Peter Sterling - US grants

Our goal is to achieve a comprehensive understanding of the neural circuitry of the cat retina. This seems realistic now that fundamental neuron "types" have been defined in cat retina, and it is realized that there are only three to five dozen. Seventeen types are already synaptically linked and twelve are associated with a specific transmitter. Our approach is to reconstruct adjacent retinal neurons from electron micrographs of serial sections. In such a series certain neurons are labeled beforehand by the accumulation of a tritiated transmitter, by a monoclonal antibody coupled to HRP, or by a marker of activity, such as the reaction product of cytochrome oxidase. The strength of this approach is in the detail gathered on adjacent neurons for they can be "typed" by multiple criteria and then linked in synaptic circuits. Data can be gathered quantitatively, permitting the development and testing of specific hypotheses regarding function. For example, we have demonstrated within a single series multiple synaptic connections between eight types of neurons. Among these is a Beta/X-on cell with input from two types of cone biolar, one of which may be inhibitory. The receptive field center of the Beta/X-on cell, we hypothesize, may be generated by excitation from a depolarizing cone bipolar and simultaneous withdrawal of inhibition from a hyperpolarizing bipolar. We shall test the qualitative anatomical predictions of this "push-pull" hypothesis using the methods described above. We shall also develop an electrontic model of the Beta/X-on cell in order to test a quantitative version of the hypothesis. We anticipate that additional hypotheses to explain the physiology will develop naturally as we delineate new circuits. When retinal circuits are truly understood, it should be possible tgo mimic them with integrated semiconductor circuits. Such feature-detecting devices may have broad industrial applications and will be medically useful, most obviously for the blind.

Our long term goal is to understand the structure, function and development of cat retina. This tissue is part of the central nervous system, yet it is relatively simple, containing ony about 60 types of neuron. We have shown by computer reconstruction from electron micrographs of serial sections that specific types are wired with extraordinary precision into may parallel circuits. This anatomical knowledge, plus information regarding the neurochemistry and physiology of certain cell types, has led to specific hypotheses regarding function. Thus, models have emerged to explain the receptive field properties of on gaglion cells (both X and Y), how these properties are maintained in day and night vision, and how the sensitivity of the circuits is adjusted to rapid changes in light intensity (gain control) and to slow changes (dark adaptation). We now propose: (1) Extend knowledge of structure by determining additional important features of the wiring: (a) identify rod and cone bipolar circuits to off ganglion cells. (b) identify amacrine circuits to on ganglion cells (c) identify input/output circuits of putative control elements, the GABA- ergic interplexiform cell and the dopaminergic amacrine cell. The method will combine cell identification by serial EM reconstruction. (II) Test theories of function that have emerged from our structural studies: (a) establish an in vitro preparation for physiological recording. (b) use this preparation to test the hypothesized roles of GABA and the interplexiform cell in gain control. (c) test the hypothesized contributions of types A and B horizontal cells to gain control and to the ganglion cell receptive fields. (III) Detemine the normal development of identified circuits. These circuits, absent at birth, develop explosively between 1-4 weeks postnatal. (a) determine whether precision wiring in adult is established without error or whether it emerges gradually from connetions that are at first imprecise. (b) determine when antigens specific for synapses in identified circuits first appear. The methods will combine immunocytochemistry (using existing monoclonal antibodies) with serial EM reconstruction. The proposed studies address fundamental questions regarding the organization and development of identified circuits in mammalian brain and are specifically releveant to organization are shared by cat and human.

Funds are requested to purchase a JEOL 1200 EX electron microscope to be housed in the EM facility of the Department of Anatomy. This instrument would be devoted primarily to studies of serial sections in analyzing: 1) local neuronal circuits in mammalian brain 2) regeneration of identified neural circuits in vertebrate spinal cord 3) macromolecular organization of the myosin filament. The technical features of the new instrument critical to these purposes are: 1) high accelerating voltage (120KV) 2) excellent performance at low magnification (approximately 1500X) as well as high magnification (10,000-100,000X) 3) advanced aids to critical focusing 4) microprocessor controlled realignment for efficient switching between different magnifications (for efficient time sharing by different users) 5) video system to permit scanning at low beam intensities (to minimize beam damage and contamination during search for corresponding regions of serial sections and during alignment of serial sections for photography) 6) capability of rotating and tilting the sections in the column (for alignment during photography and for producing stereo pairs of critical structures) 7) automatic montage photography. The instrument would also serve other members of the department in studies of motility and cell differentiation The proposed instrument would replace a 15 year old instrument in the Department of Anatomy which is technologically obsolete and increasingly unstable and unreliable.

