Exercise has been shown to positively augment adult hippocampal neurogenesis; however, the cellular and molecular pathways mediating this effect remain largely unknown. Previous studies have suggested that microglia may have the ability to differentially instruct neurogenesis in the adult brain. Here, we used transgenicCsf1r-GFPmice to investigate whether hippocampal microglia directly influence the activation of neural precursor cells. Our results revealed that an exercise-induced increase in neural precursor cell activity was mediated via endogenous microglia and abolished when these cells were selectively removed from hippocampal cultures. Conversely, microglia from the hippocampi of animals that had exercised were able to activate latent neural precursor cells when added to neurosphere preparations from sedentary mice. We also investigated the role of CX₃CL1, a chemokine that is known to provide a more neuroprotective microglial phenotype. Intraparenchymal infusion of a blocking antibody against the CX₃CL1 receptor, CX₃CR1, but not control IgG, dramatically reduced the neurosphere formation frequency in mice that had exercised. While an increase in soluble CX₃CL1 was observed following running, reduced levels of this chemokine were found in the aged brain. Lower levels of CX₃CL1 with advancing age correlated with the natural decline in neural precursor cell activity, a state that could be partially alleviated through removal of microglia. These findings provide the first direct evidence that endogenous microglia can exert a dual and opposing influence on neural precursor cell activity within the hippocampus, and that signaling through the CX₃CL1–CX₃CR1 axis critically contributes toward this process.

Adult hippocampal neurogenesis is a critical form of cellular plasticity that is greatly influenced by neural activity. Among the neurotransmitters that are widely implicated in regulating this process are serotonin and norepinephrine, levels of which are modulated by stress, depression and clinical antidepressants. However, studies to date have failed to address a direct role for either neurotransmitter in regulating hippocampal precursor activity. Here we show that norepinephrine but not serotonin directly activates self-renewing and multipotent neural precursors, including stem cells, from the hippocampus of adult mice. Mechanistically, we provide evidence that β₃-adrenergic receptors, which are preferentially expressed on a Hes5-expressing precursor population in the subgranular zone (SGZ), mediate this norepinephrine-dependent activation. Moreover, intrahippocampal injection of a selective β₃-adrenergic receptor agonistin vivoincreases the number of proliferating cells in the SGZ. Similarly, systemic injection of the β-adrenergic receptor agonist isoproterenol not only results in enhancement of proliferation in the SGZ but also leads to an increase in the percentage of nestin/glial fibrillary acidic protein double-positive neural precursorsin vivo. Finally, using a novelex vivo“slice-sphere” assay that maintains an intact neurogenic niche, we demonstrate that antidepressants that selectively block the reuptake of norepinephrine, but not serotonin, robustly increase hippocampal precursor activity via β-adrenergic receptors. These findings suggest that the activation of neurogenic precursors and stem cells via β₃-adrenergic receptors could be a potent mechanism to increase neuronal production, providing a putative target for the development of novel antidepressants.

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.

Although our understanding of adult neurogenesis has increased dramatically over the last decade, confusion still exists regarding both the identity of the stem cell responsible for neuron production and the mechanisms that regulate its activity. Here we show, using flow cytometry, that a small population of cells (0.3%) within the stem cell niche of the rat subventricular zone (SVZ) expresses the p75 neurotrophin receptor (p75^NTR) and that these cells are responsible for neuron production in both newborn and adult animals. In the adult, the p75^NTR-positive population contains all of the neurosphere-producing precursor cells, whereas in the newborn many of the precursor cells are p75^NTRnegative. However, at both ages, only the neurospheres derived from p75^NTR-positive cells are neurogenic. We also show that neuron production from p75^NTR-positive but not p75^NTR-negative precursors is greatly enhanced after treatment with brain-derived neurotrophic factor (BDNF) or nerve growth factor. This effect appears to be mediated specifically by p75^NTR, because precursor cells from p75^NTR-deficient mice show a 70% reduction in their neurogenic potentialin vitroand fail to respond to BDNF treatment. Furthermore, adult p75^NTR-deficient mice have significantly reduced numbers of PSA-NCAM (polysialylated neural cell adhesion molecule)-positive SVZ neuroblastsin vivoand a lower olfactory bulb weight. Thus, p75^NTRdefines a discrete population of highly proliferative SVZ precursor cells that are able to respond to neurotrophin activation by increasing neuroblast generation, making this pathway the most likely mechanism for the increased neurogenesis that accompanies raised BDNF levels in a variety of disease and behavioral situations.

Doublecortin (DCX) has recently been promulgated as a selective marker of cells committed to the neuronal lineage in both the developing and the adult brain. To explore the potential of DCX-positive (DCX⁺) cells more stringently, these cells were isolated by flow cytometry from the brains of transgenic mice expressing green fluorescent protein under the control of the DCX promoter in embryonic, early postnatal, and adult animals. It was found that virtually all of the cells (99.9%) expressing high levels of DCX (DCX^high) in the embryonic brain coexpressed the neuronal marker βIII-tubulin and that this population contained no stem-like cells as demonstrated by lack of neurosphere formationin vitro. However, the DCX⁺population from the early postnatal brain and the adult subventricular zone and hippocampus, which expressed low levels of DCX (DCX^low), was enriched for neurosphere-forming cells, with only a small subpopulation of these cells coexpressing the neuronal markers βIII-tubulin or microtubule-associated protein 2. Similarly, the DCX^lowpopulation from embryonic day 14 (E14) brain contained neurosphere-forming cells. Only the postnatal cerebellum and adult olfactory bulb contained some DCX^highcells, which were shown to be similar to the E14 DCX^highcells in that they had no stem cell activity. Electrophysiological studies confirmed the heterogeneous nature of DCX⁺cells, with some cells displaying characteristics of immature or mature neurons, whereas others showed no neuronal characteristics whatsoever. These results indicate that DCX^highcells, regardless of location, are restricted to the neuronal lineage or are bone fide neurons, whereas some DCX^lowcells retain their multipotentiality.

Upregulation and activation of developmental axon guidance molecules, such as semaphorins and members of the Eph receptor tyrosine kinase family and their ligands, the ephrins, play a role in the inhibition of axonal regeneration following injury to the central nervous system. Previously we have demonstrated in a knockout model that axonal regeneration following spinal cord injury is promoted in the absence of the axon guidance protein EphA4. Antagonism of EphA4 was therefore proposed as a potential therapy to promote recovery from spinal cord injury. To further assess this potential, two soluble recombinant blockers of EphA4, unclustered ephrin-A5-Fc and EphA4-Fc, were examined for their ability to promote axonal regeneration and to improve functional outcome following spinal cord hemisection in wildtype mice. A 2-week administration of either of these blockers following spinal cord injury was sufficient to promote substantial axonal regeneration and functional recovery by 5 weeks following injury. Both inhibitors produced a moderate reduction in astrocytic gliosis, indicating that much of the effect of the blockers may be due to promotion of axon growth. These studies provide definitive evidence that soluble inhibitors of EphA4 function offer considerable therapeutic potential for the treatment of spinal cord injury and may have broader potential for the treatment of other central nervous system injuries.