The Contribution of TRPA1 to Corneal Thermosensitivity and Blink Regulation in Young and Aged Mice.

The role of TRPA1 in the thermosensitivity of the corneal cold thermoreceptor nerve endings was studied in young and aged mice. The contribution of the TRPA1-dependent activity to basal tearing and thermally-evoked blink was also explored. The corneal cold thermoreceptors' activity was recorded extracellularly in young (5-month-old) and aged (18-month-old) C57BL/6WT (WT) and TRPA1-/- knockout (TRPA1-KO) mice at basal temperature (34 °C) and during cooling (15 °C) and heating (45 °C) ramps. The blink response to cold and heat stimulation of the ocular surface and the basal tearing rate were also measured in young animals using orbicularis oculi muscle electromyography (OOemg) and phenol red threads, respectively. The background activity at 34 °C and the cooling- and heating-evoked responses of the cold thermoreceptors were similar in WT and TRPA1-KO animals, no matter the age. Similar to the aged WT mice, in the young and aged TRPA1-KO mice, most of the cold thermoreceptors presented low frequency background activity, a low cooling threshold, and a sluggish response to heating. The amplitude and duration of the OOemg signals correlated with the magnitude of the induced thermal change in the WT but not in the TRPA1-KO mice. The basal tearing was similar in the TRPA1-KO and WT mice. The electrophysiological data suggest that the TRPA1-dependent nerve activity, which declines with age, contributes to detecting the warming of the ocular surface and also to integrating the thermally-evoked reflex blink.

Membrane potential instabilities in sensory neurons: mechanisms and pathophysiological relevance.

ABSTRACT: Peripheral sensory neurons transduce physicochemical stimuli affecting somatic tissues into the firing of action potentials that are conveyed to the central nervous system. This results in conscious perception, adaptation, and survival, but alterations of the firing patterns can result in pain and hypersensitivity conditions. Thus, understanding the molecular mechanisms underlying action potential firing in peripheral sensory neurons is essential in sensory biology and pathophysiology. Over the past 30 years, it has been consistently reported that these cells can display membrane potential instabilities (MPIs), in the form of subthreshold membrane potential oscillations or depolarizing spontaneous fluctuations. However, research on this subject remains sparse, without a clear conductive thread to be followed. To address this, we here provide a synthesis of the description, molecular bases, mathematical models, physiological roles, and pathophysiological implications of MPIs in peripheral sensory neurons. Membrane potential instabilities have been reported in trigeminal, dorsal root, and Mes-V ganglia, where they are believed to support repetitive firing. They are proposed to have roles also in intercellular communication, ectopic firing, and responses to tonic and slow natural stimuli. We highlight how MPIs are of great interest for the study of sensory transduction physiology and how they may represent therapeutic targets for many pathological conditions, such as acute and chronic pain, itch, and altered sensory perceptions. We identify future research directions, including the elucidation of the underlying molecular determinants and modulation mechanisms, their relation to the encoding of natural stimuli and their implication in pain and hypersensitivity conditions.

Sodium Channel Blockers Modulate Abnormal Activity of Regenerating Nociceptive Corneal Nerves After Surgical Lesion.

Purpose: To test the effect of different sodium channel blockers on the electrical activity of corneal nociceptors in intact and surgically injured corneas. Methods: In anesthetized guinea pigs, a 4-mm diameter corneal flap was performed in one eye at a midstromal depth using a custom-made microkeratome. At different times after surgery (3 hours to 15 days), the electrical activity of corneal nociceptor fibers was recorded from ciliary nerve filaments in the superfused eye in vitro. Mechanical threshold was measured using calibrated von Frey hairs; chemical stimulation was performed applying 30-second CO2 gas pulses. The characteristics of the spontaneous and stimulus-evoked activity of corneal nociceptors recorded from intact and lesioned corneas, before and after treatment with the sodium channel blockers lidocaine, carbamazepine, and amitriptyline, were compared. Results: No spontaneous or stimulus-evoked impulse activity was detected inside the flap at any of the studied time points. However, both were recorded from mechanonociceptor and polymodal nociceptors fibers in the surrounding corneal tissue, being significantly higher (sensitization) 24 to 48 hours after surgery. In these fibers, none of the tested drugs affected mechanical threshold, but they significantly reduced the CO2 response of polymodal nociceptors of intact and injured corneas. Likewise, they diminished significantly the transient increase in spontaneous and stimulus-evoked activity of sensitized polymodal nociceptors. Conclusions: Na+ channel blockers decrease the excitability of intact and sensitized corneal nociceptor fibers, thus acting as potential tools to attenuate their abnormal activity, which underlies the spontaneous pain, hyperalgesia, and allodynia often accompanying surgical corneal lesions, as occurs after photorefractive surgery.

