Lawrence C. Sincich - US grants

In primates, visual information passes from the primary visual area (V1) to the second visual area (V2) before distribution to higher cortical areas. An accurate description of the projections linking V 1 and V2 is crucial for understanding how the brain deciphers visual images. Prior studies have shown that V2 is partitioned into three compartments, known as pale, thin, and thick stripes, defined by their content of a metabolic enzyme called cytochrome oxidase (CO). Our principal goal is to describe the anatomical projections from V1 to each V2 stripe compartment. In Specific Aim #1 we will make injections of a retrograde tracer into single CO stripes in V2 of normal macaques. The resulting pattern of labeled cells in V1 will be correlated with the V2 stripe that received the injection. Our preliminary data indicate that, contrary to a previous report, layer 4B and interblobs both project to thick stripes and pale stripes. In Specific Aim #2 we will make paired injections of two different tracers into adjacent thick stripes and pale stripes to determine if different subpopulations of cells in layer 4B and interblobs project to these V2 compartments. In Specific Aim #3 we will make injections of [3H]proline into V1 to correlate patches of efferent projections with CO staining patterns in V2. In Specific Ai/s #4 we will examine the V1->V2 projections in animals raised with early monocular deprivation. These experiments will advance our knowledge of the mechanisms underlying amblyopia, an important cause of visual loss that affects 2% of the American population. We hypothesize that a selective loss of V1->V2 projections emanating from the ocular dominance columns serving the deprived eye contributes to the loss of vision in amblyopia.

One of the basic requirements for understanding how the brain works is to know what the electrical activity of each neuron "means" with respect to an animal's behavior. The electrical signals are manifested as temporal sequences of very brief electrical impulses, commonly called spikes, which are the currency of the nervous system. Neurons respond to stimuli by spiking, and they communicate with each other by spiking. Interpreting what such spiking patterns encode has been of longstanding scientific interest because it translates into knowing what each neuron does within a circuit. The aim of this project is to go one step further. The goal is to examine how the spike pattern received by one neuron is transformed into a new, output spike pattern. Although much has been learned about what spike sequences encode, the coding transformation which occurs from neuron to neuron has rarely been quantitatively investigated. Only when this recoding of spike patterns is understood can a neuron?s functional role be considered fully characterized.

The research naturally progresses in three stages. First, spike trains will be recorded from connected pairs of neurons responding to naturalistic stimuli. The investigators have chosen to record from neurons in the primate lateral geniculate nucleus because their input, provided from retinal ganglion cells, and their output can be accessed readily. Second, they will apply a novel analytical method to determine the optimal stimulus features encoded by each cell's spike train. This involves a computationally intensive search through the stimulus space to arrive at the stimulus representations which carry the maximum amount of information. By comparing the optimal representations for the input and output spike trains, the feature transformation from one neuron to the next will be revealed. Third, they will test the predictive power of their method by using the found optimal stimuli and systematically degraded stimuli to probe neural responses in a second series of recording experiments. Such a test will serve to validate the computational basis of the feature transformation.

Having chosen a problem of very fundamental interest, the methods being developed will be valuable for studying signal communication and representation in any system of connected neurons. The outcome will provide a basis for comprehending the processing capabilities of neurons with multiple inputs, which are common throughout the brain, and could be applied to engineered systems designed for interpreting visual scenes.

