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Central action of anorexic agents: Effects of amphetamine and fenfluramine in rats with lateral hypothalamic lesions
European Journal of Pharmacology 28 (1974) 81-88 © North-Holland Publishing Company
CENTRAL
ACTION OF ANOREXIC
FENFLURAMINE
AGENTS:
IN RATS WITH LATERAL
EFFECTS
OF AMPHETAMINE
HYPOTHALAMIC
AND
LESIONS
John E. BLUNDELL and Micah B. LESHEM Psychology Department, University of Leeds, Leeds, LS2 9JT, England
Received 10 December 1973, accepted 13 May 1974 J.E. BLUNDELL and M.B. LESHEM, Central action of anorexic agents: effects of amphetamine and fenfluramine in rats with lateral hypothalamic lesions, European J. Pharmacol. 28 (1974) 81-88. The effects upon food intake of three dose levels of fenfluramine and amphetamine were compared in rats with bilateral or unilateral lesions of the lateral hypothalamus. Unilateral lesions produced little modulation of drug action but bilateral lesions brought about opposite effects on amphetamine and fenfluramine anorexia. At 8 weeks after operation amphetamine anorexia was significantly diminished in bilaterally lesioned animals whereas fenfluramine anorexia was significantly enhanced. Further tests carried out at 14 and 20 weeks after operation showed that amphetamine regained its anorexic potency in lesioned animals, while the enhanced potency of fenfluramine remained. The results are consistent with the belief that these two anorexic agents operate through quite separate sites and mechanisms of action. Lateral hypothalamus
Anorexia
Amphetamine
1. Introduction It has frequently been suggested that anorexic drugs exert their effect on food intake by intervening in the lateral (LH) or ventromedial (VMH) hypothalamic regulatory mechanisms. For amphetamine, enhancement o f anorexia following VMH lesions (e.g. Epstein, 1959), the amelioration o f anorexia following LH lesions (Carlisle, 1964), the suppression of feeding elicited by LH stimulation (e.g. Miller, 1960), and the depression of feeding following direct chemical injections into the LH (Leibowitz, 1970; Booth, 1968) suggests that amphetamine anorexia is mediated via the LH and not the VMH. Like amphetamine, ~'enfluramine anorexia is not diminished by VMH lesions (Bernier et al., 1969) and the use of a hypothalamic model (Blundell, 1971) seems to confirm that fenfluramine does not act at the VMH. However, when fenfluramine is applied directly to the LH (Blundell and Leshem, 1973), it fails to bring about an immediate suppression o f food intake and this suggests that fenfluramine unlike amphetamine
Fenfluramine
Food intake
may not act via the LH. The present experiment was designed to test this possibility by comparing the effect of amphetamine and fenfluramine in rats in which the lateral hypothalamus had been destroyed. The experiment also examined the time course of the effect o f lateral hypothalamic lesions on anorexic drug action.
2. Materials and methods 2.1. Subjects and surgery The subjects were 49 male hooded rats weighing between 280 and 350 g at the time of operation. When required, lesions were produced electrolytically under ether anaesthesia b y passing a 1 m A current through a 0.5 mm diameter stainless steel anode and a rectal cathode for 10 sec. The uninsulated 0.5 mm tip o f the anode was stereotaxically positioned 8.8 mm below the skull surface through a burrhole placed 1.5 mm lateral and 0.5 m m posterior
82
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
to bregma. The incisor bar of the stereotaxic instrument was raised 5.0 mm above the interaural plane, to conform to the De Groot atlas (1959). 12 animals received unilateral lateral hypothalamic (ULH) destruction; 6 animals were given lesions on the right side of the brain, and 6 on the left. Another group of 12 animals served as a lesion control group (C); in 6 of these animals an electrode was lowered to both sides of the hypothalamus but no current was passed, and the other 6 animals simply had burrholes placed in the skull with no penetration of brain tissue. The remaining 25 animals from the original subject pool were all given bilateral lesions of the lateral hypothalamus (BLH). At the conclusion of the experiment the location and extent of the hypothalamic lesions were determined from photographic enlargements of unstained 50 t2 frozen sections. 2.2. Procedure and design
Following surgery the BLH animals received special nutritional care to counteract the period of aphagia and adipsia which follows lateral hypothalamic destruction. Rats that refused dry food pellets were given sugar-sweetened mash and where necessary they were fed intragrastically twice daily. Animals were gradually weaned from intragrastic feeding and consumption of sweetened mash to feeding on dry pellets, the changes being made contingent upon maintenance of body weight. All animals were fed on dry pellets for at least 2 weeks before drug injections were given. Feeding tests were begun two months after surgery and food intake was measured following a period of 16 hr food deprivation. Food was removed from the animals at 18.00 hr each evening and at 10.00 the following day a weighed amount of food was placed in the cages. Since brief feeding tests may give rise to ambiguous results in drug studies (e.g. Blundell et al., 1973), in the present experiment food intake was monitored periodically over 24 hr and measurements were taken 1, 4, 8 and 24 hr. Spillage was collected on a tissue paper placed beneath the wire mesh cage floors and weighings were made to the nearest 0.1 g. Three procedures were adopted in order to ensure minimal disturbance of the animals during the drug injection series. After surgical treatment all animals
were rendered docile by being handled daily when removed from their cages for weighing. In addition, the food deprivation regime was initiated 10 days before the start of chemical injections to allow time for animals to stabilize their feeding pattern, and for 7 days before drug administration animals received sham i.p. injections to allow habituation to the stress of the injection procedure before the start of the experiment proper. In the main injection series beginning 8 weeks after operation, 7 drug treatments were administered 1.0, 2.5 and 5.0 mg/kg (+)-amphetamine sulphate; 2.5, 5.0 and 10.0 mg/kg (-+)-fenfluramine hydrochloride; and 0.9% w/v NaC1. The 7 drug conditions were presented in counterbalanced order 30 rain before the start of feeding tests and 72 hr intervened between successive i.p. injections. This series was intended to show the short term effect of lateral hypothalamic lesions on anorexic drug action. In the two subsequent injection series carried out 14 and 20 weeks after operation a single dose of amphetamine (1.0 mg/kg) and fenfluramine (5.0 mg/kg) was administered together with saline control. These further tests were included to assess the long term effects of lesions on drug-induced anorexia. The results were analysed statistically by an analysis of variance procedure and by Student's t-test (2-tailed tests).
