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Selegiline protects against recognition memory impairment induced by neonatal iron treatment
Experimental Neurology 196 (2005) 177 – 183 www.elsevier.com/locate/yexnr
Selegiline protects against recognition memory impairment induced by neonatal iron treatment Maria Noemia Martins de Lima a, Daniela Comparsi Laranja b, Fa´bio Caldana b, Manoela Michelon Grazziotin b, Vanessa Athaı´de Garcia b, Felipe Dal-Pizzol c, Elke Bromberg a,b, Nadja Schro¨der a,b,* a
Programa de Po´s-Graduac¸a˜o em Gerontologia Biome´dica, Instituto de Geriatria e Gerontologia, Hospital Sa˜o Lucas, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil b Laborato´rio de Memo´ria e Neurodegenerac¸a˜o, Departamento de Cieˆncias Fisiolo´gicas, Faculdade de Biocieˆncias, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil c Laborato´rio de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense, 88806-000, Criciu´ma, SC, Brazil Received 19 April 2005; revised 5 July 2005; accepted 26 July 2005 Available online 24 August 2005
Abstract Excess of iron in the brain has been implicated in the pathogenesis of several human neurodegenerative diseases, for example Alzheimer’s disease and Parkinson’s disease. It has been shown that the neonatal period is critical for the establishment of normal iron content in the adult brain. Moreover, it is known that aging alters the cerebral distribution of this metal. We have recently described that neonatal administration of iron severely impaired novel object recognition memory in rats. The aim of the present study was to determine whether selegiline, a monoamine oxidase (MAO) inhibitor known for its neuroprotective properties, could protect rats against cognitive impairment induced by neonatal administration of iron. In the first experiment, male Wistar rats received vehicle (5% sorbitol in water) or iron (10.0 mg/kg) orally from postnatal days 12 to 14 and saline (0.9% NaCl) or selegiline (1.0 or 10.0 mg/kg) intraperitoneally for 21 days, starting 24 h before the first iron dosing. In the second experiment, rats were given either vehicle or iron (10.0 mg/kg) orally from postnatal days 12 to 14 followed by saline or selegiline (1.0 or 10.0 mg/kg) intraperitoneally for 21 days, starting when rats reached adulthood (50th day after birth). Irontreated rats given selegiline in both doses showed no deficits in recognition memory. Rats receiving iron but no selegiline presented memory deficits. This is the first study reporting the reversion of iron-induced memory impairment, supporting the view that our model can be considered as a useful tool in the search for new drugs with neuroprotective and/or memory enhancing properties. D 2005 Elsevier Inc. All rights reserved. Keywords: Iron; Recognition memory; Selegiline; Neurodegeneration; Neuroprotection; Oxidative stress; Rat
Introduction The involvement of iron in several brain metabolic processes and normal development of neurological systems during a critical perinatal period, wherein deficiencies in this metal are associated with disruptions in behavioral performance, has been indicated (Youdim and Yehuda, 2000; * Corresponding author. Laborato´rio de Memo´ria e Neurodegenerac¸a˜o, Departamento de Cieˆncias Fisiolo´gicas, Faculdade de Biocieˆncias, Pontif´ıcia Universidade Cato´lica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil. Fax: +55 51 33203612. E-mail address: [email protected] (N. Schro¨der). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.07.017
Youdim et al., 1991; Ben-Shachar et al., 1986). However, there is also accumulating evidence that excessive deposits of iron in selective regions of the brain may generate cytotoxic free radical formation and cause alterations in iron metabolism, thereby possessing implications for the etiology of neurologic disorders (Zecca et al., 2004; Thomas and Jankovic, 2004; Kaur and Andersen, 2004; Sengstock et al., 1993). Increased levels of iron in selective brain regions have been reported in several neurodegenerative disorders, such as Parkinson’s (PD), HuntIngton’s (HD), HallervordenSpatz and Alzheimer’s (AD) diseases, amyotrophic lateral sclerosis as well as in normal brain aging (Jellinger, 1999).
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In some experimental animal models of PD where degeneration of nigrostriatal dopaminergic neurons has been observed, there is evidence for iron-induced oxidative stress as a pathogenic factor (Youdim et al., 2004; Leret et al., 2002). However, these models of PD are generally focused on the motor alterations associated with this disorder. The effects of iron administration during the neonatal period on cognition have been well documented. Adult mice (Fredriksson et al., 1999, 2000) and rats (Schro¨der et al., 2001) that received iron during a critical period of development, which corresponds to the period of maximal uptake of iron by the brain, showed spatial memory deficits when tested in the radial arm maze. In addition, this treatment has proven to promote disruption in the inhibitory avoidance task, a type of aversively motivated conditioning in rats (Schro¨der et al., 2001). We have recently found that iron neonatal treatment impairs long-term recognition memory in adult rats and induces oxidative damage in brain regions implicated in memory formation, thus raising the possibility that iron-induced cognitive deficits are at least partially mediated by oxidative stress (De Lima et al., 2005b). The monoamino oxidase B (MAO-B) inhibitor selegiline has been long known by its neuroprotective properties. In animal studies, selegiline has proved to be effective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced parkinsonism (Cohen et al., 1984). In addition, selegiline pretreatment can protect neurons from other dopaminergic neurotoxins such as MPP+ and h-carbolinium (Matsubara et al., 2001). Selegiline treatment after 6-OHDA exposure in rats reduced dopaminergic sensitivity to apomorphine without a concomitant increase in striatal dopamine content, suggesting that its effects could be related to neurorescue properties (Spooren et al., 1999). The purpose of the present study was to investigate the possible protective effect of selegiline against iron-induced cognitive deficits. In order to do that, rats neonatally treated with iron or vehicle, were divided in two main groups which received selegiline in two different phases of life: either during the first month of life, starting 24 h before the first iron dosing, or after reaching adulthood. After that, animals were trained and tested in the novel object recognition task, which is based on the spontaneous tendency of rodents to explore a novel object. It has been proposed that this task provides a close analogy with recognition tests that are widely used in humans to test memory and to characterize amnesic syndromes, by providing an accurate index of the overall severity of declarative memory impairment (Dix and Aggleton, 1999; Reed and Squire, 1997).
