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Dopamine neurons in ventral mesencephalon: interactions with glia and locus coeruleus
Umeå University, Faculty of Medicine, Integrative Medical Biology, Histology and Cell Biology.
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Parkinson’s disease is a progressive neurodegenerative disorder, characterized by a depletion of the dopaminergic neurons in the substantia nigra. The cause of the disease is yet unknown but age, oxidative stress, and neuroinflammation are some of the features involved in the degeneration. In addition, substantial cell death of noradrenergic neurons occurs in the locus coeruleus (LC). Noradrenaline has been suggested to protect the dopamine neurons from oxidative stress and neuroinflammation. The main treatment of Parkinson’s disease is Levo-dopa, although severe side effects arise from this therapy. Hence, grafting fetal ventral mesencephalic (VM) tissue into the adult striatum has been evaluated as an alternative treatment for Parkinsons’s disease. However, the survival of the grafted neurons is limited, and the dopamine-denervated striatum does not become fully reinnervated. Therefore, elucidating factors that enhance dopamine nerve fiber formation and/or survival of the grafted neurons is of utmost importance.

To investigate dopamine nerve fiber formation and the interactions with glial cells, organotypic VM tissue cultures were utilized. Two morphologically different nerve fiber outgrowths from the tissue slice were observed. Nerve fibers were initially formed in the absence of migrating astrocytes, although thin vimentin-positive astrocytic processes were detected within the same area. A second, persistent nerve fiber outgrowth was observed associated with migrating astrocytes. Hence, both of these nerve fiber outgrowths were to some extent dependent on astrocytes, and appeared as a general feature since this phenomenon was demonstrated in β-tubulin, tyrosine hydroxylase (TH), and aldehyde dehydrogenase A1 (ALDH1)-positive nerve fibers. Neither oligodendrocytes (NG2-positive cells), nor microglia (Iba-1-positive cells) exerted any effect on these two neuronal growths. Since astrocytes appeared to influence the nerve fiber formation, the role of proteoglycans, i.e. extracellular matrix molecules produced by astrocytes, was investigated. β-xyloside was added to the cultures to inhibit proteoglycan synthesis. The results revealed a hampered astrocytic migration and proliferation, as well as a reduction of the glia-associated TH-positive nerve fiber outgrowth. Interestingly, the number of cultures displaying the non-glia-mediated TH-positive nerve fibers increased after β-xyloside treatment, although the amount of TH-protein was not altered. Thus, proteoglycans produced by astrocytes appeared to be important in affecting the dopamine nerve fiber formation.

The noradrenaline neurons in LC have been suggested to protect dopamine neurons from damage. Therefore, the interaction between VM and LC was evaluated. Using the intraocular grafting method, fetal VM and LC were grafted either as single grafts or as VM+LC co-grafts. Additionally, the recipient animals received 2% blueberry-enriched diet. The direct contact of LC promoted graft volume and survival of TH-positive neurons in the VM grafts. The number of dopamine neurons, derived preferably from the A9 (ALDH1/TH-positive) was increased, whereas the dopamine neurons from the A10 (calbindin/TH-positive) were not affected. A dense dopamine-β-hydroxylase (DBH)-positive innervation was correlated to the improved survival. Blueberry-enriched diet enhanced the number of TH-positive neurons in VM, although the graft size was not altered. The combination of blueberries and the presence of LC did not yield additive effects on the survival of VM grafts. The attachment of VM or the addition of blueberries did not affect the survival of TH-positive neurons in LC grafts. The number of Iba-1-positive microglia was decreased in co-grafted VM compared to single VM transplants. The addition of blueberries reduced the number of Iba-1-positive microglia in single VM transplants. Hence, the direct contact of LC or the addition of blueberries enhanced the survival of VM grafts.

Taken together, these data demonstrate novel findings regarding the importance of astrocytes for the nerve fiber formation of dopamine neurons. Further, both the direct attachment of LC or antioxidant-enriched diet promote the survival of fetal VM grafts, while LC is not affected.

Place, publisher, year, edition, pages
Umeå: Integrativ medicinsk biologi , 2008. , 68 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1181
Keyword [en]
Parkinson’s disease, ventral mesencephalon, nerve fiber formation, glia, locus coeruleus, grafting, antioxidant-enriched diet
National Category
Cell and Molecular Biology
URN: urn:nbn:se:umu:diva-1667ISBN: 978-91-7264-573-8OAI: diva2:141757
Public defence
2008-06-05, BiA201, Biologihuset, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2008-05-20 Created: 2008-05-20 Last updated: 2010-01-18Bibliographically 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. Inhibition of proteoglycan synthesis affects neuronal outgrowth and astrocytic migration in organotypic cultures of fetal ventral mesencephalon.
Open this publication in new window or tab >>Inhibition of proteoglycan synthesis affects neuronal outgrowth and astrocytic migration in organotypic cultures of fetal ventral mesencephalon.
2008 (English)In: J Neurosci Res, ISSN 0360-4012, Vol. 86, no 1, 84-92 p.Article in journal (Other academic) Published
Abstract [en]