Description (provided by applicant): To understand how the brain perceives color, it is necessary to learn how the retina creates its visual signals from the incoming flux of light. It is known that three types of cone photoreceptors are the starting point for any color signals. The cone responses to light are passaged to retinal ganglion cells, and their axons leave the eye to provide input to the visual thalamus, which in turn informs the rest of the brain. At present, there is a major controversy over the chromatic structure of the receptive fields of the "midget" class of retinal ganglion cells. To signal color, the responses of different cone types must be compared. It is uncertain if the midget ganglion cells receive comparative input from only single cone types, or from cone mixtures. The options would lead to different color coding schemes at this stage of the visual system, and thus have important consequences for how color is thought to be processed at later stages. Because the midget ganglion cells comprise about 80% of the output from the retina, it is crucial to work out their true signaling properties. The main impediments to solving this problem have been the inability to identify and stimulate individual cones in the living retina. The goal of this proposal is to overcome these limitations and develop a retinal microstimulator that can visualize the cones in a living eye, identify their spectral type, and most importantly, stimulate single cones selectively and repeatably with colored light sources. State-of-the-art adaptive optics techniques will be used to image and track the cone mosaic. The design will incorporate several convergent, confocal optical trains for multiwavelength imaging, stimulation, and cone spectral identification. To verify that the system can deliver stimuli as intended and produce wavelength-specific responses from cones, neurophysiological experiments will be conducted to map the cone fields providing input to single neurons in the visual thalamus of a trichromatic primate. These experiments are the only means of validating the stimulus precision of the instrument, and will provide the empirical foundation necessary for any future studies conducted in humans. PUBLIC HEALTH RELEVANCE: A color retinal microstimulator with unprecedented control of photoreceptor- specific stimuli will benefit ophthalmologists and physiologists studying normal and diseased photoreceptor function, as well as those interested in the neural basis of color processing in the cerebral cortex. The instrument will offer the first opportunity for probing, at a cellular level, the physiological and perceptual changes associated with cone dystrophies and colorblindness. It will also be useful for testing the effectiveness of gene therapies being developed for retinal ciliopathies.

DESCRIPTION (provided by applicant): To understand how we perceive color, it is necessary to learn how our retina creates visual signals from the incoming flux of light. It is known that 3 types of cone photoreceptors are the starting point for any color signals. The cone responses to light are passed to retinal ganglion cells, and their axons leave the eye to provide input to the thalamus, which in turn sends signals to primary visual cortex (V1). Currently, there are two persistent controversies over the chromatic structure of the receptive fields of neurons along this pathway. One controversy concerns the midget class of retinal ganglion cells near the fovea. It is uncertain if these ganglion cells receive input from only single cone types, or from mixed cone types, in their receptive field centers. The two options lead to different color coding schemes at this stage of the visual system, and thus constrain how color is processed at later stages. Because the midget ganglion cells comprise about 80% of the output from the retina, it is crucial to work out their true signaling properties. The second controversy centers on color signaling in V1. Here, it has also been unclear how sensitive are V1 neurons to cone-specific stimuli, and the difficulty has been, in part, inherited from problems associated with uncertainties in the retinal input. The main impediments to solving these problems have been the inability to identify and stimulate individual cones in the retina in vivo. The goal of this proposal is to employ a newly developed retinal microscope that overcomes these hurdles. The instrument can image the cones in a living eye, and can stimulate single cones selectively and repeatably with colored lights. We first propose to verify that cones can be mapped by their spectral sensitivity in humans, using psychophysical techniques. Using the stimulation parameters that allow cone identities to be obtained, we next propose to map physiologically the cone fields that provide input to single neurons in the visual thalamus of a trichromatic primate. With cone-sized stimulation, thalamic neurons receive input essentially from single retinal ganglion cells; thus we will learn whether ganglion cell receptive field centers are composed of pure or mixed cone types. We will focus on foveal thalamic neurons that receive input from the midget retinal ganglion cell class. Finally, we propose to map the cone fields of V1 neurons, to determine the strength of their cone-specific input. We will confirm histologically the location of these neurons in visual cortex, to learn where they are situated within the known anatomical circuits of V1. Our results will afford the first direct mapping of cone fields in vivo, and improve our understanding of how photoreceptor signals are processed by neurons subserving foveal vision.

