Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Thétiot, Melina; Ronzano, Remi; Aigrot, Marie‐Stéphane; Lubetzki, Catherine; Desmazières, Anne

doi:10.3791/59163

Cited by 8 publications

(13 citation statements)

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“…To study microglia-node interaction with an increased temporal and spatial resolution, we used the organotypic culture of mouse cerebellar slice model, which present a preserved cytoarchitecure and is highly accessible for live-imaging and pharmacological treatments 36,37 . We first showed, using CX3CR1 GFP/+ cerebellar fixed tissue, that microglia also contact nodes of Ranvier during developmental myelination, both in vivo and ex vivo (Figure S2A and S2B, respectively).…”

Section: Resultsmentioning

confidence: 99%

“…Ex vivo culture protocol was adapted from 36,37 Briefly, P8 to P10 mouse cerebella were dissected in ice cold Gey’s balanced salt solution complemented with 4.5 mg/ml D-Glucose and penicillin-streptomycin (100 IU/mL, Thermo Fisher Scientific). They were cut into 250μm parasagittal slices using a McIlwain tissue chopper and the slices placed on Millicell membrane (3-4 slices per membrane, 2 membranes per animal, 0.4 μm Millicell, Merck Millipore) in 50% BME (Thermo Fisher Scientific), 25% Earle’s Balanced Salt Solution (Sigma), 25% heat-inactivated horse serum (Thermo Fisher Scientific), supplemented with GlutaMax (2 mM, Thermo Fisher Scientific), penicillin-streptomycin (100 IU/mL, Thermo Fisher Scientific) and D-Glucose (4.5 mg/ml; Sigma).…”

Section: Methodsmentioning

confidence: 99%

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Microglia-neuron communication at nodes of Ranvier depends on neuronal activity through potassium release and contributes to myelin repair

Ronzano

Roux

Thétiot

et al. 2020

Preprint

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Microglia, the resident immune cells of the central nervous system, are key players in healthy brain homeostasis and plasticity. In neurological diseases, such as Multiple Sclerosis, activated microglia either promote tissue damage or favor neuroprotection and myelin regeneration. The mechanisms for microglia-neuron communication remain largely unkown. Here, we identify nodes of Ranvier as a direct and stable site of interaction between microglia and axons, in both mouse and human tissue. Using dynamic imaging, we highlight the preferential interaction of microglial processes with nodes of Ranvier along myelinated fibers. We show that microglianode interaction is modulated by neuronal activity and associated potassium release, with THIK-1 ensuring their microglial read-out. Disrupting axonal K+ flux following demyelination polarizes microglia towards a pro-inflammatory phenotype and decreases remyelination rate. Taken together, these findings identify the node of Ranvier as a major site for microglia-neuron communication, participating in the pro-remyelinating effect of microglia after myelin injury.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Microglia-neuron communication at nodes of Ranvier depends on neuronal activity through potassium release and contributes to myelin repair

Ronzano

Roux

Thétiot

et al. 2020

Preprint

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“…6-well plates with 0.4 µM, 30-mm diameter cell culture inserts, and 1 ml of culture media (50% MEM 10× no glutamine and with Earle’s salts, 5% BME with Earle’s salts, 25% horse serum, 1× GlutaMAX 100× [Gibco], 0.648% glucose 45%, 0.5% Penicillin/Streptomycin, 7.5% NaHCO 3 to adjust the pH to ∼7.2, and autoclaved deionized water) were previously prepared and put in the incubator (at 37°C, 5% CO 2 ) for at least 2 h before placing the slices, for acclimatization of the insert membranes and oxygenation of the media. The medium was changed 1 d after the culture was prepared and then every 3 d. Demyelination of the slices was induced as previously described ( Thetiot et al, 2019 ). Briefly, at 7 DIV the inserts containing the cerebellar slices were placed in fresh medium containing 0.5 mg/ml LPC (L-alfa-lysophosphatidylcholine from egg yolk) in PBS for 16 h in the incubator.…”

Section: Methodsmentioning

confidence: 99%

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis

Cunha

Cantuti-Castelvetri

et al. 2020

Journal of Experimental Medicine

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Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB–dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes.

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“…Primary cultures are useful models that allow the assessment of oligodendroglial lineage cell differentiation and interactions with neurons. However, other approaches have also been developed to evaluate OL functions and myelination, demyelination and remyelination from ex-vivo cerebellar organotypic slice cultures 25,26 , and in vivo studies, notably with zebrafish and tadpole models 27 which are needed in final steps of pre-clinical studies.…”

Section: Discussionmentioning

confidence: 99%

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

Mazuir

Dubessy

Wallon

et al. 2020

JoVE

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HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. L'archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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Preparation and Immunostaining of Myelinating Organotypic Cerebellar Slice Cultures

Cited by 8 publications

References 0 publications

Microglia-neuron communication at nodes of Ranvier depends on neuronal activity through potassium release and contributes to myelin repair

Microglia-neuron communication at nodes of Ranvier depends on neuronal activity through potassium release and contributes to myelin repair

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis

Generation of Oligodendrocytes and Oligodendrocyte-Conditioned Medium for Co-Culture Experiments

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