3. Results
Of the 25 animals which received bilateral lateral hypothalamic lesions, 12 failed to recover from the operation, and the following results were derived from experiments on 13 bilaterally lesioned subjects (BLH) 12 unilaterally lesioned subjects (ULH), and 12 sham-lesioned animals (C). 3.1. Histology
Examination of the photographic enlargements of the brain sections indicated that the lateral hypothalamic lesions consistently destroyed an area of the lateral hypothalamus adjacent, and slightly dorsal, to the descending column of the fornix in the De Groot planes A 5.4-4.6 (fig. 1). Some lesions also en-
J.E Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
r
A 5.0
39
A 5.0
43
50
A 5.0
22
40
47
A
5.0
37
A 5.0
A 5.4
42
A 5.0
49
83
A 5.4
A 4.6
A 5.0
Fig. 1. Coronal brain sections showing the position and extent of the bilateral lateral hypothalamic lesions (dark shaded areas) copied from photographic enlargements. Under each section the left hand numeral indicates the animal number while the right hand numeral shows the plane in the De Groot atlas corresponding to that section. The brain of one rat (38) was damaged during processing and is not shown.
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
84
Table 1 Mean body weights (g) (+ S.E.) of the 3 groups of animals at operation and at 3 stages during the recovery period. The figures in parentheses show the number of animals subjected to sham lesions (C), unilateral lateral hypothalamic lesions (ULH) and bilateral lesions (BLH). Surgery
BLH (13) ULH (12) C (12)
Operation
315.2 -+4.8 304.4 -+ 5.2 299.7 -+2.9
Post-operative period (weeks) 1
3
6
277.6 -+ 8.8 300.2 -+6.4 307.4 +- 4.2
310.5 -+ 9.3 332.9 -+ 7.1 340.1 +- 6.7
352.2 -+ 12.1 365.7 -+ 7.9 376.7 -+ 8.2
croached upon the medial edge o f the internal capsule and the ventral edge of the zona incerta.
3.2. Body weight Table 1 shows the effect of the various lesion procedures on body weight. In keeping with the histological evidence, the weight of the bilaterally lesioned animals fell precipitiously following operation but began to recover after about 1 week during the period of extra nutritional care; 6 weeks after operation animals of all groups were eating dry food and body weights had begun to stabilize. It is noticeable that the mean weight of the unilaterally lesioned group falls between that of the bilateral and sham-lesioned animals.
3.3. Brain lesions and food intake The design of the experiment conformed to a 3 (lesion groups) b y 7 (drug treatments) factorial study and the results of the feeding tests were subjected to an analysis of variance procedure for repeated measures (Winer, 1970). During the first feeding period
( 0 - 1 hr) the lateral hypothalamic lesion exerted no significant effect on food intake (F = 1.31, df 2 and 36, p > 0.05), though table 2 shows that the brain treatments influenced feeding during the subsequent feeding periods.
3.4. Food intake and drugs (short term effect) In the main experiment, which was carried out 8 weeks after operation, the analysis of variance procedure showed that the drug treatments produced a significant modulation of food intake (F = 113.16, df 6 and 216, p < 0.001), and revealed a significant d r u g - l e s i o n interaction (F = 7.75, df 12 and 216, p < 0.01). The main effect of the drug treatment was expected and shows simply that anorexic drugs reduce food intake. However, further investigation of the drug-lesion interaction showed that the anorexic effect of amphetamine was mildly attenuated in the ULH group and markedly diminished in the BLH Ss (table 3). Statistical tests confirmed that the anorexic action o f amphetamine was significantly reduced in animals with bilateral lesions o f the lateral hypothalamus when compared with control animals (t = 3.39, df 23, p < 0.01 for pooled dose levels).
Table 2 Mean food intake (g) and standard deviations for the 3 brain treatment conditions following control injections of 0.9% saline administered during tests 8 weeks after operation. Brain treatment
C (12) ULH (12) BLH (13)
Period of food intake (hr) 0-1
0-4
0-8
0-24
7.8 -+ 1.8 7.7 -+ 1.4 7.1 -+ 1.6
15.0 +- 2.1 15.2 -+ 2.2 12.4 -+ 1.6"
22.9 -+ 3.7 23.4 -+ 1.5 18.1 -+4.2*
44.0 -+4.5 42.6 +- 2.1 36.7 -+ 3.7*
* Indicates BLH intake significantly different from ULH and C; smallest t = 3.19, df 23, p < 0,01.
