Brain Research, 511 (1990) 227-233 Elsevier
227
BRES 15269
Influence of ambient temperature on sleep and body temperature after phentolamine in rats Stephen Kent I and Evelyn Satinoff 1-3 ~Department of Psychology, 2Program in Neural and Behavioral Biology and 3Department of Physiology and Biophysics, University of lUinois at Urbana-Champaign, Champaign, 1L 61820 (U.S.A.) (Accepted 8 August 1989)
Key words: Rapid eye movement sleep; Phentolamine; a-Adrenoceptor antagonist; Body temperature; Noradrenergic mechanism; Ambient temperature
Phentolamine, an a-adrenoceptor antagonist, both decreases rapid-eye-movement sleep (REMS) and causes a dose- and ambient temperature (Ta)-dependent drop in body temperature (Tb). The purpose of this paper was to examine the correlation between changes in sleep and changes in Tb after phentolamine at various Ta's. Tb and sleep were recorded in male rats for 4 h after i.p. injection of either saline or phentolamine (1 mg/kg at Ta 20 °C; 5 mg/kg at 20, 30 and 32 °C; 10 mg/kg at 20, 30, 32, and 34 °C). Changes in sleep were highly correlated with changes in Tb: when Tb dropped, amounts of sleep, especially REMS, also were decreased. The largest effects were seen at Ta 20 °C. After 5 mg/kg, Tb was below normal for 2 h and latency to REMS increased 82 + 27 min (P < 0.025). After 10 mg/kg Tb was decreased for 3 h, and latency to REMS increased 91 + 19 min (P < 0.001). While Tb was lower amounts of REMS were also decreased. Smaller effects were observed on slow wave sleep. At those conditions where phentolamine had no effect on Tb (i.e. Ta 20 °C, 1 mg/kg, and Ta 32 °C, 5 and 10 mg/kg) no differences were found in any measure of sleep. These results demonstrate that the effects of phentolamine on sleep are not caused by direct action on sleep mechanisms, but rather by its actions on thermoregulatory and possibly other mechanisms that modulate sleep. INTRODUCTION T h e n e u r o p h a r m a c o l o g y of sleep has been a topic of intense study ever since Jouvet s p r o p o s e d his m o n o a m i n ergic t h e o r y of sleep. A c c o r d i n g to this theory, noradrenergic neurons are necessary for the maintenance of r a p i d - e y e - m o v e m e n t sleep ( R E M S ) . This implies that t r e a t m e n t s that facilitate n o r a d r e n e r g i c transmission should increase R E M S and b l o c k a d e of n o r a d r e n e r g i c activity should decrease it. Since this seminal p a p e r , n u m e r o u s studies have a t t e m p t e d to test this hypothesis, but the role of n o r a d r e n e r g i c involvement in R E M S remains controversial. A m b i e n t t e m p e r a t u r e (T~) also has a strong effect on R E M S , but here there is no controversy: Ta's a b o v e or b e l o w t h e r m o n e u t r a l i t y decrease R E M S 16'18'21'25. If the t h e r m a l stress is e x t r e m e enough, slow wave sleep (SWS) is d i s r u p t e d also. In rats, a m o u n t of R E M S varies significantly even within the t h e r m o n e u t r a l zone defined by minimal and constant metabolic rate (25-31 °C), reaching a p e a k at 29 + 1 °C19. Since R E M S is so sensitive to T~, any t r e a t m e n t that alters amounts and/or distribution of R E M S m a y be doing so by m a k i n g a particular T~ m o r e or less stressful. For e x a m p l e , Szymusiak and Satinoff2° e x a m i n e d sleep in
rats after lesions in the medial p r e o p t i c area of the hypothalamus, which interfere with thermoregulation. The severity of the postlesion h y p o s o m n i a and the course of recovery of sleep were strongly d e p e n d e n t u p o n the T a at which the rats were studied. Most n o r a d r e n e r g i c agents, including all those that have been used in sleep studies, also affect t h e r m o r e g u lation (see refs. 2 and 3 for review). In general, n o r a d r e n e r g i c blocking agents that reduce R E M S lower b o d y t e m p e r a t u r e (Tb). W e have previously r e p o r t e d that after injection of the nonselective a - a d r e n o c e p t o r antagonist, p h e n t o l a m i n e , amounts of R E M S d e p e n d e d on the Ta at which sleep was m e a s u r e d . A t Ta's at which this c o m p o u n d l o w e r e d T b it inhibited R E M S ; at Ta's where it did not change T b, it had no effect on R E M S 9. The present p a p e r continues the study of the effects of this drug on Tb, waking, SWS and R E M S .
MATERIALS AND METHODS
General procedure Rats implanted for EEG and Tb recording were adapted to the sleep recording chamber for 6-10 h on 3 separate occasions at Ta 23 °C. Sleep and Tb data were recorded for 1-3 h before and 4 h after i.p. injections of phentolamine-HCl (1, 5, and 10 mg/kg in 1 ml isotonic saline) or an equivalent volume of saline alone. Two rats,
Correspondence: E. Satinoff, Psychology Department, University of Illinois, 603 E. Daniel St., Champaign, IL 61820, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
228 one injected with phentolamine and one with saline, were recorded from simultaneously. Only rats that had had at least two 1-min REMS bouts prior to injection were used in the analyses. All recordings were begun 2-5 h after lights-on. Each rat was injected once with either 1, 5, or 10 mg/kg phentolamine and once with saline, at one or more T~'s. Drug injections were spaced at least 4 days apart. No rat received more than 3 drug injections during the study.
Sleep and Body Temperature at Ta20°C #16
Saline SW~ 37
Subjects and housing Subjects were 30 male hooded rats of the Long-Evans strain. They were maintained from birth at Ta 23 + 1 °C on a 12:12 light-dark cycle, and weighed 280-445 g at the start of the experiments. Food and water were available ad libitum. For sleep recording, each rat was placed in an open-topped, Plexiglas cage (29 x 30 x 30 cm), housed in a large light- and temperature-controlled chamber (T a 23 + 0.5 °C). Food and water were not available during recording sessions. One h before recording began, the chamber Ta was set to either 20, 30, 32, or 34 °C. This took approximately 10 min at 20 °C and 40-60 min at the warmer Ta's.
36.
