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Norepinephrine modulates calcium dynamics in cortical oligodendrocyte precursor cells promoting proliferation during arousal in mice

Nature Neuroscience volume 26, pages 1739–1750 (2023)Cite this article

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Abstract

Oligodendrocytes, the myelinating cells of the central nervous system (CNS), are generated from oligodendrocyte precursor cells (OPCs) that express neurotransmitter receptors. However, the mechanisms that affect OPC activity in vivo and the physiological roles of neurotransmitter signaling in OPCs are unclear. In this study, we generated a transgenic mouse line that expresses membrane-anchored GCaMP6s in OPCs and used longitudinal two-photon microscopy to monitor OPC calcium (Ca2+) dynamics in the cerebral cortex. OPCs exhibit focal and transient Ca2+ increases within their processes that are enhanced during locomotion-induced increases in arousal. The Ca2+ transients occur independently of excitatory neuron activity, rapidly decline when OPCs differentiate and are inhibited by anesthesia, sedative agents or noradrenergic receptor antagonists. Conditional knockout of α1A adrenergic receptors in OPCs suppresses spontaneous and locomotion-induced Ca2+ increases and reduces OPC proliferation. Our results demonstrate that OPCs are directly modulated by norepinephrine in vivo to enhance Ca2+ dynamics and promote population homeostasis.

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Fig. 1: In vivo 2P imaging reveals that OPCs exhibit dynamic Ca2+ activity.
Fig. 2: OPC Ca2+ activity is significantly suppressed during anesthesia.
Fig. 3: Enforced locomotion enhances OPC Ca2+ activity in the mouse visual cortex.
Fig. 4: OPC Ca2+ activity is reduced when noradrenergic signaling is inhibited.
Fig. 5: OPC Ca2+ transients evoked by the α1 adrenergic receptor agonist are independent of synaptic activity in vitro.
Fig. 6: Deletion of Adra1a from OPCs eliminates alpha-adrenoceptor-mediated Ca2+ signaling.
Fig. 7: Spontaneous Ca2+ transients in OPCs progressively decline as they differentiate into myelinating oligodendrocytes.
Fig. 8: Selective deletion of Adra1a from OPCs reduces their proliferation.

Data availability

The gene expression dataset in Extended Data Fig. 10c was deposited to the Gene Expression Omnibus with accession number GSE226635 and is available to the public immediately (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE226635). The mouse genome assembly (GRCm38/mm10) used in this study is available via the UCSC Genome Browser Gateway (https://genome.ucsc.edu/cgi-bin/hgGateway?db=mm10). All images and videos generated during this study are available from the corresponding author upon reasonable request.

Code availability

Code used for data acquisition and analysis in this study is available on GitHub (https://github.com/Bergles-lab/Lu-et-al-NN-2023).

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Acknowledgements

We thank our colleagues for their support. T. Babola, Y. Wang and G. Yu provided helpful suggestions for analyzing OPC Ca2+ activity. C. Call provided assistance in SCoRe microscopy. D. G. Caro, R. Catenacci and M. Smith provided assistance in OPC live-cell imaging. N. Ye and A. E. Bush helped with mouse husbandry. M. Pucak and A. E. Bush provided assistance with daily operation and maintenance of the microscopes essential to this study. We appreciate the generosity of the SciDraw community, especially L. Petrucco (https://doi.org/10.5281/zenodo.3925903, used in Fig. 1a) and E. Tyler and L. Kravitz (https://doi.org/10.5281/zenodo.3925975, used in Fig. 3a). This study was supported by grants from the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the National Institute of Neurological Disorders and Stroke (R01 NS041435) and the National Institute on Aging (R01 AG072305). E.T.H. is supported by the National Science Foundation (2019278189).

Author information

Author notes

  1. Tsai-Yi Lu

    Present address: Department of Neuroscience, University of Virginia, Charlottesville, VA, USA

  2. Amit Agarwal

    Present address: Chica and Heinz Schaller Research Group, Institute for Anatomy and Cell Biology, Heidelberg University, Heidelberg, Germany

Authors and Affiliations

  1. Solomon H. Snyder Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA

    Tsai-Yi Lu, Priyanka Hanumaihgari, Eric T. Hsu, Amit Agarwal, Peter A. Calabresi & Dwight E. Bergles

  2. Department of Psychiatry & Biobehavioral Sciences, Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA, USA

    Riki Kawaguchi

  3. Department of Neurology, Division of Neuroimmunology and Neurological Infections, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Peter A. Calabresi

