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Thermogenetic changes following frontal neocortex stimulation
Brain Research Bullerin, Vol. 22, pp. 1003-1007.
0361-9230/89
0 Pergamon Press plc. 1989. Printed in the U.S.A.
$3.00 + 00
Thermogenetic Changes Following Frontal Neocortex Stimulation B. DE LUCA,’
M. MONDA,
S. AMARO
AND M. P. PELLICANO
Institute of Human Physiology and Biomedical Physics, ‘ ‘Filippo Bottazzi, ” University of Naples, 80138 Naples, Italy Received
12 May
1988
DE LUCA, B., M. MONDA, S. AMARO AND M. P. PELLICANO. Thermogenetic changesfollowingfronra[ neocortex stimulation. BRAIN RES BULL 22(6) 1003-1007, 1989.-Heat production changes were recorded in anesthetized female Sprague-Dawley rats after stimulation of orbital frontal neocortex. The results obtained show that orbital frontal neocortex stimulation significantly increases oxygen consumption, and core and brown adipose tissue temperature. The increase was more substantial after stimulation of left than right cortex. Administration of the P-blocker propranolol abolished the increase in 0, consumption, core and brown adipose tissue temperature following cortical stimulation. These results are in agreement with our previous research showing that functional ablation of cerebral cortex blocked the increase in thermogenesis following lateral hypothalamic lesion. These findings also show that the orbital frontal neocortex in rats is specifically involved in the control of thermogenesis. Brown adipose tissue
Orbital frontal neocortex
Oxygen consumption
Propranolol
Stimulation
Temperature
(4). Kolb et al. reported that orbital frontal neocortex lesioning, induced before lateral hypothalamic lesion, leads to a faster recovery in rats (16). In a previous work we reported that the functional ablation of the cerebral cortex in anesthetized rats induced by cortical spreading depression (CSD, a specific reaction of the cerebral cortex to various depolarizing stimuli) blocks the increase of 0, consumption and of central and brown adipose tissue temperatures following lateral hypothalamic damage. This block is similar to that obtained after propranolol administration (9). CSD is a phenomenon involving the whole cortex, and as reported by Kolb et al. (16), frontal cortex lesion induces changes in food intake and in body weight regulation, with the orbital frontal neocortex being particularly involved in recovery from lateral hypothalamic lesions. In addition, Shibata et al. (26) reported that when single waves of CSD entered the frontal cortex, the firing rate of ipsilateral preoptic and anterior hypothalamic thermosensitive neurons was changed in urethane anesthetized rats. The same authors (27) found that the prefrontal neurons in similarly anesthetized rats were affected by changes in scrotal skin and hypothalamic temperature. Therefore the present experiment was carried out to further evaluate the role of the cortex in affecting metabolic heat production and the final efferent pathway of this effect. Specifically, the experiment investigated: 1) the changes in 0, consumption and brown adipose tissue and colonic temperature after stimulation of left and right orbital frontal neocortex, and left parieto-occipital cortex; 2) the changes in thermogenic response after P-blocker propranolol administration.
ENERGY balance is regulated by both energy intake and energy expenditure. Underfed rats have a decreased metabolic rate (12), while rats exposed to cold or overfed reveal an increased energy expenditure (23,24). Experimental manipulations of the hypothalamus lead to changes in the energy balance; lesioning the medial hypothalamus induces an increase in food intake and a decrease in energy expenditure (5), while lateral hypothalamic injury leads to aphagia and adipsia and an increase of energy expenditure at least for the first 24 hours after lesion (28). This effect is mediated by sympathetic activation since it is blocked by the P-blocker propranolol (6). It is also reported that acute stimulation of the hypothalamus, particularly of the medial hypothalamus, increases both 0, consumption and respiratory quotient (1). Perkins et al. (19) showed that brown adipose tissue thermogenesis, monitored by recording the tissue temperature, was increased after stimulation of the ventromedial hypothalamic area, and this increase disappeared after propranolol administration. An increase in brown adipose tissue temperature was obtained by Holt et al. (13) after stimulating the ventromedial hypothalamic nucleus in lean and obese Zucker rats. The regulatory role of the hypothalamus on energy balance is not a characteristic peculiar to this region; examples of other subcortical regions involved in this balance are the entopeduncular nucleus (10) and the globus pallidus (18). The cerebral cortex also affects thermoregulation; indeed, surgically decorticated dogs, cats (20) and monkeys (8) show a failure to maintain body temperature when exposed to cold or warm environments. Rats that survive complete or anterolateral neocortical ablation display aphagia and adipsia followed by a recovery sequence similar to that found in lateral hypothalamus injured rats
‘Requests for reprints should be addressed to Bruno De Luca, M.D., Institute of Human Physiology S. Andrea della Dame, 8, 80138 Naples, Italy.
1003
and Biomedical
Physics,
University
of Naples,
DE LUCA E7‘ ,<\I.
FIG. 1. Coronal section of rat brain. The small electrolytic
METHOD
lesions and blue iron complexes
show the sites of lesioninp.
connected to a Beckman R11 dynograph.
