Psychoneuroendocrinology, Vol. 1 I, No. 4, pp. 4 3 7 - 446, 1986. 0 3 0 6 - 4 5 3 0 / 8 6 $3.00 + 0.00 Printed in Great Britain. Pergamon Journals Ltd.

D I F F E R E N T I A L EFFECT OF P O S T T R A I N I N G N A L O X O N E ,

13-ENDORPHIN, L E U - E N K E P H A L I N A N D E L E C T R O C O N V U L S I V E

S H O C K A D M I N I S T R A T I O N U P O N M E M O R Y OF A N O P E N - F I E L D

H A B I T U A T I O N A N D OF A W A T E R - F I N D I N G TASK

CARLOS ALEXANDRE NETTO, RENATO DUTRA DIAS and IVAN IZQUIERDO Laboratorio de Neuroquimica, Depar tamento de Bioquimica, lnst i tuto de Biociencias, U.F.R.G.S. (centro),

90.000 Porto Alegre, RS, Brasil

(Received 5 March 1985; in f inal form 18 November 1985)

SUMMARY Rats were trained and tested in an open field for habituat ion of rearing responses, for a water-

finding task, or for both tasks simultaneously. Training-test interval was 24 hr. The water-finding task consisted o f locating a metal tube in one o f the walls o f the box, which was attached to a water bottle on the outside; animals were water deprived between training and testing. Retention was estimated by measuring the latency to lick from the tube on the test session. Rats learned this task either with or without water deprivation, also prior to training. Habituat ion learning (reduction of the number of rearings between the training and test session) occurred either simultaneously with the water-finding task or in animals trained without the water tube, so that they could not learn the water-finding task. As happens with many other tasks, training in the open field was followed by a large decrease of hypothalamic 13-endorphin immunoreactivity, attributable to a release o f this substance. Posttraining IP naloxone (1.6 mg/kg) administrat ion facilitated, and posttraining 13-endorphin (2.0 txg/kg), leu-enkephalin (5.0 ~tg/kg), or electroconvulsive shock (15 mA, 60 Hz, 2 sec) depressed the retention of habituation; this occurred regardless of whether the animals were trained and /o r tested with or without water deprivation, and whether the task was acquired alone or simultaneously with the water-finding task. By contrast, none of these treatments had any effect on retention of the water finding task, acquired either with or without prior water deprivation. Thus , habituat ion was, and water-finding was not, sensitive to posttraining treatments known to affect endogenous opioids: the opioids themselves, their antagonist , naloxone, and electroconvulsive shock which releases brain opioids and causes naloxone-reversible retrograde amnesia. Learning of the water-finding task was merely incidental to exploration of the open field; it took place even when the animals were trained without the water tube. This suggests that the posttraining treatments that affect endogenous opioid function affect memory only of the task(s) that actually cause the release of brain I~-endorphin (in this case, probably habituation), and not of others that may occur simultaneously but are merely incidental (water-finding). A feature apparently common to the former is that they must directly involve either the recognition of novelty, or the initiation of an interaction with a new environment , or perhaps the habituation of such interaction.

Key Words--Brain 13-endorphin system; naloxone; I~-endorphin; leu-enkephalin; memory modulation; open field habituation; water-finding task; simultaneous memories.

INTRODUCTION TRAINING paradigms that involve exposure to a new environment or a new task are followed by decrease of brain I~-endorphin-like immunoreactivity, attributable to a release of this substance (Izquierdo et al., 1981, 1984; Izquierdo & Netto, 1985a; Netto et al., 1985). The [~-endorphin that is released probably plays a role in memory modulation. Posttraining administration of naloxone (Gallagher & Kapp, 1978; Messing et al., 1979; Izquierdo, 1979, 1982; Izquierdo & Dias, 1983, 1985; McGaugh, 1983; Rodgers et al., 1984; Izquierdo & Netto, 1985a,b; Izquierdo & McGaugh, 1985), or other opiate receptor

437

438 CARLOS AtEXANDRE NETTO, RENATO DUTRA DIAS and IVAN IZQUIERDO

b locke r s ( G a l l a g h e r , 1982), fac i l i ta tes r e t e n t i o n o f the tasks w h o s e t r a in ing is

a c c o m p a n i e d by a re lease o f [3-endorphin. P o s t t r a i n i n g i n t r a p e r i t o n e a l ( IP) o r

i n t r a c e r e b r o v e n t r i c u l a r ( ICV) [3-endorphin a d m i n i s t r a t i o n ( I z q u i e r d o et al. , 1981;

I z q u i e r d o & Dias , 1983, 1985; " M c G a u g h , 1983; I z q u i e r d o & N e t t o , 1985a,b) depresses

r e t en t i on , a n d its e f fec t is c o m p e t i t i v e l y a n t a g o n i z e d by n a l o x o n e ( I z q u i e r d o , 1982;

I z q u i e r d o & Dias , 1983, 1985). T h e e n k e p h a l i n s share the e f fec t o f [3-endorphin

( I z q u i e r d o et al . , 1981) b u t a r e t hemse lve s n o t re leased f r o m the b ra in d u r i n g t r a in ing

( C a r r a s c o et al . , 1982b). E l e c t r o c o n v u l s i v e shock (ECS) re leases mass ive a m o u n t s o f b ra in

