Morphological classification of radial glia-like cells in the postnatal mouse subventricular zone.

The subventricular zone (SVZ) is one of the neurogenic regions of the adult mammalian brain. Neural stem cells (NSCs) in the SVZ have certain key features: they express glial fibrillary acidic protein (GFAP), proliferate slowly, have a radial glia-like (RG-L) morphology, and are in contact with the cerebrospinal fluid (CSF). NSCs have been isolated by FACS to analyse them, but their morphology has not been systematically examined. To address this knowledge gap, we sparsely labelled RG-L cells in the SVZ of neonatal mice by introducing via electroporation a plasmid expressing fluorescent protein under the control of the GFAP promoter. We then classified RG-L cells into three types (RG-L1, 2, and 3) based on their morphologies. RG-L1 cells had a basal process with some branches and numerous fine processes. RG-L2 cells had a basal process, but fewer branches and fine processes than RG-L1 cells. RG-L3 cells had one basal process that was almost free of branches and fine processes. Importantly, regardless of the cell type, about half of their somata resided on the basal side of the SVZ. Based on changes in their proportions during postnatal development and their expression of GFAP and cell proliferation markers at the adult stage, we speculated that NSCs change their morphologies during development/maturation and not all NSCs must always be in the apical SVZ or in contact with the CSF. Our results indicate that in addition to expression of markers for NSCs, the morphology is a critical feature to identify NSCs.

Sulfatide species with various fatty acid chains in oligodendrocytes at different developmental stages determined by imaging mass spectrometry.

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. Read the Editorial Highlight for this article on page 356.

c-jun is differentially expressed in embryonic and adult neural precursor 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.

Sox2 promotes survival of satellite glial cells in vitro.

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.

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.

Cortical projection to the subventricular zone and its effect on adult neurogenesis in mice.

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.

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.

Origin and progeny of reactive gliosis: A source of multipotent cells in the injured brain.

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 (PDGFRalpha) 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.

Improvement in Double Staining With Fluoro-Jade C and Fluorescent Immunostaining: FJC Staining Is Not Specific to Degenerating Mature Neurons.

Fluoro-Jade C (FJC) staining has been used to detect degenerating neurons in tissue sections. It is a simple and easy staining procedure and does not depend on the manner of cell death. In some experiments, double staining with FJC and fluorescent immunostaining (FI) is required to identify cell types. However, pretreatment for FJC staining contains some processes that are harsh to fluorophores, and the FI signal is greatly reduced. To overcome this issue, we improved the double staining protocol to acquire clear double-stained images by introducing the labeled streptavidin-biotin system. In addition, several studies indicate that FJC can label non-degenerating glial cells, including resting/reactive astrocytes and activated microglia. Moreover, our previous study indicated that degenerating mesenchymal cells were also labeled by FJC, but it is still unclear whether FJC can label degenerating glial cells. Acute encephalopathy model mice contained damaged astrocytes with clasmatodendrosis, and 6-aminonicotinamide-injected mice contained necrotic astrocytes and oligodendrocytes. Using our improved double staining protocol with FJC and FI, we detected FJC-labeled degenerating astrocytes and oligodendrocytes with pyknotic nuclei. These results indicate that FJC is not specific to degenerating neurons in some experimental conditions.

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.

Evaluation of Fluoro-Jade C Staining: Specificity and Application to Damaged Immature Neuronal Cells in the Normal and Injured Mouse Brain.

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.

Adult neurogenesis requires Smad4-mediated bone morphogenic protein signaling in stem 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.

Conditional deletion of beta1-integrin in astroglia causes partial reactive gliosis.

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 beta1-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 beta1-integrin deletion. However, these reactive astrocytes did not divide, suggesting that the loss of beta1-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 beta1-integrin, we conclude that beta1-integrin-mediated signaling in astrocytes is required to promote their acquisition of a mature, nonreactive state. Alterations in beta1-integrin-mediated signaling may hence be implicated in eliciting specific aspects of reactive gliosis after injury.

Distinct role of growth hormone on epilepsy progression in a model of temporal lobe epilepsy.

Temporal lobe epilepsy is a common form of pharmacoresistant epilepsy, in which epileptogenic foci propagate to other regions of the brain from the area of the initial insult. The present study focused on epileptogenesis, that is, the development of the first foci inducing seizures in amygdala-kindled mice, a model of temporal lobe epilepsy, to find the molecular process promoting the formation of epileptogenic networks. The expression of growth hormone (GH) was up-regulated along neural circuits during the epileptogenesis, while there was no difference in the pituitary gland. The up-regulation was associated with increased phosphorylation/activation of signal transducer and activator of transcription 5 and expression of the Serum Response Element-regulated genes, FBJ osteosarcoma oncogene, early growth response 1, and Jun-B oncogene, suggesting that expression of GH leads to GH signaling in the hippocampus and cortex. Furthermore, the administration of the hormone into the hippocampus markedly enhanced the progression of kindling. The administration of an inhibitor of its secretion into the hippocampus elicited a delay in the progression. Our results demonstrate directly that regulation via growth hormone has a robust impact in epileptogenesis.

Impact of Hands-on Experience of a Cadaver Dissection on the Professional Identity Formation of Health Sciences Students.

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.

Developing a mouse model of acute encephalopathy using low-dose lipopolysaccharide injection and hyperthermia treatment.

IMPACT STATEMENT: Acute encephalopathy (AE), mainly reported in East Asia, is classified into four categories based on clinical and neuropathological findings. Among them, AE caused by cytokine storm is known as the severest clinical entity that causes cerebral edema with poor prognosis. Because suitable and convenient model animal of AE had not been developed, the treatment of patients with AE is not established. In the present study, we established a simple and convenient protocol to mimic AE due to cytokine storm. Our model animal should be useful to elucidate the pathogenesis of AE.

Distinct modes of neuron addition in adult mouse neurogenesis.

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.

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.

Identification of NeuN immunopositive cells in the adult mouse subventricular zone.

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.