Yasuharu Takamori

Multi-directional differentiation of doublecortin-and NG2-immunopositive progenitor cells in the adult rat neocortex in vivo

In the adult mammalian brain, multipotent stem or progenitor cells involved in reproduction of neurons and glial cells have been well investigated only in very restricted regions; the subventricular zone of the lateral ventricle and the dentate gyrus in the hippocampal formation. In the neocortex, a series of in vitro studies has suggested the possible existence of neural progenitor cells possessing neurogenic and/or gliogenic potential in adult mammals. However, the cellular properties of the cortical progenitor cells in vivo have not been fully elucidated. Using 5′‐bromodeoxyuridine labeling and immunohistochemical analysis of cell differentiation markers, we found that a subpopulation of NG2‐immunopositive cells co‐expressing doublecortin (DCX), an immature neuron marker, ubiquitously reside in the adult rat neocortex. Furthermore, these cells are the major population of proliferating cells in the region. The DCX(+)/NG2(+) cells reproduced the same daughter cells, or differentiated into DCX(+)/NG2(–) (approximately 1%) or DCX(–)/NG2(+) (approximately 10%) cells within 2 weeks after cell division. The DCX(+)/NG2(–) cells were also immunopositive for TUC‐4, a neuronal linage marker, suggesting that these cells were committed to neuronal cell differentiation, whereas the DCX(–)/NG2(+) cells showed faint immunoreactivity for glutathione S‐transferase (GST)‐pi, an oligodendrocyte lineage marker, in the cytoplasm, suggesting glial cell lineage, and thereafter the cells differentiated into NG2(–)/GST‐pi(+) mature oligodendrocytes after a further 2 weeks. These findings indicate that DCX(+)/NG2(+) cells ubiquitously exist as ‘multipotent progenitor cells’ in the neocortex of adult rats.

Differential expression of nuclear lamin, the major component of nuclear lamina, during neurogenesis in two germinal regions of adult rat brain

Lamins are major structural proteins of the nuclear envelope. Three lamin subtypes, A/C, B1 and B2, predominate in mammalian somatic cells. While the expression levels of lamins in several tissues are known to change during cell differentiation, lamin expression is poorly understood in the nervous system. To investigate the expression of lamins during neuronal differentiation in the mammalian adult brain, we performed immunohistochemical studies on lamins A/C, B1 and B2 in two neurogenic regions of rat brain: the subgranular zone of the dentate gyrus and the subventricular zone of the lateral ventricle. In particular, three types of cells were analysed using confocal microscopy: GFAP‐positive cells as primary progenitor (stem) cells, PSA‐NCAM‐positive cells as subsequent neuronal progenitor cells, and NeuN‐positive mature neurons. GFAP‐positive cells possesed lamin A/C (++), B1 (++) and B2 (++), PSA‐NCAM‐positive cells possessed lamin A/C (–), B1 (+++) and B2 (+), and mature neurons possessed lamin A/C (++), B1 (+) and B2 (+++), in both neurogenic regions. These observations showed that the compositions of expressing lamin subtypes are distinct in particular differentiation stages during neurogenesis in the adult rat brain. Our results suggest that the alteration of nuclear lamina structure is coupled with the progression of neuronal differentiation.

Nestin-positive microglia in adult rat cerebral cortex.

Nestin is a class VI intermediate filament protein, which was first identified in the early developmental stages of the nervous system. It is widely used as a stem or progenitor cell marker. In the adult mammalian brain, nestin is expressed not only in germinal cells in the neurogenic regions but also in non-germinal cells, such as reactive astrocytes, endothelial cells and pericytes. In the present study, we found another nestin-positive cell type within the adult rat cerebral cortex. We immunohistochemically analyzed which types of cells exhibit immunoreactivity for nestin, and through the use of co-immunostaining with Iba1, CD11b and GLUT5, which are known to be specific for microglia, identified these cells as microglia. Approximately >20% of the microglia were immunoreactive for nestin in the rat cerebral cortex under normal conditions. Nestin signals were not widely distributed in the microglial cytoplasm, but were restricted to the perikaryon and to parts of the cell processes. Nestin-positive microglia were also immunoreactive for the intermediate filament protein vimentin. These observations demonstrate that a subpopulation of microglia in a resting state has nestin-containing intermediate filament networks. Therefore, nestin in conjunction with vimentin might have roles in maintaining the structural integrity of the microglia.

