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Effects of anesthetic injected into brainstem sites on body temperature and behavioral thermoregulation
Physiology & Behavior, Vol. 17, pp. 667-674. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Effects of Anesthetic Injected into Brainstem Sites on Body Temperature and Behavioral Thermoregulation' R. B. HUMPHREYS, M. H A W K I N S A N D J. M. LIPTON Departments o f Psychiatry, Physiology and Neurology, The University o f Texas Health Science Center at Dallas, Dallas, Texas 75235 U.S.A. (Received 4 N o v e m b e r 1975) HUMPHREYS, R. B., M. HAWKINS AND J. M. LIPTON. Effects of anesthetic infected into brainstem sites on body temperature and behavioral thermoregulation. PHYSIOL. BEHAV. 17(4) 6 6 7 - 6 7 4 , 1 9 7 6 . - The preoptic/anterior hypothalamic region (PO/AH), the posterior (PH) and lateral hypothalamic (LH) regions, the mesencephalic reticular formation (MRF) and the medulla oblongata (MED) have all been implicated in the control of body temperature. The purpose of the present research was to learn whether differential effects upon physiological and behavioral thermoregulation are produced by temporarily depressing the activity of these regions with anesthetic. The rectal temperature (Tre) of rats with central cannulae was recorded after sodium pentobarbital injections while the animals rested in 23 °, 10 ° and 34°C environments. In other experiments the effects of central anesthetic injections on behavioral regulation against heat were measured. Anesthetic injected into the PO/AH region caused changes in Tre and behavior that are consistent with a coordinated rise in the set point of body temperature control. Injections into the MED produced transient and rapid decreases in Tre without affecting behavioral thermoregulation. Bilateral injections into the LH caused hyperthcrmia in the 10 ° and 34°C environments, hypothermia in the 23°C environment and had no effect on behavioral temperature regulation. No changes in thermoregulatory responses were observed after PH and MRF injections. These results indicate that there are differences among the 5 brain regions in relative importance to overall temperature control and specific differences in the significance of certain regions to the two forms of temperature control: physiological and behavioral temperature regulation. Central temperature controls
Body temperature
Behavioral thermoregulation
P H Y S I O L O G I C A L and behavioral t e m p e r a t u r e c o n t r o l may d e p e n d u p o n the activity o f one or m o r e of several regions of the b r a i n s t e m . The p r e o p t i c / a n t e r i o r h y p o t h a l a m i c region ( P O / A H ) is believed to c o n t a i n a r e g u l a t o r y mechanism w h i c h acts as the p r i m a r y t e m p e r a t u r e c o n t r o l , integrating central and p e r i p h e r a l t h e r m a l i n f o r m a t i o n and driving t h e r m o r e g u l a t o r y responses. However, it is clear that this brain region is not t h e sole site of central t e m p e r a t u r e c o n t r o l since n e i t h e r physiological n o r behavioral t h e r m o r e g u l a t i o n are totally o b l i t e r a t e d w h e n the region is d e s t r o y e d [ 1 6 . 1 8 ] . T h e p o s t e r i o r h y p o t h a l a m u s has been associated with t e m p e r a t u r e c o n t r o l since the early e x p e r i m e n t s of R a n s o n [28] in w h i c h bilateral lesions of the p o s t e r o l a t e r a l h y p o t h a l a m u s caused a loss of capacity to regulate b o d y t e m p e r a t u r e in the cold. Bilateral lesions in the lateral h y p o t h a l a m u s have been r e p o r t e d to disturb behavioral t h e r m o r e g u l a t i o n w i t h o u t i n t e r f e r i n g with physiological t e m p e r a t u r e c o n t r o l [ 2 9 ] . T e m p e r a t u r e sensitive n e u r o n s in the m i d b r a i n are believed to act to prevent deviations in b o d y t e m p e r a t u r e b y sensing local t e m p e r a t u r e and driving physiological t h e r m o r e g u l a t o r y
responses [ 8 ] . Finally, t h e r e is evidence t h a t the reticular s u b s t a n c e of the m e d u l l a o b l o n g a t a c o n t a i n s a s e c o n d a r y thermosensitive temperature control mechanism which drives physiological and behavioral t h e r m o r e g u l a t i o n ind e p e n d e n t of the P O / A H region [ 1 5 , 1 6 } . E x c e p t for general c o m p a r i s o n s of the t h e r m o s e n s i t i v i t y of the P O / A H region and the m e d u l l a o b l o n g a t a [ 1 6 ] , there is little data on the relative i m p o r t a n c e of specific brain regions to b o d y t e m p e r a t u r e c o n t r o l . One objective in the present exp e r i m e n t s was to c o m p a r e the effects on b o d y t e m p e r a t u r e and t h e r m o r e g u l a t o r y b e h a v i o r of acute depression of the activity of the particular b r a i n s t e m regions w h i c h have b e e n implicated in t e m p e r a t u r e c o n t r o l . A second goal was to learn w h e t h e r selective depression of these sites affects physiological or behavioral t e m p e r a t u r e c o n t r o l alone, or w h e t h e r b o t h f o r m s of t e m p e r a t u r e c o n t r o l are altered. MATERIALS AND METHOI)S The general a p p r o a c h was to inject a n e s t h e t i c i n t o a particular brain region and to record e i t h e r the effects u p o n
This research was supported by the Leland Fikes Foundation and the National Institute of Neurological and Communications Disorders and Stroke Grant No. 2-R01-NS 10046 02. The authors thank Dr. W. G. Clark for his comments on the manuscripL 667
668 rectal t e m p e r a t u r e (Tre) while the animals rested in n e u t r a l hot or cold e n v i r o n m e n t s or to n o t e the effects of central anesthetic u p o n a standard test of t h e r e m o r e g u l a t o r y behavior. A total of 45 male albino rats ( H o l t z m a n strain) were operated. The rats were maintained in individual cages in a r o o m controlled at 24 ± 2 ° C. All animals were weighed daily and b o d y weight was controlled at 3 2 0 - 4 0 0 g by regulating access to Purina Laboratory Chow. Because of cannula failure, inanition and surgical m o r t a l i t y a total of 22 animals which were in good c o n d i t i o n were used in the experiments from which the data described below were obtained.
Surgery Each rat was anesthetized with an intraperitoneal injection of sodium p e n t o b a r b i t a l (50 mg/kg) and placed in a Kopf stereotaxic instrument. A stainless steel guide cannula assembly fitted with an o b t u r a t o r and a Teflon cap [19] was implanted, using standard surgical procedures. Most animals had a single cannula implanted in one of the l o l l ' o w i n g sites: the p r e o p t i c / a n t e r i o r hypothalamic (PO/AH) region; the posterior h y p o t h a l a m i c (PH) region: the medulla oblongata (MED); or the mesencephalic reticular f o r m a t i o n (MRF). Bilateral cannulae were placed in the lateral h y p o t h a l a m i c (LH) region in other rats. At least 7 days were allowed for recovery before the animals were tested.
Injection Procedures To make an injection the o b t u r a t o r was removed, and an injection cannula was inserted so that it e x t e n d e d 0.5 mm below the tip of the guide tube. One ul of either nonpyrogenic saline or sodium pentobarbital was injected over 30 sec. The cannula was left in place for another 30 sec before it was removed and the o b t u r a t o r replaced. Injections were separated by periods of at least 48 hr with saline and anesthetic given alternately in each rat at each of the 3 ambient temperatures. In pilot work sodium pentobarbital dissolved in saline and injected into the P O / A H region was found to produce only brief changes in body t e m p e r a t u r e unless p r o p y l e n e glycol was added to the solution to reduce the rate of dispersion. F o r this reason pentobarbital sodium in salinepropylene glycol-alcohol solution ( N e m b u t a l ) was used in the main experiments. Changes in b o d y t e m p e r a t u r e produced by injecting the vehicle (saline, 50%; propylene glycol, 40%; ethanol, 10%) into the P O / A H region in animals resting in neutral, hot and cold environments were not different from those seen after saline alone was injected. Because of this finding and the need to avoid c o n t a m i n a t i o n with pyrogens whenever possible, only commercial n o n p y r o g e n i c saline ( A b b o t t L a b o r a t o r i e s ) w a s used for control injections in the main experiments.
Body Temperature Tests of the effects of central anesthetic on body temperature were p e r f o r m e d in an environmental chamber with the ambient t e m p e r a t u r e held at 10 °, 23 ° , or 34°C. Tre in these and in the behavioral experiments was recorded using a thermistor probe inserted 6 cm past the anus. After a 1 hr baseline period in which Tre was measured every 15 min an intracerebral injection was made, and the temperatures were recorded for an additional 5 hr.
