Correction: GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

[This corrects the article DOI: 10.1371/journal.pgen.1005710.].

GDNF Overexpression from the Native Locus Reveals its Role in the Nigrostriatal Dopaminergic System Function.

Degeneration of nigrostriatal dopaminergic system is the principal lesion in Parkinson's disease. Because glial cell line-derived neurotrophic factor (GDNF) promotes survival of dopamine neurons in vitro and in vivo, intracranial delivery of GDNF has been attempted for Parkinson's disease treatment but with variable success. For improving GDNF-based therapies, knowledge on physiological role of endogenous GDNF at the sites of its expression is important. However, due to limitations of existing genetic model systems, such knowledge is scarce. Here, we report that prevention of transcription of Gdnf 3'UTR in Gdnf endogenous locus yields GDNF hypermorphic mice with increased, but spatially unchanged GDNF expression, enabling analysis of postnatal GDNF function. We found that increased level of GDNF in the central nervous system increases the number of adult dopamine neurons in the substantia nigra pars compacta and the number of dopaminergic terminals in the dorsal striatum. At the functional level, GDNF levels increased striatal tissue dopamine levels and augmented striatal dopamine release and re-uptake. In a proteasome inhibitor lactacystin-induced model of Parkinson's disease GDNF hypermorphic mice were protected from the reduction in striatal dopamine and failure of dopaminergic system function. Importantly, adverse phenotypic effects associated with spatially unregulated GDNF applications were not observed. Enhanced GDNF levels up-regulated striatal dopamine transporter activity by at least five fold resulting in enhanced susceptibility to 6-OHDA, a toxin transported into dopamine neurons by DAT. Further, we report how GDNF levels regulate kidney development and identify microRNAs miR-9, miR-96, miR-133, and miR-146a as negative regulators of GDNF expression via interaction with Gdnf 3'UTR in vitro. Our results reveal the role of GDNF in nigrostriatal dopamine system postnatal development and adult function, and highlight the importance of correct spatial expression of GDNF. Furthermore, our results suggest that 3'UTR targeting may constitute a useful tool in analyzing gene function.

Magnetic resonance imaging as a tool to image neuroinflammation in a rat model of Parkinson's disease--phagocyte influx to the brain is promoted by bilberry-enriched diet.

Neuroinflammation is a chronic event in neurodegenerative disorders. In the rat model of Parkinson's disease, including a striatal injection of the neurotoxin 6-hydroxydopamine (6-OHDA), antioxidant treatment affects the inflammatory process. Despite a heavy accumulation of microglia early after the injury, dopamine nerve fibre regeneration occurs. It remains unclear why this heavy accumulation of microglia is found early after the lesion in antioxidant-treated animals, or even more, what is the origin of these microglia. In this study magnetic resonance imaging (MRI) was used to elucidate whether the inflammatory response was generated from the blood or from activated brain microglia. Superparamagnetic iron oxide (SPIO) nanoparticles were injected intravenously prior to a striatal 6-OHDA injection to tag phagocytes in the blood. Rats were fed either with bilberry-enriched or control diet. T2*-weighted MRI scans were performed 1 week after the lesion, and hypointense areas were calculated from T2*-weighted images, to monitor the presence of SPIO particles. The results revealed that feeding the animals with bilberries significantly promoted accumulation of blood-derived immune cells. Gadolinium-enhanced MRI demonstrated no difference in leakage of the blood-brain barrier independent of diets. To conclude, bilberry-enriched diet promotes an influx of periphery-derived immune cells to the brain early after injury.

Vascular pathology of 20-month-old hypercholesterolemia mice in comparison to triple-transgenic and APPSwDI Alzheimer's disease mouse models.

