Modulation of Thermoreceptor TRPM8 by Cooling Compounds

ThermoTRPs, a subset of the Transient Receptor Potential (TRP) family of cation channels, have been implicated in sensing temperature. TRPM8 and TRPA1 are both activated by cooling. TRPM8 is activated by innocuous cooling (<30 °C) and contributes to sensing unpleasant cold stimuli or mediating the effects of cold analgesia and is a receptor for menthol and icilin (mint-derived and synthetic cooling compounds, respectively). TRPA1 (Ankyrin family) is activated by noxious cold (<17 °C), icilin, and a variety of pungent compounds. Extensive amount of medicinal chemistry efforts have been published mainly in the form of patent literature on various classes of cooling compounds by various pharmaceutical companies; however, no prior comprehensive review has been published. When expressed in heterologous expression systems, such as Xenopus oocytes or mammalian cell lines, TRPM8 mediated currents are activated by a number of cooling compounds in addition to menthol and icilin. These include synthetic p-menthane carboxamides along with other class of compounds such as aliphatic/alicyclic alcohols/esters/amides, sulphones/sulphoxides/sulphonamides, heterocyclics, keto-enamines/lactams, and phosphine oxides. In the present review, the medicinal chemistry of various cooling compounds as activators of thermoTRPM8 channel will be discussed according to their chemical classes. The potential of these compounds to emerge as therapeutic agents is also discussed.     

Modulation of Na<Subscript>v</Subscript>1.8 by Lysophosphatidic Acid in the Induction of Bone Cancer Pain

Given that lysophosphatidic acid (LPA) and the tetrodotoxin-resistant sodium channel Na_v1.8 are both involved in bone cancer pain, the present study was designed to investigate whether crosstalk between the LPA receptor LPA₁ (also known as EDG2) and Na_v1.8 in the dorsal root ganglion (DRG) contributes to the induction of bone cancer pain. We showed that the EDG2 antagonist Ki16198 blocked the mechanical allodynia induced by intrathecal LPA in naïve rats and attenuated mechanical allodynia in a rat model of bone cancer. EDG2 and Na_v1.8 expression in L_4–6 DRGs was upregulated following intrathecal or hindpaw injection of LPA. EDG2 and Na_v1.8 expression in ipsilateral L_4–6 DRGs increased with the development of bone cancer. Furthermore, we showed that EDG2 co-localized with Na_v1.8 and LPA remarkably enhanced Na_v1.8 currents in DRG neurons, and this was blocked by either a protein kinase C (PKC) inhibitor or a PKCε inhibitor. Overall, we demonstrated the modulation of Na_v1.8 by LPA in DRG neurons, and that this probably underlies the peripheral mechanism by which bone cancer pain is induced.     

Connexin 36 Mediates Orofacial Pain Hypersensitivity Through GluK2 and TRPA1

Trigeminal neuralgia is a debilitating condition, and the pain easily spreads to other parts of the face. Here, we established a mouse model of partial transection of the infraorbital nerve (pT-ION) and found that the Connexin 36 (Cx36) inhibitor mefloquine caused greater alleviation of pT-ION-induced cold allodynia compared to the reduction of mechanical allodynia. Mefloquine reversed the pT-ION-induced upregulation of Cx36, glutamate receptor ionotropic kainate 2 (GluK2), transient receptor potential ankyrin 1 (TRPA1), and phosphorylated extracellular signal regulated kinase (p-ERK) in the trigeminal ganglion. Cold allodynia but not mechanical allodynia induced by pT-ION or by virus-mediated overexpression of Cx36 in the trigeminal ganglion was reversed by the GluK2 antagonist NS102, and knocking down Cx36 expression in Nav1.8-expressing nociceptors by injecting virus into the orofacial skin area of Nav1.8-Cre mice attenuated cold allodynia but not mechanical allodynia. In conclusion, we show that Cx36 contributes greatly to the development of orofacial pain hypersensitivity through GluK2, TRPA1, and p-ERK signaling.Electronic supplementary materialThe online version of this article (10.1007/s12264-020-00594-4) contains supplementary material, which is available to authorized users.     

