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Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats
Molecular Brain Research 103 (2002) 116–129 www.elsevier.com / locate / bres
Research report
Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats ´ ´ Mayka Tomas-Camardiel, Antonio J. Herrera, Jose´ L. Venero, Mari Cruz Sanchez-Hidalgo, Josefina Cano, Alberto Machado* ´ ´ y Toxicologıa ´ , Facultad de Farmacia, Universidad de Sevilla, C /Prof. Garcıa ´ Gonzalez ´ , Bromatologıa s /n, Departamento de Bioquımica Seville, Spain Accepted 14 March 2002
Abstract Recent studies have implicated chronic elevated exposures to environmental agents, such as metals (e.g. manganese, Mn) and pesticides, as contributors to neurological disease. Eighteen-month-old rats received intraperitoneal injections of manganese chloride (6 mg Mn / kg / day) or equal volume of saline for 30 days in order to study the effect of manganese on the dopamine- and GABA-neurons. The structures studied were substantia nigra, striatum, ventral tegmental area, nucleus accumbens and globus pallidus. First, we studied the enzymatic activity of mitochondrial complex II succinate dehydrogenase (SDH). We found an overall decrease of SDH in the different brain areas analyzed. We then studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra and ventral tegmental area following Mn treatment. Correspondingly, TH immunoreactivity was increased in substantia nigra and ventral tegmental area. Manganese treatment significantly decreased GAD mRNA levels in individual GABAergic neurons in globus pallidus but not in striatum. We also quantified the density of glial fibrillary acidic protein (GFAP)-labeled astrocytes and OX-42 positive cells. Reactive gliosis in response to Mn treatment occurred only in striatum and substantia nigra and the morphology of the astrocytes was different than in control animals. These results suggest that the nigrostriatal system could be specifically damaged by manganese toxicity. Thus, changes produced by manganese treatment on 18-month-old rats could play a role in the etiology of Parkinson’s disease. 2002 Elsevier Science B.V. All rights reserved. Theme: Motor system and sensorimotor integration Topic: Basal ganglia Keywords: Rat; Manganese; Nigrostriatal system; Ventral tegmental area; Nucleus accumbens
1. Introduction Parkinson’s disease (PD) is one of the most common movement disorders and is related to destruction of neurons in the substantia nigra pars compacta of the basal ganglia. While the cause of neuronal death in PD remains
*Corresponding author. Tel.: 134-95-455-6751; fax: 134-95-4556752. E-mail address: [email protected] (A. Machado).
unknown, increasing evidence has involved a variety of environmental factors in the selective dopaminergic cell loss in substantia nigra (SN) typical of nonfamilial PD patients [22,37,54]. Thus, research has been focused on the possible involvement of genetics, exogenous toxins or endogenous toxins from cellular oxidative reactions. The identification of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a synthetic heroin that destroys SN dopamine (DA) neurons, has given additional credence to an environmental factor hypothesis [33,53], although MPTP is not an environmental contaminant. However, the
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00192-4
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structural homology of the herbicide 1,19-dimethyl-4,4bipyridinium (paraquat, PQ) to 1-methyl-4phenylpyridinium (MPP1), the active metabolite of MPTP, has emerged as a putative risk factor. Occupational PQ exposures have been associated with parkinsonism [17,37]. The fungicide manganese ethylenebisdithiocarbamate (maneb; MB) decreases locomotor activity [39] and potentiates MPTP effects on locomotor activity and catalepsy [52]. At least two incidences of parkinsonism in humans have been related to MB exposure [20,38]. Moreover, it is well established that chronic exposure to manganese and manganese-related hepatic encephalopathy cause neuropsychiatric symptoms [8,58], which often manifest as extrapyramidal symptoms resembling Parkinson’s disease, including bradikynesia and distonia [1,15,18,57]. Mn is a widely used metal, and miners exposed to Mn fumes develop Parkinson syndrome. Racette et al. [43] have reported 15 parkinsonian patients who were professional welders. Of interest, chronic manganese intoxication causes specific PD-like symptoms that respond to levodopa [29]. In primates exposed to Mn for 18 months, marked degeneration of the SN has been reported [25]. In the normal human brain, the highest Mn concentrations are found in the globus pallidus, striatum, thalamus and SN. The need to better understand the effects of manganese on behavior is becoming more important due to a potential increase of environmental exposure to manganese since it has been proposed as a gasoline additive in a number of countries. However, the animal models for studying the Mn-related neurotoxic features have pointed out a great disparity of neurotoxic manifestations; manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism [5]. Other did not find any neurotoxic action, or described protective effects. The intranigral infusion of manganese (1.05–4.2 nmol) protected nigral neurons from iron-induced oxidative injury and dopamine depletion [51]. The purpose of this study was therefore to investigate whether manganese exposure influences the functioning of the nigrostriatal system in aged rats and to compare with another DA system, the ventral tegmental area (VTA) / nucleus accumbens. Manganese has been shown to inhibit mitochondrial aconitase activity, which results in disruption of brain mitochondrial energy production as well as iron metabolism [59]. In addition, previous reports have indicated that Mn intoxication is primarily confined to the globus pallidus [46]. Moreover, selective vulnerability of pallidal neurons in the early phases of manganese intoxication has been reported [47]. For this reason, we have studied the effect of Mn treatment on energetic metabolism measured as succinate dehydrogenase activity, the tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA expression as markers of dopaminergic system and also the glutamic acid decarboxylase (GAD) mRNA expression in two different structures, globus pallidus and striatum. We also analysed the glial response after Mn treatment.
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2. Material and methods
2.1. Animals, treatment and dissection Twenty 18-month-old male albino Wistar rats were used. The rats, born in our laboratory, were housed in a room maintained under constant temperature (2262 8C) and humidity conditions (60%) with a 12:12 h light–dark cycle. Food and water were available ad libitum. Animals were cared for and treated in accord to the Guidelines of the European Union Council (86 / 609 / EU), following the Spanish regulations (BOE 67 / 8509-12, 1988) for the use of laboratory animals and approved by the Scientific Committee of the University of Sevilla. Animals were divided into two groups. Rats received intraperitoneal injections of manganese chloride (MnCl 2 ) at the dose of 6 mg Mn / kg / day or equal volume of saline for 30 days. Five animals from each group (control or experimental) were used for in situ hybridization and five animals from each group (control and experimental) were used for immunohistochemistry.
2.2. Immunohistological evaluation: tyrosine hydroxylase, glial fibrillary acidic protein and OX-42 Rats were perfused through the heart under deep anaesthesia (chloral hydrate) with 150–200 ml of 4% paraformaldehyde in phosphate buffer, pH 7.4. The brains were removed, and then cryoprotected serially in sucrose in phosphate-buffered saline (PBS), pH 7.4; first in 10% sucrose for 24 h and then in 30% sucrose until sunk (2–5 days). The brains were then frozen in isopentane at 215 8C, and 25-mm sections were cut on a cryostat and mounted in gelatine-coated slices. Primary antibodies used were: mouse-derived antityrosine hydroxylase (Roche Diagnostic, Barcelona, Spain; 1:200), antiglial fibrillary acidic protein (GFAP) (Chemicon, Temecula, CA, USA; 1:300), and mousederived OX-42 (Serotec, Oxford, UK; 1:200). OX-42 is an antibody directed against the type-3 complement receptor (CR3) and recognizes macrophages and microglial cells. All incubations and washes were in Tris buffered saline (TBS), pH 7.4 unless otherwise noted. All work was done at room temperature. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 15 min, washed again, and incubated in a solution containing TBS and 1% horse serum for 60 min in a humid chamber. Slices were drained and further incubated with the primary antibody in TBS containing 1% horse serum and 0.25% Triton-X-100 for 24 h. Sections were then incubated for 2 h with biotinylated horse antimouse IgG (Vector, 1:200) followed by a second 1-h incubation with kit ABC (Immuno pure standard ultrasensitive ABC staining kit, Pierce, 1:100). The antibody was diluted in TBS containing 0.25% Triton-X-100, and its addition was preceded by three 10-min rinses in TBS. The peroxidase was
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visualized with a standard diaminobenzidine–hydrogen peroxidase chromogen reaction for 5 min.
