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Early life stress decreases hippocampal BDNF content and exacerbates recognition memory deficits induced by repeated d -amphetamine exposure
Behavioural Brain Research 224 (2011) 100–106
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Early life stress decreases hippocampal BDNF content and exacerbates recognition memory deficits induced by repeated d-amphetamine exposure Maria Noêmia Martins de Lima a,b , Juliana Presti-Torres a , Gustavo Vedana a , Luisa Azambuja Alcalde a , Laura Stertz b , Gabriel Rodrigo Fries b , Rafael Roesler c,d,g , Monica Levy Andersen e , João Quevedo f,g , Flávio Kapczinski b,g , Nadja Schröder a,g,∗ a
Neurobiology and Developmental Biology Laboratory, Faculty of Biosciences, Pontifical Catholic University, 90619-900 Porto Alegre, RS, Brazil Bipolar Disorders Program and Laboratory of Molecular Psychiatry, Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS, Brazil c Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS, Brazil d Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, Brazil e Department of Psychobiology, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04024-002 São Paulo, SP, Brazil f Laboratory of Neurosciences, Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciuma, SC, Brazil g National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS, Brazil b
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Article history: Received 11 October 2010 Received in revised form 17 May 2011 Accepted 22 May 2011 Keywords: Maternal deprivation d-Amphetamine Recognition memory Brain-derived neurotrophic factor Psychostimulant Rat
a b s t r a c t Adverse experiences early in life may have profound influences on brain development, for example, determining alterations in response to psychostimulant drugs, an increased risk of developing a substance abuse disorder, and individual differences in the vulnerability to neuropsychiatric disorders later in life. Here, we investigated the effects of exposure to an early adverse life event, maternal deprivation, combined with repeated d-amphetamine (AMPH) administration in adulthood, on recognition memory and brain-derived neurotrophic factor (BDNF) levels in rats’ brain and serum. Rats were exposed to one of the following maternal rearing conditions from postnatal days 1 to 14: non-deprived (ND) or deprived (D). In adulthood, both groups received injections of saline (SAL) or AMPH (2.0 mg/kg, i.p.) for 7 days. In Experiment I (performed 24 h after the last AMPH injection), AMPH induced long-term memory (LTM) impairments in ND and D groups. The D + AMPH group also presented short-term memory (STM) impairments, indicating that the effects of AMPH on memory were more pronounced when the animals where maternally deprived. The group exposed to D + SAL (SAL) showed only LTM impairments. In Experiment II (performed 8 days after the last injection), AMPH detrimental effects on memory persisted in ND and D groups. BDNF levels were decreased in the hippocampus of D + SAL rats. In conclusion, AMPH produces severe and persistent recognition memory impairments that were more pronounced when the animals were maternally deprived, suggesting that an early adverse life event may increase the vulnerability of cognitive function to exposure to a psychostimulant later in life. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Exposure to adverse events earlier in life may profoundly affect brain development, leading to long-lasting effects on neuronal structure and behavior and playing a role in the etiology of mood and anxiety disorders. Impairments of brain function that can be induced by early stress include cognitive disturbances later in life [1]. Maternal deprivation, a widely used model of early life stress in rodents, has been shown to produce abnormalities
∗ Corresponding author at: Department of Physiological Sciences, Faculty of Biosciences, Pontifical Catholic University, Av. Ipiranga, 6681 Prédio 12D, Sala 340, 90619-900 Porto Alegre, RS, Brazil. Tel.: +55 51 33203545; fax: +55 51 33203612. E-mail address: [email protected] (N. Schröder). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.05.022
in emotionally related behavioral responses and neuroendocrine responses to stress that can be detected in adulthood. These alterations might involve a range of mechanisms, including changes in the secretion of stress hormones, proinflammatory cytokines, and neurotransmitters, and epigenetic modifications mediated by DNA methylation, resulting in structural changes in neurons and synaptic remodeling [2–4]. Recent evidence has indicated that the neurotrophin brain-derived neurotrophic factor (BDNF) is a molecular candidate for mediating the effects triggered by early adverse events on brain structure and function [5,6]. Behavioral changes associated with early life stress include altered response to drugs. For example, maternal separation leads to increased alcohol preference [7], and repeated social defeat stress during adolescence results in reduced locomotion induced by administration of d-amphetamine (AMPH) during adulthood in
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rats [8]. Repeated AMPH exposure in rats has been used as a model to investigate behavioral and neurochemical alterations induced by psychostimulants, or associated with some aspects of neuropsychiatric disorders, such as bipolar disorder and schizophrenia [9–14]. Repeated AMPH administration in rats produces behavioral sensitization, hyperlocomotion, and alterations in memory [14–16]. In addition, AMPH leads to neurochemical and structural changes including increases in the concentration of dopamine and norepinephrine and dendritic spine density in selected brain areas [9,16,17]. Responses to AMPH might also involve BDNF. Thus, AMPH-induced locomotion was increased in BDNF heterozygous mice [18], and behavioral responses to AMPH were associated with the BDNF G196A (val66met) genotype in healthy human subjects [19]. Based on the findings reviewed above, we hypothesized that early life stress and exposure to psychostimulants such as AMPH later in life could interact to produce neurochemical and behavioral alterations related to substance abuse and psychiatric disorders, including cognitive dysfunction. In addition, the evidence suggests that alterations in BDNF levels might play a role in mediating the effects of early stress and AMPH. In order to address these questions, in the present study we have examined the effects of maternal deprivation, repeated AMPH administration, or maternal deprivation combined with AMPH on locomotor activity, recognition memory, and serum and brain levels of BDNF in rats. 2. Material and methods 2.1. Animals Pregnant Wistar rats were obtained from Fundac¸ão Estadual de Pesquisa e Produc¸ão em Saúde, Porto Alegre, RS, Brazil. After birth, each litter was adjusted within 48 h to contain eight rat pups and the same proportion of male and female individuals. Pups were maintained together with its respective mother in individually ventilated cages with sawdust bedding in a room at temperature of 22 ± 1 ◦ C and a 12 h light/dark cycle. At the age of 4 weeks the pups were weaned and the males were selected and raised in groups of 3 rats. The animals were supplied with standardized pellet food and tap water ad libitum. All behavioral experiments took place between 9:00 and 17:00. These experimental procedures were performed in accordance with the NIH guide for Care and Use of Laboratory Animals (NIH publication number 80-23 revised 1996) and approved by the Animal Use and Care Committee of the Pontifical Catholic University (09/00070-CEUA). All efforts were made to minimize the number of animals and their suffering. 