Frank S. Werblin - US grants

The objective of this research is the understanding of visual function in terms of the underlying membrane events in retinal neurons. The research will proceed in a hierarchical order, beginning with an analysis of membrane conductances in individual cells that account for cellular behavior, then building an understanding of cellular network behavior through an analysis of synaptic function. Finally we hope to be able to account for retinal visual function by analyzing cell and network interactions. The membrane currents underlying cellular and synaptic activity will be analyzed using the newly developed methods of whole cell patch clamp technology applied to living retinal slices and enzymatically-isolated cells. Our preliminary results indicate that these techniques allow a much greater resolution of recording and cause less cell damage than conventional methods. Using these methods, each retinal cell-type now appears to utilize a variety of active currents in processing the visual message, currents obscured earlier by the damaging effects of conventional recording. These studies will provide specific information about the ionic basis for individual cell function, a better understanding of synaptic function in the unique graded-potential synapses of the retina, the role of specific transmitter sybstances in mediating cellular communication, and the propagation of signals through lateral retinal networks.

This proposal focusses on providing a description of the components of neural pathways in the retina responsible for the modulation of visual signals. For example, as the outer retina the behavior of cones is modulated by rod activity. Rod and cone signals are modulated by dopamine. Gain between photoreceptors and second order cells is modulated by lateral interactions of unknown path, pharmacology and mechanism. At the inner retina transmission from bipolar to third order cells is modulated by feedback to bipolar cells via unknown pharmacological and functional mechanisms. The technique of patch recording and simultaneous staining in retinal slices provides for the first time a means for defining pathways, determining pharmacology, characterizing receptors, and testing second messenger systems, so that the neuromodulatory pathways can be defined. Synaptic currents can be measured, elicited by puffs of transmitter substances or transretinal current, activating specific synaptic accessible in the slice. Cell membranes can be polarized by steps of voltage in the presence and absence of transmitters and modulators, while isolating their effects upon specific voltage-gated membrane currents. The effects of modulation of release can be measured by recording synaptic inputs from cells postsynaptic to the subject cell thus using the postsynaptic cell as a bioassay for release. The behavior of second messenger systems can be controlled by introducing modulators and blockers into the cytoplasm via the path electrode. With these techniques the source of modulatory signals, their pharmacology, the membrane receptors, the second messenger pathways, and the sites affected by the modulation can be defined. This work is of value in health science because it attempts to define some of the fundamental mechanisms underlying visual functions. Since much of the mechanism is mediated by pharmacology to be defined in these studies, the route for medical treatment will also be clarified. Thus it may be possible to provide a link between retinal function (or dysfunction) and pharmacological intervention.

1) To understand the dynamic interactions among the processes of populations of bipolar and amacrine cells in the inner plexiform layer related to retinal movement and change analysis. The idea is to measure activity in the processes, not normally accessible to single cell recordings, with newly developed optical techniques. The work will utilize methods of video imaging of neural activity revealed by voltage and Calcium sensitive dyes. Preliminary results suggest that different dynamic processes occupy different strata. We will generate a picture of dynamic stratification of activity throughout the depth of the inner, plexiform layer. 2) To understand how the release of the transmitter glutamate is controlled at the graded potential synapse of the photoreceptors. It is the concentration of glutamate at these synapses that informs the postsynaptic cells of the graded levels of photoreceptor activity. The (hyperpolarizing) light response in initiated, not by the release, but by the uptake (via a transporter) of transmitter. This control appears to involve interactions between vesicular release and transporter uptake, and preliminary results suggest a titration between these two mechanisms. We will generate a picture of dynamic interactions between uptake and release involved in the control of glutamate concentrations. 3) To understand the role of newly-discovered neuromodulatory events in the retina, including dopamine modulation of GABA sensitivities in the inner and outer retina, and relate these to the overall scheme of visual processing. Preliminary results suggest that dopamine modulation of GABAc receptor sensitivity may complement or amplify earlier well-known neuromodulatory events. These studies will utilize patch clamp of single cells and of cells in living retinal slices, a technique developed by this laboratory and now universally implemented. In addition to looking at single cell events, we will use the newly-developed technique of multielectrode array recording to look carefully at changes in activity in populations of cells mediated by dopamine; effects such as changes in correlation of activity between units and changes in rates of spread of activity, that would not be visible recording from single cells.

