Coordination Deficits Induced in Young Adult Mice Treated with Methylmercury

Abstract

Male and female C57BL/6J mice starting at postnatal (P) day 34 were exposed orally to five divided doses totaling 1.0 or 5.0 mg/kg of methylmercury (MeHg; given as methylmercuric chloride) or sterile deionized water in moistened rodent chow. After a 5-day waiting period, control and MeHg-treated mice were subjected to a standard battery of behavior tests for balance and motor coordination. Latency to falling on the accelerating rota-rod was significantly decreased in 5.0 mg/kg MeHg-exposed mice when compared to control mice. In the open field, horizontal exploration with respect to total distance traveled during the first 5 min on the first test day was significantly reduced in 1.0 mg/kg MeHg-exposed mice when compared to control mice. Rearing activity was not affected by MeHg treatment. In the footprint analysis, angle of foot placement measured in 1.0 mg/kg MeHg-treated mice was significantly greater compared to control mice. Base stance and stride length were unaffected by MeHg treatment. On the vertical pole test, 10 mice from each treatment group fell off the pole during the time the pole was shifted from a horizontal position to a vertical position, whereas none of the control mice fell. These results indicate that short-term, low to moderate doses of MeHg in young adult mice can be detrimental to motor coordination and balance.

Mercury (Hg) is an ubiquitous and hazardous environmental contaminant found in ocean and freshwater fish, shellfish, and plants (Eisler 2004; Risher, Murray, and Prince 2002; Sanfeliu, Sebesta, and Ki 2001; Shenker, Guo, and Shapiro 1998; Siciliano et al. 2003). The organic or methylated form of Hg (methylmercury; MeHg) accounts for most of the Hg to which humans are exposed (Rice 1995). Hg as MeHg gets rapidly absorbed from the gastrointestinal tract and is readily transported into the brain where it is sequestered and gradually converted into inorganic Hg. MeHg is a potent neurotoxicant known to cause neuronal degeneration, and neocortical and cerebellar granule neurons, in particular, are very sensitive to MeHg exposure (Bakir et al. 1973; Limke and Atchison 2002; Nielsen 1992; Rikuzo and Mitsuhiro 1996; Yuan and Atchison 1999). One of the most severe intoxications ever reported in humans was due to eating contaminated fish and shellfish from the Minamata Bay in Japan in the 1950s (Clarkson 2002; Harada 1995; Matsumoto and Takeuchi 1965). Autopsies of affected adults revealed extensive damage of the cerebellum and cortical sulci (Matsumoto and Takeuchi 1965). In the early 1970s, a second significant episode of MeHg poisoning occurred in Iraq. Numerous individuals exposed to MeHg exhibited symptoms comparable to the residents of Minamata Bay (Amin-Zaki et al. 1974).

In 1997, the U.S. Environmental Protection Agency (EPA) recommended a reference dose of no more than 0.1 μg/kg body weight/day for MeHg exposure in the human population (Clarkson 2002; U.S. EPA 1972). This translates into limiting consumption of food sources with MeHg contamination, including freshwater fish and ocean fish such as tuna (Limke and Atchison 2002; Rice 1996). The persistent contamination of our environment with mercury indicates that mercury will remain bioavailable as MeHg for decades, affecting current as well as future generations (Grandjean and Weihe 1998).

Studies using rodent animal models have proven useful in simulating neurobehavioral effects of MeHg exposure (Spyker and Smithberg 1972; Spyker, Sparber, and Goldberg 1972). However, most neurological effects observed in rodents have been reported from studies that utilized prenatal exposure to MeHg (Goulet, Dore, and Mirault 2003; Kakita et al. 2000; Kim et al. 2000; Newland and Rasmussen 2000; Newland, Reile, and Langston 2004; Rasmussen and Newland 2001; Rossi et al. 1997; Sakamoto et al. 2002; Salvaterra et al. 1973; Su and Okita 1976). Still, comparatively little is known about the behavioral effects of MeHg exposure on young adults at low to moderate exposure levels.

