Nigrostriatal Dopaminergic Neurotoxicity Research Articles

The impact of selenium and magnesium on nigrostriatal dopaminergic neurotoxicity following paraquat exposure in mice.

Exposure to Paraquat (PQ) has been linked to Parkinsonism in both experimental animals and humans. PQ is widely used among other herbicides in developing countries, where its safe use has been proven to be impossible. This action has resulted in farmers' fatal poisonings with PQ and threatens public health. The present study was designed to investigate the role of PQ induced neurotoxicity in the sustantia Nigra, striatal and cerebellar neurons in addition to the neuroprotective potentials of Selenium (Se) and Magnesium (Mg) in abrogating PQ-induced neurotoxicity in mice. We used rotarod test to access PQinduced motor coordination deficit in mice. We also determined: PQ-induced mild cognitive impairment using Morris water maze to check for spatial memory in mice, PQ-induced oxidative stress by investigating lipid peroxidation level of malonyl dialdehyde (MDA) and glutathione peroxidase (GSH) and measuring the activities of superoxide dismutase (SOD), and catalase (CAT) in the brain of PQ-induced mice. The histomorphological outcome of the nigrostriatal neurons and cerebellum were also accessed in PQinduced experimental mice. Immunoreactivity of nigrostriatal dopaminergic neurons to thyroxine hydroxylase (TH) was accessed to determine dopamine loss in the nigrostriatal neurons. In addition, we determine selenium and magnesium's antioxidative effect on paraquat-induced Parkinsonism in mice by checking their effects on motor coordination, cognition, MDA, SOD, CAT, and GSH levels and assessing their ameliorating effect on dopaminergic neurons of the nigrostriatal system and in cerebellar neurons using both the neurons' histomorphological outline and their affinity to TH. Generally, our results showed that PQ induces Parkinsonism in mice by inducing oxidative stress, degenerative cell death in the SNpc, striatum, and cerebellum and by inducing dopamine depletion in SNpc and striatum which resulted in motor and cognitive deficits. Se and Mg also proved to be good antioxidants ameliorating PQ-induced neurodegenerative effects but combine dose of Se and Mg caused further neuronal degeneration on PQinduced mice. Se and Mg may be promising therapeutic targets for PD management in addition to L-dopa treatment since patients from developing countries may not afford deep brain stimulation (DBS) and optogenetics used in the management of PD.Keywords: Paraquat, Parkinsonism, Selenium, Magnesium, Oxidative stress, sustantia Nigra, striatum, cerebellum, motor coordination, cognition.

Early Postnatal Exposure to Paraquat and Maneb in Mice Increases Nigrostriatal Dopaminergic Susceptibility to a Re-challenge with the Same Pesticides at Adulthood: Implications for Parkinson’s Disease

Exposure to environmental contaminants represents an important etiological factor in sporadic Parkinson's disease (PD). It has been reported that PD could arise from events that occur early in development and that lead to delayed adverse consequences in the nigrostriatal dopaminergic system at adult life. We investigated the occurrence of late nigrostriatal dopaminergic neurotoxicity induced by exposures to the pesticides paraquat (PQ) and maneb (MB) during the early postnatal period in mice, as well as whether the exposure to pesticides during development could enhance mice vulnerability to subsequent challenges. Male Swiss mice were exposed to a combination of 0.3mg/kg PQ and 1.0mg/kgMB (PQ + MB) from postnatal (PN) day 5 to 19. PN exposure to pesticides neither induced mortally nor modified motor-related parameters. However, PN pesticides exposure decreased the number of tyrosine hydroxylase (TH)- and dopamine transporter (DAT)-positive neurons in the substantia nigra pars compacta (SNpc), as well as reduced TH and DAT immunoreactivity in the striatum. A parallel group of animals developmentally exposed to the pesticides was re-challenged at 3months of age with 10mg/kg PQ plus 30mg/kgMB (twice a week, 6weeks). Mice exposed to pesticides at both periods (PN + adulthood) presented motor deficits and reductions in the number of TH- and DAT-positive neurons in the SNpc. These findings indicate that the exposure to PQ + MB during the early PN period can cause neurotoxicity in the mouse nigrostriatal dopaminergic system, rendering it more susceptible to a subsequent adult re-challenge with the same pesticides.

