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Effects of fencamfamine on latent inhibition
Progress in Neuro-Psychopharmacology & Biological Psychiatry 26 (2002) 1089 – 1093
Effects of fencamfamine on latent inhibition Cilene R.R. Alvesa, Roberto Deluciab, M. Teresa A. Silvaa,* a
Department of Experimental Psychology, Institute of Psychology, University of Sa˜o Paulo, Av. Professor Mello Moraes 1721, 05508-900 Sa˜o Paulo, SP, Brazil b Department of Pharmacology, Instituto de Cieˆncias Biome´dicas, University of Sa˜o Paulo, 05508-900 Sa˜o Paulo, SP, Brazil Accepted 1 May 2002
Abstract The effects of fencamfamine (FCF), an indirect dopamine (DA) agent, were investigated using the latent inhibition (LI) model of schizophrenia. In the LI procedure, rats preexposed (PE) to an unreinforced stimulus show difficulty in subsequent learning of an association in which that stimulus is predictive of an unconditioned stimulus (US). FCF (1.75, 3.5 and 7.0 mg/kg ip) yielded an inverse dose – response relationship regarding LI. At 3.5 mg/kg, LI was abolished and no effect was observed at 1.75 and 7.0 mg/kg. The effect of FCF (3.5 mg/kg) on LI was blocked by the antipsychotic risperidone (RIS; 4.0 mg/kg), a D2/5HT2 antagonist. These results confirm the similarity of the behavioral profile of FCF and amphetamine (AMPH). In addition, they provide a further validation of the LI model for psychosis, since RIS was shown to prevent a psychostimulant-induced disruption of LI. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Fencamfamine; Latent inhibition; Risperidone; Schizophrenia models
1. Introduction Fencamfamine (FCF) is a psychostimulant that has complex effects in the central nervous system. It acts primarily by blocking synaptical reuptake of dopamine (DA), but it also promotes the release of catecholamines (Seyfried, 1983; DeLucia et al., 1984, 1997). In vivo studies showed that FCF, similarly to amphetamine (AMPH) and cocaine, increases DA levels in both the striatum and the nucleus accumbens (Kuczenski et al., 1991). The behavioral effects of FCF include activation of locomotor, rearing and sniffing behavior. At high doses, it induces stereotyped behavior (Aizenstein et al., 1983). It was also shown by our laboratory that FCF can act as a positive reinforcer (Planeta et al., 1995). All these effects are similar to those of AMPH and cocaine and have generally been attributed to its indirect dopaminergic action (Seyfried, 1983; DeLucia et al., 1984, 1997). Abbreviations: AMPH, amphetamine; CS, conditioned stimulus; DA, dopamine; D, dopamine receptor; FCF, fencamfamine; LI, latent inhibition; NPE, nonpreexposed; PE, preexposed; RIS, risperidone; SAL, saline; 5HT, serotonin; SR, suppression ratio; US, unconditioned stimulus. * Corresponding author. Tel.: +55-11-3091-4444x208; fax: +55-113091-4357. E-mail address: [email protected] (M.T.A. Silva).
The similarity of behavioral and neurochemical effects between FCF and AMPH suggests that FCF might have psychotogenic properties. In human beings, AMPH at high doses can give rise to behavioral symptoms indistinguishable from those of a psychotic breakdown (Angrist, 1983). In animals, the latent inhibition (LI) model of schizophrenia has been shown to be consistently sensitive to AMPH (for example, Weiner et al., 1988; Warburton et al., 1994). The LI model seems well suited to investigate this plausible similarity between FCF and AMPH. LI occurs when an animal previously exposed to a stimulus shows impaired learning of a conditioned stimulus (CS)– unconditioned stimulus (US) association in which the CS is the preexposed (PE) stimulus. In a typical procedure, suppression of a drinking response associated with an aversive CS is measured. Animals that have not been PE to the CS suppress the response, whereas preexposed animals do not. The difference between the suppression indices of PE and NPE animals is the measure of LI. Thus, animals exposed to the CS prior to conditioning show difficulty in learning that such stimulus is predictive of an US (Lubow, 1973). Because of this feature, LI has been considered a measure of the ability to ignore irrelevant simuli (Mackintosh, 1975; Lubow et al., 1982) and has been proposed as a behavioral model of cognitive abnor-
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malities in schizophrenia (Feldon and Weiner, 1991; Gray et al., 1992; Shadach et al., 2000). The purpose of this experiment was to test the responsiveness of the LI model to FCF. A subsequent experiment investigated whether the effect of FCF on LI would be blocked by the atypical antipsychotic agent risperidone (RIS), a mixed D2/5HT2 receptor antagonist that has been shown to facilitate LI (Leysen et al., 1994; Buckley and Meltzer, 1995; Alves and Silva, 2001). As it is not known whether FCF has any serotonergic action, using a mixed DA/5-HT antagonist would be a first step in distinguishing between these possible mechanisms. Also, it has not been demonstrated that FCF has psychotogenic properties, but in view of the similarities between AMPH and FCF, an eventual antagonism would suggest, in the context of the model, that FCF may have such properties.
