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Various brain regions/nuclei project axons to the subventricular zone (SVZ), a postnatal neurogenic niche. In adults, neurogenesis is controlled by neuronal activity, via neurotransmitters. Glutamate is a major excitatory neurotransmitter, and glutamate receptors are expressed in SVZ cells. Although the cerebral cortex is a major source of glutamate and the medial cortex projects axons to the medial striatum next to the SVZ, it remains unclear whether cortical neurons regulate adult neurogenesis in vivo. First, to analyze axonal projection, plasmid vector expressing DsRed was introduced to the medial cortex by in utero electroporation. At the adult stage, DsRed-labeled axons were detected in the dorsolateral, striatal, and septal areas of the SVZ, and where they were in contact with neuroblasts. Furthermore, maturation of the cortical projection and the SVZ appeared to synchronize during postnatal stages. Next, stab injuries were made in the bilateral medial cortex to interrupt cortical input to the SVZ. At 17 days post-injury, cell proliferation in the SVZ and tangential migration of neuroblasts to the olfactory bulb were not significantly affected. There were clusters of neuroblasts in the striatum close to the SVZ in all experimental groups, but the number and size of neuroblast clusters were significantly larger in the medial cortex-injured group compared with the other experimental groups. These neuroblast clusters had a morphology of tangentially migrating cells to the olfactory bulb. These results suggest that cortical input to the SVZ inhibits the radial migration of neuroblasts to converge with the migration pathway in vivo.

Astrocytes play many pivotal roles in the adult brain, including their reaction to injury. A hallmark of astrocytes is the contact of their endfeet with the basement membrane surrounding blood vessels, but still relatively little is known about the signaling mediated at the contact site. Here, we examine the role of β1‐integrin at this interface by its conditional deletion using different Cre lines. Thereby, the protein was reduced only at postnatal stages either in both glia and neurons or specifically only in neurons. Strikingly, only the former resulted in reactive gliosis, with the hallmarks of reactive astrocytes comprising astrocyte hypertrophy and up‐regulation of the intermediate filaments GFAP and vimentin as well as pericellular components, such as Tenascin‐C and the DSD‐1 proteoglycan. In addition, we also observed to a certain degree a non‐cell autonomous activation of microglial cells after conditional β1‐integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of β1‐integrin‐mediated signaling is not sufficient to elicit proliferation of these cells as observed after brain injury. Interestingly, this partial reactive gliosis appeared in the absence of cell death and blood brain barrier disturbances. As these effects did not appear after neuron‐specific deletion of β1‐integrin, we conclude that β1‐integrin‐mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in β1‐integrin‐mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury. © 2009 Wiley‐Liss, Inc.

Reactive gliosis is the universal reaction to brain injury, but the precise origin and subsequent fate of the glial cells reacting to injury are unknown. Astrocytes react to injury by hypertrophy and up-regulation of the glial-fibrillary acidic protein (GFAP). Whereas mature astrocytes do not normally divide, a subpopulation of the reactive GFAP+ cells does so, prompting the question of whether the proliferating GFAP+ cells arise from endogenous glial progenitors or from mature astrocytes that start to proliferate in response to brain injury. Here we show by genetic fate mapping and cell type-specific viral targeting that quiescent astrocytes start to proliferate after stab wound injury and contribute to the reactive gliosis and proliferating GFAP+ cells. These proliferating astrocytes remain within their lineage in vivo, while a more favorable environment in vitro revealed their multipotency and capacity for self-renewal. Conversely, progenitors present in the adult mouse cerebral cortex labeled by NG2 or the receptor for the platelet-derived growth factor (PDGFRα) did not form neurospheres after (or before) brain injury. Taken together, the first fate-mapping analysis of astrocytes in the adult mouse cerebral cortex shows that some astrocytes acquire stem cell properties after injury and hence may provide a promising cell type to initiate repair after brain injury.

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.

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.