Neuroprotective effects of umbelliferone and esculetin in a mouse model of Parkinson's disease

The production of reactive oxygen species and mitochondrial dysfunction in the brain are both associated with the progression of several neurodegenerative diseases, including Parkinson's disease. These characteristics are also observed when rodents are exposed to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a compound that causes nigrostriatal dopaminergic neurotoxicity and that has been used previously for assessing the effectiveness of neuroprotective agents. In this study, the neuroprotective effects of two coumarins, umbelliferone and esculetin, against MPTP-induced neurotoxicity were examined in C57BL/6J mice. The results show that dietary administration of umbelliferone and esculetin significantly attenuated MPTP-induced neurotoxicity in the substantia nigra pars compacta but not striatum, as measured by tyrosine hydroxylase staining. Both coumarins also prevented an MPTP-induced increase in nitrosative stress as measured by 3-nitrotyrosine immunoreactivity and also maintained glutathione levels in MPTP-exposed mice as well as in cell lines exposed to the metabolite 1-methyl-4-phenylpyridinium. Umbelliferone and esculetin also prevented MPTP-dependent caspase 3 activation, an indicator of apoptosis, but did not inhibit monoamine oxidase activity. This is the first time that the neuroprotective capabilities of these coumarins have been demonstrated, and the results indicate that umbelliferone and esculetin can protect against MPTP-induced neurotoxicity in the mouse. These compounds can cross the blood-brain barrier, so their effectiveness indicates that they have the potential to protect in neurodegenerative disease such as Parkinson's disease.

Ginkgo biloba extract (EGb 761) modulates the expression of dopamine-related genes in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinsonism in mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity and behavioral impairment in rodents similar to Parkinson’s disease. The MPTP mouse model is widely used to evaluate new protective agents. EGb 761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves according to a standardized procedure. We have shown that EGb 761 attenuates the loss of striatal dopamine levels and prevents the neurodegeneration of the nigrostriatal pathway induced by MPTP. This finding shows that neuroprotective effects of EGb 761 act, in part, on the dopamine system. Therefore, this study investigates whether EGb 761 exerts dopaminergic neuroprotection through the regulation of dopamine-related gene expression in MPTP-induced Parkinsonism. Male C57BL/6J mice were injected with MPTP (30mg/kg, i.p.) for 5days and later with EGb 761 (40mg/kg, i.p.) daily for 18days. The expression of selected genes was evaluated in the striatum and midbrain by quantitative PCR. The genes for tyrosine hydroxylase (Th), vesicular monoamine transporter 2 (Vmat2), dopamine transporter (Dat), dopamine D2 receptor (Da-d2r), and transcription factors (Pitx3 and Nurr1) related to dopamine neurotransmission were selected for the analysis. EGb 761 administration to MPTP-treated mice protected Th (41%), Vmat2 (15%), Dat (102%), Da-d2r (46%), Pitx3 (63%), and Nurr1 (148%) mRNA levels in the midbrain, all of which were up-regulated. However, EGb 761 partially reversed the MPTP effect exclusively for Th (48%) and Nurr1 (96%) mRNA in the striatum. Only Th and Nurr1 mRNA and protein levels were regulated by EGb 761 in both regions of the nigrostriatal pathway. This result could be related to the regulation of their transcription. Our results suggest that EGb 761-associated neuroprotection against MPTP neurotoxicity is related to the regulation of the dopamine genes. Moreover, this neuroprotection also involves the regulation of transcription factors such as Nurr1 that are important for the functional maintenance of dopaminergic neurons.

