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Inhibitory effects of modafinil on emotional memory in mice
Neuropharmacology 64 (2013) 365e370
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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Inhibitory effects of modafinil on emotional memory in mice Helaine A. Fernandes a, b,1, Karina A. Zanin a, b,1, Camilla L. Patti a, b, *, Raphael Wuo-Silva b, Rita C. Carvalho b, Luciano Fernandes-Santos a, b, Lia R.A. Bittencourt a, Sergio Tufik a, Roberto Frussa-Filho a, b, * a b
Departamento de Psicobiologia, Universidade Federal de São Paulo, R. Napoleão de Barros 925, São Paulo, SP, Brazil Departamento de Farmacologia, Universidade Federal de São Paulo, Rua Botucatu 862, Ed. Leal Prado, 1 andar, 04023062 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2012 Received in revised form 5 June 2012 Accepted 27 June 2012
Modafinil (MOD), a psychostimulant used to treat narcolepsy, excessive daytime sleepiness, and sleepiness due to obstructive sleep apnea, appears to promote a possible facilitatory effect on cognitive function. In the present study, we investigated the effects of the acute administration of MOD on the different steps of emotional memory formation and usage (acquisition, consolidation and retrieval) as well as the possible participation of the state-dependency phenomenon on the cognitive effects of this compound. Mice were acutely treated with 32, 64 or 128 mg/kg MOD before training or testing or immediately after training and were subjected to the plus-maze discriminative avoidance task. The results showed that although pre-training MOD administration did not exert any effects on learning, the doses of 32 or 64 mg/kg induced emotional memory deficits during testing. Still, the post-training acute administration of the higher doses of MOD (64 and 128 mg/kg) impaired associative memory consolidation. When the drug was administered pre-test, only the 32 mg/kg dose impaired the task retrieval. Importantly, the cognitive impairing effects induced by 32 mg/kg MOD were not related to the phenomenon of state-dependency. In all, our findings provide pre-clinical evidence of potential emotional memory amnesia induced by MOD. This article is part of a Special Issue entitled ‘Cognitive Enhancers’. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Modafinil Learning Consolidation Retrieval State-dependency Plus-maze discriminative avoidance task
1. Introduction Modafinil (MOD) is a psychostimulant that acts as a wakepromoting drug and has been approved for the treatment of excessive daytime sleepiness in narcolepsy, obstructive sleep apnea, and shift work syndrome. Clinical studies have pointed MOD positive effects when used to treat Parkinson’s disease (Ferraro et al., 1998; Nieves and Lang, 2002), multiple sclerosis (Kraft and Bowen, 2005), schizophrenia (Turner et al., 2004) and attention deficit hyperactivity disorder (Taylor and Russo, 2000). Additionally, MOD seems to be widely prescribed off-label to enhance alertness, attention, memory for dementia and depression (Joos et al., 2010). Of note, an illicit market exists for academic doping as well (Cakic, 2009). * Corresponding authors. Departamento de Farmacologia, Universidade Federal de São Paulo, Rua Botucatu 862, Ed. Leal Prado, 1 andar, 04023062 São Paulo, SP, Brazil. Tel.: þ55 11 5549 4122; fax: þ55 11 5549 4122x222. E-mail addresses: [email protected] (C.L. Patti), [email protected] (R. Frussa-Filho). 1 These authors contributed equally to this study. 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.06.058
There are several hypotheses that attempt to explain the mechanisms of action of MOD. Studies have suggested that it may increase the release of catecholamines (dopamine and norepinephrine), serotonin and glutamate, thereby reducing the release of GABA in various brain regions, and activating hypothalamic neurons containing hypocretin/orexin neurons of the tuberomammillary nucleus (Minzemberg and Carter, 2008). It has been demonstrated that MOD modifies the activity of brain areas involved with memory, such as the hippocampus and prefrontal cortex (Béracochéa et al., 2003). Within this context, studies indicate that MOD has cognitive-enhancing abilities in rodents performing a variety of learning/memory exercises in the T-Maze based on spontaneous alternation behavior (Béracochéa et al., 2001), and also enhanced learning (Béracochéa et al., 2003) and memory retrieval (Béracochéa et al., 2008). Still, it was reported that this drug was able to restore the memory impairments observed after a 10 h total sleep deprivation period in a contextual memory task (Piérard et al., 2007) and also prevented the 96 h of paradoxical sleep deprivation-induced memory deficits in the multiple trial inhibitory avoidance paradigm (Moreira et al., 2010).
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Concerning its chronic administration, MOD was reported to improve learning (Béracochéa et al., 2002). However many studies have reported cognitive enhancing abilities of MOD (Shuman et al., 2009), the facilitative effects of MOD on memory have not always been verified when memory consolidation was specifically investigated. In fact, a study conducted by Shuman et al. (2009) reported that the administration of MOD immediately after training had no effects on either cued or contextual fear paradigms. Although experimental evidence suggests a possible positive effect of MOD on memory, the facilitatory effects of this drug on memory formation still require systematic experimentation with distinct models of learning and memory. Indeed, most of the facilitatory effects of MOD on learning/memory have been obtained in tasks which were devoid of any emotional component (Béracochéa et al., 2001; Piérard et al., 2007, 2011) or in tasks that involve positive reinforces (Béracochéa et al., 2002, 2003). In this scenario, the investigation of the effects of MOD on the plus-maze discriminative avoidance task (PM-DAT) can be interesting because this animal model can evaluate learning and the retention of an emotional discriminative avoidance task (Gulick and Gould, 2011; Patti et al., 2006, 2010; Sanday et al., 2012; Silva et al., 1997; Silva and Frussa-Filho, 2000; Zanin et al., in press). Thus, the objective of the present study was to investigate the effects of MOD on the different steps of emotional memory formation in the PM-DAT. The present findings provide evidence of potential emotional amnestic proprieties of acutely administered MOD. 2. Material and methods 2.1. Subjects Three-month-old Swiss male mice (raised and maintained in the Centre for Development of Experimental Models in Medicine and Biology of Universidade Federal de São Paulo) were used in the experiments. Animals weighing 30e35 g were housed under controlled temperature (22e23 C) and lighting (12 h light, 12 h dark; lights on at 6:45 a.m.) conditions. Food and water were available ad libitum throughout the experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals used in this study were maintained in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications N 8023, revised 2011) and in accordance with the Brazilian Law for Procedures for Animal Scientific Use (#11794/2008). All experimental procedures were approved by the Ethics Committee under protocol #1162/08. 