Acute exposure to methylmercury at two developmental windows: Focus on neurobehavioral and neurochemical effects in rat offspring

Neuroscience 141 (2006) 1619 –1629

ACUTE EXPOSURE TO METHYLMERCURY AT TWO DEVELOPMENTAL WINDOWS: FOCUS ON NEUROBEHAVIORAL AND NEUROCHEMICAL EFFECTS IN RAT OFFSPRING M. R. CARRATÙ,a* P. BORRACCI,a A. COLUCCIA,a A. GIUSTINO,a G. RENNA,a M. C. TOMASINI,b E. RAISI,b T. ANTONELLI,b V. CUOMO,c E. MAZZONIb AND L. FERRAROb

Key words: methylmercury, neurodevelopment, motor activity, novel exploration object, glutamate, cortical neuron cultures.

a

Department of Pharmacology and Human Physiology, Medical School, University of Bari, Policlinico, Piazza Giulio Cesare 11, 70124 Bari, Italy

The most important source of methylmercury (MeHg) exposure in humans is through the consumption of fish and fish products. In view of the changing dietary habits of modern society, which advocates the benefits of fish consumption for maintenance of general health, as well as for prevention of cardiovascular disease (Bouzan et al., 2005), the possibility of MeHg intoxication through ingestion of contaminated fish and fish products is of great concern (Gochfeld and Burger, 2005). In particular, consumption of MeHg-contaminated food by pregnant women poses one of the most serious potential hazards for the offspring. While high-dose exposure may result in cerebral palsy, deafness and severe mental retardation associated with disorganization of cerebral cortex cytoarchitecture and severe atrophy of the folia of cerebellum hemispheres (Choi, 1989; National Research Council, 2000), lower MeHg doses may produce more subtle neurobehavioral changes. The most intriguing aspect is that following MeHg exposure in utero, an infant who appears normal at birth may develop psychomotor deficit as the nervous system matures (Marsh et al., 1987). Moreover, whereas adverse effects have been unequivocally demonstrated in poisoning incidents, the implications of the exposure to low levels of MeHg, such as those occurring in fish-eating populations, are still controversial (Davidson et al., 1998; Mahaffey, 1998; Cohen et al., 2005). Furthermore, the lowest dose of MeHg that might impair neurodevelopment is still unknown. Based on data from a study of 81 Iraqi children whose mothers were exposed to MeHg during pregnancy (Marsh et al., 1987), the U.S. Environmental Protection Agency established a reference dose for MeHg of 0.1 ␮g/kg per day (Environmental Protection Agency, 1997). Experimental data obtained in rodents show that the consequences of in utero exposure to MeHg range from increased rates of intrauterine death, delayed developmental growth, and altered brain cellular arrangement to more subtle effects, such as delayed reflexive behavior, impairment of locomotor activity and motor coordination or cognitive dysfunctions, depending on the duration and level of exposure at different developmental stages (Cuomo et al., 1984; Cagiano et al., 1990; Kakita et al., 2000; Doré et al., 2001; Baraldi et al., 2002; Daré et al., 2003; Goulet et al., 2003).

b

Department of Clinical and Experimental Medicine, Pharmacology Section, University of Ferrara, Via Fossato di Mortara 17-19, 44100 Ferrara, Italy c

Department of Human Physiology and Pharmacology “V. Erspamer,” University “La Sapienza,” Viale Aldo Moro 5, 00185 Rome, Italy

Abstract—The neurobehavioral and neurochemical effects produced by prenatal methylmercury exposure (8 mg/kg, gestational-days 8 or 15), were investigated in rats. On postnatal day 40, animals exposed to methylmercury and tested in the open field arena, showed a reduction in the number of rearings, whereas the number of crossings and resting time was not altered with respect to the age-matched control rats. The methylmercury-exposed groups showed a lower level of exploratory behavior as well as an impairment in habituation and working memory when subjected to the novel object exploration task. The neophobia displayed by methylmercury-exposed rats is unlikely to be attributed to a higher degree of anxiety. Prenatal methylmercury exposure did not affect motor coordination or motor learning in 40-day-old rats subjected to the balance task on a rotating rod, and it did not impair the onset of reflexive behavior in pups screened for righting reflex, cliff aversion and negative geotaxis. In cortical cell cultures from pups exposed to methylmercury during gestation, basal extracellular glutamate levels were higher, whereas the KCl-evoked extracellular glutamate levels were lower than that measured in cultures from rats born to control mothers. In addition, a higher responsiveness of glutamate release to N-methyl-D-aspartic acid receptor activation was evident in cortical cell cultures from pups born from methylmercury-treated dams than in cultures obtained from control rats. The present results suggest that acute maternal methylmercury exposure induces, in rat offspring, subtle changes in short-term memory as well as in exploratory behavior. These impairments seem to be associated to alterations of cortical glutamatergic signaling. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. *Corresponding author. Tel: ⫹39 080 5478455; fax: ⫹39 080 5478444. E-mail address: [email protected] (M. R. Carratù). Abbreviations: ANOVA, analysis of variance; ASR, acoustic startle reflex; GD, gestation day; HDIPS, exploratory head dip; MeHg, methylmercury; NMDA, N-methyl-D-aspartic acid; PND, postnatal day; PPI, prepulse inhibition; r.p.m., rotations per minute; SAP, stretched-attend posture; TO, time spent on open quadrants.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.05.017

