Number Of TH-positive Neurons Research Articles

Cyclosporine A and MnTMPyP Alleviate α-Synuclein Expression and Aggregation in Cypermethrin-Induced Parkinsonism.

Cypermethrin induces the mitochondrial dysfunction and oxidative damage to the nigrostriatal dopaminergic neurons leading to Parkinsonism in rats. Despite α-synuclein aggregation is reported to be critical in Parkinson's disease, its role and alliance with the mitochondrial dysfunction and oxidative damage leading to cypermethrin-induced Parkinsonism have not yet been deciphered. The present study aimed to examine the effect of cypermethrin on the expression and aggregation of α-synuclein and its subsequent connection with oxidative damage and mitochondrial dysfunction leading to the nigrostriatal dopaminergic neurodegeneration in the presence or absence of a mitochondrial membrane transition pore opening inhibitor, cyclosporine A and a superoxide dismutase/catalase mimetic, manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride (MnTMPyP). The expression of α-synuclein, 3-nitrotyrosine (3-NT), 4-hydroxynonenal (4-HNE)-modified proteins, mitochondrial dysfunction-dependent apoptotic proteins, nitrite content, lipid peroxidation (LPO) and number of tyrosine hydroxylase (TH)-positive neurons were estimated in the substantia nigra and dopamine content in the striatum of control and treated rats employing standard procedures. Cypermethrin augmented the expression of α-synuclein, 3-NT, 4-HNE-modified proteins, caspase-3, mitochondrial Bax and cytosolic cytochrome-c along with nitrite and LPO and reduced the expression of cytosolic Bax, mitochondrial cytochrome-c, dopamine and number of TH-positive neurons. Cyclosporine A or MnTMPyP alleviated the expression and aggregation of α-synuclein along with indicators of the mitochondrial dysfunction, oxidative damage and dopaminergic neurodegeneration. The results demonstrate that cypermethrin induces α-synuclein expression and aggregation while cyclosporine A or MnTMPyP rescues from α-synuclein over-expression and aggregation along with the mitochondrial dysfunction and oxidative damage leading to Parkinsonism in rats.

Effects of neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment on ovarian development of the sapphire devil, Chrysiptera cyanea.

In the neuroendocrine system controlling fish reproduction, dopamine (DA) acts as a gonadotropin inhibitory factor and plays a role in regulating gonadal development of certain species. The present study examined the effects of chemical destruction of dopaminergic neurons in the brain on DA production and ovarian development in the sapphire devil Chrysiptera cyanea, a reef-associated damselfish. The avidin-biotin-peroxidase complex method using an antibody against tyrosine hydroxylase (TH), a critical enzyme in the DA synthesis pathway, identified a population of dopaminergic neurons with somata in the anteroventral preoptic nucleus of the diencephalon and fibers terminating in the proximal pars distalis of the pituitary. Maintaining fish in seawater containing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) at 0.02 and 0.2 µg/mL for 2 days resulted in decreases in DA, 3,4-dihydroxyphenylacetic acid (DOPAC; DA metabolite), and DA metabolic rate in the whole brain. The number of TH-positive neurons in the diencephalon decreased after 0.02 µg/mL MPTP treatment for 2 days. These results suggest that MPTP treatment destroys TH-positive neurons in the diencephalon, thereby decreasing the synthesis and release of DA from the brain. This treatment rescued ovarian development in fish with artificially retracted ovaries during the spawning season. The gonadosomatic index of MPTP-treated fish 5 and 7 days after treatment was significantly higher than that of control fish. Oocytes in the vitellogenic stages were observed in the ovaries of MPTP-treated fish, but not in control fish. These results suggest that DA in the brain drives ovarian development in the sapphire devil.

