Chemico-Biological Interactions 159 (2006) 223–234

Effects of microcystins over short- and long-term memory andoxidative stress generation in hippocampus of rats

M. Maidanaa,b, V. Carlisa, F.G. Galhardia, J.S. Yunesa,c, L.A. Geracitanoa,b,J.M. Monserrata,b, D.M. Barrosa,b,∗

a Departamento de Ciencias Fisiologicas, Fundacao Universidade Federal do Rio Grande (FURG), Rio Grande 96201-900, Brazilb Programa de Pos-graduacao em Ciencias Fisiologicas, Fisiologia Animal Comparada (FURG), Brazil

c Departamento de Quımica, Unidade de Pesquisa em Cianobacterias, FURG, Rio Grande 96201-900, Brazil

Received 14 September 2005; received in revised form 2 December 2005; accepted 2 December 2005

Abstract

Microcystins produced by cyanobacteria are potent inhibitors of some protein phosphatases, but recent evidence also indicatesits potential to generate oxidative stress. In the present study, the effects of microcystin raw extracts (Mic; 0.01 and 20�g/L) andpurified okadaic acid (OA; 0.01 and 10�g/L) on short- and long-term memory alteration and antioxidant and oxidative damagewere investigated in hippocampus of rats. The results showed an amnesic effect with 0.01 and 20�g/L Mic on retrieval and onlywith 0.01�g/L Mic on spatial learning. Parallel to these effects oxidative damage was observed as evidenced by augmented levelsof lipid peroxides and DNA damage and the absence of antioxidant responses in terms of total oxyradical scavenging capacity.P howedp osphatasei e fact thato in brainp©

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hase II reactions catalyzed by glutathione-S-transferase were not modified after microcystins exposure. Overall this study shysiological events (retrieval and spatial learning) that can be related to the classical toxic effects of microcystins (i.e., ph

nhibition). In addition, evidence of alternative toxicity mechanisms via oxidative stress generation was also obtained. Thrganic anion transporter polypeptides (OATP) involved in microcystins uptake are expressed not only in liver but alsooints to the environmental relevance of the observed effects.2005 Elsevier Ireland Ltd. All rights reserved.

eywords: Microcystins; Phosphatase inhibitors; Amnesia; Oxidative stress; DNA damage

. Introduction

Microcystins are monocyclic heptapeptide moleculesontaining bothd- andl-aminoacids and are producedy cyanobacteria from the genusMicrocystis. These

oxins are inhibitors of serine/threonine phosphatasesPP1 and PP2A), affecting intracellular signaling in cellrowth and differentiation processes[1,2]. Classic dele-

∗ Corresponding author. Tel.: +55 53 32336855;ax: +55 53 32336850.

E-mail address: barrosdm@yahoo.com.br (D.M. Barros).

terious effects (i.e., hepatotoxicity and hyperphosphlation of proteins) are believed to be a consequenphosphatase inhibition. However, new evidences pto additional mechanisms of toxicity exerted by thtoxins. Ding et al.[3,4] reported higher reactive oxygspecies (ROS) levels in primary cultured rat hepatocexposed to extracts of lyophilized cyanobacteria[3].Higher ROS production and lipid peroxidation were aseen in rat hepatocytes exposed to microcystin ext[4]. Several authors have reported DNA damageexposure to these toxins[5–7].

Considering that microcystin seems to favor oxidastress generation, it is important to note that the tripe

009-2797/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved.doi:10.1016/j.cbi.2005.12.001

224 M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234

(�-glutamyl-l-cysteinylglicine) glutathione (GSH) isconsidered one of the main non-enzymatic antioxi-dant and constitutes the first protection line of defenseagainst ROS. Some studies reported the formation ofa microcystin-GSH conjugate in a reaction catalyzedby the enzyme glutathione-S-transferase[8]. The factthat the microcystin–GSH complex has a much lowerinhibitory potency over phosphatases is evidence thatthis conjugation reaction represents a detoxification pro-cess. However, the conjugation of microcystin with GSHshould represent a leak of this tripeptide from the intra-cellular pool, leaving the cell more susceptible to oxida-tive stress[4].

