MECHANISMS OF TROPHIC MOLECULES DURING NEURONAL DEVELOPM

Oregon Health And Science University, Portland OR

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Abstract

The long term goal of this study is to understand the molecular and cellular mechanisms of target- neuron interactions during development. We will accomplish this goal by applying a variety of methods for manipulating gene and protein expression during chicken embryo development in vivo. These studies are expected to advance the understanding of the development and maintenance of the nervous system, providing insights into the processes of neural maintenance and neurodegenerative disease. Our studies will use the avian ciliary ganglion (CG) because of its simple neuronal composition, the accessibility of the neurons and their targets throughout development, and the coincidence of synapse formation, cell death, and transmitter expression within a well-defined window of development. The CG contains two populations of neurons: ciliary neurons that innervate iris and ciliary body and choroid neurons that innervate arterial smooth muscle in the choroid layer of the eye. We have identified three molecules by their actions on CG neurons in cell culture: growth promoting activity (GPA), which supports neuronal survival; activin A, which induces the expression of somatostatin; and follistatin, an inhibitor of activin. Our working model is that availability and levels of these three target-derived molecules regulate survival and neuropeptide phenotype of CG neurons. We will determine the validity of this model by testing the following hypotheses: l). Cell death in the CG during development is controlled by GPA expressed in the iris, ciliary body, and choroid layer; 2) Specific expression of somatostatin in choroid neurons of the CG is induced by activin expressed in the choroid layer, whereas follistatin blocks the effects of activin expressed in the iris/ciliary body. The specific aims are: l) to overexpress GPA in order to determine if CG neurons are rescued from cell death; 2) to use cell cultures of CG target tissues as well as Co-cultures to assay the effectiveness of GPA blocking agents; 3) to block GPA during embryonic development in vivo to determine if CG cell death is exacerbated; 4) to identify the mechanism by which alpha bungarotoxin increases CG neuron survival; 5) to quantify and characterize GPA receptor and receptor associated molecules during development; 6) to test the effectiveness of activin and follistatin blocking agents on co-cultures of CG neurons with choroid smooth muscle and iris/ciliary body; 7) to block to the action of activin in vivo to determine if somatostatin expression in choroid neurons is prevented; 8) to inject activin in the anterior chamber of the eye to induce somatostatin in ciliary neurons; 9) to block the effects of follistatin in the iris/ciliary body to determine if somatostatin is induced in ciliary neurons.