Oligodendrocyte progenitor cells are paired with GABA neurons in the mouse dorsal cortex: Unbiased stereological analysis.

Oligodendrocyte progenitor cells (OPC) are glial cells that differentiate into myelinating oligodendrocytes during early stages of post-natal life. However, OPCs persist beyond developmental myelination and represent an important population of cycling cells in the gray and white matter of the adult brain. While adult OPCs form unique territories that are maintained through self-avoidance, some cortical OPCs appear to position their cell body very close to that of a neuron, forming what are known as OPC-neuron pairs. We used unbiased systematic stereological analysis of the NG2-CreERTM:EYFP reporter mouse to determine that close to 170,000 OPC-neuron pairs can be found in the dorsal portion of the adult neocortex, with approximately 40% of OPCs and 4% of neurons in pairs. Through stereological analysis, we also determined that reference memory training does not change the prevalence of OPC-neuron pairs or the proportion of OPCs and neurons that form them. GABAergic agent administration did not affect the proportion of OPCs and neurons that can be found in pairs. However, the GABAB-receptor agonist baclofen and the GABAA receptor antagonist picrotoxin significantly increased the estimated number of pairs when compared to the control group and the GABAB-receptor antagonist (i.e. saclofen) group. Density of OPC-neuron pairs was increased by the GABAA receptor antagonist picrotoxin. Finally, histological analysis of OPC-neuron pairs suggested that in the dorsal portion of the cortex, GABAergic interneurons represent the most common neuronal component of the pairs, and that calbindin, calretinin and parvalbumin GABAergic interneurons found in the cortex take part in these pairs. Using previous estimates of the number of GABAergic neurons in the rodent cortex, we estimate that roughly one in four GABAergic neurons are paired with an OPC.

Doublecortin in Oligodendrocyte Precursor Cells in the Adult Mouse Brain.

Key Points Oligodendrocyte precursor cells express doublecortin, a microtubule-associated protein.Oligodendrocyte precursor cells express doublecortin, but at a lower level of expression than in neuronal precursor.Doublecortin is not associated with a potential immature neuronal phenotype in Oligodendrocyte precursor cells. Oligodendrocyte precursor cells (OPC) are glial cells that differentiate into myelinating oligodendrocytes during embryogenesis and early stages of post-natal life. OPCs continue to divide throughout adulthood and some eventually differentiate into oligodendrocytes in response to demyelinating lesions. There is growing evidence that OPCs are also involved in activity-driven de novo myelination of previously unmyelinated axons and myelin remodeling in adulthood. Considering these roles in the adult brain, OPCs are likely mobile cells that can migrate on some distances before they differentiate into myelinating oligodendrocytes. A number of studies have noted that OPCs express doublecortin (DCX), a microtubule-associated protein expressed in neural precursor cells and in migrating immature neurons. Here we describe the distribution of DCX in OPCs. We found that almost all OPCs express DCX, but the level of expression appears to be much lower than what is found in neural precursor. We found that DCX is downregulated when OPCs start expressing mature oligodendrocyte markers and is absent in myelinating oligodendrocytes. DCX does not appear to signal an immature neuronal phenotype in OPCs in the adult mouse brain. Rather, it could be involved either in cell migration, or as a marker of an immature oligodendroglial cell phenotype.

Unbiased stereological analysis of the fate of oligodendrocyte progenitor cells in the adult mouse brain and effect of reference memory training.

Oligodendrocyte progenitor cells (OPCs) are glial cells that differentiate into myelinating oligodendrocytes during early stages of post-natal life. However, OPCs persist beyond developmental myelination and represent an important population of cycling cells in the gray and white matter of the adult brain. Here, we used unbiased systematic stereological analysis to determine the total number of OPCs in the neocortex and corpus callosum of the adult mouse. We found that the ratio of OPCs to neurons is of 1:10 in the adult neocortex. Likewise, the ratio of OPCs to oligodendrocytes is of 1:1 in the cortex and 1:7 in the corpus callosum. We also used BrdU labeling and the NG2-CreER™:EYFP reporter mouse to determine the proportion of proliferating adult OPCs and their fate. We show that OPCs continue to differentiate into oligodendrocytes in adulthood, with white matter OPCs being more likely to differentiate into an oligodendrocyte phenotype than gray matter OPCs. The differentiation of OPCs into an oligodendrocyte phenotype can occur either directly from a spontaneous differentiation by an OPC or following OPC cell division. We also provide evidence for the neuronal differentiation of adult OPCs in the cortical gray matter. Although activity-dependent neural network activity has been hypothesized to serve as a modulator of OPC proliferation and differentiation, we found that reference memory training did not affect the proportion of proliferating and differentiated OPCs in the adult mouse brain.

A simple histological technique to improve immunostaining when using DNA denaturation for BrdU labelling.

The typical immunohistochemistry technique used to reveal 5-bromo-2'-deoxyuridine (BrdU) incorporation requires denaturation of the DNA by heat and acid to permeabilize the cell nucleus. This treatment can damage tissue and reduce the antigenicity of several proteins, which then leads to weak immunostaining and/or false negatives. We show that an overnight post-fixation step following immunohistochemistry for antigens of interest protects immunostaining during the acid/heat denaturation treatment for subsequent BrdU staining. We used this technique to study the differentiation of recently divided oligodendrocyte progenitor cells in NG2CreER:EYFP reporter mice. We used a GFP anti-EYFP antibody to maximize visualization of the EYFP-containing oligodendrocyte progenitor cells, Olig1, and GST-pi to confirm the cell phenotype. Immunostaining for GFP, Olig1, and GST-pi is reduced by DNA denaturation. We found that incorporating a post-fixation step after double immunostaining for GFP/Olig1 and GFP/GST-pi prior to DNA denaturation prevented the fading and false negatives associated with this treatment. This simple addition to BrdU immunohistochemistry protocols extends the range of proteins that can be detected in combination with BrdU, along with the number of antibodies that can be used successfully in the study of cell proliferation.