H U M P H R E Y S , HAWKINS AND LIPTON
Behavioral Therrnoregulation The behavioral thermoregutation apparatus has been described previously [13, 14, 26]. Each behavior test unil consisted of a Plexiglas cylinder m o u n t e d in a manifold box fitted with an exhaust fan (60 cfm)+ A heat lamp (250 Wt was centered over the behavior chamber. Pressing a lever turned the heat lamp off and activated the exhaust fan which drew ambient air through the chamber. F o u r chambers of this type were used in a room where the ambient temperature was held at 23 t+ U C ) . The intensity of the lamp was gradually reduced to 125 W by controlling voltage so that the time spent depressing the lever wa.~ reduced from initial levels of 85% + 98% to 40%- 6 5 q . After reliable behavioral responding was established with the lamp set at 125 W the rats spent at least 2 additional 6 hr periods in the chamber. In the behavior tests percent time spent depressing the lever was measured for a total of 6 hr with intracerebral injections of physiological saline or Nembutal given I hr after the teq was begun. Tests on individual animals were separated hy a period of at least 48 hr. After the experiments were c o m p l e t e d the rats were killed with an intraperitoneal injection of sodium pento-barbital and the brains were perfused with saline and t 0 ~ Formalin buffered with potassium. Serial sectim~s 35 thick were cut from frozen brain tissue and stained with Luxol blue. The location of the tips of the cannulae were reconstructed on copies of line drawings taken from brain atlases [12,251 with the injection sites traced in by hand in accordance with enlarged images of the brain sections.
RESUL I5;
PO/A H R egio n In the 2 3 e n v i r o n m e n t sodium pentobarbital caused hyperthermia when placed in the PO/AH region l Fig. 11. The average m a x i m u m increase in body temperature was 1.&C which occurred 2 hr after injection. A smaller h y p e r t h e r m i c response was observed in the cold environment where the average m a x i m u m increase in Tre was 0.9°C at 3 hr after injection of anesthetic. In the saline control experiments run in the 10 ° e n v i r o n m e n t Tre showed the slight increase that was characteristic in rats exposed to cold (see Figs. 3 and 41). In the 34 ' e n v i r o n m e n t preinjection "Ires were higher ( 0 . 5 - 1 . 0 ° C ) in all rats as a result of the 1 hr exposure to heat. After saline injection, these rats showed a slight initial rise and then a decrease in Tre over the remainder of the period (Fig. I ). Injections of sodium pentobarbital caused an average m a x i m u m increase of 0+2-C 2 hr after injection. The main difference after central anesthetic was that the Tres were maintained t h r o u g h o u t the 5 hr recording period in contrast to the decrease seen after control injections. In the behavioral thermoregulation tests, injection of sodium pentobarbital into the P O / A H region caused a decrease in the a m o u n t of time spent pressing the lever to escape heat (Fig. 2). This change in responding began within one hr after the injection and was maintained for 2---3 hr, followed by a return to near preinjection levels. Saline injections caused a slight rise in the a m o u n t of time spent escaping heat which persisted throughout the test period.
BRAINSTEM AND THERMOREGULATION
669
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I 2 3 4 5 HOURS AFTER INJECTION
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FIG. 1. Effects of injecting anesthetic into the PO/AH region on Tre in rats resting in neutral, hot and cold environments. Data points are mean (+ SE) changes in 4 rats. In this and following graphs control data were obtained using the same animals that received pentobarbital injections. Injection sites are indicated by circles on coronal brain section.
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There was no significant chang0 in behavioral t h e r m o regulation after a n e s t h e t i c was injected into the MED (Table 1 ).
Lateral tIypothalarn us
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2
3
4
HOURS AFTER INJECTION
FIG. 2. Effects of anesthetic injections in the PO/AH region on behavioral regulation against heat. All animals showed a decrease in the amount of time spent responding for 2 - 3 hr after injections.
Medulla Oblongata In the neutral e n v i r o n m e n t all rats w i t h m e d u l l a r y c a n n u l a e s h o w e d e x t r e m e l y rapid a n d brief h y p o t h e r m i a after s o d i u m p e n t o b a r b i t a l injections (Fig. 3). T h e h y p o t h e r m i a reached m a x i m u m 30 rain a f t e r i n j e c t i o n and Tre r e t u r n e d to c o n t r o l levels w i t h i n an a d d i t i o n a l 60 rain. H y p o t h e r m i a was also observed in the 10 ° e n v i r o n m e n t , a l t h o u g h the m a x i m u m change in b o d y t e m p e r a t u r e was not as great. In the 3 4 ' e n v i r o n m e n t t h e r e was no clear evidence of a h y p o t h e r m i c effect after central a n e s t h e t i c when the results are c o m p a r e d w i t h c o n t r o l data. Tre showed the c h a r a c t e r i s t i c decline t h a t followed saline injections (See Figs. 1 and 4) and the decrease in Tre after central a n e s t h e t i c was a l m o s t the same.
Injections of a n e s t h e t i c i n t o the LH caused h y p o t h e r m i a in rats tested in the 23°C e n v i r o n m e n t (Fig. 4). Tre r e t u r n e d to n o r m a l by the end of the 5 hr p o s t i n j e c t i o n period. Depression of the activity of the LH in the same animals in t h e cold e n v i r o n m e n t did not p r o d u c e h y p o t h e r m i a but r a t h e r caused h y p e r t h e r m i a s with peak Tre s of 39.4 40.3°C. Shivering began in some rats w i t h i n 0.5 hr after i n j e c t i o n and recurred i n t e r m i t t e n t l y t h r o u g h o u t the r e m a i n d e r of the test period. H y p e r t h e r m i a was also recorded after s o d i u m p e n t o b a r b i t a l was placed in the LH of animals in the 34" e n v i r o n m e n t while the response to saline was again a progressive decrease in Tre. A l t h o u g h the changes in b o d y t e m p e r a t u r e in the three a m b i e n t t e m p e r a t u r e s were m a r k e d and c o n s i s t e n t , the only change in t h e r m o r e g u l a t o r y b e h a v i o r p r o d u c e d by depressing the activity in the LH region was a slight elevation in r e s p o n d i n g by one rat 3 hr after the injections were given (Table 1 ). It s h o u l d be n o t e d t h a t the results described above were derived from e x p e r i m e n t s in which 50 ug of sodium p e n t o b a r b i t a l was given bilaterally. In p r e l i m i n a r y exp e r i m e n t s on three rats unilateral injections of 50 ug sodium p e n t o b a r b i t a l had no c o n s i s t e n t effects on b o d y t e m p e r a t u r e while bilateral injections of 2 0 - 3 0 ug produced decreases in Tre ranging b e t w e e n 0 . 7 - 1 . 8 ° ( ` in animals resting in the 23"C e n v i r o n m e n t .
670
HUMPHREYS,
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FIG. 3. Effects of anesthetic in the medulla oblongata on Tre in four rats exposed to neutral, hot and cold environments.
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FIG. 4. Changes in b o d y temperature after bilateral anesthetic injections in the LH region of 4 rals exposed to neutral, h o t and cold environments.
BRAINSTEM AND THERMOREGULATION
671 TABLE 1
MEAN ( -+ SE) CHANGES (%) IN TIME SPENT RESPONDING TO ESCAPE HEAT AFTER INTRACEREBRAL INJECTIONS
Injection
1
2
Hours After Injection 3
4
5
Anesthetic Saline
-6.6 (+_ 1.4) -5.5 ( -+ 1.3)
Medulla (N =4) -9.0 ( + 3.0) -8.3 ( ± 3.1 - 6 . 9 ( ± 2.2) - 5 . 6 ( ± 2.8)
9.1 I -+ 2.9) - 8 . 7 ( ± 2.2)
-6.0 - 8.1
+ 2.9) +_ 3.4)
Anesthetic Saline
-3.3 ( -+ 0.9) 4.1 ( _4_-0.8)
Lateral Hypothalamus (N =4) 7.4 ( ± 0.9) 1.0 ( ± 2.1) 6.9 ( ± 1.4t 0.3 ( ± 2.1)
8.0 ( - 3.0) 4.8 ( ± 3.8)
7.0 2.9
_+ 3.0) _+ 4.1)
Anesthetic Saline
3.1 -1.1
-+ 2.2) +_ 1.4)
2.0 ( _+ 1.5) 3.2 ( + 1.3)
6.0 5.4
_+ 2.21 _+ 0.91
Anesthetic Saline
-3.3 -2.4
_+ 0.9) -+ 0.7)
4.2 ( +_ 2.1) 3.4 ( _+ 1.9)
1.0 5.1
= 3.3) + 2.3)
Posterior Hypothalamus (N=5) 2.0 ( _+ 1.1) 2.1 ( + 1.7) -2.0 ( -+ 1.3) 0.0 ) -4--0.6) Midbrain (N =5) -1.0 ( _+ 0.5) 0.1 ( + 3.1) -2.1 ( ± 0.7) -2.0 ( -4--0.9)
DISCUSSION
i
FIG. 5. Sites in the posterior hypothalamus (top) and mesencephalic reticular formation where anesthetic injections had no effect on body temperature or thermoregulatory behavior.
