William J. Brunken, PhD - US grants

Despite biochemical and anatomical data which indicate that serotonin or a related indoleamine is a transmitter in the mammalial retina, our understanding of the function of the endogenous indoleamine is extremely limited. The role of this transmitter will be studied using pharmacological manipulation of the rabbit retina. Both an isolated superfused eyecup and an intact arterially perfused preparation will be employed to study the effects of specific serotonergic antagonists on the responses of physiologically identified ganglion cells. Results up to this point have shown that indoleamine antagonists reduce ON responses in both ON center and OFF center brisk (X and Y) ganglion cells. Several other aspects of ganglion cell function will now be addressed. 1. What is the role of the endogenous indoleamine in the elaboration of the receptive field properties of W-cells? Specifically are only ON-pathways affected and does the indoleamine make any contribution to trigger features. (In the past the role of a given transmitter has been elucidated only when these W-cells were studied). 2. What is the role of the endogenous indoleamine in the photopic versus scoptic retina? The indoleamine-accumulating neuron is extensively connected to the rod bipolar cells, thus it is possible that the endogenous indoleamine has a specific role in scotopic retina. 3. What role does the endogenous indoleamine have in the modulation of ganglion cell responsivity and sensitivity? The hypothesis which has emerged from previous work suggests that this transmitter serves to increase the sensitivity to increments of light. This hypothesis will be tested by studying the effects of serotonergic drugs on the responsivity and sensitivity of ganglion cells. The rabbit retina has proven to be a useful model system in which to develop generalizations about transmitter function which have proven valid in other retinae and also other regions of the CNS. Thus, elucidating the role of the endogenous indoleamine in rabbit retina will contribute to our understanding of visual processing and to the understanding of the function of serotonin in the mammalian CNS.

This proposal seeks to understand the network and cellular basis of serotonin's action in the mammalian retina. Serotonin-containing neurons or serotonin-accumulating neurons are found in most retina, including human. In mammals, they are reciprocally connected to the rod bipolar cell; it has been suggested that they gate the output of the rod bipolar cell. This application seeks to critically examine several aspects of this hypothesis. Two preparations of the retina will be used: an eyecup preparation and dissociated retinal neurons. The role of serotonin in both light- and dark-adapted retinae will be examined with extracellular recording techniques from ganglion cells in the intact rabbit retina. The effects of selective antagonists and agonists on the ON and OFF responses will be observed. In addition to testing changes in the magnitude of ganglion cell responses, changes in responsitivity and sensitivity will be studied using response-intensity data and incremental threshold. The site of action of serotoninergic drugs will be confirmed with drugs whose sites of action in the rod pathway have been proven. To study the cellular basis of serotonin action on retinal neurons, a dissociated cell culture preparation will be used. In these experiments, whole-cell recordings will be used to define the currents associated with serotonin receptor activation. Specific serotoninergic agonists will be used to identify the subtypes of receptors involved. The mechanisms of coupling receptors to ion channels will be explored. Finally, the cellular localization of the neuronal high-affinity serotonin transporter will be identified by immunohistochemical and pharmacological techniques in the rabbit retina. This work has direct significance to rod information processing and thus may be relevant to clinical conditions affecting night vision. Serotonin is a widely distributed CNS neurotransmitter; it is implicated in a variety of disease states including sleeping disorders, chronic anxiety and depression. Pharmacological manipulations of the retina have been proven to be of general significance to the mechanism of neurotransmitter function in the CNS. This proposal seeks to couple a cellular understanding of transmitter action with its network role.

