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Unimpaired maintenance of a conditioned avoidance response in the rat with diabetes insipidus
Physiology & Behavior, Vol. 15, pp. 707--711. Pergamon Press and Brain Research Pubi., 1975. Printed in the U.S.A.
Unimpaired Maintenance of a Conditioned Avoidance Response in the Rat With Diabetes Insipidus JOHN F. CELESTIAN, ROBERT J. CA REY AND MYRON MILLER
Departments o f Medicine and Psychology, Veterans Administration Hospital and State University o f N e w York, Upstate Medical Center, Syracuse, New York (Received 27 January 1975) CELESTIAN, J. F., R. J. CAREY AND M. MILLER. Unimpaired maintenance ofa conditioned avoidance response in the rat with diabetes insipidus. PHYSIOL. BEHAV. 15(6) 707-711, 1975. - The ability to maintain a conditioned avoidance response (CAR) was studied in normal Long-Evans rats and rats of the Brattleboro strain which were either homozygous or heterozygous for hereditary hypothalamic diabetes insipidus (DI). Homozygous DI rats had a lower CAR acquisition rate than did normal or heterozygous DI rats. However, the homozygous DI rats exhibited significantly greater CAR retention than did the other animals over the total period of extinction testing. The greater CAR retention could not be accounted for by either increased sensitivity to the electric foot shock used as the unconditioned stimulus or by perching on the metal center barrier of the training shuttle box, a form of behavior unique to the homozygous DI rats. Since the homozygous DI rat is totally lacking in hypothalamic antidiuretic hormone (ADH), the greater CAR retention of these animals indicates that ADH is not a requirement for CAR retention. Conditioned avoidance response (CAR)
Diabetes insipidus
THE physiological role of vasopressin (ADH) in the maintenance o f water balance is well known. A number of recent studies have shown that this hormone also may be implicated in learning and memory processes [ 1, 9, 12, 13 ]. DeWied [8] found normal acquisition but faster extinction of a shuttle box avoidance response in rats subjected to posterior pituitary lobectomy. Rats subjected to hypophysectomy differed in that after training, they had a decreased ability to maintain a shuttle box avoidance response without reinforcement and were also unable to acquire a conditioned avoidance response ( C A R ) w i t h o u t hormone replacement therapy [2, 3, 6, 8, 13]. The injection of vasopressin tannate, an extract of the posterior pituitary lobe, restored performance of hypophysectomized rats to control levels [6,13]. The actions of vasopressin tannate and lysine vasopressin were also apparent in intact animals since these agents were shown to enhance retention of a conditioned avoidance response in normal rats [ 4 - 1 0 , 14,
Extinction
Retention
Vasopressin (ADH)
EXPERIMENT 1 The first experiment sought to determine if there was a difference in CAR performance between normal rats and ADH deficient rats of the Brattlebroo strain. METHOD
Animals All rats were bred and raised in our animal colony and were between 1 0 0 - 1 5 0 days old at the time of testing. The animals were housed in metabolic cages, and allowed access to food and water ad lib. Before experimentation, urine osmolality of each rat was determined using an Advanced Instruments Co. freezing point depression osmometer. DI rats with complete ADH deficiency had urine osmolalities below 400 m0sm/kg, while heterozygous rats ranged between 1200 and 1800 m0sm/kg, and urine osmolahty in normal rats was above 2300 m0sm/kg. In the first experiment, 5 naive rats homozygous for DI and 5 naive normal Long-Evans rats were used.
151. In the light of this evidence implicating ADH in CAR performance, the present study was undertaken to evaluate the CAR performance of the Brattleboro strain of LongEvans rats which have diabetes insipidus (DI) due to a specific hereditary deficiency of ADH [16]. Since these rats are ADH deficient, they provide a useful model to further study the influence of ADH on learning and retention.
Apparatus A two compartment automated shuttle box (Lehigh Valley, LVE No. 3770) was used. The floor of each of the two compartments contained a grid, consisting of parallel bars 2 mm in diameter spaced 1.1 cm apart. The compart707
708
CELESTIAN, CAREY AND MILLER
ments were separated by a 2 in. high metal barrier. The entire shuttle box was enclosed within a attenuating chamber, LVE 1488, ventilated by an electric fan. Rats were viewed through a one way mirror. The source of foot shock was a LVE 1531 constant current shocker with scrambler.
performance within 100 trials (one other DI rat reached the criterion of performance but developed inner ear disease and thus had to be dropped). Two rats from each group failed to reach the criterion of performance within the 100 trials. A marked difference in extinction of the conditioned avoidance response was found between the two groups of rats. As seen in Fig. I, the two homozygous DI rats maintained a high CAR performance thoughout extinction (100 percent at 504 hours), while the performance of the 3 normal rats gradually declined over the extinction sessions (only 15 percent CAR's at 504 hours).
