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Accelerated extinction after post-trial halothane anaesthesia in rats: An aversive effect
Physiology & Behavior, Vol. 26, pp. 223-231. Pergamon Press and Brain Research Publ., 1981. Printed in the U.S.A.
Accelerated Extinction After Post-Trial Halothane Anaesthesia in Rats: An Aversive Effect PASCALE
GISQUET-VERRIER
Centre National de la R e c h e r c h e Scientifique, Laboratoire de Physiologie N e r v e u s e , D d p a r t e m e n t de Psychophysiologie, 91190 Gif-Sur-Yvette, France R e c e i v e d l 0 M a r c h 1980 GISQUET-VERRIER, P. Accelerated extinction after post-trial halothane anaesthesia in rats: An aversive effect. PHYSIOL. BEHAV 26(2)223-231, 1981.--The effects of halothane anaesthesia immediately post-trial were investigated in three experiments in rats. The results showed that this treatment accelerated the extinction of three previously acquired, positively reinforced operant tasks: straight alley, barpress learning or modified K-maze. This acceleration was not due to any motor impairment and treatments delayed two hours had no effect on performance. No amnesic component was observed in those experiments in which treatment was administered a single time, but aversive effect appeared gradually through the extinction phase. The results are not consistent with the hypothesis of the amnesic effects of halothane anaesthesia; they are interpreted in terms of aversive effects. Rats
Halothane anaesthesia
Retrograde amnesia
Aversive effect
N U M E R O U S clinical and experimental studies indicate that retention o f recently acquired habits can be impaired by a great variety of treatments. An experimental amnesia generally occurs when such agents as electroconvulsive shock [14,20], hypothermia [29,30[ or anaesthesia [6,27] are administered immediately or shortly after acquisition. The existence of an inverse relationship between the observed behavioral deficits and the training-to-treatment interval has generally been interpreted in terms of retrograde amnesia, i.e., that these treatments impair retention by disrupting a memory trace consolidation process. Ever since Coons and Miller [11] pointed out that punishing effects of electroconvulsive shock could be expected to show the same kind of interfering gradient as amnesia in an active avoidance paradigm, experimental research in this field has developed in two major directions: (a) the attempt to discover behavioral paradigms in which amnestic and non-amnestic effects of treatments could be easily separated and (b) the use of mild, non-traumatic disruptive agents. It has been claimed that one trial passive avoidance procedures (step down [24,32], step through [13,23]), which have often been used in the investigation of retrograde amnesia, allow a clear dissociation of amnestic and punishing effects of treatments. Indeed, a number of authors using this procedure, especially in conjunction with electroconvulsive shock, have found retrograde amnesia without aversive effects after a single administration. Some data, however, make this assumption questionable. Carew [8] has demonstrated that, in both these tasks, those rats which were considered amnesic when their performances were scored in terms of passive avoidance showed no memory deficit when
Extinction
an active avoidance criterion was used. Similarly, several authors [18,26] testing the effects of a single ECS on retention o f a one-trial passive avoidance, have reported behavioral deficits in motor responses; at the same time, however, they noted considerable variations in autonomic responses (heart rate, defecation) when the animals were placed in test situations, a fact which indicates some persistence of memory. Such observations suggest that the one-trial passive avoidance procedures alone might not be the appropriate method for testing whether, and to what extent, an experimental treatment can induce retrograde amnesia. Similarly, the use of positively reinforced acquisition does not allow an analysis of data in terms of aversive versus amnestic effects, since both these hypotheses would predict a disturbance in performance. On the other hand, if the treatment to be tested is administered in an extinction paradigm, the assumptions of aversive or amnestic effects would each lead to a different prediction (see Madsen and Luttges [22], Colavita [10], Greenough and Schweitzgebel [17] for ECS, and Leukel and Quinton [21] using carbon dioxide). If the treatment produced retrograde amnesia for recent events, extinction would be prolonged, since the memory loss would be that of the extinction contingencies; if the treatment acted as an aversive stimulus, extinction would take place quite rapidly, since the response would be punished as well as not rewarded. Another direction in the attempt to rule out any interpretation in terms of aversive effects is the search for mild, non-traumatic amnesic treatments. Among a great variety of agents, volatile anaesthetics [6, 21, 27] have been frequently
223
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used. F o r example, in our laboratory and others, an experimental amnesia has been observed after halothane anaesthesia. First reported by Cherkin and Lee-Teng [9] in chicks, behavioral deficits after halothane treatment have been reported several times [3, 5, 12, 19, 28] in rats, mice or chicks, using either one-trial passive avoidance tasks or one-trial positively reinforced tasks. A gradient effect has been described by Bloch et al. [6] and Penrod and Boice [28]. However, the effects of halothane anaesthesia have been exclusively analyzed to date in terms of retrograde amnesia. The present experiments were conducted in order to investigate the possibility of aversive effects, even with this nontraumatic treatment, after one or several administrations. In order to dissociate amnestic and aversive effects, the extinction procedure was chosen. In the first experiment, the effects of halothane anaesthesia were tested in a runway alley, which is the most commonly used task in extinction studies. EXPERIMENT 1 The first experiment was run in two series using 50 subjects each. METHOD
Animals One hundred Sprague-Dawley male albino rats (supplied by Iffa Credo, St Germain sur l'Arbresle) weighing 170-180 g at the start of the experiment were used. Upon arrival in the laboratory, they were housed in individual wire mesh cages (35 ×21 × 18 cm) with ad lib food and water. After one week of familiarization with the experimenter, the daily food ration was progressively reduced until the weight loss was 15%, compared to control animals on a normal diet. The food intake was reduced over 4 days from 30 g to 15 g, and then maintained at this level. Animals were weighed daily, and the diet was adjusted individually in order to maintain a homogenous weight loss.
Apparatus The experimental apparatus, a slightly modified G r a y ' s runway [16], consisted o f a 15 cm wide alley, divided by two manually operated perspex doors opening on the side into a 20 cm long start box, a 120 cm long runway and a 28 cm long goal box. The wooden walls (except the front side which was of perspex) were 30 cm high. Photo beams placed 18 cm from the start box door, 2.5 cm before the goal box were used to measure the start latency, the total running time and the entry latency into the goal box. Halothane anaesthesia was administered over a 60 min period. Treatment was applied in a box supplied with a 4% concentration of halothane in a mixture of air and oxygen, producing unconsciousness within about 60 sec; the animals were then placed into another tank, supplied with 1 to 1.5% concentration of halothane and maintained there for the rest of the 60 min treatment period. Behavioral recovery was usually complete within 20 min after removal from the tank. No perturbation such as gastrointestinal malaise was noticed after anaesthesia. No subject died during or after the treatment.
Experimental Procedure Pretraining. F o r the first two days of pretraining, animals were allowed to explore the alley in groups of six during 30
min, with food available in the goal box. On days 3 and 4, each animal was removed individually from the home cage, placed into the closed goal box and allowed to eat for 2 min. Individual food intake latencies were recorded. Each animal was submitted to this situation 3 times in 2 days. Training. Training began on day 5 and was identical for all animals. Each rat was taken from the home cage, weighed, and placed into the start box for 30 sec. The start box door was then opened manually and the first timer started. The goal box door was closed after the rat had broken the final photo beam. The animal was allowed to eat for 20 sec and then was removed from the goal box and immediately returned to the start box for another trial. Each animal was subjected to 8 consecutively daily trials, under a continuous reinforcement schedule for the first 4 days of acquisition, and a partial reinforcement schedule (PR) for the 8 following days. During the PR phase, reinforcement was delivered randomly after 50% of trials, according to a schedule which was changed daily. This PR schedule was introduced to avoid extinction taking place too quickly in this very simple task. Training was terminated when the mean performance of animals remained at the same level for 4 consecutive days. Extinction. Extinction began on the following day. Behavioral procedures were identical to those used in acquisition, with the exception that no food was delivered in the goal box. During extinction, a rat which failed to break the final photo beam within two min was removed from the alley, but allowed to run again for subsequent trials. In the second series of this experiment, extinction period had to be reduced to 5 days due to an accidental interruption of the daily food deprivation.