Human visual performance depends on parallel functional channels established in the early stages of visual processing. Each psychophysically defined channel may depend on contributions from several types of ganglion cell. In turn, the contribution from each type of ganglion cell depends on the structure of its array and its wiring to specific types of photoreceptor. Our broad goal is to determine the retinal microanatomy that underlies these psychophysical and physiological aims are: 1) Identify in the foveal region of the primate (macaque) retina, which closely resembles the human, all the types of on ganglion cell that are present. 2) Determine the array structure of each type. 3) Determine the detailed connections of each type with rods and chromatically identified cones. 4) Incorporate this information into computational models that simulate ganglion cell performance under different conditions of luminance and spectral composition. These findings will provide a basis for linking the anatomical circuitry to the physiology and psychophysics. Our strategy is to study intensively a small patch of retina just off the foveal center, where spatial acuity is high, yet rods are also present, and where ganglion cells are stacked in multiple ranks. We will prepare the tissue in two series of ultrathin sections, one radial and the other tangential, photograph them in the electron microscope, and create complete photographic montages of each. We shall use the radial series to identify types and their circuitry and the tangential series to study the structure of the various arrays.

Human visual performance depends critically on the efficiency of circuits that connect foveal cones to ganglion cells. Our broad goal is to discover the basis for this efficiency. In the standard view all information destined for cortex is "multiplexed" by two arrays: P/midget (95% of foveal ganglion cells) and M/parasol (5%). However, our current studies suggest that non-midget cells are more prevalent than suspected (>25%) and comprise four types. Thus, we hypothesize five ganglion cell arrays: a dense array of spectrally nonselective cells (midget); two sparser arrays of spectrally opponent cells (R/G & B/Y bistratified); a moderately dense array of linear, high contrast gain cells (beta-like parasol); and a sparse array of non-linear, motion-sensitive cells (alpha-like parasol). If confirmed, such arrays suggest that signal are not multiplexed but segregated early, thus permitting maximum amplification. We now propose: 1) Test the "five array" hypothesis by determining quantitatively the circuits for all four types of non-midget ganglion cell. This should suggest possible correspondences to psychophysical channels because the number and weighting of cones connected to a ganglion cell type, plus its sampling frequency, set the information capacity of the array. 2) Test hypothesis that R and G midget ganglion cells in human receive different numbers of synapses. This will link our current studies on macaque to human circuitry and test our identification of spectrally opponent inputs to bistratified cells. 3) Determine amacrine circuits to ganglion cells. Amacrine synapses are numerous (e.g., 50% of the synapses to midget cells), but their circuits, which probably serve nonlinear mechanisms such as gain control, are completely unknown. 4) Determine which members of the ionotropic and metabotropic families of glutamate receptor are expressed on specific types of bipolar and ganglion cell. A circuit's coding capacity (i.e., signal/noise, gain, temporal bandwidth) depends critically on molecular properties of its postsynaptic receptors (e.g., binding constant, channel conductance and open time). Localizing these known properties in identified circuits will provide data essential to AIM 5. 5) Assess how identified factors (circuit structure, sampling frequency, and postsynaptic receptor properties) affect coding efficiency. Incorporate neural factors into "ideal observer" models for comparison to preneural factors and psychophysical performance. Circuits will be studied by electron microscopy and by intracellular dye injection followed by digital light microscopy; glutamate receptors will be identified by immunocytochemistry and amplification of mRNA in identified cells; compartmental models will be used to assess how circuits optimize information transfer given their constraints: to be small (few synapses; prone to saturate) and to employ noisy mechanisms for signal transfer.

