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Changes in corticosterone levels in iron deficient rats
t
Physiology & Behavior, Vol. 27, pp. 1085--1088. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
Changes in Corticosterone Levels in Iron Deficient R a t s I A . M. W I L L I A M S O N , 2 K . T. N G A N D A . R I C H D A L E
Department of Psychology, La Trobe University, Bundoora, Victoria, Australia 3083 R e c e i v e d 1 J u l y 1981 WlLLIAMSON, A. M., K. T. NG AND A. RICHDALE. Changes in corticosterone levels in iron deficient rats.
PHYSIOL. BEHAV. 27(6) 1085-1088, 1981.--The hypothesis that behavioural changes in iron deficiency are a product of stress was tested by monitoring corticosterone levels. Adult hooded rats were placed on an iron deficient diet for 2 (Stage 1), 8 (Stage 2) or 12 (Stage 3) weeks. When trained and tested on a single-trial taste aversion task, iron deficient animals showed the same performance as seen in previous experiments. Poor performance in early iron deficiency were followed by a temporary return to normal levels as the deficiency progressed, and falling below normal with continued iron depletion. Iron deficient animals failed to demonstrate the predicted stress-related changes in cortocosterone levels. Instead of an increasing in corticosterone levels after conditioning, levels fell to well below normal at all stages of deficiency following conditioning. Iron deficiency probably limits the action of certain enzymes responsible for corticosterone production. Due to the similarity of changes in catecholamines in stress and iron deficiency, it is possible that these changes will explain the pattern of behavioural response in iron depletion. Corticosterone
Iron deficiency
Adult rats
Memory
W H I L E iron deficiency is the world's most common nutritional deficiency, it remains relatively unresearched. This is particularly so for the behavioural consequences of iron deficiency. Recently, however, some evidence has appeared that a period of iron deficiency does affect the behaviour of both weanling and adult rats. In weanling rats, iron deficiency in the first weeks of life decreased responsiveness in novel and aversive situations [20]. Exploratory behaviour was also affected, with iron deficient rats showing reduced activity. In adult rats, iron deficiency produces deficits in memory [22] as well as changes in exploratory activity [21,22]. In adult animals the pattern of behavioural change as a result of iron deficiency is suggestive of a response to stress [15, 21, 22]. The initial reaction to iron depletion is alarm. Although the level of iron stores is the only physiological indicator to be affected, activity increases significantly above normal levels, while memory deteriorates. With further iron depletion the animal is able to adapt to the stressor both physiologically and psychologically. Iron stores and memory return to normal levels but activity deteriorates to below normal. Exhaustion occurs with prolonged iron depletion causing further loss of motor and cognitive functions as well as body iron. That iron deficiency may be a potent stressor is supported by a study with weanling rats [20] in which iron deficiency was found to produce higher resting corticosterone levels but a smaller stress increment than normal. The authors postulated that iron deficiency reduces the young animal's responsiveness to environmental stimuli.
Activity
Further support is given by the findings of elevated basal levels of plasma corticoids in both animals and humans who have protein-calorie malnutrition [6,16]. This study was designed to investigate whether the observed behavioural changes in iron deficient adult rats [22] constituted an adaptation to stress. It was not sufficient, however, simply to assay for corticosterone at stages in the iron deficiency process and compare the results with those of normals of the same age. Unfortunately the behaviours observed in the progression of iron deficiency may be confounded by the task used to measure them, viz a conditioned taste aversion task. Evidence suggests that injections of lithium chloride that are sufficient to produce learned taste aversions, produce sustained increases in plasma corticoid levels lasting from 2 to 4 hours [4]. Any changes in corticosterone levels could therefore be due to the nature of the task itself. For this reason corticosterone levels were measured at stages during the deficiency process and at invervals during the task.
METHOD
Subjects One hundred and six male and female Wistar-derived rats from the breeding colony of the Department of Psychology at La Trobe University were used. Animals were housed in the laboratory on a 12/12 light-dark cycle for at least one week prior to the beginning of the study. Males were housed in
1.This work was carried out with the support of a La Trobe University Postgraduate Scholarship. 2Address reprint requests to first author, now at Division of Occupational Health and Radiation Control, Health Commission Of New South Wales, P.O. Box 163, Lidcombe, New South Wales, Australia, 2141.