Fluoro-Jade C (FJC) staining is widely used for the specific detection of all degenerating mature neurons, including apoptotic, necrotic, and autophagic cells. However, whether FJC staining can detect degenerating immature neurons and neural stem/precursor cells remains unclear. In addition, some conflicting studies have shown that FJC and its ancestral dyes, Fluoro-Jade (FJ) and FJB, can label resting/activated astrocytes and microglia. In the present study, we examined the validity of FJC staining for the detection of neuronal cells in adult and embryonic mouse brains under normal and injured conditions. In the adult rodent subventricular zone (SVZ)–rostral migratory stream (RMS)–olfactory bulb (OB) system, apoptosis associated with neurogenesis occurs under normal conditions. Using this system, we detected FCJ positive (+) cells, some of which were doublecortin (DCX)(+) neuroblasts, in addition to neuronal nuclei (NeuN)(+) mature neurons. FJC negative (−) apoptotic cells expressing activated Caspase 3 were also observed, and a small number of FJC(+)/ionized calcium-binding adaptor molecule 1 (Iba1)(+) microglia and FJC(+)/glial fibrillary acidic protein (GFAP)(+) astrocytes were observed in the normal brain. Next, we analyzed embryonic brains, in which the apoptosis of neural stem/precursor cells was induced by the administration of N-ethyl-N-nitrosourea (ENU) or ethanol at embryonic day 14 or 10, respectively. In those brains, FJC(+) neural stem/precursor cells and neuroepithelial cells expressing SRY-related HMG-box 2 (Sox2) were observed. Surprisingly degenerating mesenchymal cells were also FJC(+). The present study indicates that FJC is a reliable marker for degenerating neuronal cells during all differentiation stages. However, FJC could also label degenerating non-neuronal cells under some conditions.

Status epilepticus (SE) induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3+ (PH3+) neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase)/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3+ brain regions, and that H3 phosphorylation is a suitable indicator of strong 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.

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.

HSO3‐3‐galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro‐oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4‐positive cells. In this study, we identified a sulfatide species from the pro‐oligodendroblast‐to‐myelination stage by imaging mass spectrometry. The results demonstrated that short‐chain sulfatides with 16 carbon non‐hydroxylated fatty acids (C16) and 18 carbon non‐hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18‐OH) existed in restricted regions of the early embryonic spinal cord, where pro‐oligodendroblasts initially appear, and co‐localized with Olig2‐positive pro‐oligodendroblasts. C18 and C18‐OH sulfatides also existed in isolated pro‐oligodendroblasts. C22‐OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short‐chain fatty acids in pro‐oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination.

In the adult rodent subventricular zone (SVZ), there are neural stem cells (NSCs) and the specialized neurogenic niche is critical to maintain their stemness. To date, many cellular and noncellular factors that compose the neurogenic niche and markers to identify subpopulations of Type A cells have been confirmed. In particular, neurotransmitters regulate adult neurogenesis and mature neurons in the SVZ have been only partially analyzed. Moreover, Type A cells, descendants of NSCs, are highly heterogeneous and more molecular markers are still needed to identify them. In the present study, we systematically classified NeuN, commonly used as a marker of mature and immature post‐mitotic neurons, immunopositive (+) cells within the adult mouse SVZ. These SVZ‐NeuN+ cells (SVZ‐Ns) were mainly classified into two types. One was mature SVZ‐Ns (M‐SVZ‐Ns). Neurochemical properties of M‐SVZ‐Ns were similar to those of striatal neurons, but their birth date and morphology were different. M‐SVZ‐Ns were generated during embryonic and early postnatal stages with bipolar peaks and extended their processes along the wall of the lateral ventricle. The second type was small SVZ‐Ns (S‐SVZ‐Ns) with features of Type A cells. They expressed not only markers of Type A cells, but also proliferated and migrated from the SVZ to the olfactory bulb. Furthermore, S‐SVZ‐Ns could be classified into two types by their spatial locations and glutamic acid decarboxylase 67 expression. Our data indicate that M‐SVZ‐Ns are a new component of the neurogenic niche and S‐SVZ‐Ns are newly identified subpopulations of Type A cells.

Acute encephalopathy (AE) is mainly reported in East Asia and, in most cases, results from pediatric viral infections, leading to fever, seizure, and loss of consciousness. Cerebral edema is the most important pathological symptom of AE. At present, AE is classified into four categories based on clinical and pathophysiological features, and cytokine storm-induced AE is the severest among them. The pathogenesis of AE is currently unclear; this can be attributed to the lack of a simple and convenient animal model for research. Here, we hypothesized that the induction of systemic inflammation using lipopolysaccharide (LPS) injection followed by hyperthermia (HT) treatment can be used to develop an animal model of cytokine storm-induced AE. Postnatal eight-day-old mouse pups were intraperitoneally injected with low-dose LPS (50 or 100 µg/kg) followed by HT treatment (41.5°C, 30 min). Histological analysis of their brains was subsequently performed. Fluorescein isothiocyanate assay combined with immunohistochemistry was used to elucidate blood–brain barrier (BBB) disruption. LPS (100 µg/kg) injection followed by HT treatment increased BBB permeability in the cerebral cortex and induced microglial activation. Astrocytic clasmatodendrosis was also evident. The brains of some pups exhibited small ischemic lesions, particularly in the cerebral cortex. Our results indicate that a low-dose LPS injection followed by HT treatment can produce symptoms of cytokine storm-induced AE, which is observed in diseases, such as acute necrotizing encephalopathy and hemorrhagic shock and encephalopathy syndrome. Thus, this mouse model can help to elucidate the pathogenetic mechanisms underlying AE.

