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Acute high dose of chlorpyrifos alters performance of rats in the elevated plus-maze and the elevated T-maze
NeuroToxicology 30 (2009) 1025–1029
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NeuroToxicology
Acute high dose of chlorpyrifos alters performance of rats in the elevated plus-maze and the elevated T-maze G.A. Lo´pez-Crespo a, P. Flores b, F. Sa´nchez-Santed b, M.C. Sa´nchez-Amate b,* a b
Departamento de Psicologı´a Ba´sica y Metodologı´a, Universidad de Murcia, Spain Departamento de Neurociencia y Ciencias de la Salud, Universidad de Almerı´a, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 February 2009 Accepted 16 July 2009 Available online 24 July 2009
Chlorpyrifos (CPF) is a broad spectrum organophosphate (OP) pesticide widely used in agriculture, industry and household. Several animal studies indicate emotional disturbances after CPF exposure, although the results are sometimes puzzling. Thus, both anxiolytic and anxiogenic effects of CPF have been reported in different animal models of anxiety [Sa´nchez-Amate MC, Flores P, Sa´nchez-Santed F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285–92; Sa´nchez˜ adas F, Flores P, Sa´nchez-Santed F. Chlorpyrifos shares stimulus properties with Amate MC, Da´vila E, Can pentilenetetrazol as evaluated by and operant drug discrimination task. Neurotoxicology 2002;23:795– 803; Lo´pez-Crespo G, Carvajal F, Flores P, Sa´nchez-Santed F, Sa´nchez-Amate MC. Time-course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurotoxicology 2007;28:541– 7]. On the other hand, other behavioural effects of CPF are time-dependent [Lo´pez-Crespo G, Carvajal F, Flores P, Sa´nchez-Santed F, Sa´nchez-Amate MC. Time-course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurotoxicology 2007;28:541–7], raising the question that the effects of CPF could be task and post-administration time dependent. To test this hypothesis, three groups of rats were treated with a single high dose of CPF (250 mg/kg); one of the groups was tested on day 5 on the elevated plus-maze, to complete our previous study on day 2 [Sa´nchez-Amate MC, Flores P, Sa´nchezSanted F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285–92]. The remaining groups were tested on the elevated T-maze on days 2 and 5. CPF produced an increased open arm activity on the elevated plus-maze on day 5, an increased escape latency on the elevated Tmaze on day 2 and an impaired inhibitory avoidance on day 5. Data are discussed taking together all studies carried out in our laboratory, confirming that CPF effects on emotional behaviour are dependent on both task contingencies and post-administration time. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Chlorpyrifos Elevated plus-maze Elevated T-maze Time-dependent effects
1. Introduction Chlorpyrifos (CPF) is a broad spectrum organophosphate (OP) pesticide whose wide applicability and safety relative to other OP has led to its extensive use in agriculture, industry and household (Slotkin, 2004). It has been well established that the main mechanism of OP toxicity is the persistent inhibition of acetylcholinesterase (AChE) activity (Pope, 1999; Richardson, 1995). Subcutaneous administration of CPF produces an extensive longlasting AChE inhibition, because it is delivered slowly in the organism (Richardson, 1995). Biochemical studies have shown that a single high-dose of CPF (250 mg/kg s.c.) produces an inhibition of
* Corresponding author at: Departamento de Neurociencia y Ciencias de la Salud, Facultad de Psicologı´a, Universidad de Almerı´a, 04120 Almerı´a, Spain. Tel.: +34 950 015 134; fax: +34 950 015 473. E-mail address: [email protected] (M.C. Sa´nchez-Amate). 0161-813X/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2009.07.009
brain AChE activity up to several weeks after treatment (Bushnell et al., 1993; Carvajal et al., 2007; Lo´pez-Crespo et al., 2007) leading to the accumulation of acetylcholine (ACh) in cholinergic synapses. Despite the fact that CPF exerts acute cholinergic activity, surprisingly, no overt toxicity signs are found after administration of high doses of the compound (Richardson, 1995; Sa´nchez-Amate et al., 2001). However, several reports have shown neurobehavioral and emotional deficit in animals by CPF exposure (AbouDonia, 2003; Richardson, 1995). Several studies in our laboratories point to emotional disturbances produced by one s.c. dose of 250 mg/kg of CPF. Carvajal et al. (2005) found that the only cholinoceptive area with c-fos expression 24 h after treatment with CPF was the central nucleus of the amygdala, a nucleus involved in emotional responses such as anxiety (Kalin et al., 2004). In the same way, behavioural testing has shown that an acute dose of 250 mg/kg of CPF modulates the anxiety on different animal models of anxiety. The acute administration of CPF (250 mg/kg s.c.) produced an anxiogenic-like
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effect 2 days after administration on the elevated plus-maze (Sa´nchez-Amate et al., 2001), and a generalization to the anxiogenic drug pentylenetetrazol (PTZ) from 24 h up to 6 days after treatment (Sa´nchez-Amate et al., 2002). In contrast, in a third animal model of anxiety, the open-field test, an anxiolytic-like effect was observed 2 and 5 days after exposure with the same dosage of CPF (Lo´pez-Crespo et al., 2007). Neurobiological mechanisms underlying anxiety are very complex (Millan, 2003). Preclinical investigations from lesion studies to central drug administration have shown that different brain regions and neurotransmitters control behaviour in the different animal tests of anxiety (Menard and Treit, 1996; Treit and Menard, 1997, 1999). Therefore, the effects of a given substance on anxiety levels may depend on the animal model of anxiety in which the substance is tested, and this could be the reason for the different results found in our laboratory with CPF. In order to deepen on the effects of CPF on emotional behaviour, we carried out a set of experiments employing different experimental paradigms: the elevated plus-maze and the elevated T-maze. Both are well validated experimental paradigms extensively used to screen new putative anxiolytic agents and to investigate the behavioural and neurochemical bases of anxiety (Graeff et al., 1998; Hogg, 1996; Pellow and File, 1986), and both take advantage of the innate fear of rodents to elevated open places. For some authors, the elevated plus-maze assesses mixed avoidance of and escape from open, elevated arms (McNaughton and Zangrossi, 2008). In contrast, the elevated T-maze has been proposed as an experimental paradigms distinguishing between these two types of defensive reaction related to anxiety: (1) avoidance of open arms when the rat is on one of the closed arms, and (2) escape from an open arm to enter a safer closed arm (Graeff et al., 1998; Zangrossi and Graeff, 1997). In the first experiment of this study the effect of an acute high dose of CPF was studied on the elevated plus-maze 5 days after administration, in order to complete, in this experimental paradigm, our temporal study previously carried out on drug discrimination and open-field tests (Sa´nchez-Amate et al., 2002; Lo´pez-Crespo et al., 2007). In a second experiment, the effect of the same dose of CPF at 2 and 5 days post-administration was studied on the elevated T-maze with independent groups. 2. Materials and methods 2.1. Subjects Seventy-two male Wistar albino rats purchased from Charles River (n = 24 per experiment and post-administration day), weighing 350 g, were housed in groups of four in an environmentally controlled room (22 8C temperature, 12:12 h light/dark cycle). Food and water were available ad libitum. After a period of 1 week of habituation to the housing conditions, rats were handled in the test room for 5 days prior to experiments. Handling was for 5 min the first day, 3 min on the second and third days, and 2 min on the fourth and fifth days. All the manipulations and procedures followed the standard ethical guidelines of Spanish Royal Decree 223/1988 for reducing animal pain and discomfort. 2.2. Apparatus The plus-maze was made of black metacrilate and consisted of two opposite open arms (50 cm 10 cm 1 cm) and two opposite closed arms (50 cm 10 cm 40 cm) extending from a common central platform (10 cm 10 cm) in the shape of a plus and elevated 50 cm from the floor. A 60 W spotlight placed 60 cm from the side of the closed arms illuminated the room.
