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Vasopressin analogues and spatial short-term memory in rats
Peptides, Vol. 1, pp. 261-263. Printed in the U.S.A.
Vasopressin Analogues and Spatial Short-Term Memory in Rats OLGA BURE~OVA
AND JANA ~KOPKOVA
Institute of Physiology and Institute of Organic Chemistry and Biochemistry Czechoslovak Academy of Sciences, Prague, Czechoslovakia R e c e i v e d 29 D e c e m b e r 1979 B U R E t ; O V A , O. A N D J. ~KOPKOVA. Vasopressin analogues and spatial short-term memory in rats. P E P T I D E S 1(3) 261-263, 1980.--The effect of vasopressin analogues on short-term memory was tested in the 12-arm radial maze. After the first 6 choices rats (n= 16) were removed from the apparatus and allowed to complete the remaining 6 choices 20 min later. Whereas desgly-NH2-VP, AVP, dAVP and DAVP (3.0 ~kg) administered 40 rain before or immediately after the first 6 choices did not change the incidence of errors in the second series of choices (2.0 errors under control conditions), similarly applied dDAVP deteriorated the rat's performance almost to the chance level of 3 errors. The significance of short-term memory tests for assessing the mnestic role of peptide hormones is stressed.
Pituitary peptides
Short-term memory
Spatial memory
D U R I N G the past decade various pituitary peptides and their analogues were shown to influence higher nervous activity. Neurohypophyseal hormones can repair behavioral abnormalities induced either by the extirpation of the pituitary gland [17] or by hereditary hypothalamic diabetes insipidus [4]. In intact rats, vasopressin and its analogues (even those devoid of pressor or antidiuretic activities) increase resistance to extinction of active [16, 18, 20] and passive avoidance responses [1, 3, 12]. The effect of the pituitary peptides on appetitively motivated learning is less clear. According to Garrud et al. [7] desglycinamide lysine vasopressin (DG-LVP) caused no changes in extinction of a food-rewarded straight runway task, but Hostetter et al. [9] reported facilitation of a positively rewarded discrimination. In spite of the subtlety of some of the above effects, it is generally agreed (see recent reviews [6,11]) that vasopressin and other peptide hormones affect acquisition and retrieval of long-term memories. Little is known, however, how these substances influence short-term memory. An enkephalin analogue [ 14] failed to improve the performance in a delayed response task, but facilitated learning of a discrimination reversal task in monkeys. The purpose of the present paper is to test the effect of several vasopressin analogues on the short-term working memory using the radial maze technique [5, 13, 151. METHOD
The subjects were 16 male hooded rats (Druckray strain) aged 5-6 months and weighing between 280 and 350 g. They were housed 4 to a cage with free access to water, but food was available only during daily sessions in the maze and for 40 min after the experiment. The animals were overtrained for 5 months in the 12-channel radial maze [13]. The circular choice platform (43 cm in diameter), raised 5 cm above the floor and made accessible through a central opening (8.5 cm in diameter), provided access to 12 regularly spaced tubular channels, forming the radial arms of the maze. The tubes
C o p y r i g h t © 1980 A N K H O
were 7.5 cm wide and 31 cm long, had a transparent ceiling and a recessed feeder at the far end of the channel. They were equipped with one-way entrance and exit doors, permitting only centrifugal passage of the animal through the maze arms. After entering one of the channels from the choice platform, the rat had to descend on the main floor, return below the choice platform, climb onto it through the central opening and make another choice. The apparatus was surrounded by 50-cm high Plexiglas circular wall. During each session the rat was placed into the apparatus with all channels baited with 100 mg morsels of L a r s e n ' s diet. Olfactory recognition of the baited channels was prevented by saturating the maze with food odour released by large pieces of Larson's diet placed in plastic containers, mounted on the perforated ceiling of individual maze channels. The animals were allowed to make 12 choices. After the first six choices they were removed from the maze and isolated for 20 min in a separate waiting cage. They were then placed into the apparatus again and allowed to complete the remaining 6 choices. The sequence of the channels visited before and after the 20 min interruption was recorded and the number of errors, i.e. the repeated visits of the same channel were evaluated. Since no errors occurred during the first choices only errors made after the 20 min interruption were statistically evaluated. Random choice behavior should result