Astrocytes are thought to play a variety of key roles in the adult brain, such as their participation in synaptic transmission, in wound healing upon brain injury, and adult neurogenesis. However, to elucidate these functions in vivo has been difficult because of the lack of astrocyte‐specific gene targeting. Here we show that the inducible form of Cre (CreERT2) expressed in the locus of the astrocyte‐specific glutamate transporter (GLAST) allows precisely timed gene deletion in adult astrocytes as well as radial glial cells at earlier developmental stages. Moreover, postnatal and adult neurogenesis can be targeted at different stages with high efficiency as it originates from astroglial cells. Taken together, this mouse line will allow dissecting the molecular pathways regulating the diverse functions of astrocytes as precursors, support cells, repair cells, and cells involved in neuronal information processing. © 2006 Wiley‐Liss, Inc.

c-jun, a major component of AP-1 transcription factor, has a wide variety of functions. In the embryonic brain, c-jun mRNA is abundantly expressed in germinal layers around the ventricles. Although the subventricular zone (SVZ) of the adult brain is a derivative of embryonic germinal layers and contains neural precursor cells (NPCs), the c-jun expression pattern is not clear. To study the function of c-jun in adult neurogenesis, we analyzed c-jun expression in the adult SVZ by immunohistochemistry and compared it with that of the embryonic brain. We found that almost all proliferating embryonic NPCs expressed c-jun, but the number of c-jun immunopositive cells among proliferating adult NPCs was about half. In addition, c-jun was hardly expressed in post-mitotic migrating neurons in the embryonic brain, but the majority of c-jun immunopositive cells were tangentially migrating neuroblasts heading toward the olfactory bulb in the adult brain. In addition, status epilepticus is known to enhance the transient proliferation of adult NPCs, but the c-jun expression pattern was not significantly affected. These expression patterns suggest that c-jun has a pivotal role in the proliferation of embryonic NPCs, but it has also other roles in adult neurogenesis.

Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors.

[Background] In Japan, some nursing and health science universities that train nurses and/or clinical laboratory technicians have a curriculum in which students observe medical students performing a cadaver dissection. Observing a cadaver dissection is believed to affect the formation of a student’s professional identity. This study aimed to investigate the effects of observing a cadaver dissection on the professional identity of nursing and clinical laboratory science students to find an effective educational support system for developing professional identity. [Methods] Sophomores majoring in nursing science or clinical laboratory science were asked to complete a questionnaire with a professional identity scale before and after hands-on experience of a cadaver dissection performed by medical students. After their hands-on session was complete, they responded to a free-answer question about acquiring a professional identity. [Results] The professional identity score of nursing students significantly decreased after the hands-on experience of the cadaver dissection. No significant change in professional identity score was observed in the clinical laboratory science students. However, the effect size (r) was moderate. [Conclusion] Although professional identity formation fluctuates immediately after the experience of the hands-on experience of a cadaver dissection, the findings do suggest that these hands-on sessions will be effective for developing their professional identity if educational support is provided to help them utilize what they learned through reflection.

Adult neurogenesis is restricted to two distinct areas of the mammalian brain: the olfactory bulb (OB) and the dentate gyrus (DG). Despite its spatial restriction, adult neurogenesis is of crucial importance for sensory processing and learning and memory. Although it has been shown that tens of thousands of new neurons arrive in the OB and DG every day with about half of them surviving after integration, the total contribution of adult neurogenesis to the pre-existing network remains mostly unknown. This is because of previous approaches labeling only a small proportion of adult-generated neurons. Here, we used genetic fate mapping to follow the majority of adult-generated neurons over long periods. Our data demonstrate two distinct modes of neuron addition to the pre-existing network. In the glomerular layer of the OB, there is a constant net addition of adult-generated neurons reaching a third of the total neuronal population within 9 months. In contrast, adult neurogenesis contributes to only a minor fraction of the entire neuronal network in the granular cell layer of the OB and the DG. Although the fraction of adult generated neurons can be further increased by an enriched environment, it still remains a minority of the neuronal network in the DG. Thus, neuron addition is distinct and tightly regulated in the neuronal networks that incorporate new neurons life long.