Posterior Hypothalamie tfcular Formation
Region and Mesencephalic Re-
I n j e c t i o n s of a n e s t h e t i c i n t o these t w o regions (Fig. 5) caused very little change in Tre in n e u t r a l , hot and cold e n v i r o n m e n t s (Table 2) and had no clear effects u p o n behavioral t b e r m o r e g u l a t i o n (Table 1).
Our results indicate t h a t n o r m a l activity of at least 3 of the 5 brain regions previously implicated in r e g u l a t i o n of b o d y t e m p e r a t u r e is necessary to t h e r m o r e g u l a t o r y processes. Depression of the activity of the P O / A H region, the medulla o b l o n g a t a and the lateral h y p o t h a l a m u s with a n e s t h e t i c caused changes in b o d y t e m p e r a t u r e and, in the case of the P O / A H region, changes in t h e r m o r e g u l a t o r y behavior while a n e s t h e t i c in the m i d b r a i n and in the medial posterior h y p o t h a l a m u s had no a p p r e c i a b l e effect. Injections of sodium p e n t o b a r b i t a l i n t o the p r i m a r y t e m p e r a t u r e c o n t r o l in t h e P O / A H region caused an increase in Tre in neutral and cold e n v i r o n m e n t s and a failure to show a n o r m a l decrease in the h o t e n v i r o n m e n t . Depression of activity of the P O / A H region also altered t h e r m o r e g u l a t o r y m o t i v a t i o n , causing a decrease in the a m o u n t of time spent escaping heat. The c o o r d i n a t e d changes in physiological and behavioral t h e r m o r e g u l a t i o n are c o n s i s t e n t w i t h an alt e r a t i o n in the set p o i n t a r o u n d w h i c h b o d y t e m p e r a t u r e is c o n t r o l l e d . Similar c o o r d i n a t e d changes in physiological and behavioral t e m p e r a t u r e regulation have been observed w h e n this part of the f o r e b r a i n is heated or cooled [6, 7, 11], when i n t r a c e r e b r o v e r t r i c u l a r t e t r o d o t o x i n is administered [5] and w h e n p y r o g e n s are given peripherally [ 4 , 3 1 ] . A l t h o u g h the results of the present e x p e r i m e n t s s u p p o r t the i m p o r t a n c e of the P O / A H region to t e m p e r a t u r e c o n t r o l , they differ f r o m those o b t a i n e d in e x p e r i m e n t s on animals with electrolytic d e s t r u c t i o n of the P O / A H region. The rats in the present e x p e r i m e n t did n o t show the t h e r m o l a b i l i t y in t h e heat and cold w h i c h is characteristic after P O / A H injury [ 1 8 ] . Nor did these rats increase r e s p o n d i n g to escape heat as seen after P O / A H lesions in o t h e r e x p e r i m e n t s [ 1 3 ] . The reason for these differences m a y be t h a t the sodium p e n t o b a r b i t a l injections did n o t affect the same or as m a n y n e u r o n s as the lesions. However, there is no r e a s o n to believe t h a t the a n e s t h e t i c is selective in its i n f l u e n c e on n e u r o n s . S o d i u m p e n t o b a r b i t a l is a general CNS d e p r e s s a n t [10] w h i c h alters s o d i u m and p o t a s s i u m c o n d u c t a n c e across n e u r o n a l m e m b r a n e s [ 2 ]. On the o t h e r h a n d , a l t h o u g h materials injected i n t o the brain tend to spread c o n s i d e r a b l y [ 2 1 ] , there is n o way to rule
672
HUMPHREYS, HAWKINS AND LIPTON 'FABLE 2 MEAN (---SE)CHANGES IN Tre AFTER ANESTHETICINJECTIONS IN POSTERIOR HYPOTHALAMUS AND MIDBRAIN
Injection
l
2
Hours After Injection 3
4
Posterior Hypothalamus IN=5)
Anesthetic Saline
-0.3 (+ 0.4) -0.1 (_* 0.2)
Anesthetic Saline
0.1 (± 0.2) -0.1 (m 0.2)
Anesthetic Saline
--0.2 (± 0.1) -0.1 (_+ 0.2)
Anesthetic Saline
-0.2 (_+ 0.3) -0.2 (± 0.2)
Anesthetic Saline
-0.1 (± 0.1) 0.0 (+_ 0.2)
Anesthetic Saline
-0.1 (±_ 0.2) -0.2 (± 0.2)
23°C -0.3 (+- 0.2) -0.3 (_+_0.2) -0.1 (_+ 0.2) -0.2 (+ 0.2) 10°C 0.3 (± 0.2) 0.6 (+_ 0.4) 0.0 (± 0.2) 0.1 Ix 0.3) 34°C -0.5 (m 0.2) -0,4 (_~ 0.2) -0.2 (+ 0.2) -0.3 (_+ 0.2) Midbrain (N ~5) 23°C -0.1 (± 0.1) -0.4 (± 0.2) -0.2 (+ 0.1) 0.4 (± 0.2) 10°C -0.2 (_+ 0.1) -0.5 (± 0.3) -0.6 (+_ 0.4) -0.2 (± 0.2) 34°C -0.3 (+ 0.2) -0.3 1± 0.2) --0.1 (± 0.2) -0.3 (± 0.2)
out the possibility that fewer neurons were involved in the Nembutal treatments than after PO/AH lesions. An alternative explanation of the difference between the effects of central anesthetic and lesions may lie in differences in persistence over time since the effects of central anesthetic resemble those observed immediately after the PO/AH region is destroyed, when resting body temperature increases. Similar increases in core temperature were observed when Nembutal was injected into pyrogen-sensitive sites in the PO/AH region of the squirrel monkey [ 17 ]. It is believed that depression of heat-sensitive cells, which are more densely distributed in the PO/AH region, causes reversible hyperthermia that resembles the elevated temperatures seen in infectious fever. Thermolability and compensatory behavioral responding might develop only after the activity of the region has been depressed by anesthetic for some time or it might develop over time after the region has been destroyed. Then the main difference between the effects of anesthetic treatments and lesions would be due to the difference in their persistence over time. The medulla oblongata has been shown to be thermosensitive and to drive physiological and behavioral thermoregulatory responses in much the same way they are driven by altering the temperature of the PO/AH region [ 16]. The finding that the relation between local medullary temperature and thermoregulatory responses was not as close as that found with thermal stimulation of the PO/AH region, and the observation that the effects of thermal stimulation either persisted or were enhanced after PO/AH destruction, led to the idea that the medullary reticular substance contains a separate, secondary temperature control [ 16]. The brief hypothermia after anesthetic injections in the
-0.5 (± 0.3) -0.2 (_+ 0.1)
-0.3 (._ 0.1) -0.3 (= 0.2)
0.4 (+_ 0.3) 0.0 (± 0.2)
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-0.3 (_+ 0.1) -0.3 (_+ 0.1)
-I).4 (-. 0.4) - 0 4 (~ 0.3)
-0.4 (± 0.1) -0.4 (± 0.1)
--0.5 ~± 0.1) -0.5 (~ 0.1)
-0.5 (± 0.3) -0.5 (± 0.3)
-0.4 1± 0.2) --0.6 (:~ 0.2)
-0.2 (± 0.2) -0.3 (± 0.2)
---0.3 (± 0.21 -0.2 t:~ 0.1)
present experiments may represent the major response of the medullary control mechanism which is t h e n rapidly compensated by the intact primary control in the PO/AH region. The hypothermic effect was not observed in the 34 ° C environment perhaps because of the brevity of action and the inertia of high body temperature in the heat. No changes in thermoregulatory motivation were observed after the medullary injections. It should be noted that any effect upon behavior induced by depression of medullary activity might also be rapidly compensated by temperature control mechanisms in the PO/AH region. In previous experiments [18] rats with longstanding electrolytic lesions in the lower brainstem showed low resting body temperatures, deficits in regulation against cold and development of hyperthermia after 5 - 6 hr exposure to 35 ° environment. The effects Observed after anesthetic injections are general!y consistent with the effects after medullary lesions, although the brevity of the changes after anesthetic injections suggest that considerably more medullary tissue must be depressed for clear deficits in temperature control to be revealed. q'he falls in body temperature observed after intramedullary anesthetic contrast with results of previous research in which no changes in Tre were seen after 45 #g sodium pentobarbital in saline was injected into the medulla oblongata [20]. In the earlier research there was evidence of hypothermia in some rats but the decreases in Tre were no greater than those produced by saline injections. Reasons for the discrepancy in results are not clear but, since the anesthetic solution was different and the rats were restrained in the previous study, perhaps these factors are responsible for the failure to note changes in Tre. Restraint appears to be an especially important
