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.

Differential responses of endogenous adult mouse neural precursors to excess neuronal excitation.

Adult neurogenesis in the subgranular zone of the hippocampus (SGZ) is enhanced by excess as well as mild neuronal excitation, such as chemoconvulsant-induced brief seizures. Because most studies of neurogenesis after seizures have focused on the SGZ, the threshold of neuronal excitation required to enhance neurogenesis in the subventricular zone (SVZ) is not clear. Therefore, we examined the responses of SVZ precursors to brief generalized clonic seizures induced by a single administration of the chemoconvulsant pentylenetetrazole (PTZ). Cell cycle progression of precursors was analysed by systemic administration of thymidine analogues. We found that brief seizures immediately resulted in cell cycle retardation in the SVZ. However, the same effect was not seen in the SGZ. This initial cell cycle retardation in the SVZ was followed by enhanced cell cycle re-entry after the first round of mitosis, leading to precursor pool expansion, but the cell cycle retardation and expansion of the precursor pool were transient. Cell cycle progression in the PTZ-treated group returned to normal after one cell cycle. The numbers of precursors in the SVZ and new neurons in the olfactory bulb, which are descendants of SVZ precursors, were not significantly different from those in control mice more than 2 days after seizures. Because similar effects were observed following electroconvulsive seizures, these responses are likely to be general effects of brief seizures. These results suggest that neurogenesis in the SVZ is more tightly regulated and requires stronger stimuli to be modified than that in the SGZ.

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.

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

C38 antigen is specifically expressed in neuronal cells of the retina. The purpose of this study was to isolate C38 cDNA and determine its molecular functions. Sequence analysis of C38 cDNA revealed that C38 is equivalent to rat BM88, which has been reported to induce cell-cycle arrest and neuronal differentiation in Neuro2a cells. C38 and Ki67, a marker of proliferating cells, were not colocalized during retinal development. C38 was first detected in the retinal ganglion cells at embryonic day 16, much later than the expression of doublecortin, a marker of immature neurons. Although all the horizontal cells were post-mitotic at this stage, C38 was not detected in horizontal cells until the postnatal period. In addition, C38 over-expression did not induce neuronal differentiation or cell-cycle arrest of pluripotent P19 embryonal carcinoma cells. Instead, C38 promoted maturation during neuronal differentiation of P19 embryonal carcinoma cells by down-regulating Oct-3, a pluripotent cell marker and enhancing the expressions of positive regulators of neurogenesis. In conclusion, during retinal development, C38 is first expressed in post-mitotic retinal neurons and is up-regulated during their maturation. C38 does not induce neuronal competence in pluripotent cells, but does promote maturation in already committed neuronal cells.

Involvement of PLAGL1/ZAC1 in hypocretin/orexin transcription.

The hypocretin/orexin neuropeptide system coordinates the regulation of various physiological processes. Our previous study reported that a reduction in the expression of pleomorphic adenoma gene­like 1 (Plagl1), which encodes a C2H2 zinc­finger transcription factor, occurs in hypocretin neuron­ablated transgenic mice, suggesting that PLAGL1 is co­expressed in hypocretin neurons and regulates hypocretin transcription. The present study examined whether canonical prepro­hypocretin transcription is functionally modulated by PLAGL1. Double immunostaining indicated that the majority of hypocretin neurons were positive for PLAGL1 immunoreactivity in the nucleus. Notably, PLAGL1 immunoreactivity in hypocretin neurons was altered in response to several conditions affecting hypocretin function. An uneven localization of PLAGL1 was detected in the nuclei of hypocretin neurons following sleep deprivation. Chromatin immunoprecipitation revealed that endogenous PLAGL1 may bind to a putative PLAGL1­binding site in the proximal region of the hypocretin gene, in the murine hypothalamus. In addition, electroporation of the PLAGL1 expression vector into the fetal hypothalamus promoted hypothalamic hypocretin transcription. These results suggested that PLAGL1 may regulate hypothalamic hypocretin transcription.

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.

Differential expression of nuclear lamin subtypes in the neural cells of the adult rat cerebral cortex.

