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Non-shivering thermogenesis during prostaglandin E1 fever in rats: role of the cerebral cortex
BRAIN RESEARCH ELSEVIER
Brain Research 651 (1994) 148-154
Research Report
Non-shivering thermogenesis during prostaglandin E 1 fever in rats: role of the cerebral cortex M. Monda *, S. Amaro, B. De Luca Department of Human Physiology and Integrate Biological Function 'Filippo Bottazzi', Faculty of Medicine and Surgery, Second Unit)ersity of Naples, Naples, Italy Accepted 29 March 1994
Abstract
We have tested the hypothesis that there is a role for the cerebral cortex in the control of non-shivering thermogenesis during fever induced by prostaglandin E 1 (PGE1). While under urethan anesthesia, the firing rate of nerves innervating interscapular brown adipose tissue (IBAT), IBAT and colonic temperatures (TmA x and Tc) and oxygen (0 2) consumption were monitored during the fever from PGE 1 injection (400 and 800 ng) in a lateral cerebral ventricle in controls and in functionally decorticated Sprague-Dawley rats. Rats were functionally decorticated by applying 3.3 M KCI solution on the frontal cortex which causes cortical spreading depression (CSD). Pyrogen injections caused dose-related increases in firing rate, TreAT, Tc and 0 2 consumption and CSD reduced these enhancements. Our findings indicate that the cerebral cortex could be involved in the control of non-shivering thermogenesis during PGEl-induced febrile response. Key words: Non-shivering thermogenesis; Prostaglandin E~; Cerebral cortex; Brown adipose tissue; Sympathetic activity; Oxygen
consumption; Body temperature I. Introduction
In fever there is a resetting of the thermal set point to defend a higher than normal 'core' temperature [28]. Prostaglandins of the E series (PGE) are involved in mediating this event [27]. It has been accepted that the principal mechanism of increased heat production during the development of fever is the same as during cold exposure, i.e., visible shivering and an inhibition of heat-loss systems accomplished, for example, through an increase in vasomotor tone [3]. However, the heat production unrelated to the shivering is also involved in the febrile mechanism. Brown adipose tissue (BAT) is the organ responsible for evoking 35-65% of the total increase in metabolic heat production during various experimental manipulations in rodents [8,16]. BAT occurs as numerous discrete deposits, particularly in the interscapular region of the body [1] and it is known
* Corresponding author. Address: Dipartimento di Fisiologia Umana, Seconda Universit~ di Napoli, Via Costantinopoli 16, 80138 Napoli, Italia. Fax: (39) (81) 566 58 20. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 4 2 1 - 8
to be under the peripheral neurogenic control of the sympathetic nervous system. Interscapular BAT (IBAT) is supplied by a mixed nerve, which provides five separate branches to the individual lobes [9,10]. There is evidence suggesting that brown fat contributes to PGE-evoked fever in rats. Indeed, infusion of P G E 1 into a lateral cerebral ventricle increases BAT temperature [11]. Moreover, the cerebral cortex is involved in the control of heat production. Electrical stimulation of sulcal prefrontal cortex activates warm-sensitive units and suppresses cold-sensitive units in the anterior hypothalamic area [17]. The increase of metabolic rate, due to lateral hypothalamic lesioning, fails to occur in decorticated rats [6]. Recently we demonstrated that functional decortication blocks the fever induced by i.c.v. P G E 1 (100 ng) administration in free-moving rats [21]. The aims of these experiments were: (1) to examine the effects of CSD on oxygen (O 2) consumption and 'core' temperature during PGE1 fever induced by higher doses exceeding 100 ng, and (2) to evaluate the activity of sympathetic outflow to brown fat in these phenomena.
M. Monda et al. / Brain Research 651 (1994) 148-154
2. Materials and methods 2.1. Animals We used male Sprague-Dawley rats (n = 72, 280-320 g). These were housed in pairs at controlled temperature (22+ I°C) and humidity (70%) with a 12:12-h light-dark cycle from 07:00 to 19:00. Laboratory standard food (Mil, Morini, Italy) and water were available at all times.
2.2. Apparatus Firing rate of nerves to IBAT was recorded using a pair of silver wire electrodes. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-path filters (NeuroLog System, Digitimer). T h e raw pulses were displayed on a oscilloscope (Tektronix) and sent to a window discriminator. Square waves originating from the discriminator were sent to an analog-digital converter (DAS system, Keithley) and stored on a computer (Personal Computer AT, IBM) every 2 s. Furthermore, a rate meter with a reset time of 2 s was used to observe the time course of the nerve activity recorded by pen recorder (Dynograph, Beckman). Because signal-to-noise ratio depended on the n u m b e r of nerve filaments and the condition of contact between the nerve and electrodes, the basal rates of sampled pulses were different for each rat. T h e threshold level of the event detector was fixed in each rat during the experiment at 50% of the peaks of the largest pulses and above background noise. Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (Tc and TIBAT) and the values were stored on a cart recorder. Resting 0 2 consumption was determined with an indirect calorimeter. The closed circuit apparatus used was an adaptation of Benedict and MacLeod's calorimeter. Air was continuously circulated through a drying column (CaSO4 Drierite), a respiratory chamber 2.5 1 and CO 2 trap (soda lime), by a peristaltic p u m p at a rate of 2 l / m i n . A 1-1 cylindrical metal bell fit in a concentric cylinder filled with water forming an air-tight seal served as the O 2 reservoir. The 5-ml graduated cylinder was connected to the respiratory chamber. Respiratory chamber temperature was maintained constant at 29°C by circulating water, and was monitored by an internal thermometer. The volume of O 2 consumed by each animal was corrected for temperature and pressure and was expressed as ml of 0 2 / m i n / k g b.w. 0.75 [18].
