Hollis Cline - US grants

Nerve cells communicate with one another via a slender cellular process called an axon. Each nerve cell, or neuron, has a very specific pattern of axonal connections, and normal function of the nervous system depends on this specific pattern of connections. Development of the proper pattern of connections in the brain requires a precise regulation of axonal branching in the vicinity of a neuron's target cells. The long-term goal of this project is to identify the mechanisms that regulate axonal branching in the developing nervous system. Recent studies show that neurons grow by the continuous addition and retraction of branches. An increase in axonal branching, such as that observed after activity is blocked, might occur through a decrease in the amount of branch retraction or an increase in the amount of branch addition. To resolve this issue, it is necessary to continuously monitor the living axon over time. This can now be accomplished using laser scanning confocal microscopy. This project will determine the temporal limits for modifications of axonal structure and test the hypothesis that blocking synaptic activity increases the rate of branch initiation. These results will provide important new insights into the mechanisms responsible for nerve cell form and function.

DESCRIPTION (provided by applicant): Sensory experience is required for the development of the visual system, however the direct effects of visual experience on the development of the neural circuits in visual centers have been difficult to determine. Development of neural circuits requires that neurons extend axons and dendrites into regions of the brain where they then form and maintain synaptic connections. In order to understand mechanisms controlling circuit development, it is essential to determine the mechanisms that control both the structural development of neurons and the formation of synaptic connections. The primary goal of the experiments in this proposal is to determine the activity-dependent mechanisms that govern the formation of visual system circuits. We address this question by studying the mechanisms that control the development of synaptic connections and neuronal structure in the retinotectal system of Xenopus, using imaging, electrophysiology and molecular biology methods. During the last funding period, we found that a relatively brief period of visual stimulation leads to a significant increase in growth of dendritic arbors of neurons in the optic tectum and that the increased growth of the optic tectal neurons requires glutamatergic synaptic transmission. We now have the unique opportunity to determine direct consequences of sensory experience on the structural and functional development of the visual system. We will express fluorescent proteins in presynaptic retinal axons and postsynaptic optic tectal neurons which allow us to visualize the development of neuronal structures and synapse formation in the intact brain. We propose to determine how visual stimulation and activation of postsynaptic glutamate receptors regulates growth of presynaptic retinal axons, tectal cell dendrites and formation of synaptic connections from the eye into the brain. In addition to addressing fundamental questions regarding mechanisms of synaptogenesis and stabilization, we will determine the effects of visual stimulation, synaptic transmission and glutamate receptor function on visual system development.

DESCRIPTION (Adapted from applicant's abstract): Visual activity patterns in the developing brain shape the formation of circuits which process that activity. While this is well known, the mechanisms by which this activity exerts these effects remains a critical problem in the field of visual system development. One possibility is that electrical activity in neurons triggers the synthesis of new genes and their protein products. These activity-dependent proteins could then modify the growth of neurons and the formation of synaptic connections. In this proposal, a series of experiments are proposed to test the hypothesis that activity can modify the structure of neurons in the visual system of the albino frog Xenopus laevis. This experimental system permits the in vivo observation of the growth of single neurons in the intact brain of the anesthetized animal. By combining this approach with virus-mediated heterologous expression, and electrophysiological recordings, it will be possible to test whether the expression of two recently identified activity-regulated genes have an impact on tectal development. Preliminary data indicate that these proteins will indeed produce robust changes.

During development, many brain regions establish 'maps' in which inputs are organized so that correlated activity converges on common target nerve cells, the neurons. Activity coming from the eye is crucial in regulating the formation of a spatial map of the external visual world. The incoming nerve fibers, called afferent axons, make functional contacts called synapses on the receiving parts, called dendrites, of target neurons. Physiological activity regulates how the axon terminals and the dendrites develop their detailed morphology, and regulates the strength of the synaptic connections; without vision, these connections do not form properly. This project uses pharmacological manipulations with a novel microscopic technology of time-lapse confocal imaging of cellular structures as they develop in the brain of tadpoles. Tests analyze how synaptic strength affects synaptic structure, and how the neurotransmitter molecule called GABA and a molecular receptor called the NMDA receptor regulate development of structure and function.
Results will provide valuable new information on regulatory mechanisms controlling development at the molecular level in the living brain. This project also provides substantial training for students from the high school to postdoctoral level.