0
60
120 Time (min)
180
240
Fig. 1. Sleep state progression and Tb measured every 10 min for rat 16 at Ta 20 °C for 20 min before and 240 min post saline injection (a) and post phentolamine injection (10 mg/kg) (b). S, SWS; W, Waking; R, REMS. Arrow indicates time of injection.
Surgery Each rat was anesthetized with Nembutal (sodium pentobarbital; 50 mg/kg i.p.), injected with atropine sulfate (0.5 mg/kg i.p.) and placed in a stereotaxic instrument. Four stainless-steel jeweler's screws were threaded through holes drilled in the skull to record cortical and hippocampal EEG, and either 4 single-stranded, insulated, stainless-steel wires bared at the tip or 4 thin silver plates ( = 2 x 7 mm) were implanted in the dorsal neck muscles, two on each side of the midline, to record EMG activity. A screw placed in the skull over the cerebellum served as ground. All electrode leads were soldered to a miniature 9-pin Amphenol connector, which was anchored to the skull with dental acrylic. At the same time a temperature telemetry device (1 x 2.1 cm cylinder, weight 2.3 g; Mini-mitter Co., Sunriver, OR) was implanted into the peritoneal cavity. Each rat was then returned to its home cage for at least 1 week.
Data collection Tb data were obtained through the implanted transmitter, which emits a broadband RF pulse at a rate proportional to its temperature. This signal was converted to a Tb value by an Apple II+ microcomputer. Tb values were recorded and stored on diskettes at 5 or 10 min intervals. The rat was connected, via a cable on a counterweighted slipring, to a Model 7 Grass polygraph. The bandwidths used were 1-35 Hz for the cortical EEG, 3-15 Hz for the hippocampal EEG, and 10-35 Hz for the EMG. To record movement, a magnet was hung in a wire coil attached to the cage floor. The cage was mounted on 10 light springs and any movement displaced the magnet and emitted a signal. All signals were displayed on chart paper at a speed of 5 mm/s.
changes in Tb: (1) maximal change from baseline during the first 2 h post injection, and (2) a hypothermia index (HI), which was calculated by integrating the area between the Tb curve and the extrapolated baseline. For both of these measures baseline was the mean Tb of the hour immediately pre-injection. Latency and maximal Tb change data were analyzed by within-subjects two-tailed t-tests. All other data were analyzed using 2-way ANOVA's with repeated measures on the 4 hourly blocks. Separate analyses were done for each Ta and dose. Planned post hoc comparisons were done at each hour to determine significant differences between the two treatments.
RESULTS
Overall s u m m a r y I n g e n e r a l , w h e n p h e n t o l a m i n e c a u s e d a d r o p in Tb, latency to REMS
increased
and
amounts
of REMS
d e c r e a s e d . T h e r e w a s also a m i l d e r a n d s h o r t e r - l a s t i n g d e c r e a s e in S W S . A f t e r 10 m g / k g , t h e l a r g e s t d r o p s in T b a n d t h e m o s t s e v e r e s l e e p c h a n g e s w e r e o b s e r v e d at 20 °C. T h e r e w e r e s m a l l b u t s i g n i f i c a n t d e c r e a s e s in Tb, REMS,
a n d S W S at 30 °C. A t 32 °C, t h e r e w e r e n o
differences between the groups on any measure. At Ta 34 °C, w h i c h w a s a h e a t stress, n e i t h e r g r o u p s l e p t m u c h . The 5 mg/kg dose had smaller and more variable effects
Sleep state scoring Waking, SWS and REMS were scored in 10 or 15 s epochs by experienced scorers. Waking was identified by low voltage fast activity in cortical EEG, small amounts of hippocampal theta (associated with movement), high, variable amplitude in EMG, and a high incidence of movement. SWS was characterized by an increased voltage in EEG, especially slow cortical activity, a lack of clear theta activity, an intermediate level of EMG activity, and little or no movement. REMS was identified by low voltage fast cortical activity, continuous theta, and tonically silent EMG interspersed with phasic bursts of activity.
o n Tb a n d R E M S ,
e s p e c i a l l y at 20 °C. A t 30 °C, t h e r e
w e r e slight, b u t s i g n i f i c a n t , d e c r e a s e s in T b a n d R E M S . As with the higher dose, there was no effect on any v a r i a b l e at 32 °C. 1 m g / k g p h e n t o l a m i n e h a d n o e f f e c t o n T b o r a n y s l e e p m e a s u r e a t 20 °C, t h e o n l y Ta at w h i c h this d o s e was t e s t e d . Fig. 1 s h o w s Tb c h a n g e s a n d s l e e p s t a t e p r o g r e s s i o n f o r a r a t i n j e c t e d w i t h s a l i n e (Fig. l a ) o r 10 m g / k g p h e n t o l a m i n e (Fig. l b ) at 20 °C. A t t h i s 7", n o r a t i n j e c t e d w i t h
Data analysis
phentolamine at either dose had any REMS until after Tb
The scored sleep data were stored on the hard disk of an IBM AT for data reduction. A program determined latencies to SWS and REMS, bout number and length, and duration of waking, SWS, and REMS for each hour. Two measures were used to characterize
h a d r e a c h e d its l o w e s t p o i n t a n d r e t u r n e d t o w i t h i n 0.5 °C o f p r e - i n j e c t i o n l e v e l s ( u s u a l l y 6 0 - 1 8 0 m i n ) . A t 32 °C n e i t h e r Tb, s l e e p , n o r w a k i n g w a s a f f e c t e d (Fig. 2 a , b ) .
229
Sleep and Body Temperature at Ta32°C #42 I
Saline S
38
I
1
~
W
° I'5°
Ta 20 °C
37
~
~
°
° 37 I
3.
I
0
I
I
60
I
120 Time (min)
I
I
I
180
I
240
Fig. 2. Same as Fig. 1 for rat 42 at T. 32 °C and 10 mg/kg.
