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Neurobehavioral changes in mice treated with methylmercury at two different stages of fetal development
Neurotoxicology and Teratology 23 (2001) 463 – 472
Neurobehavioral changes in mice treated with methylmercury at two different stages of fetal development F.Y. Dore´a,*, S. Gouleta, A. Gallaghera, P.-O. Harveya, J.-F. Cantina, T. D’Aigleb, M.-E. Miraultb Centre de Recherche Universite´ Laval Robert-Giffard and E´cole de Psychologie, Pavillon F.A. Savard, Universite´ Laval, Que´bec, Que´bec, Canada G1K 7P4 b Centre de Recherche du CHUL-CHUQ and Department of Medicine, Universite´ Laval, Que´bec, Que´bec, Canada
a
Received 6 March 2001; received in revised form 9 July 2001; accepted 12 July 2001
Abstract Pregnant C57BL/6 mice were orally given daily doses of 4 or 6 mg/kg of methylmercury chloride (MeHg) or vehicle during either gestational days 7 – 9 (GD7 – 9) or days 12 – 14 (GD12 – 14). Their female offspring were tested between 6 and 16 weeks of age on a variety of behavioral tasks. Motor coordination on the rotarod and visual discrimination learning in the Y maze were not affected by administration of MeHg either at GD7 – 9 or at GD12 – 14. In the open field, the total number of square crossings was lower in mice treated with 4 and 6 mg/kg of MeHg at GD12 – 14 than in control mice whether the environment was new or familiar, but prenatal administration of MeHg at GD7 – 9 had no effect on this measure. Administration of MeHg either at GD7 – 9 or at GD12 – 14 had no effect on the percentage of central square crossings or on the frequency of rearings in the open field. On spatial alternation training in the T maze, both treated groups in Condition GD7 – 9 and the group treated with 6 mg/kg at GD12 – 14 required more sessions to reach the learning criterion than their respective vehicle groups. When spatial alternation was tested with delays, treated groups did not differ from their respective control groups. In the radial arm maze, the performance of mice treated at GD7 – 9 was normal, but reference memory and working memory were impaired by administration of MeHg at GD12 – 14. In mice treated with 4 mg/kg of MeHg, reference memory was impaired only on the first block of trials, whereas in mice treated with 6 mg/kg, the deficit persisted on all blocks of trials. Overall, these results indicate that prenatal administration of MeHg at GD12 – 14 had more detrimental effects on behavioral performance than administration at GD7 – 9. It reduced locomotor activity and impaired reference memory for egocentric and allocentric spatial information as well as working memory for places. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Methylmercury; Prenatal exposure; Activity; Learning; Memory
1. Introduction The teratological and neurobehavioral effects of prenatal exposure to methylmercury (MeHg) on human and animal development have been well documented for decades (for reviews, see Refs. [3,7 –9,21,29,38]). These effects include reduced survival rate and weight gain, sensory and motor dysfunctions as well as learning and memory deficits. In mice, prenatal MeHg exposure tends to retard reflexive behavior in early development (Ref. [31], but see Ref. [36]), impairs motor coordination [13,34,36] and decreases spontaneous locomotor activity
* Corresponding author. Tel.: +1-418-656-2376; fax: +1-418656-3646. E-mail address: [email protected] (F.Y. Dore´).
[17,35]. Early experiments [34,35,38] reported longer latencies to explore the environment in the open field test and decreased [35,38] or increased [36] frequencies of rearings. In a recent experiment [19], less overall locomotor activity and more locomotion directed toward the center of the field were observed in this apparatus. Studies of learning and memory in mice showed that acquisition [16,36] and extinction [32,36] of avoidance responses were impaired by prenatal MeHg exposure and suggest that spatial long-term memory in the Morris water maze was also impaired [19]. Only a few studies [17,34] have compared the neurobehavioral consequences of repeated administrations of MeHg at different fetal stages of mice development. In the following experiment, we investigated neurobehavioral changes in female offspring of mice, which received vehicle or methylmercury chloride (MeHg) at one of two daily
0892-0362/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 6 7 - 2
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doses (4 or 6 mg/kg) and at one of two developmental windows, i.e., during days 7 –9 or days 12– 14 of gestation. Doses and treatment windows were selected on the basis of early [34,36] and more recent studies [17,19,38], as well as on the basis of neurodevelopmental landmarks. Until gestational day 12 (GD12), interference with cell proliferation can result in usual teratologies and gross defects of the central nervous system [30]. In the case of MeHg, the eighth or ninth day of gestation is especially important since it is the beginning of maximum susceptibility of the developing rodent brain to this neurotoxic agent [16,33]. From GD12 to birth, cell proliferation is marked by bursts of activity in the cerebellum, the thalamus, the striatum, the limbic structures and the cerebral cortex [18,30]. Therefore, brain damage can also occur after the critical period for malformations. Five behavioral tasks were used in our experiment. Acquisition of motor coordination and equilibrium was measured on the rotarod. In mice with cerebellar lesions [1,5,6] and in cerebellar mutant mice [14,22], fall latencies on the rotarod task have been reported to be shorter than in control mice. Activity and exploration were assessed in the open field both when the environment was new, as in most studies on developmental exposure to MeHg in rodents, and when it was becoming familiar. Although the precise neurobehavioral significance of activity in the open field is not well understood, it seems that limbic structures (amygdala, hippocampal formation and prelimbic cortex) modulate the behavioral response to novelty, whereas the nucleus accumbens mediates locomotor activity and exploration [4]. Performance was also examined in a variety of learning and memory tasks. A visual discrimination learning task was administered in the Y maze. Acquisition of a similar task was shown to be impaired in rats by selective lesions of the hippocampus [24]. Two tasks were used to test spatial learning and memory. One task was spatial alternation in the T maze, which requires the animal to choose the arm opposite to the one selected on the previous trial. Since the walls of the maze were opaque, no extramaze cues were available and the spatial alternation response could be learned by relying on egocentric information. Egocentric localization memory deficits have been reported in rodents after lesions of the caudate nucleus [10,28]. Training to spatial alternation was followed by testing with delays. Delayed spatial alternation and spatial working memory have been repeatedly shown to be impaired by frontal lesions [11,20,23,37]. The other spatial task involved place learning and memory in the radial arm maze. In this task, the maze is surrounded by a variety of extramaze stimuli, which are visible from different arms. To solve the problem, the animal relies on allocentric spatial information, i.e., information that is independent of the position of the animal. In our experiment, we used the reference working memory version of the radial arm maze task [26]. Shortterm and long-term retention for places are impaired on this task by hippocampal lesions [26] and by caudate lesions [27], respectively.
2. Materials and methods 2.1. Animals Mice of the C57BL/6 strain were obtained from a local breeder (Charles River, St. Constant, Que´bec, Canada). For mating, ninety-eight 11- to 12-week-old primigravid females were placed two per cage with one male breeder. GD1 was confirmed by the presence of a vaginal plug in the morning. The 79 females with vaginal plugs were placed into individual nesting cages and were assigned at random to two conditions. In Condition GD7 – 9, they were treated on GD7, GD8 and GD9, whereas in Condition GD12 –14, they were treated on GD12, GD13 and GD14. In each condition, plugged females received a dose of either 0 mg MeHg/kg body weight/day (GD7 – 9: n = 10; GD12 – 14: n = 12), 4 mg/kg (GD7 – 9: n = 12; GD12 – 14: n = 12) or 6 mg/kg (GD7 –9: n = 12; GD12 – 14: n = 21), for a cumulative dose of 0, 12 and 18 mg/kg, respectively. MeHg (Laboratoire MAT, Beauport, Que´bec, Canada) was diluted with sterile phosphate-buffered saline (PBS). MeHg or an equivalent volume of PBS was administered by peroral injection to treated and vehicle groups, respectively. Three females treated with 3 6 mg/kg of MeHg in Condition GD12 – 14 were sacrificed at GD15 and two were sacrificed at GD17; mercury levels were determined in pools of livers and pools of brains of fetuses from each of these females. The remaining females (n = 74) were checked every morning for the presence of newborns. They gave birth to 58 litters (GD7 – 9: 8, 10 and 9 litters treated with 0, 4 and 6 mg/kg of MeHg, respectively; GD12– 14: 11, 9 and 11 litters treated with 0, 4 and 6 mg/kg of MeHg, respectively). On the day of birth (GD19 or GD20), which was defined as postnatal day 1 (PND1), three litters treated with 3 6 mg/kg of MeHg and three vehicle litters in Condition GD12 – 14 were randomly selected. Pups from these litters were used for determinations of liver and brain levels of mercury according to a procedure similar to the one used with GD15 and GD17 fetuses, with the exception that separate pools were made for females and males, and only female pools were analyzed. In the remaining litters (n = 52), the average number of pups per litter was very similar across groups [Condition GD7– 9: 8.6 ( ± S.E.M. = 0.6) in Group 3 0 mg/kg; 9.0 ( ± S.E.M. = 0.4) in Group 3 4 mg/kg; and 8.1 ( ± S.E.M. = 0.7) in Group 3 6 mg/kg; Condition GD12 – 14: 8.9 ( ± S.E.M. = 1.0) in Group 3 0 mg/kg; 9.3 ( ± S.E.M. = 0.7) in Group 3 4 mg/kg; and 9.1 ( ± S.E.M. = 0.4) in Group 3 6 mg/kg]. These pups remained with their biological mothers until weaned at PND21. There was potential exposure to MeHg during nursing, but a previous study [16], which used a fostering procedure, showed that behavioral deficits were not significantly influenced by the treatment to the mother, which reared the pups, and were rather due to exposure of the mice in utero.
