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. 2020 Jul 30;177(1):263–280. doi: 10.1093/toxsci/kfaa104

Inducible and Conditional Stimulation of Adult Hippocampal Neurogenesis Rescues Cadmium-Induced Impairments of Adult Hippocampal Neurogenesis and Hippocampus-Dependent Memory in Mice

Hao Wang k1, Megumi T Matsushita k1, Liang Zhang k1, Glen M Abel k1, Brett C Mommer k1, Timothy F Huddy k2, Daniel R Storm k3, Zhengui Xia k1,✉
PMCID: PMC7553705  PMID: 32617577

Abstract

Cadmium (Cd) is a heavy metal and an environmental pollutant. However, the full spectrum of its neurotoxicity and the underlying mechanisms are not completely understood. Our previous studies demonstrated that Cd exposure impairs adult hippocampal neurogenesis and hippocampus-dependent memory in mice. This study aims to determine if these adverse effects of Cd exposure can be mitigated by genetically and conditionally enhancing adult neurogenesis. To address this issue, we utilized the transgenic constitutive active MEK5 (caMEK5) mouse strain we previously developed and characterized. This mouse strain enables us to genetically and conditionally activate adult neurogenesis by administering tamoxifen to induce expression of a caMEK5 in adult neural stem/progenitor cells, which stimulates adult neurogenesis through activation of the endogenous extracellular signal-regulated kinase 5 mitogen-activated protein kinase pathway. The caMEK5 mice were exposed to 0.6 mg/l Cd through drinking water for 38 weeks. Once impairment of memory was confirmed, tamoxifen was administered to induce caMEK5 expression and to activate adult neurogenesis. Behavior tests were conducted at various time points to monitor hippocampus-dependent memory. Upon completion of the behavior tests, brain tissues were collected for cellular studies of adult hippocampal neurogenesis. We report here that Cd impaired hippocampus-dependent spatial memory and contextual fear memory in mice. These deficits were rescued by the tamoxifen induction of caMEK5 expression. Furthermore, Cd inhibition of adult hippocampal neurogenesis was also reversed. This rescue experiment provides strong evidence for a direct link between Cd-induced impairments of adult hippocampal neurogenesis and hippocampus-dependent memory.

Keywords: cadmium, neurotoxicity, cognitive deficits, adult hippocampal neurogenesis, ERK5 MAP kinase

Cadmium (Cd) is a widespread, nonbiodegradable toxic heavy metal and is released into the environment through natural processes and human activities. It is one of the 10 chemicals of major public health concern listed by the World Health Organization and ranks No. 7 on the Substance Priority List of Agency for Toxic Substances and Disease Registry (ATSDR, 2019). Ingestion of Cd-contaminated food and cigarette smoking are the primary routes of Cd exposure for the general population (Jarup et al., 1998; Satarug et al., 2010). Because of its long biological half-life (15–30 years) in humans, Cd accumulates in the body and induces adverse effects in various organs, including kidney (Jarup et al., 2000; Nordberg et al., 1975), liver (Go et al., 2015; Goering et al., 1993; Habeebu et al., 1998), lung (Hart et al., 1999; Manca et al., 1994; Strauss et al., 1976), and bone (Chen et al., 2009; Trzcinka-Ochocka et al., 2010; Wallin et al., 2013).

Recent studies suggest that Cd is a potential neurotoxicant. In vitro studies have shown that Cd exposure induces cell death in various neural cells (Chen et al., 2008; Lopez et al., 2003; Wang et al., 2017; Yuan et al., 2013). Animal studies also reported that Cd exposure can induce damage to the brain and cause behavioral deficits in multiple assays, including passive avoidance (1, 5, and 25 mg/kg Cd in food), conditioned suppression and schedule controlled responding (5 mg/kg Cd in food) (Goncalves et al., 2012; Nation et al., 1983; Wong and Klaassen, 1982). Interestingly, several epidemiological studies suggested an association between Cd exposure in adults (median [IQR] of urinary Cd levels: 0.42 μg/l [0.19–0.82]; blood Cd levels: 0.35 μg/l [0.24–0.56]) and impairments of cognition (Ciesielski et al., 2013; Li et al., 2018) although others failed to find such an association (GhazaLi et al., 2013; Przybyla et al., 2017). Thus, the relationship between Cd exposure in adult human and cognitive impairment is still unclear. Our recent study has shown a clear, direct causal relationship between Cd exposure and cognitive deficits in adult male mice (Wang et al., 2018), providing experimental evidence in model organisms for Cd impairment of cognitive function. However, the potential mechanisms for the neurotoxicity of Cd on hippocampus-dependent memory are not well understood.

Adult neurogenesis is a process that generates functional new neurons from adult neural progenitor/stem cells (aNPCs). Under normal physiological conditions, this process is restricted to 2 regions in adult brains: the subgranular zone in the dentate gyrus (DG) of the hippocampus and the subventricular zone of the lateral ventricles (Ming and Song, 2005, 2011). It has been established that adult hippocampal neurogenesis plays an important role in the formation of hippocampus-dependent memory (Clelland et al., 2009; Deng et al., 2010; Pan et al., 2012a,c). Recently, we found that at exposure levels that induce cognitive deficits in male mice, Cd also impairs adult hippocampal neurogenesis, suggesting that Cd may impair hippocampus-dependent memory by affecting adult neurogenesis in the hippocampus (Wang et al., 2018, 2019). However, direct evidence proving this causal relationship is lacking.

Extracellular signal-regulated kinase 5 (ERK5) is a member of the mitogen-activated protein (MAP) kinase family (English et al., 1995; Zhou et al., 1995). MEK5 is a highly specific upstream activating kinase for ERK5 and does not activate other MAP kinases even when overexpressed (English et al., 1995; Zhou et al., 1995). All of the effects of MEK5 have been attributed to its ability to induce the activation of ERK5 (Hayashi and Lee, 2004). In the adult brain, the ERK5 MAP kinase is specifically expressed in adult neurogenic regions (Pan et al., 2012a,b,d). Deletion of erk5 in adult neurogenic regions impairs adult neurogenesis in the hippocampus (Pan et al., 2012d), suggesting that ERK5 is necessary for adult hippocampal neurogenesis. We have also generated a gain-of-function knock-in mouse model that allows inducible and conditional expression of an active form of MEK5 (caMEK5) specifically in the aNPCs upon tamoxifen administration (Wang et al., 2014, 2015). We have demonstrated that the inducible activation of endogenous ERK5 MAP kinase in aNPCs is sufficient to increase adult hippocampal neurogenesis and improve hippocampus-dependent memory in caMEK5 mice (Wang et al., 2014). Thus, this caMEK5 transgenic mouse line provides a unique tool to investigate a direct causal relationship between Cd impairment of adult neurogenesis and memory loss.

In this study, we first determined if Cd exposure at environmentally relevant exposure levels inhibits adult hippocampal neurogenesis in wild-type C57BL/6 mice. Subsequently, we exposed caMEK5 mice to Cd through drinking water. Once the memory deficit was established, we administered tamoxifen to induce caMEK5 expression and examined if this is sufficient to rescue mice from Cd-induced deficits in adult hippocampal neurogenesis as well as memory.