J.E. Blundell, M.B. Leshem, Lateral hypothalarnus and anorexic drugs
85
Table 3 Effect upon food intake of injections of amphetamine and fenfluramine in rats with bilateral lesions of the lateral hypothalamus (BLH), unilateral lesions (ULH) and sham lesions (C). The figures represent the percentage of food eaten compared with the amount consumed following control injections (0.9% saline). Unilateral lesions show little effect on anorexic drug action hut bilateral lesions diminish the potency of amphetamine and enhance the potency of fenfluramine (see text for statistical analysis). Treatments
Period of food intake (hr)
Surgery
Drug
Dose (mg/kg)
0-1
0-4
0-8
0-24
BLH
Amphetamine
1.0 2.5 5.0 2.5 5.0 10.0
100 36 2 49 22 8
101 85 54 57 29 12
100 86 76 65 40 20
100 97 88 89 75 63
1.0 2.5 5.0 2.5 5.0 i0.0
66 11 2 71 58 25
90 72 33 82 64 36
90 75 56 78 66 38
96 87 77 93 89 77
1.0 2.5 5.0 2.5 5.0 10.0
52 5 3 71 52 33
84 62 35 79 62 32
95 78 63 82 65 39
97 91 84 95 88 82
Fenfluramine
ULH
Amphetamine
Fenfluramine
C
Amphetamine
Fenfluramine
H o w e v e r , t h e effect o f f e n f l u r a m i n e o n t h e lesion p r e p a r a t i o n s s t o o d in m a r k e d c o n t r a s t t o t h a t o f amp h e t a m i n e ( t a b l e 3): in a n i m a l s w i t h bilateral lesions o f t h e lateral h y p o t h a l a m u s t h e a n o r e x i c p o t e n c y o f f e n f l u r a m i n e was significantly e n h a n c e d (t = 2.94, d f
23, p < 0 . 0 1 ) , n o t a t t e n u a t e d . T h e clearest d e m o n s t r a t i o n o f this d i s s o c i a t i o n b e t w e e n drugs c a n b e derived f r o m a c o m p a r i s o n o f t h e e f f e c t o f t h e 1.0 m g / k g dose o f a m p h e t a m i n e a n d t h e 5.0 m g / k g dose o f f e n f l u r a m i n e b o t h o f w h i c h p r o d u c e d t h e same
Table 4 Comparison of the effect upon bilaterally lesioned animals (BLH) of doses of amphetamine (1.0 mg/kg) and fenfluramine (5.0 mg/kg) which had been shown to have equivalent anorexic potency for sham-lesioned animals (C) during the 0 - 1 hr test. Figures show mean values for food intake (g) ± standard deviations. Treatments
Period of food intake (hr)
Surgery
Drug
Dose (mg/kg)
0-1
0-4
0-8
0-24
BLH(n = 13)
Amphetamine Fenfluramine Saline
1.0 5.0
7.1 ± 2.6 1.6" ± 1.5 7.1 ± 1.7
12.6 ± 2.9 3.6* ± 2.3 12.4 ± 1.7
18.2 ± 3.5 7.3* ± 3.5 18.1 ± 2.9
36.7 ± 6.1 27.8* ± 5.6 36.7 ± 4.0
C(n=12)
Amphetamine Fenfluramine Saline
1.0 5.0
4 . 1 " ± 1.9 4.1" ± 1.3 7.8 ± 1.9
12.6 ± 1.9 9.3* ± 1.5 15.0 ± 2.2
21.9 -+ 3.0 15.0" ± 3.3 22.9 ± 3.9
42.8 ± 4.5 39.1 -+ 7.6 44.0 ± 4.7
*Indicates significantly different from saline at p < 0.01.
86
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
Table 5 Effect of amphetamine and fenfluramine in animals with bilateral lateral hypothalamic lesions (BLH) and sham lesions (C) at different periods following operation. The results given are for the 0-1 hr feeding period and the first figure in each column shows the mean food intake (g) ± standard deviations, while the figure in parentheses shows intake as a percentage of consumption following saline injection. The prolonged effect of BLH lesions on fenfluramine anorexia, evident at the 14 and 20 week experimental sessions, was also apparent during the 4, 8 and 24 hr feeding tests. Treatments
Time after operation (weeks)
Drug
Surgery
8
14
20
Amphetamine (1.0 mg/kg)
BLH C
7.1 -+ 2.6 (100)* 4.1 -+ 1.9 (52)
3.7 -+ 2.4 (49) 3.5 -+ 2.0 (41)
3.3 -+ 2.2 (46) 3.3 -+ 1.9 (41)
Fenfluramine (5.0 mg/kg)
BLH C
1.6 +- 1.5 (22)* 4.1 -+ 1.3 (52)
2.3 -+ 1.6 (33)* 5.5 ± 2.0 (70)
3.3 -+ 1.7 (46)* 5.8 ± 1.4 (72)
*Indicates BLH different from C atp < 0.01. (52%) depression o f food intake in control animals during the 0 - 1 hr test. These approximate EDso doses produced quite different effects on the BLH group (table 4). At this particular dose the anorexic effect of amphetamine was completely abolished and the BLH animals responded in identical fashion to both amphetamine and saline injections. On the other hand, fenfluramine gave rise to a more severe reduction of food intake in BLH animals than in controls, and it is worth noting that this marked depression cannot be accounted for by the slightly lower baseline intake o f BLH animals. Unlike the work of Fibiger et al. (1973), not only did lesioned animals treated with fenfluramine eat less than similarly treated control animals, but the degree of anorexia exerted by fenfluramine was significantly greater in the lesioned group (t = 3.68, df 23, p < 0.01).
3.5. Food intake and drugs (long term effects) The results of the main experiment which was carried out 8 weeks after operation, together with two further drug tests carried out at 14 and 20 weeks after surgery, showed differences between the long term effects of amphetamine and fenfluramine in animals with lateral hypothalamic lesions (table 5). For amphetamine, the marked amelioration of anorexic potency in BLH Ss observed 8 weeks after operation, was not apparent at 14 or 20 weeks after surgery when the potency of amphetamine in BLH Ss was similar to that in control animals. However, the response of BLH Ss to fenfluramine was quite differ-
ent: the exacerbation of fenfluramine's anorexic potency observed in the early experiment was still apparent in the subsequent trials. There was no difference between the effect of amphetamine in BLH and control Ss at the 14 week test (t = 0.34, df 22, p > 0.7) or at the 20 week test (t = 0.14, df 22, p > 0.9), but fenfluramine maintained its enhanced anorexic potency in BLH Ss (14 week test: t = 4.21, df 22, p < 0.01 ; 20 week test: t = 3.81, d f 2 2 , p < 0.01).
