The long-term objective of the proposed research is to elucidate the mechanisms by which neuronal activity shapes synaptic connections during development of the visual system in mammals. The goal of the present application is to examine the role of nitric oxide (NO) and its downstream signaling pathways in the formation of specific connections between retinal ganglion cell axons and cells of the lateral geniculate nucleus (LGN) in ferrets, during the sorting of retinogeniculate projections into on-center and off-center sublaminae. The hypothesis is that synchronously active retinal afferents activate N-methyl-d-aspartate (NMDA) receptors on LGN cells, allow the entry of calcium into the postsynaptic cell and stimulate the production of NO by neuronal nitric oxide synthase (NOS). NO is available simultaneously to both pre and postsynaptic cells, and is particularly suited to coordinate synaptic sites in both presynaptic terminals and postsynaptic dendrites. We propose that NO diffuses to the presynaptic terminal and acts as a retrograde messenger to activate soluble guanylyl cyclase (GC), elevating levels of the cyclic nucleotide cGMP and is associated cGMP-dependent protein kinase (PKG), thus stabilizing active synapses. In the postsynaptic cell, NO, acting through pathways that include cGMP and PKG signaling, and by virtue of the close coupling between nNOS and NMDA receptor-anchoring proteins, regulates transmission at retinogeniculate synapses.
Specific aims are to examine: (1) The role in retinogeniculate arbor segregation of specific substrates that link NMDA receptor activation and the generation of NO; (2) The role of cGMP as a downstream effector of NMDA receptors and NO in retinal axon patterning; (3) The effect of NO on NMDA currents during retinogeniculate development, and on the ratio of NMDA to AMPA-mediated retinogeniculate responses; (4) The effect of NO on the developmental regulation of NMDA receptor subunits; (5) The role of NO and cGMP in retinogeniculate transmission and plasticity; (6) The role of the NO-cGMP pathway in shaping postsynaptic LGN cell morphology; (7) The dynamic regulation of pre- and postsynaptic structure by NO and cGMP. These experiments will utilize anatomical labeling of single presynaptic retinal axons and postsynaptic LGN cells, intracellular recording from LGN in slices, immunocytochemical labeling of intracellular signals and of glutamate receptor subunits, and confocal microscope imaging of pre-and postsynaptic elements in living slices. Such approaches are crucial to understanding the complex cellular mechanisms that underlie the development and refinement of connections during pattern formation.