into 3 errors in choices 7-12. The effects of desglycinamide (8-L-lysine) vasopressin (desgly-NH2-VP) [2], (8-LArginine) vasopressin (AVP), (8-L-Arginine) deaminov a s o p r e s s i n (dAVP) [10], (8-D-Arginine) v a s o p r e s s i n (DAVP) [21] and (8-D-Arginine) d e a m i n o - v a s o p r e s s i n (dDAVP) [22] were tested at 3 to 4-day intervals. DesglyNH2-VP was prepared by a tryptic splitting of (8-L-lysine) vasopressin and did not contain more than 5% of (8-L-lysine) vasopressin. The peptides were applied in the dose of 1/~g/rat in 0.5 ml of saline SC, either 40 rain before the first choice or immediately after the first 6 choices. The same volume of saline was injected in the control experiments. Radial maze training
I n t e r n a t i o n a l Inc.--0196-9781/80/030261-03500.80/0
262
BURE~OV,~ AND ~KOPKOVA APPLICATION 4OMIN BEFORE THE FIRST CHOICE
E 3.0
E 3.0'
t
2.S
APPLICATION AFTER COMPLETION OF FIRST 6 CHOICES
2.S-
2.0
1.S
C
deegly-
AVP
d AVP
DAVP
IDAVP
NH2-VP
1.S C
descjlyNHt-VP
AVF
dAVP
DAVP
dDAVP
FIG. 1. Average number of errors ( _+ SEM) in the last 6 choices after treatment with the 5 peptides applied 40 min before the experiment. C-injection of saline.
FIG. 2. Average number of errors ( _+ SEM) in the last 6 choices after treatment with the 5 peptides applied at the onset of the interruption. Other description as in Fig. 1.
continued on the remaining days when the animals were allowed to make the 12 choices without interruption.
It has been reported that substitution of the " D " isomer for the natural " L " isomer reverses the behavioral effects of some ACTH peptides [19]. Analogous results were obtained in the present study with dDAVP (D-isomer) and dAVP (Lisomer). The first peptide had a marked deteriorating effect whereas the second one produced the lowest error scores. The failure of the tested peptides to improve the shortterm memory might be due to inadequate dosages. A more definite answer requires a detailed analysis of the dose effect relationship of the various peptides taking into account their postinjection concentration in the central nervous system. The adverse effect of dDAVP on short-term memory in a paradigm which is formally similar to the tests used for evaluation of human memory deficits contrasts with the positive results reviewed in the introduction. Such controversial findings are not so surprising, however, if we take into account the differences between working and reference memory [8]. Efficient working memory requires that a particular stimulus is forgotten or extinguished as soon as the trial is completed, so that its memory does not interfere with remembering a new stimulus set on the next trial. Slower extinction induced by vasopressin, which is usually interpreted as improvement of memory, indicates at the same time reduced flexibility of behavior manifested by maladaptive perseverance of responding in absence of punishment or reward. Slower extinction is a disadvantage rather than an asset in the working memory task. Such considerations stress the necessity to increase the array of tests employed for assessing the effect of peptide hormones on learning and memory.
RESULTS The data obtained with the 5 peptides tested are summarized in Fig. 1 and Fig. 2. The 20-min interruption increased the number of errors in choices 7 to 12 from 1.25 _+ 0.23 to 2.0 _ 0.24 under control conditions, but significant differences against the predicted random choice behavior, t(31)=4.02, p<0.001 indicated persistence of the spatial short-term memory. The analysis of variance revealed significant differences between treatments, F(5,75)=2.53, p<0.05, but failed to show significant differences between animals, F(15,75)= 1.75, n.s., and application times, F(1,75)=1.72, n.s. Neither of the first order interactions was significant. Multiple comparison tests of the treatments showed that dDAVP significantly deteriorated performance with respect to dAVP, t(6.75)=4.26, p<0.05. The dDAVP difference against controls almost reached significance, t(5,75)=3.75, tabulated value 3.86 for p =0.05. Other differences were not statistically significant. DISCUSSION None of the five peptides used in this study improved short-term memory in the 12-arm radial maze. It must be pointed out, however, that the effect of other psychoactive drugs on this task has not yet been studied and that it is not clear whether the short-term memory of this type can be improved at all. Absence of significant differences between the effect of peptides applied 40 min before the first choice and immediately after the sixth choice indicates that the hormones do not influence the acquisition mechanism differently than the retention and retrieval processes.
ACKNOWLEDGEMENT AVP, dAVP, DAVP and dDAVP were kindly donated by Dr. M. Flegel from LI~IVA Mod~any.
