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Effects of cyclazocine and scopolamine on swim-to-platform performance in rats
Brain Research 922 (2001) 229–233 www.elsevier.com / locate / bres
Research report
Effects of cyclazocine and scopolamine on swim-to-platform performance in rats a
b
G. Buckton , E.M. Zibrowski , C.H. Vanderwolf
a,c ,
*
a Department of Psychology, University of Western Ontario, London, Ontario N6 A 5 C2, Canada Department of Epidemiology and Biostatistics, University of Western Ontario, London, Ontario N6 A 5 C3, Canada c Graduate Program in Neuroscience, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6 G 2 V4, Canada b
Accepted 11 September 2001
Abstract DL-Cyclazocine (0.5–2.0 mg / kg, i.p.) produced no impairment in rats’ acquisition and retention of the behavior of swimming to a large visible platform in a water tank. However, cyclazocine produced a significant enhancement or potentiation of the impairment in swim-to-platform behavior produced by scopolamine. Since cyclazocine has previously been shown to abolish serotonin-dependent electrocortical activation (enabling it, in combination with central muscarinic blockade, to block all cortical activation), the results lend further support to the hypothesis that blockade of electrocortical activation produces dementia rather than sleep or coma as was previously believed. 2001 Published by Elsevier Science B.V.
Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Behavioral pharmacology Keywords: Cyclazocine; Dementia; Psychotomimetic; Scopolamine; Water maze
1. Introduction Generalized activation of the cerebral cortex, including the production of rhythmical slow activity in the hippocampal formation and low voltage fast activity in the neocortex, can be elicited by a cholinergic input from the basal forebrain or a serotonergic input from the brainstem. These inputs are active in relation to behavior in somewhat different ways and can be blocked selectively by antimuscarinic drugs such as atropine or scopolamine (blocking the cholinergic pathway) or parachlorophenylalanine (blocking the serotonergic pathway). If both pathways are blocked simultaneously, all cerebral cortical activation is abolished. Other pathways or transmitters which can produce cortical activation appear to act indirectly, their
*Corresponding author. Department of Psychology, University of Western Ontario, London, Ontario N6A 5C2, Canada. Tel.: 11-5196612-111, ext. 84627; fax: 11-519-6613-961. E-mail address: [email protected] (C.H. Vanderwolf).
effects mediated by the cholinergic and serotonergic inputs [4,21]. Blockade of cholinergic cortical activation by systemic injections of atropine or scopolamine produces a generalized impairment of both learned and instinctive behavior. This impairment is potentiated by prior treatment with parachlorophenylalanine, producing a behavioral syndrome that has been proposed as an experimental model of Alzheimer’s disease [25]. Since the behavioral impairment produced by a combination of parachlorophenylalanine and scopolamine is similar in many ways to the effect of surgical decortication, it may be that the drugs act primarily by a blockade of normal neocortical and hippocampal function [19]. Vanderwolf [20] reported that the psychotomimetic drug cyclazocine suppressed serotonergic (atropine-resistant) hippocampal and neocortical activation but appeared to have little effect on cholinergic (atropine-sensitive) activation. Since this is similar to the effect of parachlorophenylalanine, it may be that cyclazocine potentiates the behavioral impairment produced by central muscarinic
0006-8993 / 01 / $ – see front matter 2001 Published by Elsevier Science B.V. PII: S0006-8993( 01 )03176-6
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G. Buckton et al. / Brain Research 922 (2001) 229 – 233
blockade in much the same way that parachlorophenylalanine does. This hypothesis was tested.
2. Materials and methods
2.1. Subjects Male hooded rats (n578), weighing between 250 to 600 g, were maintained in standard laboratory cages, a 12:12 light–dark cycle, and with continuous access to food (Agway rat chow) and water. All testing was conducted during the light phase.
2.2. Drugs DL-Cyclazocine was dissolved in a few drops of glacial acetic acid, diluted in saline to yield a solution with a pH of 4.0 or slightly higher, and injected intraperitoneally in doses of 0.5–2.0 mg / kg. Larger doses were not used since they tend to produce ataxia [20]. Scopolamine hydrobromide was dissolved in saline and given subcutaneously in the nape of the neck in a dose of 5.0 mg / kg. This dose was chosen because it is sufficient to produce a substantial impairment in behavioral swim-to-platform performance and it completely blocks cortical activation dependent on cholinergic muscarinic transmission [21,23]. Equivalent volumes of the drug vehicle were also injected intraperitoneally or subcutaneously as a control procedure.