2.3. Preparation of the riboprobe TH antisense transcript was generated from a plasmid kindly provided by Dr. Sokoloff (INSERM, Paris, France), which contains a 282-nucleotide fragment of the 39 end of the rat TH gene cloned in PGEM-4Z (Promega) (including 13 nucleotides of 39 untranslated sequence), which corresponds to nucleotides 1240–1521 of the rat TH DNA sequence [23]. This plasmid was linearized with EcoRI and used as template with the T7 RNA polymerase to get the antisense riboprobe [16]. DAT antisense riboprobe was generated from a plasmid kindly provided by Dr. M.P. Martres (INSERM), which contains the rat DAT cDNA 800-bp XbaI–HindIII fragment cloned in pRc / CMV (Invitrogen). To prepare the DAT antisense riboprobe this plasmid was linearized with XbaI and used as a template with the T7 RNA polymerase [16]. pBbluescript SK plasmids, containing the cDNA sequence for GAD67 as a 3.2-kb EcoRI insert (clones 14 and 18), were kindly provided by Dr. A. Tobin (UCLA, Los Angeles, CA, USA). The GAD67 cDNA was isolated from a 1 gt-11 cDNA library made from poly(A) RNA from adult rat brain [19]. To prepare the GAD67 antisense transcript, clone 14 was digested with SalI and used as a template with the T3RNA polymerase. To prepare the GAD67 sense transcript, clone 18 was digested with SalI and used as a template with the T3 RNA polymerase. Single-strand antisense cRNA probes were synthesized with T7 and T3 RNA polymerases according to a protocol provided by the RNA polymerase supplier (Bethesda Research Laboratories, Bethesda, MD, USA). The reaction mixture contained the reaction buffer provided with the RNA polymerase; ATP, CTP and GTP each at 1 mM; and 30 mM [ 35 S] UTP (1300 Ci / mmol).
SSC at 25 8C followed by 0.13 SSC at 60 8C for 1 h, dehydrated in a series of ethanols, air-dried and processed for emulsion autoradiography. Autoradiograms were generated by opposing the labelled tissue to bmax Hyperfilm for 2 weeks. After looking at the dry film autoradiography, slides were dipped in Amersham LM1 emulsion (diluted 1:1 with water) and exposed in the dark for 10–15 days at 4 8C. Slides were then developed in D-19 (Kodak) at 15 8C for 2.5 min fixed for 10 min in fixer (Kodak), and counterstained with cresyl violet (Sigma–Aldrich).
2.5. In situ hybridization data analysis For quantitation of dry film autoradiography, the films were scanned at high resolution and the TIF image was analyzed by densitometry using the computer program ANALYSIS (Germany) based on calibrated gray scale densities. Calibration was performed by means of [ 3 H] microscales. The region of interest was delineated and the optical density obtained. Additional measurements of mRNA levels were obtained by quantifying the number of silver grains per neuron using the same computer program. Emulsion-dipped slides were used for these measurements. Labeling was considered specific when grain accumulation over cells with a large nucleus exceeded five times the background value. The area with high grain density over individual cell body was delineated and the number of grains within this field was counted by means of the computerized image analysis system with a 403 magnification lens. Because of the high levels of labeling, the typical disposition of the clusters over the cell body and the high magnification, the delineation was unequivocal. Only well-separated cells were selected for quantification.
2.4. In situ hybridization histochemistry
2.6. Immunohistochemistry data analysis
In situ hybridization on brain sections was carried out following a modification of a procedure described in detail elsewhere [41]. Thaw-mounted 12-mm sections were postfixed for 30 min in 4% paraformaldehyde, followed by three 10-min washes in phosphate-buffered saline (pH 7.4). Sections were treated for 1 min in 0.1 M triethanolamine, followed by 10 min in acetic anhydride–0.1 M triethanolamine in order to decrease nonspecific binding. Following a 1-min wash in 23 standard saline citrate (SSC), sections were dehydrated in a series of increasing concentrations of ethanol and then air-dried. The sections were hybridized for 3 h at 50 8C with the [ 35 S] cRNA, rinsed in 43 SSC–20 mM DTT, then 43 SSC alone. Sections were subjected to 30 min of RNase digestion at 37 8C (20 mg / ml RNase A in 0.5 M NaCl, 0.01 M Tris–HCl, 1 mM EDTA; pH 8.0), washed for 2 h in 23
Reactive astrocytes detected as GFAP immunopositive cells and microglia detected by OX-42 were counted in SN and VTA at one specific rostrocaudal level (plate 39 of the rat brain atlas of Swanson [50]). The rationale for this is the heterogeneity of these brain areas. In SN, the quantifications of astrocytes and microglia were performed in three different nigral locations corresponding to medial, central and lateral aspects of this brain area, whereas in the VTA area the quantification was restricted to one location. All quantifications were done at 403 magnification. In striatum and nucleus accumbens, the GFAP and OX-42 immunohistochemistries were counted every seventh serial frontal section. Counting of striatal astrocytes and microglia was performed in three different locations corresponding to medial, central, and lateral striatum.
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2.7. Tyrosine hydroxylase staining intensity in substantia nigra, ventral tegmental area, striatum and nucleus accumbens Sections were stained as described under Immunological evaluation. Quantitative estimates of TH immunoreactivity were made in the bilateral SN pars compacta, VTA, striatum and nucleus accumbens. Intensity measurements were done with the experimenter blinded to the treatment and in random order to avoid any bias. A total of ten representative sections per animal were used to determine TH density within the substantia nigra pars compacta,
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VTA, striatum and nucleus accumbens. Each treatment group was represented by five animals. The analysis was done in each sampled section at a magnification of 403, permitting representative visualization of each region. The average of the density for each section was added and divided by the total number of sections analyzed to give the mean TH density for each animal. The results are expressed as percentage compared to the control animals.
2.8. Succinate dehydrogenase histochemistry Slides were incubated for 45 min at 37 8C in 50 ml of
Fig. 1. Succinate dehydrogenase (SDH) histochemistry in Control and MnCl 2 -treated animals. (A,C) Control; (B,D) MnCl 2 treatment; (E) measurement of optical density, expressed as percentage of Control. Numbers are mean6S.D. of five independent experiments. Statistical significance (Student’s t test): *, P,0.05; **, P,0.01. Scale bars51 mm.
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0.06 M phosphate buffer (pH 7.0) containing 20 mg of nitro blue tetrazolium and 0.68 mg of succinate [31]. The specificity of the reaction was confirmed by incubating the sections with increasing concentrations of 3-nitropropionic acid, a selective inhibitor of the enzyme. Enzyme activity of controls and experimental animals was determined by densitometry based on calibrated grey scale values.
2.9. Statistical analyses Statistical analyses of the differences between groups were performed by using Student’s t test. The difference between two means was considered significant if P values were #0.05.
3. Results
3.1. Changes in SDH activity Analysis demonstrated an overall decrease of SDH activity throughout the brain. Most remarkable decreases were found in striatum, SN, VTA and subthalamic nucleus, which are shown in Fig. 1. We were unable to reliably measure SDH activity in globus pallidus due to the constitutive low activity in this brain area.
3.2. Changes in TH immunoreactivity Levels of TH immunoreactivity (TH-IR) were significantly increased in the cell body regions in SN (144%
Fig. 2. Tyrosine hydroxylase (TH) immunoreactivity in Control and MnCl 2 -treated animals. (A) Control; (B) MnCl 2 treatment. The immunoreactivity increased in treated animals as compared to the Control. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. Scale bar5500 mm. (C) TH immunoreactivity quantification. Numbers are mean6S.D. of five independent experiments, and are expressed as optical density (% of control). Statistical significance (Student’s t test): *, P,0.01.
