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Illness-induced taste aversions in normal and bulbectomized hamsters
Physiology & Behavior, Vol. 17, pp. 235--238. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.
Illness-Induced Taste Aversions in Normal and Bulbectomized Hamsters STEPHEN H. HOBBS
Department o f Psychology, Augusta College, Augusta, Georgia 30904 HAROLD CLINGERMAN AND RALPH L. ELKINS
Veterans Administration Hospital, Augusta, Georgia 55555
(Received 27 May 1975) HOBBS, S. H., H. CLINGERMAN AND R. L. ELKINS. Illness-induced taste aversions in normal and bulbectomized hamsters. PHYSIOL. BEHAV. 17(1) 235-238, 1976. - Baitshyness acquisition and extinction in male Syrian golden hamsters was evaluated using saccharin solution as the target flavor and 75 or 150 mg/kg injections of cyclophosphamide as the illness-inducing agent. Conditioned aversions were obtained in drug-injected animals, but extinction was rapid and complete within twelve days of two-bottle preference testing. A second experiment using the same animals found that bilateral aspiration of the olfactory bulbs disrupted the subsequent acquisition of an aversion to milk. Baitshyness appears to have advantages over other tasks producing avoidance behavior in the hamster, and the hamster may be useful in neural investigations of conditioned taste aversions which have previously concentrated on the rat. Taste aversions
Bulbectomy
Hamsters
illness could artificially increase the final effective dose level. The present study was undertaken to further explore the consumatory avoidance behavior of hamsters which had not received previous exposure to the target flavor or illness-inducing drug.
A NUMBER of studies have previously examined the performance of hamsters on various avoidance tasks based upon electrical shock [3, 16, 23, 25, 28]. The avoidance efficiency of these rodents has generally reflected nonassociative factors such as operant levels of bar pressing, propensity to climb, or ability to transverse grid floors. These findings underscore the need to consider the nature of the avoidance behavior demanded by the experimental situation. Bolles [5] stressed this point when he proposed that species-specific defense reactions determine the ease with which various avoidance tasks are learned. In a broader perspective, Seligman [27] has suggested that some associations are more readily acquired simply because they have greater survival value in the natural environment. One type of learning for which a number of species appear to be prepared is the avoidance of a particular food which has been paired with internal malaise [17]. This learning, commonly referred to as taste aversion conditioning or baitshyness, has been extensively studied in the rat and may offer a more natural means by which to assess the acquisition of avoidance behavior in the hamster. Our initial study with the hamster [8] indicated that high dosages of a commonly used illness-inducing drug (cyclophosphamide) were required to produce either observable signs of illness or detectable flavor aversion. The data were somewhat ambiguous, however, since multiple injections of increasingly higher dosages were given the same animals until an effective dosage was obtained. Previous experiments with rats (e.g. [12,14]), suggest that successive familiarization with either the taste stimulus or with toxic
EXPERIMENT 1
Method Animals. The animals were 24 experimentally naive male Syrian golden hamsters (Mesocricetus auratus; ARS/SpragueDawley, Madison, Wisconsin) housed in individual cages and provided with paper nesting materials and free access to Purina Laboratory Chow. Animal maintenance, weighing, and behavioral testing occurred during the last 3 hours of the light portion of the cycle (lights on at 0600 hr, off at 1800 hr EST). Animals were randomly assigned to experimental and control conditions. Procedure. When the hamsters reached 90 days of age they were put on a restricted water-access schedule to assure drinking of the target fluid on the day of conditioning. Two bottles of room-temperature tap water were placed on the cage front for 20 min each day for 4 consecutive days. On the 5th day a single bottle of 0.10% solution (w/v) of sodium saccharin in tap water was presented to each animal for I hour. Five minutes after the onset of drinking, six hamsters were given a 150 mg/k_g intraperitoneal injection of cyclophosphamide (Cytoxan r~, Meade-Johnson, Evansville, Indiana). A lower dosage of 75 mg/kg was administered to an additional six animals. The 235
236 control groups consisted of six hamsters given injections of isotonic saline and six that received no injections. Groups of animals receiving drug injections in the absence of saccharin solution were not included since non-associative effects of cyclophosphamide on fluid intake had not been obtained in previous experiments [8]. Preferences were determined over the next 6 days using a two-bottle procedure pitting tap water against the saccharin solution. The animals were fluid deprived except during the 20-min daily testing period. This restricted-access schedule was selected to minimize spillage errors resulting from the high manipulative activity of hamsters. Nondeprived preference measures were made, however, in a final 6 days of testing in which hamsters were allowed continuous 24 hr access to the two fluids. Throughout two-bottle testing, the positions of the bottles on the cage front were alternated daily. Data analysis. Fluid intake measures were converted to saccharin preference scores reflecting the percentage of total daffy fluid accounted for by the saccharin solution. Experimental effects were analyzed with the Kruskal-Wallis ranked sums test and appropriate multiple comparisons where indicated [22]. Saline- and noninjected groups were combined for graphical and statistical purposes since they did not differ on postconditioning fluid or body-weight measures. Results The effectiveness of the drug in inducing