It is well established that the main target of micro-cystin is the liver, due primarily to toxin uptake bythe bile acid transport system[9]. However, in 1996 ahemodialysis unit in Caruaru, Brazil, employed watercontaminated with microcystins that resulted in deathof 60 of 126 patients. The symptoms included liverfailure but also neurotoxicity[10,11], indicating thatsomehow these toxins can enter through the blood–brainbarrier. Recently Fischer et al.[12] reported that mem-bers of the organic anion transporting polypeptidesuper family involved in microcystin uptake (includ-ing the human OATP1A2 transporter) are expressedboth in liver and in brain. Therefore, this indicates thatthe target of microcystins is not only liver but alsobrain.

Several reports have suggested the existence ofendogenous molecular suppressors that negatively con-

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Taking into account that cyanotoxins like micro-cystins alter the balance between phosphorylation anddephosphorylation of several proteins and also generateoxidative stress, the goals of this study were to evalu-ate the effect of microcystin raw extracts over short- andlong-term memory across bilateral hippocampal infu-sions of rats and several oxidative damage parameters.As a positive control, assays were performed employingpure okadaic acid, a toxin produced by marine spongesthat potently and specifically inhibits PP-1c and PP-2A[1].

2. Materials and methods

2.1. Chemicals

Cells of Microcystis RST 9501 were cultured inBG11 (±8.82 mM of NaNO3) medium at 25± 1◦Cand employed as toxic cell extract source. Character-ization of microcystins produced by the strain RST9501 was previously reported by Matthiensen et al.[23].These authors found that methanolic extracts of strainRST 9501 analyzed by HPLC/UV (238–239 nm) pre-sented an UV spectra characteristic of microcystins,with a major and three minor peaks. Interestingly, theretention time of the major peak was different to thatof commercially acquired microcystin-LR (Sigma®).Further analysis confirmed that the most abundantmicrocystin produced by strain RST 9501 is a [d-Leu1]microcystin-LR, a variant with a similar potency

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trol the efficacy of neuronal transmission and mory formation [13]. Between them it can be metioned protein phosphatases and oxygen reactive spThese molecules, together with protein kinases, rlate many cellular processes by the reversible phosrylation/dephosphorylation of specific substrates[13].Memory in the hippocampus can last for minutes (shterm) to days (long-term)[14]. Previous studies hashown that an increase in synaptic strength, i.e., lterm potentiation (LTP), is accompanied by an increin the activity of various protein kinases (for a review,Smirnova et al.[15]) and a decrease in basal protein phphatase activity[16]. Calcium influx through NMDARtriggers a serine–threonine kinase signaling paththat results in phosphorylation of existing AMPA rectors and insertion of new AMPA receptors in the psynaptic membrane[17–19], processes that result inreversible form of LTP. In contrast, a decrease in synstrength, i.e., long-term depression (LTD), is depenon protein phosphatases (PPs)[20,21] and is accompanied by an increase in the activity of serine/threoprotein phosphatases[22].

.

of microcystin-LR in terms of phosphatases inbition.

Cells were frozen and thawed three times andtrifuged (12,000× g) at room temperature for 10 miThe supernatant was collected and stored at−20◦C.Microcystin concentration was estimated as 143.1�g/Lusing a commercial enzyme-linked immunoas(ELISA) with polyclonal antibodies (EnviroLogix IncPortland, ME). The stock solution of microcystins (Mwas diluted with physiological human saline to obtfinal concentrations of 0.01 and 20�g/L (Mic 0.01 andMic 20, respectively). Okadaic acid (OA; Sigma; pu>80%) was diluted with dimethylsulfoxide (DMSO)obtain final concentrations of 0.01 and 10�g/L (OA 0.01and OA 10, respectively).

2.2. Laboratory animals and surgery

Wistar rats (age 2.5 months old; weight 220–28were housed in plastic cages, five to a cage, withaccess to water and food under a 12-h light/12-hcycle (lights on at 07:00 h) and constant tempera

M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234 225

(23± 1◦C). Animals were bilaterally implanted underdeep thiopental (40 mg/kg) anesthesia with 27-gaugeguides aimed 1.0 mm above the pyramidal cell layerof the CA1 (Cornu Ammonis) sub area of the dor-sal hippocampus. Coordinates wereA =−4.3; L = +4.0;V = +3.4 [24]. Cannulae for intrahippocampal injectionof solutions were fixed to the skull with dental acrylic[25]. In the first few hours following surgery, animalswere kept in individual cages under a heating lamp untilthey were able to walk. The percentage of survival ani-mals to the surgical procedure was 95%. Then theywere placed back in their home cages. For infusion (seebelow), a tight-fitting inner probe was placed throughthe indwelling cannulae so that its tip protruded 1.0 mmbeyond that of the latter and reached the pyramidal celllayer of CA1. The infusions were all at a volume of 1�Land were carried out slowly at a constant speed through-out 30 s, after which the infusion cannula was left in itsplace for an additional 15 s in order to avoid backflow.Infusions were carried out first on one side and then onthe other, so total infusion time were slightly over 90 s.[25].

