Physiology & Behavior, Vol. 18, pp. 1121--1125. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.

Defense of a Lowered Weight Maintenance Level by Lateral Hypothamically Lesioned Rats: Evidence from a Restriction-Refeeding Regimen' J O E L S. M I T C H E L A N D R I C H A R D E. K E E S E Y 2

University o f Wisconsin, Madison, WI 53706 (Received 8 January 1977) MITCHEL, J. S. AND R. E. KEESEY. Defense of a lowered weight maintenance level by lateral hypothalamically lesioned rats: evidence from a restriction-refeeding regimen. PHYSIOL. BEHAV. 18(6) 1121-1125, 1977. - Male rats maintained their body weight at approximately 85% that of sham-lesioned controls following lesions of the lateral hypothalamus (LH). One month following surgery, the food intake of half the LH-lesioned animals was restricted until their body weight had declined to 80% that of nonrestricted LH animals. Half the sham-lesioned animals were similarly restricted until their body weight fell to 80% that of nonrestricted control animals. When returned to an ad lib feeding schedule, both restricted groups were initially hyperphagic and quickly restored their body weights to the level of the nonrestricted group from which they were originally selected. In doing so, the LH animals increased their food intake by the same amount and took the same number of days to restore their weight to control levels as the sham-lesioned animals. These observations provide further evidence of the vigor and effectiveness with which LH animals defend their reduced level of maintained body weight. Lateral hypothalamus

Body weight regulation

Food intake

IT HAS B E E N p r o p o s e d t h a t t h e c h r o n i c a l l y r e d u c e d level of weight m a i n t e n a n c e b y l a t e r a l - h y p o t h a l a m i c a l l y (LH) lesioned rats is indicative of a l o w e r e d r e g u l a t i o n level or s e t - p o i n t for b o d y weight [ 1 0 ] . S u p p o r t for such a view derives p r i n c i p a l l y f r o m o b s e r v a t i o n s i n d i c a t i n g t h a t LH a n i m a l s n o t o n l y m a i n t a i n t h e i r b o d y w e i g h t at a r e d u c e d p e r c e n t a g e o f n o r m a l for p e r i o d s up to h a l f a y e a r p o s t l e s i o n [ 7 ] , b u t t h a t t h e y also d e f e n d this lowered level m u c h as a n o n - l e s i o n e d a n i m a l d e f e n d s a n o r m a l b o d y weight. If challenged b y changes in a d i e t ' s caloric d e n s i t y [1,10] or p a l a t a b i l i t y [ 1 , 4 ] , for e x a m p l e , LH a n i m a l s display a d j u s t m e n t s in f o o d i n t a k e t h a t are n e a r l y i d e n t i c a l to t h o s e o f n o n l e s i o n e d animals. T h e y likewise m a i n t a i n b o d y weight at t h e same r e d u c e d p e r c e n t a g e of c o n t r o l levels t h r o u g h o u t such d i e t a r y challenges. O t h e r e v i d e n c e of a n altered weight r e g u l a t i o n in LH-lesioned a n i m a l s c o m e s f r o m w o r k involving experim e n t a l m a n i p u l a t i o n s o f t h e i r b o d y weight. E x p e r i m e n t a l l y l o w e r i n g t h e b o d y weight of an a n i m a l prior to lesioning the lateral h y p o t h a l a m u s can, for e x a m p l e , s h o r t e n or even

