Golo Kronenberg

Milestones of neuronal development in the adult hippocampus

Adult hippocampal neurogenesis originates from precursor cells in the adult dentate gyrus and results in new granule cell neurons. We propose a model of the development that takes place between these two fixed points and identify several developmental milestones. From a presumably bipotent radial-glia-like stem cell (type-1 cell) with astrocytic properties, development progresses over at least two stages of amplifying lineage-determined progenitor cells (type-2 and type-3 cells) to early postmitotic and to mature neurons. The selection process, during which new neurons are recruited into function, and other regulatory influences differentially affect the different stages of development.

Early determination and long-term persistence of adult-generated new neurons in the hippocampus of mice

New neurons are continually generated in the adult hippocampus, but the important question, whether adult neurogenesis is transient or leads to the lasting presence of new neurons, has not yet been answered. Dividing cells were labeled with bromodeoxyuridine (BrdU) and were investigated by means of immunofluorescence and confocal microscopy at several time-points 1 day to 11 months thereafter. BrdU-labeled neurons remained stable in number and in their relative position in the granule cell layer over at least 11 months. This finding implies that the addition of new neurons is not transient and that their final number and localization are determined early. By contrast, expression of immature markers β-III-tubulin and doublecortin in BrdU-labeled cells, peaked early after division and was not detectable after 4 weeks. In transgenic mice expressing enhanced green fluorescent protein under the nestin promoter none of the BrdU/nestin-positive cells early after division expressed the mature marker NeuN, confirming that no dividing neurons were detected. These new data suggest that new neurons are recruited early from the pool of proliferating progenitor cells and lead to a lasting effect of adult neurogenesis.

Subpopulations of proliferating cells of the adult hippocampus respond differently to physiologic neurogenic stimuli.

To study how adult hippocampal neurogenesis might originate from the proliferation of stem or progenitor cells in vivo, we have used transgenic mice expressing green fluorescent protein (GFP) under the nestin promoter to identify these cells. Having described an astrocyte‐like type 1 cell with low proliferative activity, a characteristic morphology, vascular end feet, and passive electrophysiological properties, we focused here on the large population of nestin‐GFP‐expressing type 2 cells, which lack all these features. Type 2 cells were highly proliferative and showed signs suggestive of their involvement in the neuronal lineage. They could be subclassified by the absence (type 2a) or presence (type 2b) of a coexpression of the early neuronal marker doublecortin. A third type of proliferating cells was doublecortin positive but nestin‐GFP negative (type 3). We believe that type 2a, 2b, and 3 cells mirror a marker progression during earliest neuronal development. This view is supported by the increasing coexpression of the early granule cell‐specific marker Prox‐1. The low proliferative activity of type 1 cells showed little change over time or under “neurogenic interventions,” such as a challenge by environmental complexity (ENR) or voluntary physical activity (RUN). However, RUN led to a significant increase of type 2 cells labeled with the proliferation marker bromodeoxyuridine (BrdU). ENR did not cause increased cell proliferation or an increased number of BrdU‐labeled type 2 cells, but both ENR and RUN resulted in more newly generated cells lacking nestin‐GFP immunoreactivity and expressing Prox‐1. These findings allow us to break down what was broadly perceived as “proliferation” in earlier experiments into the relative contribution of several cell types, representing the earliest steps of neuronal development. J. Comp. Neurol. 467:455–463, 2003.