Animals Adult female Sprague-Dawley rats, bred in the laboratory, were used throughout the experiment. They had a body weight ranging from 200 to 250 g. During the experiment the rats were housed singly in plastic cages, in a controlled room kept at 22 4 1°C with 70% humidity and a 12 light-dark cycle. All the experiments were performed during the light period of the circadian cycle. Unless otherwise stated, pellets and water were available in the cage at all times. Apparatus Oxygen consumption was measured in a closed circuit apparatus, an adaptation of the Benedict and McLeod calorimeter (3). The temperature in the respiratory chamber was maintained constant by circulating water. Oxygen consumption was obtained by taking the difference between two readings made every 5 min. The volume of 0, consumed corrected for temperature and pressure was expressed as ml of O,/min/kg’-‘* b.wt. (15). The temperature of the respiratory chamber was kept constant at 29°C. Sine wave stimuli were delivered by an autotransformer. The current was monitored with an oscilloscope indicating the voltage drop across a known resistor placed in series with the animal. Colonic and brown adipose tissue temperatures (Tc, Tbat) were recorded with a thermistor (Yellow Springs Instrument)
One week before the experiment the animals of the first group were anesthetized with ether, were fixed in a stereotaxic apparatus with lambda 1 mm lower than the bregma, and two openings were drilled on symmetrical points of the frontal cortex. Two twisted stainless steel wires 125 km in diameter coated with teflon were subsequently positioned at the following coordinates: 4.5 anterior to the bregma, 3.7 lateral to midline and 3.5 below the surface of the skull (11). Before insertion in the brain the wires were soldered to a 4 pin miniature socket. An anchoring bolt was placed in the frontal bone and the whole implant was fixed to the skull with acrylate. Rats of a second group were implanted with bipolar electrodes in the left parieto-occipital cortex at the following coordinates: 5.0 mm posterior to bregma, 3.7 mm lateral to midline and 1.7 mm from the surface of the skull. Subsequently a second experiment was performed on a new group of rats (third group), using the same procedure as the first group, but the animals were implanted in the left hemisphere only. Procedure The animals of the first group, deprived of food for 24 hr and anesthetized with urethane 0.9 g/kg b.wt., were put in the
1005
THERMOGENESIS AND CORTICAL STIMULATION TABLE 2
TABLE 1 Time (mm)
Time (mm)
0
5
10
15
20
2.5
0
5
10
15
20
25
13.89 20.70
11.77 20.86
11.44 -co.79
9.97 20.54
10.78 20.82
COlttr.
(N= 14)
9.85 kO.36
(N=9)
9.80 to.32
13.34 20.51
11.78 kO.81
11.10 1-0.61
11.77 to.65
10.32 rto.50
Dx (N= 14)
9.48 50.53
11.23 ~~0.84
9.88 kO.57
9.03 20.63
9.20 21.14
8.90 rtO.88
Propr. (N=9)
8.54 kO.55
9.20 20.91
9.54 21.04
8.62 eO.60
8.72 20.56
8.16 kO.45
occ (N= 14)
9.77 20.24
10.28 20.25
10.13 20.23
9.55 20.27
9.88 to.32
9.51 rto.33
Tbat Contr. fN=9)
35.57 to.22
35.98 to.21
35.82 to. 17
35.66 20.19
35.56 to.21
35.48 kO.21
Tbat Sn (N= 14)
36.04 20.22
36.47 20.23
36.24 to.20
36.20 r0.19
36.19 to.20
36.10 rto.21
Propr. (N=9)
34.97 to.36
34.91 to.37
34.93 50.34
34.83 -to.33
34.81 20.33
34.73 20.32
(N= _ 14)
occ
35.97 kO.18 35.87
36.23 kO.21 35.90
36.06 20.16 35.81
35.98 rtro.15 35.86
35.94 20.15 35.85
35.93 to.17 35.84
Corm. (N=9)
36.02 20.13
36.08 kO.10
36.10 to.12
36.03 to.10
35.94 to.13
35.91 20.14
(N=14)
20.14
20.18
20.21
co.19
20.19
r0.20
FYopr.
35.54 lr0.38
35.43 to.36
35.42 kO.33
35.38 to.33
35.36 to.32
35.30 k0.31
0,
4 Sn
DX
Tc
fN=9) IC
Sn
36.51
36.74
36.70
(N=14)
kO.21
kO.19
ltrO.20
to.13
36.49
20.22
36.64
20.23
36.54
Dx (N=14)
36.37 20.30
36.54 20.30
36.33 20.31
36.29 20.29
36.22 20.30
36.18 +-0.29
occ (N=14)
36.18 kO.15
36.14 zkO.14
36.08 20.15
36.08 kO.14
36.06 k0.13
35.98 k0.16
Means 2 SEM of changes over time of O2 consumption (ml/min/kg b.wt.‘.“), brown adipose tissue temperature (Tbat, “C) and colonic temperature (Tc, “C), after stimulation of left (Sn) or right (Dx) orbital frontal cortex or occipital cortex @cc). Time 0= baseline value, N= number of animals.
metabolic chamber and two pins of the socket were connected to the stimulator. One thermistor probe was inserted into a small incision of the skin in the inter-scapular region and secured with a stitch to the skin; the other was inserted 7 cm into the anus of the rat. The 0, consumption, Tbat and Tc were recorded for 60 mitt (base-line period); after that the animals were stimulated for 20 set with sine wave current 50 Hz, 300 PA. The 0, consumption was recorded every 5 minutes, and temperatures were recorded continuously for 25 min. Five days later all measurements were repeated with the same procedure, but the stimulus site was the opposite hemisphere. The rats were divided into two subgroups. Subgroup 1 was fiit stimulated in the left hemisphere, while subgroup 2 in the right hemicortex. The data of the two subgroups were pooled together, since no significant differences were found between subgroups 1 and 2. At the end of the two stimulations small electrolytic lesions were made to mark the stimulus sites. Then the rats were injected with an overdose of Nembutal and were intracardially perfused with physiological saline followed by phosphate buffered formalin containing 5% (w/v) potassium ferrocyanide. The brains were removed, left in formalin for 10 days, and serial 50 Frn thick coronal frozen sections were then made on a Leitz microtome. Sections were subsequently stained with cresyl violet. The animals of the second group implanted in the parietooccipital cortex were similarly stimulated, and O2 consumption, Tc and Tbat were recorded in the same way.