Experimental Design At the end of the training period, each of the 100 animals were assigned to one of the six following groups:Group E 8 (first experimental group), subjected to an 8-trial daily session, immediately followed by halothane anaesthesia. Group AC 8, in which the animals were immediately returned to their home cages after the 8-trial session and anaesthetized 2 hours later. Group NAC 8 (control group), in which the animals were not anaesthetized. They were placed, immediately after each session, in a box identical to the halothane tank, supplied with air, for 1 rain and then returned to their cages. Group E 4 (the second experimental group), subjected to a 4-trial daily session, immediately followed by anaesthesia. Group AC 4, in which the animals were subjected to 4 daily extinction trials, then returned to their home cages, and anaesthetized 2 hours later. Group NAC 4 (the second control group), in which the animals were not anaesthetized, but only submitted to a pseudo-treatment (as in Group NAC 8). Twenty subjects were assigned to each of the experimental (E) and each of the "anaesthetized control" (AC) conditions; 10 animals were assigned to each of one of the "non-anaesthetized control" (NAC) conditions. In summary, each animal could be subjected to one of 3 treatments (immediate anaesthesia, delayed anaesthesia, pseudo-anaesthesia), under one of 2 experimental conditions (4 or 8 daily trials). The number of daily extinction trials was varied in order to estimate whether the effects of halothane anaesthesia were related to the quantity of information available in each session.
ACCELERATED EXTINCTION--HALOTHANE
225
ext n _ ext i r : ~ (median) ext_l
I
(median)
NAC S
W
AC8
E~ Ac,
E8 t
NAC 4 E4
treatment
t
treatment
20
10
10-
extlt
ext2t
ext3t
ext4t
ext5~
ext6
extl t ext2 t
ext3 t ext4 t
ext5 t ext6
FIG. 1. Median "loss" score for extinction sessions in animals subjected to 8 (a) or 4 dally trials (b). Halothane anaesthesia (arrows) was administered immediately after extinction in experimental animals (E 8 and E 4), and was delayed 2 hr in anaesthetized control animals (AC 8 and AC 4). Animals in non-anaesthetized control group (NAC 8) were subjected to a pseudo-treatment only.
RESULTS
This experiment was conducted using two series of 50 animals each, under the same learning procedures; however, at the end of acquisition period, a statistical comparison of running times yielded a significant difference between the two samples. A parameter which allowed the comparison of the 100 individual animals despite this difference was used; for each subject and extinction session, performances were compared to the performance in the last training trial, by means of a "loss" score, r=(Extn - Ext-0Ext_l (where r is the "loss" score, Extn the running time measured on the n th extinction session, and Ext_l the running time observed on the last training trial) which is similar in principle to the savings score of Ebbinghaus. These data, summarized in Fig. la and lb, were subjected to intra- and inter-group analyses.
lntra-Group Comparisons The evolution of " r " scores from day to day showed that extinction actually occurred in all groups. The individual performances in each group, indexed in r scores, were subjected to Wilcoxon comparisons. These comparisons
showed that extinction began in the second extinction session, i.e., after one application of the treatment (the T values, calculated between the Ist and the 2nd extinction session were, respectively, T=0, p<0.01 in Group NAC 8; T=6, p<0.005 in Group AC 8; T=0, p<0.005 in Group E 8; T=7, p<0.03 in Group NAC 4; T=35,p<0.005 in Group AC 4, and T=12, p<0.005 in Group E 4). Other intra-group comparisons showed that extinction continued in all groups from session 2 to session 6. It should be noted, however, that during and after the 3rd session, running times were much longer for experimental (immediately anaesthetized) animals than for animals in the other groups.
Inter-Group Comparisons For each extinction session, r scores were subjected to inter-group comparisons. A Kruskall-Wallis analysis of variance showed no difference in performance in the second extinction session (H=3.77, n.s. for animals subjected to 8 trials; H = I , n.s., for animals subjected to 4 trials). A different picture emerged during and after the 3rd extinction session: Kruskall-Wallis comparisons yielded significant differences among groups, both in 8-daily trial groups (H=15.76, p<0.001 on 3rd session; H=22.14, p<0.001 on
226
GISQUET-VERRIER TABLE 1 Days of Extinction Es/NAC8
Es/AC8
NACs/AC,
EdNAC4
EJAC4
NAC4/AC4
Ext 3
U=20 p<0.002
U=88 p<0.002
U=51 NS
U=24 p<0.002
U=90 p<0.02
U=59 NS
Ext 4
U= 7 p<0.002
U=51 p<0.002
U=70 NS
U= 3 p<0.002
U=20 p<0.02
U=91 NS
Ext 5
U=13 p<0.002
U=22 p <0.002
U=93 NS
U=I5 p<0.002
U=40 p<0.02
U=74 NS
Ext 6
U=ll p<0.02
U= 8 p<0.002
U=53 NS
U=21 p<0.05
E8 n=20, AC8 n=20, NAC8 n=10; E4 n=20, AC4 n=20, NAC4 n=10. Individual Mann Whitney U comparisons (two tailed). 4th session; H=29.92, p<0.001 on session 5; H=12.45, p<0.01 in 6th session) and in 4-daily trials groups ( H = 11.02, p<0.01 on 3rd session; H=26.89, p<0.001 on 4th session; H=29.41, p<0.001 on 5th session; U=21, p<0.025 on 6th session). Individual Mann-Whitney comparisons showed that these differences were due to a much more rapid extinction in experimental (E 8 and E 4) groups (see Table 1). Many more animals in Group E 8 than in the other groups did not break the final photo beam within 2 min (cut-off latency) during the last extinction sessions. A chi-square analysis yielded significant differences from sessions 3 to 6 (3rd session, X2=72.54; 4th session, X2= 107.80; 5th session, X2= 179.69; 6th session, X2=81.92; df=2;p
thane applications is strengthened by visual observation of the animals' behavior in the alley, which showed that experimental animals clearly avoided the goal box from the 3rd session to the end of extinction. In the last extinction session, 70 to 90% of the experimental animals avoided entering the goal box within the 2 min cut-off latency. After two administrations of the post-trial treatment, they seemed able to associate the apparatus cues with the aversive properties of anaesthesia. However, at the end of this experiment the possibility of the results being due to the particularities of the experimental situation--very simple learning task, PR schedule at the end of the acquisition period--could not be completely ruled out. F o r this reason, similar experiments were conducted with two other learning tasks: the first was extinction of bar-pressing in a Skinner box, previously used by Deweer [12] in our laboratory (see Experiment 2); the second was extinction in a 4-choice maze (see Experiment 3). This latter task was chosen because it has some similarities with a straight alley (linear maze), but also calls for discriminative responses. EXPERIMENT 2 METHOD
Animals Twenty-two Sprague-Dawley albino rats, weighing 170-180 g at the start of the experiment, were used. Eight days after their arrival, the animals were placed under a 23 hr water deprivation schedule, and maintained approximately at a 10% weight loss, compared with control animals with water ad lib.
Apparatus The apparatus consisted of a 40x30x30 cm Skinner box, provided with a 1.5x3.5 cm lever and a water cup on the same wall. For each animal, two parameters were recorded: the total number of bar presses, and the first press latency. Halothane anaesthesia was delivered in the same conditions as in Experiment 1.
Procedure Pretraining. F o r the first two days of pretraining, the animals were allowed to explore the box, in groups of three,
ACCELERATED
EXTINCTION--HALOTHANE
227
MEDIAN FIRST PRESS LATENCY (sec) ACQUISITION I
EXTINCTION
I I I I
loo
5o.
a I
-'-E
I I I
J J j
L 1
1
I
]
I
I
lo-
NAC I'-'J AC
t
treatment
I
I
~ extl t
I day,
ext2 t
ext3 t
ex,. t
extS
MEDIAN NUMBER OF PRESSES
b
~ NAC [ ~ AC mWJE t treatment
ext4
t
EXTINCTION
ACQUISITION
i:i',.,, 2o-
!!i
!!