VASOPRESSIN
AND MEMORY
263
REFERENCES 1. Ader, R. and D. de Wied. Effects of lysine vasopressin on passive avoidance learning. Psychon. Sci. 29: 46--48, 1972. 2. Barth, T. and I. Kluh. Des-glycinamideg-vasopressin and deslysineS-glycinamideg-vasopressin: some pharmacological properties. Collection Czech. chem. Commun. 39" 506-508, 1974. 3. Bohus, B., R. Ader and D. de Wied. Effects of vasopressin on active and passive avoidance behavior. Hormones Behav. 3: 191-197, 1972. 4. Bohus, B., Tj. B. van Wiersma Greidanus and D. de Wied. Behavioral and endocrine responses of rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain). Physiol. Behay. 14: 609-615, 1975. 5. Bure~ov,q, O. Spatial memory and instrumental conditioning. Acta Neurobiol. exp. 40: 51-65, 1980. 6. Dunn, A. J. Neurochemistry of learning and memory: An evaluation of recent data. A. Rev. Psychol. 31: 343-390, 1980. 7. Garrud, P., J. A. Gray and D. de Wied. Pituitary-adrenal hormones and extinction of rewarded behaviour in the rat. Physiol. Behav. 12: 109-119, 1974. 8. Honig, W. K. Studies of working memory in the pigeon. In: Cognitive Aspects o f Animal Behavior, edited by S. H. Hulse, W. K. Honig and H. Fowler. Hillsdale: Lawrence Erlbaum Associates, 1978, pp. 211-248. 9. Hostetter, G., S. L. Jubb and G. P. Kozlowski. Vasopressin affects the behavior of rats in a positively-rewarded discrimination task. Life Science 21: 1323-1328, 1977. 10. Huguenin, R. L. and R. A. Boissonnas. S y n t h ~ e de la d6saminol-ArgS-vasopressine et de la d6saminol-PhC-Arg svasopressine, deux analogues poss6dant une activit6 antidiur6tique plus 61ev6e et plus s61ective que ceile des vasopressines natureiles. Heir. chim. Acta 49: 695-698, 1966. 11. Kovfics, G. L., B. Bohus and D. H. G. Versteeg. The effects of vasopressin on memory processes: The role of noradrenergic neurotransmission. Neuroscience 4: 1529-1537, 1979. 12. Krej~f, I. and B. Kupkov~i. Effects of vasopressin analogues on passive avoidance behavior. Activitas nerv. sup. 20: 11-12, 1978.
13. Magni, S., I. Krekule and J. BureL Radial maze type as a determinant of the choice behavior of rats. J. Neurosci. Metho. 1: 343-354, 1979. 14. Olson, G. A., R. D. Olson, A. J. Kastin, M. T. Green, R. RoigSmith, C. W. Hill and D. H. Coy. Effects of an enkephalin analog on complex learning in the rhesus monkey. Pharmac. Biochem. Behav. 11: 341-345, 1979. 15. Olton, D. S. and R. L. Samuelson. Remembrance of places passed: Spatial memory in rats. J. Exp. Psychol.: Anita. Behav. Proc. 2: 97-116, 1976. 16. Schulz, H., G. L. Kovacs and G. Telegdy. The effect of vasopressin and oxytocin on avoidance behavior in rats. In: Cellular and Molecular Bases o f Neuroendocrine Processes. edited by E. Endr6czi. Budapest: Akedemiai Kiado, 1976, pp. 555-564. 17. Wied, D. de. 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. 18. Wied, D. de. Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature (Lond.) 232: 58-60, 1971. 19. Wied, D. de. Pituitary-adrenal system hormones and behavior. In: The Neurosciences, Third Study Program, edited by F. O. Schmitt and E. G. Worden. Cambridge, MA: MIT Press, 1974, pp. 653-666. 20. Wied, D. de and B. Bohus. Long term and short term effects on retention of a conditioned response in rats by treatment with long acting pitressin and a-MSH. Nature, Lond. 212: 14841486, 1966. 21. Zaoral, M., J. Kolc and F. ~orm. Synthesis of D-Arg-8- and D-Lys-8-vasopressin. Collection Czech. chem. Commun. 32: 1242-1249, 1967. 22. Zaoral, M., J. Kolc and F. ~orm. Synthesis of l-deamino8-D-gamma-aminobutyrine-vasopressin, l-deamino-8-D-lysinevasopressin and l-deamino-8-D-arginine-vasopressin. Collection Czech. chem. Commun. 32: 1250-1257, 1967.
