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Attenuation of anoxia-induced retrograde amnesia in rats by a pretraining placebo injection
Physiology and Behavior, Vol. 10, pp. 693-696. Brain Research Publications Inc., 1973. Printed in the U.S.A.
Attenuation of Anoxia-Induced Retrograde Amnesia in Rats by a Pretraining Placebo Injection M. DAVID-REMACLE
Centre de Psychologie, Experimtale et Comparee, Universitb Catholique de Louvain Chateau de Pellenberg, Belgique- BEL GIUM
(Received 8 December 1972) DAVID-REMACLE, M. Attenuation of anoxia-induced retrograde amnesia in rats by a pretraining placebo injection. PHYSIOL. BEHAV. 10(4) 693-696, 1973.-Rats were given an oral injection of distilled water 35 rain before one-trial passive avoidance learning, achieved by punishing a bar-press performance with a single footshock. Immediately after training the rats were submitted to asphyxia-induceA anoxia. While the noninjected control group failed to perform an avoidance response 24 hr after the anoxic treatment, the injected group, whose normal bar-press performance remained unchanged after the injection, showed a good retention for the shock event despite the anoxic treatment. These data demonstrate that an injection like other environmental events effect memory elaboration and they do not support the consolidation hypothesis which does not account for such variables. Anoxia Retrograde amnesia Injection-handling
Memory consolidation
ACCORDING to the consolidation hypothesis, memory trace of a new event is not spontaneously established but requires a period of time to consolidate [7,19]. However a variety of pretraining [17, 18, 22] as well as posttraining environmental events [4, 23, 26, 281 have been shown to effect the results of memory studies, which could partly account for the lack of agreement among the authors as to the effectiveness of some amnesic agents [ 11 ] and the vast differences in temporal gradient of consolidation [ 14, 18, 251. Recently, Jensen and Riccio reported that preexposure to shock outside the training environment prevents hypothermia induced retrograde amnesia (RA) in passive avoidance learning [12]. It has been supposed that preshock, like familiarization in Lewis's studies [ 17,181, acts by association of the training situation with such manipulations, presenti'ng similar cues [12]. On the other hand, posttraining shock occurring before the retention test has also been found to effect RA by restoring memory loss induced by different agents [1, 15, 32]. Furthermore this retrieval facilitation is observed even if the reminder shock is quite different from training stimuli such as water immersion or water reward reinforcements [1,32]. These recent results indicate that it is not necessary for the reminder treatment to be directly related to the learning experience in order to facilitate retrieval of memory and that presumably many other manipulations can have reminder properties. For example, memory return due to an intrecranial injection of saline has been previously reported by Flexner and Flexner [5].
Environmental events
Perhaps as in posttraining manipulations, pretraining manipulations not related to the subsequent training reinforcement can influence RA. One of the most frequent pretraining treatments which occurs in experimental design in the study of memory is the injection of a drug. Indeed, recently, many investigators focused on drug effects as a research tool to provide clues to the nature of the physiological basis mechanism of memory [21,35]. As a consequence of this methodological approach, injection was introduced as a systematic experimental manipulation. Therefore, it would be useful to know what role, if any, the injection per se can play in the elaboration of a memory trace. This variable has remained relatively unexplored. Flexner and Flexner [5] found that an intracranial injection of saline is able to trigger recovery of memory deficit induced by an intracranial injection of puromycin. Recently, Bauer [2] reported a small effect of 20 intraperitoneal injections of saline on an avoidance performance. The purpose of this experiment is to determine if a water injection per os, as environmental event, is able to interfere with anoxia-induced retrograde amnesia in a one trial avoidance learning. Anoxia was choosen as amnesic agent because it reliably produces RA [6, 9, 24, 31, 33] and has been explored less than ECS. METHOD
A 12imals Sixty-three females rats of the Wistar strain were purchased from the colony of Universit~ Catholique de 693
694 Louvain. Seven days prior training the animals were brought to the laboratory and group housed with food and water ad tib. They were handled a few minutes each day. All the rats were four months old at the start of the experiment.
Apparatus The rats were trained in two identical Skinner boxes equipped with a lever, a food-cup and a dispenser of pellets (45 mg) made from the Trouw and Co. Muracon usual food. A 600 uA shock could be distributed through the grid-floor and the lever, by means of G.S. generator and scrambler.
