The behavior of vasopressin-deficient rats (Brattleboro strain)

Physiology & Behavior, Vol. 30, pp. 29-34. PergamonPress, 1983. Printedin the U.S.A.

The Behavior of Vasopressin-Deficient Rats (Brattleboro Strain) G I L B E R T O N. O. B R I T O

Center for Brain Research, University o f Rochester Medical Center, Rochester, N Y 14642 R e c e i v e d 10 M a y 1982 BRITO, G. N. O. The behavior ofvasopressin-deficient rats (Brattleboro strain). PHYSIOL BEHAV 30(1) 29--34, 1983.Seven Brattleboro rats homozygous for diabetes insipidus (DI) and seven normal Long-Evans (LE) rats were tested on a neuropsychological test battery comprised of the following tasks: time-spent-eating in two adaptation boxes, time-toemerge into an open field, adaptation to a T-maze, contingently reinforced T-maze alternation, olfactory and visual discrimination, runway learning, approach-avoidance conflict, step-through passive avoidance, prod burying, and stressinduced interference. It was found that D1 rats adapted more slowly than LE rats to novel environments (e.g., adaptation box, T-maze, and runway), and DI rats were slower to emerge into an open field. However, DI rats performed as well as LE rats on all other tasks. These results suggest that DI rats have altered temperamental dispositions (timidity or cautiousness), normal working and reference memory, and similar susceptibility (compared to LE rats) to the interfering effects of inescapable stress. Brattleboro rat

Vasopressin

Working memory

THE behavioral functions of the neurohormone vasopressin (VP) have been viewed mostly in terms of memory [11]. This view has been supported by reports that vasopressindeficient rats (Brattleboro strain--DI) are impaired in memory [4], and that administration of vasopressin ameliorates their memory impairment [10]. However, research in other laboratories has not demonstrated that normal Long-Evans (LE) rats show better memory than DI rats for shockmotivated behavior [2, 7, 18]. It is interesting that most of the research on the memory capabilities of DI rats used shock-motivated behavioral tasks. This approach may not provide a conclusive assessment regarding behavioral capabilities of DI rats. Therefore, a neuropsychological test battery was used to determine the behavioral characteristics of DI rats, and it was found that DI rats do not have a general impairment in memory [6]. Instead, that study suggested that DI rats have altered temperamental dispositions and impaired reference memory [16] compared with LE rats. Consequently, our previous study [6] did not support the idea that DI rats have general impairment of memory [4,10]. In the present paper, the neuropsychological battery used to assess the behavior of DI rats has been expanded, and it includes measurement of water intake, and food- and shock-motivated tasks involving different hypothetical mechanisms such as temperamental dispositions [6], reference and working memory [5,16], species-specific behavior [19], and stress-induced interference [14]. It was found that DI rats have altered temperamental dispositions (timidity or

Reference memory

Temperament

Stress

cautiousness) and apparently intact reference and workingmemory processes, and they are at least as susceptible as LE rats to the interfering effects of preshock on subsequent avoidance behavior. METHOD

Subjects Seven young-adult male Long-Evans rats homozygous for diabetes insipidus (DI) and seven normal Long-Evans rats (LE), matched for weight, were used in this study. Since DI rats are impaired in growth [4], LE rats were about four weeks younger than DI rats. The younger LE rats weighed between 175 and 191 g and the weights of DI rats ranged from 161 to 208 g at the beginning of experimentation. The median 24-hr water intake of LE rats was 15.5 ml/100 g of body weight (13.0-19.5) and that of DI rats was 92.5 ml/100 g body wt. (60.0-113.5). Two Dis and one LE rat died toward the end of the study so data for only 5 Dis and 6 LE rats are presented for the last behavioral task. The rats were housed individually in wire-mesh cages and given food and water ad lib. After 7 days, the rats were put on a reduced-food regimen of 7 g of powdered food as wet mash per day until their weights decreased to about 85% ad lib weight. Thereafter, the rats received a ration of 7 g of wet mash each day after testing. The light cycle in the vivarium room was from 07:00 to 19:00 hours throughout the study. In a previous report [6], DI and LE rats were obtained from a colony maintained in the Medical Center, and they

~Part of these results was presented at the International Symposium on the Brattleboro Rat held at Dartmouth Medical School, Hanover, New Hampshire, September 1981. 2Supported by NIH grant NS---17543. ZRequests for reprints should be addressed to Gilberto N. O. Brito, now at the Departamento de Ciencias Fisiologicas, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Avenida 28 de Setembro, 87-Fundos, Rio de Janeiro, RJ 20551, Brasil.