Methods Subjects Pregnant Wistar rats were obtained from Fundac¸a˜o Estadual de Pesquisa e Produc¸a˜o em Sau´de, Porto Alegre,
RS, Brazil. After birth, each litter was adjusted within 48 h to eight rat pups and to contain offspring of both genders in about equal proportions. Each pup was kept together with its mother in a plastic cage with sawdust bedding in a room temperature of 22 T 1-C and a 12/12 h light/dark cycle. At the age of 4 weeks, pups were weaned and the males were selected and raised in groups of three to five rats. For postnatal treatments, animals were supplied with standardized pellet food and tap water ad libitum. Behavioral testing started when animals reached the age of 3 months. All experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care. The protocol for this research was approved by the Institutional Ethics Committee of the Pontificia Universidade Catolica do Rio Grande do Sul (307/03-CEP). Treatments Pharmacological treatments are depicted in Fig. 1. The neonatal iron treatment has been described in detail elsewhere (Fredriksson et al., 1999; Schro¨der et al., 2001). Briefly, 12-day-old rat pups received orally a single daily dose (10.0 ml/kg solution volume) of vehicle (5% sorbitol in water) (control group) or 10.0 mg/kg of body weight of Fe2+ (Ferromyn\, AB Ha¨ssle, Go¨teborg, Sweden; iron concentration in the solution was 1.0 mg/ml) via a metallic gastric tube, over 3 days (postnatal days 12 –14). In this model, iron is given orally during the period of maximal iron uptake by the brain, so that the model correlates with dietary iron supplementation to infants. We have previously characterized that this treatment protocol induces a selective accumulation of iron in the rat basal ganglia (Schro¨der et al., 2001). In Experiment I, selegiline (0.0, 1.0 or 10.0 mg/ kg of body weight) (Sigma-Aldrich, SP, Brazil) was administered intraperitoneally during 21 days starting 24 h before the first iron dosing. The doses of selegiline, as well as the method of injection and treatment duration, were chosen on the basis of previous studies (Brandeis et al., 1991; Yavich et al., 1993; Stoll et al., 1994; Head et al., 1996; Kiray et al., 2004; Maia et al., 2004; De Lima et al., 2005a; Kiray et al., 2005). In Experiment II, selegiline at the same doses used in Experiment I was administered for 21 days starting when animals were 50-day old. Selegiline was dissolved in saline in a 1.0 ml/kg injection volume. Novel object recognition A rectangular open field (45 40 60 cm) with sawdust covering its floor was used in the novel object recognition task. On the first day, rats were submitted to a habituation session during which they were placed in the empty open field for 5 min. On the following day, rats were given one 5-min training trial in which they were exposed to two identical objects (A1 and A2). All objects were made of
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Fig. 1. (A) Scheme showing groups treated with vehicle or iron (orally) and saline or selegiline (i.p.) in the neonatal period. Iron or vehicle was given from postnatal days 12 to 14. Selegiline treatment started 24 h before iron treatment and was extended for 21 days. (B) Scheme showing groups treated with vehicle or iron (orally) from the 12th to the 14th postnatal day followed by saline or selegiline (i.p.) at adulthood (50th day after birth). Selegiline was administered for 21 days starting at the 50th day after birth. (n = 10 – 12 animals per group).
plastic Duplo Lego Toys and had a height of about 10 cm. Objects presented similar textures, colors and sizes, but distinctive shapes. The objects were positioned in two adjacent corners, 9 cm from the walls. Between trials, the objects were washed with a 10% ethanol solution. On the short-term memory (STM) testing trial (90 min 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. These were placed in the same locations as in the training session. 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. In STM and LTM retention test trials, the novel object was placed in 50% trials in the right side and 50% trials in the left side of the open field. The same animals were used to evaluate STM and LTM. Object exploration was measured by two experimenters blind to group treatment assignments, using two stopwatches to record the time spent exploring the objects during the experimental sessions. Exploration was defined as follows: sniffing or touching the object with the nose. Sitting on the object was not considered as exploration. A recognition index calculated for each animal was expressed by the ratio TB / (TA + TB) [TA = time spent exploring the object A;
TB = time spent exploring the object B] (Schro¨der et al., 2003; Tang et al., 1999). The possibility that selegiline affected sensorimotor function inducing alterations in locomotion, anxiety or motivation was assessed by evaluating the total time exploring objects during the training trials (Schro¨der et al., 2003; De Lima et al., 2005a). Other side effects of selegiline that could have affected functions unrelated to memory and behavior were not a concern for the present study. Statistical analysis Comparisons among groups were performed with a Kruskal – Wallis analysis of variance followed by Mann – Whitney U tests when necessary. Comparisons between sessions within the same group were performed with a Wilcoxon test. P values of less than 0.05 were considered to indicate statistical significance.
