Central injections of arginine vasopressin prolong extinction of active avoidance

Peptides, Vol. 7, pp. 213-218, 1986. ©Ankho InternationalInc. Printed in the U.S.A.

0196-9781/86 $3.00 + .00

Central Injections of Arginine Vasopressin Prolong Extinction of Active Avoidance G E O R G E F. K O O B , R O B E R T D A N T Z E R , * R O S E - M A R I E B L U T H I ~ , * C H R I S T I N E L E B R U N , F L O Y D E. B L O O M A N D M I C H E L L E M O A L *

Division of Preclinical Neuroscience and Endocrinology, Scripps Clinic and Research Foundation 10666 North Torrey Pines Road, La Jolla, CA 92037 and *INSERM UnitO 259, Psychobiologie des Comportements Adaptatifs, Domaine de Carreire Rue Camille Saint-Saens, 33077 Bordeaux, France R e c e i v e d 16 O c t o b e r 1985 KOOB, G. F., R. DANTZER, R.-M. BLUTHI~, C. LEBRUN, F. E. BLOOM AND M. LE MOAL. Centralinjections of arginine vasopressinprolong extinctionof active avoidance. PEPTIDES 7(2) 213-218, 1986.--Behavioral and physiological effects of arginine vasopressin (AVP) were examined following intracerebroventricular (ICV) injection in the rat. ICV injections prolonged extinction of active avoidance at doses of 1.0 and 10.0 ng/rat and this effect was blocked by peripheral injection of the vasopressor antagonist of vasopressin [dPtyr(Me)AVP] at a dose of 30/zg/kg (SC). However, 1.0 ng of AVP ICV failed to alter systemic blood pressure and also failed to produce taste aversions in a one or two bottle test. Results suggest that central AVP has a central action independent of systemic changes in blood pressure, but that the receptor mediating this action is functionally similar to the AVP VI (vasopressor) receptor. Arginine vasopressin

Active avoidance

Extinction

E X T E N S I V E work exists to show that systemic administration of arginine vasopressin (AVP) and vasopressin analogs can delay extinction or improve learning consolidation [6, 7, 15, 18]. AVP prolongs extinction of active avoidance whether injected during extinction [6,15] or whether injected immediately after acquisition [7]. AVP also improves performance in inhibitory (passive) avoidance tasks [3,18] and in simple appetitive learning situations [12]. These results have generally been interpreted as an effect of AVP on the central nervous system since much smaller amounts of AVP administered by intracerebroventricular (ICV) injections produce similar effects [7]. However, recent studies using a vasopressor antagonist of vasopressin [1] have challenged the notion of a central action for systemically administered AVP [20]. In brief, the doses of the AVP antagonist required to antagonize the effects of systemically administered AVP are the same whether the antagonist is administered systemically [20] or intracerebroventricularly [19]. Moreover, these doses of the antagonist are identical to those required to block the increases in blood pressure [19] and the unconditioned aversive effects of AVP [14]. These observations suggested that: (a) peripheral sites mediate the action of AVP when injected systemically; and (b) the vasopressor antagonist (which is significantly more lipophilic than AVP itself) readily leaves the central nervous system to act at those peripheral sites. However, those experiments cannot determine whether there may also be additional centrally mediated behavioral

Blood pressure

effects of AVP or whether the antagonist may be able to act on those putative central receptors. Unknown at this time is whether the effects of centrally administered AVP may also be mediated by systemic physiological changes. The purpose of the present study was to test this hypothesis by examining the effects of very low doses of AVP administered ICV on avoidance behavior, blood pressure and conditioned taste aversion and to examine the effects of systemic administration of the AVP antagonist on the behavioral actions of centrally injected AVP. We confirm here that ICV injection of AVP in doses above 1 ng/rat does prolong extinction of active avoidance and we report that this prolongation of extinction can be blocked by systemic administration of an AVP pressor antagonist. However, this same dose of AVP does not raise systemic blood pressure nor does it produce a conditioned taste aversion. These results suggest that the action of centrally administered AVP is directly in the central nervous system on receptors similar to the VI vasopressor receptor. METHOD

Subjects The subjects were male Wistar rats weighing between 120--200 g at the time of surgery. All animals were group housed in a temperature controlled room under a 12 hr light/ dark cycle (0800--2000 hr light). Rats initially were subjected

213

KOOB ET AL.

