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Journal of Thermal Biology 37 (2012) 392396

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Ambient temperature-dependent thermoregulatory role of REM sleep

Deependra Kumar 1, Velayudhan Mohan Kumar 2, Hruda Nanda Mallick n,1
Department of Physiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India

a r t i c l e i n f o
Article history: Received 31 October 2011 Accepted 14 February 2012 Available online 3 March 2012 Keywords: Capsaicin Sleep Body temperature Ambient temperature Thermal preference

a b s t r a c t
Changes in ambient temperature produce complex effects on sleepwakefulness. In order to nd out the mechanisms involved in temperature-sensitive changes in sleep in rats, their thermal preference, body temperature and sleep were studied before and after the destruction of both peripheral and central warm receptors, by systemic administration of 375 mg/kg capsaicin. Though the pre-treated rats preferred to stay mostly at the ambient temperature of 27 1C, post-treated rats strayed freely into chambers having ambient temperature of 30 1C and 33 1C. Sleep and body temperature of these rats were studied for six hours each, when they were kept at an ambient temperature of 1836 1C. Total sleep time, especially REM sleep, was maximum at 30 1C in pre-treated rats, but this REM sleep peak at 30 1C disappeared after capsaicin administration. Body temperature increased sharply in post-treated rats, at ambient temperatures above 30 1C. Apart from the ability to defend body temperature at high ambient temperature, avoidance of warm ambient temperature and increase in REM sleep are the behavioral measures which are lost in post-treated rats. Results of this study suggest that the ambient temperature-related increase in REM sleep at 30 1C could be part of the thermoregulatory measures. & 2012 Elsevier Ltd. All rights reserved.

1. Introduction Ambient temperature (Tamb) does inuence sleep in animals, including rats. It is generally assumed that one sleeps more at a comfortable temperature. When rats are given the choice of different Tamb to select from, they prefer to stay in 27 1C Tamb (Gulia et al., 2005; Kumar et al., 2009; Ray et al., 2004, 2005). However, they had maximum total sleep time (TST), when they were kept at 30 1C (Gulia et al., 2005; Kumar et al., 2009; Mahapatra et al., 2005; Sakaguchi et al., 1979; Szymusiak and Satinoff, 1981). This inuence of warm Tamb on sleep is probably mediated by warm receptors, as the increased Tamb does not produce a signicant change in sleep of rats with destroyed warm receptors (Benedek et al., 1980; Obal et al., 1983). The temperatures preferred by the rats before and after the destruction of warm receptors were not studied in these reports. It is possible that the change in thermal preference of the rats was responsible for the lack of sleep changes in rats with destroyed warm

Abbreviations: Ambient temperature, Tamb; Body temperature, Tb; Sleepwakefulness, SW; Total sleep time, TST; Slow wave sleep, SWS n Corresponding author. Tel.: 91 11 26594881; fax: 91 11 26588641, 91 11 26588663. E-mail addresses: (D. Kumar), (V.M. Kumar), (H.N. Mallick). 1 Tel.: 91 11 26594623. 2 Present address: Comprehensive Centre for Sleep Disorders, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Thiruvananthapuram 695012, India. Tel.: 91 471 2328004; fax: 91 471 2341814. 0306-4565/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2012.02.005

receptors. Moreover, those reports are based on the results obtained from two separate groups of rats (normal vs. capsaicin-treated) and the animals were exposed only to two Tamb. It is also necessary to test the changes in different components of sleep before and after the destruction of warm receptors. The interrelationship between the changes in sleep and body temperature (Tb) is yet another area which has not been studied in capsaicin-treated animals. Capsaicin, an active substance of hot peppers, can stimulate the warm receptors, when given in small doses, and can selectively destroy the warm receptors, when given in large doses. So, nyi and it is widely used in studies on thermoregulation (Szolcsa -Ga bor, 1973). It produces a steep fall in body temperature Jancso (Tb) when small quantity (test dose) is subcutaneously injected in -Ga bor et al., 1970a). However, after the warm rats (Jancso receptors are destroyed with large systemic doses of capsaicin, no hypothermic action is produced on administration of a test -Ga bor et al., 1970a, 1970b). In the dose of this drug (Jancso present study, a small quantity of capsaicin was subcutaneously injected in rats to nd out the functional integrity of warm receptors, and large doses were administered to destroy all the warm receptors. The thermal preferences of the animals were studied before and after warm receptors were destroyed. Capsaicin sensitivity test was also performed before and after large doses of capsaicin injection. TST, REM sleep, slow wave sleep (SWS) and Tb at wide ranging Tamb were analyzed in order to understand the interrelationship between Tamb, sleep, and Tb in these animals.

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2. Materials and methods Ten adult male Wistar rats, weighing between 250 and 275 g, were chronically implanted with electrodes for electroencephalogram (EEG), electromyogram (EMG) and electrooculogram (EOG), as described elsewhere (Kumar et al., 1984). A Radio-transmitter, TA10TA-F40 (Data Science International, USA), was implanted in the abdomen of the animals to record Tb (Vetrivelan et al., 2003). All procedures were conducted in accordance with the rules of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and were approved by the Institutional Animal Ethics Committee, AIIMS, New Delhi, India. The rats were maintained at 14 h light and 10 h dark conditions with light on from 06:00 h, with controlled temperature (25 7 1 1C), and ad libitum access to food and water. The recording of various parameters on different days is shown in Fig. 1. After a ten-day recovery period and two-day habituation in the recording chamber, the functional integrity of the warm receptors in these rats was assessed using the capsaicin sensitiv -Ga bor et al., 1970a, 1970b). This was done by ity test (Jancso injecting capsaicin (8-Methyl-N-Vanillyl-6-Nonenamide, Sigma, USA) subcutaneously at a dose of 2 mg/kg body weight and measuring the fall in Tb. The thermal preference of the rats was studied for six hours (11:0017:00 h) in an environmental chamber with three compartments, tted with activity monitoring systems (Habitest response sensing module, model E45-04, Coulbourn Instrument,

USA). Vibration-sensing platforms were kept below the oors of all the compartments of the environmental chamber (Fig. 2, upper panel). The vibration produced by the movement of the rat was monitored using E24-72 resistive bridge transducer monitor. The sensitivity and threshold of the transducer were adjusted with the animals weight. All transducers were connected to the interface card in the computer (controlling the three vibration sensing platforms) and nally to the Graphic state program. Each movement of the animal was registered as a count (from that particular platform/compartment) in Graphic state software. Thus, the software assessed the time interval between rst count from one compartment and rst count in other compartment i.e. time spent by the animals in each of the compartments. The three compartments of the chamber were maintained at 271, 301, and 33 1C respectively. These three Tamb were selected on the basis of the existing literature; 27 1C being the Tamb preferred by the rats as per the earlier report (Gulia et al., 2005; Kumar et al., 2009; Ray et al., 2004, 2005) and 30 1C and 33 1C being the temperatures shown to induce maximum sleep (Gulia et al., 2005; Kumar et al., 2009). Sleepwakefulness (SW) and Tb were recorded for six hours (11:0017:00 h) in each of the rats at 181, 211, 241, 271, 301, 331 and 36 1C Tamb, on alternate days. Flexible cables with connectors were plugged to the rats heads (Fig. 2, lower panel). Their outputs were connected to a digital recorder (BSL PRO 36, BIOPAC system Inc., USA) for recordings EOG, EEG, and EMG, along with telemetrically assed Tb (DATA QUEST 1.1, Data Science International, and USA). The conditions of the recording cage were kept identical to that of the thermal preference recording chamber, except for Tamb, which varied on different days of exposure. The temperature regulating system of the recording cage was capable of obtaining the altered Tamb within ve minutes. The relative humidity varied from 70% to 80%. Capsaicin was systemically administered to destroy the warm receptors throughout the body. Total dose of 375 mg/kg capsaicin was given subcutaneously over a period of four days, under light ether anesthesia. Two injections each were given at intervals of 2030 min, on all the four days. On the rst day, the rats were given two injections of capsaicin at the dose of 25 mg/kg. They received 25 mg/kg and 50 mg/kg, on the second day. Two injections of 50 mg/kg were given on the third day, and on the fourth day 50 mg/kg and 100 mg/kg were administered. The rst injection of capsaicin on each day was preceded by an intraperitoneal injection of atropine (25 mg/kg). Results mentioned in this study are based on the ndings from ve rats that survived the injection procedures. Three weeks after the last capsaicin injection, the destruction of warm receptors was assessed (functionally) on the basis of Tb response to capsaicin test dose. The thermal preferences, SW and Tb of the capsaicin-treated animals were studied at Tamb similar to those of the pre-treated animals. At the end of the study, capsaicin sensitivity was reassessed in the treated animals. The SW records were split into 15 s epochs and visually classied on the basis of EEG, EMG and EOG, as described earlier (John et al., 1994). The Tb and percentage TST, REM sleep and SWS (calculated as percentage of total recording time) were compared by non-parametric two-way analysis of variance (Friedmans test). The SW and Tb data at 27 1C were taken as the control reading for comparison (using Wilcoxon Signed Rank Test) with the data of these parameters obtained at 181, 211, 241, 301, 331 and 36 1C. Data from two consecutive Tamb, and before and after destruction, were also compared using the same test.