15-endorphin (Dias et al . , 1981) and e n k e p h a l i n ( C a r r a s c o et al . , 1982b) a n d p r o d u c e s

n a l o x o n e - r e v e r s i b l e r e t r o g r a d e a m n e s i a ( C a r r a s c o et al. , 1982a). M o s t l ea rn ing e x p e r i m e n t s i nvo lve the spec i f ic ana lys is o f j u s t o n e task, bu t it is

poss ib le tha t m a n y t r a in ing s i tua t ions m a y i n v o l v e the s i m u l t a n e o u s a c q u i s i t i o n o f m o r e

t h a n one b e h a v i o r ( I z q u i e r d o & C a v a l h e i r o , 1976). T h e w a t e r - f i n d i n g p a r a d i g m desc r ibed

by T e n e n (1965) a n d L e M o a l et al. (1984) c o u l d be one such e x a m p l e . In this p a r a d i g m ,

a n i m a l s a r e a l l o w e d to e x p l o r e an o p e n f ie ld such as is c o m m o n l y used fo r h a b i t u a t i o n

s tudies (e.g. R o d g e r s et al . , 1984). In the c o u r s e o f the i r e x p l o r a t i o n they de tec t a wa t e r

t u b e p l a c e d s o m e w h e r e in the f ield. A n i m a l s t hen a re tes ted u n d e r c o n d i t i o n s o f wa t e r

d e p r i v a t i o n to see h o w qu i ck ly they r each the tube (Le M o a l et al . , 1984). O b v i o u s l y , s ince

the a n i m a l s a re t r a ined in an o p e n f ield, they m a y also a c q u i r e h a b i t u a t i o n , wh ich can be

m e a s u r e d by the r e d u c t i o n in the n u m b e r o f r ea r ings be tween the t r a in ing and the test

sess ion ( R o d g e r s et al . , 1984).

T h e p re sen t s tudy e x a m i n e d the e f fec t o f t r a in ing in the o p e n f ie ld on h y p o t h a l a m i c

6 - e n d o r p h i n a n d the e f fec t o f the p o s t t r a i n i n g a d m i n i s t r a t i o n o f n a l o x o n e , f3-endorphin ,

l e u - e n k e p h a l i n , a n d E C S on r e t e n t i o n o f the w a t e r - f i n d i n g task and o f the h a b i t u a t i o n o f

r ea r ing r e sponses to the o p e n field.

MATERIALS & METHODS

A total of 392 female Wistar rats from our own breeding stock were used (age 70 - 90 days, weight I l0 - 190 g). Behavioral procedures were carried out in a 50 cm high, 40 cm deep, and 60 cm wide open field made of brown plywood with a frontal glass wall. In the middle of the rear wall there was a 5 cm deep, l0 cm wide, and 50 cm high recess with a metal water tube protruding into the box l0 cm above floor level. The tube was attached to a water container placed on the outside and was identical to the tubes from which the rats normally drank water in their home cages. The apparatus was similar to that described by LeMoal et al. 0984).

Two hundred and thirty-two animals were trained and tested with the water tube present in the box, so that they could learn both the water-finding task and habituation (Experiments l, 2, 3 and 4); l0 animals were trained without, and tested with, the water tube (Experiment 1); and the remaining 150 animals were trained and tested without the water tube, so that they could learn just the habituation (Experiment 5).

In training sessions, rats were taken directly from their home cage to the open field, placed on its left frontal corner, and left to explore the arena for either a fixed (Experiments l, 2, 3, 5) or a variable (Experiments l, 4) period of time. Some animals were deprived of water prior to training (Experiments l, 3, 5), and others were not (Experiments 1, 2, 4, 5). Except for one of the groups in Experiment 5, animals had free access to water in their home cages in the 10 min immediately following training, and then there was a 24 hi" period of water deprivation up to the time of testing. The incidence of rearing responses was measured in the training session of all experi- ments, and latency to touch the tube on the training session was measured in Experiments 1 to 4.

On the test session the animals were again placed on the left frontal corner of the box, and their latency to lick the water tube (Experiments 1, 2, 3, 4), and/or the incidence of rearing responses (Experiments 3, 4, 5) was measured. When the tube was present (Experiments 1 to 4), test sessions were terminated as soon as the rats started to lick the tube.

OPIOID-SENSITIVE AND INSENSITIVE MEMORIES 439

In Experiment 2, animals were killed for 13-endorphin radioimmunoassay six min after either the training or the test session. In Experiments 3, 4 and 5, the animals received, less than one min after training, an IP injection of saline (1 ml/kg), naloxone HCI (Endo) (1.6 mg/kg), human 13-endorphin (Sigma) (2.0 Ixg/kg), or leu- enkephalin (synthesized by Prof. A. C. M. Paiva, University of Sao Paulo)(5.0 lxg/kg); or a 15.0 mA, 60 Hz,2.0 sec transcorneal electroconvulsive shock (ECS) (Dias et al., 1981; Carrasco et al., 1982a). Drugs were dissolved in saline to a vol of 1 ml/kg. The doses were chosen from among those found to be effective in many previous studies (Izquierdo, 1979; 1982; Messing et al., 1979; Gallagher et al., 1983; Izquierdo & Dias, 1983; 1985; Rodgers et al., 1984; lzquierdo & Netto, 1985a, 1985b; Izquierdo & McGaugh, 1985a; 1985b). The ECS caused a full tonic-clonic convulsion in all animals that were submitted to it.