214 to 2 min of handling at 2-5 days after arrival in the laboratory.

Peptides Arginine vasopressin and an analog of vasopressin, [l-deaminopenicillamine-2-(0-methyl)tyrosine]-argininevasopressin [dPtyr(Me)AVP] were synthesized at the Clayton Foundation Laboratory for Peptide Biology of the Salk Institute by Dr. Jean Rivier. This peptide analog of AVP has been fully characterized as a specific antagonist of the pressor actions of exogenous vasopressin in urethane-anesthetized phenoxybenzamine-pretreated rats [1]. In the present study, before injection, peptides were first dissolved in 50/xl of 0.01 M HCI and then further diluted in 0.9% saline.

Surgery and Injection Procedure For the subcutaneous injections, rats were injected in the neck with a constant volume of 0.5 ml of peptide solution. Control rats received saline alone. For the intracerebroventricular (ICV) injections, rats were equipped with a cannula aimed above the lateral ventricle. For this surgery, rats were anesthetized with chloral hydrate anesthesia (6.5 ml/kg 6% solution) and secured in a Kopf stereotaxic instrument. A stainless steel guide cannula, made of 23 gauge stainless steel tubing (in diameter) and 7 mm 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 the tooth bar 5 mm above interaural zero, 0.6 mm posterior to bregma, 2.0 mm lateral and 3.2 mm below skull surface at the point of entry. 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 I mm beyond its tip. One p~l of peptide was injected by gravity over a 30-sec period simply by raising the tubing above the head of the rat until flow began. Volume was measured by marks on the PE 10 tubing previously calibrated with a 5/zl Hamilton syringe. Only those rats whose cannulae flowed easily with this technique were used in the experiment. All experiments were performed using a blind procedure where the experimenter testing the rats was unaware of the subject's treatment.

Active Avoidance The animals were trained and tested in a Skinner box (BRS/LVE) 25 x 30x 27 cm that contained an electrified grid floor for administering shock and had been modified for the "pole-jump" active avoidance task. The grid floor was composed of 16 bars, 0.5 cm in diameter and spaced 2 cm apart. A wooden pole, darkened with ink and 1.67 cm in diameter, extended from the floor to the center of the cage ceiling. A 40 watt incandescent light bulb was centered just above the clear Plexiglas ceiling and served as the conditioned stimulus (CS). For acquisition each rat was subjected to 10 trials per day for three days. The intertrial interval was 40, 60, or 80 sec with an average of 60 sec. A trial began with illumination of the CS for 5 sec; 5 seconds after the onset ofCS a scrambled AC shock (0.30-0.5 mA; 0.3 mA day 1, 0.4 mA day 2 and 0.5 mA day 3) was delivered to the grid floor for 30 sec or until

the rat jumped on the pole. During the first 3 trials on the first day of acquisition, rats failing to jump on the pole within 5-10 sec were gently placed on the pole by the experimenter. Further, throughout training, rats failing to climb down the pole within 30 sec after jumping were gently turned around on the pole and thus forced to return to the grid floor. All testing was conducted in a darkened room between 1000 and 1900 hr. For evaluation of extinction, the same procedure was employed except that the shock was always omitted and the rats were subjected to 10 trials every 2 hr during the course of the 4th day. Before utilizing rats for drug injections a selection procedure was employed similar to that reported by De Wied and associates [30]. Only rats that made 15 or more avoidance responses during acquisition and 7 or more successful avoidances during the first 10 extinction trials were treated with peptide or placebo immediately after these first 10 extinction trials and continued in the experiment through further extinction trials. Peptide was only injected after the first 10 extinction trials. This procedure was used to correspond to earlier studies using peripheral (systemic) administration of AVP [14, 18-20]. Two experiments were conducted, the first to determine a dose effect function for ICV AVP and the second to test the hypothesis that the effects of centrally administered AVP could be reversed by dPtyr(Me)AVP. In the first experiment, 47 rats were divided into 4 groups, each group receiving a different dose of AVP: 0, 0.1, 1.0 and 10.0 ng/rat. In the second experiment, 66 rats were divided into 3 groups, one group receiving saline ICV and saline subcutaneously (SC), the second group receiving AVP-I ng ICV and saline SC and the third group receiving AVP-1 ng ICV and dPtyr(Me)AVP6/.tg/kg SC.

