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The molecular mechanisms and evolutionary changes accompanying synapse development are still poorly understood. Here, we generated a cross-species proteomic map of synapse development in the human, macaque, and mouse neocortex. By tracking the changes of >1,000 postsynaptic density (PSD) proteins from midgestation to young adulthood, we found that PSD maturation in humans separates into three major phases that are dominated by distinct pathways. Cross-species comparisons reveal that the human PSD matures about two to three times slower than other species and contains higher levels of Rho guanine nucleotide exchange factors (RhoGEFs) in the perinatal period. Enhancement of the RhoGEF signaling in human neurons delays the morphological maturation of dendritic spines and the functional maturation of synapses, potentially contributing to the neotenic traits of human brain development. In addition, PSD proteins can be divided into four modules that exert stage- and cell type-specific functions, possibly explaining their differential associations with cognitive functions and diseases. Together, our proteomic map of synapse development provides a blueprint for studying the molecular basis and evolutionary changes of synapse maturation.

To address the inefficiency of passive diffusion for antibody penetration in thick tissue samples, which limits clearing-technique applications, we developed a versatile and simple device to perform antibody incubation under increased barometric pressure. Pressurized immunohistochemistry greatly improves the uniformity, intensity, and depth of fluorescent immunostaining in thick human and mouse brain samples. Furthermore, pressurized immunohistochemistry substantially decreases the time required for classic staining of thin sections.SUBMISSION CATEGORYNew Results

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc.

New neurons continue to be generated in the subgranular zone of the dentate gyrus of the adult mammalian hippocampus(1-5). This process has been linked to learning and memory, stress and exercise, and is thought to be altered in neurological disease(6-10). In humans, some studies have suggested that hundreds of new neurons are added to the adult dentate gyrus every day(11), whereas other studies find many fewer putative new neurons(12-14). Despite these discrepancies, it is generally believed that the adult human hippocampus continues to generate new neurons. Here we show that a defined population of progenitor cells does not coalesce in the subgranular zone during human fetal or postnatal development. We also find that the number of proliferating progenitors and young neurons in the dentate gyrus declines sharply during the first year of life and only a few isolated young neurons are observed by 7 and 13 years of age. In adult patients with epilepsy and healthy adults (18-77 years; n = 17 post-mortem samples from controls; n = 12 surgical resection samples from patients with epilepsy), young neurons were not detected in the dentate gyrus. In the monkey (Macaca mulatta) hippocampus, proliferation of neurons in the subgranular zone was found in early postnatal life, but this diminished during juvenile development as neurogenesis decreased. We conclude that recruitment of young neurons to the primate hippocampus decreases rapidly during the first years of life, and that neurogenesis in the dentate gyrus does not continue, or is extremely rare, in adult humans. The early decline in hippocampal neurogenesis raises questions about how the function of the dentate gyrus differs between humans and other species in which adult hippocampal neurogenesis is preserved.

Somatic stem cells have been identified in multiple adult tissues. Whether self-renewal occurs symmetrically or asymmetrically is key to understanding long-term stem cell maintenance and generation of progeny for cell replacement. In the adult mouse brain, neural stem cells (NSCs) (B1 cells) are retained in the walls of the lateral ventricles (ventricular-subventricular zone [V-SVZ]). The mechanism of B1 cell retention into adulthood for lifelong neurogenesis is unknown. Using multiple clonal labeling techniques, we show that the vast majority of B1 cells divide symmetrically. Whereas 20%-30% symmetrically self-renew and can remain in the niche for several months before generating neurons, 70%-80% undergo consuming divisions generating progeny, resulting in the depletion of B1 cells over time. This cellular mechanism decouples self-renewal from the generation of progeny. Limited rounds of symmetric self-renewal and consuming symmetric differentiation divisions can explain the levels of neurogenesis observed throughout life.