After 10 mg/kg phentolamine, Tb started to drop within 20 min and reached a minimum between 30 and 80 rain post-injection. It decreased a maximum of 1.3 + 0.3 °C (+ S.E.M.) (P < 0.001) compared to the Tb of saline-injected controls. The hypothermia index (HI) was significantly higher for 3 h (Fig. 3A). The rats were awake more (P < 0.01), due mainly to increased waking during the first hour (31 + 2 rain post saline vs 46 + 3 min post drug, P < 0.005) and the second hour (21 _+ 3 min vs 30 + 4 min, P < 0.005; Fig. 3A). Bout length increased from 1.3 + 0.1 to 3.8 + 0.8 (P < 0.02) during the first hour. Controls actually had more waking bouts (26 + 2 vs 15
Changes in Sleep and Tb after Phentolamine (10 mg/kg) Ta =30°C
T a = 20°C Waking
N = 11
Waking
T a = 34°C
Ta =32°C N = 10
Waking
N= 8
Waking
N = 4
60[ -'Z-
*t Z 0 SWS
sws
SWS
SWS
40 (1)
t-"
lO REMS
REMS
REMS
REMS
t~9 C
.~-1 .o[ •~t- - o5!.
*
•-
* ~
Saline Phentolamine
I
2
3
Hours
A
4
1
2
3
4
2
3
Hours
Hours
B
C
4
2
3
4
Hours
D
Fig. 3. Hourly analysis of levels of waking, SWS, REMS and change in Tb after injection of phentolamine (10 mg/kg) or saline at 20 °C (A), 30 °C (B), 32 °C (C), and 34 °C (D) for 4 h post-injection.'P < 0.05; "*P < 0.01.
230 Latency to REMS increased by 91 + 19 min (P < 0.001) compared to saline-injected controls. REMS latency was correlated both with the maximal drop in T0 (r = -0.60, P < 0.05) and with the H I 3 (r = 0.66, P < 0.025). Amounts of REMS were decreased for the first 3 h (P < 0.01; Fig. 3A), due to a decrease in bout number (P < 0.001). Consequently, total amounts during the 4 h recording time were reduced (P < 0.001). For the first 3 h, rats injected with phentolamine had only 28% of the REMS of controls: by the fourth hour, REMS levels were equivalent. Total REMS time was also correlated with both the drop in To (r = 0.76, P < 0.01) and the HI 3 (r = -0.79, P < 0.005). After 5 mg/kg, Tb decreased 0.8 + 0.2 °C (P < 0.01) and the HI was higher for only 2 h (Fig. 4A). There were
+ 3, P < 0.02), but since they were so much shorter it did not affect total wake time. The increase during the second hour was due to a non-significant increase in bout length in the rats injected with phentolamine. Latency to SWS was increased by 16 + 5 min (P < 0.01). Consequently, SWS amounts during the first hour dropped from 25 + 2 to 13 + 3 min (P < 0.01; Fig. 3A). This was due to a decrease in bout number from 26 + 2 to 15 + 3 (P < 0.02). Amounts were still decreased during the second hour (P < 0.05; Fig. 3A). Latency to SWS post drug was correlated with the cumulative HI for the first 3 h (HI3) (r = 0.75, P < 0.01). Total SWS time during the first 2 h post phentolamine was correlated with both H I 3 (r = -0.69, P < 0.02) and the maximal drop in T b (r = 0.64, P < 0.05,).
Changes in Sleep and Tb after Phentolamine (5 mg/kg) Ta =30°C
T a = 20°C
60 50 ~
._
Waking
N= 7
Waking
Ta =32°C
N=8
Waking
N=5
30 20
10 0
SWS
SWS
3040
~
SWS
~
cyJ ._c
20 10 0
REMS
REMS
REMS
*
10
.c_
I
-- -0.5
1°I U
~_ o.o I I
1.0 1
**
*
2
3
~ 2
3
Hours
4
1
4
A
B 30 °C,
1
2
3
Hour s
Hour s
Fig. 4. Same as Fig. 3 for phentolamine (5 mg/kg). (A) 20 °C, (B)
l
C and (C) 32 °C.
4
231 no significant differences in waking or SWS. Latency to REMS increased by 82 + 27 min (P < 0.025) and amounts of REMS were reduced during the second and fourth hours post injection (P < 0.01). During the first hour only one phentolamine-injected rat had any REMS, whereas 5/8 controls did. As with 10 mg/kg, these decreases were primarily caused by a reduction in bout number. Neither To nor any sleep parameter was affected by phentolamine (1 mg/kg). T,, 30 °C
Phentolamine (10 mg/kg) increased the HI for the first 3 h (Fig. 3B): To decreased a maximum of 0.8 + 0.2 °C (P < 0.005). Waking was increased only during the first hour post injection (P < 0.005; Fig. 3B). SWS latency was increased by 8 + 2 min (P < 0.005) and amounts decreased during the first hour post injection, from 31 + 2 to 26 + 2 min (P < 0.02; Fig. 3B). REMS latency increased 24 + 9 min (P < 0.025) and REMS duration did not return to normal until the second hour (Fig. 3B). However, total REMS for the 4 h period was decreased (P < 0.05). After 5 mg/kg, the HI was increased compared to controls during the second and third hour post injection (Fig. 4B). Since T0 decreased a maximum of only 0.3 + 0.1 °C (P < 0.02), we do not believe this to be of any functional importance. Waking was increased for the 4 h recording period (P < 0.025), but only the change in the third hour post injection reached significance (Fig. 4B). The increase in waking was due to a rise in bout number during the first hour post injection (P < 0.05) and a non-significant increase in bout length during all 4 h. There were no differences in SWS latency or amounts. REMS amounts also decreased for the 4 h period (P < 0.05), but only the third hour showed a significant drop (Fig. 4B). T,,32°C
There were no differences in To or any measure of sleep between phentolamine- and saline-injected rats (Figs. 3C and 4C). T a 34 °C
There were no differences in T0 at this T~: Tb's of both saline- and phentolamine-injected rats rose by over 0.6 °C (Fig. 3D). Amounts of SWS were very low, but equivalent for both groups (Fig. 3D). REMS was severely inhibited for both groups (Fig. 3D). There were no differences in SWS or REMS latency. DISCUSSION These results demonstrate that phentolamine produces a dose- and Ta-dependent fall in To. When there was a
large drop in Tb, there was a large decrease in REMS, with lesser effects on waking and SWS. REMS was inhibited only as long as Tb was lowered. As Tb returned to baseline levels, amounts of REMS also returned to control amounts. At higher Ta's or lower doses, where phentolamine did not lower Tb, there were no changes in sleep or waking. The 'equilibrium' Ta for our rats after 5 and 10 mg/kg was 32 °C. At 32 °C all effects of phentolamine on Tb and sleep were eliminated. The effects of phentolamine on Tb and sleep at 20 °C agree with earlier reports. At 20 °C and 10 mg/kg, this compound lowered the Tb of rats a mean of 1.6 + 0.3 °C24. With different doses and T~'s, it was reported that the higher the dose or the lower the Ta, the larger the drop in Tb, and that at warmer Ta's or low doses no drop occurred ml. M/ikel/i and Hilakivi 12 found that phentolamine (10 mg/kg) reduced REMS for the first 2-3 h postinjection. This study was conducted at 22-24 °C, which is a low enough Ta to produce a drop in Tb. In cats, phentolamine (20 mg/kg) increased