title: Depolarizing chloride gradient in developing cochlear nucleus neurons: underlying mechanism and implication for calcium signaling.
abstract: Precise regulation of the chloride homeostasis crucially determines the action of inhibitory transmitters GABA and glycine and thereby endows neurons or even discrete neuronal compartments with distinct physiological responses to the same transmitters. In mammals, the signaling mediated by GABAA/glycine receptors shifts during early postnatal life from depolarization to hyperpolarization, due to delayed maturation of the chloride homeostasis system. While the activity of the secondary active, K(+)-Cl(-)-extruding cotransporter KCC2, renders GABA/glycine hyperpolarizing in auditory brainstem nuclei of altricial rodents, the mechanisms contributing to the initially depolarizing transmembrane gradient for Cl(-) in respective neurons remained unknown. Here we used gramicidin-perforated patch recordings, non-invasive Cl(-) and Ca(2+) imaging, and immunohistochemistry to identify the Cl(-)-loading transporter that renders depolarizing effects of GABA/glycine in early postnatal life of spherical bushy cells in the cochlear nucleus of gerbil. Our data identify the 1Na(+):1K(+):2Cl(-) cotransporter 1 (NKCC1) as the major Cl(-)-loader responsible for depolarizing action of GABA/glycine at postnatal days 3-5 (P3-5). Extracellular GABA/muscimol elicited calcium signaling through R-, L-, and T-type channels, which was dependent on bumetanide- and [Na(+)]e-sensitive Cl(-) accumulation. The "adult like", low intracellular Cl(-) concentration is established during the second postnatal week, through a mechanism engaging the NKCC1-down regulation between P5 and P15 and ongoing KCC2-mediated Cl(-)-extrusion.
authors.affiliation: Institute of Biology, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany; Institute of Neuroanatomy, University Medical Center Göttingen, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Witte
authors.affiliation: Institute of Biology, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Reinert
authors.affiliation: Institute of Biology, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Dietz
authors.affiliation: Institute of Biology, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Nerlich
authors.affiliation: Institute of Biology, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany.
authors.forename: R
authors.initials: R
authors.lastname: Rübsamen
authors.affiliation: Institute of Biology, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Germany. Electronic address: mili@uni-leipzig.de.
authors.forename: I
authors.initials: I
authors.lastname: Milenkovic
mesh_terms: D000818:Animals; D002034:Bumetanide / Q000494:pharmacology; D015220:Calcium Channels / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002713:Chlorine / Q000378:metabolism; D017626:Cochlear Nucleus / Q000187:drug effects / Q000254:growth & development* / Q000502:physiology*; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005260:Female; D058785:GABA-A Receptor Agonists / Q000494:pharmacology; D005849:Gerbillinae; D066298:In Vitro Techniques; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008959:Models, Neurological; D009118:Muscimol / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D012964:Sodium / Q000378:metabolism; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D064506:Solute Carrier Family 12, Member 2 / Q000378:metabolism; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D058785:GABA-A Receptor Agonists; D049994:Sodium Potassium Chloride Symporter Inhibitors; D064506:Solute Carrier Family 12, Member 2; D002034:Bumetanide; D009118:Muscimol; D002713:Chlorine; D005680:gamma-Aminobutyric Acid; D012964:Sodium
keywords: KCC2; NKCC1; bushy cells; chloride homeostasis; imaging; perforated patch