C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/121085-04502.00/0
1086
W I L L I A M S O N , NG A N D R I C H D A L E
wire cages in pairs and females in triplets. Food and water were available ad lib and access to the laboratory by personnel was kept to a minimum.
100, c
.O
Apparatus The conditioned taste aversion apparatus that was used has been described elsewhere [22].
80
> 60
Diet
C
Iron deficiency was produced by a diet of skim milk, sucrose, salt mixture, added vitamins, minerals and essential amino acids, with 2-3 mg/kg iron [9]. Control animals received the same diet but with 70 mg/kg iron.
t-
40
U U ta
20
Experimental Design The design was essentially a 2×3 factorial design with sample sizes varying from 5 to 8, and balanced as far as possible with respect to the sex variable. Beginning at 90 days after birth, experimental animals were placed on the iron deficient diet for 14 (Stage 1), 56 (Stage 2) or 84 (Stage 3) days. Animals were tested in a no-delay taste aversion conditioning paradigm in which performance measures were taken 24 hr after conditioning. Blood samples were taken at intervals throughout the procedure.
Procedure The behavioural task has already been reported in the literature [19,22]. Pretraining phase. Animals were water deprived 24 hr before experimentation began. On each of the following 3 days each animal was placed in the test box for 10 min and allowed to drink tap water from the two spouts. At all other times only food was available. Training phase. On the fourth day 0.1% sodium saccharin solution instead of water was available from both spouts and each animal was allowed to drink for 10 minutes. Immediately after, a solution of 0.6% (0.15 M) lithium chloride (LiCl) calculated on a dosage of 10 ml/kg bodyweight was injected intragastrically. Testing phase. In the 10 min drinking session on day 5 saccharin solution was available from one of the two spouts and tap water from the other, with position randomized for each animal. Performance measures. The total number of licks and the amount of fluid consumed from each spout was monitored in each 20 rain test box exposure. Activity was monitored on a Dopler activity counter. Blood samples were taken from one-third of all animals on day 3 (final day of pretraining), day 4 (conditioning day) and day 5 (test day). Sampling occurred within ten minutes of removal from the behavioural testing apparatus. Animals were no longer used after this sample was taken. Memory in the conditioning task was indexed by a saccharin aversion score defined as: % SA =
number of licks of water in test session number of licks in test session
× 100
Saccharin aversion scores and activity measures were taken for animals which went through to day 5.
Corticosterone Analysis After anaesthetization with ether, approximately 3 ml of
weeks
on
diet
FIG. 1. Performance of iron deficient ( I ) and normal (e) rats on the conditioned taste aversion task (full association, 24 hour retention interval). Mean saccharin aversion scores are given. Significantly Tower saccharin aversion scores are shown by iron deficient rats compared with their controls after 2 and 12 weeks of no-iron diet.
blood was collected by the orbital eye bleed technique [14]. This whold process took less than 3 min to complete. The blood was centrifuged, plasma collected and frozen immediately. Blood collection always occurred between 12 and 2 p. m. Corticosterone analysis was performed on the plasma [11]. Florisil was used as the absorbent material and human plasma CBG (corticosteroid binding globulin) as the binder. For female rats progesterone was precipitated out of the plasma before corticosterone analysis was performed. RESULTS Saccharin aversion results are shown in Fig. 1. Analysis of variance revealed a significant difference between deficient and control groups, F(5,32)=3.35,p<0.025. Subsequent Scheffe tests showed that both Stage 1 and Stage 3 animals were significantly different from their own controls. Stage 2 animals did not differ from controls. Analysis of variance of activity scores revealed no significant differences between deficient and control animals, F(5,32)=2.31, N.S. Activity results are shown in Fig. 2. A significant negative correlation was found between saccharin aversion and activity scores, r= -0.36, t =3.27, p<0.01. The results of corticosterone analysis are shown in Table 1. Mann Whitney U tests were performed on this data due to the small and unequal sample sizes. Corticosterone levels of all deficient groups were significantly lower than control levels on day 5 of the conditioned taste aversion task (Stage I compared with control U = 3 , p<0.002; Stage 2 with control U = 2 , p<0.006; Stage 3 with control, U = 4 , p<0.01). Iron deficient animals had significantly lower corticosterone levels on day 5 than on day 3 (Stage l, U--13, p<0.02; Stage 2, U = 6 , p<0.04; Stage 3, U = l , p<0.008). F o r control
C O R T I C O S T E R O N E L E V E L S A N D IRON D E F I C I E N C Y
1087 TABLE 1 VARIATIONSIN CORTICOSTERONE LEVELS (ng/100ml PLASMA)OF IRON DEFICIENTAND NORMALRATSDURINGTHE CONDITIONED TASTE AVERSIONTASK
g~ SO0 t
E 4001 0
E aoo Stage 1
(~ 2oc
Stage 1, control
m
~) lOO ,D
-'Q-
.......