The elevated T-maze is derived from the elevated plus-maze by closing the entry of one of its two closed arms. It consisted of tree arms of equal dimensions (50 10) elevated 50 cm from the floor with one of the arms enclosed by 40 cm-high walls and was oriented perpendicularly to two opposed open arms. To avoid falls, the open arms were surrounded by a 1-cm black metacrilate. A 60 W spotlight placed 60 cm from the side of the closed arm illuminated the room. 2.3. Procedure Two days after handling, rats were divided into two groups for s.c. injection of vehicle (olive oil, 1 ml/kg) or CPF (O,O0 -diethyl-O-[3,5,6-tricholoro-2-pyridyl] phosphorothioate, 99,5%, Riedel-de-Hae¨n, Seelze, Germany, dissolved in olive oil, 250 mg/kg in 1 ml/kg). All animals were naı¨ve to the behavioural procedure. The behavioural tests began 2 or 5 days after treatment. 2.3.1. Elevated plus-maze The procedure was identical to the one previously employed in our lab by Sa´nchez-Amate et al. (2001). Five days after CPF administration, the experiment began by transferring the rats to the test room, where they were allowed to rest for 30 min and then placed individually in the centre of the plus-maze facing an open arm, and allowed 5 min of free exploration. An observer, sitting quietly 1 m from the maze, measured the number of open- and closed-arm entries (arm entry being defined as all four paws entering an arm) and time spent in the open and closed arm. The maze was cleaned with ethanol (10%, v/v) to avoid possible bias due to odors and/or residues left by rats tested earlier. For the purpose of analysis, open-arm activity was quantified as the time spent on the open arms relative to the total time spent in both arms (open/total 100), and number of entries into open arms relative to the total number of entries into any arm (open/total 100). In addition, the total number of entries as well as the closed arm entries were used as an index of locomotor activity (Hogg, 1996). 2.3.2. Elevated T-maze The procedure was adapted from the one validated by Graeff et al. (Graeff et al., 1998; Zangrossi and Graeff, 1997). Two or five days after CPF administration, the experiment began with transfer of the rats to the test room, where they were allowed to rest for 30 min. For the inhibitory avoidance task, each animal was placed at the distal end of the closed arm, facing the intersection of the arms. The time taken to leave this arm with all four paws (Baseline Latency) was recorded by a trained observer. This procedure was repeated in two subsequent trials (Avoidance 1 and 2) with a 30-s interval. Learning is indicated by the increase of the withdrawal latency along the trials. Baseline latency (time to first withdrawal from the closed arm) was used as a secondary index of locomotor activity (Texeira et al., 2000). Thirty seconds after the last trial of inhibitory avoidance, the rats were placed at the end of one open arm and the latency to enter the enclosed arm (one-way escape) was recorded. A cutoff time of 300 s was established for inhibitory avoidance and one-way escape. After each trial the maze was cleaned with ethanol (10%, v/v) to avoid possible bias due to odors and/or residues left by rats tested earlier. 2.4. Statistical analyses Data from the animals that fell off the mazes during testing (1 vehicle in experiment 1 and 1 vehicle and 1 CPF in experiment 2) where excluded from the statistical analyses. Data obtained on the
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elevated plus-maze and the escape trial of the elevated T-maze were analysed by a one-way variance analysis (ANOVA), with treatment (VEH and CPF) as between-group factor. Data obtained on the three avoidance trials on the elevated T-maze were analysed through a repeated-measures ANOVA, with Treatment (VEH and CPF) and Trial (Base-Line, Avoidance 1 and Avoidance 2) as between-group and within-subject factors, respectively. Where appropriate, post hoc comparisons were carried out using the Newman–Keuls test. The accepted significance level for all tests was p < 0.05. 3. Results 3.1. Experiment 1: effects of CPF on the elevated plus-maze test 5 days after administration Fig. 1 shows the effect of CPF administration on percentage of open-arm entries and percentage of time spent on the open-arm 5 days after CPF treatment. As can be seen in Fig. 1A, CPF produces an increased in the percentage of open-arm entries (treatment effect [F(1,21) = 13.48, p < 0.01]) and in the percentage of open arm time (treatment effect [F(1,21) = 6.65, p < 0.05]; Fig. 1B). These results indicate a specific anxiolytic-like effect of CPF at day 5 postadministration. Table 1 shows the total arm entries as well as the closed- and open-arm entries for VEH and CPF treated rats recorded 5 days after treatment. The statistical analyses showed that CPF treatment did not change the total arm entries [F < 1] although it decreased the number of closed arms entries (treatment effect [F(1,21) = 6.13, p < 0.05]). The analyses also indicated that the reduction on closed arm entries on day 5 was due to a concomitant increase of open arm entries (treatment effect [F(1,21) = 5.2, p < 0.05]). Therefore, CPF produced a redistribution of arm entries rather than a locomotor effect, as confirmed by the data on total arm entries and by previous results found in our laboratory employing the open-field test (Lo´pez-Crespo et al., 2007).