Several studies have shown that elevated plasma cholesterol levels (i.e. hypercholesterolemia) serve as a risk factor for late-onset Alzheimer's disease (AD). However, it remains unclear how hypercholesterolemia may contribute to the onset and progression of AD pathology. In order to determine the role of hypercholesterolemia at various stages of AD, we evaluated the effects of high cholesterol diet (5% cholesterol) in wild-type (WT; C57BL6) and triple-transgenic AD (3xTg-AD; Psen1, APPSwe, tauB301L) mice at 7, 14, and 20 months. The transgenic APP-Swedish/Dutch/Iowa AD mouse model (APPSwDI) was used as a control since these animals are more pathologically-accelerated and are known to exhibit extensive plaque deposition and cerebral amyloid angiopathy. Here, we describe the effects of high cholesterol diet on: (1) cognitive function and stress, (2) AD-associated pathologies, (3) neuroinflammation, (4) blood­brain barrier disruption and ventricle size, and (5) vascular dysfunction. Our data show that high dietary cholesterol increases weight, slightly impairs cognitive function, promotes glial cell activation and complement-related pathways, enhances the infiltration of blood-derived proteins and alters vascular integrity, however, it does not induce AD-related pathologies. While normal-fed 3xTg-AD mice display a typical AD-like pathology in addition to severe cognitive impairment and neuroinflammation at 20 months of age, vascular alterations are less pronounced. No microbleedings were seen by MRI, however, the ventricle size was enlarged. Triple-transgenic AD mice, on the other hand, fed a high cholesterol diet do not survive past 14 months of age. Our data indicates that cholesterol does not markedly potentiate AD-related pathology, nor does it cause significant impairments in cognition. However, it appears that high cholesterol diet markedly increases stress-related plasma corticosterone levels as well as some vessel pathologies. Together, our findings represent the first demonstration of prolonged high cholesterol diet and the examination of its effects at various stages of cerebrovascular- and AD-related disease.

Dopamine release from serotonergic nerve fibers is reduced in L-DOPA-induced dyskinesia.

L-DOPA is the most commonly used treatment for symptomatic control in patients with Parkinson's disease. Unfortunately, most patients develop severe side-effects, such as dyskinesia, upon chronic l-DOPA treatment. The patophysiology of dyskinesia is unclear; however, involvement of serotonergic nerve fibers in converting l-DOPA to dopamine has been suggested. Therefore, potassium-evoked dopamine release was studied after local application of l-DOPA in the striata of normal, dopamine- and dopamine/serotonin-lesioned l-DOPA naïve, and dopamine-denervated chronically l-DOPA-treated dyskinetic rats using in vivo chronoamperometry. The results revealed that local l-DOPA administration into normal and intact hemisphere of dopamine-lesioned l-DOPA naïve animals significantly increased the potassium-evoked dopamine release. l-DOPA application also increased the dopamine peak amplitude in the dopamine-depleted l-DOPA naïve striatum, although these dopamine levels were several-folds lower than in the normal striatum, whereas no increased dopamine release was found in the dopamine/serotonin-denervated striatum. In dyskinetic animals, local l-DOPA application did not affect the dopamine release, resulting in significantly attenuated dopamine levels compared with those measured in l-DOPA naïve dopamine-denervated striatum. To conclude, l-DOPA is most likely converted to dopamine in serotonergic nerve fibers in the dopamine-depleted striatum, but the dopamine release is several-fold lower than in normal striatum. Furthermore, l-DOPA loading does not increase the dopamine release in dyskinetic animals as found in l-DOPA naïve animals, despite similar density of serotonergic innervation. Thus, the dopamine overflow produced from the serotonergic nerve fibers appears not to be the major cause of dyskinetic behavior.

Chronic intermittent L-DOPA treatment induces changes in dopamine release.

