Caspase-3 and caspase-9 mediate developmental apoptosis in the mouse olfactory system

Neuronal apoptosis is a key component in the sculpting of tissues during embryonic and postnatal development and is driven largely by the action of caspases. In the mouse olfactory system, caspase-3 and -9 are expressed in olfactory receptor neurons (ORNs) of adult mice, and their selective retrograde activation drives ORN apoptosis following ablation of their target, the olfactory bulb (OB). Here, we show that both of these caspases are expressed at the earliest stages of ORN embryonic development, and their expression is concentrated in outgrowing ORN axons. The retention, in null mice for both caspases, of a population of ORNs that would normally undergo developmental apoptosis beginning at E13 of development, results in a permanently expanded population of ORNs. In turn, in some caspase-3 null mice, the ORN target organ, the OB, also develops abnormally, resulting in the formation of secondary, apparently functional, extracranial ectopic OBs.     

Decreased neurogenesis after cholinergic forebrain lesion in the adult rat

Adult neurogenesis has been shown to be regulated by a multitude of extracellular cues, including hormones, growth factors, and neurotransmitters. The cholinergic system of the basal forebrain is one of the key transmitter systems for learning and memory. Because adult neurogenesis has been implicated in cognitive performance, the present work aims at defining the role of cholinergic input for adult neurogenesis by using an immunotoxic lesion approach. The immunotoxin 192IgG-saporin was infused into the lateral ventricle of adult rats to selectively lesion cholinergic neurons of the cholinergic basal forebrain (CBF), which project to the two main regions of adult neurogenesis: the dentate gyrus and the olfactory bulb. Five weeks after lesioning, neurogenesis, defined by the number of cells colocalized for bromodeoxyuridine (BrdU) and the neuronal nuclei marker NeuN, declined significantly in the granule cell layers of the dentate gyrus and olfactory bulb. Furthermore, immunotoxic lesions to the CBF led to increased numbers of apoptotic cells specifically in the subgranular zone, the progenitor region of the dentate gyrus, and within the periglomerular layer of the olfactory bulb. We propose that the cholinergic system plays a survival-promoting role for neuronal progenitors and immature neurons within regions of adult neurogenesis, similar to effects observed previously during brain development. As a working hypothesis, neuronal loss within the CBF system leads not only to cognitive deficits but may also alter on a cellular level the functionality of the dentate gyrus, which in turn may aggravate cognitive deficits.     

The rostral migratory stream in adult squirrel monkeys: contribution of new neurons to the olfactory tubercle and involvement of the antiapoptotic protein Bcl-2

The subventricular zone (SVZ) lying along the ependymal layer of lateral ventricle is known to generate neural progenitor cells throughout adulthood in specific areas of the mammalian brain. In rodents, the anterior region of the SVZ produces neuroblasts that migrate in chain toward the olfactory bulb along the so-called rostral migratory stream (RMS). In the present study, the organization of the RMS in a representative of New World primates - the squirrel monkey (Saimiri sciureus) - was studied by using bromodeoxyuridine (BrdU), a thymidine analogue that incorporates itself into the DNA of cells undergoing mitotic division. Double and triple immunofluorescence labelling with a confocal microscope served to visualize cells that expressed BrdU as well as molecular markers of neurogenesis. Numerous newborn (BrdU+) cells, many ensheated in glial (GFAP+) tubes, were scattered along the entire RMS in squirrel monkeys. Some of these BrdU+ cells expressed molecular markers for early committed neurons (TuJ1), postmitotic granular neuroblasts (TUC-4) or mature neurons (MAP-2, NeuN), and virtually all of them expressed the antiapoptotic protein Bcl-2. A significant number of BrdU+ cells were found to deviate from the main stream of the RMS. Instead of reaching the olfactory bulb, these cells migrated ventrally into the olfactory tubercle, where they expressed a mature neuronal phenotype (MAP-2). These findings reveal that the RMS in New World monkeys is mitotically robust and markedly extended and suggest that Bcl-2 might play a role in the survival and/or differentiation of newborn neurons destined to olfactory bulb and olfactory tubercle in primates.     

