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Effects of early malnutrition on tail pinch-induced behavior of the rat
Physioh,gy & Beharior, Vol. 22. pp. 149--155. Pergamon Press and Brain Research Publ., 1979. Printed in the U.S.A.
Effects of Early Malnutrition on Tail Pinch-Induced Behavior of the Rat T H O M A S A. P I C O N E " A N D R O B E R T D. H A L L :~
Worcester Foutuhttion for Experimental Biology, Shrewsbury, MA 01545 ( R e c e i v e d 25 May 1978) PICONE, T. A. AND R. D. HALL. F~ff'ects o f early mahmtrition on tail pinch-induced behavior o f the rat. PHYSIOL. BEHAV. 22( I1 149.-155, 1979.--To determine if rats malnourished early in life are hyperresponsive to aversive stimulation their responses to tail pinch were measured in three experiments. Early malnutrition was induced by feeding the mothers an 8~,4 casein diet from 5 weeks before mating until the pups were weaned at 21 days of age. Well-nourished offspring were born to mothers fed a 25~ casein diet. At weaning half of the pups in each group were switched to the other diet. They were 60-100 days old when tested. Neither the latency to the first response elicited by tail pinch nor the total duration of stimulus-bound behavior indicated that the rats malnourished as preweanlings were hyperresponsive. In all three experiments, however, rats subjected to early malnutrition responded differently than well-nourished animals, irrespective of the dietary treatment at the time of testing. Malnourished rats gnawed more than they licked food and other objects; wellnourished rats did the opposite. Measurements of food weights confirmed the observation that eating was an infrequent rather than a predominant behavior.
Tail-pinch behavior
Early malnutrition
Ingestion
SEVERAL lines of evidence have suggested that animals subjected to early protein or protein-calorie malnutrition are hyperemotional, even after dietary rehabilitation. They have been found to be less active and to defecate more in open fields than well-nourished rats 14, 8, 191. These findings, however, have not been supported by other open-field studies [13, 20, 21]. Much of the other evidence is based on an apparent hyperresponsiveness to aversive stimulation, mainly electric shock used in studies of conditioned avoidance behavior [7, 13, 17]. For example, the faster acquisition of passive avoidance behavior by malnourished as compared to well-nourished rats has been interpreted as evidence of a stronger fear response to the shock stimulus in the malnourished animal [71. Levine and Wiener [12] have pointed out, however, that hyperresponsiveness is not necessarily indicative of hyperemotionality. Nor is it clear that the results of shock avoidance studies indicate a hyperresponsiveness to shock. The reported differences in the behavior of malnourished and well-nourished rats in such situations are open to question because it is uncertain that effective shock intensities were equal for animals whose sizes were different because of different nutritional histories [5]. Even after several months of rehabilitation rats malnourished as preweanlings remain significantly smaller than rats that have never been deprived, and there is evidence that the effectiveness of foot shock is indirectly related to body size [161. It seemed desirable to reexamine the question of hyperresponsiveness in rats subjected to early malnutrition using
Stimulus-bound behavior aversive stimulation other than electric shock. If malnourished rats are hyperresponsive to other kinds of aversive stimulation the question of hypereraotionality could be subjected to further analysis. The behavior induced by tail pinch is one kind of aversive behavior we have examined in the malnourished rat. Under the stress induced by tail pinch rats engage in various kinds of behavior, mostly oral behavior such as eating, gnawing, licking, or drinking when a very palatable fluid is available [1, 2, 3]. We reasoned that if malnourished rats are hyperresponsive to aversive stimuli they should exhibit more tail pinch-induced behavior than wellnourished rats. EXPERIMENT 1
Method Animals. The subjects were 24 male and 29 female Sprague-Dawley rats of the Charles River CD strain bred in this laboratory as described below. They were approximately 70 days old at the beginning of the experiment. Dietary conditions. The rats were reared under 4 dietary conditions. Two groups were born to dams fed a low protein diet (8% casein) from 5 weeks before mating until their pups were weaned at 21 days of age. One of these groups (LL) was fed the same diet after weaning; the other (LH) was switched at weaning to an isocaloric high protein diet (25% casein). Rats in the other two groups were born to dams maintained on the high protein diet before mating and throughout gestation and lactation. The rats in one group (HH) were fed the same diet postweaning; rats in the other group (HL) were
~This research was supported by National Institutes of Health grant HDO6364. The authors are grateful to William B. Forbes and Wendy M. Robertson for helpful suggestions about an earlier draft of the paper. :Thomas A. Picone is now at the University of Connecticut. :~Requests for reprints should be sent to Robert D. Hall, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Copyright ' 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/010149-07502.00/0
150
PICONE AND HALL TABLE 1 MEAN WEIGHTS OF RATS IN EXPERIMENTS 1-3 Diet LL HH LH HL
Experiment 1 Male Female 121 414 264 195
(5)* (7) (5) (7)
100 264 196 144
(6) (7) (9) (7)
Experiment 2 Male Female 70 367 248 161
(9) (9) (8) (10)
79 235 171 180
(6) (7) (8) (6)
Experiment 3 Male Female 113 412 307 206
(7) (8) (8) (7)
118 253 199 187
(9) (7) (6) (8)
*Number of rats is shown in parentheses. Weights are in grams.