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At the age of 5 weeks, one female per litter was selected for behavioral testing. Each female pup was individually weighed and coded to make the experimenters blind to the nature of their treatment. Mice were maintained on a 12:12 h light – dark photoperiod and the tests were administered during the light phase of the cycle. Because food reinforcement (pieces of Fruit Loops cereals, Kellogg) was used in three of the five behavioral tasks, the normal daily food ration was restricted to reduce body weight to 85% of freefeeding level and therefore, mice had to be housed individually in standard plastic cages. In order to reduce body weight to 85% of free-feeding level while allowing growth, the weights of the mice under experiment were compared and adjusted to the average weight of mice of the same age (n = 15) receiving normal food ration. Water was provided ad libitum. Behavioral tests were administered 7 days/week. All mice of Groups 3 0 (n = 8), 3 4 (n = 10) and 3 6 mg/kg (n = 9) in Condition GD7 –9 and all mice of Groups 3 0 (n = 8) and 3 4 mg/kg (n = 9) in Condition GD12 – 14 completed the five tests. In Group 3 6 mg/kg of Condition GD12 –14 (n = 8), three mice had to be replaced by another female in the course of testing — one because of small weight and weakness and two because of injuries. 2.2. Mercury determinations For total mercury determinations, each tissue was digested in concentrated nitric acid prior to reduction of mercury to its metal state by stannous chloride and quantification of mercury vapour by UV [12]. A certified reference material (CRM) was used in the analytic process for quality control purposes. The CRM used was a homogenised dogfish liver tissue sample (DOLT-2-) obtained from the National Research Council of Canada. The obtained value for mercury was 2.2 mg/g. The certified value is 2.0 mg/g. The coefficient of variation for 50 determinations on different days was 4.9%. 2.3. Motor coordination on the rotarod At 6 weeks of age, the female offspring were tested on the rotarod. The balance rod was a 45-cm-long cylinder of 3 cm diameter wrapped with masking tape and suspended at the top and center of a wooden enclosure (35 45 115 cm). It was surrounded by three 30-cm-high black walls to prevent animals from climbing off the rod and was divided in the middle by a 30-cm-high vertical black screen, so that two mice could be tested simultaneously. The cylinder was connected to a DC motor (115 V, 33 A, 1/50 hp; Fisher Scientific Canada), which supplied power to a gear box as a function of electrical output from a variable transformer. The floor of the enclosure was covered with a 40-cm-thick cotton cushion to prevent any harm when the mice fell off the rod. Each daily session included five trials during which the mice were required to maintain balance on the rod for
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120 s. A fall occurring in the first 10 s of a trial was defined as a false start, which was a rare occurrence, and the trial was resumed 30 s later. Each trial was administered on squads of eight mice, so the intertrial interval was approximately 8 –10 min. Mice tested simultaneously were timed on two separate chronometers and if one of the two mice fell off the rotarod, only the chronometer for this mouse was restarted. On the first session, the mice were placed on the stationary rod (0 rpm) and on the second session, the rod rotated at a constant speed of 3 rpm. On the following four daily sessions, the rod rotated at a constant speed of 20 rpm. Fall latencies were recorded and results from the four sessions at 20 rpm were analyzed. 2.4. Spatial alternation training and delay testing in the T maze The mice were 7 weeks old at the beginning of the T-maze task, which was administered in two identical T mazes made of opaque acrylic with 30-cm-high walls and 10-cm-wide corridors. The stem was divided into a start box (30 cm) and a runway (50 cm). At the end of each choice arm (30 cm), food reward could be concealed in an opaque food cup. Black guillotine doors separated the start box and the choice arms from the runway. On the first day, the mice were familiarized to the apparatus. Several reinforcers were scattered on the stem, on the choice arms and in the food cups. The mouse was placed in the start box and was free to move in the maze while the experimenter periodically opened and closed the guillotine doors. It was allowed to explore the maze for a total of 10 min or until all the reinforcers had been consumed, whichever occurred first. Training to spatial alternation began the day after familiarization. Each daily session consisted of 11 trials. On the first trial (trial 0) of each session, both food cups in the choice arms were baited with a food reinforcer and the mouse was allowed to choose one arm. For the next 10 trials, the reinforcer was placed in the arm opposite to that chosen by the mouse on the previous trial. Criterion for ending training was eight successes out of 10 trials averaged over two consecutive days. A maximum of 30 sessions was administered. Once criterion was reached, delays of 30, 60 and 120 s were interposed between trials. Each delay was used for two consecutive days and then was increased to the next delay value. 2.5. Activity in the open field The mice were 10 weeks old at the beginning of testing in the open field. The floor of the apparatus measured 100 100 cm and was divided into 25 equal squares. The floor was surrounded by opaque acrylic 30-cm-high walls; squares adjacent to walls were referred to as periphery, and the nine remaining squares were referred to as center. On each of five consecutive days, each mouse was moved from its home cage and placed in the open field, facing the lower
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left corner. They were allowed to move freely for 15 min but data were taken only in the first and in the last 5 min. The following behavioral measures were recorded during these 10 min: number of peripheral and central square crossings and frequency of rearings. Total number of square crossings, percentage of central square crossings and frequency of rearings were averaged for the first session (new environment) and for the following four daily sessions (familiar environment). 2.6. Visual discrimination learning in the Y maze The mice were 11 weeks old at the beginning of this task. Two identical Y mazes made of opaque acrylic with 30-cm-high walls were used for visual discrimination learning. In each maze, the start box (10 30 cm) led to a stem choice area (20 15 cm), which led itself to two parallel runways (10 30 cm) with opaque food cups placed at their ends. Black guillotine doors separated the start box, the stem choice area and the runways. Each runway and half of the stem choice area leading to it were covered with a white or a black interchangeable acrylic floor insert (10 45 cm). The floor inserts were thus visible to the mouse as soon as it emerged from the start box. On each trial, the runway covered with the black insert was baited (S+), whereas the runway covered with the white insert was not (S ). The left and right positions of the inserts were determined by a Gellerman pseudorandom sequence. Each daily session consisted of 20 trials separated by a 10-s intertrial interval. On the first two sessions, a correction procedure was used: if the mouse chose the S , an error was recorded and the trial was immediately repeated with only the S+ runway opened (forced successful choice). On the following sessions, no correction procedure was used and each trial was administered only once. Criterion for completion of the task was 17 successes out of 20 trials averaged over two consecutive days. 2.7. Spatial working reference memory in the radial arm maze The mice were 14 weeks old at the beginning of this task. The eight-arm radial maze was made of wood and painted flat gray. The maze was 60 cm from the floor. Each arm was 60 cm long and 9 cm wide. A recessed food well was located at the end of each arm. The center platform was 40 cm in diameter and was surrounded by a 40-cm-high wooden wall. The entrance to each arm was blocked by a gray acrylic guillotine door. The maze was centrally located in a room providing a number of large objects (a window, a poster, a vertical grid, etc.) and surrounded on two sides by 160-cm-high folding screens decorated with posters. The animals’ behavior was monitored with a video camera that was suspended over the maze. The camera was connected to a video monitor that was located outside the folding screens where the experimenter raised and lowered the
guillotine doors by pulling on strings in order to provide access to the radial arms. The working reference memory version of the radial arm maze task was used [26]. In this version, half of the arms, always the same, is baited on each daily trial and the other half is unbaited. As the animal learns the rules of the task and/or features which are constant from trial to trial (reference or long-term memory), it becomes able to discriminate baited from unbaited arms and avoids entering unbaited arms. It also becomes able to avoid baited arms it has already visited within a trial (working or short-term memory). The first five daily trials served to familiarize the animals with the apparatus. On the first day of familiarization, several reinforcers were scattered on the central platform and on four of the eight arms. The mouse was placed on the central platform and was free to move in the maze for 5 min while the experimenter periodically opened and closed the guillotine doors. If the reinforcers in the four baited arms were not found and consumed in the first 5 min, the mouse was placed at the end of each baited arm and had to walk to the central platform; then, from the central platform, it was allowed to explore the maze for 5 min. On the following 4 days of familiarization, four baited arms were randomly selected each day and the number of reinforcers was gradually reduced until only the food wells of the four selected arms were baited. Training began the day after the last familiarization session. There was one daily trial for 15 consecutive days. On each trial, only four arms, always the same so they could be maintained in reference memory, were baited. At the beginning of a trial, the mouse was placed on the central platform with all guillotine doors closed. After 10 s, the doors were opened and the mouse was allowed to choose an arm. When the mouse returned to the central platform, the doors were closed and a 10-s waiting period began. After the waiting period, all the doors were opened and the mouse was allowed again to choose an arm. This procedure continued until one of the following criteria was reached, whichever occurred first: the four baited arms were visited, 16 choices were made or 10 min had elapsed. A reference memory error was recorded each time the mouse visited for the first time one of the four never baited arms and a working memory error was recorded each time it revisited an arm within a trial, whether this arm was originally baited or not. 2.8. Statistical analysis Within each condition (GD7 – 9 or GD12 – 14), results of Groups 3 0, 3 4 and 3 6 mg/kg were subjected to ANOVAs with Group as a between-subject factor. On the rotarod and delayed spatial alternation tasks, Session and Delay were within-subject factors, respectively. Analyses of simple main effects of the interaction (with Satterthwaite’s correction for the error term and its degrees of freedom [15]) and Newman– Keuls tests served to locate specific signific-
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ant effects. On the radial arm maze task, the total numbers of correct choices, of reference memory errors and of working memory errors made by mice in Condition GD7 –9 were also subjected to an ANOVA with Group as a betweensubject factor and Block of five daily trials as a withinsubject factor. As for Condition GD12 – 14, it appeared that mice exposed to MeHg made fewer reference memory errors, but also fewer choices, than control mice. Since the total number of first choices made in 10 min differed in treated and control mice, the measures in the radial arm maze had to be subjected to special analyses. In each group of Condition GD12 –14, the numbers of visits to baited arms (correct choices) and to unbaited arms (reference memory errors) were compared by an ANOVA with Type of arm and Block of five trials as within-subject factors; the numbers of working memory errors in the first and third blocks of five trials were compared by a unilateral paired t test.
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age of 5 weeks in mice treated with 3 6 mg/kg of MeHg at GD7 – 9 and at GD12 –14. 3.2. Motor coordination on the rotarod task In the first 2 days of testing, all mice were able to maintain balance on the rod in at least four of the five trials when the rod was stationary or rotated at a speed of 3 rpm. In the following four daily sessions, when the rod rotated at a constant speed of 20 rpm (Fig. 1), fall latencies increased across sessions in all groups and thus, there was some learning of motor coordination. However, prenatal exposure to MeHg had no effect on motor learning. In Condition GD7 – 9 [ F(3, 72) = 14.73, P < .0001] and in Condition GD12 – 14 [ F(3, 66) = 19.49, P < .0001], the factor Session was significant but the factor Group [Condition GD7 – 9: F(2, 24) = 1.09; Condition GD12 –14: F(2, 22) = 1.25] and the interaction were not significant [Condition GD7 – 9: F(6, 72) = 0.59; Condition GD12 – 14: F(6, 66) = 0.58].
