Glycine triggers an intracellular calcium influx in oligodendrocyte progenitor cells which is mediated by the activation of both the ionotropic glycine receptor and Na⁺-dependent transporters

Introduction

During recent years, evidence has emerged that oligodendroglial cells express a complex array of functional receptors to a variety of neuroligands previously known to affect mostly neurons. These include not only neurotransmitter but also neuropeptide and neurohormone receptors (Belachew et al. 1998a; Verkhratsky et al. 1998) which, via several pathways activate molecular cascades regulating intracellular calcium ([Ca²⁺]_i). Actually, [Ca²⁺]_i increase is already known to be induced in oligodendroglial cells by stimulation of AMPA/kainate-3 type glutamate (Holtzclaw et al. 1995), ATP (P2y and/or P2u; Kirischuk et al. 1995a,b), adrenergic (Cohen & Almazan 1993) and muscarinic (Cohen & Almazan 1994) receptors. Interestingly, the expression by oligodendrocytes of carbachol and ATP receptors is controlled by oligodendrocyte–neuronal contacts (He et al. 1996). Taken together, these data suggest that oligodendroglial calcium signalling could be a target on which converge various signals involved in the neuronal regulation of the oligodendroglial growth and fate.

Regarding these signals, it is now established that glycine receptors (GlyR) are expressed in oligodendroglial lineage cells (Kirchhoff et al. 1996; Belachew et al. 1998a,b). The peak of glycine-induced chloride current is observed at the progenitor stage (OP), thus suggesting a maximum expression of the GlyR during a transient developmental period which takes place just before the O4-positive stage (Belachew et al. 1998b). The pharmacological properties and presumably the molecular structure of the oligodendroglial GlyR are specific (Belachew et al. 1998b) but the functional consequences of GlyR activation in OP cells still remain to be elucidated.

Spinal cord oligodendrocytes are known to possess a furosemide-sensitive Na⁺/K⁺/2Cl^- uptake system that maintains an unusually high intracellular chloride concentration (Hoppe & Kettenmann 1989). As a consequence of this chloride uptake, the calculated Nernst chloride equilibrium potential (E_Cl = −35 mV) is more positive in oligodendrocytes than neurons and therefore, as already demonstrated for GABA_A (Gilbert et al. 1984), GlyR activation-induced increase of chloride conductance should result in a depolarization of oligodendrocytes. Moreover, in cultures derived from cortex, oligodendrocytes express both low-voltage- (T-type) and high-voltage (presumably L-type)-gated calcium channels (Von Blankenfeld et al. 1992). In cultured murine precursor cells of oligodendrocyte lineage, GABA-induced depolarization of the cells exceeds the opening threshold for voltage-gated calcium channels (VGCC) and thereby produces Ca²⁺ influx and a measurable [Ca²⁺]_i increase which exclusively result from a Ca²⁺ entry via VGCC as removal of extracellular calcium ([Ca²⁺]_o) inhibits this [Ca²⁺]_i rise (Kirchhoff & Kettenmann 1992). Here, we demonstrate that the activation of GlyR, the other major ligand-gated anion channel, also induces calcium influx in cultured OP cells derived from newborn rat cerebral cortex as is the case for developing cortical neurons (Flint et al. 1998). Furthermore, we also provide evidence that this depolarization-induced calcium increase could be in part the consequence of the activation of a Na⁺-dependent electrogenic uptake, most probably the 2Na⁺/Cl^-/glycine uptake system (Aragon et al. 1987) through the glycine transporters 1 (GLYT1) and/or 2 (GLYT2) which are both expressed by cultured OP cells. To date, two different glycine transporter genes have been cloned: (i) three isoforms of GLYT1 (a, b and c) with different amino terminals can be produced by alternative splicing and/or alternative promoter usage and are thought to be exclusively present in astroglial processes surrounding N-methyl-d-aspartate and glycinergic synapses (Guastella et al. 1992; Liu et al. 1992; Smith et al. 1992; Borowsky et al. 1993; Kim et al. 1994; Adams et al. 1995; Zafra et al. 1995a); and (ii), GLYT2, expressed mainly in presynaptic nerve terminals but also in glial elements selectively in the brainstem and spinal cord where it could specifically modulate glycinergic neurotransmission (Liu et al. 1993; Zafra et al. 1995b; Morrow et al. 1998).

The demonstration of glycine transporter expression in OP cells allows us to hypothesize that GLYT1 and GLYT2 also regulate, together with GlyR, glycine-mediated neurono–oligodendroglial interactions.

Materials and methods

Results

Glycine induces an increase of intracellular calcium concentration in OP cells through the opening of voltage-gated calcium channels

The cultured cells were studied in the outgrowth zone of expanded oligospheres 3 days after switching to DMEM-N₁. We have previously shown that, in this growth factor-free medium, the cultures contain mainly A2B5-positive cells with many of them already coexpressing a galactocerebroside immunophenotype (Belachew et al. 1998a). We also demonstrated that there was virtually no neuronal or glial fibrillary acidic protein-positive cells (< 1%) in such cultures (Belachew et al. 1998a).

