Purchase this article with an account.
T. C. Jakobs, A. Koizumi, G. Zeck, R. H. Masland; Manipulating Gene Expression in Functioning Retinal Ganglion Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2007;48(13):240.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
An explant culture preparation of adult rabbit retina was established to provide a platform for genetic manipulation, electrophysiological recording, and cell imaging of individual retinal neurons within their normal neuronal network.
Pieces of adult rabbit retina were mounted ganglion cell side up on a filter and kept for up to 6 days in an interphase culture. Retinal ganglion cells were transfected biolistically with plasmids encoding EGFP or HcRed, or fusion proteins from EGFP with PSD95, synaptophysin, and rapsyn. At the end of the incubation period the retina was used for patch-clamp recording and immunocytochemistry.
Although there was axonal retraction and occasional swelling of ganglion cell dendrites, the morphology of individual ganglion, amacrine, bipolar, and Mueller cells revealed no major abnormality. We recorded from 14 ganglion cells in whole-cell patch configuration. The ganglion cells produced action potentials and increased their firing rate in response to depolarizing current steps. Responses to light could be elicited after four days and, less reliably, after six days. Transfection of ganglion cells by gene gunning showed that fusion proteins of the presynaptic marker PSD95 localize correctly in ganglion cells. PSD95-GFP fusion proteins were distributed as bright puncta in the dendritic arbor. Immunocytochemistry with antibodies against synaptic ribbon components, revealed that most of the PSD95 puncta are in close proximity to synaptic ribbons. Starburst amacrine cells transfected by the same method show PSD95-GFP puncta, indicating the localization of input synapses to the cell, over the whole extent of their dendrites, whereas synaptophysin-GFP puncta, indicating the starburst cell’s output synapses, are localized exclusively in the outer third of the dendrites. This agrees with the distribution observed by electron microscopy.
The method described here provides a flexible way to manipulate gene expression in individual neurons without the need to design viral vectors or generate transgenic animals. As the tissue can then be used for electrophysiological recording and imaging, it is useful for experiments that seek to combine these approaches.
This PDF is available to Subscribers Only