2. Methods 2.1. Animals Naı¨ve male Wistar rats weighing approximately 300 g at the beginning of the experiment were used. They were housed singly under a 12-h/12-h light/dark cycle (lights off at 20:00 h) under controlled temperature (21±1 °C). Animals were kept on a water restriction schedule of approximately 23 h throughout the experiment. Food was freely available in the home cage. 2.2. Drugs FCF (Merck) was dissolved in a 0.9% NaCl solution. RIS (Research Biochemicals International) was dissolved in a small amount of acetic acid and was added to a 5.5% glucose solution. Control solutions were prepared with the corresponding vehicle. All solutions were injected by intraperitoneal route in a volume of 1 ml/kg. 2.3. Apparatus Four operant conditioning chambers encased in soundattenuating isolation boxes (all equipment were from Med Associates) were used. A ventilation fan provided background noise. A removable drinking bottle was located on one wall of the box. Licks were detected by a lickometer circuit. Tone stimuli (5 s, 70 dB, 2.8 kHz) were generated by a Sonalert module. Shock stimuli (1.0 mA, 1 s) were supplied by a shock generator and scrambler. A 486 IBM personal computer was programmed to control stimulus presentation and data recording. 2.4. Experimental procedure The experimental procedure was based on Weiner et al. (1987). It consisted of four phases, conducted consistently at
the same time of the day. Half of the subjects were run on the AM period and the other half on the PM period. 2.4.1. Baseline training (Days 1– 5) Animals were individually placed in the experimental chamber and remained there until they had completed 600 licks. The subject was then returned to its home cage and was allowed to drink for 30 min. 2.4.2. Preexposure (Day 6) The bottle was removed, and each subject was placed in the experimental chamber. The PE animals received 30 presentations of a 5-s tone, with an intertrial interval (ITI) of 30 s. The nonpreexposed (NPE) animals were confined to the chamber for an identical length of time, but they did not receive the tone. 2.4.3. Conditioning (Day 7) Each animal was again placed in the experimental chamber with the water bottle removed. Five minutes later, the subject was given two tone shock pairings, 5 min apart. The tone was identical to that used in the preexposure. Each tone presentation was immediately followed by a scrambled foot shock (1 s, 1.0 mA). Animals were removed from the box 5 min after the second shock. 2.4.4. Testing (Day 8) The water bottle was replaced, and each animal was allowed to drink freely. When the rat had completed 90 licks, the tone was presented. The tone continued until additional 10 licks had been made. If the subject failed to complete these 10 licks within 300 s, the session was terminated. A suppression ratio (SR) was calculated as the time between licks 80– 90 (pre-CS period) divided by the time between licks 80– 100 (pre-CS period+ CS period). 2.5. Experimental design 2.5.1. Effect of FCF on LI In order to study the effect of FCF on LI, three experiments were conducted, testing three different doses of the drug: 1.75, 3.5 and 7.0 mg/kg. Each FCF dose group was subdivided into PE and NPE groups. Each one of these groups had a corresponding control group (saline, SAL) which received the drug vehicle. FCF or SAL were administered 15 min prior to the preexposure and conditioning phases. 2.5.2. Pretreatment with RIS In the study aimed at examining the effect of pretreatment with RIS on the 3.5 mg/kg FCF effect on LI, two experiments were conducted, testing two different doses of RIS: 2.0 and 4.0 mg/kg. Each RIS+FCF dose group was subdivided into PE and NPE animals. RIS was administered 45 min prior to FCF, and FCF was adminis-
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3. Results 3.1. Effect of FCF on LI Fig. 1 presents the mean SR of drug and vehicle groups in each of the three experiments. Panels A, B and C show the results of the 1.75, 3.5 and 7.0 mg/kg FCF doses, respectively. As expected, when a high level of stimulus preexposure is employed, there was a marked difference between PE and NPE animals in all SAL groups, that is, a control LI effect was clearly observed. At the lower and higher FCF dose (1.75 and 7.0 mg/kg) the LI effect was also present: the 22 ANOVA revealed a significant main effect of Preexposure [ F(1,26)=9.622, P<.5 and F(1,39)=4.131, P=.5, respectively] but no significant effect of Drug condition [ F(1,26)=1.129, NS and F(1,39)=0.069, NS], as well as no significant interaction between the Preexposure and Drug conditions [ F(1,26)=1.312, NS and F (1,39)=0.398, NS]. At the dose of 3.5 mg/kg, however, the difference between PE and NPE groups is abolished, that is, no LI is shown. Statistical analysis revealed a significant effect of the Preexposure [ F(1,29)=10.464, P<.05] and Drug [ F(1,29)=5.447, P<.05] conditions, as well as a significant PreexposureDrug interaction [ F(1,29)=8.881, P<.05]. 3.2. Pretreatment with RIS Fig. 2 presents the mean SR of drug and vehicle groups in the two blocking experiments. Panel A (top) shows the results of the 2.0 mg/kg RIS plus 3.5 mg/kg FCF group (RIS2.0+FCF), whereas panel B presents the SR of groups receiving 4.0 mg/kg of RIS plus 3.5 mg/kg of FCF
Fig. 1. Effect of FCF on LI. Mean and S.E. of SR for groups of rats given FCF or SAL. (A, top panel) FCF 1.75 mg/kg; (B, middle panel) FCF 3.5 mg/kg; (C, bottom panel) FCF 7 mg/kg; n for each group is shown inside the corresponding bar.
