Methylmercury exposure at dosage conditions that do not affect growth can impair memory in adolescent mice

Abstract

Methylmercury is an environmental pollutant that can induce serious central nervous system damage. Its ubiquitous presence in the environment in trace amounts has raised concerns about potential adverse effects on human health. Although many studies have evaluated the effects of methylmercury on neural development in fetal and neonatal mice, there has been less focus on studies using adolescent mice. Therefore, in this study, the effects of methylmercury on brain neurodevelopment and maturation were evaluated by various neurobehavioral trials using adolescent mice exposed to 30 ppm methylmercuric chloride (approximately 24 ppm methylmercury) for up to 8 weeks. Under these administration conditions, weight gain in adolescent mice was unaffected by methylmercury exposure. Furthermore, methylmercury exposure in adolescent mice had no effect on sociability as assessed by the social interaction test, impulsivity as assessed by the cliff avoidance reaction test, depressive behavior as assessed by the tail-suspension test, or locomotor activity as assessed using the Supermex system. In contrast, short-term memory assessed by the Y-maze test, as well as long-term memory assessed by novel object recognition and passive avoidance tests, revealed impairments induced by methylmercury exposure in adolescent mice. These results suggest that long-term exposure to methylmercury during adolescence potentially impairs memory function, and the nervous pathway of brain areas involved in learning and memory are particularly vulnerable to the adverse effects of methylmercury.

Supplementary Information

The online version contains supplementary material available at 10.1007/s43188-024-00239-y.

Introduction

Methylmercury is the causative agent of Minamata disease and can induce severe central nervous system damage. Produced from inorganic mercury by microorganisms, methylmercury is ubiquitous in the environment and accumulates in particularly high concentrations in large fish (e.g., tuna) because of bioaccumulation in seawater [1]. Modern humans are primarily exposed to methylmercury through seafood consumption. For this reason, potential impacts on human health due to continuous exposure to small amounts of methylmercury has become an international concern, however the mechanisms involved in its toxicity remain largely unknown. The brain is a noteworthy target organ for methylmercury toxicity, with mammalian fetuses also exhibiting high sensitivity to methylmercury-induced neurotoxicity [2]. Against this background, there are concerns that low-level methylmercury exposure of women during pregnancy may have irreversible effects on the neurobehavioral development of their unborn children. In recent years, many studies have reported memory impairment and decreased locomotor activity in rodents exposed to low-level methylmercury during fetal and neonatal periods [3–7]. Additionally, methylmercury exposure equivalent to water containing 0.5–5.0 ppm methylmercury during fetal and neonatal periods has been linked to neurodevelopmental disorders such as attention-deficit/hyperactivity disorder and autism, as well as depression [8–11].

In methylmercury toxicity studies using adolescent or adult rodents exposed to relatively high concentrations of methylmercury (e.g., modeling Minamata disease), higher brain dysfunction such as motor impairment was observed under all administration conditions; in addition, weight loss was observed as an early sign of such impairment [12–15]. However, considering the low amounts of methylmercury to which humans are exposed in their daily lives, the concentrations used to assess methylmercury toxicity in adolescence and adulthood should also be set as low as possible. Indeed, there is substantial interest in the health effects of exposure to concentrations lower than those that induce higher brain dysfunction such as motor impairment. Fujimura et al. previously reported observing neuropathological abnormalities in the cerebrum of C57BL/6 mice fed ad libitum water containing methylmercury at a final concentration of 30 ppm for 8 weeks [16]. However, no neuropathological changes were observed in the cerebrum of C57BL/6 mice fed ad libitum water containing methylmercury at a final concentration of 20 ppm for 10 weeks. We also conducted preliminary experiments in which C57BL/6 mice were given 20, 30, and 40 ppm methylmercuric chloride (approximately 16–32 ppm methylmercury) ad libitum for 8 weeks and found that 40 ppm methylmercuric chloride caused neuronal damage in the parietal lobes of mice, while 30 ppm methylmercuric chloride caused only minimal damage (unpublished data). Therefore, in this study, adolescent mice were exposed to methylmercuric chloride (approximately 24 ppm methylmercury) for 6–8 weeks at a final concentration of 30 ppm, which was expected to have no effect on growth, and observed for neurobehavioral effects associated with methylmercury exposure.