Intravenous mesenchymal stem cell administration exhibits therapeutic effects against 6-hydroxydopamine-induced dopaminergic neurodegeneration and glial activation in rats

To explore a novel therapy against Parkinson's disease (PD), we evaluated the therapeutic effects of human bone marrow-derived mesenchymal stem cells (hBM-MSCs), pluripotent stromal cells with secretory potential of various neurotrophic and anti-inflammatory factors, in a hemi-parkinsonian rat model. The unilateral intrastriatal 6-hydroxydopamine (6-OHDA)-lesioned rats were injected hBM-MSCs (1.0×107cells) or PBS intravenously 16 days after lesioning. Administration of hBM-MSCs inhibited methamphetamine-stimulated rotational behavior at 7, 14, 21 and 28 days after transplantation. Immunohistochemical analysis also showed that number of TH-positive neurons in the substantia nigra pars compacta was significantly preserved in hBM-MSCs-transplanted rats compared to sham-operated rats, whereas the immunoreactivity of ionized calcium binding adaptor molecule 1 was markedly inhibited. In this study, we demonstrated the therapeutic effects of intravenous hBM-MSCs administration in parkinsonian model rats presenting distinct parkinsonian phenotype at 16 days after 6-OHDA lesioning. The favorable findings raise the possibility that hBM-MSCs could be a novel therapeutic option to promote survival of dopaminergic neurons in PD.

Sorting nexin 27 regulation of G protein-gated inwardly rectifying K⁺ channels attenuates in vivo cocaine response.

The subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.

Morphine regulates Argonaute 2 and TH expression and activity but not miR-133b in midbrain dopaminergic neurons

Epigenetic changes such as microRNAs (miRs)/Ago2-induced gene silencing represent complex molecular signature that regulate cellular plasticity. Recent studies showed involvement of miRs and Ago2 in drug addiction. In this study, we show that changes in gene expression induced by morphine and morphine withdrawal occur with concomitant epigenetic modifications in the mesolimbic dopaminergic (DA) pathway [ventral tegmental area (VTA)/nucleus accumbens (NAc) shell], which is critically involved in drug-induced dependence. We found that acute or chronic morphine administration as well as morphine withdrawal did not modify miR-133b messenger RNA (mRNA) expression in the VTA, whereas Ago2 protein levels were decreased and increased in morphine-dependent rats and after morphine withdrawal, respectively. These changes were paralleled with enhanced and decreased NAc tyrosine hydroxylase (TH) protein (an early DA marker) in morphine-dependent rats and after withdrawal, respectively. We also observed changes in TH mRNA expression in the VTA that could be related to Ago2-induced translational repression of TH mRNA during morphine withdrawal. However, the VTA number of TH-positive neurons suffered no alterations after the different treatment. Acute morphine administration produced a marked increase in TH activity and DA turnover in the NAc (shell). In contrast, precipitated morphine withdrawal decreased TH activation and did not change DA turnover. These findings provide new information into the possible correlation between Ago2/miRs complex regulation and DA neurons plasticity during opiate addiction.

Lack of CCR5 modifies glial phenotypes and population of the nigral dopaminergic neurons, but not MPTP-induced dopaminergic neurodegeneration

Constitutive expression of C–C chemokine receptor (CCR) 5 has been detected in astrocytes, microglia and neurons, but its physiological roles in the central nervous system are obscure. The bidirectional interactions between neuron and glial cells through CCR5 and its ligands were thought to be crucial for maintaining normal neuronal activities. No study has described function of CCR5 in the dopaminergic neurodegeneration in Parkinson's disease. In order to examine effects of CCR5 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration, we employed CCR5 wild type (WT) and knockout (KO) mice. Immunostainings for tyrosine hydroxylase (TH) exhibited that CCR5 KO mice had lower number of TH-positive neurons even in the absence of MPTP. Difference in MPTP (15mg/kg×4 times, 2hr interval)-mediated loss of TH-positive neurons was subtle between CCR5 WT and KO mice, but there was larger dopamine depletion, behavioral impairments and microglial activation in CCR5 deficient mice. Intriguingly, CCR5 KO brains contained higher immunoreactivity for monoamine oxidase (MAO) B which was mainly localized within astrocytes. In agreement with upregulation of MAO B, concentration of MPP+ was higher in the substantia nigra and striatum of CCR5 KO mice after MPTP injection. We found remarkable activation of p38 MAPK in CCR5 deficient mice, which positively regulates MAO B expression. These results indicate that CCR5 deficiency modifies the nigrostriatal dopaminergic neuronal system and bidirectional interaction between neurons and glial cells via CCR5 might be important for dopaminergic neuronal survival.

