Number Of Tyrosine Hydroxylase-immunoreactive Neurons Research Articles

Expression of magnesium transporter SLC41A1 in the striatum of 6-hydroxydopamine-induced parkinsonian rats

ObjectivesThe aim of this study was to investigate the expression of the mammalian Mg2+ transporter solute carrier family 41, member 1 (SLC41A1) in the striatum (STR) of a 6-hydroxydopamine (6-OHDA)-induced rat model, and its response to magnesium before the degenerative process commenced. Materials and methodsA unilateral parkinsonian rat model was induced by injection of 6-OHDA into the right medial forebrain bundle. Some rats received MgSO4 (90 mg/kg/day, intraperitoneal injection) for 7 or 14 days starting from the day following the 6-OHDA injection. The extent of dopamine depletion was determined by assessing the numbers of tyrosine hydroxylase (TH)-immunoreactive neurons in the substantia nigra pars compacta (SNc) and the apomorphine (APO)-induced rotational behavior. The mRNA and protein expression of SLC41A1 in STR were evaluated by real-time RT-PCR and western blotting respectively. ResultsAPO-induced rotations increased significantly in the 6-OHDA lesioned rats compared with the controls, with MgSO4 administration significantly improving the behavior. The numbers of TH-immunoreactive neurons in SNc were significantly lower in the lesioned side than in the unlesioned side. Administration of MgSO4 for 14 days partly ameliorated the loss of TH-positive neurons. The mRNA and protein expressions of SLC41A1 in the lesioned STR were lower in the 6-OHDA lesioned rats than in the controls at both 7 and 14 days post-lesion induction. MgSO4 supplementation partly reversed the mRNA and protein expressions of SLC41A1. ConclusionThe regulation of SLC41A1 expression is responsive to magnesium in a 6-OHDA-induced rat model, wherein 6-OHDA may alter magnesium transport in the brain.

Aminochrome induces microglia and astrocyte activation

Aminochrome has been suggested as a more physiological preclinical model capable of inducing five of the six mechanisms of Parkinson's Disease (PD). Until now, there is no evidence that aminochrome induces glial activation related to neuroinflammation, an important mechanism involved in the loss of dopaminergic neurons. In this study, the potential role of aminochrome on glial activation was studied in primary mesencephalic neuron-glia cultures and microglial primary culture from Wistar rats. We demonstrated that aminochrome induced a reduction in the number of viable cells on cultures exposed to concentration between 10 and 100μM. Moreover, aminochrome induces neuronal death determined by Fluoro-jade B. Furthermore, we demonstrated that aminochrome induced reduction in the number of TH-immunoreactive neurons and reactive gliosis, featured by morphological changes in GFAP+ and Iba1+ cells, increase in the number of OX-42+ cells and increase in the number of NF-κB p50 immunoreactive cells. These results demonstrate aminochrome neuroinflammatory ability and support the hypothesis that it may be a better PD preclinical model to find new pharmacological treatment that stop the development of this disease.

Subcutaneous rotenone rat model of Parkinson's disease: Dose exploration study

Subcutaneous administration of rotenone has recently attracted attention because of its convenience, simplicity and efficacy in replicating features of Parkinson's disease (PD) in animal models. However, the wide range of doses reported in the literature makes it difficult to evaluate the effectiveness of this technique objectively. The aim of the present study was to identify the optimum dose of subcutaneous rotenone for establishing a model of PD. We injected male Wistar rats subcutaneously with one of three doses of rotenone (1.5, 2, or 2.5mg/kg) daily for 5 weeks. Rotenone caused a dose-dependent increase in α-synuclein in the substantia nigra. Furthermore, at 2 and 2.5mg/kg, rotenone caused a significant decrease in the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra, and dopamine in the striatum. However, mortality at 2.5mg/kg was 46.7%, compared with just 6.7% at 2mg/kg; the high mortality observed at 2.5mg/kg would limit its application. The 2mg/kg dose showed no detrimental effect on body weight after 5 weeks of daily injections. Furthermore, rats in the 2mg/kg group showed a longer latency to descend from a horizontal bar and a grid wall, decreased rearing, and shorter latency to fall from a rotarod than rats that received vehicle or saline. Mitochondrial damage, observed by transmission electron microscopy, was also evident at this dose. Together, our data indicate that daily subcutaneous injection of 2mg/kg rotenone in rats facilitates the formation of α-synuclein and reproduces the typical features of PD, while maintaining low mortality.