Food deprivation

e l i m i n a t e t h e n o r m a l p o s t l e s i o n periods of aphagia and a n o r e x i a [ 1 0 ] . Conversely, h o l d i n g an LH a n i m a l ' s b o d y weight at c o n t r o l levels b y force-feeding i m m e d i a t e l y a f t e r lesioning will p r o l o n g LH aphagia a n d a n o r e x i a [ 6 ] . Or, force-feeding an LH a n i m a l so t h a t its b o d y weight is r e s t o r e d to t h e level o f c o n t r o l a n i m a l s will r e i n s t a t e a p a t t e r n of aphagia a n d / o r a n o r e x i a [ 5 ] . T h e a b o v e o b s e r v a t i o n s , t h o u g h all c o n s i s t e n t w i t h the view t h a t LH a n i m a l s regulate b o d y weight at r e d u c e d levels, d o n o t d i r e c t l y address the q u e s t i o n of w h e t h e r or n o t t h e s e n s o r i m o t o r deficits of t h e sort o t h e r s [2, 3, 11 ] have o b s e r v e d in LH animals c o n t r i b u t e to this weight effect. Is it possible, for e x a m p l e , t h a t such deficits c o n t r i b u t e to a l o w e r e d w e i g h t m a i n t e n a n c e level by r e n d e r i n g t h e LH a n i m a l i n c a p a b l e of increasing its food i n t a k e to levels sufficient to restore b o d y w e i g h t to c o n t r o l levels? The p r e s e n t e x p e r i m e n t addresses this issue b y assessing t h e c a p a c i t y o f LH-lesioned a n i m a l s to r e c o v e r b o d y weight following an e n f o r c e d weight loss. B o t h t h e i r level of

1This research was supported by National Institute of Mental Health Research Grant MH-08909 and the Research Committee of the University of Wisconsin Graduate School. The authors wish to thank both Peter C. Boyle and Leonard H. Storlien for their helpful criticisms of an earlier version of this manuscript. 2 Requests for reprints should be addressed to Richard E. Keesey, Department of Psychology, University of Wisconsin, Madison, WI 53706. 1121

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h y p e r p h a g i a and t h e rate at w h i c h t h e y restore the lost weight are e x a m i n e d and c o m p a r e d to t h a t of n o n l e s i o n e d animals w h i c h have u n d e r g o n e a c o m p a r a b l e weight loss. METHOD

Animals and Maintenance The animals, 35 male a l b i n o rats of the Sprague-Dawley strain ( H o l t z m a n , Madison, WI), were a p p r o x i m a t e l y 70 days old at the time of surgery. T h e y were individually h o u s e d u n d e r a 1 2 / 1 2 l i g h t - d a r k schedule and fed ad lib o n a wet mash diet of Wayne lab c h o w p r e p a r e d daily b y mixing w a t e r and g r o u n d c h o w in a ratio o f 2:1 b y weight.

Surgical Procedures U n d e r sodium p e n t o b a r b i t a l a n e s t h e s i a (40 mg/kg), stereotaxically-placed e l e c t r o l y t i c lesions were p r o d u c e d t h r o u g h e l e c t r o d e s c o n s t r u c t e d from Size 00 insect pins insulated with E p o x y l y t e e x c e p t for 0.4 to 0.5 m m b a r e d at the tip. With the e l e c t r o d e passing p e r p e n d i c u l a r to the plane b e t w e e n b r e g m a and l a m b d a , t h e c o o r d i n a t e s for the e l e c t r o d e p l a c e m e n t in the lesioned group were 2.8 m m p o s t e r i o r to bregma, 1.85 m m lateral to the midline, and 8.4 m m ventral to the skull surface. A 1.0 m A a n o d a l c u r r e n t was passed for 9 sec w i t h a saline tail b a t h serving as the c a t h o d e . C o n t r o l a n i m a l s received sham lesions w i t h the e l e c t r o d e lowered 6.4 m m v e n t r a l to the skull surface.

