Organotypic Slice Cultures to Study Oligodendrocyte Proliferation, Fate, and Myelination

doi:10.1007/978-1-4939-7862-5_11

. 2018:1791:145-156.

doi: 10.1007/978-1-4939-7862-5_11.

Organotypic Slice Cultures to Study Oligodendrocyte Proliferation, Fate, and Myelination

Amin Sherafat¹, Robert A Hill^{2

1

3}, Akiko Nishiyama^{4

5

6}

Affiliations

¹ Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA.
² Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.
³ Department of Neurology, Yale School of Medicine, New Haven, CT, USA.
⁴ Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA. akiko.nishiyama@uconn.edu.
⁵ Institute of Systems Genomics, University of Connecticut, Storrs, CT, USA. akiko.nishiyama@uconn.edu.
⁶ Institute of Brain and Cognitive Science, University of Connecticut, Storrs, CT, USA. akiko.nishiyama@uconn.edu.

PMID: 30006707
PMCID: PMC6372239
DOI: 10.1007/978-1-4939-7862-5_11

Organotypic Slice Cultures to Study Oligodendrocyte Proliferation, Fate, and Myelination

Amin Sherafat et al. Methods Mol Biol. 2018.

. 2018:1791:145-156.

doi: 10.1007/978-1-4939-7862-5_11.

Authors

Amin Sherafat¹, Robert A Hill^{2

1

3}, Akiko Nishiyama^{4

5

6}

Affiliations

¹ Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA.
² Department of Biological Sciences, Dartmouth College, Hanover, NH, USA.
³ Department of Neurology, Yale School of Medicine, New Haven, CT, USA.
⁴ Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA. akiko.nishiyama@uconn.edu.
⁵ Institute of Systems Genomics, University of Connecticut, Storrs, CT, USA. akiko.nishiyama@uconn.edu.
⁶ Institute of Brain and Cognitive Science, University of Connecticut, Storrs, CT, USA. akiko.nishiyama@uconn.edu.

PMID: 30006707
PMCID: PMC6372239
DOI: 10.1007/978-1-4939-7862-5_11

Abstract

Oligodendrocyte development and myelination are processes in the central nervous system that are regulated by cell intrinsic and extrinsic mechanisms. Organotypic slice cultures provide a simple method for studying factors that affect oligodendrocyte proliferation, differentiation, and myelination in the context of the local cellular environment. Here we show that major glial cell types and neurons are preserved in slice cultures from postnatal mouse forebrain, and their morphological characteristics are retained. We further demonstrate that cellular processes requiring interactions with neighboring cells such as myelination can proceed in slice culture.

Keywords: Myelin; NG2; Oligodendrocyte; Oligodendrocyte precursor; Organotypic slice culture; PDGF; Pdgfrα; Proliferation.

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Figures

**Fig. 1**
Setup for making organotypic slice cultures from P8 mouse brain. (**a–e**) Tools. (f) Tissue chopper. (g, h) Treatment of tools with 70% ethanol. (i) Bubbling dissection buffer with 90% O₂/5% CO₂ gas mixture and chilling it on ice. (j) Cutting the dissected brain into two hemispheres. (k) Preparation of the stage for slicing using Whatman paper to hold the brain in place. (l) Picking up sliced hemisphere with a spatula. (m, n) Sliding a slice onto the membrane of a Millicell insert. O: Removing extra dissection buffer from around the slice using a transfer pipet