Conditioned Taste A version The test used in the first experiment was a one bottle test involving the presentation of a novel solution of sweetened milk to mildly deprived rats. Twenty-seven rats weighing 200 g were implanted with ICV cannulas. Seven days later they were deprived of water and food for 24 hr only on the first day and subsequently were allowed ad lib food and water with water deprivation 30 rain prior to the test. For the first 5 days the rats were allowed to drink a 20% saccharin solution for 15 min. The conditioning began on day 6 when the rats were allowed 15 min access to the sweetened milk solution ( 1 volume of NestlEs concentrated sweetened milk to 2 volumes of water). Immediately after the test the rats received an injection of I btl ICV containing 0 (n=7), 0.1 ng (n=7), 1.0 ng (n=7), or 10.0 ng of AVP (n=6). In the second experiment, a two bottle test was used. Rats were water deprived and habituated for 7 days to drinking water for 30 min per day in a test cage. On the eighth day the rats were presented with a 0.1% saccharin solution for 30 min and then immediately injected ICV with 1 /zl saline (n=6) or 1 ng of AVP in 1~1 of saline (n=6). On the ninth day they were presented with water. The same cycle was repeated with the rats being presented with saccharin and injected with ICV AVP on day 10 and presented with water on day I I. On day 12 the rats were given a two bottle test, one bottle containing water, the other 0.1% saccharin.

Measurement of Blood Pressure The rats were anesthetized with sodium pentobarbital (50

AVP EFFECTS ON ACTIVE AVOIDANCE

EXTINCTION

215

TABLE I injections s.c.~ ~i.c.v.

EFFECTS OF AVP ICV ON EXTINCTION OF ACTIVE AVOIDANCE

[ ~ SALINE + SALINE (N=24) SALINE+AVP I n9 i.c.v. (N=I8) dPTyr(Me) AVP 30.ug/k9 + AVP 1 no i.c.v. (N=24)

Extinction Session (10 trials) in hours

Saline n=12 0.1 ng n=12 1.0 ng n=12 10.0 ng n = 11

I0-

0t

2

4

9.3 ± 0.9

5.0 ± 0.8

2.0 ± 0.8

0.7 ± 0.6

o~

9.7 ± 0.7

4.8 ± 0.8

2.2 ± 0.8

1.7 ± 0.6

m~

9.9 _+ 0.3

7.7 ± 0.9*

4.7 ± 0.9*

2.9 ± 0.7*

9.8 + 0.4

7.4 _+ 0.6*

3.9 ± 1.1"

2.8 ± 0.8*

~U z z z,~

1"injection 0

2

4

6

EXTINCTION (hrs)

Values represent mean ± S.E.M. *Significantly different from saline control group, p<0.05 Duncan's Multiple Range test following analysis of variance (significant dose effect, but no dose x hours interaction). +Rats were injected immediately after the first extinction session.

FIG. 1. The effects of AVP ICV and AVP 1CV plus dPtyr(Me)AVP SC on extinction of active avoidance. *Significantly different from saline and antagonist group, p<0.05, Duncan's Multiple Range test following analysis of variance.

