Peter B. Detwiler - US grants

Our long term goal is to understand the cellular physiology of vertebrate photoreceptors. The research during this grant period focuses on membrane and intracellular events involved in the generation of the rod light response. The specific aims are: 1) to use patch clamp recording techniques to study the properties of the light-sensitive channels in the outer segment surface membrane. The emphasis of this work will be to investigate how the channels are influenced by external Ca, light and voltage. 2) To use the same recording technique to document the changes in the properties of outer segment membrane as it ages and undergoes modifications associated with the shedding process. 3) To study the influence of sulfhydral reagents on the ionic currents in excised patches of outer segment membrane. Particular attention will be placed on understanding the basis of the single channel currents evoked by dithiothreitol and their relationship to other outer segment ion channels. 4) To understand the control conditions necessary for stable long term recording from rods during internal dialysis. The motivation is to find a method to study the internal pharmacology of the transduction process. 5) To produce in vivo alterations in rod disks through intraoccular injection of compounds. The goal of these experiments is to study the intradiskal events evoked by light. 6) To use the above technique in conjunction with metallochromic Ca indicators to measure the light-evoked changes in intradiskal Ca. The outlined research will provide fundamental information about the physiology of rods in general and about the properties of outer segment surface membrane in particular. This will further our basic understanding of photoreception and enlarge our view of receptor biology. The latter is crucial for a full understanding of the pathophysiology of blindness caused by degenerative retinal diseases such as retinitis pigmentosa.

Our long term goal is to understand the cellular physiology of vertebrate photoreceptors. The research during this grant period focuses on membrane and intracellular events involved in the generation of the rod light response. The specific aims are: 1) to use patch clamp recording techniques to study the properties of the light-sensitive channels in the outer segment surface membrane. The emphasis of this work will be to investigate how the channels are influenced by external Ca, light and voltage. 2) To use the same recording technique to document the changes in the properties of outer segment membrane as it ages and undergoes modifications associated with the shedding process. 3) To study the influence of sulfhydral reagents on the ionic currents in excised patches of outer segment membrane. Particular attention will be placed on understanding the basis of the single channel currents evoked by dithiothreitol and their relationship to other outer segment ion channels. 4) To understand the control conditions necessary for stable long term recording from rods during internal dialysis. The motivation is to find a method to study the internal pharmacology of the transduction process. 5) To produce in vivo alterations in rod disks through intraoccular injection of compounds. The goal of these experiments is to study the intradiskal events evoked by light. 6) To use the above technique in conjunction with metallochromic Ca indicators to measure the light-evoked changes in intradiskal Ca. The outlined research will provide fundamental information about the physiology of rods in general and about the properties of outer segment surface membrane in particular. This will further our basic understanding of photoreception and enlarge our view of receptor biology. The latter is crucial for a full understanding of the pathophysiology of blindness caused by degenerative retinal diseases such as retinitis pigmentosa.

The outer segment region of the vertebrate photoreceptor is the only light- sensitive element in the entire visual system. The ultimate goal of this research is to understand how it works. The highly specialized purpose of the outer segment is to couple the absorption of light to the reduction of a standing cation current that steadily flows into the outer segment in darkness. Whole-cell voltage clamp is used to measure inward dark current through light-regulated ion channels in the plasma membrane of functionally intact isolated rod outer segments that have been mechanically detached from the rest of the receptor cell. Diffusional exchange between outer segment cytoplasm and whole-cell pipet filling solution is used to study the effects of specific changes in the biochemical composition of the intracellular compartment on the generation, maintenance and light- regulation of recorded dark current. The underlying processes are mediated by changes in two intracellular messengers, cGMP and Ca. The investigation will describe the reciprocal interactions between cGMP and Ca during the generation of the light response in darkness and adapting background light. More direct information about the molecular events that couple light to a change in outer segment dark current will be obtained by combining whole- cell voltage clamp with optical measurements: fluorescence, IR-light scattering, and light absorption. Optical methods will be used to assess the extent of cytoplasmic manipulations produced by internal dialysis and to study light-evoked changes in internal Ca, G protein state and intradiskal membrane potential. Hypotheses about the mechanism of phototransduction and its regulation in dark and light adaptation will be tested further using light-sensitive excised patches of outer segment plasma membrane. The presence of the transduction machinery in inside-out excised patches makes it accessible to bath applied reagents and the effects of specific molecular components will be studied by reconstitution. Basic research on the physiology, biochemistry and cell biology of the rod outer segment provides a more clear understanding of "how it works" in health and in so doing expands the foundation for understanding "how it does not work" in disease.