3. Results 3.1. Liver and brain levels of Hg and percentages of survival at the age of 5 weeks At GD15, GD17 and at birth, liver levels of mercury in mice treated with 6 mg/kg of MeHg at GD12 –14 were 12.70 ( ± S.E.M. = 1.79), 15.67 ( ± S.E.M. = 0.27) and 15.76 mg/g ( ± S.E.M. = 0.54), respectively, whereas brain levels were 16.74 ( ± S.E.M.=.1.56), 13.26 ( ± S.E.M. = 3.76) and 12.09 mg/g ( ± S.E.M. = 0.89). In control mice, liver and brain levels of mercury at birth were 0.17 ( ± S.E.M. = 0.003) and 0.13 mg/g ( ± S.E.M. = 0.01), respectively. Although liver and brain levels of mercury were measured in a small number of mice and only in Condition GD12 – 14, it is clear that they were very high shortly after treatment (GD15 and GD17) as well as at birth [3]. In each litter, the number of offspring which survived to the age of 5 weeks, i.e., 1 week before the beginning of behavioral testing, was divided by the number of offspring at birth and the result was multiplied by 100 to give the percentage of survival. In Condition GD7 –9, the percentages of survival in Groups 3 0, 3 4 and 3 6 mg/kg were 90.3% ( ± S.E.M. = 3.4), 86.0% ( ± S.E.M. = 3.8) and 61.8% ( ± S.E.M. = 11.9), respectively. These percentages significantly differed [ F(2, 24) = 4.12, P < .05] and the Newman – Keuls test ( P < .05) showed that the percentage of survival in Group 3 6 mg/kg was significantly lower than in Groups 3 0 and 3 4 mg/kg. In Condition GD12 –14, the percentages of survival were 86.5% ( ± S.E.M. = 5.6) for Group 3 0 mg/kg, 68.7% ( ± S.E.M. = 11.2) for Group 3 4 mg/kg and 60.5% ( ± S.E.M. = 7.4) for Group 3 6 mg/kg. These percentages also significantly differed [ F(2, 22) = 11.34, P < .01] and the Newman– Keuls test ( P < .01) showed that the percentage of survival in Group 3 6 mg/kg was significantly lower than in Group 3 0 mg/kg. Thus, prenatal exposure to MeHg reduced the percentage of survival at the
3.3. Spatial alternation training and delay testing in the T maze Prenatal exposure to MeHg impaired training to spatial alternation in the T maze (Fig. 2A). The number of sessions to reach the learning criterion [ F(2, 24) = 3.93, P < .05] significantly differed in the groups of Condition GD7 – 9. The Newman– Keuls test ( P < .05) showed that mice treated with 4 and 6 mg/kg of MeHg required more sessions than control mice. In Condition GD12 –14, there was also a
Fig. 1. Fall latencies on the rotarod task. Data points are group means ± S.E.M. for four consecutive daily sessions. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Data of each condition were analyzed by ANOVA with Group as a between-subject factor and Sessions (1 – 4) as a within-subject factor. The factor Group [Condition GD7 – 9: F(2, 24) = 1.09; Condition GD12 – 14: F(2, 22) = 1.25] and its interaction with the factor Session [Condition GD7 – 9: F(6, 72) = 0.59; Condition GD12 – 14: F(6, 66) = 0.58] were not significant, but the factor Session was significant [Condition GD7 – 9: F(3, 72) = 14.73, P < .0001; Condition GD12 – 14: F(3, 66) = 19.49, P < .0001].
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all mice were able to produce 80% correct responses, but in Condition GD7 – 9, 4/10 mice treated with 3 4 mg/kg of MeHg and 2/9 mice treated with 3 6 mg/kg failed to attain this level of performance within the maximum of 30 sessions. Those mice were not tested in the delayed spatial alternation test. When delays of 30, 60 and 120 s were interposed between trials, spatial alternation performance deteriorated compared to the last two sessions of training (Fig. 2B), but it was not impaired by prenatal administration of MeHg at GD7– 9 or GD12 – 14. In both conditions, the factor Delay had a significant effect [Condition GD7 – 9: F(3, 54) = 22.77, P < .0001; Condition GD12 – 14: F(3, 66) = 18.33, P < .001], whereas the factor Group [Condition GD7 – 9: F(2, 18) = 0.10; Condition GD12 –14: F(2, 22) = 0.16] and the interaction [Condition GD7 –9: F(6, 54) = 0.55; Condition GD12 – 14: F(6, 66) = 0.52] were not significant. However, differences between treated and control mice might have been masked by a floor effect because at a 30-s delay, the number of correct choices was already between 55% and 70%. 3.4. Activity in the open field
Fig. 2. Training and delay testing on spatial alternation in the T maze. (A) Number of sessions to reach criterion. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Each histogram represents the mean ± S.E.M. Data of each condition were analyzed by a one-way ANOVA followed by a posteriori comparisons with the Newman – Keuls test. In Condition GD7 – 9, the factor Group was significant [ F(2, 24) = 3.93, P < .05]: mice treated with 4 and 6 mg/kg of MeHg required more sessions to reach criterion than control mice ( P < .05). In Condition GD12 – 14, the factor Group was also significant [ F(2, 22) = 4.17, P < .05] and Group 3 6 mg/kg required more sessions to reach criterion than Groups 3 0 and 3 4 mg/kg ( P < .05). (B) Frequency of correct choices in delay testing. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 6); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Data points are group means ± S.E.M. for each delay (0-s delay: data from the two criterion sessions). Data of each condition were analyzed by ANOVA with Group as a betweensubject factor and Delay as a within-subject factor. The factor Group [Condition GD7 – 9: F(2, 18) = 0.10; Condition GD12 – 14: F(2, 22) = 0.16] and the interaction [Condition GD7 – 9: F(6, 54) = 0.55; Condition GD12 – 14: F(6, 66) = 0.52] were not significant, but the factor Delay was significant [Condition GD7 – 9: F(3, 54) = 22.77, P < .0001; Condition GD12 – 14: F(3, 66) = 18.33, P < .001].
significant difference between groups in the number of sessions to reach criterion [ F(2, 22) = 4.17, P < .05] and the Newman– Keuls test ( P < .05) revealed that Group 3 6 mg/kg required more sessions to reach criterion than Groups 3 0 and 3 4 mg/kg. In Condition GD12 –14,
Fig. 3A presents the total number of square crossings in the open field during the 10 min of recording in the first session and in the following four sessions. Only mice treated with MeHg at GD12 –14 were significantly hypoactive. When the environment was new (Session 1), the groups in Condition GD7 – 9 did not differ [ F(2, 24) = 0.60], but the groups in Condition GD12 –14 did [ F(2, 22) = 5.98, P < .01]: Groups 3 6 and Group 3 4 mg/kg crossed significantly fewer squares than Group 3 0 mg/kg (Newman –Keuls at P < .01). In Sessions 2– 5, when the environment was becoming familiar, similar results were observed. The groups in Condition GD7 – 9 did not differ [ F(2, 24) = 0.16], but the groups in Condition GD12 – 14 did [ F(2, 22) = 9.01, P < .005]: Groups 3 4 and 3 6 mg/kg crossed significantly fewer squares than Group 3 0 mg/kg (Newman –Keuls at P < .01). Fig. 3B presents the percentage of central square crossings in 10 min. If this behavioral measure is an index of fear or emotionality as it has frequently been described [19], there was no evidence of altered function in mice with prenatal exposure to MeHg. In both conditions, the groups did not differ whether the environment was new [Session 1: Condition GD7 – 9: F(2, 24) = 0.51; Condition GD12 – 14: F(2, 22) = 0.11] or familiar [Sessions 2 – 5: Condition GD7 – 9: F(2, 24) = 0.57; Condition GD12 – 14: F(2, 22) = 0.97]. Finally, Fig. 3C presents the frequency of rearings in the open field. Again, in both conditions, the groups did not differ whether the environment was new [Session 1: Condition GD7 – 9: F(2, 24) = 0.55; Condition GD12 – 14: F(2, 22) = 0.02] or familiar [Sessions 2 – 5: Condition GD7 – 9: F(2, 24) = 0.39; Condition GD12 – 14: F(2, 22) = 0.82].
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3.5. Visual discrimination learning in the Y maze Prenatal exposure to MeHg had no effect on learning the visual discrimination in the Y maze (Fig. 4). The number of sessions to reach criterion did not significantly differ in the three groups of mice either in Condition GD7 –9 [ F(2, 24) = 1.29] or in Condition GD12 –14 [ F(2, 22) = 2.33]. For an unknown reason, all groups in Condition GD12– 14 seemed to require more sessions than groups in Condition GD7 –9. 3.6. Spatial working-reference memory in the radial arm maze In Condition GD7 – 9 (Fig. 5A), the performance in the radial maze was similar in treated and control mice. The frequency of correct choices was high and there was no significant difference between groups [Group: F(2. 24) = 0.02; Group Block: F(4, 48) = 0.07] or across blocks of trials [ F(2, 48) = 0.00]. The frequency of reference memory errors significantly decreased across blocks of trials [ F(2, 48) = 5.01, P < .01], but there was no significant difference between groups [Group: F(2, 24) = 0.13; Group Block: F(4, 48) = 0.29]. Similarly, the frequency of working memory errors significantly decreased across blocks [ F(2, 48) = 3.85, P < .05] and there was no significant difference between groups [Group: F(2, 24) = 1.57; Group Block: F(4, 48) = 0.32]. In Condition GD12 – 14, the results on the radial arm maze task were more complex. In the course of testing, it became clear that mice of Groups 3 4 and 3 6 mg/kg behaved differently than control mice of the same condition (Fig. 5B): a trial had frequently to be ended after the 10-min criterion was reached, before the four baited arms were
Fig. 3. Behavior in the open field. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Each histogram represents the mean ± S.E.M. Data of each condition were analyzed by a one-way ANOVA followed by a Newman – Keuls test. (A) Total number of square crossings during the 10-min of recording when the environment was new (Session 1) and familiar (Session 2 – 5). In Session 1, the groups did not differ in Condition GD7 – 9 [ F(2, 24) = 0.60], but they significantly differed in Condition GD12 – 14 [ F(2, 22) = 5.98, P < .01], with Group 3 0 mg/kg crossing more squares than Group 3 6 mg/kg ( P < .01). In Sessions 2 – 5, the groups did not differ in Condition GD7 – 9 [ F(2, 24) = 0.16], but they significantly differed in Condition GD12 – 14 [ F(2, 22) = 9.01, P < .005], with Group 3 0 mg/kg crossing more squares than Groups 3 4 and 3 6 mg/kg ( P < .01). (B) Percentage of central square crossings when the environment was new (Session 1) and familiar (Session 2 – 5). The groups in both conditions did not differ in Session 1 [Condition GD7 – 9: F(2, 24) = 0.51; Condition GD12 – 14: F(2, 22) = 0.11] or in Sessions 2 – 5 [Condition GD7 – 9: F(2, 24) = 0.57; Condition GD12 – 14: F(2, 22) = 0.97]. (C) Frequency of rearings when the environment was new (Session 1) and familiar (Session 2 – 5). The groups in both conditions did not differ in Session 1 [Condition GD7 – 9: F(2, 24) = 0.55; Condition GD12 – 14: F(2, 22) = 0.02] or in Sessions 2 – 5 [Condition GD7 – 9: F(2, 24) = 0.39; Condition GD12 – 14: F(2, 22) = 0.82].