Procedure Before the start of experimentation, all rats were handled for 5 min on each of 3 consecutive days. On the first and subsequent days of CAR testing the rats were allowed to adapt to the shuttle box for a 5 min period before the conditioning commenced. During this 5 min pretest adaptation period, measurements of each rats spontaneous crossing rate were compiled. The conditioned stimulus (CS) was a light of 42.4 DC watts, situated in the upper corner of the sound attenuating chamber and was presented for 5 sec. If the rat did not cross the barrier within this 5 sec CS period, a 1.0 mA electric footshock (unconditioned stimulus, (UCS) was delivered through the appropriate grid floor until the rat crossed the barrier, or for a 10 sec period if it failed to cross the barrier. Twenty trials were given per day with variable inter-trial intervals from 25 to 80 sec duration separating trials in a predetermined sequence in order to reduce the likelihood of temporal conditioning. The daily training trials continued until rats reached the criterion of 80 percent or more avoidance responses for at least 3 consecutive sessions. Furthermore, those rats not reaching the criterion of performance within 100 trials were dropped from the experiment. Twenty-four hours after reaching the criterion avoidance level, the rats were given 20 extinction (No Shock) trials. During extinction sessions the rat again had to cross the barrier within the 5 sec CS period to score an avoidance, but was not shocked if it failed to score an avoidance. In addition, 8 subsequent extinction sessions were run 2, 4, 24, 48, 72, 168, 336, and 504 hr later. Inter-trial crossings during each session were measured. Rats were also observed through a one way mirror.
EXPERIMENT 2 In view of the unexpected greater CAR retention exhibited by the DI rats in experiment one, Experiment 2 sought to substantiate this finding by utilizing a greater number of rats. METHOD
Animals In Experiment two, 3 naive rat populations were tested: 11 normal rats, 14 rats heterozygous for DI and 35 rats homozygous for DI. Heterozygous rats have vasopressin levels midway between those of normal and homozygous DI rats [16].
Apparatus The rats were run in the same shuttle box apparatus used in Experiment 1.
Procedure The CAR test procedure was identical to Experiment 1, except that the criterion of performance was reduced to one session of 80 percent or more avoidances to better conform to the one used by earlier experimenters [ 17]. RESULTS
RESULTS
With the greater number of animals being used, a large percentage of animals was detected within the homozygous DI rat population which failed to reach criterion. Avoid-
Three normal rats reached the criterion of performance within 70 trials, while two DI rats reached the criterion of
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FIG. 1. Individual extinction scores following acquisition of a conditioned avoidance response in normal rats as compared to those of homozygous DI rats.
CAR IN THE RAT WITH DIABETES INSIPIDUS
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FIG. 2. Mean extinction scores over 168 hours of homozygous DI rats as compared to heterozygous DI rats and normal rats.
ance responses in the homozygous DI rats increased from a mean of only 1.8 at Session 1 to 8.2 on Session 5. The normal rats started at Session 1 with a mean of 5.0 avoidances, and reached a mean of 14.0 avoidances on Session 5. The heterozygous DI rats increased from a mean of 10.3 to 18.3 avoidances from Session 1 to Session 5. CAR acquisition results showed that only 30 percent (11) of the DI rats reached criterion compared with 78 percent (11) for the heterozygous rats and 64 percent (7) for normals. Since extinction testing involved only rats which reached the 80 percent criterion of performance~ all groups had the same level of CAR's on completion of acquisition training: homozygous DI rats 90 percent, normal rats 87 percent, and heterozygous DI rats 86 percent CAR's. Significant differences between the homozygous DI rat population and the normal and heterozygous rat populations were seen from the second extinction session throughout the remaining extinction sessions (Fig. 2). On Session 2, the homozygous DI rats maintained 80 percent CAR's while the heterozygous DI rats maintained only 12 percent CAR's, and the normal rats 12 percent CAR's. The homozygous DI rats continued at a higher level of CAR retention than the normal and heterozygous DI rats throughout the remaining extinction sessions. These differences among groups were highly significant statistically, the two way analysis of variance yielded F values of F(2,12) = 14.61, p<0.01, for the groups effects, F(6,72) = 20.34, p<0.01, for the trials effect, and F(12,72) = 2.91, p<0.01, for the groups and trials effect.
METHOD
Animals Twenty-nine naive rats were used: 9 normal, 9 heterozygous for DI and 11 homozygous for DI. The homozygous DI rats were divided into 2 groups, as were the normal and heterozygous DI rats.
Apparatus All apparatus was the same as in Experiment 1, except some studies were performed without the 2 in. metal center barrier.
Procedure Eleven homozygous DI rats were studied under 2 conditions: (1) with the two inch metal center barrier removed, (2) with the 2 in. metal center barrier intact. Normal and heterozygous DI rats also were studied with the 2 in. barrier removed and intact. Test procedure was identical to Experiment 2. RESULTS
In agreement with Experiment 2, the homozygous DI rats exhibited the lowest n u m b e r of avoidance responses by Day 5 of the acquisition sessions (Fig. 3). No significant improvement in the acquisition of a conditioned avoidance was found in any of the 3 groups of rats as a consequence of removing the 2 in. metal center barrier.
EXPERIMENT 3
EXPERIMENT 4
Observations of the homozygous DI rats which failed to reach the criterion of performance during acquisition revealed that of the nonlearners 60 percent perched on the 2 in. metal center barrier to avoid shock for the duration of the experimental sessions. Experiment 3 sought to determine if this behavior could account for the poorer CAR acquisition rate exhibited by the homozygous DI rats.