Results Statistical comparison of recognition indexes showed that groups treated neonatally with iron and receiving saline either neonatally (Fig. 2) or in adulthood (Fig. 3) showed
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impaired long-term retention of recognition memory, revealed by comparisons between groups treated with iron followed by saline and the respective control groups given vehicle followed by saline. Short-term retention was not affected by iron (Figs. 2A and 3A). Comparisons in recognition indexes between training and the long-term memory trial within each group indicated that animals given iron showed no significant difference between training and retention test performances, suggesting that these animals had a complete memory blockade revealed by the lack of preference towards the novel object in the long-term retention test trial (Figs. 2B and 3B). Iron-treated rats given selegiline (1.0 and 10.0 mg/kg) either in the neonatal period (Fig. 2) or in the adult life (Fig. 3) showed normal short- and long-term recognition memory. Animals given both iron and selegiline showed a significant difference in retention test performance when compared to animals given iron and saline, but not when compared to the respective control groups treated with vehicle and saline. This finding indicates that selegiline was able to fully protect against iron-induced recognition memory deficits. In
Fig. 3. Effect of selegiline on iron-induced recognition memory deficits in rats treated with vehicle or iron in the neonatal period followed by saline or selegiline in adulthood. Iron or vehicle was given from postnatal days 12 to 14. Selegiline was administered for 21 days starting at the 50th day after birth. Data are median (interquartile ranges) recognition indexes in (A) short- (1.5 h after training) and (B) long-term retention (24 h after training) test trials in a novel object recognition task. Behavioral testing was carried out when animals were 3 months old. The proportion of the total exploration time that the animal spent investigating the novel object was the ‘‘Recognition Index’’ expressed by the ratio TB / (TA + TB), TA = time spent exploring the familiar object and TB = time spent exploring the novel object; n = 10 – 12 animals per group; **P < 0.01 compared to the control group.
Fig. 2. Effect of selegiline on iron-induced recognition memory deficits in rats treated with vehicle or iron concomitantly with saline or selegiline in the neonatal period. Iron or vehicle was given from postnatal days 12 to 14. Selegiline treatment started 24 h before iron treatment and was extended for 21 days. Data are median (interquartile ranges) recognition indexes in (A) short- (1.5 h after training) and (B) long-term retention (24 h after training) test trials in a novel object recognition task. Behavioral testing was carried out when animals were 3 months old. The proportion of the total exploration time that the animal spent investigating the novel object was the ‘‘Recognition Index’’ expressed by the ratio TB / (TA + TB), TA = time spent exploring the familiar object and TB = time spent exploring the novel object; n = 10 – 12 animals per group; *P < 0.05 compared to the control group.
addition, results showed that selegiline by itself had no effect on recognition memory in adult rats, as revealed by comparisons between the groups given vehicle and selegiline and the respective control groups treated with saline and vehicle. There were no significant differences among groups in the total time exploring both objects during acquisition (training session), indicating that the treatments with iron and/or selegiline did not affect sensorimotor parameters such as locomotion, motivation and anxiety (overall mean T SE time exploring both objects during the training trial was 57.60 T 2.39 in Experiment I and 34.80 T 1.64 in Experiment II).
Discussion In the present study, iron-treated rats presented long-term recognition memory deficits consistent with those seen in a
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previous study from our laboratory (De Lima et al., 2005b). As previously reported, no significant differences were seen among the experimental groups in short-term memory, indicating that iron effects are restricted to the consolidation of long-term memory. However, iron-treated rats given selegiline showed no deficits in recognition memory. Taken together, these results suggest that selegiline, regardless the period in which it was administered (concomitantly with iron treatment or in adulthood), was able to protect against recognition memory deficits induced by neonatal iron exposure. Most importantly, this study reports for the first time the reversion of iron-induced memory impairment. The precise mechanisms underlying the effects of iron on cognition remain to be clarified. It is well known that iron content in the brain overlaps with the distribution of dopaminergic neurons (Roskams and Connor, 1994). Accordingly, we have previously reported that our iron neonatal treatment induces a selective increase in iron levels in the substantia nigra and the basal ganglia in rats (Schro¨der et al., 2001) and mice (Fredriksson et al., 1999). It has been proposed that iron accumulation in these areas can induce damage by interacting with hydrogen peroxide originated from dopamine metabolism. Moreover, it has been hypothesized that this mechanism could be involved in cell death during normal aging as well as in neurodegenerative disorders such as Parkinson’s disease (Youdim et al., 2005; Floyd and Hensley, 2002). It is generally accepted that iron accumulates in the brain as a function of age (Martin et al., 1998; Zecca et al., 2004). Besides, it has been found that neurotoxins such as MPTP and 6-OHDA, used to investigate the mechanism of dopaminergic neurodegeneration, increase iron concentration in the substantia nigra of rats, mice and monkeys (Oestreicher et al., 1994; Temlett et al., 1994). We have chosen two different time-periods to administer selegiline to iron-treated rats in order to determine whether iron-induced effect on cognition was permanent or it could be antagonized by a neuroprotective strategy. Previous studies have indicated that selegiline reverses memory impairments associated with aging (Brandeis et al., 1991; Stoll et al., 1994; Head et al., 1996; Kiray et al., 2004; De Lima et al., 2005a; Kiray et al., 2005), ischemia (Maia et al., 2004) or drug administration (Yavich et al., 1993), although several studies have suggested that selegiline fails to induce cognitive enhancement under normal conditions (Yavich et al., 1993; Stoll et al., 1994; Barbelivien et al., 2001). Assuming that the deleterious effects of iron were related to neurotoxicity and neuronal death, we could hypothesize that selegiline prevented memory deficits through a neuroprotective mechanism, as seen in Experiment I, when selegiline treatment started simultaneously with iron. However, results of Experiment II showed that selegiline was able to prevent iron-induced cognitive impairment even when given later in life, thus raising the possibility that ironinduced damage is not restricted to the neonatal period. Another possibility is that selegiline acted through other