The roles of TRPV1, TRPA1 and TRPM8 channels in chemical and thermal sensitivity of the mouse oral mucosa

Spices in food and beverages and compounds in tobacco smoke interact with sensory irritant receptors of the transient receptor potential (TRP) cation channel family. TRPV1 (vanilloid type 1), TRPA1 (ankyrin 1) and TRPM8 (melastatin 8) not only elicit action potential signaling through trigeminal nerves, eventually evoking pungent or cooling sensations, but by their calcium conductance they also stimulate the release of calcitonin gene‐related peptide (CGRP). This is measured as an index of neuronal activation to elucidate the chemo‐ and thermosensory transduction in the isolated mouse buccal mucosa of wild types and pertinent knockouts. We found that the lipophilic capsaicin, mustard oil and menthol effectively get access to the nerve endings below the multilayered squamous epithelium, while cigarette smoke and its gaseous phase were weakly effective releasing CGRP. The hydrophilic nicotine was ineffective unless applied unprotonated in alkaline (pH9) solution, activating TRPA1 and TRPV1. Also, mustard oil activated both these irritant receptors in millimolar but only TRPA1 in micromolar concentrations; in combination (1 mm ) with heat (45 °C), it showed supraadditive, that is heat sensitizing, effects in TRPV1 and TRPA1 knockouts, suggesting action on an unknown heat‐activated channel and mustard oil receptor. Menthol caused little CGRP release by itself, but in subliminal concentration (2 mm ), it enabled a robust cold response that was absent in TRPM8^?/? but retained in TRPA1^?/? and strongly reduced by TRPM8 inhibitors. In conclusion, all three relevant irritant receptors are functionally expressed in the oral mucosa and play their specific roles in inducing neurogenic inflammation and sensitization to heat and cold.     

MK-801, an NMDA receptor antagonist, in the rostroventromedial medulla attenuates development of neuropathic symptoms in the rat.

Segmental ligation of spinal nerves in the rat induces a long-lasting hyperalgesia and allodynia that mimicks neuropathic conditions in humans. In the present study we attempted to determine whether supraspinal NMDA receptors contribute to the induction of the long-lasting hypersensitivity to noxious and innocuous mechanical stimulation following segmental ligation of spinal nerves in the rat. MK-801, an NMDA receptor antagonist, was microinjected into the rostroventromedial medulla (RVM) 15 min before or 25 min after the ligation of spinal nerves and mechanical hypersensitivity was assessed at various time points following surgery by determining the hindlimb withdrawal threshold to noxious and innocuous mechanical stimulation. A single dose of MK-801 administered prior to nerve ligation into the RVM significantly attenuated the development of mechanical hypersensitivity throughout the 2 week postoperative observation period, whereas corresponding administration of MK-801 immediately after the nerve ligation attenuated the development of mechanical hypersensitivity only during the first postoperative day but not later. The results indicate that NMDA receptors in the RVM are involved in triggering the enhanced sensitivity to mechanical stimulation induced by a nerve injury.     

Antisense knock down of TRPA1, but not TRPM8, alleviates cold hyperalgesia after spinal nerve ligation in rats

Patients with neuropathic pain frequently experience hypersensitivity to cold stimulation. However, the underlying mechanisms of this enhanced sensitivity to cold are not well understood. After partial nerve injury, the transient receptor potential ion channel TRPV1 increases in the intact small dorsal root ganglion (DRG) neurons in several neuropathic pain models. In the present study, we precisely examined the incidence of cold hyperalgesia and the changes of TRPA1 and TRPM8 expression in the L4 and L5 DRG following L5 spinal nerve ligation (SNL), because it is likely that the activation of two distinct populations of TRPA1- and TRPM8-expressing small neurons underlie the sensation of cold. We first confirmed that L5 SNL rats developed cold hyperalgesia for more than 14 days after surgery. In the nearby uninjured L4 DRG, TRPA1 mRNA expression increased in trkA-expressing small-to-medium diameter neurons from the 1st to 14th day after the L5 SNL. This upregulation corresponded well with the development and maintenance of nerve injury-induced cold hyperalgesia of the hind paw. In contrast, there was no change in the expression of the TRPM8 mRNA/protein in the L4 DRG throughout the 2-week time course of the experiment. In the injured L5 DRG, on the other hand, both TRPA1 and TRPM8 expression decreased over 2 weeks after ligation. Furthermore, intrathecal administration of TRPA1, but not TRPM8, antisense oligodeoxynucleotide suppressed the L5 SNL-induced cold hyperalgesia. Our data suggest that increased TRPA1 in uninjured primary afferent neurons may contribute to the exaggerated response to cold observed in the neuropathic pain model.     

