Physiology & Behavior, Vol. 19, pp. 2 3 9 - 2 4 4 . Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.
Saccharin Aversion in the Rat Following Adrenalectomy M. T E R E S A A. S I L V A
Department o f Psychology, FacuMade de Filosofia, Ciencias e Letras de Ribeirflo Preto, University o f Sfto Paulo, 14.100 Ribeir~o, SP, Brazil ( R e c e i v e d 15 April 1 9 7 7 ) SILVA, M. T. A. Saccharin aversion in the rat following adrenalectomy. PHYSIOL. BEHAV. 19(2) 239-244, 1 9 7 7 . Changes in saccharin preference as a function of concentration were studied in rats after either adrenalectomy or sham surgery. A 48 hr two-bottle preference test was used. Under two different sets of testing conditions, adrenalectomized animals rejected saccharin solutions at concentrations that were highly acceptible to controls and did not show an appetitive behavior towards any of the concentrations tested. This aversion pattern persisted even when preference tests were conducted for as long as l0 days. However, it was abolished when the adrenalectomized rats were exposed to saccharin for 6 days prior to surgery, or when they were administered the glucocorticoid dexamethasone. Results are discussed with respect to the effects of adrenal hormones on sensory systems and to taste-aversion learning. Saccharin preference
Adrenalectomy
Taste aversion
Adrenal hormones
A N I M A L studies o n the i n f l u e n c e o f adrenal h o r m o n e s o n b e h a v i o r have dealt e x t e n s i v e l y w i t h m o t i v a t i o n a l a n d l e a r n i n g aspects [3, 35, 3 6 ] . Less a t t e n t i o n has b e e n paid t o t h e effects o f these h o r m o n e s o n p e r c e p t i o n . Y e t , a d r e n a l h o r m o n e s c a n p r o f o u n d l y affect p e r c e p t u a l s y s t e m s in h u m a n s [ 1 0 ] , a n d m a n y of the a n i m a l b e h a v i o r effects o b s e r v e d a f t e r a l t e r a t i o n s o f t h e adrenal s y s t e m m i g h t reflect e v e n t u a l p e r c e p t u a l changes. H e n k i n [10] has d e m o n s t r a t e d t h a t a d r e n a l h o r m o n e s are i n v o l v e d in a u d i t o r y , o l f a c t o r y a n d taste p e r c e p t i o n . He f o u n d i n c r e a s e d a c u i t y a n d decreased d i s c r i m i n a t i o n ability in t h e s e sensory m o d a l i t i e s in p a t i e n t s suffering f r o m u n t r e a t e d a d r e n a l i n s u f f i c i e n c y . These a l t e r a t i o n s were reverted by the administration of glucocorticoids. T h e species g e n e r a l i t y o f such findings h a s b e e n little e x p l o r e d . One s t u d y o n a u d i t o r y sensitivity in chinchillas a n d rats m e a s u r e d a u d i t o r y e v o k e d p o t e n t i a l s u n d e r cond i t i o n s o f a d r e n a l cortical i n s u f f i c i e n c y , a n d o b s e r v e d a l t e r a t i o n s c o n s i s t e n t w i t h t h o s e o b s e r v e d in h u m a n adrenal hypofunction [2]. Another report indicated that olfactory a c u i t y o f a d r e n a l e c t o m i z e d rats is h i g h e r t h a n n o r m a l a n d is r e s t o r e d b y c o r t i c o s t e r o n e [ 2 7 ] . The p r e s e n t s t u d y investigates taste p r e f e r e n c e in a d r e n a l e c t o m i z e d rats. If adrenal i n s u f f i c i e n c y p r o d u c e s changes in s e n s o r y acuity a n d r e c o g n i t i o n in r o d e n t s a n a l o g o u s to t h o s e o b s e r v e d in h u m a n s , taste p r e f e r e n c e m i g h t be e x p e c t e d to be a f f e c t e d by p r o c e d u r e s t h a t r e d u c e adrenal cortical f u n c t i o n . A l t e r a t i o n s in taste sensitivity related t o adrenal funct i o n in rats were earlier p r o p o s e d b y R i c h t e r [ 2 3 ] . In r e p o r t i n g his classical d e m o n s t r a t i o n s o n r e d u c e d preference t h r e s h o l d a n d e n h a n c e d p r e f e r e n c e f o r NaC1 s o l u t i o n s over w a t e r in a d r e n a l e c t o m i z e d rats, he suggested t h a t
