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Time-dependent effects of post-trial reinforcement, punishment or ECS on passive avoidance learning
Physiology & Behavior, Vol. 18, pp. 1103-1109. Pergamon Press and Brain Research Publ., 1977. Printed in the U.S.A.
Time-Dependent Effects of Post-Trial Reinforcement, Punishment or ECS on Passive Avoidance Learning' C E S A R E M O N D A D O R I , P E T E R G. W A S E R A N D J O S E P H P. H U S T O N 2
Institute o f Pharmacology, University o f Zurich, Zurich, Switzerland (Received 1 1 November, 1976) MONDADORI, C., P. G. WASER AND J. P. HUSTON. Time-dependent effects ofposttrial reinforcement, punishment or ECS on passive avoidance learning. PHYSIOL. BEHAV. 18(6) 1103-1109, 1977. - Three studies were performed to assess the effects of posttrial food reinforcement, ice-water punishment, and ECS on step-down passive avoidance learning in mice. The treatments were each administered at <5, 30, 60 and 90 sec after the footshock. One-half min of feeding beginning either 10 or 30 sec after the footshock increased the step-down latencies 24 hr later. This was considered to support the hypothesis that reinforcement facilitates learning by a direct influence on short-term memory processes. Posttrial ice-water punishment (10 sec immersion) disrupted learning only in the 60 sec postshock delay group. ECS (5.4 mA, 0.35 sec, across the ears) disrupted learning at all posttrial delays except 90 sec, with maximal effects immediately and at 60 sec, and minimal disruption at 30 sec. Similarities in the topography of the ECS and punishment curves suggested that some of the effects of posttrial ECS could be due to aversive properties of the ECS (acting directly on memory processes). Memory
Passive avoidance
Reinforcement
Punishment
ECS
Mice
p u n i s h m e n t w o u l d act in a m a n n e r reciprocal to posttrial r e i n f o r c e m e n t , a n d t h u s i n h i b i t a v o i d a n c e learning. In t h r e e e x p e r i m e n t s w i t h mice we t e s t e d the effects on passive a v o i d a n c e l e a r n i n g of (a) p o s t t r i a l food r e i n f o r c e m e n t , in a m o d i f i c a t i o n of t h e p r e v i o u s s t u d y [ 1 0 ] , ( b ) i c e - w a t e r p u n i s h m e n t , p r e s e n t e d at the same p o s t s h o c k intervals, and (c) p o s t t r i a l e l e c t r o c o n v u l s i v e s h o c k (ECS) a d m i n i s t e r e d at the same delays.
S E V E R A L studies have s h o w n t h a t p o s t t r i a l r e i n f o r c e m e n t can facilitate passive a v o i d a n c e learning. In o n e e x p e r i m e n t [ 10] mice were given 1 m i n of food r e i n f o r c e m e n t at various delays a f t e r the f o o t s h o c k in a s t e p - d o w n passive a v o i d a n c e task and tested 24 hr later. Facilitation of learning was o b t a i n e d in the groups a d m i n i s t e r e d reinforcem e n t w i t h i n 20 a n d 50 sec a f t e r the f o o t s h o c k b u t n e i t h e r b e f o r e n o r a f t e r this interval. In a s u b s e q u e n t s t u d y [18] rats were t r a i n e d o n a o n e - w a y s t e p - t h r o u g h active a v o i d a n c e task a n d t h e n s u b j e c t e d to a reversal p r o c e d u r e , i.e. t h e y were given a f o o t s h o c k in t h e previously safe c h a m b e r . T h e rats t h a t received r e i n f o r c i n g h y p o t h a l a m i c s t i m u l a t i o n 30 sec l a t e r avoided this c h a m b e r b e t t e r t h a n t h e n o n s t i m u l a t e d controls 24 h r later. We i n t e r p r e t e d these results in t e r m s of a direct a c t i o n o f r e i n f o r c e m e n t o n s h o r t - t e r m m e m o r y processes, in s u p p o r t o f t h e t h e o r y [ 11 ] t h a t r e i n f o r c e m e n t in the i n s t r u m e n t a l l e a r n i n g s i t u a t i o n acts o n ( m a i n t a i n s ) an i m m e d i a t e m e m o r y trace t h a t is left b y t h e o p e r a n t behavior. A c c o r d i n g to this t h e o r y r e i n f o r c e m e n t acts as such by p r e v e n t i n g this trace f r o m fading, and t h e r e f o r e , it s h o u l d be possible to facilitate l e a r n i n g b y p r e s e n t i n g r e i n f o r c e m e n t d u r i n g a n y labile m e m o r y process, i n c l u d i n g s h o r t - t e r m m e m o r y processes o f a v o i d a n c e learning. The p r e s e n t s t u d y s o u g h t to d e t e r m i n e w h e t h e r p o s t t r i a l
GENERAL METHOD T h e a n i m a l s were male a l b i n o mice, weighing 2 0 - 2 5 g, of t h e i n b r e d C 3 H / H e / G i f COB strain, o u t b r e d f r o m Charles River Mouse F a r m s ICR COBS. T h e y were h o u s e d in groups of 20 w i t h free access to food and w a t e r u n d e r a n o r m a l d a y / n i g h t regime. The h o m e cages were of Makralon, m e a s u r i n g 4 2 x 26 x 15 cm w i t h ceilings. T h e passive a v o i d a n c e s t e p - d o w n a p p a r a t u s consisted o f a 50 x 50 x 50 cm b o x w i t h an electrifiable grid floor (6 m m dia. stainless steel rods placed 13 m m apart). A 10 m m high, 67 m m dia. r o u n d w o o d e n p l a t f o r m was s i t u a t e d in the m i d d l e o f t h e grid. Enclosing this p l a t f o r m was a 20 m m long, 68 m m dia. plastic tube. The f o o t s h o c k was a s c r a m b l e d 1 sec d u r a t i o n 1 m A c u r r e n t , l i m i t e d b y a c o n s t a n t c u r r e n t unit. A trial c o n s i s t e d in placing an a n i m a l o n t o the w o o d e n
Supported by Swiss National Science Foundation Grant 3. 6610. 75. We thank Mrs. H. Koerber, Miss E. Pichler, Mr. W. Frei, Mr. A. Gonser, Mr. M. Gygax, and Mr. R. Lehner for their help in running experiments. Reprints may be obtained from Joseph P. Huston, Institute of Pharmacology, University of Zurich, Gloriastrasse 32a, 8006 Zurich, Switzerland. 1103
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M O N D A D O R I , W A S E R AND H U S T O N
p l a t f o r m within the enclosing cylinder. A f t e r 15 sec the cylinder was r e m o v e d and the latency to descend u p o n the grid floor was measured. The f o o t s h o c k was a d m i n i s t e r e d as soon as the animal's four paws t o u c h e d the grid. The e x p e r i m e n t a l animals t h e n received the various posttrial m a n i p u l a t i o n s ( r e i n f o r c e m e n t , p u n i s h m e n t , ECS), as described b e l o w in the respective m e t h o d sections. The test for learning was always p e r f o r m e d 24 hr later. EXPERIMENT
1
In a previous e x p e r i m e n t [ 10] we r e p o r t e d that 1 min of access to f o o d given within 2 0 - 5 0 sec after the f o o t s h o c k in a passive avoidance s t e p - d o w n task led to i m p r o v e d learning u p o n retest 24 hr later in mice. The i n t e n t o f the p r e s e n t e x p e r i m e n t was to c o n f i r m this finding u n d e r various d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s , which are s u m m a r i z e d in the discussion. Me tit od In addition to the materials described above u n d e r General Method, plastic reward boxes (12 x 6 x 5.5 cm), with doors in f r o n t and metal lids, were used to a d m i n i s t e r posttrial f o o d r e i n f o r c e m e n t . Beginning 1 4 - 1 6 hr prior to the e x p e r i m e n t 300 mice were f o o d deprived. At various times after t h e y received the f o o t s h o c k in the s t e p - d o w n apparatus the animals were placed into the reward boxes, where t h e y were allowed to eat for 30 sec. The delays b e t w e e n f o o t s h o c k and access to f o o d were 10, 30, 60 and 90 sec. During the interval b e t w e e n f o o t s h o c k and f o o d r e i n f o r c e m e n t the animals ( e x c e p t the 10 sec delay group) were placed into a waiting b o x (a ceilingless box similar to the h o m e cage). All animals were t h e n i n t r o d u c e d i n t o the reward b o x e s 10 sec prior to the a n t i c i p a t e d onset time o f eating. Eating o n s e t was timed with a s t o p - w a t c h . Only those animals were used who c o m m e n c e d eating within + 5 sec of the e x p e c t e d o n s e t time, and w h o s e feeding was n o t i n t e r r u p t e d for more than 5 sec. Due to these rigorous criteria, only 123 o f the total 300 animals could be included in the e x p e r i m e n t . The e x p e r i m e n t a l animals were placed i n t o n e w h o m e cages i m m e d i a t e l y after the f o o d r e i n f o r c e m e n t period. The c o n t r o l animals were placed into new h o m e cages i m m e d i a t e l y after the f o o t s h o c k . One h o u r t h e r e a f t e r all animals received free access to food and water. Six h o u r s later their food was w i t h d r a w n again in o r d e r to establish a similar level o f f o o d deprivation during the time o f retesting on the n e x t day. The test for learning in the s t e p - d o w n apparatus was carried out at the same time of the day as the initial learning trial. During this retest the s t e p - d o w n latencies were r e c o r d e d again. A trial was t e r m i n a t e d if an animal r e m a i n e d on the p l a t f o r m for 150 sec. To eliminate any bias, a double blind p r o c e d u r e was used, Results To our delight 30 sec o f f o o d - r e i n f o r c e m e n t , c o m mencing either 1 0 or 30 sec after the f o o t s h o c k , facilitated learning o f the s t e p - d o w n avoidance. These t w o groups showed significantly longer s t e p - d o w n latencies (means 95 and 96 sec) c o m p a r e d to the c o n t r o l group (44 sec). Two tailed Mann-Whitney U-tests yielded p < 0 . 0 5 for the 10 sec group and p < 0 . 0 0 3 for the 30 sec delay group c o m p a r e d to the controls. These groups also had significantly longer mean s t e p - d o w n latencies than the 60 sec and 90 sec posttrial r e i n f o r c e m e n t delay groups (see Table 1). N e i t h e r
