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Adult neurogenesis occurs in brain subventricular zone (SVZ). Our recent data reveal an elevated proliferation of BrdU(+) cells in SVZ following subchronic manganese (Mn) exposure in rats. This study was designed to distinguish Mn effect on the critical stage of adult neurogenesis, ie, proliferation, migration, survival and differentiation from the SVZ via the rostral migratory stream to the olfactory bulb (OB). Adult rats received a single ip-dose of BrdU at the end of 4-week Mn exposure to label proliferating cells. Immunostaining and cell-counting showed a 48% increase of BrdU(+) cells in Mn-exposed SVZ than in controls (P < .05). These BrdU(+) cells were identified as a mixed population of mainly GFAP(+) type-B neural stem cells, Nestin(+) type-C transit progenitor cells, DCX(+) migratory neuroblasts and Iba1(+) microglial cells. Another group of adult rats received 3 daily ip-injections of BrdU followed by subchronic Mn exposure. By 4-week post BrdU labeling, most of the surviving BrdU(+) cells in the OB were differentiated into NeuN(+) matured neurons. However, survival rates of BrdU/NeuN/DAPI triple-labeled cells in OB were 33% and 64% in Mn-exposed and control animals, respectively (P < .01). Infusion of Cu directly into the lateral ventricle significantly decreased the cell proliferation in the SVZ. Taken together, these results suggest that Mn exposure initially enhances the cell proliferation in adult SVZ. In the OB, however, Mn exposure significantly reduces the surviving adult-born cells and markedly inhibits their differentiation into mature neurons, resulting in an overall decreased adult neurogenesis in the OB.
Neurogenesis in adult mammalian brain is a complex process involving cell proliferation, migration, differentiation, and integration of newborn cells into the existing neuronal network. The adult neurogenesis occurs throughout life in two restricted brain regions, ie, the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus (Altman and Das, 1965; Eriksson et al., 1998; Lois and Alvarez-Buylla, 1994). Neural stem/progenitor cells (NSPCs) in the adult SVZ have the capability to self-renew and generate migratory neuroblasts; the latter can migrate anteriorly and tangentially through the rostral migratory stream (RMS) to reach their final destination, ie, the olfactory bulb (OB), where they mature and integrate into local neuronal circuitry (Gage, 2000; Lois et al., 1996; Luskin, 1993; Yang, 2008). In response to abnormal insults, such as brain injury, ischemia, epilepsy, tumors, psychiatric disorders, or neurodegenerative damages, the newborn neuroblasts are able to escape from the normal migratory pathway, attracted toward the impaired sites and involved in neural repair and regeneration (Cayre et al., 2009; Curtis et al., 2007; Winner and Winkler, 2015). Therefore, the association between the adult neurogenesis and brain functions under physical and pathological conditions has been strongly suggested.
Recent published studies from this laboratory using synchrotron x-ray fluorescence (XRF) microscopy and atomic absorption spectrophotometry reveal an extraordinarily high copper (Cu) content in the SVZ along the external walls of rat brain lateral ventricles, which is about 20- to 30-folds higher than those in other brain regions (Fu et al., 2015a,b; Pushkar et al., 2013). A decreased Cu level in the rat SVZ has been associated with age (Fu et al., 2015b, Pushkar et al., 2013). Cu is an essential cofactor for a number of cuproenzymes, such as cytochrome C oxidase, superoxide dismutase, ceruloplasmin, and tyrosinase, that are critical to cellular biochemical reactions (Gambling et al., 2011; Lorraine et al., 2011; Turski and Thiele, 2009; Uriu-Adams et al., 2010; Zheng and Monnot, 2012). A disrupted Cu homeostasis in brain has been associated with the pathogenesis of known genetic diseases such as Wilson’s disease and Menkes disease and neurodegenerative disorders such as Parkinson’s disease, Alzheimer’s disease, familial amyotrophic lateral sclerosis, and prion disease (Gaggelli et al., 2006; Matés et al., 2010; Zheng and Monnot, 2012). Limited data also support a role of Cu in regulating embryonic stem cell differentiation (El Meskini et al., 2007; Haremaki et al., 2007; Niciu et al., 2007). Further studies from this laboratory have also shown that subchronic exposure to manganese (Mn) causes a significant increase in Mn and Cu concentrations in the cerebrospinal fluid (CSF), choroid plexus (CP), striatum, hippocampus, and frontal cortex (Fu et al., 2014; Zheng et al., 2009). Moreover, in the same animal model, we have found that Mn exposure, while increasing Cu in many other brain regions, greatly reduces the Cu content in the SVZ, accompanying with an upregulated expression of DMT1 and increased proliferating cells positive to BrdU and glial fibrillary acidic protein (GFAP) in this neurogenic niche (Fu et al., 2015a). Since BrdU labels all proliferating cells, it remained unclear what cell type(s) were in response to Mn insults to proliferate and whether these proliferating cells would migrate, survive, and ultimately differentiate into neurons.
Recent literature data have suggested that Mn exposure can cause apoptosis in immature granule cells, misguide neuronal migration, increase immature reelin-synthesizing GABAergic interneurons, and disrupt the epigenetic gene regulation in the dentate gyrus of hippocampus following maternal exposure to Mn (Wang et al., 2012, 2013). A reduction in type 2b and type 3 progenitor cells, as well as parvalbumin (Pvalb)+-GABAergic interneurons in the dentate hilus is evident in oral Mn-exposed adult mice (Kikuchihara et al., 2015). However, neither do these studies employ BrdU or other useful markers to label newly proliferating cells, nor do they follow appropriate study protocols to trace, distinguish, and quantify the key parameters in the adult neurogenesis process, ie, the proliferation, migration, survival, and differentiation of newborn neuroblasts in the SVZ and/or SGZ, not to mention the lack of studies on the dynamic changes of NSPCs in the SVZ-RMS-OB system.
