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Scopolamine induces recovery of shuttle box avoidance behavior after frontal cortex ablation
ELSEVIER
Behavioural Brain Research 62 (1994) 135-141
BEHAVIOURAL BRAIN RESEARCH
Research Report
Scopolamine induces recovery of shuttle box avoidance behavior after frontal cortex ablation Manuel A. Castro-Alamancos*, Jos6 Borrell Cajal bTstitute [CS1C], Avenida Dr. Arce 37. 28002-Madrid, Spain Received 8 October 1993: revised 20 January 1994: accepted 24 January 1994
Abstract
The learning and reversal of shuttle box active avoidance behavior in animals with a bilateral frontal cortex ablation was investigated during and after scopolamine or pilocarpine treatment. Scopolamine facilitated the performance of the avoidance task in normal animals and in those with frontal cortex lesions and also increased the number of intertrial responses, while pilocarpine increased the deleterious effects of the lesions. Furthermore, in the absence of scopolamine, the animals previously treated with the drug showed that its beneficial effects persisted while the number of intertrial responses were no longer increased. The results indicate that the beneficial effects of scopolamine treatment on active avoidance behavior are independent from the effects observed on intertrial activity since only the former are observed after drug withdrawal. Therefore, scopolamine treatment seems to induce a long lasting recovery process in frontal cortex ablated animals. Key words. Scopolamine; Functional recovery; Brain damage; Shuttle box; Frontal cortex
1. Introduction Frontal cortex lesions lead to a variety of deficits measured in different behavioral tasks [7]. Active avoidance learning has been reported to be impaired to some extent after lesions of the frontal cortex [1,6,3]. Also, the reversal of previously learned behaviors is clearly impaired after frontal cortex ablation [6], and this has been shown to be the case in the reversal training of a shuttle box avoidance task [3]. The response inhibition deficit of these animals may account for the impairment observed in reversal tasks, where the animals must shift their response from a previously reinforced stimulus to a new reinforced one [7]. Cholinergic transmission blockers at the muscarinic receptor, such as scopolamine, have been shown to impair the performance of a wide variety of working memory tasks [ 13,15]. Nevertheless, these same agents have been shown to facilitate performance in a reference memory task, such as shuttle box avoidance behavior [8]. Moreover, the effects of these cholinergic agonists and antagonists have been widely studied after lesions of the major
* Corresponding author. Present address: Department of Neuroscience, Box G-M+ Brown University, Providence, RI 02912, USA. Fax: ( 1) (401 ) 863-2537. E-mail: [email protected] 0166-4328;94 $7.00 © 1994 Elsevier Scicnce B.V. All rights reserved SSIH 01 6 6 - 4 3 2 8 ( 9 4 ) 0 0 0 2 7 - D
cholinergic projection to the cortex; the nucleus basalis of Meynert (nbM). The results have consistently shown that cholinomimetics, probably acting at the frontal cortex [5 ], greatly attenuate the behavioral deficits induced by n b M lesions [ 11 ]. Also, the facilitatory effect of scopolamine on shuttle box avoidance performance has been shown to dissipate after n b M lesions [8]. While the effects of cholinergic agonists and antagonists have been widely studied after n b M lesions, this is not the case for frontal cortex lesions. Thus, the possible beneficial effect of scopolamine after frontal cortex lesions in the performance of shuttle box avoidance behavior has not been studied. Knowledge of the effects of cholinergic agonists and antagonists upon the behavioral consequences of frontal cortex ablation would further serve to advance in our understanding of the processes which are affected after frontal cortex injury. Thus, if cholinergic agonists or antagonists have a clear beneficial effect on the behavioral performance of frontal cortex injured animals, this would indicate the involvement of the cholinergic system in the behavioral consequences of a frontal cortex ablation. We used a shuttle box avoidance task due to several reasons. First, some aspects of this task are clearly and consistently impaired after a frontal cortex ablation. Second, it has been described that shuttle box avoidance is
136
M . A . Castro-Alamancos and J. Borrell : Beha vioural Brain Research 62 ;1994
facilitated by scopolamine in normal animals indicating the possibility that it may also have a beneficial effect in injured animals. Finally, this task allows us to differentiate between the facilitatory effects of the drugs, when they are present, from a long lasting recovery process which would be reflected in an adequate performance of the animals when the drug is no longer present. Thus, we decided to investigate if scopolamine would have a beneficial effect in the performance of this task after a cortical ablation and if this effect would persist after retrieval of the treatment.
