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Rinke, Ilka (2010): Chloride regulatory mechanisms and their influence on neuronal excitability. Dissertation, LMU München: Faculty of Biology
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Abstract

The chloride concentration in neurons is in general established by the precise functional expression of the sodium-potassium-chloride cotransporter one (NKCC1) and the potassium-chloride cotransporter two (KCC2). NKCC1 raises the intracellular chloride concentration, while KCC2 extrudes chloride. The intracellular chloride concentration determines the strength and direction of γ-aminobutyric acid (GABA) receptor-mediated transmission. In general, the intracellular chloride concentration in neurons is low and causes GABA-mediated inhibition. However, the intracellular chloride concentration in immature neurons is high leading to GABAergic depolarization, which can cause excitation. The effects of excitatory GABA signaling in early development is still unclear. It has been speculated that excitatory GABA, causing general depolarization in neurons, has profound effects on neuronal activity and neuronal maturation. Therefore, I studied in collaboration with Carsten Pfeffer the development of the hippocampal network during the early phase of postnatal development under conditions when excitatory GABA action is abolished. Here, sodium-potassium-chloride cotransporter one (NKCC1) knockout mice (Nkcc1-/-) were used to reduce the intracellular chloride concentration in immature neurons. Young CA1 pyramidal neurons of Nkcc1-/- mice showed diminished GABAergic depolarization. I found that this reduction was sufficient to cause a delay in the maturation of glutamatergic and GABAergic synapses. This suggests that GABAergic excitation during early postnatal development, increasing the network activity, facilitates the maturation of synaptic networks. GABAergic depolarization in Nkcc1-/- mice was reduced but not completely abolished; suggesting that additional chloride loading mechanisms might exist. As the anionexchanger three (AE3) was proposed to contribute to chloride accumulation, AE3 knockout (Ae3-/-) mice were also studied. I could not detect any changes in intracellular chloride concentration after loss of AE3 at postnatal day one (P1). However at P5, the disruption of AE3 affected the early network activity pattern, indicating an effect of reduced intracellular chloride concentration. These data showed that NKCC1 establishes high intracellular chloride concentration in neurons providing the basis for GABAergic excitation. The role of AE3 is still not clear; it might contribute to the chloride accumulation in neurons. In addition to the function of chloride transporters, chloride conductive channels are likely to modulate the intracellular chloride concentration, and therefore could influence neuronal excitability. Especially ClC-2 has been suggested to contribute to chloride extrusion. I investigated the functional role of the voltage-gated chloride channel ClC-2. As specific blockers for ClC-2 are not available, I used ClC-2 knockout (Clcn2-/-) mice. It has been proposed that ClC-2 constitutes a pathway for chloride extrusion to maintain the inhibitory action of GABA in mature neurons. My data provide direct evidence that ClC-2 mediates fast chloride extrusion preventing chloride accumulation. Chloride extrusion by ClC-2 seemed to be important especially in adult hippocampal pyramidal neurons where GABAA receptor activation occurs in high frequency bursts. Interestingly, the chloride-conductance of ClC-2 occurs first in the second postnatal week of developing mice, suggesting that ClC-2 is important in fully developed neurons, but might not be important in immature neurons. Surprisingly, neurons in Clcn2-/- mice have a very high membrane resistance compared to WT animals, indicating that ClC-2 is active during the resting membrane potential. This might be a general feature of neurons, as I recorded the chloride conductance of ClC-2 in various neuron types. I showed that the resting conductance of ClC-2 affects resting membrane properties, which determine the neuronal excitability. As a consequence, the loss of ClC-2 increases the excitability of a neuron; however, it does not cause hyperexcitation of the hippocampal network. Even more, the network excitability is reduced in ClC-2 KO mice in comparison to the WT. This reduction is caused by an increased inhibition. I found that ClC-2 expressing interneurons increased their inhibitory action onto pyramidal cells after loss of ClC-2. Taken together, my data reveal that ClC-2 plays a dual role in adult neurons. First, ClC-2 contributes a fast mechanism to extrude chloride after chloride accumulation. Second, ClC-2 provides the chloride leak conductance under resting conditions. The loss of ClC-2 leads to a higher excitability of the neuron due to a strongly increased membrane resistance. Importantly, hyper-excitability of the neuronal network is circumvented by a parallel enhanced inhibition, which can explain the absence of an epileptic phenotype in mice.