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On dopamine neurons: nerve fiber outgrowth and L-DOPA effects
Umeå University, Faculty of Medicine, Integrative Medical Biology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Parkinson’s disease is a disorder mainly characterized by progressive degeneration of dopamine producing neurons in the substantia nigra of the midbrain. The most commonly used treatment strategy is to pharmacologically restore the lost function by the administration of the dopaminergic precursor L-DOPA. Another treatment strategy is to replace the degenerated neurons with immature fetal ventral mesencephalic tissue, or ultimately stem cell-derived tissue. Grafting trials have, however, revealed poor reinnervation capacity of the grafts, leaving much of the striata dopamine-denervated. An additional drawback is the upcoming of dyskinesia (involuntary movements), a phenomenon also observed during L-DOPA treatment of Parkinson’s disease patients. Attempts to characterize nerve fiber formation from dopamine neurons have demonstrated that the nerve fibers are formed in two morphologically diverse outgrowth patterns, one early outgrowth seen in the absence of astrocytes and one later appearing outgrowth seen in co-existence with astrocytes.

The overall objective of this thesis has been to study the dopaminergic outgrowth including guidance of nerve fiber formation, and to look into the mechanisms of L-DOPA-induced dyskinesia. The first paper in this thesis characterizes the different outgrowth patterns described above and their relation to different glial cells. The study demonstrated the two different outgrowth patterns to be a general phenomenon, applying not only to dopamine neurons. Attempts of characterization revealed no difference of origin in terms of dopaminergic subpopulations, i.e. A9 or A10, between the outgrowth patterns. Furthermore, the “roller-drum” technique was found optimal for studying the dual outgrowth sequences.

The second and the third paper also utilized the “roller-drum” technique in order to promote both patterns of neuronal fiber formation. The effects of glial cell line-derived neurotrophic factor (GDNF) on the formation of dopamine nerve fibers, was investigated. Cultures prepared from gdnf knockout mice revealed that dopaminergic neurons survive and form nerve fiber outgrowth in the absence of GDNF. The dopaminergic nerve fibers exhibited an outgrowth pattern consistent with that previous observed in rat. GDNF was found to exert effect on the glial-associated outgrowth whereas the non-glial-associated was not affected. Astrocytic proliferation was inhibited using cytosine β-D-arabinofuranoside, resulting in reduced glial-associated outgrowth. The non-glial-associated dopaminergic outgrowth was on the other hand promoted, and was retained over longer time in culture. Furthermore, the non-glial-associated nerve fibers were found to target the fetal frontal cortex. Different developmental stages were shown to promote and affect the outgrowths differently. Taken together, these data indicate and state the importance of astrocytes and growth factors for neuronal nerve fiber formation and guidance. It also stresses the importance of fetal donor age at the time for transplantation.

The fourth and fifth studies focus on L-DOPA dynamics and utilize in vivo chronoamperometry. In study four, 6-OHDA dopamine-depleted rats were exposed to chronic L-DOPA treatment and then rated as dyskinetic or non-dyskinetic. The electrochemical recordings demonstrated reduced KCl-evoked release in the intact striatum after chronic L-DOPA treatment. Time for maximal dopamine concentration after L-DOPA administration was found to be shorter in dyskinetic animals than in non-dyskinetic animals. The serotonergic nerve fiber content in the striatum was evaluated and brains from dyskinetic animals were found to exhibit significantly higher nerve fiber density compared to non-dyskinetic animals. Furthermore, the mechanisms behind the conversion of L-DOPA to dopamine in 6-OHDA dopamine-depleted rats were studied. Local administration of L-DOPA in the striatum increased the KCl-evoked dopamine release in the intact striatum. Acute application of L-DOPA resulted sometimes in a rapid conversion to dopamine, probably without vesicle packaging. This type of direct conversion is presumably occurring in non-neuronal tissue. Furthermore, KCl-evoked dopamine releases were present upon local application of L-DOPA in the dopamine-depleted striatum, suggesting that the conversion to dopamine took place elsewhere, than in dopaminergic nerve fibers. In conclusion, these studies state the importance of astrocytes for neuronal nerve fiber formation and elucidate the complexity of L-DOPA conversion in the brain.