To investigate whether GlyR activation could affect intracellular oligodendroglial calcium concentration, we used 3-day-old cultures because the peak density of GlyR expression occurs at this intermediate stage of differentiation (Belachew et al. 1998b).

OP cells were imaged using confocal microscopy and the calcium indicator dye fluo-3 in Locke standard extracellular solution. All the recordings were made in the presence of bicuculline (100 μm) to avoid any nonspecific increase of intracellular calcium that would result from a glycine-induced cross-activation of GABA_A receptors. In such conditions, the application of glycine (0.5 mm) results in a prolonged rise of [Ca²⁺]_i in around 50% of the cells (30 out of 61 tested cells). A glycine-evoked change in [Ca²⁺]_i was not observed in these OP cells (16 out of 61 tested cells) which do not exhibit an intracellular calcium response to depolarization induced by high extracellular K⁺ concentration (50 mm). The glycine-elicited [Ca²⁺]_i increase in OP cells (n = 7; Fig. 1A) is completely abolished in a calcium-free extracellular solution supplemented with EGTA (2 mm). Moreover, nifedipine (10 μm) an l-type calcium channel blocker, reversibly inhibits the glycine-induced [Ca²⁺]_i rise in all the tested OP cells (n = 41; Fig. 1B). These data thus suggest that, in OP cells, glycine triggers a depolarization-induced calcium entry through VGCC.

Glycine-induced calcium influx in OP cells is mediated by both GlyR and Na⁺-dependent transporters, most probably GLYTs

Strychnine (30 μm), a competitive antagonist of GlyR, produces a reversible but variable inhibition of glycine-induced calcium responses (Fig. 1D). In three of eight OP cells, the strychnine block of [Ca²⁺]_i increase was complete, whereas the inhibition was only partial in the other tested OP cells, despite a virtually full blockade of OP GlyR at such strychnine concentrations (Belachew et al. 1998b). This suggested that the glycine-induced depolarization in OP cells and its subsequent calcium entry is only partly due to GlyR activation. As it was recently demonstrated that the glial glycine uptake transport systems mediated by GLYT1b and GLYT2 induce inward depolarizing uptake currents in HEK 293 cells transfected with these transporters (López-Corcuera et al. 1998), we suspected the presence of such transporters in oligodendroglial cells. Immunocytochemical stainings (Fig. 2) indeed show that all the A2B5-positive (98 ± 2%, mean ± SEM) and the majority of O4-positive OP cells (76 ± 5%, mean ± SEM) express GLYT1 in vitro. We also observed the expression of GLYT2 in most of the A2B5-positive OP cells (90 ± 3%, mean ± SEM). These two transporters are thus colocalized with GlyR because, as we previously shown, GlyR is expressed in more than 90% of the cells at this stage after 3 days of differentiation in DMEM-N₁ (Belachew et al. 1998b).

In order to investigate the role of the GLYTs and/or less specific electrogenic transporters in glycine-induced [Ca²⁺]_i increase in OP cells, we blocked these Na⁺-dependent transporters by using Na⁺-free Locke solution. In such conditions, a partial inhibition of glycine-evoked calcium responses was observed (n = 7; Fig. 1D). Finally, if strychnine (30 μm) and external Na⁺ removal are combined, a full inhibition of the glycine response is obtained (n = 11; 1, 3), thus demonstrating that a glycine-triggered [Ca²⁺]_i increase in OP cells is actually due to the activation of not only GlyRs but also Na⁺-dependent transporters, most probably GLYTs.

It is noteworthy that the inhibition in Na⁺-free solution is greater (65%) than that expected based on strychnine-resistant inhibition (40%). This discrepancy could arise from inhibition of Na⁺-dependent chloride transporters affecting the chloride gradient and thus influencing GlyR signalling.

Discussion

Glial cells, obviously non-excitable according to the classical view of excitability (i.e. the ability to generate action potentials), express voltage-gated Ca²⁺ channels that constitute an important pathway for calcium entry in oligodendroglial cells (Kirischuk et al. 1995b). Such calcium influx through VGCCs and the resulting increase of [Ca²⁺]_i are likely to regulate various intracellular events in OP cells (including metabolic reactions, gene expression or ion transport systems) that could ultimately be linked to the myelinating function. In this study, we report that exogenously applied glycine induces an important elevation of [Ca²⁺]_i in cortical OP cells. We show that this effect is observed only in cells which express functional VGCCs, as demonstrated by [Ca²⁺]_i increase in response to depolarization. Glycine-evoked [Ca²⁺]_i rise results from a depolarization-induced calcium influx as it is completely suppressed by VGCC blockade or removal of extracellular calcium. However, we cannot exclude that plasmalemnal calcium entry could secondarily trigger a calcium release from intracellular stores but, so far, such a Ca²⁺-induced Ca²⁺-release mechanism has not been demonstrated in oligodendroglial cells (Kirischuk et al. 1995b).

