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Participation of preoptic area TRPV4 ion channel in regulation of body temperature
Journal of Thermal Biology 66 (2017) 81–86
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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Participation of preoptic area TRPV4 ion channel in regulation of body temperature Rajesh Yadav, Ashok Kumar Jaryal, Hruda Nanda Mallick
MARK
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Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Preoptic area TRPV4 ion channel GSK1016790A RN 1734 Body temperature
Transient receptor potential vanilloid 4 (TRPV4) ion channel is a non-selective cation channel and its role in cutaneous thermosensation is emerging. It is expressed in many areas of the brain including the preoptic area (POA)/anterior hypothalamus which is the key neural site for thermoregulation. The present study was conducted to find out the role of TRPV4 ion channel in the POA in thermoregulation. Rats preimplanted with guide cannulae with indwelling styli 2.0 mm above the POA received TRPV4 agonist/antagonist/isotonic saline injections bilaterally in the POA using an injector cannula in three separate groups of six rats each. Body temperature (Tb) was recorded telemetrically by preimplanted radio transmitter in the peritoneal cavity. The injection of TRPV4 agonist (GSK1016790A) in the POA decreased Tb while its antagonist (RN1734) increased Tb. Immunohistochemical localization showed presence of TRPV4 ion channel in the POA. The results of the present study suggest that TRPV4 ion channels in the POA may play an important role in thermoregulation.
1. Introduction The role of preoptic area (POA) in thermoregulation comes from studies involving direct thermal stimulation of this neural structure. Magoun et al. (1938) observed that localized brain heating evokes panting in anesthetized cats. The POA warming produced cutaneous vasodilatation, sweating, panting and other heat loss behavioral responses (Adair, 1977; Boulant et al., 1980; Hellstorm and Hammel, 1967). Both electrolytic and chemical lesions of the POA produced hyperthermia in rats and cats (Gamble and Patton, 1953; Satinoff et al., 1982; Szymusiak et al., 1985). Intracerebral injections of various neurotransmitters in the POA also influenced Tb e.g. local application of norepinephrine at mPOA produced hypothermia (Datta et al., 1987; Mallick et al., 1988). Recently, transient receptor potential (TRP) channels super family have received considerable attention. The TRPV subfamily TRPV (1−4) initially located in sensory nerve and skin is proposed to be involved in peripheral thermotransduction mechanism (Acs et al., 1996; Nedungadi et al., 2012; Peier et al., 2002). In vitro study shows TRPV (1−4) channels responding to a broad range of temperature from warm to hot (Caterina et al., 1997, 1999; Güler et al., 2002; Peier et al., 2002; Smith et al., 2002; Watanabe et al., 2002a, 2000b; Xu et al., 2002). TRPV (1−4)channels are expressed in number of brain areas including the POA (Acs et al., 1996; Caterina et al., 1997; Güler et al., 2002; Nedungadi et al., 2012). The role of POA TRPV1 channel in thermo-
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Corresponding author. E-mail address: [email protected] (H.N. Mallick).
http://dx.doi.org/10.1016/j.jtherbio.2017.04.001 Received 26 December 2016; Received in revised form 5 April 2017; Accepted 5 April 2017 Available online 06 April 2017 0306-4565/ © 2017 Elsevier Ltd. All rights reserved.
regulation has been well studied. Peripheral or central administration of TRPV1 agonist, capsaicin induces hypothermia (Hori et al., 1984; Jancso-Gabor et al., 1970a, 1970b; Kumar et al., 2011). TRPV4 channel, another member of subfamily TRPV may too have a role in thermoregulation, as they are activated by temperature ranging from 25 to 34 °C as studied in HEK 293 cell line (Güler et al., 2002; Watanabe et al., 2002a). Besides temperature, TRPV4 ion channels are activated by a variety of physical (cell swelling, mechanical stimuli, moderate heat) and chemical stimuli (Nilius et al., 2003, 2004; Watanabe et al., 2002a, 2002b). Initially the role of TRPV4 in osmotic regulation was observed in knockout mouse model of TRPV4 (Mizuno et al., 2003). TRPV4 ion channels are widely expressed in heart, endothelium, liver, kidney, urine bladder, keratinocytes, placenta, lung, trachea, salivary glands including brain (Strotmann et al., 2000; Liedtke et al., 2000; Wissenbach et al., 2000; Delany et al., 2001). Latest studies show the presence of TRPV4 channel in the POA (Güler et al., 2002). Role of TRPV4 ion channels in thermal selection behavior has also been shown in knockout mice (Lee et al., 2005). However, the role of TRPV4 ion channels of the POA in thermoregulation has not been addressed. The present study was undertaken to examine the effect of microinjection of TRPV4 ion channel agonist and its antagonist in the POA on Tb along with localization of TRPV4 channel in the POA.
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2. Materials & methods
injector cannula to mark the injection sites. The rats were perfused transcardially first with 100 ml of isotonic saline (0.9%) and then with 10% formaldehyde with 0.3% potassium permanganate to fix the brain tissue as described earlier (Bagga et al., 1981). Brains were removed from the skull. 10 µm thick paraffin sections of the brains were made and stained with hematoxylin-eosin, for histological examination of the injection sites which appeared blue on histological sections as ferric chloride reacted with potassium permanganate and formed Prussian blue (Fe4 [Fe (CN) 6 ]3·xH2O).
2.1. Animals Adult male Wistar rats (n=24) weighing between 200 and 250 g (breed, reared and maintained in Central Animal Facility of All India Institute of Medical Sciences, New Delhi, India, 623/1AEAC/11) were used for the experiments. The animals were housed in separate polypropylene cages (40×28×15 cm), in an animal room with controlled room temperature (25 ± 1 °C) and having 14 h light period (light above 200 lx) and 10 h dark period (dark below 5 lx) schedule with light on at 6:00 h till 20:00 h. Food and water were provided ad libitum. All procedure were conducted in accordance with the rules of the Committee for the purpose of Control and Supervision for Experiments on Animals (CPCSEA), India and approved by the Institutional Animal Ethics Committee, AIIMS, New Delhi, India. The experimental procedures were also in compliance with the Directive 2010/63/EU of the European parliament and of the Council of 22 September 2010.