Grafting fetal ventral mesencephalon has been utilized to alleviate the symptoms of Parkinson's disease. One obstacle in using this approach is the limited outgrowth from the transplanted dopamine neurons. Thus, it is important to evaluate factors that promote outgrowth from fetal dopamine neurons. Proteoglycans (PGs) are extracellular matrix molecules that modulate neuritic growth. This study was performed to evaluate the role of PGs in dopamine nerve fiber formation in organotypic slice cultures of fetal ventral mesencephalon. Cultures were treated with the PG synthesis inhibitor methyl-umbelliferyl-beta-D-xyloside (beta-xyloside) and analyzed using antibodies against tyrosine hydroxylase (TH) to visualize dopamine neurons, S100beta to visualize astrocytes, and neurocan to detect PGs. Two growth patterns of TH-positive outgrowth were observed: nerve fibers formed in the presence of astrocytes and nerve fibers formed in the absence of astrocytes. Treatment with beta-xyloside significantly reduced the distance of glial-associated TH-positive nerve fiber outgrowth but did not affect the length of the non-glial-associated nerve fibers. The addition of beta-xyloside shifted the nerve fiber growth pattern from being mostly glial-guided to being non-glial-associated, whereas the total amount of TH protein was not affected. Further, astrocytic migration and proliferation were impaired after beta-xyloside treatment, and levels of non-intact PG increased. beta-Xyloside treatment changed the distribution of neurocan in astrocytes, from being localized in vesicles to being diffusely immunoreactive in the processes. To conclude, inhibition of PG synthesis affects glial-associated TH-positive nerve fiber formation in ventral mesencephalic cultures, which might be an indirect effect of impaired astrocytic migration. (c) 2007 Wiley-Liss, Inc.

National Category
Medical and Health Sciences
urn:nbn:se:umu:diva-8136 (URN)doi:10.1002/jnr.21465 (DOI)17868150 (PubMedID)
Available from: 2008-01-15 Created: 2008-01-15 Last updated: 2012-04-10Bibliographically approved
3. Locus coeruleus promotes survival of dopamine neurons in ventral mesencephalon: An in oculo grafting study
Open this publication in new window or tab >>Locus coeruleus promotes survival of dopamine neurons in ventral mesencephalon: An in oculo grafting study
2009 (English)In: Experimental Neurology, ISSN 0014-4886, E-ISSN 1090-2430, Vol. 216, no 1, 158-165 p.Article in journal (Refereed) Published
Abstract [en]

Parkinson's disease is a neurodegenerative disorder where dopamine neurons in the substantia nigra of ventral mesencephalon undergo degeneration. In addition to the loss of dopamine neurons, noradrenaline neurons in the locus coeruleus degenerate, actually to a higher extent than the dopamine neurons. The interaction between these two nuclei is yet not fully known, hence this study was undertaken to investigate the role of locus coeruleus during development of dopamine neurons utilizing the intraocular grafting model. Fetal ventral mesencephalon and locus coeruleus were implanted either as single grafts or co-grafts, placed in direct contact or at a distance. The results revealed that the direct attachment of locus coeruleus to ventral mesencephalon enhanced graft volume and number of tyrosine hydroxylase (TH)-positive neurons in ventral mesencephalic grafts. Cell counts of subpopulations of TH-positive neurons also immunoreactive for aldehyde dehydrogenase 1-A1 (ALDH1) or calbindin, revealed improved survival of ALDH1/TH-positive neurons. However, the number of calbindin/TH-positive neurons was not affected. High density of dopamine-beta-hydroxylase (DBH)-positive innervation in the ventral mesencephalon placed adjacent to locus coeruleus was correlated to the improved survival. Ventral mesencephalic tissue, implanted at a distance to locus coeruleus, did not demonstrate improved survival, although DBH-positive nerve fibers were detected. In conclusion, the direct contact of locus coeruleus resulting in dense noradrenergic innervation of ventral mesencephalon is beneficial for the survival of ventral mesencephalic grafts. Thus, when trying to rescue dopamine neurons in Parkinson's disease, improving the noradrenergic input to the substantia nigra might be worth considering.

National Category
urn:nbn:se:umu:diva-19403 (URN)10.1016/j.expneurol.2008.11.029 (DOI)19150447 (PubMedID)
Available from: 2009-03-05 Created: 2009-03-05 Last updated: 2012-04-10Bibliographically approved
4. Blueberry-enriched diet enhances the survival of fetal ventral mesencephalic intraocular grafts
Open this publication in new window or tab >>Blueberry-enriched diet enhances the survival of fetal ventral mesencephalic intraocular grafts
(English)Manuscript (Other academic)
urn:nbn:se:umu:diva-3238 (URN)
Available from: 2008-05-20 Created: 2008-05-20 Last updated: 2010-06-23Bibliographically approved

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