EXPERIMENTAL
NEUROLOGY
49,
174-188
Effect of Neonatal during Starvation K. E. BIGNALL, Department
Decerebration on Thermogenesis and Cold Exposure in the Rat
F. W. HEGGENESS,
of Physiology,
University
(1975)
of Rochester,
Received
AND J. E. PALMER
School of Medicine Rochester, New April
1
and Dentistry, York 14642
9, 1975
Rat pups, 5 and 10 days old, were decerebrated at the midpontine level, or the hypothalamus was removed. Their metabolic responses to cold exposure (elevated 02 consumption) was then measured after Z-10 hr of starvation, and compared with that of intact animals starved for the same length of time. The 0, uptake of the unoperated pups declined after Z-4 hr and disappeared by 8-10 hr, whereas the decerebrate rats maintained near maximal Oa consumption either in the cold or at thermoneutrality. Colonic temperatures averaged 5 C higher by 10 hr of starvation than in the intact animals. Blood glucose was also measured. It declined progressively in the intact pups, but remained near normal in the decerebrate preparations. These findings suggest that the low basal metabolism, and suppression by starvation of the metabolic response to cold exposure in the infant rat, are the result of descending inhibition of a heat generating system caudal to the transection, which is released by severing the connections. Aspiration of the hypothalamus caused only slight impairment of the responses to cold at this age, suggesting a critical thermoregulatory role for the more caudal brain stem and spinal structures early in life, with dependency on descending influences developing during maturation. A similar mechanism is also proposed to account for the maintained high blood glucose despite starvation in the decerebrate animals, and a possible relation between the two is discussed.
INTRODUCTION A few hours or days of nutritional deprivation severely impairs the ability of the young of many speciesto regulate body temperature during cold stress (9, 21, 23, 36, 37, 39). Metabolic studies on newborn rats and rabbits (4, 9, 12, 31, 40, 41) indicate that this is probably due in large part to impaired ability to maintain and increase heat production in response to cold exposure. In a previous report (9), it was suggested 1 This research was supported by NIH grants NS 05713 and SR 05403. 174 Copyright All rights
1975 by Academic Press, Inc. oP reproduction in any form reserved.
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that under many conditions this may be the result of active neural suppression of metabolism to conserve energy in the absence of food, even during the stimulating influence of cold exposure, rather than depletion of energy stores or failure of the thermoregulatory system. As a test of this hypothesis, it should be possible, by appropriately placed transections of the brain, to effect release of inhibition of thermogenesis during starvation. The effects of these lesions are presented in this report. As postulated, the starvation-induced suppression of metabolic responses to cold exposure was released by midpontine brain stem transection. Oxygen uptake rose to “summit” (1, 15) levels within 4 hr after the transection, and continued unabated even though the animals were starved. Of perhaps much broader significance, however, was the unexpected finding that metabolism rose and remained high even at the thermoneutral temperature, which raises the possibility that the cut produced the autonomic equivalent of Sherrington’s decerebrate rigidity in the motor system ; release of descending inhibition of an otherwise tonically active excitatory system situated caudal to the cut and projecting to effector neurons. Pursuit of this analogy in the newborn may lead to further understanding of the similarities between autonomic and motor function. The neural control of blood sugar concentration may be similarIy organized in the infant rat, and interact with the thermoregulatory system, since starvation hypoglycemia, which is common to the starved infant (4, 21, 37, 40, 41, SO), was not present in these midpontine animals. METHODS The experiments were conducted on Sprague-Dawley rat pups, 5 and 10 days old. Litter size was adjusted to 8-11 animals so that weights would be uniform. Sufficient pups were used during each experimental procedure to assure a confidence level of at least 90% for each observation. The animals were allowed to nurse until taken for the experiment. They were then anesthetized, either by cryo-anesthesia at 5 days of age, or ether at 10 days. In one group (the “midpontine” or “decerebrate” group), the brain stem was transected at the pontine level (Fig. 1) by insertion of a blunt spatula through a transverse slit drilled approximately 3 mm caudal to the lambdoidal crest. To determine the effects of surgery alone, sham operations were performed. These included all procedures performed on the experimental group, except that the spatula was inserted only into overlying cerebellum. In another group, the hypothalamus was removed by suction (Fig. 1). For this, the suction tip was inserted through an opening made in the skull dorsal to the hypothalamus just rostra1 to the superior colliculi. Control operations in these experiments consisted of aspirating neural tissue dorsal to the hypothalamus, but not
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A
FIG. 1. Examples of parasagittal transections through the pons in the 5 day old (A) and 10 day o!d (B) rat pup, showing typical level and angle of cut, and hypothalamic ablation in the 5 day old pup (C) showing rostrocaud,al and dorsoventral extent of lesion. Luxol fast blue cresyl violet stain. Magnification approximately 4X.