Means it SEM of changes over time of 0, consumption (ml/minkg b.wt..75), brown adipose tissue (That, “C) and colonic temperature (Tc, “C) after stimulation of orbital frontal cortex in control (Contr.) and propranolo1 (Propr.)-treated rats. Time 0 = baseline value, N = number of animals.
The rats with the stimulating electrodes in the left hemisphere only (third group), after the same fasting period, were anesthetized with urethane and were stimulated like the rats of the Fist group, following a base-line recording of 30 min; changes in 0, consumption, Tc and Tbat were similarly recorded for 25 min. Five days later the same procedure was repeated, with the exception that propranolol 10 mg&g b.wt. was injected intraperitoneally 15 min after anesthetic administration. After a recording period of 30 min the animals were stimulated in the left hemisphere only, and changes in O,, Tc and Tbat were recorded for 25 min. Subsequent procedures were the same as those for the fist group.
Results were expressed as mean 2 SEM; statistics were computed using analysis of variance for repeated measurements: multiple comparisons were performed by Newman-Keuls post hoc tests (29). RESULTS
Histological analysis of the sections reveals that the electrodes were well positioned in the orbital frontal neocortex. Figure 1 reports a coronal section, stained with cresyl violet, showing the blue brown complex developed at the stimulation points. The effect of electrical stimulation of the left and right orbito~ont~ cortex on 0, consumption, Tc and Tbat are shown in Table 1, while a recording of both temperatures is shown in Fig. 2. The changes in both temperatures and O2 consumption after electrical stimulation in the left or right orbitofrontal cortex or in parieto-occipital cortex are shown in Fig. 3. The analysis of variance on the changes of 0, consumption shows a significant effect of stimulation site, F(2,39)=3.640, pcO.05, and of time, F(5,195)=9.629, p
1006
DE LUCA ET AL.
AT
PIG. 2. Brown adipose tissue and colonic temperaturerecordings before and after left orbital frontal cortex stimulation. S = stimulation.
hoc tests show significant differences between left frontal and parieto-occipital cortex and between the basal value and that recorded 5 min after stimulation in the right cortex, and between basal value and those obtained at 5 and 10 min after stimulation in the left cortex. No significant differences were found after parieto-occipital cortex stimulation. ANOVA performed on the changes of Tbat shows the significant effect of time, F(5,195) = 3.3 11, p
AT dC 0.5 f% 4
0.3
_.
0
2 0
I
05
,
15 min
,
25
__
05
15
r
--is--
min
FIG. 3. Changes in 0, consumption, brown adipose tissue and colonic temperatmz(Tbat, Tc) after left or right orbital frontal cortex or pa&tooccipitalcortex stimulation, 0,: A-left, O-right, Ci-parieto-occipital; T: A-That left, 0-Tbat right, q -That parieto-occipital;A-Tc left, l -Tc right, n -Tc par&o-occipital.
‘C
0 5
15
25 min
05
15
25 min
FIG. 4. Changes in 0, consumption, brown adipose tissue and colonic temperature (Tbat, Tc) after left orbital-frontal cortex stimulation with (P+) or without (P-) propranoiol injection, 0,: A-P-, O-P+; T: A-Tbat P-, C-Tbat P+, A-Tc P-, l -Tc P+.
propranolol is between the basal and the 10 min values. Statistical analysis on the Tbat reveals a significant difference for time, F(5,80) = 5.571, p
The results obtained in the first experiment show that the orbital frontal neocortex is involved in the control of thermogenesis. In fact, thermogenesis is specifically dependent on the orbital frontal stimulation, since the stimulation of the parieto-occipital cortex produced no changes in the parameters measured. The enhancement of heat production is due, at least in part, to the activation of brown adipose tissue. Indeed, both groups of rats showed an immediate increase in 0, consumption, Tc and Tbat following stimulation, and between the two temperatures the increase was larger for Tbat man for Tc. The changes in all three parameters were more substantial after left than after right orbital frontal stimulation. The increase in 0, consumption after orbital frontal neocortex stimulation is similar to that found by Atrens ef at. (1) after hypo~~~ic simulation. The changes in Tbat are more similar to those obtained by Perkins (19) aFter stimulation of ventromedial hypothalamus than to those of Holt et al.(13)who stimulated the same structure in urethane anesthetized rats. Nevertheless, heat production following cortical stimulation is not in agreement with the previously cited results obtained by Shibata et ~2. (26), who found that when a single wave of CSD, elicited in one hemisphere, entered the frontal cortex, the firing rate of preoptic anterior hypothaIamic warm-sensitive neurons was decreased, while that of cold-sensitive neurons in the same regions was increased. Our results obtained after cortical stimulation, consistent with our previous findings obtained after application of bilaterat prolonged CSD, differ from those of Shibata’s group who recorded unit activity fobowing eliciting of a single wave of CSD. Therefore, taking into account the diverse techniques used in functional cortical ablation, both groups produced thermoregulation results consistent with consummatory behavior results. In-
1007
THERMOGENESIS AND CORTICAL STIMULATION
deed, whereas the bilateral repeated waves of CSD (7) cause inhibition of eating, single waves of unilateral CSD (14) are accompanied by an activation of feeding and drinking. The difference in thermogenesis after left or right orbital frontal cortex stimulation could be due to the functional heterogeneity between the two hemispheres also observed in other regulatory activities (2). The administration of propranolol before cortical stimulation completely prevents the increase in 0, consumption, Tc and Tbat. This last finding agrees with the results of Perkins (19), who did not find any changes in Tbat following medial hypothalamic stimulation in rats pretreated with propranolol and demonstrates that the sympathetic nervous system is the final common path for both structures. The administration of P-blocker does not significantly decrease the basal level of all considered values; this finding is in agreement with previous experiments performed by our group (9) and by others (6). The present results were obtained in rats anesthetized with urethane, to avoid heat production due to shivering, because we were interested in the cortical influence on metabolic heat production. Urethane is widely utilized in acute experiments for its minor changes in respiration, small influence on the neuronal activity (17) particularly at the level of the rat frontal cortex (21) and, as
recently reported, for the lack in metabolic changes with doses less than 1.4 g/kg b.wt. (25). The same drug is not commonly utilized in chronic experiments because of its prolonged anesthesia and for its slow breakdown. Nevertheless, in the present experiment it was found that the changes in the values considered following orbital frontal cortex stimulation were no different after the second than after the first urethane anesthesia, with 5 days interval between experiments. In conclusion, the results of the present experiment confirm our previous data about the involvement of the cerebral cortex in the enhancement of thermogenesis. They also demonstrate that the orbital frontal neocortex, which is also involved in brain stimulation rewards (22) as well as in motivational behavior (7,14) such as feeding and drinking, also affects thermoregulation, mainly because of the activation of brown adipose tissue mediated by the sympathetic nervous system. ACKNOWLEDGEMENTS Supported by the Minister0 della Pubblica Istruzion Roma (40% grant), by the University of Naples, Naples Italy (60% grant) and by an Italian National Research Council grant.