ACTH
a d r e n a l e c t o m y increased s e n s o r y acuity for NaCI [ 2 3 , 2 5 ] . It is also k n o w n t h a t any s o d i u m salt is p r e f e r r e d to n o n s o d i u m salts after a d r e n a l e c t o m y [ 1 9 , 2 5 ] . Since the taste s u b s t a n c e s in all o f these cases were related to t h e s o d i u m d e f i c i e n c y of a d r e n a l e c t o m i z e d rats, a m o t i v a t i o n a l r a t h e r t h a n sensory i n t e r p r e t a t i o n c a n n o t easily be discarded. A n o t h e r change in g u s t a t o r y responses o b s e r v e d in a d r e n a l e c t o m i z e d rats was a decreased p r e f e r e n c e for a d e x t r o s e s o l u t i o n [ 2 4 ] . H o w e v e r , since d e x t r o s e is a n u t r i e n t , this result was a t t r i b u t e d to an i m b a l a n c e in c a r b o h y d r a t e m e t a b o l i s m . In o r d e r to s t u d y the adrenal role in taste sensitivity in animals, a s u b s t a n c e u n r e l a t e d to t h e b o d i l y needs b r o u g h t a b o u t b y adrenal a l t e r a t i o n s w o u l d be desirable. T h e objective o f this s t u d y was to verify w h e t h e r a d r e n a l h o r m o n e s i n f l u e n c e a n i m a l taste p r e f e r e n c e w h e n a n o n n u t r i t i v e sweet s u b s t a n c e is used as a g u s t a t o r y stimulus. Specifically, a d r e n a l e c t o m i z e d rats have b e e n o b s e r v e d f o r changes in p r e f e r e n c e as a f u n c t i o n o f saccharin c o n c e n t r a tion. The time course of such a p r e f e r e n c e has b e e n e x p l o r e d as well as some o f the factors t h a t m i g h t a f f e c t it: p r i o r e x p o s u r e to the flavor a n d h o r m o n a l r e p l a c e m e n t with a glucocorticoid.
METHOD
Animals T h e animals were e x p e r i m e n t a l l y naive male Wistar rats, weighing b e t w e e n 2 0 0 - 2 5 0 g at the b e g i n n i n g o f t h e e x p e r i m e n t s . T h r e e days b e f o r e surgery, each rat was placed in an individual cage, w i t h f o o d a n d w a t e r ad lib. A f t e r
The author is grateful to Graqa A. Cicillini for her assistance in this experiment. 239
240
SILVA
surgery, NaC1 crystals in the form of pellets were m a d e available ad lib to the rats.
Solutions All saccharin s o l u t i o n s were m a d e w i t h s o d i u m saccharin ( S q u i b b ) dissolved in tap water. C o n c e n t r a t i o n s are expressed as w e i g h t / v o l u m e p e r c e n t a g e .
Surgery Rats were a n e s t h e t i z e d w i t h 35 m g / k g s o d i u m p e n t o barbital. Bilateral a d r e n a l e c t o m y was p e r f o r m e d b y exposing the k i d n e y s and r e m o v i n g the adrenals w i t h as m u c h of the s u r r o u n d i n g adipose tissue as possible. Previous w o r k in this l a b o r a t o r y h a d s h o w n t h a t this p r o c e d u r e allows for fairly c o m p l e t e a n d lasting r e m o v a l of t h e adrenals, as assessed by f l u o r i m e t r i c d e t e r m i n a t i o n of plasma c o r t i c o s t e rone levels [ 3 0 , 3 1 ] . C o n t r o l rats were s u b m i t t e d to the same surgical p r o c e d u r e , e x c e p t for r e m o v a l of t h e adrenal glands.