-0
E
Stage 2 Stage 2, control
c
Stage 3 weeks
on diet
FIG. 2. Variations in the activity levels (mean number of large movements) or iron deficient (11) and normal (O) rats. Activity levels of iron deficient rats were no different from those of their normal controls at any stage of deficiency.
animals, this same comparison revealed no significant differences (Stage, 1 U = 5 , p > 0 . 0 5 ; Stage 2, U = 8 , p > 0 . 0 5 ; Stage 3, U=14, p>0.05). DISCUSSION Iron deficient animals in this study show the same conditioned taste aversion performance as seen in previous studies [22]. Stage 1 animals performed the task very poorly compared with controls. Stage 2 performed as well as normal animals and Stage 3 animals again showed a performance deficit relative to controls and to Stage 2 animals (Fig. 1). It is difficult to compare activity results in the same way due to the variance in instruments used to measure them. The Dopier activity counter was changed between the previous studies and this one. Unfortunately, it is not possible to equate the sensitivities of these counters, so the activity measures in this experiment are considerably less sensitive than those of the previous ones. The fact that iron deficiency appeared not to affect activity levels in this study is in conflict with our previous findings [21,22]. The pattern of results however is not. Performance of the taste aversion task was found to be inversely related to activity. Higher activity levels were associated with poor taste aversion performance and vice versa, thus supporting previous research. The corticosterone results were not as predicted. Although the general trend in this experiment was for hormone levels of normal animals to be higher on the final day of the task when the rat was required to choose between saccharin, the aversant, and water, than on day 3, during acquisition, the difference was not significant. In previous studies [1,4] the corticosterone levels of normal rats were significantly increased following exposure to a taste to which an aversion had been produced. This may be due to an amalgamation of factors; the nature of iron deficiency as a stressor, the time of day when blood sampling occurred and the great individ-
Stage 3, control
Before Conditioning
After Conditioning
After Test
(Day 3)
(Day 4)
(Day 5)
30.35* 31.26t 20.60* 14.1 It
28.2* 24.87t 33.25* 19.00t
4.53* 3.34t 58.75* 35.86t
24.22* 17.00t 62.12" 45.60t
16.18" 9.28t 44.13" 47.61t
4.85* 2.54t 46.25* 24.89t
42.96* 27.30t 45.79* 43.57t
37.74* 12.0t 24.67* 9.97t
3.76* 3.28t 53.33* 24.65 t
Mean saccharin aversion scores (*) and their standard deviations (t) are shown.