149
After recovery, six animals (1st group) were anesthetized with urethane (1.2 g / k g b.w.i.p.) and m o u n t e d in a stereotaxic instrument (Stoelting). The level of anesthesia was kept constant on a stage III, plane 1 level as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli [26]. Nerve activity was recorded by small nerve bundles dissected from the nerve branches innervating the right side of IBAT. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope to record the efferent activity with a pair of silver wire electrodes. The nerve filaments were covered with a mixture of vaseline and liquid petroleum at 37°C to avoid dehydration. The firing rate was recorded 30-40 min (base-line period) before i.c.v, injection of 400 ng P G E t and 40 min after injection of pyrogen. The PGE1, dissolved in a vol. of 5 p.1, was delivered into the left cerebral ventricle by gravity flow over 1 min, using a cannula 0.4 m m longer than the guide cannula. Furthermore, Tc and TmAT were monitored at the same time as the nerve activity record. Tc was measured by inserting the thermocouple into the colon at 7 cm from the anus, while TmAT was monitored by inserting the thermocouple in the left side of IBAT estimated region. T h e application of 3.3 M KC1 solution upon the frontal cerebral cortex (which causes CSD) [2] preceded the i.c.v. injection by 10 min. The same variables at the same time were recorded in the other six animals (2nd group), but instead of KCI solution 3.3 M NaCI solution was applied to the cortex, which did not affect the cortical functions [2]~ The same procedure as the 1st group was carried out in the other six animals (3rd group) except for higher drug dose, using 800 ng of P G E r A n o t h e r group of six rats (4th group) was treated like the animals of the 2nd group, using 800 ng of pyrogen. The base-line values of Tc from all animals used were maintained constant by a heating pad. The electrical energy supplied to which was not altered during the experimental period. The second 24 rats were anesthetized with urethan (1.2 g / k g b.w.) and divided in four groups as the first 24 animals. In six animals (5th group), 0 2 consumption was measured over 30-40 min (base-line values) before i.c.v, injection of 400 ng P G E 1 and 40 min after injection of the drug. The application of 3.3 M KCI solution upon the frontal cortex preceded the i.c.v, injection by 10 min. The same variable at the same time was recorded in the 6th group, but KCI solution was substituted for 3.3 M NaCI solution. The same procedure as the 5th group was carried out in the 7th group, using 800 ng of P G E 1. The rats of the 8th group were treated like the animals of the 6th group, using 800 ng of pyrogen.
2.5. Preliminary experiments 2.3. Drug P G E I was purchased from Sigma (St Louis, MO). Doses of 400 and 800 ng were dissolved in 5 p,l of 0.9% NaCI sterile pyrogen-free solution.
2. 4. Procedure The animals were anesthetized with i.p. pentobarbital (50 m g / k g b.w.) and a 20-gauge stainless guide cannula was positioned stereotaxically [22] over the left lateral cerebral ventricle (1.5 m m lateral to the midline, 1.3 m m posterior to the bregma, 3.0 m m from the cranial theca). Stylets were inserted into the guide tubes and removed only when drug administration occurred. In addition, two symmetrical craniotomies (4 m m in diameter) were performed in the frontal bone and two polyethylene wells were placed upon the frontal cortex. These wells were filled with saline solution and capped until the experiment began. The guide cannulas and polyethylene wells were secured to the skull by m e a n s of screws and dental cement. Rats were given 7-10 days to recover from surgery as judged by recovery of preoperative body weight.
In preliminary experiments, we used four groups of six rats to test the effect of 3.3 M KC1 or 3.3 M NaCl solution alone (without the pyrogen injection) on the cortical electroencephalogram (EEG), the slow potential and the variables considered above (firing rate of nerves to IBAT, T¢, TIBAT and O 2 consumption). The surgical preparation was similar to that described above. After recovery, two groups were anesthetized with urethan (1.2 g / k g b.w.). Firing rate of nerves to IBAT, Tc and TIBAT were recorded 30-40 min before and 40 min after the i.c.v, injection of 5 p,l of 0.9% NaCl sterile solution. The i.c.v, injection was preceded by an application of 3.3 M KCI (in the first six rats) or 3.3 M NaCl (in the second six animals) solution upon the frontal cortex. In addition, E E G and slow potential changes were recorded at the same time with two wick calomel cell electrodes applied on the parietal cortex. This was previously exposed before the experiment. The reference electrode was placed on the frontal bone. The three electrodes were connected to a Beckman R-611 dynograph recorder. The last two groups of rats were used to test the effect of KCI or NaC1 solution, applied upon the cortex, on the 0 2 consumption. After surgical recovery, 0 2 consumption was measured in the ure-
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M. Monda et al. / B r a i n Research 651 (1994) 148-154
CHANGES IN FIRING RATE OF IBAT NERVES
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Fig. 1. Means_+ S.E. of changes in firing rate of nerves innervating interscapular brown adipose tissue. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10. than-anesthetized rats for 30-40 min before and 40 min after i.c.v. injection of 5 /xl of 0.9% NaCI sterile solution. The i.c.v, injection was preceded by an application of 3.3 M KCI or 3.3 M NaCI solution. 2.6. Histology At the end of the experiment the rats were injected with an overdose of pentobarbital (500 m g / k g b.w.) and the location of the cannula was identified histologically. The animals were perfused with 0.9% NaCI followed by 10% (v/v) formalin solution. The brain was removed and stored in formalin solution. After few days, 50 /zm coronal sections of the fixed brain were cut and stained with Cresyl violet.
2. 7. Statistical analysis The values are presented as means_+ S.E. of difference starting at the i.c.v, injection. Statistical analysis was performed using factorial A N O V A for repeated m e a s u r e m e n t s [7].
101
i0 min
Fig. 2. Firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 400 ng P G E p
400 and 800 ng PGE r The pyrogen injection elicited increases in all groups, with the peak at 10 min. This enhancement was attenuated in the decorticated groups. A N O V A showed a significant effect of CSD (Fl,~0 = 9.582, P < 0.01), of d o s e (F1,2o = 17.067, P < 0.01) and of time (F8,16 0 = 61.672, P < 0.01). The absolute value at the P G E 1 injection time is illustrated in Table 1. Fig. 2 shows an example of firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 400 ng PGEL. Another example of firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 800 ng PGE~ is reported in Fig. 3. The effect of 400 and 800 ng PGE~ i.c.v, injection on IBAT temperature in control and decorticated animals is illustrated in Fig. 4. CSD reduced the increase 2O %
3. Results Fig. 1 shows the changes in firing rate of IBAT nerves in control and decorticated rats receiving i.c.v. Table 1 Absolute values_+ S.E. of firing rate (FR) ( s p i k e s / 2 s), temperature of brown adipose tissue ( T m a T) (°C), colonic temperature (Tc) (°C) and oxygen consumption (O z) ( m l / m i n / k g bw 0.75) at time of pyrogen injection Group
2nd
3rd
4th
Tc
11.31_+2.42 36.22_+0.11 37.21_+0.14
10.53_+1.84 36.15_+0.13 37.16_+0.18
12.14_+1.95 36.06_+0.14 37.09_+0.19
10.83_+2.12 36.25_+0.15 37.20_+0.12
Group
5th
6th
7th
8th
0 2
9.78 _+ 1.82
11.46 _+1.31
10.54 _+ 1.58
10,68 _+ 1.52
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10 min 2O
PGE1 80~ng
Absolute values at time of pyrogen administration 1st
FR
n
r. n o
T KCI
10 min
Fig. 3. Firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 800 ng PGE~.
