Efficacy of Synaptic Inhibition Depends on Multiple, Dynamically Interacting Mechanisms Implicated in Chloride Homeostasis

Abstract

Chloride homeostasis is a critical determinant of the strength and robustness of inhibition mediated by GABA _A receptors (GABA _ARs). The impact of changes in steady state Cl ⁻ gradient is relatively straightforward to understand, but how dynamic interplay between Cl ⁻ influx, diffusion, extrusion and interaction with other ion species affects synaptic signaling remains uncertain. Here we used electrodiffusion modeling to investigate the nonlinear interactions between these processes. Results demonstrate that diffusion is crucial for redistributing intracellular Cl ⁻ load on a fast time scale, whereas Cl ⁻extrusion controls steady state levels. Interaction between diffusion and extrusion can result in a somato-dendritic Cl ⁻ gradient even when KCC2 is distributed uniformly across the cell. Reducing KCC2 activity led to decreased efficacy of GABA _AR-mediated inhibition, but increasing GABA _AR input failed to fully compensate for this form of disinhibition because of activity-dependent accumulation of Cl ⁻. Furthermore, if spiking persisted despite the presence of GABA _AR input, Cl ⁻ accumulation became accelerated because of the large Cl ⁻ driving force that occurs during spikes. The resulting positive feedback loop caused catastrophic failure of inhibition. Simulations also revealed other feedback loops, such as competition between Cl ⁻ and pH regulation. Several model predictions were tested and confirmed by [Cl ⁻] _i imaging experiments. Our study has thus uncovered how Cl ⁻ regulation depends on a multiplicity of dynamically interacting mechanisms. Furthermore, the model revealed that enhancing KCC2 activity beyond normal levels did not negatively impact firing frequency or cause overt extracellular K ⁻ accumulation, demonstrating that enhancing KCC2 activity is a valid strategy for therapeutic intervention.

Author Summary

Fast synaptic inhibition relies on chloride current to hyperpolarize the neuron or to prevent depolarization caused by concurrent excitatory input. Both scenarios necessarily involve chloride flux into the cell and, thus, a change in intracellular chloride concentration. The vast majority of models neglect changes in ion concentration despite experimental evidence that such changes occur and are not inconsequential. The importance of considering chloride homeostasis mechanisms is heightened by evidence that several neurological diseases are associated with deficient chloride extrusion capacity. Steady state chloride levels are altered in those disease states. Fast chloride dynamics are also likely affected, but those changes have yet to be explored. To this end, we built an electrodiffusion model that tracks changes in the concentration of chloride plus multiple other ion species. Simulations in this model revealed a multitude of complex, nonlinear interactions that have important consequences for the efficacy of synaptic inhibition. Several predictions from the model were tested and confirmed with chloride imaging experiments.