The ventromedial hypothalamus and metabolic adjustments of feeding behavior

BEHAVIORAL BIOLOGY, 9, 65-75 (1973), Abstract No. 266R

The Ventromedial Hypothalamus and Metabolic Adjustments of Feeding Behavior 1

JAAK PANKSEPP

Department of Psychology, Bowling Green State University, Bowling Green, Ohio 43403 Rats with bilateral lesions in the VMH did not show the usual preference reversal seen in intact rats during free access to concentrated (25 or 50%) vs dilute (5 or 10%) glucose solutions. In addition, unlike intact rats or rats with bilateral lesions of the lateral hypothalamus, rats with medial hypothalamic lesions did not exhibit prolonged depression of food intake following intraperitoneal injection of glucose. Finally, intraperitoneal injections of [14C]'-labeled glucose which inhibit food intake more than identical intragastric loads produce larger absolute and relative levels of radioactivity in the medial hypothalamus than lateral hypothalamus. These findings indicate the medial hypothalamus modulates feeding behavior in response to metabolic states, and suggests that these adjustments may be due to differential handling of incoming nutrients by the medial hypothalamus as compared to other parts of the brain.

Regulatory control of feeding behavior implies appropriate changes in food intake in response to both increases and decreases of the regulated factor. A recent proposal that the ventromedial hypothalamus (VMH)mediates long-term regulation of energy balance rather than short-term satiety (Panksepp, 1971b) derived support from the finding that VMH-lesioned rats fail to increase feeding in response to food deprivation. This was taken to indicate VMH participation in excitatory control of feeding in response to depletion of body nutrient stores. Unfortunately, aside from the propensity of VMH-lesioned animals to overeat and become obese, empirical evidence for the loss of an inhibitory function after VMH lesions has never been found. In fact, the evidence indicates that VMH animals can adjust food intake appropriately when the caloric density of their diet is decreased (Thomas and Mayer, 1968) or increased (Carlisle and Stellar, 1969). Still, it is possible that these findings only reflect the capacity of VMH-lesioned animals to respond to short-term postprandial satiety signals (Panksepp, 1971a) and accordingly, do 1This research was partially supported by NIGMS postdoctoral Fellowship (1 FO GM 44047-01) and NIMH Grant TOt MH 10625. I thank J. Jalowiec for valuable suggestions on the manuscript. 65 Copyright © 1973 by Academic Press, Inc. All rights of reproduction in any form reserved.

66

PANKSEPP

not constitute critical evidence for the intact presence of a long-term regulatory system. Only certain experiments, designed to minimize focus on short-term signals, can indicate that finer adjustments of a longer-term mechanism no longer intervene after VMH damage-leading to cumulative errors rather than stable states of energy balance. In other words, operation of the long-term mechanism may be difficult to observe directly because it manifests itself in behavior by modulating the effectiveness of short-term satiety signals (Panksepp, 1973). The following experiments were designed with such difficulties in mind. In general, the results supported the idea that VMH-lesioned rats do not adjust food intake appropriately in response to metabolic signals which modify feeding in normal rats.

GENERAL METHODOLOGY Mature (120-180 days old) Sprague-Dawley male albino rats were used in all experiments. Rats were individually housed in standard wire cages and had free access to lab chow (powdered Spillers Small Animal Diet presented in 1-1b glass jars with metal lids having 2 in. diam feeding holes) and water except as indicated. Food intakes were measured to 0.1 g. Lighting was on a 12-hr light, 12-hr dark cycle in all experiments. For surgery, rats were anesthetized with 40 mg/kg Nembutal. Lesions were produced by passing 1-2mA current for 15-30sec between a brain anode (a stereotaxically positioned stainless steel No. 0 insect pin insulated except for 3/4 mm at the tip) and a rectal or ear-bar cathode. LHA-lesioned rats, all of which exhibited total aphagia and adipsia after surgery (Mean + SD: 11 (+ 9) days and 12 (+ 7) days, respectively), were given conventional nursing care (i.e., intragastric feeding and watering) until spontaneous feeding and drinking returned. The data collected in these experiments (Expt 2) were obtained when animals had resumed eating and drinking water for approximately 3.5 too. During that interval they served in other experiments related to feeding behavior. VMH-lesioned rats were tested approximately 2 mo after surgery-during what is traditionally considered the static phase. It should be noted, however, that the immediate postoperative hyperphagia was not large in the group of animals used (ranging approx 20-50% above control levels) possibly because males and a powdered diet were used. After testing, all rats were anesthetized with an overdose of Nembutal and perfused with physiological saline and 10% formalin. Brains were removed and fixed. Frozen sections were cut through the extent of brain damage and stained with either cresylecht violet or by the Kl~iver-Barrera method.