NG2 cells are distinct from neurogenic cells in the postnatal mouse subventricular zone

NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet‐derived growth factor receptor‐α but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA‐NCAM, beta‐tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells. J. Comp. Neurol. 512:702–716, 2009. © 2008 Wiley‐Liss, Inc.     

Effects of developmental age,brain region,and time in culture on long‐term proliferation and multipotency of neural stem cell populations

Neural stem cells (NSCs) in the murine subventricular zone (SVZ) niche allow life‐long neurogenesis. During the first postnatal month and throughout aging, the decrease of neuroblasts and the rise of astrocytes results in diminished neurogenesis and increased astrocyte:neuron ratio. Also, a different neurogenic activity characterizes the SVZ periventricular region (LV, lateral ventricle) as compared to its rostral extension (RE). In order to investigate whether and to what extent these physiological modifications may be ascribed to intrinsic changes of the endogenous NSC/progenitor features, we performed a functional analysis on NSCs isolated and cultured from LV and RE tissues at distinct postnatal stages that are marked by striking modifications to the SVZ niche in vivo. We evaluated the effect of age and brain region on long‐term proliferation and multipotency, and characterized the cell type composition of NSC‐derived progeny, comparing this make‐up to that of region‐ and age‐matched primary neural cultures. Furthermore, we analyzed the effect of prolonged in vitro expansion on NSC functional properties. We documented age‐ and region‐dependent differences on the clonogenic efficiency and on the long‐term proliferative capacity of NSCs. Also, we found age‐ and region‐dependent quantitative changes in the cell composition of NSC progeny (decreased quantity of neurons and oligodendrocytes; increased amount of astroglial cells) and these differences were maintained in long‐term cultured NSC populations. Overall, these data strengthen the hypothesis that age‐ and region‐dependent differences in neurogenesis (observed in vivo) may be ascribed to the changes in the intrinsic developmental program of the NSC populations. J. Comp. Neurol. 517:333–349, 2009. © 2009 Wiley‐Liss, Inc.     

A quantitative study of the effects of early unilateral olfactory deprivation on the number and distribution of mitral and tufted cells and of glomeruli in the rat olfactory bulb

Anatomic effects of early unilateral olfactory deprivation on the developing olfactory bulb were investigated in the albino rat. Unilateral anosmia was experimentally induced by neonatal cauterization of the left or right nare; regenerating tissue permanently blocked the nostril. The anosmic olfactory bulb (ipsilateral to the blocked nare) and its contralateral counterpart, serving as the normal control, were compared for the following quantitative anatomic parameters: total number and distribution of mitral and tufted cells and olfactory glomeruli; average diameters of mitral cells and glomeruli; relative dimensions of the bulb and its layers. The effects on mitral cells and glomeruli were studied at the ages of 25 and 60 days and at 2 years; other studies were carried out in the 25-day-old animals only. In the normal mature bulb, the number of mitral cells, tufted cells and glomeruli was found to be about 70,000, 160,000 and 3000, respectively. It was found that the absence of early olfactory stimulation was invariably correlated with a significant and permanent loss of mitral cells, amounting to more than a quarter of the total number. This loss apparently occurred rapidly during the first 3 week, as it was already evident by day 25 and did not increase appreciably with prolongation of deprivation. Tufted cells were apparently even more susceptible, because their number decreased by about 45%. As evident from their distribution profiles, the loss of mitral and tufted cells occurred uniformly throughout the bulb. It is shown that these differences were due neither to inherent interbulbar differences, nor to a hyperplasia in the normal bulb. Early anosmia had no significant effects on the number or average diameter of the glomeruli. Morphometric studies revealed that the dimensions and thickness of layers (internal and external plexiform and granular) of the anosmic bulb were significantly reduced. It is suggested that early olfactory stimulation is necessary for survival of the developing mitral and tufted cells; thus the first 2–3 postnatal weeks, covering the final developmental stages of these cells, would constitute a vulnerable period in the development of the rat olfactory system.     

