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Developmental Cognitive Neuroscience 3 (2013) 1– 10

Contents lists available at SciVerse ScienceDirect

Developmental Cognitive Neuroscience

j ourna l ho me pag e: ht t p: / /www.e lsev ier .com/ locate /dcn

thanolic extract from bulbs of Cipura paludosa reduced long-lastingearning and memory deficits induced by prenatal methylmercuryxposure in rats

reice M. Lucenaa, Rui D. Predigerb, Mônica V. Silvaa, Setsuko N. Santosa,oão F. Bomfim Silvaa, Adair R.S. Santosb, Mariangela S. Azevedoc, Vania M. Ferreiraa,∗

Faculdade de Ciências da Saúde, Universidade de Brasília (UnB), DF 70910-900, BrazilCentro de Ciências Biológicas, Universidade Federal de Santa Catarina (UFSC), Campus Universitário, Trindade, Florianópolis, SC 88049-900, BrazilDepartamento de Química, Universidade Federal de Rondônia (UNIR), Porto Velho, RO 78980-500, Brazil

r t i c l e i n f o

rticle history:eceived 25 January 2012eceived in revised form 16 August 2012ccepted 17 August 2012

eywords:ipura paludosaearning and memoryethylmercury (MeHg)eurological deficits

a b s t r a c t

Previous studies from our group have indicated important biological properties of theethanolic extract (EE) and isolated compounds from the bulbs of Cipura paludosa (Iridaceae),a native plant widely distributed in northern Brazil. In the present study, the effects ofchronic treatment with the EE on the memory of adult rats exposed to methylmercury(MeHg) during early development were assessed. Pregnant rats were treated by gavagewith a single dose of MeHg (8 mg/kg) on gestational day 15, the developmental stage crit-ical for cortical neuron proliferation. Adult offspring were administered orally with theEE of C. paludosa (1, 10 or 100 mg/kg) over 14 consecutive days. EE improved short-termsocial memory in a specific manner and facilitated the step-down inhibitory avoidance ofshort- and long-term memory. MeHg exposure induced pronounced long-lasting impair-ments in social recognition memory that were improved by EE. Moreover, EE significantlyincreased the step-down latencies specifically during the short-term session in prenatal

MeHg-exposed rats. These results demonstrate that EE reduced the long-lasting short-termlearning and memory deficits induced by MeHg exposure. These findings may encour-age further studies evaluating the cognitive enhancing properties of C. paludosa and itscomponents on neuropathological conditions associated with exposure to environmentalcontaminants.

© 2012 Elsevier Ltd. All rights reserved.

. Introduction

Consumption of methylmercury (MeHg)-contaminated

ood by pregnant women represents one of the most seri-us potential hazards for their offspring. While high-dosexposure may result in cerebral palsy, deafness, and severe

∗ Corresponding author at: Universidade de Brasília (UnB), Campusniversitário Darcy Ribeiro (Asa Norte), 70910-900 Brasília, DF, Brazil.el.: +55 61 8122 0005.

E-mail address: vmmf@unb.br (V.M. Ferreira).

878-9293/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.ttp://dx.doi.org/10.1016/j.dcn.2012.08.003

mental retardation associated with disorganisation of cere-bral cortex cyto-architecture and atrophy of the folia ofcerebellum hemispheres, lower MeHg doses may producemore subtle neurobehavioural changes (Choi, 1989; Lianget al., 2009; National Research Council, 2000).

Experimental data obtained in rodents show that theconsequences of in utero exposure to MeHg includeincreased intrauterine mortality of offspring, delayed

developmental growth, altered cellular brain arrange-ments, and more subtle effects such as delayed reflex-ive behaviours, impairment of locomotor activity andmotor coordination, cognitive and emotional dysfunctions,

ntal Cog

need to be further clarified. All procedures used in the

2 G.M. Lucena et al. / Developme

depending on the duration and level of MeHg exposureat different developmental stages of offspring (Ferraroet al., 2009; Lucena et al., 2010; Maia et al., 2009).Moreover, as recently reviewed by Farina et al. (2011),experimental studies have been pivotal in elucidating themolecular mechanisms that mediate MeHg-induced neu-rotoxicity that include the impairment of intracellularcalcium homeostasis (Sirois and Atchison, 2000), oxida-tive stress (Franco et al., 2007; Lucena et al., 2010; Ouet al., 1999), and the alteration of glutamate homeosta-sis (Aschner et al., 2000; Farina et al., 2003; Manfroi et al.,2004).

Although MeHg-induced neurotoxicity is an extensivelyreported phenomenon, there are no effective treatmentsavailable that completely abolish its toxic effects; thus,supportive care is often needed to maintain vital func-tions. In addition, chelating agents that aid the body’sability to eliminate mercury from tissues are of limiteduse because of their adverse side effects (Tchounwou et al.,2003). Interestingly, more recent studies have indicated thepotential of some purified phytochemicals or plant extractsto confer protection against excitotoxicity and oxidativestress following co-exposure with MeHg (Lucena et al.,2007a; Farina et al., 2005; Yongjin et al., 2008). Possiblemechanisms involved in the mitigation of MeHg effects byphytochemicals may include the reduction of reactive oxy-gen species, activation of enzymatic antioxidant systems,restoration of the mitochondrial membrane potential, andmodulation of cell signalling pathways (Campos-Esparzaet al., 2009; Farina et al., 2011; Franco et al., 2010).

In this context, previous findings from our group havedemonstrated the protective effects of the ethanolic extract(EE) of Cipura paludosa Aubl. against the MeHg-inducedneurotoxicity in adult mice (Lucena et al., 2007a). C. palu-dosa, a member of the large family Iridaceae, is principallycharacterised by a bulbous rootstock (Goldblatt, 1990).It is a native plant, widely distributed in the north ofBrazil, popularly known as ‘batata roxa’, ‘alho-do-mato’and ‘cebolinha-do-campo’. During the last years, we haveextensively studied the pharmacological properties of theEE, fractions and isolated compounds from the bulbs ofthis plant. Our previous studies indicated a series of bio-logical activities of C. paludosa including antinociceptiveand anti-inflammatory (Lucena et al., 2007b), and antiox-idant and antiglutamatergic (Lucena et al., 2007a) effects.More recently, we also demonstrated the in vivo effects ofthe major compounds (eleutherine and isoeleutherine) indifferent models of hypernociception and inflammation inmice (Tessele et al., 2011), justifying, at least in part, itspopular therapeutic use for treating conditions related toinflammation and dolorous processes.

Of high interest, we observed recently that chronictreatment with the EE of C. paludosa attenuated theanxiety- and depression-like behaviours and the antioxi-dant deficits induced by prenatal MeHg exposure in rats(Lucena et al., 2010). Taking into account the absence ofeffective treatments for neurological deficits associated

with MeHg exposure and the existence of previous findingsdemonstrating that C. paludosa may represent a valuabletool to attenuate long-lasting emotional defects inducedby prenatal MeHg exposure, we investigated in the current

nitive Neuroscience 3 (2013) 1– 10

study the effects of chronic administration of the EE of C.paludosa on the short- and long-term learning and mem-ory impairments in adult rats born to dams treated withMeHg.