/ 3 5 - i 4 ,'
Forty-two male Wistar rats (250-300 g b.wt.) were used in this study. The rats were housed in a controlled environment (temperature: 22-24 °C; humidity: 50 + 10~0) with a non-reversed cycle. Food and water were freely available.
was placed and 10 s of escape interval in which a 0.2 mA footshock was delivered through the grid floor mad the light remained on. The animals were submitted to 70 trials of this sequence on one day (,learning procedure). The following da~ the animals were submitted to reversal training. During reversal training the sequence of trials and the number of trials was the same. but the light did not appear in the compartment where the animal was but in the other compartment. Therefore. in order to avoid or escape fiom the aversive stimulus the animals would havc to enter the compartment where the light ~xas (reversal procedure). During this reversal training the animals received the samc drug treatments as the previous day, One week latter all the animals were submitted to the ~ame real sequence in the shuttle box. with the onl~ difference thal the first 35 trials were normal training trials (avoidance or escape by leaving the compartment were the light appeared I and the following 35 were reversal training trials lavoidancc or escape bv entering the compartment were the light appeared). In this day none of the animals were administered anx of the drug treatments (traimng-reversal procedure J,
2.2. Surgery: lesions of the frontal cortex
2.4. Histology
Rats were anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic instrument (Stoelting 51200). The skull was drilled bilaterally 2-5 mm anterior to bregma and 1-4 lateral to the midline. The dura and pia were carefully incised and removed and the underlying cortex was removed with delicate suction. The lesions were then filled with gelatin hemostatic sponge [3 ].
After the completion of the behavioral studies the animals were decapitated and the brain was rapidly removed and left in paraformaldehyde (4~o) for several days. Sections were cut with a cryostat ~md then stained with Cresyl violet, In order to compare the lesion sites and sizes. stained sections were placed in a microscope and the lesions were drawn and subsequently compared between animals. Comparisons were performed qualitatively by directly comparing lesions from individual animals of each injured group.
2. Materials and methods
2. I. Animab
2.3. Behavioral procedure Half of the animals in this study were submitted to bilateral ablation of the frontal cortex while the other half were submitted to sham surgery or to no surgery. Seven days after surgery six groups were formed. Three of the six groups had been submitted to surgery and received lesions of the frontal cortex. The other three groups were noninjured control animals. The three injured groups formed were injected (i.p.) either with saline 10 min prior to performance (FR group; n l0), scopolamine -1 mg/kg - 2 0 min prior to performance (FR + Scopolamine group; n 10) or pilocarpine - 1 mg/kg - 1 0 min prior to performance (FR + Pilocarpine group; n5). The three control groups were either injected saline (control group; n7), scopolamine (Scopolamine group; n5) or pilocarpine (Pilocarpine group; n5) at the same doses and times. The animals were then introduced in standard shuttle boxes. All the animals were trained with a trial sequence consisting of 30 s of intertrial interval, 10 s of avoidance interval signaled by a light in the ceiling of the compartment in which the animal
3. Results ANOVA revealed that the number of" avoidance responses per blocks of ten trials in the learning procedure (Fs.3~,=8.54: P<0.0001J and reversal procedure (Fs.36 = 11.13: P<0.0001J, or per blocks of five trials in the training ((F5.36=1t.26: P<0.0001) plus reversal (F5.36=8.07: P<0.0001) procedure differed between groups (Fig. 1 and Fig. 2). A multiple comparison t-test showed the significant differences between the specific groups as described below. Also. ANOVA revealed that the total number of intertrial responses m the learning procedure (Fs.36 = 18.9: P<0.0001) and in the reversal procedure (Fs.36=13.5: P<0.0001), but not in the training-reversal procedure differed between groups (Fig. 3). The significant differences between the groups are described below.
M.A. ('astro-Alamancos and J, Borrell / Behavioural Brain Research 62 (1994) 135-141
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Fig. ]. Mean number of avoidance responses ( + S.E.M.) per blocks of ten trials (top and middle panels) or five trials (bottom panel) of animals with a lesion in the frontal cortex (FR lesion group), control animals (Control group), animals with a lesion in the frontal cortex and administered scopolamine (FR + Scopolamine group) and animals with a lesion in the frontal cortex and administered pilocarpine (FR + Pilocarpine group). The animals were submitted to a learning session 7 days after surgery (A), to reversal training the following day (B) and to normal training plus reversal training in the task a week latter (C). in the traning plus reversal session (C) the blocks are formed of five trials, and the animals in the injected groups were not administered any drug.
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Fig. 2. Mean number of avoidance responses t -+ S.E.M.) per blocks of ten trials (A and B ) or five trials [C) of control animals (Control group t. control animals administered scopolamine (Scopolamine group) and control animals administered pilocarpine (Pilocarpine group). The animals were submitted to a learning session seven days after surgery (A), to reversal training the following day (B) and to normal training plus reversal training m the task a week latter (C). In the training plus reversal session ( C / t h e blocks are formed of five trials, and the animals in the injected groups were not administered any drug. The animals in the Control group of this figure are the same as in Fig. 1.