Procedure Training. Passive avoidance training was adapted from the method previously described by Pearlman et al. [25] and Weissman [34], involving the suppression of a previously learned lever-press response by administration of a single electric shock through the lever. Three days prior to learning, all the rats were placed on a food deprivation schedule consisting of 14 mg of Trouw and Co Muracon usual food every 24 hr. Then the animals were shaped 20 min each day, in the Skinner boxes, to press a lever for pellet reinforcemerit on a continous reinforcement schedule (CRF). After completion of the acquisition phase, the animals were returned to the rat-chamber for a month until there was a total recuperation of their initial weight. Then the rats were returned to the laboratory and placed on the same feeding schedule as during acquisition. On the day following the return to the laboratory, the rats were tested 20 min for the bar-press response. On the 4th, 5th and 6th days after the onset of the food deprivation program, performance during 10 rain for each rat was recorded. On the seventh day, the first response following 5 min of CRF schedule was punished with a 600 uA, 2 sec footshock delivered through the grids and the lever. Then the rat was removed from the training apparatus and assigned randomly to 'one of the following treatment groups.
DAVI D-REMAC L L: injection of 1 ml of distilled water per os, ] h e y were submitted to a shock and anoxia treatment similar to the FSAN group.
Retention Test Twenty-four hours after the treatment, the rats were tested for retention of the shock event. Retention was expressed as a percentage of the bar-pressing responses emitted on the first 5 rain of the retention test session compared with the number emitted just before the footshock on the preceeding day. RESULTS Friedman two-way analysis of variance indicates no significant difference among the three days of bar-press performance testing preceding the footshock treatment (N=63, Xr2=6.8; dr=2, p>0.05). It can be concluded that performance was stable. Response rate of the injected group recorded over the 5 min just before shock (i.e. 30 min after injection) was compared with the baseline performance of this group calcualted by averaging the response rate over the first 5 min on the 3 consecutives preceding sessions. Wilcoxson matched-pairs signed-ranks indicates that the response rate before and after the injection handling do not significantly differ (N=8. t=9. p>0.05 two-tailed). It can be concluded that the injection treatment has not perturbed the normal lever-press response. Kruskal-Wallis one-way analysis of variance shows that there was no significant difference between the bar-pressing responses rates in the 5 groups, emitted just before shock (H=t.797, d)'-4, p>0.70). It can be concluded that all the groups were equivalent before the treatment The statistical evaluation of the data of the retention test was made by the Mann-Whitney U test. Median percentages of bar-pressing responses (retention scores) of the different groups are shown in Fig. 1 Percentages of
9o
Treatment Footshock + A noxia (FSAN). Five to 10 sec after shock, the 18 rats of this group were submitted to a severe anoxia induced by a 60 sec compression of the thorax. Similar technique has previously been shown by Hayes [9] to produce amnesia. Anoxia convulsant behavioral pattern includes a short atoni¢ phase followed by an exaggerated clonic p h a s e ' w i t h subsequent excitability followed by lethargy. This behavioral seizure pattern presumably corresponds to the triphasic effect of carbon dioxide anoxia on brain excitability outlined by Woodbury and Karler [36]. Anoxia only (ANO). The 11 animals in this group received the same treatment as the FSAN group except that no footshock preceeded anoxia. Footshock only (FSO). The 15 animals of this group were returned to their home cage after the shock producing bar-press response without any amnesic treatment following the shock. Control group (00). The 10 rats in this group were removed from the training apparatus after 5 min without any shock or amnesic treatment. In/ection + Footshock + Anoxia (1FSAN). Thirty-five rain before shock the c~ rats of this group received a single
~-
8O O~
0.
M.
o
<
k-
~20 a.
Z~lo FSAN
FSO
ANO
O0
II IFSAN
FIG. 1. Median percentages of responses ~RA scores) on the retention-test day. Note: A high percentage indicates a failure of avoidance or a loss of retention for the shock.