•o
lo-
iii
i:i days
extl
t
ext2
t
ext3
t
ext5
FIG. 2. Performances in Skinner box for the last two training sessions and extinction sessions, scored in terms of median first press latency (a) of median number of presses (b). Halothane anaesthesia (arrows) was administered immediately after extinction sessions in Group E, was delayed 2 hr in anaesthetized control group (AC); NAC group was only subjected to a pseudo-treatment. Note that the effects of anaesthesia depend on the index of performance used: first press latencies showed a faster extinction (a); no effect could be observed concerning number of bar presses
(b).
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GISQUET-VERRIER
with water continuously present in the cup. They were then subjected to 3 consecutive 10 min individual shaping sessions. Training. Training began on day 6 and was identical for all animals. Each rat was taken from the home cage and placed into the Skinner box every day at the same time. The training sessions lasted 4 min during the first 10 days and were then reduced to 2 min for the last two days. This reduction in the duration of training sessions was planned in order to slow down the extinction. A partial reinforcement schedule was not considered necessary since the preliminary experiment showed that extinction period following this type of learning was long enough to observe the effect of the treatment. Extinction. Extinction began on day 18, and all behavioral procedures were identical to those used in acquisition, except for the absence of water. The daily extinction sessions lasted 2 min. Each animal was subjected to 5 consecutive extinction sessions. In training, individual scores were noted in terms of total number of bar presses and first press latency.
Experimental Design At the end of the training period, the mean number of bar presses in training sessions 11-12 was calculated for each rat, and the animals were assigned to one of the following groups: Group E (n=7), in which each extinction session was immediately followed by halothane anaesthesia. Group AC (n=7), in which animals were subjected to a 2 hr delayed anaesthesia after each extinction session. Group NAC (n= 8), in which animals were not subjected to any treatment; they were merely placed, immediately after extinction sessions, into a box identical to the halothane box for 1 min. RESULTS
The data from the individual performances underwent an intra- and an inter-group analysis.
Intra-Group Analysis Performances in each group were compared, for every extinction session, to the level attained at the end of the acquisition period, by means of Wilcoxon comparisons. These comparisons showed that extinction took place in the second session, i.e., after one administration of the treatment, both for total numbers of bar presses (T=0, p <0.01 in Group NAC; T=2.5, p <0.17 in Group AC; T =4, p <0.055 in Group E) and for first bar press latencies (T= 1, p<0.016 in Group NAC; T = 0 , p<0.004 in Group AC; T = 3 , p <0.039 in Group E), and continued during subsequent sessions (see Fig. 2a and b).
Inter-Group Analysis As can be seen in Fig. 2b, no difference could be observed among the three groups during the extinction period, when performances were measured in terms of total numbers of bar presses. The results are quite different when the first bar press latency is considered. After the first two days of extinction, a difference between the experimental (immediately anaesthetized) group and the two control groups appeared (see Fig. 2a). The hypothesis of the acceleration of extinction by immediate anaesthesia was tested by a Kruskall-Wallis analysis of variance, which showed a non-significant tendency on the 3rd session and significant differences among the 3 groups on sessions 4 (H=6.23, p<0.05) and 5 (H=7.58, p<0.05).
Comparisons of individual groups by Mann-Whitney U tests confirmed (a) that there was no difference between the two control Groups AC and NAC (respectively, for sessions 4 and 5, U=31 and U=27, n.s.), and (b) that first bar press latencies were significantly greater in Group E than in Groups NAC (4th session, U = 8 , p<0.01; 5th session, U = 6 , p<0.005) and AC (4th session, U=10, p<0.02; 5th session, U = 3 , p<0.002).
Behavioral Observations In spite of the fact that the total number of bar presses was identical in the three groups, it seems important to note that the strategies were quite different between these groups. Control animals performed with regularity in both delayed anaesthetized and non-treated groups; each lever press was generally followed by a visit to the water cup. In the experimental group, on the other hand, the latency of the first bar press was much more variable and longer; the subsequent bar presses took place in clusters of 3-5 presses, irregularly spaced, and either followed by a visit to the cup or not. These observations might explain why Deweer [12], who only noted the total number of bar presses during longer (6 min) extinction sessions, did not obtain the same results as reported here, although the same task and the same treatment were used. EXPERIMENT 3 METHOD
Animals Thirty-eight Sprague-Dawley male albino rats, weighing 170-180 g at the start of the experiment, served as subjects. After one week of familiarization, the daily food ration was progressively reduced until the weight loss reached 15%, compared to control animals. The deprivation schedule was then maintained in the same conditions as in Experiment 1.
Apparatus The maze consisted of four T shape units. A start box (25 x 25 x 35 cm) communicated with the first maze unit by a manually opened sliding door. Each unit measured 50x40x35 cm. The choice point, or the stem of the T, was located 25 crn from the entrance of each unit. The true path led into the next unit by a 10 cm opening and the other led into a blind. The barriers in each unit of the maze could be changed from one side to the other. The maze configuration was R L L R or LRRL. The goal box, of the same dimensions as the start box, could be closed by a sliding door. Halothane anaesthesia was delivered in the same conditions as in Experiments 1 and 2.
Procedure Pretraining. During the pretraining period, rats were habituated to the experimental conditions. On day 1, individual rats were allowed to feed in the goal box for two 20 min periods. Day 2 involved 30 min free exploration of the maze, in groups of 6, with the barriers removed and with food pellets in the goal box. Days 3 and 4 consisted of training by pairs for departure from the start box, entering the goal box, using only one unit of the maze between the start box and the goal box. Procedure on day 5 was the same, but for individual rats. Training. Training began 24 hr later. Barriers were placed
ACCELERATED EXTINCTION--HALOTHANE
229
MEDIAN TOTAL RUNNING TIME (sec/
ACQUISITION
NAC E treatment
t
EXTINCTION
20(
I01
days extl text2 text3 text4 text5
ext 6
FIG. 3. Median times to run the maze for the last two acquisition trials, and extinction trials. Animals subjected to immediate halothane anaesthesia (arrows) immediately after each trial extinguished much faster than non-anaesthetized control (NAC) animals. at the appropriate points, and in order to prevent animals from following the scent of the preceding subject, the path was changed to its mirror configuration for every other rat, and the maze was cleaned several times a day. Each animal was removed from the home cage and placed into the start box; the door was opened, and the time for the rat to arrive at the goal box was recorded, as was the number of incorrect choices and the number of retracings. The animals were allowed to eat for 1 min in the goal box, before being removed and returned to their home cages. The animals were subjected to 12 consecutive daily trials. At this time, they had attained an asymptote performance for 4 consecutive days.