including it. The wound was sutured, sealedwith collodion and anesthesia discontinued. The pups did not suckle after decerebration or hypothalamic ablation, so for all tests following these operations they were necessarily in the postabsorptive state, i.e., food and water deprived or “starved.” After surgery, the animals were placed in an incubator set at the neutral temperature appropriate for their age (35 C for the 5 day olds and 32 C for the 10 day olds) ( 14, 51), to minimize caloric expenditures before testing their ability to respond metabolically to cold exposure. After 2-10 hr of fasting at thermoneutral temperature the response to cold exposure was tested by the following method. The animals were transferred into individual glass jars containing soda lime to absorb CO*. The jars were flushed to establish an approximately 50% oxygen gas mixture, to avoid previously noted (14, 51) adverse effects of hypoxia on the metabolic response to cold stress. They were then sealed except for one opening to the air, and immersed in a constant temperature water bath for measurement of 02 consumption by the volume displacement technique (56). The temperature of the water
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hath was set at levels established by previous studies (14, 51) as being sufficiently cold (30 C for the 5 day olds and 27 C for 10 day olds) to evoke maximal metabolic response (0, consumption), but not so cold as to overwhelm the fragile thermoregulatory capacity of the newborn rat: i.e., the “summit” metabolism (1, 15). After allowing approximately 1 hr for thermal equilibration, Oa uptake was measured and recorded (as ml/min/kg body wt, corrected to standard temperature and pressure). The animals were then removed from the jars, and colonic temperatures immediately recorded. They were then decapitated to obtain blood samples for determination of blood glucose by the glucose oxidase method (5). To determine the effects of anesthesia alone on blood glucose, the same paradigm was followed except that no surgery was performed. The results were the same as from the untreated animals. To determine the location and extent of the lesions, the brains were fixed in Bouin’s solution, embedded in paraffin, then cut into 15 pm sagittal sections and stained hy the Luxol Fast Blue-cresyl violet method. Examples are shown in Fig. 1. For brain transection, the spatula entered the pons through the cerebellum. within 1 mm of the caudal border of the inferior colliculi, then slanted rostrally to exit at the base of the brain near the level of nucleus pontis, in both the 5 and 10 day old rats (Fig. 1 A, B) . The cuts were verified histologically to be complete laterally for animals from which the data reported herein were obtained. For ablation of the hypothalamus, tissue destruction extended from the rostra1 border of the superior colliculi rostrally to the fornix, dorsoventrally from the cortical surface to the base of the brain (Fig. 1C) and laterally 1.5-2.5 mm from the midline on both sides. In a few animals, histological examination revealed sparing of some posterior or extreme hypothalamus. Data from these did not differ significantly from those in which the hypothalamus was completely destroyed, so they will be reported as such. In some experiments, 6-hydroxydopamine (6-OHDA : 25 mg/kg, dissolved in 0.9% saline solution containing 1% ascorbic acid) was injected subcutaneously in the region of the interscapular fat pad, to modify sympathetic responsiveness (2, 19, 42, 43, 47) and thus 02 uptake during starvation, cold exposure, and/or decerebration (manuscript in preparation). A single injection of 6-OHDA caused an initial (l-2 hr) large increase in 02 consumption, followed by long lasting depression of the response to cold stress. Ascorbic acid in saline injected alone produced no response or block. RESULTS Efects of Midpontine Transection on Metabolism. Figure 2 compares metabolic rates (as reflected by 02 uptake), and changes in colonic tem-
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AND
PALMER
TA
350 -_---0
INTACT
4
2
IO
TIME (HOk
STARVE;)
FIG. 2. Effect of midpontine transection on metabolism (oxygen consumption: ML/MIN/KG) and colonic temperature at the thermoneutral ambient temperature (Ta = 3.5 C; open symbols and dashed lines) and in the cold (T, = 30 C; closed symbols and solid lines) at 5 days of age. Time in “HOURS STARVED” denotes interval after removal from mother for surgery (TRANSECTED) or to serve as unoperated controls (INTACT). SE values are indicated by vertical bars in the lower graph, and were too small (0.3 C) to be illustrated for colonic temperatures. Level of transection illustrated in Fig. 1A.
in the intact and midpontine 5 day old rat tested at the thermoneutral and moderately cold temperatures for that age (35 and 30 C respectively: ref. 14, 51) after 2-10 hr of starvation. In the unoperated animals (circles) O2 consumption at neutral temperature was minimal (approximately 25 ml/min/kg) and remained essentially constant throughout the 10 hr starvation period (dashed line). This served to maintain core (colonic) temperature at approximately 34.8 C throughout the test period, as shown. Exposure to cold elicited the well known metabolic responseto cold (IS), in this case a twofold rise in OS consumption (filled circles, solid lines) sufficient to maintain core temperature S-6 C above ambient temporarily. Then, as previously described in detail (9), the
perature,
metabolic
response
declined
during
food
and
water
deprivation,
10 hr of starvation it was no longer detectable (Fig. 2).
until
by
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This pattern of high initial metabolic response to cold exposure and subsequent decline during starvation was not observed in the midpontine preparations. Instead, these animals, although also starved, displayed an elevated 02 consumption nearly equal to that during cold exposure when tested at the neutral temperature shortly (2 hr) after surgery (Fig. 2, square symbols). Then, rather than declining during starvation as in the intact preparations, Oz uptake continued to rise (after an intermediate fall at 4-6 hr postoperatively in the cold) until by the end of the test period (10 hr of food and water deprivation) the metabolic rate in both neutral and cold environments was equivalent to that (45-50 mI/min/ kg) of the fed rat plop responding to cold stress (9). These elevated metabolic rates were reflected as high core temperatures ; starvation did not lead to hypothermia in the cold as in intact animals, and at the neutral temperature the transected pups became hyperthermic (Fig. 2). The hyperthermia was probably not due to excessive heat generated by rigidity or hyperactivity as is the case in the decerebrate adult mammal (3, 58, 60)) since in agreement with previous reports on neonates (S, 34, 57) the transection did not result in tonic rigidity or hyperactivity. Overall activity was below normal; the pups usually lay quietly, with only brief episodes of spasmodic muscle contraction or running movements. Each animal was watched for changes in coloration, precipitous drops in 02 consumption, labored breathing or other indication of deteriorating physical condition. When this occurred, or if the animal died during the test period, data from it were not used. By 10 days of age (Fig. 3) the basal metabolic rate of the intact pups was higher, and the initial elevated 02 intake during cold exposure (27 C in this case) greater than in the 5 day old rats, but the metabolic response to cold stress declined as rapidly with food deprivation as it did in the younger animals. This deterioration was simiIarIy prevented by midpontine transection, although as is illustrated in Fig. 2 the elevated 02 consumption in decerebrate 10 day old rats was preceded by 2-4 hr of postsurgical depression of metabolism. The subsequent increase in Oz intake was more pronounced in the thermoneutral environment (open squares) than in the cold (filled squares), and resulted in hyperthermia in the absence of cold stress and reversal of the starvation-induced drop in body temperature in the cold. Animals older than 10 days were not tested, because by 15 days of age there is little suppression of cold-induced metabolism by starvation (9), so there should be little or no inhibition to be released by the transection at this age or older. Effects of Transection on Blood Glucose During Starvation. In a previous report (9) the metabohc response to cold exposure was shown to
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@ -----0 INTACT 0 TRANSECTED 270 . .
2oL
2
INTACT TRANSECTED
--- Y JH 4
!if!ggg 6
TIME.(HOURS
0
- + 10
STARVED)
FIG. 3. Effects of midpontine transection on 02 consumption (ML/MIN/KG) and colonic temperature at thermoneutrality (32 C) and in the cold (27 C) in the 10 day old rat. Starvation times, symbols and other notations as in Fig. 2. Level of transection shown in Fig. 1B.
be inversely correlated with blood glucose levels, when the latter was varied by starvation, glucose by gavage or insulin injection. With this possible causal relation between blood sugar level and thermoregulation in mind, glucose measurements were made on the rats with the brain transected, which were generating abnormally high quantities of heat even when thermogenesis should have been suppressed by starvation. Figure 4 shows that the transection produced a transitory hyperglycemia in the 5 day old pups, which leveled off after 3-5 hr postoperatively, so that the midpontine animals then maintained essentially normal glucose levels (110 +- 10 mg/lOO ml) for the remainder of the test period. This contrasts sharply with the intact rats, which became severely hypoglycemic (60-70 mg/lOO ml) by 10 hr of starvation. By 10 days of age, blood glucose was elevated only slightly by the surgery, and declined progressively during starvation in a course paralieling that for the intact animals but lagging it for the first 8 hr of the test
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Changes in blood glucose levels (mg/lOO ml serum) during starvation in day old rats (circles) and after pontine section (squares) at thermo(T, = 35 C: dashed lines) or in the cold (30 C : solid lines). Other as in Fig. 2. Level of transection shown in Fig. 1A.