4. Discussion The results of the main experiment confirmed previous reports (Carlisle, 1964; Russek et al., 1973) that bilateral lesions of the lateral hypothalamus ameliorate the anorexic effect o f amphetamine, possibly by destroying certain terminals of the ventral noradrenergic bundle (Ahlskog and Hoebel, 1973). On the other hand, BLH lesions produced an opposite effect on fenfluramine anorexia: a more pronounced depression of feeding was apparent at all dosages and at every feeding period. The experiment demonstrates clearly that the mechanisms of action of these anorexic drugs are quite different: the lateral hypothalamus seems to play a crucial role in the action o f amphetamine but is apparently not critical in the mediation of fenfluramine anorexia. The results of experirnents in which amphetamine has been administered to animals with various hypothalamic lesions conform to a coherent pattern. Since the action of amphetamine is diminished by BLH le-
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
sions, it has been argued that anorexia is produced by a blocking of the lateral hypothalamic feeding centre; and since the effectiveness of amphetamine is increased by ventromedial hypothalamic lesions it follows that the inhibitory ventromedial zone induces the lateral hypothalamus to become hypersensitive according to a 'release from restraint' hypothesis (Cole, 1973). This explanation is consistent with known hypothalamic mechanisms for the regulation of food intake, and embodies the notion that lateral hypothalamic lesions destroy most of the receptor sites where amphetamine may directly (Leibowitz, 1970) or indirectly (Holtzman and Jewett, 1971) suppress feeding. If the results with fenfluramine obtained in the present experiment are to be incorporated into this pattern then it must be inferred that since the effectiveness of fenfluramine is increased by lateral hypothalamic lesions then this zone is inhibitory and its destruction leads to the 'release from restraint' of some other structure. Such an interpretation is cumbersome and the implication that the lateral hypothalamus exerts inhibitory influence has only limited experimental support (e.g. Margules, 1970). So far, the results of hypothalamic lesion studies have failed to demonstrate a critical hypothalamic zone for the mediation of fenfluramine anorexia. Both VMN lesions (Bernier et al., 1969) and BLH lesions (present experiment)enhance the severity of fenfluramine anorexia, while anterior hypothalamic lesions (Blundell and Leshem, in preparation) produce only a weak and insignificant diminution in anorexic potency. A hypothalamic area has not yet been demarcated which, when lesioned, will abolish fenfluramine anorexia. Unlike amphetamine, the anorexic effect of fenfluramine cannot be accounted for by action of the drug at a neuroanatomical locus within the hypothalamus. However, elimination of fenfluramine anorexia has been achieved by lesions of the midbrain raphe nuclei (Samanin et al., 1972) which deplete brain serotonin. These findings suggest that, although hypothalamic mechanisms may participate in the action of fenfluramine on feeding behaviour, a full explanation of fenfluramine anorexia will have to take into account the effect of this drug on widespread aminergic systems regulating food intake. The marked dissociation of the effects of amphetamine and fenfluramine, noted by other authors and evident in the present experiment, is quite consistent
87
with the further new observation that in lateral hypothalamic animals the decline in anorexic potency of amphetamine is only temporary, whereas the enhanced potency of fenfluramine remains at least until 20 weeks after surgery. This difference in the rate of recovery of drug effect following lesioning again suggests a different mechanism of action for each of the drugs. The restoration of amphetamine anorexia is in keeping with the notion that recovery of feeding following lateral hypothalamic lesions recapitulates ontogeny of feeding (Teitelbaum et al., 1969), and with the observed gradual onset of amphetamine anorexia in developing neonatal rats (Lytle et al., 1971). On the other hand, the continued enhancement of fenfluramine anorexia cannot readily be incorporated into this pattern. It seems more likely that an explanation for the effect of BLH lesions on fenfluramine anorexia may be found through investigation of the long-term alterations in brain amine systems brought about by BLH lesions (Oltmans and Harvey, 1972).
Acknowledgements The authors are indebted to Mrs. Janet Maxwell for assistance with the testing procedure, and to Mr. Alan English for help with histology and photography. The work was financed by a research grant from Servier Laboratories.
References Ahlskog, J.E. and B.G. Hoebel, 1973, Overeating and obesity from damage to noradrenergic system in the brain, Science 182,166. Bernier, A., N. Sicot and J.C. Le Douarec, 1969, Action compar~e de la fenfluramine et de l'amphetamine chez le rats ob~ses hypothalamiques, Rev. Franc. Et. Chim. Biol. 14, 762. Blundell, J.E., 1971, Possible mechanism for the effect of anorexic agents on feeding and hoarding behaviour in rats, Psychopharmacol. 22,224. BlundeU, J.E. and M.B. Leshem, 1973, Dissociation of the anorexic effects of fenfluramine and amphetamine following intrahypothalamic injection, Brit. J. Pharmacol. 47, 183. Blundell, J.E., C.J. Latham and M.B. Leshem, 1973, Biphasic action of a 5-hydroxytryptamine inhibitor on fenfluramine-induced anorexia, J. Pharm. Pharmacol. 25,492. Booth, D.A., 1968, Amphetamine anorexia by direct action on the adrenergic feeding system of the rat hypothalamus, Nature, London 217,869.
