Neurogenesis normally only occurs in limited areas of the adult mammalian brain--the hippocampus, olfactory bulb and epithelium, and at low levels in some regions of macaque cortex. Here we show that endogenous neural precursors can be induced in situ to differentiate into mature neurons, in regions of adult mammalian neocortex that do not normally undergo any neurogenesis. This differentiation occurs in a layer- and region-specific manner, and the neurons can re-form appropriate corticothalamic connections. We induced synchronous apoptotic degeneration of corticothalamic neurons in layer VI of anterior cortex of adult mice and examined the fates of dividing cells within cortex, using markers for DNA replication (5-bromodeoxyuridine; BrdU) and progressive neuronal differentiation. Newly made, BrdU-positive cells expressed NeuN, a mature neuronal marker, in regions of cortex undergoing targeted neuronal death and survived for at least 28 weeks. Subsets of BrdU+ precursors expressed Doublecortin, a protein found exclusively in migrating neurons, and Hu, an early neuronal marker. Retrograde labelling from thalamus demonstrated that BrdU+ neurons can form long-distance corticothalamic connections. Our results indicate that neuronal replacement therapies for neurodegenerative disease and CNS injury may be possible through manipulation of endogenous neural precursors in situ.

Neurogenesis continues throughout adulthood in the mammalian olfactory bulb and hippocampal dentate gyrus, suggesting the hypothesis that recently generated, adult-born neurons contribute to neural plasticity and learning. To explore this hypothesis, we examined whether olfactory experience modifies the responses of adult-born neurons to odorants, using immediate early genes (IEGs) to assay the response of olfactory granule neurons. We find that, shortly after they differentiate and synaptically integrate, the population of adultborn olfactory granule neurons has a greater population IEG response to novel odors than mature, preexisting neurons. Familiarizing mice with test odors increases the response of the recently incorporated adult-born neuron population to the test odors, and this increased responsiveness is long lasting, demonstrating that the response of the adult-born neuron population is altered by experience. In contrast, familiarizing mice with test odors decreases the IEG response of developmentally generated neurons, suggesting that recently generated adult-born neurons play a distinct role in olfactory processing. The increased IEG response is stimulus specific; familiarizing mice with a set of different, "distractor" odors does not increase the adult-born neuron population response to the test odors. Odor familiarization does not influence the survival of adult-born neurons, indicating that the changes in the population response of adultborn neurons are not attributable to increased survival of odor-stimulated neurons. These results demonstrate that recently generated adult-born olfactory granule neurons and older, preexisting granule neurons undergo contrasting experience-dependent modifications in vivo and support the hypothesis that adult-born neurons are involved in olfactory learning.

The adult mammalian CNS shows a very limited capacity to regenerate after injury. However, endogenous precursors, or stem cells, provide a potential source of new neurons in the adult brain. Here, we induce the birth of new corticospinal motor neurons (CSMN), the CNS neurons that die in motor neuron degenerative diseases, including amyotrophic lateral sclerosis, and that cause loss of motor function in spinal cord injury. We induced synchronous apoptotic degeneration of CSMN and examined the fates of newborn cells arising from endogenous precursors, using markers for DNA replication, neuroblast migration, and progressive neuronal differentiation, combined with retrograde labeling from the spinal cord. We observed neuroblasts entering the neocortex and progressively differentiating into mature pyramidal neurons in cortical layer V. We found 20 -30 new neurons per mm 3 in experimental mice vs. 0 in controls. A subset of these newborn neurons projected axons into the spinal cord and survived >56 weeks. These results demonstrate that endogenous precursors can differentiate into even highly complex long-projection CSMN in the adult mammalian brain and send new projections to spinal cord targets, suggesting that molecular manipulation of endogenous neural precursors in situ may offer future therapeutic possibilities for motor neuron degenerative disease and spinal cord injury. The identification of populations of neural precursors, or stem cells, in the adult mammalian CNS raises the possibility of future repair of neuronal loss from neurodegenerative diseases or neuronal damage from brain and spinal cord injuries resulting from trauma or stroke (1-5). In the adult brain, new neurons are continuously generated, but such neurogenesis is normally restricted to two evolutionarily primitive regions: the olfactory bulb, from precursors in the subventricular zone (SVZ) (6), and the hippocampal dentate gyrus (1, 7), from local precursors in the subgranular zone.A variety of factors, ranging from genetics (8) to environmental modifications, can modulate neurogenesis in these regions. Exercise (9), environmental enrichment (10), pregnancy (11), and even seizure activity (12, 13) promote neurogenesis, whereas depression (14) and aging (15) reduce it. Modulating the cellular and molecular factors that control adult neurogenesis could yield new approaches to replacing neurons lost to injury or disease.Recently, our laboratory and those of others have demonstrated that neurogenesis can be induced from endogenous precursors by manipulating the microenvironment in regions of the adult CNS that are normally nonneurogenic (16-21). Selective neuronal death due to targeted apoptosis or ischemia induces endogenous precursors to divide and differentiate into neurons in mammalian neocortical layer VI (16), hippocampal region CA1 (17), striatum (18,19), substantia nigra (20), and the high vocal center in songbirds (21). These neurons express neuron-specific proteins and adopt appropriate morphologies, and in some cases their recruitment cor...