Fig. 2. The effects of acute administration of CPF on the avoidance (A) (BL: BaseLine; AVOID1: first avoidance trial; AVOID2: second avoidance trial) and escape latencies (B) recorded on the elevated T-maze 2 days after treatment. Administration of 250 mg/kg s.c. of CPF significantly increased the escape latencies. Data represent mean SEM. *p < 0.05 vs. vehicle (VEH).
3.2. Experiment 2: effects of CPF on the elevated T-maze 2 and 5 days after administration Fig. 2 shows the avoidance and escape latencies of VEH and CPF treated rats recorded 2 days after treatment. The statistical analyses showed a main effect of trial [F(2,40) = 9.34, p < 0.001] on the avoidance latencies (A) recorded 2 days after CPF treatment. Post-hoc test revealed that latencies on the Avoidance 2 trial were higher than latencies on the Base-Line Trial (p < 0.001). There was no main effect of Treatment [F(1,20) = 4.76; p = 0.06] and the Treatment by Trial interaction did not reach statistical significance [F < 1]. For the escape latencies (B), the ANOVA showed that the latencies were higher on CPF-treated rats than in VEH-rats [F(1,20) = 4.69; p < 0.05]. There were no differences between VEH and CPF-treated rats on Baseline avoidance latency [F < 1], indicating no gross motor impairment on this day. Thus, CPF did not alter anxiety measures on the elevated T-maze avoidance trials on day 2 although the data on escape latencies pointed to an anxiolytic-like effect of CPF on this time-point. Fig. 3 shows the avoidance and escape latencies of VEH and CPF treated rats recorded 5 days after treatment. The statistical analyses showed main effects of Treatment [F(1,22) = 7.34, p < 0.05] and Trials [F(2,44) = 7.61, p < 0.005] on the avoidance trials (A). The interaction of Treatment by Trial was also statistically significant [F(2,44) = 4.96, p < 0.05]. Post hoc analyses
Fig. 1. The effects of acute administration of CPF on the percentage of open arms entries (A) and open arm time (B) recorded on the elevated plus-maze 5 days after treatment. Administration of 250 mg/kg s.c. of CPF significantly increased both the percentage of open arm entries and percentage of time spent on the open arms of the maze. Data represent mean SEM. *p < 0.05; **p < 0.01 vs. vehicle (VEH).
Table 1 The effects of acute administration of CPF on total- closed- and open-arm entries recorded on the elevated plus-maze 5 days after treatment. Administration of 250 mg/kg s.c. of CPF did not change total entries, but significantly decreased the closed arm entries and also increased the number of open arm entries. Drug
Vehicle (olive oil) CPF (250 mg/kg) *
Number of arm entries Total
Open
Closed
13.25 1.29 12.5 0.82
2.58 0.82 4.83 0.56*
10.09 0.81 7.67 0.57*
p < 0.05 vs. vehicle (VEH).
Fig. 3. The effects of acute administration of CPF on the avoidance (A) (BL: BaseLine; AVOID1: first avoidance trial; AVOID2: second avoidance trial) and escape latencies (B) recorded on the elevated T-maze 5 days after treatment. Administration of 250 mg/kg s.c. of CPF significantly decreased the second trial avoidance latency. Data represent mean SEM. *p < 0.05 vs. vehicle (VEH) #p < 0.05 vs. Base-Line Trial (BL).
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showed that the latencies of CPF-treated rats were lower than those of VEH animals in Avoidance 2 (p < 0.05). The VEH animals increased their avoidance latencies along trials (p < 0.05 and p < 0.001 for the comparison between Base-Line Trial and Avoidance 1 and 2 respectively). However, CPF treated rats did not increase their avoidance latencies along trials (p > 0.1). For the escape latencies (B), the ANOVA showed no effect of Treatment [F < 1]. There were no differences between VEH- and CPF-treated rats on Baseline avoidance latency [F(1,22) = 1.97, p > 1]. Therefore, these results point to an anxiolytic-like effect of CPF 5 days after administration on the avoidance latencies of the elevated Tmaze, but no effect on the anxiety measured on escape trials. 4. Discussion The present study assessed the effect of a single high dose of CPF (250 mg/kg s.c.) on the anxiety response of rats assessed through different experimental paradigms: the elevated T-maze (avoidance and escape trials) and the elevated plus-maze. The behavioural assessment was carried out on days 2 and 5 for the elevated Tmaze and on day 5 for the elevated plus-maze since a previous study in our lab tested the effects of CPF on day 2 on this experimental paradigm (Sa´nchez-Amate et al., 2001). Results obtained in the first experiment show that an acute administration of a high dose of CPF (250 mg/kg s.c.) produced an anxiolytic-like effect in the elevated plus-maze 5 days after administration. The same effect has been also found by Aldridge et al. (2005) in rat exposed postnatally to subtoxic doses of CPF. Surprisingly, in this experiment the anxiogenic-like effect obtained previously in our laboratory 2 days after CPF administration (Sa´nchez-Amate et al., 2001) was not observed. The difference of effects observed in both studies could not be attributed to methodological differences, since both experiments were carried out in identical conditions. Results obtained on the elevated T-maze showed that CPF increased escape latencies 2 days after administration, suggesting a panicolytic-like effect of CPF, since pharmacological studies have shown that the one-way escape is related to panic (Texeira et al., 2000; Poltronieri et al., 2003; Zanoveli et al., 2005). Nevertheless, the increased escape latencies produced by CPF may be due to motor impairment, because an identical dose of CPF significantly decreased locomotion on the open-field test 2 days after administration (Lo´pez-Crespo et al., 2007). However, a motor impairment of CPF should have produced an increase of the avoidance latencies on the elevated T-maze, but no such effect was observed; additionally, no significant difference was seen in the baseline avoidance, a secondary measure of locomotor activity (Texeira et al., 2000). Thus, CPF 2 days after administration produced only an increased escape latency. On the contrary, 5 days after administration, CPF impaired only inhibitory avoidance, suggesting anxiolytic-like effect, whereas no effect was observed on escape latency. In addition to the time-dependent effects produced on the elevated T-maze, CPF also produces a bimodal effect on the elevated plus-maze in function of the time since its administration: anxiogenic-like effect at day 2 (Sa´nchez-Amate et al., 2001) and anxiolytic-like effect 5 days after administration. An anxiolytic-like effect was observed on the open-field test at 2 and 5 days post-administration of CPF (Lo´pez-Crespo et al., 2007). In contrast, in the drug discrimination procedure, CPF produced a generalization to the anxiogenic drug pentylenetetrazol (PTZ) from 24 h up to 6 days after treatment (Sa´nchez-Amate et al., 2002). Taken together these results, we could say that the effect of CPF depends on the experimental paradigm used to assess anxiety and the time lapsed from its administration. A possible explanation to this pattern of changes produced by CPF on anxiety would be related to its simultaneous actions on different neurochemical systems, and/