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mechanisms of action (for instance, a drug-induced increase in extracellular monoamine content). In humans, selegiline has been investigated in a clinical trial called DATATOP Study, which has examined whether it could delay the disease progression in patients with PD. The results showed that selegiline slowed progression of the signs and symptoms, but did not stop disease progression (Parkinson Study Group, 1989; Tretud and Langston, 1989; Parkinson Study Group, 1993). Dixit et al. (1999) reported that selegiline can improve the motor symptoms and memory deficits in patients with PD. Besides the beneficial effects on PD, selegiline also increases cognitive performance in patients with AD. Schulzer et al. (1992) proposed that the beneficial effects of selegiline are symptomatic and mediated by an enhancement of dopaminergic neurotransmission, since MAO-B inhibition could increase the concentration of dopamine in the synaptic cleft and selegiline could also promote the accumulation of phenylethylamine, which potentiates the action of dopamine. This mechanism by itself would not account for the effects of selegiline in the group of rats given selegiline during the first month of life. The present results suggest that the beneficial effects of selegiline could be at least partially attributed to neuroprotection, since the group of rats that received selegiline during the first month of life had their iron-induced memory deficits prevented by the treatment. A large body of evidence has established that selegiline increases neuronal survival independently of MAO-B inhibition by interfering with apoptosis signaling pathways. Paterson et al. (1997) reported that selegiline treatment reduces delayed neuronal death of hippocampal pyramidal cells induced by hypoxia – ischemia. In another study, results indicated that ( )deprenyl increased hippocampal neuronal survival compared to saline-matched controls following kainic acid insult (Gelowitz and Paterson, 1999). In vitro studies indicated that selegiline protected dopaminergic SH-SYSY cells from apoptosis in a dosedependent way (Maruyama and Naoi, 1999). Another possibility is that antioxidant effects are involved in the neuroprotective actions of selegiline (Mytilineou et al., 1997; Tatton and Chalmers-Redman, 1996). There is evidence that administration of selegiline increases the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the nigrostriatum (Carrilo et al., 1992; Kitani et al., 2002). Interestingly, we have recently reported (De Lima et al., 2005b) that iron neonatal treatment induces oxidative damage in cortex, hippocampus and substantia nigra in rats exhibiting memory deficits, thus we can consider the possibility that selegiline could be exerting its neuroprotective effects against iron by increasing the antioxidant defenses. Oxidative stress has been implicated in the cognitive decline associated with aging and neurodegenerative disorders, and potential clinical applications of antioxidants in the treatment of age-related learning impairments have been proposed (Socci et al., 1995; Fukui et al.,
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2001, 2002; Liu et al., 2003; Kiray et al., 2004; Serrano and Klann, 2004). A recent study (Kiray et al., 2005) has indicated that beneficial effects of selegiline on memory in aged rats might be mediated by drug-induced reduction in lipid peroxidation. It is likely that different mechanisms are involved in mediating the effects of selegiline in animals treated during the neonatal period and adulthood. Future studies should further evaluate the possible mechanisms underlying the beneficial effects of selegiline, for instance by verifying whether these effects could be reversed by administration of dopamine receptor antagonists. In summary, young adult rats treated with iron for 3 days during the neonatal period show consistent and reproducible neurofunctional deficits. The reversion of ironinduced memory deficits can provide insights on the elucidation of the mechanisms underlying the effects of iron. Most importantly, the neonatal iron administration model can be an important tool in identifying drugs with neuroprotective or cognitive enhancing properties since it can be related to cognitive decline associated with either normal aging or neurodegenerative disorders involving brain iron accumulation. Acknowledgments This research was supported by CNPq-MCT-Brazil (grant 307265/2003-0 to N.S.) and PUCRS. References Barbelivien, A., Nyman, L., Haapalinna, A., Sirvio, J., 2001. Inhibition of MAO-A activity enhances behavioural activity of rats assessed using water maze and open arena tasks. Pharmacol. Toxicol. 88, 304 – 312. Ben-Shachar, D., Ashkenazi, R., Youdim, M.B., 1986. Long-term consequence of early iron-deficiency on dopaminergic neurotransmission in rats. Int. J. Dev. Neurosci. 4, 81 – 88. Brandeis, R., Sapir, M., Kapon, Y., Borelli, G., Cadel, S., Valsecchi, B., 1991. Improvement of cognitive function by MAO-B inhibitor ldeprenyl in aged rats. Pharmacol. Biochem. Behav. 39, 297 – 304. Carrilo, M.C., Kanai, S., Nokubo, M., Ivy, G.O., Sato, Y., Kitani, K., 1992. ( )Deprenyl increases activities of superoxido dismutase and catalase in striatum but not in hippocampus: the sex and age-related differences in the optimal dose in the rat. Exp. Neurol. 116, 286 – 294. Cohen, G., Pasik, P., Cohen, B., Leist, A., Mytilineou, C., Yahr, M.D., 1984. Pargyline and deprenyl prevent the neurotoxicity of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) in monkeys. Eur. J. Pharmacol. 106, 209 – 210. De Lima, M.N., Laranja, D.C., Caldana, F., Bromberg, E., Roesler, R., Schroder, N., 2005a. Reversal of age-related deficits in object recognition memory in rats with l-deprenyl. Exp. Gerontol. 40, 506 – 511. De Lima, M.N.M., Polydoro, M., Laranja, D.C., Bonnato, F., Bromberg, E., Moreira, J.C.F., Dal-Pizzol, F., Schro¨der, N., 2005b. Recognition memory impairment and brain oxidative stress induced by postnatal iron administration. Eur. J. Neurosci. 21, 2521 – 2528. Dix, S.L., Aggleton, J.P., 1999. Extending the spontaneous preference test of recognition: evidence of object-location and object-location and object-context recognition. Behav. Brain Res. 99, 191 – 200.
