Different patterns of neuronal activity trigger distinct responses of oligodendrocyte precursor cells in the corpus callosum

<div><p>In the developing and adult brain, oligodendrocyte precursor cells (OPCs) are influenced by neuronal activity: they are involved in synaptic signaling with neurons, and their proliferation and differentiation into myelinating glia can be altered by transient changes in neuronal firing. An important question that has been unanswered is whether OPCs can discriminate different patterns of neuronal activity and respond to them in a distinct way. Here, we demonstrate in brain slices that the pattern of neuronal activity determines the functional changes triggered at synapses between axons and OPCs. Furthermore, we show that stimulation of the corpus callosum at different frequencies in vivo affects proliferation and differentiation of OPCs in a dissimilar way. Our findings suggest that neurons do not influence OPCs in “all-or-none” fashion but use their firing pattern to tune the response and behavior of these nonneuronal cells.</p></div

The rate and the time course of delayed glutamate release at axon-oligodendrocyte precursor cell (OPC) synapses depend on the stimulation paradigm.

<p><b>(A)</b> Average peak rate of the delayed events after the stimulation with 2 pulses (<i>n</i> = 6 cells), 5 pulses (<i>n</i> = 6 cells), or 20 pulses (<i>n</i> = 8 cells) at 25 Hz. The box “Spont.” shows the frequency of the spontaneous events recorded before each stimulation train. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s019" target="_blank">S7 Data</a>). <b>(B)</b> Average rate of delayed events after the stimulation with 2, 5, or 25 pulses at 25 Hz. Solid lines indicate monoexponential fits to the events rate. The same cells used as in <b>(A)</b>. <b>(C)</b> Average decay time constants of the delayed events rate after the stimulation with 2, 5, or 20 pulses at 25 Hz. The same cells used as in <b>(A).</b> One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s019" target="_blank">S7 Data</a>). <b>(D)</b> Data and statistical comparisons are as in <b>(A)</b> but for the stimulation paradigms of 5 pulses at 5 Hz (<i>n</i> = 7 cells), 25 Hz (<i>n</i> = 7 cells), and 100 Hz (<i>n</i> = 6 cells). One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s019" target="_blank">S7 Data</a>). <b>(E)</b> Data as in <b>(B)</b> but for the stimulation paradigms of 5 pulses at 5, 25, and 100 Hz. The same cells used as in <b>(D)</b>. <b>(F)</b> Data and statistical comparisons are as in <b>(C)</b> but for the stimulation paradigms of 5 pulses at 5, 25, and 100 Hz. The same cells used as in <b>(D)</b>. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s019" target="_blank">S7 Data</a>). <b>(G)</b> Data and statistical comparisons are as in <b>(A)</b> but for the stimulation paradigms of 20 pulses at 25 Hz (<i>n</i> = 8 cells) and 100 Hz (<i>n</i> = 13 cells). One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s019" target="_blank">S7 Data</a>). <b>(H)</b> Data as in <b>(B)</b> but for the stimulation paradigms of 20 pulses at 25 and 100 Hz. The same cells used as in <b>(G)</b>. <b>(I)</b> Data and statistical comparisons are as in <b>(C)</b> but for the stimulation paradigms of 20 pulses at 25 and 100 Hz. The same cells used as in <b>(G)</b>. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s019" target="_blank">S7 Data</a>). Box and whisker plots: the bottom and top of each box represent 25th and 75th percentiles of the data, respectively, while whiskers represent 10th and 90th percentiles. The midline represents the median. The numerical data used in A, C, D, F, G, and I are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s020" target="_blank">S8 Data</a>.</p

The time course and the amount of synaptic charge transfer through alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors at axon–oligodendrocyte precursor cell (OPC) synapses depend on the stimulation paradigm.

<p><b>(A)</b> Average synaptic charge transfer upon stimulation of callosal axons with trains of four different frequencies and durations plotted versus real time. Each color (blue, red, dark red, or black) represents mean charge transfer from <i>n</i> = 5 cells (5 pulses at 5 Hz), <i>n</i> = 5 cells (5 pulses at 25 Hz), <i>n</i> = 5 cells (20 pulses at 25 Hz), and <i>n</i> = 10 cells (20 pulses at 100 Hz). Charge transfer is shown in 5-ms bins. Note that plotting charge transfer versus real time allows observing not only the amount of synaptic facilitation but also its distribution over time, and it differs dramatically depending on the stimulation paradigm (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.g003" target="_blank">Fig 3A & 3D</a>). <b>(B)</b> Total average synaptic charge transfer during the stimulation trains of different frequencies and durations. Both phasic and asynchronous charge transfer during the trains are considered for these bar graphs. The same cells used as in <b>(A)</b>. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s021" target="_blank">S9 Data</a>). <b>(C)</b> Total synaptic charge transferred by the delayed currents occurring after the stimulation trains of different frequencies and durations. The same cells used as in <b>(A)</b>. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s021" target="_blank">S9 Data</a>). <b>(D)</b> Percentage contribution of synaptic charge transferred during (phasic + asynchronous charge) and after (delayed charge) the stimulation trains of different frequencies and durations. White numbers on the bars indicate the proportion of charge transferred during the train. Box and whisker plots: the bottom and top of each box represent 25th and 75th percentiles of the data, respectively, while whiskers represent 10th and 90th percentiles. The midline represents the median. The numerical data used in B–C are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s022" target="_blank">S10 Data</a>.</p

Activity-dependent changes at axon-oligodendrocyte precursor cell (OPC) synapses are determined by the stimulation paradigm also in the adult (P50–53) mice.