Experiment 2 showed that the homozygous DI rats differed in two important aspects from the normal and heterozygous DI rats. First homozygous DI rats are less likely to acquire a CAR, but secondly if the response is acquired it is more resistant to extinction. Experiment 4 was undertaken to ascertain whether the homozygous DI rats are more sensitive to shock since a greater sensitivity to
710
CELESTIAN, CAREY AND MILLER
Procedure
20
Each rat was placed in the testing apparatus and given 5 rain to adapt to the box. The rats were given 4 consecutive sets of unavoidable electric shocks. The shocks were presented in alternating increasing and decreasing series, ranging from 0.1 to 1.0 m A in increments of 0.1 m A for 0.2 sac duration. T w o i n d e p e n d e n t raters recorded either a nonresponse (0), a crouch-flinch-jerk (10) or a j u m p - r u n response (20).
18
16
RESULTS
No significant difference in sensitivity to shock was seen a m o n g the 3 populations of rats, with all 3 groups uniformly exhibiting a jump-run response when the shock level e x c e e d e d 0.4 mA (Fig. 4). An over all rating mean score o f 18.1 was observed for the normal rats, with scores o f 17.6 for the heterozygous DI rats, and 17.6 for the h o m o z y g o u s DI rats.
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FIG. 4. Mean score for the sets of unavoidable electric foot shocks in normal, heterozygous DI and homozygous DI rats.
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FIG. 3. Acquisition of a conditioned avoidance response in homozygous DI rats, heterozygous DI rats and normal rats with the two inch metal center barrier intact (BI) and removed (BO). shock might account for the different performance of these rats. METHOD
Animals Eighteen naive rats were used: 6 normal, 6 heterozygous for DI, and 6 h o m o z y g o u s for DI.
Apparatus The apparatus used was an Evans Flinch J u m p Box, which consisted of a 8 × 14 in. box with a metal grid floor and clear plastic walls and top [ 11 ].
In these studies, rats with c o m p l e t e deficiency of A D H were found to differ in two ways in C A R performance from rats with either normal a m o u n t s of h y p o t h a l a m i c ADH or with partial A D H deficiency. First, the normal and heterozygous DI rat showed a greater increase in avoidance acquisition during most stages of learning, in contrast to the h o m o z y g o u s DI rat which did not achieve the high levels reached by the normal and heterozygous DI rats. Second, h o m o z y g o u s DI rats which did reach C A R criterion retained significantly more conditioned avoidance responses than did their counterparts which had endogenous hypothalamic ADH. If we compare the extinction rate of h o m o z y g o u s DI rats with that of normal rats, then the lack of vasopressin appears to retard the rate of extinction of a conditioned avoidance response, which would seem to show A D H as a negative factor in CAR retention. This result is c o n t r a d i c t o r y to previous findings which have shown that A D H deficiency produced by h y p o p h y s e c t o m y or posterior pituitary l o b e c t o m y increases the rate of extinction of an avoidance response [ 6]. This dramatic difference in findings in A D H deficient rats may be the result of various factors. (1) The present study differs from previous experiments in that no surgery is involved with the associated trauma to the h y p o t h a l a m u s which could be a factor in previous observations [6]. (2) Many previous experiments used Pitressin which contains other substances in addition to ADH [7]. (3) When LVP,
C A R IN T H E R A T W I T H D I A B E T E S I N S I P I D U S
711
Pitressin o r vasopressin analogs were used, t h e a m o u n t s were s u p r a p h y s i o l o g i c a l [6, 7, 10, 1 8 ] . (4) D i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s were used w i t h d i f f e r e n t s e l e c t i o n factors. T h u s , in s o m e e x p e r i m e n t s De Wied used pole j u m p i n g c o n d i t i o n i n g , a n d selected o n l y rats w h i c h m a d e 80 p e r c e n t or m o r e a v o i d a n c e s in the first e x t i n c t i o n session t o c o n t i n u e in t h e e x p e r i m e n t [ 9 ] . O n e or all o f t h e s e factors c o u l d play a p a r t in t h e d i f f e r e n c e s f o u n d b e t w e e n o u r results a n d t h o s e f o u n d b y o t h e r e x p e r i m e n t ers. In a d d i t i o n , it is possible t h a t t h e b e h a v i o r a l d i f f e r e n c e s b e t w e e n rat p o p u l a t i o n s in t h e p r e s e n t s t u d y are due t o genetic d i f f e r e n c e s in t h e rat strains. T h e p h e n o m e n o n o f b a r r i e r climbing, f o u n d in t h e h o m o z y g o u s DI rat p o p u l a t i o n , suggests t h e p r e s e n c e o f d i f f e r e n t c h a n n e l s o f learning b e h a v i o r in these animals, since b a r r i e r c l i m b i n g was n o t o b s e r v e d in e i t h e r o f t h e o t h e r rat p o p u l a t i o n s . A n o t h e r d i f f e r e n c e in b e h a v i o r o b s e r v e d in the h o m o z y g o u s DI rats was t h a t w i t h t h e b a r r i e r r e m o v e d , in m o s t cases t h e rats e x h i b i t e d p r e p a r a t o r y c r o u c h i n g w h e n the c o n d i t i o n e d s t i m u l u s was p r e s e n t e d a n d t h e n leaped just as t h e electric s h o c k was p r e s e n t e d . Thus, while this b e h a v i o r was n o t