Vasopressin Analogues and Spatial Short-Term Memory in Rats OLGA BURE~OVA
AND JANA ~KOPKOVA
Institute of Physiology and Institute of Organic Chemistry and Biochemistry Czechoslovak Academy of Sciences, Prague, Czechoslovakia R e c e i v e d 29 D e c e m b e r 1979 B U R E t ; O V A , O. A N D J. ~KOPKOVA. Vasopressin analogues and spatial short-term memory in rats. P E P T I D E S 1(3) 261-263, 1980.--The effect of vasopressin analogues on short-term memory was tested in the 12-arm radial maze. After the first 6 choices rats (n= 16) were removed from the apparatus and allowed to complete the remaining 6 choices 20 min later. Whereas desgly-NH2-VP, AVP, dAVP and DAVP (3.0 ~kg) administered 40 rain before or immediately after the first 6 choices did not change the incidence of errors in the second series of choices (2.0 errors under control conditions), similarly applied dDAVP deteriorated the rat's performance almost to the chance level of 3 errors. The significance of short-term memory tests for assessing the mnestic role of peptide hormones is stressed.
Pituitary peptides
Short-term memory
Spatial memory
D U R I N G the past decade various pituitary peptides and their analogues were shown to influence higher nervous activity. Neurohypophyseal hormones can repair behavioral abnormalities induced either by the extirpation of the pituitary gland [17] or by hereditary hypothalamic diabetes insipidus [4]. In intact rats, vasopressin and its analogues (even those devoid of pressor or antidiuretic activities) increase resistance to extinction of active [16, 18, 20] and passive avoidance responses [1, 3, 12]. The effect of the pituitary peptides on appetitively motivated learning is less clear. According to Garrud et al. [7] desglycinamide lysine vasopressin (DG-LVP) caused no changes in extinction of a food-rewarded straight runway task, but Hostetter et al. [9] reported facilitation of a positively rewarded discrimination. In spite of the subtlety of some of the above effects, it is generally agreed (see recent reviews [6,11]) that vasopressin and other peptide hormones affect acquisition and retrieval of long-term memories. Little is known, however, how these substances influence short-term memory. An enkephalin analogue [ 14] failed to improve the performance in a delayed response task, but facilitated learning of a discrimination reversal task in monkeys. The purpose of the present paper is to test the effect of several vasopressin analogues on the short-term working memory using the radial maze technique [5, 13, 151. METHOD
The subjects were 16 male hooded rats (Druckray strain) aged 5-6 months and weighing between 280 and 350 g. They were housed 4 to a cage with free access to water, but food was available only during daily sessions in the maze and for 40 min after the experiment. The animals were overtrained for 5 months in the 12-channel radial maze [13]. The circular choice platform (43 cm in diameter), raised 5 cm above the floor and made accessible through a central opening (8.5 cm in diameter), provided access to 12 regularly spaced tubular channels, forming the radial arms of the maze. The tubes
C o p y r i g h t © 1980 A N K H O
were 7.5 cm wide and 31 cm long, had a transparent ceiling and a recessed feeder at the far end of the channel. They were equipped with one-way entrance and exit doors, permitting only centrifugal passage of the animal through the maze arms. After entering one of the channels from the choice platform, the rat had to descend on the main floor, return below the choice platform, climb onto it through the central opening and make another choice. The apparatus was surrounded by 50-cm high Plexiglas circular wall. During each session the rat was placed into the apparatus with all channels baited with 100 mg morsels of L a r s e n ' s diet. Olfactory recognition of the baited channels was prevented by saturating the maze with food odour released by large pieces of Larson's diet placed in plastic containers, mounted on the perforated ceiling of individual maze channels. The animals were allowed to make 12 choices. After the first six choices they were removed from the maze and isolated for 20 min in a separate waiting cage. They were then placed into the apparatus again and allowed to complete the remaining 6 choices. The sequence of the channels visited before and after the 20 min interruption was recorded and the number of errors, i.e. the repeated visits of the same channel were evaluated. Since no errors occurred during the first choices only errors made after the 20 min interruption were statistically evaluated. Random choice behavior should result into 3 errors in choices 7-12. The effects of desglycinamide (8-L-lysine) vasopressin (desgly-NH2-VP) [2], (8-LArginine) vasopressin (AVP), (8-L-Arginine) deaminov a s o p r e s s i n (dAVP) [10], (8-D-Arginine) v a s o p r e s s i n (DAVP) [21] and (8-D-Arginine) d e a m i n o - v a s o p r e s s i n (dDAVP) [22] were tested at 3 to 4-day intervals. DesglyNH2-VP was prepared by a tryptic splitting of (8-L-lysine) vasopressin and did not contain more than 5% of (8-L-lysine) vasopressin. The peptides were applied in the dose of 1/~g/rat in 0.5 ml of saline SC, either 40 rain before the first choice or immediately after the first 6 choices. The same volume of saline was injected in the control experiments. Radial maze training
I n t e r n a t i o n a l Inc.--0196-9781/80/030261-03500.80/0
262
BURE~OV,~ AND ~KOPKOVA APPLICATION 4OMIN BEFORE THE FIRST CHOICE
E 3.0
E 3.0'
t
2.S
APPLICATION AFTER COMPLETION OF FIRST 6 CHOICES
2.S-
2.0
1.S
C
deegly-
AVP
d AVP
DAVP
IDAVP
NH2-VP
1.S C
descjlyNHt-VP
AVF
dAVP
DAVP
dDAVP
FIG. 1. Average number of errors ( _+ SEM) in the last 6 choices after treatment with the 5 peptides applied 40 min before the experiment. C-injection of saline.
