Persistent activation of GABA(A) receptor/Cl(-) channels by astrocyte-derived GABA in cultured embryonic rat hippocampal neurons

• Published in: Journal of neurophysiology Vol. 84; no. 3; pp. 1392 - 1403
• Main Authors: Liu, Q Y, Schaffner, A E, Chang, Y H, Maric, D, Barker, J L
• Format: Journal Article
• Language: English
• Published: United States 01.09.2000
• Subjects: 3-Mercaptopropionic Acid - pharmacology; Animals; Animals, Newborn; Astrocytes - metabolism; Bicuculline - pharmacology; Calcium - metabolism; Cell Differentiation - drug effects; Cells, Cultured; Chloride Channels - antagonists & inhibitors; Chloride Channels - metabolism; Chlorides - metabolism; Coculture Techniques; Culture Media, Conditioned - chemistry; Culture Media, Conditioned - pharmacology; Egtazic Acid - analogs & derivatives; GABA Antagonists - pharmacology; GABA-A Receptor Antagonists; gamma-Aminobutyric Acid - metabolism; Glycine Agents - pharmacology; Hippocampus - cytology; Hippocampus - embryology; Hippocampus - metabolism; Membrane Potentials - physiology; Neurons - cytology; Neurons - metabolism; Patch-Clamp Techniques; Rats; Receptors, GABA-A - metabolism; Strychnine - pharmacology
• ISSN: 0022-3077; 1522-1598
• DOI: 10.1152/jn.2000.84.3.1392

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.