Materials and methods

Animals

Male C57BL/6 mice (aged 6–7 weeks) were purchased from Japan SLC (Shizuoka, Japan). Mice were housed in cages (3 or 4 mice per cage) with free access to food and water under controlled conditions (temperature, 22 ± 2ºC; humidity, 55% ± 5%) and a 12/12-h light/dark cycle (lights on: 07:00 to 19:00). Groups of 10 mice were used for behavioral trials. All experiments were conducted following approval from the Ethics Committee of Animal Experiment at Tohoku Medical and Pharmaceutical University. Additionally, all procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Efforts were made to use the smallest possible number of animals and minimize suffering.

Methylmercury exposure

Mice (aged 6–7 weeks) were exposed to methylmercury for 6–8 weeks via drinking water containing 30 ppm of methylmercuric chloride (Tokyo Chemical Industry, Tokyo, Japan)-reduced glutathione (GSH) (Nacalai Tesque, Kyoto, Japan) (1:1) complex, which is approximately 24 ppm methylmercury as previously described [16]. The control group (vehicle-treated mice) was provided the same amount of GSH-containing water.

Y-maze test

Y-maze testing was performed as previously described [17]. The Y-maze apparatus, made of black plastic, consisted of three compartments (40 cm L × 10 cm W × 10 cm H) radiating out from the center platform (3 cm × 3 cm × 3 cm triangle). Mice were placed in one of the compartments and allowed to move freely for 8 min. The light intensity of the platform was kept at 35 lx. An arm entry was defined as three legs entering one of the arms, and the sequence of entries was manually recorded. An alternation was defined as entry into all three arms on consecutive choices. Thus, the maximum number of alterations was the total number of entries minus 2, and the percent alteration was calculated as (actual alterations/maximum alterations) × 100. Percent alteration was designated as the spontaneous alternation behavior of the mouse and taken as a measure of spatial short-term memory.

Novel object recognition (NOR) test

The NOR test is a behavioral trial that assesses short- or long-term recognition memory in mice by relying on the innate preference of rodents for novelty. The experiment was carried out in a plastic chamber (25 cm L × 17 cm W × 12 cm H) between 10:00 and 15:00 for three consecutive days, as previously described [18]. Mice were habituated to the measuring room 1 h before experiments. On Day 1, mice were allowed to freely explore the chamber for 30 min. On Day 2 (sample phase), two identical objects were placed equally spaced in the chamber and the mice were allowed to explore freely for 10 min. Twenty-four hours later, mice were allowed to explore a familiar (old) object and a novel object for 10 min (test phase). Mice used in the NOR test were different from mice used in the passive avoidance test. Each trial was recorded by camera and the exploration time for both objects was analyzed by an experimenter blinded to the mouse information. The discrimination index on Day 2 (D) was calculated using the following equation: D = (exploration time on right object − exploration time on left object)/total exploration time for two identical objects. The discrimination index on Day 3 (D′) was calculated using the following equation: D′ = (exploration time on novel object − exploration time on familiar object)/total exploration time for novel and familiar objects.

Passive avoidance test

Passive avoidance testing was performed as previously described [17]. In brief, methylmercury-exposed mice (or control mice) that performed training trials were placed in the light compartment and their latency to enter the dark compartment was recorded (retention trial). Training and retention trials for the passive avoidance task were conducted in a plexiglass box that contained dark (25 cm × 25 cm × 25 cm) and light (14 cm × 10 cm × 25 cm) compartments. The floor was constructed of stainless-steel rods. The floor rods in the dark compartment were connected to an electronic stimulator. In the training trials, mice were placed in the light compartment of the box and as soon as the mouse had completely entered the dark compartment, an electric shock (1.0 mA for 500 ms) was supplied to the floor bars. In the retention trial, each mouse for each time point (2, 4 and 6 weeks after the training trial) was placed in the light compartment and their latency to enter the dark compartment was once again recorded. The maximum cut-off time was set to 10 min. Behavior in the passive avoidance task is generally considered to reflect long-term memory.