A N O X I A - I N D U C E D R E T R O G R A D E AMNESIA bar-pressing were significantly greater in the OO group than in the FSO group indicating that footshock is an effective suppressor (n, =10, n 2 15; U=6, p<0.001 one-tailed). Comparison of percentages of bar-pressing in FSAN group and FSO group showed a highly significant difference (n, =15," n 2 = 18, U=26.5, p< 0.001 one-tailed) suggesting that anoxia is effective in blocking the bar-press avoidance response. Anoxia treatment alone does not effect performance since percentage of responses in ANO group does not differ significantly from the percentage of responses in OO group (n I --10; n 2 =11 ; U=52.5; p > 0 . 1 0 two-tailed). IFSAN group emitted significantly fewer responses than did the FSAN group (n, =9; n~ =18; U=8.5, p < 0 . 0 0 2 two-tailed). Injection appears to prevent RA induced by anoxia. DISCUSSION In addition to confirming previous results about the ability of anoxia to induce amnesia [6, 9, 24, 3 1 , 3 3 ] , this study clearly demonstrates that a single pretraining water injection is able to prevent the anoxia-amnesic effect. It is not certain whether this amnesic effect is due to oxygen lack or CO7 accumulation in the brain. Since there is some disagreement in the experimental findings [6,33] this point remains controversial. These data provide additional evidence that pretraining manipulations interfere with the establishment of RA. How injection handling, familiarization with training apparatus [ 17,18], or preexposure to the conditioned stimulus [12] have similar preventive effects on RA cannot be determinated from this experiment. However, a speculation gives rise to several plausible hypotheses. Since th~ present experiment uses a pretraining event quite different from the learning situation, the prevention o f RA cannot be explained in terms of Lewis familiarization concept which is restricted to preexposure to elements involved in the subsequent experimental situation. Jensen and Riccio [12] reaffirming Lewis, assumed that preshock attenuates RA "in view of a possibility of associative mediation due to a variety" of stimulus similarities from preshock to the training situation". Even though our present results suggest that a prior event totally dissimilar from the subsequent task is also effective in reducing m e m o r y loss, it is not incongruous with the above reported findings, if familiarization is taken in the broader sense of organism's prior learned response. Injection handling can have sufficient aversive effect to trigger an organism's latent predisposition to avoid. Such organism familiarized with the appropriate response demanded by the learning task is no longer naive and consequently has less to learn, thereby possibly reducing the time required for consolidation. A possible alternative explanation considers the injection effect in terms of arousal. Most authors use the "arousal response" to refer to electrophysiological changes elecited by a novel stimulus or by changes in normally present stimuli of all sensory modalities [8]. It can be supposed that injection, a sudden peripheral stimulation, produces sufficient changes to elicit such an arousal response b y the mediation of an adrenergic system [13]. Involvement of adrenergic substances and other arousal related biogenic amines in m e m o r y has been recently suggested by Kety [13] who from recent findings on norepinephrine emphasized that "some chemical components of affective states should be able to facilitate protein synthesis" and so subserve the consolidation of the trace. The author presents a highly speculative model of neural process whereby this
695 could be achieved. Recently, the participation of arousal in consolidation has been demonstrated. Bloch [3] points out that subliminary stimulation of the mesencephalic reticular formation accelerates both acquisition and extinction and eliminates RA effect of fluothane. With regard to these recent findings it can be suggested that the preventive action of the injection's peripheral stimulation is related to its arousal properties. Unexpected stimulus produces a different electrophysiological response than an expected stimulus [8]. By alerting the organism to environmental changes, the injection might have enhanced the significance of the subsequent shock event, a novel stimulus superimposed to a well-established habit (bar-pressing). The anticipating organism's state, provided b y injection, could have facilitated the elaboration of memorY trace for a new event, which has become less vulnerable to the amnesic treatment. Conversely, if further studies show that habituation to repeated injections eliminates this preventive effect, the arousal hypothesis will be supported. It can be argued that this experiment does not indicate if injection has improved acquisition or if it has intensified memorY consolidation of shock event. Such an objection implicitly supposes that memory trace elaboration occurs in the postacquisition period. This assumption is derived from the classical consolidation hypothesis stating that memory is a time-dependent process [191. However a growing b o d y of literature challenges this view (see Lewis's review [ 16 ] ) showing that memory trace of an event depends not only on time but on prior and subsequent experience. Such an event is elaborated and integrated into a preestablished long-term memory of past experience which never consolidates but evolves by a continual dynamic reorganization [29]. So, to try to separate effects on acquisition from effects on consolidation becomes a false problem. An alternative model more concordant with the facts will consider that a m e m o r y trace proceeds both from a dynamic reciprocal modulation of environment demands and a functional system (the ontogenetic brain's potential to organize stimuli) in order to p r o m o t e a further adaptarive reaction. This s u p p o r t s ' t h e elaboration or integrativedistributive-elaborative-facilitative memory process stated by Irwin, e t al. [ 10]. This new perspective implies the need for new approach in the study of drugs effect on memory. Psychopharmacological research must develop a suitable methodology accounting of the experimental history o f the laboratory animal. As shown by the present experiment, a pretraining injection handling can change vulnerability of memory to amnesic treatment. Several psychopharmacological studies, focusing on the consolidation aspects of the trace, provide strong evidence that psychoactive drugs such as strychnine, pemoline, amphetamine, pentylenetatrazol .... in low concentration can improve learning or reduce retrograde amnesia, despite the fact that at the convulsive level they may be disruptive [20,35]. The list of drugs found to strengthen memory processes continues to grow. Previous analysis in this laboratory demonstrated that Piracetam, a nontoxic and nonanaleptic drug protects rats against retrograde amnesia induced by sudden as well as progressive nitrogen hypoxia [31]. However, since injection handling can also have per se some psychoactive properties, it is presumed that it is able to interfere, i.e. potentiate or inhibit, the action of drugs on memory. If drugs can act on the injection produced effects as well as on the memory process itself or on their synergetic interaction, it is necessary to be
696
DAVID-REMACLE
able t o d e t e r m i n e o n w h i c h p h e n o m e n o n t h e drug specifically acts. C h a n g e s in t h e classical e x p e r i m e n t a l design are required: the addition of a group to control the placebo effect is r e q u i r e d p a r t i c u l a r l y for d r u g s w h o s e m e c h a n i s m o f a c t i o n is n o t well u n d e r s t o o d .