tered 15 min prior to the preexposure and conditioning phases. Each one of these groups had a corresponding control group (SAL+SAL) of animals that received an equivalent volume of SAL at the same time intervals as the experimental groups. The 2.0 mg/kg RIS dose was chosen for the first experiment because it had been shown in our laboratory to facilitate LI (Alves and Silva, 2001). Since this dose proved ineffective in antagonizing the FCF effect, it was increased by a factor of two in the second experiment. 2.6. Statistical analysis SRs were analysed by a 22 ANOVA with main factors of Preexposure and Drug.
Fig. 2. Effect of RIS on FCF disruption of latent inhibition. Mean and S.E. of SR for groups of rats given risperidone plus fencamfamine (RIS+FCF) or saline (SAL+SAL). (A, top panel) RIS 2.0+FCF 3.5 mg/kg; (B, bottom panel) RIS 4.0+FCF 3.5 mg/kg; n for each group is shown inside the corresponding bar.
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(RIS4.0+FCF). Again, a LI effect was observed in all of the SAL groups. At the dose of 2.0 mg/kg RIS, statistical analysis revealed a significant PreexposureDrug interaction [ F(1,36)= 11.570, P<.05] but no significant effect of the Preexposure [ F(1,36)=0.923, NS] or Drug [ F(1,36)=1.410, NS] conditions. Thus, at the RIS2.0+FCF condition, there was no LI effect, that is, the FCF-induced abolition of LI was not prevented by this dose of RIS. However, 4.0 mg/kg RIS blocked the FCF-induced disruption of LI. The 22 ANOVA revealed a significant main effect of Preexposure [ F(1,32)=4.671, P<.5] but no significant effect of Drug condition [ F(1,32)=0.045, NS], as well as no significant interaction between the Preexposure and Drug conditions [ F(1,32)=0.066, NS], demonstrating a LI effect.
4. Discussion 4.1. Effect of FCF on LI The present results show that FCF at 3.5 mg/kg abolished LI. In contrast, LI was present in the experiments employing doses below and above 3.5 mg/kg (1.75 and 7.0 mg/kg). The 1.75 mg/kg result suggests that this dose was below the drug efficacy threshold. On the other hand, the difference in the effects of the intermediate and high doses is consistent with other behavioral effects of FCF. For instance, an inverse FCF dose – response relationship has been shown to occur in conditioned place preference and avoidance retention (Planeta et al., 1995; DeLucia et al., 1997). A similar inverse pattern was observed on LI after AMPH administration (Weiner et al., 1987; Gray, 1996). The abolition of LI by 3.5 mg/kg FCF is probably mediated by the dopaminergic system. Several lines of evidence indicate that LI is dependent upon the mesolimbic dopaminergic system (Gray, 1996). As already mentioned, FCF blocks DA synaptical reuptake and causes increased DA levels in the nucleus accumbens and striatum. On the other hand, the higher FCF dose employed (7.0 mg/kg) did not affect LI. Since FCF at high doses produces stereotyped behavior (Aizenstein et al., 1983), it is possible that unconditioned sterotyped responses elicited by this dose interfered with behavioral processes occurring at the conditioning phase. Intermediate doses do not produce stereotyped behavior, and, thus, it was possible for the conditioning effect to take place when FCF 3.5 mg/kg was used. Another explanation for the permanence of LI under 7.0 mg/kg is also plausible. The intermediate and high doses of FCF employed could have produced a differential pattern of activation of the DA system at the nucleus accumbens and striatum, in an analogous manner to the differential effect of low and high doses of AMPH on these structures (e.g., (Groves and Rebec, 1976; Hitzemann et al., 1980; Joyce and Iversen, 1984; Porrino et al., 1984; Weiner et al., 1987). If so, it may be suggested that at the highest FCF dose used
here there was a decrease in the activation of the mesolimbic system, preserving the LI phenomenon at 7.0 mg/kg. 4.2. Pretreatment with RIS The 2.0 mg/kg RIS dose was used because it has been shown to facilitate LI (Alves and Silva, 2001). However, this dose was unable to antagonize the FCF blocking effect on LI. Since RIS activity is primarily directed to 5HT2 receptors, it is probable that this dose was ineffective in counteracting FCF-induced DA release. At the highest dose used (4.0 mg/kg), RIS blocked FCF-induced abolition of LI. This antagonism was probably due to the high D2 receptor blocking action of RIS at this dose (Leysen et al., 1994). In fact, antagonism of D2 receptors has already been shown to counteract a FCF-induced behavioral response: FCFinduced locomotor hyperactivity was prevented by metoclopramide and pimozide (Planeta et al., 1995). Serotonergic activity is also involved in LI (Moser et al., 1996; Moser et al., 2000). Thus, considering the potent anti5HT2 effect of RIS, the interference of serotonergic transmission on the observed RISFCF antagonism cannot be discarded. In spite of the action of serotonergic compounds on LI being still controversial (Moser et al., 2000), serotonergic agonists such as DOI (5-HT2A/2C) (Hitchcock et al., 1997) and RU 24969 (5-HT1B) (Cassaday et al., 1993) have been shown to disrupt LI. Also, the selective 5-HT2A antagonist MDL 100,907 reversed the blocking effect of AMPH on LI (Moser et al., 1996; Hitchcock et al.,1997). Such effect has been attributed to an indirect serotonergic mechanism leading to reduced DA transmission (Moser et al., 1996). In an analogous manner, the 5-HT2A antagonist action of RIS could be involved in reversing the FCF disruptive effect on LI, considering that FCF shares so many of the AMPH effects. A study aimed at investigating this point is being conducted in this laboratory. Since AMPH’s disruption of LI is considered as an experimental model of human positive psychotic symptoms (Gray, 1996), both the LI blocking effect of FCF and its antagonism by RIS suggest that FCF might have a psychotogenic effect in humans. FCF effect on LI may reflect either impaired loss of selective attention to the CS during preexposure or a breakdown in the ability of past environment – behavior relations to guide present responses (Hemsley, 1993). In either case, it could be predicted that FCF would model psychotic symptoms in the same way as AMPH.