2.3. Apparatus and procedure Rats were trained on a simple swim-to-platform test in a glass tank measuring 43390345 cm deep and filled with clean water at a temperature of 208C to a depth of 25 cm. A wire mesh rat cage provided a platform measuring 21.5318.5 cm, located in the center of the tank, and raised about 1 cm above the surface of the water. Acquisition training was begun by dropping a rat in the water facing one corner of the tank and recording the time taken to climb up on the platform with all four feet. An escape time greater than 10 s was considered an error. The number of times a rat swam past the half-way mark of the long axis of the tank was also recorded. If a rat failed to climb up on the platform within 60 s, it was placed there manually. After a 10–30 s intertrial interval, in which the rat remained on the platform, a second trial was begun. After 10 such training trials a rat was removed from the tank, the rectal temperature was recorded (6.5 cm penetration), the fur was dried with paper towels and the rat was allowed to warm up for 15 min under a 100-W incandescent light bulb. An additional 10 trials of retention testing was given when the rectal temperature had returned to 36–378C. Rectal temperatures were also recorded during acquisition or retention training in rats that were performing
poorly and were, therefore, exposed to the water for long periods of time. If the body temperature fell below about 308C, the rats were dried off and warmed for 10 min after trial five. It has been shown that a core temperature below 308C has a severe detrimental effect on swim-to-platform performance [22]. Drug or control injections were given either 15 min prior to acquisition training or immediately after acquisition training. Six different groups were used in the first experiment: group I, saline vehicle before acquisition, scopolamine (5.0 mg / kg) before retention testing; group II, cyclazocine (0.5 mg / kg) before acquisition, scopolamine (5.0 mg / kg) before retention testing; group III, cyclazocine (1.0 mg / kg) before acquisition, scopolamine (5.0 mg / kg) before retention testing; group IV, cyclazocine (2.0 mg / kg) before acquisition, scopolamine (5.0 mg / kg) before retention testing; group V, cyclazocine (2.0 mg / kg) before acquisition, saline vehicle before retention testing; group VI, saline vehicle before acquisition, saline vehicle before retention testing. In a second experiment, scopolamine (5.0 mg / kg) was administered alone before acquisition (group VII) or in combination with cyclazocine (2.0 mg / kg) before acquisition (group VIII). In these two groups, no further injections were made after acquisition.
2.4. Statistical tests All data are expressed as means6standard errors of the means. Data were analyzed using one-way analyses of variance (ANOVAs). Significant main effects were further analyzed by means of Fisher’s protected least significant difference tests (PLSD), which corrects probabilities in proportion to the number of comparisons made. Additionally, t-tests and the Pearson product moment correlation coefficient were used in the data analysis. Statistical tests were conducted by means of the Statview computer software program [9].
3. Results Fig. 1 shows that the administration of cyclazocine failed to produce a significant increase in the errors made during acquisition [F (5, 52)51.12; P50.36]. However, a highly significant main effect of scopolamine was found in the experimental groups during retention testing [F (5, 52)57.90; P,0.0001). Fisher’s PLSD revealed that the vehicle plus scopolamine group (group I, Fig. 1) made significantly fewer errors than groups II–IV (P,0.05 in two comparisons; P,0.001 in one comparison) which received cyclazocine plus scopolamine. The post hoc tests also revealed that the three groups (groups II–IV) receiving cyclazocine plus scopolamine combinations made significantly more errors than the rats administered
G. Buckton et al. / Brain Research 922 (2001) 229 – 233
Fig. 1. Effect of cyclazocine and scopolamine on errors and passes in swim-to-platform performance. Different groups (n510 or n58) are identified by Roman numerals. (A) Injection given 15 min prior to acquisition; (B) injection given 15 min prior to retention; SAL, saline vehicle; (C) cyclazocine in doses ranging from 0.5 to 2.0 mg / kg; SCOP, scopolamine 5.0 mg / kg. The saline / saline control group scored 010 (no errors) on the retention test. The following groups differed with respect to errors as indicated using Fisher’s protected least significant difference tests. Acquisition: no significant differences between the groups. Retention: I vs. II, P,0.05; I vs. III, P,0.05; I vs. IV, P,0.001; II vs. V, P,0.01; II vs. VI, P,0.01; III vs. V, P,0.01; III vs. VI, P,0.001; IV vs. V, P,0.001; IV vs. VI, P,0.001. The following groups differed with respect to passes as indicated. Acquisition: no significant differences between the groups. Retention: I vs. III, P,0.05; I vs. IV, P,0.05; II vs. V, P,0.05; II vs. VI, P,0.01; III vs. V, P,0.01; III vs. VI, P,0.001; IV vs. V, P,0.01; IV vs. VI, P,0.01.