DESCRIPTION (Adapted from applicant's abstract): The goal of the proposed work is to understand retinal processing through a careful analysis of the dynamic (space-time) patterns of activity that are generated at each retinal level. The spatiotemporal patterns will be generated in two complementary ways: (1) patterns will be measured and constructed through a novel method of physiological recording of excitation and inhibition over a large region for every cell type at each retinal level and extending the result to a population of that cell type, and (2) the patterns will be generated as the emergent properties of retinal models constructed by incorporating the space, time pharmacological and morphological properties derived from single cell studies. The measured and modeled patterns will be compared to evaluate the quantitative hypothesis of retinal embodied in the model. The modeling infrastructure, the Cellular Neural Network, (CNN) is now well-established massively parallel analog array processor designed with an architecture quite similar to the vertebrate retina. These studies will show how both simple and complex stimuli are represented in terms of physical, electrical activity (excitatory and inhibitory membrane currents and voltages) across arrays of thousands of elements at each retinal level. Analyses of these data will yield insights into the underlying neural mechanisms that mediate visual function and provide some clues about the strategies undertaken via neuronal interactions at each retinal level.

[unreadable] DESCRIPTION (provided by applicant): Our deepest understanding of retinal activity has been derived from studies that show how identified amacrine cell types mediate specific visual functions. Saccadic suppression and figure-ground separation by the polyaxonal amacrine cells, directional selective movement detection by the starburst amacrine cells, and the processing of rod vision by the All amacrine cells are examples of sophisticated function mediated by well-defined retinal circuitry involving specific amacrine cell types. The strategy in those studies was to determine how excitatory and inhibitory signals generated by amacrine cells are integrated to form meaningful retinal activity. There exist an additional two dozen amacrine cells in the retina whose morphology and stratification has been well defined, but whose visual functional roles still remain obscure. It is the goal of this proposal to investigate the visual functional role of many of these less defined amacrine cells. Our strategy is to utilize our recent studies of ganglion cell function that have defined a dozen different "feature detectors" formed by the neatly stratified dendrites of a dozen different ganglion cell types distributed throughout the depth of the inner plexiform layer. These studies define the space, time, stratification and phase properties of excitation and inhibition arriving at the dendrites of each ganglion cell type. The inhibitory components of these responses reflect the space, time, stratification and phase properties of different amacrine cell types whose processes costratify with the dendrites of each ganglion cell type. We will record from specific amacrine cell types in retinal slices and correlate their space time phase and stratification properties with the inhibitory properties of the ganglion cells with which they costratify. Through this correlation process we hope to be able to describe the role of specific amacrine cells in generating different components of visual function. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Our long-term goal is to uncover the fundamental circuit design rules that govern retinal visual processing. We focus in this proposal upon a recently discovered but ubiquitous form of circuitry that utilizes push-pull interactions. This circuit motif exists throughout the visual pathway from ganglion cells to cells in the LGN and visual cortex but has never been fully investigated. Push-pull interactions occur when inhibition decreases while excitation increases at a given neuron, or vice versa. We have recently discovered that push pull interactions represent a dominant form of circuitry in bipolar, amacrine and ganglion cells. In all cases, push pull interactions utilize the convergence of complementary, activity from the ON and OFF pathways. When excitation from the ON pathway increases, there is concomitant decrease in inhibition from the OFF pathway and vice versa. Push pull crossover inhibition is manifest in many different circuitries: It is expressed as feedback inhibition between ON and OFF bipolar cells, as feedforward inhibition to ON and OFF retinal ganglion cells, and recursive inhibition between ON and OFF amacrine cells. [unreadable] [unreadable] These crossover inhibitory interactions underlie a form of parallel processing where the integration of ON and OFF visual signals compensate for signal degradation and enhance signal processing functions such as common mode rejection, drift reduction, and noise reduction and non-linearity corrections. Similar circuitry is known to be used extensively in modern electronic circuit design. The overall goal of the studies is to extract principles of functional organization in the circuitry of the retina that will set precedents for processing of visual information in the retina and at higher visual centers as well as in other sensory systems. Crossover pathways will be studied using isolated retina and retinal slices, using patch clamp recording. Synaptic pathways will be dissected and evaluated using pharmacological agonists and antagonists. Correlation with previously established cell types will be implemented through morphological analysis using confocal microscopy of intracellularly-stained and patch-recorded neurons. [unreadable] [unreadable] The push-pull, crossover pathways constitute a fundamental paradigm for signal transmission and processing throughout the visual system. A thorough understanding of these circuitries will help us decipher the strategies used by the retina and higher visual centers to process the visual message. This understanding will enhance our ability to diagnose visual signal processing anomalies in the retina and higher visual centers, to treat disorders of the visual system, and to design devises for enhancing vision with prosthetic devices. [unreadable] [unreadable] [unreadable]