,14. ,4. Castro-A lamancos and J. Borrell Behavioural Brain Research 62 (1994) I35-141
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Fig. 3. Mean of the total number of intertrial responses ( + S.E.M.) of control animals (Control group), animals with a lesion in the frontal cortex (FR lesion group), animals with a lesion in the frontal cortex and administered scopolamine (FR + Scopolamine group), animals with a lesion in the frontal cortex and administered pilocarpine (FR + Pilocarpine group), control animals administered scopolamine (Scopolamine group) and control animals administered pilocarpine (Pilocarpine group). The number of intertrial responses correspond to the learning session 7 days after surgery (A), to the reversal training session of the following day (B) and to the normal training plus reversal training session in the task a week latter (C). In the training plus reversal session (C)the animals were not administered any drug. (* P < 0.0001, ** P < 0.02 significant difference with respect to the other groups).
3.1. E[:fect o/frontal cortex ablation The animals with frontal cortex lesions (FR lesion group) showed an impairment in the performance of
the learning procedure (P=0.03), reversal procedure (P= 0.005) and in the training (P<0.0001) plus reversal (P< 0.0001) procedure as compared to the control group (Fig. 1). Also, the animals in the FR lesion group did not
140
M . A . C a s t r o - A l a m a n c o s a n d ,I. Borrell : Behavioural Brain R e s e a r c h 62 1994 ~ 135--; ¢ :
differ significantly from the control group in the number of intertrial responses for any of the behavioral procedures (Fig. 3).
3.2. Effect of scopolamine The animals in the FR + Scopolamine group did not differ significantly in the number of avoidance responses from the control group in any of the behavioral procedures (Fig. 1). Thus, the FR + Scopolamine group showed a significantly larger number of avoidance responses than the FR lesion group in the learning procedure (P--0.04), reversal procedure (P = 0.005) and training ( P < 0.0001) plus reversal (P = 0.0001) procedure (Fig. 1). The Scopolamine group showed a clear tendency, in both the learning and the reversal procedures, to perform a larger number of avoidance responses than the control group (Fig. 2A and B). This difference was significant in the reversal procedure (P = 0.02). Finally, both the FR + Scopolamine group (P < 0.0001 ) and the Scopolamine group (P < 0.02) showed a significantly larger number of intertrial responses than the rest of the groups in the learning and in the reversal procedures (Fig. 3A and B), but not in the training-reversal procedure (Fig. 3C). Also, the FR + Scopolamine group showed a significantly larger number ofintertrial responses than the Scopolamine group (P < 0,01) in both procedures (Fig. 3A and B).
3.3. Effect of pilocarpine The animals in the FR + Pilocarpine group showed a significantly lower number of avoidance responses than the rest of the groups (P<0.005), except the Pilocarpine group, during the learning procedure (Fig. IA and Fig. 2A). In the reversal procedure (Fig. 1B and Fig. 2B) this difference was significant compared to all the groups (P < 0.005). During the training-reversal procedure (Fig. 1C and Fig. 2C) the FR + Pilocarpine group showed a lower number of avoidance responses than all the groups except the FR lesion group. The Pilocarpine group differed significantly from the control group during the learning (P=0.001) and reversal (P=0.01) procedures (Fig. 2A and B), but not in the training-reversal procedure (Fig. 2C). The effect of pilocarpine was not mediated by the number of intertrial responses since both groups treated with pitocarpine did not differ significantly from the control groups in the number of intertrial responses (Fig. 3).
3.4. Histology The histological qualitative analysis showed very small variability in lesion site and size between the injured animals, which indicated that the site and size of the frontal
cortex ablation did not differ between the groups. This was also evident due to the fact that animals from one injured group could be matched in lesion size bv animals m the other injured group. Also. comparisons between the individual lesions and the animals behavior indicated that the size of the lesions could not account for the behaviora performance of the animals since there was no correlation between lesion size and behavioral performancc. The lesions were similar to those previous/~ described {31 and included all the frontal cortex sparing most ~1" 1he caudal orbital cortex and the frontal pole of the motor cortex.
