Ryanodine receptor- and sodium-calcium exchanger-mediated spontaneous calcium activity in immature oligodendrocytes in cultures

Highlights

• Immature OLCs but not OPCs or mature OLCs demonstrate spontaneous Ca²⁺ activity.

• Spontaneous Ca²⁺ transients occur predominantly in OLC processes.

• Spontaneous Ca²⁺ transients depend on Ca²⁺ release from the endoplasmic reticulum.

• Spontaneous Ca²⁺ transients depend on Na⁺, Ca²⁺ exchanger.

Abstract

Myelination in the central nervous system depends on interactions between axons and oligodendrocyte precursor cells (OPCs). Action potentials in an axon can be followed by release of biologically active substances, like glutamate, which can instruct OPCs to start myelination. Myelin Basic Protein (MBP) is an “executive molecule of myelin” required for the formation of compact myelin. As cells of the oligodendrocyte lineage (OLCs) are capable of producing MBP in pure oligodendrocyte cultures, i.e. without neurons, we investigated Ca²⁺ signaling in developing OLCs in cultures. We show that spontaneous Ca²⁺ transients (CTs) occur at very low frequency in both bipolar OPCs and mature oligodendrocytes. In contrast immature OLCs (imOLCs), cells with several thick processes, demonstrate a relatively high frequency of CTs. Moreover, CT frequency in imOLC processes is much higher as compared with the somatic CT frequency. Somatic CTs are almost completely blocked by thapsigargin, an antagonist of sarco-(endo-) plasmic reticulum Ca²⁺ ATPase, and ryanodine, a blocker of ryanodine receptors, indicating an involvement of Ca²⁺ release from the endoplasmic reticulum. Ryanodine strongly reduces CT frequency in imOLC processes. Ouabain, an antagonist of Na⁺, K⁺-ATPase (NKA), applied at low concentration increases CT frequency, while KB-R7943, a blocker of reverse mode of Na⁺, Ca²⁺ exchanger (NCX), decreases CT frequency. We suggest that local RyR-NCX-(NKA?) interaction might underlie the generation of CTs in imOLC in the absence of neurons, and this activity influences oligodendrocyte maturation.

Introduction

Oligodendrocytes are glial cells in the central nervous system. One of their main functions is to myelinate neuronal axons, which is vital for efficient and rapid saltatory conduction of action potentials. Before becoming myelinating oligodendrocytes, oligodendrocyte precursor cells (OPCs) have to migrate to their proper destinations and differentiate. Developing cells of the oligodendrocytic linage (OLCs) express a variety of receptors linked to intracellular Ca²⁺ signaling (for review [9,23]). It has been shown in vitro and in vivo that those receptors can be activated by mediators released from neurons [13,16,24] and astrocytes [10]. There is strong evidence that various intracellular processes in OLCs are regulated by patterns of intracellular Ca²⁺ activity rather than [Ca²⁺]_i levels. For example, elongation or shortening of myelin sheaths in the spinal cord of zebrafish is determined by the frequency of Ca²⁺ transients (CTs) in OLCs [12]. Furthermore, distinct patterns of neuronal activity in the corpus callosum in vivo affect proliferation and differentiation in different way [18].

Interestingly, OLCs in pure oligodendrocyte cultures, i.e. in the physical absence of neurons, can build specific sheets filled with several components of myelin, including Myelin Basic Protein (MBP) [7]. As compact myelin cannot be formed in the absence of MBP, the latter was called the ‘executive molecule of myelin’ [3]. Before local translation RNA granules containing MBP mRNA are transported from nucleus to proper cytoplasmic translation site [17]. MBP translation and myelination in vivo appears to be initiated by neuronal activity [14,21,22]. But OLCs are capable to produce MBP in pure oligodendrocyte cultures, and MBP synthesis appears to be modulated by intracellular ionic signaling [4,7,11]. Spontaneous CTs were observed in cultured OLCs. Voltage-gated Ca²⁺ channels (Ca_v) [5,19] and sodium-calcium exchanger (NCX, [4]) have been suggested to contribute to CT generation. But a stimulus that could depolarize OLCs to activate Ca_v and/or reverse NCX in neuron-free OLC cultures remains elusive. Ca²⁺ release from intracellular stores represents an alternative source of Ca²⁺ ions. OLCs express both ryanodine (RyR3) and inositol 1,4,5-trisphosphate (IP₃R2) receptors which can mediate Ca²⁺ release from the endoplasmic reticulum. However, under resting conditions CT frequency at OLC somata remains very low [8,15]. As local MBP synthesis takes place at OLC processes and calcium/calmodulin-dependent kinase type II (CaMKII) contributes to myelination [20], we asked whether spontaneous CTs in OLC processes occur independently on the activity in OLC soma.

In this study we investigated spontaneous CTs in developing OLCs in pure oligodendrocyte culture. We report that the frequency of spontaneous CTs is the highest in the processes of immature OLCs (imOLCs), while CT frequency in both bipolar OPCs and mature oligodendrocytes is very low. RyR-mediated Ca²⁺ release from the endoplasmic reticulum and plasmalemmal sodium-calcium exchanger (NCX) contribute to CT generation. We suggest that local Ca²⁺-mediated interaction between RyR and NCX may represent an amplification mechanism to generate CT pattern(s) required for oligodendrocyte differentiation in cultures.