2.6. Immunohistochemistry The immunohistochemistry was performed to localize the TRPV4 ion channel in the POA as described earlier (Tóth et al., 2005). The rats of Group 4 were perfused with formalin transcardially. Brains were removed and placed in 10% formalin for 24 h and transferred into 15% and 30% sucrose solution consequently for 48 h. Brains were placed in the cryomedia with OCT (−20 °C) for 15 min and 16 µm coronal cryosection were taken. The sections were placed on poly-L-Lysine coated glass slides (Sigma Aldrich, USA) and stored at 4 °C. Before processing slides, the sections were rehydrated three times each for 20 min by phosphate buffer saline (PBS) and then preincubated for 2 h at room temperature with 10% bovine serum albumin (Sigma Aldrich, USA) to prevent binding of unspecific antibody to receptors. The sections were incubated for 24 h at 4 °C with rabbit polyclonal antibody that recognizes N-Terminus of TRPV4 (Sigma Chemical Co. USA), diluted in 1:100 in PBS). After three round of washing with PBSTX (Phosphate Buffer Saline TritonX) for 5 min each, the sections were again incubated for 2 h with secondary antibody, anti- rabbit IgG-Atto 488. After 2 h fluoroshield media (Sigma Aldrich, USA) was added and covered with cover slips. The brain sections were captured on fluorescent and confocal microscopes (Leica microsystem, Germany) at 10X and 20X magnifications respectively.
2.2. Surgical procedure Surgery was performed under aseptic conditions using pentobarbitone sodium anaesthesia (Aldrich Thomas Co, USA, 40 mg/kg BW, iP) to implant peritoneal transmitter and guide cannulae in the brain. For intracerebral microinjection bilateral guide cannulae made from stainless steel tube 24 G with indwelling styli were implanted aimed 2.0 mm above the POA, according to stereotaxic coordinate (AP 7.8, V 7.5, L ± 0.6) as per D Groot's atlas (De Groot, 1959). A radio transmitter TA10TAF40 (Data Science International, USA) was implanted in peritoneal cavity to record Tb. The rats were allowed seven days to recover from the surgical trauma. 2.3. Drugs and vehicle for microinjection
2.7. Statistical analysis The drugs used in this study TRPV4 agonist GSK1016790A and TRPV4 antagonist, RN1734 was purchased from Sigma-Aldrich, Co. USA. They were dissolved in 0.9% pyrogen-free saline having 6% Tween 80+1% ethanol which was also used as the control/ vehicle (Jancso-Gabor et al., 1970). Anti-TRPV4 polyclonal antibody (produced in rabbit), was used as primary antibody while anti rabbit IgG- Atto 488 antibody (produced in goat) was used as secondary antibody in immunohistochemistry for localization of TRPV4 channel in the POA. All the antibodies were procured from Sigma-Aldrich Co. USA.
The pre and post injection data, after giving TRPV4 agonist, antagonist and vehicle microinjection in Groups 1, 2 and 3, were compared with repeated measure ANOVA test using Graph Pad Prism 6 software. The 2 h pre injection data (Mean ± S.D) of Tb was compared with every 15 min (Mean ± S.D) post injection data of body temperature for 6 h. 3. Results
2.4. Recording procedure and experimental schedule
Effect of microinjection of TRPV4 channel agonist, antagonist and vehicle in the POA on Tb in freely moving rats and localization of thermo TRPV4 channels in the POA are described in the results.
The study was conducted in four groups of 6 rats in each. Animals of Group 1 received TRPV4 agonist GSK1016790A, Group 2 received TRPV4 antagonist RN 1734, and Group 3 received vehicle while immunohistochemistry was performed in Group 4. The Tb was measured from 10.00 to 16.00 h and injection was given at 12.00 h. Three control recordings of Tb were taken on alternate days. Microinjection studies were done on 4th alternative day after control studies. Injections (0.4 µg/0.2 µl) were given bilaterally into the POA at a rate of 0.1 micro litre /min through a 32 G injector cannula inserted through the guide cannulae. The injector cannula was 2 mm longer than the guide cannulae. Tb was recorded telemetrically (DSI, USA) at 15 s interval. Temperature data of 15 min epochs were averaged for statistical analysis.
3.1. Effect of TRPV4 agonist microinjection in the POA on body temperature In Group1, effect of TRPV4 agonist (GSK1016790A, 0.4 µg/0.2 µl) microinjection in the POA in 6 rats on Tb is shown in Fig. 1A. The Tb during 2 h pre injection period was 37.47 ± 0.2 °C (Mean ± SD). After the microinjection of TRPV4 agonist, GSK1016790A, there was significant fall in the Tb during first one hour of injection. The temperature started falling 15 min after the injection and the effect lasted for more than one hour. The maximum fall in Tb was about 0.9 °C and it was observed around 75 min of injection.
2.5. Histology and perfusion 3.2. Effect of TRPV4 antagonist microinjection in the POA on body temperature
At the end of the experiments the rats of Group 1, 2 and 3 were anaesthetized with pentobarbitone sodium (50–60 mg/kg B.W, I.P) and 2% ferric chloride (0.4 μg/0.2 µl) was injected in the POA through the
The effect of TRPV4 antagonist RN 1734 microinjection in the POA 82
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Fig. 1. Shows body temperature (Mean ± S.D) at 15 min interval during 2 h pre injection recording (10:00–12:00 h) and 4 h post injection recording (12:00–16:00 h) of 6 rats in each groups. A. Effect of TRPV4 agonist (GSK1016790A, 0.4 µg/0.2 µl) on body temperature (n=6). B. Effect of TRPV4 antagonist (RN1734, 0.4 µg/0.2 µl) on body temperature (n=6) C. Effect of Vehicle (0.2 µl of 0.9%) on body temperature (n=6). Microinjection was made at 12 h indicated by (↑). The pre and post injection data were analyzed with repeated measure analysis of variance (ANOVA). * P < 0.05, ** P < 0.01, *** P < 0.005 represent level of significance. Solid line (-) represents body temperature recording. X- Axis shows time in hours. Y-Axis shows temperature in °C.
