations of the calcineurin-binding sites in the channel (PQAAAS-AQAP) did not influence the PMA-induced increase of potassium current. In sharp contrast, the mutations of the target residue of calcineurin in TRESK, S264A, and S264E prevented the effect of PMA. The enforced phosphorylation of S264 by coexpression of a microtubule-affinity regulating kinase construct (MARK2Δ) also abolished the PKC-dependent TRESK activation. These results suggest that, in addition to calcineurin, PKC regulates TRESK by changing the phosphorylation status of S264. Coexpression of PKC slowed recovery of the K+ current to the resting state after the calcineurin-dependent dephosphorylation of TRESK. Therefore, the likely mechanism of action is the PKC-dependent inhibition of the kinase responsible for the (re)phosphorylation of the channel at S264. The PKC-dependent dephosphorylation of TRESK protein was also detected by the Phos-tag SDS-PAGE method. In summary, the activation of novel-type PKC results in the slow (indirect) dephosphorylation of TRESK at the regulatory residue S264 in a calcineurin-independent manner.

The Role of Voltage-Gated Sodium Channels in Pain Signaling.

Acute pain signaling has a key protective role and is highly evolutionarily conserved. Chronic pain, however, is maladaptive, occurring as a consequence of injury and disease, and is associated with sensitization of the somatosensory nervous system. Primary sensory neurons are involved in both of these processes, and the recent advances in understanding sensory transduction and human genetics are the focus of this review. Voltage-gated sodium channels (VGSCs) are important determinants of sensory neuron excitability: they are essential for the initial transduction of sensory stimuli, the electrogenesis of the action potential, and neurotransmitter release from sensory neuron terminals. Nav1.1, Nav1.6, Nav1.7, Nav1.8, and Nav1.9 are all expressed by adult sensory neurons. The biophysical characteristics of these channels, as well as their unique expression patterns within subtypes of sensory neurons, define their functional role in pain signaling. Changes in the expression of VGSCs, as well as posttranslational modifications, contribute to the sensitization of sensory neurons in chronic pain states. Furthermore, gene variants in Nav1.7, Nav1.8, and Nav1.9 have now been linked to human Mendelian pain disorders and more recently to common pain disorders such as small-fiber neuropathy. Chronic pain affects one in five of the general population. Given the poor efficacy of current analgesics, the selective expression of particular VGSCs in sensory neurons makes these attractive targets for drug discovery. The increasing availability of gene sequencing, combined with structural modeling and electrophysiological analysis of gene variants, also provides the opportunity to better target existing therapies in a personalized manner.

Melanocortin type 4 receptor–mediated inhibition of A-type K+ current enhances sensory neuronal excitability and mechanical pain sensitivity in rats

α-Melanocyte-stimulating hormone (α-MSH) has been shown to be involved in nociception, but the underlying molecular mechanisms remain largely unknown. In this study, we report that α-MSH suppresses the transient outward A-type K+ current (IA) in trigeminal ganglion (TG) neurons and thereby modulates neuronal excitability and peripheral pain sensitivity in rats. Exposing small-diameter TG neurons to α-MSH concentration-dependently decreased IA This α-MSH-induced IA decrease was dependent on the melanocortin type 4 receptor (MC4R) and associated with a hyperpolarizing shift in the voltage dependence of A-type K+ channel inactivation. Chemical inhibition of phosphatidylinositol 3-kinase (PI3K) with wortmannin or of class I PI3Ks with the selective inhibitor CH5132799 prevented the MC4R-mediated IA response. Blocking Gi/o-protein signaling with pertussis toxin or by dialysis of TG neurons with the Gβγ-blocking synthetic peptide QEHA abolished the α-MSH-mediated decrease in IA Further, α-MSH increased the expression levels of phospho-p38 mitogen-activated protein kinase, and pharmacological or genetic inhibition of p38α abrogated the α-MSH-induced IA response. Additionally, α-MSH significantly increased the action potential firing rate of TG neurons and increased the sensitivity of rats to mechanical stimuli applied to the buccal pad area, and both effects were abrogated by IA blockade. Taken together, our findings suggest that α-MSH suppresses IA by activating MC4R, which is coupled sequentially to the Gβγ complex of the Gi/o-protein and downstream class I PI3K-dependent p38α signaling, thereby increasing TG neuronal excitability and mechanical pain sensitivity in rats.

