- Email: [email protected]
Centrally injected arginine vasopressin (AVP) facilitates social memory in rats
Neuroscience Letters, 77 (1987) 353 359
353
Elsevier Scientific Publishers Ireland Ltd. NSL 04645
Centrally injected arginine vasopressin (AVP) facilitates social memory in rats M . L e M o a l 1, R . D a n t z e r l, B. M i c h a u d I a n d G . F . K o o b 2 ZlNRA, Psychobiologie des Comporternents Adaptatifs. U259 INSERM, Bordeaux (France) and "Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla. CA 92037 (U.S.A.)
(Received 28 November 1986; Revised version received and accepted 28 February 1987) Key word~." Arginine vasopressin; Social memory; Rat
"Memory' for a juvenile conspecific in male rats can be measured by variation in duration of investigation times when the same juvenile is presented at different intervals. Typically, exposure of an adult male rat to a juvenile results in transient investigatory activity that rapidly declines with repeated exposures at short interexposure intervals (30 min). Longer interexposure intervals (120 rain) result in re-investigation with durations similar or greater than the original investigation. Arginine vasopressin (AVP) injected into adult male rats intracerebroventricularly in doses of 0.5 2.0 ng immediately after investigation of the juvenile decreased social investigation of the same juvenile at the long (120 min) interexposure interval. This decrease in investigatory time was similar to that observed after a 30-min interexposure interval in untreated animals. These results support the hypothesis that increasing the availability of AVP in the central nervous system can improve the consolidation of olfactory information and improve conspecific recognition in rats.
S u b s t a n t i a l e v i d e n c e n o w s u p p o r t s the h y p o t h e s i s t h a t a r g i n i n e v a s o p r e s s i n ( A V P ) a n d related n e u r o p e p t i d e s are i n v o l v e d in a d a p t i v e b e h a v i o r s a n d even in l e a r n i n g a n d m e m o r y processes [1, 4, 7]. T h e s e effects h a v e b e e n d e m o n s t r a t e d b o t h after adm i n i s t r a t i o n o f m i c r o g r a m a m o u n t s o f A V P i n j e c t e d p e r i p h e r a l l y a n d after i n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) a d m i n i s t r a t i o n o f n a n o g r a m a m o u n t s o f the p e p t i d e s [4, 7, 8]. These s t u d i e s i n d i c a t e a f a c i l i t a t i o n o f c o n s o l i d a t i o n a n d recall processes in aversively m o t i v a t e d t a s k s after p e r i p h e r a l a d m i n i s t r a t i o n o f the h o r m o n e , b u t o n l y a few h a v e d e m o n s t r a t e d such effects in a p p e t i t i v e l y m o t i v a t e d tasks, a n d p a r t i c u l a r l y after central a d m i n i s t r a t i o n [9]. O n e o f the p r o b l e m s m e t by such studies c o n c e r n s the a n i m a l m o d e l s g e n e r a l l y used in the e x p e r i m e n t a l s t u d y o f l e a r n i n g a n d m e m o r y [6]. A f o r m o f m e m o r y , very similar to f a c t u a l m e m o r y in h u m a n s a n d w h i c h has received little a t t e n t i o n , is recog-
Correspondence: M. Le Moal, INRA, Psychobiologie des Comportements Adaptatifs, U259 INSERM,
Rue Camille Saint-Saens, 33077 Bordeaux Cedex, France. 0304-3940/87/$ 03.50 (D 1987 Elsevier Scientific Publishers Ireland Ltd.