EXPERIMENT 1. SOME CHARACTERISTICS OF THE WATER-FINDING TASK

N o n d e p r i v e d a n i m a l s w e r e p l a c e d in t h e t r a i n i n g a p p a r a t u s f o r 2, 3 o r 5 m i n . N o n e o f

t h e m t o u c h e d t h e t u b e in t h e t r a i n i n g se s s ion ; h o w e v e r , w h e n t e s t e d a f t e r 24 h r o f w a t e r

d e p r i v a t i o n , a l l o f t h e m w e n t t o t h e t u b e a n d d r a n k f r o m it f a s t e r t h a n u n t r a i n e d r a t s j u s t

s u b m i t t e d to a 24 h r w a t e r d e p r i v a t i o n (0 m i n g r o u p s ) . T h i s h a p p e n e d r e g a r d l e s s o f

w h e t h e r t h e a n i m a l s w e r e t r a i n e d w i t h o r w i t h o u t t h e w a t e r t u b e ( T a b l e I ) . S i n c e t e s t

l a t e n c i e s w e r e s i m i l a r a m o n g g r o u p s t r a i n e d w i t h t h e w a t e r t u b e f o r 2, 3 o r 5 m i n , in al l

s u b s e q u e n t e x p e r i m e n t s w a t e r - u n m o t i v a t e d t r a i n i n g s e s s ions w e r e l i m i t e d t o 2 m i n

( E x p e r i m e n t s 2, 4, 5). I n u n t r a i n e d a n i m a l s (0 r a i n g r o u p s ) , t h e I P i n j e c t i o n o f s a l i ne ,

n a l o x o n e (1 .6 m g / k g ) , f l - e n d o r p h i n (2 .0 lxg/kg), o r l e u - e n k e p h a l i n (5 .0 Ixg/kg), or t h e

TABLE I. MEAN (± SEM) WATEI~-LICK LATENCY IN TEST SESSIONS OF THE WATER-FINDING TASK. (A) WITHOUT WATER

DEPRIVATION PRIOR TO TRAINING: (B) 24 hr WATER DEPRIVATION PRIOR TO TRAINING. N = l 0 RATS PER GROUP

Duration of training Treatment 24 hr Water-lick latency session (min) prior to testing in test session

(A) No water deprivation prior to training

0* - - 135.6 ± 13.4 2t - - 57.7 ± 17.9§ 2 - - 52.0 ___ 7.1§ 3 - - 56.5 ± 13.4§ 5 - - 55.9± 11.2§ 0* Saline (1 ml/kg) 156.6 ± 13.6 0* Naloxone (1.6 mg/kg) 162.5 ± 14.9 0* I~-endorphin (2.0 I.tg/kg) 137.8 ± 14.8 0* Leu-enkephalin (5.0 I.tg/kg) 145.0 ± 12.5 0* ECS (15.0 mA, 2 sec) 141.4 ± 13.0

(B) 24 hr of water deprivation prior to training

0* - - 76.5 ± 12.1 132.5 ± 14.4:~ - - 13.1 ± 2.711

*Untrained; since all animals were water deprived for 24 hr prior to testing, " tes t" session latencies of these groups actually represent training session latencies preceded by 24 hr of water deprivation in panel A and by 48 hr of water deprivation in panel B.

tTrained without the water tube. ~tMean + S.E.M. training session latency; this value was not significantly different from "test" session

latencies of the 0 min groups (see above) in a Duncan multiple range test (p > 0.1). Differences in test session latencies among groups in panel A were significant: F (8, 81) = 10.77, p < 0.01.

§Significant difference from 0 min group at p < 0.01 level in a Duncan multiple range test. USignificant difference from training session latency and from "test" session latency of the 0 min group in

panel B in a Duncan multiple range test (17 < 0.01).

440 CARLOS ALEXANDRE NETTO, RENATO DUTRA DIAS a n d IVAN IZQUIERDO

administration of ECS carried out 24 hr prior to testing had no effect on the latency to reach the tube (Table I).

In contrast to non-deprived animals, rats deprived of water for 24 hr prior to training went to the tube and drank f rom it on the training session, and presented much lower test session latencies than either animals trained without thirst or animals deprived for 48 hr and placed in the apparatus for the first time (Table I). Therefore, it can be concluded that thirst was not essential to learn the water-finding paradigm, but it served a facilitatory role. In addition, the presence of the tube during the training session was also not essential for learning the water-finding task. This indicates that mere exploration of the box is a much more important factor for learning that task than any specific cues about the tube shape or location.

Naloxone, ~-endorphin, leu-enkephalin, or ECS given one day before had no effect on water-lick latencies.