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As far as the mechanism implicated in developmental MeHg neurotoxicity is concerned, experimental data support the involvement of glutamate-mediated excitotoxicity (Miyamoto et al., 2001; Juarez et al., 2005). It is well known that in different neurodegenerative disorders, excitatory amino acids may mediate neuronal injury and exert deleterious consequences on brain functional development, depending on the levels. Moreover, it is worth noting that NMDA receptor activation is involved in the plastic processes during brain development. Since it has been reported that MeHg preferentially accumulates in astrocytes where it induces cell swelling and specifically inhibits excitatory amino acid uptake (Aschner et al., 2000; Allen et al., 2002; Juarez et al., 2002; Fonfria et al., 2005; Morken et al., 2005), it seems likely that the consequent elevation of glutamate levels in the extracellular space may trigger or accelerate processes of excitotoxic neurodegeneration. In order to further investigate on the deleterious effect of MeHg on brain development, the purpose of the present study was to evaluate the possible neurobehavioral and neurochemical changes induced in male rat offspring by acute maternal exposure to MeHg (8 mg/kg) at two different developmental windows (gestation days (GDs) 8 or 15). The single dose model was adopted taking into account that it is useful in evaluating mechanisms of toxicity in ways not possible with humans. More importantly, since the pregnant rat does not excrete much mercury until lactation, a single dose provides a constant mercury exposure to the fetus as well as a steady exposure during lactation (Magos et al., 1980). The developmental windows have been chosen taking into account that the GD 8 or 9 is considered the beginning of maximum susceptibility of the developing rodent brain to MeHg, and that brain damage can also occur after the critical period for malformations (Rodier, 1980; Choi, 1989; Rice and Barone, 2000). The MeHg dose level has been selected on the basis of previous studies showing that following maternal administration of 8 mg/kg on GD 15 the brain concentration of MeHg in the offspring at birth is 60-fold higher than in the brain of saline-treated rats, remaining four-fold higher until postnatal day (PND) 21 with a decline to the normal range on PND 60 (Cagiano et al., 1990). Since the fetus may act as a “sink” sequestering mercury away from the mother (Shimai and Satoh, 1985) and because of the slow movement of MeHg from fetus to mother (Reynolds and Pitkin, 1975), also administration of this compound on GD 8 will result in a greater accumulation in the fetal brain, the peak concentration reaching the maximum within 48 h (Lewandowski et al., 2002) with a decline to the normal range after weaning. In order to assess possible neurobehavioral outcomes of developmental MeHg exposure, the following endpoints were evaluated during adolescence: (1) motor coordination and motor learning (rotarod/accelerod test); (2) locomotor activity (open field); (3) exploratory behavior, habituation, and visual discrimination (novel object exploration task); and (4) inhibition of the response to the acoustic startling stimulus by a weak prestimulus (prepulse inhibition, PPI). The adolescence period was selected taking

into account that the consequence of errors in developmental processes, such as possible impairment in working memory, could become manifest at the end of adolescence (Frangou and Murray, 1996), and that young rats learn to handle a higher working memory load as testing progresses (Bimonte et al., 2003). Moreover, the ontogeny of reflexive behavior was assessed during early postnatal life. In order to identify possible mechanisms underlying neurofunctional deficit, experiments have been performed to evaluate whether prenatal MeHg exposure could alter glutamatergic signaling in primary neuronal cultures from cerebral cortex. Finally, the effects of gestational exposure to MeHg on reproduction parameters have been also investigated.

EXPERIMENTAL PROCEDURES Animals and treatment schedule The experiments have been conducted in accordance with guidelines released by Italian Ministry of Health (D.L. 116/92), the Declaration of Helsinki and the “Guide for the Care and Use of Laboratory Animals” as adopted and promulgated by the National Institutes of Health. The minimum number of animals has been used. All animals have been treated humanely according to institutional guidelines, with due consideration to the alleviation of distress and discomfort. Primiparous Sprague–Dawley female rats (Harlan, San Pietro al Natisone, Udine, Italy) weighing 250 –280 g were used. The animals were allowed free access to food and water, were housed at constant room temperature (20 –22 °C) and exposed to a light cycle of 12 h/day (08:00 h-20:00 h) for two weeks before the experiment. Pairs of females were placed with single male rats in the late afternoon. Vaginal smears were taken the following morning at 09:00 h. The day on which sperms were present was designated GD 0. Pregnant rats were then randomly assigned to three groups treated with i ) saline (i.e. control), ii ) 8 mg/kg MeHg on GD 8 and iii ) 8 mg/kg MeHg on GD 15, respectively. Solutions were administered by means of intragastric intubation in volumes of 1 ml/kg of body weight. All litters were reduced to a standard size of eight male pups per litter (when possible) within 24 h after birth. Pups were weaned at 21 days of age. One male pup per litter from different litters per treatment group was used. Each pup was used only for a single behavioral test and tested once.

Reproduction data Body weights of dams (control n⫽10; MeHg 8 mg/kg on GD 8 n⫽9; MeHg 8 mg/kg on GD 15 n⫽9) were taken from GD 0 to GD 20. The number of dams giving birth as well as the length of pregnancy was determined. Litter size at birth and postnatal mortality (number of male pups died before weaning) were evaluated. Body weights of male rats (one pup per litter from 10 control litters, nine treated with MeHg 8 mg/kg on GD 8 and nine treated with MeHg 8 mg/kg on GD 15) were taken.

Onset of reflexive behavior From PND 2– 8, one pup per litter from each treatment group (control n⫽9; MeHg 8 mg/kg on GD 8 n⫽9; MeHg 8 mg/kg on GD 15 n⫽9) was tested for the development of righting reflex and cliff aversion (Altman and Sudarshan, 1975). The following scoring procedure was used for each behavioral variable: 0⫽no response; 1⫽uncertain response; 2⫽incomplete response; 3⫽full response. Furthermore, negative geotaxis was performed on PNDs 2, 4, 6, 8, 10 and 12. In this test, each pup was placed on a 30° angle

M. R. Carratù et al. / Neuroscience 141 (2006) 1619 –1629 slope with its head pointing down the incline and the latency before turning around and crawling up the slope (cutoff time⫽180 s) was measured.