Overexpression of Parkin Ameliorates Dopaminergic Neurodegeneration Induced by 1- Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine in Mice

Mutations in the parkin gene are currently thought to be the most common cause of recessive familial Parkinsonism. Parkin functions as an E3 ligase to regulate protein turnover, and its function in mitochondrial quality control has been reported recently. Overexpression of parkin has been found to prevent neuronal degeneration under various conditions both in vivo and in vitro. Here, we generated a transgenic mouse model in which expression of wild type parkin was driven by neuron-specific enolase (NSE) promoter. We reported that both young and old parkin transgenic mice exhibited less reduction of striatal TH protein and number of TH positive neurons in the substantia nigra induced by 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP), compared to wild type littermates. MPTP-induced mitochondrial impairment in the substantia nigra was improved in young parkin transgenic mice. Decreased striatal α-synuclein was demonstrated in old parkin transgenic mice. These results provide reliable evidence from the transgenic mouse model for parkin that overexpression of parkin may attenuate dopaminergic neurodegeneration induced by MPTP through protection of mitochondria and reduction of α-synuclein in the nigrostriatal pathway.

Nifedipine Prevents Iron Accumulation and Reverses Iron-Overload-Induced Dopamine Neuron Degeneration in the Substantia Nigra of Rats

The mechanisms of iron accumulation in substantia nigra (SN) of Parkinson's diseases remain unclear. The objective of this study was to investigate effects of nifedipine on iron-overload-induced iron accumulation and neurodegeneration in SN of rats. By high performance liquid chromatography-electrochemical detection, tyrosine hydroxylase (TH) immunohistochemistry, and iron content array, we first quantified iron content and the number of dopamine neurons in SN of experimental rats treated with iron dextran. We further assessed effects of treatment with nifedipine. Our results showed that nifedipine treatment prevents iron dextran-induced dopamine depletion in the striatum. Consistently, we found that nifedipine restores the number of TH-positive neurons reduced by iron dextran overload and prevents increase of iron content in the SN. These results suggested that nifedipine may suppress iron toxicity in dopamine neurons and prevent neurodegeneration.

Neuroprotective Effects of San-Huang-Xie-Xin-Tang in the MPP+/MPTP Models of Parkinson’s DiseaseIn VitroandIn Vivo

San-Huang-Xie-Xin-Tang (SHXT), composed of Coptidis rhizoma, Scutellariae radix, and Rhei rhizoma, is a traditional Chinese medicine used for complementary and alternative therapy of cardiovascular and neurodegenerative diseases via its anti-inflammatory and antioxidative effects. The aim of this study is to investigate the protective effects of SHXT in the 1–methyl–4–phenylpyridinium (MPP+)/1–methyl–4–phenyl–1,2,3,6–tetrahydropyridine (MPTP) models of Parkinson's disease. Rat primary mesencephalic neurons and mouse Parkinson disease model were used in this study. Oxidative stress was induced by MPP+ in vitro and MPTP in vivo. In MPP+-treated mesencephalic neuron cultures, SHXT significantly increased the numbers of TH-positive neurons. SHXT reduced apoptotic signals (cytochrome and caspase) and apoptotic death. MPP+-induced gp91phox activation and ROS production were attenuated by SHXT. In addition, SHXT increased the levels of GSH and SOD in MPP+-treated neurons. In MPTP animal model, SHXT markedly increased TH-positive neurons in the substantia nigra pars compacta (SNpc) and improved motor activity of mice. In conclusion, the present results reveal the evidence that SHXT possesses beneficial protection against MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative and antiapoptotic effects. SHXT might be a potentially alternative and complementary medicine for neuroprotection.