3,4-Dihydroxyphenyl-l-alanine (l-DOPA)-induced dyskinesia often develops as a side effect of chronic l-DOPA therapy. This study was undertaken to investigate dopamine (DA) release upon l-DOPA treatment. Chronoamperometric measurements were performed in unilaterally DA-depleted rats, chronically treated with l-DOPA, resulting in dyskinetic and non-dyskinetic animals. Normal and lesioned l-DOPA naïve animals were used as controls. Potassium-evoked DA releases were significantly reduced in intact sides of animals undertaken chronic l-DOPA treatment, independent on dyskinetic behavior. Acute l-DOPA further attenuated the amplitude of the DA release in the control sides. In DA-depleted striata, no difference was found in potassium-evoked DA releases, and acute l-DOPA did not affect the amplitude. While immunoreactivity to serotonin uptake transporter was higher in lesioned striata of animals displaying dyskinetic behavior, no correlation could be documented between serotonin transporter-positive nerve fiber density and the amplitude of released DA. In conclusions, the amplitude of potassium-evoked DA release is attenuated in intact striatum after chronic intermittent l-DOPA treatment. No change in amplitude was found in DA-denervated sides of either dyskinetic or non-dyskinetic animals, while release kinetics were changed. This indicates the importance of studying DA release dynamics for the understanding of both beneficial and adverse effects of l-DOPA replacement therapy.

Noradrenaline is crucial for the substantia nigra dopaminergic cell maintenance.

In Parkinson's disease, degeneration of substantia nigra dopaminergic neurons is accompanied by damage on other neuronal systems. A severe denervation is for example seen in the locus coerulean noradrenergic system. Little is known about the relation between noradrenergic and dopaminergic degeneration, and the effects of noradrenergic denervation on the function of the dopaminergic neurons of substantia nigra are not fully understood. In this study, N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4) was injected in rats, whereafter behavior, striatal KCl-evoked dopamine and glutamate releases, and immunohistochemistry were monitored at 3 days, 3 months, and 6 months. Quantification of dopamine-beta-hydroxylase-immunoreactive nerve fiber density in the cortex revealed a tendency towards nerve fiber regeneration at 6 months. To sustain a stable noradrenergic denervation throughout the experimental timeline, the animals in the 6-month time point received an additional DSP4 injection (2 months after the first injection). Behavioral examinations utilizing rotarod revealed that DSP4 reduced the time spent on the rotarod at 3 but not at 6 months. KCl-evoked dopamine release was significantly increased at 3 days and 3 months, while the concentrations were normalized at 6 months. DSP4 treatment prolonged both time for onset and reuptake of dopamine release over time. The dopamine degeneration was confirmed by unbiased stereology, demonstrating significant loss of tyrosine hydroxylase-immunoreactive neurons in the substantia nigra. Furthermore, striatal glutamate release was decreased after DSP4. In regards of neuroinflammation, reactive microglia were found over the substantia nigra after DSP4 treatment. In conclusion, long-term noradrenergic denervation reduces the number of dopaminergic neurons in the substantia nigra and affects the functionality of the nigrostriatal system. Thus, locus coeruleus is important for maintenance of nigral dopaminergic neurons.

Dual effects of TNFalpha on nerve fiber formation from ventral mesencephalic organotypic tissue cultures.

Tumor necrosis factor alpha (TNFalpha) is toxic to dopamine neurons and increased levels of TNFalpha are observed in Parkinson's disease. Dopamine nerve fiber outgrowth in organotypic cultures of fetal ventral mesencephalon occurs in two waves. The early appearing nerve fibers are formed in the absence of astroglia, while migrating astrocytes guide the late appearing dopamine nerve fibers. TNFalpha (40 ng/ml) was added to the medium of organotypic ventral mesencephalic tissue cultures between days 4-7 and 11-14. The cultures were evaluated at days 7 or 19 to study the effects of TNFalpha on both types of nerve fiber formation. Tyrosine hydroxylase (TH)-immunohistochemistry demonstrated that the number of cultures showing non-glial-guided TH-positive outgrowth was reduced compared to controls, when TNFalpha was added at day 4. By contrast, the glial-guided TH-positive nerve fiber outgrowth and the astrocytic migration reached significantly longer distances by early TNFalpha treatment. Ki67-immunohistochemistry revealed that TNFalpha did not affect proliferation of astrocytes. Treatment with TNFalpha and antibodies against TNFalpha receptor 1 between days 4 and 7 revealed that the non-glial-guided TH-positive outgrowth reappeared. TNFalpha treatment between days 11 and 14 triggered neither the TH-positive glial-guided outgrowth, nor promoted the astrocytic migration to reach longer distances. The number of microglia was significantly increased after the late but not early TNFalpha treatment. In conclusion, TNFalpha is toxic for the non-glial dopaminergic nerve fiber outgrowth but stimulates the glial-guided outgrowth and the migration of astrocytes at an early time point. TNFalpha increased the number of microglia in VM tissue cultures after late but not after early treatment.

Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures.

Tyrosine hydroxylase-positive nerve fiber formation occurs in two diverse morphological patterns in rat fetal ventral mesencephalic slice cultures; one is non-glial-associated and the other is glial-associated. The aim of this study was to characterize the non-glial-associated nerve fibers and its relation to migration of astrocytes. Organotypic slice cultures were prepared from embryonic days 12, 14, and 18 rat fetuses and maintained for 5, 7 or 14 days in vitro. Inhibition of cell proliferation using cytosine beta-D-arabinofuranoside was conducted in embryonic day 14 ventral mesencephalic cultures. The treatment impaired astrocytic migration at 7 and 14 days in vitro. The reduced migration of astrocytes exerted a negative effect on the glial-associated tyrosine hydroxylase-positive nerve fibers, reducing the outgrowth from the tissue slice. The non-glial-associated outgrowth was, however, positively affected by reduced astrocytic migration, reaching distances around 3mm in 2 weeks, and remained for longer time in culture. Co-cultures of fetal ventral mesencephalon and frontal cortex revealed the cortex as a target for the non-glial-associated tyrosine hydroxylase-positive outgrowth. The age of the fetal tissue at plating affected the astrocytes such that older tissue increased the length of astrocyte migration. Younger tissue at plating promoted the presence of non-glial-associated outgrowth and long radial-glia-like processes, while older tissue promoted migration of neurons instead of formation of nerve fiber network. In conclusion, inhibition of astrocytic proliferation promotes the persistence of long-distance growing tyrosine hydroxylase-positive nerve fibers in ventral mesencephalic slices cultures. Furthermore, the long-distance growing nerve fibers target the frontal cortex and are absent in cultures derived from older tissue.

Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures.

Rat fetal ventral mesencephalic organotypic cultures have demonstrated two morphologically different dopamine nerve fiber growth patterns, in which the initial nerve fibers are formed in the absence of astrocytes and the second wave is guided by astrocytes. In this study, the presence of subpopulations of dopamine neurons, other neuronal populations, and glial cells was determined. We used "roller-drum" organotypic cultures, and the results revealed that beta-tubulin-positive/tyrosine hydroxylase (TH)-negative nerve fibers were present as early as 1 day in vitro (DIV). A similar growth pattern produced by TH-positive neurons was present from 2 DIV. These neurites grew to reach distances over 4 mm and over time appeared to be degenerating. Thin, vimentin-positive processes were found among these nerve fibers. As the first growth was retracted, a second outgrowth was initiated and formed on migrating astrocytes. TH- and aldehyde dehydrogenase-1 (ALDH1)-positive nerve fibers formed both the nonglia-associated and the glia-associated outgrowth. In cultures with membrane inserts, only the glia-associated outgrowth was found. Vimentin-positive cells preceded migration of NG2-positive oligodendrocytes and Iba-1-positive microglia. Oligodendrocytes appeared not to be involved in guiding neuritic growth, but microglia was absent over areas dense with TH-positive neurons. In conclusion, in "roller-drum" cultures, nerve fibers are generally formed in two sequences. The early-formed nerve fibers grow in the presence of thin, vimentin-positive processes. The second nerve fiber outgrowth is formed on astroglia, with no correlation to the presence of oligodendrocytes or microglia. ALDH1-positive nerve fibers, presumably derived from A9 dopamine neurons, participate in formation of both sequences of outgrowth.