Rotarod/accelerod test The procedure has been described previously (Coluccia et al., 2004). Experiments were performed by using a device consisting of a computerized electronically controlled system (TSE System, Bad Homburg, Germany), composed of a four-lane rotating drum (6 cm in diameter) suspended 14 cm above the stainless steel floor grid, whose surface was manufactured to provide grip for the animal. Animal falls were detected by light-beam sensors mounted into each compartment and with the unit equipped with an escape-preventing transparent cover. The apparatus was set up in an environment with minimal disturbances. Two experimental paradigms were performed: a) a single session at constant rotation speed mode; b) repeated daily sessions at accelerating rotation speed mode. Rats were acclimated to the stationary rod for 3 min on the day before the test. In the first procedure (constant speed session), animals were placed on the rotating rod at the following speed: 0 (stationary), five, 10 and 15 rotations per minute (r.p.m.). At each rotation speed, four trials were conducted and the latency to fall was monitored for 180 s. Inter-trial interval for each animal was 20 min. In the second procedure (accelerating rotation speed mode), rats previously trained during the different constant speed sessions were tested as follows: during each test session, each rat was placed on the stationary rod for approximately 30 s; then rod rotation accelerated from 4 to 40 r.p.m. over 300 s. The time from the beginning of acceleration to the animal fall was automatically recorded. Animals were subjected to four consecutive daily sessions. The test was performed on 40 days of age. Each experimental group consisted of the following number of animals: control n⫽10; MeHg 8 mg/kg on GD 8 n⫽9; MeHg 8 mg/kg on GD 15 n⫽8. Since performance on a rotating rod requires an intact muscular strength, MeHg-exposed rats were also subjected to the grip strength test. Grip strength meter (UGO Basile, Italy) consisting of a grasping-trapeze attached to a force transducer (model 47105– 002), peak amplifier (model 47105– 001) was used. After adjusting the height of the grasping trapeze, the animal was allowed to grasp the trapeze and then was pulled by the tail. The peak pulling force (grip strength) was recorded from the digital display on the amplifier (Tariq et al., 2002). The test was performed on 40 days of age. Each experimental group consisted of the following number of animals: control n⫽6; MeHg 8 mg/kg on GD 8 n⫽8; MeHg 8 mg/kg on GD 15 n⫽6.

Acoustic startle reflex (ASR) test and PPI The apparatus and procedures have been described previously (Anisman et al., 2000). Briefly, startle measurements were made using an animal startle reflex system (MED Associates, St. Albans, VT, USA). This system consisted of one ventilated, sound attenuating startle chamber (30⫻55⫻50 cm), a rack mounted operating station and a standard IBM compatible PC. In each chamber, a wideband speaker (1–16 kHz) provided the audio source for the startle stimuli, as well as background noise, while a startle sensor platform, signal transducer and load cell amplifier served to measure the animal’s startle response. When downward pressure was exerted by the animal in response to a startle stimulus, the resultant force was converted to an analog signal by the sensor platform, amplified by the load cell amplifier and then digitized on a scale of 0 –2048 arbitrary units by a Dig-729ADC card (MED Associates). The presentation and ordering of all stimuli were controlled by the startle reflex system software (version 5, MED Associates). Procedure. Rats were taken from their home cage and placed in a clear, cylindrical acrylic holder (7.6⫻14 cm) (ENV-

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262B, MED Associates) which was attached to a rectangular base that could be fastened to the startle platform. The holders were ventilated by a long slot running the length of the tube, as well as by slots placed at both ends of the tube, adjacent to the position of the animal’s head. Once the animal had been secured in the holder using two removable panels, placed at opposite ends of the tube, the holder was secured to the startle platform, which was positioned approximately 20 cm for the audio source. The chamber was then sealed and each animal allowed to acclimatize to the startle chamber for a period of 5 min. Throughout the length of the experiment, low levels of ambient light were generated in each chamber by a 3-W red-filtered light. Additionally, background white noise, with an intensity of 55 dB, was maintained to minimize the impact of acoustic stimuli outside of the chamber environment. The 20 min startle test session consisted of a 5 min acclimatization period (with a background noise of 55 dB) and three different types of trials: 1) prepulse stimulus only (85 dB presented for 20 ms); 2) startle stimulus only (110 dB presented for 50 ms); 3) prepulse and startle stimuli with a 100 ms interstimulus interval. Each trial was presented 10 times (for a total of 30 trials) with an intertrial of 30 s. Basal startle amplitude was determined as the mean amplitude of 10 startle trials. Suppression of the acoustic startle response by a weak prestimulus (PPI) has been examined in order to ascertain that possible treatment-induced differences in startle were unrelated to attentional factors. The amount of PPI was calculated as %PPI⫽100⫺(startle amplitude for pulse⫹prepulse trial/startle amplitude for pulse only trial)⫻100. The test was carried out at 40 days of age. Each experimental group consisted of the following number of animals: control n⫽10; MeHg 8 mg/kg on GD 8 n⫽9; MeHg 8 mg/kg on GD 15 n⫽9.

Locomotor activity The technique was previously described by Laviola et al. (1988). Briefly, the apparatus was an open field arena (60⫻60 cm) made of black Plexiglas, with a light green bottom subdivided by black lines into 12⫻12 cm squares. Crossings of square limits with both forepaws were recorded by a counter mechanically activated by the experimenter. Moreover, resting time and rearings were recorded. All tests were carried out in a sound-attenuating cabin (3⫻2⫻2 m) illuminated by a 20-W white light suspended 2 m above the apparatus. Background noise of 42 dB was produced by a fan. Animals were subjected to a 10-min session at 40 days of age. The test started by placing the animal at the center of the arena. Immediately after each test, the apparatus was thoroughly cleaned by cotton pads wetted with 96% ethanol solution. No recourse was made to simultaneous scoring by two experimenters or to blind procedures, because no experimenter biases were found in previous control experiments in the case of the response considered. Each experimental group consisted of the following number of animals: control n⫽10; MeHg 8 mg/kg on GD 8 n⫽9; MeHg 8 mg/kg on GD 15 n⫽9.

Novel exploration object test The novel exploration object test used in the present study was a modified version (Giustino et al., 1999) of that previously described by Ennaceur and Delacour (1988). Briefly, 40-day-old male rats (control n⫽10; MeHg 8 mg/kg on GD 8 n⫽9; MeHg 8 mg/kg on GD 15 n⫽9) were submitted to two habituation sessions (intersession interval: 24 h) where they were allowed 5 min to explore the apparatus (circular arena, 75-cm diameter). Twenty-four hours after the last habituation session, each rat was placed in the arena and given two 3-min trials with a 1-min intertrial interval. In the first trial (T1) rats were exposed to two identical objects (black and white striped plastic bottles), which constituted samples A1 and A2. During the second trial (T2) rats were exposed again to two objects, of which one sample was

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familiar and the other, a new object, B. Object exploration was quantified as: (1) exploratory activity: total time spent exploring both objects during each trial (T1 and T2); (2) index of global habituation: it is measured by comparing the total time spent exploring the two objects in T1 to that spent in T2; (3) discrimination between the new and the familiar objects: it is measured in T2 by comparing the time spent exploring the familiar to that spent exploring the new object. At the end of experiments, each tested rat was killed and brain weight was measured.

