Abstract
(Englisch)
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It is established that neurogenesis occurs in discrete regions of the mammalian central nervous system throughout adulthood. Neurogenesis is particularly intense in the dentate gyrus, giving rise continuously to an important pool of granular cells. It is unclear whether these newly-born cells functionally integrate into the pre-existing local neuronal circuitry. Organotypic slice cultures are very well suited to address these questions.
During the last year, efforts were continued to induce and modulate neurogenesis in 2 to 8-week-old hippocampal organotypic cultures. Cultures were prepared from 6-day-old mice and treated with 5-bromo-2'-deoxyuridine (BrdU) at different time points to quantify proliferation. Double and triple staining experiments using neuronal and glial markers allowed us to define the phenotype of the proliferating cells. Our results show that robust proliferation of glial cells, but not neurogenesis, occurs in standard hippocampal slice cultures (i.e. cultured with medium containing a high concentration of serum). However, discrete neurogenesis can be observed after transferring the hippocampal slices to serum-free culture medium (i.e. neurobasal medium with B27 complement).
To further increase the intensity of neurogenesis in our cultures, potential neurogenic factors (i.e. EGF, FGF-2, IGF-I, 5-HT, CPP) are being tested. For this purpose, transgenic mice (i.e. Ngn2-GFP knock-in mice) were obtained from the group of François Guillemot in Strasbourg. Neurogenin-2 (Ngn2) is a key transcription factor for the postnatal development of the dentate gyrus and may therefore be an adequate molecular marker of neuronal progenitors in our preparation. Ngn2 indeed shows high levels of expression at the time of culturing. In slices maintained in culture medium containing serum, this expression decreases dramatically, disappearing within a few days. It persists, although at low levels, under serum-free conditions. This latter finding clearly establishes that our approach to use Ngn2-GFP as a molecular marker for neurogenesis in the postnatal dentate gyrus is a valid one.
In further morphological studies, the degree of differentiation and maturation new-born neurons can undergo in organotypic slices are analyzed. Synaptic markers and injection of the retrograde tracer fluororuby in the CA3 area, i.e. the normal target area of dentate granular cells, are used to address this question.
For the characterization of the electrophysiological properties of new-born neurons, their direct visualization (i.e. by expression of a fluorescent marker) within the hippocampal network is required. Unfortunately, the GFP expression driven by Ngn2 is too weak and too transitory to be useful without further amplification. We are, therefore, now establishing a Cre approach to induce strong and persistent expression of GFP in cells expressing Ngn2. We have obtained a Ngn2-CreER knock-in mouse from the group of David Anderson (Caltech Institute) that we are breeding with a Cre-reporter mouse. These mice may allow us to study in more detail the progeny of Ngn2-expressing cells and facilitate the analysis of their potential for physiological integration.
In parallel to the study of endogenous neurogenesis, we have continued our efforts to characterize the incorporation of exogenous stem cells (i.e. stem cells isolated at various developmental stages from different CNS regions) into the hippocampal network. We have received different cell types from various research groups in Zurich and from abroad (i.e. Partner 3A of the European contract). Optimal culture conditions have been established for the survival of these cell types in organotypic cultures. We have preliminary evidence that 3 out of 5 types of exogenous stem cells penetrate into the slice and differentiate into neurons.
One cell type (i.e. the adult hippocampal progenitors) has been shown to present strong integrative capabilities and has been studied in more detail. These cells show clear electrophysiological signs of maturation as a function of time in culture, with the gradual appearance of voltage-gated currents. However, GFP expression in these cells decreases dramatically during their maturation, thus preventing us from fully characterizing their properties. To partially circumvent this limitation, stable transfection of these cells has been achieved with an enhanced GFP transgene under control of an ecdyson response element. This construct may enable us to temporally control the GFP expression in adult hippocampal progenitors, thereby allowing us to follow both anatomically and electrophysiologically their full maturation and integration into the hippocampal circuitry.
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