Jeffrey D. Macklis

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.

Neuronal subtype specification in the cerebral cortex

In recent years, tremendous progress has been made in understanding the mechanisms underlying the specification of projection neurons within the mammalian neocortex. New experimental approaches have made it possible to identify progenitors and study the lineage relationships of different neocortical projection neurons. An expanding set of genes with layer and neuronal subtype specificity have been identified within the neocortex, and their function during projection neuron development is starting to be elucidated. Here, we assess recent data regarding the nature of neocortical progenitors, review the roles of individual genes in projection neuron specification and discuss the implications for progenitor plasticity.

Mena is required for neurulation and commissure formation.

Mammalian enabled (Mena) is a member of a protein family thought to link signal transduction pathways to localized remodeling of the actin cytoskeleton. Mena binds directly to Profilin, an actin-binding protein that modulates actin polymerization. In primary neurons, Mena is concentrated at the tips of growth cone filopodia. Mena-deficient mice are viable; however, axons projecting from interhemispheric cortico-cortical neurons are misrouted in early neonates, and failed decussation of the corpus callosum as well as defects in the hippocampal commissure and the pontocerebellar pathway are evident in the adult. Mena-deficient mice that are heterozygous for a Profilin I deletion die in utero and display defects in neurulation, demonstrating an important functional role for Mena in regulation of the actin cytoskeleton.

Fezl Is Required for the Birth and Specification of Corticospinal Motor Neurons

The molecular mechanisms controlling the differentiation of neural progenitors into distinct subtypes of neurons during neocortical development are unknown. Here, we report that Fezl is required for the specification of corticospinal motor neurons and other subcerebral projection neurons, which are absent from Fezl null mutant neocortex. There is neither an increase in cell death in Fezl(-/-) cortex nor abnormalities in migration, indicating that the absence of subcerebral projection neurons is due to a failure in fate specification. In striking contrast, other neuronal populations in the same and other cortical layers are born normally. Overexpression of Fezl results in excess production of subcerebral projection neurons and arrested migration of these neurons in the germinal zone. These data indicate that Fezl plays a central role in the specification of corticospinal motor neurons and other subcerebral projection neurons, controlling early decisions regarding lineage-specific differentiation from neural progenitors.

MECP2 is progressively expressed in post-migratory neurons and is involved in neuronal maturation rather than cell fate decisions.

Rett syndrome is a neurodevelopmental disorder and one of the causes of mental retardation and autistic behavior in girls, as well as in a small group of boys. It was recently discovered that mutation of the methyl-CpG-binding protein 2 (MECP2) gene encoding a transcriptional repressor on the X chromosome causes Rett syndrome. Although it is evident that phenotypes of MECP2 mutant mice that resemble those of Rett syndrome are attributable to lack of the MECP2 gene in the central nervous system (CNS), there is little understanding of the neuropathological abnormalities in the CNS of MECP2-null mice. Here, we investigated the developmental regulation and specific cellular expression of MECP2 during neural development both in vitro and in vivo. MECP2 is expressed in mature neurons, but not in astroglia or oligodendroglia, and is increasingly expressed during development of the mouse neocortex. In addition, in vitro culture studies suggest that MECP2 is expressed in more differentiated neurons rather than in less differentiated neuroblasts. Under in vitro conditions using neural precursor cultures, we find that MECP2 mutant neural precursors differentiate into morphologically mature neurons and glia, and no significant differences in differentiation are detected between cells from wild-type and MECP2 mutant mice, suggesting that MECP2 may play a different role in mice than it does in Xenopus embryos. In agreement with this hypothesis, neocortical projection layers in MECP2 -/y mice are thinner than those in wild-type mice, and pyramidal neurons in layer II/III in MECP2 -/y mice are smaller and less complex than those in wild-type mice. Taken together, our results indicate that MECP2 is involved in the maturation and maintenance of neurons, including dendritic arborization, rather than in cell fate decisions.

Targeted Neuronal Death Affects Neuronal Replacement and Vocal Behavior in Adult Songbirds

In the high vocal center (HVC) of adult songbirds, increases in spontaneous neuronal replacement correlate with song changes and with cell death. We experimentally induced death of specific HVC neuron types in adult male zebra finches using targeted photolysis. Induced death of a projection neuron type that normally turns over resulted in compensatory replacement of the same type. Induced death of the normally nonreplaced type did not stimulate their replacement. In juveniles, death of the latter type increased recruitment of the replaceable kind. We infer that neuronal death regulates the recruitment of replaceable neurons. Song deteriorated in some birds only after elimination of replaceable neurons. Behavioral deficits were transient and followed by variable degrees of recovery. This raises the possibility that induced neuronal replacement can restore a learned behavior.