Extinction. Twenty-four hours later the extinction period began. Behavioral procedures were identical to those used in the acquisition period, except that food was no longer delivered. During this period, if a rat failed to enter the goal-box within 4 min, it was removed from the maze and subjected to the treatment of its group. Experimental Design At the end of the training period, animals were divided into 2 equivalent groups, according to total run time, number of errors and number of retracings: Group E, in which animals were immediately anaesthetized after each extinc-
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tion trial. Group N A C , non-anaesthetized control group, in which animals were returned to their home cages after spending 1 min in a box identical to the halothane box. The Group AC, anaesthetized control group, was not used in this experiment, since the two preceding ones showed no difference between Groups AC and NAC. RESULTS Total Running Times As in the two preceding experiments, an intra-group analysis showed that extinction took place in the second session, i.e., after one administration of the treatment. Wilcoxon comparisons showed significant increases in running times from the last training trial to the second extinction trial (respectively, T=38.5, p<0.038 in NAC group, and T=24.5, p<0.006 in Group E). This extinction continued in nonanaesthetized control animals (see Fig. 3); running times in this group, on the last extinction session, were multiplied by 7, 8, 9 or 10, compared to the last training trial. In the experimental (immediately anaesthetized) group, animals ran much slower than controls from the 3rd to the last extinction trial (see Fig. 3). Inter-group comparisons (Mann-Whitney U tests, one-tailed) confirmed the acceleration of extinction after successive halothane administrations (3rd trial, U = 120, p <0.05; 4th trial, U = 87, p <0.01, 5th trial, U=41.5, p<0.001; 6th trial, U=25, p<0.001). Errors and Retracings As regards running times, experimental animals showed accelerated extinction, compared to controls, when the numbers of errors and retracings were considered. MannWhitney U comparisons yielded significant differences between the two groups, from the 3rd trial for retracings (3rd trial, U=98, p<0.01; 4th trial, U=84.5, p<0.01; 5th trial, U=46.5, p<0.001; 6th trial, U=38.5, p<0.001) and from the 4th trial for errors (4th trial, U = 106.5, p<0.05; 5th trial, U=38.5, p<0.001; 6th trial, U=32, p<0.001 one-tailed tests). It should be noted, however, that these results were not due to "forgetting" the correct pathway, since the animals in the experimental group generally performed a first run without errors, then avoided entering the goal box and came back towards the start box, exploring then the entire maze with many retracings and incorrect choices. G E N E R A L DISCUSSION Several tentative conclusions can be drawn from these three experiments. First--and this is probably the most important conclusiorr--extinction was observed in experimental animals as well as in control subjects. Furthermore, in our experimental conditions, intra-group analysis showed extinction in all groups after a single administration of the treatment. All groups, including the experimental group, memorized the new information available during the first session (i.e., the absence of positive reinforcement). In the second extinction session, no effect of halothane anaesthesia, neither amnestic nor aversive, could be detected. However, with repeated administrations, the extinction rate was markedly increased in experimental animals. Such an effect could not result from a proactive effect of halothane anaesthesia on animals' motor behavior, since there was no difference between delayed anaesthetized and non-treated control animals. Finally, experimental animals
avoided the response reinforced during the acquisition period (entering the goal box in Experiments 1 and 3, bar pressing in Experiment 2). However, in every experiment, this avoidance behavior, only appeared after two successive treatments (during and after the 3rd extinction session), strongly suggesting an aversive action with repetition, an hypothesis already proposed in several experiments, particularly with ECS [25]. Several authors, however, (Bloch et al. [6], Alexinsky and Chapouthier [1,2]) have reported effects of halothane anaesthesia after a singh, application. This discrepancy might be attributed to differences in experimental conditions: Bloch et al. used a one-trial, positively reinforced discriminative transfer; Alexinsky and Chapouthier, a 3-choice discrimination box. In retention tests, in both situations, the animals could respond correctly, incorrectly or not at all. In our situations involving extinction procedures, the only alternative was to respond or not to respond. It seems reasonable to suggest that if halothane anaesthesia has aversive properties, an avoidance behavior will rapidly appear when alternative responses are possible. On the other hand, if the only choice left to the animals is to give no response, it will take them longer to find the appropriate strategy. In an additional experiment [15] we examined whether the beginning of anaesthesia could be the actual aversive factor, as demonstrated by Leukel and Quinton [21] for CO2. A group was subjected to a pseudo-anaesthesia for 30 sec after each extinction trial and compared to anaesthetized (1 hr) and non-anaesthetized animal. A slight increase in the extinction rate was observed, which led to intermediate performances, as compared to those of anaesthetized and nonanaesthetized animals (comparisons between Groups NAC and E30 s e c showed a significant difference in the last extinction trial for running times (U=56, p<0.05), and in the 4th and 5th extinction trials for retracings (respectively, U=68, p<0.025 and U=65, p<0.025). The same comparison between groups Er0 ~c,:and E30 ..... yielded a significant difference for running times in the 4th extinction session (U=68, p<0.01). The beginning of anaesthesia seems, therefore, to play a role in the development of aversion; but other factors, such as chilling, or sudden drop in arterial pressure, might also be important. To date, the time-dependent nature of the effects of halothane anaesthesia (i.e., the fact that the closer the administration follows upon the end of acquisition, the greater are the effects) has been considered as a demonstration of the amnesic properties of this treatment [6,28], and referred to a "retrograde amnesia gradient". However, the development of such gradients can also be observed after administration of aversive treatments and explained in terms of laws of learning: when a treatment is administered shortly after the end of acquisition, the animal easily associates the response it has just made with the subsequent traumatic event; whereas increasing the temporal distance between the two events will make this association more difficult to establish. Halothane has recently been found to be aversive by other investigators. Alexinsky and Chapouthier [2] used a 3-choice discrimination task, where the animals were placed into a "signal" box and had subsequently to choose the identical box to obtain reinforcement. If an 8-min anaesthesia was administered between the presentation of the signal and the choice trial, animals systematically avoided the box associated with anaesthesia. Similarly, Schmaltz [31] has recently shown that association between the experimental apparatus and halothane anaesthesia can disturb perform-
ACCELERATED EXTINCTION--HALOTHANE
231
ance in this apparatus 24 hr later. Similarly, our o w n experiments allow the conclusion that halothane anaesthesia acts on subsequent p e r f o r m a n c e through a v e r s i v e rather than a m n e s t i c effects. A v e r s i v e interpretation o f amnesic treatment, which is not n e w [11], has already b e e n p r o p o s e d not only w h e n it is applied repeatedly but also after a single administration for m o s t agents such as E C S [7,8], ether anaesthesia [4], c a r b o n
dioxide [21,33] and KC1 cortical spreading d e p r e s s i o n [34]. O u r o w n w o r k leads us to c o n c l u d e that it cannot be too strongly e m p h a s i z e d that rigorous experimental control for motivational effects is required before any amnesic action can be unequivocally attributed to a treatment: the fact that deficits are found in retention tests does not necessarily m e a n that these are deficits in retention.
REFERENCES
1. Alexinsky, T. and G. Chapouthier. Nature des perturbations mn6siques induites par la narcose au fluothane chez le Rat. J. Physiol., Paris 73: 127, 1976. 2. Alexinsky, T. and G. Chapouthier. Halothane anaesthesia and DMS performance in rats: memory impairment or avoidance behavior? Physiol. Behav. 22: 99-105, 1979. 3. Angel, C., H. M. Bounds and A. Perry. A comparison of the effects of halothane on blood brain barrier and memory consolidation. Dis. Nerv. Syst. 33: 87-93, 1972. 4. Black, M., M. D. Subovski and N. L. Freedman. Effects of cortical spreading depression and ether following one-trial discriminated avoidance learning. Psychon. Sci. 9: 597-598, 1967. 5. Bloch, V. and B. Deweer. Facilitation of memory consolidation processes by stimulation of the reticular formation of the brain stem. Proc. Int. Un. Physiol. Sci. 7: 48, 1968. 6. Bloch, V., B. Deweer and E. Hennevin. Suppression de l'amnrsie rrtrograde et consolidation d'un apprentissage ~ essai unique par stimulation rrticulaire. Physiol. Behav. 5: 1235-1241, 1970. 7. Brennan, M. J. and W. C. Gordon. Modification of the effects of ECS as a function of prior footshock: the aversive properties of ECS. Physiol. Behav. 18: 819-824, 1977. 8. Carew, T. J. Do passive avoidance tasks permit assessment of retrograde amnesia in rats? J. comp. physiol. Psychol. 72: 267271, 1970. 9. Cherkin, A. and E. Lee-Teng. Interruption by halothane of memory consolidation in chicks. Fedn Proc. 24: 328, 1965. 10. Colavita, F. B. Decreased resistance to extinction of a running response following electroconvulsive shock. Psychon. Sci. 3: 487-488, 1965. 11. Coons, E. E. and N. E. Miller. Conflict versus consolidation of memory traces to explain retrograde amnesia produced by ECS. J. comp. physiol. Psychol. 53: 524-531, 1960. 12. Deweer, B. Accrlrration de l'extinction d'un conditionnement par stimulation rrticulaire chez le Rat. J. Physiol., Paris 62: 270-271, 1970. 13. Dorfman, L. J. and M. E. Jarvik. A parametric study of electroshock induced retrograde amnesia in mice. Neuropsychologia 6: 373-380, 1968. 14. Duncan, C. P. The retroactive effect of electroshock on learning. J. comp. physiol. Psychol. 42: 32-44, 1949. 15. Gisquet-Verrier, P. Etude cin6tique de l'amnrsie rrtrograde exprrimentale: rrle aversif d'un agent inducteur, le Fluothane. Thrse 3e Cycle, Paris VI, 1978, pp. 1-167. 16. Gray, J. A. Effects of septal driving of the hippocampal theta rhythm on resistance to extinction. Physiol. Behav. 8: 481-490, 1972. 17. Greenough, W. T. and R. L. Schweitzgebel. Effect of a single ECS on extinction of a bar press. Psychol. Rep. 19: 122%1230, 1966.