354 nition of conspecific identity that may be considered as an ethological model of social memory [2, 11]. For instance, adult male rats spend a great amount of time investigating novel juveniles; the rats exposed to the same juvenile 30 min alter the initial exposure display less investigatory behavior, but if the re-exposure occurs 2 h later, the juvenile is not recognized and is thoroughly investigated [I I]. We have recently demonstrated that this transient m e m o r y for a juvenile was sensitive to retroactive facilitation and retroactive interference, and was modulated by peripheral administration of microgram amounts of hypophyseai neuropeptides [3]. The present study was undertaken to see if such memory processes were modulated by central administration of AVP. The results indicate that AVP injected i.c.v, in nanogram amounts enhances the retention of such conspecific social interaction. The subjects were retired breeder male rats of the Wistar strain, 6 months of age. For the purpose of i.c.v, injections, they received at their arrival in the laboratory a cannula aimed above the lateral ventricle. For this surgery, rats were anesthetized with chloral hydrate (6 ml/kg, 6% solution) and secured in a K o p f stereotaxic instrument. A 23-gauge stainless steel guide cannula, 7 m m long, was lowered to within 1 mm of the ventricle and anchored to the skull with two stainless-steel screws and dental cement. Coordinates were with tooth bars 5 m m above interaural zero, 0.6 m m posterior to the bregma, 2.0 m m lateral and 3.2 m m below the skull surface at the point of entry. After surgery, the rats were housed individually in a 30 x 45 × 19 cm transparent plastic cage and maintained for two weeks before the onset of the experiments on ad libitum food and water in a temperature-controlled room under a reverse d a r k light cycle (light on: 20:30 to 8:30 h), Laboratory-reared juvenile Wistar male rats, 25-30 days old, were used as social stimulus. For each session, all the rats under investigation were placed 2 h before the test in an experimental room equipped with the same light cycle and with a background white noise. All the observations were made between 11.00 and 16.00 h and were carried out with a video camera using an infrared light. The test was conducted in the home cage of the male and consisted of a 5-min exposure to a given juvenile, followed by a second exposure of the same duration to the same juvenile, at given intertrial intervals for each session. Between the two successive presentations, the juveniles were kept individually in small glass jars containing fresh wood litter. The procedure was conducted according to a previous study which indicated that interexposure intervals of 60 and 120 min did not affect investigation durations, i.e. no recognition was noticed, while for interexposure intervals less than 60 min, the investigation time was reduced indicating short-term memory for a particular individual [3]. During each 5 min test, total duration of the investigatory behavior of the subject was measured by typing pre-set keys on the keyboard of an Apple II computer. To check the reliability of the scoring, test sessions were video-taped at regular intervals and scored by another observer. Investigations were defined as direct contacts of the adult with the juvenile: nosing, grooming, sniffing, pawing and close following [3]. Only the rats that reliably investigated the juveniles and did not display aggressive behavior were retained for the experiments and treat-
355 ments. Arginine vasopressin (Clin-Midy-Montpellier) was dissolved in 50 #1 of 0.01 M HC1 and then diluted in 0.9% saline. Before treatment, 8 rats were subjected to 5 sessions~ one per day, with intertrial intervals of 5, 10, 30, 120 and 120 rain, respectively. This procedure was undertaken to validate the behavioral model and to make sure that the rats showed normal recognition for intervals less than 30 min and no recognition for 120-rain intervals. Two days later, the treatment sessions began, 5 sessions, with an intersession delay of 2 days. The treatments were given immediately after the 5 min of the initial presentation as follows: 1st day half of the rats received saline, half0.5 ng of AVP, the treatments being reversed on the 3rd day, then the same procedure was given on the 5th and 7th day with the 1.0 and 2.0 ng doses of AVP. This increasing dosage schedule was selected in preference to a random dosage procedure to avoid problems of possible sensitization or toxicity resulting from injection of higher doses. On the 9th day, all the rats received 0.25 ng. For an injection, the dummy stylet was removed and a 30-gauge stainless-steel cannula with 30 cm of PE 10 tubing attached was inserted through the guide to 2 mm beyond the tip. One #1 of the peptide was injected by gravity over a 30-s period. Volume was measured by a calibrated PE l0 tubing. All experiments were performed using a blind procedure where the experimenter testing the rat was unaware of the treatment. At the end of the experiment, the placement of the cannula was controlled by injection of a dye solution. Seven rats in which injection was located in the ventricle and which had performed in all the experiments were included in the results. One rat died of unknown causes before the completion of the study. The results were computed for each session so that the investigation duration of the second exposure was compared to the investigation duration of the first exposure. Comparisons were also made between each of the treatment conditions for the second exposure. These data were compared using a two-way analysis of variance with repeated measures on two factors (first-exposure vs second exposure and no treatments vs treatments). Individual means comparisons within each treatment day were made using a simple main effects test, and individual means comparisons between different treatment days were made using the Newman Keuls test. These comparisons to the no-treatment baseline allow an assessment of any placebo effect which cannot be dismissed in any behavioral test that is sensitive to peptide effects, and the comparison to the placebo baseline allows one to assess the effects of treatment (AVP) per se. To test the hypothesis that motor activation might contribute to any behavioral changes observed in the social interaction task, separate groups of rats (n = 6 8 per group) were tested in photocell activity cages either during the light (16.00) or dark (24.00) period of the activity cycle. Male Wistar rats weighing 180 220 g were implanted with i.c.v, cannulas and then were habituated for 90 rain to photocell activity cages measuring 20 x 25 x 36 cm with twin infrared photocell beams, across the long axis 2 cm above the cage floor. After a 90-min habituation period the rats were removed from the cage and injected with 0, 0.5, 1.0 or 2.0 ng o f A V P i.c.v, and immediately placed back in the cages. Locomotor activity was measured every 5 rain for
356
SOCIALMEMORY
IO0
100
80
80
~
60
60
= ._o
40
40
~
20
20
C 0
~ C
._o
.N= 0 Inter-~'euntation Interval (rain)
l
+30
t
I
+120 +120 No Treatment
I
+120 SAL
I
I
I
+120 +120 +120 0.25 0.5 1.0 Dose of AVP ICV (panogramslrat)
I
+120 2.0
0
Fig. 1. Solid circles represent mean _ S.E.M. investigation duration for the second exposure on non-treatment and treatment days (n = 7). The solid line represents overall mean investigation duration during the first exposure of a given non-treatment or treatment day and the shaded area represents the overall average S.E.M. Note that all comparisons for statistical reliability involved within subject comparisons and the variance decribed by the S.E.M. reflects only the between-subject variance. Overall analysis of variance revealed a significant day × treatment interaction (F= 6.152, df = 7,42, P < 0.05) and simple main effects revealed no difference between the treatment days for the first exposure (F< 1) but a significant effect of treatments for the second exposure (F=2.518, df= 7,42, P<0.05). *Significant difference between the second exposure duration and the first exposure duration for a given treatment day, P < 0.05 simple main effects. *Significantly different from saline treatment for the second exposure only, P<0.05 Newmann Keuls test.
