Takashi Namba

GABAergic Excitation Promotes Neuronal Differentiation in Adult Hippocampal Progenitor Cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.

Roles of disrupted-in-schizophrenia 1-interacting protein girdin in postnatal development of the dentate gyrus.

Disrupted-In-Schizophrenia 1 (DISC1), a susceptibility gene for major psychiatric disorders, regulates neuronal migration and differentiation during mammalian brain development. Although roles for DISC1 in postnatal neurogenesis in the dentate gyrus (DG) have recently emerged, it is not known how DISC1 and its interacting proteins govern the migration, positioning, and differentiation of dentate granule cells (DGCs). Here, we report that DISC1 interacts with the actin-binding protein girdin to regulate axonal development. DGCs in girdin-deficient neonatal mice exhibit deficits in axonal sprouting in the cornu ammonis 3 region of the hippocampus. Girdin deficiency, RNA interference-mediated knockdown, and inhibition of the DISC1/girdin interaction lead to overextended migration and mispositioning of the DGCs resulting in profound cytoarchitectural disorganization of the DG. These findings identify girdin as an intrinsic factor in postnatal development of the DG and provide insights into the critical role of the DISC1/girdin interaction in postnatal neurogenesis in the DG.

Repetitive Cocaine Administration Decreases Neurogenesis in Adult Rat Hippocampus

Abstract: Cocaine HCl (20 mg/kg) was administered to adult male rats to investigate the effects of cocaine on neurogenesis in the hippocampus. Proliferation of granule cells in the dentate gyrus was measured by in vivo labeling with 5‐bromo‐2′‐deoxyuridine (BrdU). Rats that received repetitive cocaine treatment for 14 days showed 26% fewer BrdU‐positive cells relative to control rats, while no difference was observed in the rats that received a single injection of cocaine. Differentiation of newly born cells was not influenced. The present experiment is the first to demonstrate the influence of cocaine on hippocampal neurogenesis. These data suggest that the regulation of hippocampal neurogenesis may be involved in the emergence of certain symptoms of cocaine addiction, such as cognitive impairment and behavioral sensitization.

The fate of neural progenitor cells expressing astrocytic and radial glial markers in the postnatal rat dentate gyrus

In the dentate gyrus neurons continue to be generated from late embryonic to adult stage. Recent extensive studies have unveiled several key aspects of the adult neurogenesis, but only few attempts have so far been made on the analysis of the early postnatal neurogenenesis, a transition state between the embryonic and adult neurogenesis. Here, we focus on the early postnatal neurogenesis and examine the nature and development of neural progenitor cells in Wistar rats. Immunohistochemistry for Ki67, a cell cycle marker, and 5‐bromo‐2‐deoxyuridine (BrdU) labelling show that cell proliferation occurs mainly in the hilus and partly in the subgranular zone. A majority of the proliferating cells express S100β and astrocyte‐specific glutamate transporter (GLAST) and the subpopulation are also positive for glial fibrillary acidic protein (GFAP) and nestin. Tracing with BrdU and our modified retrovirus vector carrying enhanced green fluorescent protein (GFP) indicate that a substantial population of the proliferating cells differentiate into proliferative neuroblasts and immature neurons in the hilus, which then migrate to the granule cell layer (66.8%), leaving a long axon‐like process behind in the hilus, and the others mainly become star‐shaped astrocytes (12.0%) and radial glia‐like cells (4.7%) in the subgranular zone. These results suggest that the progenitors of the granule cells expressing astrocytic and radial glial markers, proliferate and differentiate into neurons mainly in the hilus during the early postnatal period.

Clustering, Migration, and Neurite Formation of Neural Precursor Cells in the Adult Rat Hippocampus

Adult neurogenesis occurs in the subgranular zone and innermost part of the dentate granule cell layer. To examine how neural precursor cells proliferate, migrate, and extend their neurites, we performed BrdU‐ and improved retrovirus‐green fluorescence protein (GFP)‐labeling analyses. Soon after labeling the majority of BrdU+ cells and GFP+ cells expressed Ki67, a cell cycle marker, and formed clusters together with PSA+ neuroblasts. Most of the Ki67+ proliferating cells expressed Hu, an immature and mature neuronal marker, and the subpopulation expressed both Hu+ and GFAP+. In the clusters, Ki67+ and PSA+ cells strongly expressed β‐catenin and N‐cadherin, but PSA+ cells outside the clusters did not. Therefore, it was mainly Hu+ neuronal precursor cells that proliferated within clusters in which the cluster cells are closely associated via cell adhesion molecules, such as N‐cadherin/β‐cateninIn and PSA. The newly generated cells appeared to stay in the clusters for a few days and then disperse around the clusters. The findings of this in vivo analysis and in vitro time‐lapse imaging of early postnatal hippocampal slices support the notion that most postmitotic neuroblasts migrate tangentially from clusters, extending tangentially oriented processes, one of which often retains close contact with the clusters, and finally extend radial processes, or prospective apical dendrites. These results suggest that the clustering cells and tangentially migrating cells have a systematic cellular arrangement and intercellular interaction. J. Comp. Neurol. 502:275–290, 2007.

Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brains cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.