To understand the mechanism whereby Mn influences the adult neurogenesis, we designed 3 experiments in the current report using different labeling schedules to trace and analyze the fate of BrdU-labeled cells originated from the SVZ. In Experiment 1 (Figure 1A), we labeled the proliferating cells with a single dose of BrdU and traced them within a 4-h time window after animals had undergone a 4-week subchronic Mn exposure. Experiment 2 (Figure 1B) was carried out by labeling newly generated NSPCs with BrdU prior to 4-week’s subchronic Mn exposure; these cells were then traced for their migration, survival, and differentiation in SVZ, RMS, and OB at different time points. Finally, to investigate the mechanism whereby Cu and Mn affected the SVZ neurogenesis, we conducted an intracerebroventricular (icv) perfusion experiment coupled with a short-term BrdU labeling (Figure 1C). The results of these studies provide the first hand evidence on Mn detrimental effect on the various phases of adult neurogenesis; more importantly, our data help establish the concept of the CP-CSF-SVZ axis in regulating neurogenesis in adult brain.
Chemical reagents were purchased from the following sources: Rabbit anti-rat DMT1 antibody was obtained from Alpha Diagnostic (San Antonio, California), mouse monoclonal anti-BrdU antibody from Santa Cruz Biotechnology (Dallas, Texas), ProLong Gold anti-fade reagent, Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (Catalog no. A11008), and Alexa Fluor 555 goat anti-mouse IgG (H+L) antibody (A21424) from Life Technologies (Carlsbad, California); rabbit polyclonal anti-nestin (Catalog no. ab92391), anti-doublecortin (DCX) (ab18723), and anti-NeuN (ab177487) antibodies, and chicken polyclonal anti-GFAP antibodies from Abcam (Cambrdge, Massachusetts); polyclonal rabbit anti-ionizing calcium-binding adaptor molecule 1 (Iba1) antibody (Catalog no. WEE4506) from Wako Chemicals USA Inc (Richmond, Virginia); manganese chloride tetra-hydrate (MnCl2·4H2O) from Fisher scientific (Pittsburgh, Pennsylvania); Protease Inhibitor Cocktail from Calbiochem (San Diego, California); paraformaldehyde (PFA) from ACROS Organics (NJ); bovine serum albumin (BSA) from AMRESCO (Solon, Ohio); and normal goat serum from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania). All reagents were of analytical grade, high performance liquid chromatography (HPLC) grade, or the best available pharmaceutical grade.
Male Sprague-Dawley rats were purchased from Harlan Sprague Dawley Inc (Indianapolis, Indiana). At the time of use, the rats were 10 weeks old weighing 220–250g. Upon arrival, the rats were housed in a temperature-controlled room under a 12-h light/12-h dark cycle and allowed to acclimate for 1-week prior to experimentation. They had free access to deionized water and pellet Purina semipurified rat chow (Purinal Mills Test Diest, 5755C. Purina Mills, Richmond, Inc). The study was conducted in compliance with standard animal use practices and approved by the Animal Care and Use Committee of Purdue University.
MnCl2·4H2O dissolved in sterile saline. Rats received intraperitoneal (ip) injections of Mn solution (1ml/kg body weight) at the dose of 6mg Mn/kg, once daily, 5 days/week for 4 consecutive weeks. The daily equivalent volume of sterile saline was given to the animals in the control group.
BrdU was administered by ip. injection(s) to Mn-exposed or control rats. Three different experimental protocols were designed to investigate the Mn toxicity on different states of adult neurogenesis, including proliferation, migration, survival, and differentiation. Experiment 1 was designed to characterize Mn effect on the cell proliferation in the SVZ. Rats underwent subchronic Mn exposure as described above. Twenty-four hour after the last Mn dose, rats received a single dose of BrdU (50mg/kg, ip.) and were sacrificed 4 h later for cardiac perfusion fixation (Figure 1A). Experiment 2 was designed to determine whether Mn exposure disrupted the migration, survival and differentiation of NSPCs along RMS and in OB. Rats received ip. injections of BrdU at 50mg/kg once daily for 3 consecutive days to label the total population of newly adult-born progenitors in the SVZ prior to the first Mn dose. Animals were sacrificed at 0day, 14 days, and 28 days after Mn exposure (Figure 1B). Experiment 3 was designed to investigate the direct effects of Mn and Cu on the proliferation of NSPCs in the SVZ. A well-established icv infusion technique was applied (see below). A total volume 6μl of saline, 50μg/ml MnCl2, and 30μg/ml CuCl2 were icv-infused directly into the right lateral ventricle of rats. At 24 h after the icv infusion, the rats received 1 ip. injection of BrdU at 50mg/kg and were subjected to cardiac perfusion fixation 4 h later (Figure 1C).
Prior to surgery, animals were fully anesthetized with 3%–5% isoflurane gas and placed in a stereotaxic frame. A longitudinal incision was made in the scalp to expose the skull; a cranial burr hole (1mm) was drilled into the skull of the right hemisphere with the coordinates of 0.8mm posterior to bregma and 1.4mm to lateral using the Paxinos and Watson Atlas. This was followed by an insertion of a sterilized cannula at 3.5mm ventral from the skull surface with a 26-gauge needle connected to a 10 µl Hamilton syringe (Hamilton Company, Reno, Nevada). The syringe was filled with 10 µl of saline, 10 µl of 50μg Mn/ml as MnCl2, or 10µl of 30μg Cu/ml as CuCl2 solution, which was slowly infused at a rate of 12 µl/min for 0.5min. The needle was allowed to remain inside the ventricle for an additional 5min to allow for proper absorption. After withdrawal of the cannula, the bone wax was used to fill the hole and the surgery site was stitched. Core body temperature was maintained at 37°C during the surgery using a rectal probe feedback-controlled heating pad.