2.2. Maternal deprivation Maternal deprivation was performed as previously described [20–22]. Rat pups were exposed to one of the following maternal rearing conditions from post-natal day 1 to 14: (1) non-deprived (ND), animals were exposed to a daily 15 min period in which the dam was removed and the litter was weighed or (2) deprived (D), animals were exposed to a daily 180 min period in which the dam was removed and the litter was weighed. During the separation period, rat pups of each litter were maintained together in a plastic cage with standard bedding material in an adjacent room to their dams on an incubator at the temperature of 35 ◦ C to avoid hypothermia. After the separation period, pups were returned to the nest and rolled in home cage bedding material, and the dam was returned. It is known that in the rat species, the mother is routinely off the litter for periods of 20–25 min [23]. Thus, only the group exposed to a 180 min period of separation (deprived), but not the group exposed to a 15 min period of separation (non-deprived), results in a deprivation of maternal care. Sixteen non-deprived and 16 deprived male rats were used in the experiments performed in the present study. 2.3. Pharmacological treatment When non-deprived and deprived male rats reached the age of 3 months, they were further divided into two subgroups that received daily intraperitoneal injections (1.0 ml/kg solution volume) of saline (0.9% NaCl, SAL) (n = 8, non-deprived group; n = 8, deprived group) or d-amphetamine (AMPH) (2.0 mg/kg; Sigma–Aldrich, St. Louis, MO, USA) dissolved in SAL (n = 8, non-deprived group; n = 8, deprived group) for 7 consecutive days. Each treatment group consisted of rats derived from, at least, three different litters. The dose of AMPH was selected on the basis of previous studies from our laboratories [10–13,24]. We assessed open-field behavior 2 h (Experiment I) and 7 days (Experiment II) after the last injection. Recognition memory was assessed 24 h after open-field exploration in both Experiments I and
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II in the same apparatus used to assess open-field behavior, in the same groups of animals. 2.4. Open-field behavior Open-field behavior was measured as previously described [11,13]. It was assessed 2 h (Experiment I) and 7 days (Experiment II) after the last injection in the same animals. The open field was a 40 cm × 45 cm × 60 cm arena surrounded by 50 cm high walls, made of plywood with a frontal glass wall. The floor of the arena was divided into 12 equal squares by black lines. Animals were placed in the rear left corner and left to explore the field freely for 5 min. The number of line crossings and rearings were measured using two manual counters during the experimental sessions by an investigator unaware of treatment group of the animals. 2.5. Novel object recognition The novel object recognition task was performed as previously described [25,26]. Recognition memory was assessed 24 h after open-field behavior evaluations in the same apparatus except that the arena floor was covered with sawdust from bedding material during the recognition memory training and test trials. The open field exploration was thus used as a context habituation trial for the recognition memory task. On the first day, rats were given one training trial in which they were exposed to two identical objects: A1 and A2 (Duplo Lego toys). The objects were positioned in two adjacent corners, 9 cm from the walls. The rats were allowed to freely explore the objects until they had accumulated 30 s of total inspection time or for a maximum of 20 min. On the short-term memory (STM) testing trial (1.5 h after the training session), rats were allowed to explore the open field for 5 min in the presence of two objects: the familiar object A and a novel object B. On the long-term memory (LTM) testing trial (24 h after the training session), rats were allowed to explore the open field for 5 min in the presence of two objects: the familiar object A and a third novel object C. These were placed in the same locations as in the training session. All objects presented similar textures, colors, and sizes, but distinctive shapes. Between trials the objects were washed with 10% ethanol solution. In Experiment II animals were retrained and tested using a distinct set of objects (i.e., objects D, E and F) eight days after AMPH or SAL treatments. Object exploration was measured using two stopwatches to record the time spent exploring the objects during the experimental sessions by an investigator unaware of treatment group of the animals. Exploration was defined as follows: sniffing or touching the object with the nose. Sitting on the object was not considered as exploration. Animals that did not explore the objects during the training or retention testing sessions were excluded from the study. In this study, all animals reached the criterion in the training session. A recognition index calculated for each animal was expressed by the ratio TN /(TF + TN ) [TF = time spent exploring the familiar object; TN = time spent exploring the novel object]. 2.6. BDNF measurement The measurement of BDNF levels was performed as previously described [13]. Twenty-four hours after the last day of Experiment II (i.e., 10 days after the last injection), rats were rapidly decapitated, the blood was collected in EDTA-containing tubes, skulls were removed and prefrontal cortex, hippocampus, striatum and amygdala were dissected and stored at −80 ◦ C for biochemical analyses. The blood was centrifuged for 10 min at 4000 rpm and serum was stored at −80 ◦ C until biochemical analyses. BDNF levels were measured by anti-BDNF sandwich-ELISA, according to the manufacturer’s instructions (Chemicon International Inc., Temecula, CA, USA). Briefly, tissue samples were homogenized in phosphate-buffered solution with 1 mM phenylmethylsulfonyl fluoride and 1 mM ethyleneglycoltetraacetic acid. Microtiter plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in sample diluent and standard curve ranged from 7.8 to 500 pg/ml of BNDF. The plates were then washed four times with sample diluent and a monoclonal antiBNDF rabbit antibody diluted 1:1000 in sample diluent was added to each well and incubated for 3 h at room temperature. After washing, a peroxidase-conjugated anti-rabbit antibody (horseradish peroxidase enzyme; diluted 1:1000) was added to each well and incubated at room temperature for 1 h. After addition of streptavidin enzyme, substrate (3,3 ,5,5 -tetramethylbenzidine) and stop solution, the amount of BDNF was determined by absorbance in 450 nm. The standard curve demonstrates a direct relationship between optical density and BDNF concentration. BDNF was expressed as picogram of BDNF per ml of serum obtained from tissues homogenate. Total protein was measured by Lowry’s method using bovine serum albumin as a standard. 2.7. Statistical analysis The behavioral and biochemical effects of early maternal deprivation on subsequent administration of AMPH were analyzed using two-way analysis of variance (ANOVA). The model includes two fixed factors each with two levels corresponding to maternal deprivation (yes versus no) and subsequent AMPH administration (yes versus no). To test the differences between the experimental groups we used one-way ANOVA followed by Tukey HSD post hoc tests. P values less than 0.05 were considered to indicate statistical significance. All data are presented as mean ± S.E.M.
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revealed a significant interaction between the effects of maternal deprivation and AMPH administration in number of crossings (F = 5.224; df = 1; P = 0.030), but not rearings (F = 0.220; df = 1; P = 0.643). Post hoc analysis has indicated that both ND and D animals that received d-AMPH in adulthood showed an increase in number of crossings (both Ps < 0.001; Tukey HSD) and number of rearings (P = 0.003 and P = 0.039, respectively; Tukey HSD) when compared to ND animals that received SAL. The results indicate that AMPH increased locomotor activity 2 h after administration irrespective of exposure to early stress (Fig. 1A and B). In Experiment II (performed 7 days after the last injection), there were no statistically significant differences among all experimental groups regarding the parameters evaluated (Fig. 1C and D). 3.2. Novel object recognition
Fig. 1. Effects of maternal deprivation and d-amphetamine (AMPH) treatment on open-field behavior. In Experiment I, the number of (A) crossings and (B) rearings was evaluated in an open-field apparatus 2 h after AMPH or SAL treatments. In Experiment II, the number of crossings (C) and rearings (D) was evaluated 7 days after AMPH or SAL treatments in the same animals. Data are expressed as mean ± S.E.M., n = 8 per group. Differences between ND + SAL group versus other groups are indicated as: *P < 0.05 and **P < 0.01.