VMH AND METABOLICADJUSTMENTOF FEEDING

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EXPERIMENT 1 When given free access to two glucose solutions, one of high concentration and the other low, normal rats initially prefer the more concentrated solution. After several days, however, they begin to take more and more of the weaker solution and eventually exhibit a prolonged reversal in preference (Booth, Lovett, and McSherry, 1972; Jacobs, 1958). This shift may reflect matabolic readjustment of preference behavior. The following experiment was run to determine whether VMH-lesioned rats behave likewise. Method. Six normal and six VMH-lesioned rats were used. All animals had free access to food and water for 2 wk prior to testing. At the start of testing the mean weight of normal rats was 426 g and of VMH-lesioned rats, 593 g. Each animal was given 11 days of free access to 25% w/v and 5% w/v d-glucose dissolved in tap water. Solutions were given in graduated drinking bottles and daily intakes were measured to 1.0 ml. Solutions were changed daily and the position of bottles was reversed each day. At the end of this ll-day period, all rats were shifted to a choice between 50% w/v and 10% w/v glucose for an additional 7 days. Water was not available during either test period. Results. Mean daily intakes of glucose solutions are depicted in Fig. 1. Normal rats exhibited an initial preference for 25% glucose (p < .05-.001 for days 1-5). By days 6-11 no differences in volume intake from the two solutions was observed (p < .10 for all days). The VMH-lesioned rats exhibited an unmodified preference for 25% glucose for the duration of testing (p < .001, for days 1-11). During the second phase, normal rats immediately preferred the 10% solution (p < .001, for days 12-18), while lesioned rats continued to imbibe the more concentrated solution (p < .01-.001, for days 12-14). By days 15-18, however, the two solutions were almost equally preferred. Discussion. When given access to two glucose solutions of different concentrations, the preference behavior of VMH-lesioned rats did not change in a manner similar to that of normal rats. If the preference shift in normal animals is due to some metabolic consequence of high levels of incoming glucose, it would be reasonable to conclude that VMH lesions abolish the capacity to respond to that metabolic effect. Alternatively, of course, it could be concluded that the differential effect is due to lesion-induced hyperreactivity to taste. Whether this alternative hypothesis really embodies an empirically testable distinction is uncertain. According to one theory both are subsumed under a unitary mechanism which governs food intake (Panksepp, 1971b).

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EXPERIMENT 2 Intraperitoneal (ip) injections of glucose depress feeding considerably more than comparable intragastric (ig) loads (Booth, Lovett, and Simpson, 1970). The mechanism underlying this differential is not known, but for the purposes of the present experiment, the possibility was entertained that ip loads depress feeding b.y having an unusually prolonged or powerful influence on mechanisms which mediate long-term regulation of energy balance. Accordingly, it was hypothesized that at some point in time ip loads of glucose would depress feeding more in normal than in VMH-lesioned animals. Three experiments were run. In the first, the effects of ip and ig injections of glucose were compared in normal rats. In the second, the effect of ip injections of glucose were compared in normal and VMH-lesioned rats. In the third, the same comparison was made in VMH-lesioned, LHAqesioned, and control animals. Procedure. Ad lib. food intake of 12 normal rats was measured for 3 days. Then six of the animals were deprived for 1 hr and injected ip with 8 ml of 25% w/v glucose. Similarly, the other six were loaded ig with the same amount. The injections were given just before the onset of the dark cycle. Food intakes were measured after 1,4,7, and 23 hr on the first day. On the

