Gary Matthews - US grants

The human visual system has achieved the theoretical physical limit of sensitivity to light: absorption of a single quantum of light is an event that can be detected by the cells in the retina. This remarkable sensitivity is due in part to properties of the light-sensitive cells themselves, the photoreceptors, and in part to the ways in which other retinal cells process the electrical signals generated by the photoreceptors. The overall aim of the proposal research is to gather quantitative information about the generation and analysis of these electrical signals in the vertebrate retina. The first step on vision is the translation of light energy into an electrical signal that can be passed along to other neurons in the retina. Understanding of this process of visual transduction is fundamental to an understanding of human vision, and a number of proposed experiments are aimed at the mechanism of transduction. In most experiments, the focus is on inferences about the transduction process drawn from analysis of the electrical responses to light in individuals photoreceptors. These responses will be monitored by recording changes in the electrical current flowing across the membrane of the photoreceptor, a technique that is suffciently sensitive to detect the absorption of a single quantum of light. Orther experiments will test the degree to which the scheme for photoreceptor function emerging from studies of cold-blooded vertebrates can be extended to mammalian retina. Another line of research will investigate the ways in which the responses of photoreceptors are modified as they are passed on through the retina for eventual relay to the brain.

This project will investigate the physiology and pharmacology of individual epithelial cells of the eye. The focus will be on cells of the ciliary body, which secretes the aqueous humor, and of the retinal pigment epithelium, which is necessary for the survival of the photoreceptors. The overall aim is to understand the cellular actions of neural and hormonal signals that regulate the physiology of the epithelial cells, including secretion and phagocytosis. To date, there is little information at the cellular level about these processes, but because of the central role of ocular epithelial cells in diseases of the human eye, including glaucoma and retinal degenerative disorders, an understanding of the basic physiology of these cells and how it is regulated will be vital to better understanding and treatment of ocular disease. The proposed studies will be done on single cells. Electrical techniques will be used to measure transmembrane ionic currents and to determine how the modulation of these currents controls both secretion and phagocytosis in ocular epithelial. Changes in cell membrane capacitance will be employed to monitor the changes in membrane area that accompany exocytosis and phagocytosis. In addition, fluorescent indicators will be used to measure changes in important intracellular ions, such as calcium. The electrical and fluorescence measurements can be combined within a single cell, providing powerful tools for answering questions of cellular physiology. In the ciliary body, the experiments will give information about how the secretion of the aqueous humor is regulated and thus shed light on the cellular actions of drugs used to control glaucoma. In the retinal pigment epithelium, a major goal is to understand the cellular events that lead to phagocytosis of the shed tips of photoreceptor outer segments, an event that is defective in an animal model of blindness caused by hereditary retinal degeneration.

The goal of this project is to understand how electrical signals are generated and processed in the neurons of the retina. The approach to this goal is to study ionic conductances of single, isolated neurons and to determine how those conductances are modulated by neurotransmitters. Then, the information obtained from these studies of single neurons will be used to make testable predictions about the effects of transmitters on light responses in intact retina. In that way, fundamental information can be obtained about how the neural circuitry of the retina extracts information about the visual world. A particular focus of the work will be the regulation of intracellular calcium in individual synaptic terminals of retinal neurons. The intracellular calcium concentration controls the release of chemical transmitter from neurons, and thus factors that regulate internal calcium will be important in modulating synaptic transmission. These factors include neurotransmitters released by other neurons, which can either potentiate or inhibit the calcium channels through which external calcium can enter the synaptic terminal. To study this neurotransmitter modulation of calcium channels, combined electrical measurements of ionic current and fluorescent-indicator measurements of internal calcium concentration will be made simultaneously in single synaptic terminals of retinal neurons. The resulting information will be valuable not only in understanding the retina, but also the rest of the central nervous system, where modulation of presynaptic calcium current is thought to be an important mechanism by which neurotransmitters modulate synaptic transmission.

DESCRIPTION (Adapted from applicant's abstract): The secretion of fluid by the ciliary body requires transepithelial transport of solutes, with water following osmotically. The experiments proposed in this project will examine the ionic conductances that control the movement of ions across the cell membrane, and thus control the secretion of fluid by the ciliary body. The principal focus is to determine how neurotransmitters and hormones affect these ionic conductances which may help explain the regulation of intraocular pressure by these agents. The intracellular signaling pathways that link neurotransmitter or hormone receptors to the ion channels of the ciliary epithelial cells will also be investigated. Because transport of chloride ions is a vital component of the transepithelial fluid transport, one aspect of the proposal focuses on chloride channels and their regulation. The ionic mechanisms will be investigated in both the nonpigmented and the pigmented epithelial cell layers. The overall goal is to establish the cellular mechanisms governing epithelial transport in the ciliary body in order to gain a greater understanding of the control of intraocular pressure and of the signaling pathways which may provide important targets for drugs that can reduce aqueous inflow.

