Imaging of Free Cytosolic Calcium Dynamics in Spines

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Dendritic spines are tiny membranous compartments that are the receiving ends of excitatory synapses. Spines contain synaptic receptors, channels, signaling molecules eytoskeletal proteins, and smooth endoplasmic reticulum. Synaptic plasticity expressed at spines may underlie the formation of some kinds of memories, and many forms of mental disorders are associated with spine pathologies. Of particular interest is [Ca 2+] signaling in spines. Spines compartmentalize Ca 2+ ions that enter the spine cytoplasm. Spine Ca 2+ plays a crucial role in the induction of most forms synaptic plasticity by regulating postsynaptic enzymes that trigger rapid modifications of synaptic strength, and also to activate transcription factors that facilitate long-term maintenance of these modifications. An important question is how Ca 2+ can encode all of these functions with any kind of specificity? The answer must lie in the details: [Ca 2+] signals with different amplitudes, time courses or in different locations will have distinct biochemical meanings for the cell. We will measure spine [Ca 2+] signals and the mechanisms that shape them, focusing on Ca 2+ sources and extrusion mechanisms. We will measure the trial-to-trial fluctuations in Ca 2+ influx and count Ca 2+ channels and synaptic receptors that serve as Ca 2+ sources. Using fluorescent indicators of ealmodulin (CAM) activation we will characterize patterns of [Ca 2+] elevation in terms of their ability to activate CaM, allowing us to construct a detailed kinetic model of CaM activation in situ. We will also study plasticity of Ca 2+ channels in spines mediated by Ca2+-CaM dependent kinase (CaMKII). Parameters derived from our measurements will be incorporated into a quantitative model of [Ca 2+] signaling and CaMKII in spines, coded in MCell. In addition, our measurements will serve as important benchmarks for the model. The MCell model will allow us to explore the mechanisms shaping [Ca 2+] dynamics at resolutions beyond our current experimental methods. We will use the MCell model to gain an-understanding of [Ca 2+] dependent plasticity of Ca 2+ channels, which may be triggered by microdomains of high [Ca2+], beyond the resolution of optical techniques.