Tyrosine Hydroxylase-immunopositive Cells Research Articles

Endogenous orexin and hyperacute autonomic responses after resuscitation in a preclinical model of cardiac arrest.

The study of autonomic responses to cardiac arrest (CA) resuscitation deserves attention due to the impact of autonomic function on survival and arousal. Orexins are known to modulate autonomic function, but the role of endogenous orexin in hyperacute recovery of autonomic function post-resuscitation is not well understood. We hypothesized that endogenous orexin facilitates hyperacute cardiovascular sympathetic activity post-resuscitation, and this response could be attenuated by suvorexant, a dual orexin receptor antagonist. A well-established 7-min asphyxial CA rat model was studied. Heart rate (HR) and blood pressure were monitored from baseline to 90-min post-resuscitation. Autonomic function was evaluated by spectral analysis of HR variability, whereby the ratio of low- and high-frequency components (LF/HF ratio) represents the balance between sympathetic/parasympathetic activities. Plasma orexin-A levels and orexin receptors immunoreactivity in the rostral ventrolateral medulla (RVLM), the key central region for regulating sympathetic output, were measured post-resuscitation. Neurological outcome was assessed via neurologic-deficit score at 4-h post-resuscitation. A significant increase in HR was found over 25-40 min post-resuscitation (p < 0.01 vs. baseline), which was attenuated by suvorexant significantly (p < 0.05). Increased HR (from 15-to 25-min post-resuscitation) was correlated with better neurological outcomes (rs = 0.827, p = 0.005). There was no evident increase in mean arterial pressure over 25-40 min post-resuscitation, while systolic pressure was reduced greatly by suvorexant (p < 0.05). The LF/HF ratio was higher in animals with favorable outcomes than in animals injected with suvorexant over 30-40 min post-resuscitation (p < 0.05). Plasma orexin-A levels elevated at 15-min and peaked at 30-min post-resuscitation (p < 0.01 vs. baseline). Activated orexin receptors-immunoreactive neurons were found co-stained with tyrosine hydroxylase-immunopositive cells in the RVLM at 2-h post-resuscitation. Together, increased HR and elevated LF/HF ratio indicative of sympathetic arousal during a critical window (25-40 min) post-resuscitation are observed in animals with favorable outcomes. The orexin system appears to facilitate this hyperacute autonomic response post-CA.

Tyrosine Hydroxylase-Immunopositive Cells and Melanin in the Mesencephalon of Yugan Black-Bone Fowl.

Background:The Yugan blackbone fowl (YBF) is a special poultry with hyperpigmentation in various organs, including feather. However, the mechanism of hyperpigmentation is limited, and the melanic information of other organs except skin is rare.Aims and Objectives:In this study, we attempt to get an insight of the mechanism of melanogenesis of birds.Materials and Methods:The mesencephalon of YBF was observed by light microscopy with hematoxylineosin and tyrosine hydroxylase (TH) immunohistochemistry.Results:The TH immunopositive cells were found in the mesencephalon. Moreover, the melanin was also observed in the connective tissue of the mesencephalon.Conclusion:Our results confirmed the existence of melanin andTH immunopositive cells in the mesencephalon of YBF. These results provide a reference for further study on the mechanism of melanogenesis/hyperpigmentation in birds.

Methyl jasmonate ameliorates rotenone-induced motor deficits in rats through its neuroprotective activity and increased expression of tyrosine hydroxylase immunopositive cells.