Optical Assessment of Nociceptive TRP Channel Function at the Peripheral Nerve Terminal.

Free nerve endings are key structures in sensory transduction of noxious stimuli. In spite of this, little is known about their functional organization. Transient receptor potential (TRP) channels have emerged as key molecular identities in the sensory transduction of pain-producing stimuli, yet the vast majority of our knowledge about sensory TRP channel function is limited to data obtained from in vitro models which do not necessarily reflect physiological conditions. In recent years, the development of novel optical methods such as genetically encoded calcium indicators and photo-modulation of ion channel activity by pharmacological tools has provided an invaluable opportunity to directly assess nociceptive TRP channel function at the nerve terminal.

TRP Channels as Sensors of Chemically-Induced Changes in Cell Membrane Mechanical Properties.

Transient Receptor Potential ion channels (TRPs) have been described as polymodal sensors, being responsible for transducing a wide variety of stimuli, and being involved in sensory functions such as chemosensation, thermosensation, mechanosensation, and photosensation. Mechanical and chemical stresses exerted on the membrane can be transduced by specialized proteins into meaningful intracellular biochemical signaling, resulting in physiological changes. Of particular interest are compounds that can change the local physical properties of the membrane, thereby affecting nearby proteins, such as TRP channels, which are highly sensitive to the membrane environment. In this review, we provide an overview of the current knowledge of TRP channel activation as a result of changes in the membrane properties induced by amphipathic structural lipidic components such as cholesterol and diacylglycerol, and by exogenous amphipathic bacterial endotoxins.

TRP Channels as Sensors of Bacterial Endotoxins.

The cellular and systemic effects induced by bacterial lipopolysaccharides (LPS) have been solely attributed to the activation of the Toll-like receptor 4 (TLR4) signalling cascade. However, recent studies have shown that LPS activates several members of the Transient Receptor Potential (TRP) family of cation channels. Indeed, LPS induces activation of the broadly-tuned chemosensor TRPA1 in sensory neurons in a TLR4-independent manner, and genetic ablation of this channel reduced mouse pain and inflammatory responses triggered by LPS and the gustatory-mediated avoidance to LPS in fruit flies. LPS was also shown to activate TRPV4 channels in airway epithelial cells, an effect leading to an immediate production of bactericidal nitric oxide and to an increase in ciliary beat frequency. In this review, we discuss the role of TRP channels as sensors of bacterial endotoxins, and therefore, as crucial players in the timely detection of invading gram-negative bacteria.

TRPV4 activation triggers protective responses to bacterial lipopolysaccharides in airway epithelial cells.

Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca2+ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.

How Azobenzene Photoswitches Restore Visual Responses to the Blind Retina.

Azobenzene photoswitches confer light sensitivity onto retinal ganglion cells (RGCs) in blind mice, making these compounds promising candidates as vision-restoring drugs in humans with degenerative blindness. Remarkably, photosensitization manifests only in animals with photoreceptor degeneration and is absent from those with intact rods and cones. Here we show that P2X receptors mediate the entry of photoswitches into RGCs, where they associate with voltage-gated ion channels, enabling light to control action-potential firing. All charged photoswitch compounds require permeation through P2X receptors, whose gene expression is upregulated in the blind retina. Photoswitches and membrane-impermeant fluorescent dyes likewise penetrate through P2X receptors to label a subset of RGCs in the degenerated retina. Electrophysiological recordings and mapping of fluorescently labeled RGC dendritic projections together indicate that photosensitization is highly selective for OFF-RGCs. Hence, P2X receptors are a natural conduit allowing cell-type-selective and degeneration-specific delivery of photoswitches to restore visual function in blinding disease.

Biosorption of cadmium (II) from aqueous solutions by natural and modified non-living leaves of Posidonia oceanica.