ual variation between rats in absolute corticosterone levels and their dally rhythm. No simple explanation is immediately evident. Iron deficient animals also failed to demonstrate the predicted changes in corticosterone levels. Rather than showing the increased plasma corticosterone levels that were predicted according to the stress-adaptation hypothesis, iron deficient animals were no different from normals on either day 3 or day 4 of the task. On day 5 they were significantly lower than normals, at each stage of deficiency. Iron deficient animals appear therefore to have normal corticosterone levels until they are stressed when the levels fall to well below normal. These lowered corticosterone levels seen in stress may be a product of depressed adrenocortical activity. While corticosterone production is sufficient to produce normal levels in nonstressed conditions, it may not be adequate to cope with the demands of stress. Consequently, corticosterone levels fail. A number of studies have explained depressed corticoid levels as a result of chronic stress in this fashion [3, 5, 12]. The fall in corticosterone levels in this study is most likely the result of impaired functioning of particular enzymes in the pathway of steroid synthesis. In the formation of cholesterol, a precursor of corticosterone, the steps, synthesis of squalene, then squalene to lanosterol require the microsomal enzyme NADPH. Levels of microsomal N A D P H are known to be reduced in early iron deficiency [2]. It is likely then that cholesterol synthesis is decreased in iron deficiency and consequently corticosterone levels suffer. Stress, as measured by circulating corticosterone levels, did not provide an explanation for the observed behavioural changes in iron deficiency. The stress-adaptation hypothesis cannot be entirely discounted however. Similar changes in catecholamines are known to occur in stress and iron deficiency.
1088
WlLLIAMSON,
T y r o s i n e h y d r o x y l a s e levels i n c r e a s e a n d m o n o a m i n e o x i d a s e levels d e c r e a s e in b o t h stress [7,8] a n d iron defic i e n c y [13,17]. S i n c e t y r o s i n e h y d r o x y l a s e is t h e ratelimiting e n z y m e in c a t e c h o l a m i n e s y n t h e s i s [18] a n d m o n o a m i n e o x i d a s e is a n i m p o r t a n t e n z y m e in the d e g r a d a -
NG AND RICHDALE
tion o f c a t e c h o l a m i n e s [10] i n c r e a s e d c a t e c h o l a m i n e s in iron deficiency a n d in s t r e s s are to b e e x p e c t e d . T h u s it is n o t surprising t h a t in b o t h stress a n d iron d e f i c i e n c y c a t e c h o l a m i n e levels are e n h a n c e d . T h e b e h a v i o u r a l r e s u l t s in iron deficiency m a y b e a p r o d u c t o f c h a n g e s s u c h as these.
REFERENCES 1. Ader, R. Conditioned adrenocortical steroid elevations in the rat. J. comp. physiol. Psychol. 90: 1156-1163, 1976. 2. Bailey-Wood, R., L. M. Blayney, J. R. Muir and A. Jacobs. The effects of iron deficiency on rat liver enzymes. Br. J. exp. Pathol. 56: 193-198, 1975. 3. Collins, K. J. and J. S. Weiner. Endocrine aspects of exposure to high environmental temperatures. Physiol. Rev. 48: 785-839, 1968. 4. Hennessy, J. W., W. P. Smotherman and S. Levine. Conditioned taste aversion and the pituitary-adrenal system. Behav. Biol. 16: 413--424, 1976. 5. Hess, J. L., V. M. Denenberg, M. X. Zarrow and W. D. Pfeifer. Modification of the plasma corticosterone response curve as a function of stimulation in infancy. Physiol. Behav. 4: 10%115, 1969. 6. Kadival, G. V. and A. M. Samuel. Effect of experimental malnutrition on corticosteroids and liver cytoplasmic receptors in rats. Biochem. Med. 18: 353-359, 1977. 7. Kennessy, A. and Z. Muzti. The effect of monoamine oxidase inhibitors on the synthesis and degradation ofcatecholamines in immobilised rats. In: Catecholamines and Stress, edited by E. Usdin, R. Kvetnansky and I. J. Kopin. Oxford: Pergamon Press, 1976. 8. Kvetnansky, R., V. K. Weise and I. J. Kopin. Elevation of adrenal tyrosine hydroxylase and phenylethanolamine-Nmethyl transferase by repeated immobilisation of rats. Endocrinolology. 87: 744, 1970. 9. McCall, M. G., G. E. Newman, J. R. P. O'Brien, L. S. Valberg and L. J. Witts. Studies in iron metabolism I. The experimental production of iron deficiency in the growing rat. Br. J. Nutr. 16: 297-304, 1962. 10. Pavez, H. and S. Pavez. Activity of catechol-o-methyl transferase and monoamine oxidase enzymes in different body organs of the rat following metopirone administration. Pharmac. Res. Commun. 4: 36%375, 1972.