Fig. 2. Shows the sites of injection of TRPV4 agonist/TRPV4 antagonist /Saline control in three groups of rats. A. Photomicrograph of a rat brain section at the level of POA, showing the injection site (indicate by horizontal arrow). AC –Anterior commissure; OCOptic chiasma; Scale bar- 100 µm. B. Shows the sites of drug injection (•) in 18 rats 6 in each groups which reconstructed from the coronal histological sections and drawn at coordinates of A 7.8 as per De Groot's atlas.
dorsoventrally from about 5.5 mm below the below the anterior commissure level of anterior commissure. A representative Prussian blue spot indicated by arrow for confirmation of microinjection site is shown in Fig. 2A. The microinjection sites confirmed by Prussian blue spots in 18 rats in group1, 2, 3 are redrawn and shown in Fig. 2B.
in 6 rats on Tb in Group 2 is shown in Fig. 1B. Average of 2 h preinjection recording of Tb for TRPV4 antagonist group was 37.4 ± 0.18 °C. There was increase in Tb after microinjection of RN 1734, which was significant during 12.45–14.45 h. The magnitude of maximum increase in Tb was about 0.98 °C. The Tb did not return to its baseline value till 16.00 h.
3.5. Localization of TRPV4 channel in the preoptic area The images obtained by fluorescent and confocal microscope at 10× (Fig. 3 A) and 20× (Fig. 3B) magnification showed green fluorescent reaction in black background indicating the presence of TRPV4 channel in the POA (Fig. 3). Fig. 3C and D showed confocal images indicating TRPV4 channel in the POA at 10 X magnification.
3.3. Effect of vehicle microinjection in the POA on body temperature In Group 3, the pre and post-injection effects of vehicle microinjection in the POA in 6 rats on Tb is presented in Fig. 1C. The average of 2 h pre-injection recording of Tb for vehicle group was 37.46 ± 0.11 °C. After vehicle microinjection, there was no significant change in Tb.
4. Discussion The microinjection of TRPV4 agonist, GSK1016790A in the POA produced fall in the Tb. TRPV4 is a non-selective cation channel, and it was first described as an osmosensor as activated by decrease in osmolarity (Liedtke et al., 2000; Nilius et al., 2004; Nilius and Voets, 2004; Strotmann et al., 2000) Recent studies suggest that TRPV4 ion channels may be a polymodal receptor since it can be activated by heat
3.4. Histological confirmation of microinjection site All the 18 rats of the Group 1, 2 and 3 showed Prussian blue spot at the site of microinjections, which extended in the antero-posterior direction from 7.8 to 8.4 from the vertical plane as per De Groot's atlas. Microinjection sites extended upto 0.6 mm from the midline and 83
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Fig. 3. Shows fluorescent microscopic images of rat brain coronal sections at the level of preoptic area. Immunofluorescent reaction to TRPV4 ion channel shown at 10 X magnification in A and 20 X in B while C and D shows confocal microscopic images of the sections of brain showing immunofluorescent reaction to TRPV4 ion channel in the POA at 10× magnification. E and F shows fluorescent microscopic images of brain section without using primary antibody (control). 3 V, third ventricle, arrow shows TRPV4 channel. Anti TRPV4 antibody dilution 1:100.
capsaicin in the rat POA produced hypothermia (Jancso-Gabor et al., 1970a, 1970b). Repeated injection of capsaicin produced desensitization of the response and desensitized rats lost the ability to regulate the Tb when placed in heated environment. Local heating of anterior hypothalamus normally leads to hypothermia. It was absent in rats desensitized with capsaicin. Such studies provided evidence that, the capsaicin responsive cells are necessary for normal heat sensing; although it is now known that capsaicin is an agonist of TRPV1 ion channels which is found in the POA (Acs et al., 1996). In addition, the hypothermic response to capsaicin is almost entirely lost in TRPV1 knock out mice, emphasizing the role of TRPV1 channels in thermoregulation (Toth et al., 2011). Our result shows presence TRPV4 ion
(Güler et al., 2002; Watanabe et al., 2002a, 2002b), low pH and citrate (Tóth et al., 2005) in addition to osmotic stimulation. Most studies on TRPV4 ion channels activation and function have been performed in transfected cells or cells expressing TRPV4 ion channel (Liedtke et al., 2000; Strotmann et al., 2000; Watanabe et al., 2002). To the best of our knowledge this is the first evidence to show in vivo activation of TRPV4 ion channel in the POA leading to fall in Tb. The POA contains 40% thermosensitive neurons out of which 30% are warm sensitive and 10% are cold sensitive (Boulant et al., 1973; Hardy et al., 1964; Hellon, 1967; Nakayama et al., 1963). The activation of warm sensitive neurons in warm environment downregulate the body temperature by heat dissipation mechanism. Long ago it was shown that injection of 84
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5. Conclusion
channels in the POA and its activation produced hypothermia which is similar to the effect that is produced by TRPV1. However this requires the presence of TRPV4 ion channel on the warm sensitive neurons. Punctate staining of TRPV4 has been observed in the neuropil of the POA and anterior hypothalamus (Wechselberger et al., 2006). Although the molecular identity of warm sensitive neuron remains unknown, a recent study reveals that about 2/3rd of parvalbumin (PV) positive neurons in the anterior hypothalamus area can be activated by heat and other remaining neurons are inhibited by heat. Strikingly, more than half of the PV cells express TRPV4 ion channels (Wang et al., 2015). The significance of TRPV4 ion channel in thermotransduction mechanism in cutaneous thermoreceptor is better understood. TRPV4 is highly expressed in epidermal keratinocytes that are able to detect modest temperature changes, contributing to warmth perception and cutaneous thermoregulation (Chung et al., 2003, 2004). It is still not clear to date what exactly is the signal transduction mechanism between the skin cells and thermo sensory nerve fibres. It is hypothesized that TRPV4 activation releases ATP which might activate purinergic receptor in the free sensory nerve ending (Chung et al., 2004). Lee et al. (2005) showed that TRPV4-/- mice demonstrate preference for warmer temperature compared to wild type mice undergoing thermal gradient or temperature selection test while chemical activation of TRPV4 ion channel in the skin leads to selection of cooler temperature. This effect was blocked by intervention of TRPV4 antagonist. Taking into consideration the role of TRPV4 ion channel in the peripheral and central regulation of Tb, it is proposed that TRPV4 ion channels play a crucial role in maintaining the Tb within the innocuous temperature range either by behavioral thermoregulation or by involving heat dissipation mechanism. In the recent study, Vizin et al. (2015) showed that chemical activation of TRPV4 ion channels by topical application of TRPV4 agonist, RN1747 on the skin leads to hypothermia and this effect is blocked by the pretreatment with the selective antagonist of the channel. However intracerebroventricular treatment with RN1747 did not cause hypothermia, indicating that the observed response was indeed due to activation of TRPV4 ion channels in the periphery and not due to central thermosensors. However, in the present study microinjection of TRPV4 agonist, GSK1016790A in the POA produced hypothermia. In this study TRPV4 channel was also localized in the POA, which supports earlier finding of Guler et al. (2002). TRPV4 ion channels which are activated in vitro at temperature in the range of approx. 