Stewart A. Bloomfield - US grants

Amacrine cells constitute a unique class of axonless neurons whose pre- and postsynaptic contacts subserve visual processing in the inner retina. Although the amacrine cells have been the subject of numerous morphological and pharmacological studies, there have been surprisingly few studies which directly examined their physiology. As a result, the full range of amacrine cell response properties has yet to be determined and much of our knowledge of amacrine cell function has been attained indirectly, from physiological work directed at the ganglion cells. A multidisciplinary approach is proposed to study the physiology, morphology, and pharmacological interactions of amacrine cells in the rabbit retina. The long-term goal of this research is to elucidate how amacrine cells influence bipolar to ganglion cell transmission, and how these interactions contribute to the formation of ganglion cell responses in the mammalian retina. Three major specific aims are proposed. The first aim is to determine the different light-evoked response and receptive field properties displayed by rabbit amacrine cells. This will be accomplished by obtaining intracellular recordings from amacrine cells in the rabbit retina-eyecup during presentation of various photic stimuli. Amacrine cell receptive fields will be described utilizing the same criteria used in prior studies to characterize ganglion cells, including complex response properties. My preliminary data indicate that rabbit amacrine cells exhibit some of the complex receptive field properties formerly thought to be limited to ganglion cells, such as direction and orientation selectivity. The second aim is to determine the morphology of amacrine cells and correlate this with their response properties. Physiologically-characterized amacrine cells will be labelled with horseradish peroxidase (HRP) to visualize their dendritic architectures. The neuronal profiles obtained will then be compared to amacrine cells described in Golgi and histochemical studies whose synaptic connections and/or neurotransmitters are known. The third aim is to examine the actions of the putative amacrine cell transmitter, GABA and acetylcholine, on amacrine cell responses. This work will elucidate the extent and specifically of the interconnections between amacrine cells subserved by these transmitters. Comparison of these results with those of prior pharmacological studies of rabbit ganglion cells will be made to localize more precisely the sites of specific transmittter effects, and to provide insights into which ganglion cell response properties are dependent on amacrine cell input.

DESCRIPTION (provided by applicant): Like other CNS loci, the major mode of cellular communication in the mammalian retina is via chemically-mediated synaptic transmission. However, work over the last decade indicates that electrical synaptic transmission, via gap junctions, forms a second significant mode of neuronal interaction in the retina. It is now clear that gap junctions are ubiquitous throughout the retina, occurring between cells within each of the five major cell classes. In addition, retinal gap junctions have been shown to be dynamically regulated by changes in ambient illumination and circadian rhythms acting through light-activated neuromodulators such as dopamine and nitric oxide. These data suggest that gap junctions play a key role in light adaptation. The networks formed by electrically coupled retinal neurons thus provide plastic, reconfigurable circuits for the flow of visual signals. Overall, direct intercellular communication via electrical coupling is positioned to play key and diverse roles in the transmission and integration of visual information at every retinal level. The long-term goal of this research is to define the distribution, function and regulation of the gap junctions in the mammalian retina so as to understand their roles in the transmission of visual information. Accordingly, the specific aims of this proposal include: (1) to determine the roles of the different gap junctions that form crucial elements in the different rod pathways; (2) to determine the roles of ganglion-to-ganglion cell and ganglion-to-amacrine cell electrical coupling in the synchronization of the spike activity of neighboring alpha ganglion cells and whether this is regulated by light; and (3) to elucidate the different subtypes of amacrine and ganglion cells that form distinct and stereotypic coupled networks in the proximal mammalian retina. A final aim is to define the structure and function of amacrine cell types, long a focus of the work in our lab, to provide a framework to understand the role of their electrical junctions. The functions of gap junctions will be assayed electrophysiologically by recording from retinal neurons under conditions in which gap junctions are disrupted either pharamcologically or in a connexin36 knockout mouse model. In addition, the biotinylated tracer Neurobiotin, which can pass through gap junctions, will be used to morphologically assay changes in the extent of coupling so as to determine how it is regulated by light or disrupted in the experimental models. Gap junctions have been implicated in a number of neurological diseases including X-linked Charcot-Marie-Tooth disease, nonsyndromic autosomal deafness as well as having a role in neuroprotection and cell loss following stroke or trauma. Although focused on the function and regulation of gap junctions in the mammalian retina, the proposed work should nevertheless provide important insights into the roles and plasticity of gap junctions throughout the brain.