Project Summary We are asking for support to continue to develop and enhance three state-of-the-art optical instruments that will be used to answer questions about the most important and the most challenging region in the retina to study, the fovea. The instruments are built upon two key technical strengths - adaptive optics scanning laser ophthalmoscope (AOSLO) systems and accurate, high-speed eye-motion tracking. Adaptive optics technology corrects the imperfections in the eye and can be used to generate microscopic views of the living retina and deliver ultra-sharp images to the retina. Eye tracking is used to measure and compensate for ever-present eye motion. Together, these allow for visualization, tracking and delivery of light to retinal features as small as single cone photoreceptors, enabling measurements of properties of spatial and color vision on an unprecedented scale. Although the three systems will be identical, the scope of study for each system will be very different. The AOSLO at in Alabama will be used to test vision in non-human primates, the AOSLO in Berkeley will be used to perform advanced vision testing on healthy human eyes, and the AOSLO in San Francisco will be used to study patients with eye disease. The key advantage of having the BRP manage three identical systems is that it will facilitate hardware innovations plus rapid translation of knowledge and innovative testing from animal models to the clinic. Briefly, the specific aims are: Aim 1: Advanced AOSLO display capabilities for color vision: We propose a series of technical developments will expand the scope of AOSLO experiments, not just for color vision, but also spatial vision and clinical applications. Specifically, we will (i) add 2-photon stimulation (ii) develop new methods to display large stimuli that are fixed in world-coordinates (iii) integrate dichoptic displays to enable experiments that distinguish retinal from cortical visual processing (iv) develop I-TRACK (improved software tools for retina- contingent vision testing) and (v) invisible imaging and tracking. These tools will enable a series of experiments to learn how the visual system extracts color and spatial information from its sensory inputs. Aim 2: Enhanced AOSLO systems and modeling for spatial vision: In this aim we will (i) develop advanced wavefront propagation tools to model light-cone interactions (ii) integrate AOSLO microstimulation with a system for 2-photon functional brain imaging in non-human primates. We aim to use these tools to greatly enhance our understanding of receptive fields at and near the fovea. Aim 3: Clinical translation: We will integrate the new technology into the system at UCSF to (i) study rod vision in patients with rod-cone degenerations (ii) measure the time course, structure and function of dysflective cones (iii) investigate the structure and function of the preferred retinal locus in diseases that affect the fovea and (iv) assess inner retinal function in eye disease.

Project Summary Vision forms a prominent part of our daily lives, and this sensory process begins with the capture of light by a particular class of photoreceptor called cones, a class well adapted to daylight conditions, to high acuity vision, and to color perception. The basic response properties of cone photoreceptors have been largely described in studies conducted in isolated cells or retinal tissue. Surprisingly, many of these response properties, such as temporal dynamics and intensity sensitivity range, remain controversial in the literature. Moreover, some of them conflict with data acquired in vivo and with human visual performance when measured psychophysically. The primary aims of this grant are to generate a more comprehensive characterization of cone photoreceptors in cone-dominated animals, including primates. This will be achieved by conjoint experiments conducted in intact retinal explants in parallel with in vivo recordings where it is now possible to target single cones with adaptive optics microstimulation. Similar stimulation paradigms will be employed in each experimental approach to the measure intensity response functions, the rapidity of time-to-peak, the decay kinetics following saturating stimuli, and the adaptation rate in response to changing background light levels. These data will be used to develop a complete theoretical model of response properties based on cone phototransduction which will be of great use to vision scientists and may delineate the limits and boundaries of in vivo vs. in vitro experiments on the retina. An additional aim will be to detail how a novel form of cone activation?via pulsed infrared 2-photon excitation?may be used for improved spatial stimulation of cones. The 2-photon experiments are best done interleaved with traditional 1- photon stimulation in order to examine how the 2-photon excitation mechanism differs from 1- photon absorption, and also to determine how much endogenous fluorescence may be generated by such stimuli. As 2-photon absorption may be used to measure the health of the cone visual cycle, understanding the basic response properties of infrared stimulation will be foundational for future studies of cone function and disease.