La zona subventricular-ventricular (ZSV-V) es un extenso nicho neurogénico que contiene células madre neurales (astrocitos B) en las paredes de los ventrículos laterales del cerebro adulto de mamíferos. Las células del subtipo B1 han sido identificadas clásicamente como las verdaderas células madre, tienen un origen embrionario y se mantienen quiescentes hasta que se reactivan post-natalmente. En el presente estudio, mostramos que una subpoblación de células B de la ZSV-V presenta una estructura nuclear denominada “lámina de cromatina de la envoltura nuclear” (ELCS, del inglés envelope-limited chromatin sheets), previamente descrita en células normales y cancerosas. Esta estructura se caracteriza por presentar una lámina de heterocromatina de 30 nm limitada en ambos lados por las membranas nucleares interna y externa. A través de marcadores moleculares, estudios de proliferación con 3H-Timidina y la administración de la droga antimitótica Ara-C, encontramos que las células B1 con ELCS corresponden a células madre neurales quiescentes GFAP+, BLBP+, GLAST+, Nestina- y EGFR-. Además, los estudios de microscopía electrónica revelaron que las ELCS empiezan a formarse en las células de glía radial en los estadios embrionarios y se mantienen presentes durante las etapas postnatales tempranas en una subpoblación de células tipo B que disminuye drásticamente con la edad. Adicionalmente, las ELCS representan un compartimento nuclear que contiene modificaciones epigenéticas específicas y telómeros. Estas estructuras también se encuentran presentes en la zona subgranular del giro dentado de roedores y en la ZSV-V de primates no humanos y humanos. Estos resultados muestran que las células madre neurales quiescentes presentan un compartimento muy bien definido estructuralmente y molecularmente en su núcleo, característica que contribuirá a identificar estos progenitores primarios y estudiar como funciona su regulación génica The ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (B1 astrocytes) in the walls of the lateral ventricles of the adult brain. B1 cells have an embryonic origin and remain largely quiescent until they become reactivated postnatally. In this study, we show that a subset of adult V-SVZ B1 cells has nuclear envelope limited chromatin sheets or ELCS, previously described in other normal and cancer cells. Using molecular markers, 3H-thymidine birthdating and the antimitotic drug Ara-C, we found that type B1 cells with ELCS correspond to GFAP+, BLBP+, GLAST+, Nestin- and EGFR- quiescent neural stem cells. TEM reveals that nuclear ELCS begin forming in the embryo radial glia cells and remain during early postnatal stages in a subpopulation of type B cells that drastically decreases with age. Additionally, ELCS represent a specific nuclear compartment that contains specific epigenetic modifications and telomeres. Notably, we found that ELCS are a preserved nuclear structure in the subgranular zone of the rodent dentate gyrus and in the ZSV-V of nonhuman and human primate neural stem cells. These results reveal that quiescent neural stem cells have a unique structurally and molecularly defined compartment in their nuclei, a feature that may help to identify these primary progenitors and study their gene regulation.

Summary Neural stem cells (B1 astrocytes; NSCs) in the adult ventricular-subventricular-zone (V-SVZ) originate in the embryo. Surprisingly, recent work has shown that B1 cells remain largely quiescent. They are reactivated postnatally to function as primary progenitors for neurons destined for the olfactory bulb and some corpus callosum oligodendrocytes. The cellular and molecular properties of quiescent B1 cells remain unknown. Here we found that a subpopulation of B1 cells has a unique nuclear envelope invagination specialization similar to envelope-limited chromatin sheets (ELCS), reported in certain lymphocytes and some cancer cells. Using molecular markers, [3H]thymidine birth-dating, and Ara-C, we found that B1 cells with ELCS correspond to quiescent NSCs. ELCS begin forming in embryonic radial glia cells and represent a specific nuclear compartment containing particular epigenetic modifications and telomeres. These results reveal a unique nuclear compartment in quiescent NSCs, which is useful for identifying these primary progenitors and study their gene regulation., Highlights • A subpopulation of V-SVZ B cells presents nuclear ELCS • ELCS appear in RGCs at E14.5 and remain in a subpopulation of B cells postnatally • Type B cells with ELCS exhibit characteristics of quiescent NSCs • ELCS present characteristic chromatin modifications, In this article, García-Verdugo and colleagues show that a subpopulation of quiescent B cells in the ventricular-subventricular zone of the brain show a unique nuclear envelope structure identified as nuclear envelope-limited chromatin sheets (ELCS). This structure, which appears in embryonic stages, represents a nuclear compartment with specific chromatin and epigenetic modifications.

