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Cell cycle length of olfactory bulb neuronal progenitors in the rostral migratory stream
Developmental Dynamics
Abstract
The anterior portion of the neonatal telencephalic subventricular zone (SVZa) contains proliferating cells that generate an immense number of neurons destined to become the granule and periglomerular cells of the olfactory bulb. In contrast to other immature neurons in the central nervous system, cells arising in the SVZa maintain the ability to divide as they traverse the rostral migratory stream to their final destinations despite expressing an antigenic marker of differentiated neurons (Menezes et al. [1995] Molec. Cell. Neurosci. 6:496-508). Because of their considerable proliferative capacities and unusual mitotic behavior, we decided to determine the cell cycle length of proliferating cells within the SVZa and within the migratory pathway used by SVZa-derived cells. Following the methodology of Nowakowski et al. [1989](J. Neurocytol. 18:311-318), postnatal day 2 rat pups were exposed to 5'-bromo-2'deoxyuridine (BrdU) for increasing periods of time before perfusion. By plotting the percentage of nuclei undergoing DNA synthesis in the SVZa at each time versus the BrdU labeling interval, we determined that approximately 15% of the SVZa population is actively dividing and that these cells have a cycle length of approximately 14 hr, significantly less than the 18.6 hr determined to be the cycle length of dividing cells in more posterior, glia-generating regions of the subventricular zone (Thomaidou et al. [1997] J. Neurosci. 17:1075-1085). The cycle length of cells dividing in the mid portion of the rostral migratory stream, however, is considerably longer: 17.3 hr. This may reflect the need for these cells to coordinate the processes of migration and division. Our studies also suggest that there may be regional differences in the types of descendants produced by the proliferating cells. Retroviral lineage tracing studies showed that those cells that divide within the rostral migratory stream, like proliferating cells within the SVZa, make cells destined for the olfactory bulb. Unlike the progenitors that divide within the SVZa and generate more granule cells than periglomerular cells, the proliferating cells within the migratory pathway generate more periglomerular cells than granule cells. Collectively the proliferating cells of the SVZa and migratory pathway produce a large number of olfactory bulb interneurons. Our work suggests that this may be achieved in part by the relatively rapid divisions of progenitor cells within the SVZa and in part by the ongoing division of migrating cells en route to the olfactory bulb.
... The cells of the olfactory bulb originate from the subventricular zone (SVZ) neuronal progenitors that differentiate into periglomerular and granule cells of the bulb. 1,2 These cells are different form the other central nervous system neurons because of their ability to divide after the expression of adult neuronal markers. 3,4 Several studies have reported the unusual cell cycle and mitotic behavior of these cells during the development of the nervous system, although the molecular mechanism responsible for the control of the prolonged cell cycle and adult neurogenesis remains elusive. ...
... 3,4 Several studies have reported the unusual cell cycle and mitotic behavior of these cells during the development of the nervous system, although the molecular mechanism responsible for the control of the prolonged cell cycle and adult neurogenesis remains elusive. 1,5,6 Tumor suppressor protein (p53) and cell cycle exit protein (Bax) largely participates in control of cell proliferation in the SVZ neuronal stream. 7,8 In vitro experiments involving knock out mice models have shown that over expression of p53 or Bax reduces the rate of cell proliferation and renewal in the developing olfactory bulb. ...
... It involves the repopulation of neuronal stem cells migrating from the SVZ into the granule and periglomerular cell layers of the olfactory bulb to participate on the formation of inter neurons required for the integration of newly formed cells of the olfactory circuit. 1,44 Despite the importance or neuronal survival in the olfactory bulb, the regulatory mechanism remains elusive. Hypothetically, the prominent players marked for study are the cell cycle and apoptosis protein-P53 and Bax. ...
The granule and periglomerular cells of the olfactory bulb migrate from the sub-ventricular zone (SVZ) as progenitor cell forming the neuronal stream of the rostral olfactory bulb. These cells are characterized by their ability to divide while expressing adult proteins; a phenomenon attributed to the prolonged cell cycle and the regulatory activities of proteins which modulates apoptosis and proliferation in the developing nervous system. Of interest are the proteins concerned with tumor suppression (p53) and cell cycle exit (Bax) and how they regulate survivability of these neurons in the adult system after an induced oxidative stress.
This study sets to investigate the interplay between p53 and Bax in the adult olfactory bulb (periglomerular and granule cell layer), and how these proteins determine proliferation and neuronal survival after Cytochrome C induced-oxidative stress. Also, we demonstrate the effect of the induced-stress threshold on such regulation in vivo.
Adult Wistar rats were segregated into three groups. 10 and 20 mg/Kg BW of potassium cyanide (KCN) was administered to the treatment groups for 15 days while the control received normal saline for the same duration. The olfactory bulb was dissected and processed for general histology and immunohistochemistry of p53/Bax in the periglomerular and granule cell layers. Total (Histology) and immunopositive (p53 and Bax) cell count was done using Image J. Subsequently, we determined the analysis of variance with significance set at *P<0.05.
We observed an increase in cell count for the 10 mg/KgBW treatment; this was characterized by a significant decrease in Bax expression and no change in p53 expression when this treatment group was compared to the control. However, no change was observed in the total cell count for 20 mg/Kg BW treatment for the same duration of exposure. Interestingly, there was also no significant change in Bax and p53 for this treatment when compared with the control.
Although p53 plays an important role in development of the olfactory bulb neurons, our findings suggests it has little contribution in neuronal cell viability and proliferation in the adult olfactory bulb. No significant change in p53 was observed irrespective of treatment dose and cell count while Bax expression was reduced at 10 mg/Kg BW treatment and was associated with an increased cell count. We conclude that regulation of survival of neurons in the adult olfactory bulb, following induced-oxidative stress was more dependent of the expression of Bax and the threshold of the induced stress rather than p53 expression.
... While proliferation markers such as Ki67 label type-B and mainly type-C cells that are both sessile [26], they also label a small population of migrating neuroblasts (∼15%; [4]). Due to this non-specificity, we next focused our attention to specific markers of SVZ-NSCs and of progenitors of independent lineages (Tbr2 and Dlx2; see below). ...
... Our data’s reveal a clear rostral enrichment of cycling cells compared to more caudal regions of the LV. This distribution might be partly due to the rostral migration of neuroblasts, a fraction of which proliferate (∼15%; [4]). In comparison to cycling cells, detailed analysis of SVZ-NSC numbers and of their dissemination through the rostro-caudal axis of the LV is scarce. ...
... Accumulating evidence underlines major differences in the time and place of origin of distinct olfactory neuronal subtypes (reviewed in [2], [3]). Original work that fuelled these observations was the demonstration, by retroviral labelling, of progenitors in the LV or the RMS resulting in the generation of granular or periglomerular cells respectively [4]–[6]. Subsequent studies showed that distinct olfactory neuronal subtypes originate from defined regions of the LV, both in the early postnatal [7], [8] and adult forebrain [8]–[12]. ...
Recent studies suggest that the subventricular zone (SVZ) of the lateral ventricle is populated by heterogeneous populations of stem and progenitor cells that, depending on their exact location, are biased to acquire specific neuronal fates. This newly described heterogeneity of SVZ stem and progenitor cells underlines the necessity to develop methods for the accurate quantification of SVZ stem and progenitor subpopulations. In this study, we provide 3-dimensional topographical maps of slow cycling "stem" cells and progenitors based on their unique cell cycle properties. These maps revealed that both cell populations are present throughout the lateral ventricle wall as well as in discrete regions of the dorsal wall. Immunodetection of transcription factors expressed in defined progenitor populations further reveals that divergent lineages have clear regional enrichments in the rostro-caudal as well as in the dorso-ventral span of the lateral ventricle. Thus, progenitors expressing Tbr2 and Dlx2 were confined to dorsal and dorso-lateral regions of the lateral ventricle, respectively, while Mash1+ progenitors were more homogeneously distributed. All cell populations were enriched in the rostral-most region of the lateral ventricle. This diversity and uneven distribution greatly impede the accurate quantification of SVZ progenitor populations. This is illustrated by measuring the coefficient of error of estimates obtained by using increasing section sampling interval. Based on our empirical data, we provide such estimates for all progenitor populations investigated in this study. These can be used in future studies as guidelines to judge if the precision obtained with a sampling scheme is sufficient to detect statistically significant differences between experimental groups if a biological effect is present. Altogether, our study underlines the need to consider the SVZ of the lateral ventricle as a complex 3D structure and define methods to accurately assess neural stem cells or progenitor diversity and population sizes in physiological or experimental paradigms.
... growth fraction (GF; proportion of proliferating cells) and the length of cell cycle (TC) (Nowakowski et al., 1989;Takahashi et al., 1993;Caviness et al., 2003). Studies in neonatal and postnatal rats show that the cell cycle length of the SVZ ranges from 14 to 18.6 h (Schultze and Korr, 1981;Smith and Luskin, 1998). Increase in the proliferating cell population in the SVZ after EAE could result from increase of GF and/or shortening of TC of progenitor cells in the SVZ. ...
... These data suggest that changes in the cell cycle kinetics contribute to EAE-induced neurogenesis in the adult rat brain. Our observation that approximately 20% of the SVZ population in the adult rat was actively dividing is consistent with the proliferating population (15% to 21%) in postnatal animals (Schultze and Korr, 1981;Smith and Luskin, 1998). EAE increases the proliferating cell population to 27%. ...
... Data from cumulative BrdU labeling method indicated that the cell cycle length in the adjuvant controls was 19 h. The cell cycle length in the pseudostratified ventricular epithelium of E14 mice is 15.1 h (Takahashi et al., 1993) and 18 to 21 h in the SVZ of adult rat (Schultze and Korr, 1981;Smith and Luskin, 1998). In adult rodents, the cell cycle length in the SVZ remains relatively constant throughout the animal lifetime (Smith and Luskin, 1998). ...
Cytokinetics regulating cell cycle division can be modulated by several endogenous factors. EAE (experimental autoimmune encephalomyelitis) increases proliferation of progenitor cells in the subventricular zone (SVZ). Using cumulative and single S phase labeling with 5-bromo-2-deoxyuridine, we examined cell cycle kinetics of neural progenitor cells in the SVZ after EAE. 20% of the SVZ cell population was proliferating in adjuvant control rats. However, EAE significantly increased them up to 27% and these cells had a cell cycle length (TC) of 15.6h, significantly (P<0.05) shorter than the 19 h TC in non EAE SVZ cells. Few TUNEL (+) cells were detected in the SVZ cells of adjuvant controls. EAE increased (P<0.05) TUNEL (+) nuclei in SVZ suggesting early stage progenitor cell death. Cell cycle phase analysis revealed that EAE substantially shortened the length of the G1 phase (9.6h) compared with the G1 phase of 12.25 h in adjuvant control SVZ cells (P<0.05). This reduction in G1 contributes to EAE-induced reduction of TC because no significant changes were detected on the length of S, G2 and M phases between the two groups. Our results show a surge in proliferating progenitor cells in the SVZ with concomitant increase in apoptotic cell death after EAE. Furthermore, increase in the SVZ proliferation contributes to EAE-induced neurogenesis and this increase is regulated by shortening the G1 phase. Our investigation suggests the activation of quiescent cells in SVZ to generate actively proliferating progenitors. Moreover, the increase in the cell death in proliferating population may contribute towards negative regulation of proliferative cell number and hence diminished regenerative capacity of CNS following EAE.
... Cumulative labeling is extensively applied for determining key parameters of the cell cycle and is conducted via either continuous nucleotide analogue delivery (usually for in vitro labeling) (98,99) or via repeated pulse labeling (for in vivo labeling) (100)(101)(102)(103)(104)(105)(106)(107)(108)(109)(110). Cumulative labeling is aimed to mark all cells of the proliferative population and to track the kinetics of label incorporation into cells passing the cell cycle ( Figure 3A). ...
... Pulse-chase labeling with the extended chase period (7,45,90) Pulse-chase labeling with two temporally discriminated labels (20,54) Pulse-chase labeling with three temporally discriminated labels (88 Cumulative labeling (100)(101)(102)(103)(104)(105)(106)(107)(108)(109)(110) and percent labeled mitoses method (100,110) Pulse-chase labeling with two labels delivered at variable time intervals (64,112,113) Pulse-chase labeling with three temporally discriminated labels (41) Determination of the G1-and G2-phase durations (14,16,117) ...
Detection of thymidine analogues after their incorporation into replicating DNA represent a powerful tool for the study of cellular DNA synthesis, progression through the cell cycle, cell proliferation kinetics, chronology of cell division, and cell fate determination. Recent advances in the concurrent detection of multiple such analogues offer new avenues for the investigation of unknown features of these vital cellular processes. Combined with quantitative analysis, temporal discrimination of multiple labels enables elucidation of various aspects of stem cell life cycle in situ, such as division modes, differentiation, maintenance, and elimination. Data obtained from such experiments are critically important for creating descriptive models of tissue histogenesis and renewal in embryonic development and adult life. Despite the wide use of thymidine analogues in stem cell research, there is a number of caveats to consider for obtaining valid and reliable labeling results when marking replicating DNA with nucleotide analogues. Therefore, in this review, we describe critical points regarding dosage, delivery, and detection of nucleotide analogues in the context of single and multiple labeling, outline labeling schemes based on pulse-chase, cumulative and multilabel marking of replicating DNA for revealing stem cell proliferative behaviors and determining cell cycle parameters, and discuss preconditions and pitfalls in conducting such experiments. The information presented in our review is important for rational design of experiments on tracking dividing stem cells by marking replicating DNA with thymidine analogues.