E X P E R I M E N T 2. E F F E C T O F T R A I N I N G A N D T E S T I N G IN T H E O P E N F I E L D O N H Y P O T H A L A M 1 C ~ - E N D O R P H I N L I K E I M M U N O R E A C T I V I T Y

In numerous previous studies (Izquierdo et al., 1981, 1984; Izquierdo, 1982; Izquierdo & Netto, 1985a; Netto et aL, 1985), it was reported that exposure to a novel experience, such as a new environment or a new task, caused a decrease of hypothalamic ~-endorphin immunoreactivity in rats. The present experiment was designed to find out whether this also would be the case with training in the apparatus described above.

Twenty-two animals were used. Eight were taken out of their home cages and killed immediately; seven were killed six min after a 2 min exposure to the open field without prior water deprivation, and seven were killed 6 min after a test session. Killing was by decapitation. The brain was quickly removed and the hypothalamus dissected on an inverted Petri-dish placed on ice. The tissue was homogenized, extracted with hot acetic acid, and processed for fi-endorphin radioimmunoassay as recommended by Rossier et aL (1977a,b) and as extensively studied in our laboratory (Carrasco et aL, 1982; Dias et aL, 1981; Izquierdo & Netto, 1985a; Izquierdo et aL, 1981, 1984; Netto et aL, 1985). A I~-endorphin radioimmunoassay kit donated by the National Institute of Drug Abuse (U.S.A.) was used.

Results are shown in Table II. As in all other tasks studied so far (Izquierdo et al., 198 l, 1984), the first exposure to the task (training) was followed by a large decrease of hypothalamic 13-endorphin-like immunoreactivity, whereas a second exposure (testing) was not. As discussed elsewhere (Rossier et aL, 1977b; Dias et al., 1981; Izquierdo et aL,

TABLE ll . ~-ENDORPHIN-LIKE IMMUNOREACTIVITY MEASURED IN THE HYPOTHALAMUS OF RAIS KILLED 6 MIN AFTER TRAINING OR TEST SESSIONS OF A WATER-FINDING TASK

I ] -Endorph in - l ike i m m u n o r e a c t i v i t y G r o u p N ( M e a n _+ S . E . M . n g o f 13-endorphin per m g o f wet tissue)

In t ac t c o n t r o l s 8 342 _+ 30 T r a i n i n g 7 244 _+ 33* Test 7 328 + 30

*Signi f ican t d i f f e r ence f r o m the o t h e r t w o g r o u p s in a D u n c a n mul t ip le r a n g e test at p < 0.001 level.

OPIOID-SENSITIVE AND INSENSITIVE MEMORIES 441

1981, 1984; Carrasco et ai., 1982a; Izquierdo & Netto, 1984a; Netto et al., 1985), the decrease of hypothalamic 13-endorphin immunoreactivity can best be explained by a release followed by degradation of the substance; it cannot be explained by synthesis inhibition, because the ratio of I~-endorphin to precursor is high in this region and its rate of synthesis is very slow (hours). Thus, synthesis inhibition cannot account for such large changes developing in 6 min.

E X P E R I M E N T 3. P O S T T R A I N I N G T R E A T M E N T S A F F E C T T H E R E T E N T I O N O F H A B I T U A T I O N , BUT N O T O F T H E S I M U L T A N E O U S L Y A C Q U I R E D W A T E R - F I N D I N G TASK, IN R A T S T R A I N E D

W I T H O U T P R I O R W A T E R D E P R I V A T I O N

Rats were submitted to a training and a test session in the open field as described above; training was without prior water deprivation. As mentioned in Experiment 1, none of the animals touched the tube on the training session, but all did, and drank from it, on the test session (Table III). Rearing responses were counted in both sessions; in the test session, this was possible only up to the moment that the animals started licking the tube. The incidence of rearings per minute was lower in the test session in control animals (Table III). Therefore, it can be concluded that animals trained without thirst acquire both the water-finding task (LeMoal et al., 1984) and habituation to the open field (Rodgers et al., 1984).

Posttraining naloxone administration enhanced, and 13-endorphin, leu-enkephalin and ECS reduced, the retention of habituation, as measured by their effect on the training-test difference in performance of rearing responses. None of the treatments affected retention of the water-finding task, as measured by the latency to reach the tube in the test session (Table III).

Therefore, in spite of the fact that both tasks were acquired simultaneously during a training session in which there was a release of brain [3-endorphin, one of the tasks was, and the other was not, sensitive to posttraining treatments known to affect endogenous opioid mechanisms.

TABLE II1. MEAN (--+ S .E .M. ) LATENCY TO LICK WATER TUBE IN THE TEST SESSION, AND PERFORMANCE OF REARING RESPONSES IN THE TRAINING AND TEST SESSIONS, OF RATS GIVEN A POSTTRAINING IP INJECTION OF SALINE, NALOXONE, [~-ENDORF'HIN, LEU-ENKEPHALIN, OR AN ECS. ANIMALS NOT WATER DEPRIVED PRIOR TOTRAINING. N = l0 PER GROUP

Test N u m b e r of rear ings T r e a t m e n t Dose Water - l i ck latencies Tra in ing session Test session

Sal ine 1 m l / k g 74 _+ 14 12.0 _+ 1.4 5.4 _+ 0.8* N a l o x o n e 1.6 m g / k g 65 ~_ 17 13.2 _+ 1.7 2.3 _+ 1.0"~ 13-Endorphin 2.0 l ag /kg 74 _+ 13 10.2 _+ 0.8 8.7 _+ 0 . 8 t Leu -enkepha l in 5.0 p .g/kg 82 _+ 15 8.6 _+ 1.3 8.3 + 0.9:~ ECS (15 mA, 2 sec) - - 68 _+ 9 10.2 _+ 0.9 8.1 _+ 0.6:~

D u r a t i o n of test sessions was equa l to test session la tency. La tency d i f ferences a m o n g g roups not s igni f icant in a one -way ana lys i s o f var iance , F (4 ,45 ) = 0.44, p > 0.1 Dif ferences in rear ings a m o n g groups , no t s igni f icant in the t r a in ing session, F (4, 45) = 2,43, p > 0.1; bu t s igni f icant in the test session, F (4, 45) = 8.48, p < 0.01.