2.2. Drug MOD (CephalonÒ) was dissolved in 0.5% Arabic gum and intraperitoneally (i.p.) administered in a volume of 10 ml/kg body weight at doses of 32 (MOD32), 64 (MOD64), or 128 mg/kg (MOD128). MOD vehicle was used as the control solution and administered i.p. This dose range was selected based on previous work of our group (Wuo-Silva et al., 2011). 2.3. Behavioral test: PM-DAT The apparatus employed in the PM-DAT is a modified elevated plus-maze made of wood. The apparatus has two enclosed arms with sidewalls and no top (28.5 7 18.5 cm). The enclosed arms are opposite to two open arms (28.5 7 cm). A non-illuminated, 100-W lamp and a hair dryer were placed over the center of one of the enclosed arms (aversive enclosed arm). In the training session, each mouse was placed at the center of the apparatus, and during a 10-min period, the aversive stimuli were administered every time the animal entered the enclosed arm containing the lamp and the hair dryer and was continued until the animal left the arm. The aversive stimuli consisted of both the illumination of the 100-W light and cold air blow produced by the hair dryer. In the test session, which was performed in the same room 10 days after the training, mice were again placed in the center of the apparatus and were observed for 3 min; however, the mice did not receive the aversive stimuli when they entered the aversive enclosed arm even though the non-illuminated lamp and the hair dryer were still placed on the middle of this arm to help distinguish between the aversive and non-aversive arms. In all experiments, the animals were observed in a blind manner, and the apparatus was cleaned with a 5% alcohol solution after each behavioral session. The percent time spent in the aversive enclosed arm (time spent in aversive enclosed arm/time spent
in both enclosed arms 100) was calculated. Learning and memory were evaluated by the percent time spent in the aversive enclosed arm during training and testing, respectively. All the measures taken during the PM-DAT were obtained manually. 2.4. Statistical analysis The comparisons were made using the 1-way analysis of variance (ANOVA) followed by Duncan’s test when necessary. Significance was accepted at p-values less than 0.05. 2.5. Experimental design 2.5.1. Experiment 1: effect of the acute pre-training administration of MOD in mice subjected to the PM-DAT Forty-eight animals were randomly assigned to one of the following groups (n ¼ 12): vehicle, 32 (MOD32), 64 (MOD64) or 128 mg/kg MOD (MOD128). Mice received an acute i.p. administration of vehicle or MOD. Thirty min after the injection, animals were trained in the PM-DAT. Ten days later, they were tested. This experiment was designed to evaluate the effects of acute MOD administration on acquisition and encoding of the emotional discriminative avoidance memory. 2.5.2. Experiment 2: effects of the acute post-training administration of MOD in mice subjected to the PM-DAT Forty-eight animals were randomly assigned to one of the following groups (n ¼ 12): vehicle, 32 (MOD32), 64 (MOD64) or 128 mg/kg MOD (MOD128). Mice were trained in the PM-DAT and, immediately afterwards, received an i.p. administration of vehicle or MOD at different doses. Ten days later, animals were tested. In this experiment, we aimed to investigate the effects of acute MOD administration exclusively on the encoding phase of memory formation. 2.5.3. Experiment 3: effects of the acute pre-test administration of MOD in mice subjected to the PM-DAT Forty-eight animals were randomly assigned to one of the following groups (n ¼ 12): vehicle, 32 (MOD32), 64 (MOD64) or 128 mg/kg MOD (MOD128). Mice were trained in the PM-DAT. Ten days later, the animals received an acute i.p. administration of vehicle or different doses of MOD. Thirty min after the injection, the animals were tested. With pre-test administration, we investigated the effects of acute MOD administration in the retrieval of the emotional memory task. 2.5.4. Experiment 4: role of the state-dependency phenomenon on the cognitive effects of 32 mg/kg MOD in mice subjected to the PM-DAT Forty-eight mice were randomly assigned to one of the following groups (n ¼ 12): pre-training/pre-test administration of vehicle (VEHeVEH), pre-training administration of MOD (MODeVEH), pre-test administration of MOD (VEHeMOD), or pre-training/pre-test administration of MOD (MODeMOD). Groups of 24 mice received vehicle or MOD. Thirty min after the injection, all animals were trained in the PM-DAT. Ten days after the training, 12 animals from the pre-training vehicle group received another vehicle injection, whereas the other 12 mice from the same group received an injection of MOD. Similarly, 12 animals from the pretraining MOD group received a vehicle injection, whereas the other 12 mice received another 32 mg/kg MOD injection. The test session was performed 30 min after the 2nd injection.
3. Results 3.1. Experiment 1: effect of the acute pre-training administration of MOD in mice subjected to the PM-DAT In the training session, the ANOVA revealed that there were no significant differences among groups in the percent time spent in the aversive enclosed arm [F(3,44) ¼ 0.17; p > 0.05] (Fig. 1A). In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test showed that the MOD32 and MOD64 groups spent a significantly longer percent time in the aversive enclosed arm compared to the other groups (the vehicle and MOD128 groups) [F(3,44) ¼ 6.91; p < 0.05] (Fig. 1B). 3.2. Experiment 2: effects of the acute post-training administration of MOD in mice subjected to the PM-DAT In the training session, as expected, the ANOVA revealed that there were no significant basal differences among the groups for the percent time spent in the aversive enclosed arm [F(3,44) ¼ 1.88; p > 0.05] (Fig. 1C).
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Fig. 1. Effects of the acute administration of modafinil on emotional memory of a discriminative avoidance task. Mice received vehicle or 32, 64 or 128 mg/kg modafinil (MOD) before training (A and B), immediately after it (C and D) or before testing (E and F). Results are presented as the mean S.E. of percent time spent in the aversive enclosed arm in the p < 0.05 compared to the VEH group, ◙ p < 0.05 compared to the MOD32 group and p < 0.05 compared to the MOD128 training (A, C and E) and test (B, D and F) sessions. group (ANOVA and Duncan’s test).
In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test for the percent time spent in the aversive enclosed arm revealed that the MOD64 and MOD128 groups presented an increase in this parameter when compared to the vehicle and MOD32 groups [F(3,44) ¼ 5.36; p < 0.05] (Fig. 1D).
In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test revealed that the MOD32 group presented an increased percent time spent in the aversive enclosed when compared to the vehicle group [F(3,44) ¼ 2.37; p < 0.05] (Fig. 1F).