or development of dynamic compensatory and/or adaptive response on the neurochemical systems. CPF is a compound with both cholinergic and non-cholinergic activity (Pope, 1999; Richardson, 1995). Acute exposure to a single high dose of CPF can lead to several neurochemical changes in the brain of the exposed animal that vary with time and may account for some of the time-dependent behavioural effects of CPF. Karanth et al. (2006), using in vivo microdialysis, found that the levels of ACh in rat striatum are higher at 4 and 7 days after dosing (84, 156 and 279 mg/kg of CPF, s.c.) than at day 1. Won et al. (2001) assessed the ACh release on striatum and cortex at different time-points after CPF exposure. They found that ACh release decreased 24 h after a dose of 136 mg/kg in both striatum and cortex, but increased 96 h after treatment. Nevertheless other neurochemical systems could also explain the different effect produced by CPF. Recent research indicated the involvement of other systems as GABAergic and cytokine, in the effects of a single high dose of CPF (Lo´pez-Grancha et al., 2006; Carvajal et al., 2005; Sa´nchez-Amate et al., 2002; Ray and Richards, 2001). The monoaminergic systems are also modified by CPF. Neurodevelopmental studies have found dopaminergic as well as serotoninergic alterations in brains after pre- and/or postnatal exposure to CPF (Aldridge et al., 2004, 2005; Dam et al., 1999; Raines et al., 2001). In agreement with these studies, Moreno et al. (2008) found different patterns of monoaminergic activity changes over time on different brain structures after CPF treatment on adult rats. An acute dose of 250 mg/kg of CPF produced changes in dopamine metabolism after 2 days and changes in serotonin turnover 7 and 15 days after its administration on the striatum but not on the nucleus accumbens. Nevertheless, another possible interpretation is possible to explain the behaviour effects produced by CPF on the different experimental paradigms used. Specifically, CPF, 5 days after administration, produced an anxiolytic-like effect on task involving a conflicting situation between the natural tendencies of the rodent to explore novel locations vs. the inhibition of exploration produced by their aversion to open (and elevated) spaces (Dawson and Tricklebank, 1995; Rodgers and Dalvi, 1997; Rodgers et al., 1997). Behavioural disinhibition in experimental paradigms which invoke a conflict situation, like elevated plus-maze and elevated Tmaze, may rather reflect a loss of impulsive control with or without a concomitant alleviation of anxiety (Olausson et al., 2001a,b; Soubrie´, 1986). In this later case, it is possible that the behavioural disinhibition produced by CPF on experimental paradigms that involve a conflicting situation could be reflecting the impulsive properties of this compound, rather than a selective action over the anxiety systems. Studies carried out in our laboratory have shown that an acute high dose of CPF (250 mg/kg) increased impulsive behaviour (rats prefer immediate reward and exhibit a more impulsive choice) in a delay discounting task 6 months after treatment (Cardona et al., 2006) as well as 30 days after exposure (unpublished data). In summary, taking together all the studies carried out in our laboratory, two possible interpretations could explain the effects produced by a single high dose of CPF at different postadministration timing and on different experimental paradigms used to assess anxiety. On one hand, CPF could be modulating different neurochemical systems at different timing and on discrete brain area to produce different effects on animal models of anxiety. On the other hand, CPF could be simultaneously modifying different neurochemical systems underlying different behaviour, such as anxiety and/or impulsivity. More studies are guaranteed to elucidate this question. Conflict of interest The authors declare that there are no conflicts of interest.
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Menard J, Treit D. Lateral and medial septal lesions reduce anxiety in the plus-maze and probe-burying tests. Physiol Behav 1996;60(3):845–53. Millan MJ. The neurobiology and control of anxious states. Prog Neurobiol 2003;70: 83–244. Moreno M, Can˜adas F, Cardona D, Sun˜ol C, Campa L, Sa´nchez-Amate MC, Flores P, Sa´nchez-Santed F. Long-term monoamine changes in the striatum and nucleus accubemns after acute chlorpyrifos exposure. Toxicol Lett 2008;176:162–7. Olausson P, Akesson P, Engel JA, So¨derpalm B. Effects of 5-HT1A and 5-HT2 receptor agonists on the behavioral and neurochemical consequences of repeated nicotine treatment. Eur J Pharmacol 2001a;420(1):45–54. Olausson P, Akesson P, Petersson A, Engel JA, So¨derpalm B. Behavioral and neurochemical consequences of repeated nicotine treatment in the serotonin-depleted rat. Psychopharmacology (Berl) 2001b;155(4):348–61. Pellow S, File SE. Anxiolytic and anxiogenic drug effects on exploratory activity in an elevated plus-maze: a novel test of anxiety in the rat. Pharmacol Biochem Behav 1986;24:525–9. Poltronieri SC, Zangrossi H Jr, Viana MB. Antipanic-like effect of serotonin reuptake inhibition in the elevated T-maze. Behav Brain Res 2003;147:185–92. Pope CN. Organophosphorus pesticides: do they all have the same mechanism of toxicity? J Toxicol Environ Health B 1999;2:161–81. Ray DE, Richards PG. The potential for toxic effects of chronic, low-dose exposure to organophospates. Toxicol Lett 2001;120:343–51. Raines KW, Seidler FJ, Slotkin TA. Alterations