Social interaction test

The measurement of social interaction was performed by video recording mice without any observer in the room, as previously described [19]. Briefly, mice to be measured were kept individually for 1 h before unfamiliar guest mice of comparable size were placed simultaneously in opposite corners of the arena and the behavior of each mouse was observed for 10 min. The behavior of mice sniffing at a distance of less than 2 cm from each other was measured as a social interaction.

Cliff avoidance reaction (CAR) test

The CAR test to assess impulsivity was performed according to previously described methods [20]. Briefly, mice were placed on a circular platform (an inverted glass container with a 13-cm diameter and 20-cm height) and the time until they fell was captured by a video camera for 7 min. When the mouse did not fall within the measurement time, it was assumed to have fallen at 7 min.

Tail-suspension (TS) test

Tail-suspension testing was performed as previously described to evaluate depression-like behavior [21]. Mice were taped from the tail tip and suspended 30 cm from the floor. Mice were considered immobile only when they were hanging completely and passively motionless. The duration of time spent immobile was quantified during a total test period of 10 min, including the 2 min immediately after suspension as a habituation period.

Locomotor activity

Locomotor activity was determined using the Supermex multichannel activity-counting system. The details of this device have been described previously [22]; briefly, this system can automatically count vertical movements such as jumping, as well as horizontal movements such as walking and running. Locomotor activity was measured during the light phase, between 11:00 and 15:00. For adaptation, each mouse was placed in an activity box for 15 min and locomotor activity was recorded for 90 min.

Statistical analyses

Data are expressed as mean ± SEM. Significant differences were analyzed using Student's unpaired t test and two-way analysis of variance. Statistical analysis was performed using GraphPad Prism (ver. 9.5.1). In all comparisons, p < 0.05 was considered statistically significant.

Results

Methylmercury exposure at dosage conditions that do not affect growth can impair short-term memory in adolescent mice.

To determine the effects of methylmercury exposure on the behavior of adolescent mice, C57BL/6 male mice were allowed to access water containing a final concentration of 30 ppm methylmercuric chloride (approximately 24 ppm methylmercury) ad libitum for up to 8 weeks. In addition, with the aim of reducing the number of animals used in the experiment and preventing interference between behavioral trials, we conducted a series of behavioral trials on mice according to the following schedule (Fig. 1A). Under these administration conditions, the weight gain of mice was unaffected by methylmercury (Fig. 1B) and no noticeable abnormal behaviors were observed. First, we assessed the short-term memory of mice using the Y-maze test and found that the alternation rate of mice exposed to methylmercury was significantly lower than that of vehicle-treated mice (Fig. 2A). In addition, measurements of total sniffing time (Fig. 2B), time to fall from the platform (Fig. 2C), and immobility time (Fig. 2D) assessed by the social interaction test, CAR test, and tail suspension test, respectively, show methylmercury-exposed mice displayed no significant behavioral changes compared with vehicle-treated mice. In addition, when the spontaneous locomotor activity time of mice was measured using an automated locomotor measurement system, no significant difference was observed between vehicle-treated and methylmercury-exposed mice (Fig. 2E). The evaluation conditions for behavioral trials used in this study were developed by us and other research groups, and their usefulness has been confirmed. In other words, we found that methylmercury exposure, which does not affect growth, only impaired short-term memory without affecting social interaction, impulsivity, depression-like behavior, or locomotor activity in adolescent mice. These results suggest that methylmercury may readily cause functional impairment by damaging specific areas in the brains of mice, particularly those involved in memory and learning.

Fig. 1.

Effects of long-term exposure to methylmercury on growth of adolescent mice. a The experimental schedule consisted of five successive behavioral trials (see Methods for details). The maximum exposure period to 30 ppm methylmercuric chloride (MeHgCl) was 8 weeks, and behavioral trials were conducted from 6 weeks of exposure. b Body weights were measured weekly during the administration period. Values represent mean ± SEM for 9 mice per group. The absence of a bar indicates that the SEM falls within the symbol

Fig. 2.