switched to the low protein diet at weaning. The diets, made by Teklad Mills, Madison, Wisconsin, are described elsewhere [6]. At birth the pups were randomly mixed with pups born the mine day to mothers on the same diet and culled to litters ofeijht. Mean weights of the rats in the four diet groups are ~ in Table 1. Procedure. Tail pinch was administered by means of an alligator clip with jaws covered by tubber after the teeth were filed away. The clip was suspended by a wire and a hook that slid ~ a rod which was mounted 26 cm high across the center of the cooler chest (51x28x35 cm) in which the rats were observed. In this way the tail was held high, at a point approximately 14.5 cm above the floor, which discouraged behavior directed at the clip, To ensure that the pinch was applied with equal force to rats of ~ r ent sizes it was applied at a position nearest the tip of the tail where the tail was 1/8 in. (3.2 ram) in dia. The force required to open the jaws of the clip this far was 1 1 ~ g. This ~ t i o n was ~ the day before a test and marked with a felt tipped marker. In each test there were 2 trials in ~ tail pinch was ~ for 2 rain. The first trial occurred alter the rat was given 5 rain to adapt to the test situation, and the 2 trials were separated by 4 min. The rat remained in the cooler chest during the intertrial interval, A large window was installed in the lid of the cooler chest which was i l ~ by a single 7.5-W bulb nmunted on an end wall, The room was illuminated by one 60.W red bulb. A mirror mounted over the chest made it possible for the observer to view a rat without being in the animal's direct view. The time spent in each of 5 kinds of behavior was measured on counters pulsed at 0.5-sec intervals. The latency of the fwst response to tail pinch on each trial was also recorded. The rats were kept on a 12-hr li,.~tt-dark cycle and tested d ~ the light phase. Water and the appropriate diet were available at all times in their cages. In the first test several objects were present: lab chow pallets, the rejular mash diet, wood blocks and a bottle of water, all plaeed at the lighted end of the chest. In a second test 8 days later only lab chow pellets were present, and the amount of food inll~ted by rats that gnawed on the ~ s was estimated b y w e i ~ n g the pellets before and after the test. Results
In the first test the total duration of all tail pinch-induced behavior was actually less in rats mak'muri~a~ed prior to weaning than it was in rats well-nourished during gestation and lactation. Mean durations (2 trials combined) for the four
diet groups were as follows: HH, 74.3 sec; HL, 77.2 sec; LL, 30.7 sec; LH, 50.8 sec. A three-way ANOVA (preweaning diet x postweaning diet x sex) indicated that only the preweaning diet had a significant effect on the amount of tail pinch-induced behavior, F(!,45)=6.91, p<0.025. In the second test the total amount of tail pinch-induced behavior did not differ for rats in the diflk,~t diet groups. Latencies of the behavior did not differ signifr.antly as a result of dietary treatment in either test. In the second test there was a prominent difference related to diet in the kind of behavior elicited by tail pinch. Rats subjected to the r ~ t ~ protein conditions as preweanlings were more likely to gnaw than to-lick o b j ~ t $ such as food pellets and feces. Rats that had been fed the high protein diet before weaning did just the opposite: they licked the pellets, feces and pans of the apparatus more than they gnawed on them. For two reasons it was convenient to express this difference in behavior in a "lick-gaaw ratio" based on the total amount of licking and 8hawing ~ t i v e ofxhe object. First, this memmre of p r e f i ~ n c e for one ~ a v i o r or the other is laqleiy independent of the large individualdiffer. ences in the absolute amount of behavior induced by tail pinch. Second, it summariz~ in a sintlle index a preference that is sensitive to the dietary treatments bUt cannot always be demonstrated for a particular goat object because too few animals in a given test respond to that object. The ratio is defined as:
total amount of ii~kijng-totat amount of gnawing total amount of licking+total amount of gnawing ,
=
It can vary from + 1.0 for a rat that licks but never gnaws to - 1.0 for a rat that gnaws but never licks. Differences in the ratio associated with the dietary treatments are shown in Fig. I where it is clear that the preweaniog nutritional experience, not the current diet, determined the sign of the ratio. For the preweaning diet variable F(1,45)=72.2, p<0.001. Because these diet-related differences were not ~ n t in the first test, Experiment 2 was undertaken to determine if they were replicable. Estimates of the amount of food ingested were obtained for onty 16 rats that actually chewed the Ilediets. The mean difference in w ~ l h t of the food before and after the test was actually a net gain of 0.06 g. The crumbled pellets were heavier in a number of cases because they were wet with saliva. In only two cases were the losses in weight as great as 0.1 g. This apparent lack of ingestion might have resulted from the use of a novel and not especially palatable food. A second purpose of Experiment 2 was to examine this possibility, and sugar cubes rather than lab chow pellets
MALNUTRITION AND TAIL-PINCH BEHAVIOR
UI each group during the tall-pinch test. Lab chow pellets were present for the other half. Sugar cubes or pellets were weighed before and after the two trials given each animal.
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LL LH DIET FIG. 1. Relative amounts of licking and gnawing induced by tail pinch in rats of four diet groups expressed as mean lick-gnaw ratios. Measures used to compute ratios included all licking and gnawing irrespective of the object. See Method for description of dietary conditions.
were present during tail-pinch tests for half of the rats in the following experiment. EXPERIMENT 2
Sixty-three rats, bred and reared in the laboratory as described in Experiment I, were approximately 60 days old at the time they were tested. They were divided among the four dietary conditions as shown in Table 1. The stimulus, apparatus, nutritional conditions and test procedures were like those of the second test of Experiment 1, except that sugar cubes were present for half of the rats in
As in the first experiment rats subjected to early malnutrition (LL and LH groups) gnawed more than they licked in response to tall pin.oh, and rats that had not been deprived as preweanlings did just the opposite (Table 2). A four-way ANOVA (preweaning diet × postweaning diet × sex × food type) indicated that preweaning diet was the only variable having a significant effect on lick-paw ratios, F(1,46)= I26.8, p<0.001. Rats given sugar eul~s in the t ~ t s exhibited the same nutritkm-related differences in licking and gnawing as the animals tested with lab chow pellets. Although there was appreciably more gnawing by the HH rats given sugar cubes than by those given pellets, their mean lick-gnaw ratios were not significantly different. There was no convincing evidence of ingestion in the weights of pellets and sugar cubes. The ~ difference in pellet weights for the 12 rats that chewed them was -0.02 g, the largest reduction being 0.06 g. The mean diffm'ence in sugar cube weights for the 19 rats that gnaw~l them was larger, -0.11 g, but this larger difference was primarily a r~flection of the difficulty in recovering the crumbled sugar in a number of cases where it was partially dissolved and stuck to the floor. In 11 of 19 cases the losses in sugar cube weights were less than 0.1 g. Sugar cubes signitieantly increased the total amount of tall pinch-induced behavior in all four diet groups by an average of 38% above that of rats given pellets, F(1,46)=6.53, p<0.025. These increases resulted mainly from increases in the licking and gnawing of the sugm" cubes. Again the total amounts and latencies of tall-pinch behavior provided no evidence that rats subjected to early malnutrition were more responsive than rats well-nourished before weaning. It seemed possible that the differences in lick-gnaw ratios resulted from differences in effective stimulus intensity associated, in part at least, with diet-related differences in body size. Although the pressures applied to the tail were the same for rats in the different diet groups, the positions where
TABLE 2 EFFECTS OF DIET ON LATENCY, DURATION AND LICK-GNAWRATIOS OF TAIL PINCH-INDUCEDBEHAVIOR* i
Diet Lab Chow HH HL LL LH
Latency
i
Grooming
i
Licking Feces
PelletsPresent During Test 39.7 3.6 26.3 38.3 7.9 11.1 50.9 0 0 39.4 0.9 0
Sugar Cubes Present During Test HH 33.1 4.4 HL 26.3 6.3 LL 34.1 0.9 LH 40.9 4.4
7.0 16.4 0 0
Licking Licking Gnawing Food Floor Feces
i
i
i
Gnawing Food
Total Gnawin~
Total Licking
Total Behavior
LGR¢
34.3 12.1 0 0
27.1 41.8 4.6 9.7
0 6.8 34.2 25.1
3.4 3.4 35.4 50.3
3.4 10.1 69.6 75.4
91.3 72.9 4.6 10.6
94.7 83.0 74.1 86.0
+0.95 +0.79 -0.47 -0.80
43.8 38.1 0 0
31.8 57.2 12.9 9.6
0 1.7 17.6 24.1
42.9 14.8 65.6 75.9
42.9 16.5 83.2 100
87.0 118.0 13.8 14.0
129.9 134.5 97.0 114.0
+0.48 +0.83 -0.66 -0.80
*All measures except median latencies and lick-gnaw ratios (LGR) are mean durations of the various kinds of behavior. Latencies and durations are in seconds. +Total gnawing included gnawing of food and feces; total licking included licking of food, feces and floor and grooming. :~Group LGRs were computed from LGRs of individual rats, not from group means of total gnawing and total licking.