successful in avoiding f o o t s h o c k e x p o s u r e , the s h o c k d u r a t i o n was m i n i m i z e d . Again, this b e h a v i o r was n o t e x h i b i t e d b y t h e n o r m a l rats a n d did reveal t h a t t h e h o m o z y g o u s DI rats s h o w e d a d a p t a t i o n t o the C A R p r o c e d u r e , b u t an a d a p t a t i o n w h i c h was n o t generally r e f l e c t e d in t h e scoring p r o c e d u r e used. It will be of i n t e r e s t t o f u r t h e r s t u d y t h e processes b y w h i c h t h e h o m o z y g o u s DI rat learns c o m p a r e d to n o r m a l rats. It is a p p a r e n t t h a t l o n g t e r m r e t e n t i o n in t h e h o m o z y gous DI rat does n o t require vasopressin since t h e absence o f this p e p t i d e appears t o facilitate r e t e n t i o n o f learned responses. It can be c o n c l u d e d t h a t A D H is n o t a r e q u i r e m e n t for C A R r e t e n t i o n since rats genetically lacking A D H r e t a i n C A R b e t t e r t h a n rats w i t h e i t h e r n o r m a l a m o u n t s o f h y p o t h a l a m i c A D H or w i t h partial A D H deficiency. ACKNOWLEDGEMENTS The authors wish to thank Mr. A. Albert for his many hours of assistance and suggestions regarding this work. The expert technical assistance of Mr. G. Procopio is gratefully acknowledged.
REFERENCES 1. Ader, R. and D. DeWied. Effects of lysine vasopressin on passive avoidance learning. Psychon. Sci. 29: 4 6 - 4 8 , 1972. 2. Applezweig, M. H. and F. D. Baundry. The pituitary-adrenocortical system in avoidance learning. Psychol. Rep. 1: 4 1 7 - 4 2 0 , 1955. 3. Applezwieg, M. H. and G. Moeller. The pituitary-adrenocortical system and anxiety in avoidance learning. Acta Psychol. (Amst.) 15: 6 0 2 - 6 0 3 , 1959. 4. Beatty, B. A., W. W. Beatty, R. E. Bowman and J. C. Gilchrist. The effects of ACTH, adrenalectomy and dexamethasone on the acquisition of an avoidance response in rats. Physiol. Behav. 5: 939-944, 1970. 5. DeWied, D. The influence of the anterior pituitary on avoidance learning and escape behavior. J. Physiol. 207: 255-259, 1964. 6. DeWied, D. The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response in rats. Int. J. Neuropharmac. 4: 157-167, 1965. 7. DeWied, D. Long term and short term effects on retention of a conditioned avoidance response in rats by treatment with long acting pitressin and MSH. Nature, Lond. 212: 1484-1486, 1966. 8. DeWied, D. Effects of peptide hormones on behavior. In: Frontiers in Neuroendocrinology, edited by L. Martini and W. F. Ganong. London: Oxford University Press, 1969, pp. 97-140. 9. DeWied, D. Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature, Lond. 232: 5 8 - 6 0 , 1971.
10. DeWied, D., A. M. L. Van Delft, W. H. Gispen, J. A. W. M. Weijnen and Tj. B. van Wimersma Greidanus. The role of Pituitary-adrenal system hormones in active avoidances conditioning. In: Hormones and Behavior, edited by S. Levine. New York: Academic Press, 1972, pp. 135-171. 11. Evans, W. O. A new technique for the investigation of some analgesic drugs on a reflexive behavior in the rat. Psychopharmacologia 2: 318-325, 1961. 12. Lande, S., J. B. Flexner and L. B. Flexner. Effect of corticortropin and desglycinamide-lysine vasopressin on suppression of memory by puromycin. Proc. natn. Acad. Sci. U.S.A. 69: 558-560, 1972. 13. Lande, S., A. Witter and D. DeWied. An octapeptide that stimulates conditioned avoidance acquisition in hypophysectomized rats. J. biol. Chem. 246: 2058-2062, 1971. 14. Levine, S. and F. R. Brush. Adrenocortical activity and avoidance learning as a function of time after avoidance training. Physiol. Behav. 2: 385-388, 1967. 15. Levine, S. and L. E. Jones. ACTH and passive avoidance learning. J. comp. physiol. Psychol. 59: 357-360, 1960. 16. Miller, M. and A. M. Moses. Radioimmunoassay of urinary antidiuretic hormone with application to study of the Brattleboro rat. Endocrinology 88: 1389-1396, 1971. 17. Murphy, J. V. and R. E. Miller. The Effect of Adrenocorticotrophic Hormone (ACTH) on Avoidance Conditioning in the Rat. J. comp. Psychol. 48: 4 7 - 4 9 , 1955. 18. Wang, Su-Sun. Synthesis of desglycinamide lysine vasopressin and its behavioral activity in rats. Biochem. biophys. Res. Commun. 48: 1511-1515, 1972.