Fig. 4. Number of sessions to reach criterion on the visual discrimination in the Y maze. Each histogram represents the mean ± S.E.M. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Data of each condition were analyzed by a one-way ANOVA. In Conditions GD7 – 9 [ F(2, 24) = 1.29] and GD12 – 14 [ F(2, 22) = 2.33], the groups did not differ.
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visited and before 16 choices were made. ANOVAs made on the overall results of the 15 trials revealed that although the groups did not differ in terms of working memory errors [ F(2, 22) = 1.72], they significantly differed in terms of correct choices [ F(2, 22) = 4.45, P < .05] and reference memory errors [ F(2, 22) = 5. 29, P < .05]. Newman –Keuls tests ( P < .05) showed that Groups 3 4 and 3 6 mg/kg made fewer reference memory errors but they also made fewer correct choices. Because the number of first entries into arms was lower in prenatally exposed groups than in control mice, direct intergroup comparisons were not possible. An intragroup strategy of analysis was adopted. Long-term retention or reference memory of the rules and/ or constant features of the task implies that the animal gradually learned to discriminate baited and unbaited arms. Thus, reference memory was evaluated, within each group, by comparing the number of visits to baited arms (correct choices) and the number of visits to unbaited arms (reference memory errors). An ANOVA with Type of arm and Block of five trials as within-subject factors was used for these comparisons. In Group 3 0 mg/kg, the factor Type of arm was significant [ F(1, 7) = 34.97, P < .001], and the factor Block [ F(2, 14) = 1.43] and the interaction [ F(2, 14) = 2.78] were not significant. Thus, control mice visited more baited
arms than unbaited arms on all three blocks of trials. Treatment with MeHg in Condition GD12 – 14 impaired discrimination of baited and unbaited arms and this impairment was more severe at the highest dose. In Group 3 4 mg/kg, the factor Type of arm [ F(1, 8) = 29.07, P < .001] and the interaction [ F(2, 16) = 5.61, P < .02] were significant, but the factor Block [ F(2, 16) = 0.19] was not significant. The analysis of simple main effects showed that in Group 3 4 mg/kg, more visits were made to baited arms than to unbaited arms on the second [ F(1, 16) = 15.11, P < .002] and on the third blocks of trials [ F(1, 16) = 44.20, P < .0001], but not on the first block [ F(1, 16) = 3.02]. In Group 3 6 mg/kg, the frequency of visits to baited and unbaited arms did not differ on any block: the factors Type of arm [ F(1, 7) = 3.25] and Block [ F(2, 14) = 1.85], as well as the interaction [ F(2, 14) = 0.11], were not significant. Whereas Group 3 4 mg/ kg was initially slower to learn the rules and/or constant features of the task than the vehicle group, Group 3 6 mg/ kg did not discriminate baited and unbaited arms on any of the three blocks of five trials. In Condition GD12 – 14, working memory errors were analyzed by comparing, within each group, the numbers of reentries on the first and third blocks of trials. In normal
Fig. 5. Frequency of correct choices (visits to baited arms), of reference memory errors (first visits to unbaited arms) and working memory errors (reentries in baited or unbaited arms) in the radial arm maze. Data points are group means ± S.E.M. for each block of five trials. (A) Data of Condition GD7 – 9. Number of mice in each group: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9). Data were analyzed by ANOVAs with Group as a between-subject factor and Blocks of five trials as a within-subject factor. There was no significant difference between groups or across blocks of trials in the number of correct choices [Group: F(2, 24) = 0.02; Block: F(2, 48) = 0.00; Group Block: F(4, 48) = 0.07]. The frequency of reference memory errors significantly decreased [ F(2, 48) = 5.01, P < .01] and there was no significant difference between groups [Group: F(2, 24) = 0.13; Group Block: F(4, 48) = 0.29]. The frequency of working memory errors significantly decreased [ F(2, 48) = 3.85. P < .05] and there was no significant difference between groups [Group: F(2, 24) = 1.57; Group Block: F(4, 48) = 0.32]. (B) Data of Condition GD12 – 14. Number of mice in each group: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). In order to compare correct choices and reference memory errors, data of each group were analyzed by ANOVAs with Type of arm and Block of five trials as a within-subject factors. In Group 3 0 mg/kg, the factor Type of arm was significant [ F(1, 7) = 34.97, P < .001], and the factor Block [ F(2. 14) = 1.43] and the interaction [ F(2, 14) = 2.78] were not significant. In Group 3 4 mg/kg, the factor Type of arm [ F(1, 8) = 29.07, P < .001] and the interaction [ F(2, 16) = 5.61, P < .02] were significant, but the factor Block [ F(2, 16) = 0.19] was not significant. The analysis of simple main effects showed that in Group 3 4 mg/kg, more visits were made to baited arms than to unbaited arms on the second [ F(1, 16) = 15.11. P < .002] and third blocks [ F(1, 18) = 44.20, P < .0001], but not on the first block [ F(1, 16) = 3.02]. Finally, in Group 3 6 mg/kg, the frequency of visits to baited and unbaited arms did not differ on any block because the factor Type of arm [ F(1, 7) = 3.25], Block [ F(2, 14) = 1.85] and the interaction [ F(2, 14) = 0.11] were not significant. For each group, the frequencies of working memory errors on the first and third blocks of trials were compared by a unilateral Student’s t test for paired samples. The frequency of working memory errors significantly decreased in Group 3 0 mg/kg [t (7) = 1.91, P < .05], but not in Group 3 4 mg/kg [t(8) = 0.46] or in Group 3 6 mg/kg [t(7) = 0.40].
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animals, these errors usually decrease across trials as they learn to avoid revisiting an arm. In fact, the number of working memory errors significantly decreased in Group 3 0 mg/kg [t (7) = 1.91, P < .05], but not in Groups 3 4 [t (8) = 0.46] and 3 6 mg/kg [t (7) = 0.40].
4. Discussion The mercury determinations in brain and liver of mice treated at GD12 – 14 indicate that high exposure to MeHg lasted from very shortly after treatment until birth. A weakness of the present experiment is that mercury levels were not measured in mice treated at GD7 –9. However, an indication of the effects of administration of MeHg in this condition is that, as in Condition GD12– 14, survival to the postnatal age of 5 weeks was significantly decreased by administration of 6 mg/kg of MeHg. Observations made during the experiment did not reveal any obvious sign of motor deficit and testing on the rotarod did not show any impairment in learning motor coordination, even in mice treated with 3 6 mg/kg. Previous studies on rodents did find gross impairments of motor function after prenatal exposure to MeHg, but these studies examined reflexive behavior in early development [25] or swimming ability [13,34,36]. On the other hand, our results in the open field are consistent with early [34,35,38] and with more recent [19] reports, which showed that prenatal exposure to MeHg significantly decreases locomotor activity. In our experiment, this effect was observed only in mice treated at GD12 – 14 and frequency of rearings was not affected. Contrary to the results of Kim et al., there was no evidence that hypoactivity in the open field was related to fear or to changes in emotional status: the percentage of central square crossings in treated mice and control mice did not differ. Hypoactivity in mice treated with MeHg at GD12 – 14 was not specific to novelty and also appeared when the open field was familiar. This result suggests that the decrease in locomotor activity was related to damage to the nucleus accumbens rather than to limbic structures [4]. In order to discriminate the black and white floors in the Y maze, to acquire the spatial alternation response in the T maze and to discriminate baited and unbaited arms in the radial maze, mice had to learn the basic rules and/or constant features of the task. Thus, the three tasks required intact reference memory or long-term retention of either an individual cue (black floor in the visual discrimination task) or of spatial information (T maze and radial maze). In all mice treated with MeHg, learning on the visual discrimination task was normal and this result is consistent with previous experiments on rodents [2,13]. On spatial tasks, reference memory was impaired by prenatal exposure to MeHg whether the task involved egocentric or allocentric information. However, the effects of treatment windows on the two spatial tasks differed.
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On spatial alternation training, both treated groups of Condition GD7 – 9 required more sessions to reach the learning criterion and in Condition GD12 – 14, only Group 6 mg/kg was impaired. This result suggests that reference memory for egocentric spatial information was affected by a lower dose of MeHg when treatment occurred at GD7 –9 than at GD12 – 14. In the radial arm maze, reference memory for allocentric spatial information was impaired only in Condition GD12 –14 and this impairment was more severe after prenatal exposure to the highest dose. Whereas discrimination of baited and unbaited arms was impaired on all three blocks after prenatal exposure to 6 mg/kg, it was impaired only on the first block of trials in mice exposed to 4 mg/kg. Reference memory for egocentric spatial information [10,28] and reference memory for allocentric spatial information [27] have been both associated with the function of the caudate nucleus and yet, exposure to MeHg at two fetal developmental stages had different effects on spatial alternation training and on the radial arm maze task. One possible explanation is that treatment at GD7 –9 and GD12 –14 altered the function of different neurotransmitter systems in the caudate nucleus. Delay testing of spatial alternation in the T maze suggests that working memory for egocentric spatial information and frontal function were not affected by prenatal exposure to MeHg. However, this conclusion can only be tentative. First, working memory errors on this task were already low at the shortest delay and the possibility of a floor effect cannot be definitely excluded. Second, in Condition GD7– 9, some treated mice did not reach criterion and could not be tested with delays although their performance on this task was probably more affected by exposure to MeHg than in Condition GD12 – 14. In contrast, the analysis of reentries in the radial arm maze revealed that in Condition GD12 – 14, but not in Condition GD7 –9, working memory errors did not decrease between the first and the third blocks of trials in mice prenatally exposed to either dose of MeHg. Thus, working memory for places was impaired and this deficit is usually associated with dysfunctions of the hippocampus and/or adjacent entorhinal cortex. In summary, treatment with MeHg at two different stages of fetal development had different effects. In mice treated with MeHg at GD7 – 9, reference memory for egocentric spatial information was impaired, whereas in mice treated with MeHg at GD12 – 14, a variety of neurobehavioral functions were altered. Damages to the ventral striatum, the dorsal striatum and the hippocampal formation are suggested by the hypoactivity observed in the open field, by reference memory deficits on spatial alternation training and on the radial maze task, and by working memory deficits in the radial maze, respectively.
Acknowledgments The authors thank Mr. Alain Tremblay and Ms. Micheline Noel for their excellent technical assistance. This
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research was supported by funds from Fonds de Recherche en Sante´ du Que´bec (FRSQ)/Hydro-Que´bec (Programme de recherche en sante´ de l’enfant) and by Health Canada (Programme Saint-Laurent Vision 2000). The research received approval from the Comite´ de protection des animaux de laboratoire de l’Universite´ Laval, which is responsible for the application and enforcement of the rules of the Canadian Council on Animal Care.
