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The ability to discriminate between diverse types of sensation is mediated by heterogeneous populations of peripheral sensory neurons. Human peripheral sensory neurons are inaccessible for research and efforts to study their development and disease have been hampered by the availability of relevant model systems. The in vitro differentiation of peripheral sensory neurons from human embryonic stem cells therefore provides an attractive alternative since an unlimited source of biological material can be generated for studies that specifically address development and injury. The work presented in this study describes the derivation of peripheral sensory neurons from human embryonic stem cells using small molecule inhibitors. The differentiated neurons express canonical- and modality-specific peripheral sensory neuron markers with subsets exhibiting functional properties of human nociceptive neurons that include tetrodotoxin-resistant sodium currents and repetitive action potentials. Moreover, the derived cells associate with human donor Schwann cells and can be used as a model system to investigate the molecular mechanisms underlying neuronal death following peripheral nerve injury. The quick and efficient derivation of genetically diverse peripheral sensory neurons from human embryonic stem cells offers unlimited access to these specialised cell types and provides an invaluable in vitro model system for future studies.

Maintenance of a low intraneuronal Cl- concentration, [Cl-](i), is critical for inhibition in the CNS. Here, the contribution of passive, conductive Cl- flux to recovery of [Cl-](i) after a high load was analyzed in mature central neurons from rat. A novel method for quantifying the resting Cl- conductance, important for [Cl-](i) recovery, was developed and the possible contribution of GABAA and glycine receptors and of ClC-2 channels to this conductance was analyzed. The hypothesis that spontaneous, action potential-independent release of GABA is important for [Cl-](i) recovery was tested. [Cl-](i) was examined by gramicidin-perforated patch recordings in medial preoptic neurons. Cells were loaded with Cl- by combining GABA or glycine application with a depolarized voltage, and the time course of [Cl-](i) was followed by measurements of the Cl- equilibrium potential, as obtained from the current recorded during voltage ramps combined with GABA or glycine application. The results show that passive Cl- flux contributes significantly, in the same order of magnitude as does K+-Cl- cotransporter 2 (KCC2), to [Cl-](i) recovery and that Cl- conductance accounts for similar to 6% of the total resting conductance. A major fraction of this resting Cl- conductance is picrotoxin (PTX)-sensitive and likely due to open GABAA receptors, but ClC-2 channels do not contribute. The results also show that when the decay of GABAA receptor-mediated miniature postsynaptic currents (minis) is slowed by the neurosteroid allopregnanolone, such minis may significantly quicken [Cl-](i) recovery, suggesting a possible steroid-regulated role for minis in the control of Clhomeostasis.

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. 

The relation between current and voltage, I-V relation, is central to functional analysis of membrane ion channels. A commonly used method, since the introduction of the voltage-clamp technique, to establish the I-V relation depends on the interpolation of current amplitudes recorded at different steady voltages. By a theoretical computational approach as well as by experimental recordings from GABA(A) receptor mediated currents in mammalian central neurons, we here show that this interpolation method may give reversal potentials and conductances that do not reflect the properties of the channels studied under conditions when ion flux may give rise to concentration changes. Therefore, changes in ion concentrations may remain undetected and conclusions on changes in conductance, such as during desensitization, may be mistaken. In contrast, an alternative experimental approach, using rapid voltage ramps, enable I-V relations that much better reflect the properties of the studied ion channels.