period (Fig. 5). During this time, in the cold but not at thermoneutrality, midpontine animals maintained blood glucose levels close to normal. This was not the casein the intact pups. Hypothalallzic Lesions. The hypothalamus was removed in 5 day old pups to test the possibility that the high metabolic rate despite starvation of the midpontine animals was due to release of descending inhibition from the hypothalamic temperature regulating areas. If this were the case, removal of the hypothalamus should produce an effect similar to pontine transection. On the other hand, if the hypothalamus were essential for cold-evoked thermogenesis, as transection studies on adult mammals indicate (3, 58, 60). removal of the hypothalamus should abolish the responseeven in the unstarved pups. Measurements of 02 uptake at 2 and
FIG. 5. Changes in blood glucose levels during starvation in the 10 day old rat at thermoneutrality (32 C) and in the cold (27 C), either intact (circles) or after midpontine transection (squares). Level of transection shown in Fig. 1B.
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8 hr after hypothalamic ablation revealed responses intermediate between these extremes. The unstarved animals were able to elevate their O2 uptake to 37 f 2 ml/min/kg in the cold (30 C), from a value of 24 * 2 at thermoneutrality. This is significantly below the response capacity of the intact pups tested 2 hr postabsorptively (45 * 3 ml/min/kg at 30 C : Fig. 2), but equal to the ability of those starved for 6 or more hr. Then, when tested at 8 hr after removal of the hypothalamus, 0s consumption was not elevated as in the midpontine preparations at this time, but was down to 30 ml/min/kg, slightly lower than that of the intact pups starved for the same time. After removal of the hypothalamus, mean colonic temperatures in the cold were 34 +- 0.2 C after 8 hr, one degree higher than the starved rats, and one degree lower than the midpontine ones were at this time (Fig. 2). Blood glucose at 8 hr of starvation was within the range of the fed, intact pups, i.e., not depressed as in starvation alone. Sham Operations. The transitory nature of the initial hyperglycemia following midpontine transection in all rat pups (Figs. 4, S), and depressed 0s consumption postoperatively-especially in the 10 day old pups (Figs. 2, 3)-suggested that postsurgical trauma might be a contributory factor, so sham operations (Methods) were performed in these animals. It was found that blood glucose was initially elevated to a value (130-150 mg/lOO ml) approximating that following brain stem transection at both ages (Figs. 4, 5) and then declined to a low level, IO-15% above that for the intact starved rats. In contrast, unlike the transections, sham operations had no appreciable effect on 02 uptake, initially or during starvation, at either age. It follows that the surgical approach contributes to the rise in blood sugar, but not to the initial depression of metabolism. DISCUSSION In some species and under certain circumstances, the decline of the metabolic response to cold exposure after a few hours of starvation shortly after birth may be accounted for by exhaustion of heat producing fuel stores (15). Th is is probably true for newborn rabbits starved in the cold. They lose the response when their brown adipose tissue, the main energy source of the additional heat production in this species (26, 29), falls to low levels (12, 23, 31). However, the response also fails despite ample remaining available fuel, if they are kept warm during starvation (23). In a previous study (9), this was confirmed for infant rats, and it was suggested that rather than exhaustion of available fuel being the limiting factor, the decline of caloric output in the cold may reflect intervention by the central nervous system to conserve energy when no food is available. Although the purpose of the CNS intervention cannot be decided
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experimentally and remains conjecture, its existence and nature can be, and it was to this end that the present investigation was directed. Before the transection studies were initiated, further evidence was obtained that the nervous system of the rat pup suppresses the metabolic response to cold stress if starvation continues for very long. For this, pups were starved in the warm for a time sufficient to abolish this response (9), and then injected with 6-hydroxydopamine (6-OHDA), in an attempt to stimulate metabolism by the known action (2, 19, 42, 43, 47) of this drug of initially releasing norepinephrine from sympathetic noradrenergic terminals. A single injection eIicited a sharp transient increase in 02 uptake, having a peak amplitude (50 * 3 ml/min/kg) mimicking that reached during brief exposure to cold without starvation (unpublished observations, see Methods). So evidentally the stores are there and available for use, but cease to be mobilized by the nervous system during cold stress, if food deprivation is prolonged. Consistent with this is the finding (Figs. 2, 3) that after decerebration and without drug injection, summit metabolism continued unabated after periods of starvation which abolished it in intact rats. The sustained high metabolism in the cold despite starvation in the midpontine ratlets indicates a tonically active system caudal to the cut, stimulatory to metabolism and subject to inhibition by structures rostra1 to the pons. Since the neural path by which the metabolic response to cold exposure in the infant is mediated is sympathetic (15, 26, 30), the unrestrained metabolism following the transection is apparently due to inhibited sympathetic discharge associated with temperature regulation-an autonomic equivalent in the newborn of Sherrington’s decerebrate rigidity in the motor system of the adult. There are indications of such disinhibited sympathetic discharge in the poikilothermy of decerebrate adult preparations in that they are often hyperthermic (3, 58, 60), but this is usually obscured by or attributed to heat generated by muscle tension or hyperactivity. In the newborn rat, however, the somatic motor system is very poorly developed ( 11, 53) and as in the kitten (7, 34), d oes not become hyperactive following decerebration (S), whereas the sympathetic nervous system is well developed and functional at birth (2, 1.5, 30). Thus the detection and interpretation of autonomic reactions is not nearly so much complicated by superimposed motor activity in the rat pup as it is after the motor system matures, and this was probably not the cause of the high metabolic rate following decerebration in these experiments. Evidence for this is an observation that depleting the sympathetic terminals of norepinephrine by injecting 6-OHDA prevented the rise in metabolism after decerebration without visibly altering behavior. It is more likely that the immaturity of the