The persistence of proliferative cells, which could correspond to progenitor populations or potential cells of origin for tumors, has been extensively studied in the adult mammalian forebrain, including human and nonhuman primates. Proliferating cells have been found along the entire ventricular system, including around the central canal, of rodents, but little is known about the primate spinal cord. Here we describe the central canal cellular composition of the Old World primate Macaca fascicularis via scanning and transmission electron microscopy and immunohistochemistry and identify central canal proliferating cells with Ki67 and newly generated cells with bromodeoxyuridine incorporation 3 months after the injection. The central canal is composed of uniciliated, biciliated, and multiciliated ependymal cells, astrocytes, and neurons. Multiciliated ependymal cells show morphological characteristics similar to multiciliated ependymal cells from the lateral ventricles, and uniciliated and biciliated ependymal cells display cilia with large, star-shaped basal bodies, similar to the Ecc cells described for the rodent central canal. Here we show that ependymal cells with one or two cilia, but not multiciliated ependymal cells, proliferate and give rise to new ependymal cells that presumably remain in the macaque central canal. We found that the infant and adult human spinal cord contains ependymal cell types that resemble those present in the macaque. Interestingly, a wide hypocellular layer formed by bundles of intermediate filaments surrounded the central canal both in the monkey and in the human, being more prominent in the stenosed adult human central canal.

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence.

Adult neural stem cells (aNSCs) of the forebrain are GFAP-expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an inducible transgenic mouse line (hGFAP-CreERT2) to conditionally label GFAP-expressing cells in the adult SVZ and SC periaqueduct, and directly and systematically compared their self-renewal and multipotential properties in vitro. We demonstrate that a population of GFAP+ cells that share the morphology and the antigenic properties of SVZ-NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non-proliferative in the intact spinal cord, but incorporate the S-phase marker EdU following spinal cord injury. Multipotent, clonal YFP-expressing neurospheres (i.e., deriving from recombined GFAP-expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self-renewal properties when compared with SVZ-neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non-neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP+ cells co-exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self-renewal properties. GLIA 2013;61:2100–2113

The possibility of obtaining large numbers of cells with potential to become functional neurons implies a great advance in regenerative medicine. A source of cells for therapy is the subventricular zone (SVZ) where adult neural stem cells (NSCs) retain the ability to proliferate, self-renew, and differentiate into several mature cell types. The neurosphere assay, a method to isolate, maintain, and expand these cells has been extensively utilized by research groups to analyze the biological properties of aNSCs and to graft into injured brains from animal models. In this review we briefly describe the neurosphere assay and its limitations, the methods to optimize culture conditions, the identity and the morphology of aNSC-derived neurospheres (including new ultrastructural data). The controversy regarding the identity and “stemness” of cells within the neurosphere is revised. The fine morphology of neurospheres, described thoroughly, allows for phenotypical characterization of cells in the neurospheres and may reveal slight changes that indirectly inform about cell integrity, cell damage, or oncogenic transformation. Along this review we largely highlight the critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere-derived cells to the clinical setting. Anat Rec, 296:1435-1452, 2013. © 2013 Wiley Periodicals, Inc.

Neurogenesis decreases during aging and following cranial radiotherapy, causing a progressive cognitive decline that is currently untreatable. However, functional neural stem cells remained present in the subventricular zone of high dose-irradiated and aged mouse brains. We therefore investigated whether alterations in the neurogenic niches are perhaps responsible for the neurogenesis decline. This hypothesis was supported by the absence of proliferation of neural stem cells that were engrafted into the vascular niches of irradiated host brains. Moreover, we observed a marked increase in TGF-β1 production by endothelial cells in the stem cell niche in both middle-aged and irradiated mice. In co-cultures, irradiated brain endothelial cells induced the apoptosis of neural stem/progenitor cells via TGF-β/Smad3 signalling. Strikingly, the blockade of TGF-β signalling in vivo using a neutralizing antibody or the selective inhibitor SB-505124 significantly improved neurogenesis in aged and irradiated mice, prevented apoptosis and increased the proliferation of neural stem/progenitor cells. These findings suggest that anti-TGF-β-based therapy may be used for future interventions to prevent neurogenic collapse following radiotherapy or during aging.

