Kwoon Y. Wong - US grants

DESCRIPTION (provided by candidate): All organisms display daily cyclical changes known as circadian rhythms in their physiological processes, and in mammals such rhythms are driven internally by the suprachiasmatic nucleus (SCN). These internal rhythms are reset ("entrained") daily by the solar cycle so that they are in phase with the external light/dark cycle. Desynchronization between the internal and external rhythms results in "jet lag" symptoms which can have serious health consequences. Besides the classical photoreceptors, that is, rods and cones, the recently identified intrinsically photosensitive retinal ganglion cells (ipRGCs) are also involved in entrainment. While the rods/cones and the ipRGCs are known to be physiologically distinct, their differential roles in entrainment are poorly understood. In addition, interactions between the rod/cone system and the ipRGCs have not been explored. In this project, both issues will be studied at the retinal and the SCN levels, using patch-clamp and extracellular recording techniques combined with pharmacological manipulations. These experiments may enable a better understanding of how the mammalian circadian pacemaker is regulated, which in turn may lead to better therapies and workplace policies combating/preventing jet lag problems.

[unreadable] DESCRIPTION (provided by applicant): One function of the visual system is to enable conscious visual perception of details in the environment; this requires the rod and cone photoreceptor cells in the retina. The visual system also mediates subconscious photic regulation of various physiological functions such as resetting of our body clocks to the solar cycle. Failure to reset these clocks results in "jet lag" symptoms which can have serious health consequences including sleep disorders and cancer. Subconscious photoreception requires not only rods and cones but also the recently discovered ganglion-cell photoreceptors, i.e. the intrinsically photosensitive retinal ganglion cells (ipRGCs). ipRGCs interact bidirectionally with rod/cone-driven bipolar-cell and amacrine-cell circuits. This proposal uses electrophysiological and imaging methods to study such interactions in rodent retinas and comprises three goals: (1) To characterize the functions and morphologies of bipolar and amacrine cells innervating the ipRGCs, through dual intracellular recordings from a bipolar/amacrine cell and an ipRGC followed by fluorescence imaging of dyes injected into these cells; (2) To examine the influence of dopamine and other amacrine-cell neuromodulators on ipRGC light responses by analyzing the effects of receptor agonists and antagonists; and (3) To investigate which bipolar and amacrine cell types receive synaptic inputs from ipRGCs, through dual intracellular recordings followed by fluorescence imaging. Findings from these studies will enable a better understanding of how rod/cone signals regulate our body clocks, and will also provide clues as to how the ipRGCs may modulate rod/cone circuits and thus conscious visual perception. LAY SUMMARY: Neuronal circuits for conscious vision and for subconscious visual functions (e.g. regulation of sleep timing) interact in the retina. This proposal will study the functional roles of such interactions and may help design better therapies and workplace policies combating or preventing jet lag problems. CANDIDATE: My #1 career goal is to get a faculty position and continue to explore neuronal mechanisms for both conscious and subconscious vision. This K99/R00 award will help me achieve this goal, since I will learn imaging and anatomical techniques from my sponsor, and the award topic is highly relevant to my long-term research interests and will be a basis for future ROVs. The excellent research facilities and mentor support at Brown University will greatly facilitate execution of the planned experiments. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The retina mediates various subconscious, photoadaptive responses to light, including pupil constriction, acute enhancement of alertness, regulating hormone secretion from the brain, and synchronizing the body clock to the light/dark cycle. Inadequate or mistimed induction of these responses, such as occurs in shift workers or the blind, can lead to jet lag symptoms, winter depression, sleep disorders, headache, and even breast and prostate cancer. Retinal input to the subconscious visual system is mediated by the intrinsically photosensitive retinal ganglion cells (ipRGCs), which generate intrinsic, melanopsin-based light responses as well as extrinsic, rod/cone-driven photoresponses. These neurons were discovered recently and much remains to be learned about how they respond to different kinds of light, and how they interact with other cells in the retina. This application's long-term objective is to fill both of these knowledge gaps and use this knowledge to help develop lighting technologies that promote health and productivity, light therapies for winter depression, better diagnostic tests for eye disorders, and medical innovations that ameliorate conditions of the visually impaired. This grant proposal consists of three Specific Aims. In aim 1, whole-cell recordings will be made from ipRGCs identified using two-photon microscopy, and various pharmacological tools will be used to dissect the molecular and cellular mechanisms underlying these ganglion cells' rod/cone-driven responses to light. In aim 2, whole-cell recording, immunohistochemistry and confocal imaging will be used to identify retinal amacrine cells that receive input from ipRGCs, characterize their light-evoked responses, and determine their potential physiological functions. Aim 3 will use multielectrode-array recording, pharmacological manipulation and behavioral assays to determine the role of the retinal pigment epithelium (RPE) in the generation of ipRGCs' melanopsin-based light responses. The RPE is known to be crucial for the photosensitivity of the classical photoreceptors but their importance for ganglion-cell photoreceptors has not been fully investigated.