cyclazocine alone (group V) or the saline vehicle only (group VI; P,0.01 or better in all cases). Analysis of the data on swimming past the platform without climbing up (i.e., passes, Fig. 1) revealed that cyclazocine failed to exert a significant effect upon the number of passes during acquisition [F (5, 52)51.60; P50.18]. Retention testing after scopolamine had been given to groups I–IV (Fig. 2) produced a clearer effect [F (5,52)55.08; P50.0007]. Rats tested with cyclazocine
231
Fig. 2. Effect of scopolamine and scopolamine and cyclazocine together on acquisition and retention in swim-to-platform performance. Group VII (n510) received scopolamine (5.0 mg / kg) 15 min prior to acquisition; group VIII (n510) received scopolamine (5.0 mg / kg) plus cyclazocine (2.0 mg / kg) 15 min prior to acquisition. In both groups, retention testing occurred 15 min after the end of acquisition training. Errors: *, differs from group VII retention performance, P,0.005, t-test. The retention scores differed significantly from the acquisition scores in group VII (paired t-test, P,0.02). Other differences are not significant. Passes: *, differs from group VII acquisition performance, P,0.005, t-test. The retention scores differed significantly from the acquisition scores in group VII (paired t-test, P,0.02). Other differences are not significant.
plus scopolamine made more than twice as many passes as rats tested with saline plus scopolamine. Rats tested with cyclazocine plus saline did not differ significantly from rats treated with only the saline vehicle (Fig. 1). When scopolamine (5.0 mg / kg, group VII) or cyclazocine (2.0 mg / kg) plus scopolamine (5.0 mg / kg, group VIII) were given prior to acquisition, performance was very poor, both in terms of errors and passes (Fig. 2). During acquisition there were no significant differences between the two groups in errors but group VIII made fewer passes than group VII owing to a tendency to swim persistently while trapped in a corner. During retention testing, the scopolamine only group (group VII) improved
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significantly in terms of both errors and passes but the cyclazocine plus scopolamine group (group VIII) performed at much the same level during retention as during acquisition. There was a close correlation between errors and passes in the data shown in Fig. 1. The Pearson correlation was r50.78 (n558) during acquisition and r50.90 (n558) during retention (P,0.0001 in both cases). A high correlation between errors and passes was also observed in group VII which received scopolamine prior to acquisition [during acquisition, r50.88 (n510), P,.003; during retention, r50.99 (n510), P,0.0001]. However, in group VIII which received a combination of cyclazocine and scopolamine prior to acquisition the correlation between errors and passes was not significant [during acquisition, r50.14 (n510); P50.71; during retention r50.51 (n510); P50.13]. As already noted, group VIII, unlike all other groups, tended to swim persistently while trapped with the nose in a corner of the tank.
4. Discussion Previous studies of the effects of low doses of cyclazocine have not revealed a consistent impairment of behavior. Doses of 0.125–2.0 mg / kg improved the performance of rats working on a lever press shock avoidance task [11] but doses of 1.0 mg / kg or more depressed the performance of rats pressing a lever to obtain a food reward on a fixed ratio schedule [10]. In the present study, DL-cyclazocine alone, in doses of 0.5–2.0 mg / kg had no consistent effect on either errors or passes during acquisition of swim-to-platform behavior. However, when experimental animals were given scopolamine, the effect of the prior treatments with cyclazocine became apparent. Both errors and passes increased to 2–3 times control levels during retention testing in the combined drug groups. Groups III and IV, in comparison to group I, deteriorated significantly after scopolamine treatment. In contrast, performance in rats tested for retention following cyclazocine alone or scopolamine alone, given after acquisition, did not differ significantly from performance in control rats tested with the drug vehicle alone. A combination of cyclazocine and scopolamine given prior to acquisition training produced an impairment of performance roughly equivalent to the effect of scopolamine alone. However, during an additional 10 training trials the scopolamine only rats improved significantly while the scopolamine plus cyclazocine rats showed no sign of improvement. In this case also, cyclazocine further increased the behavioral impairment produced by scopolamine. Thus, low doses of cyclazocine alone do not produce severe impairments of behavior, but the drug does appear to potentiate the behavioral impairment produced by scopolamine.