<p><b>(A–C)</b> Normalized average <b>(A)</b> current amplitude including failures, <b>(B)</b> probability of responses, and <b>(C)</b> absolute potency of responses upon each stimulus of the minimal stimulation train of 20 pulses at 25 Hz (<i>n</i> = 4 cells), 100 Hz (<i>n</i> = 6 cells), or 300 Hz (<i>n</i> = 5 cells). One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s023" target="_blank">S11 Data</a>). 25 Hz versus 300 Hz stimulation: *<i>p</i> < 0.05, **0.001 < <i>p</i> < 0.01. 100 Hz versus 300 Hz stimulation: #<i>p</i> < 0.05. 25 Hz versus 100 Hz stimulation: ^<i>p</i> < 0.05 <b>(D)</b> Example traces showing average currents (including failures) in 3 OPCs in response to the 1st, 5th, 10th, and 20th stimulus of the minimal stimulation train of 20 pulses at 25 Hz, 100 Hz, and 300 Hz. At least 15 trials were averaged to generate each example trace. <b>(E)</b> Original example traces showing delayed currents in callosal OPC (holding potential [V_h] = −80 mV) occurring after the stimulation train of 20 pulses at 25 Hz. <b>(F)</b> Average rate of delayed currents occurring after the stimulation train of 20 pulses at 25 Hz, 100 Hz, or 300 Hz. Solid lines indicate monoexponential fits to the events rate. <b>(G)</b> Average decay time constants of the delayed event rate after the stimulation train with 20 pulses at 25 Hz, 100 Hz, or 300 Hz. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s023" target="_blank">S11 Data</a>). <b>(H)</b> Average peak rate of the delayed events after the stimulation with 20 pulses at 25 Hz, 100 Hz, or 300 Hz. The box “Spont.” shows the frequency of the spontaneous events recorded before each stimulation train. One-way ANOVA (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s023" target="_blank">S11 Data</a>). Box and whisker plots: the bottom and top of each box represent 25th and 75th percentiles of the data, respectively, while whiskers represent 10th and 90th percentiles. The midline represents the median. The numerical data used in A–C and G–H are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s024" target="_blank">S12 Data</a>.</p

Transient stimulation of callosal axons in vivo at 5 Hz but not at 25 Hz or 300 Hz promotes differentiation of oligodendrocyte precursor cells (OPCs) into oligodendrocytes (OLs).

<p><b>(A)</b> Scheme describing experimental design for studying effects of callosal stimulation in vivo on proliferation and differentiation of OPCs. <b>(B)</b> Scheme showing sagittal section of mouse brain and the position of an electrode array used for electrical stimulation of the corpus callosum. <b>(C)</b> Schematic drawings of the investigated cell types. For cell counting, OPCs were identified as NG2⁺CC1^- cells, premyelinating OLs (pre-OLs) as NG2⁺CC1⁺ cells, and myelinating OLs as NG2^- cells expressing CC1 in their soma. Within the OL lineage, 5-ethynyl-2´-deoxyuridine (EdU) only labels proliferating OPCs. However, the progeny of an EdU⁺ OPC will be EdU⁺. <b>(D)</b> Coronal sections of the corpus callosum. Maximum intensity projection (from 14 successive confocal planes) showing triple channel immunofluorescent labelling with DAPI (top left, blue), NG2 (top right, green), CC1 (bottom left, red), and the overlay of 3 channels (bottom right). White dashed line denotes the middle region of the corpus callosum used for cell counting. White arrow indicates midline of the brain. Scale bars: 100 μm. <b>(E)</b> As in <b>(D)</b>, but higher magnification example of one NG2⁺CC1^- OPC (arrow) and three NG2^-CC1⁺ OLs (arrowheads). Maximum intensity projection was generated from a z-stack of 3 successive confocal planes. Note that some processes of an NG2⁺ OPC are clearly visible. Scale bars: 10 μm. <b>(F)</b> As in <b>(E)</b>, but an example of one NG2⁺CC1⁺ pre-OL (arrow). Note that the expression level of NG2 is weaker than in OPCs, and the expression level of CC1 is weaker than in OLs (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.g007" target="_blank">Fig 7E</a>). Scale bars: 10 μm. <b>(G–I)</b> Average density of <b>(G)</b> OPCs, (<b>H)</b> pre-OLs, and (<b>I)</b> OLs in corpus callosum upon electrical stimulation of callosal axons at 5 Hz (<i>n</i> = 5 mice, total 13 slices), 25 Hz (<i>n</i> = 5 mice, total 16 slices), or 300 Hz (<i>n</i> = 5 mice, total 17 slices) versus sham-stimulated controls (<i>n</i> = 7 mice, total 25 slices). Note that differentiation rate was significantly increased by 5 Hz but not by 25 Hz or 300 Hz stimulation <b>(H)</b>. Nested ANOVA and post hoc Tukey were used for statistical analysis (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s025" target="_blank">S13 Data</a>). Box and whisker plots: the bottom and top of each box represent 25th and 75th percentiles of the data, respectively, while whiskers represent 10th and 90th percentiles. The midline represents the median. The numerical data used in G–I are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s026" target="_blank">S14 Data</a>.</p