In the present study, male and female C57BL/6J mice starting at postnatal day (P) 34 were exposed to MeHg via food using a total dose of either 1.0 or 5.0 mg/kg body weight. They were compared with age-matched control (vehicle-only) mice using a standard battery of behavior tests for motor coordination, equilibrium, and open field activity, starting 5 days after the last dose of MeHg. It is well known from the literature that the central nervous system (CNS) effects of MeHg are typically delayed in onset (Rice 1996). In our preliminary testing we observed some behavioral effects of MeHg exposure using a 5-day waiting period after cessation of exposure and we chose to maintain that waiting time for the remainder of this study.

MATERIALS AND METHODS

Animals

Adult C57BL/6J wild type (+/+) male and female mice, originally obtained from The Jackson Laboratory (Bar Harbor, MA, USA), were bred to produce wild-type offspring. A total of 43 male and 34 female mice were used in this study. Mice were further divided into three treatment groups as follows: control (22), 1.0 mg/kg methylmercuric chloride (MMC) (26), and 5.0 mg/kg MMC (29). All mice were housed at the Laboratory Animal Research and Resource building, Texas A&M University, in a constant temperature (21°C to 22°C) and humidity (45% to 50%) room with a 12-h light-dark cycle. The mice were weaned at 29 days of age and housed individually for the duration of training, dosing, and behavior testing. All procedures were carried out in accordance with the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised 1996).

Chemicals and Dosing

Methylmercury (MeHg) provided as methylmercuric chloride (MMC; 95% purity; Alfa Aesar, Ward Hill, MA, USA) was initially prepared as a stock solution of 1.0 mg/ml in sterile deionized water and further diluted with sterile deionized water for addition to food. All MMC solutions were stored at 4°C until used. Starting at postnatal day (P) 29 all mice were trained to eat moistened rodent chow for 5 days as described earlier by Bellum et al. (2007). Starting at P34, one group of mice received 0.2 mg/kg and a second group of mice received 1.0 mg/kg MMC, orally in 2.0 g moistened rodent chow, given daily for 5 consecutive days to achieve a total dose of 1.0 or 5.0 mg/kg body weight, respectively. The control mice received only vehicle (0.1 ml sterile deionized water) in 2.0 g moistened rodent chow for 5 consecutive days. The mice were given moistened rodent chow with MMC or vehicle only during the dark phase of light-dark cycle and had access to dry rodent chow ad libitum only during the light phase of the light-dark cycle. All mice had access to water 24 h every day.

Rota-rod

Quantitative measurements of motor coordination in control, 1.0 and 5.0 mg/kg MeHg-treated mice were performed using an accelerating rota-rod (model 7650, UGO Basile, Comerio, VA, Italy). The rota-rod consisted of a plastic rod (diameter = 3 cm; length = 30 cm) partitioned off with round plates to prevent the mice from escaping from the sides of the rod. The rod was covered with smooth plastic tubing and suspended 16 cm above five plastic levers attached to timers that stop when mice land on the lever surface. The mice were placed on the rod that rotated at 3 rpm at 0 min and accelerated to 26 rpm after 3 min of run time. Mice were oriented perpendicular to the long axis of the rod, such that the mice had to make forward walking movements to avoid falling. Starting at P44, the latency time to fall were measured for 3 consecutive days (days 6, 7, and 8 after the last dose of MeHg), with each mouse experiencing four trials per day with an intertrial interval of 10 min. The first three trials on each day were considered training; data from the fourth trial of each day were analyzed.

Open Field Activity

Horizontal and vertical exploration were analyzed in all mice by measuring total distance traveled and rearing activity in an open field using Versa Max System activity chambers (Accuscan Instruments, Columbus, OH, USA). Each activity chamber was equipped with photocell beams that use x-, y-, and z-coordinates to detect and digitally record horizontal and vertical movements. The Plexiglas chambers (42 × 42 cm) had walls that were 30 cm in height. Each chamber was further divided into four equal compartments of 21 × 21 cm. Two compartments (second of top row and first of bottom row) in each chamber were used for testing to prevent overlap of beam pathways. The mice were placed in the same corner of each chamber at the beginning of each trial and activity levels for four mice at a time were recorded for 30 min per day, on days 9 and 10 after the last dose of MeHg.