3.3. Experiment 3: effects of the acute pre-test administration of MOD in mice subjected to the PM-DAT
3.4. Experiment 4: role of the state-dependency phenomenon on the cognitive effects of MOD using the PM-DAT
As expected, the ANOVA revealed that there were no significant basal differences among groups for the percent time spent in the aversive enclosed arm in the training session [F(3,44) ¼ 0.43; p > 0.05] (Fig. 1E).
In the training session, the ANOVA revealed that there were no significant differences among groups when the percent time spent in the aversive enclosed arm was analyzed [F(3,44) ¼ 1.10; p > 0.05] (Fig. 2A).
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Fig. 2. Effects of pre-training and/or pre-test administration of modafinil in a discriminative avoidance task. Mice were treated with vehicle (VEH) or 32 mg/kg modafinil (MOD) 30 min before the training session and tested, 10 days later, 30 min after an injection of VEH or 32 mg/kg (MOD). Results are presented as the mean S.E. of percent time spent in the aversive enclosed arm in the training (A) and test (B) sessions. p < 0.05 compared to VEHeVEH group (ANOVA and Duncan’s test).
In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test showed that the MODeVEH, VEHeMOD and MODeMOD groups had a significant increase in the percent time in the aversive enclosed arm compared to the VEH-treated group [F(3,44) ¼ 3.51; p < 0.05] (Fig. 2B). 4. Discussion The main findings of the present study were the following: 1) acute pre-training administration of MOD (32, 64 or 128 mg/kg) did not impair learning but it hindered retention at doses of 32 and 64 mg/kg; 2) acutely administered 64 or 128 mg/kg MOD, but not 32 mg/kg, impaired the consolidation of the emotional discriminative avoidance task in mice; 3) when the drug was given before testing, only the lowest dose (32 mg/kg) was able to impair retrieval; 4) finally, the emotional memory deficits induced by the pre-training or pre-test administrations of 32 mg/kg MOD were not state-dependent. In the PM-DAT, learning can be assessed by the magnitude of the avoidance of the aversive enclosed arm in the training session (Patti et al., 2010). The storage of the task (and, consequently, the processes of consolidation and recall) is detected by the percent time spent in the aversive enclosed arm in the testing. In this context, the avoidance of the aversive enclosed arm upon testing has been validated as a measurement of retention because amnestic manipulations decrease it (Alvarenga et al., 2008; Claro et al., 1999; Kameda et al., 2007; Patti et al., 2010; Sanday et al., 2012; Silva and Frussa-Filho, 2000, 2002; Silva et al., 1999, 2002, 2004; Zanin et al., in press). In contrast, memory-improving treatments increase such avoidance (Claro et al., 1999; Silva et al., 1997, 1999, 2000). In the 1st experiment, we aimed to evaluate the effects of the acute administration of MOD on learning of an emotional discriminative task. Previous work has shown that the pre-training administration of 75 mg/kg MOD improved performance of rats in the Morris water maze (Shuman et al., 2009). Additionally, mounting evidence suggests that MOD is able to facilitate learning depending on the complexity of the task. Accordingly, Béracochéa et al. (2001) found that the administration of 64 mg/kg MOD (but not 8 or 32 mg/kg) improved the T-maze performance in mice. They also described that the greater the interval between training and testing, which makes the tasks more complex, the better the performance in animals treated with MOD, suggesting an interaction between task complexity and the possible cognitive effects of MOD (Béracochéa et al., 2002, 2003). In the present study, no learning modifications were induced by any doses of MOD. In this
sense, we could raise 2 possibilities: 1) as the complexity of the task is a critical factor for learning, the aversive nature of the discriminative avoidance could have critically contributed to an absence of a hypothetical facilitatory effect; 2) such hypothetical facilitatory effect of MOD on learning cannot be discarded because of a possible floor effect. In other words, the absence of a facilitatory effect could have resulted from a low percent time spent in the aversive enclosed arm presented by the VEH groups, preventing a decrement of the exploration of the aversive arm displayed by MODtreated mice. The pre-training administration of 32 or 64 mg/kg (but not 128 mg/kg) MOD impaired emotional memory. In fact, animals acutely treated with the lower doses of the drug showed an increased exploration of the aversive enclosed arm in the test session, suggesting amnestic effects of MOD. Conversely, the dose of 128 mg/kg MOD did not affect the pattern of exploration of the aversive arm. These findings suggest that the pre-training acute administration of MOD promoted emotional memory deficits in an inverted-U shape fashion. Notwithstanding our findings are not in agreement with previous studies in rodents (Béracochéa et al., 2001, 2002, 2003, 2008; Morgan et al., 2007; Piérard et al., 2006, 2007, 2011; Tsanov et al., 2010; Ward et al., 2004), but they seem to be in line with other studies describing no effects (Waters et al., 2005) or even MOD-induced deleterious effects (Burgos et al., 2010; Shuman et al., 2009) on memory. In this way, Shuman et al. (2009) have verified that pre-training 75 mg/kg MOD disrupted contextual-fear whereas it did not impair cued-fear conditioning or benefitted performance of mice in the Morris water maze. In addition, the memory-enhancing effect of MOD has been demonstrated on working or episodic-like memory tasks (Béracochéa et al., 2001), even after sleep deprivation (Piérard et al., 2007, 2011), which were devoid of any emotional aversive components and which involve flexible cognitive processes. Moreover, the same authors found that the memory enhancingeffect of MOD was modulated by interaction with the stressinduced corticosterone increase (Piérard et al., 2006). Taken together, these results suggest that the cognitive effects of MOD seem to be dependent on the task employed. Specifically, this apparent discrepancy could be due to the difference between the emotional component of the tasks (positive or aversive reinforces) and the nature of the task. When drug administration occurs before training, it can affect memory by altering learning or consolidation processes. On the other hand, when the drug is given exclusively after training, it can only exert its effects on the consolidation phase. Once MOD induced emotional memory impairment in experiment 1 without