**Fig. 2**
CNS structures and cell types identified in slice cultures from P8 NG2cre:zeg mice after 7 DIV. (a) A schematic showing the culture strategy. (b) Low-magnification image of a coronal anterior forebrain slice depicting GFP⁺ cells and MBP immunostaining in both cortex (ctx; gray matter) and corpus callosum (cc; white matter) in forebrain cultures by 7 DIV. Scale bar, 100 μm. (c, c′) High-magnification images taken from the boxed region of the white matter in (b) showing GFP⁺ cells and their processes extending into parallel MBP+ myelin sheaths. (c′) represents area shown in box in (c). Since myelination in the mouse corpus callosum does not begin until P11 [26], this likely represents myelination that occurred during slice culture. Scale bars, 20 μm. (d, d′) GFP⁺ PDGFRα+ NG2 cells (arrowheads) and GFP⁺ PDGFRα-negative cells, which are presumably oligodendrocytes (arrows). Scale bars, 20 μm. (e, e′) GFP⁺ oligodendrocyte lineage cells in the cortex are distinct from GFAP+ astrocytes that exhibit the typical stellate morphology. (f, f′) GFP+ oligodendrocyte lineage cells are distinct from Iba1+ microglial cells but the two cell types are closely associated with each other. (g, g′) NeuN+ neurons in the cortex. GFP⁺ oligodendrocyte lineage cells are often found in satellite position to neuronal cell bodies. (h, h′) Slice cultures from Olig2flfl:NG2cre:zeg mice showing GFAP+ bushy protoplasmic astrocytes (arrows) that are GFP+ and hence are the progeny of NG2 cells. Arrowheads in (h) and (h′) denote cells with polydendrocyte morphology that are likely to be NG2 cells. Scale, 20 μm

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References

1. Dawson MR, Polito A, Levine JM, Reynolds R (2003) NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS. Mol Cell Neurosci 24(2):476–488 - PubMed
1. Hill RA, Nishiyama A (2014) NG2 cells (poly-dendrocytes): listeners to the neural network with diverse properties. Glia 62(8):1195–1210. 10.1002/glia.22664 - DOI - PMC - PubMed
1. Nishiyama A (2007) Polydendrocytes: NG2 cells with many roles in development and repair of the CNS. Neuroscientist 13(1):62–76. 10.1177/1073858406295586 - DOI - PubMed
1. Nishiyama A, Komitova M, Suzuki R, Zhu X (2009) Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity. Nat Rev Neurosci 10(1):9–22 - PubMed
1. Dimou L, Simon C, Kirchhoff F, Takebayashi H, Gotz M (2008) Progeny of Olig2-expressing progenitors in the gray and white matter of the adult mouse cerebral cortex. J Neurosci 28(41):10434–10442 - PMC - PubMed

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[1] Dawson MR, Polito A, Levine JM, Reynolds R (2003) NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS. Mol Cell Neurosci 24(2):476–488 - PubMed

[2] Dawson MR, Polito A, Levine JM, Reynolds R (2003) NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS. Mol Cell Neurosci 24(2):476–488 - PubMed

[3] Hill RA, Nishiyama A (2014) NG2 cells (poly-dendrocytes): listeners to the neural network with diverse properties. Glia 62(8):1195–1210. 10.1002/glia.22664 - DOI - PMC - PubMed

[4] Hill RA, Nishiyama A (2014) NG2 cells (poly-dendrocytes): listeners to the neural network with diverse properties. Glia 62(8):1195–1210. 10.1002/glia.22664 - DOI - PMC - PubMed

[5] Nishiyama A (2007) Polydendrocytes: NG2 cells with many roles in development and repair of the CNS. Neuroscientist 13(1):62–76. 10.1177/1073858406295586 - DOI - PubMed

[6] Nishiyama A (2007) Polydendrocytes: NG2 cells with many roles in development and repair of the CNS. Neuroscientist 13(1):62–76. 10.1177/1073858406295586 - DOI - PubMed

[7] Nishiyama A, Komitova M, Suzuki R, Zhu X (2009) Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity. Nat Rev Neurosci 10(1):9–22 - PubMed

[8] Nishiyama A, Komitova M, Suzuki R, Zhu X (2009) Polydendrocytes (NG2 cells): multifunctional cells with lineage plasticity. Nat Rev Neurosci 10(1):9–22 - PubMed

[9] Dimou L, Simon C, Kirchhoff F, Takebayashi H, Gotz M (2008) Progeny of Olig2-expressing progenitors in the gray and white matter of the adult mouse cerebral cortex. J Neurosci 28(41):10434–10442 - PMC - PubMed

[10] Dimou L, Simon C, Kirchhoff F, Takebayashi H, Gotz M (2008) Progeny of Olig2-expressing progenitors in the gray and white matter of the adult mouse cerebral cortex. J Neurosci 28(41):10434–10442 - PMC - PubMed

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Organotypic Slice Cultures to Study Oligodendrocyte Proliferation, Fate, and Myelination

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Organotypic Slice Cultures to Study Oligodendrocyte Proliferation, Fate, and Myelination

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