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of ≈70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with thesein vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provid

In the mammalian brain, neurogenesis continues only in few regions of the forebrain. The molecular signals governing neurogenesis in these unique neurogenic niches, however, are still ill defined. Here, we show that bone morphogenic protein (BMP)-mediated signaling is active in adult neural stem cells and is crucial to initiate the neurogenic lineage in the adult mouse subependymal zone. Conditional deletion of Smad4 in adult neural stem cells severely impairs neurogenesis, and this is phenocopied by infusion of Noggin, an extracellular antagonist of BMP. Smad4 deletion in stem, but not progenitor cells, as well as Noggin infusion lead to an increased number of Olig2-expressing progeny that migrate to the corpus callosum and differentiate into oligodendrocytes. Transplantation experiments further verified the cell-autonomous nature of this phenotype. Thus, BMP-mediated signaling via Smad4 is required to initiate neurogenesis from adult neural stem cells and suppress the alternative fate of oligodendrogliogenesis.

Acute encephalopathy (AE) is mainly reported in East Asia and, in most cases, results from pediatric viral infections, leading to fever, seizure, and loss of consciousness. Cerebral edema is the most important pathological symptom of AE. At present, AE is classified into four categories based on clinical and pathophysiological features, and cytokine storm-induced AE is the severest among them. The pathogenesis of AE is currently unclear; this can be attributed to the lack of a simple and convenient animal model for research. Here, we hypothesized that the induction of systemic inflammation using lipopolysaccharide (LPS) injection followed by hyperthermia (HT) treatment can be used to develop an animal model of cytokine storm-induced AE. Postnatal eight-day-old mouse pups were intraperitoneally injected with low-dose LPS (50 or 100 µg/kg) followed by HT treatment (41.5°C, 30 min). Histological analysis of their brains was subsequently performed. Fluorescein isothiocyanate assay combined with immunohistochemistry was used to elucidate blood–brain barrier (BBB) disruption. LPS (100 µg/kg) injection followed by HT treatment increased BBB permeability in the cerebral cortex and induced microglial activation. Astrocytic clasmatodendrosis was also evident. The brains of some pups exhibited small ischemic lesions, particularly in the cerebral cortex. Our results indicate that a low-dose LPS injection followed by HT treatment can produce symptoms of cytokine storm-induced AE, which is observed in diseases, such as acute necrotizing encephalopathy and hemorrhagic shock and encephalopathy syndrome. Thus, this mouse model can help to elucidate the pathogenetic mechanisms underlying AE.

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.

In the adult rodent subventricular zone (SVZ), there are neural stem cells (NSCs) and the specialized neurogenic niche is critical to maintain their stemness. To date, many cellular and noncellular factors that compose the neurogenic niche and markers to identify subpopulations of Type A cells have been confirmed. In particular, neurotransmitters regulate adult neurogenesis and mature neurons in the SVZ have been only partially analyzed. Moreover, Type A cells, descendants of NSCs, are highly heterogeneous and more molecular markers are still needed to identify them. In the present study, we systematically classified NeuN, commonly used as a marker of mature and immature post‐mitotic neurons, immunopositive (+) cells within the adult mouse SVZ. These SVZ‐NeuN+ cells (SVZ‐Ns) were mainly classified into two types. One was mature SVZ‐Ns (M‐SVZ‐Ns). Neurochemical properties of M‐SVZ‐Ns were similar to those of striatal neurons, but their birth date and morphology were different. M‐SVZ‐Ns were generated during embryonic and early postnatal stages with bipolar peaks and extended their processes along the wall of the lateral ventricle. The second type was small SVZ‐Ns (S‐SVZ‐Ns) with features of Type A cells. They expressed not only markers of Type A cells, but also proliferated and migrated from the SVZ to the olfactory bulb. Furthermore, S‐SVZ‐Ns could be classified into two types by their spatial locations and glutamic acid decarboxylase 67 expression. Our data indicate that M‐SVZ‐Ns are a new component of the neurogenic niche and S‐SVZ‐Ns are newly identified subpopulations of Type A cells.