Experirn en tal Design The animals were divided into two groups, m a t c h e d on the basis of b o d y weight. One group (LH, N = 20) received LH lesions while the r e m a i n i n g g r o u p (CON, N = 15) received sham lesions. T h i r t y - o n e days following surgery, the LH-lesioned group was subdivided to form o n e group ( L H - R E S , N = 10) whose food intake was restricted so as to r e d u c e its body weight to 8 0 ~ t h a t o f a second group ( L H - C O N , N = 10) which c o n t i n u e d to receive food ad lib. The neurally i n t a c t a n i m a l s were subdivided into one ad lib fed group (CON CON, N = 5) and t w o food restricted groups. One of these groups (CON RES, N = 5) has its food i n t a k e restricted so as to l o w e r its b o d y weight to 80% t h a t of the n o n r e s t r i c t e d c o n t r o l group ( C O N - C O N ) while the restriction regimen for the second group ( C O N - R E S - R E S , N = 5) was such as to reduce its b o d y weight to the level of animals which had b e e n b o t h lesioned and food deprived (i.e., the LH RES group). The C O N - R E S - R E S group was included in o r d e r to d e t e r m i n e w h e t h e r lowering an a n i m a l ' s b o d y weight to a level as low as t h a t prescribed for the L H - R E S group m i g h t c h r o n i c a l l y depress b o d y weight or o t h e r w i s e i m p a i r t h a t a n i m a l ' s ability to restore b o d y weight to its p r o p e r level. The restricted feeding period was of 14 days d u r a t i o n . Following this period (i.e., 45 days postlesion), the f o o d restricted groups were again given food ad lib. B o t h b o d y weight and food i n t a k e were t h e n r e c o r d e d for an additional 33 days before the e x p e r i m e n t was t e r m i n a t e d .

Food Restriction The food restricted animals were initially placed on the feeding regimen described b y M o s k o w i t z [ 8 ] , w i t h the l i m i t a t i o n s t h a t no a n i m a l received less t h a n 10 g of wet mash (3.3 g o f dry meal) o n a n y day. A typical s e q u e n c e

for the L H - R E S and C O N - R E S groups consisted of decreasing i n t a k e f r o m 4 0 g of wet mash on the first day o f r e s t r i c t i o n to 10 g o n the f o u r t h or fifth day, and t h e n gradually increasing to an a m o u n t w h i c h w o u l d m a i n t a i n a n e a r - n o r m a l weight gain ( a p p r o x i m a t e l y 55 g per d a y ) by the t e n t h d a y of the r e s t r i c t i o n period. The C O N - R E S - R E S g r o u p was t r e a t e d similarly e x c e p t t h a t it was necessary to hold the daily i n t a k e level at 10 20 g u n t i l t h e t w e l f t h d a y of restriction. By the end of the food r e s t r i c t i o n period, a p p r o x i m a t e l y 45 g of wet mash were sufficient to m a i n t a i n a near n o r m a l weight gain for this group.

Histological Preparation and Analysis At the c o n c l u s i o n of t h e e x p e r i m e n t , e a c h rat was given an overdose o f s o d i u m p e n t o b a r b i t a l and p e r f u s e d t h r o u g h the h e a r t with 0.9% saline followed by 10% formal-saline. The brain was t h e n r e m o v e d and fixed before f r o z e n sections were t a k e n at 4 0 u thickness. A l t e r n a t e sections were m o u n t e d o n slides and stained w i t h a luxol blue-cresyl violet t e c h n i q u e . The lesions were m a p p e d as described previously [ 7 ] . Data from animals having less t h a n 20% of the critical LH area d e s t r o y e d [9] or having lesions w h i c h i n t r u d e d i n t o the v e n t r o - m e d i a l h y p o t h a l a m i c area were discarded. One L H - C O N and 2 L H - R E S animals failed to m e e t the a b o v e histological criterion. With t h e e l i m i n a t i o n of these animals, the e x t e n t o f h y p o t h a l a m i c d a m a g e did n o t significantly differ b e t w e e n the LH CON and the L H - R E S groups (31.1% vs. 30.0%, respectively; t = 0.37, 0 . 7 < p < 0 . 8 ) . RESULTS