Effects of glial cell line-derived neurotrophic factor deletion on ventral mesencephalic organotypic tissue cultures.

Glial cell line-derived neurotrophic factor (GDNF) is potent for survival and promotion of nerve fibers from midbrain dopamine neurons. It is also known to exert different effects on specific subpopulations of dopamine neurons. In organotypic tissue cultures, dopamine neurons form two diverse nerve fiber growth patterns, targeting the striatum differently. The aim of this study was to investigate the effect of GDNF on the formation of dopamine nerve fibers. Organotypic tissue cultures of ventral mesencephalon of gdnf gene-deleted mice were studied. The results revealed that dopamine neurons survive in the absence of GDNF. Tyrosine hydroxylase immunoreactivity demonstrated, in gdnf knockout and wildtype cultures, nerve fiber formation with two separate morphologies occurring either in the absence or the presence of astrocytes. The outgrowth that occurred in the absence of astrocytes was unaffected by gdnf deletion, whereas nerve fibers guided by the presence of astrocytes were affected in that they reached significantly shorter distances from the gdnf gene-deleted tissue slice, compared to those measured in wildtype cultures. Treatment with GDNF reversed this effect and increased nerve fiber density independent of genotype. Furthermore, migration of astrocytes reached significantly shorter distances from the tissue slice in GDNF knockout compared to wildtype cultures. Exogenous GDNF increased astrocytic migration in gdnf gene-deleted tissue cultures, comparable to lengths observed in wildtype tissue cultures. In conclusion, cultured midbrain dopamine neurons survive in the absence of GDNF, and the addition of GDNF improved dopamine nerve fiber formation - possibly as an indirect effect of astrocytic stimulation.

Embryonic and mature astrocytes exert different effects on neuronal growth in rat ventral mesencephalic slice cultures.

One obstacle with grafting of dopamine neurons in Parkinson's disease is the insufficient ability of the transplant to reinnervate the host striatum. Another issue is the prospective interaction between the donor fetal tissue and the adult astrocytes of the host. To study nerve fiber growth and its interaction with immature/mature astrocytes, ventral mesencephalic (VM) organotypic rat tissue cultures from embryonic days (E) 12, E14, and E18 were studied up to 35 days in vitro (DIV), and co-cultures of E14 VM tissue and mature green fluorescent protein (GFP)-positive astrocytes were performed. Generally, nerve fibers grew from the tissue slice either in association with a monolayer of migrated astroglia surrounding the tissue (glial-associated), or distal to the astroglia as non-glial-associated outgrowth. The tyrosine hydroxylase (TH)-positive glial-associated nerve fiber outgrowth reached a plateau at 21 DIV in E12 and E14 cultures. In E18 cultures, TH-positive neurons displayed short processes and migrated onto the astrocytes. While the non-glial-associated nerve fiber outgrowth dominated the E14 cultures, it was found absent in E18 cultures. The GFP-positive cells in the VM and GFP-positive astrocyte co-cultures were generally located distal to the monolayer of migrated fetal astrocytes, a few GFP-positive cells were however observed within the astrocytic monolayer. In those cases TH-positive neurons migrated towards the GFP-positive cells. Both the non-glial- and glial-associated nerve fibers grew onto the GFP-positive cells. Taken together, the glial-associated growth has limited outgrowth compared to the non-glial-associated nerve fibers, while none of the outgrowth types were hampered by the mature astrocytes.

Single injection of small-molecule amyloid accelerator results in cell death of nigral dopamine neurons in mice.

