Abstract

Neurogenesis in the adult dentate gyrus occurs in the subgranular zone where newborn neurons (NNs) migrate a short distance into the granule cell layer and extend their rudimentary apical dendritic processes upon a radial glial scaffold. Using doublecortin (DCX) immunocytochemistry, these growing dendrites can be visualized because dendritic growth cones, including filipodia and lamellipodia, are labeled in both light and electron microscopic preparations. To study the rate of dendritic outgrowth of newborn dentate granule cells, single injections of 5-bromo-2-deoxyuridine (BrdU) with different survival times were combined with double immunolabeling for BrdU and DCX. At the earliest time points (4 and 12 h after BrdU injections), a rudimentary process can be observed to emanate from BrdU/DCX double-labeled cells. By 48 h the dendrites first appeared in the molecular layer. By 96 h after BrdU injection, these apical dendrites extended into the middle of the molecular layer where they ramified. The calculated rate of dendritic growth for NNs was about 15 μm per day for the first 3 days, and then a doubling in length occurred at 4 and 5 days that coincided with a retraction of the basal dendrite. In addition, electron microscopy of DCX-labeled apical dendrites showed that they were much thinner (1/4 to 1/3 the size) in diameter than unlabeled, mature apical dendrites and that they had developing synapses on them in the molecular layer.

Highlights

  • The subgranular zone of the dentate gyrus is one of two neurogenic zones in the adult mammalian brain (Cameron and McKay, 2001; Alvarez-Buylla and Lim, 2004)

  • At 4 h after a BrdU injection, BrdU/DCX double-labeled cells were found in the subgranular zone and at the base of the granule cell layer (Fig. 1A)

  • The spatiotemporal profile of dendritic outgrowth for NNs in the adult dentate gyrus was examined in this study using double-labeling immunocytochemistry for BrdU and DCX following single BrdU injections

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Summary

Introduction

The subgranular zone of the dentate gyrus is one of two neurogenic zones in the adult mammalian brain (Cameron and McKay, 2001; Alvarez-Buylla and Lim, 2004) At this site, radial glial cells are the progenitor cells (Seri et al, 2001, 2004; Garcia et al, 2004), and non-radial processes of the radial glial cells cradle the neurons born in the subgranular zone (Shapiro et al, 2005a). Confocal microscopy was used to examine double-immunolabeled preparations for both BrdU and DCX Using this double-labeling method, the mean lengths of the dendrites emanating from BrdU/DCX double-labeled cells were measured to determine the in vivo rate of dendritic process outgrowth for NNs in the adult dentate gyrus of rats. Electron microscopy was used to examine the fine structural features of the DCX-labeled apical dendrites in the molecular layer

Results
Statistical analysis of apical dendritic growth
Electron microscopic features of DCX-labeled apical dendrites
Discussion
Rate of dendritic growth compared between adult and development
Implications of synaptogenesis on DCX-labeled dendrites
Perfusions and sectioning
Immunocytochemistry
Electron microscopy
Dendritic outgrowth analysis
Full Text
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