4. Discussion
The results of the present study show that the impairmerit in learning and in the reversal of an active avoidance task induced by the bilateral ablation of the frontal cortex is eliminated by scopolamine. 111 the presence of scopolamine facilitation of behavioral performance is accompanied by an increase in the number of intertrial responses the animals perform. Moreover, the animals which have been treated with scopolamine show a long lasting beneficial recovery process after the drug is no longer present. This process is not accompanied by an increase 111 the number of intertrial responses of" the recovered animals. Thus, after frontal cortex ablation the presence of scopolamine facilitates shuttle box avoidance behavior, increases locomotor activity (i.e. as indicated by the number of intertrial responses) and induces a long lasting recovery process This long lasting recover3 process is reflected in the adequate performance of the animals in the absence of the drug, which seems to be independent of the occurrence of an increase in locomotor activity. The results also show that in the presence of pilocarpme the animals with a frontal cortex ablation show a potenuation of their deficits. but this effect is not long lasting since in the absence of pilocarpine these animals show a deficit similar to frontal cortex ablated animals. As we have shown previously, the bilateral ablation of" the frontal cortex induces deficits in learnin~ and especially in the reversal of a shuttle box avoidance task [3], The effects of scopolamine on the facilitation of behavioral performance in the presence of the drug could be interpreted as the consequence of an increase in the acuvit~ of the animals, which masks the deficit induced by the lesion. However this does not seem to be the case since in the absence of the drug the facilitation persists while the increase m activity is no longer present indicating the independence between both effects. Thus, the long-term effects observed in the scopolamine injected animals when evaluated in the absence of the drug arc especially interesting; frontal cortex ablated animals treated with scopolamine
M.A. Castro-Alamancos and J. Borrell / Behavioural Brain Research 62 (1994) 135-141
show an adequate performance in the task a week after scopolamine administration, and do not show an increase in the number of intertrial responses. Since animals with frontal cortex ablation and previously treated with scopolamine do not show an increase in the number of intertrial responses in the training-reversal procedure, the performance level can not be attributed to an increase in activity. Thus, the recovery observed in this performance period is probably the consequence of a long-term recovery process induced by scopolamine. Other drugs (i.e. amphetamine) which increase the performance levels of non injured animals, have been shown to produce the same effect after frontal cortex lesions [14]. These drugs produce an effect which is maintained after the drug is discontinued. The mechanism through which the drug induces this effect is unknown, but a likely hypothesis is that different neurotransmitter systems which are depressed or potentiated after the lesion become regulated by these drugs. Thus, the effects of amphetamine have been attributed to the stimulation of functionally depressed cathecolamine systems [4]. Therefore, scopolamine may regulate the effects of acetylcholine in the basal forebrain or in other subsystems. It has been shown that the cholinergic input to the cortex is essential in controlling the response properties of cortical cells to environmental stimuli [12]. This mechanism may be essential in avoidance learning and scopolamine may influence it since this drug has been shown to regulate cortical activity. Moreover, scopolamine does not exert its activating effect through a target in the frontal cortex, since it has this effect in frontal cortex injured animals. This activating effect is even potentiated in injured animals. The sites at which scopolamine acts to produce its beneficial effect seem to be the nbM projection sites [8]. Recent work has also shown that scopolamine induces a beneficial behavioral effect after traumatic brain injury if administered immediately after the trauma [ 10]. However, this effect which has been attributed to the blockade of excessive neuronal excitation caused by the lesion [9], is most probably not mediating the effects observed in the present study since in the present work scopolamine was administered for the first time one week after the lesion. Nevertheless, both results agree in the beneficial consequences of muscarinic receptor blockade after frontal cortex damage. Finally, since reorganizational processes taking place in the cerebral cortex after cortical damage have been shown to mediate functional recovery [2], it is possible that similar processes are induced by cholinergic blockade at the muscarinic receptor in cortical areas adjacent to the lesion and thus account for the beneficial effects observed in behavioral performance after frontal cortex ablation.
141
In conclusion, frontal cortex ablation produces a deficit in the learning and reversal of an active avoidance task. Performance in this behavioral task and locomotor activity (as indicated by the number of intertrial responses) is facilitated by scopolamine treatment. Furthermore, animals previously treated with scopolamine show, in the absence of the drug, an adequate performance in the behavioral task which is not followed by an increase in locomotor activity. This indicates that a long-lasting recovery process may have occurred in these animals.
