the effects of anorexic doses of dextro-amphetamine on the ventromedial-hypothalamic hyperphagic rat

The Effects of Anorexic Doses of dextro-Amphetamine on the Ventromedial-Hypothalamic Hyperphagic Rat l

1)epcirtrnc'ttt c!f'Psychology, University c~Srzskritc~hevt~czn, Suskntoon, Sciskatche,t*rtn S7N OW0

Received Novernber 22, I972

WISHAKT, T. B., and WALLS, E. K. 1973. The effects of anorexic doses of dextrs-amphetamine on the ventromedial-hypothalamic hyperphagic rat. Can. J. Physiol. Pharmacol. 51, 354-359.

Rats were made hyperphagic by the production of electrolytic lesions in the ventromedial hy pothalamu,. Following a recovery period during which a rapid weight gain was observed, d-amphetamine was adminis- tered intraperitoneally in various dosages, and food intake\, water intakes, and body weight changes were measured after a 3 h feeding period. '4 dosage of 0.2 mglkg d-amphetamine had no observable effect. Increasing the do\age to 0.5 or 1 .O mglkg. however, significantly depressed food and water intakes, and resulted in reduced weight gains in control and experimental animals. Furthermore, the dose-response curves for both experimental and control groups were similar, indicating that cf-amphetamine has the same effect in the hyperphagic rat as in the control. It is concluded that amphetamine anorexia is not mediated by the ventromedial hypothalamus.

WISHART, '1'. B. , et W41.r.s. E. K. 1973. The effects of anorexic doses of dextro-amphetamine on the ventromedial-hypothalamic hyperphagic rat. Can. J . Physiol. Pharmacol. 51, 354-359.

Des rats sont rendus hyperphagiques par lisioll electrolytique de l'hypothalamus ventromedian. AplZs une pkriode de recuperation, durant laquelle nous avons note une importante augmentation du poids cor- pore!, nous avons administre diffkrentes doses de d-amphetamine i.p. La prise alimentaire et aqueuse de mkme que les variations de poids coi-porel ont ete mesurees apres une periode de 3 h d'alimentation. Nous n'avons pas note d'effets aprks une dose de 8.2 mg/kg de d-amphktamine; toutefois aprks tine dose de 0.5 ou de 1 mglkg, nous avons note une diminution de l'augmentation de poids corporel nussi bien chez les animaux experimentaux que temoins. De plus, les courbes dose-reponses des rats experilnentaux et temoins sont semblables, indiquant ainsi que la d-amphetamine a le meme effet chez le rat hyperphagique que chez le rat normal. Nous concluons que l'anordxie indanite par I'amphetamine n'est pas medite par I'hypothalamus ventromddian. ['l'raduit par le journal]

Introduction tion of this area inhibits food consumption even

Research conducted over the past several decades has generally tended to show that the hypothalamic region of the brain, in most species, is thc critical neural structure responsible for the regulation of energy balance (Anand and Brobeck 195 1 a ) . Thc hypothalamus, furthermore. appears to be differentiated into medial and lateral regions according to function. The lateral hypothaIamus controls the initiation of food intake since lesions in this region producc aphagia (Anand and Brobeck 195 1 b ) whereas clectrical stimrllation or adrcn- ergic chemical stiml~lation may induce eating (Coons 1964; Cirossman 1960, 1962). The medial portion of the hypothalamrls in the region of the vcntromedial nucleus (V.M.H.) , on the other hand, seems to be involved in the termination of fccding since e1ectric:il stimula-

'This study was supported by the Non-Medical Use of Drugs Directorate, Department of National Hcalth and Welfare (Project No. 121 5-7-1 2).

in the food-deprived animal ( ~ a r g u l e s and Olds 1962), and lesions in the area result in chronic overeating and obcsity (Hetherington and Ranson 19440, 1942). The control of moti- vated, consummatory behavior is, therefore, a neural control located primarily within the hypothalamus with, no doubt, sensory receptors in the mouth, gastrointestinal tract, blood, and perhaps in the hypothalamus itself, providing information regarding the state of energy balance.

A nurrlbcr of chemical agcnts, amphetamine being the most notable, have been found to produce an anorexic (hunger-reducing) effect whcn taken orrilly or adrninistcred intrave- nously. Presumably, these drugs act via the central control mech:inism to produce thcir effects. The prcsent experiment, one of a series, was designed to determine whether or not amphetanline anorcxia is mediated by the hunger satiety system.

