Persistent activation of GABA(A) receptor/Cl(-) channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons.
Whole cell patch-clamp recordings using Cl(-)-filled pipettes revealed more negative levels of baseline current and associated current variance in embryonic rat hippocampal neurons co-cultured on a monolayer of astrocytes than those cultured on poly-D-lysine. These effects were mimicked by culturing neurons on poly-D-lysine in astrocyte-conditioned medium (ACM). The baseline current and variance decreased immediately in all cells after either local perfusion with saline or exposure to bicuculline, an antagonist of GABA at GABA(A) receptor/Cl(-) channels. Baseline current and variance in all cells reached a nadir at approximately 0 mV, the calculated equilibrium potential for Cl(-). Perfusion of ACM rapidly induced a sustained current in neurons, which also reversed polarity at approximately 0 mV. Bicuculline attenuated or eliminated the ACM-induced current at a concentration that completely blocked micromolar GABA-induced current. Quantitative analyses of spontaneously occurring fluctuations superimposed on the ACM-induced current revealed estimated unitary properties of the underlying channel activity similar to those calculated for GABA's activation of GABA(A) receptor/Cl(-) channels. Bicuculline-sensitive synaptic-like transients, which reversed at approximately 0 mV, were also detected in neurons cultured in ACM, and these were immediately eliminated along with the negative baseline current and superimposed current fluctuations by perfusion. Furthermore bicuculline-sensitive synaptic-like transients were rapidly and reversibly triggered when ACM was acutely applied. ACM induced an increase in cytoplasmic Ca(2+) in cultured embryonic hippocampal neurons that was completely blocked by bicuculline and strychnine. We conclude that astrocytes release diffusible substances, most likely GABA, that persistently activate GABA(A) receptor/Cl(-) channels in co-cultured neurons.
Liu
Q Y
QY
Laboratory of Neurophysiology, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-4066, USA.
Schaffner
A E
AE
Chang
Y H
YH
Maric
D
D
Barker
J L
JL
D015097:3-Mercaptopropionic Acid / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000378:metabolism*; D001640:Bicuculline / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000737:chemistry / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives*; D018756:GABA Antagonists / Q000494:pharmacology; D058787:GABA-A Receptor Antagonists; D018684:Glycine Agents / Q000494:pharmacology; D006624:Hippocampus / Q000166:cytology / Q000196:embryology / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013331:Strychnine / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*
D018118:Chloride Channels; D002712:Chlorides; D017077:Culture Media, Conditioned; D018756:GABA Antagonists; D058787:GABA-A Receptor Antagonists; D018684:Glycine Agents; D011963:Receptors, GABA-A; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D005680:gamma-Aminobutyric Acid; D015097:3-Mercaptopropionic Acid; D013331:Strychnine; D002118:Calcium; D001640:Bicuculline