Elevated zero-maze test Apparatus. According to the technique previously described by (Shepherd and coworkers 1994) and modified by (Bickerdike and coworkers 1994), the maze comprised a black Perspex annular platform (105 cm diameter, 10 cm width) elevated to 65 cm above ground level, divided equally into four quadrants. Two opposite quadrants were enclosed by black Perspex walls (27 cm high) on both the inner and outer edges of the platform, while the remaining two opposite quadrants were surrounded only by a Perspex “lip” (1 cm high) which served as a tactile guide to animals on these open areas. A video camera, connected to video equipment in a separate observation room, was mounted overhead in order to record behavior on the maze for subsequent analysis. Procedure. Subjects were placed on a closed quadrant and a 5-min test period was recorded on videotape for subsequent analysis. The maze was cleaned with 96% ethanol solution and dried thoroughly between test sessions. Behavioral measures were as follows: A. Percent time spent on the “open” quadrants (% TO) expressed as the percentage of the total time of the test. Time on the open quadrants was timed from the moment all four paws of the rat were placed on an open section and ended when all four paws re-entered a closed quadrant. B. Number of exploratory head dips (HDIPS) made over the edge of the platform, either from the exit of the “closed” quadrant, or while on the open quadrant. C. Number of stretched-attend postures (SAP) made from the exit of a closed quadrant toward an open quadrant. This exploratory posture can be described as a forward elongation of the body, with static hindquarters, followed by a retraction to the original position. The test was carried out at 40 days of age. Each experimental group consisted of the following number of animals: control n⫽9; MeHg 8 mg/kg on GD 8 n⫽7; MeHg 8 mg/kg on GD 15 n⫽9.

Primary cultures of cerebral cortex neurons Neurons have been obtained from the cerebral cortex of one-dayold Sprague–Dawley rats (Alho et al., 1988) born to control and MeHg-exposed dams. After the resuspension in the plating medium, the cells were counted and then plated on poly-L-lysine (5 ␮/ml) -coated dishes at a density of 2.5⫻106 cells/dish (NUNC dishes). The culture medium consisted of Eagle’s basal medium supplemented with inactivated fetal calf serum 10%, 25 mM KCl, 2 mM glutamine, and 100 ␮g/ml gentamycine. Thereafter, the cultures have been kept at 37 °C in an incubator under a humidified atmosphere (5% CO2/95% air). Glial cell proliferation was inhibited by cytosine arabinoside (10 ␮M) to obtain a vast dominance of neuronal cells (90 –97%; Alho et al., 1988).

Basal and evoked cortical extracellular glutamate levels On the day of the release experiment, the cells were rinsed twice by replacing the culture medium with warmed (37 °C) Krebs Ringer-bicarbonate buffer (in mM: NaCl 118.5, KCl 4.8, CaCl2 2.5,

MgSO4 1.2, NaHCO3 25, NaH2PO4 1.2, glucose 11, pH 7.4). Thereafter, five consecutive fractions were collected renewing this solution (400 ␮l) every 30 min. The first two samples were used to assess basal glutamate levels while, to evoke endogenous glutamate, the cells were treated with an isotonic Krebs solution containing 20 mM KCl, applied 15 min before the end of the third fraction. A pharmacological challenge with the glutamate receptor agonist N-methyl-D-aspartic acid (NMDA) 0.1 ␮M, was carried out during the third fraction by applying the compound 10 min before the end of the collection period.

Endogenous glutamate analysis Reverse-phase high-performance liquid chromatography HPLC coupled with fluorometric detection (wavelengths: emission, 450 nm; excitation, 370 nm) was used to assay endogenous glutamate. Briefly, after a precolumn derivatization with an o-phtaldialdehyde/␤-mercaptoethanol reagents followed by separation on an analytical Chromsep 5 (C18) column perfused under isocratic conditions at a flow rate of 1 ml/min the amino acid was quantified in a Beckman fluorescence spectrometer. The mobile phase consisted of 0.1 M sodium acetate, 10% methanol and 2.5% tetrahydrofurane, pH 6.5. The limit of detection for glutamate was 30 fmol/sample. External standards were used to identify and calibrate the peaks resulting from the injection of the samples.

Statistical analysis The general reproduction data, grip strength, ASR, PPI, locomotor activity, body and brain weights (PND 40) were analyzed by one-way ANOVA. Offspring body weight gain (PND 1 to PND 21), negative geotaxis, motor coordination (pooling data of the four trials for each rotation speed) and motor learning were analyzed by two-way ANOVA for repeated measures. Individual comparisons were performed by Tukey’s multiple comparisons tests. Fisher’s exact-test was used where appropriate. The original response variables related to righting reflex and cliff aversion were transformed considering the first day of adultlike response. For each response variable, the score (2 or 3) corresponding to an adult-like response on the basis of the developmental profile expected in control animals was chosen. The resulting new variables were analyzed by non parametric test, Kruskal-Wallis ANOVA, followed by post hoc test (Dunn’s multiple comparison test). Data relative to the novel exploration object test were evaluated by using non-parametric statistics due to highly significant heteroscedasticity in all instances. Statistical analysis was based on Kruskal-Wallis ANOVA followed by post hoc tests (Wilcoxon paired signed-rank test for within-group comparisons and Dunn’s multiple comparison test for between-group comparisons). According to previous studies (Bickerdike et al., 1994), % TO produced normal data, whereas both SAP and HDIPS produced skewed non-parametric data (Poisson distribution). Therefore, square root transformations of SAP and HDIPS were carried out, thereby producing normal distribution of data and allowing parametric analyses. % TO and transformed SAP and HDIPS data were analyzed by one-way ANOVAs. For brevity, in the Results section square root head dips and square root SAP are referred to simply as head dips and SAP. In the in vitro study, the effects of the treatments on the endogenous extracellular glutamate levels during the third fraction are reported and expressed as percent changes of the basal values, as calculated by the means of the two fractions collected prior the treatment. The statistical analysis was carried out by one-way analysis of variance (ANOVA) followed by the NewmanKeuls test for multiple comparisons.

M. R. Carratù et al. / Neuroscience 141 (2006) 1619 –1629

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Table 1. Effects of prenatal MeHg exposure on reproductive parameters MeHg concentration (mg/kg)

Dam weight gain (%)a (mean values⫾S.E.M.)

