Neurotransmitter binding to specific integral membrane receptors results in a diversity of cellular responses. The spatial extent of these different responses may vary tremendously. Inhibitory inputs on the somata of neurons can have dramatic effects on the electrical properties of the whole cell. Conversely, 1P3 mediated release of intracellular stores of calcium can be limited to a single dendritic spine. Thus, spatial localization is an especially crucial feature of synaptic chemical signaling and it is believed to be responsible for the input specificity of certain forms of synaptic plasticity (viz, long-term potentiation or depression). More generally, spatially localized signaling underlies a wide range of neuronal function in development and adulthood, e.g. cell migration, axon guidance and neural computation. Many of these processes are disturbed in pathological states. Before an adequate description of these disease states can be given, a more complete understanding of non-disease states should be accomplished. The proposed studies will contribute to a greater understanding of the normal functioning of these processes. The long-term goal of this grant is the development and application of new chromophores designed for highly localized photorelease of neurotransmitters at synapses. The development of technologies such as the patch clamp technique and laser-scanning confocal microscopy has revolutionized our understanding of many cellular functions. These techniques enable us to monitor the intracellular environment in real time. Caged compounds (i.e. photosensitive, biologically inert signaling molecules) complement these electrical and optical techniques as they provide control of cellular chemistry, in both temporal and spatial domains. During the past ten years a new type of solid-state laser technology has become readily available (Ti:sapphire), which allows for infrared excitation of UV-absorbing chromophores. Uncaging is produced by simultaneous absorption of two red photons of equivalent energy to one blue photon. However, the currently available caged compounds are insensitive to 2-photon photolysis. It is the intent of this grant to develop and test new chromophores that will be effective for 2-photon photolysis. Uncaging using these compounds will provide 3-D spatial control of the release of neurotransmitters onto living cells. There are four general themes concerning the functioning of neurons that will be studied: 1. receptor localization; 2. receptor density; 3. receptor trafficking; and 4. synaptic plasticity. This project will further our understanding of the basic mechanisms of signaling within the brain and will pave the way toward understanding the etiology of neurological disorders that act by altering the central synaptic transmission.