Of the many possibilities for the neural struc-

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WISHART A N D WAI,I,S: ANOREXIC DOSES OF d-AMPIIETAMINE 355

TABLE 1. Experimental paradigm

Phase Duration Access to food Access to water Daily records - A

Habituation to laboratory 14 days Unrestricted Unrestricted Elody weights Recovery-from-surgery 24 h None Unrestricted Body weights Hyperphagia test 20 days Unrestricted Unrestricted Body weights Habituation to food 13 days 3 h daily Unrestricted Body weights, and food

deprivation and water consumptions Drug test 27 days 3 h daily Unrcstricted Body weights, and food

and water consumptions - - - - - . - - -- - - - - -- - - -

ture responsible for amphetamine anorexia, perhaps the ventromedial hypothalamus is the best candidate. As indicated above, the V.M.H. is usually considered part of an inhibitory or "satiety" system responsible for the cessation of food intake. The drug may therefore activate or excite the cells in this region and the net effect of this stimulation would be an inhibition of food intake.

A sinlple test of the hypothesis outlined in the preceding paragraph is to destroy the cells in the region of the V.M.H. through the use of electrolytic lesions, to administer the drug in various dosages, and to test for evidence of anorexia. If amphetamine anorexia is mediated by the cells in this area, the normal effect of the drug should be absent ill animals with lesions of the V.M.H.

Methods Male, black-hooded rats were employed in the

experiment. After 2 weeks habituation to the lahora- tory, the animals were divided into two groups (control, n :- 8; experimental, n = 16) equal in average body weight (see Table 2, preoperative weights). Each animal was housed individually in a standard stainless-steel rodent cage supplied by the Wahmann Manufacturing Co. Ltd. (Baltimore).

S~trgical Procrdrrre Under 45 m g k g sodium pentobarbital anesthesia,

each animal in the first group (hereinafter called the experimental or V.M.H. lesion group) was given bilateral electrolytic lesions of the ventromedial nuclei of the hypothalarna~s. Employing stereotaxic pro- cedures, insulated stainless-steel electrodes manufac- tiared from No. 7 insect pins were lowered into the region of the V.M.H. (coordinates: 1.5 mm posterior to bregrna, 0.8 mm lateral to the midline suture, and 8.5 mrn ventral to the skull surface). Electrolytic lesions were made by passing a 1.5 mA anodal current for 20 s.

continuously in all phases of the experiment. All subjects were then placed on an ad libitrun feeding schedule (hyperphagia test) lasting for 20 days, during which body weights were recorded daily. Throughout the experiment, food was available in the form of standard lab chow pellets (Purina) and monkey chow pellets ( Purina) .

Both groups were then placed on a 21 h food- deprivation schedule (habituation to food deprivation), and food consumptions, water intakes, and body weight changes were recorded for the remaining 3 11 in which food was available.

Following the habituation to food deprivation period, drug treatment began. Thirty minutes prior to the daily feeding period, each animal received an intraperitoneal injection of 0.2. 0.5, or 1.0 mg/kg d-amphetamine. Food consumptions, water intakes, and body weight changes were again recorded at the termination of the 3 h feeding period. Food was then made available for a 24 h period to allow the animal to regain the , ,eight loss sustained as a result of the drug treatment. 0 1 1 ;he two remaining days of each control period, d3,3 were collected as described above for purposes of comparison. Each subject received two injections at each dosage lcvel, with a minimum of 3 days between injections to permit recovery.

Results Immediately after surgery and during re-

covery from anesthesia, those animals given bilateral V.M.H. lesions exhibited the symptoms most oftcn observed following such treatment 4i.e. hyperactivity and stereotyped compulsive behavior). In the 24 h period foilowing surgery, six of the experimental subjects dicd, possibly due to excessive hyperactivity, hypothermia, or wntcr intoxication (postmortem body weights had increased by an average of 44 g or 16.5% body weight in four of these animals despite the fact that only water was available during this pcriod) .

Table 2 presents the body weight changes Tc~til ig Procedrrrcls

.I.he experimental design is in wllich occurred during the period beginning at During the 24 h reco\rery-from-surgery period, all the time of surgery and ending with the onset subjects wcre deprived of food. Water was available of food restriction (hyperphagia test). The

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CAN. J . PHYSIOI.. PHAKMACOL. VOL. 51. 1973

TABLE 2. Weight gain following lesions of the ventromedial hypothala~nus*

Postoperative weight, Preoperative weight 20 days after surgery

-- - - (6) (6)

- - - --

Control group 288.4 + 3.7 352.4 + 4.3 Experimental group 284.9 4 4.2 -

385.6 4 8.1 --

*Values are mertns f standard errors s f means.

drug dosage ( rng / kg body weight 1

FIG. 1. Effect of &amphetamine on 3 h food consumption in the V.M.H. lesion rat. ( 0 ) Mean for control group; (v) mean for experimental group.

average weight gain by the aninlals in the V.M.H. group was 108.7 g whereas that for the controls was 64.0 g, a first indication that the lesions had been properly placed.