TABLE 2 C O N D I T I O N E D T A S T E A V E R S I O N P R O D U C E D BY ICV I N J E C T I O N OF V A S O P R E S S I N

Groups Amt. of milk consumed first day (ml)

Saline n=7

0.1 ng n=7

1.0 ng

n=7

10.0 ng n=6

9.01 ± 1.30

6.89 ± 0.76

7.04 _+ 2.17

11.50 ± 1.85

Amt. of milk consumed--difference between Day 1 and each subsequent day. Day 2 3 4 5

4.95 5.85 7.10 10.12

+ ± ± ±

1.64 1.64 1.76 1.50

7.15 7.67 8.08 9.59

_+ 1.26 ± 1.21 ± 1.10 ± 1.14

3.64 4.02 6.15 8.50

_+ 1.19 ± 1.15 ± 1.00 ± 1.20

2.09 2.07 3.45 3.95

_+ 0.91" ± 1.13" ± 1.29" ± 0.99*

Values represent mean ± S.E.M. *Significantly different from the other groups, p<0.05, Duncan's Multiple Range test following analysis of variance.

mg/kg) i n t r a p e r i t o n e a l l y . F o r m e a s u r e m e n t s o f b l o o d pressure in c o n s c i o u s a n i m a l s , the a b d o m i n a l a o r t a w a s c a n n u lated with PE-10 p o l y e t h y l e n e t u b i n g t h r o u g h the left femoral a r t e r y a n d tied in place a b o u t 2.5 c m a b o v e the e n t r a n c e to the a b d o m i n a l cavity. T h e t u b i n g filled w i t h h e p a r i n i z e d saline (20 units/ml) w a s t h e n plugged distally a n d led t h r o u g h the s u b c u t a n e o u s s p a c e to exit t h r o u g h a d o r s a l incision at the i n t e r s c a p u l a r level. B e g i n n i n g 24 h r or m o r e a f t e r c a n n u l a t i o n , e a c h rat was placed in a s o u n d a t t e n u a t e d e x p e r i m e n t a l c h a m b e r and the c a n n u l a from the left f e m o r a l a r t e r y c o n n e c t e d to a S t a t h a m p r e s s u r e t r a n s d u c e r (P2310) a n d m o n i t o r e d with a B u x c o ( Q u i n c y , MA) c a r d i o v a s c u l a r a n a l y z e r for h e a r t rate, a n d systolic, diastolic a n d m e a n arterial p r e s s u r e . A f t e r recording at least 15 min, 1/zl of a solution containing 1 ng o f A V P was injected ICV. In a n o t h e r group, 5/zg/kg A V P was injected SC. R e c o r d i n g w a s c o n t i n u e d for 60 m i n a f t e r the injections. In a s e c o n d e x p e r i m e n t , to replicate the c o n d i t i o n s o f the b e h a v ioral e x p e r i m e n t s , A V P (1 ng) w a s injected ICV after a 15 min r e c o r d i n g s e s s i o n a n d t h e n m o n i t o r e d for 15 min at 2 h r i n t e r v a l s for 6 hr.

Statistical Analysis F o r the b l o o d p r e s s u r e e x p e r i m e n t s , d a t a for 3 min r e a d i n g s were e x p r e s s e d as the m e a n - S . E . M , and d i f f e r e n c e s b e t w e e n p r e - i n j e c t i o n a n d p o s t - i n j e c t i o n were e v a l u a t e d using a t w o f a c t o r r e p e a t e d m e a s u r e s a n a l y s i s o f v a r i a n c e ( A N O V A ) w i t h r e p e a t e d m e a s u r e s o n t w o factors, b l o c k (I b l o c k o f t h r e e time p o i n t s p r e - i n j e c t i o n a n d 6 b l o c k s o f t h r e e time p o i n t s p o s t - i n j e c t i o n ) and time ( e a c h 3 min m e a s u r e ) . F o r the b e h a v i o r a l e x p e r i m e n t s r e s u l t s were a n a l y z e d using a t w o f a c t o r a n a l y s i s o f v a r i a n c e w i t h r e p e a t e d m e a s u r e s o n o n e factor, h o u r s . S u b s e q u e n t individual m e a n s c o m p a r i s o n s were m a d e u s i n g a single f a c t o r A N O V A a n d D u n c a n ' s Multiple R a n g e a posteriori test [11]. RESULTS

A V P injected I C V p r o d u c e d a p r o l o n g a t i o n o f e x t i n c t i o n at d o s e s of 1.0 a n d 10 ng/rat (see T a b l e 1). A t the z e r o h o u r time p o i n t t h e r e w a s n o significant difference b e t w e e n any o f

216

KOOB E T A L . s.c. %ug/kg (n=6) uJ n~ lu n. 0.