MATERIALS AND METHODS

Animals

Female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, Massachusetts ) at 9 weeks of age. We previously generated an ERK5-activating knock-in mouse line in which a constitutive active MEK5 (caMEK5) sequence tagged with eGFP was inserted into the Gt(ROSA)26Sor genomic locus (ROSA26) through homologous recombination in embryonic stem cells derived from a hybrid of 129S6/SvEvTac X C57BL/6Ncr mice (Wang et al., 2014). This caMEK5-eGFPloxP/loxP mouse line was then crossed with Nestin-CreER mice, an inducible Cre line driven by the Nestin promoter in the C57BL/6 background (Kuo et al., 2006) to generate the Nestin-CreER:caMEK5-eGFPloxP/loxP mouse line used in this study, which we designated as caMEK5 mice for simplicity. To obtain caMEK5 mice, we bred female caMEK5-eGFPloxP/loxP with male Nestin-CreER:caMEK5-eGFPloxP/loxP mice because female Nestin-CreER:caMEK5-eGFPloxP/loxP mice are infertile.

All mice were housed (4–5 animals per cage) under standard conditions (12 h light/dark cycle) with food (Picolab Rodent Diet 20, LabDiet, St Louis, Missouri) and water (tap water purified by reverse osmosis, acidified with 2.4–2.8% HCl, and autoclaved) provided ad libitum. The C57BL/6 mice started to receive normal drinking water or drinking water with 0.6 mg/l Cd (in the form of CdCl2, Cat: 202980; MilliporeSigma, Burlington, Massachusetts) at 12 weeks of age. When we started the experiment, there was a 2-week age range in the experimental caMEK5 female mice cohort. We evenly assigned animals to each group such that every treatment subgroup of caMEK5 mice had a similar age distribution. The caMEK5 mice started receiving Cd exposure at 15–17 weeks of age. The drinking water for animals containing CdCl2 was prepared from a stock solution and replaced every 1–2 weeks. The preparation, use, and disposal of hazardous reagents were conducted according to the guidelines from the Environmental Health and Safety Office at the University of Washington. All animal care and treatments were preapproved by the University of Washington Institutional Animal Care and Use Committee.

Tamoxifen administration and 5-bromo-2′-deoxyuridine labeling

To induce Nestin-CreER-mediated recombination of caMEK5-eGFP in aNPCs in vivo, caMEK5 mice were given freshly made tamoxifen (200 mg/kg, dissolved in corn oil with 2% glacial acetic acid; Cat: T5648; MilliporeSigma) by oral gavage once a day for 4 consecutive days in each cycle, for a total of 3 cycles with a 2-week interval between each tamoxifen treatment cycle, as previously described (Wang et al., 2014). Tamoxifen induces expression of caMEK5-eGFP in adult neural stem cells in caMEK5 mice. Upon tamoxifen treatment, Cre-mediated recombination and expression of caMEK5-eGFP in the mice occurs specifically in Nestin-expressing aNPCs and their progenies. This allows inducible and conditional activation of adult neurogenesis via caMEK5 activation of the endogenous ERK5 MAP kinase (Wang et al., 2014, 2015).

5-Bromo-2′-deoxyuridine (BrdU; Cat: B9285; MilliporeSigma) was dissolved in sterile saline and administered by intraperitoneal injection (100 mg/kg). To identify BrdU-labeled proliferating cells, mice received 5 BrdU injections in 1 day (every 2 h) and were euthanized 2 h after the last BrdU injection (2-h labeling). To identify BrdU-retaining, adult-born cells, mice received 2 BrdU injections per day for 3 consecutive days, starting the day before and ending the day after the 2 h labeling day. Animals were euthanized 2.5 weeks after the last BrdU injection (2.5-week labeling). Mice were anesthetized (ketamine/xylazine) and euthanized by decapitation. Brain tissues were collected; one brain hemisphere was quickly frozen in liquid nitrogen for biochemical and Cd analysis. The other brain hemisphere was fixed by immersion in 4% Paraformaldehyde in PBS overnight, followed by 30% (w/v) sucrose/PBS (pH 7.4) at 4°C for 2–3 days, and used for immunostaining.

Immunohistochemistry

Immunohistochemistry of mice brain tissues was performed as previously described (Pan et al., 2012a; Wang et al., 2014). Coronal brain sections (30 μm) were used for free-floating immunohistochemistry. All primary and secondary antibodies were diluted in the blocking buffer (10% goat serum/donkey serum [Cat: G9023/D9663, MilliporeSigma] and 1% Bovine serum albumin [Cat: BAC61, Equitech-Bio, Kerrville, Texas]). The primary and secondary antibodies and dilutions used in immunohistochemistry were rat monoclonal anti-BrdU (1:500; Cat: MCA2060, Bio-Rad, Hercules, California), mouse monoclonal anti-NeuN (1:1000; Cat: MAB377, MilliporeSigma), goat polyclonal anti-DCX (1:200; Cat: SC-8066, Santa Cruz, Dallas, Texas), rabbit polyclonal anti-GFP (1:500; Cat: A11122, Invitrogen, Carlsbad, California), Alexa Fluor-conjugated Secondary antibody (1:2000; Cat: A11077, A11001, A11055, and A21209, Invitrogen), and biotinylated goat anti-rabbit secondary antibody (1:250; Cat: BA-1000, Vector Labs, Burlingame, California). After blocking and incubation of primary and secondary antibodies, tissues were incubated with 2.5 μg/ml Hoechst 33342 (Cat: H3570, Invitrogen) for 30 min, washed once again with PBS, then mounted onto slides using anti-fade Aqua Poly/Mount (Cat: 18606, Polysciences, Warrington, Pennsylvania) solution. For BrdU staining, the tissues underwent additional processing before blocking: 10 min in 1 N HCl at 4°C, 30 min in 2 N HCl at 37°C, and 2× 30 min in 0.1 M borate buffer. For eGFP staining, a tyramide signal amplification kit (TSA; Cat: NEL701A001KT, PerkinElmer, Waltham, Massachusetts) was used as previously described (Wang et al., 2014).

Imaging and quantification of immunostained cells

Immunostained cells from the brain tissues were quantified as previously described (Pan et al., 2012a; Wang et al., 2014). Every eighth serial coronal section (a total of 9–12 sections per brain) encompassing the entire hippocampus was immunostained for each marker (or each combination of markers) and quantified by an experimenter blinded to animal treatment. This number was multiplied by 8 to estimate the total number of marker positive cells. The colocalization of positive markers was defined as overlapping fluorescent signals in a single cell using a Z-series stack. All images were captured with an Olympus (Tokyo, Japan) Fluoview-1000 laser scanning confocal microscope with these lenses: numerical aperture (NA) 0.75, 10×, 20×, or an NA 1.35, 60× oil-immersion lenses. Optical Z-sections were collected and processed using ImageJ software (NIH, Bethesda, Maryland). Images were uniformly adjusted for color, brightness, and contrast with ImageJ software. Analysis of dendritic morphology was performed using Simple Neurite Tracer (NIH) as previously described (Li et al., 2013), with 10 individual neurons randomly chosen and analyzed from each mouse brain (a total of 40 neurons per treatment group). Sholl analysis was completed using the Sholl analysis function in ImageJ.