the next 2 h. D a t a were a n a l y z e d using a t w o - f a c t o r analysis o f v a r i a n c e ( A N O V A ) with r e p e a t e d m e a s u r e s on one factor, time. The results with the r e p e a t e d j u v e n i l e e x p o s u r e s h o w e d t h a t i n t e r p r e s e n t a t i o n intervals o f 30 min a n d less led to a s h o r t e r i n v e s t i g a t i o n d u r a t i o n , i n d i c a t i n g t h a t the stimulus a n i m a l was recognized (see Fig. 1, left side). F o r the 30-min interval, investig a t i o n d u r a t i o n decreased f r o m 6 9 _ 10 s for the first e x p o s u r e to 43-t-10 s for the second e x p o s u r e (Fig. 1, simple m a i n effects, F = 19.6, df, 1,42; P < 0 . 0 5 ) . In c o n t r a s t , when the i n t e r t r i a l interval was o f 120 min, the j u v e n i l e was no longer r e c o g n i z e d a n d the rats a c t u a l l y spent m o r e time in social investigation, t h a n in the initial e x p o sure (Fig. 1, s e c o n d 120 min). Here, the s e c o n d e x p o s u r e is significantly higher t h a n the first e x p o s u r e (simple m a i n effects, F = 8.486, d f 1,42, P < 0.05). A V P injected j u s t after the first e x p o s u r e p r o d u c e d a r e t r o a c t i v e facilitating effect o n social r e c o g n i t i o n so t h a t at the doses o f 0.5, 1.0 a n d 2 ng, the d u r a t i o n o f investig a t i o n for the s e c o n d e x p o s u r e was significantly lower t h a n the d u r a t i o n c o m p u t e d for the first e x p o s u r e ( P < 0.05, simple m a i n effects, respectively) even with a 120-min i n t e r e x p o s u r e interval, t n the case o f 1.0 ng, the s e c o n d e x p o s u r e d u r a t i o n o f investi-
357
gation was significantly lower than the mean duration of investigation after the saline treatment for the same second exposure (P < 0.05, N e w m a n - K e u l s test). There was a tendency for the 2-ng dose to be less effective than the l-rig dose, which is in accordance with the classical U-shaped curve dose-effect relationship generally obtained with behaviorally active peptides. While no toxic effects such as barrel rolling or increases in blood pressure have been observed in doses up to 1 ng per rat i.c.v., doses of 2 ng and above are at the threshold for non-specific effects and this may explain the present data. The performance induced by the 1-ng AVP administration was similar to the performance obtained after a short interexposure interval such as 30 min. Also, in previous studies this was the most effective dose of AVP for prolonging extinction of active avoidance [8] and in producing changes in electroencephalographic activity [5]. It is interesting to note that the saline administration induced by itself some reduction in the investigation duration compared to the pre-injection measures, but not compared to the first presentation in the same session. Also, the last dose injected was 0.25 ng, on the last day of the experiment for all the rats, and it did not modify significantly investigation duration at the second exposure. Finally, there was no significant change in the duration of the first investigation over the repeated sessions ( F = 1.0, dr-- 7,42). These doses of AVP did not modify locomotor activity as measured in photocell activity cages. Here a similar dose range (0.5 2.0 ng/rat) failed to alter activity when the rats were tested in both the quiescent (light) and active (dark) parts of their diurnal cycle, see Table I. Also previous results have shown that doses of AVP as high as 1.0 ng/rat in freely moving Wistar rats do not increase systemic blood pressure [8]. While these results do not rule out subtle changes in intervening variables such as 'arousal' [5], they do show that increases in locomotor activity or blood pressure are not likely candidates. The aim of this study was to investigate if AVP injected centrally had an effect on the 'social m e m o r y ' as measured by duration of investigation exhibited by adult rats during exposure to a juvenile. When tested in a retroactive enhancement procedure, AVP decreased the duration of investigation of the juvenile presented 120 min later, at a time when the juvenile was normally no longer recognized. These investigation times were of the same magnitude as those displayed when the interinvestigation TABLE I L O C O M O T O R A C T I V I T Y C O U N T S F O R 120 M I N F O L L O W I N G I.C.V. AVP *A two-way A N O V A with repeated measures over time revealed no significant dose or dose x time interaction ( F < 1.2, for all 4 comparisons). N u m b e r in parentheses refers to number of rats per group.
