The Immunosuppressant Macrolide Tacrolimus Activates Cold-Sensing TRPM8 Channels.

TRPM8 is a polymodal, nonselective cation channel activated by cold temperature and cooling agents that plays a critical role in the detection of environmental cold. We found that TRPM8 is a pharmacological target of tacrolimus (FK506), a macrolide immunosuppressant with several clinical uses, including the treatment of organ rejection following transplants, treatment of atopic dermatitis, and dry eye disease. Tacrolimus is an inhibitor of the phosphatase calcineurin, an action shared with cyclosporine. Tacrolimus activates TRPM8 channels in different species, including humans, and sensitizes their response to cold temperature by inducing a leftward shift in the voltage-dependent activation curve. The effects of tacrolimus on purified TRPM8 in lipid bilayers demonstrates conclusively that it has a direct gating effect. Moreover, the lack of effect of cyclosporine rules out the canonical signaling pathway involving the phosphatase calcineurin. Menthol (TRPM8-Y745H)- and icilin (TRPM8-N799A)-insensitive mutants were also activated by tacrolimus, suggesting a different binding site. In cultured mouse DRG neurons, tacrolimus evokes an increase in intracellular calcium almost exclusively in cold-sensitive neurons, and these responses were drastically blunted in Trpm8 KO mice or after the application of TRPM8 antagonists. Cutaneous and corneal cold thermoreceptor endings are also activated by tacrolimus, and tacrolimus solutions trigger blinking and cold-evoked behaviors. Together, our results identify TRPM8 channels in sensory neurons as molecular targets of the immunosuppressant tacrolimus. The actions of tacrolimus on TRPM8 resemble those of menthol but likely involve interactions with other channel residues.SIGNIFICANCE STATEMENT TRPM8 is a polymodal TRP channel involved in cold temperature sensing, thermoregulation, and cold pain. TRPM8 is also involved in the pathophysiology of dry eye disease, and TRPM8 activation has antiallodynic and antipruritic effects, making it a prime therapeutic target in several cutaneous and neural diseases. We report the direct agonist effect of tacrolimus, a potent natural immunosuppressant with multiple clinical applications, on TRPM8 activity. This interaction represents a novel neuroimmune interface. The identification of a clinically approved drug with agonist activity on TRPM8 channels could be used experimentally to probe the function of TRPM8 in humans. Our findings may explain some of the sensory and anti-inflammatory effects described for this drug in the skin and the eye surface.

Inhibitory Effect of Amitriptyline on the Impulse Activity of Cold Thermoreceptor Terminals of Intact and Tear-Deficient Guinea Pig Corneas.

PURPOSE: Chronic dryness of the ocular surface evokes sensitization of corneal cold-sensitive neurons through an increase of sodium currents and a decrease of potassium currents, leading to the unpleasant dryness and pain sensations typical of dry eye disease. Here, we explored the effects of amitriptyline, a voltage-gated Na+ channel blocker used for the treatment of depression and chronic pain, on nerve terminal impulse (NTI) activity of cold-sensitive nerve terminals recorded in intact and tear-deficient guinea pig corneas. METHODS: Main lachrymal gland was surgically removed in anesthetized guinea pigs to induce chronic tear deficiency. Four to 6 weeks afterward, animals were sacrificed and both corneas placed in a perfusion chamber superfused at 34°C. Thermal stimuli were induced by changing the solution temperature from 34°C to 20°C (cooling ramp) and from 34°C to 50°C (heating ramp). Spontaneous and stimulus-evoked NTIs of cold-sensitive nerve terminals were recorded before, during, and after perfusion with solutions containing amitriptyline at different concentrations (3-30 µM). RESULTS: Perfusion with amitriptyline inhibited irreversibly and in a concentration-dependent manner the spontaneous NTI activity of cold thermoreceptors of intact corneas. This effect was less evident in tear-deficient corneas. In addition, amitriptyline (10 µM) attenuated the maximal response to cooling ramps without changing cold threshold in intact but not in tear-deficient corneas. Only cold thermoreceptors with low cooling threshold values were sensitive to amitriptyline. CONCLUSION: Amitriptyline effectively reduces the activity of cold thermoreceptors, although its efficacy is different in intact and tear-deficient corneas, which might be due to the changes induced by ocular dryness in the expression of the various voltage-gated Na+ channels responsible of the action potential generation and propagation.