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control levels, P,0.01) and VTA (150% control levels, P,0.01) after Mn treatment (Fig. 2). In contrast, Mn treatment failed to alter the density of TH-IR in their respective dopaminergic projecting areas in striatum and nucleus accumbens (Fig. 2).
3.3. Expression of TH mRNA and DAT mRNA in substantia nigra and ventral tegmental area In situ hybridization of brains from control- and Mntreated animals with the antisense riboprobes for TH mRNA and DAT mRNA resulted in specific labeling in the ventral mesencephalon (Fig. 3). The typical disposition of the grain clusters over the cell bodies and the precise location of these neurons in the SN pars compacta and
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VTA clearly indicate that these TH and DAT mRNA expressing neurons were dopaminergic. For quantification, as a first step, we performed analysis of optical density from dry film autoradiographs. Densitometric analysis revealed a significant elevation in TH mRNA levels in SN and VTA after Mn treatment as compared to controls (169 and 159% of control levels for SN and VTA, respectively, P,0.01; Figs. 3 and 4A). On the contrary, DAT mRNA levels remained at control levels following Mn-treatment in both structures (Figs. 3 and 4A). Analysis of cellular expression of TH mRNA in the SN and VTA by silver grains counting confirmed the Mninducing effect over TH mRNA expression. Cellular levels of TH mRNA within individual dopaminergic neurons in
Fig. 3. Expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNAs in substantia nigra from Control and MnCl 2 -treated animals. (A) Control TH; (B) TH after MnCl 2 treatment; (C) control DAT; (D) DAT after MnCl 2 treatment; (E,F) high magnifications of silver grains from (A) and (B), respectively. Scale bars: A–D, 1 mm; E–F, 50 mm.
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Fig. 4. Quantification of tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA in situ hybridization in Control and MnCl 2 treated animals. (A) Optical density measured from dry films expressed as percentage of Control. Numbers are mean6S.D. of five independent experiments; (B) silver grain counting. TH mRNA expression in SN and VTA of Control and MnCl 2 -treated animals were expressed as silver grains per cell. Numbers are mean6S.D. of five independent experiments. Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.05; **, P,0.01.
response to Mn treatment were as follow: SN, 161% of control levels, P,0.01; VTA: 123% of control levels, P,0.05. (Figs. 3 and 4B).
3.4. Glial fibrillary acidic protein and OX-42 immunoreactivity As seen in Fig. 5, counting of reactive astrocytes in striatum and SN was performed in three different locations from the medial to the lateral part of the structures considering the heterogeneity in projection patterns of these brain areas. VTA, globus pallidus and nucleus accumbens were analyzed at a single location. We found a medial to lateral gradient decrease in the distribution pattern of GFAP immunoreactive astrocytes in the striatum of control and Mn-treated animals. Thus, the density of reactive astrocytes in the medial striatum was about 2-fold of that found in the lateral part of this brain area. In contrast, SN did not show a noticeable topographical gradient. In animals treated with Mn, the density of GFAP-
immunoreactive astrocytes significantly increased above controls in striatum and SN. This effect was seen in all striatal and nigral locations analyzed (Figs. 6–8). The increases of GFAP-IR were similar in the three striatal and nigral locations analyzed following Mn treatment as compared with controls (Figs. 6–8). The reactive astrocytes were never isolated. The morphology of reactive astrocytes were different in Mn-treated rats as compared with controls. They showed more abundant and larger processes than resting astrocytes and became hypertrophic, losing their characteristic stellate morphology (Figs. 6 and 7). In contrast to striatum and SN, Mn treatment failed to alter the density of GFAP-IR astrocytes in VTA and nucleus accumbens (data not shown). Counting of OX-42 positive cells also demonstrated a slight medial to lateral gradient decrease in the density of microglial cells in striatum and SN of both control and Mn-treated animals (Fig. 9). Following Mn treatment, there was a significant increase in the density of OX-42 immunoreactive microglia in the striatum and SN (Figs. 6, 7 and 9). OX-42 antibody stained ramified microglial cells in both areas. After Mn treatment, microglial reactivation was more intense than in the control animals in both structures (Figs. 6 and 7). Mn treatment failed to alter the density of OX-42 immunoreactive microglia in VTA and nucleus accumbens (data not shown).
3.5. Expression of GAD67 mRNA in striatum and globus pallidum after Mn treatment Once we found an inducing effect of Mn treatment on TH mRNA levels, the step-limiting enzyme in catecholamine biosynthesis, we wanted to know whether this effect was restricted to the dopaminergic system. For that, we analysed GAD mRNA, the biosynthetic enzyme for GABA. Within the striatum and in the globus pallidus, the riboprobe used for detecting GAD mRNA labeled numerous cell bodies in both structures (Fig. 10). Quantifications performed on dry film autoradiographs demonstrated that following Mn treatment there was a significant decrease in GAD mRNA levels in globus pallidus (68.2% of control, P,0.01; Fig. 10). In contrast, no differences were found in striatum in terms of GAD mRNA levels (Fig. 10).
4. Discussion It has long been established that Mn disrupts different markers of monoaminergic activity in the brainstem, this effect being more evident in aged animals [13]. Consequently, we have used aged rats in our study. Mn has been shown to impair mitochondrial energy metabolism at different levels [4,59]. We now demonstrate that Mn treatment significantly decreases SDH activity in nearly all the structures studied. These decreases were of nearly 30%
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Fig. 5. Locations of reactive astrocytes counting in several brain structures in Control and MnCl 2 -treated animals. (A) Counting of reactive astrocytes in striatum were performed in three different locations from the medial to the lateral part of this brain area (numbered dots); the asterisk marks the nucleus accumbens. Section level was 0.95 mm anterior to bregma (B); counting of reactive astrocytes in substantia nigra were also performed in three different locations from the medial to the lateral part of this brain area (numbered dots); the asterisk marks the VTA. Section level was 6.06 mm posterior to bregma.
in SN and striatum. This decrease in energetic metabolism could be one action that explains the extrapyramidal symptoms described in Mn intoxications. It is in agreement with the fact that manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism [5]. Afterwards, we measured reactive microglia and astroglia in response to Mn treatment, which are good indexes of degenerative-related events. Typical morphological features of reactive microglia and phagocytic cells were observed in response to Mn administration in SN and striatum, especially in the former. These Mninduced changes were regionally-specific since no significant changes were observed in other brain areas analyzed including VTA, nucleus accumbens and globus pallidus. The Mn-induced activation of microglial cells in the
nigrostriatal system could be a consequence of tissuespecific damage, a specific activation of glial cells or both. High levels of manganese have been shown to potentiate microglial nitric oxide production in response to bacterial lipopolysaccharide, a potent inducer of inflammation [9]. This potent activity of manganese is not shared by other transition metals, including iron, cobalt, cooper and zinc [9]. Within this context, it is interesting to note the presence of reactive microglia in the SN of patients with PD [21]. This response has been also reported in animal models of PD [7,28,32]. Interestingly, the SN contains the highest concentration of microglia in the brain [34], and it contains a specific vasculature that results in an ineffective blood–brain barrier [45]. With regard to the astroglial population, the density of
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Fig. 6. Glial fibrillary acidic protein (GFAP) and OX-42 immunoreactivity in striata from Control and MnCl 2 -treated animals. (A,B) GFAP immunostaining in Control and MnCl 2 -treated animals, respectively. Astrocytes in MnCl 2 -treated animals appear hyperthrophic, with abundant and large processes. (C,D) OX-42 immunostaining in Control and MnCl 2 -treated animals, respectively. Immunostaining is more intense in treated animals. Scale bar5200 mm.