0 (Saline) 0.5 ng 1.0 ng 2.0 ng
Light (16.00 h)*
Dark (24.00h)*
330±54 413±93 417±93 356±35
1219±262 9 2 0 ± 163 1055± 114 1248± 154
(6) (6) (7) (7)
(6) (8) (7) (6)
358 interval was 30 min, for which the hypothesized transient ' m e m o r y ' was still persistent and objective. The present findings also support the validity o f this behavioral model as a powerful and reliable tool for learning and m e m o r y investigations in behavioral p h a r m a c o l o g y [3]. It has been shown that the duration o f social interaction is based on chemosensory characteristics o f the urine o f the juvenile [10] and is easily facilitated by further exposure to the same juvenile or impaired by exposure to another juvenile, i.e. it is sensitive to retroactive facilitation or interference [3]. These results reflect similar results as those observed with A V P when injected subcutaneously. In a previous study, A V P was behaviorally active at a dose which increased blood pressure and the behavioral effects were blocked by administration o f a V1-AVP receptor antagonist [3]. Other studies have shown that the behavioral actions o f central administration o f A V P are similar to those o f peripherally injected A V P but are not mediated by systemic physiological changes, although these actions are readily blocked by the V I - A V P receptor antagonist [8]. Using the same rat strain and measurements for up to 2 h post i.c.v, injection o f A V P (1 ng) revealed no changes in systemic blood pressure [8]. Further, these doses o f A V P do not produce significant effects on gross m o t o r activity. The present study confirms these results and reinforces the hypothesis we have formulated that a large range o f behavioral effects can be obtained f r o m both peripheral and central administration o f A V P at different doses (micrograms vs nanograms), but both m o d e s o f administration m a y act through different mechanisms. Moreover, because o f the nature o f the behavioral model used, this study focuses more precisely on a possible role o f central A V P on m e m o r y processes. Further studies ar required to reveal the specific neuronal site o f action for these changes in social interaction effects. This w o r k was supported by an I N S E R M grant to M . L . M . , R.D. and B.M., N I N C D S G r a n t 20912 to G . F . K . , C N R S G r a n t 920232 to M . L . M . and R.D., and N S F G r a n t BNS-8514490 to G . F . K . We thank Rose Marie Bluthe for her advice on the experimental procedure and the Basic and Clinical Research W o r d Processing Center at Scripps Clinic for manuscript preparation. This is publication n u m b e r BCR4587 from the Research Institute o f Scripps Clinic. 1 Bohus, G., Kovacs, G.L. and De Wied, D., Oxytocin, vasopressin and memory: opposite effects on consolidation and retrieval processes, Brain Res., 157 (1978) 414417. 2 Carr, W.J., Yee, L., Gable, D. and Marasco, E., Olfactory recognition ofconspecifics in domestic Norway rats, J. Comp. Physiol. Psychol., 90 (1976) 821 828. 3 Dantzer, R., Bluthe, R.M., Koob, G.F. and Le Moal, M., Modulation of social memory in male rats by neurohypophyseal peptides, Psychopharmacology, 91 (1987) 363--368. 4 De Wied, D., Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments, Life Sci., 19 (1976) 685-690. 5 Ehlers, C.E., Reed. T.K., Wang, M., Lebrun, C.J. and Koob, G.F., EEG effects of subcutaneous and intracerebroventricular injections of arginine vasopressin in the rat, Psychopharmacology, 87 (1985) 43~433. 6 Heise, G.A., Learning and memory facilitators: experimental definition and current status, Trends Pharmacol. Sci., 2 (1981) 158 160.
359 7 Koob, G.F., Le Moal, M., Gaffori, O., Manning, M., Sawyer, W.H., Rivier, J. and Bloom, F.E., Arginine wtsopressin and a vasopressin antagonist peptide: opposite effects in extinction of active avoidance in rats, Regul. Peptides, 2 (1981) 153 163. 8 Koob, G.F., Dantzcr, R., Bluthe, R.M., Lcbrun, C., Bloom, F.E. and Le Moal, M., Central injections of arginine vasopressm prolong extinction of active avoidance, Peptides, 7 (1986) 213 218. 9 Le Moal, M., Dantzer, R., Mormede, P., Baduel, A., Lebrun, C.J., Ettenberg, A., van der Kooy, D., Wenger, J., Deyo, S., Koob, G.F. and Bloom, F.E., Behavioral effects of peripheral administration of arginine vasopressin. A review of our search for a mode of action and a hypothesis, Psychoncuroendocrinology, 9 (1984) 319 341. 10 Sawycr, T.F., Hengehold, A.K. and Perez, W.A., Chemosensory and hormonal mediation of social memory in male rats, Behav. Neurosci., 98 (1984) 908 913. I I Thor, D.H. and Holloway, W.R., Social memory of the male laboratory rat, J. Comp. Physiol. Psychol., 96 (1982) 1000 1006.