References [1] Brito, G.N.O. and Brito, L.S.O., Septohippocampal system and the prelimbic sector of frontal cortex: a neuropsychological battery analysis in the rat, Behav. Brain Res., 36 (1990) 127-146. [2] Castro-Alamancos, M.A., Garcia-Segura, L.M. and Borrell, J., Transfer of function to a specific area of the cortex after induced recovery from brain damage, Eur. J. Neurosci., 4 (1992) 853-863. [3] Castro-Alamancos, M.A. and Borrell, J., Facilitation and recovery of shuttle box avoidance behavior after frontal cortex lesions is induced by a contingent electrical stimulation in the ventral tegmental nucleus, Behav. Brain Res.~ 50 (1992) 69-76. [4] Feeney, D.M., Gonzalcz, A. and Law, W.A., Amphetamine, haloperidol and experience interact to affect rate of recovery after motor cortex injury, Science, 217 (1982) 855-857. [5] Haroutunian, V.. Mantin, R. and Kanof, P.D., Frontal cortex as the site of action of physostigmine in nbM-lesioncd rats, Physiol. Behav., 47 (1990) 203-206. [6] Irle, E. and Markowithsch, H.J., Differential effects of prefrontal lesions and combined prefrontal and limbic lesions on subsequent learning performance in the cat, Behav. Neurosci., 98 (1984) 884897. [7] Kolb, B., Functions of the frontal cortex of the rat: a comparative review, Brain Res. Rev., 8 (1984) 65-98. [8] Lo Conte, G., Bartolini, L., Casamenti, F., Marconcini-Pepeu, I. and Pepeu, G., Lesions of cholinergic forebrain nuclei: changes in avoidance behavior and scopolamine actions, Pharmacol. Biochem. Behav., 17 (1982) 933-937. [9] Lyeth, B.G. and Hayes, R.L., Cholinergic and opioid mediation of traumatic brain injury, J. Neurotrauma, 9 (1992) 463-474. [ 10] Lyeth, B.G., Ray, M., Hamm, R.J., Schnabel, J., Saady, J.J., Poklis, A., Jenkins, L.W., Gudeman, S.K. and Hayes, R.L., Postinjury scopolamine administration in experimental traumatic brain injury, Bra#7 Res., 569 (1992) 281-286. [11] Murray, C.L. and Fibiger, H.C., Learning and memory deficits after lesions of the nucleus basalis magnocellularis: reversal by physostigmine, Neuroscience, 14 (1985) 1025-1032. [12] Rigdon G.C. and Pirsch, J.H., Nucleus basalis involvement in conditioned neuronal responses in the rat frontal cortex, J. Neurosci., 6 (1986) 2535-2542. [13] Spangler, E.L., Rigby, P. and Ingrain, D.K., Scopolamine impairs learning performance of rats in a 14-unit T-maze, Pharmacol. Biochem. Behav., 25 (1986)673-679. [14] Sutton, R.L., Hovda, D.A. and Feeney, D.M., Amphetamine acceleratcs recovery of locomotor function following bilateral frontal cortex ablation in cats, Behav. Neurosci., 103 (1989) 837-841. [15] Viscardi, A.P. and Heise, G.A., Effects of scopolamine on components of delayed response performance in the rat, Pharmacol. Biochem. Behav., 25 (1986) 633-639.
Behavioural Brain Research 62 (1994) 135-141
BEHAVIOURAL BRAIN RESEARCH
Research Report
Scopolamine induces recovery of shuttle box avoidance behavior after frontal cortex ablation Manuel A. Castro-Alamancos*, Jos6 Borrell Cajal bTstitute [CS1C], Avenida Dr. Arce 37. 28002-Madrid, Spain Received 8 October 1993: revised 20 January 1994: accepted 24 January 1994
Abstract
The learning and reversal of shuttle box active avoidance behavior in animals with a bilateral frontal cortex ablation was investigated during and after scopolamine or pilocarpine treatment. Scopolamine facilitated the performance of the avoidance task in normal animals and in those with frontal cortex lesions and also increased the number of intertrial responses, while pilocarpine increased the deleterious effects of the lesions. Furthermore, in the absence of scopolamine, the animals previously treated with the drug showed that its beneficial effects persisted while the number of intertrial responses were no longer increased. The results indicate that the beneficial effects of scopolamine treatment on active avoidance behavior are independent from the effects observed on intertrial activity since only the former are observed after drug withdrawal. Therefore, scopolamine treatment seems to induce a long lasting recovery process in frontal cortex ablated animals. Key words. Scopolamine; Functional recovery; Brain damage; Shuttle box; Frontal cortex
1. Introduction Frontal cortex lesions lead to a variety of deficits measured in different behavioral tasks [7]. Active avoidance learning has been reported to be impaired to some extent after lesions of the frontal cortex [1,6,3]. Also, the reversal of previously learned behaviors is clearly impaired after frontal cortex ablation [6], and this has been shown to be the case in the reversal training of a shuttle box avoidance task [3]. The response inhibition deficit of these animals may account for the impairment observed in reversal tasks, where the animals must shift their response from a previously reinforced stimulus to a new reinforced one [7]. Cholinergic transmission blockers at the muscarinic receptor, such as scopolamine, have been shown to impair the performance of a wide variety of working memory tasks [ 13,15]. Nevertheless, these same agents have been shown to facilitate performance in a reference memory task, such as shuttle box avoidance behavior [8]. Moreover, the effects of these cholinergic agonists and antagonists have been widely studied after lesions of the major
* Corresponding author. Present address: Department of Neuroscience, Box G-M+ Brown University, Providence, RI 02912, USA. Fax: ( 1) (401 ) 863-2537. E-mail: [email protected] 0166-4328;94 $7.00 © 1994 Elsevier Scicnce B.V. All rights reserved SSIH 01 6 6 - 4 3 2 8 ( 9 4 ) 0 0 0 2 7 - D