*Signi f icant d i f fe rence f rom t r a in ing session at p < 0.01 level by t test. i -Signif icant d i f fe rence f rom cont ro l a t p < 0.05 level in Duncan mul t ip le range test. *Same, at p < 0.01 level.

442 CARLOS ALEXANDRE NET70, RENATO DUTRA DIAS and IVAN IZQUIERDO

EXPERIMENT 4. IMPOSSIBILITY TO MEASURE HABITUATION, AND LACK OF EFFECT OF POSTTRAINING TREATMENTS ON THE WATER-FINDING TASK, IN ANIMALS TRAINED WITH

PRIOR WATER DEPRIVATION In conf i rmat ion of the f indings of Exper iment 1, animals deprived of water for 24 hr

prior to t ra in ing showed a much shorter test session latency to reach the tube than animals t rained without thirst (Table IV). In fact, test latencies were so short that any measure of rearing responses in this session became meaningless. Most of the animals performed just one or zero rearings in the test session (median 1, interquart i le range, 0 to 3, n = 50). Thus, it was not possible to measure retent ion of hab i tua t ion in the animals t rained with

prior water deprivat ion. In these animals , the post t ra in ing t reatments also had no effect on retent ion of the

water- f inding task (Table IV). Therefore, the lack of effect of these t reatments on this

task observed in Exper iment 3 was not due to the absence of thirst at the time of t raining, and may be considered as representing an insensit ivity of the water-f inding task to the

treatments.

EXPERIMENT 5. POSTTRAINING TREATMENTS AFFECT HABITUATION ACQUIRED INDEPENDENTLY OF THE WATER-FINDING TASK

Exper iment 3 shows that post t ra ining naloxone, 13-endorphin, leu-enkephal in, and ECS altered retent ion of hab i tua t ion but not of the s imul taneously acquired water-f inding task in animals trained without prior water deprivat ion. This suggests that memory of hab i tua t ion is, and memory of the water-f inding task is not, amenable to post t ra in ing modu la t ion by manipu la t ions of endogenous opioid systems. Of course, one al ternative possibility is that the effects on hab i tua t ion shown in Exper iment 3 could have been due to interact ions with the concomi tan t ly acquired water-f inding task a n d / o r with the dissociation of mot iva t ional states between the two sessions (no thirst at the t ime of t raining, thirst at the time of testing).

One hundred and fifty rats were submit ted to hab i tua t ion t ra ining and testing in the open field without the water tube, so that they could not learn the water f inding task. Each session consisted of a 2 min exposure to the field; the training-test interval was 24 hr.

Fifty animals were trained and tested without water deprivat ion; 50 were trained without

TABLE IV. MEAN (_+ S E M ) LATENCY FO LICK WAFER TUBE AND PERFORMANCE OF REARING RESPONSES IN r i l e

TRAINING AND TEST SESSIONS OF RATS GIVEN A POSTTRAINING IP INJECTION OF SAtENE. NALOXONE, 13-ENDORPHIN. OR

LEU-ENKEPHALIN, OR AN E C S . ANIMAt.S DEPRIVED OF WATER [-OR 24 h r PRIOR TO TRAINING SAME DOSES AND

E C S PARAMETERS AS IN TABLE I l l . N = l 0 PER GROUP

Water-lick latencies Number of rearings Treatment Training Test Training Test

Saline 124.8 +_ 18.4 14.2 ÷ 2.5 11.2 _+ 1.3 1.5 _+ 0.2 Naloxone 143.6 ± 18.6 17.7 ± 4.6 16.2 ÷ 0.9 1.3 +_ 0.3 I~-Endorphin 129.6 ± 19.5 13.0 _+ 2.1 12.2 + 0.8 1.2 _+ 0.2 Leu-enkephalin 120.8 _+ 19.5 10.8 ± 1.4 11.1 _+ 0.6 1.4 ± 0.2 ECS 141.1 ± 20.5 12.7 _+ 2.5 13.6 ± 0.9 1.3 _+ 0.1

Duration of training and test sessions was equal to water-lick latency in both sessions. Differences in water- lick latencies in training (F(4, 45) = 0.241) or test sessions (F(4, 45) = 0.735) or in rearing responses in training (F (4, 45) = 2.048) or test sessions (F (4, 45) = 0.266) were not significant (p > 0.1).

OPIOID-SENSITIVE AND INSENSITIVE MEMORIES 443

a n d tes ted w i t h a p r e c e d i n g 24 h r p e r i o d o f d e p r i v a t i o n ; a n d 50 were w a t e r d e p r i v e d f o r 24

hr b e f o r e b o t h t r a i n i n g a n d tes t ing.

