E ffect of Ambient Temperature on Sleep-Wakefulness in Normal and Medial Preoptic Area Lesioned Rats

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1 Sleep Research Online 3(3): , Printed in the USA. All rights reserved X 2000 WebSciences E ffect of Ambient Temperature on SleepWakefulness in Normal and Medial optic Area Lesioned Rats Thannickal C. Thomas and Velayudhan Mohan Kumar D e p a rtment of Physiology, All India Institute of Medical Sciences, New Delhi , India The changes in sleepwakefulness were studied in rats during their exposure to different ambient temperatures of 18 C, 24 C and 30 C, before and after the destruction of the medial preoptic area neurons by NMethyl Daspartic acid. In normal rats, there was an increase in paradoxical sleep and slow wave sleep and a decrease in wakefulness at higher ambient temperatures. The increase in sleep was primarily due to an increase in the duration of sleep episodes. Destruction of the medial preoptic area neurons produced a decrease in sleep at all three different ambient temperatures. But, there was a linear increase in sleep with higher temperatures in the lesioned rats that was qualitatively different from that in the normal animals, as the increase in sleep was associated with an increase in the number of short duration slow wave sleep episodes. The findings indicate that the medial preoptic area is essential for sleep maintenance and improving the quality of sleep with higher ambient temperatures. It is possible that the medial preoptic area serves as a finetuning mechanism to regulate sleep for energy homeostasis, including thermoregulation. CURRENT CLAIM: The medial preoptic area is essential for sleep maintenance and improving the quality of sleep with higher ambient temperatures. The medial preoptic area (mpoa) participates in the regulation of sleep and body temperature (Nauta, 1946; Hammel et al., 1963; Satinoff, 1983; John et al., 1994; John and Kumar, 1998; Kumar and Khan, 1998). Changes in sleep on manipulations of the environmental and preoptic area (POA) temperatures gave rise to the hypothesis that sleep is modulated by thermosensitive elements of the POA (Kawamura and Sawyer, 1965; Valatx et al., 1973; Parmeggiani et al., 1975; Obal et al., 1983; Obal, 1984; Satinoff, 1983; McGinty and Szymusiak, 1990; Finn et al., 1991). It was suggested that the thermoreceptors in the POA may provide the input to the sleepregulating mechanisms situated in this area (Roberts and Robinson, 1969). As many thermosensitive neurons of the POAshowed alterations in their firing rate with changes in the vigilance states, their involvement in the regulation of both slow wave sleep (SWS) and body temperature has been suggested (Alam et al., 1996) A moderate increase in the ambient temperature () can enhance sleep (Obal, 1984; Finn et al., 1991). When normal cats were exposed to 13 C, 23 C and 33 C, they slept the maximum at 23 C. But, after the lesion of the mpoa, sleep was maximum at 33 C (Szymusiak et al., 1991). The study also showed that sleep was restored in the lesioned animals when they were exposed to a higher atmospheric temperature. But, according to an earlier report, the rats had maximum SWS and paradoxical sleep () at 30 C and least at 20 C, when they were exposed to of 20 C, 25 C and 30 C (Szymusiak and Satinoff, 1984). Following a lesion of the POA, SWS and were highest at 25 C. The apparent differences in these two reports on these two species need to be studied further. The purpose of the present study was also to see whether the hyposomnia, produced by NMethyl Daspartic acid (NMDA) lesion of the mpoa in rats, could be compensated for, both qualitatively and quantitatively, by exposure to diff e r e n t temperatures. METHODS In this study, sleepwakefulness (SW) in rats was monitored before and after chronic lesion of the mpoa, when they were kept in different. Six male Wistar rats were used for this study, weighing between 225 g and 300 g each, maintained in an animal room with a controlled temperature (24±1 C), and 14 h light (illumination above 200 lux) and 10 h dark (illumination below 5 lux) cycle, with lights on from 0500 h. Food and water were provided ad libitum. Surgery was conducted under pentobarbital sodium anesthesia (40 mg/kg ip). Electrodes for electroencephalogram (EEG), electromyogram (EMG) and electrooculogram (EOG) were chronically implanted, as described earlier (Kumar et al., 1984; John et al., 1994). The electrodes were soldered to connectors of a plug that was fixed to the skull with dental cement. After a sevenday recovery period, the rats were trained for three days to move around freely in the recording cage with the attached cables. Flexible cables with connectors, plugged to the rats heads and taken out through a microswivel, were connected to the input of the polygraph. EEG, EMG and EOG were recorded continuously for 5 h ( h) at 18 C, 24 C and 30 C, on three alternate days. The animals were allowed to habituate to the in the recording chamber for 2 h C o rre s p o n d e n c e : Prof. V. Mohan Kumar, Department of Physiology, All India Institute of Medical Sciences, New Delhi , India, Tel: , Fax: , mohankum@medinst.ernet.in.