Since the foregoing results are quite similar to those previously obtained with scopolamine and parachlorophenylalanine [19,27] it might be supposed that cyclazocine interferes with serotonergic function in some way. Large doses of cyclazocine result in elevated levels of 5-hydroxyindoleacetic acid in several brain areas, suggesting that the drug may enhance the release of serotonin [5]. The fact that metergoline blocks both the diuretic and some behavioral effects of cyclazocine has further suggested that cyclazocine may be a serotonin agonist [6,10]. However, since cyclazocine also reduces brain levels of dopamine and norepinephrine and increases the brain levels of metabolites of these neurotransmitters [5,11] it is probable that cyclazocine also stimulates the release of catecholamines. This effect, together with the release of serotonin [18,26] may be responsible for the pronounced behavioral stimulation produced by cyclazocine in doses of 4–32 mg / kg [11]. There is much evidence supporting the view that cyclazocine interacts with opioid receptors including the kappa, mu and sigma receptors [2,7,13,15,16]. These effects have been suggested as the basis of the well-known psychotomimetic effect of cyclazocine [8–14] and may also be involved in the behavioral impairments studied here. Finally, there is also strong evidence that cyclazocine acts as an antagonist at N-methyl-D-aspartate (NMDA) receptors [2,3,17]. Since Cain et al. [1] has demonstrated that other NMDA receptor antagonists can increase the behavioral impairment produced by scopolamine, it may be that NMDA antagonism is responsible for some of the behavioral effects studied here. Since cyclazocine has multiple actions on different neurotransmitter systems it is not possible, at this time, to determine the exact mechanism by which it suppresses atropine-resistant cortical activation [20] and produces impairments in behavior. Our finding that cyclazocine increases or potentiates the behavioral impairment produced by scopolamine, however, lends further support to the view that blockade of electrographic activation of the cerebral cortex produces a dementia-like condition rather than sleep or coma as had been previously thought [25]. It should be noted, however, that the ability to increase or potentiate the behavioral effects of antimuscarinic drugs is not restricted to agents which block atropine-resistant (serotonin-dependent) cortical activation. Large lesions of: (a) the hippocampal formation and posterior neocortex; (b) the medial thalamus; and (c) 6-hydroxydopamine-induced loss of ascending noradrenergic projections all increase the behavioral impairment produced by scopolamine even though none of these treatments blocks cortical activation [24]. Although we do not understand the basis of the enhanced behavioral impairment produced by combining scopolamine with a variety of other drugs or brain lesions it is clear that some treatments that impair brain function do not produce additive or potentiative effects when they
G. Buckton et al. / Brain Research 922 (2001) 229 – 233
are combined. Parachlorophenylalanine, for example, potentiates the effect of scopolamine but does not increase the behavioral effect of large lesions of the medial thalamus or of the hippocampal formation and posterior neocortex [27]. Finally, it should be mentioned that scopolamine given after acquisition (group I) has a far smaller effect on swim-to-platform behavior than scopolamine given prior to acquisition (group VII). This effect has been discussed previously [19,27].
[12]
[13]
[14]
[15]
Acknowledgements [16]
The experiments reported here were supported by an operating grant to C.H.V. from the Natural Sciences and Engineering Research Council of Canada.