Resu l t s a r e s h o w n in T a b l e V. R e t e n t i o n was s imi la r in al l g r o u p s . I n al l cases, n a l o x o n e

e n h a n c e d , a n d the o t h e r t r e a t m e n t s d i s r u p t e d , r e t e n t i o n o f t he h a b i t u a t i o n task .

T h e r e f o r e , t h e sens i t iv i ty o f h a b i t u a t i o n to the p o s t t r a i n i n g t r e a t m e n t s c a n be e x p l a i n e d

by an i n f l u e n c e o f these u p o n o p i o i d - m o d u l a t e d p rocesses r e l evan t to m e m o r y o f this

task , as has b e e n s h o w n f o r m a n y o t h e r s ( G a l l a g h e r & K a p p , 1978; I z q u i e r d o , 1979, 1982;

M e s s i n g et al., 1979; I z q u i e r d o & Dias , 1983, 1985; I z q u i e r d o & N e t t o , 1985a,b) , a n d n o t

to an i n t e r a c t i o n wi th e i the r th i r s t o r w i th t he w a t e r - f i n d i n g task .

DISCUSSION

T h e cen t r a l f i nd ings o f th is s t udy a re t h o s e o f E x p e r i m e n t s 2 a n d 3. T r a i n i n g in t he o p e n

f ie ld r e l eased b r a i n 13-endorphin ( E x p e r i m e n t 2) a n d caused l e a rn ing o f h a b i t u a t i o n to the

o p e n f ie ld a n d o f t he w a t e r - f i n d i n g task . P o s t t r a i n i n g t r e a t m e n t s t ha t a l t e r ed e n d o g e n o u s

o p i o i d f u n c t i o n a f f e c t e d the r e t e n t i o n o f t he f o r m e r b u t n o t o f the l a t t e r ( E x p e r i m e n t 3).

TABLE V. MEAN (_+ S . E . M . ) REARING IN 2 MIN TRAINING AND TEST SESSIONS IN RA.TS GIVEN A POSTTRAIN1NG IP

INJECTION OF SALINE. NALOXONE, [~-ENDORPHIN, OR LEU-ENKEPHALIN, OR AN E C S . SAME DOSES AND E C S

PARAMETERS AS IN TABLE 111. N = l0 ANIMALS PER GROUP

Number of rearings Treatment Training session Test session

(A) No water deprivation prior to either training or testing

Saline 13.4 _+ 0.8 9.9 _+ 0.6* Naloxone 13.4 _+ 0.5 6.3 _+ 0.5"~ [~-Endorphin 13.4 _+ 0.8 13.0 _+ 1.2t Leu-enkephalin 14.5 +_ 0.8 14.4 +_ 1.0t ECS 13.2 _+ 0.9 12.9 _+ 0.6t

(B) 24 hr water deprivation prior to both training and testing

Saline 13.9 _+ 0.7 10.6 _+ 0.6* Naloxone 15.1 _+ 0.7 7.8 _+ 1.0*:~ [~-Endorphin 14.2 _+ 0.5 14.1 +_ 0.4:~ Leu-enkephalin 13.1 _+ 0.7 12.9 _+ 0.8, ECS 13.3 _+ 0.7 12.8 _+ 0.4~

(C) 24 hr water deprivation prior to testing only

Saline 14.1 _+ 0.8 10.9 _+ 0.7* Naloxone 14.5 _+ 0.8 8.3 _+ 0.6*t 13-Endorphin 14.7 _+ 0.7 14.3 +_ 0.8t Leu-enkephalin 14.3 _+ 0.8 13.8 _+ 1.0t ECS 14.1 _+ 0.6 13.7 _+ 0.6t

Duration of both sessions was 2 min. Differences among groups in rearing responses in training sessions were not significant: (A) F (4, 45) = 0.434; (B) F (4, 45) = 1.201; (C) F (4, 45) = 0.098; all p's> 0.1. Differences among groups in rearing responses in test sessions were significant: (A) F(4, 45) = 13.852, p < 0.01; (B) F(4, 45) = 12.115, p < 0.01; (C) F (4,45) = 10.317, p < 0.01.

*Significant difference from training session performance by t test, p < 0.01. tSignificant difference from test session performance of saline group at p < 0.05 level in a Duncan multiple

range test. ~:Same, at p < 0.01 level.

444 CARLOS ALEXANDRE NETTO, RENATO DU]RA DIAS and IVAN IZQUIERDO

Many other tasks, including several forms of habituation and of inhibitory avoidance, active avoidance, and exposure to escapable or inescapable footshock, also are accompanied by a release of brain 13-endorphin (Rossier et al., 1977b; Izquierdo et al., 1981, 1984; Netto et al., 1985; Izquierdo and Netto, 1985a). Retention of these tasks is facilitated by posttraining naloxone and depressed by posttraining opioid or ECS ad- ministration (Izquierdo, 1979, 1982; Messing et al., 1979; Gallagher, 1982; Gallagher et al., 1983; McGaugh, 1983; Izquierdo & Dias, 1983, 1985; Izquierdo & McGaugh, 1985a; Izquierdo & Netto, 1985a,b). ECS releases a massive amount of brain fi-endorphin (Dias et al., 1981) and enkephalin (Carrasco et al., 1982a), and its amnestic effect is reversed by naloxone (Carrasco et al., 1982b), which suggests that it is mediated by the opioid release.