Long-term effect of intraventricular injection of low-dose lipopolysaccharide on behavior, microglias and dopaminergic neurons in the substantia nigra of rats

Objective To investigate the long-term effect of inflammation on behavior,microglias and dopaminergic （DA） neurons in the substantia nigra of intracephalic inflammation rat models induced by intracerebroventricular injection of low-dose（10μg） lipopolysaccharide （LPS）. To analyze the relationship between activa- tion of microglias and DA neurons degeneration in order to explore the mechanism of inflammation in the progres- sive process of Parkinson＇s disease （PD）. Methods 50 healthy male SD rats were randomly assigned into sa- line-injected control group and 10μg LPS-injected group. All injections were made intracerebroventricularly on right side of rats with saline or LPS. Moving speed was measured at different time points. At 24 weeks and 40 weeks after saline or LPS injection, specific antibodies of OX-42 and OX-6 were used separately to detect the changes of microglia in the substantia nigra of rat. The changes in morphology and numbers of substantia nigra DA neurons were observed by tyrosine hydroxylase（TH） immunohistochemical staining. The expression and distribution of the degenerated neurons in substantia nigra were detected by using Fluoro-Jade B （FJB）. Results （1）Analysis of moving speed showed that the moving speed of 10μg LPS-injected group rats and saline-injected group rats was similar from 4 weeks to 36 weeks after injection. At 40 weeks post injection, moving speed of 10μg LPS-injected group rats decreased by 24.6% compared with that of saline-injected group rats （P〉 0.05 ）. （2）At 24 weeks and 40 weeks after injection,there were many activated 0X-42 positive microglias in the substantia nigra of 10μg LPS-in- jected group rats, but there was almost no significant activated OX-42 positive microglia in saline-injected group. OX-6 positive microglias were not found in the substantia nigra of both of two groups. （1）At 24 weeks and 40 weeks post injection, the number of TH-positive neurons in the substantia nigra of 10μg LPS-iniected rout3 rats decreasedby 24.2% （ t=4.803, P〈0.01 ） and 27.6% （ t= 3. 212, P〈0.01 ） respectively compared with those of salineinjected group. （4） There was no FJB positive neurons in the suhstantia nigra of the two group rats. Conclusion Intraventricular injection of low-dose LPS （10μg） in rats may induce long-term activation of microglias and chronic degeneration of DA neurons in the substantia nigra of rats although the necrosis are not occurs to DA neurons till 40 weeks post LPS injection. Intravenlricular injection of low-dose LPS in rats could he ideal model to study the mechanism of chronic degeneration of DA neurons in PD. Key words: Parkinson＇ s disease ; Inflammation ; Behaviour ; Microglia ; Dopaminergic neurons

Chronic Dichlorvos Exposure: Microglial Activation, Proinflammatory Cytokines and Damage to Nigrostriatal Dopaminergic System

Dopaminergic cells in the substantia nigra are highly vulnerable to the neurodegenerative process of Parkinson's disease. Therefore, mechanisms that enhance their susceptibility to injury bear important implications for disease pathogenesis. We have previously shown that chronic dichlorvos exposure caused nigrostriatal dopaminergic degeneration and significant behavioral impairments. In this study, we analyzed the relationship between microglial activation and dopaminergic neurodegeneration to examine the possibility that neuroinflammation may induce dopaminergic neuronal loss in the nigrostriatal system. Chronic dichlorvos exposure causes microglial activation including induction of NADPH oxidase and a selective loss of dopaminergic neurons in rat. Microglial marker expression was increased at transcription as well as translational levels in the substantia nigra (SN) and corpus striatum (CS) of rats exposed to dichlorvos. Activated microglia were seen in SN and CS of dichlorvos-treated animals but were rarely observed in controls. Immunostaining revealed lesser number of TH-positive neurons and higher number of microglia in SN and CS regions after dichlorvos treatment. The mRNA and protein levels of the NADPH oxidase main subunit gp91(phox) were significantly increased after dichlorvos administration. Dichlorvos exposure also leads to increased level of microglial noxious mediators such as IL-1β, TNF-α and IL-6 in ventral midbrain and CS at transcription as well as translational levels. Data indicate that microglial activation and consequent induction of NADPH oxidase and proinflammatory cytokines such as TNF-α, IL-1β and IL-6 may act as risk factors for Parkinson's disease by increasing the vulnerability of dopaminergic cells to dichlorvos toxic injury.