PICONE AND HALL
152
they were applied on the tail might not have been comparable. One result arguing against such an explanation is that rats in the LH and HL groups mainly gnawed or licked. respectively, although their weights were similar. Nevertheless, a third experiment was undertaken to determine the extent to which the differences in lick-gnaw ratios depend on stimulus intensity.
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Thirty male and 30 female rats bred and reared as in the previous experiments were approximately 75 days old at the beginning of the experiment. Mean weights of the animals in the 4 diet groups are shown in Table 1, One rat became ill and was discarded. Three battery clamps were modified to provide tail pinch of three intensities: 680 g, 1060 g, and 1480 g. The medium intensity was similar to that of the alligator clip used in the first two experiments, 1100 g. The different intensities were obtained by using different springs in the clamps. The forces were measured by determining the weights required to open the jaw 1/8 in. (3.2 mm). In all other respects the procedures were like those of Experiment 2, except that each rat was given three tests, one at each stimulus intensity, two days apart. The order of stimulus presentation was according to a Latin square design. Only food pellets were present, and they were weighed before and after each test. Resutts
Lick-gnaw ratios were influenced by the dietary treatments in the same way they had been in the previous experiments (Fig. 2). The effect of the preweaning diet was again significant, F(1,51)=89.5, p
-J -0.5 z ~ -1.0
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Low
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Medium STIMULUS INTENSITY
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FIG. 2. Mean lick-gnaw ratios of four diet groups at three intensities of tail-pinch stimulation: 680 g, 1060g, and 1480g.
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than did rats in the other groups (Newman-Keuis comparisons, p<0.01). There were no other significant differences in latency, nor did the duration of the tail pinch-induced behavior show any effects of the dietary treatments. Repeated testing led to a progressive increase in the amount of behavior elicited by tail pinch as shown in Fig. 4. F(2,110)=21.3, p<0.001. Onset latencies diminished in a corresponding way, F(2,110)= 15.3, p <0.001, and were again influenced by the postweaning dietary treatment. F(1,55)=4.35, p<0.05. The diet effect seems marginal, however, and Newman-Keuis comparisons yielded no significant differences between the groups in any session, not even for the HH rats in the first session. Six rats de£mitely ate the lab chow pellets in this experiment. This was clear from their behavior and from the measurements of pellet weights. The weight differences for all of the tests on which rats gnawed the pellets are shown in Fig. 5 where measurements designated by hatohing are for the 10 trials in which the six rats ingested food. When eating, the rats held the pellets just below the chin, took small bites, spilling very little, and chewed the food in an unhurried way
MALNUTRITION AND TAIL-PINCH BEHAVIOR
c
had been malnourished before weaning, four LL rats and two LH rats. It is unlikely that the six were just by chance in the LL and LH groups and none were in the HH and HL groups, Xz (df= I) = 4.83, p<0.05. It seemed possible that ingestion was rare in the experiments described above because the rats had been reared on special mash diets. As a check on this possibility the behavior of rats reared on lab chow pellets in the Charles River Breeding Labs (Wilmington, Mass.) was examined under the same conditions used in the previous experiments.
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FIG. 4. Changes in the latency and amount of behavior (total duration) induced by tail pinch as a function of repeated testing of four diet groups. The three stimulus intensities that were used, counterbalanced in their order of presentation, were ignored in this summary.
50 40
Method
Eleven male Sprague-Dawley rats of the Charles River CD strain were purchased from the breeder as adults. Their mean weight at the beginning of the experiment was 275 g. Tail pinch was applied by means of the alligator clip used in Experiments 1 and 2. The test procedures were like those of the previous experiments except, of course, there was only one dietary treatment. The 11 rats of this experiment, like the HH rats in the earlier experiments, were wellnourished throughout their lives.
['7] INGESTION
Results
r'l
There was no ingestion of food in either test. Only one rat gnawed on the food, and there was no reduction in the weight of the food as a result of that gnawing. On only four other occasions during the two tests was there any gnawing, and in each case it involved feces. The mean lick-gnaw ratios for the group were +0.72 and +0.78 for the two tests. These results suggest that rearing on the special mash diets had not been responsible for the occurrence of so little eating in the previous experiments--or for the predominance of licking rather than gnawing in rats well-nourisbed early in life.