2 142 THOMAS AND KUMAR ble 1. Sleep Stage/Total Sleep Time (TST) Ratio in Percentage (mean±sd) Before (pre) and A f t e r (post) N M D A Lesion of the Medial optic A rea at Diff e rent Ambient Te m p e r a t u res ( ) S * * * * * *p<0.05, significance compared to prelesion values, + p<0.05, + + p<0.01. Significance compared to. S1 light slow wave sleep/tstratio, deep slow wave sleep/tst ratio, paradoxical sleep/tst ratio. lesion and values at and were not compared for effect as corresponding had only zero values ble 2. The Percentage of SleepWakefulness Stages (mean+sd) Before (pre) and After (post) NMDA Lesion of the Medial optic Area at Different Ambient Temperatures () ** * * * * * *p<0.05, **p<0.01, significance compared to prelesion values, + p<0.05, + + p<0.01, significance compared to. W1 active wakefulness, W2 quiet wakefulness, S1 light slow wave sleep, deep slow wave sleep, paradoxical sleep. lesion and values at and were not compared for effect as there were only zero values at corresponding. before each recording session. The 5 h sleepwakefulness (S W) recordings were split into epochs of 30 second duration each and visually scored as described earlier (John et al., 1994). The wakeful period was classified into two stages, viz, active wakefulness (W1) and quiet wakefulness (W2). The sleep period was classified into light slow wave sleep (S1), deep slow wave sleep () and paradoxical sleep (). After control recording of SW at different, the mpoa neurons were destroyed by intracerebral injection of NMDA (Sigma, St. Louis, USA). The rats were anesthetized with sodium pentobarbitone (40 mg/kg, body weight) and 5 µg NMDA in 0.2 µl distilled water, neutralized with NaOH, was injected bilaterally into the mpoa with the help of a slow injector (Palmer, England), at coordinates A7.8, H1.5 and L 0.6, as per DeGroot atlas (DeGroot, 1959). The injector cannula was kept in place for five minutes to facilitate better diffusion of the drug. All the parameters for the assessment of SW were again recorded continuously for 5 h ( h) at, and, on three alternate days, starting from the tenth day after the NMDA injection. No two rats had the same sequence of exposure to different temperatures. At the end of the experiment, all the rats were perfused with 10% formalin under sodium pentobarbitone anesthesia (45 mg/kg, bw). The brains were then removed and processed for histological examination of the lesion site, in cresyl violet stained slides. The percentage of SW stages, frequency and duration of the episodes of different SW stages, frequency of the SWS episodes of different durations, and sleep stage/total sleep time (TST) ratio at different were calculated before and after the lesion of the mpoa. Twoway analysis of variance (ANOVA) was used to compare all the values of these parameters. The pre and postlesion values were compared using Student's paired t test for those parameters showing significant diff e r e n c e s between columns in ANOVA. For studying the effect of temperature on SW parameters, the values at were compared with those of and. RESULTS Effect of on SW in Normal Rats When the normal rats were exposed to different, there was a decrease in S1/TST ratio at, as compared to that at (ble 1). The total amount of and /TST ratio was increased at (bles 1 and 2). There was an increase in SWS episodes of long duration ( min) on exposure to higher (ble 3). There was an increase in the total amount of, /TST ratio, and the number of episodes at higher (bles 1, 2 and 4). The mean duration of episodes was also higher at (ble 5). High () produced a decrease in the frequency of W1 and W2 (ble 4). Effect of mpoa Lesion on SW The mpoalesion produced a decrease in the total amount of S1 (except at ), though there was an increase in the S l / T S T ratios (bles 1 and 2). The was totally abolished after the lesion, at, and the frequency and duration of episodes were reduced at the rest of the (bles 2, 4 and 5). The SWS episodes of shortest duration ( min) were increased at, and long duration episodes at min were decreased at (ble 3). The /TST and /TST ratios were reduced at and respectively, after the mpoa lesion (ble 1). The was totally abolished at, and reduced at the rest of the (ble 2). The duration and frequency of episodes were also reduced, as compared to prelesion values, though there was no significant reduction in the duration at (bles 4 and 5). There was an increase in the percentage of recorded W 1 (except at ) after the mpoa lesion (ble 2). In addition, there was an increase in the frequency of W1 episodes and a reduction of W2 at (ble 4).