Restricted role of CRF1 receptor for the activity of brainstem catecholaminergic neurons in the negative state of morphine withdrawal

Evidence suggests that corticotropin-releasing factor (CRF) system is an important mediator in the negative symptoms of opioid withdrawal. We used genetically engineered mice lacking functional CRF receptor-1 (CRF1R) levels to study the role for CRF/CRF1R pathways in the negative affective states of opioid withdrawal. Wild-type and CRF1R(-/-) offspring of CRF1R(+/-) breeders were identified by PCR analysis of tail DNA and were rendered dependent on morphine via intraperitoneal injection of increasing doses of morphine (10-60mg/kg). Negative state associated with opioid withdrawal was examined by using conditioned place aversion (CPA), TH expression and TH phosphorylation were measured in different brain regions involved in addictive behaviours using immunohistochemistry. The weight loss in morphine withdrawn CRF1R(-/-) animals was significantly (p < 0.05) lower versus wild-type. The aversion for environmental cues paired with opioid withdrawal was lower (p < 0.001) in the CRF1R-deficient versus wild-type. Using dual immunolabeling for c-Fos, data show that naloxone-induced withdrawal increases the number of TH positive neurons phosphorylated at Ser40 or Ser31 that coexpress c-Fos in the nucleus of tractus solitarius (NTS)-A2 from wild-type and CRF(-/-) deficient mice. By contrast, the number of phospho-Ser40 or phospho-Ser31 positive neurons expressing c-Fos was lower in the ventrolateral medulla (VLM)-A1 in CRF(-/-)-deficient mice. Our study demonstrates an increased activity of brainstem catecholaminergic neurons after CPA induced by morphine withdrawal suggesting that CRF1R is implicated in the activation of A1 neurons and provides evidence that this receptor is involved in the body weight loss and in the negative aversive effects of morphine withdrawal.

The effect of olfactory ensheathing cell transplantation combined with walking training on neurofunction recovery in rats after spinal cord contusion

Objective To explore the effect of olfactory ensheathing cell （OEC） transplantation combined with walking training on neurofunction recovery in rats after spinal cord contusion. Methods Forty adult female rats aged （75 ± 1 ） days were subjected to experimental spinal cord contusion at the T10 level using a New York University impactor at a height of 25 mm. They were then divided into 4 groups： （ 1 ） an OEC transplantation combined with walking training （OEC-walking training） group, （2） an OEC transplantation （OEC） group, （3） a walking training combined with Dulbecco＇s modified Eagle medium injection （DMEM） （walking training-DMEM） group, and （4） aDMEM injection （SCI-DMEM） group. The OEC transplants and DMEM injections were performed 2 weeks post-injury. Walking training began at the 7th day post-injury and consisted of daily sessions （once daily, 5 days a week for 10 weeks） of quadrupedal treadmill training, starting from 15 min and gradually increasing to 30 min daily, at speeds starting from 3 m/min and gradually increasing in accordance to the condition of the rats. Locomotor function recovery of the rats＇ hindlimbs was evaluated weekly using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale.The expression of tyrosine hydroxylase （ TH ） was detected in the injured region of the lumbar spinal cord. Results The BBB scores of rats in the OEC-walking training group and the walking training-DMEM group improved significantly from the 4th week post-injury compared to the SCI-DMEM injection group. Rats in the OEC transplantation group had a significant improvement in BBB scores at the 5th to 8th weeks post-injury. At the end of the 11th week post-injury, the average BBB scores were 13.14 ± 0.24 in the OEC-walking training group, 11. 64 ± 0.56 in the OEC transplantation group, 12.29 ±0.64 in the walking training-DMEM group and 11.07 ± 0.84 in the SCI-DMEM group.The OEC-walking training group scored significantly higher than the other 3 groups. Although the number of TH-positive neurons in the lumbar spinal cord was not significantly different among the groups, the morphology of TH-positiveneurons in the OEC-walking training group and the walking training-DMEM group was different from those in the OEC transplantation group and the SCI-DMEM group. Conclusions OEC transplantation combined with walking training can effectively promote the functional recovery of the hindlimb. The plasticity of the descending TH system and of motoneurons of the ventral horn of the lumbar spinal cord might mediate the changes. Key words: Spinal cord contusion; Olfactory ensheathing cells transplantation; Walking training; Neurofunction; Tyrosine hydroxylase