5. Conclusion The administration of the indirect DA agent FCF blocked LI; this effect was prevented by the antipsychotic RIS. These results confirm the similar behavioral profile of FCF and AMPH. In addition, they provide a further validation of the LI model for antipsychotic testing, since RIS was shown to prevent a psychostimulant-induced abolition of LI.
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Acknowledgements This research was supported by FAPESP (Doctoral fellowship C.R.R.A.) and CNPq (M.T.A.S, Proc. 520591/96-8). References Aizenstein, M.L., Scavone, C., Bernardi, M.M., DeLucia, R., 1983. Comparative biochemical and behavioural effects of fencamfamine and DLamphetamine in rats. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 7, 187 – 194. Alves, C.R.R., Silva, M.T.A., 2001. Facilitation of latent inhibition by the atypical antipsychotic risperidone. Pharmacol. Biochem. Behav. 68, 503 – 506. Angrist, B., 1983. Psychoses induced by central nervous system stimulants and related drugs. In: Creese, I. (Ed.), Stimulants: Neurochemical, Behavioral And Clinical Perspectives. Raven Press, New York, pp. 1 – 30. Buckley, P.F., Meltzer, H.Y., 1995. Treatment of schizophrenia. In: Schatzberg, A.F., Nemeroff, C.B. (Eds.), Textbook of Psychopharmacology. American Psychiatric Press, London, pp. 615 – 639. Cassaday, H.J., Hodges, H., Gray, J.A., 1993. The effects of ritanserin, RU24969 and 8-OH-DPAT on latent inhibition in the rat. J. Psychopharmacol. 7, 63 – 71. Delucia, R., Bernardi, M.M., Scavone, C., Aizenstein, M.L., 1984. On the mechanism of central stimulation action of fencamfamine. Gen. Pharmacol. 15, 407 – 410. Delucia, R., Planeta, C.S., Aizenstein, M.L., Scavone, C., 1997. Repeated administration intensifies the reinforcing effect of fencamfamine in rats. Gen. Pharmacol. 29, 265 – 267. Feldon, J., Weiner, I., 1991. The latent inhibition model of schizophrenic attention disorder: haloperidol and sulpiride enhance rats’ ability to ignore irrelevant stimuli. Biol. Psychiatry 29, 635 – 646. Gray, J.A., 1996. Dopamine release in the nucleus accumbens: the perspective from aberrations of consciousness in schizophrenia. Neuropsychology 33, 1143 – 1153. Gray, N.S., Pickering, A.D., Hemsley, D.R., Dawling, S., Gray, J.A., 1992. Abolition of latent inhibition by a single 5 mg dose of D-amphetamine in man. Psychopharmacology 107, 425 – 430. Groves, P.M., Rebec, G.V., 1976. Biochemistry and behavior: some central actions of amphetamine and antipsychotic drugs. Annu. Rev. Psychol. 27, 91 – 127. Hemsley, D.R., 1993. A simple (or simplistic?) cognitive model for schizophrenia. Behav. Res. Ther. 31, 633 – 645. Hitchcock, J.M., Lister, S., Fischer, T.R., Wettstein, J.G., 1997. Disruption of latent inhibition in the rat by the 5-HT2 agonist DOI: effects of MDL 100,907, clozapine, risperidone and haloperidol. Behav. Brain Res. 88, 43 – 49.
