John E. Dowling - US grants

This project is concerned with the structure, function, chemistry and development of synapses within the vertebrate retina. The processing of visual information within the retina depends on, and derives from, the synaptic interactions occurring within the retinal plexiform layers. An understanding of these synaptic junctions is prerequisite for an understanding of retinal function. For the next five years of the project, five sub-projects are planned: 1) Synaptic organization of physiological subtypes of ganglion cells. The synaptic contacts made onto catfish and rabbit ganglion cells, stained intracellularly with horseradish peroxidase, will be determined by electron microscopy. 2) Synaptic organization of pharmacological subtypes of retinal elements. The synapses made by an onto identified retinal elements in fish and rabbit, stained with transmitter-specific monoclonal antibodies, will be examined by electron microscopy. 3) The production and characterization of monoclonal antibodies against retinal neurons. Monoclonal antibodies specific to the surfaces of fish retinal cells will be produced and the antibodies used to probe for functional groups on retinal cell membranes. 4) Isolation and biochemical characterization of horizontal cell gap junctions. Gap junctional membranes will be isolated from the white perch retina, the junctional proteins extracted and separated, and the possible phosphorylation of the junctional proteins by cyclic AMP dependent kinase tested. 5) Formation of synapses between fish retinal cells in culture. The factors underlying retinal cell longevity, process growth and synaptogenesis in culture will be studied.

The overall goal of this project is to gain an understanding of retinal function. For the next 5 years, we are focusing our efforts on attempting to identify the neurotransmitters used by the retinal neurons and especially on the effects these substances have on retinal cells. Much of the work planned will utilize isolated retinal cells maintained in culture for periods of days to weeks. Five specific projects are proposed: 1) Patch and voltage clamping of isolated horizontal and bipolar cells of the carp and skate retina. This project is studying by patch clamping techniques the response of the horizontal cell membrane to L-glutamate and its analogues. We plan in particular to use patch clamp electrodes to voltage clamp the isolated cells in order to study the ion flows that occur in these cells in response to neurotransmitter substances. 2) An analysis of the ionic mechanisms underlying the responses of isolated and cultured horizontal and bipolar cells of the carp and skate retinas to various neurotransmitter agents, and a quantitative evaluation of the effects of neurotransmitter agonists and antagonists on these neurons. For these experiments, drugs or altered Ringer's solution will be applied to the isolated cells by a microperfusion technique. 3) A physiological and pharmacological study of neurons in culture other than horizontal and bipolar cells, and a study of isolated neurons maintained in culture that are derived from animals other than fish. We are particularly interested in maintaining amacrine cells in culture and studying their physiological and pharmacological properties and also studying isolated retinal neurons from a mammal, perhaps rabbit, in culture. 4) A study of synapse formation between neurons maintained in culture and a physiological and pharmacological analysis of such synapses. Initially we plan to study electrical synapse formation between cultured horizontal cells, but eventually we hope to induce and study chemical synapse formation between a variety of retinal cells. 5) An analysis of the effects of drugs and altered Ringer's solution on neurons in the intact perfused retina. These experiments are intended to complement the isolated cell studies by comparing the effects of drugs and/or altered Ringer's solution on isolated cells with the effects of the same treatments on cells in the intact retina.

This project is attempting to learn more about retinal synaptic mechanisms. In particular, we are seeking to discover what substances are used at specific synaptic junctions and what effects these substances have on retinal cells. It is now generally believed that two basic types of substances are released at brain synapses; neurotransmitters which initiate fast excitatory and inhibitory responses in neural cells, and neuromodulators which mediate slow, long lasting events in nerve cells. The next five years of this project will focus on the action of, and interaction between, glutamate, a classic neurotransmitter,and dopamine, a classic neuromodulator, on bipolar, amacrine and ganglion cells of the fish (white perch retina). We also will study the pharmacology and molecular biology of a newly identified GABA receptor/channel, the GABA-c receptor, from white perch or hybrid bass retinas. Finally, recordings will be made from cone photoreceptors of zebrafish that absorb maximally in the ultraviolet region of the spectrum. Specific projects planned for the next five years include: 1) A study of amino acid-gated channels in retinal bipolar, amacrine and ganglion cells and their modulation by dopamine and other neuroactive substances. This project is focusing initially on the glutamate-mediated responses of bipolar cells. Bipolar cells in retinal slices are voltage- clamped with patch electrodes and the glutamate responses characterized. 2) A study of the pharmacological and molecular biological properties of GABA-c receptors in retinal horizontal cells and in Xenopus oocytes. This project will examine the conformation of GABA that best activates these novel receptors. This part of the project will employ conformationally- restricted GABA analogues and isolated rod (H4) horizontal cells from the white perch or hybrid bass retinas. Other studies will involve expressing in Xenopus oocytes the gene(s) encoding the subunits that make up these novel receptors. 3) An investigation of the response properties of ultraviolet-sensitive photoreceptors. This project will explore the response properties of UV- sensitive cones isolated from the zebrafish. Suction electrodes will record the currents generated by these photoreceptors.