TABLE1 POSTTRIAL REINFORCEMENT. MEANS AND STANDARD DEVIATIONS FOR ONSET TIMES OF EATING AND STEP-DOWN LATENCIES (SDL) DURING BASELINE AND RETEST TRIALS FOR THE EXPERIMENTAL AND CONTROL GROUPS
Group
Eating Onset (sec)
SDL Baseline (sec)
SDL Retest (sec)
10 (sec) 30 60 90 control
10.6 _+ 1.8 28.2 _+ 4.2 58.4 _+ 2.9 88.9 +_ 4.1 --
8.9 -+ 4.9 11.0 -+ 4.9 9.3 _+ 3.2 10.5 _+ 3.6 9.9 _+ 2.9
Mann-Whitney 10 vs control 30 vs control 10 vs 60 30 vs 60 10 vs 90 30 vs 90
U-test (two-tailed) retest SDL comparisons p<0.03 p<0.003 p<0.06 p<0.0(ll p<0.05 p<0.003
94.9 96.5 55.9 57.0 44.2
n
_+ 56.5 +_ 57.2 _+ 54.8 +_ 52.9 _+ 40.3
9 28 30 27 29
90 "G 8O in
- 70
O
c 60 tll
E 50
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t0
t
I
I
10
30
I
I
60 90sec delay after trial
HG. l. Mean step-down latencies of" the four groups given 30 sec of posttrial food reinforcement and of the control group. the 60 n o r the 90 sec delay group differed significantly from the c o n t r o l group. Table 1 p r e s e n t s the m e a n s and standard deviations of the o n s e t latencies for eating, as well as o f the baseline and retest s t e p - d o w n latencies for all groups. It also summarizes the results of the various statistical c o m p a r i s o n s b e t w e e n groups. Figure 1 depicts the mean s t e p - d o w n latencies o f all o f the groups. Discussion The results c o n f i r m the findings of the previous study
POST-TRIAL REINFORCEMENT, PUNISHMENT OR ECS that posttrial food reinforcement can facilitate learning of a passive avoidance response in a delay-dependent manner in mice. Whereas in the present study a facilitation was obtained with the 10 and 30 sec posttrial reinforcement groups, in the previous study reinforcement facilitated learning when presented at 20, 30 and 50 sec delays (maximally at 30 sec), but not when given immediately after the footshock (<5 sec). The major procedural differences between the two studies can be summarized as follows: (a) The present study employed a lower intensity footshock (1 vs 2 mA). (b) The mice were food deprived during the retest trial, whereas in the earlier study free access to food was reinstated 1 hr after the posttrial reinforcement. (c) In the present experiment they had a shorter access (30 vs 60 sec) to food reinforcement, (d) the delay groups were established on the basis of onset of eating, rather than time of access to food. (e) The experiment was run during the lights-on phase of the day/night cycle. It should be noted that from conventional considerations of the action of reinforcement, one might have rather predicted that the posttrial reinforcement would have increased the probability of stepping down from the platform (poorer learning of the avoidance). Our finding of longer step-down latencies (improved learning), therefore allows an interpretation of these results in terms of a direct action of reinforcement on short-term memory processes [li].
1105 ted from Jacobs and Sorenson [12], who used shorter delays (until 30 sec) between footshock and ice-water. To show the direct influence of ice-water punishment not on memory processing, but on stepping-down behavior, 245 mice were randomly split into 5 groups. The groups received 10 sec ice-water punishment either immediately, 30, 60 or 90 sec after stepping off the platform. Hence the procedure was identical to the preceeding experiment except that the animals did not receive a footshock after stepping down. Results
Figure 2 illustrates the major results. It shows, for one, that the step-down latencies of the immediate (<5 sec) posttrial ice-water group were somewhat shorter than those of the 30 sec delay group, but not significantly (p<0.2). Neither was this group significantly different from the nontreatment control group @<0.42). Table 2 summarizes the statistical results of comparisons between various groups using the two-tailed Mann-Whitney U-test.
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EXPERIMENT 2 The purpose of the present experiment was to test the influence of posttrial punishment on step-down passive avoidance learning. We wished to determine whether posttrial punishment would have opposite effects to posttrial reinforcement on avoidance learning; i.e. whether a second punishment (10 sec swimming in ice water)following the footshock would disrupt learning, in which case it would be interpretable in terms of a direct influence on short-term memory processes. Hence 10 sec of ice water punishment were administered after the footshock at the same delays used in the preceeding posttrial reinforcement study. Secondly to determine the direct effects of ice-water punishment on the stepping-down behavior, the experiment was replicated, except that the animals only received the ice-water treatment at the same delays after stepping off the platform, but without being administered a footshock. Method
In addition to the materials described under General Method above, two plastic boxes (42 × 26 x 15 cm) were used, one of which was filled with ice-water (I°C). The 323 mice were randomly split into four posttrial punishment delay groups and one control group. At various delays (< 5, 30, 60 or 90 sec) after stepping off the platform and receiving the footshock, the experimental animals were inserted into the ice-water for 10 sec. During the interval between footshock and ice-water the animals (except for the <5 sec delay group) were placed into a waiting box (identical to the home cage). The control animals were placed into new home cages immediately after the footshock, the experimental animals immediately after the ice-water bath. The posttrial ice-water punishment procedure was adop-
u 30 C
E
20 10 1
I
I
10
30
60
I
90 sec delay after trial
FIG. 2. Mean step-down latencies of the four groups given l0 sec of posttrial ice-water punishment and of the control group. Interestingly, the 60 sec delay group showed significantly shorter step-down latencies than the 30 sec delay group (p<0.02) and 90 sec group (p<0.004) and differed from the nontreatment control group, as well (p<0.11). Hence, posttrial ice-water punishment was most effective in disrupting learning when presented 60 sec after the footshock. Table 2 presents the means and standard deviations of the baseline and retest step-down latencies for all groups. The replication without the footshock showed that the ice-water punishment influenced stepping-down behavior upon retest in a time-dependent manner. The result was a gradient of gradually decreasing step-down latencies as a function of the delay between stepping off the platform and ice-water punishment (See Fig. 3 for illustration of the retest latencies). Only immediate punishment, however, led to a significant increase in step-down latencies (to learning), compared to the controls (p<0.001, two-tailed Mann and
1106
M O N D A D O R I , W A S E R AND HUSTON
TABLE 3
TABLE2 POSTTRIAL ICE-WATER PUNISHMENT. MEANS AND STANDARD D E V I A T I O N S FOR STEP-DOWN LATENCIES (SDL) D U R I N G BASELINE AND RETEST TRIALS FOR THE EXPERIMENTAL AND
CONTROL GROUPS SDL
SDL
SDL
SDL
Baseline
Retest
Baseline
Retest
(sec)
(sec)
(sec)
(sec)
Group <5 (sec) 30 60 90 control
10.1 10.7 9.8 10.5 11.8
Mann-Whitney <5 vs control 60 vs control <5 vs 30 30 vs 60 60 vs 90
_+ 4.2 _+ 4.7 _+ 4.4 _+ 5.3 _+ 5.8
39.9 51.9 33.3 60.5 50.8
+ 44.3 _+ 52.9 +_ 41.1 +_ 56.9 _+ 56.0
n
Group
49 49 49 49 49
<5 (sec) 30 60 90 control
U-test (two-tailed) retest SDL comparisons p<0.42 p<0.11 p<0.2 p<0.02 p<0.004
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20
E 10
il
10
EFFECTS OF 10 SEC OF ICE-WATER PUNISHMENT ON STEPDOWN LATENCIES. MEANS A N D S T A N D A R D DEVIATIONS DURING BASELINE A N D RETEST TRIALS FOR THE EXPERIMENTAL AND CONTROL GROUPS
I
30
I
I
60 90 sec detay after trial
HG. 3. Mean step-down latencies of the four groups given only 10 sec of ice-water punishment (without footshock) after stepping off the platform and of the control group.