11. Pearson Murphy, B. Some studies of the protein-binding of steroids and their application to the routine micro and ultra micro measurement of various steroids in body fluids by competitive protein-binding radioassay. J. clin. Endocr. Metab. 27: 973-990, 1%7. 12. Petrovic, V. M., V. Davidovic and V. Janc-Sibalic. Catecholamine synthesis release and excretion and adrenocortical activity in the heatstressed rat. In: Catecholamines and Stress, edited by E. Usdin, R. Kvetnansky and I. J. Kopin. Oxford: Pergamon Press, 1976. 13. Quik, M. and T. L. Sourkes. The effect of chronic iron deficiency on tyrosine hydroxylase activity. Can. J. Biochem. 55: 60-65, 1976. 14. Schermer, S. The Blood Morphology o f Laboratory Animals. Philadelphia: F. A. Davis Co., 1%7. 15. Selye, M. Stress. Montreal: Acta Inc., Medical Publications, 1950. 16. Smith, S. R., T. Bledsoe and M. K. Chhetrie. Cortisol metabolism and the pituitary-adrenal axis in adults with protein-calorie malnutrition. J. clin. Endrocr. Metab. 40: 43-52, 1975. 17. Symes, A. L., M. B. H. Sourkes, G. Youdim, G. Gregoriadis and M. Birnbaum. Decreased monoamine oxidase activity in the liver of iron deficient rats. Can. J. Biochem. 47: 999-1002, 1%9. 18. Theonen, H. Comparison between the effect of neuronal activity and nerve growth factor on the enzymes involved in the synthesis of norepinephrine. Pharmac. Rev. 24: 255-267, 1972. 19. Tucker, A. and M. E. Gibbs. Cycloheximide-amnesia for taste aversion memory in rats. Pharmac. Biochem. Behav. 4: 181184, 1976. 20. Weinberg, J., P. R. Dallman and S. Levine. Iron deficiency during early development in the rat: behavioural and physiological consequences. Pharmac. Biochem. Behav. 12: 493-502, 1980. 21. WiUiamson, A. M. and K. T. Ng. Activity and t-maze performance in iron deficient rats. Physiol. Behav. 24:1157-1160, 1980. 22. Williamson, A. M. and K. T. Ng. Behavioural effects of iron deficiency in adult rats. Physiol. Behav. 24: 561-567, 1980.
Physiology & Behavior, Vol. 27, pp. 1085--1088. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
Changes in Corticosterone Levels in Iron Deficient R a t s I A . M. W I L L I A M S O N , 2 K . T. N G A N D A . R I C H D A L E
Department of Psychology, La Trobe University, Bundoora, Victoria, Australia 3083 R e c e i v e d 1 J u l y 1981 WlLLIAMSON, A. M., K. T. NG AND A. RICHDALE. Changes in corticosterone levels in iron deficient rats.
PHYSIOL. BEHAV. 27(6) 1085-1088, 1981.--The hypothesis that behavioural changes in iron deficiency are a product of stress was tested by monitoring corticosterone levels. Adult hooded rats were placed on an iron deficient diet for 2 (Stage 1), 8 (Stage 2) or 12 (Stage 3) weeks. When trained and tested on a single-trial taste aversion task, iron deficient animals showed the same performance as seen in previous experiments. Poor performance in early iron deficiency were followed by a temporary return to normal levels as the deficiency progressed, and falling below normal with continued iron depletion. Iron deficient animals failed to demonstrate the predicted stress-related changes in cortocosterone levels. Instead of an increasing in corticosterone levels after conditioning, levels fell to well below normal at all stages of deficiency following conditioning. Iron deficiency probably limits the action of certain enzymes responsible for corticosterone production. Due to the similarity of changes in catecholamines in stress and iron deficiency, it is possible that these changes will explain the pattern of behavioural response in iron depletion. Corticosterone
Iron deficiency
Adult rats
Memory
W H I L E iron deficiency is the world's most common nutritional deficiency, it remains relatively unresearched. This is particularly so for the behavioural consequences of iron deficiency. Recently, however, some evidence has appeared that a period of iron deficiency does affect the behaviour of both weanling and adult rats. In weanling rats, iron deficiency in the first weeks of life decreased responsiveness in novel and aversive situations [20]. Exploratory behaviour was also affected, with iron deficient rats showing reduced activity. In adult rats, iron deficiency produces deficits in memory [22] as well as changes in exploratory activity [21,22]. In adult animals the pattern of behavioural change as a result of iron deficiency is suggestive of a response to stress [15, 21, 22]. The initial reaction to iron depletion is alarm. Although the level of iron stores is the only physiological indicator to be affected, activity increases significantly above normal levels, while memory deteriorates. With further iron depletion the animal is able to adapt to the stressor both physiologically and psychologically. Iron stores and memory return to normal levels but activity deteriorates to below normal. Exhaustion occurs with prolonged iron depletion causing further loss of motor and cognitive functions as well as body iron. That iron deficiency may be a potent stressor is supported by a study with weanling rats [20] in which iron deficiency was found to produce higher resting corticosterone levels but a smaller stress increment than normal. The authors postulated that iron deficiency reduces the young animal's responsiveness to environmental stimuli.