Pioneering Axons Regulate Neuronal Polarization in the Developing Cerebral Cortex

The polarization of neurons, which mainly includes the differentiation of axons and dendrites, is regulated by cell-autonomous and non-cell-autonomous factors. In the developing central nervous system, neuronal development occurs in a heterogeneous environment that also comprises extracellular matrices, radial glial cells, and neurons. Although many cell-autonomous factors that affect neuronal polarization have been identified, the microenvironmental cues involved in neuronal polarization remain largely unknown. Here, we show that neuronal polarization occurs in a microenvironment in the lower intermediate zone, where the cell adhesion molecule transient axonal glycoprotein-1 (TAG-1) is expressed in cortical efferent axons. The immature neurites of multipolar cells closely contact TAG-1-positive axons and generate axons. Inhibition of TAG-1-mediated cell-to-cell interaction or its downstream kinase Lyn impairs neuronal polarization. These results show that the TAG-1-mediated cell-to-cell interaction between the unpolarized multipolar cells and the pioneering axons regulates the polarization of multipolar cells partly through Lyn kinase and Rac1.

Glial fibrillary acidic protein-expressing neural progenitors give rise to immature neurons via early intermediate progenitors expressing both glial fibrillary acidic protein and neuronal markers in the adult hippocampus

Adult neurogenesis occurs in the subgranular zone (SGZ) of the dentate gyrus, where primary neuronal progenitors that express glial fibrillary acidic protein (GFAP) develop into granule neurons. Here, we used transgenic mice with mouse GFAP promoter-controlled enhanced green fluorescent protein (mGFAP-EGFP Tg mice) to examine how astrocyte-like progenitors differentiate into neuron-committed progenitors. Bromodeoxyuridine (BrdU) analysis indicated that proliferating cells in the neurogenic SGZ transiently expressed EGFP and GFAP, and finally differentiated into cells positive for the neuronal marker, Hu (Hu+). Most proliferating EGFP+ cells showed expression of the stem cell marker, Sox2, and formed clusters of two to four cells containing GFAP+/EGFP+ and GFAP-/EGFP+ cells. No GFAP-/EGFP+ cells were detected in non-neurogenic regions, such as CA1 and CA3 of the pyramidal cell layer. Together with the assumption that exogeneous EGFP has a higher stability than that of endogenous GFAP in the degradation process, it is highly probable that the GFAP-/EGFP+ cells were daughter cells or immediate progeny derived from GFAP+/EGFP+ cells. The subpopulation of proliferating GFAP+/EGFP+ cells expressed proneural protein Mash1 and neuronal marker Hu, while the proliferating GFAP-/EGFP+ cells expressed additional immature neuronal markers, such as polysialic acid-neural cell adhesion molecule (PSA-NCAM) and doublecortin. Therefore, these results suggest that through a few cell divisions, GFAP+ progenitors give rise to neuronal progenitors via neuron-committed early intermediate progenitors that express both GFAP and Hu (and/or Mash1). The findings of the present study also indicated that mGFAP-EGFP Tg mice are useful animals for identifying the daughter cells or immediate progeny derived from GFAP+ neural progenitors.

Girdin Is an Intrinsic Regulator of Neuroblast Chain Migration in the Rostral Migratory Stream of the Postnatal Brain

In postnatally developing and adult brains, interneurons of the olfactory bulb (OB) are continuously generated at the subventricular zone of the forebrain. The newborn neuroblasts migrate tangentially to the OB through a well defined pathway, the rostral migratory stream (RMS), where the neuroblasts undergo collective migration termed “chain migration.” The cell-intrinsic regulatory mechanism of neuroblast chain migration, however, has not been uncovered. Here we show that mice lacking the actin-binding Akt substrate Girdin (a protein that interacts with Disrupted-In-Schizophrenia 1 to regulate neurogenesis in the dentate gyrus) have profound defects in neuroblast chain migration along the RMS. Analysis of two gene knock-in mice harboring Girdin mutants identified unique amino acid residues in Girdins C-terminal domain that are responsible for the regulation of neuroblast chain migration but revealed no apparent requirement of Girdin phosphorylation by Akt. Electron microscopic analyses demonstrated the involvement of Girdin in neuroblast cell–cell interactions. These findings suggest that Girdin is an important intrinsic factor that specifically governs neuroblast chain migration along the RMS.

NMDA receptor antagonist memantine promotes cell proliferation and production of mature granule neurons in the adult hippocampus

Memantine, which is used clinically for the treatment of Alzheimers disease (AD), is classified as an N-methyl-d-aspartate (NMDA) receptor antagonist. Since previous studies have shown that NMDA receptor antagonists promote neurogenesis in the adult brain, we examined the effect of memantine on neurogenesis in the adult mouse hippocampus. We intraperitoneally injected 3-month-old mice with memantine (at 10 or 50 mg/kg body weight) followed by 5-bromo-2-deoxyuridine (BrdU) injections (3x) after 3 days. We then examined the number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus at different time points. The number of BrdU+ cells in the 50 mg/kg memantine-injected group increased by 2.1-fold (1 day after BrdU-injection), 3.4-fold (after 7 days), and 6.8-fold (after 28 days), whereas the 10 mg/kg dose of memantine had little effect on labeling compared to the control group. Immunohistochemical staining at 28 days after BrdU-injection revealed that the newly generated cells in the 50 mg/kg memantine-group had differentiated into mature granule neurons. Moreover, when 12-month-old mice were injected with memantine, cell proliferation was promoted in the DG (3.7-fold). These findings demonstrate that memantine promotes the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus.