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.

La consommation d’alcool pendant la grossesse peut entraîner des conséquences néfastes sur le développement de l’enfant et mener aux troubles du spectre de l’alcoolisation fœtale (TSAF). Cependant on en connaît peu sur son effet durant la période de préimplantation. Cette période est reconnue comme étant sensible à l’environnement, majoritairement dû au fait qu’elle est caractérisée par la reprogrammation dynamique des profils de méthylation de l’ADN. L’alcool est reconnu pour altérer les mécanismes impliqués dans les processus de méthylation de l’ADN (e.g., cycle du folate, actions des DNMTs). Récemment, nous avons démontré qu’une exposition prénatale à l’alcool éthylique durant la préimplantation altère les futurs profils de méthylation de l’ADN du cerveau et augmente le nombre de défauts morphologiques à la mi-gestation. Il n’y a présentement aucun traitement pour les TSAF, mais certaines études suggèrent qu’une diète enrichie en donneurs de groupement méthyles (e.g., folate, choline), nécessaires aux réactions de méthylation, pourrait atténuer les effets d’une exposition prénatale à l’alcool. C’est pourquoi nous avons voulu déterminer si une diète enrichie en donneurs de groupements méthyles pouvait apporter une protection aux embryons ayant subi une exposition prénatale à l’alcool durant la préimplantation. Pour ce faire, une diète standard ou enrichie a été donné à des souris 4 semaines avant la gestation et pendant celle-ci. Les femelles gestantes ont reçu une dose d’alcool au jour E2.5, puis nous avons récolté les embryons à E10.5 et E18.5 pour évaluer les anomalies physiques et faire l’analyse de la méthylation des cerveaux antérieurs. Nos résultats démontrent que la diète enrichie prévient ou élimine un certain nombre de défauts morphologiques à E10.5 et E18.5. Nous avons aussi observé que la diète enrichie n’affectait pas la mise en place et le maintien de la méthylation et l’expression des gènes à empreintes durant le développement. La diète enrichie en donneurs de groupements méthyles aide donc à prévenir certains effets nuisibles de l’exposition prénatale à l’alcool survenant durant la préimplantation. / Alcohol consumption during pregnancy can have a significant impact on the development of the child and lead to fetal alcohol spectrum disorders (FASD). Nonetheless, little is known about its effect during the pre-implantation period. However, we know that this period is very sensitive to the environment, mainly because of major reprogramming of DNA methylation patterns. Moreover, alcohol can alter the mechanisms involved in DNA methylation processes (e.g., folate cycle, actions of DNMTs). Recently, we have shown that prenatal alcohol exposure (PAE) during preimplantation alters future DNA methylation patterns of the young embryo brain and increases the number of morphological defects. There is currently no treatment for FASD, but studies suggest that a diet enriched in methyl donors (e.g., folate, choline), which are necessary for methylation reactions, may mitigate the effects of PAE. Therefore, we wanted to determine if a diet enriched in methyl group donors could provide protection to embryos against a PAE during preimplantation. To do so, a standard or enriched diet was given to mice 4 weeks before and during gestation. Pregnant females were exposed to alcohol at day E2.5, then we harvested embryos (E10.5, E18.5) to assess physical abnormalities and analyse forebrain methylation. Our results demonstrate that the enriched diet reduces or eliminate defects at E10.5 and E18.5. We also observed that the fortified diet did not affect the establishment and maintenance of methylation and expression of imprinted genes during development. Thus, our results show that a methyl enriched diet can prevent some of the adverse effects of prenatal alcohol exposure occurring during preimplantation.