Eric A. Newman - US grants

The long-term objectives of this research project are to characterize the membrane properties of muller cells of the vertebrate retina and to relate these properties to possible Muller cell functions, including the regulation of extracellular K+ and pH within the retina and the generation of the electroretinogram. Studies conducted during previous grant periods have demonstrated that the membrane conductance of Muller cells is distributed in a highly non-uniform manner over the cell surface. Conductance is localized to cell endfeet and, perhaps, to cell processes in contact with retinal blood vessels. Specific aims for the coming grant period are as follows: 1) Describe the types and spatial distribution of ion channels present in muller cells. This research will test the hypothesis that the high K+ conductance of the Muller cell endfoot is mediated by the same K+ channel(s) that are present in other cell regions.. 2) Describe changes in the ion channels of Muller cells during development, during retinal degeneration, and following retinal injury. This research will test two hypotheses: (i) That blood vessels induce "endfoot-like" properties in Muller cell processes contacting them. (ii) That significant changes occur in Muller cell ion channels and transport processes during retinal degeneration and following retinal injury produced by a penetrating wound. 3) Characterize the electrogenic Na+-HCO3- cotransport system of Muller cells. The stoichiometry of this transport system and its distribution over the cell surface will be studied. 4) Test the hypothesis that K+ and HCO3- are released preferentially from Muller cell endfeet and from processes in contact with blood vessels. Such ion fluxes may help regulate K+ and pH levels in the retina. 5) Test the hypothesis that glial cells regulate blood flow in the retina. 6) Test hypotheses of Muller cell function using computer simulations of Muller cells and retinal K+ dynamics. Electrophysiological investigations on mouse (normal and retinal degeneration mutant), rabbit, salamander and dogfish Muller cells will be conducted using the following preparations: (i) enzymatically dissociated Muller cells, (ii) retinal slices, and (iii) isolated retinas. Whole-cell voltage-and current-clamp, patch-clamp, and ion-selective microelectrode recording techniques will be employed. Elucidating Muller cell membrane properties is critical for understanding the role that these cells play in retinal function. Knowledge of the changes that occur in Muller cells during retinal degeneration and injury will help us understand such clinical conditions as retinitis pigmentosa and retinal trauma.

The long term objectives of this research are to characterize the membrane properties of Muller cells and retinal astrocytes and to relate these properties to possible glial cell functions in the retina, including the regulation of extracellular pH and [K]o, the control of retinal blood flow, and the generation of the electroretinogram. Specific aims for the project period differ from previous periods and examine, primarily, pH regulation in retinal glial cells. The aims are; (1) Characterize the acid/base transport systems of amphibian and mammalian Muller cells. Hypotheses to be tested include; (i) Muller cells possess an electrogenic Na+/HCO-3 cotransport system and a Cl-/HCO-3 exchanger which are both localized tot he cell endfoot. (ii) Muller cells possess a Na+/H+ exchanger and a H+-ATPase. (2) Test the hypothesis that muller cells modulate extracellular pH (pHo) by the action of the electrogenic Na+/HCO-3 cotransporter. The hypothesis will be tested in two preparations; (i) dissociated Muller cells (Salamander and rat) and (ii) Muller cells in situ in the isolated rat retina. (3) Test the hypothesis that Muller cells regulate pHo in the retina by functioning as CO2 buffers, transporting CO2, in the form of H- and HCO-3, to the vitreous humor. (4) Characterize the acid/base transport systems of mammalian astrocytes in situ. Specific hypotheses to be tested include: (i) the acid/base transport systems of mammalian astroytes in situ have properties similar to those found in Muller cells and cultured astrocytes. (ii) the Na+/HCO-3 cotransport system of mammalian astrocytes in situ is localized in cell endfeet. (5) Test the hypothesis that glial cells regulate blood flow in the mammalian retina. Blood vessel diameter will be monitored in the isolated rat retina as the membrane potential of glial cells is modulated by current injection. Acid/base transport systems will be characterized by monitoring intracellular and extracellular pH with ratio imaging of pH-sensitive indicator eyes. Measurements in dissociated salamander and rat muller cells will be made by imaging the dye BCECF with inverted fluorescence microscopy. In situ measurements in the isolated rat retina will be made by confocal imaging of the dye SNARF-1. Elucidating the basic membrane properties of Muller cells and retinal astrocytes is necessary for understanding the ole that these cells play in retinal function under normal and pathological condiitons, including retinitis pigmentosa and macular dystrophy.