GFAP-immunolabeled astrocytes also increased in the terminal field and cell body region in the nigrostriatal system in response to Mn treatment. The induction of astrocytosis by Mn has also been reported [49]. Mn administered intranasally in rats for 3 weeks increased levels of GFAP and S-100 in the olfactory bulbs [27]. Variations of GFAP immunoreactivity is thought to be a sensitive index of astrocyte reaction to any damage occurring in the brain tissue. Taken together, our results are consistent with the view that Mn triggers an inflammatory stimulus for SN and striatum, which could be deleterious to the nigrostriatal system. Reactive microglia and astrocytes are known to secrete different proinflammatory cytokines and cytotoxic molecules, such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and nitric oxide (NO) [2,9,10,35]. Similar effects have been reported for Mn, which induced the iNOS of microglia [9] and astrocytes
[49], although Mn did not appear to be cytotoxic to glial cells. As a next step, we sought to determine whether the dopaminergic system was affected, since most specific relevant changes were confined to the nigrostriatal system. In fact, the activation of glial cells in response to Mn administration could be related to DA toxicity, taking into account that dopaminergic areas were specially affected. DA may exert neurotoxic effects via enzymatic metabolism forming H 2 O 2 that can be broken down to free radicals species in the presence of metals [40,48] or via autooxidation forming various reactive compounds [26]. Linert et al. [36] have shown that Mn(II) significantly enhances the rate of dopamine auto-oxidation in vitro. We studied different markers of dopaminergic activity and, contrary to expectations, neither of them decreased in response to Mn administration. On the contrary, TH
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Fig. 7. Glial fibrillary acidic protein (GFAP) and OX-42 immunoreactivity in substantiae nigrae from Control and MnCl 2 -treated animals. (A,B) GFAP immunostaining in Control and MnCl 2 -treated animals, respectively. The immunostaining is much more intense in both the substantia nigra pars compacta (SNc) and pars reticulata (SNr). (C,D) OX-42 immunostaining in Control and MnCl 2 -treated animals, respectively. Immunostaining appears more intense in treated animals. Scale bar5500 mm.
mRNA expression and TH immunoreactivity increased in SN pars compacta and VTA in experimental animals. These results are in agreement with those showing that rats chronically treated with a high oral load of MnCl 2 showed a significant increase in the activity of TH in neostriatum, midbrain and hippocampus [3]. Moreover, a recent report [6] also failed to find differences in terms of striatal TH-positive nerve fibers and nigral TH-positive neurons in the nigrostriatal system. However, our data contrast with other studies. Thus, intrathecal Mn decreased DA content with no change of HVA and DOPAC, increased Mn concentration in SN and striatum and decreased spontaneous motor activity [30]. Besides, Mn has been reported to induce biphasic changes in the striatal levels of DA and its metabolites [14]. At lower doses, Mn increased DA and its metabolite levels, while the opposite effect was seen at higher doses [14]. These discrepancies could be related to different factors, including the age of the experimental
animals, the Mn concentration employed and the method of administration. The increase in TH mRNA and protein in response to Mn administration could be of clinical interest since human manganese intoxication starts with a psychiatric phase bearing similarities to schizophrenia, in which the primary disturbance has been suggested to be an overactivity of dopamine neurons [3]. The question that arises deals with the mechanisms by which TH mRNA expression and translation increased in response to Mn administration. Within this context, an upregulation of TH mRNA in nonmelanised A10 midbrain dopaminergic neurons from parkinsonian patients has been reported, suggesting the existence of a compensatory mechanism at presynaptic level [55]. Alternatively, Mn administration has been shown to decrease striatal GSH levels in aged rats [14]. We have demonstrated that a Se-deficient diet, which decreased GPx enzyme and consequently GSH levels, in-
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Fig. 8. Quantification of glial fibrillary acidic protein (GFAP) immunopositive cells in striatum and substantia nigra from Control and MnCl 2 treated animals. (A) GFAP imunoreactivity quantification in striatum. Numbers are mean6S.D. of five independent experiments, expressed as GFAP immunopositive cells / mm 2 . A medial to lateral gradient decrease can be observed in both Control and MnCl 2 -treated animals. (B) GFAP imunoreactivity quantification in substantia nigra. Numbers are mean6S.D. of five independent experiments, expressed as GFAP immunopositive cells / mm 2 . Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.01.
creased TH mRNA expression in SN [44], thus suggesting a link between oxidative status and TH mRNA expression. In this context, TH mRNA expression has been shown to be regulated by the oxygen status in cultured mesencephalic dopaminergic neurons. Thus, dopaminergic mesencephalic neurons in culture exhibits remarkable higher DA levels and / or increase in TH-expressing neurons when exposed to either anoxic (0% O 2 ) [24] or hypoxic (5% O 2 ) conditions [12]. Within the same context, hypoxic conditions favor expression of the TH gene [11,42]. Availability of O 2 may depend upon efficiency of oxidative phosphorylation. It has been proposed that within the synaptic milieu, Mn may influence mitochondrial functioning leading to a decreased oxidative phosphorylation [56]. These results are in agreement with the decrease in the SDH found by us and described above. Mn treatment also produced a decrease of GAD mRNA expression in globus pallidus, which was not accompanied by gliosis. This finding is in good agreement with studies
Fig. 9. Quantification of OX-42 immunopositive cells in striatum and substantia nigra from Control and MnCl 2 -treated animals. (A) OX-42 immunoreactivity quantification in striatum. Numbers are mean6S.D. of five independent experiments, expressed as OX-42 immunopositive cells / 2 mm . A medial to lateral gradient decrease can be observed in both Control and MnCl 2 -treated animals. (B) OX-42 imunoreactivity quantification in substantia nigra. Numbers are mean6S.D. of five independent experiments, expressed as GFAP immunopositive cells / mm 2 . A medial to lateral gradient decrease is apparent in both Control and treated animals. Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.05.
showing that manganese intoxication (20 mg / ml in drinking water for 3 months) failed to induce either neuronal loss or gliosis in globus pallidus of rats [47]. Mn intoxication has been also shown to induce behavioral disinhibition in the absence of major motor alterations [6]; experimental rats were significantly more active than control animals in the empty open field. These behavioral changes could be associated with the significant decrease of GAD mRNA expression in globus pallidus. We conclude that the chronic (1 month, i.p.) Mn treatment (6 mg Mn / kg / day) in aged animals induced the appearance of typical features of cell damage, including activation of microglia and astroglia. These Mn-induced changes were mostly restricted to the nigrostriatal system. In this system, it was remarkable the increase found in TH mRNA and protein in response to Mn administration. All these changes were accompanied by an overall decrease in the energetic metabolism along with a decrease in GAD
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Fig. 10. Expression of GAD67 mRNA in in striatum and globus palidus from Control and MnCl 2 -treated animals. (A) Control; (B) MnCl 2 treatment; (C) optical density measured from dry films expressed as percentage of Control. Numbers are mean 6S.D. of five independent experiments. Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.01. Scale bar51 mm.
mRNA expression in globus pallidus. Overall, these Mninduced changes may underlie some effects in movement ascribed to Mn administration.
Acknowledgements This work was supported by a grant from CICYT (PM98-0160). M.T-C. and M.C.S-H. thank MEC for Becas of FPU and FPI, respectively. We are grateful to E. Fontiveros and J.P. Calero for their invaluable technical assistance. The computer-generated figure presented in this article has been adapted from Swanson, L.M. (1998) Brain Maps: Structure of the rat brain, 2nd edition, Elsevier, Amsterdam.