The assembly process of α-synuclein toward amyloid fibers is linked to neurodegeneration in Parkinson's disease. In the present study, we capitalized on the in vitro discovery of a small-molecule accelerator of α-synuclein amyloid formation and assessed its effects when injected in brains of normal mice. An accelerator and an inhibitor of α-synuclein amyloid formation, as well as vehicle only, were injected into the striatum of normal mice and followed by behavioral evaluation, immunohistochemistry, and metabolomics up to six months later. The effects of molecules injected into the substantia nigra of normal and α-synuclein knock-out mice were also analyzed. When accelerator or inhibitor was injected into the brain of normal mice no acute compound toxicity was found. However, 6 months after single striatal injection of accelerator, mice sensorimotor functions were impaired, whereas mice injected with inhibitor had no dysfunctions. Injection of accelerator (but not inhibitor or vehicle) into the substantia nigra revealed significant loss of tyrosine hydroxylase (TH)-positive neurons after 3 months. No loss of TH-positive neurons was found in α-synuclein knock-out mice injected with accelerator into the substantia nigra. Metabolic serum profiles from accelerator-injected normal mice matched those of newly diagnosed Parkinson's disease patients, whereas the profiles from inhibitor-injected normal mice matched controls. Single inoculation of a small-molecule amyloid accelerator may be a new approach for studies of early events during dopamine neurodegeneration in mice.

Guidance of dopaminergic neuritic growth by immature astrocytes in organotypic cultures of rat fetal ventral mesencephalon.

Astrocytes, with their many functions in producing and controlling the environment in the brain, are of great interest when it comes to studying regeneration after injury and neurodegenerative diseases such as in grafting in Parkinson's disease. This study was performed to investigate astrocytic guidance of growth derived from dopaminergic neurons using organotypic cultures of rat fetal ventral mesencephalon. Primary cultures were studied at different time points starting from 3 days up to 28 days. Cultures were treated with either interleukin-1 beta (IL-1 beta), which has stimulating effects on astrocytic proliferation, or the astrocytic inhibitor cytosine arabinoside (Ara-C). Tyrosine hydroxylase (TH)-immunohistochemistry was used to visualize dopaminergic neurons, and antibodies against glial fibrillary acidic protein (GFAP) and S100 beta were used to label astrocytes. The results revealed that a robust TH-positive nerve fiber production was seen already at 3 days in vitro. These neurites had disappeared by 5 days. This early nerve fiber outgrowth was not guided by direct interactions with glial cells. Later, at 7 days in vitro, a second wave of TH-positive neuritic outgrowth was clearly observed. GFAP-positive astrocytic processes guided these neurites. TH-positive neurites arborized overlying S100 beta-positive astrocytes in an area distal to the GFAP-positive astrocytic processes. Treatment with IL-1 beta resulted in an increased area of TH-positive nerve fiber network. In cultures treated with Ara-C, neither astrocytes nor outgrowth of dopaminergic neurites were observed. In conclusion, this study shows that astrocytes play a major role in long-term dopaminergic outgrowth, both in axonal elongation and branching of neurites. The long-term nerve fiber growth is preceded by an early transient outgrowth of dopamine neurites.

Fetal lateral ganglionic eminence attracts one of two morphologically different types of tyrosine hydroxylase-positive nerve fibers formed by cultured ventral mesencephalon.

The purpose of this study was to investigate the influence of fetal lateral ganglionic eminence (LGE) on nerve fiber outgrowth formed by fetal ventral mesencephalon (VM). Organotypic tissue cultures of fetal VM and LGE plated as single or cocultures were employed. Survival time was 3-21 days in vitro. Nerve fiber outgrowth and migration of astrocytes were analyzed using immunohistochemistry for tyrosine hydroxylase (TH) and S100. In addition, cultures were labeled with the TUNEL technique and with antibodies directed against neurofilament (NF) in order to study apoptosis and retraction of nerve fibers, respectively. The results revealed two morphologically different types of TH-positive outgrowth growing into the substrate. The initially formed TH-positive outgrowth radiated continuously without changing direction, while a second wave of TH-positive outgrowth became obvious when the initial growth already had reached a distance of approximately 1000 microm. The second wave of TH-positive outgrowth radiated from the tissue, but at a certain distance changed direction and formed a network surrounding the culture. The initially formed TH-positive growth was not associated with the presence of S100-positive astrocytes and avoided to grow into the LGE. At longer time points the first wave of TH-positive nerve fibers appeared dotted, with disrupted NF-immunoreactive fibers and in most cultures these long distance growing fibers had disappeared at 21 days in vitro. The second wave of TH-positive nerve fibers was growing onto a layer of glia and never reached the distance of the first wave. LGE became innervated by TH-positive fibers at the time point for when the second wave of TH-positive growth had been initiated, and the innervation appeared in TH-dense patches that also showed a high density of S100-positive astrocytes. Significantly increased TUNEL activity within LGE portion of cocultures was observed when TH-positive fibers entered the LGE and formed patches. In conclusion, two morphologically different types of TH-positive outgrowth were found and the initially formed fibers neither targeted the LGE nor were they guided by glial cells, but their potential to grow for long distances was high.