Doublecortin expression continues into adulthood in horizontal cells in the rat retina.

The doublecortin (DCX) protein is associated with microtubules, and is essential for neuronal migration, differentiation, and plasticity. In mammals, it is expressed in developing neurons and new immature neuroblasts in the adult brain, but not generally in mature neurons. In the retina, doublecortin is detectable as early as embryonic day 15 (E15), is highly expressed between E18 and E20, and is poorly expressed postnatally. In this study, we investigated immunohistochemically the expression and cellular localization of doublecortin in the adult rat retina. Doublecortin was expressed in the outer plexiform layer (OPL), and in cells in the outer border of the inner nuclear layer (INL). No other layers were labeled by anti-doublecortin antibodies. In double-labeling experiments, doublecortin expression co-localized with the expression of the marker for horizontal cells, calbindin D. By contrast, the marker for immature neuroblasts, polysialylated neural cell-adhesion molecule, was not expressed in horizontal cells. These results suggest that either horizontal cells have the capacity to continuously remodel their neurites or doublecortin has a different function in horizontal cells from the control of neuronal plasticity that it is known to modulate other neurites. In addition, doublecortin might be an alternative molecular marker for horizontal cells in the adult rat retina.

Phenotype Analysis and Quantification of Proliferating Cells in the Cortical Gray Matter of the Adult Rat

In intact adult mammalian brains, there are two neurogenic regions: the subependymal zone and the subgranular layer of the hippocampus. Even outside these regions, small numbers of proliferating precursors do exist. Many studies suggest that the majority of these are oligodendrocyte precursors that express NG2, a chondroitin sulfate proteoglycan, and most of the residual proliferating cells seem to be endothelial cells. However, it is still unclear whether NG2-immunonegative proliferating precursors are present, because previous studies have neglected their possible existence. In this study, we systematically analyzed the phenotypes of the proliferating cells in the intact adult rat cortical gray matter. We improved our techniques and carefully characterized the proliferating cells, because there were several problems with identifying and quantifying the proliferating cells: the detection of NG2-expressing cells was dependent on the fixation condition; there were residual proliferating leukocytes in the blood vessels; and two anti-NG2 antibodies gave rise to different staining patterns. Moreover, we used two methods, BrdU and Ki67 immunostaining, to quantify the proliferating cells. Our results strongly suggest that in the intact adult cerebral cortical gray matter, there were only two types of proliferating cells: the majority were NG2-expressing cells, including pericytes, and the rest were endothelial cells.

Sox2 in the adult rat sensory nervous system

Sex-determining region Y (SRY)-box 2 (Sox2) is a member of the Sox family transcription factors. In the central nervous system, Sox2 is expressed in neural stem cells from neurogenic regions, and regulates stem cell proliferation and differentiation. In the peripheral nervous system, Sox2 is found only in the immature and dedifferentiated Schwann cells, and is involved in myelination inhibition or N-cadherin redistribution. In the present immunohistochemical study, we found that Sox2 is also expressed in other cells of the adult rat peripheral nervous system. Nuclear Sox2 was observed in all satellite glial cells, non-myelinating Schwann cells, and the majority of terminal Schwann cells that form lamellar corpuscles and longitudinal lanceolate endings. Sox2 was not found in myelinating Schwann cells and terminal Schwann cells of subepidermal free nerve endings. Satellite glial cells exhibit strong Sox2 immunoreactivity, whereas non-myelinating Schwann cells show weak immunoreactivity. RT-PCR confirmed the presence of Sox2 mRNA, indicating that the cells are likely Sox2 expressors. Our findings suggest that the role of Sox2 in the peripheral nervous system may be cell-type-dependent.