... The RMS is primarily recognized as a migratory pathway, and its proliferative role is less recognized; proliferation is slower in the RMS than in the SVZ, generating most of the olfactory bulb interneuron subtypes calretinin granular and periglomerular cells [61]. Cell proliferation in the RMS is higher in the posterior and medial regions than in the anterior part [61,62]. ...
... The RMS is primarily recognized as a migratory pathway, and its proliferative role is less recognized; proliferation is slower in the RMS than in the SVZ, generating most of the olfactory bulb interneuron subtypes calretinin granular and periglomerular cells [61]. Cell proliferation in the RMS is higher in the posterior and medial regions than in the anterior part [61,62]. The medial and anterior regions of the RMS show a higher glial proliferation than the posterior region [63]. ...
Microtus ochrogaster is a rodent with a monogamous reproductive strategy characterized by strong pair bond formation after 6 h of mating. Here, we determine whether mating-induced pair bonding increases cell proliferation in the subventricular zone (SVZ), rostral migratory stream (RMS), and dentate gyrus (DG) of the hippocampus in male voles. Males were assigned to one of the four groups: (1) control: males were placed alone in a clean cage; (2) social exposure to a female (SE m/f): males that could see, hear, and smell a sexually receptive female but where physical contact was not possible, because the animals were separated by an acrylic screen with small holes; (3) social exposure to a male (SE m/m): same as group 2 but males were exposed to another male without physical contact; and (4) social cohabitation with mating (SCM): males that mated freely with a receptive female for 6 h. This procedure leads to pair bond formation. Groups 2 and 3 were controls for social interaction. Male prairie voles were injected with 5-bromo-2'-deoxyuridine (BrdU) during the behavioral tests and were sacrificed 48 h later. Brains were processed to identify the new cells (BrdU-positive) and neuron precursor cells (neuroblasts). Our principal findings are that in the dorsal region of the SVZ, SCM and SE m/f and m/m increase the percentage of neuron precursor cells. In the anterior region of the RMS, SE m/f decreases the percentage of neuron precursor cells, and in the medial region SE m/f and m/m decrease the number of new cells and neuron precursor cells. In the infrapyramidal blade of the subgranular zone of the DG, SE m/m and SCM increase the number of new neuron precursor cells and SE m/m increases the percentage of these neurons. Our data suggests that social interaction, as well as sexual stimulation, leads to pair bonding in male voles modulating cell proliferation and differentiation to neuronal precursor cells at the SVZ, RMS, and DG.
... The OB interneurons do not exhibit full morphological maturity until they reach the OB. Retroviral lineage tracing studies showed, that proliferating cells within the SVZa generate more granule neurons and the cells that divide within the RMS generate more periglomerular neurons [10]. Further retroviral study of [11] described five stages and the timing for the maturation of granular neurons: tangentially migrating neuroblasts (days 2-7), radially migrating young neurons (days 5-7), the neurons with a simple unbranched dendrite (days 9-13), the neurons with branched dendrite (days [11][12][13][14][15][16][17][18][19][20][21][22] and the mature granular neurons (days [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. ...
... Earlier studies of irradiation effects in normal brain tissue have been mostly focused on studies of large scale of single radiation doses [12][13][14][15][16][17][18]. Single whole brain irradiation (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) led to reduced numbers of proliferating cells and immature neurons from the SVZ in a dose-dependent pattern, up to several months after treatment, but these changes were reversible [13,19]. However, a large single dose of radiation is not commonly used in the clinical practice. ...
Ionizing radiation induces altered brain tissue homeostasis and can lead to morphological and functional deficits. In this study, adult male Wistar rats received whole-body exposure with fractionated doses of gamma rays (a total dose of 5 Gy) and were investigated 30 and 60 days later. Immunohistochemistry and confocal microscopy were used to determine proliferation rate of cells residing or derived from the forebrain anterior subventricular zone (SVZa) and microglia distributed along and/or adjacent to subventricular zone-olfactory bulb axis. Cell counting was performed in four anatomical parts along the well-defined pathway, known as the rostral migratory stream (RMS) represented by the SVZa, vertical arm, elbow and horizontal arm of the RMS. Different spatiotemporal distribution pattern of cell proliferation was seen up to 60 days after irradiation through the migratory pathway. A population of neuroblasts underwent less evident changes up to 60 days after treatment. Fractionated exposure led to decline or loss of resting as well as reactive forms of microglia until 60 days after irradiation. Results showed that altered expression of the SVZa derived cells and ultimative decrease of microglia may contribute to development of radiation-induced late effects.
... First, there is a decreasing gradient of proliferation in the SVZ from posterior to anterior (Coskun and Luskin, 2001). The different populations of neuroblasts also leave the cell cycle at different locations, as shown by the labelling of proliferative cells through retrovirus injection in the anterior SVZ (Luskin, 1993), and in the intersection between the descending and horizontal limbs of the rostral migratory stream (RMS) (Smith and Luskin, 1998; Suzuki and Goldman, 2003; Hack et al., 2005). Although labelled cells in the anterior SVZ produce mostly granular neurons (65%, compared with 25% found in the glomerular layer), 75% of the labelled cells after RMS injections go to the glomerular layer (Luskin, 1993; Smith and Luskin, 1998). ...
... The different populations of neuroblasts also leave the cell cycle at different locations, as shown by the labelling of proliferative cells through retrovirus injection in the anterior SVZ (Luskin, 1993), and in the intersection between the descending and horizontal limbs of the rostral migratory stream (RMS) (Smith and Luskin, 1998; Suzuki and Goldman, 2003; Hack et al., 2005). Although labelled cells in the anterior SVZ produce mostly granular neurons (65%, compared with 25% found in the glomerular layer), 75% of the labelled cells after RMS injections go to the glomerular layer (Luskin, 1993; Smith and Luskin, 1998). These data indicate that different neuronal types have different timings of maturation. ...
During embryonic development, the telencephalon is specified along its axis through morphogenetic gradients, leading to the positional-dependent generation of multiple neuronal types. After embryogenesis, however, the fate of neuronal progenitors becomes more restricted, and they generate only a subset of neurons. Here, we review studies of postnatal and adult neurogenesis, challenging the notion that fixed genetic programs restrict neuronal fate. We hypothesize that the adult brain maintains plastic neural stem cells that are capable of responding to changes in environmental cues and generating diverse neuronal types. Thus, the limited diversity of neurons generated under normal conditions must be actively maintained by the adult milieu.
... Neuroblasts migrate tangentially to the OB via the rostral migratory stream (RMS), a restricted route that is approximately 5 mm in length in mice [34,66]. During migration neuroblasts retain the ability to divide but have a longer cell cycle than proliferative cells in the SVZ (17.3 vs 12.5-14 hr), while maintaining an average migration rate of 23 μm/h [39,44,48,53,59]. ...
... Longer cell cycles have been reported with aging [22,64]. If the average migration rate of 23 μm/h is maintained with aging, neuroblasts with a longer cell cycle may migrate through the VA before dividing, supporting our data showing decreased proliferation in the VA and stable proliferation in the elbow and HA [39,48,59,64]. ...
Throughout life the subventricular zone (SVZ) is a source of new olfactory bulb (OB) interneurons. From the SVZ, neuroblasts migrate tangentially through the rostral migratory stream (RMS), a restricted route approximately 5 mm long in mice, reaching the OB within 10-14 days. Within the OB, neuroblasts migrate radially to the granule and glomerular layers where they differentiate into granule and periglomerular (PG) cells and integrate into existing synaptic circuits. SVZ neurogenesis decreases with age, and might be a factor in age-related olfactory deficits. However, the effect of aging on the RMS and on the differentiation of interneuron subpopulations remains poorly understood. Here, we examine RMS cytoarchitecture, neuroblast proliferation and clearance from the RMS, and PG cell subpopulations at 6, 12, 18, and 23 months of age. We find that aging affects the area occupied by newly generated cells within the RMS and regional proliferation, and the clearance of neuroblasts from the RMS and PG cell subpopulations and distribution remain stable.
... By selective killing of type C cells or by cell fate mapping, it has been shown that type B cells are the ancestor of type C and type A cells (Doetsch et al., 1999; Garcia et al., 2004 ) and that type A neuroblasts and their descendant OB differentiated neurons originate from Ascl1 transit-amplifying type C progenitors (Kim et al., 2007 Gene transfer into SVZ neural stem cells before their migration has been proposed with the purpose of recruiting these cells for repair of CNS lesions (Fallon et al., 2000; Chmielnicki et al., 2004; Cho et al., 2007; Henry et al., 2007). Retroviral and lentiviral vectors, which integrate into the cellular genome, have been used for tracing and fate analysis of SVZ neural stem cells (Smith and Luskin, 1998). Because of their requirement for cell division, retroviral vectors (Goldman et al., 1993) have allowed the specific labeling of type C cells and, to a lesser extent, slowly proliferating GFAP-positive type B cells. ...
... We show that the percentage of migrating Dcx + cells relative to the pool of Mash1 + neural precursors was unaltered by viral transduction. Furthermore, the newly generated neurons were located in the OB granular layer, expressed the GAD67 marker of GABAergic neurons, and were TH negative, as expected for cells originating in the SVZ (Smith and Luskin, 1998). The here-described inhibition of proliferation of immature neuronal cells should be taken into account when interpreting experiments aiming at the recruitment of SVZ neural stem cells by AAV-mediated gene transfer (Henry et al., 2007). ...
... Therefore, it is likely that the expression of cell cycle regulatory molecules in distinct cell populations accounts for differences in cell cycle kinetics. In agreement with this model, studies on cell cycle length in the neonatal rat brain (Schultze and Korr, 1981; Menezes et al., 1995; , 1998; Smith and Luskin, 1998) have indicated that differences in cell cycle kinetics between cells in the neonatal anterior SVZ (that have a fast cell cycle time) and the migratory cells in the RMS with a slower kinetics of cell division (Smith and Luskin, 1998), correlate with the levels of expression of the G1 inhibitor p19INK4d (Coskun and Luskin, 2001). Indeed cell cycle length and the probability to exit from the cell cycle are both affected by cell cycle regulatory molecules and transcription factors whose expression can be modulated by genetic factors, epigeneticmodifications of chromatin and by the integration of extracellular signals. ...
... Therefore, it is likely that the expression of cell cycle regulatory molecules in distinct cell populations accounts for differences in cell cycle kinetics. In agreement with this model, studies on cell cycle length in the neonatal rat brain (Schultze and Korr, 1981; Menezes et al., 1995; , 1998; Smith and Luskin, 1998) have indicated that differences in cell cycle kinetics between cells in the neonatal anterior SVZ (that have a fast cell cycle time) and the migratory cells in the RMS with a slower kinetics of cell division (Smith and Luskin, 1998), correlate with the levels of expression of the G1 inhibitor p19INK4d (Coskun and Luskin, 2001). Indeed cell cycle length and the probability to exit from the cell cycle are both affected by cell cycle regulatory molecules and transcription factors whose expression can be modulated by genetic factors, epigeneticmodifications of chromatin and by the integration of extracellular signals. ...
Although stem cell therapy has been proposed for therapeutic strategies aimed at repairing functions, it is important to realize
that as yet, relatively little is known about the behavior of embryonic and adult stem cells in terms of responsiveness to
extracellular cues and intracellular signaling molecules.
The challenge that awaits ahead is to define possible differences in intracellular signaling molecules between embryonic and
adult derived neural stem cells that may underlie the distinctive responsiveness of these different cell types to external
signals. A better understanding of the mechanisms regulating proliferation and differentiation of multipotent progenitors
into differentiated neurons, astrocyte and oligodendrocytes is, therefore, essential for developing a realistic frame of therapeutic
intervention while preventing undesirable - and yet possible-neoplastic transformation of adult neural stem cells.
... By selective killing of type C cells or by cell fate mapping, it has been shown that type B cells are the ancestor of type C and type A cells (Doetsch et al., 1999; Garcia et al., 2004 ) and that type A neuroblasts and their descendant OB differentiated neurons originate from Ascl1 transit-amplifying type C progenitors (Kim et al., 2007 Gene transfer into SVZ neural stem cells before their migration has been proposed with the purpose of recruiting these cells for repair of CNS lesions (Fallon et al., 2000; Chmielnicki et al., 2004; Cho et al., 2007; Henry et al., 2007). Retroviral and lentiviral vectors, which integrate into the cellular genome, have been used for tracing and fate analysis of SVZ neural stem cells (Smith and Luskin, 1998). Because of their requirement for cell division, retroviral vectors (Goldman et al., 1993) have allowed the specific labeling of type C cells and, to a lesser extent, slowly proliferating GFAP-positive type B cells. ...