2. Materials and methods

2.1. Breeding and prenatal treatment

Three-month-old male and female Wistar rats, obtainedfrom the Animal Facility of Centro Universitário deBrasília (UNICEUB, Brazil), were housed in groups offive animals/sex in polycarbonate cages under controlledconditions of temperature (23 ± 1 ◦C) and photoperiod(light:dark, 12:12 h) with free access to food and water.For mating, individual females were placed overnightwith a single male. Detection of a sperm plug the nextmorning denoted pregnancy, indicating gestation day one.Pregnant rats were housed individually in polycarbonatecages and assigned randomly to receive tap water or MeHg(8 mg/kg) on gestational day 15. This day represents a crit-ical developmental stage when the forebrain ventricularzone produces neurons in the embryonic cerebral cortex(DiCicco-Bloom and Sondell, 2005). Solutions were admin-istered by means of intragastric intubation (gavage) at avolume of 1 ml/kg of body weight dissolved in NaCl 0.9%(saline). The schedule of MeHg treatment was selectedaccording to previous literature (Lucena et al., 2010; Maiaet al., 2009; Zanoli et al., 1994) and it represents a model forthe study of long-lasting behavioural impairments of acutepoisoning during pregnancy.

2.2. Pregnancy outcome

The day of birth was designated as postnatal day one.After birth, the litters were culled to ten pups per litterand returned to their mothers until weaning on postnatalday 21, when they were then grouped (n = 5 animals) bysex and regimen of treatment. During the lactation period,offspring were maintained with their respective dams toavoid interference related to maternal deprivation, whichhas been linked to behavioural alterations including anx-iety, depression and cognitive dysfunction (Daniels et al.,2004; Huot et al., 2002).

All behavioural experiments were carried out withtwo-month-old male rats to avoid the well-described fluc-tuations of learning and memory performance of femalerats across the oestrous cycle (Korol et al., 2004; Pompiliet al., 2010). Moreover, previous epidemiological studieswith children (Grandjean et al., 1998) and experimen-tal studies with laboratory animals (Gimenez-Llort et al.,2001; Onishchenko et al., 2007; Rossi et al., 1997) on MeHgexposure have also reported more pronounced develop-mental effects in males than in females, although themechanisms underlying such gender-related differences

present study were in compliance with the guidelines onanimal care of the UnB Ethics Committee on the Use of Ani-mals, which follows the “Principles of laboratory animalcare” from NIH publication no. 85-23.

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.3. Preparation of the EE of C. paludosa and treatment

Plant material was collected at Porto Velho, State ofondônia, Brazil. A voucher specimen was deposited at theerbarium Dr. Ary Tupinambá Penna Pinheiro (Porto Velho,razil) under number 1782. Bulbs from C. paludosa wereried in a stove at 40 ◦C and subsequently powdered using aortar and pestle. The powder was incubated with ethanol

95%, v/v) at room temperature for approximately sevenays. The supernatant was filtered on filter paper, and theulb powder was submitted to a second extraction withthanol. The supernatants from both ethanol extractionsere combined, and the solvent was evaporated under

educed pressure at 40 ◦C to obtain the crude EE.EE of C. paludosa was dissolved in NaCl 0.9% (saline) plus

ween 80 (Merck, Darmstadt, Germany). The final concen-ration of Tween 80 did not exceed 2% and did not causeny per se effects. The control solution consisted of salineith 2% Tween 80 (vehicle). The doses of the EE of C. palu-

osa (1, 10 or 100 mg/kg), selected according to previousiterature (Lucena et al., 2007a, 2010), were administeredrally in a volume of 1 ml/kg of body weight to adult maleffspring rats (two months old) for 14 consecutive days. Allehavioural experiments were carried out 1 h following the

ast extract administration. The present treatment sched-le was selected according to a previous study from ourroup (Lucena et al., 2010) and was designed to investigatehe effects of chronic extract treatment on the long-lastingognitive deficits induced by prenatal MeHg exposure.

.4. Behavioural tests

Animals were acclimated to the experimental room fort least 2 h before beginning the experimental procedures,hich were carried out between 8 a.m. and 12 p.m. in order

o avoid circadian influence and any kind of stress thatould have interfered with the animals’ behaviour. Theehavioural tests were conducted in independent groupsf animals (n = 10 animals in each group).

.4.1. Social recognition taskShort-term social recognition memory was assessed

ith the social recognition task as previously describedDantzer et al., 1987; Prediger et al., 2005). Adultats were housed individually in plastic cages42 cm × 34 cm × 17 cm) and were used only after ateast seven days of habituation to their new environment.uvenile rats (25–30 days old, 100–150 g) were kept inroups of 10 per cage and served as social stimuli forhe adult rats. Animals were habituated for at least 1 hefore the beginning of the experiment. All juvenile ratsere isolated in individual cages for 20 min prior to the

eginning of the experiment.The social recognition task consisted of two successive

resentations (5 min each) separated by a delay period,uring which a juvenile rat was placed in the home cagef the adult rat, and the time spent by the adult investi-

ating the juvenile (nosing, sniffing, grooming or pawing)as recorded. At the end of the first presentation, the juve-ile was removed and kept in an individual cage duringhe delay period and re-exposed to the adult rat after 30 or

nitive Neuroscience 3 (2013) 1– 10 3

120 min. In this kind of test, if the delay period is less than40 min, adult male rats display recognition of this juvenileas indicated by a significant reduction of the social investi-gation time during the second presentation (Prediger andTakahashi, 2003; Prediger et al., 2005). However, whenthe same juvenile is re-exposed for a longer period oftime (more than 60 min) after the first presentation, theadult rat no longer recognises this juvenile; i.e. the socialinvestigation time in the second presentation is similarto that observed during the first. Thus, 30-min and 120-min intervals were selected as temporal windows suitablefor testing memory-disrupted and memory-enhancingtreatments, respectively. An additional experiment wasperformed to discard non-memory related effects of theextract administration. In this experiment, an unfamiliarjuvenile rat (i.e. different from that used in the first pre-sentation) was exposed to the adult rat during the secondencounter, with a similar expected duration of social inves-tigation time (Prediger and Takahashi, 2003; Prediger et al.,2005).

2.4.2. Inhibitory avoidance taskThe inhibitory avoidance apparatus was an acrylic box

(50 cm × 25 cm × 25 cm), the floor of which consisted ofparallel stainless steel bars (1 mm diameter) spaced 1 cmapart. A platform (7-cm wide × 2.5-cm high) was placedon the floor against the left wall. Animals were placedon the platform, and the time from latency to step-downon the grid with four paws was measured with an auto-matic device. Animals were submitted to the inhibitoryavoidance task using a protocol similar to that describedpreviously (Prediger et al., 2008). During training sessions,immediately after stepping down on the grid, the animalsreceived a 0.4-mA, 1.0-s scrambled foot shock. During testsessions, no foot shock was administered, and the step-down latency (maximum 180 s) was used as measure ofretention. Animals were submitted to a single trainingsession. In order to evaluate short- and long-term memory,test sessions were performed 1.5 and 24 h after training,respectively. The last administration of EE or saline wasperformed by oral route 1 h before training in the apparatusof inhibitory avoidance.