Activation of the TRPV1 Thermoreceptor Induced by Modulated or Unmodulated 1800 MHz Radiofrequency Field Exposure.

The existence of effects of radiofrequency field exposure at environmental levels on living tissues and organisms remains controversial, in particular regarding potential "nonthermal" effects produced in the absence of temperature elevation. Therefore, we investigated whether TRPV1, one of the most studied thermosensitive channels, can be activated by the heat produced by radiofrequency fields and by some specific nonthermal interaction with the fields. We have recently shown that TRPV1 activation can be assessed in real-time on live cells using the bioluminescence resonance energy transfer technique. Taking advantage of this innovative assay, we monitored TRPV1 thermal and chemical modes of activation under radiofrequency exposure at 1800 MHz using different signals (CW, GSM, UMTS, LTE, Wi-Fi and WiMAX) at specific absorption rates between 8 and 32 W/kg. We showed that, as expected, TRPV1 channels were activated by the heat produced by radiofrequency field exposure of transiently-transfected HEK293T cells, but found no evidence of TRPV1 activation in the absence of temperature elevation under radiofrequency field exposure. There was no evidence either that, at fixed temperature, radiofrequency exposure altered the maximal efficacy of the agonist Capsaicin to activate TRPV1.

Effect of stimulation of cold receptors with menthol on EMG activity of quadriceps muscle during low load contraction.

PURPOSE: Facilitatory and inhibitory responses of spinal motor neurons are influenced by somatosensory input from the skin. The purpose of this study, employing electromyography, was to examine the neuromuscular changes that occur with menthol applied to the skin over the quadriceps muscle. METHODS: Forty-two healthy volunteers performed isometric knee extensions at 35% maximum voluntary contraction (MVC) in three groups (Adult Placebo, Adult Menthol, Older Adult Menthol). Stimulation used was application of 5% menthol gel to the skin. Surface electromyography (sEMG) from the vastus lateralis (VL), vastus medialis (VM), and rectus femoris (RF) was recorded using miniature pair electrodes. RESULTS: Root mean square electromyography (rmsEMG) in VL and VM significantly increased with menthol stimulation both in Adult and Older Adult, but no significant difference was observed between Adult Menthol and Older Adult Menthol. There was a significant decrease in mean power frequency (MPF) in VM with menthol stimulation in Older Adult, but no significant changes were observed in Adult Menthol. CONCLUSION: Neuromuscular modulation was observed with the application of menthol gel at low loads in the present study. These findings could lead to a new method of muscular training that targets the recruitment of fast type muscle safe for older adults.

Two different avian cold-sensitive sensory neurons: Transient receptor potential melastatin 8 (TRPM8)-dependent and -independent activation mechanisms.

Sensing the ambient temperature is an important function for survival in animals. Some TRP channels play important roles as detectors of temperature and irritating chemicals. There are functional differences of TRP channels among species. TRPM8 in mammals is activated by cooling compounds and cold temperature, but less information is available on the functional role of TRPM8 in avian species. Here we investigated the pharmacological properties and thermal sensitivities of chicken TRPM8 (cTRPM8) and cold-sensitive mechanisms in avian sensory neurons. In heterologously expressed cTRPM8, menthol and its derivative, WS-12 elicited [Ca2+]i increases, but icilin did not. In chicken sensory neurons, icilin increased [Ca2+]i, in a TRPA1-dependent manner. Icilin selectively stimulated heterologously expressed chicken TRPA1 (cTRPA1). Similar to mammalian orthologue, cTRPM8 was activated by cold. Both heterologous and endogenous expressed cTRPM8 were sensitive to mammalian TRPM8 antagonists. There are two types of cold-sensitive cells regarding menthol sensitivity in chicken sensory neurons. The temperature threshold of menthol-insensitive neurons was significantly lower than that of menthol-sensitive ones. The population of menthol-insensitive neurons was large in chicken but almost little in mammals. The cold-induced [Ca2+]i increases were not abolished by the external Ca2+ removal or by blockades of PLC-IP3 pathways and ryanodine channels. The cold stimulation failed to evoke [Ca2+]i increases after intracellular Ca2+ store-depletion. These results indicate that cTRPM8 acts as a cold-sensor similar to mammals. It is noteworthy that TRPM8-independent cold-sensitive neurons are abundant in chicken sensory neurons. Our results suggest that most of the cold-induced [Ca2+]i increases are mediated via Ca2+ release from intracellular stores and that these mechanisms may be specific to avian species.