Neuronal or inducible nitric oxide synthase (NOS) expression level is not involved in the different susceptibility to nigro-striatal dopaminergic neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) between C57BL/6 and BALB/c mice

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces severe degeneration of dopaminergic (DA-ergic) neurons when administrated to C57BL/6 mice, but such lesions are not observed in BALB/c mice. To clarify the factors which influence such marked strain differences in the susceptibility to MPTP, the involvement of neuronal NOS (nNOS) and inducible NOS (iNOS) was investigated. MPTP was intraperitoneally (ip) administrated to adult C57BL/6 (highly sensitive) and BALB/c (resistant) mice. Immunohistochemical analysis using an antibody to tyrosine hydroxylase (TH) showed a significant decrease in TH-immunopositive areas in the striatum and TH-positive cells in the substantia nigra pars compacta (SNpc) of MPTP-treated C57BL/6 mice at 1 and 7 days (d) after administration, compared to control C57BL/6 mice. On the other hand, MPTP-treated BALB/c mice showed no significant changes. By Western blot analysis, TH, MAO-B, DAT, nNOS and iNOS protein expression levels were examined in intact and MPTP-treated mice. Intact BALB/c mice showed higher DAT protein expression in the striatum and TH protein expression in the midbrain than intact C57BL/6 mice. In addition, MPTP-treated BALB/c mice showed a more significant increase of MAO-B expression than MPTP-treated C57BL/6 mice at 12 h. The increase of nNOS and iNOS protein expressions in MPTP-treated BALB/c mice was more pronounced in the striatum and midbrain than in MPTP-treated C57BL/6 mice at 12 h and 2 d. These results indicate that MAO-B, DAT, nNOS or iNOS expression levels do not influence the different strain susceptibility to MPTP.

S-Allylcysteine, a garlic compound, protects against oxidative stress in 1-methyl-4-phenylpyridinium-induced parkinsonism in mice

S-Allylcysteine (SAC), the most abundant organosulfur compound in aged garlic extract, has multifunctional activity via different mechanisms and neuroprotective effects that are exerted probably via its antioxidant or free radical scavenger action. The 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. 1-Methyl-4-phenylpyridinium (MPP +) is the stable metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and it causes nigrostriatal dopaminergic neurotoxicity. Previous studies suggest that oxidative stress, via free radical production, is involved in MPP +-induced neurotoxicity. Here, we report on the neuroprotective effect of SAC against oxidative stress induced by MPP + in the striatum of C57BL/6J mice. Mice were pretreated with SAC (125 mg/kg ip) daily for 17 days, followed by administration of MPP + (0.72 mg/kg icv), and were sacrificed 24 h later to evaluate lipid peroxidation, different antioxidant enzyme activities, spontaneous locomotor activity and dopamine (DA) content. MPP + administration resulted in a significant decrease in DA levels in the striatum. Mice receiving SAC (125 mg/kg ip) had significantly attenuated MPP +-induced loss of striatal DA levels (32%). The neuroprotective effect of SAC against MPP + neurotoxicity was associated with blocked (100% of protection) of lipid peroxidation and reduction of superoxide radical production — indicated by an up-regulation of Cu-Zn-superoxide dismutase activity — both of which are indices of oxidative stress. Behavioral analyses showed that SAC improved MPP +-induced impairment of locomotion (35%). These findings suggest that in mice, SAC attenuates MPP +-induced neurotoxicity in the striatum and that an antioxidant effect against oxidative stress may be partly responsible for its observed neuroprotective effects.

Exendin-4 protects dopaminergic neurons by inhibition of microglial activation and matrix metalloproteinase-3 expression in an animal model of Parkinson's disease

Exendin-4 is a naturally occurring more potent and stable analog of glucagon-like peptide-1 (GLP-1) that selectively binds at the GLP-1 receptor. It has been recently demonstrated that GLP-1 receptor stimulation preserves dopaminergic neurons in cellular and rodent models of Parkinson's disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity in rodents; previous studies suggest that activated microglia actively participate in the pathogenesis of PD neurodegeneration. However, the role of microglia in the neuroprotective properties of exendin-4 is still unknown. Here, we show that, in the mouse MPTP PD model, systemic administration of exendin-4 significantly attenuates the loss of substantia nigra pars compacta (SNpc) neurons and the striatal dopaminergic fibers. Exendin-4 prevents MPTP-induced microglial activation in the SNpc and striatum, and the expression of matrix metalloproteinase-3. In addition, exendin-4 also suppressed MPTP-induced expression of pro-inflammatory molecules and tumor necrosis factor alpha and interleukin-1 beta. Our data indicate that exendin-4 may act as a survival factor for dopaminergic neurons by functioning as a microglia-deactivating factor and suggest that exendin-4 may be a valuable therapeutic agent for neurodegenerative diseases such as PD.