REFERENCES 1. Atrens, D. M.; Sinden, J. D.; Penicand, L.; Devos, M.; Le Magnen, J. Hypothalamic modulation of energy expenditure. Physiol. Behav. 35:15-20; 1985. 2. Bameoud, P.; Neven, P. J.; Vitiello, S.; Le Moal, M. Functional heterogeneity of the right and left cerebral neocortex in the modulation of the immune system. Physiol. Behav. 41:525-530; 1987. 3. Benedict, F. G.; MacLeod, M. The heat production of the albino rat. J. Nutr. 1:346-366; 1928. 4. Braun, J. J. Neocortex and feeding behavior in the rat. J. Comp. Physiol. Psychol. 89:507-522; 1975. 5. Bray, G. A.; York, D. A. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis. Physiol. Rev. 59:719-809; 1979. 6 Corbett, S. W.; Kaufman, L. M.; Keesey, R. E. Effects of Badrenergic blockade on lateral hypothalamic lesion-induced thermogenesis. Am. J. Phvsiol. 245(8):E535-E541: 1983. 7 Cytawa, J.; Teitelbaum, P. Spreading depression and recovery of subcortical functions. Acta. Biol. Exp. 27:345-353; 1967. 8 Delgado, J. M. R.; Livingston, R. B. Some respiratory, vascular and thermal responses to stimulation of orbital surface of frontal lobe. J. Neurophysiol. 11:39-55; 1948. 9 De Luca, B.; Monda, M.; Pellicano, M. P.; Zenga, A. Cortical control of thermogenesis induced by lateral hypothalamic lesion and overeating. Am. J. Physiol. 23:R62&R633; 1987. 10. De Luca, B.; Monda, M.; Amaro, S.; Pellicano, M. P. Heat production and motor deficit in rats lesioned in globus pallidus, entopeduncular nucleus and lateral hypothalamus. Physiol. Behav. 45:119-126; 1989. 11. F&ova, E.; Marsala, J. Stereotaxic atlases for the cat, rabbit and rat. In: Bures, J.; Petran, M.; Zachar, J., eds. Electrophysiological methods in biological research. New York: Academic Press; 1967. 12. Forsum, E.; Hillman, P. E.; Nesheim, M. C. Effect of energy restriction on total heat production, basal metabolic rate, and specific dynamic action of food in rats. J. Nutr. 111:1691-1697; 1981. 13. Holt, S. J.; Wheal, M. V.; York, D. A. Hypothalamic control of brown adipose tissue in zucker lean and obese rats. Effect of electrical stimulation of the ventromedial nucleus and other hypothalamic centres. Brain Res. 405:227-233; 1987. 14 Huston, J. P.: Bures, J. Drinking and eating elicited bv cortical spreading depression. Science 169:?02-704; 1970. I 15. Kleiber, M. The fire of life. An introduction to animal energetics. Huntington, New York: R. E. Krieger Publishing Company; 1975.
