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Effects of moderate zinc deficiency on cognitive performance in young adult rats
Physiology & Behavior, Vol. 25, pp. 117-121. Pergamon Press, 1982. Printed in the U.S.A.
Effects of Moderate Zinc Deficiency on Cognitive Performance in Young Adult Rats THOMAS
M. M O H S A N D G. F O S M I R E
F. M A S S A R O ,
Nutrition Program, The Pennsylvania State University 219 H u m a n D e v e l o p m e n t Building, University Park, P A 16802 R e c e i v e d 13 O c t o b e r 1981 MASSARO, T. F., M. MOHS AND G. FOSMIRE. Effects of moderate zinc deficiency on cognitive performance in young adult rats. PHYSIOL. BEHAV. 29(1)117-121, 1982.--Two experiments were conducted to establish a dietary zinc level which approximates a moderate deficiency in the young adult rat and to determine if a concurrent zinc deficiency affects cognitive performance. Male rats were fed varying levels of zinc in diet throughout a 17-day period. The lowest dietary level that depressed serum and bone zinc without influencing food consumption or body weight gains was observed to be 5.8/,~g Zn/g diet. Young adult rats maintained on either a zinc adequate (24.4/~g Zn/g) or low-zinc (5.3/zg Zn/g) diet were tested in a modified Skinner Box involving tests of visual, auditory, association, and discrimination learning. No differences were observed in the visual discrimination performance of the zinc deficient animals when compared with control counterparts. Deficits in the ability to transfer a learned association between visual and auditory stimuli were observed, however, in the deficient group during the transfer test phase. The latter performed better during the final auditory discrimination task in transferring a learned food-relevant cue. Moderate zinc deficiency
Association learning
Rat
A SEVERE zinc deficiency ( < l mg/kg diet), imposed either during gestation or lactation in the rat, adversely affects the behavioral outcome of the offspring. In the past decade, numerous studies demonstrated the long-term effects of preand postnatal zinc deficiency on various aspects of behavior, including avoidance conditioning [8], maze performance [14], conditional emotional response performance [10], discrimination learning [8], consummatory behavior [6,17], and several aspects o f social behavior [7,16]. To our knowledge, no studies exist in the literature which have examined the effects of a concurrent moderate zinc deficiency on cognitive performance. The present study was undertaken, therefore, to (1) establish a dietary level of zinc which would result in a moderate deficiency in the young adult rat, and (2) examine the functional consequences of a concurrent zinc deficiency on several aspects of discrimination and association learning. EXPERIMENT 1: DETERMINATIONOF MODERATE ZINC DEFICIENCY This experiment established a dietary zinc level that would depress serum and bone zinc concentration in the rat without adversely affecting either food consumption or body weight gain.
Method From a total of 50 male Wistar rats, aged 45 days and weighing approximately 178 grams, 10 were randomly assigned to each of five dietary groups. Rats in group 1 were fed a diet containing 5.8/xg Zn/g diet, group 2, 8.2; group 3, 11.8; group 4, 15.1; group 5, 24.5. All animals within each group were fed ad lib the respective powder diet (see Table
l) throughout a 17-day period and had free access to double distilled H20 from glass bottles fitted with stainless steel sipper tubes and silicon stoppers. Body weight of each animal was recorded at 3-day intervals, food intake, adjusted for spillage, was recorded daily. On Day 18, each animal was exsanguinated (5 ml of blood was removed via heart puncture) and right tibia removed for determination of zinc content by atomic absorption spectrophotometry. All data were treated statistically by analysis of variance using repeated measure designs [20]; individual group comparisons were c a r d e d out employing Ducans Multiple Range Test.
Results As evidenced by a nonsignificant Group × Days interaction, weight gain throughout the 18-day period was similar for all groups irrespective of the dietary zinc level, F(20,224)=0.811, p>0.05. The only significant main effect was that of Days, F(6,224)= 1080.5, p<0.001, indicating that all groups gained weight throughout the experimental period. Likewise, mean daily food intake was similar for all treatment groups throughout the experimental period, F(4,45)=0.579, p>0.05. As shown in Table 2, and as revealed by analysis of variance, both serum zinc, F(4,44)=9.57, p<0.001, and bone zinc values, F(4,43)=29.06, p<0.001, were significantly altered by dietary treatment. Compared to groups fed 5.8 and 8.2/~g Zn/g diet, serum and bone zinc values were significantly greater (o<0.01) in those animals fed 24.5, 15.1, and 11.8/~g Zn/g diet. Nonsignificant differences were observed among the latter three groups with respect to either serum or bone zinc (0>0.05).