c-fos antisense oligonucleotides increase firing rate of striatal neurons in the anaesthetized rat.

Extracellular electrophysiological recordings were used to study the firing rate of striatal neurons before and up to 4 h after intrastriatal c-fos antisense oligonucleotide injections in urethane-anesthetized rats. A four-fold increase from baseline neuronal activity was observed between 1 and 3 h upon antisense treatment, but not after control oligonucleotide injections. We conclude that, under urethane anesthesia, which here does not affect c-fos expression in the striatum by itself, neuronal activity appears to be tonically suppressed by basal striatal c-fos expression.

Generation of tyrosine hydroxylase-immunoreactive neurons in ventral mesencephalic tissue of Nurr1 deficient mice.

Nurr1 is an orphan nuclear receptor belonging to the family of evolutionary conserved steroid/thyroid hormone receptors. It has been shown that Nurr1 is required for development of ventral mesencephalic dopaminergic cells in vivo and that Nurr1 regulates the tyrosine hydroxylase (TH) gene. The aim of this study was to investigate the possibility of finding ventral mesencephalic TH-positive neurons in Nurr1 deficient tissue when developed in the presence of wild type (WT) striatum. Therefore, fetal ventral mesencephalic tissue from embryonic day (E) 9.5-10.5 fetuses from Nurr1 mutant mice was co-cultured with lateral ganglionic eminence (LGE) from WT fetuses using the 'roller-drum' culture technique. TH-immunohistochemistry revealed similar number of positive neurons in WT, heterozygous, and Nurr1 deficient tissue, respectively. When ventral mesencephalon, dissected from E10.5 fetuses, was cultured alone without the presence of LGE, significantly more TH-immunoreactive neurons were found in WT and Nurr1 +/- than that seen in Nurr1 -/- cultures. In single ventral mesencephalic cultures dissected from E15.5, TH-positive neurons were found in all tissue cultures derived from knockout animals. Interestingly, the formation of TH-positive nerve fiber bundles was obvious in WT cultures while not observed in cultures of knockout tissue. When ventral mesencephalon was cultured alone in serum-free medium, almost no TH-positive neurons were found in cultures of knockout tissue. The addition of the growth factors epidermal growth factor and fibroblast growth factor-8 did not induce TH-immunoreactivity in serum-free Nurr1 -/- tissue cultures. In conclusion, TH-positive neurons may be generated in ventral mesencephalic tissue of Nurr1 deficient mice, suggesting that Nurr1 is not required for TH gene expression in ventral midbrain in vitro.

Magnetic resonance imaging (MRI) to study striatal iron accumulation in a rat model of Parkinson's disease.

Abnormal accumulation of iron is observed in neurodegenerative disorders. In Parkinson's disease, an excess of iron has been demonstrated in different structures of the basal ganglia and is suggested to be involved in the pathogenesis of the disease. Using the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease, the edematous effect of 6-OHDA and its relation with striatal iron accumulation was examined utilizing in vivo magnetic resonance imaging (MRI). The results revealed that in comparison with control animals, injection of 6-OHDA into the rat striatum provoked an edematous process, visible in T2-weighted images that was accompanied by an accumulation of iron clearly detectable in T2*-weighted images. Furthermore, Prussian blue staining to detect iron in sectioned brains confirmed the existence of accumulated iron in the areas of T2* hypointensities. The presence of ED1-positive microglia in the lesioned striatum overlapped with this accumulation of iron, indicating areas of toxicity and loss of dopamine nerve fibers. Correlation analyses demonstrated a direct relation between the hyperintensities caused by the edema and the hypointensities caused by the accumulation of iron.