16 Kolb, B.; Nonneman, A. J.; Whishaw, I. R. Influence of frontal neocortex lesions and body weight manipulation on the severity of lateral hypothalamic aphagia. Physiol. Behav. 21:541-547; 1978. 17. Maggi, C. A.; Meli, A. Suitability of urethane anesthesia for physiopharmacological investigations in various system. Part 1: General considerations. Experientia 42:109-l 14; 1986. 18. Morgane, P. J. Alteration in feeding and drinking behavior of rats with lesion in globi pallidi. Am. J. Physiol. 201:420-428; 1961. 19. Perkins, M. N.; Rothwell, N. J.; Stock, M. J.; Stone, T. W. Activation of brown adipose tissue thermogenesis by ventromedial hypothalamus. Nature 289401-402; 1981. 20. Pinkston, J. 0.; Bard, P.; Rioch, D. M. The response to changes in environmental temperature after removal of portion of the forebrain. Am. J. Physiol. 109:515-531; 1934. 21. Pirch, J. H.; Corbus, M. J.; Rigdon, G. C. Conditioning-related single unit activity in the frontal cortex of urethane anesthetized rats. Int. J. Neurosci. 25:236-271; 1985. 22. Rolls, E. T.; Cooper, S. J. Activation of neurones in the prefrontal cortex by brain-stimulation reward in rat. Brain Res. 60:35 l-368; 1973. 23. Rothwell, N. J.; Stock, M. J. Energy expenditure of ‘cafeteria’-fed rats determined from measurement of energy balance and indirect calorimetry. J. Physiol. Lond. 328:371-377; 1982. 24. Rothwell, N. J.; Stock, M. J. Similarities between cold- and dietinduced thermogenesis in the rat. Can. J. Physiol. Pharmacol. 58:842-848; 1980. 25. Sanchez-Pozo, A.; Alados, J. C.; Sanchez-Medina, F. Metabolic changes induced by urethane anesthesia in rats. Gen. Pharmacol. 19:281-284; 1988. 26. Shibata, M.; Hori, T.; Kiyohara, T.; Nakashima, T. Activity of hypothalamic thermosensitive neurons during cortical spreading depression in the rat. Brain Res. 308:255-262; 1984. 27. Shibata, M.; Hori, T.; Kiyohara, T.; Nakashima, T. Convergence of skin and hypothalamic temperature signals on the sulcal p&frontal cortex in the rats. Brain Res. 443:37-46; 1988. 28. Tordoff, M. G.; Grijalva, C. V.; Novin, D.; Butcher, L. L.; Walsh, J. H.; Pi-Sunyer, F. X.; VanderWeele, D. A. Influence of sympathectomy on the lateral hypothalamic lesion syndrome. Behav. Neurosci. 98:1039-1059: 1984. 29. Winer, B. J. Statistical principles in experimental design. New York: McGraw-Hill; 1971.
0361-9230/89
0 Pergamon Press plc. 1989. Printed in the U.S.A.
$3.00 + 00
Thermogenetic Changes Following Frontal Neocortex Stimulation B. DE LUCA,’
M. MONDA,
S. AMARO
AND M. P. PELLICANO
Institute of Human Physiology and Biomedical Physics, ‘ ‘Filippo Bottazzi, ” University of Naples, 80138 Naples, Italy Received
12 May
1988
DE LUCA, B., M. MONDA, S. AMARO AND M. P. PELLICANO. Thermogenetic changesfollowingfronra[ neocortex stimulation. BRAIN RES BULL 22(6) 1003-1007, 1989.-Heat production changes were recorded in anesthetized female Sprague-Dawley rats after stimulation of orbital frontal neocortex. The results obtained show that orbital frontal neocortex stimulation significantly increases oxygen consumption, and core and brown adipose tissue temperature. The increase was more substantial after stimulation of left than right cortex. Administration of the P-blocker propranolol abolished the increase in 0, consumption, core and brown adipose tissue temperature following cortical stimulation. These results are in agreement with our previous research showing that functional ablation of cerebral cortex blocked the increase in thermogenesis following lateral hypothalamic lesion. These findings also show that the orbital frontal neocortex in rats is specifically involved in the control of thermogenesis. Brown adipose tissue
Orbital frontal neocortex
Oxygen consumption
Propranolol
Stimulation
Temperature
(4). Kolb et al. reported that orbital frontal neocortex lesioning, induced before lateral hypothalamic lesion, leads to a faster recovery in rats (16). In a previous work we reported that the functional ablation of the cerebral cortex in anesthetized rats induced by cortical spreading depression (CSD, a specific reaction of the cerebral cortex to various depolarizing stimuli) blocks the increase of 0, consumption and of central and brown adipose tissue temperatures following lateral hypothalamic damage. This block is similar to that obtained after propranolol administration (9). CSD is a phenomenon involving the whole cortex, and as reported by Kolb et al. (16), frontal cortex lesion induces changes in food intake and in body weight regulation, with the orbital frontal neocortex being particularly involved in recovery from lateral hypothalamic lesions. In addition, Shibata et al. (26) reported that when single waves of CSD entered the frontal cortex, the firing rate of ipsilateral preoptic and anterior hypothalamic thermosensitive neurons was changed in urethane anesthetized rats. The same authors (27) found that the prefrontal neurons in similarly anesthetized rats were affected by changes in scrotal skin and hypothalamic temperature. Therefore the present experiment was carried out to further evaluate the role of the cortex in affecting metabolic heat production and the final efferent pathway of this effect. Specifically, the experiment investigated: 1) the changes in 0, consumption and brown adipose tissue and colonic temperature after stimulation of left and right orbital frontal neocortex, and left parieto-occipital cortex; 2) the changes in thermogenic response after P-blocker propranolol administration.
ENERGY balance is regulated by both energy intake and energy expenditure. Underfed rats have a decreased metabolic rate (12), while rats exposed to cold or overfed reveal an increased energy expenditure (23,24). Experimental manipulations of the hypothalamus lead to changes in the energy balance; lesioning the medial hypothalamus induces an increase in food intake and a decrease in energy expenditure (5), while lateral hypothalamic injury leads to aphagia and adipsia and an increase of energy expenditure at least for the first 24 hours after lesion (28). This effect is mediated by sympathetic activation since it is blocked by the P-blocker propranolol (6). It is also reported that acute stimulation of the hypothalamus, particularly of the medial hypothalamus, increases both 0, consumption and respiratory quotient (1). Perkins et al. (19) showed that brown adipose tissue thermogenesis, monitored by recording the tissue temperature, was increased after stimulation of the ventromedial hypothalamic area, and this increase disappeared after propranolol administration. An increase in brown adipose tissue temperature was obtained by Holt et al. (13) after stimulating the ventromedial hypothalamic nucleus in lean and obese Zucker rats. The regulatory role of the hypothalamus on energy balance is not a characteristic peculiar to this region; examples of other subcortical regions involved in this balance are the entopeduncular nucleus (10) and the globus pallidus (18). The cerebral cortex also affects thermoregulation; indeed, surgically decorticated dogs, cats (20) and monkeys (8) show a failure to maintain body temperature when exposed to cold or warm environments. Rats that survive complete or anterolateral neocortical ablation display aphagia and adipsia followed by a recovery sequence similar to that found in lateral hypothalamus injured rats
‘Requests for reprints should be addressed to Bruno De Luca, M.D., Institute of Human Physiology S. Andrea della Dame, 8, 80138 Naples, Italy.