Dams giving birth (%)

Pregnancy length (days) (mean values⫾S.E.M.)

Litter size at birth (mean values⫾S.E.M.)

Postnatal mortalityb

Control 8 (on GD 8) 8 (on GD 15)

56.96⫾1.96 52.03⫾2.63 50.89⫾2.66

100 100 100

21.4⫾0.16 21.0⫾0.00 20.9⫾0.20

14.2⫾0.83 12.9⫾1.15 13.3⫾0.41

3/139 11/110* 21/119**

a

From day 0 to day 20 of pregnancy. From birth to weaning. * P⬍0.02,** P⬍0.0001 significantly different from control (Fisher’s exact test). b

over, an overall two-way ANOVA for repeated measures of negative geotaxis (latency to rotate) gave the following results: Ftreatments⫽3.70, df⫽2/24, P⬍0.05; Fages⫽54.66, df⫽5/120, P⬍0.0001; Ftreatments⫻ages⫽1.18, df⫽10/120, n.s. In the absence of significant interaction between treatment and ages, selective post hoc tests (Tukey’s multiple comparison test), performed on the basis of data inspection as suggested by Wilcox (1987), did not show any significant difference.

RESULTS Reproduction data General reproduction data are reported in Table 1. Overall one-way ANOVAs showed that dam weight gain (F⫽1.85, df⫽2/25, n.s.), pregnancy length (F⫽3.28, df⫽2/25, n.s.) and litter size at birth (F⫽0.63, df⫽2/25, n.s.) were not significantly affected by prenatal treatment with MeHg. Moreover, statistical analysis (Fisher’s-exact test) showed that prenatal treatment with MeHg did not influence the number of dams giving birth. The Fisher’s-exact test revealed that MeHg at the dose of 8 mg/kg on both GDs 8 and 15 significantly increased postnatal mortality of male pups with respect to control animals (on GD 8: P⬍0.02; on GD 15: P⬍0.0001) (Table 1). An overall two-way ANOVA for repeated measures of male pup weight gain gave the following results: Ftreatments⫽4.11, df⫽2/25, P⬍0.05; Fages⫽830.87, df⫽7/175, P⬍0.0001; Ftreatments⫻ages⫽3.68, df⫽14/175, P⬍0.0001. Individual comparisons (Tukey’s multiple comparison test) revealed that prenatal treatment with MeHg at the dose of 8 mg/kg on GD 15 significantly decreased male pup weight gain on PND 21 with respect to the agematched control rats (P⬍0.01) (Table 2). Finally, an overall one-way ANOVA showed that body weight on PND 40 (F⫽0.27, df⫽2/25, n.s.) was not significantly affected by prenatal treatment with MeHg (data not shown).

Rotarod/accelerod test Motor coordination, balance and ataxia were tested with rotarod which measures the ability of the rat to maintain balance on a rotating rod. This task requires an intact cerebellar function and motor coordination (Carter et al., 1999). In MeHg-exposed offspring no change in the performance of this task was observed (data not shown). An overall two-way ANOVA for repeated measures of the latency to fall in single sessions at constant speed mode (which measures motor coordination) did not show any significant difference between MeHg-exposed rats and the agematched control animals (Ftreatments⫽0.77, df⫽2/24, n.s.; Fsessions⫽3.25, df⫽3/72, P⬍0.05; Ftreatments⫻sessions⫽0.62, df⫽6/72, n.s.). Furthermore, an overall two-way ANOVA for repeated measures of the latency to fall in accelerating rotation speed mode (which provides an indication of motor learning ability) did not reveal significant differences between MeHg-exposed offspring and the age-matched control group (Ftreatments⫽0.15, df⫽2/24, n.s.; Fsessions⫽0.68, df⫽3/72, n.s.; Ftreatments⫻sessions⫽1.61, df⫽6/72, n.s.). An overall one-way ANOVA shows that prenatal MeHg exposure did not affect the muscular strength (F⫽0.65, df⫽2/17; n.s.) (data not shown).

Onset of reflexive behavior This was assessed by evaluating the onset of the following reflexes: righting reflex, cliff aversion and negative geotaxis. In particular, the negative geotaxis test allows the evaluation of cerebellar function with special regard to the labyrinthic integrity (Hermans et al., 1993). Prenatal MeHg exposure did not affect the maturation of the reflexive behavior (data not shown). Nonparametric analysis (Kruskal-Wallis ANOVA) showed the following results: righting reflex: H⫽4.10, df⫽2, n.s.; cliff-aversion: H⫽1.48, df⫽2, n.s. More-

ASR test and PPI The unconditioned reflexive response to a sudden acoustic stimulus and its suppression by a weak prestimulus was

Table 2. Effects of prenatal MeHg exposure on body weight of male offspring from PND 1 to PND 21 MeHg concentration (mg/kg)

PND1

PND3

PND6

PND9

PND12

PND15

PND18

PND21

Control 8 (on GD 8) 8 (on GD 15)

6.5⫾0.15 6.6⫾0.18 5.9⫾0.19

8.5⫾0.20 8.6⫾0.39 7.3⫾0.43

12.6⫾0.31 12.9⫾0.72 10.6⫾0.95

18.1⫾0.69 18.3⫾1.14 15.0⫾1.75

25.4⫾0.81 26.2⫾1.14 21.2⫾2.24

33.7⫾0.92 34.0⫾1.11 28.2⫾2.55

40.8⫾1.04 41.8⫾1.14 34.2⫾3.02

51.2⫾1.37 51.8⫾1.48 42.0⫾4.08*

Data represent mean values (g)⫾S.E.M. * P⬍0.01 significantly different from the control group (Tukey’s multiple comparison test).

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P⬍0.01; on GD 15: P⬍0.05) (Fig. 1), thus suggesting an impaired exploratory behavior. Novel exploration object test Kruskal-Wallis ANOVA for total exploratory activity during T1 showed the following significant difference: H⫽8.9; df⫽2; P⬍0.05. Between-group comparisons showed that the time spent exploring the objects during T1 was significantly affected by prenatal exposure to MeHg (on GDs 8 and 15: P⬍0.05, Dunn’s multiple comparison test). As regards global habituation, within-group comparisons showed that control rats exhibited lower levels of exploratory activity during T2 compared with those found in T1 (P⬍0.005), whereas no significant difference in the exploration time between T1 and T2 was found in 40 day old rats exposed to MeHg (Fig. 2A). However, a further statistical analysis (Kruskal-Wallis ANOVA: H⫽10.6; df⫽2; P⬍0.01 followed by betweengroup comparisons) showed an altered index of global habituation (T1–T2) in rats exposed to MeHg (on GDs 8 and 15: P⬍0.05, Dunn’s multiple comparison test) (Fig. 2A; inset).