This proposal seeks to understand the network and cellular basis of serotonin's action in the mammalian retina. Serotonin-containing neurons or serotonin-accumulating neurons are found in most retina, including human. In mammals, they are reciprocally connected to the rod bipolar cell; it has been suggested that they gate the output of the rod bipolar cell. This application seeks to critically examine several aspects of this hypothesis. Two preparations of the retina will be used: an eyecup preparation and dissociated retinal neurons. The role of serotonin in both light- and dark-adapted retinae will be examined with extracellular recording techniques from ganglion cells in the intact rabbit retina. The effects of selective antagonists and agonists on the ON and OFF responses will be observed. In addition to testing changes in the magnitude of ganglion cell responses, changes in responsitivity and sensitivity will be studied using response-intensity data and incremental threshold. The site of action of serotoninergic drugs will be confirmed with drugs whose sites of action in the rod pathway have been proven. To study the cellular basis of serotonin action on retinal neurons, a dissociated cell culture preparation will be used. In these experiments, whole-cell recordings will be used to define the currents associated with serotonin receptor activation. Specific serotoninergic agonists will be used to identify the subtypes of receptors involved. The mechanisms of coupling receptors to ion channels will be explored. Finally, the cellular localization of the neuronal high-affinity serotonin transporter will be identified by immunohistochemical and pharmacological techniques in the rabbit retina. This work has direct significance to rod information processing and thus may be relevant to clinical conditions affecting night vision. Serotonin is a widely distributed CNS neurotransmitter; it is implicated in a variety of disease states including sleeping disorders, chronic anxiety and depression. Pharmacological manipulations of the retina have been proven to be of general significance to the mechanism of neurotransmitter function in the CNS. This proposal seeks to couple a cellular understanding of transmitter action with its network role.

biomedical equipment purchase;

DESCRIPTION (provided by applicant): Laminins are biologically active molecules that function as cell adhesion molecules, regulate various aspects of development, and serve to stabilize complex anatomical structures. They are large extracellular matrix molecules which are composed of three subunit chains, designated alpha, beta and gamma. Five alpha, three beta and three gamma chains have been identified. Laminins are widely expressed in the CNS; as are their receptors. Several disorders of the nervous system are linked to laminin genes: some congenital muscular dystrophies involve the alpha2 chain (merosin); the beta2 chain is reduced in Walker-Warburg syndrome; and a complex group of CNS developmental disorders (muscle-brain-eye disease; retinitis pigmentosa with deafness (RP21 with deafness); Walker-Warburg syndrome) maps to the site of the gamma3 gene. Genetic disruptions in some laminin-related genes also result in human disease and in dysmorphogenesis in animal models. We have identified two novel CNS laminins, alpha4beta2gamma3 and alpha5beta2gamma3 (LN 14 & LN 15, respectively); these are found in the interphotoreceptor matrix and in the matrix of the outer plexiform layer (OPL). These laminins appear to play important roles in the morphogenesis of photoreceptors. First, these chains are expressed prior to the onset of rod genesis and persist into adulthood. Second, ablation of the gene encoding one of the beta2 chains results in the production of dysmorphic photoreceptors; specifically, photoreceptor outer segments are reduced in length and the photoreceptor terminals in the OPL are disrupted. Finally the amplitude of the ERG b-wave is drastically diminished suggesting that transmission from photoreceptors to second order cells is disrupted by loss of beta2-containing laminins. We hypothesize that LN 14 and 15 are critical mediators of synapse assembly and stabilization. Furthermore, we hypothesize that LN14 and 15 form unique substrates with which photoreceptor terminals interact. Specifically, we hypothesize that the molecular assembly and structure of the photoreceptor synapse is dependent on the interactions between these laminins and their receptors. We propose to test several aspects of this hypothesis. We will ask two specific questions: 1) What is the functional composition of the laminin complex in the OPL? 2) How does disruption of the laminin complex alter the functional organization of the OPL? With these studies, we will: gain insight into the molecular mechanisms of synaptic assembly in the outer retina; define the role of the ECM in this process; and shed light on the basis of a series of genetic disorders in humans.