ACKNOWLEDGEMENT The author is indebted to her colleague S. J. Sara for her helpful comments. The author wishes also to thank E. Delwaert for his technical assistance.
REFERENCES 1. Barondes, B. H. and H. D. Cohen. Arousal and the conversion of "short-term" to "long-term" memory. Prec. natn Acad. Sci. 61: 9 2 3 - 9 2 9 , 1968. 2. Bauer, R. H. Twenty-four hour proactive facilitation of avoidance and discrimination by pentylenetetrazol. Psychopharmacologia. (Berl.) 24: 2 7 5 - 2 9 5 , 1972. 3. Bloch, V. Facts and hypotheses concerning memory consolidation processes. Brain Res. 2 4 : 5 6 1 - 5 7 5 , 1970. 4. Davis, R. E. and P. O. Klinger. Environmental control of amnesic effects of various agents in goldfish. Physiol. 8ehav. 4: 2 6 9 - 2 7 1 , 1969. 5. Flexner, L. B. and J. B. Flexner. Intracerebral saline: Effect on memory of trained mice tested with puromycin. Science 159: 3 3 0 - 3 3 1 , 1968. 6. Giurgea, C., D. Lefevre, C. Lescrenier and M. David-Remacle. Pharmacological protection against hypoxia induced Amnesia in rats. Psychopharmacoiogia {Berl.) 20: 160-168, 1971. 7. Glickman, S: E. Perseverative neural processes and consolidation of the neural trace. Psychol. Bull. 58: 2 t 8 - 2 3 3 , 1961. 8. Grossman, S. P. A Testbook o f Physiological. New York: John Wiley and Sons, Inc. 6 2 3 - 6 4 0 , 1967. 9. Hayes, K. J. Anoxia and convulsive amnesia in rats. J. comp. physiol. Psychol. 46: 2 1 6 - 2 1 7 , 1953. 10. Irwin, S., A. Banuazizi, S. Kalsner and A. Curtis. One-trial learning in the mouse: its characteristics and modifications by experimental seasonal variables. In: Neurophysiological and Behavioral Aspects of Psychotropic Drugs, edited by A. G. Karczmar and W. P. Koella. Springfield, Illinois: C. C. Thomas. 1969, pp. 115-131. l l . Jarvik, M. E. Consolidation of memory. In: Psychopharmacologia, a Review o f Progress, edited by D. H. Efron. Public Health Service Publication. 8 8 5 - 8 9 0 , 1968. 12. Jensen, R. A. and D. Riccio. Effect of prior experience upon retrograde amnesia produced by hypothermia. Physiol. Behav. 5: 1291-1294, 1970. 13. Kety, S. S. Norepinephrine in central nervous system and its correlation with behavior. In: Brain and Human Behavior. edited by A. G. Karczmar and J. C. Eccles. Berlin: Springer Veflag. 1972, pp. 115-129. 14. Kopp, R., Z. Bohdanecky and M. E. Jarvik. Long temporal gradient of retrograde amnesia for a well-discriminated stimulus. Science 153: 1547-1549, 1966. 15. Koppenaal, K: J., and E. Jogoda and J. A. J. Cruce. Recovery from ECS produced amnesia following a reminder. Psychon. Sci. 9: 2 9 3 - 2 9 4 , 1967. 16. Lewis, D. J. Source of experimental amnesia. Psychol. Rev. 76: 4 6 1 - 4 7 2 , 1969. 17. Lewis, J. D., R. R. Miller and J. R. Misanin. Control of retrograde amnesia. J. comp. physiol. Psychol. 66: 4 4 - 5 2 . 1968. 18. Lewis, J. D., R. R. Miller and J. R. Mi~nin. Selective amnesia in rats produced by electroconvulsive shock. J. comp. physiol. Psychol. 69: 136-140, 1969.