[17]
References [18] [1] D.P. Cain, K. Ighanian, F. Boon, Individual and combined manipulation of muscarinic, NMDA, and benzodiazopine receptor activity in the water maze task: implication for a rat model of Alzheimer dementia, Behav. Brain Res. 111 (2000) 125–137. [2] J. Church, D. Lodge, Cyclazocine and pentazocine as N-methylaspartate antagonists on cat and rat spinal neurons in vivo, J. Pharmacol. Exp. Ther. 253 (1990) 636–645. [3] J. Church, J.D. Millar, M.G. Jones, D. Lodge, NMDA receptor antagonist effects of the stereoisomers of b-cyclazocine in rats in vivo and in vitro, Eur. J. Pharmacol. 192 (1991) 337–342. [4] H.C. Dringenberg, C.H. Vanderwolf, Involvement of direct and indirect pathways in electrocorticographic activation, Neurosci. Biobehav. Rev. 22 (1998) 243–257. [5] M.M. Gavend, F. Serre, M.R. Gavend, C. Ragoucy, P. Caron, Effects of acute administration of cyclazocine on the metabolism of biogenic amines in different regions of rat brain, Psychopharmacology 75 (1981) 79–83. [6] M. Gavend, M. Mallaret, F. Caron, G. Bargatti, Antagonism of metergoline on the diuretic effect of cyclazocine and U-50488 drugs with a kappa agonist activity, Biomed. Pharmacother. 47 (1993) 337–344. [7] P.E. Gilbert, W.R. Martin, The effects of morphine and nalorphinelike drugs on the nondependent, morphine-dependent and cyclazocine-dependent chronic spinal dog, J. Pharmacol. Exp. Ther. 198 (1976) 66. [8] C.A. Haertzen, Subjective effects of narcotic antagonists cyclazocine and nalorphine on the Addiction Research Centre Inventory (ARCI), Psychopharmacologia 18 (1970) 366–377. [9] K.A. Haycock, J. Roth, J. Gagnon, W.F. Finzer, C. Soper, Abacus Concepts, Statview, Abacus Concepts, Berkeley, CA, 1992. [10] J.W. Henck, D.J. Mokler, R.L. Commissaris, R.H. Rech, Cyclazocine disruption of operant behavior is antagonized by naloxone and metergoline, Pharmacol. Biochem. Behav. 18 (1983) 41–45. [11] S.G. Holtzman, R.E. Jewett, Stimulation of behavior in the rat by
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Research report
Effects of cyclazocine and scopolamine on swim-to-platform performance in rats a
b
G. Buckton , E.M. Zibrowski , C.H. Vanderwolf
a,c ,
*
a Department of Psychology, University of Western Ontario, London, Ontario N6 A 5 C2, Canada Department of Epidemiology and Biostatistics, University of Western Ontario, London, Ontario N6 A 5 C3, Canada c Graduate Program in Neuroscience, Siebens-Drake Research Institute, University of Western Ontario, London, Ontario N6 G 2 V4, Canada b
Accepted 11 September 2001
Abstract DL-Cyclazocine (0.5–2.0 mg / kg, i.p.) produced no impairment in rats’ acquisition and retention of the behavior of swimming to a large visible platform in a water tank. However, cyclazocine produced a significant enhancement or potentiation of the impairment in swim-to-platform behavior produced by scopolamine. Since cyclazocine has previously been shown to abolish serotonin-dependent electrocortical activation (enabling it, in combination with central muscarinic blockade, to block all cortical activation), the results lend further support to the hypothesis that blockade of electrocortical activation produces dementia rather than sleep or coma as was previously believed. 2001 Published by Elsevier Science B.V.
Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Behavioral pharmacology Keywords: Cyclazocine; Dementia; Psychotomimetic; Scopolamine; Water maze
1. Introduction Generalized activation of the cerebral cortex, including the production of rhythmical slow activity in the hippocampal formation and low voltage fast activity in the neocortex, can be elicited by a cholinergic input from the basal forebrain or a serotonergic input from the brainstem. These inputs are active in relation to behavior in somewhat different ways and can be blocked selectively by antimuscarinic drugs such as atropine or scopolamine (blocking the cholinergic pathway) or parachlorophenylalanine (blocking the serotonergic pathway). If both pathways are blocked simultaneously, all cerebral cortical activation is abolished. Other pathways or transmitters which can produce cortical activation appear to act indirectly, their
*Corresponding author. Department of Psychology, University of Western Ontario, London, Ontario N6A 5C2, Canada. Tel.: 11-5196612-111, ext. 84627; fax: 11-519-6613-961. E-mail address: [email protected] (C.H. Vanderwolf).
effects mediated by the cholinergic and serotonergic inputs [4,21]. Blockade of cholinergic cortical activation by systemic injections of atropine or scopolamine produces a generalized impairment of both learned and instinctive behavior. This impairment is potentiated by prior treatment with parachlorophenylalanine, producing a behavioral syndrome that has been proposed as an experimental model of Alzheimer’s disease [25]. Since the behavioral impairment produced by a combination of parachlorophenylalanine and scopolamine is similar in many ways to the effect of surgical decortication, it may be that the drugs act primarily by a blockade of normal neocortical and hippocampal function [19]. Vanderwolf [20] reported that the psychotomimetic drug cyclazocine suppressed serotonergic (atropine-resistant) hippocampal and neocortical activation but appeared to have little effect on cholinergic (atropine-sensitive) activation. Since this is similar to the effect of parachlorophenylalanine, it may be that cyclazocine potentiates the behavioral impairment produced by central muscarinic
0006-8993 / 01 / $ – see front matter 2001 Published by Elsevier Science B.V. PII: S0006-8993( 01 )03176-6
230
G. Buckton et al. / Brain Research 922 (2001) 229 – 233
blockade in much the same way that parachlorophenylalanine does. This hypothesis was tested.