General Procedure for Preference Tests A t w o - b o t t l e choice p r o c e d u r e was used. T w o 100-ml R i c h t e r t u b e s were m o u n t e d in f r o n t of each cage a n d filled with tap w a t e r before the saccharin p r e f e r e n c e tests began. Preference tests s t a r t e d t h r e e days a f t e r surgery. The r o u t i n e p r o c e d u r e was as follows: the t w o R i c h t e r tubes, one c o n t a i n i n g tap w a t e r and the o t h e r a saccharin s o l u t i o n , were c o n t i n u o u s l y accessible to the animals. Every 24 hr, fluid i n t a k e was m e a s u r e d , b o t t l e s were cleaned a n d refilled, and t h e i r p o s i t i o n i n v e r t e d in o r d e r to c o u n t e r balance possible p o s i t i o n h a b i t effects. Fluid i n t a k e readings were c o r r e c t e d for b o d y weight and are expressed as m l / 1 0 0 g. S a c c h a r i n p r e f e r e n c e (SP) is expressed b y the index : SP =
saccharin s o l u t i o n i n t a k e
x 100.
saccharin s o l u t i o n + w a t e r i n t a k e Fluid i n t a k e a n d saccharin p r e f e r e n c e data refer to the daily c o n s u m p t i o n averaged over t w o c o n s e c u t i v e days. Where m e d i a n s are s h o w n , statistical analysis was m a d e by the M a n n - W h i t n e y U test; w h e n m o r e t h a n two g r o u p s were c o m p a r e d , the U test was p e r f o r m e d w h e n i n d i c a t e d by a Kruskal-Wallis analysis of variance. Where m e a n s are s h o w n , c o m p a r i s o n s are based o n t-tests. Experiment I. In o r d e r to measure changes in p r e f e r e n c e as a f u n c t i o n of saccharin c o n c e n t r a t i o n , d i f f e r e n t animals were studied u n d e r t h r e e sets of e x p e r i m e n t a l c o n d i t i o n s . The first one i n c l u d e d 6 a d r e n a l e c t o m i z e d ( A D X ) a n d 5 s h a m - o p e r a t e d animals (SHAM). Three days a f t e r surgery, they were e x p o s e d to preference tests b e t w e e n w a t e r a n d saccharin s o l u t i o n s w h i c h were o f f e r e d at increasing conc e n t r a t i o n s : 0.008%, 0.03%, 0.15%, 0.5% a n d 4.0%. Each c o n c e n t r a t i o n was available for 48 hr. In the s e c o n d e x p e r i m e n t a l s i t u a t i o n , 82 rats were divided into 7 groups, each including 7 A D X and 5 SHAM animals. Each g r o u p was e x p o s e d to p r e f e r e n c e tests b e t w e e n w a t e r a n d one of the following saccharin c o n c e n trations: 0.004%, 0.006%, 0.008%, 0.03%, 0.15%, 0.5%, 1.5%, for 48 hr. In t h e t h i r d e x p e r i m e n t a l c o n d i t i o n , 5 A D X rats a n d 5 SHAM c o n t r o l s were e x p o s e d to the t w o - b o t t l e p r e f e r e n c e test d u r i n g 10 consecutive days. The saccharin s o l u t i o n
c o n c e n t r a t i o n was c o n s t a n t at 0.5% t h r o u g h o u t this test. This c o n c e n t r a t i o n was c h o s e n because it yielded large differences in p r e f e r e n c e scores b e t w e e n ADX and SHAM groups. Experiment2. T w e n t y - f o u r rats were t e s t e d in the p r e f e r e n c e s i t u a t i o n for 6 days prior to surgery, A 0.5% saccharin s o l u t i o n was used. The animals were t h e n divided i n t o t w o g r o u p s m a t c h e d for m e d i a n SP scores; 13 animals were a d r e n a l e c t o m i z e d , and the r e m a i n i n g were s h a m - o p e r ated. F o r the first t h r e e days a f t e r surgery, all animals