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Research report
Differential regulation of glutamic acid decarboxylase mRNA and tyrosine hydroxylase mRNA expression in the aged manganese-treated rats ´ ´ Mayka Tomas-Camardiel, Antonio J. Herrera, Jose´ L. Venero, Mari Cruz Sanchez-Hidalgo, Josefina Cano, Alberto Machado* ´ ´ y Toxicologıa ´ , Facultad de Farmacia, Universidad de Sevilla, C /Prof. Garcıa ´ Gonzalez ´ , Bromatologıa s /n, Departamento de Bioquımica Seville, Spain Accepted 14 March 2002
Abstract Recent studies have implicated chronic elevated exposures to environmental agents, such as metals (e.g. manganese, Mn) and pesticides, as contributors to neurological disease. Eighteen-month-old rats received intraperitoneal injections of manganese chloride (6 mg Mn / kg / day) or equal volume of saline for 30 days in order to study the effect of manganese on the dopamine- and GABA-neurons. The structures studied were substantia nigra, striatum, ventral tegmental area, nucleus accumbens and globus pallidus. First, we studied the enzymatic activity of mitochondrial complex II succinate dehydrogenase (SDH). We found an overall decrease of SDH in the different brain areas analyzed. We then studied the mRNA levels for tyrosine hydroxylase (TH) and the dopamine transporter (DAT) by in situ hybridization. TH mRNA but not DAT mRNA was significantly induced in substantia nigra and ventral tegmental area following Mn treatment. Correspondingly, TH immunoreactivity was increased in substantia nigra and ventral tegmental area. Manganese treatment significantly decreased GAD mRNA levels in individual GABAergic neurons in globus pallidus but not in striatum. We also quantified the density of glial fibrillary acidic protein (GFAP)-labeled astrocytes and OX-42 positive cells. Reactive gliosis in response to Mn treatment occurred only in striatum and substantia nigra and the morphology of the astrocytes was different than in control animals. These results suggest that the nigrostriatal system could be specifically damaged by manganese toxicity. Thus, changes produced by manganese treatment on 18-month-old rats could play a role in the etiology of Parkinson’s disease. 2002 Elsevier Science B.V. All rights reserved. Theme: Motor system and sensorimotor integration Topic: Basal ganglia Keywords: Rat; Manganese; Nigrostriatal system; Ventral tegmental area; Nucleus accumbens
1. Introduction Parkinson’s disease (PD) is one of the most common movement disorders and is related to destruction of neurons in the substantia nigra pars compacta of the basal ganglia. While the cause of neuronal death in PD remains
*Corresponding author. Tel.: 134-95-455-6751; fax: 134-95-4556752. E-mail address: [email protected] (A. Machado).
unknown, increasing evidence has involved a variety of environmental factors in the selective dopaminergic cell loss in substantia nigra (SN) typical of nonfamilial PD patients [22,37,54]. Thus, research has been focused on the possible involvement of genetics, exogenous toxins or endogenous toxins from cellular oxidative reactions. The identification of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a synthetic heroin that destroys SN dopamine (DA) neurons, has given additional credence to an environmental factor hypothesis [33,53], although MPTP is not an environmental contaminant. However, the
0169-328X / 02 / $ – see front matter 2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X( 02 )00192-4
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structural homology of the herbicide 1,19-dimethyl-4,4bipyridinium (paraquat, PQ) to 1-methyl-4phenylpyridinium (MPP1), the active metabolite of MPTP, has emerged as a putative risk factor. Occupational PQ exposures have been associated with parkinsonism [17,37]. The fungicide manganese ethylenebisdithiocarbamate (maneb; MB) decreases locomotor activity [39] and potentiates MPTP effects on locomotor activity and catalepsy [52]. At least two incidences of parkinsonism in humans have been related to MB exposure [20,38]. Moreover, it is well established that chronic exposure to manganese and manganese-related hepatic encephalopathy cause neuropsychiatric symptoms [8,58], which often manifest as extrapyramidal symptoms resembling Parkinson’s disease, including bradikynesia and distonia [1,15,18,57]. Mn is a widely used metal, and miners exposed to Mn fumes develop Parkinson syndrome. Racette et al. [43] have reported 15 parkinsonian patients who were professional welders. Of interest, chronic manganese intoxication causes specific PD-like symptoms that respond to levodopa [29]. In primates exposed to Mn for 18 months, marked degeneration of the SN has been reported [25]. In the normal human brain, the highest Mn concentrations are found in the globus pallidus, striatum, thalamus and SN. The need to better understand the effects of manganese on behavior is becoming more important due to a potential increase of environmental exposure to manganese since it has been proposed as a gasoline additive in a number of countries. However, the animal models for studying the Mn-related neurotoxic features have pointed out a great disparity of neurotoxic manifestations; manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism [5]. Other did not find any neurotoxic action, or described protective effects. The intranigral infusion of manganese (1.05–4.2 nmol) protected nigral neurons from iron-induced oxidative injury and dopamine depletion [51]. The purpose of this study was therefore to investigate whether manganese exposure influences the functioning of the nigrostriatal system in aged rats and to compare with another DA system, the ventral tegmental area (VTA) / nucleus accumbens. Manganese has been shown to inhibit mitochondrial aconitase activity, which results in disruption of brain mitochondrial energy production as well as iron metabolism [59]. In addition, previous reports have indicated that Mn intoxication is primarily confined to the globus pallidus [46]. Moreover, selective vulnerability of pallidal neurons in the early phases of manganese intoxication has been reported [47]. For this reason, we have studied the effect of Mn treatment on energetic metabolism measured as succinate dehydrogenase activity, the tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA expression as markers of dopaminergic system and also the glutamic acid decarboxylase (GAD) mRNA expression in two different structures, globus pallidus and striatum. We also analysed the glial response after Mn treatment.
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2. Material and methods
2.1. Animals, treatment and dissection Twenty 18-month-old male albino Wistar rats were used. The rats, born in our laboratory, were housed in a room maintained under constant temperature (2262 8C) and humidity conditions (60%) with a 12:12 h light–dark cycle. Food and water were available ad libitum. Animals were cared for and treated in accord to the Guidelines of the European Union Council (86 / 609 / EU), following the Spanish regulations (BOE 67 / 8509-12, 1988) for the use of laboratory animals and approved by the Scientific Committee of the University of Sevilla. Animals were divided into two groups. Rats received intraperitoneal injections of manganese chloride (MnCl 2 ) at the dose of 6 mg Mn / kg / day or equal volume of saline for 30 days. Five animals from each group (control or experimental) were used for in situ hybridization and five animals from each group (control and experimental) were used for immunohistochemistry.
2.2. Immunohistological evaluation: tyrosine hydroxylase, glial fibrillary acidic protein and OX-42 Rats were perfused through the heart under deep anaesthesia (chloral hydrate) with 150–200 ml of 4% paraformaldehyde in phosphate buffer, pH 7.4. The brains were removed, and then cryoprotected serially in sucrose in phosphate-buffered saline (PBS), pH 7.4; first in 10% sucrose for 24 h and then in 30% sucrose until sunk (2–5 days). The brains were then frozen in isopentane at 215 8C, and 25-mm sections were cut on a cryostat and mounted in gelatine-coated slices. Primary antibodies used were: mouse-derived antityrosine hydroxylase (Roche Diagnostic, Barcelona, Spain; 1:200), antiglial fibrillary acidic protein (GFAP) (Chemicon, Temecula, CA, USA; 1:300), and mousederived OX-42 (Serotec, Oxford, UK; 1:200). OX-42 is an antibody directed against the type-3 complement receptor (CR3) and recognizes macrophages and microglial cells. All incubations and washes were in Tris buffered saline (TBS), pH 7.4 unless otherwise noted. All work was done at room temperature. Sections were washed and then treated with 0.3% hydrogen peroxide in methanol for 15 min, washed again, and incubated in a solution containing TBS and 1% horse serum for 60 min in a humid chamber. Slices were drained and further incubated with the primary antibody in TBS containing 1% horse serum and 0.25% Triton-X-100 for 24 h. Sections were then incubated for 2 h with biotinylated horse antimouse IgG (Vector, 1:200) followed by a second 1-h incubation with kit ABC (Immuno pure standard ultrasensitive ABC staining kit, Pierce, 1:100). The antibody was diluted in TBS containing 0.25% Triton-X-100, and its addition was preceded by three 10-min rinses in TBS. The peroxidase was
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visualized with a standard diaminobenzidine–hydrogen peroxidase chromogen reaction for 5 min.