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J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
Carlisle, J., 1964, Differential effects of amphetamine on food and water intake in rats with lateral hypothalamic lesions, J. Comp. Physiol. Psychol. 58, 47. Cole, S.O., 1973, Hypothalamic feeding mechanisms and amphetamine anorexia, Psychol. Bull. 79, 13. De Groot, J., 1959, The Rat Forebrain in Stereotaxic Coordinates (North-Holland, Amsterdam). Epstein, A.N., 1959, Suppression of eating and drinking by amphetamine and other drugs in normal and hyperphagic rats, J. Comp. Physiol. Psychol. 52, 37. Fibiger, H.C., A.P. Zis and E.G. McGeer, 1973, Feeding and drinking deficits after 6-hydroxydopamine administration in the rat: similarities to the lateral hypothalamic syndrome, Brain Research 55, 135. Holtzman, S.G. and R.E. Jewett, 1971, The role of brain norepinephrine in the anorexic effects of dextroamphetamine and monoamine oxidase inhibitors in the rat, Psychopharmacol. 22, 151. Leibowitz, S.F., 1970, Reciprocal hunger regulating circuits involving alpha- and beta-adrenergic receptors located respectively in the ventromedial and lateral hypothalamus, Proc. Nat. Acad. Sci. 67, 1063. Lytle, L.D., W.H. Moorcroft and B.A. Campbell, 1971, Ontogeny of amphetamine anorexia and insulin hyperphagia
in the rat, J. Comp. Physiol. Psychol. 77,388. Margules, D.L., 1970, Alpha-adrenergic receptors in hypothalamus for the suppression of feeding behaviour by satiety, J. Comp. Physiol. Psychol. 73, 1. Miller, N.E., 1960, Motivational effects of brain stimulation and drugs, Federation Proc. 19,846. Oltmans, G.A. and J.A. Harvey, 1972, LH syndrome and brain catecholamine levels after lesions of the nigrostriatal bundle, Physiol. Behav. 8, 69. Russek, M., A.M. Rodriguez-Zendejas and P. Teitelbaum, 1973, The action of adrenergic anorexic substances on rats recovered from lateral hypothalamic lesions, Physiol. Behav. 10,329. Samanin, R., D. Ghezzi, L. Valzelli and S. Garattini, 1972, The effects of selective lesioning of brain serotonin or catecholamine containing neurones on the anorectic activity of fenfluramine and amphetamine, European J. Pharmacol. 19,318. Teitelbaum, P., M. Cheng and P. Rozin, 1969, Development of feeding parallels its recovery after hypothalamic damage, J. Comp. Physiol. Psychol. 67,430. Winer, B.J., 1970, Statistical Principles in Experimental Design (McGraw-Hill, New York).
CENTRAL
ACTION OF ANOREXIC
FENFLURAMINE
AGENTS:
IN RATS WITH LATERAL
EFFECTS
OF AMPHETAMINE
HYPOTHALAMIC
AND
LESIONS
John E. BLUNDELL and Micah B. LESHEM Psychology Department, University of Leeds, Leeds, LS2 9JT, England
Received 10 December 1973, accepted 13 May 1974 J.E. BLUNDELL and M.B. LESHEM, Central action of anorexic agents: effects of amphetamine and fenfluramine in rats with lateral hypothalamic lesions, European J. Pharmacol. 28 (1974) 81-88. The effects upon food intake of three dose levels of fenfluramine and amphetamine were compared in rats with bilateral or unilateral lesions of the lateral hypothalamus. Unilateral lesions produced little modulation of drug action but bilateral lesions brought about opposite effects on amphetamine and fenfluramine anorexia. At 8 weeks after operation amphetamine anorexia was significantly diminished in bilaterally lesioned animals whereas fenfluramine anorexia was significantly enhanced. Further tests carried out at 14 and 20 weeks after operation showed that amphetamine regained its anorexic potency in lesioned animals, while the enhanced potency of fenfluramine remained. The results are consistent with the belief that these two anorexic agents operate through quite separate sites and mechanisms of action. Lateral hypothalamus
Anorexia
Amphetamine
1. Introduction It has frequently been suggested that anorexic drugs exert their effect on food intake by intervening in the lateral (LH) or ventromedial (VMH) hypothalamic regulatory mechanisms. For amphetamine, enhancement o f anorexia following VMH lesions (e.g. Epstein, 1959), the amelioration o f anorexia following LH lesions (Carlisle, 1964), the suppression of feeding elicited by LH stimulation (e.g. Miller, 1960), and the depression of feeding following direct chemical injections into the LH (Leibowitz, 1970; Booth, 1968) suggests that amphetamine anorexia is mediated via the LH and not the VMH. Like amphetamine, ~'enfluramine anorexia is not diminished by VMH lesions (Bernier et al., 1969) and the use of a hypothalamic model (Blundell, 1971) seems to confirm that fenfluramine does not act at the VMH. However, when fenfluramine is applied directly to the LH (Blundell and Leshem, 1973), it fails to bring about an immediate suppression o f food intake and this suggests that fenfluramine unlike amphetamine
Fenfluramine
Food intake
may not act via the LH. The present experiment was designed to test this possibility by comparing the effect of amphetamine and fenfluramine in rats in which the lateral hypothalamus had been destroyed. The experiment also examined the time course of the effect o f lateral hypothalamic lesions on anorexic drug action.
