We hypothesize that perturbations of protein trafficking into and out of synapses contribute to the manifestations of human neurological diseases. In order to test this hypothesis, new methodologies need to be developed that allow real-time monitoring of protein movement in small cellular compartments of living neurons. The specific aims are to:
1. Develop a method to directly visualize the movement of signaling and structural proteins into and out of synapses in living neurons.
2. Identify sets of stimuli that trigger rapid rearrangements of protein constituents of the synapse.
3. Determine if perturbation of synaptic protein movement are present in a mouse model of the human disease Tuberous Sclerosis Complex.
We propose to accomplish these aims using a novel microscope that makes use of precisely controlled laser pulses to simultaneously manipulate and monitor synaptically relevant proteins and signaling cascades deep in brain tissue. The microscope focuses and scans two lasers independently over the specimen. One laser is tuned such that its activation triggers a chemical reaction that results in the release of bioactive molecules or the activation of fluorescent proteins. The second laser is used to image the structure of the neuron, follow the movement of synaptic proteins, or monitor intracellular signaling cascades. Both the imaging and photoactivation properties of the microscope rely on 2- photon excitation and therefore have high spatial and temporal resolution. Thus they can be used to study synaptic function in small parts of the neuron that are not accessible with other techniques.
We will generate a panel of plasmids encoding photoactivatable green fluorescent protein (PAGFP) fused to proteins known to be associated with the synapse. The dynamics of each protein at the synapse will be determined by activating the linked PAGFP using 720 nm laser light and following the movement of the activated fluorophore using 910 nm excitation. The regulation of protein dynamics by action potential firing and synaptic activation will be investigated. Lastly, neurons lacking Tsc1 will be examined to understand if the defects in spine morphology seen in this mouse model of Tuberous Sclerosis Complex perturb the dynamics and trafficking of synaptic proteins.