Callosal stimulation in vivo enhances proliferation rate of oligodendrocyte precursor cells (OPCs) and promotes differentiation of newly born OPCs into oligodendrocytes (OLs).

<p><b>(A)</b> Coronal sections of corpus callosum. Maximum intensity projection image (from 14 successive confocal planes) showing fluorescent 5-ethynyl-2´-deoxyuridine (EdU) labelling in corpus callosum. White dashed line marks the region of interest used for cell counting. Top panel: overview. Scale bar: 100 μm. Bottom panels: higher magnification of the area marked by the white square at the top panel; left, EdU (false color look-up table (LUT) “Green Fire Blue”); right, overlay of EdU and DAPI (blue) channels. Arrowheads indicate EdU⁺ cells. Scale bar: 10 μm. <b>(B)</b> Density of EdU⁺ cells in corpus callosum. <b>(C)</b> Proportion of OL lineage cells (OPCs + pre-OLs + OLs) labelled with EdU, within the total population of EdU⁺ cells. <b>(D)</b> Proportion of EdU⁺ OPCs (green), premyelinating OLs (pre-OLs) (red-green) and OLs (red) within the total population of EdU⁺ cells. <b>(E)</b> Single layer confocal image showing quadruple channel fluorescent labelling with DAPI (blue), NG2 (green), CC1 (red), and EdU (false color LUT “Green Fire Blue”), and the overlay of four channels (top right). The arrowhead indicates an EdU⁺ OPC. Scale bars: 10 μm. The box plot shows proportion of EdU⁺ OPCs within the total OPCs population. <b>(F)</b> Single layer confocal image showing quadruple-channel fluorescent labelling with DAPI (blue), NG2 (green), CC1 (red), and EdU (false color LUT “Green Fire Blue”), and the overlay of four channels (top right). The arrowhead points to an EdU⁺ OL. Scale bar: 10 μm. The box plot shows proportion of EdU⁺ OLs within the total OLs population. Throughout this figure, the same mice and slices were analyzed as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.g007" target="_blank">Fig 7</a>. Nested ANOVA and post hoc Tukey test were used for statistical analysis (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s027" target="_blank">S15 Data</a>). Box and whisker plots: the bottom and top of each box represent 25th and 75th percentiles of the data, respectively, while whiskers represent 10th and 90th percentiles. The midline represents the median. The numerical data used in B–F are included in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2001993#pbio.2001993.s026" target="_blank">S14 Data</a>.</p

Z score values of samples calculated by three different previously published methods.

<p><b>□</b> –trisomic samples, <b>○</b> –euploid samples, horizontal line—mean z score value of trisomic samples. Dotted lines represent the standard limit for identification of a trisomic sample (z score = 3). A—Ion Torrent PGM analyzed samples, B—MiSeq analyzed samples.</p

Z score values of trisomic and euploid samples before and after <i>in silico</i> size selection of sequencing reads.

<p><b>□</b> –trisomic samples, <b>○</b> –euploid samples, horizontal line—mean z score value). Dotted lines represent the standard limit for identification of a trisomic sample (z score = 3). A—Ion Torrent PGM analyzed samples, B—MiSeq analyzed samples.</p

Determination of optimal <i>in silico</i> size selection limit for sequencing reads to be used in z score calculation.

<p>Dotted lines represent the standard limit for identification of a trisomic sample (z score = 3). A—Ion Torrent PGM analyzed samples, B—MiSeq analyzed samples.</p

Z score values calculated from reads without size selection (all), after <i>in silico</i> size selection (IS) and after physical size selection (P).

<p>Horizontal lines represent mean z score value calculated from 3, 2 and 1 million raw reads (3m, 2m, 1m). Dotted lines represent the standard limit for identification of a trisomic sample (z score = 3). A—Ion Torrent PGM analyzed samples, B—MiSeq analyzed samples.</p