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Neurobehavioral changes in mice treated with methylmercury at two different stages of fetal development F.Y. Dore´a,*, S. Gouleta, A. Gallaghera, P.-O. Harveya, J.-F. Cantina, T. D’Aigleb, M.-E. Miraultb Centre de Recherche Universite´ Laval Robert-Giffard and E´cole de Psychologie, Pavillon F.A. Savard, Universite´ Laval, Que´bec, Que´bec, Canada G1K 7P4 b Centre de Recherche du CHUL-CHUQ and Department of Medicine, Universite´ Laval, Que´bec, Que´bec, Canada
a
Received 6 March 2001; received in revised form 9 July 2001; accepted 12 July 2001
Abstract Pregnant C57BL/6 mice were orally given daily doses of 4 or 6 mg/kg of methylmercury chloride (MeHg) or vehicle during either gestational days 7 – 9 (GD7 – 9) or days 12 – 14 (GD12 – 14). Their female offspring were tested between 6 and 16 weeks of age on a variety of behavioral tasks. Motor coordination on the rotarod and visual discrimination learning in the Y maze were not affected by administration of MeHg either at GD7 – 9 or at GD12 – 14. In the open field, the total number of square crossings was lower in mice treated with 4 and 6 mg/kg of MeHg at GD12 – 14 than in control mice whether the environment was new or familiar, but prenatal administration of MeHg at GD7 – 9 had no effect on this measure. Administration of MeHg either at GD7 – 9 or at GD12 – 14 had no effect on the percentage of central square crossings or on the frequency of rearings in the open field. On spatial alternation training in the T maze, both treated groups in Condition GD7 – 9 and the group treated with 6 mg/kg at GD12 – 14 required more sessions to reach the learning criterion than their respective vehicle groups. When spatial alternation was tested with delays, treated groups did not differ from their respective control groups. In the radial arm maze, the performance of mice treated at GD7 – 9 was normal, but reference memory and working memory were impaired by administration of MeHg at GD12 – 14. In mice treated with 4 mg/kg of MeHg, reference memory was impaired only on the first block of trials, whereas in mice treated with 6 mg/kg, the deficit persisted on all blocks of trials. Overall, these results indicate that prenatal administration of MeHg at GD12 – 14 had more detrimental effects on behavioral performance than administration at GD7 – 9. It reduced locomotor activity and impaired reference memory for egocentric and allocentric spatial information as well as working memory for places. D 2001 Elsevier Science Inc. All rights reserved. Keywords: Methylmercury; Prenatal exposure; Activity; Learning; Memory
1. Introduction The teratological and neurobehavioral effects of prenatal exposure to methylmercury (MeHg) on human and animal development have been well documented for decades (for reviews, see Refs. [3,7 –9,21,29,38]). These effects include reduced survival rate and weight gain, sensory and motor dysfunctions as well as learning and memory deficits. In mice, prenatal MeHg exposure tends to retard reflexive behavior in early development (Ref. [31], but see Ref. [36]), impairs motor coordination [13,34,36] and decreases spontaneous locomotor activity
* Corresponding author. Tel.: +1-418-656-2376; fax: +1-418656-3646. E-mail address: [email protected] (F.Y. Dore´).
[17,35]. Early experiments [34,35,38] reported longer latencies to explore the environment in the open field test and decreased [35,38] or increased [36] frequencies of rearings. In a recent experiment [19], less overall locomotor activity and more locomotion directed toward the center of the field were observed in this apparatus. Studies of learning and memory in mice showed that acquisition [16,36] and extinction [32,36] of avoidance responses were impaired by prenatal MeHg exposure and suggest that spatial long-term memory in the Morris water maze was also impaired [19]. Only a few studies [17,34] have compared the neurobehavioral consequences of repeated administrations of MeHg at different fetal stages of mice development. In the following experiment, we investigated neurobehavioral changes in female offspring of mice, which received vehicle or methylmercury chloride (MeHg) at one of two daily
0892-0362/01/$ – see front matter D 2001 Elsevier Science Inc. All rights reserved. PII: S 0 8 9 2 - 0 3 6 2 ( 0 1 ) 0 0 1 6 7 - 2
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doses (4 or 6 mg/kg) and at one of two developmental windows, i.e., during days 7 –9 or days 12– 14 of gestation. Doses and treatment windows were selected on the basis of early [34,36] and more recent studies [17,19,38], as well as on the basis of neurodevelopmental landmarks. Until gestational day 12 (GD12), interference with cell proliferation can result in usual teratologies and gross defects of the central nervous system [30]. In the case of MeHg, the eighth or ninth day of gestation is especially important since it is the beginning of maximum susceptibility of the developing rodent brain to this neurotoxic agent [16,33]. From GD12 to birth, cell proliferation is marked by bursts of activity in the cerebellum, the thalamus, the striatum, the limbic structures and the cerebral cortex [18,30]. Therefore, brain damage can also occur after the critical period for malformations. Five behavioral tasks were used in our experiment. Acquisition of motor coordination and equilibrium was measured on the rotarod. In mice with cerebellar lesions [1,5,6] and in cerebellar mutant mice [14,22], fall latencies on the rotarod task have been reported to be shorter than in control mice. Activity and exploration were assessed in the open field both when the environment was new, as in most studies on developmental exposure to MeHg in rodents, and when it was becoming familiar. Although the precise neurobehavioral significance of activity in the open field is not well understood, it seems that limbic structures (amygdala, hippocampal formation and prelimbic cortex) modulate the behavioral response to novelty, whereas the nucleus accumbens mediates locomotor activity and exploration [4]. Performance was also examined in a variety of learning and memory tasks. A visual discrimination learning task was administered in the Y maze. Acquisition of a similar task was shown to be impaired in rats by selective lesions of the hippocampus [24]. Two tasks were used to test spatial learning and memory. One task was spatial alternation in the T maze, which requires the animal to choose the arm opposite to the one selected on the previous trial. Since the walls of the maze were opaque, no extramaze cues were available and the spatial alternation response could be learned by relying on egocentric information. Egocentric localization memory deficits have been reported in rodents after lesions of the caudate nucleus [10,28]. Training to spatial alternation was followed by testing with delays. Delayed spatial alternation and spatial working memory have been repeatedly shown to be impaired by frontal lesions [11,20,23,37]. The other spatial task involved place learning and memory in the radial arm maze. In this task, the maze is surrounded by a variety of extramaze stimuli, which are visible from different arms. To solve the problem, the animal relies on allocentric spatial information, i.e., information that is independent of the position of the animal. In our experiment, we used the reference working memory version of the radial arm maze task [26]. Shortterm and long-term retention for places are impaired on this task by hippocampal lesions [26] and by caudate lesions [27], respectively.
2. Materials and methods 2.1. Animals Mice of the C57BL/6 strain were obtained from a local breeder (Charles River, St. Constant, Que´bec, Canada). For mating, ninety-eight 11- to 12-week-old primigravid females were placed two per cage with one male breeder. GD1 was confirmed by the presence of a vaginal plug in the morning. The 79 females with vaginal plugs were placed into individual nesting cages and were assigned at random to two conditions. In Condition GD7 – 9, they were treated on GD7, GD8 and GD9, whereas in Condition GD12 –14, they were treated on GD12, GD13 and GD14. In each condition, plugged females received a dose of either 0 mg MeHg/kg body weight/day (GD7 – 9: n = 10; GD12 – 14: n = 12), 4 mg/kg (GD7 – 9: n = 12; GD12 – 14: n = 12) or 6 mg/kg (GD7 –9: n = 12; GD12 – 14: n = 21), for a cumulative dose of 0, 12 and 18 mg/kg, respectively. MeHg (Laboratoire MAT, Beauport, Que´bec, Canada) was diluted with sterile phosphate-buffered saline (PBS). MeHg or an equivalent volume of PBS was administered by peroral injection to treated and vehicle groups, respectively. Three females treated with 3 6 mg/kg of MeHg in Condition GD12 – 14 were sacrificed at GD15 and two were sacrificed at GD17; mercury levels were determined in pools of livers and pools of brains of fetuses from each of these females. The remaining females (n = 74) were checked every morning for the presence of newborns. They gave birth to 58 litters (GD7 – 9: 8, 10 and 9 litters treated with 0, 4 and 6 mg/kg of MeHg, respectively; GD12– 14: 11, 9 and 11 litters treated with 0, 4 and 6 mg/kg of MeHg, respectively). On the day of birth (GD19 or GD20), which was defined as postnatal day 1 (PND1), three litters treated with 3 6 mg/kg of MeHg and three vehicle litters in Condition GD12 – 14 were randomly selected. Pups from these litters were used for determinations of liver and brain levels of mercury according to a procedure similar to the one used with GD15 and GD17 fetuses, with the exception that separate pools were made for females and males, and only female pools were analyzed. In the remaining litters (n = 52), the average number of pups per litter was very similar across groups [Condition GD7– 9: 8.6 ( ± S.E.M. = 0.6) in Group 3 0 mg/kg; 9.0 ( ± S.E.M. = 0.4) in Group 3 4 mg/kg; and 8.1 ( ± S.E.M. = 0.7) in Group 3 6 mg/kg; Condition GD12 – 14: 8.9 ( ± S.E.M. = 1.0) in Group 3 0 mg/kg; 9.3 ( ± S.E.M. = 0.7) in Group 3 4 mg/kg; and 9.1 ( ± S.E.M. = 0.4) in Group 3 6 mg/kg]. These pups remained with their biological mothers until weaned at PND21. There was potential exposure to MeHg during nursing, but a previous study [16], which used a fostering procedure, showed that behavioral deficits were not significantly influenced by the treatment to the mother, which reared the pups, and were rather due to exposure of the mice in utero.