13.0 12.0 11.0 11.8 12.4
_+ 6.4 + 6.2 _+ 4.5 _+ 4.7 _+ 6.7
50.9 35.2 34.3 26.0 25.9
_+ 44.2 + 36.0 _+ 32.0 + 23.7 _+ 23.7
n 49 49 49 49 49
and S o r e n s o n [12] w h o f o u n d s h o r t e r s t e p - d o w n latencies with i m m e d i a t e p o s t s h o c k ice-water p u n i s h m e n t , and a lack o f e f f e c t with 30 sec delayed ice-water. Hence the results of the p o s t s h o c k p u n i s h m e n t s t u d y can p r o b a b l y n o t be i n t e r p r e t e d simply in t e r m s o f an additive effect o f two p u n i s h m e n t s on s t e p p i n g d o w n behavior, w h i c h leaves o p e n the possibility o f additional t i m e - d e p e n d e n t e f f e c t s o f the p o s t s h o c k ice-water directly on the m e m o r y processing for the s t e p - d o w n f o o t s h o c k c o n t i n g e n c y . The results, h o w e v e r , provide only meager s u p p o r t for the h y p o t h e s i s that posttrial p u n i s h m e n t could influence s h o r t - t e r m m e m o r y , reciprocal to the e f f e c t s o f posttrial r e i n f o r c e m e n t . Only the 60 sec delay group s h o w e d s o m e w h a t , b u t n o t significantly, p o o r e r learning c o m p a r e d to c o n t r o l s ( p < 0 . 1 1 ) , and significantly p o o r e r learning c o m p a r e d to the 30 and 90 sec group. This could be i n t e r p r e t e d in t e r m s of a direct influence on m e m o r y processes. This 60 sec delay e f f e c t d e p a r t s f r o m the maximal facilitation at 30 sec f o u n d in the posttrial r e i n f o r c e m e n t e x p e r i m e n t s ([ 10,18] and Exp. 1). However it should be n o t e d that the d u r a t i o n of the posttrial r e i n f o r c e m e n t s in the e x p e r i m e n t s was either 30 sec ([ 18], E x p e r i m e n t 1) or one rain [ 10], thus e x t e n d i n g i n t o the 60 sec delay period. The results are interesting from a n o t h e r perspective. When the curve o f Fig. 2 is c o m p a r e d to the effects o f posttrial ECS on the same task in Fig. 4, some c o r r e s p o n d e n c e b e t w e e n the t w o curves is obvious, w h i c h suggests a relationship b e t w e e n ECS and p u n i s h m e n t . EXPERIMENT 3
Whitney U-test). Table 3 s u m m a r i z e s the m e a n s and standard deviations of the s t e p - d o w n latencies during baseline and retest trials for the e x p e r i m e n t a l and c o n t r o l groups. Discussion Ten sec o f ice-water p u n i s h m e n t alone led to significant s t e p - d o w n avoidance learning w h e n p r e s e n t e d i m m e d i a t e l y u p o n s t e p p i n g o f f the p l a t f o r m . Hence, we e x p e c t e d an additive effect o f the posttrial ice-water p u n i s h m e n t with the f o o t s h o c k on learning, at least with the i m m e d i a t e p o s t s h o c k ice-water t r e a t m e n t . The results however, as s h o w n in Fig. 2, do n o t indicate such a simple additive effect. On the c o n t r a r y , the i m m e d i a t e p o s t s h o c k ice-water group did n o t differ significantly f r o m the c o n t r o l group, and s h o w e d s o m e w h a t p o o r e r learning t h a n the 30 sec delay group. This c o r r e s p o n d s with the results o f J a c o b s
Given our a n o m a l o u s results with posttrial p u n i s h m e n t in the p r e c e e d i n g e x p e r i m e n t , we wished to test the effects o f posttrial ECS p r e s e n t e d at exactly the same delays, using the same paradigm. Some evidence suggests that ECS has aversive p r o p e r t i e s [3, 4, 13, 16, 2 2 ] ; h o w e v e r , we could n o t find any one study with mice in w h i c h ECS was a d m i n i s t e r e d at the various posttrial delays which were o f interest to us. Method The same p r o c e d u r e as in E x p e r i m e n t 2 was used, e x c e p t that the mice were a d m i n i s t e r e d ECS instead o f ice-water either at <5, 30, 60 or 90 sec after the f o o t s h o c k . The n o n t r e a t e d c o n t r o l animals were placed into new h o m e cages i m m e d i a t e l y after the f o o t s h o c k . Of a total of 350 mice 297 were used. The rest was discarded for failure to
P O S T - T R I A L R E I N F O R C E M E N T , P U N I S H M E N T O R ECS
1107
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30 O3
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I
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I
10
30
60
I
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detay after trial FIG. 4. Mean step-down latencies of the four groups given posttrial ECS (lower curve), the four handling control groups (upper curve), and the non-handled control group (C). adhere to the criteria for an effective ECS t r e a t m e n t (convulsions). The ECS was o f 5.4 m A i n t e n s i t y and 0.35 sec d u r a t i o n . It was applied w i t h t w o t h i m b l e - e l e c t r o d e s , w h i c h were gloved over the t h u m b and forefinger, and a d m i n i s t e r e d across the ears (see Fig. 5). To c o n t r o l f o r the e f f e c t s o f handling, 4 o t h e r groups ( < 5 , 30, 60 and 90 sec) were t r e a t e d in e x a c t l y the same m a n n e r as t h e ECS groups (their h e a d s were grabbed b e t w e e n t h u m b and f o r e f i n g e r e l e c t r o d e ) , e x c e p t t h a t ECS was n o t a d m i n i s t e r e d .
Results The
posttrial
ECS d i s r u p t e d learning o f the passive
~b
/'i
FIG. 5. Thimble electrodes for ECS constructed of meshed brass wire. Insulation provided by rubber glove. avoidance. When c o m p a r e d to the n o n - h a n d l e d c o n t r o l s p o o r e r learning ( s h o r t e r s t e p - d o w n latencies) was s h o w n by the <5 sec, ( p < 0 . 0 2 ) , 30 sec ( p < 0 . 0 8 ) , and 60 sec ( p < 0 . 0 1 ) delay groups, but n o t by the 90 sec group ( M a n n - W h i t n e y U-test, 2-tailed). Interestingly, the 60 sec delay group s h o w e d the m o s t drastic d i s r u p t i o n o f learning, similar to the e f f e c t s o f
TABLE 4 POSTTRIAL ECS. MEANS AND STANDARD DEVIATIONS OF STEP-DOWN LATENCIES (SDL) DURING BASELINE AND RETEST TRIALS FOR THE ECS AND CONTROL GROUPS Group
<5 (sec) 30 60 90 Cnh*
SDL experiment baseline 11.1 ± 5.4 11.7 ± 5.9 i 1.2 +-- 4.8 11.9 ___ 5.9
retest 26.6 _ 30.3 36.1 ± 41.3 24.8 --- 29.3 40.8 ___45.7
SDL control baseline 11.9 ± 4.6 11.3 ± 4.7 10.3 ___ 5.0 I 1.0 ± 4.8 11.1 ± 5.6
*Nonhandled controls. Mann-Whitney U-test (two-tailed) retest SDL comparisons <5 vs c<5p<0.05 30 vs c30 p<0.05 60 vs c60 p<0.02 90 vs c90 p<0.5 <5 vs Cnh p<0.02 30 vs Cnh p<0.08 60 vs Cnh p<0.01 <5 VS 30 p<0.3 30 VS 60 p<0.25 60 VS 90 p<0.02 C5 VS %0 p<0.25
retest 41.0 ± 46.7 54.6 ± 54.9 48.9 ___ 52.8 50.9 ± 54.4 50.8 -----54.8
n exp. 58 59 58 59
contr. 69 69 69 69 60
1108
MONDADORI, WASER AND HUSTON
posttrial p u n i s h m e n t in E x p e r i m e n t 2. A l t h o u g h the 60 sec delay group was n o t significantly d i f f e r e n t f r o m the 30 sec group ( p < 0 . 2 5 ) , it differed significantly f r o m the 90 sec delay g r o u p ( p < 0 . 0 2 , two-tailed U-test). The < 5 , 30, and 60 sec delay ECS groups, b u t n o t the 90 sec group, also s h o w e d significantly p o o r e r learning c o m p a r e d to their respective h a n d l i n g - c o n t r o l groups (see Table 4). Interestingly, the < 5 sec h a n d l i n g - c o n t r o l group learned s o m e w h a t p o o r e r t h a n the 30 sec h a n d l i n g - c o n t r o l group. Discussion The results of the ECS e x p e r i m e n t s t r e n g t h e n the finding of E x p e r i m e n t 2 t h a t 60 sec is s o m e h o w a critical period for p o s t t r i a l effects for the s t e p - d o w n avoidance learning in mice. The close c o r r e s p o n d a n c e b e t w e e n the 2 curves (Figs. 2 and 3) suggests t h a t some of the effects of ECS could be a c c o u n t e d for in t e r m s of a direct punishm e n t of posttrial m e m o r y processes, at least at the 60 sec delay. It is i n t e r e s t i n g t h a t the h a n d l i n g c o n t r o l group in E x p e r i m e n t 3 (Fig. 4) also s h o w e d similar d e l a y - d e p e n d e n t differences in learning, and t h a t the 5 sec h a n d l i n g - c o n t r o l group s h o w e d p o o r e r learning t h a n the 30 sec h a n d l i n g group. It should be n o t e d t h a t the h a n d l i n g c o n t r o l p r o c e d u r e was m o s t likely aversive, and h e n c e , it is