C o p y r i g h t © 1983 P e r g a m o n Press--O031-9384/83/010029-06503.00/0

30 were matched for age. Even though the rats from the present study differed from rats in that previous report as regards source and matching procedures, the previous finding [6] that DI rats have altered temperamental dispositions (e.g., timidity) compared to LE rats was replicated in the present study.

Apparatus Adaptation boxes. Time-spent-eating was measured in two boxes. The first box is made of wood and measures 29x30×31 cm. The second box is made of cardboard and measures 26x27x27 cm. T-maze. Contingently reinforced T-maze alternation and discrimination learning were conducted in the trough T-maze described before [5]. Briefly, the maze consists of a start box (10x14x37 cm), a choice area, and two goal boxes (10x 14x43 cm). Two compartments located at the end of each goal box, from which they were separated by fine wire-mesh made of brass, contained 7.5 W light bulbs or dishes with stimuli for olfactory discrimination. A gentle stream of air was continuously pulled through the maze from the distal ends of the goal boxes to the back end of the start box to carry the olfactory discriminative stimuli. Straight runway. Approach-avoidance conflict was tested in the straight runway described previously [6]. In brief, the start box is 27 cm, the runway is 114 cm, and the goal box is 48 cm long. Width is 10 cm and height is 20 cm throughout the runway. Step-through passive-avoidance device. Passive avoidance training and testing was conducted in a 30 x 30 × 25 cm box made of plywood with inside walls painted black and with a grid floor. The grid of the floor is comprised of 15 brass rods (3.5 mm in diameter) spaced 2 cm apart (center to center); even-numbered rods are connected together, as are odd-numbered rods, for presenting foot shock. The circular door of the box is 7.5 cm in diameter and adjoined by an elevated platform (30.5x 11.5 cm), 105 cm above the floor of the test room and painted white. Prod-burying box. The prod-burying test was conducted in a wooden box measuring 29x31 x31 cm and painted gray. The front of the box is made of Plexiglas to allow observation of rats in the box. Two brass rods, spaced 2 mm apart, are located 5.5 cm above the floor and protrude 5.5 cm from the center of the right sidewall of the box. The floor of the box is covered with sawdust up to the level just below the rods. Inescapable-shock box. Stress-induced interference consisted of testing the rats on a one-way active-avoidance task following a session of inescapable shock. Inescapable shock was given in a ~rid box 30x25x30 cm with walls made of Plexiglas. This box was housed within a sound attenuating chamber with inside walls painted white. The grid of the floor is made of 16 rods spaced 2 cm apart (center to center). Active-avoidance box. A guillotine door separates two grid compartments 29x30×43 cm made of plywood and painted black. In order to make the two compartments distinctive from each other, the grid floor of the "safe" compartment was covered with a flat piece of nonpainted wood, and its walls were covered with white cardboard. The grid floor of the "dangerous" compartment is comprised of rods spaced 1.7 cm apart (center to center). One 15 W light bulb is placed on the ceiling of each of the two compartments. The rats were observed through tilted mirrors placed underneath the box.