REMS in the first 4 h postinjection 17. However, without knowing the effect of this compound on Tb in cats, it is impossible to speculate on the reasons for this difference. Any drug can produce Tb changes in one of two ways: (1) by changing the thermal set-point, or preferred Tb, or (2) by altering one or more thermoregulatory effector mechanisms. Either mechanism of action will alter the thermoneutral zone, that range of Ta's in which thermoregulatory responses are minimal. In untreated rats this is generally 25-31 °C. However, even within this range amounts of REMS vary greatly. Decreases of 6 °C, from 29 to 23 °C (the high end of the T~ of many laboratories), decreased REMS by over 50%. Raising the T~ only 2 °, from 29 to 31 °C, reduced REMS by o v e r 60% 19. Thus, small and seemingly inconsequential changes in T~ can produce large changes in REMS. If phentolamine were lowering thermal set-point, then warmer Ta's should be more stressful and the rats should sleep less. This is not what we found. On the other hand, if phentolamine lowers T0 by altering thermoregulatory effectors (and, in fact, it does cause peripheral vasodilation in rats 11, humans 22, and dogs 7, and decreases metabolic rate in rats11), then the thermoneutral zone, or zone of thermal comfort, should be raised, and maximal amounts of sleep should occur at higher than normal Ta's. Lower T~'s and the drop in Tb that accompanies them should be more stressful and sleep should be decreased, especially REMS. This is exactly what we found. We are currently verifying this mechanism of action using a behavioral thermoregulation paradigm. One way of determining if a drug has altered thermal set-point is by measuring operant responses to thermal reinforcement. Preliminary data from this lab indicate that
232 p h e n t o l a m i n e does not alter the thermal set-point 1°. Rats injected with 10 mg/kg work harder than controls to w a r m themselves in a cold environment, thereby preventing a d r o p in T b. In the heat, T o does not fall and there is no difference in response rate between drug and control groups. These results, together with the present results on sleep, strongly suggest that phentolamine does not alter thermal set-point. As M c G i n t y and Siegel ~3 have pointed out, R E M S is a fragile state: several non-specific stressors can disrupt it. In this p a p e r we have only addressed thermal stress. A t 20 °C, after 10 mg/kg of phentolamine, the drop in T o only accounted for about half of the variance in sleep measures (56% of SWS latency and 48% of SWS amounts during the first 2 h; 44% of R E M S latency and 62% of total R E M S amounts). This does not mean that the residual effects are due to a direct action of the drug on sleep mechanisms. Phentolamine, like other noradrenergic agents, has a variety of effects which may contribute to changes in R E M S . These include, but may not be limited to, hypotension, peripheral vasodilation, tachycardia and/or palpitations, gastrointestinal irritability (i.e. diarrhea and nausea), and insulin secretion 5-7,11,14,15,22,23
O n e of these effects - - peripheral vasodilation - causes an increase in skin t e m p e r a t u r e . Skin t e m p e r a ture, like Tb, affects amounts of R E M S . Brief changes in skin t e m p e r a t u r e t o w a r d t h e r m o n e u t r a l i t y trigger R E M S , whereas changes away from it inhibit R E M S 21. With this in mind we are beginning to measure changes in skin t e m p e r a t u r e after p h e n t o l a m i n e . W e expect that adding this measure will enable us to account for a larger percentage of the variance in our data. In conclusion, we have shown that p h e n t o l a m i n e acts on sleep indirectly. Its action on T b is responsible for much of its effect on sleep. W h e n this effect is eliminated, by raising the T a at which the rats are tested, phentolamine has no effect on sleep. Consequently, this drug and p r o b a b l y others do not act directly on sleep mechanisms, but alter sleep through t h e r m o r e g u l a t i o n . A n y a t t e m p t to d e t e r m i n e the n e u r o p h a r m a c o l o g y of sleep needs to be u n d e r t a k e n with this point in mind. Acknowledgements. Support to E.S. from NSF Grant BNS 8311466 and NIMH Grant 1 RO1 MH 41138 is gratefully acknowledged. S.K. was partially supported by NIMH training Grant 1 T32 MH18412. We thank Hua Ma and Rick Kaplan for technical assistance. We also thank CIBA-Geigy for their generous gift of phentolamine.
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16 17 18 19 20 21
phentolamine on behavioral thermoregulation, Soc. Neurosci. Abstr., 14 (1988) 529. Lin, M.T., Chandra, A. and Fung, T.C., Effects of phentolamine on thermoregulatory functions of rats to different ambient temperatures, Can. J. Physiol. Pharmacol., 57 (1979) 14011406. M/ikel/i, J.P. and Hilakivi, I.T., Effect of alpha-adrenoceptor blockade on sleep and wakefulness in the rat, Pharmacol. Biochem. Behav., 24 (1986) 613-616. McGinty, D.J. and Siegel, J.M., Sleep states. In E. Satinoff and P. Teitelbaum (Eds.), Handbook of Behavioral Neurobiology. Motivation, Plenum Press, New York, 1983, pp. 105-181. Meier, R., Yonkman, E E , Craver, B.N. and Gross, E, A new imidazoline derivative with marked adrenolytic properties, Proc. Soc. Exp. Biol. Med., 71 (1949) 70-72. Moyer, J.H. and Caplovitz, C., The clinical results of oral and parenteral administration of 2-(N'p-tolyl-N'-m-hydroxyphenylaminomethyl) imidazoline hydrochloride (Regitine) in the treatment of hypertension and an evaluation of the celebral hemodynamic effects, Am. Heart J., 45 (1953) 602-610. Parmeggiani, P.L. and Rabini, C., Sleep and environmental temperature, Arch. ltal. Biol., 108 (1970) 369-387. Putkonen, P.T.S. and Leppvuori, A., Increase in paradoxical sleep in the cat after phentolamine, an alpha-adrenoceptor antagonist, Acta. Physiol. Scand., 100 (1977)488-490. Schmidek, W.R., Hoshino, K., Schmidek, M. and Timo-Iaria, C., Influence of environmental temperature on the sleepwakefulness cycle in the rat, Physiol. Behav., 8 (1972) 363-371. Szymusiak, R. and Satinoff, E., Maximal REM sleep time defines a narrower thermoneutral zone than does minimal metabolic rate, Physiol. Behav., 26 (1981) 687-690. 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. Szymusiak, R., Satinoff, E., Schallert, T. and Whishaw, I.Q., Brief skin temperature changes towards thermoneutrality trigger REM sleep in rats, Physiol. Behav., 25 (1980) 305-311.