2. Materials and methods
2.1. Subjects Male hooded rats (n578), weighing between 250 to 600 g, were maintained in standard laboratory cages, a 12:12 light–dark cycle, and with continuous access to food (Agway rat chow) and water. All testing was conducted during the light phase.
2.2. Drugs DL-Cyclazocine was dissolved in a few drops of glacial acetic acid, diluted in saline to yield a solution with a pH of 4.0 or slightly higher, and injected intraperitoneally in doses of 0.5–2.0 mg / kg. Larger doses were not used since they tend to produce ataxia [20]. Scopolamine hydrobromide was dissolved in saline and given subcutaneously in the nape of the neck in a dose of 5.0 mg / kg. This dose was chosen because it is sufficient to produce a substantial impairment in behavioral swim-to-platform performance and it completely blocks cortical activation dependent on cholinergic muscarinic transmission [21,23]. Equivalent volumes of the drug vehicle were also injected intraperitoneally or subcutaneously as a control procedure.
2.3. Apparatus and procedure Rats were trained on a simple swim-to-platform test in a glass tank measuring 43390345 cm deep and filled with clean water at a temperature of 208C to a depth of 25 cm. A wire mesh rat cage provided a platform measuring 21.5318.5 cm, located in the center of the tank, and raised about 1 cm above the surface of the water. Acquisition training was begun by dropping a rat in the water facing one corner of the tank and recording the time taken to climb up on the platform with all four feet. An escape time greater than 10 s was considered an error. The number of times a rat swam past the half-way mark of the long axis of the tank was also recorded. If a rat failed to climb up on the platform within 60 s, it was placed there manually. After a 10–30 s intertrial interval, in which the rat remained on the platform, a second trial was begun. After 10 such training trials a rat was removed from the tank, the rectal temperature was recorded (6.5 cm penetration), the fur was dried with paper towels and the rat was allowed to warm up for 15 min under a 100-W incandescent light bulb. An additional 10 trials of retention testing was given when the rectal temperature had returned to 36–378C. Rectal temperatures were also recorded during acquisition or retention training in rats that were performing
poorly and were, therefore, exposed to the water for long periods of time. If the body temperature fell below about 308C, the rats were dried off and warmed for 10 min after trial five. It has been shown that a core temperature below 308C has a severe detrimental effect on swim-to-platform performance [22]. Drug or control injections were given either 15 min prior to acquisition training or immediately after acquisition training. Six different groups were used in the first experiment: group I, saline vehicle before acquisition, scopolamine (5.0 mg / kg) before retention testing; group II, cyclazocine (0.5 mg / kg) before acquisition, scopolamine (5.0 mg / kg) before retention testing; group III, cyclazocine (1.0 mg / kg) before acquisition, scopolamine (5.0 mg / kg) before retention testing; group IV, cyclazocine (2.0 mg / kg) before acquisition, scopolamine (5.0 mg / kg) before retention testing; group V, cyclazocine (2.0 mg / kg) before acquisition, saline vehicle before retention testing; group VI, saline vehicle before acquisition, saline vehicle before retention testing. In a second experiment, scopolamine (5.0 mg / kg) was administered alone before acquisition (group VII) or in combination with cyclazocine (2.0 mg / kg) before acquisition (group VIII). In these two groups, no further injections were made after acquisition.
2.4. Statistical tests All data are expressed as means6standard errors of the means. Data were analyzed using one-way analyses of variance (ANOVAs). Significant main effects were further analyzed by means of Fisher’s protected least significant difference tests (PLSD), which corrects probabilities in proportion to the number of comparisons made. Additionally, t-tests and the Pearson product moment correlation coefficient were used in the data analysis. Statistical tests were conducted by means of the Statview computer software program [9].