Two hours after the end of the behavioral proce-dures, 0.5�L of a 4% methylene blue solution wasinfused as indicated above into the implanted sites. Ani-mals were killed by decapitation 1 h later and theirbrains withdrawn and stored in formalin for histologi-cal localization of infusion sites as explained elsewhere[25]. Infusion placements were correctly placed (i.e.,within 1.5 mm3 of the intended site) in all but seveno ataf werec

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analyzed by non-parametric statistics (Kruskal–Wallisanalysis of variance for each experiment and two-tailedMann–WhitneyU-tests, when applicable).

In Experiment 1, infusions of toxins were performed15 min before training session (n = 10 per group). Short-term memory (STM) was measured 1.5 h after train-ing and long-term memory (LTM) was measured 24 hafter training. In Experiment 2, infusions of toxins werecarried out 15 min prior to the behavioral test ses-sion (n = 10 per group). LTM was measured 24 h aftertraining.

2.3.2. Radial arm mazeIn brief, the radial arm maze consisted of a clear Plexi-

glas apparatus with a center octagonal platform (20 cm indiameter) and eight arms (90 cm long, 10 cm wide). Eacharm contained a food cup (0.5 cm deep) placed 1 cm fromthe end. The entire apparatus was elevated 90 cm fromthe floor and was placed in a room that was well lightwith visual cues (pictures)[27]. The radial arm test wasperformed during 8 days of testing and 2 days of habitu-ation (10 min per day in the maze with all arms baited).Seven days before the stereotaxic surgery the rats werefood deprived to 80% of their ad libitum consumption.Two days after surgery, the rats were evaluated in theradial arm maze every day for 8 days. Fifteen minutesbefore each trial the animals received intrahippocampalinfusion of microcystin or okadaic acid. Control groupsreceived saline or vehicle. Each animal was placed indi-vidually in the center of the maze and subjected to a

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s noten tohav-and

ar-.pre-sue

ined

f the animals implanted in CA1. Only behavioral drom the animals with correct cannulae placementsonsidered.

.3. Behavioral testing

.3.1. Step-down inhibitory avoidance taskAfter recovery from surgery, the animals w

ubmitted to one-trial step-down inhibitory avonce training[25,26]. Rats were placed on a 2m high, 7.0-cm wide platform at the left of0.0 cm× 25.0 cm× 25.0 cm box, whose floor waseries of a parallel 0.1-cm width stainless steelpaced 1.0 cm apart. The rats were individually plan the platform facing its rear left corner. Latencytep-down, placing the four paws on the grid, was mured with an automatic device. In the training sessmmediately upon stepping-down, the animals rece

0.5 mA, 1.0 s scrambled foot shock. No foot shas given during the test session, and step-down laas cut off at 180 s; values equal to or higher than 1ere counted as 180 s. Because of this limit, data

working and reference memory task[27], in which thesame four arms (1, 3, 5, 7) were baited for each dtraining trial. The training trial continued until all fobaits in the food cup had been consumed or until 10had elapsed. The number of working memory er(entering an arm containing food previously entered)reference memory errors (entering an arm that wabaited), were measured for each rat. The time takconsume all four baits was also recorded. After beioral assessment, rats were killed by decapitationtheir hippocampus were removed and kept at−80◦Cfor biochemical analyses.

2.4. Evaluation of oxidative stress

2.4.1. Lipid peroxidation (TBARS assay)Lipid peroxidation was quantified by the thiob

bituric acid (TBA) method[28] with modificationsBriefly, hippocampus tissue homogenates (1:9) werepared in 1.15% KCl in phosphate buffer (pH 7.4). Tisextract was centrifuged at 1000× g for 10 min at 4◦Cand the pellet discarded. The reaction mixture conta

226 M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234

125�L of supernatant, 50�L of 8.1% sodium dodecylsulfate (SDS) (w/v), 25�L of 0.2% butylated hydroxy-toluene (BHT), 250�L of 40% acetic acid solution (v/v)and 500�L of 0.8% aqueous solution of thiobarbituricacid (w/v). The mixture was heated at 95◦C for 60 min.After cooling, 1.25 ml ofn-butanol was added, and themixture was shaken vigorously. After centrifugation at4000× g for 10 min, the absorbance of organic layer(upper layer) was measured at 532 nm. The results wereexpressed in micromoles of malondialdeyide equivalentsper gram of wet weight tissue.