We consider that glycine-induced depolarization and calcium influx in OP cells are mediated by the simultaneous activation of the ionotropic GlyR and the Na⁺-dependent transporters, the best candidates being glycine transporters, since: (i) we demonstrate by immunostaining the presence of GLYT1 and GLYT2 in the majority of OP cells, and these transporters (2Na⁺/Cl^–/glycine) are known to be electrogenic, and thus their activation produces an inward depolarizing uptake current (López-Corcuera et al. 1998); (ii) we have previously shown the presence of GlyR in OP cells (Belachew et al. 1998b), the activation of which should induce a depolarization as is the case after GABA_A receptor activation; (iii) glycine-evoked [Ca²⁺]_i increase is significantly, but only partly, inhibited either by application of saturating concentrations of strychnine or by choline substitution of extracellular Na⁺, which are conditions that block the GlyR and Na⁺-dependent transporters, respectively; and, (iv) glycine-evoked [Ca²⁺]_i increase is totally abolished by the simultaneous blockade of oligodendroglial GlyR and Na⁺-dependent transporters, therefore demonstrating that both types of membrane proteins transduce the effect of glycine leading to an increased OP [Ca²⁺]_i. Due to the lack of availability of pharmacological agents that would specifically block both GLYT1 and GLYT2, we cannot provide conclusive, but only circumstantial, evidence demonstrating that GLYTs are the sole Na⁺-dependent transporters implied.

The coexpression of glycine receptors and transporters and the consequences of their stimulation on calcium homeostasis has not been reported in oligodendroglial cells. This oligodendroglial colocalization of GLYT1 and GlyR is, however, not surprising considering the in situ hybridization data that show a temporal and spatial coincidence of GLYT1 and GlyR β-subunit mRNA expression (Zafra et al. 1995b). Contrary to GLYT1, GLYT2 expression is prominently neuronal and restricted to the brainstem, the spinal cord and the cerebellum, where it colocalizes with GlyR and glycine immunoreactivity (Jursky & Nelson 1995; Luque et al. 1995; Poyatos et al. 1997). Such correlating distributions of GLYTs and GlyR suggest that GLYTs reuptake activity could regulate the glycinergic neurotransmission by modulating glycine concentrations locally bathing presynaptic elements of glycinergic synapses in vivo. It is also known that in cultured astroglial cells, the initiation and the maintenance of GLYT1 expression require the presence of neurons whereas we show that it is not the case for oligodendroglial GLYTs (Zafra et al. 1997). Consequently, we consider that glycine, via its receptors and transporters, could be involved in a bidirectional neuron–oligodendrocyte dialogue that could regulate oligodendroglial development through glycine-induced [Ca²⁺]_i increase. There is currently good evidence that neurons influence oligodendrogliogenesis through the release of growth factors (Calver et al. 1998) and electrical activity (Demerens et al. 1996). Neurotransmitters may also play a role in this neuronal regulation of oligodendrocyte behaviour. However, as oligodendroglial calcium signalling can be triggered by numerous membrane receptor systems (Verkhratsky et al. 1998), the question of the specificity of such effects arises. In other words do oligodendrocytes distinguish, for instance through spatiotemporal cell-specific pattern of [Ca²⁺]_i rise, between different stimulations that all increase [Ca²⁺]_i? At present, the functional responses of oligodendroglial cells that are triggered by an elevation of [Ca²⁺]_i has still to be investigated. In cortical OP cells, calcium influx can induce the phosphorylation of the cAMP response element binding protein (CREB, Pende et al. 1997) that is known to be a crucial factor for Ca²⁺-dependent gene expression. Very recent data support the idea that CREB phosphorylation, which is increased when [Ca²⁺]_i rises, could mediate neuronal signals that, coupled to specific transduction cascades, may play different regulatory roles at specific stages of oligodendrocyte differentiation (Sato-Bigbee et al. 1999). Interestingly, OP cells also express a G-protein-coupled [Ca²⁺]_o-sensing receptor (CaR, Chattopadhyay et al. 1998). The stimulation of CaR by [Ca²⁺]_o elevation increases OP proliferation by triggering a [Ca²⁺]_i rise which is likely to activate an outward K⁺ channel (Chattopadhyay et al. 1998) whose role is essential for the regulation of OP cell cycle (Knutson et al. 1997). Considering this emerging evidence for a central role of [Ca²⁺]_i in oligodendroglial development, we would expect that glycine-induced [Ca²⁺]_i elevation in OP cells could be one of several neuronal signals modulating proliferation, migration or differentiation of OP cells, and therefore ultimately myelination and possibly remyelination, thus opening therapeutic prospects.

Acknowledgements

We thank Dr Frank Kirchhoff (Max Delbrueck Center for Molecular Medicine, Berlin, Germany) for his helpful comments on the manuscript. This work was supported by the Fonds National de la Recherche Scientifique (FNRS), the Concerted Action of the Government of the French Community of Belgium, the Fondation Médicale Reine Elisabeth (FMRE) and the Ligue Belge de la Sclérose en Plaques. We thank P. Ernst-Gengoux for her technical support and expertise. B. Rogister and S. Belachew are, respectively, Research Associate and Research Assistant of the FNRS.

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