  4. Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Peter A. Calabresi

  5. Kavli Neuroscience Discovery Institute, Johns Hopkins University, Baltimore, MD, USA

    Dwight E. Bergles

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Contributions

T.-Y.L. and D.E.B. designed the research and experiments. T.-Y.L. performed and analyzed all the experiments. P.H. contributed to the analysis of the longitudinal OPC Ca2+ activity and helped to perform the OPC proliferation and fate-mapping experiments in vivo. E.T.H. constructed the enforced locomotion rig. A.A. designed and generated the Rosa26-lsl-mGCaMP6s and Rosa26-lsl-GCaMP6s transgenic mouse lines. R.K. performed the bulk RNA sequencing analysis. P.A.C. provided instruments for live-cell imaging and feedback to the manuscript. T.-Y.L., P.H., A.A. and D.E.B. co-wrote and edited the manuscript.

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Correspondence to Dwight E. Bergles.

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Extended data

Extended Data Fig. 1 Expressing membrane-anchored GCaMP6s (mGCaMP6s) in OPCs using Rosa26-lsl-mGCaMP6s knockin transgenic mice.

a, The design of the Rosa26-lsl-mGCaMP6s and Rosa26-lsl-GCaMP6s knockin transgenic mice. MARCKS: the N-terminal myristoylation sequence of myristoylated alanine-rich C-kinase substrate. b, Representative confocal images from 3 independent experiments showing the expression of mGCaMP6s (anti-GFP) in the cortical OPCs (anti-NG2) following immunohistochemistry 4 weeks after tamoxifen injection. c, Quantification of b (n = 3 mice). Error bars represent SEM. d, Representative confocal images from 3 independent experiments showing the expression of cytosolic GCaMP6s (anti-GFP) in the cortical OPCs (anti-NG2) following immunohistochemistry 4 weeks after tamoxifen injection. e, Quantification of d (n = 3 mice). Error bars represent s.e.m. f,g, Representative confocal images from 3 independent experiments showing single mGCaMP6s- (f) and GCaMP6s-expressing (g) OPCs with magnified views of their distal processes (yellow arrowheads) in the dotted squares, respectively. Note the lack of cytosolic GCaMP6s expression in the processes.

Extended Data Fig. 2 Basic properties of OPC Ca2+ events detected by mGCaMP6s and GCaMP6s.

a, Dot plots comparing the average (avg.) event frequency (# of events/min), active area, frequency normalized to active area, event area, amplitude, duration (the time between 50% onset time point – 50% offset time point), rise time (onset duration from 10% to 90% of the peak amplitude) and decay time (offset duration from 90% to 10% of the peak amplitude) between mGCaMP6s- and GCaMP6s-expressing OPCs. Each data dot represents an OPC. n = 6 OPCs from 6 mice. Black filled circles and error bars represent mean ± s.e.m. For data passed the Shapiro-Wilk normality test at the 0.05 level, Student’s t-test (two-sided) was further performed. For data that did not pass the Shapiro-Wilk normality test at the 0.05 level, including avg. event area and avg. rise time, Mann-Whitney test (two-sided) was performed instead to determine if the mean values between mGCaMP6s- and GCaMP6s-expressing OPCs are different or not. b, Quantification of the event origins from mGCaMP6s- and GCaMP6s-expressing OPCs (n = 6 OPCs from 6 mice). Student’s t-test, two-sided.

Extended Data Fig. 3 Propagation of OPC Ca2+ transients is independent of site of event origin, event amplitude, and somatic Ca2+ activity.

a, Average percentage of stationary (having an overall propagation score < 10 µm) and propagating (having an overall propagation score ≥ 10 µm) OPC membrane Ca2+ events (n = 6 OPCs from 6 mice). Black filled circles and error bars represent mean ± s.e.m. Student’s t-test, two-sided. b, A plot of the distance between event origin and soma (Origin from soma) versus overall propagation score. R: Pearson’s r. n = 1,100 propagating events from 6 mice. c, Plotting event amplitude against event overall propagation score. R: Pearson’s r. n = 1,100 propagating events from 6 mice. d, Directions of event propagation 10 s before and after the onset of a soma event. Event travelling direction was determined by total voxels travelled away from soma minus total voxels travelled toward soma. n = 911 propagating events from 6 mice. e, ΔF/F traces of the process events (thin gray lines) peaked 10 s before and after soma event onset in mGCaMP6s-expressing and GCaMP6s-expressing OPCs, respectively. Mean ΔF/F (solid blue and brown lines) is the average ΔF/F of 165 events in the mGCaMP6s-expressing mice (6 cells from 6 mice), and 509 events in the GCaMP6s-expressing mice (6 cells from 6 mice). Shuffled mean (dotted purple lines) is the average value after shuffling ΔF/F values of each event. Shaded areas indicate standard deviation.