3. Results The subcutaneous injection of 2 mg/kg of capsaicin (capsaicin sensitivity test) in normal rats produced a fall of  3 1C in Tb and

Fig. 1. Schematic diagram shows experimental steps conducted on different days.

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394 D. Kumar et al. / Journal of Thermal Biology 37 (2012) 392396

Fig. 2. Schematic diagram shows recording setup used for assessing thermal preference (upper panel), sleepwakefulness and body temperature (lower panel).

Fig. 3. The gure shows the mean 7 S.D. of body temperature of pre-treated and post-treated rats (n 5) when 2 mg/kg subcutaneous capsaicin was injected. n p o 0.05, signicant change after test dose injection compare with before Tb in pre-treated rats. zp o 0.01, signicant change compared between pre and posttreatment of capsaicin.

Fig. 4. The gure shows thermal preference of rats (n 5) recorded for 6 h (11:00 17:00 h). Y-axis shows the duration of stay (mean 7 S.D.) in each compartment, pre and post-injection of capsaicin. X-axis shows temperatures in the three compartments. np o 0.05, signicant change between thermal preference at 27 1C and 30 1C in pre-treated rats. np o 0.05, signicant change between thermal preference at 27 1C and 33 1C in pre-treated rats. yp o 0.05, signicant change between pre- and post-injection of capsaicin.

the hypothermia persisted for about an hour (Fig. 3). Three weeks after the destruction of warm receptors, with systemic injection of high dose of capsaicin, subcutaneous injection of 2 mg/kg of capsaicin did not produce any fall in Tb. These rats remained nonresponsive to capsaicin test till the end of the study.

The pre-treated rats preferred to stay at 27 1C, (compared to 30 1C, z 2.023, p 0.043, and 33 1C, z 2.023, p 0.043, two sided) during 11:0017:00 h, when they were provided a choice of three different temperatures of 271, 301, and 33 1C (Fig. 4). After a high dose injection of capsaicin, there was a signicant decrease (z 2.023, p 0.043, two sided) in the preference for 27 1C, and an

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D. Kumar et al. / Journal of Thermal Biology 37 (2012) 392396 395

Fig. 5. The gure shows the mean 7 S.D. of REM sleep, slow wave sleep and total sleep time (bar diagram) and body temperature (line diagram) of rats (n 5) pre- and post-treatment of capsaicin when they are exposed to various ambient temperatures. X-axis shows the ambient temperatures. Y-axis shows percentage recording time (bar diagram) and body temperature (line diagram). np o 0.05, signicant change compared to 27 1C. yp o 0.05, zp o 0.01, signicant change between pre- and post-treatment of capsaicin. np o 0.05, signicant change in pre-treated rats when compared between two consecutive ambient temperatures. mp o 0.05, signicant change in post-treated rats when compared between two consecutive ambient temperatures.

increase (z 2.023, p 0.043) in the preference for 30 1C. But the increase in preference of 33 1C was not signicant (z 1.753, p 0.080). In the pre-treated rats, the average TST at 27 1C Tamb was 67.91 7 7.8% of 11:0017:00 h recording time (Fig. 5). TST was the maximum at 30 1C, (69.89 7 10.8% z 2.023, p 0.043, two sided); it decreased above (at 36 1C, 49.71 7 6.27%, z 2.023, p 0.043) and below (at 18 1C, z 2.023, p 0.043 and 21 1C, z 2.023, p 0.043) this Tamb. There were no signicant differences in TST at 24 1C (z 0.674, p 0.500) and 33 1C (z 0.674, p 0.500) when compared with 27 1C. The TST peak at 30 1C was primarily due to the increase (16.08 7 3.1%, z 2.023, p 0.043) in REM sleep (Fig. 5). The REM sleep peak at 30 1C disappeared (10.26 7 2.4%, z 0.944, p 0.345) in post-treated rats. In addition, the TST peak was observed at 33 1C (69.43 7 8.1%, z 2.023, p 0.043) in these rats. Though the mean Tb increased signicantly (37.76 7 0.3 1C, z 2.023, p 0.043) at 36 1C in pre-treated rats, there was a steep rise in Tb above 30 1C (at 33 1C, Tb 38.14 7 0.6 1C, z 2.023, p 0.043 and 36 1C, Tb 38.93 7 0.4 1C, z 2.023, p 0.043) after treatment (Fig. 5).