1003
and Biomedical
Physics,
University
of Naples,
DE LUCA E7‘ ,<\I.
FIG. 1. Coronal section of rat brain. The small electrolytic
METHOD
lesions and blue iron complexes
show the sites of lesioninp.
connected to a Beckman R11 dynograph.
Animals Adult female Sprague-Dawley rats, bred in the laboratory, were used throughout the experiment. They had a body weight ranging from 200 to 250 g. During the experiment the rats were housed singly in plastic cages, in a controlled room kept at 22 4 1°C with 70% humidity and a 12 light-dark cycle. All the experiments were performed during the light period of the circadian cycle. Unless otherwise stated, pellets and water were available in the cage at all times. Apparatus Oxygen consumption was measured in a closed circuit apparatus, an adaptation of the Benedict and McLeod calorimeter (3). The temperature in the respiratory chamber was maintained constant by circulating water. Oxygen consumption was obtained by taking the difference between two readings made every 5 min. The volume of 0, consumed corrected for temperature and pressure was expressed as ml of O,/min/kg’-‘* b.wt. (15). The temperature of the respiratory chamber was kept constant at 29°C. Sine wave stimuli were delivered by an autotransformer. The current was monitored with an oscilloscope indicating the voltage drop across a known resistor placed in series with the animal. Colonic and brown adipose tissue temperatures (Tc, Tbat) were recorded with a thermistor (Yellow Springs Instrument)
One week before the experiment the animals of the first group were anesthetized with ether, were fixed in a stereotaxic apparatus with lambda 1 mm lower than the bregma, and two openings were drilled on symmetrical points of the frontal cortex. Two twisted stainless steel wires 125 km in diameter coated with teflon were subsequently positioned at the following coordinates: 4.5 anterior to the bregma, 3.7 lateral to midline and 3.5 below the surface of the skull (11). Before insertion in the brain the wires were soldered to a 4 pin miniature socket. An anchoring bolt was placed in the frontal bone and the whole implant was fixed to the skull with acrylate. Rats of a second group were implanted with bipolar electrodes in the left parieto-occipital cortex at the following coordinates: 5.0 mm posterior to bregma, 3.7 mm lateral to midline and 1.7 mm from the surface of the skull. Subsequently a second experiment was performed on a new group of rats (third group), using the same procedure as the first group, but the animals were implanted in the left hemisphere only. Procedure The animals of the first group, deprived of food for 24 hr and anesthetized with urethane 0.9 g/kg b.wt., were put in the
1005
THERMOGENESIS AND CORTICAL STIMULATION TABLE 2
TABLE 1 Time (mm)
Time (mm)
0
5
10
15
20
2.5
0
5
10
15
20
25
13.89 20.70
11.77 20.86
11.44 -co.79
9.97 20.54
10.78 20.82
COlttr.
(N= 14)
9.85 kO.36
(N=9)
9.80 to.32
13.34 20.51
11.78 kO.81
11.10 1-0.61
11.77 to.65
10.32 rto.50
Dx (N= 14)
9.48 50.53
11.23 ~~0.84
9.88 kO.57
9.03 20.63
9.20 21.14
8.90 rtO.88
Propr. (N=9)
8.54 kO.55
9.20 20.91
9.54 21.04
8.62 eO.60
8.72 20.56
8.16 kO.45
occ (N= 14)
9.77 20.24
10.28 20.25
10.13 20.23
9.55 20.27
9.88 to.32
9.51 rto.33
Tbat Contr. fN=9)
35.57 to.22
35.98 to.21
35.82 to. 17
35.66 20.19
35.56 to.21
35.48 kO.21
Tbat Sn (N= 14)
36.04 20.22
36.47 20.23
36.24 to.20
36.20 r0.19
36.19 to.20
36.10 rto.21
Propr. (N=9)
34.97 to.36
34.91 to.37
34.93 50.34
34.83 -to.33
34.81 20.33
34.73 20.32
(N= _ 14)
occ
35.97 kO.18 35.87
36.23 kO.21 35.90
36.06 20.16 35.81
35.98 rtro.15 35.86
35.94 20.15 35.85
35.93 to.17 35.84
Corm. (N=9)
36.02 20.13
36.08 kO.10
36.10 to.12
36.03 to.10
35.94 to.13
35.91 20.14
(N=14)
20.14
20.18
20.21
co.19
20.19
r0.20
FYopr.
35.54 lr0.38
35.43 to.36
35.42 kO.33
35.38 to.33
35.36 to.32
35.30 k0.31
0,
4 Sn
DX
Tc
fN=9) IC
Sn
36.51
36.74
36.70
(N=14)
kO.21
kO.19
ltrO.20
to.13
36.49
20.22
36.64
20.23
36.54
Dx (N=14)
36.37 20.30
36.54 20.30
36.33 20.31
36.29 20.29
36.22 20.30
36.18 +-0.29
occ (N=14)
36.18 kO.15
36.14 zkO.14
36.08 20.15
36.08 kO.14
36.06 k0.13
35.98 k0.16
Means 2 SEM of changes over time of O2 consumption (ml/min/kg b.wt.‘.“), brown adipose tissue temperature (Tbat, “C) and colonic temperature (Tc, “C), after stimulation of left (Sn) or right (Dx) orbital frontal cortex or occipital cortex @cc). Time 0= baseline value, N= number of animals.