A novel laminin subunit chain, gamma3, has been recently identified. Biochemical studies of gamma3 containing laminins show that gamma3 can associate with at least alpha2, alpha4, beta1, and beta2 subunit chains. The tissue distribution of gamma3 shows it to be present in regions not containing ultrastructurally defined basement membranes. These regions include the brain matrix and the apical surface of ciliated epithelial cells. The following specific aims describe proposed intentions to test the hypothesis that gamma3 containing laminins form a unique matrix at these non-basement membrane sites, the function of which is to stabilize the cytoskeletal arrangements required for the assembly and stabilization of cilia, and to also provide a distensible matrix stabilizing the structure of the CNS. To test this hypothesis specific aims are proposed: (1) Carefully describe the anatomical distribution of gamma3 and other selected laminin chains, as well as of known matrix macromolecules and receptors in the brain and at ciliated epithelial surfaces during development and in the adult mouse; (2) Determine the molecular composition of gamma3 containing laminins in the brain and at ciliated epithelial surfaces. Chemical quantities of the appropriate laminins will be produced in cell culture and purified. (3) Potential interactions between molecules which colocalized spatially and temporally will be tested in vitro. Binding to potential receptors will be determined by cell binding studies. (4) A gamma3 null mouse will be generated by homologous recombination and the phenotype characterized. We will cross the gamma3 and beta2 heterozygous mice to produce the double KO. It is likely that these studies will provide the foundations for further study of a novel extracellular matrix. It is also possible that these studies will provide novel insights into the pathophysiology of neurodegenerative diseases and infertility.

Synapses have traditionally been thought to be fixed structural elements but work during the past decade has revealed the dynamic nature of synaptic endings in the brain. This project is to study a unique synapse residing in the mammalian retina, the ribbon synapse. This synapse resides in the photoreceptors, which transduce light energy into neural signals. The photoreceptor ribbon has an exceptionally large active zone and one of the highest rates of vesicular transmission of any synapse in the nervous system. It was first described over 50 years ago as a fixed structural member of the photoreceptor presynaptic terminal. The precise function of the ribbon remains unclear. A monoclonal antibody that recognizes synaptic ribbons in the retina (B16) has been developed in the PIs laboratory and used to isolate a family of retinal specific proteins. The function of these proteins in vesicular trafficking and signal transduction pathways at the photoreceptor ribbon synapse will be studied, the identity of the B16's epitope on the synaptic ribbon will be determined and determination of the amino acid sequence of the proteins composing the synaptic ribbon will be attempted. It is also proposed to study the circadian control of the morphological dynamics observed in the synaptic ribbon.
NSF funding will continue to support a rich educational environment promoting hands-on research experience for high school students from the New England area, undergraduate and graduate students at Boston College, and undergraduates from other east coast universities has been established.

? DESCRIPTION (provided by applicant): The immediate goal of our project is to understand the role of the extracellular matrix in retinal development and disease. In prior funding periods, we identified unique isoforms of laminins, containing either the ß2 or ?3 chains that are expressed in the eye and brain. Mutations in these two laminin genes, in humans, result in autism, ocular dysgenesis, and kidney dysfunction. Ablation of these genes, in mouse, produce cortical and ocular dysgenesis; the latter includes disruptions of: 1) retinal ganglion cell development; 2) astrocyte migration and subsequent vascular development; 3) the sub-cellular organization of the Müller cell; 4) the photoreceptor- bipolar synapse. Our fundamental hypothesis is that laminins are critical for establishing the three dimensional structure of the retina. Specifically, we hypothesize that laminins provide environmental cues that are essential for angiogenesis and neurogenesis. Our first aim explores the contributions of laminin signaling in formation of the template for angiogenic development. The working hypothesis is that RGCs drive astrocyte migration; then, interactions between astrocytes and microglia regulate endothelial development. We will use a reverse genetic approach, deleting Lamb2 or Lamc3 genes alone, or together, to disrupt the signaling among these cells. The first set of experiments will focus on the spatial patterning in Lamb2-/- and Lamc3-/- animals. Our second set of experiments will address the role of laminin-mediated recruitment and activation of microglia. The third set of experiments will examine the effectors of laminin signaling in endothelial cells during angiogenesis. Our current data suggest that ß2-containing laminins are pro-angiogenic and ¿3-containing laminins are anti-angiogenic. Our second aim is focused on the role of laminins in neurogenesis. We will examine the hypothesis that laminin regulates apical-basal polarity of the radially organized progenitor. Our published data demonstrate that Müller cell compartmentalization is disrupted in the Lamb2-/- retina. Moreover, our preliminary data demonstrate that the cell cycle is dysregulated in both Lamb2-/- and Lamc3-/- mice. Our first set of experiments will focus on the regulation of symmetric versus asymmetric division in the Lamb2-/- and Lamc3-/- retina. Next, we will turn to a study of the pattern of inheritance of important cell cycle regulators in these same mice. Last, we will measure directly the cell cycle regulation in Lamb2-/- and Lamc3-/- retina. Our preliminary data suggest that ß2- and ?3-containing laminins are necessary to preserve the proliferative state. Our work is relevant to an understanding of the pathobiology of retinal neovascular disease, gliosis and proliferative vitreoretinopathy because astrocytes and microglia play critical roles in retinal vascularization and remodeling. Our work on retinal progenitor cells will improve our fundamental understanding of retinal development and our understanding of the regulation of the cell cycle in CNS progenitors and will influence the development of 3D culture systems designed to grow retina ex vivo.