Fig. 1. Effects of prenatal MeHg exposure on crossings (A), resting time (B) and rearings (C) of 40-day-old offspring during locomotor activity. Each column represents the mean values⫾S.E.M. * P⬍0.05; ** P⬍0.01 significantly different from the control group (Tukey’s multiple comparison test).

assessed. The results show that prenatal MeHg exposure did not affect this sensorimotor gating reflex (i.e. auditory gating). Overall one-way ANOVAs did not show any significant difference for acoustic startle response (F⫽0.43, df⫽2/25, n.s.) and PPI (F⫽0.71, df⫽2/25, n.s.) between 40-day-old rats born to MeHg-exposed dams and the agematched control animals (data not shown). Locomotor activity The results obtained in the open field test failed to show any general motor deficit in MeHg-exposed rats. Overall one-way ANOVAs did not show any significant difference for crossings (F⫽2.17, df⫽2/25, n.s) and resting time (F⫽1.63, df⫽2/25, n.s.) between control and MeHgtreated groups. However, an overall one-way ANOVA of rearings showed the following difference: F⫽6.18, df⫽2/25, P⬍ 0.01. Individual comparisons (Tukey’s multiple comparison test) showed that prenatal treatment with MeHg at the dose of 8 mg/kg on both GDs 8 and 15 significantly decreased rearings with respect to control rats (on GD 8:

Fig. 2. Effects of prenatal MeHg exposure on exploratory activity (A) during trial 1 (T1) and trial 2 (T2) of 40-day-old rats. Data are expressed as median values and interquartiles (dashed lines). ° P⬍0.05 significantly different from T1 controls (Dunn’s multiple comparison test); * P⬍0.005 significantly different from T1 of the same treatment group (Wilcoxon paired signed-rank test). Inset: bars represent T1–T2 values. # P⬍0.05 significantly different from control. Effects of prenatal MeHg exposure on exploratory activity (B) during trial 2 of 40-day-old rats. Data are expressed as median values and interquartiles (dashed lines). * P⬍0.005 vs. novel object (Wilcoxon paired signed-rank test).

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Kruskal-Wallis ANOVA for discrimination showed the following difference: H⫽10.0; df⫽2; P⬍0.01. Within-group comparisons showed that time spent by control rats in exploring the familiar object during T2 was significantly lower than that spent in exploring the novel object (P⬍0.005). Conversely, rats exposed to 8 mg/kg of MeHg on both GDs 8 and 15 did not show any significant difference in the exploration time of the novel with respect to the familiar object (Fig. 2B). Finally, one-way ANOVA did not show any significant difference in brain weight between MeHg-exposed offspring and the age-matched control rats (F⫽0.59; df⫽2/ 25; n.s.) (data not shown). Elevated zero-maze test Overall one-way ANOVAs show that prenatal MeHg exposure did not affect % TO (F⫽0.13, df⫽2/22; n.s.), HDIPS (F⫽0.14, df⫽2/22; n.s.) and SAP (F⫽0.9, df⫽2/22; n.s.) (data not shown). In vitro cortical cell cultures Basal and K⫹-evoked extracellular glutamate levels. In cortical cell cultures obtained from 1 day-old pups born from control mothers, basal extracellular glutamate levels evaluated as the mean of first two samples were 122⫾ 8.5 nM) and remained essentially stable over the duration of the experiment (five collected fractions; 150 min). As shown in Fig. 3A, extracellular glutamate levels were found significantly higher in cortical cell cultures of pups born from mothers exposed to MeHg at the dose of 8 mg/kg on both GDs 8 and 15 (F⫽4.94, df⫽2/123; P⬍0.01) than in those born from control mothers. A 20-min treatment with high K⫹ (20 mM) solution increased extracellular glutamate levels in all groups of cell cultures. However, the enhancement found in cultures of rats born from mothers exposed to MeHg at the dose of 8 mg/kg on both GDs 8 and 15 (F⫽9.32, df⫽2/38; P⬍0.01) was significantly lower than in those of rats born from control mothers (Fig. 3B). Effect of NMDA on basal extracellular glutamate levels To evaluate whether the prenatal exposure to MeHg affected cortical NMDA receptor function, the effects of NMDA on the endogenous extracellular glutamate levels were investigated. A 10-min treatment with NMDA (0.1 and 10 ␮M) solution increased extracellular glutamate levels in all groups of cell cultures. However, the enhancement found in cultures of rats born from mothers exposed to MeHg at the dose of 8 mg/kg on GD 8 was significantly higher than that observed in cell cultures of rats born from control mothers and further increased in cultures of rats born from mothers exposed to MeHg at the dose of 8 mg/kg on GD 15 (F⫽17.88, df⫽2/26; P⬍0.01; Fig. 4A and F⫽26.15, df⫽2/ 17; P⬍0.01; Fig. 4B).

Fig. 3. Effects of prenatal MeHg exposure on basal (A) and KClevoked (B) extracellular glutamate levels in cortical cell cultures. The MeHg groups represent cortical cell cultures obtained from pups born from mothers treated with MeHg at a dose of 8 mg/kg at GDs 8 and 15. The control group represents cortical cell cultures obtained from pups born from mothers treated with saline. Each value represents the mean⫾S.E.M. * P⬍0.05, ** P⬍0.01 significantly different from the respective control group (ANOVA followed by Newman-Keuls test for multiple comparisons).

DISCUSSION The present findings show that prenatal MeHg exposure induces, in rat offspring, subtle behavioral abnormalities at dose levels below those associated with overt symptoms of neurotoxicity or maternal toxicity. In this context, it is worth noting that children with evident neurological symptoms are born to asymptomatic mothers exposed to this neurotoxic compound.