BRAINSTEM AND T H E R M O R E G U L A T I O N influence on central temperature controls since morphine sulfate (50 ug) injected into the PO/AH region causes hyperthermia in unrestrained rats and falls in body temperature when the animals are restrained [30]. The effects of bilateral injections of sodium pentobarbital into lateral hypothalamus suggest that this region is also required for normal thermoregulatory processes. When there was little opportunity for behavioral thermoregulation the animals showed hypothermia in the neutral environment and hyperthermia in the extreme ambient temperatures after central anesthetic. On the other hand, behavioral thermoregulation was not significantly affected. These results contrast with the conclusion of Satinoff and Shan [29] that the lateral hypothalamus mediates behavioral thermoregulation primarily and is not required for physiological temperature control. The disruption of normal body temperature seen in the present experiment, together with deficits in the cold after parasaggital cuts which sever communication between the LH and more medial structures [18], suggests that the integrity of the lateral hypothalamus is indeed necessary to normal physiological temperature control. This idea is supported by the data of Satinoff and Shah [29] who found, in addition to the main result of decreased thermoregulatory responding after lateral hypothalamic lesions, evidence of severe impairment of physiological thermoregulation which outlasted short-term disturbances of behavior. The occurrence of hypothermia in the neutral environment and hyperthermia in the extreme ambient temperatures after injections of anesthetic into the lateral hypothalamus suggests that the nature of the role of this brain region in physiological temperature regulation is complex. The fact that the lateral hypothalamus is a nodal point of ascending and descending brainstem pathways and that it contains major connections between medial hypothalamic regions important ~o temperature control and other central structures [24,27] may account for the complexity of the results. Although we found no significant change in thermoregulatory behavior in our tests we cannot rule out the possibility that compensatory behavioral responding might occur if the lateral hypothalamus were depressed for a prolonged period. The posterior hypothalamus has been proposed to be the major site of convergence of temperature signals and the origin of the central effector signal [9]. If this conception, which is based primarily on results of experiments in which not only the posterior hypothalamus but all ventrolateral pathways in the brainstem were destroyed, were true then it would be expected that injection of a nonspecific anesthetic into the region would result in major thermoregulatory deficits. According to another theory the mechanism for controlling body temperature around a given level depends upon a constant, uniform balance between sodium and calcium ions within the posterior hypothalamus which provides Ihe reference input to the PO/AH temperature control [22,231. Again, if it were true that the posterior hypothalamus plays a crucial role in the control of body temperature one would expect injections into this region of an anesthetic which alters sodium conductance to cause disturbance of major thermoregulatory processes. Using injections of sodium pentobarbital into the posterior hypothalamic region, at a dose level which was effective in altering body temperature when placed in the PO/AH region, we were unable ~o demonstrate significant effects
673 on thermoregulatory processes and thus to support the idea that the normal activity of the posterior hypothalamus is essential in temperature control. If our observations are correct, then the nature of the influence of the posterior hypothalamus may be different from that of a crucial integrating control. Rather, it would appear more likely that the region can drive or influence thermoregulatory processes when it is manipulated (e.g., by altering ionic balance) but that, when the activity of the region is depressed, sufficient temperature control persists such that no clear deficits can be demonstrated. Cabanac and Hardy [3] found that shivering occurred when the mesencephalon of the rabbit was cooled. In another report Hardy [8] noted that rabbits 'in a cold environment show changes in heat production, and some alteration in vasomotor state, in response to midbrain heating and cooling. However, altering the temperature of the mesencephalic reticular substance does not influence behavioral thermoregulation of the squirrel monkey [11 or the rat [15]. In the present experiments temporary depression of neural activity at sites in this brain region did not disrupt temperature control. It appears that either normal activity in mesencephalic sites is not essential to normal temperature control in the rat or that effects upon thermoregulation caused by local injections of anesthetic in this region can be compensated by other central control mechanisms. A major accounting of the central controls of body temperature must include many circuits and, in the case of behvioral thermoregulation, must encompass not only autonomic components but sensory and motor components with cortical representation as well. The technique of acute depression of activity at a specific brain site reveals not the exact role of the anesthetized tissue but rather whether a function persists through mediation by unaffected tissue or whether the function is dependent upon the integrity of the depressed region. Using this method we have found that unaffected brain mechanisms are capable of regulating body temperature when the posterior hypothalamus and mesencephalic reticular formation are temporarily depressed. However, in the case of the PO/Att region, depression of activity caused changes in physiological and behavioral thermoregulation which suggest that the remaining control system has lost part of the input from heat-sensitive cells. These findings confirm again the importance of the PO/AH to normal temperature control. We found thai undepressed mechanisms in the brain are capable of rapidly compensating for depression of activity in the medulla oblongata so that hypothermia is rapidly reversed. If there is an effect of medullary depression on behavioral temperature control, as one would expect from previous studies on the driving of thermoregulatory behavior by thermal stinmlation of the medulla [16], it is compensated so rapidly that it is not observable with the behavioral techniques used here. It seems that the unaffected mechanisms are not capable of compensating the effects of body temperature of lateral hypothalamic depression but that behavioral regulation can continue without disruption. Although the results indicate that the lateral hypothalamic region is essential to normal temperature control, the nature of its role is not obvious from the uncompensated alterations in thermoregulation which are observed when the region is depressed.
674
H U M P H R E Y S , H A W K I N S A N D LIPTON
REFERENCES 1. Adair, E. R. and J. T. Stitt. Behavioral temperature regulation in the squirrel monkey: effects of midbrain temperature displacements. J. Physiol. Paris. 6 3 : 1 9 1 - 194, 1971. 2. Blaustein, M. P. Barbiturates block sodium and potassium conductance increases in voltage-clamped lobster axons. Z gen. Physiol. 51: 2 9 3 - 3 0 7 , 1968. 3. Cabanac, M. and J. D. Hardy. R6ponses unitaire et thermor6gulatrices lors de rechauffements et refroidissements localis6s de la region pr6optique et de m4senc6phale chez le lapin. J. Physiol. Paris61: 3 3 1 - 3 4 7 , 1969. 4. Cabanac, M., R. Duclaux and A. Giltet. Thermoregulation comportementale chez le chien: effets de la fievre et de la thyroxine. Physiol. Behav. 5: 6 9 7 - 7 0 4 , 1970. 5. Clark, W. G. and J. M. Lipton. Complementary lowering of behavioral and physiological thermoregulatory set-points by tetrodotoxin and saxitoxin in the cat. J. PhysioL 238: 181-191, 1974. 6. Gale, C. C., M. Matthews and J. Young. Behavioral thermoregulatory responses to hypothalamic cooling and warming in baboons. Physiol. Behav. 5: 1 - 6 , 1970. 7. Hammel, H. T. Regulation of internal body temperature. Ann. Rev. Physiol. 30: 6 4 1 - 7 1 0 , 1968. 8. Hardy, J. D. Thermoregulatory responses to temperature changes in the midbrain of the rabbit. Fedn Proc. 28: 713, 1969. 9. Hardy, J. D. Posterior hypothalamus and the regulation of body temperature. Fedn Proc. 32: 1564-1571, 1973. 10. Harvey, S. C. Hypnotics and sedatives: the barbiturates. In: The Pharmacological Basis of Therapeutics, edited by L. S. Goodman and A. Gilman. New York: Macmillan Publishing Co., 1975, pp. 1 0 2 - t 2 3 . 11. Hensel, H. Neural processes in thermoregulation. Physiol. Rev. 53: 9 4 8 - 1 0 1 7 , 1973. 12. Konig, J. F. R. and R. A. Klippel. The Rat Brain. Baltimore: Williams and Wilkins, 1963. 13. Lipton, J. M. Effects of preoptic lesions on heat escape responding and colonic temperature in the rat. Physiol. Behav. 3: 165-169, 1968. 14. Lipton, J. M. Effects of high-fat diets on caloric intake, body weight and heat-escape responses in normal and hyperphagic rats. J. comp. physiol. Psyehol. 6 8 : 5 0 7 - 5 1 5 , 1969. 15. Lipton, J. M. Behavioral temperature regulation in the rat. Effects of thermal stimulation of the medulla. Z Ph.vsiol. Paris 63: 3 2 5 - 3 2 8 , 1971. 16. Lipton, J. M. Thermosensitivity of medulla oblongata in control of body temperature. Am. J. Physiol. 224: 8 9 0 - 8 9 7 . 1973.