NO INGESTION
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PRETEST-POSTTEST DIFFERENCE IN FOOD WEIGHT (g). FIG. 5. Distribution of differences in food weights measured before and after tail-pinch tests in Experiment 3. Measurements are from all tests in which rats gnawed on pellets, which was not every test. Measures indicated by hatching are from rats that were seen to ingest the food.
before taking an additional bite. This behavior was quite different from the frenzied uninterrupted gnawing that was seen throughout most of this study, with near total crumbling of a pellet in a matter of seconds. Weight measurements for the unambiguous cases of eating form a distinct limb of the distribution in Fig. 5. It seems likely that nearly all, if not all of the other small differences, most less than 0.1 g, are no more than errors of measurement due mainly to difficulties in recovering all of the crumbled pellets and to contamination by fluids. The six rats that ingested food were all animals that
The experiments described above have established three things: (1) Under the stress induced by tail pinch rats that have been malnourished early in life respond differently than rats that have never known such deprivation; they are more likely to gnaw than to lick food and other objects, whereas well-nourished rats are likely to do just the opposite. (2) Neither the amount of tail pinch-induced behavior nor the rapidity of its onset was any greater in rats subjected to early protein malnutrition than in well-nourished ones. (3) Under some conditions eating is an infrequent response to tail pinch rather than a predominant one. We have no ready explanation for the diet-related differences in lick-gnaw ratios; but tail pinch-induced behavior is similar in many ways to behavior elicited by hypothalamic stimulation [1], which suggests several ways of thinking about the problem. Both kinds of behavior are influenced by the stimulus properties of objects present in the test situation. Sugar cubes in the present study stimulated more licking and gnawing than did lab chow pellets, but lick-gnaw ratios were not affected by food type. Moreover, the differences between diet groups were apparent in the licking and gnawing of not just the food, but other objects as well. All of this suggests that the response itself was important, not the object of the response. One possibility is that differences in lick-gnaw ratios reflect "natural" behavioral propensities in
154
PICONE AND H A L L
the way that hypothalamically elicited behavior does [9,15]. Our rats malnourished early in life may generally gnaw more and lick less than well-nourished rats, and we are currently studying ways to check this possibility. The prepotency of gnawing in malnourished rats is also suggested, albeit indirectly, by the present findings on tail pinch-induced eating--or the lack of it. The only rats that clearly ingested food in these experiments were six animals in Experiment 3 that had been malnourished as preweanlings. Four o f them in the L L group were malnourished at the time of testing. Our best guess at present is that eating acquires a prepotency as a result of early deprivation and that the gnawing which was so prevalent in the malnourished rats is a fractional component of such a prepotent response. This explanation says nothing, of course, about the predominance of licking in well-nourished rats. At another level o f analysis it is possible that the differences in lick-gnaw ratios reflect a difference in the functioning o f brain catecholamine systems. Tail pinch-induced behavior apparently depends on the dopaminergic nigrostriatai system [2,3] and is accompanied by an increased turnover of cortical norepinephrine [3]. It has been shown that catecholamine metabolism is altered in our malnourished animals, as indicated by their abnormally high levels o f brain norepinephrine [22]. It was also shown that these animals are less sensitive than well-nourished rats to the stereotypyinducing effects of apomorphine, a dopamine agonist [I1]. Whether the diet related differences in behavior elicited by tail pinch are in any way related to these changes in catecholamine functions remains to be seen. We were surprised to find so little ingestion because several investigators have reported that eating is the predominant response to tail pinch [2, 10, 18]. The measurement of food weights merely confirmed what seemed obvious to us from watching the animals; those that chewed the pellets did so not to eat them but, apparently, to derive whatever relief they could from the gnawing. It must also be remembered that nearly half of the animals, most of those that had been well-nourished as preweanlings, gnawed the pellets very little but licked them instead.
It seems unlikely that tail pinch induced so little eating because the lab chow pellets were novel or unpalatable to the rats reared on special mash diets. Sugar cubes were eaten no more than the pellets, and the rats raised on lab chow in Experiment 4 did not eat under the stress of tail pinch. Part of the answer may lie in the tail-pinch stimulus. In some informal experiments we were more successful in eliciting eating when tail pinch was applied with a 25-cm hemostat like that used by Antelman and his coworkers. It seemed, however, that the intensity of the stimulus (at around t h e first notch on the hemostat) was appreciably greater than the intensities used in the present experiments. All six rats that ingested food in Experiment 3 did so only in response to the most intense stimulus or to other stimuli only after they had experienced the most intense one. However, the data from these same 6, as discussed above, suggest that tail pinchinduced eating may also depend on nutritional status: all 6 had been malnourished as preweanlings. The question with which this study began seems to have been answered unambiguously. Rats malnourished early in life are no more responsive to tail pinch than well-nourished rats. There was some indication in Experiment 3 that rats fed the low protein diet at the time of testing responded with shorter latencies than rats on the high protein diet, but this effect has little bearing on the question of whether preweaning nutritional deprivation produces rats that are hyperresponsive later in life. It is conceivable that gnawing is indicative o f a stronger response to tail pinch than licking is, but the data do not support such a notion. Gnawing increased no more than licking did as lick-gnaw ratios underwent nonmonotonic changes with increases in stimulus strength, even though response duration and latency clearly indicated increasing response strength with increasing stimulus intensity. Whether the rats in our model of malnutrition are hyperresponsive to other kinds of aversive stimulation remains to be seen. It seems clear in any case that any hyperresponsiveness that might be interpreted as hyperemotionality in rats subjected to early malnutrition must be specific to certain kinds of stimuli or situations whose essential attributes are not yet understood.
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17: 743-748, 1976. 2. Anteiman, S. M. and H. Szechtman. Tail pinch induces eating in sated rats which appears to depend on niEp'ostriatai dopamine. Science 189: 731-733, 1975. 3. Antelman, S. M., H. Szechtman, P. Chin and A. E. Fisher. Tail pinch-induced eating, gnawing and licking behavior in rats: dependence on the niBrostriatal dopamine system. Br~lin Res. 99: 319--337, 1975. 4. Cowley, J. J. and R. D. Griesel. Low protein diet "and emotionality in the albino rat. J. genet. Psycho/. 104: 89-98, 1964. 5. Crnic, L. S. Effects ofinfantibe undernutrition on adult beaming in rats: methodological and design problems. Psycho/. Bull. 83: 715-728, 1976. 6. Forbes, W. B., C. Tracy, O. Resnick and P. J. Morgane. Effects of maternal dietary protein restriction on growth of the brain and body in the rat. Bruin Res. Bull. 2: I31-135, 1977. 7. Fr-'ankov~i,S. and R. H. Barnes. Effect of malnutrition in early life on avoidance conditioning and behavior of adult rats. J. Nutr. 96: 485-493, 1968.