Body Weight Ef[ects o f LH lesions. F o l l o w i n g surgery, t h e LHlesioned animals were aphagic a n d / o r a n o r e x i c for several days (Mean = 2.9, range = I to 7), d u r i n g w h i c h t i m e the group m e a n weight declined 49 g (see Fig. 1). T h e shamlesioned a n i m a l s were a n o r e x i c for only o n e day following surgery and the m e a n weight of this group was a b o v e the p r e o p e r a t i v e level b y the second day following surgery. T h o u g h n o longer a n o r e x i c , the b o d y weight of the LH animals r e m a i n e d well below, a n d at n e a r l y a c o n s t a n t p e r c e n t a g e of, the n o n l e s i o n e d c o n t r o l s t h r o u g h o u t the r e m a i n d e r of the e x p e r i m e n t . On Days 25, 50, and 75, respectively, t h e L H - C O N g r o u p was 62 ( t = 5.370, p < 0 . 0 0 1 ) , 80 (t = 6.847, p < 0 . 0 0 1 ) , a n d 88 ( t = 4 . 9 9 2 , p < 0 . 0 0 1 ) g b e l o w the C O N - C O N group. On these days, the LH animals were at 85.9, 84.1 and 83.6% of controls, respectively. kTfects o f deprivation and re.feeding. The b o d y weights of b o t h the L H - R E S and C O N - R E S were r e d u c e d to the desired level by the r e s t r i c t i o n regimen (see Fig. 1). By the last t h r e e days o f the r e s t r i c t i o n period (Days 4 3 - 4 5 ) , the mean b o d y weights of the L H - R E S and CON RES h a d been r e d u c e d to 79.5% and 79.1% t h a t o f the L H - C O N and C O N - C O N groups, respectively. The m e a n b o d y weight of b o t h restricted groups was significantly ( p < 0 . 0 0 1 ) l o w e r at this p o i n t t h a n t h a t of t h e i r respective n o n r e s t r i c t e d c o n t r o l groups. When r e t u r n e d to an ad lib feeding schedule, the weight of b o t h restricted groups was o b s e r v e d to r e t u r n q u i c k l y to the level o f t h e i r n o n r e s t r i c t e d c o n t r o l group (see Fig. 1). The m e a n weight o f the C O N - R E S group ceased to be

L A T E R A L H Y P O T H A L A M I C L E S I O N S AND BODY W E I G H T

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(425.3 vs. 427.6 g). Likewise, the increase in intake shown by the restricted LH and control groups in the first two weeks following restoration of ad lib food conditions are also quite similar (71.4 g vs. 77.7 g).

CON-RES

R E S Group

The food restriction procedure e m p l o y e d with this group reduced its mean b o d y weight to essentially the level of the L H - R E S group (337.2 g vs. 327.4 g, respectively). At this level (i.e., 337.2 g), the mean body weight of the C O N - R E S - R E S group was 68.9% of normal. When ad lib feeding conditions were restored, this group added weight rapidly and its mean b o d y weight was no longer significantly different from the mean of the C O N - C O N group after 17 days of free-feeding ( p > 0 . 0 5 ) . By the end of the observation period (i.e., Day 32 of ad lib feeding), the b o d y weight of this group was 98% that of the unrestricted control ( C O N - C O N ) group. It was also observed that the food intake of this group was significantly elevated relative to the C O N - C O N group across the first 2-week period of refeeding (M = 519 g, t = 5.82, p < 0 . 0 0 1 ) , though it was not significantly greater than that of the C O N - R E S group (t = 0.959, p > 0 . 2 ) . Thus, lowering an animal's b o d y weight by 30% (i.e., an a m o u n t similar to that experienced by the LH animals subject to food restriction) neither impairs its capacity to increase its f o o d intake significantly nor precludes the restoration of b o d y weight to the n o r m a l control level.

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LA T ER AL HYPOTHALAMIC LESIONS AND BODY WEIGHT DISCUSSION The results presented here confirm earlier reports of weight maintenance at a chronically-reduced level by LH-lesioned rats [5]. As in previous experiments, the lesioned animals were aphagic andfor anorexic for up to a week following surgery, during which time body weight declined sharply. Food intake then returned to normal levels, but body weight remained at a reduced percentage of normal. The body weight of the nonrestricted LH group in this experiment, for example, remained at 84.8 -+ 1.3% of the nonlesioned, nondeprived ( C O N - C O N ) g r o u p from the third week postlesion (Day 21) to the 11 th week postlesion (Day 77). Previous work has also suggested that LH animals will successfully defend this reduced weight level when challenged by experimental manipulations of their body weight [6,10] or their diet [ 1,4]. The present observations provide further evidence of this defense. After a weight loss due to caloric restriction, LH animals quickly restored their body weight to the level of LH animals fed ad lib. Compared to similarly-treated nonlesioned animals they took nearly an identical number of days to return to their normal level and made up equivalent percentages of their lost weight per day. The food intake data of the restricted LH and nonlesioned animals display a similar parallel. Equivalent degrees of hyperphagia were shown by the LH RES and C O N - R E S groups following their return to ad lib feeding conditions (see Fig. 3). Then, as the weights of these two groups approached the level of their respective controls,