... We show that the percentage of migrating Dcx + cells relative to the pool of Mash1 + neural precursors was unaltered by viral transduction. Furthermore, the newly generated neurons were located in the OB granular layer, expressed the GAD67 marker of GABAergic neurons, and were TH negative, as expected for cells originating in the SVZ (Smith and Luskin, 1998). The here-described inhibition of proliferation of immature neuronal cells should be taken into account when interpreting experiments aiming at the recruitment of SVZ neural stem cells by AAV-mediated gene transfer (Henry et al., 2007). ...
The adult rat brain subventricular zone (SVZ) contains proliferative precursors that migrate to the olfactory bulb (OB) and differentiate into mature neurons. Recruitment of precursors constitutes a potential avenue for brain repair. We have investigated the kinetics and cellular specificity of transgene expression mediated by AAV2/1 vectors (i.e., adeno-associated virus type 2 pseudotyped with AAV1 capsid) in the SVZ. Self-complementary (sc) and single-stranded (ss) AAV2/1 vectors mediated efficient GFP expression, respectively, at 17 and 24 hr postinjection. Transgene expression was efficient in all the rapidly proliferating cells types, that is, Mash1(+) precursors (30% of the GFP(+) cells), Dlx2(+) neuronal progenitors (55%), Olig2(+) oligodendrocyte progenitors (35%), and doublecortin-positive (Dcx(+)) migrating cells (40%), but not in the slowly proliferating glial fibrillary acidic protein-positive (GFAP(+)) neural stem cell pool (5%). Because cell cycle arrest by wild-type and recombinant AAV has been described in primary cultures, we examined SVZ proliferative activity after vector injection. Indeed, cell proliferation was reduced immediately after vector injection but was normal after 1 month. In contrast, migration and differentiation of GFP(+) precursors were unaltered. Indeed, the proportion of Dcx(+) cells was similar in the injected and contralateral hemispheres. Furthermore, 1 month after vector injection into the SVZ, GFP(+) cells, found, as expected, in the OB granular cell layer, were mature GABAergic neurons. In conclusion, the rapid and efficient transgene expression in SVZ neural precursors mediated by scAAV2/1 vectors underlines their potential usefulness for brain repair via recruitment of immature cells. The observed transient precursor proliferation inhibition, not affecting their migration and differentiation, will likely not compromise this strategy.
... The proportion of proliferating cells and the length of the cell cycle are two critical parameters of the cytokinetics for neocortical neurogenesis (Nowakowski et al., 1989; Takahashi et al., 1993; Caviness et al., 2003). In the adult rat, approximately 15 to 21% of the SVZ cell population is actively dividing (Schultze and Korr, 1981; Smith and Luskin, 1998; Zhang et al., 2006b). The cell cycle length of actively dividing SVZ cells in the adult rat is 18–21 hr and remains relatively constant throughout the animal's lifetime (Schultze and Korr, 1981; Smith and Luskin, 1998; Zhang et al., 2006b). ...
... In the adult rat, approximately 15 to 21% of the SVZ cell population is actively dividing (Schultze and Korr, 1981; Smith and Luskin, 1998; Zhang et al., 2006b). The cell cycle length of actively dividing SVZ cells in the adult rat is 18–21 hr and remains relatively constant throughout the animal's lifetime (Schultze and Korr, 1981; Smith and Luskin, 1998; Zhang et al., 2006b). Stroke changes the length of the cell cycle and the proportion of proliferating SVZ cells, which contribute to stroke-induced neurogenesis (Zhang et al., 2006b; Zhang et al., 2007b). ...
The subventricular zone (SVZ) of the lateral ventricle contains neural stem and progenitor cells that generate neuroblasts, which migrate to the olfactory bulb where they differentiate into interneurons. Ischemic stroke induces neurogenesis in the SVZ and these cells migrate to the boundary of the ischemic lesion. This article reviews current data on cytokinetics, signaling pathways and vascular niche that are involved in processes of proliferation, differentiation, and migration of neural progenitor cells after stroke.
... Evidence for a link between cell migration and differentiation also is compelling in the postnatal SVZ-olfactory bulb pathway. SVZ neuroblasts continue to divide as they migrate to the olfactory bulb (Smith and Luskin, 1998). In neonatal rats, cell cycle length is 14 hours in the SVZ and 17.3 hours in the RMS (Smith and Luskin, 1998). ...
... SVZ neuroblasts continue to divide as they migrate to the olfactory bulb (Smith and Luskin, 1998). In neonatal rats, cell cycle length is 14 hours in the SVZ and 17.3 hours in the RMS (Smith and Luskin, 1998). The cell cycle length of neural progenitors undergoing neurogenic, differentiative divisions is significantly longer than those undergoing proliferative divisions (Calegari et al., 2005). ...
Neural progenitor cells persist throughout life in the forebrain subventricular zone (SVZ). They generate neuroblasts that migrate to the olfactory bulb and differentiate into interneurons, but mechanisms underlying these processes are poorly understood. Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic factor that influences cell motility, proliferation and morphogenesis in neural and non-neural tissues. HGF and its receptor, c-Met, are present in the rodent SVZ-olfactory bulb pathway. Using in vitro neurogenesis assays and in vivo studies of partially HGF-deficient mice, we find that HGF promotes SVZ cell proliferation and progenitor cell maintenance, while slowing differentiation and possibly altering cell fate choices. HGF also acts as a chemoattractant for SVZ neuroblasts in co-culture assays. Decreased HGF signaling induces ectopic SVZ neuroblast migration and alters the timing of migration to the olfactory bulb. These results suggest that HGF influences multiple steps in postnatal forebrain neurogenesis. HGF is a mitogen for SVZ neural progenitors, and regulates their differentiation and olfactory bulb migration.
... As I stated above, the two principal regions (niches) with high density of dividing (precursor/progenitor) cells within the adult mammals CNS are the sub granular zone (SGZ) of the dentate gyrus of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles (Kaplan and Hinds, 1977;Smith and Luskin, 1998;Rietze et aI., 2001). The SVZ is a layer of dividing cells that extends along the lateral walls of the lateral ventricles (Figure 1.1) (Doetsch, 2003). ...
p>To understand the mechanisms underlying neuronal control of hippocampal neurogenesis, we are investigating the hypothesis that VIP modulates the activity of postnatal hippocampal stem/progenitor cells.
We have investigated VIP effects in hippocampal neuronal cultures from postnatal rats (P7-9). BrdU and Ki-67 were used to measure cell proliferation. Quantification of cell death was achieved by DAPI and propidium iodide. Immunohistochemistry was used to determine cell-specific phenotypes. PCR assay was carried out to study the expression of VIP receptors.
We have shown that VIP at nanomolar concentrations has a trophic and self-renewal effect on nestin hippocampal cells in vitro , particularly the amplifying cell population, through enhanced symmetrical cell division. This effect is specific for dentate gyrus progenitor cells. Using immunohistochemistry and PCR techniques, we demonstrate the expression of VPAC1 and VPAC2 receptors and their mRNAs in hippocampal progenitor cultures. Pharmacologically, we have shown that VIP survival and self-renewal effects are VPAC2 mediated. We confirm that VPAC2 knockout adult mice have a significantly reduced number of newly-born neurons in the granule cell layer of the dentate gyrus.
We conclude that VIP is a trophic and self-renewal factor that may play a key role in hippocampal neurogenesis. This work demonstrates a novel mechanism by which neuronal activity can influence trophism and fate of precursor cells in the hippocampus.</p
... We established that clonally related NBs migrate in small clusters, and are widespread through the rostrocaudal RMS axis. Previous research has shown that some NBs in the RMS retain their ability to proliferate (Poon et al., 2010;Smith and Luskin, 1998). Upon reaching the OB, NBs begin to radially migrate to their final position where they integrate into the pre-existing neural olfactory circuit (Belluzzi et al., 2003;Carleton et al., 2003), replacing granular and periglomerular cells (Imayoshi et al., 2008). ...
Understanding the contribution of adult neural progenitor cells (NPCs) and their lineage potential is a great challenge in neuroscience. To reveal progenitor diversity and cell-lineage relationships of postnatal NPCs in the subventricular zone (SVZ), we performed in vivo lineage-tracing genetic analysis using the UbC-StarTrack. We determined the progeny of single SVZ-NPCs, the number of cells per clone, the dispersion of sibling cells, and the cell types within clones. Long-term analysis revealed that both the cell-dispersion pattern and number of cells comprising clones varied depending on the glial/neuronal nature of sibling cells. Sibling-olfactory interneurons were primarily located within the same layer, while sibling-glial cells populated SVZ-adjacent areas. Sibling astrocytes and interneurons did not form big clones, whereas oligodendroglial-lineage clones comprised the largest clones originated in adult brains. These results demonstrate the existence of SVZ postnatal bipotential progenitors that give rise to clones widely dispersed across the olfactory bulb and SVZ-adjacent areas.
... [17,18] Actively dividing cell population in the SVZ of adult rat is approximately 15% to 21%. [19][20][21] Previous study indicated that stroke substantially increased dividing SVZ cells up to 31% in mice model. [22] Though supplementing on survival and proliferation of intrinsic NSCs could assist to repair the damaged tissues, the efficacy of this supplementation has been shown to be limited. ...
Background
Chinese herbal medicine (CHM) has been used to treat stroke for thousands of years. The objective of the study is to assess the current evidence for bioactive components of CHM as neurogenesis agent in animal models of ischemic stroke.
Methods
We searched PubMed, China National Knowledge Infrastructure, WanFang Database, and VIP Database for Chinese Technical Periodicals published from the inception up to November 2015. The primary measured outcome was one of neurogenesis biomarker, including Bromodeoxyuridine (BrdU), Nestin, doublecortin (DCX), polysialylated form of the neural cell adhesion molecule (PSA-NCAM), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP).
Results
Thirty eligible studies were identified. The score of quality assessment ranged from 2 of 10 to 7 of 10. Compared with controls, 10 studies conducting neurobehavioral evaluation showed significant effects on bioactive components of CHM for improving neurological deficits score after ischemic insults (P < 0.01 or P < 0.05); 6 studies in Morris water-maze test showed bioactive components of CHM significantly decreased escape latency and increased residence time (P < 0.05); 5 studies demonstrated that bioactive components of CHM significantly reduced infarct volume after ischemic stroke (P < 0.05); 25 of 26 studies showed that bioactive components of CHM significantly increased the expression of BrdU and/or Nestin markers in rats/mice brain after ischemic injury (P < 0.05, or P < 0.01); 4 of 5 studies for promoting the expression of PSA-NCAM or DCX biomarker (P < 0.05); 5 studies for improving the expression of NeuN biomarker (P < 0.05); 6 of 7 studies for promoting the expression of GFAP biomarker in brain after ischemic stroke (P < 0.05).
Conclusion
The findings suggest that bioactive components of CHM may improve neurological function, reduce infarct volume, and promote endogenous neurogenesis, including proliferation, migration, and differentiation of neural stem cells after ischemic stroke. However, evidences are supported but limited because only a few studies were available for each descriptive analysis. Further rigor study is still needed.
... The progenitors located along different points of dorso-ventral and antero-posterior axes of the ventricular wall display different cell cycle kinetics and cell type density (Falcão et al., 2012). Retroviral labeling of the proliferative progenitors in the anterior SVZ (Luskin, 1993) or at the horizontal limb of the rostral migratory stream (Smith and Luskin, 1998), demonstrated that progenitors of granular neurons leave the cell cycle earlier (more posteriorly) than the ones that will generate the periglomerular (PG) neurons. These data, later reproduced by other group (Hack et al., 2005), show that different populations of newly generated OB neurons segregate very early, while still migrating. ...
The adult mammalian brain harbors a population of cells around their lateral ventricles capable of giving rise to new neurons throughout life. The so-called subventricular zone (SVZ) is a heterogeneous germinative niche in regard to the neuronal types it generates. SVZ progenitors give rise to different olfactory bulb (OB) interneuron types in accordance to their position along the ventricles. Here, I review data showing the difference between progenitors located along different parts of the SVZ axes and ages. I also discuss possible mechanisms for the origin of this diversity.
... As they migrate from the SVZ through the RMS towards the olfactory bulb, neuroblasts proliferate, then exit the cell cycle, and stop expressing proliferative markers such as Ki67 (Smith and Luskin 1998). Our SVZ expression analysis ( Table 2) had shown that all of the proliferating neuroblasts investigated, and the majority of those that were no longer proliferating, expressed NFIX. ...
Transcription factors of the nuclear factor one (NFI) family play a pivotal role in the development of the nervous system.
One member, NFIX, regulates the development of the neocortex, hippocampus, and cerebellum. Postnatal Nfix−/− mice also display abnormalities within the subventricular zone (SVZ) lining the lateral ventricles, a region of the brain
comprising a neurogenic niche that provides ongoing neurogenesis throughout life. Specifically, Nfix−/− mice exhibit more PAX6-expressing progenitor cells within the SVZ. However, the mechanism underlying the development of this
phenotype remains undefined. Here, we reveal that NFIX contributes to multiple facets of SVZ development. Postnatal Nfix−/− mice exhibit increased levels of proliferation within the SVZ, both in vivo and in vitro as assessed by a neurosphere assay.
