Neuronal input triggers Ca2+ influx through AMPA receptors and voltage‐gated Ca2+ channels in oligodendrocytes

Abstract Communication between neurons and developing oligodendrocytes (OLs) leading to OL Ca2+ rise is critical for axon myelination and OL development. Here, we investigate signaling factors and sources of Ca2+ rise in OLs in the mouse brainstem. Glutamate puff or axon fiber stimulation induces a Ca2+ rise in pre‐myelinating OLs, which is primarily mediated by Ca2+‐permeable AMPA receptors. During glutamate application, inward currents via AMPA receptors and elevated extracellular K+ caused by increased neuronal activity collectively lead to OL depolarization, triggering Ca2+ influx via P/Q‐ and L‐type voltage‐gated Ca2+ (Cav) channels. Thus, glutamate is a key signaling factor in dynamic communication between neurons and OLs that triggers Ca2+ transients via AMPARs and Cav channels in developing OLs. The results provide a mechanism for OL Ca2+ dynamics in response to neuronal input, which has implications for OL development and myelination.


| INTRODUCTION
Oligodendrocytes (OLs) ensheath axons with their myelinating processes, providing protection and maintenance of axonal integrity and enabling saltatory conduction of action potentials. Dynamic interactions between neurons and OLs modulate development of OL lineage cells and myelination to control conduction speed in the central nervous system (CNS; Pajevic, Basser, & Fields, 2014;Sinclair et al., 2017). Recent studies demonstrated that a dynamic Ca 2+ rise in OLs is associated with myelin sheath refinement in vivo in the zebrafish spinal cord (Baraban, Koudelka, & Lyons, 2018;Krasnow, Ford, Valdivia, Wilson, & Attwell, 2018). Ca 2+ is an important second messenger that induces cellular processes that can lead to local translation and process extension in OL lineage cells (Baraban et al., 2018;Krasnow et al., 2018;Wake, Lee, & Fields, 2011). However, little is known about signaling factors from active axons that facilitate activitydependent neuron-OL communication leading to Ca 2+ transients in OLs, as well as the sources of OL Ca 2+ rise in response to neuronal inputs.
Previous studies have reported the expression of L-, N-, and R-type Ca v channels and the presence of their currents in OL lineage cells (Berger, Schnitzer, Orkand, & Kettenmann, 1992;Butt, 2006). In NG2+ glial cells, integration of synaptic input resulted in Ca 2+ signals by recruiting low voltage-activated R-type or T-type Ca v channels (Sun, Matthews, Nicolas, Schoch, & Dietrich, 2016). In addition, high voltage-activated Ltype Ca v channels are known to modulate OL development and migration (Cheli et al., 2016;Cheli, Santiago González, Spreuer, & Paez, 2015;Paez, Fulton, Colwell, & Campagnoni, 2009). Despite functional implications of Ca v channels in OL lineage cells, there is a lack of evidence for the functional link between glutamatergic inputs and Ca 2+ influx through Ca v channels in developing OLs beyond the precursor stage.
Here, we investigate the mechanism by which glutamate induces a Ca 2+ rise in OLs in the medial nucleus of the trapezoid body (MNTB) of the auditory brainstem. The heavily myelinated axon fibers in the MNTB, which are important for ensuring the reliability and temporal fidelity of conduction, undergo activity-dependent myelination during development (Kim, Renden, & von Gersdorff, 2013;Kim, Turkington, Kushmerick, & Kim, 2013;Sinclair et al., 2017). In the MNTB, we find glutamatergic input-induced Ca 2+ transients and Ca 2+ currents via Ca v channels in developing OLs using electrophysiology and Ca 2+ imaging in mice expressing OL-specific GCaMP6f, a genetically encoded Ca 2+ indicator with fast kinetics (Chen et al., 2013). We identify AMPARs and L-type and P/Q-type
Stack images were acquired at a digital size of 1,024 × 1,024 pixels with optical section separation (z interval) of 0.5 μm and were later cropped to the relevant part of the field without changing the resolution.

| Ca 2+ imaging
Ca 2+ imaging was performed in normal aCSF at room temperature (22-24 C). Normal aCSF was the same as slicing aCSF, but with 1 mM
Statistical significance was determined using paired Student's t test  OLs was replicable and consistent in amplitude after a two-minute recovery period. Morphologically, the processes of GCaMP6f + cells did not align with axons to generate myelin sheaths ( Figure 1d). In wholecell voltage-clamp recordings, these GCaMP6f + cells displayed outwardly rectifying K + currents and, and 37.5% of cells showed transient inward Na + currents ( Figure 1e). This electrophysiological profile aligns with what has previously been observed in pre-myelinating OLs in the MNTB (Berret et al., 2017).