The outer segment region of the vertebrate photoreceptor is the only light- sensitive element in the entire visual system. The ultimate goal of this research is to understand how it works. The highly specialized purpose of the outer segment is to couple the absorption of light to the reduction of a standing cation current that steadily flows into the outer segment in darkness. Whole-cell voltage clamp is used to measure inward dark current through light-regulated ion channels in the plasma membrane of functionally intact isolated rod outer segments that have been mechanically detached from the rest of the receptor cell. Diffusional exchange between outer segment cytoplasm and whole-cell pipet filling solution is used to study the effects of specific changes in the biochemical composition of the intracellular compartment on the generation, maintenance and light- regulation of recorded dark current. The underlying processes are mediated by changes in two intracellular messengers, cGMP and Ca. The investigation will describe the reciprocal interactions between cGMP and Ca during the generation of the light response in darkness and adapting background light. More direct information about the molecular events that couple light to a change in outer segment dark current will be obtained by combining whole- cell voltage clamp with optical measurements: fluorescence, IR-light scattering, and light absorption. Optical methods will be used to assess the extent of cytoplasmic manipulations produced by internal dialysis and to study light-evoked changes in internal Ca, G protein state and intradiskal membrane potential. Hypotheses about the mechanism of phototransduction and its regulation in dark and light adaptation will be tested further using light-sensitive excised patches of outer segment plasma membrane. The presence of the transduction machinery in inside-out excised patches makes it accessible to bath applied reagents and the effects of specific molecular components will be studied by reconstitution. Basic research on the physiology, biochemistry and cell biology of the rod outer segment provides a more clear understanding of "how it works" in health and in so doing expands the foundation for understanding "how it does not work" in disease.

DESCRIPTION (provided by applicant): The goal of the proposal is to exploit advanced optical recording methods to study the basic properties of retinal dendrites and the role they play in the collection, integration and processing of visual information. Dendrites are the most dominant feature of a retinal neuron;they cover more surface area and are functional more complex than any other neuronal component, yet they are the least understood. They are too small for routine electrical recording, but it is possible to record from dendrites optically using two-photon laser scanning fluorescent microscopy to excite a Ca2+ -dependent fluorophore and measure changes in the fluorescence signal in response to visual stimulation. The investigator is a pioneer in the use of this method and has demonstrated its unique ability to monitor signals from dendritic compartments too small to be recorded form in any other way. In this research optical recording will be used to study the mechanisms responsible for the generation of light-evoked dendritic Ca2+ signals and their spatio-temporal spread through the dendritic arbor. This includes determining the relationship between the properties of the Ca2+ signal and visual stimulus that evoked it under dark and light adapted conditions. The retina is an image processor that is made up of neurons whose function as computational units depends on their dendrites. Hence in order to replace an eye lost to disease or trauma with a prosthetic devise it is necessary to first know how the neurons work and this requires knowing how their dendrites work.

The research goal is a novel application of multiphoton laser scanning microscopy to determine neural coding mechanisms of diverse cell types and local circuits in the vertebrate retina that have been previously inaccessible by current experimental methods. Our specific request is to acquire a scanning laser microscope with specialized features that uniquely permit optical recording of neural signals from retinal neurons while simultaneously stimulating the retina with complex visual stimuli. The requested instrument is a BioRad 2000 MP two-photon scanning laser microscope coupled to a femtosecond laser light source and an open optical architecture that allows optical recording of calcium signals evoked by light stimulation of the intact retina maintained in vitro. The critical application of this instrument to vision research is dependent on the confinement of fluorescence activation to a spatially restricted femtoliter-sized volume at infrared wavelength with insufficient energy to directly excite the light sensitive retinal photoreceptors. The requested instrument is a newly developed tool that is specialized for retinal research and will be used almost exclusively by three major investigators whose currently funded specific aims are focused on mechanisms of signal processing by identified retinal neurons. Each investigator will use the instrument to determine the link between calcium signals in morphologically identified dendritic processes and specific features of retinal and visual function. Detwiler proposes to further investigate the basic properties of the light-evoked calcium signals. This will include understanding their dependence on visual stimuli, their biophysical properties and the molecular mechanisms responsible for their generation. Rieke proposes to determine how mechanisms in the synaptic processes of rods, bipolar cells and amacrine cells transform the rod-mediated light response. Dacey proposes to determine how a color code in the primate retina is computed at the dendritic tree of newly identified retinal cell types. Because of the specialized application of this research to visual neuroscience the instrument will be linked to existing equipment needed for physiological recording and light stimulation. The instrument will be heavily used by each of the three investigators since the microscope would be engaged on a daily basis in experiments that will run for several hours.