Dennis M. Dacey - US grants

The proposed research will determine the morphology, topographic distribution, transmitter candidates and synaptic relations of three distinct neuronal cell types that have been observed for the first time in an in vitro preparation of the cat's retina. During the previous project period it was demonstrated that after intraocular injections of dopamine and the indoleaminergic transmitter analogue 5,7-dihydroxytryptamine (5,7-DHT), an intense yellow-green fluorescence (resembling formaldehyde-induced histofluorescence) appears in distinct subpopulations of neurons in the inner plexiform layer and the ganglion cell layer of the living retina. Although the chemical basis for the in vitro fluorescence is not understood, its discovery permitted the identification of the fluorescing cells by intracellular injections of horseradish peroxidase (HRP) under direct microscopic control. Three distinct monoamine-accumulating (Ma) cell types were identified: the first an amacrine cell, the second a ganglion cell and the third a cone bipolar cell type. Morphological analysis of the HRP injected cells demonstrated that the processes of the three cell types costratify in a narrow stratum at the outer border of the inner plexiform layer. The proposed research will be divided into five related projects that exploit the in vitro fluorescence to further characterize these cell types. 1) Following intracellular injections of HRP, an analysis of the detailed morphology, stratification, and mosaic organization of the dendritic trees of each type by will be completed. 2) The topographic distribution of each type will be determined by a method that permits systematic sampling form the living retina. 3) Neurotransmitter candidates for the Ma cell types will be investigated by colocalizing immunoreactivity for tyrosine hydroxylase, serotonin or GABA with intracellular injections of the fluorescent dye Lucifer Yellow. 4) The central projections of the Ma ganglion cell type will be examined by colocalizing retrogradely transported fluorescent tracers that have been injected into the superior colliculus or the C-laminae of the dorsal lateral geniculate nucleus, with the in vitro fluorescence. 5) The hypothesis that the three Ma cell types are functionally linked will be tested; potential synaptic contacts between HRP-injected Ma cells identified by light microscopy will be subsequently analyzed by electron microscopy. Taken together, the first three projects will provide a detailed, light microscopic characterization of each of the cell types. The last two projects will test the hypothesis that they are in direct synaptic contact, and comprise the major intraretinal elements in a newly identified visual pathway that projects to the superior colliculus and/or the C-laminae of the dorsal lateral geniculate nucleus.

The long term goal is to understand the structure and function of the diverse ganglion cell types in human and macaque retina and the cellular mechanisms and circuits that give rise to parallel visual pathways. Our specific goals for the next period are to determine the origin and mechanisms of separate blue/yellow and red/green color opponent pathways. A unique in vitro preparation of the intact macaque monkey retina will be used to record intracellularly form anatomically identified ganglion and amacrine cell types. The proposed research has four specific aims: 1) to determine the locus and cellular mechanisms for blue/yellow spectral oppenency in the small bistratified ganglion cell type. We will test the novel hypothesis that blue-ON/yellow-OFF spectral antagonism derives from combined excitatory ON- and OFF-pathway cone bipolar inputs to the bistratified dendritic tree. 2) To determine the locus and retinal mechanism for red-green spectral opponency in the midget ganglion cell type. We will test the hypothesis that the relative weightings of L- and M-cone input to the excitatory center and inhibitory surround of the midget ganglion cell type determines the presence and degree of red/green opponent signals. 3) To measure the color opponent and non-opponent properties of the newly identified ganglion cell types that project to the lateral geniculate nucleus (LGN). We will characterize in detail the morphology and physiology of novel ganglion cell types that project to the LGN and determine their roles in color-opponent and non-opponent pathways to primary visual cortex. Tracer injection will be made into physiologically identified sites in the LGN and retrogradely labelled ganglion cells will be targeted for intracellular recording and analysis in the in vitro retina. 4) To determine the light responses and functional cone connections of identified amacrine cell types in macaque. We will continue our analysis of primate amacrine cell physiology and test the hypothesis that distinctive small-field cell types contribute to blue/yellow, red/green and non-opponent cone signal pathways. Many aspects of macaque vision and visual pathway organization are comparable to the human counterpart; our results therefore will contribute to the best and most detailed structure-function model of the cell types and functional architecture of the human retina. Primate retinal cell types have traditionally been inaccessible to physiological analysis and their functional significance and relevance to human retinal disease and visual disorders have remain unexplored. Taken together, the proposed projects will contribute to clarifying the retinal origins and circuits for color-opponent pathways, the evolution of color vision in primates, the cellular basis for psychophysical measures of human color vision and mechanisms by which retinal disease affects human color vision.