Arsenite induces neurotoxic effects on AFD neurons via oxidative stress in Caenorhabditis elegans.

Arsenic permeates our environment. As a result, humans are continually exposed to it. This study investigates the possible roles of oxidative stress in arsenite (As(III))-induced neurotoxicity in Caenorhabditis elegans. Exposure to As(III), at the concentrations examined, caused a decrease in locomotor behaviors (frequencies of body bends, head thrashes, and reversals) of C. elegans. In addition, As(III) exposure (100 µM) decreased thermotactic behaviors, and induced severe deficits in the structural properties of AFD sensory neurons. Exposure to As(III) (100 µM) also caused an elevated production of intracellular reactive oxygen species (ROS) in wild-type C. elegans. Pretreatment with the antioxidant curcumin ameliorated the decrease in locomotor and thermotactic behavior, the formation of deficits in the structural properties of AFD sensory neurons, and intracellular ROS in As(III)-exposed nematodes. Our study suggests that oxidative stress plays a crucial role in the As(III)-induced neurotoxic effects on locomotor behavior and the structures and function of AFD sensory neurons in As(III)-exposed nematodes.

Spraying with 0.20% L-menthol does not enhance 5 km running performance in the heat in untrained runners.

BACKGROUND: L-Menthol stimulates cutaneous thermoreceptors and induces cool sensations improving thermal comfort but has also been linked to heat storage responses. Therefore, L-Menthol application could lead to a conflict in behavioural and thermoregulatory drivers improving comfort but leading to a higher rate of deep body temperature rise; the present study examined this possibility. METHODS: Six untrained male participants (age 21 [1] years; height 1.80 [0.07] m; mass 78.9 [6.9] kg; surface area 1.98 [0.13] m2) took part. They completed three trials in hot conditions (34 °C) where their clothing was sprayed (CONTROL-SPRAY or MENTHOL-SPRAY) or not sprayed (CONTROL) after a fixed intensity exercise period (15-minutes), which induced thermal discomfort, before completing a 5 km treadmill time trial (TT). Thermal perception (thermal sensation and comfort; TS, TC), thermal responses (aural temperature [Tau], skin temperature [Tskin]), perceived exertion (RPE), heart rate, pacing (1 km split time) and performance (TT completion time) were measured. RESULTS: MENTHOL-SPRAY induced improvements in TS (up to 3 km of TT) and TC (up to 1 km) with Tau showing a tendency to be higher than CONTROL-SPRAY (+0.20 [0.29] °C) and CONTROL condition (0.30 [0.34] °C); this was not statistically significant and the rate of rise in Tau was linear. Tau was continuing to rise between the 4th and 5th kilometre of the TT. The other variables were unchanged. TT completion time and pace were not different: CONTROL 27.92 [1.65], CONTROL-SPRAY 28.10 [1.12], MENTHOL-SPRAY 27.53 [2.85] minutes. CONCLUSION: Spraying L-MENTHOL prior to exercise in the heat culminated in improved perception but not altered performance.

Thermal taste and anti-aspiration drugs: a novel drug discovery against pneumonia.