Neuroprotective Effect of Ghrelin in the 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine Mouse Model of Parkinson’s Disease by Blocking Microglial Activation

Ghrelin is an endogenous ligand for growth hormone (GH) secretagogue receptor 1a (GHS-R1a) and is produced and released mainly from the stomach. It was recently demonstrated that ghrelin can function as a neuroprotective factor by inhibiting apoptotic pathways. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity in rodents; previous studies suggest that activated microglia actively participate in the pathogenesis of Parkinson's disease (PD) neurodegeneration. However, the role of microglia in the neuroprotective properties of ghrelin is still unknown. Here we show that, in the mouse MPTP PD model generated by an acute regimen of MPTP administration, systemic administration of ghrelin significantly attenuates the loss of substantia nigra pars compacta (SNpc) neurons and the striatal dopaminergic fibers through the activation of GHS-R1a. We also found that ghrelin reduced nitrotyrosine levels and improved the impairment of rota-rod performance. Ghrelin prevents MPTP-induced microglial activation in the SNpc and striatum, the expression of pro-inflammatory molecules tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1beta), and the activation of inducible nitric oxide synthase. The inhibitory effect of ghrelin on the activation of microglia appears to be indirect by suppressing matrix metalloproteinase-3 (MMP-3) expression in stressed dopaminergic neurons because GHS-R1a is not expressed in SNpc microglial cells. Finally, in vitro administration of ghrelin prevented 1-methyl-4-phenylpyridinium-induced dopaminergic cell loss, MMP-3 expression, microglial activation, and the subsequent release of TNF-alpha, IL-1beta, and nitrite in mesencephalic cultures. Our data indicate that ghrelin may act as a survival factor for dopaminergic neurons by functioning as a microglia-deactivating factor and suggest that ghrelin may be a valuable therapeutic agent for neurodegenerative diseases such as PD.

Testosterone enhances dopamine depletion by methamphetamine in male, but not female, mice

We examined the effects of varying concentrations of testosterone propionate (T) treatment within intact and gonadectomized male and female mice with regard to its capacity to alter striatal dopamine (DA) depletion in response to a neurotoxic regimen of methamphetamine (MA). Administration of T at 24h prior to MA significantly increased striatal DA depletion in intact and gonadectomized male mice. Similar treatments administered to intact and gonadectomized female mice failed to alter striatal DA concentrations in response to MA. These results demonstrate that T can enhance MA-induced neurotoxicity in male, but not in female, mice. Such findings have important implications with regard to sex differences in nigrostriatal dopaminergic function, in general, and, in specific, to sex differences related to nigrostriatal dopaminergic neurotoxicity and neurodegeneration like that in response to MA and in Parkinson’s disease, where a greater incidence is typically reported for males.

EGb761 protects against nigrostriatal dopaminergic neurotoxicity in 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine‐induced Parkinsonism in mice: role of oxidative stress