17. Lipton, J. M. and D. E. Fossler. Fever produced in the squirrel monkey by intravenous and intraeerebral endotroxin. Am. Z PhysioL 226: 1022-1027, 1974. 18. Lipton, J. M., P. E. Dwyer and D. E. t"ossler. Effects of brainstem lesions on temperature regulation in hot and cold environments. Am. Z Physiol. 226: 1356-1365, 1974. 19. Lipton, J. M., J. P. Welch and W. G. Clark. Changes in body temperature produced by injecting prostaglandin E 1 , EGTA and bacterial endotoxins into the PO/AH region and the medulla oblongata of the rat. L;xperientia 29: 806-808, 1973. 20. Lomax, P. The hypothermic efteel of pentobarbital in the rat: sites and mechanisms of action. Brain Res. 1: 296--302, 1966. 21. Myers, R. D. Injection of solutions into cerebral tissue: relation between volume and diffusion. Physiol. Behar. I: 171-174, 1966. 22. Myers, R. D. and W. L. Veale. Body temperature: possible ionic mechanism in the hypothalamus controlling the set point. Science 170: 9 5 - 9 7 , 1970. 23. Myers, R. D. and T. L. Yaksh. Thermoregulation around a new "set point" established in the monkey by altering the ratio of sodium to calcium ions within the hypothalamus. Z PhysioL 218: 6 0 9 - 6 3 3 , 1971. 24. Nauta, W. J. H. and W. Haymaker. ttypothalamic nuclei and fiber connections. In: The Hypothalamus. edited by W. Haymaker, E. Anderson and W. J. tl. Nauta. Springfield, Ill.: C C. Thomas, 1969, pp. 136-209. 25. Pellegrino, L. J. and A. J. Cushman. A Stereotaxic Atlas of the Rat Brain. New York: Appleton-Century-Crofts, 1967. 26. Polk, D. L. and J. M. Lipton. Effects of sodium salicylate, aminopyrine and chlorpromazine on behavioral temperature regulation. Pharmac. Biochem. Behar. 3: 167-172, 1975. 27. Raisman, G. Some aspects of the neural connections of the hypothalamus. In: The Hypothalamus, edited by L. Martini, M. Malta and F. Fraschini. New York: Academic Press, t 970, pp. 1-15. 28. Ranson, S. W. The hypothalamus a~ a thernmstat regulating body temperature. Psychosom. Med. 1: 4 8 6 - 4 9 6 , 1939. 29. Satinoff. E. and S. Y. Y. Sham Loss of behavioral thermoregulation after lateral hypothalamic lesions in rats. J. comp. physiol. Psvchol. 7 7 : 3 0 2 - 3 1 2 , 1971. 30. Trzcinka, G. P., J. M. Lipton, M. Hawkins and W. G. Clark. Changes in body temperature induced by microinjection of morphine, chlorpromazine and tetrodotoxin into the preoptic/anterior hypothalamic region and the medulla oblongata of the rat. Fedn Proc. 35: 481, 1976. 31. Weiss, B., V. G. Laties and A. B. Weiss. Behavioral thermoregulation by cats with pyrogen-induced fever. ,4rchs Int. Pharmacodyn. ThOr. 165:467 -475, 1967.
Effects of Anesthetic Injected into Brainstem Sites on Body Temperature and Behavioral Thermoregulation' R. B. HUMPHREYS, M. H A W K I N S A N D J. M. LIPTON Departments o f Psychiatry, Physiology and Neurology, The University o f Texas Health Science Center at Dallas, Dallas, Texas 75235 U.S.A. (Received 4 N o v e m b e r 1975) HUMPHREYS, R. B., M. HAWKINS AND J. M. LIPTON. Effects of anesthetic infected into brainstem sites on body temperature and behavioral thermoregulation. PHYSIOL. BEHAV. 17(4) 6 6 7 - 6 7 4 , 1 9 7 6 . - The preoptic/anterior hypothalamic region (PO/AH), the posterior (PH) and lateral hypothalamic (LH) regions, the mesencephalic reticular formation (MRF) and the medulla oblongata (MED) have all been implicated in the control of body temperature. The purpose of the present research was to learn whether differential effects upon physiological and behavioral thermoregulation are produced by temporarily depressing the activity of these regions with anesthetic. The rectal temperature (Tre) of rats with central cannulae was recorded after sodium pentobarbital injections while the animals rested in 23 °, 10 ° and 34°C environments. In other experiments the effects of central anesthetic injections on behavioral regulation against heat were measured. Anesthetic injected into the PO/AH region caused changes in Tre and behavior that are consistent with a coordinated rise in the set point of body temperature control. Injections into the MED produced transient and rapid decreases in Tre without affecting behavioral thermoregulation. Bilateral injections into the LH caused hyperthcrmia in the 10 ° and 34°C environments, hypothermia in the 23°C environment and had no effect on behavioral temperature regulation. No changes in thermoregulatory responses were observed after PH and MRF injections. These results indicate that there are differences among the 5 brain regions in relative importance to overall temperature control and specific differences in the significance of certain regions to the two forms of temperature control: physiological and behavioral temperature regulation. Central temperature controls
Body temperature
Behavioral thermoregulation
P H Y S I O L O G I C A L and behavioral t e m p e r a t u r e c o n t r o l may d e p e n d u p o n the activity o f one or m o r e of several regions of the b r a i n s t e m . The p r e o p t i c / a n t e r i o r h y p o t h a l a m i c region ( P O / A H ) is believed to c o n t a i n a r e g u l a t o r y mechanism w h i c h acts as the p r i m a r y t e m p e r a t u r e c o n t r o l , integrating central and p e r i p h e r a l t h e r m a l i n f o r m a t i o n and driving t h e r m o r e g u l a t o r y responses. However, it is clear that this brain region is not t h e sole site of central t e m p e r a t u r e c o n t r o l since n e i t h e r physiological n o r behavioral t h e r m o r e g u l a t i o n are totally o b l i t e r a t e d w h e n the region is d e s t r o y e d [ 1 6 . 1 8 ] . T h e p o s t e r i o r h y p o t h a l a m u s has been associated with t e m p e r a t u r e c o n t r o l since the early e x p e r i m e n t s of R a n s o n [28] in w h i c h bilateral lesions of the p o s t e r o l a t e r a l h y p o t h a l a m u s caused a loss of capacity to regulate b o d y t e m p e r a t u r e in the cold. Bilateral lesions in the lateral h y p o t h a l a m u s have been r e p o r t e d to disturb behavioral t h e r m o r e g u l a t i o n w i t h o u t i n t e r f e r i n g with physiological t e m p e r a t u r e c o n t r o l [ 2 9 ] . T e m p e r a t u r e sensitive n e u r o n s in the m i d b r a i n are believed to act to prevent deviations in b o d y t e m p e r a t u r e b y sensing local t e m p e r a t u r e and driving physiological t h e r m o r e g u l a t o r y
responses [ 8 ] . Finally, t h e r e is evidence t h a t the reticular s u b s t a n c e of the m e d u l l a o b l o n g a t a c o n t a i n s a s e c o n d a r y thermosensitive temperature control mechanism which drives physiological and behavioral t h e r m o r e g u l a t i o n ind e p e n d e n t of the P O / A H region [ 1 5 , 1 6 } . E x c e p t for general c o m p a r i s o n s of the t h e r m o s e n s i t i v i t y of the P O / A H region and the m e d u l l a o b l o n g a t a [ 1 6 ] , there is little data on the relative i m p o r t a n c e of specific brain regions to b o d y t e m p e r a t u r e c o n t r o l . One objective in the present exp e r i m e n t s was to c o m p a r e the effects on b o d y t e m p e r a t u r e and t h e r m o r e g u l a t o r y b e h a v i o r of acute depression of the activity of the particular b r a i n s t e m regions w h i c h have b e e n implicated in t e m p e r a t u r e c o n t r o l . A second goal was to learn w h e t h e r selective depression of these sites affects physiological or behavioral t e m p e r a t u r e c o n t r o l alone, or w h e t h e r b o t h f o r m s of t e m p e r a t u r e c o n t r o l are altered. MATERIALS AND METHOI)S The general a p p r o a c h was to inject a n e s t h e t i c i n t o a particular brain region and to record e i t h e r the effects u p o n
This research was supported by the Leland Fikes Foundation and the National Institute of Neurological and Communications Disorders and Stroke Grant No. 2-R01-NS 10046 02. The authors thank Dr. W. G. Clark for his comments on the manuscripL 667
668 rectal t e m p e r a t u r e (Tre) while the animals rested in n e u t r a l hot or cold e n v i r o n m e n t s or to n o t e the effects of central anesthetic u p o n a standard test of t h e r e m o r e g u l a t o r y behavior. A total of 45 male albino rats ( H o l t z m a n strain) were operated. The rats were maintained in individual cages in a r o o m controlled at 24 ± 2 ° C. All animals were weighed daily and b o d y weight was controlled at 3 2 0 - 4 0 0 g by regulating access to Purina Laboratory Chow. Because of cannula failure, inanition and surgical m o r t a l i t y a total of 22 animals which were in good c o n d i t i o n were used in the experiments from which the data described below were obtained.
