Neurobehavioral effect of chronic and bolus doses of methylmercury following prenatal exposure in C57BL/6 weanling mice

Neurotoxicology and Teratology 31 (2009) 372–381

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Neurotoxicology and Teratology

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Neurobehavioral effect of chronic and bolus doses of methylmercury followingprenatal exposure in C57BL/6 weanling mice

Jacky Liang a, Mike Inskip b, Deborah Newhook b, Claude Messier a,⁎a School of Psychology, University of Ottawa, 200 Lees, Building E, 2nd floor, Room 260 J, Ottawa, ON, Canada K1N 6N5b Healthy Environments and Consumer Safety Branch, Health Canada, 0801B3, Environmental Health Center, Promenade de la Columbine, Tunney's Pasture, Ottawa, ON, Canada K1A 0K9

⁎ Corresponding author. School of Psychology, UnBuilding E, 2nd floor, Room 260 J, Ottawa, ON, Canad5800x4562; fax: +1 613 562 5147.

E-mail address: [email protected] (C. Messier).

0892-0362/$ – see front matter © 2009 Elsevier Inc. Aldoi:10.1016/j.ntt.2009.08.007

a b s t r a c t
a r t i c l e i n f o
Article history:Received 5 March 2009Received in revised form 10 July 2009Accepted 16 August 2009Available online 23 August 2009

Keywords:MethylmercuryMotricityLearningMemoryAnxietyIn utero

Several studies with animals have shown that even low and medium prenatal and postnatal exposure tomethylmercury (MeHg) can result in locomotor, motor coordination and learning deficits. However, somebehavioural effects of MeHg remain controversial and themethods tomodel humanMeHg exposure in animalstill remain to be optimized. We investigated the neurobehavioral effects of two different patterns of MeHgexposure. MeHg was givenmixed in palatable food that mice readily ate. For the first pattern (chronic group),C57BL/6 mice dams were given 1.4 μg/g body weight (BW)/day (n=20) throughout gestation mixed inpalatable food. For the second pattern (bolus) dams were given 6.0 μg/g BW/day mixed in palatable food ongestation day 12 and 16 together with a lower chronic dose of 0.85 μg/g BW/daymixed in palatable food on allremaining gestation days (n=20). Day 12 and 16 were chosen because neuron proliferation and the start ofmigration for many brain regions occur during that period. Behavioural testing on weanling animals started at8 weeks. Both the chronic and bolus groups showed an impairment of working memory and visual spatialability in the radial arm maze task. Other tests did not provide clear evidence that methylmercury exposurehad significant adverse effects on locomotor activity,motor coordination or emotional reactivity. However, thechronic groups had a tendency for lower performance inmost tests including activity in Skinner box and openfield trials, as well as a higher number of anxiety-like behaviors. Chronic exposure to lower levels of MeHgcombined to acute exposure with high levels of a few days during gestation appears to be less damaging thanchronic exposure to slightly higher levels without acute MeHg exposure even though, equal amounts wereadministered during gestation. Possibly, as indicated by preliminary data, the relatively larger impact ofchronic administration of a higher daily dose could be the consequence of a higher brainMeHg burden in pupscompared to brain MeHg levels in the pups from dams receiving a smaller daily dose with 2 large doses ongestation day 12 and 16. Alternatively, bolus MeHg could have had a larger impact if administered on differentgestation days when some neural development processes are more sensitive to MeHg.

© 2009 Elsevier Inc. All rights reserved.

1. Introduction

Methylmercury (MeHg) is a particularly toxic form ofmercury thatexists naturally in the environment — albeit in small amounts — andhas been shown to result in neurological deficits in animals and inhumans [1,6,7,19]. Particularly germane are issues such as the in uterosusceptibility of the developing nervous system to MeHg [27] and theexistence of periods (‘windows’) of critical vulnerability of the brainduring in utero development [28].

In some Northern communities in Canada, fish and marinemammals used as food sources can be contaminated with high

iversity of Ottawa, 200 Lees,a K1N 6N5. Tel.: +1 613 562

l rights reserved.

MeHg levels [38]: this is particularly true of predator species that cancontain elevated MeHg concentrations in the ppm range (Alan Pennpersonal communication). Consumption of such fish would theneffectively constitute an intake of infrequent but high doses of MeHgtogether with more constant lower doses of MeHg exposure from therest of the environment. This example illustrates the need for moremodels of human MeHg exposure in animal research and differentpatterns of exposure may be the source of variability in the previouslyobserved behavioural effects of human MeHg exposure.

For example, in some of the earlier debates on why differentresults were seen in the Faeroe Islands and Seychelles Islands studies,the ‘bolus dose effect’ — the result of a meal of whale meat with highMeHg levels— has sometimes been suggested as a possible explainingfactor for the differences observed (Clarkson, personal communica-tion). Indeed, it has been estimated that the bolus doses in the Faroes(and in an earlier New Zealand study) might have been ten times thatin the Seychelles study [7].

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Diagram 1. ADiagramof schematic for steady state vs bolus dosemethylmercury intake.

373J. Liang et al. / Neurotoxicology and Teratology 31 (2009) 372–381

A number of recent experiments have addressed the behaviouraleffects of prenatal MeHg using two patterns of administration: acutehigh doses on particular days during gestation and smaller dosesadministered every day during gestation. Two of those examined theimpact of a single administration of MeHg (8 μg/g body weight (BW))in pregnant rats on gestational day 15. In the first one, when tested on60 days postnatal, the offspring presented with active avoidancedeficits [5]. In the second one, this group used a similar protocol toexamine the effect of the same dose of MeHg (8 μg/g BW) but atgestational day 8 or 15 [4]. When tested at postnatal day 40, MeHg theexposed rats showed no motor impairments as measured by theRotarod, and the development of the righting reflex, cliff aversion andnegative geotaxis and no effect on anxiety as measured by theelevated zero maze. However, they produced less rearing in the openfield task and measures of learning, habituation and exploration in anovel object exploration task suggesting impairment. The same groupalso found that the offspring of rat dams treated with doses of 8 butnot 4 μg/g BW MeHg on gestational day 15 resulted in passiveavoidance deficits [14].