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Selegiline protects against recognition memory impairment induced by neonatal iron treatment Maria Noemia Martins de Lima a, Daniela Comparsi Laranja b, Fa´bio Caldana b, Manoela Michelon Grazziotin b, Vanessa Athaı´de Garcia b, Felipe Dal-Pizzol c, Elke Bromberg a,b, Nadja Schro¨der a,b,* a
Programa de Po´s-Graduac¸a˜o em Gerontologia Biome´dica, Instituto de Geriatria e Gerontologia, Hospital Sa˜o Lucas, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil b Laborato´rio de Memo´ria e Neurodegenerac¸a˜o, Departamento de Cieˆncias Fisiolo´gicas, Faculdade de Biocieˆncias, Pontifı´cia Universidade Cato´lica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil c Laborato´rio de Fisiopatologia Experimental, Universidade do Extremo Sul Catarinense, 88806-000, Criciu´ma, SC, Brazil Received 19 April 2005; revised 5 July 2005; accepted 26 July 2005 Available online 24 August 2005
Abstract Excess of iron in the brain has been implicated in the pathogenesis of several human neurodegenerative diseases, for example Alzheimer’s disease and Parkinson’s disease. It has been shown that the neonatal period is critical for the establishment of normal iron content in the adult brain. Moreover, it is known that aging alters the cerebral distribution of this metal. We have recently described that neonatal administration of iron severely impaired novel object recognition memory in rats. The aim of the present study was to determine whether selegiline, a monoamine oxidase (MAO) inhibitor known for its neuroprotective properties, could protect rats against cognitive impairment induced by neonatal administration of iron. In the first experiment, male Wistar rats received vehicle (5% sorbitol in water) or iron (10.0 mg/kg) orally from postnatal days 12 to 14 and saline (0.9% NaCl) or selegiline (1.0 or 10.0 mg/kg) intraperitoneally for 21 days, starting 24 h before the first iron dosing. In the second experiment, rats were given either vehicle or iron (10.0 mg/kg) orally from postnatal days 12 to 14 followed by saline or selegiline (1.0 or 10.0 mg/kg) intraperitoneally for 21 days, starting when rats reached adulthood (50th day after birth). Irontreated rats given selegiline in both doses showed no deficits in recognition memory. Rats receiving iron but no selegiline presented memory deficits. This is the first study reporting the reversion of iron-induced memory impairment, supporting the view that our model can be considered as a useful tool in the search for new drugs with neuroprotective and/or memory enhancing properties. D 2005 Elsevier Inc. All rights reserved. Keywords: Iron; Recognition memory; Selegiline; Neurodegeneration; Neuroprotection; Oxidative stress; Rat
Introduction The involvement of iron in several brain metabolic processes and normal development of neurological systems during a critical perinatal period, wherein deficiencies in this metal are associated with disruptions in behavioral performance, has been indicated (Youdim and Yehuda, 2000; * Corresponding author. Laborato´rio de Memo´ria e Neurodegenerac¸a˜o, Departamento de Cieˆncias Fisiolo´gicas, Faculdade de Biocieˆncias, Pontif´ıcia Universidade Cato´lica do Rio Grande do Sul, 90619-900 Porto Alegre, RS, Brazil. Fax: +55 51 33203612. E-mail address: [email protected] (N. Schro¨der). 0014-4886/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2005.07.017
Youdim et al., 1991; Ben-Shachar et al., 1986). However, there is also accumulating evidence that excessive deposits of iron in selective regions of the brain may generate cytotoxic free radical formation and cause alterations in iron metabolism, thereby possessing implications for the etiology of neurologic disorders (Zecca et al., 2004; Thomas and Jankovic, 2004; Kaur and Andersen, 2004; Sengstock et al., 1993). Increased levels of iron in selective brain regions have been reported in several neurodegenerative disorders, such as Parkinson’s (PD), HuntIngton’s (HD), HallervordenSpatz and Alzheimer’s (AD) diseases, amyotrophic lateral sclerosis as well as in normal brain aging (Jellinger, 1999).
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In some experimental animal models of PD where degeneration of nigrostriatal dopaminergic neurons has been observed, there is evidence for iron-induced oxidative stress as a pathogenic factor (Youdim et al., 2004; Leret et al., 2002). However, these models of PD are generally focused on the motor alterations associated with this disorder. The effects of iron administration during the neonatal period on cognition have been well documented. Adult mice (Fredriksson et al., 1999, 2000) and rats (Schro¨der et al., 2001) that received iron during a critical period of development, which corresponds to the period of maximal uptake of iron by the brain, showed spatial memory deficits when tested in the radial arm maze. In addition, this treatment has proven to promote disruption in the inhibitory avoidance task, a type of aversively motivated conditioning in rats (Schro¨der et al., 2001). We have recently found that iron neonatal treatment impairs long-term recognition memory in adult rats and induces oxidative damage in brain regions implicated in memory formation, thus raising the possibility that iron-induced cognitive deficits are at least partially mediated by oxidative stress (De Lima et al., 2005b). The monoamino oxidase B (MAO-B) inhibitor selegiline has been long known by its neuroprotective properties. In animal studies, selegiline has proved to be effective against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced parkinsonism (Cohen et al., 1984). In addition, selegiline pretreatment can protect neurons from other dopaminergic neurotoxins such as MPP+ and h-carbolinium (Matsubara et al., 2001). Selegiline treatment after 6-OHDA exposure in rats reduced dopaminergic sensitivity to apomorphine without a concomitant increase in striatal dopamine content, suggesting that its effects could be related to neurorescue properties (Spooren et al., 1999). The purpose of the present study was to investigate the possible protective effect of selegiline against iron-induced cognitive deficits. In order to do that, rats neonatally treated with iron or vehicle, were divided in two main groups which received selegiline in two different phases of life: either during the first month of life, starting 24 h before the first iron dosing, or after reaching adulthood. After that, animals were trained and tested in the novel object recognition task, which is based on the spontaneous tendency of rodents to explore a novel object. It has been proposed that this task provides a close analogy with recognition tests that are widely used in humans to test memory and to characterize amnesic syndromes, by providing an accurate index of the overall severity of declarative memory impairment (Dix and Aggleton, 1999; Reed and Squire, 1997).