Verbascoside promotes the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra.

Tyrosine hydroxylase is a key enzyme in dopamine biosynthesis. Change in tyrosine hydroxylase expression in the nigrostriatal system is closely related to the occurrence and development of Parkinson's disease. Verbascoside, an extract from Radix Rehmanniae Praeparata has been shown to be clinically effective in treating Parkinson's disease. However, the underlying mechanisms remain unclear. It is hypothesized that the effects of verbascoside on Parkinson's disease are related to tyrosine hydroxylase expression change in the nigrostriatal system. Rat models of Parkinson's disease were established and verbascoside (60 mg/kg) was administered intraperitoneally once a day. After 6 weeks of verbascoside treatment, rat rotational behavior was alleviated; tyrosine hydroxylase mRNA and protein expression and the number of tyrosine hydroxylase-immunoreactive neurons in the rat right substantia nigra were significantly higher than the Parkinson's model group. These findings suggest that the mechanism by which verbascoside treats Parkinson's disease is related to the regeneration of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra.

Transcriptional and structural plasticity of tyrosine hydroxylase expressing neurons in both striatum and nucleus accumbens following dopaminergic denervation

Mice that express green fluorescent protein (GFP) under the control of tyrosine hydroxylase (TH) gene promoter were used to visualize transcriptional as well as structural regulation of TH cells following prolonged dopaminergic denervation.A unilateral lesion of the medial forebrain bundle was induced by 6-hydroxydopamine. In the unlesioned contralateral striatum and nucleus accumbens surprisingly high numbers of resident GFP-positive neurons (about 2653 and 422 per striatum and accumbens, respectively) were observed while only much lower TH-positive neurons (about 214 and 102 per striatum and accumbens, respectively) were detectable. In the lesioned hemisphere the number of GFP neurons was slightly increased already at day 4 by 16% and more at day 40 by 47% while the number of TH-immunoreactive neurons was dramatically increased by 848% at day 4 and by 1139% at day 40 over the control side. Additionally and particularly pronounced in the nucleus accumbens, GFP-positive neurons demonstrated increased sprouting of their projections over time, stronger than observed by TH-immunostaining.The load in TH protein may be essentially determined by post-transcriptional suppression/degradation while GFP may rather reflect the gross transcriptional activity. Thus, permanent dopaminergic pathway injury induces both transcriptional as well as structural plasticity of TH expressing neurons in striatal and accumbal target areas of ventral midbrain dopaminergic neurons.

Collapsin response mediator protein 4 affects the number of tyrosine hydroxylase‐immunoreactive neurons in the sexually dimorphic nucleus in female mice

In the sexually dimorphic anteroventral periventricular nucleus (AVPV) of the hypothalamus, females have a greater number of tyrosine hydroxylase-immunoreactive (TH-ir) and kisspeptin-immunoreactive (kisspeptin-ir) neurons than males. In this study, we used proteomics analysis and gene-deficient mice to identify proteins that regulate the number of TH-ir and kisspeptin-ir neurons in the AVPV. Analysis of protein expressions in the rat AVPV on postnatal day 1 (PD1; the early phase of sex differentiation) using two-dimensional fluorescence difference gel electrophoresis followed by MALDI-TOF-MS identified collapsin response mediator protein 4 (CRMP4) as a protein exhibiting sexually dimorphic expression. Interestingly, this sexually differential expressions of CRMP4 protein and mRNA in the AVPV was not detected on PD6. Prenatal testosterone exposure canceled the sexual difference in the expression of Crmp4 mRNA in the rat AVPV. Next, we used CRMP4-knockout (CRMP4-KO) mice to determine the in vivo function of CRMP4 in the AVPV. Crmp4 knockout did not change the number of kisspeptin-ir neurons in the adult AVPV in either sex. However, the number of TH-ir neurons was increased in the AVPV of adult female CRMP4-KO mice as compared with the adult female wild-type mice. During development, no significant difference in the number of TH-ir neurons was detected between sexes or genotypes on embryonic day 15, but a female-specific increase in TH-ir neurons was observed in CRMP4-KO mice on PD1, when the sex difference was not yet apparent in wild-type mice. These results indicate that CRMP4 regulates the number of TH-ir cell number in the female AVPV.