illness was initially assessed through observation of the animals by one of the experimenters naive to group designations. Obvious signs of illness were noted in all six animals in the 150 mg/kg group. Three hamsters in the 75 mg/kg group and three in the saline-injected group were also classified as exhibiting some behavioral indications of illness. Most of the control animals returned to their nests, ate, and appeared to sleep within a few minutes after their drinking bout, which always lasted less than 5 min. Drug-injected animals typically did not return to the nest, but were observed scattering food and nest materials about the cage and attempting (unsuccessfully) to urinate. These animals were characterized by squinted or closed eyes, distended testicles, and hyperreactivity to a puff of air. Such symptoms had~ disappeared in most animals by the following day, although a few failed to completely open one or both eyes for several days. The 150 mg/kg group had the least amount of weight gain in the 2 weeks following conditioning, but a statistically significant difference in body weight among the groups was not obtained. During extinction testing, drug-injected animals showed more aversion to the saccharin-flavored water than controls. Some initial saccharin rejection was noted in several salineand noninjected animals which minimized the degree to which they differed from drug-injected animals. During the first two-bottle test period mean saccharin intakes for the control, 75 mg/kg, and 150 mg/kg groups were 2.1 ml, 1.3 ml, and 0.7 ml, respectively. This compares with mean water intakes for the three groups of 2.7 ml, 3.2 ml, and 2.9 ml, respectively. Converting these and subsequent days' data to saccharin preference scores (Fig. 1), significant overall group differences were obtained for each of the first 4 extinction days (H values ranging from 6.16 to 7.92; all ps<0.05). Individual comparisons revealed significant differences (p<0.05) during this period only between the
HOBBS, CLINGERMAN AND ELKINS
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FIG. l. Median group saccharin preference scores over the 12 days of extinction testing. control and 150 mg/kg groups. While total fluid intake for all groups predictably increased during the 6 days of nondeprived testing, preference scores continued to show an orderly rate of extinction. By the tenth day of testing all hamsters clearly demonstrated preference for the saccharin solution. EXPERIMENT
2
While the first experiment confirmed that hamsters, like rats, are capable of developing taste aversions, it is unclear as to whether the underlying processes of the phenomenon are the same in the two rodents. Brain lesion procedures, particularly of the olfactory and limbic systems, have recently been employed to investigate the neural mechanisms associated with baitshyness development [ 17]. While some contradictory lesion effects have been obtained, bilateral ablation of the olfactory bulbs of rats has consistently resulted in at least partial disruption of conditioned taste aversions (e.g. [ 10,20] ). In his recent review, Cain [7] noted differences in the behavioral effects of olfactory bulbectomy among several rodent species. Experiment 2 therefore was conducted as an initial inquiry into the role of olfactory and limbic structures in baitshyness in a species other than the rat. It was our intent to aspirate the anterior two-thirds of the olfactory bulbs since this restricted lesion has been found effective in producing baitshyness deficits in rats [ 15] and since this would minimize the likelihood of incidental damage to the frontal cortex. Method Animals. Hamsters from the previous experiment were used. Five of these animals were subsequently lost to surgical death or illness. Assignment of animals to surgical and drug conditions was made randomly with the restriction that previously conditioned animals be equally distributed among the groups. Seven weeks elapsed between the end of Experiment 1 and surgery for Experiment 2, during which time the animals were maintained as before with free access to food, water, and nesting materials.
ILLNESS-INDUCED TASTE AVERSIONS Procedure. Nine hamsters receiving olfactory bulb ablations were anesthetized with Nembutal (90 mg/kg) and positioned in a stereotaxic instrument. A 3 mm hole 9¢as drilled in the skull to allow aspiration of the olfactory bulbs through a glass pipette. The resulting cavity was lightly packed with gelfoam soaked in Zephiran solution. Sham surigcal procedures were identical except that no neural tissue was aspirated. The control group consisted of six hamsters receiving the sham procedure and four which remained unoperated. After a 3 week recovery period, the animals were introduced, as in the first experiment, to a restricted drinking schedule. For 4 consecutive days, two bottles, each containing tap water, were placed on the cage front for 20 min per day. On the 5th day access was given for 1 hour to a single bottle containing Carnation Instant Nonfat Dry Milk in tap water (8.33% w/v). Five minutes after the onset of drinking, five hamsters from the control group and five from the bulbectomized group were given an intraperitoneal injection of cyclophosphamide (150 mg/kg) and returned to the home cage. Remaining animals were each injected with isotonic saline. In order to additionally separate conditioning and testing exposure to the milk, two bottles containing tap water were available during the drinking period on the following day. Fluid preferences were assessed over the next 2 drinking periods using two-bottle tests, one bottle containing tap water and another the milk solution. The positions of the milk and water bottles were reversed for the second testing period. When testing was completed the animals were sacrificed with an overdose of Nembutal and perfused with saline and Formalin. The brains were then removed for determination of olfactory destruction or incidental neural damage.