Intravenous administration of mesenchymal stem cells exerts therapeutic effects on parkinsonian model of rats: Focusing on neuroprotective effects of stromal cell-derived factor-1α

BackgroundMesenchymal stem cells (MSCs) are pluripotent stem cells derived from bone marrow with secretory functions of various neurotrophic factors. Stromal cell-derived factor-1α (SDF-1α) is also reported as one of chemokines released from MSCs. In this research, the therapeutic effects of MSCs through SDF-1α were explored. 6-hydroxydopamine (6-OHDA, 20 μg) was injected into the right striatum of female SD rats with subsequent administration of GFP-labeled MSCs, fibroblasts, (i.v., 1 × 107 cells, respectively) or PBS at 2 hours after 6-OHDA injection. All rats were evaluated behaviorally with cylinder test and amphetamine-induced rotation test for 1 month with consequent euthanasia for immunohistochemical evaluations. Additionally, to explore the underlying mechanisms, neuroprotective effects of SDF-1α were explored using 6-OHDA-exposed PC12 cells by using dopamine (DA) assay and TdT-mediated dUTP-biotin nick-end labeling (TUNEL) staining.ResultsRats receiving MSC transplantation significantly ameliorated behaviorally both in cylinder test and amphetamine-induced rotation test compared with the control groups. Correspondingly, rats with MSCs displayed significant preservation in the density of tyrosine hydroxylase (TH)-positive fibers in the striatum and the number of TH-positive neurons in the substantia nigra pars compacta (SNc) compared to that of control rats. In the in vitro study, SDF-1α treatment increased DA release and suppressed cell death induced by 6-OHDA administration compared with the control groups.ConclusionsConsequently, MSC transplantation might exert neuroprotection on 6-OHDA-exposed dopaminergic neurons at least partly through anti-apoptotic effects of SDF-1α. The results demonstrate the potentials of intravenous MSC administration for clinical applications, although further explorations are required.

Zonisamide Attenuates MPTP Neurotoxicity in Marmosets

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) induces parkinsonism in humans and animals. The effects of zonisamide on dopamine neurons were studied in MPTP-treated common marmosets (Callithrix jacchus). Groups of animals (n = 3) were treated with MPTP (2.5 mg/kg, every 24 h x 3); MPTP plus zonisamide (40 mg/kg administered 1 h before each MPTP dose); MPTP plus selegiline (a known MAO-B inhibitor) (2 mg/kg administered 1 h before each MPTP dose); and saline controls. An immunohistochemical study of the substantia nigra was performed 14 days after MPTP treatment in each group. MPTP reduced the mean number of tyrosine hydroxylase (TH)-positive neurons to 10% of the normal control group and mean cell size was significantly (P < 0.001) reduced from 424 to 159 µm². In the group pre-treated with zonisamide, the mean number of TH-positive neurons was reduced to 26% of that in the normal control group and the mean neuron size was significantly (P < 0.05) increased from 159 to 273 µm² compared with the group treated with MPTP alone. Moreover, in the group pre-treated with selegiline, the mean number of TH-positive neurons was 47% of that in the normal control group and the mean neuron size was increased significantly (P < 0.01) from 159 to 319 µm² compared to the group treated with MPTP alone. This observation suggests that zonisamide reduces MPTP toxicity.