The Role of Autophagy Dysregulation in Manganese-Induced Dopaminergic Neurodegeneration

The etiological role of dysregulated autophagy in neurodegenerative diseases has been a subject of intense investigation. While manganese (Mn) is known to cause dopaminergic (DAergic) neurodegeneration, it has yet to be determined whether the dysregulation of autophagy plays a role in Mn-induced neuronal injury. In this study, we investigated the effect of Mn on autophagy in a rat model of manganism, a neurodegenerative disease associated with excessive exposure to Mn. After a single intrastriatal injection of Mn, the short- (4–12 h) and long-term (1–28 days) effect of Mn on DAergic neurons and autophagy were examined. Marked reduction in the number of TH-immunoreactive neurons in the substantia nigra pars compacta (SNpc) as well as TH protein expression, and a significant increase of apomorphine-induced rotations were observed in rats after Mn injection. Manganese also induced the down-regulation of dopamine levels and D1 dopamine receptor expression. In addition, autophagy was dysregulated and inhibited, as evidenced by increased number of abnormal lysosomes, decreased protein expression of Beclin1, and decreased ratio of microtubule-associated protein 1 light chain 3 (LC3) II over LC3 I, concomitant with activated mammalian target of rapamycin (mTOR)/p70 ribosomal protein S6 kinase (p70s6k) signaling. In contrast, in the early phase of Mn exposure, the level of autophagy was not be suppressed but compensatorily activated. Although the morphology of the DAergic neuron was intact in the early phase, Mn caused a significant decrease in TH-immunoreactivity and a significant increase in apomorphine-induced rotations in the presence of wortmannin, an inhibitor of autophagy. Taken together, these results demonstrate, for the first time, that autophagy may play a protective role against Mn-induced neuronal damage, whilst dysregulation of autophagy at later phases may mediate DAergic neurodegeneration.

Knockdown of tyrosine hydroxylase in the nucleus of the solitary tract (NTS) reduces elevated blood pressure during chronic intermittent hypoxia (CIH)

We tested the hypothesis that tyrosine hydroxylase (TH) knockdown in NTS reduces cardiovascular responses to CIH, a model of the arterial hypoxemia observed during sleep apnea. Adult male Sprague‐Dawley rats were implanted with radiotelemetry transmitters and AAV constructs with a GFP reporter having either short hairpin RNA for TH (shRNA) or scrambled virus (scrambled) were injected into caudal NTS. Virus injected rats were exposed to 7 days CIH (alternating 6 min periods of 10% O2 and 4 min of 21% O2 from 8am to 4pm; from 4pm to 8am rats were exposed to 21% O2). CIH increased MAP and HR during the day in both the scrambled (n= 14) and shRNA (n=13) groups (all p < 0.001). During the night MAP and HR remained elevated in the scrambled rats (both p<0.001) but not in the shRNA group. The number of TH‐immunoreactive neurons per section was reduced by 20% in sections with GFP fluorescence; shRNA 28±1 vs. scrambled 35±1 (p=.005) without altering the number of dopamine â‐hydroxylase (DBH) – immunoreactive neurons; shRNA 45±3 cells/section vs. scrambled 45±3 cells/section. Western blot analysis showed reductions in TH protein levels of 30% and 10% in caudal and sub‐postremal NTS respectively. Exposure to CIH increased MAP beyond the period of exposure to CIH. Knockdown of TH in the NTS reduces this persistent increase in MAP. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH. Supported by HL‐088052