The goal of the Core Grant is to further the research aims of 16 Vision Investigators at the University of Minnesota by establishing an integrated series of image acquisition and analysis facilities and associated services. The Grant consists of four research and service modules. The first module, the Image Acquisition and Processing Facility, is the central "anchor" module of the grant and consists of three components. Two of these, the confocal microscope facility and the deconvolution microscope facility, will be used by Core Grant Investigators in acquiring images from histological sections and from live tissue. The third component of the module, the media graphics facility, will be used in analyzing, processing and producing hard copies of images acquired on the confocal and deconvolution microscopes. A technician will supervise the confocal and media graphics facilities. The second module, Network and Microcomputer Support, will provide essential technical support for the Image Acquisition and Processing Facility as well as for individual Investigators using laboratory microcomputers. A microcomputer specialist who is proficient in microcomputer operating systems and networks will serve as the primary resource of the module and will be responsible for maintaining the Core Grant network server. The third module, the Histology Facility, will provide support for histological procedures utilized heavily by Core Grant Investigators. Slide-based in situ hybridization and PCR and histological sectioning of paraffin and cryostat sections will be supported. A histology technician will conduct routine procedures and will maintain the facility. The fourth module, the Machine Shop, will support a skilled machinist and will serve Investigators who require custom machining of equipment and components needed for their research. The modules will be functionally linked together by a dedicated local area computer network that will facilitate Investigator access to and will enhance the capabilities of the facilities. Each module will be headed by an established NEI-funded Investigator. The Core Grant will be administered by an advisory' committee comprised of the Grant PI, module heads, and two additional Department of Ophthalmology representatives. The Core Grant, by enhancing facilities for acquiring and processing images, will further the research efforts and collaborative projects of vision researchers at the University of Minnesota.

computer network; computer program /software; computer system design /evaluation; biomedical facility; vision;

DESCRIPTION (provided by applicant): The long term objective of this project is to determine the functions of glial cells (Muller cells and astrocytes) in the mammalian retina. Preliminary studies from our laboratory show that light-evoked neuronal activity can induce calcium increases in retinal gila and that activation of glial cells can either facilitate or depress synaptic transmission onto ganglion cells. The specific aims for the project period are: 1) Identify the mechanisms by which light-evoked neuronal activity elicits calcium increases in retinal glial cells. Hypotheses to be tested include: i) neurotransmitters released from synaptic terminals evoke glial calcium increases, ii) transmitters released from extra-synaptic regions of ganglion cells evoke glial calcium increases. 2) Determine the cellular mechanism by which glial cells hyperpolarize retinal ganglion cells. We will test the hypothesis that glial cells inhibit ganglion cells by opening ganglion cell G-protein coupled inwardly rectifying K+ channels (GIRKs). 3) Identify the mechanisms by which glial cells facilitate synaptic transmission. Hypotheses to be tested include: Gila facilitate synaptic transmission i) by releasing ATP and activating presynaptic P2X, P2Y or A2 receptors, ii) by releasing glutamate and activating presynaptic mGluRs, iii) by releasing D-serine and facilitating postsynaptic NMDA receptors. 4) Identify the mechanisms by which glial cells depress synaptic transmission. Hypotheses to be tested include: Gila depress synaptic transmission i) by releasing ATP and activating presynaptic A1 receptors, ii) by releasing glutamate and activating presynaptic mGluRs, iii) by releasing glutamate and desensitizing postsynaptic GluRs. Glial cells have been implicated in many types of retinal pathology, including diabetic retinopathy, glaucoma and macular degeneration. Knowledge of the basic physiological properties of retinal gila and their interactions with neurons will add to our understanding of how these cells contribute to retinal pathology. The research outlined in this proposal will provide significant progress towards this goal.