237 TABLE 1 MEAN, MEDIAN, AND RANGE OF MILK PREFERENCE SCORES AVERAGEDACROSSTHE 2 DAYSOF TESTING Group
Mean
Median
Range
CONT-SALINE (n = 5) CONT-DRUG (n = 5) BULB-SALINE (n = 4) BULB-DRUG (n = 5)
76.4 24.2 72.0 75.0
90.0 25.0 74.5 88.0
42-95 18-30 52-88 50-92
phosphamide than are commonly required for the rat. As reported with the rat [4], there was considerable variability in symptomatic severity of drug-induced illness, but no within-group correlation between these observations and subsequent aversion strength. Surprisingly, the aversions to saccharin which did develop, even with the higher dose level, were not particularly strong and extinguished very rapidly relative to aversions observed with rats (cf., [13]). It appeared (although no direct measures were made) that the hamsters sampled the saccharin more often and perhaps sooner after illness than rats given similar training. Thus the fluid may have been additionally paired with recovery from illness, a procedure which has been shown in rats to result in flavor enhancement (e.g. [19]). Delaying the reintroduction of the conditioned stimulus and/or comparisons of rats and hamsters using continuous drinkometer recordings would seem appropriate in exploring this further.
Results Aspiration of the olfactory bulbs in hamsters was found to clearly disrupt the acquisition of a flavor aversion to milk (Table 1). As in the first experiment, a dosage of 150 mg/kg of cyclophosphamide was effective in producing baitshyness in the control animals. There was no overlap in the preference scores between the bulbectomized and control animals injected with cyclophosphamide, obviating the necessity of statistical comparisons. Average daily milk and water intakes, respectively, for the four groups were: control-saline, 4.1 and 1.2 ml; control-drug, 1.2 and 3.7 ml; bulbectomized-saline, 4.4 and 1.3 ml; bulbectomized-drug, 2.5 and 0.7 ml. Thus, while olfactory bulb removal alone had no measurable effect on fluid intake or percentage preference for the milk solution, the additional trauma of drug-induced illness may have reduced overall drinking. Hamsters in the present study received bilateral destruction of the anterior bulbar layers as illustrated in Fig. 2. One animal (in the saline-injected group) sustained complete bulb destruction. Other lesions were at or slightly larger than the minimum-size lesion depicted in Fig. 2, presumably sparing the anterior olfactory nucleus and leaving all or part of the accessory olfactory bulb intact. Damage to the frontal poles was not observed. DISCUSSION The present experiments confirmed our earlier observation [8] that conditioned taste aversions can be induced in hamsters, although with much higher dosages of cyclo-
FIG. 2. Schematic representation of the minimum extent of lesion damage (diagonal lines) and maximum extent of lesion damage (diagonal lines plus solid area). Davison, Corwin, and McGowan [9] have recently been more successful in obtaining strong conditioned aversions in hamsters. Their experimental animals were given multiple injections of ethanol (1.17 mg/kg or 1.76 mg/kg) following 20 min of saccharin-solution access. Subsequent two-bottle preference measures revealed that the group receiving the higher drug dosage had developed strong aversions to the saccharin solution which did not completely extinguish in the 25 days of testing. Still another report of taste aversion conditioning using hamsters came to our attention following the acceptance of this manuscript for publication. In a series of interesting studies, Zahorik and Johnston [29] found clear aversions to several flavors, including vaginal secretions, following lithium chloride poisoning. It may be important that, in contrast with the present procedures, Zahorik and Johnston conducted all experimentation during the dark portion of the light-dark cycle. Since hamsters have been shown to be strongly nocturnal [2, 6, 21], time of conditioning and/or testing may be significant factors in demonstrating baitshyness in these rodents. Together these several studies suggest that taste-aversion conditioning can be readily obtained in the hamster, at least under some
238
HOBBS, C L I N G E R M A N A N D E L K I N S
experimental paradigms, and that it m a y be of value in future investigations of avoidance behavior in this species. It would appear, however, that the relative i m p o r t a n c e of using different training procedures and conditioning agents in producing baitshyness in the hamster is in need of additional evaluation. Other authors have demonstrated that prior toxicosis may influence acceptance of subsequently presented novel or distinctive flavors [ 11,24]. While we found n e o p h o b i a to the saccharin solution in some u n c o n d i t i o n e d animals in E x p e r i m e n t 1, little evidence o f similar phobic responding to the milk solution in E x p e r i m e n t 2 was observed. F u r t h e r m o r e , no interactions b e t w e e n previous conditioning history and preference scores in E x p e r i m e n t 2 could be detected, although it is quite possible that such relationships may have been observed had this been specifically investigated using larger groups of animals. The present study did determine that the rostral portions of the o l f a c t o r y bulbs are involved in the acquisition of conditioned taste aversions in hamsters as in rats [ 1 5 ] . Previous experiments using rats have suggested that the disruptive effect of b u l b e c t o m y on baitshyness is not related to the sensory f u n c t i o n of the olfactory bulbs. For instance, Hankins e t al. [20] have shown that
b u l b e c t o m y , but not peripheral anosmia, disrupts baitshyness acquisition. It may be i m p o r t a n t to c o n d u c t a similar comparison with hamsters since peripherally-induced anosmia (relative to b u l b e c t o m y ) has been reported to have a greater effect on social behaviors in the hamster than in the rat (see Cain [7] for a review). Such differences are suggestive of significant species variations in the c o m p l e x olfactory organ system [1]. While the comparative neuroanatomical connections of the rat and hamster are not well known, similar degenerative patterns following olfactory bulb lesions have been reported [26]. A l t h o u g h hamsters in the present investigation suffered damage largely restricted to the rostral portions of the olfactory bulb, they showed several of the behavioral changes characteristic of hamsters receiving m o r e extensive bulbar damage [ 1 8 ] . In particular, we found lesioned animals to be more reactive to handling than controls and to have more poorly constructed nests and food piles. It is tempting to speculate on the basis of these observations that such behavioral changes are i n d e p e n d e n t of insult to the accessory o l f a c t o r y nucleus which may have been largely spared by the a n t e r i o r p l a c e m e n t of our lesions. Such a conclusion must await additional study using histological analysis of tissue damage.