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M. Monda et al. /Brain Research 651 (1994) 148-154
CHANGES IN IBAT TEMPERATURE
CHANGES IN OXYGEN CONSUMPTION ml/mln/bw'7S
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Fig. 4. Means+S.E. of changes in temperature of interscapular brown adipose tissue. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10.
in TIBAT. In the control rats, the p e a k was obtained at 15 and 20 min, respectively, for 400 and 800 ng. In the decorticated rats, the m a x i m u m value was at 20 and 25 min, respectively, for low and high dose. A N O V A showed a significant effect of C S D (Fl,20 = 16.690, P < 0.01), of d o s e (F1,20 = 18.856, P < 0.01) and of time (F8,160 = 52.653, P < 0.01). T h e absolute value at P G E 1 injection time is shown in Table 1. Fig. 5 illustrates the changes in Tc of rats receiving 400 and 800 ng P G E 1 i.c.v, in the presence or absence of CSD. In the rats with intact cortex, P G E 1 injection caused a rapid increase in Tc that p e a k e d at 15 min postinjection for 400 and 800 ng. In contrast, C S D a t t e n u a t e d the PGE~-elicited t e m p e r a t u r e increase
CHANGES IN COLONIC TEMPERATURE oc
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Fig. 6. Means + S.E. of changes in oxygen consumption. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10.
with both doses. A N O V A showed a significant effect of C S D (F1,20 = 18.711, P < 0 . 0 1 ) , of dose (F1,20 = 14.267, P < 0.01) and of time (Fs,160 = 40.792, P < 0.01). T h e absolute value at P G E 1 injection time is reported in Table 1. Changes in 0 2 c o n s u m p t i o n are illustrated in Fig. 6. T h e C S D r e d u c e d the upshifted metabolic rate with both doses of P G E r A N O V A showed a significant effect of C S D (F1,20 = 36.154, P < 0.01), of dose (F1,20 = 57.107, P < 0.01) and of time (F8,16 0 = 46.149, P < 0.01). T h e absolute value at P G E 1 injection time is r e p o r t e d in Table 1. Figs. 7 and 8 show E E G and slow potential changes induced by the application of 3.3 M KC1 or NaC1 solution u p o n the frontal cortex. KC1 modified the electrical activity of the cortex. This was not affected by the NaCI solution. The a p p e a r a n c e of r e p e a t e d changes of slow potentials indicated episodes of spreading depression. These waves persisted for the duration of all experiment. T h e effect of C S D alone, tested in the preliminary experiment, on firing rate of I B A T nerves, Tc and TIBAT temperatures and 0 2 consumption are not presented in the figures, because C S D alone did not modify these parameters.
,
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Fig. 5. Means + S.E. of changes in colonic temperature. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10.
O u r experiment is one of the first to show that C S D reduces the t h e r m o g e n i c changes in anaesthetized rats during P G E 1 fever. Indeed, o u r previous findings [21] showed that C S D blocks P G E 1 fever in non-anaesthetized rats. Anaesthesia with u r e t h a n maintains the rat's
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M. Monda et al. /Brain Research 651 (1994) 148-154
2mV AO
~oopv AO
1
2mVBo
tni
n
~--
},O0~V BO
Fig. 7. EEG and slow potential changes induced by application of 3.3 M KCI solution to frontal cortex. Electrode position (O,A,B) and holes for KCI application (A',B') are shown in inset brain diagram.
ability to develop fever [20] and removes the possible interferences due to motor activity. The significant role of BAT in the fever induced by PGE 1 [11] is confirmed. BAT is a major effector of non-shivering thermogenesis [14] and diet-induced thermogenesis [23] in rodents and is therefore involved in the maintenance of body temperature and energy
balance. The activity of BAT is controlled by the sympathetic nervous system [19], and factors, which influence thermogenesis, appear to act on the central nervous system to modify the sympathetic outflow to BAT [24]. Throughout this experiment, we report direct evidence of increased sympathetic tone in nerves innervating BAT after i.c.v. PGE1 injection. The
2iv AO
zoopv AO
1
m~n
ZmV
BO
zooHv BO
Fig. 8. EEG and slow potential changes induced by application of 3.3 M NaC1 solution to frontal cortex. Brain diagram is the same of Fig. 7.