3. Results 3.1. Open-field behavior Results for open-field behavior are shown in Fig. 1. In Experiment I (performed 2 h after the last injection), two-way ANOVA
Results for recognition memory are shown in Fig. 2. In Experiment I (performed 24 h after the last injection, Fig. 2A), two-way ANOVA revealed a significant interaction between the effects of maternal deprivation and AMPH administration in exploratory preferences in STM (F = 16.446; df = 1; P < 0.001) and in LTM retention trials (F = 6.778; df = 1; P = 0.015), but not in training trial (F = 0.649; df = 1; P = 0.427). Post hoc analysis has indicated that ND (P = 0.019; Tukey HSD) and D (P = 0.045; Tukey HSD) animals treated with AMPH in adulthood presented LTM impairments when compared with ND animals that received SAL. Interestingly, the group given maternal deprivation plus AMPH was the only group showing impaired STM, indicating that early stress might produce a more pronounced deficit in some aspects of cognitive function when combined with AMPH (P < 0.001; Tukey HSD), and D animals that received SAL (P < 0.001; Tukey HSD) or AMPH (P < 0.001; Tukey HSD). D animals that received SAL showed only LTM impairments when compared to ND animals that received SAL (P = 0.013; Tukey HSD). One-way ANOVA revealed no differences among all groups regarding the latency to reach the criterion of 30 s of total time exploring the objects in the training trial (F = 2.466; df = 3; P = 0.083, data not shown). In order to determine if the detrimental effects of AMPH on the central nervous system persists more than a week after the end of the treatment, rats were retrained and tested 8 days after the last injection (Experiment II, Fig. 2B). Two-way ANOVA revealed a significant interaction between the effects of maternal deprivation and AMPH administration in exploratory preferences in STM (F = 5.582; df = 1; P < 0.025) and in LTM retention trials (F = 5.425; df = 1; P = 0.026), but not in training trial (F = 0.908; df = 1; P = 0.349). Post hoc analysis indicated that ND and D animals treated with AMPH in adulthood still presented STM and LTM impairments when compared to ND animals that received SAL (ND group: P = 0.001 for LTM; D group: P < 0.001 for STM and P = 0.001 for LTM; Tukey HSD), and to D animals that received SAL (D group: P < 0.001 for STM; Tukey HSD) or AMPH (D group: P < 0.001 for STM; Tukey HSD). D animals that received SAL also preserved the LTM impairments when compared to ND animals that received SAL (P = 0.015; Tukey HSD). One-way ANOVA revealed no differences among all groups regarding the latency to reach the criterion of 30 s of total time exploring the objects in the training trial (F = 0.822; df = 3, P = 0.493, data not shown). 3.3. BDNF measurement Results for the measurement of BDNF levels are shown in Fig. 3. Two-way ANOVA revealed a significant interaction between the effects of maternal deprivation and AMPH administration in BDNF levels in hippocampus (F = 16.350; df = 1; P = 0.001). Post hoc analysis indicated that BDNF levels were decreased in the hippocampus of D animals that received SAL in adulthood in comparison to
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Fig. 2. Effects of maternal deprivation and d-amphetamine (AMPH) treatment on recognition memory. Recognition memory was evaluated (A) 24 h and (B) 8 days after AMPH or SAL treatments. Short-term memory (STM) and long-term memory (LTM) retention tests were performed 1.5 and 24 h after training, respectively. The proportion of the total exploration time that the animal spent investigating the novel object was the “Recognition Index” expressed by the ratio TN /(TF + TN ), TF = time spent exploring the familiar object and TN = time spent exploring the novel object. Data are expressed as mean ± S.E.M., n = 8 per group. Differences between ND + SAL group versus other groups are indicated as: **P < 0.01; differences between D + SAL group versus other groups are indicated as: ++ P < 0.01; differences between ND + AMPH group versus D + AMPH are indicated as: ## P < 0.01.
ND animals that received SAL in adulthood (Fig. 3B, P = 0.012; Tukey HSD) and to D animals that received AMPH in adulthood (Fig. 3B, P = 0.019; Tukey HSD). No statistically significant differences among groups were observed in prefrontal cortex (Fig. 3A), striatum (Fig. 3C), amygdala (Fig. 3D), or serum (Fig. 3E).
4. Discussion The present findings are in agreement with our previous results indicating that AMPH treatment induces locomotor hyperactivity in rats [13,24,27]. Here, we show for the first time that the increased locomotion is accompanied by severe recognition memory impairments, which persist at least for 8 days after the last AMPH injection in both ND and D groups. Importantly, the present results indicate that short-term retention was impaired only in rats given both early stress and AMPH, suggesting that the adverse experiences early in life may sensitize specific neurocircuits to subsequent exposure to drugs or other stressors. The training protocol used in the present study, in which animals are trained to meet the criterion of 30 s exploring objects was chosen in order to overcome the possible motor and motivational/exploratory effects induced by the treatments. Comparison of the time required to reach criterion among groups was not statistically different, suggesting that the recognition memory impairment found in the present study is likely related to memory formation/consolidation processes rather than acquisition.
Although we have not measured biochemical parameters related to hypothalamo–pituitary–adrenal (HPA) axis activity in the rats used in the present study, we have previously shown that depressive-like behavior in rats exposed to maternal deprivation is accompanied by an increase in plasma adrenocorticotrophin hormone (ACTH) levels in adulthood [28]. These findings are consistent with several other reports of HPA activation and persistent increases in plasma levels of corticosterone and adrenocorticotropin hormone (ACTH) in deprived rats [29–31]. For example, a recent study described elevated plasma levels of ACTH and corticosterone measured in adulthood (69–90 days of age) in rats deprived from their mothers at postnatal day 9 [32]. Moreover, both early maternal deprivation and AMPH administration have been shown to alter brain monoamine levels [8,33,34]. Maternal deprivation and AMPH have also been shown to induce structural alterations (i.e., reorganization of dendritic spine density) in several brain regions. In rats given maternal deprivation plus AMPH in adulthood, deprivation blocked the AMPH-induced alterations in spine density without altering behavioral sensitization [35]. A number of preclinical studies have investigated the correlation between exposure to early adverse life events and cognitive performance in adult life [36–38]. It has been suggested that environmental factors along the neurodevelopmental trajectory could influence neurotrophin expression and learning and memory later in life. In fact, Choy et al. [39] examined the combined long-term effect of an early stress, in the form of maternal deprivation, and a
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later stress, simulated by chronic young–adult treatment with the stress hormone, corticosterone, on cognition and BDNF expression in the hippocampus of rats. They demonstrated that rats exposed to stress in early and late life exhibited learning and memory deficits and reduced BDNF expression in the hippocampus. Consistent with these findings, Aisa et al. [40] demonstrated that maternal deprivation produces impairments in learning and memory function and a significant increase in the susceptibility to cholinergic neurons lesioning in adult rats. They also showed that NGF expression in the hippocampus is decreased in maternally deprived animals. Interestingly, Pham et al. [41] demonstrated that mild chronic stress in the adult life enhances cognitive function and hippocampal NGF levels in rats that were not submitted to maternal deprivation, producing opposite effects in maternally deprived animals. Most importantly, they observed that non-deprived and mildly stressed groups that performed well in behavioral tests had higher levels of hippocampal NGF than the non-stressed and non-deprived rats that performed poorly. Consistently with previous findings, hippocampal BDNF content was not affected by AMPH treatment itself [11,13]. BDNF levels were decreased in the hippocampus of the D group that received SAL, as expected. Interestingly, BDNF levels in the D group that received AMPH were higher than BDNF levels in D animals that received SAL, although no differences were found when this group was compared to ND animals that received SAL. While we do not know why BDNF levels seem to be restored in D animals that received AMPH, we may consider the possibility that it results from a modulatory interaction between AMPH exposure and neonatal stress that differentially influenced BDNF expression, since ND animals that received AMPH did not present alterations in BDNF levels. Indeed, it has been shown that neurotrophins have relevant action on neurons involved in mediating the effects of psychostimulants, such as dopaminergic neurons, and can play dual roles: first, in neuronal survival and death, and, second, in activity-dependent plasticity [42]. Moreover, recent studies have indicated that activation of dopamine receptors (the main neurotransmitter involved in AMPH psychostimulant effect) induces an increase in BDNF mRNA and protein expression in neuronal cultures and in selected brain regions, such as the striatum and hippocampus [43–45]. Further experiments using intracerebral administration of BDNF and strategies to inhibit BDNF signaling (for instance, using RNA interference) are needed to clarify what the role of BDNF in mediating the effects of early stress and AMPH. Over the years, preclinical and clinical studies have shown that repeated exposure to amphetamine derivatives, such as methamand 3,4-methylenedioxy-N-methamphetamine phetamine, (MDMA), impairs recognition memory as well as dopaminergic neural transmission [46–49]. Bisagno et al. [15] have reported that chronic AMPH impairs short-term recognition memory, tested seven days after drug treatment. Long-term memory was not evaluated in that study. Our results indicate that a seven-day regimen of AMPH was able to significantly impair long-term recognition memory. Most importantly, here we show that the combination of maternal deprivation and adult AMPH administration produces a synergistic deleterious effect on cognitive performance assessed by recognition memory.
