Abstracts

Rationale: The cytoplasmic concentration of chloride ([Cl-]i) is actively managed by neurons. [Cl-]i varies both developmentally and spatially along the dendrites of neurons. Because [Cl-]i is maintained by equilibrative transporters, variance in [Cl-]i raises the possibility of corresponding variance in extracellular chloride concentration [Cl-]o. Small changes in transmembrane chloride gradient may profoundly affect the response to GABA-A receptor (GABAAR) activation and thus affect the principle mechanism of many anti-epileptic drugs (AEDs). While the action of cation-chloride cotransporters (CCCs) NKCC1 and KCC2 and their differential expression during early development is surely important, data from multiple laboratories now demonstrate that CCCs do not set EGABA. Rather, [Cl-]i and [Cl-]o are determined by a Donnan system of which the CCCs comprise the membrane permeability. Cytoplasmic perimembranous proteins and extracellular glycoproteins comprise the fixed charges of the Donnan system. We propose that this Donnan system determines [Cl-]i and [Cl-]o. Such a system would also be influenced by osmotic forces because the CCCs cotransport water with cations and chloride. As the neuron integrates electrical signals and osmotic conditions the Donnan potential is adjusted dynamically. Should there be a deficit in fixed intracellular anions, as in status epilepticus and hypoxic-ischemic injury where cytoskeletal depolymerization is known to occur, one would expect a compensatory increase in [Cl-]i. Elucidating this mechanism would be an important step toward improving AED function or finding new AED targets. Methods: We measure [Cl^-]_i and [Cl^-]_o directly in live cells using the genetically encoded ratiometric reporter Super-Chlomeleon (SClm) and the extracellular fluorescence lifetime imaging (FLIM) reagent SBiQ (or MEQ), respectively. In fixed samples we use fluorescently labeled phalloidin and paclitaxel to visualize actin and microtubules, respectively, and surfen to visualize extracellular glycoproteins. The work presented here focuses on dissociated cell culture isolated from P2-6 mice and imaged DIV 12-24.  Results: Here we present our work studying the relationship between ‘fixed’ anionic and osmotically active moieties inside and outside the cell and their relationships with [Cl^-]_i and [Cl^-]_o. Early results have demonstrated local differences within neurite [Cl^-]_i. Pharmacological manipulation of cytoskeletal regulatory mechanisms or direct influence over cytoskeletal polymer dynamics experimentally alter the distribution of [Cl^-]_i and we continue to work to quantify these changes. Conclusions: [Cl-]i is of particular importance for neuronal hyperpolarization via GABAergic interneuron input and thus for neural circuit function and prevention of seizures. Our findings suggest that the non-uniform distribution of [Cl-]i is locally influenced by fixed anions. This suggests that both neuronal volume and EGABA also vary with fixed anion content, and we are working to test these hypotheses on either side of the cellular membrane. Funding: Not applicable