Abstract 635: Knockdown Of Tyrosine Hydroxylase In The Nucleus Of The Solitary Tract (NTS) Reduces Persistent Increase In Blood Pressure During Chronic Intermittent Hypoxia (CIH)

A2 neurons in caudal NTS express tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, and respond to stressors such as hypoxia. We tested the hypothesis that TH knockdown in NTS reduces cardiovascular responses to CIH, an animal model that mimics the arterial hypoxemia observed during sleep apnea in humans. Adult male Sprague-Dawley rats were implanted with radiotelemetry transmitters and adeno-associated viral constructs (GeneDetect, Inc) with a GFP reporter having either short hair-pin RNA for TH (shRNA) or scrambled virus (scRNA) were injected into caudal NTS. A group of no injection, no CIH (control) rats were also used. Baseline mean arterial pressure (MAP) and heart rate (HR) were collected from all 3 groups for at least 4 days after which the virus injected rats were exposed to 7 days CIH (alternating 3 min periods of 10% O2 and 21% O2 from 8am to 4pm; from 4pm to 8am the rats were exposed to 21% O2). In shRNA injected rats (n=9) 7 day exposure to CIH increased MAP and HR during the day by 6.6±1.8 mmHg and 49.9±7.7 bpm and during the night by 0.3±1.8 mmHg and -4.5±1.3 bpm compared to baseline. In scRNA injected rats (n=10) 7 day exposure to CIH increased MAP and HR during the day by 7.7±1.2 mmHg and 65.3±7.9 bpm and during the night by 4.8±1.5 mmHg and 0.0±4.9 bpm compared to baseline. In control rats (n=8) MAP and HR were 1.5±1.0 mmHg and -8.4±3.8 bpm during the day and 1.8±0.9 mmHg and -10.8±2.9 bpm during the night compared to baseline. CIH induced significant changes in MAP and HR during the day in both the scRNA (p=.006 MAP; p<.001 HR) and shRNA (p=.007 MAP; p<.001 HR) groups. Compared to control rats, during the night CIH induced significant changes in MAP in the scRNA rats (p=0.02) but not in the shRNA group and no significant changes in HR in either group. The number of TH-immunoreactive neurons was reduced by 20% in sections with GFP fluorescence; shRNA 28±1 cells/section compared to scRNA 35±1 cells/section (p=.005). CIH induces an increase in MAP that persists beyond the period of exposure to CIH. Knockdown of TH in the NTS reduces this persistent increase in MAP induced by exposure to CIH. This indicates that NTS A2 neurons play a role in the cardiovascular responses to CIH and suggests they may play a similar role in the pathology of sleep apnea in humans.

Effects of cannabinoids Δ(9)-tetrahydrocannabinol, Δ(9)-tetrahydrocannabinolic acid and cannabidiol in MPP+ affected murine mesencephalic cultures

Cannabinoids derived from Cannabis sativa demonstrate neuroprotective properties in various cellular and animal models. Mitochondrial impairment and consecutive oxidative stress appear to be major molecular mechanisms of neurodegeneration. Therefore we studied some major cannabinoids, i.e. delta-9-tetrahydrocannabinolic acid (THCA), delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in mice mesencephalic cultures for their protective capacities against 1-methyl-4-phenyl pyridinium (MPP+) toxicity. MPP+ is an established model compound in the research of parkinsonism that acts as a complex I inhibitor of the mitochondrial respiratory chain, resulting in excessive radical formation and cell degeneration. MPP+ (10μM) was administered for 48h at the 9th DIV with or without concomitant cannabinoid treatment at concentrations ranging from 0.01 to 10μM.All cannabinoids exhibited in vitro antioxidative action ranging from 669±11.1 (THC), 16±3.2 (THCA) to 356±29.5 (CBD) μg Trolox (a vitamin E derivative)/mg substance in the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Cannabinoids were without effect on the morphology of dopaminergic cells stained by tyrosine hydroxylase (TH) immunoreaction. THC caused a dose-dependent increase of cell count up to 17.3% at 10μM, whereas CBD only had an effect at highest concentrations (decrease of cell count by 10.1–20% at concentrations of 0.01–10μM). It influenced the viability of the TH immunoreactive neurons significantly, whereas THCA exerts no influence on dopaminergic cell count.Exposure of cultures to 10μM of MPP+ for 48h significantly decreased the number of TH immunoreactive neurons by 44.7%, and shrunken cell bodies and reduced neurite lengths could be observed. Concomitant treatment of cultures with cannabinoids rescued dopaminergic cells. Compared to MPP+ treated cultures, THC counteracted toxic effects in a dose-dependent manner. THCA and CBD treatment at a concentration of 10μM lead to significantly increased cell counts to 123% and 117%, respectively. Even though no significant preservation or recovery of neurite outgrowth to control values could be observed, our data show that cannabinoids THC and THCA protect dopaminergic neurons against MPP+ induced cell death.