Other studies have used a repeated administration of MeHg overparts of the prenatal period. One study examined the impact of in uterodaily doses of 6 μg/g BWduring either gestational day 7 to 9 or 12 to 14.It was found that, in general, the deleterious effectswere greaterwhenMeHg was administered at day 12–14: deficits included reducedlocomotion in the open field and reduced spatial memory [16].

Another experiment examined the impact of repeated in uteroexposure toMeHg fromsmall daily doseof 0.01 μg/g BWgiven toC57BL/6J pregnant mice during gestational days 8 to 18 [24]. The resultsindicated significant deficits in the adult offsprings for motor abilities,coordination, and overall activity, as measured by Rotarod, footprintanalysis and open field. In addition, reference memory was impaired inthe Morris water maze task. Taken together, these studies suggest thatan acute dose of MeHg in the 6–10 μg/g BW range administered aroundgestational days 15 to 18 produce the greatest deficits while muchsmaller daily doses (0.01 μg/g BW) given through the gestational periodappear also be deleterious. These days lie within the most susceptibleperiod from the stand point of neuronal proliferation and distribution indeveloping fetal brain [27]. As stated earlier, these studies try to modelan acute exposure to a high dose ofMeHg (bolus) on the one hand and amuch smaller but constant exposure.

In the current study, advice from the University of Rochesterinvestigators (Clarkson and Weiss, personal communication) hasbeen adopted, in order to attempt to probe — in an animal model —this issue of “bolus” or “pulse” dose exposures in pregnancy. An earlierpilot toxicokinetic study in mice [17] had indicated that stable Hgisotopes could be used to distinguish tissue levels in the maternal andfetal brain arising from such bolus doses administered to the damsduring gestation, as compared to tissue concentrations arising fromchronic exposure only. Consequently, we examined the impact onbehavioural outcome of similar cumulative doses of MeHg but ad-ministered in different patterns.

In our study, we compared two models of prenatal exposure —

chronic and bolus dose (see Diagram 1). Thus, C57BL/6 mice wereexposed to MeHg throughout the gestational period (GD): from day 1to 18) but to model different exposure patterns, two in uterotreatments were devised. The first one (“chronic” group) consistedof a chronic or steady state (1.4 μg/g BW/day) exposure (for a totalexposure of 25.2 μg/g BW). The second one (“bolus” group) consistedof two bolus doses (6.0 μg/g BW/day) on gestation day 12 and 16,together with a reduced chronic dose (0.85 μg/g BW/day) during theremaining days of gestation (for a total exposure of 25.6 μg/g BW). Inthese two groups, animals are exposed to similar levels of MeHg butadministered in a way to model constant exposure versus constantexposure together with peak exposure on 2 occasions to model mealswith a high MeHg contamination. This is the first study that comparesthe impact of constant exposure with or without acute doses.

2. Materials and methods

2.1. Animals and dose administration

All procedures were approved by the Animal care committees ofHealth Canada and the University of Ottawa and followed theguidelines of the Canadian Council on Animal Care.

2.1.1. AnimalsFemale C57BL/6mice (Charles River, Quebec, Canada) were kept at

Health Canada's Animal Research facility under a 14/10hour light:darkcycle. Paired females were placed in with provenmales overnight, andpregnancy (GD0) was assumed on observation of a genital plug on thefollowing day. Housing was in individual cages with free access towater. Food, for all animals, consisted of the Teklad 2018C dietcontaining no fishmeal. This diet was chosen because other rodentdiets can contain fishmeal that could indirectly raise methylmercuryexposure [36]. The roomwas suppliedwithHepafiltered-air in a Class-100 clean room (Biobubble, Inc., CO) and with an additional ac-tivated carbon pre-filter. Standard temperature (21±2°C) andhumidity (40±10% RH) were maintained. Because breeding successis highly dependent on stress levels and disruption of pregnant damsin C57BL/6 mice, we added a single-entrance aluminum nesting boxand supply of nesting material which ensured pregnant and rearingdams had control over their micro-environment conditions includingprivacy and light.

2.1.2. Methylmercury administrationMeHg was administered by dissolving MeHgCl (methylmercury

chloride) in ethanol, then incorporating it into cookie treats (TeddyGrahams, Nabisco, Canada). Alcohol was allowed to evaporate beforegiving the cookie morsel to mice. Mice were previously acclimatizedto this food and readily ate it. MeHgCl was added to a standard cookiemorsel according to the dosage given and the weight of the mouse.Animals always ate the cookie morsel immediately as confirmed by avisual inspection. In the chronic condition, 1.4 μg/g (BW/day wasgiven from gestation day 1 to 18 (n=20) in palatable food. In thebolus condition, dams were given a slightly reduced chronic dose of0.85 μg/g BW/day in palatable food on all days except gestation day 12and 16, when a bolus dose of 6.0 μg/g (BW/day was administered inpalatable food (n=20). These doses resulted in the same totalamount of MeHgCl administered during the pregnancy (with slightanimal to animal variations due to different body weight gain during

374 J. Liang et al. / Neurotoxicology and Teratology 31 (2009) 372–381

pregnancy). Measurement of MeHg in the Teklad diet confirmed thepresence of very low concentrations of MeHg (meanb0.015 μg/g dryweight). At 6 weeks of age, equal numbers of male and femaleweanling mice (2 male and 2 female were chosen at random fromeach litter) were transported to the University of Ottawa forbehavioral testing, under blind conditions. Weanling mice werehoused in individual standard plastic hepa-filtered cages in atemperature-controlled room (21±1 °C) and kept under a 12/12 hlight–dark cycle with free access to food and water for a two-weekacclimatization period before behavioural testing started at postnatalday (PN) 57.