cholinergic projection to the cortex; the nucleus basalis of Meynert (nbM). The results have consistently shown that cholinomimetics, probably acting at the frontal cortex [5 ], greatly attenuate the behavioral deficits induced by n b M lesions [ 11 ]. Also, the facilitatory effect of scopolamine on shuttle box avoidance performance has been shown to dissipate after n b M lesions [8]. While the effects of cholinergic agonists and antagonists have been widely studied after n b M lesions, this is not the case for frontal cortex lesions. Thus, the possible beneficial effect of scopolamine after frontal cortex lesions in the performance of shuttle box avoidance behavior has not been studied. Knowledge of the effects of cholinergic agonists and antagonists upon the behavioral consequences of frontal cortex ablation would further serve to advance in our understanding of the processes which are affected after frontal cortex injury. Thus, if cholinergic agonists or antagonists have a clear beneficial effect on the behavioral performance of frontal cortex injured animals, this would indicate the involvement of the cholinergic system in the behavioral consequences of a frontal cortex ablation. We used a shuttle box avoidance task due to several reasons. First, some aspects of this task are clearly and consistently impaired after a frontal cortex ablation. Second, it has been described that shuttle box avoidance is
136
M . A . Castro-Alamancos and J. Borrell : Beha vioural Brain Research 62 ;1994
facilitated by scopolamine in normal animals indicating the possibility that it may also have a beneficial effect in injured animals. Finally, this task allows us to differentiate between the facilitatory effects of the drugs, when they are present, from a long lasting recovery process which would be reflected in an adequate performance of the animals when the drug is no longer present. Thus, we decided to investigate if scopolamine would have a beneficial effect in the performance of this task after a cortical ablation and if this effect would persist after retrieval of the treatment.
/ 3 5 - i 4 ,'
Forty-two male Wistar rats (250-300 g b.wt.) were used in this study. The rats were housed in a controlled environment (temperature: 22-24 °C; humidity: 50 + 10~0) with a non-reversed cycle. Food and water were freely available.
was placed and 10 s of escape interval in which a 0.2 mA footshock was delivered through the grid floor mad the light remained on. The animals were submitted to 70 trials of this sequence on one day (,learning procedure). The following da~ the animals were submitted to reversal training. During reversal training the sequence of trials and the number of trials was the same. but the light did not appear in the compartment where the animal was but in the other compartment. Therefore. in order to avoid or escape fiom the aversive stimulus the animals would havc to enter the compartment where the light ~xas (reversal procedure). During this reversal training the animals received the samc drug treatments as the previous day, One week latter all the animals were submitted to the ~ame real sequence in the shuttle box. with the onl~ difference thal the first 35 trials were normal training trials (avoidance or escape by leaving the compartment were the light appeared I and the following 35 were reversal training trials lavoidancc or escape bv entering the compartment were the light appeared). In this day none of the animals were administered anx of the drug treatments (traimng-reversal procedure J,
2.2. Surgery: lesions of the frontal cortex
2.4. Histology
Rats were anesthetized with sodium pentobarbital (50 mg/kg) and placed in a stereotaxic instrument (Stoelting 51200). The skull was drilled bilaterally 2-5 mm anterior to bregma and 1-4 lateral to the midline. The dura and pia were carefully incised and removed and the underlying cortex was removed with delicate suction. The lesions were then filled with gelatin hemostatic sponge [3 ].
After the completion of the behavioral studies the animals were decapitated and the brain was rapidly removed and left in paraformaldehyde (4~o) for several days. Sections were cut with a cryostat ~md then stained with Cresyl violet, In order to compare the lesion sites and sizes. stained sections were placed in a microscope and the lesions were drawn and subsequently compared between animals. Comparisons were performed qualitatively by directly comparing lesions from individual animals of each injured group.
2. Materials and methods
2. I. Animab
2.3. Behavioral procedure Half of the animals in this study were submitted to bilateral ablation of the frontal cortex while the other half were submitted to sham surgery or to no surgery. Seven days after surgery six groups were formed. Three of the six groups had been submitted to surgery and received lesions of the frontal cortex. The other three groups were noninjured control animals. The three injured groups formed were injected (i.p.) either with saline 10 min prior to performance (FR group; n l0), scopolamine -1 mg/kg - 2 0 min prior to performance (FR + Scopolamine group; n 10) or pilocarpine - 1 mg/kg - 1 0 min prior to performance (FR + Pilocarpine group; n5). The three control groups were either injected saline (control group; n7), scopolamine (Scopolamine group; n5) or pilocarpine (Pilocarpine group; n5) at the same doses and times. The animals were then introduced in standard shuttle boxes. All the animals were trained with a trial sequence consisting of 30 s of intertrial interval, 10 s of avoidance interval signaled by a light in the ceiling of the compartment in which the animal
3. Results ANOVA revealed that the number of" avoidance responses per blocks of ten trials in the learning procedure (Fs.3~,=8.54: P<0.0001J and reversal procedure (Fs.36 = 11.13: P<0.0001J, or per blocks of five trials in the training ((F5.36=1t.26: P<0.0001) plus reversal (F5.36=8.07: P<0.0001) procedure differed between groups (Fig. 1 and Fig. 2). A multiple comparison t-test showed the significant differences between the specific groups as described below. Also. ANOVA revealed that the total number of intertrial responses m the learning procedure (Fs.36 = 18.9: P<0.0001) and in the reversal procedure (Fs.36=13.5: P<0.0001), but not in the training-reversal procedure differed between groups (Fig. 3). The significant differences between the groups are described below.