2.3. Preparation of the riboprobe TH antisense transcript was generated from a plasmid kindly provided by Dr. Sokoloff (INSERM, Paris, France), which contains a 282-nucleotide fragment of the 39 end of the rat TH gene cloned in PGEM-4Z (Promega) (including 13 nucleotides of 39 untranslated sequence), which corresponds to nucleotides 1240–1521 of the rat TH DNA sequence [23]. This plasmid was linearized with EcoRI and used as template with the T7 RNA polymerase to get the antisense riboprobe [16]. DAT antisense riboprobe was generated from a plasmid kindly provided by Dr. M.P. Martres (INSERM), which contains the rat DAT cDNA 800-bp XbaI–HindIII fragment cloned in pRc / CMV (Invitrogen). To prepare the DAT antisense riboprobe this plasmid was linearized with XbaI and used as a template with the T7 RNA polymerase [16]. pBbluescript SK plasmids, containing the cDNA sequence for GAD67 as a 3.2-kb EcoRI insert (clones 14 and 18), were kindly provided by Dr. A. Tobin (UCLA, Los Angeles, CA, USA). The GAD67 cDNA was isolated from a 1 gt-11 cDNA library made from poly(A) RNA from adult rat brain [19]. To prepare the GAD67 antisense transcript, clone 14 was digested with SalI and used as a template with the T3RNA polymerase. To prepare the GAD67 sense transcript, clone 18 was digested with SalI and used as a template with the T3 RNA polymerase. Single-strand antisense cRNA probes were synthesized with T7 and T3 RNA polymerases according to a protocol provided by the RNA polymerase supplier (Bethesda Research Laboratories, Bethesda, MD, USA). The reaction mixture contained the reaction buffer provided with the RNA polymerase; ATP, CTP and GTP each at 1 mM; and 30 mM [ 35 S] UTP (1300 Ci / mmol).
SSC at 25 8C followed by 0.13 SSC at 60 8C for 1 h, dehydrated in a series of ethanols, air-dried and processed for emulsion autoradiography. Autoradiograms were generated by opposing the labelled tissue to bmax Hyperfilm for 2 weeks. After looking at the dry film autoradiography, slides were dipped in Amersham LM1 emulsion (diluted 1:1 with water) and exposed in the dark for 10–15 days at 4 8C. Slides were then developed in D-19 (Kodak) at 15 8C for 2.5 min fixed for 10 min in fixer (Kodak), and counterstained with cresyl violet (Sigma–Aldrich).
2.5. In situ hybridization data analysis For quantitation of dry film autoradiography, the films were scanned at high resolution and the TIF image was analyzed by densitometry using the computer program ANALYSIS (Germany) based on calibrated gray scale densities. Calibration was performed by means of [ 3 H] microscales. The region of interest was delineated and the optical density obtained. Additional measurements of mRNA levels were obtained by quantifying the number of silver grains per neuron using the same computer program. Emulsion-dipped slides were used for these measurements. Labeling was considered specific when grain accumulation over cells with a large nucleus exceeded five times the background value. The area with high grain density over individual cell body was delineated and the number of grains within this field was counted by means of the computerized image analysis system with a 403 magnification lens. Because of the high levels of labeling, the typical disposition of the clusters over the cell body and the high magnification, the delineation was unequivocal. Only well-separated cells were selected for quantification.
2.4. In situ hybridization histochemistry
2.6. Immunohistochemistry data analysis
In situ hybridization on brain sections was carried out following a modification of a procedure described in detail elsewhere [41]. Thaw-mounted 12-mm sections were postfixed for 30 min in 4% paraformaldehyde, followed by three 10-min washes in phosphate-buffered saline (pH 7.4). Sections were treated for 1 min in 0.1 M triethanolamine, followed by 10 min in acetic anhydride–0.1 M triethanolamine in order to decrease nonspecific binding. Following a 1-min wash in 23 standard saline citrate (SSC), sections were dehydrated in a series of increasing concentrations of ethanol and then air-dried. The sections were hybridized for 3 h at 50 8C with the [ 35 S] cRNA, rinsed in 43 SSC–20 mM DTT, then 43 SSC alone. Sections were subjected to 30 min of RNase digestion at 37 8C (20 mg / ml RNase A in 0.5 M NaCl, 0.01 M Tris–HCl, 1 mM EDTA; pH 8.0), washed for 2 h in 23
Reactive astrocytes detected as GFAP immunopositive cells and microglia detected by OX-42 were counted in SN and VTA at one specific rostrocaudal level (plate 39 of the rat brain atlas of Swanson [50]). The rationale for this is the heterogeneity of these brain areas. In SN, the quantifications of astrocytes and microglia were performed in three different nigral locations corresponding to medial, central and lateral aspects of this brain area, whereas in the VTA area the quantification was restricted to one location. All quantifications were done at 403 magnification. In striatum and nucleus accumbens, the GFAP and OX-42 immunohistochemistries were counted every seventh serial frontal section. Counting of striatal astrocytes and microglia was performed in three different locations corresponding to medial, central, and lateral striatum.
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2.7. Tyrosine hydroxylase staining intensity in substantia nigra, ventral tegmental area, striatum and nucleus accumbens Sections were stained as described under Immunological evaluation. Quantitative estimates of TH immunoreactivity were made in the bilateral SN pars compacta, VTA, striatum and nucleus accumbens. Intensity measurements were done with the experimenter blinded to the treatment and in random order to avoid any bias. A total of ten representative sections per animal were used to determine TH density within the substantia nigra pars compacta,
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VTA, striatum and nucleus accumbens. Each treatment group was represented by five animals. The analysis was done in each sampled section at a magnification of 403, permitting representative visualization of each region. The average of the density for each section was added and divided by the total number of sections analyzed to give the mean TH density for each animal. The results are expressed as percentage compared to the control animals.
2.8. Succinate dehydrogenase histochemistry Slides were incubated for 45 min at 37 8C in 50 ml of
Fig. 1. Succinate dehydrogenase (SDH) histochemistry in Control and MnCl 2 -treated animals. (A,C) Control; (B,D) MnCl 2 treatment; (E) measurement of optical density, expressed as percentage of Control. Numbers are mean6S.D. of five independent experiments. Statistical significance (Student’s t test): *, P,0.05; **, P,0.01. Scale bars51 mm.
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0.06 M phosphate buffer (pH 7.0) containing 20 mg of nitro blue tetrazolium and 0.68 mg of succinate [31]. The specificity of the reaction was confirmed by incubating the sections with increasing concentrations of 3-nitropropionic acid, a selective inhibitor of the enzyme. Enzyme activity of controls and experimental animals was determined by densitometry based on calibrated grey scale values.
2.9. Statistical analyses Statistical analyses of the differences between groups were performed by using Student’s t test. The difference between two means was considered significant if P values were #0.05.
3. Results
3.1. Changes in SDH activity Analysis demonstrated an overall decrease of SDH activity throughout the brain. Most remarkable decreases were found in striatum, SN, VTA and subthalamic nucleus, which are shown in Fig. 1. We were unable to reliably measure SDH activity in globus pallidus due to the constitutive low activity in this brain area.
3.2. Changes in TH immunoreactivity Levels of TH immunoreactivity (TH-IR) were significantly increased in the cell body regions in SN (144%
Fig. 2. Tyrosine hydroxylase (TH) immunoreactivity in Control and MnCl 2 -treated animals. (A) Control; (B) MnCl 2 treatment. The immunoreactivity increased in treated animals as compared to the Control. VTA, ventral tegmental area; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata. Scale bar5500 mm. (C) TH immunoreactivity quantification. Numbers are mean6S.D. of five independent experiments, and are expressed as optical density (% of control). Statistical significance (Student’s t test): *, P,0.01.