2.1.3. Behavioural testingA total of eight behavioural tests were administered during a 30-day

test period. All tests were performed during the light phase of the cycle(8 h00 to 16 h00). From PN63 to PN87, all animals were maintained atabout 85% of their free-feeding bodyweight tomotivate them to obtainfood rewards in the radial armmaze and the operant bar-pressing task.To that end,micewereweighed everymorning and the quantity of foodwas adjusted individually throughout the diet period. During the teststhat involved food deprivation, the daily food rationwas given after thecompletion of the test. Tests were performed in a following order: Openfield (PN57 to PN62 and PN83 to PN88), emergence (at the beginning ofeach open field task), elevated plus maze (PN57 to PN62, following theopen field task), radial arm maze (PN63 to PN82) and operant bar-pressing task (day PN83 to PN87). The following tests were donebetween days PN63 to PN82, and each test was administrated in fiveconsecutive days at least 30min after the animals had been fed: Balancebeam, climbing task and Rotarod. All animals were euthanized at HeathCanada at the end of behavioural tests to eventually perform selectedtissue residue analysis and brain ROS measurements (not presentedhere).

2.2. Open field locomotor activity

The automatedmeasurement of activity was carried out in an openfield every day from postnatal day PN57 to PN62 and to PN83 to PN88.The apparatus floor measured 90.5×90.5 cm and was divided intofour equal quadrants (45×45 cm). Each quadrant had an opaque greyacrylic floor and was surrounded by opaque grey acrylic 30.5 cm highwalls. Four mice were placed in random order in this apparatus, oneanimal per quadrant, inside a small cardboard housing unit (seeSection 2.4.1, Emergence). Once the mouse emerged from the housingunit, it was left to explore its quadrant for 30 min. An overheadcamera recorded the animal's movements that were later analyzedusing the video analysis program Ethovision 3.2 (Noldus). Thefollowing measures were extracted: rearing frequency, total distancetraveled, proportion of time spent in the center of the open field arenaand maximal speed reached by the mice.

2.3. Motor coordination

2.3.1. Balance beamMice were tested once daily for five consecutive days. Mice were

placed in themiddle of a 110 cm long and 1 cmwide beam, suspended21.8 cm above a wooden depression. The beam was divided into 11equal segments. During a 5-min period, the number of segmentscrossed by the animal was recorded. If applicable, the time spent onthe beam before the mouse fell was also recorded.

2.3.2. ClimbingThe mouse was placed in the middle of a 50 cm high by 32 cmwide

vertical mesh head-down above a wooden depression. Four measure-ments were recorded during the shortest time of either 10 min or thetime taken to reach the top of the mesh: time required orientingupwards, timeuntil climbing started and time to reach thebottomor the

top of the mesh. If applicable, the time spent on the mesh before a fallinto the depression was recorded. Mice underwent two trials per day(each trial was separated by at least 15 min) for five consecutive days.

2.3.3. Acceleration on the RotarodMice underwent five trials per day (each trial separated by at least

15min) for five consecutive days. The Rotarod (San Diego Instruments,CA) consisted of a rotating plastic cylinder (diameter=3.34 cm;length=45.6 cm). The cylinder was suspended 45.7 cm above acushioned depression and divided into four chambers (11.4 cm wide,71.1 cmhigh, and49.5 cmdeep). Aphotocell beamsituated between therod and the cushioned depression recorded the individual falling timeofeach mouse. Four mice were placed on the stationary cylinder for thefirst 2 min of each trial (habituation phase). If the animal fell inside thisperiod, the time was recorded, and this particular trial ended. After thehabituation phase, the rod started to rotate at a constant speed of 2 rpm,which was gradually increased by 2 rpm every 30 s until the mousecould no longer keep up and fell; the time of the fall was recorded.

2.4. Emotional reactivity

2.4.1. EmergenceThis task was performed immediately before each open field test.

The mouse was placed in one quadrant of the open field (seeSection 2.2 Locomotive activity in the open field) in a small housingunit (similar to one placed in their home cage), which was made ofgrey cardboard 11 cm×11 cm and 6.5 cm high with a small opening(diameter=2.5 cm) on each side. The length of time taken to emergefrom the housing unit into the open area was recorded.

2.4.2. Elevated plus mazeOne 10-min trial per day was administered from PN57 to PN62.

Themousewas placed in the center of the elevated plusmaze, 43.5 cmabove ground. Two opposite 28.5 cm long corridors had 17-cm highblack walls while the other two opposite 27.6 cm long corridors didnot have walls. Five measurements were recorded: time required forthe first entry into an open corridor, time required for the first entryinto a closed corridor, time spent in the center, the open corridors andthe closed corridors.