1125

mean daily intake declined and eventually returned to the level of the nonrestricted groups. Interest in the LH syndrome, both past and present, has generally tended to focus upon the various feeding deficits or dysfunctions that lesions of this hypothalamic area can produce (e.g., see [ 2, 3, 1 I ]). Consideration of this work inevitably raises the question of whether the weight loss we see in LH animals might not be the result of an impaired ability to feed rather than a response to a lowered regulation level for body weight. The present results address themselves directly to this issue. First, the LH animals observed here quickly restore their body weight to the exact level of nonrestricted LH animals following the enforced weight loss. More to the point, however, they increased their food intake by the same amount, and gained weight just as fast, as did nonlesioned animals experiencing a comparable weight loss. Clearly, animals capable of these levels of hyperphagia and weight gain do not remain at chronically reduced weight levels due to an impaired capacity to feed. This is not to question that lesions of the LH and/or surrounding areas can and often do produce a variety of feeding dysfunctions. Rather, it is to say that our LH animals are not seriously impaired in their ability to maintain themselves, and that our observations concerning the effects of LH lesions upon body weight are not confounded to any significant extent by feeding impairments. In our opinion, such an LH preparation could be particularly useful in studies of the neural contributions to the achievement of energy balance.

REFERENCES 1. Boyle, P. C. and R. E. Keesey. Chronically reduced body weight in LH-lesioned rats maintained upon palatable diets and drinking solutions. J. comp. physiol. Psychol. 88: 218-223, 1975. 2. Epstein, A. N. The lateral hypothalamic syndrome: Its implications for the physiological psychology of hunger and thirst. In: Progress in Physiological Psychology, edited by E. Stellar and J. Sprague. New York: Academic Press, 1971. 3. Grossman, S. P. Role of the hypothalamus in the regulation of food and water intake. P~ychol. Rev. 82:210-218, 1975. 4. Keesey, R. E. and P. C. Boyle. Effects of quinine adulturation upon the body weight of LH-lesioned and intact male rats. J. comp. physiol. Psychol. 84: 38-46, 1973. 5. Keesey, R. E., P. C. Boyle, J. W. Kemnitz and J. S. Mitchel. The role of the lateral hypothalamus in determining the body weight set point. In: Hunger: Basic Mechanisms and Clinical Implications, edited by D. Novin, W. Wyrwicka and G. Bray. New York: Raven Press, 1976.

6. Keesey, R. E., T. L. Powley and J. W. Kemnitz. Prolonging lateral hypothalamic anorexia by tube-feeding. PhysioL Behav. 17:367 371, 1976. 7. Mitchel, J. S. and R. E. Keesey. The effects of lateral hypothalamic lesions and castration upon the body weight and composition of male rats. Behav. Biol. 11: 69-82, 1974. 8. Moskowitz, M. J. Running-wheel activity in the white rat as a function of combined food and water deprivation. J. comp. physiol. Psychol. 52: 621-625, 1959. 9. Powley, T. L. Reduction of body weight set-point by lateral hypothalamic lesions: Implications for an analysis of the lateral hypothalamic feeding syndrome. Ph.D. Thesis, University of Wisconsin, 1970. 10. Powley, T. L. and R. E. Keesey. Relationship of body weight to the lateral hypothalamic feeding syndrome. J. comp. physioL Psychol. 70: 25-36, 1970. 11. Teitelbaum, P. and A. N. Epstein. The lateral hypothalamic syndrome: Recovery of feeding and drinking after lateral hypothalamic lesions. PsychoL Rev. 69: 74-94, 1962.