Thus, the major question is what makes water-finding different from all those other tasks. Insensitivity of the water-finding task to the posttraining treatments is certainly not because of the lack of brain I$-endorphin release (Experiment 2) or because of the absence of thirst at the time of training (Experiment 4). It is also not due to the fact that it is an alimentary task; other alimentary tasks are affected by one or more of the treatments used here (e.g., Gallagher et al., 1983), and even water-finding can be affected if a 10 times larger ECS and a multi-trial training design are used (Tenen, 1965). The water-finding task is not generally insensitive to modulatory treatments either; using very similar training parameters to those of Experiment 3, LeMoal et al. (1984) observed posttraining memory facilitation of this task with vasopressin.

One peculiarity of the water-finding task that makes it different from all the others is that, as shown in Experiment 1, it is merely incidental to the exploration of and subsequent habituation to the open field. It does not depend on specific information about the water tube or its location; rats trained with or without the tube showed similar test session latencies (Table I). Thus, acquisition of the water-finding task does not imply learning anything really new about the environment, but merely the use of general information about it casually obtained in the course of exploration and habituation. Getting general information about a new environment is something that animals learn how to do probably very early in life (it may even be an innate behavior), and may then apply to any situation. Therefore, the water-finding task does not really involve an interaction with novelty; it is merely a by-product of the scanning of the environment.

This is in sharp contrast to all the other tasks that are affected by posttraining naloxone or opioid administration which directly imply, one way or another, the recognition of novelty, or a reaction to novelty, or the habituation of that reaction (Netto et al., 1985; Izquierdo & Netto, 1985a; Izquierdo & McGaugh, 1985b). The release of hypothalamic 13-endorphin caused by training is seen only when animals are exposed for the first time to an environment, or to a change in the meaning of environmental cues (Izquierdo et al., 1981, 1984; Netto et al., 1985; Izquierdo & Netto, 1985a,b) (Experiment 2). It is not known whether the release results from the recognition of novelty (lzquierdo & Netto, 1985a,b; Netto et al., 1985), or from an initial reaction to novelty (Izquierdo & McGaugh, 1985b), or from habituation.

The present results suggests that posttraining naloxone, opioid administration, and ECS affect only those tasks that involve the processing of novelty-related information and therefore are causally related to brain I$-endorphin release, and not other tasks that may develop secondarily or incidentally, even if they are acquired at the same time.

OPIOID-SENSITIVE AND INSENSITIVE MEMORIES 445

H a b i t u a t i o n obviously belongs to the former, and water- f inding to the latter, category. As discussed elsewhere in detail ( Izquierdo, 1982; Izquierdo et al . , 1984; Izquierdo &

Dias, 1985; Izquierdo & McGaugh , 1985a,b; Izquierdo & Netto, 1985a,b), the [5-endorphin released by t ra in ing induces state dependency for the tasks that caused the release; re ten t ion scores of ~hese tasks are enhanced by the pre-test admin i s t r a t ion of ~-endorphin . Also as shown elsewhere ( Izquierdo & McGaugh , 1985b), the state dependency is restricted to the task that caused the release in the first place, and does no t extend to other tasks that are acquired short ly after. The present results show that this also holds true for s imul taneous ly learned behaviors; pos t t ra in ing opioid memory

m o d u l a t i o n affects only the re ten t ion of the task that is causally l inked to the ~l-endorphin

release. In conclus ion, pos t t ra in ing naloxone, opioid or ECS t rea tments affect re tent ion of

hab i tua t ion to an open field bu t no t of a s imul taneous ly acquired water- f inding task. The t rea tments also affect the re tent ion of hab i tua t ion when it is acquired alone. The effect is independen t of the presence of thirst at the t ime of t ra in ing or testing. Tra in ing in the open field is accompanied , like other tasks, by bra in •-endorphin release. The present results show that this release is no t a necessary condi t ion for the under ly ing behavior(s) to be amenab le to endogenous opioid man ipu la t ion . Possibly the release of b ra in ~ -endorph in results f rom elements present in the hab i tua t ion task (recogni t ion of novelty, react ion to novelty, inh ib i t ion of that reaction) and unre la ted to water-f inding. The water- f inding task is insensit ive to pos t t ra in ing opioid man ipu la t ion , p robab ly because it does not involve the processing of i n f o r m a t i o n related to novelty, and is merely a by-product of the other task,

The work is supported by funds from FINEP and CNPq, Brazil. We are indebted to Dr William Pollin, Director of the U.S. National Institute of Drug Abuse, for the donation of a 13-endorphin radioimmunoassay kit manufactured by that agency.

REFERENCES Carrasco M A, Dias R D, Izquierdo 1 (1982a) Naloxone reverses amnesia induced by electroconvulsive shock.