M.A. ('astro-Alamancos and J, Borrell / Behavioural Brain Research 62 (1994) 135-141
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Fig. ]. Mean number of avoidance responses ( + S.E.M.) per blocks of ten trials (top and middle panels) or five trials (bottom panel) of animals with a lesion in the frontal cortex (FR lesion group), control animals (Control group), animals with a lesion in the frontal cortex and administered scopolamine (FR + Scopolamine group) and animals with a lesion in the frontal cortex and administered pilocarpine (FR + Pilocarpine group). The animals were submitted to a learning session 7 days after surgery (A), to reversal training the following day (B) and to normal training plus reversal training in the task a week latter (C). in the traning plus reversal session (C) the blocks are formed of five trials, and the animals in the injected groups were not administered any drug.
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,14. ,4. Castro-A lamancos and J. Borrell Behavioural Brain Research 62 (1994) I35-141
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Fig. 3. Mean of the total number of intertrial responses ( + S.E.M.) of control animals (Control group), animals with a lesion in the frontal cortex (FR lesion group), animals with a lesion in the frontal cortex and administered scopolamine (FR + Scopolamine group), animals with a lesion in the frontal cortex and administered pilocarpine (FR + Pilocarpine group), control animals administered scopolamine (Scopolamine group) and control animals administered pilocarpine (Pilocarpine group). The number of intertrial responses correspond to the learning session 7 days after surgery (A), to the reversal training session of the following day (B) and to the normal training plus reversal training session in the task a week latter (C). In the training plus reversal session (C)the animals were not administered any drug. (* P < 0.0001, ** P < 0.02 significant difference with respect to the other groups).
3.1. E[:fect o/frontal cortex ablation The animals with frontal cortex lesions (FR lesion group) showed an impairment in the performance of
the learning procedure (P=0.03), reversal procedure (P= 0.005) and in the training (P<0.0001) plus reversal (P< 0.0001) procedure as compared to the control group (Fig. 1). Also, the animals in the FR lesion group did not
140
M . A . C a s t r o - A l a m a n c o s a n d ,I. Borrell : Behavioural Brain R e s e a r c h 62 1994 ~ 135--; ¢ :
differ significantly from the control group in the number of intertrial responses for any of the behavioral procedures (Fig. 3).
3.2. Effect of scopolamine The animals in the FR + Scopolamine group did not differ significantly in the number of avoidance responses from the control group in any of the behavioral procedures (Fig. 1). Thus, the FR + Scopolamine group showed a significantly larger number of avoidance responses than the FR lesion group in the learning procedure (P--0.04), reversal procedure (P = 0.005) and training ( P < 0.0001) plus reversal (P = 0.0001) procedure (Fig. 1). The Scopolamine group showed a clear tendency, in both the learning and the reversal procedures, to perform a larger number of avoidance responses than the control group (Fig. 2A and B). This difference was significant in the reversal procedure (P = 0.02). Finally, both the FR + Scopolamine group (P < 0.0001 ) and the Scopolamine group (P < 0.02) showed a significantly larger number of intertrial responses than the rest of the groups in the learning and in the reversal procedures (Fig. 3A and B), but not in the training-reversal procedure (Fig. 3C). Also, the FR + Scopolamine group showed a significantly larger number ofintertrial responses than the Scopolamine group (P < 0,01) in both procedures (Fig. 3A and B).
3.3. Effect of pilocarpine The animals in the FR + Pilocarpine group showed a significantly lower number of avoidance responses than the rest of the groups (P<0.005), except the Pilocarpine group, during the learning procedure (Fig. IA and Fig. 2A). In the reversal procedure (Fig. 1B and Fig. 2B) this difference was significant compared to all the groups (P < 0.005). During the training-reversal procedure (Fig. 1C and Fig. 2C) the FR + Pilocarpine group showed a lower number of avoidance responses than all the groups except the FR lesion group. The Pilocarpine group differed significantly from the control group during the learning (P=0.001) and reversal (P=0.01) procedures (Fig. 2A and B), but not in the training-reversal procedure (Fig. 2C). The effect of pilocarpine was not mediated by the number of intertrial responses since both groups treated with pitocarpine did not differ significantly from the control groups in the number of intertrial responses (Fig. 3).