Natural, HCl-treated, and formaldehyde-treated non-living leaves of Posidonia oceanica, a marine plant, were investigated as potential biosorbents to remove Cd2+ from aqueous solutions. The studied biosorbents were characterized by elemental analysis and Fourier transform infrared spectroscopy (FTIR) and it was observed that the adsorption capacity of the biosorbents strongly depended on the pH, increasing as the pH rises. The adsorption process was fast. The adsorption kinetic was analyzed using five kinetic models: pseudo-first order, pseudo-second order, Elovich, intraparticle diffusion, and Bangham models. The adsorption isotherms were analyzed by the Langmuir, Freundlich, Dubinin-Radushkevich, Sips, Redlich-Peterson, and Toth models. The maximum biosorption capacity was attained by the biosorbent treated with HCl (1.11 mmol g-1). The distribution equilibrium constant and the Gibbs free energy change were calculated. The effects of the presence of Na+, K+, Mg2+, and Ca2+ ions in the solution on Cd2+ uptake were studied. Results indicate that non-living leaves of P. oceanica, natural or treated, can be considered as effective and low-cost biosorbents for the removal of cadmium from aqueous solutions.

Serotonin modulates spike probability in the axon initial segment through HCN channels.

The axon initial segment (AIS) serves as the site of action potential initiation in most neurons, but difficulties in isolating the effects of voltage-gated ion channels in the AIS from those of the soma and dendrites have hampered understanding how AIS properties influence neural coding. Here we have combined confocal microscopy, patch-clamp recordings and light-sensitive channel blockers ('photoswitches') in binaural auditory gerbil neurons to show that hyperpolarization and cyclic-nucleotide-gated (HCN) channels are expressed in the AIS and decrease spike probability, in a manner distinct from that of HCN channels in the soma and dendrites. Furthermore, the control of spike threshold by HCN channels in the AIS can be altered through serotonergic modulation of 5-hydroxytryptamine 1A (5-HT1A) receptors, which hyperpolarizes the activation range of HCN channels. As release of serotonin signals changes in motivation and attention states, axonal HCN channels provide a mechanism to translate these signals into changes in the threshold for sensory stimuli.

Abnormal activity of corneal cold thermoreceptors underlies the unpleasant sensations in dry eye disease.

Dry eye disease (DED) affects >10% of the population worldwide, and it provokes an unpleasant sensation of ocular dryness, whose underlying neural mechanisms remain unknown. Removal of the main lachrymal gland in guinea pigs caused long-term reduction of basal tearing accompanied by changes in the architecture and density of subbasal corneal nerves and epithelial terminals. After 4 weeks, ongoing impulse activity and responses to cooling of corneal cold thermoreceptor endings were enhanced. Menthol (200 µM) first excited and then inactivated this augmented spontaneous and cold-evoked activity. Comparatively, corneal polymodal nociceptors of tear-deficient eyes remained silent and exhibited only a mild sensitization to acidic stimulation, whereas mechanonociceptors were not affected. Dryness-induced changes in peripheral cold thermoreceptor responsiveness developed in parallel with a progressive excitability enhancement of corneal cold trigeminal ganglion neurons, primarily due to an increase of sodium currents and a decrease of potassium currents. In corneal polymodal nociceptor neurons, sodium currents were enhanced whereas potassium currents remain unaltered. In healthy humans, exposure of the eye surface to menthol vapors or to cold air currents evoked unpleasant sensations accompanied by increased blinking frequency that we attributed to cold thermoreceptor stimulation. Notably, stimulation with menthol reduced the ongoing background discomfort of patients with DED, conceivably due to use-dependent inactivation of cold thermoreceptors. Together, these data indicate that cold thermoreceptors contribute importantly to the detection and signaling of ocular surface wetness, and develop under chronic eye dryness conditions an injury-evoked neuropathic firing that seems to underlie the unpleasant sensations experienced by patients with DED.

TRPA1 channels mediate acute neurogenic inflammation and pain produced by bacterial endotoxins.

Gram-negative bacterial infections are accompanied by inflammation and somatic or visceral pain. These symptoms are generally attributed to sensitization of nociceptors by inflammatory mediators released by immune cells. Nociceptor sensitization during inflammation occurs through activation of the Toll-like receptor 4 (TLR4) signalling pathway by lipopolysaccharide (LPS), a toxic by-product of bacterial lysis. Here we show that LPS exerts fast, membrane delimited, excitatory actions via TRPA1, a transient receptor potential cation channel that is critical for transducing environmental irritant stimuli into nociceptor activity. Moreover, we find that pain and acute vascular reactions, including neurogenic inflammation (CGRP release) caused by LPS are primarily dependent on TRPA1 channel activation in nociceptive sensory neurons, and develop independently of TLR4 activation. The identification of TRPA1 as a molecular determinant of direct LPS effects on nociceptors offers new insights into the pathogenesis of pain and neurovascular responses during bacterial infections and opens novel avenues for their treatment.

Methodological considerations to understand the sensory function of TRP channels.