DESCRIPTION: Visual experience modulates the development circuits in the brain that process visual information. One way that such neuronal activity can modify neuronal development is by changing the structure of the component parts, the individual neurons themselves. Considerable evidence supports the hypothesis that neuronal growth can be increased or decreased by changes in the pattern of electrical activity experienced by the neuron. The biochemical mechanisms by which such changes in neuronal growth occur are likely to be mediated through enzymes which themselves are controlled by neuronal activity. One such class of enzymes is the calcium-sensitive protein kinases, including. This protein kinase has been implicated in neuronal development, synapse formation and synaptic plasticity in the visual system. Unfortunately the pharmacological agents used in earlier studies could not clearly distinguish CaMKII and another calcium-sensitive protein kinase, protein kinases C. To address the problem of poor specificity of pharmacological agents, we now propose to use Vaccinia virus to introduce into neurons of the frog visual system a gene for a form of CaMKII that is no longer regulated by calcium and calmodulin. With this method, we can obtain expression of the constitutively active kinase only in the postsynaptic neurons, but not in the presynaptic retinal cells. Using confocal microscopy, we will then take time-lapse images of dye-labeled retinal axons or optic tectal neurons to observe their pattern of growth over a period of up to 4 days This latter method offers the unique opportunity to observe neurons grow in their normal complex environment. Previous experiments in fixed tissue have provided static images of neuronal development, from which active processes were inferred. Thus far, our observations of neuronal growth in situ have shown that in fact neuronal structure is much more dynamic than previously recognized and that retinal axonal structure can be modified by activity at earlier developmental stages than previously thought. By combining these 2 techniques, viral transfection of neurons in the optic tectum and confocal imaging of developing neurons in situ we will be able to test the hypothesis that postsynaptic CaMKII activity can modify the development of neuronal structure and synaptic circuits in the visual system.

Cold Spring Harbor Laboratory's Animal Shared Resource consists of two separate facilities that house and support research animals for use by the Cancer Center's research investigators. These facilities consist of the Harris Research Support Shared Resource, located on the Laboratory's main campus, and the Woodbury Research Support Shared Resource, currently under construction and located within the Laboratory's Woodbury Genome Center. This modern facility is specifically designed to maintain animals under pathogen free conditions. The CSHL Cancer Center Animal Shared Resource is used by most of the scientific staff at the Laboratory. Usage of the Shared Resource can be subdivided into three categories based on the research needs of the individual investigators: antibody production, the generation of transgenic animals, and housing animals. The Resource was used at about 90% capacity over the last year. Approximately 80% of the use of the Harris Animal Shared Resource was by members of the Cancer Center. The CCSG provided 24% of the income to the Shared Resource, while 76% of the income was provided by core billing charge backs. The use of the Animal Shared Resource divided almost evenly among members of the three different programs (Gene expression, Cell Biology and Cancer Genetics)) in the Cancer Center). Significant improvements to the Animal Shared Resource over the net funding period will be the expansion into the second Woodbury Research Support Shared Resource and the provision of additional services relating to the use of animal models for cancer research.

DESCRIPTION (provided by applicant): Activity-dependent mechanisms regulate multiple aspects of visual system development. One way that activity can influence the development of neuronal connections is by the induction of activity-regulated or "candidate plasticity" genes (CPGs). The protein products of these genes may then affect the morphological development of neurons and the formation of synaptic connections. We propose to study the function of two activity-regulated genes, homer and cpgl5, in the development of the visual system of the frog, Xenopus. We do this by expressing either wild type or mutant genes in the optic tectum, combined with in vivo time-lapse confocal imaging and electrophysiological studies of retinotectal connectivity. CPG15 enhances the morphological development of tectal cell dendrites and retinal axons and it promotes the development of retinotectal synaptic connections. We now propose to determine the mechanisms that regulate the expression, trafficking and function of endogenous CPG15. Our studies on Homer are also quite exciting. Xenopus has 2 Homer isoforms, one (Homer la) is induced by activity and the other (Homer 1b) is not. Homer 1b is a cytosolic anchoring protein that is thought to link cell surface receptors to intracellular calcium stores. Our studies suggest that Homer 1b is required for axon guidance in optic tectal neurons. We propose to test the hypothesis that Homer 1b regulates calcium signaling in growth cones in response to axon guidance cues in the environment. We will determine the generality of the role of Homer 1b in axon guidance by testing its function in tectal cell axons and retinal ganglion cell axons. The Xenopus visual system is ideal for these experiments because one can collect images of the developing visual system under control or experimentally altered conditions. We will continue to use our expertise in in vivo imaging of neuronal morphology and intracellular calcium dynamics. The addition of two-photon laser scanning imaging to our imaging repertoire has significantly increased our ability to resolve detailed structural dynamics deep in the brain and over a wide range of time scales not permitted by traditional confocal microscopy. We will apply these powerful approaches to test the function of activity-regulated genes in the development of visual system connectivity.

Abstract Not Provided