Distinct expression of TRPM8, TRPA1, and TRPV1 mRNAs in rat primary afferent neurons with adelta/c-fibers and colocalization with trk receptors

The transient receptor potential (TRP) superfamily of cation channels contains four temperature-sensitive channels, named TRPV1-4, that are activated by heat stimuli from warm to that in the noxious range. Recently, two other members of this superfamily, TRPA1 and TRPM8, have been cloned and characterized as possible candidates for cold transducers in primary afferent neurons. Using in situ hybridization histochemistry and immunohistochemistry, we characterized the precise distribution of TRPA1, TRPM8, and TRPV1 mRNAs in the rat dorsal root ganglion (DRG) and trigeminal ganglion (TG) neurons. In the DRG, TRPM8 mRNA was not expressed in the TRPV1-expressing neuronal population, whereas TRPA1 mRNA was only seen in some neurons in this population. Both A-fiber and C-fiber neurons expressed TRPM8, whereas TRPV1 was almost exclusively seen in C-fiber neurons. All TRPM8-expressing neurons also expressed TrkA, whereas the expression of TRPV1 and TRPA1 was independent of TrkA expression. None of these three TRP channels were coexpressed with TrkB or TrkC. The TRPM8-expressing neurons were more abundant in the TG compared with the DRG, especially in the mandibular nerve region innervating the tongue. Our data suggest heterogeneity of TRPM8 and TRPA1 expression by subpopulations of primary afferent neurons, which may result in the difference of cold-sensitive primary afferent neurons in sensitivity to chemicals such as menthol and capsaicin and nerve growth factor.     

The Cav2.3 calcium channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain

Neuropathic pain is a difficult state to treat, characterized by alterations in sensory processing that can include allodynia (touch-evoked pain). Evidence exists for nerve damage-induced plasticity in both transmission and modulatory systems, including changes in voltage-dependent calcium channel (VDCC) expression and function; however, the role of Ca(v)2.3 calcium channels has not clearly been defined. Here, the effects of SNX-482, a selective Ca(v)2.3 antagonist, on sensory transmission at the spinal cord level have been investigated in the rat. The spinal nerve ligation (SNL) model of chronic neuropathic pain [Kim & Chung, (1992)Pain, 50, 355-363] was used to induce mechanical allodynia, as tested on the ipsilateral hindpaw. In vivo electrophysiological measurements of dorsal horn neuronal responses to innocuous and noxious electrical and natural stimuli were made after SNL and compared to sham-operated animals. Spinal SNX-482 (0.5-4 microg/50 microL) exerted dose-related inhibitions of noxious C-fibre- and Adelta-fibre-mediated neuronal responses in conditions of neuropathy, but not in sham-operated animals. Measures of spinal cord hyperexcitability and nociception were most susceptible to SNX-482. In contrast, non-noxious Abeta-mediated responses were not affected by SNX-482. Moreover, responses to innocuous mechanical and also thermal stimuli were more sensitive to SNX-482 in SNL than control animals. This study is the first to demonstrate an antinociceptive role for SNX-482-sensitive channels in dorsal horn neurons during neuropathy. These data are consistent with plasticity in Ca(V)2.3 calcium channel expression and suggest a potential selective target to reduce nociceptive transmission during conditions of nerve damage.     

A novel type of cold-sensitive neuron in rat dorsal root ganglia with rapid adaptation to cooling stimuli

Cold sensing in mammals is heterogeneous and more than one type of receptor molecule is likely to be involved in the transduction process. Most features of innocuous cold receptors have been explained by TRPM8, the cold and menthol receptor, but their fast adaptation to cooling has not yet been reproduced in cellular systems. In this study we have used a newly developed system for applying fast thermal stimuli to dissociated dorsal root ganglia (DRG) neurons from young rats (150-200 g) in primary culture. We describe a novel type of cold-sensitive rat DRG neuron with rapid adaptation to cooling. These cells (4.3% of the total DRG population) do not express either TRPM8 or the other cold-activated TRP channel, TRPA1, and the epithelial sodium channel (ENaC) is not involved in their transduction. Increases in intracellular calcium induced by cooling in rapidly adapting neurons are caused by calcium entry. These neurons express a large and rapidly adapting cold-induced inward current with a time constant of adaptation in the seconds range, and may correspond to the rapidly adapting cold receptors described in vivo.     

