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anterior hypothalamic lesions
BRAIN RESEARCH Brain Research 703 (1995) 93-99
ELSEVIER
Research report
Sleep deprivation in rats with preoptic/anterior hypothalamic lesions Ping-Fu Feng, Bernard M. Bergmann, Allan Rechtschaffen * Sleep Research Laboratory, Departments of Psychiatry and Psychology, The University of Chicago, 5743 S. Drexel Avenue, Chicago, IL 60637, USA
Accepted 8 August 1995
Abstract
Chronic total sleep deprivation (TSD) of rats by the disk-over-water method reliably produces initial increases and subsequent decreases in waking intraperitoneal (Tip) and hypothalamic (Thy) temperatures, progressive increases in energy expenditure, skin lesions on the tail and plantar surface,,;, debilitated appearance, and eventual death. We investigated the possible role of the preoptic/anterior hypothalamus (POAH) in the mediation of the TSD effects by comparing these effects in POAH-lesioned and unlesioned rats. Bilateral POAH lesions sufficient in size to impair homeothermic responses to changes in ambient temperature did not produce TSD-like temperature changes under baseline ambient temperatures of 28-29°C, implying that the thermoregulatory changes produced by TSD do not result from impairment of tlhe lesioned area. However, the possibility remains that the TSD effects are mediated by damage to POAH areas that were not lesioned. During TSD, lesioned and unlesioned rats showed similar progressive increases in energy expenditure, but the lesioned rats showed earlier, steeper, and eventually greater declines in Tip and Thy. This result suggests that in unlesioned rats the POAH may counter-regulate against, and thereby attenuate, the reduction in heat retention caused by TSD. This failure of regulation in lesioned rats is consistent with their impaired response to ambient temperature change and implies that, in unlesioned rats, some POAH thermoregulatory mechanisms continue to function normally during TSD. Lesioned rats did not show the characteristic TSD-induced early increases in Tip and Thy. This result could imply either that heat retention was so compromised that body temperatures did not rise in spite of a TSD-induced increases in thermoregulatory setpoint, or that the setpoint increase in unlesioned rats is POAH-mediated. Notwithstanding the greater Tit' and Thy declines in lesioned rats, they survived the TSD procedure longer than the unlesioned rats, thus supporting previous indications that death did not result from hypothermia. Keywords: Sleep deprivation; The~moregulation; Preoptic/anterior hypothalamus
I. Introduction
Rats subjected to total sleep deprivation (TSD) by the disk-over- water method show initial increases in intraperitoneal (Tip) and hypothalamic (Tny) temperatures followed by later falls to below baseline [13]. Two thermoregulatory changes have been identified. (1) An increased temperature setpoint (Tset) is indicated by behavioral attempts at warming even while Tip and Thy are above baseline, a more sustained rise in Thy than in Tip, and an increase in energy expenditure (EE) as these temperatures decline towards baseline. (2) A heat retention deficit is indicated by the late temperature declines and behavioral warming [16,19] in spite of greatly increased EE, the accelerated decline of Tip relative to Thy [13], and a hormonal profile consistent with a response to cold exposure [1]. Presumably, both ther-
* Corresponding author. Fax: (1) (312) 702-0277. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01071-8
moregulatory deficits obtain over the course of deprivation, while temperature levels vary with the relative strengths of the two deficits and the mobilization of calorigenic and behavioral responses to them. The preoptic/anterior hypothalamus (POAH) is an important structure for the mediation of both sleep and temperature regulation [4,9,12]. Lesions of the POAH induce thermoregulatory deficits a n d / o r reduced sleep in cats [11,17], rats [12] and dogs [10]. We used the neurotoxin N-methyl-DL-aspartic acid (NMDA) to make lesions of this area to evaluate the role of the POAH in TSD-induced thermoregulatory effects. Our histological examinations by both light microscopy [8] and electron microscopy [7] have not revealed anatomical damage to the POAH in TSD rats. Nevertheless, it is possible that TSD-induced functional impairment of the POAH caused some of the observed temperature and energy changes. If this were the case, one would expect that experimentally-induced lesions of the POAH might induce similar changes in rats that
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were not sleep deprived. Even in the absence of such TSD-like lesion effects in normally-sleeping rats, the lesions might exacerbate changes during TSD if the POAH normally counter-regulates TSD-induced malfunctions of other thermoregulatory centers. A third possibility (although we know of no precedent for it) is that the TSD effects were mediated by hyperactivation of hypothalamic thermoregulatory mechanisms, e.g., heat defense, in which case one might expect a reduction of thermoregulatory changes in POAH-lesioned TSD rats. The POAH is a multifunctional regulatory region with only partial localization of function. Since our goal was to disrupt thermoregulatory function without otherwise incapacitating the rat, we attempted to produce lesions as small and well-localized as possible and still produce measurable thermoregulatory deficits. In particular we wished to avoid lesions large enough or so-positioned that they would kill or completely incapacitate the rats or would by themselves produce large deficits in sleep under our experimental conditions.
2. M a t e r i a l s
and methods
2.1. Subjects and surgical preparation Subjects were eight male Sprague-Dawley rats (550-680 g) that were kept in constant light prior to surgery and throughout the experiment to flatten their circadian rhythms. Electrodes for recording EEG, EMG and hippocampal theta, transducers for recording Thy and Tip, and guide tubes for bilateral POAH lesions were implanted under Nembutal anesthesia (50 mg/kg). A thermistor encased in a 10 mm by 0.5 mm diam. glass rod (Model P20-BA-130-M, Thermometrics) was stereotaxically implanted into the left lateral posterior hypothalamus (P = 1.5 mm, L = 1.5 mm, H = 8.5 mm) [14]. Guide tubes for neurotoxin injection (C315G, 26G, Plastics One) were implanted bilaterally at a lateral-to-medial angle to avoid the sagittal sinus and were targeted to 1.0 mm above the POAH (P = 0.4, L = 0.6, H = 8.5). Dummy cannulae were inserted into the guide tubes to keep them from blocking. A thermosensitive transmitter (Model T-SLO, W.T. Barrows Co.) was placed into the peritoneal cavity to record Tip.
2.2. Apparatus for sleep deprivation The apparatus used for TSD has been described previously in detail [2]. Briefly, a pair of rectangular plastic cages were placed in parallel over a common disk which formed a partial floor for each cage (Fig. 1). Under the disk were water-containing stainless steel pans which extended on the cage walls. A TSD rat and a yoked control rat occupied the two cages. During deprivation, whenever
Plexlgless cage over disk .'.'-'----.----'---'.'.%'.-.-.-.'. ".-.-.-.'.'.'.'.'." i , E". . . .W . . a. .t.e. .r. . .pan . . . . . . u. *n. d: .e- r. - .dlsk - . - . - . ..*-'-'-'.'-'-'-.'-'~, .: , %%%..%%* %%% .%* %%%%
MOTOR~
, i
DISK
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~
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Fig. 1. Schematic illustration of top view of the deprivation apparatus. When sleep onset is detected in the TSD (or TSDL) rat, the disk is automatically rotated and the rat must walk opposite to disk rotation to avoid being carried into the water. A yoked control rat on the other side of the disk can sleep when the TSD rat is spontaneously awake and the disk is still.
the TSD rat started to sleep, the disk would rotate and both rats would move to avoid being carried into the water. The yoked control rat, however, could sleep when the TSD rat was spontaneously awake. In this study, all the yoked controls died either as a result of surgery or POAH lesions (if a potential TSD rat died, its yoked control was promoted to be a TSD rat), so no yoked control data were available. Because the major loci in this study were comparisons between lesioned vs. unlesioned TSD rats during baseline and TSD, the unavailability of yoked control lesioned rats was not critical.
