And Beta-Adrenergic Receptors Located, Respectively, in the Ventromedial and Lateral Hypothalamus* Sarah Fryer Leibowitz

Proceedings of the National Academy of Sciences Vol. 67, No. 2, pp. 1063-1070, October 1970

Reciprocal Hunger-Regulating Circuits Involving Alpha- and Beta-Adrenergic Receptors Located, Respectively, in the Ventromedial and Lateral Hypothalamus* Sarah Fryer Leibowitz

THE ROCKEFELLER UNIVERSITY, NEW YORK, N.Y. 10021 Communicated by Neal E. Miller, July 30, 1970 Abstract. The injection of adrenergic and adrenolytic drugs directly into the brain through permanently implanted cannulas has yielded results showing that food consumption in the rat is regulated by a hypothalamic alpha-adrenergic "hunger" system and a hypothalamic beta-adrenergic "satiety" system. The rats' differential responses to alpha-adrenergic and beta-adrenergic drugs injected into different hypothalamic sites indicate the following: (1) the lateral hypothalamic "feeding" center contains beta receptors, the activation of which produces satiation, presumably by inhibition of the lateral "feeding" cells; (2) the ventromedial hypothalamic "satiety" center contains alpha receptors, the activation of which produces eating, presumably by inhibition of the ventromedial "satiety" cells; and (3) the medio-lateral perifornical area of the hypothalamus contains both alpha and beta receptors, which lead to inhibition of the ventromedial or lateral hypothalamic centers respectively. It is suggested that the ventromedial and lateral hypothalamus are connected by reciprocal circuits, so that activation of the ventromedial center results in stimulation of the lateral beta receptors which inhibit the lateral "feeding" cells, and activation of the lateral center results in stimulation of the ventromedial alpha receptors which inhibit the ventromedial "satiety" cells.

In the rat hypothalamus there appears to exist an adrenergic mechanism con- sisting of both alpha and beta receptors which function antagonistically in the regulation of hunger in rats.'-' This antagonistic relationship exists between an alpha-adrenergic "hunger" system, which elicits eating behavior, and a beta- adrenergic "satiety" system, which suppresses eating behavior. A great deal of pharmacological evidence has accumulated in this laboratory to support the concept of a hypothalamic alpha- and beta-adrenergic hunger- regulating system. Grossman4 first showed that norepinephrine, when injected directly into the hypothalamus, induced an eating response in satiated rats. This norepinephrine eating response was found to be blocked by an alpha, but not a beta, antagonist. 6 In a recent series of studies," 3 I have tested other alpha-adrenergic agonists in the rat hypothalamus and have found that these drugs, like norepinephrine, enhance food consumption, both in satiated and food-deprived rats. However, when beta-adrenergic agonists were injected into the same hypothalamic site, they were found to suppress food consumption. The 1063 Downloaded by guest on September 26, 2021 1064 PHYSIOLOGY: S. F. LEIBOWITZ PRoc. N. A. S.