Nestin-expressing cells and their relationship to mitotically active cells in the subventricular zones of the adult rat

Nestin is a protein that is thought to be expressed in neural stem cells; however, there is a paucity of data on nestin expression in vivo, and little is known of the relationship between nestin and mitotically active cell populations in the subventricular zones (SVZ). In this study, the subventricular zone of the third ventricle contained a high proportion of cells that expressed nestin, while there were significantly fewer cells that expressed nestin in the SVZ of the lateral ventricles. In contrast, bromodeoxyuridine (BrdU) immunoreactivity was the diametric opposite, being higher in the SVZ of the lateral ventricle than in the SVZ of the third ventricle. Morphological and anatomical evidence suggests that nestin-expressing cells in these two areas may be different cell types. In a separate set of experiments, an acute localized lesion was induced adjacent to one of the ventricles. While the number of BrdU cells and Ki-67 cells in the SVZs increased with this manipulation, the number of nestin-expressing cells did not change significantly. These data indicate that the expression of nestin does not correlate with mitotic activity in cells of the SVZs under either normal or inflammatory conditions. It is hypothesized that nestin-expressing cells in the SVZs may give way to transit amplifying cells that in turn give way to immature neurons or glia. These transit-amplifying cells may have a much higher rate of mitosis than nestin-positive cells and may react to neural damage by increasing their rate of proliferation.     

干细胞因子及其受体在胎脑不同区域的表达

目的　检测干细胞因子 ( stem cell factor,SCF)及其受体 C-kit在胎脑中的表达 ,并探讨其生理意义。方法　利用 RT-PCR半定量法 ,检测人胎脑不同分区和体外培养的人神经干细胞 ( NSC)中 SCF/C-kit m RNA的表达。结果　在大脑、小脑、海马、脑干、延髓、纹状体和垂体均见 SCF/C-kit基因表达。除检测到脑中占主要部分的可溶性片段外 ,还见一约 75 0 bp的未知片段。在体外培养的 NSC中 ,不同因素作用组之间 SCF/C-kit的表达量极不相同。上皮生长因子 ( EGF)诱导的 NSC中 ,SCF/C-kit的表达量高于碱性成纤维细胞生长因子 ( b FGF)单独或与 EGF联合诱导的 NSC。NSC分化后 ,各因素作用组之间差异无显著性。结论　SCF/C-kit可在体内和体外表达 ,提示这一配基 -受体信号系统在脑发育中具有一定生理功能。     

Review: Adult neurogenesis and its role in neuropsychiatric disease,brain repair and normal brain function

Neural stem/progenitor cells (NSPCs) in the mammalian brain retain the ability to generate new neurones throughout life in discrete brain regions, through a process called adult neurogenesis. Adult neurogenesis, a dramatic form of adult brain circuitry plasticity, has been implicated in physiological brain function and appears to be of pivotal importance for certain forms of learning and memory. In addition, failing or altered neurogenesis has been associated with a variety of brain diseases such as major depression, epilepsy and age‐related cognitive decline. Here we review recent advances in our understanding of the basic biology underlying the neurogenic process in the adult brain, focusing on mechanisms that regulate quiescence, proliferation and differentiation of NSPCs. In addition, we discuss how neurogenesis influences normal brain function, and in particular its role in memory formation, as well as its contribution to neuropsychiatric diseases. Finally, we evaluate the potential of targeting endogenous NSPCs for brain repair.     

Light microscope differentiation of two populations of rat olfactory bulb granule cells

Examination of glycolmethacrylate embedded olfactory bulbs of normal rats revealed that the granule cells of the accessory olfactory bulb were dissimilar from the majority of main olfactory bulb granule cells. Cells from these structures can be discriminated on the basis of spherical form. nuclear diameter, nucleoplasm staining, basal dendritic arborizations, and susceptibility to loss after neonatal X-irradiation. Based on their staining qualities, we have called these cells light and dark granule cells. In the normal rat the dark granules make up about 85% of granule cells in the main olfactory bulb and is the cell type preferentially killed by neonatal X-irradiation. Timing of postnatal X-irradiation and consequent differential loss of the dark type of main olfactory bulb granule cell suggests that the light type is largely prenatally formed.     

Light microscopic differentiation of two populations of rat olfactory bulb granule cells

Examination of glycolmethacrylate embedded olfactory bulbs of normal rats revealed that the granule cells of the accessory olfactory bulb were dissimilar from the majority of main olfactory bulb granule cells. Cells from these structures can be discriminated on the basis of spherical form, nuclear diameter, nucleoplasm staining, basal dendritic arborizations, and susceptibility to loss after neonatal X-irradiation. Based on their staining qualities, we have called these cells light and dark granule cells. In the normal rat the dark granules make up about 85% of granule cells in the main olfactory bulb and is the cell type preferentially killed by neonatal X-irradiation. Timing of postnatal X-irradiation and consequent differential loss of the dark type of main olfactory bulb granule cell suggests that the light type is largely prenatally formed.     