19. McGaugh, J. L. Time~lependent processes in memory storage. Science 153: 1351-1358, 1966. 20. McGaugh, J. L. Drugs facilitation of memory and learning. In: Psychopharmacologia, a Review of Progress, edited by D. H. Efron. Public Health Service Publication, 891-904, 1967. 21. McGaugh, J. L. and L. F. Petrinovich. Effects of drugs in learning and memory. Int. Rev. Neurobiol. 8: 139-196, 1965. 22. Miller, R. R. Effects of environmental complexity on amnesia induced by electroconvulsive shock in rats. J. comp. physiol. Psychol. 71: 267-275, 1970. 23. Miller, R. R., J. R. Misanin and D. J. Lewis. Amnesia as a function of events during the learning-ECS interval. J. comp. physiol. Psychol. 67: 145-148, 1969. 24. Paolino, R. M., O. Quatermain and N. E. Miller. Different temporal gradients of retrograde amnesia produced by carbon dioxide anesthesia and electroconvulsive shock. J. temp. physiol. Psychol. 62: 2 7 0 - 2 7 4 , 1966. 25. Pearlman, C. A., S. K. Sharpless and M. E. Jamik. Retrograde amnesia produced by anesthesia and convulsive agents. J~ comp. physiol. Psychol. 5 4 : 1 0 9 - 1 1 2 . 1961. 26. Potts, W. J. The effect of different environments prior the electroconvulsive shock on the gradient of retrograde amnesia. Physiol. Behav. 7: 161 - 164, 1971. 27. Quatermain, D., R. M. Paolino and N. E. Miller. A brief temporal gradient of retrograde amnesia independent of situational change. Science 160: 330-332. 1968. 28. Robusteili, F. and M. E. Jarvik. Retrograde amnesia from detention. Physiol. Behav. 3: 543-547. 1968. 29. Sara, S. ]. Recovery from hypoxia and ECS-induced amnesia. Physiol. Behav. lO: 8 5 - 8 9 , 1973. 30: Sara, S. J. and D. Lefevre. A reexamination of the role of familiarization (FAM) in retrograde amnesia in rats. J. cornp. physiol. Psychol., in press. 31. Sara, S. J. and D. Lefevre. Hypoxie-induced amnesia in one-trial learning and pharmacological protection by pitacetam. Psychopharmacoligia, IBerl.) 25: 32--40, 1972. 32. Springer, A. D. and R. R, Miller. Retrieval failure induced by electroconvuisive shock: reversal with dissimilar training and recovery agents. Science 139: 6 2 8 - 6 3 0 , 1972. 33. Taber, R. A. and A. Banuazizi. CO2-induced retrograde amnesia in a one-trial learning situation. Psychopharmacologia (Berl.) 9: 3 8 2 - 3 9 1 , 1 9 7 2 . 34. Weissman, A. Effect of electroconvulsive shock intensity and seizure pattern on retrograde amnesia in rats. J. comp. physiol. Psychol. 56: 8 0 6 - 8 1 0 , 1963. 35. Weissman, A. Drugs and retrograde amnesia. Int. Rev. Neurobiol. i0: 167-198, 1967. 36. Woodbury, D. M. and R. Karler. The role of carbon dioxide in the nervous system. Anesthesiology. 21 : 686 -705. 1960.