All animals were deeply anesthetized using ketamine/xylazine (75:10mg/kg, 1mg/kg ip.), and perfused transcardially with ice-cold saline followed by 4% PFA in phosphate buffered saline (PBS). Brains were then removed from the skull and postfixed in 4% PFA for 24h followed by dehydration process in 30% sucrose for 7 days. Serial 30 µm thick coronal or sagittal sections were cut using a microtome and stored in cryoprotectant solution at −20°C. Specifically, For coronal and sagittal sectioning, the brains were sectioned from Bregma 2.0mm to −6.0mm and Lateral 0.2mm to 4.0mm/hemisphere, respectively, according to the rat brain atlas, in order to cover the entire distance of the lateral ventricles, which yielded about 250–270 sections/brain (coronal) and 125–135 sections/hemisphere (sagittal). All the coronal sections were placed in a 12-well plate in serial order, and all of the sagittal sections collected from one hemisphere were placed in 6 wells of the 12-well plate in serial order. Each well contains about 18–22 sections of both coronal and sagittal sections, which accounts for 1/12 of the total brain sections. The 18–22 sections within the same well and with the same well number order across all animals were then processed for immunohistochemical analysis.
Every 12th section (360 µm interval), covering the distance of the lateral ventricle, was processed for immunohistochemistry (IHC) analysis. Free-floating sections were washed with PBS (3×10min), incubated in 2N hydrogen chloride (HCl) for 2h at room temperature (RT), and then blocked in 0.1 M borate buffer for 15min (pH 8.4). After 3 washes with PBS (10min/wash), sections were incubated in blocking solution (0.3% Triton X-100, 1% BSA, and 5% normal goat serum in PBS) for 1.5 h at RT, followed by overnight incubation with primary antibodies at 4°C. The sections were washed with PBS (3×10min) and incubated with secondary antibodies for 2 h at RT. After incubation with DAPI (4′, 6-diamidino-2-phenylindole) for 15min at RT, sections were rinsed with PBS (3×10min) and mounted using Fluorescent Mount G.
The procedure to combine the IHC staining with various primary antibodies was as followed: (1) to examine the proliferation of newly born progenitor cells, brain sections were stained with mouse anti-BrdU (1:500) and rabbit anti-Nestin or rabbit anti-GFAP primary antibodies (1:1000), followed by incubation with Alexa Fluor 555 goat anti-mouse IgG (H+L) antibody (1:500) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (1:1000); (2) to identify whether the BrdU(+) cells were DCX(+) neuroblasts, brain sections were labeled with mouse anti-BrdU primary antibody (1:500) and rabbit anti-DCX primary antibodies (1:1000), followed by treatment with Alexa Fluor 555 goat anti-mouse IgG (H+L) antibody (1:500) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (1:1000); and (3) to verify whether BrdU(+) cells were further differentiated to become the interneurons being integrated into the OB circuitry, brain sections were incubated with mouse anti-BrdU primary antibody (1:500) and rabbit anti-NeuN primary antibodies (1:1000), followed by incubation with Alexa Fluor 555 goat anti-mouse IgG (H+L) antibody (1:500) and Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (1:1000).
After IHC processing, coronal brain sections from experimental design protocol 1 and 3 were examined using an inverted microscopy system (Zeiss Axiovert 200 M) combined with apotome and interfaced with a digital camera (Zeiss Axio Cam MRc5) controlled by a computer. Images were captured using apotome in software (AxioVision, v4.8). Due to the large scan areas of SVZ, RMS, and OB, sagittal brain sections from experimental design protocol 2 were examined using a Nikon TE2000-U inverted microscope equipped with a Nikon A1 confocal system (Nikon Instruments, Melville, New York). Images were taken using the software NIS Elements AR (v4.20). All images were assembled and labeled in Photoshop CC.
All sections were analyzed with appropriate filter or laser combinations under an objective lens of 20×/0.75 (DIC N2, ∞0.17 WD). Large image plus Z-Stack scanning was employed to confine the entire SVZ, RMS, and OB from either coronal or sagittal sections. Z-Stacking images with higher magnifications were captured under the objective lens of 60×/1.49 oil (DIC N2, ∞0.13-0.21) to verify the colocalizations of different cellular markers.
IHC was performed simultaneously on sections from different groups to detect the target cells. Series of every 12th section (30µm thickness, 360 µm apart) through each lateral ventricle were processed. The cell density was determined through a blinded quantitative histological analysis. A profile count method was used. Every single BrdU(+) cells (including partial of BrdU(+) nuclei at the border of section), or BrdU and specific cell marker (Nestin for NSPCs, GFAP for astrocytes, DCX for neuroblasts, NeuN for mature neurons, and Iba1 for microglial cells) double-labeled cells in the different subregion of SVZ, RMS, and OB in the multiplanes throughout the entire 30µm section, were counted under the fluorescent or confocal microscope using the large Z-stacking images through a whole series of sections.