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modifying learning ability, we conducted experiment 2 to investigate the effects of acute post-training MOD administration on memory consolidation. In this case, MOD was given immediately after training. The results showed that the post-training acute administration of 64 or 128 mg/kg MOD promoted amnesia because the animals spent considerable more time in the aversive enclosed arm during the testing. To the best of our knowledge, only the study of Shuman et al. (2009) systematically investigated the effects of MOD on memory consolidation. These authors have reported that MOD was ineffective at modifying the consolidation of context- or cued-conditioned fear tasks in mice. The discrepant findings could lie on the memory tasks employed and the MOD doses used in both studies. While we have examined 32, 64 or 128 mg/kg MOD, in that study they examined the effects of 0.075e75 mg/kg of this drug. In the analysis of a possible effect of MOD on retrieval (experiment 3), only the pre-test acute administration of 32 mg/kg MOD lead to retrieval deficits. The reported effects of MOD on retrieval in the literature are controversial. Thus, while an absence of effect of MOD on retrieval was observed in the Morris water maze task (Shuman et al., 2009), pre-test MOD administration was effective in restoring sleep-deprivation induced memory deficits (Moreira et al., 2010; Piérard et al., 2011). While both 64 and 128 mg/kg MOD doses negatively affect the consolidation of the emotional memory, the lower studied dose did not exert any effect on this phase. Conversely, the dose of 32 mg/kg MOD induced memory impairment when given before training or testing only. Within this context, it could be argued that the impairing effects of 32 mg/kg MOD on emotional memory would be state-dependent. In this vein, memory can be state-dependent, in that a response that has been acquired in a given (i.e., drug-induced) state may not be retrieved when the organism is in a different state (Colpaert et al., 2001). Our data have indicated that the dose of 32 mg/kg MOD did not impair consolidation (experiment 2) but it did induce memory deficits when administered exclusively before training (experiment 1) or testing (experiment 3), suggesting a possible state-dependent effect. In this concern, in experiment 4 we attempted to investigate the effects of 32 mg/kg MOD administered before both training and test sessions. It was shown that the pre-test administration of 32 mg/kg MOD did not counteract the pre-training-induced amnestic effects of this drug, discarding the involvement of the state-dependency phenomenon on MOD-induced emotional memory impairment. Several previous studies have shown that stress exposure has complex effects on cognition. For the acquisition/consolidation of information, the effects of stress and/or glucocorticoids follow an inverted U-shaped doseeresponse relationship, in which high levels impair memory and moderate activation seems to be a prerequisite for the long-term storage of information (Akirav et al., 2004; de Quervain et al., 2009; Kogan and Richter-Levin, 2010; Sandi and Pinelo-Nava, 2007). In parallel, stress and/or glucocorticoids are likely to be involved in the mechanism of MOD action on memory processes. In fact, Piérard et al. (2006) demonstrated that corticosterone was significantly increased after 16 or 32 mg/kg MOD administration in non-stressed mice, while under stress conditions, the corticosterone level was decreased to 8 mg/kg and remained unchanged in response to 16 or 32 mg/kg MOD. Importantly, while the exposure to the traditional elevated plus-maze is a stressful stimulus that enhances plasma corticosterone concentration in rodents (File et al., 1994), the PM-DAT possesses additional intrinsic stress levels because of the aversive stimulation (light and air blow). Thus, the fact that pre-training, post-training and pre-test MOD administration produced impairment of emotional memory at different range doses could be related to the specific stress levels during these
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different experimental moments (i.e., before/during the training, after it and before/during testing) and their interactions with MOD effects. Alternatively, it has been demonstrated that MOD inhibits the dopamine transporter with exceptional selectivity (Mignot et al., 1994). The initial report, which was obtained in guinea pig striatum, was reproduced in the monkey brain in vivo and in human embryonic kidney cells in vitro (Madras et al., 2006). Additionally, recent evidence indicates that MOD increases extracellular dopamine in the rat (Zolkowska et al., 2009), monkey (Andersen et al., 2010) and human brain (Volkow et al., 2009). As hippocampal dopamine modulates long-term memory encoding and consolidation during or early after training (Jay, 2003; O’Carroll et al., 2006), it could be hypothesized that after MOD administration, the dopamine-dependent mechanisms in the hippocampus that favor memory encoding during or early after training might be attenuated, thereby leading to long-term memory deficits in the present emotional discriminative avoidance task. Surprisingly, the present findings provide evidence of potential amnesic properties of MOD. This unusual finding could be explained by the aversive character of the PM-DAT and the associative conditioning nature of the task. Within this context, the MOD-induced amnesia of aversive emotional memory may lend a new perspective for posttraumatic stress disorder (PTSD) treatment. Specifically, because PTSD is characterized by a pathological reverberation of aversive and stressful memories, MOD could become a potential treatment for this disorder. Collectively, our data suggest that MOD has amnestic effects that are dramatically dependent on the dose and the timing of administration. Importantly, these observed emotional memory deficits were not related to state-dependency. These results point to the need of caution in prescribing this drug. Disclosure statement All authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work that could inappropriately influence (bias) the present work. All authors critically reviewed content and approved final version for publication. Authorship HAF, CLP, RWS and RF-F were responsible for the study concept and design. HAF, LFS and KAZ contributed to the acquisition of animal data. HAF, KAZ, RWS, LFS and RF-F assisted with data analysis and interpretation of findings. HAF, CLP, KAZ and RF-F drafted the manuscript. LRAB, RCC and ST provided critical revision of the manuscript for important intellectual content. Acknowledgments This research was supported by fellowships from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP e CEPID grant #1998/14303-3), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Associação Fundo de Incentivo à Pesquisa (AFIP). The English language in this manuscript has been proofread and edited by American Journal Experts (AJE), but we are entirely responsible for the scientific content of the paper. The authors would like to thank Ms. Teotila R. R. Amaral, Ms. Claudenice M. dos Santos, Mr. Cleomar S. Ferreira and Mr. Antonio Rodrigues dos Santos for their capable technical assistance. R.F.F., S.T. and L.R.A.B. are recipients of the CNPq fellowship.