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somatic motor system in itself accounts in part for the lesser effect of the transection on its function, according to recent proposals (7, 49) that the severity and nature of disorder is in direct consequence of the amount of synaptic connectivity to the intrinsic system that has emerged, and hence on the degree of pharmacologic dependency on the existence of such extrinsic bombardment that has built up, at the time of the transection. In this case, the lesion is visualized as having been inflicted prior to the development of most such dependencies in the motor system, and subsequent to their emergence in autonomic organization. Similar transient imbalances in the chemical milieu of affected neurons, and subsequent readjustments (13, 22) in existing or genetically predetermined (48) emerging physicochemical relations during development may account for much of the remarkable retention or recovery of function following neurological injury inflicted early in ontogeny (cf. 7, 8, 13, 16, 18, 20, 33, 49, 54). Integration of the decerebration and drug injection data with the findings of others from various species provides the following tentative scheme for the neural restrictions imposed on thermogenesis by starvation in the newborn rat. Considerable thermoregulation probably occurs at the spinal (10, 46, 59) and pontomedullary (lo? 35) level. The sympathetic nervous system plays a prominent role (15, 30)) acting directly or indirectly via hormones to mobilize brown fat and other fuel stores (27, 29-32). Without starvation, and in the warm, sympathetic discharge may be under inhibition from mesencephalic, hypothalamic (10, 24) and perhaps other thermoregulatory areas, resulting in resting metabolic rate. With cold stress, the descending inhibition lessens in the fed animal, and the metabolic response to cold exposure ensues. The onset of starvation apparently triggers reinstatement of the inhibition, resulting in decline of the response with conservation of energy supplies, as food deprivation continues. Midpontine transection releases the inhibition even of the animals in the warm and starved, and of course with prolonged starvation, especially in the cold, fuel for extra thermogenesis in the cold becomes exhausted and it ceases (12, 23,29, 30). The hypothalamus seems to play little role in cold-induced thermogenesis in the infant rat, since removing it neither increased metabolism as in the case of midpontine transection, or abolished it. Consistent with this is the fact that decerebration, which severs hypothalamic input to lower centers, does not severely impair the development of thermoregulation in the newborn kitten (S), but renders the adult animal essentially poikilothermic. The inhibition of metabolism at low environmental temperatures during starvation appears to be triggered largely by rapidly declining glucose
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levels (9, 23). If this is correct, the disinhibition (i.e., maintained 02 consumption and core temperature despite starvation) that appears after brain stem transection should be reflected in sustained blood sugar concentration, rather than the sharp decrease (Figs. 4, 5) that occurs with nutritional deprivation alone. The results reported above show that this is indeed the case ; with midpontine transection, no hypoglycemia was detected within the 10 hr test period. This supports the contention that the level of blood glucose is of primary importance for maintenance of thermogenesis in the face of starvation, but it leaves open the question of why transecting the brain stem should alleviate hypoglycemia. The data, and reports of others (6, 44) suggest that the low pons and medulla (and perhaps even spinal cord) are capable of activating organs which release glucose into the vascular system during starvation, but this remains for future experiments to decide. Neurohumoral control via the isolated hypothalamus may be involved. A problem with involving blood glucose as a modulator during starvation is that brain glucose itself seems to be very loosely regulated, and in z~iz~obrain glucose utilization is very low in the rat pup (17, 38, 45). The low utilization, however, and reports that the brain of the suckling rat utilizes ketone bodies (25) and amino acids (52, 55) rather than depending solely on glucose for respiration (28) indicate that starvation hypoglycemia (4, 7, 21, 37, 39-41) leading to malfunction of the thermoregulatory system of the brain is probably not the explanation for the decline in thermogenesis during starvation. The sustained blood glucose levels after midpontine transection and despite starvation is reminiscent of Claude Bernard’s piqfire diabttique (6)) in which insertion of a spatula into the floor of the fourth ventricle, at approximately the same level as in our experiments, produced hyperglycemia. Although we did not observe the excessive blood glucose levels as in his report, the still undefined neural determinants may be the same. This also remains to be decided by future experimentation. REFERENCES G. 1962.Temperatureregulationin the newbornlamb. V. Summit metabolism. A&r. J. Agric. Res. 13 : 100-121. 2. ANGELETTI, P. V., and R. LEVI-M• NTALCINI. 1970.Sympatheticnerve cell destruction in newbornmammalsby 6-hydroxydopamine.Proc. Nut. Acad. Sci. 65: 114-121. 3. BARD, P., and M. B. MACHT. 1958.The behavior of chronically decerebratecats, pp. 55-71.In “The NeurologicalBasisof Behavior.” Ciba Symposium,London, Little, Brown, Boston. 4. BARIC, I. 1953.La consummation d’oxyg&nedu rat nouveau-n&au tours du jeune. Acad. Serbe. Sci. 12 : 71-76. 5. BERGMEYER, H. 1963.“Methods of Enzymatic Analysis.” AcademicPress,New York. 1. ALEXANDER,
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H. E. 1964. Historical review, pp. Z-44. In “Developmental Neurobiology.” W. A. Himwich [Ed.]. Thomas, Springfield, 111. HULL, D., and M. M. SEGALL. 1965a. The contribution of brown adipose tissue to heat production in the newborn rabbit. J. Physiol. (Londorl) 181: 449-457. HULL, D., and M. M. SEGALL. l%Sb. Sympathetic nervous control of brown adipose tissue and heat production in the newborn rabbit. I. Physiol. (London) 181: 458-467. HULL, D., and M. M. SEGALL. 1965c. Heat production in the newborn rabbit and the fat control of the brown adipose tissue. J. Physiol. (London) 181: 468-477. KACIUBA-USCILKO, H. 1972. Hormonal regulation of thermogenesis in the newborn pig. Biol. Neonate 21: 245-258. KENNARD, M. A. 1942. Cortical reorganization of motor function: Studies on a series of monkeys of various ages from infancy to maturity. AMA Arch. Neural. Psychiat. 8 : 227-240. LANGWORTHY, 0. R. 1929. A correlated study of the development of reflex activity in fetal and young kittens and the myelinization of tracts in the nervous system. Contrib. Embrol. Carnegie Inst. Washington 20: 127-171. LIPTON, J. M. 1973. Thermosensitivity of medulla oblongata in control of body temperature. Amer. J. Physiol. 224 : 890-897. MANN, T. P., and R. I. K. ELLIOTT. 1957. Neonatal cold injury due to accidental exposure to cold. Lancet 272 : 229-234. MCCANCX, R. A., and E. M. WIDDOWSON. 1959. The effect of lowering the ambient temperature on the metabolism of the newborn pig. J. Physiol. (London) 147 : 124-134. MOORE, T. J., A. P. LIONE, D. M. REGEN, H. L. TARPLEY, and P. L. RAINES. 1971. Brain glucose metabolism in the newborn rat. Amer. J. Physiol. 221: 1761753. MORRILL, C. C. 1952. Studies on baby pig mortality. Influence of environmental temperature on fasting newborn pigs. Amer. J. Vet. Res. 13 : 322-324. MOUREK, J. 1958. The effect of starvation on oxygen consumption following hypoxia during ontogenesis in rats. Physiol. Bohemoslov. 7: 431-436. MOUREK, J. 1959. Oxygen consumption during ontogenesis in rats in environments with a high and low oxygen content. Physiol. Bohemoslov. 8: 106-111. PAPPAS, B. A., and L. V. DICARA. 1973. Neonatal sympathectomy by 6-hydroxydopamine: Cardiovascular responses in the paralyzed rat. Physiol. Behav. 10:
28. HIMWICH,
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and W. DE JONG. 1974. Neonatal chemical sympathectomy: functional control of denervation of the vascular system in tissue noradrenaline level in the rat after 6-hydroxydopamine. NaunywSchmiede-
berg’s Arch. Pharmacol. 284 : 353-363. 44. ROSENBERG, F. J., and V. DISTEFANO. 1962. A central nervous of epinephrine hyperglycemia. Amer. J. Physiol. 203 : 782-788. 45. SAMSON, F. E., W. M. BALFOUR, and N. A. DAHL. 1958. The
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46. SIMON,
E. 1969. Kreislaufwirkungen
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49. STELZNER, D. J., W. B. ERSHLER, and E. D. WEBER. 1975. Effects of spinal transection in neonatal and weanling rats: survival of function. Exp. Nruroi. 46: 156-177. 50. SWIATEK, K. R., D. M. KIPNIS, G. MASON, K.-L. CHAO, and M. CORNBLATH. 1968. Starvation hypoglycemia in newborn pigs. Amer. J. Physiol. 214: 400405.