Methods Subjects Pregnant Wistar rats were obtained from Fundac¸a˜o Estadual de Pesquisa e Produc¸a˜o em Sau´de, Porto Alegre,
RS, Brazil. After birth, each litter was adjusted within 48 h to eight rat pups and to contain offspring of both genders in about equal proportions. Each pup was kept together with its mother in a plastic cage with sawdust bedding in a room temperature of 22 T 1-C and a 12/12 h light/dark cycle. At the age of 4 weeks, pups were weaned and the males were selected and raised in groups of three to five rats. For postnatal treatments, animals were supplied with standardized pellet food and tap water ad libitum. Behavioral testing started when animals reached the age of 3 months. All experimental procedures were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care. The protocol for this research was approved by the Institutional Ethics Committee of the Pontificia Universidade Catolica do Rio Grande do Sul (307/03-CEP). Treatments Pharmacological treatments are depicted in Fig. 1. The neonatal iron treatment has been described in detail elsewhere (Fredriksson et al., 1999; Schro¨der et al., 2001). Briefly, 12-day-old rat pups received orally a single daily dose (10.0 ml/kg solution volume) of vehicle (5% sorbitol in water) (control group) or 10.0 mg/kg of body weight of Fe2+ (Ferromyn\, AB Ha¨ssle, Go¨teborg, Sweden; iron concentration in the solution was 1.0 mg/ml) via a metallic gastric tube, over 3 days (postnatal days 12 –14). In this model, iron is given orally during the period of maximal iron uptake by the brain, so that the model correlates with dietary iron supplementation to infants. We have previously characterized that this treatment protocol induces a selective accumulation of iron in the rat basal ganglia (Schro¨der et al., 2001). In Experiment I, selegiline (0.0, 1.0 or 10.0 mg/ kg of body weight) (Sigma-Aldrich, SP, Brazil) was administered intraperitoneally during 21 days starting 24 h before the first iron dosing. The doses of selegiline, as well as the method of injection and treatment duration, were chosen on the basis of previous studies (Brandeis et al., 1991; Yavich et al., 1993; Stoll et al., 1994; Head et al., 1996; Kiray et al., 2004; Maia et al., 2004; De Lima et al., 2005a; Kiray et al., 2005). In Experiment II, selegiline at the same doses used in Experiment I was administered for 21 days starting when animals were 50-day old. Selegiline was dissolved in saline in a 1.0 ml/kg injection volume. Novel object recognition A rectangular open field (45 40 60 cm) with sawdust covering its floor was used in the novel object recognition task. On the first day, rats were submitted to a habituation session during which they were placed in the empty open field for 5 min. On the following day, rats were given one 5-min training trial in which they were exposed to two identical objects (A1 and A2). All objects were made of
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Fig. 1. (A) Scheme showing groups treated with vehicle or iron (orally) and saline or selegiline (i.p.) in the neonatal period. Iron or vehicle was given from postnatal days 12 to 14. Selegiline treatment started 24 h before iron treatment and was extended for 21 days. (B) Scheme showing groups treated with vehicle or iron (orally) from the 12th to the 14th postnatal day followed by saline or selegiline (i.p.) at adulthood (50th day after birth). Selegiline was administered for 21 days starting at the 50th day after birth. (n = 10 – 12 animals per group).
plastic Duplo Lego Toys and had a height of about 10 cm. Objects presented similar textures, colors and sizes, but distinctive shapes. The objects were positioned in two adjacent corners, 9 cm from the walls. Between trials, the objects were washed with a 10% ethanol solution. On the short-term memory (STM) testing trial (90 min 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. These were placed in the same locations as in the training session. 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. In STM and LTM retention test trials, the novel object was placed in 50% trials in the right side and 50% trials in the left side of the open field. The same animals were used to evaluate STM and LTM. Object exploration was measured by two experimenters blind to group treatment assignments, using two stopwatches to record the time spent exploring the objects during the experimental sessions. Exploration was defined as follows: sniffing or touching the object with the nose. Sitting on the object was not considered as exploration. A recognition index calculated for each animal was expressed by the ratio TB / (TA + TB) [TA = time spent exploring the object A;
TB = time spent exploring the object B] (Schro¨der et al., 2003; Tang et al., 1999). The possibility that selegiline affected sensorimotor function inducing alterations in locomotion, anxiety or motivation was assessed by evaluating the total time exploring objects during the training trials (Schro¨der et al., 2003; De Lima et al., 2005a). Other side effects of selegiline that could have affected functions unrelated to memory and behavior were not a concern for the present study. Statistical analysis Comparisons among groups were performed with a Kruskal – Wallis analysis of variance followed by Mann – Whitney U tests when necessary. Comparisons between sessions within the same group were performed with a Wilcoxon test. P values of less than 0.05 were considered to indicate statistical significance.