REFERENCES 1. Alberts, J. R. Producing and interpreting experimental olfactory deficits. Physiol. Behav. 12: 657-670, 1974. 2. Aschoff, J. and K. Honma. Art-und Individual Muster der Tagesperiodik. Z. vergl. Physiol. 42: 383-392, 1959. 3. Babbini, M. and W. M. Davis. Active avoidance learning in hamsters. Psychonom. Sci. 9: 149-150, 1967. 4. Barker, L. M., E. M. Suarez and D. Gray. Backward conditioning of taste aversions in rats using cyclophosphamide as the US. Physiol. Psychol. 2: 117-119, 1974. 5. Bolles, R. C. Species-specific defense reactions and avoidance learning. Psychol. Rev. 77: 32-48, 1970. 6. Borer, K. T., J. B. Powers, S. S. Winans and E. S. Valenstein. Influence of olfactory bulb removal on ingestive behaviors, activity levels, and self-stimulation in hamsters. J. comp. physiol. Psychol. 86: 396-403, 1974. 7. Cain, D. P. The role of the olfactory bulb in limbic mechanisms. Psychol. Bull. 81: 654-671, 1974. 8. Clingerman, H., and S. H. Hobbs. Taste aversion in hamsters: Role o f dosage, age, and sex. Paper presented at the meeting of the Georgia Psychological Association, Savannah, 1974. 9. Davison, C. S., G. Corwin and T. McGowan. Alcohol-induced taste aversion in the golden hamster. J. Stud. Alcohol 37: 606-610, 1976. Atlanta, 1975. 10. Dinc, H. I., and J. C. Smith. Role of the olfactory bulbs in the detection of ionizing radiation by the rat. Physiol. Behav. 1: 139-144, 1966. 11. Domjan, M. Poison-induced neophobia in the rats: Role of stimulus generalization of conditioned taste aversions. Anim. Learn. Behav. 3: 205-211, 1975. 12. Elkins, R. L. Attenuation of drug-induced bait shyness to a palatable solution as an increasing function of its availability prior to conditioning. Behav. Biol. 9 : 2 2 1 - 2 2 6 , 1973. 13. Elkins, R. L. Individual differences in bait shyness: Effects of drug dose and measurement technique. Psychol. Rec. 23: 349-358, 1973. 14. Elkins, R. L. Bait-shyness acquisition and resistance to extinction as functions of US exposure prior to conditioning. Physiol. Psychol. 2: 341-343, 1974.
15. Elkins, R. L., J. Fraser, and S. H. Hobbs. Dissociation o f shock-motivated avoidance and drug-induced bait shyness following selective olfactory system lesions. Paper presented at the meeting of the Southeastern Psychological Association, Hollywood, Florida, 1974. 16. Ewing, D. R. Avoidance conditioning in two varieties o f the Syrian hamster. Unpublished master's thesis, Iowa State University, 1964. 17. Garcia, J., W. G. Hankins, and K. W. Rusiniak. Behavioral regulation of the milieu interne in man and rat. Science 185: 824-831, 1974. 18. Goodman, E. D., and M. I. Firestone. Olfactory bulb lesions: Nest reinforcement and handling reactivity in hamsters. Physiol. Behav. 10: 1-8, 1973. 19. Green, K. F., and J. Garcia. Recuperation from illness: Flavor enhancement for rats. Science 173:749-751,1971. 20. Hankins, W. G., J. Garcia, and K. W. Rusiniak. Dissociation of odor and taste in bait shyness. Behav. Biol. 8:407 -419, 1973. 21. Hobbs, S. H., C. R. Miller, B. N. Bunnell, and L. J. Peacock. General activity in hamsters with septal lesions. Physiol. Behav. 9: 349-352, 1972. 22. Hollander, M., and D. A. Wolfe. Nonparametric Statistical Methods. New York: John Wiley & Sons, 1973. 23. Pearl, J. Avoidance learning in rodents: A comparative study. Psychol. Rep. 12: 139-145, 1963. 24. Rozin, P. Specific aversions and neophobia resulting from vitamin deficiency or poisoning in half-wild and domestic rats. J. comp. physiol. Psychol. 66: 82-88, 1968. 25. Sandier, B. E., and G. G. Karas. Acquisition of a jumping avoidance response in hamsters. Psychon. Sci. 10: 191-192, 1968. 26. Scott, J. W. and C. M. Leonard. The olfactory connections of the lateral hypothalamus in the rat, mouse and hamster. J. comp. Neurol. 141: 331-344, 1971. 27. Seligman, M. E. P. On the generality of the laws of learning. Psychol. Rev. 77: 406-418, 1970. 28. Williams, R. D. A comparative study o f golden hamsters and albino rats in an avoidance conditioning situation. Unpublished master's thesis, Iowa State University, 1963. 29. Zahorik, D. M., and R. E. Johnston. Taste aversions to food flavors and vaginal secretion in golden hamsters. 3". comp. physiol. Psychol. 90: 57-66, 1976.