M. Monda et al. /Brain Research 651 (1994) 148-154
changes in firing rate of BAT nerves precedes the change in temperature, confirming the important role of the sympathetic nervous system on BAT activity [13]. The actual TmAT is below Tc in agreement with the results of other experiments [6,20]. Changes in 0 2 consumption and Tc during the fever in urethan-anaesthetized rats are dose-dependent and in agreement with the literature [20], corroborating the validity of this experimental model in investigating febrile reactions. CSD reduces the fever induced by PGE 1 and this effect depends on the pyrogen dose. On the evidence from these experiments we cannot draw any conclusions about heat loss, but we can say that cortex depression influences the thermogenic events during the fever evoked by PGE~. In other words, these findings are the first to show that CSD reduces 'core' body temperature through reduced neuronal activation of BAT as a means of reducing 0 2 consumption. Depression of the cortex acts on the pyrogen response by reducing the activity of sympathetic nervous system. This effect on sympathetic activity has also been shown with other experimental models. Bilateral lesions of hypothalamic nuclei cause a marked increase in heat production with hyperthermia for many hours [15] and this phenomenon is mediated by the adrenergic system [4]. We showed that like beta-blockers CSD impairs the thermogenic increases following electrolytic damage of the lateral hypothalamus in rats [6]. In the present experiment, CSD does not modify body temperature and metabolic rate under basal conditions, the effect occurs only after pyrogen administration. This suggests a phasic influence of the cerebral cortex in rising thermogenesis due to PGE~ and seems to exclude a tonic cortical activity on thermogenic mechanisms. The cerebral cortex may participate in adaptative responses by activating the sympathetic nervous system when body temperature is far from the thermal set point, as well as after injection of pyrogens or after lesioning of the lateral hypothalamus. Conversely, the cortex is not involved when the body temperature is close to the set point. A less likely interpretation would be that the cortex per se does not cause such an effect, but these changes are caused by secondary or tertiary effect of CSD through cortical-subcortical neural pathways. Regardless of its cause, this is why CSD is able to attenuate increased heat production via activation of the cephalic sympathetic system. CSD is a peculiar reaction of the cerebral cortex to various depolarizing stimuli. It is characterized by the depression of electroencephalographic activity and by the appearance of surface negative slow potential change [2]. This cortical reaction involves the whole cortex arising from the stimulus area and, for this reason, the area of the cortex implicated in the control
153
of the febrile reaction cannot be located with precision. The prefrontal cortex is a likely candidate. There is evidence showing a convergence of skin and hypothalamic temperature signals on the prefrontal cortex [25]. Furthermore, we demonstrated that electrical stimulation of the prefrontal cortex enhances the metabolic rate through sympathetic nervous system activation and these effects are not evident after stimulation of the parietal or occipital cortex [5]. Finally, there is anatomical evidence showing pathways from the prefrontal cortex to the basal forebrain [12]. In conclusion, these findings suggest that the cerebral cortex is certainly involved in non-shivering thermogenesis, through the control on sympathetic activity during febrile reaction evoked by PGEt.
Acknowledgments. This study was supported by Italian National Research Council Grant 89.299.75.
References [1] Alexander, G., Cold thermogenesis. In D. Robertshaw (Ed.), International Review of Physiology, Environmental Physiology III, VoL 20, University Park Press, Baltimore, MD, 1979, pp. 43-155. [2] Bures, J. and Buresova, O., Inducing cortical spreading depression. In R.D. Myers (Ed.), Method in Psychology, Academic, New York, NY, 1972, pp. 319-343. [3] Cooper, K.E., Veale, W.L. and Kasting, N.W., Temperature regulation, fever, and antipyretics. In H.J.M. Barnet et al. (Eds.), Acetylsalicylic Acid: New Uses for an Old Drug, Raven, New York, NY, 1982, pp. 153-160. [4] Corbett, S.W., Kaufman, L.M. and Keesey, E., Effects of 13adrenergic blockade on lateral hypothalamic lesion-induced thermogenesis, Am. J. PhysioL, 245 (1983) E535-E541. [5] De Luca, B., Monda, M., Amaro, S., Pellicano M.P. and Cioffi, L.A., Thermogenic changes following frontal neocortex stimulation, Brain Res. Bull., 22 (1989) 1003-1007. [6] De Luca, B., Monda, M., Pellicano, M.P. and Zenga, A., Cortical control of thermogenesis induced by lateral hypothalamic lesion and overeating, Am. J. Physiol., 253 (1987) R626-R633. [7] Edwards, A.L., Statistical Methods, Holt, Rinehart & Winston, New York, NY, 1967. [8l Foster, D.O., Quantitative contribution of brown adipose tissue to overall metabolism, Can. J. Biochem. Cell. BioL, 62 (1984) 618-622. [9] Foster, D.O., Depocas, F. and Zaror-Behrens, G., Unilaterality of the sympathetic innervation of each pad of rat interscapular brown adipose tissue, Can. J. PhysioL PharmacoL, 60 (1982) 107-113. [10] Foster, D.O., Depocas, F. and Zuker, M., Heterogeneity of the sympathetic innervation of rat interscapular brown adipose tissue via intercostal nerves, Can. J. Physiol. Pharmacol., 60 (1982) 747-754. [11] Fyda, D.M., Cooper, K.E. and Veale, W.L., Contribution of brown adipose tissue to central PGE1-evoked hyperthermia in rats, Am. J. Physiol., 260 (1991) R59-R66.
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[12] Gaykema, R.P.A., Van Weeghel, R., Hersh, L.B. and Luiten, P.G.M., Prefrontal cortical projections to the cholinergic neurons in the basal forebrain, J. Cornp. NeuroL, 303 (1991) 563-583. [13] Geloen, A. and Trayhurn, P., Regulation of the level of uncoupling protein in brown adipose tissue by insulin requires the mediation of the sympathetic nervous system, FEBS Lett., 267 (1990) 265-267. [14] Girardier, L., Brown fat: an energy dissipating tissue. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenesis, Chapman and Hall, London, UK, 1983, pp. 50-98 [15] Grijalva, C.V., Aphagia, gastric pathology, hyperthermia and sensomotor dysfunction following lateral hypothalamic lesions: effects of insulin pretreatment, Physiol. Behal'., 25 (1980) 931937. [16] Himmis-Hagen, J., Non-shivering thermogenesis, Brain Res. Bull., 12 (1984) 151-16/). [17] Hori, T., Shibata, M., Kyohara. T. and Nakashima, T., Prefrontal cortical influences on behavioural thermoregulation and thermosensitive neurons, J. Therm. Biol., 9 (1984)27-31. [18] Kteiber, M. (Ed.), The Fire of Life. An hztroduction to Animal Energeti~s, R.E. Kreiger Publishing Company, Huntington, 1975. [19] Landsberg, L. and Young, J.B., Autonomic regulation of thermogenesis. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenesis, Chapman and Hall, London. UK, 1983, pp. 99-140.