Unimpaired Maintenance of a Conditioned Avoidance Response in the Rat With Diabetes Insipidus JOHN F. CELESTIAN, ROBERT J. CA REY AND MYRON MILLER
Departments o f Medicine and Psychology, Veterans Administration Hospital and State University o f N e w York, Upstate Medical Center, Syracuse, New York (Received 27 January 1975) CELESTIAN, J. F., R. J. CAREY AND M. MILLER. Unimpaired maintenance ofa conditioned avoidance response in the rat with diabetes insipidus. PHYSIOL. BEHAV. 15(6) 707-711, 1975. - The ability to maintain a conditioned avoidance response (CAR) was studied in normal Long-Evans rats and rats of the Brattleboro strain which were either homozygous or heterozygous for hereditary hypothalamic diabetes insipidus (DI). Homozygous DI rats had a lower CAR acquisition rate than did normal or heterozygous DI rats. However, the homozygous DI rats exhibited significantly greater CAR retention than did the other animals over the total period of extinction testing. The greater CAR retention could not be accounted for by either increased sensitivity to the electric foot shock used as the unconditioned stimulus or by perching on the metal center barrier of the training shuttle box, a form of behavior unique to the homozygous DI rats. Since the homozygous DI rat is totally lacking in hypothalamic antidiuretic hormone (ADH), the greater CAR retention of these animals indicates that ADH is not a requirement for CAR retention. Conditioned avoidance response (CAR)
Diabetes insipidus
THE physiological role of vasopressin (ADH) in the maintenance o f water balance is well known. A number of recent studies have shown that this hormone also may be implicated in learning and memory processes [ 1, 9, 12, 13 ]. DeWied [8] found normal acquisition but faster extinction of a shuttle box avoidance response in rats subjected to posterior pituitary lobectomy. Rats subjected to hypophysectomy differed in that after training, they had a decreased ability to maintain a shuttle box avoidance response without reinforcement and were also unable to acquire a conditioned avoidance response ( C A R ) w i t h o u t hormone replacement therapy [2, 3, 6, 8, 13]. The injection of vasopressin tannate, an extract of the posterior pituitary lobe, restored performance of hypophysectomized rats to control levels [6,13]. The actions of vasopressin tannate and lysine vasopressin were also apparent in intact animals since these agents were shown to enhance retention of a conditioned avoidance response in normal rats [ 4 - 1 0 , 14,
Extinction
Retention
Vasopressin (ADH)
EXPERIMENT 1 The first experiment sought to determine if there was a difference in CAR performance between normal rats and ADH deficient rats of the Brattlebroo strain. METHOD
Animals All rats were bred and raised in our animal colony and were between 1 0 0 - 1 5 0 days old at the time of testing. The animals were housed in metabolic cages, and allowed access to food and water ad lib. Before experimentation, urine osmolality of each rat was determined using an Advanced Instruments Co. freezing point depression osmometer. DI rats with complete ADH deficiency had urine osmolalities below 400 m0sm/kg, while heterozygous rats ranged between 1200 and 1800 m0sm/kg, and urine osmolahty in normal rats was above 2300 m0sm/kg. In the first experiment, 5 naive rats homozygous for DI and 5 naive normal Long-Evans rats were used.
151. In the light of this evidence implicating ADH in CAR performance, the present study was undertaken to evaluate the CAR performance of the Brattleboro strain of LongEvans rats which have diabetes insipidus (DI) due to a specific hereditary deficiency of ADH [16]. Since these rats are ADH deficient, they provide a useful model to further study the influence of ADH on learning and retention.
Apparatus A two compartment automated shuttle box (Lehigh Valley, LVE No. 3770) was used. The floor of each of the two compartments contained a grid, consisting of parallel bars 2 mm in diameter spaced 1.1 cm apart. The compart707
708
CELESTIAN, CAREY AND MILLER
ments were separated by a 2 in. high metal barrier. The entire shuttle box was enclosed within a attenuating chamber, LVE 1488, ventilated by an electric fan. Rats were viewed through a one way mirror. The source of foot shock was a LVE 1531 constant current shocker with scrambler.
performance within 100 trials (one other DI rat reached the criterion of performance but developed inner ear disease and thus had to be dropped). Two rats from each group failed to reach the criterion of performance within the 100 trials. A marked difference in extinction of the conditioned avoidance response was found between the two groups of rats. As seen in Fig. I, the two homozygous DI rats maintained a high CAR performance thoughout extinction (100 percent at 504 hours), while the performance of the 3 normal rats gradually declined over the extinction sessions (only 15 percent CAR's at 504 hours).