Surgery Each rat was anesthetized with an intraperitoneal injection of sodium p e n t o b a r b i t a l (50 mg/kg) and placed in a Kopf stereotaxic instrument. A stainless steel guide cannula assembly fitted with an o b t u r a t o r and a Teflon cap [19] was implanted, using standard surgical procedures. Most animals had a single cannula implanted in one of the l o l l ' o w i n g sites: the p r e o p t i c / a n t e r i o r hypothalamic (PO/AH) region; the posterior h y p o t h a l a m i c (PH) region: the medulla oblongata (MED); or the mesencephalic reticular f o r m a t i o n (MRF). Bilateral cannulae were placed in the lateral h y p o t h a l a m i c (LH) region in other rats. At least 7 days were allowed for recovery before the animals were tested.
Injection Procedures To make an injection the o b t u r a t o r was removed, and an injection cannula was inserted so that it e x t e n d e d 0.5 mm below the tip of the guide tube. One ul of either nonpyrogenic saline or sodium pentobarbital was injected over 30 sec. The cannula was left in place for another 30 sec before it was removed and the o b t u r a t o r replaced. Injections were separated by periods of at least 48 hr with saline and anesthetic given alternately in each rat at each of the 3 ambient temperatures. In pilot work sodium pentobarbital dissolved in saline and injected into the P O / A H region was found to produce only brief changes in body t e m p e r a t u r e unless p r o p y l e n e glycol was added to the solution to reduce the rate of dispersion. F o r this reason pentobarbital sodium in salinepropylene glycol-alcohol solution ( N e m b u t a l ) was used in the main experiments. Changes in b o d y t e m p e r a t u r e produced by injecting the vehicle (saline, 50%; propylene glycol, 40%; ethanol, 10%) into the P O / A H region in animals resting in neutral, hot and cold environments were not different from those seen after saline alone was injected. Because of this finding and the need to avoid c o n t a m i n a t i o n with pyrogens whenever possible, only commercial n o n p y r o g e n i c saline ( A b b o t t L a b o r a t o r i e s ) w a s used for control injections in the main experiments.
Body Temperature Tests of the effects of central anesthetic on body temperature were p e r f o r m e d in an environmental chamber with the ambient t e m p e r a t u r e held at 10 °, 23 ° , or 34°C. Tre in these and in the behavioral experiments was recorded using a thermistor probe inserted 6 cm past the anus. After a 1 hr baseline period in which Tre was measured every 15 min an intracerebral injection was made, and the temperatures were recorded for an additional 5 hr.
H U M P H R E Y S , HAWKINS AND LIPTON
Behavioral Therrnoregulation The behavioral thermoregutation apparatus has been described previously [13, 14, 26]. Each behavior test unil consisted of a Plexiglas cylinder m o u n t e d in a manifold box fitted with an exhaust fan (60 cfm)+ A heat lamp (250 Wt was centered over the behavior chamber. Pressing a lever turned the heat lamp off and activated the exhaust fan which drew ambient air through the chamber. F o u r chambers of this type were used in a room where the ambient temperature was held at 23 t+ U C ) . The intensity of the lamp was gradually reduced to 125 W by controlling voltage so that the time spent depressing the lever wa.~ reduced from initial levels of 85% + 98% to 40%- 6 5 q . After reliable behavioral responding was established with the lamp set at 125 W the rats spent at least 2 additional 6 hr periods in the chamber. In the behavior tests percent time spent depressing the lever was measured for a total of 6 hr with intracerebral injections of physiological saline or Nembutal given I hr after the teq was begun. Tests on individual animals were separated hy a period of at least 48 hr. After the experiments were c o m p l e t e d the rats were killed with an intraperitoneal injection of sodium pento-barbital and the brains were perfused with saline and t 0 ~ Formalin buffered with potassium. Serial sectim~s 35 thick were cut from frozen brain tissue and stained with Luxol blue. The location of the tips of the cannulae were reconstructed on copies of line drawings taken from brain atlases [12,251 with the injection sites traced in by hand in accordance with enlarged images of the brain sections.
RESUL I5;
PO/A H R egio n In the 2 3 e n v i r o n m e n t sodium pentobarbital caused hyperthermia when placed in the PO/AH region l Fig. 11. The average m a x i m u m increase in body temperature was 1.&C which occurred 2 hr after injection. A smaller h y p e r t h e r m i c response was observed in the cold environment where the average m a x i m u m increase in Tre was 0.9°C at 3 hr after injection of anesthetic. In the saline control experiments run in the 10 ° e n v i r o n m e n t Tre showed the slight increase that was characteristic in rats exposed to cold (see Figs. 3 and 41). In the 34 ' e n v i r o n m e n t preinjection "Ires were higher ( 0 . 5 - 1 . 0 ° C ) in all rats as a result of the 1 hr exposure to heat. After saline injection, these rats showed a slight initial rise and then a decrease in Tre over the remainder of the period (Fig. I ). Injections of sodium pentobarbital caused an average m a x i m u m increase of 0+2-C 2 hr after injection. The main difference after central anesthetic was that the Tres were maintained t h r o u g h o u t the 5 hr recording period in contrast to the decrease seen after control injections. In the behavioral thermoregulation tests, injection of sodium pentobarbital into the P O / A H region caused a decrease in the a m o u n t of time spent pressing the lever to escape heat (Fig. 2). This change in responding began within one hr after the injection and was maintained for 2---3 hr, followed by a return to near preinjection levels. Saline injections caused a slight rise in the a m o u n t of time spent escaping heat which persisted throughout the test period.
BRAINSTEM AND THERMOREGULATION
669
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FIG. 1. Effects of injecting anesthetic into the PO/AH region on Tre in rats resting in neutral, hot and cold environments. Data points are mean (+ SE) changes in 4 rats. In this and following graphs control data were obtained using the same animals that received pentobarbital injections. Injection sites are indicated by circles on coronal brain section.
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There was no significant chang0 in behavioral t h e r m o regulation after a n e s t h e t i c was injected into the MED (Table 1 ).
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FIG. 2. Effects of anesthetic injections in the PO/AH region on behavioral regulation against heat. All animals showed a decrease in the amount of time spent responding for 2 - 3 hr after injections.
Medulla Oblongata In the neutral e n v i r o n m e n t all rats w i t h m e d u l l a r y c a n n u l a e s h o w e d e x t r e m e l y rapid a n d brief h y p o t h e r m i a after s o d i u m p e n t o b a r b i t a l injections (Fig. 3). T h e h y p o t h e r m i a reached m a x i m u m 30 rain a f t e r i n j e c t i o n and Tre r e t u r n e d to c o n t r o l levels w i t h i n an a d d i t i o n a l 60 rain. H y p o t h e r m i a was also observed in the 10 ° e n v i r o n m e n t , a l t h o u g h the m a x i m u m change in b o d y t e m p e r a t u r e was not as great. In the 3 4 ' e n v i r o n m e n t t h e r e was no clear evidence of a h y p o t h e r m i c effect after central a n e s t h e t i c when the results are c o m p a r e d w i t h c o n t r o l data. Tre showed the c h a r a c t e r i s t i c decline t h a t followed saline injections (See Figs. 1 and 4) and the decrease in Tre after central a n e s t h e t i c was a l m o s t the same.
Injections of a n e s t h e t i c i n t o the LH caused h y p o t h e r m i a in rats tested in the 23°C e n v i r o n m e n t (Fig. 4). Tre r e t u r n e d to n o r m a l by the end of the 5 hr p o s t i n j e c t i o n period. Depression of the activity of the LH in the same animals in t h e cold e n v i r o n m e n t did not p r o d u c e h y p o t h e r m i a but r a t h e r caused h y p e r t h e r m i a s with peak Tre s of 39.4 40.3°C. Shivering began in some rats w i t h i n 0.5 hr after i n j e c t i o n and recurred i n t e r m i t t e n t l y t h r o u g h o u t the r e m a i n d e r of the test period. H y p e r t h e r m i a was also recorded after s o d i u m p e n t o b a r b i t a l was placed in the LH of animals in the 34" e n v i r o n m e n t while the response to saline was again a progressive decrease in Tre. A l t h o u g h the changes in b o d y t e m p e r a t u r e in the three a m b i e n t t e m p e r a t u r e s were m a r k e d and c o n s i s t e n t , the only change in t h e r m o r e g u l a t o r y b e h a v i o r p r o d u c e d by depressing the activity in the LH region was a slight elevation in r e s p o n d i n g by one rat 3 hr after the injections were given (Table 1 ). It s h o u l d be n o t e d t h a t the results described above were derived from e x p e r i m e n t s in which 50 ug of sodium p e n t o b a r b i t a l was given bilaterally. In p r e l i m i n a r y exp e r i m e n t s on three rats unilateral injections of 50 ug sodium p e n t o b a r b i t a l had no c o n s i s t e n t effects on b o d y t e m p e r a t u r e while bilateral injections of 2 0 - 3 0 ug produced decreases in Tre ranging b e t w e e n 0 . 7 - 1 . 8 ° ( ` in animals resting in the 23"C e n v i r o n m e n t .