The goal of this research is to understand the structure and function of ganglion cells-the output neurons-in macaque retina. Certain cells in the retina-the photoreceptors-are stimulated by long-, medium-, or short-wavelength light. The photoreceptors pass information to bipolar cells, which in turn relay to ganglion cells, which then output to visual centers of the brain. There are also cells within the retina-horizontal and amacrine-that "hook up" to these circuits and share information with other cells within the retina. Some monkey species, like humans, share color vision based on three primaries-red, green and blue-which feed into two color-opponent channels in the brain. Our experiments are directed at determining which combinations of cell types are responsible for coding information about blue/yellow and red/green channels. We use intracellular recording and imaging in a unique preparation of living retina, combined with a three-color light, to test sensiti vity and c onnectivity of individual cells. FUNDING NIH grants RR00166, EY09625, EY06678, and EY03221. Peterson, B. and Dacey, D.M. Morphology of wide-field, monostratified ganglion cells of the human retina. Invest. Ophthal. Vis. Sci., Suppl. 39 S563, 1998 (abstract). Verweji, J., Dacey, D.M., and Buck, S.L.Receptive field size, sensitivity and dynamics of rod signals in macaque HI horizontal cells. Invest. Ophthal. Vis. Sci., Suppl. 39 S209, 1998 (abstract). Smith, V.C., Dacey, D.M., Lee, B.B., and Pokorny, J. Time dependent adaptation in primate horizontal cells. Invest. Ophthal. Vis. Sci., Suppl. 39 S209, 1998 (abstract). Lee, B.B., Dacey, D.M., Smith, V.C., and Pokorny, J. Spatial independence of adaptation in primate outer retina. Invest. Ophthal. Vis. Sci., Suppl. 39:S210, 1998 (abstract). [This project was also reported in the Neuroscience Core section.]

ABSTRACT NOT AVAILABLE

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this project is determine how the receptive field surround is established at the level of the ganglion cell and how the surround contributes to color-coding by identifying the cone inputs to the surround. The presynaptic hypothesis predicts that the creation of the cone bipolar center-surround organization is the critical locus for cone opponency. We showed for the first time in a mammalian retina that cone bipolar cells have robust center-surround organization comparable to their ganglion cell counterparts. We then undertook a series of experiments to attempt to selectively block ganglion cell surrounds. We found, in parasol cells, that either cobalt or the gap-junction blocker, carbenoxolone, selectively reduces surround antagonism. We are now using these drugs to assess the receptive field surround contribution to color-opponency in a number of ganglion cell classes. In a second series of experiments, we developed a new stimulus protocol to measure the strength and sign of L, M and S-cone inputs to the receptive field surrounds of midget and parasol ganglion cell types. The specific goal was to assess how cone signal weighting may be altered during transmission from outer to inner retinal circuitry. We showed that there are no gain changes from the horizontal-bipolar cell level to the ganglion cell surround and concluded that cone type-specific changes in synaptic gain are not a likely mechanism for generating color-opponent signals.

neurophysiology; neuroanatomy; ganglion cell; cell biology; animal colony; Primates; visual photoreceptor; clinical research;

Abscission; Blood Coagulation Factor IV; CRISP; Ca++ element; Calcium; Calcium Ion Signaling; Calcium Signaling; Cell Body; Cells; Characteristics; Coagulation Factor IV; Collaborations; Color; Computer Retrieval of Information on Scientific Projects Database; Cone; Cones (Eye); Cones (Retina); Dendrites; Development; Diffuse; Electromagnetic, Laser; Excision; Extirpation; Factor IV; Funding; Goals; Grant; Image; Inner Limiting Membrane; Institution; Investigators; Laser Scanning Microscopy; Lasers; Light; Macaca; Macaque; Mammals, Mice; Measures; Mechanics; Mice; Microscope; Modeling; Murine; Mus; NIH; National Institutes of Health; National Institutes of Health (U.S.); Photons; Photoradiation; Photoreceptors, Cone; Physiologic pulse; Pulse; Pulse taking; Radiation, Laser; Removal; Research; Research Personnel; Research Resources; Researchers; Resources; Retina; Retinal Cone; Salamander; Scanning; Scanning Microscopy, Laser; Signal Pathway; Source; Students; Surgical Removal; Synapses; Synaptic; System; System, LOINC Axis 4; Testing; Trees; United States National Institutes of Health; Whole-Cell Recordings; calcium green; cell body (neuron); cone cell; digital; gangliocyte; ganglion cell; imaging; neural cell body; neuronal cell body; resection; response; retinal neuron; soma; visual stimulus