24–34 °C (Güler et al., 2002; Kauer et al., 2009). Consistent with their thermal sensitivity in vitro, the TRPV4 ion channels were located at some anatomical location compatible with thermoregulatory function which include primary sensory neurons, keratinocytes, endothelial cells sweat glands sympathetic neurons and POA (AlessandriHaber et al., 2003; Delany et al., 2001; Güler et al., 2002; Liedtke et al., 2000; Watanabe et al., 2002a). In fact TRPV4 ion channels have been proposed to act as thermoreceptor as genetic ablation of TRPV4 mice lead to selection of warmer temperature than control animals when exposed to thermal gradient or when they had to choose between two temperatures (Lee et al., 2005). However whether this channel is activated in warmth condition in vivo in a non- genetically modified animal and whether it recruits autonomic and /or behavioral thermoregulatory mechanism were not assessed. Microinjection of TRPV4 antagonist RN 1734 in the POA produced significant rise in Tb and the effect was long lasting. TRPV4 ion channels are activated in vitro at temperature in the range of approximately 24–34 °C (Güler et al., 2002; Kauer et al., 2009). We did all the recording at an ambient temperature of 25 °C. Intravenous blockade of TRPV4 ion channel with HC-067047 caused an increased body temperature at ambient temperature of 26 °C and 30 °C but not 22 °C and 32 °C (Vizin et al., 2015). Taking into consideration results of all these studies including ours, TRPV4 ion channels in the POA and outside the POA may play a role in homeostatic regulation of Tb by both thermoregulatory as well as behavioral regulations.
The TPV4 ion channel in the POA is an important component of thermoregulatory mechanism. Activation of TPV4 ion channel decreased body temperature whereas its blockade produced hyperthermia. These results along with others suggest the role of the TRPV4 ion channel not only in thermotransduction but also in thermoregulation. Conflicts of Interest None. Disclosure Study concept and design: All authors; Acquisition of data: Rajesh Yadav; Analysis and data interpretation: Rajesh Yadav; Drafting of the manuscript: All authors; Critical revision of the manuscript for important intellectual content: All authors; Administrative, technical or material support: Dr. Hruda Nanda Mallick; Study supervision: Dr. Hruda Nanda Mallick and Dr. Ashok Kumar Jaryal. Acknowledgements The study was supported by Indian Council of Medical Research, New Delhi, India (BMS 55/3/2011). Statistical help rendered by Dr. Bhasker, Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, is acknowledged. References Acs, G., Palkovits, M., Blumberg, P.M., 1996. Specific binding of [3H]resiniferatoxin by human and rat preoptic area, locus coeruleus, medial hypothalamus, reticular formation and ventral thalamus membrane preparations. Life Sci. 59, 1899–1908. Adair, E.R., 1977. Skin, preoptic, and core temperatures influence behavioral thermoregulation. J. Appl. Physiol. Respir. Environ. Exerc Physiol. 42, 559–564. Alessandri-Haber, N., Yeh, J.J., Boyd, A.E., Parada, C.A., Chen, X., Reichling, D.B., Levine, J.D., 2003. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron 39, 497–511. Bagga, N., Chhina, G.S., Kumar, V.M., Singh, B., 1981. Mechanism of participation of medial preoptic area in the hippocampal inhibition of ovulation. Brain Res. 216, 444–448. Boulant, J.A., Bignall, K.E., 1973. Brain neuronal responses to peripheral and deep-body temperatures. Am. J. Physiol. 225, 1371–1374. Boulant, J.A., 1980. Brain baseline of thermoregulation. Neurophysiological basis. In: Morgane, P.J., Panksepp, J. (Eds.), Handbook of hypothalamus. Marcel Dekker Inc, New York, pp. 1–82. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824. Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J., Julius, D., A., 1999. Capsaicinreceptor homologue with a high threshold for noxious heat. Nature 398, 436–441. Chung, M.K., Lee, H., Caterina, M.J., 2003. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J. Biol. Chem. 278, 32037–32046. Chung, M.K., Lee, H., Mizuno, A., Suzuki, M., Caterina, M.J., 2004. 2aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J. Neurosci. 24, 5177–5182. Datta, S., Kumar, V.M., Chhina, G.S., Singh, B., 1987. Effect of application of serotonin in medial preoptic area on body temperature and sleep-wakefulness. Indian J. Exp. Biol. 25, 681–685. De, G.R.O.O.T., 1959. The rat hypothalamus in stereotaxic coordinates. J. Comp. Neurol. 113, 389–400. Delany, N.S., Hurle, M., Facer, P., Alnadaf, T., Plumpton, C., Kinghorn, I., See, C.G., Costigan, M., Anand, P., Woolf, C.J., Crowther, D., Sanseau, P., Tate, S.N., 2001. Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol. Genom. 4, 165–174. Gamble, J.E., Patton, H.D., 1953. Pulmonary edema and haemorrhage from preoptic lesions in rats. Am. J. Physiol. 172, 623–631. Güler, A.D., Lee, H., Iida, T., Shimizu, I., Tominaga, M., Caterina, M., 2002. Heat-evoked activation of the ion channel, TRPV4. J. Neurosci. 22, 6408–6414. Hardy, J.D., Hellon, R.F., Sutherland, K., 1964. Temperature-sensitive neurons in the dog's hypothalamus. J. Physiol. 175, 242–253. Hellon, R.F., 1967. Thermal stimulation of brain neurons in anaesthetized rabbits. J. Physiol. 19, 381–395. Hellstrom, B., Hammel, H.T., 1967. Some characteristics of temperature regulation in the unanesthetized dog. Am. J. Physiol. 213, 547–556. Hori, T., 1984. Capsaicin and central control of thermoregulation. Pharmacol. Ther. 26,
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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Participation of preoptic area TRPV4 ion channel in regulation of body temperature Rajesh Yadav, Ashok Kumar Jaryal, Hruda Nanda Mallick
MARK
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Department of Physiology, All India Institute of Medical Sciences, New Delhi 110029, India
A R T I C L E I N F O
A B S T R A C T
Keywords: Preoptic area TRPV4 ion channel GSK1016790A RN 1734 Body temperature
Transient receptor potential vanilloid 4 (TRPV4) ion channel is a non-selective cation channel and its role in cutaneous thermosensation is emerging. It is expressed in many areas of the brain including the preoptic area (POA)/anterior hypothalamus which is the key neural site for thermoregulation. The present study was conducted to find out the role of TRPV4 ion channel in the POA in thermoregulation. Rats preimplanted with guide cannulae with indwelling styli 2.0 mm above the POA received TRPV4 agonist/antagonist/isotonic saline injections bilaterally in the POA using an injector cannula in three separate groups of six rats each. Body temperature (Tb) was recorded telemetrically by preimplanted radio transmitter in the peritoneal cavity. The injection of TRPV4 agonist (GSK1016790A) in the POA decreased Tb while its antagonist (RN1734) increased Tb. Immunohistochemical localization showed presence of TRPV4 ion channel in the POA. The results of the present study suggest that TRPV4 ion channels in the POA may play an important role in thermoregulation.