The double-labeled cells were determined as follows. BrdU was as an indicator. When the BrdU(+) cell was identified, the channel was switched to those matching the cell-specific markers. Those double-labeled cells were considered the target cells for cell counting. The total number of quantified cells was justified by correction (Coggeshall and Lekan, 1996). The total number of BrdU(+) or double-labeled cells were then calculated by the equation (the total cell number = [the sum of actual cell counting number] × 12), and expressed as total number/target region/brain (n = 3 or 4 brains for each group).
All data are presented as mean ± SD. Statistical analyses of the differences between control and Mn-exposed groups were carried out by Student’s t tests. Comparisons of differences among the negative control baseline group (0-day), the control and Mn-exposed groups within the 2- and 4-week time points were analyzed by 1-way ANOVA with post hoc comparisons by the Dunnett’s test. Comparisons of differences between control and Mn-exposed groups within the 2- and 4-week time points were analyzed by 2-way ANOVA with post hoc comparisons by the Tukey test. All the statistical analyses were conducted using IBM SPSS for Windows (version 22.0). The differences between 2 means were considered significant for P ≤ .05.
Our previous studies by repeated injections of BrdU for 5 days in the last stage of Mn exposure have revealed a significantly increased BrdU fluorescent intensity in both SVZ and RMS (Fu et al., 2015a). Two issues remained unsolved, ie, (1) the cell type(s) being induced in proliferation were unknown and (2) the exact cell numbers (but not the fluorescent signal) in proliferation were not counted. The study designed in (Figure 1A) allowed us to assess the proliferating cells within a 4-h time window following 4-week subchronic Mn exposure. In the saline control group, the newly proliferating cells labeled by the BrdU were mainly located in the external wall of the lateral ventricle where the SVZ is located (Figure 2A). After Mn exposure for 4 weeks, the density of BrdU(+) cells along the SVZ region increased greatly (Figure 2B). By counting BrdU(+)/DAPI(+) cells, there was a 48.2% increase in Mn-treated SVZ over controls (89.9 ± 17.5 × 103 in Mn-treated animals versus 60.6 ± 2.4 ×103 in controls, P < .05, n = 4 for each group) (Figure 2C). The data suggested that subchronic Mn exposure stimulated cell proliferation in the adult SVZ, which is consistent with our previous findings (Fu et al., 2015a).
As a major source of NSPCs in the process of adult neurogenesis, the SVZ consists of 4 major cell types including type-E ependymal cells that have direct contact with the CSF, DCX(+) type-A migratory neuroblasts, GFAP(+) type-B neural stem cells, and Nestin(+) type-C transit amplifying cells (Doetsch et al., 1997). BrdU labels all of the cell types in proliferation, but does not distinguish one from another. The current study used a triple-staining technique to colocalize the BrdU(+) nuclei with DAPI and other cellular markers (GFAP, Nestin, and DCX) in the SVZ. Under the high magnification, newborn cells were identified morphologically by their round or oval shape and their size of about 10 µm in diameter (Figs. 3A–C). Merged images in (Figs. 3A-a) with 3-dimentional (3-D) reconstruction in the molecular layer of Mn-exposed SVZ indicated that the round BrdU-labeled nuclei in red color (Fig. 3A-a2) was co-localized with nuclei marker DAPI in blue color (Figs. 3A-a1, a4) and surrounded by the green GFAP signals (Figs. 3A-a, a3, a5). The data supported the view that a large portion of these newborn cells were astrocytic type-B stem cells.
Generally, GFAP(+) type-B stem cells are capable of remaining in a quiescent state with a slow amplifying rate until they are activated by either intrinsic or extrinsic stimuli. Once activated, type-B cells quickly develop into Nestin(+) type-C active proliferating transient cells, which further turn into DCX(+) type-A migratory neuroblasts. Thus, a triple-staining strategy with BrdU/Nestin/DAPI or BrdU/DCX/DAPI antibodies was used to distinguish the proliferating type-C or migrating type-A cells following Mn exposure. Data in Figure 3B (white dash line box) showed that some BrdU(+) cells in the temporal horn of SVZ were colocalized with Nestin. In addition, the data in Figure 3C displayed that a great number of BrdU(+) nuclei in Mn-exposed SVZ were surrounded by the green fluorescent DCX signals. Fluorescent merged images with 3-D reconstruction confirmed the colocalization of BrdU with DCX (Figs. 3C-c, c1-5). Together, these evidences suggested that the active proliferating cells in the adult SVZ induced by subchronic Mn exposure were not a pure population with a singular cell type, but rather a mixed population with all of the type-B stem cells, type-C transit amplifying stem cells as well as type-A migratory neuroblasts.
Literature data suggest that Mn exposure results in microglial activation with the subsequent release of inflammatory factors (Verina et al., 2011; Zhao et al., 2009). We hypothesized that the increased cell proliferation in adult SVZ following Mn exposure may be a consequence of Mn-induced microglial activation. To test this hypothesis, the Iba1, a well-known marker for microglia under either quiescent or active states (Ohsawa et al., 2000), was double-stained with BrdU. By quantifying the cells positive for both BrdU and Iba1 markers, there were a total of 1.09±0.08 ×103 newborn microglia in the control adult SVZ (Figure 4A). Compared with a total of 60.6±2.4 ×103 BrdU(+) cells in control adult SVZ (Figure 2C), these activated microglia constituted only a small fraction, about 1.8% of newborn cells, in the SVZ. After Mn exposure, the BrdU(+)/Iba1(+) microglial cells were increased to 2.42 ± 0.31 ×103, about 2.2-fold increase compared with controls (P < .01, Figure 4A). Although Mn exposure did significantly increase the number of activated microglia, the percentage over the total newborn cell population in Mn-exposed SVZ (89.9±17.5 ×103, Figure 2C) remained low, about 2.69% of newborn NSPCs in the SVZ. Because of the small percentage of microglia in the SVZ, very few BrdU(+)/Iba1(+) microglia were observed in each brain section. A representative confocal image with 3-D reconstruction in Figs. 4B-b confirmed the colocalization of BrdU with Iba1. These results suggested that Mn exposure appeared to increase newly born microglial cells in adult SVZ; however, there was a mild but not substantial increase in microglia proliferation. This small increase may not contribute significantly to the overall cell proliferation in the SVZ under Mn influence.