Neurorescue Effect of Rosmarinic Acid on 6-Hydroxydopamine-Lesioned Nigral Dopamine Neurons in Rat Model of Parkinson's Disease

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. It has been reported that RA possessed antioxidant and anti-inflammatory properties. Our previous study showed that RA could protect MES23.5 dopaminergic cells against 6-hydroxydopamine (6-OHDA)-induced neurotoxicity in vitro. The purpose of this study was to explore the neuroreparative (neurorescue) effect of RA on 6-OHDA-lesioned rat model of Parkinson's disease (PD) in vivo. In this study, the rats were given RA orally after intrastriatal 6-OHDA lesion. Results showed that the dopamine content in the striatum decreased and the numbers of tyrosine hydroxylase-immunoreactive neurons reduced after 6-OHDA treatment. RA treatment after 6-OHDA administration could restore these changes. Further studies demonstrated that 6-OHDA treatment increased the iron-staining positive cells, which were markedly decreased by RA treatment. Moreover, RA suppressed the increased ratio of Bax/Bcl-2 at gene level induced by 6-OHDA. This indicates that the neurorescue effects of RA against 6-ODHA-induced degeneration of the nigrostriatal dopaminergic system were achieved by decreasing nigral iron levels and regulating the ratio of Bcl-2/Bax gene expression.

The impact of maternal separation on the number of tyrosine hydroxylase-expressing midbrain neurons during different stages of ontogenesis

Early life stressors have life-long functional and anatomical consequences. Though many neurotransmitters are involved in the functional impact of early life stress, dopamine seems to be important because of its roles in motor control, adaptation to stressful conditions, mood, cognition, attention and reward. Thus, in the present study, we investigated the way that early life stress, in the form of maternal separation (MS), affects the populations of tyrosine hydroxylase-immunoreactive (TH-IR) dopaminergic neurons in rat midbrain structures during ontogenesis. We included in the study the sub-regions of the substantia nigra (SN) and the ventral tegmental area (VTA). In both the control and MS rats, we found that the estimated total number of TH-expressing neurons fluctuated during ontogenesis. Moreover, MS influenced the number of TH-IR cells, especially in the SN pars reticulata (SNr) and VTA. Shortly after the termination of MS, on postnatal day (PND) 15, a decrease in the estimated total number of TH-IR neurons was observed in the SNr and VTA (in both males and females). On PND 35, MS caused a transient increase in the number of TH-IR cells only in the SNr of female rats. On PND 70, MS affected the number of TH-IR neurons in the VTA of females; specifically, an increase in the number of these cells was observed. Additionally, MS did not alter TH-IR cell sizes or the total levels of TH (measured by Western blot analysis) in the SN and VTA for all stages of ontogenesis in both males and females. The results from the study herein indicate that early life stress has enduring effects on the populations of midbrain TH-expressing dopaminergic neurons (especially in female rats), which are critically important for dopamine-regulated brain function throughout ontogenesis.