Decreased tyrosine hydroxylase (TH) activity, due to degeneration of dopaminergic neurons contributes to the low dopamine content and the motor deficits that characterized Parkinson's disease (PD). This study examines the effect of methyl jasmonate (MJ), a neuroprotective bioactive compound isolated from jasminum grandiflorum, on motor functions, immunopositive cells of TH, dendritic neurons and dopamine contents in rotenone (Rot)-treated rats. Rats pretreated daily with MJ (100mg/kg, i.p) for 21days also received Rot (2.5mg/kg, i.p.) 30min after each pretreatment for every 48h for 21days. Motor functions were assessed on day 22. The specific brain regions of the rats were processed for determination of dopamine contents, immunopositive cells of TH, neuronal cell morphology and dendritic aborizations. Rot impaired locomotion and rearing behavior, and decreased dopamine content in the striatum, prefrontal cortex and midbrain. It further reduced the expression of TH in the substantia nigra and striatum relative to vehicle-control (p < 0.05). Histopathologic studies revealed that Rot-treated rats had degenerated neurons with pyknotic nuclei and loss of nigrostriatal neuronal cells. Rot also altered the nigrostriatal dendritic neuronal networks, decreased the dendritic length and spine density. However, pretreatment with MJ improved motor deficits, increased TH activity and dopamine contents in the specific brain regions of Rot-treated rats. MJ also attenuated the cyto-architectural distortions, loss of neuronal cells and dendritic aborizations of the striatum of Rot-treated rats. These findings suggest that MJ may reverse the motor deficits associated with PD by modifying the key pathological abnormalities involved in the disease progression.

Loss of Midbrain Dopamine Neurons and Altered Apomorphine EEG Effects in the 5xFAD Mouse Model of Alzheimer's Disease.

Cognitive malfunction, synaptic dysfunction, and disconnections in neural networks are core deficits in Alzheimer's disease (AD). 5xFAD mice, a transgenic model of AD, are characterized by an enhanced level of amyloid-β and abnormal neurotransmission. The dopaminergic (DA) system has been shown to be involved in amyloid-β transformations and neuronal plasticity; however, its role in functional network changes in familial AD still remains unclear. In 5xFAD and non-transgenic freely moving mice, electroencephalograms (EEGs) were simultaneously recorded from the secondary motor cortex (MC), superficial layers of the hippocampal CA1 area (HPC), substantia nigra (SN), and ventral tegmental area (VTA). EEGs and their frequency spectra were analyzed before and after systemic injection of a DA receptor agonist, apomorphine (APO). In the baseline EEG from MC and HPC of 5xFAD mice, delta and alpha oscillations were enhanced and beta activity was attenuated, compared to control mice. In VTA and SN of 5xFAD mice, delta-theta activity was decreased and beta oscillations dominated. In control mice, APO suppressed delta activity in VTA to a higher extent than in MC, whereas in 5xFAD mice, this difference was eliminated due to attenuation of the delta suppression in VTA. APO increased beta activity in MC of mice from both groups while significant beta suppression was observed in VTA of 5xFAD mice. These mice were characterized by significant decrease of tyrosine hydroxylase immunopositive cells in both VTA and SN and of DA transporter in MC and hippocampal dentate gyrus. We suggest that the EEG modifications observed in 5xFAD mice are associated with alterations in dopaminergic transmission, resulting in adaptive changes in the cerebral networks in the course of familial AD development.

The organization of tyrosine hydroxylase-immunopositive cells in the sparrow retina

The purpose of this study was to identify tyrosine hydroxylase-immunopositive (TH+) cells in the sparrow retina using immunocytochemistry and quantitative analysis. All TH+ cells were conventional amacrine cells. Based on dendritic morphology, at least two types were observed. The first type had a single thick primary process that descended from the cell body and many densely beaded processes in substrata (s) 1, less beaded processes in s3, and spiny processes in s4/5 of the inner plexiform layer. The dendrites of the second type appeared similar in each layer, but it displayed several primary processes that spread laterally away from the soma before descending to the inner plexiform layer. The average density of TH+ cells was 37.48 ± 1.97 cells/mm2 (mean ± standard deviation; n = 4), and the estimated total number of TH+ cells was 3,061.25 ± 192.79. The highest and lowest densities of TH+ cells were located in the central dorsotemporal retina and periphery of the ventronasal retina, respectively. TH+ cells did not express calbindin-D28 K, calretinin, or parvalbumin. These results suggest that all TH+ cells in specific amacrine cell subpopulations are involved in retinal information processing in both the ON and OFF sublaminae in sparrow retina.