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control levels, P,0.01) and VTA (150% control levels, P,0.01) after Mn treatment (Fig. 2). In contrast, Mn treatment failed to alter the density of TH-IR in their respective dopaminergic projecting areas in striatum and nucleus accumbens (Fig. 2).
3.3. Expression of TH mRNA and DAT mRNA in substantia nigra and ventral tegmental area In situ hybridization of brains from control- and Mntreated animals with the antisense riboprobes for TH mRNA and DAT mRNA resulted in specific labeling in the ventral mesencephalon (Fig. 3). The typical disposition of the grain clusters over the cell bodies and the precise location of these neurons in the SN pars compacta and
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VTA clearly indicate that these TH and DAT mRNA expressing neurons were dopaminergic. For quantification, as a first step, we performed analysis of optical density from dry film autoradiographs. Densitometric analysis revealed a significant elevation in TH mRNA levels in SN and VTA after Mn treatment as compared to controls (169 and 159% of control levels for SN and VTA, respectively, P,0.01; Figs. 3 and 4A). On the contrary, DAT mRNA levels remained at control levels following Mn-treatment in both structures (Figs. 3 and 4A). Analysis of cellular expression of TH mRNA in the SN and VTA by silver grains counting confirmed the Mninducing effect over TH mRNA expression. Cellular levels of TH mRNA within individual dopaminergic neurons in
Fig. 3. Expression of tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNAs in substantia nigra from Control and MnCl 2 -treated animals. (A) Control TH; (B) TH after MnCl 2 treatment; (C) control DAT; (D) DAT after MnCl 2 treatment; (E,F) high magnifications of silver grains from (A) and (B), respectively. Scale bars: A–D, 1 mm; E–F, 50 mm.
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Fig. 4. Quantification of tyrosine hydroxylase (TH) and dopamine transporter (DAT) mRNA in situ hybridization in Control and MnCl 2 treated animals. (A) Optical density measured from dry films expressed as percentage of Control. Numbers are mean6S.D. of five independent experiments; (B) silver grain counting. TH mRNA expression in SN and VTA of Control and MnCl 2 -treated animals were expressed as silver grains per cell. Numbers are mean6S.D. of five independent experiments. Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.05; **, P,0.01.
response to Mn treatment were as follow: SN, 161% of control levels, P,0.01; VTA: 123% of control levels, P,0.05. (Figs. 3 and 4B).
3.4. Glial fibrillary acidic protein and OX-42 immunoreactivity As seen in Fig. 5, counting of reactive astrocytes in striatum and SN was performed in three different locations from the medial to the lateral part of the structures considering the heterogeneity in projection patterns of these brain areas. VTA, globus pallidus and nucleus accumbens were analyzed at a single location. We found a medial to lateral gradient decrease in the distribution pattern of GFAP immunoreactive astrocytes in the striatum of control and Mn-treated animals. Thus, the density of reactive astrocytes in the medial striatum was about 2-fold of that found in the lateral part of this brain area. In contrast, SN did not show a noticeable topographical gradient. In animals treated with Mn, the density of GFAP-
immunoreactive astrocytes significantly increased above controls in striatum and SN. This effect was seen in all striatal and nigral locations analyzed (Figs. 6–8). The increases of GFAP-IR were similar in the three striatal and nigral locations analyzed following Mn treatment as compared with controls (Figs. 6–8). The reactive astrocytes were never isolated. The morphology of reactive astrocytes were different in Mn-treated rats as compared with controls. They showed more abundant and larger processes than resting astrocytes and became hypertrophic, losing their characteristic stellate morphology (Figs. 6 and 7). In contrast to striatum and SN, Mn treatment failed to alter the density of GFAP-IR astrocytes in VTA and nucleus accumbens (data not shown). Counting of OX-42 positive cells also demonstrated a slight medial to lateral gradient decrease in the density of microglial cells in striatum and SN of both control and Mn-treated animals (Fig. 9). Following Mn treatment, there was a significant increase in the density of OX-42 immunoreactive microglia in the striatum and SN (Figs. 6, 7 and 9). OX-42 antibody stained ramified microglial cells in both areas. After Mn treatment, microglial reactivation was more intense than in the control animals in both structures (Figs. 6 and 7). Mn treatment failed to alter the density of OX-42 immunoreactive microglia in VTA and nucleus accumbens (data not shown).
3.5. Expression of GAD67 mRNA in striatum and globus pallidum after Mn treatment Once we found an inducing effect of Mn treatment on TH mRNA levels, the step-limiting enzyme in catecholamine biosynthesis, we wanted to know whether this effect was restricted to the dopaminergic system. For that, we analysed GAD mRNA, the biosynthetic enzyme for GABA. Within the striatum and in the globus pallidus, the riboprobe used for detecting GAD mRNA labeled numerous cell bodies in both structures (Fig. 10). Quantifications performed on dry film autoradiographs demonstrated that following Mn treatment there was a significant decrease in GAD mRNA levels in globus pallidus (68.2% of control, P,0.01; Fig. 10). In contrast, no differences were found in striatum in terms of GAD mRNA levels (Fig. 10).
4. Discussion It has long been established that Mn disrupts different markers of monoaminergic activity in the brainstem, this effect being more evident in aged animals [13]. Consequently, we have used aged rats in our study. Mn has been shown to impair mitochondrial energy metabolism at different levels [4,59]. We now demonstrate that Mn treatment significantly decreases SDH activity in nearly all the structures studied. These decreases were of nearly 30%
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Fig. 5. Locations of reactive astrocytes counting in several brain structures in Control and MnCl 2 -treated animals. (A) Counting of reactive astrocytes in striatum were performed in three different locations from the medial to the lateral part of this brain area (numbered dots); the asterisk marks the nucleus accumbens. Section level was 0.95 mm anterior to bregma (B); counting of reactive astrocytes in substantia nigra were also performed in three different locations from the medial to the lateral part of this brain area (numbered dots); the asterisk marks the VTA. Section level was 6.06 mm posterior to bregma.
in SN and striatum. This decrease in energetic metabolism could be one action that explains the extrapyramidal symptoms described in Mn intoxications. It is in agreement with the fact that manganese injection into the rat striatum produces excitotoxic lesions by impairing energy metabolism [5]. Afterwards, we measured reactive microglia and astroglia in response to Mn treatment, which are good indexes of degenerative-related events. Typical morphological features of reactive microglia and phagocytic cells were observed in response to Mn administration in SN and striatum, especially in the former. These Mninduced changes were regionally-specific since no significant changes were observed in other brain areas analyzed including VTA, nucleus accumbens and globus pallidus. The Mn-induced activation of microglial cells in the
nigrostriatal system could be a consequence of tissuespecific damage, a specific activation of glial cells or both. High levels of manganese have been shown to potentiate microglial nitric oxide production in response to bacterial lipopolysaccharide, a potent inducer of inflammation [9]. This potent activity of manganese is not shared by other transition metals, including iron, cobalt, cooper and zinc [9]. Within this context, it is interesting to note the presence of reactive microglia in the SN of patients with PD [21]. This response has been also reported in animal models of PD [7,28,32]. Interestingly, the SN contains the highest concentration of microglia in the brain [34], and it contains a specific vasculature that results in an ineffective blood–brain barrier [45]. With regard to the astroglial population, the density of
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Fig. 6. Glial fibrillary acidic protein (GFAP) and OX-42 immunoreactivity in striata from Control and MnCl 2 -treated animals. (A,B) GFAP immunostaining in Control and MnCl 2 -treated animals, respectively. Astrocytes in MnCl 2 -treated animals appear hyperthrophic, with abundant and large processes. (C,D) OX-42 immunostaining in Control and MnCl 2 -treated animals, respectively. Immunostaining is more intense in treated animals. Scale bar5200 mm.