51. TAYLOR, P. M. 1960. Oxygen consumption in new-born rats. J. Phgsiol. (London) 154 : 153-168. 52. TEWS, J. K., S. H. CARTER, and W. E. STONE. 1965. Chemical changes in the brain during insulin hypoglycaemia and recovery. J. Neurochem. 12: 670693. 53. TILNEY, F. 1933. Behavior in its relation to the development of the brain. Bull. Neural. 54. TWITCHELL,
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59. W~~NNERBERG,
60.
NEUROLOGY
49,
174-188
Effect of Neonatal during Starvation K. E. BIGNALL, Department
Decerebration on Thermogenesis and Cold Exposure in the Rat
F. W. HEGGENESS,
of Physiology,
University
(1975)
of Rochester,
Received
AND J. E. PALMER
School of Medicine Rochester, New April
1
and Dentistry, York 14642
9, 1975
Rat pups, 5 and 10 days old, were decerebrated at the midpontine level, or the hypothalamus was removed. Their metabolic responses to cold exposure (elevated 02 consumption) was then measured after Z-10 hr of starvation, and compared with that of intact animals starved for the same length of time. The 0, uptake of the unoperated pups declined after Z-4 hr and disappeared by 8-10 hr, whereas the decerebrate rats maintained near maximal Oa consumption either in the cold or at thermoneutrality. Colonic temperatures averaged 5 C higher by 10 hr of starvation than in the intact animals. Blood glucose was also measured. It declined progressively in the intact pups, but remained near normal in the decerebrate preparations. These findings suggest that the low basal metabolism, and suppression by starvation of the metabolic response to cold exposure in the infant rat, are the result of descending inhibition of a heat generating system caudal to the transection, which is released by severing the connections. Aspiration of the hypothalamus caused only slight impairment of the responses to cold at this age, suggesting a critical thermoregulatory role for the more caudal brain stem and spinal structures early in life, with dependency on descending influences developing during maturation. A similar mechanism is also proposed to account for the maintained high blood glucose despite starvation in the decerebrate animals, and a possible relation between the two is discussed.
INTRODUCTION A few hours or days of nutritional deprivation severely impairs the ability of the young of many speciesto regulate body temperature during cold stress (9, 21, 23, 36, 37, 39). Metabolic studies on newborn rats and rabbits (4, 9, 12, 31, 40, 41) indicate that this is probably due in large part to impaired ability to maintain and increase heat production in response to cold exposure. In a previous report (9), it was suggested 1 This research was supported by NIH grants NS 05713 and SR 05403. 174 Copyright All rights
1975 by Academic Press, Inc. oP reproduction in any form reserved.
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that under many conditions this may be the result of active neural suppression of metabolism to conserve energy in the absence of food, even during the stimulating influence of cold exposure, rather than depletion of energy stores or failure of the thermoregulatory system. As a test of this hypothesis, it should be possible, by appropriately placed transections of the brain, to effect release of inhibition of thermogenesis during starvation. The effects of these lesions are presented in this report. As postulated, the starvation-induced suppression of metabolic responses to cold exposure was released by midpontine brain stem transection. Oxygen uptake rose to “summit” (1, 15) levels within 4 hr after the transection, and continued unabated even though the animals were starved. Of perhaps much broader significance, however, was the unexpected finding that metabolism rose and remained high even at the thermoneutral temperature, which raises the possibility that the cut produced the autonomic equivalent of Sherrington’s decerebrate rigidity in the motor system ; release of descending inhibition of an otherwise tonically active excitatory system situated caudal to the cut and projecting to effector neurons. Pursuit of this analogy in the newborn may lead to further understanding of the similarities between autonomic and motor function. The neural control of blood sugar concentration may be similarIy organized in the infant rat, and interact with the thermoregulatory system, since starvation hypoglycemia, which is common to the starved infant (4, 21, 37, 40, 41, SO), was not present in these midpontine animals. METHODS The experiments were conducted on Sprague-Dawley rat pups, 5 and 10 days old. Litter size was adjusted to 8-11 animals so that weights would be uniform. Sufficient pups were used during each experimental procedure to assure a confidence level of at least 90% for each observation. The animals were allowed to nurse until taken for the experiment. They were then anesthetized, either by cryo-anesthesia at 5 days of age, or ether at 10 days. In one group (the “midpontine” or “decerebrate” group), the brain stem was transected at the pontine level (Fig. 1) by insertion of a blunt spatula through a transverse slit drilled approximately 3 mm caudal to the lambdoidal crest. To determine the effects of surgery alone, sham operations were performed. These included all procedures performed on the experimental group, except that the spatula was inserted only into overlying cerebellum. In another group, the hypothalamus was removed by suction (Fig. 1). For this, the suction tip was inserted through an opening made in the skull dorsal to the hypothalamus just rostra1 to the superior colliculi. Control operations in these experiments consisted of aspirating neural tissue dorsal to the hypothalamus, but not
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A
FIG. 1. Examples of parasagittal transections through the pons in the 5 day old (A) and 10 day o!d (B) rat pup, showing typical level and angle of cut, and hypothalamic ablation in the 5 day old pup (C) showing rostrocaud,al and dorsoventral extent of lesion. Luxol fast blue cresyl violet stain. Magnification approximately 4X.