had only w a t e r available to drink, being t h e n offered b o t h w a t e r and a 0.5% saccharin s o l u t i o n up to the fifth day. Experiment 3. F o r 6 c o n s e c u t i v e days, s t a r t i n g 3 days a f t e r surgery, A D X rats received IP injections of 0.2 m g / k g d e x a m e t h a s o n e acetate ( D E X , N = 7) or vehicle (N = 6) daily; SHAM c o n t r o l s were also i n j e c t e d with the same dose of t h e drug (N = 5) or w i t h the vehicle (N = 5). A n o t h e r group of A D X rats was injected w i t h the vehicle o n i n j e c t i o n Days 1 and 2, and with DEX on i n j e c t i o n Days 3 to 6. DEX was dissolved in the p r o p o r t i o n of 1 mg of the salt to 0.125 ml e t h a n o l a n d 10 ml saline. P r e f e r e n c e tests were carried o u t for 6 days b e g i n n i n g o n the first i n j e c t i o n day, using the 0.5% saccharin s o l u t i o n . RESULTS
Changes in Saccharin Preference Following Adrenalectomy Experiment 1. Figure I shows the m e a n fluid i n t a k e a n d the m e d i a n saccharin p r e f e r e n c e (SP) values for the 11 animals t h a t were e x p o s e d to increasing c o n c e n t r a t i o n s of saccharin s o l u t i o n versus water. Each value is based on daily i n t a k e averaged over 48 hr, t h u s taking i n t o a c c o u n t the reversal of b o t t l e position. The SHAM group displayed the n o r m a l p r e f e r e n c e p a t t e r n for saccharin solutions: t h e m e d i a n SP i n d e x c h a n g e d as a f u n c t i o n of c o n c e n t r a t i o n , increasing f r o m a n e u t r a l p o i n t to a peak as c o n c e n t r a t i o n increased, up t o a p o i n t where the s o l u t i o n was rejected. ADX animals, h o w e v e r , did n o t show any p r e f e r e n c e for the saccharin solutions, and at the highest c o n c e n t r a t i o n s (0.5% and 4.0%) t h e y e x h i b i t e d s t r o n g rejection. Differences in SP i n d e x e s b e t w e e n ADX and SHAM animals were significant for all c o n c e n t r a t i o n s tested e x c e p t the m o s t dilute (U = O, p < 0 . 0 0 2 for 0.03%, 0.5% and 4.0%; U = 2, p < 0 . 0 0 9 for 0.15%). Total fluid intake, as s h o w n in the u p p e r part of Fig. 1, was slightly b u t n o t significantly higher for the SHAM group in all b u t the m o s t c o n c e n t r a t ed saccharin solution. Figure 2 shows the fluid i n t a k e and saccharin p r e f e r e n c e for 7 d i f f e r e n t groups of rats tested for p r e f e r e n c e w i t h d i f f e r e n t c o n c e n t r a t i o n s of saccharin solution. Again, the m e d i a n SP i n d e x of rats in SHAM groups c h a n g e d as a f u n c t i o n o f c o n c e n t r a t i o n of the s o l u t i o n : it increased f r o m a neutral p o i n t a r o u n d 50 to a peak near 90, at 0.15%. This characteristic f u n c t i o n was n o t o b t a i n e d for the A D X animals, w h i c h t e n d e d to e x h i b i t an increasingly higher rejection for saccharin as the c o n c e n t r a t i o n rose. The differences b e t w e e n the SP i n d e x e s of A D X and SHAM groups were significant at the following c o n c e n t r a t i o n s : 0.03% (U = 2, p < 0 . 0 0 9 ) ; 0.15% (U = 7, p < 0 . 0 5 ) ; and 0.5% (U = 1, p < 0 . 0 0 4 ) . Fluid i n t a k e was n o t significantly differe n t b e t w e e n ADX and SHAM groups, e x c e p t at 0.5% (p
Saccharin Aversion in the Rat Following Adrenalectomy M. T E R E S A A. S I L V A