c-jun, a major component of AP-1 transcription factor, has a wide variety of functions. In the embryonic brain, c-jun mRNA is abundantly expressed in germinal layers around the ventricles. Although the subventricular zone (SVZ) of the adult brain is a derivative of embryonic germinal layers and contains neural precursor cells (NPCs), the c-jun expression pattern is not clear. To study the function of c-jun in adult neurogenesis, we analyzed c-jun expression in the adult SVZ by immunohistochemistry and compared it with that of the embryonic brain. We found that almost all proliferating embryonic NPCs expressed c-jun, but the number of c-jun immunopositive cells among proliferating adult NPCs was about half. In addition, c-jun was hardly expressed in post-mitotic migrating neurons in the embryonic brain, but the majority of c-jun immunopositive cells were tangentially migrating neuroblasts heading toward the olfactory bulb in the adult brain. In addition, status epilepticus is known to enhance the transient proliferation of adult NPCs, but the c-jun expression pattern was not significantly affected. These expression patterns suggest that c-jun has a pivotal role in the proliferation of embryonic NPCs, but it has also other roles in adult neurogenesis.

HSO3‐3‐galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro‐oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4‐positive cells. In this study, we identified a sulfatide species from the pro‐oligodendroblast‐to‐myelination stage by imaging mass spectrometry. The results demonstrated that short‐chain sulfatides with 16 carbon non‐hydroxylated fatty acids (C16) and 18 carbon non‐hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18‐OH) existed in restricted regions of the early embryonic spinal cord, where pro‐oligodendroblasts initially appear, and co‐localized with Olig2‐positive pro‐oligodendroblasts. C18 and C18‐OH sulfatides also existed in isolated pro‐oligodendroblasts. C22‐OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short‐chain fatty acids in pro‐oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination.

Sox2 is a transcriptional factor expressed in neural stem cells. It is known that Sox2 regulates cell differentiation, proliferation and survival of the neural stem cells. Our previous study showed that Sox2 is expressed in all satellite glial cells of the adult rat dorsal root ganglion. In this study, to examine the role of Sox2 in satellite glial cells, we establish a satellite glial cell-enriched culture system. Our culture method succeeded in harvesting satellite glial cells with the somata of neurons in the dorsal root ganglion. Using this culture system, Sox2 was downregulated by siRNA against Sox2. The knockdown of Sox2 downregulated ErbB2 and ErbB3 mRNA at 2 and 4 days after siRNA treatment. MAPK phosphorylation, downstream of ErbB, was also inhibited by Sox2 knockdown. Because ErbB2 and ErbB3 are receptors that support the survival of glial cells in the peripheral nervous system, apoptotic cells were also counted. TUNEL-positive cells increased at 5 days after siRNA treatment. These results suggest that Sox2 promotes satellite glial cell survival through the MAPK pathway via ErbB receptors.

Status epilepticus (SE) induces pathological and morphological changes in the brain. Recently, it has become clear that excessive neuronal excitation, stress and drug abuse induce chromatin remodeling in neurons, thereby altering gene expression. Chromatin remodeling is a key mechanism of epigenetic gene regulation. Histone H3 phosphorylation is frequently used as a marker of chromatin remodeling and is closely related to the upregulation of mRNA transcription. In the present study, we analyzed H3 phosphorylation levels in vivo using immunohistochemistry in the brains of mice with pilocarpine-induced SE. A substantial increase in H3 phosphorylation was detected in neurons in specific brain structures. Increased H3 phosphorylation was dependent on neuronal excitation. In particular, a robust upregulation of H3 phosphorylation was detected in the caudate putamen, and there was a gradient of phosphorylated H3+ (PH3+) neurons along the medio-lateral axis. After unilateral ablation of dopaminergic neurons in the substantia nigra by injection of 6-hydroxydopamine, the distribution of PH3+ neurons changed in the caudate putamen. Moreover, our histological analysis suggested that, in addition to the well-known MSK1 (mitogen and stress-activated kinase)/H3 phosphorylation/c-fos pathway, other signaling pathways were also activated. Together, our findings suggest that a number of genes involved in the pathology of epileptogenesis are upregulated in PH3+ brain regions, and that H3 phosphorylation is a suitable indicator of strong neuronal excitation.

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.

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.

