The objective of this project is an understanding of the neurotrophic support responsible for the development and maintenance of the nigrostriatal dopamine system. A central working hypothesis of Project 4 states that glial cell line-derived neurotrophic factor (GDNF) is the endogenous dopaminotrophic factor. This hypothesis leads to four postulates; 1) Exogenous GDNF must exert dopaminotrophic activity in vivo: This postulate is tested by a series of intraocular brain tissue grafting experiments. We will test the effect of neuron age as well as the effect of GDNF on target specificity and target=-elicited fiber growth in the intraocular model. 2a) GDNF must be expressed in the appropriate dopamine target areas: This postulate is tested by mapping the expression of GDNF mRNA by in situ hybridization and the presence of GDNF protein by immunohistochemistry. Treatment with neuroexcitatory drugs will test if GDNF expression in striatum is regulated. 2b) GDNF must be taken up by dopamine nerve terminals and transported retrogradely to their cell bodies of origin; The postulate is tested by transport studies using radiolabeled and unlabelled GDNF and autoradiographic and immunohistochemical detection methods, respectively. 3) Absence of GDNF must lead to a disturbed development and/or maintenance of the nigrostriatal dopamine system: This postulate is tested by a set of gene targeting experiments. We will generate """"""""classical"""""""" knockouts of the GDNF gene. Several ways to handle the possibility that a """"""""classical"""""""" GDNF knockout leads to early lethality, such as grafting mutated embryonic tissue to healthy animals, cross- breeding with animals overexpressing GDNF under a muscle-specific promoter (to rescue motor neurons) or studying heterozygous animals, which might have a milder phenotype, are also proposed. Using the Cre-loxP system, promoter-driven specific knockouts of the GDNF gene will also be generated. To cause a null-mutation of the GDNF gene in only neurons, only astrocytes or only striatum, respectively, we will use the nestin, thy-1 or neurofilament promoter (neurons), the GFAP promoter (astrocytes) or specific RARbeta or RXR gamma transcription factor promotors (striatum). 4) GDNF should have unique dopaminotrophic properties: This postulate is tested by studying the cellular distribution of another cloned TGFbeta superfamily member, OP-1. Additionally, the cloned OP01 receptor BMPR-type II will be mapped. Taken together, these experiments will provide new knowledge about the normal trophic support of the nigrostriatal dopamine system.
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