Activity
Further support is given by the findings of elevated basal levels of plasma corticoids in both animals and humans who have protein-calorie malnutrition [6,16]. This study was designed to investigate whether the observed behavioural changes in iron deficient adult rats [22] constituted an adaptation to stress. It was not sufficient, however, simply to assay for corticosterone at stages in the iron deficiency process and compare the results with those of normals of the same age. Unfortunately the behaviours observed in the progression of iron deficiency may be confounded by the task used to measure them, viz a conditioned taste aversion task. Evidence suggests that injections of lithium chloride that are sufficient to produce learned taste aversions, produce sustained increases in plasma corticoid levels lasting from 2 to 4 hours [4]. Any changes in corticosterone levels could therefore be due to the nature of the task itself. For this reason corticosterone levels were measured at stages during the deficiency process and at invervals during the task.
METHOD
Subjects One hundred and six male and female Wistar-derived rats from the breeding colony of the Department of Psychology at La Trobe University were used. Animals were housed in the laboratory on a 12/12 light-dark cycle for at least one week prior to the beginning of the study. Males were housed in
1.This work was carried out with the support of a La Trobe University Postgraduate Scholarship. 2Address reprint requests to first author, now at Division of Occupational Health and Radiation Control, Health Commission Of New South Wales, P.O. Box 163, Lidcombe, New South Wales, Australia, 2141.
C o p y r i g h t © 1981 B r a i n R e s e a r c h P u b l i c a t i o n s Inc.--0031-9384/81/121085-04502.00/0
1086
W I L L I A M S O N , NG A N D R I C H D A L E
wire cages in pairs and females in triplets. Food and water were available ad lib and access to the laboratory by personnel was kept to a minimum.
100, c
.O
Apparatus The conditioned taste aversion apparatus that was used has been described elsewhere [22].
80
> 60
Diet
C
Iron deficiency was produced by a diet of skim milk, sucrose, salt mixture, added vitamins, minerals and essential amino acids, with 2-3 mg/kg iron [9]. Control animals received the same diet but with 70 mg/kg iron.
t-
40
U U ta
20
Experimental Design The design was essentially a 2×3 factorial design with sample sizes varying from 5 to 8, and balanced as far as possible with respect to the sex variable. Beginning at 90 days after birth, experimental animals were placed on the iron deficient diet for 14 (Stage 1), 56 (Stage 2) or 84 (Stage 3) days. Animals were tested in a no-delay taste aversion conditioning paradigm in which performance measures were taken 24 hr after conditioning. Blood samples were taken at intervals throughout the procedure.