Lamins are type V intermediate filament proteins that are located beneath the inner nuclear membrane. In mammalian somatic cells, LMNB1 and LMNB2 encode somatic lamins B1 and B2, respectively, and the LMNA gene is alternatively spliced to generate somatic lamins A and C. Mutations in lamin genes have been linked to many human hereditary diseases, including neurodegenerative disorders. Knowledge about lamins in the nervous system has been accumulated recently, but a precise analysis of lamin subtypes in glial cells has not yet been reported. In this study we investigated the composition of lamin subtypes in neurons, astrocytes, oligodendrocyte-lineage cells, and microglia in the adult rat cerebral cortex using an immunohistochemical staining method. Lamin A was not observed in neurons and glial cells. Lamin C was observed in astrocytes, mature oligodendrocytes and neurons, but not observed in oligodendrocyte progenitor cells. Microglia also did not stain positive for lamin C which differed from macrophages, with lamin C positive. Lamin B1 and B2 were observed in all glial cells and neurons. Lamin B1 was intensely positive in oligodendrocyte progenitor cells compared with other glial cells and neurons. Lamin B2 was weakly positive in all glial cells compared to neurons. Our current study might provide useful information to reveal how the onset mechanisms of human neurodegenerative diseases are associated with mutations in genes for nuclear lamin proteins.

Crossreaction with an anti-Bax antibody reveals novel multi-endocrine cellular antigen.

We found a novel protein that has crossreactivity with a polyclonal anti-Bax antibody (SCBAX antibody). The protein was localized exclusively in the endocrine cells of hypothalamus, pituitary gland, and pancreatic islets. Immunohistochemical (IHC) double labeling revealed that the cells showing crossreactivity with this antibody corresponded precisely to oxytocin neurons and ACTH, alpha-MSH, and glucagon cells in rat and gerbil. By immunoelectron microscopy, the protein was localized predominantly in and just around the secretory granules in the cytoplasm but not in the mitochondria. Double-labeling IHC with the anti-Bax SCBAX antibody and two anti-Bax monoclonal antibodies (MAbs) showed that cells stained with the anti-Bax SCBAX antibody were not stained with anti-Bax MAbs except for very few cells (probably apoptotic cells). Western blotting analysis revealed that the molecular mass of the protein was approximately 55 kD, which differs from that of Bax protein (21 kD). These findings indicate that the anti-Bax SCBAX antibody recognizes not only pro-apoptotic Bax protein (a 21-kD mitochondrial protein) but also an unknown substance present in one endocrine cell group in each endocrine organ. Therefore, the protein is designated as multi-endocrine cellular antigen (MECA). MECA is probably a 55-kD protein secreted from the particular differentiated cell groups of endocrine tissues.

Organization and cellular arrangement of two neurogenic regions in the adult ferret (Mustela putorius furo) brain.

In the adult mammalian brain, two neurogenic regions have been characterized, the subventricular zone (SVZ) of the lateral ventricle (LV) and the subgranular zone (SGZ) of the dentate gyrus (DG). Despite remarkable knowledge of rodents, the detailed arrangement of neurogenic regions in most mammals is poorly understood. In this study, we used immunohistochemistry and cell type-specific antibodies to investigate the organization of two germinal regions in the adult ferret, which belongs to the order Carnivora and is widely used as a model animal with a gyrencephalic brain. From the SVZ to the olfactory bulb, doublecortin-positive cells tended to organize in chain-like clusters, which are surrounded by a meshwork of astrocytes. This structure is homologous to the rostral migratory stream (RMS) described in other species. Different from rodents, the horizontal limb of the RMS emerges directly from the LV, and the anterior region of the LV extends rostrally and reached the olfactory bulb. In the DG, glial fibrillary acidic protein-positive cells with long radial processes as well as doublecortin-positive cells are oriented in the SGZ. In both regions, doublecortin-positive cells showed characteristic morphology and were positive for polysialylated-neural cell adhesion molecule, beta-III tubulin, and lamin B1 (intense staining). Proliferating cells were detected in both regions using antibodies against proliferating cell nuclear antigen and phospho-histone H3. These observations demonstrate that the two neurogenic regions in ferrets have a similar cellular composition as those of other mammalian species despite anatomical differences in the brain.

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.

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.

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.

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.

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 microm 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.