Objective:To identify cell-surface antibodies in patients with neuromyotonia and to describe the main clinical implications.Methods:Sera of 3 patients with thymoma-associated neuromyotonia and myasthenia gravis were used to immunoprecipitate and characterize neuronal cell-surface antigens using reported techniques. The clinical significance of antibodies against precipitated proteins was assessed with sera of 98 patients (neuromyotonia 46, myasthenia gravis 52, thymoma 42; 33 of them with overlapping syndromes) and 219 controls (other neurologic diseases, cancer, and healthy volunteers).Results:Immunoprecipitation studies identified 3 targets, including the Netrin-1 receptors DCC (deleted in colorectal carcinoma) and UNC5A (uncoordinated-5A) as well as Caspr2 (contactin-associated protein-like 2). Cell-based assays with these antigens showed that among the indicated patients, 9 had antibodies against Netrin-1 receptors (7 with additional Caspr2 antibodies) and 5 had isolated Caspr2 antibodies. Only one of the 219 controls had isolated Caspr2 antibodies with relapsing myelitis episodes. Among patients with neuromyotonia and/or myasthenia gravis, the presence of Netrin-1 receptor or Caspr2 antibodies predicted thymoma (p < 0.05). Coexisting Caspr2 and Netrin-1 receptor antibodies were associated with concurrent thymoma, myasthenia gravis, and neuromyotonia, often with Morvan syndrome (p = 0.009). Expression of DCC, UNC5A, and Caspr2 proteins was demonstrated in paraffin-embedded thymoma samples (3) and normal thymus.Conclusions:Antibodies against Netrin-1 receptors (DCC and UNC5a) and Caspr2 often coexist and associate with thymoma in patients with neuromyotonia and myasthenia gravis.Classification of evidence:This study provides Class III evidence that antibodies against Netrin-1 receptors can identify patients with thymoma (sensitivity 21.4%, specificity 100%).

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc.

Increased neurogenesis has been reported in neurodegenerative disease, but its significance is unclear. In a mouse model of prion disease, Gomez-Nicola et al. detect increased neurogenesis in the dentate gyrus that partially counteracts neuronal loss. Targeting neurogenesis may have therapeutic potential., The study of neurogenesis during chronic neurodegeneration is crucial in order to understand the intrinsic repair mechanisms of the brain, and key to designing therapeutic strategies. In this study, using an experimental model of progressive chronic neurodegeneration, murine prion disease, we define the temporal dynamics of the generation, maturation and integration of new neurons in the hippocampal dentate gyrus, using dual pulse-chase, multicolour γ-retroviral tracing, transmission electron microscopy and patch-clamp. We found increased neurogenesis during the progression of prion disease, which partially counteracts the effects of chronic neurodegeneration, as evidenced by blocking neurogenesis with cytosine arabinoside, and helps to preserve the hippocampal function. Evidence obtained from human post-mortem samples, of both variant Creutzfeldt-Jakob disease and Alzheimer’s disease patients, also suggests increased neurogenic activity. These results open a new avenue into the exploration of the effects and regulation of neurogenesis during chronic neurodegeneration, and offer a new model to reproduce the changes observed in human neurodegenerative diseases.

SummaryThe ventricular-subventricular zone (V-SVZ) is an extensive germinal niche containing neural stem cells (NSCs) in the walls of the lateral ventricles of the adult brain. How the adult brain’s neural activity influences the behavior of adult NSCs remains largely unknown. We show that serotonergic (5HT) axons originating from a small group of neurons in the raphe form an extensive plexus on most of the ventricular walls. Electron microscopy revealed intimate contacts between 5HT axons and NSCs (B1) or ependymal cells (E1) and these cells were labeled by a transsynaptic viral tracer injected into the raphe. B1 cells express the 5HT receptors 2C and 5A. Electrophysiology showed that activation of these receptors in B1 cells induced small inward currents. Intraventricular infusion of 5HT2C agonist or antagonist increased or decreased V-SVZ proliferation, respectively. These results indicate that supraependymal 5HT axons directly interact with NSCs to regulate neurogenesis via 5HT2C.