Department o f Psychology, FacuMade de Filosofia, Ciencias e Letras de Ribeirflo Preto, University o f Sfto Paulo, 14.100 Ribeir~o, SP, Brazil ( R e c e i v e d 15 April 1 9 7 7 ) SILVA, M. T. A. Saccharin aversion in the rat following adrenalectomy. PHYSIOL. BEHAV. 19(2) 239-244, 1 9 7 7 . Changes in saccharin preference as a function of concentration were studied in rats after either adrenalectomy or sham surgery. A 48 hr two-bottle preference test was used. Under two different sets of testing conditions, adrenalectomized animals rejected saccharin solutions at concentrations that were highly acceptible to controls and did not show an appetitive behavior towards any of the concentrations tested. This aversion pattern persisted even when preference tests were conducted for as long as l0 days. However, it was abolished when the adrenalectomized rats were exposed to saccharin for 6 days prior to surgery, or when they were administered the glucocorticoid dexamethasone. Results are discussed with respect to the effects of adrenal hormones on sensory systems and to taste-aversion learning. Saccharin preference
Adrenalectomy
Taste aversion
Adrenal hormones
A N I M A L studies o n the i n f l u e n c e o f adrenal h o r m o n e s o n b e h a v i o r have dealt e x t e n s i v e l y w i t h m o t i v a t i o n a l a n d l e a r n i n g aspects [3, 35, 3 6 ] . Less a t t e n t i o n has b e e n paid t o t h e effects o f these h o r m o n e s o n p e r c e p t i o n . Y e t , a d r e n a l h o r m o n e s c a n p r o f o u n d l y affect p e r c e p t u a l s y s t e m s in h u m a n s [ 1 0 ] , a n d m a n y of the a n i m a l b e h a v i o r effects o b s e r v e d a f t e r a l t e r a t i o n s o f t h e adrenal s y s t e m m i g h t reflect e v e n t u a l p e r c e p t u a l changes. H e n k i n [10] has d e m o n s t r a t e d t h a t a d r e n a l h o r m o n e s are i n v o l v e d in a u d i t o r y , o l f a c t o r y a n d taste p e r c e p t i o n . He f o u n d i n c r e a s e d a c u i t y a n d decreased d i s c r i m i n a t i o n ability in t h e s e sensory m o d a l i t i e s in p a t i e n t s suffering f r o m u n t r e a t e d a d r e n a l i n s u f f i c i e n c y . These a l t e r a t i o n s were reverted by the administration of glucocorticoids. T h e species g e n e r a l i t y o f such findings h a s b e e n little e x p l o r e d . One s t u d y o n a u d i t o r y sensitivity in chinchillas a n d rats m e a s u r e d a u d i t o r y e v o k e d p o t e n t i a l s u n d e r cond i t i o n s o f a d r e n a l cortical i n s u f f i c i e n c y , a n d o b s e r v e d a l t e r a t i o n s c o n s i s t e n t w i t h t h o s e o b s e r v e d in h u m a n adrenal hypofunction [2]. Another report indicated that olfactory a c u i t y o f a d r e n a l e c t o m i z e d rats is h i g h e r t h a n n o r m a l a n d is r e s t o r e d b y c o r t i c o s t e r o n e [ 2 7 ] . The p r e s e n t s t u d y investigates taste p r e f e r e n c e in a d r e n a l e c t o m i z e d rats. If adrenal i n s u f f i c i e n c y p r o d u c e s changes in s e n s o r y acuity a n d r e c o g n i t i o n in r o d e n t s a n a l o g o u s to t h o s e o b s e r v e d in h u m a n s , taste p r e f e r e n c e m i g h t be e x p e c t e d to be a f f e c t e d by p r o c e d u r e s t h a t r e d u c e adrenal cortical f u n c t i o n . A l t e r a t i o n s in taste sensitivity related t o adrenal funct i o n in rats were earlier p r o p o s e d b y R i c h t e r [ 2 3 ] . In r e p o r t i n g his classical d e m o n s t r a t i o n s o n r e d u c e d preference t h r e s h o l d a n d e n h a n c e d p r e f e r e n c e f o r NaC1 s o l u t i o n s over w a t e r in a d r e n a l e c t o m i z e d rats, he suggested t h a t