Furthermore, we show that the migration of SVZ-derived neuroblasts to the olfactory bulb is impaired, and that the olfactory
bulbs of postnatal Nfix−/− mice are smaller. We also demonstrate that gliogenesis within the rostral migratory stream is delayed in the absence of Nfix, and reveal that Gdnf (glial-derived neurotrophic factor), a known attractant for SVZ-derived neuroblasts, is a target for transcriptional activation
by NFIX. Collectively, these findings suggest that NFIX regulates both proliferation and migration during the development
of the SVZ neurogenic niche.
... As a physiological process, SVZ cells migrate to the olfactory bulb via the rostral migratory stream to differentiate into granule interneurons [14]. After ischemic stroke, chemoattractants such as SDF-1a in the injury are upregulated and divert neural progenitors from the rostral migratory stream toward the ischemic infarct [13,15,16]. Cerebral ischemia dramatically increases SVZ neural progenitor cell proliferation, migration to the infarct, and differentiation into neurons [9,[17][18][19][20][21]. ...
Limited treatments are available for perinatal/neonatal stroke. Induced pluripotent stem cells (iPSCs) hold therapeutic promise for stroke treatment, but the benefits of iPSC transplantation in neonates are relatively unknown. We hypothesized that transplanted iPS cell-derived neural progenitors (iPSC-NPCs) would increase regeneration after stroke.Mouse pluripotent iPSCs were differentiated into neural progenitors using a retinoic acid protocol. Differentiated neural cells were characterized by using multiple criteria and assessments. Ischemic stroke was induced in postnatal day 7 (P7) rats by occluding the right middle cerebral artery and right common carotid artery. iPSC-NPCs (400,000 in 4 µl) were transplanted into the penumbra via intracranial injection 7d after stroke. Trophic factor expression in the peri-infarct tissue was measured using Western blot analysis. Animals received daily BrdU injections and were sacrificed 21d after stroke for immunohistochemistry. The vibrissae-elicited forelimb placement test was used to evaluate functional recovery.Differentiated iPSCs expressed mature neuronal markers, functional sodium and potassium channels, and fired action potentials. Several angiogenic and neurogenic trophic factors were identified in iPSC-NPCs. Animals that received iPSC-NPC transplantation had greater expression of SDF-1α and VEGF in the peri-infarct region. iPSC-NPCs stained positive for NeuN or GFAP 14 days after transplantation. iPSC-NPC-transplanted animals showed greater numbers of BrdU/NeuN and BrdU/Collagen IV co-labeled cells in the peri-infarct area compared to stroke controls, and performed better in a sensorimotor functional test after stroke. iPSC-NPC therapy may play multiple therapeutic roles after stroke by providing trophic factors, increasing angiogenesis and neurogenesis, and providing new cells for tissue repair. Stem Cells 2014
... Therefore, the number of proliferating cells at a certain time point in the SVZ results from the balance between proliferation and migration. In this context, our data obtained in sham surgery animals showing higher values to those present in the ischemic group, despite the positive correlation with lesion size at 7 d, also support this affirmation and are consistent with the proliferating population (15-21%) described in postnatal animals (19,20). However, no correlation was found between SVZ cell proliferation and neuroblast migration, suggesting that the number of BrdU ϩ cells at the SVZ at the times studied is not only affected by migration but also by modulation of proliferation. ...
Toll-like receptor 4 (TLR4) mediates brain damage after stroke. Now our objective is to determine TLR4 involvement in stroke-induced neurogenesis. Stroke was induced by permanent middle cerebral artery occlusion in wild-type and TLR4-deficient mice. Stereological and densitometric analysis of immunofluorescence-labeled brain sections and FACS analysis of cell suspensions were performed. Our results show that subventricular zone (SVZ) cell proliferation after stroke depends on infarct size. Second, when comparing brains with similar lesions, TLR4 attenuated SVZ proliferation, as shown by a decrease in prominin-1(+)/EGFR(+)/nestin(-) cells (type-C cells) at 1-2 d, and in BrdU(+) cells at 7 d, in TLR4(+/+) vs. TLR4(-/-) mice. Interestingly, 7 d after the infarct, neuroblasts in TLR4(+/+) mice migrated farther distances, reaching areas closer to the lesion than those in TLR4-deficient mice. However, at 14 d, TLR4-deficient mice presented a higher number of neuroblasts in all migratory zones than the TLR4(+/+) counterparts, which suggests that TLR4 deficiency delays neuroblast migration. Consistently, TLR4(+/+) mice showed an increased number of interneurons (NeuN(+)/BrdU(+)/GAD67(+) cells) in peri-infarct cortex 14-28 d after stroke. Our data indicate that, despite a negative effect on SVZ cell proliferation, TLR4 plays an important role in stroke-induced neurogenesis by promoting neuroblasts migration and increasing the number of new cortical neurons after stroke.-Moraga, A., Pradillo, J. M., Cuartero, M. I., Hernández-Jiménez, M., Oses, M., Moro, M. A., Lizasoain, I. Toll-like receptor 4 modulates cell migration and cortical neurogenesis after focal cerebral ischemia.
... Thus, neuroblasts migrate tangentially from the SVZ and incorporate into the OB. Neuroblasts in the RMS retain their ability to proliferate (Smith and Luskin, 1998;Poon et al., 2010), but once they reach the OB, neuroblasts begin radial migration and mature into interneurons that integrate in preexisting functional circuits (Lois and Alvarez-Buylla, 1994;Luskin et al., 1997;Carleton et al., 2003;Imayoshi et al., 2008;Kelsch et al., 2010;Lazarini and Lledo, 2011). Although most SVZ precursor cells generate neuroblasts to support OB neurogenesis, a small subpopulation of precursors gives rise to cells in the oligodendroglial lineage, which are able to migrate toward the corpus callosum, striatum, or septum to differentiate into myelinating oligodendrocytes (Nait-Oumesmar et al., 1999;Menn et al., 2006;Gonzalez-Perez et al., 2009;Gonzalez-Perez and Alvarez-Buylla, 2011;Capilla-Gonzalez et al., in press). ...
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.
... Proliferation, however, is much slower than in the SVZ and the cell cycle duration is lengthened. Interestingly, unlike the progenitors that divide within the SVZ and generate more granule cells than periglomerular cells, the proliferating cells within the migratory pathway generate more periglomerular cells than granule cells (Smith and Luskin, 1998). The presence of neural precursors within the MOB has been also reported in mice (Gritti et al., 2002), sheep (Brus et al., 2010Brus et al., , 2013), macaque monkey (Kornack and Rakic, 2001) and humans (Pagano et al., 2000; Bedard and Parent, 2004), suggesting a contribution to the addition of newborn neurons to the olfactory system. ...
Production of new neurons continues throughout life in most invertebrates and vertebrates like crustaceans, fishes, reptiles, birds, and mammals including humans. Most studies have been carried out on rodent models and demonstrated that adult neurogenesis is located mainly in two structures, the dentate gyrus (DG) of the hippocampus and the sub-ventricular zone (SVZ). If adult neurogenesis is well preserved throughout evolution, yet there are however some features which differ between species. The present review proposes to target similarities and differences in the mechanism of mammalian adult neurogenesis by comparing selected species including humans. We will highlight the cellular composition and morphological organization of the SVZ in primates which differs from that of rodents and may be of functional relevance. We will particularly focus on the dynamic of neuronal maturation in rodents, primates, and humans but also in sheep which appears to be an interesting model due to its similarities with the primate brain.
... With the single injection of BrdU, only S-phase cells were labelled. Since the cell cycle length for proliferating cells in the RMS is approximately 17.3 h [22], injecting mice 26 h before their perfusion implies that cells could be in S-phase at most twice. Therefore, cells could be labelled with BrdU twice. ...
The rostral migratory stream is a highly proliferative region of the central adult nervous system that extends from the lateral ventricles to the olfactory bulb. Through the rostral migratory stream, progenitor cells originated in subventricular zones migrate tangentially to the subependymal zone of the olfactory bulb, even during adult life. During their migration to the olfactory bulb these cells divide, and hence this zone is characterized by a high rate of proliferation. Here we have developed optimized protocols to detect this cell proliferation using two markers: 5-bromo-2’-deoxyuridine (BrdU) and proliferating cell nuclear antigen (PCNA). We carried out assays with different fixatives, sectioning methods, and an antigen retrieval technique to obtain the best signal-to-noise ratio. Using the optimal protocol, it is possible to detect PCNA and BrdU simultaneously, providing a clear immunocytochemical identification of proliferating cells by use of optical or confocal microscopy.
... Neuroblasts maintain the ability to proliferate and express many genes that are involved in different aspects of cell division and cell cycle regulation (Fig. 2 ). However, their proliferative potential is limited by and large to the posterior part of the RMS (Coskun and Luskin, 2002; Menezes et al., 1995; Smith and Luskin, 1998). Thus, neuroblasts labeled by a short pulse of BrdU (5-bromo-2 0 -deoxyuridine, proliferation marker ) exhibited a posterior-anterior gradient, with significantly more labeled neuroblast in the proximity of the SVZ and a gradual decrease towards the bulb (Coskun and Luskin, 2002; Menezes et al., 1995). ...
The subventricular zone (SVZ) of the lateral ventricles is a major neurogenic region in the postnatal mammalian brain. Thousands of neuroblasts are generated daily throughout the life of an animal. Newly born neuroblasts migrate via the rostral migratory stream (RMS) into the olfactory bulb where they mature into distinct neuronal subtypes. Neuroblasts exiting the SVZ retain the ability to proliferate, however, proliferation declines in the course of migration to the olfactory bulb. While migrating in the RMS, neuroblasts receive a plethora of stimuli that modify transcription according to the local microenvironment, and eventually modulate neuroblast migration. In the target area, the olfactory bulb, neuroblasts develop into mature neurons. In this review, we discuss dynamic changes of the transcriptome that occur during the "lifetime" of a neuroblast, thereby governing the activation or inhibition of distinct genes/pathways that are responsible for proliferation, migration and differentiation.
... Neuroblasts derived from SVZ stem cells express immature neuronal markers DCX, PSA-NCAM, and Type III β-tubulin (TuJI) and tangentially migrate along the RMS to the olfactory bulb [13, 14]. During the migration, these cells continue to divide, but the cell cycle is lengthened [20]. When they arrive at the end of the RMS, residing in the core of olfactory bulb, they exit from the RMS, switch to a radial migration pattern, and differentiate into mature interneurons in the granule layer and periglomerular layer of the olfactory bulb [13, 14]. ...
The discovery of adult neurogenesis has greatly advanced our knowledge of the human brain. During the past 50 years, the regulatory
mechanisms and potential functions of this intriguing process have been extensively investigated. Our current knowledge supports
the model that adult neurogenesis is regulated by both intrinsic genetic and epigenetic programs and extrinsic microenvironment
and stimuli. This intricate molecular network has profound roles in controlling the self-renewal and multipotency of neural
stem cells, the cellular basis of adult neurogenesis. In this review, we will summarize the current knowledge and our recent
work in understanding adult neurogenesis with emphasis on answering two questions: how intrinsic epigenetic mechanisms, mediated
through histone modifications, non-coding RNAs, and DNA methylation, define the signature of adult neural stem cells, and
how extrinsic effects of growth factors, cytokines, and chemokines contribute to the adult neurogenic niche.
... Regarding the radiation-induced damage of DNA and resulting inhibition of cell-cycle progress (Dasika et al., 1999; Melo and Toczyski, 2002) we suppose that the DNA damage might contribute to slowing down of cell migration and thus induces accumulation of BrdU positive cells in the RMS. Our opinion can be supported by findings of Smith and Luskin (1998) concerning different cell cycle length of the neuronal progenitors in the RMS, which may reflect the need to coordinate the process of migration and division. ...
One of the few areas of the adult CNS, that are known to be competent for neuronal proliferation, is the subventricular zone (SVZ) lining the brain lateral ventricles. Cells proliferating in the SVZ migrate along a defined pathway, the rostral migratory stream (RMS), where their proliferation continues until reaching the olfactory bulb.
1. In relation to the fact that brain is, in general, regarded as a radioresistant organ composed from non dividing cells, the aim of the present study was to investigate effect of ionizing radiation on proliferating cell numbers in the RMS of adult rats.
2. Male Wistar rats were investigated 25 and 80 days after whole body gamma irradiation with the dose of 3 Gy. Dividing cells were labeled by bromodeoxyuridine (BrdU). BrdU-positive cells were counted by Disector program. The mean number of BrdU+ cells in the whole RMS and in its individual parts (vertical arm, elbow, and horizontal arm) was evaluated.
3. Temporary increase in proliferating cell number (by 30%) was seen in the whole RMS at the 25th day after irradiation.
4. The most expressive increase occurred in the vertical arm (by 60%) and elbow (about 37%). The values reduced till the 80th day after exposure.
Our results show that ionizing irradiation significantly influences the extent of cell proliferation and migration in the adult rat RMS.
... During migration, type-A cells continue to divide and initiate neuronal maturation [6], but their rate of proliferation is drastically reduced and the cell cycle time is lengthened [7]. The migration is predominantly towards the OB, but many parts of the individual paths can point in other directions. ...
