H. Georg Kuhn

Transient expression of doublecortin during adult neurogenesis

During development of the central nervous system, expression of the microtubule binding protein doublecortin (DCX) is associated with migration of neuroblasts. In addition to this developmental role, expression of DCX remains high within certain areas of the adult mammalian brain. These areas, mainly the dentate gyrus and the lateral ventricle wall in conjunction with the rostral migratory stream and olfactory bulb, retain the capacity to generate new neurons into adulthood. Adult neurogenesis is typically detected by incorporation of bromodeoxyuridine (BrdU) into dividing cells and colabeling of BrdU‐positive cells with markers for mature neurons. To elucidate whether DCX could act as an alternative indicator for adult neurogenesis, we investigated the temporal expression pattern of DCX in neurogenic regions of the adult brain. Analysis of newly generated cells showed that DCX is transiently expressed in proliferating progenitor cells and newly generated neuroblasts. As the newly generated cells began expressing mature neuronal markers, DCX immunoreactivity decreased sharply below the level of detection and remained undetectable thereafter. The transient expression pattern of DCX in neuronal committed progenitor cells/neuroblasts indicates that DCX could be developed into a suitable marker for adult neurogenesis and may provide an alternative to BrdU labeling. This assumption is further supported by our observation that the number of DCX‐expressing cells in the dentate gyrus was decreased with age according to the reduction of neurogenesis in the aging dentate gyrus previously reported. J. Comp. Neurol. 467:1–10, 2003.

Enriched environment and physical activity stimulate hippocampal but not olfactory bulb neurogenesis.

Exposure to an enriched environment and physical activity, such as voluntary running, increases neurogenesis of granule cells in the dentate gyrus of adult mice. These stimuli are also known to improve performance in hippocampus‐dependent learning tasks, but it is unclear whether their effects on neurogenesis are exclusive to the hippocampal formation. In this study, we housed adult mice under three conditions (enriched environment, voluntary wheel running and standard housing), and analysed proliferation in the lateral ventricle wall and granule cell neurogenesis in the olfactory bulb in comparison to the dentate gyrus. Using bromodeoxyuridine to label dividing cells, we could not detect any difference in the number of newly generated cells in the ventricle wall. When giving the new cells time to migrate and differentiate in the olfactory bulb, we observed no changes in the number of adult‐generated olfactory granule cells; however, voluntary running and enrichment produced a doubling in the amount of new hippocampal granule cells. The discrepancy between the olfactory bulb and the dentate gyrus suggests that these living conditions trigger locally through an as yet unidentified mechanism specific to neurogenic signals in the dentate gyrus.

Temporal and Spatial Dynamics of Brain Structure Changes during Extensive Learning

The current view regarding human long-term memory as an active process of encoding and retrieval includes a highly specific learning-induced functional plasticity in a network of multiple memory systems. Voxel-based morphometry was used to detect possible structural brain changes associated with learning. Magnetic resonance images were obtained at three different time points while medical students learned for their medical examination. During the learning period, the gray matter increased significantly in the posterior and lateral parietal cortex bilaterally. These structural changes did not change significantly toward the third scan during the semester break 3 months after the exam. The posterior hippocampus showed a different pattern over time: the initial increase in gray matter during the learning period was even more pronounced toward the third time point. These results indicate that the acquisition of a great amount of highly abstract information may be related to a particular pattern of structural gray matter changes in particular brain areas.

Long‐term survival and cell death of newly generated neurons in the adult rat olfactory bulb

In the adult rat olfactory bulb, neurons are continually generated from progenitors that reside in the lateral ventricle wall. This study investigates long‐term survival and cell death of newly generated cells within the adult olfactory bulb. After injecting rats at 2 months of age with 5‐bromodeoxyuridine (BrdU), the newly generated cells were quantified over a period of 19 months. A peak of BrdU‐positive cells was reached in the olfactory bulb 1 month after BrdU injection, when all new cells have finished migrating from the ventricle wall. Thereafter, a reduction of BrdU‐positive cells to about 50% was observed and it was confirmed by dUTP‐nick end‐labelling (TUNEL) that progenitors and young neurons undergo programmed cell death. However, cells that survived the first 3 months after BrdU injection persisted for up to 19 months. The majority of the BrdU‐positive cells that reach the olfactory bulb differentiate into granule cells, but a small fraction migrate further into the glomerular layer. These newborn cells differentiate more slowly into periglomerular interneurons, with a delay of more than 1 month when compared to the granule cells. The newly generated periglomerular neurons, among them a significant fraction of dopaminergic cells, showed a similar decline in number compared to the granule cell layer and long‐term survival for the remaining new neurons of up to 19 months. Rather than replacing old neurons, this data suggests that adult olfactory bulb neurogenesis utilizes the overproduction and turnover of young neurons, which is reminiscent of the cellular dynamics observed during brain development.

Direct stimulation of adult neural stem cells in vitro and neurogenesis in vivo by vascular endothelial growth factor.