[20] Malkinson, T.J., Cooper, K.E. and Veale, W.L., Physiological changes during thermoregulation and fever in urethananaesthetized rats, Am. Z Physiol., 255 (1988) R73-R81. [21] Monda, M. and Pittman, Q.J., Cortical spreading depression blocks prostaglandin E] and endotoxin fever in rats, Am..L PhysioL, 264 (1993) R456-R459. [22] Paxinos, G. and Watson, C. (Eds.), The Rat Brain in Stereotaxic Coordinates, Academic, New York, NY, 1986. [23] Rothwell, N.J. and Stock, M.J., Diet-induced thermogenesis. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenes&. Chapman and Hall, London, UK, 1983, pp. 208-233. [24] Rothwell, N.J. and Stock, M.J., Sympathetic and adrenocorticoid influence on diet-induced thermogenesis and brown fat activity in the rat, Comp. Biochem. PhysioL, 79A (1984) 575-579. [25] Shibata, M, Hori, T., Kiyohara, T. and Nakashima, T., Convergence of skin and hypothalamic temperature signals on the sulcal prefrontal cortex in the rat, Brain Res., 443 (1988) 37-46. [26] Soma, L.R. (Ed.), Textbook o]" Veterinary Surgeo', Depth O[ GeneralAnesthesia, Williams and Wilkins, Baltimore, MD, 1971, pp. 178-187. [27] Stitt, J.T., Prostaglandin E as the neural mediator of thc febrile response, Yale J. Biol. Med., 59 (1986) 137-149. [28] Stitt, J.T., Fever versus hyperthermia, b),d. P~vc., 38 (1979) 39-43.
Brain Research 651 (1994) 148-154
Research Report
Non-shivering thermogenesis during prostaglandin E 1 fever in rats: role of the cerebral cortex M. Monda *, S. Amaro, B. De Luca Department of Human Physiology and Integrate Biological Function 'Filippo Bottazzi', Faculty of Medicine and Surgery, Second Unit)ersity of Naples, Naples, Italy Accepted 29 March 1994
Abstract
We have tested the hypothesis that there is a role for the cerebral cortex in the control of non-shivering thermogenesis during fever induced by prostaglandin E 1 (PGE1). While under urethan anesthesia, the firing rate of nerves innervating interscapular brown adipose tissue (IBAT), IBAT and colonic temperatures (TmA x and Tc) and oxygen (0 2) consumption were monitored during the fever from PGE 1 injection (400 and 800 ng) in a lateral cerebral ventricle in controls and in functionally decorticated Sprague-Dawley rats. Rats were functionally decorticated by applying 3.3 M KCI solution on the frontal cortex which causes cortical spreading depression (CSD). Pyrogen injections caused dose-related increases in firing rate, TreAT, Tc and 0 2 consumption and CSD reduced these enhancements. Our findings indicate that the cerebral cortex could be involved in the control of non-shivering thermogenesis during PGEl-induced febrile response. Key words: Non-shivering thermogenesis; Prostaglandin E~; Cerebral cortex; Brown adipose tissue; Sympathetic activity; Oxygen
consumption; Body temperature I. Introduction
In fever there is a resetting of the thermal set point to defend a higher than normal 'core' temperature [28]. Prostaglandins of the E series (PGE) are involved in mediating this event [27]. It has been accepted that the principal mechanism of increased heat production during the development of fever is the same as during cold exposure, i.e., visible shivering and an inhibition of heat-loss systems accomplished, for example, through an increase in vasomotor tone [3]. However, the heat production unrelated to the shivering is also involved in the febrile mechanism. Brown adipose tissue (BAT) is the organ responsible for evoking 35-65% of the total increase in metabolic heat production during various experimental manipulations in rodents [8,16]. BAT occurs as numerous discrete deposits, particularly in the interscapular region of the body [1] and it is known
* Corresponding author. Address: Dipartimento di Fisiologia Umana, Seconda Universit~ di Napoli, Via Costantinopoli 16, 80138 Napoli, Italia. Fax: (39) (81) 566 58 20. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 0 4 2 1 - 8
to be under the peripheral neurogenic control of the sympathetic nervous system. Interscapular BAT (IBAT) is supplied by a mixed nerve, which provides five separate branches to the individual lobes [9,10]. There is evidence suggesting that brown fat contributes to PGE-evoked fever in rats. Indeed, infusion of P G E 1 into a lateral cerebral ventricle increases BAT temperature [11]. Moreover, the cerebral cortex is involved in the control of heat production. Electrical stimulation of sulcal prefrontal cortex activates warm-sensitive units and suppresses cold-sensitive units in the anterior hypothalamic area [17]. The increase of metabolic rate, due to lateral hypothalamic lesioning, fails to occur in decorticated rats [6]. Recently we demonstrated that functional decortication blocks the fever induced by i.c.v. P G E 1 (100 ng) administration in free-moving rats [21]. The aims of these experiments were: (1) to examine the effects of CSD on oxygen (O 2) consumption and 'core' temperature during PGE1 fever induced by higher doses exceeding 100 ng, and (2) to evaluate the activity of sympathetic outflow to brown fat in these phenomena.
M. Monda et al. / Brain Research 651 (1994) 148-154
2. Materials and methods 2.1. Animals We used male Sprague-Dawley rats (n = 72, 280-320 g). These were housed in pairs at controlled temperature (22+ I°C) and humidity (70%) with a 12:12-h light-dark cycle from 07:00 to 19:00. Laboratory standard food (Mil, Morini, Italy) and water were available at all times.
2.2. Apparatus Firing rate of nerves to IBAT was recorded using a pair of silver wire electrodes. The electrical pulses were amplified by a condenser-coupled amplifier and were filtered by band-path filters (NeuroLog System, Digitimer). T h e raw pulses were displayed on a oscilloscope (Tektronix) and sent to a window discriminator. Square waves originating from the discriminator were sent to an analog-digital converter (DAS system, Keithley) and stored on a computer (Personal Computer AT, IBM) every 2 s. Furthermore, a rate meter with a reset time of 2 s was used to observe the time course of the nerve activity recorded by pen recorder (Dynograph, Beckman). Because signal-to-noise ratio depended on the n u m b e r of nerve filaments and the condition of contact between the nerve and electrodes, the basal rates of sampled pulses were different for each rat. T h e threshold level of the event detector was fixed in each rat during the experiment at 50% of the peaks of the largest pulses and above background noise. Thermocouples (Ellab) were used to monitor colonic and IBAT temperatures (Tc and TIBAT) and the values were stored on a cart recorder. Resting 0 2 consumption was determined with an indirect calorimeter. The closed circuit apparatus used was an adaptation of Benedict and MacLeod's calorimeter. Air was continuously circulated through a drying column (CaSO4 Drierite), a respiratory chamber 2.5 1 and CO 2 trap (soda lime), by a peristaltic p u m p at a rate of 2 l / m i n . A 1-1 cylindrical metal bell fit in a concentric cylinder filled with water forming an air-tight seal served as the O 2 reservoir. The 5-ml graduated cylinder was connected to the respiratory chamber. Respiratory chamber temperature was maintained constant at 29°C by circulating water, and was monitored by an internal thermometer. The volume of O 2 consumed by each animal was corrected for temperature and pressure and was expressed as ml of 0 2 / m i n / k g b.w. 0.75 [18].