1. Introduction The role of preoptic area (POA) in thermoregulation comes from studies involving direct thermal stimulation of this neural structure. Magoun et al. (1938) observed that localized brain heating evokes panting in anesthetized cats. The POA warming produced cutaneous vasodilatation, sweating, panting and other heat loss behavioral responses (Adair, 1977; Boulant et al., 1980; Hellstorm and Hammel, 1967). Both electrolytic and chemical lesions of the POA produced hyperthermia in rats and cats (Gamble and Patton, 1953; Satinoff et al., 1982; Szymusiak et al., 1985). Intracerebral injections of various neurotransmitters in the POA also influenced Tb e.g. local application of norepinephrine at mPOA produced hypothermia (Datta et al., 1987; Mallick et al., 1988). Recently, transient receptor potential (TRP) channels super family have received considerable attention. The TRPV subfamily TRPV (1−4) initially located in sensory nerve and skin is proposed to be involved in peripheral thermotransduction mechanism (Acs et al., 1996; Nedungadi et al., 2012; Peier et al., 2002). In vitro study shows TRPV (1−4) channels responding to a broad range of temperature from warm to hot (Caterina et al., 1997, 1999; Güler et al., 2002; Peier et al., 2002; Smith et al., 2002; Watanabe et al., 2002a, 2000b; Xu et al., 2002). TRPV (1−4)channels are expressed in number of brain areas including the POA (Acs et al., 1996; Caterina et al., 1997; Güler et al., 2002; Nedungadi et al., 2012). The role of POA TRPV1 channel in thermo-
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Corresponding author. E-mail address: [email protected] (H.N. Mallick).
http://dx.doi.org/10.1016/j.jtherbio.2017.04.001 Received 26 December 2016; Received in revised form 5 April 2017; Accepted 5 April 2017 Available online 06 April 2017 0306-4565/ © 2017 Elsevier Ltd. All rights reserved.
regulation has been well studied. Peripheral or central administration of TRPV1 agonist, capsaicin induces hypothermia (Hori et al., 1984; Jancso-Gabor et al., 1970a, 1970b; Kumar et al., 2011). TRPV4 channel, another member of subfamily TRPV may too have a role in thermoregulation, as they are activated by temperature ranging from 25 to 34 °C as studied in HEK 293 cell line (Güler et al., 2002; Watanabe et al., 2002a). Besides temperature, TRPV4 ion channels are activated by a variety of physical (cell swelling, mechanical stimuli, moderate heat) and chemical stimuli (Nilius et al., 2003, 2004; Watanabe et al., 2002a, 2002b). Initially the role of TRPV4 in osmotic regulation was observed in knockout mouse model of TRPV4 (Mizuno et al., 2003). TRPV4 ion channels are widely expressed in heart, endothelium, liver, kidney, urine bladder, keratinocytes, placenta, lung, trachea, salivary glands including brain (Strotmann et al., 2000; Liedtke et al., 2000; Wissenbach et al., 2000; Delany et al., 2001). Latest studies show the presence of TRPV4 channel in the POA (Güler et al., 2002). Role of TRPV4 ion channels in thermal selection behavior has also been shown in knockout mice (Lee et al., 2005). However, the role of TRPV4 ion channels of the POA in thermoregulation has not been addressed. The present study was undertaken to examine the effect of microinjection of TRPV4 ion channel agonist and its antagonist in the POA on Tb along with localization of TRPV4 channel in the POA.
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2. Materials & methods
injector cannula to mark the injection sites. The rats were perfused transcardially first with 100 ml of isotonic saline (0.9%) and then with 10% formaldehyde with 0.3% potassium permanganate to fix the brain tissue as described earlier (Bagga et al., 1981). Brains were removed from the skull. 10 µm thick paraffin sections of the brains were made and stained with hematoxylin-eosin, for histological examination of the injection sites which appeared blue on histological sections as ferric chloride reacted with potassium permanganate and formed Prussian blue (Fe4 [Fe (CN) 6 ]3·xH2O).
2.1. Animals Adult male Wistar rats (n=24) weighing between 200 and 250 g (breed, reared and maintained in Central Animal Facility of All India Institute of Medical Sciences, New Delhi, India, 623/1AEAC/11) were used for the experiments. The animals were housed in separate polypropylene cages (40×28×15 cm), in an animal room with controlled room temperature (25 ± 1 °C) and having 14 h light period (light above 200 lx) and 10 h dark period (dark below 5 lx) schedule with light on at 6:00 h till 20:00 h. Food and water were provided ad libitum. All procedure were conducted in accordance with the rules of the Committee for the purpose of Control and Supervision for Experiments on Animals (CPCSEA), India and approved by the Institutional Animal Ethics Committee, AIIMS, New Delhi, India. The experimental procedures were also in compliance with the Directive 2010/63/EU of the European parliament and of the Council of 22 September 2010.