FIG. 2. Average number of errors ( _+ SEM) in the last 6 choices after treatment with the 5 peptides applied at the onset of the interruption. Other description as in Fig. 1.
continued on the remaining days when the animals were allowed to make the 12 choices without interruption.
It has been reported that substitution of the " D " isomer for the natural " L " isomer reverses the behavioral effects of some ACTH peptides [19]. Analogous results were obtained in the present study with dDAVP (D-isomer) and dAVP (Lisomer). The first peptide had a marked deteriorating effect whereas the second one produced the lowest error scores. The failure of the tested peptides to improve the shortterm memory might be due to inadequate dosages. A more definite answer requires a detailed analysis of the dose effect relationship of the various peptides taking into account their postinjection concentration in the central nervous system. The adverse effect of dDAVP on short-term memory in a paradigm which is formally similar to the tests used for evaluation of human memory deficits contrasts with the positive results reviewed in the introduction. Such controversial findings are not so surprising, however, if we take into account the differences between working and reference memory [8]. Efficient working memory requires that a particular stimulus is forgotten or extinguished as soon as the trial is completed, so that its memory does not interfere with remembering a new stimulus set on the next trial. Slower extinction induced by vasopressin, which is usually interpreted as improvement of memory, indicates at the same time reduced flexibility of behavior manifested by maladaptive perseverance of responding in absence of punishment or reward. Slower extinction is a disadvantage rather than an asset in the working memory task. Such considerations stress the necessity to increase the array of tests employed for assessing the effect of peptide hormones on learning and memory.
RESULTS The data obtained with the 5 peptides tested are summarized in Fig. 1 and Fig. 2. The 20-min interruption increased the number of errors in choices 7 to 12 from 1.25 _+ 0.23 to 2.0 _ 0.24 under control conditions, but significant differences against the predicted random choice behavior, t(31)=4.02, p<0.001 indicated persistence of the spatial short-term memory. The analysis of variance revealed significant differences between treatments, F(5,75)=2.53, p<0.05, but failed to show significant differences between animals, F(15,75)= 1.75, n.s., and application times, F(1,75)=1.72, n.s. Neither of the first order interactions was significant. Multiple comparison tests of the treatments showed that dDAVP significantly deteriorated performance with respect to dAVP, t(6.75)=4.26, p<0.05. The dDAVP difference against controls almost reached significance, t(5,75)=3.75, tabulated value 3.86 for p =0.05. Other differences were not statistically significant. DISCUSSION None of the five peptides used in this study improved short-term memory in the 12-arm radial maze. It must be pointed out, however, that the effect of other psychoactive drugs on this task has not yet been studied and that it is not clear whether the short-term memory of this type can be improved at all. Absence of significant differences between the effect of peptides applied 40 min before the first choice and immediately after the sixth choice indicates that the hormones do not influence the acquisition mechanism differently than the retention and retrieval processes.
ACKNOWLEDGEMENT AVP, dAVP, DAVP and dDAVP were kindly donated by Dr. M. Flegel from LI~IVA Mod~any.