known for a long time that systemic injection of high dose of capsaicin in the animals produced insensitivity to subsequent test -Ga bor et al., 1970a; Szolcsa nyi dose injections for months (Jancso -Ga bor, 1973). Moreover, electrophysiological and and Jancso morphological studies have shown that high dose of subcutaneous capsaicin administration produces long-lasting impairment of peripheral and central warm receptors (Hori, 1981; Hori and -Ga bor et al., 1970a, 1970b; Oba l et al., Tsuzuki, 1981; Jancso nyi, 1977; Szolcsa nyi et al., 1971; Szolcsa nyi 1979, 1980; Szolcsa -Ga bor, 1973). In the present study also, these animals and Jancso were unable to defend Tb, as evident from the sharp increase in Tb at Tamb above 30 1C. Similar ndings were reported earlier where rats succumbed to heat stroke at Tamb that the normal rats could l et al., 1979). easily cope with (Benedek et al., 1980; Oba Capsaicin-treated animals also showed some fall in Tb in cold Tamb as reported in an earlier study (Benedek et al., 1983). Thus, a high dose of capsaicin administration that destroyed warm receptors, grossly reduced the Tb regulation at higher Tamb, though thermoregulation in lower Tamb was also affected. 4.2. Effect of capsaicin treatment on thermal preference

4. Discussion 4.1. Effect of capsaicin treatment on Tb regulation It can be safely assumed that the warm receptors are substantially destroyed in the rats treated with high dose of capsaicin, as there was no fall in Tb to capsaicin sensitivity test. It was

Control rats did not venture into Tamb above 27 1C, but those with destroyed warm receptors moved freely into 30 1C and 33 1C Tamb. Preference for Tamb above 27 1C after capsaicin treatment can be considered as an impairment of thermal selection behavior l et al., 1981). Earlier studies have shown that of the animals (Oba the thermal selection by the animals is preserved after selective destruction of peripheral warm receptors (Gulia et al., 2005).

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In the light of this earlier report, it can be assumed that the central warm receptors are primarily responsible for the changed thermal selection behavior of the rats in the present study. 4.3. Effect of capsaicin treatment on SW regulation Several studies suggested that SW in rats is sensitive to Tamb (Gulia et al., 2005; Kumar et al., 2009; Schmidek et al., 1972; Szymusiak and Satinoff, 1981). A moderately warmer Tamb (30 1C) promotes sleep in normal rats (Gulia et al., 2005; Kumar et al., 2009; Sakaguchi et al., 1979; Szymusiak and Satinoff, 1981). This increase in sleep is probably a thermoregulatory response to thermal load (Obal et al., 1983). It is interesting to note that TST increased relatively (69.43 7 8.1%, p 0.043) at 33 1C Tamb, along with a steep increase in Tb, after destruction of warm receptors. In the absence of peripheral and central warm receptors, induction of sleep at 33 1C should be attributed to a direct effect of increase in Tb, and brain temperature. Lower Tamb did not signicantly change sleep, despite the small reduction in Tb. Thus it is sleep at higher Tamb that is mainly affected in capsaicin-treated rats. The effect of Tamb on REM sleep was more marked, and it was maximum at 30 1C in control rats. REM sleep has been considered as a state where thermoregulation ceases (Permeggiani et al., 1977). But it is possible that Tamb-related increase in REM sleep in control rats, specically at 30 1C, could be part of thermoregulatory measure. Decreased muscle tone during REM sleep leads to decreased heat production by the muscles, which may help in decreasing the thermal load (Aristakesian and Vataev, 1993). The selective increase in REM sleep (REM sleep peak) at 30 1C disappears after the destruction of the warm receptors. This indicates that the animal lost REM sleep-related heat dissipating mechanism at warm Tamb. Our nding is in agreement with various studies on capsaicin-treated rats in which heat defense -Ga bor responses such as skin vasodilation (Dib, 1983; Jancso l et al., 1982), salivation (Jancso -Ga bor et al., et al., 1970a, Oba l et al., 1979), prone body extension, heat escape 1970a; Oba l et al., 1979), skin cooling operant behavior (Dib, locomotion (Oba l et al., 1983; Hori and Tsuzuki, 1981) and water bathing (Oba 1980) are lost after capsaicin treatment.

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5. Conclusion Sleep, especially REM sleep, was increased at 30 1C in the normal rats, though they preferred 27 1C Tamb. It can be assumed from the present study that thermal selection behavior and increase in REM sleep at 30 1C Tamb are dependent on central warm receptors. Both these responses are used for thermoregulation, but they are not interrelated physiological responses. Increase in REM sleep at 30 1C Tamb in normal rats could be considered as a thermoregulatory function of warm receptors, which does not involve conscious perception of warm sensation.

Acknowledgment This study was supported by the Defense Research and Development Organization of India.