C o p y r i g h t © 1982 P e r g a m o n Press---0031-9384/82/070117-05503.00/0
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MASSARO, MOHS AND FOSMIRE TABLE 2 MEAN VALUES -+ S.D. OF SERUMAND BONE ZINC
TABLE 1 COMPOSITIONOF BASALDIETS
Egg white solids, spray dried* Dextrose* Salts? Vitamin mix~ Corn Oily Alphocel hydrolyzed* Biotin§ Calcium stearate, technical¶
Percentage by Weight
Dietary Zinc /xg Zn/gram
Mean Serum Zinc (/zg/ml)
Mean Bone Zinc (pg/g)
20.00 55.49 3.50 1.00 10.00 10.00 0.00002 1.00
5.8 (n=9) 8.2 (n=10) 11.8 (n=10) 15.1 (n=10) 24.5 (n=10)
0.973 _+ 0.211" 1.403 _+ 0.426* 1.824 _+ 0.618 1.836 _+ 0.377 1.977 _+ 0.267
156.5 -+ 13.6" 188.5 - 18.0" 222.6 _+ 28.5 229.2 _+ 10.3 249.7 - 31.1
*Teklad Test Diets, Madison, WI. tAmerican Institute of Nutrition zinc free mineral mix. Zinc carbonate added to mixture after zinc free mineral mix was weighed for each dietary level. Total zinc content of diets determined by atomic absorption spectophotometry. For Experiment 1: 5.8, 8.2, 11.8, 15.1, and 24.5 ~g Zn/gram diet. For Experiment 2: before and after pelleting, 5.3 /zg Zn/gram and 24.4 rig/gram diet for low zinc and control group, respectively. SAmerican Organization of Agricultural Chemist vitamin mix, Teklad Test diets, Madison, WI. §Biotin, purissima: Tridom Chemical Inc., Hauppauge, NY. ¶Fisher Scientific Co., Fairlawn, NJ; added as a lubricant for pelleting purposes.
The behavioral effects of moderate zinc deficiency, employing the lowest dietary treatment ( - 5 . 8 /zg Zn/g diet), were determined in Experiment 2. EXPERIMENT 2: MODERATEZINC DEFICIENCY--COGNITIVE PERFORMANCE
Method Male Wistar rats, aged 45 days and weighing approximately 180 grams, were randomly assigned to either a zinc adequate (ZA) or zinc deficient (ZD) dietary group and fed ad lib a pelleted diet containing 24.4/~g Zn/g and 5.3/~g Zn/g diets, respectively. An additional 20 animals were randomly chosen, separated into two equal groups, and sacrificed at the initiation of the experiment in order to establish baseline values for both serum and bone zinc. Thereafter, randomly chosen nontested animals from each dietary group were sacrificed on Days 13 and 18 to provide continual bone and serum zinc data. Each animal chosen for behavioral testing was individually housed in a stainless steel laboratory cage and fed ad lib their respective diet prior to being placed in the testing chamber on Day 13. Following completion of testing, all animals were sacrificed on Day 18, and a single sample of blood was removed by heart puncture, and the right tibia removed for determination of zinc content by atomic absorption spectrophotometry. Food intake, body weight, serum zinc, and bone zinc data were statistically analyzed by analysis of variance.
Behavioral Testing Apparatus Four environmental test chambers were used for the measurement of association learning [ 15]. The basic operant
*Statistical difference from Group 3, 4 and 5 p<0.01.
conditioning apparatus consisted of a modified Skinner Box (Plexiglas chamber 26 × 26x 26 cm) enclosed in a sound-proof cubicle. A metal press bar, which protrudes 4 cm into the cage, was located on one wall of the chamber. On the opposite wall were two press bars, located 4 cm above the floor, and separated by 8 cm. A one-inch yellow lamp jewel, illuminated by a 71/2 watt lamp, was located 3 cm above each bar. A small cup, into which 45 mg food pellets (Noyes Precision Food Pellets) were mechanically dispensed, was located below the dual press bars. Two three-inch speakers were located in the lid of the chamber over the food cup area and were connected to an audio generator. Each speaker emitted a tone pattern at a distinctly different frequency. The complete system, including test chambers and Coulbourn digital programming units, was fully automated to facilitate stimuli presentation and data collection.
Behavioral Testing Procedure During Phase 1 of training, to obtain a food pellet, each animal was required to solve a visual discrimination problem. The animal was first required to depress the single press bar to initiate the learning trial. One of the two lights, located on the opposite side of the cage, was randomly (50%) illuminated. If the animal attended to the visual cue and depressed the bar corresponding to the illuminated light, it received a single food pellet. If an incorrect bar was pressed, the animal was required to press the single bar again to initiate another learning trial. All trials were initiated by the animal rather than the experimenter, and it was unnecessary either to food or water-deprive the animals to insure acquisition of the visual discrimination. Each animal remained in the initial phase of training until a minimum of 300 trials had been completed and an asymptotic performance achieved (i>80% correct for five consecutive blocks of trials; 10 trials/block). Approximately 8 hours (range 5-10) were allotted to complete Phase I of testing. Each animal which failed to meet the established criteria was classified as a nonperformer. Since the animals remained in the testing chamber throughout the study, all meals (ZA;ZD pellets) were obtained through successful completion of trials. Phase 2 of training, the association phase, commenced following attainment of the selected Phase l performance level. Two behavioral training groups were established within each dietary treatment. Each animal assigned to the experimental training condition (E) was exposed to a redundant auditory stimulus, simultaneously paired with a corresponding visual cue for 100 consecutive trials. Thus the
119
ZINC D E F I C I E N C Y ON C O G N I T I V E P E R F O R M A N C E TABLE 3 MEAN VALUES _+S.D. OF SERUM AND BONE ZINC
Day 0 Treatment ZA ZD
Day 13
Day 18
Serum Zinc (/zg/dl)
Bone Zinc (/zg/g)
Serum Zinc (/zg/dl)
Bone Zinc (/zg/g)
Serum Zinc (/zl/dl)
Bone Zinc (/zg/g)
161.60 ± 24.80 (n= 10) 160.60 ± 27.52 (n= 10)
186.30 ± 15.27 (n= 10) 195.60 ± 14.60 (n= 10)
177.25 ± 14.24 (n=4) 118.75 ± 10.01 (n=8)
234.50 ± 14.61 (n=4) 158.12 ± 10.24 (n=8)