Neurobehavioral changes in adolescent mice exposed long-term to methylmercury. a–e Mice exposed to at least 30 ppm methylmercuric chloride (MeHgCl) for 6 weeks were used to measure a the percentage of alternation, b sniffing time when confronted with unfamiliar mice, c time to fall from the platform, d immobility time when the tail was suspended, and e locomotor activity. Values represent mean ± SEM for 9 mice per group. ^**p < 0.01 compared with vehicle-treated mice

Methylmercury exposure at dosage conditions that do not affect growth can impair long-term memory in adolescent mice.

As shown in Fig. 2A, methylmercury dosages that do not affect growth can impair short-term memory in adolescent mice. The hippocampus is particularly involved in short-term memory and it is known that new memories are first stored in the hippocampus. However, many research groups have previously reported that the hippocampus is not a target site for methylmercury toxicity in the brain [16]. Therefore, we reevaluated the effect of methylmercury on short-term memory in adolescent mice according to the schedule shown in Fig. 3A, and evaluated long-term memory using the NOR test. Y-maze test results reconfirmed that the alternation rate of mice was reduced by methylmercury exposure (Fig. 3B), as previously shown in Fig. 2A. The NOR test, a behavioral trial used to evaluate memory and learning in mice, was used in this study as an evaluation system for long-term memory 24 h after learning. When mice explored the novel object longer than the familiar (old) object, it indicates that they recognized (remembered) the old object; the larger this proportion, the more positive the discrimination index. As a result, when mice were given objects of the same shape on test Day 1 (sample phase), the preference for objects did not change between vehicle-treated and methylmercury-exposed mice (Fig. 3C). In contrast, when one of the familiar objects was replaced by a novel object with a different shape on Day 2 (test phase), vehicle-treated mice preferred to explore the novel object, whereas methylmercury-exposed mice showed no preference for it (Fig. 3D). Collectively, our results suggest that methylmercury exposure at a dosage that does not affect growth can impair both short- and long-term memory in adolescent mice.

Fig. 3.

Effects of long-term exposure to methylmercury on memory in adolescent mice. a The experimental schedule consisted of two successive behavioral trials (see Methods for details). Behavioral trials began 6 weeks after 30 ppm methylmercuric chloride (MeHgCl) exposure. The Y-maze test reflects short-term memory performance, while the NOR test reflects long-term memory performance. b Percentage of alternation in the Y-Maze test and discrimination index in the c sample phase and d test phase of the NOR test. Values represent mean ± SEM for 10 mice per group. ^*p < 0.05, ^**p < 0.01 compared with vehicle-treated mice

Next, we used another method of examining long-term memory, the passive avoidance test, to examine long-term memory according to the schedule shown in Fig. 4A and explore the timing of memory impairment caused by methylmercury exposure (Fig. 4A). In this test, methylmercury administration was started after the first training trial, and retention trials were conducted at 2, 4, and 6 weeks after exposure to measure the latency of mice to enter the dark room. In this test, when mice remain in the light room, it indicates that aversive (long-term) memory is retained. Up to 4 weeks under experimental conditions, both vehicle-treated and methylmercury-exposed mice remained in the light room at cutoff times up to 10 min. However, at 6 weeks under experimental conditions, vehicle-treated mice remained in the light room, whereas half of methylmercury-exposed mice entered the dark room (Fig. 4B). That is, under the conditions of this test, adolescent mice remembered and avoided the aversive (electrical) stimulus once experienced, even after 6 weeks had passed; however, this avoidance behavior was inhibited by methylmercury exposure. These results strongly suggest that methylmercury exposure at dosages that do not affect growth can impair long-term memory in adolescent mice.

Fig. 4.