Subpopulation of nestin-expressing progenitor cells in the adult murine hippocampus shows electrophysiological and morphological characteristics of astrocytes

Based on the expression of glial fibrillary acidic protein (GFAP), a recent hypothesis considered stem or progenitor cells in the adult hippocampus to be a type of astrocyte. In a complementary approach, we used transgenic mice expressing green fluorescent protein (GFP) under the promoter for nestin, an intermediate filament present in progenitor cells, to demonstrate astrocytic features in nestin-GFP-positive cells. Morphologically, two subpopulations of nestin-GFP-positive cells were distinguishable; one had an elaborate tree of processes in the granule cell layer and expression of GFAP (but not of S100beta, another astrocytic marker). Electron microscopy revealed vascular end feet of nestin-positive cells, further supporting astrocytic differentiation. Electrophysiological examination of nestin-GFP-positive cells on acutely isolated hippocampal slices showed passive current characteristics of astrocytes in one subset of cells. Among the nestin-GFP-expressing cells with lacking astrocytic features, two cell types could be identified electrophysiologically: cells with delayed-rectifying potassium currents and a very small number of cells with sodium currents, potentially representing signs of the earliest steps of neuronal differentiation.

Transient calretinin expression defines early postmitotic step of neuronal differentiation in adult hippocampal neurogenesis of mice

We here show that the early postmitotic stage of granule cell development during adult hippocampal neurogenesis is characterized by the transient expression of calretinin (CR). CR expression was detected as early as 1 day after labeling dividing cells with bromodeoxyuridine (BrdU), but not before. Staining for Ki-67 confirmed that no CR-expressing cells were in cell cycle. Early after BrdU, CR colocalized with immature neuronal marker doublecortin; and later with persisting neuronal marker NeuN. BrdU/CR-labeled cells were negative for GABA and GABAA1 receptor, but early on expressed granule cell marker Prox-1. After 6 weeks, no new neurons expressed CR, but all contained calbindin. Stimuli inducing adult neurogenesis have limited (enriched environment), strong (voluntary wheel running), and very strong effects on cell proliferation (kainate-induced seizures). In these models the induction of cell proliferation was paralleled by an increase of CR-positive cells, indicating the stimulus-dependent progression from cell division to a postmitotic stage.

Physical exercise prevents age-related decline in precursor cell activity in the mouse dentate gyrus

Physical activity induces adult hippocampal neurogenesis. We here show that the acute up-regulating effect of voluntary wheel running on precursor cell proliferation decreases with continued exercise, but that continued exercise reduces the age-dependent decline in adult neurogenesis. Cell proliferation peaked at 3 days of running. After 32 days of exercise this response returned to baseline. Running-induced proliferation of transiently amplifying progenitor cells led to a consecutive increase in the number of more mature cells. Increasing age reduced adult neurogenesis at 9 months to 50% of the value at 6 weeks and to 17% at the age of 2 years. At both 1 and 2 years, precursor cell divisions remained inducible by physical activity. Exercise from 3 to 9 months of age significantly reduced the age-dependent decline in cell proliferation but (presumably in the absence of additional stimuli) did not maintain net neurogenesis at levels corresponding to a younger age. We propose that physical activity might contribute to successful aging by increasing the potential for neurogenesis represented by the pool of proliferating precursor cells.

Depressed new Neurons?—Adult hippocampal neurogenesis and a cellular plasticity hypothesis of major depression

In a novel theory, a failure of adult hippocampal neurogenesis has been proposed to provide the biological and cellular basis of major depression. The as yet unresolved function of the new hippocampal neurons will have to be in the center of any attempt to prove this hypothesis. Only knowledge of normal functional relevance of new neurons will allow an assessment of their potential role in disturbed hippocampal function in depression; however, major depression is not primarily a hippocampal disorder. We therefore propose that consideration of the neurogenesis hypothesis of depression be the most prominent aspect of a more general cellular plasticity hypothesis.

Differential regulation of gliogenesis in the context of adult hippocampal neurogenesis in mice.