2. Materials and methods 2.1. Subjects and surgery The subjects were 49 male hooded rats weighing between 280 and 350 g at the time of operation. When required, lesions were produced electrolytically under ether anaesthesia b y passing a 1 m A current through a 0.5 mm diameter stainless steel anode and a rectal cathode for 10 sec. The uninsulated 0.5 mm tip o f the anode was stereotaxically positioned 8.8 mm below the skull surface through a burrhole placed 1.5 mm lateral and 0.5 m m posterior
82
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
to bregma. The incisor bar of the stereotaxic instrument was raised 5.0 mm above the interaural plane, to conform to the De Groot atlas (1959). 12 animals received unilateral lateral hypothalamic (ULH) destruction; 6 animals were given lesions on the right side of the brain, and 6 on the left. Another group of 12 animals served as a lesion control group (C); in 6 of these animals an electrode was lowered to both sides of the hypothalamus but no current was passed, and the other 6 animals simply had burrholes placed in the skull with no penetration of brain tissue. The remaining 25 animals from the original subject pool were all given bilateral lesions of the lateral hypothalamus (BLH). At the conclusion of the experiment the location and extent of the hypothalamic lesions were determined from photographic enlargements of unstained 50 t2 frozen sections. 2.2. Procedure and design
Following surgery the BLH animals received special nutritional care to counteract the period of aphagia and adipsia which follows lateral hypothalamic destruction. Rats that refused dry food pellets were given sugar-sweetened mash and where necessary they were fed intragrastically twice daily. Animals were gradually weaned from intragrastic feeding and consumption of sweetened mash to feeding on dry pellets, the changes being made contingent upon maintenance of body weight. All animals were fed on dry pellets for at least 2 weeks before drug injections were given. Feeding tests were begun two months after surgery and food intake was measured following a period of 16 hr food deprivation. Food was removed from the animals at 18.00 hr each evening and at 10.00 the following day a weighed amount of food was placed in the cages. Since brief feeding tests may give rise to ambiguous results in drug studies (e.g. Blundell et al., 1973), in the present experiment food intake was monitored periodically over 24 hr and measurements were taken 1, 4, 8 and 24 hr. Spillage was collected on a tissue paper placed beneath the wire mesh cage floors and weighings were made to the nearest 0.1 g. Three procedures were adopted in order to ensure minimal disturbance of the animals during the drug injection series. After surgical treatment all animals
were rendered docile by being handled daily when removed from their cages for weighing. In addition, the food deprivation regime was initiated 10 days before the start of chemical injections to allow time for animals to stabilize their feeding pattern, and for 7 days before drug administration animals received sham i.p. injections to allow habituation to the stress of the injection procedure before the start of the experiment proper. In the main injection series beginning 8 weeks after operation, 7 drug treatments were administered 1.0, 2.5 and 5.0 mg/kg (+)-amphetamine sulphate; 2.5, 5.0 and 10.0 mg/kg (-+)-fenfluramine hydrochloride; and 0.9% w/v NaC1. The 7 drug conditions were presented in counterbalanced order 30 rain before the start of feeding tests and 72 hr intervened between successive i.p. injections. This series was intended to show the short term effect of lateral hypothalamic lesions on anorexic drug action. In the two subsequent injection series carried out 14 and 20 weeks after operation a single dose of amphetamine (1.0 mg/kg) and fenfluramine (5.0 mg/kg) was administered together with saline control. These further tests were included to assess the long term effects of lesions on drug-induced anorexia. The results were analysed statistically by an analysis of variance procedure and by Student's t-test (2-tailed tests).
3. Results
Of the 25 animals which received bilateral lateral hypothalamic lesions, 12 failed to recover from the operation, and the following results were derived from experiments on 13 bilaterally lesioned subjects (BLH) 12 unilaterally lesioned subjects (ULH), and 12 sham-lesioned animals (C). 3.1. Histology
Examination of the photographic enlargements of the brain sections indicated that the lateral hypothalamic lesions consistently destroyed an area of the lateral hypothalamus adjacent, and slightly dorsal, to the descending column of the fornix in the De Groot planes A 5.4-4.6 (fig. 1). Some lesions also en-
J.E Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
r
A 5.0
39
A 5.0
43
50
A 5.0
22
40
47
A
5.0
37
A 5.0
A 5.4
42
A 5.0
49
83
A 5.4
A 4.6
A 5.0
Fig. 1. Coronal brain sections showing the position and extent of the bilateral lateral hypothalamic lesions (dark shaded areas) copied from photographic enlargements. Under each section the left hand numeral indicates the animal number while the right hand numeral shows the plane in the De Groot atlas corresponding to that section. The brain of one rat (38) was damaged during processing and is not shown.
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
84
Table 1 Mean body weights (g) (+ S.E.) of the 3 groups of animals at operation and at 3 stages during the recovery period. The figures in parentheses show the number of animals subjected to sham lesions (C), unilateral lateral hypothalamic lesions (ULH) and bilateral lesions (BLH). Surgery
BLH (13) ULH (12) C (12)
Operation
315.2 -+4.8 304.4 -+ 5.2 299.7 -+2.9
Post-operative period (weeks) 1
3
6
277.6 -+ 8.8 300.2 -+6.4 307.4 +- 4.2
310.5 -+ 9.3 332.9 -+ 7.1 340.1 +- 6.7
352.2 -+ 12.1 365.7 -+ 7.9 376.7 -+ 8.2
croached upon the medial edge o f the internal capsule and the ventral edge of the zona incerta.
3.2. Body weight Table 1 shows the effect of the various lesion procedures on body weight. In keeping with the histological evidence, the weight of the bilaterally lesioned animals fell precipitiously following operation but began to recover after about 1 week during the period of extra nutritional care; 6 weeks after operation animals of all groups were eating dry food and body weights had begun to stabilize. It is noticeable that the mean weight of the unilaterally lesioned group falls between that of the bilateral and sham-lesioned animals.
3.3. Brain lesions and food intake The design of the experiment conformed to a 3 (lesion groups) b y 7 (drug treatments) factorial study and the results of the feeding tests were subjected to an analysis of variance procedure for repeated measures (Winer, 1970). During the first feeding period
( 0 - 1 hr) the lateral hypothalamic lesion exerted no significant effect on food intake (F = 1.31, df 2 and 36, p > 0.05), though table 2 shows that the brain treatments influenced feeding during the subsequent feeding periods.
3.4. Food intake and drugs (short term effect) In the main experiment, which was carried out 8 weeks after operation, the analysis of variance procedure showed that the drug treatments produced a significant modulation of food intake (F = 113.16, df 6 and 216, p < 0.001), and revealed a significant d r u g - l e s i o n interaction (F = 7.75, df 12 and 216, p < 0.01). The main effect of the drug treatment was expected and shows simply that anorexic drugs reduce food intake. However, further investigation of the drug-lesion interaction showed that the anorexic effect of amphetamine was mildly attenuated in the ULH group and markedly diminished in the BLH Ss (table 3). Statistical tests confirmed that the anorexic action o f amphetamine was significantly reduced in animals with bilateral lesions o f the lateral hypothalamus when compared with control animals (t = 3.39, df 23, p < 0.01 for pooled dose levels).