Footprint Analysis

Changes in motor coordination and balance were assessed using footprint analysis. A plastic-lined walkway, 100 cm long, 6 cm wide, 15 cm tall, and closed off at one end, was used. The hind paws of each mouse were dipped in nontoxic glass paint and the mouse was allowed to walk on a strip of paper placed on the floor of the walkway. Stride length (in mm), angle of foot placement (in degrees) and distance between the hind feet (base stance, in mm) were measured for five consecutive strides and averaged for each mouse.

Vertical Pole Test

Coordination and balance also were assessed using the vertical pole test. Mice were individually placed on a 60 cm long, 2.5 cm diameter plastic rod covered with tape to provide adequate grip. The rod was held horizontally 50 cm above the surface of a soft cushion to prevent injuries to the mice when falling. Mice were placed midway on the rod facing the end that was lifted up. Keeping one end of the rod stationary the other end was raised to a 45° angle over a 10-s time period. An additional 5 s was taken to shift the rod from 45° to 90°. Whether mice stayed on or fell off the rod during the 15-s testing period was recorded.

Statistical Analysis

Rota-rod data were analyzed with general linear model (GLM) repeated-measure analysis (α = 0.05), using SPSS (Windows version 11.0). Open field and footprint data were analyzed using GLM multivariate analysis of variance (ANOVA) and included treatment, gender, and interaction effects (two-way ANOVA). In all cases where no gender effects were observed, data from male and female mice were combined. Significant differences among treated and control groups were interpreted using the Tukey’s honest significant difference (HSD) post hoc test at α = 0.05. Chi-square tests were used to analyze vertical pole test data.

RESULTS

The total doses of 1.0 and 5.0 mg/kg MeHg did not produce overt signs of toxicity in treated mice. The MeHg-treated mice did not show any signs of significantly altered behavior or obvious ataxia compared to control mice and no differences in body weight were observed between control and treated mice (Table 1). We then evaluated the mice for more subtle changes in activity levels and coordination using a standard battery of behavior tests: rota-rod, open field activity, foot placement, and coordination on a vertical pole.

Rota-rod Performance

All mice tended to increase the length of time they stayed on the rota-rod when trials 1 to 4 were compared on each day of testing, with the mice tending to stay on the rota-rod the longest time on the fourth trial (data not shown). We also observed that the differences observed when comparing the fourth trials on the rota-rod were also reflected in the differences on trials 1 to 3 for each day (data not shown). On the fourth trial of all 3 days, GLM repeated-measure analysis indicated a significant treatment effect (p = .031) (Figure 1). No significant differences were observed between males and females (p = .145), and the interaction between treatment and sex (p = .553) was not significant. On the fourth trial of the third day, GLM univariate analysis of variance and the Tukey’s HSD post hoc test indicated the latency to falling exhibited by 5.0 mg/kg MeHg-exposed mice was significantly lower when compared to age-matched control mice (p = .001). However, 1.0 mg/kg MeHg-treated mice were not significantly different from control mice or 5.0 mg/kg MeHg-treated mice (Figure 1). GLM multivariate analysis of variance and the Tukey’s HSD post hoc test indicated a significant increase in latency to falling in control mice on day 3 when compared to day 1. Latency to falling on day 2 for control mice was not significantly different from day 1 or day 3. There was no significant increase in latency to falling in 1.0 mg/kg MeHg-exposed mice when data from all 3 days were compared (p = 0.335). The 5.0 mg/kg MeHg-exposed mice exhibited a significant increase in latency to falling on day 2 when compared to day 1 (p = .033). The latency to falling observed on day 3 for the 5.0 mg/kg MeHg-exposed mice was not significantly different from day 1 or day 2.

Open Field Activity

Analysis of total distance traveled and rearing activity during a 30-min session on 2 consecutive days did not reveal any significant differences (data not shown). We then focused on the first 5 min of the first day to assess exploratory behavior in a novel environment. Generally, a 5-min test session in an open field chamber is sufficient for preliminary assessment of motor activity or to evaluate gross abnormalities in locomotion (Crawley 1999). Figure 2 presents spontaneous locomotion activity in the open field during the first 5 min of recording on day 1. GLM multivariate analysis of variance indicated a significant difference in total distance traveled (p = .015) (Figure 2). There were no significant differences between males and females (p = .153). The interaction between MeHg exposure and sex was not significant (p = .239). Vertical exploration (i.e., rearing activity) did not show any significant differences between treated and nontreated mice (data not shown).