670
HUMPHREYS,
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Ta= lOoC
HAWKINS
AND LIPTON
Ta= B4oC
I., %
.
}-
~ z
-1.0"
0
I
I
I
I
1
2
3
4
I
I
50
I
I
,, I
I
1 2 3 4 50 HOURS AFTER INJECTION
I
I
I
I
I
1
2
3
4
5
FIG. 3. Effects of anesthetic in the medulla oblongata on Tre in four rats exposed to neutral, hot and cold environments.
Ta : 1O* C
Ta= 23"C
Ta : B4°C
+1.5"
,,,
F ÷1.0"
i -r
o
o.---,.e
-1.0° L
0
I
I
I
2
I
5
I
4
J
SALINE
I
I
1
1
..._.L
J
50 HOURS AFTER INJECTION
FIG. 4. Changes in b o d y temperature after bilateral anesthetic injections in the LH region of 4 rals exposed to neutral, h o t and cold environments.
BRAINSTEM AND THERMOREGULATION
671 TABLE 1
MEAN ( -+ SE) CHANGES (%) IN TIME SPENT RESPONDING TO ESCAPE HEAT AFTER INTRACEREBRAL INJECTIONS
Injection
1
2
Hours After Injection 3
4
5
Anesthetic Saline
-6.6 (+_ 1.4) -5.5 ( -+ 1.3)
Medulla (N =4) -9.0 ( + 3.0) -8.3 ( ± 3.1 - 6 . 9 ( ± 2.2) - 5 . 6 ( ± 2.8)
9.1 I -+ 2.9) - 8 . 7 ( ± 2.2)
-6.0 - 8.1
+ 2.9) +_ 3.4)
Anesthetic Saline
-3.3 ( -+ 0.9) 4.1 ( _4_-0.8)
Lateral Hypothalamus (N =4) 7.4 ( ± 0.9) 1.0 ( ± 2.1) 6.9 ( ± 1.4t 0.3 ( ± 2.1)
8.0 ( - 3.0) 4.8 ( ± 3.8)
7.0 2.9
_+ 3.0) _+ 4.1)
Anesthetic Saline
3.1 -1.1
-+ 2.2) +_ 1.4)
2.0 ( _+ 1.5) 3.2 ( + 1.3)
6.0 5.4
_+ 2.21 _+ 0.91
Anesthetic Saline
-3.3 -2.4
_+ 0.9) -+ 0.7)
4.2 ( +_ 2.1) 3.4 ( _+ 1.9)
1.0 5.1
= 3.3) + 2.3)
Posterior Hypothalamus (N=5) 2.0 ( _+ 1.1) 2.1 ( + 1.7) -2.0 ( -+ 1.3) 0.0 ) -4--0.6) Midbrain (N =5) -1.0 ( _+ 0.5) 0.1 ( + 3.1) -2.1 ( ± 0.7) -2.0 ( -4--0.9)
DISCUSSION
i
FIG. 5. Sites in the posterior hypothalamus (top) and mesencephalic reticular formation where anesthetic injections had no effect on body temperature or thermoregulatory behavior.
Posterior Hypothalamie tfcular Formation
Region and Mesencephalic Re-
I n j e c t i o n s of a n e s t h e t i c i n t o these t w o regions (Fig. 5) caused very little change in Tre in n e u t r a l , hot and cold e n v i r o n m e n t s (Table 2) and had no clear effects u p o n behavioral t b e r m o r e g u l a t i o n (Table 1).
Our results indicate t h a t n o r m a l activity of at least 3 of the 5 brain regions previously implicated in r e g u l a t i o n of b o d y t e m p e r a t u r e is necessary to t h e r m o r e g u l a t o r y processes. Depression of the activity of the P O / A H region, the medulla o b l o n g a t a and the lateral h y p o t h a l a m u s with a n e s t h e t i c caused changes in b o d y t e m p e r a t u r e and, in the case of the P O / A H region, changes in t h e r m o r e g u l a t o r y behavior while a n e s t h e t i c in the m i d b r a i n and in the medial posterior h y p o t h a l a m u s had no a p p r e c i a b l e effect. Injections of sodium p e n t o b a r b i t a l i n t o the p r i m a r y t e m p e r a t u r e c o n t r o l in t h e P O / A H region caused an increase in Tre in neutral and cold e n v i r o n m e n t s and a failure to show a n o r m a l decrease in the h o t e n v i r o n m e n t . Depression of activity of the P O / A H region also altered t h e r m o r e g u l a t o r y m o t i v a t i o n , causing a decrease in the a m o u n t of time spent escaping heat. The c o o r d i n a t e d changes in physiological and behavioral t h e r m o r e g u l a t i o n are c o n s i s t e n t w i t h an alt e r a t i o n in the set p o i n t a r o u n d w h i c h b o d y t e m p e r a t u r e is c o n t r o l l e d . Similar c o o r d i n a t e d changes in physiological and behavioral t e m p e r a t u r e regulation have been observed w h e n this part of the f o r e b r a i n is heated or cooled [6, 7, 11], when i n t r a c e r e b r o v e r t r i c u l a r t e t r o d o t o x i n is administered [5] and w h e n p y r o g e n s are given peripherally [ 4 , 3 1 ] . A l t h o u g h the results of the present e x p e r i m e n t s s u p p o r t the i m p o r t a n c e of the P O / A H region to t e m p e r a t u r e c o n t r o l , they differ f r o m those o b t a i n e d in e x p e r i m e n t s on animals with electrolytic d e s t r u c t i o n of the P O / A H region. The rats in the present e x p e r i m e n t did n o t show the t h e r m o l a b i l i t y in t h e heat and cold w h i c h is characteristic after P O / A H injury [ 1 8 ] . Nor did these rats increase r e s p o n d i n g to escape heat as seen after P O / A H lesions in o t h e r e x p e r i m e n t s [ 1 3 ] . The reason for these differences m a y be t h a t the sodium p e n t o b a r b i t a l injections did n o t affect the same or as m a n y n e u r o n s as the lesions. However, there is no r e a s o n to believe t h a t the a n e s t h e t i c is selective in its i n f l u e n c e on n e u r o n s . S o d i u m p e n t o b a r b i t a l is a general CNS d e p r e s s a n t [10] w h i c h alters s o d i u m and p o t a s s i u m c o n d u c t a n c e across n e u r o n a l m e m b r a n e s [ 2 ]. On the o t h e r h a n d , a l t h o u g h materials injected i n t o the brain tend to spread c o n s i d e r a b l y [ 2 1 ] , there is n o way to rule
672
HUMPHREYS, HAWKINS AND LIPTON 'FABLE 2 MEAN (---SE)CHANGES IN Tre AFTER ANESTHETICINJECTIONS IN POSTERIOR HYPOTHALAMUS AND MIDBRAIN
Injection
l
2
Hours After Injection 3
4
Posterior Hypothalamus IN=5)
Anesthetic Saline
-0.3 (+ 0.4) -0.1 (_* 0.2)
Anesthetic Saline
0.1 (± 0.2) -0.1 (m 0.2)
Anesthetic Saline
--0.2 (± 0.1) -0.1 (_+ 0.2)
Anesthetic Saline
-0.2 (_+ 0.3) -0.2 (± 0.2)
Anesthetic Saline
-0.1 (± 0.1) 0.0 (+_ 0.2)
Anesthetic Saline
-0.1 (±_ 0.2) -0.2 (± 0.2)
23°C -0.3 (+- 0.2) -0.3 (_+_0.2) -0.1 (_+ 0.2) -0.2 (+ 0.2) 10°C 0.3 (± 0.2) 0.6 (+_ 0.4) 0.0 (± 0.2) 0.1 Ix 0.3) 34°C -0.5 (m 0.2) -0,4 (_~ 0.2) -0.2 (+ 0.2) -0.3 (_+ 0.2) Midbrain (N ~5) 23°C -0.1 (± 0.1) -0.4 (± 0.2) -0.2 (+ 0.1) 0.4 (± 0.2) 10°C -0.2 (_+ 0.1) -0.5 (± 0.3) -0.6 (+_ 0.4) -0.2 (± 0.2) 34°C -0.3 (+ 0.2) -0.3 1± 0.2) --0.1 (± 0.2) -0.3 (± 0.2)
out the possibility that fewer neurons were involved in the Nembutal treatments than after PO/AH lesions. An alternative explanation of the difference between the effects of central anesthetic and lesions may lie in differences in persistence over time since the effects of central anesthetic resemble those observed immediately after the PO/AH region is destroyed, when resting body temperature increases. Similar increases in core temperature were observed when Nembutal was injected into pyrogen-sensitive sites in the PO/AH region of the squirrel monkey [ 17 ]. It is believed that depression of heat-sensitive cells, which are more densely distributed in the PO/AH region, causes reversible hyperthermia that resembles the elevated temperatures seen in infectious fever. Thermolability and compensatory behavioral responding might develop only after the activity of the region has been depressed by anesthetic for some time or it might develop over time after the region has been destroyed. Then the main difference between the effects of anesthetic treatments and lesions would be due to the difference in their persistence over time. The medulla oblongata has been shown to be thermosensitive and to drive physiological and behavioral thermoregulatory responses in much the same way they are driven by altering the temperature of the PO/AH region [ 16]. The finding that the relation between local medullary temperature and thermoregulatory responses was not as close as that found with thermal stimulation of the PO/AH region, and the observation that the effects of thermal stimulation either persisted or were enhanced after PO/AH destruction, led to the idea that the medullary reticular substance contains a separate, secondary temperature control [ 16]. The brief hypothermia after anesthetic injections in the
-0.5 (± 0.3) -0.2 (_+ 0.1)
-0.3 (._ 0.1) -0.3 (= 0.2)
0.4 (+_ 0.3) 0.0 (± 0.2)
I).3 (z 0.2) 0.1 ~e 0.2)
-0.3 (_+ 0.1) -0.3 (_+ 0.1)
-I).4 (-. 0.4) - 0 4 (~ 0.3)
-0.4 (± 0.1) -0.4 (± 0.1)
--0.5 ~± 0.1) -0.5 (~ 0.1)
-0.5 (± 0.3) -0.5 (± 0.3)
-0.4 1± 0.2) --0.6 (:~ 0.2)
-0.2 (± 0.2) -0.3 (± 0.2)
---0.3 (± 0.21 -0.2 t:~ 0.1)
