Marla B. Feller - US grants

DESCRIPTION: (provided by the applicant) The long-term objective is to delineate the molecular mechanisms by which neural activity regulates the efficacy and structural integrity of central synapses, and to identify processes that ultimately determine the synaptic connectivity patterns. Many drugs of abuse modulate synaptic activity, and their long-term use is likely to promote remodeling of neural circuitry that contributes to the neurological dysfunction. This proposal seeks to facilitate a search for therapeutic intervention of neural remodeling that accompany substance abuse. Cultured neurons provide a valuable system to study activity-dependent behavior of individual synapses. Long-term observation of individual synapses for correlating activity and structural modifications, however, is hampered by procedural limitations. Stimulation protocols either lack spatial specificity, or require Substantial physiological perturbation to the neuron. The goal of this proposal is to develop a novel non-invasive stimulation technique to examine activity-dependent synapse remodeling of cultured neurons and to implement the technique. I. Photoconductive stimulation of cultured hippocampal neurons The ability of light to alter the conductivity of silicon will be exploited to achieve targeted photoconductive stimulation of dissociated hippocampal neurons cultured on silicon surface. Photoconductive stimulation parameters will be optimized using Ca(2+)-sensitive fluorescent dyes and activity-dependent fluorescent synapse markers. II. Mechanisms of activity-dependent morphological plasticity of synapses formed on silicon The role of activity in inducing short- and long-term morphological changes of hippocampal synapses will be addressed by targeted photoconductive stimulation. Our working model is that stimulus paradigms that are used to elicit long-term potentiation induce new synapse formation. The behavior of synaptic actin dynamics, synaptic vesicle distribution and the stability of postsynaptic density will be examined by real-time video imaging of fluorescent markers, and pharmacological requirements of morphological plasticity will be established. The role of actin dynamics as the potential downstream target of synaptic activation, which initiate the actual process of synapse remodeling, will also be addressed.

Immature retinal neurons spontaneously generate correlated activity in the form of waves of action potentials that sweep across the retinal ganglion cell layer. These retinal waves occur during the developmental period when functional circuits within the retina are emerging and retinal projections to the brain are undergoing a tremendous amount of refinement. Though significant progress in elucidating the circuits that mediate these waves has been achieved, the mechanisms that underlie the reliable generation of retinal waves is not fully understood. Here we explore the circuits that robustly generate retinal waves. Retinal waves are mediated by different circuits at different stages of development. In the first postnatal week, a circuit consisting of cholinergic interneurons, called starburst amacrine cells (SACs), mediates waves. During the second postnatal week, glutamatergic interneurons called bipolar cells mediate waves. Although cholinergic and glutamatergic waves are mediated by distinct circuits, they share one characteristic: the depolarization propagates across cells that do not have direct synaptic connections. Using two-photon calcium imaging and state-of-art optical sensors that measure[?] extrasynaptic release of ACh and glutamate, we will test the hypothesis that these waves propagate via volume transmission. In addition, we will study the influence of intrinsically photosensitive retinal ganglion cells (ipRGCs) on retinal waves. While ipRGCs do modulate waves in WT mice, they more dramatically modulate the compensatory waves found in knockout mice lacking the ¿2 subunit of the nicotinic acetylcholine receptor (¿2KO). ¿2KO mice exhibit significantly different patterns of spontaneous activity than WT mice, and therefore have served as a primary model system for understanding the role of retinal waves in visual system development. We propose using multielectrode and targeted recordings from WT and ¿2KO mice expressing GFP in ipRGCs to explore the novel hypothesis that ipRGCs regulate firing patterns in the developing retina by altering the dopamine level. This work will address the principles of general organization that are responsible for generating the activity patterns that drive activity-dependent developmental processes. It should also elucidate the principles that govern the normal development of the human nervous system, thus making it possible to understand the origin of neurological birth defects and to devise strategies that enable the nervous system to regenerate functioning neural circuits after injury.

[unreadable] DESCRIPTION (provided by applicant): This is an application for partial funding for the conference on "Retinal Neurobiology and Visual Processing", held under the auspices of the Federation of American Societies for Experimental Biology (FASEB) to be held in Saxtons River, Vermont, July 17-22, 2004. This will be the seventh in a series of biannual conferences that has established a unique forum for researchers with a variety of backgrounds to discuss vision. The participants have traditionally drawn from many areas of neurobiology and vision research with the goal of applying state-of-the-art techniques to understanding the neurobiological basis of vision. A second goal of this meeting is to provide opportunities for graduate and postdoctoral students to meet and share their ideas with established investigators. The proposed conference program addresses questions that are of high interest to the vision community. The major session topics will be 1) retinal implants 2) dendritic processing, 3) relative role of genetic factors and neural activity in retinal development 4) visual signaling from retina to brain 5) interneuron signaling 6) models for retinal disease, 7) plasticity in the retina, and 8) the biophysics of release. In addition, there will be a workshop on transfection techniques and two 2-day poster sessions. Approximately 180 participants will be chosen from among scientists most likely to participate in discussion in a stimulating free exchange of ideas. This conference will allow for critical review of recent research as well as an evaluation of future directions. [unreadable] [unreadable]

The long-term objective is to delineate the cellular mechanisms by which spontaneous correlated neural activity is generated in the developing mammalian retina. Very early in brain development, before sensory experience is possible, both electrical and chemical activity is generated spontaneously throughout the immature nervous system. There is growing evidence that this early activity is critical for the appropriate development of neural circuits. Developing a detailed understanding of the organizing principles that govern the normal development of the human nervous system may make it possible to understand the origin of neurological birth defects. In addition, it will provide critical insights into devising strategies that allow the nervous system to rewire normal functioning neural circuits in response to developmental abnormalities such as amblyopia (lazy eye). The cellular basis of spontaneous activity in the retina has been studied primarily by electrophysiology and calcium imaging. Spontaneous retinal activity is characterized by depolarizations that occur with a period on the order of minutes. Indeed, we can reproduce this slow periodicity in dissociated retina neurons, indicating that the pacemaker underlying this periodicity may be cell autonomous. Here we propose to test the hypothesis that this slow periodicity is set by oscillations in the second messenger, cAMP. The goals of this proposal are two fold. First, we propose to implement in retinal neurons the use of two indicators - one sensitive to levels of the second-messenger cAMP levels, and the second sensitive to activity of protein kinase-A. Second, we will then use these indicators to determine whether spontaneous oscillations in cAMP underlie the periodicity observed in both the intact retina as well spontaneously active networks formed by dissociated retinal neurons.