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Hitzemann, R., Wu, J., Hom, D., Loh, H., 1980. Brain locations controlling the behavioural effects of chronic amphetamine intoxication. Psychopharmacology 72, 92 – 101. Joyce, E.M., Iversen, S.D., 1984. Dissociable effects of 6-OHDA-induced lesions of neostriatum on anorexia, locomotor activity and stereotypy: the role of behavioural competition. Psychopharmacology 83, 363 – 366. Kuczenski, R., Segal, D.S., Aizenstein, M.L., 1991. Amphetamine, cocaine and fencamfamine: relationship between locomotor and stereotypy response profiles and caudate and accumbens dopamine dynamics. J. Neurosci. 11, 2703 – 2712. Leysen, J.E., Janssen, P.M.F., Megens, A.H.P., Schotte, A., 1994. Risperidone: a novel antipsychotic with balanced serotonin – dopamine antagonism, receptor occupancy profile, and pharmacologic activity. J. Clin. Psychiatry 55, 5 – 12. Lubow, R.E., 1973. Latent inhibition. Psychol. Bull. 79, 398 – 407. Lubow, R.E., Weiner, I., Feldon, J., 1982. An animal model of attention. In: Spiegelstein, M.Y., Levy, A. (Eds.), Behavioral Models and Analysis of Drug Action. Elsevier, Amsterdam, pp. 89 – 107. Mackintosh, N.J., 1975. A theory of attention: variations in the associability of stimuli with reinforcement. Psychol. Rev. 82, 276 – 298. Moser, P.C., Moran, P.M., Frank, R.A., Kehne, J.H., 1996. Reversal of amphetamine-induced behaviours by MDL 100,907, a selective 5HT2A antagonist. Behav. Brain Res. 73, 163 – 167. Moser, P.C., Hitchcock, J.M., Lister, S., Moran, P.M., 2000. The pharmacology of latent inhibition as an animal model of schizophrenia. Brain Res. Rev. 33, 275 – 307. Planeta, C.S., Aizenstein, M.L., Delucia, R., 1995. Reinforcing properties of fencamfamine: involvement of dopamine and opioid receptors. Pharmacol. Biochem. Behav. 50, 35 – 40. Porrino, L.J., Lucignani, G., Dow-Edwards, D., Sokoloff, L., 1984. Correlation of dose-dependent effects of acute amphetamine on behaviour and local cerebral metabolism in rats. Brain Res. 307, 311 – 320. Seyfried, C.A., 1983. Dopamine uptake inhibiting versus dopamine releasing properties of fencamfamine: an in vitro study. Biochem. Pharmacol. 32, 2329 – 2331. Shadach, E., Gaisler, I., Schiller, D., Weiner, I., 2000. The latent inhibition model dissociates between clozapine, haloperidol and ritanserin. Neuropsychopharmacology 23, 151 – 161. Warburton, E.C., Joseph, M.H., Feldon, J., Weiner, I., Gray, J.A., 1994. Antagonism of amphetamine-induced disruption of latent inhibition in rats by haloperidol and ondansetron: implications for a possible antipsychotic action of ondansetron. Psychopharmacology 114, 657 – 664. Weiner, I., Izraeli-Telerant, A., Feldon, J., 1987. Latent inhibition is not affected by acute or chronic administration of 6 mg/kg DL-amphetamine. Psychopharmacology 91, 345 – 351. Weiner, I., Lubow, R.E., Feldon, J., 1988. Disruption of latent inhibition by acute administration of low doses of amphetamine. Pharmacol. Biochem. Behav. 30, 871 – 878.
Effects of fencamfamine on latent inhibition Cilene R.R. Alvesa, Roberto Deluciab, M. Teresa A. Silvaa,* a
Department of Experimental Psychology, Institute of Psychology, University of Sa˜o Paulo, Av. Professor Mello Moraes 1721, 05508-900 Sa˜o Paulo, SP, Brazil b Department of Pharmacology, Instituto de Cieˆncias Biome´dicas, University of Sa˜o Paulo, 05508-900 Sa˜o Paulo, SP, Brazil Accepted 1 May 2002
Abstract The effects of fencamfamine (FCF), an indirect dopamine (DA) agent, were investigated using the latent inhibition (LI) model of schizophrenia. In the LI procedure, rats preexposed (PE) to an unreinforced stimulus show difficulty in subsequent learning of an association in which that stimulus is predictive of an unconditioned stimulus (US). FCF (1.75, 3.5 and 7.0 mg/kg ip) yielded an inverse dose – response relationship regarding LI. At 3.5 mg/kg, LI was abolished and no effect was observed at 1.75 and 7.0 mg/kg. The effect of FCF (3.5 mg/kg) on LI was blocked by the antipsychotic risperidone (RIS; 4.0 mg/kg), a D2/5HT2 antagonist. These results confirm the similarity of the behavioral profile of FCF and amphetamine (AMPH). In addition, they provide a further validation of the LI model for psychosis, since RIS was shown to prevent a psychostimulant-induced disruption of LI. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Fencamfamine; Latent inhibition; Risperidone; Schizophrenia models
1. Introduction Fencamfamine (FCF) is a psychostimulant that has complex effects in the central nervous system. It acts primarily by blocking synaptical reuptake of dopamine (DA), but it also promotes the release of catecholamines (Seyfried, 1983; DeLucia et al., 1984, 1997). In vivo studies showed that FCF, similarly to amphetamine (AMPH) and cocaine, increases DA levels in both the striatum and the nucleus accumbens (Kuczenski et al., 1991). The behavioral effects of FCF include activation of locomotor, rearing and sniffing behavior. At high doses, it induces stereotyped behavior (Aizenstein et al., 1983). It was also shown by our laboratory that FCF can act as a positive reinforcer (Planeta et al., 1995). All these effects are similar to those of AMPH and cocaine and have generally been attributed to its indirect dopaminergic action (Seyfried, 1983; DeLucia et al., 1984, 1997). Abbreviations: AMPH, amphetamine; CS, conditioned stimulus; DA, dopamine; D, dopamine receptor; FCF, fencamfamine; LI, latent inhibition; NPE, nonpreexposed; PE, preexposed; RIS, risperidone; SAL, saline; 5HT, serotonin; SR, suppression ratio; US, unconditioned stimulus. * Corresponding author. Tel.: +55-11-3091-4444x208; fax: +55-113091-4357. E-mail address: [email protected] (M.T.A. Silva).