Fig. 3. Effects of maternal deprivation and d-amphetamine (AMPH) treatment on brain-derived neurotrophic factor (BDNF) levels. The levels of BDNF are expressed in: (A) prefrontal cortex; (B) hippocampus; (C) striatum; (D) amygdale; and (E) serum. Data are expressed as mean ± S.E.M. picogram of BDNF/g of protein, n = 5–8 animals per group. Rats were euthanized for BDNF measurement 10 days after the last drug injection. Differences between ND + SAL versus other groups are indicated as: *P < 0.05; differences between D + SAL group versus other groups are indicated as: + P < 0.05.
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5. Conclusions Taken together, the present results support the view that exposure to adverse events early in life might increase the susceptibility to the adverse effects of exposure to psychostimulant drugs during adulthood. This type of effect may have implications for the onset of a mood disorder [50–52]. In addition, a decrease in BDNF levels in the hippocampus might be involved in the long-lasting effects of maternal deprivation on memory and response to drugs. It has been suggested that alterations in BDNF signaling may contribute to the pathogenesis of mood and psychiatric disorders [6,53]. Since recognition memory changes were also seen in rats that had either been maternally deprived or received AMPH treatment in adulthood, it is reasonable to assume that other factors in addition to alterations in BDNF content may have been influenced by early stress. Further experiments will be required in order to characterize the molecular mechanisms by which exposure to an early environmental stressor produces long-lasting detrimental effects on cognition, and the possible implications for the development of mood and anxiety disorders. Acknowledgements Funding for this study was provided by CAPES-MEC, Brazil (AUXPE-Procad grant 657/2008). J.P.T. and V.A.G. are supported by a CAPES/MEC fellowship. L.H.H. is supported by an IC/CNPq fellowship. L.A.A. is supported by a PIBIC/CNPq fellowship. L.S. and G.R.F. are supported by an HCPA/CNPq fellowship. R.R., M.L.A., J.Q., F.K., and N.S. are CNPq research fellows. References [1] Bremner JD, Narayan M. The effects of stress on memory and the hippocampus throughout the life cycle: implications for childhood development and aging. Dev Psychopathol 1998;10:871–85. [2] Hennessy MB, Deak T, Schiml-Webb PA. Early attachment-figure separation and increased risk for later depression: potential mediation by proinflammatory processes. Neurosci Biobehav Rev 2010;34:782–90. [3] Kaffman A, Meaney MJ. Neurodevelopmental sequelae of postnatal maternal care in rodents: clinical and research implications of molecular insights. J Child Psychol Psychiatry 2007;48:224–44. [4] Veenema AH. Early life stress, the development of aggression and neuroendocrine and neurobiological correlates: what can we learn from animal models? Front Neuroendocrinol 2009;30:497–518. [5] Cirulli F, Francia N, Berry A, Aloe L, Alleva E, Suomic SJ. Early life stress as a risk factor for mental health: role of neurotrophins from rodents to non-human primates. Neurosci Biobehav Rev 2009;33:573–85. [6] Cowansage KK, LeDoux JE, Monfils MH. Brain-derived neurotrophic factor: a dynamic gatekeeper of neural plasticity. Curr Mol Pharmacol 2010;3:12–29. [7] Huot RL, Thrivikraman KV, Meaney MJ, Plotsky PM. Development of adult ethanol preference and anxiety as a consequence of neonatal maternal separation in long Evans rats and reversal with antidepressant treatment. Psychopharmacology (Berlin) 2001;158:366–73. [8] Burke AR, Renner KJ, Forster GL, Watt MJ. Adolescent social defeat alters neural, endocrine and behavioral responses to amphetamine in adult male rats. Brain Res 2010;1352:147–56. [9] Camp DM, DeJonghe DK, Robinson TE. Time-dependent effects of repeated amphetamine treatment on norepinephrine in the hypothalamus and hippocampus assessed with in vivo microdialysis. Neuropsychopharmacology 1997;17:130–40. [10] Frey BN, Martins MR, Petronilho FC, Dal-Pizzol F, Quevedo J, Kapczinski F. Increased oxidative stress after repeated amphetamine exposure: possible relevance as a model of mania. Bipolar Disord 2006;8:275–80. [11] Frey BN, Andreazza AC, Ceresér KM, Martins MR, Valvassori SS, Réus GZ, et al. Effects of mood stabilizers on hippocampus BDNF levels in an animal model of mania. Life Sci 2006;79:281–6. [12] Frey BN, Valvassori SS, Réus GZ, Martins MR, Petronilho FC, Bardini K, et al. Effects of lithium and valproate on amphetamine-induced oxidative stress generation in an animal model of mania. J Psychiatry Neurosci 2006;31:326–32. [13] Kauer-Sant’Anna M, Andreazza AC, Valvassori SS, Martins MR, Barbosa LM, Schwartsmann G, et al. A gastrin-releasing peptide receptor antagonist blocks d-amphetamine-induced hyperlocomotion and increases hippocampal NGF and BDNF levels in rats. Peptides 2007;28:1447–52. [14] Peleg-Raibstein D, Yee BK, Feldon J, Hauser J. The amphetamine sensitization model of schizophrenia: relevance beyond psychotic symptoms? Psychopharmacology (Berlin) 2009;206:603–21.