Illness-Induced Taste Aversions in Normal and Bulbectomized Hamsters STEPHEN H. HOBBS
Department o f Psychology, Augusta College, Augusta, Georgia 30904 HAROLD CLINGERMAN AND RALPH L. ELKINS
Veterans Administration Hospital, Augusta, Georgia 55555
(Received 27 May 1975) HOBBS, S. H., H. CLINGERMAN AND R. L. ELKINS. Illness-induced taste aversions in normal and bulbectomized hamsters. PHYSIOL. BEHAV. 17(1) 235-238, 1976. - Baitshyness acquisition and extinction in male Syrian golden hamsters was evaluated using saccharin solution as the target flavor and 75 or 150 mg/kg injections of cyclophosphamide as the illness-inducing agent. Conditioned aversions were obtained in drug-injected animals, but extinction was rapid and complete within twelve days of two-bottle preference testing. A second experiment using the same animals found that bilateral aspiration of the olfactory bulbs disrupted the subsequent acquisition of an aversion to milk. Baitshyness appears to have advantages over other tasks producing avoidance behavior in the hamster, and the hamster may be useful in neural investigations of conditioned taste aversions which have previously concentrated on the rat. Taste aversions
Bulbectomy
Hamsters
illness could artificially increase the final effective dose level. The present study was undertaken to further explore the consumatory avoidance behavior of hamsters which had not received previous exposure to the target flavor or illness-inducing drug.
A NUMBER of studies have previously examined the performance of hamsters on various avoidance tasks based upon electrical shock [3, 16, 23, 25, 28]. The avoidance efficiency of these rodents has generally reflected nonassociative factors such as operant levels of bar pressing, propensity to climb, or ability to transverse grid floors. These findings underscore the need to consider the nature of the avoidance behavior demanded by the experimental situation. Bolles [5] stressed this point when he proposed that species-specific defense reactions determine the ease with which various avoidance tasks are learned. In a broader perspective, Seligman [27] has suggested that some associations are more readily acquired simply because they have greater survival value in the natural environment. One type of learning for which a number of species appear to be prepared is the avoidance of a particular food which has been paired with internal malaise [17]. This learning, commonly referred to as taste aversion conditioning or baitshyness, has been extensively studied in the rat and may offer a more natural means by which to assess the acquisition of avoidance behavior in the hamster. Our initial study with the hamster [8] indicated that high dosages of a commonly used illness-inducing drug (cyclophosphamide) were required to produce either observable signs of illness or detectable flavor aversion. The data were somewhat ambiguous, however, since multiple injections of increasingly higher dosages were given the same animals until an effective dosage was obtained. Previous experiments with rats (e.g. [12,14]), suggest that successive familiarization with either the taste stimulus or with toxic
EXPERIMENT 1
Method Animals. The animals were 24 experimentally naive male Syrian golden hamsters (Mesocricetus auratus; ARS/SpragueDawley, Madison, Wisconsin) housed in individual cages and provided with paper nesting materials and free access to Purina Laboratory Chow. Animal maintenance, weighing, and behavioral testing occurred during the last 3 hours of the light portion of the cycle (lights on at 0600 hr, off at 1800 hr EST). Animals were randomly assigned to experimental and control conditions. Procedure. When the hamsters reached 90 days of age they were put on a restricted water-access schedule to assure drinking of the target fluid on the day of conditioning. Two bottles of room-temperature tap water were placed on the cage front for 20 min each day for 4 consecutive days. On the 5th day a single bottle of 0.10% solution (w/v) of sodium saccharin in tap water was presented to each animal for I hour. Five minutes after the onset of drinking, six hamsters were given a 150 mg/k_g intraperitoneal injection of cyclophosphamide (Cytoxan r~, Meade-Johnson, Evansville, Indiana). A lower dosage of 75 mg/kg was administered to an additional six animals. The 235
236 control groups consisted of six hamsters given injections of isotonic saline and six that received no injections. Groups of animals receiving drug injections in the absence of saccharin solution were not included since non-associative effects of cyclophosphamide on fluid intake had not been obtained in previous experiments [8]. Preferences were determined over the next 6 days using a two-bottle procedure pitting tap water against the saccharin solution. The animals were fluid deprived except during the 20-min daily testing period. This restricted-access schedule was selected to minimize spillage errors resulting from the high manipulative activity of hamsters. Nondeprived preference measures were made, however, in a final 6 days of testing in which hamsters were allowed continuous 24 hr access to the two fluids. Throughout two-bottle testing, the positions of the bottles on the cage front were alternated daily. Data analysis. Fluid intake measures were converted to saccharin preference scores reflecting the percentage of total daffy fluid accounted for by the saccharin solution. Experimental effects were analyzed with the Kruskal-Wallis ranked sums test and appropriate multiple comparisons where indicated [22]. Saline- and noninjected groups were combined for graphical and statistical purposes since they did not differ on postconditioning fluid or body-weight measures. Results The effectiveness of the drug in inducing illness was initially assessed through observation of the animals by one of the experimenters naive to group