TGF‐beta Signalling and Development of Mouse Midbrain Dopaminergic Neurons and Hindbrain Serotonergic Neurons

Tyrosine hydroxylase (TH) positive, midbrain dopaminergic neurons (mDA) and hindbrain serotonergic neurons (5‐HT) represent clinically important ventral neuronal subpopulations. Dysregulation of mDA has been linked to the development of depression and drug addiction, while their degeneration leads to the characteristic symptoms of Parkinson's disease. Dysfunction of hindbrain 5HT neurons has been associated with several neurological and psychiatric diseases. In previous studies we have shown that members of the transforming growth factor beta family are regulators of development and maintenance of mDA neurons. In this work we aim to understand the role that TGF‐beta signalling plays on the development of mDA and hindbrain 5HT neurons. For this purpose, we have generated a conditional knockout mouse model in which a region of the midbrain and hindbrain, including the mDA and rostral 5HT neurons, lacks the TGF‐beta Receptor II (TGFBR2). This model was generated using the Cre‐lox system by crossing the En1‐Cre line, in which the expression of the recombinase Cre is controlled by the Engrailed 1 promoter, and the TGFBR2‐flox line, in which the TGFBR2 gene is floxed. Through the use of immunohistochemistry on brain cryosections, and counts of TH immunopositive cells we found significant reduced numbers in both substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) of the mutants at E18 and even at E16. In contrast, at E14 comparable numbers of TH immunopositive mDA neurons in cKO and WT were obtained. Using in situ hybridization we have calculated the volume of Cck and Ahd2 expression at E14 and our results show dramatic loss of Cck and Ahd2 expression. Development of rostral and caudal 5‐HT subpopulations was also affected by loss of TGF‐beta signaling. The number of dorsal raphe, median raphe, and paramedian rahe 5‐HT neurons was significantly reduced in the conditional knock out, compared to the wildtype, accompanied by decreased expression of Pet and Gata2.In order to understand the molecular mechanism by which TGF‐beta acts in this process, we performed a cDNA microarray to analyse the transcriptome of the ventral midbrain of conditional knockout mice and compared it with that of wild type mice. Among the genes differentially regulated by the loss of the TGFBR2, we found genes that fall into the category of receptors, ion transporters, phosphatases and molecular chaperones, among others. Some of the differentially regulated genes have been shown to be implicated in processes like inflammation and endosomal sorting. Future analysis will be focused on understanding the function these genes have during the generation of the midbrain DA neurons, in order to shed some light onto the mechanism by which TGF‐beta acts on these process.Support or Funding InformationDFG

Somatic and neuritic spines on tyrosine hydroxylase-immunopositive cells of rat retina.

Dopamine- and tyrosine hydroxylase-immunopositive cells (TH cells) modulate visually driven signals as they flow through retinal photoreceptor, bipolar, and ganglion cells. Previous studies suggested that TH cells release dopamine from varicose axons arborizing in the inner and outer plexiform layers after glutamatergic synapses depolarize TH cell dendrites in the inner plexiform layer and these depolarizations propagate to the varicosities. Although it has been proposed that these excitatory synapses are formed onto appendages resembling dendritic spines, spines have not been found on TH cells of most species examined to date or on TH cell somata that release dopamine when exposed to glutamate receptor agonists. By use of protocols that preserve proximal retinal neuron morphology, we have examined the shape, distribution, and synapse-related immunoreactivity of adult rat TH cells. We report here that TH cell somata, tapering and varicose inner plexiform layer neurites, and varicose outer plexiform layer neurites all bear spines, that some of these spines are immunopositive for glutamate receptor and postsynaptic density proteins (viz., GluR1, GluR4, NR1, PSD-95, and PSD-93), that TH cell somata and tapering neurites are also immunopositive for a γ-aminobutyric acid (GABA) receptor subunit (GABAA Rα1 ), and that a synaptic ribbon-specific protein (RIBEYE) is found adjacent to some colocalizations of GluR1 and TH in the inner plexiform layer. These results identify previously undescribed sites at which glutamatergic and GABAergic inputs may stimulate and inhibit dopamine release, especially at somata and along varicose neurites that emerge from these somata and arborize in various levels of the retina. J. Comp. Neurol. 525:1707-1730, 2017. © 2016 Wiley Periodicals, Inc.