178.70 -+- 23.25 (n= 18) 98.47 ± 19.40 (n=23)
229.90 ± 29.25 (n= 18) 136.56 - 11.36 (n=23)
sound pattern, emitted from the right speaker, was presented simultaneously with illumination of the right visual cue, and the sound pattern emitted from the left speaker presented simultaneously with the left visual cue. The other group, control training (C), received n o redundant exposure to auditory stimuli--only an additional 100 trials of light presentation. Phase 3, the transfer of learning t e s t phase, involved random presentation of one of two auditory stimuli in the absence of the visual stimulus. Both groups were required to complete 450 trials during Phase 3. Phase 4, the final t e s t phase, involved presentation for a second time of auditory stimuli for 300 consecutive trials. During Phase 4, each randomized tone was emitted simultaneously from both speakers while still being paired with the same press bar it had been associated with during Phases 2 and 3. In order to obtain a food pellet, each animal was required to associate a particular tone with its correct press bar regardless of the location of the tone. The difference in performance (number o f correct responses/10 trials during Phase 3 - - t e s t phase) between those groups previously exposed to the simultaneous visual and auditory cues, plus those exposed to only visual cues, was taken as a measure of transfer learning. Differences in performance between groups in all other phases were taken as a measure of discrimination learning. Behavioral data were subjected to nonparametric analysis employing the Wilcoxon Mann-Whitney W test [21]. Results
Prior to entering behavioral testing on Day 13, no significant differences were observed in food consumption of moderately zinc deficient animals when compared to control, F(3,34)=0.441, p>0.05. Within each diet group a subgroup of animals, identified as nonperformers, failed to complete the behavioral testing sequence. Mean food intake of ZA nonperformers (23.0___2.8 g) on Day 12 was similar to ZD nonperformers (23.6___3.7 g) and did not differ (p >0.05) from those animals who completed the behavioral testing sequence (ZA 21.5___3.1; ZD 24.2_+3.8 g). The mean body weight on Day 12 was likewise comparable among ZA (242.0_+6.9 g) and ZD (239.9_+18.7 g) nonperformers and performers (ZA 249.4_+8.7 g; ZD 249.6___8.4 g). As shown in Table 3, bone zinc concentration (/zg Zn/g bone) gradually increased in the ZA group from Day 0 through Day 13 and remained relatively stable thereafter. In contrast, a reduction in bone zinc was observed in the ZD group throughout this interval as evidenced by a significant
Diet × Time interaction, F(2,62)--46.57, p<0.01. Expressed on an absolute weight basis (/zg Zn/bone), the zinc content per tibia remained static throughout the experimental period in the ZD group as evidenced by an average gain of only 1.75 /zg. The ZA group exhibited a steady increase in zinc content of bone, averaging 24.05/zg. As shown in Table 3, a similar effect was seen in serum zinc concentrations which gradually increased in the ZA group by Day 13 and remained relatively stable thereafter. In contrast, a reduction in serum zinc was observed in ZD groups throughout this interval, as evidenced by significant D i e t x T i m e interaction, F(2,61)=20.63, p<0.01. One of the most striking behavioral characteristics displayed by the moderately deficient animals was the increased incidence of " n o n p e r f o r m a n c e . " Nonperformers were defined as those animals which did not reach the specified criteria during training (Phase 1) and failed to complete the subsequent phases of tests. Of the 22 ZA animals that began the testing sequence, 2 (9%) were classified as nonperformers. In comparison, of the 26 ZD animals that began the testing sequence, 8 (31%) were classified as nonperformers. Twenty ZA animals and 18 ZD animals completed all phases of training and testing. Behavioral performance of 20 ZA and 18 ZD animals met the specified criteria during Phase 1 of visual discrimination. No significant differences were observed in the mean number of trials to reach criteria between the ZA (152.5) and ZD (158.9) groups, respectively. Likewise, as shown in Table 4, performance in Phase 2 (association phase) was similar for all treatment groups. Compounding the visual stimulus with an auditory cue has no detrimental effect on performance of either the ZA of ZD-E during this critical phase of training. Differences in performance were observed, however, during Phase 3, the transfer of learning test phase. Zinc adequate-experimental animals, having prior experience with auditory cues, reached criteria in significantly (p <0.01) less trials when compared to all other groups (ZA-C, ZD-C, ZD-E). In contrast, the performance of ZD-E, having prior exposure to the auditory cue, did not significantly differ (0>0.05) from either the ZA-C or ZD-C, having n o previous exposure to the test stimulus. As shown in Table 4, ZA-C animals performed better during Phase 4 (simultaneous tone presentation), reaching specified criteria in significantly less trials (215.5) when compared to the ZA-E (323.0) animals (p<0.01), whereas ZD-C (97.8) did not differ from ZD-E (176.7) in the number of trials to reach criteria (0>0.05). Of interest was the obser-
120
MASSARO, MOHS A N D F O S M I R E TABLE 4 MEAN NUMBER OF TRAILS TO CRITERION* _+ S.D. Behavioral Treatment Zinc Adequate Control (n= 10) Experimental (n= 10) Zinc Deficient Control (n=9) Experimental (n =9)
Phase 2
Phase 3
Phase 4
51.0_+ (3.2)
182.0-+ (111.5)
215.5-+ (117.3)
53.0 ± (6.8)
118.0 ± (93.4)
323.0 ± (149.4)
65.6 ± (34.3)
210.0 ± (151.9)
97.8 ± (93.6)
50.0 ± (0.0)
242.3 ± (114.7)
176.7 ± (163.2)
*/>80% correct for 5 consecutive blocks of trials; 10 trials/block.
vation that ZD animals, regardless of previous training and or performance during previous phases, made significantly fewer errors and reached criteria with less trials during this final discrimination task (p<0.01).