This Core Grant will further the research aims of 17 vision investigators at the University of Minnesota by supporting four research and service modules. The Digital Imaging module, consists of two components, the Confocal Microscope facility and the Image Analysis facility. The Confocal facility will be used by Core Grant investigators to acquire images from histological sections and from live tissue. The Image Analysis facility will be used to process, analyze and produce hard copies of images acquired on the confocal microscope and images produced in investigator laboratories. The Digital Imaging manager will supervise the Confocal and Image Analysis facilities and train investigators and staff on use of the equipment The Information Technology module will provide essential technical support for the Digital Imaging module as well as for individual investigators using microcomputers. The Information Technology manager will maintain the Core Grant network server, will provide networking and microcomputer support to investigators, and will create archival backups of investigator data. The Histology module will provide a fully equipped histology laboratory and the services of the module histologist to Core Grant investigators. Procedures offered by the facility include paraffin and frozen sections of ocular tissue, hematoxylin and eosin staining of sections, immunohistochemistry, PCR, in situ hybridization, nuclease protection assays, RNA extraction, plasmid design and construction, and sequence analysis. The Machine Shop module will provide the services of a skilled machinist to Core Grant investigators who require custom design and machining of equipment and components of their research. The modules will be linked together by a dedicated computer network that will facilitate investigator access and will enhance the capabilities of the facilities. Each module will be directed by an established NEI-funded investigator. The Core Grant will be administered by an advisory group comprised of the grant PI and module directors. The Core Grant, by providing research and service facilities, will further the research efforts of vision researchers at the University of Minnesota.

DESCRIPTION (provided by applicant): Stimulating the eye with light results in an increase in retinal blood flow, a response termed functional hyperemia. This response, which supplies active neurons with oxygen and nutrients, is essential for healthy retinal function. Functional hyperemia is disrupted in several retinal pathologies, including diabetic retinopathy, where the response is dramatically reduced. The mechanisms mediating functional hyperemia are not well understood. The goals of the proposed project are to determine i) how blood flow is regulated in the retina, ii) why functional hyperemia is disrupted in the diabetic retina, and iii) whether functional hyperemia can be restored by inhibiting inducible nitric oxide synthase (iNOS). Experiments will be conducted on the rat intact-globe in vivo preparation. The specific aims for the project period are: Aim 1. Determine whether capillaries contribute to the regulation of blood flow in the retina. Blood flow through capillaries will be monitored to test whether a greater fraction of capillaries are functionally perfused during photopic stimulation. Capillary control of blood flow is of particular interest as pericytes, the contractile cells that control blood flow through capillaries, are one of the first retinal cells to die in diabetic retinopathy. Aim 2. Test the hypothesis that glial cells mediate functional hyperemia in the retina. Glial cells will be selectively stimulated by photolysis of caged Ca2+ while monitoring blood flow to determine whether these cells evoke vasomotor responses. Light-evoked changes in blood flow will be monitored to determine whether functional hyperemia is blocked when neuron-to-glia signaling is interrupted by addition of a purinergic signaling antagonist. Determining whether glial cells mediate functional hyperemia will help in the development of therapies for preventing or reversing the loss of function hyperemia in the diabetic retina. Aim 3. Determine whether inhibiting iNOS reverses the loss of functional hyperemia in the diabetic retina. Using a streptozotocin rat model of type 1 diabetes, light- and glial-evoked changes in blood flow will be characterized in control and diabetic animals. iNOS will be inhibited with aminoguanidine to test whether functional hyperemia can be restored in diabetic animals. PUBLIC HEALTH RELEVANCE: Regulation of retinal blood flow is disrupted in diabetic retinopathy, which is one of the leading causes of blindness. This project will investigate the mechanisms responsible for blood flow regulation in the retina and test a therapy for restoring normal blood flow in the diabetic retina.

