The Journal of Neuroscience, February 1, 2002, 22(3):619–623 Neurogenesis in Adult Mammals: Some Progress and Problems Elizabeth Gould and Charles G. Gross Department of Psychology, Princeton University, Princeton, New Jersey 08544 Approximately 40 years after its first report, the addition of addition, the growing evidence that glia communicate among neurons to the brains of adult mammals has now been generally themselves electrically and have many functional properties that accepted. We now know that several endocrine and experiential interact and overlap with neurons (Rose and Konnerth, 2001; variables modulate adult neurogenesis. However, several meth- Bezzi and Volterra, 2001) suggests that new glia may play a odological problems in its quantitative study remain. One is the significant role. use of low doses of the exogenous marker of cell proliferation, In the 1990s there were several developments that finally es- bromodeoxyuridine (BrdU). A second is the transient lifetime of tablished neurogenesis in the adult rodent. The first was the most of the adult-generated cells. A third is that the survival of introduction of cell type-specific markers for immunohistochem- new neurons may depend on stimuli that are lacking in standard ical identification of the phenotype of newly generated cells laboratory conditions. This review considers these issues as well (Cameron et al., 1993; Okano et al., 1993; Seki and Arai, 1993). as the possible functions of new neurons. These could be combined with 3H-thymidine labeling to deter- From the beginnings of modern neuroscience in the late 19th mine the identity of the new cells. The second was the introduc- century, it was assumed that the mammalian CNS became struc- tion of the thymidine analog BrdU as another in vivo marker of turally stable soon after birth and remained that way throughout proliferating cells (Miller and Nowakowski, 1988; Seki and Arai life. A fundamental aspect of this stability was that no new 1995; Kuhn et al., 1996). An advantage of BrdU is that it can be neurons were added to the brain in adulthood (Gross, 2000). In visualized with immunocytochemical techniques and quantita- the 1960s, studies using the newly introduced methods of 3H- tively assessed with stereological counting techniques. Further- thymidine autoradiography challenged this view. 3H-thymidine is more, confocal microscopy can be used to show unequivocal taken up by cells undergoing DNA synthesis in preparation for double labeling with BrdU and cell type-specific markers when mitosis, and thus can be used as a marker for proliferating cells (at the cells are rotated in orthogonal planes (Fig. 1). With these short survival times) and their progeny (at longer survival times). techniques, several laboratories confirmed that new neurons are Using this method, Altman (1962, 1963, 1966, 1967, 1969) and added to the granule cell layer of the dentate gyrus of adult Altman and Das (1965, 1966) reported new neurons in a variety rodents (Cameron et al., 1993; Seki and Arai, 1993; Kuhn et al., of structures in the adult rat and cat including the olfactory bulb, 1996). In addition, adult neurogenesis was found in the dentate hippocampus, and cerebral cortex. Fifteen years later, Kaplan gyrus of tree shrews, marmoset monkeys, macaque monkeys, and examined the ultrastructure of 3H-thymidine-labeled cells in the humans (Gould et al., 1997, 1998, 1999b; Eriksson et al., 1998; olfactory bulb, hippocampus, and neocortex of adult rats and Kornack and Rakic, 1999). In the dentate gyrus of both rodents confirmed that they were neurons (Kaplan and Hinds, 1977; and monkeys, adult-generated cells appear to arise from progen- Kaplan, 1981, 1985; Kaplan and Bell, 1984). Further support that itor cells in the hilus or subgranular zone and migrate the short the cells added to the dentate gyrus were neurons came from distance to the granule cell layer where they differentiate into studies by Stanfield and Trice (1988) and Gue´neau et al. (1982). neurons. Overall, the pioneering work of these investigators was greeted Recently, we have found new cells with neuronal characteristics with considerable skepticism and had little impact on the field in the neocortex of adult rats and macaques, consistent with the (Gross, 2000; Kaplan, 2001). In contrast, demonstrations of adult earlier claims of Altman (1963) and Kaplan (1981). In the mon- neurogenesis in nonmammalian vertebrates such as fish, reptiles, key, these adult-generated cells were found in prefrontal cortex, and birds appear to have been more readily accepted (Anderson temporal cortex, and parietal cortex (Gould et al., 1999c, 2001; and Waxman, 1985; Nottebohm, 1985, 1989; Lopez-Garcia et al., Bedard et al., 2001). The density of adult generated cells with 1988), but their potential relevance to mammals was not generally neuronal characteristics is much lower in cortical areas than in the acknowledged. dentate gyrus in both the monkey and the