During the first 3 days on the fcrod-deprivation schedule, the animals in the experimental group lost an average of 24.9 g whereas those in the control group lost an average of 19.9 g. Prior to the first injection trial the V.M.H. animals had lost an average of 35.9 g of body weight whereas thc controls had lost ran average of 30.5 g. All of the subjects had adapted to the food-deprivation schedule by the time of the first injection and were maintaining their body weights although at lower levels than those of the ad libitum feeding period.

The data of particular interest are presented in Figs. 1, 2, and 3, and Table 3. Whethcr or not an injection was given. th6 V.M.H. group subjects ate lcss food, drank less water, and gained fewer grams body weight than did the controls in the 3 h period in which food was available ( p < 8.005, no significant interac- tions).

A subseyuea~t analysis (Newman-Keuls) was performed on this data. Food intake was not inhibited by a dosage of 0.2 rng/kg of d-arn- yhetamine ( p > 0.05). 111 fact, both groups of animals actually increased their food consumption under this dosage, although the increase was nonsignificant. A dosage of 0.5 mg/kg, howex~er, reduced food intake in both contrsH

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WISHART AN11 WAI,L,S: ANOKEXIC IIOSES OF d-AMPHETAMINE

drug dosage (rng/kg body weight)

PIG. 2. Effect of d-amphetamine on 3 k water consumption in the V.M.H. lesion rat. ( @ ) Mean for control group; (V) mean for experimental group.

0.1 0.2 0 3 0.4 0.5 0.8 0.7 0.8 0.0 1.0

drug dosage (mg/kg body weight)

PIG. 3. Effect of d-amphetamine on 3 h body weight change in the V.M.H. lesion rat. ( a ) Mean for control group; ( v ) mean for experimental group.

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358 C A N . J . PHYSIOI.. PHARMACOL.. VOL.. 51, 1973

TABLE 3. Standard errors of measurement - -- -- --

Dosage (mg/kg)

Food consumption data Control 0.8 0.8 1.0 0.7 Experimental 0.6 0.6 0 .6 0.5

Water consumption data Control 0.9 0.8 1.1 1.1 Experimen ta I 0.9 1.4 0.9 1.0

Body weight change data Control 1.3 1.4 1.3 1.9 Experimental 1.2 1.3 1.2 1.2

and V.M.H. animals below normal levels (p < 0.05). Increasing the dosage to 1.0 mg/kg inhibited food intake in both groups even further (17 < 0.05).

As was the case with food intake, water intake was unaffected by the injection of 0.2 mg/kg d-amphetamine (p > 0.05). The animals drank less water at 0.5 mg/kg than they did at 0.2 mg/kg ( p < 0.05), but the difference from non-drug levels did not reach significance (17 > 0.05). At a drug dosage level of 1.0 mg/kg, water intake was significantly depressed below non-drug lcvels (17 < 0.05 ) .

Body weight changes closely paralleled the decreases in food and water consumptions which occurred with increasing dosages of amphetamine. The lowest drug dosage did not change the amount of weiglat gained by the animals during the 3 h feeding period (p > 0.05). An injection of 0.5 mg/kg d-amghet- amine, however, resulted in a significant reduc- tion in weight gain during the feeding period ( 1 1 < 0.851, and an increase in dosage up to 1.0 mg/kg further enhanced this effect ( p < 0.05). In fact, under tlae two larger dosage levels, some subjects (two in the control group, four in the V.M.H. group) actually lost weight during the 3 la feeding period. This was tlae only instance in whicla this effect was observed.

Histological examination of the brains of the experimental subjects revealed damage in and around the area of the ventromediall nucleus of the hypothalamus. Most of the lesions produced were fairly symmetrical and approxiinately 1.0-2.0 mm in diameter. Tn some cases, the lesion extendcd either anteriorly to involve the anterior hypothalamus or posteriorly and later- ally to involve the medial, anterior portio~a of

the lateral hypothalamus. In the latter cases, however, such damage was not so severe as to produce any indication of aphagia. In all cases the grcatest damagc was in the region of the V.M.H. and, since all of the experimental subjects were affected similarly by food deprivation and drug treatment, the lesions were considered to be effective; thus, the data for all srrbjects were included in the analysis.

Discussion Thc postoperative hyperactivity and compul-

sive behavior, thc rapid weight gain, and thc histological data together indicate that effective V.M.H. lesions were produced in all of the cxperiinental animals. Further support for this conclusion is provided by the data of food intake under food restriction conditions. The V.M.H. lesion subjects consumed less food during the 3 h period in which food was available daily than did normal controls. Similar observations were reported by Epstein ( 1959).