Open field test

The open field test was used to examine locomotor activity and anxiety. During the test, mice were placed into a 10-in. (width) × 10-in. (depth) × 16-in. (height) TruScan Photo Beam Tracking arena (Coulbourn Instruments, Holliston, Massachusetts) with clear Plexiglas sidewalls and their movement was monitored by 2 sets of infrared breams spaced 0.6 in. apart. Mice were allowed to explore in the arena without prehabituation for 20 min, and the data were collected and analyzed by TruScan 2.0 software (Coulbourn Instruments). The total move distance, total move time, and average speed were used to examine locomotor activity. The distance and time spent in margin and center, as well as center entries, were used to assess anxiety.

Elevated plus maze test

The elevated plus maze test was used to examine anxiety. The elevated plus maze apparatus (26 in. × 26 in. × 15.25 in.) consists of 2 open arms, 2 closed arms, and a center area (San Diego Instruments, San Diego, California). Each closed arm has a 7-in. wall on both sides, and the center area of the maze measures 2 in. × 2 in. In the test, mice were placed into the center of the apparatus facing an open arm and allowed to explore freely for 5 min. A video camera and ANYmaze software (San Diego Instruments) were used to track and analyze the movement of mice during the test.

Novel object location test

The novel object location (NOL) test was used to examine hippocampus-dependent spatial working memory. This assay was performed as previously described (Wang et al., 2018). Briefly, each mouse was placed into an open field arena (Coulbourn Instruments) with 2 identical objects placed in 2 different corners. During the training session, the mouse was allowed to explore the 2 identical objects freely for 5 min and then returned to its homecage. The testing session was performed 1 h after training. The animal was returned to the arena with the same 2 objects; one object remained in its original location, and the other moved to a new location. During the whole process, alternating corners were used for object presentation to exclude preference of specific location. The time the mouse spent investigating each object during the training and testing session was video recorded and quantified manually by an experimenter blinded to the treatment of animals.

Cued and contextual fear conditioning tests

In this study, a modified cued and contextual fear conditioning test using a weak foot shock conditioning paradigm (3× 0.3 mA, 2-s shocks with 2-min intertrial intervals) was used as previously described (Engstrom et al., 2017) to examine the contextual fear memory. For the conditioning session, the mouse was placed into the foot shock context (10-in. × 10-in. × 16-in. arena with grid shock floor; Coulbourn Instruments) and allowed to explore in the arena for 2 min before the presentation of a 90-dB, 30-s tone (conditioned stimulus, CS). During the last 2 s of the tone, a 0.3-mA foot shock (unconditioned stimulus, US) was delivered. This cycle was repeated 2 more times before the mouse was returned to its cage. The CS and US were delivered automatically by the TruScan software (Coulbourn Instruments). The contextual fear memory test was conducted 24 h after the conditioning session. The mouse was placed back into the same foot shock context for 2 min in the absence of tone or foot shock. For the cued test, which was performed 2 h after the context test, the mouse was placed into a novel context (new room; hexagonal Plexiglas arena) for 2 min. A 2-min CS (tone) was then presented. For the novel context test, which was performed 2 h after the cued test, the animal was placed into another novel context (new room; rat cage) and allowed to explore in the novel context for 2 min without presentation of either tone or foot shock. In all 3 tests, persistent freezing behavior (no head or body movement besides breathing, 4 paws on the ground) was recorded and manually quantified by an experimenter blinded to animal treatment.

Blood and brain Cd analysis

The Environmental Health Laboratory at the University of Washington measured blood (whole blood) and brain (whole brain) Cd levels using inductively coupled plasma mass spectrometry (ICP-MS). We did not measure the hippocampal Cd levels because according to our previous study under similar exposure conditions, the level of Cd in each mouse hippocampus is below the detection limit of the instrument (data not shown). The experimenters who measured the Cd concentrations were blinded to the treatment. The final blood and brain Cd analysis was conducted using an Agilent 7900 ICP-MS (Agilent Technologies, Santa Clara, California) with a detection limit of 0.08 μg/l for blood or 0.15 ng/g for brain tissue (200 mg sample mass).

Statistical analysis

All statistical analyses were conducted using GraphPad Prism software (GraphPad Software Inc, La Jolla, California). Shapiro-Wilk test (α = .05) was performed to test the normality of data before any further analysis. For blood Cd concentrations in C57BL/6 mice, Mann Whitney test (α = .05) was used to analyze the data. For immunohistochemical studies in C57BL/6 mice, Student’s two-tailed t test (α = .05) was used to analyze the data. For Sholl analysis, Student’s two-tailed t test (α = .05) was used to analyze the total dendritic length, and Mann Whitney test (α = .05) was used to analyze the number of crossing between control and Cd-treat mice. Mixed-effects linear regression was used for longitudinal analysis (α = .05) of caMEK5 mice body weights and water consumption. For the NOL test, Student’s two-tailed t test (α = .05) was used for comparisons within each treatment group. For open field test, Kruskal-Wallis test (α = .05) was used to analyze the data. For elevated plus maze test, contextual fear memory test, blood, and brain Cd levels of caMEK5 mice, two-way ANOVA test was performed to account for the main effect of Cd (Control vs Cd), tamoxifen (Vehicle [Veh] vs Tamoxifen [Tam]), and interactions between the 2 variables. When two-way ANOVA test showed significant results, post hoc multiple comparisons with Sidak correction (α = .05) was used to further analyze the data. All data are presented as mean ± SEM. *p < .05, **p < .01, and ***p < .001.

RESULTS

Cd Exposure Impairs Adult Hippocampal Neurogenesis in Female C57BL/6 Mice by Inhibiting Neuronal Differentiation

Our recent study in humanized transgenic mice expressing human ApoE3 or ApoE4 genes demonstrated that animals exposed to drinking water containing 0.6 mg/l CdCl2 yielded 0.3–0.4 μg/l of blood Cd (Zhang et al., 2020), which are within the blood Cd levels found in the general US population (men: 0.21–0.40 μg/l; women: 0.26–0.42 μg/l) (Centers for Disease Control and Prevention, 2019). In order to examine if Cd exposure at environmentally relevant exposure levels impairs adult hippocampal neurogenesis in female wild-type mice, we first exposed 12-week-old female C57BL/6 mice to drinking water containing 0.6 mg/l Cd (in the form of CdCl2) for up to 18.5 weeks. Mice were euthanized at the end of 16- or 18.5-week exposure, and blood samples were collected for Cd concentration analysis (Figure 1A). The peak blood Cd concentrations at the end of either 16- or 18.5-week exposure were similar (Supplementary Figure S1), and 0.32 ± 0.06 μg/l when data were combined (Figure 1B). This is significantly higher than the control group of 0.098 ± 0.01 μg/l.

Figure 1.

Figure 1.

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Cd exposure increased blood Cd concentrations in adult female C57BL/6 mice. A, Experiment design and the timeline for adult neurogenesis cellular studies. Twelve-week-old female C57BL/6 mice were exposed to 0.6 mg/l Cd via drinking water for 16–18.5 weeks. Mice received 5-bromo-2′-deoxyuridine (BrdU) injection at 16 weeks into Cd exposure. Half of the cohort (n = 4) was euthanized on the same day of BrdU injection to investigate the effects of Cd on cell proliferation. The remaining half of the cohort (n = 4) was euthanized 2.5 weeks after BrdU injection (at experimental week 18.5) to examine the effects of Cd on 2.5-week-old adult-born cells. B, Blood Cd concentrations in female C57BL/6 mice after 16–18.5 weeks of Cd exposure. n = 8 in each group, including both BrdU-injected subgroups. Data are presented as mean ± SEM. **p < .01.