Persistent MDMA‐induced dopaminergic neurotoxicity in the striatum and substantia nigra of mice

Acute administration of repeated doses of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) dramatically reduces striatal dopamine (DA) content, tyrosine hydroxylase (TH), and DA transporter-immunoreactivity in mice. In this study, we show for the first time the spatiotemporal pattern of dopaminergic damage and related molecular events produced by MDMA administration in mice. Our results include the novel finding that MDMA produces a significant decrease in the number of TH-immunoreactive neurons in the substantia nigra (SN). This decrease appears 1 day after injection, remains stable for at least 30 days, and is accompanied by a dose-dependent long-lasting decrease in TH- and DA transporter-immunoreactivity in the striatum, which peaked 1 day after treatment and persisted for at least 30 days, however, some recovery was evident from day 3 onwards, evidencing sprouting of TH fibers. No change is observed in the NAc indicating that MDMA causes selective destruction of DA-containing neurons in the nigrostriatal pathway, sparing the mesolimbic pathway. The expression of Mac-1 increased 1 day after MDMA treatment and glial fibrillary acidic protein increased 3 days post-treatment in the striatum and SN but not in the NAc, in strict anatomical correlation with dopaminergic damage. These data provide the first evidence that MDMA causes persistent loss of dopaminergic cell bodies in the SN.

Manganese Induces Dopaminergic Neurodegeneration via Microglial Activation in a Rat Model of Manganism

Manganese is an essential trace element required for normal development and bodily functions. However, exposure of the brain to excessive amounts of manganese results in neurotoxicity. Although previous studies examining manganese neurotoxicity have focused on neuronal injury, especially direct injury to dopaminergic neurons, the effects of manganese-induced neurotoxicity on glial cells have not been reported. The current study was designed to examine the effect of manganese on microglial activation, and the underlying mechanism of manganese-induced dopaminergic neuronal injury in vivo. We established an animal model of manganism by intrastriatal injection of MnCl(2).4H(2)O into male Sprague-Dawley rats. One day after administration of manganese, a few microglial cells in the substantia nigra (SN) were activated, although the number of tyrosine hydroxylase (TH)-immunoreactive neurons in the SN was unaffected. Seven days after administration of manganese, a marked reduction in the number of TH-immunoreactive neurons was observed in the SN, and the majority of microglial cells were activated. We found that manganese upregulated inducible nitric oxide synthase (iNOS) and tumor necrosis factor alpha (TNF-alpha) gene expression, as well as iNOS, TNF-alpha, and interleukin-1beta (IL-1beta) protein levels in the SN. Furthermore, treatment with minocycline, an inhibitor of microglial activation, attenuated microglial activation and mitigated IL-1beta, TNF-alpha, and iNOS production as well as dopaminergic neurotoxicity induced by manganese. These results suggested that dopaminergic neurons could be damaged by manganese neurotoxicity, and that the activated microglial cells and their associated activation products played an important role in this neurodegenerative process.

脂多糖预处理改善脂多糖诱导的中脑脑片神经元损伤

To identify the protective effect of lipopolysaccharide (LPS) preconditioning against LPS-induced inflammatory damage in dopaminergic neurons of midbrain slice culture and the possible mechanisms. After cultured in vitro for 14 d, the rat organotypic midbrain slices were pretreated with different concentrations (0, 1, 3, 6 or 10 ng/mL) of LPS for 24 h followed by treatment with 100 ng/mL LPS for 72 h. The whole slice viability was determined by measurement of the activity of lactic acid dehydrogenase (LDH). Tyrosine hydroxylase-immunoreactive (TH-IR) neurons and CD11b/c equivalent-immunoreactive (OX-42-IR) microglia in the slices were observed by immunohistochemical method, and tumor necrosis factor-alpha (TNF-alpha) levels in the culture media were detected by enzyme-linked immunosorbent assays (ELISA). In the slices treated with 100 ng/mL LPS for 72 h, the number of TH-IR neurons reduced from 191+/-12 in the control slices to 46+/-4, and the LDH activity elevated obviously (P < 0.01), along with remarkably increased number of OX-42-IR cells and production of TNF-alpha (P < 0.01). Preconditioning with 3 or 6 ng/mL LPS attenuated neuron loss (the number of TH-IR neurons increased to 126+/-12 and 180+/-13, respectively) and markedly reduced LDH levels (P < 0.05), accompanied by significant decreases of OX-42-IR microglia activation and TNF-alpha production (P < 0.05). Low-dose LPS preconditioning could protect dopaminergic neurons against inflammatory damage in rat midbrain slice culture, and inhibition of microglial activation and reduction of the proinflammatory factor TNF-alpha production may contribute to this protective effect. Further understanding the underlying mechanism of LPS preconditioning may open a new window for treatment of Parkinson's disease.