18. Hine, B. and R. M. Paolino. Retrograde amnesia: production of skeletal but not cardiac response gradient by electroconvulsive shock. Science 169: 1224-1226, 1970. 19. Lecanuet, J. P., B. Deweer and V. Bloch. Evidence for a consolidation process following imprinting in the one-day-old chick. Brain Res. 37: 363, 1972. 20. Leukel, F. A comparison of the effect of ECS and anaesthesia on acquisition of the maze habit. J. comp. physiol. Psychol. 50: 300-306, 1957. 21. Leukel, F. and E. Quinton. Carbon dioxide effects on acquisition and extinction of avoidance behavior. J. comp. physiol. Psychol. 57: 267-270, 1964. 22. Madsen, M. C. and M. W. Luttges. Effect of electroconvulsive shock (ECS) on extinction of an approach response. Psychol. Rep. 13: 225-266, 1963. 23. Madsen, M. C. and J. L. McGaugh. The effect of electroconvulsive shock on one trial avoidance learning. J. comp. physiol. Psychol. 54: 522-523, 1961. 24. McGaugh, J. L. and H. P. Alpern. Effects of electroshock on memory: amnesia without convulsions. Science 152: 665-666, 1966. 25. McGaugh, J. L. and M. C. Madsen. Amnesic and punishing effects ofelectroconvulsive shock. Science 144: 182-183, 1964. 26. Mendoza, J. E. and H. E. Adams. Does electroconvulsive shock produce retrograde amnesia? Physiol. Behav. 4: 307-309, 1969. 27. Pearlman, C. A., S. K. Sharpless and M. E. Jarvik. Retrograde amnesia produced by anesthetic and convulsant agents. J. comp. physiol. Psychol. 54: 109-112, 1961. 28. Penrod, W. C. and R. Boice. Effects of halothane anaesthesia on the retention of passive avoidance task in rats. Psychon. Sci. 23: 205-207, 1971. 29. Ransmeier, R. E. and R. W. Gerard. Effects of temperature, convulsion and metabolic factors on rodent memory and EEG. Am. J. Physiol. 179: 663-664, 1954. 30. Riccio, D. C. and E. R. Stikes. Persistent but modifiable retrograde amnesia produced by hypothermia. Physiol. Behav. 4: 649-652, 1969. 31. Schmaltz, G. Can halothane anaesthesia have any aversive effects on the rats? Physiol. Behav. 22: 25-29, 1979. 32. Sprott, R. L. Retrograde amnesia in two strains of mice. Psychol. Rep. 19: 1247-1250, 1966. 33. Van Eys, G., H. Rigter and B. E. Leonard. Time-dependant aspects of CO2 induced amnesia and hippocampal monoamine metabolism in rats. Pharmac. Biochem. Behav. 3: 787-793, 1975. 34. Winn, F. J., M. A. Kent and T. H. Libkuman. Learned taste aversion induced by cortical spreading depression. Physiol. Behav. 15: 21-24, 1975.
Accelerated Extinction After Post-Trial Halothane Anaesthesia in Rats: An Aversive Effect PASCALE
GISQUET-VERRIER
Centre National de la R e c h e r c h e Scientifique, Laboratoire de Physiologie N e r v e u s e , D d p a r t e m e n t de Psychophysiologie, 91190 Gif-Sur-Yvette, France R e c e i v e d l 0 M a r c h 1980 GISQUET-VERRIER, P. Accelerated extinction after post-trial halothane anaesthesia in rats: An aversive effect. PHYSIOL. BEHAV 26(2)223-231, 1981.--The effects of halothane anaesthesia immediately post-trial were investigated in three experiments in rats. The results showed that this treatment accelerated the extinction of three previously acquired, positively reinforced operant tasks: straight alley, barpress learning or modified K-maze. This acceleration was not due to any motor impairment and treatments delayed two hours had no effect on performance. No amnesic component was observed in those experiments in which treatment was administered a single time, but aversive effect appeared gradually through the extinction phase. The results are not consistent with the hypothesis of the amnesic effects of halothane anaesthesia; they are interpreted in terms of aversive effects. Rats
Halothane anaesthesia
Retrograde amnesia
Aversive effect
N U M E R O U S clinical and experimental studies indicate that retention o f recently acquired habits can be impaired by a great variety of treatments. An experimental amnesia generally occurs when such agents as electroconvulsive shock [14,20], hypothermia [29,30[ or anaesthesia [6,27] are administered immediately or shortly after acquisition. The existence of an inverse relationship between the observed behavioral deficits and the training-to-treatment interval has generally been interpreted in terms of retrograde amnesia, i.e., that these treatments impair retention by disrupting a memory trace consolidation process. Ever since Coons and Miller [11] pointed out that punishing effects of electroconvulsive shock could be expected to show the same kind of interfering gradient as amnesia in an active avoidance paradigm, experimental research in this field has developed in two major directions: (a) the attempt to discover behavioral paradigms in which amnestic and non-amnestic effects of treatments could be easily separated and (b) the use of mild, non-traumatic disruptive agents. It has been claimed that one trial passive avoidance procedures (step down [24,32], step through [13,23]), which have often been used in the investigation of retrograde amnesia, allow a clear dissociation of amnestic and punishing effects of treatments. Indeed, a number of authors using this procedure, especially in conjunction with electroconvulsive shock, have found retrograde amnesia without aversive effects after a single administration. Some data, however, make this assumption questionable. Carew [8] has demonstrated that, in both these tasks, those rats which were considered amnesic when their performances were scored in terms of passive avoidance showed no memory deficit when
Extinction
an active avoidance criterion was used. Similarly, several authors [18,26] testing the effects of a single ECS on retention o f a one-trial passive avoidance, have reported behavioral deficits in motor responses; at the same time, however, they noted considerable variations in autonomic responses (heart rate, defecation) when the animals were placed in test situations, a fact which indicates some persistence of memory. Such observations suggest that the one-trial passive avoidance procedures alone might not be the appropriate method for testing whether, and to what extent, an experimental treatment can induce retrograde amnesia. Similarly, the use of positively reinforced acquisition does not allow an analysis of data in terms of aversive versus amnestic effects, since both these hypotheses would predict a disturbance in performance. On the other hand, if the treatment to be tested is administered in an extinction paradigm, the assumptions of aversive or amnestic effects would each lead to a different prediction (see Madsen and Luttges [22], Colavita [10], Greenough and Schweitzgebel [17] for ECS, and Leukel and Quinton [21] using carbon dioxide). If the treatment produced retrograde amnesia for recent events, extinction would be prolonged, since the memory loss would be that of the extinction contingencies; if the treatment acted as an aversive stimulus, extinction would take place quite rapidly, since the response would be punished as well as not rewarded. Another direction in the attempt to rule out any interpretation in terms of aversive effects is the search for mild, non-traumatic amnesic treatments. Among a great variety of agents, volatile anaesthetics [6, 21, 27] have been frequently
223
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GISQUET-VERRIER
used. F o r example, in our laboratory and others, an experimental amnesia has been observed after halothane anaesthesia. First reported by Cherkin and Lee-Teng [9] in chicks, behavioral deficits after halothane treatment have been reported several times [3, 5, 12, 19, 28] in rats, mice or chicks, using either one-trial passive avoidance tasks or one-trial positively reinforced tasks. A gradient effect has been described by Bloch et al. [6] and Penrod and Boice [28]. However, the effects of halothane anaesthesia have been exclusively analyzed to date in terms of retrograde amnesia. The present experiments were conducted in order to investigate the possibility of aversive effects, even with this nontraumatic treatment, after one or several administrations. In order to dissociate amnestic and aversive effects, the extinction procedure was chosen. In the first experiment, the effects of halothane anaesthesia were tested in a runway alley, which is the most commonly used task in extinction studies. EXPERIMENT 1 The first experiment was run in two series using 50 subjects each. METHOD
Animals One hundred Sprague-Dawley male albino rats (supplied by Iffa Credo, St Germain sur l'Arbresle) weighing 170-180 g at the start of the experiment were used. Upon arrival in the laboratory, they were housed in individual wire mesh cages (35 ×21 × 18 cm) with ad lib food and water. After one week of familiarization with the experimenter, the daily food ration was progressively reduced until the weight loss was 15%, compared to control animals on a normal diet. The food intake was reduced over 4 days from 30 g to 15 g, and then maintained at this level. Animals were weighed daily, and the diet was adjusted individually in order to maintain a homogenous weight loss.