Despite the development of strong antibiotics, the pneumonia death is increasing all over the world in these decades. Among the people who died of pneumonia, the majority were 65 years old or over. Although pneumonia is recently categorized into several entities, aspiration pneumonia includes all entities. Therefore, targeting dysphagia and aspiration to treat pneumonia is a promising strategy and anti-aspiration drugs will be a part of pneumonia treatment. The swallowing reflex in elderly people was temperature-sensitive and the improvement of swallowing reflex by temperature stimuli could be mediated by the thermosensing TRP channels at pharynx. The administration of capsaicin as an agonist stimulus of TRPV1, a warm temperature receptor, decreased the delay in swallowing reflex. Red wine polyphenols improved swallowing reflex by enhancing TRPV1 response. Food with menthol, agonist of TRPM8 which is a cold temperature receptor, also decreased the delay in swallowing reflex. Olfactory stimulation such as black pepper was useful to improve the swallowing reflex for people with low ADL levels or with decreased consciousness. Thus, recent advancement of geriatrics found several anti-aspiration drugs such as thermosensing TRP channel agonists, black pepper odor, amantadine, cilostazol, theophylline and angiotensin- converting enzymes inhibitors. Thermosensing TRP channel agonists include capsaicin, capsiate, menthol, and red wine polyphenols. Controls of swallowing are mediated by various stages of neural system from peripheral sensory nerves to the entire cerebral cortex. Each anti-aspiration drug acts on various sites of neural axis of swallowing reflex. The combination of various anti-aspiration drugs may improve dysphagia and prevent aspiration pneumonia.

Phthalates induce neurotoxicity affecting locomotor and thermotactic behaviors and AFD neurons through oxidative stress in Caenorhabditis elegans.

BACKGROUND: Phthalate esters are ubiquitous environmental contaminants and numerous organisms are thus exposed to various levels of phthalates in their natural habitat. Considering the critical, but limited, research on human neurobehavioral outcomes in association with phthalates exposure, we used the nematode Caenorhabditis elegans as an in vivo model to evaluate phthalates-induced neurotoxicity and the possible associated mechanisms. PRINCIPAL FINDINGS: Exposure to phthalates (DEHP, DBP, and DIBP) at the examined concentrations induced behavioral defects, including changes in body bending, head thrashing, reversal frequency, and thermotaxis in C. elegans. Moreover, phthalates (DEHP, DBP, and DIBP) exposure caused toxicity, affecting the relative sizes of cell body fluorescent puncta, and relative intensities of cell bodies in AFD neurons. The mRNA levels of the majority of the genes (TTX-1, TAX-2, TAX-4, and CEH-14) that are required for the differentiation and function of AFD neurons were decreased upon DEHP exposure. Furthermore, phthalates (DEHP, DBP, and DIBP) exposure at the examined concentrations produced elevated intracellular reactive oxygen species (ROS) in C. elegans. Finally, pretreatment with the antioxidant ascorbic acid significantly lowered the intracellular ROS level, ameliorated the locomotor and thermotactic behavior defects, and protected the damage of AFD neurons by DEHP exposure. CONCLUSIONS: Our study suggests that oxidative stress plays a critical role in the phthalate esters-induced neurotoxic effects in C. elegans.

The effect of menthol application to the skin on sweating rate response during exercise in swimmers and controls.

We tested the hypothesis that menthol application would reduce the magnitude and initiation of sweating via excitation of cold-sensitive afferent pathways and concurrently via a cross-inhibition of heat loss pathways in acclimatized (swimmers, SW) and non acclimatized (control, CON) subjects in cool water. It was expected this effect to be exaggerated in SW subjects. Eight SW and eight CON subjects cycled at 60% of their VO(2)max, as long as to reach 38 degrees C in rectal temperature (Tre), without or with (4.6 g per 100 ml of water) all-body application of menthol sediment. Heart rate (HR), Tre, sweating rate (SwR), the proximal-distal skin temperature gradient (TSk(f-f)), and oxygen consumption (VO(2)) were measured continuously. VO(2) and HR were similar between groups and conditions. Menthol increased TSk(f-f), Tre threshold for SwR [+0.32 (0.01) degrees C] and Tre gain, while menthol reduced exercise time by 8.1 (4.1) min. SW group showed higher changes in Tre threshold for SwR [+0.50 (0.01) degrees C for SW vs. +0.13 (0.03) degrees C for CON], higher Tre gain, lower time for Tre increase and shorter exercise time [-10.7 (7) min for SW vs. -4.9 (4) min for CON] in menthol condition. Upon exercise initiation, previously applied menthol on the skin seems to induce vasoconstriction, results in a delayed sweating, which in turn affects the rectal temperature. Acclimatized subjects showed higher delay in SwR and earlier rise in Tre, which most probably is due to the inter-group differences in cold receptors activity.