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Fig. 4. Effects of prenatal exposure to MeHg on NMDA (0,1 ␮M, panel A; 10 ␮M, panel B) -evoked extracellular glutamate levels in cortical cell cultures. The MeHg groups represent cortical cell cultures obtained from pups born from mothers treated with MeHg at a dose of 8 mg/kg at GDs 8 and 15. The control group represents cortical cell cultures obtained from pups born from mothers treated with saline. Each value represents the mean⫾S.E.M. ** P⬍0.01 significantly different from the respective control group; °° P⬍0.01 significantly different from the respective MeHg at GD 8 group (ANOVA followed by Newman-Keuls test for multiple comparisons).

To the purpose of the present study, the behavior of MeHg-exposed offspring has been assessed by analyzing motor and cognitive functions together with other developmental, reproduction and neurochemical parameters. The results of the present study show that acute exposure to 8 mg/kg on both GDs 8 and 15 decreases significantly the survival of rat pups to the PND 21. A

significant reduction in pups’ weight gain was observed on PND 21 only following MeHg administration on GD 15, whereas no difference was found on PND 40. In this regard, it should be pointed out that high concentrations of MeHg can be detected in the offspring until PND 21 with a decline to the normal range on PND 60 (Cagiano et al., 1990), thus suggesting that a nutritional deficiency could be a factor in the offspring outcome. Accordingly, literature data show a significant increase in postnatal mortality and a reduction of body weight gain in mice exposed to MeHg during the gestational period (Choi, 1989; Doré et al., 2001). Percentage of successful pregnancies, duration of pregnancy, dam weight gain and litter size were not modified by the MeHg treatment. Furthermore, no difference in brain weight was found between control and MeHg-treated rats on PND 40, thus excluding that MeHg can induce gross alterations of brain growth. Several studies in rodents reported delayed or abnormal maturation of the cerebellum after developmental treatment with MeHg (Sager et al., 1984; Choi, 1989; Markowski et al., 1998). Therefore, possible cerebellar dysfunctions were assessed by analyzing the sensorimotor development (Altman and Sudarshan, 1975; Hua and Houk, 1997) and the performance on the rotarod (Carter et al., 1999). The results show that the onset of specific reflexes (righting reflex, cliff aversion, negative geotaxis) was not delayed in MeHg-exposed offspring (PND 2–12), thus excluding a gross impairment in the cerebellar function. The performance of MeHg-treated rats (PND 40) on the rotarod/accelerod task showed no signs of alteration in the motor coordination or motor learning ability. The results are consistent with literature data showing that prenatal or perinatal treatment with MeHg does not impair this neuromotor performance in mice offspring (Doré et al., 2001; Goulet et al., 2003). Since the rotarod is a very sensitive test in detecting motor function deficit (Karl et al., 2003), and since this task requires an unaltered cerebellar function and motor coordination (Carter et al., 1999), it can be further stated that the acute low-level exposure to MeHg during early or late gestational periods does not result in a gross impairment of cerebellar functions. The absence of motor deficits was also confirmed by using the open field paradigm. The performance of MeHg-treated rats (PND 40) in the open field test was not significantly different from that of the age-matched controls. No significant difference was found in the number of crossing and resting time between control and MeHg-treated rats. However, a significant reduction in the number of rearings was found in rats exposed to 8 mg/kg on both GDs 8 and 15, thus reflecting a possible impairment in the exploratory behavior. In this regard, it is worth noting that rearings are thought to represent an appropriate index of exploratory behavior involving cerebellar function (Caston et al., 1998; Dubois et al., 2002). Therefore, reduction in rearings could represent a functional consequence of very subtle alterations in the cerebellar circuitry. The results are consistent with other reports showing no impairment of locomotor behavior but a significant reduction in the number of rearings in adult rats exposed to MeHg (8 mg/kg) on GD 8 (Baraldi et al., 2002).

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Concerning the novel exploration object test, object recognition, which is assessed from the preference normal rats display for investigating novel rather than familiar objects, provides a valid and relatively pure measure of nonspatial working memory (Ennaceur and Delacour, 1988; Giustino et al., 1999). The present results show a marked difference in time spent exploring novel object between MeHg-treated and untreated rats. The lower exploratory activity of MeHg-exposed offspring could be partly attributed to an altered responsiveness to situations requiring adaptation to novel environmental stimuli. Specifically, the neophobia displayed by MeHg rats in the present experiments could be attributed to a higher degree of anxiety with respect to control ones, although such explanation does not seem to be plausible since no change in % TO, HDIPS and SAP was found in MeHg-exposed rats subjected to the elevated zero-maze test. Furthermore, the present experiments show lack of habituation (lower levels of exploratory activity during T2 compared with those found in T1) in MeHg-exposed rats with respect to control ones. Habituation failure exhibited by MeHg-treated offspring could be the consequence of altered mechanisms involved in attention and/or short-term memory. Further, control rats were able to discriminate between the novel and the familiar object, whereas MeHg offspring lost this discrimination capacity. As far as the possible relationship between the habituation failure and attentional dysfunctions is concerned, it is unlikely that prenatal MeHg exposure may affect the attentional processes, since inhibition of the response to the acoustic startling stimulus by a weak prestimulus (PPI) was not affected in MeHg-exposed rats. In this regard, it should be pointed out that deficits in PPI may measure attentional dysfunctions (Anisman et al., 2000), and therefore the habituation failure in MeHg-exposed rats is more likely due to altered mechanisms involved in short-term memory. Interestingly, the PPI is one of the few paradigms in which humans and rodents can be tested in a similar fashion, thus allowing a reliable extrapolation of experimental data to man. Neurobehavioral data show no difference between the two developmental windows. In this regard, previous studies (Lewandowski et al., 2002) have shown that the peak of mercury concentration after an acute exposure can be found in embryonic tissues within 48 h of dosing, suggesting a fairly rapid transfer of MeHg from the dam to the embryo. Therefore, in our experimental model, we can assume that the peak of MeHg concentration in embryos can be reached on GD10 after administration on GD8, and on GD17 after administration on GD15. Taking into account that neural tube formation is complete at approximately GD10.5–11 in rats (Rice and Barone, 2000), prenatal MeHg treatment either on GD8 or on GD15 would not impair neurulation and, therefore, would not cause any severe abnormalities. Moreover, by GD15, the first cells are beginning to arrive in the area that will ultimately form the laminae of the cortical plate, layers VI and V being readily distinguishable only on PND 5 (Rice and Barone, 2000). Therefore, on the basis of the above consider-