To examine the effect of Cd on cell proliferation, we injected half of the mice with BrdU (100 mg/kg, 5 injections in one day, every 2 h) 16 weeks into Cd exposure. Animals were euthanized 2 h after the last BrdU injection to label actively proliferating, S-phase cells (2-h labeling) (Figure 2A). We did not find a significant difference between control and Cd-treated mice in the number of 2-h BrdU+ cells (Figure 2B), suggesting that Cd exposure does not affect cell proliferation.

Figure 2.

Figure 2.

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Cd exposure did not affect cell proliferation in the dentate gyrus (DG) of adult female C57BL/6 mice. 5-Bromo-2′-deoxyuridine (BrdU; 100 mg/kg, 5 injections in 1 day, every 2 h) was administered after 16 weeks of Cd exposure. Animals were euthanized 2 h after the last injection. A, Representative images of BrdU (red) and Hoechst (blue) staining in the DG of control and Cd-treated mice. Scale bar, 50 μm. B, Quantification of BrdU+ cells per DG in control and Cd-treated mice. n = 4 in each group. Data are presented as mean ± SEM.

To investigate the effects of Cd exposure on adult-born cell survival, BrdU was administered to the remaining half of the Cd-exposed and control cohorts, and animals were euthanized 2.5 weeks later to label 2.5-week-old, adult-born cells in the DG of the hippocampus (2.5-week labeling) (Figure 3A). BrdU immunostaining showed that Cd-treated mice had a lower number of surviving BrdU+ cells in the DG than control mice, however, this decrease was not statistically significant (p = .07) (Figure 3B). Therefore, the effects of Cd exposure on adult-born cell survival are inconclusive.

Figure 3.

Figure 3.

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Cd exposure decreased the number and proportion of adult-born cells that differentiated into mature neurons in the dentate gyrus (DG) of adult female C57BL/6 mice. Twelve-week-old female C57BL/6 mice were exposed to 0.6 mg/l Cd for 18.5 weeks and then euthanized. 5-Bromo-2′-deoxyuridine (BrdU) was administered 2.5 weeks before euthanasia. A, Representative images of BrdU (red) and NeuN (green) staining in the DG of control and Cd-treated mice. Boxed areas were enlarged and shown as orthogonal views of confocal z-stack images. Arrows point to cells that are both BrdU+ and NeuN+, whereas arrowheads point to cells that are BrdU+ but NeuN−. Scale bar, 50 μm. B, Quantification of BrdU+ cells per DG in control and Cd-treated mice. C, Quantification of BrdU+ and NeuN+ cells per DG in control and Cd-treated mice. D, Quantification of the percentage of total BrdU+ cells that were both BrdU+ and NeuN+ per DG in control and Cd-treated mice. n = 4 in each group. Data are presented as mean ± SEM. *p < .05 and ***p < .001.

To examine the effect of Cd on neuronal differentiation of adult-born cells in the DG, we performed coimmunostaining of BrdU with NeuN (Figure 3A) or DCX (Figure 4A), markers for mature (Sarnat et al., 1998) or immature neurons (Brown et al., 2003; Couillard-Despres et al., 2005), respectively. Cd treatment significantly reduced the number of adult-born mature neurons (BrdU+ and NeuN+ cells) and the proportion of adult-born cells that expressed NeuN+ (BrdU+ NeuN+/total BrdU+) in the DG (Figs. 3C and 3D). In addition, Cd decreased the number of adult-born immature neurons (BrdU+ and DCX+ cells) and the proportion of adult-born cells that expressed DCX+ (BrdU+ DCX+/total BrdU+) in the DG (Figs. 4B and 4C). These data suggest that Cd inhibits neuronal differentiation during adult hippocampal neurogenesis.

Figure 4.

Figure 4.

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Cd exposure decreased the number and proportion of adult-born cells that differentiated into immature neurons in the dentate gyrus (DG) of adult female C57BL/6 mice. Twelve-week-old female C57BL/6 mice were exposed to 0.6 mg/l Cd for 18.5 weeks and then euthanized. 5-Bromo-2′-deoxyuridine (BrdU) was administered 2.5 weeks before euthanasia. A, Representative images of Hoechst (blue), DCX (green), and BrdU (red) staining in the DG of control and Cd-treated mice. Boxed areas were enlarged and shown as orthogonal views of confocal z-stack images. Arrows point to cells that are both BrdU+ and DCX+, whereas arrowheads point to cells that are BrdU+ but DCX−. Scale bar, 50 μm. B, Quantification of the total number of DCX+ cells per DG in control and Cd-treated mice. C, Quantification of the percentage of total BrdU+ that were both BrdU+ and DCX+ per DG in control and Cd-treated mice. n = 4 in each group. Data are presented as mean ± SEM. *p < .05 and ***p < .001.

Cd Exposure Decreased the Dendritic Complexity of Adult-Born Immature Neurons in the DG of Female C57BL/6 Mice

We also assessed the dendritic morphology to measure neuronal maturation of adult-born neurons in the DG. Because the acid treatment in BrdU staining damages the neuronal processes, we performed separate DCX staining without costaining for BrdU to better visualize neuronal processes of newly generated immature neurons (Figure 5A). The results of neurite tracing and Sholl analysis show that Cd exposure reduced the total dendritic length (Figure 5B) and the number of dendritic crossings of immature neurons (Figure 5C) in the DG. These data indicate that Cd exposure impairs neuronal maturation of adult-born cells in the DG of female C57BL/6 mice. Collectively, these results suggest that Cd exposure at environmentally relevant exposure levels impairs adult hippocampal neurogenesis in female mice.

Figure 5.

Figure 5.

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Cd exposure reduced the dendritic complexity of immature neurons in the dentate gyrus (DG) of adult female C57BL/6 mice. Animals were treated as in Figure 4. A, Representative images of Hoechst (blue) and DCX (green) staining in the DG and examples of DCX+ neurons traced by the ImageJ Simple Neurite Tracer. Arrows point to traced cells. Scale bar, 50 μm. B, Quantification of the total dendritic length of DCX+ cells per DG in control and Cd-treated mice. C, Sholl analysis for the number of neurite crossings of DCX+ neurons per DG in control and Cd-treated mice. n = 4 in each group. Data are presented as mean ± SEM. **p < .01 and ***p < .001.

Inducible and Conditional Expression of caMEK5 Rescued Female caMEK5 Mice From Cd-Induced Impairments of Hippocampus-Dependent Spatial Working Memory in the NOL Test

To establish a direct causal relationship between Cd-induced impairments of adult neurogenesis and memory loss, we conducted a rescue experiment using transgenic caMEK5 mice that we previously generated and characterized (Wang et al., 2014). We exposed young adult female caMEK5 mice (15–17 weeks old) to 0.6 mg/l Cd (in the form of CdCl2) through drinking water for 38 weeks, and conducted behavior tests before and during Cd exposure (Figs. 6A and 6B). Animals housed without Cd in their drinking water were used as controls for Cd treatment. Once we confirmed Cd-induced impairments of hippocampus-dependent memory, tamoxifen was administered, still in the presence of Cd in drinking water, to induce caMEK5 expression. We recorded body weights and water consumption of mice during the entire Cd-exposure period and did not observe any significant effect of Cd or tamoxifen on body weights (Figure 6C) or water consumption (Supplementary Figure S2).