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity and behavioral impairment in rodents. Previous studies suggest that oxidative stress, via free radical production, is involved in MPTP-induced neurotoxicity. The MPTP-treated mouse has been the most widely used model for assessing neuroprotective agents for Parkinson's disease. It has been reported previously that EGb761 prevents dopaminergic neurotoxicity of MPTP. This compound is multifunctional via different mechanisms. Here, we report the neuroprotective effect of EGb761 against oxidative stress induced by MPTP in C57BL/6J mice. EGb761 is a patented and well-defined mixture of active compounds extracted from Ginkgo biloba leaves, with neuroprotective effects, exerted probably via its antioxidant or free radical scavenger action. MPTP administration resulted in a significant decrease in striatal dopamine levels and tyrosine hydroxylase immunostaining in the striatum and substantia nigra pars compacta. Mice receiving EGb761 had significantly attenuated MPTP-induced loss of striatal dopamine levels and tyrosine hydroxylase immunostaining in the striatum and substantia nigra pars compacta. The neuroprotective effect of EGb761 against MPTP neurotoxicity is associated with blockade of lipid peroxidation and reduction of superoxide radical production (indicated by a down-regulation of Mn-superoxide dismutase activity), both of which are indices of oxidative stress. Behavioral analyses showed that EGb761 improved MPTP-induced impairment of locomotion in a manner that correlated with enhancement of striatal dopamine levels. These findings suggest that, in mice, EGb761 attenuates MPTP-induced neurodegeneration of the nigrostriatal pathway and that an inhibitory effect against oxidative stress may be partly responsible for its observed neuroprotective effects.

Evidence for Hydroxyl Radical Scavenging Action of Nitric Oxide Donors in the Protection Against 1-Methyl-4-phenylpyridinium-induced Neurotoxicity in Rats

In the present study we provide evidence for hydroxyl radical (*OH) scavenging action of nitric oxide (NO*), and subsequent dopaminergic neuroprotection in a hemiparkinsonian rat model. Reactive oxygen species are strongly implicated in the nigrostriatal dopaminergic neurotoxicity caused by the parkinsonian neurotoxin, 1-methyl-4-phenylpyridinium (MPP+). Since the role of this free radical as a neurotoxicant or neuroprotectant is debatable, we investigated the effects of some of the NO* donors such as S-nitroso-N-acetylpenicillamine (SNAP), 3-morpholinosydnonimine hydrochloride (SIN-1), sodium nitroprusside (SNP) and nitroglycerin (NG) on in vitro *OH generation in a Fenton-like reaction involving ferrous citrate, as well as in MPP+-induced *OH production in the mitochondria. We also tested whether co-administration of NO* donor and MPP+ could protect against MPP+-induced dopaminergic neurotoxicity in rats. While NG, SNAP and SIN-1 attenuated MPP+-induced *OH generation in the mitochondria, and in a Fenton-like reaction, SNP caused up to 18-fold increase in *OH production in the latter reaction. Striatal dopaminergic depletion following intranigral infusion of MPP+ in rats was significantly attenuated by NG, SNAP and SIN-1, but not by SNP. Solutions of NG, SNAP and SIN-1, exposed to air for 48 h to remove NO*, when administered similarly failed to attenuate MPP+-induced neurotoxicity in vivo. Conversely, long-time air-exposed SNP solution when administered in rats intranigrally, caused a dose-dependent depletion of the striatal dopamine. These results confirm the involvement of *OH in the nigrostriatal degeneration caused by MPP+, indicate the *OH scavenging ability of NO*, and demonstrate protection by NO* donors against MPP+-induced dopaminergic neurotoxicity in rats.

Edaravone (3-Methyl-1-phenyl-2-pyrazolin-5-one), a Radical Scavenger, Prevents 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-Induced Neurotoxicity in the Substantia Nigra but Not the Striatum