DESCRIPTION (provided by applicant): Diabetic retinopathy is the leading cause of blindness in working age Americans. However, the initial factors leading to development of the disease remain unclear. Blood flow in retinal vessels is compromised in early stages of diabetic retinopathy. The proposed research will test the hypothesis that this decrease in retinal blood flow, coupled with the increased metabolic demand of rod photoreceptors that occurs during the night, when the retina is dark-adapted, results in retinal hypoxia and to the development of diabetic retinopathy. Key predictions of this hypothesis remain untested. Notably, retinal pO2 (oxygen partial pressure) has never been directly measured during early stages of diabetic retinopathy. The hypothesis will be tested by measuring retinal pO2 and by determining whether light exposure to prevent elevated retinal metabolism during the night slows the progression of retinopathy in diabetic rats. The aims of the proposal are: Aim 1. Test the hypothesis that elevated retinal metabolism in the dark, coupled with decreased blood flow associated with diabetic retinopathy, results in retinal hypoxia. Retinal blood flow and retinal pO2 will be measured at 0.5, 3, and 6 months following induction of diabetes. Measurements will be made under dark-adapted and light-adapted conditions. Aim 2. Test the hypothesis that light exposure to prevent elevated retinal metabolism at night slows the progression of diabetic retinopathy. This hypothesis will be tested by maintaining animals in constant light from the time diabetes is induced. The 30-lux light level to be used during subjective night is high enough to saturate the rod system but not high enough to cause light-induced retinal damage. The progression of retinopathy will be assessed at 0.5, 3, 6, and 12 months following induction of diabetes.

Project Summary Neuronal activity triggers increases in blood flow in the central nervous system. This hemodynamic response, termed functional hyperemia, supplies active neurons with needed oxygen and nutrients and is essential for the health and proper function of the CNS. However, the neurovascular coupling mechanisms that mediate blood flow regulation remain controversial. A prominent hypothesis of neurovascular coupling holds that active neurons stimulate glial cells, evoking cytosolic Ca2+ increases and releasing vasodilating agents. However, this hypothesis has recently been challenged. Preliminary experiments from our laboratory offer a resolution to this controversy, suggesting that glial Ca2+ increases mediate capillary but not arteriole dilation. This hypothesis will be tested in the following aims. Aim 1. Characterize Ca2+ signaling in Müller glial cell endfeet that terminate on capillaries and arterioles. We will test the hypothesis that flicker-evoked neuronal activity in the retina evokes rapid Ca2+ increases in the endfeet of Müller cells terminating on capillaries but not on arterioles. Aim 2. Test the hypothesis that Müller cell Ca2+ signaling evokes capillary dilation. We will test this hypothesis by correlating changes in capillary diameter with spontaneous Ca2+ transients in Müller cell endfeet and with Ca2+ increases produced by intercellular Ca2+ waves in Müller cells. Aim 3. Test the hypothesis that light-evoked capillary dilation is blocked in the absence of Müller cell Ca2+ signaling. We will test the hypothesis that capillary dilation is blocked in IP3R2 null mice, which lack glial cell Ca2+ signaling. Aim 4. Determine the neurovascular coupling signaling pathways responsible for capillary dilation. We will test the hypothesis that neurovascular coupling onto capillaries is mediated by glial release of vasoactive arachidonic acid metabolites, including prostacyclin, prostaglandin E2, epoxyeicosatrienoic acids, and 20-hydroxy-eicosatetraenoic acid.