2.5. Learning and memory

2.5.1. Radial arm mazeTherewas one trial each day from PN63 to PN82. The labyrinthwas

made of a central platform (12.5 cm diameter) surrounded by eight30.5 cm long corridors at a 45° angle of each other. Each 5.3 cm widecorridor was enclosed in 15 cm high walls. The maze was made ofsolid wood covered by clear Plexiglas. At the end of each corridor,there was a 0.5 cm thick wall made of clear Plexiglas allowing themouse to see outside. The maze was baited with one small 3 mg pieceof pastamade of durumwheat placed at the end of each corridor. Micewere initially placed in the central platform and the animals' goal wasto navigate in each corridor and take the food located near the endwithout returning to a previously visited corridor. Each entry into apreviously visited corridorwas noted as an error. The time required bythe animal to visit each arm and the number of errors were recorded.

2.5.2. Operant-learning task (Skinner box)Mice were tested for five consecutive days from PN86 to PN90. The

operant test cage (13×13×18.4 cm)wasmade of three black Plexiglaswalls, one front clear Plexiglas wall, and a grid floor. A metal bar(5.7×3.5 cm) and a food cup (1.3×3.8 cm) extended from the frontwall andwere separated by awhite Plexiglas partition (5 cm long, 1 cmwide and 18.4 cm high). Photocell beams in front of the foodcup, thebar and the back of the chamber recorded the animal's movement.After a bar-press, themouse had to go around the partition to reach the

Table 1Total distance (in centimetres) traveled by the mice in the open field task.

Control Chronic Bolus

Day 1 10230±325 8803±386 8956±601Day 2 10186±438 10420±1183 8644±297Day 3 10344±634 9054±371 8814±367Day 4 9596±448 8923±250 8493±334Day 5 10017±528 9485±272 9418±524Day 6 10720±463 9438±308 9421±316Day 7 11214±562 9701±323 9224±331Day 8 11107±458 10109±484 13847±4622Day 9 11481±513 10002±464 9590±393Day 10 11359±486 10721±307 9415±355


food cup and receive the reinforcement. The test cage controls wereprogrammed for continuous reinforcement (CRF 1). Mice wereallowed to bar-press for 30 min. A reinforced trial was defined as abar press followedwithin 30 s by the consumption of the reinforcer (asmall 3 mg piece of pasta made of durum wheat). Mice were givensome of these reinforcers in the days preceding the training so thatthey readily consumed these reinforcers during training.

2.6. Statistical analysis

Measures were analyzed using a multivariate analysis for repeatedmeasures. When the sphericity assumption was not met, Greenhouse–Geisser correction of degrees of freedom was applied and reported foreach corrected F statistic. Multiple comparisons were performed usingTukey's HSD test with Bonferroni correction for significance level.

3. Results

3.1. Locomotor activity in the open-field

Rearing frequency, ameasureof verticalmovements did not showaneffect of groups (F(2,56)=0.47). However, therewas a significant effectof days of testing (F(6,353)=9.31 pb0.001). This difference was due toa smaller rearing frequency on the first day and a higher one on the lastday in comparison to the other days. The day by group interaction didnot reach significance (F(13,353)=1.7 p=0.06). Fig. 1 shows that therewas a tendency for all groups to have higher number of rearing as thedays progressed. Also, the chronic group tended to make slightly lessrearing during the last 2 days of testing. The second measure, anindicator of animals' general activity, was the total distance traveled bythe animal during the test (Table 1). There was no significant differenceeither for group (F(2,56)=1.9), day (F(1,73)=1.85) or day by groupinteraction (F(3,73)=0.8). The next measure was the proportion oftime spent in the center of the open field (Table 2), whereas the centerarea was defined as an area excluding a 3-cm wide area along the 4walls. This indirectmeasure of emotional reactivitydidnot showagroupeffect (F(2,56)=0.002) but a significant effect of day (F(6,357)=11.98,pb0.001). Post-hoc tests revealed that all groups spent less time in thecenter of the maze during days 1, 4 and 5. There was no day by groupinteraction (F(13,357)=0.89). The final measure was maximalspeed reached by mice (Table 3). This measure was highly variableand there were no significant effects of either group (F(2,56)=1.95),day (F(7,413)=0.81) or day by group interaction (F(15,413)=0.342).Althoughnoneof the comparisonswere significant, inspection of Table 3

Fig. 1. Mice rearing frequency in locomotive activity testing in open field task. Datapoints are group means±S.E.M. ⁎pb0.05 different from control group.

reveals that animals in the bolus group tended to have a lesser meanmaximal speed than the control group on most days except two whilethe chronic group maximal speed was lower than that of the controlgroup every day. However, the high variability of that measureprecludes any conclusion. Overall, the open field results yielded amodest indication that the chronic group had slightly decreased verticalmovement on the last 2 days of testing but no othermeasure in that testfurther supported an impact of methylmercury.

3.2. Motor coordination

3.2.1. Balance beamTwo performance measures were taken. The first one was the

time to complete the task (Fig. 2). For this measure, there were nogroup (F(2,56)=1.73), day (F(2,129)=0.73) or day by group effects(F(5,129)=1.86 p=0.11), although therewas a tendency for controlsto complete the test faster in the last 3 days. The second measure wasthe distance traveled on the beam (Fig. 3). There was no group effect(F(2,56)=2.29) but a day effect (F(3,175)=4.33 p=0.005) indicat-ing an increased distance traveled over time, however there was noday by group interaction (F(5,175)=1.043). The chronic grouptended to travel less than the control group on the last 3 days oftesting. During testing, we noted the possibility of a ceiling effect inthat the apparatus used may not have provided a sufficientlychallenging task for the mice because the beam's width may havebeen too large and may not have discriminated more subtle deficits.