Results Statistical comparison of recognition indexes showed that groups treated neonatally with iron and receiving saline either neonatally (Fig. 2) or in adulthood (Fig. 3) showed
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impaired long-term retention of recognition memory, revealed by comparisons between groups treated with iron followed by saline and the respective control groups given vehicle followed by saline. Short-term retention was not affected by iron (Figs. 2A and 3A). Comparisons in recognition indexes between training and the long-term memory trial within each group indicated that animals given iron showed no significant difference between training and retention test performances, suggesting that these animals had a complete memory blockade revealed by the lack of preference towards the novel object in the long-term retention test trial (Figs. 2B and 3B). Iron-treated rats given selegiline (1.0 and 10.0 mg/kg) either in the neonatal period (Fig. 2) or in the adult life (Fig. 3) showed normal short- and long-term recognition memory. Animals given both iron and selegiline showed a significant difference in retention test performance when compared to animals given iron and saline, but not when compared to the respective control groups treated with vehicle and saline. This finding indicates that selegiline was able to fully protect against iron-induced recognition memory deficits. In
Fig. 3. Effect of selegiline on iron-induced recognition memory deficits in rats treated with vehicle or iron in the neonatal period followed by saline or selegiline in adulthood. Iron or vehicle was given from postnatal days 12 to 14. Selegiline was administered for 21 days starting at the 50th day after birth. Data are median (interquartile ranges) recognition indexes in (A) short- (1.5 h after training) and (B) long-term retention (24 h after training) test trials in a novel object recognition task. Behavioral testing was carried out when animals were 3 months old. The proportion of the total exploration time that the animal spent investigating the novel object was the ‘‘Recognition Index’’ expressed by the ratio TB / (TA + TB), TA = time spent exploring the familiar object and TB = time spent exploring the novel object; n = 10 – 12 animals per group; **P < 0.01 compared to the control group.
Fig. 2. Effect of selegiline on iron-induced recognition memory deficits in rats treated with vehicle or iron concomitantly with saline or selegiline in the neonatal period. Iron or vehicle was given from postnatal days 12 to 14. Selegiline treatment started 24 h before iron treatment and was extended for 21 days. Data are median (interquartile ranges) recognition indexes in (A) short- (1.5 h after training) and (B) long-term retention (24 h after training) test trials in a novel object recognition task. Behavioral testing was carried out when animals were 3 months old. The proportion of the total exploration time that the animal spent investigating the novel object was the ‘‘Recognition Index’’ expressed by the ratio TB / (TA + TB), TA = time spent exploring the familiar object and TB = time spent exploring the novel object; n = 10 – 12 animals per group; *P < 0.05 compared to the control group.
addition, results showed that selegiline by itself had no effect on recognition memory in adult rats, as revealed by comparisons between the groups given vehicle and selegiline and the respective control groups treated with saline and vehicle. There were no significant differences among groups in the total time exploring both objects during acquisition (training session), indicating that the treatments with iron and/or selegiline did not affect sensorimotor parameters such as locomotion, motivation and anxiety (overall mean T SE time exploring both objects during the training trial was 57.60 T 2.39 in Experiment I and 34.80 T 1.64 in Experiment II).
Discussion In the present study, iron-treated rats presented long-term recognition memory deficits consistent with those seen in a
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previous study from our laboratory (De Lima et al., 2005b). As previously reported, no significant differences were seen among the experimental groups in short-term memory, indicating that iron effects are restricted to the consolidation of long-term memory. However, iron-treated rats given selegiline showed no deficits in recognition memory. Taken together, these results suggest that selegiline, regardless the period in which it was administered (concomitantly with iron treatment or in adulthood), was able to protect against recognition memory deficits induced by neonatal iron exposure. Most importantly, this study reports for the first time the reversion of iron-induced memory impairment. The precise mechanisms underlying the effects of iron on cognition remain to be clarified. It is well known that iron content in the brain overlaps with the distribution of dopaminergic neurons (Roskams and Connor, 1994). Accordingly, we have previously reported that our iron neonatal treatment induces a selective increase in iron levels in the substantia nigra and the basal ganglia in rats (Schro¨der et al., 2001) and mice (Fredriksson et al., 1999). It has been proposed that iron accumulation in these areas can induce damage by interacting with hydrogen peroxide originated from dopamine metabolism. Moreover, it has been hypothesized that this mechanism could be involved in cell death during normal aging as well as in neurodegenerative disorders such as Parkinson’s disease (Youdim et al., 2005; Floyd and Hensley, 2002). It is generally accepted that iron accumulates in the brain as a function of age (Martin et al., 1998; Zecca et al., 2004). Besides, it has been found that neurotoxins such as MPTP and 6-OHDA, used to investigate the mechanism of dopaminergic neurodegeneration, increase iron concentration in the substantia nigra of rats, mice and monkeys (Oestreicher et al., 1994; Temlett et al., 1994). We have chosen two different time-periods to administer selegiline to iron-treated rats in order to determine whether iron-induced effect on cognition was permanent or it could be antagonized by a neuroprotective strategy. Previous studies have indicated that selegiline reverses memory impairments associated with aging (Brandeis et al., 1991; Stoll et al., 1994; Head et al., 1996; Kiray et al., 2004; De Lima et al., 2005a; Kiray et al., 2005), ischemia (Maia et al., 2004) or drug administration (Yavich et al., 1993), although several studies have suggested that selegiline fails to induce cognitive enhancement under normal conditions (Yavich et al., 1993; Stoll et al., 1994; Barbelivien et al., 2001). Assuming that the deleterious effects of iron were related to neurotoxicity and neuronal death, we could hypothesize that selegiline prevented memory deficits through a neuroprotective mechanism, as seen in Experiment I, when selegiline treatment started simultaneously with iron. However, results of Experiment II showed that selegiline was able to prevent iron-induced cognitive impairment even when given later in life, thus raising the possibility that ironinduced damage is not restricted to the neonatal period. Another possibility is that selegiline acted through other