Apparatus The experimental apparatus, a slightly modified G r a y ' s runway [16], consisted o f a 15 cm wide alley, divided by two manually operated perspex doors opening on the side into a 20 cm long start box, a 120 cm long runway and a 28 cm long goal box. The wooden walls (except the front side which was of perspex) were 30 cm high. Photo beams placed 18 cm from the start box door, 2.5 cm before the goal box were used to measure the start latency, the total running time and the entry latency into the goal box. Halothane anaesthesia was administered over a 60 min period. Treatment was applied in a box supplied with a 4% concentration of halothane in a mixture of air and oxygen, producing unconsciousness within about 60 sec; the animals were then placed into another tank, supplied with 1 to 1.5% concentration of halothane and maintained there for the rest of the 60 min treatment period. Behavioral recovery was usually complete within 20 min after removal from the tank. No perturbation such as gastrointestinal malaise was noticed after anaesthesia. No subject died during or after the treatment.
Experimental Procedure Pretraining. F o r the first two days of pretraining, animals were allowed to explore the alley in groups of six during 30
min, with food available in the goal box. On days 3 and 4, each animal was removed individually from the home cage, placed into the closed goal box and allowed to eat for 2 min. Individual food intake latencies were recorded. Each animal was submitted to this situation 3 times in 2 days. Training. Training began on day 5 and was identical for all animals. Each rat was taken from the home cage, weighed, and placed into the start box for 30 sec. The start box door was then opened manually and the first timer started. The goal box door was closed after the rat had broken the final photo beam. The animal was allowed to eat for 20 sec and then was removed from the goal box and immediately returned to the start box for another trial. Each animal was subjected to 8 consecutively daily trials, under a continuous reinforcement schedule for the first 4 days of acquisition, and a partial reinforcement schedule (PR) for the 8 following days. During the PR phase, reinforcement was delivered randomly after 50% of trials, according to a schedule which was changed daily. This PR schedule was introduced to avoid extinction taking place too quickly in this very simple task. Training was terminated when the mean performance of animals remained at the same level for 4 consecutive days. Extinction. Extinction began on the following day. Behavioral procedures were identical to those used in acquisition, with the exception that no food was delivered in the goal box. During extinction, a rat which failed to break the final photo beam within two min was removed from the alley, but allowed to run again for subsequent trials. In the second series of this experiment, extinction period had to be reduced to 5 days due to an accidental interruption of the daily food deprivation.
Experimental Design At the end of the training period, each of the 100 animals were assigned to one of the six following groups:Group E 8 (first experimental group), subjected to an 8-trial daily session, immediately followed by halothane anaesthesia. Group AC 8, in which the animals were immediately returned to their home cages after the 8-trial session and anaesthetized 2 hours later. Group NAC 8 (control group), in which the animals were not anaesthetized. They were placed, immediately after each session, in a box identical to the halothane tank, supplied with air, for 1 rain and then returned to their cages. Group E 4 (the second experimental group), subjected to a 4-trial daily session, immediately followed by anaesthesia. Group AC 4, in which the animals were subjected to 4 daily extinction trials, then returned to their home cages, and anaesthetized 2 hours later. Group NAC 4 (the second control group), in which the animals were not anaesthetized, but only submitted to a pseudo-treatment (as in Group NAC 8). Twenty subjects were assigned to each of the experimental (E) and each of the "anaesthetized control" (AC) conditions; 10 animals were assigned to each of one of the "non-anaesthetized control" (NAC) conditions. In summary, each animal could be subjected to one of 3 treatments (immediate anaesthesia, delayed anaesthesia, pseudo-anaesthesia), under one of 2 experimental conditions (4 or 8 daily trials). The number of daily extinction trials was varied in order to estimate whether the effects of halothane anaesthesia were related to the quantity of information available in each session.
ACCELERATED EXTINCTION--HALOTHANE
225
ext n _ ext i r : ~ (median) ext_l
I
(median)
NAC S
W
AC8
E~ Ac,
E8 t
NAC 4 E4
treatment
t
treatment
20
10
10-
extlt
ext2t
ext3t
ext4t
ext5~
ext6
extl t ext2 t
ext3 t ext4 t
ext5 t ext6
FIG. 1. Median "loss" score for extinction sessions in animals subjected to 8 (a) or 4 dally trials (b). Halothane anaesthesia (arrows) was administered immediately after extinction in experimental animals (E 8 and E 4), and was delayed 2 hr in anaesthetized control animals (AC 8 and AC 4). Animals in non-anaesthetized control group (NAC 8) were subjected to a pseudo-treatment only.
RESULTS
This experiment was conducted using two series of 50 animals each, under the same learning procedures; however, at the end of acquisition period, a statistical comparison of running times yielded a significant difference between the two samples. A parameter which allowed the comparison of the 100 individual animals despite this difference was used; for each subject and extinction session, performances were compared to the performance in the last training trial, by means of a "loss" score, r=(Extn - Ext-0Ext_l (where r is the "loss" score, Extn the running time measured on the n th extinction session, and Ext_l the running time observed on the last training trial) which is similar in principle to the savings score of Ebbinghaus. These data, summarized in Fig. la and lb, were subjected to intra- and inter-group analyses.
lntra-Group Comparisons The evolution of " r " scores from day to day showed that extinction actually occurred in all groups. The individual performances in each group, indexed in r scores, were subjected to Wilcoxon comparisons. These comparisons
showed that extinction began in the second extinction session, i.e., after one application of the treatment (the T values, calculated between the Ist and the 2nd extinction session were, respectively, T=0, p<0.01 in Group NAC 8; T=6, p<0.005 in Group AC 8; T=0, p<0.005 in Group E 8; T=7, p<0.03 in Group NAC 4; T=35,p<0.005 in Group AC 4, and T=12, p<0.005 in Group E 4). Other intra-group comparisons showed that extinction continued in all groups from session 2 to session 6. It should be noted, however, that during and after the 3rd session, running times were much longer for experimental (immediately anaesthetized) animals than for animals in the other groups.
