Effect of fenfluramine on the electrical activity of the hypothalamic feeding centers

Physiology and Behavior. Vol. 7, pp. 433--438. Pergamon Press, 1971. Printed in Great Britain

Effect of Fenfluramine on the Electrical Activity of the Hypothalamic Feeding Centers ' G. S. C H H I N A , H. K. K A N G , B. S I N G H A N D B. K. A N A N D

Department o f Physiology, All-India Institute of Medical Sciences, New Delhi-16, India (Received 23 April 1971) CHHI~A, G. S., H. K. KANG,B. SINGHANDB. K. ANAND. Effect offenfluramine on the electrical activity of the hypothalamic feeding centers. PHYSIOL.BEHAV.7 (3) 433-438, 1971.--Electrodes were stere~taxically implanted in the lateral hypothalamic feeding center and medial satiety center as well as in other hypothalamic and cerebral regions in male rhesus monkeys. Electrical activity of these regions was recorded electroencephalographically, before and following iv injections of Fenfluramine given dailY for 7 -10 days in doses of 1.5 mg]kg and 3 mg/kg body-weightrespectively. In addition changes in the eating behavior, daily food intake, body weight and general behavior were observed. In another set of animals arteriovenous glucose differences (to provide indices of glucose utilization in the body) in response to Fenfluramine injections were estimated. Fenfluramine in doses of 1.5 mg/kg gradually resulted in slow wave activity in the feeding center, which became more pronounced after subsequent injections thus demonstrating a cumulative effect. This coincided with anorexic behavior and decrease in food intake. The activity of the satiety center changed to low voltagefast response, specially in starving animals. Arteriovenous glucose estimations suggest that the effects of Fenfluramine may be due to the increased level of glucose utilization in the body. Injections in doses of 3 mR/kShowever produced a generalizeddrowsy response. Appetite

Depressingdrugs

EEG activity

hypothalamic feeding centers

THE ROLE of hypothalamus in the regulation of feeding behavior is now well recognised. It contains two opposing mechanisms; a mechanism in the lateral hypothalamus which initiates feeding and has been designated as feeding center and another one in the medial hypothalamus which brings about satiation and has been named as satiety center [4, 8]. Various suggestions have been put forward to explain the activation of these hypothalamic centers both after food has been taken (satiety), and in hunger after the food has been disposed [3]. In addition to the stimuli arising from the digestive tract [10], certain changes occurring in the milieu interieur as a result of feeding, act as signals to these centers. It has been demonstrated that there is activation of the satiety center and inhibition of the feeding center in response to an increase in the level of glucose utilization in the body [5-7]. A number of pharmacological preparations influence appetite and some of these as preludin [9], cyproheptadine [12] and 2-deoxy-D-glucose [13] have been shown to affect appetite by changing the activity of the hypothalamic feeding centers in response to changes in the level of glucose utilization. Amphetamine, another commonly used appetite depressing drug, however has been shown to directly influence the activity of the satiety center [11] without possibly any change in the level of glucose utilization. Fenfluramine, an amphetamine derivative, has a strong

appetite depressing action, without producing any increase in sympathetic activity. The present investigations were undertaken to find out the effects of this compound on the electrical activity of the hypothalamic feeding centers. MATERIALS AND METHODS

A total of 8 male rhesus monkeys, weighing between 4.5-5 kg have been used for these studies. These were fed ad lib a diet consisting of bread, bananas and ground-nuts. Records of daily food intake, body weight and general and feeding behavior of these animals were kept throughout the period of study, both before and after the administration of drug. These animals were given daily injections of Fenfluramine and the effects of these on the electroencephalographically recorded activity of the hylSothalamic and other brain regions were observed.

Implantation of Electrodes and Recording The animals were anesthetized with intraperitoneal nembutal 35 mg/kg body weight and bipolar electrodes, non-insulated at their tips and joined together by Plexiglas with their tips 2 mm apart, were implanted stereotaxicaUy [1] in different hypothalamic and other brain regions. In each animal about 5 electrodes were implanted, 2-3 in different

1Fenfluramine 1-(metatrifluoro methyl-phenyl)-2ethylamino propane (Chlorohydrate) was kindly supplied by Selphram Laboratories Ltd., London, U.K. 433

434

CHHINA, KANG, SINGH AND ANAND

areas of the hypothalamus and the remaining in extrahypothalamic regions. The distribution of these electrodes is shown in Table 1. The upper ends of the electrodes were connected to a miniature radio tube socket which was embedded with dental cement on to skull. Bipolar EEG recording was made through these electrodes in unanesthetized animals restrained on a monkey chair. Before giving the drug, recordings were taken for a few days mostly from animals which had been starved overnight and in some others after normal feeding. Daily drug injections were then given and EEG recordings continued for about a week, before as well as for 3-5 hr after administration of the drug on any day. The recordings were further continued after stoppage of drug injections.