designations. Obvious signs of illness were noted in all six animals in the 150 mg/kg group. Three hamsters in the 75 mg/kg group and three in the saline-injected group were also classified as exhibiting some behavioral indications of illness. Most of the control animals returned to their nests, ate, and appeared to sleep within a few minutes after their drinking bout, which always lasted less than 5 min. Drug-injected animals typically did not return to the nest, but were observed scattering food and nest materials about the cage and attempting (unsuccessfully) to urinate. These animals were characterized by squinted or closed eyes, distended testicles, and hyperreactivity to a puff of air. Such symptoms had~ disappeared in most animals by the following day, although a few failed to completely open one or both eyes for several days. The 150 mg/kg group had the least amount of weight gain in the 2 weeks following conditioning, but a statistically significant difference in body weight among the groups was not obtained. During extinction testing, drug-injected animals showed more aversion to the saccharin-flavored water than controls. Some initial saccharin rejection was noted in several salineand noninjected animals which minimized the degree to which they differed from drug-injected animals. During the first two-bottle test period mean saccharin intakes for the control, 75 mg/kg, and 150 mg/kg groups were 2.1 ml, 1.3 ml, and 0.7 ml, respectively. This compares with mean water intakes for the three groups of 2.7 ml, 3.2 ml, and 2.9 ml, respectively. Converting these and subsequent days' data to saccharin preference scores (Fig. 1), significant overall group differences were obtained for each of the first 4 extinction days (H values ranging from 6.16 to 7.92; all ps<0.05). Individual comparisons revealed significant differences (p<0.05) during this period only between the
HOBBS, CLINGERMAN AND ELKINS
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FIG. l. Median group saccharin preference scores over the 12 days of extinction testing. control and 150 mg/kg groups. While total fluid intake for all groups predictably increased during the 6 days of nondeprived testing, preference scores continued to show an orderly rate of extinction. By the tenth day of testing all hamsters clearly demonstrated preference for the saccharin solution. EXPERIMENT
2
While the first experiment confirmed that hamsters, like rats, are capable of developing taste aversions, it is unclear as to whether the underlying processes of the phenomenon are the same in the two rodents. Brain lesion procedures, particularly of the olfactory and limbic systems, have recently been employed to investigate the neural mechanisms associated with baitshyness development [ 17]. While some contradictory lesion effects have been obtained, bilateral ablation of the olfactory bulbs of rats has consistently resulted in at least partial disruption of conditioned taste aversions (e.g. [ 10,20] ). In his recent review, Cain [7] noted differences in the behavioral effects of olfactory bulbectomy among several rodent species. Experiment 2 therefore was conducted as an initial inquiry into the role of olfactory and limbic structures in baitshyness in a species other than the rat. It was our intent to aspirate the anterior two-thirds of the olfactory bulbs since this restricted lesion has been found effective in producing baitshyness deficits in rats [ 15] and since this would minimize the likelihood of incidental damage to the frontal cortex. Method Animals. Hamsters from the previous experiment were used. Five of these animals were subsequently lost to surgical death or illness. Assignment of animals to surgical and drug conditions was made randomly with the restriction that previously conditioned animals be equally distributed among the groups. Seven weeks elapsed between the end of Experiment 1 and surgery for Experiment 2, during which time the animals were maintained as before with free access to food, water, and nesting materials.
ILLNESS-INDUCED TASTE AVERSIONS Procedure. Nine hamsters receiving olfactory bulb ablations were anesthetized with Nembutal (90 mg/kg) and positioned in a stereotaxic instrument. A 3 mm hole 9¢as drilled in the skull to allow aspiration of the olfactory bulbs through a glass pipette. The resulting cavity was lightly packed with gelfoam soaked in Zephiran solution. Sham surigcal procedures were identical except that no neural tissue was aspirated. The control group consisted of six hamsters receiving the sham procedure and four which remained unoperated. After a 3 week recovery period, the animals were introduced, as in the first experiment, to a restricted drinking schedule. For 4 consecutive days, two bottles, each containing tap water, were placed on the cage front for 20 min per day. On the 5th day access was given for 1 hour to a single bottle containing Carnation Instant Nonfat Dry Milk in tap water (8.33% w/v). Five minutes after the onset of drinking, five hamsters from the control group and five from the bulbectomized group were given an intraperitoneal injection of cyclophosphamide (150 mg/kg) and returned to the home cage. Remaining animals were each injected with isotonic saline. In order to additionally separate conditioning and testing exposure to the milk, two bottles containing tap water were available during the drinking period on the following day. Fluid preferences were assessed over the next 2 drinking periods using two-bottle tests, one bottle containing tap water and another the milk solution. The positions of the milk and water bottles were reversed for the second testing period. When testing was completed the animals were sacrificed with an overdose of Nembutal and perfused with saline and Formalin. The brains were then removed for determination of olfactory destruction or incidental neural damage.