Figure 6.

Figure 6.

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Experimental design for the functional rescue experiment. A, Schematic of the rescue experiment in female caMEK5 mice for Figures 7–11. Young adult female caMEK5 mice (15–17 weeks old) were exposed to 0.6 mg/l Cd in drinking water for 38 weeks. Novel object location (NOL) test was conducted to probe the onset of Cd-induced memory loss. Once the memory loss was confirmed in 3 consecutive weeks, animals were given 3 cycles of tamoxifen or vehicle control by oral gavage to conditionally and selectively induce caMEK5 expression in adult neural stem/progenitor cells (once a day, 4 days in each cycle, with 2 weeks intervals between each cycle). Novel object location test was performed 2 weeks after the last dose of tamoxifen. After Cd-induced memory loss in the NOL test was reversed in the tamoxifen-treated group, Cd treatment was stopped, and all animals were given normal drinking water. Additional behavior tests were performed to assess motor function, anxiety, and fear conditioning. At the end of the behavior tests, animals were injected with 5-bromo-2′-deoxyuridine to label adult-born cells before euthanasia. Blood and brain tissues were collected for Cd measurement and cellular studies at the time of euthanasia, which corresponded to 29.5 or 32 weeks after the cessation of Cd exposure. B, The timeline of the rescue experiment for Figures 7–11. C, Cd and tamoxifen treatment did not affect the body weights during the entire Cd-exposure period. n = 9–10 in each group. Data are presented as mean ± SEM.

We performed 1-h NOL test to assess hippocampus-dependent spatial memory (Figure 7A). At each time point before and during Cd exposure, there was no difference in the exploration time of the 2 identical objects at location A versus B in the training session among the 4 treatment groups of mice (Figs. 7B–D), suggesting that none of the groups exhibited a preference for either object location.

Figure 7.

Figure 7.

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Inducible and conditional caMEK5 expression rescued mice from Cd-impaired hippocampus-dependent spatial working memory in the novel object location (NOL) test. Tamoxifen or vehicle control was given by oral gavage to the caMEK5 mice during weeks 19, 22, and 25 of the 38-week-long Cd treatment. The NOL tests were conducted before and during Cd exposure. A, Schematic of 1-h NOL test (A and B: old locations; C: novel location). B–D, During training sessions, animals at all time points examined spent equal time investigating the 2 identical objects placed at location A or B, demonstrating no bias for either location. E, Before they were exposed to Cd, all animals had spatial memory for the object placed at old location A and spent more time exploring the object placed at the novel location C in the testing session. F, After 17 weeks of Cd exposure, the Cd-treated animals lost their spatial memory and spent equal time exploring objects at locations A and C in the testing session. G, Tamoxifen treatment, which induces caMEK5 expression, restored the spatial memory loss even in the continued presence of Cd exposure. n = 8–10 in each group. Data are presented as mean ± SEM. **p < .01 and ***p < .001.

Before Cd exposure, all groups of mice spent significantly more time exploring the object in the new location (location C) versus old location (location A) in the testing session, suggesting that all mice had memory for the original object locations (Figure 7E). Seventeen weeks into the Cd exposure, although the control groups without Cd exposure (Control + Veh and Control + Tam) still spent significantly more time exploring the object in the novel location C, the Cd-treated groups (Cd + Veh and Cd + Tam) spent equal amounts of time exploring the 2 objects in both old and new locations in the testing session (Figure 7F). Thus, the Cd-treated mice did not discriminate between the old and new object locations, which is indicative of impaired short-term spatial memory.

After we confirmed Cd-induced memory deficit in the NOL test in 3 consecutive weeks (tested once per week), we administered tamoxifen (Tam) dissolved in corn oil, in the continued presence of Cd exposure, to the animals via oral gavage to conditionally and selectively induce caMEK5 expression in aNPCs. Control animals received vehicle (corn oil) treatment via oral gavage as controls for tamoxifen. Six weeks after the last tamoxifen treatment (31 weeks into Cd exposure), the 2 control treatment groups of mice (Control + Veh and Control + Tam) continued to exhibit normal memory for the old object location, whereas the Cd + Veh group of mice continued to exhibit a deficit in spatial memory as expected (Figure 7G). Interestingly, the Cd + Tam group of mice spent significantly more time exploring the object in the new location (location C) in the testing session. This result suggests that the inducible and conditional expression of caMEK5 rescued female caMEK5 mice from Cd-induced impairment of hippocampus-dependent spatial working memory.

Inducible and Conditional Expression of caMEK5 Rescued Female caMEK5 Mice From Cd-Induced Impairments of Hippocampus-Dependent Contextual Fear Memory

We also performed the cued and contextual fear conditioning test to assess the effects of Cd treatment on contextual fear memory, another form of hippocampus-dependent memory, and on cued fear memory, which is dependent on both hippocampus and amygdala function. We stopped Cd treatment after 38 weeks of total exposure to test whether the deficits in hippocampus-dependent memory persist postexposure. A challenging training paradigm in which animals received a 0.3-mA, 2-s foot shock 3 times during the fear conditioning session (Figure 8A) was used in the current study because this form is sensitive to changes in adult neurogenesis according to our previous findings (Engstrom et al., 2017; Pan et al., 2012a). In the context test performed 24 h after the training session, only the Cd + Veh group exhibited significantly reduced freezing, whereas the Cd + Tam group exhibited similar freezing as the Control + Veh group (Figure 8B, two-way ANOVA: Cd, F(1,31) = 2.093, p = .158; Tam, F(1,31) = 3.769, p = .061; interaction, F(1,31) = 7.674, p = .009; Sidak’s: Control + Veh vs Cd + Veh, p = .029; Cd + Veh vs Cd + Tam, p = .01; Control + Veh vs Cd + Tam, p = .983). There was no difference among the 4 groups in freezing behavior in auditory-cued memory test or novel context test, suggesting that neither auditory-cued memory nor general freezing behavior was affected by Cd or tamoxifen treatment. Collectively, these results indicate that inducible and conditional expression of caMEK5 rescued female caMEK5 mice from Cd-induced impairments of hippocampus-dependent contextual fear memory.

Figure 8.

Figure 8.

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Inducible and conditional caMEK5 expression rescued female caMEK5 mice from Cd-impaired hippocampus-dependent contextual fear memory. A, Schematic of cued and contextual fear conditioning test, performed at 23 weeks after the cessation of Cd exposure. B, The performance (freezing behavior) of 4 treatment groups of mice in preshock, context test, cued test, and novel context test. n = 8–10 in each group. Data are presented as mean ± SEM. *p < .05.

Cd and Tamoxifen Treatment Did Not Affect the Locomotor Activity or Cause Anxiety in caMEK5 Mice

We conducted the open field test and elevated plus maze test to assess locomotor activity and anxiety. We did not observe significant differences among the 4 groups of mice in the total distance traveled, moving time, and speed (Figure 9A), both before and after Cd treatment, suggesting that Cd and tamoxifen treatment did not affect locomotor activity.

Figure 9.

Figure 9.

Figure 9.