149
After recovery, six animals (1st group) were anesthetized with urethane (1.2 g / k g b.w.i.p.) and m o u n t e d in a stereotaxic instrument (Stoelting). The level of anesthesia was kept constant on a stage III, plane 1 level as evaluated by skeletal muscle relaxation, eye and palpebral responses to stimuli [26]. Nerve activity was recorded by small nerve bundles dissected from the nerve branches innervating the right side of IBAT. Nerve filaments were isolated from the central cut end of these nerve bundles under a dissecting microscope to record the efferent activity with a pair of silver wire electrodes. The nerve filaments were covered with a mixture of vaseline and liquid petroleum at 37°C to avoid dehydration. The firing rate was recorded 30-40 min (base-line period) before i.c.v, injection of 400 ng P G E t and 40 min after injection of pyrogen. The PGE1, dissolved in a vol. of 5 p.1, was delivered into the left cerebral ventricle by gravity flow over 1 min, using a cannula 0.4 m m longer than the guide cannula. Furthermore, Tc and TmAT were monitored at the same time as the nerve activity record. Tc was measured by inserting the thermocouple into the colon at 7 cm from the anus, while TmAT was monitored by inserting the thermocouple in the left side of IBAT estimated region. T h e application of 3.3 M KC1 solution upon the frontal cerebral cortex (which causes CSD) [2] preceded the i.c.v. injection by 10 min. The same variables at the same time were recorded in the other six animals (2nd group), but instead of KCI solution 3.3 M NaCI solution was applied to the cortex, which did not affect the cortical functions [2]~ The same procedure as the 1st group was carried out in the other six animals (3rd group) except for higher drug dose, using 800 ng of P G E r A n o t h e r group of six rats (4th group) was treated like the animals of the 2nd group, using 800 ng of pyrogen. The base-line values of Tc from all animals used were maintained constant by a heating pad. The electrical energy supplied to which was not altered during the experimental period. The second 24 rats were anesthetized with urethan (1.2 g / k g b.w.) and divided in four groups as the first 24 animals. In six animals (5th group), 0 2 consumption was measured over 30-40 min (base-line values) before i.c.v, injection of 400 ng P G E 1 and 40 min after injection of the drug. The application of 3.3 M KCI solution upon the frontal cortex preceded the i.c.v, injection by 10 min. The same variable at the same time was recorded in the 6th group, but KCI solution was substituted for 3.3 M NaCI solution. The same procedure as the 5th group was carried out in the 7th group, using 800 ng of P G E 1. The rats of the 8th group were treated like the animals of the 6th group, using 800 ng of pyrogen.
2.5. Preliminary experiments 2.3. Drug P G E I was purchased from Sigma (St Louis, MO). Doses of 400 and 800 ng were dissolved in 5 p,l of 0.9% NaCI sterile pyrogen-free solution.
2. 4. Procedure The animals were anesthetized with i.p. pentobarbital (50 m g / k g b.w.) and a 20-gauge stainless guide cannula was positioned stereotaxically [22] over the left lateral cerebral ventricle (1.5 m m lateral to the midline, 1.3 m m posterior to the bregma, 3.0 m m from the cranial theca). Stylets were inserted into the guide tubes and removed only when drug administration occurred. In addition, two symmetrical craniotomies (4 m m in diameter) were performed in the frontal bone and two polyethylene wells were placed upon the frontal cortex. These wells were filled with saline solution and capped until the experiment began. The guide cannulas and polyethylene wells were secured to the skull by m e a n s of screws and dental cement. Rats were given 7-10 days to recover from surgery as judged by recovery of preoperative body weight.
In preliminary experiments, we used four groups of six rats to test the effect of 3.3 M KC1 or 3.3 M NaCl solution alone (without the pyrogen injection) on the cortical electroencephalogram (EEG), the slow potential and the variables considered above (firing rate of nerves to IBAT, T¢, TIBAT and O 2 consumption). The surgical preparation was similar to that described above. After recovery, two groups were anesthetized with urethan (1.2 g / k g b.w.). Firing rate of nerves to IBAT, Tc and TIBAT were recorded 30-40 min before and 40 min after the i.c.v, injection of 5 p,l of 0.9% NaCl sterile solution. The i.c.v, injection was preceded by an application of 3.3 M KCI (in the first six rats) or 3.3 M NaCl (in the second six animals) solution upon the frontal cortex. In addition, E E G and slow potential changes were recorded at the same time with two wick calomel cell electrodes applied on the parietal cortex. This was previously exposed before the experiment. The reference electrode was placed on the frontal bone. The three electrodes were connected to a Beckman R-611 dynograph recorder. The last two groups of rats were used to test the effect of KCI or NaC1 solution, applied upon the cortex, on the 0 2 consumption. After surgical recovery, 0 2 consumption was measured in the ure-
150
M. Monda et al. / B r a i n Research 651 (1994) 148-154
CHANGES IN FIRING RATE OF IBAT NERVES
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Fig. 1. Means_+ S.E. of changes in firing rate of nerves innervating interscapular brown adipose tissue. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10. than-anesthetized rats for 30-40 min before and 40 min after i.c.v. injection of 5 /xl of 0.9% NaCI sterile solution. The i.c.v, injection was preceded by an application of 3.3 M KCI or 3.3 M NaCI solution. 2.6. Histology At the end of the experiment the rats were injected with an overdose of pentobarbital (500 m g / k g b.w.) and the location of the cannula was identified histologically. The animals were perfused with 0.9% NaCI followed by 10% (v/v) formalin solution. The brain was removed and stored in formalin solution. After few days, 50 /zm coronal sections of the fixed brain were cut and stained with Cresyl violet.