including it. The wound was sutured, sealedwith collodion and anesthesia discontinued. The pups did not suckle after decerebration or hypothalamic ablation, so for all tests following these operations they were necessarily in the postabsorptive state, i.e., food and water deprived or “starved.” After surgery, the animals were placed in an incubator set at the neutral temperature appropriate for their age (35 C for the 5 day olds and 32 C for the 10 day olds) ( 14, 51), to minimize caloric expenditures before testing their ability to respond metabolically to cold exposure. After 2-10 hr of fasting at thermoneutral temperature the response to cold exposure was tested by the following method. The animals were transferred into individual glass jars containing soda lime to absorb CO*. The jars were flushed to establish an approximately 50% oxygen gas mixture, to avoid previously noted (14, 51) adverse effects of hypoxia on the metabolic response to cold stress. They were then sealed except for one opening to the air, and immersed in a constant temperature water bath for measurement of 02 consumption by the volume displacement technique (56). The temperature of the water
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hath was set at levels established by previous studies (14, 51) as being sufficiently cold (30 C for the 5 day olds and 27 C for 10 day olds) to evoke maximal metabolic response (0, consumption), but not so cold as to overwhelm the fragile thermoregulatory capacity of the newborn rat: i.e., the “summit” metabolism (1, 15). After allowing approximately 1 hr for thermal equilibration, Oa uptake was measured and recorded (as ml/min/kg body wt, corrected to standard temperature and pressure). The animals were then removed from the jars, and colonic temperatures immediately recorded. They were then decapitated to obtain blood samples for determination of blood glucose by the glucose oxidase method (5). To determine the effects of anesthesia alone on blood glucose, the same paradigm was followed except that no surgery was performed. The results were the same as from the untreated animals. To determine the location and extent of the lesions, the brains were fixed in Bouin’s solution, embedded in paraffin, then cut into 15 pm sagittal sections and stained hy the Luxol Fast Blue-cresyl violet method. Examples are shown in Fig. 1. For brain transection, the spatula entered the pons through the cerebellum. within 1 mm of the caudal border of the inferior colliculi, then slanted rostrally to exit at the base of the brain near the level of nucleus pontis, in both the 5 and 10 day old rats (Fig. 1 A, B) . The cuts were verified histologically to be complete laterally for animals from which the data reported herein were obtained. For ablation of the hypothalamus, tissue destruction extended from the rostra1 border of the superior colliculi rostrally to the fornix, dorsoventrally from the cortical surface to the base of the brain (Fig. 1C) and laterally 1.5-2.5 mm from the midline on both sides. In a few animals, histological examination revealed sparing of some posterior or extreme hypothalamus. Data from these did not differ significantly from those in which the hypothalamus was completely destroyed, so they will be reported as such. In some experiments, 6-hydroxydopamine (6-OHDA : 25 mg/kg, dissolved in 0.9% saline solution containing 1% ascorbic acid) was injected subcutaneously in the region of the interscapular fat pad, to modify sympathetic responsiveness (2, 19, 42, 43, 47) and thus 02 uptake during starvation, cold exposure, and/or decerebration (manuscript in preparation). A single injection of 6-OHDA caused an initial (l-2 hr) large increase in 02 consumption, followed by long lasting depression of the response to cold stress. Ascorbic acid in saline injected alone produced no response or block. RESULTS Efects of Midpontine Transection on Metabolism. Figure 2 compares metabolic rates (as reflected by 02 uptake), and changes in colonic tem-
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AND
PALMER
TA
350 -_---0
INTACT
4
2
IO
TIME (HOk
STARVE;)
FIG. 2. Effect of midpontine transection on metabolism (oxygen consumption: ML/MIN/KG) and colonic temperature at the thermoneutral ambient temperature (Ta = 3.5 C; open symbols and dashed lines) and in the cold (T, = 30 C; closed symbols and solid lines) at 5 days of age. Time in “HOURS STARVED” denotes interval after removal from mother for surgery (TRANSECTED) or to serve as unoperated controls (INTACT). SE values are indicated by vertical bars in the lower graph, and were too small (0.3 C) to be illustrated for colonic temperatures. Level of transection illustrated in Fig. 1A.
in the intact and midpontine 5 day old rat tested at the thermoneutral and moderately cold temperatures for that age (35 and 30 C respectively: ref. 14, 51) after 2-10 hr of starvation. In the unoperated animals (circles) O2 consumption at neutral temperature was minimal (approximately 25 ml/min/kg) and remained essentially constant throughout the 10 hr starvation period (dashed line). This served to maintain core (colonic) temperature at approximately 34.8 C throughout the test period, as shown. Exposure to cold elicited the well known metabolic responseto cold (IS), in this case a twofold rise in OS consumption (filled circles, solid lines) sufficient to maintain core temperature S-6 C above ambient temporarily. Then, as previously described in detail (9), the
perature,
metabolic
response
declined
during
food
and
water
deprivation,
10 hr of starvation it was no longer detectable (Fig. 2).
until
by
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This pattern of high initial metabolic response to cold exposure and subsequent decline during starvation was not observed in the midpontine preparations. Instead, these animals, although also starved, displayed an elevated 02 consumption nearly equal to that during cold exposure when tested at the neutral temperature shortly (2 hr) after surgery (Fig. 2, square symbols). Then, rather than declining during starvation as in the intact preparations, Oz uptake continued to rise (after an intermediate fall at 4-6 hr postoperatively in the cold) until by the end of the test period (10 hr of food and water deprivation) the metabolic rate in both neutral and cold environments was equivalent to that (45-50 mI/min/ kg) of the fed rat plop responding to cold stress (9). These elevated metabolic rates were reflected as high core temperatures ; starvation did not lead to hypothermia in the cold as in intact animals, and at the neutral temperature the transected pups became hyperthermic (Fig. 2). The hyperthermia was probably not due to excessive heat generated by rigidity or hyperactivity as is the case in the decerebrate adult mammal (3, 58, 60)) since in agreement with previous reports on neonates (S, 34, 57) the transection did not result in tonic rigidity or hyperactivity. Overall activity was below normal; the pups usually lay quietly, with only brief episodes of spasmodic muscle contraction or running movements. Each animal was watched for changes in coloration, precipitous drops in 02 consumption, labored breathing or other indication of deteriorating physical condition. When this occurred, or if the animal died during the test period, data from it were not used. By 10 days of age (Fig. 3) the basal metabolic rate of the intact pups was higher, and the initial elevated 02 intake during cold exposure (27 C in this case) greater than in the 5 day old rats, but the metabolic response to cold stress declined as rapidly with food deprivation as it did in the younger animals. This deterioration was simiIarIy prevented by midpontine transection, although as is illustrated in Fig. 2 the elevated 02 consumption in decerebrate 10 day old rats was preceded by 2-4 hr of postsurgical depression of metabolism. The subsequent increase in Oz intake was more pronounced in the thermoneutral environment (open squares) than in the cold (filled squares), and resulted in hyperthermia in the absence of cold stress and reversal of the starvation-induced drop in body temperature in the cold. Animals older than 10 days were not tested, because by 15 days of age there is little suppression of cold-induced metabolism by starvation (9), so there should be little or no inhibition to be released by the transection at this age or older. Effects of Transection on Blood Glucose During Starvation. In a previous report (9) the metabohc response to cold exposure was shown to
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AND
PALMER
@ -----0 INTACT 0 TRANSECTED 270 . .