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ations, it is not surprising that MeHg exposure on GD8 and GD15 would cause similar effects on neurobehavioral parameters. Because multiple neurotransmitter systems modulate the behavioral endpoints affected by prenatal MeHg exposure, experiments have been carried out in order to explore the neurochemical mechanisms underlying these behavioral changes. The demonstration that higher MeHg concentrations have been found in the cerebral cortex during the first postnatal life period (Sakamoto and Nakano, 1995), suggests that this brain region could be especially susceptible to the neurotoxic effects of MeHg during gestation. The results of the present study provide new evidence that the exposure to MeHg during pregnancy affects the cortical glutamatergic neurotransmission and NMDA receptor function. In particular, basal extracellular glutamate levels measured in cortical cell cultures obtained from pups prenatally exposed to MeHg were higher than those measured in cortical cell cultures obtained from control pups. This increase of extracellular glutamate levels after prenatal MeHg treatment, is in agreement with recent studies that show an inhibition of glutamic acid transporters by MeHg, with consequent reduction of glutamate uptake (Aschner et al., 2000; Fonfria et al., 2005; Morken et al., 2005). The enhancement of extracellular concentration of glutamate will lead to a major sensitivity of neurons to excitotoxic injury. Furthermore, in view of the recent demonstration of behavioral deficits in adult rats treated neonatally with glutamate (Hlinak et al., 2005), it could be speculated that the increase in glutamate levels in pups born to mothers exposed to MeHg during pregnancy could be responsible for the behavioral impairments observed in the present study. Another relevant alteration in the function of glutamatergic signaling in cortical cells obtained from pups prenatally exposed to MeHg, was observed in the presence of an activation induced by a chemical depolarizing stimulus. In fact, the KCl-evoked increase of extracellular glutamate levels in the cortical cell cultures obtained from pups prenatally exposed to MeHg is compromised, since the effect of KCl in these cultures is less pronounced with respect to that observed in cortical cell cultures obtained from control pups. Working memory relies on the prefrontal cortex and evoked persistent synaptic activity in this area is mediated by a complex interplay of glutamatergic and GABAergic currents, with glutamatergic currents providing sustained depolarization (Seamans et al., 2003). In view of this, it could be speculated that the observed reduction in secretory activity of cortical glutamatergic transmission induced by prenatal MeHg exposure might be involved, at least in part in the cognitive deficit affecting the offspring. In view of the implication of the glutamatergic neurotransmission system in the neurological brain dysfunction associated with MeHg exposure, recent studies report that MeHg exposure changes the characteristics of NMDA receptors (Cagiano et al., 1990; Miyamoto et al., 2001; Baraldi et al., 2002), that are important for the toxic actions of glutamate. In fact, the substantial elevation in extracellular glutamate and, consequently, the excessive stimulation of glutamate receptors, espe-

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cially NMDA receptors, are involved in the neuronal cell death during degenerative processes. In the present study, an increased responsiveness of the NMDA receptors after prenatal MeHg exposure, has been observed. In fact, the NMDA-induced increase of extracellular glutamate levels observed in the cortical cell cultures obtained from pups prenatally exposed to MeHg is higher than that in cultures obtained from control pups. These results are in apparent contradiction with those demonstrating a less pronounced effect of KCl on glutamate levels in cell cultures obtained from pups prenatally exposed to MeHg, suggesting the involvement of a different mechanism that is not simply related to neuron depolarization. Thus, although other mechanisms cannot be excluded, it could be speculated that prenatal MeHg exposure induced an increase in the affinity of NMDA receptor for its agonist and/or changes in intracellular events that lead to glutamate efflux following receptor activation. This point remains to be elucidated in further studies. Finally, although in our cell culture preparation glial cell proliferation was inhibited by cytosine arabinoside to obtain a vast dominance of neuronal cells (90 –97%; Alho et al., 1988), it is not possible to exclude an involvement of astrocytes in the variation of glutamate release we observed in the present study. In fact, several observations indicate that astrocytes are targets for MeHg toxicity and that MeHg causes modifications in excitatory amino acid release and uptake in astrocytes (Aschner et al., 2000; Allen et al., 2002; Mutkus et al., 2005). The present results suggest that an enhanced sensitivity of NMDA receptors could make the developing cortical neurons more susceptible to MeHg neurotoxicity (Miyamoto et al., 2001). This hypothesis is further supported by recent data demonstrating that in adult rats prenatally exposed to MeHg (8 mg/kg), the cognitive deficits found at 60 days of age paralleled particularly an increase in mRNA levels of a NMDA receptor subunit (NR-2B) in the hippocampus (Baraldi et al., 2002). Furthermore, when MK801 (0.1 mg/kg), a non-competitive antagonist of NMDA receptors, is administered intraperitoneally with MeHg during brain development, MeHg-induced neurodegeneration is markedly ameliorated (Miyamoto et al., 2001). The observation that the increase in NMDA receptor responsivity is more evident following prenatal treatment with MeHg at the GD 15 suggests that in this period, NMDA cortical receptor seems to be more vulnerable to the action of the compound. Further experiments will be necessary to elucidate this point.

CONCLUSION In conclusion, the results point out that acute low-level exposure to MeHg during gestation induces subtle cognitive dysfunctions associated with an altered glutamate signaling pathway in the cerebral cortex, and further underline that consumption of MeHg-contaminated food by pregnant women poses one of the most serious potential hazards for the offspring. Indeed, the consequence of errors in developmental processes, such as impairment in working memory, becoming manifest at the end of adolescence could

have a relevant societal impact when amortized across the entire population and across the lifespan of humans (Rice and Barone, 2000), thus requiring attention to the neurobiological substrates and processes which may be perturbed by this environmental agent. Acknowledgments—This study was supported by grants from MIUR (PRIN-COFIN 2003) and “Fondo Ateneo” (2002, 2004). The authors thank “Fondazione Cassa di Risparmio di Ferrara,” Ferrara, Italy. Prof. Zoltan Annau and Prof. Mineshi Sakamoto are gratefully acknowledged for their helpful criticism.

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(Accepted 4 May 2006) (Available online 15 June 2006)