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M A L N U T R I T I O N AND T A I L - P I N C H B E H A V I O R 14. Levitsky, D. A. and R. H. Barnes. Nutritional and environmental interactions in the behavioral development of the rat: long-term effects. Science 176: 68-71, 1972. 15. Madlafousek0 J., K. Freund and I. Grofowi. Variables determining the effects of electrostimulation in the lateral preoptic area on the sexual behavior of male rats. J. comp. phyMol. Psychol. 70: 28--44, 1970. 16. Par~, W. P. Age, sex and strain differences in the aversive threshold to grid shock in the rat. J. ('omp. physiol. Psycho/. 69: 214-218, 1969. 17. Rider, A. A. and M. Simonson. The relationship between maternal diet, birth weight, and behavior of the offspring in the rat. Nutr. Rep. Int. 10: 19-24, 1974. 18. Sahakian, B. J. and T. W. Robbins. lsolatiou-rearing enhances tail-pinch induced oral behavior in rats. Physiol. Behav. lg: 53-58, 1977.
155
19. Simonson, M., J. K. Stephan, H. M. Hanson and B. F. Chow. Open field studies in offspring of underfed mother rats. J. Nutr. 101: 331-336, 1971. 20, Slob, A. K., C. E. Snow and E. Natris-Mathot. Absence of behavioral deficits following neonatal undernutrition in the rat. Devl Psychobiol. 6: 177-186, 1973. 21. Smart, J. L. Activity and exploratory behavior of adult offspring of undernourished mother rats. Dev/ Psychohiol. 7: 315-321, 1974. 22. Stern, W. C., M. Miller, W. B. Forbes, P. J. Morgane and O. Resnick. Ontogeny of the levels of biogenic amines in various parts of the brain and in peripheral tissues in normal and protein malnourished rats. Expl Neurol. 49: 314-326, 1975.
Effects of Early Malnutrition on Tail Pinch-Induced Behavior of the Rat T H O M A S A. P I C O N E " A N D R O B E R T D. H A L L :~
Worcester Foutuhttion for Experimental Biology, Shrewsbury, MA 01545 ( R e c e i v e d 25 May 1978) PICONE, T. A. AND R. D. HALL. F~ff'ects o f early mahmtrition on tail pinch-induced behavior o f the rat. PHYSIOL. BEHAV. 22( I1 149.-155, 1979.--To determine if rats malnourished early in life are hyperresponsive to aversive stimulation their responses to tail pinch were measured in three experiments. Early malnutrition was induced by feeding the mothers an 8~,4 casein diet from 5 weeks before mating until the pups were weaned at 21 days of age. Well-nourished offspring were born to mothers fed a 25~ casein diet. At weaning half of the pups in each group were switched to the other diet. They were 60-100 days old when tested. Neither the latency to the first response elicited by tail pinch nor the total duration of stimulus-bound behavior indicated that the rats malnourished as preweanlings were hyperresponsive. In all three experiments, however, rats subjected to early malnutrition responded differently than well-nourished animals, irrespective of the dietary treatment at the time of testing. Malnourished rats gnawed more than they licked food and other objects; wellnourished rats did the opposite. Measurements of food weights confirmed the observation that eating was an infrequent rather than a predominant behavior.
Tail-pinch behavior
Early malnutrition
Ingestion
SEVERAL lines of evidence have suggested that animals subjected to early protein or protein-calorie malnutrition are hyperemotional, even after dietary rehabilitation. They have been found to be less active and to defecate more in open fields than well-nourished rats 14, 8, 191. These findings, however, have not been supported by other open-field studies [13, 20, 21]. Much of the other evidence is based on an apparent hyperresponsiveness to aversive stimulation, mainly electric shock used in studies of conditioned avoidance behavior [7, 13, 17]. For example, the faster acquisition of passive avoidance behavior by malnourished as compared to well-nourished rats has been interpreted as evidence of a stronger fear response to the shock stimulus in the malnourished animal [71. Levine and Wiener [12] have pointed out, however, that hyperresponsiveness is not necessarily indicative of hyperemotionality. Nor is it clear that the results of shock avoidance studies indicate a hyperresponsiveness to shock. The reported differences in the behavior of malnourished and well-nourished rats in such situations are open to question because it is uncertain that effective shock intensities were equal for animals whose sizes were different because of different nutritional histories [5]. Even after several months of rehabilitation rats malnourished as preweanlings remain significantly smaller than rats that have never been deprived, and there is evidence that the effectiveness of foot shock is indirectly related to body size [161. It seemed desirable to reexamine the question of hyperresponsiveness in rats subjected to early malnutrition using
Stimulus-bound behavior aversive stimulation other than electric shock. If malnourished rats are hyperresponsive to other kinds of aversive stimulation the question of hypereraotionality could be subjected to further analysis. The behavior induced by tail pinch is one kind of aversive behavior we have examined in the malnourished rat. Under the stress induced by tail pinch rats engage in various kinds of behavior, mostly oral behavior such as eating, gnawing, licking, or drinking when a very palatable fluid is available [1, 2, 3]. We reasoned that if malnourished rats are hyperresponsive to aversive stimuli they should exhibit more tail pinch-induced behavior than wellnourished rats. EXPERIMENT 1
Method Animals. The subjects were 24 male and 29 female Sprague-Dawley rats of the Charles River CD strain bred in this laboratory as described below. They were approximately 70 days old at the beginning of the experiment. Dietary conditions. The rats were reared under 4 dietary conditions. Two groups were born to dams fed a low protein diet (8% casein) from 5 weeks before mating until their pups were weaned at 21 days of age. One of these groups (LL) was fed the same diet after weaning; the other (LH) was switched at weaning to an isocaloric high protein diet (25% casein). Rats in the other two groups were born to dams maintained on the high protein diet before mating and throughout gestation and lactation. The rats in one group (HH) were fed the same diet postweaning; rats in the other group (HL) were
~This research was supported by National Institutes of Health grant HDO6364. The authors are grateful to William B. Forbes and Wendy M. Robertson for helpful suggestions about an earlier draft of the paper. :Thomas A. Picone is now at the University of Connecticut. :~Requests for reprints should be sent to Robert D. Hall, Worcester Foundation for Experimental Biology, Shrewsbury, MA 01545.