Neurogenesis has been the subject of active research in recent years and many authors have explored the phenomenology of the process, its regulation and its purported purpose. Recent developments in bioluminescent imaging (BLI) allow direct in vivo imaging of neurogenesis, and in order to interpret the experimental results, mathematical models are necessary. This study proposes such a mathematical model that describes adult mammalian neurogenesis occurring in the subventricular zone and the subsequent migration of cells through the rostral migratory stream to the olfactory bulb (OB). This model assumes that a single chemoattractant is responsible for cell migration, secreted both by the OB and in an endocrine fashion by the cells involved in neurogenesis. The solutions to the system of partial differential equations are compared with the physiological rodent process, as previously documented in the literature and quantified through the use of BLI, and a parameter space is described, the corresponding solution to which matches that of the rodent model. A sensitivity analysis shows that this parameter space is stable to perturbation and furthermore that the system as a whole is sloppy. A large number of parameter sets are stochastically generated, and it is found that parameter spaces corresponding to physiologically plausible solutions generally obey constraints similar to the conditions reported in vivo. This further corroborates the model and its underlying assumptions based on the current understanding of the investigated phenomenon. Concomitantly, this leaves room for further quantitative predictions pertinent to the design of future proposed experiments.
... BrdU-ir cells were also observed in the SVZ, particularly at the innermost ependymal cell layer lining the lateral ventricle. As these cells are better quantified in sagittal brain sections (Fowler et al., 2002;Smith and Luskin, 1998), we focused on the DG and the limbic brain regions mentioned above. Due to a low overall cell count, Ki67-and TUNEL-labeled cells were quantified using a modified version of the optical fractionator method (West et al., 1991). ...
Disruptions in the social environment, such as social isolation, are distressing and can induce various behavioral and neural changes in the distressed animal. We conducted a series of experiments to test the hypothesis that long-term social isolation affects brain plasticity and alters behavior in the highly social prairie vole (Microtus ochrogaster). In Experiment 1, adult female prairie voles were injected with a cell division marker, 5-bromo-2'-deoxyuridine (BrdU), and then same-sex pair-housed (control) or single-housed (isolation) for 6weeks. Social isolation reduced cell proliferation, survival, and neuronal differentiation and altered cell death in the dentate gyrus of the hippocampus and the amygdala. In addition, social isolation reduced cell proliferation in the medial preoptic area and cell survival in the ventromedial hypothalamus. These data suggest that long-term social isolation affects distinct stages of adult neurogenesis in specific limbic brain regions. In Experiment 2, isolated females displayed higher levels of anxiety-like behaviors in both the open field and elevated plus maze tests and higher levels of depression-like behavior in the forced swim test than controls. Further, isolated females showed a higher level of affiliative behavior than controls, but the two groups did not differ in social recognition memory. Together, our data suggest that social isolation not only impairs cell proliferation, survival, and neuronal differentiation in limbic brain areas, but also alters anxiety-like, depression-like, and affiliative behaviors in adult female prairie voles. These data warrant further investigation of a possible link between altered neurogenesis within the limbic system and behavioral changes.
... These latter processes are ATP-dependent and occur on a minute-to-hour time scale131415. At any given time, about 15–20% of SVZ cells are actively cycling as assessed by labeling with the nucleoside analog bromodeoxyuridine (BrdU), which is taken up during S phase (e.g. [16,17]). Nutrients and metabolites supplied through blood perfusion may be sufficient to maintain basal cell proliferation. ...
The postnatal subventricular zone (SVZ) contains proliferating neural progenitor cells in close proximity to blood vessels. Insults and drug treatments acutely stimulate cell proliferation in the SVZ, which was assessed by labeling cells entering S phase. Although G1-to-S progression is metabolically demanding on a minute-to-hour time scale, it remains unknown whether increased SVZ cell proliferation is accompanied by a local hemodynamic response. This neurovascular coupling provides energy substrates to active neuronal assemblies. Transcardial dye perfusion revealed the presence of capillaries throughout the SVZ that constrict upon applications of the thromboxane A(2) receptor agonist U-46119 in acute brain slice preparations. We then monitored in vivo blood flow using laser Doppler flowmetry via a microprobe located either in the SVZ or a mature network. U-46119 injections into the lateral ventricle decreased blood flow in the SVZ and the striatum, which are near the ventricle. A 1-hour ventricular injection of epidermal and basic fibroblast growth factor (EGF and bFGF) significantly increased the percentage of Sox2 transcription factor-positive cells in S phase 1.5 hours post-injection. This increase was accompanied by a sustained rise in blood flow in the SVZ but not in the striatum. Direct growth factor injections into the cortex did not alter local blood flow, ruling out direct effects on capillaries. These findings suggest that an acute increase in the number of G1-to-S cycling SVZ cells is accompanied by neurometabolic-vascular coupling, which may provide energy and nutrient for cell cycle progression.
... " Steady-state " in the crayfish niche is, therefore, 2-4 cells (seeFigure 2A). As more than 4 cells rarely accumulate in the niche in crayfish exposed to control conditions, the usual method for determining the length of the cell cycle (T C ) by defining the percentage of cells relative to steady state that incorporate BrdU during incremental increases in BrdU exposure time (e.g., 1 hr, 2 hr, 3 hr, 4 hr)[39], is not applicable. Such a graph changes sharply from 0% to 100% (3-4 cells) within 6 hr for small crayfish (<15 mm CL). ...
Adult neurogenesis, the production and integration of new neurons into circuits in the brains of adult animals, is a common feature of a variety of organisms, ranging from insects and crustaceans to birds and mammals. In the mammalian brain the 1st-generation neuronal precursors, the astrocytic stem cells, reside in neurogenic niches and are reported to undergo self-renewing divisions, thereby providing a source of new neurons throughout an animal's life. In contrast, our work shows that the 1st-generation neuronal precursors in the crayfish (Procambarus clarkii) brain, which also have glial properties and lie in a neurogenic niche resembling that of vertebrates, undergo geometrically symmetrical divisions and both daughters appear to migrate away from the niche. However, in spite of this continuous efflux of cells, the number of neuronal precursors in the crayfish niche continues to expand as the animals grow and age. Based on these observations we have hypothesized that (1) the neuronal stem cells in the crayfish brain are not self-renewing, and (2) a source external to the neurogenic niche must provide cells that replenish the stem cell pool.
In the present study, we tested the first hypothesis using sequential double nucleoside labeling to track the fate of 1st- and 2nd-generation neuronal precursors, as well as testing the size of the labeled stem cell pool following increasing incubation times in 5-bromo-2'-deoxyuridine (BrdU). Our results indicate that the 1st-generation precursor cells in the crayfish brain, which are functionally analogous to neural stem cells in vertebrates, are not a self-renewing population. In addition, these studies establish the cycle time of these cells. In vitro studies examining the second hypothesis show that Cell Tracker™ Green-labeled cells extracted from the hemolymph, but not other tissues, are attracted to and incorporated into the neurogenic niche, a phenomenon that appears to involve serotonergic mechanisms.
These results challenge our current understanding of self-renewal capacity as a defining characteristic of all adult neuronal stem cells. In addition, we suggest that in crayfish, the hematopoietic system may be a source of cells that replenish the niche stem cell pool.
... During tangential migration through the RMS, cells continue to divide and initiate neuronal maturation (Menezes et al., 1995; Coskun et al., 2007). Retroviral studies demonstrate that cells dividing in the RMS have a much longer cell cycle time than those dividing in the SVZ (Smith and Luskin, 1998). It will be interesting to study whether daughter cells pause during or after division or whether daughter cells migrate immediately. ...
Neuroblasts born in the adult subventricular zone (SVZ) migrate long distances in the rostral migratory stream (RMS) to the olfactory bulbs where they integrate into circuitry as functional interneurons. As very little was known about the dynamic parameters of SVZ neuroblast migration, we used two-photon time-lapse microscopy to analyze migration in acute slices. This involved analyzing 3D stacks of images over time and uncovered several novel aspects of SVZ migration: chains remain stable, cells can be immotile for extensive periods, morphology does not necessarily correlate with motility, neuroblasts exhibit local exploratory motility, dorsoventral migration occurs throughout the striatal SVZ, and neuroblasts turn at distinctive angles. We investigated these novel findings in the SVZ and RMS from the population to the single cell level. In this review we also discuss some technical considerations when setting up a two-photon microscope imaging system. Throughout the review we identify several unsolved questions about SVZ neuroblast migration that might be addressed with current or emerging techniques.
... The type C cells differentiate into type A cells (neuroblasts), which migrate to the olfactory bulb (OB) through the rostral migratory stream (RMS) and then differentiate into local interneurons [1,2]. Several reports have demonstrated that these neuroblasts, which are committed to the neuronal fate, retain their proliferative capacity while migrating toward the OB [3][4][5], suggesting that they are inhibited from exiting the cell cycle. However, the molecular mechanisms underlying this unique feature of adult neuroblasts are only partly understood. ...
The subventricular zone (SVZ) is the largest neurogenic region in the adult rodent brain. In the adult SVZ, unlike in the embryonic brain, neuronally committed precursor cells (neuroblasts) maintain their proliferative activity while migrating toward the olfactory bulb (OB), suggesting that they are inhibited from exiting the cell cycle. Little is known about the mechanisms underlying the unique ability of adult neuroblasts to proliferate during migration. Here, we studied the expression and function of Diversin, a component of the Wnt signaling pathways. In the neonatal and adult mouse brain, Diversin expression was observed in neuroblasts and mature neurons in the SVZ and hippocampus. Retrovirus-mediated overexpression of Diversin promoted the proliferation of neuroblasts and increased the number of neuroblasts that reached the OB. Conversely, the knockdown of Diversin decreased the proliferation of neuroblasts. Our results indicate that Diversin plays an important role in the proliferation of neuroblasts in the SVZ of the adult brain.
... SVZ cells can also switch to behavioral repertoires associated with their progenitors. Despite the evidence for distinct proliferative and migratory populations described above, there is evidence that migrating neuroblasts occasionally proliferate [44,45]. Remarkably, magnetic bead sorted PSA-NCAM+ SVZ neuroblasts become gliogenic when transplanted, suggesting they lose their fate commitment and acquire stem cell-like characteristics [46]. ...
The adult subventricular zone (SVZ) contains stem and progenitor cells that generate neuroblasts throughout life. Although it is well accepted that SVZ neuroblasts are migratory, recent evidence suggests their progenitor cells may also exhibit motility. Since stem and progenitor cells are proliferative and multipotential, if they were also able to move would have important implications for SVZ neurogenesis and its potential for repair.
We studied whether SVZ stem and/or progenitor cells are motile in transgenic GFP+ slices with two photon time lapse microscopy and post hoc immunohistochemistry. We found that stem and progenitor cells; mGFAP-GFP+ cells, bright nestin-GFP+ cells and Mash1+ cells were stationary in the SVZ and rostral migratory stream (RMS). In our search for motile progenitor cells, we uncovered a population of motile betaIII-tubulin+ neuroblasts that expressed low levels of epidermal growth factor receptor (EGFr). This was intriguing since EGFr drives proliferation in the SVZ and affects migration in other systems. Thus we examined the potential role of EGFr in modulating SVZ migration. Interestingly, EGFr(low) neuroblasts moved slower and in more tortuous patterns than EGFr-negative neuroblasts. We next questioned whether EGFr stimulation affects SVZ cell migration by imaging Gad65-GFP+ neuroblasts in the presence of transforming growth factor alpha (TGF-alpha), an EGFr-selective agonist. Indeed, acute exposure to TGF-alpha decreased the percentage of motile cells by approximately 40%.
In summary, the present study directly shows that SVZ stem and progenitor cells are static, that EGFr is retained on some neuroblasts, and that EGFr stimulation negatively regulates migration. This result suggests an additional role for EGFr signaling in the SVZ.
... However, we are currently unable to draw a definitive conclusion about the presence of stem cells within the amygdala. It has been estimated that stem cells proliferate approximately once every four weeks (Morshead et al., 1994), producing progenitor cells that may continue progress through subsequent divisions ~17 hr while migrating (Smith and Luskin, 1998). Thus, it is possible that stem cells are located in the better-known proliferative population, such as the SVZ, and produce progenitors which migrate to the amygdala and continue to proliferate locally within this region. ...
Text (Electronic thesis) in PDF format. Mode of access: World Wide Web. Advisor: Dr. Zuoxin Wang, Florida State University, College of Arts and Sciences, Dept. of Psychology. Title and description from dissertation home page (viewed Sept. 22, 2004). Thesis (Ph. D.)--Florida State University, 2004. Includes bibliographical references.
... that acts neurogenic and anti-proliferative in cortical precursors (Haubst et al., 2004;). Indeed, the endogenous gradient of Pax6-positive precursors increasing rostrally within the RMS correlates to a reduced cell proliferation with increasing distance from the SEZ ( Smith and Luskin, 1998). ...
München, Univ., Diss., 2005 (Nicht für den Austausch).
The olfactory system consists of two parallel systems, the main olfactory system and the accessory olfactory system. This chapter focuses upon the neurophysiology of the main olfactory bulb (MOB), the first brain structure in the main olfactory system that processes olfactory signals relayed from olfactory receptor neurons (ORNs) in the nasal epithelium. The MOB has long been an attractive model to study cellular mechanisms underlying the encoding, transfer, processing, and decoding of sensory information. The field of olfaction has undergone explosive growth over the last decade. In large part, this growth has been driven by the discovery of odorant receptor genes, how receptor neurons expressing specific odorant receptors map onto the MOB, and the development of mammalian MOB slice preparations. This chapter focuses primarily on neurophysiological studies of the mammalian MOB from the last decade. Readers are referred to other sources that review earlier work on MOB neurophysiology (Mori, K., 1987; Nickell, W. T. and Shipley, M. T., 1992).