[unreadable] DESCRIPTION: The photoreceptor cells of the retina carry out the essential task of translating light into electrical signals, which are then transmitted to retinal bipolar neurons for further analysis before being communicated to the rest of the visual system. All visual information must pass through this obligatory pathway, which involves two synaptic relays, one from photoreceptors to bipolar cells, and another from bipolar cells to third-order neurons of the inner retina. Both of these through-conducting synapses are specialized for continuous release of chemical neurotransmitter, which is required for the proper transmission of visual information. Despite their crucial role in vision, these unique photoreceptor and bipolar-cell synapses remain poorly understood, and it is not yet known how they are able to support high rates of neurotransmitter release for prolonged periods, while other synapses in the brain fatigue rapidly during sustained stimulation. This project will use a combination of cellular electrophysiology and high-resolution fluorescence imaging to provide the first complete picture of the synaptic vesicle cycle at the special synapses of retinal bipolar neurons and photoreceptor cells. The vesicle cycle can be broken down into several components: rapid fusion of synaptic vesicles to initiate transmitter release, resupply of releasable vesicles to support continued release, movement of vesicles to and from reserve and releasable pools, compensatory endocytosis to retrieve fused vesicles, and regeneration of new releasable vesicles after endocytosis. The specific aims of the project exploit the ability to tag synaptic vesicles and synaptic active zones with distinct fluorescent labels to observe directly the vesicle movements that underlie all these components of the vesicle cycle. Then, genetic manipulations and selective protein interference will be used to unravel the molecular machinery that gives retinal synapses the unique ability to release neurotransmitter continuously and allows them to perform their central role in transmitting visual signals. The results will provide essential new information about how the visual signals arising in the photoreceptor cells are ultimately transmitted to the rest of the brain. [unreadable] [unreadable] [unreadable]

DESCRIPTION (Applicant's Description): The goal of this project is to understand the molecular mechanisms of neurotransmitter responses in retinal bipolar neurons, with a focus on the roles of cyclic nucleotide-gated (CNG) channels. Visual signals from the photoreceptors are funneled to the rest of the brain exclusively through bipolar neurons, which therefore occupy a central position in retinal information processing. The separation of visual information into the ON pathway (in which cells depolarize in response to light) and the OFF pathway (in which cells hyperpolarize in response to light) occurs in bipolar neurons, at the first synapse in the visual system. The proposed research project will explore the cellular mechanisms that generate the ON pathway at the synapse between photoreceptor cells and ON bipolar neurons. At this synapse, glutamate released from the photoreceptor hyperpolarizes ON bipolar cells by activating a transduction cascade culminating in closure of cation channels. These cation channels may be members of the family of CNG channels, which open when cyclic GMP binds directly to the intracellular portion of the channel. A combination of electrical recording and fluorescence imaging techniques will be used to establish the role of CNG channels and cyclic GMP in the glutamate response of ON bipolar cells. Other potential roles of CNG channels will also be explored, such as modulation of neurotransmitter release in the synaptic terminal of the bipolar cell. Molecular biological techniques will be used to identify and characterize the CNG channel subtypes expressed in bipolar neurons. From amino acid sequences derived from complementary DNA encoding CNG channel subtypes, selective antibodies will be produced that can be used to localize the channel subtypes in isolated cells and retinal tissue. Biophysical characterization of CNG channel subtypes found in bipolar neurons will be carried out both in bipolar neurons and in cells transfected with full-length complementary DNA encoding a specific channel subtype. The proposed experiments will provide new information about the synaptic responses of an important class of retinal neurons and will specify physiological roles for specific types of CNG channels expressed in these neurons. Because cyclic nucleotide-gated channels are found in a variety of cell types throughout the nervous system, the results of the proposed project will also have general implications for cellular communication in the other parts of the brain.

DESCRIPTION (provided by applicant): The goal of this project is to develop techniques for the transgenic expression of altered proteins in retinal neurons. These techniques will be important in establishing the functional role of individual proteins in the retina. With the rapid success of genome sequencing projects, there is increasing information about gene sequences but little information about the functional roles of the proteins encoded by those genes. Transgenic techniques are particularly suitable for the task of deciphering those functional roles. Targeted expression of altered proteins or peptide fragments in particular cell types will allow the physiological action of a protein of interest to be tested in an individual cell. Also, the incorporation of a transgene in which expression of a marker protein is controlled by a target gene's regulatory region can be used to determine the cell types that express a particular gene. To accomplish these goals, methods will be developed that will allow the expression of engineered proteins in retinal neurons. Both conventional bacterial plasmid vectors and bacterial artificial chromosomes (BACs) will be explored for producing DNA constructs that are suitable for transgenesis. The transgenic techniques that are the goal of this project can be combined with electrophysiological and imaging techniques to yield a powerful way of attacking fundamental questions of retinal neurobiology. More broadly, completion of the proposed project will lead to new experimental tools that will be generally useful to retina researchers for the next level of functional analysis, after the completion of genome sequencing initiatives.

DESCRIPTION (provided by applicant): The photoreceptor cells of the retina carry out the essential task of translating light into electrical signals that can be passed on and analyzed by the rest of the visual system. Diseases that cause the photoreceptor cells to degenerate lead to irreversible blindness, because the remaining neurons of the retina are not sensitive to light and therefore cannot substitute for the loss of the photoreceptor cells. The goal of the proposed research is to induce sensitivity to light in the surviving retinal bipolar neurons, which would normally receive synaptic inputs from photoreceptors, as a strategy to restore useful vision after photoreceptor degeneration. The approach is to use genetic engineering to induce bipolar neurons to express two different light-activated ion channels, channelrhodopsin-2 and halorhodopsin, that will mimic the naturally occurring light response of ON and OFF bipolar cells, respectively. To drive expression in bipolar neurons, the project will make use of transgenic animals that incorporate DNA encoding channelrhodopsin-2 or halorhodopsin under the control of gene regulatory elements that will confer expression selectively in each of the two subtypes of bipolar neurons. The results will establish the feasibility of using these genetically encoded photosensor proteins as a functional replacement for missing photoreceptors in the diseased retina. PUBLIC HEALTH RELEVANCE: Degeneration of the photoreceptor cells in the retina is a leading cause of blindness, and at present, there is no treatment. The proposed research explores a new approach for restoration of vision despite the loss of photoreceptor cells. The strategy uses molecular engineering to make the remaining neurons of the retina sensitive to light, thereby replacing the function of the missing photoreceptors.