Gynostemma pentaphyllum Ethanolic Extract Protects Against Memory Deficits in an MPTP-Lesioned Mouse Model of Parkinson's Disease Treated with L-DOPA.

This study investigated the effects of ethanol extract from Gynostemma pentaphyllum (GP-EX) on memory deficits in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mouse model of Parkinson's disease (PD) (MPTP-lesioned mice). MPTP (30 mg/kg/day, 5 days)-lesioned mice showed deficits of habit learning memory and spatial memory, which were further aggravated by treatment with L-3,4-dihydroxyphenylalanine (L-DOPA) (25 mg/kg, 21 days). However, treatment with GP-EX (50 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice treated with L-DOPA (25 mg/kg): GP-EX prevented the decreases in retention latency time in the passive avoidance test and tyrosine hydroxylase-immunopositive cells and dopamine levels in the nigrostriatum. GP-EX also reduced increases in retention transfer latency time of the elevated plus-maze test and expression of N-methyl-D-aspartate (NMDA) receptor and improved decreases in phosphorylation of extracellular signal-regulated kinase (ERK1/2) and cyclic AMP-response element binding protein (CREB) in the hippocampus in the same models. By contrast, L-DOPA treatment (10 mg/kg, 21 days) ameliorated memory deficits in MPTP-lesioned mice, which were further improved by GP-EX treatment. These results suggest that GP-EX ameliorates habit learning memory deficits by activating dopaminergic neurons and spatial memory deficits by modulating NMDA receptor-ERK1/2-CREB system in MPTP-lesioned mice treated with L-DOPA. GP-EX may serve as an adjuvant phytonutrient for memory deficits in PD.

Effects of striatal transplantation of cells transfected with GDNF gene without pre- and pro-regions in mouse model of Parkinson's disease.

BackgroundPreviously, we have shown that transgenic cells bearing the GDNF gene with deleted pre- and pro-regions (mGDNF) can release transgenic GDNF. The medium conditioned by transgenic cells with mGDNF induced axonal growth in rat embryonic spinal ganglion in vitro. Here we demonstrate a neurotrophic effect of mGDNF on PC12 cells in vitro as well as its neuroprotective effect on dopaminergic neurons in the substantia nigra pars compacta in vivo as indicated by improved motor coordination and sleep-wakefulness cycle in the MPTP mouse model of Parkinson’s disease.ResultsHEK293 cells were transfected with a vector encoding an isoform of the human GDNF gene with deleted pre- and pro-regions (mGDNF). This factor in the medium conditioned by the transfected cells was shown to induce axonal growth in PC12 cells. The early Parkinson’s disease model was established by injection of the dopaminergic pro-neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into C57Bl/6 mice. Transgenic HEK293/mGDNF/GFP cells were transplanted into the striatum (caudate-putamen) of experimental mice. The sleep-wakefulness cycle was studied by continuous EEG and motor activity monitoring 1 and 2 weeks after MPTP injection. After the experiment, the motor coordination of experimental animals was evaluated in the rotarod test, and dopaminergic neurons in the substantia nigra pars compacta were counted in cross-sections of the midbrain. MPTP administration lowered the number of tyrosine hydroxylase immunopositive cells in the substantia nigra pars compacta, decreased motor coordination, and increased the total wake time during the dark period. The transplantation of HEK293/mGDNF cells into the caudate-putamen 3 days prior to MPTP injection smoothed these effects, while the control transplantation of HEK293 cells showed no notable impact.ConclusionsTransplantation of transgenic cells with the GDNF gene lacking the pre- and pro-sequences can protect dopaminergic neurons in the mouse midbrain from the subsequent administration of the pro-neurotoxin MPTP, which is confirmed by polysomnographic, behavioral and histochemical data. Hence it is released from transfected cells and preserves the differentiation activity and neuroprotective properties.