possible that the results with this t r e a t m e n t reflect p o s t t r i a l punishm e n t effects, similar to these in E x p e r i m e n t 2. The effects of i m m e d i a t e and 30 sec delayed ECS c o r r e s p o n d s closely with the results of C h o r o v e r and Schiller [ 2] w i t h rats. The d e p a r t u r e a f t e r 30 sec f r o m the e x p e c t e d delayd e p e n d e n t g r a d i e n t of amnesia suggests t h a t m e m o r y processes (be t h e y c o n s o l i d a t i o n or retrival processes) are i n f l u e n c e d by ECS in a more c o m p l e x m a n n e r t h a n is usually assumed. GENERAL DISCUSSION All t h r e e e x p e r i m e n t s s u p p o r t the n o t i o n of a critical posttrial interval d u r i n g w h i c h m e m o r y processing can be i n f l u e n c e d . T h i r t y sec of food r e i n f o r c e m e n t facilitated learning w h e n initiated up to a delay of 60 sec a f t e r the f o o t s h o c k . ECS as well as ice-water p u n i s h m e n t r e t a r d e d learning w h e n a d m i n i s t e r e d w i t h i n 60 sec a f t e r the trial. A perusal of t h e ECS literature on mice shows t h a t a vast range o f i n t e n s i t i e s and d u r a t i o n s of ECS are b e i n g used. The i n t e n s i t i e s vary f r o m 4 m A [7] to 50 m A [ 1 9 ] ; and the d u r a t i o n f r o m 200 msec [19] to 800 msec [ 2 1 ] . As s h o w n by B u c k h o l z and B o w m a n [ 1 ] , these d i f f e r e n c e s could a c c o u n t for some of the discrepancies b e t w e e n the
results. We used a r a t h e r low i n t e n s i t y (5.4 m A ) ECS in o r d e r to m i n i m i z e irreversible damage to the brain. Nevertheless o u r results are in a g r e e m e n t w i t h some previous data in mice. As a rule ECS a d m i n i s t e r e d up to 15 sec after the trial results in retrograde amnesia irrespective of the i n t e n s i t y and d u r a t i o n of the shock. Our data w i t h i n this delay are in a c c o r d a n c e w i t h this rule. O u r results at the 30 sec ECS delay ( n o amnesia w h e n c o m p a r e d to the n o n - h a n d l e d c o n t r o l group, and partial amnesia w h e n c o m p a r e d to the h a n d l e d c o n t r o l s ) agree w i t h t h e findings of D a w s o n and M c G a u g h [5] at a 20 sec delay ( n o a m n e s i a w i t h o u t familiarisation trials) and of McGaugh, et al. [17] at a 30 sec delay (partial amnesia). Less c o m p a r a b l e data is available at larger ECS delays. D o r f m a n and Jarvik [6] failed to find amnesia at 45 sec delay w i t h a 5 m A s h o c k intensity. R o b u s t e l l i and Jarvik [20] r e p o r t e d amnesia at a 60 sec delay, in a c c o r d a n c e w i t h o u r results. A m n e s i a has also b e e n r e p o r t e d at 75 sec [9] and 80 sec [ 1 4 ] . Hence, a l t h o u g h n o o t h e r studies seem to have investigated the effects of ECS at all of the intervals we have used, o u r finding of a lack of a linear delayd e p e n d e n t g r a d i e n t of amnesia seems to c o n f o r m to s c a t t e r e d results in the literature. The idea of a linear g r a d i e n t of retrograde amnesia in mice seems to be based m a i n l y on data using e i t h e r widely spaced [14] or very closely spaced [8] posttrial ECS intervals. Our data suggest t h a t the v u l n e r a b i l i t y of posttrial m e m o r y processes changes drastically w i t h i n 90 sec. T h e c o n c o r d a n c e b e t w e e n o u r ECS and ice-water p u n i s h m e n t results suggests t h a t , a l t h o u g h ECS has m o r e drastic a m n e s t i c p r o p e r t i e s t h a n p u n i s h m e n t , the m e c h a n i s m of a c t i o n of these t r e a t m e n t s o n m e m o r y processes m a y be similar. The possibility of a direct a c t i o n of p u n i s h m e n t on m e m o r y processes seems to have achieved little or n o a t t e n t i o n . J a c o b s and S o r e n s o n [12] f o u n d t h a t 10 sec of ice-water p u n i s h m e n t i n h i b i t e d learning of a passive avoidance w h e n a d m i n i s t e r e d i m m e d i a t e l y after the f o o t s h o c k . This effect w e a k e n e d with increasing delays b e t w e e n f o o t s h o c k a n d ice-water, and at 30 sec delay ice-water n o longer d i s r u p t e d learning. This result c o r r e s p o n d s with o u r findings w i t h i n t h a t interval. A n o t h e r s t u d y w h i c h m i g h t have relevance to the issue of p u n i s h m e n t of m e m o r y processes is t h a t of Leukel [ 15], w h o f o u n d d i s r u p t i o n of learning in rats t h a t received a painful legshock one m i n u t e a f t e r each trial of a o n e - w a y active a v o i d a n c e task. The p r o b l e m of posttrial p u n i s h m e n t and its r e l a t i o n s h i p to ECS a n d o t h e r a m n e s t i c t r e a t m e n t s obviously requires f u r t h e r investigation.
REFERENCES 1. Buckholtz, N. S. and R. E. Bowman. Incubation and retrograde amnesia studied with various ECS intensities and durations. Physiol. Behav. 8: 113-117, 1972. 2. Chorover, S. L. and P. H. Schiller. Short-term retrograde amnesia in rats. J. eomp. physiol. Psychol. 59: 7 3 - 7 8 , 1960. 3. Coons, E. E. and N. E. Miller. Conflict versus consolidation of memory traces to explain "retrograde amnesia" produced by ECS. J. comp. physiol. Psyehol. 53: 5 2 4 - 5 3 1 , 1960. 4. Dawson, R. G. and G. T. Pryor. Onset vs recovery in the aversive effects of electroconsulsive shock. Psychon. ScL 3: 273-274, 1965.
5. Dawson, R. G. and J. L. McGaugh. Electroconvulsive shockproduced retrograde amnesia: Analysis of the familiarisation effect. Communs. behav. Biol. 4: 9 1 - 9 5 , 1969. 6. Dorfman, L. J. and M. E. Jarvik. Comparative amnesic effects of transcorneal and transspinnate ECS in mice. PhysioL Behav. 3: 815-818, 1968a. 7. Dorfman, L. J. and M. E. Jarvik. A parametric study of electroshock-induced retrograde amnesia in mice. Neuropsyehologia 6: 373-380, 1968b.
P O S T - T R I A L R E I N F O R C E M E N T , P U N I S H M E N T O R ECS 8. Grosser, G. S., H. E. Percy and L. E. Pierce. Short footshockelectroconvulsive shock intervals and retrograde amnesia in mice. Psychon. Sci. 25: 26-28, 1971. 9. Herz, M. J. and H. V. S. Peeke. Permanence of retrograde amnesia produced by electroconvulsive shock. Science 156: 1396-1397, 1967. 10. Huston, J. P., C. Mondadori and P. G. Waser. Facilitation of learning by reward of posttrial memory processes. Experientia 30: 1038-1040, 1974. 11. Huston, J. P. and C. Mondadori. Memory and reinforcement: a model. In: Proc. 2nd lnt. Congr. CLANS, Prague, 1975,Activ. Nerv. Sup. in press. 12. Jacobs, B. L. and C. A. Sorenson. Memory disruption in mice by brief posttrial immersion in hot or cold water. J. comp. physioL Psychol. 68: 239-244, 1969. 13. Kesner, R. P. ECS as a punishing stimulus: Dependency on retrograde amnesia, duration of anterograde amnesia, and intensity of pain. J. comp. physiol. Psychol. 74: 398-406, 1971. 14. Kopp, R., Z. Bohdanecky and M. E. Jarvik. Long temporal gradients of retrograde amnesia for a well discriminated stimulus. Science 153: 1547-1549, 1966. 15. Leukel, F. A. A comparison of the effects of ECS and anaesthesia on acquisition of the maze habit. J. comp. physioL PsychoL 50: 300-306, 1957.
1109 16. McGaugh, J. L. and M. C. Madsen. Amnesic and punishing effects of electroconvulsive shock. Science 144: 182-183, 1964. 17. McGaugh, J. L., R. G. Dawson, R. Coleman and J. Rawie. Electroshock effects on memory in Diethyl-Ether-treated mice: Analysis of the CER-incubation hypothesis of retrograde amnesia. Communs. behav. Biol. 6: 227-232, 1971. 18. Mondadori, C., K. Ornstein, P. G. Waser and J. P. Huston. Posttrial reinforcing hypothalamic stimulation can facilitate avoidance learning. NeuroscL Letters 2 : 1 8 3 - 1 8 7 , 1976. 19. Ray, O. S. and L. W. Bivens. Reinforcement magnitude as a determinant of performance decrement after electroconvulsive shock. Science 160: 330-332, 1968. 20. Robustelli, F. and M. E. Jarvik. Retrograde amnesia from detention. Physiol. Behav. 3: 543-547, 1968. 21. Zornetzer, S. F. and J. L. McGaugh. Effects of electroconvulsive shock upon inhibitory avoidance: The persistence and stability of amnesia. Communs. Behav. Biol. 3: 173-180, 1969. 22. Zornetzer, S. F. and J. L. McGaugh. Retrograde amnesia and brain seizures in mice. Physiol. Behav. 7: 401-408, 1971.