in serotonin transporter expression in brain regions of rats exposed neonatally to chlorpyrifos. Dev Brain Res 2001;130: 65–72. Richardson RJ. Assessment of the neurotoxic potential of chlorpyrifos relative of other organophosphorus compounds: a critical review of the literature. J Toxicol Environ Health 1995;44:135–65. Rodgers RJ, Cao BJ, Dalvi A, Holmes A. Animal models of anxiety: an ethological perspective. Braz J Med Biol Res 1997;30:289–304. Rodgers RJ, Dalvi A. Anxiety, defence and the elevated plus-maze. Neurosci Biobehav Rev 1997;21(6):801–10. Sa´nchez-Amate MC, Da´vila E, Can˜adas F, Flores P, Sa´nchez-Santed F. Chlorpyrifos shares stimulus properties with pentilenetetrazol as evaluated by and operant drug discrimination task. Neurotoxicology 2002;23:795–803. Sa´nchez-Amate MC, Flores P, Sa´nchez-Santed F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285–92. Slotkin TA. Guidelines for developmental neurotoxicity and their impact on organophosphates pesticides: a personal view from an academic perspective. Neurotoxicology 2004;25:631–40. Soubrie´ P. Reconciling the role of central serotonin neurons in human and animal behavior. Behav Brain Sci 1986;9:319–64. Texeira RC, Zangrosi H Jr, Graeff FG. Behavioral effects of acute and chronic imipramine in the elevated T-maze model of anxiety. Pharmacol Biochem Behav 2000;65: 571–6. Treit D, Menard J. Dissociations among the anxiolytic effects of septal, hippocampal, and amygdaloid lesions. Behav Neurosci 1997;111(3):653–8. Treit D, Menard J. Effects of centrally administered anxiolytic compounds in animal models of anxiety. Neurosci Biobehav Rev 1999;23(4):591–613. Won YK, Liu J, Olivier K, Zheng Q, Pope CN. Age-related effects of chlorpyrifos on acetylcholine release in rat brain. Neurotoxicology 2001;22:39–48. Zangrossi H, Graeff FG. Behavioral validation of the elevated T-maze, a new animal model of anxiety. Brain Res Bull 1997;44:1–5. Zanoveli JM, Nogueira RL, Zangrossi H Jr. Chronic imipramine treatment sensitize 5-HT1A and 5-HT2A receptors in the dorsal periaqueductal gray matter: evidences from the elevated T-maze test of anxiety. Behav Pharmacol 2005;16:543–52.
Contents lists available at ScienceDirect
NeuroToxicology
Acute high dose of chlorpyrifos alters performance of rats in the elevated plus-maze and the elevated T-maze G.A. Lo´pez-Crespo a, P. Flores b, F. Sa´nchez-Santed b, M.C. Sa´nchez-Amate b,* a b
Departamento de Psicologı´a Ba´sica y Metodologı´a, Universidad de Murcia, Spain Departamento de Neurociencia y Ciencias de la Salud, Universidad de Almerı´a, Spain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 February 2009 Accepted 16 July 2009 Available online 24 July 2009
Chlorpyrifos (CPF) is a broad spectrum organophosphate (OP) pesticide widely used in agriculture, industry and household. Several animal studies indicate emotional disturbances after CPF exposure, although the results are sometimes puzzling. Thus, both anxiolytic and anxiogenic effects of CPF have been reported in different animal models of anxiety [Sa´nchez-Amate MC, Flores P, Sa´nchez-Santed F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285–92; Sa´nchez˜ adas F, Flores P, Sa´nchez-Santed F. Chlorpyrifos shares stimulus properties with Amate MC, Da´vila E, Can pentilenetetrazol as evaluated by and operant drug discrimination task. Neurotoxicology 2002;23:795– 803; Lo´pez-Crespo G, Carvajal F, Flores P, Sa´nchez-Santed F, Sa´nchez-Amate MC. Time-course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurotoxicology 2007;28:541– 7]. On the other hand, other behavioural effects of CPF are time-dependent [Lo´pez-Crespo G, Carvajal F, Flores P, Sa´nchez-Santed F, Sa´nchez-Amate MC. Time-course of biochemical and behavioural effects of a single high dose of chlorpyrifos. Neurotoxicology 2007;28:541–7], raising the question that the effects of CPF could be task and post-administration time dependent. To test this hypothesis, three groups of rats were treated with a single high dose of CPF (250 mg/kg); one of the groups was tested on day 5 on the elevated plus-maze, to complete our previous study on day 2 [Sa´nchez-Amate MC, Flores P, Sa´nchezSanted F. Effects of chlorpyrifos in the plus-maze model of anxiety. Behav Pharmacol 2001;12:285–92]. The remaining groups were tested on the elevated T-maze on days 2 and 5. CPF produced an increased open arm activity on the elevated plus-maze on day 5, an increased escape latency on the elevated Tmaze on day 2 and an impaired inhibitory avoidance on day 5. Data are discussed taking together all studies carried out in our laboratory, confirming that CPF effects on emotional behaviour are dependent on both task contingencies and post-administration time. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Chlorpyrifos Elevated plus-maze Elevated T-maze Time-dependent effects
1. Introduction Chlorpyrifos (CPF) is a broad spectrum organophosphate (OP) pesticide whose wide applicability and safety relative to other OP has led to its extensive use in agriculture, industry and household (Slotkin, 2004). It has been well established that the main mechanism of OP toxicity is the persistent inhibition of acetylcholinesterase (AChE) activity (Pope, 1999; Richardson, 1995). Subcutaneous administration of CPF produces an extensive longlasting AChE inhibition, because it is delivered slowly in the organism (Richardson, 1995). Biochemical studies have shown that a single high-dose of CPF (250 mg/kg s.c.) produces an inhibition of
* Corresponding author at: Departamento de Neurociencia y Ciencias de la Salud, Facultad de Psicologı´a, Universidad de Almerı´a, 04120 Almerı´a, Spain. Tel.: +34 950 015 134; fax: +34 950 015 473. E-mail address: [email protected] (M.C. Sa´nchez-Amate). 0161-813X/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2009.07.009