Attenuation of Anoxia-Induced Retrograde Amnesia in Rats by a Pretraining Placebo Injection M. DAVID-REMACLE
Centre de Psychologie, Experimtale et Comparee, Universitb Catholique de Louvain Chateau de Pellenberg, Belgique- BEL GIUM
(Received 8 December 1972) DAVID-REMACLE, M. Attenuation of anoxia-induced retrograde amnesia in rats by a pretraining placebo injection. PHYSIOL. BEHAV. 10(4) 693-696, 1973.-Rats were given an oral injection of distilled water 35 rain before one-trial passive avoidance learning, achieved by punishing a bar-press performance with a single footshock. Immediately after training the rats were submitted to asphyxia-induceA anoxia. While the noninjected control group failed to perform an avoidance response 24 hr after the anoxic treatment, the injected group, whose normal bar-press performance remained unchanged after the injection, showed a good retention for the shock event despite the anoxic treatment. These data demonstrate that an injection like other environmental events effect memory elaboration and they do not support the consolidation hypothesis which does not account for such variables. Anoxia Retrograde amnesia Injection-handling
Memory consolidation
ACCORDING to the consolidation hypothesis, memory trace of a new event is not spontaneously established but requires a period of time to consolidate [7,19]. However a variety of pretraining [17, 18, 22] as well as posttraining environmental events [4, 23, 26, 281 have been shown to effect the results of memory studies, which could partly account for the lack of agreement among the authors as to the effectiveness of some amnesic agents [ 11 ] and the vast differences in temporal gradient of consolidation [ 14, 18, 251. Recently, Jensen and Riccio reported that preexposure to shock outside the training environment prevents hypothermia induced retrograde amnesia (RA) in passive avoidance learning [12]. It has been supposed that preshock, like familiarization in Lewis's studies [ 17,181, acts by association of the training situation with such manipulations, presenti'ng similar cues [12]. On the other hand, posttraining shock occurring before the retention test has also been found to effect RA by restoring memory loss induced by different agents [1, 15, 32]. Furthermore this retrieval facilitation is observed even if the reminder shock is quite different from training stimuli such as water immersion or water reward reinforcements [1,32]. These recent results indicate that it is not necessary for the reminder treatment to be directly related to the learning experience in order to facilitate retrieval of memory and that presumably many other manipulations can have reminder properties. For example, memory return due to an intrecranial injection of saline has been previously reported by Flexner and Flexner [5].
Environmental events
Perhaps as in posttraining manipulations, pretraining manipulations not related to the subsequent training reinforcement can influence RA. One of the most frequent pretraining treatments which occurs in experimental design in the study of memory is the injection of a drug. Indeed, recently, many investigators focused on drug effects as a research tool to provide clues to the nature of the physiological basis mechanism of memory [21,35]. As a consequence of this methodological approach, injection was introduced as a systematic experimental manipulation. Therefore, it would be useful to know what role, if any, the injection per se can play in the elaboration of a memory trace. This variable has remained relatively unexplored. Flexner and Flexner [5] found that an intracranial injection of saline is able to trigger recovery of memory deficit induced by an intracranial injection of puromycin. Recently, Bauer [2] reported a small effect of 20 intraperitoneal injections of saline on an avoidance performance. The purpose of this experiment is to determine if a water injection per os, as environmental event, is able to interfere with anoxia-induced retrograde amnesia in a one trial avoidance learning. Anoxia was choosen as amnesic agent because it reliably produces RA [6, 9, 24, 31, 33] and has been explored less than ECS. METHOD
A 12imals Sixty-three females rats of the Wistar strain were purchased from the colony of Universit~ Catholique de 693
694 Louvain. Seven days prior training the animals were brought to the laboratory and group housed with food and water ad tib. They were handled a few minutes each day. All the rats were four months old at the start of the experiment.
Apparatus The rats were trained in two identical Skinner boxes equipped with a lever, a food-cup and a dispenser of pellets (45 mg) made from the Trouw and Co. Muracon usual food. A 600 uA shock could be distributed through the grid-floor and the lever, by means of G.S. generator and scrambler.
Procedure Training. Passive avoidance training was adapted from the method previously described by Pearlman et al. [25] and Weissman [34], involving the suppression of a previously learned lever-press response by administration of a single electric shock through the lever. Three days prior to learning, all the rats were placed on a food deprivation schedule consisting of 14 mg of Trouw and Co Muracon usual food every 24 hr. Then the animals were shaped 20 min each day, in the Skinner boxes, to press a lever for pellet reinforcemerit on a continous reinforcement schedule (CRF). After completion of the acquisition phase, the animals were returned to the rat-chamber for a month until there was a total recuperation of their initial weight. Then the rats were returned to the laboratory and placed on the same feeding schedule as during acquisition. On the day following the return to the laboratory, the rats were tested 20 min for the bar-press response. On the 4th, 5th and 6th days after the onset of the food deprivation program, performance during 10 rain for each rat was recorded. On the seventh day, the first response following 5 min of CRF schedule was punished with a 600 uA, 2 sec footshock delivered through the grids and the lever. Then the rat was removed from the training apparatus and assigned randomly to 'one of the following treatment groups.
DAVI D-REMAC L L: injection of 1 ml of distilled water per os, ] h e y were submitted to a shock and anoxia treatment similar to the FSAN group.