BRITO

Behavioral Testing The neuropsychological battery consisted of the tasks described below in the order administered. All behavioral testing began at 13:00 hours, and the rats were tested in a different order every day. Time-spent-eating. Following housing, general adaptation to the laboratory and measurement of water intake [6], the rats were placed in a box, once a day, for 1 rain, and timespent-eating from a dish of wet mash was measured with a stopwatch. Testing continued for 9 days. After the rats were eating with alacrity in the first box, time-spent-eating was measured in a second box. The rats were tested in a second box because it was hypothesized that the timidity of DI rats would preclude transfer of habituation from the first to the second box. Testing in the second box was conducted as in the first box and continued for 5 days. Time-to-emerge test. Details of the procedures for the time-to-emerge test have been reported previously [6]. Briefly, latencies to emerge from home cage into an open field were measured with a stopwatch for each rat. The open-field box consisted of a square wooden box 91 cm on a side. The rats received one daily trial of 5-min duration on each of 5 days. T-maze adaptation. Each rat was placed in the start box of the T-maze and forced to go right or left by raising only one of the goal-box doors according to a balanced sequence [13]. Time to enter the goal box (GB) was measured with a stopwatch. Because DI rats adapt more slowly than LE rats, they received 16 sessions, 1 session per day, of 12 trials, whereas LE rats received only 12 sessions. T-maze alternation. Contingently reinforced T-maze alternation was conducted with the "two-run trial" procedure. On the first run of each trial, the rats were forced (by raising the door to only one arm) to go to one of the two GBs of the maze in order to obtain reinforcement (wet mash), and the direction of the forced run was determined by a balanced sequence [13]. On the second run of the trial, doors to both arms of the T-maze were raised and the rats were reinforced only if they chose the GB opposite the one they were forced to go on the previous run. Each rat received 6 trials (6 forced and 6 choice runs) each day for 6 days. The interrun interval for each trial was as brief as practicable (ca 8 sec) and the intertrial i n t e r v a l - - I T I - - w a s determined by the time it took to run the other rats in the squad of 7 (ca 3.5 min). The "two-run trial" procedure probably represents a more appropriate index of working memory (as defined in 5) than the rerun-correction method of delayed T-maze alternation used in a previous report [6]. Olfactory discrimination. Odors of cloves and mineral oil were used in this discrimination. On each trial one odor stimulus was placed in one arm of the T-maze and the other in the opposite arm. Reward (wet mash) was presented only when the rat entered the positive arm. The positive stimulus was the one less frequently chosen in a stimulus-preference session the rats received the day before olfactory discrimination training began. A spaced-trials procedure (ITIs ca 3.5 min; rats were run in squads of 7) was used, and the rats received 14 sessions, 1 session per day, of 12 trials each. The position of the positive stimulus followed a balanced sequence [13]. Visual discrimination. As in the olfactory discrimination task, the rats received one stimulus-preference session, and the positive stimulus subsequently used in discrimination training was the stimulus less frequently chosen on that ses-