DISCUSSION The present study demonstrates that a moderate, concurrent zinc deficiency alters cognitive performance. Zinc deficient animals reached criteria on both the initial visual discrimination task and the final auditory task. This observation agrees with numerous reports providing evidence that simple stimulus-response learning if not impaired via dietary manipulations [12]. The better performance of both ZD and ZA-C animals during the final discrimination phase probably reflects a g r a d u a l increase in motivation whereby the new auditory cues become better predictors of food reinforcement. Similar effects of diet-induced enhancement of appetitive learning have likewise been observed in the concurrently and previously malnourished primate and rodent [11, 12, 22]. The deficit in association learning observed in the present study is not a unique characteristic of zinc deficiency. Others have shown this basic style of learning to be impaired by early protein or caloric restriction in the rat [13], early infantile malnutrition in the primate [5], and, more recently, moderate, concurrent iron deficiency in the young adult rat [15]. The present study provides further evidence that nutrient deficiencies of short duration interfere with normal processes involved in association learning. The common mechanism(s), behavioral or biochemical, that are responsible for this observed deficit in performance remain speculative. Acute nutritional insults may force an animal to adopt a learning strategy for processing of biologically meaningful information at the expense of other kinds of
information [13], or nutritional deprivations may attenuate curiosity itself, which is a necessary component for development of cognitive potential [5]. However, the extent to which motivationally based changes in behavior are operational under conditions of moderate nutrient deficiencies that produce no discernible effect on either food intake or utilization remain to be determined. One may explain the above findings through examination of behavioral characteristics displayed by ZD nonperformers. These animals although comparable to ZA and ZD performers with respect to food intake and resultant weight gain patterns, were over-reactive to simple handling procedures, were often immobile in the testing apparatus, and frequently entered into a prolonged food and water deprivation (>72 hours), with no attempt to initiate the reinforcement contingency. Similar behavioral disturbances have been documented, not only in protein-calorie malnourished (unpublished observations) and zinc deficient animals [2,12], but also in animals with excess adrenal steroid levels [19]. Both zinc deficiency and protein restriction have also been shown to produce adrenal hypertrophy, hypersensitivity to ACTH, and increase in level of circulating corticosteroids [ 1, 2, 4, 18]. In light of these observations, it has recently been suggested that many of the behavioral manifestations associated with zinc deficiency may be attributed to altered pituitary-adrenal function [9]. Unfortunately, however, little information is available depicting the effects of moderate zinc deficiency on these parameters. It is reasonable to suggest that a similar perturbation in pituitary-adrenal function may result from a moderate nutrient deficiency. This altered function may lead to behaviors that block performance of an animal in a particular task (nonperformers) or impair performance of an animal in tasks of association learning. Importantly, such a mechanism could help to explain many of the coincidental behavioral similarities observed to result from dietary manipulations.
REFERENCES 1. Apgar, J. Effect of zinc deprivation from day 12, 15 or 18 of gestation on parturition in the rat. J. Nutr. 102: 343-348, 1972.
2. Bajaj, J. S., R. Khardori, M. G., Deo and D. D. Bansal. Adrenocortical function in experimental protein malnutrition. Metabolism 28: 54%598, 1979.
ZINC DEFICIENCY
ON COGNITIVE PERFORMANCE
3. Chesters, J. K. and M. Will. Some factors controlling food intake by zinc-deficient rats. Br. J. Nutr. 30: 555-566, 1973. 4. DePasquale-Jardiew, P. and P. J. Fraher. Further characterization of the role of corticosterone in the loss of humorol immunity in zinc-deficient A/J mice as determined by adrenalectomy. J. lmmunol. 124: 2650-2655, 1980. 5. Goldberger, L. I., L. M. Ausman and R. C. Boelkins. Incidental learning, attention, and curiosity in squirrel monkeys caloriedeprived as infants. Learn. Motivat. 11: 185-207, 1980. 6. Halas, E. S., P. A. Burger and H. H. Sandstead. Food motivation of rehabilitated malnourished rats: Implications for learning studies. Anita. Learn. Behav. 8: 152-158, 1980. 7. Halas, E. S., M. J. Hanlon and H. H. Sandstead. Intrauterine nutrition and aggression. Nature 257: 221-222, 1975. 8. Halas, E. S. and H. H. Sandstead. Some effects of prenatal zinc deficiency on behavior of the adult rat. Pediat. Res. 9: 94-97, 1975. 9. Hesse, G. W., K. A. Frank-Hesse and F. A. Catalanotto. Behavioral characteristics of rats experiencing chroniczinc deficiency. Physiol. Behav. 22: 211-215, 1979. 10. Hsueh, A. M., M. K. Simonson, M. J. Kellum and B. F. Chow. Perinatal undernutrition and metabolic and behavioral development of offspring. Nutr. Rep. Int. 7: 437-455, 1973. 1 I. Katz, H. B., R. E. Rosett and R. Ostwald. The compensatory role of food motivation in the maze learning performance of lactationally undernourished rats. DevlPsychobiol. 12: 305-315, 1979. 12. Levitsky, D. A., L. Goldberger and T. F. Massaro. Malnutrition, learning, and animal models of cognition. In: Human Nutrition: A Comprehensive Treatise, vol. 1, Nutrition: Pre- and Postnatal Development, edited by M. Winick. New York: Plenum Press, 1979, pp. 273-286.