The similarity of behavioral and neurochemical effects between FCF and AMPH suggests that FCF might have psychotogenic properties. In human beings, AMPH at high doses can give rise to behavioral symptoms indistinguishable from those of a psychotic breakdown (Angrist, 1983). In animals, the latent inhibition (LI) model of schizophrenia has been shown to be consistently sensitive to AMPH (for example, Weiner et al., 1988; Warburton et al., 1994). The LI model seems well suited to investigate this plausible similarity between FCF and AMPH. LI occurs when an animal previously exposed to a stimulus shows impaired learning of a conditioned stimulus (CS)– unconditioned stimulus (US) association in which the CS is the preexposed (PE) stimulus. In a typical procedure, suppression of a drinking response associated with an aversive CS is measured. Animals that have not been PE to the CS suppress the response, whereas preexposed animals do not. The difference between the suppression indices of PE and NPE animals is the measure of LI. Thus, animals exposed to the CS prior to conditioning show difficulty in learning that such stimulus is predictive of an US (Lubow, 1973). Because of this feature, LI has been considered a measure of the ability to ignore irrelevant simuli (Mackintosh, 1975; Lubow et al., 1982) and has been proposed as a behavioral model of cognitive abnor-
0278-5846/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 8 - 5 8 4 6 ( 0 2 ) 0 0 2 4 1 - 5
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malities in schizophrenia (Feldon and Weiner, 1991; Gray et al., 1992; Shadach et al., 2000). The purpose of this experiment was to test the responsiveness of the LI model to FCF. A subsequent experiment investigated whether the effect of FCF on LI would be blocked by the atypical antipsychotic agent risperidone (RIS), a mixed D2/5HT2 receptor antagonist that has been shown to facilitate LI (Leysen et al., 1994; Buckley and Meltzer, 1995; Alves and Silva, 2001). As it is not known whether FCF has any serotonergic action, using a mixed DA/5-HT antagonist would be a first step in distinguishing between these possible mechanisms. Also, it has not been demonstrated that FCF has psychotogenic properties, but in view of the similarities between AMPH and FCF, an eventual antagonism would suggest, in the context of the model, that FCF may have such properties.
2. Methods 2.1. Animals Naı¨ve male Wistar rats weighing approximately 300 g at the beginning of the experiment were used. They were housed singly under a 12-h/12-h light/dark cycle (lights off at 20:00 h) under controlled temperature (21±1 °C). Animals were kept on a water restriction schedule of approximately 23 h throughout the experiment. Food was freely available in the home cage. 2.2. Drugs FCF (Merck) was dissolved in a 0.9% NaCl solution. RIS (Research Biochemicals International) was dissolved in a small amount of acetic acid and was added to a 5.5% glucose solution. Control solutions were prepared with the corresponding vehicle. All solutions were injected by intraperitoneal route in a volume of 1 ml/kg. 2.3. Apparatus Four operant conditioning chambers encased in soundattenuating isolation boxes (all equipment were from Med Associates) were used. A ventilation fan provided background noise. A removable drinking bottle was located on one wall of the box. Licks were detected by a lickometer circuit. Tone stimuli (5 s, 70 dB, 2.8 kHz) were generated by a Sonalert module. Shock stimuli (1.0 mA, 1 s) were supplied by a shock generator and scrambler. A 486 IBM personal computer was programmed to control stimulus presentation and data recording. 2.4. Experimental procedure The experimental procedure was based on Weiner et al. (1987). It consisted of four phases, conducted consistently at