The retina core will fill three scientific needs: 1) Provision of unique equipment and expertise in specialized protocols not available in the laboratories of individual investigators. Examples are the EM, fundus angiography, 3D imaging, retinal electrophysiology; 2) Undertaking procedures that various NYPD-PRC investigators require only periodically but are nonetheless essential for their work. Methods that are used infrequently in an individual laboratory are not only handled inefficiently but generate low quality data. In a core lab, such work is done daily, and, we believe, with better consistency and quality. Examples are ERG recording, and EM; and 3) Performing services that are formulaic and time-consuming yet vital to research projects. Removing these routine activities from the investigative staff reduces their fatigue; maximizes their time for intellectually or technically demanding work; and importantly saves time and money. A good example of this is the histology services. Their choice specifically reflects the considerable strength the SEI has in molecular, cellular biology of ocular disease and development as well as very solid presence in cortical and visual processing. The services offered by the NYPD-PRC Cores will be reviewed every six months to ensure that they are cost-effective and state-of-the-art. And importantly are serving the needs of the investigative staff. These needs are expected to evolve over time and with the addition of other funded projects. Thus, we will set in place plans to develop additional shared core facilities. Of particular interest to our membership is the development of a clinical or translation core that will facilitate bench to bedside therapeutics. In order to achieve this, the NYPD-PRC will partner with another of the SUNY REACH initiatives - the Clinical Trial and Translation project headed up by Dr. Nachman (SUNY Stony Brook); Dr. Aranda is a member of their steering committee. This effort is particularly focused on participation in pharmaceutical trials. However, within the Brooklyn Neonatal network we have a complementary expertise and strength in clinical studies. Our patient base (39,000 births per year and 11 NICUs) represents a unique and valuable resource is assessing ocular disease in the new born as well as evaluating best practices. Thus, our future goal is to develop this expertise into a core that will support funded research in this area.

DESCRIPTION (provided by applicant): The State University of New York (SUNY) Eye Institute (SEI) is a multi-center, multidisciplinary research institute, which embodies a novel and exciting model for vision research - that of a truly integrated multi-center institute that serves he broad and diverse constituency of our nation's third most populous state. The instrumentation funded by this S10 application will provide additional support for the SEI. The overall objective of this S10 application is to provide core support for NIH- funded research in the SUNY Eye Institute (SEI) at its SUNY Downstate Medical Center site, as well as to provide investigators the ability to obtain data for additional NIH funding. The SEI, a novel entity established in 2009, integrates the complementary strengths in vision research of the four SUNY medical universities and SUNY Optometry into a single research consortium. The SEI model represents a paradigm shift, built on the traditional academic infrastructure, using high-speed communication to generate a state-wide research- focused institute. It unites the three core constituencies of the National Eye Institute (NEI) - basic, ophthalmic, and optometric research - into a collaborative institute whose mission is to elucidate pathobiologies of the visual system. This collaborative faculty effort strongly supports the NEI program goals to develop novel insights into, and therapeutic avenues for, visual dysfunction. The goals of this S10 application are to provide instrumentation to: ¿ improve state-of-the-art eye research core facilities to serve R01-funded and other NIH-funded SEI researchers; ¿ reduce duplication of research expenses and consolidate resources for NIH-funded SEI researchers; ¿ promote and foster collaborative research interactions among NIH-funded SEI scientists; ¿ enhance the research environment for vision research in the SUNY system generally and at SUNY Downstate Medical Center specifically.