brain AChE activity up to several weeks after treatment (Bushnell et al., 1993; Carvajal et al., 2007; Lo´pez-Crespo et al., 2007) leading to the accumulation of acetylcholine (ACh) in cholinergic synapses. Despite the fact that CPF exerts acute cholinergic activity, surprisingly, no overt toxicity signs are found after administration of high doses of the compound (Richardson, 1995; Sa´nchez-Amate et al., 2001). However, several reports have shown neurobehavioral and emotional deficit in animals by CPF exposure (AbouDonia, 2003; Richardson, 1995). Several studies in our laboratories point to emotional disturbances produced by one s.c. dose of 250 mg/kg of CPF. Carvajal et al. (2005) found that the only cholinoceptive area with c-fos expression 24 h after treatment with CPF was the central nucleus of the amygdala, a nucleus involved in emotional responses such as anxiety (Kalin et al., 2004). In the same way, behavioural testing has shown that an acute dose of 250 mg/kg of CPF modulates the anxiety on different animal models of anxiety. The acute administration of CPF (250 mg/kg s.c.) produced an anxiogenic-like
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effect 2 days after administration on the elevated plus-maze (Sa´nchez-Amate et al., 2001), and a generalization to the anxiogenic drug pentylenetetrazol (PTZ) from 24 h up to 6 days after treatment (Sa´nchez-Amate et al., 2002). In contrast, in a third animal model of anxiety, the open-field test, an anxiolytic-like effect was observed 2 and 5 days after exposure with the same dosage of CPF (Lo´pez-Crespo et al., 2007). Neurobiological mechanisms underlying anxiety are very complex (Millan, 2003). Preclinical investigations from lesion studies to central drug administration have shown that different brain regions and neurotransmitters control behaviour in the different animal tests of anxiety (Menard and Treit, 1996; Treit and Menard, 1997, 1999). Therefore, the effects of a given substance on anxiety levels may depend on the animal model of anxiety in which the substance is tested, and this could be the reason for the different results found in our laboratory with CPF. In order to deepen on the effects of CPF on emotional behaviour, we carried out a set of experiments employing different experimental paradigms: the elevated plus-maze and the elevated T-maze. Both are well validated experimental paradigms extensively used to screen new putative anxiolytic agents and to investigate the behavioural and neurochemical bases of anxiety (Graeff et al., 1998; Hogg, 1996; Pellow and File, 1986), and both take advantage of the innate fear of rodents to elevated open places. For some authors, the elevated plus-maze assesses mixed avoidance of and escape from open, elevated arms (McNaughton and Zangrossi, 2008). In contrast, the elevated T-maze has been proposed as an experimental paradigms distinguishing between these two types of defensive reaction related to anxiety: (1) avoidance of open arms when the rat is on one of the closed arms, and (2) escape from an open arm to enter a safer closed arm (Graeff et al., 1998; Zangrossi and Graeff, 1997). In the first experiment of this study the effect of an acute high dose of CPF was studied on the elevated plus-maze 5 days after administration, in order to complete, in this experimental paradigm, our temporal study previously carried out on drug discrimination and open-field tests (Sa´nchez-Amate et al., 2002; Lo´pez-Crespo et al., 2007). In a second experiment, the effect of the same dose of CPF at 2 and 5 days post-administration was studied on the elevated T-maze with independent groups. 2. Materials and methods 2.1. Subjects Seventy-two male Wistar albino rats purchased from Charles River (n = 24 per experiment and post-administration day), weighing 350 g, were housed in groups of four in an environmentally controlled room (22 8C temperature, 12:12 h light/dark cycle). Food and water were available ad libitum. After a period of 1 week of habituation to the housing conditions, rats were handled in the test room for 5 days prior to experiments. Handling was for 5 min the first day, 3 min on the second and third days, and 2 min on the fourth and fifth days. All the manipulations and procedures followed the standard ethical guidelines of Spanish Royal Decree 223/1988 for reducing animal pain and discomfort. 2.2. Apparatus The plus-maze was made of black metacrilate and consisted of two opposite open arms (50 cm 10 cm 1 cm) and two opposite closed arms (50 cm 10 cm 40 cm) extending from a common central platform (10 cm 10 cm) in the shape of a plus and elevated 50 cm from the floor. A 60 W spotlight placed 60 cm from the side of the closed arms illuminated the room.
The elevated T-maze is derived from the elevated plus-maze by closing the entry of one of its two closed arms. It consisted of tree arms of equal dimensions (50 10) elevated 50 cm from the floor with one of the arms enclosed by 40 cm-high walls and was oriented perpendicularly to two opposed open arms. To avoid falls, the open arms were surrounded by a 1-cm black metacrilate. A 60 W spotlight placed 60 cm from the side of the closed arm illuminated the room. 2.3. Procedure Two days after handling, rats were divided into two groups for s.c. injection of vehicle (olive oil, 1 ml/kg) or CPF (O,O0 -diethyl-O-[3,5,6-tricholoro-2-pyridyl] phosphorothioate, 99,5%, Riedel-de-Hae¨n, Seelze, Germany, dissolved in olive oil, 250 mg/kg in 1 ml/kg). All animals were naı¨ve to the behavioural procedure. The behavioural tests began 2 or 5 days after treatment. 2.3.1. Elevated plus-maze The procedure was identical to the one previously employed in our lab by Sa´nchez-Amate et al. (2001). Five days after CPF administration, the experiment began by transferring the rats to the test room, where they were allowed to rest for 30 min and then placed individually in the centre of the plus-maze facing an open arm, and allowed 5 min of free exploration. An observer, sitting quietly 1 m from the maze, measured the number of open- and closed-arm entries (arm entry being defined as all four paws entering an arm) and time spent in the open and closed arm. The maze was cleaned with ethanol (10%, v/v) to avoid possible bias due to odors and/or residues left by rats tested earlier. For the purpose of analysis, open-arm activity was quantified as the time spent on the open arms relative to the total time spent in both arms (open/total 100), and number of entries into open arms relative to the total number of entries into any arm (open/total 100). In addition, the total number of entries as well as the closed arm entries were used as an index of locomotor activity (Hogg, 1996). 