Abstract
Current advances in imaging techniques have extended the possibility of visualizing small structures within large volumes of both fixed and live specimens without sectioning. These techniques have contributed valuable information to study neuronal plasticity in the adult brain. However, technical limits still hamper the use of these approaches to investigate neurogenic regions located far from the ventricular surface such as parenchymal neurogenic niches, or the scattered neuroblasts induced by brain lesions. Here, we present a method to combine confocal laser scanning microscopy (CLSM) and serial section reconstruction in order to reconstruct large volumes of brain tissue at cellular resolution. In this method a series of thick sections are imaged with CLSM and the resulting stacks of images are registered and 3D reconstructed. This approach is based on existing freeware software and can be performed on ordinary laboratory personal computers. By using this technique we have investigated the morphology and spatial organization of a group of doublecortin (DCX)+ neuroblasts located in the lateral striatum of the late post-natal guinea pig. The 3D study unraveled a complex network of long and poorly ramified cell processes, often fascicled and mostly oriented along the internal capsule fiber bundles. These data support CLSM serial section reconstruction as a reliable alternative to the whole mount approaches to analyze cyto-architectural features of adult germinative niches.
Highlights
Generated neuronal cells are constantly added to the mammalian olfactory bulb (OB) and dentate gyrus (DG) during adult life (Kriegstein and Alvarez-Buylla, 2009)
The cell processes of the individual elements were often fascicled and closely associated with the internal capsule fiber bundles. This neurogenic system, which will be described in detail in a separate study (Luzzati et al, unpublished results), offers a good opportunity to show how the method of confocal laser scanning microscopy (CLSM) serial section reconstruction we developed is suitable to investigate the distribution, organization, and morphological features of immunolabeled newborn neurons located deep within the brain parenchyma
Newborn neuronal cells have been described in the mature striatum of different mammalian species, raising hopes to exploit this neurogenic activity for brain repair (Arvidsson et al, 2002; Bedard et al, 2002; Luzzati et al, 2006, 2007)
Summary
Generated neuronal cells are constantly added to the mammalian olfactory bulb (OB) and dentate gyrus (DG) during adult life (Kriegstein and Alvarez-Buylla, 2009). Recent advances in light microscopy and in molecular and genetic manipulations have greatly extended the possibility of imaging large volumes of both fixed and live neural tissue at cellular resolution, enabling the visualization of complex 3D objects such as neuronal or vascular networks (Mizrahi, 2007; Lu et al, 2009; Tsai et al, 2009; Wilt et al, 2009; Khairy and Keller, 2011). The whole mount preparation is less suitable to study neurogenic regions located far from the ventricular surface such as parenchymal neurogenic niches (Luzzati et al, 2006, 2007), or scattered neuroblasts induced by a brain lesion (Arvidsson et al, 2002; Parent et al, 2002) In these latter cases, reconstruction from serial sections represents a reliable alternative. The obtained results support the CLSM serial section reconstruction method as a useful tool to analyze neuroanatomical features of adult germinative niches
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