2. 7. Statistical analysis The values are presented as means_+ S.E. of difference starting at the i.c.v, injection. Statistical analysis was performed using factorial A N O V A for repeated m e a s u r e m e n t s [7].
101
i0 min
Fig. 2. Firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 400 ng P G E p
400 and 800 ng PGE r The pyrogen injection elicited increases in all groups, with the peak at 10 min. This enhancement was attenuated in the decorticated groups. A N O V A showed a significant effect of CSD (Fl,~0 = 9.582, P < 0.01), of d o s e (F1,2o = 17.067, P < 0.01) and of time (F8,16 0 = 61.672, P < 0.01). The absolute value at the P G E 1 injection time is illustrated in Table 1. Fig. 2 shows an example of firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 400 ng PGEL. Another example of firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 800 ng PGE~ is reported in Fig. 3. The effect of 400 and 800 ng PGE~ i.c.v, injection on IBAT temperature in control and decorticated animals is illustrated in Fig. 4. CSD reduced the increase 2O %
3. Results Fig. 1 shows the changes in firing rate of IBAT nerves in control and decorticated rats receiving i.c.v. Table 1 Absolute values_+ S.E. of firing rate (FR) ( s p i k e s / 2 s), temperature of brown adipose tissue ( T m a T) (°C), colonic temperature (Tc) (°C) and oxygen consumption (O z) ( m l / m i n / k g bw 0.75) at time of pyrogen injection Group
2nd
3rd
4th
Tc
11.31_+2.42 36.22_+0.11 37.21_+0.14
10.53_+1.84 36.15_+0.13 37.16_+0.18
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Group
5th
6th
7th
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Absolute values at time of pyrogen administration 1st
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Fig. 3. Firing rate changes of nerves to IBAT in a control (panel A) and decorticated (panel B) rat receiving i.c.v. 800 ng PGE~.
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M. Monda et al. /Brain Research 651 (1994) 148-154
CHANGES IN IBAT TEMPERATURE
CHANGES IN OXYGEN CONSUMPTION ml/mln/bw'7S
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Fig. 4. Means+S.E. of changes in temperature of interscapular brown adipose tissue. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10.
in TIBAT. In the control rats, the p e a k was obtained at 15 and 20 min, respectively, for 400 and 800 ng. In the decorticated rats, the m a x i m u m value was at 20 and 25 min, respectively, for low and high dose. A N O V A showed a significant effect of C S D (Fl,20 = 16.690, P < 0.01), of d o s e (F1,20 = 18.856, P < 0.01) and of time (F8,160 = 52.653, P < 0.01). T h e absolute value at P G E 1 injection time is shown in Table 1. Fig. 5 illustrates the changes in Tc of rats receiving 400 and 800 ng P G E 1 i.c.v, in the presence or absence of CSD. In the rats with intact cortex, P G E 1 injection caused a rapid increase in Tc that p e a k e d at 15 min postinjection for 400 and 800 ng. In contrast, C S D a t t e n u a t e d the PGE~-elicited t e m p e r a t u r e increase
CHANGES IN COLONIC TEMPERATURE oc
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Fig. 6. Means + S.E. of changes in oxygen consumption. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10.
with both doses. A N O V A showed a significant effect of C S D (F1,20 = 18.711, P < 0 . 0 1 ) , of dose (F1,20 = 14.267, P < 0.01) and of time (Fs,160 = 40.792, P < 0.01). T h e absolute value at P G E 1 injection time is reported in Table 1. Changes in 0 2 c o n s u m p t i o n are illustrated in Fig. 6. T h e C S D r e d u c e d the upshifted metabolic rate with both doses of P G E r A N O V A showed a significant effect of C S D (F1,20 = 36.154, P < 0.01), of dose (F1,20 = 57.107, P < 0.01) and of time (F8,16 0 = 46.149, P < 0.01). T h e absolute value at P G E 1 injection time is r e p o r t e d in Table 1. Figs. 7 and 8 show E E G and slow potential changes induced by the application of 3.3 M KC1 or NaC1 solution u p o n the frontal cortex. KC1 modified the electrical activity of the cortex. This was not affected by the NaCI solution. The a p p e a r a n c e of r e p e a t e d changes of slow potentials indicated episodes of spreading depression. These waves persisted for the duration of all experiment. T h e effect of C S D alone, tested in the preliminary experiment, on firing rate of I B A T nerves, Tc and TIBAT temperatures and 0 2 consumption are not presented in the figures, because C S D alone did not modify these parameters.
,
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4. Discussion C80400
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Fig. 5. Means + S.E. of changes in colonic temperature. Pyrogen (400 or 800 ng) was injected at t = 0. KCI or NaCI solution was applied to cortex of decorticated (CSD) or control (Contr) rats at t = - 10.
O u r experiment is one of the first to show that C S D reduces the t h e r m o g e n i c changes in anaesthetized rats during P G E 1 fever. Indeed, o u r previous findings [21] showed that C S D blocks P G E 1 fever in non-anaesthetized rats. Anaesthesia with u r e t h a n maintains the rat's
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M. Monda et al. /Brain Research 651 (1994) 148-154
2mV AO
~oopv AO
1
2mVBo
tni
n
~--
},O0~V BO
Fig. 7. EEG and slow potential changes induced by application of 3.3 M KCI solution to frontal cortex. Electrode position (O,A,B) and holes for KCI application (A',B') are shown in inset brain diagram.
ability to develop fever [20] and removes the possible interferences due to motor activity. The significant role of BAT in the fever induced by PGE 1 [11] is confirmed. BAT is a major effector of non-shivering thermogenesis [14] and diet-induced thermogenesis [23] in rodents and is therefore involved in the maintenance of body temperature and energy
balance. The activity of BAT is controlled by the sympathetic nervous system [19], and factors, which influence thermogenesis, appear to act on the central nervous system to modify the sympathetic outflow to BAT [24]. Throughout this experiment, we report direct evidence of increased sympathetic tone in nerves innervating BAT after i.c.v. PGE1 injection. The
2iv AO
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m~n
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zooHv BO
Fig. 8. EEG and slow potential changes induced by application of 3.3 M NaC1 solution to frontal cortex. Brain diagram is the same of Fig. 7.
