The modification of synaptic connections between neurons is thought to underlie the ability to form memories and learn new behaviors. In the central nervous system, most of excitatory synapses terminate on dendritic spines, tiny (~0.5 µm in diameter) protrusions emanating from the dendritic surface. Calcium influx into spines activates various signaling pathways that underlie synaptic plasticity. The small GTPase protein Ras couples calcium influx to many forms of synaptic plasticity, such as rapid synaptic potentiation and new synapse formation. Ras activation can also trigger protein synthesis and gene transcription important for the long-term maintenance of synaptic plasticity and for many other neuronal responses, including cell survival, death, and differentiation. Consistent with many essential roles of Ras signaling in neuronal plasticity, mutations in the Ras signaling pathway are associated with diseases causing cognitive impairments and learning deficits such as autism, X-linked mental retardation and neurofibromatosis 1.
The extensive branching of the Ras pathway raises a question of how Ras can control such diverse downstream effects, including local synapse-specific plasticity and global neuron-wide responses. Our hypothesis is that particular spatial and temporal patterns of calcium elevation result in specific patterns of Ras activation, which subsequently turn on distinct sets of downstream proteins, leading to specific effects. However, most of our knowledge of Ras signaling comes from classical biochemical experiments that lack spatial resolution. For a deeper understanding of molecular mechanisms of synaptic plasticity, and ultimately learning and memory, measurements of Ras activity at the level of individual spines are crucial.
To study Ras signaling in spines and dendrites, we have been developing a Ras activity sensor based on Fluorescence Resonance Energy Transfer (FRET). Since FRET occurs only when the two fluorescent molecules are within a few nanometers of each other, it can be used as a sensitive detector of interactions between proteins labeled with fluorophores. We use fluorescence lifetime, which is defined as the time elapsed between fluorophore excitation and photon emission, as a robust and quantitative measure of FRET.
Although these optical techniques have been implemented in other systems, there are obstacles to applying them to the study of signaling in individual spines. Since often only a few copies of each protein exist in each spine, single molecule sensitivity is required. In addition, strong light-scattering of the brain tissue makes high resolution imaging impossible with conventional microscopy. Because 2-photon laser scanning microscopy (2pLSM) allows robust signal detection necessary for single molecule sensitivity and sufficient spatial resolution, we combine 2pLSM with fluorescence lifetime measurements (2-photon fluorescence lifetime imaging microscopy, or 2pFLIM). This microscopy technique enables us to image Ras activity by measuring interactions between Ras fused with green fluorescence protein and Ras binding domain of Raf, which binds selectively to active Ras, fused with red fluorescence protein.
Using this Ras imaging technique, we will measure the spatial and temporal dynamics of Ras activity in response to physiological stimuli causing different calcium localization, and examine how the Ras pathway processes the dynamics of calcium elevation spatially and temporally. Specifically, we will ask to what extent Ras signaling is restricted in spines in response to spine specific calcium elevation, and what kind of stimulation causes more global Ras activation. Through these experiments, this project is expected to reveal how Ras couples particular patterns of calcium signaling to specific cellular responses.
A deep understanding of the Ras pathway will provide immediate insights into treatments for Ras related diseases. Furthermore, our method will open new possibilities to analyze how mutations associated with mental diseases affect normal Ras activity and neuronal plasticity at the single synapse level. This will significantly impact the development of therapeutics for mental diseases.