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Cd and tamoxifen treatments did not affect locomotor activity or anxiety status in adult female caMEK5 mice. A, The floor plane move distance, time, and speed of animals in the open field tests conducted before and after Cd and tamoxifen treatments. B, Open field tests to monitor animals’ movement along the margin or toward the center of the arena. An increased tendency to stay along the margins and/or decreased tendency to explore the center are signs of increased anxiety, and vice versa. n = 8–10 in each group. C, Elevated plus maze test. More time spent or distance traveled in closed arms reflects increased anxiety, and vice versa. n = 8–10 in each group. Data are presented as mean ± SEM.

In addition, all groups traveled similar distance along the margin and to the center, spent a similar amount of time in the margin and the center, and had similar center entries in the open field test both before and after Cd treatment (Figure 9B). These results suggest no overt anxiety in any of the treatment groups. The elevated plus maze was also performed to further examine the effects of Cd and tamoxifen treatment on anxiety. There was no significant difference between the 4 groups in the time and distance traveled in the closed arm, the open arm, or the center zone (Figure 9C). Together, the data of open field test and elevated plus maze test suggest that Cd and tamoxifen treatment did not decrease or increase anxiety in female caMEK5 mice. Thus, the differences in hippocampus-dependent memory observed in the 4 treatment groups of animals are unlikely due to changes in motor function or anxiety.

Persistent Cd Accumulation in the Brains of Female caMEK5 Mice After Oral Exposure

We measured blood and brain Cd concentrations from tissues collected when mice were euthanized at the end of the experiments, at 29.5 or 32 weeks after Cd exposure was stopped. There was no difference among the 4 treatment groups in the blood Cd concentrations (Figure 10A). However, the Cd-treated groups had significantly higher Cd concentrations in the brains than their controls, whereas the tamoxifen treatment had no effects on the brain Cd concentrations (Figure 10B, two-way ANOVA: Cd, F(1,14) = 117.3, p < .0001; Tam, F(1,14) = 0.364, p = .556; interaction, F(1,14) = 0.6656, p = .4282; Sidak’s: Control vs Cd, Veh, p < .0001; Tam, p < .0001). These data indicate that although the blood Cd concentrations in Cd-treated groups had returned to normal levels, brain Cd levels were still higher at 29.5–32 weeks postexposure. Furthermore, it suggests that the functional rescue of Cd-induced memory loss upon tamoxifen administration was not due to the effects of tamoxifen on brain Cd accumulation.

Figure 10.

Figure 10.

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Cd exposure led to Cd accumulation in the brains of female caMEK5 mice that persisted after cessation of Cd exposure. A, Blood Cd concentrations at the end of all experiments, 29.5–32 weeks after the cessation of Cd exposure. n = 7–9 in each group. B, Brain Cd concentrations at the end of all experiments. n = 4–5 in each group. Data are presented as mean ± SEM. ***p < .001.

Inducible and Conditional Expression of caMEK5 in Female Mice Rescued Animals From Cd-Induced Impairments of Adult Hippocampal Neurogenesis

We investigated the effects of tamoxifen-induced expression of caMEK5 on adult hippocampal neurogenesis. Because caMEK5 is sequence tagged with eGFP in this transgenic mouse strain, we performed eGFP immunostaining of brain tissues collected at the end of all behavior experiments. eGFP staining was found in the DG of tamoxifen-treated groups (Control + Tam and Cd + Tam), but not in the vehicle-treated controls (Control + Veh and Cd + Veh) (Figure 11A). These results confirm the conditional and selective induction of caMEK5 expression in aNPCs in the DG only upon tamoxifen administration.

Figure 11.

Figure 11.

Figure 11.

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Inducible and conditional caMEK5 expression rescued female caMEK5 mice from Cd-impaired adult hippocampal neurogenesis. A, Representative images of Hoechst (blue) and eGFP (green) staining to detect caMEK5-eGFP fusion protein expression in the dentate gyrus (DG). n = 4 in each group. B, Representative images of Hoechst (blue) and 5-bromo-2′-deoxyuridine (BrdU) (red) staining in the DG. 5-Bromo-2′-deoxyuridine was administered at experimental week 67.5 (29.5 weeks after Cd exposure) and animals were euthanized 2 h after the last BrdU injection (5 injections, 2 h apart). Scale bar, 50 μm. C, Quantification of the number of BrdU+ cells from (B) data. n = 3 in each group. D, Representative images of NeuN (green) and BrdU (red) staining in the DG. 5-Bromo-2′-deoxyuridine was administered at experimental week 67.5 and euthanized 2.5 weeks later (32 weeks after Cd exposure). Boxed areas were enlarged and shown as orthogonal views of confocal z-stack images. Arrows point to cells that are both BrdU+ and NeuN+, whereas arrowheads point to cells that are BrdU+ but NeuN−. Scale bar, 50 μm. E, Quantification of the total number of BrdU+ cells from (D) data. n = 5–7 in each group. F, Quantification of the number of BrdU+ and NeuN+ cells from (D) data. n = 5–7 in each group. Data are presented as mean ± SEM. *p < .05 and **p < .01.

To investigate the effects of Cd treatment and caMEK5 expression on cell proliferation, BrdU was administered at 29.5 weeks after Cd exposure (67.5-week of experimental age). Half of the animals were euthanized 2 h after the last injection to capture the proliferating, S-phase cells (Figs. 11B and 11C), whereas the other half was euthanized 2.5 weeks later (32 weeks after Cd exposure, which equals experimental week 70) to determine the number of surviving adult-born cells (BrdU+) or adult-born neurons (BrdU+ and NeuN+) (Figs. 11D–F). We did not find significant differences among the 4 treatment groups in the number of BrdU+ proliferating cells in the DG (Figure 11C). These data suggest that Cd treatment and tamoxifen-induced caMEK5 expression did not affect neural progenitor/stem cell proliferation in the hippocampus at this time point. However, there was a significantly lower number of 2.5-week-old, surviving BrdU+ cells (Figure 11E) as well as surviving adult-born neurons (BrdU+ and NeuN+) (Figure 11F) in the DG of the Cd + Veh group when compared with the Control + Veh group. Importantly, the Cd + Tam group had a significant higher number of 2.5-week-old, surviving adult-born cells (BrdU+) when compared with the Cd + Veh group (Figure 11E, two-way ANOVA: Cd, F(1,18) = 5.801, p = .027; Tam, F(1,18) = 10.83, p = .004; interaction, F(1,18) = 3.052, p = .098; Sidak’s: Control vs Cd, Veh, p = .022; Tam, p = .865; Veh vs Tam, Control, p = .519, Cd, p = .003). Similarly, tamoxifen treatment significantly increased the number of 2.5-week-old, surviving adult-born neurons (BrdU+ and NeuN+) in Cd-treated mice (Figure 11F, two-way ANOVA: Cd, F(1,18) = 7.636, p = .013; Tam, F(1,18) = 11.82, p = .003; interaction, F(1,18) = 2.191, p = .156; Sidak’s: Control vs Cd, Veh, p = .019; Tam, p = .587; Veh vs Tam, Control, p = .358, Cd, p = .004). Together, these data suggest that Cd treatment inhibits the survival of adult-born cells and decreases the total number of adult-born neurons in the caMEK5 mouse line at least partially by impairing cell survival. Furthermore, tamoxifen-induced caMEK5 expression in aNPCs restored adult hippocampal neurogenesis in female caMEK5 mice that was suppressed by Cd treatment.