Roles of Cav3.2 and TRPA1 channels targeted by hydrogen sulfide in pancreatic nociceptive processing in mice with or without acute pancreatitis

Hydrogen sulfide (H₂S), formed by multiple enzymes, including cystathionine‐γ‐lyase (CSE), targets Ca_v3.2 T‐type Ca²⁺ channels (T channels) and transient receptor potential ankyrin‐1 (TRPA1), facilitating somatic pain. Pancreatitis‐related pain also appears to involve activation of T channels by H₂S formed by the upregulated CSE. Therefore, this study investigates the roles of the Ca_v3.2 isoform and/or TRPA1 in pancreatic nociception in the absence and presence of pancreatitis. In anesthetized mice, AP18, a TRPA1 inhibitor, abolished the Fos expression in the spinal dorsal horn caused by injection of a TRPA1 agonist into the pancreatic duct. As did mibefradil, a T‐channel inhibitor, in our previous report, AP18 prevented the Fos expression following ductal NaHS, an H₂S donor. In the mice with cerulein‐induced acute pancreatitis, the referred hyperalgesia was suppressed by NNC 55‐0396 (NNC), a selective T‐channel inhibitor; zinc chloride; or ascorbic acid, known to inhibit Ca_v3.2 selectively among three T‐channel isoforms; and knockdown of Ca_v3.2. In contrast, AP18 and knockdown of TRPA1 had no significant effect on the cerulein‐induced referred hyperalgesia, although they significantly potentiated the antihyperalgesic effect of NNC at a subeffective dose. TRPA1 but not Ca_v3.2 in the dorsal root ganglia was downregulated at a protein level in mice with cerulein‐induced pancreatitis. The data indicate that TRPA1 and Ca_v3.2 mediate the exogenous H₂S‐induced pancreatic nociception in naïve mice and suggest that, in the mice with pancreatitis, Ca_v3.2 targeted by H₂S primarily participates in the pancreatic pain, whereas TRPA1 is downregulated and plays a secondary role in pancreatic nociceptive signaling. © 2014 Wiley Periodicals, Inc.     

Glycolytic metabolite methylglyoxal inhibits cold and menthol activation of the transient receptor potential melastatin type 8 channel

Methylglyoxal (MG) is a reactive dicarbonyl compound involved in protein modifications linked to diabetes mellitus. The plasma level of MG is elevated in diabetic patients, particularly those with painful diabetic neuropathy. Diabetic neuropathy is often associated with spontaneous pain and altered thermal perception. This study assesses effects of MG on TRPM8, an ion channel involved in innocuous cold sensing and cold allodynia and also in cold‐mediated analgesia. Acute treatment with MG inhibited the activation of recombinant rat and human transient receptor potential melastatin type 8 (TRPM8) by cold and chemical agonists. A similar effect was observed when native TRPM8 was investigated in cultured rat dorsal root ganglion (DRG) neurons. DRG neurons treated with MG for 16–24 hr displayed a significant reduction in the fraction of cold‐ and menthol‐sensitive neurons, most likely expressing TRPM8. The fraction of allyl isothiocyanate‐sensitive neurons was also reduced, and the coexpression among different neuronal populations was affected. The same prolonged exposure to MG significantly reduced the expression of TRPM8 at the mRNA level. Overall, our data provide evidence for decreased activity and expression level of TRPM8 in the presence of MG, which may be linked to some of the alterations in pain and temperature sensing reported by diabetic patients. © 2015 Wiley Periodicals, Inc.     

Contribution of the activation of satellite glia in sensory ganglia to pathological pain

Peripheral tissue injury/inflammation can alter the properties of somatic sensory pathways, resulting in behavioral hypersensitivity and pathological and/or chronic pain, including increased responses to pain caused by both noxious stimuli (hyperalgesia) and normally innocuous stimuli (allodynia). Although there are increasing reports that glia in the spinal cord contribute to the maintenance of pathological pain, recent evidence suggests that activation of satellite glia in sensory ganglia may also play an important role in the development of hyperalgesia and allodynia. There is evidence that non-synaptically released chemical mediators derived from both neurons and satellite glia may trigger chronic pain via autocrine and/or paracrine mechanisms and that augmented excitability of primary afferent neurons results in changes in central pain-signaling neurons (central sensitization). The focus of the present review is on the contribution of the activation of satellite glia in sensory ganglia to pathological pain. In addition, we discuss potential therapeutic targets in satellite glia–neuronal interactions for the prevention of pathological pain.     