According to literature, a complete adult neurogenesis from the SVZ, via RMS, to OB in rodent includes: (1) 2–6 days for SVZ-derived neuroblasts to undergo tangential migration along the RMS towards the OB; (2) 5–7 days for neuroblasts to reach the core of the OB and subsequently switch to a radial migration toward their final destination in the granular cell layer (GCL), mitral cell layer (MCL), external plexiform layer (EPL), and glomerular layer (GL) of the OB; and (3) 15–30 days within the OB subcellular layers for these cells to complete the maturation to be the functional interneurons integrating into the local neuronal network (Abrous et al., 2005; Lledo et al., 2006; Ming and Song, 2005; Petreanu and Alvarez-Buylla, 2002). To evaluate the effects of Mn exposure on the entire process of adult neurogenesis from SVZ, along RMS, to OB, the total newly adult-born cell population in the SVZ were labeled with BrdU (50mg/kg, ip. injection for 3 days) prior to Mn exposure; these newborn progenitors were then traced at 0day, 14 days, and 28 days after Mn exposure to investigate their migration, survival, and ultimate differentiation in the OB (Figure 1B).
After BrdU administration for 3 days (at day 0 without Mn exposure), a large population (49.0±12.5 ×103) of newborn proliferating cells were labeled, and these BrdU(+) cells were primarily distributed within the SVZ and RMS in the control rats (Figs. 5A and F). Double-staining with BrdU/DCX confirmed that the newborn cells derived from the SVZ became the migrating neuroblasts (Figs. 5A-a). Within the RMS, a total of 67.8±16.0 ×103 BrdU(+)/DCX(+) cells were counted, suggesting an active migration of newborn neuroblasts along the RMS during the 3-day BrdU administration (Figure 5G).
At 2 weeks after saline treatment in the control group, the total BrdU(+) cells in SVZ and RMS were reduced to 2.55 ± 0.48 ×103 and 1.76 ± 0.56 ×103, respectively, indicating that about 95% and 98% of the BrdU-labeled cells had migrated away from the SVZ (Figs. 5B, D, and F). These numbers became even less in the control SVZ and RMS at 4 weeks (1.10+0.18 ×103 and 0.72±0.22 ×103) (Figs. 5F and G). In the meantime, the Mn-exposed animals showed a similar time-dependent reduction in BrdU(+) newborn cells in the SVZ (2-week: 1.07±0.09×103; 4-week: 0.63±0.11 ×103) and RMS (2-week: 0.85±0.16×103; 4-week: 0.36±0.11 ×103). The total BrdU-labeled cells were significantly less than those in controls at the same time point in the respective SVZ or RMS region (P < 0.01, n = 4 each group, Figs. 5F and G). These data suggested that Mn exposure may either prompt the migration of neuroblasts toward the OB or decreased the survival of residual newborn cells within the SVZ and RMS.
Noticeably, our observations of a time-dependent reduction of BrdU(+) cells in control SVZ and later in RMS are in a good agreement with the literature reports, indicating that the neuroblasts derived from the SVZ can complete the migration along the RMS in about 2 weeks and reach the destination of OB for further integration (Abrous et al., 2005; Ming and Song, 2005; Petreanu and Alvarez-Buylla, 2002).
Since migration of SVZ-derived adult-born neuroblasts to the center of OB takes about 2 weeks, there were barely any BrdU(+) cells in the control OB after 3-day BrdU administration at day 0 without Mn dose administration (Figure 6A). At 2 weeks, however, these BrdU(+) cells arrived in the OB. In the control rats, most of these BrdU(+) cells were scattered within the GCL; a rather small portion of the cells were distributed in the MCL, EPL, and GL (Figs. 6B/a-c). As shown in Figs. 6B/d-f, double-staining with BrdU/DCX revealed that the BrdU(+) cells were DCX(+) immature neurons. Confocal images with 3-D reconstruction in Figs. 6B/g (g1-5) confirmed the colocalization of BrdU with DCX signals. Moreover, in the control OB, quantitation of BrdU(+) cells indicated a total of 108.6±12.4×103 of surviving BrdU(+) NSPCs (Figure 6H), which gave rise to a survival rate about 92%, taking into account the total BrdU-labeled newborn cell population (117.7×103) initially generated within the SVZ, RMS and OB in control rats (Figure 6I).
In the 2-week Mn-exposed OB, most of the surviving BrdU(+) cells were distributed within the GCL but with dramatically lower numbers of the BrdU(+) cells observed than those of the 2-week control (Figs. 6C/a-c). A total number of 57.4±8.8×103 surviving cells were counted to be positive for both BrdU and DCX markers in the GCL of 2-week Mn-exposed OB, which was significantly lower than those in the correspondent control OB (P < .01, Figure 6H). Furthermore, the survival rate of the immature neurons in the GCLS of the Mn-exposed OB was calculated at 49%, which was significantly lower than that of the control (P < .01, Figure 6I). Confocal images in Figs. 6C/g, g1-5 confirmed the colocalization of BrdU with DCX in the GCL of the Mn-exposed animals. These results indicated that most of the surviving BrdU(+) cells in GCL were DCX(+) immature neurons, and at 2-week after Mn exposure, the survival rate of these newborn neurons was significantly reduced.