2.4.2. Lipid peroxidation (FOX assay)Lipid peroxides (LPO) were also measured accord-

ing to Hermes-Lima et al.[29] and modified formicroplate reader (Biotek ELx 800, Winooski, VT)according to Monserrat et al.[30]. Hippocampi from ratswere homogenized (1:15) in cold methanol 100%. Thehomogenates were centrifuged at 1000× g for 10 min at4◦C and the supernatants kept for assay. Lipid peroxideswere determined using 90�L of FeSO4 (1 mM) preparedimmediately before use, 35�L of H2SO4 (250 mM),35�L of xylenol orange (1 mM), 185�L of deionizedwater, and 5�L of the extract. All the reagents wereadded following the sequential order mentioned above.Samples were incubated for 5 h at room temperature andthe absorbance measured at 550 nm. Cumene hidroper-oxide (CHP; 0.1 mM; Sigma) was employed as standard.Results were expressed as CHP equivalents per mg ofwet weight (w/w) tissue.

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e))(pHing a

Shimadzu GC-17A gas chromatographer equipped witha DB-5 capillary columm (30 m× 0.25 mm× 0.25�m)(Agilent Technologies) and a flame ionization detector(FID). TOSC values were calculated according toRegoli and Winston[31] and standardized by the totalprotein content in the homogenates.

Glutathione-S-transferase (GST) activity was mea-sured by evaluating the conjugation of glutathione(25 mM; Sigma) with 1-chloro-2,4-dinitro benzene(CDNB) (50 mM; from Sigma) at 340 nm during 1 min[32]. Aliquots (5�l) of hippocampus homogenates wereincubated (37◦C) in phosphate buffer 0.1 M at pH 7.0prior to addition of CDNB. An extinction coefficient of9.6 mM cm−1 was employed in order to calculate themoles of CDNB conjugated. All determinations weredone at least in duplicate.

2.5. Alkaline single cell electrophoresis (comet)assay

The DNA damage was evaluated through the alka-line single cell electrophoresis (comet) assay accordingto the methodology of Tice et al.[33], with minor modi-fications. Hippocampus from five rats for each treatment(saline; DMSO; OA 0.01; OA 10; Mic 0.01 and Mic 20)were dissected. The tissues were homogenized in 500�Lof cold (4◦C) phosphate-buffered saline solution (PBS).Aliquots (0.3�L) of these homogenates were diluted in80�L of low melting point agarose (0.65%) and added tofully frozen slides, which had been covered with a layer

des%thylein toer-he5),ricro-satedam-

wererved

waswasests

2.4.3. Total oxyradical scavenging capacity andglutathione-S-transferase activity

Total oxyradical scavenging capacity (TOSC)determined according to Regoli and Winston[31]. Hip-pocampi from rats were homogenized (1:4) in phphate buffer 50 mM (pH 7.50), plus NaCl (1.8%). Thomogenates were centrifuged at 13,500× g (4◦C) for25 min, and the supernatant centrifuged at 33,000× g at4◦C during 1 h. The supernatant obtained was colleand stored at−80◦C.

For TOSC measurements, alpha-keto�-methiolbutyric acid (KMBA; 2 mM; Sigma), whichused as substrate, reacts with ROS, producing ethyTOSC values reflect the sample capability to scavthe oxyradicals produced in vitro, thus inhibitethylene formation[31]. TOSC capability againperoxyl radicals were analyzed after thermal (35◦C)decomposition of 2,2′-azobis (2 methylpropionamidindihydrochloride (ABAP; 200 mM; from Aldrichdissolved in potassium phosphate buffer 100 mM7.4). Ethylene gas produced was measured us

of 0.65% normal melting point agarose. Cells in sliwere lysed (2.5 M NaOH, 0.1 M EDTA, 0.01 M Tris, 1sodium sarcocinate, 1% Triton X-100, and 10% dimesulfoxide, pH 10,0) over night at 4◦C. Samples werthen placed in the electrophoresis solution for 30 mallow DNA unwinding. Then, electrophoresis was pformed during 35 min at 25 V and 280 mA. Finally, tslides were neutralized with 0.4 M Tris buffer (pH 7.stained with 15�L of Sybr Gold (1:10000; MoleculaProbes) and analyzed using an epifluorescence mscope Zeiss Axioplan (400×). The DNA damage wameasured in 50 randomly selected cells in duplicslides, considering five-damage categories (0: undaged to 4: complete damaged). The final scoresmade multiplying the category number by the obsefrequency.