2.6. Immunohistochemistry The immunohistochemistry was performed to localize the TRPV4 ion channel in the POA as described earlier (Tóth et al., 2005). The rats of Group 4 were perfused with formalin transcardially. Brains were removed and placed in 10% formalin for 24 h and transferred into 15% and 30% sucrose solution consequently for 48 h. Brains were placed in the cryomedia with OCT (−20 °C) for 15 min and 16 µm coronal cryosection were taken. The sections were placed on poly-L-Lysine coated glass slides (Sigma Aldrich, USA) and stored at 4 °C. Before processing slides, the sections were rehydrated three times each for 20 min by phosphate buffer saline (PBS) and then preincubated for 2 h at room temperature with 10% bovine serum albumin (Sigma Aldrich, USA) to prevent binding of unspecific antibody to receptors. The sections were incubated for 24 h at 4 °C with rabbit polyclonal antibody that recognizes N-Terminus of TRPV4 (Sigma Chemical Co. USA), diluted in 1:100 in PBS). After three round of washing with PBSTX (Phosphate Buffer Saline TritonX) for 5 min each, the sections were again incubated for 2 h with secondary antibody, anti- rabbit IgG-Atto 488. After 2 h fluoroshield media (Sigma Aldrich, USA) was added and covered with cover slips. The brain sections were captured on fluorescent and confocal microscopes (Leica microsystem, Germany) at 10X and 20X magnifications respectively.
2.2. Surgical procedure Surgery was performed under aseptic conditions using pentobarbitone sodium anaesthesia (Aldrich Thomas Co, USA, 40 mg/kg BW, iP) to implant peritoneal transmitter and guide cannulae in the brain. For intracerebral microinjection bilateral guide cannulae made from stainless steel tube 24 G with indwelling styli were implanted aimed 2.0 mm above the POA, according to stereotaxic coordinate (AP 7.8, V 7.5, L ± 0.6) as per D Groot's atlas (De Groot, 1959). A radio transmitter TA10TAF40 (Data Science International, USA) was implanted in peritoneal cavity to record Tb. The rats were allowed seven days to recover from the surgical trauma. 2.3. Drugs and vehicle for microinjection
2.7. Statistical analysis The drugs used in this study TRPV4 agonist GSK1016790A and TRPV4 antagonist, RN1734 was purchased from Sigma-Aldrich, Co. USA. They were dissolved in 0.9% pyrogen-free saline having 6% Tween 80+1% ethanol which was also used as the control/ vehicle (Jancso-Gabor et al., 1970). Anti-TRPV4 polyclonal antibody (produced in rabbit), was used as primary antibody while anti rabbit IgG- Atto 488 antibody (produced in goat) was used as secondary antibody in immunohistochemistry for localization of TRPV4 channel in the POA. All the antibodies were procured from Sigma-Aldrich Co. USA.
The pre and post injection data, after giving TRPV4 agonist, antagonist and vehicle microinjection in Groups 1, 2 and 3, were compared with repeated measure ANOVA test using Graph Pad Prism 6 software. The 2 h pre injection data (Mean ± S.D) of Tb was compared with every 15 min (Mean ± S.D) post injection data of body temperature for 6 h. 3. Results
2.4. Recording procedure and experimental schedule
Effect of microinjection of TRPV4 channel agonist, antagonist and vehicle in the POA on Tb in freely moving rats and localization of thermo TRPV4 channels in the POA are described in the results.
The study was conducted in four groups of 6 rats in each. Animals of Group 1 received TRPV4 agonist GSK1016790A, Group 2 received TRPV4 antagonist RN 1734, and Group 3 received vehicle while immunohistochemistry was performed in Group 4. The Tb was measured from 10.00 to 16.00 h and injection was given at 12.00 h. Three control recordings of Tb were taken on alternate days. Microinjection studies were done on 4th alternative day after control studies. Injections (0.4 µg/0.2 µl) were given bilaterally into the POA at a rate of 0.1 micro litre /min through a 32 G injector cannula inserted through the guide cannulae. The injector cannula was 2 mm longer than the guide cannulae. Tb was recorded telemetrically (DSI, USA) at 15 s interval. Temperature data of 15 min epochs were averaged for statistical analysis.
3.1. Effect of TRPV4 agonist microinjection in the POA on body temperature In Group1, effect of TRPV4 agonist (GSK1016790A, 0.4 µg/0.2 µl) microinjection in the POA in 6 rats on Tb is shown in Fig. 1A. The Tb during 2 h pre injection period was 37.47 ± 0.2 °C (Mean ± SD). After the microinjection of TRPV4 agonist, GSK1016790A, there was significant fall in the Tb during first one hour of injection. The temperature started falling 15 min after the injection and the effect lasted for more than one hour. The maximum fall in Tb was about 0.9 °C and it was observed around 75 min of injection.
2.5. Histology and perfusion 3.2. Effect of TRPV4 antagonist microinjection in the POA on body temperature
At the end of the experiments the rats of Group 1, 2 and 3 were anaesthetized with pentobarbitone sodium (50–60 mg/kg B.W, I.P) and 2% ferric chloride (0.4 μg/0.2 µl) was injected in the POA through the
The effect of TRPV4 antagonist RN 1734 microinjection in the POA 82
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Fig. 1. Shows body temperature (Mean ± S.D) at 15 min interval during 2 h pre injection recording (10:00–12:00 h) and 4 h post injection recording (12:00–16:00 h) of 6 rats in each groups. A. Effect of TRPV4 agonist (GSK1016790A, 0.4 µg/0.2 µl) on body temperature (n=6). B. Effect of TRPV4 antagonist (RN1734, 0.4 µg/0.2 µl) on body temperature (n=6) C. Effect of Vehicle (0.2 µl of 0.9%) on body temperature (n=6). Microinjection was made at 12 h indicated by (↑). The pre and post injection data were analyzed with repeated measure analysis of variance (ANOVA). * P < 0.05, ** P < 0.01, *** P < 0.005 represent level of significance. Solid line (-) represents body temperature recording. X- Axis shows time in hours. Y-Axis shows temperature in °C.