0

AVP

160

0 i.c.v. 1 ng/rat (n=6)

170

160

150

._I

120

I

I

I

-16.5 -10.5 -4.5

I

I

0

4.5

I

I

I

I

I

I

I

I

I

10.5 16.5 22.5 28.,5 34.5 40.5 46.5 52.5 58.5 TI ME (rain}

FIG. 2. Effects of AVP injected 1CV and systemically on systolic blood pressure. Rats were monitored for at least 15 min before injection, injected with AVP and 9 min later monitored for I hr. Values represent the mean and S.E.M. ANOVA revealed a significant block effect (I block of three measures pre-injection versus six blocks postinjection) in the rats treated with AVP SC, see text. Abscissa begins at +4.5 min because readings were taken every 3 rain continuously except for a 9 min period during which the rat received an ICV injection. 4.5 min was the first 3 min reading post-injection and only every other reading is shown for graphic purposes.

I

VP

i.c.v.

injections

! ng/rot (n=8)

130 w I1= ~ 120

o~II0 ..I >- I 0 0 ¢/) MINUTES

HOURS

l

I

i

¢

0

3

6

9 0

.

,

12 15

0

i

I

,

3

6

9 2

.

i

12 15

I

I

I

I

0

3

6

9 4

I

I

12 15

I

I

I

I

0

3

6

9

I

I

12 15

6

FIG. 3. Blood pressure in rats monitored for 15 min prior to and at 2 hr intervals after injection with l ng of A V P 1CV. Values represent mean_+S.E.M. Zero time points refers to the time period 0-3 rain post-injection.

the doses, F(3,43)=2.07, p >0.05. Analysis of the time points post-injection (2, 4 and 6 hr) using a two factor analysis of variance revealed a significant dose effect, F(3,43)=3.36, p<0.05, and a significant time effect, F(2,86)=91.50, p<0.05. Subsequent individual means comparisons collapsed across time revealed a significant difference between the 1.0 and I0.0 nanogram doses and saline (p<0.05, Duncan's Multiple Range test). In the second experiment, 1 ng of AVP injected ICV again reliably prolonged extinction of active avoidance (see Fig. 1). This prolongation of extinction was blocked by simultaneous SC injection of 30 /zg/kg of dPtyr(Me)AVP. These effects were evident only at the 4 and 6 hr time points when the saline injected rats had extinguished (one way ANOVAs, 4 hours: F(2,62)=6.117, p<0.05; 6 hours: F(2,62)= 16.551, p<0.05). AVP injected ICV produced a taste aversion in the one bottle test only at the 10 ng dose, F(3,23)=4.06, p<0.05 (see Table 2). The 0.1 and 1.0 ng dose failed to alter reliably the

amount of sweetened milk consumed. The taste aversion produced by 10.0 ng was accompanied by barrel-rolling immediately following injection in 5 out of 6 rats injected. In the two bottle test, two pairings of I ng of AVP failed to decrease saccharin intake relative to controls. On the test day the saline injected animals drank 4.3_ + 1.9 ml water and 14.4-+1.7 ml (mean-+S.E.M.) of saccharin and the AVP injected rats drank 3.9-+1.6 ml water and 14.4-+2.3 ml of saccharin (means_+S.E.M.s) ( F < 1 for both group effect and group × days interaction, analysis of variance). AVP injected systemically in a dose of 6/.~g/kg produced a strong taste aversion in an identical 2 pairing procedure [2]. AVP at the 1 ng dose failed to alter systemic blood pressure immediately after the injection, F(6,30)=1.01, p>0.05 (see Fig. 2). Two rats injected with saline 1CV showed exactly the same pattern observed with the 6 rats injected with AVP ICV. In contrast, AVP administered systemically (5 /xg/kg SC) produced a large and prolonged increase in blood pressure (see Fig. 2). There were no