Progressive noradrenergic deficits in the locus coeruleus of Mecp2 deficient mice

Methyl-CpG binding protein 2 (MeCP2) is a transcriptional regulator. Mutations in this gene cause a wide range of neurological disorders. Mecp2 deficiency has been previously associated to catecholaminergic dysfunctions leading to autonomic defects in the brainstem and the sympathoadrenergic system of the mouse. The present study was undertaken to determine if the locus coeruleus (LC), the main noradrenergic cell group of the brain, is affected. Using real type PCR, we found a reduction of the tyrosine hydroxylase (Th) mRNA level, the rate-limiting enzyme in catecholamine synthesis, in the whole pons of P15 (-36%), P30 (-47%) and P50 (-42%) Mecp2 null male as well as in adult heterozygous female (-44%) mice. Using immunoquantification we did not observe any difference of the Th staining level in P30 null male mice. However at P50, we demonstrated a significant decrease in both the Th staining level (-24%), and the number of Th-positive neurons (-23%). We subsequently characterized a reduction (-28%) of the dendritic density of the Th-positive fibers surrounding the LC in P50 null male mice. In heterozygous female mice immunoquantification did not revealed significant modifications, but only a tendency towards reduction. Finally, we did not found any apoptotic neurons in the pons indicating that LC neurons are not dying but are more likely loosing their catecholaminergic phenotype. In conclusion, our results showing a progressive catecholaminergic deficit in the LC of Mecp2 deficient null male mice could open new perspectives to better understand the autonomic and cognitive deficits due to the lack of Mecp2.

The substantia nigra pars compacta of the Göttingen minipig: an anatomical and stereological study

Parkinson disease (PD) is a neurodegenerative disorder resulting from progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Despite advances in medical and surgical therapies, many PD patients experience progression of their symptoms and medical side effects over time. To explore new treatments, new animal models mimicking the progressive PD nature are needed. The pig is well suited for this purpose with its large gyrated brain, sensitive to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The objective of this study was to provide the anatomical foundation for such a model, describing in detail the SNc in normal Göttingen minipigs and estimating the volume and total number of tyrosine hydroxylase (TH)-positive neurons. The brain stems of 6 Göttingen minipigs were paraffin embedded and serially cut before Nissl staining and immunohistochemical visualization of TH. The volume of the SNc and the total number of TH-positive neurons were estimated by design-based stereology. The substantia nigra was located at the dorsal rim of the crus cerebri extending throughout the mesencephalon. A dorsal pars compacta and a ventral pars reticulata were demonstrated. The SNc merged with the ventral tegmental area medially and the retro-rubral field dorsocaudolaterally. The total number of TH-positive neurons in the SNc unilaterally was estimated to 80,700 [74,100;87,300], and the volume estimate was 26.4 mm(3) [25.0;27.8]. We conclude that the anatomy of the SNc in the Göttingen minipig corresponds well with that of higher primates, and is well suited for further studies aimed at optimizing this non-primate large animal model for PD.

Effect of Intranigral Injection of GDNF and EGF on the Survival and Possible Differentiation Fate of Progenitors and Immature Neurons in 6-OHDA-Lesioned Rats