A new method to study sensory modulation of locomotor networks by activation of thermosensitive cutaneous afferents using a hindlimb attached spinal cord preparation.

The use of isolated in vitro spinal cord preparations to examine the underlying networks that control locomotion has become popular. It is also well known that afferent feedback can excite and modulate these networks. However, it is often difficult to selectively activate classes of afferents that subserve specific modalities using in vitro preparations. Here, we describe a technique where afferent receptors that detect temperature were selectively activated. To accomplish this we used an in vitro preparation of the mouse where the spinal cord was isolated (T5-cauda equina) with one hind limb left attached. We designed a special chamber allowing the hind paw to be placed in such a way that it remained attached to the spinal cord but received a separate supply of artificial cerebrospinal fluid (aCSF). This allowed us to alter the temperature of the hind limb compartment without affecting the temperature of the central compartment containing the spinal cord. We also demonstrate using this approach that agonists which activate receptors which detect noxious heat could be intradermally injected into the hind limb without it diffusing into the central compartment.

Inhibition by the cold receptor agonists menthol and icilin of airway smooth muscle contraction.

Menthol, known as a cold receptor agonist, has widely been used in the relief of respiratory symptoms such as coughing and chest congestion. Previous studies have demonstrated that menthol reduces bronchoconstriction and airway hyperresponsiveness. The aim of this study was to examine the effects of menthol and icilin, another cold receptor agonist, on airway smooth muscle contraction. Isometric force was monitored using epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. Intracellular Ca(2+) concentrations were assessed by fura-2 fluorescence. (-)Menthol (0.01-1mM) inhibited contraction induced by methacholine (MCh, 0.01-10microM) and high extracellular K(+) concentrations (20-60mM) in a concentration-dependent manner. Moreover, the increases of intracellular Ca(2+) concentrations induced by MCh or high K(+) were significantly reduced by (-)menthol. Icilin (100microM) also significantly attenuated contraction induced by MCh or high K(+). The inhibitory effect of 1mM (-)menthol on MCh-induced contraction was significantly higher at cool temperature (24-26 degrees C) than at 37 degrees C. The present results demonstrate that inhibition of Ca(2+) influx plays an important role in the menthol-mediated inhibition of contraction in airway smooth muscle. Furthermore, our findings indicate that stimulation of unknown cold receptors may be involved in these mechanisms. These findings suggest that the use of menthol is beneficial for reducing respiratory symptoms because of its inhibitory effects on airway smooth muscle contraction.

Topical capsaicin application causes cold hypersensitivity in awake monkeys.

Recent animal studies have demonstrated that many trigeminal ganglion neurons co-express TRPV1 and TRPA1 receptors following peripheral inflammation. In the present study, we examined whether cold receptors were sensitized by capsaicin in awake monkeys. Two monkeys were trained to detect a change in cold stimulus temperature (30 degrees C to 0.5, 1.0, 1.5 or 2.0 degrees C) applied to the facial skin. A total of 589 trials were studied, and the number of escape and hold-through trials and detection latency were measured. The number of escape trials was increased after capsaicin treatment, whereas that of hold-through trials was decreased. Detection latency was significantly decreased after capsaicin treatment. The present findings suggest that topical application of capsaicin to the facial skin induces reversible hypersensitivity to a facial cold stimulus in behaving monkeys.

Human lung epithelial cells express a functional cold-sensing TRPM8 variant.

Several transient receptor potential (TRP) ion channels sense and respond to changes in ambient temperature. Chemical agonists of TRP channels, including menthol and capsaicin, also elicit sensations of temperature change. TRPM8 is a cold- and menthol-sensing ion channel that converts thermal and chemical stimuli into neuronal signals and sensations of cooling/cold. However, the expression and function of TRPM8 receptors in non-neuronal cells and tissues is a relatively unexplored area. Results presented here document the expression and function of a truncated TRPM8 variant in human bronchial epithelial cells. Expression of the TRPM8 variant was demonstrated by RT-PCR, cloning, and immunohistology. Receptor function was characterized using the prototypical TRPM8 agonist, menthol, and exposure of cells to reduced temperature (18 degrees C). The TRPM8 variant was expressed primarily within endoplasmic reticulum membranes of lung epithelial cells and its activation was attenuated by thapsigargin, the cell-permeable TRPM8 antagonist N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide, and shRNA-induced suppression of TRPM8 expression. Activation of the TRPM8 variant in lung cells was coupled with enhanced expression of the inflammatory cytokines IL-6 and IL-8. Collectively, our results suggest that this novel TRPM8 variant receptor may function as a modulator of respiratory physiology caused by cold air, and may partially explain asthmatic respiratory hypersensitivity to cold air.