In the mammalian brain, neurogenesis continues only in few regions of the forebrain. The molecular signals governing neurogenesis in these unique neurogenic niches, however, are still ill defined. Here, we show that bone morphogenic protein (BMP)-mediated signaling is active in adult neural stem cells and is crucial to initiate the neurogenic lineage in the adult mouse subependymal zone. Conditional deletion of Smad4 in adult neural stem cells severely impairs neurogenesis, and this is phenocopied by infusion of Noggin, an extracellular antagonist of BMP. Smad4 deletion in stem, but not progenitor cells, as well as Noggin infusion lead to an increased number of Olig2-expressing progeny that migrate to the corpus callosum and differentiate into oligodendrocytes. Transplantation experiments further verified the cell-autonomous nature of this phenotype. Thus, BMP-mediated signaling via Smad4 is required to initiate neurogenesis from adult neural stem cells and suppress the alternative fate of oligodendrogliogenesis.

Adult neurogenesis is restricted to two distinct areas of the mammalian brain: the olfactory bulb (OB) and the dentate gyrus (DG). Despite its spatial restriction, adult neurogenesis is of crucial importance for sensory processing and learning and memory. Although it has been shown that tens of thousands of new neurons arrive in the OB and DG every day with about half of them surviving after integration, the total contribution of adult neurogenesis to the pre-existing network remains mostly unknown. This is because of previous approaches labeling only a small proportion of adult-generated neurons. Here, we used genetic fate mapping to follow the majority of adult-generated neurons over long periods. Our data demonstrate two distinct modes of neuron addition to the pre-existing network. In the glomerular layer of the OB, there is a constant net addition of adult-generated neurons reaching a third of the total neuronal population within 9 months. In contrast, adult neurogenesis contributes to only a minor fraction of the entire neuronal network in the granular cell layer of the OB and the DG. Although the fraction of adult generated neurons can be further increased by an enriched environment, it still remains a minority of the neuronal network in the DG. Thus, neuron addition is distinct and tightly regulated in the neuronal networks that incorporate new neurons life long.

Astrocytes are thought to play a variety of key roles in the adult brain, such as their participation in synaptic transmission, in wound healing upon brain injury, and adult neurogenesis. However, to elucidate these functions in vivo has been difficult because of the lack of astrocyte‐specific gene targeting. Here we show that the inducible form of Cre (CreERT2) expressed in the locus of the astrocyte‐specific glutamate transporter (GLAST) allows precisely timed gene deletion in adult astrocytes as well as radial glial cells at earlier developmental stages. Moreover, postnatal and adult neurogenesis can be targeted at different stages with high efficiency as it originates from astroglial cells. Taken together, this mouse line will allow dissecting the molecular pathways regulating the diverse functions of astrocytes as precursors, support cells, repair cells, and cells involved in neuronal information processing. © 2006 Wiley‐Liss, Inc.

To elucidate the role of astrocytes in the stress response of the central nervous system to ischemia, early gene expression was evaluated in cultured rat astrocytes subjected to hypoxia/reoxygenation. Using differential display, a novel putative vesicle transport-related factor (RA410) was cloned from reoxygenated astrocytes. Analysis of the deduced amino acid sequence showed RA410 to be composed of domains common to vesicle transport-related proteins of the Sec1/Unc18 family, including Sly1p and Sec1p (yeast), Rop (Drosophila), Unc18 (Caenorhabditis elegans), and Munc18 (mammalian), suggesting its possible role in vesicular transport. Northern analysis of normal rat tissues showed the highest expression of RA410 transcripts in testis. When astrocyte cultures were subjected to a period of hypoxia followed by reoxygenation, induction of RA410 mRNA was observed within 15 min of reoxygenation, reaching a maximum by 60 min. At the start of reoxygenation, the addition of diphenyl iodonium, an NADPH oxidase inhibitor, blocked in parallel astrocyte generation of reactive oxygen intermediates and expression of RA410 message. In contrast, cycloheximide did not affect RA410 mRNA levels, indicating that RA410 is an immediate-early gene in the setting of reoxygenation. Using polyclonal antibody raised against an RA410-derived synthetic peptide, Western blotting of lysates from reoxygenated astrocytes displayed an immunoreactive band of ≈70 kDa, the expression of which followed induction of the mRNA. Fractionation of astrocyte lysates on sucrose gradients showed RA410 antigen to be predominantly in the plasma membrane. Immunoelectron microscopic analysis demonstrated RA410 in large vesicles associated with the Golgi, but not in the Golgi apparatus itself, consistent with its participation in post-Golgi transport. Consistent with thesein vitro data, RA410 expression was observed in rat brain astrocytes following transient occlusion of the middle cerebral artery. These data provid