Peripheral noxious stimulation induces phosphorylation of the NMDA receptor NR1 subunit at the PKC-dependent site, serine-896, in spinal cord dorsal horn neurons

The N‐methyl‐D ‐aspartate receptor (NMDAR) contributes to central sensitization in the spinal cord and the generation of pain hypersensitivity. NMDAR function is modulated by post‐translational modifications including phosphorylation, and this is proposed to underlie its involvement in the production of pain hypersensitivity in the spinal cord. We now show that a noxious heat stimulus applied to the rat hindpaw induces phosphorylation of the NMDAR NR1 subunit at a protein kinase C (PKC)‐dependent site, serine‐896, in superficial dorsal horn neurons. Phosphorylation of NR1 serine‐896 is essentially absent in the superficial dorsal horn laminae of naïve rats, but there is rapid (< 2 min) induction following a noxious but not innocuous heat stimulus. The number of pNR1‐immunoreactive neuronal profiles in the superficial dorsal horn peaks 30 min after noxious heat stimulation and persists for up to 1 h. pNR1serine896 induction occurs in the endoplasmic reticulum, suggesting that it contributes to trafficking of the receptor from intracellular stores to the membrane. The phosphorylation of the subunit is attenuated by intrathecal injection of the NMDAR antagonist, MK801, suggesting that the NMDAR is involved via a feed‐forward mechanism in its own phosphorylation. The pNR1serine896‐positive neurons are highly co‐localized with PKCdelta and only rarely with PKCgamma. These data provide evidence for an activity‐dependent NMDAR phosphorylation at the PKC‐dependent site, serine‐896, in spinal cord dorsal horn neurons initiated by peripheral noxious stimuli.     

Melittin,the Major Pain-Producing Substance of Bee Venom

Melittin is a basic 26-amino-acid polypeptide that constitutes 40–60% of dry honeybee(Apis mellifera)venom.Although much is known about its strong surface activity on lipid membranes,less is known about its painproducing effects in the nervous system.In this review,we provide lines of accumulating evidence to support the hypothesis that melittin is the major pain-producing substance of bee venom.At the psychophysical and behavioral levels,subcutaneous injection of melittin causes tonic pain sensation and pain-related behaviors in both humans and animals.At the cellular level,melittin activates primary nociceptor cells through direct and indirect effects.On one hand,melittin can selectively open thermal nociceptor transient receptor potential vanilloid receptor channels via phospholipase A2-lipoxygenase/cyclooxygenase metabolites,leading to depolarization of primary nociceptor cells.On the other hand,algogens and inflammatory/proinflammatory mediators released from the tissue matrix by melittin's pore-forming effects can activate primary nociceptor cells through both ligand-gated receptor channels and the G-protein-coupled receptor-mediated opening of transient receptor potential canonical channels.Moreover,subcutaneous melittin up-regulates Nav1.8 and Nav1.9subunits,resulting in the enhancement of tetrodotoxinresistant Na~+currents and the generation of long-term action potential firing.These nociceptive responses in the periphery finally activate and sensitize the spinal dorsal horn pain-signaling neurons,resulting in spontaneous nociceptive paw flinches and pain hypersensitivity to thermal and mechanical stimuli.Taken together,it is concluded that melittin is the major pain-producing substance of bee venom,by which peripheral persistent pain and hyperalgesia(or allodynia),primary nociceptive neuronal sensitization,and CNS synaptic plasticity(or metaplasticity) can be readily induced and the molecular and cellular mechanisms underlying naturally-occurring venomous biotoxins can be experimentally unraveled.     