ACTH
a d r e n a l e c t o m y increased s e n s o r y acuity for NaCI [ 2 3 , 2 5 ] . It is also k n o w n t h a t any s o d i u m salt is p r e f e r r e d to n o n s o d i u m salts after a d r e n a l e c t o m y [ 1 9 , 2 5 ] . Since the taste s u b s t a n c e s in all o f these cases were related to t h e s o d i u m d e f i c i e n c y of a d r e n a l e c t o m i z e d rats, a m o t i v a t i o n a l r a t h e r t h a n sensory i n t e r p r e t a t i o n c a n n o t easily be discarded. A n o t h e r change in g u s t a t o r y responses o b s e r v e d in a d r e n a l e c t o m i z e d rats was a decreased p r e f e r e n c e for a d e x t r o s e s o l u t i o n [ 2 4 ] . H o w e v e r , since d e x t r o s e is a n u t r i e n t , this result was a t t r i b u t e d to an i m b a l a n c e in c a r b o h y d r a t e m e t a b o l i s m . In o r d e r to s t u d y the adrenal role in taste sensitivity in animals, a s u b s t a n c e u n r e l a t e d to t h e b o d i l y needs b r o u g h t a b o u t b y adrenal a l t e r a t i o n s w o u l d be desirable. T h e objective o f this s t u d y was to verify w h e t h e r a d r e n a l h o r m o n e s i n f l u e n c e a n i m a l taste p r e f e r e n c e w h e n a n o n n u t r i t i v e sweet s u b s t a n c e is used as a g u s t a t o r y stimulus. Specifically, a d r e n a l e c t o m i z e d rats have b e e n o b s e r v e d f o r changes in p r e f e r e n c e as a f u n c t i o n o f saccharin c o n c e n t r a tion. The time course of such a p r e f e r e n c e has b e e n e x p l o r e d as well as some o f the factors t h a t m i g h t a f f e c t it: p r i o r e x p o s u r e to the flavor a n d h o r m o n a l r e p l a c e m e n t with a glucocorticoid.
METHOD
Animals T h e animals were e x p e r i m e n t a l l y naive male Wistar rats, weighing b e t w e e n 2 0 0 - 2 5 0 g at the b e g i n n i n g o f t h e e x p e r i m e n t s . T h r e e days b e f o r e surgery, each rat was placed in an individual cage, w i t h f o o d a n d w a t e r ad lib. A f t e r
The author is grateful to Graqa A. Cicillini for her assistance in this experiment. 239
240
SILVA
surgery, NaC1 crystals in the form of pellets were m a d e available ad lib to the rats.
Solutions All saccharin s o l u t i o n s were m a d e w i t h s o d i u m saccharin ( S q u i b b ) dissolved in tap water. C o n c e n t r a t i o n s are expressed as w e i g h t / v o l u m e p e r c e n t a g e .
Surgery Rats were a n e s t h e t i z e d w i t h 35 m g / k g s o d i u m p e n t o barbital. Bilateral a d r e n a l e c t o m y was p e r f o r m e d b y exposing the k i d n e y s and r e m o v i n g the adrenals w i t h as m u c h of the s u r r o u n d i n g adipose tissue as possible. Previous w o r k in this l a b o r a t o r y h a d s h o w n t h a t this p r o c e d u r e allows for fairly c o m p l e t e a n d lasting r e m o v a l of t h e adrenals, as assessed by f l u o r i m e t r i c d e t e r m i n a t i o n of plasma c o r t i c o s t e rone levels [ 3 0 , 3 1 ] . C o n t r o l rats were s u b m i t t e d to the same surgical p r o c e d u r e , e x c e p t for r e m o v a l of t h e adrenal glands.