DISCUSSION

We previously reported that Cd exposure results in the loss of several forms of hippocampus-dependent memory in male mice (Wang et al., 2018). However, the underlying mechanisms have not been fully elucidated. Adult hippocampal neurogenesis is a process in which neuronal stem cells in the hippocampus of the adult brain continuously proliferate and differentiate into neurons that functionally integrate into the existing neuron network. Consequently, it plays a critical role in the formation of hippocampus-dependent memory. Although we discovered impaired adult hippocampal neurogenesis in Cd-treated male mice, there was no direct evidence confirming that the inhibition of adult hippocampal neurogenesis causes Cd-induced impairments of hippocampus-dependent memory. Furthermore, because our previous studies were conducted in male mice (Wang et al., 2018, 2019), it was not clear if Cd elicits similar adverse effects at cellular and behavioral levels in female mice. Here, we report that oral Cd exposure in adult female mice yielded blood Cd levels commonly found in the general US population and impaired adult hippocampal neurogenesis. Moreover, inducible and conditional expression of caMEK5 in aNPCs rescued Cd-induced loss of hippocampus-dependent memory in female mice. This rescue was not due to changes in brain Cd accumulation, general motor function, or anxiety. Rather, it correlated with restoration of adult hippocampal neurogenesis that was inhibited by Cd treatment. Together, our study provides strong evidence that inhibition of adult hippocampal neurogenesis underlies Cd-induced hippocampus-dependent memory loss.

We previously reported the results of oral Cd exposure through 3 mg/l Cd in the drinking water elevated peak blood Cd to 2.25 ± 0.48 μg/l (Wang et al., 2018). This is close to the blood Cd levels of smokers in the US (men: 0.58–0.94 μg/l; women: 0.69–1.17 μg/l) and lower than the OSHA’s standard trigger level of Cd (5 μg/l) for medical surveillance. This level of exposure inhibited adult hippocampal neurogenesis and hippocampus-dependent memory in wild-type, male C57BL/6 mice (Wang et al., 2018, 2019). Recently, we discovered that Cd exposure at 0.6 mg/l CdCl2 in drinking water for 14 weeks yielded peak blood Cd concentrations of 0.3–0.4 μg/l, a range which is within the blood Cd levels found in the general US population (men: 0.21–0.40 µg/l; women: 0.26–0.42 µg/l, 1999–2016) (Centers for Disease Control and Prevention, 2019). This exposure paradigm also impaired adult hippocampal neurogenesis in mice expressing the human ApoE3 or ApoE4 gene (Zhang et al., 2020). However, the effects of Cd exposure on adult hippocampal neurogenesis in wild-type, female mice were still unknown. In the current study, we found that 16–18.5 weeks of exposure in young adult female C57BL/6 mice to 0.6 mg/l Cd similarly resulted in a peak blood Cd level of 0.32 ± 0.06 μg/l, which is likewise within the range of Cd blood concentrations in the general nonsmoker US population.

In Cd-treated female C57BL/6 mice, we did not find any change in the number of 2-h BrdU+ labeled, actively proliferating cells, suggesting that Cd does not interfere with the proliferation of aNPCs in the hippocampus. However, the total number and proportion of adult-born cells that differentiated into immature (BrdU+ and DCX+ cells) and mature (BrdU+ and NeuN+ cells) neurons were decreased in Cd-treated female C57BL/6 mice. Furthermore, there was a significant reduction in the dendritic complexity of immature neurons in the hippocampus of Cd-treated female C57BL/6 mice. These findings are similar to those we reported in male C57BL/6 mice exposed to 3 mg/l Cd (Wang et al., 2019). Together our data demonstrate that, like in male mice, Cd exposure impairs critical processes of adult hippocampal neurogenesis in female mice, including neuronal differentiation and maturation.

We previously generated a line of knock-in transgenic mice in which endogenous ERK5 in aNPCs can be activated by tamoxifen-induced conditional expression of a constitutively active MEK5 in Nestin-expressing aNPCs (Wang et al., 2014). We confirmed that the caMEK5-eGFP fusion protein used in this mouse line specifically activates endogenous ERK5 when expressed and that the expression of caMEK5-eGFP protein in the brain is tightly controlled by Cre-mediated recombination and is specific to aNPCs and their progenies after tamoxifen treatment (Wang et al., 2014, 2015). We also demonstrated that this caMEK5 expression enhances adult hippocampal neurogenesis by promoting cell survival and neuronal differentiation but does not affect cell proliferation (Wang et al., 2014). In the current study, we found that Cd does not impair proliferation of aNPCs in the DG of caMEK5 mice. Moreover, there is no difference in aNPCs proliferation between any of the 4 groups of female caMEK5 animals treated with Cd and/or tamoxifen, consistent with our previous finding that caMEK5 expression does not affect cell proliferation. In contrast, Cd treatment significantly lowered the number of surviving adult-born cells (2.5-week BrdU+ cells) and adult-born neurons (BrdU+ and NeuN+ cells) in caMEK5 mice, and these reductions were reversed after tamoxifen treatment. Together, these results suggest that Cd exposure decreases the number of adult-born neurons in caMEK5 mice at least partially by impairing cell survival. Furthermore, inducible and targeted caMEK5 expression is sufficient to rescue mice from Cd-induced impairments of adult hippocampal neurogenesis. That these effects were observed long after the cessation of Cd exposure and tamoxifen treatment suggests that the toxic effects of Cd and rescuing effects of tamoxifen-induced caMEK5 expression on adult neurogenesis are persistent.

In the NOL test, the short-term spatial memory for locations was impaired in Cd-treated (Cd + Veh and Cd + Tam) female caMEK5 mice at 17 weeks of Cd exposure. This impairment was reversed 6 weeks after the last day of tamoxifen that was administered in the continued presence of Cd exposure (Cd + Tam). The cued and contextual fear memory test was conducted 23 weeks after Cd exposure had stopped and 36 weeks after the last day of tamoxifen treatment. Cd exposure also impaired the hippocampus-dependent contextual fear memory in Cd-treated (Cd + Veh) mice, however, we did not observe this deficit in the Cd + Tam group. Cd treatment did not affect the auditory-cued memory or general freezing behavior in any of the treatment groups. According to Hunsaker and Kesner’s study (Hunsaker and Kesner, 2008), the hippocampus plays a very small role in the retrieval of auditory-cued fear, which may explain why the cued fear memory was not impaired in Cd-treated mice. Together our data demonstrate that Cd exposure impairs adult hippocampal neurogenesis, hippocampus-dependent spatial working memory, and contextual fear memory in female caMEK5 mice. These findings are similar to those from male mice (Wang et al., 2018). Furthermore, inducible and conditional enhancement of adult hippocampal neurogenesis via tamoxifen-induced caMEK5 expression in aNPCs rescues these deficits.

We measured the blood and brain Cd concentrations in female caMEK5 mice at 29.5–32 weeks after the cessation of Cd exposure and after all behavior experiments were concluded. We did not find any significant difference in the blood Cd levels among the 4 treatment groups. However, brain Cd concentrations were significantly higher in Cd-treated groups (Cd + Veh and Cd + Tam). These results indicate that Cd accumulates in the mouse brain even after it has been cleared systemically, and it induces persistent memory impairments long after exposure has ended.