hunger-stimulating effect of the alpha agonists was found to be blocked by a hypothalamically-injected alpha-adrenergic antagonist and enhanced by a hypothalamically-injected beta-adrenergic antagonist, and the hunger-suppressing effect of the beta agonists was found to be blocked by the beta-adrenergic antagonist and enhanced by the alpha-adrenergic antagonist. Further evidence in support of these opposing alpha and beta hunger-regulating systems was obtained when the alpha- and beta-adrenergic antagonists were injected alone into the rat hypothalamus in relatively high doses, and they were found to affect food consumption in the expected direction.' The alpha antagonist suppressed food intake, presumably by blocking the hypothalamic alpha "hunger" system, and the beta antagonist enhanced food intake, presumably by blocking the hypothalamic beta "satiety" system. Problem. In the above studies, which demonstrated a neurochemical differentiation between a hypothalamic "hunger" system and a hypothalamic "satiety" system, all drugs were injected into the same anatomical site, namely, the perifornical region of the hypothalamus at the level of the anterior portion of the ventromedial nucleus. However, a great deal of both lesion and electrical stimulation work has shown that hunger and satiety in the rat are anatomically differentiated in other parts of the hypothalamus, with the ventromedial hypothalamus as a "satiety center" and the lateral hypothalamus as a "hunger center."7 The question arises as to whether there is a functional association between the hunger/satiety systems found to be differentiated anatomically and the hunger/satiety systems found to be differentiated neurochemically. It is suggested here that the two hypothalamic areas, lateral and ventromedial, which have been associated with hunger and satiety, respectively, are neurochemically different, and that the interaction that occurs between these hypothalamic hunger-regulating areas is mediated by alpha- and beta-adrenergic nerve connections. In an attempt to localize the alpha and beta hunger-regulating systems within specific areas of the hypothalamus, a study was carried out which varied the anatomical site of injection of several different alpha- and beta-adrenergic drugs. This localization study, in addition to providing information concerning the neurochemistry of the receptors in these areas, may answer questions concerning the location of the central site of action of particular hunger-regulating drugs, such as amphetamine, which are normally administered peripherally; the physio- logical role that the perifornical region plays in hunger regulation; and the significance and extent of drug spread after central injection. Five hypothalamic injection sites were chosen for the study, in which a series of alpha- and beta- adrenergic agonists and antagonists were tested. These sites, which were approximately at the same anterior-posterior level, were the ventromedial area, the dorsomedial area, the medio-lateral perifornical area, and two areas in the lateral hypothalamus. The results clearly demonstrated a neurochemical-anatomical relationship. Briefly, we found that the medial hypothalamus was sensitive only to the alpha drugs; that the lateral hypothalamus was sensitive only to the beta drugs; and, confirming our previous findings, that the perifornical region was sensitive to both the alpha and beta drugs. Downloaded by guest on September 26, 2021 VOL. 67, 1970 HYPOTHALAMIC HUNGER-REGULATING CIRCUITS 1065