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Early life stress decreases hippocampal BDNF content and exacerbates recognition memory deficits induced by repeated d-amphetamine exposure Maria Noêmia Martins de Lima a,b , Juliana Presti-Torres a , Gustavo Vedana a , Luisa Azambuja Alcalde a , Laura Stertz b , Gabriel Rodrigo Fries b , Rafael Roesler c,d,g , Monica Levy Andersen e , João Quevedo f,g , Flávio Kapczinski b,g , Nadja Schröder a,g,∗ a
Neurobiology and Developmental Biology Laboratory, Faculty of Biosciences, Pontifical Catholic University, 90619-900 Porto Alegre, RS, Brazil Bipolar Disorders Program and Laboratory of Molecular Psychiatry, Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, RS, Brazil c Laboratory of Neuropharmacology and Neural Tumor Biology, Department of Pharmacology, Institute for Basic Health Sciences, Federal University of Rio Grande do Sul, 90050-170 Porto Alegre, RS, Brazil d Cancer Research Laboratory, University Hospital Research Center (CPE-HCPA), Federal University of Rio Grande do Sul, 90035-003 Porto Alegre, Brazil e Department of Psychobiology, Universidade Federal de São Paulo, Escola Paulista de Medicina, 04024-002 São Paulo, SP, Brazil f Laboratory of Neurosciences, Postgraduate Program in Health Sciences, Health Sciences Unit, University of Southern Santa Catarina, 88806-000 Criciuma, SC, Brazil g National Institute for Translational Medicine (INCT-TM), 90035-003 Porto Alegre, RS, Brazil b
a r t i c l e
i n f o
Article history: Received 11 October 2010 Received in revised form 17 May 2011 Accepted 22 May 2011 Keywords: Maternal deprivation d-Amphetamine Recognition memory Brain-derived neurotrophic factor Psychostimulant Rat
a b s t r a c t Adverse experiences early in life may have profound influences on brain development, for example, determining alterations in response to psychostimulant drugs, an increased risk of developing a substance abuse disorder, and individual differences in the vulnerability to neuropsychiatric disorders later in life. Here, we investigated the effects of exposure to an early adverse life event, maternal deprivation, combined with repeated d-amphetamine (AMPH) administration in adulthood, on recognition memory and brain-derived neurotrophic factor (BDNF) levels in rats’ brain and serum. Rats were exposed to one of the following maternal rearing conditions from postnatal days 1 to 14: non-deprived (ND) or deprived (D). In adulthood, both groups received injections of saline (SAL) or AMPH (2.0 mg/kg, i.p.) for 7 days. In Experiment I (performed 24 h after the last AMPH injection), AMPH induced long-term memory (LTM) impairments in ND and D groups. The D + AMPH group also presented short-term memory (STM) impairments, indicating that the effects of AMPH on memory were more pronounced when the animals where maternally deprived. The group exposed to D + SAL (SAL) showed only LTM impairments. In Experiment II (performed 8 days after the last injection), AMPH detrimental effects on memory persisted in ND and D groups. BDNF levels were decreased in the hippocampus of D + SAL rats. In conclusion, AMPH produces severe and persistent recognition memory impairments that were more pronounced when the animals were maternally deprived, suggesting that an early adverse life event may increase the vulnerability of cognitive function to exposure to a psychostimulant later in life. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Exposure to adverse events earlier in life may profoundly affect brain development, leading to long-lasting effects on neuronal structure and behavior and playing a role in the etiology of mood and anxiety disorders. Impairments of brain function that can be induced by early stress include cognitive disturbances later in life [1]. Maternal deprivation, a widely used model of early life stress in rodents, has been shown to produce abnormalities
∗ Corresponding author at: Department of Physiological Sciences, Faculty of Biosciences, Pontifical Catholic University, Av. Ipiranga, 6681 Prédio 12D, Sala 340, 90619-900 Porto Alegre, RS, Brazil. Tel.: +55 51 33203545; fax: +55 51 33203612. E-mail address: [email protected] (N. Schröder). 0166-4328/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbr.2011.05.022
in emotionally related behavioral responses and neuroendocrine responses to stress that can be detected in adulthood. These alterations might involve a range of mechanisms, including changes in the secretion of stress hormones, proinflammatory cytokines, and neurotransmitters, and epigenetic modifications mediated by DNA methylation, resulting in structural changes in neurons and synaptic remodeling [2–4]. Recent evidence has indicated that the neurotrophin brain-derived neurotrophic factor (BDNF) is a molecular candidate for mediating the effects triggered by early adverse events on brain structure and function [5,6]. Behavioral changes associated with early life stress include altered response to drugs. For example, maternal separation leads to increased alcohol preference [7], and repeated social defeat stress during adolescence results in reduced locomotion induced by administration of d-amphetamine (AMPH) during adulthood in
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rats [8]. Repeated AMPH exposure in rats has been used as a model to investigate behavioral and neurochemical alterations induced by psychostimulants, or associated with some aspects of neuropsychiatric disorders, such as bipolar disorder and schizophrenia [9–14]. Repeated AMPH administration in rats produces behavioral sensitization, hyperlocomotion, and alterations in memory [14–16]. In addition, AMPH leads to neurochemical and structural changes including increases in the concentration of dopamine and norepinephrine and dendritic spine density in selected brain areas [9,16,17]. Responses to AMPH might also involve BDNF. Thus, AMPH-induced locomotion was increased in BDNF heterozygous mice [18], and behavioral responses to AMPH were associated with the BDNF G196A (val66met) genotype in healthy human subjects [19]. Based on the findings reviewed above, we hypothesized that early life stress and exposure to psychostimulants such as AMPH later in life could interact to produce neurochemical and behavioral alterations related to substance abuse and psychiatric disorders, including cognitive dysfunction. In addition, the evidence suggests that alterations in BDNF levels might play a role in mediating the effects of early stress and AMPH. In order to address these questions, in the present study we have examined the effects of maternal deprivation, repeated AMPH administration, or maternal deprivation combined with AMPH on locomotor activity, recognition memory, and serum and brain levels of BDNF in rats. 2. Material and methods 2.1. Animals Pregnant Wistar rats were obtained from Fundac¸ão Estadual de Pesquisa e Produc¸ão em Saúde, Porto Alegre, RS, Brazil. After birth, each litter was adjusted within 48 h to contain eight rat pups and the same proportion of male and female individuals. Pups were maintained together with its respective mother in individually ventilated cages with sawdust bedding in a room at temperature of 22 ± 1 ◦ C and a 12 h light/dark cycle. At the age of 4 weeks the pups were weaned and the males were selected and raised in groups of 3 rats. The animals were supplied with standardized pellet food and tap water ad libitum. All behavioral experiments took place between 9:00 and 17:00. These experimental procedures were performed in accordance with the NIH guide for Care and Use of Laboratory Animals (NIH publication number 80-23 revised 1996) and approved by the Animal Use and Care Committee of the Pontifical Catholic University (09/00070-CEUA). All efforts were made to minimize the number of animals and their suffering. 