Probing cortical excitability using rapid frequency tagging

Frequency tagging has been widely used to study the role of visual selective attention. Presenting a visual stimulus flickering at a specific frequency generates so-called steady-state visually evoked responses. However, frequency tagging is mostly done at lower frequencies (<30 Hz). This produces a visible flicker, potentially interfering with both perception and neuronal oscillations in the theta, alpha and beta band. To overcome these problems, we used a newly developed projector with a 1440 Hz refresh rate allowing for frequency tagging at higher frequencies. We asked participants to perform a cued spatial attention task in which imperative pictorial stimuli were presented at 63 Hz or 78 Hz while measuring whole-head magnetoencephalography (MEG). We found posterior sensors to show a strong response at the tagged frequency. Importantly, this response was enhanced by spatial attention. Furthermore, we reproduced the typical modulations of alpha band oscillations, i.e., decrease in the alpha power contralateral to the attentional cue. The decrease in alpha power and increase in frequency tagged signal with attention correlated over subjects. We hereby provide proof-of-principle for the use of high-frequency tagging to study sensory processing and neuronal excitability associated with attention.

Inhibition of multiple voltage-gated calcium channels may contribute to spinally mediated analgesia by epipregnanolone in a rat model of surgical paw incision

ABSTRACTVoltage-activated calcium channels play an important role in excitability of sensory nociceptive neurons in acute and chronic pain models. We have previously shown that low-voltage-activated calcium channels, or T-type channels (T-channels), increase excitability of sensory neurons after surgical incision in rats. We have also found that endogenous 5β-reduced neuroactive steroid epipregnanolone [(3β,5β)-3-hydroxypregnan-20-one] blocked isolated T-currents in dorsal root ganglion (DRG) cells in vitro, and reduced nociceptive behavior in vivo, after local intraplantar application into the foot pads of heathy rats and mice. Here, we investigated if epipregnanolone exerts an antinociceptive effect after intrathecal (i.t.) application in healthy rats, as well as an antihyperalgesic effect in a postsurgical pain model. We also studied if this endogenous neurosteroid blocks currents originating from high voltage-activated (HVA) calcium channels in rat sensory neurons. In in vivo studies, we found that epipregnanolone alleviated thermal and mechanical nociception in healthy rats after i.t. administration without affecting their sensory-motor abilities. Furthermore, epipregnanolone effectively reduced mechanical hyperalgesia after i.t application in rats after surgery. In subsequent in vitro studies, we found that epipregnanolone blocked isolated HVA currents in nociceptive sensory neurons with an IC50 of 3.3 μM in a G-protein-dependent fashion. We conclude that neurosteroids that have combined inhibitory effects on T-type and HVA calcium currents may be suitable for development of novel pain therapies during the perioperative period.

Migraine-Associated TRESK Mutations Increase Neuronal Excitability through Alternative Translation Initiation and Inhibition of TREK

It is often unclear why some genetic mutations to a given gene contribute to neurological disorders and others do not. For instance, two mutations have previously been found to produce a dominant negative for TRESK, a two-pore-domain K+ channel implicated in migraine: TRESK-MT, a 2-bp frameshift mutation, and TRESK-C110R. Both mutants inhibit TRESK, but only TRESK-MT increases sensory neuron excitability and is linked to migraine. Here, we identify a new mechanism, termed frameshift mutation-induced alternative translation initiation (fsATI), that may explain why only TRESK-MT is associated with migraine. fsATI leads to the production of a second protein fragment, TRESK-MT2, which co-assembles with and inhibits TREK1 and TREK2, two other two-pore-domain K+ channels, to increase trigeminal sensory neuron excitability, leading to a migraine-like phenotype in rodents. These findings identify TREK1 and TREK2 as potential molecular targets in migraine and suggest that fsATI should be considered as a distinct class of mutations.

Role of nitric oxide in the induction of the behavioral and cellular changes produced by a common aversive stimulus in Aplysia

Although it is well documented that exposure to aversive stimuli induces modulation of neural circuits and subsequent behavioral changes, the means by which an aversive stimulus concomitantly alters behaviors of different natures (e.g., defensive and appetitive) remains unclear. Here, we addressed this issue by using the learning-induced concurrent modulation of defensive and appetitive behaviors that occurs when the mollusk Aplysia is exposed to aversive stimuli. In Aplysia, aversive stimuli concomitantly enhance withdrawal reflexes (i.e., sensitization) and suppress feeding. Sensitization and feeding suppression, which are expressed in the short term and long term, depending on the training protocol, are accompanied by increased excitability of the tail sensory neurons (TSNs) controlling the withdrawal reflexes, and by decreased excitability of feeding decision-making neuron B51, respectively. Serotonin (5-HT) has been shown to mediate sensitization, but not feeding suppression. In this study, we examined which other neurotransmitter might be responsible for feeding suppression and its underlying cellular changes. Our results indicate that nitric oxide (NO) contributes to both short-term and long-term feeding suppression, as well as to the underlying decreased B51 excitability. NO was also necessary for the induction of long-term sensitization and for the expression of short-term increased TSN excitability in vitro, revealing a previously undocumented interaction between 5-HT and NO signaling cascades in sensitization. Overall, these results revealed a scenario in which multiple modulators contribute to the widespread changes induced by sensitizing stimuli in Aplysia.