121 13. Levitsky, D. A. Malnutrition and the hunger to learn. In: Malnutrition and Behavior: An Assessment o f Recent Research, edited by D. A. Levitsky. New York: Cornell University Press, 1979, pp. 161-179. 14. Lokken, P. M., E. S. Halas and H. H. Sandstead. Influence of zinc deficiency on behavior. Proc. Soc. exp. Biol. Med. 144: 680-682, 1973. 15. Massaro, T. F. and P. Widmayer. The effect of iron deficiency on cognitive performance in the rat. Am. J. clin. Nutr. 34: 864870, 1981. 16. Peters, D. P. Effects of prenatal nutritional deficiency on affiliation and aggression in rats. Physiol. Behav. 20: 359-362, 1978. 17. Peters, D. P. Effects of prenatal nutrition on learning and motivation in rats. Physiol. Behav. 22: 1067-1071, 1979. 18. Quarterman, J. and W. R. Humphries. Effect of zinc deficiency and zinc supplementation on adrenals, plasma steroids and thymus in rats. Life Sci. 24: 177-184, 1979. 19. Stevenson, J. A. F. and C. Franklin. Effects of ACTH and corticosteroids in the regulation of food and water intake. Prog. Brain Res. 32: 141-151, 1970. 20. Winer, B. J. Statistical Principles in Experimental Design. New York: McGraw-Hill, 1962, p. 539. 21. Wonnacott, T. and R. J. Wonnocott. Intridoctory Statistics for Business and Economics. New York: John Wiley & Sons, 1977, p. 481. 22. Zimmerman, R. R. Effects of age, experience, and malnourishment on object retention in learning set. Percept. Mot. Skills 28: 867-876, 1969.
Effects of Moderate Zinc Deficiency on Cognitive Performance in Young Adult Rats THOMAS
M. M O H S A N D G. F O S M I R E
F. M A S S A R O ,
Nutrition Program, The Pennsylvania State University 219 H u m a n D e v e l o p m e n t Building, University Park, P A 16802 R e c e i v e d 13 O c t o b e r 1981 MASSARO, T. F., M. MOHS AND G. FOSMIRE. Effects of moderate zinc deficiency on cognitive performance in young adult rats. PHYSIOL. BEHAV. 29(1)117-121, 1982.--Two experiments were conducted to establish a dietary zinc level which approximates a moderate deficiency in the young adult rat and to determine if a concurrent zinc deficiency affects cognitive performance. Male rats were fed varying levels of zinc in diet throughout a 17-day period. The lowest dietary level that depressed serum and bone zinc without influencing food consumption or body weight gains was observed to be 5.8/,~g Zn/g diet. Young adult rats maintained on either a zinc adequate (24.4/~g Zn/g) or low-zinc (5.3/zg Zn/g) diet were tested in a modified Skinner Box involving tests of visual, auditory, association, and discrimination learning. No differences were observed in the visual discrimination performance of the zinc deficient animals when compared with control counterparts. Deficits in the ability to transfer a learned association between visual and auditory stimuli were observed, however, in the deficient group during the transfer test phase. The latter performed better during the final auditory discrimination task in transferring a learned food-relevant cue. Moderate zinc deficiency
Association learning
Rat
A SEVERE zinc deficiency ( < l mg/kg diet), imposed either during gestation or lactation in the rat, adversely affects the behavioral outcome of the offspring. In the past decade, numerous studies demonstrated the long-term effects of preand postnatal zinc deficiency on various aspects of behavior, including avoidance conditioning [8], maze performance [14], conditional emotional response performance [10], discrimination learning [8], consummatory behavior [6,17], and several aspects o f social behavior [7,16]. To our knowledge, no studies exist in the literature which have examined the effects of a concurrent moderate zinc deficiency on cognitive performance. The present study was undertaken, therefore, to (1) establish a dietary level of zinc which would result in a moderate deficiency in the young adult rat, and (2) examine the functional consequences of a concurrent zinc deficiency on several aspects of discrimination and association learning. EXPERIMENT 1: DETERMINATIONOF MODERATE ZINC DEFICIENCY This experiment established a dietary zinc level that would depress serum and bone zinc concentration in the rat without adversely affecting either food consumption or body weight gain.
Method From a total of 50 male Wistar rats, aged 45 days and weighing approximately 178 grams, 10 were randomly assigned to each of five dietary groups. Rats in group 1 were fed a diet containing 5.8/xg Zn/g diet, group 2, 8.2; group 3, 11.8; group 4, 15.1; group 5, 24.5. All animals within each group were fed ad lib the respective powder diet (see Table
l) throughout a 17-day period and had free access to double distilled H20 from glass bottles fitted with stainless steel sipper tubes and silicon stoppers. Body weight of each animal was recorded at 3-day intervals, food intake, adjusted for spillage, was recorded daily. On Day 18, each animal was exsanguinated (5 ml of blood was removed via heart puncture) and right tibia removed for determination of zinc content by atomic absorption spectrophotometry. All data were treated statistically by analysis of variance using repeated measure designs [20]; individual group comparisons were c a r d e d out employing Ducans Multiple Range Test.
