NKCC-1 mediated Cl- uptake in immature CA3 pyramidal neurons is sufficient to compensate phasic GABAergic inputs.

Activation of GABAA receptors causes in immature neurons a functionally relevant decrease in the intracellular Cl- concentration ([Cl-]i), a process termed ionic plasticity. Amount and duration of ionic plasticity depends on kinetic properties of [Cl-]i homeostasis. In order to characterize the capacity of Cl- accumulation and to quantify the effect of persistent GABAergic activity on [Cl-]i, we performed gramicidin-perforated patch-clamp recordings from CA3 pyramidal neurons of immature (postnatal day 4-7) rat hippocampal slices. These experiments revealed that inhibition of NKCC1 decreased [Cl-]i toward passive distribution with a time constant of 381 s. In contrast, active Cl- accumulation occurred with a time constant of 155 s, corresponding to a rate of 15.4 µM/s. Inhibition of phasic GABAergic activity had no significant effect on steady state [Cl-]i. Inhibition of tonic GABAergic currents induced a significant [Cl-]i increase by 1.6 mM, while activation of tonic extrasynaptic GABAA receptors with THIP significantly reduced [Cl-]i.. Simulations of neuronal [Cl-]i homeostasis supported the observation, that basal levels of synaptic GABAergic activation do not affect [Cl-]i. In summary, these results indicate that active Cl--uptake in immature hippocampal neurons is sufficient to maintain stable [Cl-]i at basal levels of phasic and to some extent also to compensate tonic GABAergic activity.

Spines slow down dendritic chloride diffusion and affect short-term ionic plasticity of GABAergic inhibition.

Cl(-) plays a crucial role in neuronal function and synaptic inhibition. However, the impact of neuronal morphology on the diffusion and redistribution of intracellular Cl(-) is not well understood. The role of spines in Cl(-) diffusion along dendritic trees has not been addressed so far. Because measuring fast and spatially restricted Cl(-) changes within dendrites is not yet technically possible, we used computational approaches to predict the effects of spines on Cl(-) dynamics in morphologically complex dendrites. In all morphologies tested, including dendrites imaged by super-resolution STED microscopy in live brain tissue, spines slowed down longitudinal Cl(-) diffusion along dendrites. This effect was robust and could be observed in both deterministic as well as stochastic simulations. Cl(-) extrusion altered Cl(-) diffusion to a much lesser extent than the presence of spines. The spine-dependent slowing of Cl(-) diffusion affected the amount and spatial spread of changes in the GABA reversal potential thereby altering homosynaptic as well as heterosynaptic short-term ionic plasticity at GABAergic synapses in dendrites. Altogether, our results suggest a fundamental role of dendritic spines in shaping Cl(-) diffusion, which could be of relevance in the context of pathological conditions where spine densities and neural excitability are perturbed.