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic neurotoxicity and behavioral impairment in rodents, and previous studies suggest that nitric oxide and reactive oxygen species are involved in MPTP-induced neurotoxicity. The present study examines the effect of edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one), a radical scavenger, on MPTP-induced neurotoxicity in the striatum and substantia nigra pars compacta (SNc) of C57BL/6J mice. MPTP treatment (10 mg/kg s.c. x 4 with 2-h intervals) decreased dopamine levels and tyrosine hydroxylase immunostaining in the striatum and SNc. Pretreatment with edaravone (1 and 3 mg/kg i.p.) significantly reduced the neurotoxicity in the SNc but not striatum. An immunohistochemical study showed that MPTP caused microglial activation both in the striatum and SNc, whereas it increased 3-nitrotyrosine immunoreactivity, an in vivo biomarker of peroxynitrite production, in the SNc but not the striatum. Furthermore, MPTP increased lipid peroxidation product thiobarbituric acid reactive substance in the midbrain, but not the striatum. Edaravone inhibited activation of the microglia and the increased 3-nitrotyrosine immunoreactivity in the SNc but not the striatum, and it also inhibited thiobarbituric acid reactive substance levels in the midbrain. Behavioral analyses showed that edaravone improved MPTP-induced impairment of locomotion and Rotorod performance. These results suggest that edaravone protects against MPTP-induced neurotoxicity in the SNc by blocking the production of reactive oxygen species or peroxynitrite and imply that dopaminergic degeneration in the SNc may play an important role in MPTP-induced motor dysfunction of mice.

Pesticide Exposure Exacerbates α-Synucleinopathy in an A53T Transgenic Mouse Model

The factors initiating or contributing to the pathogenesis of Parkinson's disease and related neurodegenerative synucleinopathies are still largely unclear, but environmental factors such as pesticides have been implicated. In this study, A53T mutant human alpha-synuclein transgenic mice (M83), which develop alpha-synuclein neuropathology, were treated with the pesticides paraquat and maneb (either singly or together), and their effects were analyzed. Immunohistochemical and biochemical analyses showed that chronic treatment of M83 transgenic mice with both pesticides (but not with either pesticide alone) drastically increased neuronal alpha-synuclein pathology throughout the central nervous system including the hippocampus, cerebellum, and sensory and auditory cortices. alpha-Synuclein-associated mitochondrial degeneration was observed in M83 but not in wild-type alpha-synuclein transgenic mice. Because alpha-synuclein inclusions accumulated in pesticide-exposed M83 transgenic mice without a motor phenotype, we conclude that alpha-synuclein aggregate formation precedes disease onset. These studies support the notion that environmental factors causing nitrative damage are closely linked to mechanisms underlying the formation of alpha-synuclein pathologies and the onset of Parkinson's-like neurodegeneration.

Local proteins associated with methamphetamine‐induced nigrostriatal dopaminergic neurotoxicity

The present study aimed to examine the proteins involved in the methamphetamine (MA)-induced nigrostriatal dopaminergic toxicity. Infusion of anisomycin into striatum and substantia nigra both abolished the MA-induced striatal dopamine (DA) and dihydroxyphenylacetic acid (DOPAC) depletions, indicating a critical role of local protein synthesis in determining such dopaminergic toxicity. Moreover, local protein synthesis blockade reversed this neurotoxicity via a temperature-independent mechanism. We then employed a proteomic approach, two-dimensional gel electrophoresis (2-DE) in conjunction with mass spectrometry analysis, to identify the protein candidates associated with the MA-induced neurotoxicity. In striatal samples, 2-DE analysis revealed that the intensities of nine protein spots were altered by MA treatment. Mass spectrometry analysis allowed us to identify five proteins, including an up-regulated protein, alpha-synuclein, and four down-regulated proteins, ATPase, F-actin capping protein beta subunit, ubiquitin carboxy-terminal hydrolase/PGP 9.5, and peroxidase. MA-altered expression levels of alpha-synuclein and ubiquitin carboxy-terminal hydrolase/PGP 9.5 in striata were confirmed by western blotting analysis. Taken together, these results suggest that local up-regulation of alpha-synuclein and down-regulation of ubiquitin carboxy-terminal hydrolase/PGP 9.5 could be linked to the MA-induced dopaminergic terminal toxicity.