Materials and Methods. Placement of cannulas: A total of 73 male albino Sprague-Dawley rats (approx 320 g) were used in these experiments. They were all stereotaxically implanted with unilateral chronic cannulas under Nembutal anesthesia according to the procedure described by Slangen and Miller.' All placements were aimed at one of five areas in the hypothalamus: two in the medial hypothalamus, two in the lateral hypothalamus, and one in between. Coordinates for the five placements were as follows. (1) The ventromedial hypothalamus (n = 21), with skull flat, was 2.7 mm behind bregma, 0.4 mm lateral, and 9.5 mm vertical. (2) The dorsomedial hypothalamus (n = 5), with skull flat, was 2.9 mm behind bregma, 0.7 mm lateral, and 8.7 mm vertical. (3) The medio-lateral perifornical area (n = 20), with the teeth bar 3.1 mm above the intra-aural line, was 0.0 mm (bregma), 1.3 mm lateral, and 8.2 mm vertical. (4) The first lateral hypothalamic placement (n = 21), with skull flat, was 2.8 mm behind bregma, 1.5 mm lateral, and 8.5 mm vertical. This placement has been found to be effective in experiments on self-stimulation and stimulus-bound eating. (5) The second lateral hypothalamic placement (n = 6), which is slightly more posterior, had DeGroot coordinates of 5.4 mm anterior, 1.8 mm lateral, and -2.7 vertical. All five placements, which were histologically verified at the completion of the experiments, were found to be at roughly the same anterior-posterior level. General procedure: All rats were maintained and tested on Purina lab chow pellets. The first series of tests was carried out on the rats while they were food- and water-satiated. The test, which was given in the morning every 2-3 days, consisted of measuring the rats' food intake during the first 90 min immediately after drug injection. After an interval of several days, the second series of tests was begun. These tests were carried out on the rats while they were food-deprived. The deprivation period, which lasted for 18 hr, extended from 4:00 p.m. on the day before the test day until 10:00 a.m. the next morning, when they were tested. These tests were given every 3-4 days and also consisted of measuring the rats' food intake during the first 90 min immediately after drug injection. Upon completion of each test, the rats received food and water ad lib. until their next period of deprivation. Drugs: The following drugs were used in these experiments. (1) l-Norepinephrine bitartrate (0.01, 0.02, and 0.04,mol). Peripheral experiments show this drug to have primarily alpha-adrenergic activity except with respect to the heart, where it acts as a beta-adrenergic agonist.8 In the central nervous system, norepinephrine also appears to be primarily alpha-adrenergic, although under special circumstances, such as in the presence of an alpha antagonist and at higher doses, some beta-adrenergic activity has been identified.3 Because of the relatively low doses used here, the present study is concerned only with norepinephrine's alpha-adrenergic action. (2) l-Epinephrine bitartrate (0.02 and 0.04 pmol). In the periphery, this drug is known to have both alpha- and beta- adrenergic activity.8 In the brain, it also appears to have both alpha and beta activity, depending upon dose.',3 In the present study, epinephrine is injected at the relatively low doses which were found to have primarily alpha-adrenergic effects. (3) l-Isopro- terenol bitartrate (0.10 and 0.05 umol), which, in the periphery as well as in the central nervous system, appears-to have only beta-adrenergic activity." 8 (4) d-Amphetamine sulfate (0.1 and 0.2 gmol). Amphetamine is known to have both alpha- and beta-receptor activity in the periphery and excitatory effects in the brain.8 Only recently has amphetamine been shown to have a potent beta-adrenergic action in the brain, which apparently mediates this drug's hunger-suppressing effect.2 In addition to its potent hypothalamic beta anorexic activity, amphetamine, at the same dose, appears to have a weaker alpha- adrenergic hunger-stimulating effect which was detected in the perifornical area of the hypothalamus in the presence of a beta antagonists The present study is concerned with both alpha and beta actions of amphetamine. (5) Phentolamine hydrochloride (0.32 /mol), an alpha-adrenergic antagonist. (6) Propranolol hydrochloride (0.35 JAmol), a beta-adrenergic antagonist. All drugs, except phentolamine, were dissolved in normal saline and injected directly into the hypothalamus via the same cannula in volumes of 0.2-0.5 1A. Phentolamine was Downloaded by guest on September 26, 2021 1066 PHYSIOLOGY: S. F. LEIBOWITZ PROC. N. A. S.