DESCRIPTION (provided by applicant): We propose to continue a broad, interdisciplinary Vision Training Program now in its 27th year. The program includes 23 preceptors in nine departments and spans many areas: signal transduction (biophysics and biochemistry); retina (circuitry, computation, neurochemistry, cell biology, developmental genetics); eye (cataract, myopia, tears); central pathways (physiology and behavior); higher processes (psychophysics, cognitive neuroscience, computation), and "neuromorphic" devices (silicon retina). Predoctoral students are selected for excellence via a centralized office of Biomedical Graduate Studies and join a particular "graduate group", mainly Neuroscience or Bioengineering. On Penn's centralized campus, graduate education is organized across Departments and Schools in order to foster multi-disciplinary training and collaborative research. Students are trained broadly during the first two years and are attracted specifically to vision research via: (1) formal lectures and laboratory training; (2) weekly "lunch seminars" with research talks by intramural and external speakers; (3) research rotations in three different laboratories (each followed by a 15 minute public talk); (4) annual research "retreat" with student talks and visiting scholar. At this level all students are exposed to key modem methods, such as patch clamp, molecular biology, computation, and FMRI. Neuroscience and Bioengineering admit about 15 students annually, and of these, about 10 rotate through vision labs. Currently, about 23 have settled in a vision lab for the dissertation and are thus eligible for support by the Vision Training Program. In the past 10 years the Vision Training Program has graduated 45 PhD/MD-PhDs. Most have continued training at excellent labs, and many have subsequently assumed positions at excellent research institutions. Based on this growth in both faculty and students, we request support for 6 predoctoral students per year. We also request funds for one postdoctoral student per year to help promising candidates shift into vision research from other areas while seeking independent support for further career development.

Our long term goal is to understand the functional architecture of neural circuits in cat retina. Here we focus on the cone circuits to on-beta (X) and on-alpha (Y) ganglion cells. These types (with their off counterparts) account for greater that 90% of the optic nerve cross- section and divide spatio-temporal frequency space. Beta cells (narrow field, densely distributed) serve higher spatial and lower temporal frequencies; alpha cells (wide field, sparsely distributed) serve the reverse. Thus far, we have determined for both types the complete sets of parallel bipolar circuits that connect them to rods and cones. This has led to specific hypotheses regarding the basis for receptive field structure at different luminances. We now propose: 1) Complete the structure of the beta circuit. Determine amacrine cell types that supply the last 60 unidentified synapses; quantitate cone contacts to the three bipolar types with input to the beta cell (intracellular filling + confocal and electron microscopy). 2) Probe function at outer plexiform by localizing molecules that serve GABA and glutamate transmission. For GABA, localize the synthetic enzyme, transporter, and specific subunits of the GABAA receptor. For glutamate, localize (in on bipolar cells) components of G protein system possibly corresponding to the phototransduction cascade: transducins, phosphodiesterase, arrestins, cGMP ion channels (immuno- and in situ hybridization histochemistry). 3) Determine how known circuit gains for alpha and beta relate to the number of bipolar-to-ganglion cell synapses per retinal area. Measure bipolar synapses/ganglion cell membrane area at different eccentricities (EM) and dendritic membrane area/retinal area (fill + confocal). 4) Determine how the unique 3-D architectures of the alpha and beta dendritic meshworks are accounted for by known functional constraints (such as density, coverage, membrane area/retinal area, dendritic volume) and constraints on dendritic spacing. Quantitate these factors (fill neighbors of same type, confocal + 3-D graphics). 5) Assess how identified factors (optical + neural) account for beta ganglion cell performance. Construct compartmental models and ideal observer models; compare these to actual performance; assess which factors contribute to the difference. The proposed studies address fundamental mechanisms responsible for "image processing" by identified circuits in mammalian brain. These studies will help to understand human retina because many features of retinal organization are shared across species.

Our broad goal is to understand the circuits that link foveal cones to ganglion cells. Structural studies during the current period show the fovea to harbor multiple ganglion cell arrays. We suggest that each array signals a particular aspect of the visual scene and propose the following correspondences: high spatial frequency= midget (P) cell; low temporal frequency= parasol (M) cell; high temporal frequency= garland cell (M); hue= blue-yellow cell, red-green cell. We now plan to elucidate circuits for the remaining ganglion cells in our EM library and thus identify all the parallel channels from fovea to brain. We also plan to test these hypotheses by functional imaging of identified synapses: incubate retina with peroxidase tracer and briefly present a specific spatio-temporal-chromatic pattern to evoke exocytosis. As fused synaptic vesicles are retrieved by endocytosis, they sequester tracer which is fixed, and then visualized by confocal and electron microscopy. The ratio of labeled vesicles in different types of bipolar and amacrine synapse measures their relative release to a given stimulus. This approach, now being developed for a color-opponent circuit in guinea pig retina, will be applied to homologous circuits in primate retina. Finally, we plan to determine how the cGMP-gated channel contributes to a ganglion cell s visual response. Current through this channel is enhanced by nitric oxide and suppressed by glutamate through a novel mechanism (AMPA receptor coupled to a G-protein). We will identify the types of ganglion cell that express this current and determine which visual stimuli evoke and suppress it. The fovea occupies only two mm2 (0.3 percent) of the retina, but it supplies half of the visual cortex and is thus key to human vision. Knowing the functional architecture of its multiple, parallel circuits will help understand visual impairment (from conditions such as macular edema and age-related macular degeneration) and will ultimately contribute to the design of a retinal prosthesis.