Time-Dependent Effects of Post-Trial Reinforcement, Punishment or ECS on Passive Avoidance Learning' C E S A R E M O N D A D O R I , P E T E R G. W A S E R A N D J O S E P H P. H U S T O N 2
Institute o f Pharmacology, University o f Zurich, Zurich, Switzerland (Received 1 1 November, 1976) MONDADORI, C., P. G. WASER AND J. P. HUSTON. Time-dependent effects ofposttrial reinforcement, punishment or ECS on passive avoidance learning. PHYSIOL. BEHAV. 18(6) 1103-1109, 1977. - Three studies were performed to assess the effects of posttrial food reinforcement, ice-water punishment, and ECS on step-down passive avoidance learning in mice. The treatments were each administered at <5, 30, 60 and 90 sec after the footshock. One-half min of feeding beginning either 10 or 30 sec after the footshock increased the step-down latencies 24 hr later. This was considered to support the hypothesis that reinforcement facilitates learning by a direct influence on short-term memory processes. Posttrial ice-water punishment (10 sec immersion) disrupted learning only in the 60 sec postshock delay group. ECS (5.4 mA, 0.35 sec, across the ears) disrupted learning at all posttrial delays except 90 sec, with maximal effects immediately and at 60 sec, and minimal disruption at 30 sec. Similarities in the topography of the ECS and punishment curves suggested that some of the effects of posttrial ECS could be due to aversive properties of the ECS (acting directly on memory processes). Memory
Passive avoidance
Reinforcement
Punishment
ECS
Mice
p u n i s h m e n t w o u l d act in a m a n n e r reciprocal to posttrial r e i n f o r c e m e n t , a n d t h u s i n h i b i t a v o i d a n c e learning. In t h r e e e x p e r i m e n t s w i t h mice we t e s t e d the effects on passive a v o i d a n c e l e a r n i n g of (a) p o s t t r i a l food r e i n f o r c e m e n t , in a m o d i f i c a t i o n of t h e p r e v i o u s s t u d y [ 1 0 ] , ( b ) i c e - w a t e r p u n i s h m e n t , p r e s e n t e d at the same p o s t s h o c k intervals, and (c) p o s t t r i a l e l e c t r o c o n v u l s i v e s h o c k (ECS) a d m i n i s t e r e d at the same delays.
S E V E R A L studies have s h o w n t h a t p o s t t r i a l r e i n f o r c e m e n t can facilitate passive a v o i d a n c e learning. In o n e e x p e r i m e n t [ 10] mice were given 1 m i n of food r e i n f o r c e m e n t at various delays a f t e r the f o o t s h o c k in a s t e p - d o w n passive a v o i d a n c e task and tested 24 hr later. Facilitation of learning was o b t a i n e d in the groups a d m i n i s t e r e d reinforcem e n t w i t h i n 20 a n d 50 sec a f t e r the f o o t s h o c k b u t n e i t h e r b e f o r e n o r a f t e r this interval. In a s u b s e q u e n t s t u d y [18] rats were t r a i n e d o n a o n e - w a y s t e p - t h r o u g h active a v o i d a n c e task a n d t h e n s u b j e c t e d to a reversal p r o c e d u r e , i.e. t h e y were given a f o o t s h o c k in t h e previously safe c h a m b e r . T h e rats t h a t received r e i n f o r c i n g h y p o t h a l a m i c s t i m u l a t i o n 30 sec l a t e r avoided this c h a m b e r b e t t e r t h a n t h e n o n s t i m u l a t e d controls 24 h r later. We i n t e r p r e t e d these results in t e r m s of a direct a c t i o n o f r e i n f o r c e m e n t o n s h o r t - t e r m m e m o r y processes, in s u p p o r t o f t h e t h e o r y [ 11 ] t h a t r e i n f o r c e m e n t in the i n s t r u m e n t a l l e a r n i n g s i t u a t i o n acts o n ( m a i n t a i n s ) an i m m e d i a t e m e m o r y trace t h a t is left b y t h e o p e r a n t behavior. A c c o r d i n g to this t h e o r y r e i n f o r c e m e n t acts as such by p r e v e n t i n g this trace f r o m fading, and t h e r e f o r e , it s h o u l d be possible to facilitate l e a r n i n g b y p r e s e n t i n g r e i n f o r c e m e n t d u r i n g a n y labile m e m o r y process, i n c l u d i n g s h o r t - t e r m m e m o r y processes o f a v o i d a n c e learning. The p r e s e n t s t u d y s o u g h t to d e t e r m i n e w h e t h e r p o s t t r i a l
GENERAL METHOD T h e a n i m a l s were male a l b i n o mice, weighing 2 0 - 2 5 g, of t h e i n b r e d C 3 H / H e / G i f COB strain, o u t b r e d f r o m Charles River Mouse F a r m s ICR COBS. T h e y were h o u s e d in groups of 20 w i t h free access to food and w a t e r u n d e r a n o r m a l d a y / n i g h t regime. The h o m e cages were of Makralon, m e a s u r i n g 4 2 x 26 x 15 cm w i t h ceilings. T h e passive a v o i d a n c e s t e p - d o w n a p p a r a t u s consisted o f a 50 x 50 x 50 cm b o x w i t h an electrifiable grid floor (6 m m dia. stainless steel rods placed 13 m m apart). A 10 m m high, 67 m m dia. r o u n d w o o d e n p l a t f o r m was s i t u a t e d in the m i d d l e o f t h e grid. Enclosing this p l a t f o r m was a 20 m m long, 68 m m dia. plastic tube. The f o o t s h o c k was a s c r a m b l e d 1 sec d u r a t i o n 1 m A c u r r e n t , l i m i t e d b y a c o n s t a n t c u r r e n t unit. A trial c o n s i s t e d in placing an a n i m a l o n t o the w o o d e n
Supported by Swiss National Science Foundation Grant 3. 6610. 75. We thank Mrs. H. Koerber, Miss E. Pichler, Mr. W. Frei, Mr. A. Gonser, Mr. M. Gygax, and Mr. R. Lehner for their help in running experiments. Reprints may be obtained from Joseph P. Huston, Institute of Pharmacology, University of Zurich, Gloriastrasse 32a, 8006 Zurich, Switzerland. 1103
1104
M O N D A D O R I , W A S E R AND H U S T O N
p l a t f o r m within the enclosing cylinder. A f t e r 15 sec the cylinder was r e m o v e d and the latency to descend u p o n the grid floor was measured. The f o o t s h o c k was a d m i n i s t e r e d as soon as the animal's four paws t o u c h e d the grid. The e x p e r i m e n t a l animals t h e n received the various posttrial m a n i p u l a t i o n s ( r e i n f o r c e m e n t , p u n i s h m e n t , ECS), as described b e l o w in the respective m e t h o d sections. The test for learning was always p e r f o r m e d 24 hr later. EXPERIMENT
1
In a previous e x p e r i m e n t [ 10] we r e p o r t e d that 1 min of access to f o o d given within 2 0 - 5 0 sec after the f o o t s h o c k in a passive avoidance s t e p - d o w n task led to i m p r o v e d learning u p o n retest 24 hr later in mice. The i n t e n t o f the p r e s e n t e x p e r i m e n t was to c o n f i r m this finding u n d e r various d i f f e r e n t e x p e r i m e n t a l c o n d i t i o n s , which are s u m m a r i z e d in the discussion. Me tit od In addition to the materials described above u n d e r General Method, plastic reward boxes (12 x 6 x 5.5 cm), with doors in f r o n t and metal lids, were used to a d m i n i s t e r posttrial f o o d r e i n f o r c e m e n t . Beginning 1 4 - 1 6 hr prior to the e x p e r i m e n t 300 mice were f o o d deprived. At various times after t h e y received the f o o t s h o c k in the s t e p - d o w n apparatus the animals were placed into the reward boxes, where t h e y were allowed to eat for 30 sec. The delays b e t w e e n f o o t s h o c k and access to f o o d were 10, 30, 60 and 90 sec. During the interval b e t w e e n f o o t s h o c k and f o o d r e i n f o r c e m e n t the animals ( e x c e p t the 10 sec delay group) were placed into a waiting b o x (a ceilingless box similar to the h o m e cage). All animals were t h e n i n t r o d u c e d i n t o the reward b o x e s 10 sec prior to the a n t i c i p a t e d onset time o f eating. Eating o n s e t was timed with a s t o p - w a t c h . Only those animals were used who c o m m e n c e d eating within + 5 sec of the e x p e c t e d o n s e t time, and w h o s e feeding was n o t i n t e r r u p t e d for more than 5 sec. Due to these rigorous criteria, only 123 o f the total 300 animals could be included in the e x p e r i m e n t . The e x p e r i m e n t a l animals were placed i n t o n e w h o m e cages i m m e d i a t e l y after the f o o d r e i n f o r c e m e n t period. The c o n t r o l animals were placed into new h o m e cages i m m e d i a t e l y after the f o o t s h o c k . One h o u r t h e r e a f t e r all animals received free access to food and water. Six h o u r s later their food was w i t h d r a w n again in o r d e r to establish a similar level o f f o o d deprivation during the time o f retesting on the n e x t day. The test for learning in the s t e p - d o w n apparatus was carried out at the same time of the day as the initial learning trial. During this retest the s t e p - d o w n latencies were r e c o r d e d again. A trial was t e r m i n a t e d if an animal r e m a i n e d on the p l a t f o r m for 150 sec. To eliminate any bias, a double blind p r o c e d u r e was used, Results To our delight 30 sec o f f o o d - r e i n f o r c e m e n t , c o m mencing either 1 0 or 30 sec after the f o o t s h o c k , facilitated learning o f the s t e p - d o w n avoidance. These t w o groups showed significantly longer s t e p - d o w n latencies (means 95 and 96 sec) c o m p a r e d to the c o n t r o l group (44 sec). Two tailed Mann-Whitney U-tests yielded p < 0 . 0 5 for the 10 sec group and p < 0 . 0 0 3 for the 30 sec delay group c o m p a r e d to the controls. These groups also had significantly longer mean s t e p - d o w n latencies than the 60 sec and 90 sec posttrial r e i n f o r c e m e n t delay groups (see Table 1). N e i t h e r