Fig. 2. Shows the sites of injection of TRPV4 agonist/TRPV4 antagonist /Saline control in three groups of rats. A. Photomicrograph of a rat brain section at the level of POA, showing the injection site (indicate by horizontal arrow). AC –Anterior commissure; OCOptic chiasma; Scale bar- 100 µm. B. Shows the sites of drug injection (•) in 18 rats 6 in each groups which reconstructed from the coronal histological sections and drawn at coordinates of A 7.8 as per De Groot's atlas.
dorsoventrally from about 5.5 mm below the below the anterior commissure level of anterior commissure. A representative Prussian blue spot indicated by arrow for confirmation of microinjection site is shown in Fig. 2A. The microinjection sites confirmed by Prussian blue spots in 18 rats in group1, 2, 3 are redrawn and shown in Fig. 2B.
in 6 rats on Tb in Group 2 is shown in Fig. 1B. Average of 2 h preinjection recording of Tb for TRPV4 antagonist group was 37.4 ± 0.18 °C. There was increase in Tb after microinjection of RN 1734, which was significant during 12.45–14.45 h. The magnitude of maximum increase in Tb was about 0.98 °C. The Tb did not return to its baseline value till 16.00 h.
3.5. Localization of TRPV4 channel in the preoptic area The images obtained by fluorescent and confocal microscope at 10× (Fig. 3 A) and 20× (Fig. 3B) magnification showed green fluorescent reaction in black background indicating the presence of TRPV4 channel in the POA (Fig. 3). Fig. 3C and D showed confocal images indicating TRPV4 channel in the POA at 10 X magnification.
3.3. Effect of vehicle microinjection in the POA on body temperature In Group 3, the pre and post-injection effects of vehicle microinjection in the POA in 6 rats on Tb is presented in Fig. 1C. The average of 2 h pre-injection recording of Tb for vehicle group was 37.46 ± 0.11 °C. After vehicle microinjection, there was no significant change in Tb.
4. Discussion The microinjection of TRPV4 agonist, GSK1016790A in the POA produced fall in the Tb. TRPV4 is a non-selective cation channel, and it was first described as an osmosensor as activated by decrease in osmolarity (Liedtke et al., 2000; Nilius et al., 2004; Nilius and Voets, 2004; Strotmann et al., 2000) Recent studies suggest that TRPV4 ion channels may be a polymodal receptor since it can be activated by heat
3.4. Histological confirmation of microinjection site All the 18 rats of the Group 1, 2 and 3 showed Prussian blue spot at the site of microinjections, which extended in the antero-posterior direction from 7.8 to 8.4 from the vertical plane as per De Groot's atlas. Microinjection sites extended upto 0.6 mm from the midline and 83
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Fig. 3. Shows fluorescent microscopic images of rat brain coronal sections at the level of preoptic area. Immunofluorescent reaction to TRPV4 ion channel shown at 10 X magnification in A and 20 X in B while C and D shows confocal microscopic images of the sections of brain showing immunofluorescent reaction to TRPV4 ion channel in the POA at 10× magnification. E and F shows fluorescent microscopic images of brain section without using primary antibody (control). 3 V, third ventricle, arrow shows TRPV4 channel. Anti TRPV4 antibody dilution 1:100.
capsaicin in the rat POA produced hypothermia (Jancso-Gabor et al., 1970a, 1970b). Repeated injection of capsaicin produced desensitization of the response and desensitized rats lost the ability to regulate the Tb when placed in heated environment. Local heating of anterior hypothalamus normally leads to hypothermia. It was absent in rats desensitized with capsaicin. Such studies provided evidence that, the capsaicin responsive cells are necessary for normal heat sensing; although it is now known that capsaicin is an agonist of TRPV1 ion channels which is found in the POA (Acs et al., 1996). In addition, the hypothermic response to capsaicin is almost entirely lost in TRPV1 knock out mice, emphasizing the role of TRPV1 channels in thermoregulation (Toth et al., 2011). Our result shows presence TRPV4 ion
(Güler et al., 2002; Watanabe et al., 2002a, 2002b), low pH and citrate (Tóth et al., 2005) in addition to osmotic stimulation. Most studies on TRPV4 ion channels activation and function have been performed in transfected cells or cells expressing TRPV4 ion channel (Liedtke et al., 2000; Strotmann et al., 2000; Watanabe et al., 2002). To the best of our knowledge this is the first evidence to show in vivo activation of TRPV4 ion channel in the POA leading to fall in Tb. The POA contains 40% thermosensitive neurons out of which 30% are warm sensitive and 10% are cold sensitive (Boulant et al., 1973; Hardy et al., 1964; Hellon, 1967; Nakayama et al., 1963). The activation of warm sensitive neurons in warm environment downregulate the body temperature by heat dissipation mechanism. Long ago it was shown that injection of 84
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5. Conclusion
channels in the POA and its activation produced hypothermia which is similar to the effect that is produced by TRPV1. However this requires the presence of TRPV4 ion channel on the warm sensitive neurons. Punctate staining of TRPV4 has been observed in the neuropil of the POA and anterior hypothalamus (Wechselberger et al., 2006). Although the molecular identity of warm sensitive neuron remains unknown, a recent study reveals that about 2/3rd of parvalbumin (PV) positive neurons in the anterior hypothalamus area can be activated by heat and other remaining neurons are inhibited by heat. Strikingly, more than half of the PV cells express TRPV4 ion channels (Wang et al., 2015). The significance of TRPV4 ion channel in thermotransduction mechanism in cutaneous thermoreceptor is better understood. TRPV4 is highly expressed in epidermal keratinocytes that are able to detect modest temperature changes, contributing to warmth perception and cutaneous thermoregulation (Chung et al., 2003, 2004). It is still not clear to date what exactly is the signal transduction mechanism between the skin cells and thermo sensory nerve fibres. It is hypothesized that TRPV4 activation releases ATP which might activate purinergic receptor in the free sensory nerve ending (Chung et al., 2004). Lee et al. (2005) showed that TRPV4-/- mice demonstrate preference for warmer temperature compared to wild type mice undergoing thermal gradient or temperature selection test while chemical activation of TRPV4 ion channel in the skin leads to selection of cooler temperature. This effect was blocked by intervention of TRPV4 antagonist. Taking into consideration the role of TRPV4 ion channel in the peripheral and central regulation of Tb, it is proposed that TRPV4 ion channels play a crucial role in maintaining the Tb within the innocuous temperature range either by behavioral thermoregulation or by involving heat dissipation mechanism. In the recent study, Vizin et al. (2015) showed that chemical activation of TRPV4 ion channels by topical application of TRPV4 agonist, RN1747 on the skin leads to hypothermia and this effect is blocked by the pretreatment with the selective antagonist of the channel. However intracerebroventricular treatment with RN1747 did not cause hypothermia, indicating that the observed response was indeed due to activation of TRPV4 ion channels in the periphery and not due to central thermosensors. However, in the present study microinjection of TRPV4 agonist, GSK1016790A in the POA produced hypothermia. In this study TRPV4 channel was also localized in the POA, which supports earlier finding of Guler et al. (2002). TRPV4 ion channels which are activated in vitro at temperature in the range of approx. 24–34 °C (Güler et al., 2002; Kauer et al., 2009). Consistent with their thermal sensitivity in vitro, the TRPV4 ion channels were located at some anatomical location compatible with thermoregulatory function which include primary sensory neurons, keratinocytes, endothelial cells sweat glands sympathetic neurons and POA (AlessandriHaber et al., 2003; Delany et al., 2001; Güler et al., 2002; Liedtke et al., 2000; Watanabe et al., 2002a). In fact TRPV4 ion channels have been proposed to act as thermoreceptor as genetic ablation of TRPV4 mice lead to selection of warmer temperature than control animals when exposed to thermal gradient or when they had to choose between two temperatures (Lee et al., 2005). However whether this channel is activated in warmth condition in vivo in a non- genetically modified animal and whether it recruits autonomic and /or behavioral thermoregulatory mechanism were not assessed. Microinjection of TRPV4 antagonist RN 1734 in the POA produced significant rise in Tb and the effect was long lasting. TRPV4 ion channels are activated in vitro at temperature in the range of approximately 24–34 °C (Güler et al., 2002; Kauer et al., 2009). We did all the recording at an ambient temperature of 25 °C. Intravenous blockade of TRPV4 ion channel with HC-067047 caused an increased body temperature at ambient temperature of 26 °C and 30 °C but not 22 °C and 32 °C (Vizin et al., 2015). Taking into consideration results of all these studies including ours, TRPV4 ion channels in the POA and outside the POA may play a role in homeostatic regulation of Tb by both thermoregulatory as well as behavioral regulations.