3.2.2. ClimbingA total of three measures were taken in the climbing task. The

first measure was the time to reorient towards the top of the appa-ratus (Table 4). There was no significant difference between eithergroup (F(2,32)=0.79), day (F(3, 94)=0.97) or day by group inter-action (F(6,94)=0.96). The second measure was the time taken tostart climbing towards the top of the apparatus (Table 5). For this

Table 2Percentage of time spent in the center of the open field Arena.


Day 1 0.52±0.03 0.53±0.03 0.49±0.04Day 2 0.57±0.02 0.59±0.03 0.54±0.03Day 3 0.57±0.03 0.56±0.03 0.57±0.04Day 4 0.55±0.02 0.49±0.03 0.53±0.03Day 5 0.54±0.03 0.52±0.03 0.55±0.03Day 6 0.63±0.02 0.58±0.03 0.62±0.02Day 7 0.60±0.03 0.60±0.02 0.64±0.02Day 8 0.62±0.03 0.65±0.02 0.63±0.03Day 9 0.62±0.03 0.64±0.03 0.63±0.03Day 10 0.60±0.03 0.64±0.02 0.61±0.03

(group means±S.E.M.).

Table 3Maximal speed (cm/s) the mice reached in the open field task.


Day 1 84.2±15.6 76.3±16.9 62.2±10.9Day 2 77.8±13.2 72.2±12.5 86.0±15.1Day 3 80.0±13.9 66.2±12.4 70.0±11.3Day 4 81.9±12.3 72.9±14.2 79.8±11.8Day 5 80.4±13.7 51.8±2.3 65.8±11.5Day 6 64.1±8.6 62.1±10.7 58.0±6.6Day 7 92.7±13.9 68.8±10.9 66.3±9.4Day 8 76.6±10.6 72.1±9.0 81.4±19.2Day 9 90.8±13.5 69.8±8.1 79.8±10.4Day 10 87.4±10.8 72.5±7.1 75.1±9.5

Table 4Time (s) to reorient upwards in the climbing task (group means±S.E.M.).


Day 1 7.4±0.8 7.7±1.6 7.9±1.0Day 2 8.7±2.6 6.9±1.0 5.9±1.3Day 3 10.2±3.1 11.0±4.5 6.9±1.3Day 4 6.8±1.6 11.7±3.2 8.7±2.3Day 5 4.9±1.3 10.8±3.4 5.9±1.1

Table 5Time (s) to start climbing towards the top in the Climbing Test (group means±S.E.M.).




measure, there was no group effect (F(2,33)=1.51), a significantday effect (F(2, 62)=11.42, pb0.001) and no day by group inter-action (F(4,62)=0.82) indicating that all animals started movingtoward the top of the apparatus more quickly on the last two days incomparison to the first three. Even though themean time of the chronicgroup was consistently higher, high variability on this measureobscured this apparent difference. The last measure was the timetaken to climb to the top of the apparatus (Fig. 4). There was a groupeffect (F(2,55)=4.24pb0.001) for thismeasure. Post-hoc tests revealedthat both experimental groups took longer than the control group toreach the top. There was also a day effect (F(2, 120)=13.54, pb0.001)which revealed an increased time to reach the top as days of testing

Fig. 2. The time taken (days) to complete the balance beam task. Data points are groupmeans±S.E.M.

Fig. 3. Total distance traveled in thebalancebeamtask.Datapoints aregroupmeans±S.E.M.⁎pb0.05 different from control group.

progressed which was similar in all groups as indicated by a non-significant day by group interaction (F(4,120)=0.5).

3.2.3. Acceleration on the RotarodSince the five repeated trials on each day were showing a high

correlation for both measures taken (rN0.9), these trials wereaveraged over each day. The first measure was the time during thehabituation period where mice stayed on the drum (Table 6). Therewas no effect of group (F(2,57)=1.5), a day effect (F(2,112)=9.21,pb0.001), but no group by day interaction (F(4,112)=0.66). Theday effect however, was judged to be essentially due to some animalson day 1 who did not stay on the drum for the full 2-minutehabituation phase. The second measure was the time during whichthe mice stayed on the drum while accelerating (Fig. 5), a measure ofanimal motor coordination and endurance. There was no effect ofgroup (F(2,57)=1.33), a day effect (F(3,174)=23.0, pb0.001), butno day by group interaction (F(6,174)=0.81). Again, as above, theday effect was assessed to be essentially due to day 1, where animalsonly managed to stay a short time on the rotating drum.

Fig. 4. Time to reach the horizontal summit or the bottom of themesh the climbing task.Data points are group means±S.E.M. ⁎pb0.05 different from control group.

Table 6Time (s) mice stayed on the drum during the 2 min habituation period in the Rotarodtask (group means±S.E.M.).



Only an effect of day was found mainly due to the slightly lower time of a few animalson the first day.

Fig. 6. The time to emerge from the black box into the open field area in the emergencetask. Data points are group means±S.E.M.


In summary, in motor tasks, the significant effect of the methyl-mercury treatment was limited to the climbing task. The results of thetwo other tasks were less useful possibly due to the large variability ofthese measures even though chronic animals tended to performworse in the balance beam and climbing tasks. The Rotarod was notcompleted by all animals and did not appear to be a useful task in thecontext of testing mice for small deficits. It was unclear that thebalance beam apparatus provided a sufficiently challenging situationto reveal more subtle deficits.

3.3. Emotional reactivity

3.3.1. EmergenceIn this test, the latency to emerge from a protected space was

measured (Fig. 6), which was a measure of animals' anxiety level.Therewas no significant effect of group (F(2,57)=1.48) but an effect ofdays (F(2, 127)=14.41 pb0.001). The latency to emergewas in generallonger in the first 4 days in comparison to the following 6 days. Therewas a trend toward a day by group interaction (F(5,127)=2.16p=0.07), however no clear pattern of interaction could be observedexcept for a general tendency of the animals in the chronic group tohabituate more slowly to the situation.