Cyperi Rhizoma inhibits the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced reduction in nigrostriatal dopaminergenic neurons in estrogen-deprived mice

Ethnopharmacological relevanceCyperi Rhizoma has commonly been used for the treatment of gynecological and neuropsychiatric disorders in traditional medicine. The aim of this study was to evaluate the estrogenic properties and neuroprotective effects of Cyperi Rhizoma under estrogen-deprived condition in female mice. Materials and methodsTo determine the estrogen-like effect of Cyperi Rhizoma extract (CRE), we measured luciferase expression after transfection of a promoter construct containing an estrogen response element (ERE) and treatment of CRE. To evaluate the neuroprotective effect of CRE, we measured striatal dopamine, movement ability, tyrosine hydroxylase (TH) immunoreactivity, and apoptosis-related protein expression levels after treatment of CRE either with or without 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in ovariectomized female mice. ResultsCRE significantly induced the luciferase expression driven by an ERE in PC12 cells, a dopaminergic cell line, in a dose-dependent manner. In mice, MPTP significantly decreased the levels of dopamine in the striatum and behavior performance; in contrast, both CRE and 17β-estradiol benzoate (EB) recovered these parameters to normal levels. CRE and EB treatment also recovered TH immunopositive fibers and cells, respectively, from MPTP toxicity. Additionally, MPTP significantly down-regulated Bcl-2 expression in the mitochondria of dopaminergic cells in the SN, followed by an increase in Bax expression, cytochrome C translocation to the cytosol, and cleaved-caspase-3 expression, whereas these were inhibited by CRE or EB treatment. ConclusionsThese findings provide the first evidence that CRE has estrogen-like and neuroprotective effects on dopaminergic neurons in estrogen-deprived mice treated with MPTP-toxin.

Effects of berberine on 6-hydroxydopamine-induced neurotoxicity in PC12 cells and a rat model of Parkinson's disease

Protoberberine isoquinoline alkaloids including berberine inhibit dopamine biosynthesis and aggravate l-DOPA-induced cytotoxicity in PC12 cells. In this study, the effects of berberine on 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in PC12 cells and on unilateral 6-OHDA-lesioned rats were investigated. In PC12 cells, berberine at 10 and 30 μM associated with 6-OHDA (10, 20, and 50 μM) enhanced cytotoxicity at 48 h compared to 6-OHDA alone, indicated by an increase in apoptotic cell death. In addition, treatment with berberine (5 and 30 mg/kg, i.p.) for 21 days in 6-OHDA-lesioned rats markedly depleted tyrosine hydroxylase-immunopositive cells in the substantia nigra as compared to berberine-untreated rats. Further, the levels of dopamine and norepinephrine were also significantly decreased by berberine administration (5 and 30 mg/kg) in the striatal regions of 6-OHDA-lesioned rats. These results suggested that berberine aggravated 6-OHDA-induced cytotoxicity in PC12 cells, and led to the degeneration of dopaminergic neuronal cells in the substantia nigra of 6-OHDA-lesioned rats. It is, therefore, suggested that the use of long-term l-DOPA therapy with isoquinoline derivatives including berberine may need to be examined for the presence of adverse symptoms.

Glutamate receptor properties of human mesencephalic neural progenitor cells: NMDA enhances dopaminergic neurogenesis in vitro