metabolic chamber and two pins of the socket were connected to the stimulator. One thermistor probe was inserted into a small incision of the skin in the inter-scapular region and secured with a stitch to the skin; the other was inserted 7 cm into the anus of the rat. The 0, consumption, Tbat and Tc were recorded for 60 mitt (base-line period); after that the animals were stimulated for 20 set with sine wave current 50 Hz, 300 PA. The 0, consumption was recorded every 5 minutes, and temperatures were recorded continuously for 25 min. Five days later all measurements were repeated with the same procedure, but the stimulus site was the opposite hemisphere. The rats were divided into two subgroups. Subgroup 1 was fiit stimulated in the left hemisphere, while subgroup 2 in the right hemicortex. The data of the two subgroups were pooled together, since no significant differences were found between subgroups 1 and 2. At the end of the two stimulations small electrolytic lesions were made to mark the stimulus sites. Then the rats were injected with an overdose of Nembutal and were intracardially perfused with physiological saline followed by phosphate buffered formalin containing 5% (w/v) potassium ferrocyanide. The brains were removed, left in formalin for 10 days, and serial 50 Frn thick coronal frozen sections were then made on a Leitz microtome. Sections were subsequently stained with cresyl violet. The animals of the second group implanted in the parietooccipital cortex were similarly stimulated, and O2 consumption, Tc and Tbat were recorded in the same way.
Means it SEM of changes over time of 0, consumption (ml/minkg b.wt..75), brown adipose tissue (That, “C) and colonic temperature (Tc, “C) after stimulation of orbital frontal cortex in control (Contr.) and propranolo1 (Propr.)-treated rats. Time 0 = baseline value, N = number of animals.
The rats with the stimulating electrodes in the left hemisphere only (third group), after the same fasting period, were anesthetized with urethane and were stimulated like the rats of the Fist group, following a base-line recording of 30 min; changes in 0, consumption, Tc and Tbat were similarly recorded for 25 min. Five days later the same procedure was repeated, with the exception that propranolol 10 mg&g b.wt. was injected intraperitoneally 15 min after anesthetic administration. After a recording period of 30 min the animals were stimulated in the left hemisphere only, and changes in O,, Tc and Tbat were recorded for 25 min. Subsequent procedures were the same as those for the fist group.
Results were expressed as mean 2 SEM; statistics were computed using analysis of variance for repeated measurements: multiple comparisons were performed by Newman-Keuls post hoc tests (29). RESULTS
Histological analysis of the sections reveals that the electrodes were well positioned in the orbital frontal neocortex. Figure 1 reports a coronal section, stained with cresyl violet, showing the blue brown complex developed at the stimulation points. The effect of electrical stimulation of the left and right orbito~ont~ cortex on 0, consumption, Tc and Tbat are shown in Table 1, while a recording of both temperatures is shown in Fig. 2. The changes in both temperatures and O2 consumption after electrical stimulation in the left or right orbitofrontal cortex or in parieto-occipital cortex are shown in Fig. 3. The analysis of variance on the changes of 0, consumption shows a significant effect of stimulation site, F(2,39)=3.640, pcO.05, and of time, F(5,195)=9.629, p
1006
DE LUCA ET AL.
AT
PIG. 2. Brown adipose tissue and colonic temperaturerecordings before and after left orbital frontal cortex stimulation. S = stimulation.
hoc tests show significant differences between left frontal and parieto-occipital cortex and between the basal value and that recorded 5 min after stimulation in the right cortex, and between basal value and those obtained at 5 and 10 min after stimulation in the left cortex. No significant differences were found after parieto-occipital cortex stimulation. ANOVA performed on the changes of Tbat shows the significant effect of time, F(5,195) = 3.3 11, p
AT dC 0.5 f% 4
0.3
_.
0
2 0
I
05
,
15 min
,
25
__
05
15
r
--is--
min
FIG. 3. Changes in 0, consumption, brown adipose tissue and colonic temperatmz(Tbat, Tc) after left or right orbital frontal cortex or pa&tooccipitalcortex stimulation, 0,: A-left, O-right, Ci-parieto-occipital; T: A-That left, 0-Tbat right, q -That parieto-occipital;A-Tc left, l -Tc right, n -Tc par&o-occipital.
‘C
0 5
15
25 min
05
15
25 min
FIG. 4. Changes in 0, consumption, brown adipose tissue and colonic temperature (Tbat, Tc) after left orbital-frontal cortex stimulation with (P+) or without (P-) propranoiol injection, 0,: A-P-, O-P+; T: A-Tbat P-, C-Tbat P+, A-Tc P-, l -Tc P+.