AVP E F F E C T S ON ACTIVE A V O I D A N C E EXTINCTION changes in blood pressure during the period when the rats would be tested during extinction, F(3,21)=1.41, p>0.05 (see Fig. 3). DISCUSSION The present results replicate previous studies showing that arginine vasopressin injected in the nanogram range can prolong extinction of active avoidance in rats [7,15]. In the present study a dose of 0.1 ng was ineffective. Similar results have been reported for performance in an inhibitory (passive) avoidance task [5,8] and generally support the hypothesis that increased availability of AVP in the central nervous system stimulates memory function [29]. The results showing that systemic dPtyr(Me)AVP can reverse the effects of centrally administered AVP suggested the possibility that the effects of AVP may be related to systemic changes in blood pressure triggered by the central injection. To test this hypothesis, the blood pressure of rats was monitored after similar injections. No effects were observed at the behaviorally active dose of 1 ng of AVP either immediately after injection or at 2 hr intervals after injections. Others have observed small changes in systemic blood pressure following injection of AVP ICV in larger doses or with more local injections in the brainstem [22,24]. Also, systemically injected AVP readily produces both conditioned taste aversions and place aversions [13, 14, 21, 26] and these peripherally generated effects can apparently per se stimulate the AVP effects on memory performance. However, in the present study an injection of 1 ng of AVP ICV failed to produce conditioned taste aversions in either a one bottle or two bottle test, suggesting that this behaviorally active central dose does not trigger the same internal cues as systemically administered AVP. Thus unresolved, is how the vasopressin antagonist injected systemically can reverse the behavioral effect of intracerebroventricularly administered AVP. One possibility is that the more lipophilic antagonist readily crosses the blood brain barrier whereas AVP does not [9]. For example, the AVP antagonist dPtyr(Me)AVP, when injected intracerebroventricularly, reverses the effects of AVP administered systemically but only at doses sufficient to reverse the peripheral pressor effects [19]. Small doses of this antagonist given ICV either in combination with AVP [19] or given alone [15] have no effect on extinction of active avoidance. Similar effects have been observed for the facilitation of performance in the water finding task [14]. These results show that this antagonist can readily exit from the brain, but do not preclude central actions occurring during the efflux from central sites. These results document previous results reported in part by de Wied et a l . , but our conclusions differ dramatically. We hypothesize that there are two AVP response systems, one central and the other peripheral. Although they may influence similar behavioral actions (i.e., prolongation of extinction), they do so by different and independent

217 mechanisms. One potential problem for this hypothesis has been the repeated observation that analogs of AVP with minimal physiological activity also produce behavioral effects similar to AVP itself [5, 29, 30]. While these effects are of certain interest, their site and mechanism of action remain to be determined. F o r example, desglycinamide arginine vasopressin does not bind to central AVP receptors [10] suggesting a unique site of action for this synthetic analog. Also, its possible formation, while reported, has not been established under conditions where catabolism of AVP is prevented. One hypothesis for the site and mechanism of action of AVP located in the central nervous system [4] is that AVP acts on noradrenergic systems. AVP injected into the dorsal septal nucleus, or dorsal hippocampus facilitated consolidation in passive avoidance [16], and destruction of these structures as well as destruction of the noradrenergic projections to them prevents the facilitory effects of AVP on avoidance behavior [16, 17, 27, 28]. According to this hypothesis, AVP would presumably bind to neuronal receptors to effect its behavioral action. Further evidence for the hypothesized separation of the peripheral versus central AVP systems is the observation that lesions of the dorsal noradrenergic bundle fail to alter the aversive effects of AVP [26]. An alternative hypothesis would be that AVP acts via some physiological means, for example local vasoconstriction to alter neuronal activity. The excitation of central neurons produced by AVP is blocked by a pressor antagonist of AVP [23], and all behavioral effects of AVP studied to date are blocked by similar V-1 antagonists. Finally, a recent study has suggested that AVP can also induce local microvascular vasoconstriction in the hippocampal slice [25]. Further exploration of this hypothesis might explain how this peptide modulates neuronal activity in limbic regions and produces its memory-enhancing actions; however, even this hypothesis would not necessarily establish these effects of exogenous AVP as mediating a physiological role in memory. To definitively establish a functional role for central endogenous AVP will require a means of specifically activating or inactivating these central neuronal systems.