2.3. Data collection and experimental procedures Amplified EEG, EMG and theta signals, the detector output for the intra-peritoneal transmitter, and the bridge circuit output for the brain thermistor were fed through a data collection microcomputer (DAP 2400, Microstar Laboratories) to a personal computer (PC) in 30-s epochs. 24-h data blocks (2880 epochs) were automatically scored for sleep and temperature each epoch by a computer program [3]. Waking temperature for a day was the mean of all the temperature values for wake epochs during the day, e.g,, if a rat spent half a day awake, the wake temperature for that day was based on values for 1440 epochs. After surgery, the animal was placed in the experimental apparatus for 9 to 12 days of recovery and adaptation. During this period, a plastic floor was placed over the disk. Then water was added to the pans under the disk, the floor was removed, and 6 to 12 days of baseline recording followed. As soon as the plastic floor was taken out, ambient temperature (Tam) was adjusted to 28-29°C and, except for tests of thermal defense (see below) was maintained at this level for the entire experiment. Lesions were made at the end of baseline. Then a 5-11 day lesion-baseline started, at the end of which, thermal defense was tested. TSD was then started and continued until the
P.-F. Fenget al. /Brain Research 703 (1995) 93-99 sleep-deprived rat died or death appeared imminent. Thus, the experimental schedule was surgery; 9-12 days of recovery; 6-12 days of baseline; lesion; 5-11 days of lesion-baseline; thermal defense test; and TSD until imminent or actual death.
2.4. The POAH lesion Because NMDA has excitatory effects, lesions were made under light anesthesia (chloral hydrate, 140 mg/kg, i.p.). For each side, a cannula (C3151, 33G, Plastics One) was connected through a hand-stretched polyethylene tube (i.d. about 0.3 mm) to a 1 /zl syringe mounted on a syringe pump (Model 55-1111, HaJvard Apparatus). The cannula was inserted through the guide tube to 1.0 mm below its tip. NMDA (Sigma) was dissolved in sterile isotonic saline solution (200 ng/ml, pH = 7.4); then 0.2 /xl was injected into each side over a period of 2.0 min. To ensure that the solution was delivered into the brain, the travel distance of the solution within the PE tubing (about 3 mm, depending upon the i.d. of the tubing) was monitored via movement of an air pocket. After a 3 min wait, the interior cannula was gently removed and the dummy catheter replaced.
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2.Z Histology On completion of the experiment, rats were killed by anesthetic overdose and their brains were quickly removed and frozen on dry ice. Sections were cut at 40 p,m and stained with cresyl violet. Light microscopy was used to observe the lesion size and location.
2.8. Data presentation The effects of POAH lesions on thermoregulation during TSD were determined by comparing Tip, Thy and EE in lesioned TSD (TSDL) rats and intact TSD rats from a previous experiment that measured the same variables [13]. Similar pathology developed in all TSDL rats during deprivation, but at slightly different rates. To easily compare the results of TSDL rats with the earlier study, data collected during deprivation were time-scaled by quarters of deprivation (survival) time. Repeated measures ANOVA was used to determine the group by time interactions, which reflected differences between TSDL and TSD rats over the course of deprivation, t-Tests were used for specific comparisons of matched and unmatched samples.
2.5. Thermal challenge as a test of thermoregulation 3. Results To establish that the lesions affected thermoregulatory mechanisms, the four lesioned rats and four unlesioned rats were compared in the deprivation apparatus on cold and heat defenses. Tip and Th:, were continuously recorded while Tam w a s gradually increased from its normal level of 29°C to about 40°C and then decreased to about 14°C over a period of approximately 5 h. As measures of thermal defense, two parameters, AThy//ATamand ATip/ATam, i.e., the amounts Thy and Tip changed per change in Tam, were defined: ZIThy//ATam = ( n T h y ATip//ATar n = ( n T i p -
ZThy )//( nTam - ZTam) ZTip ) / / ( n T a m - ZTam)
HThy and HTip a r e the averages of the five 30-s epochs with highest Thy and Tip during high Tam (HTam) thermal challenge, and LThy and LTip are the averages of the five epochs with lowest temperatures during the down phase of Tam (LTam). High scores on these measures reflect poor thermal defenses, i.e., a relative inability to maintain stable body and brain temperatures as ambient temperatures varies. where
2.6. Energy expenditure (EE) EE (kcal/day) was calculated based on the caloric values of daily food consumption and change in body weight as described earlier [2]. This measure was validated as an indicator of metabolilc rate in an earlier TSD study [1].
3.1. POAH lesion anatomy Morphologically, all the lesions were highly localized; the diameter of the area with severe cell destruction was about 0.2 to 0.3 mm. In three of the four rats, the area of maximal cell body destruction was in the anterior medial hypothalamus (AMPO). In one rat, the lesion was slightly off the center of the AMPO; cell destruction was concentrated near the edge of the medial preoptic area at the border of the medial and lateral preoptic areas.
3.2. Effects of POAH lesions during baseline 3.2.1. Sleep during pre-lesion and post-lesion baseline Sleep percentages of total time during the last five days of TSDL baseline (45.2 +_ 1.5% for NREM; 6.6 ± 0.6% for PS) resembled the percentages previously reported for the TSD rats (47.3 _+ 2.1% for NREM; 5.2 ± 1.2% for PS) [13]. Although lesions were made under light anesthesia, all rats were agitated for about 1.5 h after lesion induction. In a comparison of the averages of the last five post-lesion and pre-lesion baseline days, one TSDL rat showed a 23% decrease in NREM sleep post-lesion, while the other three rats showed small increases; the group averages (pre-lesion -- 45.2%; post-lesion = 45.1%) were almost identical. Two rats showed small decreases in PS post-lesion, while the other two rats showed large decreases (from 5.7 and 6.6% to 2.6 and 2.5%, respectively). The group average of 4.6% was 30.3% lower than during pre-lesion baseline, but,
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P.-F. Feng et al. /Brain Research 703 (1995) 93-99
o• v
given the small subject sample and large post-lesion variance, the change was not statistically significant. It is also unlikely that these reductions in PS strongly affected thermoregulation; they are more typical of the modest reductions seen in yoked control rats (e.g., 47% [6]), which have relatively small temperature changes, than of the near complete reductions of PS seen in TSD rats (e.g., 96% [6]).