VASOPRESSIN AND BEHAVIOR sion. F o r some animals the lit arm of the maze was the positive stimulus and for other rats the dark arm of the T-maze served as the positive stimulus. The rats received 20 sessions, 1 session per day, of 12 spaced trials (ITI ca 3.5 min; rats were run in squads of 7). The right or left position of the positive stimulus in each session followed a balanced sequence [13]. Runway learning. The rats were trained to run in a straight runway for wet mash reinforcement presented in the goal box of the runway. Running time was measured with an electric clock that started when the side-action door of the start-box was opened and stopped when the rat stepped on a treadle in the goal box. The rats received 8 sessions, l per day, of 12 trials each. Maximum running time allowed was 60 sec (default time), and the ITIs were as brief as practicable (ca 15 sec). Approach-avoidance conflict. Procedures for this test have been described before [6]. Briefly, the rats received a 5 mA electric shock (Constant Current Shock GeneratorStoelting Co.) contingent on touching the food dish on the fifth trial of the ninth session in the runway. The rats were left in the closed goal box for 1 min, and the number of shocks they took was recorded. Immediately following the shock trial, the rats received seven additional trials on which shock was not administered. Thereafter, the rats received 6 trials a day for 7 days in the absence of shock. As in the runway learning task, running time was measured with an electric clock, and the ITIs were as brief as practicable (ca 15 sec). Trials were defaulted if the animals had not entered the goal box of the runway within 60 sec from time the startbox door was opened. Step-through passive avoidance. On the first day of this test, the rats were placed in the dark compartment of the passive-avoidance device for a 2 min adaptation period. Immediately following that the rats were placed on the elevated platform, and latency to enter the dark compartment was measured with a stopwatch. The room lights were left on throughout passive-avoidance testing in order to make the environment outside the dark compartment comparatively aversive to the animals. On the second day of testing, the rats received 3 trials, on which they were placed on the platform and latency to enter was measured. The interval between these trials was 2 rain, during which the rats were kept in their home cages. The rats received a 1 mA electric shock for 3 sec (cf. [10]) after they had entered the dark compartment on the third trial. Beginning on the following day, the rats were retested every day for 5 days. Procedures for postshock testing were as described above, except that shock was not presented in the dark compartment. Prod-burying test. The rats received 10 adaptation trials of placement in the prod-burying box (the shock prods were not present), 1 trial per day, for 30 min each. On the 1 lth day, testing began, and it consisted of placing each rat in the box, and the rats received a 5 mA electric shock contingent on touching the shock prods. The shock stayed on for 5 rain from the time the rat first received the first shock. Thereafter, the rats were left in the box for an additional 25 min. Number of shocks taken, height of highest mound of bedding, and time-spent-burying following shock presentation were recorded. Stress-induced interference. Procedures for this test were similar to those reported by Glazer and Weiss [14]. Two groups of DI rats and two groups of LE rats, matched for weight, underwent stress-induced interference testing. One group of DI (N=2) and one group of LE (N=3) rats received

31 180 inescapable footshocks (scrambled) of 4 mA intensity, 2 sec duration, and 20 sec intershock intervals. These shock parameters were identical to those of Glazer and Weiss [14] except that delivery was through the feet rather than tail. The other two groups (DI, N = 3 ; and LE, N = 3 ) received no inescapable shock, but the rats in these groups were kept in the inescapable-shock box for the same length of time (1.1 hr) as rats that received shock. Rats in all groups were tested on one-way active-avoidance 30 min after inescapable shock (or no shock) pretreatment. F o r one-way active-avoidance testing, each rat was placed into the " d a n g e r o u s " compartment of the activeavoidance box and the light in the " s a f e " compartment was turned on. After 15 sec, the guillotine door was raised and the rat had 5 sec to avoid a scrambled footshock of 0.7 mA intensity by crossing to the " s a f e " compartment. If the rat had not escaped shock 60 sec after its presentation, i.e., had not crossed to the "gafe" compartment, the trial was terminated and an ITI of 1 min ensued. Latencies to cross from the " d a n g e r o u s " to the " s a f e " compartment and durations of shock and ITIs were measured with a stopwatch. Statistics. Nonparametric statistical procedures [8] were used throughout the present report. All statistical tests were two-tailed, with the exception of the stress-lnduced interference test, which was one-tailed because interference effects were predicted to be only in one direction. RESULTS

Time-Spent-Eating The total time-spent-eating by DI rats in the first adaptation box across 9 sessions was significantly less than that of LE rats (U=5, p<0.01). However, as shown in Fig. 1 (panel A), DI rats, by the 7th session, were eating substantially. When subsequently placed in the second adaptation box (to test for transfer of habituation), DI rats spent as much time eating as L E rats, i.e., DI rats did not show neophobia to the second box presumably because the two situations were not different enough to prevent transfer of habituation.

Time-to-Emerge The DI rats were significantly slower to emerge into an open field than LE rats as indicated in Fig. 1 (panel B). When total time-to-emerge across 8 trials for DI rats is compared with that for LE rats, the difference is statistically significant ( U = 7 , p<0.02). By the last trial, only 3 DI rats had emerged whereas 6 LE rats had done so, i.e., DI rats were more reluctant to emerge than L E rats. Since it has been reported that DI rats are more active than normal Wistar rats when placed in a circular arena [4], it is unlikely that differences between DI and L E rats on the time-to-emerge test are due to differences in general activity.