Reduced Immunoreactivity of Tyrosine Hydroxylase in the Hypothalamic Paraventricular Nucleus of the Seizure Sensitive Gerbil

We compared the immunoreactivity and numbers of tyrosine hydroxylase (TH) immunoreactive neurons and neuropil in the paraventricular nucleus (PVN) of the hypothalamus between the seizure sensitive (SS) and seizure resistant (SR) gerbils. The distributional pattern of TH immunoreactivity was similar in both groups: TH immunoreactivity was seen mainly in magnocellular neurons of the PVN. However, total TH immunoreactivity in the neurons and neuropil in the SS gerbils was significantly lower than that in the SR gerbils. In addition, the number of TH immunoreactive neurons in the SS gerbils was also much lower than those in the SR gerbils. These results indicate that SS gerbils have a low TH immunoreactivity in the hypothalamic PVN compared with that in SR gerbils.

Co-Expression of Tyrosine Hydroxylase and GTP Cyclohydrolase I in Arginine Vasopressin-Synthesizing Neurons of the Human Supraoptic Nucleus Demonstrated by Laser Microdissection and Real-Time PCR

Tyrosine hydroxylase (TH), the first and limiting enzyme for catecholamine synthesis, has been identified immunohistochemically (IHC) in human neurosecretory neurons where it is found to colocalize with vasopressin (AVP) or oxytocin. TH expression shows striking interindividual variability and appears to depend on neuronal activation. Since GTP cyclohydrolase I (GCHI), the first enzyme for tetrahydrobiopterin synthesis, the essential cofactor of TH, and aromatic L-amino acid decarboxylase (AADC) have so far not been detected in neurosecretory neurons, the functional role of TH in catecholamine synthesis is still questionable. Our purpose was to investigate in postmortem hypothalamus whether GCHI and AADC mRNAs are co-expressed with TH in human AVP-synthesizing neurons. Total RNA was extracted from laser microdissected TH-IHC-identified neurons as well as from dissected parts of the dorsolateral supraoptic nucleus (dl-SON) of 12 control subjects, i.e. without known neurological, psychiatric or endocrinological illness. GCHI, AADC and TH mRNA expression was determined by real-time PCR. Our results showed that GCHI mRNA is co-expressed with TH in almost all cases that had a considerable number of TH-immunoreactive (TH-IR) neurosecretory neurons. A positive correlation was found between TH-immunohistochemical intensity and the presence of GCHI mRNA. AADC mRNA expression was detected only in microdissected areas of dl-SON in 2 cases that showed an increased number of TH-IR neurons. The co-expression of GCHI with TH indicates that TH is indeed active in human neurosecretory neurons. The apparent limited expression of AADC indicates that dopamine might be produced in human neurosecretory neurons under activation of the hypothalamoneurohypophyseal system, although the possibility that L-dopa is the final product cannot be excluded.

Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration

Inflammation is implicated in the progressive nature of neurodegenerative diseases, such as Parkinson's disease, but the mechanisms are poorly understood. A single systemic lipopolysaccharide (LPS, 5 mg/kg, i.p.) or tumor necrosis factor alpha (TNFalpha, 0.25 mg/kg, i.p.) injection was administered in adult wild-type mice and in mice lacking TNFalpha receptors (TNF R1/R2(-/-)) to discern the mechanisms of inflammation transfer from the periphery to the brain and the neurodegenerative consequences. Systemic LPS administration resulted in rapid brain TNFalpha increase that remained elevated for 10 months, while peripheral TNFalpha (serum and liver) had subsided by 9 h (serum) and 1 week (liver). Systemic TNFalpha and LPS administration activated microglia and increased expression of brain pro-inflammatory factors (i.e., TNFalpha, MCP-1, IL-1beta, and NF-kappaB p65) in wild-type mice, but not in TNF R1/R2(-/-) mice. Further, LPS reduced the number of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra (SN) by 23% at 7-months post-treatment, which progressed to 47% at 10 months. Together, these data demonstrate that through TNFalpha, peripheral inflammation in adult animals can: (1) activate brain microglia to produce chronically elevated pro-inflammatory factors; (2) induce delayed and progressive loss of DA neurons in the SN. These findings provide valuable insight into the potential pathogenesis and self-propelling nature of Parkinson's disease.