2oL
2
INTACT TRANSECTED
--- Y JH 4
!if!ggg 6
TIME.(HOURS
0
- + 10
STARVED)
FIG. 3. Effects of midpontine transection on 02 consumption (ML/MIN/KG) and colonic temperature at thermoneutrality (32 C) and in the cold (27 C) in the 10 day old rat. Starvation times, symbols and other notations as in Fig. 2. Level of transection shown in Fig. 1B.
be inversely correlated with blood glucose levels, when the latter was varied by starvation, glucose by gavage or insulin injection. With this possible causal relation between blood sugar level and thermoregulation in mind, glucose measurements were made on the rats with the brain transected, which were generating abnormally high quantities of heat even when thermogenesis should have been suppressed by starvation. Figure 4 shows that the transection produced a transitory hyperglycemia in the 5 day old pups, which leveled off after 3-5 hr postoperatively, so that the midpontine animals then maintained essentially normal glucose levels (110 +- 10 mg/lOO ml) for the remainder of the test period. This contrasts sharply with the intact rats, which became severely hypoglycemic (60-70 mg/lOO ml) by 10 hr of starvation. By 10 days of age, blood glucose was elevated only slightly by the surgery, and declined progressively during starvation in a course paralieling that for the intact animals but lagging it for the first 8 hr of the test
THERMOGENESIS
FIG. 4. intact 5 neutrality notations
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NEONATE
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Changes in blood glucose levels (mg/lOO ml serum) during starvation in day old rats (circles) and after pontine section (squares) at thermo(T, = 35 C: dashed lines) or in the cold (30 C : solid lines). Other as in Fig. 2. Level of transection shown in Fig. 1A.
period (Fig. 5). During this time, in the cold but not at thermoneutrality, midpontine animals maintained blood glucose levels close to normal. This was not the casein the intact pups. Hypothalallzic Lesions. The hypothalamus was removed in 5 day old pups to test the possibility that the high metabolic rate despite starvation of the midpontine animals was due to release of descending inhibition from the hypothalamic temperature regulating areas. If this were the case, removal of the hypothalamus should produce an effect similar to pontine transection. On the other hand, if the hypothalamus were essential for cold-evoked thermogenesis, as transection studies on adult mammals indicate (3, 58, 60). removal of the hypothalamus should abolish the responseeven in the unstarved pups. Measurements of 02 uptake at 2 and
FIG. 5. Changes in blood glucose levels during starvation in the 10 day old rat at thermoneutrality (32 C) and in the cold (27 C), either intact (circles) or after midpontine transection (squares). Level of transection shown in Fig. 1B.
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8 hr after hypothalamic ablation revealed responses intermediate between these extremes. The unstarved animals were able to elevate their O2 uptake to 37 f 2 ml/min/kg in the cold (30 C), from a value of 24 * 2 at thermoneutrality. This is significantly below the response capacity of the intact pups tested 2 hr postabsorptively (45 * 3 ml/min/kg at 30 C : Fig. 2), but equal to the ability of those starved for 6 or more hr. Then, when tested at 8 hr after removal of the hypothalamus, 0s consumption was not elevated as in the midpontine preparations at this time, but was down to 30 ml/min/kg, slightly lower than that of the intact pups starved for the same time. After removal of the hypothalamus, mean colonic temperatures in the cold were 34 +- 0.2 C after 8 hr, one degree higher than the starved rats, and one degree lower than the midpontine ones were at this time (Fig. 2). Blood glucose at 8 hr of starvation was within the range of the fed, intact pups, i.e., not depressed as in starvation alone. Sham Operations. The transitory nature of the initial hyperglycemia following midpontine transection in all rat pups (Figs. 4, S), and depressed 0s consumption postoperatively-especially in the 10 day old pups (Figs. 2, 3)-suggested that postsurgical trauma might be a contributory factor, so sham operations (Methods) were performed in these animals. It was found that blood glucose was initially elevated to a value (130-150 mg/lOO ml) approximating that following brain stem transection at both ages (Figs. 4, 5) and then declined to a low level, IO-15% above that for the intact starved rats. In contrast, unlike the transections, sham operations had no appreciable effect on 02 uptake, initially or during starvation, at either age. It follows that the surgical approach contributes to the rise in blood sugar, but not to the initial depression of metabolism. DISCUSSION In some species and under certain circumstances, the decline of the metabolic response to cold exposure after a few hours of starvation shortly after birth may be accounted for by exhaustion of heat producing fuel stores (15). Th is is probably true for newborn rabbits starved in the cold. They lose the response when their brown adipose tissue, the main energy source of the additional heat production in this species (26, 29), falls to low levels (12, 23, 31). However, the response also fails despite ample remaining available fuel, if they are kept warm during starvation (23). In a previous study (9), this was confirmed for infant rats, and it was suggested that rather than exhaustion of available fuel being the limiting factor, the decline of caloric output in the cold may reflect intervention by the central nervous system to conserve energy when no food is available. Although the purpose of the CNS intervention cannot be decided
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experimentally and remains conjecture, its existence and nature can be, and it was to this end that the present investigation was directed. Before the transection studies were initiated, further evidence was obtained that the nervous system of the rat pup suppresses the metabolic response to cold stress if starvation continues for very long. For this, pups were starved in the warm for a time sufficient to abolish this response (9), and then injected with 6-hydroxydopamine (6-OHDA), in an attempt to stimulate metabolism by the known action (2, 19, 42, 43, 47) of this drug of initially releasing norepinephrine from sympathetic noradrenergic terminals. A single injection eIicited a sharp transient increase in 02 uptake, having a peak amplitude (50 * 3 ml/min/kg) mimicking that reached during brief exposure to cold without starvation (unpublished observations, see Methods). So evidentally the stores are there and available for use, but cease to be mobilized by the nervous system during cold stress, if food deprivation is prolonged. Consistent with this is the finding (Figs. 2, 3) that after decerebration and without drug injection, summit metabolism continued unabated after periods of starvation which abolished it in intact rats. The sustained high metabolism in the cold despite starvation in the midpontine ratlets indicates a tonically active system caudal to the cut, stimulatory to metabolism and subject to inhibition by structures rostra1 to the pons. Since the