3. Results Fig. 1 shows that the administration of cyclazocine failed to produce a significant increase in the errors made during acquisition [F (5, 52)51.12; P50.36]. However, a highly significant main effect of scopolamine was found in the experimental groups during retention testing [F (5, 52)57.90; P,0.0001). Fisher’s PLSD revealed that the vehicle plus scopolamine group (group I, Fig. 1) made significantly fewer errors than groups II–IV (P,0.05 in two comparisons; P,0.001 in one comparison) which received cyclazocine plus scopolamine. The post hoc tests also revealed that the three groups (groups II–IV) receiving cyclazocine plus scopolamine combinations made significantly more errors than the rats administered
G. Buckton et al. / Brain Research 922 (2001) 229 – 233
Fig. 1. Effect of cyclazocine and scopolamine on errors and passes in swim-to-platform performance. Different groups (n510 or n58) are identified by Roman numerals. (A) Injection given 15 min prior to acquisition; (B) injection given 15 min prior to retention; SAL, saline vehicle; (C) cyclazocine in doses ranging from 0.5 to 2.0 mg / kg; SCOP, scopolamine 5.0 mg / kg. The saline / saline control group scored 010 (no errors) on the retention test. The following groups differed with respect to errors as indicated using Fisher’s protected least significant difference tests. Acquisition: no significant differences between the groups. Retention: I vs. II, P,0.05; I vs. III, P,0.05; I vs. IV, P,0.001; II vs. V, P,0.01; II vs. VI, P,0.01; III vs. V, P,0.01; III vs. VI, P,0.001; IV vs. V, P,0.001; IV vs. VI, P,0.001. The following groups differed with respect to passes as indicated. Acquisition: no significant differences between the groups. Retention: I vs. III, P,0.05; I vs. IV, P,0.05; II vs. V, P,0.05; II vs. VI, P,0.01; III vs. V, P,0.01; III vs. VI, P,0.001; IV vs. V, P,0.01; IV vs. VI, P,0.01.
cyclazocine alone (group V) or the saline vehicle only (group VI; P,0.01 or better in all cases). Analysis of the data on swimming past the platform without climbing up (i.e., passes, Fig. 1) revealed that cyclazocine failed to exert a significant effect upon the number of passes during acquisition [F (5, 52)51.60; P50.18]. Retention testing after scopolamine had been given to groups I–IV (Fig. 2) produced a clearer effect [F (5,52)55.08; P50.0007]. Rats tested with cyclazocine
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Fig. 2. Effect of scopolamine and scopolamine and cyclazocine together on acquisition and retention in swim-to-platform performance. Group VII (n510) received scopolamine (5.0 mg / kg) 15 min prior to acquisition; group VIII (n510) received scopolamine (5.0 mg / kg) plus cyclazocine (2.0 mg / kg) 15 min prior to acquisition. In both groups, retention testing occurred 15 min after the end of acquisition training. Errors: *, differs from group VII retention performance, P,0.005, t-test. The retention scores differed significantly from the acquisition scores in group VII (paired t-test, P,0.02). Other differences are not significant. Passes: *, differs from group VII acquisition performance, P,0.005, t-test. The retention scores differed significantly from the acquisition scores in group VII (paired t-test, P,0.02). Other differences are not significant.
plus scopolamine made more than twice as many passes as rats tested with saline plus scopolamine. Rats tested with cyclazocine plus saline did not differ significantly from rats treated with only the saline vehicle (Fig. 1). When scopolamine (5.0 mg / kg, group VII) or cyclazocine (2.0 mg / kg) plus scopolamine (5.0 mg / kg, group VIII) were given prior to acquisition, performance was very poor, both in terms of errors and passes (Fig. 2). During acquisition there were no significant differences between the two groups in errors but group VIII made fewer passes than group VII owing to a tendency to swim persistently while trapped in a corner. During retention testing, the scopolamine only group (group VII) improved
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significantly in terms of both errors and passes but the cyclazocine plus scopolamine group (group VIII) performed at much the same level during retention as during acquisition. There was a close correlation between errors and passes in the data shown in Fig. 1. The Pearson correlation was r50.78 (n558) during acquisition and r50.90 (n558) during retention (P,0.0001 in both cases). A high correlation between errors and passes was also observed in group VII which received scopolamine prior to acquisition [during acquisition, r50.88 (n510), P,.003; during retention, r50.99 (n510), P,0.0001]. However, in group VIII which received a combination of cyclazocine and scopolamine prior to acquisition the correlation between errors and passes was not significant [during acquisition, r50.14 (n510); P50.71; during retention r50.51 (n510); P50.13]. As already noted, group VIII, unlike all other groups, tended to swim persistently while trapped with the nose in a corner of the tank.