M. Monda et al. /Brain Research 651 (1994) 148-154
changes in firing rate of BAT nerves precedes the change in temperature, confirming the important role of the sympathetic nervous system on BAT activity [13]. The actual TmAT is below Tc in agreement with the results of other experiments [6,20]. Changes in 0 2 consumption and Tc during the fever in urethan-anaesthetized rats are dose-dependent and in agreement with the literature [20], corroborating the validity of this experimental model in investigating febrile reactions. CSD reduces the fever induced by PGE 1 and this effect depends on the pyrogen dose. On the evidence from these experiments we cannot draw any conclusions about heat loss, but we can say that cortex depression influences the thermogenic events during the fever evoked by PGE~. In other words, these findings are the first to show that CSD reduces 'core' body temperature through reduced neuronal activation of BAT as a means of reducing 0 2 consumption. Depression of the cortex acts on the pyrogen response by reducing the activity of sympathetic nervous system. This effect on sympathetic activity has also been shown with other experimental models. Bilateral lesions of hypothalamic nuclei cause a marked increase in heat production with hyperthermia for many hours [15] and this phenomenon is mediated by the adrenergic system [4]. We showed that like beta-blockers CSD impairs the thermogenic increases following electrolytic damage of the lateral hypothalamus in rats [6]. In the present experiment, CSD does not modify body temperature and metabolic rate under basal conditions, the effect occurs only after pyrogen administration. This suggests a phasic influence of the cerebral cortex in rising thermogenesis due to PGE~ and seems to exclude a tonic cortical activity on thermogenic mechanisms. The cerebral cortex may participate in adaptative responses by activating the sympathetic nervous system when body temperature is far from the thermal set point, as well as after injection of pyrogens or after lesioning of the lateral hypothalamus. Conversely, the cortex is not involved when the body temperature is close to the set point. A less likely interpretation would be that the cortex per se does not cause such an effect, but these changes are caused by secondary or tertiary effect of CSD through cortical-subcortical neural pathways. Regardless of its cause, this is why CSD is able to attenuate increased heat production via activation of the cephalic sympathetic system. CSD is a peculiar reaction of the cerebral cortex to various depolarizing stimuli. It is characterized by the depression of electroencephalographic activity and by the appearance of surface negative slow potential change [2]. This cortical reaction involves the whole cortex arising from the stimulus area and, for this reason, the area of the cortex implicated in the control
153
of the febrile reaction cannot be located with precision. The prefrontal cortex is a likely candidate. There is evidence showing a convergence of skin and hypothalamic temperature signals on the prefrontal cortex [25]. Furthermore, we demonstrated that electrical stimulation of the prefrontal cortex enhances the metabolic rate through sympathetic nervous system activation and these effects are not evident after stimulation of the parietal or occipital cortex [5]. Finally, there is anatomical evidence showing pathways from the prefrontal cortex to the basal forebrain [12]. In conclusion, these findings suggest that the cerebral cortex is certainly involved in non-shivering thermogenesis, through the control on sympathetic activity during febrile reaction evoked by PGEt.
Acknowledgments. This study was supported by Italian National Research Council Grant 89.299.75.
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[12] Gaykema, R.P.A., Van Weeghel, R., Hersh, L.B. and Luiten, P.G.M., Prefrontal cortical projections to the cholinergic neurons in the basal forebrain, J. Cornp. NeuroL, 303 (1991) 563-583. [13] Geloen, A. and Trayhurn, P., Regulation of the level of uncoupling protein in brown adipose tissue by insulin requires the mediation of the sympathetic nervous system, FEBS Lett., 267 (1990) 265-267. [14] Girardier, L., Brown fat: an energy dissipating tissue. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenesis, Chapman and Hall, London, UK, 1983, pp. 50-98 [15] Grijalva, C.V., Aphagia, gastric pathology, hyperthermia and sensomotor dysfunction following lateral hypothalamic lesions: effects of insulin pretreatment, Physiol. Behal'., 25 (1980) 931937. [16] Himmis-Hagen, J., Non-shivering thermogenesis, Brain Res. Bull., 12 (1984) 151-16/). [17] Hori, T., Shibata, M., Kyohara. T. and Nakashima, T., Prefrontal cortical influences on behavioural thermoregulation and thermosensitive neurons, J. Therm. Biol., 9 (1984)27-31. [18] Kteiber, M. (Ed.), The Fire of Life. An hztroduction to Animal Energeti~s, R.E. Kreiger Publishing Company, Huntington, 1975. [19] Landsberg, L. and Young, J.B., Autonomic regulation of thermogenesis. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenesis, Chapman and Hall, London. UK, 1983, pp. 99-140.
[20] Malkinson, T.J., Cooper, K.E. and Veale, W.L., Physiological changes during thermoregulation and fever in urethananaesthetized rats, Am. Z Physiol., 255 (1988) R73-R81. [21] Monda, M. and Pittman, Q.J., Cortical spreading depression blocks prostaglandin E] and endotoxin fever in rats, Am..L PhysioL, 264 (1993) R456-R459. [22] Paxinos, G. and Watson, C. (Eds.), The Rat Brain in Stereotaxic Coordinates, Academic, New York, NY, 1986. [23] Rothwell, N.J. and Stock, M.J., Diet-induced thermogenesis. In L. Girardier and M.J. Stock (Eds.), Mammalian Thermogenes&. Chapman and Hall, London, UK, 1983, pp. 208-233. [24] Rothwell, N.J. and Stock, M.J., Sympathetic and adrenocorticoid influence on diet-induced thermogenesis and brown fat activity in the rat, Comp. Biochem. PhysioL, 79A (1984) 575-579. [25] Shibata, M, Hori, T., Kiyohara, T. and Nakashima, T., Convergence of skin and hypothalamic temperature signals on the sulcal prefrontal cortex in the rat, Brain Res., 443 (1988) 37-46. [26] Soma, L.R. (Ed.), Textbook o]" Veterinary Surgeo', Depth O[ GeneralAnesthesia, Williams and Wilkins, Baltimore, MD, 1971, pp. 178-187. [27] Stitt, J.T., Prostaglandin E as the neural mediator of thc febrile response, Yale J. Biol. Med., 59 (1986) 137-149. [28] Stitt, J.T., Fever versus hyperthermia, b),d. P~vc., 38 (1979) 39-43.