T-Maze Adaptation Figure 1 (panel C) shows that DI rats adapted to the T-maze more slowly than L E rats. Total latency to enter the goal box of the maze summed across 12 sessions was significantly greater for the DI rats compared with L E rats ( U = 0 , p<0.002). By the 6th session, L E rats were performing at asymptote and continued to do so through their last (12th) session. Although panel C shows that the median latency for DI rats on the last adaptation session is similar to that for L E rats, the latencies of DI rats were still significantly longer than those for the LE rats ( U = 7 , p<0.02).

32

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SESSIONS

FIG. 1. Performance of DI and LE rats on four tasks of the neuropsychological battery. Panel A shows median time-spent-eating as a function of sessions in the first adaptation box. Panel B illustrates the results for the time-to-emerge test. Panel C shows median latency as a function of adaptation sessions in the T-maze. Panel D indicates median latency to enter goal box of the straight runway as a function of sessions.

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T h e r e was no significant difference b e t w e e n DI and L E rats in performance o f T - m a z e alternation. DI rats a c h i e v e d a median percent alternation score across 6 sessions of 83.3% whereas the median percent alternation of L E rats was 77.7%.

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FIG. 2. The upper panel illustrates the results for the approachavoidance conflict test and the lower panel shows the results for the step-through passive avoidance task.

Olfactory Discrimination DI rats did not differ significantly from L E rats in total n u m b e r of correct responses in the olfactory discrimination task. Both DI and L E rats made a median o f 54% correct responses during the first half of the discrimination. During the second half, the median percentage of correct responses made by DI rats was 81% and that of L E rats was 76%. This difference was not statistically significant.

Visual Discrimination As in olfactory discrimination, DI rats p e r f o r m e d the visual discrimination task as well as L E rats. The median percentage of correct responses made by DI rats during the first (49%) and second (77%) half o f the visual discrimination task did not differ from that of L E rats (first half, 48%; second half, 82%).

Runway Learning As shown in Fig. 1 (panel D), it took DI rats significantly longer than L E rats to reach the goal box o f the straight runway on the first session (U =4, p <0.01). The p e r f o r m a n c e o f DI rats was much i m p r o v e d on the last session c o m p a r e d with the first, and it approached that of L E rats, although, statistically, DI rats were still performing w o r s e than L E rats ( U = 4 . 5 , p<0.01).

Approach-Avoidance Conflict Figure 2 (top panel) indicates that the performance of D1 rats on trials before the introduction o f shock was similar to that of L E rats, e v e n though the difference b e t w e e n the two groups was statistically significant (nonparametric procedures may reveal statistical significance when the ranges of two distributions do not overlap e v e n though the medians of the distributions are very similar, as is the case here). H o w ever, on the post-shock sessions, DI rats showed response iatencies that were as high as those of L E rats. (Although the top panel of Fig. 2 shows a difference between the two groups on the last three post-shock sessions with DI rats showing better " m e m o r y " than L E rats, this difference was only marginally significant, p<0.10).

Passive Avoidance As shown in the bottom panel of Fig. 2, DI rats entered the dark box from an elevated platform across 4 pre-shock sessions significantly more slowly than L E rats ( U = 6 , p<0.02). On the post-shock sessions, DI rats were at least as reluctant as L E rats to enter the dark box. On the last post-

VASOPRESSIN AND BEHAVIOR

33

TABLE 1 TOTAL LATENCIES OF INDIVIDUAL DI AND LE RATS ON THE STRESS-INDUCED INTERFERENCE TEST

DI

LE p(two-tailed)

No Preshock

Preshock

p(one-tailed)

56.5 62.1 70.8 75.6 76.5 114.2 0.05

98.4 145.2

0.05

104.2 131.7 152.5 n.s.

0.05

shock session, only 1 DI rat entered the dark compartment, whereas 4 LE rats did so.

