Properties of GABAA receptors in cultured rat oligodendrocyte progenitor cells
Introduction
Cells of the oligodendroglial lineage were once considered as relatively simple ‘myelination machines’, passively facilitating the axonal propagation of action potentials within the central nervous system (CNS). More recently, however, these cells have been shown to possess a variety of important signalling molecules. These include, in addition to numerous G-protein coupled receptors, neurotransmitter receptors coupled to the gating of intrinsic ion channels and kinase moieties plus a broad range of voltage-gated ion channels (Barres et al., 1990a, Berger et al., 1992a, Berger et al., 1992b, Sontheimer, 1994). This indicates that these cells, like other glia, are well equipped to utilise various inter- and intracellular signalling pathways to tune their behaviour to respond to a range of developmental, functional and pathological demands.
The development of the oligodendroglial lineage is characterised by a number of intermediate stages, each possessing a unique repertoire of ion channels and receptors (Sontheimer et al., 1989, von Blankenfeld et al., 1991, von Blankenfeld et al., 1992). In vivo, the first cell type committed to the oligodendroglia lineage is the oligodendrocyte progenitor. In in vitro systems, the equivalent cell is known as the oligodendrocyte-type 2 astrocyte (O-2A) progenitor, so named because of its ability to differentiate into either an oligodendrocyte or a type 2 astrocyte (Raff et al., 1983, Levison and Goldman, 1993).
O-2A progenitor cells are small electrically excitable bipolar cells which possess voltage-activated Na+, Ca2+, K+ and Cl− channels (Bevan et al., 1987, Sontheimer et al., 1989, Verkhratsky et al., 1990, Barres et al., 1990b), as well as ionotropic neurotransmitter receptors (Barres et al., 1990a, von Blankenfeld et al., 1991, Kirchoff and Kettenmann, 1992, Borges et al., 1994, Holtzclaw et al., 1994). Beyond the maintenance of the membrane potential and production of action potentials, the functional importance of the numerous ion channels in these cells has remained largely mysterious.
Previous studies have indicated that O-2A progenitors possess functional GABAA receptors. As in neurones these are coupled to an inherent Cl− selective pore. Thus the net current flux observed following receptor activation reverses polarity close to the Cl− equilibrium potential. In mature neurones this usually produces inhibitory (i.e. hyperpolarising) responses. In glia and certain immature neurones, in contrast, GABAA receptor activation produces depolarisation (von Blankenfeld et al., 1991). This occurs in oligodendroglia because a high intracellular concentration of Cl− is maintained by an inwardly directed Cl− pump (Hoppe and Kettenmann, 1989). Via the subsequent activation of voltage-gated Ca2+ channels, such GABA-induced depolarisations can produce substantial increases in intracellular [Ca2+] in oligodendrocyte progenitors (Kirchoff and Kettenmann, 1992).
In addition to the simple electrochemical actions of membrane potential on the amplitude and direction of current fluxes, ligand-gated channels often exhibit voltage-dependent kinetics. These are thought to arise from the effects of the membrane field on charge movements associated with state-to-state transitions of the receptor (Hille, 1992). In neuronal GABAA receptors such voltage-dependent kinetics produce inhibitory synaptic currents with voltage-dependent lifetimes (e.g. Mellor and Randall, 1998).
As well as sensitivity to the cells membrane potential GABAA receptor gating can be altered by wide variety of allosteric modulators. These include neurosteroids, protons, various di and trivalent ions, barbiturates and benzodiazepines (BDZs) (Sieghart, 1995). Studies of recombinant GABAA receptors have indicated that the ability of these agents to affect receptor function critically depends on the subunit composition of the receptor. Benzodiazepine receptor agonists produce two seemingly separable responses at GABAA receptors. Firstly, they increase the amplitude of responses to submaximally effective concentrations of GABA (Sieghart, 1995). Secondly, they slow the deactivation of responses to both maximally and sub-maximally effective concentrations of GABA (Mellor and Randall, 1997a).
In the study presented here we have evaluated the properties of GABA responses in O-2A progenitors using rapid agonist application techniques. The use of these methods allows previously undescribed insights into the kinetic properties of GABA responses in these cells. Our experiments reveal that O-2A progenitors exhibit GABA responses that are somewhat different from those characterised in neurones using similar methods.
Section snippets
Tissue culture
Cultures enriched in O-2A progenitor cells were prepared from the brains of 1–2 day old Sprague–Dawley rats as described elsewhere (Webb et al., 1995). In short, following humane killing by rapid decapitation, the brain was removed, minced and incubated for 20 min with 0.1% trypsin at 37°C in Ca2+/Mg2+ free phosphate-buffered saline solution. The tissue was subsequently triturated in the presence of 0.05% trypsin inhibitor (Sigma) and 0.001% DNAse in Hanks Buffered Salts until a fine cell
Concentration-dependent GABA currents in O-A progenitors
Forty nine of 57 cells with O-2A progenitor morphology exhibited detectable current responses (IGABA) in response to application of 1 or 10 mM GABA. At a holding potential of −70 mV, the mean amplitude of the response to 1 mM GABA was 257±54 pA (n=27). Contrary to a previous report (von Blankenfeld et al., 1991) at GABA concentrations ≤10 μM either very small or no responses at all were observed. To further characterise the actions of GABA on O-2A progenitors we collected dose-response data
Discussion
The experiments described above indicate that GABAA receptor-mediated responses in O-2A progenitors are somewhat atypical in comparison to those seen in most neurones. Notable features of the GABA responses in O-2A progenitors include a low apparent affinity for GABA (Fig. 2B), a slow current activation rate (even at high agonist doses, Fig. 2C), slight inward rectification (Fig. 6), monoexponential deactivation (Fig. 2D and 7B) and insensitivity to β-carbolines and benzodiazepines (Fig. 5).
One
Acknowledgements
We would like to thank Dr Bill Wisden for his comments on the manuscript. JRM and ALG were MRC funded PhD students. We also are indebted to Dr Bill Sather for allowing AVW to return to the UK to complete her part of the work shown herein.
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Present address: Department of Pharmacology, Campus Box B-138, 420 East Ninth Avenue, Denver, Colorado 80262, USA.