Retention Test Twenty-four hours after the treatment, the rats were tested for retention of the shock event. Retention was expressed as a percentage of the bar-pressing responses emitted on the first 5 rain of the retention test session compared with the number emitted just before the footshock on the preceeding day. RESULTS Friedman two-way analysis of variance indicates no significant difference among the three days of bar-press performance testing preceding the footshock treatment (N=63, Xr2=6.8; dr=2, p>0.05). It can be concluded that performance was stable. Response rate of the injected group recorded over the 5 min just before shock (i.e. 30 min after injection) was compared with the baseline performance of this group calcualted by averaging the response rate over the first 5 min on the 3 consecutives preceding sessions. Wilcoxson matched-pairs signed-ranks indicates that the response rate before and after the injection handling do not significantly differ (N=8. t=9. p>0.05 two-tailed). It can be concluded that the injection treatment has not perturbed the normal lever-press response. Kruskal-Wallis one-way analysis of variance shows that there was no significant difference between the bar-pressing responses rates in the 5 groups, emitted just before shock (H=t.797, d)'-4, p>0.70). It can be concluded that all the groups were equivalent before the treatment The statistical evaluation of the data of the retention test was made by the Mann-Whitney U test. Median percentages of bar-pressing responses (retention scores) of the different groups are shown in Fig. 1 Percentages of
9o
Treatment Footshock + A noxia (FSAN). Five to 10 sec after shock, the 18 rats of this group were submitted to a severe anoxia induced by a 60 sec compression of the thorax. Similar technique has previously been shown by Hayes [9] to produce amnesia. Anoxia convulsant behavioral pattern includes a short atoni¢ phase followed by an exaggerated clonic p h a s e ' w i t h subsequent excitability followed by lethargy. This behavioral seizure pattern presumably corresponds to the triphasic effect of carbon dioxide anoxia on brain excitability outlined by Woodbury and Karler [36]. Anoxia only (ANO). The 11 animals in this group received the same treatment as the FSAN group except that no footshock preceeded anoxia. Footshock only (FSO). The 15 animals of this group were returned to their home cage after the shock producing bar-press response without any amnesic treatment following the shock. Control group (00). The 10 rats in this group were removed from the training apparatus after 5 min without any shock or amnesic treatment. In/ection + Footshock + Anoxia (1FSAN). Thirty-five rain before shock the c~ rats of this group received a single
~-
8O O~
0.
M.
o
<
k-
~20 a.
Z~lo FSAN
FSO
ANO
O0
II IFSAN
FIG. 1. Median percentages of responses ~RA scores) on the retention-test day. Note: A high percentage indicates a failure of avoidance or a loss of retention for the shock.
A N O X I A - I N D U C E D R E T R O G R A D E AMNESIA bar-pressing were significantly greater in the OO group than in the FSO group indicating that footshock is an effective suppressor (n, =10, n 2 15; U=6, p<0.001 one-tailed). Comparison of percentages of bar-pressing in FSAN group and FSO group showed a highly significant difference (n, =15," n 2 = 18, U=26.5, p< 0.001 one-tailed) suggesting that anoxia is effective in blocking the bar-press avoidance response. Anoxia treatment alone does not effect performance since percentage of responses in ANO group does not differ significantly from the percentage of responses in OO group (n I --10; n 2 =11 ; U=52.5; p > 0 . 1 0 two-tailed). IFSAN group emitted significantly fewer responses than did the FSAN group (n, =9; n~ =18; U=8.5, p < 0 . 0 0 2 two-tailed). Injection appears to prevent RA induced by anoxia. DISCUSSION In addition to confirming previous results about the ability of anoxia to induce amnesia [6, 9, 24, 3 1 , 3 3 ] , this study clearly demonstrates that a single pretraining water injection is able to prevent the anoxia-amnesic effect. It is not certain whether this amnesic effect is due to oxygen lack or CO7 accumulation in the brain. Since there is some disagreement in the experimental findings [6,33] this point remains controversial. These data provide additional evidence that pretraining manipulations interfere with the establishment of RA. How injection handling, familiarization with training apparatus [ 17,18], or preexposure to the conditioned stimulus [12] have similar preventive effects on RA cannot be determinated from this experiment. However, a speculation gives rise to several plausible hypotheses. Since th~ present experiment uses a pretraining event quite different from the learning situation, the prevention o f RA cannot be explained in terms of Lewis familiarization concept which is restricted to preexposure to elements involved in the subsequent experimental situation. Jensen and Riccio [12] reaffirming Lewis, assumed that preshock attenuates RA "in view of a possibility of associative mediation due to a variety" of stimulus similarities from preshock to the training situation". Even though our present results suggest that a prior event totally dissimilar from the subsequent task is also effective in reducing m e m o r y loss, it is not incongruous with the above reported findings, if familiarization is taken in the broader sense of organism's prior learned response. Injection handling can have