Treatment with Cd and tamoxifen did not affect gross motor function in the open field test, nor did it cause any changes in the anxiety behavior in the open field test or elevated plus maze test. Furthermore, both auditory-cued fear memory and generalized freezing in the novel context were not affected. Finally, we did not find a significant difference between the Cd-vehicle group and the Cd-tamoxifen group in brain Cd concentrations, precluding the possibility that somehow tamoxifen affects the brain Cd accumulation, thus reversing the memory loss. Taken together, the reversal of Cd-induced memory loss in the NOL test and the contextual fear memory test after tamoxifen administration reflects functional rescue as a result of induced caMEK5 expression and enhanced adult neurogenesis. These findings provide clear and persuasive evidence supporting a causal relationship between Cd-impaired adult hippocampal neurogenesis and the decline in hippocampus-dependent learning and memory in female caMEK5 mice.

There is an age-related decline of adult hippocampal neurogenesis associated with memory decline during aging (Bizon et al., 2004; Lazarov et al., 2010). In the current study, the female caMEK5 mice were euthanized at age 82.5–87 weeks, whereas female C57BL/6 mice were euthanized at age 28–30 weeks. This age gap and difference in Cd-exposure durations may have impacted our results for adult neurogenesis from C57BL/6 versus caMEK5 mice. For example, this could explain why the caMEK5 mice had a lower number of total adult-born cells (BrdU+ cells) and adult-born neurons (BrdU+ and NeuN+cells) compared with C57BL/6 mice. The age-related decline of adult neurogenesis may also explain why we only found a trend but not statistically significant increase of adult neurogenesis in the Control + Tam group compared with the Control + Veh group.

In this study, tamoxifen was administered at the 18-week point in the continued Cd exposure and lasted for 6 weeks. Because we wanted to examine whether the deficits in hippocampus-dependent memory and adult neurogenesis persist postexposure, Cd exposure was terminated after 38 weeks of total exposure, while animals were not euthanized until week 67.5 or 70. In theory, this 29.5–32-week recovery time without Cd exposure, when BrdU administrations occurred, could contribute to the attenuative effects observed in neurobehavior tests and adult hippocampal neurogenesis in addition to the tamoxifen rescue effect. However, in the contextual fear memory test, which was conducted late in the study at week 61, Cd + Veh mice froze significantly less than Control + Veh mice, suggesting that the detrimental effect of Cd on hippocampus-dependent memory is persistent. Furthermore, Cd + Tam mice froze significantly more than Cd + Veh mice, suggesting that tamoxifen treatment clearly rescued this memory deficit. Moreover, we found that adult hippocampal neurogenesis was impaired in the Cd + Veh group compared with the Control + Veh group, and this deficit was rescued in the Cd + Tam group at the very end of the study. Therefore, it does not seem likely that the recovery time contributed to the attenuative effects observed in behavior tests or adult hippocampal neurogenesis in these female mice. Rather, the observed rescue effect is likely due to tamoxifen induction of caMEK5 expression. Nevertheless, it would be interesting in the future to determine the immediate impacts of tamoxifen induction and 38-week Cd exposure on caMEK5 expression in aNPCs, adult hippocampal neurogenesis, and neurobehavior.

Experience is another important regulator of adult neurogenesis (Opendak and Gould, 2015). Intense stress experiences, such as predator odor exposure, sleep deprivation, and social defeat, have been found to reduce adult hippocampal neurogenesis in animals (Burgado et al., 2014; Mohammadi et al., 2014; Mueller et al., 2008; Schoenfeld and Gould, 2012). In contrast, exercise and enriched environment increase adult hippocampal neurogenesis (Brandt et al., 2010; Leuner et al., 2010; Monteiro et al., 2014; Nilsson et al., 1999; Schoenfeld et al., 2013). In this study, the caMEK5 mice had experienced a series of different behavior tests before BrdU injection. Most of these tests were not stressful except the cued and contextual fear memory test which was conducted at the end of the behavior tests. Unfortunately, despite considerable effort, we did not have enough caMEK5 animals to include as controls in the cellular studies that did not go through behavioral testing in each treatment group.

Currently, whether sex differences exist in Cd neurotoxicity is not well established. Epidemiology studies have yielded conflicting results. Although Zhou et al. found that childhood Cd exposure may adversely affect cognition, particularly in girls (Zhou et al., 2020), other studies suggest that Cd exposure affects the cognitive functions more significantly in boys (Gustin et al., 2018; Rodríguez-Barranco et al., 2014). We recently reported that there is a significant gene-environment interaction between Cd exposure and the ApoE4 gene in memory impairment in male, but not in female transgenic mice (Zhang et al., 2020). Whether a sex difference exists in basal adult neurogenesis in mammalian brains remains controversial, with evidence both for (Galea and McEwen, 1999; Spritzer et al., 2017; Tanapat et al., 1999) and against the possibility (Amrein et al., 2004; Lagace et al., 2007). We originally planned on conducting the rescue experiments in both male and female caMEK5 mice generated from the same breeding cohort so we could also examine any sex differences. After several rounds of breeding to scale up our colony size, we had enough animals to set up 50 breeding pairs. Unfortunately, despite this considerable breeding effort, we only obtained 38 female caMEK5 offspring and even fewer male caMEK5 mice. Because our recent studies investigating the toxic effects of Cd on cognition and adult neurogenesis were conducted in male mice (Wang et al., 2018; Zhang et al., 2020), we decided that it was important to utilize these female caMEK5 mice to fill the critical gap of our knowledge regarding the neurotoxic effects of Cd on females even though we cannot directly evaluate any potential sex differences. We showed in this study that similar to male animals, female mice are also susceptible to Cd-induced impairment in adult hippocampal neurogenesis and hippocampus-dependent memory. Future studies conducted in animals with both sexes in the same cohort are needed to systematically investigate any potential sex differences in Cd neurotoxicity.

In summary, our data demonstrate that chronic, low-level Cd exposure impairs adult hippocampal neurogenesis in female mice. Furthermore, inducible and conditional stimulation of adult hippocampal neurogenesis rescues female mice from Cd-induced memory loss. These findings provide evidence for a direct causal relationship between inhibition of adult hippocampal neurogenesis and Cd-induced cognitive deficits in mice. Our results provide new insight into how exposure to environmental toxicants may accelerate cognitive decline and lead to neurodegeneration. Although the existence of adult neurogenesis occurs in the human hippocampus is still unresolved (Duque and Spector, 2019; Kempermann et al., 2018; Paredes et al., 2018), our findings have the exciting translational implications that impaired cognitive functions resulting from environmental exposure may be rejuvenated by promoting endogenous adult hippocampal neurogenesis in humans.

SUPPLEMENTARY DATA

Supplementary data are available at Toxicological Sciences online.

DECLARATION OF CONFLICTING INTERESTS

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

FUNDING

University of Washington Superfund Research Program (National Institute of Environmental Health Sciences [NIEHS] P42ES004696 to Z.X.).

Supplementary Material

kfaa104_supplementary_data
Click here for additional data file. (114.4KB, pdf)

ACKNOWLEDGMENTS

We thank the Center on Human Development and Disability (CHDD) at the University of Washington for the use of the confocal microscope. We also thank the Environmental Health Laboratory at the University of Washington for providing ICP-MS services to measure blood and brain Cd.

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