233 22 Taylor, S.H., Sutherland, G.R., MacKenzie, G.J., Staunton, H.P. and Donald, K.W., The circulatory effects of phentolamine in man with particular respect to changes in forearm blood flow, Clin. Sci., 28 (1965) 265-284. 23 Trapold, J.H., Warren, M.R. and Woodbury, R.A., Pharmacological and toxicological studies on 2-(N-p'-tolyl-N-(m'-hydroxyphenyi)-aminomethyl)-imidazoline (C-7337), a new adrenergic blocking agent, J. Pharmacol. Exp. Ther., 100 (1950) 119-127.
24 Tsoucaris-Kupfer, D. and Schmitt, H., Hypothermic effect of a-sympathomimetie agents and their antagonism by adrenergic and cholinergic blocking drugs, Neuropharmacology, 11 (1972) 625-635. 25 Valatx, J.L., Roussel, B. and Cur6, M., Sommeil et temperature c6r6brale du rat au cours de l'exposition chronique en ambiance chaude, Brain Research, 55 (1973) 107-122.
227
BRES 15269
Influence of ambient temperature on sleep and body temperature after phentolamine in rats Stephen Kent I and Evelyn Satinoff 1-3 ~Department of Psychology, 2Program in Neural and Behavioral Biology and 3Department of Physiology and Biophysics, University of lUinois at Urbana-Champaign, Champaign, 1L 61820 (U.S.A.) (Accepted 8 August 1989)
Key words: Rapid eye movement sleep; Phentolamine; a-Adrenoceptor antagonist; Body temperature; Noradrenergic mechanism; Ambient temperature
Phentolamine, an a-adrenoceptor antagonist, both decreases rapid-eye-movement sleep (REMS) and causes a dose- and ambient temperature (Ta)-dependent drop in body temperature (Tb). The purpose of this paper was to examine the correlation between changes in sleep and changes in Tb after phentolamine at various Ta's. Tb and sleep were recorded in male rats for 4 h after i.p. injection of either saline or phentolamine (1 mg/kg at Ta 20 °C; 5 mg/kg at 20, 30 and 32 °C; 10 mg/kg at 20, 30, 32, and 34 °C). Changes in sleep were highly correlated with changes in Tb: when Tb dropped, amounts of sleep, especially REMS, also were decreased. The largest effects were seen at Ta 20 °C. After 5 mg/kg, Tb was below normal for 2 h and latency to REMS increased 82 + 27 min (P < 0.025). After 10 mg/kg Tb was decreased for 3 h, and latency to REMS increased 91 + 19 min (P < 0.001). While Tb was lower amounts of REMS were also decreased. Smaller effects were observed on slow wave sleep. At those conditions where phentolamine had no effect on Tb (i.e. Ta 20 °C, 1 mg/kg, and Ta 32 °C, 5 and 10 mg/kg) no differences were found in any measure of sleep. These results demonstrate that the effects of phentolamine on sleep are not caused by direct action on sleep mechanisms, but rather by its actions on thermoregulatory and possibly other mechanisms that modulate sleep. INTRODUCTION T h e n e u r o p h a r m a c o l o g y of sleep has been a topic of intense study ever since Jouvet s p r o p o s e d his m o n o a m i n ergic t h e o r y of sleep. A c c o r d i n g to this theory, noradrenergic neurons are necessary for the maintenance of r a p i d - e y e - m o v e m e n t sleep ( R E M S ) . This implies that t r e a t m e n t s that facilitate n o r a d r e n e r g i c transmission should increase R E M S and b l o c k a d e of n o r a d r e n e r g i c activity should decrease it. Since this seminal p a p e r , n u m e r o u s studies have a t t e m p t e d to test this hypothesis, but the role of n o r a d r e n e r g i c involvement in R E M S remains controversial. A m b i e n t t e m p e r a t u r e (T~) also has a strong effect on R E M S , but here there is no controversy: Ta's a b o v e or b e l o w t h e r m o n e u t r a l i t y decrease R E M S 16'18'21'25. If the t h e r m a l stress is e x t r e m e enough, slow wave sleep (SWS) is d i s r u p t e d also. In rats, a m o u n t of R E M S varies significantly even within the t h e r m o n e u t r a l zone defined by minimal and constant metabolic rate (25-31 °C), reaching a p e a k at 29 + 1 °C19. Since R E M S is so sensitive to T~, any t r e a t m e n t that alters amounts and/or distribution of R E M S m a y be doing so by m a k i n g a particular T~ m o r e or less stressful. For e x a m p l e , Szymusiak and Satinoff2° e x a m i n e d sleep in
rats after lesions in the medial p r e o p t i c area of the hypothalamus, which interfere with thermoregulation. The severity of the postlesion h y p o s o m n i a and the course of recovery of sleep were strongly d e p e n d e n t u p o n the T a at which the rats were studied. Most n o r a d r e n e r g i c agents, including all those that have been used in sleep studies, also affect t h e r m o r e g u lation (see refs. 2 and 3 for review). In general, n o r a d r e n e r g i c blocking agents that reduce R E M S lower b o d y t e m p e r a t u r e (Tb). W e have previously r e p o r t e d that after injection of the nonselective a - a d r e n o c e p t o r antagonist, p h e n t o l a m i n e , amounts of R E M S d e p e n d e d on the Ta at which sleep was m e a s u r e d . A t Ta's at which this c o m p o u n d l o w e r e d T b it inhibited R E M S ; at Ta's where it did not change T b, it had no effect on R E M S 9. The present p a p e r continues the study of the effects of this drug on Tb, waking, SWS and R E M S .
