Richard H. Masland - US grants

The goal of this project is to learn the effect of neurotransmitter release by identified mammalian amacrine cells on the activity of the retinal ganglion cells. A group of methods has been developed in which the neurons of intact retinas are labeled with fluorescent nucleic acid stains. The staining makes it possible to recognize the cell bodies of subclasses of amacrine or ganglion cell under a fluorescence microscope. The neurons are then injected under visual control with lucifer yellow, which fills the dendrites. The yield of completely stained cells is an order of magnitude greater than has been previously available. Two types of experiment will be carried out. The first is anatomical in purpose; for this, dye-stained cells will be injected with lucifer yellow in lightly fixed whole mounts. An amacrine cell will be injected, and then a second, nearby ganglion or amacrine cell will be injected as well. The cells will be identified on the basis of their shapes. In some cases identification with known neurotransmitter classes will be made, by comparison of the shape of the injected amacrine cell with published immunohistochemical staining. Special attention will be paid to the amacrine cells which contain somatostatin or substance P. The spatial relations between the dendrites of the injected cell pairs will be studied by classical methods and computer aided reconstruction. The second experiment is physiological, and will study living retinas in vitro. The same methods of fluorescent staining and cell injection will be used. The activity of a retinal ganglion cell will be recorded extracellularly. A preselected amacrine cell will be penetrated by micropipette. The action of the amacrine cell upon the ganglion cell will be learned by observing the effect on ganglion cell activity of passing electrical currents into the amacrine cell. After recording, the identities and dendritic geometries of both neurons will be confirmed by lucifer injection.

The long-term objective of the proposed research is to understand how the internal circuits of mammalian retinas shape the receptive fields and response properties of the retinal ganglion cells. The specific aim is to complete the census of the rabbit retina's major cell populations. Mammalian retinas have been known for a long time to contain many cell types and subtypes. Although it is unlikely that we can analyze all of the retina's rare (infrequent) cells, we believe that we are now able to identify and map all of the major ones. The initial question is: what are the total numbers of horizonal, bipolar, and amacrine cells? The question will be approached by two independent sets of experiments. In the first, populations of retinal cells will be sequentially identified by staining. Generally available stains, fluorescent labels and antibodies will be used to distinguish individual classes of cell. In the second, the three major classes of retinal cells will be distinguished by confocal microscopy. The distribution of each cell type across the retinal surface will be mapped quantitatively. These experiments will make available (for the first time) an estimate of the total number of amacrine cells. By comparing that number with the total numbers of the amacrine cells that have been stained by histochemical and autoradiographic methods, we will learn how many unidentified amacrine cells must still exist. The shapes of the unidentified cells will then be sought by experiments in which amacrine cells are injected with lucifer yellow. Injection will be combined with fluorescent, histochemical or autoradiographic labeling of cell bodies to give the population densities of the injected cells.

[unreadable] DESCRIPTION (provided by applicant): The ganglion cells of mammalian retinas exist in roughly a dozen types, each transmitting a different encoding of the visual scene to the brain. The long-term goal of this research is to learn the mechanism by which specific types of ganglion cells achieve these codlings. We have developed an interface incubation system that allows maintenance of multiple samples of adult rabbit retinas for several days in an unsupervised, culture-like system. Genes coding for RNAi or tagged synapse proteins are biolistically transfected into individual retinal ganglion cells. [unreadable] [unreadable] Two questions involve the synaptic events underlying directional selectivity in certain retinal ganglion cells. First, we propose to use RNAi to knock down GABAergic or cholinergic responsiveness in individual ganglion cells. Recording will reveal the contribution of these direct (postsynaptic) inputs to direction selectivity. Second, we will investigate the co-release of GABA and Ach by the starburst amacrine cells. Are the two neurotransmitters released from the same cellular sites or different ones? This will be studied by localizing their vesicular transport proteins. [unreadable] [unreadable] The third question is a more general one. Our recent experiments suggest that the excitatory inputs to ganglion cell dendrites are spatially distributed according to an even-spacing law: the synapses seem to repel each other. In addition, they systematically avoid branch points in the dendritic arbor. We now propose to see if these rules apply to all types of ganglion cells; to model their physiological consequences; and to see if the same rules apply to inhibitory synapses. [unreadable] [unreadable] The methods introduced here can be used in adult animals of any species. They obviate the need for transgenic animals, increase the number of samples that can be simultaneously studied, and allow labeling of proteins that have long half-lives. They can be used in normal tissue or in disease models. We hope that they will be useful to laboratories studying both basic and clinical problems. [unreadable] [unreadable] [unreadable]

Project Summary In both humans and mice, astrocytes form the immediate cellular environment of the ganglion cell axons in the optic nerve head. They occupy up to 50% of the total tissue volume at that location. When the axons of retinal ganglion cells are damaged, they become reactive. In the proposed research we investigate the signaling between optic axons and the astrocyte that triggers this response. The first question is the nature and timing of the changes in gene expression that follow damage to the optic axons. Our preliminary results indicate that very different sets of glial genes are up or down regulated at different times following axonal injury. The experiments of Specific Aim 1 seek to confirm this finding, using an improved model of axonal damage. Specific Aim 2 uses the results of our existing and future expression profiling to identify candidates for the signaling systems that communicate from axons to astrocytes. The candidate signals will be overexpressed, using viral vectors, in retinal ganglion cells. The prediction is that overexpression of an actual signal will lead to the changes in astrocyte gene expression and morphology characteristic of astrocyte reactivity. Specific Aim 3 investigates the hypothesis that the initial response of the astrocytes to axonal damage is a protective one, i.e. that retinal ganglion cells threatened with injury send a distress signal to the ensheathing astrocytes, which then initiate a neuroprotective response. We will test this hypothesis by stressing the optic nerve in transgenic mice carrying mutations that compromise astrocyte communication and motility. The prediction is that the compromise of astrocyte function will worsen the damage to the axons resulting from this stress.