Resilience to Pain: A Peripheral Component Identified Using Induced Pluripotent Stem Cells and Dynamic Clamp.

Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing NaV1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, KCNQ in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain.SIGNIFICANCE STATEMENT Individual-to-individual differences in pain are well documented, but not well understood. In this study, we show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing and dynamic clamp, we show that it is possible to pinpoint a specific gene variant that modulates pain signaling and contributes to interindividual differences in pain.

Channeling metastasis pain with VEGF

Pain Metastatic cancer in the bone is painful. Yang et al. found that vascular endothelial growth factor (VEGF) promotes tumor angiogenesis and also contributes to this pain. In a rat model of bone-metastatic breast cancer, tumor-secreted VEGF repressed the expression of the potassium (K+) channel TRESK. Loss of K+ current through TRESK increased the excitability of sensory neurons and made the animals hypersensitive to heat and touch near the bone lesion. Blocking this pathway restored channel activity and alleviated pain. Sci. Signal. 11 , eaao5150 (2018).

CC chemokine ligand 2 (CCL2) enhances TTX-sensitive sodium channel activity of primary afferent neurons in the complete Freud adjuvant-induced inflammatory pain model.

CC chemokine ligand 2 (CCL2) has been implicated in pathological pain, but the mechanism underlying the pronociceptive effect of CCL2 is not fully understood. Voltage-gated sodium (Nav) channels are important determinants of the excitability of sensory neurons. Hence we tested the hypothesis that CCL2 contributes to inflammatory pain via modulating Nav channel activity of primary afferent neurons. Chronic inflammatory pain was induced in rats by intraplantar injection of the complete Freud adjuvant (CFA) to one of the hind paws. Control rats received intraplantar injection of equal volume of saline. A significant increase of CCL2 mRNA and CCL2 receptor (CCR2) protein expression was detected in the ipsilateral dorsal root ganglion (DRG) in CFA-treated rats. Intraplantar injection of CCL2 protein in the control rats had minimal effect on the paw withdrawal threshold (PWT) in response to mechanical stimulation. However, in CFA-treated rats, intraplantar CCL2 led to an increase in pain responses. Patch-clamp recording of acutely dissociated DRG neurons revealed that CCL2 had minimum effect on the excitability of sensory neurons from control rats. However, CCL2 directly depolarized a large proportion of small to medium-sized sensory neurons from CFA-treated rats. In addition, CCL2 was found to enhance whole-cell TTX-sensitive sodium currents without significantly affecting the TTX-resistant sodium currents and the potassium currents. These results are in agreement with previous reports concerning the involvement of CCL2-CCR2 signaling in inflammatory hyperalgesia and further indicate that enhanced TTX-sensitive channel activity may partly underlie the pronociceptive effects of CCL2.

Anionic Phospholipids Bind to and Modulate the Activity of Human TRESK Background K+ Channel.

The background K+ channel TRESK regulates sensory neuron excitability, and changes in its function/expression contribute to neuronal hyperexcitability after injury/inflammation, making it an attractive therapeutic target for pain-related disorders. Factors that change lipid bilayer composition/properties (including volatile anesthetics, chloroform, chlorpromazine, shear stress, and cell swelling/shrinkage) modify TRESK current, but despite the importance of anionic phospholipids (e.g., PIP2) in the regulation of many ion channels, it remains unknown if membrane lipids affect TRESK function. We describe that both human and rat TRESK contain potential anionic phospholipid binding sites (apbs) in the large cytoplasmic loop, but only the human channel is able to bind to multilamellar vesicles (MLVs), enriched with anionic phospholipids, suggesting an electrostatically mediated interaction. We mapped the apbs to a short stretch of 14 amino acids in the loop, located at the membrane-cytosol interface. Disruption of electrostatic lipid-TRESK interactions inhibited hTRESK currents, while subsequent application of Folch Fraction MLVs or a PIP2 analog activated hTRESK, an effect that was absent in the rat ortholog. Strikingly, channel activation by anionic phospholipids was conferred to rTRESK by replacing the equivalent rat sequence with the human apbs. Finally, in the presence of a calcineurin inhibitor, stimulation of a Gq/11-linked GPCR reduced hTRESK current, revealing a likely inhibitory effect of membrane lipid hydrolysis on hTRESK activity. This novel regulation of hTRESK by anionic phospholipids is a characteristic of the human channel that is not present in rodent orthologs. This must be considered when extrapolating results from animal models and may open the door to the development of novel channel modulators as analgesics.