IGF-I specifically enhances axon outgrowth of corticospinal motor neurons

Corticospinal motor neurons (CSMN) are among the most complex CNS neurons; they control voluntary motor function and are prototypical projection neurons. In amyotrophic lateral sclerosis (ALS), both spinal motor neurons and CSMN degenerate; their damage contributes centrally to the loss of motor function in spinal cord injury. Direct investigation of CSMN is severely limited by inaccessibility in the heterogeneous cortex. Here, using new CSMN purification and culture approaches, and in vivo analyses, we report that insulin-like growth factor-1 (IGF-I) specifically enhances the extent and rate of murine CSMN axon outgrowth, mediated via the IGF-I receptor and downstream signaling pathways; this is distinct from IGF-I support of neuronal survival. In contrast, brain-derived neurotrophic factor (BDNF) enhances branching and arborization, but not axon outgrowth. These experiments define specific controls over directed differentiation of CSMN, indicate a distinct role of IGF-I in CSMN axon outgrowth during development, and might enable control over CSMN derived from neural precursors.

SOX5 Controls the Sequential Generation of Distinct Corticofugal Neuron Subtypes

The molecular mechanisms controlling the development of distinct subtypes of neocortical projection neurons, and CNS neuronal diversity more broadly, are only now emerging. We report that the transcription factor SOX5 controls the sequential generation of distinct corticofugal neuron subtypes by preventing premature emergence of normally later-born corticofugal neurons. SOX5 loss-of-function causes striking overlap of the identities of the three principal sequentially born corticofugal neuron subtypes: subplate neurons, corticothalamic neurons, and subcerebral projection neurons. In Sox5(-/-) cortex, subplate neurons aberrantly develop molecular hallmarks and connectivity of subcerebral projection neurons; corticothalamic neurons are imprecisely differentiated, while differentiation of subcerebral projection neurons is accelerated. Gain-of-function analysis reinforces the critical role of SOX5 in controlling the sequential generation of corticofugal neurons--SOX5 overexpression at late stages of corticogenesis causes re-emergence of neurons with corticofugal features. These data indicate that SOX5 controls the timing of critical fate decisions during corticofugal neuron production and thus subtype-specific differentiation and neocortical neuron diversity.The molecular mechanisms controlling the development of distinct subtypes of neocortical projection neurons, and CNS neuronal diversity more broadly, are only now emerging. We report that the transcription factor SOX5 controls the sequential generation of distinct corticofugal neuron subtypes by preventing premature emergence of normally later-born corticofugal neurons. SOX5 loss-of-function causes striking overlap of the identities of the three principal sequentially born corticofugal neuron subtypes: subplate neurons, corticothalamic neurons, and subcerebral projection neurons. In Sox5(-/-) cortex, subplate neurons aberrantly develop molecular hallmarks and connectivity of subcerebral projection neurons; corticothalamic neurons are imprecisely differentiated, while differentiation of subcerebral projection neurons is accelerated. Gain-of-function analysis reinforces the critical role of SOX5 in controlling the sequential generation of corticofugal neurons--SOX5 overexpression at late stages of corticogenesis causes re-emergence of neurons with corticofugal features. These data indicate that SOX5 controls the timing of critical fate decisions during corticofugal neuron production and thus subtype-specific differentiation and neocortical neuron diversity.

Progressive incorporation of propidium iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity

We describe a visual assay of neuronal electrophysiologic status for use with cultured neurons, based on the exclusion of propidium iodide (PI) by intact cellular membranes. We use this fluorescent dye, which binds to nucleic acids, at concentrations suitable for long-term exposure to neurons without toxicity. We correlate the progressive loss of resting membrane potential and the progressive inability to generate stimulated action potentials by cultured mouse dorsal root ganglion neurons with increasing incorporation of PI. The scoring system used to gauge incorporation of PI is rapid and highly reproducible using a standard fluorescence microscope. Applications exist for studies of neuronal toxicity, survival, and electrophysiology in vitro.

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.