ACKNOWLEDGEMENTS Preliminary reports of portions of these experiments were presented at the 12th and 13th Annual Meeting of the Society for Neuroscience (Le Moal, Koob, Mormede, Dantzer and Bloom. Soc Neurosci Abstr 8: 368, 1982; Koob, Dantzer, Mormede, Bluthe, Bloom and Le Moal. Soc N e u r o s c i Abstr 9: 202, 1983). We thank Dr. Jean Rivier for providing both AVP and dPtyr(Me)AVP and the Basic and Clinical Research Word Processing Center for manuscript preparation. This work was supported by NINCDS grant NS 20912-01 to G.F.K.; by CNRS/NSF grant INT 8215308) to M.L.M. and G.F.K. and INSERM CL grant to M.L.M. This is publication 4129BCR from the Research Institute of Scripps Clinic, La Jolla, CA.

REFERENCES

1. Bankowski, K., M. Manning, J. Haldar and W. H. Sawyer. Design of potent antagonists of the vasopressor response to arginine vasopressin. J M e d Chem 21: 850-853, 1978. 2. Bluth~, R.-M., R. Dantzer and M. Le Moal. Peripheral injection of vasopressin controls behavior by way ofinteroceptive signals for hypertension. Behav Brain Res 18: 31-39, 1985.

3. Bohus, G., G. L. Kovacs and D. De Wied. Oxytocin, vasopressin and memory: Opposite effects on consolidation and retrieval processes. Brain R e s 157: 414-417, 1978. 4. Buijs, R. M. Vasopressin and oxytocin: their role in neurotransmission. P h a r m a c o l Ther 22: 127-141, 1983.

218 5. Burbach, J. P. H., G. L. Kovacs, D. De Wied, J. W. van Gispen and H. M. Greven. A major metabolite or arginine vasopressin in the brain is a highly potent neuropeptide. Science 221: 1310-1312, 1983. 6. De Wied, D. Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature 232: 58--60, 1971. 7. De Wied, D. Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments. Life Sci 46: 27-29, 1976. 8. De Wied, D., O. Gaffori, J. M. van Ree and W. deJong. Central target for the behavioral effects of vasopressin neuropeptides. Nature 305: 276--278, 1984. 9. Deyo, S. N., W. J. Shoemaker, A. Ettenberg, F. E. Bloom and G. F. Koob. Subcutaneous administration of behaviorally effective doses of arginine vasopressin change brain AVP content only in median eminence. Neuroendocrinology 42: 260-266, 1986. 10. Dorsa, D. M., F. M. Petracca, D. G. Baskin and L. E. Cornett. Localization and characterization of vasopressin binding sites in the amygdala of the cat brain. J Neurosci 4: 1764-1770, 1984. 11. Duncan, D. B. Multiple range and multiple F tests. Biometrics 11: 1-42, 1955. 12. Ettenberg, A., M. Le Moal, G. F. Koob and F. E. Bloom. Vasopressin potentiation in the performance of a learned appetitive task: Reversal by a pressor antagonist analog of vasopressin. Pharmacol Biochem Behav 18: 645-647, 1983. 13. Ettenberg, A., K. van der Kooy, M. Le Moal, G. F. Koob and F. E. Bloom. Can aversive properties of peripherally-injected vasopressin account for its putative role in memory? Behav Brain Res 7: 331-350, 1983. 14. Ettenberg, A. Intracerebroventricular application of a vasopressin antagonist prevents the 'memory' and 'aversive' actions of vasopressin. Behav Brain Res 14:201-211, 1984. 15. Koob, G. F., M. Le Moal, O. Gaffori, M. Manning, W. H. Sawyer, J. Rivier and F. E. Bloom. Arginine vasopressin and a vasopressin antagonist peptide: Opposite effects in extinction of active avoidance in rats. Regul Pept 2: 153-163, 1981. 16. Kovacs, G. L., B. Bohus and D. H. G. Versteeg. Facilitation of memory consolidation by vasopressin: mediation by terminals of the dorsal noradrenergic bundle? Brain Res 172: 73-85, 1979. 17. Kovacs, G. L., B. Bohus, D. H. G. Versteeg, E. R. De Kloet and D. De Wied. Effect of oxytocin and vasopressin on memory consolidation sites of action and catecholaminergic correlates after microinjection into limic-midbrain structures. Brain Res 175: 303-314, 1979. 18. Lebrun, C. J., J. L. Rigter, J. Martinez, Jr., G. F. Koob, M. Le Moal and F. E. Bloom. Antagonism of effects of vasopressin (AVP) an inhibitory avoidance by a vasopressin antagonist peptide [(dPtyr(Me)AVP]. Life Sci 35: 1505-1512, 1984.