Expression of the tight junction protein ZO-1 in the olfactory system: Presence of ZO-1 on olfactory sensory neurons and glial cells

The olfactory system is a unique part of the central nervous system since it retains neuronal turnover and regenerative capacities in adulthood. Thus it provides an ideal model to study plasticity of membrane moities involved in cell-cell interactions. One structure particularly involved in cell-cell interaction is the tight junction, which establishes polarization of epithelial cells and creates diffusion barriers to paracellular passages. ZO-1 is a phosphoprotein peripherally associated with tight junctions. We have studied expression of ZO-1 protein in the developing and adult olfactory system of the mouse in order to get information about the localization and developmental expression of this tight junction component. ZO-1 expression has also been determined in cell cultures of olfactory bulbs. ZO-1 was present in the olfactory placode prior to formation of tight junctions. ZO-1 was localized in the developing and mature olfactory epithelium at heterotypic contacts between supporting cells and olfactory neurons as well as at homotypic contacts between both these cell types. Confocal microscopy showed quantitative differences in the ZO-1 expression among different olfactory dendrites. In the olfactory nerves ZO-1 immunolabeling was detectable between olfactory ensheathing cells. From the seventh postnatal day ZO-1 immunolabeling was detected at the mitral cell layer of the bulb on cells tentatively identified as oligodendrocytes. Myelinated tracts of the bulb were ZO-1 negative. Cell cultures of olfactory bulbs showed ZO-1 immunoreaction, mostly localized on glial fibrillary acidic protein (GFAP)-positive cells. Our results provide further evidence that ZO-1 serves functions unrelated to the tight junction complex and indicate molecular heterogeneity of these cell-cell contacts.     

Changes in granule cells of the ferret olfactory bulb associated with imprinting on prey odours

The maturation of the granule cells of the ferret olfactory bulb around the time of odour imprinting has been examined. Rapid Golgi impregnation studies revealed a temporal overshoot in the development of the spines on the external and internal dendrites of the granule cells. In contrast, the number of somatic spines decreased continuously. Electron microscopical examinations of the synaptic contacts in the external plexiform layer revealed that the time course of synapse and reciprocal synapse formation was similar to that of the formation of the spines on the external dendrites. The results were taken as evidence that both the Golgi and the electron microscopical investigations described the same developmental process of postnatal synaptic rearrangement.     

Selective sensitivity of early postmitotic retinal cells to apoptosis induced by inhibition of protein synthesis

In previous work we showed that apoptosis in retinal tissue from developing rats can be induced by inhibition of protein synthesis (Rehen et al. 1996, Development, 122, 1439-1448). Here we show that recent postmitotic cells are the cells sensitive to apoptosis triggered by blockade of protein synthesis. To label all proliferating cells in the retina, a series of injections of the nucleotide analogue, bromo-deoxy-uridine (BrdU, 60 mg/kg b.w.), was given in rat pups. Then, explants of the retina were incubated in vitro with the inhibitor of protein synthesis anisomycin (1.0-3.2 microg/mL) for 1 day to induce apoptosis. Detection of apoptotic bodies under differential interference contrast microscopy was combined with immunocytochemistry for BrdU, proliferating cell nuclear antigen (PCNA) or for various markers of retinal cell differentiation. Despite the large number of BrdU- and PCNA-labelled cells in the tissue, the vast majority of the cells that underwent apoptosis were postmitotic cells which have left the mitotic cycle 3-4 days before. However, these cells were not labelled with antibodies to calretinin, calbindin, rhodopsin or to a Muller glial cell marker, suggesting that these are early postmitotic neurons. We suggest that during migration and initial differentiation, the apoptotic machinery is blocked by suppressor proteins, thus allowing recent postmitotic cells to find their final positions and differentiate while protected from apoptosis.     