DESCRIPTION (provided by applicant): Neural signaling employs many stochastic processes (transmitter release, receptor binding, channel opening) that might add noise and thus lose information. Loss has been assessed by measuring neural efficiency, the ratio of sensitivities for a human and an "ideal observer", which is a computational model that includes the human preneural losses (blur, photon noise, etc.) but no neural loss. Neural efficiency has seemed to reach at most about 0.5, implying considerable loss (50%); however, neural efficiency might be much higher (about 1) and have been underestimated because test stimuli were ill-matched to the neural pathway that mediates the discrimination. To test these alternative hypotheses, AIM 1 will measure neural efficiency for "brisk" ganglion cells that encode by precise spike timing and supply the geniculo-striate system. We record a cell's response to an optimal stimulus (i.e., matched to the spatiotemporal impulse response); then we compare the cell's sensitivity to that of an ideal observer subject only to preneural loss. Ganglion cell efficiency (the ratio of these measures) may well be >0.5. We will also record from pairs of adjacent brisk ganglion cells and use their combined responses to compute overall retinal efficiency, expecting values >0.50. Finally, we will test psychophysical sensitivity to similar stimuli, which might show that the brain and retina are equally efficient. Roughly half of all ganglion cells use a different coding strategy. They fire "sluggishly" (fewer spikes, less precise timing) to signal complex features of the visual scene, such as local edges. We hypothesize that a "sluggish code", although less effective than the "brisk code" at transmitting high temporal frequencies, is more efficient with respect to metabolic energy and wire volume. To evaluate this hypothesis AIM 2 will compare the coding properties, energy budgets, and wire volumes for brisk-sustained and "local-edge" cells, which have similar receptive field size and are the most numerous of the brisk and sluggish types. Finally, AIM 3 will investigate local circuits that may lend efficiency to both types of coding. Certain bipolar terminals release glutamate quanta in "bursts" whose timing resembles the spike bursts in brisk ganglion cells. We hypothesize that the bursts are caused by inhibitory amacrine feedback onto the terminals of brisk but not sluggish bipolar cells. We will test this by identifying the bipolar types that contact brisk and sluggish cells (morphology and function) and reconstructing their local amacrine circuits. The proposed studies address fundamental mechanisms of retinal function critical to early stages of human vision.

DESCRIPTION (provided by applicant): Our broad goal is to investigate key 'design' principles by which the primate retina transfers large amounts of information to the brain. Its 106 axons are strikingly heterogeneous. They comprise 15 channels, spanning a 50-fold range in axon diameter and a 10-fold range in spike rate. Noting that foveal circuits are strongly constrained for space and energy, we hypothesize that multiple channels exist in order to relay information at least cost in "wire volume' and metabolic energy. Preliminary studies suggest that channels transmitting at low information rates (few bits/second) are more efficient (more bits/spike) and physically smaller, thus using less space and probably less energy per bit. Thus central nuclei, which acquire information at different rates (e.g. geniculate M vs. K layers), can receive their messages at least cost. To test this, AIM 1 will measure for several ganglion cell types: (a) 'natural information rate' (bits/spike and bits/s in response to natural images); (b) total wire volume (soma + dendrites + axon + terminal arbor)(cell density); (c) relative energy costs, i.e., mitochondrial content. We predict that natural information rates differ strongly across channels and that 'low-rate' channels use less space and energy per bit. Noting that OFF channels are spatially finer and denser than ON channels and that 'blue/yellow' channels also occur on two spatial scales, we hypothesize that receptive field sizes and sampling rates are tuned to the distribution of information in natural scenes. To test this, AIM 2 will measure achromatic and chromatic information in natural images on scales corresponding to known receptive fields. We predict that in nature dark regions occupy higher spatial frequencies and contain more information per retinal area than bright regions and thus require finer channels with more synapses; there are analogous predictions for the blue/yellow channels. Noting that information transfer through the retina relies on 'ribbon' synapses, we hypothesize that they release and retrieve vesicles at very high rates. To test this, AIM 3 will measure release rates at the cone synapse (2-photon + electron microscopy of FM1-43 dye), and AIM 4 will measure the rates at the bipolar synapse (electrophysiology + EM of ferritin). Glaucoma, a major cause of blindness, has been attributed to both ganglion cell anoxia and reduced axonal transport. Our studies will relate ganglion cell signaling to both oxidative capacity and axonal transport capacity, and thus should extend the basic foundation for future clinical studies.