Extended Data Fig. 4 Activation of visual cortex by visual stimulation with light does not alter OPC Ca2+ events in vivo.

a, Schematic illustration of the experiment setup. A customized 3D-printed objective shield was used to prevent LED light from entering the objective. The bottom part of the objective shield is not depicted in the illustration in order to display the cranial window. See Methods for details. b, Schematic illustration of the experiment design. Baseline OPC Ca2+ activity was recorded for 60 seconds (s) followed by 3 brief LED stimulations 30 s apart that lasted for 0.1 s each. An infrared (IR) camera was on throughout the experiment to observe mouse behaviors during image acquisition. c, Representative heat maps showing the ΔF/F value and duration of OPC Ca2+ events sorted according to the time of event onset. OPC Ca2+ events that occurred during 10 s of quiescence or 10 s after LED stimulation were overlaid onto a single frame (Maximum projection), respectively. d, Averaging the OPC Ca2+ activity during 20 s of quiescence (gray) and around LED stimulation (blue) suggests that LED stimulation does not influence OPC Ca2+ activity in vivo. Shaded areas represent standard deviation. n = 4 mice. e, Quantification of OPC Ca2+ event frequency, area, duration and amplitude during 10 s of quiescence and 10 s post LED stimulation. n = 8 randomly-selected quiescent periods and 10 LED trials in 4 mice (color-coded). Black filled circles and error bars represent mean ± s.e.m. Student’s t-test, two-sided.

Extended Data Fig. 5 Exposure to carrier (DMSO) does not significantly alter OPC Ca2+ activity.

Quantification of OPC Ca2+ event frequency (# of events/min), area, duration, and amplitude before (Baseline) and 20 minutes after DMSO injection (DMSO). Black filled circles and error bars represent mean ± s.e.m. n = 5 OPCs from five mice each. Paired sample Student’s t-test, two-sided.

Extended Data Fig. 6 α1A adrenergic receptors mRNA is enriched in cortical OPCs relative to pre-myelinating oligodendrocytes.

a, Representative confocal images of an adult B6 visual cortex hybridized with probes recognizing the OPC marker, Pdgfra (green) and Adra1a (red) mRNA. DAPI (blue) stains cell nuclei. Adra1a mRNA is found around Pdgfra+ nuclei, suggesting that cortical OPCs express ADRA1A (n = 19 cells, 3 mice). b, Representative confocal images of an adult B6 visual cortex hybridized with probes recognizing the pre-myelinating oligodendrocyte marker, lncOL1 (green), and Adra1a (red) mRNA. DAPI stains cell nuclei (n = 6 cells, 2 mice). c, Quantification of a and b. Black filled circles and error bars represent mean ± s.e.m.

Extended Data Fig. 7 An example of an mGCaMP6s-expressing OPC undergoing cell death.

The mGCaMP6s-expressing OPC is highlighted in yellow. Note the round-shaped and intensely bright cell body (red arrowhead) as well as the fragmented processes on Day 9. We could not identify any Ca2+ events in the fragmented processes. Representative data from 2 independent experiments.

Extended Data Fig. 8 Local myelin profiles do not change around stable OPCs.

The mGCaMP6s-expressing OPC (highlighted in green) was followed for 16 days and local myelin profile was recorded by SCoRE microscopy concurrently. Local myelin profile remained unchanged from Day 0 (green) to Day 16 (magenta). Representative data from 4 independent experiments that yielded similar results.

Extended Data Fig. 9 Myelinating oligodendrocytes exhibit Ca2+ events in only a select few myelin sheaths.

a, Local calcium events detected (randomly pseudocolored by AQuA) in the same imaging plane where the traced OPC (Fig. 7f, highlighted in blue) became undetectable on Day 0. We did not observe persistent or enhanced Ca2+ events that can be attributed to the pre-myelinating OPC during this stage of maturation. Representative data from 4 independent experiments that yielded similar results. b, Representative confocal images from 3 independent experiments showing the expression of mGCaMP6s (anti-GFP) in the cortical myelinating oligodendrocytes (anti-MBP) using oligodendrocyte-specific and tamoxifen-inducible Cre transgenic line, Mobp-iCreER. c, The magnified views of the dotted squares in b. d, Schematic illustrations of the research design. The expression of mGCaMP6s in myelinating oligodendrocytes was induced between P60–80. Oligodendrocyte Ca2+ activity in the visual cortex of head-fixed, awake mice was observed and recorded using the same condition as the recording of OPC Ca2+ activity (see Fig. 1). e, Representative images showing the Ca2+ activity detected using 2P microscopy (Sum of Ca2+ activity from a 6-minute recording) corresponds to local myelin sheath detected using SCoRe. Cyan arrowhead indicates auto-fluorescent vascular structures. f, Ca2+ events detected in e. g, Distribution of the Ca2+ event numbers detected in myelin sheaths within 6 minutes (n = total 215 sheaths from 3 mice). Note that about 85% of the myelin sheath did not generate any Ca2+ event during the recording. h, Example ΔF/F traces of oligodendrocyte membrane Ca2+ events in f. i, Quantification of average Ca2+ event frequency, size, duration, and amplitude (n = 3 mice). Black filled circles and error bars represent mean ± s.e.m.

Extended Data Fig. 10 PE promotes OPC proliferation in vitro.

a, Gene ontology (GO) terms that were significantly up-regulated (adjusted p value < 0.05) in primary OPCs after 1 hour of PE treatment (20 µM, n = 3 independent biological repeats, differential gene analysis by EdgeR 3.15 with k = 2, adjusted for multiple comparisons). Numbers in the bars indicate the number of genes that were significantly up-regulated after PE treatment within each GO term. If more than 5 GO terms were significantly enriched within the subontology (BP: Biological process; MF: Molecular function. CC: Cellular component), only the top 5 GO terms were shown. b, GO terms that were significantly down-regulated (adjusted p value < 0.05) in a. c, Volcano plot showing differential gene expression in OPCs treated with PE for 1 hour compared to control (no treatment). FDR: false discovery rate. FC: fold change. Total variables: 19,820. d, Representative live cell tracking of primary cultured OPCs for 24 hours after PE treatment (+ PE) and without treatment (Ctrl). OPCs that did not proliferate within 24 hours were labeled in blue. OPCs that proliferated at least once within 24 hours were labeled in red. e, Quantification of d. n = 3 independent biological repeats. Black filled circles and error bars represent mean ± s.e.m. Student’s t-test, two-sided. f, The experimental design of OPC differentiation assay, and representative confocal images of the OPC/oligodendrocytes mixed cultures 2 days after PDGF-AA withdrawal (Day 4) in the absence (Ctrl) or with the presence of PE (+PE). Green arrow indicates an example of fully differentiated oligodendrocytes with strong MBP expression (MBPS). White arrow indicates an example of differentiating OPCs that have weak expression of both NG2 and MBP (NG2WMBPW). Magenta arrow indicates an example of OPCs that remain undifferentiated with strong NG2 expression. g, Quantification of f. n = 3 independent biological repeats. Black filled circles and error bars represent mean ± s.e.m. Student’s t-test, two-sided.

Supplementary information

Supplementary Information

Supplementary Fig. 1, Supplementary Video Legend 1, Supplementary Video Legend 2, Supplementary Video Legend 3, Supplementary Video Legend 4 and Supplementary Video Legend 5

Reporting Summary

Supplementary Video 1

Supplementary Video 1. Cytosolic OPC Ca2+ activity in the visual cortex in vivo. Left, OPC Ca2+ activity detected by 2P microscopy; right, output video from AQuA with randomly pseudo-colored Ca2+ events (5× speed).

Supplementary Video 2

Supplementary Video 2. Membrane OPC Ca2+ activity in the visual cortex in vivo. Left, OPC Ca2+ activity detected by 2P microscopy; right, output video from AQuA with randomly pseudo-colored Ca2+ events (5× speed).

Supplementary Video 3

Supplementary Video 3. Enforced locomotion stimulates OPC Ca2+ activity in the mouse visual cortex. The red dot indicates when the platter began to rotate (movie played at 5× speed).

Supplementary Video 4

Supplementary Video 4. PE evokes Ca2+ influx in OPCs in acute cortical slices. PE was superfused ~3 min after the recording begins (movie shown at 50× speed).

Supplementary Video 5

Supplementary Video 5. Myelinating oligodendrocytes exhibit infrequent Ca2+ activity in the visual cortex in vivo. Left, oligodendrocyte near membrane Ca2+ activity detected by 2P microscopy; right, output video from AQuA with randomly pseudo-colored Ca2+ events (5× speed).

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Lu, TY., Hanumaihgari, P., Hsu, E.T. et al. Norepinephrine modulates calcium dynamics in cortical oligodendrocyte precursor cells promoting proliferation during arousal in mice. Nat Neurosci 26, 1739–1750 (2023). https://doi.org/10.1038/s41593-023-01426-0

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