Network-Based Drug Discovery: Coupling Network Pharmacology with Phenotypic Screening for Neuronal Excitability

Diseases such as chronic pain with complex etiologies are unlikely to respond to single, target-specific therapeutics but rather require intervention at multiple points within a perturbed disease system. Such approaches are being enabled by the rise of computational methods to identify key points of intervention and by new screening techniques that focus on a relevant condition or phenotype, rather than a specific target. Here we apply an in silico network pharmacology approach to identify small-molecule compounds with the potential to selectively disrupt the structure of a chronic-pain specific disease network, which we validate using a novel phenotypic screen that recapitulates key aspects of neuronal and pain biology by measuring changes in neuronal excitability in native sensory neurons. The combination of network pharmacology with a phenotypic screen is a powerful approach; we show that hit rates increase from 26% to 42%. This represents a rational approach to the discovery of compounds with a poly-pharmacology based therapeutic value, which will be vital for the discovery of treatments for complex disease.

Whole-transcriptome changes in gene expression accompany aging of sensory neurons in Aplysia californica

BackgroundLarge-scale molecular changes occur during aging and have many downstream consequences on whole-organism function, such as motor function, learning, and memory. The marine mollusk Aplysia californica can be used to study transcriptional changes that occur with age in identified neurons of the brain, because its simplified nervous system allows for more direct correlations between molecular changes, physiological changes, and their phenotypic outcomes. Behavioral deficits in the tail-withdrawal reflex of aged animals have been correlated with reduced excitation in sensory neurons that control the reflex. RNASeq was used to investigate whole-transcriptome changes in tail-withdrawal sensory neurons of sexually mature and aged Aplysia to correlate transcriptional changes with reduced behavioral and physiological responses.ResultsPaired-end sequencing resulted in 210 million reads used for differential expression analysis. Aging significantly altered expression of 1202 transcripts in sensory neurons underlying the tail-withdrawal reflex, with an approximately equal number of these genes up- and down regulated with age. Despite overall bidirectionality of expression changes, > 80% of ion channel genes that were differentially expressed had decreased expression with age. In particular, several voltage-gated K+ and Ca2+ channels were down regulated. This marked decrease in ion channel expression may play an important role in previously observed declines in aged sensory neuron excitability. We also observed decreased expression of genes and pathways involved in learning and memory. Genes involved in the stress response showed increased expression in aged Aplysia neurons.ConclusionsSignificantly altered expression of many genes between sexually mature and aged Aplysia suggests large molecular changes that may impact neuronal function. Decreased ion channel mRNA observed could mean fewer receptors present in aged neurons, resulting in reduced excitability of PVC sensory neurons, ultimately leading to reduced tail-withdrawal reflex observed in aged Aplysia. Significant changes in other genes and pathways, such as stress response and learning and memory, have previously been shown to occur with age in many vertebrate organisms. This suggests that some effects of aging are common across many animal phyla.

Epigenetic Modifications Associated to Neuroinflammation and Neuropathic Pain After Neural Trauma.

Accumulating evidence suggests that epigenetic alterations lie behind the induction and maintenance of neuropathic pain. Neuropathic pain is usually a chronic condition caused by a lesion, or pathological change, within the nervous system. Neuropathic pain appears frequently after nerve and spinal cord injuries or diseases, producing a debilitation of the patient and a decrease of the quality of life. At the cellular level, neuropathic pain is the result of neuronal plasticity shaped by an increase in the sensitivity and excitability of sensory neurons of the central and peripheral nervous system. One of the mechanisms thought to contribute to hyperexcitability and therefore to the ontogeny of neuropathic pain is the altered expression, trafficking, and functioning of receptors and ion channels expressed by primary sensory neurons. Besides, neuronal and glial cells, such as microglia and astrocytes, together with blood borne macrophages, play a critical role in the induction and maintenance of neuropathic pain by releasing powerful neuromodulators such as pro-inflammatory cytokines and chemokines, which enhance neuronal excitability. Altered gene expression of neuronal receptors, ion channels, and pro-inflammatory cytokines and chemokines, have been associated to epigenetic adaptations of the injured tissue. Within this review, we discuss the involvement of these epigenetic changes, including histone modifications, DNA methylation, non-coding RNAs, and alteration of chromatin modifiers, that have been shown to trigger modification of nociception after neural lesions. In particular, the function on these processes of EZH2, JMJD3, MeCP2, several histone deacetylases (HDACs) and histone acetyl transferases (HATs), G9a, DNMT, REST and diverse non-coding RNAs, are described. Despite the effort on developing new therapies, current treatments have only produced limited relief of this pain in a portion of patients. Thus, the present review aims to contribute to find novel targets for chronic neuropathic pain treatment.