Q t-
J~ o ~
3.2.3. Thermal defense The thermal challenge comparisons were made between the four TSDL rats (after lesion) and four unlesioned rats of comparable age and weight which had been surgically prepared for the TSDL experiment and subjected to the adaptation and baseline schedules, but whose post-lesion data were not used. In the unlesioned rats, thermal challenges induced mostly small, unpredictable increases or decreases for both increases and decreases in Tam. TSDL rats after POAH lesions clearly showed impaired homeothermic mechanisms; both Why and Tip of these animals tended to vary in the same direction as Tam. During exposure to the cold ambient temperatures, mean Tip fell by 1.8°C and mean Thy fell by 1.5°C. During exposure to warm ambient temperatures, m e a n Tip in-
0 -1
L ....,,-- Tip T S D L ' ~ ' % ' ~
-2
\
Tip TSD
Q1
\ b
Thv TSD L
~, \
- Thy TSDL
~" -4 E 0 I-- -5
3.2.2. Temperatures during pre- and post-lesion baselines Because the major focus of the study was on temperature changes during sleep deprivation, when only waking temperatures could be reliably evaluated, only waking temperatures were evaluated during baseline as well. During the last five days of pre-lesion baseline, mean waking Tip of TSDL rats was 37.3 _+ 0.27°C; during the last five days of post-lesion baseline, it was 37.4 + 0.28°C. Over the same time periods, Thy changed from 38.7 + 0.34°C to 38.5 + 0.41°C. Thus, at the relatively warm 29°C ambient temperature in the experimental apparatus, the POAH lesions had little effect on Tip or Thy. Substantial changes were seen only immediately following lesion induction when both temperatures were increased for 3 to 7 h in most rats; the maximal increase during this period was about 1.5°C
1
Q2
Q3
Q4
Time Fig. 3. Changes from baseline Thy and Tip in TSD and TSDL rats during post-lesion baseline (L, TSDL rats only) and successive quarters (Q1-Q4) of sleep deprivation.
creased by 1.1°C and m e a n Zhy increased by 1.0°C. The m e a n AThy//z~Tam and ATip/ATam were 0.04 and 0.05 respectively in unlesioned rats (Fig. 2). The values in lesioned rats were 0.15 for Thy and 0.16 for Tip. Both AThy//ATam and ATip//ATam were significantly larger in lesioned rats ( P = 0.04 for Thy and P = 0.005 for Tip).
3.2.4. Energy expenditure, appearance, and skin condition during baseline As part of the general issue of whether POAH lesions alone could produce changes like those which result from TSD, EE, appearance and skin condition were evaluated during the baseline periods. Mean EE of the TSDL rats was 93.9 + 9.2 kcal/day during the last five days of pre-lesion baseline and remained unchanged at 94.4 + 17.4 kcal/day during the last five days of post-lesion baseline. During post-lesion baseline none of the TSDL rats developed any signs of the debilitated appearance or skin lesions on the tail and plantar surfaces that are characteristic of TSD rats. It could be reasonably argued that even if POAH lesions produced changes like those of TSD, because appearance and skin changes develop gradually over time, the 5-11 day post-lesion baseline period may not have been sufficiently long to permit those symptoms to fully emerge. This caveat does not apply in the case of EE, which generally shows increases of about 50-100% over similar time periods in TSD rats [1,6,13].
0.204
3.3. Effects of POAH lesions on the major pathology of
o.1~
TSD 3.3.1. Body temperature during TSD <~ Tip
Thy
[]
Unlesioned
•
Lesioned
Fig. 2. Mean+S.E.M. of AT/ATam, a measure of body temperature change relative to ambient temperature change (see text), for Thy and Tip during baseline thermal challenge tests in rats with and without POAH lesions.
Tip of intact TSD rats shows a typical pattern, verified by several studies in our laboratory [1,13,22], of an initial rise and then a fall below baseline. The pattern in TSDL rats was obviously different. Instead of the initial rise, mean Tip of TSDL rats dropped shortly after TSD began (Fig. 3). Then it showed a progressive decline that was much steeper than in intact TSD rats. The lowest mean waking Tip was 3.39°C below baseline (Fig. 3), which represented more than twice the decrease in intact TSD
P.-F. Feng et al. / Brain Research 703 (1995) 93-99
rats. Repeated measures ANOVA on Tip showed a significant group × time interaction ( P = 0.028). Compared with baseline, Tip changes of TSDL rats in quarters 3 and 4 of deprivation were highly significant ( P < 0.01).
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days. A t-test showed that the survival time for TSDL rats was significantly longer than for intact TSD rats ( P = 0.043).
3.5. Appearance changes and lesions 3.3.2. Hypothalamic temperature during TSD In intact TSD rats, Thy increases above baseline shortly after TSD is initiated. This elevation is maintained for about 90% of the deprivation period and is followed by a decrease below baseline in the last one or two days of the TSD (Fig. 3). In TSDL rats mean Thy never rose above baseline. As with intact TSD rats, a level plateau was maintained over the first three quarters of deprivation, although the level was lower. As with TSD rats, Thy of TSDL rats showed an accelerated decline during the last quarter of deprivation, but the decline was much steeper and progressed to much lower levels in the TSDL rats. Repeated measures ANOVA showed a significant group × time interaction for Zhy ( P = 0.008). ThUS the shapes of both the Thy and Tip curves were similar in TSD and TSDL, except that the TSDL curves were at a lower level and showed steeper progressive declines.
3.3.3. Energy expenditure (EE) during TSD Mean baseline EE was 93.9 _ 9.23 kcal/day in TSDL rats and 98.8 _ 22.0 kcal/day in intact TSD rats. The two groups showed similar large increases during the first quarter of deprivation and more gradually progressive increases thereafter (Fig. 4). In both conditions, EE values were very similar. Repeated measures ANOVA showed no significant group × time interaction ( P > 0.1).
3.4. Survival time Of the four TSDL rats, one died during the period of deprivation, two were sacrificed when the amplitude of EEG decreased and large EEG spikes appeared, and one died during the second day of recovery. The mean survival time of the four TSDL rats was 24.3 + 4.0 days. Our previous data from three TSD experiments with otherwise untreated rats showed a mean survival time as 17.1 + 7.2 20O% 160% 120% J:
u
80%
uJ UJ 40% 0%, L
I;11
Q2
Q3
Q4
Time
Fig. 4. Percentage changes from baseline (mean+S.E.M.) in energy expenditure (EE) in TSD and TSDL rats during post-lesion baseline (L, TSDL rats only) and successive quarters of sleep deprivation.
Typically, TSD rats show a progressively debilitated appearance with yellowing, disheveled fur. They also show stereotypic ulcerative and hyperkeratotic skin lesions localized to the plantar surfaces and the tail. The TSDL rats developed the same appearance changes and lesions at similar rates.