VASOPRESSIN
AND MEMORY
263
REFERENCES 1. Ader, R. and D. de Wied. Effects of lysine vasopressin on passive avoidance learning. Psychon. Sci. 29: 46--48, 1972. 2. Barth, T. and I. Kluh. Des-glycinamideg-vasopressin and deslysineS-glycinamideg-vasopressin: some pharmacological properties. Collection Czech. chem. Commun. 39" 506-508, 1974. 3. Bohus, B., R. Ader and D. de Wied. Effects of vasopressin on active and passive avoidance behavior. Hormones Behav. 3: 191-197, 1972. 4. Bohus, B., Tj. B. van Wiersma Greidanus and D. de Wied. Behavioral and endocrine responses of rats with hereditary hypothalamic diabetes insipidus (Brattleboro strain). Physiol. Behay. 14: 609-615, 1975. 5. Bure~ov,q, O. Spatial memory and instrumental conditioning. Acta Neurobiol. exp. 40: 51-65, 1980. 6. Dunn, A. J. Neurochemistry of learning and memory: An evaluation of recent data. A. Rev. Psychol. 31: 343-390, 1980. 7. Garrud, P., J. A. Gray and D. de Wied. Pituitary-adrenal hormones and extinction of rewarded behaviour in the rat. Physiol. Behav. 12: 109-119, 1974. 8. Honig, W. K. Studies of working memory in the pigeon. In: Cognitive Aspects o f Animal Behavior, edited by S. H. Hulse, W. K. Honig and H. Fowler. Hillsdale: Lawrence Erlbaum Associates, 1978, pp. 211-248. 9. Hostetter, G., S. L. Jubb and G. P. Kozlowski. Vasopressin affects the behavior of rats in a positively-rewarded discrimination task. Life Science 21: 1323-1328, 1977. 10. Huguenin, R. L. and R. A. Boissonnas. S y n t h ~ e de la d6saminol-ArgS-vasopressine et de la d6saminol-PhC-Arg svasopressine, deux analogues poss6dant une activit6 antidiur6tique plus 61ev6e et plus s61ective que ceile des vasopressines natureiles. Heir. chim. Acta 49: 695-698, 1966. 11. Kovfics, G. L., B. Bohus and D. H. G. Versteeg. The effects of vasopressin on memory processes: The role of noradrenergic neurotransmission. Neuroscience 4: 1529-1537, 1979. 12. Krej~f, I. and B. Kupkov~i. Effects of vasopressin analogues on passive avoidance behavior. Activitas nerv. sup. 20: 11-12, 1978.
13. Magni, S., I. Krekule and J. BureL Radial maze type as a determinant of the choice behavior of rats. J. Neurosci. Metho. 1: 343-354, 1979. 14. Olson, G. A., R. D. Olson, A. J. Kastin, M. T. Green, R. RoigSmith, C. W. Hill and D. H. Coy. Effects of an enkephalin analog on complex learning in the rhesus monkey. Pharmac. Biochem. Behav. 11: 341-345, 1979. 15. Olton, D. S. and R. L. Samuelson. Remembrance of places passed: Spatial memory in rats. J. Exp. Psychol.: Anita. Behav. Proc. 2: 97-116, 1976. 16. Schulz, H., G. L. Kovacs and G. Telegdy. The effect of vasopressin and oxytocin on avoidance behavior in rats. In: Cellular and Molecular Bases o f Neuroendocrine Processes. edited by E. Endr6czi. Budapest: Akedemiai Kiado, 1976, pp. 555-564. 17. Wied, D. de. 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. 18. Wied, D. de. Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature (Lond.) 232: 58-60, 1971. 19. Wied, D. de. Pituitary-adrenal system hormones and behavior. In: The Neurosciences, Third Study Program, edited by F. O. Schmitt and E. G. Worden. Cambridge, MA: MIT Press, 1974, pp. 653-666. 20. Wied, D. de and B. Bohus. Long term and short term effects on retention of a conditioned response in rats by treatment with long acting pitressin and a-MSH. Nature, Lond. 212: 14841486, 1966. 21. Zaoral, M., J. Kolc and F. ~orm. Synthesis of D-Arg-8- and D-Lys-8-vasopressin. Collection Czech. chem. Commun. 32: 1242-1249, 1967. 22. Zaoral, M., J. Kolc and F. ~orm. Synthesis of l-deamino8-D-gamma-aminobutyrine-vasopressin, l-deamino-8-D-lysinevasopressin and l-deamino-8-D-arginine-vasopressin. Collection Czech. chem. Commun. 32: 1250-1257, 1967.