Axons of both central and peripheral neurons in adult mammals have the intrinsic potential to regenerate after crush or transection, as indicated by the fact that they initiate sprouting after such injuries. A large body of evidence suggests that whether or not axons regenerate depends, in large part, on their associated glial cells. Spinal nerves are organized into three structural levels. The fundamental unit is the endoneurial unit, composed of an axon; its associated Schwann cell sheath; and surrounding connective tissue sheath, the endoneurium. The cell bodies of the motor axons are located in the ventral horn of the cord and synapse with descending tracts of motor axons from the motor centers of the brain. Motor neurons send axons peripherally to join sensory axons distal to dorsal root ganglia. During regeneration, axotomized neurons switch from a transmitting mode to a growth mode and express growth-associated proteins. The distal part of an injured axon is degraded by a process called Wallerian degeneration.
Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt-qPCR was most prominent in the SVZ and in the developing white matter, around the first 1-2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA-014418 in mice aged postnatal day (P) 8-11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt-qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β-catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt-signaling. GLIA 2014.
Neonates are particularly susceptible to coxsackievirus infections of the central nervous system (CNS), which can cause meningitis, encephalitis, and long-term neurological deficits. However, viral tropism and mechanism of spread in the CNS have not been examined. Here we investigate coxsackievirus B3 (CVB3) tropism and pathology in the CNS of neonatal mice, using a recombinant virus expressing the enhanced green fluorescent protein (eGFP). Newborn pups were extremely vulnerable to coxsackievirus CNS infection, and this susceptibility decreased dramatically by 7 days of age. Twenty-four hours after intracranial infection of newborn mice, viral genomic RNA and viral protein expression were detected in the choroid plexus, the olfactory bulb, and in cells bordering the cerebral ventricles. Many of the infected cells bore the anatomical characteristics of type B stem cells, which can give rise to neurons and astrocytes, and expressed the intermediate filament protein nestin, a marker for progenitor cells. As the infection progressed, viral protein was identified in the brain parenchyma, first in cells expressing neuron-specific class III β-tubulin, an early marker of neuronal differentiation, and subsequently in cells expressing NeuN, a marker of mature neurons. At later time points, viral protein expression was restricted to neurons in specific regions of the brain, including the hippocampus, the entorhinal and temporal cortex, and the olfactory bulb. Extensive neuronal death was visible, and appeared to result from virus-induced apoptosis. We propose that the increased susceptibility of the neonatal CNS to CVB infection may be explained by the virus' targeting neonatal stem cells; and that CVB is carried into the brain parenchyma by developing neurons, which continue to migrate and differentiate despite the infection. On full maturation, some or all of the infected neurons undergo apoptosis, and the resulting neuronal loss can explain the longer-term clinical picture.
Cerebral ischemia induces neurogenesis, including proliferation and differentiation of neural progenitor cells and migration of newly generated neuroblasts. MicroRNAs (miRNAs) are small noncoding RNAs that decrease gene expression through mRNA destabilization and/or translational repression. Emerging data indicate that miRNAs have a role in mediating processes of proliferation and differentiation of adult neural progenitor cells. This article reviews recent findings on miRNA profile changes in neural progenitor cells after cerebral infarction and the contributions of miRNAs to their ischemia-induced proliferation and differentiation. We highlight interactions between the miR-124 and the miR17-92 cluster and the Notch and Sonic hedgehog signaling pathways in mediating stroke-induced neurogenesis.
Neurogenesis has been the subject of active research in recent years. Although the majority of neurons form during the embryonic period, neurogenesis continues in restricted regions of the mammalian brain well into adulthood. In rodent brains, neuronal migration is present in the rostral migratory stream (RMS), connecting the subventricular zone to the olfactory bulb (OB). The migration in the RMS is characterised by a lack of dispersion of neuroblasts into the surrounding tissues and a highly directed motion towards the OB. This study uses a simple mathematical model to investigate several theories of migration of neuroblasts through the RMS proposed in the literature, including chemo-attraction, chemorepulsion, general inhibition and the presence of a migration-inducing protein. Apart from the general inhibition model, all the models were able to provide results in good qualitative correspondence with the experimental observations.
Significance
The time taken by neural stem cells and intermediate progenitor cells to transit through the cell cycle, and number of times they divide, is essential information to understand how new neurons are produced in the adult rodent brain.
Faithful progression through the cell cycle is crucial to the maintenance and developmental potential of stem cells. Here, we demonstrate that neural stem cells (NSCs) and intermediate neural progenitor cells (NPCs) employ a zinc-finger transcription factor specificity protein 2 (Sp2) as a cell cycle regulator in two temporally and spatially distinct progenitor domains. Differential conditional deletion of Sp2 in early embryonic cerebral cortical progenitors, and perinatal olfactory bulb progenitors disrupted transitions through G1, G2 and M phases, whereas DNA synthesis appeared intact. Cell-autonomous function of Sp2 was identified by deletion of Sp2 using mosaic analysis with double markers, which clearly established that conditional Sp2-null NSCs and NPCs are M phase arrested in vivo. Importantly, conditional deletion of Sp2 led to a decline in the generation of NPCs and neurons in the developing and postnatal brains. Our findings implicate Sp2-dependent mechanisms as novel regulators of cell cycle progression, the absence of which disrupts neurogenesis in the embryonic and postnatal brain.
In the postnatal forebrain, the subventricular zone (SVZ) contains a pool of undifferentiated cells, which proliferate and migrate along the rostral migratory stream (RMS) to the olfactory bulb and differentiate into granule cells and periglomerular cells. Plexin-B2 is a semaphorin receptor previously known to act on neuronal proliferation in the embryonic brain and neuronal migration in the cerebellum. We show here that, in the postnatal and adult CNS, Plexin-B2 is expressed in the subventricular zone lining the telencephalic ventricles and in the rostral migratory stream. We analyzed Plxnb2(-/-) mice and found that there is a marked reduction in the proliferation of SVZ cells in the mutant. Plexin-B2 expression is downregulated in the olfactory bulb as interneurons initiate radial migration. BrdU labeling and GFP electroporation into postnatal SVZ, in addition to time-lapse videomicroscopy, revealed that neuroblasts deficient for Plexin-B2 migrate faster than control ones and leave the RMS more rapidly. Overall, these results show that Plexin-B2 plays a role in postnatal neurogenesis and in the migration of SVZ-derived neuroblasts.
Many aspects of animal and human behavior are guided by, or dependent on, the sense of smell‐olfaction. This chapter summarizes
the major facts of the anatomy, neurochemistry, molecular biology and physiology of the olfactory system. We emphasize the
mammalian olfactory system, particularly rodents, because of their widespread use as a laboratory model and the rich database
related to this species.
The retinal pigment epithelium (RPE) plays a key role in supporting photoreceptor survival and function, and RPE loss and
dysfunction in age-related macular degeneration (AMD) leads to photoreceptor death. AMD is one of the leading causes of blindness,
yet there is neither a cure nor a way to prevent it. In this chapter, we discuss recent progress in the study of AMD, summarize
what is known about RPE development, and review recent progress in deriving RPE from human embryonic stem cells (hESC). The
RPE is derived from the optic vesicle, which in turn is derived from the eye field of the anterior neural plate. Multiple
extracellular signaling factors and transcriptional regulators have been identified that are crucial to RPE development. Knowledge
of these events is guiding efforts to understand how hESC differentiate into RPE cells that might be used therapeutically
for AMD. RPE derived from hESC are remarkably similar to fetal RPE with respect to gene expression and cellular function.
Future research on these cells will lead to a better understanding of RPE development and the advancement of cellular therapy
for AMD.
A great deal of progress has been made in the identification and use of markers in the study of neural progenitor and stem
cells. The use of individual proteins or genes as highly specific markers, however, must always be made with a great deal
of caution-no one antigen or gene is always likely to be only expressed by one specific cell type. Additionally, recent studies
depicting the complexity and heterogeneity of neural stem cells will call for newer and better markers as well as new ways
of thinking about cell identity.
The objective of this study was to investigate whether stressful experience during early postnatal period may influence morphological characteristics of the rat neurogenic pathway--the rostral migratory stream (RMS) and proliferation of neuronal precursors in three successive areas of the RMS: in the vertical arm, the elbow and the horizontal arm. To induce stress, the pups were subjected to repeated maternal deprivation during the first postnatal week after birth. Brains were analyzed at the seventh postnatal day. The controls matched the age of maternally deprived animals. Observation of hematoxylin-eosin stained sections showed that maternal deprivation did not affect the general morphological appearance of the RMS. The shape of the RMS of maternally deprived rats resembles the RMS of control animals. Maternal deprivation caused slight, not significant increase in the RMS thickness in comparison with control rats. Significant difference between the control and maternally deprived rats concerns the olfactory ventricle. While in seven days old control rats the olfactory ventricle is completely closed, in maternally deprived rats of the same age the olfactory ventricle was regularly visible as a narrow lumen at the axis of the RMS horizontal arm. This finding indicates delayed maturation of the migratory pathway as a consequence of stress. Proliferation activity has been assessed by immunoreactivity of the endogenous cell cycle protein Ki-67. The results of Ki-67 immunohistochemistry showed that seven days' maternal separation for 3 h daily induces significant quantitative changes in the number of proliferating cells within the RMS. The response of Ki-67-positive cells to stress differed in individual part of the RMS, with a marked decrease in the vertical arm and a significant increase in the elbow, suggesting heterogeneity of neural stem cells along the RMS; while in the RMS vertical arm the number of dividing cells significantly decreased, there was a marked increase of Ki-67-positive cells in the RMS elbow. This suggests heterogeneity of neural stem cells along the RMS.
The early development of the mammalian forebrain involves the massive proliferation of the ventricular zone cells lining the lateral ventricles. A remnant of this highly proliferative region persists into adult life, where it is known as the subependymal layer. We examined the proliferation kinetics and fates of the mitotically active cells in the subependyma of the adult mouse. The medial edge, the lateral edge, and the dorsolateral corner of the subependymal layer of the rostral portion of the lateral ventricle each contained mitotically active cells, but the dorsolateral region had the highest percentage of bromodeoxyuridine (BrdU)-labeled cells per unit area. Repeated injections of BrdU over 14 hr revealed a proliferation curve for the dorsolateral population with a growth fraction of 33%, indicating that 33% of the cells in this subependymal region make up the proliferating population. The total cell cycle time in this population was approximately 12.7 hr, with an S-phase of 4.2 hr. To examine the fate of these proliferating cells, we injected low concentrations of a replication-deficient, recombinant retrovirus directly into the lateral ventricles of adult mice for uptake by mitotically active subependymal cells. Regardless of the survival time postinjection (10 hr, 1 d, 2 d, or 8 d), the number of retrovirally labeled cells per clone remained the same (1 or 2 cells/clone). This suggests that one of the progeny from each cell division dies. Moreover, the clones remained confined to the subependyma and labeled cells were not seen in the surrounding brain tissue. Thus, while 33% of the dorsolateral subependymal cells continue to proliferate in adult life, the fate of the postmitotic progeny is death.
A cumulative labelling protocol using 5-bromo-2'-deoxyuridine (BUdR) was followed to determine: (1) the growth fraction (i.e., the proportion of cells that comprise the proliferating population), (2) the length of the cell cycle, and (3) the length of the DNA-synthetic phase (S-phase) for proliferative cells in the dentate gyrus of the mouse. On postnatal day 20 (P20), C57BL/6J mice were injected with BUdR at two hour intervals for a total period of 12 hours. Animals were sacrificed at selected intervals, and the brains were processed for immunohistochemistry using a monoclonal antibody directed against single-stranded DNA containing BUdR. The numbers of BUdR-labelled and unlabelled cells in sections through the hilus of the dentate gyrus were counted. The number of BUdR-labelled cells increased linearly from an initial value of about 12% of the total number of cells to a maximum value of just over 24% of the total. These findings indicate that, at P20, a maximum of 24.2 +/- 1.2% of the cells in the dentate hilus are part of the proliferating population. The calculated length of the cell cycle of the cells comprising the intrahilar proliferative zone was estimated to be 16.1 +/- 0.8 h. The length of the S-phase was estimated at 8.0 +/- 0.4 h. In addition, mathematical analysis, using one and two population models, indicates that over 90% of the proliferating cells in the dentate hilus at this age comprise a single population at least in terms of the lengths of the cell cycle and the S-phase.(ABSTRACT TRUNCATED AT 250 WORDS)
It is possible on the basis of their proliferative properties to divide the various cell types in the brain into two groups. Proliferation of a first group of cells (neurons, microglia, pericytes, ependymal cells and epithelial cells of the choroid plexus) occurs mainly prenatally but may continue into the early postnatal period (2-3 weeks after birth in the mouse and rat). In adult animals neurons do not proliferate at all and the same is true for microglial cells, pericytes and plexus epithelial cells. Ependymal cells show very slight proliferative activity in the adult animal. The cells of a second group (neuroglia, endothelial cells and cells of the subependymal layer) continue to proliferate throughout adult life. For these quite different cell types which are of ecto- and mesodermal origin, the cell cycle parameters and the mode of proliferation, i.e., proliferation with cell loss and permanent exchange of cells between the growth fraction and the non-growth fraction, appear to be very similar. This suggests that there might be a common principle of the regulatory mechanism of cell proliferation in the brain of the adult animal. The cell cycle parameters of these cells are consistent and surprisingly short [T(c)c about 20 hr, T(s)c about 10 hr]. During ontogeny the precursor cells of neurons, ependymal cells and epithelial cells of the choroid plexus (first group) as well as of neuroglia (second group) i.e., the neural epithelial cells start proliferation with very short cycle times of about 8 hr and durations of the S phase of about 5 hr during early embryonic development. These values increase towards the end of foetal development to about 20 hr and 10 hr respectively. These phase durations then remain approximately constant for those cells (second group) that continue to proliferate during adult life. The agreement of these values with those measured by Wilson et al. (1972) for a transplantable glioma in the brain of the rat [T(c)=20 hr, T(s)=10hr] is of interest. Similar values for the S phase duration have been reported for human gliomas (Hoshino et al., 1972).