3.3.2. Elevated plus mazeThreemeasureswere obtained and are shown on Fig. 7. The first one

was the time spent in theopenarmsof themaze that isusually takenasameasure of anxiety. There was no group effect (F(2,56)=1.68), butthere was an effect of day (F(3,137)=5.29 pb0.003) which indicatedthat all groups remained a little longer in the open arms as the test daysprogressed. Post-hoc tests suggested that days 2 and 3 had the lowestproportion of time spent in the open arm. There was no group by day

Fig. 5. Timemice stayed on the drumwhile accelerating in the Rotarod task. Data pointsare group means±S.E.M.

interaction (F(5,137)=1.16). However, it was notable that, in general,very little time was spent by all mice in the open arms suggesting thepossibility that the test situation was highly anxiogenic for all animalsand thusmight not have allowed to observe small differences in anxiety.Two complementary measures were also used: The time spent in theclosed arm and the center of the maze (because these were highlycorrelated, only the closed armdatawas analyzed). For the time spent inclosed arms, there was no group effect (F(2,56)=1.07) but a day effect(F(3,156)=3.2 pb0.03). Post-hoc tests indicated that days 1 and 3weresignificantly different but no interaction was found (F(6,156)=1.71,p=0.13). Only the chronic group showed a tendency of increasedproportion of time spent in closed arms (by staying less time in thecenter area). That measure for the other two groups was stableover days. A final measure was analyzed: the time to first enter anopen arm (Table 7). For thismeasure, therewas a trend towards a groupeffect (F(2,57)=2.64 p=0.08), a day effect (F(3,184)=4.7 pb0.005)

Fig. 7. Percentage of time spent in various sections in the elevated plus maze. Datapoints are group means. The percentage of time spent in the closed arms (top threelines), center (three middle lines) and open arms (bottom three lines) are showed.

Table 7Time (s) to the first entry in open arm in the cross labyrinth task (group means±S.E.M.).



There was a non-significant trend for the experimental groups, particularly the chronicgroup to have longer latencies.

Fig. 9. Time to complete the radial arm maze task. Data points are group means.⁎pb0.05 different from control group.


but no day by group interaction (F(7,184)=0.97). The chronic groupshowed a non-significant trend for an increased latency to enter in anopen arm. Overall, no substantial differences were observed but therewas a small (not significant) tendency for the chronic group to showincreased anxiety as measured by an increased time spent in the closedarm and also a slightly higher latency to enter the open arm for the firsttime.

3.4. Learning and memory

3.4.1. Radial arm mazeTwo measures were obtained from the radial maze task. The first

one was the number of errors made (Fig. 8) by animals. There was agroup effect (F(2,55)=4.27 pb0.02) and post-hoc tests revealed asignificant difference between the chronic and the control group. Asimilar trend was observed for the bolus group but that difference didnot reach significance because these animals performed as well orbetter than the control group on 10 of 20 days of testing. There was aday effect (F(11,612)=9.1 pb0.001) indicating that less errors weremade as mice learned the task but there was no day by groupinteraction (F(22,612)=0.95). The second measure was the time tocomplete the maze (Fig. 9). There was again a group effect (F(2,55)=10.2 pb0.001) which indicated that overall both experimental groupstook longer than the control group to complete the task. There was aday effect (F(5,295)=6.23 pb0.001) indicating that overall, animalstook less time to complete the maze as they learned (particu-larly up until days 8–10) but there was no day by group interaction(F(11,295)=0.742).

3.4.2. Operant learning task (Skinner box)Two measures were obtained from this task. The first one was the

number of reinforced bar presses (Fig. 10) by animals, which is a

Fig. 8.Number of errors made in the radial armmaze task. Data points are groupmeans.⁎pb0.05 different from control group.

measure of learning andmemory. Therewas no group effect (F(2,50)=1.25) but a day effect (F(3,171)=11.01 pb0.001), which was anindication that all animals improved with time. However, there was anabsence of group by day interaction (F(7,171)=1.0). The control groupappeared slightly better than the twoMeHg groups but high variabilityobscured this apparent difference. The second measure was thelocomotor activity in the operant box (Fig. 11). There was a groupeffect (F(2,50)=4.23 p=0.02) and post-hoc tests suggested that micein both experimental groups, particularly the bolus group, were lessactive than control animals. There was a day effect (F(3,120)=29.34pb0.001) suggesting an increased locomotion over days of testing butno day by group interaction (F(5,120)=0.996). These results suggestedrelatively unimpaired learning and memory abilities in the twomethylmercury groups but a reduced activity in the methylmercurygroups. This is a potential indication of a reduction of exploratorybehaviour. However, because mice were deprived in this task, theresults could also indicate a reduction of the typical increase in motoractivity associated with food deprivation.

Overall, the learning ability appeared to be reduced in tasksrequiring spatial navigation (radial arm maze) while operant tasksthat are less based on spatial abilities remained mostly unaltered.However, it was noted in both tasks that both groups of methylmer-cury treated pups were less active.

Fig. 10. Number of reinforced bar-presses in the operant learning task. Data points aregroup means±S.E.M.

Fig. 11. General activity in the operant learning task. Activity was measured by thenumber of times an animal broke photocell beams in the operant cage. Data points aregroup means±S.E.M. ⁎pb0.05 different from control group.