Spatial and temporal assessment of orofacial somatosensory sensitivity: a methodological study.

AIMS: To evaluate the sensitivity and reproducibility of a multimodal psychophysical technique for the assessment of both spatial and temporal changes in somatosensory function after an infraorbital nerve block. METHODS: Sixteen healthy volunteers with a mean (+/- SD) age of 22.5 +/- 3.4 years participated in 2 identical experimental sessions separated by 2 weeks. The subjects rated the perceived intensity of standardized nonpainful tactile, painful pinprick, warm, and cold stimuli applied to 25 points in 5 x 5 matrices in the infraorbital region of each side. The reproducibility of single points was tested, and a mean difference of 1.4 +/- 0.5 was found. A 0-50-100 numerical rating scale (NRS) with 50 denoting "just barely painful" was used. A modified ice hockey mask with adjustable settings was developed as a template to allow stimulation of the same points in the 2 sessions. Assessment of somatosensory function was carried out before the injection (baseline) and after 30 and 60 minutes on both the anesthetized and contralateral (control) side. In addition, the applicability of the psychophysical techniques was tested in pilot experiments in 2 patients before maxillary osteotomy and 3 months afterward. RESULTS: The overall analysis of mean NRS scores, number of points, and center-of-gravity coordinates for all stimulus modalities showed no significant main effects of session. Post-hoc tests for all stimulus modalities demonstrated significantly lower mean NRS scores and significantly more points (hyposensitivity) at 30 and 60 minutes postinjection compared to baseline values on the injection side (Tukey tests: P < .002). In the 2 maxillary osteotomy patients, the psychophysical techniques could successfully be applied, and bilateral hyposensitivity to all stimulus modalities was demonstrated at the 3-month follow-up. CONCLUSION: The present findings indicate that the psychophysical method is sufficiently reproducible, with no major differences between sessions in healthy subjects. All stimulus modalities demonstrated adequate sensitivity. Furthermore, measurement of points in 5 x 5 matrices allowed a spatial description of somatosensory sensitivity. This method may be valuable for studies on changes in somatosensory sensitivity following trauma or orthognathic surgery on the maxilla.

Bidirectional shifts of TRPM8 channel gating by temperature and chemical agents modulate the cold sensitivity of mammalian thermoreceptors.

TRPM8, a member of the melastatin subfamily of transient receptor potential (TRP) cation channels, is activated by voltage, low temperatures and cooling compounds. These properties and its restricted expression to small sensory neurons have made it the ion channel with the most advocated role in cold transduction. Recent work suggests that activation of TRPM8 by cold and menthol takes place through shifts in its voltage-activation curve, which cause the channel to open at physiological membrane potentials. By contrast, little is known about the actions of inhibitors on the function of TRPM8. We investigated the chemical and thermal modulation of TRPM8 in transfected HEK293 cells and in cold-sensitive primary sensory neurons. We show that cold-evoked TRPM8 responses are effectively suppressed by inhibitor compounds SKF96365, 4-(3-chloro-pyridin-2-yl)-piperazine-1-carboxylic acid (4-tert-butyl-phenyl)-amide (BCTC) and 1,10-phenanthroline. These antagonists exert their effect by shifting the voltage dependence of TRPM8 activation towards more positive potentials. An opposite shift towards more negative potentials is achieved by the agonist menthol. Functionally, the bidirectional shift in channel gating translates into a change in the apparent temperature threshold of TRPM8-expressing cells. Accordingly, in the presence of the antagonist compounds, the apparent response-threshold temperature of TRPM8 is displaced towards colder temperatures, whereas menthol sensitizes the response, shifting the threshold in the opposite direction. Co-application of agonists and antagonists produces predictable cancellation of these effects, suggesting the convergence on a common molecular process. The potential for half maximal activation of TRPM8 activation by cold was approximately 140 mV more negative in native channels compared to recombinant channels, with a much higher open probability at negative membrane potentials in the former. In functional terms, this difference translates into a shift in the apparent temperature threshold for activation towards higher temperatures for native currents. This difference in voltage-dependence readily explains the high threshold temperatures characteristic of many cold thermoreceptors. The modulation of TRPM8 activity by different chemical agents unveils an important flexibility in the temperature-response curve of TRPM8 channels and cold thermoreceptors.