Cytogenesis is the critical determinant of the total number of neurons that contribute to the formation of the cerebral cortex and the rate at which the cells are produced. Two distinct cell populations constitute the proliferative population, a pseudostratified ventricular epithelium (PVE) lying within the ventricular zone (VZ) at the margin of the ventricle, and a secondary proliferative population that is intermixed with the PVE within the VZ but also is distributed through the overlying subventricular and intermediate zones of the cerebral wall. The present analysis, based upon cumulative S-phase labeling of the proliferative cells with 5-bromo-2'-deoxyuridine, is principally concerned with the PVE of the gestational-day-14 (E14) murine cerebral wall. It has immediate but also more far reaching general objectives. The most immediate objective, essential to the design and interpretation of later experiments, is to provide estimates of critical parameters of cytogenesis for the PVE. The growth fraction is virtually 100%. The lengths of the overall cell cycle, S-, G2+M-m, and G1-phases are 15.1 hr, 3.8 hr, 2 hr, and 9.3 hr, respectively. The PVE is homogeneous with respect to cell cycle length. For methodological considerations, these estimates are more accurate than estimates of the same parameters obtained in earlier analyses based upon S-phase labeling with tritiated thymidine. It is particularly with respect to a shorter length of S-phase determined here that the present values are different from those obtained with thymidine. At a more innovative level, the temporal and spatial resolution of nuclear movement made possible by the methods developed here will allow, in a way not previously attempted, a fine-grained tracking of nuclear movement as cells execute the successive stages of the cell cycle or exit the cycle subsequent to mitosis. Such observations are pertinent to our understanding of the regulatory mechanisms of neocortical histogenesis and the cell biological mechanisms that govern the proliferative cycle of the ventricular epithelium itself. It is known that the velocity of nuclear movement in the PVE is maximum in G2 (fourfold increase from S-phase) and minimum in M and early G1.(ABSTRACT TRUNCATED AT 400 WORDS)
Large numbers of dying cells are found in proliferating tissues, suggesting a link between cell death and cell division. We detected and quantified dying cells during pre- and early postnatal development of the rat cerebral cortex using in situ end labeling of DNA fragmentation [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)] and electron microscopy. The proliferative zones that give rise to the neuronal and glial cell types of the cortex, the ventricular and, to a larger extent, the subventricular zones showed higher incidence of cell death than other regions of the developing cortex during the period of neurogenesis. Gel electrophoresis of DNA isolated from the subventricular zone of newborn animals showed a ladder pattern that is characteristic of apoptosis. The number of apoptotic cells remained high in this zone for at least 2 weeks, during which period cells continued to divide. The correlation between cell division and cell death was studied in the subventricular zone of newborn rats; cumulative labeling with bromodeoxyuridine showed that 71% of TUNEL-labeled cells had taken up this S-phase marker before undergoing cell death. Using bromodeoxyuridine and [3H]-thymidine in succession to identify a cohort of proliferating cells, we found that the clearance time of TUNEL-positive nuclei was 2 hr and 20 min. A comparison between the number of mitotic figures and that of TUNEL-positive nuclei showed that cell death affects one in every 14 cells produced by dividing ventricular zone cells at embryonic day 16 and one in every 1.5 cells produced in the subventricular zone of newborn rats. In addition, we found that most of TUNEL-positive cells were in the G1 phase of their cell cycle. We conclude that apoptosis is prominent in the proliferating neuroepithelium of the developing rat cerebral cortex and that it is related to the progression of the cell cycle.
The neurons of the glomerular layer of the rat olfactory bulb have been studied using Nissl staining and Golgi-Kopsch impregnation in light microscopy to define the size, shape and morphological features of individual cell somata, dendrites and axons; these have been correlated with electron-microscopic material in which fine-structural characteristics were also noted for each cell type, particularly synaptic specializations. Three neuron types are described: the external tufted and periglomerular cells of classical microscopy, and additional, superficial short-axon cells; a description of the glomerular arborizations of the mitral and deep tufted cells is also included. The tufted and mitral cells show large, non-spiny glomerular dendritic arborizations, having terminal varicosities, the external tufted cells being more limited in their branching than the deeper cells. External tufted cells have large somata and abundant cytoplasm containing stacks of Nissl material; their main dendrites are characterized by pale cytoplasm and a regular array of neurotubules. Reciprocal dendro-dendritic and somato-dendritic synapses are commonly found, the tufted/mitral cells containing spherical vesicles and contacting by means of asymmetrical membrane thickenings; the other profile involved is a gemmule containing large flattened vesicles and associated with a symmetrical thickening. The periglomerular cells are smaller, with a spiny glomerular arborization, as well as some other dendrites; all the dendrites of these cells tend to be of irregular outline. They have a dark nucleus and very little somatic cytoplasm; somatic and dendritic appendages are common and often contain large flattened vesicles. Synapses oriented from the dendritic shaft or gemmule also show such vesicles, invariably associated with symmetrical thickenings. The superficial short-axon cells are characterized by the entirely periglomerular distribution of their dendrites, which are varicose and rarely branch. Of intermediate soma dimensions, but containing dispersed Nissl material, these cells and their stem dendrites show no regions that can be designated as presynaptic.
Features of axon initial segments, axo-somatic and axo-dendritic synapses are also described for each cell, as well as some unusual glial relationships. Reasons are adduced for relating the superficial short-axon cell to the axon terminal type containing small flattened vesicles, as well as for considering that the external tufted and periglomerular cells show the same synaptic specializations at their axon terminals as at their dendritic and somatic synapses. The cells of the glomerular layer are compared with those of the deeper layers of the bulb and atypical synaptic specializations discussed; some physiological implications of these findings are considered.
Thymidine-H3 was injected intraperitoneally into 6- and 13-day old rats and they lived afterwards for periods ranging from one hour to 60 days. Autoradiographic data obtained from animals surviving for short periods were used to estimate rates of regional cell proliferation. Animals with longer survival were used to deduce the movements of new cells from germinal sites, through migratory channels, to target areas, and to determine their mode of differentiation.
The formation and differentiation of microneurons goes on during infancy, though in most structures at a declining rate. In the wall of the olfactory ventricle cell multiplication continued at a high rate at six days, with a decline at 13 days, and the new cells migrated to the layers of the olfactory bulb. The migration of labeled cells from the lateral ventricle was traced, by way of the fimbria, to the polymorph cell layer of the dentate gyrus, and from there to the granular layer. The “older” granule cells were located in the upper part of the granular layer, the “younger” cells at its base. Cell multiplication continued at a very high rate in the external granular layer of the cerebellar cortex, whence cells migrated to the molecular layer and internal granular layer. Speed of migration was approximately 50 μU/day in the olfactory bulb, and 60–70 μ/day in the cerebellum. The possible significance of these findings was discussed.
The properties and fate of the cells of the subependymal layer of the anterior lateral ventricle and its rostral extension into the olfactory bulb were examined. In one experiment, histological analysis was made of this structure in a large group of rats, ranging in age from newborn to adults. It was established that the ventricular subependymal layer and its rostral extension are present as proliferative and migratory matrices throughout the period studied, with relatively little reduction in size from birth to adulthood. In another, autoradiographic study, the proliferation and migration of cells of this system, and their destination and mode of differentiation, were studied in rats that were injected with thymidine-H3 at 30 days of age and killed at intervals ranging from 1 hour to 180 days. There was a declining gradient in cell proliferation in a caudorostral direction from a high level near the lateral ventricle to the absence of cell proliferation in the olfactory bulb. The labeled cells that were present in high proportion near the lateral ventricle in the rats killed 1–24 hours after injection had further multiplied and moved to the middle portion of the “rostral migratory stream” by the third day, and were located in the subependymal layer of the olfactory bulb by the sixth day after injection. By the twentieth day the labeled cells disappeared from the subependymal layer of the olfactory bulb and were distributed throughout the internal granular layer. The differentiated cells were tentatively identified as granular nerve cells and neuroglia cells. These results established that the major target structure of cell production in the subependymal layer of the lateral ventricle in young-adult rats is the olfactory bulb, with only moderate contribution made to the anterior neocortex and basal ganglia. It was postulated that the function of cell migration to the olfactory bulb is the renewal of its cell population.
Thesis (Ph. D.)--University of Virginia, 1991. Includes bibliographical references (leaves 262-281).
A quantitative lifespan study of changes in cell number, cell division and cell death in various regions of the mouse forebrain
A quantitative study of changes in total cell number was carried out in the indusium griseum and anterior commissure from fetal life to old age in the mouse brain. The changes in the number of mitotic and pyknotic cells were recorded in the indusium griseum, anterior commissure, subependymal and ependymal layers over the same period. The number of neurons which are produced and which migrate to the indusium griseum are in excess of the number eventually required and the surplus neurons are lost by cell death in late gestation and early postnatal life while synapto-genesis and neuronal differentiation is taking place. This neuronal loss is associated with a rapid turnover of glia. Most first generation glia, or their immediate precursors, are produced prenatally, in parallel but one day behind neurons. There is no large burst of mitotic activity in the postnatal brain which gives rise to the myelination gliosis which is probably largely a migratory phenomenon. Cell division continues throughout life in all parts of the brain studied. The greatest mitotic activity is centred in the subependymal layer where mitotic cells substantially outnumber pyknotic ones. There is a gradual decrease in mitotic activity in the subependymal layer up to 9 months of age with fairly constant mitotic activity thereafter. Mitotic activity in the indusium griseum levels out at 3 months post-natum with mitotic and pyknotic cells present in roughly equal numbers thereafter. Mitotic activity in all parts of the anterior commissure levels out at 6 months postnatum and remains constant thereafter. Mitotic and pyknotic cells are present in similar numbers except for a peak in pyknotic cells at 9 months. Cell number in the indusium griseum and anterior commissure is fairly constant between 3 and 9 months, but glial number begins to decrease in all parts of the anterior commissure from 12 to 22 months. In the indusium griseum the number of glia increased slightly between 6 and 22 months. The number of neurons fluctuated during the first week after birth then remained constant until 18 months. There was a significant decrease in the number of neurons between 18 and 22 months.
This chapter summarizes the recent advances on postnatal gliogenesis obtained by electron microscope and radioautography. The electron microscope has provided precise criteria for the identification of glial cells, and has given several indications of their possible function. Radioautography has permitted investigators to label immature cells through incorporation of labeled nucleotides, and to follow their ultimate fate. Evidence of protracted gliogenesis throughout life, in several mammalian species, has been firmly established, and a common origin for astrocytes and oligodendrocytes from a neuroectodermal precursor has been demonstrated. It has also been shown that the production of either type from the matrix layer is not random, and that astrocytes are generated earlier than oligodendrocytes, although both are still produced, at different rates, in adult rats. The origin of microglial cells is not so evident; nevertheless, several points have been clarified. Brain macrophages may originate from several sources and, when extensive necrosis occurs, blood leukocytes are always involved. In other cases indigenous elements are responsible for phagocytosis, and they most likely represent microglial cells. These might in turn originate from mesenchymal elements of the brain, or from neuroectodermal cells. The neuroectodermal origin of microglial cells needs to be unequivocally demonstrated. The possibility that apparently mature macroglial cell types transform into each other or into microglial cells needs to be explored.
Five beta-tubulin isotypes are expressed differentially during chicken brain development. One of these isotypes is encoded by the gene c beta 4 and has been assigned to an isotypic family designated as Class III (beta III). In the nervous system of higher vertebrates, beta III is synthesized exclusively by neurons. A beta III-specific monoclonal antibody was used to determine when during chick embryogenesis c beta 4 is expressed, the cellular localization of beta III, and the number of charge variants (isoforms) into which beta III can be resolved by isoelectric focusing. On Western blots, beta III is first detectable at stages 12-13. Thereafter, the relative abundance of beta III in brain increases steadily, apparently in conjunction with the rate of neural differentiation. The isotype was not detectable in non-neural tissue extracts from older embryos (days 10-14) and hatchlings. Western blots of protein separated by two-dimensional gel electrophoresis (2D-PAGE) reveal that the number of beta III isoforms increases from one to three during neural development. This evidence indicates that beta III is a substrate for developmentally regulated, multiple-site posttranslational modification. Immunocytochemical studies reveal that while c beta 4 expression is restricted predominantly to the nervous system, it is transiently expressed in some embryonic structures. More importantly, in the nervous system, immunoreactive cells were located primarily in the non-proliferative marginal zone of the neural epithelia. Regions containing primarily mitotic neuroblasts were virtually unstained. This localization pattern indicates that c beta 4 expression occurs either during or immediately following terminal mitosis, and suggests that beta III may have a unique role during early neuronal differentiation and neurite outgrowth.