Inter-Group Comparisons For each extinction session, r scores were subjected to inter-group comparisons. A Kruskall-Wallis analysis of variance showed no difference in performance in the second extinction session (H=3.77, n.s. for animals subjected to 8 trials; H = I , n.s., for animals subjected to 4 trials). A different picture emerged during and after the 3rd extinction session: Kruskall-Wallis comparisons yielded significant differences among groups, both in 8-daily trial groups (H=15.76, p<0.001 on 3rd session; H=22.14, p<0.001 on
226
GISQUET-VERRIER TABLE 1 Days of Extinction Es/NAC8
Es/AC8
NACs/AC,
EdNAC4
EJAC4
NAC4/AC4
Ext 3
U=20 p<0.002
U=88 p<0.002
U=51 NS
U=24 p<0.002
U=90 p<0.02
U=59 NS
Ext 4
U= 7 p<0.002
U=51 p<0.002
U=70 NS
U= 3 p<0.002
U=20 p<0.02
U=91 NS
Ext 5
U=13 p<0.002
U=22 p <0.002
U=93 NS
U=I5 p<0.002
U=40 p<0.02
U=74 NS
Ext 6
U=ll p<0.02
U= 8 p<0.002
U=53 NS
U=21 p<0.05
E8 n=20, AC8 n=20, NAC8 n=10; E4 n=20, AC4 n=20, NAC4 n=10. Individual Mann Whitney U comparisons (two tailed). 4th session; H=29.92, p<0.001 on session 5; H=12.45, p<0.01 in 6th session) and in 4-daily trials groups ( H = 11.02, p<0.01 on 3rd session; H=26.89, p<0.001 on 4th session; H=29.41, p<0.001 on 5th session; U=21, p<0.025 on 6th session). Individual Mann-Whitney comparisons showed that these differences were due to a much more rapid extinction in experimental (E 8 and E 4) groups (see Table 1). Many more animals in Group E 8 than in the other groups did not break the final photo beam within 2 min (cut-off latency) during the last extinction sessions. A chi-square analysis yielded significant differences from sessions 3 to 6 (3rd session, X2=72.54; 4th session, X2= 107.80; 5th session, X2= 179.69; 6th session, X2=81.92; df=2;p
thane applications is strengthened by visual observation of the animals' behavior in the alley, which showed that experimental animals clearly avoided the goal box from the 3rd session to the end of extinction. In the last extinction session, 70 to 90% of the experimental animals avoided entering the goal box within the 2 min cut-off latency. After two administrations of the post-trial treatment, they seemed able to associate the apparatus cues with the aversive properties of anaesthesia. However, at the end of this experiment the possibility of the results being due to the particularities of the experimental situation--very simple learning task, PR schedule at the end of the acquisition period--could not be completely ruled out. F o r this reason, similar experiments were conducted with two other learning tasks: the first was extinction of bar-pressing in a Skinner box, previously used by Deweer [12] in our laboratory (see Experiment 2); the second was extinction in a 4-choice maze (see Experiment 3). This latter task was chosen because it has some similarities with a straight alley (linear maze), but also calls for discriminative responses. EXPERIMENT 2 METHOD
Animals Twenty-two Sprague-Dawley albino rats, weighing 170-180 g at the start of the experiment, were used. Eight days after their arrival, the animals were placed under a 23 hr water deprivation schedule, and maintained approximately at a 10% weight loss, compared with control animals with water ad lib.
Apparatus The apparatus consisted of a 40x30x30 cm Skinner box, provided with a 1.5x3.5 cm lever and a water cup on the same wall. For each animal, two parameters were recorded: the total number of bar presses, and the first press latency. Halothane anaesthesia was delivered in the same conditions as in Experiment 1.
Procedure Pretraining. F o r the first two days of pretraining, the animals were allowed to explore the box, in groups of three,
ACCELERATED
EXTINCTION--HALOTHANE
227
MEDIAN FIRST PRESS LATENCY (sec) ACQUISITION I
EXTINCTION
I I I I
loo
5o.
a I
-'-E
I I I
J J j
L 1
1
I
]
I
I
lo-
NAC I'-'J AC
t
treatment
I
I
~ extl t
I day,
ext2 t
ext3 t
ex,. t
extS
MEDIAN NUMBER OF PRESSES
b
~ NAC [ ~ AC mWJE t treatment
ext4
t
EXTINCTION
ACQUISITION
i:i',.,, 2o-
!!i
!!
•o
lo-
iii
i:i days
extl
t
ext2
t
ext3
t
ext5
FIG. 2. Performances in Skinner box for the last two training sessions and extinction sessions, scored in terms of median first press latency (a) of median number of presses (b). Halothane anaesthesia (arrows) was administered immediately after extinction sessions in Group E, was delayed 2 hr in anaesthetized control group (AC); NAC group was only subjected to a pseudo-treatment. Note that the effects of anaesthesia depend on the index of performance used: first press latencies showed a faster extinction (a); no effect could be observed concerning number of bar presses
(b).
228
GISQUET-VERRIER
with water continuously present in the cup. They were then subjected to 3 consecutive 10 min individual shaping sessions. Training. Training began on day 6 and was identical for all animals. Each rat was taken from the home cage and placed into the Skinner box every day at the same time. The training sessions lasted 4 min during the first 10 days and were then reduced to 2 min for the last two days. This reduction in the duration of training sessions was planned in order to slow down the extinction. A partial reinforcement schedule was not considered necessary since the preliminary experiment showed that extinction period following this type of learning was long enough to observe the effect of the treatment. Extinction. Extinction began on day 18, and all behavioral procedures were identical to those used in acquisition, except for the absence of water. The daily extinction sessions lasted 2 min. Each animal was subjected to 5 consecutive extinction sessions. In training, individual scores were noted in terms of total number of bar presses and first press latency.
Experimental Design At the end of the training period, the mean number of bar presses in training sessions 11-12 was calculated for each rat, and the animals were assigned to one of the following groups: Group E (n=7), in which each extinction session was immediately followed by halothane anaesthesia. Group AC (n=7), in which animals were subjected to a 2 hr delayed anaesthesia after each extinction session. Group NAC (n= 8), in which animals were not subjected to any treatment; they were merely placed, immediately after extinction sessions, into a box identical to the halothane box for 1 min. RESULTS
The data from the individual performances underwent an intra- and an inter-group analysis.
Intra-Group Analysis Performances in each group were compared, for every extinction session, to the level attained at the end of the acquisition period, by means of Wilcoxon comparisons. These comparisons showed that extinction took place in the second session, i.e., after one administration of the treatment, both for total numbers of bar presses (T=0, p <0.01 in Group NAC; T=2.5, p <0.17 in Group AC; T =4, p <0.055 in Group E) and for first bar press latencies (T= 1, p<0.016 in Group NAC; T = 0 , p<0.004 in Group AC; T = 3 , p <0.039 in Group E), and continued during subsequent sessions (see Fig. 2a and b).
Inter-Group Analysis As can be seen in Fig. 2b, no difference could be observed among the three groups during the extinction period, when performances were measured in terms of total numbers of bar presses. The results are quite different when the first bar press latency is considered. After the first two days of extinction, a difference between the experimental (immediately anaesthetized) group and the two control groups appeared (see Fig. 2a). The hypothesis of the acceleration of extinction by immediate anaesthesia was tested by a Kruskall-Wallis analysis of variance, which showed a non-significant tendency on the 3rd session and significant differences among the 3 groups on sessions 4 (H=6.23, p<0.05) and 5 (H=7.58, p<0.05).
Comparisons of individual groups by Mann-Whitney U tests confirmed (a) that there was no difference between the two control Groups AC and NAC (respectively, for sessions 4 and 5, U=31 and U=27, n.s.), and (b) that first bar press latencies were significantly greater in Group E than in Groups NAC (4th session, U = 8 , p<0.01; 5th session, U = 6 , p<0.005) and AC (4th session, U=10, p<0.02; 5th session, U = 3 , p<0.002).
Behavioral Observations In spite of the fact that the total number of bar presses was identical in the three groups, it seems important to note that the strategies were quite different between these groups. Control animals performed with regularity in both delayed anaesthetized and non-treated groups; each lever press was generally followed by a visit to the water cup. In the experimental group, on the other hand, the latency of the first bar press was much more variable and longer; the subsequent bar presses took place in clusters of 3-5 presses, irregularly spaced, and either followed by a visit to the cup or not. These observations might explain why Deweer [12], who only noted the total number of bar presses during longer (6 min) extinction sessions, did not obtain the same results as reported here, although the same task and the same treatment were used. EXPERIMENT 3 METHOD
Animals Thirty-eight Sprague-Dawley male albino rats, weighing 170-180 g at the start of the experiment, served as subjects. After one week of familiarization, the daily food ration was progressively reduced until the weight loss reached 15%, compared to control animals. The deprivation schedule was then maintained in the same conditions as in Experiment 1.
Apparatus The maze consisted of four T shape units. A start box (25 x 25 x 35 cm) communicated with the first maze unit by a manually opened sliding door. Each unit measured 50x40x35 cm. The choice point, or the stem of the T, was located 25 crn from the entrance of each unit. The true path led into the next unit by a 10 cm opening and the other led into a blind. The barriers in each unit of the maze could be changed from one side to the other. The maze configuration was R L L R or LRRL. The goal box, of the same dimensions as the start box, could be closed by a sliding door. Halothane anaesthesia was delivered in the same conditions as in Experiments 1 and 2.