4. Discussion

The present results suggest that prenatal exposure to MeHgproduces behavioral deficits that were captured by some tests but notothers. Overall, the most consistent effects were found for the time toclimb a vertical mesh. The other motor coordination tasks did notsignificantly differentiate the MeHg-dosed groups from the controlanimals. In terms of general motor activity, both experimental groupswere significantly less active when food deprived but not in the openfield test that did not involve any food restriction. There was a small butconsistent increase in the behavioural expression of anxiety-relatedbehaviours in the chronic group. Finally, both experimental groups hadimpaired spatial learning abilities in the radialmazewhile learning of anoperant task was not impaired. Taken together, the results generallyrevealed that chronic exposure produced the largest impairments in alltasks while the performance of the bolus group was closer but notequivalent to the control group. These results demonstrate that theadministration of the same MeHg amounts but in different dosingpatterns led to slightly different patterns of deficits.

The consensus concerning the minimum level of MeHg in the braincapable of producing toxic effects has been in the range of 1–2 µg/g BWin both humans and experimental animals [19]. Although brain levels ofMeHg were not measured in the present study, preliminary analysesshowed that newborn pups, from dams that received the same bolusregimen as in the current study, had brain levels of 13.3 mg/g wetweight (male) and 12.8 µg/g wet weight (female) [18]. This is clearly inthe range of brain concentrations where effects would be expected. Theequivalent maternal brain MeHg concentration measured for thisparticular dam was 5.9 µg/g of wet weight. Although equivalent datafor MeHg brain concentrations are not yet available for the pups born ofthe chronic dosed dams, a preliminary pilot phase study [17] reportedhigher brain concentrations under this dosing administration than forthe bolus-dosed group.

Because there is a wide variety in prior research for the administra-tion procedure, length of exposure to MeHg, animal strain used andbehavioral tests used, comparison between the present results andprevious experiments is not straightforward. The following paragraphswill analyse the current results in light of previous research thatadministered MeHg during the gestational period or shortly thereafter.Gestational critical periods appear to center around day 15 whenforebrain ventricular zone is producing neurons in the embryoniccerebral cortex [10] and when cell migration begins for the cortex [26]and cerebellum [22,31]. As reported by these studies, MeHg exposuredecreases cell proliferation associated with new neurons [13,29] andthese new neurons can have abnormal final destinations and/or

organization. These effects might be due to interactions will the cellcycle [3,29], migration mechanisms [20] or early exotoxicity due toincreased susceptibility to glutamate [4] or enhanced sensitivity ofNMDA receptors [23] that may make neurons of P16 animals mostsusceptible to MeHg neurotoxicity. In turn, abnormal brain develop-ment can lead to behavioural deficits in the adult organism.

Several studies in rodents reported delayed or abnormal maturationof the cerebellum after developmental treatment with MeHg [6,22,30],which led to many studies of the impact of MeHg onmotor behaviours.In the present experiment, a number of behavioural deficits wereobserved that were consistent with previous experiments. Generallocomotor activity in the open field was essentially not affected. Othermeasures of locomotor activity in the open field test, such as the totaldistance traveled and themaximum speed reached by animals, also didnot support a general effect of MeHg exposure on locomotion aspreviously reported in some experiments [11,21]. However, locomotoractivity measures obtained in the Skinner box and radial maze suggestthat goal-directed locomotor activity was reduced in food-deprivedmice that were exposed to prenatal methylmercury. These last resultsare consistent with other experiments that have reported eitherdecreased rearing and total distance traveled in the open field forC57BL/6 mice receiving very small doses of MeHg (0.01 μg/g BW) fromgestational day 8 to 18 [24], lesser activity in the home cage during thelight phase in C57BL/6 receiving 3 μg/g BW of methylmercury ongestational day 14–16 [21] or reduction in the number of rearings in ratsadministered0.75mg/kgBW/day(MeHg) frompostnatal day (PD)14 toPD23 in theopenfieldwithout altering thedistance traveled, the restingtime and the time spent in the central part of the arena.Onepossibility isthat effect on motor behaviour is more easily observable in food-restricted animals as during the operant task and radial maze testsbecause food deprivation increases general locomotor activity. Thishypothesis is consistentwith the observation that in the last five days ofthe open field task, when mice were on a restricted diet, the chronic-dose group tended to have a lower rearing frequency than the controlgroup. A similar decrease in rearing was observed after a MeHg bolusdose of 8 μg/g BW [2,4].

The motor coordination tests used in the present experiment havebeen used extensively to evaluate motor deficits, and our results areconsistent with many previous studies [11,16] where there was nostrong evidence of adverse effects of prenatal methylmercuryexposure. In our experiment, the only significant difference betweencontrol and experimental groups was the time taken to climb avertical grid. Only the recent study by Montgomery observed Rotaroddeficits [24]. We had the impression that in our experiment, theclimbing task was the most challenging in terms of muscle strength.Therefore possibly, prenatal MeHg exposure does produces motorcoordination deficits but the tests commonly used may not challengethe motor systems sufficiently, and thus deficits remain undetected.To determine which alternative is correct, more challenging tests mayneed to be used. One such test could be one in which mice are placedupside down hanging from amesh or metal grid and the time taken tofall from the mesh recorded. A similar task is the “tight wire” testwhere the mouse is suspended upside down from a wire. The samelatency to fall is taken as a measure of motor strength. Finally, in theRotarod task, a smaller diameter drum may be useful to increasedifficulty. Overall, although we observed a small effect of methyl-mercury in the climbing task, other motor coordination measures didnot provide strong evidence indicating an effect of prenatal MeHgexposure at these administered levels.