2.5. Statistical analysis

Data on the inhibitory avoidance task are shown asthe medians (interquartile range) of step-down latencies.Comparisons of test session step-down latencies betweengroups were performed with a Kruskal–Wallis test fol-lowed by a Dunn’s multiple comparison test. Data on thesocial recognition task were expressed as means ± S.E.M,and statistical comparisons were carried out using two-way analysis of variance (ANOVA) with pre-treatment(MeHg or control) and treatment (EE of C. paludosa orvehicle) as independent variables. Following significant

using a Newman–Keuls test. The accepted level of signifi-cance for the tests was p ≤ 0.05. All tests were performedusing the Statistica® software package (StatSoft Inc., Tulsa,OK, USA).

4 G.M. Lucena et al. / Developmental Cognitive Neuroscience 3 (2013) 1– 10

Fig. 1. Effects of repeated oral administration of the ethanolic extract ofCipura paludosa (1, 10 or 100 mg/kg, once daily for 14 days) or vehicle(V) on social investigation times of prenatally methylmercury (MeHg)-exposed adult rats when the same juvenile was re-exposed at 30 minafter the first presentation (n = 10 per group). Bars represent investigationtimes (mean ± S.E.M., in seconds) in the first (white) and second presenta-

Fig. 2. Effects of repeated oral administration of the ethanolic extract ofCipura paludosa (1, 10 or 100 mg/kg, once daily for 14 days) or vehicle(V) on social investigation times of prenatally methylmercury (MeHg)-exposed adult rats when the same juvenile was re-exposed at 120 minafter the first presentation (n = 10 per group). Bars represent investigationtimes (mean ± S.E.M., in seconds) in the first (white) and second presenta-

tion (grey). *p ≤ 0.05 compared to the first presentation of the same group.#p ≤ 0.05 compared to the second presentation of the vehicle-treated ani-mals of the control group (Newman–Keuls test).

3. Results

3.1. Social recognition task

The results for the effects of chronic oral adminis-tration of the EE of C. paludosa (1, 10 or 100 mg/kg)or vehicle on the social recognition memory of prenatalMeHg-exposed rats evaluated when the same juvenile ratwas re-exposed after 30 min are shown in Fig. 1. One-wayANOVA revealed a significant effect of the treatment factorwith EE [F(15,159) = 27.47, p < 0.0001] on the investigationtime during the second presentation of the familiar juve-nile rat. Subsequent Newman–Keuls tests indicated that,at this time point, MeHg-treated rats were not able torecognise the juvenile rat because no significant reduc-tion in the investigation time was observed during thesecond encounter. Importantly, repeated oral administra-tion of the EE of C. paludosa (10 or 100 mg/kg) reduced theinvestigation time of MeHg-treated rats during the secondpresentation of the familiar juvenile, indicating that treat-ment with the extract of C. paludosa was able to reverse thesocial recognition memory deficits associated with prena-tal MeHg exposure.

The effects of repeated oral administration of EE orvehicle on the social recognition memory of prenatalMeHg-exposed rats evaluated when the same juvenile ratwas re-exposed after 120 min are shown in Fig. 2. One-way ANOVA revealed a significant effect of the treatmentfactor with EE [F(15,159) = 21.04; p < 0.0001] on the investi-

gation time during the second presentation of the familiarjuvenile rat. Subsequent post hoc comparisons indicatedthat, at this time point, vehicle- or prenatal MeHg-exposedrats were not able to recognise the juvenile rat because

tion (grey). *p ≤ 0.05 compared to the first presentation of the same group(Newman–Keuls test).

no significant reduction in the investigation time wasobserved during the second encounter. Interestingly, alltested doses of the EE of C. paludosa (1, 10 or 100 mg/kg)significantly decreased the investigation time displayed byprenatal vehicle-exposed rats in the forgetting procedure(exposure 120 min later to a familiar juvenile), suggest-ing that EE facilitates short-term social memory. Moreover,repeated oral administration of the EE of C. paludosa (10or 100 mg/kg) reduced the investigation time of MeHg-treated rats during the second presentation of the familiarjuvenile, suggesting that the social memory-enhancingproperties of EE also extend to intrauterine MeHg-exposedrats.

In addition, as shown in Fig. 3, when a different juve-nile from the first encounter was used during the secondpresentation, no significant reduction in the investigationtime was observed in the groups [F(15,159) = 1.14; p = 0.33].These results suggest that the EE of C. paludosa given atthe indicated doses specifically improves the long-lastingsocial recognition memory deficits of adult rats exposed tointrauterine MeHg.

3.2. Inhibitory avoidance task

The effects of oral chronic treatment with the EE ofC. paludosa (1, 10 or 100 mg/kg) or vehicle on the short-and long-term memory of prenatal MeHg-exposed ratsevaluated in the step-down inhibitory avoidance task areshown in Fig. 4. The Kruskal–Wallis non-parametric testdid not reveal any significant effects of EE treatment on the

step-down latencies during the training session (p > 0.05).However, there was a significant effect of EE treatment onstep-down latencies during the short-term [H(8, N = 62.70);

G.M. Lucena et al. / Developmental Cog

Fig. 3. Effects of repeated oral administration of the ethanolic extract ofCipura paludosa (1, 10 or 100 mg/kg, once daily for 14 days) or vehicle (V)on social investigation times of adult rats exposed prenatally methylmer-cury (MeHg)-exposed adult rats when a different juvenile was re-exposedais

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t 120 min after the first presentation (n = 10 per group). Bars representnvestigation times (mean ± S.E.M., in seconds) in the first (white) andecond presentation (hatched).

< 0.0001] and long-term [H(8, N = 56.10); p < 0.0001] testessions.

The Dunn’s test indicated that the administration of theE of C. paludosa (10 or 100 mg/kg), 60 min before the train-ng session, significantly increased the step-down latenciesuring the short- and long-term test session performed.5 h and 24 h after the training session, respectively. Theseesults suggest that the extract of C. paludosa at the indi-ated doses specifically enhances the short- and long-term

etention of a step-down inhibitory avoidance test in rats.oreover, the EE of C. paludosa (10 mg/kg) significantly

ncreased the step-down latencies specifically during thehort-term session in prenatal MeHg-exposed rats. These

ig. 4. Effects of repeated oral administration of the ethanolic extract ofipura paludosa (1, 10 or 100 mg/kg, once daily for 14 days) or vehicleV) on the short-term (1.5 h) and long-term (24 h) memory evaluated inhe step-down inhibitory avoidance task in adult rats exposed prenatallyo methylmercury (MeHg). Data are shown as the medians (interquar-ile ranges) of latencies to step-down in the training (white) and test1.5 h: grey; 24 h: hatched) sessions (n = 10 animals per group). *p ≤ 0.05ompared to the respective session of the control-treated group (Dunn’sest).

nitive Neuroscience 3 (2013) 1– 10 5

results reinforce previous findings obtained in the socialrecognition task that the extract of C. paludosa specificallyimproves the long-lasting short-term memory deficits ofadult rats exposed to MeHg during intrauterine life.