At the end of the 4-week treatment, most of the BrdU(+) cells in the control group were distributed within the GCL region of the OB (Figs. 6D/a-c). The numbers of BrdU(+) cells in the OB were significantly lower than those in the 2-week control OB (Figure 6H). Noticeably, costaining the OB with anti-BrdU and anti-DCX antibodies did not identify the colocalization of BrdU with DCX in the 4-week control GCL (Figs. 6D/d-f); this was further confirmed by 3-D reconstruction of confocal images at the high magnification (Figs. 6D/g, g1-5). The data suggested that the surviving BrdU(+) cells were no longer the DCX(+) immature neurons; they may have differentiated into mature neurons and integrated into the local circuity.
In the 4-week Mn-exposed animals, the BrdU(+) cell density in the OB was significantly lower than that of the correspondent control OB, although most of them remained within the GCL region (P< .01, Figs. 6E/a-c). Similar to the control group, no colocalization of BrdU and DCX was observed in the GCL region of 4-week Mn-exposed OB (Figs. 6E/g, g1-5).
To determine whether the surviving BrdU(+) cells were ultimately differentiated into mature neurons, the OB were double-stained for BrdU and NeuN; the latter is an established marker for mature neurons. The 3-D confocal images of the GCL at a high magnification revealed that the round BrdU(+) nuclei were colocalized with NeuN marker in both 4-week control and Mn-exposed groups (Figs. 6F/g, g1-5, and Figs. 6G/g, g1-5), suggesting the maturation of newborn neurons in adult OB after 4-week BrdU tracing. Quantification of the surviving cells positive for both BrdU and NeuN within the GCL indicated that following 4-week treatment with saline or Mn, a total of 75.6±4.81 ×103 surviving BrdU(+) cells became the mature neurons in the control OB, whereas a total of 39.4±9.4 ×103 BrdU(+) cells turned into the mature neurons in the Mn-exposed OB, which gave rise to 64% and 33% survival rates of mature neurons in the control and Mn-treated animals, respectively (Figs. 6H-I). The difference between the control and Mn-treated groups was statistically significant (P < .01, Figs. 6H-I), suggesting that after subchronic Mn exposure, fewer BrdU(+) cells survived and differentiated into mature neurons in the OB.
Previous studies using XRF microscopy discover an extraordinary high level of Cu accumulates within the SVZ and RMS (Pushie et al., 2011; Pushkar et al., 2013). Our recent studies also reveal a significant reduction of Cu content in the SVZ in contrast to an overall increase of Cu in other brain regions following in vivo subchronic Mn exposure (Fu et al., 2015a). An age-related increase of Cu level in the SVZ has also been reported (Fu et al., 2015b). We hypothesized that the Mn-Cu interaction in the SVZ may underlie Mn-induced alteration of the adult neurogenesis. An icv infusion technique was used to deliver a single dose (6μl) of saline, 30μg/ml CuCl2, or 50μg/ml MnCl2 directly into the right lateral ventricle to investigate the cell proliferation outcomes using a 4-h BrdU labeling regimen (Figure 1C). Confocal imaging data showed that the density of BrdU(+) cells in the SVZ of the left hemisphere from control rats receiving the icv infusion of saline (Figure 7B) was markedly higher than those of control animals without receiving the icv infusion (Figure 7A), suggesting that the procedure with a physical insertion could induce the adult neurogenesis. In contrast, rats receiving the icv infusion with either MnCl2 (Figure 7C) or CuCl2 (Figure 7D) produced much less BrdU signals as compared to the controls with the procedure (Figure 7B).
Quantitation of BrdU(+) proliferating cells showed that the control SVZ with and without the icv procedure had a total of 45.79±0.48×103 and 33.52±2.79×103 BrdU(+) proliferating cells, respectively. This difference was statistically significant (P < .01, Figure 7E), confirming that similar to an invasive brain surgery to cause traumatic brain injury, the procedure of icv infusion had stimulated the cell proliferation in adult SVZ. In Mn- and Cu-exposed animals, the total numbers of BrdU(+) cells in the SVZ were 34.24±3.54×103 and 37.77±2.89×103, respectively. Both were significantly lower than that of the saline control (P < .01, Figure 7E). Thus, these results indicate that the presence of a high level of Mn or Cu in the CSF may inhibit the cell proliferation in adult SVZ.
The data from the current study confirm our previous observation that subchronic Mn exposure stimulates the cell proliferation in the SVZ (Fu et al., 2015a). By in-depth analysis of major cell types involving in the adult neurogenesis in the SVZ, our data reveal that the proliferated cells under Mn influence are the mixed cell population, including GFAP(+) type-B neural stem cells, Nestin(+) type-C transit amplifying stem cells, DCX(+) type-A migratory neuroblasts, and Iba1(+) microglial cells. In addition, our time-course study to trace BrdU-labeled cells from their initial proliferation in the SVZ to subsequent migration via the RMS and ultimate survival and differentiation in the OB demonstrate that in vivo Mn exposure in fact reduces the survival of BrdU(+) cells and inhibits their differentiation into matured neurons in the OB. Direct in-situ exposure of SVZ cells with Cu in the ventricular CSF further discovers a suppressed cell proliferation in the SVZ, suggesting a role of Cu in mediating the cell proliferation in the SVZ.