The results of the present experiment demon- strate rather conclusively that the central ner- vous system site of action for amphetanline anorexia is not at the level of the vc~~tromcdial nucleus of the hypothralamus. This nucleus and surrounding tissue has, as one of its functions, the inhibition of food intake and it would be cxpccted that in an animal Backing this strue- ture, alnphetanaine would fail to suppress food consumption. Such was not the case. Adminis- tration of 0.5 or 1.0 rng/kg of d-amphetamine to animals with bilateral lcsions of this nucleus significantly depressed food intake to the same degree as corresponding injections given to control aninaals.

Previous research with regard to this hypothesis employing the samc preparation has provided similar results (Stowe and Miller 1957: Epsteiil 1959; Reynolds 1959). In these earlier studies. however, much laigl~er dosrages (2.0--5.0 mg/kg) were generally employed, and it might be argued that the observed rsduction in food consumption may have arisen as a by- l~roduct of the hyperactivity and compulsive behavior ~sroduced by such high dosages. Thc present study attempted to control these alternate motor behaviors through the use of much Bower dosage Bevels at which these alternate responses are absent or lcss prevalent.

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WISHAKT AND WAL,I,S: AN0Kt:XLC TIOSES (.IF d-AMPHETAMINE 359

Earlier researchers also reported that the AN AND, B. K., and BROBECK, J. K. 1951~ . Hypothalamic L

anorexic effect of amphetamine is exaggerated in animals with lesions of the V.M.H. (Epstein 1959; Reynolds 3959). Such an effect was not observed in the present experiment. Food and water intakes as well as body weight gains of the V.M.H. lesion animals paralleled almost cxactly those of the control subjects, indicating that the drug had siinilar effects in both experimental and control rats. The differences in results between this and previous experiments is no st likely due to the lower dosage Icvels employed here. It is conceivable that high dosages produce more adverse motor or other effects in the V.M.H. lesion rat than in the control although observations on this point are lacking. At low dosage levels, however, there is a marked similarity in the dose-response curves oE both groups.

It is thus concluded that amphetamine does not produce its anorexic effect through an acti- vation of the inhibitory or satiety system for food intake. Support is thus provided for the conclusion drawn by Carlisle ( 1964) who sug- gested that amphetamine affects either the lateral hypothalamus (i.e. direct inhibition of the ex- citatory feeding system) or same other neural region such as the amygdala, which is known to be involved in the control of energy balance (Grossman and Grossman 19631, and which exerts descending influences upon the hypothalamic homeostatic system. Further research is required to determine the exact site or system where amphetamine exerts its anorexic effect.

control of food intake in rats and cats. Yale J. Biol. Med. 24, 123.

1951h. Localization of a feeding center in the hypothalamus of the rat. Proc. Soc. Exp. Biol. Med. 77, 323-324.

CARL ISI E, H. J. 1964. Differential effects of amphetamine on food and water intake in rats with lateral hypothalamic lesions. J . Comp. Physiol. Pbychol. 58, 44-54.

COONS, E. E. 1964. Motivational correlates of eating elicited by electrical stimulation in the hypothalamic feeding area. Doctoral Dissertation. Yale University, University hlicrofilrns Inc., Ann Arbor, Mich., No. 64-13, 166.

GROSSMAN, S. P. 1960. Eating or drinking elicted by direct adrenergic or cholinergic stimulation of hypothalamus. Science, 132, 301-302.

1962. Direct adrenergic and cholinergic stimulation of hypothalamic mechanibins. Am. J. Physiol. 202, 842-882.

GROSSMAN, S. P., and GROSSMAN, L. 1963. Food and water intake following lesions tsr electrical stimulation of the amygdala. 4m. J. Physiol. 285, 461-765.

Ers I LIN , A. N. 1959. Suppression of eating and drinking by amphetamine and other drugs in normal and hy perphagic rats. J. Comp. Physiol . Psychol. 52, 37-4s.

H E I H F R I N G T ~ N , ,4. W., and RANSON, S . W. 1940. Hypothalamic lesions and adiposity in the rat. Anat. Rec. 78, 149-172.

1942. Effect of early hypophysectonly on hypothalamic obesity. Endocrinology, 31. 3CL34.

MARGLJLES, D. L., and OLDS, J. 1962. Identical "feeding" and "reward~ng" qystems in the lateral hypothalamus of rats. Science, 135, 374-375.

RFYNOJ DS, R. W. 1959. The effect of amphetamine on food intake in normal and hypothalamic hyperphagic rats. J. Comp. Physiol. Psychol. 52, 682-684.

Srowi-, F. R., and MILLER, A. T. 1957. The effect of amphetamine on food intake in rats with hypothalamic hyperphagia. Experientia, 13, 114-1 15.

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the effects of anorexic doses of dextro-amphetamine on the ventromedial-hypothalamic hyperphagic rat

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