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At the age of 5 weeks, one female per litter was selected for behavioral testing. Each female pup was individually weighed and coded to make the experimenters blind to the nature of their treatment. Mice were maintained on a 12:12 h light – dark photoperiod and the tests were administered during the light phase of the cycle. Because food reinforcement (pieces of Fruit Loops cereals, Kellogg) was used in three of the five behavioral tasks, the normal daily food ration was restricted to reduce body weight to 85% of freefeeding level and therefore, mice had to be housed individually in standard plastic cages. In order to reduce body weight to 85% of free-feeding level while allowing growth, the weights of the mice under experiment were compared and adjusted to the average weight of mice of the same age (n = 15) receiving normal food ration. Water was provided ad libitum. Behavioral tests were administered 7 days/week. All mice of Groups 3 0 (n = 8), 3 4 (n = 10) and 3 6 mg/kg (n = 9) in Condition GD7 –9 and all mice of Groups 3 0 (n = 8) and 3 4 mg/kg (n = 9) in Condition GD12 – 14 completed the five tests. In Group 3 6 mg/kg of Condition GD12 –14 (n = 8), three mice had to be replaced by another female in the course of testing — one because of small weight and weakness and two because of injuries. 2.2. Mercury determinations For total mercury determinations, each tissue was digested in concentrated nitric acid prior to reduction of mercury to its metal state by stannous chloride and quantification of mercury vapour by UV [12]. A certified reference material (CRM) was used in the analytic process for quality control purposes. The CRM used was a homogenised dogfish liver tissue sample (DOLT-2-) obtained from the National Research Council of Canada. The obtained value for mercury was 2.2 mg/g. The certified value is 2.0 mg/g. The coefficient of variation for 50 determinations on different days was 4.9%. 2.3. Motor coordination on the rotarod At 6 weeks of age, the female offspring were tested on the rotarod. The balance rod was a 45-cm-long cylinder of 3 cm diameter wrapped with masking tape and suspended at the top and center of a wooden enclosure (35 45 115 cm). It was surrounded by three 30-cm-high black walls to prevent animals from climbing off the rod and was divided in the middle by a 30-cm-high vertical black screen, so that two mice could be tested simultaneously. The cylinder was connected to a DC motor (115 V, 33 A, 1/50 hp; Fisher Scientific Canada), which supplied power to a gear box as a function of electrical output from a variable transformer. The floor of the enclosure was covered with a 40-cm-thick cotton cushion to prevent any harm when the mice fell off the rod. Each daily session included five trials during which the mice were required to maintain balance on the rod for
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120 s. A fall occurring in the first 10 s of a trial was defined as a false start, which was a rare occurrence, and the trial was resumed 30 s later. Each trial was administered on squads of eight mice, so the intertrial interval was approximately 8 –10 min. Mice tested simultaneously were timed on two separate chronometers and if one of the two mice fell off the rotarod, only the chronometer for this mouse was restarted. On the first session, the mice were placed on the stationary rod (0 rpm) and on the second session, the rod rotated at a constant speed of 3 rpm. On the following four daily sessions, the rod rotated at a constant speed of 20 rpm. Fall latencies were recorded and results from the four sessions at 20 rpm were analyzed. 2.4. Spatial alternation training and delay testing in the T maze The mice were 7 weeks old at the beginning of the T-maze task, which was administered in two identical T mazes made of opaque acrylic with 30-cm-high walls and 10-cm-wide corridors. The stem was divided into a start box (30 cm) and a runway (50 cm). At the end of each choice arm (30 cm), food reward could be concealed in an opaque food cup. Black guillotine doors separated the start box and the choice arms from the runway. On the first day, the mice were familiarized to the apparatus. Several reinforcers were scattered on the stem, on the choice arms and in the food cups. The mouse was placed in the start box and was free to move in the maze while the experimenter periodically opened and closed the guillotine doors. It was allowed to explore the maze for a total of 10 min or until all the reinforcers had been consumed, whichever occurred first. Training to spatial alternation began the day after familiarization. Each daily session consisted of 11 trials. On the first trial (trial 0) of each session, both food cups in the choice arms were baited with a food reinforcer and the mouse was allowed to choose one arm. For the next 10 trials, the reinforcer was placed in the arm opposite to that chosen by the mouse on the previous trial. Criterion for ending training was eight successes out of 10 trials averaged over two consecutive days. A maximum of 30 sessions was administered. Once criterion was reached, delays of 30, 60 and 120 s were interposed between trials. Each delay was used for two consecutive days and then was increased to the next delay value. 2.5. Activity in the open field The mice were 10 weeks old at the beginning of testing in the open field. The floor of the apparatus measured 100 100 cm and was divided into 25 equal squares. The floor was surrounded by opaque acrylic 30-cm-high walls; squares adjacent to walls were referred to as periphery, and the nine remaining squares were referred to as center. On each of five consecutive days, each mouse was moved from its home cage and placed in the open field, facing the lower
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left corner. They were allowed to move freely for 15 min but data were taken only in the first and in the last 5 min. The following behavioral measures were recorded during these 10 min: number of peripheral and central square crossings and frequency of rearings. Total number of square crossings, percentage of central square crossings and frequency of rearings were averaged for the first session (new environment) and for the following four daily sessions (familiar environment). 2.6. Visual discrimination learning in the Y maze The mice were 11 weeks old at the beginning of this task. Two identical Y mazes made of opaque acrylic with 30-cm-high walls were used for visual discrimination learning. In each maze, the start box (10 30 cm) led to a stem choice area (20 15 cm), which led itself to two parallel runways (10 30 cm) with opaque food cups placed at their ends. Black guillotine doors separated the start box, the stem choice area and the runways. Each runway and half of the stem choice area leading to it were covered with a white or a black interchangeable acrylic floor insert (10 45 cm). The floor inserts were thus visible to the mouse as soon as it emerged from the start box. On each trial, the runway covered with the black insert was baited (S+), whereas the runway covered with the white insert was not (S ). The left and right positions of the inserts were determined by a Gellerman pseudorandom sequence. Each daily session consisted of 20 trials separated by a 10-s intertrial interval. On the first two sessions, a correction procedure was used: if the mouse chose the S , an error was recorded and the trial was immediately repeated with only the S+ runway opened (forced successful choice). On the following sessions, no correction procedure was used and each trial was administered only once. Criterion for completion of the task was 17 successes out of 20 trials averaged over two consecutive days. 2.7. Spatial working reference memory in the radial arm maze The mice were 14 weeks old at the beginning of this task. The eight-arm radial maze was made of wood and painted flat gray. The maze was 60 cm from the floor. Each arm was 60 cm long and 9 cm wide. A recessed food well was located at the end of each arm. The center platform was 40 cm in diameter and was surrounded by a 40-cm-high wooden wall. The entrance to each arm was blocked by a gray acrylic guillotine door. The maze was centrally located in a room providing a number of large objects (a window, a poster, a vertical grid, etc.) and surrounded on two sides by 160-cm-high folding screens decorated with posters. The animals’ behavior was monitored with a video camera that was suspended over the maze. The camera was connected to a video monitor that was located outside the folding screens where the experimenter raised and lowered the
guillotine doors by pulling on strings in order to provide access to the radial arms. The working reference memory version of the radial arm maze task was used [26]. In this version, half of the arms, always the same, is baited on each daily trial and the other half is unbaited. As the animal learns the rules of the task and/or features which are constant from trial to trial (reference or long-term memory), it becomes able to discriminate baited from unbaited arms and avoids entering unbaited arms. It also becomes able to avoid baited arms it has already visited within a trial (working or short-term memory). The first five daily trials served to familiarize the animals with the apparatus. On the first day of familiarization, several reinforcers were scattered on the central platform and on four of the eight arms. The mouse was placed on the central platform and was free to move in the maze for 5 min while the experimenter periodically opened and closed the guillotine doors. If the reinforcers in the four baited arms were not found and consumed in the first 5 min, the mouse was placed at the end of each baited arm and had to walk to the central platform; then, from the central platform, it was allowed to explore the maze for 5 min. On the following 4 days of familiarization, four baited arms were randomly selected each day and the number of reinforcers was gradually reduced until only the food wells of the four selected arms were baited. Training began the day after the last familiarization session. There was one daily trial for 15 consecutive days. On each trial, only four arms, always the same so they could be maintained in reference memory, were baited. At the beginning of a trial, the mouse was placed on the central platform with all guillotine doors closed. After 10 s, the doors were opened and the mouse was allowed to choose an arm. When the mouse returned to the central platform, the doors were closed and a 10-s waiting period began. After the waiting period, all the doors were opened and the mouse was allowed again to choose an arm. This procedure continued until one of the following criteria was reached, whichever occurred first: the four baited arms were visited, 16 choices were made or 10 min had elapsed. A reference memory error was recorded each time the mouse visited for the first time one of the four never baited arms and a working memory error was recorded each time it revisited an arm within a trial, whether this arm was originally baited or not. 2.8. Statistical analysis Within each condition (GD7 – 9 or GD12 – 14), results of Groups 3 0, 3 4 and 3 6 mg/kg were subjected to ANOVAs with Group as a between-subject factor. On the rotarod and delayed spatial alternation tasks, Session and Delay were within-subject factors, respectively. Analyses of simple main effects of the interaction (with Satterthwaite’s correction for the error term and its degrees of freedom [15]) and Newman– Keuls tests served to locate specific signific-
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ant effects. On the radial arm maze task, the total numbers of correct choices, of reference memory errors and of working memory errors made by mice in Condition GD7 –9 were also subjected to an ANOVA with Group as a betweensubject factor and Block of five daily trials as a withinsubject factor. As for Condition GD12 – 14, it appeared that mice exposed to MeHg made fewer reference memory errors, but also fewer choices, than control mice. Since the total number of first choices made in 10 min differed in treated and control mice, the measures in the radial arm maze had to be subjected to special analyses. In each group of Condition GD12 –14, the numbers of visits to baited arms (correct choices) and to unbaited arms (reference memory errors) were compared by an ANOVA with Type of arm and Block of five trials as within-subject factors; the numbers of working memory errors in the first and third blocks of five trials were compared by a unilateral paired t test.
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age of 5 weeks in mice treated with 3 6 mg/kg of MeHg at GD7 – 9 and at GD12 –14. 3.2. Motor coordination on the rotarod task In the first 2 days of testing, all mice were able to maintain balance on the rod in at least four of the five trials when the rod was stationary or rotated at a speed of 3 rpm. In the following four daily sessions, when the rod rotated at a constant speed of 20 rpm (Fig. 1), fall latencies increased across sessions in all groups and thus, there was some learning of motor coordination. However, prenatal exposure to MeHg had no effect on motor learning. In Condition GD7 – 9 [ F(3, 72) = 14.73, P < .0001] and in Condition GD12 – 14 [ F(3, 66) = 19.49, P < .0001], the factor Session was significant but the factor Group [Condition GD7 – 9: F(2, 24) = 1.09; Condition GD12 –14: F(2, 22) = 1.25] and the interaction were not significant [Condition GD7 – 9: F(6, 72) = 0.59; Condition GD12 – 14: F(6, 66) = 0.58].