Mesencephalic human neural progenitor cells transplanted into the neonatal hemiparkinsonian rat striatum differentiate into neurons and improve motor behaviour

Neural stem cell transplantation is a promising strategy for the treatment of neurodegenerative diseases. To evaluate the differentiation potential of human neural progenitor cells (hNPCs) as a prerequisite for clinical trials, we intracerebrally transplanted in vitro expanded fetal mesencephalic hNPCs into hemiparkinsonian rats. On postnatal day one (P1), 17 animals underwent a unilateral intraventricular 6-hydroxydopamine injection into the right lateral ventricle. At P3, animals (n = 10) received about 100,000 hNPCs (1 microL) in the right striatum. Five weeks after birth, animals underwent behaviour tests prior to fixation, followed by immunohistochemistry on brain slices for human nuclei, glial fibrillary acidic protein, S100beta, neuronal nuclei antigen, neuron-specific enolase and tyrosine hydroxylase. Compared with the apomorphine-induced rotations in the lesioned-only group (7.4 +/- 0.5 min(-1)), lesioned and successfully transplanted animals (0.3 +/- 0.1 min(-1)) showed a significant therapeutic improvement. Additionally, in the cylinder test, the lesioned-only animals preferred to use the ipsilateral forepaw. Conversely, the lesioned and transplanted animals showed no significant side bias similar to untreated control animals. Transplanted human nuclei-immunoreactive cells were found to survive and migrate up to 2000 microm into the host parenchyma, many containing the pan-neuronal markers neuronal nuclei antigen and neuron-specific enolase. In the striatum, tyrosine hydroxylase-immunoreactive somata were also found, indicating a dopaminergic differentiation capacity of transplanted hNPCs in vivo. However, the relative number of tyrosine hydroxylase-immunoreactive neurons in vivo seemed to be lower than in corresponding in vitro differentiation. To minimize donor tissue necessary for transplantation, further investigations will aim to enhance dopaminergic differentiation of transplanted cells in vivo.

Parkinson??s disease-like syndrome in rats induced by 2,9-dimethyl-??-carbolinium ion, a ??-carboline occurring in the human brain

Regarding the pathogenesis of Parkinson's disease, a neurotoxin hypothesis was proposed following the discovery that 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) produces a Parkinson-like syndrome in humans and primates. Since then, researchers have searched for endogenous and exogenous compounds that are structurally similar to this neurotoxin. Such compounds include beta-carbolines, formed from tryptophan and its derivatives. beta-carbolines are present naturally in the human brain and cerebrospinal fluid. The present study examined the effect of bilateral, intranigral administration of 2,9-dimethyl-beta-carbolinium ion on muscle tone, electromyographic activity, dopamine metabolism in the striatum, and the number of tyrosine hydroxylase-immunoreactive neurons and volume of the substantia nigra in rats. We found that the beta-carbolinium ion (15 or 40 nmol per side) caused a significant decrease in the striatal levels of dopamine and its metabolites, which was accompanied by an enhancement of muscle tone and electromyographic activity. Stereological counting revealed that the beta-carbolinium caused a significant decrease in the total number of tyrosine hydroxylase-immunoreactive neurons and shrinkage of the substantia nigra. The findings suggest that the methylated beta-carbolinium ion produces a dose-dependent degeneration of nigrostriatal neurons, leading to deficits in dopaminergic neurotransmission and an increase of muscle resistance and electromyographic activity, a syndrome equivalent to muscle rigidity in Parkinson's disease.