sufficient aversive effect to trigger an organism's latent predisposition to avoid. Such organism familiarized with the appropriate response demanded by the learning task is no longer naive and consequently has less to learn, thereby possibly reducing the time required for consolidation. A possible alternative explanation considers the injection effect in terms of arousal. Most authors use the "arousal response" to refer to electrophysiological changes elecited by a novel stimulus or by changes in normally present stimuli of all sensory modalities [8]. It can be supposed that injection, a sudden peripheral stimulation, produces sufficient changes to elicit such an arousal response b y the mediation of an adrenergic system [13]. Involvement of adrenergic substances and other arousal related biogenic amines in m e m o r y has been recently suggested by Kety [13] who from recent findings on norepinephrine emphasized that "some chemical components of affective states should be able to facilitate protein synthesis" and so subserve the consolidation of the trace. The author presents a highly speculative model of neural process whereby this
695 could be achieved. Recently, the participation of arousal in consolidation has been demonstrated. Bloch [3] points out that subliminary stimulation of the mesencephalic reticular formation accelerates both acquisition and extinction and eliminates RA effect of fluothane. With regard to these recent findings it can be suggested that the preventive action of the injection's peripheral stimulation is related to its arousal properties. Unexpected stimulus produces a different electrophysiological response than an expected stimulus [8]. By alerting the organism to environmental changes, the injection might have enhanced the significance of the subsequent shock event, a novel stimulus superimposed to a well-established habit (bar-pressing). The anticipating organism's state, provided b y injection, could have facilitated the elaboration of memorY trace for a new event, which has become less vulnerable to the amnesic treatment. Conversely, if further studies show that habituation to repeated injections eliminates this preventive effect, the arousal hypothesis will be supported. It can be argued that this experiment does not indicate if injection has improved acquisition or if it has intensified memorY consolidation of shock event. Such an objection implicitly supposes that memory trace elaboration occurs in the postacquisition period. This assumption is derived from the classical consolidation hypothesis stating that memory is a time-dependent process [191. However a growing b o d y of literature challenges this view (see Lewis's review [ 16 ] ) showing that memory trace of an event depends not only on time but on prior and subsequent experience. Such an event is elaborated and integrated into a preestablished long-term memory of past experience which never consolidates but evolves by a continual dynamic reorganization [29]. So, to try to separate effects on acquisition from effects on consolidation becomes a false problem. An alternative model more concordant with the facts will consider that a m e m o r y trace proceeds both from a dynamic reciprocal modulation of environment demands and a functional system (the ontogenetic brain's potential to organize stimuli) in order to p r o m o t e a further adaptarive reaction. This s u p p o r t s ' t h e elaboration or integrativedistributive-elaborative-facilitative memory process stated by Irwin, e t al. [ 10]. This new perspective implies the need for new approach in the study of drugs effect on memory. Psychopharmacological research must develop a suitable methodology accounting of the experimental history o f the laboratory animal. As shown by the present experiment, a pretraining injection handling can change vulnerability of memory to amnesic treatment. Several psychopharmacological studies, focusing on the consolidation aspects of the trace, provide strong evidence that psychoactive drugs such as strychnine, pemoline, amphetamine, pentylenetatrazol .... in low concentration can improve learning or reduce retrograde amnesia, despite the fact that at the convulsive level they may be disruptive [20,35]. The list of drugs found to strengthen memory processes continues to grow. Previous analysis in this laboratory demonstrated that Piracetam, a nontoxic and nonanaleptic drug protects rats against retrograde amnesia induced by sudden as well as progressive nitrogen hypoxia [31]. However, since injection handling can also have per se some psychoactive properties, it is presumed that it is able to interfere, i.e. potentiate or inhibit, the action of drugs on memory. If drugs can act on the injection produced effects as well as on the memory process itself or on their synergetic interaction, it is necessary to be
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able t o d e t e r m i n e o n w h i c h p h e n o m e n o n t h e drug specifically acts. C h a n g e s in t h e classical e x p e r i m e n t a l design are required: the addition of a group to control the placebo effect is r e q u i r e d p a r t i c u l a r l y for d r u g s w h o s e m e c h a n i s m o f a c t i o n is n o t well u n d e r s t o o d .
ACKNOWLEDGEMENT The author is indebted to her colleague S. J. Sara for her helpful comments. The author wishes also to thank E. Delwaert for his technical assistance.
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