Results As evidenced by a nonsignificant Group × Days interaction, weight gain throughout the 18-day period was similar for all groups irrespective of the dietary zinc level, F(20,224)=0.811, p>0.05. The only significant main effect was that of Days, F(6,224)= 1080.5, p<0.001, indicating that all groups gained weight throughout the experimental period. Likewise, mean daily food intake was similar for all treatment groups throughout the experimental period, F(4,45)=0.579, p>0.05. As shown in Table 2, and as revealed by analysis of variance, both serum zinc, F(4,44)=9.57, p<0.001, and bone zinc values, F(4,43)=29.06, p<0.001, were significantly altered by dietary treatment. Compared to groups fed 5.8 and 8.2/~g Zn/g diet, serum and bone zinc values were significantly greater (o<0.01) in those animals fed 24.5, 15.1, and 11.8/~g Zn/g diet. Nonsignificant differences were observed among the latter three groups with respect to either serum or bone zinc (0>0.05).
C o p y r i g h t © 1982 P e r g a m o n Press---0031-9384/82/070117-05503.00/0
118
MASSARO, MOHS AND FOSMIRE TABLE 2 MEAN VALUES -+ S.D. OF SERUMAND BONE ZINC
TABLE 1 COMPOSITIONOF BASALDIETS
Egg white solids, spray dried* Dextrose* Salts? Vitamin mix~ Corn Oily Alphocel hydrolyzed* Biotin§ Calcium stearate, technical¶
Percentage by Weight
Dietary Zinc /xg Zn/gram
Mean Serum Zinc (/zg/ml)
Mean Bone Zinc (pg/g)
20.00 55.49 3.50 1.00 10.00 10.00 0.00002 1.00
5.8 (n=9) 8.2 (n=10) 11.8 (n=10) 15.1 (n=10) 24.5 (n=10)
0.973 _+ 0.211" 1.403 _+ 0.426* 1.824 _+ 0.618 1.836 _+ 0.377 1.977 _+ 0.267
156.5 -+ 13.6" 188.5 - 18.0" 222.6 _+ 28.5 229.2 _+ 10.3 249.7 - 31.1
*Teklad Test Diets, Madison, WI. tAmerican Institute of Nutrition zinc free mineral mix. Zinc carbonate added to mixture after zinc free mineral mix was weighed for each dietary level. Total zinc content of diets determined by atomic absorption spectophotometry. For Experiment 1: 5.8, 8.2, 11.8, 15.1, and 24.5 ~g Zn/gram diet. For Experiment 2: before and after pelleting, 5.3 /zg Zn/gram and 24.4 rig/gram diet for low zinc and control group, respectively. SAmerican Organization of Agricultural Chemist vitamin mix, Teklad Test diets, Madison, WI. §Biotin, purissima: Tridom Chemical Inc., Hauppauge, NY. ¶Fisher Scientific Co., Fairlawn, NJ; added as a lubricant for pelleting purposes.
The behavioral effects of moderate zinc deficiency, employing the lowest dietary treatment ( - 5 . 8 /zg Zn/g diet), were determined in Experiment 2. EXPERIMENT 2: MODERATEZINC DEFICIENCY--COGNITIVE PERFORMANCE
Method Male Wistar rats, aged 45 days and weighing approximately 180 grams, were randomly assigned to either a zinc adequate (ZA) or zinc deficient (ZD) dietary group and fed ad lib a pelleted diet containing 24.4/~g Zn/g and 5.3/~g Zn/g diets, respectively. An additional 20 animals were randomly chosen, separated into two equal groups, and sacrificed at the initiation of the experiment in order to establish baseline values for both serum and bone zinc. Thereafter, randomly chosen nontested animals from each dietary group were sacrificed on Days 13 and 18 to provide continual bone and serum zinc data. Each animal chosen for behavioral testing was individually housed in a stainless steel laboratory cage and fed ad lib their respective diet prior to being placed in the testing chamber on Day 13. Following completion of testing, all animals were sacrificed on Day 18, and a single sample of blood was removed by heart puncture, and the right tibia removed for determination of zinc content by atomic absorption spectrophotometry. Food intake, body weight, serum zinc, and bone zinc data were statistically analyzed by analysis of variance.
Behavioral Testing Apparatus Four environmental test chambers were used for the measurement of association learning [ 15]. The basic operant
*Statistical difference from Group 3, 4 and 5 p<0.01.
conditioning apparatus consisted of a modified Skinner Box (Plexiglas chamber 26 × 26x 26 cm) enclosed in a sound-proof cubicle. A metal press bar, which protrudes 4 cm into the cage, was located on one wall of the chamber. On the opposite wall were two press bars, located 4 cm above the floor, and separated by 8 cm. A one-inch yellow lamp jewel, illuminated by a 71/2 watt lamp, was located 3 cm above each bar. A small cup, into which 45 mg food pellets (Noyes Precision Food Pellets) were mechanically dispensed, was located below the dual press bars. Two three-inch speakers were located in the lid of the chamber over the food cup area and were connected to an audio generator. Each speaker emitted a tone pattern at a distinctly different frequency. The complete system, including test chambers and Coulbourn digital programming units, was fully automated to facilitate stimuli presentation and data collection.