4. Discussion
Were the lesions successful? Our POAH lesions did not produce the large decreases in sleep previously reported for the rat [12], probably because of the small size of the lesions and the high Tam (28-29°C) during recording. Previous studies in rats [21] and cats [20] have shown that sleep reduction only occurs with large lesions and low ambient temperatures. Szymusiak et al. [20] reported that cats with POAH lesions produced by NMDA showed an impairment of warm defense only, in effect raising the thermoregulatory setpoint. The TSDL rats more closely resembled rats with electrolytic POAH lesions insofar as they showed impaired defenses against both heat and cold (summarized in [18]). Like the POAH-lesioned rats of Satinoff et al. [18], the TSDL rats were able to maintain stable body temperatures at an ambient temperature near 29°C. The strong and reliable effects of TSD on temperature regulation and the role of the POAH in temperature regulation and other regulatory processes raise the question of whether the physiological effects of TSD are mediated by damage it inflicts on the POAH. Under baseline conditions with Tam at 28-29°C, the POAH lesions failed to produce any of the thermal, metabolic, or dermatological effects reliably produced by TSD, which would argue against a mediation of TSD effects by POAH impairment, at least for the lesioned area. On the other hand, the induced lesions were small, and it is not known whether TSD effects might be mediated by POAH sites not affected by the lesions. Older studies of rats with large POAH lesions (e.g., [18]) have revealed such a diverse mixture of thermoregulatory changes and such marked variations in survival time that comparisons with TSD rats are problematic. It is of interest, however, that some POAH-lesioned rats show a 'paradoxical hyperthermia', i.e., body temperatures are elevated, but the rats are unable to defend against cold stress [18]. This condition is remindful of TSD rats which have an elevated Tset but impaired heat retention. Might TSD effects result from some anomalous, TSDinduced hyperactivity of POAH mechanisms? If that were the case, we might expect some mitigation of these effects
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in the lesioned rats during TSD. Yet most of the TSD effects (including the Tip and Thy declines, EE increase, skin lesions, debilitated appearance, and actual or imminent death) were apparent in all the lesioned rats after deprivation. Exceptions to the above pattern were the early increases of Tip and Thy, which are characteristic of TSD rats, but were not seen in the lesioned rats during deprivation, and the increased survival time. These exceptions will be discussed below. The appearance of TSD effects in TSDL rats does not mean that the POAH played no role in their development. A role is clearly evident in the earlier and greater declines of Tip and Thy during deprivation in the lesioned rats than in the TSD rats. Since EE was similar in the two groups, ability to retain heat must have been reduced more in the lesioned rats. The lesions alone did not decrease heat retention, because they did not result in temperature declines or elevated EE during baseline. Rather, the TSD effect on heat retention was exaggerated in the lesioned rats. A parsimonious explanation for this interaction is readily available. Just as the lesioned rats evidenced impaired cold defense to thermal challenge during baseline, they likely had an impaired cold defense to the drop in body temperatures induced by TSD. Presumably, in unlesioned rats, intact POAH mechanisms counter-regulate and attenuate the potential TSD-induced heat retention deficit. As noted above, lesioned TSD rats did not show the characteristic early increases in body temperatures. Two explanations are available. One is that TSD-induced increases in Tset are mediated by POAH mechanisms that are no longer available in the lesioned rats. This is consistent with the well-documented role of the POAH in adjusting Tset [9]. A second explanation is that Tset may have increased as in the unlesioned rats, but heat retention may have been so impaired that the body temperatures could not increase to above baseline levels. The exaggeration of this impairment in the lesioned rats was noted above. The second explanation is more consistent with other data. Another group of TSD rats which failed to show an initial rise in Tip but showed exaggerated Tip declines consisted of rats whose EE increase was blunted by making them hypothyroid [15]. In this case, the absence of temperature increases was more likely due to a failure of energy compensation for the heat retention deficit, since such rats had an intact POAH. However, it is also possible that the rise in T~t in TSD rats requires thyroid hormone. The survival time of TSDL rats was longer than that of intact TSD rats. It is unlikely that the longer survival times in the TSDL rats resulted from their relatively large size at the start of the experiment, since they survived longer than similarly large rats in other TSD experiments [13,16]. Furthermore, the within-experiment relationship between initial weight and survival has been shown to be negative (r = - 0 . 9 1 ) [5]. Since the TSDL rats had lower temperatures, this suggests that, as we have previously argued [15], within a limited range, hypothermia does not reduce sur-
vival time. The longer survival time of TSDL rats is not readily explained. Although overall metabolic rate was not reduced by the hypothermia, it is possible that some specific, harmful metabolic process or processes were slowed by the Q10 effect. However, survival was not similarly extended in hypothyroid TSD rats with similarly low Tip [15]. Given the numerous possible differences between the TSDL rats and previously run TSD rats, the extension of survival needs to be replicated before it can be given a convincing explanation. In summary, although the POAH-lesioned rats showed impaired heat and cold defenses, the lesion itself did not produce effects that are reliably produced by sleep deprivation. It is not clear whether larger lesions might have produced such effects. TSD-induced decreases in heat retention are exaggerated in POAH-lesioned rats, suggesting that the POAH normally regulates temperatures within a narrow range against the excessive heat loss produced by TSD. POAH-lesioned rats did not show the characteristic increases in body temperatures early in sleep deprivation, which could mean either that TSD-induced increases in T~t are mediated by the POAH, or that heat retention was so impaired that temperatures could not rise above baseline in spite of increases in T~et. A small, but statistically significant increase in survival time in lesioned TSD rats compared to unlesioned TSD rats remains unexplained.
Acknowledgements This work was supported by NIMH grants MH4151 and MH18428 to A.R.
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[9] Hammel, H.T., Jackson, D.C., Stolwijk, J.A.J., Hardy, J.D. and Stromme, S.B'., Temperature regulation by hypothalamic proportional control with an adjustable set point, J. Appl. Physiol., 18 (1963) Rl146-Rl154. [10] Keller, A.D., Temperature regulation disturbances in dogs following hypothalamic ablations. In J.D. Hardy (Ed.), Temperature: Its Measurement and .Control in Science and Industry, Reinhold, New York, 1963, pp. 181-215. [11] McGinty, Dennis and Sterman, M.B., Sleep suppression after basal forebrain lesions in the cat, Science, 160 (1968) 1253-1255. [12] Nauta, W.J.H., Hypothalamie regulation of sleep in rats. An experimental study, J. Neurophysid., 9 (1946) 285-316. [13] Obermeyer, W., Bergmann, B.M. and Rechtschaffen, A., Sleep deprivation in the rat: XIV. Comparison of waking hypothalamic and peritoneal temperatures, ,Sleep, 14 (1991) 285-293. [14] Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986, pp. 1-156. [15] Pilcher, J.J., Bergmann, ILM., Refetoff, S., Fang, V.S. and Rechtschaffen, A., Sleep deprivation in the rat: XIII. The effect of hypothyroidism on sleep deprivation symptoms, Sleep, 14 (1991) 201-210. [16] Prete, F.R., Bergmann, B.M., Holtzman, P., Obermeyer, W. and
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Rechtschaffen, A., Sleep deprivation in the rat: XII. Effect on ambient temperature choice, Sleep, 14 (1991) 109-115. [17] Sallanon, M., Denoyer, M., Kitahama, K., Aubert, C., Gay, N. and Jouvet, M., Long-lasting insomnia induced by preoptic neuron lesions and its transient reversal by muscimol injection into the posterior hypothalamus in the cat, Neuroscience, 32 (1989) 669-683. [18] Satinoff, E., Valentino, D. and Teitelbaum, P., Thermoregulatory cold-defense deficits in rats with preoptic/anterior hypothalamic lesions, Brain. Res. BulL, 1 (1976) 553-565. [19] Shaw, P.J., Bergmann, Bernard M. and Rechtschaffen, A., Operant control of ambient temperature during sleep deprivation, Sleep Res., 23 (1994) 424 (Abstract). [20] Szymusiak, R., Danowski, J. and McGinty, D., Exposure to heat restores sleep in cats with preoptic/anterior hypothalamic cell loss, Brain Res., 541 (1991) 134-138. [21] Szymusiak, R. and Satinoff, E., Ambient temperature-dependence of sleep disturbances produced by basal forebrain damage in rats, Brain Res. Bull., 12 (1984) 295-305. [22] Tsai, L.L., Bergmann, B.M. and Rechtschaffen, A., Sleep deprivation in the rat: XVI. Effects in a light-dark cycle, Sleep, 15 (1992) 537-544.