Procedure Before the start of experimentation, all rats were handled for 5 min on each of 3 consecutive days. On the first and subsequent days of CAR testing the rats were allowed to adapt to the shuttle box for a 5 min period before the conditioning commenced. During this 5 min pretest adaptation period, measurements of each rats spontaneous crossing rate were compiled. The conditioned stimulus (CS) was a light of 42.4 DC watts, situated in the upper corner of the sound attenuating chamber and was presented for 5 sec. If the rat did not cross the barrier within this 5 sec CS period, a 1.0 mA electric footshock (unconditioned stimulus, (UCS) was delivered through the appropriate grid floor until the rat crossed the barrier, or for a 10 sec period if it failed to cross the barrier. Twenty trials were given per day with variable inter-trial intervals from 25 to 80 sec duration separating trials in a predetermined sequence in order to reduce the likelihood of temporal conditioning. The daily training trials continued until rats reached the criterion of 80 percent or more avoidance responses for at least 3 consecutive sessions. Furthermore, those rats not reaching the criterion of performance within 100 trials were dropped from the experiment. Twenty-four hours after reaching the criterion avoidance level, the rats were given 20 extinction (No Shock) trials. During extinction sessions the rat again had to cross the barrier within the 5 sec CS period to score an avoidance, but was not shocked if it failed to score an avoidance. In addition, 8 subsequent extinction sessions were run 2, 4, 24, 48, 72, 168, 336, and 504 hr later. Inter-trial crossings during each session were measured. Rats were also observed through a one way mirror.
EXPERIMENT 2 In view of the unexpected greater CAR retention exhibited by the DI rats in experiment one, Experiment 2 sought to substantiate this finding by utilizing a greater number of rats. METHOD
Animals In Experiment two, 3 naive rat populations were tested: 11 normal rats, 14 rats heterozygous for DI and 35 rats homozygous for DI. Heterozygous rats have vasopressin levels midway between those of normal and homozygous DI rats [16].
Apparatus The rats were run in the same shuttle box apparatus used in Experiment 1.
Procedure The CAR test procedure was identical to Experiment 1, except that the criterion of performance was reduced to one session of 80 percent or more avoidances to better conform to the one used by earlier experimenters [ 17]. RESULTS
RESULTS
With the greater number of animals being used, a large percentage of animals was detected within the homozygous DI rat population which failed to reach criterion. Avoid-
Three normal rats reached the criterion of performance within 70 trials, while two DI rats reached the criterion of
20~
t4 ~
I0 B
~4 2 I I I
I
I
I
I
0 4
24
4B
72
168 Hours
m& = DI
of
/¢(
./.7
336
504
Extinction
[] AO= N o r m a l
FIG. 1. Individual extinction scores following acquisition of a conditioned avoidance response in normal rats as compared to those of homozygous DI rats.
CAR IN THE RAT WITH DIABETES INSIPIDUS
709
18
16
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~" f2 oo
DI
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Normal ,
I ~,'t I t
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214
48
72 Hours
168 of
Extinction
Day of S h o c k T r a i n i n g
FIG. 2. Mean extinction scores over 168 hours of homozygous DI rats as compared to heterozygous DI rats and normal rats.
ance responses in the homozygous DI rats increased from a mean of only 1.8 at Session 1 to 8.2 on Session 5. The normal rats started at Session 1 with a mean of 5.0 avoidances, and reached a mean of 14.0 avoidances on Session 5. The heterozygous DI rats increased from a mean of 10.3 to 18.3 avoidances from Session 1 to Session 5. CAR acquisition results showed that only 30 percent (11) of the DI rats reached criterion compared with 78 percent (11) for the heterozygous rats and 64 percent (7) for normals. Since extinction testing involved only rats which reached the 80 percent criterion of performance~ all groups had the same level of CAR's on completion of acquisition training: homozygous DI rats 90 percent, normal rats 87 percent, and heterozygous DI rats 86 percent CAR's. Significant differences between the homozygous DI rat population and the normal and heterozygous rat populations were seen from the second extinction session throughout the remaining extinction sessions (Fig. 2). On Session 2, the homozygous DI rats maintained 80 percent CAR's while the heterozygous DI rats maintained only 12 percent CAR's, and the normal rats 12 percent CAR's. The homozygous DI rats continued at a higher level of CAR retention than the normal and heterozygous DI rats throughout the remaining extinction sessions. These differences among groups were highly significant statistically, the two way analysis of variance yielded F values of F(2,12) = 14.61, p<0.01, for the groups effects, F(6,72) = 20.34, p<0.01, for the trials effect, and F(12,72) = 2.91, p<0.01, for the groups and trials effect.
METHOD
Animals Twenty-nine naive rats were used: 9 normal, 9 heterozygous for DI and 11 homozygous for DI. The homozygous DI rats were divided into 2 groups, as were the normal and heterozygous DI rats.
Apparatus All apparatus was the same as in Experiment 1, except some studies were performed without the 2 in. metal center barrier.
Procedure Eleven homozygous DI rats were studied under 2 conditions: (1) with the two inch metal center barrier removed, (2) with the 2 in. metal center barrier intact. Normal and heterozygous DI rats also were studied with the 2 in. barrier removed and intact. Test procedure was identical to Experiment 2. RESULTS
In agreement with Experiment 2, the homozygous DI rats exhibited the lowest n u m b e r of avoidance responses by Day 5 of the acquisition sessions (Fig. 3). No significant improvement in the acquisition of a conditioned avoidance was found in any of the 3 groups of rats as a consequence of removing the 2 in. metal center barrier.
EXPERIMENT 3
EXPERIMENT 4
Observations of the homozygous DI rats which failed to reach the criterion of performance during acquisition revealed that of the nonlearners 60 percent perched on the 2 in. metal center barrier to avoid shock for the duration of the experimental sessions. Experiment 3 sought to determine if this behavior could account for the poorer CAR acquisition rate exhibited by the homozygous DI rats.