This application is for a new, integrated neuroscience training program at New York University. Historically, there have been two neuroscience graduate program at NYU located at the Washington Square and School of Medicine campuses. However, there has been an effort over the past two years to seamlessly integrate the graduate programs and thereby offer training with far greater breadth and depth than each offers individually. Moreover, it has formed a highly complementary research efforts given the emphasis of systems, cognitive, and computational neuroscience at Washington Square and cellular, molecular, developmental, translational, and clinical neuroscience at the medical school. The proposed training program strives to continue the integration ofthe extensive neuroscience community at NYU and provide the next step towards full collaboration and cooperative between the two sister campuses. The specific goals ofthe neuroscience training program are: (1) To provide a rigorous and broad-based graduate education in neuroscience within an interactive and collegial research environment. (2) To increase the number of high caliber students that apply to and participate in the program, including active recruitment of underrepresented minorities. (3) To provide students with guidance by a rigorous mentoring system through a series of milestones to a doctoral degree typically in 5-6 years. (4) To build critical awareness by open discussion of problems associated with the scientific method and the interpretation of results as well as discussion ofthe ethical problems associated with scientific research. (5) To provide a broad perspective on how training in neuroscience can allow students to make contributions in basic, translational, and clinical research. This includes access to detailed information concerning post-graduate career choices. The program brings together 69 faculty members with diverse research interests to ensure broad training graduate students in fundamental principles of neuroscience and their application to basic and clinical research problems. Cohesiveness in the program is achieved through a comprehensive core course curriculum required for our students, as well as seminars, symposia, journal clubs, tutorials, retreats, and extensive laboratory training. Each student will be carefully mentored throughout their training tenure to ensure that

Electrical synaptic transmission via gap junctions (GJs) is an important mode of neuronal communication in the CNS. An elegant example is the retina in which each of the five main neuronal types is electrically coupled via GJs. The broad distribution, diverse connexin subunit structure, and regulation of retinal GJs suggest a diversity of functional roles in visual processing; elucidating these roles forms the long-term goal of our experimental program. Here we propose to study the GJs in the inner mouse retina, which subserve a rich and complex variety of electrical circuits. The first aim of this proposal is to determine the role of cell-to-cell vs. neuron ensemble interactions in creating the robust, correlated activity displayed by retinal ganglion cells (RGCs). Correlated RGC activity is believed to have a number of functions, including enhancement of signal saliency and encoding of specific information about visual stimuli such as intensity, size, and motion. While it is now clear that GJs are critical to the creation of the robust concerted activity between RGC neighbors, the exact mechanism remains unclear. While it has been posited that reciprocal drive between coupled RGC neighbors can produce concerted firing, our preliminary data suggest that this does not occur. Rather, it appears that coherent activity within neuronal ensembles is necessary to recruit additional RGCs and produce their coherent activity. We propose a multidisciplinary approach combining electrophysiological, pharmacological, and optogenetic techniques applied to transgenic and knockout mouse lines to differentiate the circuits responsible for correlated RGC activity. The second aim will focus on the coupling between RGCs and amacrine cells (ACs). This type of electrical coupling occurs extensively across the retina, but how this affects neuronal activity has not been studied comprehensively due to a lack of an experimental platform to visualize and target coupled RGC-AC pairs for recording. We will target coupled RGC-AC pairs using two techniques: (1) labeling cells with the GJ-permenat dye Po-Pro-1; and (2) using the transgenic Grik4 mouse line in which ON ?-RGCs and coupled ACs express fluorescent markers. For both, we will record from coupled pairs of RGCs and ACs to determine the role that this electrical interaction has on the response activity of inner retinal neurons. In the third aim we will study the novel idea that RGCs can alter intraretinal activity by signaling back to AC through interconnecting GJs. We posit that RGCs can alter the activity of coupled ACs, which, in turn, inhibit other ganglion cells via conventional chemical synapses. This form of intraretinal signaling thereby creates a circuit providing a novel form of lateral inhibition. Deficits in GJ communication have been implicated in a number of brain neuropathies, including visual impairments associated with retinitis pigmentosa, glaucoma and ischemic retinopathy. The experimental program proposed here will extend our understanding of the distribution and physiological roles of GJs, which forms an important prerequisite for determining how GJ dysfunction affects neural function so as to indicate novel targets for the treatment of human diseases.