Transient Receptor Potential channels are exquisite molecular transducers of multiple physical and chemical stimuli, hence the raising interest to study their relevance to Sensory Biology. Here we discuss a number of aspects of the biophysical and pharmacological properties of TRP channels, which we consider essential for a clear understanding of their sensory function in vivo. By examining concrete examples extracted from recent literature we illustrate that TRP channel research is a field in motion, and that many established dogmas on biophysical properties, drug specificity and physiological role are continuously reshaped, and sometimes even dismantled.

Nicotine activates the chemosensory cation channel TRPA1.

Topical application of nicotine, as used in nicotine replacement therapies, causes irritation of the mucosa and skin. This reaction has been attributed to activation of nicotinic acetylcholine receptors (nAChRs) in chemosensory neurons. In contrast with this view, we found that the chemosensory cation channel transient receptor potential A1 (TRPA1) is crucially involved in nicotine-induced irritation. We found that micromolar concentrations of nicotine activated heterologously expressed mouse and human TRPA1. Nicotine acted in a membrane-delimited manner, stabilizing the open state(s) and destabilizing the closed state(s) of the channel. In the presence of the general nAChR blocker hexamethonium, nociceptive neurons showed nicotine-induced responses that were strongly reduced in TRPA1-deficient mice. Finally, TRPA1 mediated the mouse airway constriction reflex to nasal instillation of nicotine. The identification of TRPA1 as a nicotine target suggests that existing models of nicotine-induced irritation should be revised and may facilitate the development of smoking cessation therapies with less adverse effects.

Characteristics and physiological role of hyperpolarization activated currents in mouse cold thermoreceptors.

Hyperpolarization-activated currents (I(h)) are mediated by the expression of combinations of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel subunits (HCN1-4). These cation currents are key regulators of cellular excitability in the heart and many neurons in the nervous system. Subunit composition determines the gating properties and cAMP sensitivity of native I(h) currents. We investigated the functional properties of I(h) in adult mouse cold thermoreceptor neurons from the trigeminal ganglion, identified by their high sensitivity to moderate cooling and responsiveness to menthol. All cultured cold-sensitive (CS) neurons expressed a fast activating I(h), which was fully blocked by extracellular Cs(+) or ZD7288 and had biophysical properties consistent with those of heteromeric HCN1-HCN2 channels. In CS neurons from HCN1(-/-) animals, I(h) was greatly reduced but not abolished. We find that I(h) activity is not essential for the transduction of cold stimuli in CS neurons. Nevertheless, I(h) has the potential to shape the excitability of CS neurons. First, I(h) blockade caused a membrane hyperpolarization in CS neurons of about 5 mV. Furthermore, impedance power analysis showed that all CS neurons had a prominent subthreshold membrane resonance in the 5-7 Hz range, completely abolished upon blockade of I(h) and absent in HCN1 null mice. This frequency range matches the spontaneous firing frequency of cold thermoreceptor terminals in vivo. Behavioural responses to cooling were reduced in HCN1 null mice and after peripheral pharmacological blockade of I(h) with ZD7288, suggesting that I(h) plays an important role in peripheral sensitivity to cold.

Lipid raft segregation modulates TRPM8 channel activity.

Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterol-rich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn(934) residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol- and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.

TRPA1 channels: novel targets of 1,4-dihydropyridines.

Transient receptor potential type A1 (TRPA1) channels are cation permeable channels activated by irritant chemicals and pungent natural compounds. Their location in peptidergic sensory terminals innervating the skin and blood vessels makes them important effectors of vasodilator responses of neural origin. 1,4-dihydropyridines are a class of L-type calcium channel antagonists commonly used in the treatment of hypertension and ischemic heart disease. Here we show that four different 1,4-dihydropyridines (nifedipine, nimodipine, nicardipine and nitrendipine), and the structurally related L-type calcium channel agonist BayK8644, exert powerful excitatory effects on TRPA1 channels. The activation does not depend on elevated Ca2+ levels and cross-desensitizes with that produced by other TRPA1 agonists. The activation produced by nifedipine was reduced by camphor and the selective TRPA1 antagonist HC03001. In a subclass of mouse nociceptors expressing TRPA1 channels, assessed by responses to the TRPA1 agonist mustard oil, nifedipine also produced large elevations in [Ca2+](i). These responses were fully abrogated in TRPA1(-/-) mice. These findings identify TRPA1 channels as a new molecular target for the 1,4-dihydropyridine class of calcium channel modulators.

TRPA1 channels mediate cold temperature sensing in mammalian vagal sensory neurons: pharmacological and genetic evidence.