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Patti, C.L., Kameda, S.R., Carvalho, R.C., Takatsu-Coleman, A.L., Lopez, G.B., Niigaki, S.T., Abílio, V.C., Frussa-Filho, R., Silva, R.H., 2006. Effects of morphine on the plus-maze discriminative avoidance task: role of state-dependent learning. Psychopharmacology (Berl) 184, 1e12. Patti, C.L., Zanin, K.A., Sanday, L., Kameda, S.R., Fernandes-Santos, L., Fernandes, H.A., Andersen, M.L., Tufik, S., Frussa-Filho, R., 2010. Effects of sleep deprivation on memory in mice: role of state-dependent learning. Sleep 33, 1669e1679. Piérard, C., Liscia, P., Chauveau, F., Coutan, M., Corio, M., Krazem, A., Beracochea, D., 2011. Differential effects of total sleep deprivation on contextual and spatial memory: modulatory effects of modafinil. Pharmacol. Biochem. Behav. 97, 399e405. Piérard, C., Liscia, P., Philippin, J.N., Mons, N., Lafon, T., Chauveau, F., Beers, P.V., Drouet, I., Serra, A., Jouanin, J.C., Béracochéa, D., 2007. Modafinil restores memory performance and neural activity impaired by sleep deprivation in mice. Pharmacol. Biochem. Behav. 88, 55e63. Piérard, C., Liscia, P., Valleau, M., Drouet, I., Chauveau, F., Huart, B., Bonneau, D., Jouanin, J.C., Beaumont, M., Béracochéa, D., 2006. Modafinil-induced modulation of working memory and plasma corticosterone in chronically-stressed mice. Pharmacol. Biochem. Behav. 83, 1e8. Sanday, L., Zanin, K.A., Patti, C.L., Tufik, S., Frussa-Filho, R., 2012. Role of statedependency in memory impairment induced by acute administration of midazolam in mice. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 37, 1e7. Sandi, C., Pinelo-Nava, M.T., 2007. Stress and memory: behavioral effects and neurobiological mechanisms. Neural Plast. 2007, 78970. Shuman, T., Wood, S.C., Anagnostaras, S.G., 2009. Modafinil and memory: effects of modafinil on Morris water maze learning and Pavlovian fear conditioning. Behav. Neurosci. 123, 257e266. 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Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm
Inhibitory effects of modafinil on emotional memory in mice Helaine A. Fernandes a, b,1, Karina A. Zanin a, b,1, Camilla L. Patti a, b, *, Raphael Wuo-Silva b, Rita C. Carvalho b, Luciano Fernandes-Santos a, b, Lia R.A. Bittencourt a, Sergio Tufik a, Roberto Frussa-Filho a, b, * a b
Departamento de Psicobiologia, Universidade Federal de São Paulo, R. Napoleão de Barros 925, São Paulo, SP, Brazil Departamento de Farmacologia, Universidade Federal de São Paulo, Rua Botucatu 862, Ed. Leal Prado, 1 andar, 04023062 São Paulo, SP, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 March 2012 Received in revised form 5 June 2012 Accepted 27 June 2012
Modafinil (MOD), a psychostimulant used to treat narcolepsy, excessive daytime sleepiness, and sleepiness due to obstructive sleep apnea, appears to promote a possible facilitatory effect on cognitive function. In the present study, we investigated the effects of the acute administration of MOD on the different steps of emotional memory formation and usage (acquisition, consolidation and retrieval) as well as the possible participation of the state-dependency phenomenon on the cognitive effects of this compound. Mice were acutely treated with 32, 64 or 128 mg/kg MOD before training or testing or immediately after training and were subjected to the plus-maze discriminative avoidance task. The results showed that although pre-training MOD administration did not exert any effects on learning, the doses of 32 or 64 mg/kg induced emotional memory deficits during testing. Still, the post-training acute administration of the higher doses of MOD (64 and 128 mg/kg) impaired associative memory consolidation. When the drug was administered pre-test, only the 32 mg/kg dose impaired the task retrieval. Importantly, the cognitive impairing effects induced by 32 mg/kg MOD were not related to the phenomenon of state-dependency. In all, our findings provide pre-clinical evidence of potential emotional memory amnesia induced by MOD. This article is part of a Special Issue entitled ‘Cognitive Enhancers’. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Modafinil Learning Consolidation Retrieval State-dependency Plus-maze discriminative avoidance task
1. Introduction Modafinil (MOD) is a psychostimulant that acts as a wakepromoting drug and has been approved for the treatment of excessive daytime sleepiness in narcolepsy, obstructive sleep apnea, and shift work syndrome. Clinical studies have pointed MOD positive effects when used to treat Parkinson’s disease (Ferraro et al., 1998; Nieves and Lang, 2002), multiple sclerosis (Kraft and Bowen, 2005), schizophrenia (Turner et al., 2004) and attention deficit hyperactivity disorder (Taylor and Russo, 2000). Additionally, MOD seems to be widely prescribed off-label to enhance alertness, attention, memory for dementia and depression (Joos et al., 2010). Of note, an illicit market exists for academic doping as well (Cakic, 2009). * Corresponding authors. Departamento de Farmacologia, Universidade Federal de São Paulo, Rua Botucatu 862, Ed. Leal Prado, 1 andar, 04023062 São Paulo, SP, Brazil. Tel.: þ55 11 5549 4122; fax: þ55 11 5549 4122x222. E-mail addresses: [email protected] (C.L. Patti), [email protected] (R. Frussa-Filho). 1 These authors contributed equally to this study. 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.06.058
There are several hypotheses that attempt to explain the mechanisms of action of MOD. Studies have suggested that it may increase the release of catecholamines (dopamine and norepinephrine), serotonin and glutamate, thereby reducing the release of GABA in various brain regions, and activating hypothalamic neurons containing hypocretin/orexin neurons of the tuberomammillary nucleus (Minzemberg and Carter, 2008). It has been demonstrated that MOD modifies the activity of brain areas involved with memory, such as the hippocampus and prefrontal cortex (Béracochéa et al., 2003). Within this context, studies indicate that MOD has cognitive-enhancing abilities in rodents performing a variety of learning/memory exercises in the T-Maze based on spontaneous alternation behavior (Béracochéa et al., 2001), and also enhanced learning (Béracochéa et al., 2003) and memory retrieval (Béracochéa et al., 2008). Still, it was reported that this drug was able to restore the memory impairments observed after a 10 h total sleep deprivation period in a contextual memory task (Piérard et al., 2007) and also prevented the 96 h of paradoxical sleep deprivation-induced memory deficits in the multiple trial inhibitory avoidance paradigm (Moreira et al., 2010).