Table 2 Mean food intake (g) and standard deviations for the 3 brain treatment conditions following control injections of 0.9% saline administered during tests 8 weeks after operation. Brain treatment
C (12) ULH (12) BLH (13)
Period of food intake (hr) 0-1
0-4
0-8
0-24
7.8 -+ 1.8 7.7 -+ 1.4 7.1 -+ 1.6
15.0 +- 2.1 15.2 -+ 2.2 12.4 -+ 1.6"
22.9 -+ 3.7 23.4 -+ 1.5 18.1 -+4.2*
44.0 -+4.5 42.6 +- 2.1 36.7 -+ 3.7*
* Indicates BLH intake significantly different from ULH and C; smallest t = 3.19, df 23, p < 0,01.
J.E. Blundell, M.B. Leshem, Lateral hypothalarnus and anorexic drugs
85
Table 3 Effect upon food intake of injections of amphetamine and fenfluramine in rats with bilateral lesions of the lateral hypothalamus (BLH), unilateral lesions (ULH) and sham lesions (C). The figures represent the percentage of food eaten compared with the amount consumed following control injections (0.9% saline). Unilateral lesions show little effect on anorexic drug action hut bilateral lesions diminish the potency of amphetamine and enhance the potency of fenfluramine (see text for statistical analysis). Treatments
Period of food intake (hr)
Surgery
Drug
Dose (mg/kg)
0-1
0-4
0-8
0-24
BLH
Amphetamine
1.0 2.5 5.0 2.5 5.0 10.0
100 36 2 49 22 8
101 85 54 57 29 12
100 86 76 65 40 20
100 97 88 89 75 63
1.0 2.5 5.0 2.5 5.0 i0.0
66 11 2 71 58 25
90 72 33 82 64 36
90 75 56 78 66 38
96 87 77 93 89 77
1.0 2.5 5.0 2.5 5.0 10.0
52 5 3 71 52 33
84 62 35 79 62 32
95 78 63 82 65 39
97 91 84 95 88 82
Fenfluramine
ULH
Amphetamine
Fenfluramine
C
Amphetamine
Fenfluramine
H o w e v e r , t h e effect o f f e n f l u r a m i n e o n t h e lesion p r e p a r a t i o n s s t o o d in m a r k e d c o n t r a s t t o t h a t o f amp h e t a m i n e ( t a b l e 3): in a n i m a l s w i t h bilateral lesions o f t h e lateral h y p o t h a l a m u s t h e a n o r e x i c p o t e n c y o f f e n f l u r a m i n e was significantly e n h a n c e d (t = 2.94, d f
23, p < 0 . 0 1 ) , n o t a t t e n u a t e d . T h e clearest d e m o n s t r a t i o n o f this d i s s o c i a t i o n b e t w e e n drugs c a n b e derived f r o m a c o m p a r i s o n o f t h e e f f e c t o f t h e 1.0 m g / k g dose o f a m p h e t a m i n e a n d t h e 5.0 m g / k g dose o f f e n f l u r a m i n e b o t h o f w h i c h p r o d u c e d t h e same
Table 4 Comparison of the effect upon bilaterally lesioned animals (BLH) of doses of amphetamine (1.0 mg/kg) and fenfluramine (5.0 mg/kg) which had been shown to have equivalent anorexic potency for sham-lesioned animals (C) during the 0 - 1 hr test. Figures show mean values for food intake (g) ± standard deviations. Treatments
Period of food intake (hr)
Surgery
Drug
Dose (mg/kg)
0-1
0-4
0-8
0-24
BLH(n = 13)
Amphetamine Fenfluramine Saline
1.0 5.0
7.1 ± 2.6 1.6" ± 1.5 7.1 ± 1.7
12.6 ± 2.9 3.6* ± 2.3 12.4 ± 1.7
18.2 ± 3.5 7.3* ± 3.5 18.1 ± 2.9
36.7 ± 6.1 27.8* ± 5.6 36.7 ± 4.0
C(n=12)
Amphetamine Fenfluramine Saline
1.0 5.0
4 . 1 " ± 1.9 4.1" ± 1.3 7.8 ± 1.9
12.6 ± 1.9 9.3* ± 1.5 15.0 ± 2.2
21.9 -+ 3.0 15.0" ± 3.3 22.9 ± 3.9
42.8 ± 4.5 39.1 -+ 7.6 44.0 ± 4.7
*Indicates significantly different from saline at p < 0.01.
86
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
Table 5 Effect of amphetamine and fenfluramine in animals with bilateral lateral hypothalamic lesions (BLH) and sham lesions (C) at different periods following operation. The results given are for the 0-1 hr feeding period and the first figure in each column shows the mean food intake (g) ± standard deviations, while the figure in parentheses shows intake as a percentage of consumption following saline injection. The prolonged effect of BLH lesions on fenfluramine anorexia, evident at the 14 and 20 week experimental sessions, was also apparent during the 4, 8 and 24 hr feeding tests. Treatments
Time after operation (weeks)
Drug
Surgery
8
14
20
Amphetamine (1.0 mg/kg)
BLH C
7.1 -+ 2.6 (100)* 4.1 -+ 1.9 (52)
3.7 -+ 2.4 (49) 3.5 -+ 2.0 (41)
3.3 -+ 2.2 (46) 3.3 -+ 1.9 (41)
Fenfluramine (5.0 mg/kg)
BLH C
1.6 +- 1.5 (22)* 4.1 -+ 1.3 (52)
2.3 -+ 1.6 (33)* 5.5 ± 2.0 (70)
3.3 -+ 1.7 (46)* 5.8 ± 1.4 (72)
*Indicates BLH different from C atp < 0.01. (52%) depression o f food intake in control animals during the 0 - 1 hr test. These approximate EDso doses produced quite different effects on the BLH group (table 4). At this particular dose the anorexic effect of amphetamine was completely abolished and the BLH animals responded in identical fashion to both amphetamine and saline injections. On the other hand, fenfluramine gave rise to a more severe reduction of food intake in BLH animals than in controls, and it is worth noting that this marked depression cannot be accounted for by the slightly lower baseline intake o f BLH animals. Unlike the work of Fibiger et al. (1973), not only did lesioned animals treated with fenfluramine eat less than similarly treated control animals, but the degree of anorexia exerted by fenfluramine was significantly greater in the lesioned group (t = 3.68, df 23, p < 0.01).