237 TABLE 1 MEAN, MEDIAN, AND RANGE OF MILK PREFERENCE SCORES AVERAGEDACROSSTHE 2 DAYSOF TESTING Group
Mean
Median
Range
CONT-SALINE (n = 5) CONT-DRUG (n = 5) BULB-SALINE (n = 4) BULB-DRUG (n = 5)
76.4 24.2 72.0 75.0
90.0 25.0 74.5 88.0
42-95 18-30 52-88 50-92
phosphamide than are commonly required for the rat. As reported with the rat [4], there was considerable variability in symptomatic severity of drug-induced illness, but no within-group correlation between these observations and subsequent aversion strength. Surprisingly, the aversions to saccharin which did develop, even with the higher dose level, were not particularly strong and extinguished very rapidly relative to aversions observed with rats (cf., [13]). It appeared (although no direct measures were made) that the hamsters sampled the saccharin more often and perhaps sooner after illness than rats given similar training. Thus the fluid may have been additionally paired with recovery from illness, a procedure which has been shown in rats to result in flavor enhancement (e.g. [19]). Delaying the reintroduction of the conditioned stimulus and/or comparisons of rats and hamsters using continuous drinkometer recordings would seem appropriate in exploring this further.
Results Aspiration of the olfactory bulbs in hamsters was found to clearly disrupt the acquisition of a flavor aversion to milk (Table 1). As in the first experiment, a dosage of 150 mg/kg of cyclophosphamide was effective in producing baitshyness in the control animals. There was no overlap in the preference scores between the bulbectomized and control animals injected with cyclophosphamide, obviating the necessity of statistical comparisons. Average daily milk and water intakes, respectively, for the four groups were: control-saline, 4.1 and 1.2 ml; control-drug, 1.2 and 3.7 ml; bulbectomized-saline, 4.4 and 1.3 ml; bulbectomized-drug, 2.5 and 0.7 ml. Thus, while olfactory bulb removal alone had no measurable effect on fluid intake or percentage preference for the milk solution, the additional trauma of drug-induced illness may have reduced overall drinking. Hamsters in the present study received bilateral destruction of the anterior bulbar layers as illustrated in Fig. 2. One animal (in the saline-injected group) sustained complete bulb destruction. Other lesions were at or slightly larger than the minimum-size lesion depicted in Fig. 2, presumably sparing the anterior olfactory nucleus and leaving all or part of the accessory olfactory bulb intact. Damage to the frontal poles was not observed. DISCUSSION The present experiments confirmed our earlier observation [8] that conditioned taste aversions can be induced in hamsters, although with much higher dosages of cyclo-
FIG. 2. Schematic representation of the minimum extent of lesion damage (diagonal lines) and maximum extent of lesion damage (diagonal lines plus solid area). Davison, Corwin, and McGowan [9] have recently been more successful in obtaining strong conditioned aversions in hamsters. Their experimental animals were given multiple injections of ethanol (1.17 mg/kg or 1.76 mg/kg) following 20 min of saccharin-solution access. Subsequent two-bottle preference measures revealed that the group receiving the higher drug dosage had developed strong aversions to the saccharin solution which did not completely extinguish in the 25 days of testing. Still another report of taste aversion conditioning using hamsters came to our attention following the acceptance of this manuscript for publication. In a series of interesting studies, Zahorik and Johnston [29] found clear aversions to several flavors, including vaginal secretions, following lithium chloride poisoning. It may be important that, in contrast with the present procedures, Zahorik and Johnston conducted all experimentation during the dark portion of the light-dark cycle. Since hamsters have been shown to be strongly nocturnal [2, 6, 21], time of conditioning and/or testing may be significant factors in demonstrating baitshyness in these rodents. Together these several studies suggest that taste-aversion conditioning can be readily obtained in the hamster, at least under some
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experimental paradigms, and that it m a y be of value in future investigations of avoidance behavior in this species. It would appear, however, that the relative i m p o r t a n c e of using different training procedures and conditioning agents in producing baitshyness in the hamster is in need of additional evaluation. Other authors have demonstrated that prior toxicosis may influence acceptance of subsequently presented novel or distinctive flavors [ 11,24]. While we found n e o p h o b i a to the saccharin solution in some u n c o n d i t i o n e d animals in E x p e r i m e n t 1, little evidence o f similar phobic responding to the milk solution in E x p e r i m e n t 2 was observed. F u r t h e r m o r e , no interactions b e t w e e n previous conditioning history and preference scores in E x p e r i m e n t 2 could be detected, although it is quite possible that such relationships may have been observed had this been specifically investigated using larger groups of animals. The present study did determine that the rostral portions of the o l f a c t o r y bulbs are involved in the acquisition of conditioned taste aversions in hamsters as in rats [ 1 5 ] . Previous experiments using rats have suggested that the disruptive effect of b u l b e c t o m y on baitshyness is not related to the sensory f u n c t i o n of the olfactory bulbs. For instance, Hankins e t al. [20] have shown that
b u l b e c t o m y , but not peripheral anosmia, disrupts baitshyness acquisition. It may be i m p o r t a n t to c o n d u c t a similar comparison with hamsters since peripherally-induced anosmia (relative to b u l b e c t o m y ) has been reported to have a greater effect on social behaviors in the hamster than in the rat (see Cain [7] for a review). Such differences are suggestive of significant species variations in the c o m p l e x olfactory organ system [1]. While the comparative neuroanatomical connections of the rat and hamster are not well known, similar degenerative patterns following olfactory bulb lesions have been reported [26]. A l t h o u g h hamsters in the present investigation suffered damage largely restricted to the rostral portions of the olfactory bulb, they showed several of the behavioral changes characteristic of hamsters receiving m o r e extensive bulbar damage [ 1 8 ] . In particular, we found lesioned animals to be more reactive to handling than controls and to have more poorly constructed nests and food piles. It is tempting to speculate on the basis of these observations that such behavioral changes are i n d e p e n d e n t of insult to the accessory o l f a c t o r y nucleus which may have been largely spared by the a n t e r i o r p l a c e m e n t of our lesions. Such a conclusion must await additional study using histological analysis of tissue damage.