Dopamine transporter function differences between male and female CD-1 mice

It has been reported that male mice are more susceptible to the neurotoxic effects of methamphetamine (MA) upon the nigrostriatal dopaminergic (NSDA) system. Since MA utilizes the dopamine transporter (DAT) to exert its effects, in the present study, we tested for differences in the dynamics of DAT function between male and female mice as an approach to understand some of the bases for this sex difference in MA-induced NSDA neurotoxicity. To accomplish this goal, in Experiment 1, the amount of dopamine (DA) obtained following DA infusion into the superfused striatal tissue fragments of male and female mice was measured while in Experiment 2 responses to the DA uptake blocker, nomifensine (NMF), were assessed in these preparations. The differences obtained to these treatments demonstrate that marked differences in DA transporter activity exist between male and female mice. When combining the DA and DOPAC measures from these two experiments, the data suggest that the female mice show a more active and efficient recovery and vesicular packaging of extracellular DA. These findings have important implications for sex differences in NSDA functions and responses to neurotoxins which enter the neurons via the DAT.

Effects of neonatal and prepubertal hormonal manipulations upon estrogen neuroprotection of the nigrostriatal dopaminergic system within female and male mice

Estrogen (E) can function as a neuroprotectant of the nigrostriatal dopaminergic (NSDA) system against methamphetamine (MA) neurotoxicity in female, but not male, mice. In the present report we examined whether the organizational effects of gonadal steroid hormones, as exerted in the early postnatal period, or developmental effects, as exerted during the pubertal period, would contribute to this sexually dimorphic neuroprotectant action of E. Neonatal gonadectomy and treatment with testosterone of female mice, retained the ability to show an E neuroprotectant response when tested as adults. However, females not treated with gonadal steroids failed to show an E-dependent neuroprotectant response. Neonatal gonadectomy of male mice, failed to result in the display of an E neuroprotectant response when tested as adults. Prepubertal gonadectomy of female mice, with or without testosterone treatment, abolished the capacity for E to produce neuroprotection against MA-induced NSDA neurotoxicity. Nor did prepubertal gonadectomy enable male mice to show an E neuroprotectant response. Taken together these results demonstrate that none of the manipulations performed within male mice enabled them to show an E-dependent neuroprotective response against MA-induced neurotoxicity of the NSDA system when tested as adults. For the female, it appears that the presences of gonadal steroids at these two developmental periods are needed for the display of an E-dependent neuroprotectant response within the adult.

Gender differences in modulatory effects of tamoxifen upon the nigrostriatal dopaminergic system

It has been demonstrated that the gonadal steroid hormone estrogen can exert neuroprotective effects upon the nigrostriatal dopaminergic (NSDA) system against methamphetamine (MA)-induced neurotoxicity in female, but not male, mice. In contrast, the anti-estrogen, tamoxifen (TMX) can function as a NSDA neuroprotectant within both female and male mice. In an attempt to understand these effects of TMX, the effects of this anti-estrogen upon both behavioral and neurochemical indices of NSDA function were examined within female and male mice following treatment with MA. In general, TMX exerted markedly different (bi-directional) effects upon NSDA function between female and male mice. Notably, treatment with TMX resulted in a relative decrease in striatal dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations within male mice and a relative increase in female mice when treated with MA to produce a significant gender difference. Similar effects were obtained for locomotor behaviors related with NSDA function. That is, TMX produced increases in horizontal activity, number of movements and total distance traveled within MA-treated female mice resulting in statistically significant gender differences for the two former parameters. For non-locomotor behaviors, like time occupying the center and margin of the cage, TMX-treated male mice showed statistically significant increases and decreases compared within TMX-treated female mice, respectively. These results show that in contrast to the similar neuroprotective effects of TMX upon MA-induced NSDA neurotoxicity, a number of other NSDA indices induced by MA show markedly different response profiles between TMX-treated female and male mice.