2.3.2. Elevated T-maze The procedure was adapted from the one validated by Graeff et al. (Graeff et al., 1998; Zangrossi and Graeff, 1997). Two or five days after CPF administration, the experiment began with transfer of the rats to the test room, where they were allowed to rest for 30 min. For the inhibitory avoidance task, each animal was placed at the distal end of the closed arm, facing the intersection of the arms. The time taken to leave this arm with all four paws (Baseline Latency) was recorded by a trained observer. This procedure was repeated in two subsequent trials (Avoidance 1 and 2) with a 30-s interval. Learning is indicated by the increase of the withdrawal latency along the trials. Baseline latency (time to first withdrawal from the closed arm) was used as a secondary index of locomotor activity (Texeira et al., 2000). Thirty seconds after the last trial of inhibitory avoidance, the rats were placed at the end of one open arm and the latency to enter the enclosed arm (one-way escape) was recorded. A cutoff time of 300 s was established for inhibitory avoidance and one-way escape. After each trial the maze was cleaned with ethanol (10%, v/v) to avoid possible bias due to odors and/or residues left by rats tested earlier. 2.4. Statistical analyses Data from the animals that fell off the mazes during testing (1 vehicle in experiment 1 and 1 vehicle and 1 CPF in experiment 2) where excluded from the statistical analyses. Data obtained on the
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elevated plus-maze and the escape trial of the elevated T-maze were analysed by a one-way variance analysis (ANOVA), with treatment (VEH and CPF) as between-group factor. Data obtained on the three avoidance trials on the elevated T-maze were analysed through a repeated-measures ANOVA, with Treatment (VEH and CPF) and Trial (Base-Line, Avoidance 1 and Avoidance 2) as between-group and within-subject factors, respectively. Where appropriate, post hoc comparisons were carried out using the Newman–Keuls test. The accepted significance level for all tests was p < 0.05. 3. Results 3.1. Experiment 1: effects of CPF on the elevated plus-maze test 5 days after administration Fig. 1 shows the effect of CPF administration on percentage of open-arm entries and percentage of time spent on the open-arm 5 days after CPF treatment. As can be seen in Fig. 1A, CPF produces an increased in the percentage of open-arm entries (treatment effect [F(1,21) = 13.48, p < 0.01]) and in the percentage of open arm time (treatment effect [F(1,21) = 6.65, p < 0.05]; Fig. 1B). These results indicate a specific anxiolytic-like effect of CPF at day 5 postadministration. Table 1 shows the total arm entries as well as the closed- and open-arm entries for VEH and CPF treated rats recorded 5 days after treatment. The statistical analyses showed that CPF treatment did not change the total arm entries [F < 1] although it decreased the number of closed arms entries (treatment effect [F(1,21) = 6.13, p < 0.05]). The analyses also indicated that the reduction on closed arm entries on day 5 was due to a concomitant increase of open arm entries (treatment effect [F(1,21) = 5.2, p < 0.05]). Therefore, CPF produced a redistribution of arm entries rather than a locomotor effect, as confirmed by the data on total arm entries and by previous results found in our laboratory employing the open-field test (Lo´pez-Crespo et al., 2007).
Fig. 2. The effects of acute administration of CPF on the avoidance (A) (BL: BaseLine; AVOID1: first avoidance trial; AVOID2: second avoidance trial) and escape latencies (B) recorded on the elevated T-maze 2 days after treatment. Administration of 250 mg/kg s.c. of CPF significantly increased the escape latencies. Data represent mean SEM. *p < 0.05 vs. vehicle (VEH).
3.2. Experiment 2: effects of CPF on the elevated T-maze 2 and 5 days after administration Fig. 2 shows the avoidance and escape latencies of VEH and CPF treated rats recorded 2 days after treatment. The statistical analyses showed a main effect of trial [F(2,40) = 9.34, p < 0.001] on the avoidance latencies (A) recorded 2 days after CPF treatment. Post-hoc test revealed that latencies on the Avoidance 2 trial were higher than latencies on the Base-Line Trial (p < 0.001). There was no main effect of Treatment [F(1,20) = 4.76; p = 0.06] and the Treatment by Trial interaction did not reach statistical significance [F < 1]. For the escape latencies (B), the ANOVA showed that the latencies were higher on CPF-treated rats than in VEH-rats [F(1,20) = 4.69; p < 0.05]. There were no differences between VEH and CPF-treated rats on Baseline avoidance latency [F < 1], indicating no gross motor impairment on this day. Thus, CPF did not alter anxiety measures on the elevated T-maze avoidance trials on day 2 although the data on escape latencies pointed to an anxiolytic-like effect of CPF on this time-point. Fig. 3 shows the avoidance and escape latencies of VEH and CPF treated rats recorded 5 days after treatment. The statistical analyses showed main effects of Treatment [F(1,22) = 7.34, p < 0.05] and Trials [F(2,44) = 7.61, p < 0.005] on the avoidance trials (A). The interaction of Treatment by Trial was also statistically significant [F(2,44) = 4.96, p < 0.05]. Post hoc analyses
Fig. 1. The effects of acute administration of CPF on the percentage of open arms entries (A) and open arm time (B) recorded on the elevated plus-maze 5 days after treatment. Administration of 250 mg/kg s.c. of CPF significantly increased both the percentage of open arm entries and percentage of time spent on the open arms of the maze. Data represent mean SEM. *p < 0.05; **p < 0.01 vs. vehicle (VEH).
Table 1 The effects of acute administration of CPF on total- closed- and open-arm entries recorded on the elevated plus-maze 5 days after treatment. Administration of 250 mg/kg s.c. of CPF did not change total entries, but significantly decreased the closed arm entries and also increased the number of open arm entries. Drug
Vehicle (olive oil) CPF (250 mg/kg) *
Number of arm entries Total
Open
Closed
13.25 1.29 12.5 0.82
2.58 0.82 4.83 0.56*
10.09 0.81 7.67 0.57*
p < 0.05 vs. vehicle (VEH).
Fig. 3. The effects of acute administration of CPF on the avoidance (A) (BL: BaseLine; AVOID1: first avoidance trial; AVOID2: second avoidance trial) and escape latencies (B) recorded on the elevated T-maze 5 days after treatment. Administration of 250 mg/kg s.c. of CPF significantly decreased the second trial avoidance latency. Data represent mean SEM. *p < 0.05 vs. vehicle (VEH) #p < 0.05 vs. Base-Line Trial (BL).