? DESCRIPTION (provided by applicant): We request a continuation of the T35 Training Program at the State University of New York College of Optometry that has existed for over 30 years. The purpose of the program is to introduce optometry students to basic, translational, and clinical optometric and vision science research by participating full-time for ten weeks in a research project mentored by one of the fifteen members of our distinguished research faculty. Research programs of the training faculty include cell biology, ocular pharmacology, visual psychophysics, computational modeling, visual neuroscience, optics, and clinical vision science. In addition to research, trainees will attend research colloquia, graduate seminars, journal clubs, and a course in Scientific Ethics and the Responsible Conduct of Research. It is expected that this experience will provide trainees with both technical and problem solving skills that will inspire them to include vision research as one important component of their future career as optometrists. In the most favorable scenario, highly motivated trainees will choose to continue their research by entering the combined O.D.-M.S. or O.D.-Ph.D. graduate programs in Vision Science and thereby obtain both a clinical and a research degree. The training will be conducted within the 50,000 sq. ft. of state-of-the-art basic, translational, and clinical research space at the College. The College also maintains computer, electronic, graphic, and extensive library facilities that are freely available to the trainees. Trainees will consist of optometry students from any of the Colleges of Optometry in the US who have completed their first or second year of the professional program and have shown high academic achievement. The Training Program strives to provide trainees the clinician/researcher interface that is essential t bringing the latest scientific discoveries to the clinic. This experience is aimed at providing students, and later to be doctors, the knowledge and skillset to both continue in research and to provide the latest and most optimal vision treatment and care to the patient.

? DESCRIPTION (provided by applicant): Glaucoma, the second leading cause of blindness worldwide, is a neurodegenerative disease often associated with elevated intraocular pressure (IOP) and characterized by the progressive loss of retinal ganglion cells (RGCs) leading to visual loss. Among the various types of cell death associated with neuropathologies, the major cellular pathways underlying apoptosis and necrosis have been well characterized. However, in addition to these intrinsic mechanisms underlying primary cell death, intercellular communication via gap junctions (GJs) appear to play a major, yet poorly understand, role in secondary or `bystander' cell death. GJs, proteins that form cytoplasmic bridges between neighboring cells, act as conduits by which toxic materials are passed from dying cells to their neighbors leading to their death. Studies in CNS suggest that progressive secondary cell death mediated by GJs may, in fact, account for the majority of cell death associated with a number of insults, including ischemia, excitotoxicity, and trauma. Consistent with these findings, our preliminary data in retina show that blockade or ablation of GJs can significantly reduce the loss of RGCs and amacrine cells (ACs) in experimental glaucoma. We therefore posit that GJ-mediated secondary cell death is a crucial mechanistic element of glaucomatous injury, resulting in the majority of RGC and amacrine cell (AC) loss, and thereby offers a novel target for neuroprotective treatment. To test this hypothesis, we propose to use mouse models of experimental glaucoma to show directly that pharmacological blockade of GJs or genetic ablation of their constituent connexin subunits results in a significant reduction in in the loss o RGCs and the ACs to which they are coupled. Different GJs, based on the composition of their connexins, can have selective permeabilities, suggesting that the makeup of a GJ may determine whether it promotes cell death following injury. We will therefore use knockout mice in which selective connexins are ablated to determine which GJ cohorts support cell loss in glaucoma and should thus be targeted to promote neuroprotection. Finally, we will carry out electrophysiological and behavioral experiments to assess whether increasing RGC and AC survivability in glaucoma by blocking GJs results in preservation of visual function. In these experiments, we will record the electroretinogram (ERG), and use patch-clamp and microelectrode array recording techniques to assess retinal function. We will also record visual evoked potentials (VEP) and measure the optokinetic response (OKR) to assess central visual function. The results of this study should elucidate a new and important mechanism of RGC degeneration associated with glaucoma and, in so doing, reveal a novel target for neuroprotective treatment. While the proposed work will be directed at glaucoma, the therapeutic strategies that emerge should be applicable to the treatment of neurodegenerative diseases seen in other parts of the CNS.