69

VMH AND METABOLIC ADJUSTMENT OF FEEDING

second and third days they were again deprived for the last hour of the light cycle and food intakes were measured after 1 and 23 hr. Three different animals were injected with the same amount of glucose ip and 24 hr later they were anesthetized and disemboweled. Peritoneal fluid was collected and assayed for glucose by the glucose-oxidase method. In the second experiment eight normal (mean wt 441 g) and eight VMH-lesioned animals (580 g) having continual access to food and water, were injected ip with 8 ml of 25% w/v d-glucose. Food intakes were monitored for 5 days thereafter. In the third experiment, 12 normal (449 g), 8 VMH-lesioned (591 g) and 10 LHA-lesioned (400 g) rats were injected ip with 4 ml of 50% w/v glucose and food intakes were measured for 3 postinjection days. The animals used in the previous experiment were also used in this one. Results. In normal animals ip loads of glucose depressed feeding more than ig loads (Fig. 2). In fact, food intake after ip loads was still depressed on the second and third days after treatment (p < .01, for each day).

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The amount of glucose remaining within the peritoneal cavity 24 hr after ip injection ranged from 2 to 4 rag. Since each animal had been injected with 2 g of glucose, more than 99% of the glucose had been absorbed by the start of the second postinjection d a y - a point in time when food intake was still inhibited. The VMH-lesioned rats exhibited as much depression o f food intake as controls on the first day after ip injection of glucose (Fig. 3), but on the second to fifth days they were eating normal amounts of food whereas controls still exhibited some hypophagia (p < .01, for days 4-5). This effect was replicated in the third experiment (Fig. 4). In contrast to VMH-lesioned rats, however, animals with bilateral LHA damage responded in a manner similar to controls. Discussion. The present data replicate results reported by Booth et al. (1971): ip injections of glucose depress feeding more than ig injections. Although the reasons for this effect are not dear, undoubtedly part of the effect is due to distress. Rats which received large ip injections of glucose 8 ml 25% Glucose i.p. 100

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DAYS Fig. 4. Daily food intakes as percentage of control after intragastric injection of 4 ml of 50% w/v glucose. The mean 100% intake level of normal rats was 30.0 g, of VMH-lesioned rats 28.1 g, and of LHA-lesioned rats 24.6 g. show signs of discomfort for about an hour after injection. A day later, however, the animals appear normal, and as indicated by analysis of peritoneal fluid, negligible amounts of glucose remained at the site of injection. At that point in time, normal animals still exhibited depressed feeding while intake of VMH lesioned rats was back to control levels. It is unlikely that the maintained hypophagia in normal rats was caused by lingering malaise-for by that reasoning the hyperemotional VMH-lesioned animals (see Grossman, 1967) should have exhibited the most intense inhibition of feeding. The data thus support the idea that VMH-lesioned rats respond deficiently to a metabolic signal arising from prior nutrient administration.