353
Elsevier Scientific Publishers Ireland Ltd. NSL 04645
Centrally injected arginine vasopressin (AVP) facilitates social memory in rats M . L e M o a l 1, R . D a n t z e r l, B. M i c h a u d I a n d G . F . K o o b 2 ZlNRA, Psychobiologie des Comporternents Adaptatifs. U259 INSERM, Bordeaux (France) and "Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla. CA 92037 (U.S.A.)
(Received 28 November 1986; Revised version received and accepted 28 February 1987) Key word~." Arginine vasopressin; Social memory; Rat
"Memory' for a juvenile conspecific in male rats can be measured by variation in duration of investigation times when the same juvenile is presented at different intervals. Typically, exposure of an adult male rat to a juvenile results in transient investigatory activity that rapidly declines with repeated exposures at short interexposure intervals (30 min). Longer interexposure intervals (120 rain) result in re-investigation with durations similar or greater than the original investigation. Arginine vasopressin (AVP) injected into adult male rats intracerebroventricularly in doses of 0.5 2.0 ng immediately after investigation of the juvenile decreased social investigation of the same juvenile at the long (120 min) interexposure interval. This decrease in investigatory time was similar to that observed after a 30-min interexposure interval in untreated animals. These results support the hypothesis that increasing the availability of AVP in the central nervous system can improve the consolidation of olfactory information and improve conspecific recognition in rats.
S u b s t a n t i a l e v i d e n c e n o w s u p p o r t s the h y p o t h e s i s t h a t a r g i n i n e v a s o p r e s s i n ( A V P ) a n d related n e u r o p e p t i d e s are i n v o l v e d in a d a p t i v e b e h a v i o r s a n d even in l e a r n i n g a n d m e m o r y processes [1, 4, 7]. T h e s e effects h a v e b e e n d e m o n s t r a t e d b o t h after adm i n i s t r a t i o n o f m i c r o g r a m a m o u n t s o f A V P i n j e c t e d p e r i p h e r a l l y a n d after i n t r a c e r e b r o v e n t r i c u l a r (i.c.v.) a d m i n i s t r a t i o n o f n a n o g r a m a m o u n t s o f the p e p t i d e s [4, 7, 8]. These s t u d i e s i n d i c a t e a f a c i l i t a t i o n o f c o n s o l i d a t i o n a n d recall processes in aversively m o t i v a t e d t a s k s after p e r i p h e r a l a d m i n i s t r a t i o n o f the h o r m o n e , b u t o n l y a few h a v e d e m o n s t r a t e d such effects in a p p e t i t i v e l y m o t i v a t e d tasks, a n d p a r t i c u l a r l y after central a d m i n i s t r a t i o n [9]. O n e o f the p r o b l e m s m e t by such studies c o n c e r n s the a n i m a l m o d e l s g e n e r a l l y used in the e x p e r i m e n t a l s t u d y o f l e a r n i n g a n d m e m o r y [6]. A f o r m o f m e m o r y , very similar to f a c t u a l m e m o r y in h u m a n s a n d w h i c h has received little a t t e n t i o n , is recog-
Correspondence: M. Le Moal, INRA, Psychobiologie des Comportements Adaptatifs, U259 INSERM,
Rue Camille Saint-Saens, 33077 Bordeaux Cedex, France. 0304-3940/87/$ 03.50 (D 1987 Elsevier Scientific Publishers Ireland Ltd.
354 nition of conspecific identity that may be considered as an ethological model of social memory [2, 11]. For instance, adult male rats spend a great amount of time investigating novel juveniles; the rats exposed to the same juvenile 30 min alter the initial exposure display less investigatory behavior, but if the re-exposure occurs 2 h later, the juvenile is not recognized and is thoroughly investigated [I I]. We have recently demonstrated that this transient m e m o r y for a juvenile was sensitive to retroactive facilitation and retroactive interference, and was modulated by peripheral administration of microgram amounts of hypophyseai neuropeptides [3]. The present study was undertaken to see if such memory processes were modulated by central administration of AVP. The results indicate that AVP injected i.c.v, in nanogram amounts enhances the retention of such conspecific social interaction. The subjects were retired breeder male rats of the Wistar strain, 6 months of age. For the purpose of i.c.v, injections, they received at their arrival in the laboratory a cannula aimed above the lateral ventricle. For this surgery, rats were anesthetized with chloral hydrate (6 ml/kg, 6% solution) and secured in a K o p f stereotaxic instrument. A 23-gauge stainless steel guide cannula, 7 m m long, was lowered to within 1 mm of the ventricle and anchored to the skull with two stainless-steel screws and dental cement. Coordinates were with tooth bars 5 m m above interaural zero, 0.6 m m posterior to the bregma, 2.0 m m lateral and 3.2 m m below the skull surface at the point of entry. After surgery, the rats were housed individually in a 30 x 45 × 19 cm transparent plastic cage and maintained for two weeks before the onset of the experiments on ad libitum food and water in a temperature-controlled room under a reverse d a r k light cycle (light on: 20:30 to 8:30 h), Laboratory-reared juvenile Wistar male rats, 25-30 days old, were used as social stimulus. For each session, all the rats under investigation were placed 2 h before the test in an experimental room equipped with the same light cycle and with a background white noise. All the observations were made between 11.00 and 16.00 h and were carried out with a video camera using an infrared light. The test was conducted in the home cage of the male and consisted of a 5-min exposure to a given juvenile, followed by a second exposure of the same duration to the same juvenile, at given intertrial intervals for each session. Between the two successive presentations, the juveniles were kept individually in small glass jars containing fresh wood litter. The procedure was conducted according to a previous study which indicated that interexposure intervals of 60 and 120 min did not affect investigation durations, i.e. no recognition was noticed, while for interexposure intervals less than 60 min, the investigation time was reduced indicating short-term memory for a particular individual [3]. During each 5 min test, total duration of the investigatory behavior of the subject was measured by typing pre-set keys on the keyboard of an Apple II computer. To check the reliability of the scoring, test sessions were video-taped at regular intervals and scored by another observer. Investigations were defined as direct contacts of the adult with the juvenile: nosing, grooming, sniffing, pawing and close following [3]. Only the rats that reliably investigated the juveniles and did not display aggressive behavior were retained for the experiments and treat-
355 ments. Arginine vasopressin (Clin-Midy-Montpellier) was dissolved in 50 #1 of 0.01 M HC1 and then diluted in 0.9% saline. Before treatment, 8 rats were subjected to 5 sessions~ one per day, with intertrial intervals of 5, 10, 30, 120 and 120 rain, respectively. This procedure was undertaken to validate the behavioral model and to make sure that the rats showed normal recognition for intervals less than 30 min and no recognition for 120-rain intervals. Two days later, the treatment sessions began, 5 sessions, with an intersession delay of 2 days. The treatments were given immediately after the 5 min of the initial presentation as follows: 1st day half of the rats received saline, half0.5 ng of AVP, the treatments being reversed on the 3rd day, then the same procedure was given on the 5th and 7th day with the 1.0 and 2.0 ng doses of AVP. This increasing dosage schedule was selected in preference to a random dosage procedure to avoid problems of possible sensitization or toxicity resulting from injection of higher doses. On the 9th day, all the rats received 0.25 ng. For an injection, the dummy stylet was removed and a 30-gauge stainless-steel cannula with 30 cm of PE 10 tubing attached was inserted through the guide to 2 mm beyond the tip. One #1 of the peptide was injected by gravity over a 30-s period. Volume was measured by a calibrated PE l0 tubing. All experiments were performed using a blind procedure where the experimenter testing the rat was unaware of the treatment. At the end of the experiment, the placement of the cannula was controlled by injection of a dye solution. Seven rats in which injection was located in the ventricle and which had performed in all the experiments were included in the results. One rat died of unknown causes before the completion of the study. The results were computed for each session so that the investigation duration of the second exposure was compared to the investigation duration of the first exposure. Comparisons were also made between each of the treatment conditions for the second exposure. These data were compared using a two-way analysis of variance with repeated measures on two factors (first-exposure vs second exposure and no treatments vs treatments). Individual means comparisons within each treatment day were made using a simple main effects test, and individual means comparisons between different treatment days were made using the Newman Keuls test. These comparisons to the no-treatment baseline allow an assessment of any placebo effect which cannot be dismissed in any behavioral test that is sensitive to peptide effects, and the comparison to the placebo baseline allows one to assess the effects of treatment (AVP) per se. To test the hypothesis that motor activation might contribute to any behavioral changes observed in the social interaction task, separate groups of rats (n = 6 8 per group) were tested in photocell activity cages either during the light (16.00) or dark (24.00) period of the activity cycle. Male Wistar rats weighing 180 220 g were implanted with i.c.v, cannulas and then were habituated for 90 rain to photocell activity cages measuring 20 x 25 x 36 cm with twin infrared photocell beams, across the long axis 2 cm above the cage floor. After a 90-min habituation period the rats were removed from the cage and injected with 0, 0.5, 1.0 or 2.0 ng o f A V P i.c.v, and immediately placed back in the cages. Locomotor activity was measured every 5 rain for
356
SOCIALMEMORY
IO0
100
80
80
~
60
60
= ._o
40
40
~
20
20
C 0
~ C
._o
.N= 0 Inter-~'euntation Interval (rain)
l
+30
t
I
+120 +120 No Treatment
I
+120 SAL
I
I
I
+120 +120 +120 0.25 0.5 1.0 Dose of AVP ICV (panogramslrat)
I
+120 2.0
0
Fig. 1. Solid circles represent mean _ S.E.M. investigation duration for the second exposure on non-treatment and treatment days (n = 7). The solid line represents overall mean investigation duration during the first exposure of a given non-treatment or treatment day and the shaded area represents the overall average S.E.M. Note that all comparisons for statistical reliability involved within subject comparisons and the variance decribed by the S.E.M. reflects only the between-subject variance. Overall analysis of variance revealed a significant day × treatment interaction (F= 6.152, df = 7,42, P < 0.05) and simple main effects revealed no difference between the treatment days for the first exposure (F< 1) but a significant effect of treatments for the second exposure (F=2.518, df= 7,42, P<0.05). *Significant difference between the second exposure duration and the first exposure duration for a given treatment day, P < 0.05 simple main effects. *Significantly different from saline treatment for the second exposure only, P<0.05 Newmann Keuls test.