MATERIALS AND METHODS
General procedure Rats implanted for EEG and Tb recording were adapted to the sleep recording chamber for 6-10 h on 3 separate occasions at Ta 23 °C. Sleep and Tb data were recorded for 1-3 h before and 4 h after i.p. injections of phentolamine-HCl (1, 5, and 10 mg/kg in 1 ml isotonic saline) or an equivalent volume of saline alone. Two rats,
Correspondence: E. Satinoff, Psychology Department, University of Illinois, 603 E. Daniel St., Champaign, IL 61820, U.S.A. 0006-8993/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
228 one injected with phentolamine and one with saline, were recorded from simultaneously. Only rats that had had at least two 1-min REMS bouts prior to injection were used in the analyses. All recordings were begun 2-5 h after lights-on. Each rat was injected once with either 1, 5, or 10 mg/kg phentolamine and once with saline, at one or more T~'s. Drug injections were spaced at least 4 days apart. No rat received more than 3 drug injections during the study.
Sleep and Body Temperature at Ta20°C #16
Saline SW~ 37
Subjects and housing Subjects were 30 male hooded rats of the Long-Evans strain. They were maintained from birth at Ta 23 + 1 °C on a 12:12 light-dark cycle, and weighed 280-445 g at the start of the experiments. Food and water were available ad libitum. For sleep recording, each rat was placed in an open-topped, Plexiglas cage (29 x 30 x 30 cm), housed in a large light- and temperature-controlled chamber (T a 23 + 0.5 °C). Food and water were not available during recording sessions. One h before recording began, the chamber Ta was set to either 20, 30, 32, or 34 °C. This took approximately 10 min at 20 °C and 40-60 min at the warmer Ta's.
36.
0
60
120 Time (min)
180
240
Fig. 1. Sleep state progression and Tb measured every 10 min for rat 16 at Ta 20 °C for 20 min before and 240 min post saline injection (a) and post phentolamine injection (10 mg/kg) (b). S, SWS; W, Waking; R, REMS. Arrow indicates time of injection.
Surgery Each rat was anesthetized with Nembutal (sodium pentobarbital; 50 mg/kg i.p.), injected with atropine sulfate (0.5 mg/kg i.p.) and placed in a stereotaxic instrument. Four stainless-steel jeweler's screws were threaded through holes drilled in the skull to record cortical and hippocampal EEG, and either 4 single-stranded, insulated, stainless-steel wires bared at the tip or 4 thin silver plates ( = 2 x 7 mm) were implanted in the dorsal neck muscles, two on each side of the midline, to record EMG activity. A screw placed in the skull over the cerebellum served as ground. All electrode leads were soldered to a miniature 9-pin Amphenol connector, which was anchored to the skull with dental acrylic. At the same time a temperature telemetry device (1 x 2.1 cm cylinder, weight 2.3 g; Mini-mitter Co., Sunriver, OR) was implanted into the peritoneal cavity. Each rat was then returned to its home cage for at least 1 week.
Data collection Tb data were obtained through the implanted transmitter, which emits a broadband RF pulse at a rate proportional to its temperature. This signal was converted to a Tb value by an Apple II+ microcomputer. Tb values were recorded and stored on diskettes at 5 or 10 min intervals. The rat was connected, via a cable on a counterweighted slipring, to a Model 7 Grass polygraph. The bandwidths used were 1-35 Hz for the cortical EEG, 3-15 Hz for the hippocampal EEG, and 10-35 Hz for the EMG. To record movement, a magnet was hung in a wire coil attached to the cage floor. The cage was mounted on 10 light springs and any movement displaced the magnet and emitted a signal. All signals were displayed on chart paper at a speed of 5 mm/s.
changes in Tb: (1) maximal change from baseline during the first 2 h post injection, and (2) a hypothermia index (HI), which was calculated by integrating the area between the Tb curve and the extrapolated baseline. For both of these measures baseline was the mean Tb of the hour immediately pre-injection. Latency and maximal Tb change data were analyzed by within-subjects two-tailed t-tests. All other data were analyzed using 2-way ANOVA's with repeated measures on the 4 hourly blocks. Separate analyses were done for each Ta and dose. Planned post hoc comparisons were done at each hour to determine significant differences between the two treatments.
RESULTS
Overall s u m m a r y I n g e n e r a l , w h e n p h e n t o l a m i n e c a u s e d a d r o p in Tb, latency to REMS
increased
and
amounts
of REMS
d e c r e a s e d . T h e r e w a s also a m i l d e r a n d s h o r t e r - l a s t i n g d e c r e a s e in S W S . A f t e r 10 m g / k g , t h e l a r g e s t d r o p s in T b a n d t h e m o s t s e v e r e s l e e p c h a n g e s w e r e o b s e r v e d at 20 °C. T h e r e w e r e s m a l l b u t s i g n i f i c a n t d e c r e a s e s in Tb, REMS,
a n d S W S at 30 °C. A t 32 °C, t h e r e w e r e n o
differences between the groups on any measure. At Ta 34 °C, w h i c h w a s a h e a t stress, n e i t h e r g r o u p s l e p t m u c h . The 5 mg/kg dose had smaller and more variable effects
Sleep state scoring Waking, SWS and REMS were scored in 10 or 15 s epochs by experienced scorers. Waking was identified by low voltage fast activity in cortical EEG, small amounts of hippocampal theta (associated with movement), high, variable amplitude in EMG, and a high incidence of movement. SWS was characterized by an increased voltage in EEG, especially slow cortical activity, a lack of clear theta activity, an intermediate level of EMG activity, and little or no movement. REMS was identified by low voltage fast cortical activity, continuous theta, and tonically silent EMG interspersed with phasic bursts of activity.
o n Tb a n d R E M S ,
e s p e c i a l l y at 20 °C. A t 30 °C, t h e r e
w e r e slight, b u t s i g n i f i c a n t , d e c r e a s e s in T b a n d R E M S . As with the higher dose, there was no effect on any v a r i a b l e at 32 °C. 1 m g / k g p h e n t o l a m i n e h a d n o e f f e c t o n T b o r a n y s l e e p m e a s u r e a t 20 °C, t h e o n l y Ta at w h i c h this d o s e was t e s t e d . Fig. 1 s h o w s Tb c h a n g e s a n d s l e e p s t a t e p r o g r e s s i o n f o r a r a t i n j e c t e d w i t h s a l i n e (Fig. l a ) o r 10 m g / k g p h e n t o l a m i n e (Fig. l b ) at 20 °C. A t t h i s 7", n o r a t i n j e c t e d w i t h
Data analysis
phentolamine at either dose had any REMS until after Tb
The scored sleep data were stored on the hard disk of an IBM AT for data reduction. A program determined latencies to SWS and REMS, bout number and length, and duration of waking, SWS, and REMS for each hour. Two measures were used to characterize
h a d r e a c h e d its l o w e s t p o i n t a n d r e t u r n e d t o w i t h i n 0.5 °C o f p r e - i n j e c t i o n l e v e l s ( u s u a l l y 6 0 - 1 8 0 m i n ) . A t 32 °C n e i t h e r Tb, s l e e p , n o r w a k i n g w a s a f f e c t e d (Fig. 2 a , b ) .