We investigated the survival and the possible differentiation fate of the progenitors and immature neurons in the pars compacta of the substantia nigra (SNc) by intranigral injection of a glial cell line-derived neurotropic factor (GDNF) or glial cell line-derived neurotropic factor plus epidermal growth factor (EGF + GDNF) in 6-hydroxydopamine (6-OHDA)-lesioned rats. First, we performed behavioral tests by postural asymmetry and forelimb akinesia on the rats injected with 6-OHDA in striatumat day 7, and selected the qualified model according to the results. Then, intranigral GDNF or EGF + GDNF treatment was administered in the qualified PD model rats. On day 21, behavioral tests were performed with these rats; and then the rats were sacrificed for analyses of β-tubulin isotype-III (Tuj1), nestin, glial fibrillary acidic protein (GFAP), and tyrosine hydroxylase (TH) by immunohistochemistry and Western blotting. The results indicated that GDNF could promote the survival of the progenitor cells and immature neurons in rat SNc following 6-OHDA lesion. Moreover, EGF is capable of enhancing the survival effect of GDNF on the progenitor cells and immature neurons in SNc. On day 21, rapid functional recovery from the lesion-induced behavioral asymmetries was observed in the GDNF or EGF + GDNF treated rats, and the numbers of TH-positive neurons increased in SNc, suggesting that the rats might generate new dopaminergic neurons. Thus, our study provides the new insight that the progenitors and immature neurons in SNc of 6-OHDA-lesioned rats might be able to differentiate toward the dopaminergic neurons fate subsequent to treatment with GDNF or EGF + GDNF.

Generation of dopaminergic neurons from human fetal mesencephalic progenitors after co-culture with striatal-conditioned media and exposure to lowered oxygen

Mature dopaminergic neurons were generated from human fetal mesencephalic progenitors by co-culture with human fetal striatal-conditioned media (SCM) and exposure to lowered oxygen. Immunofluorescence analysis showed that cells cultured in differentiation media with embryonic SCM had a greater number of TH-positive neurons than those cultured in traditional media without SCM addition. The TH-positive neurons cultured in lowered oxygen (3–5% O 2) had more branches and those branches were longer than those of neurons cultured under 20% O 2 culture conditions. Furthermore, more TH-positive cells with mature morphology were observed in the culture containing combined SCM addition and lowered oxygen. We also observed higher levels of dopamine release under the combined media conditions than observed under any individual media condition when depolarized by high K+ as determined by high-performance liquid chromatography (HPLC). Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA expressions of sonic hedgehog (SHH), glial cell derived neurotrophic factor (GDNF) and tyrosine hydroxylase (TH), which are linked to the generation of dopaminergic neurons, were increased after co-culture with SCM. Lowered O 2 significantly induced mRNA expression of erythropoietin (EPO) and P27, which affect neuronal differentiation, and was accompanied by upregulation of Nurr1, Pitx3 and dopamine transporter (DAT) mRNAs, which are correlated to the maturation of dopaminergic neurons. This study makes an important contribution to understanding the effects of hypoxia and SCM on the differentiation and maturation of TH-positive neurons derived from human fetal mesencephalic progenitors in vitro.

A combined behavioral and morphological study on the effects of fetal asphyxia on the nigrostriatal dopaminergic system in adult rats

Fetal asphyxic insults in the brain are known to be associated with developmental neurological problems like neuromotor disorders. However, little is known about the long-term consequences of fetal asphyxia (FA). For that reason, the present study investigated the long-term effects of FA on motor behavior and dopaminergic circuitry. FA was induced at embryonic day 17 by 75-minute clamping of the uterine circulation. SHAM animals underwent the same procedure except for the clamping. This was followed by full-term vaginal delivery of animals in all groups (FA, SHAM and untreated controls). At 6 months, basal and amphetamine-induced locomotor activity was measured during open field testing. Brain sections were stained for tyrosine hydroxylase (TH) and glial fibrillary acidic protein (GFAP). TH-positive cells and GFAP-positive cells in substantia nigra pars compacta (SN C) and striatum were counted using design-based stereology. Moreover, TH-immunoreactivity in the striatum was assessed by grey value measurements. Behavioral analysis demonstrated that SHAM and FA showed less basal and amphetamine-induced activity than controls. Histochemically, FA decreased the number of TH-positive neurons in the SN C and lowered TH-positive in the striatum. Furthermore, more GFAP-positive cells were found in the SN C and striatum in FA than in either control and SHAM groups. Additionally, FA animals showed ventriculomegaly associated with smaller white matter as well as grey matter volumes. The data show that FA was associated with deficits in both dopamine-related motor behavior and biochemistry. These alterations were associated with nigrostriatal astrogliosis. The present study demonstrates the sensitivity of the dopaminergic system towards FA.