the next 2 h. D a t a were a n a l y z e d using a t w o - f a c t o r analysis o f v a r i a n c e ( A N O V A ) with r e p e a t e d m e a s u r e s on one factor, time. The results with the r e p e a t e d j u v e n i l e e x p o s u r e s h o w e d t h a t i n t e r p r e s e n t a t i o n intervals o f 30 min a n d less led to a s h o r t e r i n v e s t i g a t i o n d u r a t i o n , i n d i c a t i n g t h a t the stimulus a n i m a l was recognized (see Fig. 1, left side). F o r the 30-min interval, investig a t i o n d u r a t i o n decreased f r o m 6 9 _ 10 s for the first e x p o s u r e to 43-t-10 s for the second e x p o s u r e (Fig. 1, simple m a i n effects, F = 19.6, df, 1,42; P < 0 . 0 5 ) . In c o n t r a s t , when the i n t e r t r i a l interval was o f 120 min, the j u v e n i l e was no longer r e c o g n i z e d a n d the rats a c t u a l l y spent m o r e time in social investigation, t h a n in the initial e x p o sure (Fig. 1, s e c o n d 120 min). Here, the s e c o n d e x p o s u r e is significantly higher t h a n the first e x p o s u r e (simple m a i n effects, F = 8.486, d f 1,42, P < 0.05). A V P injected j u s t after the first e x p o s u r e p r o d u c e d a r e t r o a c t i v e facilitating effect o n social r e c o g n i t i o n so t h a t at the doses o f 0.5, 1.0 a n d 2 ng, the d u r a t i o n o f investig a t i o n for the s e c o n d e x p o s u r e was significantly lower t h a n the d u r a t i o n c o m p u t e d for the first e x p o s u r e ( P < 0.05, simple m a i n effects, respectively) even with a 120-min i n t e r e x p o s u r e interval, t n the case o f 1.0 ng, the s e c o n d e x p o s u r e d u r a t i o n o f investi-
357
gation was significantly lower than the mean duration of investigation after the saline treatment for the same second exposure (P < 0.05, N e w m a n - K e u l s test). There was a tendency for the 2-ng dose to be less effective than the l-rig dose, which is in accordance with the classical U-shaped curve dose-effect relationship generally obtained with behaviorally active peptides. While no toxic effects such as barrel rolling or increases in blood pressure have been observed in doses up to 1 ng per rat i.c.v., doses of 2 ng and above are at the threshold for non-specific effects and this may explain the present data. The performance induced by the 1-ng AVP administration was similar to the performance obtained after a short interexposure interval such as 30 min. Also, in previous studies this was the most effective dose of AVP for prolonging extinction of active avoidance [8] and in producing changes in electroencephalographic activity [5]. It is interesting to note that the saline administration induced by itself some reduction in the investigation duration compared to the pre-injection measures, but not compared to the first presentation in the same session. Also, the last dose injected was 0.25 ng, on the last day of the experiment for all the rats, and it did not modify significantly investigation duration at the second exposure. Finally, there was no significant change in the duration of the first investigation over the repeated sessions ( F = 1.0, dr-- 7,42). These doses of AVP did not modify locomotor activity as measured in photocell activity cages. Here a similar dose range (0.5 2.0 ng/rat) failed to alter activity when the rats were tested in both the quiescent (light) and active (dark) parts of their diurnal cycle, see Table I. Also previous results have shown that doses of AVP as high as 1.0 ng/rat in freely moving Wistar rats do not increase systemic blood pressure [8]. While these results do not rule out subtle changes in intervening variables such as 'arousal' [5], they do show that increases in locomotor activity or blood pressure are not likely candidates. The aim of this study was to investigate if AVP injected centrally had an effect on the 'social m e m o r y ' as measured by duration of investigation exhibited by adult rats during exposure to a juvenile. When tested in a retroactive enhancement procedure, AVP decreased the duration of investigation of the juvenile presented 120 min later, at a time when the juvenile was normally no longer recognized. These investigation times were of the same magnitude as those displayed when the interinvestigation TABLE I L O C O M O T O R A C T I V I T Y C O U N T S F O R 120 M I N F O L L O W I N G I.C.V. AVP *A two-way A N O V A with repeated measures over time revealed no significant dose or dose x time interaction ( F < 1.2, for all 4 comparisons). N u m b e r in parentheses refers to number of rats per group.