Effects of long-term methylmercury exposure on long-term memory in adolescent mice. a Experimental schedule for the passive avoidance test (see Methods for details). Retention trials began 2, 4, or 6 weeks after 30 ppm methylmercuric chloride (MeHgCl) exposure. b Top: Schematic diagram of the passive avoidance test. Bottom: Latency (sec) of entry from the light box to the dark box after passive avoidance training. Values represent mean ± SEM for 10 mice per group. ^*p < 0.05 compared with vehicle-treated mice

Discussion

In this study, when adolescent mice were exposed to 30 ppm methylmercury chloride (approximately 24 ppm methylmercury) for 6 weeks, weight gain was unaffected and growth was normal. Furthermore, under these administration conditions, social interaction, impulsivity, depression-like behavior, and locomotor activity were unaffected in adolescent mice-only memory impairment occurred. These findings suggest that methylmercury adversely affects memory in mice during adolescence, as well as fetal and neonatal periods.

Previously, methylmercury exposure during the fetal and neonatal periods was reported to affect all the aforementioned behaviors, including memory [8–11, 23]. Moreover, rats exposed to low concentrations of methylmercury during fetal and neonatal periods are known to exhibit decreased motor activity and short- and long-term memory impairments [24]. These suggest that various regions of the brain are susceptible to the adverse effects of methylmercury in an active state of neurodevelopment. In contrast, mice exposed to methylmercury during adolescence may be more susceptible to damage in the central nervous system associated with memory than in the system associated with locomotor activity. Thus, it cannot be denied that extending the period of methylmercury exposure or changing the behavioral test conditions used in this study may induce behavioral impairments such as motor impairment and depression.

Information that comes in through the eyes, ears, nose, and other sensory organs is processed throughout the cerebral cortex (primarily the prefrontal cortex) and stored as memory. Previous studies confirmed the presence of neuronal damage in somatosensory, auditory, and visual cortices in patients with Minamata disease and methylmercury-exposed mice [16, 25–27]. In addition to these areas, neuronal damage in the motor cortex and striatum has been reported. Interestingly, while methylmercury strongly damages neurons in certain areas of the cerebral cortex, hippocampal neurons appear to be unaffected by methylmercury [16, 28–30]. Indeed, this study did not observe methylmercury-induced neuronal damage in the hippocampal dentate gyrus of adolescent mice with memory impairment (data not shown). This result suggests that methylmercury may cause memory impairment in adolescent mice without damaging hippocampal neurons. On the other hand, rats exposed long-term to low concentrations of methylmercury have been reported to show neurodegeneration in the hippocampal parenchyma, in addition to short-term and long-term memory impairments [31]. Recent evaluations of short-term memory using working memory as an index revealed short-term memory impairment in Amazonian indigenous people exposed to methylmercury [32]. The Y-maze test used in this study is also a behavioral trial to evaluate spatial working memory, which depends on function of both the prefrontal cortex and hippocampus [33]. In addition, as mentioned above, the present study revealed that both short-term memory, which depends on the hippocampus, and long-term memory, which depends on the cerebral cortex, are adversely affected by methylmercury exposure. Because the hippocampus is involved in both short-term memory storage and long-term memory transfer, methylmercury may cause memory impairment by suppressing various information signaling pathways entering the hippocampus.

Taken together, long-term exposure to methylmercury during adolescence may adversely affect memory performance by causing more damage to cerebral cortical neurons than hippocampal neurons. In the future, we expect to determine the brain areas involved in memory impairment (i.e., areas with methylmercury-induced neuronal damage) through neurohistological analysis of mouse brain, which will provide clues to elucidate the mechanisms involved in this impairment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Ryota Yamagata, Ai Saito, Ryoko Fukushima, Naoya Yamashita and Gi-Wook Hwang. The first draft of the manuscript was written by Ryota Yamagata and supervised by Gi-Wook Hwang. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Funding

This work was partially supported by the Japan Society for the Promotion of Science (JSPS) (JSPS KAKENHI Grant No. JP22H03752) and the Study of the Health Effects of Heavy Metals organized by the Ministry of the Environment of Japan.

Data availability

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Conflict of interest

The authors have no relevant financial or non-financial interests to disclose.

Ethical approval

All animal procedures were approved by the Ethics Committee of Animal Experiment at Tohoku Medical and Pharmaceutical University (Animal Experiment Protocols: p5-9).