Barium ions inhibit the dynamic response of guinea-pig corneal cold receptors to heating but not to cooling.

An in vitro preparation of the guinea-pig cornea was used to study the effects of the K+ channel blockers 4-aminopyridine (4-AP), tetraethylammonium (TEA) and Ba2+ on nerve terminal impulses (NTIs) recorded extracellularly from cold sensory receptors. These receptors have an ongoing discharge of NTIs that is increased by cooling and decreased by heating. The K+ channel blocker 4-AP reduced the negative amplitude of the diphasic (positive-negative) NTIs, whereas TEA and Ba2+ prolonged the duration of the negative component. As the shape of the NTI is determined by the first derivative (dV/dt) of the membrane voltage change, these changes in the negative component are consistent with the blockade of K+ channels that contribute to action potential repolarization. Only TEA changed the basal activity of the receptors, increasing the likelihood of burst discharges. Ba2+ selectively reduced the response of the receptors to heating, whereas neither 4-AP nor TEA modified the response to heating or to cooling. The findings indicate that K+ channels blocked by 4-AP, TEA and Ba2+ contribute to action potential repolarization in corneal cold receptors, and that ionic mechanisms that underlie the reduction in NTI frequency in response to heating differ from those that increase activity in response to cooling.

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior.

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22 degrees C) and non-thermal hot-plate stress (30 degrees C) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50 degrees C) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50 degrees C; N = 9 in each group). The results (means +/- SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 +/- 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 +/- 3.19 s), 5.0 mg/kg (11.53 +/- 1.64 s) and 7.5 mg/kg naloxone (7.38 +/- 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 +/- 8.53 s) and saline groups (29.1 +/- 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64% in the saline group and maximum 83.2% decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.

The terrestrial Gastropoda Megalobulimus abbreviatus as a useful model for nociceptive experiments: effects of morphine and naloxone on thermal avoidance behavior

We describe the behavior of the snail Megalobulimus abbreviatus upon receiving thermal stimuli and the effects of pretreatment with morphine and naloxone on behavior after a thermal stimulus, in order to establish a useful model for nociceptive experiments. Snails submitted to non-functional (22°C) and non-thermal hot-plate stress (30°C) only displayed exploratory behavior. However, the animals submitted to a thermal stimulus (50°C) displayed biphasic avoidance behavior. Latency was measured from the time the animal was placed on the hot plate to the time when the animal lifted the head-foot complex 1 cm from the substrate, indicating aversive thermal behavior. Other animals were pretreated with morphine (5, 10, 20 mg/kg) or naloxone (2.5, 5.0, 7.5 mg/kg) 15 min prior to receiving a thermal stimulus (50°C; N = 9 in each group). The results (means ± SD) showed an extremely significant difference in response latency between the group treated with 20 mg/kg morphine (63.18 ± 14.47 s) and the other experimental groups (P < 0.001). With 2.5 mg/kg (16.26 ± 3.19 s), 5.0 mg/kg (11.53 ± 1.64 s) and 7.5 mg/kg naloxone (7.38 ± 1.6 s), there was a significant, not dose-dependent decrease in latency compared to the control (33.44 ± 8.53 s) and saline groups (29.1 ± 9.91 s). No statistically significant difference was found between the naloxone-treated groups. With naloxone plus morphine, there was a significant decrease in latency when compared to all other groups (minimum 64 percent in the saline group and maximum 83.2 percent decrease in the morphine group). These results provide evidence of the involvement of endogenous opioid peptides in the control of thermal withdrawal behavior in this snail, and reveal a stereotyped and reproducible avoidance behavior for this snail species, which could be studied in other pharmacological and neurophysiological studies.