Human midbrain-derived neural progenitor cells (NPCs) may serve as a continuous source of dopaminergic neurons for the development of novel regenerative therapies in Parkinson's disease. However, the molecular and functional characteristics of glutamate receptors in human NPCs are largely unknown. Here, we show that differentiated human mesencepahlic NPCs display a distinct pattern of glutamate receptors. In whole-cell patch-clamp recordings, l-glutamate and NMDA elicited currents in 93% of NPCs after 3 weeks of differentiation in vitro. The concentration-response plots of differentiated NPCs yielded an EC(50) of 2.2 microM for glutamate and an EC(50) of 36 microM for NMDA. Glutamate-induced currents were markedly inhibited by memantine in contrast to 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) suggesting a higher density of functional NMDA than alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)/kainate receptors. NMDA-evoked currents and calcium signals were blocked by the NR2B-subunit specific antagonist ifenprodil indicating functional expression of NMDA receptors containing subunits NR1 and NR2B. In calcium imaging experiments, the blockade of voltage-gated calcium channels by verapamil abolished AMPA-induced calcium responses but only partially reduced NMDA-evoked transients suggesting the expression of calcium-impermeable, GluR2-containing AMPA receptors. Quantitative real-time PCR showed a predominant expression of subunits NR2A and NR2B (NMDA), GluR2 (AMPA), GluR7 (kainate), and mGluR3 (metabotropic glutamate receptor). Treatment of NPCs with 100 microM NMDA in vitro during proliferation (2 weeks) and differentiation (1 week) increased the amount of tyrosine hydroxylase-immunopositive cells significantly, which was reversed by addition of memantine. These data suggest that NMDA receptors in differentiating human mesencephalic NPCs are important regulators of dopaminergic neurogenesis in vitro.

Ziram Causes Dopaminergic Cell Damage by Inhibiting E1 Ligase of the Proteasome

The etiology of Parkinson disease (PD) is unclear but may involve environmental toxins such as pesticides leading to dysfunction of the ubiquitin proteasome system (UPS). Here, we measured the relative toxicity of ziram (a UPS inhibitor) and analogs to dopaminergic neurons and examined the mechanism of cell death. UPS (26 S) activity was measured in cell lines after exposure to ziram and related compounds. Dimethyl- and diethyldithiocarbamates including ziram were potent UPS inhibitors. Primary ventral mesencephalic cultures were exposed to ziram, and cell toxicity was assessed by staining for tyrosine hydroxylase (TH) and NeuN antigen. Ziram caused a preferential damage to TH+ neurons and elevated alpha-synuclein levels but did not increase aggregate formation. Mechanistically, ziram altered UPS function through interfering with the targeting of substrates by inhibiting ubiquitin E1 ligase. Sodium dimethyldithiocarbamate administered to mice for 2 weeks resulted in persistent motor deficits and a mild reduction in striatal TH staining but no nigral cell loss. These results demonstrate that ziram causes selective dopaminergic cell damage in vitro by inhibiting an important degradative pathway implicated in the etiology of PD. Chronic exposure to widely used dithiocarbamate fungicides may contribute to the development of PD, and elucidation of its mechanism would identify a new potential therapeutic target.

Neuroprotective effects of human mesenchymal stem cells on dopaminergic neurons through anti‐inflammatory action

Parkinson's disease (PD) is a common, progressive neurodegenerative disorder caused by the loss of dopaminergic neurons in the substantia nigra (SN). Numerous studies have provided evidence suggesting that neuroinflammation plays an important role in the pathogenesis of PD. In this study, we used lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models to investigate whether human mesenchymal stem cells (hMSCs) have a protective effect on the dopaminergic system through anti-inflammatory mechanisms. The hMSC treatment significantly decreased LPS-induced microglial activation, tumor necrosis factor (TNF)-alpha, inducible nitric oxide synthase (iNOS) mRNA expression, and production of NO and TNF-alpha compared with the LPS-only treatment group. In co-cultures of microglia and mesencephalic dopaminergic neurons, hMSC treatment significantly decreased the loss of tyrosine hydroxylase-immunopositive (TH-ip) cells. The hMSC treatment in rats showed that TH-ip neuronal loss induced by LPS stimulation in the SN was considerably decreased and was clearly accompanied by a decrease in activation of microglia, as well as TNF-alpha and iNOS mRNA expression and production of TNF-alpha. These data suggest that hMSCs have a neuroprotective effect on dopaminergic neurons through anti-inflammatory actions mediated by the modulation of microglial activation. Along with various trophic effects and trans-differentiational potency, the anti-inflammatory properties of MSCs could have major therapeutic implications in the treatment of PD.