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Concerning its chronic administration, MOD was reported to improve learning (Béracochéa et al., 2002). However many studies have reported cognitive enhancing abilities of MOD (Shuman et al., 2009), the facilitative effects of MOD on memory have not always been verified when memory consolidation was specifically investigated. In fact, a study conducted by Shuman et al. (2009) reported that the administration of MOD immediately after training had no effects on either cued or contextual fear paradigms. Although experimental evidence suggests a possible positive effect of MOD on memory, the facilitatory effects of this drug on memory formation still require systematic experimentation with distinct models of learning and memory. Indeed, most of the facilitatory effects of MOD on learning/memory have been obtained in tasks which were devoid of any emotional component (Béracochéa et al., 2001; Piérard et al., 2007, 2011) or in tasks that involve positive reinforces (Béracochéa et al., 2002, 2003). In this scenario, the investigation of the effects of MOD on the plus-maze discriminative avoidance task (PM-DAT) can be interesting because this animal model can evaluate learning and the retention of an emotional discriminative avoidance task (Gulick and Gould, 2011; Patti et al., 2006, 2010; Sanday et al., 2012; Silva et al., 1997; Silva and Frussa-Filho, 2000; Zanin et al., in press). Thus, the objective of the present study was to investigate the effects of MOD on the different steps of emotional memory formation in the PM-DAT. The present findings provide evidence of potential emotional amnestic proprieties of acutely administered MOD. 2. Material and methods 2.1. Subjects Three-month-old Swiss male mice (raised and maintained in the Centre for Development of Experimental Models in Medicine and Biology of Universidade Federal de São Paulo) were used in the experiments. Animals weighing 30e35 g were housed under controlled temperature (22e23 C) and lighting (12 h light, 12 h dark; lights on at 6:45 a.m.) conditions. Food and water were available ad libitum throughout the experiments. All efforts were made to minimize animal suffering and to reduce the number of animals used. The animals used in this study were maintained in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications N 8023, revised 2011) and in accordance with the Brazilian Law for Procedures for Animal Scientific Use (#11794/2008). All experimental procedures were approved by the Ethics Committee under protocol #1162/08. 2.2. Drug MOD (CephalonÒ) was dissolved in 0.5% Arabic gum and intraperitoneally (i.p.) administered in a volume of 10 ml/kg body weight at doses of 32 (MOD32), 64 (MOD64), or 128 mg/kg (MOD128). MOD vehicle was used as the control solution and administered i.p. This dose range was selected based on previous work of our group (Wuo-Silva et al., 2011). 2.3. Behavioral test: PM-DAT The apparatus employed in the PM-DAT is a modified elevated plus-maze made of wood. The apparatus has two enclosed arms with sidewalls and no top (28.5 7 18.5 cm). The enclosed arms are opposite to two open arms (28.5 7 cm). A non-illuminated, 100-W lamp and a hair dryer were placed over the center of one of the enclosed arms (aversive enclosed arm). In the training session, each mouse was placed at the center of the apparatus, and during a 10-min period, the aversive stimuli were administered every time the animal entered the enclosed arm containing the lamp and the hair dryer and was continued until the animal left the arm. The aversive stimuli consisted of both the illumination of the 100-W light and cold air blow produced by the hair dryer. In the test session, which was performed in the same room 10 days after the training, mice were again placed in the center of the apparatus and were observed for 3 min; however, the mice did not receive the aversive stimuli when they entered the aversive enclosed arm even though the non-illuminated lamp and the hair dryer were still placed on the middle of this arm to help distinguish between the aversive and non-aversive arms. In all experiments, the animals were observed in a blind manner, and the apparatus was cleaned with a 5% alcohol solution after each behavioral session. The percent time spent in the aversive enclosed arm (time spent in aversive enclosed arm/time spent
in both enclosed arms 100) was calculated. Learning and memory were evaluated by the percent time spent in the aversive enclosed arm during training and testing, respectively. All the measures taken during the PM-DAT were obtained manually. 2.4. Statistical analysis The comparisons were made using the 1-way analysis of variance (ANOVA) followed by Duncan’s test when necessary. Significance was accepted at p-values less than 0.05. 2.5. Experimental design 2.5.1. Experiment 1: effect of the acute pre-training administration of MOD in mice subjected to the PM-DAT Forty-eight animals were randomly assigned to one of the following groups (n ¼ 12): vehicle, 32 (MOD32), 64 (MOD64) or 128 mg/kg MOD (MOD128). Mice received an acute i.p. administration of vehicle or MOD. Thirty min after the injection, animals were trained in the PM-DAT. Ten days later, they were tested. This experiment was designed to evaluate the effects of acute MOD administration on acquisition and encoding of the emotional discriminative avoidance memory. 2.5.2. Experiment 2: effects of the acute post-training administration of MOD in mice subjected to the PM-DAT Forty-eight animals were randomly assigned to one of the following groups (n ¼ 12): vehicle, 32 (MOD32), 64 (MOD64) or 128 mg/kg MOD (MOD128). Mice were trained in the PM-DAT and, immediately afterwards, received an i.p. administration of vehicle or MOD at different doses. Ten days later, animals were tested. In this experiment, we aimed to investigate the effects of acute MOD administration exclusively on the encoding phase of memory formation. 2.5.3. Experiment 3: effects of the acute pre-test administration of MOD in mice subjected to the PM-DAT Forty-eight animals were randomly assigned to one of the following groups (n ¼ 12): vehicle, 32 (MOD32), 64 (MOD64) or 128 mg/kg MOD (MOD128). Mice were trained in the PM-DAT. Ten days later, the animals received an acute i.p. administration of vehicle or different doses of MOD. Thirty min after the injection, the animals were tested. With pre-test administration, we investigated the effects of acute MOD administration in the retrieval of the emotional memory task. 2.5.4. Experiment 4: role of the state-dependency phenomenon on the cognitive effects of 32 mg/kg MOD in mice subjected to the PM-DAT Forty-eight mice were randomly assigned to one of the following groups (n ¼ 12): pre-training/pre-test administration of vehicle (VEHeVEH), pre-training administration of MOD (MODeVEH), pre-test administration of MOD (VEHeMOD), or pre-training/pre-test administration of MOD (MODeMOD). Groups of 24 mice received vehicle or MOD. Thirty min after the injection, all animals were trained in the PM-DAT. Ten days after the training, 12 animals from the pre-training vehicle group received another vehicle injection, whereas the other 12 mice from the same group received an injection of MOD. Similarly, 12 animals from the pretraining MOD group received a vehicle injection, whereas the other 12 mice received another 32 mg/kg MOD injection. The test session was performed 30 min after the 2nd injection.
3. Results 3.1. Experiment 1: effect of the acute pre-training administration of MOD in mice subjected to the PM-DAT In the training session, the ANOVA revealed that there were no significant differences among groups in the percent time spent in the aversive enclosed arm [F(3,44) ¼ 0.17; p > 0.05] (Fig. 1A). In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test showed that the MOD32 and MOD64 groups spent a significantly longer percent time in the aversive enclosed arm compared to the other groups (the vehicle and MOD128 groups) [F(3,44) ¼ 6.91; p < 0.05] (Fig. 1B). 3.2. Experiment 2: effects of the acute post-training administration of MOD in mice subjected to the PM-DAT In the training session, as expected, the ANOVA revealed that there were no significant basal differences among the groups for the percent time spent in the aversive enclosed arm [F(3,44) ¼ 1.88; p > 0.05] (Fig. 1C).