OL lineage cells express ionotropic glutamate receptors (e.g., AMPA and NMDA receptors) and receive glutamatergic input from neurons (Berret et al., 2017;Micu et al., 2016 reducing the amplitude of the Ca 2+ response to 55.4 ± 6.70% of the control response from the same cell (n = 16 cells from four animals; p < .0001, paired t test; Figure 3a). The additional application of CdCl 2 , which blocks Ca v channels, reduced the amplitude of the remaining Ca 2+ response to 5.7 ± 2.34% of the control response from the same cell (n = 26 cells from four animals; p < .0001, paired t test, Figure 3b). CdCl 2 alone reduced the amplitude of the Ca 2+ response to 33.4 ± 5.31% of   (Figure 3c). In whole-cell recordings of OLs with Ca 2+ indicator Fluo-4, glutamate puff elicited a depolarization from −65 mV tõ − 40 mV (20.8 ± 2.00 mV change, n = 6; Figure 3d). Glutamateinduced depolarization resulted in a Ca 2+ rise (0.12 ± 0.021 ΔF/F, n = 6 cells from four animals; Figure 3e). By adding Fluo-4 and Alexa 568 to the internal pipette solution, OL processes within 20 μm from the OL soma, which were not myelin sheaths, were clearly visible and showed detectable glutamate-induced Ca 2+ responses. The Ca 2+ rise in the processes was 187.6 ± 22.76% of the response in the soma of the same cell (n = 6 cells from four animals; p = .0085, paired t test; Figure 3f). In the presence of CdCl 2 , the Ca 2+ rise was significantly reduced in both processes and soma (n = 6 cells from four animals; p = .0012 for soma, p = .0018 for process, paired t test; Figure 3g), while glutamate-induced depolarization was unaffected, indicating that glutamate puff is able to depolarize pre-myelinating OLs and consequently activate Ca v channels.

| Increased neuronal activity increases extracellular K + and glutamate, resulting in OL Ca 2+ rise
During increased neuronal activity, K + accumulates in the extracellular space and can depolarize OLs (Battefeld, Klooster, & Kole, 2016;Larson et al., 2018). We next tested the capacity of OL depolarization to recruit Ca v channels allowing Ca 2+ influx independent of OL AMPAR activation. KCl puff applied to OLs and surrounding tissue, which mimics elevated extracellular K + when neuronal activity increases, resulted in a Ca 2+ rise in GCaMP6f + OLs (Figure 4a). In the presence of Naspm and CNQX to isolate the OL depolarizationinduced Ca 2+ entry, KCl-induced Ca 2+ rise was 70.5 ± 0.06% of control responses (n = 10 cells from three animals; p = .0008; paired t test). This remaining Ca 2+ response was significantly reduced by the application of CdCl 2 reduced to 24.3 ± 6.30% of responses in the presence of CNQX and Naspm (n = 10 cells from three animals; p < .0001, paired t test; Figure 4b). Together, these data indicate that OL depolarization is sufficient for Ca 2+ influx via Ca v channels.
We next determined whether increased neuronal activity and  which was a slight decrease from control (depolarization was 21.44 ± 1.88 mV in control; n = 6 from three animals; p = .0021; paired t test; Figure 4c,e). The reduction in the presence of TTX is assumed to be caused by the inhibited neuronal activity, although voltage-activated Na + channels in OLs could be also involved in the TTX-sensitive response (Berret et al., 2017). The addition of Naspm and CNQX blocked the remaining Ca 2+ response (4.5 ± 2.26% of control response; n = 17 cells from five animals; p < .0001, paired t test) and depolarization (2.8 ± 0.64 mV; n = 6 cells from three animals; p < .0001, paired t test). These results suggest that elevated extracellular K + due to increased firing rate of neurons partially contributes to depolarization and Ca 2+ rise in OLs, and that the larger remaining response is due to AMPAR-mediated currents.

| OL Ca 2+ rise through P/Q-type and L-type Ca v channels
Furthermore, we identified the types of Ca v channels involved in Ca 2+ influx in OLs in response to glutamate puff. We specifically examined L-type and P/Q-type channels, which are expressed in OL lineage cells (Cheli et al., 2015(Cheli et al., , 2016Paez et al., 2009;Zhang et al., 2014). After from four animals; p < .0001, paired t tests; Figure 5a,b). Combined, agatoxin and nifedipine nearly abolished the glutamate-induced Ca 2+ rise, reducing the amplitude to 6.2 ± 1.33% of control responses (n = 10 cells from three animals; p < .0001, paired t test). The combination of agatoxin and CdCl 2 reduced the Ca 2+ -impermeable AMPARmediated Ca 2+ response to 6.4 ± 2.26% of control responses in the same cell (n = 10 cells from three animals; p < .0001, paired t test).