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mechanisms of action (for instance, a drug-induced increase in extracellular monoamine content). In humans, selegiline has been investigated in a clinical trial called DATATOP Study, which has examined whether it could delay the disease progression in patients with PD. The results showed that selegiline slowed progression of the signs and symptoms, but did not stop disease progression (Parkinson Study Group, 1989; Tretud and Langston, 1989; Parkinson Study Group, 1993). Dixit et al. (1999) reported that selegiline can improve the motor symptoms and memory deficits in patients with PD. Besides the beneficial effects on PD, selegiline also increases cognitive performance in patients with AD. Schulzer et al. (1992) proposed that the beneficial effects of selegiline are symptomatic and mediated by an enhancement of dopaminergic neurotransmission, since MAO-B inhibition could increase the concentration of dopamine in the synaptic cleft and selegiline could also promote the accumulation of phenylethylamine, which potentiates the action of dopamine. This mechanism by itself would not account for the effects of selegiline in the group of rats given selegiline during the first month of life. The present results suggest that the beneficial effects of selegiline could be at least partially attributed to neuroprotection, since the group of rats that received selegiline during the first month of life had their iron-induced memory deficits prevented by the treatment. A large body of evidence has established that selegiline increases neuronal survival independently of MAO-B inhibition by interfering with apoptosis signaling pathways. Paterson et al. (1997) reported that selegiline treatment reduces delayed neuronal death of hippocampal pyramidal cells induced by hypoxia – ischemia. In another study, results indicated that ( )deprenyl increased hippocampal neuronal survival compared to saline-matched controls following kainic acid insult (Gelowitz and Paterson, 1999). In vitro studies indicated that selegiline protected dopaminergic SH-SYSY cells from apoptosis in a dosedependent way (Maruyama and Naoi, 1999). Another possibility is that antioxidant effects are involved in the neuroprotective actions of selegiline (Mytilineou et al., 1997; Tatton and Chalmers-Redman, 1996). There is evidence that administration of selegiline increases the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) in the nigrostriatum (Carrilo et al., 1992; Kitani et al., 2002). Interestingly, we have recently reported (De Lima et al., 2005b) that iron neonatal treatment induces oxidative damage in cortex, hippocampus and substantia nigra in rats exhibiting memory deficits, thus we can consider the possibility that selegiline could be exerting its neuroprotective effects against iron by increasing the antioxidant defenses. Oxidative stress has been implicated in the cognitive decline associated with aging and neurodegenerative disorders, and potential clinical applications of antioxidants in the treatment of age-related learning impairments have been proposed (Socci et al., 1995; Fukui et al.,
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2001, 2002; Liu et al., 2003; Kiray et al., 2004; Serrano and Klann, 2004). A recent study (Kiray et al., 2005) has indicated that beneficial effects of selegiline on memory in aged rats might be mediated by drug-induced reduction in lipid peroxidation. It is likely that different mechanisms are involved in mediating the effects of selegiline in animals treated during the neonatal period and adulthood. Future studies should further evaluate the possible mechanisms underlying the beneficial effects of selegiline, for instance by verifying whether these effects could be reversed by administration of dopamine receptor antagonists. In summary, young adult rats treated with iron for 3 days during the neonatal period show consistent and reproducible neurofunctional deficits. The reversion of ironinduced memory deficits can provide insights on the elucidation of the mechanisms underlying the effects of iron. Most importantly, the neonatal iron administration model can be an important tool in identifying drugs with neuroprotective or cognitive enhancing properties since it can be related to cognitive decline associated with either normal aging or neurodegenerative disorders involving brain iron accumulation. Acknowledgments This research was supported by CNPq-MCT-Brazil (grant 307265/2003-0 to N.S.) and PUCRS. References Barbelivien, A., Nyman, L., Haapalinna, A., Sirvio, J., 2001. Inhibition of MAO-A activity enhances behavioural activity of rats assessed using water maze and open arena tasks. Pharmacol. Toxicol. 88, 304 – 312. Ben-Shachar, D., Ashkenazi, R., Youdim, M.B., 1986. Long-term consequence of early iron-deficiency on dopaminergic neurotransmission in rats. Int. J. Dev. Neurosci. 4, 81 – 88. Brandeis, R., Sapir, M., Kapon, Y., Borelli, G., Cadel, S., Valsecchi, B., 1991. Improvement of cognitive function by MAO-B inhibitor ldeprenyl in aged rats. Pharmacol. Biochem. Behav. 39, 297 – 304. Carrilo, M.C., Kanai, S., Nokubo, M., Ivy, G.O., Sato, Y., Kitani, K., 1992. ( )Deprenyl increases activities of superoxido dismutase and catalase in striatum but not in hippocampus: the sex and age-related differences in the optimal dose in the rat. Exp. Neurol. 116, 286 – 294. Cohen, G., Pasik, P., Cohen, B., Leist, A., Mytilineou, C., Yahr, M.D., 1984. Pargyline and deprenyl prevent the neurotoxicity of 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP) in monkeys. Eur. J. Pharmacol. 106, 209 – 210. De Lima, M.N., Laranja, D.C., Caldana, F., Bromberg, E., Roesler, R., Schroder, N., 2005a. Reversal of age-related deficits in object recognition memory in rats with l-deprenyl. Exp. Gerontol. 40, 506 – 511. De Lima, M.N.M., Polydoro, M., Laranja, D.C., Bonnato, F., Bromberg, E., Moreira, J.C.F., Dal-Pizzol, F., Schro¨der, N., 2005b. Recognition memory impairment and brain oxidative stress induced by postnatal iron administration. Eur. J. Neurosci. 21, 2521 – 2528. Dix, S.L., Aggleton, J.P., 1999. Extending the spontaneous preference test of recognition: evidence of object-location and object-location and object-context recognition. Behav. Brain Res. 99, 191 – 200.
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