4. Discussion Previous studies of the effects of low doses of cyclazocine have not revealed a consistent impairment of behavior. Doses of 0.125–2.0 mg / kg improved the performance of rats working on a lever press shock avoidance task [11] but doses of 1.0 mg / kg or more depressed the performance of rats pressing a lever to obtain a food reward on a fixed ratio schedule [10]. In the present study, DL-cyclazocine alone, in doses of 0.5–2.0 mg / kg had no consistent effect on either errors or passes during acquisition of swim-to-platform behavior. However, when experimental animals were given scopolamine, the effect of the prior treatments with cyclazocine became apparent. Both errors and passes increased to 2–3 times control levels during retention testing in the combined drug groups. Groups III and IV, in comparison to group I, deteriorated significantly after scopolamine treatment. In contrast, performance in rats tested for retention following cyclazocine alone or scopolamine alone, given after acquisition, did not differ significantly from performance in control rats tested with the drug vehicle alone. A combination of cyclazocine and scopolamine given prior to acquisition training produced an impairment of performance roughly equivalent to the effect of scopolamine alone. However, during an additional 10 training trials the scopolamine only rats improved significantly while the scopolamine plus cyclazocine rats showed no sign of improvement. In this case also, cyclazocine further increased the behavioral impairment produced by scopolamine. Thus, low doses of cyclazocine alone do not produce severe impairments of behavior, but the drug does appear to potentiate the behavioral impairment produced by scopolamine.
Since the foregoing results are quite similar to those previously obtained with scopolamine and parachlorophenylalanine [19,27] it might be supposed that cyclazocine interferes with serotonergic function in some way. Large doses of cyclazocine result in elevated levels of 5-hydroxyindoleacetic acid in several brain areas, suggesting that the drug may enhance the release of serotonin [5]. The fact that metergoline blocks both the diuretic and some behavioral effects of cyclazocine has further suggested that cyclazocine may be a serotonin agonist [6,10]. However, since cyclazocine also reduces brain levels of dopamine and norepinephrine and increases the brain levels of metabolites of these neurotransmitters [5,11] it is probable that cyclazocine also stimulates the release of catecholamines. This effect, together with the release of serotonin [18,26] may be responsible for the pronounced behavioral stimulation produced by cyclazocine in doses of 4–32 mg / kg [11]. There is much evidence supporting the view that cyclazocine interacts with opioid receptors including the kappa, mu and sigma receptors [2,7,13,15,16]. These effects have been suggested as the basis of the well-known psychotomimetic effect of cyclazocine [8–14] and may also be involved in the behavioral impairments studied here. Finally, there is also strong evidence that cyclazocine acts as an antagonist at N-methyl-D-aspartate (NMDA) receptors [2,3,17]. Since Cain et al. [1] has demonstrated that other NMDA receptor antagonists can increase the behavioral impairment produced by scopolamine, it may be that NMDA antagonism is responsible for some of the behavioral effects studied here. Since cyclazocine has multiple actions on different neurotransmitter systems it is not possible, at this time, to determine the exact mechanism by which it suppresses atropine-resistant cortical activation [20] and produces impairments in behavior. Our finding that cyclazocine increases or potentiates the behavioral impairment produced by scopolamine, however, lends further support to the view that blockade of electrographic activation of the cerebral cortex produces a dementia-like condition rather than sleep or coma as had been previously thought [25]. It should be noted, however, that the ability to increase or potentiate the behavioral effects of antimuscarinic drugs is not restricted to agents which block atropine-resistant (serotonin-dependent) cortical activation. Large lesions of: (a) the hippocampal formation and posterior neocortex; (b) the medial thalamus; and (c) 6-hydroxydopamine-induced loss of ascending noradrenergic projections all increase the behavioral impairment produced by scopolamine even though none of these treatments blocks cortical activation [24]. Although we do not understand the basis of the enhanced behavioral impairment produced by combining scopolamine with a variety of other drugs or brain lesions it is clear that some treatments that impair brain function do not produce additive or potentiative effects when they
G. Buckton et al. / Brain Research 922 (2001) 229 – 233
are combined. Parachlorophenylalanine, for example, potentiates the effect of scopolamine but does not increase the behavioral effect of large lesions of the medial thalamus or of the hippocampal formation and posterior neocortex [27]. Finally, it should be mentioned that scopolamine given after acquisition (group I) has a far smaller effect on swim-to-platform behavior than scopolamine given prior to acquisition (group VII). This effect has been discussed previously [19,27].
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Acknowledgements [16]
The experiments reported here were supported by an operating grant to C.H.V. from the Natural Sciences and Engineering Research Council of Canada.
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