2.6. Statistical analysis

When the assumption of variance homogeneityverified, a one-way or repeated-measures ANOVAfollowed by Student–Newman–Keuls post hoc t

M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234 227

or planned comparisons. If the assumption of vari-ance homogeneity was violated, Kruskal–Wallis non-parametric ANOVA followed by Mann–Whitney com-parisons was performed. Behavioral data are shown asmedian an interquartile range. The other data are shownas means± 1S.E. In all cases, significance level adoptedwas 5%[34].

3. Results

3.1. Step-down inhibitory avoidance task

Training session step-down latency differ-ences among groups were not significant followedKruskal–Wallis analyses (χ2 = 3.2;p > 0.1). The median

(interquartile range) training step-down latency for allanimals was 5.1 s (3.5–7.5) (n = 120).

3.1.1. Experiment 1: Effects of pre traininginfusions on STM and LTM

STM and LTM test session latency data from the ani-mals that received microcystin (Mic; 0.01 and 20�g/L)and purified okadaic acid (OA; 0.01 and 10�g/L) 15 minprior training are shown inFig. 1. The Kruskal–WallisANOVA revealed a significant group effect for LTM(χ2 > 27; p < 0.01). Microcystin at the highest con-centration and okadaic acid at both concentrationsenhanced LTM. The lowest concentration of okadaicacid (0.01�g/L) caused retrograde amnesia for STM(two tailed Mann–WhitneyU-test;p < 0.05) (Fig. 1a).

Fb(wSs

ig. 1. (a) Effect of bilateral intrahippocampal infusions of microcystinefore step-down inhibitory avoidance training session. (b) Effect of bOA) on memory retrieval when given 15 min before test session. Conhich Mic or OA were diluted, respectively. Columns indicate mediansTM and LTM test for each group. Asterisks indicate significant statisession (n = 8–10 per group).

(0.01 or 20�g/L) (Mic) and okadaic acid (0.01 or 10�g/L) (OA) 15 minilateral intrahippocampal infusions of microcystin (Mic) and okadaic acidtrol group received saline or 20% dimethylsulfoxide (DMSO) in saline in(interquartile ranges) of step-down latencies in seconds, of training (TR),

tical difference (p < 0.05) level from the respective control groups at each

228 M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234

3.1.2. Experiment 2: Effects of pre test infusions onLTM

Treatments with microcystin (0.01 and 20�g/L)(Fig. 1b) given prior to testing impaired retrieval of LTM(Mann–WhitneyU-test;p < 0.05). Okadaic acid was noteffective in altering LTM retrieval at both concentrations(Mann–WhitneyU-test;p > 0.05).

3.2. Effect of microcystin and okadaic acid onspatial memory in the radial arm maze

Spatial memory was tested 15 min after intrahip-pocampal infusion of Mic (0.01 and 20�g/L) and OA(0.01 and 10�g/L), saline or DMSO once per day in theradial arm maze task. As shown inFig. 2a, the numberof working memory errors increased at day 8 in ani-mals treated with microcystins at both concentrations(p < 0.05). Only the animals treated with OA 0.01 had asignificant increase in working memory errors (p < 0.05)compared with DMSO (control group;Fig. 2b). For ref-erence memory (Fig. 2c), it was also revealed that thenumber of errors increased significantly at day 8 in ratsfrom groups Mic 0.01 and 20 compared with salinetreated rats (Mann–WhitneyU-test;p < 0.05) and only

the lowest concentration of OA increased the referenceerrors at day 8 (Mann–WhitneyU-test,p < 0.05) whencompared with DMSO treated animals (Fig. 2d). Finally,the time spent to consume all four baits increased inrats from groups Mic 0.01 and OA 0.01 when comparedto saline and DMSO groups (Mann–WhitneyU-test;p < 0.01) (Fig. 3a and b).