Activation of medullary dorsal horn γ isoform of protein kinase C interneurons is essential to the development of both static and dynamic facial mechanical allodynia

The γ isoform of protein kinase C (PKCγ), which is concentrated in a specific class of interneurons within inner lamina II (II_i) of the spinal dorsal horn and medullary dorsal horn (MDH), is known to be involved in the development of mechanical allodynia, a widespread and intractable symptom of inflammatory or neuropathic pain. However, although genetic and pharmacological impairment of PKCγ were shown to prevent mechanical allodynia in animal models of pain, after nerve injury or reduced inhibition, the functional consequences of PKCγ activation alone on mechanical sensitivity are still unknown. Using behavioural and anatomical approaches in the rat MDH, we tested whether PKCγ activation in naive animals is sufficient for the establishment of mechanical allodynia. Intracisternal injection of the phorbol ester, 12,13‐dibutyrate concomitantly induced static as well as dynamic facial mechanical allodynia. Monitoring neuronal activity within the MDH with phospho‐extracellular signal‐regulated kinases 1 and 2 immunoreactivity revealed that activation of both lamina I–outer lamina II and II_i–outer lamina III neurons, including lamina II_i PKCγ‐expressing interneurons, was associated with the manifestation of mechanical allodynia. Phorbol ester, 12,13‐dibutyrate‐induced mechanical allodynia and associated neuronal activations were all prevented by inhibiting selectively segmental PKCγ with KIG31‐1. Our findings suggest that PKCγ activation, without any other experimental manipulation, is sufficient for the development of static and dynamic mechanical allodynia. Lamina II_i PKCγ interneurons have been shown to be directly activated by low‐threshold mechanical inputs carried by myelinated afferents. Thus, the level of PKCγ activation within PKCγ interneurons might gate the transmission of innocuous mechanical inputs to lamina I, nociceptive output neurons, thus turning touch into pain.     

The cannabinoid WIN 55,212‐2‐mediated protection of dentate gyrus granule cells is driven by CB1 receptors and modulated by TRPA1 and Cav2.2 channels

Cannabinoids regulate numerous physiological and pathological events like inflammation or neurodegeneration via CB₁ and CB₂ receptors. The mechanisms behind cannabinoid effects show a high variability and may also involve transient receptor potential channels (TRP) and N‐type voltage‐gated Ca²⁺ channels (Ca_v2.2). In the present study we investigated the neuroprotective effects of the synthetic cannabinoid WIN 55,212–2 (WIN) on dentate gyrus (DG) granule cells and elucidated the involvement of TRP and Ca_v2.2 that are shown to participate in inflammatory processes. Organotypic hippocampal slice cultures were excitotoxically lesioned using NMDA and subsequently incubated with different WIN concentrations (0.001–10 μM). WIN showed neuroprotective properties in an inverse concentration‐dependent manner, most effectively at 0.01 μM. The CB₁ receptor antagonist AM251 blocked neuroprotection mediated by WIN whereas the CB₂ receptor antagonist AM630 showed no effects. Application of the TRPA1 blocker HC‐030031 enhanced the neuroprotective efficacy of high (10 μM) WIN concentrations and the number of degenerating neurons became equal to that seen after application of the most effective WIN dose (0.01 μM). In contrast, the application of TRPA1 agonist icilin or allyl isothiocyanate (AITC) led to a stronger neurodegeneration. The use of TRPV1 blocker 6‐iodo‐nordihydrocapsaicin did not affect WIN‐mediated neuroprotection. The selective Ca_v2.2 blocker ω‐conotoxin (GVIA) completely blocked neuroprotection shown by 10 μM WIN. GVIA and HC‐030031 exerted no effects at WIN concentrations lower than 10 μM. Our data show that WIN protects dentate gyrus granule cells in a concentration dependent manner by acting upon CB₁ receptors. At high (10 μM) concentrations WIN additionally activates TRPA1 and Ca_v2.2 within the hippocampal formation that both interfere with CB₁ receptor‐mediated neuroprotection. This leads to the conclusion that physiological and pharmacological effects of cannabinoids strongly depend on their concentration and the neuroprotective efficacy of cannabinoids may be determined by interaction of activated CB₁ receptor, TRPA1, and Ca_v2.2. © 2010 Wiley‐Liss, Inc.     