Procedure The behavioural task has already been reported in the literature [19,22]. Pretraining phase. Animals were water deprived 24 hr before experimentation began. On each of the following 3 days each animal was placed in the test box for 10 min and allowed to drink tap water from the two spouts. At all other times only food was available. Training phase. On the fourth day 0.1% sodium saccharin solution instead of water was available from both spouts and each animal was allowed to drink for 10 minutes. Immediately after, a solution of 0.6% (0.15 M) lithium chloride (LiCl) calculated on a dosage of 10 ml/kg bodyweight was injected intragastrically. Testing phase. In the 10 min drinking session on day 5 saccharin solution was available from one of the two spouts and tap water from the other, with position randomized for each animal. Performance measures. The total number of licks and the amount of fluid consumed from each spout was monitored in each 20 rain test box exposure. Activity was monitored on a Dopler activity counter. Blood samples were taken from one-third of all animals on day 3 (final day of pretraining), day 4 (conditioning day) and day 5 (test day). Sampling occurred within ten minutes of removal from the behavioural testing apparatus. Animals were no longer used after this sample was taken. Memory in the conditioning task was indexed by a saccharin aversion score defined as: % SA =
number of licks of water in test session number of licks in test session
× 100
Saccharin aversion scores and activity measures were taken for animals which went through to day 5.
Corticosterone Analysis After anaesthetization with ether, approximately 3 ml of
weeks
on
diet
FIG. 1. Performance of iron deficient ( I ) and normal (e) rats on the conditioned taste aversion task (full association, 24 hour retention interval). Mean saccharin aversion scores are given. Significantly Tower saccharin aversion scores are shown by iron deficient rats compared with their controls after 2 and 12 weeks of no-iron diet.
blood was collected by the orbital eye bleed technique [14]. This whold process took less than 3 min to complete. The blood was centrifuged, plasma collected and frozen immediately. Blood collection always occurred between 12 and 2 p. m. Corticosterone analysis was performed on the plasma [11]. Florisil was used as the absorbent material and human plasma CBG (corticosteroid binding globulin) as the binder. For female rats progesterone was precipitated out of the plasma before corticosterone analysis was performed. RESULTS Saccharin aversion results are shown in Fig. 1. Analysis of variance revealed a significant difference between deficient and control groups, F(5,32)=3.35,p<0.025. Subsequent Scheffe tests showed that both Stage 1 and Stage 3 animals were significantly different from their own controls. Stage 2 animals did not differ from controls. Analysis of variance of activity scores revealed no significant differences between deficient and control animals, F(5,32)=2.31, N.S. Activity results are shown in Fig. 2. A significant negative correlation was found between saccharin aversion and activity scores, r= -0.36, t =3.27, p<0.01. The results of corticosterone analysis are shown in Table 1. Mann Whitney U tests were performed on this data due to the small and unequal sample sizes. Corticosterone levels of all deficient groups were significantly lower than control levels on day 5 of the conditioned taste aversion task (Stage I compared with control U = 3 , p<0.002; Stage 2 with control U = 2 , p<0.006; Stage 3 with control, U = 4 , p<0.01). Iron deficient animals had significantly lower corticosterone levels on day 5 than on day 3 (Stage l, U--13, p<0.02; Stage 2, U = 6 , p<0.04; Stage 3, U = l , p<0.008). F o r control
C O R T I C O S T E R O N E L E V E L S A N D IRON D E F I C I E N C Y
1087 TABLE 1 VARIATIONSIN CORTICOSTERONE LEVELS (ng/100ml PLASMA)OF IRON DEFICIENTAND NORMALRATSDURINGTHE CONDITIONED TASTE AVERSIONTASK
g~ SO0 t
E 4001 0
E aoo Stage 1
(~ 2oc
Stage 1, control
m
~) lOO ,D
-'Q-
.......
-0
E
Stage 2 Stage 2, control
c
Stage 3 weeks
on diet
FIG. 2. Variations in the activity levels (mean number of large movements) or iron deficient (11) and normal (O) rats. Activity levels of iron deficient rats were no different from those of their normal controls at any stage of deficiency.