Contrary to two previous publications [9,21], where emotionalreactivity was affected by methylmercury, we did not find anysignificant difference between experimental and control groups.Although the chronic group shown a tendency for increasing anxietyas measured by an increased time spent in closed arms and a higherlatency to enter the open arm of the elevated plusmaze, these data didnot reach significance. We also observed in all groups a reduction of


anxiety-like behaviours as tests were repeated, which suggest asimilar habituation to anxiogenic situations. The only reservation tothis conclusion was the very small proportion of time spent in theopen arms by all groups suggesting a floor effect for the C57BL/6mouse strain in the elevated plus maze test. A consideration for thefuture might be to create a reduced anxiogenic situation by includinga small 1-cm wall around the open arms.

The involvement of the hippocampus in MeHg neurotoxicity isstrongly supported by recent data in rats [12], showing that a singleperinatal injection of a moderate dose of MeHg (5 μg/g BW) leads to areduction in hippocampal size and cell number, particularly in thegranule cell layer and hilus of the dentate gyrus. This study andprevious behavioural ones suggest that learning and memoryprocesses may be altered by MeHg prenatal exposure.

Even if there was no evidence of learning impairment of theoperant task, in the radial arm maze, both experimental groups mademore errors and took consequently more time to complete the taskthan the control group, a result consistent with previous studies[8,15,16,21,24,25,34]. This finding suggested the capacity of theMeHg-exposed animals to navigate in the maze using visual clues(spatial navigation) and/or the ability to memorize which arm hadalready been entered (working memory) was impaired.

In addition, the extensive testing regimen in the presentexperiment could have introduced a new variable. The dailymanipulation and testing of all animals is somewhat equivalent toan enrichment procedure. Such environmental enrichment haspreviously been shown to improve the functional outcome of brainlesions [35]. In the present experiment, enrichment may have servedto reduce the deficit produced by MeHg exposure. The presentexperiment was not designed to address this hypothesis. However,there was little indication that tests performed in the early part oftesting (compared to the later part) discriminated between controland experimental groups. A more definite test of this hypothesiswould need to be carefully designed.

In summary, only the impairment of prenatal methylmercuryexposure on memory of visio-spatial and working information wasclearly observed in this study. Although the locomotor activity, motorcoordination and emotional reactivity in various tests did not showclear evidence that MeHg exposure had adverse effect, the chronicgroup had a general tendency for lower activity and higher anxiety-like behaviors. Given the two in utero exposure patterns (essentiallyboth chronic exposures but one at a lower steady state dose pattern),it appears that a chronic dosing regimen of MeHg with a highersteady-state dose is more damaging to the fetus than an occasionalbolus MeHg exposure superimposed on a lower steady-state doseregimen. Further studies will be necessary to determine if a bolus doseat a critical time during brain development could be as damaging (ormore so) as steady state (i.e. chronic) exposure. For example, when aparticular endpoint occurring on a particular gestation day has beencharacterized. For example one study showed that a single exposureto methamphetamine during fetal development caused long-termdamage as measured by oxidative DNA damage in the brain [37].

The current animal study was designed (projected to a humanmodel) to test the effects of the consumption of two large meals of fish(i.e., ‘bolus’ doses) during the course of pregnancy, since these couldpotentially result in an intake of severalmilligrams ofmethylmercury, ifthe species of fish was one known to contain elevated MeHg. However,critical periods of susceptibility of each neurotoxin during thedevelopment of the fetal nervous system may differ [27]. Thus, thetiming of administration may be a key factor, so that, in the reportedstudy, the bolus doses given on GD 12 and GD 16 might not havecorresponded to the most ‘critical’ period for MeHg. The substantialdifferences in gestation times between humans andmice also add to thedifficulty in selecting equivalent periods of brain development. This isparticularly the case for rodents if some of the brain development thatoccurs prenatally in humans happens postnatally in mice. However,

the chronic administration in the present experiment may haveincreased the likelihood of observing deleterious behavioural effectsby lengthening the period during which the foetus was exposed toMeHg albeit to a smaller dosage.

Behavioural studies on humans are complex because the environ-ment and individual pattern of exposures are difficult to control [16].In that respect, animal models have proven useful to approximate theeffect of MeHg exposure on the human brain [32,33]. The presentexperiment adds to this enquiry by showing that MeHg exposureduring the prenatal period produces functional deficits postnatally. Italso was found that chronic exposure to lower levels is moredamaging than infrequent acute exposure to higher levels eventhough total MeHg exposure was equivalent.

Conflict of interest

Nothing declared.

Acknowledgments

Thanks are extended to Kevin Teutenberg, Angela L. Allen, JasonPolak, Alexia de Jong, Maya Elias, Julie Chabot and Rebecca Holby, fortheir excellent assistance, and to Sylvie Emond for taking car of theanimals at Universiy of Ottawa. Thanks are also expressed to the staff ofthe Animal Resources Division of Health Canada, including veterinarianDr. Martha Navarro, managers Bill Pearce and Wendy Lezama, andanimal health technologists including: Gail Merrikin, Kevin Kittle,Dominique Patry, Don Demers and Genevieve L'Ecuyer. Special thanksare extended to Dr. David Blakey, Director, Environmental HealthScience and Research Bureau, to Russ Shearer, Director, NorthernContaminants Program of Indian and Northern Affairs Canada, andDr. Constantine Tikhonov of the First Nations and Inuit Health Branch, ofHealth Canada., for theirfinancial and administrative support and toDr. JVan Oostdam and Dr. S Donaldson, Health Canada, for encouragementthroughout. Thanks are also extended to Dr.W. Bowers, Health Canada,for loan of the Rotarod equipment.

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Neurobehavioral effect of chronic and bolus doses of methylmercury following prenatal exposure in C57BL/6 weanling mice