DESCRIPTION (provided by applicant): The retina mediates various subconscious, photoadaptive responses to light, including pupil constriction, acute enhancement of alertness, regulating hormone secretion from the brain, and synchronizing the body clock to the light/dark cycle. Inadequate or mistimed induction of these responses, such as occurs in shift workers or the blind, can lead to jet lag symptoms, winter depression, sleep disorders, headache, and even breast and prostate cancer. Retinal input to the subconscious visual system is mediated by the intrinsically photosensitive retinal ganglion cells (ipRGCs), which generate intrinsic, melanopsin-based light responses as well as extrinsic, rod/cone-driven photoresponses. These neurons were discovered recently and much remains to be learned about how they respond to different kinds of light, and how they interact with other cells in the retina. This application's long-term objective is to fill both of these knowledge gaps and use this knowledge to help develop lighting technologies that promote health and productivity, light therapies for winter depression, better diagnostic tests for eye disorders, and medical innovations that ameliorate conditions of the visually impaired. This grant proposal consists of three Specific Aims. In aim 1, whole-cell recordings will be made from ipRGCs identified using two-photon microscopy, and various pharmacological tools will be used to dissect the molecular and cellular mechanisms underlying these ganglion cells' rod/cone-driven responses to light. In aim 2, whole-cell recording, immunohistochemistry and confocal imaging will be used to identify retinal amacrine cells that receive input from ipRGCs, characterize their light-evoked responses, and determine their potential physiological functions. Aim 3 will use multielectrode-array recording, pharmacological manipulation and behavioral assays to determine the role of the retinal pigment epithelium (RPE) in the generation of ipRGCs' melanopsin-based light responses. The RPE is known to be crucial for the photosensitivity of the classical photoreceptors but their importance for ganglion-cell photoreceptors has not been fully investigated.

SUMMARY/ABSTRACT - ELECTRONICS AND COMPUTER MODULE The Electronics and Computer Module, staffed by a full-time electrical engineer, has long been an integral part of a variety of NEI R01-funded research projects at the University of Michigan. Electronic instrumentation and computer-assisted data acquisition and analysis are essential to many areas of vision research, ranging from visual neuroscience to retinal development and regeneration. Although much of the electronic instrumentation used in vision science is commercially available, there remains a high demand for: modification of commercially available equipment; custom equipment such as light sources, filters, shutters, controllers, power supplies and heaters; and technical support to ensure the smooth, coordinated operation of electronic instrumentation, computers, and software. A second way in which the Electronics and Computer Module benefits investigators is by providing assistance with data acquisition and analysis (including programming), as well as hardware and software support for research computers and data backup systems. Finally, this Module provides a rapid repair service for electronic instrumentation including such laboratory equipment as freezers, centrifuges and incubators, which allows investigators to avoid costly delays in their experimental timeline. The Module, housed in a dedicated room in the Brehm Tower at the W.K. Kellogg Eye Center, has a full complement of electronic test equipment, light meters, computer hardware and software to support research, including Tektronix oscilloscopes, general purpose pulse/function generators, power supplies and breadboards, programmers for programmable memory and logic devices, multimeters and logic probes, Windows and Macintosh computers, a spectrophotometer, and A/D & D/A boards for program development, measurement, and testing purposes. Programming software includes C, LabVIEW, and MATLAB. The Electronics and Computer Module was heavily or moderately utilized by 8 participating investigators and their lab members during the past funding period, and 14 participating investigators anticipate using the Module to a moderate or extensive degree in the next funding period. This Module has also provided support for computers in the other Core Modules and will continue to do so in the next funding period.

Classically, photic information was assumed to flow only from rod and cone photoreceptors through bipolar, horizontal and amacrine interneurons to retinal ganglion cells (RGCs), which were assumed to signal only to higher visual centers of the brain. Unexpectedly, the PI and colleagues discovered that a subset of RGCs, namely intrinsically photosensitive retinal ganglion cells (ipRGCs), transmit light-evoked responses intraretinally to several types of amacrine cells (ACs). The functional significance of this counterintuitive retrograde signaling is largely unknown. Since ACs are well-known to regulate the physiology of all classes of retinal neurons, the central hypothesis for this project is that retrograde ipRGC signaling serves to regulate visual processing in the retina, ultimately shaping vision. In the previous funding period the PI?s team identified gap junction (GJ) coupling as one mechanism by which ipRGCs signal to ACs. To begin to assess the roles of retrograde ipRGC signaling, the team genetically knocked out one type of GJ protein specifically in ipRGCs, and an optokinetic behavioral assay showed a reduction in the mice?s ability to track moving stripes of high spatial frequencies or low contrasts. Thus, a functional significance of gap junctional ipRGC-to-AC signaling is that it improves spatial acuity and contrast sensitivity at the behavioral level. The proposed project will test three hypothesized mechanisms by which gap junctional ipRGC-to-AC signaling could enhance acuity and contrast sensitivity. Aim 1 will test the hypothesis that gap junctional ipRGC-to-AC signaling potentiates retinal light responses. The PI?s team will use field-potential recording of bipolar cells and recording of individual RGCs to measure how these cells? responses to single light pulses are altered when GJ-mediated ipRGC-to-AC signaling is manipulated. Aim 2 will test the hypothesis that gap junctional ipRGC-to-AC signaling improves the ability of retinal cells to resolve repetitive changes in light intensity. The team will determine whether manipulating GJ-mediated ipRGC-to-AC signaling alters bipolar and ganglion cells? responses to flickering light. Aim 3 will test the hypothesis that gap junctional ipRGC-to-AC signaling modulates the receptive field properties of RGCs. The team will test whether manipulating GJ-mediated ipRGC-to-AC signaling alters the sizes of RGC receptive fields and the strength of center/surround antagonism in these receptive fields. The research team has strong preliminary data in support of each Aim. The impact of this study will be to: 1) illuminate the roles of a previously overlooked but functionally important retinal signaling pathway; 2) further the appreciation of ipRGCs? contribution to image-forming vision, besides their well-known roles in nonimage-forming photoresponses such as the pupil reflex and circadian photoentrainment; and 3) elucidate the functions of GJ coupling between amacrine and ganglion cells, which remain poorly understood. A potential long-term impact of understanding retrograde ipRGC signaling, which persists following rod/cone degeneration, is to inform new strategies for restoring sight in patients suffering photoreceptor dystrophy.