Maternal Separation Induced Alterations of Neurogenesis in the Rat Rostral Migratory Stream

1. The aim of our study was to investigate the possibility that maternal separation, an experimental model for studies of early environmental influences, has an effect on postnatal neurogenesis in neurogenic pathway—the rostral migratory stream (RMS). 2. Rat pups were subjected to maternal separation daily for 3 h, starting from the first postnatal day (P1) till P14 or P21. In the first two groups, brains were analyzed at the age of P14 and P21, respectively. In the third group, after 3 weeks of maternal separation, 1 week of normal rearing was allowed, and the brains were analyzed at P28. The controls matched the age of maternally separated animals. Dividing cells were labeled by bromodeoxyuridine; dying cells were visualized by Fluoro-Jade C and nitric oxide (NO) producing cells by NADPH-diaphorase histochemistry. 3. Quantitative analysis of proliferating cells in the RMS showed that maternal separation decreased the number of dividing cells in all experimental groups. This decrease was most prominent in the caudal part of the RMS. The amount of dying cells was increased at the end of 3 weeks of maternal separation as well as 1 week later. The number of differentiated nitrergic cells in the RMS was increased at the end of 2 or 3 weeks of maternal separation, respectively. Besides quantitative changes, maternally separated animals showed an accelerated maturation of nitrergic cells. 4. Our results indicate that an exposure of rats to adverse environmental factors in early postnatal periods may induce acute site-specific changes in the RMS neurogenesis.     

Delayed maturation and altered proliferation within the rat rostral migratory stream following maternal deprivation

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 Number of Proliferating Cells in the Rostral Migratory Stream of Rat During the First Postnatal Month

SUMMARY The objective of this study was to analyze neurogenesis in the rat rostral migratory stream (RMS) during the first postnatal month.1. During the early postnatal development some morphological changes, concerning the RMS thickness, shape, and the olfactory ventricle persistence at P0 were observed.2. Bromodeoxyuridine (BrdU) immunohistochemistry and subsequent quantification of proliferating cells showed significant age-dependent changes. The highest number of proliferating cells was found at P3 and significant decrease of BrdU-positive cells at P7 rats. At P28, the number of proliferating cells reached the level of P0 rats.     

Age-Related Changes of NADPH-Diaphorase Positivity in the Rat Rostral Migratory Stream

Summary Accumulating evidence confirms that nitric oxide (NO), a versatile diffusible signaling molecule, contributes to controling of adult neurogenesis. We have previously shown the timing of NADPH-diaphorase (NADPH-d) positivity within the rat rostral migratory stream (RMS) during the first postnatal month. The present study was designed to describe further age-related changes of NO presence in this neurogenic region. The presence of NO synthesizing cells in the RMS was shown by NADPH-d histochemistry and neuronal nitric oxide synthase (nNOS) immunohistochemistry. The phenotypic identity of nitrergic cells was examined by double labeling with GFAP and NeuN. Systematic qualitative and quantitative analysis of NADPH-d-positive cells was performed in the neonatal (P14), adult(5 months) and aging (20 months) rat RMS. 1. Nitrergic cells with different distribution pattern and morphological characteristics were present in the RMS at all ages examined. In neonatal animals, small, moderately stained NADPH-d-positive cells were identified in the RMS vertical arm and in the RMS elbow. In adult and aging rats a few labeled cells could be also detected in the RMS horizontal arm. NADPH-d-positive cells in adult and aging rats were characterized by long varicose processes and displayed dark labeling in comparison to the neonatal group. 2. Double immunolabeling has revealed that nNOS-immunoreactivity co-localized with that of NeuN. This indicates that nitrergic cells within the RMS are neurons. 3. Quantitative analysis showed that the number of NADPH-d-positive cells increases with advancing age. The presence of NO producing cells in the RMS of neonatal adult and aging rats indicates, that this proliferating and migratory area is under the influence of NO throughout the entire life of the animals.     

Trypsin inhibitor and maternal reunion increase plasma cholecystokinin in neonatal rats.

Intragastric soybean trypsin inhibitor increased plasma CCK bioactivity by 87% in nondeprived, 9-12-day-old rat pups. Reunion with the dam for 1 h after overnight maternal deprivation also increased plasma CCK significantly. These results demonstrate that CCK can be released from the small intestine of rats as early as postnatal day 9.     

Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells

Secreted proteoglycan molecule Tsukushi (TSK) regulates various developmental processes, such as early body patterning and neural plate formation by interacting with major signaling pathways like Wnt, BMP, Notch etc. In central nervous system, TSK inhibits Wnt signaling to control chick retinal development. It also plays important roles for axon guidance and anterior commissure formation in mouse brain. In the present study, we investigated the role of TSK for the development and proper functioning of mouse hippocampus. We found that TSK expression is prominent at hippocampal regions of early postnatal mouse until postnatal day 15 and gradually declines at later stages. Hippocampal dimensions are affected in TSK knockout mice (TSK-KO) as shown by reduced size of hippocampus and dentate gyrus (DG). Interestingly, neural stem cell (NSC) density at the neural niche of DG was higher in TSK-KO compared with wild-type. The ratio of proliferating NSCs as well as the rate of overall cell proliferation was also higher in TSK-KO hippocampus. Our in vitro study also suggests an increased number of neural stem/progenitor cells residing in TSK-KO hippocampus. Finally, we found that the terminal differentiation of NSCs in TSK-KO was disturbed as the differentiation to neuronal cell lineage was increased while the percentages of astrocytes and oligodendrocytes were decreased. Overall, our study establishes the involvement of TSK in hippocampal development, NSC maintenance and terminal differentiation at perinatal stages.     

Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.     

Immunohistochemical Evidence for the Presence of Synaptic Connections of Nitrergic Neurons in the Rat Rostral Migratory Stream

The rostral migratory stream (RMS) is a migration route for neuroblasts originating in the richest neurogenic niche of the adult mammalian brain—the subventricular zone. Most studies are focused on cellular dynamics of migrating neuroblasts and interactions between neuroblasts and astrocytes which both represent the major cellular component of the RMS. Our previous experiments have brought evidence about the existence of a small population of mature neurons in the adult rat RMS with capacity to produce nitric oxide (NO). In order to further support functional significance of nitrergic cells, the aim of the present study was to determine whether NO producing neurons could form synapses. Sagittal sections from the adult rat brain were processed for simultaneous immunohistochemical detection of neuronal nitric oxide synthase (nNOS), the enzyme present in NO producing cells and synaptophysin, a glycoprotein found in synaptic vesicles. Synaptophysin positivity in the RMS was significantly lower in comparison with other brain areas, but its colocalization with nNOS-positive neurons was obvious. Our results suggest that nitrergic neurons in the RMS could be involved in a neuronal circuitry with potential impact on regulation of neurogenesis in the RMS.     

Ionizing Radiation Perturbs Cell Cycle Progression of Neural Precursors in the Subventricular Zone Without Affecting Their Long-Term Self-Renewal

Damage to normal human brain cells from exposure to ionizing radiation may occur during the course of radiotherapy or from accidental exposure. Delayed effects may complicate the immediate effects resulting in neurodegeneration and cognitive decline. We examined cellular and molecular changes associated with exposure of neural stem/progenitor cells (NSPs) to ¹³⁷Cs γ-ray doses in the range of 0 to 8 Gy. Subventricular zone NSPs isolated from newborn mouse pups were analyzed for proliferation, self-renewal, and differentiation, shortly after irradiation. Strikingly, there was no apparent increase in the fraction of dying cells after irradiation, and the number of single cells that formed neurospheres showed no significant change from control. Upon differentiation, irradiated neural precursors did not differ in their ability to generate neurons, astrocytes, and oligodendrocytes. By contrast, progression of NSPs through the cell cycle decreased dramatically after exposure to 8 Gy (p < .001). Mice at postnatal day 10 were exposed to 8 Gy of γ rays delivered to the whole body and NSPs of the subventricular zone were analyzed using a four-color flow cytometry panel combined with ethynyl deoxyuridine incorporation. Similar flow cytometric analyses were performed on NSPs cultured as neurospheres. These studies revealed that neither the percentage of neural stem cells nor their proliferation was affected. By contrast, γ-irradiation decreased the proliferation of two classes of multipotent cells and increased the proliferation of a specific glial-restricted precursor. Altogether, these results support the conclusion that primitive neural precursors are radioresistant, but their proliferation is slowed down as a consequence of γ-ray exposure.     

Jagged1 is necessary for postnatal and adult neurogenesis in the dentate gyrus

Understanding the mechanisms that control the maintenance of neural stem cells is crucial for the study of neurogenesis. In the brain, granule cell neurogenesis occurs during development and adulthood, and the generation of new neurons in the adult subgranular zone of the dentate gyrus contributes to learning. Notch signaling plays an important role during postnatal and adult subgranular zone neurogenesis, and it has been suggested as a potential candidate to couple cell proliferation with stem cell maintenance. Here we show that conditional inactivation of Jagged1 affects neural stem cell maintenance and proliferation during postnatal and adult neurogenesis of the subgranular zone. As a result, granule cell production is severely impaired. Our results provide additional support to the proposal that Notch/Jagged1 activity is required for neural stem cell maintenance during granule cell neurogenesis and suggest a link between maintenance and proliferation of these cells during the early stages of neurogenesis.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

Early Postnatal In Vivo Gliogenesis From Nestin-Lineage Progenitors Requires Cdk5