ELSEVIER
Research report
Sleep deprivation in rats with preoptic/anterior hypothalamic lesions Ping-Fu Feng, Bernard M. Bergmann, Allan Rechtschaffen * Sleep Research Laboratory, Departments of Psychiatry and Psychology, The University of Chicago, 5743 S. Drexel Avenue, Chicago, IL 60637, USA
Accepted 8 August 1995
Abstract
Chronic total sleep deprivation (TSD) of rats by the disk-over-water method reliably produces initial increases and subsequent decreases in waking intraperitoneal (Tip) and hypothalamic (Thy) temperatures, progressive increases in energy expenditure, skin lesions on the tail and plantar surface,,;, debilitated appearance, and eventual death. We investigated the possible role of the preoptic/anterior hypothalamus (POAH) in the mediation of the TSD effects by comparing these effects in POAH-lesioned and unlesioned rats. Bilateral POAH lesions sufficient in size to impair homeothermic responses to changes in ambient temperature did not produce TSD-like temperature changes under baseline ambient temperatures of 28-29°C, implying that the thermoregulatory changes produced by TSD do not result from impairment of tlhe lesioned area. However, the possibility remains that the TSD effects are mediated by damage to POAH areas that were not lesioned. During TSD, lesioned and unlesioned rats showed similar progressive increases in energy expenditure, but the lesioned rats showed earlier, steeper, and eventually greater declines in Tip and Thy. This result suggests that in unlesioned rats the POAH may counter-regulate against, and thereby attenuate, the reduction in heat retention caused by TSD. This failure of regulation in lesioned rats is consistent with their impaired response to ambient temperature change and implies that, in unlesioned rats, some POAH thermoregulatory mechanisms continue to function normally during TSD. Lesioned rats did not show the characteristic TSD-induced early increases in Tip and Thy. This result could imply either that heat retention was so compromised that body temperatures did not rise in spite of a TSD-induced increases in thermoregulatory setpoint, or that the setpoint increase in unlesioned rats is POAH-mediated. Notwithstanding the greater Tit' and Thy declines in lesioned rats, they survived the TSD procedure longer than the unlesioned rats, thus supporting previous indications that death did not result from hypothermia. Keywords: Sleep deprivation; The~moregulation; Preoptic/anterior hypothalamus
I. Introduction
Rats subjected to total sleep deprivation (TSD) by the disk-over- water method show initial increases in intraperitoneal (Tip) and hypothalamic (Tny) temperatures followed by later falls to below baseline [13]. Two thermoregulatory changes have been identified. (1) An increased temperature setpoint (Tset) is indicated by behavioral attempts at warming even while Tip and Thy are above baseline, a more sustained rise in Thy than in Tip, and an increase in energy expenditure (EE) as these temperatures decline towards baseline. (2) A heat retention deficit is indicated by the late temperature declines and behavioral warming [16,19] in spite of greatly increased EE, the accelerated decline of Tip relative to Thy [13], and a hormonal profile consistent with a response to cold exposure [1]. Presumably, both ther-
* Corresponding author. Fax: (1) (312) 702-0277. 0006-8993/95/$09.50 © 1995 Elsevier Science B.V. All rights reserved SSDI 0006-8993(95)01071-8
moregulatory deficits obtain over the course of deprivation, while temperature levels vary with the relative strengths of the two deficits and the mobilization of calorigenic and behavioral responses to them. The preoptic/anterior hypothalamus (POAH) is an important structure for the mediation of both sleep and temperature regulation [4,9,12]. Lesions of the POAH induce thermoregulatory deficits a n d / o r reduced sleep in cats [11,17], rats [12] and dogs [10]. We used the neurotoxin N-methyl-DL-aspartic acid (NMDA) to make lesions of this area to evaluate the role of the POAH in TSD-induced thermoregulatory effects. Our histological examinations by both light microscopy [8] and electron microscopy [7] have not revealed anatomical damage to the POAH in TSD rats. Nevertheless, it is possible that TSD-induced functional impairment of the POAH caused some of the observed temperature and energy changes. If this were the case, one would expect that experimentally-induced lesions of the POAH might induce similar changes in rats that
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were not sleep deprived. Even in the absence of such TSD-like lesion effects in normally-sleeping rats, the lesions might exacerbate changes during TSD if the POAH normally counter-regulates TSD-induced malfunctions of other thermoregulatory centers. A third possibility (although we know of no precedent for it) is that the TSD effects were mediated by hyperactivation of hypothalamic thermoregulatory mechanisms, e.g., heat defense, in which case one might expect a reduction of thermoregulatory changes in POAH-lesioned TSD rats. The POAH is a multifunctional regulatory region with only partial localization of function. Since our goal was to disrupt thermoregulatory function without otherwise incapacitating the rat, we attempted to produce lesions as small and well-localized as possible and still produce measurable thermoregulatory deficits. In particular we wished to avoid lesions large enough or so-positioned that they would kill or completely incapacitate the rats or would by themselves produce large deficits in sleep under our experimental conditions.
2. M a t e r i a l s
and methods
2.1. Subjects and surgical preparation Subjects were eight male Sprague-Dawley rats (550-680 g) that were kept in constant light prior to surgery and throughout the experiment to flatten their circadian rhythms. Electrodes for recording EEG, EMG and hippocampal theta, transducers for recording Thy and Tip, and guide tubes for bilateral POAH lesions were implanted under Nembutal anesthesia (50 mg/kg). A thermistor encased in a 10 mm by 0.5 mm diam. glass rod (Model P20-BA-130-M, Thermometrics) was stereotaxically implanted into the left lateral posterior hypothalamus (P = 1.5 mm, L = 1.5 mm, H = 8.5 mm) [14]. Guide tubes for neurotoxin injection (C315G, 26G, Plastics One) were implanted bilaterally at a lateral-to-medial angle to avoid the sagittal sinus and were targeted to 1.0 mm above the POAH (P = 0.4, L = 0.6, H = 8.5). Dummy cannulae were inserted into the guide tubes to keep them from blocking. A thermosensitive transmitter (Model T-SLO, W.T. Barrows Co.) was placed into the peritoneal cavity to record Tip.