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal is to characterize a unique ganglion cell population that contains a novel photopigment and is intrinsically photosensitive;these ganglion cells play a key role in entraining human circadian rhythms, driving the pupillary light reflex and may also participate in color perception. This project marks the first steps in the analysis of novel visual pathway and photopigment, not previously recognized in the primate visual system. We have started a collaborative project with King-Wai Yau, employing a human antibody to the putative photopigment melanopsin, and have now determined the detailed morphology and spatial distribution of these ganglion cells in both macaque and human retina. Using retrograde photodynamics we have begun to define the central targets of these cells, showing that these cells project widely to both the superior colliculus and lateral geniculate nucleus as well as the pretectal olivary nucleus. We have targeted these cells in vitro for detailed physiological analysis and have characterized cone inputs, rod inputs and the spectral tuning and dynamics of the intrinsic light response. Intracellular recordings show that both the intrinsic and cone-mediated signals converge to confer unique luminance coding and chromatic properties to this ganglion cell.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Experimental evidence in other animal models has demonstrated that retinal function can be restored using gene therapy in adults. However, the effects on normal retinal physiology following gene therapy has not been investigated at the cellular level. This is an important question that is central to determining whether ocular gene therapy can ultimately be used to treat human vision anomalies and disease. The model system we will test is non-human primate retinal physiology following incorporation of new genetic material via viral vector. Following expression of a gene for green fluorescence in retinal cells, physiology and morphology will be assessed in vitro. Modifications to the promoter genes will be made so as to potentially target specific retinal cell subpopulations.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Color perception is one of the key features of our visual system that permits an appropriate interaction with our environment. Hence, the investigation of neuronal mechanisms forming the basis for color vision is one of the major goals in the field of retinal neuroscience. Processing of color signals starts already at the level of the retina, where the light responses of the different cone photoreceptor types are 'compared'and modulated. Among other mammalian species, humans and many of the non-human primates show one of the most highly developed systems for color vision. This system is based on three chromatically tuned cone types (red, green, and blue). Retinal ganglion cells receive and process the cones'signals and send them to higher visual centers of the brain. Up to date, research on color-coded ganglion cells of the primate retina focused on two distinct cell types: the midget ganglion cell (red-green processing) and the small bistratified ganglion cell (blue-yellow processing). However, further primate ganglion cell types have been discovered, which showed color-opponent responses to chromatic light stimuli. Though a detailed analysis is still missing, one can expect that these cell types also play a fundamental role in primate color vision. These cell types include the large bistratified ganglion cell, which is supposed to show blue-yellow opponent responses, similar to what has been observed in the small bistratified cell. The aim of this project is to provide a comprehensive study on the anatomical and physiological properties of the large bistratified ganglion cell in the primate retina. The main focus will be directed at the analysis of the neuronal pathways and the synaptic mechanisms, underlying the chromatically tuned responses of this cell type.

A major research challenge for neurobiology is to understand the neural mechanisms that give rise to an extreme diversity of parallel visual pathways and ultimately the contributions that these pathways make to our perception of motion, form and color. For motion perception the cell types, circuits and synaptic mechanisms that mediate selectivity to the direction of moving stimuli have been intensively studied in the non-primate mammal for decades and over a dozen distinct direction selective pathways are recognized in the mouse retina together with growing evidence for similarly diverse underlying neural mechanisms. The great complexity of the visual pathways found in the mouse is mirrored in the primate, yet surprisingly the abundant direction selective ganglion cells have not been previously identified. The broad long-term objective of this new research program is to elucidate for the first time the cell types, circuits, synaptic organization and underlying cellular mechanisms for direction selectivity in the macaque monkey retina, as an ideal model for human visual processing centered around the fovea. Our proposed research plan arises from a series of discoveries that opens a door to the first detailed study of both the visual physiology and synaptic organization of direction selective circuitry in the macaque retina. In preliminary studies we have identified the primate ON-OFF direction selective ganglion cell as the recursive bistratified type and have developed new methods that permit systematic targeting of this cell type for analysis. The synaptic physiology and directional tuning of this ganglion cell type are the focus of Aim 1 where we test the hypothesis that directional selectivity in the primate is radially aligned with respect to the fovea. Second, we have developed reliable methods for targeting the starburst amacrine cell type, the key retinal interneuron in the direction selective circuit, for both physiological analysis and connectomic circuit reconstruction for the first time. Preliminary data reveal novel features of starburst receptive field structure, directional tuning and connectivity providing the focus for Aim 2 where we test new hypotheses for the cellular origins of direction selectivity and its synaptic transfer to ganglion cells. Finally, we have discovered direction selectivity in the poly-axonal spiking A1 amacrine cell type and evidence for a functional link to ON-OFF direction selective ganglion cells. The focus of Aim 3 therefore is to test the hypotheses that the A1 cells unique axonal component provides synaptic input to both starburst and ON-OFF direction selective ganglion cells, and determine the role of the A1 cells unique dendro-axonal structure in direction selectivity. In sum the broad aim is to characterize the directional tuning properties of these three cell types, and to use connectomics for the first time to determine the underlying synaptic interactions that create direction selectivity in the primate retina. Outcomes will thus have a specific impact on understanding of mechanisms motion processing in human vision and more broadly on growing applications of the primate model for the development of tools and methods for vision restoration.