present experiments may represent the major response of the medullary control mechanism which is t h e n rapidly compensated by the intact primary control in the PO/AH region. The hypothermic effect was not observed in the 34 ° C environment perhaps because of the brevity of action and the inertia of high body temperature in the heat. No changes in thermoregulatory motivation were observed after the medullary injections. It should be noted that any effect upon behavior induced by depression of medullary activity might also be rapidly compensated by temperature control mechanisms in the PO/AH region. In previous experiments [18] rats with longstanding electrolytic lesions in the lower brainstem showed low resting body temperatures, deficits in regulation against cold and development of hyperthermia after 5 - 6 hr exposure to 35 ° environment. The effects Observed after anesthetic injections are general!y consistent with the effects after medullary lesions, although the brevity of the changes after anesthetic injections suggest that considerably more medullary tissue must be depressed for clear deficits in temperature control to be revealed. q'he falls in body temperature observed after intramedullary anesthetic contrast with results of previous research in which no changes in Tre were seen after 45 #g sodium pentobarbital in saline was injected into the medulla oblongata [20]. In the earlier research there was evidence of hypothermia in some rats but the decreases in Tre were no greater than those produced by saline injections. Reasons for the discrepancy in results are not clear but, since the anesthetic solution was different and the rats were restrained in the previous study, perhaps these factors are responsible for the failure to note changes in Tre. Restraint appears to be an especially important
BRAINSTEM AND T H E R M O R E G U L A T I O N influence on central temperature controls since morphine sulfate (50 ug) injected into the PO/AH region causes hyperthermia in unrestrained rats and falls in body temperature when the animals are restrained [30]. The effects of bilateral injections of sodium pentobarbital into lateral hypothalamus suggest that this region is also required for normal thermoregulatory processes. When there was little opportunity for behavioral thermoregulation the animals showed hypothermia in the neutral environment and hyperthermia in the extreme ambient temperatures after central anesthetic. On the other hand, behavioral thermoregulation was not significantly affected. These results contrast with the conclusion of Satinoff and Shan [29] that the lateral hypothalamus mediates behavioral thermoregulation primarily and is not required for physiological temperature control. The disruption of normal body temperature seen in the present experiment, together with deficits in the cold after parasaggital cuts which sever communication between the LH and more medial structures [18], suggests that the integrity of the lateral hypothalamus is indeed necessary to normal physiological temperature control. This idea is supported by the data of Satinoff and Shah [29] who found, in addition to the main result of decreased thermoregulatory responding after lateral hypothalamic lesions, evidence of severe impairment of physiological thermoregulation which outlasted short-term disturbances of behavior. The occurrence of hypothermia in the neutral environment and hyperthermia in the extreme ambient temperatures after injections of anesthetic into the lateral hypothalamus suggests that the nature of the role of this brain region in physiological temperature regulation is complex. The fact that the lateral hypothalamus is a nodal point of ascending and descending brainstem pathways and that it contains major connections between medial hypothalamic regions important ~o temperature control and other central structures [24,27] may account for the complexity of the results. Although we found no significant change in thermoregulatory behavior in our tests we cannot rule out the possibility that compensatory behavioral responding might occur if the lateral hypothalamus were depressed for a prolonged period. The posterior hypothalamus has been proposed to be the major site of convergence of temperature signals and the origin of the central effector signal [9]. If this conception, which is based primarily on results of experiments in which not only the posterior hypothalamus but all ventrolateral pathways in the brainstem were destroyed, were true then it would be expected that injection of a nonspecific anesthetic into the region would result in major thermoregulatory deficits. According to another theory the mechanism for controlling body temperature around a given level depends upon a constant, uniform balance between sodium and calcium ions within the posterior hypothalamus which provides Ihe reference input to the PO/AH temperature control [22,231. Again, if it were true that the posterior hypothalamus plays a crucial role in the control of body temperature one would expect injections into this region of an anesthetic which alters sodium conductance to cause disturbance of major thermoregulatory processes. Using injections of sodium pentobarbital into the posterior hypothalamic region, at a dose level which was effective in altering body temperature when placed in the PO/AH region, we were unable ~o demonstrate significant effects
673 on thermoregulatory processes and thus to support the idea that the normal activity of the posterior hypothalamus is essential in temperature control. If our observations are correct, then the nature of the influence of the posterior hypothalamus may be different from that of a crucial integrating control. Rather, it would appear more likely that the region can drive or influence thermoregulatory processes when it is manipulated (e.g., by altering ionic balance) but that, when the activity of the region is depressed, sufficient temperature control persists such that no clear deficits can be demonstrated. Cabanac and Hardy [3] found that shivering occurred when the mesencephalon of the rabbit was cooled. In another report Hardy [8] noted that rabbits 'in a cold environment show changes in heat production, and some alteration in vasomotor state, in response to midbrain heating and cooling. However, altering the temperature of the mesencephalic reticular substance does not influence behavioral thermoregulation of the squirrel monkey [11 or the rat [15]. In the present experiments temporary depression of neural activity at sites in this brain region did not disrupt temperature control. It appears that either normal activity in mesencephalic sites is not essential to normal temperature control in the rat or that effects upon thermoregulation caused by local injections of anesthetic in this region can be compensated by other central control mechanisms. A major accounting of the central controls of body temperature must include many circuits and, in the case of behvioral thermoregulation, must encompass not only autonomic components but sensory and motor components with cortical representation as well. The technique of acute depression of activity at a specific brain site reveals not the exact role of the anesthetized tissue but rather whether a function persists through mediation by unaffected tissue or whether the function is dependent upon the integrity of the depressed region. Using this method we have found that unaffected brain mechanisms are capable of regulating body temperature when the posterior hypothalamus and mesencephalic reticular formation are temporarily depressed. However, in the case of the PO/Att region, depression of activity caused changes in physiological and behavioral thermoregulation which suggest that the remaining control system has lost part of the input from heat-sensitive cells. These findings confirm again the importance of the PO/AH to normal temperature control. We found thai undepressed mechanisms in the brain are capable of rapidly compensating for depression of activity in the medulla oblongata so that hypothermia is rapidly reversed. If there is an effect of medullary depression on behavioral temperature control, as one would expect from previous studies on the driving of thermoregulatory behavior by thermal stinmlation of the medulla [16], it is compensated so rapidly that it is not observable with the behavioral techniques used here. It seems that the unaffected mechanisms are not capable of compensating the effects of body temperature of lateral hypothalamic depression but that behavioral regulation can continue without disruption. Although the results indicate that the lateral hypothalamic region is essential to normal temperature control, the nature of its role is not obvious from the uncompensated alterations in thermoregulation which are observed when the region is depressed.
674
H U M P H R E Y S , H A W K I N S A N D LIPTON
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