Astrocytes, one of the most common cell types in the brain, are essential for processes ranging from neural development through potassium homeostasis to synaptic plasticity. Surprisingly, the developmental origins of astrocytes in the neocortex are still controversial. To investigate the patterns of astrocyte development in the neocortex we examined cortical development in a transgenic mouse in which a random, sparse subset of neural progenitors undergoes CRE/lox recombination, permanently labeling their progeny. We demonstrate that neural progenitors in neocortex generate discrete columnar structures that contain both projection neurons and protoplasmic astrocytes. Ninety five percent of developmental cortical columns labeled in our system contained both astrocytes and neurons. The astrocyte to neuron ratio of labeled cells in a developmental column was 1:7.4, similar to the overall ratio of 1:8.4 across the entire grey matter of the neocortex, indicating that column-associated astrocytes account for the majority of protoplasmic astrocytes in neocortex. Most of the labeled columns contained multiple clusters of several astrocytes. Dividing cells were found at the base of neuronal columns at the beginning of gliogenesis, and later within the cortical layers, suggesting a mechanism by which astrocytes could be distributed within a column. These data indicate that radial glia are the source of both neurons and astrocytes in the neocortex, and that these two cell types are generated in a spatially restricted manner during cortical development.

Neurogenesis continues through adulthood in the hippocampus and olfactory bulb of mammals. Adult neurogenesis has been implicated in learning and memory, and linked with depression. Hippocampal neurogenesis is increased in response to a number of stimuli, including exposure to an enriched environment, increased locomotor activity, and administration of antidepressants. Adult neurogenesis is depressed in response to aging, stress and sleep deprivation. Intriguingly, caffeine modulates a number of these same stimuli in a dose-dependent manner. We examined the dose and duration dependent effects of caffeine on the proliferation, differentiation, and survival of newly generated hippocampal neurons in adult mice. Extended, 7-day caffeine administration, alters the proliferation of adult hippocampal precursors in the mouse in a dose dependent manner; moderate to high doses (20-30mg/kg/day) of caffeine depress proliferation while supraphysiological doses (60mg/kg/day) increase proliferation of neuronal precursors. Acute, 1-day administration had no affect on proliferation. Caffeine administration does not affect the expression of early or late markers of neuronal differentiation, or rates of long-term survival. However, neurons induced in response to supraphysiological levels of caffeine have a lower survival rate than control cells; increased proliferation does not yield an increase in long-term neurogenesis. These results demonstrate that physiologically relevant doses of caffeine can significantly depress adult hippocampal neurogenesis.

Bromodeoxyuridine, variously abbreviated as BrdU, BudR, and BrdUrd, is a halogenated thymidine analog that is permanently integrated into the DNA of dividing cells during DNA synthesis in S phase. BrdU can be immunocytochemically detected in vitro and in vivo, allowing the identification of cells that were dividing the period of BrdU exposure. In vivo, it has been used to identify the "birthdate" of cells during development, to examine the fate of postnatally generated cells, and to label cells before transplantation, for subsequent identification.

Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. During the past 3 decades, research exploring potential neuronal replacement therapies has focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. However, in the last decade, the development of new techniques has resulted in an explosion of new research showing that neurogenesis, the birth of new neurons, normally occurs in two limited and specific regions of the adult mammalian brain and that there are significant numbers of multipotent neural precursors in many parts of the adult mammalian brain. Recent advances in our understanding of related events of neural development and plasticity, including the role of radial glia in developmental neurogenesis and the ability of endogenous precursors present in the adult brain to be induced to produce neurons and partially repopulate brain regions affected by neurodegenerative processes, have led to fundamental changes in the views about how the brain develops as well as to approaches by which endogenous precursors might be recruited to repair the adult brain. Recruitment of new neurons can be induced in a region-specific, layer-specific and neuronal-type-specific manner, and, in some cases, newly recruited neurons can form long-distance connections to appropriate targets. Elucidation of the relevant molecular controls may both allow control over transplanted precursor cells and potentially allow the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that do not require transplantation of exogenous cells.

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