2.2. Apparatus for sleep deprivation The apparatus used for TSD has been described previously in detail [2]. Briefly, a pair of rectangular plastic cages were placed in parallel over a common disk which formed a partial floor for each cage (Fig. 1). Under the disk were water-containing stainless steel pans which extended on the cage walls. A TSD rat and a yoked control rat occupied the two cages. During deprivation, whenever
Plexlgless cage over disk .'.'-'----.----'---'.'.%'.-.-.-.'. ".-.-.-.'.'.'.'.'." i , E". . . .W . . a. .t.e. .r. . .pan . . . . . . u. *n. d: .e- r. - .dlsk - . - . - . ..*-'-'-'.'-'-'-.'-'~, .: , %%%..%%* %%% .%* %%%%
MOTOR~
, i
DISK
%%%%* *
~
l i
Fig. 1. Schematic illustration of top view of the deprivation apparatus. When sleep onset is detected in the TSD (or TSDL) rat, the disk is automatically rotated and the rat must walk opposite to disk rotation to avoid being carried into the water. A yoked control rat on the other side of the disk can sleep when the TSD rat is spontaneously awake and the disk is still.
the TSD rat started to sleep, the disk would rotate and both rats would move to avoid being carried into the water. The yoked control rat, however, could sleep when the TSD rat was spontaneously awake. In this study, all the yoked controls died either as a result of surgery or POAH lesions (if a potential TSD rat died, its yoked control was promoted to be a TSD rat), so no yoked control data were available. Because the major loci in this study were comparisons between lesioned vs. unlesioned TSD rats during baseline and TSD, the unavailability of yoked control lesioned rats was not critical.
2.3. Data collection and experimental procedures Amplified EEG, EMG and theta signals, the detector output for the intra-peritoneal transmitter, and the bridge circuit output for the brain thermistor were fed through a data collection microcomputer (DAP 2400, Microstar Laboratories) to a personal computer (PC) in 30-s epochs. 24-h data blocks (2880 epochs) were automatically scored for sleep and temperature each epoch by a computer program [3]. Waking temperature for a day was the mean of all the temperature values for wake epochs during the day, e.g,, if a rat spent half a day awake, the wake temperature for that day was based on values for 1440 epochs. After surgery, the animal was placed in the experimental apparatus for 9 to 12 days of recovery and adaptation. During this period, a plastic floor was placed over the disk. Then water was added to the pans under the disk, the floor was removed, and 6 to 12 days of baseline recording followed. As soon as the plastic floor was taken out, ambient temperature (Tam) was adjusted to 28-29°C and, except for tests of thermal defense (see below) was maintained at this level for the entire experiment. Lesions were made at the end of baseline. Then a 5-11 day lesion-baseline started, at the end of which, thermal defense was tested. TSD was then started and continued until the
P.-F. Fenget al. /Brain Research 703 (1995) 93-99 sleep-deprived rat died or death appeared imminent. Thus, the experimental schedule was surgery; 9-12 days of recovery; 6-12 days of baseline; lesion; 5-11 days of lesion-baseline; thermal defense test; and TSD until imminent or actual death.
2.4. The POAH lesion Because NMDA has excitatory effects, lesions were made under light anesthesia (chloral hydrate, 140 mg/kg, i.p.). For each side, a cannula (C3151, 33G, Plastics One) was connected through a hand-stretched polyethylene tube (i.d. about 0.3 mm) to a 1 /zl syringe mounted on a syringe pump (Model 55-1111, HaJvard Apparatus). The cannula was inserted through the guide tube to 1.0 mm below its tip. NMDA (Sigma) was dissolved in sterile isotonic saline solution (200 ng/ml, pH = 7.4); then 0.2 /xl was injected into each side over a period of 2.0 min. To ensure that the solution was delivered into the brain, the travel distance of the solution within the PE tubing (about 3 mm, depending upon the i.d. of the tubing) was monitored via movement of an air pocket. After a 3 min wait, the interior cannula was gently removed and the dummy catheter replaced.
95
2.Z Histology On completion of the experiment, rats were killed by anesthetic overdose and their brains were quickly removed and frozen on dry ice. Sections were cut at 40 p,m and stained with cresyl violet. Light microscopy was used to observe the lesion size and location.
2.8. Data presentation The effects of POAH lesions on thermoregulation during TSD were determined by comparing Tip, Thy and EE in lesioned TSD (TSDL) rats and intact TSD rats from a previous experiment that measured the same variables [13]. Similar pathology developed in all TSDL rats during deprivation, but at slightly different rates. To easily compare the results of TSDL rats with the earlier study, data collected during deprivation were time-scaled by quarters of deprivation (survival) time. Repeated measures ANOVA was used to determine the group by time interactions, which reflected differences between TSDL and TSD rats over the course of deprivation, t-Tests were used for specific comparisons of matched and unmatched samples.
2.5. Thermal challenge as a test of thermoregulation 3. Results To establish that the lesions affected thermoregulatory mechanisms, the four lesioned rats and four unlesioned rats were compared in the deprivation apparatus on cold and heat defenses. Tip and Th:, were continuously recorded while Tam w a s gradually increased from its normal level of 29°C to about 40°C and then decreased to about 14°C over a period of approximately 5 h. As measures of thermal defense, two parameters, AThy//ATamand ATip/ATam, i.e., the amounts Thy and Tip changed per change in Tam, were defined: ZIThy//ATam = ( n T h y ATip//ATar n = ( n T i p -
ZThy )//( nTam - ZTam) ZTip ) / / ( n T a m - ZTam)
HThy and HTip a r e the averages of the five 30-s epochs with highest Thy and Tip during high Tam (HTam) thermal challenge, and LThy and LTip are the averages of the five epochs with lowest temperatures during the down phase of Tam (LTam). High scores on these measures reflect poor thermal defenses, i.e., a relative inability to maintain stable body and brain temperatures as ambient temperatures varies. where
2.6. Energy expenditure (EE) EE (kcal/day) was calculated based on the caloric values of daily food consumption and change in body weight as described earlier [2]. This measure was validated as an indicator of metabolilc rate in an earlier TSD study [1].
3.1. POAH lesion anatomy Morphologically, all the lesions were highly localized; the diameter of the area with severe cell destruction was about 0.2 to 0.3 mm. In three of the four rats, the area of maximal cell body destruction was in the anterior medial hypothalamus (AMPO). In one rat, the lesion was slightly off the center of the AMPO; cell destruction was concentrated near the edge of the medial preoptic area at the border of the medial and lateral preoptic areas.