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PANKSEPP EXPERIMENT 3

After intragastric loads of [14 C] -labeled glucose, the VMH contains higher levels of radioactivity than the rest of the brain (Panksepp, 1972). If this differential is the manifestation of an error signal by which nutrient intake is regulated, and if ip injections of glucose generate a larger error signal than ig loads of glucose, then ip injections of [14C]-labeled glucose should lead to larger differences in radioactivity between VMH and LHA than identical intragastric loads. Procedure. Mature male ablino rats (34) were used; 17 animals received ig loads of 18 ArC; of uniformly labeled [d-14C]-labeledglucosein 4.5 rnl of 50% w/v unlabeled d-glucose. Fifteen animals re~ceived the same load via the intraperitoneal route. All rats were sacrificed 30 hr later with an overdose of Nembutal (approx 100 mg/kg), brains and liver samples were removed, and promptly frozen on dry ice. Two milliliters of blood were drawn from the heart and after centrifugation, 20-30 mg plasma samples were taken. Smaller samples were also dissected from brain and liver and weighed to 0.1 (+ 0.1) mg on a microtorsion balance. VMH and LHA samples were taken from each brain. The LHA samples consisted of bilateral tissue pieces between the fornices medially, cerebral peduncles laterally, and zona inserta dorsally. The anterior-posterior extents of both samples extended from the premammillary area caudally to the border of the optic chiasma rostrally. Immediately after weighing, tissue samples were placed in counting vials containing 0.3 ml of tissue solvent~(Solune®) and allowed to dissolve for 24 hr, whereupon 3 ml of toluene scintillation fluid was added to each vial (4 g PPO and 0.2 g POPOP per liter of toluene). Samples were counted for 20-50 min in a Beckman scintillation counter to 3-5% accuracy. Counts per minute were corrected for background radiation and expressed as cpm/lO mg wet wt tissue. Results. Counts per minute/10 mg tissue are summarized in Table 1. After both treatments the VMH contained more radioactivity than the LHA Co < .002, in both cases). Furthermore, the mean percentage of increase after intragastric injections was 12.8% whereas the increase after intraperitoneal injections was 21.7% (t9 < .001). Absolute levels of radioactivity were higher after ip injections than after ig injections. The plasma also had significantly higher levels of radioactivity after ip injections than after ig injections ( p < .01) but the absolute levels were considerably lower than brain levels. Liver showed no differences in levels after the two treatments although absolute levels were the highest of all the tissues. HISTOLOGY The VMH lesions were similar to the ones that have been previously used in this laboratory. In general, they were large and symetrical, situated at

VMH AND METABOLICADJUSTMENTOF FEEDING

73

TABLE1 CPM/10 mg Tissue (± SEM) 30 hr after Intragastric or Intraperitoneal Loads of 18 #Ci of [i 4C]-Labeled Glucose in 4.5 ml of 50% w/v d-Glucose w/v D-Glucose Tissue

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the base of the brain, similar to those depicted in Panksepp (1971b). Damage usually extended from the caudal border of the optic chiasma to the premammiUary region, laterally to the fornices and dorsally to the zona inserta. The LHA lesions were immediately medial to the internal capsule and optic tract, extending throughout the rostrocaudal extent of the hypothalamus. Dorsally they extended through the zona inserta, often including parts of the ventral nucleus of the thalamus. Ventrally, they extended to the level of the fornix, usually reaching the base of the brain in the caudal hypothalamus.

GENERAL DISCUSSION These data are consistent with the idea that the VMH participates in adjusting ingestive behavior in response to a metabolic signal. VMH lesions attenuate preference shifts normally exhibited by rats given simultaneous long-term access to two glucose solutions of different concentrations. Whereas normal rats tend to shift from the concentrated to the dilute glucose solution after several days of access, VMH-lesioned rats continue taking the concentrated solution. Although the nature of the signal which mediates the preference change in intact animals is not yet certain, it would be reasonable to suppose that an internal consequence of high glucose intake may produce a strong signal of body nutrient repletion leading to learned and unlearned adjustments of feeding behavior which tend to reduce that signal. This could be accomplished either by reducing absolute intake of the concentrated solution while maintaining a preference for it, or by the shift of preference to a more dilute solution. The normal rat tends to choose the second option; the VMH-lesioned animal chooses neither.