2.2. Maternal deprivation Maternal deprivation was performed as previously described [20–22]. Rat pups were exposed to one of the following maternal rearing conditions from post-natal day 1 to 14: (1) non-deprived (ND), animals were exposed to a daily 15 min period in which the dam was removed and the litter was weighed or (2) deprived (D), animals were exposed to a daily 180 min period in which the dam was removed and the litter was weighed. During the separation period, rat pups of each litter were maintained together in a plastic cage with standard bedding material in an adjacent room to their dams on an incubator at the temperature of 35 ◦ C to avoid hypothermia. After the separation period, pups were returned to the nest and rolled in home cage bedding material, and the dam was returned. It is known that in the rat species, the mother is routinely off the litter for periods of 20–25 min [23]. Thus, only the group exposed to a 180 min period of separation (deprived), but not the group exposed to a 15 min period of separation (non-deprived), results in a deprivation of maternal care. Sixteen non-deprived and 16 deprived male rats were used in the experiments performed in the present study. 2.3. Pharmacological treatment When non-deprived and deprived male rats reached the age of 3 months, they were further divided into two subgroups that received daily intraperitoneal injections (1.0 ml/kg solution volume) of saline (0.9% NaCl, SAL) (n = 8, non-deprived group; n = 8, deprived group) or d-amphetamine (AMPH) (2.0 mg/kg; Sigma–Aldrich, St. Louis, MO, USA) dissolved in SAL (n = 8, non-deprived group; n = 8, deprived group) for 7 consecutive days. Each treatment group consisted of rats derived from, at least, three different litters. The dose of AMPH was selected on the basis of previous studies from our laboratories [10–13,24]. We assessed open-field behavior 2 h (Experiment I) and 7 days (Experiment II) after the last injection. Recognition memory was assessed 24 h after open-field exploration in both Experiments I and
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II in the same apparatus used to assess open-field behavior, in the same groups of animals. 2.4. Open-field behavior Open-field behavior was measured as previously described [11,13]. It was assessed 2 h (Experiment I) and 7 days (Experiment II) after the last injection in the same animals. The open field was a 40 cm × 45 cm × 60 cm arena surrounded by 50 cm high walls, made of plywood with a frontal glass wall. The floor of the arena was divided into 12 equal squares by black lines. Animals were placed in the rear left corner and left to explore the field freely for 5 min. The number of line crossings and rearings were measured using two manual counters during the experimental sessions by an investigator unaware of treatment group of the animals. 2.5. Novel object recognition The novel object recognition task was performed as previously described [25,26]. Recognition memory was assessed 24 h after open-field behavior evaluations in the same apparatus except that the arena floor was covered with sawdust from bedding material during the recognition memory training and test trials. The open field exploration was thus used as a context habituation trial for the recognition memory task. On the first day, rats were given one training trial in which they were exposed to two identical objects: A1 and A2 (Duplo Lego toys). The objects were positioned in two adjacent corners, 9 cm from the walls. The rats were allowed to freely explore the objects until they had accumulated 30 s of total inspection time or for a maximum of 20 min. On the short-term memory (STM) testing trial (1.5 h after the training session), rats were allowed to explore the open field for 5 min in the presence of two objects: the familiar object A and a novel object B. On the long-term memory (LTM) testing trial (24 h after the training session), rats were allowed to explore the open field for 5 min in the presence of two objects: the familiar object A and a third novel object C. These were placed in the same locations as in the training session. All objects presented similar textures, colors, and sizes, but distinctive shapes. Between trials the objects were washed with 10% ethanol solution. In Experiment II animals were retrained and tested using a distinct set of objects (i.e., objects D, E and F) eight days after AMPH or SAL treatments. Object exploration was measured using two stopwatches to record the time spent exploring the objects during the experimental sessions by an investigator unaware of treatment group of the animals. Exploration was defined as follows: sniffing or touching the object with the nose. Sitting on the object was not considered as exploration. Animals that did not explore the objects during the training or retention testing sessions were excluded from the study. In this study, all animals reached the criterion in the training session. A recognition index calculated for each animal was expressed by the ratio TN /(TF + TN ) [TF = time spent exploring the familiar object; TN = time spent exploring the novel object]. 2.6. BDNF measurement The measurement of BDNF levels was performed as previously described [13]. Twenty-four hours after the last day of Experiment II (i.e., 10 days after the last injection), rats were rapidly decapitated, the blood was collected in EDTA-containing tubes, skulls were removed and prefrontal cortex, hippocampus, striatum and amygdala were dissected and stored at −80 ◦ C for biochemical analyses. The blood was centrifuged for 10 min at 4000 rpm and serum was stored at −80 ◦ C until biochemical analyses. BDNF levels were measured by anti-BDNF sandwich-ELISA, according to the manufacturer’s instructions (Chemicon International Inc., Temecula, CA, USA). Briefly, tissue samples were homogenized in phosphate-buffered solution with 1 mM phenylmethylsulfonyl fluoride and 1 mM ethyleneglycoltetraacetic acid. Microtiter plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in sample diluent and standard curve ranged from 7.8 to 500 pg/ml of BNDF. The plates were then washed four times with sample diluent and a monoclonal antiBNDF rabbit antibody diluted 1:1000 in sample diluent was added to each well and incubated for 3 h at room temperature. After washing, a peroxidase-conjugated anti-rabbit antibody (horseradish peroxidase enzyme; diluted 1:1000) was added to each well and incubated at room temperature for 1 h. After addition of streptavidin enzyme, substrate (3,3 ,5,5 -tetramethylbenzidine) and stop solution, the amount of BDNF was determined by absorbance in 450 nm. The standard curve demonstrates a direct relationship between optical density and BDNF concentration. BDNF was expressed as picogram of BDNF per ml of serum obtained from tissues homogenate. Total protein was measured by Lowry’s method using bovine serum albumin as a standard. 2.7. Statistical analysis The behavioral and biochemical effects of early maternal deprivation on subsequent administration of AMPH were analyzed using two-way analysis of variance (ANOVA). The model includes two fixed factors each with two levels corresponding to maternal deprivation (yes versus no) and subsequent AMPH administration (yes versus no). To test the differences between the experimental groups we used one-way ANOVA followed by Tukey HSD post hoc tests. P values less than 0.05 were considered to indicate statistical significance. All data are presented as mean ± S.E.M.
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revealed a significant interaction between the effects of maternal deprivation and AMPH administration in number of crossings (F = 5.224; df = 1; P = 0.030), but not rearings (F = 0.220; df = 1; P = 0.643). Post hoc analysis has indicated that both ND and D animals that received d-AMPH in adulthood showed an increase in number of crossings (both Ps < 0.001; Tukey HSD) and number of rearings (P = 0.003 and P = 0.039, respectively; Tukey HSD) when compared to ND animals that received SAL. The results indicate that AMPH increased locomotor activity 2 h after administration irrespective of exposure to early stress (Fig. 1A and B). In Experiment II (performed 7 days after the last injection), there were no statistically significant differences among all experimental groups regarding the parameters evaluated (Fig. 1C and D). 3.2. Novel object recognition
Fig. 1. Effects of maternal deprivation and d-amphetamine (AMPH) treatment on open-field behavior. In Experiment I, the number of (A) crossings and (B) rearings was evaluated in an open-field apparatus 2 h after AMPH or SAL treatments. In Experiment II, the number of crossings (C) and rearings (D) was evaluated 7 days after AMPH or SAL treatments in the same animals. Data are expressed as mean ± S.E.M., n = 8 per group. Differences between ND + SAL group versus other groups are indicated as: *P < 0.05 and **P < 0.01.