Thus, P/Q-type and L-type Ca v channels play a significant role in OL Ca 2+ rise in response to glutamate puff.
To confirm the presence of functional P/Q-type Ca v channel currents in OLs, voltage-activated Ca 2+ currents (I Ca ) were elicited from OLs in whole-cell voltage-clamp recordings. I Ca was shown in response to depolarization steps from −80 mV to 60 mV and was completely inhibited by CdCl 2 (Figure 5c). Partial Ca v channel inactivation was observed, as expected for P/Q-type Ca v channels. Moreover, a portion of I Ca was inhibited by agatoxin, suggesting that P/Q-type Ca v channels are involved in Ca 2+ influx due to depolarization   (Figure 6a,b). Blocking Ca 2+ -permeable AMPARs with Naspm reduced the Ca 2+ response to axon fiber stimulation to 58.2 ± 3.36% (n = 20 cells from four animals; p < .0001, paired t tests), and the combination of Naspm and CNQX reduced the response to 28.4 ± 3.16% of control responses in the same cell (n = 20 cells from four animals; p < .0001, paired t tests; Figure 6c,d), indicating that neuronal activity provides sufficient glutamate to induce Ca 2+ rise.
The Ca 2+ response, which was not blocked by Naspm and CNQX, was significantly inhibited by Ca v channel blocker CdCl 2 (5.1 ± 0.99% of control responses in the same cell, n = 10 cells from two animals; p < .0001, paired t test). During axon fiber stimulation, increased neuronal activity leads to the accumulation of extracellular K + and consequently OL depolarization (Battefeld et al., 2016;Larson et al., 2018).
Indeed, we demonstrated that increasing extracellular K + concentration using KCl puff depolarized and induced Ca 2+ influx through Ca v channels in OLs without AMPAR activation (Figure 4a). These data reveal that, when neuronal activity-induced Ca 2+ rise in OLs is mediated by AMPARs and Ca v channels as sources of Ca 2+ influx.

| DISCUSSION
During development, neuronal activity drives physiological responses in OL lineage cells that influence OL maturation and myelination. It is proposed that neuronal activity triggers release of signaling factors to elicit OL Ca 2+ transients and initiate downstream signaling processes. Here, we studied what factors mediate the dynamic communication between neurons and OLs leading to OL Ca 2+ rise and the primary source of OL Ca 2+ transients in response to neuronal input. The results reveal that increased neuronal activity leads to Ca 2+ influx in OLs through glutamatergic signaling, mediated by AMPARs, depolarization, and L-type and P/Q-type Ca v channels. These findings support that glutamatergic signaling is a key mechanism for neuron-OL interaction that could lead to various downstream Ca 2+ signaling pathways important for myelination and OL function in the mammalian nervous system.
Due to the overlap in expression of channels and receptors in both neurons and glia in the brain slice, it was difficult to isolate effects on single OLs and to quantify the contribution of each cell type to OL Ca 2+ responses using pharmacological approaches. For example, bath application of CdCl 2 can block axonal or presynaptic Ca 2+ channels, inhibiting vesicular glutamate release, as well as oligodendroglial Ca 2+ channels, and thus a mixed response could be observed. Glutamate puff could be activating AMPARs on neighboring cells, resulting in increased firing and release of other molecules or ions to trigger OL Ca 2+ transients. During periods of increased action potential firing, the concentration of K + in the extracellular area increases, which in turn depolarizes OLs (Battefeld et al., 2016;Larson et al., 2018). Our findings in Figure 4 showed that increased extracellular K + release from neighboring cells partially contributes to OL Ca 2+ transients. This depolarization could then activate Ca v channels in OLs, resulting in a Ca 2+ rise. In addition, ATP and adenosine are other possible molecules, which are released in response to axon stimulation and have been shown to induce a Ca 2+ rise in OL lineage cells Hamilton, Vayro, Wigley, & Butt, 2010;Stevens, Porta, Haak, & Fields, 2002). Activation of neighboring cells may impact OL Ca 2+ transients and explain the slow and prolonged kinetics of the Ca 2+ rise in OLs in response to electrical stimulation of axons seen in Figure 6. In physiological conditions, Ca 2+ dynamics in OLs are a result of a summation of various inputs over time when neuronal activity increases.