3 AMBIENTTEMPERATURE AND mpoaregulation OF SLEEP ± ± ±6.6 ble 3. Number of Slow Wave Sleep (SWS) Episodes (mean±sd) of Different Duration Before (pre) and After (post) NMDA Lesion of the Medial optic Area at Different Ambient Temperatures () ± ± ±7.8 * 7.8± ± ± ± ± ± ± ± ±3.1 SWS Episode Duration (in min) 3.1± ± ± ± ± ± ± ± ± ± ± ± ± ±1.3 * 2.7± ± ± ± ± ±0.9 *p<0.05, significance compared to prelesion values, + p<0.05, significance compared to. lesion SWS values for min at and were not compared for effect as corresponding had only zero values ± ± ± ble 4. Frequency of SleepWakefulness Stages (mean±sd) Before (pre) and After (post) NMDA Lesion of the Medial optic Area at Different Ambient Temperatures () 47.20± ± ±11.97 * 42.56± ± ± ±3.32* 28.01± ± ± ± ± ± ± ± ± ± ± ±1.20 * 1.33 ±1.41 * 2.66± ± ± ±1.40 ** 2.66±1.41 * *p<0.05, **p<0.01, significance compared to prelesion values, + p<0.05, ++ p<0.01, +++ p<0.001, significance compared to. Abbreviations of sleepwakefulness stages same as ble 2. lesion and values at and were not compared for effect as corresponding had only zero values. 2.8± ± ±1.1 ble 5. Episode Duration (in minutes) of SleepWakefulness Stages (mean±sd) Before (pre) and After (post) NMDALesion of the Medial optic Area at Different Ambient Temperatures () 4.4± ± ± ± ± ± ± ± ± ± ± ±1.2 induced Changes in SWAfter the mpoa Lesion After the mpoa lesion, the S1 was increased at, as compared to its percentage at (ble 2). But, there was a reduction in S1/TST ratios at higher (ble 1). Though and were recorded at higher ( and ), they were totally abolished at after the lesion (bles 2, 4 and 5). So, the values of these two SW stages at and were not stastically compared with that at, in the lesioned rats. The W1 was decreased at in the lesioned rats (ble 2). Though the lesion per se produced no significant change in the amount of W2, it was increased at as compared to (unlike the trend that was observed in normal rats). 2.6± ± ± ± ± ± ±0.35 * 0.3 ±0.5 * 0.8± ± ± ±0.4 ** 1.4±1.0 *p<0.05, **p<0.01, significance compared to prelesion values, + p<0.05, significance compared to. Abbreviations of sleepwakefulness stages same as ble 2. lesion and values at and were not compared for effect as corresponding had only zero values. Histology There was an extensive destruction of neurons in the mpoa in all the rats. Though there was a variation in the size and extent of the lesion, all the rats had bilateral lesions at the mpoa (Figure 1). Though the lesions were primarily confined to the mpoa, the brain regions along the needle tract, above the mpoa, were also destroyed in some rats. Figure 1. The NMDA lesion sites in six rats shown diagramatically. Hatched area shows the brain regions with severe and moderate neuronal loss. Lesioned area was drawn with the help of camera lucida. Coordinates shown are as per DeGroot atlas.

4 144 THOMAS AND KUMAR DISCUSSION Effect of on SW in Normal Rats It was known for a long time that SW in rats is sensitive to (Schmidek et al., 1972; Szymusiak and Satinoff, 1981, 1984). Decrease in S1/TST ratio at high () resulted from an increase in the deeper stages of sleep ( and ) in normal rats. In addition to the /TSTratio, the total amount of was also increased at. The increase in SWS episodes of long duration ( min), on exposure to and, indicates an improvement in the SWS maintenance at higher. The effect of on was more marked as there was an increase in the total amount of, /TST ratio, and the number of episodes at and. The mean duration of episodes was also higher at. The increase in sleep, especially and, at higher, is in agreement with the earlier report in rats (Valatx et al., 1973; Szymusiak and Satinoff, 1981, 1984; Sazonov and Pastukhov, 1985; Alfoldi et al., 1990; Rosenthal and Vogel, 1993). The increase in sleep with increased may be considered as an adaptation to thermal load aimed at energy conservation (Obal et al., 1983). The increase in TST and the ratio of sleep stages are believed to be adaptations to the sleepstate related changes in the characteristics of the sleep regulatory system (Schmidek et al., 1972). Significant increase in the episode frequency with higher and increase in the duration of episodes at could be interpreted as a stimulation of the generating and maintaining mechanism by the warm environment. has been shown to be very sensitive to slight variations in the thermal environment and it varies significantly even within 25ºC and, which has been defined as the thermoneutral zone for rats on the basis of the minimal metabolic rate (Roussel et al., 1976). There was a decrease in the frequency of awake (W1 and W2) episodes at. In other words, there was increase in these episodes at low () as compared to. This could mean that there was an inhibition of the awakening mechanism at high, or stimulation of it at low. It was suggested that when the is low, the central nervous system has to call for an increase in the relative amount of arousal at the expense of the sleep stages, especially desynchronized sleep, in order to maintain the body temperature (Parmeggiani and Rabini, 1970). An increase in arousal in cold is necessary for the production of more heat by increasing motor activity. Thus, the functional state of wakefulness enables the organism to optimize thermoregulation. On the other hand, the, in which the regulation of the body temperature is said to be suspended, is incompatible with