3.3. Analyses of oxidative stress

Evaluation of TOSC values showed the absence ofsignificant effect of exposure to either Mic or OA(p > 0.05;Fig. 4a and b). Higher GST activity (p < 0.05),however, was registered in Mic 0.01 respect Mic 20group (Fig. 4c), and no effect was verified after OA expo-sure (p > 0.05;Fig. 4d).

After exposure to both kinds of toxins, the hip-pocampi were analyzed to determine the lipid perox-idation product (LPO). The highest concentration ofmicrocystins (Mic 20) increased LPO levels measuredby TBARS assay respect the control group (p < 0.05).No differences (p > 0.05) were evident after OA treat-ments (Fig. 5a). Using the FOX assay, higher LPO lev-els were only observed at the lowest concentration of

ic; a an arm mazers in ra 1) injectionin salin .respe

Fig. 2. Effects of bilateral hippocampal injection of microcystin (Mconsidering working memory errors and reference memory erroof saline or Mic; (2) injection of 20% dimethylsulfoxide (DMSO)Asterisks indicate significant statistical difference (p < 0.05) from the

d c) and okadaic acid (OA; b and d) on performance in the radialts, respectively. The behavioral test was started 15 min after: (e or OA. Each value represents the mean± 1S.E. (n = 8–10 per group)

ctive control groups at each trial.

M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234 229

Fig. 3. Effects of bilateral hippocampal injection of microcystin (Mic;a) and okadaic acid (OA; b) on performance in the radial arm mazetask (time spent during trials) in rats. The behavioral test was started15 min after: (1) injection of saline or Mic; (2) injection of 20%dimethylsulfoxide (DMSO) in saline or OA. Each value representsthe mean± 1S.E. (n = 8–10 per group). Asterisks indicate significantstatistical difference (p < 0.05) from the respective control groups ateach trial.

microcystins (Mic 0.01) when compared with the controlgroup (p < 0.05). Again, no differences (p > 0.05) wereobserved after OA exposure (Fig. 5b).

Concerning DNA damage, microcystins exposureinduced a significant increase (p < 0.05) in the scores ofthe comet assay compared to control group (Fig. 5c).The treatment with OA presented significant damage(p < 0.01) only at the highest concentration (Fig. 5d).

4. Discussion

When the phosphatase inhibitors were infused 15 minbefore test session only the microcystins at both concen-trations blocked memory retrieval. Some, but not all, ofthe molecular mechanisms involved in LTM acquisitionand consolidation are also crucial for retrieval[25]. This

situation could be occurring in the present study sinceOA enhanced LTM when administrated before train-ing but showed no effect when given 15 min before testsession. It is possible that OA affects different cellularprocesses governed by protein phosphorylation in thehippocampus. However, the intrahippocampal infusionsof Mic (0.01 and 20�g/L) and OA (0.01 and 10�g/L)15 min before training increased significantly LTM, butonly OA 0.01 impaired STM. It is clear that short- andlong-term memory is processed by separate but parallelmolecular mechanisms[35]. The results indicate that OA0.01 blocked short-term memory and enhanced LTM,confirming previous reports of different mechanismsinvolved in STM and LTM. It has been hypothesized thatneural input may decreased, as well as increased, synap-tic efficacy [36,37]. Parallel studies (data not shown)indicated that the highest dose of Mic that influencedretrieval was ineffective in alter locomotion activity orto promote pro- or anti-conflict behavior.

Increased synaptic efficacy is considered to be asso-ciated with increased protein phosphorylation due toactivation of protein kinase activity, while decreasedsynaptic efficacy is considered to result from increaseddephosphorylation as result from activation of phos-phatases[36]. Alternatively, kinases and phosphatasesmay be involved in relatively distinct cellular mecha-nisms underlying different memory stages. Protein phos-phorylation is a key biochemical process involved insynaptic plasticity that operates through a tight balancebetween the action of protein kinases and protein phos-

ajor

ftenoteinctorsffec-ho-thats andbal-d-andolo-m-PP1ro-entPApeti-

ti-ry.