Newly proliferated cells can be labeled with BrdU within hours before they migrate and turn into other cell types (Duan et al., 2008). To precisely investigate the cell proliferation, the current study adapted an approach to trace newly labeled BrdU(+) cells for only 4h. It became apparent that within a short time frame of BrdU labeling, all major cell types in the SVZ underwent the proliferation after Mn exposure; their characteristic cellular markers were colocalized with BrdU and their numbers were increased. These results are consistent with our previous findings of enhanced expressions of BrdU, Gfap, Nestin, and Dcx in the Mn-exposed SVZ (Fu et al., 2015a). Interestingly, an acute, direct exposure of the SVZ with a high Mn concentration in the CSF (by the icv infusion) did not cause an increased, but rather a suppressed, proliferation. Thus, these observations appear to suggest that an increased proliferation under the in vivo subchronic Mn exposure regimen may represent brain’s initial neural repair mechanism in response to Mn-induced overall neurotoxicity. The Mn-exposed rat model used in current studies has been found to result in significant Mn accumulations in the motor cortex, CP, hippocampus, striatum, and SVZ (Fu et al., 2014, 2015a; Robison et al., 2012; Zheng et al., 2009). Our recent studies with this animal model also demonstrate a Mn-induced dysfunction in the striatal dopaminergic system (O’Neal et al., 2014). Moreover, Mn exposure is known to causes the olfactory injury both in human subjects and in animal models (Elder et al., 2006; Sen et al., 2011). These neuronal injuries could trigger the cell proliferation in the adult neurogenic niches, although the increased cells may not eventually turn into the matured neurons in the late stage of adult neurogenesis (see Discussion below).
Among five major cell types studied, the GFAP(+) type-B neural stem cells are the progenitor cells in the SVZ to give rise to type-C and type-A cells. With abundant high-affinity/capacity transporting proteins, Mn is known to accumulate preferentially in astrocytes, about 60-fold higher than in neurons (Aschner et al., 1992; Wedler et al., 1989). Astrocytes play a pivotal role in maintaining normal neuronal functions and regulating neuronal survival, proliferation, differentiation, and neurogenesis in the neurogenic niches (Bak et al., 2006; Blondel et al., 2000; Farina et al., 2007; Liebner et al., 2011). Currently there is no evidence to suggest to what degree Mn may accumulate in the type-B cells in reference to its accumulation in other cell types in brain neurogenic niches and whether there is a correlation between Mn and Cu in each cell type. However, the data by the XRF microscopy with subcellular resolution has established that Cu is preferentially localized in the GFAP(+) cells in the SVZ (Pushkar et al., 2013). Increased Cu levels have been associated with a decreased neurogenesis in aging (Fu et al., 2015b; Pushkar et al., 2013). An inverse association between Mn and Cu has also been observed in hippocampal dentate gyrus, another neurogenic niche in the brain (Robison et al., 2013). As stated above, it is possible that Mn-induced general neurotoxicity may incite the neurogenesis; yet a direct interaction between Mn and Cu in the type-B astrocytic cells may disturb the normal function of these cells in the SVZ, leading to an elevated cell proliferation. These hypotheses require additional experiments to test.
In the brain, microglial cells serve as immune macrophages that abundantly reside in the basal ganglia and hippocampus (Lawson et al., 1990). Several lines of evidence indicate that Mn exposure can lead to microglial activation with an ensuing release of reactive oxygen species, nitric oxide, prostaglandin E2, and inflammatory cytokines such as TNF-α, IL-1β, and IL-6 (Block et al., 2007; Liu et al., 2009; Tansey and Goldberg, 2010; Zhang et al., 2007). The current study with dual labeling of BrdU and Iba1 confirms that the proliferating cells in the SVZ include microglial cells. However, the limited percentage of these microglia over the total proliferating cell population (approximately 3%) does not appear to support a major role of Mn-induced microglia activation in the cell proliferation in adult SVZ following Mn exposure.
In contrast to the initial increase in cell proliferation, Mn exposure apparently inhibits the cell migration, survival, and differentiation in the late stage of adult neurogenesis. After 2-week Mn exposure, fewer BrdU(+) cells were present in the RMS and a much lower density of BrdU/DCX(+) cells was observed in the OB in the Mn group than those in the control. Clearly, Mn exposure impairs the migration of SVZ-derived neuroblasts along the RMS. The process for adult-born neuroblasts to migrate through the RMS is strictly regulated by a complex of intrinsic and extrinsic factors that involve in dynamic cell-cell communication, cell-extracellular matrix interactions, chemorepellent, and chemoattractant signaling (Christie and Turnley, 2012; Kaneko et al., 2010; Sawamoto et al., 2006; Vukovic et al., 2011). It is still unclear how exactly Mn impacts the adult neurogenesis in the SVZ-RMS-OB system. However, Mn has been shown to disrupt the Gln/Glu-GABA cycle in astrocytes (Lee et al., 2009; Milatovic et al., 2007; Sidoryk-Wegrzynowicz et al., 2009) and altering GABA transport and receptor proteins which are implicated in neuroblasts migration in the RMS (Anderson et al., 2008; Bolteus and Bordey 2004; Dydak et al., 2011). Noticeably, the migration of neuroblasts in the integrated architecture of RMS is guided by the “astroglial tube,” formed by the network of astrocytic processes (Bozoyan et al., 2012; Jankovski and Sotelo, 1996; Lois et al., 1996; Peretto et al., 1997). This astroglial tube not only creates a physical barrier, but also regulates the signaling molecules such as GABA. It is likely that Mn may disturb the neuroblasts migration by influencing the structure and function of these ensheathing astrocytes as well as the GABA level within the SVZ and RMS.