Fenfluramine Administration The experimental animals were divided into two batches of four each. One batch received daily injections of Fenfturamine in doses of 3 rng/kg body weight, dissolved in 20 cm 3 saline. As this dose resulted in pronounced drowsy behavior in the animals, the other batch of four animals received daily injections of drug in doses of 1.5 mg/kg body weight dissolved in 10 cm 3 saline. Injections were given intravenously in the morning and repeated daily for about 7-10 days. EEG recordings were taken on all these days, as well as for another week after stoppage of injections.

Blood Glucose Estimation Blood glucose estimations could not be carried out in the experimental animals, in view of the difficulty of arterial and venous punctures in the unanesthetized state. In another set of anesthetized animals Fenfluramine was given intravenously in doses of 1.5 mg/kg body weight and samples of arterial and venous blood drawn from femoral vessels, before injection as well as at intervals of 20-30 min after injection for about 3--4 hr. Blood samples were estimated for their glucose content by modified King's method [14] and arteriovenous glucose differences worked out to provide indices of glucose utilization in the body.

Disposal of Aninlals and Locating Electrodes After completion of the study anesthetized animals were killed by intracardiac formaline injection, brain removed, serially sectioned and examined for exact location of electrodes. RESULTS

EEG Characteristics of Hypothalamic Centers EEG responses from the medial satiety area in fed and awake animals normally consisted of low voltage fast activity (Fig. 3) while lateral feeding area activity was slower as com-

pared with the medial region. Other regions such as anterior hypothalamus, posterior hypothalamus, amygdala and cerebral cortex also showed low voltage fast activity. In starved animals however activity of the medial hypothalamic region was slower (Fig. 1) as compared to that of lateral hypothalamus, which was low voltage fast. Thus the physiological activation of these areas is represented by fast (low voltage) activity while slow wave activity denotes inactivation. (1) Effects of 1.5 mg/kg Fenfluramine on EEG Activity (a) Effect of first injection. Within about 2-3 min of the first injection changes in the electroencephatographically recorded activity of the hypothalamic centers appeared. These changes were most pronounced in the lateral hypothalamus (feeding center) resulting in slowing in that area (Figs. 1 and 2). Simultaneously, in starved animals, which had some slow wave activity in the ventromedial area (satiety center), it gradually became faster (Figs. I and 2). On the other hand, in fed animals in which medial hypothalamic activity was already fast, no further significant change was observed with Fenfluramine (Fig. 3). The activity of other areas of brain also sometimes showed slight slowing after about 5-10 rain of the injection (Fig. 1). Cortical activity, however, showed some slowing only after about half an hour. The changes in the activity of hypothalamic centers persisted for about 1½ hr before returning to original pattern, while the cortical slowing lasted for only about 15 min. (b) Effect of subsequent daily injections. The second injection produced EEG changes in the hypothalamic centers and other areas, similar to the first injection but a bit more pronounced. These also lasted for over 2 hr following the injection and even continued beyond. The EEG activity after the third injection was also similar, but more pronounced and in the lateral hypothalamus intermittent slow bursts lasting for 3-20 sec also appeared. The slowing in the other areas of brain including the cortex was much less pronounced as compared to the lateral hypothalamus. On subsequent daily injections, slowing in the lateral hypothalamus commenced almost immediately after the injection and could be continuously recorded from this region (Figs. 1-4). The other brain areas also showed some slowing which appeared to some extent even in the records taken from medial hypothalamus. By about the fifth day of continued daily i ~ i o n s , EEG activity of all the brain areas showed varying degrees of slowing, maximum slowing however being present in the lateral hypothalamic region. A certain degree of slowing was observed even before giving the injection which became more pronounced after the injection. It thus appeared that Fenfluramine injections produced some cumulative effects.

TABLE 1 DISTRIBUTION OF ELECTRODES IN DIFFERENT REGIONS OF BRAIN

Hypothalamus No. of Monkeys

Anterior

8

3

Satiety Center 5

Feeding Center 5

Posterior 2

Orbital Surface of Frontal Lobe 2

Anterior Perforate Caudate Substance Nucleus 2

1

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FIG. 1. Bipolar EEG recordings from hypothalamus and cortex of a male monkey, taken on different days 16 hr after the last meal, before starting injections of Fenfluramine, and at various intervals after giving the first, third and fourth daily injection of Fenfluramine. Vertical lines against each tracing represent 50 ~.u calibration.

FIG. 3. Bipolar recordings from a male monkey, taken on each day 2-3 hr after the last meal, before starting injections of Fenfluramine, and at different intervals after giving the fourth and fifth daily injection of Fenfluramine. The last record was taken 5 days after discontinuation of the injections. Vertical lines against each tracing represent 50 ~.V calibration.

