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The overall aim of the present thesis is to clarify the control of intracellular chloride homeostasis in central neurons, because of the critical role of chloride ions (Cl^–) for neuronal function. Normal function of the central nervous system (CNS) depends on a delicate balance between neuronal excitation and inhibition. Inhibition is, in the adult brain, most often mediated by the neurotransmitter γ-aminobutyric acid (GABA). GABA may, however, in some cases cause excitation. GABA acts by activating GABA type A receptors (GABA_ARs), which are ion channels largely permeable to Cl^–. The effect of GABA_AR-mediated neuronal signaling - inhibitory or excitatory - is therefore mainly determined by the Cl^– gradient across the membrane. This gradient varies with neuronal activity and may be altered in pathological conditions. Thus, understanding Cl^– regulation is important to comprehend neuronal function. This thesis is an attempt to clarify several unknown aspects of neuronal Cl^– regulation. For such clarification, a sufficiently sensitive method for measuring the intracellular Cl^– concentration, [Cl^–]_i, is necessary. In the first study of this thesis, we examined two electrophysiological methods commonly used to estimate [Cl^–]_i. Both methods, here called the interpolation and the voltage-ramp method, depend on an estimate of the Cl^– equilibrium potential from the current-voltage relation of GABA- or glycine-evoked Cl^– currents. Both methods also provide an estimate of the membrane Cl^– conductance, g_Cl. With a combination of computational and electrophysiological techniques, we showed that the most common (interpolation) method failed to detect changes in [Cl^–]_i and g_Cl during prolonged GABA application, whereas the voltage-ramp method accurately detected such changes. Our analysis also provided an explanation as to why the two methods differ. In a second study, we clarified the role of the extracellular matrix (ECM) for the distribution of Cl^– across the cell membrane of neurons from rat brain. It was recently proposed that immobile charges located within the ECM, rather than as previously thought cation-chloride transporter proteins, determine the low [Cl^–]_i which is critical to GABA_AR-mediated inhibition. By using electrophysiological techniques to measure [Cl^–]_i, we showed that digestion of the ECM decreases the expression and function of the neuron-specific K⁺ Cl^– cotransporter 2 (KCC2), which normally extrudes Cl^- from the neuron, thus causing an increase in resting [Cl^–]_i. As a result of ECM degradation, the action of GABA may be transformed from inhibitory to excitatory. In a third study, we developed a method for quantifying the largely unknown resting Cl^– (leak) conductance, g_Cl, and examined the role of g_Cl for the neuronal Cl^– homeostasis. In isolated preoptic neurons from rat, resting g_Cl was about 6 % of total resting conductance, to a major part due to spontaneously open GABA_ARs and played an important role for recovery after a high Cl^– load. We also showed that spontaneous, impulse-independent GABA release can significantly enhance recovery when the GABA responses are potentiated by the neurosteroid allopregnanolone. In a final commentary, we formulated the mathematical relation between Cl^– conductance, KCC2-mediated Cl^– extrusion capacity and steady-state [Cl^–]_i. In summary, the present thesis (i) clarifies how well common electrophysiological methods describe [Cl^–]_i and g_Cl, (ii) provides a novel method for quantifying g_Cl in cell membranes and (iii) clarifies the roles of the ECM, ion channels and ion transporters in the control of [Cl^–]_i homeostasis and GABA_AR-mediated signaling in central neurons.