Prior to birth, before there is communication with the outside world, the nervous system is spontaneously generating highly patterned activity. Dr. Feller studies this phenomenon in the developing retina. Before vision is possible, neighboring retinal ganglion cells, the neurons of the retina that project to the brain, spontaneously fire correlated bursts of action potentials that propagate across the retina in the form of waves. This correlated activity, termed retinal waves, is required for the proper refinement of retinal projections to targets in the brain.
There is a stage of retinal development immediately preceding eye-opening during which retinal circuits gradually transition from generating retinal waves to mediating light-evoked responses that are the first stage of visual processing. During this transition, these two functional circuits coexist for several days. Two questions regarding this critical stage of development, when the retina itself may be highly plastic are addressed. First, how are retinal waves generated? Sophisticated physiology and imaging experiments will be conducted on acutely isolated mouse retina are used to identify the circuit that underlies retinal waves. Second, how do retinal waves and light-evoked responses interact to determine the firing pattern in the retina? A specialized array of electrodes will be used to record simultaneously from dozens of retinal ganglion cells to determine the interaction between the circuits that mediate waves and light-evoked activity. These experiments will help to better understand the role of spontaneous vs. sensory driven activity in the assembly of neural circuits.
In addition, the funds from this project will support the training of two young scientists who are new to Neurobiology. The first is a postdoctoral researcher, Anastasia Anishchenko, who received her Ph. D. in theoretical physics. The second is graduate student, Aaron Hamby, who is trained as a molecular biologist. In addition, the results of this research will be used as material for an undergraduate course on developmental neurobiology that emphasizes the construction of neural circuits.

Direction-selective ganglion cells respond strongly to an image moving in the preferred direction and weakly to an image moving in the opposite, or null direction, and are critical for driving ocular-motor reflexes that stabilize images on the retina as we move through a visual scene. The preferred direction of direction-selective ganglion cells cluster along the cardinal directions (up, down, left and right) and the direction-selective ganglion cells sensitive to each cardinal direction are organized into mosaics such that at each point in space, each direction of motion is represented. The predominant model for the generation of direction selectivity in the retina is that a particular class of interneurons form inhibitory synapses on the null side of the dendritic tree of directionselective ganglion cells. The mechanisms that instruct the emergence of mosaics comprised of cells that receive an asymmetric distribution of inhibitory inputs during development are unknown. Our long-term goal is to determine the mechanisms that underlie the development of these two essential features of direction-selectivity - the circuits that underlie the null side inhibition and the existence of direction-selective ganglion cells mosaics. In particular, we will determine whether spontaneous retinal activity plays a critical role in the formation of these circuits. Here we propose to use a combination of state-of-the-art electrophysiological and imaging techniques to determine the age at which directional circuits are established.

DESCRIPTION (provided by applicant): Direction-selective ganglion cells respond strongly to an image moving in the preferred direction and weakly to an image moving in the opposite, or null direction, and are critical for driving ocular-motor reflexes that stabilize images on the retina as we move through a visual scene. The preferred direction of direction-selective ganglion cells cluster along the cardinal directions (up, down, left and right) and the direction-selective ganglion cells sensitive to each cardinal direction are organized into mosaics such that at each point in space, each direction of motion is represented. The predominant model for the generation of direction selectivity in the retina is that a particular class of interneurons forms inhibitory synapses on the null side of the dendritic tree of direction- selective ganglion cells. The mechanisms that instruct the emergence of mosaics comprised of cells that receive an asymmetric distribution of inhibitory inputs during development are unknown. Here we propose to use a combination of state-of-the-art electrophysiological and imaging techniques to determine the mechanisms that underlie the development of these two essential features of direction-selectivity - the circuits that underlie the null side inhibition and the existence of direction-selective ganglion cells mosaics. In particular, we will determine whether spontaneous retinal activity plays a critical role in the formation of these circuits.

Our research goal is to determine the factors that instruct the development of visual responses in the mammalian retina. In particular, we are studying the developmental period when the retina transitions from generating retinal waves to mediating visual responses by forming functional circuits. Utilizing a high-speed volumetric two-photon microscope will enable the first description of spontaneous firing patterns across identified microcircuits, such as those that mediate direction selectivity. Our work will determine what role early spontaneous activity plays in wiring up circuits during development.  

A major focus of modern neuroscience is understanding the role of neuromodulators in neural circuit function and development, in behavior, and their dysfunction in neurological disease. Neural circuit research has been revolutionized by powerful new optical techniques. Here we propose to use new near-infrared optical nanosensor technology to image dopamine. As a proof of principle, we will use this sensor to monitor dopamine release under physiological conditions in both the developing and adult retinas. The ability to combine neuromodulator imaging with other physiological measures in a well-defined neural circuit will greatly advance our understanding of neuromodulation, allowing us to elucidate its many impacts on normal and pathological circuits.  

See abstract in Overall Component.