C38, equivalent to BM88, is developmentally expressed in maturing retinal neurons and enhances neuronal maturation

J. Neurochem. (2010) 112, 1235–1248.

Nuclear lamins are differentially expressed in retinal neurons of the adult rat retina

Lamins are type V intermediate filament proteins that support nuclear membranes. They are divided into A-type lamins, which include lamin A and C, and B-type lamins, which include lamin B1 and B2. In the rat brain, lamin A and C are expressed in relatively equal amounts, while the expressions of lamin B1 and B2 vary depending on the cell type. Lamins play important roles in normal morphogenesis and function. In the nervous system, their abnormal expression causes several neurodegenerative diseases such as peripheral neuropathy, leukodystrophy and lissencephaly. The retina belongs to the central nervous system (CNS) and has widely been used as a source of CNS neurons. We investigated the expression patterns of lamin subtypes in the adult rat retina by immunohistochemistry and found that the staining patterns differed when compared with the brain. All retinal neurons expressed lamin B1 and B2 in relatively equal amounts. In addition, horizontal cells and a subpopulation of retinal ganglion cells expressed lamin A and C, while photoreceptor cells expressed neither lamin A nor C, and all other retinal neurons expressed lamin C only. This differential expression pattern of lamins in retinal neurons suggests that they may be involved in cellular differentiation and expression of cell-specific genes in individual retinal neurons.

Perineuronal germinal cells in the rat cerebral cortex.

The generation and renewal of cells in the adult mammalian central nervous system maintains brain functions, including plasticity. Even in the cerebral cortex of adult mammals, glial cells are thought to be replaced with newly generated cells every 100 days. Recently, we demonstrated that this proliferation is stimulated by neural activity. However, whether any germinal areas exist in the cortical parenchyma is unknown. Here, we examined the proliferating cell dynamics in the cerebral cortex of adult rats using BrdU labeling and immunohistochemistry for NeuN and lamin B1. At 2 h after a single injection of BrdU, more than 80% of BrdU-labeled cells were observed in the perineuronal territory in which the BrdU-labeled nuclei were located within 5 µm from neuronal nuclei. The ratio of perineuronal cells to nonperineuronal cells in BrdU-labeled cells gradually decreased over the 2 weeks following BrdU injection. These observations indicate that numerous cortical cells proliferate in the perineuronal territory, the germinal soil, and that part of these newly generated cells migrate from the perineuronal territory into the surrounding areas during the 2 weeks following mitosis.

Effects of sensory denervation by neonatal capsaicin administration on experimental pancreatitis induced by dibutyltin dichloride.

Increase in the number of intrapancreatic sensory nerve fibers has been implicated in the generation of pain in chronic pancreatitis. Because some sensory neurotransmitters (e.g., substance P) are known to have proinflammatory effects, we hypothesized that denervation of intrapancreatic nerves might influence not only pain generation but also inflammation. Neonatal Lewis rats were injected with capsaicin (50 mg/kg or 0 mg/kg), a neurotoxin, to induce denervation of primary sensory neurons. When rats reached 170–190 g body weight, experimental pancreatitis was induced by a single administration of dibutyltin dichloride (7 mg/mg). The severity of pancreatitis was evaluated in both groups in the acute phase (at 3 and 7 days) and chronic phase (at 28 days). At day 7, the sensory denervation induced by neonatal capsaicin administration inhibited pancreatic inflammation on both histological (determination of interstitial edema, expansion of interlobular septa and intercellular spaces, and inflammatory cell infiltration) and biochemical (intrapancreatic myeloperoxidase activity) evaluation. Furthermore, at day 28, glandular atrophy, pseudotubular complexes, and rate of fibrosis were each significantly lower in the capsaicin-pretreated group than in the vehicle-pretreated group. Our findings provide in vivo evidence that primary sensory neurons play important roles in both acute pancreatitis and chronic pancreatic inflammation with fibrosis.