We describe a cell-lineage marking system applicable to the vertebrate nervous system. The basis of the technique is gene transfer using the retroviral vector system. We used Escherichia coli beta-galactosidase as a marker gene and demonstrate a high level of expression of this marker from the viral long terminal repeat promoter, with simultaneous expression of the Tn5 neo gene from the simian virus 40 early promoter. This expression has allowed us to detect individual infected cells histochemically. We applied this marking technique to the study of lineage relationships in the developing vertebrate nervous system, both in vivo and in culture. In the rat retina, we injected virus in vivo and histochemically identified clones of marked neural cells. In addition, we used this virus to infect cultures of rat cerebral cortex and have analyzed the clonal relationships of morphologically different neural cell types. The host range of the marking system extends to avian as well as mammalian species. Thus, this system should have broad applicability as a means of gene transfer and expression in the nervous system.
SUMMARY The neuropil of the glomeruli of the rat olfactory bulb has been studied with the electron microscope with a view to elucidating the type of processes involved - dendrites, appendages and axons - their cellular identity, and the synaptic relationships they establish. The problems encountered in defining these are considered and criteria based on the previous study of neuron types and on examination of serial sections are put forward. The glomeruli are large structures containing many thousands of processes and are the sole site of termination of the olfactory receptor axons. The terminals of the latter are characteristically electron-dense, allowing identification in normal material; they run through the glomeruli making many synapses by means of spherical vesicles and asymmetrical thickenings on to all types of dendritic profile. The glomerular dendritic arborizations of mitral and tufted cells, which are indistinguishable from each other, start as large, fairly regular, pale profiles but become increasingly varicose as they branch and diminish in size. They regularly show groups of spherical vesicles, often in association with asymmetrical synaptic thickenings directed from the dendrite; these are typically associated with return, reciprocal, synapses of the symmetrical type from profiles con- taining large flattened vesicles. These latter profiles are those of the dendrites and gemmules of periglomerular cells; the dendrites are of irregular outline and give rise to many appendages, mostly gemmules making synaptic contact with mitral or tufted cell dendrites. A small number of pale axon terminals containing either small or large flattened vesicles, derived from short- axon and periglomerular cells respectively, synapse with symmetrical thickenings on to the periglomerular cell dendritic processes. Close associations of particular types of axo-dendritic and dendro-dendritic synapses on interconnecting processes, termed synaptic patterns, are described and their significance considered. The nature of the glomerular interactions is dis- cussed and then placed in the context of other, smaller glomeruli in the central nervous system; certain common principles of glomeruli are suggested.
The glial fibrillary acidic (GFA) protein, a brain specific protein extracted from severely gliosed human tissue, is not species specific; cross-reaction occurs between anti-human GFA protein antibodies and brain extracts of rabbit, guinea pig, rat and dog. Using anti-GFA protein antiserum, astrocytes are selectively stained with the indirect immunofluorescence technique in both normal and pathological (gliosed) brain tissue.
Neonate rats were injected systemically with thymidine-H3 and killed after different periods of survival. Cell proliferation, migration and transformation in the brain were studied autoradiographically. It was established that cells multiplying in the ependymal and subependymal walls of the olfactory ventricle migrate outward into the olfactory bulb, where they become differentiated into granule cells. These postnatally formed granule cells contribute to the formation of the granular and several other layers of the olfactory bulb. Cells multiplying at a high rate in the wall of the lateral ventricle migrate to the hippocampus and contribute to the formation of the granule cells in the granular layer of the dentate gyrus. Cells multiplying at a high rate in the external and internal granular layers of the cerebellum become differentiated into granule cells, and, to a lesser extent, other types of nerve cells of the cerebellar cortex. Evidence was also obtained of the postnatal origin of many of the granule cells of the cochlear nucleus. Postnatal neurogenesis is restricted to these short-axoned granule cells or microneurons; the long-axoned nerve cells or macroneurons of the brain are formed prenatally.
Neurogenesis in the rat olfactory bulb was examined with 3H-thymidine-radiography. For the animals in the prenatal groups, the initial 3H-thymidine exposures were separated by 24 h; they were the offspring of pregnant females given two injections on consecutive embryonic (E) days (E12-E13, E13-E14, . . . E21-E22). For the animals in the postnatal (P) groups, the initial 3H-thymidine injections were separated by 48 h, each group receiving either four (PO-P3, P2-P4, . . . P6-P9) or two (P8-P9, P10-P11, . . . P20-P21) consecutive daily injections. On P60, the percentage of labeled cells and the proportion of cells added during either 24 h or 48 h periods were quantified at several anatomical levels for each neuronal population in the main olfactory bulb (mitral cells, tufted cells, granule cells, interneurons in the external plexiform layer, periglomerular granule cells) and accessory olfactory bulb (output neurons, granule cells, periglomerular granule cells). The total time span of neurogenesis extends from E12 to beyond P20. Output neurons are prenatally generated over 5-9 day periods (with most neurogenesis occurring over 2-4 days) in a strict sequential order beginning with the accessory bulb output neurons (E13-E14) and ending with the interstitial tufted cells lying between the glomeruli in the main bulb (E20-E22). These data are correlated with the main and accessory bulb projection fields in the amygdala and with the chronology of amygdala neurogenesis. With the exception of the granule cells in the accessory bulb (88% generated between E15-E22), the rest of the interneuronal populations are generated postnatally and nearly simultaneously. While most neurons (75-80%) originate during the first three weeks of life, all interneuronal populations, including accessory bulb granule cells, show some neurogenesis beyond P20. Injections of 3H-thymidine in juvenile and adult rats indicates neurogenesis up to P60 in the accessory bulb and up to P180 in the main bulb, especially in the main bulb granule cell population. There is circumstantial evidence for turnover of main bulb granule cells during adult life.
A spatially discrete region of the anterior part of the postnatal telencephalic subventricular zone, referred to as the SVZa generates vast numbers of lineally-related neurons destined for the olfactory bulb (Luskin, 1993). The cells originating in the SVZa migrate to the olfactory bulb along a highly restricted pathway which is in a direction orthogonal to the orientation of radial glial fibers. In this study we analysed the number, distribution, orientation and rate of migration of SVZa-derived cells as they approach the olfactory bulb. In order to track the SVZa-derived cells, a retroviral lineage tracer, encoding the reporter gene E. coli beta-galactosidase (lacZ) was injected precisely into the rat SVZa at postnatal day 1 (P1). The lacZ-positive cells were visualized 1, 2 and 3 days later by X-Gal histochemistry in cryostat sections. As the number of SVZa-derived cells in the pathway increased with survival time, their distribution changed systematically. The distribution pattern of lacZ-positive cells by 2 and 3 days postinjection suggested that some of the progeny of infected progenitor cells were undergoing neurogenesis as they proceeded to the olfactory bulb; a large percentage of the lacZ-positive cells were substantially displaced from the SVZa injection site. To investigate whether lacZ-positive cells migrate in a directed fashion, their orientation preference was scored. For the majority of lacZ-positive cells (> 94%), their leading process was directed toward the olfactory bulb, possibly reflecting a response to migratory cues present along the pathway. The estimated average rate of cell migration to the olfactory bulb was 23 mu m/h, which is approximately twice the speed of radially directed neuronal migration from the telencephalic ventricular zone to the cortical plate (O'Rourke et al., 1992). Collectively, these results suggest that SVZa-derived interneurons en route to the olfactory bulb may employ a novel mode of tangential migration.
Dissection of the subependyma from the lateral ventricle of the adult mouse forebrain is necessary and sufficient for the in vitro formation of clonally derived spheres of cells that exhibit stem cell properties such as self-maintenance and the generation of a large number of progeny comprising the major cell types found in the central nervous system. Killing the constitutively proliferating cells of the subependyma in vivo has no effect on the number of stem cells isolated in vitro and induces a complete repopulation of the subependyma in vivo by relatively quiescent stem cells found within the subependyma. Depleting the relatively quiescent cell population within the subependyma in vivo results in a corresponding decrease in spheres formed in vitro and in the final number of constitutively proliferating cells in vivo, suggesting that a relatively quiescent subependymal cell is the in vivo source of neural stem cells.
The subventricular zone of the postnatal forebrain produces mainly glia, although it supports limited neurogenesis. To determine whether the subventricular zone is positionally specified, the phenotype and destination of the progeny of subventricular zone cells along the anterior-posterior axis of the lateral ventricles were analyzed. A retroviral lineage tracer containing the E. coli reporter gene lacZ was injected into different parts of the subventricular zone of neonatal rat pups, and at various times thereafter, the expression of beta-galactosidase was detected histochemically or immunohistochemically in the descendants of infected cells. A discrete region of the anterior part of the subventricular zone (SVZa) generated an immense number of neurons that differentiated into granule cells and periglomerular cells of the olfactory bulb-the two major types of interneurons. Thus, the SVZa appears to constitute a specialized source of neuronal progenitor cells. To reach the olfactory bulb, neurons arising in the SVZa migrate several millimeters along a highly restricted route. Guidance cues must be involved to prohibit widespread dispersion of these migrating neurons.
The cells arising in the anterior part of the subventricular zone (SVZa) migrate along a well-demarcated pathway which lacks radial glial fibers to the olfactory bulb where they differentiate into interneurons of the granule cell layer or glomerular layer (Luskin, 1993, Neuron 11, 173). To analyze the mechanisms underlying this highly directed migration, we have compared the migratory behavior of unmanipulated SVZa-derived cells to that of homotopically transplanted SVZa cells and of heterotopically transplanted telencephalic ventricular zone (VZ) cells that ordinarily migrate in association with radial glial fibers. To identify the phenotype of the SVZa progenitor cells prior to their transplantation, we characterized them in vitro using cell type-specific markers. After 1 day in culture nearly all the SVZa cells were stained with TuJ1, a neuron-specific marker; only an occasional cell exhibited a glial phenotype as judged by the presence of GFAP-immunoreactivity. This indicates that SVZa cells express a neuronal phenotype. To reveal the spatiotemporal distribution of homotopically transplanted neonatal SVZa cells in a host brain, dissociated SVZa cells from Postnatal Day 0 (P0)-P2 animals were labeled with the lipophilic dye PKH26 or the cell proliferation marker BrdU and implanted into the SVZa of host animals of the same age. Within the first week after transplantation there were vast numbers of labeled cells throughout the pathway. Over the next 2 weeks the labeled cells migrated into the overlying cellular layer of the olfactory bulb and began to differentiate, and within 4 weeks the transplanted cells had reached their final positions in the granule cell and glomerular layers of the olfactory bulb in the same proportions as for unmanipulated SVZa-derived cells. While en route to the olfactory bulb the homotopically transplanted cells never strayed from the migratory pathway. In contrast, heterotopically transplanted VZ cells from the embryonic telencephalon did not undergo migration although they did differentiate. These results demonstrate that the homotopically transplanted SVZa-derived cells adopt a mode of migration indistinguishable from that ordinarily utilized by SVZa-derived neurons and that the VZ cells are unable to decipher the same set of guidance cues.
The rostral migratory stream consists of a large number of cells migrating from the lateral ventricles to the rostral telencephalon, primarily the olfactory bulb. The pathway continually provides neuro- and glioblasts throughout life. The present paper indicates that a considerable number of cells undergo apoptotic cell death en route, even in young (day 3) rats when presumably many vacant sites are still available in the developing brain.
In the mammalian forebrain most neurons originate from proliferating cells in the ventricular zone lining the lateral ventricles. These neurons become postmitotic before they undergo migration to their final destinations. In this study we examined the proliferative and migratory properties of cells destined for the olfactory bulb that arise postnatally from progenitor cells situated at the anterior extent of the subventricular zone (SVZa). The SVZa-derived cells migrate along a stereotypical pathway to the olfactory bulb where they become interneurons. Using lineage tracers and the cell proliferation marker BrdU, we have demonstrated that SVZa-derived cells in the rat retain the capacity for division after migrating away from their initial site of generation. These cells also express a neuron-specific tubulin, recognized by the antibody TuJ1. These results suggest that, unlike other immature neurons, these SVZa-derived cells have made a commitment to become neurons before becoming postmitotic.
The division of neuronal progenitor cells during migration in the neonatal mammalian forebrain
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The expression and posttranslational modification of a neuron-specific -tubulin isotype during chick embryogenesis
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The division of neuronal progenitor cells during migration in the neonatal mammalian forebrain
- Jrl Menezes
- C M Smith
- K C Nelson
- M B Luskin
Menezes JRL, Smith CM, Nelson KC, Luskin MB. The division of
neuronal progenitor cells during migration in the neonatal mammalian forebrain. Molec. Cell. Neurosci. 1995;6:496-508.