The TPV4 ion channel in the POA is an important component of thermoregulatory mechanism. Activation of TPV4 ion channel decreased body temperature whereas its blockade produced hyperthermia. These results along with others suggest the role of the TRPV4 ion channel not only in thermotransduction but also in thermoregulation. Conflicts of Interest None. Disclosure Study concept and design: All authors; Acquisition of data: Rajesh Yadav; Analysis and data interpretation: Rajesh Yadav; Drafting of the manuscript: All authors; Critical revision of the manuscript for important intellectual content: All authors; Administrative, technical or material support: Dr. Hruda Nanda Mallick; Study supervision: Dr. Hruda Nanda Mallick and Dr. Ashok Kumar Jaryal. Acknowledgements The study was supported by Indian Council of Medical Research, New Delhi, India (BMS 55/3/2011). Statistical help rendered by Dr. Bhasker, Department of Biostatistics, All India Institute of Medical Sciences, New Delhi, is acknowledged. References Acs, G., Palkovits, M., Blumberg, P.M., 1996. Specific binding of [3H]resiniferatoxin by human and rat preoptic area, locus coeruleus, medial hypothalamus, reticular formation and ventral thalamus membrane preparations. Life Sci. 59, 1899–1908. Adair, E.R., 1977. Skin, preoptic, and core temperatures influence behavioral thermoregulation. J. Appl. Physiol. Respir. Environ. Exerc Physiol. 42, 559–564. Alessandri-Haber, N., Yeh, J.J., Boyd, A.E., Parada, C.A., Chen, X., Reichling, D.B., Levine, J.D., 2003. Hypotonicity induces TRPV4-mediated nociception in rat. Neuron 39, 497–511. Bagga, N., Chhina, G.S., Kumar, V.M., Singh, B., 1981. Mechanism of participation of medial preoptic area in the hippocampal inhibition of ovulation. Brain Res. 216, 444–448. Boulant, J.A., Bignall, K.E., 1973. Brain neuronal responses to peripheral and deep-body temperatures. Am. J. Physiol. 225, 1371–1374. Boulant, J.A., 1980. Brain baseline of thermoregulation. Neurophysiological basis. In: Morgane, P.J., Panksepp, J. (Eds.), Handbook of hypothalamus. Marcel Dekker Inc, New York, pp. 1–82. Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D., Julius, D., 1997. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389, 816–824. Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J., Julius, D., A., 1999. Capsaicinreceptor homologue with a high threshold for noxious heat. Nature 398, 436–441. Chung, M.K., Lee, H., Caterina, M.J., 2003. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J. Biol. Chem. 278, 32037–32046. Chung, M.K., Lee, H., Mizuno, A., Suzuki, M., Caterina, M.J., 2004. 2aminoethoxydiphenyl borate activates and sensitizes the heat-gated ion channel TRPV3. J. Neurosci. 24, 5177–5182. Datta, S., Kumar, V.M., Chhina, G.S., Singh, B., 1987. Effect of application of serotonin in medial preoptic area on body temperature and sleep-wakefulness. Indian J. Exp. Biol. 25, 681–685. De, G.R.O.O.T., 1959. The rat hypothalamus in stereotaxic coordinates. J. Comp. Neurol. 113, 389–400. Delany, N.S., Hurle, M., Facer, P., Alnadaf, T., Plumpton, C., Kinghorn, I., See, C.G., Costigan, M., Anand, P., Woolf, C.J., Crowther, D., Sanseau, P., Tate, S.N., 2001. Identification and characterization of a novel human vanilloid receptor-like protein, VRL-2. Physiol. Genom. 4, 165–174. Gamble, J.E., Patton, H.D., 1953. Pulmonary edema and haemorrhage from preoptic lesions in rats. Am. J. Physiol. 172, 623–631. Güler, A.D., Lee, H., Iida, T., Shimizu, I., Tominaga, M., Caterina, M., 2002. Heat-evoked activation of the ion channel, TRPV4. J. Neurosci. 22, 6408–6414. Hardy, J.D., Hellon, R.F., Sutherland, K., 1964. Temperature-sensitive neurons in the dog's hypothalamus. J. Physiol. 175, 242–253. Hellon, R.F., 1967. Thermal stimulation of brain neurons in anaesthetized rabbits. J. Physiol. 19, 381–395. Hellstrom, B., Hammel, H.T., 1967. Some characteristics of temperature regulation in the unanesthetized dog. Am. J. Physiol. 213, 547–556. Hori, T., 1984. Capsaicin and central control of thermoregulation. Pharmacol. Ther. 26,
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