How organized patterns of neuronal connections develop early in life is a fundamental question in the development of the nervous system. Communication between neurons in the retina occurs even before the photoreceptors respond to light. This leads to patterns of coordinated activity which can guide the development of precise connections within the retina, and between the retina and visual centers in the brain. Without such precision in neural connectivity, visual image processing may never reach its adult level of maturity and sophistication. This proposal addresses the role of coordinated activity and intercellular communication in the retina of the early postnatal ferret. In the first project, changes in intracellular calcium in vitro will be used to monitor how spontaneous waves of excitation may lead to correlations in activity among neurons of various types, and across various regions of the neonatal ferret retina. The identity of the active cells will be obtained by combining Fura-2 imaging with intracellular dye- filling. Recordings from dissociated cells will be made in order to determine whether the rhythmic bursting activity of retinal cells is a physiological property of the cell, rather than the network of cells. The role of neurotransmitter release and gap junctions in mediating the propagation of the waves will be assessed using pharmacological agents to disrupt the waves, and intracellular injection of a tracer that crosses gap junctions, to determine whether coactive cells are intercellularly coupled. The early appearance of neurotransmitters and their receptors in the retina suggests a role for these molecules in the development of retinal structure and function. In the second project, we wish to determine whether transmitters mediate local communication between neurons of the neonatal retina, in a manner which could be instructive for synapse formation. In acute neonatal retinal slices, amacrine and bipolar cells will be stimulated, and the intracellular calcium levels of their neighbors will be monitored optically. The means by which the cells communicate (transmitters versus gap junctions) will be assessed using pharmacological agents and intracellular Neurobiotin (a tracer which crosses gap junctions) injections. In the third project, the interactions of cells in the ventricular zone of neonatal ferrets will be studied. In particular, the role of muscarinic cholinergic receptors in intracellular and intercellular signaling will be examined pharmacologically. The outcome of these interactions on cellular proliferation will be determined by examining whether in vivo intraocular injections of muscarinic receptor antagonists reduces mitosis. By monitoring the physiological interactions between large numbers of developing retinal neurons, we hope to gain a better understanding of how these interactions might lead to the formation of precise connections of the retina, and between the retina and its central targets. Furthermore, these experiments would enhance our understanding of what common strategies are employed to ensure the normal development of neuronal pathways in the central nervous system.
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