4. Discussion

There is an increasing number of studies investigat-ing the protective effects of plants or natural compoundson various neuropathological conditions. Remarkably, ithas been demonstrated that plants/natural compoundsare able to counteract metal-induced neurotoxicity underin vivo conditions (Farina et al., 2005; Franco et al., 2010;Gupta and Flora, 2006; Xu et al., 2005). For instance, Farinaet al. (2005) showed the beneficial effects of the hydroal-coholic extract of plants of the genus Polygala againstMeHg-induced neurotoxicity in mice. Interestingly, Blacket al. (2011) have recently used a rat perinatal exposuremodel to investigate the modulation of developmentalMeHg toxicity by an extract of Rhododendron tomento-sum ssp. subarcticum, generally known as Labrador Tea, aplant rich in antioxidants traditionally consumed by Cana-dia Inuit. The authors observed that R. tomemtosum extractattenuated MeHg’s effects on oxidative stress and brainglutamate N-methyl-d-aspartate (NMDA) NMDA receptorlevels. However, despite the modulation of these molecularendpoints, R. tomemtosum extract did not lead to significantalterations of MeHg’s effects on rat neurobehaviour (Blacket al., 2011).

In addition, an in vitro study showed that quercetin,a well known flavonoid with antioxidant properties,prevented the mitotoxic effects of MeHg by inhibitingMeHg-induced reactive oxygen species formation (Francoet al., 2007). In the same study, other compounds suchas coumarins and xanthones, did not display protectiveeffects, suggesting that flavonoids may represent promis-ing neuroprotective agents to counteract MeHg-inducedneurotoxicity. Corroborating these findings, Franco et al.(2010) have demonstrated that the co-incubation ofmouse brain mitochondria with two flavonoids (myricetinand myricitrin) derived from medicinal plants causeda concentration-dependent decrease of MeHg-inducedmitochondrial dysfunction and oxidative stress. There-fore, consumption of these plants or natural compounds,which are already part of the cultural background ofmany communities, may therefore represent a conve-nient and practical method to increase phytochemical-based antioxidant intake in populations exposed toMeHg.

In this regard, previous findings from our group (Lucenaet al., 2007a) indicated the protective effects of the EE ofC. paludosa against MeHg-induced neurotoxicity in adultmice. Additionally, we observed recently that chronic treat-ment with the EE of C. paludosa attenuated the anxiety-and depression-like behaviours and the antioxidant deficitsinduced by prenatal MeHg exposure in rats (Lucena et al.,2010). Taking into account the absence of effective treat-

ments for neurological deficits associated with MeHgexposure and the existence of previous findings demon-strating that C. paludosa may represent a valuable toolto attenuate long-lasting emotional changes induced by

ntal Cog

6 G.M. Lucena et al. / Developme

prenatal MeHg exposure, we investigated in the currentstudy the effects of chronic administration of the EE of C.paludosa on the short- and long-term learning and memoryimpairments in adult rats born to dams treated with MeHg.

Behaviour is an important endpoint in studies onthe effects of environmental agents on the nervoussystem of mammals and can be a useful tool for explor-ing whether toxic agents can interact to exacerbatebehavioural aberrations such as learning and mem-ory impairments (for review see Farina et al., 2011).The present findings show that a single oral dose ofMeHg (8 mg/kg) during intrauterine development inter-feres with long-lasting neurobehavioural responses inadult rat offspring, inducing marked learning and mem-ory impairments, which reinforces previous findings fromour group (Lucena et al., 2010; Maia et al., 2009, 2010)and others (Carratù et al., 2006; Ferraro et al., 2009;Onishchenko et al., 2007). Moreover, in the present studywe extend the potential pharmacological properties of C.paludosa, demonstrating its cognitive enhancing activityon the social recognition memory and inhibitory avoid-ance short- and long-term memory of adult rats. Of highimportance, the results of the current study demonstrate,for the first time, that the chronic oral treatment withthe EE of C. paludosa reduced the long-lasting short-term learning and memory deficits induced by prenatalMeHg.

There is increasing concern about neurological defectsin humans, and environmental contaminants have beenproposed as possible causes of learning and emotionaldisturbances that manifest at a young age and neurode-generative diseases later in life (Grandjean and Herz,2011; Landrigan et al., 2005; National Research Council,2000). MeHg is known to be an environmental neurotoxinthat potentially causes neurological abnormalities, cogni-tive impairment, and behavioural disturbances in humansand laboratory animals (Bourdineaud et al., 2011; Yorifujiet al., 2011). More recently, developmental exposure to lowdoses of MeHg found in seafood was found to be a risk factorfor cognitive and emotional disorders (e.g. attention, mem-ory and emotional deficits) in children and adolescents inthe fish-eating population of the Faroe Islands (Debes et al.,2006; Grandjean et al., 1997). In comparing the relevanceof different animal models to human MeHg exposure, itshould be noted that models of low-level exposure aremore appropriate to simulate neurotoxic effects occurringin high fish-consuming populations. The most intriguingaspect is that, following MeHg exposure in utero, an infantwho appears normal at birth may develop psychomotordeficits as the nervous system matures (Marsh et al., 1987).Indeed, the foetus appears to be more sensitive to toxiceffects of MeHg than the mother, and adverse neurode-velopmental effects have been reported in the offspring ofwomen showing little or no overt toxicity (Choi et al., 1978;Marsh et al., 1987).

The MeHg dose used in this study was selected on thebasis of previous studies showing that, following mater-

nal administration of 8 mg/kg on gestational day 15, thebrain concentration of MeHg in the offspring at birth is60-fold higher than that in the brain of vehicle-treatedrats, and it remains three-fold higher until postnatal day

nitive Neuroscience 3 (2013) 1– 10

60 (Cagiano et al., 1990; Maia et al., 2009, 2010). More-over, using this same schedule of MeHg administration,we demonstrated recently that developmental exposureto low levels of MeHg induces long-lasting alterations inanxiety- and depression-like behaviour as well as inhi-bition of the antioxidant enzymes catalase (CAT) andglutathione peroxidase (GPx) in the cortex and cerebel-lum of rats (Lucena et al., 2010). These findings reinforcethe notion of early exposure to environmental contam-inants as a possible risk factor for neurodevelopmentaldisorders.

It has been well established that memories can be clas-sified differently according to their duration in workingmemory (immediate memory lasting seconds or a few min-utes), short-term memory (developing in a few secondsor minutes and lasting for several hours) and long-termmemory (consolidating slowly and remaining relativelypermanent) (for a review see Izquierdo et al., 1999). Short-and long-term memories are identified as separate entities(Izquierdo et al., 1999). These distinct types of memory canbe evaluated in animal studies using different behaviouraltasks. Thorpe et al. (2004) highlighted the importance ofhaving more than one potential measure of memory sinceit is possible that null results are due to the test not beingsensitive enough, rather than a failure in learning and/ormemory. Therefore, a battery of behavioural assays, ratherthan a singe test, should be included in the investigation ofthe effects of substances and procedures on learning andmemory.

