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Contribution of Resting Conductance, GABA(A)-Receptor Mediated Miniature Synaptic Currents and Neurosteroid to Chloride Homeostasis in Central Neurons
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
Umeå University, Faculty of Medicine, Department of Integrative Medical Biology (IMB).
2017 (English)In: eNeuro, ISSN 2373-2822, Vol. 4, no 2, e0019Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2017. Vol. 4, no 2, e0019
Keyword [en]
chloride homeostasis, GABA(A) receptor, KCC2, miniature postsynaptic current, neurosteroid, resting loride conductance
National Category
Physiology
Identifiers
URN: urn:nbn:se:umu:diva-133788DOI: 10.1523/ENEURO.0019-17.2017ISI: 000397567900003OAI: oai:DiVA.org:umu-133788DiVA: diva2:1090289
Note

Originally published in manuscript form

Available from: 2017-04-24 Created: 2017-04-24 Last updated: 2017-04-24Bibliographically approved
In thesis
1. Chloride Homeostasis in Central Neurons
Open this publication in new window or tab >>Chloride Homeostasis in Central Neurons
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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 (GABAARs), which are ion channels largely permeable to Cl. The effect of GABAAR-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, gCl. With a combination of computational and electrophysiological techniques, we showed that the most common (interpolation) method failed to detect changes in [Cl]i and gCl 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 GABAAR-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, gCl, and examined the role of gCl for the neuronal Cl homeostasis. In isolated preoptic neurons from rat, resting gCl was about 6 % of total resting conductance, to a major part due to spontaneously open GABAARs 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 gCl, (ii) provides a novel method for quantifying gCl in cell membranes and (iii) clarifies the roles of the ECM, ion channels and ion transporters in the control of [Cl]i homeostasis and GABAAR-mediated signaling in central neurons. 

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 43 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1861
Keyword
chloride concentration, current-voltage relation, interpolation, voltage ramp, reversal potential, [Cl–]i recovery, KCC2, extracellular matrix, GABAA receptor, chloride leak conductance, neurosteroid
National Category
Physiology Neurosciences
Research subject
Physiology
Identifiers
urn:nbn:se:umu:diva-127655 (URN)978-91-7601-602-2 (ISBN)
Public defence
2016-12-15, KB3A9, KBC-huset, Linnéus väg 9, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Research Council, 22292
Available from: 2016-11-24 Created: 2016-11-16 Last updated: 2017-04-24Bibliographically approved

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