Molecular Genetics Of Early Eye Development

National Eye Institute

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Abstract

Olfactomedin 1 (Olfm1), Olfm2 and Olfm3, together with neuritin, Brorin (Vwc2) and brorin-like (Vwc2l) proteins, are extracellular components of the AMPA receptor (AMPAR) complex. AMPARs are the major ionotropic glutamate receptors within both the brain and the retina and play a major role in synaptic formation and communication. Co-immunoprecipitation experiments have demonstrated that Olfm1, Olfm2, Olfm3, GluR1, GluR2, Vwc2 and Vwc2l proteins are involved in complex physical interactions, and individual elimination of these proteins leads to changes in the composition of the AMPAR complex. We continued our analysis of the retinal phenotype of knockout (KO) mice for genes encoding these proteins. Brorin KO mice were analyzed using retinal cell cultures, Ca2+ imaging analysis, GluR1/2 internalization assays, immunofluorescence, and patch clamp experiments. In the retina, horizontal cells exhibited the highest level of Brorin expression, followed by a moderate level of expression in amacrine cells and weak expression in retinal ganglion cells, suggesting its role in the inhibitory interneurons. Amacrine cell activity was analyzed by patch clamp techniques using retinal cell cultures from postnatal mouse retinas. The results demonstrated that the amplitude of spontaneous AMPAR current was reduced and the decay time of the responses was extended in Brorin KO amacrine cells. The I-V relation in glutamate evoked current on outside out patch clamp revealed a more inward rectified nature of the AMPARs in the KO amacrine cells. These data suggested that the AMPAR complex in Brorin KO cells contains less GluR2 and is more Ca2+ permeable than in control cells. Intracellular Ca2+ concentrations in amacrine cells were measured by Fura-2 AM fluorescence and the fluctuation in Ca2+ was determined by stimulation with AMPA. The resting and AMPA-stimulated concentrations of Ca2+ were significantly higher in amacrine cells from Brorin KO mice than those from wild type mice. These changes in Ca2+ permeability resulted from changes in the expression the GluR2 core subunit. The presence of this subunit makes AMPARs Ca2+ impermeable. The surface expression of GluR2 was significantly reduced in amacrine cells of Brorin KO mice compared with wild type. At the same time, the expression of the GluR1 subunit was not different in the KO cells compared to wild type cells. These data confirm that Brorin modulates the composition of the AMPAR complex by affecting the Ca2+ impermeable GluR2 subunit in amacrine cells in the mouse retina. There were no detectable changes in the activities of retinal horizontal cells in Brorin KO compared with wild type mice as judged in electrophysiology experiments. However, immunofluorescence staining with calbindin, a horizontal cell marker, showed a smaller number of horizontal cells and their reduced arborization to the outer plexiform layer in the Brorin KO retina compared with the wild type retina. The number of amacrine cells was also reduced in the Brorin KO retina compared with the wild type. This suggests that Brorin may regulate development of those inhibitory neurons in the retina.