Abstracts

Rationale: Cation-chloride cotransporters (CCCs) are critical salt and water transporters that contribute to neuronal volume and chloride concentration. Sodium-potassium-chloride cotransporter (NKCC1) is highly expressed in immature neurons, while potassium-chloride cotransporter (KCC2) is highly expressed in mature neurons. These cotransporters balance the transmembrane water and ion gradients. KCC2 transport often comes to equilibrium with an excess of free water outside the neuron. In contrast, NKCC1 equilibrium is frequently associated with an excess of free water inside the neuron.

Brain injury damages the neuronal cytoplasmic membrane and increases its permeability to small ions and water. We propose that once the membrane becomes nonspecifically leaky, chloride accumulates in the cytoplasm, shifting the GABA_A equilibrium potential toward positive values, reducing inhibition, and enabling seizure activity. However, the direction of water movement across permeabilized membranes depends on the prior expression of KCC2 vs NKCC1. KCC2-expressing neurons gain water and swell while NKCC1-expressing neurons lose water and shrink.

Methods: We used oxygen-glucose deprivation (OGD) in organotypic hippocampal slice cultures as a model of hypoxic-ischemic brain injury. The slices are prepared from mice transgenically expressing a neuronal chloride-sensitive fluorophore (Clomeleon). Slices were submerged in a chamber continuously perfused in oxygenated (95% O₂ and 5% CO₂) media. OGD was induced, for 15 minutes, by perfusion of anoxic, glucose-free media saturated with 95% N₂ and 5% CO₂. Intracellular chloride concentration ([Cl^-]_i) and neuronal volume prior, during, and after OGD were assessed in slices at days in vitro 3-13 (DIV3-13) by multi-photon imaging.

Results: 1) Neuronal [Cl^-]_i increased during OGD and remained slightly elevated two hours after OGD. 2) The majority of immature neurons shrank in response to OGD. In contrast most mature neurons swelled. 3) Post-experiment immunohistochemistry confirmed higher NKCC1 expression in young slices in which the majority of neurons shrank. Older slices, in which the majority of neurons swelled after OGD, had higher KCC2 expression.

Conclusions: The hippocampal organotypic slice combined with multi-photon imaging and immunohistochemistry create a controlled environment to study the role of CCCs in injury-induced changes in neuronal volume and chloride regulation. Our findings indicate that the ratio of NKCC1 vs KCC2 expressing neurons changes during development. This age-dependent change in CCC expression may contribute to the age dependence of the neuronal volume response to injury. Therefore, modulation of membrane permeability after injury might be an effective intervention not only to prevent acute seizures, but also to prevent the secondary injuries associated with brain swelling and shrinkage.

Funding: Please list any funding that was received in support of this abstract.: R35NS116852.