neural path by which the metabolic response to cold exposure in the infant is mediated is sympathetic (15, 26, 30), the unrestrained metabolism following the transection is apparently due to inhibited sympathetic discharge associated with temperature regulation-an autonomic equivalent in the newborn of Sherrington’s decerebrate rigidity in the motor system of the adult. There are indications of such disinhibited sympathetic discharge in the poikilothermy of decerebrate adult preparations in that they are often hyperthermic (3, 58, 60), but this is usually obscured by or attributed to heat generated by muscle tension or hyperactivity. In the newborn rat, however, the somatic motor system is very poorly developed ( 11, 53) and as in the kitten (7, 34), d oes not become hyperactive following decerebration (S), whereas the sympathetic nervous system is well developed and functional at birth (2, 1.5, 30). Thus the detection and interpretation of autonomic reactions is not nearly so much complicated by superimposed motor activity in the rat pup as it is after the motor system matures, and this was probably not the cause of the high metabolic rate following decerebration in these experiments. Evidence for this is an observation that depleting the sympathetic terminals of norepinephrine by injecting 6-OHDA prevented the rise in metabolism after decerebration without visibly altering behavior. It is more likely that the immaturity of the
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AND
PALMER
somatic motor system in itself accounts in part for the lesser effect of the transection on its function, according to recent proposals (7, 49) that the severity and nature of disorder is in direct consequence of the amount of synaptic connectivity to the intrinsic system that has emerged, and hence on the degree of pharmacologic dependency on the existence of such extrinsic bombardment that has built up, at the time of the transection. In this case, the lesion is visualized as having been inflicted prior to the development of most such dependencies in the motor system, and subsequent to their emergence in autonomic organization. Similar transient imbalances in the chemical milieu of affected neurons, and subsequent readjustments (13, 22) in existing or genetically predetermined (48) emerging physicochemical relations during development may account for much of the remarkable retention or recovery of function following neurological injury inflicted early in ontogeny (cf. 7, 8, 13, 16, 18, 20, 33, 49, 54). Integration of the decerebration and drug injection data with the findings of others from various species provides the following tentative scheme for the neural restrictions imposed on thermogenesis by starvation in the newborn rat. Considerable thermoregulation probably occurs at the spinal (10, 46, 59) and pontomedullary (lo? 35) level. The sympathetic nervous system plays a prominent role (15, 30)) acting directly or indirectly via hormones to mobilize brown fat and other fuel stores (27, 29-32). Without starvation, and in the warm, sympathetic discharge may be under inhibition from mesencephalic, hypothalamic (10, 24) and perhaps other thermoregulatory areas, resulting in resting metabolic rate. With cold stress, the descending inhibition lessens in the fed animal, and the metabolic response to cold exposure ensues. The onset of starvation apparently triggers reinstatement of the inhibition, resulting in decline of the response with conservation of energy supplies, as food deprivation continues. Midpontine transection releases the inhibition even of the animals in the warm and starved, and of course with prolonged starvation, especially in the cold, fuel for extra thermogenesis in the cold becomes exhausted and it ceases (12, 23,29, 30). The hypothalamus seems to play little role in cold-induced thermogenesis in the infant rat, since removing it neither increased metabolism as in the case of midpontine transection, or abolished it. Consistent with this is the fact that decerebration, which severs hypothalamic input to lower centers, does not severely impair the development of thermoregulation in the newborn kitten (S), but renders the adult animal essentially poikilothermic. The inhibition of metabolism at low environmental temperatures during starvation appears to be triggered largely by rapidly declining glucose
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levels (9, 23). If this is correct, the disinhibition (i.e., maintained 02 consumption and core temperature despite starvation) that appears after brain stem transection should be reflected in sustained blood sugar concentration, rather than the sharp decrease (Figs. 4, 5) that occurs with nutritional deprivation alone. The results reported above show that this is indeed the case ; with midpontine transection, no hypoglycemia was detected within the 10 hr test period. This supports the contention that the level of blood glucose is of primary importance for maintenance of thermogenesis in the face of starvation, but it leaves open the question of why transecting the brain stem should alleviate hypoglycemia. The data, and reports of others (6, 44) suggest that the low pons and medulla (and perhaps even spinal cord) are capable of activating organs which release glucose into the vascular system during starvation, but this remains for future experiments to decide. Neurohumoral control via the isolated hypothalamus may be involved. A problem with involving blood glucose as a modulator during starvation is that brain glucose itself seems to be very loosely regulated, and in z~iz~obrain glucose utilization is very low in the rat pup (17, 38, 45). The low utilization, however, and reports that the brain of the suckling rat utilizes ketone bodies (25) and amino acids (52, 55) rather than depending solely on glucose for respiration (28) indicate that starvation hypoglycemia (4, 7, 21, 37, 39-41) leading to malfunction of the thermoregulatory system of the brain is probably not the explanation for the decline in thermogenesis during starvation. The sustained blood glucose levels after midpontine transection and despite starvation is reminiscent of Claude Bernard’s piqfire diabttique (6)) in which insertion of a spatula into the floor of the fourth ventricle, at approximately the same level as in our experiments, produced hyperglycemia. Although we did not observe the excessive blood glucose levels as in his report, the still undefined neural determinants may be the same. This also remains to be decided by future experimentation. REFERENCES G. 1962.Temperatureregulationin the newbornlamb. V. Summit metabolism. A&r. J. Agric. Res. 13 : 100-121. 2. ANGELETTI, P. V., and R. LEVI-M• NTALCINI. 1970.Sympatheticnerve cell destruction in newbornmammalsby 6-hydroxydopamine.Proc. Nut. Acad. Sci. 65: 114-121. 3. BARD, P., and M. B. MACHT. 1958.The behavior of chronically decerebratecats, pp. 55-71.In “The NeurologicalBasisof Behavior.” Ciba Symposium,London, Little, Brown, Boston. 4. BARIC, I. 1953.La consummation d’oxyg&nedu rat nouveau-n&au tours du jeune. Acad. Serbe. Sci. 12 : 71-76. 5. BERGMEYER, H. 1963.“Methods of Enzymatic Analysis.” AcademicPress,New York. 1. ALEXANDER,
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