3. Results 3.1. Open-field behavior Results for open-field behavior are shown in Fig. 1. In Experiment I (performed 2 h after the last injection), two-way ANOVA
Results for recognition memory are shown in Fig. 2. In Experiment I (performed 24 h after the last injection, Fig. 2A), two-way ANOVA revealed a significant interaction between the effects of maternal deprivation and AMPH administration in exploratory preferences in STM (F = 16.446; df = 1; P < 0.001) and in LTM retention trials (F = 6.778; df = 1; P = 0.015), but not in training trial (F = 0.649; df = 1; P = 0.427). Post hoc analysis has indicated that ND (P = 0.019; Tukey HSD) and D (P = 0.045; Tukey HSD) animals treated with AMPH in adulthood presented LTM impairments when compared with ND animals that received SAL. Interestingly, the group given maternal deprivation plus AMPH was the only group showing impaired STM, indicating that early stress might produce a more pronounced deficit in some aspects of cognitive function when combined with AMPH (P < 0.001; Tukey HSD), and D animals that received SAL (P < 0.001; Tukey HSD) or AMPH (P < 0.001; Tukey HSD). D animals that received SAL showed only LTM impairments when compared to ND animals that received SAL (P = 0.013; Tukey HSD). One-way ANOVA revealed no differences among all groups regarding the latency to reach the criterion of 30 s of total time exploring the objects in the training trial (F = 2.466; df = 3; P = 0.083, data not shown). In order to determine if the detrimental effects of AMPH on the central nervous system persists more than a week after the end of the treatment, rats were retrained and tested 8 days after the last injection (Experiment II, Fig. 2B). Two-way ANOVA revealed a significant interaction between the effects of maternal deprivation and AMPH administration in exploratory preferences in STM (F = 5.582; df = 1; P < 0.025) and in LTM retention trials (F = 5.425; df = 1; P = 0.026), but not in training trial (F = 0.908; df = 1; P = 0.349). Post hoc analysis indicated that ND and D animals treated with AMPH in adulthood still presented STM and LTM impairments when compared to ND animals that received SAL (ND group: P = 0.001 for LTM; D group: P < 0.001 for STM and P = 0.001 for LTM; Tukey HSD), and to D animals that received SAL (D group: P < 0.001 for STM; Tukey HSD) or AMPH (D group: P < 0.001 for STM; Tukey HSD). D animals that received SAL also preserved the LTM impairments when compared to ND animals that received SAL (P = 0.015; Tukey HSD). One-way ANOVA revealed no differences among all groups regarding the latency to reach the criterion of 30 s of total time exploring the objects in the training trial (F = 0.822; df = 3, P = 0.493, data not shown). 3.3. BDNF measurement Results for the measurement of BDNF levels are shown in Fig. 3. Two-way ANOVA revealed a significant interaction between the effects of maternal deprivation and AMPH administration in BDNF levels in hippocampus (F = 16.350; df = 1; P = 0.001). Post hoc analysis indicated that BDNF levels were decreased in the hippocampus of D animals that received SAL in adulthood in comparison to
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Fig. 2. Effects of maternal deprivation and d-amphetamine (AMPH) treatment on recognition memory. Recognition memory was evaluated (A) 24 h and (B) 8 days after AMPH or SAL treatments. Short-term memory (STM) and long-term memory (LTM) retention tests were performed 1.5 and 24 h after training, respectively. The proportion of the total exploration time that the animal spent investigating the novel object was the “Recognition Index” expressed by the ratio TN /(TF + TN ), TF = time spent exploring the familiar object and TN = time spent exploring the novel object. Data are expressed as mean ± S.E.M., n = 8 per group. Differences between ND + SAL group versus other groups are indicated as: **P < 0.01; differences between D + SAL group versus other groups are indicated as: ++ P < 0.01; differences between ND + AMPH group versus D + AMPH are indicated as: ## P < 0.01.
ND animals that received SAL in adulthood (Fig. 3B, P = 0.012; Tukey HSD) and to D animals that received AMPH in adulthood (Fig. 3B, P = 0.019; Tukey HSD). No statistically significant differences among groups were observed in prefrontal cortex (Fig. 3A), striatum (Fig. 3C), amygdala (Fig. 3D), or serum (Fig. 3E).
4. Discussion The present findings are in agreement with our previous results indicating that AMPH treatment induces locomotor hyperactivity in rats [13,24,27]. Here, we show for the first time that the increased locomotion is accompanied by severe recognition memory impairments, which persist at least for 8 days after the last AMPH injection in both ND and D groups. Importantly, the present results indicate that short-term retention was impaired only in rats given both early stress and AMPH, suggesting that the adverse experiences early in life may sensitize specific neurocircuits to subsequent exposure to drugs or other stressors. The training protocol used in the present study, in which animals are trained to meet the criterion of 30 s exploring objects was chosen in order to overcome the possible motor and motivational/exploratory effects induced by the treatments. Comparison of the time required to reach criterion among groups was not statistically different, suggesting that the recognition memory impairment found in the present study is likely related to memory formation/consolidation processes rather than acquisition.
Although we have not measured biochemical parameters related to hypothalamo–pituitary–adrenal (HPA) axis activity in the rats used in the present study, we have previously shown that depressive-like behavior in rats exposed to maternal deprivation is accompanied by an increase in plasma adrenocorticotrophin hormone (ACTH) levels in adulthood [28]. These findings are consistent with several other reports of HPA activation and persistent increases in plasma levels of corticosterone and adrenocorticotropin hormone (ACTH) in deprived rats [29–31]. For example, a recent study described elevated plasma levels of ACTH and corticosterone measured in adulthood (69–90 days of age) in rats deprived from their mothers at postnatal day 9 [32]. Moreover, both early maternal deprivation and AMPH administration have been shown to alter brain monoamine levels [8,33,34]. Maternal deprivation and AMPH have also been shown to induce structural alterations (i.e., reorganization of dendritic spine density) in several brain regions. In rats given maternal deprivation plus AMPH in adulthood, deprivation blocked the AMPH-induced alterations in spine density without altering behavioral sensitization [35]. A number of preclinical studies have investigated the correlation between exposure to early adverse life events and cognitive performance in adult life [36–38]. It has been suggested that environmental factors along the neurodevelopmental trajectory could influence neurotrophin expression and learning and memory later in life. In fact, Choy et al. [39] examined the combined long-term effect of an early stress, in the form of maternal deprivation, and a
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later stress, simulated by chronic young–adult treatment with the stress hormone, corticosterone, on cognition and BDNF expression in the hippocampus of rats. They demonstrated that rats exposed to stress in early and late life exhibited learning and memory deficits and reduced BDNF expression in the hippocampus. Consistent with these findings, Aisa et al. [40] demonstrated that maternal deprivation produces impairments in learning and memory function and a significant increase in the susceptibility to cholinergic neurons lesioning in adult rats. They also showed that NGF expression in the hippocampus is decreased in maternally deprived animals. Interestingly, Pham et al. [41] demonstrated that mild chronic stress in the adult life enhances cognitive function and hippocampal NGF levels in rats that were not submitted to maternal deprivation, producing opposite effects in maternally deprived animals. Most importantly, they observed that non-deprived and mildly stressed groups that performed well in behavioral tests had higher levels of hippocampal NGF than the non-stressed and non-deprived rats that performed poorly. Consistently with previous findings, hippocampal BDNF content was not affected by AMPH treatment itself [11,13]. BDNF levels were decreased in the hippocampus of the D group that received SAL, as expected. Interestingly, BDNF levels in the D group that received AMPH were higher than BDNF levels in D animals that received SAL, although no differences were found when this group was compared to ND animals that received SAL. While we do not know why BDNF levels seem to be restored in D animals that received AMPH, we may consider the possibility that it results from a modulatory interaction between AMPH exposure and neonatal stress that differentially influenced BDNF expression, since ND animals that received AMPH did not present alterations in BDNF levels. Indeed, it has been shown that neurotrophins have relevant action on neurons involved in mediating the effects of psychostimulants, such as dopaminergic neurons, and can play dual roles: first, in neuronal survival and death, and, second, in activity-dependent plasticity [42]. Moreover, recent studies have indicated that activation of dopamine receptors (the main neurotransmitter involved in AMPH psychostimulant effect) induces an increase in BDNF mRNA and protein expression in neuronal cultures and in selected brain regions, such as the striatum and hippocampus [43–45]. Further experiments using intracerebral administration of BDNF and strategies to inhibit BDNF signaling (for instance, using RNA interference) are needed to clarify what the role of BDNF in mediating the effects of early stress and AMPH. Over the years, preclinical and clinical studies have shown that repeated exposure to amphetamine derivatives, such as methamand 3,4-methylenedioxy-N-methamphetamine phetamine, (MDMA), impairs recognition memory as well as dopaminergic neural transmission [46–49]. Bisagno et al. [15] have reported that chronic AMPH impairs short-term recognition memory, tested seven days after drug treatment. Long-term memory was not evaluated in that study. Our results indicate that a seven-day regimen of AMPH was able to significantly impair long-term recognition memory. Most importantly, here we show that the combination of maternal deprivation and adult AMPH administration produces a synergistic deleterious effect on cognitive performance assessed by recognition memory.