propranolol is between the basal and the 10 min values. Statistical analysis on the Tbat reveals a significant difference for time, F(5,80) = 5.571, p
The results obtained in the first experiment show that the orbital frontal neocortex is involved in the control of thermogenesis. In fact, thermogenesis is specifically dependent on the orbital frontal stimulation, since the stimulation of the parieto-occipital cortex produced no changes in the parameters measured. The enhancement of heat production is due, at least in part, to the activation of brown adipose tissue. Indeed, both groups of rats showed an immediate increase in 0, consumption, Tc and Tbat following stimulation, and between the two temperatures the increase was larger for Tbat man for Tc. The changes in all three parameters were more substantial after left than after right orbital frontal stimulation. The increase in 0, consumption after orbital frontal neocortex stimulation is similar to that found by Atrens ef at. (1) after hypo~~~ic simulation. The changes in Tbat are more similar to those obtained by Perkins (19) aFter stimulation of ventromedial hypothalamus than to those of Holt et al.(13)who stimulated the same structure in urethane anesthetized rats. Nevertheless, heat production following cortical stimulation is not in agreement with the previously cited results obtained by Shibata et ~2. (26), who found that when a single wave of CSD, elicited in one hemisphere, entered the frontal cortex, the firing rate of preoptic anterior hypothaIamic warm-sensitive neurons was decreased, while that of cold-sensitive neurons in the same regions was increased. Our results obtained after cortical stimulation, consistent with our previous findings obtained after application of bilaterat prolonged CSD, differ from those of Shibata’s group who recorded unit activity fobowing eliciting of a single wave of CSD. Therefore, taking into account the diverse techniques used in functional cortical ablation, both groups produced thermoregulation results consistent with consummatory behavior results. In-
1007
THERMOGENESIS AND CORTICAL STIMULATION
deed, whereas the bilateral repeated waves of CSD (7) cause inhibition of eating, single waves of unilateral CSD (14) are accompanied by an activation of feeding and drinking. The difference in thermogenesis after left or right orbital frontal cortex stimulation could be due to the functional heterogeneity between the two hemispheres also observed in other regulatory activities (2). The administration of propranolol before cortical stimulation completely prevents the increase in 0, consumption, Tc and Tbat. This last finding agrees with the results of Perkins (19), who did not find any changes in Tbat following medial hypothalamic stimulation in rats pretreated with propranolol and demonstrates that the sympathetic nervous system is the final common path for both structures. The administration of P-blocker does not significantly decrease the basal level of all considered values; this finding is in agreement with previous experiments performed by our group (9) and by others (6). The present results were obtained in rats anesthetized with urethane, to avoid heat production due to shivering, because we were interested in the cortical influence on metabolic heat production. Urethane is widely utilized in acute experiments for its minor changes in respiration, small influence on the neuronal activity (17) particularly at the level of the rat frontal cortex (21) and, as
recently reported, for the lack in metabolic changes with doses less than 1.4 g/kg b.wt. (25). The same drug is not commonly utilized in chronic experiments because of its prolonged anesthesia and for its slow breakdown. Nevertheless, in the present experiment it was found that the changes in the values considered following orbital frontal cortex stimulation were no different after the second than after the first urethane anesthesia, with 5 days interval between experiments. In conclusion, the results of the present experiment confirm our previous data about the involvement of the cerebral cortex in the enhancement of thermogenesis. They also demonstrate that the orbital frontal neocortex, which is also involved in brain stimulation rewards (22) as well as in motivational behavior (7,14) such as feeding and drinking, also affects thermoregulation, mainly because of the activation of brown adipose tissue mediated by the sympathetic nervous system. ACKNOWLEDGEMENTS Supported by the Minister0 della Pubblica Istruzion Roma (40% grant), by the University of Naples, Naples Italy (60% grant) and by an Italian National Research Council grant.
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16 Kolb, B.; Nonneman, A. J.; Whishaw, I. R. Influence of frontal neocortex lesions and body weight manipulation on the severity of lateral hypothalamic aphagia. Physiol. Behav. 21:541-547; 1978. 17. Maggi, C. A.; Meli, A. Suitability of urethane anesthesia for physiopharmacological investigations in various system. Part 1: General considerations. Experientia 42:109-l 14; 1986. 18. Morgane, P. J. Alteration in feeding and drinking behavior of rats with lesion in globi pallidi. Am. J. Physiol. 201:420-428; 1961. 19. Perkins, M. N.; Rothwell, N. J.; Stock, M. J.; Stone, T. W. Activation of brown adipose tissue thermogenesis by ventromedial hypothalamus. Nature 289401-402; 1981. 20. Pinkston, J. 0.; Bard, P.; Rioch, D. M. The response to changes in environmental temperature after removal of portion of the forebrain. Am. J. Physiol. 109:515-531; 1934. 21. Pirch, J. H.; Corbus, M. J.; Rigdon, G. C. Conditioning-related single unit activity in the frontal cortex of urethane anesthetized rats. Int. J. Neurosci. 25:236-271; 1985. 22. Rolls, E. T.; Cooper, S. J. Activation of neurones in the prefrontal cortex by brain-stimulation reward in rat. Brain Res. 60:35 l-368; 1973. 23. Rothwell, N. J.; Stock, M. J. Energy expenditure of ‘cafeteria’-fed rats determined from measurement of energy balance and indirect calorimetry. J. Physiol. Lond. 328:371-377; 1982. 24. Rothwell, N. J.; Stock, M. J. Similarities between cold- and dietinduced thermogenesis in the rat. Can. J. Physiol. Pharmacol. 58:842-848; 1980. 25. Sanchez-Pozo, A.; Alados, J. C.; Sanchez-Medina, F. Metabolic changes induced by urethane anesthesia in rats. Gen. Pharmacol. 19:281-284; 1988. 26. Shibata, M.; Hori, T.; Kiyohara, T.; Nakashima, T. Activity of hypothalamic thermosensitive neurons during cortical spreading depression in the rat. Brain Res. 308:255-262; 1984. 27. Shibata, M.; Hori, T.; Kiyohara, T.; Nakashima, T. Convergence of skin and hypothalamic temperature signals on the sulcal p&frontal cortex in the rats. Brain Res. 443:37-46; 1988. 28. Tordoff, M. G.; Grijalva, C. V.; Novin, D.; Butcher, L. L.; Walsh, J. H.; Pi-Sunyer, F. X.; VanderWeele, D. A. Influence of sympathectomy on the lateral hypothalamic lesion syndrome. Behav. Neurosci. 98:1039-1059: 1984. 29. Winer, B. J. Statistical principles in experimental design. New York: McGraw-Hill; 1971.