For the next five years of this project, we shall focus on experiments designed to elucidate the role of dopamine in the inner retina. In particular, we shall carry out experiments designed to uncover to role of dopamine in mediating rod signal transmission through the inner plexiform layer of the zebrafish. We shall also develop more sophisticated electrophysiological tests, based on electroretinographic (ERG) recordings, to analyze zebrafish retinal mutations. With behavioral tests, we have identified both recessive and dominant mutations that affect retinal function in zebrafish. A number of the mutations resemble inherited retinal diseases in man including dominantly- inherited retinitis pigmentosa (our nba mutant) protanopia (our pob mutant) and congenital stationary night blindness (our noa mutant). Further, electrophysiological testing of these mutants will help identify where in the retina a specific defect resides and more about the nature of the defect. Specific sub-projects planned for the next five years include: 1) An analysis of ganglion cell responses in normal zebrafish and zebrafish deprived of dopamine by the intraocular injection of 6- hydroxydopamine. Ganglion cells will be patch-clamped and synaptic currents evoked in the cells by puffs of K+ applied in the inner plexiform layer. 2) The electroretinogram (ERG) of the zebrafish will be dissected pharmacologically by drugs that selectively block transmission from photoreceptor cells to all second-order cells, transmission from rod photoreceptor cells to ON-bipolar cells, and transmission from rod and cone photoreceptor cells to OFF-bipolar cells. As part of this sub- project, the effects of these pharmacological manipulations on the visual performance of zebrafish will be tested behaviorally. 3) The ERG responses of selected zebrafish mutants will be further studied using the pharmacological manipulations described in (2) above. In particular, the effects of the mutations on photoreceptor (ERG a-wave) function will be examined.

[unreadable] DESCRIPTION (provided by applicant): To examine in more detail the developmental processes in the vertebrate retina, new screening methods will be used in zebrafish. Specifically, transgenic zebrafish expressing the green fluorescent protein (GFP) under a rod photoreceptor specific promoter will be used in two ways: A) A forward genetic screen to identify recessive mutations affecting rod development. Homozygous male transgenic zebrafish will be mutagenized by exposure to ethylnitrosourea. Mutagenized zebrafish will be bred to homozygous transgenic females to produce F2 generation families that are homozygous for the transgene. To identify recessive mutations, F3 generation fish will be examined by light and fluorescence microscopy for changes in GFP expression, indicating an alteration in rod photoreceptor number and/or retinal differentiation. B) Chemical genetic screens to identify molecles that disrupt pathways involved in proper retinal development and rod differentiation. Transgenic embryos will be arrayed at a density of 3 embryos/well of a 96-well plate and exposed to small molecules from libraries of known bioactive substances. The small molecule libraries consist of 2489 compounds and were assembled to contain molecules with known biological activity and with previously identified protein targets. Embryos will be exposed to small molecules at three timepoints significant in eye development: 9 hours post fertilization (hpf), when optic cup formation begins; 24 hpf, a time of retinoblast proliferation but prior to cellular differentiation; 48 hpf, the onset of rod photoreceptor differentiation. Embryos will be examined by standard light microscopy to screen for general morphological defects, differential interference contrast (DIC) microscopy to screen for defects in retinal lamination, and fluorescence microscopy to screen for defects in rod photoreceptor differentiation. Upon identification of specific mutations or small molecules, the phenotypes will be characterized by histology, immunohistochemistry, in situ hybridization, and mosaic analysis, when necessary. The goal of these studies will be to identify pathways in retinal development that could be missed in routine forward genetic screens. [unreadable] [unreadable]

Project Summary/Abstract: Little is known about the cellular interactions within the human macula/fovea. This is especially true for interactions between retinal glial cells and retinal neurons ? interactions likely important to understanding retinal degenerative diseases including Macular Telangiectasia (MacTel), a form of age-related macular degeneration (AMD) in which it has been proposed that a defect in the Müller glial cells may be at play. Müller glia cells are relied upon to regulate ionic balance and neurotransmission, maintain metabolic stasis, constitute the blood-retinal barrier, among multiple other essential neuroprotective functions. An increasing focus has been on the dependence of retinal neurons on Müller cell based L-serine biosynthesis since retinal neurons (as do neurons throughout the CNS) lack the rate limiting biosynthetic enzyme PHGDH. L-serine synthesis is essential for lipid metabolism and maintenance of mitochondrial function. Not only have metabolomics studies implicated alterations in the L-serine metabolic pathway in the development of MacTel but genome-wide association/GWAS studies have linked alterations in the PHGDH gene with early onset MacTel. Not surprisingly, alterations in the normal relationships between neurons and glial cells feature prominently in maintaining normal retinal function and are implicated in the etiology of multiple forms of retinal degeneration. In recent years, electron microscopic (EM) techniques have been developed such that it is now possible to reconstruct pieces of retinal tissue down to the membrane level. Termed connectomics, it is possible to cut and collect on tape, thousands of serial sections, and image specific regions of the sections with an EM that has multiple beams, allowing for 61 images to be obtained simultaneously. Software methods for aligning the images into a single 3D volume have also been developed. To date, this connectomics approach has not been applied to gaining an understanding of pathological changes that underlie retinal degenerative diseases. The first aim in our current proposal is to determine the structural features/changes in glial-neuronal relationships and mitochondrial health involved in MacTel. These studies will focus especially on the cristae structure of mitochondria, mitochondrial degradation, and the relationship between Müller cells and the photoreceptor axons and synaptic terminals. We have made significant progress in making the typically lengthy connectomics workflow more efficient and tailored to analyzing the basis of a neurodegenerative disease. Using our targeted high-throughput connectomics approach our second aim is to perform parallel analysis of our genetically similar 79-year-old donor eye and other retinas using methods refined from our experience working with our 48-year-old donor eye. In summary, we are confident that our targeted high-throughput connectomics approach will enable us to efficiently extract relevant ultrastructural data from multiple diseased and control retinal samples. In future, having the ability to perform efficient large-scale ultrastructural studies will provide a pathway to understand the cellular basis of retinal degenerative diseases.