The early postnatal period is a unique time of brain development, as diminishing amounts of neurogenesis coexist with waves of gliogenesis. Understanding the molecular regulation of early postnatal gliogenesis may provide clues to normal and pathological embryonic brain ontogeny, particularly in regards to the development of astrocytes and oligodendrocytes. Cyclin dependent kinase 5 (Cdk5) contributes to neuronal migration and cell cycle control during embryogenesis, and to the differentiation of neurons and oligodendrocytes during adulthood. However, Cdk5’s function in the postnatal period and within discrete progenitor lineages is unknown. Therefore, we selectively removed Cdk5 from nestin-expressing cells and their progeny by giving transgenic mice (nestin-CreERT2/R26R-YFP/CDK5^flox/flox [iCdk5] and nestin-CreERT2/R26R-YFP/CDK5^wt/wt [WT]) tamoxifen during postnatal (P) days P2-P 4 or P7-P 9, and quantified and phenotyped recombined (YFP+) cells at P14 and P21. When Cdk5 gene deletion was induced in nestin-expressing cells and their progeny during the wave of cortical and hippocampal gliogenesis (P2-P4), significantly fewer YFP+ cells were evident in the cortex, corpus callosum, and hippocampus. Phenotypic analysis revealed the cortical decrease was due to fewer YFP+ astrocytes and oligodendrocytes, with a slightly earlier influence seen in oligodendrocytes vs. astrocytes. This effect on cortical gliogenesis was accompanied by a decrease in YFP+ proliferative cells, but not increased cell death. The role of Cdk5 in gliogenesis appeared specific to the early postnatal period, as induction of recombination at a later postnatal period (P7-P9) resulted in no change YFP+ cell number in the cortex or hippocampus. Thus, glial cells that originate from nestin-expressing cells and their progeny require Cdk5 for proper development during the early postnatal period.     

Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development

Prematurely born babies are often treated with glucocorticoids. We studied the consequences of an early postnatal and short dexamethasone treatment (0.1-0.01 microg/g, days 1-4) on lung development in rats, focusing on its influence on peaks of cell proliferation around day 4 and of programmed cell death at days 19-21. By morphological criteria, we observed a dexamethasone-induced premature maturation of the septa (day 4), followed by a transient septal immatureness and delayed alveolarization leading to complete rescue of the structural changes. The numbers of proliferating (anti-Ki67) and dying cells (TdT-mediated dUTP nick end labeling) were determined and compared with controls. In dexamethasone-treated animals, both the peak of cell proliferation and the peak of programmed cell death were reduced to baseline, whereas the expression of tissue transglutaminase (transglutaminase-C), another marker for postnatal lung maturation, was not significantly altered. We hypothesize that a short neonatal course of dexamethasone leads to severe but transient structural changes of the lung parenchyma and influences the balance between cell proliferation and cell death even in later stages of lung maturation.     

Effects of unilateral olfactory deprivation in the developing opossum,Monodelphis domestica

Unilateral naris closure in young rodents leads to striking alterations in the development of the ipsilateral olfactory system. One of the most pronounced effects is a 25% reduction in the size of the experimental olfactory bulb, a change that stems in part from decreased cell survival. Since naris occlusion in rodents alters the system more during development than in adulthood, we investigated the consequences of olfactory deprivation in a species that is born in a very immature state, Monodelphis domestica. In this pouchless marsupial, offspring are born after a short 14-day gestation. In the present study, the thymidine analogue bromodeoxyuridine was used to examine early postnatal neurogenesis in the olfactory bulb. Unlike rats and mice, neurogenesis of the main output neurons (the mitral cells) continues into postnatal life. Unilateral naris closure was begun on postnatal day 4 (P4) or P5 in Monodelphis and continued for 30 or 60 days. Laminar volume measurements revealed a significant reduction in the size of the experimental bulb following 60, but not 30, days of early olfactory deprivation. Mitral cell number estimates indicated a significant reduction after both 30 and 60 days of naris closure. The immaturity of Monodelphis offspring may render the population of mitral cells susceptible to the effects of olfactory deprivation. These findings suggest that afferent activity plays a role in the survival of all bulb neurons, irrespective of cell class. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 429–438, 1997     

Cell Proliferation in the Adult Rat Rostral Migratory Stream Following Exposure to Gamma Irradiation

Summary 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.     

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice

Ezrin is a member of the ezrin–radixin–moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell–matrix recognition, cell–cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.     

Maternal perinatal undernutrition impairs chromaffin cells proliferation in the postnatal rat.

Numerous data show that malnutrition during early life programs chronic diseases in adulthood. Many of these disorders may result from alterations in the development of neuroendocrine systems, such as the hypothalamo-pituitary-adrenal axis and the sympathoadrenal system. We have previously reported that maternal 50% food restriction during late pregnancy and lactation reduces adrenal weight and impairs chromaffin cell differentiation in male rats at weaning. In addition, maternal undernutrition modifies the expression of several genes involved in proliferation and apoptosis. This study therefore investigated the impact of maternal food restriction on adrenal cell growth in the late postnatal rat. Histological analysis showed that the number of proliferating chromaffin cells assessed by nuclear labelling with BrdU was reduced by 45%, whereas the level of apoptosis visualised by caspase-3 immunoreactivity was increased by 340% in adrenal medulla of offspring from undernourished mothers. In contrast, maternal food restriction did not affect proliferation and apoptosis in cortical cells of rats. These developmental changes were associated with overexpression of TGFbeta2. These data show that perinatal undernutrition impairs the balance between chromaffin cell proliferation and apoptosis. These modifications may lead to "malprogramming" of adrenal medulla development, which could contribute to the pathogenesis of chronic diseases in adulthood.