3.2. Effects of POAH lesions during baseline 3.2.1. Sleep during pre-lesion and post-lesion baseline Sleep percentages of total time during the last five days of TSDL baseline (45.2 +_ 1.5% for NREM; 6.6 ± 0.6% for PS) resembled the percentages previously reported for the TSD rats (47.3 _+ 2.1% for NREM; 5.2 ± 1.2% for PS) [13]. Although lesions were made under light anesthesia, all rats were agitated for about 1.5 h after lesion induction. In a comparison of the averages of the last five post-lesion and pre-lesion baseline days, one TSDL rat showed a 23% decrease in NREM sleep post-lesion, while the other three rats showed small increases; the group averages (pre-lesion -- 45.2%; post-lesion = 45.1%) were almost identical. Two rats showed small decreases in PS post-lesion, while the other two rats showed large decreases (from 5.7 and 6.6% to 2.6 and 2.5%, respectively). The group average of 4.6% was 30.3% lower than during pre-lesion baseline, but,
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P.-F. Feng et al. /Brain Research 703 (1995) 93-99
o• v
given the small subject sample and large post-lesion variance, the change was not statistically significant. It is also unlikely that these reductions in PS strongly affected thermoregulation; they are more typical of the modest reductions seen in yoked control rats (e.g., 47% [6]), which have relatively small temperature changes, than of the near complete reductions of PS seen in TSD rats (e.g., 96% [6]).
Q t-
J~ o ~
3.2.3. Thermal defense The thermal challenge comparisons were made between the four TSDL rats (after lesion) and four unlesioned rats of comparable age and weight which had been surgically prepared for the TSDL experiment and subjected to the adaptation and baseline schedules, but whose post-lesion data were not used. In the unlesioned rats, thermal challenges induced mostly small, unpredictable increases or decreases for both increases and decreases in Tam. TSDL rats after POAH lesions clearly showed impaired homeothermic mechanisms; both Why and Tip of these animals tended to vary in the same direction as Tam. During exposure to the cold ambient temperatures, mean Tip fell by 1.8°C and mean Thy fell by 1.5°C. During exposure to warm ambient temperatures, m e a n Tip in-
0 -1
L ....,,-- Tip T S D L ' ~ ' % ' ~
-2
\
Tip TSD
Q1
\ b
Thv TSD L
~, \
- Thy TSDL
~" -4 E 0 I-- -5
3.2.2. Temperatures during pre- and post-lesion baselines Because the major focus of the study was on temperature changes during sleep deprivation, when only waking temperatures could be reliably evaluated, only waking temperatures were evaluated during baseline as well. During the last five days of pre-lesion baseline, mean waking Tip of TSDL rats was 37.3 _+ 0.27°C; during the last five days of post-lesion baseline, it was 37.4 + 0.28°C. Over the same time periods, Thy changed from 38.7 + 0.34°C to 38.5 + 0.41°C. Thus, at the relatively warm 29°C ambient temperature in the experimental apparatus, the POAH lesions had little effect on Tip or Thy. Substantial changes were seen only immediately following lesion induction when both temperatures were increased for 3 to 7 h in most rats; the maximal increase during this period was about 1.5°C
1
Q2
Q3
Q4
Time Fig. 3. Changes from baseline Thy and Tip in TSD and TSDL rats during post-lesion baseline (L, TSDL rats only) and successive quarters (Q1-Q4) of sleep deprivation.
creased by 1.1°C and m e a n Zhy increased by 1.0°C. The m e a n AThy//z~Tam and ATip/ATam were 0.04 and 0.05 respectively in unlesioned rats (Fig. 2). The values in lesioned rats were 0.15 for Thy and 0.16 for Tip. Both AThy//ATam and ATip//ATam were significantly larger in lesioned rats ( P = 0.04 for Thy and P = 0.005 for Tip).
3.2.4. Energy expenditure, appearance, and skin condition during baseline As part of the general issue of whether POAH lesions alone could produce changes like those which result from TSD, EE, appearance and skin condition were evaluated during the baseline periods. Mean EE of the TSDL rats was 93.9 + 9.2 kcal/day during the last five days of pre-lesion baseline and remained unchanged at 94.4 + 17.4 kcal/day during the last five days of post-lesion baseline. During post-lesion baseline none of the TSDL rats developed any signs of the debilitated appearance or skin lesions on the tail and plantar surfaces that are characteristic of TSD rats. It could be reasonably argued that even if POAH lesions produced changes like those of TSD, because appearance and skin changes develop gradually over time, the 5-11 day post-lesion baseline period may not have been sufficiently long to permit those symptoms to fully emerge. This caveat does not apply in the case of EE, which generally shows increases of about 50-100% over similar time periods in TSD rats [1,6,13].
0.204
3.3. Effects of POAH lesions on the major pathology of
o.1~
TSD 3.3.1. Body temperature during TSD <~ Tip
Thy
[]
Unlesioned
•
Lesioned
Fig. 2. Mean+S.E.M. of AT/ATam, a measure of body temperature change relative to ambient temperature change (see text), for Thy and Tip during baseline thermal challenge tests in rats with and without POAH lesions.
Tip of intact TSD rats shows a typical pattern, verified by several studies in our laboratory [1,13,22], of an initial rise and then a fall below baseline. The pattern in TSDL rats was obviously different. Instead of the initial rise, mean Tip of TSDL rats dropped shortly after TSD began (Fig. 3). Then it showed a progressive decline that was much steeper than in intact TSD rats. The lowest mean waking Tip was 3.39°C below baseline (Fig. 3), which represented more than twice the decrease in intact TSD
P.-F. Feng et al. / Brain Research 703 (1995) 93-99
rats. Repeated measures ANOVA on Tip showed a significant group × time interaction ( P = 0.028). Compared with baseline, Tip changes of TSDL rats in quarters 3 and 4 of deprivation were highly significant ( P < 0.01).
97
days. A t-test showed that the survival time for TSDL rats was significantly longer than for intact TSD rats ( P = 0.043).
3.5. Appearance changes and lesions 3.3.2. Hypothalamic temperature during TSD In intact TSD rats, Thy increases above baseline shortly after TSD is initiated. This elevation is maintained for about 90% of the deprivation period and is followed by a decrease below baseline in the last one or two days of the TSD (Fig. 3). In TSDL rats mean Thy never rose above baseline. As with intact TSD rats, a level plateau was maintained over the first three quarters of deprivation, although the level was lower. As with TSD rats, Thy of TSDL rats showed an accelerated decline during the last quarter of deprivation, but the decline was much steeper and progressed to much lower levels in the TSDL rats. Repeated measures ANOVA showed a significant group × time interaction for Zhy ( P = 0.008). ThUS the shapes of both the Thy and Tip curves were similar in TSD and TSDL, except that the TSDL curves were at a lower level and showed steeper progressive declines.