Behav Neural Biol 34:352 - 357. Carrasco M A, Perry M L S, Dias R D, Wofchuk S T, Izquierdo 1 (1982b) Effect of tones, footshocks, shuttle

avoidance, and electroconvulsive shock on met-enkephalin immunoreactivity of rat brain. Behav Neural Bio! 34:1 - 4 ,

Dias R D, Perry M L S, Carrasco M A, Izquierdo 1 (1981) Effect of electroconvulsive shock on ~-endorphin immunoreactivity of rat brain, pituitary gland and plasma. Behav Neural Biol 32:265 - 268.

Gallagher M (1982) Naloxone enhancement of memory processes: effects of other opiate antagonists. Behav Neural Biol 35:375 - 382.

Gallagher M, Kapp B S (1978) Opiate administration into the amygdala. Effects on memory processes. Life Sci 23:1973 - 1978.

Gallagher M, King R A, Young N B (1983) Opiate antagonists improve spatial memory. Science 221:975 - 976. Izquierdo l (1979) Effect of naloxone and morphine on various forms of memory in the rat: possible role of

endogenous opiate mechanisms in memory consolidation. Psychopharmacology 66: 199- 203. Izquierdo I (1982) The role of an endogenous mechanism mediated by brain ~-endorphin in memory

modulation. Braz J Med Biol Res 15: 119- 134. Izquierdo I, Cavalheiro E A (1976) Three main factors in rat shuttle behavior: their pharmacology and

sequential entry in operation during a two way avoidance session. Psychopharmacology 49:145 - 157. Izquierdo 1, Dias R D (1983) Effect of ACTH, epinephrine, [~-endorphin, naloxone, and of the combination

of naloxone or ~-endorphin with ACTH or epinephrine on memory consolidation. Psychoneuroendo- crinology g: 81 - 87.

Izquierdo I, Dias R D (1985) Influence on memory of posttraining and pre-test injections of ACTH, vasopressin, epinephrine, or ~-endorphin, and their interaction with naloxone. Psychoneuroendocrinology 10: 165- 172.

446 CARLOS ALEXANDRE NETTO, RENATO DUTRA DIAS and IVAN IZQUIERD()

lzquierdo I, McGaugh J L (1985a) Delayed onset of the amnestic effect of posttraining I)-endorphin: effects of propranolol administered prior to retention testing. Eur J Pharmacol ! 13: 105- 108.

Izquierdo I, McGaugh J L (1985b) Effect of a novel experience prior to training or testing on retention of an inhibitory avoidance response in mice; involvement of an opioid system. Behav Neural Biol 44:228 - 238.

Izquierdo I, Netto C A (1985a) The role of 13-endorphin in behavioral regulation. Ann N Y ,4cad Sci 444: 162- 177.

lzquierdo I, Netto C A (1985b) Factors that influence retrieval measured 0, 3 or 6 hours after inhibitory avoid- ance training. Behav Neural Biol 43: 2 6 0 - 273.

lzquierdo I, Perry M L S, Dias R D, Orsingher O A, Carrasco M A (1981) Effect of training and testing rats in two different behavioral paradigms on brain beta-endorphin immunoreactivity. Arq Biol Tecnol 24: 327 - 331.

lzquierdo 1, Souza D O, Dias R D, Carrasco M A, Volkmer N, Perry M L S, Netto C A (1984) Effect of various behavioral training and testing procedures on brain 13-endorphin-like immunoreactivity, and the possible role of 13-endorphin in behavioral regulation. Psychoneuroendocrinology 9:381 389.

Le Moal M, Dantzer R, Mormede P, Baduel A, Lebrun C, Ettenberg A, Van der Kooy D, Wenger J, Deyo S, Koob G F, Bloom F E (1984) Behavioral effects of peripheral administrat ion of arginine vasopressin: a review of our search for a mode of action and a hypothesis. Pyschoneuroendocrinology 9: 3 1 9 - 341.

McGaugh J L (1983) Hormonal influences on memory storage. J A m Psychol Assoc 38:161 - 174. Messing R B, Jensen R A, Martinez J L Jr, Spiehler V R, Vasquez B J, Somireu-Mourat B, Liang K C,

McGaugh J L (1979) Naloxone enhancement of memory. Behav Neural Biol 27: 2 6 6 - 275. Netto C A, Cavalheiro E A, Carrasco M A, Volkmer N, Dias R D, Izquierdo I (1985) Response of the rat brain

13-endorphin system to novelty: importance of the fornix connection. Behav Neural Biol 43:37 - 4 6 . Rodgers R J, Richards C, Precious J 1 (1984) Naloxone administration following brief exposure to novelty

reduces activity and rearing in mice upon 24-hr retest: a conditioned aversion? Psychopharmacology 82: 322 - 326.

Rossier J, Bayon A, Vargo T M, Ling N, Guillemin R, Bloom F (1977a) Radioimmunoassay of brain peptides: evaluation of a methodology for the assay of fl-endorphin and enkephalin. Life Sci 21; 841 - 852.

Rossier J, French E D, Rivier C, Ling N, Guillemin R, Bloom F E (1977b) Foot-shock induced stress increases 13-endorphin levels in blood but not brain. Nature 270: 6 1 8 - 6 2 0 .

Tenen S S (1965) Retrograde amnesia from electroconvulsive shock in a one-trial appetitive learning task. Science 148: 1248- 1250.