low during which appropriate thermoregulatory responses are needed to protect the animals from hypothermia (Schmidek et al., 1972). Effect of mpoa Lesion on SW Changes in SW after the mpoa lesion, namely a decrease in the frequency and duration of episodes, decrease in the total duration of (in addition to a reduction in frequency and duration of episodes at most of the ), an increase in the percentage of W1, and the frequency of W1 episodes at certain, are generally in agreement with the earlier reports (McGinty and Sterman, 1968; Sallanon et al., 1989; Asala et al., 1990; Szymusiak et al., 1991; John et al., 1994; John and Kumar, 1998). The decrease in deeper stages of sleep has also resulted in an increase in the S1/TST ratios, in spite of a decrease in the total amount of S1 (except at ), after the mpoa lesion. An increase in the SWS episodes of shortest duration at and a decrease in the long duration ( min) episodes at, are also generally in agreement with the earlier report (John and Kumar, 1998). The decrease in the durations of and after the mpoa lesion shows that the mpoa is important for the maintenance of deeper stages of sleep (John and Kumar, 1998). The decrease in the frequency of and episodes after the mpoalesion suggests that this area may also be involved in the initiation of these phases of sleep. induced Changes in SW After the mpoalesion optic anterior hypothalamus damaged cats have been shown to exhibit reduced sleep at 23ºC. However, this sleep deficit was dramatically attenuated when the cats were exposed to 33ºC (Szymusiak et al., 1991). This report is in partial agreement with the present finding where, after the mpoa lesion, the S1 was increased and W1 was decreased at, as compared to their percentage at. On the other hand, according to an earlier report, the at which maximal amounts of REM sleep occurred shifted from to 25ºC after electrolytic lesion of the mpoa in rats (Szymusiak and Satinoff, 1984). The difference in this observation from the present finding could possibly be due to the difference in the techniques used for lesion in the two studies. Though there was a reduction in S1/TST ratios at higher, in both normal and lesioned rats, the SW stages were not altered in an identical manner before and after the lesion of the mpoa. There was an increase in long duration ( min) SWS episodes on exposure to high in normal rats, but no similar change in SWS episode was produced by high in the lesioned rats. There was an increase in S1 and a decrease in W1 at high () in the lesioned rats. Similar responses were not seen in normal rats. As the values of and at and were not statistically compared with that at in the lesioned rats, it is difficult to comment on the induced changes in these S W phases after the lesion. But, it was observed that the and were recorded at and, though they were totally abolished at after the lesion. The reduction in S1/TST ratios at higher primarily resulted from the appearance of and at and. From the results of this study, it can be concluded that the mpoais essential to sufficiently increase the duration of SWS episodes by the thermal stimulus, though sleep could be induced through structures other than this area. In other words, the mpoa is essential for organizing the sleep architecture as per the thermoregulatory requirement. An increase in the amount of sleep with increased may be a thermoregulatory requirement, and it can be considered as an adaptation to the thermal load aimed at energy conservation (Obal et al., 1983). It may be mentioned here that one suggested function of the mpoa is to provide a fine tuning of the energy balance (John and Kumar, 1998; Kumar, 1999a, l999b).

5 AMBIENTTEMPERATURE AND mpoaregulation OF SLEEP The amount of W2 increased at in lesioned animals, unlike the trend that was observed in normal rats. It may be argued that the lesioned rats shifted to a less thermogenic state of W2 (compared to W1) when they were exposed to thermal load. Thus, the regulation of wakefulness activity for thermoregulation may be possible without the mpoa. It is also true that this response was not observed in normal rats. It would be pertinent here to point out that a study in which SW changes were recorded for 24 h could be undertaken to substantiate further the findings of this study. Simultaneous assessment of the thermal status of the animal would also help in a better interpretation of the data. But, as the present findings are in tune with the earlier reports of changes in SW after the mpoaneuronal lesion (based on 24 h recordings), the present data from a 5 h recording can be taken to indicate the changes in sleep under different thermal conditions. Though there was a variation in the extent of the lesion, the observed effects could be attributed to the destruction of the mpoa, as all the rats had bilateral loss of neurons in this area. ACKNOWLEDGMENTS This study was supported by the Department of Science and Technology. REFERENCES 1. Alam MN, McGinty D, Szymusiak R. optic/anterior hypothalamic neurons: Thermosensitivity in wakefulness and non rapid eye movement sleep. Brain Res 1996; 29: Alfoldi P, Rubicsek G, Cserni G, Obal F Jr. Brain and core temperature and peripheral vasomotion during sleep and wakefulness at various ambient temperatures in the rat. Pflugers Arch 1990; 417: Asala SA, Okano Y, Honda K, Inoue S. 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