Copyright ' 1979 Brain R e s e a r c h Publications Inc.--0031-9384/79/010149-07502.00/0
150
PICONE AND HALL TABLE 1 MEAN WEIGHTS OF RATS IN EXPERIMENTS 1-3 Diet LL HH LH HL
Experiment 1 Male Female 121 414 264 195
(5)* (7) (5) (7)
100 264 196 144
(6) (7) (9) (7)
Experiment 2 Male Female 70 367 248 161
(9) (9) (8) (10)
79 235 171 180
(6) (7) (8) (6)
Experiment 3 Male Female 113 412 307 206
(7) (8) (8) (7)
118 253 199 187
(9) (7) (6) (8)
*Number of rats is shown in parentheses. Weights are in grams.
switched to the low protein diet at weaning. The diets, made by Teklad Mills, Madison, Wisconsin, are described elsewhere [6]. At birth the pups were randomly mixed with pups born the mine day to mothers on the same diet and culled to litters ofeijht. Mean weights of the rats in the four diet groups are ~ in Table 1. Procedure. Tail pinch was administered by means of an alligator clip with jaws covered by tubber after the teeth were filed away. The clip was suspended by a wire and a hook that slid ~ a rod which was mounted 26 cm high across the center of the cooler chest (51x28x35 cm) in which the rats were observed. In this way the tail was held high, at a point approximately 14.5 cm above the floor, which discouraged behavior directed at the clip, To ensure that the pinch was applied with equal force to rats of ~ r ent sizes it was applied at a position nearest the tip of the tail where the tail was 1/8 in. (3.2 ram) in dia. The force required to open the jaws of the clip this far was 1 1 ~ g. This ~ t i o n was ~ the day before a test and marked with a felt tipped marker. In each test there were 2 trials in ~ tail pinch was ~ for 2 rain. The first trial occurred alter the rat was given 5 rain to adapt to the test situation, and the 2 trials were separated by 4 min. The rat remained in the cooler chest during the intertrial interval, A large window was installed in the lid of the cooler chest which was i l ~ by a single 7.5-W bulb nmunted on an end wall, The room was illuminated by one 60.W red bulb. A mirror mounted over the chest made it possible for the observer to view a rat without being in the animal's direct view. The time spent in each of 5 kinds of behavior was measured on counters pulsed at 0.5-sec intervals. The latency of the fwst response to tail pinch on each trial was also recorded. The rats were kept on a 12-hr li,.~tt-dark cycle and tested d ~ the light phase. Water and the appropriate diet were available at all times in their cages. In the first test several objects were present: lab chow pallets, the rejular mash diet, wood blocks and a bottle of water, all plaeed at the lighted end of the chest. In a second test 8 days later only lab chow pellets were present, and the amount of food inll~ted by rats that gnawed on the ~ s was estimated b y w e i ~ n g the pellets before and after the test. Results
In the first test the total duration of all tail pinch-induced behavior was actually less in rats mak'muri~a~ed prior to weaning than it was in rats well-nourished during gestation and lactation. Mean durations (2 trials combined) for the four
diet groups were as follows: HH, 74.3 sec; HL, 77.2 sec; LL, 30.7 sec; LH, 50.8 sec. A three-way ANOVA (preweaning diet x postweaning diet x sex) indicated that only the preweaning diet had a significant effect on the amount of tail pinch-induced behavior, F(!,45)=6.91, p<0.025. In the second test the total amount of tail pinch-induced behavior did not differ for rats in the diflk,~t diet groups. Latencies of the behavior did not differ signifr.antly as a result of dietary treatment in either test. In the second test there was a prominent difference related to diet in the kind of behavior elicited by tail pinch. Rats subjected to the r ~ t ~ protein conditions as preweanlings were more likely to gnaw than to-lick o b j ~ t $ such as food pellets and feces. Rats that had been fed the high protein diet before weaning did just the opposite: they licked the pellets, feces and pans of the apparatus more than they gnawed on them. For two reasons it was convenient to express this difference in behavior in a "lick-gaaw ratio" based on the total amount of licking and 8hawing ~ t i v e ofxhe object. First, this memmre of p r e f i ~ n c e for one ~ a v i o r or the other is laqleiy independent of the large individualdiffer. ences in the absolute amount of behavior induced by tail pinch. Second, it summariz~ in a sintlle index a preference that is sensitive to the dietary treatments bUt cannot always be demonstrated for a particular goat object because too few animals in a given test respond to that object. The ratio is defined as:
total amount of ii~kijng-totat amount of gnawing total amount of licking+total amount of gnawing ,
=
It can vary from + 1.0 for a rat that licks but never gnaws to - 1.0 for a rat that gnaws but never licks. Differences in the ratio associated with the dietary treatments are shown in Fig. I where it is clear that the preweaniog nutritional experience, not the current diet, determined the sign of the ratio. For the preweaning diet variable F(1,45)=72.2, p<0.001. Because these diet-related differences were not ~ n t in the first test, Experiment 2 was undertaken to determine if they were replicable. Estimates of the amount of food ingested were obtained for onty 16 rats that actually chewed the Ilediets. The mean difference in w ~ l h t of the food before and after the test was actually a net gain of 0.06 g. The crumbled pellets were heavier in a number of cases because they were wet with saliva. In only two cases were the losses in weight as great as 0.1 g. This apparent lack of ingestion might have resulted from the use of a novel and not especially palatable food. A second purpose of Experiment 2 was to examine this possibility, and sugar cubes rather than lab chow pellets
MALNUTRITION AND TAIL-PINCH BEHAVIOR
UI each group during the tall-pinch test. Lab chow pellets were present for the other half. Sugar cubes or pellets were weighed before and after the two trials given each animal.
+1.0
Results
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iY
o I
i
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HL
LL LH DIET FIG. 1. Relative amounts of licking and gnawing induced by tail pinch in rats of four diet groups expressed as mean lick-gnaw ratios. Measures used to compute ratios included all licking and gnawing irrespective of the object. See Method for description of dietary conditions.