the same time of the day. Half of the subjects were run on the AM period and the other half on the PM period. 2.4.1. Baseline training (Days 1– 5) Animals were individually placed in the experimental chamber and remained there until they had completed 600 licks. The subject was then returned to its home cage and was allowed to drink for 30 min. 2.4.2. Preexposure (Day 6) The bottle was removed, and each subject was placed in the experimental chamber. The PE animals received 30 presentations of a 5-s tone, with an intertrial interval (ITI) of 30 s. The nonpreexposed (NPE) animals were confined to the chamber for an identical length of time, but they did not receive the tone. 2.4.3. Conditioning (Day 7) Each animal was again placed in the experimental chamber with the water bottle removed. Five minutes later, the subject was given two tone shock pairings, 5 min apart. The tone was identical to that used in the preexposure. Each tone presentation was immediately followed by a scrambled foot shock (1 s, 1.0 mA). Animals were removed from the box 5 min after the second shock. 2.4.4. Testing (Day 8) The water bottle was replaced, and each animal was allowed to drink freely. When the rat had completed 90 licks, the tone was presented. The tone continued until additional 10 licks had been made. If the subject failed to complete these 10 licks within 300 s, the session was terminated. A suppression ratio (SR) was calculated as the time between licks 80– 90 (pre-CS period) divided by the time between licks 80– 100 (pre-CS period+ CS period). 2.5. Experimental design 2.5.1. Effect of FCF on LI In order to study the effect of FCF on LI, three experiments were conducted, testing three different doses of the drug: 1.75, 3.5 and 7.0 mg/kg. Each FCF dose group was subdivided into PE and NPE groups. Each one of these groups had a corresponding control group (saline, SAL) which received the drug vehicle. FCF or SAL were administered 15 min prior to the preexposure and conditioning phases. 2.5.2. Pretreatment with RIS In the study aimed at examining the effect of pretreatment with RIS on the 3.5 mg/kg FCF effect on LI, two experiments were conducted, testing two different doses of RIS: 2.0 and 4.0 mg/kg. Each RIS+FCF dose group was subdivided into PE and NPE animals. RIS was administered 45 min prior to FCF, and FCF was adminis-
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3. Results 3.1. Effect of FCF on LI Fig. 1 presents the mean SR of drug and vehicle groups in each of the three experiments. Panels A, B and C show the results of the 1.75, 3.5 and 7.0 mg/kg FCF doses, respectively. As expected, when a high level of stimulus preexposure is employed, there was a marked difference between PE and NPE animals in all SAL groups, that is, a control LI effect was clearly observed. At the lower and higher FCF dose (1.75 and 7.0 mg/kg) the LI effect was also present: the 22 ANOVA revealed a significant main effect of Preexposure [ F(1,26)=9.622, P<.5 and F(1,39)=4.131, P=.5, respectively] but no significant effect of Drug condition [ F(1,26)=1.129, NS and F(1,39)=0.069, NS], as well as no significant interaction between the Preexposure and Drug conditions [ F(1,26)=1.312, NS and F (1,39)=0.398, NS]. At the dose of 3.5 mg/kg, however, the difference between PE and NPE groups is abolished, that is, no LI is shown. Statistical analysis revealed a significant effect of the Preexposure [ F(1,29)=10.464, P<.05] and Drug [ F(1,29)=5.447, P<.05] conditions, as well as a significant PreexposureDrug interaction [ F(1,29)=8.881, P<.05]. 3.2. Pretreatment with RIS Fig. 2 presents the mean SR of drug and vehicle groups in the two blocking experiments. Panel A (top) shows the results of the 2.0 mg/kg RIS plus 3.5 mg/kg FCF group (RIS2.0+FCF), whereas panel B presents the SR of groups receiving 4.0 mg/kg of RIS plus 3.5 mg/kg of FCF
Fig. 1. Effect of FCF on LI. Mean and S.E. of SR for groups of rats given FCF or SAL. (A, top panel) FCF 1.75 mg/kg; (B, middle panel) FCF 3.5 mg/kg; (C, bottom panel) FCF 7 mg/kg; n for each group is shown inside the corresponding bar.
tered 15 min prior to the preexposure and conditioning phases. Each one of these groups had a corresponding control group (SAL+SAL) of animals that received an equivalent volume of SAL at the same time intervals as the experimental groups. The 2.0 mg/kg RIS dose was chosen for the first experiment because it had been shown in our laboratory to facilitate LI (Alves and Silva, 2001). Since this dose proved ineffective in antagonizing the FCF effect, it was increased by a factor of two in the second experiment. 2.6. Statistical analysis SRs were analysed by a 22 ANOVA with main factors of Preexposure and Drug.
Fig. 2. Effect of RIS on FCF disruption of latent inhibition. Mean and S.E. of SR for groups of rats given risperidone plus fencamfamine (RIS+FCF) or saline (SAL+SAL). (A, top panel) RIS 2.0+FCF 3.5 mg/kg; (B, bottom panel) RIS 4.0+FCF 3.5 mg/kg; n for each group is shown inside the corresponding bar.