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Fig. 1. Effects of the acute administration of modafinil on emotional memory of a discriminative avoidance task. Mice received vehicle or 32, 64 or 128 mg/kg modafinil (MOD) before training (A and B), immediately after it (C and D) or before testing (E and F). Results are presented as the mean S.E. of percent time spent in the aversive enclosed arm in the p < 0.05 compared to the VEH group, ◙ p < 0.05 compared to the MOD32 group and p < 0.05 compared to the MOD128 training (A, C and E) and test (B, D and F) sessions. group (ANOVA and Duncan’s test).
In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test for the percent time spent in the aversive enclosed arm revealed that the MOD64 and MOD128 groups presented an increase in this parameter when compared to the vehicle and MOD32 groups [F(3,44) ¼ 5.36; p < 0.05] (Fig. 1D).
In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test revealed that the MOD32 group presented an increased percent time spent in the aversive enclosed when compared to the vehicle group [F(3,44) ¼ 2.37; p < 0.05] (Fig. 1F).
3.3. Experiment 3: effects of the acute pre-test administration of MOD in mice subjected to the PM-DAT
3.4. Experiment 4: role of the state-dependency phenomenon on the cognitive effects of MOD using the PM-DAT
As expected, the ANOVA revealed that there were no significant basal differences among groups for the percent time spent in the aversive enclosed arm in the training session [F(3,44) ¼ 0.43; p > 0.05] (Fig. 1E).
In the training session, the ANOVA revealed that there were no significant differences among groups when the percent time spent in the aversive enclosed arm was analyzed [F(3,44) ¼ 1.10; p > 0.05] (Fig. 2A).
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Fig. 2. Effects of pre-training and/or pre-test administration of modafinil in a discriminative avoidance task. Mice were treated with vehicle (VEH) or 32 mg/kg modafinil (MOD) 30 min before the training session and tested, 10 days later, 30 min after an injection of VEH or 32 mg/kg (MOD). Results are presented as the mean S.E. of percent time spent in the aversive enclosed arm in the training (A) and test (B) sessions. p < 0.05 compared to VEHeVEH group (ANOVA and Duncan’s test).
In the test session, performed 10 days after training, the ANOVA followed by Duncan’s test showed that the MODeVEH, VEHeMOD and MODeMOD groups had a significant increase in the percent time in the aversive enclosed arm compared to the VEH-treated group [F(3,44) ¼ 3.51; p < 0.05] (Fig. 2B). 4. Discussion The main findings of the present study were the following: 1) acute pre-training administration of MOD (32, 64 or 128 mg/kg) did not impair learning but it hindered retention at doses of 32 and 64 mg/kg; 2) acutely administered 64 or 128 mg/kg MOD, but not 32 mg/kg, impaired the consolidation of the emotional discriminative avoidance task in mice; 3) when the drug was given before testing, only the lowest dose (32 mg/kg) was able to impair retrieval; 4) finally, the emotional memory deficits induced by the pre-training or pre-test administrations of 32 mg/kg MOD were not state-dependent. In the PM-DAT, learning can be assessed by the magnitude of the avoidance of the aversive enclosed arm in the training session (Patti et al., 2010). The storage of the task (and, consequently, the processes of consolidation and recall) is detected by the percent time spent in the aversive enclosed arm in the testing. In this context, the avoidance of the aversive enclosed arm upon testing has been validated as a measurement of retention because amnestic manipulations decrease it (Alvarenga et al., 2008; Claro et al., 1999; Kameda et al., 2007; Patti et al., 2010; Sanday et al., 2012; Silva and Frussa-Filho, 2000, 2002; Silva et al., 1999, 2002, 2004; Zanin et al., in press). In contrast, memory-improving treatments increase such avoidance (Claro et al., 1999; Silva et al., 1997, 1999, 2000). In the 1st experiment, we aimed to evaluate the effects of the acute administration of MOD on learning of an emotional discriminative task. Previous work has shown that the pre-training administration of 75 mg/kg MOD improved performance of rats in the Morris water maze (Shuman et al., 2009). Additionally, mounting evidence suggests that MOD is able to facilitate learning depending on the complexity of the task. Accordingly, Béracochéa et al. (2001) found that the administration of 64 mg/kg MOD (but not 8 or 32 mg/kg) improved the T-maze performance in mice. They also described that the greater the interval between training and testing, which makes the tasks more complex, the better the performance in animals treated with MOD, suggesting an interaction between task complexity and the possible cognitive effects of MOD (Béracochéa et al., 2002, 2003). In the present study, no learning modifications were induced by any doses of MOD. In this
sense, we could raise 2 possibilities: 1) as the complexity of the task is a critical factor for learning, the aversive nature of the discriminative avoidance could have critically contributed to an absence of a hypothetical facilitatory effect; 2) such hypothetical facilitatory effect of MOD on learning cannot be discarded because of a possible floor effect. In other words, the absence of a facilitatory effect could have resulted from a low percent time spent in the aversive enclosed arm presented by the VEH groups, preventing a decrement of the exploration of the aversive arm displayed by MODtreated mice. The pre-training administration of 32 or 64 mg/kg (but not 128 mg/kg) MOD impaired emotional memory. In fact, animals acutely treated with the lower doses of the drug showed an increased exploration of the aversive enclosed arm in the test session, suggesting amnestic effects of MOD. Conversely, the dose of 128 mg/kg MOD did not affect the pattern of exploration of the aversive arm. These findings suggest that the pre-training acute administration of MOD promoted emotional memory deficits in an inverted-U shape fashion. Notwithstanding our findings are not in agreement with previous studies in rodents (Béracochéa et al., 2001, 2002, 2003, 2008; Morgan et al., 2007; Piérard et al., 2006, 2007, 2011; Tsanov et al., 2010; Ward et al., 2004), but they seem to be in line with other studies describing no effects (Waters et al., 2005) or even MOD-induced deleterious effects (Burgos et al., 2010; Shuman et al., 2009) on memory. In this way, Shuman et al. (2009) have verified that pre-training 75 mg/kg MOD disrupted contextual-fear whereas it did not impair cued-fear conditioning or benefitted performance of mice in the Morris water maze. In addition, the memory-enhancing effect of MOD has been demonstrated on working or episodic-like memory tasks (Béracochéa et al., 2001), even after sleep deprivation (Piérard et al., 2007, 2011), which were devoid of any emotional aversive components and which involve flexible cognitive processes. Moreover, the same authors found that the memory enhancingeffect of MOD was modulated by interaction with the stressinduced corticosterone increase (Piérard et al., 2006). Taken together, these results suggest that the cognitive effects of MOD seem to be dependent on the task employed. Specifically, this apparent discrepancy could be due to the difference between the emotional component of the tasks (positive or aversive reinforces) and the nature of the task. When drug administration occurs before training, it can affect memory by altering learning or consolidation processes. On the other hand, when the drug is given exclusively after training, it can only exert its effects on the consolidation phase. Once MOD induced emotional memory impairment in experiment 1 without