REFERENCES 1. Alberts, J. R. Producing and interpreting experimental olfactory deficits. Physiol. Behav. 12: 657-670, 1974. 2. Aschoff, J. and K. Honma. Art-und Individual Muster der Tagesperiodik. Z. vergl. Physiol. 42: 383-392, 1959. 3. Babbini, M. and W. M. Davis. Active avoidance learning in hamsters. Psychonom. Sci. 9: 149-150, 1967. 4. Barker, L. M., E. M. Suarez and D. Gray. Backward conditioning of taste aversions in rats using cyclophosphamide as the US. Physiol. Psychol. 2: 117-119, 1974. 5. Bolles, R. C. Species-specific defense reactions and avoidance learning. Psychol. Rev. 77: 32-48, 1970. 6. Borer, K. T., J. B. Powers, S. S. Winans and E. S. Valenstein. Influence of olfactory bulb removal on ingestive behaviors, activity levels, and self-stimulation in hamsters. J. comp. physiol. Psychol. 86: 396-403, 1974. 7. Cain, D. P. The role of the olfactory bulb in limbic mechanisms. Psychol. Bull. 81: 654-671, 1974. 8. Clingerman, H., and S. H. Hobbs. Taste aversion in hamsters: Role o f dosage, age, and sex. Paper presented at the meeting of the Georgia Psychological Association, Savannah, 1974. 9. Davison, C. S., G. Corwin and T. McGowan. Alcohol-induced taste aversion in the golden hamster. J. Stud. Alcohol 37: 606-610, 1976. Atlanta, 1975. 10. Dinc, H. I., and J. C. Smith. Role of the olfactory bulbs in the detection of ionizing radiation by the rat. Physiol. Behav. 1: 139-144, 1966. 11. Domjan, M. Poison-induced neophobia in the rats: Role of stimulus generalization of conditioned taste aversions. Anim. Learn. Behav. 3: 205-211, 1975. 12. Elkins, R. L. Attenuation of drug-induced bait shyness to a palatable solution as an increasing function of its availability prior to conditioning. Behav. Biol. 9 : 2 2 1 - 2 2 6 , 1973. 13. Elkins, R. L. Individual differences in bait shyness: Effects of drug dose and measurement technique. Psychol. Rec. 23: 349-358, 1973. 14. Elkins, R. L. Bait-shyness acquisition and resistance to extinction as functions of US exposure prior to conditioning. Physiol. Psychol. 2: 341-343, 1974.
15. Elkins, R. L., J. Fraser, and S. H. Hobbs. Dissociation o f shock-motivated avoidance and drug-induced bait shyness following selective olfactory system lesions. Paper presented at the meeting of the Southeastern Psychological Association, Hollywood, Florida, 1974. 16. Ewing, D. R. Avoidance conditioning in two varieties o f the Syrian hamster. Unpublished master's thesis, Iowa State University, 1964. 17. Garcia, J., W. G. Hankins, and K. W. Rusiniak. Behavioral regulation of the milieu interne in man and rat. Science 185: 824-831, 1974. 18. Goodman, E. D., and M. I. Firestone. Olfactory bulb lesions: Nest reinforcement and handling reactivity in hamsters. Physiol. Behav. 10: 1-8, 1973. 19. Green, K. F., and J. Garcia. Recuperation from illness: Flavor enhancement for rats. Science 173:749-751,1971. 20. Hankins, W. G., J. Garcia, and K. W. Rusiniak. Dissociation of odor and taste in bait shyness. Behav. Biol. 8:407 -419, 1973. 21. Hobbs, S. H., C. R. Miller, B. N. Bunnell, and L. J. Peacock. General activity in hamsters with septal lesions. Physiol. Behav. 9: 349-352, 1972. 22. Hollander, M., and D. A. Wolfe. Nonparametric Statistical Methods. New York: John Wiley & Sons, 1973. 23. Pearl, J. Avoidance learning in rodents: A comparative study. Psychol. Rep. 12: 139-145, 1963. 24. Rozin, P. Specific aversions and neophobia resulting from vitamin deficiency or poisoning in half-wild and domestic rats. J. comp. physiol. Psychol. 66: 82-88, 1968. 25. Sandier, B. E., and G. G. Karas. Acquisition of a jumping avoidance response in hamsters. Psychon. Sci. 10: 191-192, 1968. 26. Scott, J. W. and C. M. Leonard. The olfactory connections of the lateral hypothalamus in the rat, mouse and hamster. J. comp. Neurol. 141: 331-344, 1971. 27. Seligman, M. E. P. On the generality of the laws of learning. Psychol. Rev. 77: 406-418, 1970. 28. Williams, R. D. A comparative study o f golden hamsters and albino rats in an avoidance conditioning situation. Unpublished master's thesis, Iowa State University, 1963. 29. Zahorik, D. M., and R. E. Johnston. Taste aversions to food flavors and vaginal secretion in golden hamsters. 3". comp. physiol. Psychol. 90: 57-66, 1976.