Behavioral Testing Procedure During Phase 1 of training, to obtain a food pellet, each animal was required to solve a visual discrimination problem. The animal was first required to depress the single press bar to initiate the learning trial. One of the two lights, located on the opposite side of the cage, was randomly (50%) illuminated. If the animal attended to the visual cue and depressed the bar corresponding to the illuminated light, it received a single food pellet. If an incorrect bar was pressed, the animal was required to press the single bar again to initiate another learning trial. All trials were initiated by the animal rather than the experimenter, and it was unnecessary either to food or water-deprive the animals to insure acquisition of the visual discrimination. Each animal remained in the initial phase of training until a minimum of 300 trials had been completed and an asymptotic performance achieved (i>80% correct for five consecutive blocks of trials; 10 trials/block). Approximately 8 hours (range 5-10) were allotted to complete Phase I of testing. Each animal which failed to meet the established criteria was classified as a nonperformer. Since the animals remained in the testing chamber throughout the study, all meals (ZA;ZD pellets) were obtained through successful completion of trials. Phase 2 of training, the association phase, commenced following attainment of the selected Phase l performance level. Two behavioral training groups were established within each dietary treatment. Each animal assigned to the experimental training condition (E) was exposed to a redundant auditory stimulus, simultaneously paired with a corresponding visual cue for 100 consecutive trials. Thus the
119
ZINC D E F I C I E N C Y ON C O G N I T I V E P E R F O R M A N C E TABLE 3 MEAN VALUES _+S.D. OF SERUM AND BONE ZINC
Day 0 Treatment ZA ZD
Day 13
Day 18
Serum Zinc (/zg/dl)
Bone Zinc (/zg/g)
Serum Zinc (/zg/dl)
Bone Zinc (/zg/g)
Serum Zinc (/zl/dl)
Bone Zinc (/zg/g)
161.60 ± 24.80 (n= 10) 160.60 ± 27.52 (n= 10)
186.30 ± 15.27 (n= 10) 195.60 ± 14.60 (n= 10)
177.25 ± 14.24 (n=4) 118.75 ± 10.01 (n=8)
234.50 ± 14.61 (n=4) 158.12 ± 10.24 (n=8)
178.70 -+- 23.25 (n= 18) 98.47 ± 19.40 (n=23)
229.90 ± 29.25 (n= 18) 136.56 - 11.36 (n=23)
sound pattern, emitted from the right speaker, was presented simultaneously with illumination of the right visual cue, and the sound pattern emitted from the left speaker presented simultaneously with the left visual cue. The other group, control training (C), received n o redundant exposure to auditory stimuli--only an additional 100 trials of light presentation. Phase 3, the transfer of learning t e s t phase, involved random presentation of one of two auditory stimuli in the absence of the visual stimulus. Both groups were required to complete 450 trials during Phase 3. Phase 4, the final t e s t phase, involved presentation for a second time of auditory stimuli for 300 consecutive trials. During Phase 4, each randomized tone was emitted simultaneously from both speakers while still being paired with the same press bar it had been associated with during Phases 2 and 3. In order to obtain a food pellet, each animal was required to associate a particular tone with its correct press bar regardless of the location of the tone. The difference in performance (number o f correct responses/10 trials during Phase 3 - - t e s t phase) between those groups previously exposed to the simultaneous visual and auditory cues, plus those exposed to only visual cues, was taken as a measure of transfer learning. Differences in performance between groups in all other phases were taken as a measure of discrimination learning. Behavioral data were subjected to nonparametric analysis employing the Wilcoxon Mann-Whitney W test [21]. Results
Prior to entering behavioral testing on Day 13, no significant differences were observed in food consumption of moderately zinc deficient animals when compared to control, F(3,34)=0.441, p>0.05. Within each diet group a subgroup of animals, identified as nonperformers, failed to complete the behavioral testing sequence. Mean food intake of ZA nonperformers (23.0___2.8 g) on Day 12 was similar to ZD nonperformers (23.6___3.7 g) and did not differ (p >0.05) from those animals who completed the behavioral testing sequence (ZA 21.5___3.1; ZD 24.2_+3.8 g). The mean body weight on Day 12 was likewise comparable among ZA (242.0_+6.9 g) and ZD (239.9_+18.7 g) nonperformers and performers (ZA 249.4_+8.7 g; ZD 249.6___8.4 g). As shown in Table 3, bone zinc concentration (/zg Zn/g bone) gradually increased in the ZA group from Day 0 through Day 13 and remained relatively stable thereafter. In contrast, a reduction in bone zinc was observed in the ZD group throughout this interval as evidenced by a significant
Diet × Time interaction, F(2,62)--46.57, p<0.01. Expressed on an absolute weight basis (/zg Zn/bone), the zinc content per tibia remained static throughout the experimental period in the ZD group as evidenced by an average gain of only 1.75 /zg. The ZA group exhibited a steady increase in zinc content of bone, averaging 24.05/zg. As shown in Table 3, a similar effect was seen in serum zinc concentrations which gradually increased in the ZA group by Day 13 and remained relatively stable thereafter. In contrast, a reduction in serum zinc was observed in ZD groups throughout this interval, as evidenced by significant D i e t x T i m e interaction, F(2,61)=20.63, p<0.01. One of the most striking behavioral characteristics displayed by the moderately deficient animals was the increased incidence of " n o n p e r f o r m a n c e . " Nonperformers were defined as those animals which did not reach the specified criteria during training (Phase 1) and failed to complete the subsequent phases of tests. Of the 22 ZA animals that began the testing sequence, 2 (9%) were classified as nonperformers. In comparison, of the 26 ZD animals