Long-term survival and cell death of newly generated neurons in the adult rat olfactory bulb

In the adult rat olfactory bulb, neurons are continually generated from progenitors that reside in the lateral ventricle wall. This study investigates long-term survival and cell death of newly generated cells within the adult olfactory bulb. After injecting rats at 2 months of age with 5-bromodeoxyuridine (BrdU), the newly generated cells were quantified over a period of 19 months. A peak of BrdU-positive cells was reached in the olfactory bulb 1 month after BrdU injection, when all new cells have finished migrating from the ventricle wall. Thereafter, a reduction of BrdU-positive cells to about 50% was observed and it was confirmed by dUTP-nick end-labelling (TUNEL) that progenitors and young neurons undergo programmed cell death. However, cells that survived the first 3 months after BrdU injection persisted for up to 19 months. The majority of the BrdU-positive cells that reach the olfactory bulb differentiate into granule cells, but a small fraction migrate further into the glomerular layer. These newborn cells differentiate more slowly into periglomerular interneurons, with a delay of more than 1 month when compared to the granule cells. The newly generated periglomerular neurons, among them a significant fraction of dopaminergic cells, showed a similar decline in number compared to the granule cell layer and long-term survival for the remaining new neurons of up to 19 months. Rather than replacing old neurons, this data suggests that adult olfactory bulb neurogenesis utilizes the overproduction and turnover of young neurons, which is reminiscent of the cellular dynamics observed during brain development.     

Chain formation and glial tube assembly in the shift from neonatal to adult subventricular zone of the rodent forebrain

The subventricular zone (SVZ) is regarded as an embryonic germinal layer persisting at the end of cerebral cortex neurogenesis and capable of generating neuronal precursors throughout life. The two distinct compartments of the adult rodent forebrain SVZ, astrocytic glial tubes and chains of migrating cells, are not distinguishable in the embryonic and early postnatal counterpart. In this study we analyzed the SVZ of mice and rats around birth and throughout different postnatal stages, describing molecular and morphological changes which lead to the typical structural arrangement of adult SVZ. In both species studied, most changes occurred during the first month of life, the transition being slightly delayed in mice, in spite of their earlier development. Important modifications affected the glial cells, eventually leading to glial tube assembly. These changes involved an overall reorganization of glial processes and their mutual relationships, as well as gliogenesis occurring within the SVZ which gives rise to glial cell subpopulations. The neuroblast cell population remained qualitatively quite homogeneous throughout all the stages investigated, changes being restricted to the relationships among cells and consequent formation of chains at about the third postnatal week. Electron microscopy showed that chain formation is not directly linked to glial tube assembly, generally preceding the occurrence of complete glial ensheathment. Moreover, chain and glial tube formation is asymmetric in the medial/lateral aspect of the SVZ, being inversely related. The attainment of an adult SVZ compartmentalization, on the other hand, seems linked to the pattern of expression of adhesion and extracellular matrix molecules.     

Enhanced neurogenesis in the olfactory bulb in adult mice after injury induced by acute treatment with trimethyltin

In adults, the subventricular zone is known to contain undifferentiated neural progenitor cells that proliferate and generate the olfactory bulb (OB) interneurons throughout life. We earlier showed that trimethyltin (TMT) causes neuronal damage in the granular cell layer of the OB in adult mice. In the current study, we examined neurogenesis in the OB in adult mice after injury induced by acute treatment with TMT. On day 2 post‐TMT treatment, enhanced incorporation of 5‐bromo‐2′‐deoxyuridine (BrdU) was seen in the granular cell layer of the OB. Many of the BrdU‐labeled cells were undifferentiated cells on day 2 post‐treatment. On day 30 post‐TMT treatment, BrdU‐labeled neuronal cells were dramatically increased in number in the granular cell layer of the OB. However, TMT treatment was ineffective in affecting the migration of BrdU‐labeled cells from the subventricular zone to the OB. The results of a neurosphere assay revealed that the number of neurospheres derived from the OB was significantly increased on day 2 post‐TMT treatment. The neurosphere‐forming neural progenitor cells derived from the OB of TMT‐treated animals were capable of differentiating into neuronal cells as well as into astrocytes. Taken together, our data suggest that the OB has the ability to undergo enhanced neurogenesis following TMT‐induced neuronal injury in adult mice. © 2009 Wiley‐Liss, Inc.