GDNF and BDNF are two important factors for development, maintenance, and aging of ventral mesencephalic dopamine neurons. It is, however, not possible to study aging effects of complete null mutations of either GDNF or BDNF, since null mutated animals die shortly after birth. In project 0005 we propose to use organotypic tissue culture and grafting techniques to study the development, maintenance, and aging of ventral mesencephalic dopamine neurons in tissue derived from GDNF or BDNF knockout and heterozygous animals. Thus, organotypic tissue cultures of fetal ventral mesencephalon from GDNF-/- and BDNF-/- as well as their heterozygous counterparts will be utilized to study growth factor effects on dopamine nerve fiber formation in comparison with wildtype and heterozygous tissue cultures (Aim 1). To examine aging effects of GDNF or BDNF complete or partial knockout on midbrain dopamine neurons in isolation, fetal ventral mesencephalon and lateral ganglionic eminence will be implanted simultaneously into the lateral ventricle of wildtype mice, so that a nigrostriatal microcircuit will be created of null mutated tissue. Recipients of transplants will then be sacrificed at 3, 12, 18, and 24 months post-grafting, to determine if there are time-dependent detrimental alterations occurring in transplanted tissues during aging as a result of the GDNF or BDNF null mutation. CSF samples will be obtained from the host animals to determine potential influence of the environment on grafted tissues. GDNF and BDNF will be added to the incubation bath prior to grafting in one group of all transplant types to determine if supplementation with the appropriate trophic factors may reverse potential effects of GDNF or BDNF deletion (Aim 2). Combined effects of GDNF or BDNF depletion and early neuroinflammation by prenatal exposure to lipopolysaccharide (LPS) will be studied using organotypic tissue culture (for development studies) and grafting (for aging studies). Finally, exposure to an environmental toxin (methamphetamine) in GDNF or BDNF null mutated nigrostriatal cografts will be studied (Aim 3). The overall goal with Project 0005 is to examine the fate of midbrain dopamine neurons developed and aged in the absence of GDNF or BDNF combined with effects of neuroinflammation and environmental toxins. Furthermore, Project 0005 provides two levels of control studies for the other projects: A) null mutated animals may be studied in grafts or tissue culture to determine the effects of a complete depletion of BDNF or GDNF, and B) we will be able to determine if the effects observed in Project 0001-0004 in vivo are due to intrinsic, brain specific, alterations or to systemic effects, since we will isolate the brain regions in question in intracranial transplants and tissue cultures.
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