229
Sleep and Body Temperature at Ta32°C #42 I
Saline S
38
I
1
~
W
° I'5°
Ta 20 °C
37
~
~
°
° 37 I
3.
I
0
I
I
60
I
120 Time (min)
I
I
I
180
I
240
Fig. 2. Same as Fig. 1 for rat 42 at T. 32 °C and 10 mg/kg.
After 10 mg/kg phentolamine, Tb started to drop within 20 min and reached a minimum between 30 and 80 rain post-injection. It decreased a maximum of 1.3 + 0.3 °C (+ S.E.M.) (P < 0.001) compared to the Tb of saline-injected controls. The hypothermia index (HI) was significantly higher for 3 h (Fig. 3A). The rats were awake more (P < 0.01), due mainly to increased waking during the first hour (31 + 2 rain post saline vs 46 + 3 min post drug, P < 0.005) and the second hour (21 _+ 3 min vs 30 + 4 min, P < 0.005; Fig. 3A). Bout length increased from 1.3 + 0.1 to 3.8 + 0.8 (P < 0.02) during the first hour. Controls actually had more waking bouts (26 + 2 vs 15
Changes in Sleep and Tb after Phentolamine (10 mg/kg) Ta =30°C
T a = 20°C Waking
N = 11
Waking
T a = 34°C
Ta =32°C N = 10
Waking
N= 8
Waking
N = 4
60[ -'Z-
*t Z 0 SWS
sws
SWS
SWS
40 (1)
t-"
lO REMS
REMS
REMS
REMS
t~9 C
.~-1 .o[ •~t- - o5!.
*
•-
* ~
Saline Phentolamine
I
2
3
Hours
A
4
1
2
3
4
2
3
Hours
Hours
B
C
4
2
3
4
Hours
D
Fig. 3. Hourly analysis of levels of waking, SWS, REMS and change in Tb after injection of phentolamine (10 mg/kg) or saline at 20 °C (A), 30 °C (B), 32 °C (C), and 34 °C (D) for 4 h post-injection.'P < 0.05; "*P < 0.01.
230 Latency to REMS increased by 91 + 19 min (P < 0.001) compared to saline-injected controls. REMS latency was correlated both with the maximal drop in T0 (r = -0.60, P < 0.05) and with the H I 3 (r = 0.66, P < 0.025). Amounts of REMS were decreased for the first 3 h (P < 0.01; Fig. 3A), due to a decrease in bout number (P < 0.001). Consequently, total amounts during the 4 h recording time were reduced (P < 0.001). For the first 3 h, rats injected with phentolamine had only 28% of the REMS of controls: by the fourth hour, REMS levels were equivalent. Total REMS time was also correlated with both the drop in To (r = 0.76, P < 0.01) and the HI 3 (r = -0.79, P < 0.005). After 5 mg/kg, Tb decreased 0.8 + 0.2 °C (P < 0.01) and the HI was higher for only 2 h (Fig. 4A). There were
+ 3, P < 0.02), but since they were so much shorter it did not affect total wake time. The increase during the second hour was due to a non-significant increase in bout length in the rats injected with phentolamine. Latency to SWS was increased by 16 + 5 min (P < 0.01). Consequently, SWS amounts during the first hour dropped from 25 + 2 to 13 + 3 min (P < 0.01; Fig. 3A). This was due to a decrease in bout number from 26 + 2 to 15 + 3 (P < 0.02). Amounts were still decreased during the second hour (P < 0.05; Fig. 3A). Latency to SWS post drug was correlated with the cumulative HI for the first 3 h (HI3) (r = 0.75, P < 0.01). Total SWS time during the first 2 h post phentolamine was correlated with both H I 3 (r = -0.69, P < 0.02) and the maximal drop in T b (r = 0.64, P < 0.05,).
Changes in Sleep and Tb after Phentolamine (5 mg/kg) Ta =30°C
T a = 20°C
60 50 ~
._
Waking
N= 7
Waking
Ta =32°C
N=8
Waking
N=5
30 20
10 0
SWS
SWS
3040
~
SWS
~
cyJ ._c
20 10 0
REMS
REMS
REMS
*
10
.c_
I
-- -0.5
1°I U
~_ o.o I I
1.0 1
**
*
2
3
~ 2
3
Hours
4
1
4
A
B 30 °C,
1
2
3
Hour s
Hour s
Fig. 4. Same as Fig. 3 for phentolamine (5 mg/kg). (A) 20 °C, (B)
l
C and (C) 32 °C.