Footprint Analysis

Average angle of foot placement (Figure 3) was highly significantly different in 1.0 mg/kg MeHg-treated mice when compared to control mice (p = .003). Although there was a trend for the 5.0 mg/kg MeHg-treated mice to also show an increased angle of foot placement compared to control mice, the Tukey’s HSD post hoc test indicated that 5.0 mg/kg MeHg-treated mice were not significantly different from control or 1.0 mg/kg MeHg-treated mice. There was no gender effect (p = .087) or interaction effect between MeHg treatment and gender (p = .130). The stride length (p = .123) and base stance (i.e., distance between two hind paws) (p = .532) did not differ between control and MeHg-exposed mice (data not shown).

Vertical Pole Test

A chi-square test indicated a significant difference between control and MeHg-treated mice at α = 0.05 for the vertical pole test. Using degrees of freedom (df) set at 4, the chi-square calculated value was 14.34. The chi-square table value at df 4 was 9.488 (Table 1). No significant differences were observed when the chi-square test was performed with males and females separated within each treatment group (chi-square calculated value = 13.82; chi-square table value = 18.31 at α = 0.05; df = 10), so data from males and females within each treatment group were combined. The chi-square test was significantly different for control mice compared to 1.0 mg/kg MeHg-treated mice (chi-square calculated value = 10.73; chi-square table value = 5.991 at α = 0.05; df 2) and between control and 5.0 mg/kg MeHg-treated mice (chi-square calculated value = 10.38; chi-square table value = 5.991 at α = 0.05; df 2).

DISCUSSION

Based on these results it is clear that MeHg consumed at doses that do not produce overt ataxia can still have subtle but significant effects on activity and coordination in exposed mice. We did not observe any difference in food consumption patterns between control and treated mice. All mice readily consumed all of the moistened rodent chow provided to them during the dark phase of the light-dark cycle. Furthermore, previous data from our laboratory have shown that the feeding protocol used in this study produces very consistent brain mercury levels (Bellum et al. 2007).

Motor deficits described in the literature consist of salient neurobehavioral-type effects that occurred after developmental exposure to MeHg (Dore et al. 2001; Elsner et al. 1988; Inouye, Murao, and Kajiwara 1985; Kim et al. 2000; Rice 1996; Rossi et al. 1997; Spyker and Smithberg 1972). Most of the early studies using rodents showed behavior changes following a single or at most a few doses of MeHg (Baraldi et al. 2002; Dore et al. 2001; Inouye, Murao, and Kajiwara 1985; Kim et al. 2000; Lown et al. 1977; Pereira et al. 1999; Salvaterra et al. 1973; Su and Okita 1976; Vicente et al. 2004; Watanabe et al. 1999; Yin et al. 1997). Several recent studies have reported behavior defects following chronic exposure to MeHg during prenatal or early postnatal development (Goulet, Dore, and Mirault 2003; Kakita et al. 2000; Newland and Rasmussen 2000; Newland et al. 2004; Rasmussen and Newland 2001; Rossi et al. 1997; Sakamoto et al. 2002). Sakamoto et al. (1993) demonstrated apparent deficits in rota-rod performance in rats exposed to a total MeHg dose of 71.4 mg/kg starting from postnatal day (P) 35 and a dose-dependent inhibition in rota-rod performance in rats at P14 exposed to total dose of 26 mg/kg given over a period of 10 days. However, the doses used in these studies were much higher than those commonly encountered in the environment.