GFAP-immunolabeled astrocytes also increased in the terminal field and cell body region in the nigrostriatal system in response to Mn treatment. The induction of astrocytosis by Mn has also been reported [49]. Mn administered intranasally in rats for 3 weeks increased levels of GFAP and S-100 in the olfactory bulbs [27]. Variations of GFAP immunoreactivity is thought to be a sensitive index of astrocyte reaction to any damage occurring in the brain tissue. Taken together, our results are consistent with the view that Mn triggers an inflammatory stimulus for SN and striatum, which could be deleterious to the nigrostriatal system. Reactive microglia and astrocytes are known to secrete different proinflammatory cytokines and cytotoxic molecules, such as interleukin-1 (IL-1), tumor necrosis factor (TNF) and nitric oxide (NO) [2,9,10,35]. Similar effects have been reported for Mn, which induced the iNOS of microglia [9] and astrocytes
[49], although Mn did not appear to be cytotoxic to glial cells. As a next step, we sought to determine whether the dopaminergic system was affected, since most specific relevant changes were confined to the nigrostriatal system. In fact, the activation of glial cells in response to Mn administration could be related to DA toxicity, taking into account that dopaminergic areas were specially affected. DA may exert neurotoxic effects via enzymatic metabolism forming H 2 O 2 that can be broken down to free radicals species in the presence of metals [40,48] or via autooxidation forming various reactive compounds [26]. Linert et al. [36] have shown that Mn(II) significantly enhances the rate of dopamine auto-oxidation in vitro. We studied different markers of dopaminergic activity and, contrary to expectations, neither of them decreased in response to Mn administration. On the contrary, TH
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Fig. 7. Glial fibrillary acidic protein (GFAP) and OX-42 immunoreactivity in substantiae nigrae from Control and MnCl 2 -treated animals. (A,B) GFAP immunostaining in Control and MnCl 2 -treated animals, respectively. The immunostaining is much more intense in both the substantia nigra pars compacta (SNc) and pars reticulata (SNr). (C,D) OX-42 immunostaining in Control and MnCl 2 -treated animals, respectively. Immunostaining appears more intense in treated animals. Scale bar5500 mm.
mRNA expression and TH immunoreactivity increased in SN pars compacta and VTA in experimental animals. These results are in agreement with those showing that rats chronically treated with a high oral load of MnCl 2 showed a significant increase in the activity of TH in neostriatum, midbrain and hippocampus [3]. Moreover, a recent report [6] also failed to find differences in terms of striatal TH-positive nerve fibers and nigral TH-positive neurons in the nigrostriatal system. However, our data contrast with other studies. Thus, intrathecal Mn decreased DA content with no change of HVA and DOPAC, increased Mn concentration in SN and striatum and decreased spontaneous motor activity [30]. Besides, Mn has been reported to induce biphasic changes in the striatal levels of DA and its metabolites [14]. At lower doses, Mn increased DA and its metabolite levels, while the opposite effect was seen at higher doses [14]. These discrepancies could be related to different factors, including the age of the experimental
animals, the Mn concentration employed and the method of administration. The increase in TH mRNA and protein in response to Mn administration could be of clinical interest since human manganese intoxication starts with a psychiatric phase bearing similarities to schizophrenia, in which the primary disturbance has been suggested to be an overactivity of dopamine neurons [3]. The question that arises deals with the mechanisms by which TH mRNA expression and translation increased in response to Mn administration. Within this context, an upregulation of TH mRNA in nonmelanised A10 midbrain dopaminergic neurons from parkinsonian patients has been reported, suggesting the existence of a compensatory mechanism at presynaptic level [55]. Alternatively, Mn administration has been shown to decrease striatal GSH levels in aged rats [14]. We have demonstrated that a Se-deficient diet, which decreased GPx enzyme and consequently GSH levels, in-
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Fig. 8. Quantification of glial fibrillary acidic protein (GFAP) immunopositive cells in striatum and substantia nigra from Control and MnCl 2 treated animals. (A) GFAP imunoreactivity quantification in striatum. Numbers are mean6S.D. of five independent experiments, expressed as GFAP immunopositive cells / mm 2 . A medial to lateral gradient decrease can be observed in both Control and MnCl 2 -treated animals. (B) GFAP imunoreactivity quantification in substantia nigra. Numbers are mean6S.D. of five independent experiments, expressed as GFAP immunopositive cells / mm 2 . Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.01.
creased TH mRNA expression in SN [44], thus suggesting a link between oxidative status and TH mRNA expression. In this context, TH mRNA expression has been shown to be regulated by the oxygen status in cultured mesencephalic dopaminergic neurons. Thus, dopaminergic mesencephalic neurons in culture exhibits remarkable higher DA levels and / or increase in TH-expressing neurons when exposed to either anoxic (0% O 2 ) [24] or hypoxic (5% O 2 ) conditions [12]. Within the same context, hypoxic conditions favor expression of the TH gene [11,42]. Availability of O 2 may depend upon efficiency of oxidative phosphorylation. It has been proposed that within the synaptic milieu, Mn may influence mitochondrial functioning leading to a decreased oxidative phosphorylation [56]. These results are in agreement with the decrease in the SDH found by us and described above. Mn treatment also produced a decrease of GAD mRNA expression in globus pallidus, which was not accompanied by gliosis. This finding is in good agreement with studies
Fig. 9. Quantification of OX-42 immunopositive cells in striatum and substantia nigra from Control and MnCl 2 -treated animals. (A) OX-42 immunoreactivity quantification in striatum. Numbers are mean6S.D. of five independent experiments, expressed as OX-42 immunopositive cells / 2 mm . A medial to lateral gradient decrease can be observed in both Control and MnCl 2 -treated animals. (B) OX-42 imunoreactivity quantification in substantia nigra. Numbers are mean6S.D. of five independent experiments, expressed as GFAP immunopositive cells / mm 2 . A medial to lateral gradient decrease is apparent in both Control and treated animals. Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.05.
showing that manganese intoxication (20 mg / ml in drinking water for 3 months) failed to induce either neuronal loss or gliosis in globus pallidus of rats [47]. Mn intoxication has been also shown to induce behavioral disinhibition in the absence of major motor alterations [6]; experimental rats were significantly more active than control animals in the empty open field. These behavioral changes could be associated with the significant decrease of GAD mRNA expression in globus pallidus. We conclude that the chronic (1 month, i.p.) Mn treatment (6 mg Mn / kg / day) in aged animals induced the appearance of typical features of cell damage, including activation of microglia and astroglia. These Mn-induced changes were mostly restricted to the nigrostriatal system. In this system, it was remarkable the increase found in TH mRNA and protein in response to Mn administration. All these changes were accompanied by an overall decrease in the energetic metabolism along with a decrease in GAD
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Fig. 10. Expression of GAD67 mRNA in in striatum and globus palidus from Control and MnCl 2 -treated animals. (A) Control; (B) MnCl 2 treatment; (C) optical density measured from dry films expressed as percentage of Control. Numbers are mean 6S.D. of five independent experiments. Statistical significance (Student’s t test, comparing MnCl 2 -treated vs. Control animals): *, P,0.01. Scale bar51 mm.
mRNA expression in globus pallidus. Overall, these Mninduced changes may underlie some effects in movement ascribed to Mn administration.
Acknowledgements This work was supported by a grant from CICYT (PM98-0160). M.T-C. and M.C.S-H. thank MEC for Becas of FPU and FPI, respectively. We are grateful to E. Fontiveros and J.P. Calero for their invaluable technical assistance. The computer-generated figure presented in this article has been adapted from Swanson, L.M. (1998) Brain Maps: Structure of the rat brain, 2nd edition, Elsevier, Amsterdam.
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