that began the testing sequence, 8 (31%) were classified as nonperformers. Twenty ZA animals and 18 ZD animals completed all phases of training and testing. Behavioral performance of 20 ZA and 18 ZD animals met the specified criteria during Phase 1 of visual discrimination. No significant differences were observed in the mean number of trials to reach criteria between the ZA (152.5) and ZD (158.9) groups, respectively. Likewise, as shown in Table 4, performance in Phase 2 (association phase) was similar for all treatment groups. Compounding the visual stimulus with an auditory cue has no detrimental effect on performance of either the ZA of ZD-E during this critical phase of training. Differences in performance were observed, however, during Phase 3, the transfer of learning test phase. Zinc adequate-experimental animals, having prior experience with auditory cues, reached criteria in significantly (p <0.01) less trials when compared to all other groups (ZA-C, ZD-C, ZD-E). In contrast, the performance of ZD-E, having prior exposure to the auditory cue, did not significantly differ (0>0.05) from either the ZA-C or ZD-C, having n o previous exposure to the test stimulus. As shown in Table 4, ZA-C animals performed better during Phase 4 (simultaneous tone presentation), reaching specified criteria in significantly less trials (215.5) when compared to the ZA-E (323.0) animals (p<0.01), whereas ZD-C (97.8) did not differ from ZD-E (176.7) in the number of trials to reach criteria (0>0.05). Of interest was the obser-
120
MASSARO, MOHS A N D F O S M I R E TABLE 4 MEAN NUMBER OF TRAILS TO CRITERION* _+ S.D. Behavioral Treatment Zinc Adequate Control (n= 10) Experimental (n= 10) Zinc Deficient Control (n=9) Experimental (n =9)
Phase 2
Phase 3
Phase 4
51.0_+ (3.2)
182.0-+ (111.5)
215.5-+ (117.3)
53.0 ± (6.8)
118.0 ± (93.4)
323.0 ± (149.4)
65.6 ± (34.3)
210.0 ± (151.9)
97.8 ± (93.6)
50.0 ± (0.0)
242.3 ± (114.7)
176.7 ± (163.2)
*/>80% correct for 5 consecutive blocks of trials; 10 trials/block.
vation that ZD animals, regardless of previous training and or performance during previous phases, made significantly fewer errors and reached criteria with less trials during this final discrimination task (p<0.01).
DISCUSSION The present study demonstrates that a moderate, concurrent zinc deficiency alters cognitive performance. Zinc deficient animals reached criteria on both the initial visual discrimination task and the final auditory task. This observation agrees with numerous reports providing evidence that simple stimulus-response learning if not impaired via dietary manipulations [12]. The better performance of both ZD and ZA-C animals during the final discrimination phase probably reflects a g r a d u a l increase in motivation whereby the new auditory cues become better predictors of food reinforcement. Similar effects of diet-induced enhancement of appetitive learning have likewise been observed in the concurrently and previously malnourished primate and rodent [11, 12, 22]. The deficit in association learning observed in the present study is not a unique characteristic of zinc deficiency. Others have shown this basic style of learning to be impaired by early protein or caloric restriction in the rat [13], early infantile malnutrition in the primate [5], and, more recently, moderate, concurrent iron deficiency in the young adult rat [15]. The present study provides further evidence that nutrient deficiencies of short duration interfere with normal processes involved in association learning. The common mechanism(s), behavioral or biochemical, that are responsible for this observed deficit in performance remain speculative. Acute nutritional insults may force an animal to adopt a learning strategy for processing of biologically meaningful information at the expense of other kinds of
information [13], or nutritional deprivations may attenuate curiosity itself, which is a necessary component for development of cognitive potential [5]. However, the extent to which motivationally based changes in behavior are operational under conditions of moderate nutrient deficiencies that produce no discernible effect on either food intake or utilization remain to be determined. One may explain the above findings through examination of behavioral characteristics displayed by ZD nonperformers. These animals although comparable to ZA and ZD performers with respect to food intake and resultant weight gain patterns, were over-reactive to simple handling procedures, were often immobile in the testing apparatus, and frequently entered into a prolonged food and water deprivation (>72 hours), with no attempt to initiate the reinforcement contingency. Similar behavioral disturbances have been documented, not only in protein-calorie malnourished (unpublished observations) and zinc deficient animals [2,12], but also in animals with excess adrenal steroid levels [19]. Both zinc deficiency and protein restriction have also been shown to produce adrenal hypertrophy, hypersensitivity to ACTH, and increase in level of circulating corticosteroids [ 1, 2, 4, 18]. In light of these observations, it has recently been suggested that many of the behavioral manifestations associated with zinc deficiency may be attributed to altered pituitary-adrenal function [9]. Unfortunately, however, little information is available depicting the effects of moderate zinc deficiency on these parameters. It is reasonable to suggest that a similar perturbation in pituitary-adrenal function may result from a moderate nutrient deficiency. This altered function may lead to behaviors that block performance of an animal in a particular task (nonperformers) or impair performance of an animal in tasks of association learning. Importantly, such a mechanism could help to explain many of the coincidental behavioral similarities observed to result from dietary manipulations.
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