Experiment 2 showed that the homozygous DI rats differed in two important aspects from the normal and heterozygous DI rats. First homozygous DI rats are less likely to acquire a CAR, but secondly if the response is acquired it is more resistant to extinction. Experiment 4 was undertaken to ascertain whether the homozygous DI rats are more sensitive to shock since a greater sensitivity to
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CELESTIAN, CAREY AND MILLER
Procedure
20
Each rat was placed in the testing apparatus and given 5 rain to adapt to the box. The rats were given 4 consecutive sets of unavoidable electric shocks. The shocks were presented in alternating increasing and decreasing series, ranging from 0.1 to 1.0 m A in increments of 0.1 m A for 0.2 sac duration. T w o i n d e p e n d e n t raters recorded either a nonresponse (0), a crouch-flinch-jerk (10) or a j u m p - r u n response (20).
18
16
RESULTS
No significant difference in sensitivity to shock was seen a m o n g the 3 populations of rats, with all 3 groups uniformly exhibiting a jump-run response when the shock level e x c e e d e d 0.4 mA (Fig. 4). An over all rating mean score o f 18.1 was observed for the normal rats, with scores o f 17.6 for the heterozygous DI rats, and 17.6 for the h o m o z y g o u s DI rats.
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DISCUSSION
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FIG. 3. Acquisition of a conditioned avoidance response in homozygous DI rats, heterozygous DI rats and normal rats with the two inch metal center barrier intact (BI) and removed (BO). shock might account for the different performance of these rats. METHOD
Animals Eighteen naive rats were used: 6 normal, 6 heterozygous for DI, and 6 h o m o z y g o u s for DI.
Apparatus The apparatus used was an Evans Flinch J u m p Box, which consisted of a 8 × 14 in. box with a metal grid floor and clear plastic walls and top [ 11 ].
In these studies, rats with c o m p l e t e deficiency of A D H were found to differ in two ways in C A R performance from rats with either normal a m o u n t s of h y p o t h a l a m i c ADH or with partial A D H deficiency. First, the normal and heterozygous DI rat showed a greater increase in avoidance acquisition during most stages of learning, in contrast to the h o m o z y g o u s DI rat which did not achieve the high levels reached by the normal and heterozygous DI rats. Second, h o m o z y g o u s DI rats which did reach C A R criterion retained significantly more conditioned avoidance responses than did their counterparts which had endogenous hypothalamic ADH. If we compare the extinction rate of h o m o z y g o u s DI rats with that of normal rats, then the lack of vasopressin appears to retard the rate of extinction of a conditioned avoidance response, which would seem to show A D H as a negative factor in CAR retention. This result is c o n t r a d i c t o r y to previous findings which have shown that A D H deficiency produced by h y p o p h y s e c t o m y or posterior pituitary l o b e c t o m y increases the rate of extinction of an avoidance response [ 6]. This dramatic difference in findings in A D H deficient rats may be the result of various factors. (1) The present study differs from previous experiments in that no surgery is involved with the associated trauma to the h y p o t h a l a m u s which could be a factor in previous observations [6]. (2) Many previous experiments used Pitressin which contains other substances in addition to ADH [7]. (3) When LVP,
C A R IN T H E R A T W I T H D I A B E T E S I N S I P I D U S
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Pitressin o r vasopressin analogs were used, t h e a m o u n t s were s u p r a p h y s i o l o g i c a l [6, 7, 10, 1 8 ] . (4) D i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s were used w i t h d i f f e r e n t s e l e c t i o n factors. T h u s , in s o m e e x p e r i m e n t s De Wied used pole j u m p i n g c o n d i t i o n i n g , a n d selected o n l y rats w h i c h m a d e 80 p e r c e n t or m o r e a v o i d a n c e s in the first e x t i n c t i o n session t o c o n t i n u e in t h e e x p e r i m e n t [ 9 ] . O n e or all o f t h e s e factors c o u l d play a p a r t in t h e d i f f e r e n c e s f o u n d b e t w e e n o u r results a n d t h o s e f o u n d b y o t h e r e x p e r i m e n t ers. In a d d i t i o n , it is possible t h a t t h e b e h a v i o r a l d i f f e r e n c e s b e t w e e n rat p o p u l a t i o n s in t h e p r e s e n t s t u d y are due t o genetic d i f f e r e n c e s in t h e rat strains. T h e p h e n o m e n o n o f b a r r i e r climbing, f o u n d in t h e h o m o z y g o u s DI rat p o p u l a t i o n , suggests t h e p r e s e n c e o f d i f f e r e n t c h a n n e l s o f learning b e h a v i o r in these animals, since b a r r i e r c l i m b i n g was n o t o b s e r v e d in e i t h e r o f t h e o t h e r rat p o p u l a t i o n s . A n o t h e r d i f f e r e n c e in b e h a v i o r o b s e r v e d in the h o m o z y g o u s DI rats was t h a t w i t h t h e b a r r i e r r e m o v e d , in m o s t cases t h e rats e x h i b i t e d p r e p a r a t o r y c r o u c h i n g w h e n the c o n d i t i o n e d s t i m u l u s was p r e s e n t e d a n d t h e n leaped just as t h e electric s h o c k was p r e s e n t e d . Thus, while this b e h a v i o r was n o t