4
231 no significant differences in waking or SWS. Latency to REMS increased by 82 + 27 min (P < 0.025) and amounts of REMS were reduced during the second and fourth hours post injection (P < 0.01). During the first hour only one phentolamine-injected rat had any REMS, whereas 5/8 controls did. As with 10 mg/kg, these decreases were primarily caused by a reduction in bout number. Neither To nor any sleep parameter was affected by phentolamine (1 mg/kg). T,, 30 °C
Phentolamine (10 mg/kg) increased the HI for the first 3 h (Fig. 3B): To decreased a maximum of 0.8 + 0.2 °C (P < 0.005). Waking was increased only during the first hour post injection (P < 0.005; Fig. 3B). SWS latency was increased by 8 + 2 min (P < 0.005) and amounts decreased during the first hour post injection, from 31 + 2 to 26 + 2 min (P < 0.02; Fig. 3B). REMS latency increased 24 + 9 min (P < 0.025) and REMS duration did not return to normal until the second hour (Fig. 3B). However, total REMS for the 4 h period was decreased (P < 0.05). After 5 mg/kg, the HI was increased compared to controls during the second and third hour post injection (Fig. 4B). Since T0 decreased a maximum of only 0.3 + 0.1 °C (P < 0.02), we do not believe this to be of any functional importance. Waking was increased for the 4 h recording period (P < 0.025), but only the change in the third hour post injection reached significance (Fig. 4B). The increase in waking was due to a rise in bout number during the first hour post injection (P < 0.05) and a non-significant increase in bout length during all 4 h. There were no differences in SWS latency or amounts. REMS amounts also decreased for the 4 h period (P < 0.05), but only the third hour showed a significant drop (Fig. 4B). T,,32°C
There were no differences in To or any measure of sleep between phentolamine- and saline-injected rats (Figs. 3C and 4C). T a 34 °C
There were no differences in T0 at this T~: Tb's of both saline- and phentolamine-injected rats rose by over 0.6 °C (Fig. 3D). Amounts of SWS were very low, but equivalent for both groups (Fig. 3D). REMS was severely inhibited for both groups (Fig. 3D). There were no differences in SWS or REMS latency. DISCUSSION These results demonstrate that phentolamine produces a dose- and Ta-dependent fall in To. When there was a
large drop in Tb, there was a large decrease in REMS, with lesser effects on waking and SWS. REMS was inhibited only as long as Tb was lowered. As Tb returned to baseline levels, amounts of REMS also returned to control amounts. At higher Ta's or lower doses, where phentolamine did not lower Tb, there were no changes in sleep or waking. The 'equilibrium' Ta for our rats after 5 and 10 mg/kg was 32 °C. At 32 °C all effects of phentolamine on Tb and sleep were eliminated. The effects of phentolamine on Tb and sleep at 20 °C agree with earlier reports. At 20 °C and 10 mg/kg, this compound lowered the Tb of rats a mean of 1.6 + 0.3 °C24. With different doses and T~'s, it was reported that the higher the dose or the lower the Ta, the larger the drop in Tb, and that at warmer Ta's or low doses no drop occurred ml. M/ikel/i and Hilakivi 12 found that phentolamine (10 mg/kg) reduced REMS for the first 2-3 h postinjection. This study was conducted at 22-24 °C, which is a low enough Ta to produce a drop in Tb. In cats, phentolamine (20 mg/kg) increased REMS in the first 4 h postinjection 17. However, without knowing the effect of this compound on Tb in cats, it is impossible to speculate on the reasons for this difference. Any drug can produce Tb changes in one of two ways: (1) by changing the thermal set-point, or preferred Tb, or (2) by altering one or more thermoregulatory effector mechanisms. Either mechanism of action will alter the thermoneutral zone, that range of Ta's in which thermoregulatory responses are minimal. In untreated rats this is generally 25-31 °C. However, even within this range amounts of REMS vary greatly. Decreases of 6 °C, from 29 to 23 °C (the high end of the T~ of many laboratories), decreased REMS by over 50%. Raising the T~ only 2 °, from 29 to 31 °C, reduced REMS by o v e r 60% 19. Thus, small and seemingly inconsequential changes in T~ can produce large changes in REMS. If phentolamine were lowering thermal set-point, then warmer Ta's should be more stressful and the rats should sleep less. This is not what we found. On the other hand, if phentolamine lowers T0 by altering thermoregulatory effectors (and, in fact, it does cause peripheral vasodilation in rats 11, humans 22, and dogs 7, and decreases metabolic rate in rats11), then the thermoneutral zone, or zone of thermal comfort, should be raised, and maximal amounts of sleep should occur at higher than normal Ta's. Lower T~'s and the drop in Tb that accompanies them should be more stressful and sleep should be decreased, especially REMS. This is exactly what we found. We are currently verifying this mechanism of action using a behavioral thermoregulation paradigm. One way of determining if a drug has altered thermal set-point is by measuring operant responses to thermal reinforcement. Preliminary data from this lab indicate that
232 p h e n t o l a m i n e does not alter the thermal set-point 1°. Rats injected with 10 mg/kg work harder than controls to w a r m themselves in a cold environment, thereby preventing a d r o p in T b. In the heat, T o does not fall and there is no difference in response rate between drug and control groups. These results, together with the present results on sleep, strongly suggest that phentolamine does not alter thermal set-point. As M c G i n t y and Siegel ~3 have pointed out, R E M S is a fragile state: several non-specific stressors can disrupt it. In this p a p e r we have only addressed thermal stress. A t 20 °C, after 10 mg/kg of phentolamine, the drop in T o only accounted for about half of the variance in sleep measures (56% of SWS latency and 48% of SWS amounts during the first 2 h; 44% of R E M S latency and 62% of total R E M S amounts). This does not mean that the residual effects are due to a direct action of the drug on sleep mechanisms. Phentolamine, like other noradrenergic agents, has a variety of effects which may contribute to changes in R E M S . These include, but may not be limited to, hypotension, peripheral vasodilation, tachycardia and/or palpitations, gastrointestinal irritability (i.e. diarrhea and nausea), and insulin secretion 5-7,11,14,15,22,23
O n e of these effects - - peripheral vasodilation - causes an increase in skin t e m p e r a t u r e . Skin t e m p e r a ture, like Tb, affects amounts of R E M S . Brief changes in skin t e m p e r a t u r e t o w a r d t h e r m o n e u t r a l i t y trigger R E M S , whereas changes away from it inhibit R E M S 21. With this in mind we are beginning to measure changes in skin t e m p e r a t u r e after p h e n t o l a m i n e . W e expect that adding this measure will enable us to account for a larger percentage of the variance in our data. In conclusion, we have shown that p h e n t o l a m i n e acts on sleep indirectly. Its action on T b is responsible for much of its effect on sleep. W h e n this effect is eliminated, by raising the T a at which the rats are tested, phentolamine has no effect on sleep. Consequently, this drug and p r o b a b l y others do not act directly on sleep mechanisms, but alter sleep through t h e r m o r e g u l a t i o n . A n y a t t e m p t to d e t e r m i n e the n e u r o p h a r m a c o l o g y of sleep needs to be u n d e r t a k e n with this point in mind. Acknowledgements. Support to E.S. from NSF Grant BNS 8311466 and NIMH Grant 1 RO1 MH 41138 is gratefully acknowledged. S.K. was partially supported by NIMH training Grant 1 T32 MH18412. We thank Hua Ma and Rick Kaplan for technical assistance. We also thank CIBA-Geigy for their generous gift of phentolamine.
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