This grant proposes to generate protein-based, genetically-encodable sensors for the primary excitatory neurotransmitter glutamate and to express these fusion proteins in situ to study spatiotemporal aspects of excitatory synaptic signaling. A series of constructs based on fusion of a neuronal glutamate transporter (EAAT3 or EAAT4) and ion-sensitive variants of GFP will be generated. Sensor constructs will be evaluated in three stages. First, each will be characterized in HEK cells to establish whether glutamate elicits optical signals, and if so to determine the pH and chloride sensitivity and the kinetic properties of those signals (Aim 1). Next, viable constructs will be inserted into viral vectors suitable for expression in rat brain. The ability of constructs to generate optical signals in response to excitatory synaptic activity will be tested (Aim 2). Combining optical and electrophysiological measurements, constructs will be used to map the spatial pattern of active synaptic inputs to neurons under various conditions (Aim 3). Together these experiments will develop and establish the practical usefulness of a class of genetically-encodable indicators of excitatory synaptic transmission. Once developed, such indicators are expected to have an important impact on the study of information processing within neural circuits, allowing minimally invasive measurement of subthreshold synaptic signals. ? ? ?

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21HD049238-01
Application #
6858131
Study Section
Special Emphasis Panel (ZRG1-MDCN-B (55))
Program Officer
Vitkovic, Ljubisa
Project Start
2004-12-15
Project End
2006-11-30
Budget Start
2004-12-15
Budget End
2005-11-30
Support Year
1
Fiscal Year
2005
Total Cost
$230,876
Indirect Cost
Name
University of California Los Angeles
Department
Neurosciences
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Lutz, Christoph; Otis, Thomas S; DeSars, Vincent et al. (2008) Holographic photolysis of caged neurotransmitters. Nat Methods 5:821-7