Dramatic new insights into the functioning of neural networks have been made possible by our ability to visualize neural function with calcium-sensitive fluorophores, and biology has been revolutionized by the ability to sequence and manipulate DNA, RNA, and proteins. Both of these tremendous advances have unexplored flip sides. Our understanding of neural network function remains limited by our inability see GABA-mediated synaptic activity: we can't measure the output of the remarkable diversity of interneuron structure and function. Similarly, the methods for studying templated biopolymers such as DNA, RNA, and protein are not applicable to untemplated biopolymers such as polyglutamylated intracellular tubulin, and the variably sulfated glycosaminoglycans (GAGs) that comprise the extracellular matrix. The glutamate and sulfate moieties displace chloride, thereby defining chloride microdomains. These microdomains thus provide a photographic negative of the information that may be stored in local untemplated biopolymers. Several compelling lines of evidence suggest that chloride microdomains alter the direction and magnitude of currents flowing through open GABAA receptors, providing a mechanism to read out information stored in the anion distribution of the biopolymers. This implies a unique direction and size of chloride current at each GABAA receptor, so that these critical determinants must be measured to understand the contribution of interneurons to network operation. To visualize chloride microdomains, we have assembled a uniquely qualified team to create and fully characterize a series of reporters of chloride concentration placed at the most critical location: the intra and extracellular faces of GABAA receptor subunits. Super Clomeleon, a new ratiometric chloride- sensitive fluorophore, will be fused to g2 and d subunits of the GABAA receptor. g2-linked Super Clomeleon will report synaptic chloride microdomains that drive synaptic GABA signaling, while d-linked Super Clomeleon will report chloride domains subserving extrasynaptic GABA signaling. Both intra and extracellular chloride microdomains will be analyzed by the Super Clomeleon - GABAA subunit fusion constructs, so that the direction and amplitude of chloride currents can be precisely mapped. The GABAA subunit-fluorophore fusion products will be parallel-processed in four labs so that in the three- year time window of this RFA we can assess receptor assembly, membrane trafficking, receptor conductance, chloride sensitivities, gains, pH dependence, and stability in vitro and in vivo. We can then make timely go / no go decisions regarding the utility of going forward to create transgenic mice.
We will fuse fluorescent protein-based chloride sensors to the intra- and extra- cellular faces of GABAA receptor subunits. This will provide a high-resolution view of the output activity of interneurons, and allow us to test the idea that functional chloride microdomains link the brain's extracellular matrix to memory.