dissolved in distilled water and, because of its low solubility, had to be injected in a volume of 4 A1. Experimental tests: Most of the rats were carried through the entire series of drug injections. Control tests, in which the subjects were tested with only the control medium (saline), were interspersed between the drug tests according to a Latin square sequence. The rats, which were separated into five groups according to their hypothalamic injection placement, were subjected to the following five tests. (1) While food- satiated, they were injected with rather low doses of norepinephrine and epinephrine, which at these doses are potent alpha agonists. (2) While food-deprived, they were injected with isoproterenol, a potent beta agonist with essentially no alpha activity. (3) For their third test, the rats, while food-deprived, were injected with amphetamine, a potent beta agonist with weaker alpha activity. (4) They were then injected, while food-deprived, with a rather high dose of propranolol, the beta antagonist. (5) Finally, the rats were injected, while food-deprived, with a rather high dose of phentolamine, the alpha antagonist. Results. The medial hypothalamus, the perifornical hypothalamus, and the lateral hypothalamus were found to be differentially sensitive to the alpha- and beta-adrenergic drugs. The medial hypothalamus, especially the ventromedial area, was found to be reliably sensitive to the alpha agonists (which increased food intake) and the alpha antagonist (which suppressed food intake), but was essentially insensitive to both the beta agonists and the beta antagonist. Con- versely, the lateral hypothalamus was found to be reliably sensitive to the beta agonists (which suppressed food intake) and the beta antagonist (which increased food intake), but insensitive to the alpha agonists and the alpha antagonist. the perifornical area was unique in that it was found to be reliably sensitive to all of the alpha and beta drugs, which confirms our earlier findings.'-3 The results for all five hypothalamic injection sites are presented in Table 1. In the perifornical area, the alpha agonists, norepinephrine and epinephrine (at several relatively low doses), reliably enhanced food intake in satiated rats. In the same perifornical site, the food intake of hungry rats was reliably suppressed by the alpha antagonist, phentolamine, and the beta agonist, isoproterenol. Amphetamine, which has potent beta and weaker alpha activity, also reliably suppressed food intake in the perifornical rats. Finally, in these same rats, the beta antagonist, propranolol, reliably enhanced food consumption.9 In summary, all of these perifornical results support the hypothesis that the hypothalamus contains two antagonistic hunger-regulating systems, namely, an alpha-adrenergic "hunger" system, which elicits eating behavior, and a beta- adrenergic "satiety" system, which suppresses eating behavior. The ventromedial area was found to be reliably sensitive, in the same direction as the perifornical area, to the alpha drugs; the agonists increased food intake in satiated rats, and the antagonist suppressed food intake in hungry rats. How- ever, in contrast to the perifornical area, the ventromedial area was found to be insensitive to the beta-adrenergic hunger-suppressing effects of isoproterenol and amphetamine and to the hunger-stimulating effect of the beta antagonist, propranolol.9 Although amphetamine failed to exhibit its potent beta anorexic effect in the ventromedial area, it did exhibit the opposite effect-namely, a small but reliable enhancement of food intake in hungry rats. The dorsomedial area, which lies directly above the ventromedial area, was Downloaded by guest on September 26, 2021 VOL. 67, 1970 HYPOTHALAMIC HUNGER-REGULATING C1RCUITS 1067 TABLE 1. Dependence on site of hypothalamic injection of the effect of drugs on food intake. Amount eaten (g) in 90 min after hypothalamic injection Medio- Medial lateral Lateral Dose VMA DMA PA LA1 LA2 Group Drug (/Lmol) (n = 21) (n = 5) (n = 20) (n = 21) (n = 6) 1. Alphasadrenergicago- NS ... 0.6 0.9 0.2 0.7 0.7 nists in satiated rats NE 0.01 1. 9* 1.0 1. 0* NT NT 0.02 2.2* NT 1.9* 0.7 0.5 0.04 1.8* 1.9 2.7* 0.9 0.5 EPI 0.02 2.8* 0.6 1.9* NT NT 0.04 2.9* NT 2.8* 1.4 0.4 2. Alpha-adrenergic NS ... 10.8 7.1 8.8 9.4 6.2 antagonist in food- PHT 0.32 7.7* 4.6* 6.6* 10.1 6.2 deprived rats 3. Betaadrenergic ago- NS ... 8.4 4.9 8.4 7.7 9.8 nist in food-deprived ISOP 0.10 7.3 NT 4.6* 5.1* NT rats 0.15 NT 4.2 3.6* 3.9* 4.5* 4. Beta-adrenergic NS ... 9.3 7.1 8.6 6.0 6.3 antagonistinfood- PROP 0.35 8.4 7.0 10.5* 7.1t 7.0t deprived rats 5. Beta-adrenergic ago- NS ... 8.4 4.9 8.4 7.7 9.5 nist, withsome AMPH 0.1 9.1 NT 6.7* 6.1* NT alpha-adrenergic 0.2 9.9* 5.8 5.6* 5.6* 7.1* activity, in food- deprived rats Drugs were injected (in saline) into the ventromedial area (VMA), dorsomedial area (DMA), perifornical area (PA), or one of two lateral areas, one of them (LA,) slightly anterior to the other (LA2). NS, normal saline; NE, norepinephrine; EPI, epinephrine; PHT, phentolamine; ISOP, isoproterenol; PROP, propranolol; AMPH, amphetamine; n, number of animals in the group. NT, not tested. * Significantly different from NS control score at P < 0.01. t Significantly different from NS control score at P < 0.05. As in all other tests, each animal served as its own control. Thus, the appropriate statistic for assessing significance was a correlated T. The fact that such small differences in the mean between NS and Drug (PROP) condition were found to be significant was because the variance for within-animal comparisons was also small.