Mn exposure selectively inhibits the survival and differentiation of BrdU-labeled newly generated NSPCs. This is supported by the following evidences: (1) at the 4th week, Mn exposure significantly reduced the surviving BrdU(+) cells in SVZ and RMS; (2) the surviving rate of BrdU-labeled cells in the destination OB was greatly diminished following Mn exposure; and (3) the cell differentiation to the ultimate NeuN(+) mature interneurons in the OB was also significantly decreased in Mn-treated animals as compared to controls. Several factors, such as insulin-like growth factor (IGF-I), brain derived neurotrophic factor (BDNF), vascular endothelial growth factor, epidermal growth factor, and fibroblast growth factor are essential to the normal proliferation, neuroblasts migration, and differentiation in the SVZ-RMS-OB system (Bath et al., 2008; Grade et al., 2013; Hurtado-Chong et al., 2009; Snapyan et al., 2009; Zigova et al., 1998). In primary cultured cerebellar neurons, Mn treatment has been shown to decrease insulin receptor, IGF-I receptor, and IGF-II receptor expression (Tong et al., 2009). A recent in vivo study reports a decreased BDNF level in the striatum of Mn-exposed nonhuman primates (Stansfield et al., 2014). Zou et al. (2014) report a significantly lower plasma BDNF level in a cohort of 819Mn-exposed smelters than in 293 control workers. These findings imply that in addition to its direct effect on cell survival and differentiation, Mn may interfere with the neurogenesis in the RMS and OB by acting on these critical molecules.
It should be pointed out that cell proliferation in the SVZ could be affected by both the dose and time of toxic exposure. An initial increase in cell proliferation observed in Expt-1 could be the consequence of the brain injury under the systemic Mn exposure as discussed above. It is also possible that the increased SVZ proliferation in Exp-1 could be due to the prolonged exposure scheme. Thus, a more detailed investigation on the dose-time-response relationship is well warranted.
Our early studies have shown that subchronic exposure to Mn significantly increases Mn and Cu concentrations in the CSF, CP, striatum, hippocampus, and other brain regions, but a decrease in Cu levels in the SVZ (Fu et al., 2014, 2015a; Zheng et al., 2009). To understand whether changes in chemical components of the CSF would influence the adult neurogenesis in adjacent SVZ, we used a well-established icv infusion technique to deliver Cu and Mn directly to brain lateral ventricles. Our data demonstrate a marked decrease in the number of BrdU(+) proliferating cells in the SVZ (or a suppressed neurogenesis), after perfusion with a high concentration of Cu ions into the brain ventricles. A naturally high Cu content within the rat SVZ has been identified (Pushkar et al., 2013). It is therefore tempting to believe that a high Cu level in the CSF or SVZ may be necessary to prevent the unnecessary neurogenesis in adult brain under the normal life condition; this notion is partially supported by the current icv data.
At the cellular level, Pushkar et al. (2013) observed that Cu ions predominantly accumulate in the GFAP(+) type B cells in the SVZ, while their signals are absent within actively dividing cells. Metallothioneins are found to have the highest mRNA expression in the SVZ among other proteins analyzed (ie, Cox17, Ccs, Atox1, Ctr1, DMT1, etc.) and positively correlate with age and SVZ Cu contents (Fu et al., 2015b). Our early study also shows a positive correlation between Cu concentrations and GFAP mRNA levels in the SVZ (Fu et al., 2015b). Since Mn exposure reduces the Cu level in the adult SVZ (Fu et al., 2015a) and increases cell proliferation, it seems likely that the Cu level in the SVZ may function as a sensor, which in response to surrounding environmental changes, may “switch on” (to promote) or “switch off” (to restrain) the signaling pathway that regulates the neurogenesis process in the SVZ. Mn exposure, by triggering this Cu-sensor, causes an aberrant neurogenesis within this niche. These hypotheses, however, require additional investigations.
It should be pointed out that the homeostasis of Cu and Mn is tightly regulated by the CP, a physical barrier between the blood and CSF (Zheng and Monnot 2012). The CP also secretes Slit2, the critical molecule in guiding NSPCs’ migration in the SVZ, into the CSF (Hu 1999; Wu et al., 1999). The type-B cells in the SVZ extend their primary cilium into the ventricle space and thus are in direct contact with the CSF (Gil-Perotin et al., 2009; Han et al., 2008; Ming and Song, 2005; Mirzadeh et al., 2008). Considering the unique anatomical connection between the CP and SVZ through the CSF, we propose that there is a “CP-CSF-SVZ” epithelia-ventricle-interactive lineage axis. Such an axis functions to maintain the chemical stability that is required for normal adult neurogenesis. Mn exposure, by acting on the CP (increased accumulation of Mn and Cu) and altering the CSF chemistry, may disrupt the delicate balance in the CP-CSF-SVZ axis, leading to abnormal neurogenesis.
In conclusion, the data presented in the current study demonstrate that subchronic exposure to Mn leads to an increase in cell proliferation within the adult SVZ, a reduction in surviving SVZ-derived neuroblasts and an inhibition of neuroblast maturation in the OB. Excessive Mn or Cu in the CSF decreases the BrdU(+) proliferating cells in the adult SVZ. Our findings are the beginning of our understanding of the relationship between Mn exposure and adult neurogenesis in the SVZ-RMS-OB system. Future in-depth investigations are strongly recommended to explore the underlying mechanisms as well as the role of Cu in adult neurogenesis.
This study was supported by NIH/National Institute of Environmental Health Sciences Grants Number ES008146.