Fig. 3. Effects of maternal deprivation and d-amphetamine (AMPH) treatment on brain-derived neurotrophic factor (BDNF) levels. The levels of BDNF are expressed in: (A) prefrontal cortex; (B) hippocampus; (C) striatum; (D) amygdale; and (E) serum. Data are expressed as mean ± S.E.M. picogram of BDNF/g of protein, n = 5–8 animals per group. Rats were euthanized for BDNF measurement 10 days after the last drug injection. Differences between ND + SAL versus other groups are indicated as: *P < 0.05; differences between D + SAL group versus other groups are indicated as: + P < 0.05.
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5. Conclusions Taken together, the present results support the view that exposure to adverse events early in life might increase the susceptibility to the adverse effects of exposure to psychostimulant drugs during adulthood. This type of effect may have implications for the onset of a mood disorder [50–52]. In addition, a decrease in BDNF levels in the hippocampus might be involved in the long-lasting effects of maternal deprivation on memory and response to drugs. It has been suggested that alterations in BDNF signaling may contribute to the pathogenesis of mood and psychiatric disorders [6,53]. Since recognition memory changes were also seen in rats that had either been maternally deprived or received AMPH treatment in adulthood, it is reasonable to assume that other factors in addition to alterations in BDNF content may have been influenced by early stress. Further experiments will be required in order to characterize the molecular mechanisms by which exposure to an early environmental stressor produces long-lasting detrimental effects on cognition, and the possible implications for the development of mood and anxiety disorders. Acknowledgements Funding for this study was provided by CAPES-MEC, Brazil (AUXPE-Procad grant 657/2008). J.P.T. and V.A.G. are supported by a CAPES/MEC fellowship. L.H.H. is supported by an IC/CNPq fellowship. L.A.A. is supported by a PIBIC/CNPq fellowship. L.S. and G.R.F. are supported by an HCPA/CNPq fellowship. R.R., M.L.A., J.Q., F.K., and N.S. are CNPq research fellows. References [1] Bremner JD, Narayan M. The effects of stress on memory and the hippocampus throughout the life cycle: implications for childhood development and aging. Dev Psychopathol 1998;10:871–85. [2] Hennessy MB, Deak T, Schiml-Webb PA. Early attachment-figure separation and increased risk for later depression: potential mediation by proinflammatory processes. Neurosci Biobehav Rev 2010;34:782–90. [3] Kaffman A, Meaney MJ. Neurodevelopmental sequelae of postnatal maternal care in rodents: clinical and research implications of molecular insights. J Child Psychol Psychiatry 2007;48:224–44. [4] Veenema AH. Early life stress, the development of aggression and neuroendocrine and neurobiological correlates: what can we learn from animal models? Front Neuroendocrinol 2009;30:497–518. [5] Cirulli F, Francia N, Berry A, Aloe L, Alleva E, Suomic SJ. Early life stress as a risk factor for mental health: role of neurotrophins from rodents to non-human primates. Neurosci Biobehav Rev 2009;33:573–85. [6] Cowansage KK, LeDoux JE, Monfils MH. Brain-derived neurotrophic factor: a dynamic gatekeeper of neural plasticity. Curr Mol Pharmacol 2010;3:12–29. [7] Huot RL, Thrivikraman KV, Meaney MJ, Plotsky PM. Development of adult ethanol preference and anxiety as a consequence of neonatal maternal separation in long Evans rats and reversal with antidepressant treatment. Psychopharmacology (Berlin) 2001;158:366–73. [8] Burke AR, Renner KJ, Forster GL, Watt MJ. Adolescent social defeat alters neural, endocrine and behavioral responses to amphetamine in adult male rats. Brain Res 2010;1352:147–56. [9] Camp DM, DeJonghe DK, Robinson TE. Time-dependent effects of repeated amphetamine treatment on norepinephrine in the hypothalamus and hippocampus assessed with in vivo microdialysis. Neuropsychopharmacology 1997;17:130–40. [10] Frey BN, Martins MR, Petronilho FC, Dal-Pizzol F, Quevedo J, Kapczinski F. Increased oxidative stress after repeated amphetamine exposure: possible relevance as a model of mania. Bipolar Disord 2006;8:275–80. [11] Frey BN, Andreazza AC, Ceresér KM, Martins MR, Valvassori SS, Réus GZ, et al. Effects of mood stabilizers on hippocampus BDNF levels in an animal model of mania. Life Sci 2006;79:281–6. [12] Frey BN, Valvassori SS, Réus GZ, Martins MR, Petronilho FC, Bardini K, et al. Effects of lithium and valproate on amphetamine-induced oxidative stress generation in an animal model of mania. J Psychiatry Neurosci 2006;31:326–32. [13] Kauer-Sant’Anna M, Andreazza AC, Valvassori SS, Martins MR, Barbosa LM, Schwartsmann G, et al. A gastrin-releasing peptide receptor antagonist blocks d-amphetamine-induced hyperlocomotion and increases hippocampal NGF and BDNF levels in rats. Peptides 2007;28:1447–52. [14] Peleg-Raibstein D, Yee BK, Feldon J, Hauser J. The amphetamine sensitization model of schizophrenia: relevance beyond psychotic symptoms? Psychopharmacology (Berlin) 2009;206:603–21.
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