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modifying learning ability, we conducted experiment 2 to investigate the effects of acute post-training MOD administration on memory consolidation. In this case, MOD was given immediately after training. The results showed that the post-training acute administration of 64 or 128 mg/kg MOD promoted amnesia because the animals spent considerable more time in the aversive enclosed arm during the testing. To the best of our knowledge, only the study of Shuman et al. (2009) systematically investigated the effects of MOD on memory consolidation. These authors have reported that MOD was ineffective at modifying the consolidation of context- or cued-conditioned fear tasks in mice. The discrepant findings could lie on the memory tasks employed and the MOD doses used in both studies. While we have examined 32, 64 or 128 mg/kg MOD, in that study they examined the effects of 0.075e75 mg/kg of this drug. In the analysis of a possible effect of MOD on retrieval (experiment 3), only the pre-test acute administration of 32 mg/kg MOD lead to retrieval deficits. The reported effects of MOD on retrieval in the literature are controversial. Thus, while an absence of effect of MOD on retrieval was observed in the Morris water maze task (Shuman et al., 2009), pre-test MOD administration was effective in restoring sleep-deprivation induced memory deficits (Moreira et al., 2010; Piérard et al., 2011). While both 64 and 128 mg/kg MOD doses negatively affect the consolidation of the emotional memory, the lower studied dose did not exert any effect on this phase. Conversely, the dose of 32 mg/kg MOD induced memory impairment when given before training or testing only. Within this context, it could be argued that the impairing effects of 32 mg/kg MOD on emotional memory would be state-dependent. In this vein, memory can be state-dependent, in that a response that has been acquired in a given (i.e., drug-induced) state may not be retrieved when the organism is in a different state (Colpaert et al., 2001). Our data have indicated that the dose of 32 mg/kg MOD did not impair consolidation (experiment 2) but it did induce memory deficits when administered exclusively before training (experiment 1) or testing (experiment 3), suggesting a possible state-dependent effect. In this concern, in experiment 4 we attempted to investigate the effects of 32 mg/kg MOD administered before both training and test sessions. It was shown that the pre-test administration of 32 mg/kg MOD did not counteract the pre-training-induced amnestic effects of this drug, discarding the involvement of the state-dependency phenomenon on MOD-induced emotional memory impairment. Several previous studies have shown that stress exposure has complex effects on cognition. For the acquisition/consolidation of information, the effects of stress and/or glucocorticoids follow an inverted U-shaped doseeresponse relationship, in which high levels impair memory and moderate activation seems to be a prerequisite for the long-term storage of information (Akirav et al., 2004; de Quervain et al., 2009; Kogan and Richter-Levin, 2010; Sandi and Pinelo-Nava, 2007). In parallel, stress and/or glucocorticoids are likely to be involved in the mechanism of MOD action on memory processes. In fact, Piérard et al. (2006) demonstrated that corticosterone was significantly increased after 16 or 32 mg/kg MOD administration in non-stressed mice, while under stress conditions, the corticosterone level was decreased to 8 mg/kg and remained unchanged in response to 16 or 32 mg/kg MOD. Importantly, while the exposure to the traditional elevated plus-maze is a stressful stimulus that enhances plasma corticosterone concentration in rodents (File et al., 1994), the PM-DAT possesses additional intrinsic stress levels because of the aversive stimulation (light and air blow). Thus, the fact that pre-training, post-training and pre-test MOD administration produced impairment of emotional memory at different range doses could be related to the specific stress levels during these
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different experimental moments (i.e., before/during the training, after it and before/during testing) and their interactions with MOD effects. Alternatively, it has been demonstrated that MOD inhibits the dopamine transporter with exceptional selectivity (Mignot et al., 1994). The initial report, which was obtained in guinea pig striatum, was reproduced in the monkey brain in vivo and in human embryonic kidney cells in vitro (Madras et al., 2006). Additionally, recent evidence indicates that MOD increases extracellular dopamine in the rat (Zolkowska et al., 2009), monkey (Andersen et al., 2010) and human brain (Volkow et al., 2009). As hippocampal dopamine modulates long-term memory encoding and consolidation during or early after training (Jay, 2003; O’Carroll et al., 2006), it could be hypothesized that after MOD administration, the dopamine-dependent mechanisms in the hippocampus that favor memory encoding during or early after training might be attenuated, thereby leading to long-term memory deficits in the present emotional discriminative avoidance task. Surprisingly, the present findings provide evidence of potential amnesic properties of MOD. This unusual finding could be explained by the aversive character of the PM-DAT and the associative conditioning nature of the task. Within this context, the MOD-induced amnesia of aversive emotional memory may lend a new perspective for posttraumatic stress disorder (PTSD) treatment. Specifically, because PTSD is characterized by a pathological reverberation of aversive and stressful memories, MOD could become a potential treatment for this disorder. Collectively, our data suggest that MOD has amnestic effects that are dramatically dependent on the dose and the timing of administration. Importantly, these observed emotional memory deficits were not related to state-dependency. These results point to the need of caution in prescribing this drug. Disclosure statement All authors disclose any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations within three years of beginning the work that could inappropriately influence (bias) the present work. All authors critically reviewed content and approved final version for publication. Authorship HAF, CLP, RWS and RF-F were responsible for the study concept and design. HAF, LFS and KAZ contributed to the acquisition of animal data. HAF, KAZ, RWS, LFS and RF-F assisted with data analysis and interpretation of findings. HAF, CLP, KAZ and RF-F drafted the manuscript. LRAB, RCC and ST provided critical revision of the manuscript for important intellectual content. Acknowledgments This research was supported by fellowships from Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP e CEPID grant #1998/14303-3), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Associação Fundo de Incentivo à Pesquisa (AFIP). The English language in this manuscript has been proofread and edited by American Journal Experts (AJE), but we are entirely responsible for the scientific content of the paper. The authors would like to thank Ms. Teotila R. R. Amaral, Ms. Claudenice M. dos Santos, Mr. Cleomar S. Ferreira and Mr. Antonio Rodrigues dos Santos for their capable technical assistance. R.F.F., S.T. and L.R.A.B. are recipients of the CNPq fellowship.
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