In the present study, we monitored motor coordination and balance changes in young adult mice exposed to two different doses of MeHg and compared to age-matched control mice. We observed significant differences for all four of the behavior tasks used in this study when MeHg-treated mice were compared to control mice. Specifically with rota-rod performance, we observed improved performance for most mice over the four trials each day, with the fourth trial generally eliciting the longest time to stay on the rota-rod on any given day. Therefore, we examined the fourth trial time for each mouse on each day and we observed a significantly shorter latency to falling in mice exposed to 5.0 mg/kg MeHg compared to age-matched control mice. In a study where Wistar rats were exposed to a moderate dose of MeHg (5 ppm/day) throughout gestation and lactation periods, Sakamoto et al. (2002) observed significant differences on rota-rod performance between control and MeHg-exposed rats at 5 weeks of age. In another study using rats, Sakamoto et al. (1993) demonstrated a dose-dependent decrease in latency to falling with a total oral dose of 26.0 or 71.4 mg/kg MeHg. However, the concentrations used in the Sakamoto (1993) study were 5 to 14 times higher than the 5.0 mg/kg MeHg total dose used in this study. In contrast, Goulet, Dore, and Mirault (2003) found no differences in latency to falling in 6-week-old mice chronically exposed to 4, 6, or 8 ppm MMC via drinking water during prenatal and early postnatal life. Mice in these experiments were subjected to a constant speed of 20 rpm, whereas in our experiments mice were subjected to acceleration from 3 to 26 rpm over 3 min. The diminished rota-rod performance exhibited by MeHg-exposed mice in this study indicates these mice experienced deficits in motor coordination and balance.

Results from our rota-rod experiment also revealed substantial learning effects in control mice, as demonstrated by improved performance on day 3 compared to day 1. Mice exposed to 1.0 mg/kg MeHg did not show the same improvement in performance as the control mice. On the other hand the 5.0 mg/kg MeHg-treated mice showed initial improvement in performance on day 2 compared to day 1 but did not improve on day 3 compared to day 2. These data indicate that moderate MeHg exposure in adult mice can affect cognitive as well as motor function. Additional testing of MeHg-exposed adult mice for deficits in cognitive function, such as use of the Morris water maze, is warranted.

Mice, when exposed to a novel environment, will typically actively explore the new environment during the first few minutes to get acquainted with the new space and/or try to find ways to escape (Crawley 1999). In our experiments, the first 5 min of open field testing revealed decreased horizontal exploration for 1.0 mg/kg MeHg-treated mice and a trend towards the same behavior in 5.0 mg/kg MeHg-exposed mice. These data are consistent with other rodent studies reported in the literature that examined horizontal exploration after exposure to MeHg (Dore et al. 2001; Goulet, Dore, and Mirault 2003; Kim et al. 2000; Lown et al. 1977; Pereira et al. 1999; Su and Okita 1976). Furthermore, consistent with some studies (Dore et al. 2001; Goulet, Dore, and Mirault 2003; Rossi et al. 1997) and contrary to others (Baraldi et al. 2002; Kim et al. 2000; Lown et al. 1977; Morganti et al. 1976; Salvaterra et al. 1973), we did not see any significant difference in rearing activity between control mice and mice exposed to MeHg. Although the precise behavioral significance of open field activity is not known, one can conclude that it measures complex interactions between the cerebellum, motor cortex, and limbic system that work together to mediate locomotion and exploration.

Coordination was assessed using both footprint analysis and the vertical pole test. Footprint analysis is used extensively to assess gait abnormalities especially in spinal cord injury (Hammers et al. 2001) and with animal models of Huntington’s disease and Parkinson’s disease (Carter et al. 1999; Fernagut et al. 2002). The vertical pole test was performed with slight modification as described in the literature (Fernagut et al. 2003; Kurosaki et al. 2003; Matsuura et al. 1997). This test is used extensively to assess motor deficits in animal models of movement disorders such as Parkinson’s disease (Arai, Misugi, and Goshima 1990; Ohno et al. 1994). Angle of foot placement was significantly altered in 1.0 mg/kg MeHg-treated mice when compared to controls. We observed that nearly 40% of all mice exposed to MeHg were not able to stay on the pole as it was shifted from a horizontal to a vertical position, whereas control mice experienced no difficulty staying on the pole. These results are indicative of subtle but significant coordination deficits resulting from MeHg exposure to young adult mice.

In conclusion, our findings suggest that these four behavior tests can be used as effective tools to measure subtle motor and coordination deficits that result from exposure to moderate to low doses of neurotoxicants. We found that MeHg produces adverse effects in individuals that were exposed to the neurotoxicant during early adulthood. It would be interesting in future experiments to determine the affect of such exposures in aged populations, as we know that fish forms a major source of protein in the aging population, and fish is a major source of MeHg.