(facing page

434)

FENFLURAMINE AND HYPOTHALAMIC ACTIVITY

435

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(c) Effect of stopping the injections. If the daily drug injections were stopped at this stage, the generalized slowing tended to disappear from other areas becoming localised to the lateral hypothalamic area by about the third day after stopping the drug. It, however, took about 4--6 days for the EEG changes in the hypothalamic centers to completely reverse to the original pattern. This again demonstrated the cumulative effect of the drug. Fenfluramine in doses of 1.5 rng/kg body weight thus produces gradually increasing slow activity in the lateral hypothalamus, which after about throe to four injections becomes almost persistent. Simultaneously, medial hypothalamic activity gradually becomes faster. Its pronounced

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anorectic effect could, therefore, be explained on the basis of these changes. Its continuous use even in this dosage results in some generalised slowing, which may result in a certain amount of drowsiness. This effect is much pronounced with higher dosages. (2) Effect of 3 mg/kg Fenfluramine on EEG Activity (a) Effect offirst injection. Within about 3 rain a generalised slowing appeared simultaneously in all the subcortical areas. Shortly after that cortex also developed slow waves of lower voltage (Figs. 5 and 6). The activity of the various subcortical areas further became slower with some increase in amplitude, but the cortical activity remained slow but of low voltage. These effects gradually disappeared after about I t hr when only some intermittent bursts of slow activity were seen to occur in the subcortical areas for some more hours. (b) Effect of subsequent daily injections. The second injection on the next day produced a similar but more lasting effect. The slow activity continued unchanged for about 4-6 hr. The slow activity becomes more or less generalised after subsequent injections and persists to some extent throughout the period before the next injection, which makes it further pronounced. (3) Effects of Fenfluramine on Glucose Utilization Fenfluramine injections in doses of 1.5 mg/kg body weight given to normal animals, generally resulted in increase in the arteriovenous glucose differences (glucose utilization) which were noticeable within about 30 min of the injection and increased subsequently for about 4-6 hr. Injections repeated on subsequent days resulted in a slightly more increased A-V glucose differences. (4) Effect of Fenfluramine on Food Intake. Body Weight and

Behavior (a) Effects of 1.5 rng/kg Fenfluramine. The animals showed

436

CHHINA, KANG, SINGH AND ANAND

some behavioral drowsiness and lethargy generally from the second or third injection onwards, which progressively increased following subsequent injections. They showed diminishing aggressive behavior and increased docility. Daily food intake started progressively decreasing from second injection onwards (Fig. 7) and by about fifth day it was reduced by 33-50 per cent. The body weight reduced by about 20 per cent at the end of a drug course of 1 week (Fig. 8). (b) Effects of 3 mg/kg Fenfluramine. Daily food intake decreased even with the first injection and progressively became more marked. Decrease in body weight was also noticeable earlier. Animals were lethargic and had diminished aggressive behavior after the very first injection.

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After the second and subsequent injections they became progressively more drowsy and this state continued till few days after the drug administration was stopped. The marked decrease in food intake may in part be due to this behavioral drowsiness.

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FENFLURAMINE AND HYPOTHALAMIC ACTIVITY

437

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(c) Effects o f stopping the drug. The changes observed in the feeding and general behavior tended to wear off in about one week following the discontinuation of the drug. DISCUSSION

Fenfluramine given intravenously in doses of 3 mg/kg body weight produced generalised EEG responses from most of the brain regions indicating early sleep and drowsy behavior of the animal. It was, therefore, not possible to observe any specific changes in the activity of the hypothalamic feeding centers or even in the feeding behavior of these animals. On the other hand, Fenfluramine in doses of 1.5 mg/kg body weight produced specific changes in the electroencephalographically recorded activity of the hypothalamic feeding centers. This resulted in gradually increasing slow wave activity in the lateral hypothalamic feeding center which could be observed even after the first injection, but became more pronounced and persistent after the subsequent daily injections. This was also accompanied by gradually decreasing food intake from second injection onwards. When this daily administration of the drug was persisted with for 4-5 days, it resulted in a certain degree of drowsy behavior with generalised slow wave activity in the brain. If drug administration was discontinued at this stage, the EEG and behavioral drug effects gradually disappeared in about a week's time. In the studies carried out in fed animals, changes in the electrical activity of the medial hypothalamic satiety region, in response to Fenfluramine, were minimal, as in such animals the satiety area showed a low voltage fast activity which was

