Abstracts

Rationale: GABAA receptors (GABAARs) open to allow the flow of chloride ions that underpins synaptic inhibition, but this flow is not always in the direction expected.  In neonates there is a significantly larger [Cl-]i that increases the probability of outward flow of chloride upon GABAAR activation (i.e. a shift in GABAAR reversal potential, EGABA).  While developmental changes in the expression of cation-chloride cotransporters (CCCs) offer one possible explanation for this difference, there is significant evidence that CCCs are not sufficient to explain differences in EGABA, including the wide range of reported inter- and intra-cellular EGABAs, the modest effects of pharmacological inhibition of CCCs on EGABA in healthy neurons, and the persistence of subcellular differences in EGABA after blockade of CCCs.We propose that intracellular and extracellular macromolecular anions ([A-]i and [A-]o, respectively) form a balanced Donnan equilibrium system of which chloride is a membrane-permeant component, and cytoskeletal elements are the major components of [A-]i.  In order to measure [Cl-]i we are developing tools based on Super Chlomeleon (SClm), a ratiometric CFP-YFP based FRET reporter of [Cl-].  SClm linked to cytoskeletal proteins including filamentous actin and microtubules are clarifying the cytoskeleton’s contribution to [A-]i and [Cl-]i microdomains.  Methods: Utilizing SCM fixed to cytoskeletal components of [A-]I, we are characterizing their effects on local EGABA both in dissociated cell culture as well as in organotypic hippocampal slice cultures using confocal and multiphoton microscopy. Dendritic [Cl-]i microdomains are visible at highest subcellular resolution in dissociated culture, while slice cultures enable realistic stratification by cell type.   Results: Early results support the concept of dendritic [Cl-]i microdomains.  Pharmacological manipulation of cytoskeletal regulatory mechanisms brings about changes in the distribution of [Cl-]i , as well as the [A-]o that balance the Donnan effect of [A-]i: the sulfated glycosaminoglycans of the extracellular matrix.  Efforts are now directed to measurement of cytoskeletal components relative to [Cl-]i , and to the extent that the extracellular matrix components of [A-]o mirror the distribution of cytoskeletal components of [A-]i to balance local Donnan effects. Conclusions: [Cl-]i is of particular importance for neuronal signaling via GABAergic interneurons as well as volume control.  Our findings to date support the existence of [Cl-]i microdomains and a role for intra- and extracellular macromolecular anions in their definition.  These findings will provide insight not only into the subcellular distribution of EGABA , but also how EGABA is altered in pathological conditions such as status epilepticus and hypoxic-ischemic injury, during which cytoskeletal and extracellular matrix components are damaged, neuronal volume regulation is deranged and seizures are engendered.   Funding: CURE Innovator Award, 2016.