[unreadable] DESCRIPTION (provided by applicant): The broad goal of this proposal is to provide core support services for 39 principle investigators and 11 associated investigators to: (1) enhance the quality and quantity of their research; (2) facilitate collaborations between investigators with different backgrounds and skills; and (3) recruit new investigators to vision research. The PIs sort into several groups: psychophysics/systems neuroscience, visual transduction/retinal degeneration/gene therapy, eye (lens/ myopia/glaucoma), and clinical research. PIs hold individual research grants from NEI, and Associates hold individual grants from other sources. The Core includes 5 modules: [unreadable] Biostatistics provides expert assistance in experimental design and data analysis particularly for clinical studies. [unreadable] Computation-Illustration provides resources for scientific computing as well as expert computer support. [unreadable] Instrumentation provides for design and construction of optical and electrophysiological instruments that are unavailable from commercial sources. [unreadable] Molecular Biology provides DNA sequencing and gene mapping. [unreadable] Physiological Imaging, proposed here as a new module, would provide a new facility for multi-unit recording and an adjacent facility for optical recording of various functional signals from neural tissue in vitro, such as calcium, while imaging the whole cell by confocal microscopy. This module will greatly enhance the capabilities of the systems investigators and will provide new and efficient functional assays for investigators of molecular genetics and gene therapy. [unreadable] Altogether the Core accelerates our progress, improves opportunities for collaboration and helps translate basic research into clinical application. [unreadable] [unreadable] [unreadable]

1H-Benz(de)isoquinoline-2(3H)-carboxylic acid, 6-amino-1,3-dioxo-5,8-disulfo-, 2-hydrazide, dilithium salt; Blood Coagulation Factor IV; Body Tissues; Ca++ element; Calcium; Cell Communication and Signaling; Cell Signaling; Chromosome Pairing; Coagulation Factor IV; Collaborations; Coloring Agents; Communities; Complex; Computer Programs; Computer software; Count; Data; Data Set; Dataset; Dendrites; Dimensions; Dyes; Electrodes; Electron Microscopy; Electrophysiology; Electrophysiology (science); Equipment; Factor IV; Fluorescence Agents; Fluorescent Agents; Fluorescent Dyes; Hour; Image; Imagery; In Vitro; Individual; Injection of therapeutic agent; Injections; Intracellular Communication and Signaling; Investigators; Knowledge; Laboratories; Life; Methods; Microscope; Mitochondria; Morphology; Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; Neurophysiology / Electrophysiology; Numbers; Optics; Physiologic; Physiological; Physiology; Programs (PT); Programs [Publication Type]; Proteins; Purpose; Research; Research Personnel; Researchers; Resolution; Retina; Retinal; Science of neurophysiology; Sight; Signal Transduction; Signal Transduction Systems; Signaling; Software; Staining method; Stainings; Stains; Stains, Tissue; Standards; Standards of Weights and Measures; Surface; Synapses; Synapsis; Synapsis, Chromosomal; Synaptic; Synaptic Vesicles; System; System, LOINC Axis 4; Testing; Time; Tissue Stains; Tissues; VESCL; Vesicle; Vision; Visualization; base; biological signal transduction; calcium indicator; computer program/software; design; designing; experience; fluorescent dye/probe; gene product; imaging; immunocytochemistry; instrument; lucifer yellow; mitochondrial; neuronal; neurophysiology; new approaches; novel approaches; novel strategies; novel strategy; patch clamp; programs; size