In adult hippocampal neurogenesis, new neurons appear to originate from a cell with astrocytic properties expressing glial fibrillary acidic protein (GFAP). Also, new astrocytes are generated in the adult dentate gyrus. Whereas the putative astrocyte‐like progenitor cells are consistently S‐100β‐negative, many new astrocytes are S‐100β‐positive. Thus, it is unclear whether the GFAP‐positive progenitor cells are astrocytes in a general sense or rather neural progenitor cells with certain astrocytic characteristics. We therefore investigated the development of GFAP‐expressing cells in the context of adult hippocampal neurogenesis. Proliferating cells could be either GFAP‐positive or doublecortin‐positive (DCX), but never both, indicating two independent populations of dividing cells in the glial and neuronal lineages. Two distinct populations of cells with astroglial properties were detected—one expressing GFAP, the other co‐expressing GFAP and S‐100β. We never found S‐100β‐cells to be in S‐phase. No overlap between neuronal and glial markers was seen at any time point. Thus, astrogenesis occurred in parallel and to some degree independent of adult neurogenesis. The uninterrupted GFAP expression in this lineage, and neuronal markers in the other lineage, argue against a late common precursor for neurogenesis and gliogenesis in the adult hippocampus. Very few newly generated microglia and no new oligodendrocytes were detected. Environmental enrichment and voluntary wheel running—two experimental paradigms with robust stimulatory effects on adult hippocampal neurogenesis—affected hippocampal astrogenesis differentially: Running, but not enrichment, strongly induced net astrogenesis (GFAP/S‐100β), but also GFAP‐positive S‐100β‐negative cells, which thus appear to be a transiently amplifiable intermediate population within the glial lineage.

Cognitive and physical activity differently modulate disease progression in the amyloid precursor protein (APP)-23 model of Alzheimer's disease

BACKGROUND In aging mice, activity maintains hippocampal plasticity and adult hippocampal neurogenesis at a level corresponding to a younger age. Here we studied whether physical exercise and environmental enrichment would also affect brain plasticity in a mouse model of Alzheimers disease (AD). METHODS Amyloid precursor protein (APP)-23 mice were housed under standard or enriched conditions or in cages equipped with a running wheel. We assessed beta-amyloid plaque load, adult hippocampal neurogenesis, spatial learning, and mRNA levels of trophic factors in the brain. RESULTS Despite stable beta-amyloid plaque load, enriched-living mice showed improved water maze performance, an up-regulation of hippocampal neurotrophin (NT-3) and brain-derived neurotrophic factor (BDNF) and increased hippocampal neurogenesis. In contrast, despite increased bodily fitness, wheel-running APP23 mice showed no change in spatial learning and no change in adult hippocampal neurogenesis but a down-regulation of hippocampal and cortical growth factors. CONCLUSIONS We conclude that structural and molecular prerequisites for activity-dependent plasticity are preserved in mutant mice with an AD-like pathology. Our study might help explain benefits of activity for the aging brain but also demonstrates differences between physical and more cognitive activity. It also suggests a possible cellular correlate for the dissociation between structural and functional pathology often found in AD.

Variability of doublecortin-associated dendrite maturation in adult hippocampal neurogenesis is independent of the regulation of precursor cell proliferation

BackgroundIn the course of adult hippocampal neurogenesis most regulation takes place during the phase of doublecortin (DCX) expression, either as pro-proliferative effect on precursor cells or as survival-promoting effect on postmitotic cells. We here obtained quantitative data about the proliferative population and the dynamics of postmitotic dendrite development during the period of DCX expression. The question was, whether any indication could be obtained that the initiation of dendrite development is timely bound to the exit from the cell cycle. Alternatively, the temporal course of morphological maturation might be subject to additional regulatory events.ResultsWe found that (1) 20% of the DCX population were precursor cells in cell cycle, whereas more than 70% were postmitotic, (2) the time span until newborn cells had reached the most mature stage associated with DCX expression varied between 3 days and several weeks, (3) positive or negative regulation of precursor cell proliferation did not alter the pattern and dynamics of dendrite development. Dendrite maturation was largely independent of close contacts to astrocytes.ConclusionThese data imply that dendrite maturation of immature neurons is initiated at varying times after cell cycle exit, is variable in duration, and is controlled independently of the regulation of precursor cell proliferation. We conclude that in addition to the major regulatory events in cell proliferation and selective survival, additional micro-regulatory events influence the course of adult hippocampal neurogenesis.