3.4. Histology The histological qualitative analysis showed very small variability in lesion site and size between the injured animals, which indicated that the site and size of the frontal
cortex ablation did not differ between the groups. This was also evident due to the fact that animals from one injured group could be matched in lesion size bv animals m the other injured group. Also. comparisons between the individual lesions and the animals behavior indicated that the size of the lesions could not account for the behaviora performance of the animals since there was no correlation between lesion size and behavioral performancc. The lesions were similar to those previous/~ described {31 and included all the frontal cortex sparing most ~1" 1he caudal orbital cortex and the frontal pole of the motor cortex.
4. Discussion
The results of the present study show that the impairmerit in learning and in the reversal of an active avoidance task induced by the bilateral ablation of the frontal cortex is eliminated by scopolamine. 111 the presence of scopolamine facilitation of behavioral performance is accompanied by an increase in the number of intertrial responses the animals perform. Moreover, the animals which have been treated with scopolamine show a long lasting beneficial recovery process after the drug is no longer present. This process is not accompanied by an increase 111 the number of intertrial responses of" the recovered animals. Thus, after frontal cortex ablation the presence of scopolamine facilitates shuttle box avoidance behavior, increases locomotor activity (i.e. as indicated by the number of intertrial responses) and induces a long lasting recovery process This long lasting recover3 process is reflected in the adequate performance of the animals in the absence of the drug, which seems to be independent of the occurrence of an increase in locomotor activity. The results also show that in the presence of pilocarpme the animals with a frontal cortex ablation show a potenuation of their deficits. but this effect is not long lasting since in the absence of pilocarpine these animals show a deficit similar to frontal cortex ablated animals. As we have shown previously, the bilateral ablation of" the frontal cortex induces deficits in learnin~ and especially in the reversal of a shuttle box avoidance task [3], The effects of scopolamine on the facilitation of behavioral performance in the presence of the drug could be interpreted as the consequence of an increase in the acuvit~ of the animals, which masks the deficit induced by the lesion. However this does not seem to be the case since in the absence of the drug the facilitation persists while the increase m activity is no longer present indicating the independence between both effects. Thus, the long-term effects observed in the scopolamine injected animals when evaluated in the absence of the drug arc especially interesting; frontal cortex ablated animals treated with scopolamine
M.A. Castro-Alamancos and J. Borrell / Behavioural Brain Research 62 (1994) 135-141
show an adequate performance in the task a week after scopolamine administration, and do not show an increase in the number of intertrial responses. Since animals with frontal cortex ablation and previously treated with scopolamine do not show an increase in the number of intertrial responses in the training-reversal procedure, the performance level can not be attributed to an increase in activity. Thus, the recovery observed in this performance period is probably the consequence of a long-term recovery process induced by scopolamine. Other drugs (i.e. amphetamine) which increase the performance levels of non injured animals, have been shown to produce the same effect after frontal cortex lesions [14]. These drugs produce an effect which is maintained after the drug is discontinued. The mechanism through which the drug induces this effect is unknown, but a likely hypothesis is that different neurotransmitter systems which are depressed or potentiated after the lesion become regulated by these drugs. Thus, the effects of amphetamine have been attributed to the stimulation of functionally depressed cathecolamine systems [4]. Therefore, scopolamine may regulate the effects of acetylcholine in the basal forebrain or in other subsystems. It has been shown that the cholinergic input to the cortex is essential in controlling the response properties of cortical cells to environmental stimuli [12]. This mechanism may be essential in avoidance learning and scopolamine may influence it since this drug has been shown to regulate cortical activity. Moreover, scopolamine does not exert its activating effect through a target in the frontal cortex, since it has this effect in frontal cortex injured animals. This activating effect is even potentiated in injured animals. The sites at which scopolamine acts to produce its beneficial effect seem to be the nbM projection sites [8]. Recent work has also shown that scopolamine induces a beneficial behavioral effect after traumatic brain injury if administered immediately after the trauma [ 10]. However, this effect which has been attributed to the blockade of excessive neuronal excitation caused by the lesion [9], is most probably not mediating the effects observed in the present study since in the present work scopolamine was administered for the first time one week after the lesion. Nevertheless, both results agree in the beneficial consequences of muscarinic receptor blockade after frontal cortex damage. Finally, since reorganizational processes taking place in the cerebral cortex after cortical damage have been shown to mediate functional recovery [2], it is possible that similar processes are induced by cholinergic blockade at the muscarinic receptor in cortical areas adjacent to the lesion and thus account for the beneficial effects observed in behavioral performance after frontal cortex ablation.
141
In conclusion, frontal cortex ablation produces a deficit in the learning and reversal of an active avoidance task. Performance in this behavioral task and locomotor activity (as indicated by the number of intertrial responses) is facilitated by scopolamine treatment. Furthermore, animals previously treated with scopolamine show, in the absence of the drug, an adequate performance in the behavioral task which is not followed by an increase in locomotor activity. This indicates that a long-lasting recovery process may have occurred in these animals.
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