rat (Gould et al., 2001). A major source of difficulty in early adult neurogenesis studies The site of origin of adult-generated cells in the neocortex is was uncertainty whether the adult-generated cells were glia or unclear. One possibility is the subventricular zone (SVZ), which neurons. Recent evidence suggests that these categories may not is the source of new olfactory bulb neurons in the adult rodent be as fixed as previously believed. In the adult brain, some glia and monkey (Luskin, 1993; Bernier and Parent, 1998; Kornack may be a source of neurons, and some progenitors give rise to and Rakic, 2001a). In the macaque we observed adult-generated both neurons and glia (Alvarez-Buylla et al., 2001). In fact, recent cells in the white matter that may have been immature neurons evidence in the adult rat indicates that the new granule neurons migrating from the SVZ to the cerebral cortex. New cortical cells arise from cells with glial characteristics (Seri et al., 2001). In may also arise from local division of progenitors or from progen- itors in the white matter (Gould et al., 2001) We thank H. A. Cameron, P. Tanapat, and W. T. Clark for help and the James S. Methodological problems with BrdU labeling McDonnell Foundation and National Institutes of Health for support. Correspondence should be addressed to Elizabeth Gould, Department of Psy- The BrdU technique for in vivo labeling of new cells in the brain chology, Princeton University, Princeton, NJ 08544. E-mail: [email protected]. was first applied to developing animals (Miller and Nowakowski, Copyright © 2002 Society for Neuroscience 0270-6474/02/220619-05$15.00/0 1988; Takahashi et al., 1992). The techniques used in these studies 620 J. Neurosci., February 1, 2002, 22(3):619–623 Gould and Gross • Adult Neurogenesis: Some Progress and Problems Figure 2. Methodological considerations related to BrdU labeling in adults. Top panel, A short pulse of BrdU labels in a dose-dependent manner, raising the possibility that changes in the number of BrdU- labeled cells could result from differences in BrdU availability. The graph shows the total number of BrdU-labeled cells in the dentate gyrus of adult rats after a single injection of BrdU 30 min earlier. *Indicates significant difference from all other groups, p Ͻ 0.05. **Indicates significant differ- ence from 300 mg/kg group, p Ͻ 0.05. Adapted from Cameron and McKay (2001) with permission. Bottom panel, Left, BrdU-labeled progen- itor cells in the subgranular zone (s) of the dentate gyrus. BrdU is an exogenous marker of proliferating cells. Right, Dividing cell undergoing Figure 1. Some new cells in the adult rodent and primate brain have cytokinesis labeled with phosphorylated histone H3 in the subgranular neuronal characteristics. Top panel, Confocal image of a cell (arrow)in zone. p histone H3 is an endogenous marker of cells in M phase (Hendzel the anterior cortex of an adult rat double-labeled with NeuN ( green et al., 1997). Endogenous markers can be used to verify that changes in nuclear and cytoplasmic stain; a marker for mature neurons) and BrdU the number of BrdU-labeled cells are attributable to differences in the (red nuclear stain; a marker of DNA synthesis). The image is rotated in number of dividing cells, as opposed to difference in BrdU availability. g, orthogonal planes (x, y, z) to verify double labeling throughout its extent. Granule cell layer; h, hilus. The rat was perfused 3 weeks after the BrdU injection. Bottom panel, Confocal image of a cell (arrow) in prefrontal cortex of an adult macaque double-labeled with NeuN ( green) and BrdU (red). The image is rotated There are several implications of these findings. The first is that in orthogonal planes (x, y, z) to verify double labeling throughout its most previous studies of adult neurogenesis have underestimated extent. The monkey was perfused 2 weeks after the BrdU injection (adapted from Gould et al., 2001). the number of adult-generated cells. Thus, using higher doses of BrdU, Cameron and McKay (2001) found that ϳ9000 progenitor cells exist in the dentate gyrus that have a cell cycle time of ϳ24 were then assumed to be applicable to studies of the adult brain. hr, yielding ϳ9000 new cells every day, most of which have the For example, it was assumed that BrdU would label all dividing immunocytochemical characteristics of neurons. They calculate cells in the brain at low doses and would be toxic or label that over 270,000 new cells are generated in the dentate gyrus of nonspecifically when applied at high doses. Recently, Cameron the adult rat every month. This is remarkable, considering the and McKay (2001) have shown that none of these assumptions fact that estimates of total granule cell number in the dentate applies to adult rats. They showed that the most commonly used gyrus have ranged from 1.0–2.0 million in the adult rat (Boss et dose (50 mg/kg) labels only a fraction of the cells in S phase in the al., 1985).