Production of GABA by cultured hippocampal glial cells
Introduction
GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the brain (Krnjevic, 1974). Its importance in the regulation of normal brain function has been clearly demonstrated by drugs that modulate GABA receptors. Inhibition of GABA receptors by bicuculline and picrotoxin induces seizure activity (Dichter and Ayala, 1987). Conversely, enhancement of GABA receptor activity by benzodiazepenes and barbituates, or inhibition of GABA uptake and degradation by tiagabine and vigabatrin, respectively, produces anxiolytic and anticonvulsant activity (Lloyd et al., 1992, Rogawski, 1995, Kocsis and Mattson, 1996).
In brain, GABA is primarily produced by inhibitory neurons and released during the firing of action potentials (Hall et al., 1970, Fitzpatrick et al., 1984, Kunkel et al., 1986). This generates the phasic inhibition seen in many neurons. However, there is also tonic inhibition that results from continuous activation of GABA receptors by low concentrations of extracellular GABA (Brickley et al., 1996, Semyanov et al., 2003). The inhibitory current produced by tonic inhibition is much greater than that produced by phasic inhibition and is largely responsible for suppressing neuronal excitability in the brain. Although, some tonic inhibition is caused by spillover of GABA from synaptic terminals to extrasynaptic receptors, there appears to be another source of GABA involved in tonic inhibition. Liu et al. (2000) have reported that cultured astrocytes produce a factor that activates GABA currents in rat hippocampal neurons. We have also observed that conditioned medium from glial cultures contain a factor that hyperpolarizes neurons and inhibits their activity. In this paper, we present biochemical, electrophysiological, pharmacological and physical evidence demonstrating that the conditioned medium factor is GABA. The amount of GABA produced by glia, particularly those from hippocampus, is substantial and may contribute to regulation of neuronal activity and tonic inhibition in the brain.
Section snippets
Cultures
Hippocampal neuronal cultures were prepared using methods modified from Furshpan and Potter (1989). Hippocampi were dissected from neonatal rat pups (Charles River) and incubated at 37 °C in a papain solution (Worthington) for 60 min. This was followed by incubation in an enzyme inhibitor solution for 30 min. The hippocampi were dissociated into a suspension of single cells using a 2 ml sterile pipette (Falcon). Cells were plated on glass coverslips treated with polylysine and laminin in 35 mm
Identification of GABA as the inhibitory factor in conditioned medium
Hippocampal neurons grown for several weeks in culture exhibit spontaneous activity when perfused with saline. Under voltage clamp, this activity can be seen as large inward currents that result from the activation of postsynaptic glutamate receptors. If left uninterrupted for prolonged periods, the activity can eventually depolarize and kill neurons through excitotoxicity (Furshpan and Potter, 1989). We found that medium conditioned by glia can inhibit neuronal activity and prevent
Discussion
Results from electrophysiological, pharmacological, biochemical and mass spectrometry experiments demonstrate that cultured hippocampal glial cells can produce and release significant amounts of GABA, up to hundreds of micromolar in 1 week. Large concentrations of GABA are found only in conditioned medium from glial cultures without neurons. In the presence of neurons, extracellular GABA is removed via neuronal GABA transporters. Prior to being taken up by neurons, GABA released from glia can
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