3. Results 3.1. Liver and brain levels of Hg and percentages of survival at the age of 5 weeks At GD15, GD17 and at birth, liver levels of mercury in mice treated with 6 mg/kg of MeHg at GD12 –14 were 12.70 ( ± S.E.M. = 1.79), 15.67 ( ± S.E.M. = 0.27) and 15.76 mg/g ( ± S.E.M. = 0.54), respectively, whereas brain levels were 16.74 ( ± S.E.M.=.1.56), 13.26 ( ± S.E.M. = 3.76) and 12.09 mg/g ( ± S.E.M. = 0.89). In control mice, liver and brain levels of mercury at birth were 0.17 ( ± S.E.M. = 0.003) and 0.13 mg/g ( ± S.E.M. = 0.01), respectively. Although liver and brain levels of mercury were measured in a small number of mice and only in Condition GD12 – 14, it is clear that they were very high shortly after treatment (GD15 and GD17) as well as at birth [3]. In each litter, the number of offspring which survived to the age of 5 weeks, i.e., 1 week before the beginning of behavioral testing, was divided by the number of offspring at birth and the result was multiplied by 100 to give the percentage of survival. In Condition GD7 –9, the percentages of survival in Groups 3 0, 3 4 and 3 6 mg/kg were 90.3% ( ± S.E.M. = 3.4), 86.0% ( ± S.E.M. = 3.8) and 61.8% ( ± S.E.M. = 11.9), respectively. These percentages significantly differed [ F(2, 24) = 4.12, P < .05] and the Newman – Keuls test ( P < .05) showed that the percentage of survival in Group 3 6 mg/kg was significantly lower than in Groups 3 0 and 3 4 mg/kg. In Condition GD12 –14, the percentages of survival were 86.5% ( ± S.E.M. = 5.6) for Group 3 0 mg/kg, 68.7% ( ± S.E.M. = 11.2) for Group 3 4 mg/kg and 60.5% ( ± S.E.M. = 7.4) for Group 3 6 mg/kg. These percentages also significantly differed [ F(2, 22) = 11.34, P < .01] and the Newman– Keuls test ( P < .01) showed that the percentage of survival in Group 3 6 mg/kg was significantly lower than in Group 3 0 mg/kg. Thus, prenatal exposure to MeHg reduced the percentage of survival at the
3.3. Spatial alternation training and delay testing in the T maze Prenatal exposure to MeHg impaired training to spatial alternation in the T maze (Fig. 2A). The number of sessions to reach the learning criterion [ F(2, 24) = 3.93, P < .05] significantly differed in the groups of Condition GD7 – 9. The Newman– Keuls test ( P < .05) showed that mice treated with 4 and 6 mg/kg of MeHg required more sessions than control mice. In Condition GD12 –14, there was also a
Fig. 1. Fall latencies on the rotarod task. Data points are group means ± S.E.M. for four consecutive daily sessions. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Data of each condition were analyzed by ANOVA with Group as a between-subject factor and Sessions (1 – 4) as a within-subject factor. The factor Group [Condition GD7 – 9: F(2, 24) = 1.09; Condition GD12 – 14: F(2, 22) = 1.25] and its interaction with the factor Session [Condition GD7 – 9: F(6, 72) = 0.59; Condition GD12 – 14: F(6, 66) = 0.58] were not significant, but the factor Session was significant [Condition GD7 – 9: F(3, 72) = 14.73, P < .0001; Condition GD12 – 14: F(3, 66) = 19.49, P < .0001].
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all mice were able to produce 80% correct responses, but in Condition GD7 – 9, 4/10 mice treated with 3 4 mg/kg of MeHg and 2/9 mice treated with 3 6 mg/kg failed to attain this level of performance within the maximum of 30 sessions. Those mice were not tested in the delayed spatial alternation test. When delays of 30, 60 and 120 s were interposed between trials, spatial alternation performance deteriorated compared to the last two sessions of training (Fig. 2B), but it was not impaired by prenatal administration of MeHg at GD7– 9 or GD12 – 14. In both conditions, the factor Delay had a significant effect [Condition GD7 – 9: F(3, 54) = 22.77, P < .0001; Condition GD12 – 14: F(3, 66) = 18.33, P < .001], whereas the factor Group [Condition GD7 – 9: F(2, 18) = 0.10; Condition GD12 –14: F(2, 22) = 0.16] and the interaction [Condition GD7 –9: F(6, 54) = 0.55; Condition GD12 – 14: F(6, 66) = 0.52] were not significant. However, differences between treated and control mice might have been masked by a floor effect because at a 30-s delay, the number of correct choices was already between 55% and 70%. 3.4. Activity in the open field
Fig. 2. Training and delay testing on spatial alternation in the T maze. (A) Number of sessions to reach criterion. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Each histogram represents the mean ± S.E.M. Data of each condition were analyzed by a one-way ANOVA followed by a posteriori comparisons with the Newman – Keuls test. In Condition GD7 – 9, the factor Group was significant [ F(2, 24) = 3.93, P < .05]: mice treated with 4 and 6 mg/kg of MeHg required more sessions to reach criterion than control mice ( P < .05). In Condition GD12 – 14, the factor Group was also significant [ F(2, 22) = 4.17, P < .05] and Group 3 6 mg/kg required more sessions to reach criterion than Groups 3 0 and 3 4 mg/kg ( P < .05). (B) Frequency of correct choices in delay testing. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 6); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Data points are group means ± S.E.M. for each delay (0-s delay: data from the two criterion sessions). Data of each condition were analyzed by ANOVA with Group as a betweensubject factor and Delay as a within-subject factor. The factor Group [Condition GD7 – 9: F(2, 18) = 0.10; Condition GD12 – 14: F(2, 22) = 0.16] and the interaction [Condition GD7 – 9: F(6, 54) = 0.55; Condition GD12 – 14: F(6, 66) = 0.52] were not significant, but the factor Delay was significant [Condition GD7 – 9: F(3, 54) = 22.77, P < .0001; Condition GD12 – 14: F(3, 66) = 18.33, P < .001].
significant difference between groups in the number of sessions to reach criterion [ F(2, 22) = 4.17, P < .05] and the Newman– Keuls test ( P < .05) revealed that Group 3 6 mg/kg required more sessions to reach criterion than Groups 3 0 and 3 4 mg/kg. In Condition GD12 –14,
Fig. 3A presents the total number of square crossings in the open field during the 10 min of recording in the first session and in the following four sessions. Only mice treated with MeHg at GD12 –14 were significantly hypoactive. When the environment was new (Session 1), the groups in Condition GD7 – 9 did not differ [ F(2, 24) = 0.60], but the groups in Condition GD12 –14 did [ F(2, 22) = 5.98, P < .01]: Groups 3 6 and Group 3 4 mg/kg crossed significantly fewer squares than Group 3 0 mg/kg (Newman –Keuls at P < .01). In Sessions 2– 5, when the environment was becoming familiar, similar results were observed. The groups in Condition GD7 – 9 did not differ [ F(2, 24) = 0.16], but the groups in Condition GD12 – 14 did [ F(2, 22) = 9.01, P < .005]: Groups 3 4 and 3 6 mg/kg crossed significantly fewer squares than Group 3 0 mg/kg (Newman –Keuls at P < .01). Fig. 3B presents the percentage of central square crossings in 10 min. If this behavioral measure is an index of fear or emotionality as it has frequently been described [19], there was no evidence of altered function in mice with prenatal exposure to MeHg. In both conditions, the groups did not differ whether the environment was new [Session 1: Condition GD7 – 9: F(2, 24) = 0.51; Condition GD12 – 14: F(2, 22) = 0.11] or familiar [Sessions 2 – 5: Condition GD7 – 9: F(2, 24) = 0.57; Condition GD12 – 14: F(2, 22) = 0.97]. Finally, Fig. 3C presents the frequency of rearings in the open field. Again, in both conditions, the groups did not differ whether the environment was new [Session 1: Condition GD7 – 9: F(2, 24) = 0.55; Condition GD12 – 14: F(2, 22) = 0.02] or familiar [Sessions 2 – 5: Condition GD7 – 9: F(2, 24) = 0.39; Condition GD12 – 14: F(2, 22) = 0.82].
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3.5. Visual discrimination learning in the Y maze Prenatal exposure to MeHg had no effect on learning the visual discrimination in the Y maze (Fig. 4). The number of sessions to reach criterion did not significantly differ in the three groups of mice either in Condition GD7 –9 [ F(2, 24) = 1.29] or in Condition GD12 –14 [ F(2, 22) = 2.33]. For an unknown reason, all groups in Condition GD12– 14 seemed to require more sessions than groups in Condition GD7 –9. 3.6. Spatial working-reference memory in the radial arm maze In Condition GD7 – 9 (Fig. 5A), the performance in the radial maze was similar in treated and control mice. The frequency of correct choices was high and there was no significant difference between groups [Group: F(2. 24) = 0.02; Group Block: F(4, 48) = 0.07] or across blocks of trials [ F(2, 48) = 0.00]. The frequency of reference memory errors significantly decreased across blocks of trials [ F(2, 48) = 5.01, P < .01], but there was no significant difference between groups [Group: F(2, 24) = 0.13; Group Block: F(4, 48) = 0.29]. Similarly, the frequency of working memory errors significantly decreased across blocks [ F(2, 48) = 3.85, P < .05] and there was no significant difference between groups [Group: F(2, 24) = 1.57; Group Block: F(4, 48) = 0.32]. In Condition GD12 – 14, the results on the radial arm maze task were more complex. In the course of testing, it became clear that mice of Groups 3 4 and 3 6 mg/kg behaved differently than control mice of the same condition (Fig. 5B): a trial had frequently to be ended after the 10-min criterion was reached, before the four baited arms were
Fig. 3. Behavior in the open field. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Each histogram represents the mean ± S.E.M. Data of each condition were analyzed by a one-way ANOVA followed by a Newman – Keuls test. (A) Total number of square crossings during the 10-min of recording when the environment was new (Session 1) and familiar (Session 2 – 5). In Session 1, the groups did not differ in Condition GD7 – 9 [ F(2, 24) = 0.60], but they significantly differed in Condition GD12 – 14 [ F(2, 22) = 5.98, P < .01], with Group 3 0 mg/kg crossing more squares than Group 3 6 mg/kg ( P < .01). In Sessions 2 – 5, the groups did not differ in Condition GD7 – 9 [ F(2, 24) = 0.16], but they significantly differed in Condition GD12 – 14 [ F(2, 22) = 9.01, P < .005], with Group 3 0 mg/kg crossing more squares than Groups 3 4 and 3 6 mg/kg ( P < .01). (B) Percentage of central square crossings when the environment was new (Session 1) and familiar (Session 2 – 5). The groups in both conditions did not differ in Session 1 [Condition GD7 – 9: F(2, 24) = 0.51; Condition GD12 – 14: F(2, 22) = 0.11] or in Sessions 2 – 5 [Condition GD7 – 9: F(2, 24) = 0.57; Condition GD12 – 14: F(2, 22) = 0.97]. (C) Frequency of rearings when the environment was new (Session 1) and familiar (Session 2 – 5). The groups in both conditions did not differ in Session 1 [Condition GD7 – 9: F(2, 24) = 0.55; Condition GD12 – 14: F(2, 22) = 0.02] or in Sessions 2 – 5 [Condition GD7 – 9: F(2, 24) = 0.39; Condition GD12 – 14: F(2, 22) = 0.82].
