Sanjay S. P. Magavi

Induction of neurogenesis in the neocortex of adult mice.

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.

Adult-Born and Preexisting Olfactory Granule Neurons Undergo Distinct Experience-Dependent Modifications of their Olfactory Responses In Vivo

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 adult-born 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 adult-born 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.

Induction of neuronal type-specific neurogenesis in the cerebral cortex of adult mice: manipulation of neural precursors in situ

Over the past 3 decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain [Nat. Neurosci. 3 (2000) 67-78]. Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. 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 [Mol. Cell. Neurosci. 19 (1999) 474-486]. Recent findings from our laboratory demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons [Nature 405 (2000) 951-955; Neuron 25 (2000) 481-492]. Recruitment of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and 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 central nervous system injuries that do not require transplantation of exogenous cells.

Manipulation of neural precursors in situ: induction of neurogenesis in the neocortex of adult mice.

Over the past three decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain (Bjorklund and Lindvall 2000). Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. 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 (Palmer et al. 1995). Recent findings from our lab demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons (Scharff et al. 2000; Magavi et al. 2000). Elucidation of the relevant molecular controls may allow the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that do not require transplantation of exogenous cells.

Constitutive and induced neurogenesis in the adult mammalian brain: manipulation of endogenous precursors toward CNS repair.

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.

The repair of complex neuronal circuitry by transplanted and endogenous precursors

SummaryDuring the past three 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 novel approaches 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 transplanted or endogenous precursors might be used to repair the adult brain. For example, 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 for the development of neuronal replacement therapies for neurodegenerative disease and other CNS injuries that might not require transplantation of exogenous cells.

Immunocytochemical analysis of neuronal differentiation.

Fully understanding the phenotype of neurons in vivo involves examining their morphology, immunocytochemically analyzing their protein expression, examining their afferent and efferent integration into neuronal microcircuitry, and functionally examining their activity. This task is significantly more difficult when you are attempting to determine whether multipotent precursor cells, often referred to as stem cells, differentiate into neurons in vivo. Transplanted or endogenous precursor cells often produce relatively small numbers of new neurons in the adult brain, making electron microscopy or electrophysiological analysis extremely challenging, and functional analysis difficult. Studying such cells usually depends heavily on immunocytochemical approaches. We review a range of immunocytochemical techniques for identifying whether transplanted or endogenous neural precursors have differentiated into mature neurons. We provide immunocytochemical protocols for the migratory neuronal marker Doublecortin (Dcx), the early expressed marker Hu, and mature neuronal marker NeuN. In Chapters 25 and 27 of Part IV, we provide protocols for identifying newborn cells by using the mitotic label bromodeoxyuridine and for examining axonal projections by using the retrograde label FluoroGold.

Molecular manipulation of neural precursors in situ: induction of adult cortical neurogenesis

Over the past three decades, research exploring potential neuronal replacement therapies have focused on replacing lost neurons by transplanting cells or grafting tissue into diseased regions of the brain. Over most of the past century of modern neuroscience, it was thought that the adult brain was completely incapable of generating new neurons. 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 findings from our lab demonstrate that it is possible to induce neurogenesis de novo in the adult mammalian brain, particularly in the neocortex where it does not normally occur, and that it may become possible to manipulate endogenous multipotent precursors in situ to replace lost or damaged neurons. Recruitment of new neurons can be induced in a region-specific, layer-specific, and neuronal type-specific manner, and 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.

Neuroanatomical tracing of neuronal projections with Fluoro-Gold.

The study of neuronal connectivity requires the ability to trace axons from the neuronal cell body to its axon terminal (anterograde tracing) and from the terminal back to the soma (retrograde tracing). Such neuroanatomical tracing is frequently used to identify neurons on the basis of their pre- or postsynaptic connections. Neuroanatomical tracing has become particularly important in nervous system regeneration and repair, allowing investigators to follow the axon projections of newly born, transplanted, or axotomized neurons in lesioned or neurodegenerative environments. To allow further study of neurons identified and labeled in this way, it is particularly important that tracers are compatible with other tissue processing such as immunocytochemistry. Fluoro-Gold (Fluorochrome Inc., Denver CO) is one such highly flexible fluorescent retrograde marker commonly used for neuronal labeling and neuroanatomical tracing.

Cellular Repair of Complex Cortical Circuitry

The mammalian neocortex is a highly complex structure that consists of a large number of distinct cell types and connections unparalleled throughout the rest of the mammalian central nervous system (CNS). The neocortex is responsible for high-level functions including sensorimotor integration, associative behavior, and cognition. Although many studies demonstrate that embryonic tissue or precursor cells transplanted into the embryonic or neonatal cortex can integrate and connect with a wide variety of targets, such connectivity in most studies using adult recipients has been quite limited (1, 2). In contrast, relatively recent advances in transplantation paradigms from our laboratory and other laboratories have made apparent the possibility of successful reconstruction of complex cortical circuitry in the adult mammalian brain by transplantation or by manipulation of endogenous precursors in situ. These advances include the ability to isolate increasingly specific populations of immature neurons and precursors from donor animals, and the establishment of a highly selective model of targeted degeneration in the neocortex (3-10, Leavitt, unpublished observations). Such advances now allow both the transplantation of more defined and specific populations of immature neocortical neuroblasts and precursors, and also make it possible to study the cellular, anatomic, and functional efficacy of both transplantation and manipulation of endogenous precursors in situ (9, 11) in increasingly refined models of neocortical degeneration. The neocortex has become a model system for studying circuitry formation, regional specification, cell specification, and cell autonomous versus environmental influences on neuronal development and function in the CNS. Although neocortical transplantation paradigms have proven very useful for elucidating how complex circuits and CNS cytoarchitecture evolve in the mammalian brain, more recent work using cell transplantation has made apparent the possibility of cell replacement and complex circuit repair in the neocortex. Our most recent results (9, 11) suggest that neuronal replacement therapies for neurodegenerative disease and other CNS injury may even some day be possible via molecular manipulation of endogenous neural precursors in situ, without transplantation. Although only fanciful just several years ago, it now appears that studies of cellular repopulation and circuitry reconstruction in the neocortex may provide a foundation toward developing cellular therapies for degenerative or acquired disease in neocortex and other regions of the CNS.