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(RIS4.0+FCF). Again, a LI effect was observed in all of the SAL groups. At the dose of 2.0 mg/kg RIS, statistical analysis revealed a significant PreexposureDrug interaction [ F(1,36)= 11.570, P<.05] but no significant effect of the Preexposure [ F(1,36)=0.923, NS] or Drug [ F(1,36)=1.410, NS] conditions. Thus, at the RIS2.0+FCF condition, there was no LI effect, that is, the FCF-induced abolition of LI was not prevented by this dose of RIS. However, 4.0 mg/kg RIS blocked the FCF-induced disruption of LI. The 22 ANOVA revealed a significant main effect of Preexposure [ F(1,32)=4.671, P<.5] but no significant effect of Drug condition [ F(1,32)=0.045, NS], as well as no significant interaction between the Preexposure and Drug conditions [ F(1,32)=0.066, NS], demonstrating a LI effect.
4. Discussion 4.1. Effect of FCF on LI The present results show that FCF at 3.5 mg/kg abolished LI. In contrast, LI was present in the experiments employing doses below and above 3.5 mg/kg (1.75 and 7.0 mg/kg). The 1.75 mg/kg result suggests that this dose was below the drug efficacy threshold. On the other hand, the difference in the effects of the intermediate and high doses is consistent with other behavioral effects of FCF. For instance, an inverse FCF dose – response relationship has been shown to occur in conditioned place preference and avoidance retention (Planeta et al., 1995; DeLucia et al., 1997). A similar inverse pattern was observed on LI after AMPH administration (Weiner et al., 1987; Gray, 1996). The abolition of LI by 3.5 mg/kg FCF is probably mediated by the dopaminergic system. Several lines of evidence indicate that LI is dependent upon the mesolimbic dopaminergic system (Gray, 1996). As already mentioned, FCF blocks DA synaptical reuptake and causes increased DA levels in the nucleus accumbens and striatum. On the other hand, the higher FCF dose employed (7.0 mg/kg) did not affect LI. Since FCF at high doses produces stereotyped behavior (Aizenstein et al., 1983), it is possible that unconditioned sterotyped responses elicited by this dose interfered with behavioral processes occurring at the conditioning phase. Intermediate doses do not produce stereotyped behavior, and, thus, it was possible for the conditioning effect to take place when FCF 3.5 mg/kg was used. Another explanation for the permanence of LI under 7.0 mg/kg is also plausible. The intermediate and high doses of FCF employed could have produced a differential pattern of activation of the DA system at the nucleus accumbens and striatum, in an analogous manner to the differential effect of low and high doses of AMPH on these structures (e.g., (Groves and Rebec, 1976; Hitzemann et al., 1980; Joyce and Iversen, 1984; Porrino et al., 1984; Weiner et al., 1987). If so, it may be suggested that at the highest FCF dose used
here there was a decrease in the activation of the mesolimbic system, preserving the LI phenomenon at 7.0 mg/kg. 4.2. Pretreatment with RIS The 2.0 mg/kg RIS dose was used because it has been shown to facilitate LI (Alves and Silva, 2001). However, this dose was unable to antagonize the FCF blocking effect on LI. Since RIS activity is primarily directed to 5HT2 receptors, it is probable that this dose was ineffective in counteracting FCF-induced DA release. At the highest dose used (4.0 mg/kg), RIS blocked FCF-induced abolition of LI. This antagonism was probably due to the high D2 receptor blocking action of RIS at this dose (Leysen et al., 1994). In fact, antagonism of D2 receptors has already been shown to counteract a FCF-induced behavioral response: FCFinduced locomotor hyperactivity was prevented by metoclopramide and pimozide (Planeta et al., 1995). Serotonergic activity is also involved in LI (Moser et al., 1996; Moser et al., 2000). Thus, considering the potent anti5HT2 effect of RIS, the interference of serotonergic transmission on the observed RISFCF antagonism cannot be discarded. In spite of the action of serotonergic compounds on LI being still controversial (Moser et al., 2000), serotonergic agonists such as DOI (5-HT2A/2C) (Hitchcock et al., 1997) and RU 24969 (5-HT1B) (Cassaday et al., 1993) have been shown to disrupt LI. Also, the selective 5-HT2A antagonist MDL 100,907 reversed the blocking effect of AMPH on LI (Moser et al., 1996; Hitchcock et al.,1997). Such effect has been attributed to an indirect serotonergic mechanism leading to reduced DA transmission (Moser et al., 1996). In an analogous manner, the 5-HT2A antagonist action of RIS could be involved in reversing the FCF disruptive effect on LI, considering that FCF shares so many of the AMPH effects. A study aimed at investigating this point is being conducted in this laboratory. Since AMPH’s disruption of LI is considered as an experimental model of human positive psychotic symptoms (Gray, 1996), both the LI blocking effect of FCF and its antagonism by RIS suggest that FCF might have a psychotogenic effect in humans. FCF effect on LI may reflect either impaired loss of selective attention to the CS during preexposure or a breakdown in the ability of past environment – behavior relations to guide present responses (Hemsley, 1993). In either case, it could be predicted that FCF would model psychotic symptoms in the same way as AMPH.
5. Conclusion The administration of the indirect DA agent FCF blocked LI; this effect was prevented by the antipsychotic RIS. These results confirm the similar behavioral profile of FCF and AMPH. In addition, they provide a further validation of the LI model for antipsychotic testing, since RIS was shown to prevent a psychostimulant-induced abolition of LI.
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