3.3.3. Energy expenditure (EE) during TSD Mean baseline EE was 93.9 _ 9.23 kcal/day in TSDL rats and 98.8 _ 22.0 kcal/day in intact TSD rats. The two groups showed similar large increases during the first quarter of deprivation and more gradually progressive increases thereafter (Fig. 4). In both conditions, EE values were very similar. Repeated measures ANOVA showed no significant group × time interaction ( P > 0.1).
3.4. Survival time Of the four TSDL rats, one died during the period of deprivation, two were sacrificed when the amplitude of EEG decreased and large EEG spikes appeared, and one died during the second day of recovery. The mean survival time of the four TSDL rats was 24.3 + 4.0 days. Our previous data from three TSD experiments with otherwise untreated rats showed a mean survival time as 17.1 + 7.2 20O% 160% 120% J:
u
80%
uJ UJ 40% 0%, L
I;11
Q2
Q3
Q4
Time
Fig. 4. Percentage changes from baseline (mean+S.E.M.) in energy expenditure (EE) in TSD and TSDL rats during post-lesion baseline (L, TSDL rats only) and successive quarters of sleep deprivation.
Typically, TSD rats show a progressively debilitated appearance with yellowing, disheveled fur. They also show stereotypic ulcerative and hyperkeratotic skin lesions localized to the plantar surfaces and the tail. The TSDL rats developed the same appearance changes and lesions at similar rates.
4. Discussion
Were the lesions successful? Our POAH lesions did not produce the large decreases in sleep previously reported for the rat [12], probably because of the small size of the lesions and the high Tam (28-29°C) during recording. Previous studies in rats [21] and cats [20] have shown that sleep reduction only occurs with large lesions and low ambient temperatures. Szymusiak et al. [20] reported that cats with POAH lesions produced by NMDA showed an impairment of warm defense only, in effect raising the thermoregulatory setpoint. The TSDL rats more closely resembled rats with electrolytic POAH lesions insofar as they showed impaired defenses against both heat and cold (summarized in [18]). Like the POAH-lesioned rats of Satinoff et al. [18], the TSDL rats were able to maintain stable body temperatures at an ambient temperature near 29°C. The strong and reliable effects of TSD on temperature regulation and the role of the POAH in temperature regulation and other regulatory processes raise the question of whether the physiological effects of TSD are mediated by damage it inflicts on the POAH. Under baseline conditions with Tam at 28-29°C, the POAH lesions failed to produce any of the thermal, metabolic, or dermatological effects reliably produced by TSD, which would argue against a mediation of TSD effects by POAH impairment, at least for the lesioned area. On the other hand, the induced lesions were small, and it is not known whether TSD effects might be mediated by POAH sites not affected by the lesions. Older studies of rats with large POAH lesions (e.g., [18]) have revealed such a diverse mixture of thermoregulatory changes and such marked variations in survival time that comparisons with TSD rats are problematic. It is of interest, however, that some POAH-lesioned rats show a 'paradoxical hyperthermia', i.e., body temperatures are elevated, but the rats are unable to defend against cold stress [18]. This condition is remindful of TSD rats which have an elevated Tset but impaired heat retention. Might TSD effects result from some anomalous, TSDinduced hyperactivity of POAH mechanisms? If that were the case, we might expect some mitigation of these effects
98
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in the lesioned rats during TSD. Yet most of the TSD effects (including the Tip and Thy declines, EE increase, skin lesions, debilitated appearance, and actual or imminent death) were apparent in all the lesioned rats after deprivation. Exceptions to the above pattern were the early increases of Tip and Thy, which are characteristic of TSD rats, but were not seen in the lesioned rats during deprivation, and the increased survival time. These exceptions will be discussed below. The appearance of TSD effects in TSDL rats does not mean that the POAH played no role in their development. A role is clearly evident in the earlier and greater declines of Tip and Thy during deprivation in the lesioned rats than in the TSD rats. Since EE was similar in the two groups, ability to retain heat must have been reduced more in the lesioned rats. The lesions alone did not decrease heat retention, because they did not result in temperature declines or elevated EE during baseline. Rather, the TSD effect on heat retention was exaggerated in the lesioned rats. A parsimonious explanation for this interaction is readily available. Just as the lesioned rats evidenced impaired cold defense to thermal challenge during baseline, they likely had an impaired cold defense to the drop in body temperatures induced by TSD. Presumably, in unlesioned rats, intact POAH mechanisms counter-regulate and attenuate the potential TSD-induced heat retention deficit. As noted above, lesioned TSD rats did not show the characteristic early increases in body temperatures. Two explanations are available. One is that TSD-induced increases in Tset are mediated by POAH mechanisms that are no longer available in the lesioned rats. This is consistent with the well-documented role of the POAH in adjusting Tset [9]. A second explanation is that Tset may have increased as in the unlesioned rats, but heat retention may have been so impaired that the body temperatures could not increase to above baseline levels. The exaggeration of this impairment in the lesioned rats was noted above. The second explanation is more consistent with other data. Another group of TSD rats which failed to show an initial rise in Tip but showed exaggerated Tip declines consisted of rats whose EE increase was blunted by making them hypothyroid [15]. In this case, the absence of temperature increases was more likely due to a failure of energy compensation for the heat retention deficit, since such rats had an intact POAH. However, it is also possible that the rise in T~t in TSD rats requires thyroid hormone. The survival time of TSDL rats was longer than that of intact TSD rats. It is unlikely that the longer survival times in the TSDL rats resulted from their relatively large size at the start of the experiment, since they survived longer than similarly large rats in other TSD experiments [13,16]. Furthermore, the within-experiment relationship between initial weight and survival has been shown to be negative (r = - 0 . 9 1 ) [5]. Since the TSDL rats had lower temperatures, this suggests that, as we have previously argued [15], within a limited range, hypothermia does not reduce sur-
vival time. The longer survival time of TSDL rats is not readily explained. Although overall metabolic rate was not reduced by the hypothermia, it is possible that some specific, harmful metabolic process or processes were slowed by the Q10 effect. However, survival was not similarly extended in hypothyroid TSD rats with similarly low Tip [15]. Given the numerous possible differences between the TSDL rats and previously run TSD rats, the extension of survival needs to be replicated before it can be given a convincing explanation. In summary, although the POAH-lesioned rats showed impaired heat and cold defenses, the lesion itself did not produce effects that are reliably produced by sleep deprivation. It is not clear whether larger lesions might have produced such effects. TSD-induced decreases in heat retention are exaggerated in POAH-lesioned rats, suggesting that the POAH normally regulates temperatures within a narrow range against the excessive heat loss produced by TSD. POAH-lesioned rats did not show the characteristic increases in body temperatures early in sleep deprivation, which could mean either that TSD-induced increases in T~t are mediated by the POAH, or that heat retention was so impaired that temperatures could not rise above baseline in spite of increases in T~et. A small, but statistically significant increase in survival time in lesioned TSD rats compared to unlesioned TSD rats remains unexplained.
Acknowledgements This work was supported by NIMH grants MH4151 and MH18428 to A.R.
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