TABLE1 POSTTRIAL REINFORCEMENT. MEANS AND STANDARD DEVIATIONS FOR ONSET TIMES OF EATING AND STEP-DOWN LATENCIES (SDL) DURING BASELINE AND RETEST TRIALS FOR THE EXPERIMENTAL AND CONTROL GROUPS
Group
Eating Onset (sec)
SDL Baseline (sec)
SDL Retest (sec)
10 (sec) 30 60 90 control
10.6 _+ 1.8 28.2 _+ 4.2 58.4 _+ 2.9 88.9 +_ 4.1 --
8.9 -+ 4.9 11.0 -+ 4.9 9.3 _+ 3.2 10.5 _+ 3.6 9.9 _+ 2.9
Mann-Whitney 10 vs control 30 vs control 10 vs 60 30 vs 60 10 vs 90 30 vs 90
U-test (two-tailed) retest SDL comparisons p<0.03 p<0.003 p<0.06 p<0.0(ll p<0.05 p<0.003
94.9 96.5 55.9 57.0 44.2
n
_+ 56.5 +_ 57.2 _+ 54.8 +_ 52.9 _+ 40.3
9 28 30 27 29
90 "G 8O in
- 70
O
c 60 tll
E 50
©c
t0
t
I
I
10
30
I
I
60 90sec delay after trial
HG. l. Mean step-down latencies of" the four groups given 30 sec of posttrial food reinforcement and of the control group. the 60 n o r the 90 sec delay group differed significantly from the c o n t r o l group. Table 1 p r e s e n t s the m e a n s and standard deviations of the o n s e t latencies for eating, as well as o f the baseline and retest s t e p - d o w n latencies for all groups. It also summarizes the results of the various statistical c o m p a r i s o n s b e t w e e n groups. Figure 1 depicts the mean s t e p - d o w n latencies o f all o f the groups. Discussion The results c o n f i r m the findings of the previous study
POST-TRIAL REINFORCEMENT, PUNISHMENT OR ECS that posttrial food reinforcement can facilitate learning of a passive avoidance response in a delay-dependent manner in mice. Whereas in the present study a facilitation was obtained with the 10 and 30 sec posttrial reinforcement groups, in the previous study reinforcement facilitated learning when presented at 20, 30 and 50 sec delays (maximally at 30 sec), but not when given immediately after the footshock (<5 sec). The major procedural differences between the two studies can be summarized as follows: (a) The present study employed a lower intensity footshock (1 vs 2 mA). (b) The mice were food deprived during the retest trial, whereas in the earlier study free access to food was reinstated 1 hr after the posttrial reinforcement. (c) In the present experiment they had a shorter access (30 vs 60 sec) to food reinforcement, (d) the delay groups were established on the basis of onset of eating, rather than time of access to food. (e) The experiment was run during the lights-on phase of the day/night cycle. It should be noted that from conventional considerations of the action of reinforcement, one might have rather predicted that the posttrial reinforcement would have increased the probability of stepping down from the platform (poorer learning of the avoidance). Our finding of longer step-down latencies (improved learning), therefore allows an interpretation of these results in terms of a direct action of reinforcement on short-term memory processes [li].
1105 ted from Jacobs and Sorenson [12], who used shorter delays (until 30 sec) between footshock and ice-water. To show the direct influence of ice-water punishment not on memory processing, but on stepping-down behavior, 245 mice were randomly split into 5 groups. The groups received 10 sec ice-water punishment either immediately, 30, 60 or 90 sec after stepping off the platform. Hence the procedure was identical to the preceeding experiment except that the animals did not receive a footshock after stepping down. Results
Figure 2 illustrates the major results. It shows, for one, that the step-down latencies of the immediate (<5 sec) posttrial ice-water group were somewhat shorter than those of the 30 sec delay group, but not significantly (p<0.2). Neither was this group significantly different from the nontreatment control group @<0.42). Table 2 summarizes the statistical results of comparisons between various groups using the two-tailed Mann-Whitney U-test.
6op
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50
EXPERIMENT 2 The purpose of the present experiment was to test the influence of posttrial punishment on step-down passive avoidance learning. We wished to determine whether posttrial punishment would have opposite effects to posttrial reinforcement on avoidance learning; i.e. whether a second punishment (10 sec swimming in ice water)following the footshock would disrupt learning, in which case it would be interpretable in terms of a direct influence on short-term memory processes. Hence 10 sec of ice water punishment were administered after the footshock at the same delays used in the preceeding posttrial reinforcement study. Secondly to determine the direct effects of ice-water punishment on the stepping-down behavior, the experiment was replicated, except that the animals only received the ice-water treatment at the same delays after stepping off the platform, but without being administered a footshock. Method
In addition to the materials described under General Method above, two plastic boxes (42 × 26 x 15 cm) were used, one of which was filled with ice-water (I°C). The 323 mice were randomly split into four posttrial punishment delay groups and one control group. At various delays (< 5, 30, 60 or 90 sec) after stepping off the platform and receiving the footshock, the experimental animals were inserted into the ice-water for 10 sec. During the interval between footshock and ice-water the animals (except for the <5 sec delay group) were placed into a waiting box (identical to the home cage). The control animals were placed into new home cages immediately after the footshock, the experimental animals immediately after the ice-water bath. The posttrial ice-water punishment procedure was adop-
u 30 C
E
20 10 1
I
I
10
30
60
I
90 sec delay after trial
FIG. 2. Mean step-down latencies of the four groups given l0 sec of posttrial ice-water punishment and of the control group. Interestingly, the 60 sec delay group showed significantly shorter step-down latencies than the 30 sec delay group (p<0.02) and 90 sec group (p<0.004) and differed from the nontreatment control group, as well (p<0.11). Hence, posttrial ice-water punishment was most effective in disrupting learning when presented 60 sec after the footshock. Table 2 presents the means and standard deviations of the baseline and retest step-down latencies for all groups. The replication without the footshock showed that the ice-water punishment influenced stepping-down behavior upon retest in a time-dependent manner. The result was a gradient of gradually decreasing step-down latencies as a function of the delay between stepping off the platform and ice-water punishment (See Fig. 3 for illustration of the retest latencies). Only immediate punishment, however, led to a significant increase in step-down latencies (to learning), compared to the controls (p<0.001, two-tailed Mann and
1106
M O N D A D O R I , W A S E R AND HUSTON
TABLE 3
TABLE2 POSTTRIAL ICE-WATER PUNISHMENT. MEANS AND STANDARD D E V I A T I O N S FOR STEP-DOWN LATENCIES (SDL) D U R I N G BASELINE AND RETEST TRIALS FOR THE EXPERIMENTAL AND
CONTROL GROUPS SDL
SDL
SDL
SDL
Baseline
Retest
Baseline
Retest
(sec)
(sec)
(sec)
(sec)
Group <5 (sec) 30 60 90 control
10.1 10.7 9.8 10.5 11.8
Mann-Whitney <5 vs control 60 vs control <5 vs 30 30 vs 60 60 vs 90
_+ 4.2 _+ 4.7 _+ 4.4 _+ 5.3 _+ 5.8
39.9 51.9 33.3 60.5 50.8
+ 44.3 _+ 52.9 +_ 41.1 +_ 56.9 _+ 56.0
n
Group
49 49 49 49 49
<5 (sec) 30 60 90 control
U-test (two-tailed) retest SDL comparisons p<0.42 p<0.11 p<0.2 p<0.02 p<0.004
5O
CO tin
3o
©c
20
E 10
il
10
EFFECTS OF 10 SEC OF ICE-WATER PUNISHMENT ON STEPDOWN LATENCIES. MEANS A N D S T A N D A R D DEVIATIONS DURING BASELINE A N D RETEST TRIALS FOR THE EXPERIMENTAL AND CONTROL GROUPS
I
30
I
I
60 90 sec detay after trial
HG. 3. Mean step-down latencies of the four groups given only 10 sec of ice-water punishment (without footshock) after stepping off the platform and of the control group.