Neuroprotective effect of nicotine on dopaminergic neurons by anti‐inflammatory action

Epidemiological studies have reported that smoking is associated with a lower incidence of Parkinson's disease (PD), leading to theories that smoking in general and nicotine in particular might be neuroprotective. Recent studies suggested cholinergic anti-inflammatory pathway-regulating microglial activation through alpha7 nicotinic receptors. In the present study, we used lipopolysaccharide (LPS)-induced in vitro and in vivo inflammation models to investigate whether nicotine has a protective effect on the dopaminergic system through an anti-inflammatory mechanism. Nicotine pretreatment considerably decreased microglial activation with significant reduction of tumour necrosis factor (TNF)-alpha mRNA expression and TNF-alpha release induced by LPS stimulation. In co-cultures of microglia and mesencephalic neurons, nicotine pretreatment significantly decreased the loss of tyrosine hydroxylase-immunopositive (TH-ip) cells, approximately twice more than the LPS-only treatment. alpha-Bungarotoxin, an alpha7 nicotinic acetylcholine receptor subunit-selective blocker, considerably blocked the inhibitory effects of nicotine on microglial activation and TH-ip neuronal loss. Chronic nicotine pretreatment in rats showed that TH-ip neuronal loss induced by LPS stimulation in the substantia nigra was dramatically decreased, which was clearly accompanied by a reduction in the formation of TNF-alpha. The present study demonstrated that nicotine has a neuroprotective effect on dopaminergic neurons via an anti-inflammatory mechanism mediated by the modulation of microglial activation. Along with various neuroprotective effects of nicotine, the anti-inflammatory mechanism of nicotine could have a major therapeutic implication in the preventive treatment of PD.

Specific alterations of tyrosine hydroxylase immunopositive cells in the retina of NT-4 knock out mice

To assess the effect of NT-4 deprivation on maturation of retinal circuitry, we investigated a mouse with targeted deletion of the gene encoding nt-4 ( nt-4 −/−). In particular, we studied neurons immunostained by an antibody recognizing tyrosine hydroxylase (TH), the rate limiting enzyme for dopamine (DA) synthesis. We found that TH immunopositive processes were altered in the retina of nt-4 −/−. Alteration of TH immunopositive processes in nt-4 −/− mice resulted in changes of DA turnover, as assessed by high-pressure liquid chromatography measurements. These findings suggest that retinal NT-4 plays a role in the morphological maturation of dopaminergic retinal cells.

Re‐examining the ontogeny of substantia nigra dopamine neurons

Recently, the need to detail the precise ontogeny of nigrostriatal dopamine neurons has grown significantly. It is now thought that the gestational day on which the majority of these neurons are born is important not only for maximizing the yield of primary cells for transplantation but also for extracting suitable dopamine neural precursors (as stem cells) for expansion in vitro. Historically, peak ontogeny of substantia nigra pars compacta (SNc) dopamine neurons in the rat has been considered to occur around embryonic day (E)14. However, such a concept is at odds with recent studies that reveal not only that substantial numbers of tyrosine hydroxylase-immunopositive cells reside in the ventral mesencephalic region of rats at E14 but that many of these cells have matured extensive axonal projections to the ventral forebrain. Here, then, the ontogeny of SNc neurons in rats commonly used as a source of donor tissue for experimental cell transplantation in animal models of Parkinson's disease has been re-examined. Using a combination of bromodeoxyuridine (BrdU) administration at E11, E12, E13 or E14 with immunocytochemical stainings for both BrdU and tyrosine hydroxylase after 4 weeks of postnatal development, this characterization reveals that the vast majority (perhaps 80%) of SNc dopamine neurons are probably born on E12 in Sprague-Dawley rats. Such findings are important in refining the use of embryonic tissues for primary cell transplantation and may provide more precise timing for identifying the cellular and molecular events that drive neural stem cells toward a dopaminergic phenotype during development.