phatases. This process is widely believed to be a mcellular basis of learning and memory[14]. The roleof protein phosphatases in biological systems is oconsidered secondary compared to the role of prkinases simply because kinases are the primary effeof phosphorylation, while phosphatases are the etors of dephosphorylation. However, protein phosprylation/dephosphorylation is a dynamic processrelies equally on protein kinases and phosphatasemore specifically, on a delicate and concertedance between these proteins[37]. Several recent stuies have investigated the role of PP1 in learningmemory by taking advantage of transgenic methodgies in mice. The genetic inhibition of PP1 by teporally controlled expression of the endogenousinhibitor I (I1), an ubiquitously expressed 28 kDa ptein, which upon phosphorylation by cAMP-dependprotein kinase A, results in potent inhibition of P[37]. It was demonstrated a connection between retive learning and phosphatases in forebrain neurons[38].Malleret et al.[38] demonstrated the effect of genecally inhibiting calcineurin on plasticity and memo

230 M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234

Fig. 4. Effects of bilateral hippocampal injection of: saline or microcystin (Mic) (a and c); and 20% dimethylsulfoxide (DMSO) in saline orokadaic acid (OA) (b and d) on total oxyradical scavenging capacity (TOSC) and glutathione-S-transferase activity (GST). Data are expressed asmeans + 1S.E. (n = 3–8). Similar letters indicate absence of statistical difference (p > 0.05) between experimental groups.

Fig. 5. Effects of bilateral hippocampal injection of: saline or microcystin (Mic) (a and c); and 20% dimethylsulfoxide (DMSO) in saline or okadaicacid (OA) (b and d) on lipid peroxidation (LPO) and DNA damage. Data are expressed as means + 1S.E. (n = 5). Similar letters indicate absence ofstatistical difference (p > 0.05) between experimental groups.

M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234 231

Using the doxycycline-dependent rtTA system to expressa reversible inhibitor of calcineurin in the mouse brain,they find that the transient reduction of calcineurin activ-ity facilitates long-term potentiation (LTP) in vitro andin vivo. It is accompanied by enhanced learning andstrengthened short- and long-term memory in severalhippocampal-dependent spatial and no spatial tasks. TheLTP and memory improvements are reversed fully bysuppression of transgenic expression[38].

Spatial memory deficits induced by intrahippocam-pal injections of OA and microcystin were measuredin an eight arm radial maze. The data revealed signifi-cant impairment of both working and reference memoryonly after 8 days of exposure to the lower concentra-tions of OA 0.01 and Mic 0.01. As the infusion wasgiven 15 min prior to each trial at the eight arm radialmaze, the observed responses should be considered con-sequence of the cumulative dose of OA and Mic thatthe rats received during 8 days. It is likely that memoryimpairment in OA and microcystin treated rats is dueto a secondary effect of OA and microcystin exposure,such as neuronal damage that can be directly related tooxidative stress, as lipid peroxidation or DNA damage.

Oxidative stress has been implicated in the cog-nitive decline observed in ageing animals, includinghumans. Experiments with 8–11 month-old mice exhib-ited impairments in fear-conditioning tests and increasedbrain oxidative damage that was reverted by chronicsystemic administration of synthetic reactive oxygenspecies scavenger[39]. Further support for the involve-m hasc -i bmit-t linesi ita-m anst eu-r andP hato oret bril-l n[ lates dur-i TMi Mic( ilita-t tiont .g.M n-ts ing

8 days should promote an oxidative stress situation, lead-ing to neuronal damage, as registered in terms of lipidperoxidation and DNA damage (Fig. 5) and a paralleleffect in behavioral parameters (Fig. 3). The neuronaldamage may occur because the infusion was given dur-ing 8 days, 15 min prior to each trial at the eight armradial maze task. In this way, rats were submitted to acumulative dose of microcystins and this can explain theobserved effects at the biochemical and behavioral level.

Previous studies reported augmented TOSC valuesagainst peroxyl radicals after microcystins exposure,indicating a protective response against oxidative stressin terms of LPO[46]. However, the response of hip-pocampus tissue was different, since no variation in termof TOSC was evident. This lack of antioxidant protec-tion resulted in damage that can be directly related tooxidative stress (e.g. LPO).