General Procedure for Preference Tests A t w o - b o t t l e choice p r o c e d u r e was used. T w o 100-ml R i c h t e r t u b e s were m o u n t e d in f r o n t of each cage a n d filled with tap w a t e r before the saccharin p r e f e r e n c e tests began. Preference tests s t a r t e d t h r e e days a f t e r surgery. The r o u t i n e p r o c e d u r e was as follows: the t w o R i c h t e r tubes, one c o n t a i n i n g tap w a t e r and the o t h e r a saccharin s o l u t i o n , were c o n t i n u o u s l y accessible to the animals. Every 24 hr, fluid i n t a k e was m e a s u r e d , b o t t l e s were cleaned a n d refilled, and t h e i r p o s i t i o n i n v e r t e d in o r d e r to c o u n t e r balance possible p o s i t i o n h a b i t effects. Fluid i n t a k e readings were c o r r e c t e d for b o d y weight and are expressed as m l / 1 0 0 g. S a c c h a r i n p r e f e r e n c e (SP) is expressed b y the index : SP =
saccharin s o l u t i o n i n t a k e
x 100.
saccharin s o l u t i o n + w a t e r i n t a k e Fluid i n t a k e a n d saccharin p r e f e r e n c e data refer to the daily c o n s u m p t i o n averaged over t w o c o n s e c u t i v e days. Where m e d i a n s are s h o w n , statistical analysis was m a d e by the M a n n - W h i t n e y U test; w h e n m o r e t h a n two g r o u p s were c o m p a r e d , the U test was p e r f o r m e d w h e n i n d i c a t e d by a Kruskal-Wallis analysis of variance. Where m e a n s are s h o w n , c o m p a r i s o n s are based o n t-tests. Experiment I. In o r d e r to measure changes in p r e f e r e n c e as a f u n c t i o n of saccharin c o n c e n t r a t i o n , d i f f e r e n t animals were studied u n d e r t h r e e sets of e x p e r i m e n t a l c o n d i t i o n s . The first one i n c l u d e d 6 a d r e n a l e c t o m i z e d ( A D X ) a n d 5 s h a m - o p e r a t e d animals (SHAM). Three days a f t e r surgery, they were e x p o s e d to preference tests b e t w e e n w a t e r a n d saccharin s o l u t i o n s w h i c h were o f f e r e d at increasing conc e n t r a t i o n s : 0.008%, 0.03%, 0.15%, 0.5% a n d 4.0%. Each c o n c e n t r a t i o n was available for 48 hr. In the s e c o n d e x p e r i m e n t a l s i t u a t i o n , 82 rats were divided into 7 groups, each including 7 A D X and 5 SHAM animals. Each g r o u p was e x p o s e d to p r e f e r e n c e tests b e t w e e n w a t e r a n d one of the following saccharin c o n c e n trations: 0.004%, 0.006%, 0.008%, 0.03%, 0.15%, 0.5%, 1.5%, for 48 hr. In t h e t h i r d e x p e r i m e n t a l c o n d i t i o n , 5 A D X rats a n d 5 SHAM c o n t r o l s were e x p o s e d to the t w o - b o t t l e p r e f e r e n c e test d u r i n g 10 consecutive days. The saccharin s o l u t i o n
c o n c e n t r a t i o n was c o n s t a n t at 0.5% t h r o u g h o u t this test. This c o n c e n t r a t i o n was c h o s e n because it yielded large differences in p r e f e r e n c e scores b e t w e e n ADX and SHAM groups. Experiment2. T w e n t y - f o u r rats were t e s t e d in the p r e f e r e n c e s i t u a t i o n for 6 days prior to surgery, A 0.5% saccharin s o l u t i o n was used. The animals were t h e n divided i n t o t w o g r o u p s m a t c h e d for m e d i a n SP scores; 13 animals were a d r e n a l e c t o m i z e d , and the r e m a i n i n g were s h a m - o p e r ated. F