found to be somewhat sensitive to the alpha drugs, although to a considerably lesser extent than the ventromedial. The dorsomedial area, like the ventromedial, was found to be unresponsive to the beta agonists and the beta antagonist. The two lateral hypothalamic areas were found to be reliably sensitive, in the same direction as the perifornical area, to the beta drugs; the agonist, isoproterenol, suppressed food intake and the antagonist, propranolol, produced a very small but reliable enhancement of food intake.9 In the lateral areas, as in the perifornical area, amphetamine exhibited its potent beta hunger-suppressing effect, in contrast to the ventromedial area where amphetamine enhanced food intake. In contrast to both the perifornical and the ventromedial areas, the two lateral areas were found to be insensitive to the alpha agonists and the alpha antagonist. Discussion. The results of these localization experiments, in which the ventromedial, perifornical, and lateral areas of the hypothalamus were found to be different with respect to their responsiveness to alpha and beta drugs, strongly Downloaded by guest on September 26, 2021 1068 PHYSIOLOGY: S. F. LEIBOWITZ PROC. N. A. S.

suggest that the two opposing adrenergic systems, the alpha-adrenergic "hunger" and the beta-adrenergic "satiety" systems, are localized within different areas of the hypothalamus. Assuming that hypothalamically-injected drugs are act- ing on the receptors at or near the injection site, the localization results show that the ventromedial area, which responds only to the alpha-adrenergic and alpha- adrenolytic drugs, contains primarily the alpha-adrenergic "hunger" receptors; the lateral area, which responds only to the beta-adrenergic and beta-adrenolytic drugs, contains primarily the beta-adrenergic "satiety" receptors; and the perifornical area, which responds to all alpha and beta drugs, contains both the alpha- and beta-adrenergic receptors. Studies using lesioning and electrical-stimulation techniques have yielded considerable evidence for a dual hypothalamic hunger-regulating mechanism in which the ventromedial hypothalamus is the "satiety center" and the lateral hypothalamus is the "feeding center."7 Our pharmacological studies place the alpha "hunger" receptors in the ventromedial "satiety" area and the beta "satiety" receptors in the lateral "hunger" area. These results, which clearly reveal a close association between the anatomically differentiated "lateral- hunger" and "ventromedial-satiety" centers and the neurochemically differentiated "alpha-hunger" and "beta-satiety" systems, strongly suggest that the actions of the alpha and the beta receptors are both inhibitory. This provides a basis for postulating the following neurochemical hunger-regulating circuit. It is first suggested that the beta-adrenergic receptor system, which suppresses food consumption, mediates the inhibitory effect that the ventromedial "satiety" area is thought to exert on the lateral "feeding" area.7 This means that the beta-adrenergic "satiety" cells are located in, or more probably just medial to, what is called the ventromedial nucleus. (Fluorescence studies'0 have provided some preliminary evidence for adrenergic cells just medial to the ventromedial nucleus.) Presumably, these medial beta "satiety" cells send their axons to inhibit the lateral hypothalamic "feeding" cells, on which the beta "satiety" receptors are located, and thereby induce anorexia. It is further suggested that the alpha-adrenergic "hunger" receptors located in the ventromedial area stimulate hunger by inhibiting the hypothesized ventromedial beta-adrenergic "satiety" cells. Thus, eating induced by an alpha drug is a consequence of the inhibition of an inhibitory effect, or, in other words, dis- inhibition. A beautiful and symmetrical completion to this hypothesized neurochemical hunger-regulating circuit would be a direct link from the lateral hypothalamic "feeding" cells, which would presumably be alpha-adrenergic, to the ventromedial hypothalamic beta-adrenergic "satiety" cells. However, since the lateral hypothalamus appears to have no adrenergic cell bodies, it is suggested that the alpha-adrenergic "hunger-stimulating" cells, whose axon terminals inhibit the ventromedial beta-adrenergic "satiety" cells, are located in the lower brainstem (medulla and pons), where adrenergic cells have been identified." In addition to receiving "hunger-related" information from the peripheral nervous system, these lower-brainstem alpha cells might also receive information from the lateral hypothalamic "feeding" cells. This interneuronal link would serve as a relay Downloaded by guest on September 26, 2021 VOL. 67, 1970 HYPOTHALAMIC HUNGER-REGULATING CIRCUITS 1069