The different methods to quantify lipid peroxides hasbeen the subject of much debate. Usually, spectrofluo-rometric methodologies are employed to determine con-jugated dines or malondialdehyde. This last oxidativeproduct is measured by TBARS, although some previousstudies[29] have raised concerns about the specificityand accuracy of this method. The FOX method, dif-ferent from those cited above, specifically detects lipidhydroperoxides[29]. Thus, in light of the specificityof the different methods, both the TBARS and FOXmethods were employed in this study. The differencesobserved between the two methodologies (i.e., higherTBARS values at the highest dose of microcystins and

e ofthe

Mal-of

ay.O)hole

ymestheiroped

theffer-lud-

rox-

SThydesayedy thetwo

stins.logy

ent of oxidative stress in cognitive impairmentome from studies from Fukui et al.[40]. These stud

es have demonstrated that 5 days after being sued to oxidative stress, young rats presented decn learning and memory, which was prevented by V

in E supplementation. It has been shown in humhat oxidative stress is implicated in a number of nodegenerative disorders such as Alzheimer’s (AD)arkinson’s diseases[41]. Recent evidence indicates txidative stress occurs early in AD, significantly bef

he development of the pathology hallmarks (neurofiary tangles and senile plaquet)[42]. Serrano and Klan43] considered that a mild ROS production moduynaptic plasticity, acting as a cellular messengerng LTP. In this context, the results that showed Lmprovement, suggest that a unique application ofat higher dose) or OA (both doses) induced a facion that could be mediated through kinases activahrough ROS[44] and/or by phosphatase inhibitors (eic and OA) (seeFig. 1). Several authors have me

ioned that kinases activation facilitate LTM[45]. At theame time, a continuous application of Mic or OA dur

higher FOX reactive substances at the lowest dosmicrocystins) could be reflecting the dynamics ofoxidative by products that each method detects.ondyaldehyde (MDA) is one of the end productslipid peroxidation, being identified by the TBARS assSince MDA is one of many lipid peroxidation (LPproducts, its determination may underestimate the wprocess. Also is important to stress that some enzlike aldehyde desidrogenases can degrade MDA tocorresponding alcohol. The FOX assay was develto measure lipid hydroperoxides not detected byTBARS assay. Interestingly, there are at least two dient enzymes that degrade lipid hydroperoxides, incing the phospholipid hydroperoxide glutathione peidase (PHGPx) and the glutathione-S-transferase[47].Although in the present study no induction of Gwas observed, different responses in terms of aldedesidrogenases and PHGPx activities at the two asdoses of microcystins could be in part responsible bdifferent peaks of LPO products measured by themethods at the lowest and highest doses of microcyHowever, independently of the dose or methodo

232 M. Maidana et al. / Chemico-Biological Interactions 159 (2006) 223–234

considered, these results indicated that microcystins canpromote oxidative stress, as previously observed[3–5].The fact that not only LPO levels but also higher DNAdamage was registered after exposure to these toxinsshows its ability to induce oxidative damage. To ourknowledge, the mechanisms of how this damage is gen-erated is still unknown, but as mentioned in the Intro-duction, a loss of glutathione being involved in phaseII reactions should represent a process that leave thecells prone to suffer oxidative stress, although the lackof responsiveness of GST in the present study seems tosuggest otherwise (see below).

In terms of GST activity, the treatment with micro-cystins was ineffective to augment its activity, contrary tothe report of Beattie et al.[48]. The fact that organismsfrom Mic 20 group showed a lower GST activity thatorganism from Mic 0.01 group could be explained con-sidering the results of Srivastava et al.[49] who reportedinhibition of� human GST isoenzyme by a metabolite ofbenzo[a]pyrene, indicating that some conjugation prod-ucts catalyzed by GST may hinder this process. Otherorganic peroxides such as cumene hydroperoxide areknown to inhibit � GST [50], meaning that oxidativeproducts can in fact reduce the conjugation capabilitiesmediated by GST.

The present study showed that intrahippocampal infu-sions with solutions of phosphatase inhibitors alteredseveral behavioral parameters and these effects wereparalleled by oxidative stress generation. As organicanion transporters such as OATP1A2 are expressed in

edi-fbio-envi-s theratshe

ationffer

tiveram-

ep-suchessstins

zil).

Laura A. Geracitano is a PRODOC fellow from CAPES(Brazil). The logistical support from Pos-Graduac¸aoem Ciencias Fisiologicas–Fisiologia Animal Comparada(FURG) and Dr. Paulo C. Abreu for the use of epifluo-rescence microscopy is also acknowledged. We wish tothank Dr. Alexandre Matthiensen and Dr. Gilberto Fill-mann by the English suggestion to this manuscript. Thisstudy was supported by CNPq and FAPERGS.

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