Prod-Burying Performance on this task proved to be extremely variable thus providing an insensitive behavioral index to compare the performance of DI with that of LE rats. There were no significant differences between DI and L E rats in number of shocks taken during presentation of shock (DI: median=2, range, 0-4; LE: m e d i a n = l , range, 0-3). In addition, the species-specific behavioral disposition of burying a "dangerous" object was shown by both DI and L E rats in terms of time-spent-burying (DI: mediarr--19 sec, range, 0--247; LE: median--158, range, 0-574 sec) and height of highest mound of bedding that was piled up by the rats (DI: median--7.9 cm; LE: median--7.5 cm). Thus, DI rats " r e m e m b e r e d " as well as LE rats that the prod represented a " d a n g e r o u s " object.

Stress-Induced Interference Table 1 shows total latencies across 25 trials on the active-avoidance task for individual DI and LE rats. Both DI and LE rats showed impairment in performance of one-way active avoidance following a session of inescapable shock. Also, it is interesting to note that DI rats that had not been preshocked performed better than their L E counterparts. This finding contradicts reports from other investigators [4]. Similar conclusions were drawn from analysis (not shown) of number of avoidance responses (responses within 5 sec of presentation of shock). Although the small size of the samples preclude any definitive conclusion as regards the theoretical population of DI and L E rats, it is clear that both DI and LE rats can show stress-induced interference effects to a similar degree. DISCUSSION The results of the present study suggest that vasopressin-deficient rats have altered temperamental dispositions (e.g., timidity or cautiousness) compared with

normal rats. However, DI rats seem to have intact workingand reference-memory processes [5,16] as evaluated by contingently reinforced T-maze alternation and discrimination learning, respectively. Moreover, DI rats remembered exposure to shock as well as LE rats in several shock-motivated tasks, and DI rats showed at least as much stress-induced interference as L E rats. The present data, therefore, do not support the hypothesis that vasopressin deficiency is associated with memory impairment as suggested by other investigators [4,10]. A possible explanation for the discrepancy between these results and those from other laboratories is that the genetic background of the DI rats is different from that of DI rats used by other investigators [4,10]. In this regard, it is interesting to note that the present study did not replicate some previous findings [6] as, for example, impairment of DI rats in reference-memory tasks. Since in the previous paper [6], DI rats came from a colony maintained in the Medical Center, and, in the present study, commercially obtained DI rats were used, it is possible that differences in genetic background is a more critical determinant of behavioral abnormalities in DI rats than vasopressin deficiency per se. Also DI rats were shown to be as susceptible as LE rats to the deleterious effects of inescapable shock on subsequent performance of a one-way active-avoidance task. This stress-induced interference effect has been suggested to be mediated by changes in the metabolism of monoamines in the brain (see [22]). Since DI rats seem to have altered monoamine metabolism [17,21], it was hypothesized that they would be less sensitive than L E rats to the interfering effects of stress. The fact that they were not raises interesting questions regarding the neurochemical effects of inescapable stress in DI rats, a direction now being pursued. It should be emphasized that DI rats have several other endocrinological abnormalities in addition to vasopressin deficiency. Compared with LE rats, DI rats have lower levels of leu-enkephalin in the neurointermediate lobe [20], higher plasma levels of oxytocin [ 12], lower pituitary content of growth hormone [1], decreased binding of corticosterone to hippocampal cytosol receptors [9], lower plasma levels of corticosterone during retention testing of passive-avoidance behavior [4], and higher plasma renin concentration, increased isorenin levels in adrenal gland and hypothalamus, and decreased isorenin concentration in neurohypophysis [15]. It is yet to be definitively determined to what extent the constellation of neurochemical and endocrinological abnormalities found in DI rats is secondary to vasopressin deficiency. In any case, it is probable that imbalance in neurochemical and neuroendocrinological mechanisms in the brain of DI rats is related to their altered temperamental dispositions (a similar hypothesis was advanced in [3]). ACKNOWLEDGMENTS The author wishes to thank Mary Lee Stein and Mary G. Capozzi for typing the manuscript and Sue D. Connor for technical assistance. The author is also grateful to Dr. Garth J. Thomas for providing laboratory facilities, and to Dr. Mark E. Stanton and Linda C. Stopp for criticisms regarding the manuscript.

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