Developmental and Genetic Influences upon Gender Differences in Methamphetamine‐Induced Nigrostriatal Dopaminergic Neurotoxicity

The gonadal steroid hormone estrogen (E) may play an important role in sex differences in methamphetamine (MA)-induced neurotoxicity of the nigrostriatal dopaminergic (NSDA) system because E can serve as a neuroprotectant in female, but not male, mice. Gonadal steroid hormones also exert important organizational/developmental effects upon the brain at critical developmental periods. In Part 1 we assessed whether organizational (neonatal) or developmental (prepubertal) effects of gonadal steroids would alter gender/E-dependent neuroprotection of MA-induced NSDA neurotoxicity. Attempts to feminize male mice by gonadectomy at either the neonatal or prepubertal period failed to enable E to function as a neuroprotectant within the adult male mouse. Attempts to masculinize the female by testosterone administration at the neonatal period did not abolish the capacity for E to function as a neuroprotectant. However, prepubertal gonadectomy of female mice did disrupt E's capacity to serve as a neuroprotectant. These results suggest that genetic sex may prove the primary determinant for the sex differences observed in response to MA-induced NSDA neurotoxicity. In Part 2 we examined whether gender differences in response to MA-induced NSDA neurotoxicity would interact with a specific genetic alteration in a neurotrophic factor, brain-derived neurotrophic factor (BDNF). Female and male mice that were either deficient (+/- BDNF) or overexpressing (DBH:BDNF+) BDNF were treated with MA. Sex differences in MA-induced NSDA neurotoxicity remained present in +/- BDNF mice and were less severe as compared with their wild-type controls. A similar result was obtained in mice that overexpress BDNF, with female and mutant mice showing less NSDA neurotoxicity. In both BDNF-deficient mice and mice that overexpress BDNF, the relative degree of MA-induced NSDA neurotoxicity was lower in males. Taken together, these results suggest that a selective alteration in BDNF expression offers some neuroprotective potential against MA-induced NSDA neurotoxicity, and the relative degree of this neuroprotection may interact with the gender of the subject.

The effect of gender and the neurotrophin, BDNF, upon methamphetamine-induced neurotoxicity of the nigrostriatal dopaminergic system in mice

The interactive effects between gender and a selective alteration in the neurotrophin, brain-derived neurotrophic factor (BDNF) upon methamphetamine (MA)-induced neurotoxicity of the nigrostriatal dopaminergic (NSDA) system were assessed. MA treatment produced a greater degree of NSDA neurotoxicity (indicated by greater reductions in corpus striatal dopamine levels) in wild type control BDNF male versus female mice. This sex difference was unaltered in heterozygous mutant BDNF (BDNF+/−) mice and in mice which overexpress BDNF (DBH:BDNF+). Both BDNF mutant conditions resulted in preservation of corpus striatal dopamine levels following MA treatment as compared with their respective MA-treated wild type controls. The relative amount of this preservation was greater in male BDNF mutants, with values being significantly greater than females in the BDNF+/− condition. These results suggest that alterations in BDNF do not alter basic gender differences in MA-induced NSDA neurotoxicity, but may produce a neuroprotection against MA which is relatively greater in males.

Acute effects of estrogen upon methamphetamine induced neurotoxicity of the nigrostriatal dopaminergic system

Estrogen acts as a neuroprotectant of the nigrostriatal dopaminergic system when given chronically to female mice prior to Methamphetamine (MA) insult. In this report, we tested the acute effects of Estradiol Benzoate (EB-10 microg in Oil) in ovariectomized CD-1 mice to function as a neuroprotectant when administered prior to (Experiment 1) or after (Experiment 2) MA treatment. Striatal dopamine (DA) concentrations and DOPAC/DA ratios were measured to assess the neuroprotective effects of EB. In Experiment 1, we observed that EB exerted a neuroprotective effect upon striatal dopamine concentrations when administered at 24 and 12, but not at 0.5, hours prior to MA injection and upon DOPAC/DA ratios when administered at 24, 12 and 0.5 hours prior to MA. In Experiment 2, no evidence for estrogen to protect the striatum from MA insult was obtained when EB was administered at 15, 30, 60 or 120 minutes after MA. These results show that EB can act as a modulator of MA-induced nigrostriatal dopaminergic neurotoxicity suggestive of a neuroprotectant, when administered within 0.5 hour of MA insult as assessed by measures of DOPAC/DA, but fails to prevent depletion of DA when given after MA insult. The data suggest that estrogen may exert this rapid neuroprotective effect through a non-genomic mechanism.