3.5. Food intake and drugs (long term effects) The results of the main experiment which was carried out 8 weeks after operation, together with two further drug tests carried out at 14 and 20 weeks after surgery, showed differences between the long term effects of amphetamine and fenfluramine in animals with lateral hypothalamic lesions (table 5). For amphetamine, the marked amelioration of anorexic potency in BLH Ss observed 8 weeks after operation, was not apparent at 14 or 20 weeks after surgery when the potency of amphetamine in BLH Ss was similar to that in control animals. However, the response of BLH Ss to fenfluramine was quite differ-
ent: the exacerbation of fenfluramine's anorexic potency observed in the early experiment was still apparent in the subsequent trials. There was no difference between the effect of amphetamine in BLH and control Ss at the 14 week test (t = 0.34, df 22, p > 0.7) or at the 20 week test (t = 0.14, df 22, p > 0.9), but fenfluramine maintained its enhanced anorexic potency in BLH Ss (14 week test: t = 4.21, df 22, p < 0.01 ; 20 week test: t = 3.81, d f 2 2 , p < 0.01).
4. Discussion The results of the main experiment confirmed previous reports (Carlisle, 1964; Russek et al., 1973) that bilateral lesions of the lateral hypothalamus ameliorate the anorexic effect o f amphetamine, possibly by destroying certain terminals of the ventral noradrenergic bundle (Ahlskog and Hoebel, 1973). On the other hand, BLH lesions produced an opposite effect on fenfluramine anorexia: a more pronounced depression of feeding was apparent at all dosages and at every feeding period. The experiment demonstrates clearly that the mechanisms of action of these anorexic drugs are quite different: the lateral hypothalamus seems to play a crucial role in the action o f amphetamine but is apparently not critical in the mediation of fenfluramine anorexia. The results of experirnents in which amphetamine has been administered to animals with various hypothalamic lesions conform to a coherent pattern. Since the action of amphetamine is diminished by BLH le-
J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
sions, it has been argued that anorexia is produced by a blocking of the lateral hypothalamic feeding centre; and since the effectiveness of amphetamine is increased by ventromedial hypothalamic lesions it follows that the inhibitory ventromedial zone induces the lateral hypothalamus to become hypersensitive according to a 'release from restraint' hypothesis (Cole, 1973). This explanation is consistent with known hypothalamic mechanisms for the regulation of food intake, and embodies the notion that lateral hypothalamic lesions destroy most of the receptor sites where amphetamine may directly (Leibowitz, 1970) or indirectly (Holtzman and Jewett, 1971) suppress feeding. If the results with fenfluramine obtained in the present experiment are to be incorporated into this pattern then it must be inferred that since the effectiveness of fenfluramine is increased by lateral hypothalamic lesions then this zone is inhibitory and its destruction leads to the 'release from restraint' of some other structure. Such an interpretation is cumbersome and the implication that the lateral hypothalamus exerts inhibitory influence has only limited experimental support (e.g. Margules, 1970). So far, the results of hypothalamic lesion studies have failed to demonstrate a critical hypothalamic zone for the mediation of fenfluramine anorexia. Both VMN lesions (Bernier et al., 1969) and BLH lesions (present experiment)enhance the severity of fenfluramine anorexia, while anterior hypothalamic lesions (Blundell and Leshem, in preparation) produce only a weak and insignificant diminution in anorexic potency. A hypothalamic area has not yet been demarcated which, when lesioned, will abolish fenfluramine anorexia. Unlike amphetamine, the anorexic effect of fenfluramine cannot be accounted for by action of the drug at a neuroanatomical locus within the hypothalamus. However, elimination of fenfluramine anorexia has been achieved by lesions of the midbrain raphe nuclei (Samanin et al., 1972) which deplete brain serotonin. These findings suggest that, although hypothalamic mechanisms may participate in the action of fenfluramine on feeding behaviour, a full explanation of fenfluramine anorexia will have to take into account the effect of this drug on widespread aminergic systems regulating food intake. The marked dissociation of the effects of amphetamine and fenfluramine, noted by other authors and evident in the present experiment, is quite consistent
87
with the further new observation that in lateral hypothalamic animals the decline in anorexic potency of amphetamine is only temporary, whereas the enhanced potency of fenfluramine remains at least until 20 weeks after surgery. This difference in the rate of recovery of drug effect following lesioning again suggests a different mechanism of action for each of the drugs. The restoration of amphetamine anorexia is in keeping with the notion that recovery of feeding following lateral hypothalamic lesions recapitulates ontogeny of feeding (Teitelbaum et al., 1969), and with the observed gradual onset of amphetamine anorexia in developing neonatal rats (Lytle et al., 1971). On the other hand, the continued enhancement of fenfluramine anorexia cannot readily be incorporated into this pattern. It seems more likely that an explanation for the effect of BLH lesions on fenfluramine anorexia may be found through investigation of the long-term alterations in brain amine systems brought about by BLH lesions (Oltmans and Harvey, 1972).
Acknowledgements The authors are indebted to Mrs. Janet Maxwell for assistance with the testing procedure, and to Mr. Alan English for help with histology and photography. The work was financed by a research grant from Servier Laboratories.
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J.E. Blundell, M.B. Leshem, Lateral hypothalamus and anorexic drugs
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