Short Report An in vitro model to study adult neurogenesis in mammals MARGARITA PÉREZ MARTÍN, LUIS M. RODRÍGUEZ-PÉREZ, JAVIER BERMÚDEZ-SILVA, NÉSTOR ACOSTA, PEDRO F. LLEBREZ-ZAYAS, MANUEL CIFUENTES and PEDRO FERNÁNDEZ-LLEBREZ* Laboratorio de Fisiología Animal, Facultad de Ciencias, Universidad de Málaga, Spain Int. J. Dev. Biol. 45 (S1): S85-S86 (2001) *Address correspondence to: Pedro Fernández-Llebrez. Laboratorio de Fisiología Animal, Facultad de Ciencias, Universidad de Málaga, 29071 Málaga, Spain. e-mail: llebrez@uma.es ABSTRACT The wall of the lateral ventricle of bovine brains comprised three distinct layers: ependyma, subependyma (SE) and astroglial net. The wall lining white matter had narrow SE and thin, compacted astroglial net. The wall of the striatum and the anterior horn had a wide SE and a thick and slack astroglial net. The ependyma was S-100 positive and, in rostro-lateral regions, it developed basal processes and reactivity to GFAP and vimentin. The SE and the subjacent astroglial net displayed β3 tubulin small cells and some PCNA positive nuclei. These features suggested that adult neurogenesis takes place in the bovine ventricular walls specially in the striatum and the anterior horn. Explants of the ependyma-SE were cultured in serum free medium. The ependymal cells developed a net of intermingled basal processes that became increasingly GFAP, vimentin and BLBP positive. At the same time SE cells proliferated to produce a population of β3 tubulin-positive cells. These cells synthesised IGF-1 that acted as a survival factor. These explants represent good models to study adult neurogenesis. Introduction Neural stem cells persist in the walls of the brain ventricles and the spinal central canal of adult vertebrate central nervous sys- tem. But, only in circumscribed regions, the newly generated cells migrate and become functionally active neurons. In these regions it should exist a propitious environment for cell survival, migration and differentiation (Scheffler et al., 1999). In rodents, neural stems have characteristics of ependymal cells (Johansson et al., 1999) and/or SE astrocytes (Doetsch et al. 1999). Here a rostral migratory stream of chains of newly formed neurons move through an astroglial net toward the olfactory bulb where they integrate as granular interneurons (Lois et al., 1996)). Experimentally, rodent adult neurogenesis has been shown to be increased by EGF, FGF and αTGF (Craig et al., 1996). Neural stem cells have been reported to exist in other mammals and in humans. However there are not appropriate knowledge of the architecture of the germina- tive regions in mammals other than rodents. Here we report the histology and immunocytochemical characteristics of the wall of the lateral ventricle of a large mammal, the bovine. By dissecting and culture the ependyma and a part of the SE we developed an in vitro model system to study neurogenesis. Materials and Methods Bovine brains were obtained from a local slaughterhouse, sliced and put in Bouin fixative. Pieces containing the wall of the lateral ventricles of different regions were dissected, embedded in paraffin, cut and processed for PAP immunocytochemistry. For explants, brains were put in Hank´s solution at 4°C, and the wall of the lateral ventricles exposed. The ependyma was separated and thin sheets were put in fresh DMEM medium without serum or growth factors. At different culture times, explants fixed in Bouin and processed for immunocytochemistry. In some wells BrdU were added. We used primary antibodies against the following substances: S-100 protein, vimentin, glial fibrillary acidic protein (GFAP), brain lipid binding protein (BLBP), β3 tubulin, nestin, proliferating cell nuclear antigen (PCNA) and BrdU. Results and Discussion Bovine lateral ventricle displayed anterior, inferior and poste- rior horns and a central body. The anterior horn ended close to the olfactory bulb. The wall lining white matter, such as the corpus callosum, fornix and septum, corresponding to the dorsomedial walls of the central body and of the posterior horn, displayed similar features as did the walls lining the striatum and the rostral region of the anterior horn. In general, three layers could be distinguished in the ventricular wall: a ciliated cubic ependyma, a SE and a subjacent astroglial layer (Fig. 1a). The structural and immunocytochemical characteristics of these layers varied de- pending on the region studied. Those lining white matter showed a narrow SE and a thin, compacted astroglial net. At variance, the wall of the striatum and the anterior horn displayed a wide SE and a thick and slack astroglial net. The ependyma was ciliated, cubic, with short basal processes and S-100 (Fig. 1b) and nestin positivity. In the striatum, the basal processes of the ependymal cells were more evident and they became increasingly longer and GFAP, vimentin and BLBP positive in the rostral horn. The SE displayed heterogeneous population of cells. Many of them were positive to anti-β3 tubulin (Fig. 1d) and were numerous in the walls of the striatum and the anterior horn. In the SE of all lateral ventricle regions, but especially in the striatum and the anterior horn, PCNA-positive nuclei were found (Fig. 1e). The subjacent astroglial layer was GFAP (Fig. 1c) and vimentin positive. In the striatum, this layer is a thick astroglial net containing bipolar β3 tubulin positive cells. In the cephalic end of the rostral horn, a SE is virtually absent. Here, the ependyma displayed the longest basal processes that penetrated a wide net of GFAP positive fibers intermingled with β3 tubulin positive cells. This net ex- tended ventrally toward the olfactory bulb. According to this description, we think that in the adult bovine lateral ventricles, neurogenesis takes place especially in the walls covering the striatum and the rostral horn. We hypothesise that subventricular