animals, this same comparison revealed no significant differences (Stage, 1 U = 5 , p > 0 . 0 5 ; Stage 2, U = 8 , p > 0 . 0 5 ; Stage 3, U=14, p>0.05). DISCUSSION Iron deficient animals in this study show the same conditioned taste aversion performance as seen in previous studies [22]. Stage 1 animals performed the task very poorly compared with controls. Stage 2 performed as well as normal animals and Stage 3 animals again showed a performance deficit relative to controls and to Stage 2 animals (Fig. 1). It is difficult to compare activity results in the same way due to the variance in instruments used to measure them. The Dopier activity counter was changed between the previous studies and this one. Unfortunately, it is not possible to equate the sensitivities of these counters, so the activity measures in this experiment are considerably less sensitive than those of the previous ones. The fact that iron deficiency appeared not to affect activity levels in this study is in conflict with our previous findings [21,22]. The pattern of results however is not. Performance of the taste aversion task was found to be inversely related to activity. Higher activity levels were associated with poor taste aversion performance and vice versa, thus supporting previous research. The corticosterone results were not as predicted. Although the general trend in this experiment was for hormone levels of normal animals to be higher on the final day of the task when the rat was required to choose between saccharin, the aversant, and water, than on day 3, during acquisition, the difference was not significant. In previous studies [1,4] the corticosterone levels of normal rats were significantly increased following exposure to a taste to which an aversion had been produced. This may be due to an amalgamation of factors; the nature of iron deficiency as a stressor, the time of day when blood sampling occurred and the great individ-
Stage 3, control
Before Conditioning
After Conditioning
After Test
(Day 3)
(Day 4)
(Day 5)
30.35* 31.26t 20.60* 14.1 It
28.2* 24.87t 33.25* 19.00t
4.53* 3.34t 58.75* 35.86t
24.22* 17.00t 62.12" 45.60t
16.18" 9.28t 44.13" 47.61t
4.85* 2.54t 46.25* 24.89t
42.96* 27.30t 45.79* 43.57t
37.74* 12.0t 24.67* 9.97t
3.76* 3.28t 53.33* 24.65 t
Mean saccharin aversion scores (*) and their standard deviations (t) are shown.
ual variation between rats in absolute corticosterone levels and their dally rhythm. No simple explanation is immediately evident. Iron deficient animals also failed to demonstrate the predicted changes in corticosterone levels. Rather than showing the increased plasma corticosterone levels that were predicted according to the stress-adaptation hypothesis, iron deficient animals were no different from normals on either day 3 or day 4 of the task. On day 5 they were significantly lower than normals, at each stage of deficiency. Iron deficient animals appear therefore to have normal corticosterone levels until they are stressed when the levels fall to well below normal. These lowered corticosterone levels seen in stress may be a product of depressed adrenocortical activity. While corticosterone production is sufficient to produce normal levels in nonstressed conditions, it may not be adequate to cope with the demands of stress. Consequently, corticosterone levels fail. A number of studies have explained depressed corticoid levels as a result of chronic stress in this fashion [3, 5, 12]. The fall in corticosterone levels in this study is most likely the result of impaired functioning of particular enzymes in the pathway of steroid synthesis. In the formation of cholesterol, a precursor of corticosterone, the steps, synthesis of squalene, then squalene to lanosterol require the microsomal enzyme NADPH. Levels of microsomal N A D P H are known to be reduced in early iron deficiency [2]. It is likely then that cholesterol synthesis is decreased in iron deficiency and consequently corticosterone levels suffer. Stress, as measured by circulating corticosterone levels, did not provide an explanation for the observed behavioural changes in iron deficiency. The stress-adaptation hypothesis cannot be entirely discounted however. Similar changes in catecholamines are known to occur in stress and iron deficiency.
1088
WlLLIAMSON,
T y r o s i n e h y d r o x y l a s e levels i n c r e a s e a n d m o n o a m i n e o x i d a s e levels d e c r e a s e in b o t h stress [7,8] a n d iron defic i e n c y [13,17]. S i n c e t y r o s i n e h y d r o x y l a s e is t h e ratelimiting e n z y m e in c a t e c h o l a m i n e s y n t h e s i s [18] a n d m o n o a m i n e o x i d a s e is a n i m p o r t a n t e n z y m e in the d e g r a d a -
NG AND RICHDALE
tion o f c a t e c h o l a m i n e s [10] i n c r e a s e d c a t e c h o l a m i n e s in iron deficiency a n d in s t r e s s are to b e e x p e c t e d . T h u s it is n o t surprising t h a t in b o t h stress a n d iron d e f i c i e n c y c a t e c h o l a m i n e levels are e n h a n c e d . T h e b e h a v i o u r a l r e s u l t s in iron deficiency m a y b e a p r o d u c t o f c h a n g e s s u c h as these.
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