between the lateral "feeding" and ventromedial "satiety" areas; the axons from the activated lateral "feeding" cells would excite the lower-brainstem alpha "hunger-stimulating" cells, which would then increase their inhibition of the ventromedial beta "satiety" cells and thereby induce eating. One might think that the sensitivity of the perifornical area of the hypothalamus to both alpha and beta drugs is an indication that the drugs injected into this medio-lateral area diffuse to the alpha and beta receptors located, respectively, more medial and lateral to the fornix. If this were true, one would expect the perifornical effects to have a longer latency and to be smaller in magnitude. This was definitely not so. In fact, in some rats (not reported here), the perifornical area, as compared to any of the other hypothalamic areas, exhibited effects which were larger, had shorter latencies, and became apparent at smaller doses. It is therefore suggested that the unique sensitivity of the perifornical area to all drugs indicates the presence of both alpha and beta receptors in this area. This suggestion is supported by fluorescence evidence which shows the adrenergic terminals in the perifornical region to be even more dense than the adrenergic terminals located in, or medial to, the ventromedial area and in the lateral area.1' It would be premature to conclude that this medio-lateral site actually represents an important hunger-regulating center; however, we do suggest that the perifornical area interacts with the neurochemically-differentiated medial and lateral hypothalamic hunger-regulating centers, possibly via connections which allow its alpha receptors to inhibit the ventromedial "satiety" cells and its beta receptors to inhibit the lateral "feeding" cells. The results with amphetamine present an intriguing picture which not only increases our knowledge of amphetamine's central actions but also supports our hypothesized lateral-medial neurochemical differentiation. In the lateral hypothalamus, where beta but essentially no alpha receptors are located, amphetamine exhibits a potent beta effect of hunger suppression, which can be blocked by a beta antagonist.3 In the perifornical area, where both alpha and beta receptors are located, amphetamine exhibits a potent beta anorexic effect which can be blocked and converted into a weak alpha hunger-stimulating effect by prior treatment of the area with a beta antagonist.2 Finally, in the ventromedial area, where alpha but essentially no beta receptors are located, amphetamine exhibits a small alpha effect of hunger stimulation, which can be blocked by an alpha antagonist.3 In this region, a beta antagonist is not needed to unmask amphetamine's weaker alpha effect, since this area is essentially devoid of beta receptors. The following statements can, thus, be made concerning the central action of amphetamine. In the hypothalamic hunger-regulating system, the drug amphetamine has two adrenergic effects: an alpha hunger-stimulating effect and a beta hunger-suppressing effect. The beta-receptor action of amphetamine, which takes place in the lateral part of the hypothalamus, is found to be more potent than its alpha-receptor action, which takes place in the medial part of the hypothalamus. It is suggested that the lateral hypothalamic beta-receptor mechanism provides a neurochemical-anatomical basis for the anorexic effects Downloaded by guest on September 26, 2021 1070 PHYSIOLOGY: S. F. LEIBOWITZ PROC. N. A. S.

produced by systemically-injected amphetamine. Systemic amphetamine acts on these lateral beta receptors, which then leads to inhibition of the lateral "feeding" cells and consequently to reduced hunger. This dominant beta anorexic effect masks the less potent alpha effect which amphetamine has in the medial hypothalamus. I am grateful to Miss Sandra Schnall and Mrs. Lilian Quintao for their excellent technical assistance and to Dr. N. E. Miller for his helpful criticisms of the manuscript. I also thank Smith Kline and French Laboratories, Ayerst Laboratories, Sterling-Winthrop Research Institute, and Ciba Pharmaceuticals for their generous supply of drugs.

* This research was supported by USPHS grant MH 13189 and by a grant from Hoffmann- La Roche. 1Leibowitz, S. F., Nature, 226, 963 (1970). 2 Leibowitz, S. F., Proceedings, 78th Annual Convention, American Psychological Asociation, 1970 p. 813. 3 Leibowitz, S. F., manuscript in preparation. 4 Grossman, S. P., Amer. J. Physiol., 202, 872 (1962). 6 Booth, D. A., J. Pharmacol. Exp. Therap., 160, 336 (1968). 6 Slangen, J. L., and N. E. Miller, Physiol. Behav., 4, 543 (1969). 7Mayer, J., and D. W. Thomas, Science, 156, 328 (1967). 8 Goodman, L. S., and A. Gilman, The Pharmacological Basis of Therapeutics, 3rd edition (New York: Macmillan, 1965). 9 Another experiment was carried out in which we tested the effectiveness of centrally- injected propranolol, as a function of hypothalamic injection site, in blocking the anorexic effects which have been shown to be produced by the beta agonist, isoproterenol, when peripherally injected.12 Perifornical or lateral injection of a small dose of propranolol reliably reduced anorexia induced by systemic isoproterenol, whereas ventromedial injection of propranolol did not have this counteracting effect. 10 Steiner, S., and P. Longa, personal communication. 11 Fuxe, K., Acta Physiol. Scand., 64 (Suppl. 247), 37 (1965). 12 Conte, M., D. Lehr, W. Goldman, and M. Krukowski, Pharmacologist, 10, 180 (1968). Downloaded by guest on September 26, 2021