Procedure Pretraining. During the pretraining period, rats were habituated to the experimental conditions. On day 1, individual rats were allowed to feed in the goal box for two 20 min periods. Day 2 involved 30 min free exploration of the maze, in groups of 6, with the barriers removed and with food pellets in the goal box. Days 3 and 4 consisted of training by pairs for departure from the start box, entering the goal box, using only one unit of the maze between the start box and the goal box. Procedure on day 5 was the same, but for individual rats. Training. Training began 24 hr later. Barriers were placed
ACCELERATED EXTINCTION--HALOTHANE
229
MEDIAN TOTAL RUNNING TIME (sec/
ACQUISITION
NAC E treatment
t
EXTINCTION
20(
I01
days extl text2 text3 text4 text5
ext 6
FIG. 3. Median times to run the maze for the last two acquisition trials, and extinction trials. Animals subjected to immediate halothane anaesthesia (arrows) immediately after each trial extinguished much faster than non-anaesthetized control (NAC) animals. at the appropriate points, and in order to prevent animals from following the scent of the preceding subject, the path was changed to its mirror configuration for every other rat, and the maze was cleaned several times a day. Each animal was removed from the home cage and placed into the start box; the door was opened, and the time for the rat to arrive at the goal box was recorded, as was the number of incorrect choices and the number of retracings. The animals were allowed to eat for 1 min in the goal box, before being removed and returned to their home cages. The animals were subjected to 12 consecutive daily trials. At this time, they had attained an asymptote performance for 4 consecutive days.
Extinction. Twenty-four hours later the extinction period began. Behavioral procedures were identical to those used in the acquisition period, except that food was no longer delivered. During this period, if a rat failed to enter the goal-box within 4 min, it was removed from the maze and subjected to the treatment of its group. Experimental Design At the end of the training period, animals were divided into 2 equivalent groups, according to total run time, number of errors and number of retracings: Group E, in which animals were immediately anaesthetized after each extinc-
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tion trial. Group N A C , non-anaesthetized control group, in which animals were returned to their home cages after spending 1 min in a box identical to the halothane box. The Group AC, anaesthetized control group, was not used in this experiment, since the two preceding ones showed no difference between Groups AC and NAC. RESULTS Total Running Times As in the two preceding experiments, an intra-group analysis showed that extinction took place in the second session, i.e., after one administration of the treatment. Wilcoxon comparisons showed significant increases in running times from the last training trial to the second extinction trial (respectively, T=38.5, p<0.038 in NAC group, and T=24.5, p<0.006 in Group E). This extinction continued in nonanaesthetized control animals (see Fig. 3); running times in this group, on the last extinction session, were multiplied by 7, 8, 9 or 10, compared to the last training trial. In the experimental (immediately anaesthetized) group, animals ran much slower than controls from the 3rd to the last extinction trial (see Fig. 3). Inter-group comparisons (Mann-Whitney U tests, one-tailed) confirmed the acceleration of extinction after successive halothane administrations (3rd trial, U = 120, p <0.05; 4th trial, U = 87, p <0.01, 5th trial, U=41.5, p<0.001; 6th trial, U=25, p<0.001). Errors and Retracings As regards running times, experimental animals showed accelerated extinction, compared to controls, when the numbers of errors and retracings were considered. MannWhitney U comparisons yielded significant differences between the two groups, from the 3rd trial for retracings (3rd trial, U=98, p<0.01; 4th trial, U=84.5, p<0.01; 5th trial, U=46.5, p<0.001; 6th trial, U=38.5, p<0.001) and from the 4th trial for errors (4th trial, U = 106.5, p<0.05; 5th trial, U=38.5, p<0.001; 6th trial, U=32, p<0.001 one-tailed tests). It should be noted, however, that these results were not due to "forgetting" the correct pathway, since the animals in the experimental group generally performed a first run without errors, then avoided entering the goal box and came back towards the start box, exploring then the entire maze with many retracings and incorrect choices. G E N E R A L DISCUSSION Several tentative conclusions can be drawn from these three experiments. First--and this is probably the most important conclusiorr--extinction was observed in experimental animals as well as in control subjects. Furthermore, in our experimental conditions, intra-group analysis showed extinction in all groups after a single administration of the treatment. All groups, including the experimental group, memorized the new information available during the first session (i.e., the absence of positive reinforcement). In the second extinction session, no effect of halothane anaesthesia, neither amnestic nor aversive, could be detected. However, with repeated administrations, the extinction rate was markedly increased in experimental animals. Such an effect could not result from a proactive effect of halothane anaesthesia on animals' motor behavior, since there was no difference between delayed anaesthetized and non-treated control animals. Finally, experimental animals
avoided the response reinforced during the acquisition period (entering the goal box in Experiments 1 and 3, bar pressing in Experiment 2). However, in every experiment, this avoidance behavior, only appeared after two successive treatments (during and after the 3rd extinction session), strongly suggesting an aversive action with repetition, an hypothesis already proposed in several experiments, particularly with ECS [25]. Several authors, however, (Bloch et al. [6], Alexinsky and Chapouthier [1,2]) have reported effects of halothane anaesthesia after a singh, application. This discrepancy might be attributed to differences in experimental conditions: Bloch et al. used a one-trial, positively reinforced discriminative transfer; Alexinsky and Chapouthier, a 3-choice discrimination box. In retention tests, in both situations, the animals could respond correctly, incorrectly or not at all. In our situations involving extinction procedures, the only alternative was to respond or not to respond. It seems reasonable to suggest that if halothane anaesthesia has aversive properties, an avoidance behavior will rapidly appear when alternative responses are possible. On the other hand, if the only choice left to the animals is to give no response, it will take them longer to find the appropriate strategy. In an additional experiment [15] we examined whether the beginning of anaesthesia could be the actual aversive factor, as demonstrated by Leukel and Quinton [21] for CO2. A group was subjected to a pseudo-anaesthesia for 30 sec after each extinction trial and compared to anaesthetized (1 hr) and non-anaesthetized animal. A slight increase in the extinction rate was observed, which led to intermediate performances, as compared to those of anaesthetized and nonanaesthetized animals (comparisons between Groups NAC and E30 s e c showed a significant difference in the last extinction trial for running times (U=56, p<0.05), and in the 4th and 5th extinction trials for retracings (respectively, U=68, p<0.025 and U=65, p<0.025). The same comparison between groups Er0 ~c,:and E30 ..... yielded a significant difference for running times in the 4th extinction session (U=68, p<0.01). The beginning of anaesthesia seems, therefore, to play a role in the development of aversion; but other factors, such as chilling, or sudden drop in arterial pressure, might also be important. To date, the time-dependent nature of the effects of halothane anaesthesia (i.e., the fact that the closer the administration follows upon the end of acquisition, the greater are the effects) has been considered as a demonstration of the amnesic properties of this treatment [6,28], and referred to a "retrograde amnesia gradient". However, the development of such gradients can also be observed after administration of aversive treatments and explained in terms of laws of learning: when a treatment is administered shortly after the end of acquisition, the animal easily associates the response it has just made with the subsequent traumatic event; whereas increasing the temporal distance between the two events will make this association more difficult to establish. Halothane has recently been found to be aversive by other investigators. Alexinsky and Chapouthier [2] used a 3-choice discrimination task, where the animals were placed into a "signal" box and had subsequently to choose the identical box to obtain reinforcement. If an 8-min anaesthesia was administered between the presentation of the signal and the choice trial, animals systematically avoided the box associated with anaesthesia. Similarly, Schmaltz [31] has recently shown that association between the experimental apparatus and halothane anaesthesia can disturb perform-
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ance in this apparatus 24 hr later. Similarly, our o w n experiments allow the conclusion that halothane anaesthesia acts on subsequent p e r f o r m a n c e through a v e r s i v e rather than a m n e s t i c effects. A v e r s i v e interpretation o f amnesic treatment, which is not n e w [11], has already b e e n p r o p o s e d not only w h e n it is applied repeatedly but also after a single administration for m o s t agents such as E C S [7,8], ether anaesthesia [4], c a r b o n
dioxide [21,33] and KC1 cortical spreading d e p r e s s i o n [34]. O u r o w n w o r k leads us to c o n c l u d e that it cannot be too strongly e m p h a s i z e d that rigorous experimental control for motivational effects is required before any amnesic action can be unequivocally attributed to a treatment: the fact that deficits are found in retention tests does not necessarily m e a n that these are deficits in retention.
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