Fig. 4. Number of sessions to reach criterion on the visual discrimination in the Y maze. Each histogram represents the mean ± S.E.M. Number of mice in each group: Condition GD7 – 9: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9); Condition GD12 – 14: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). Data of each condition were analyzed by a one-way ANOVA. In Conditions GD7 – 9 [ F(2, 24) = 1.29] and GD12 – 14 [ F(2, 22) = 2.33], the groups did not differ.
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visited and before 16 choices were made. ANOVAs made on the overall results of the 15 trials revealed that although the groups did not differ in terms of working memory errors [ F(2, 22) = 1.72], they significantly differed in terms of correct choices [ F(2, 22) = 4.45, P < .05] and reference memory errors [ F(2, 22) = 5. 29, P < .05]. Newman –Keuls tests ( P < .05) showed that Groups 3 4 and 3 6 mg/kg made fewer reference memory errors but they also made fewer correct choices. Because the number of first entries into arms was lower in prenatally exposed groups than in control mice, direct intergroup comparisons were not possible. An intragroup strategy of analysis was adopted. Long-term retention or reference memory of the rules and/ or constant features of the task implies that the animal gradually learned to discriminate baited and unbaited arms. Thus, reference memory was evaluated, within each group, by comparing the number of visits to baited arms (correct choices) and the number of visits to unbaited arms (reference memory errors). An ANOVA with Type of arm and Block of five trials as within-subject factors was used for these comparisons. In Group 3 0 mg/kg, the factor Type of arm was significant [ F(1, 7) = 34.97, P < .001], and the factor Block [ F(2, 14) = 1.43] and the interaction [ F(2, 14) = 2.78] were not significant. Thus, control mice visited more baited
arms than unbaited arms on all three blocks of trials. Treatment with MeHg in Condition GD12 – 14 impaired discrimination of baited and unbaited arms and this impairment was more severe at the highest dose. In Group 3 4 mg/kg, the factor Type of arm [ F(1, 8) = 29.07, P < .001] and the interaction [ F(2, 16) = 5.61, P < .02] were significant, but the factor Block [ F(2, 16) = 0.19] was not significant. The analysis of simple main effects showed that in Group 3 4 mg/kg, more visits were made to baited arms than to unbaited arms on the second [ F(1, 16) = 15.11, P < .002] and on the third blocks of trials [ F(1, 16) = 44.20, P < .0001], but not on the first block [ F(1, 16) = 3.02]. In Group 3 6 mg/kg, the frequency of visits to baited and unbaited arms did not differ on any block: the factors Type of arm [ F(1, 7) = 3.25] and Block [ F(2, 14) = 1.85], as well as the interaction [ F(2, 14) = 0.11], were not significant. Whereas Group 3 4 mg/ kg was initially slower to learn the rules and/or constant features of the task than the vehicle group, Group 3 6 mg/ kg did not discriminate baited and unbaited arms on any of the three blocks of five trials. In Condition GD12 – 14, working memory errors were analyzed by comparing, within each group, the numbers of reentries on the first and third blocks of trials. In normal
Fig. 5. Frequency of correct choices (visits to baited arms), of reference memory errors (first visits to unbaited arms) and working memory errors (reentries in baited or unbaited arms) in the radial arm maze. Data points are group means ± S.E.M. for each block of five trials. (A) Data of Condition GD7 – 9. Number of mice in each group: 3 0 (n = 8); 3 4 (n = 10); 3 6 mg (n = 9). Data were analyzed by ANOVAs with Group as a between-subject factor and Blocks of five trials as a within-subject factor. There was no significant difference between groups or across blocks of trials in the number of correct choices [Group: F(2, 24) = 0.02; Block: F(2, 48) = 0.00; Group Block: F(4, 48) = 0.07]. The frequency of reference memory errors significantly decreased [ F(2, 48) = 5.01, P < .01] and there was no significant difference between groups [Group: F(2, 24) = 0.13; Group Block: F(4, 48) = 0.29]. The frequency of working memory errors significantly decreased [ F(2, 48) = 3.85. P < .05] and there was no significant difference between groups [Group: F(2, 24) = 1.57; Group Block: F(4, 48) = 0.32]. (B) Data of Condition GD12 – 14. Number of mice in each group: 3 0 (n = 8); 3 4 (n = 9); 3 6 mg (n = 8). In order to compare correct choices and reference memory errors, data of each group were analyzed by ANOVAs with Type of arm and Block of five trials as a within-subject factors. In Group 3 0 mg/kg, the factor Type of arm was significant [ F(1, 7) = 34.97, P < .001], and the factor Block [ F(2. 14) = 1.43] and the interaction [ F(2, 14) = 2.78] were not significant. In Group 3 4 mg/kg, the factor Type of arm [ F(1, 8) = 29.07, P < .001] and the interaction [ F(2, 16) = 5.61, P < .02] were significant, but the factor Block [ F(2, 16) = 0.19] was not significant. The analysis of simple main effects showed that in Group 3 4 mg/kg, more visits were made to baited arms than to unbaited arms on the second [ F(1, 16) = 15.11. P < .002] and third blocks [ F(1, 18) = 44.20, P < .0001], but not on the first block [ F(1, 16) = 3.02]. Finally, in Group 3 6 mg/kg, the frequency of visits to baited and unbaited arms did not differ on any block because the factor Type of arm [ F(1, 7) = 3.25], Block [ F(2, 14) = 1.85] and the interaction [ F(2, 14) = 0.11] were not significant. For each group, the frequencies of working memory errors on the first and third blocks of trials were compared by a unilateral Student’s t test for paired samples. The frequency of working memory errors significantly decreased in Group 3 0 mg/kg [t (7) = 1.91, P < .05], but not in Group 3 4 mg/kg [t(8) = 0.46] or in Group 3 6 mg/kg [t(7) = 0.40].
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animals, these errors usually decrease across trials as they learn to avoid revisiting an arm. In fact, the number of working memory errors significantly decreased in Group 3 0 mg/kg [t (7) = 1.91, P < .05], but not in Groups 3 4 [t (8) = 0.46] and 3 6 mg/kg [t (7) = 0.40].
4. Discussion The mercury determinations in brain and liver of mice treated at GD12 – 14 indicate that high exposure to MeHg lasted from very shortly after treatment until birth. A weakness of the present experiment is that mercury levels were not measured in mice treated at GD7 –9. However, an indication of the effects of administration of MeHg in this condition is that, as in Condition GD12– 14, survival to the postnatal age of 5 weeks was significantly decreased by administration of 6 mg/kg of MeHg. Observations made during the experiment did not reveal any obvious sign of motor deficit and testing on the rotarod did not show any impairment in learning motor coordination, even in mice treated with 3 6 mg/kg. Previous studies on rodents did find gross impairments of motor function after prenatal exposure to MeHg, but these studies examined reflexive behavior in early development [25] or swimming ability [13,34,36]. On the other hand, our results in the open field are consistent with early [34,35,38] and with more recent [19] reports, which showed that prenatal exposure to MeHg significantly decreases locomotor activity. In our experiment, this effect was observed only in mice treated at GD12 – 14 and frequency of rearings was not affected. Contrary to the results of Kim et al., there was no evidence that hypoactivity in the open field was related to fear or to changes in emotional status: the percentage of central square crossings in treated mice and control mice did not differ. Hypoactivity in mice treated with MeHg at GD12 – 14 was not specific to novelty and also appeared when the open field was familiar. This result suggests that the decrease in locomotor activity was related to damage to the nucleus accumbens rather than to limbic structures [4]. In order to discriminate the black and white floors in the Y maze, to acquire the spatial alternation response in the T maze and to discriminate baited and unbaited arms in the radial maze, mice had to learn the basic rules and/or constant features of the task. Thus, the three tasks required intact reference memory or long-term retention of either an individual cue (black floor in the visual discrimination task) or of spatial information (T maze and radial maze). In all mice treated with MeHg, learning on the visual discrimination task was normal and this result is consistent with previous experiments on rodents [2,13]. On spatial tasks, reference memory was impaired by prenatal exposure to MeHg whether the task involved egocentric or allocentric information. However, the effects of treatment windows on the two spatial tasks differed.
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On spatial alternation training, both treated groups of Condition GD7 – 9 required more sessions to reach the learning criterion and in Condition GD12 – 14, only Group 6 mg/kg was impaired. This result suggests that reference memory for egocentric spatial information was affected by a lower dose of MeHg when treatment occurred at GD7 –9 than at GD12 – 14. In the radial arm maze, reference memory for allocentric spatial information was impaired only in Condition GD12 –14 and this impairment was more severe after prenatal exposure to the highest dose. Whereas discrimination of baited and unbaited arms was impaired on all three blocks after prenatal exposure to 6 mg/kg, it was impaired only on the first block of trials in mice exposed to 4 mg/kg. Reference memory for egocentric spatial information [10,28] and reference memory for allocentric spatial information [27] have been both associated with the function of the caudate nucleus and yet, exposure to MeHg at two fetal developmental stages had different effects on spatial alternation training and on the radial arm maze task. One possible explanation is that treatment at GD7 –9 and GD12 –14 altered the function of different neurotransmitter systems in the caudate nucleus. Delay testing of spatial alternation in the T maze suggests that working memory for egocentric spatial information and frontal function were not affected by prenatal exposure to MeHg. However, this conclusion can only be tentative. First, working memory errors on this task were already low at the shortest delay and the possibility of a floor effect cannot be definitely excluded. Second, in Condition GD7– 9, some treated mice did not reach criterion and could not be tested with delays although their performance on this task was probably more affected by exposure to MeHg than in Condition GD12 – 14. In contrast, the analysis of reentries in the radial arm maze revealed that in Condition GD12 – 14, but not in Condition GD7 –9, working memory errors did not decrease between the first and the third blocks of trials in mice prenatally exposed to either dose of MeHg. Thus, working memory for places was impaired and this deficit is usually associated with dysfunctions of the hippocampus and/or adjacent entorhinal cortex. In summary, treatment with MeHg at two different stages of fetal development had different effects. In mice treated with MeHg at GD7 – 9, reference memory for egocentric spatial information was impaired, whereas in mice treated with MeHg at GD12 – 14, a variety of neurobehavioral functions were altered. Damages to the ventral striatum, the dorsal striatum and the hippocampal formation are suggested by the hypoactivity observed in the open field, by reference memory deficits on spatial alternation training and on the radial maze task, and by working memory deficits in the radial maze, respectively.
Acknowledgments The authors thank Mr. Alain Tremblay and Ms. Micheline Noel for their excellent technical assistance. This
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research was supported by funds from Fonds de Recherche en Sante´ du Que´bec (FRSQ)/Hydro-Que´bec (Programme de recherche en sante´ de l’enfant) and by Health Canada (Programme Saint-Laurent Vision 2000). The research received approval from the Comite´ de protection des animaux de laboratoire de l’Universite´ Laval, which is responsible for the application and enforcement of the rules of the Canadian Council on Animal Care.
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