KOOB ET AL. 19. Lebrun, C. J., M. Le Moal, G. F. Koob and F. E. Bloom. Vasopressin pressor antagonist injected centrally reverses behavioral effects of peripheral injection of vasopressin, but only at doses that reverse increase in blood pressure. Regul Pept 11: 173-181, 1985. 20. Le Moal, M., G. F. Koob, L. Y. Koda, F. E. Bloom, M. Manning, W. H. Sawyer and J. Rivier. Vasopressor receptor antagonist prevents behavioral effects of vasopressin. Nature 291: 491-493, 1981. 21. Le Moal, M., R. Dantzer, P. Mormede, A. Baduel, C. J. Lebrun, A. Ettenberg, D. van der Kooy, J. Wenger, S. N. Deyo, G. F. Koob and F. E. Bloom. Behavioral effects of peripheral administration of arginine vasopressin. A review of our search for a mode of action and a hypothesis. Psyehoneuroendoerinology 3: 319-341, 1984. 22. Matsuguchi, H., F. M. Sharabi, F. J. Gordon, A. K. Johnson and P. G. Schmid. Blood pressure and heart rate responses to microinjection of vasopressin into the nucleus tract'us solitarius region of the rat. Neuropharmacology 21: 687-693, 1982. 23. Muhlethaler, M., J. J. Dreifuss and B. H. Gahwiler. Vasopressin excites hippocampal neurons. Nature 196: 74% 751, 1982. 24. Pittman, O. J., D. Lawrence and L. McLean. Central effects of arginine vasopressin on blood pressure in rats. Endocrinology ll0: 1058--1060, 1982. 25. Smock, T. and A. Topple. Action of vasopressin on neurons and microvessels in the rat hippocampal slice. Soc Neurosci Abstr 11: 708, 1985. 26. Tam, F. W. K., C. Chen, J. E. Alpert and S. D. Iversen. Aversive effects of subcutaneously injected vasopressin in the rat: independence of the ascending dorsal noradrenergic bundle. Brain Res 337: 133-137, 1985. 27. van Wimersma Greidanus, T. B. and D. De Wied. Dorsal hippocampus: a site of action of neuropeptides on avoidance behavior. Pharmacol Biochem Behav 5: 2%33, 1976. 28. van Wimersma Greidanus, T. B., G. Croiset, I. Bakker and H. Bowman. Amygdaloid lesions block the effect of neuropeptides (vasopressin, ACTH 4-10) on avoidance behavior. Physiol Behav 22: 291-295, 1979. 29. van Wimersma Greidanus, T. B., J. M. van Ree and D. De Wied. Vasopressin and memory. Pharmacol Ther 20: 437-458, 1983. 30. Walter, R., J. M. van Ree and D. De Wied. Modification of conditioned behavior of rats by neurohypophyseal hormones and analogs. Proc Natl Acad Sci USA 75: 2493-2496, 1978.