were present during tail-pinch tests for half of the rats in the following experiment. EXPERIMENT 2
Sixty-three rats, bred and reared in the laboratory as described in Experiment I, were approximately 60 days old at the time they were tested. They were divided among the four dietary conditions as shown in Table 1. The stimulus, apparatus, nutritional conditions and test procedures were like those of the second test of Experiment 1, except that sugar cubes were present for half of the rats in
As in the first experiment rats subjected to early malnutrition (LL and LH groups) gnawed more than they licked in response to tall pin.oh, and rats that had not been deprived as preweanlings did just the opposite (Table 2). A four-way ANOVA (preweaning diet × postweaning diet × sex × food type) indicated that preweaning diet was the only variable having a significant effect on lick-paw ratios, F(1,46)= I26.8, p<0.001. Rats given sugar eul~s in the t ~ t s exhibited the same nutritkm-related differences in licking and gnawing as the animals tested with lab chow pellets. Although there was appreciably more gnawing by the HH rats given sugar cubes than by those given pellets, their mean lick-gnaw ratios were not significantly different. There was no convincing evidence of ingestion in the weights of pellets and sugar cubes. The ~ difference in pellet weights for the 12 rats that chewed them was -0.02 g, the largest reduction being 0.06 g. The mean diffm'ence in sugar cube weights for the 19 rats that gnaw~l them was larger, -0.11 g, but this larger difference was primarily a r~flection of the difficulty in recovering the crumbled sugar in a number of cases where it was partially dissolved and stuck to the floor. In 11 of 19 cases the losses in sugar cube weights were less than 0.1 g. Sugar cubes signitieantly increased the total amount of tall pinch-induced behavior in all four diet groups by an average of 38% above that of rats given pellets, F(1,46)=6.53, p<0.025. These increases resulted mainly from increases in the licking and gnawing of the sugm" cubes. Again the total amounts and latencies of tall-pinch behavior provided no evidence that rats subjected to early malnutrition were more responsive than rats well-nourished before weaning. It seemed possible that the differences in lick-gnaw ratios resulted from differences in effective stimulus intensity associated, in part at least, with diet-related differences in body size. Although the pressures applied to the tail were the same for rats in the different diet groups, the positions where
TABLE 2 EFFECTS OF DIET ON LATENCY, DURATION AND LICK-GNAWRATIOS OF TAIL PINCH-INDUCEDBEHAVIOR* i
Diet Lab Chow HH HL LL LH
Latency
i
Grooming
i
Licking Feces
PelletsPresent During Test 39.7 3.6 26.3 38.3 7.9 11.1 50.9 0 0 39.4 0.9 0
Sugar Cubes Present During Test HH 33.1 4.4 HL 26.3 6.3 LL 34.1 0.9 LH 40.9 4.4
7.0 16.4 0 0
Licking Licking Gnawing Food Floor Feces
i
i
i
Gnawing Food
Total Gnawin~
Total Licking
Total Behavior
LGR¢
34.3 12.1 0 0
27.1 41.8 4.6 9.7
0 6.8 34.2 25.1
3.4 3.4 35.4 50.3
3.4 10.1 69.6 75.4
91.3 72.9 4.6 10.6
94.7 83.0 74.1 86.0
+0.95 +0.79 -0.47 -0.80
43.8 38.1 0 0
31.8 57.2 12.9 9.6
0 1.7 17.6 24.1
42.9 14.8 65.6 75.9
42.9 16.5 83.2 100
87.0 118.0 13.8 14.0
129.9 134.5 97.0 114.0
+0.48 +0.83 -0.66 -0.80
*All measures except median latencies and lick-gnaw ratios (LGR) are mean durations of the various kinds of behavior. Latencies and durations are in seconds. +Total gnawing included gnawing of food and feces; total licking included licking of food, feces and floor and grooming. :~Group LGRs were computed from LGRs of individual rats, not from group means of total gnawing and total licking.
PICONE AND HALL
152
they were applied on the tail might not have been comparable. One result arguing against such an explanation is that rats in the LH and HL groups mainly gnawed or licked. respectively, although their weights were similar. Nevertheless, a third experiment was undertaken to determine the extent to which the differences in lick-gnaw ratios depend on stimulus intensity.
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Thirty male and 30 female rats bred and reared as in the previous experiments were approximately 75 days old at the beginning of the experiment. Mean weights of the animals in the 4 diet groups are shown in Table 1, One rat became ill and was discarded. Three battery clamps were modified to provide tail pinch of three intensities: 680 g, 1060 g, and 1480 g. The medium intensity was similar to that of the alligator clip used in the first two experiments, 1100 g. The different intensities were obtained by using different springs in the clamps. The forces were measured by determining the weights required to open the jaw 1/8 in. (3.2 mm). In all other respects the procedures were like those of Experiment 2, except that each rat was given three tests, one at each stimulus intensity, two days apart. The order of stimulus presentation was according to a Latin square design. Only food pellets were present, and they were weighed before and after each test. Resutts
Lick-gnaw ratios were influenced by the dietary treatments in the same way they had been in the previous experiments (Fig. 2). The effect of the preweaning diet was again significant, F(1,51)=89.5, p
-J -0.5 z ~ -1.0
I
i
Low
I
Medium STIMULUS INTENSITY
High
FIG. 2. Mean lick-gnaw ratios of four diet groups at three intensities of tail-pinch stimulation: 680 g, 1060g, and 1480g.
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than did rats in the other groups (Newman-Keuis comparisons, p<0.01). There were no other significant differences in latency, nor did the duration of the tail pinch-induced behavior show any effects of the dietary treatments. Repeated testing led to a progressive increase in the amount of behavior elicited by tail pinch as shown in Fig. 4. F(2,110)=21.3, p<0.001. Onset latencies diminished in a corresponding way, F(2,110)= 15.3, p <0.001, and were again influenced by the postweaning dietary treatment. F(1,55)=4.35, p<0.05. The diet effect seems marginal, however, and Newman-Keuis comparisons yielded no significant differences between the groups in any session, not even for the HH rats in the first session. Six rats de£mitely ate the lab chow pellets in this experiment. This was clear from their behavior and from the measurements of pellet weights. The weight differences for all of the tests on which rats gnawed the pellets are shown in Fig. 5 where measurements designated by hatohing are for the 10 trials in which the six rats ingested food. When eating, the rats held the pellets just below the chin, took small bites, spilling very little, and chewed the food in an unhurried way
MALNUTRITION AND TAIL-PINCH BEHAVIOR
c
had been malnourished before weaning, four LL rats and two LH rats. It is unlikely that the six were just by chance in the LL and LH groups and none were in the HH and HL groups, Xz (df= I) = 4.83, p<0.05. It seemed possible that ingestion was rare in the experiments described above because the rats had been reared on special mash diets. As a check on this possibility the behavior of rats reared on lab chow pellets in the Charles River Breeding Labs (Wilmington, Mass.) was examined under the same conditions used in the previous experiments.
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FIG. 4. Changes in the latency and amount of behavior (total duration) induced by tail pinch as a function of repeated testing of four diet groups. The three stimulus intensities that were used, counterbalanced in their order of presentation, were ignored in this summary.