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showed that the latencies of CPF-treated rats were lower than those of VEH animals in Avoidance 2 (p < 0.05). The VEH animals increased their avoidance latencies along trials (p < 0.05 and p < 0.001 for the comparison between Base-Line Trial and Avoidance 1 and 2 respectively). However, CPF treated rats did not increase their avoidance latencies along trials (p > 0.1). For the escape latencies (B), the ANOVA showed no effect of Treatment [F < 1]. There were no differences between VEH- and CPF-treated rats on Baseline avoidance latency [F(1,22) = 1.97, p > 1]. Therefore, these results point to an anxiolytic-like effect of CPF 5 days after administration on the avoidance latencies of the elevated Tmaze, but no effect on the anxiety measured on escape trials. 4. Discussion The present study assessed the effect of a single high dose of CPF (250 mg/kg s.c.) on the anxiety response of rats assessed through different experimental paradigms: the elevated T-maze (avoidance and escape trials) and the elevated plus-maze. The behavioural assessment was carried out on days 2 and 5 for the elevated Tmaze and on day 5 for the elevated plus-maze since a previous study in our lab tested the effects of CPF on day 2 on this experimental paradigm (Sa´nchez-Amate et al., 2001). Results obtained in the first experiment show that an acute administration of a high dose of CPF (250 mg/kg s.c.) produced an anxiolytic-like effect in the elevated plus-maze 5 days after administration. The same effect has been also found by Aldridge et al. (2005) in rat exposed postnatally to subtoxic doses of CPF. Surprisingly, in this experiment the anxiogenic-like effect obtained previously in our laboratory 2 days after CPF administration (Sa´nchez-Amate et al., 2001) was not observed. The difference of effects observed in both studies could not be attributed to methodological differences, since both experiments were carried out in identical conditions. Results obtained on the elevated T-maze showed that CPF increased escape latencies 2 days after administration, suggesting a panicolytic-like effect of CPF, since pharmacological studies have shown that the one-way escape is related to panic (Texeira et al., 2000; Poltronieri et al., 2003; Zanoveli et al., 2005). Nevertheless, the increased escape latencies produced by CPF may be due to motor impairment, because an identical dose of CPF significantly decreased locomotion on the open-field test 2 days after administration (Lo´pez-Crespo et al., 2007). However, a motor impairment of CPF should have produced an increase of the avoidance latencies on the elevated T-maze, but no such effect was observed; additionally, no significant difference was seen in the baseline avoidance, a secondary measure of locomotor activity (Texeira et al., 2000). Thus, CPF 2 days after administration produced only an increased escape latency. On the contrary, 5 days after administration, CPF impaired only inhibitory avoidance, suggesting anxiolytic-like effect, whereas no effect was observed on escape latency. In addition to the time-dependent effects produced on the elevated T-maze, CPF also produces a bimodal effect on the elevated plus-maze in function of the time since its administration: anxiogenic-like effect at day 2 (Sa´nchez-Amate et al., 2001) and anxiolytic-like effect 5 days after administration. An anxiolytic-like effect was observed on the open-field test at 2 and 5 days post-administration of CPF (Lo´pez-Crespo et al., 2007). In contrast, in the drug discrimination procedure, CPF produced a generalization to the anxiogenic drug pentylenetetrazol (PTZ) from 24 h up to 6 days after treatment (Sa´nchez-Amate et al., 2002). Taken together these results, we could say that the effect of CPF depends on the experimental paradigm used to assess anxiety and the time lapsed from its administration. A possible explanation to this pattern of changes produced by CPF on anxiety would be related to its simultaneous actions on different neurochemical systems, and/
or development of dynamic compensatory and/or adaptive response on the neurochemical systems. CPF is a compound with both cholinergic and non-cholinergic activity (Pope, 1999; Richardson, 1995). Acute exposure to a single high dose of CPF can lead to several neurochemical changes in the brain of the exposed animal that vary with time and may account for some of the time-dependent behavioural effects of CPF. Karanth et al. (2006), using in vivo microdialysis, found that the levels of ACh in rat striatum are higher at 4 and 7 days after dosing (84, 156 and 279 mg/kg of CPF, s.c.) than at day 1. Won et al. (2001) assessed the ACh release on striatum and cortex at different time-points after CPF exposure. They found that ACh release decreased 24 h after a dose of 136 mg/kg in both striatum and cortex, but increased 96 h after treatment. Nevertheless other neurochemical systems could also explain the different effect produced by CPF. Recent research indicated the involvement of other systems as GABAergic and cytokine, in the effects of a single high dose of CPF (Lo´pez-Grancha et al., 2006; Carvajal et al., 2005; Sa´nchez-Amate et al., 2002; Ray and Richards, 2001). The monoaminergic systems are also modified by CPF. Neurodevelopmental studies have found dopaminergic as well as serotoninergic alterations in brains after pre- and/or postnatal exposure to CPF (Aldridge et al., 2004, 2005; Dam et al., 1999; Raines et al., 2001). In agreement with these studies, Moreno et al. (2008) found different patterns of monoaminergic activity changes over time on different brain structures after CPF treatment on adult rats. An acute dose of 250 mg/kg of CPF produced changes in dopamine metabolism after 2 days and changes in serotonin turnover 7 and 15 days after its administration on the striatum but not on the nucleus accumbens. Nevertheless, another possible interpretation is possible to explain the behaviour effects produced by CPF on the different experimental paradigms used. Specifically, CPF, 5 days after administration, produced an anxiolytic-like effect on task involving a conflicting situation between the natural tendencies of the rodent to explore novel locations vs. the inhibition of exploration produced by their aversion to open (and elevated) spaces (Dawson and Tricklebank, 1995; Rodgers and Dalvi, 1997; Rodgers et al., 1997). Behavioural disinhibition in experimental paradigms which invoke a conflict situation, like elevated plus-maze and elevated Tmaze, may rather reflect a loss of impulsive control with or without a concomitant alleviation of anxiety (Olausson et al., 2001a,b; Soubrie´, 1986). In this later case, it is possible that the behavioural disinhibition produced by CPF on experimental paradigms that involve a conflicting situation could be reflecting the impulsive properties of this compound, rather than a selective action over the anxiety systems. Studies carried out in our laboratory have shown that an acute high dose of CPF (250 mg/kg) increased impulsive behaviour (rats prefer immediate reward and exhibit a more impulsive choice) in a delay discounting task 6 months after treatment (Cardona et al., 2006) as well as 30 days after exposure (unpublished data). In summary, taking together all the studies carried out in our laboratory, two possible interpretations could explain the effects produced by a single high dose of CPF at different postadministration timing and on different experimental paradigms used to assess anxiety. On one hand, CPF could be modulating different neurochemical systems at different timing and on discrete brain area to produce different effects on animal models of anxiety. On the other hand, CPF could be simultaneously modifying different neurochemical systems underlying different behaviour, such as anxiety and/or impulsivity. More studies are guaranteed to elucidate this question. Conflict of interest The authors declare that there are no conflicts of interest.
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