Degradation of proteoglycans affects astrocytes and neurite formation in organotypic tissue cultures.

Chondroitin sulfate proteoglycans (CSPGs) promote nerve growth during development, and inhibit axonal growth in the adult CNS after injury. Chondroitinase ABC (ChABC) and methyl-umbelliferyl-ß-d-xyloside (ß-xyloside), two compounds that degrade CSPGs, promote regeneration after injury, however, they demonstrate opposing results in tissue culture. To elucidate the effect of the two compounds, organotypic tissue cultures, treated with ChABC or ß-xyloside, were employed to monitor nerve fiber outgrowth and astrocytic migration. Rat ventral mesencephalon (VM) and spinal cord (SC) from embryonic day (E) 14 and E18 were treated early, from the plating day for 14 days in vitro, or late where treatment was initiated after being cultured for 14 days. In the early treatment of E14 VM and SC cultures, astrocytic migration and nerve fiber outgrowth were hampered using both compounds. Early treatment of E18 cultures reduced the astrocytic migration, while nerve growth was promoted by ß-xyloside, but not by ChABC. In the late treated cultures of both E14 and E18 cultures, no differences in distances that astrocytes migrated or nerve fiber growth were observed. However, in ß-xyloside-treated cultures, the confluency of astrocytic monolayer was disrupted. In E18 cultures both early and late treatments, neuronal migration was present in control cultures, which was preserved using ChABC but not ß-xyloside. In conclusion, ChABC and ß-xyloside had similar effects and hampered nerve fiber growth and astrocytic migration in E14 cultures. In E18 cultures nerve fiber growth was stimulated and neuronal migration was hampered after ß-xyloside treatment while ChABC treatment did not exert these effects.

Striatal glutamate release in L-DOPA-induced dyskinetic animals.

L-DOPA-induced dyskinesia is a common side effect developed after chronic treatment with 3,4-dihydroxyphenyl-l-alanine (l-DOPA) in Parkinson's disease. The biological mechanisms behind this side effect are not fully comprehended although involvement of dopaminergic, serotonergic, and glutamatergic systems has been suggested. The present study utilizes in vivo amperometry to investigate the impact from unilateral 6-hydroxydopamine lesions and l-DOPA (4 mg/kg, including benserazide 15 mg/kg) -induced dyskinetic behavior on striatal basal extracellular glutamate concentration and potassium-evoked glutamate release in urethane-anesthetized rats. Recordings were performed before and after local L-DOPA application in the striatum. In addition, effects from the 5-HT(1A) receptor agonist (2R)-(+)-8-hydroxy-2-(di-n-propylamino)tetralin hydrobromide (8-OHDPAT; 1 mg/kg) was assessed on glutamate release and on dyskinetic behavior. The results revealed a bilateral ≈ 30% reduction of basal extracellular glutamate concentration and attenuated potassium-evoked glutamate release after a unilateral dopamine-depletion in L-DOPA naïve animals. In dyskinetic subjects, basal glutamate concentration was comparable to normal controls, although potassium-evoked glutamate release was reduced to similar levels as in drug naïve dopamine-lesioned animals. Furthermore, acute striatal L-DOPA administration attenuated glutamate release in all groups, except in the dopamine-lesioned striatum of dyskinetic animals. Co-administration of 8-OHDPAT and L-DOPA decreased dyskinesia in dopamine-lesioned animals, but did not affect potassium-evoked glutamate release, which was seen in normal animals. These findings indicate altered glutamate transmission upon dopamine-depletion and dyskinesia.