In the present study we employed two widely usedbehavioural tasks (social recognition and inhibitory avoid-ance) for the investigation of the effects C. paludosa onlearning and memory of rodents. As stated by Dantzeret al. (1987), a form of memory very similar to factualmemory in humans that has received little attention inbehavioural pharmacology is the so-called “social memory”or “recognition paradigm”, which is mainly generated fromolfactory cues. This memory model is based on the fact thatrodents spend more time investigating unfamiliar juve-nile conspecifics more intensely than familiar ones. Whenthe same juvenile is presented twice, the social investiga-tion time decreases in the second presentation (Dantzeret al., 1987). Social memory is prolonged by repeated expo-sure to the stimulus of the juvenile rat and is impairedby retroactively interfering stimuli. It can be facilitatedby memory-enhancing drugs and disrupted by pharma-cological and pathophysiological models known to impairmemory in rodents (Prediger et al., 2008; Prediger andTakahashi, 2005). In contrast to most other forms of learn-ing and memory assessed in rodents, social recognitionmemory is longer than most tests of working memory butis shorter than various forms of declarative, emotional, orspatial memories, which can be detected 24 h later. Addi-tionally, the effects of the EE of C. paludosa on the short- andlong-term spatial memories of rats were also investigatedusing the step-down inhibitory avoidance task, which isbased on the animal’s behaviour to refrain from execut-

ing a previous response. Several brain regions are involvedin the learning of this task, including hippocampus, amyg-dala and prefrontal cortex (for review see Izquierdo et al.,2002).

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G.M. Lucena et al. / Developme

The present results demonstrate that repeated oralreatment with the EE of C. paludosa (1, 10 or 100 mg/kg)uring 14 consecutive days decreases the investigationime of the same juvenile rat in the forgetting proce-ure (exposure 120 min later to adult rats), indicating thathe extract enhances short-term social memory in rats.his response cannot be attributed to non-memory relatedffects of the extract because no significant reduction inhe investigation time was observed when a different juve-ile was used for the second presentation. Furthermore, theositive effects of EE cannot be explained by a direct alter-tion in locomotor performance of the animals becauseo significant effects were observed in the total ambula-ion during the open field test in rats submitted to theame schedule of C. paludosa EE treatment (Lucena et al.,010). It is important to note that the facilitation of theocial recognition memory of adult rats induced by thextract cannot be solely explained by the improvementf the olfactory discrimination performance because EEdministration did not alter the investigation/explorationime during the first presentation of the juvenile rat in theocial recognition experiments. However, the fact that thepecific neuroanatomical structures and neurochemicalechanisms underlying rodent social recognition remain

nknown and the influence of motivational status onnimals’ degree of interest in the investigation of bothamiliar and unfamiliar conspecifics represent importantimitations of the social recognition task (Ferguson et al.,002).

Consistent with the results obtained in the social recog-ition paradigm, a repeated pre-training administrationf intermediate doses of the EE of C. paludosa (10 and00 mg/kg, gavage) significantly increased the step-down

atencies during test sessions performed 1.5 h and 24 h afterhe training session, indicating a facilitation of the step-own inhibitory avoidance short- and long-term memory,espectively. As reviewed by Izquierdo and Medina (1997),he step-down inhibitory avoidance task constitutes an ani-

al model of aversively motivated learning and memory.owever, since the fear of foot shock is the main moti-ation of the step-down inhibitory avoidance task, otheractors such as shock sensitivity, anxiety, and exploratoryehaviour including sniffing, rearing and locomotor activ-

ty could confound partly the interpretation of the resultsf this task. Moreover, it must be acknowledged that it isot possible to determine the exact site of action or theolecular mechanisms underlying the ability of C. palu-

osa EE to improve short- and long-term memory in rats.evertheless, a hypothesis can be speculated. Ongoing

tudies from our group have pointed to the cognitive-nhancing properties of the EE of C. paludosa in adultats and mice. These results have also shown a synergisticesponse following the co-administration of ‘sub-effective’oses of the EE of C. paludosa with caffeine or the selectivedenosine A2A receptor antagonist ZM241385 in the socialecognition and step-down inhibitory avoidance tasks, sug-esting that the blockage of adenosine receptors may be

esponsible for the present results. Although the role ofdenosine receptor agonists and antagonists in learningnd memory still needs to be elucidated, there is con-iderable evidence supporting our hypothesis (reviewed

nitive Neuroscience 3 (2013) 1– 10 7

in Takahashi et al., 2008). Additional research is certainlynecessary for determining the exact molecular mecha-nisms and anatomical sites involved in the mediation ofthe effects of the EE of C. paludosa on learning and mem-ory, and this constitutes a very interesting field for futurestudies.

In the present study, we also demonstrate that a singleoral dose of MeHg (8 mg/kg) induces pronounced long-lasting impairments in the social recognition memory ofadult male rats because they were not able to recognise ajuvenile familiar rat after a short period of time (30 min).Importantly, we observed that repeated administration ofthe EE of C. paludosa (10 and 100 mg/kg, gavage) improvesthis cognitive impairment associated with prenatal MeHgexposure in rats, as indicated by a significant reduction inthe investigation time during the second presentation ofthe juvenile rat. Consistent with the results obtained inthe social recognition test, we also observed that repeatedadministration of the EE of C. paludosa (10 or 100 mg/kg,gavage) was able to increase the step-down latencies dur-ing the short-term session in adult male rats exposed toMeHg. Reinforcing the current findings, previous studieshave reported that other plants of the Iridaceae family mayconfer protection against cerebral ischaemia and learningand memory impairments in rodents (Papandreou et al.,2011; Pitsikas et al., 2007).

The neurochemical basis of MeHg-induced behaviouralalterations may involve disturbances in a number of neu-rotransmitter systems, occurring initially during exposureand followed by long-lasting changes in brain function(Castoldi et al., 2001; Farina et al., 2011; Grandjean andHerz, 2011). Among the best characterised mechanisms ofMeHg neurotoxicity are excitotoxicity and oxidative stress(Aschner et al., 2007; Farina et al., 2011; Yin et al., 2007,2011). Perturbation of glutamate transport in astrocytesby MeHg can lead to over-stimulation and dysfunctionof NMDA receptors (Yin et al., 2007). NMDA receptorsare part of the glutamatergic system which is crucialfor neuronal plasticity, learning and memory (Scheetzand Constantine-Paton, 1994). During foetal development,NMDA receptors are also involved in the establishment ofneuronal circuitry (Haberny et al., 2002; Martel et al., 2009),which might constitute an additional target of MeHg tox-icity. While NMDA receptors mediated excitotoxicity canlead to the generation of reactive oxygen species (Gassoet al., 2001), MeHg can also generate oxidative stressthrough direct perturbation of mitochondrial functions(Aschner et al., 2007; Franco et al., 2007). The resultantoxidative stress can lead to cell membrane damage, cal-cium deregulation, enzyme and cell signalling interference,microtubule disassembly, and ultimately, cell death (Farinaet al., 2011).