Cold thermoreceptors have been described in different territories of the vagus nerve. Application of cold temperature to these visceral afferents can evoke major protective reflexes and thermoregulatory responses. However, virtually nothing is known about the transduction mechanisms underlying cold sensitivity in vagal afferents. Here, we investigated the effects of cold stimulation on intracellular calcium responses and excitability of cultured vagal sensory neurons in the rat nodose ganglion. A large fraction of vagal neurons were activated by cold, with a mean threshold of approximately 24 degrees C. Cooling was accompanied by development of a small inward current and the firing of action potentials. Most cold-sensitive neurons were also activated by heat and capsaicin, suggesting a nociceptive function. The pharmacological response to TRPM8 and TRPA1 agonists and antagonists suggested that, unlike results observed in somatic tissues, TRPA1 is the major mediator of cold-evoked responses in vagal visceral neurons. Thus, most cold-evoked responses were potentiated by cinnamaldehyde, menthol, icilin, and BCTC [4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide], agonists of TRPA1, and were inhibited by ruthenium red, camphor, and HC03001 [2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopropylphenyl)acetamide]. Results in mouse nodose neurons revealed a similar pharmacological profile of cold-evoked responses. Furthermore, experiments in TRPA1 knock-out mice showed a large reduction in the percentage of cold-sensitive neurons compared with wild-type animals. Together, these results support an important role of TRPA1 channels in visceral thermosensation and indicate major differences in the transduction of temperature signals between somatic and visceral sensory neurons.

Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators.

The transient receptor potential channel of the ankyrin-binding repeat subfamily, TRPA1, is a Ca(2+)-permeable non-selective cation channel that depolarizes the plasma membrane and causes Ca(2+) influx. A typical feature of TRPA1 is its rapid desensitization following activation by agonists such as mustard oil (MO), cinnamaldehyde, and a high intracellular Ca(2+) concentration. In whole-cell recordings on Chinese hamster ovary (CHO) cells expressing TRPA1, desensitization was delayed when phosphatidylinositol 4,5-biphosphate (PIP(2)) was supplemented via the patch pipette, whereas the PIP(2) scavenger neomycin accelerated desensitization. Preincubation with the PI-4 kinase inhibitor wortmannin reduced both constitutive TRPA1 channels activity and the response to MO. Run down was also accelerated by high intracellular Mg(2+) concentrations, whereas chelating intracellular Mg(2+) with 10 mM ethylenedinitrilotetraacetic acid (EDTA) increased the basal channel activity. In inside-out patches, we observed a rapid run down of TRPA1 activity, which could be prevented by application of diC8-PIP(2) or 2 mM Mg-ATP but not Na(2)-ATP to the cytosolic side of the excised patches. In isolated trigeminal ganglion neurons, preincubation with wortmannin resulted in inhibition of endogenous TRPA1 activation by MO. Taken together, our data indicate that PIP(2) modulates TRPA1, albeit to a lesser extent than other known PIP(2)-dependent TRP channels, and that tools modifying the plasma membrane PIP(2) content often have direct effects on this channel.

Transient receptor potential channels in sensory neurons are targets of the antimycotic agent clotrimazole.

Clotrimazole (CLT) is a widely used drug for the topical treatment of yeast infections of skin, vagina, and mouth. Common side effects of topical CLT application include irritation and burning pain of the skin and mucous membranes. Here, we provide evidence that transient receptor potential (TRP) channels in primary sensory neurons underlie these unwanted effects of CLT. We found that clinically relevant CLT concentrations activate heterologously expressed TRPV1 and TRPA1, two TRP channels that act as receptors of irritant chemical and/or thermal stimuli in nociceptive neurons. In line herewith, CLT stimulated a subset of capsaicin-sensitive and mustard oil-sensitive trigeminal neurons, and evoked nocifensive behavior and thermal hypersensitivity with intraplantar injection in mice. Notably, CLT-induced pain behavior was suppressed by the TRPV1-antagonist BCTC [(N-(-4-tertiarybutylphenyl)-4-(3-cholorpyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide)] and absent in TRPV1-deficient mice. In addition, CLT inhibited the cold and menthol receptor TRPM8, and blocked menthol-induced responses in capsaicin- and mustard oil-insensitive trigeminal neurons. The concentration for 50% inhibition (IC50) of inward TRPM8 current was approximately 200 nM, making CLT the most potent known TRPM8 antagonist and a useful tool to discriminate between TRPM8- and TRPA1-mediated responses. Together, our results identify TRP channels in sensory neurons as molecular targets of CLT, and offer means to develop novel CLT preparations with fewer unwanted sensory side effects.