0 (Saline) 0.5 ng 1.0 ng 2.0 ng
Light (16.00 h)*
Dark (24.00h)*
330±54 413±93 417±93 356±35
1219±262 9 2 0 ± 163 1055± 114 1248± 154
(6) (6) (7) (7)
(6) (8) (7) (6)
358 interval was 30 min, for which the hypothesized transient ' m e m o r y ' was still persistent and objective. The present findings also support the validity o f this behavioral model as a powerful and reliable tool for learning and m e m o r y investigations in behavioral p h a r m a c o l o g y [3]. It has been shown that the duration o f social interaction is based on chemosensory characteristics o f the urine o f the juvenile [10] and is easily facilitated by further exposure to the same juvenile or impaired by exposure to another juvenile, i.e. it is sensitive to retroactive facilitation or interference [3]. These results reflect similar results as those observed with A V P when injected subcutaneously. In a previous study, A V P was behaviorally active at a dose which increased blood pressure and the behavioral effects were blocked by administration o f a V1-AVP receptor antagonist [3]. Other studies have shown that the behavioral actions o f central administration o f A V P are similar to those o f peripherally injected A V P but are not mediated by systemic physiological changes, although these actions are readily blocked by the V I - A V P receptor antagonist [8]. Using the same rat strain and measurements for up to 2 h post i.c.v, injection o f A V P (1 ng) revealed no changes in systemic blood pressure [8]. Further, these doses o f A V P do not produce significant effects on gross m o t o r activity. The present study confirms these results and reinforces the hypothesis we have formulated that a large range o f behavioral effects can be obtained f r o m both peripheral and central administration o f A V P at different doses (micrograms vs nanograms), but both m o d e s o f administration m a y act through different mechanisms. Moreover, because o f the nature o f the behavioral model used, this study focuses more precisely on a possible role o f central A V P on m e m o r y processes. Further studies ar required to reveal the specific neuronal site o f action for these changes in social interaction effects. This w o r k was supported by an I N S E R M grant to M . L . M . , R.D. and B.M., N I N C D S G r a n t 20912 to G . F . K . , C N R S G r a n t 920232 to M . L . M . and R.D., and N S F G r a n t BNS-8514490 to G . F . K . We thank Rose Marie Bluthe for her advice on the experimental procedure and the Basic and Clinical Research W o r d Processing Center at Scripps Clinic for manuscript preparation. This is publication n u m b e r BCR4587 from the Research Institute o f Scripps Clinic. 1 Bohus, G., Kovacs, G.L. and De Wied, D., Oxytocin, vasopressin and memory: opposite effects on consolidation and retrieval processes, Brain Res., 157 (1978) 414417. 2 Carr, W.J., Yee, L., Gable, D. and Marasco, E., Olfactory recognition ofconspecifics in domestic Norway rats, J. Comp. Physiol. Psychol., 90 (1976) 821 828. 3 Dantzer, R., Bluthe, R.M., Koob, G.F. and Le Moal, M., Modulation of social memory in male rats by neurohypophyseal peptides, Psychopharmacology, 91 (1987) 363--368. 4 De Wied, D., Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments, Life Sci., 19 (1976) 685-690. 5 Ehlers, C.E., Reed. T.K., Wang, M., Lebrun, C.J. and Koob, G.F., EEG effects of subcutaneous and intracerebroventricular injections of arginine vasopressin in the rat, Psychopharmacology, 87 (1985) 43~433. 6 Heise, G.A., Learning and memory facilitators: experimental definition and current status, Trends Pharmacol. Sci., 2 (1981) 158 160.
359 7 Koob, G.F., Le Moal, M., Gaffori, O., Manning, M., Sawyer, W.H., Rivier, J. and Bloom, F.E., Arginine wtsopressin and a vasopressin antagonist peptide: opposite effects in extinction of active avoidance in rats, Regul. Peptides, 2 (1981) 153 163. 8 Koob, G.F., Dantzcr, R., Bluthe, R.M., Lcbrun, C., Bloom, F.E. and Le Moal, M., Central injections of arginine vasopressm prolong extinction of active avoidance, Peptides, 7 (1986) 213 218. 9 Le Moal, M., Dantzer, R., Mormede, P., Baduel, A., Lebrun, C.J., Ettenberg, A., van der Kooy, D., Wenger, J., Deyo, S., Koob, G.F. and Bloom, F.E., Behavioral effects of peripheral administration of arginine vasopressin. A review of our search for a mode of action and a hypothesis, Psychoncuroendocrinology, 9 (1984) 319 341. 10 Sawycr, T.F., Hengehold, A.K. and Perez, W.A., Chemosensory and hormonal mediation of social memory in male rats, Behav. Neurosci., 98 (1984) 908 913. I I Thor, D.H. and Holloway, W.R., Social memory of the male laboratory rat, J. Comp. Physiol. Psychol., 96 (1982) 1000 1006.