Hypoxia as well as global and focal ischemia are strong activators of neurogenesis in the adult mammalian central nervous system. Here we show that the hypoxia‐inducible vascular endothelial growth factor (VEGF) and its receptor VEGFR‐2/Flk‐1 are expressed in clonally‐derived adult rat neural stem cells in vitro. VEGF stimulated the expansion of neural stem cells whereas blockade of VEGFR‐2/Flk‐1‐kinase activity reduced neural stem cell expansion. VEGF was also infused into the lateral ventricle to study changes in neurogenesis in the ventricle wall, olfactory bulb and hippocampus. Using a low dose (2.4 ng/d) to avoid endothelial proliferation and changes in vascular permeability, VEGF stimulated adult neurogenesis in vivo. After VEGF infusion, we observed reduced apoptosis but unaltered proliferation suggesting a survival promoting effect of VEGF in neural progenitor cells. Strong expression of VEGFR‐2/Flk‐1 was detected in the ventricle wall adjacent to the choroid plexus, a site of significant VEGF production, which suggests a paracrine function of endogenous VEGF on neural stem cells in vivo. We propose that VEGF acts as a trophic factor for neural stem cells in vitro and for sustained neurogenesis in the adult nervous system. These findings may have implications for the pathogenesis and therapy of neurodegenerative diseases.

Decreased neurogenesis after cholinergic forebrain lesion in the adult rat.

Adult neurogenesis has been shown to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic system of the basal forebrain is one of the key transmitter systems for learning and memory. Because adult neurogenesis has been implicated in cognitive performance, the present work aims at defining the role of cholinergic input for adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG‐saporin was infused into the lateral ventricle of adult rats to selectively lesion cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the two main regions of adult neurogenesis: the dentate gyrus and the olfactory bulb. Five weeks after lesioning, neurogenesis, defined by the number of cells colocalized for bromodeoxyuridine (BrdU) and the neuronal nuclei marker NeuN, declined significantly in the granule cell layers of the dentate gyrus and olfactory bulb. Furthermore, immunotoxic lesions to the CBF led to increased numbers of apoptotic cells specifically in the subgranular zone, the progenitor region of the dentate gyrus, and within the periglomerular layer of the olfactory bulb. We propose that the cholinergic system plays a survival‐promoting role for neuronal progenitors and immature neurons within regions of adult neurogenesis, similar to effects observed previously during brain development. As a working hypothesis, neuronal loss within the CBF system leads not only to cognitive deficits but may also alter on a cellular level the functionality of the dentate gyrus, which in turn may aggravate cognitive deficits.

Adult neurogenesis: a compensatory mechanism for neuronal damage

Abstract It is now evident that the adult vertebrate brain including the human brain is efficiently and continuously generating new neurons. In the first part we describe the current view of how neurons are generated in the adult brain and the possible compensatory reactions to pathological situations in which neuronal damage might stimulate neural stem cell activity. In the second part, we discuss the current knowledge on the signals and cells involved in the process of neurogenesis. This knowledge is important because any neuronal replacement strategy depends on our ability to induce or modulate each step on the way to a new neuron: stem cell proliferation, cell fate determination, progenitor migration, and differentiation into specific neuronal phenotypes. Identification of the molecular signals that control these events are essential for the application of neural stem cell biology to develop repair strategies for neurodegenerative disorders.

14 The Balance of Trophic Support and Cell Death in Adult Neurogenesis

The fact that continuous proliferation of stem cells and progenitors, as well as the production of neurons, occurs in the adult CNS raises several basic questions concerning the number of neurons required in a particular system: Can we observe a continued growth of brain regions that sustain neurogenesis? Or does an elimination mechanism exist that keeps the number of cells constant? If so, are the old ones replaced or are the new neurons competing for limited network access? What signals would support their survival and integration and what factors are responsible for their elimination? This chapter addresses these and other questions regarding regulatory mechanisms affecting adult neurogenesis by controlling cell survival. ARE NEUROGENIC BRAIN REGIONS EXPANDING DESPITE SPACE LIMITATIONS? This question was initially addressed several decades ago, following the first evidence that adult mammalian neurogenesis exists. Total neuronal cell counts of the olfactory bulb (OB) and dentate gyrus (DG) at different ages revealed that in both regions, a continued growth of the granule cell layer occurs throughout adult life. From 1 month of age, when the developmental production of granule cells can be considered complete, until 1 year of age, the number of DG granule cells doubles in the rat (Bayer 1982; Bayer et al. 1982). A rise in total volume and increased cell density due to reduced cell diameter both contribute to this phenomenon. In the rat OB, a linear growth of the granule cell layer was observed with age (Kaplan et al. 1985), with the number of olfactory...

3 Detection and Phenotypic Characterization of Adult Neurogenesis

Advances in our understanding of the extent and regulation of adult neurogenesis have been dependent on continued improvements in the detection and quantification of critical events in neurogenesis. To date, no specific and exclusive stem cell marker has been described that would allow for prospective studies of neurogenesis. As a result, detection of neurogenic events has depended on a combination of labeling approaches that document the two critical events in neurogenesis: the generation of new cells and their subsequent progression through lineage commitment to a mature neuron. Detection of neurogenesis in vivo requires the ability to image at a cellular resolution. Although advances in noninvasive imaging approaches, such as magnetic resonance imaging (MRI), show promise for longitudinal studies of neurogenesis, the lack of suitable resolution to characterize individual cells limits the information that can be obtained. In vivo microscopy, using deeply penetrating UV illumination with mulitphoton microscopy or by the recently available endoscopic confocal microscopy, may provide new opportunities for longitudinal studies of neurogenesis in the living animal with single-cell resolution. These latter microscopy approaches are particularly compelling when coupled with transgenic mice expressing phenotype-specific fluorescent reporter genes. However, at present, the predominant approach for studies of neurogenesis relies on traditional histological methods of fixation, production of tissue sections, staining, and microscopic analysis. This chapter discusses methodological considerations for in vivo detection of neurogenesis in the adult brain according to our current state of knowledge. First, detection of newly generated cells is evaluated and the strengths of using exogenous or...