Neuroprotective effects of metabotropic glutamate receptor ligands in a 6‐hydroxydopamine rodent model of Parkinson's disease

Increasing evidence implicates glutamate-mediated excitotoxicity as a contributory factor in dopaminergic cell death in the substantia nigra pars compacta (SNc) in Parkinson's disease (PD). Previous studies have suggested that metabotropic glutamate receptor (mGluR) ligands are neuroprotective against excitotoxicity in vitro. In the present study, the neurotoxin 6-hydroxydopamine (6-OHDA) produced a significant loss (61.2 +/- 8.9%; P < 0.01) of tyrosine hydroxylase-immunopositive (TH+) cells in both the SNc and striatal dopamine (58.02 +/- 1.27%; P < 0.05) in control male Sprague-Dawley rats. Both losses were significantly attenuated by sub-chronic (7 day) treatment with the Group I mGluR antagonists, 2-methyl-6(phenylethynyl)-pyridine (MPEP) or (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385); the Group II mGluR agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC); or the Group III mGluR agonist, L(+)-2-amino-4-phosphonobutyric acid (L-AP4). These data demonstrate a neuroprotective action of mGluR ligands in vivo against 6-OHDA toxicity that has important implications for the treatment of PD.

The regional and cellular expression profile of the melatonin receptor MT1 in the central dopaminergic system

The physiological effects of pineal melatonin are primarily mediated by melatonin receptors located in the brain and periphery. Even though there are a number of studies demonstrating the regulatory role of melatonin in the development of dopaminergic behaviors, such as psychostimulant-induced diurnal locomotor sensitization or drug seeking, little is known about the contribution of melatonin receptors (i.e., MT1) to this role. Therefore, as a first step in understanding the functional role of melatonin receptors in dopaminergic behaviors, we focused on determining the expression pattern of MT1 receptors in the dopaminergic system of the human and rodent brain. Regional (e.g., nucleus accumbens shell) and cellular (e.g., tyrosine hydroxylase immunopositive cells) expression of MT1 mRNA was characterized by applying the immuno-laser capture microdissection (immuno-LCM) technique coupled with nested RT-PCR. Moreover, employing quantitative Western immunoblotting and RT-PCR, we found that the mouse MT1 receptor expression presents diurnal variations (i.e., low mRNA and high protein levels at night, ZT21). The dopaminergic system-based presence of MT1 receptor proteins was not limited to rodents; we found these receptors in postmortem human brain as well. Further research is needed to understand the regional/cellular functional role of melatonin receptors in the regulation of dopaminergic behaviors, using models such as melatonin receptor knockout mice.

Postnatal Inorganic Lead Exposure Reduces Midbrain Dopaminergic Impulse Flow and Decreases Dopamine D1 Receptor Sensitivity in Nucleus Accumbens Neurons

Lead treatment via drinking water for 3 to 6 weeks at 250 ppm was found to significantly decrease the number of spontaneously active dopamine (DA) neurons in both the substantia nigra and ventral tegmental area that were recorded using standard extracellular electrophysiological recording techniques. Lead exposure did not affect the discharge rate or discharge pattern of these DA neurons. No significant decrease in the number of tyrosine hydroxylase immunopositive cells was detected in lead-treated animals relative to controls even though the length of lead exposure was extended beyond that of the electrophysiological studies. The significant lead-induced decrease in spontaneously active cells observed in the electrophysiological studies was, therefore, not due to cell death. An acute drug challenge with the DA receptor agonist apomorphine at a dose known to hyperpolarize midbrain DA neurons (50 mug/kg i.v.) was used to determine whether hyperpolarization would normalize the number of spontaneously active DA neurons. The results suggest that depolarization inactivation was most likely not the cause for this lead effect. The D(1) receptor agonist SKF-38393 [1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol] was iontophoretically applied to type I nucleus accumbens (Nacb) neurons. The results demonstrated that type I Nacb neurons have a significantly lower basal discharge rate in lead-treated animals relative to controls and that the Nacb DA D(1) receptors were significantly less sensitive to SKF-38393 in the lead-treated animals. Therefore, lead exposure decreases DA neuron impulse flow presynaptically and decreases DA D(1) receptor sensitivity postsynaptically in the nucleus accumbens.