In this study, we examined OL Ca 2+ rise to delineate sources of Ca 2+ influx in OLs in response to glutamatergic input. An alternative mediator of Ca 2+ transient duration may be a secondary Ca 2+ source, such as release from internal stores in OLs. Amplitude and duration of Ca 2+ transients in OLs induced by AMPA application were reduced after blocking ryanodine receptors (Ruiz et al., 2010), implicating a role for CICR from internal stores in AMPAR-mediated Ca 2+ transients.
We tested the effect of dantrolene, a blocker of CICR, on glutamateinduced Ca 2+ rise in OLs, but there was no significant effect ( Figure 2). In hippocampal NG2+ cells, Haberlandt et al. (2011)  What is the functional relevance of Ca 2+ rise in OLs in response to glutamatergic input? Glutamate has been repeatedly shown to induce pathological damage to cultured OLs (Alberdi, Sánchez-Gómez, Marino, & Matute, 2002). Similar to what occurs in neurons, excitotoxicity due to over-activation of AMPARs leads to Ca 2+ rise and downstream cascades leading to mitochondrial damage and apoptosis (Leuchtmann, Ratner, Vijitruth, Quab, & McDonald, 2003;Ruiz et al., 2010;Sánchez-Gómez, Alberdi, Ibarretxe, Torre, & Matute, 2003). An irreversible elevation of Ca 2+ through either AMPAR or Ca v channels could induce necrosis of OLs in pathological conditions. However, dynamic Ca 2+ transients are beneficial for signaling cascades of physiological processes of OLs, such as increased OL lineage cell survival and migration, as well as myelination (Gautier et al., 2015;Gudz, Komuro, & Macklin, 2006;Kougioumtzidou et al., 2017). Neuronal activity elicited a Ca 2+ response in OLs in zebrafish, which was associated with myelin elongation or retraction, depending on the dynamics of the Ca 2+ response (Baraban et al., 2018;Krasnow et al., 2018). In co-cultures with dorsal root ganglion neurons, vesicular release of glutamate from axons stimulates local translation of myelin basic protein in OPCs and stimulates myelin induction through activation of NMDARs and mGluRs, rather than AMPARs (Wake et al., 2011(Wake et al., , 2015. In the mouse brainstem, we identified AMPARs and Ca v channels as an important Ca 2+ influx pathway for Ca 2+ dynamics in developing OLs. Recent studies suggest the potential role of AMPARs in OL development and myelination (Chen et al., 2018;Kougioumtzidou et al., 2017). Alterations in AMPAR expression in OPCs impact OL lineage cell differentiation, survival, and myelination in the corpus callosum (Chen et al., 2018;Kougioumtzidou et al., 2017). In addition, the electrical changes of the OL membrane are also important for OL development, because the knockdown of Ca v channels or Na v channels throughout the OL lineage impaired OL development and myelination (Berret et al., 2017;Cheli et al., 2015Cheli et al., , 2016. These studies support that AMPARs and Ca v channels, as well as other signaling mechanisms, can play a role in OL Ca 2+ dynamics as important mediators of glutamatemediated signaling to impact OL development. Dynamic interaction between active axons and OLs in the auditory brainstem is particularly relevant to auditory function, because this brain region is heavily myelinated and operates at the upper limits of action potential frequency and speed required for sound localization (Kim, Renden, & von Gersdorff, 2013;Kim, Turkington, et al., 2013;Sinclair et al., 2017). Still, neuronal glutamate-induced Ca 2+ rise in OLs has been observed in brain areas other than the MNTB. Neuronal activity induced myelination-associated Ca 2+ rises in OLs in the zebrafish spinal cord (Baraban et al., 2018;Krasnow et al., 2018). Furthermore, OPC Ca 2+ rise in response to glutamate or depolarization has been observed in the rodent optic nerve and hippocampus (Haberlandt et al., 2011;Hamilton et al., 2010;Sun et al., 2016). The present study provides a mechanistic explanation for neuronal activity-dependent Ca 2+ rise in pre-myelinating OLs in the MNTB, but could be relevant for Ca 2+ transients in developing OLs in other brain areas. Understanding the physiological properties of OLs and mechanisms of neuron-OL interaction throughout the OL lineage and throughout the brain is critical for further comprehension of their roles in brain function. The mechanism of Ca 2+ rise in pre-myelinating OLs resulting from depolarization and neuronal input demonstrated in this study provides important insight into the dynamic physiological properties of OLs.