Place, publisher, year, edition, pages
Umeå: Integrativ medicinsk biologi , 2008. , 73 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1180
Keyword [en]
dopamine, nerve fiber outgrowth, astrocytes, ventral mesencephalon, GDNF, L-DOPA, L-DOPA induced dyskinesia
National Category
URN: urn:nbn:se:umu:diva-1634ISBN: 978-91-7264-567-7OAI: diva2:141624
Public defence
2008-05-23, BiA201, Biologihuset, Umeå, 10:00 (English)
Available from: 2008-05-02 Created: 2008-05-02 Last updated: 2009-03-16Bibliographically approved
List of papers
1. Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures.
Open this publication in new window or tab >>Glial influence on nerve fiber formation from rat ventral mesencephalic organotypic tissue cultures.
2007 (English)In: Journal of Comparative Neurology, ISSN 0021-9967, Vol. 501, no 3, 431-42 p.Article in journal (Refereed) Published
Abstract [en]

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.

Aldehyde Dehydrogenase/metabolism, Animals, Dopamine/metabolism, Glutamate Decarboxylase/metabolism, Immunohistochemistry, Isoenzymes/metabolism, Mesencephalon/*cytology/embryology, Nerve Fibers/*physiology, Neuroglia/*cytology/physiology, Neurons/*physiology, Organ Culture Techniques, Rats, Rats; Sprague-Dawley, Stem Cells/cytology/metabolism, Tubulin/metabolism, Vimentin/metabolism
urn:nbn:se:umu:diva-12270 (URN)17245706 (PubMedID)
Available from: 2008-01-11 Created: 2008-01-11 Last updated: 2010-06-21Bibliographically approved
2. Effects of glial cell line-derived neurotrophic factor deletion on ventral mesencephalic organotypic tissue cultures.
Open this publication in new window or tab >>Effects of glial cell line-derived neurotrophic factor deletion on ventral mesencephalic organotypic tissue cultures.
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2007 (English)In: Brain Research, ISSN 0006-8993, Vol. 1133, no 1, 10-9 p.Article in journal (Refereed) Published
Abstract [en]

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.

urn:nbn:se:umu:diva-12266 (URN)doi:10.1016/j.brainres.2006.11.052 (DOI)17184739 (PubMedID)
Available from: 2008-01-08 Created: 2008-01-08 Last updated: 2011-01-11Bibliographically approved
3. Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures
Open this publication in new window or tab >>Inhibition of astrocytes promotes long-distance growing nerve fibers in ventral mesencephalic cultures
2008 (English)In: International Journal of Developmental Neuroscience, ISSN 0736-5748, E-ISSN 1873-474X, Vol. 26, no 7, 683-691 p.Article in journal (Refereed) Published
Abstract [en]

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.

urn:nbn:se:umu:diva-19397 (URN)10.1016/j.ijdevneu.2008.07.014 (DOI)18718519 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2010-06-21Bibliographically approved
4. Chronic intermittent L-DOPA treatment induces changes in dopamine release
Open this publication in new window or tab >>Chronic intermittent L-DOPA treatment induces changes in dopamine release
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2009 (English)In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 108, no 4, 998-1008 p.Article in journal (Refereed) Published
Abstract [en]

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.

3, 4-dihydroxyphenyl-l-alanine, 3, 4-dihydroxyphenyl-l-alanine induced dyskinesia, 5-hydroxytryptamine or serotonin, chronoamperometry, dopamine, serotonin transporter
National Category
Medical and Health Sciences
urn:nbn:se:umu:diva-3140 (URN)10.1111/j.1471-4159.2008.05848.x (DOI)19196428 (PubMedID)
Available from: 2008-05-02 Created: 2008-05-02 Last updated: 2012-06-14Bibliographically approved
5. L-DOPA conversion to dopamine in the rat dopamine-depleted striatum
Open this publication in new window or tab >>L-DOPA conversion to dopamine in the rat dopamine-depleted striatum
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(English)Manuscript (Other academic)
urn:nbn:se:umu:diva-3141 (URN)
Available from: 2008-05-02 Created: 2008-05-02 Last updated: 2010-06-21Bibliographically approved

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