Neurogenesis persists in the adult subventricular zone (SVZ) of the mammalian brain. During aging, the SVZ neurogenic capacity undergoes a progressive decline, which is attributed to a decrease in the population of neural stem cells (NSCs). However, the behavior of the NSCs that remain in the aged brain is not fully understood. Here we performed a comparative ultrastructural study of the SVZ niche of 2-month-old and 24-month-old male C57BL/6 mice, focusing on the NSC population. Using thymidine-labeling, we showed that residual NSCs in the aged SVZ divide less frequently than those in young mice. We also provided evidence that ependymal cells are not newly generated during senescence, as others studies suggest. Remarkably, both astrocytes and ependymal cells accumulated a high number of intermediate filaments and dense bodies during aging, resembling reactive cells. A better understanding of the changes occurring in the neurogenic niche during aging will allow us to develop new strategies for fighting neurological disorders linked to senescence.

The subventricular zone (SVZ) is the largest source of newly generated cells in the adult mammalian brain. SVZ-derived neuroblasts migrate via the rostral migratory stream (RMS) to the olfactory bulb (OB), where they differentiate into mature neurons. Additionally, a small proportion of SVZ-derived cells contribute to the generation of myelinating oligodendrocytes. The production of new cells in the SVZ decreases during aging, affecting the incorporation of new neurons into the OB. However, the age-related changes that occur across the RMS are not fully understood. In this study we evaluate how aging affects the cellular organization of migrating neuroblast chains, the proliferation, and the fate of the newly generated cells in the SVZ-OB system. By using electron microscopy and immunostaining, we found that the RMS path becomes discontinuous and its cytoarchitecture is disorganized in aged mice (24-month-old mice). Subsequently, OB neurogenesis was impaired in the aged brain while the production of oligodendrocytes was not compromised. These findings provide new insight into oligodendrocyte preservation throughout life. Further exploration of this matter could help the development of new strategies to prevent neurological disorders associated with senescence.

Adult neural stem cells (aNSCs) of the forebrain are GFAP-expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an induci- ble transgenic mouse line (hGFAP-CreERT2) to conditionally label GFAP-expressing cells in the adult SVZ and SC periaque- duct, and directly and systematically compared their self-renewal and multipotential properties in vitro. We demonstrate that a population of GFAP1 cells that share the morphology and the antigenic properties of SVZ-NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non-proliferative in the intact spinal cord, but incorporate the S-phase marker EdU following spinal cord injury. Multipotent, clonal YFP-expressing neurospheres (i.e., deriv- ing from recombined GFAP-expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self-renewal properties when compared with SVZ-neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non- neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP1 cells co-exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self-renewal properties.

The possibility of obtaining large numbers of cells with potential to become functional neurons implies a great advance in regenerative medicine. A source of cells for therapy is the subventricular zone (SVZ) where adult neural stem cells (NSCs) retain the ability to proliferate, self-renew, and differentiate into several mature cell types. The neurosphere assay, a method to isolate, maintain, and expand these cells has been extensively utilized by research groups to analyze the biological properties of aNSCs and to graft into injured brains from animal models. In this review we briefly describe the neurosphere assay and its limitations, the methods to optimize culture conditions, the identity and the morphology of aNSC-derived neurospheres (including new ultrastructural data). The controversy regarding the identity and "stemness" of cells within the neurosphere is revised. The fine morphology of neurospheres, described thoroughly, allows for phenotypical characterization of cells in the neurospheres and may reveal slight changes that indirectly inform about cell integrity, cell damage, or oncogenic transformation. Along this review we largely highlight the critical points that researchers have to keep in mind before extrapolating results or translating experimental transplantation of neurosphere-derived cells to the clinical setting.

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.