13.0 12.0 11.0 11.8 12.4
_+ 6.4 + 6.2 _+ 4.5 _+ 4.7 _+ 6.7
50.9 35.2 34.3 26.0 25.9
_+ 44.2 + 36.0 _+ 32.0 + 23.7 _+ 23.7
n 49 49 49 49 49
and S o r e n s o n [12] w h o f o u n d s h o r t e r s t e p - d o w n latencies with i m m e d i a t e p o s t s h o c k ice-water p u n i s h m e n t , and a lack o f e f f e c t with 30 sec delayed ice-water. Hence the results of the p o s t s h o c k p u n i s h m e n t s t u d y can p r o b a b l y n o t be i n t e r p r e t e d simply in t e r m s o f an additive effect o f two p u n i s h m e n t s on s t e p p i n g d o w n behavior, w h i c h leaves o p e n the possibility o f additional t i m e - d e p e n d e n t e f f e c t s o f the p o s t s h o c k ice-water directly on the m e m o r y processing for the s t e p - d o w n f o o t s h o c k c o n t i n g e n c y . The results, h o w e v e r , provide only meager s u p p o r t for the h y p o t h e s i s that posttrial p u n i s h m e n t could influence s h o r t - t e r m m e m o r y , reciprocal to the e f f e c t s o f posttrial r e i n f o r c e m e n t . Only the 60 sec delay group s h o w e d s o m e w h a t , b u t n o t significantly, p o o r e r learning c o m p a r e d to c o n t r o l s ( p < 0 . 1 1 ) , and significantly p o o r e r learning c o m p a r e d to the 30 and 90 sec group. This could be i n t e r p r e t e d in t e r m s of a direct influence on m e m o r y processes. This 60 sec delay e f f e c t d e p a r t s f r o m the maximal facilitation at 30 sec f o u n d in the posttrial r e i n f o r c e m e n t e x p e r i m e n t s ([ 10,18] and Exp. 1). However it should be n o t e d that the d u r a t i o n of the posttrial r e i n f o r c e m e n t s in the e x p e r i m e n t s was either 30 sec ([ 18], E x p e r i m e n t 1) or one rain [ 10], thus e x t e n d i n g i n t o the 60 sec delay period. The results are interesting from a n o t h e r perspective. When the curve o f Fig. 2 is c o m p a r e d to the effects o f posttrial ECS on the same task in Fig. 4, some c o r r e s p o n d e n c e b e t w e e n the t w o curves is obvious, w h i c h suggests a relationship b e t w e e n ECS and p u n i s h m e n t . EXPERIMENT 3
Whitney U-test). Table 3 s u m m a r i z e s the m e a n s and standard deviations of the s t e p - d o w n latencies during baseline and retest trials for the e x p e r i m e n t a l and c o n t r o l groups. Discussion Ten sec o f ice-water p u n i s h m e n t alone led to significant s t e p - d o w n avoidance learning w h e n p r e s e n t e d i m m e d i a t e l y u p o n s t e p p i n g o f f the p l a t f o r m . Hence, we e x p e c t e d an additive effect o f the posttrial ice-water p u n i s h m e n t with the f o o t s h o c k on learning, at least with the i m m e d i a t e p o s t s h o c k ice-water t r e a t m e n t . The results however, as s h o w n in Fig. 2, do n o t indicate such a simple additive effect. On the c o n t r a r y , the i m m e d i a t e p o s t s h o c k ice-water group did n o t differ significantly f r o m the c o n t r o l group, and s h o w e d s o m e w h a t p o o r e r learning t h a n the 30 sec delay group. This c o r r e s p o n d s with the results o f J a c o b s
Given our a n o m a l o u s results with posttrial p u n i s h m e n t in the p r e c e e d i n g e x p e r i m e n t , we wished to test the effects o f posttrial ECS p r e s e n t e d at exactly the same delays, using the same paradigm. Some evidence suggests that ECS has aversive p r o p e r t i e s [3, 4, 13, 16, 2 2 ] ; h o w e v e r , we could n o t find any one study with mice in w h i c h ECS was a d m i n i s t e r e d at the various posttrial delays which were o f interest to us. Method The same p r o c e d u r e as in E x p e r i m e n t 2 was used, e x c e p t that the mice were a d m i n i s t e r e d ECS instead o f ice-water either at <5, 30, 60 or 90 sec after the f o o t s h o c k . The n o n t r e a t e d c o n t r o l animals were placed into new h o m e cages i m m e d i a t e l y after the f o o t s h o c k . Of a total of 350 mice 297 were used. The rest was discarded for failure to
P O S T - T R I A L R E I N F O R C E M E N T , P U N I S H M E N T O R ECS
1107
©c
. . . . .
30 O3
c20 1 E 10
I
I
I
10
30
60
I
90see
/
detay after trial FIG. 4. Mean step-down latencies of the four groups given posttrial ECS (lower curve), the four handling control groups (upper curve), and the non-handled control group (C). adhere to the criteria for an effective ECS t r e a t m e n t (convulsions). The ECS was o f 5.4 m A i n t e n s i t y and 0.35 sec d u r a t i o n . It was applied w i t h t w o t h i m b l e - e l e c t r o d e s , w h i c h were gloved over the t h u m b and forefinger, and a d m i n i s t e r e d across the ears (see Fig. 5). To c o n t r o l f o r the e f f e c t s o f handling, 4 o t h e r groups ( < 5 , 30, 60 and 90 sec) were t r e a t e d in e x a c t l y the same m a n n e r as t h e ECS groups (their h e a d s were grabbed b e t w e e n t h u m b and f o r e f i n g e r e l e c t r o d e ) , e x c e p t t h a t ECS was n o t a d m i n i s t e r e d .
Results The
posttrial
ECS d i s r u p t e d learning o f the passive
~b
/'i
FIG. 5. Thimble electrodes for ECS constructed of meshed brass wire. Insulation provided by rubber glove. avoidance. When c o m p a r e d to the n o n - h a n d l e d c o n t r o l s p o o r e r learning ( s h o r t e r s t e p - d o w n latencies) was s h o w n by the <5 sec, ( p < 0 . 0 2 ) , 30 sec ( p < 0 . 0 8 ) , and 60 sec ( p < 0 . 0 1 ) delay groups, but n o t by the 90 sec group ( M a n n - W h i t n e y U-test, 2-tailed). Interestingly, the 60 sec delay group s h o w e d the m o s t drastic d i s r u p t i o n o f learning, similar to the e f f e c t s o f
TABLE 4 POSTTRIAL ECS. MEANS AND STANDARD DEVIATIONS OF STEP-DOWN LATENCIES (SDL) DURING BASELINE AND RETEST TRIALS FOR THE ECS AND CONTROL GROUPS Group
<5 (sec) 30 60 90 Cnh*
SDL experiment baseline 11.1 ± 5.4 11.7 ± 5.9 i 1.2 +-- 4.8 11.9 ___ 5.9
retest 26.6 _ 30.3 36.1 ± 41.3 24.8 --- 29.3 40.8 ___45.7
SDL control baseline 11.9 ± 4.6 11.3 ± 4.7 10.3 ___ 5.0 I 1.0 ± 4.8 11.1 ± 5.6
*Nonhandled controls. Mann-Whitney U-test (two-tailed) retest SDL comparisons <5 vs c<5p<0.05 30 vs c30 p<0.05 60 vs c60 p<0.02 90 vs c90 p<0.5 <5 vs Cnh p<0.02 30 vs Cnh p<0.08 60 vs Cnh p<0.01 <5 VS 30 p<0.3 30 VS 60 p<0.25 60 VS 90 p<0.02 C5 VS %0 p<0.25
retest 41.0 ± 46.7 54.6 ± 54.9 48.9 ___ 52.8 50.9 ± 54.4 50.8 -----54.8
n exp. 58 59 58 59
contr. 69 69 69 69 60
1108
MONDADORI, WASER AND HUSTON
posttrial p u n i s h m e n t in E x p e r i m e n t 2. A l t h o u g h the 60 sec delay group was n o t significantly d i f f e r e n t f r o m the 30 sec group ( p < 0 . 2 5 ) , it differed significantly f r o m the 90 sec delay g r o u p ( p < 0 . 0 2 , two-tailed U-test). The < 5 , 30, and 60 sec delay ECS groups, b u t n o t the 90 sec group, also s h o w e d significantly p o o r e r learning c o m p a r e d to their respective h a n d l i n g - c o n t r o l groups (see Table 4). Interestingly, the < 5 sec h a n d l i n g - c o n t r o l group learned s o m e w h a t p o o r e r t h a n the 30 sec h a n d l i n g - c o n t r o l group. Discussion The results of the ECS e x p e r i m e n t s t r e n g t h e n the finding of E x p e r i m e n t 2 t h a t 60 sec is s o m e h o w a critical period for p o s t t r i a l effects for the s t e p - d o w n avoidance learning in mice. The close c o r r e s p o n d a n c e b e t w e e n the 2 curves (Figs. 2 and 3) suggests t h a t some of the effects of ECS could be a c c o u n t e d for in t e r m s of a direct punishm e n t of posttrial m e m o r y processes, at least at the 60 sec delay. It is i n t e r e s t i n g t h a t the h a n d l i n g c o n t r o l group in E x p e r i m e n t 3 (Fig. 4) also s h o w e d similar d e l a y - d e p e n d e n t differences in learning, and t h a t the 5 sec h a n d l i n g - c o n t r o l group s h o w e d p o o r e r learning t h a n the 30 sec h a n d l i n g group. It should be n o t e d t h a t the h a n d l i n g c o n t r o l p r o c e d u r e was m o s t likely aversive, and h e n c e , it is possible that the results with this t r e a t m e n t reflect p o s t t r i a l punishm