o r the first t h r e e days a f t e r surgery, all animals had only w a t e r available to drink, being t h e n offered b o t h w a t e r and a 0.5% saccharin s o l u t i o n up to the fifth day. Experiment 3. F o r 6 c o n s e c u t i v e days, s t a r t i n g 3 days a f t e r surgery, A D X rats received IP injections of 0.2 m g / k g d e x a m e t h a s o n e acetate ( D E X , N = 7) or vehicle (N = 6) daily; SHAM c o n t r o l s were also i n j e c t e d with the same dose of t h e drug (N = 5) or w i t h the vehicle (N = 5). A n o t h e r group of A D X rats was injected w i t h the vehicle o n i n j e c t i o n Days 1 and 2, and with DEX on i n j e c t i o n Days 3 to 6. DEX was dissolved in the p r o p o r t i o n of 1 mg of the salt to 0.125 ml e t h a n o l a n d 10 ml saline. P r e f e r e n c e tests were carried o u t for 6 days b e g i n n i n g o n the first i n j e c t i o n day, using the 0.5% saccharin s o l u t i o n . RESULTS
Changes in Saccharin Preference Following Adrenalectomy Experiment 1. Figure I shows the m e a n fluid i n t a k e a n d the m e d i a n saccharin p r e f e r e n c e (SP) values for the 11 animals t h a t were e x p o s e d to increasing c o n c e n t r a t i o n s of saccharin s o l u t i o n versus water. Each value is based on daily i n t a k e averaged over 48 hr, t h u s taking i n t o a c c o u n t the reversal of b o t t l e position. The SHAM group displayed the n o r m a l p r e f e r e n c e p a t t e r n for saccharin solutions: t h e m e d i a n SP i n d e x c h a n g e d as a f u n c t i o n of c o n c e n t r a t i o n , increasing f r o m a n e u t r a l p o i n t to a peak as c o n c e n t r a t i o n increased, up t o a p o i n t where the s o l u t i o n was rejected. ADX animals, h o w e v e r , did n o t show any p r e f e r e n c e for the saccharin solutions, and at the highest c o n c e n t r a t i o n s (0.5% and 4.0%) t h e y e x h i b i t e d s t r o n g rejection. Differences in SP i n d e x e s b e t w e e n ADX and SHAM animals were significant for all c o n c e n t r a t i o n s tested e x c e p t the m o s t dilute (U = O, p < 0 . 0 0 2 for 0.03%, 0.5% and 4.0%; U = 2, p < 0 . 0 0 9 for 0.15%). Total fluid intake, as s h o w n in the u p p e r part of Fig. 1, was slightly b u t n o t significantly higher for the SHAM group in all b u t the m o s t c o n c e n t r a t ed saccharin solution. Figure 2 shows the fluid i n t a k e and saccharin p r e f e r e n c e for 7 d i f f e r e n t groups of rats tested for p r e f e r e n c e w i t h d i f f e r e n t c o n c e n t r a t i o n s of saccharin solution. Again, the m e d i a n SP i n d e x of rats in SHAM groups c h a n g e d as a f u n c t i o n o f c o n c e n t r a t i o n of the s o l u t i o n : it increased f r o m a neutral p o i n t a r o u n d 50 to a peak near 90, at 0.15%. This characteristic f u n c t i o n was n o t o b t a i n e d for the A D X animals, w h i c h t e n d e d to e x h i b i t an increasingly higher rejection for saccharin as the c o n c e n t r a t i o n rose. The differences b e t w e e n the SP i n d e x e s of A D X and SHAM groups were significant at the following c o n c e n t r a t i o n s : 0.03% (U = 2, p < 0 . 0 0 9 ) ; 0.15% (U = 7, p < 0 . 0 5 ) ; and 0.5% (U = 1, p < 0 . 0 0 4 ) . Fluid i n t a k e was n o t significantly differe n t b e t w e e n ADX and SHAM groups, e x c e p t at 0.5% (p
