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Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes

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Abstract

Based on the expression of glial fibrillary acidic protein (GFAP), a recent hypothesis considered stem or progenitor cells in the adult hippocampus to be a type of astrocyte. In a complementary approach, we used transgenic mice expressing green fluorescent protein (GFP) under the promoter for nestin, an intermediate filament present in progenitor cells, to demonstrate astrocytic features in nestin-GFP-positive cells. Morphologically, two subpopulations of nestin-GFP-positive cells were distinguishable; one had an elaborate tree of processes in the granule cell layer and expression of GFAP (but not of S100β, another astrocytic marker). Electron microscopy revealed vascular end feet of nestin-positive cells, further supporting astrocytic differentiation. Electrophysiological examination of nestin-GFP-positive cells on acutely isolated hippocampal slices showed passive current characteristics of astrocytes in one subset of cells. Among the nestin-GFP-expressing cells with lacking astrocytic features, two cell types could be identified electrophysiologically: cells with delayed-rectifying potassium currents and a very small number of cells with sodium currents, potentially representing signs of the earliest steps of neuronal differentiation.

Introduction

In the adult hippocampus, new neurons are constantly generated from a resident population of stem or progenitor cells, located in the subgranular zone (SGZ) at the border between the hilus and the granule cell layer (GCL) of the dentate gyrus. Ex vivo experiments have shown that these cells have stem cell properties (Palmer et al., 1997), although there is some disagreement as to which extent (Seaberg and van der Kooy, 2002). Despite numerous efforts, however, it has not yet been possible to find a specific marker that would allow the identification of neuronal stem cells in vivo (Geschwind et al., 2001). In vitro studies have demonstrated that in cells derived from the brain the intermediate filament nestin is expressed only in cells with progenitor cell qualities (Lendahl et al., 1990). The application of this insight for in vivo studies, however, has been restricted by the fact that all available antibodies against nestin have a cross-reaction with an endothelial antigen (Palmer et al., 2000). A transgenic mouse, expressing enhanced green fluorescent protein (GFP) under the nestin gene regulatory region, allowed us to bypass this problem (Yamaguchi et al., 2000). The use of the enhancer region of the nestin gene restricts reporter gene expression to neural progenitor cells (Zimmerman et al., 1994). Based on this neurally specific expression of nestin-GFP, neural progenitor cells have been successfully isolated from the same mouse line that has also been used in our experiment Sawamoto et al 2001a, Sawamoto et al 2001b. Stem cell properties of such cells have been demonstrated (Kawaguchi et al., 2001). In the context of the present study and based on these data, a stem or progenitor cell is therefore operationally defined as a nestin-GFP-expressing cell. Generally, the defining key criteria for stem cells are the ability for (unlimited) self-renewal and multipotency (Gage, 2000). A progenitor cell is a multi- or unipotent cell with the ability for limited self-renewal. As both stem and progenitor cells can be nestin expression in vitro, we use the neutral term “stem or progenitor cells” to indicate that the ultimate distinction between the two types of cells cannot be made under in vivo conditions such as in our study.

Histological analysis of brain tissue from nestin-GFP mice showed large numbers of putative stem or progenitor cells in the neurogenic and nonneurogenic regions of the brain (Yamaguchi et al., 2000), generally consistent with what would be expected from ex vivo experiments Kawaguchi et al 2001, Palmer et al 2000, Sawamoto et al 2001b.

Regarding the subventricular zone of the lateral ventricles, the second neurogenic region of the adult brain besides the hippocampus, Frisén and coworkers favored the hypothesis that ependymal cells are the stem cells of the subventricular zone (Johansson et al., 1999); Alvarez-Buylla and colleagues, in contrast, see the stem cells in astrocytes of the subependymal layer (Doetsch et al., 1999), a hypothesis supported by another study by Laywell et al. (2000). Seri et al. (2001) provided evidence for a similar identity of the putative stem or progenitor cells in the dentate gyrus with astrocytes. In both cases the cells that presumably represented progenitor cells were identified by their proliferative activity as detected by their incorporation of tritiated thymidine or thymidine analog bromodeoxyuridine (BrdU). Astrocytes were identified by their expression of glial fibrillary acidic protein (GFAP) and morphological criteria. In general, however, astrocytes are an extremely heterogeneous population of cells. GFAP expression is often used only as a minimum criterion. Therefore, one fundamental question remained open: are the putative stem or progenitor cells of the dentate gyrus truly astrocytes beyond the expression of GFAP?

The nestin-GFP-expressing mice provide a unique tool for examining putative stem or progenitor cells in the adult brain, independent of their momentary proliferative activity and of pitfalls due to antibody specificity. We used these mice to further test the hypothesis by Alvarez-Buylla and colleagues (2001) that astrocytes are the stem cells of the adult brain. Our own hypothesis was that the nestin-expressing population of putative stem or progenitor cells in the adult dentate gyrus might have astrocytic features, but also distinguishing characteristics. We used immunohistochemistry, electron microscopy, and electrophysiological techniques to investigate astrocytic features in nestin-GFP-positive cells.

Our data provide arguments in support of the view that progenitor cells in the adult hippocampus share astrocytic features, but they also show that there is no simple identity between astrocytes and progenitor cells.

Section snippets

Nestin-GFP-expressing cells of the dentate gyrus have two distinct morphologies

By morphological characteristics, especially the absence or presence of processes, nestin-GFP-expressing cells fell into two large categories. One cell population (type-1) was characterized by a long process (Fig. 1). The cell body of type-1 cells was generally located in the SGZ and the process reaching through the granule cell layer and into the molecular layer.

A second cell type (type-2) lacked long processes and had a round or ovoid nucleus and soma with scant cytoplasm. Compared to the

Discussion

One criterion to identify progenitor cells from the adult murine SGZ in vitro is their expression of intermediate filament nestin (Lendahl et al., 1990). We here show that in vivo nestin-GFP-expressing cells are not a homogenous population. Strictly speaking, we identified cells in which GFP was generated under the activity of the regulatory gene region of the nestin gene that allows expression specific to neural tissue and in which GFP was still present at the time point of examination. This

Animals

The generation of mice expressing enhanced GFP under the control of nestin gene regulatory regions has been described previously Sawamoto et al 2001a, Yamaguchi et al 2000. The mice were bred at the animal facility at the MDC. All applicable federal and local regulations of animal welfare were followed. The mice lived in standard laboratory housing conditions with a light/dark cycle of 12 h each and food and water ad libitum.

Immunohistochemistry

For immunohistochemistry, five 12-week-old animals received a single

Acknowledgements

We thank Bettina Erdmann and Christiane Nolte for help with the electron micrographs and Ruth Segner and Irene Thun for technical support. This work was funded by grants from VolkswagenStiftung and Deutsche Forschungsgemeinschaft (DFG).

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    These authors contributed equally to this study.

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