? DESCRIPTION (provided by applicant): Proper regulation of blood flow is essential for the health and function of the retina. Flicker stimulation evokes increases in blood flow in the retina a response termed functional hyperemia. This response brings additional oxygen and glucose to active neurons. We have recently demonstrated that blood flow is differentially regulated within the three capillary layers in the retina. Flickering light evokes far greater increases in capillar diameter and blood flow in the intermediate capillary layer than in the deep and superficial layers. The goal of this project is to determine the mechanisms responsible for this differential control of blood flow and to establish whether this control is altered in diabetic retinopathy, where functional hyperemia is known to be disrupted. The aims of the project are: Aim 1. Test the hypothesis that selective dilation of intermediate layer capillaries is driven by the activity f nearby neurons. The large flicker-evoked responses of intermediate layer capillaries may arise because synaptic activity near these capillaries is greater than activity near the other capillary layers, leading to greater release of vasodilating agents. This hypothesis will be tested by using local Ca2+ signals within Müller glial cells as an indicator of nearby synaptic activity. Calcium signals will be imaged in different retinal layers using transgenic mice expressing the genetically encoded Ca2+ indicator GCaMP3 in Müller cells. Aim 2. Test the hypothesis that flicker-evoked dilations of capillaries is mediated by production of arachidonic acid metabolites. Flicker-evoked capillary dilations will be monitored in the rat in vivo in the three capillary layers as the signaing of candidate vasodilators is blocked by intravitreal injection of synthesis inhibitors. Candidate vasodilators will also be injected into the vitreous humor and the resulting changes in capillary diameter in the three capillary layers will be monitored. Aim 3. Test the hypothesis that dilation of intermediate layer capillaries is compromised in diabetic retinopathy. Capillary diameter in the three capillary layers will be measured at 0.5, 1, 3 and 6 months after diabetes induction by streptozotocin injection. The effect of aminoguanidine, which reverses the loss of arteriole dilation in the diabetic retina, on capillary dilation will be investigated.

ADMINISTRATIVE CORE ? PROJECT SUMMARY The Administrative Module will support the overall management of the Vision Core Grant and the four resource and service modules. The Administrative module will convene regular meetings comprised of the Core Grant PI, the resource and service module directors, and the Administrative Manager. The Administrative module will distribute funds and determine the utilization of module resources to Core Grant Investigators and other University of Minnesota vision researchers.

VISUAL TESTING CORE ? SUMMARY Visual Testing Module. The Visual Testing module assesses visual function in rats, mice and other small animals by measuring the electroretinogram (ERG), the visual evoked potential (VEP), and the optokinetic response. Full-field (Ganzfeld flash) ERGs, multifocal ERGs, and pattern ERGs (pERGs) are used to evaluate retinal function, including the status of retinal ganglion cells as revealed by the pERG. VEPs and the optokinetic response (measured with an eye tracker) assess higher order visual function. The module is managed by a visual testing manager who maintains the test equipment, provides training to Core Grant investigators and staff on use of the equipment, and runs tests on experimental animals. A great savings in time and expense is realized by this module as investigators would otherwise would have to buy expensive visual testing equipment individually and train their staff in the use of the equipment.

PROJECT SUMMARY / ABSTRACT The development of label-free imaging technologies that directly assess neural activity remains a pressing need. Among a variety of techniques that aim to detect transient signals associated with action potential (AP) propagation, optical techniques have the potential for revealing and locating APs with high spatio- temporal resolution. For instance, differential-phase interferometry and then phase-sensitive measurements of spectral-domain optical coherence tomography (OCT) have allowed us to detect AP- related nanometer-scale transient structural changes from unmyelinated invertebrate axons. To obtain useful tests of nerve function, however, investigations on contrast enhancement methods for both myelinated and unmyelinated nerve fibers are needed. The long term goal of this project is to provide non- contact depth-resolved optical measurements of nerve function that are useful in basic scientific research. The overall objective of this project is to use multi-contrast OCT and contrast enhancement methods for depth-resolved label-free imaging of neural activity in myelinated and unmyelinated nerve models. The hypothesis behind the work is that a properly directed external static magnetic field generates Lorentz force in functioning nerve (due to ionic movements / action currents), which consequently induces a mechanical wave accompanying AP propagation and facilitates the optical imaging of neural activity. Phase-sensitive OCT is well poised to locate such transient signals with sub-nanometer sensitivity. We will also monitor the intensity (reflectivity) and birefringence (retardance) signals as additional indications of neural activity. To achieve the objective of this application, we will pursue optical imaging of neural activity based on Lorentz effect in ex-vivo preparations (Specific Aim 1) and in-vivo visual cortex (Specific Aim 2). With successful completion of the proposed work, we will achieve the following outcomes. The feasibility of using Lorentz effect to aid label-free optical imaging of APs will be revealed. This will also inform people in related imaging fields to determine whether the Lorentz effect imaging is within the capabilities of current technology. If our work is shown to be useful, it will support functional neural investigations in laboratory setting. The results may also suggest more challenging in-vivo applications that require incorporation of active tracking systems for the needed stability.