50 40
Method
Eleven male Sprague-Dawley rats of the Charles River CD strain were purchased from the breeder as adults. Their mean weight at the beginning of the experiment was 275 g. Tail pinch was applied by means of the alligator clip used in Experiments 1 and 2. The test procedures were like those of the previous experiments except, of course, there was only one dietary treatment. The 11 rats of this experiment, like the HH rats in the earlier experiments, were wellnourished throughout their lives.
['7] INGESTION
Results
r'l
There was no ingestion of food in either test. Only one rat gnawed on the food, and there was no reduction in the weight of the food as a result of that gnawing. On only four other occasions during the two tests was there any gnawing, and in each case it involved feces. The mean lick-gnaw ratios for the group were +0.72 and +0.78 for the two tests. These results suggest that rearing on the special mash diets had not been responsible for the occurrence of so little eating in the previous experiments--or for the predominance of licking rather than gnawing in rats well-nourisbed early in life.
NO INGESTION
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GENERAL DISCUSSION .0
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PRETEST-POSTTEST DIFFERENCE IN FOOD WEIGHT (g). FIG. 5. Distribution of differences in food weights measured before and after tail-pinch tests in Experiment 3. Measurements are from all tests in which rats gnawed on pellets, which was not every test. Measures indicated by hatching are from rats that were seen to ingest the food.
before taking an additional bite. This behavior was quite different from the frenzied uninterrupted gnawing that was seen throughout most of this study, with near total crumbling of a pellet in a matter of seconds. Weight measurements for the unambiguous cases of eating form a distinct limb of the distribution in Fig. 5. It seems likely that nearly all, if not all of the other small differences, most less than 0.1 g, are no more than errors of measurement due mainly to difficulties in recovering all of the crumbled pellets and to contamination by fluids. The six rats that ingested food were all animals that
The experiments described above have established three things: (1) Under the stress induced by tail pinch rats that have been malnourished early in life respond differently than rats that have never known such deprivation; they are more likely to gnaw than to lick food and other objects, whereas well-nourished rats are likely to do just the opposite. (2) Neither the amount of tail pinch-induced behavior nor the rapidity of its onset was any greater in rats subjected to early protein malnutrition than in well-nourished ones. (3) Under some conditions eating is an infrequent response to tail pinch rather than a predominant one. We have no ready explanation for the diet-related differences in lick-gnaw ratios; but tail pinch-induced behavior is similar in many ways to behavior elicited by hypothalamic stimulation [1], which suggests several ways of thinking about the problem. Both kinds of behavior are influenced by the stimulus properties of objects present in the test situation. Sugar cubes in the present study stimulated more licking and gnawing than did lab chow pellets, but lick-gnaw ratios were not affected by food type. Moreover, the differences between diet groups were apparent in the licking and gnawing of not just the food, but other objects as well. All of this suggests that the response itself was important, not the object of the response. One possibility is that differences in lick-gnaw ratios reflect "natural" behavioral propensities in
154
PICONE AND H A L L
the way that hypothalamically elicited behavior does [9,15]. Our rats malnourished early in life may generally gnaw more and lick less than well-nourished rats, and we are currently studying ways to check this possibility. The prepotency of gnawing in malnourished rats is also suggested, albeit indirectly, by the present findings on tail pinch-induced eating--or the lack of it. The only rats that clearly ingested food in these experiments were six animals in Experiment 3 that had been malnourished as preweanlings. Four o f them in the L L group were malnourished at the time of testing. Our best guess at present is that eating acquires a prepotency as a result of early deprivation and that the gnawing which was so prevalent in the malnourished rats is a fractional component of such a prepotent response. This explanation says nothing, of course, about the predominance of licking in well-nourished rats. At another level o f analysis it is possible that the differences in lick-gnaw ratios reflect a difference in the functioning o f brain catecholamine systems. Tail pinch-induced behavior apparently depends on the dopaminergic nigrostriatai system [2,3] and is accompanied by an increased turnover of cortical norepinephrine [3]. It has been shown that catecholamine metabolism is altered in our malnourished animals, as indicated by their abnormally high levels o f brain norepinephrine [22]. It was also shown that these animals are less sensitive than well-nourished rats to the stereotypyinducing effects of apomorphine, a dopamine agonist [I1]. Whether the diet related differences in behavior elicited by tail pinch are in any way related to these changes in catecholamine functions remains to be seen. We were surprised to find so little ingestion because several investigators have reported that eating is the predominant response to tail pinch [2, 10, 18]. The measurement of food weights merely confirmed what seemed obvious to us from watching the animals; those that chewed the pellets did so not to eat them but, apparently, to derive whatever relief they could from the gnawing. It must also be remembered that nearly half of the animals, most of those that had been well-nourished as preweanlings, gnawed the pellets very little but licked them instead.
It seems unlikely that tail pinch induced so little eating because the lab chow pellets were novel or unpalatable to the rats reared on special mash diets. Sugar cubes were eaten no more than the pellets, and the rats raised on lab chow in Experiment 4 did not eat under the stress of tail pinch. Part of the answer may lie in the tail-pinch stimulus. In some informal experiments we were more successful in eliciting eating when tail pinch was applied with a 25-cm hemostat like that used by Antelman and his coworkers. It seemed, however, that the intensity of the stimulus (at around t h e first notch on the hemostat) was appreciably greater than the intensities used in the present experiments. All six rats that ingested food in Experiment 3 did so only in response to the most intense stimulus or to other stimuli only after they had experienced the most intense one. However, the data from these same 6, as discussed above, suggest that tail pinchinduced eating may also depend on nutritional status: all 6 had been malnourished as preweanlings. The question with which this study began seems to have been answered unambiguously. Rats malnourished early in life are no more responsive to tail pinch than well-nourished rats. There was some indication in Experiment 3 that rats fed the low protein diet at the time of testing responded with shorter latencies than rats on the high protein diet, but this effect has little bearing on the question of whether preweaning nutritional deprivation produces rats that are hyperresponsive later in life. It is conceivable that gnawing is indicative o f a stronger response to tail pinch than licking is, but the data do not support such a notion. Gnawing increased no more than licking did as lick-gnaw ratios underwent nonmonotonic changes with increases in stimulus strength, even though response duration and latency clearly indicated increasing response strength with increasing stimulus intensity. Whether the rats in our model of malnutrition are hyperresponsive to other kinds of aversive stimulation remains to be seen. It seems clear in any case that any hyperresponsiveness that might be interpreted as hyperemotionality in rats subjected to early malnutrition must be specific to certain kinds of stimuli or situations whose essential attributes are not yet understood.
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