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PANKSEPP

Alternatively, the failure of lesioned rats to shift may only indicate the strong taste reactivity of such animals. One could argue that VMHqesioned rats still have an intact mechanism which monitors the long-term metabolic consequences of incoming glucose, but being verily "magnetized" by the sweetest taste available, they disregard those signals. Whether this conceptual alternative really embodies a viable scientific option is not clear. Until an empirical test is proposed to disentangle the two processes, the present writer prefers to view the two concepts as nonindependent ways of viewing the operation of a unitary mechanism: VMH-lesioned animals being "magnetized" only because their food intake is no longer properly modulated by body nutrient stores. A second line of evidence pointing toward VMH participation in long-term regulation of feeding is the finding that VMH-lesioned rats are somewhat less sensitive than normal rats to the long-term anorexigenic effects of glucose injected into the peritoneal cavity. Since that behavioral differential appears a considerable time after the glucose injection, it is reasonable to suppose that reactivity to a metabolic signal arising from the glucose differentiates between normal and VMH-lesioned animals. If the effect were a secondary consequence of other processes, such as thirst or emotional reactivity, differential results should have been observed during the initial postinjection day. Further, the observation of a predicted C 14 distribution differential between VMH and LHA at a point in time a behavioral differential was being observed, suggests that the metabolic transaction which governs behavioral responses to body nutrient repletion may transpire locally at the medial hypothalamus (Expt 3). To the contrary, it could be argued that the distribution differential merely reflects a midline vs laterally placed structure effect. Although that may be a contributory cause, it cannot be the whole explanation, for such reasoning should predict additive effects of different routes of nutrient administration. In comparison to the intragastric treatment, intraperitoneal injections lead to a larger increase of radioactivity in the medial hypothalamus (+ 21.7%) than in the lateral hypothalamus (+ 12.8%). Such a result is not readily explained as a passive artifact of either structure relationships or hemodynamic factors. The present author has favored characterizing the feeding control process which is elaborated in the VMH as reflective of long-term body nutrient depletion-repletion rather than of short-term satiety (Panksepp, 1971b, 1972). That conclusion coincides well with the nature of the present findings. In both behavioral experiments (Expts 1,2) differences between VMH and control groups appeared some time after institution of experimental treatments, indicating that the appearance of differential behaviors probably required ingested glucose to participate in relatively long-term metabolic events. The possibility that local metabolism of nutrients within the medial

VMH AND METABOLIC ADJUSTMENT OF FEEDING

75

hypothalamus is the critical event in regulation o f energy intake (Panksepp, 1972) is once again suggested by the findings o f Expt 3.

REFERENCES Booth, D. A., Lovett, D., and McSherry, G. M. (1972). Postingestional modulation of the sweetness preference gradient. J. Comp. Physiol. Psychol. 78, 485-512. Booth, D. A., Lovett, D., and Simpson, P. C. (1970). Subcutaneous dialysis in the study of the effects of nutrients on feeding. Physiol. Behav. 5, 1201-1203. Carlisle, H. J., and Stellar, E. (1969). Caloric regulation and food preference in normal, hyperphagic, and aphagic rats. J. Comp. Physiol. Psychol. 69, 107-114. Grossman, S~P. (1966) The VMH: a center for affective reaction, satiety or both? Physiol. Behav. 1, 1-10. Jacobs, H. L. (1958). Studies on sugar preferences: I. The preference for glucose solutions and its modification by injections of insulin. J. Comp. PhysioL Psychol. 51, 304-310. Panksepp, J. (1971a). Is satiety mediated by the ventromedial hypothalamus? Physiol. Behav. 7, 381-384. Panksepp, J. (1971b). A re-examination of the role of the ventromedial hypothalamus in feeding behavior. Physiol. Behav. 7, 385-394. Panksepp, J. (1972). Hypothalamic radioactivity after intragastric glucose-14C in rats. Amer. J. Physiol. 223, 396-401. Panksepp, J. (1973). A re-analysis of feeding patterns in the rat. J. Comp. Physiol. Psychol., 82, 78-94. Thomas, D. W., and Mayer, J. (1968). Meal taking and regulation of food intake by normal and hypothalamic hyperphagic rats. J. Comp. Physiol. Psychol. 66, 642-653.