not influenced any further by Fenfluramine injections. However, if the recordings were made in satiated animals, the EEG responses from the medial satiety area gradually changed from slow activity to low voltage fast activity. This demonstrated the inverse reciprocal effects of Fenfluramine in inhibiting the lateral hypothalamic mechanism and simultaneous activation of the medial satiety mechanism. Although no estimations on the level of glucose utilization were carried out in these experimental animals, such estimations in other normal animals have suggested that at a time when the electrical activity of the feeding and satiety regions changes in response to Fenfluramine, there is possibly a simultaneous increase in the level of glucose utilization in the body. Previous studies have provided an anatomical basis for suggesting that central mechanisms in the hypothalamus regulate food intake, the lateral feeding center providing the basic urge to eat, and the medial satiety center inhibiting it. Experimental evidence has also been provided [5, 6], suggesting that increased glucose utilization in the body activates the satiety center and decreases the activity of the feeding center, while decreased glucose utilization in the body produces the opposite effect. It thus appears that Fenfluramine depresses appetite and food intake by influencing the activity of the hypothalamic feeding centers. As in the experimental animals, in whom effects of Fenfluramine injections on the electrical activity of the hypothalamic feeding centers were studied, no blood sugar estimations were carried out, the present study cannot provide a direct correlation between the changes in the electrical activity of the feeding centers and the level of glucose utilization in the body. The effect of Fenfluramine on hypothalamic centers may possibly be a direct one as shown for amphetamine [11]. On the other hand there is suggestive evidence that this effect of Fenfluramine on the hypothalamic centers may be indirectly resulting from changes in the level of glucose utilization in the body (as observed in response to Fenfluramine injections in normal animals). Experimental studies carried out with other pharmacological preparations like preludin [9], cyproheptadine [12], and 2-deoxy-D-glucose [13] strongly suggest that the chemicals which change appetite possibly do so by changing the activity of the hypothalamic feeding centers through changes in the level of glucose utilization in the body. This may possibly be at least a part of the mechanism for the action of Fenfluramine. Since the changes produced by the drug, gradually became more prominent with repeated injections, and these persisted for few days even after the withdrawal of the drug, these are indicative of the cumulative effect of the drug. This would probably indicate its correlation with certain metabolic changes in the body. Acknowledgement--We would like to express our thanks to M/s. Selpharm Laboratories Ltd., London for supplying the drug gratis and Mrs. S. Kesar for her technical assistance.

REFERENCES 1. Anand, B. K. A method of permanent implantation of electrodes in the central nervous system of stimulation and recording of action potentials in conscious animals. Indian J. Physiol. all. Sci. 9: 1-6, 1955. 2. Anand, B. K. Nervous regulation of food intake. PhysioL Rev. 41: 677-708, 1961.

3. Anand, B. K. Central chemosensitive mechanisms related to feeding. In: Handbook of Physiology, Section 6, Alimentary Canal, Vol. 1. Washington, D.C.: American Physiological Society, 1967, p. 249. 4. Anand, B. K. and J. R. Brobeck. Localization of a Feeding Centre in the Hypothalamus of the cat. Proc. Soc. exp. Biol. Med. 77: 323-324, 1951.

438 5. Anand, B. K., G. S. Chhina and B. Singh. Effect of glucose on the activity of hypothalarnic feeding centres. Science 132: 597598, 1962. 6. Anand, B. K., G. S. Chhina, K. N. Sharma, S. Dua and B. Singh. Activity of single neurones in the hypothalamic feeding centres. Effect of glucose. Am. J. PhysioL 207:1146-1154, 1964. 7. Anand, B. K., S. Dua and B. Singh. Electrical activity of feeding centres under the effect of changes in blood chemistry. Electroenceph, clin. NeurophysioL 13: 54-59, 1961. 8. Anand, B. K., S. Dua and K. Shoenberg, Hypothalamic control of food intake in cats and monkeys. J. PhysioL 127: 143-152, 1955. 9. Anand, B. K., C. L. Malhotra, S. Dua and B. Singh. Electrical activity of hypothalamic centres under the effect of reserpin, restinon and preludin. Indian J. med. Res. 49: 82-89, 1961.

CHHINA, KANG, SINGFI AND ANAND 10. Anand, B. K. and R. V. Pillai. Activity of single neurones in the hypothalamic feeding centres: Effect of gastric distension. d. PhysioL, Lond. 192: 63-77, 1967. 11. Brobeck, J. R., S. Larsson and E. Reyes. A study of the electrical activity of the hypothalamic feeding mechanisms. J. Physiol. 132: 358-364, 1956. 12. Chakrabarty, A. S., R. V. Pillai, B. K. Anand and B. Singh. Effect of cyproheptadine on the electrical activity of hypothalamic feeding centres. Brain Res. 1: 561-569, 1967. 13. Desiraju, T., M. G. Banerjee and B. K. Anand. Activity of single neurones in the hypothalamic feeding centres: effect of 2-Deoxy-D-glucose. Physiol. Behav. 3: 757-760, 1968. 14. Sharma, N. C. and B. K. Sur. Improved method for estimating blood sugar. J. clin. Path. 19: 630-631,196t.