We have identified a neuronal-restricted precursor (NRP) cell that expresses E-NCAM (high polysialic-acid NCAM) and is morphologically distinct from multipotent neuroepithelial (NEP) cells (Kalyani et al., 1997) and spinal glial progenitors (Rao and Mayer-Proschel, 1997). NRP cells self renew over multiple passages in the presence of fibroblast growth factor (FGF) and neurotrophin-3 (NT-3) and differentiate in the presence of retinoic acid and the absence of FGF into postmitotic neurons. NRP cells can also be generated from multipotent E10.5 NEP cells. Clonal analysis shows that NRP cells arise from a NEP progenitor that generates other restricted CNS precursors. The NEP-derived NRPs undergo self renewal and can differentiate into multiple neuronal phenotypes. Thus, a direct lineal relationship exists between multipotential NEP cells and more restricted neuronal precursor cells present in vivo at E13.5 in the spinal cord.

Adherent cultures of E10.5 rat neuroepithelial cells (NEP cells) from the caudal neural tube require FGF (fibroblast growth factor) and CEE (chick embryo extract) to proliferate and maintain an undifferentiated phenotype in culture. Epidermal growth factor (EGF) does not support E10.5 NEP cells in adherent culture and NEP cells do not form EGF-dependent neurospheres. NEP cells, however, can be grown as FGF-dependent neurospheres. NEP cells express nestin and lack all lineage-specific markers for neuronal and glial sublineages, retain their pleuripotent character over multiple passages, and can differentiate into neurons, astrocytes, and oligodendrocytes when plated on laminin in the absence of CEE. In clonal culture, NEP cells undergo self-renewal and generate colonies that vary in size from single cells to several thousand cells. With the exception of a few single-cell clones, all other NEP-derived clones contain more than one identified phenotype, with over 40% of the colonies containing A2B5, beta-111 tubulin, and GFAP-immunoreactive cells. Thus, NEP cells are multipotent and capable of generating multiple neural derivatives. NEP cells also differentiate into motoneurons immunoreactive for choline acetyl transferase (ChAT) and the low-affinity neurotrophin receptor (p75) in both mass and clonal culture. Double labeling of clones for ChAT and glial, neuronal, or oligodendrocytic lineage markers shows that motoneurons always arose in mixed cultures with other differentiated cells. Thus, NEP cells represent a common progenitor for motoneurons and other spinal cord cells. The relationship of NEP cells with other neural stem cells is discussed.

Neuronal restricted precursors (NRPs) () can generate multiple neurotransmitter phenotypes during maturation in culture. Undifferentiated E-NCAM+ (embryonic neural cell adhesion molecule) immunoreactive NRPs are mitotically active and electrically immature, and they express only a subset of neuronal markers. Fully mature cells are postmitotic, process-bearing cells that are neurofilament-M and synaptophysin immunoreactive, and they synthesize and respond to different subsets of neurotransmitter molecules. Mature neurons that synthesize and respond to glycine, glutamate, GABA, dopamine, and acetylcholine can be identified by immunocytochemistry, RT-PCR, and calcium imaging in mass cultures. Individual NRPs also generate heterogeneous progeny as assessed by neurotransmitter response and synthesis, demonstrating the multipotent nature of the precursor cells. Differentiation can be modulated by sonic hedgehog (Shh) and bone morphogenetic protein (BMP)-2/4 molecules. Shh acts as a mitogen and inhibits differentiation (including cholinergic differentiation). BMP-2 and BMP-4, in contrast, inhibit cell division and promote differentiation (including cholinergic differentiation). Thus, a single neuronal precursor cell can differentiate into multiple classes of neurons, and this differentiation can be modulated by environmental signals.

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted, self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani, K. Hobson, and M. S. Rao, 1997, Dev. Biol. 186, 202-223; M. S. Rao and M. Mayer-Proschel, 1997, Dev. Biol. 188, 48-63; M. Mayer-Proschel, A. J. Kalyani, T. Mujtaba, and M. S. Rao, 1997, Neuron 19, 773-785). We now show that cells identical to rat NEPs, NRPs, and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs, respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive, undergo self-renewal in defined medium, and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus, lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells.

Young and old rats performed on a maze according to a forced-choice and then a spatial memory procedure either in the same or a different environment. Aged rats were slower to learn the spatial memory task when tested in the same, but not in a different, room. One interpretation of this pattern of results is that although old rats learn new rules as quickly as young rats, they show less flexibility with old rules and familiar spatial information. Impaired choice accuracy during asymptote performance suggests poor processing of trial-unique information by old rats. Spatial correlates of hippocampal CA1 and hilar cells varied with task demand: CA1 cells of aged rats showed more spatially selective place fields, whereas hilar cells showed more diffuse location coding during spatial memory, and not forced-choice, tests. Such representational reorganization may reflect a compensatory response to age-related neurobiological changes in hippocampus.Extensive literature indicates that normal aging is accompanied by a decline in the ability to acquire new information. Importantly, this impairment appears selective to certain types of information. For example, aged rats perform relatively poorly on complex visuospatial memory tasks (for a review, see Barnes, 1990). Past studies have ruled out the possibility that the impaired performance is due to changes in motor system function, motivation level, or visual function. The selectivity of the learning impairment has been demonstrated by many laboratories that found that aged rats perform as well as young rats on visually dependent, nonspatial maze tasks (Barnes, Green, Baldwin, & Johnson, 1987;Lowry, Ingram, Olton, Waller, Reynolds, & London, 1985;Rapp, Rosenberg, & Gallagher, 1987;Winocur, 1988). Given that hippocampal lesions often produce deficits in tasks requiring animals to learn trial-unique data, the spatial learning impairment of old rats may be mediated by age-associated neurobiological changes in hippocampus. One could also argue that old rats have difficulty learning the rules associated with particular spatial tasks. The latter interpretation suggests the involvement of nonhippocampal brain structures (e.g., frontal cortex; Winocur & Moscovitch, 1990). Therefore, we evaluated the relative contribution of rule-based and item-and event-based learning systems to the spatial navigation deficit shown by old rats. Also, given the extensive evidence indicating at least hippocampal involvement in the spatial decline of old animals (e.g., Barnes, 1979;Gallagher, Bostock, & King, 1985), we investigated possible age changes in spatial coding by hippocampal neurons.An important piece of evidence suggesting that hippocampus plays a special role in an organism's ability to navigate Sheri J. Y. Mizumori, Annette M. Lavoie, and Anjali Kalyani, Department of Psychology, University of Utah.This work was supported by National Institutes of Health Grant AG09299. We thank James G. Canfield for helpful discussions and Karen Burk and Leigh Hardy for assistance with behavioral testing ...