Neurotrophin-3 significantly reduces sodium channel expression linked to neuropathic pain states

Neuropathic pain resulting from chronic constriction injury (CCI) is critically linked to sensitization of peripheral nociceptors. Voltage gated sodium channels are major contributors to this state and their expression can be upregulated by nerve growth factor (NGF). We have previously demonstrated that neurotrophin-3 (NT-3) acts antagonistically to NGF in modulation of aspects of CCI-induced changes in trkA-associated nociceptor phenotype and thermal hyperalgesia. Thus, we hypothesized that exposure of neurons to increased levels of NT-3 would reduce expression of Na_v1.8 and Na_v1.9 in DRG neurons subject to CCI. In adult male rats, Na_v1.8 and Na_v1.9 mRNAs are expressed at high levels in predominantly small to medium size neurons. One week following CCI, there is reduced incidence of neurons expressing detectable Na_v1.8 and Na_v1.9 mRNA, but without a significant decline in mean level of neuronal expression, and similar findings observed immunohistochemically. There is also increased accumulation/redistribution of channel protein in the nerve most apparent proximal to the first constriction site. Intrathecal infusion of NT-3 significantly attenuates neuronal expression of Na_v1.8 and Na_v1.9 mRNA contralateral and most notably, ipsilateral to CCI, with a similar impact on relative protein expression at the level of the neuron and constricted nerve. We also observe reduced expression of the common neurotrophin receptor p75 in response to CCI that is not reversed by NT-3 in small to medium sized neurons and may confer an enhanced ability of NT-3 to signal via trkA, as has been previously shown in other cell types. These findings are consistent with an analgesic role for NT-3.     

Reduced expression and activation of voltage‐gated sodium channels contributes to blunted baroreflex sensitivity in heart failure rats

Voltage‐gated sodium (Na_v) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms of chronic heart failure (CHF)‐induced baroreflex dysfunction, we measured the expression and current density of Na_v channel subunits (Na_v1.7, Na_v1.8, and Na_v1.9) in the aortic baroreceptor neurons and investigated the role of Na_v channels in aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Na_v1.7 was expressed in A‐type (myelinated) and C‐type (unmyelinated) nodose neurons, but Na_v1.8 and Na_v1.9 were expressed only in C‐type nodose neurons. Real‐time RT‐PCR and Western blot data showed that CHF reduced mRNA and protein expression levels of Na_v channels in nodose neurons. In addition, using the whole‐cell patch‐clamp technique, we found that Na_v current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Na_v channel activator (rATX II, 100 nM) significantly enhanced Na_v current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Na_v channels are involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state. © 2010 Wiley‐Liss, Inc.     

Chronic Spinal Nerve Ligation Induces Changes in Response Characteristics of Nociceptive Spinal Dorsal Horn Neurons and in Their Descending Regulation Originating in the Periaqueductal Gray in the Rat

We studied whether a chronic neuropathy induced by unilateral spinal nerve ligation changes the response characteristics of spinal dorsal horn wide-dynamic range (WDR) neurons or their periaqueductal gray (PAG)-induced descending modulation. Experiments were performed in rats with behaviorally demonstrated allodynia induced by spinal nerve ligation and in a group of nonneuropathic control rats. The stimulus–response functions of WDR neurons for mechanical and thermal stimuli and the modulation of their peripherally evoked responses by electrical stimulation of the PAG were determined under pentobarbital anesthesia. The results showed that neuropathy caused a significant leftward shift in stimulus–response functions for mechanical stimuli. In contrast, stimulus–response functions for noxious heat stimuli in the neuropathic limb were, if anything, shifted rightward, although this shift was short of statistical significance. In neuropathic rats, PAG stimulation produced a significantly stronger attenuation of spinal neuronal responses induced by noxious heat in the unoperated than in the operated side. At the intensity that produced attenuation of noxious heat stimuli, PAG stimulation did not produce any significant change in spinal neuronal responses evoked by mechanical stimuli either from the operated or the nonoperated hindlimb of the neuropathic rats. Spontaneous activity of WDR neurons was higher in the operated side of neuropathic rats than in control rats. Afterdischarges evoked by peripheral stimuli were observed in 1/16 of the WDR neurons ipsilateral to spinal nerve ligation and not at all in other experimental groups. The WDR neurons studied were not activated by innocuous or noxious cold stimuli. The results indicate that spinal nerve ligation induces increased spontaneous activity and enhanced responses to mechanical stimuli in the spinal dorsal horn WDR neurons, whereas noxious heat-evoked responses are not significantly changed or if anything, attenuated. Moreover, the inhibition of noxious heat stimuli by PAG stimulation is attenuated in the neuropathic side. It is proposed that the observed changes in the response characteristics of the spinal dorsal horn WDR neurons and in their descending modulation may contribute to the neuropathic symptoms in these animals.