Of particular importance, studies from our group haveshown that C. paludosa EE abolishes MeHg-induced oxida-tive stress and the inhibition of catalase (CAT) andselenium-glutathione peroxidase (Se-GPx) activities in thecortical and cerebellar tissues in rodents (Lucena et al.,

2007a, 2010). These findings suggest that prenatal MeHgexposure, by causing oxidative stress, promotes the highspecificity of MeHg to proteins in the central nervoussystem, which can alter behavioural and biochemical

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parameters in adult offspring rats. Therefore, the antiox-idant properties of C. paludosa EE appear to represent apossible molecular mechanism involved in the observedprotective effects of EE against long-lasting learning andmemory deficits in prenatal MeHg-exposed rats. How-ever, additional research is necessary to evaluate whetherthere exists any interaction between the adenosinergic andother neurotransmitter systems in the current effects ofC. paludosa. Finally, a better evaluation of the potentialcognitive-enhancing properties of C. paludosa extract andits isolated compounds using additional tasks of spatialmemory (such as T-maze or water maze) is also indi-cated.

5. Conclusions

In conclusion, this study reinforces previous findingsshowing that acute low-level exposure to MeHg duringgestation induces subtle cognitive dysfunctions and fur-ther emphasises that consumption of MeHg-contaminatedfood by pregnant women poses one of the most seri-ous potential hazards for offspring. More importantly, thepresent results demonstrate for the first time that chronictreatment with the EE of C. paludosa reduced the long-lasting short-term learning and memory deficits inducedby prenatal MeHg exposure. Therefore, in the presentstudy we extend the potential pharmacological proper-ties of this specie, demonstrating its cognitive enhancingactivity. Experiments are currently in progress in our lab-oratory to identify the active constituents as well as themolecular mechanisms responsible for the observed pro-tective effects of C. paludosa. Finally, we hope that thisarticle may inspire other experimental research groupsto further evaluate the cognitive enhancing propertiesof C. paludosa and its components on neuropathologi-cal conditions associated with exposure to environmentalcontaminants.

Conflict of interest statement

There are no known conflicts of interest associated withthis publication and there has been no significant finan-cial support for this work that could have influenced itsoutcome.

Acknowledgements

This work was supported by grants from the ConselhoNacional de Desenvolvimento Científico e Tecnológico(CNPq) and the Coordenac ão de Aperfeic oamento de Pes-soal de Nível Superior (CAPES), both from Brazil. Theresearch was conducted in accordance with national andinstitutional guidelines for the protection of animal wel-fare. G.M.L. was supported by a scholarship from CAPES.R.D.S.P. is supported by a research fellowship from CNPq.

References

Aschner, M., Syversen, T., Souza, D.O., Rocha, J.B., Farina, M., 2007. Involve-ment of glutamate and reactive oxygen species in methylmercuryneurotoxicity. Brazilian Journal of Medical and Biological Research 40,285–291.

nitive Neuroscience 3 (2013) 1– 10

Aschner, M., Yao, C.P., Allen, J.W., Tan, K.H., 2000. Methylmercury altersglutamate transport in astrocytes. Neurochemistry International 37,199–206.

Black, P., Niu, L., Sachdeva, M., Lean, D., Poon, R., Bowers, W.J., Chan,H.M., Arnason, J.T., Pelletier, G., 2011. Modulation of the effectsof methylmercury on rat neurodevelopment by co-exposure withLabrador Tea (Rhododendron tomentosum ssp. subarcticum). Food andChemical Toxicology 49, 2336–2342.

Bourdineaud, J.P., Fujimura, M., Laclau, M., Sawada, M., Yasutake, A., 2011.Deleterious effects in mice of fish-associated methylmercury con-tained in a diet mimicking the Western populations’ average fishconsumption. Environment International 37, 303–313.

Cagiano, R., de Salvia, M.A., Renna, G., Tortella, E., Braghiroli, D., Parenti,C., Zanoli, P., Baraldi, M., Annau, Z., Cuomo, V., 1990. Evidence thatexposure to methyl mercury during gestation induces behavioral andneurochemical changes in offspring of rats. Neurotoxicology and Ter-atology 12, 23–28.

Campos-Esparza, M.R., Sanchez-Gomez, M.V., Matute, C., 2009. Molecularmechanisms of neuroprotection by two natural antioxidant polyphe-nols. Cell Calcium 45, 358–368.

Carratù, M.R., Borracci, P., Coluccia, A., Giustino, A., Renna, G., Tomasini,M.C., Raisi, E., Antonelli, T., Cuomo, V., Mazzoni, E., Ferraro, L., 2006.Acute exposure to methylmercury at two developmental windows:focus on neurobehavioral and neurochemical effects in rat offspring.Neuroscience 141, 1619–1629.

Castoldi, A.F., Coccini, T., Ceccatelli, S., Manzo, L., 2001. Neurotoxicityand molecular effects of methylmercury. Brain Research Bulletin 55,197–203.

Choi, B.H., 1989. The effects of methylmercury on the developing brain.Progress in Neurobiology 32, 447–470.

Choi, B.H., Lapham, L.W., Amin-Zaki, L., Saleem, T., 1978. Abnormal neu-ronal migration, deranged cerebral cortical organization, and diffusewhite matter astrocytosis of human fetal brain: a major effect ofmethylmercury poisoning in utero. Journal of Neuropathology andExperimental Neurology 37, 719–733.

Daniels, W.M., Pietersen, C.Y., Carstens, M.E., Stein, D.J., 2004. Maternalseparation in rats leads to anxiety-like behavior and a blunted ACTHresponse and altered neurotransmitter levels in response to a subse-quent stressor. Metabolic Brain Disease 19, 3–14.

Dantzer, R., Bluthe, R.M., Koob, G.F., Le Moal, M., 1987. Modulation of socialmemory in male rats by neurohypophyseal peptides. Psychopharma-cology 91, 363–368.

Debes, F., Budtz-Jørgensen, E., Weihe, P., White, R.F., Grandjean, P., 2006.Impact of prenatal methylmercury exposure on neurobehavioral func-tion at age 14 years. Neurotoxicology and Teratology 28, 363–375.

DiCicco-Bloom, E., Sondell, M., 2005. Neural Development and Neurogen-esis. Kaplan & Sadock’s Comprehensive Textbook of Psychiatry, vol. 8.Lippincott Williams & Wilkins, Philadelphia.

Farina, M., Dahm, K.C., Schwalm, F.D., Brusque, A.M., Frizzo, M.E., Zeni,G., Souza, D.O., Rocha, J.B., 2003. Methylmercury increases glutamaterelease from brain synaptosomes and glutamate uptake by corticalslices from suckling rat pups: modulatory effect of ebselen. Toxico-logical Sciences 73, 135–140.

Farina, M., Franco, J.L., Ribas, C.M., Meotti, F.C., Missau, F.C., Pizzolatti,M.G., Dafre, A.L., Santos, A.R., 2005. Protective effects of Polygala pan-iculata extract against methylmercury-induced neurotoxicity in mice.Journal of Pharmacy and Pharmacology 57, 1503–1508.

