Experience-Induced Neurogenesis in the Senescent Dentate Gyrus

Neurogenesis in the senescent dentate gyrus

Neurogenesis persists in the hippocampal formation of old mice. In all four groups, proliferating cells (Fig. 4A) were located in the subgranular zone, and differentiating cells migrated into the granule cell layer. BrdU-labeled cells expressed the neuronal marker NeuN 4 weeks after injection of BrdU (Fig.4B–D).

The decrease in neurogenesis with increasing age confirmed previous results from rats (Kuhn et al., 1996). We found no significant difference in the total granule cell number between the two age groups, indicating that in old age neurogenesis ceases to produce granule cells in numbers that influence the total granule cell count at a detectable rate. Comparison with data from our previous study reveals an increase in the total granule cell number between 2 and 6 months by ∼40,000 cells (Kempermann et al., 1997a). Comparable increases have been described for rats (Bayer et al., 1982).

Little is known about the role of apoptosis in the regulation of adult neurogenesis, although it is plausible that the decrease in BrdU-positive cells during 4 weeks after injection is attributable to elimination by programmed cell death. Apoptotic nuclei have been described in the subgranular zone (Bengzon et al., 1997). Quantitatively, the loss of BrdU-positive cells might reflect an overestimation of apoptosis, because BrdU itself could damage some labeled cells.

Birth dating cells by BrdU incorporation is influenced by cell cycle parameters (Cai et al., 1997). Because BrdU has a bioavailability of ∼2 hr (Takahashi et al., 1992), only cells that are in S phase in this short period incorporate BrdU. A lengthened S phase will increase the probability of a cell being labeled during each injection; however, during the period of injections fewer cell cycles are likely to occur. BrdU-positive cells that divide again after BrdU is eliminated from the bloodstream will give BrdU to their daughter cells. The rationale for 12 BrdU injections over 12 d was to minimize the impact of divisions after discontinuation of BrdU, to label more cells, and to compensate for dilution effects caused by continued divisions.

Experience-induced regulation of neurogenesis

The paradigm of environmental enrichment (Rosenzweig et al., 1962) has been applied to study the environmental effect on cellular proliferation in the adult brain (Altman and Das, 1964). We have recently shown that stimulating mice with an enriched environment results in increased neurogenesis in the dentate gyrus (Kempermann et al., 1997b).

Our present results indicate that experience-dependent regulation of adult neurogenesis occurs in the aging dentate gyrus. As in younger mice, survival of BrdU-labeled cells was promoted. The underlying mechanism could lie in an interaction with the apoptotic cascade.

In addition, we found that in enriched-living animals of both age groups relatively more BrdU-positive cells double labeled for neuronal marker NeuN, an effect that was not apparent in 3-month-old mice (Kempermann et al., 1997b). This can be interpreted as a survival-promoting effect on cells that are committed to a neuronal lineage. The absolute numbers of both BrdU-labeled astrocytes and BrdU-positive cells, which did not double label for NeuN or S100β, were not influenced by environmental stimuli—a finding that supports this hypothesis.

An alternative hypothesis that remains to be tested is that environmental stimuli could have an effect on the differentiation of progeny of the dividing progenitor cells by directly or indirectly influencing mechanisms of cell fate decisions.

The microenvironmental conditions and their experience-dependent alteration, which favor the survival of cells that have exited the cell cycle and which lead these cells toward differentiation, remain to be identified. Although gliogenesis was not significantly stimulated by experience in old animals, it remains possible that environmental influences on astrocytes contribute to the overall effects.

That there was no difference in proliferation between controls and enriched-living mice does not allow conclusions about the size of the progenitor population. In principle, our findings on proliferation would be consistent with either a smaller proliferating population with shorter cell cycles or a larger population with longer cell cycles in enriched-living mice. Theoretically, increased neurogenesis could deplete the population of progenitor cells in the subgranular zone, and experience-dependent demand for new neurons could compensate for this by shortening the cell cycle. The net result, that 4 weeks after BrdU injection significantly more BrdU-labeled neurons are present in enriched-living mice, is independent of these considerations. Future experiments will have to examine how cell cycle kinetics themselves are influenced by aging and by environmental stimulation.

Factors mediating the regulation of adult neurogenesis

Inheritable traits have different effects on three levels in the regulation of adult neurogenesis: proliferation, survival, and differentiation, suggesting the involvement of several regulatory genes (Kempermann et al., 1997a).

Neonatal handling of rats prevents age-related cell losses in CA1 and CA3 and deficits in spatial memory (Meaney et al., 1988). The hypothesized mechanism underlying this effect involves downregulation of glucocorticoid secretion. Glucocorticoids have also been shown to inhibit neurogenesis in the dentate gyrus (Gould et al., 1992). Glutamatergic afferents to the dentate gyrus also limit adult neurogenesis, as does psychosocial stress (Gould, 1994; Gould et al., 1997). In contrast, temporal lobe seizures caused an increase in neurogenesis in the dentate gyrus (Bengzon et al., 1997; Parent et al., 1997). Both results indicate a regulatory influence of glutamate on adult neurogenesis. In all of these cases effects on neurogenesis included strong stimulation of proliferation. Environmental stimulation is the first known modulator to interact with a later regulatory step of adult neurogenesis.

Mitogenic effects of growth factors have been described for progenitor cells from fetal and adult brain in vitro (Reynolds, 1992;Vescovi et al., 1993; Bartlett et al., 1994; Palmer et al., 1995; Ray et al., 1995). Epidermal growth factor, after intracerebroventricular infusion, shifted differentiation of BrdU-labeled cells in the granule cell layer to a glial fate (Kuhn et al., 1997).

Does experience-dependent regulation imply a functional role?

The extent to which spatial learning contributes to the behavioral improvements seen in our study remains undefined. However, even after living under standard conditions for 6 or 18 months, old mice stimulated by an enriched environment for as short as 68 d showed better performance in the water maze. This behavioral effect does not prove a causal link to the morphological results, but neurogenesis occurs in a context of functional effects.

Although the results might suggest that the new neurons are recruited for purposes of hippocampal function, further research is required to determine how and to what extent the neurons that are born during adulthood are integrated functionally into hippocampal circuitry. Anatomically, the new neurons extend neurites to CA3 as do other granule cells (Stanfield and Trice, 1988; Parent et al., 1997).

A functional interpretation of adult neurogenesis is supported by results from chickadees in which neurogenesis in the adult dentate gyrus has been shown to correlate with seasons in which the birds cover larger territories and have to remember numerous food storage sites (Barnea and Nottebohm, 1996).

The total numbers of neurons that can be generated in the senescent dentate gyrus, however, are low and it is important to determine whether these cells could contribute to the experience-related functional effects observed here. Various other hippocampal parameters have been shown to be inducible by living in an enriched environment (Jones and Smith, 1980). Some of these changes, such as plasticity of synaptic properties (Greenough et al., 1978), synaptic density (Saito et al., 1994), and dendritic arborization (Greenough and Volkmar, 1973), occur in older age and are likely to contribute to functional effects. However, the fact that the aging brain maintains a complex regulatory apparatus for the production of neurons in the dentate gyrus suggests that this plasticity has functional benefits. Our data support theories that place adult neurogenesis in a context of hippocampal function and shed new light on the potential for plasticity in the old brain.