successful in avoiding f o o t s h o c k e x p o s u r e , the s h o c k d u r a t i o n was m i n i m i z e d . Again, this b e h a v i o r was n o t e x h i b i t e d b y t h e n o r m a l rats a n d did reveal t h a t t h e h o m o z y g o u s DI rats s h o w e d a d a p t a t i o n t o the C A R p r o c e d u r e , b u t an a d a p t a t i o n w h i c h was n o t generally r e f l e c t e d in t h e scoring p r o c e d u r e used. It will be of i n t e r e s t t o f u r t h e r s t u d y t h e processes b y w h i c h t h e h o m o z y g o u s DI rat learns c o m p a r e d to n o r m a l rats. It is a p p a r e n t t h a t l o n g t e r m r e t e n t i o n in t h e h o m o z y gous DI rat does n o t require vasopressin since t h e absence o f this p e p t i d e appears t o facilitate r e t e n t i o n o f learned responses. It can be c o n c l u d e d t h a t A D H is n o t a r e q u i r e m e n t for C A R r e t e n t i o n since rats genetically lacking A D H r e t a i n C A R b e t t e r t h a n rats w i t h e i t h e r n o r m a l a m o u n t s o f h y p o t h a l a m i c A D H or w i t h partial A D H deficiency. ACKNOWLEDGEMENTS The authors wish to thank Mr. A. Albert for his many hours of assistance and suggestions regarding this work. The expert technical assistance of Mr. G. Procopio is gratefully acknowledged.
REFERENCES 1. Ader, R. and D. DeWied. Effects of lysine vasopressin on passive avoidance learning. Psychon. Sci. 29: 4 6 - 4 8 , 1972. 2. Applezweig, M. H. and F. D. Baundry. The pituitary-adrenocortical system in avoidance learning. Psychol. Rep. 1: 4 1 7 - 4 2 0 , 1955. 3. Applezwieg, M. H. and G. Moeller. The pituitary-adrenocortical system and anxiety in avoidance learning. Acta Psychol. (Amst.) 15: 6 0 2 - 6 0 3 , 1959. 4. Beatty, B. A., W. W. Beatty, R. E. Bowman and J. C. Gilchrist. The effects of ACTH, adrenalectomy and dexamethasone on the acquisition of an avoidance response in rats. Physiol. Behav. 5: 939-944, 1970. 5. DeWied, D. The influence of the anterior pituitary on avoidance learning and escape behavior. J. Physiol. 207: 255-259, 1964. 6. DeWied, D. The influence of the posterior and intermediate lobe of the pituitary and pituitary peptides on the maintenance of a conditioned avoidance response in rats. Int. J. Neuropharmac. 4: 157-167, 1965. 7. DeWied, D. Long term and short term effects on retention of a conditioned avoidance response in rats by treatment with long acting pitressin and MSH. Nature, Lond. 212: 1484-1486, 1966. 8. DeWied, D. Effects of peptide hormones on behavior. In: Frontiers in Neuroendocrinology, edited by L. Martini and W. F. Ganong. London: Oxford University Press, 1969, pp. 97-140. 9. DeWied, D. Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature, Lond. 232: 5 8 - 6 0 , 1971.
10. DeWied, D., A. M. L. Van Delft, W. H. Gispen, J. A. W. M. Weijnen and Tj. B. van Wimersma Greidanus. The role of Pituitary-adrenal system hormones in active avoidances conditioning. In: Hormones and Behavior, edited by S. Levine. New York: Academic Press, 1972, pp. 135-171. 11. Evans, W. O. A new technique for the investigation of some analgesic drugs on a reflexive behavior in the rat. Psychopharmacologia 2: 318-325, 1961. 12. Lande, S., J. B. Flexner and L. B. Flexner. Effect of corticortropin and desglycinamide-lysine vasopressin on suppression of memory by puromycin. Proc. natn. Acad. Sci. U.S.A. 69: 558-560, 1972. 13. Lande, S., A. Witter and D. DeWied. An octapeptide that stimulates conditioned avoidance acquisition in hypophysectomized rats. J. biol. Chem. 246: 2058-2062, 1971. 14. Levine, S. and F. R. Brush. Adrenocortical activity and avoidance learning as a function of time after avoidance training. Physiol. Behav. 2: 385-388, 1967. 15. Levine, S. and L. E. Jones. ACTH and passive avoidance learning. J. comp. physiol. Psychol. 59: 357-360, 1960. 16. Miller, M. and A. M. Moses. Radioimmunoassay of urinary antidiuretic hormone with application to study of the Brattleboro rat. Endocrinology 88: 1389-1396, 1971. 17. Murphy, J. V. and R. E. Miller. The Effect of Adrenocorticotrophic Hormone (ACTH) on Avoidance Conditioning in the Rat. J. comp. Psychol. 48: 4 7 - 4 9 , 1955. 18. Wang, Su-Sun. Synthesis of desglycinamide lysine vasopressin and its behavioral activity in rats. Biochem. biophys. Res. Commun. 48: 1511-1515, 1972.