Farina, M., Rocha, J.B., Aschner, M., 2011. Mechanisms of methylmercury-induced neurotoxicity: evidence from experimental studies. LifeSciences 89, 555–563.

Ferguson, J.N., Young, L.J., Insel, T.R., 2002. The neuroendocrine basis ofsocial recognition. Frontiers in Neuroendocrinology 23, 200–224.

Ferraro, L., Tomasini, M.C., Tanganelli, S., Mazza, R., Coluccia, A., Carratù,M.R., Gaetani, S., Cuomo, V., Antonelli, T., 2009. Developmental expo-sure to methylmercury elicits early cell death in the cerebral cortexand long-term memory deficits in the rat. International Journal ofDevelopmental Neuroscience 27, 165–174.

Franco, J.L., Braga, H.C., Stringari, J., Missau, F.C., Posser, T., Mendes, B.G.,Leal, R.B., Santos, A.R., Dafre, A.L., Pizzolatti, M.G., Farina, M., 2007.Mercurial-induced hydrogen peroxide generation in mouse brainmitochondria: protective effects of quercetin. Chemical Research inToxicology 20, 1919–1926.

Franco, J.L., Posser, T., Missau, F., Pizzolatti, M.G., Dos Santos, A.R.,Souza, D.O., Aschner, M., Rocha, J.B., Dafre, A.L., Farina, M., 2010.

Structure–activity relationship of flavonoids derived from medicinalplants in preventing methylmercury-induced mitochondrial dysfunc-tion. Environmental Toxicology and Pharmacology 30, 272–278.

Gasso, S., Cristofol, R.M., Selema, G., Rosa, R., Rodriguez-Farre, E., San-feliu, C., 2001. Antioxidant compounds and Ca(2+) pathway blockers

ntal Cog

G

G

G

G

G

G

H

H

I

I

I

K

L

L

L

L

L

M

M

M

G.M. Lucena et al. / Developme

differentially protect against methylmercury and mercuric chlorideneurotoxicity. Journal of Neuroscience Research 66, 135–145.

imenez-Llort, L., Ahlbom, E., Daré, E., Vahter, M., Ögren, S., Cec-catelli, S., 2001. Prenatal exposure to methylmercury changesdopamine-modulated motor activity during early ontogeny: age andgender-dependent effects. Environmental Toxicology and Pharmacol-ogy 9, 61–70.

oldblatt, P., 1990. Phylogeny and classification of Iridaceae. Annals of theMissouri Botanical Garden 77, 607–627.

randjean, P., Herz, K.T., 2011. Methylmercury and brain development:imprecision and underestimation of developmental neurotoxicity inhumans. Mount Sinai Journal of Medicine 78, 107–187.

randjean, P., Weihe, P., White, R.F., Debes, F., 1998. Cognitiveperformance of children prenatally exposed to “safe” levels ofmethylmercury. Environmental Research 77, 165–172.

randjean, P., Weihe, P., White, R.F., Debes, F., Araki, S., Yokoyama, K.,Murata, K., Sørensen, N., Dahl, R., Jørgensen, P.L., 1997. Cognitivedeficit in 7-year-old children with prenatal exposure to methylmer-cury. Neurotoxicology and Teratology 19, 417–428.

upta, R., Flora, S.J., 2006. Effect of Centella asiatica on arsenic-inducedoxidative stress and metal distribution in rats. Journal of Applied Tox-icology 26, 213–222.

aberny, K.A., Paule, M.G., Scallet, A.C., Sistare, F.D., Lester, D.S., Hanig, J.P.,Slikker Jr., W., 2002. Ontogeny of the N-methyl-d-aspartate (NMDA)receptor system and susceptibility to neurotoxicity. Toxicological Sci-ences 68, 9–17.

uot, R.L., Plotsky, P.M., Lenox, R.H., McNamara, R.K., 2002. Neonatalmaternal separation reduces hippocampal mossy fiber density in adultLong Evans rats. Brain Research 950, 52–63.

zquierdo, L.A., Barros, D.M., Vianna, M.R., Coitinho, A., de David e Silva, T.,Choi, H., Moletta, B., Medina, J.H., Izquierdo, I., 2002. Molecular phar-macological dissection of short- and long-term memory. Cellular andMolecular Neurobiology 22, 269–287.

zquierdo, I., Medina, J.H., 1997. Memory formation: the sequence of bio-chemical events in the hippocampus and its connection to activityin other brain structures. Neurobiology of Learning and Memory 68,285–316.

zquierdo, I., Medina, J.H., Vianna, M.R., Izquierdo, L.A., Barros, D.M., 1999.Separate mechanisms for short- and long-term memory. BehaviouralBrain Research 103, 1–11.

orol, D.L., Malin, E.L., Borden, K.A., Busby, R.A., Couper-Leo, J., 2004. Shiftsin preferred learning strategy across the estrous cycle in female rats.Hormones and Behavior 45, 330–338.

andrigan, P.J., Sonawane, B., Butler, R.N., Trasande, L., Callan, R.,Droller, D., 2005. Early environmental origins of neurodegenera-tive disease in later life. Environmental Health Perspectives 113,1230–1233.

iang, J., Inskip, M., Newhook, D., Messier, C., 2009. Neurobehavioral effectof chronic and bolus doses of methylmercury following prenatal expo-sure in C57BL/6 weanling mice. Neurotoxicology and Teratology 31,372–381.

ucena, G.M., Franco, J.L., Ribas, C.M., Azevedo, M.S., Meotti, F.C.,Gadotti, V.M., Dafre, A.L., Santos, A.R.S., Farina, M., 2007a. Cipurapaludosa extract prevent methyl mercury-induced neurotoxicityin mice. Basic and Clinical Pharmacology and Toxicology 101,127–131.

ucena, G.M., Gadotti, V.M., Maffi, L.C., Silva, G.S., Azevedo, M.S., Santos,A.R., 2007b. Antinociceptive and anti-inflammatory properties fromthe bulbs of Cipura paludosa Aubl. Journal of Ethnopharmacology 112,19–25.

ucena, G.M., Porto, F.A., Campos, E.G., Azevedo, M.S., Cechinel-Filho,V., Prediger, R.D.S., Ferreira, V.M., 2010. Cipura paludosa attenuateslong-term behavioral deficits in rats exposed to methylmercury dur-ing early development. Ecotoxicology and Environment Safety 73,1150–1158.

aia, C.D., Ferreira, V.M., Diniz, J.S., Carneiro, F.P., de Sousa, J.B., da Costa,E.T., Tomaz, C., 2010. Inhibitory avoidance acquisition in adult ratsexposed to a combination of ethanol and methylmercury during cen-tral nervous system development. Behavioural Brain Research 211,191–197.

aia, C.S., Lucena, G.M., Corrêa, P.B., Serra, R.B., Matos, R.W., Menezes, F.C.,Santos, S.N., Sousa, J.B., Costa, E.T., Ferreira, V.M., 2009. Interference ofethanol and methylmercury in the developing central nervous system.Neurotoxicology 30, 23–30.

anfroi, C.B., Schwalm, F.D., Cereser, V., Abreu, F., Oliveira, A., Bizarro, L.,Rocha, J.B., Frizzo, M.E., Souza, D.O., Farina, M., 2004. Maternal milk asmethylmercury source for suckling mice: neurotoxic effects involvedwith the cerebellar glutamatergic system. Toxicological Sciences 81,172–178.

nitive Neuroscience 3 (2013) 1– 10 9

Marsh, D.O., Clarkson, T.W., Cox, C., Myers, G.J., Amin-Zaki, L., Al-Tikriti, S.,1987. Fetal methylmercury poisoning: relationship between concen-tration in single strands of maternal hair and child effects. Archives ofNeurology 44, 1017–1022.

