Elsevier

Neuropharmacology

Volume 37, Issue 7, July 1998, Pages 859-873
Neuropharmacology

Properties of GABAA receptors in cultured rat oligodendrocyte progenitor cells

https://doi.org/10.1016/S0028-3908(98)00016-1Get rights and content

Abstract

We have studied the properties of GABA responses in oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells derived from primary cultures of the neonatal rat brain. In whole cell voltage clamp recordings, rapid application of 1–10 mM GABA elicited current responses in >85% of the cells examined. The dose-response relationship pooled from nine progenitor cells was best fit by a logistic function of EC50=113 μM and Hill coefficient=0.9. In contrast to the rate of current deactivation, the rate of current activation exhibited marked concentration-dependence. Pharmacologically, GABA, muscimol and ZAPA ((Z)-3[(aminiiminomethyl)thio]prop-2-enoic acid sulphate) produced responses with ligand-specific kinetics, whereas glycine and the GABAC receptor agonist CACA were without effect; bicuculline methochloride acted as a competitive antagonist. Neither the amplitude nor the kinetics of currents produced by 100 μM GABA were affected by the benzodiazepine flunitrazepam (1 μM). Similarly the benzodiazepine receptor inverse agonist DMCM (1 μM) was also without effect. GABA-activated currents reversed polarity within 2 mV of the calculated Cl equilibrium potential. With brief agonist pulses deactivation was monoexponential, however, unlike neurones the rate of deactivation was voltage-independent. Desensitisation of responses to 10 mM GABA was bi-exponential and accelerated at depolarised membrane potentials. Increasing the amount of GABAA receptor desensitisation (by increasing the duration of the agonist exposure) consistently produced a slowing of deactivation.

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.

References (53)

  • H. Sontheimer et al.

    Channel expression correlates with differentiation stage during the development of oligodendrocytes from their precursor cells in culture

    Neuron

    (1989)
  • S. Tia et al.

    Distinct deactivation and desensitization kinetics of recombinant GABAA receptors

    Neuropharmacology

    (1996)
  • A.N. Verkhratsky et al.

    Cultured glial precursor cells from mouse cortex express two types of calcium currents

    Neurosci. Lett.

    (1990)
  • G. White

    Heterogeneity of EC50 and nH of GABAA receptors on dorsal root ganglion neurons freshly isolated from adult rats

    Brain Res.

    (1992)
  • L.G. Aguayo et al.

    Modulation of the developing rat sympathetic GABAA receptor by Zn2+, benzodiazepines, barbiturates and ethanol

    J. Pharmacol. Exp. Ther.

    (1993)
  • G. Antonopoulos et al.

    Activation of the GABAa receptor inhibits the proliferative effects of bFGF in cortical progenitor cells

    Eur. J. Neurosci.

    (1997)
  • B.A. Barres et al.

    Ion channels in vertebrate glia

    Annu. Rev. Neurosci.

    (1990)
  • B.A. Barres et al.

    Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons

    Nature

    (1993)
  • T. Berger et al.

    Sodium and calcium currents in the glial cells of the mouse corpus callosum slice

    Eur. J. Neurosci.

    (1992)
  • T. Berger et al.

    GABA- and glutamate-activated currents in glial cells of the mouse corpus callosum slice

    J. Neurosci. Res.

    (1992)
  • S. Bevan et al.

    Voltage gated ionic channels in rat cultured astrocytes, reactive astrocytes and an astrocyte-oligodendrocyte progenitor cell

    J. Physiol. (Paris)

    (1987)
  • S.G. Brickley et al.

    Development of a tonic form of synaptic inhibition in rat cerebellar granule cells resulting from persistent activation of GABAA receptors

    J. Physiol.

    (1996)
  • B. Ebert et al.

    Molecular pharmacology of gamma-aminobutyric acid type A receptor agonists and partial agonists in oocytes injected with different alpha, beta, and gamma receptor subunit combinations

    Mol. Pharmacol.

    (1994)
  • K.J. Gingrich et al.

    Dependence of the GABAA receptor gating kinetics on the α-subunit isoform: implications for structure–function relations and synaptic transmission

    J. Physiol.

    (1995)
  • M. Gyenes et al.

    Phosphorylation factors control neurotransmitter and neuromodulator actions at the gamma-aminobutyric acid type A receptor

    Mol. Pharmacol.

    (1994)
  • B. Hille

    Ionic Channels of Excitable Membranes

    (1992)
  • Cited by (44)

    • Oligodendrocyte Differentiation and Myelination Is Potentiated via GABA<inf>B</inf> Receptor Activation

      2020, Neuroscience
      Citation Excerpt :

      GABABRs are predominantly recycled back to the plasma membrane with only a minor fraction degraded in lysosomes (Benke et al., 2012). Besides neurons, it is also known that OPCs express functional GABAARs in culture (Hoppe and Kettenmann, 1989; Von Blankenfeld et al., 1991; Kirchhoff and Kettenmann, 1992; Borges et al., 1995; Williamson et al., 1998; Cahoy et al., 2008) and in situ in hippocampal slices (Lin and Bergles, 2004), and GABABRs are also expressed in developing OLs (Luyt et al., 2007). However, sensitivity to GABA in mature OLs is greatly reduced (Berger et al., 1992).

    • Neuron-glial interactions and neurotransmitter signaling to cells of the oligodendrocyte lineage

      2020, Patterning and Cell Type Specification in the Developing CNS and PNS: Comprehensive Developmental Neuroscience, Second Edition
    • Electrophysiological properties of NG2<sup>+</sup> cells: Matching physiological studies with gene expression profiles

      2016, Brain Research
      Citation Excerpt :

      RT-PCR showed that mixed glial cultures containing 85% NG2+ cells contained mRNA for α2, α3, α4, α5, γ2, γ3, and low levels of γ1 (β sununits were not assessed) (Williamson et al., 1998). α3 is the most highly expressed subunit in culture, and α6 is absent, which is consistent with physiological data in culture and brain slices showing that NG2+ cells have low-affinity, slowly-activating receptors (Jabs et al., 2005; Lin and Bergles, 2004; Williamson et al., 1998). NG2+ cell GABAergic miniature EPSCs (mEPSCs) also have considerably slower decay kinetics than those of CA1 pyramidal neurons, consistent with expression of α5 and absence of α1 (Lin and Bergles, 2004).

    • Neuron-Glial Interactions: Neurotransmitter Signaling to Cells of the Oligodendrocyte Lineage

      2013, Comprehensive Developmental Neuroscience: Patterning and Cell Type Specification in the Developing CNS and PNS
    • Characterization of GABA<inf>A</inf> receptors expressed in glial cell membranes of adult mouse neocortex using a Xenopus oocyte microtransplantation expression system

      2011, Journal of Neuroscience Methods
      Citation Excerpt :

      Data reported in the literature on GABAA receptors expressed by glial cells are discordant. In rat oligodendrocyte progenitor cells, EC50 and nH of 113 μM and 0.9 were found (Williamson et al., 1998); in cultured astrocytes isolated from postnatal rats (Tateishi et al., 2006), nH of 1.3 and EC50 of 22 μM were reported. On the contrary, Fraser et al. (1995) found that hippocampal astrocytes bind GABA with less affinity than neurons and reported a Hill coefficient of 1.6.

    View all citing articles on Scopus
    1

    Present address: Department of Pharmacology, Campus Box B-138, 420 East Ninth Avenue, Denver, Colorado 80262, USA.

    View full text