e n t effects, similar to these in E x p e r i m e n t 2. The effects of i m m e d i a t e and 30 sec delayed ECS c o r r e s p o n d s closely with the results of C h o r o v e r and Schiller [ 2] w i t h rats. The d e p a r t u r e a f t e r 30 sec f r o m the e x p e c t e d delayd e p e n d e n t g r a d i e n t of amnesia suggests t h a t m e m o r y processes (be t h e y c o n s o l i d a t i o n or retrival processes) are i n f l u e n c e d by ECS in a more c o m p l e x m a n n e r t h a n is usually assumed. GENERAL DISCUSSION All t h r e e e x p e r i m e n t s s u p p o r t the n o t i o n of a critical posttrial interval d u r i n g w h i c h m e m o r y processing can be i n f l u e n c e d . T h i r t y sec of food r e i n f o r c e m e n t facilitated learning w h e n initiated up to a delay of 60 sec a f t e r the f o o t s h o c k . ECS as well as ice-water p u n i s h m e n t r e t a r d e d learning w h e n a d m i n i s t e r e d w i t h i n 60 sec a f t e r the trial. A perusal of t h e ECS literature on mice shows t h a t a vast range o f i n t e n s i t i e s and d u r a t i o n s of ECS are b e i n g used. The i n t e n s i t i e s vary f r o m 4 m A [7] to 50 m A [ 1 9 ] ; and the d u r a t i o n f r o m 200 msec [19] to 800 msec [ 2 1 ] . As s h o w n by B u c k h o l z and B o w m a n [ 1 ] , these d i f f e r e n c e s could a c c o u n t for some of the discrepancies b e t w e e n the
results. We used a r a t h e r low i n t e n s i t y (5.4 m A ) ECS in o r d e r to m i n i m i z e irreversible damage to the brain. Nevertheless o u r results are in a g r e e m e n t w i t h some previous data in mice. As a rule ECS a d m i n i s t e r e d up to 15 sec after the trial results in retrograde amnesia irrespective of the i n t e n s i t y and d u r a t i o n of the shock. Our data w i t h i n this delay are in a c c o r d a n c e w i t h this rule. O u r results at the 30 sec ECS delay ( n o amnesia w h e n c o m p a r e d to the n o n - h a n d l e d c o n t r o l group, and partial amnesia w h e n c o m p a r e d to the h a n d l e d c o n t r o l s ) agree w i t h t h e findings of D a w s o n and M c G a u g h [5] at a 20 sec delay ( n o a m n e s i a w i t h o u t familiarisation trials) and of McGaugh, et al. [17] at a 30 sec delay (partial amnesia). Less c o m p a r a b l e data is available at larger ECS delays. D o r f m a n and Jarvik [6] failed to find amnesia at 45 sec delay w i t h a 5 m A s h o c k intensity. R o b u s t e l l i and Jarvik [20] r e p o r t e d amnesia at a 60 sec delay, in a c c o r d a n c e w i t h o u r results. A m n e s i a has also b e e n r e p o r t e d at 75 sec [9] and 80 sec [ 1 4 ] . Hence, a l t h o u g h n o o t h e r studies seem to have investigated the effects of ECS at all of the intervals we have used, o u r finding of a lack of a linear delayd e p e n d e n t g r a d i e n t of amnesia seems to c o n f o r m to s c a t t e r e d results in the literature. The idea of a linear g r a d i e n t of retrograde amnesia in mice seems to be based m a i n l y on data using e i t h e r widely spaced [14] or very closely spaced [8] posttrial ECS intervals. Our data suggest t h a t the v u l n e r a b i l i t y of posttrial m e m o r y processes changes drastically w i t h i n 90 sec. T h e c o n c o r d a n c e b e t w e e n o u r ECS and ice-water p u n i s h m e n t results suggests t h a t , a l t h o u g h ECS has m o r e drastic a m n e s t i c p r o p e r t i e s t h a n p u n i s h m e n t , the m e c h a n i s m of a c t i o n of these t r e a t m e n t s o n m e m o r y processes m a y be similar. The possibility of a direct a c t i o n of p u n i s h m e n t on m e m o r y processes seems to have achieved little or n o a t t e n t i o n . J a c o b s and S o r e n s o n [12] f o u n d t h a t 10 sec of ice-water p u n i s h m e n t i n h i b i t e d learning of a passive avoidance w h e n a d m i n i s t e r e d i m m e d i a t e l y after the f o o t s h o c k . This effect w e a k e n e d with increasing delays b e t w e e n f o o t s h o c k a n d ice-water, and at 30 sec delay ice-water n o longer d i s r u p t e d learning. This result c o r r e s p o n d s with o u r findings w i t h i n t h a t interval. A n o t h e r s t u d y w h i c h m i g h t have relevance to the issue of p u n i s h m e n t of m e m o r y processes is t h a t of Leukel [ 15], w h o f o u n d d i s r u p t i o n of learning in rats t h a t received a painful legshock one m i n u t e a f t e r each trial of a o n e - w a y active a v o i d a n c e task. The p r o b l e m of posttrial p u n i s h m e n t and its r e l a t i o n s h i p to ECS a n d o t h e r a m n e s t i c t r e a t m e n t s obviously requires f u r t h e r investigation.
REFERENCES 1. Buckholtz, N. S. and R. E. Bowman. Incubation and retrograde amnesia studied with various ECS intensities and durations. Physiol. Behav. 8: 113-117, 1972. 2. Chorover, S. L. and P. H. Schiller. Short-term retrograde amnesia in rats. J. eomp. physiol. Psychol. 59: 7 3 - 7 8 , 1960. 3. Coons, E. E. and N. E. Miller. Conflict versus consolidation of memory traces to explain "retrograde amnesia" produced by ECS. J. comp. physiol. Psyehol. 53: 5 2 4 - 5 3 1 , 1960. 4. Dawson, R. G. and G. T. Pryor. Onset vs recovery in the aversive effects of electroconsulsive shock. Psychon. ScL 3: 273-274, 1965.
5. Dawson, R. G. and J. L. McGaugh. Electroconvulsive shockproduced retrograde amnesia: Analysis of the familiarisation effect. Communs. behav. Biol. 4: 9 1 - 9 5 , 1969. 6. Dorfman, L. J. and M. E. Jarvik. Comparative amnesic effects of transcorneal and transspinnate ECS in mice. PhysioL Behav. 3: 815-818, 1968a. 7. Dorfman, L. J. and M. E. Jarvik. A parametric study of electroshock-induced retrograde amnesia in mice. Neuropsyehologia 6: 373-380, 1968b.
P O S T - T R I A L R E I N F O R C E M E N T , P U N I S H M E N T O R ECS 8. Grosser, G. S., H. E. Percy and L. E. Pierce. Short footshockelectroconvulsive shock intervals and retrograde amnesia in mice. Psychon. Sci. 25: 26-28, 1971. 9. Herz, M. J. and H. V. S. Peeke. Permanence of retrograde amnesia produced by electroconvulsive shock. Science 156: 1396-1397, 1967. 10. Huston, J. P., C. Mondadori and P. G. Waser. Facilitation of learning by reward of posttrial memory processes. Experientia 30: 1038-1040, 1974. 11. Huston, J. P. and C. Mondadori. Memory and reinforcement: a model. In: Proc. 2nd lnt. Congr. CLANS, Prague, 1975,Activ. Nerv. Sup. in press. 12. Jacobs, B. L. and C. A. Sorenson. Memory disruption in mice by brief posttrial immersion in hot or cold water. J. comp. physioL Psychol. 68: 239-244, 1969. 13. Kesner, R. P. ECS as a punishing stimulus: Dependency on retrograde amnesia, duration of anterograde amnesia, and intensity of pain. J. comp. physiol. Psychol. 74: 398-406, 1971. 14. Kopp, R., Z. Bohdanecky and M. E. Jarvik. Long temporal gradients of retrograde amnesia for a well discriminated stimulus. Science 153: 1547-1549, 1966. 15. Leukel, F. A. A comparison of the effects of ECS and anaesthesia on acquisition of the maze habit. J. comp. physioL PsychoL 50: 300-306, 1957.
1109 16. McGaugh, J. L. and M. C. Madsen. Amnesic and punishing effects of electroconvulsive shock. Science 144: 182-183, 1964. 17. McGaugh, J. L., R. G. Dawson, R. Coleman and J. Rawie. Electroshock effects on memory in Diethyl-Ether-treated mice: Analysis of the CER-incubation hypothesis of retrograde amnesia. Communs. behav. Biol. 6: 227-232, 1971. 18. Mondadori, C., K. Ornstein, P. G. Waser and J. P. Huston. Posttrial reinforcing hypothalamic stimulation can facilitate avoidance learning. NeuroscL Letters 2 : 1 8 3 - 1 8 7 , 1976. 19. Ray, O. S. and L. W. Bivens. Reinforcement magnitude as a determinant of performance decrement after electroconvulsive shock. Science 160: 330-332, 1968. 20. Robustelli, F. and M. E. Jarvik. Retrograde amnesia from detention. Physiol. Behav. 3: 543-547, 1968. 21. Zornetzer, S. F. and J. L. McGaugh. Effects of electroconvulsive shock upon inhibitory avoidance: The persistence and stability of amnesia. Communs. Behav. Biol. 3: 173-180, 1969. 22. Zornetzer, S. F. and J. L. McGaugh. Retrograde amnesia and brain seizures in mice. Physiol. Behav. 7: 401-408, 1971.