Martel, M.A., Wyllie, D.J., Hardingham, G.E., 2009. In developing hip-pocampal neurons, NR2B-containing N-methyl-d-aspartate receptors(NMDARs) can mediate signaling to neuronal survival and synap-tic potentiation, as well as neuronal death. Neuroscience 158,334–343.

National Research Council, 2000. Toxicological Effects of Methylmercury.National Academy Press, Washington, DC.

Onishchenko, N., Tamm, C., Vahter, M., Hökfelt, T., Johnson, J.A., Johnson,D.A., Ceccatelli, S., 2007. Developmental exposure to methylmercuryalters learning and induces depression-like behavior in male mice.Toxicological Sciences 97, 428–437.

Ou, Y.C., White, C.C., Krejsa, C.M., Ponce, R.A., Kavanagh, T.J., Faustman,E.M., 1999. The role of intracellular glutathione in methylmercury-induced toxicity in embryonic neuronal cells. Neurotoxicology 20,793–804.

Papandreou, M.A., Tsachaki, M., Efthimiopoulos, S., Cordopatis, P., Lamari,F.N., Margarity, M., 2011. Memory enhancing effects of saffron in agedmice are correlated with antioxidant protection. Behavioural BrainResearch 219, 197–204.

Pitsikas, N., Zisopoulou, S., Tarantilis, P.A., Kanakis, C.D., Polissiou, M.G.,Sakellaridis, N., 2007. Effects of the active constituents of Crocus sativusL. crocins on recognition and spatial rats’ memory. Behavioural BrainResearch 183, 141–146.

Pompili, A., Tomaz, C., Arnone, B., Tavares, M.C., Gasbarri, A., 2010. Work-ing and reference memory across the estrous cycle of rat: a long-termstudy in gonadally intact females. Behavioural Brain Research 213,10–18.

Prediger, R.D., Batista, L.C., Takahashi, R.N., 2005. Caffeine reverses age-related deficits in olfactory discrimination and social recognitionmemory in rats. Involvement of adenosine A1 and A2A receptors.Neurobiology of Aging 26, 957–964.

Prediger, R.D., Fernandes, M.S., Rial, D., Wopereis, S., Pereira, V.S., Bosse,T.S., Da Silva, C.D., Carradore, R.S., Machado, M.S., Cechinel-Filho, V.,Costa-Campos, L., 2008. Effects of acute administration of the hydroal-coholic extract of mate tea leaves (Ilex paraguariensis) in animalmodels of learning and memory. Journal of Ethnopharmacology 120,465–473.

Prediger, R.D., Takahashi, R.N., 2003. Ethanol improves short-term socialmemory in rats. Involvement of opioid and muscarinic receptors.European Journal of Pharmacology 462, 115–123.

Prediger, R.D., Takahashi, R.N., 2005. Modulation of short-term socialmemory in rats by adenosine A1 and A(2A) receptors. NeuroscienceLetters 376, 160–165.

Rossi, A.D., Ahlbom, E., Ögren, S.O., Nicotera, P., Ceccatelli, S., 1997. Prenatalexposure to methylmercury alters locomotor activity of male but notfemale rats. Experimental Brain Research 117, 428–436.

Scheetz, A.J., Constantine-Paton, M., 1994. Modulation of NMDA recep-tor function: implications for vertebrate neural development. FASEBJournal 8, 745–752.

Sirois, J.E., Atchison, W.D., 2000. Methylmercury affects multiple subtypesof calcium channels in rat cerebellar granule cells. Toxicology andApplied Pharmacology 167, 1–11.

Takahashi, R.N., Pamplona, F.A., Prediger, R.D., 2008. Adenosine recep-tor antagonists for cognitive dysfunction: a review of animal studies.Frontiers in Bioscience 13, 2614–2632.

Tchounwou, P.B., Ayensu, W.K., Ninashvili, N., Sutton, D., 2003. Environ-mental exposure to mercury and its toxicopathologic implications forpublic health. Environmental Toxicology 18, 149–175.

Tessele, P.B., Delle Monache, F., Quintão, N.L., da Silva, G.F., Rocha, L.W.,Lucena, G.M., Ferreira, V.M., Prediger, R.D., Cechinel Filho, V., 2011. Anew naphthoquinone isolated from the bulbs of Cipura paludosa andpharmacological activity of two main constituents. Planta Medica 77,1035–1043.

Thorpe, C.M., Jacova, C., Wilkie, D.M., 2004. Some pitfalls in measur-ing memory in animals. Neuroscience and Biobehavioral Reviews 28,711–718.

Yongjin, L., Wei, S., Yindong, L., Yong, Z., Xuefeng, H., Chunmei, S.,Hongbo, M., Changwen, W., Yong, L., 2008. Neuroprotective effectsof chlorogenic acid against apoptosis of PC12 cells induced bymethylmercury. Environmental Toxicology and Pharmacology 26,

13–21.

Yin, Z., Lee, E., Ni, M., Jiang, H., Milatovic, D., Rongzhu, L., Farina, M.,Rocha, J.B., Aschner, M., 2011. Methylmercury-induced alterations inastrocyte functions are attenuated by ebselen. Neurotoxicology 32,291–299.

ntal Cog

10 G.M. Lucena et al. / Developme

Yin, Z., Milatovic, D., Aschner, J.L., Syversen, T., Rocha, J.B., Souza, D.O.,Sidoryk, M., Albrecht, J., Aschner, M., 2007. Methylmercury induces

oxidative injury, alterations in permeability and glutamine transportin cultured astrocytes. Brain Research 1131, 1–10.

Yorifuji, T., Tsuda, T., Inoue, S., Takao, S., Harada, M., 2011. Long-termexposure to methylmercury and psychiatric symptoms in residentsof Minamata, Japan. Environment International 37, 907–913.

nitive Neuroscience 3 (2013) 1– 10

Xu, Y., Li, G., Han, C., Sun, L., Zhao, R., Cui, S., 2005. Protective effects ofHippophae rhamnoides L. juice on lead-induced neurotoxicity in mice.

Biological and Pharmaceutical Bulletin 28, 490–494.

Zanoli, P.C., Truzzi Veneri, C., Braghiroli, D., Baraldi, M., 1994. Methylmer-cury during late gestation affects temporarily the development ofcortical muscarinic receptors in rat offspring. Pharmacology and Tox-icology 75, 261–264.